U.S. patent application number 13/786206 was filed with the patent office on 2016-06-16 for gram-positive bacteria specific binding compounds.
The applicant listed for this patent is AIMM Therapeutics B.V., Genentech, Inc.. Invention is credited to Tim BEAUMONT, Eric J. BROWN, Wouter L.W. HAZENBOS, Kimberly KAJIHARA, Mark Jeroen KWAKKENBOS, Sanjeev MARIATHASAN, John Hiroshi MORISAKI, Yi XIA.
Application Number | 20160168232 13/786206 |
Document ID | / |
Family ID | 41467048 |
Filed Date | 2016-06-16 |
United States Patent
Application |
20160168232 |
Kind Code |
A9 |
BEAUMONT; Tim ; et
al. |
June 16, 2016 |
GRAM-POSITIVE BACTERIA SPECIFIC BINDING COMPOUNDS
Abstract
The present invention provides improved binding compounds
capable of specifically binding Gram-positive bacteria. Binding
compounds are provided that are fully human, enabling therapeutic
applications in human individuals.
Inventors: |
BEAUMONT; Tim; (Ouderkerk
A/D Amstel, NL) ; KWAKKENBOS; Mark Jeroen;
(Amsterdam, NL) ; BROWN; Eric J.; (San Francisco,
CA) ; MORISAKI; John Hiroshi; (San Francisco, CA)
; HAZENBOS; Wouter L.W.; (San Francisco, CA) ;
MARIATHASAN; Sanjeev; (Millbrae, CA) ; KAJIHARA;
Kimberly; (San Francisco, CA) ; XIA; Yi;
(Cupertino, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc.
AIMM Therapeutics B.V. |
South San Francisco
Amsterdam |
CA |
US
NL |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130253175 A1 |
September 26, 2013 |
|
|
Family ID: |
41467048 |
Appl. No.: |
13/786206 |
Filed: |
March 5, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13305659 |
Nov 28, 2011 |
8617556 |
|
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13786206 |
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12837358 |
Jul 15, 2010 |
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13305659 |
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61225878 |
Jul 15, 2009 |
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Current U.S.
Class: |
530/389.5 ;
530/391.7 |
Current CPC
Class: |
A61P 37/06 20180101;
C07K 16/1278 20130101; C07K 2317/21 20130101; C07K 2317/56
20130101; G01N 33/5091 20130101; G01N 33/6854 20130101; G01N
2800/26 20130101; A61P 31/04 20180101; A61P 31/00 20180101; C07K
2317/567 20130101; A61P 37/04 20180101; G01N 2469/10 20130101; C07K
2317/565 20130101; C07K 2317/52 20130101; G01N 33/6893 20130101;
C07K 16/1271 20130101 |
International
Class: |
C07K 16/12 20060101
C07K016/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 15, 2009 |
EP |
EP 09165558.9 |
Claims
1-43. (canceled)
44. An antibody or functional part thereof capable of binding a
serine-aspartate (SD) repeat-dependent epitope.
45. The antibody of claim 44, wherein the SD repeat epitope is a
domain of an SD repeat (sdr) protein.
46. The antibody of claim 45, wherein the sdr protein is located on
the surface of a bacterium.
47. The antibody of claim 45, wherein the sdr protein is anchored
to the peptidoglycan cell wall of a gram-positive bacterium.
48. The antibody of claim 47, wherein the gram positive bacterium
is a Staphylococcus species.
49. The antibody of claim 48, wherein the bacterium is S.
aureus.
50. The antibody of claim 49, wherein the sdr protein is selected
from the group consisting of C1fA (SdrA), C1fB (SdrB), SdrC, SdrD
and SdrE.
51. The antibody of claim 48, wherein the bacterium is S.
epidermidis.
52. The antibody of claim 51, wherein the sdr protein is selected
from the group consisting of SdrF, SdrG and SdrH.
53. The antibody of claim 48, wherein the bacterium is S.
saprophyticus.
54. The antibody of claim 53, wherein the sdr protein is SdrI.
55. The antibody of claim 48, wherein the bacterium is S.
capitis.
56. The antibody of claim 55, wherein the sdr protein is SdrX.
57. The antibody of claim 48, wherein the bacterium is S.
caprae.
58. The antibody of claim 57, wherein the sdr protein is selected
from the group consisting of SdrY and SdrZ.
59. The antibody of claim 44, wherein the epitope comprises a
molecule that binds to or is associated with an Sdr protein.
60. The antibody of claim 59, wherein the molecule that binds to or
is associated with an Sdr protein is a component of a cell wall of
a gram-positive bacteria.
61. The antibody of claim 60, wherein the molecule is
peptidoglycan.
62. The antibody of claim 60, wherein the molecule is selected from
the group consisting of peptides, proteins, sugars and sugar
residues.
63. The antibody of claim 44, wherein the antibody or functional
part thereof is coupled to another moiety to form an antibody-drug
conjugates.
64. The antibody of claim 61, wherein said another moiety is a
cytotoxic agent.
65. The antibody of claim 62, wherein the cytotoxic agent is an
antibiotic.
66. The antibody of claim 44, wherein the antibody binds to cell
wall lysates from S. aureus and/or S. epidermis.
67. The antibody of claim 44, wherein the antibody is able to
compete with an antibody comprising (a) a heavy chain CDR1 sequence
RFAMS (SEQ ID NO:1), and (b) a heavy chain CDR2 sequence
SINNGNNPYYARSVQY (SEQ ID NO:2) or SINSGNNPYYARSVQY (SEQ ID NO:60),
and (c) a heavy chain CDR3 sequence DHPSSGWPTFDS (SEQ ID NO:3), and
(d) a light chain CDR1 sequence RASENVGDWLA (SEQ ID NO:4), and (e)
a light chain CDR2 sequence KTSILES (SEQ ID NO:5), and (f) a light
chain CDR3 sequence QHYXRFPYT (SEQ ID NO:6), wherein X is I or
M.
68. The antibody of claim 67, wherein the antibody binds to a
Staphylococcus species selected from the group consisting of S.
aureus, S. epidermidis, S. caprae, S. saprophyticus, S. capitis, or
methicillin-resistant S. aureus (MRSA).
69. A pharmaceutical composition comprising the antibody of claim
44.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/837,358, filed Jul. 15, 2010 and claims priority to
European Patent Application No. 09165558.9, filed Jul. 15, 2009,
and U.S. Provisional Patent Application No. 61/225,878, filed Jul.
15, 2009, the disclosures of which are incorporated herein by
reference in their entirety.
TECHNICAL FIELD
[0002] The invention relates to the fields of biology, immunology
and medicine.
BACKGROUND OF THE INVENTION
[0003] Gram-positive microorganisms cause the majority of systemic
infections. One important member of these Gram-positive pathogens
is Staphylococcus aureus (S. aureus). About 20% of the population
is a long-term carrier of S. aureus. S. aureus can cause a range of
illnesses from minor skin infections, such as pimples, impetigo
(may also be caused by Streptococcus pyogenes), boils, cellulitis
folliculitis, furuncles, carbuncles, scalded skin syndrome and
abscesses, to life-threatening diseases such as pneumonia,
meningitis, osteomyelitis, endocarditis, Toxic shock syndrome
(TSS), and septicemia. S. aureus is capable of infecting all kinds
of organs and tissues. S. aureus infections occur in
immunocompetent as well as immune compromised people. About 50% of
the infections in US intensive care units are caused by this
pathogen. Three hundred thousand S. aureus infections per year,
resulting in 12,000 deaths, are reported in the US (see also Moran
et al. NEMJ 355, 666-674 (2006)).
[0004] A major problem is the increasing antibiotic resistance of
S. aureus. Methicillin-resistant S. aureus (MRSA) emerged in the
1960s. It was initially identified in health care settings.
However, MRSA appears to be present among persons in the community
which have not been hospitalized. Treatment of MRSA is difficult
and expensive, due to the limited sensitivity of MRSA to
antibiotics. However, very few alternatives of antibiotics are
available. Vancomycin is often used to treat penicillin-resistant
MRSA. U.S. Pat. No. 6,939,543 describes a murine antibody against
lipoteichoic acid (LTA) which is capable of binding S. aureus.
Based on this murine antibody, a recombinant chimeric mouse/human
antibody was produced which contains human heavy and light chain
constant domains. However, such chimeric mouse/human antibody has
the disadvantage that murine sequences are present, which involves
the risk of serious side effects when administered to humans.
SUMMARY OF THE INVENTION
[0005] Provided herein are antibodies or functional parts thereof
or immunoglobulin chains or functional equivalents thereof, which
comprise: (a) a heavy chain CDR1 sequence comprising a sequence
which has at least 70% sequence identity to the sequence RFAMS (SEQ
ID NO:1), and/or (b) a heavy chain CDR2 sequence comprising a
sequence which has at least 70% sequence identity to the sequence
SINNGNNPYYARSVQY (SEQ ID NO:2), and/or (c) a heavy chain CDR3
sequence comprising a sequence which has at least 70% sequence
identity to the sequence DHPSSGWPTFDS (SEQ ID NO:3), and/or (d) a
light chain CDR1 sequence comprising a sequence which has at least
70% sequence identity to the sequence RASENVGDWLA (SEQ ID NO:4),
and/or, (e) a light chain CDR2 sequence comprising a sequence which
has at least 70% sequence identity to the sequence KTSILES (SEQ ID
NO:5), and/or (f) a light chain CDR3 sequence comprising a sequence
which has at least 70% sequence identity to the sequence QHYXRFPYT,
wherein X is I or M (SEQ ID NO:6). In some embodiments, the
antibody or functional part thereof or immunoglobulin chain or
functional equivalent thereof, which comprises: (a) a heavy chain
CDR1 sequence comprising a sequence which has the sequence RFAMS
(SEQ ID NO:1), and/or (b) a heavy chain CDR2 sequence comprising a
sequence which has the sequence SINNGNNPYYARSVQY (SEQ ID NO:2),
and/or (c) a heavy chain CDR3 sequence comprising a sequence which
has the sequence DHPSSGWPTFDS (SEQ ID NO:3), and/or (d) a light
chain CDR1 sequence comprising a sequence which has the sequence
RASENVGDWLA (SEQ ID NO:4), and/or, (e) a light chain CDR2 sequence
comprising a sequence which has the sequence KTSILES (SEQ ID NO:5),
and/or (f) a light chain CDR3 sequence comprising a sequence which
has the sequence QHYXRFPYT, wherein X is I or M (SEQ ID NO:6).
[0006] In some embodiments, the antibody or functional part or
immunoglobulin chain or functional equivalent has a heavy chain
sequence comprising a sequence which has at least 70% sequence
identity to the sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSV
QYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID
NO:7) and/or having a light chain sequence which has at least 70%
sequence identity to the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGS
GSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is I of M
(SEQ ID NO:8).
[0007] In some embodiments, the antibody or functional part or
immunoglobulin chain or functional equivalent has a heavy chain
sequence comprising a sequence which has at least 70% sequence
identity to the sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSV
QYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID
NO:7) and/or having a light chain sequence which has at least 70%
sequence identity to the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGS
GSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRA, wherein X is I of M
(SEQ ID NO:10).
[0008] In some embodiments, the antibody or functional part or
immunoglobulin chain or functional equivalent has a heavy chain
sequence comprising a sequence which has at least 70% sequence
identity to the sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSV
QYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID
NO:7) and/or having a light chain sequence which has at least 70%
sequence identity to the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGS
GSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIKRTV, wherein X is I of M
(SEQ ID NO:11).
[0009] In some embodiments, the antibody or functional part or
immunoglobulin chain or functional equivalent has a heavy chain
sequence comprising a sequence which has at least 70% sequence
identity to the sequence
EVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARS
VQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID
NO:9) and/or having a light chain sequence which has at least 70%
sequence identity to the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGS
GSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is I of M
(SEQ ID NO:8).
[0010] In some embodiments, the antibody or functional part or
immunoglobulin chain or functional equivalent has a heavy chain
sequence comprising a sequence which has at least 70% sequence
identity to the sequence
EVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARS
VQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID
NO:9) and/or having a light chain sequence which has at least 70%
sequence identity to the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGS
GSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRA, wherein X is I of M
(SEQ ID NO:10).
[0011] In some embodiments, the antibody or functional part or
immunoglobulin chain or functional equivalent has a heavy chain
sequence comprising a sequence which has at least 70% sequence
identity to the sequence
EVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARS
VQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID
NO:9) and/or having a light chain sequence which has at least 70%
sequence identity to the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGS
GSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIKRTV, wherein X is I of M
(SEQ ID NO:11).
[0012] In some embodiments of any of the antibodies or functional
parts or immunoglobulin chains or functional equivalents, an
asparagine has been replaced by another amino acid. In some
embodiments, the asparagine is an asparagine at position 53 of the
heavy chain, whereby the amino acid numbering is according to Kabat
(1991). In some embodiments, the other amino acid is serine.
[0013] In some embodiments of any of the antibodies or functional
parts or immunoglobulin chains or functional equivalents, at least
one amino acid other than cysteine has been replaced with cysteine.
In some embodiments, the at least one amino acid other than
cysteine is valine at light chain position 205 and/or valine at
light chain position 110, and/or alanine at heavy chain position
114, whereby the amino acid numbering is according to Kabat
(1991).
[0014] The invention provides isolated antibodies that bind to in
vivo-grown S. aureus. In some embodiments, the antibody is
human.
[0015] The invention also provides antibodies or functional parts
or immunoglobulin chains or functional equivalents capable of
binding an SD-repeat dependent epitope.
[0016] Also provided herein are antibodies or functional parts or
immunoglobulin chains or functional equivalents capable of binding
to S. aureus C1fA, C1fB, SdrC, SdrD and SdrE.
[0017] Provided herein are antibodies or functional parts or
immunoglobulin chains or functional equivalents which are capable
of competing with any antibody or functional part or immunoglobulin
chain or functional equivalent described herein for binding to a
Staphylococcus species.
[0018] In some embodiments of any of the antibodies or functional
parts or immunoglobulin chains or functional equivalents, the
antibody or functional part or immunoglobulin chain or functional
equivalent is a human antibody.
[0019] Provided herein are further isolated, synthetic or
recombinant nucleic acid sequences with a length of at least 15
nucleotides, or functional equivalents thereof, encoding at least
one CDR sequence of any of the antibodies or functional parts or
immunoglobulin chains or functional equivalents described
herein.
[0020] The invention provides isolated, synthetic or recombinant
nucleic acid sequences, or functional equivalents thereof,
comprising a sequence which has at least 70% sequence identity to a
sequence selected from the group consisting of cgctttgccatgagc (SEQ
ID NO:12), tcgatcaataatgggaataacccatactacgcacggtcggtacaatac (SEQ ID
NO:13), gatcaccctagtagtggctggcccacctttgactcc (SEQ ID NO:14),
cgggccagtgaaaacgttggtgactggttggcc (SEQ ID NO:15),
aagacatctattctagaaagt (SEQ ID NO:16) and
caacactatatacgtttcccgtacact (SEQ ID NO:17). In some embodiment, the
nucleic acid sequence or functional equivalent comprises a sequence
which has at least 70% sequence identity to at least part of a
nucleotide sequence as depicted in FIG. 1, said part having at
least 15 nucleotides and encoding at least one CDR region.
[0021] Also provided herein are isolated, synthetic or recombinant
nucleic acid sequences, or functional equivalents thereof,
comprising a sequence encoding an amino acid sequence which has at
least 70% sequence identity to the sequence RFAMS (SEQ ID NO:1),
and/or at least 70% sequence identity to the sequence
SINNGNNPYYARSVQY (SEQ ID NO:2), and/or at least 70% sequence
identity to the sequence DHPSSGWPTFDS (SEQ ID NO:3), and/or at
least 70% sequence identity to the sequence RASENVGDWLA (SEQ ID
NO:4), and/or at least 70% sequence identity to the sequence
KTSILES (SEQ ID NO:5), and/or at least 70% sequence identity to the
sequence QHYXRFPYT, wherein X is I or M (SEQ ID NO:6), and/or at
least 70% sequence identity to the sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSV
QYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID
NO:7), and/or at least 70% sequence identity to the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGS
GSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is I or M
(SEQ ID NO:8).
[0022] Provided herein are any antibodies or functional parts or an
immunoglobulin chains or functional equivalents thereof or any
nucleic acid sequence or functional equivalents thereof described
herein for use as a medicament and/or prophylactic agent.
[0023] Provided herein are also any antibodies or functional parts
or an immunoglobulin chains or functional equivalents thereof or
any nucleic acid sequences or functional equivalents thereof
described herein for use as a medicament and/or prophylactic agent
for at least in part treating and/or preventing a Gram-positive
bacterium-related disorder. Methods of treating and/or preventing a
Gram-positive bacterium-related disorder comprising administering
an effective amount of any antibodies or functional parts or an
immunoglobulin chains or functional equivalents thereof or any
nucleic acid sequences or functional equivalents thereof described
herein are further provided herein. Provided are uses of any
antibodies or functional parts or an immunoglobulin chains or
functional equivalents thereof or any nucleic acid sequences or
functional equivalents thereof described herein for the preparation
of a medicament and/or prophylactic agent for at least in part
treating and/or preventing a Gram-positive bacterium-related
disorder.
[0024] Provided herein are pharmaceutical compositions comprising
any antibodies or functional parts or an immunoglobulin chains or
functional equivalents thereof or any nucleic acid sequences or
functional equivalents thereof described herein and a
pharmaceutical acceptable carrier, diluent or excipient.
[0025] Also provided herein are isolated or recombinant antibodies
producing cell capable of producing any antibodies or functional
parts or immunoglobulin chains or functional equivalents described
herein. Provided are methods for producing any antibodies or
functional parts or immunoglobulin chains or functional equivalents
described herein, comprising providing a cell with any nucleic acid
sequences or functional equivalents described herein and allowing
said cell to translate the nucleic acid sequences or functional
equivalents described herein, thereby producing any of the
antibodies or functional parts or immunoglobulin chains or
functional equivalents described herein. In some embodiments, the
method further comprises harvesting, purifying and/or isolating any
of the antibodies or functional parts or immunoglobulin chains or
functional equivalents described herein.
[0026] Provided herein are uses of any antibodies or functional
parts or an immunoglobulin chains or functional equivalents thereof
or any nucleic acid sequences or functional equivalents thereof
described herein for diagnosis of a Staphylococcus infection.
Methods for diagnosing a Staphylococcus infection comprising
contacting a sample with any antibodies or functional parts or an
immunoglobulin chains or functional equivalents thereof or any
nucleic acid sequences or functional equivalents thereof described
herein are also provided. Provided are also antibodies or
functional parts thereof or an immunoglobulin chains or functional
equivalents thereof described herein for use in the diagnosis of a
Staphylococcus infection.
[0027] The invention also provides use of any antibodies or
functional parts or an immunoglobulin chains or functional
equivalents thereof or any nucleic acid sequences or functional
equivalents thereof described herein for detecting S. aureus and/or
S. epidermidis. Methods for detecting S. aureus and/or S.
epidermidis comprising contacting a sample with any antibodies or
functional parts or an immunoglobulin chains or functional
equivalents thereof or any nucleic acid sequences or functional
equivalents thereof described herein are also provided.
[0028] Provided are also methods for isolating S. aureus and/or S.
epidermidis bacteria comprising contacting a sample (e.g. solution)
with any antibodies or functional parts or an immunoglobulin chains
or functional equivalents thereof described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1: Heavy chain and light chain sequences of antibody
F1.
[0030] FIG. 2: F1 antibody binds to LTA preparations from several
Gram-positive bacteria. (a) LTA preparations were derived from
Sigma and tested in a capture ELISA with a mouse polyclonal
anti-LTA or (b) a highly purified set of LTA preparations derived
from W. Fischer. D25, a human anti-RSV antibody was used as
non-specific negative control and a mouse monoclonal anti-LTA
antibody as a positive control.
[0031] FIG. 3: Recombinant F1 antibody binds to S. aureus but not
to S. pneumoniae. (a) Bacteria were incubated with F1 antibody, or
a control IgG (D25, a human anti-RSV antibody) or without first
antibody (IgG-PE only). After washing the secondary antibody was
added (IgG-PE). (b) 6 clinical isolates were tested in two separate
experiments for the binding of F1. Tested were a PVL+strain (SA-1),
3 regular (SA-2 SA-3 and SA-4), and 2 MRSA strains (SA-5 and
SA-6).
[0032] FIG. 4: (A)-(H) Binding of rF1 antibody to 14 S. aureus
strains. (I)-(L) rF1 antibody binds to S. epidermidis but not to
Bacillus subtilis, Enterococcus faecalis, Listeria monocytogenes,
and Streptococcus pyogenes.
[0033] FIG. 5: A-D rF1 antibody binds to MRSA in different growth
stages, IsoC: isotype control, Med: media control (E) rF1 antibody
binds to MRSA isolated from infected tissue.
[0034] FIG. 6: rF1 antibody binds to SDR proteins. (a)
Immunoprecipitation (IP) of a commercial teichoic acid preparation
of S. aureus (Wood 46 strain) with rF1 or isotype control antibody,
followed by Western blotting with rF1 antibody (left) and mass
spectrometry analysis of fragments from the WTA preparation bound
by rF1 antibody (right), (b) Immunoprecipitation (IP) of cell wall
lysate of S. aureus (USA300 strain) with rF1 or control antibody,
followed by Western blotting (WB) with rF1 antibody (left) and mass
spectrometry analysis of cell wall fragments (USA300 strain) bound
by rF1 antibody (right) (c) Immunoprecipitation of cell wall lysate
of S. epidermidis with rF1 or isotype control antibody, followed by
Western blotting with rF1 antibody (left), mass spectrometry
analysis of cell wall fragments bound by rF1 antibody (right).
[0035] FIG. 7: Binding of rF1 to SDR proteins expressed in S.
aureus and E. coli. (a) Western blotting of S. aureus and E. coli
lysates containing overexpressed His-tagged C1fA using anti-His
(left) and rF1 (right) antibody. (b) Western Bloting of E. coli
cell lysates containing his-tagged C1fA, C1fB, SdrC, SdrD and SdrE
following incubation with S. aureus lysate with rF1 (top) or
anti-His (bottom) antibody.
[0036] FIG. 8: rF1 binds to SDR domains expressed by S. aureus.
Constructs expressed by S. aureus and tested for binding to rF1
(left), Western blots of S. aureus lysates containing the expressed
constructs with anti-MBP (maltose binding protein), anti-His and
rF1 antibody (right).
[0037] FIG. 9: Sequences of heavy chain A114C (a) and light chain
V205C (b) variants of antibody rF1. Numbering according to Kabat
(1991), boxes: CDRs.
DETAILED DESCRIPTION OF THE INVENTION
[0038] It is an object of the present invention to provide means
and methods for counteracting and/or preventing Gram-positive
bacteria-related diseases. It is a further object of the invention
to provide alternative and/or improved binding compounds against
various Gram-positive bacteria, preferably Staphylococcus species,
more preferably MRSA. It is a further object of the invention to
provide human binding compounds against various Gram-positive
bacteria, preferably Staphylococcus species, more preferably
MRSA.
[0039] The present invention provides antibodies, functional parts
thereof or immunoglobulin chains or functional equivalents thereof
capable of specifically binding Staphylococcus species in their
natural context. Therefore, they are useful for counteracting
and/or preventing and/or diagnosing disorders related to the
presence of Staphylococcus species. Preferably, S. aureus is
counteracted. In a particularly preferred embodiment, MRSA is
counteracted. Another preferred Staphylococcus species which is
counteracted is Staphylococcus epidermidis.
[0040] Staphylococcus epidermidis is usually non-pathogenic, but
patients with a compromised immune system are often at risk for
developing an infection. Infections can be both hospital and
community acquired, but they are more of a threat to hospital
patients. Most susceptible to infection with S. epidermidis are
intravenous drug users, newborns, elderly, and those using
catheters or other artificial appliances. Infections are associated
with intravascular devices (prosthetic heart valves, shunts, etc.)
but also commonly occur in prosthetic joints, catheters, and large
wounds. Symptoms include fever, headache, fatigue, anorexia and
dyspnea.
[0041] In a preferred embodiment, the present invention provides
isolated or recombinant human antibodies, functional parts thereof
or immunoglobulin chains or functional equivalents thereof capable
of specifically binding Staphylococcus species. Human antibodies
and functional parts according to the invention are capable of
binding Staphylococcus species in its natural context, so that
various Staphylococcus species are counteracted upon administration
of said antibodies, functional parts thereof or immunoglobulin
chains or functional equivalents thereof. Said human antibodies,
functional parts thereof or immunoglobulin chains or functional
equivalents thereof are therefore particularly suitable for
treatment or prevention of infection by such Staphylococcus
species. Said human antibodies, functional parts thereof or
immunoglobulin chains or functional equivalents thereof according
to the invention are more suitable for therapeutic and/or
prophylactic use for human individuals, as compared to chimeric
antibodies, because of the absence of non-human sequences. This
significantly reduces the risk of adverse side effects.
[0042] One particularly preferred antibody according to the present
invention is the antibody designated "F1", which has heavy chain
and light chain variable domain sequences as depicted in FIG. 1.
The term "F1" as used herein encompasses all F1 antibodies, for
instance isolated or recombinantly produced F1. Recombinantly
produced F1 is herein also called "rF1". The CDR sequences of F1,
which in particular contribute to the antigen-binding properties of
F1, are also depicted in FIG. 1. Antibody F1 is fully human, is
capable of specifically binding Staphylococcus species such as S.
aureus and S. epidermidis and is therefore preferred for
therapeutic use for human individuals.
[0043] Importantly, antibody F1 is capable of binding whole
bacteria in vivo as well as in vitro. Further provided is therefore
an isolated or recombinant human antibody or a functional part
thereof, which is capable of specifically binding S. aureus and/or
S. epidermidis. Furthermore, antibody F1 is capable of binding to
bacteria that have been grown in infected tissue of, for example,
an animal. Provided is therefore an isolated antibody that binds to
in vivo-grown S. aureus, wherein "in vivo-grown" is defined as
grown in infected tissue of an animal during infection with S.
aureus. Also provided is an isolated, recombinant or synthetic
immunoglobulin chain or functional equivalent thereof comprising at
least one CDR sequence of a human immunoglobulin variable region
which is specific for S. aureus and/or S. epidermidis.
[0044] A functional part of an antibody is defined as a part which
has at least one shared property as said antibody in kind, not
necessarily in amount. Said functional part is capable of binding
the same antigen as said antibody, albeit not necessarily to the
same extent. A functional part of an antibody preferably comprises
a single domain antibody, a single chain antibody, a nanobody, an
unibody, a single chain variable fragment (scFv), a Fab fragment or
a F(ab').sub.2 fragment.
[0045] A functional part of an antibody is also produced by
altering an antibody such that at least one property--preferably an
antigen-binding property--of the resulting compound is essentially
the same in kind, not necessarily in amount. This is done in many
ways, for instance through conservative amino acid substitution,
whereby an amino acid residue is substituted by another residue
with generally similar properties (size, hydrophobicity, etc), such
that the overall functioning is likely not to be seriously
affected.
[0046] As is well known by the skilled person, a heavy chain of an
antibody is the larger of the two types of chains making up an
immunoglobulin molecule. A heavy chain comprises constant domains
and a variable domain, which variable domain is involved in antigen
binding. A light chain of an antibody is the smaller of the two
types of chains making up an immunoglobulin molecule. A light chain
comprises a constant domain and a variable domain. The variable
domain is, together with the variable domain of the heavy chain,
involved in antigen binding.
[0047] Complementary-determining regions (CDRs) are the
hypervariable regions present in heavy chain variable domains and
light chain variable domains. The CDRs of a heavy chain and the
connected light chain of an antibody together form the
antigen-binding site.
[0048] A functional equivalent of an immunoglobulin chain is
defined herein as an artificial binding compound, comprising at
least one CDR sequence of an immunoglobulin chain.
[0049] Now that the present invention provides the insight that the
CDR sequences depicted in FIG. 1 provide desired binding
characteristics, a skilled person is well capable of generating
variants comprising at least one altered CDR sequence. For
instance, conservative amino acid substitution is applied. It is
also possible to alter at least one CDR sequence depicted in FIG. 1
in order to generate a variant antibody, or a functional part
thereof, with at least one altered property as compared to F1.
Preferably, an antibody or functional part is provided comprising a
CDR sequence which is at least 70% identical to a CDR sequence as
depicted in FIG. 1, so that the favorable binding characteristics
of F1 are at least in part maintained or even improved. A CDR
sequence as depicted in FIG. 1 is preferably altered such that the
resulting antibody or functional part comprises at least one
improved property, such as for instance an improved binding
affinity, selectivity and/or stability, as compared to F1. Variant
antibodies or functional parts thereof comprising an amino acid
sequence which is at least 70% identical to a CDR sequence as
depicted in FIG. 1 are therefore also within the scope of the
present invention. Various methods are available in the art for
altering an amino acid sequence. For instance, a heavy chain or
light chain sequence with a desired CDR sequence is artificially
synthesized. Preferably, a nucleic acid sequence encoding a CDR
sequence is mutated, for instance using random--or
site-directed--mutagenesis.
[0050] In one embodiment the invention therefore provides an
antibody or functional part thereof or immunoglobulin chain or
functional equivalent thereof, which comprises: [0051] a heavy
chain CDR1 sequence comprising a sequence which has at least 70%
sequence identity to the sequence RFAMS (SEQ ID NO:1), and/or
[0052] a heavy chain CDR2 sequence comprising a sequence which has
at least 70% sequence identity to the sequence SINNGNNPYYARSVQY
(SEQ ID NO:2), and/or [0053] a heavy chain CDR3 sequence comprising
a sequence which has at least 70% sequence identity to the sequence
DHPSSGWPTFDS (SEQ ID NO:3).
[0054] Further provided is an antibody or functional part thereof
or immunoglobulin chain or functional equivalent thereof, which
comprises: [0055] a light chain CDR1 sequence comprising a sequence
which has at least 70% sequence identity to the sequence
RASENVGDWLA (SEQ ID NO:4), and/or [0056] a light chain CDR2
sequence comprising a sequence which has at least 70% sequence
identity to the sequence KTSILES (SEQ ID NO:5), and/or [0057] a
light chain CDR3 sequence comprising a sequence which has at least
70% sequence identity to the sequence QHYXRFPYT, wherein X is I or
M (SEQ ID NO:6).
[0058] The above mentioned CDR sequences are the CDR sequences of
antibody F1; VH IgHV3-23 and VL IgKV1-5, and variants thereof.
Binding compounds comprising CDR sequences with at least 70%
sequence identity to F1 CDRs are particularly suitable for
counteracting and/or preventing (the effects of) infections by
S.aureus and/or S. epidermidis. It was found that a variant of F1,
comprising VH IgHV3-23 and VL IgKV1-5, in which the isoleucine in
light chain CDR3 of the light chain was altered to a methionine,
was still capable of specifically binding Staphylococcus species
such as S. aureus and S. epidermidis.
[0059] Preferably, a binding compound according to the invention
comprises a CDR sequence which is at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
86%, more preferably at least 87%, more preferably at least 88%,
more preferably at least 89%, more preferably at least 90%
identical to at least one of the CDR sequences depicted in FIG. 1.
Most preferably, a binding compound according to the invention
comprises a CDR sequence which is at least 91%, more preferably at
least 92%, more preferably at least 93%, more preferably at least
94%, more preferably at least 95% identical to at least one of the
CDR sequences depicted in FIG. 1. The particularly preferred
antibody F1, described above, comprises CDR sequences which consist
of the CDR sequences depicted in FIG. 1. A particularly preferred
embodiment according to the invention thus provides an isolated,
synthetic or recombinant antibody or a functional equivalent
thereof which is capable of specifically binding S.aureus and/or S.
epidermidis and which comprises: [0060] a heavy chain CDR1 sequence
comprising the sequence RFAMS (SEQ ID NO:1), and/or [0061] a heavy
chain CDR2 sequence comprising the sequence SINNGNNPYYARSVQY (SEQ
ID NO:2), and/or [0062] a heavy chain CDR3 sequence comprising the
sequence DHPSSGWPTFDS (SEQ ID NO:3), and/or [0063] a light chain
CDR1 sequence comprising the sequence RASENVGDWLA (SEQ ID NO:4),
and/or [0064] a light chain CDR2 sequence comprising the sequence
KTSILES (SEQ ID NO:5), and/or [0065] a light chain CDR3 sequence
comprising the sequence QHYXRFPYT, wherein X is I or M (SEQ ID
NO:6).
[0066] In one embodiment a binding compound is provided which
comprises the heavy chain CDR1 and CDR2 sequences and the light
chain CDR1 and CDR2 sequences as depicted in FIG. 1, or sequences
that are at least 70%, preferably at least 75%, more preferably at
least 80%, more preferably at least 81%, more preferably at least
82%, more preferably at least 83%, more preferably at least 84%,
more preferably at least 85% identical thereto. Further provided is
therefore an isolated, synthetic or recombinant antibody or a
functional part thereof or an immunoglobulin chain or a functional
equivalent thereof which comprises a heavy chain CDR1 sequence
comprising a sequence which is at least 70% identical to the
sequence RFAMS (SEQ ID NO:1) and a heavy chain CDR2 sequence
comprising a sequence which is at least 70% identical to the
sequence SINNGNNPYYARSVQY (SEQ ID NO:2) and a light chain CDR1
sequence comprising a sequence which is at least 70% identical to
the sequence RASENVGDWLA (SEQ ID NO:4) and a light chain CDR2
sequence comprising a sequence which is at least 70% identical to
the sequence KTSILES (SEQ ID NO:5). Said binding compound
preferably comprises CDR sequences which are at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 86%, more preferably at least 87%, more
preferably at least 88%, more preferably at least 89%, more
preferably at least 90%, more preferably at least 91%, more
preferably at least 92%, more preferably at least 93%, more
preferably at least 94%, most preferably at least 95% identical to
the above mentioned heavy chain CDR sequences and light chain CDR
sequences. Preferably, said binding compound also comprises a heavy
chain CDR3 sequence comprising a sequence which is at least 70%
identical to the sequence DHPSSGWPTFDS (SEQ ID NO:3), and/or a
light chain CDR3 sequence comprising a sequence which is at least
70% identical to the sequence QHYXRFPYT, wherein X is I or M (SEQ
ID NO:6). A binding compound comprising the above mentioned heavy
chain CDR1, CDR2 and CDR3 sequences as well as the above mentioned
light chain CDR1, CDR2 and CDR3 sequences is also provided.
[0067] Now that a human antibody capable of specifically binding
Staphylococcus species has been provided by the present invention,
it has become possible to produce an immunoglobulin chain or
functional equivalent thereof comprising at least one CDR sequence
of a human immunoglobulin variable domain which is specific for
Staphylococcus species. Further provided is thus an isolated,
recombinant or synthetic immunoglobulin chain or functional
equivalent thereof comprising at least one CDR sequence of a human
immunoglobulin variable region which is specific for Staphylococcus
species. In a preferred embodiment, a human antibody is provided.
Optionally, said at least one human CDR sequence or at least one
sequence in at least one of the framework regions is optimized,
preferably in order to improve binding efficacy or stability. This
is for instance done by mutagenesis experiments where after the
stability and/or binding efficacy of the resulting compounds are
preferably tested and an improved binding compound is selected.
[0068] Besides optimizing CDR sequences in order to improve binding
efficacy or stability, it is often advantageous to optimize at
least one sequence in at least one of the framework regions. This
is preferably done in order to improve binding efficacy or
stability. Framework sequences are for instance optimized by
mutating a nucleic acid molecule encoding such framework sequence
where after the characteristics of the resulting antibody--or
functional part--are preferably tested. This way, it is possible to
obtain improved antibodies or functional parts. In a preferred
embodiment, human germline sequences are used for framework regions
in antibodies or functional parts thereof or immunoglobulin chains
or functional equivalents according to the invention. The use of
germline sequences preferably minimizes the risk of immunogenicity
of said antibodies, immunoglobulin chains or functional equivalents
or parts, because these sequences are less likely to contain
somatic alterations which are unique to individuals from which the
framework regions are derived, and may cause an immunogenic
response when applied to another human individual.
[0069] Antibodies or functional parts thereof or immunoglobulin
chains or functional equivalents thereof comprising a heavy chain
amino acid sequence which has at least 70% sequence identity to the
heavy chain sequence as depicted in FIG. 1 are also provided. Such
heavy chain sequence provides desired binding properties, as
evidenced by antibody F1. Moreover, light chain amino acid
sequences which have at least 70% sequence identity to the light
chain sequence as depicted in FIG. 1, and a light chain sequence in
which an isoleucine in CDR3 is altered to methionine, also provide
desired binding properties, as evidenced by antibody F1, and a
variant of antibody F1 comprising said alteration. Further provided
is therefore an antibody or functional part or immunoglobulin chain
or functional equivalent according to the invention, having a heavy
chain sequence comprising a sequence which has at least 70%
sequence identity to the sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSV
QYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID
NO:7) and/or having a light chain sequence which has at least 70%
sequence identity to the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKT
SILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV,
wherein in X is I or M (SEQ ID NO:8).
[0070] Several variants of the F1 antibody have been developed,
besides the variant indicated herein above in which an isoleucine
in light chain CDR3 is altered to methionine. These variants are
capable of binding to Staphylococcus species. Examples of such
antibody variants include antibodies or functional parts or
immunoglobulin chains or functional equivalents according to the
invention, having a heavy chain sequence comprising the sequence
EVQLVESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARS
VQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID
NO:9), and/or the sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSV
QYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID
NO:7), and a light chain sequence comprising the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGS
GSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRA, wherein X is I of M
(SEQ ID NO:10), and/or the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPG
KAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIK
RTV, wherein X is I of M (SEQ ID NO:11), and/or the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGS
GSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is I of M
(SEQ ID NO:8).
[0071] An antibody or functional part or immunoglobulin chain or
functional equivalent thereof according to the invention
specifically binds to proteins comprising a serine-aspartate (SD)
repeat. SD repeat (Sdr) proteins are cell-surface-associated
proteins that are present in several bacteria such as
Staphylococcus species. Sdr proteins in general comprise an
amino-terminal signal sequence, a functional domain called the A
region, an SD repeat region, a cell wall-spanning region, a LPXTG
motif, a hydrophobic membrane-spanning domain, and a series of
positively charged residues. The LPXTG motif is the target of a
transpeptidase that cleaves the motif between threonine and glycine
residues and anchors the protein to the peptidoglycan of the cell
wall of gram-positive bacteria. Sdr proteins are thought to
interact with host molecules. Known Sdr proteins include C1fA
(SdrA), C1fB (SdrB), SdrC, SdrD and SdrE of S. aureus, SdrF, SdrG
and SdrH of S. epidermidis, SdrI of S. saprophyticus, SdrX of S.
capitis, and SdrY and SdrZ of S. caprae.
[0072] Therefore, a preferred antibody or functional part thereof
or immunoglobulin chain or functional equivalent according to the
invention specifically binds S. aureus, S. epidermidis, S.
saprophyticus, S. capitis, and S. caprae. It is preferred that said
antibody, immunoglobulin chain or functional equivalent or part
thereof binds to C1fA (SdrA), C1fB (SdrB), SdrC, SdrD and SdrE of
S. aureus, SdrF, SdrG and SdrH of S. epidermidis, SdrI of S.
saprophyticus, SdrX of S. capitis, and SdrY and SdrZ of S. caprae.
The epitope of the antibody, immunoglobulin chain or functional
equivalent or part thereof according to the invention comprises an
SD repeat-dependent epitope in Sdr proteins, for instance SD
repeat-dependent epitopes as present in S. aureus C1fA, C1fB, SdrC,
SdrD and SdrE. An SD repeat-dependent epitope is herein defined as
an epitope recognized by antibody F1, which epitope require the
presence of at least part of an SD repeat region as present in, but
not limited to, S. aureus C1fA, C1f1B, SdrC, SdrD and SdrE and S.
epidermidis SdrF, SdrG and SdrH. In one embodiment said epitope may
comprise at least part of a molecule that binds to, or is
associated with an Sdr protein. Examples of such molecules include,
but are not limited to, amino acids, peptides, proteins, sugars and
sugar residues. In another embodiment, said epitope comprises
modifications of the SD repeat region. Said modifications comprise,
for example, but are not limited to, glycosylation, amidation
and/or phosphorylation. It will be clear to a skilled person that
combination of these two embodiments is also possible.
[0073] Therefore, the invention provides an antibody or functional
part or immunoglobulin chain or functional equivalent according to
the invention capable of binding an SD repeat-dependent epitope.
Also provided is an antibody or functional part or immunoglobulin
chain or functional equivalent capable of binding to S. aureus
C1fA, C1f1B, SdrC, SdrD and SdrE. Further provided is an antibody
or functional part or immunoglobulin chain or functional equivalent
which is capable of competing with an antibody or functional part
or immunoglobulin chain or functional equivalent according to the
present invention for binding to a Staphylococcus species,
preferably S. aureus and/or S. epidermidis and/or S. saprophyticus
and/or S. capitis and/or S. caprae, more preferably MRSA.
[0074] A disadvantage of antibodies is that their stability may be
reduced, for example under harsh conditions. For instance
deamidation, the removal of a functional amide group, may occur.
Deamidation is a protein degradation pathway that may affect the
biological functions of proteins, and which occurs mainly at
asparagine residues, and to a lower extent at glutamine residues.
In one embodiment, therefore, deamidation of an antibody or
functional part thereof or immunoglobulin chain or functional
equivalent thereof according to the invention is prevented by
replacing an asparagine or glutamine by another amino acid. An
asparagine is preferably replaced by an amino acid other than
glutamine, because deamidation may also occur at a glutamine
residue. Replacement of an asparagine preferably does not
substantially affect binding affinity of an antibody according to
the invention to an antigen. In one embodiment deamidation of an
asparagine at position 53 of the heavy chain (numbering according
to Kabat, 1991) is prevented by replacing said asparagine by
another amino acid. By preventing deamidation of an asparagine at
position 53 the stability of an antibody or functional part thereof
or immunoglobulin chain or functional equivalent thereof according
to the invention is preferably increased. As is shown in the
examples, despite the fact that said asparagine is located in a
CDR, replacing an asparagine at said position does not
substantially affect the binding affinity for an antigen of an
antibody or functional part thereof or immunoglobulin chain or
functional equivalent thereof according to the invention. An
asparagine at position 53 of the heavy chain is preferably replaced
by an amino acid other than glutamine, more preferably an
asparagine at said position is replaced by serine. Therefore, also
provided by the invention is therefore an antibody or functional
part or immunoglobulin chain or functional equivalent according to
the invention, wherein an asparagine, preferably an asparagine at
position 53 of the heavy chain, is replaced by another amino acid,
preferably serine.
[0075] In one embodiment, an antibody or functional part thereof or
immunoglobulin chain or functional equivalent thereof according to
the invention is coupled to another moiety to form antibody-drug
conjugates. An antibody or functional part thereof or
immunoglobulin chain or functional equivalent thereof according to
the invention is for instance coupled to a cytotoxic agent, such as
an antibiotic. The term "cytotoxic agent" as used herein refers to
a substance that reduces or blocks the function, or growth, of
bacteria and/or causes destruction of bacteria. Said other moiety,
for example a cytotoxic agent, is preferably coupled to said
antibody or functional part thereof through a thiol group.
Therefore, preferably one or more cysteines are incorporated into
said antibody or functional part thereof or immunoglobulin chain or
functional equivalent thereof. Cysteines contain a thiol group and
therefore, incorporation of one or more cysteines into, or
replacement of one or more amino acids by one or more cysteines of
an antibody or functional part thereof according to the invention
enable coupling thereof to another moiety. Said one or more
cysteines are preferably introduced into an antibody of functional
equivalent thereof according to the invention at a position where
it does not influence folding of said antibody or functional
equivalent, and does not alter antigen binding or effector
function. The invention therefore provides an antibody or
functional part thereof or immunoglobulin chain or functional
equivalent thereof according to the invention wherein at least one
amino acid other than cysteine has been replaced by a cysteine.
Preferably at least two amino acids other than cysteine have been
replaced by cysteine. In a preferred embodiment, said at least one
amino acid other than cysteine is valine at light chain position
15, and/or alanine at light chain position 144, and/or serine at
light chain position 168, and/or valine at light chain position 205
and/or valine at light chain position 110, and/or alanine at heavy
chain position 84, and/or alanine at heavy chain position 114,
and/or alanine at heavy chain position 168, and/or serine at heavy
chain position 172, more preferably valine at light chain position
205 and/or valine at light chain position 110, and/or alanine at
heavy chain position 114 (numbering according to Kabat, 1991). A
skilled person will understand that, as an alternative or in
addition, one or more other amino acids of the heavy and/or light
chain can be replaced by cysteine if the replacement does not
influence folding of said antibody or functional equivalent, and
does not alter antigen binding or effector function.
[0076] In international patent applications WO2006/034488,
WO2008/141044, WO2009/052249, WO2009/012256, WO2009/012268 and
WO2009/099728 methods for engineering antibodies with reactive
cysteine residues are described as well as amino acid positions
suitable for cysteine engineering.
[0077] An antibody or functional part or immunoglobulin chain or
functional equivalent according to the invention preferably
comprises a variable heavy chain sequence and/or a variable light
chain sequence which is at least 75%, more preferably at least 80%,
more preferably at least 85%, more preferably at least 90%, most
preferably at least 95% identical to a heavy chain sequence and/or
the light chain sequence as depicted in FIG. 1, or a light chain
sequence as depicted in FIG. 1 in which the isoleucine in CDR3 is
altered to a methionine. The higher the identity, the more closely
said binding compound resembles antibody F1. An antibody or
functional part or immunoglobulin chain or functional equivalent
according to the invention preferably comprises a heavy chain as
well as a light chain which resemble the heavy and light chain of
F1. Further provided is therefore an antibody or functional part
thereof or immunoglobulin chain or functional equivalent thereof
comprising a heavy chain sequence and a light chain sequence which
are at least 70%, more preferably at least 80%, more preferably at
least 85%, more preferably at least 90%, most preferably at least
95% identical to the heavy chain sequence and the light chain
sequence as depicted in FIG. 1, or a light chain sequence as
depicted in FIG. 1 in which the isoleucine in CDR3 is altered to a
methionine. In one embodiment an antibody or functional part is
provided which has a heavy chain sequence as depicted in FIG. 1 and
a light chain sequence as depicted in FIG. 1 or a light chain
sequence as depicted in FIG. 1 in which the isoleucine in CDR3 is
altered to a methionine.
[0078] One embodiment provides an antibody or functional part
thereof or immunoglobulin chain or functional equivalent thereof
comprising a heavy chain sequence consisting of the heavy chain
sequence as depicted in FIG. 1, and/or comprising a light chain
sequence consisting of the light chain sequence as depicted in FIG.
1 or a light chain sequence as depicted in FIG. 1 in which the
isoleucine in CDR3 is altered to a methionine. Alternatively, as is
well known by the skilled person, it is possible to generate a
shortened heavy chain or light chain sequence while maintaining a
binding property of interest.
[0079] Preferably, such a shortened heavy chain or light chain is
generated which has a shorter constant region, as compared to the
original heavy or light chain. The variable domain is preferably
maintained. For instance, a Fab fragment or F(ab').sub.2 fragment
or a single domain antibody or a single chain antibody or a
nanobody or an unibody or a scFv fragment based on a heavy chain
sequence or light chain sequence depicted in FIG. 1 is produced. A
functional part of an antibody comprising at least a functional
part of a sequence as depicted in FIG. 1, or a light chain sequence
as depicted in FIG. 1 in which the isoleucine in CDR3 is altered to
a methionine, is therefore also provided. Said functional part has
a length of at least 20 amino acids and comprises at least one
sequence selected from the group consisting of a sequence which is
at least 70% identical to the heavy chain CDR1 sequence depicted in
FIG. 1 and a sequence which is at least 70% identical to the heavy
chain CDR2 sequence depicted in FIG. 1 and a sequence which is at
least 70% identical to the heavy chain CDR3 sequence depicted in
FIG. 1 and a sequence which is at least 70% identical to the light
chain CDR1 sequence depicted in FIG. 1 and a sequence which is at
least 70% identical to the light chain CDR2 sequence depicted in
FIG. 1 and a sequence which is at least 70% identical to the light
chain CDR3 sequence depicted in FIG. 1, or a light chain CDR3
sequence as depicted in FIG. 1 in which the isoleucine is altered
to a methionine.
[0080] The invention further provides an isolated, synthetic or
recombinant nucleic acid sequence or a functional equivalent
thereof with a length of at least 15 nucleotides, preferably at
least 30 nucleotides, more preferably at least 50 nucleotides, more
preferably at least 75 nucleotides, encoding a binding compound
according to the invention. Such nucleic acid is for instance
isolated from a B-cell which is capable of producing an antibody
according to the invention. A preferred embodiment provides a
nucleic acid sequence comprising a sequence which has at least 70%
sequence identity to at least 15 nucleotides of a nucleic acid
sequence as depicted in FIG. 1. A nucleic acid sequence according
to the invention preferably comprises a sequence which has at least
75%, more preferably at least 80%, more preferably at least 85%,
more preferably at least 90%, most preferably at least 95% sequence
identity to at least 15 nucleotides of a nucleic acid sequence as
depicted in FIG. 1. Preferably, said nucleic acid sequence as
depicted in FIG. 1 comprises at least one CDR encoding
sequence.
[0081] One preferred embodiment provides an isolated, synthetic or
recombinant nucleic acid sequence with a length of at least 15
nucleotides, or a functional equivalent thereof, encoding at least
one CDR sequence of an antibody or immunoglobulin chain according
to the invention. Said nucleic acid sequence preferably encodes at
least one CDR sequence which has at least 70% sequence identity to
a CDR region of antibody F1. Nucleic acid sequences encoding F1 CDR
regions are depicted in FIG. 1. Further provided is therefore an
isolated, synthetic or recombinant nucleic acid sequence, or a
functional equivalent thereof, comprising a sequence which has at
least 70% sequence identity to a sequence selected from the group
consisting of cgctttgccatgagc (SEQ ID NO:12),
tcgatcaataatgggaataacccatactacgcacggtcggtacaatac (SEQ ID NO:13),
gatcaccctagtagtggctggcccacctttgactcc (SEQ ID NO:14),
cgggccagtgaaaacgttggtgactggttggcc (SEQ ID NO:15),
aagacatctattctagaaagt (SEQ ID NO:16) and
caacactatatacgtttcccgtacact (SEQ ID NO:17).
[0082] Said nucleic acid sequence or functional equivalent
preferably comprises a sequence which has at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90% sequence identity to any of the above
mentioned sequences. Further provided is a nucleic acid sequence or
functional equivalent thereof comprising a sequence which has at
least 70% sequence identity to at least part of a nucleotide
sequence as depicted in FIG. 1, said part having at least 15
nucleotides and encoding at least one CDR region.
[0083] A nucleic acid sequence or functional equivalent thereof
according to the present invention preferably encodes a region
which has at least 70% sequence identity to an F1 CDR region, an F1
heavy chain and/or an F1 light chain. One embodiment thus provides
an isolated, synthetic or recombinant nucleic acid sequence, or a
functional equivalent thereof, comprising a sequence encoding an
amino acid sequence which has at least 70% sequence identity to the
sequence RFAMS (SEQ ID NO:1), and/or at least 70% sequence identity
to the sequence SINNGNNPYYARSVQY (SEQ ID NO:2), and/or at least 70%
sequence identity to the sequence DHPSSGWPTFDS (SEQ ID NO:3),
and/or at least 70% sequence identity to the sequence RASENVGDWLA
(SEQ ID NO:4), and/or at least 70% sequence identity to the
sequence KTSILES (SEQ ID NO:5), and/or at least 70% sequence
identity to the sequence QHYXRFPYT, wherein X is I or M (SEQ ID
NO:6), and/or at least 70% sequence identity to the sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWV
ASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSW
GPGTLVTVSS (SEQ ID NO:7), and/or at least 70% sequence identity to
the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGS
GSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEIKRTV, wherein X is I or M
(SEQ ID NO:8). Also provided are nucleic acid sequences or
functional equivalents thereof encoding variants of the F1 antibody
according to the invention. Provided by the invention are, for
example, nucleic acid sequences encoding a heavy chain sequence
comprising the sequence EVQLVESGGGLVQPGGSLR
LSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSVQYRFTVSRDVSQNTVSLQ
MNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID NO:9), and/or the
sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTLSRFAMSWVRQAPGRGLEWVASINNGNNPYYARSV
QYRFTVSRDVSQNTVSLQMNNLRAEDSATYFCAKDHPSSGWPTFDSWGPGTLVTVSS (SEQ ID
NO:7), and encoding a light chain sequence comprising the sequence
DIQLTQSPSALPASVGDRVSI
TCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATY
YCQHYXRFPYTFGQGTKLEIKRA, wherein X is I of M (SEQ ID NO:10), and/or
the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKPGKAPNLLIYKTSILESGVPSRFSG- S
GSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKVEIKRTV, wherein X is I of M
(SEQ ID NO:11), and/or the sequence
DIQLTQSPSALPASVGDRVSITCRASENVGDWLAWYRQKP
GKAPNLLIYKTSILESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQHYXRFPYTFGQGTKLEI
KRTV, wherein X is I of M (SEQ ID NO:8).
[0084] In one embodiment an asparagine at position 53 of the heavy
chain (numbering according to Kabat, 1991) is replaced by another
amino acid other than glutamine, in order to prevent deamidation of
the asparagine at said position. Preferably said asparagine is
replaced by a serine.
[0085] The term "% sequence identity" is defined herein as the
percentage of residues in a candidate amino acid of nucleic acid
sequence that is identical with the residues in a reference
sequence after aligning the two sequences and introducing gaps, if
necessary, to achieve the maximum percent identity. Methods and
computer programs for the alignment are well known in the art.
[0086] As used herein, a nucleic acid molecule or nucleic acid
sequence of the invention preferably comprises a chain of
nucleotides, more preferably DNA and/or RNA. In other embodiments a
nucleic acid molecule or nucleic acid sequence of the invention
comprises other kinds of nucleic acid structures such as for
instance a DNA/RNA helix, peptide nucleic acid (PNA), locked
nucleic acid (LNA) and/or a ribozyme. Such other nucleic acid
structures are referred to as functional equivalents of a nucleic
acid sequence. The term "functional equivalent of a nucleic acid
sequence" also encompasses a chain comprising non-natural
nucleotides, modified nucleotides and/or non-nucleotide building
blocks which exhibit the same function as natural nucleotides.
[0087] A nucleic acid sequence according to the present invention
is particularly useful for generating binding compounds which are
specific for S. aureus and/or S. epidermidis. This is for instance
done by introducing such nucleic acid sequence into a cell so that
the cell's nucleic acid translation machinery will produce the
encoded binding compound. In one embodiment, genes encoding a heavy
and/or light chain according to the invention are expressed in so
called producer cells, such as for instance cells of a Chinese
hamster ovary (CHO), NSO (a mouse myeloma) or 293(T) cell line,
some of which are adapted to commercial antibody production.
Proliferation of said producer cells results in a producer cell
line capable of producing antibodies or functional parts thereof
according to the present invention. Preferably, said producer cell
line is suitable for producing compounds for use in humans. Hence,
said producer cell line is preferably free of pathogenic agents
such as pathogenic micro-organisms. Most preferably, binding
compounds consisting of human sequences are generated using a
nucleic acid sequence according to the invention.
[0088] An isolated or recombinant antibody producing cell capable
of producing an antibody or functional part or immunoglobulin chain
or functional equivalent according to the invention is therefore
also provided, as well as a method for producing an antibody or
functional part or immunoglobulin chain or functional equivalent
according to the invention, comprising providing a cell with a
nucleic acid sequence or functional equivalent according to the
invention and allowing said cell to translate said nucleic acid
sequence or functional equivalent according to the invention,
thereby producing said antibody or functional part or
immunoglobulin chain or functional equivalent according to the
invention.
[0089] An antibody producing cell is defined herein as a cell which
is capable of producing and/or secreting an antibody or a
functional part thereof, and/or which is capable of developing into
a cell which is capable of producing and/or secreting antibody or a
functional part thereof. An antibody producing cell according to
the invention is preferably a producer cell which is adapted to
commercial antibody production. Preferably, said producer cell is
suitable for producing compounds for use in humans.
[0090] A method according to the invention preferably further
comprises a step of harvesting, purifying and/or isolating said
antibody or functional part or immunoglobulin chain or functional
equivalent according to the invention. The thus obtained binding
compounds according to the invention are preferably used in
diagnosis of Staphylococcus infection, isolation or detection of
Staphylococcus bacteria, distinguishing between Staphylococcus
species and other gram-positive bacteria and human therapy,
optionally after additional purification, isolation and/or other
processing steps.
[0091] An antibody or functional part thereof or immunoglobulin
chain or functional equivalent thereof according to the present
invention is particularly suitable for diagnostic uses. For
instance, a sample, such as a tissue or blood sample, can be
obtained from an individual or from any other source suspected to
be infected with a Staphylococcus bacteria, preferably S. aureus
and/or S. epidermidis, more preferably MRSA. Subsequently, said
sample can be mixed with an antibody or immunoglobulin chain or
functional equivalent or part thereof according to the invention.
Said antibody, immunoglobulin chain or functional equivalent or
part will specifically bind to Staphylococcus bacteria, preferably
S. aureus and/or S. epidermidis. Bacteria bound to an antibody,
immunoglobulin chain or functional equivalent or part can be
isolated from the sample using any method known in the art, for
example, but not limited to, isolation using magnetic beads,
streptavidin-coated beads, or isolation through the use of
secondary antibodies immobilized on a column. After washing of the
bound bacteria and antibody, immunoglobulin chain or functional
equivalent or part thereof, bacteria can be eluted from said
antibody, immunoglobulin chain or functional equivalent or part by
any method known in the art. For instance, binding between bacteria
and antibody, immunoglobulin chain or functional equivalent or part
can be disrupted by increasing the concentration of salts and/or
reducing or increasing pH, and/or by addition of excess
epitope.
[0092] Isolation of Staphylococcus bacteria, preferably S. aureus
and/or S. epidermidis, may be used for various applications. For
instance, infection with several different gram-positive bacteria
may result in overlapping symptoms in an individual. In such cases,
it can be difficult to discriminate between S. aureus and/or S.
epidermidis and other gram-positive bacteria. An antibody,
immunoglobulin chain or functional equivalent or part thereof
according to the present invention can then be used to detect the
presence of S. aureus and/or S. epidermidis, or for distinguishing
between S. aureus and/or S. epidermidis and other bacteria.
Isolation of bacteria from a sample of an individual suspected of
suffering from an infection with S. aureus and/or S. epidermidis or
from any other source, such as a bacterial culture, can facilitate
detection of said Staphylococcus bacteria, because isolation
results in an increased concentration and/or a higher purity of
said Staphylococcus bacteria.
[0093] Isolation of Staphylococcus species, preferably S. aureus
and/or S. epidermidis, more preferably MRSA, can for instance
further be used to identify the specific S. aureus and/or S.
epidermidis strain, preferably the MRSA strain in a sample.
Identification of said strain can for instance be performed by
determining the sequence of the bacterial DNA. In such case it is
preferred to first obtain an isolated S. aureus and/or S.
epidermidis.
[0094] In one embodiment of the invention, an antibody,
immunoglobulin chain or functional equivalent or part thereof
according to the present invention is labeled in order to be able
to detect said antibody, immunoglobulin chain, or functional
equivalent or part, for instance, but not limited to, fluorescently
labeled, or radioactively labeled. Alternatively, an antibody or
functional part thereof or immunoglobulin chain or functional
equivalent thereof according to the invention is detected using a
labeled secondary antibody which is directed against said antibody,
immunoglobulin chain or functional equivalent or part thereof
according to the invention. If binding of said antibody,
immunoglobulin chain or functional equivalent or part thereof is
detected, S. aureus and/or S. epidermidis is present.
[0095] Provided by the invention is therefore a use of an antibody
or functional part thereof or immunoglobulin chain or functional
equivalent thereof according to the invention for diagnosis of a
Staphylococcus infection, preferably S. aureus infection, more
preferably MRSA infection, for detecting S. aureus and/or S.
epidermidis, preferably MRSA, and for distinguishing between S.
aureus and/or S. epidermidis, preferably MRSA and other
gram-positive bacteria. Also provided is an antibody or functional
part thereof or an immunoglobulin chain or functional equivalent
thereof according to the invention for use in the diagnosis of a
Staphylococcus infection, preferably S. aureus infection, more
preferably MRSA infection.
[0096] Further provided is a method for isolating S. aureus and/or
S. epidermidis bacteria from a solution using an antibody or
functional part or an immunoglobulin chain or functional equivalent
thereof according to the invention. Said method preferably
comprises providing a sample of an individual suspected of
suffering from an infection with S. aureus and/or S. epidermidis,
preferably MRSA, or from any other source, such as a bacterial
culture, adding an antibody or functional part or an immunoglobulin
chain or functional equivalent thereof according to the invention
to said sample, allowing binding of said antibody or functional
part or an immunoglobulin chain or functional equivalent thereof
according to the invention to S. aureus and/or S. epidermidis
bacteria, when present, and isolating S. aureus and/or S.
epidermidis bacteria bound to an antibody or functional part or an
immunoglobulin chain or functional equivalent thereof according to
the invention from said sample.
[0097] Now that Gram-positive bacteria-specific binding compounds
according to the invention and nucleic acid sequences coding
therefore have been provided, including human binding compounds,
improved therapeutic applications have become available.
Gram-positive bacteria such as S. aureus and/or S. epidermidis are
counteracted by binding compounds according to the invention. A
binding compound according to the invention is therefore
particularly suitable for use as a medicine or prophylactic agent.
Preferably, binding compounds are used which consist of human
sequences, or which have at most 5% of non-human sequences, in
order to reduce the chance of adverse side effects when human
individuals are treated. An antibody or functional part or an
immunoglobulin chain or functional equivalent thereof or a nucleic
acid sequence or functional equivalent thereof according to the
invention for use as a medicament and/or prophylactic agent is
therefore also herewith provided. When a nucleic acid or functional
equivalent according to the invention is administered, it will be
translated in situ into a binding compound according to the
invention. In a particularly preferred embodiment said antibody
comprises antibody F1, or a functional part thereof. Said
medicament or prophylactic agent is preferably used for
counteracting or at least in part preventing an infection by S.
aureus and/or S. epidermidis or for counteracting or at least in
part preventing adverse effects of an infection by S. aureus and/or
S. epidermidis. Further provided is therefore an antibody or
functional part or an immunoglobulin chain or functional equivalent
thereof or a nucleic acid sequence or functional equivalent thereof
according to the invention for use as a medicament and/or
prophylactic agent for at least in part treating and/or preventing
a condition related to S. aureus and/or S. epidermidis.
Non-limiting examples of such conditions are skin infections,
pimples, impetigo, boils, cellulitis folliculitis, furuncles,
carbuncles, scalded skin syndrome, abscesses, pneumonia,
meningitis, osteomyelitis, endocarditis, toxic shock syndrome and
septicemia. Preferably, S. aureus infection is counteracted or at
least in part prevented. Most preferably, an MRSA-related condition
is counteracted, diminished and/or at least in part prevented. A
use of an antibody or functional part or an immunoglobulin chain or
functional equivalent thereof or a nucleic acid sequence or
functional equivalent thereof according to the invention for the
preparation of a medicament and/or prophylactic agent for at least
in part treating and/or preventing S. aureus and/or S. epidermidis
is therefore also provided, as well as a method for at least in
part treating or preventing a condition related to S. aureus and/or
S. epidermidis, the method comprising administering to an
individual in need thereof a therapeutically effective amount of an
antibody or functional part or an immunoglobulin chain or
functional equivalent according to the invention, preferably after
said individual has been diagnosed to be infected by S. aureus
and/or S. epidermidis. Said condition preferably comprises at least
one of the S. aureus-related conditions listed above, most
preferably an MRSA-related condition.
[0098] Said antibody preferably comprises antibody F1, or a
functional part thereof.
[0099] In order to counteract Gram-positive bacteria, a binding
compound according to the invention is preferably administered to
an individual before an infection has taken place. Alternatively, a
binding compound according to the invention is administered when an
individual is already infected. Said binding compound is preferably
administered to individuals with an increased risk of
complications, such as for instance hospitalized individuals and/or
individuals with compromised immunity. Also elderly people have an
increased risk of bacterial conditions. Binding compounds according
to the invention are preferably administered via one or more
injections. Dose ranges of binding compounds according to the
invention to be used in the therapeutic applications as described
herein before are designed on the basis of rising dose studies in
the clinic in clinical trials for which rigorous protocol
requirements exist. Typical doses are between 0.1 and 10 mg per kg
body weight. For therapeutic application binding compounds
according to the invention are typically combined with a
pharmaceutically acceptable carrier, diluent and/or excipient.
Examples of suitable carriers for instance comprise keyhole limpet
haemocyanin (KLH), serum albumin (e.g. BSA or RSA) and ovalbumin.
In one preferred embodiment said suitable carrier comprises a
solution like for example saline.
[0100] In yet another embodiment a nucleic acid encoding a binding
compound according to the invention is used. As already described,
upon administration of such nucleic acid, binding compounds are
produced by the host's machinery. Produced binding compounds are
capable of preventing and/or counteracting Gram-positive bacterial
infection and/or the adverse effects of such infection. A nucleic
acid sequence or functional equivalent according to the invention
for use as a medicament and/or prophylactic agent is therefore also
herewith provided. Said nucleic acid is preferably used for
counteracting S. aureus and/or S. epidermidis, more preferably S.
aureus, most preferably MRSA. Further provided is therefore a use
of a nucleic acid sequence or functional equivalent according to
the invention for the preparation of a medicament and/or
prophylactic agent for at least in part treating and/or preventing
a Gram-positive bacterium-related condition. Said Gram-positive
bacterium-related condition preferably comprises an infection by S.
aureus or S. epidermidis, more preferably an S. aureus infection,
most preferably an MRSA infection.
[0101] Further provided is a pharmaceutical composition comprising
an antibody or functional part or an immunoglobulin chain or
functional equivalent thereof or a nucleic acid sequence or
functional equivalent thereof according to the invention and a
pharmaceutical acceptable carrier, diluent or excipient. Said
pharmaceutical composition is preferably suitable for human
use.
[0102] The invention is further explained in the following
examples. These examples do not limit the scope of the invention,
but merely serve to clarify the invention.
[0103] All patent documents and other publications that are
referred to in the detailed description are incorporated herein by
reference in their entirety.
EXAMPLES
Example 1
Methods
B Cell Isolation
[0104] B-cells were obtained from fresh blood of adults by Ficoll
separation and CD4/CD8 negative selection with MACS microbeads as
described by the manufacturer (Miltenyi Biotech). To obtain memory
B cells, cells were sorted for
CD19.sup.+CD3.sup.-CD27.sup.+IgD.sup.-IgA.sup.- on a FACSaria
(Becton Dickinson). The use of these tissues was approved by the
medical ethical committees of the institution and was contingent on
informed consent. Donors were selected based on carrying hospital
acquired MRSA strains with genotype cluster 109 and 16.
Cell Culture
[0105] B-cells maintained in standard culture medium containing
IMDM (Gibco), 8% FBS (HyClone) and penicillin/streptomycin (Roche)
were co-cultured on .gamma.-irradiated (50Gy) mouse L cell
fibroblasts stably expressing CD40L (CD40L-L cells, 10.sup.5
cells/ml) and recombinant mouse IL-21 (25ng/ml, R&D systems).
rhIL-4 (R&D) was used at 50ng/ml. Cells were routinely tested
by PCR and were found negative for the presence of mycoplasma and
EBV (data not shown).
[0106] Bulk transduced human memory B cells double positive for
NGFR and GFP were purified by FACS cell sorting and cultured in 96
well plates at a cell density of 500 cell per well. Culture
supernatants were tested in ELISAs using bacterial lysates of the
Newman and SH1000 strains. Positive cultures were subcloned at 10
cell/well cell density in 96 well plates and again tested by ELISA.
Subsequently positive cultures were seeded at 1 cell per well and
tested again by ELISA for reactivity against S. aureus strains
Newman and SH1000.
Retroviral Transduction
[0107] The BCL6 retroviral construct has been described before
(Shvarts A. et al. Genes Dev 16, 681-686 (2002)). cDNA encoding
human Bcl-xL was kindly provided by Dr. Stanley Korsmeyer. BCL6 and
Bcl-xL were separately cloned into a BCL6-NGFR and a BclxL-GFP
construct. These constructs were transfected into the LZRS
retroviral packaging cells, phoenix as described before (Jaleco A.
C. et al. Blood 94, 2637-2646 (1999); Scheeren F. A. et al. Nat
Immunol 6, 303-313 (2005)). Memory B-cells were double transduced
by the BCL6 and Bcl-xL containing retroviruses after activation on
CD40L-L cells in the presence of rmIL-21 for 36 hrs as described
before (Diehl S. A. et al. J Immunol 180, 4805-4815 (2008);
Kwakkenbos M. et al. Nat Med in press (2009)). Transduced cells
were maintained on CD40L-L cells with rmIL-21.
ELISA
[0108] To determine antibody content ELISA plates were coated with
anti-human IgG (Jackson ImmunoResearch Laboratories) at 5 .mu.g/ml
in PBS for lhr at 37.degree. C. or o/n at 4.degree. C. and washed
in ELISA wash buffer (PBS, 0.5% Tween-20). 4% milk in PBS was used
as blocking agent, before serial dilution of cell culture
supernatants and an enzyme-conjugated detection antibody was added
(dilution 1:2500 for HRP-conjugated anti-IgG (Jackson)). TMB
substrate/stop solution (Biosource) was used for development of the
ELISAs.
[0109] For screening purposes we used lysates from the Newman and
SH1000 strains. Both were made in PBS and directly coated at 5 to
10 .mu.g ml.sup.-1 before B cell culture supernatants were tested
un- or 1:2 diluted.
[0110] To explore the antigen specificity of the human IgG1 clone
F1, an LTA detection ELISA was developed. Purified LTA preparations
from S. aureus, B. subtilis, S. faecalis and S. pyogenes (Sigma)
was added to ELISA plates coated with polyclonal IgG purified mouse
anti-LTA (1/200 stock 1 mg ml.sup.-1, QED Bioscience), before
secondary antibodies were added.
[0111] In addition, we tested several LTA preparations derived from
a library developed by W. Fischer (Institut fur Biochemie, Univ of
Erlangen, Germany) and kindly provided by B. Appelmelk (VU,
Amsterdam, Netherlands) (see for more details (Keller R. et al.
Infect Immun 60, 3664-3672 (1992), Polotsky V. Y. et al. Infection
and Immunity 64, 380-383 (1996) and Greenberg J. W. et al.
Infection and Immunity 64, 3318-3325 (1996)) and reviewed in
(Weidenmaier C. et al. Nat Rev Microbiol 6, 276-287 (2008)) in
direct ELISA's. LTA preparations were coated at 1 .mu.g ml.sup.-1
before rF1 (10 .mu.g ml.sup.-1) or control antibodies (1:5 dilution
of hybridoma supernatant) were added and further detected with
conjugated anti-human or mouse antibodies. The panel of purified
LTA preparations included: B. subtilis, S. aureus, L. lactis, L.
garvieae, B. bifidum, M. luteus, L. casei, L. mesenteroides, B.
licheniformis, L. welshimeri, E. hirae, L. raffinolactis, S.
mutans, S. pneumoniae. Several variants contain or lack alanine
residues and/or lipid anchors (not depicted here).
Binding of the F1 Antibody to Bacterial Cultures
[0112] The Newman S. aureus and the S. pneumoniae strain (serotype
3) were used. Newman was cultured in TSH 50 ml O/N, then 1 ml was
resuspendend in 100 ml for 2 to 2.5 hrs till OD:1 and bacteria were
collected. S. pneumoniae was cultured in Todd Hewitt medium mixed
with yeast medium. Before, bacteria were incubated with F1
antibody, a control IgG (D25, a human anti-RSV antibody) or only
with the secondary antibody (IgG-PE only), cells were pretreated
with 100% total mouse serum to prevent background staining. After
washing the secondary antibody was added (IgG-PE). Antibody
incubations were performed for 20 min on ice.
F1 Sequence Determination and Expression Cloning
[0113] We isolated total RNA using Trizol (Invitrogen), generated
cDNA by using superscript RT, performed PCR and cloned the heavy
and light chain variable regions into the pCR2.1 TA cloning vector
(Invitrogen). To rule out reverse transcriptase or DNA polymerase
induced mutations, we performed several independent cloning
experiments. To produce recombinant F1 mAb we cloned F1heavy and
light variable regions in frame with human IgG1 and Kappa constant
regions into a pcDNA3.1 (Invitrogen) based vector and transiently
transfected 293T cells. We purified recombinant F1 from the culture
supernatant with Protein A.
Results
Generation of the F1 Clone
[0114] From three subjects who were tested positive for MRSA but
were not sick, 50-60 ml of heparin blood was collected and
peripheral B-cells were isolated after a ficoll purification step.
B-cells from several B-cell populations were double transduced with
retroviruses containing BCL6-NGFR and Bch xL-GFP (Diehl et al and
Kwakkenbos et al). From the IgG, CD27 positive population,
polyclonal mini-cultures B-cell cultures were started in 96 wells
plate with a cell density of 500 cells/well. Supernatant of these
mini-cultures was collected and used in ELISAs to screen for the
presence of S. aureus-specific IgG antibodies (in these ELISAs the
coating was cell lysates of two S. aureus strains, SH1000 and
Newman). Mini-cultures that were screened positive in both the
SH1000 as well as the Newman S. aureus were seeded in new
mini-cultures at a density of 10 cells/well. Again, supernatant of
these mini-cultures was collected and screened in the SH1000 and
Newman ELISAs. Clones that were screened positive in both ELISAs
were seeded into monoclonal cultures (i.e. 1 cell/well). After
testing the supernatant of these monoclonal cultures, one clone
(named F1) was found to produce S. aureus specific monoclonal IgG
antibodies.
Antibodies in the Supernatant of the F1 Clone Bind to LTA
Preparations from S. aureus
[0115] Generating the F1 clone, we already found that supernatant
of F1 bound to the bacterial cell lysates of two S. aureus strains,
SH1000 and Newman. The major cell wall compound of Gram-positive
bacteria is LTA, and therefore we decided to test binding of F1
supernatant to S. aureus LTA preparations in an ELISA. As shown in
Table 1, supernatant of the F1 clone binds to bacterial cell
lysates of the SH1000 and Newman S. aureus strain but also to
commercially purchased purified S. aureus LTA preparations. We
noted, however, that the binding to the LTA preparations was
significantly lower than that observed with whole bacteria.
TABLE-US-00001 TABLE 1 Supernatant of the F1 clone binds to S.
aureus LTA preparations. As a negative control an anti-mouse-HRP
conjugated secondary antibody was used. Another control was an
anti-influenza virus antibody that only detected influenza H3
protein. mouse anti-flu F1 clone anti-LTA control SH1000 1.004
-0.010 -0.007 Newman 0.753 -0.007 0.007 SA LTA (siam 0.176 -0.005
-0.009 Flu H3 0.003 -0.002 1.523 No coat -0.013 -0.014 -0.015
indicates data missing or illegible when filed
The Recombinant Produced F1 Antibody Binds to LTA Preparations from
Multiple Sources
[0116] After cloning the antibody genes into expression vectors and
producing recombinant F1 (rF1) antibody in an expression system,
the rF1 antibody was tested on purified LTA preparations from
several bacteria. As shown in FIG. 2A, the rF1 antibody binds well
to commercial LTA samples obtained from S. aureus and B. subtilis,
and a bit less profoundly to LTA samples from S. faecalis and S.
pyogenes. The rF1 antibody did not bind to an LTA sample from S.
pneumoniae (data not shown). In addition rF1 recognized highly
purified LTA samples from B. subtilis, B. licheniformis and two
isolates of S. aureus (FIG. 2b). rF1 did not bind to LTA
preparations from S. mutans (FIG. 2b) or from L. lactis, L.
garvieae, B. bifidum, M. luteus, L. casei, L. mesenteroides, L.
welshimeri, E. hirae, L. raffinolactis, S. mutans and S. pneumoniae
(results not shown).
[0117] Recombinant F1 Antibody Binds to Living S. aureus
Bacteria
[0118] In order to study whether the rF1 antibody would also
recognize living Gram-positive bacteria, binding of the rF1
antibody to S. aureus and S. pneumoniae was tested by using flow
cytometry. As shown in FIG. 3A, the rF1 antibody binds to living S.
aureus bacteria (Newman strain), but not to S. pneumoniae. In
addition we show that rF1 recognized 6 clinical S. aureus isolates;
one is a pathogenic strain which is PVL positive, 3 regular strains
and 2 MRSA strain (FIG. 3b).
Example 2
Methods
Bacterial Strains and Culture
[0119] Methicillin-resistant S. aureus (MRSA) strains USA300
(1114), USA400, N315, USA100, USA1000, COL, MRSA252, as well as
methicillin-sensitive S. aureus (MSSA) strains Reynolds, Becker,
Smith Diffuse, MN8, and vancomycin intermediate sensitive (VISA)
strain Mu50, were all obtained from the Network on Antibiotic
Resistance in Staphylococcus aureus (NARSA); MSSA strains Newman
and Rosenbach were from ATCC. Staphylococcus epidermidis, Bacillus
subtilis, Enterococcus faecalis, and Streptococcus pyogenes were
obtained from Ward's Natural Science; Listeria monocytogenes was
from ATCC. Bacteria were grown on tryptic soy agar (TSA) plates
supplemented with 5% sheep blood for 18 h at 37.degree. C. For
liquid cultures, single colonies from TSA plates were inoculated
into tryptic soy broth (TSB) and incubated at 37.degree. C. while
shaking at 200 rpm for 18 h. Fresh 100-fold dilutions of these
cultures in fresh TSB were further subcultured for various
times.
FACS Analysis of rF1 Binding to Whole Bbacteria Grown in Vitro.
[0120] For antibody staining of whole cells, bacteria were
harvested from TSA plates or TSB cultures, and washed with Hank's
Buffered Salt Solution (HBSS) without phenol red supplemented with
0.1% BSA (IgG free; Sigma) and 10 mM Hepes, pH 7.4 (HB buffer), by
centrifugation at 1700.times.g for 20 min. Bacterial concentrations
were estimated by reading optical density at 630 nm. Bacterial
suspensions of 20.times.10.sup.8 CFU/mL in HB buffer were mixed
with equal volumes of 300 .mu.g/mL of rabbit IgG (Sigma), and
incubated for 1 h at room temperature (RT) to block nonspecific IgG
binding. Primary antibodies, including rF1 and a human IgG1 isotype
control, were added to a final concentration of 2 .mu.g/mL, and
these mixtures were incubated for 15 mM at RT. After two washes
with HB, bacterial pellets were resuspended in a solution of
fluorescent anti-human IgG secondary antibodies (Jackson
Immunoresearch) and incubated for 15 min at RT. The bacteria were
washed twice with PBS, resuspended in PBS with 1% paraformaldehyde,
and analyzed by flow cytomery.
FACS Analysis of rF1 Binding to Whole Bacteria from Infected
Tissues.
[0121] For analysis of antibody binding to bacteria from infected
tissue, 4 h subcultures of USA300 in TSB were washed with PBS. Mice
were injected intravenously with a 100 .mu.L of a suspension of
USA300 in PBS with an estimated concentration of 10.times.10.sup.8
CFU/mL. Three days later, kidneys, livers, and lungs were harvested
and homogenized using conical tissue grinder tubes (VWR). When
indicated, organs were harvested at different time points of
infection. To lyse mouse cells, the homogenates were incubated for
10 min at RT in PBS containing 0.1% Triton-X100 (Thermo), 10
.mu.g/mL of DNAseI (Roche) and Complete Mini protease inhibitor
cocktail (Roche), and passed through a 40 .mu.m filter (Falcon).
The cell suspensions were washed twice with PBS and resuspended in
HB buffer, mixed with equal volumes of 600 .mu.g/mL of human IgG
(Sigma), and incubated for 1 h at RT. Primary Abs, including rF1
and a human IgG1 isotype control, were added to a final
concentration of 2 .mu.g/mL. To differentiate bacteria from mouse
organ debris, rabbit IgG anti-S. aureus (Abcam) was added to a
concentration of 20 .mu.g/mL. After incubation for 15 min at RT,
cells were washed twice with HB buffer, and resuspended in a
mixture of anti-human IgG and anti-rabbit IgG secondary antibodies,
each labeled with a different fluorochrome (Jackson
Immunoresearch). After two washes with PBS, cells were resuspended
in PBS with 2% paraformaldehyde and analyzed by flow cytometry.
After selecting bacteria from double fluorescence plots by gating
for positive staining with rabbit IgG anti-S. aureus, overlay
histogram plots of fluorescence intensities of rF1 and isotype
control were generated.
Results
[0122] rF1 Binds Strongly to 14 S. aureus Strains and S.
epidermidis
[0123] Seven Methicillin-resistant S. aureus (MRSA) strains, six
methicillin-sensitive S. aureus (MSSA) strains, one vancomycin
intermediate sensitive S. aureus (VISA) strain, S. epidermidis and
several other gram-positive species were tested for binding to rF1
antibody. As shown in FIG. 4, rF1 strongly binds to all 14 S.
aureus strains (FIG. 4(A)-(H)) and to S. epidermidis (FIG. 4I-J),
but not to the other tested gram-positive species (FIG. 4K-L).
rF1 Binds to MRSA from Different Growth Stages and from in Vivo
Infection
[0124] We tested the ability of mAb rF1 to bind to bacteria both in
different tissues and over the course of infection. We found that
rF1 bound to bacteria isolated from murine kidney, liver and lung
tissue two days after infection and that the binding to bacteria
isolated from infected kidneys was stable, binding bacteria 2, 3
and 8 days after infection.
[0125] Binding of rF1 antibody to different growth stages of MRSA
(strain USA300), namely early logarithmical growth (2 hours) and
post exponential growth (8 hours) in TSB cultures, and growth of
solid colonies on TSA plates was tested. rF1 strongly binds to all
growth stages tested (FIG. 5A-D).
[0126] Binding of rF1 antibody to MRSA (strain USA300) to whole
bacteria from infected tissue was tested in homogenized kidneys
from mice three day after the mice were systemically infected with
MRSA. As shown in FIG. 5E rF1 binds to MRSA obtained from infected
tissue.
Example 3
[0127] Further experiments were performed to identify the epitope
bound by antibody rF1. Although binding to LTA preparations had
been observed (see, e.g., Example 1), that binding was not as
robust as binding to whole bacteria suggesting that another epitope
may be involved in rF1 binding.
Methods
[0128] Immunoprecipitation, Western blotting and Mass Spectrometry
of Cell Wall Lysates of S. aureus and S. epidermidis and Commercial
WTA Cell Wall Preparation
[0129] 40 micrograms of a commercial wall teichoic acid (WTA)
preparation from Wood46 S. aureus strain (Biodesign/ Meridian Life
Sciences, Maine) was split into two parts and immunoprecipitated
with 1 ug/ml of rF1 or isotype control human antibody. Antibodies
were captured with Protein A/G Ultralink Resin (Pierce). The
samples were then treated with 50 mM dithiothreitol, 10 mM
2-Iodoacetamide and run on an 8% Tris-glycine gel and subsequently
Western blotted with rF1.
[0130] A cell wall preparation of a 20 ml overnight culture of
USA300 S. aureus strain was prepared by treatment of the culture
(40 mg cell pellet/ml) with 100 mg/ml of lysostaphin in 30%
raffinose buffer at 37.degree. C. for 30 minutes. The entire cell
wall prep was filtered, diluted up to 10 ml with NP40 Lysis buffer
and incubated twice with anti-Flag M2 Agarose (Sigma) to deplete as
much protein-A from the cell wall prep as possible. Final cell wall
preparation was split into two parts and immunoprecipitated with 1
ug/ml of rF1 or isotype control human antibody. Antibodies were
captured with Protein A/G Ultralink Resin (Pierce). The samples
were then treated with 50 mM dithiothreitol, 10 mM 2-Iodoacetamide
and run on an 8% Tris-glycine gel and subsequently silver stained
or Western blotted with rF1.
[0131] Lysates from a 20 ml overnight culture of Staphylococcus
epidermidis was prepared by bead beating in NP40 Lysis Buffer. The
resulting lysate preparation was diluted to 10 ml with NP40 Lysis
Buffer, split into two parts and immunoprecipitated with 1 ug/ml of
rF1 or control antibody. Antibodies were captured with Protein A/G
Ultralink Resin (Pierce). The samples were then treated with 50 mM
dithiothreitol, 10 mM 2-Iodoacetamide and run on an 8% Tris-glycine
gel and subsequently silver stained or Western blotted with
rF1.
[0132] For proteomic analysis, samples were applied to a precast
SDS PAGE mini gel and the resolved proteins stained with Coomassie
Blue. Gel slices from the region of the gel corresponding to bands
visualized by rF1 western blot were excised and reduced, alkylated
with iodoacetamide, and digested in situ with trypsin. Resulting
tryptic peptides were analyzed by microcapillary reverse-phase
liquid chromatography-nano electrospray tandem mass spectrometry on
a hybrid linear ion trap Fourier transform ion cyclotron resonance
mass spectrometer (LTQ-FT; Thermo Fisher) in a data-dependent
experiment. Tandem mass spectral results were submitted for
database searching using Mascot software (Matrix Science).
Expression of Exogenous C1fA Expressed in S. aureus and E. coli
[0133] S. aureus expression of His-tagged C1fA: The Clumping
factor-A (C1fA) gene was PCR amplified from the sequence encoding
the signal sequence to the sequence encoding the glycine in the
LPXTG motif from USA300 genomic DNA. A c-terminal His Tag was
engineered at the end of the LPXTG motif and ligated into the pTet
S.aureus expression vector (pSAS10 from Genentech). The resulting
construct was then electroporated into S. aureus WT RN4220. A 20 ml
culture of either the electroporated RN4220 or RN4220 empty (not
carrying a pTet expression vector) was inoculated from an overnight
culture (starting OD.sub.600 of 0.15), grown for 1 hr in trypticase
soy broth (TSB) and then induced protein expression for 2 hrs with
anhydrotetracycline (200 ng/ml). At the end of the induction
period, the S. aureus culture was pre-lysed with lysostaphin (50
ug/ml) and then further lysed by bead beating in lysis buffer (150
mM NaCl, 20 mM Tris pH 7.5, 1% triton-X, and Roche protease
inhibitor EDTA-free tablets). Cleared lysates were then incubated
with NiNta resin (Qiagen) in the presence of 10 mM Imidazole for 1
hr at 4.degree. C. to pull-down the recombinant C1fA protein.
[0134] E.coli expression of His-tagged C1fa: The Clumping factor-A
(C1fA) gene was PCR amplified from the sequence encoding the N-term
signal sequence (with the start methionine) to the sequence
encoding the glycine in the C-term LPXTG motif from USA300 genomic
DNA. The amplified PCR product was then ligated in frame with a
c-terminal His tag into the pET 21b(+) E. coli expression vector
(Novagen). The resulting construct was transformed into E.coli
BL21-gold (DE3) Competent Cells (Stratagene) and induced for
protein expression for 3.5 hrs with IPTG according to the
manufacturer's instructions. The induced E. coli culture was lysed
by bead beating in Lysis Buffer (150 mM NaCl, 20 mM Tris pH 7.5, 1%
triton-X, and Roche protease inhibitor EDTA-free tablets). Cleared
lysates were then incubated with NiNta resin (Qiagen) in the
presence of 10 mM Imidazole for 1 hr at 4.degree. C. to pull-down
the recombinant C1fA protein.
Expression of Exogenous C1fA Expressed in E. coli and Incubation
with S. aureus Lysate.sub.--
[0135] Expression and Purification of S. aureus cell surface SDR
proteins C1fA, C1fB, SdrC, SdrD, SdrE in E.coli: The C1fA, C1fB,
SdrC, SdrD and SdrE genes were PCR amplified from the sequence
encoding the mature start of the protein to the sequence encoding
the glycine in the LPXTG motif from USA300 genomic DNA and ligated
in-frame with an N-terminal Unizyme tag into the ST239 Vector
(Genentech). The constructs were transformed into E.coli 58F3
(Genentech), induced for protein expression and subsequently
purified.
[0136] NiNta capture of NT Unizyme tagged SDR proteins: 500ug of
purified N-terminal Unizyme tagged SDR proteins (C1fA, C1fB, SdrC,
SdrD, SdrE) were diluted in PBS containing protease inhibitors
(EDTA-free) and incubated with NiNta resin (Qiagen) for 1.5 hr at
4.degree. C. NiNta resin with the captured Unizyme tagged SDR
protein was then washed once with wash buffer (50 mM
NaH.sub.2PO.sub.4, 300mM NaCl; pH 8.0).
[0137] Modification of E.coli produced SDR proteins by S. aureus
lysate: A 25 ml culture was started from an overnight culture of
APan Sdr mutant (C1fA-C1fB-SdrCDE-null; gift of Tim Foster, Trinity
College Dublin) Newman S. aureus (starting OD.sub.600 of 0.15) and
grown in TSB for 3 hrs (exponential phase) 37.degree. C., 200 rpm.
The exponential phase culture was then resuspended in 1 ml of PBS
and lysed with 200 ug/ml lysostaphin in the presence of 250 units
of Benzonase Nuclease (Novagen) at 37.degree. C. for 30 minutes.
The lysates were cleared of debris by spinning down at maximum
speed in a micro-centrifuge for 10 minutes at 4.degree. C.
Modification of NiNta captured E.coli SDR protein was carried out
by incubation with cleared APan Sdr mutant S. aureus lysates for 1
hr at 37.degree. C.
[0138] Western Blotting of modified E.coli SDR protein:
Non-modified or modified NiNta captured E.coli SDR protein samples
were then washed 3.times. with wash buffer (50 mM
NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM Imidazole; pH 8.0), prepared
for Western Blotting and run on an 8% Tris Glycine Gel
(Invitrogen). Western blots were blotted with either the rF1
antibody or an anti-Unizyme antibody (Genentech).
Identification of SDR Domain as Antigen for rF1
[0139] S. aureus expression of MBP-SD Constructs: The maltose
binding protein (MBP) gene was PCR amplified from the pMAL-c5x
vector (New England BioLabs [NEB], Ipswich, Me., USA) from the
sequence encoding the start of the mature protein until the
sequence encoding the end of the Factor Xa cleavage site. The
varying lengths of c-terminal His tagged SD (SD, SDS, DSD, SDSD,
SDSDS, SDSDSD) were synthesized as single stranded oligonucleotides
and annealed together to make double stranded dna. The Sdr region
of the C1fA gene was PCR amplified from the sequence encoding the
beginning (560D) of the Sdr region and included up to either the
sequence encoding 618A or 709S of the SD region followed by the dna
sequence coding for a His Tag from the respective plasmids
pTet.C1fA.SD618A or pTet.C1fA.SD709S (Genentech). As a control, the
sequence encoding the A domain of the C1fA gene from the beginning
of the mature protein until the sequence encoding the end of the A
domain (538G) followed by the sequence coding for a His Tag was PCR
amplified from the plasmid pTet.C1fA.Adom.538G (Genentech). The MBP
insert along with one of the various SD inserts or C1fA A domain
were ligated into the pTet S.aureus expression vector (pSAS10 from
Genentech). The resulting constructs were then electroporated into
the S. aureus RN4220 A sortase (sortase deletion mutant strain in
the RN4220 background).
[0140] A 20 ml culture of either the electroporated RN4220 A
sortase or RN4220 A sortase empty (not carrying a pTet expression
vector) was inoculated from an overnight culture (starting
OD.sub.600 of 0.15), grown for 1 hr in trypticase soy broth (TSB)
supplemented with glucose (2 g/L) and then induced for protein
expression for 2 hrs with anhydrotetracycline (200 ng/ml). At the
end of the induction period, the S. aureus culture was resuspended
in Column Buffer (150 mM NaCl, 20mM Tris pH 7.5 and Roche protease
inhibitor EDTA-free tablets) and lysed with 200 ug/ml lysostaphin
in the presence of 250 units of Benzonase Nuclease (Novagen) at
37.degree. C. for 30 minutes. The lysates were cleared of debris by
spinning down at maximum speed in a micro-centrifuge for 10 minutes
at 4.degree. C. The cleared lysates were then incubated with
amylose resin (NEB) along with a final EDTA concentration of 1 mM
for 1.5 hrs at 4.degree. C. to capture the expressed MBP-SD
proteins.
[0141] Western Blotting of S. aureus expressed MBP-SD Constructs:
Amylose resin with the captured MBP-SD proteins were washed three
times with Column Buffer, further prepared for Western blotting
analysis and run on an 8% Tris-glycine gel. Western blots were
blotted with either the rF1 antibody, an anti-penta His antibody
(Qiagen) or an anti-MBP antibody (NEB).
Results
[0142] rF1 Reacts to a Unique Family of SDR (Ser-Asp-repeat)
Proteins in S. aureus and S. epidermidis
[0143] A commercial teichoic acid preparation and cell wall lysate
(WTA) of S. aureus (USA300 strain) was tested for binding to rF1
after immunoprecipitation. rF1 binds to several components of the
commercial teichoic acid preparation (FIG. 6A, left panel) and of
the cell wall lysate of S. aureus (FIG. 6B, left panel). Using mass
spectrometry these components of the WTA preparation and cell wall
lysate were identified as C1fA (SdrA), C1fB (SdrB), SdrC, SdrD and
SdrE (FIG. 6A, right panel and FIG. 6B, right panel)
[0144] A cell wall lysate of S. epidermidis was tested for binding
to rF1 after immunoprecipitation. FIG. 6C (left panel) shows
binding of rF1 to several components of a cell wall lysate of S.
epidermidis. These components were not identified with a control
antibody. Using mass spectrometry these cell wall components were
identified as SdrF, SdrG and SdrH (FIG. 6C right panel).
Exogenous C1fA Expression in S. aureus and E.coli
[0145] Exogenous C1fA expressed in S. aureus is reactive to rF1,
whereas C1fA expressed in E. coli was not (FIG. 7A). However,
incubation of E. coli expressed Sdr proteins C1fA (SdrA), C1fB
(SdrB), SdrC, SdrD and SdrE with S. aureus lysate regained rF1
reactivity (FIG. 7B).
rF1 Binds to SDR Domains Expressed in S. aureus
[0146] rF1 antibody binds to C1fA Sdr regions consisting of the
C1fA 560D-618S and C1fA 560D-709S (FIG. 8) expressed in S. aureus.
rF1 does not bind to the A-domain of C1fA or to small peptide
sequences consisting of up to three SD repeats.
Example 4
Methods
Cloning of the Variable Regions of rF1 Into pRK Vector
[0147] Phusion DNA polymerase, restriction enzymes EcoRV, KpnI,
PvuII, ApaI, AgeI, and AhdI, T4 DNA ligase were purchased from New
England BioLabs, Ipswich, Mass., USA. Pfu DNA polymerase, Quick
change II Site-Directed Mutagenesis Kit were purchased from
Stratagene/Agilent Technologies, Santa Clara, Calif., USA. 2%
agarose gel was purchased from Invitrogen, Carlsbad, Calif., USA.
pRK vectors pRK.LPG3.HuKappa and pRK.LPG4.HumanHC were obtained
from Genentech/Roche, South San Francisco, Calif., USA.
[0148] The variable domains of heavy and light chains of rF1 Mab
from the pCPEO vectors were placed into the pRK mammalian
expression vectors. Polymerase chain reaction (PCR) was performed
in a total volume of 50 .mu.l using Pfu DNA polymerase according to
standard PCR procedures. V.sub.H was amplified with the primers
YiHCF) 5'-ATG GCT GAG GTG CAG CTG GTG GAG TCT G-3' (SEQ ID NO:18)
and YiHCR2 5'-GAA CAC GCT GGG GCC CTT GGT GCT GGC ACT CGA GAC TGT
GAC CAG GGT GCC AGG T*CC CCA G-3' (SEQ ID NO:19) (the restriction
sites of PvuII and ApaI are underlined and the * indicates the
single nucleotide G to T change to eliminate the internal ApaI
site) and V.sub.L was amplified with the primers YiLCF 5'-CGG CTC
GAC CGA TAT CCA GCT GAC CCA GAG-3' (SEQ ID NO:20) and YiLCR 5'-GAT
TTC CAG CTT GGT ACC CTG GCC G-3' (SEQ ID NO:21) (the EcoRV and KpnI
are underlined). Unique PCR products with 393 (V.sub.H) and 328
(V.sub.L) bp, respectively, were directly digested by PvuII and
Apal for V.sub.H, EcoRV and KpnI for V.sub.L, followed by Gel
purification (Invitrogen, Catalog# K2100-12). V.sub.H and V.sub.L
were individually ligated into pRK vector fragments of pRK/PvuII,
Apal for Heavy chain and pRK/EcoRV, KpnI for light chain using T4
DNA ligase. The DNA plasmids were confirmed by restriction enzyme
digestion patterns, i.e. rF1 V.sub.H released .about.300 by and 5.8
kb bands by AgeI, whereas rF1V.sub.L shown 2.3 and 3.1 kb bands by
AhdI on 2% agarose gel.
Chemical Stress Testing
[0149] By comparing antibody primary sequence data to
experimentally observed degradation events, it has become clear
that certain sequence motifs may be prone to degradation (i.e,
aspartate isomerization at DD, DG, and DS sequences and asparagine
deamidation at NG sequences). If such a degradation "hot spot"
appears in the CDR of an antibody, binding and potency may be
negatively impacted. For molecules containing hot spots, chemical
stress testing provides a way to assess the susceptibility of these
motifs to degradation by putting the antibody into a platform
formulation buffer and comparing a control sample to one stressed
at 40.degree. C. for two weeks. If degradation is observed the
primary sequence can be re-engineered to remove the hot spot.
[0150] After the sample is stressed for two weeks at 40.degree. C.,
a variety of analytical assays are performed to assess where and
how much degradation occurred. An imaged capillary isoelectric
focusing (icIEF) analysis was performed to examine charge variants
and the intact and reduced antibody mass was verified using mass
spectrometry, and an LC-MS/MS peptide map was performed to obtain
site-specific degradation information.
Mutagenesis
[0151] The rF1 pRK plasmids underwent a series of site-directed
mutagenesis to stabilize the rF1 Mab by N (AAC) 53S (AGC) in heavy
chain CDR2 with using FHV 5'-GGT GGC CAG CAT CAA CAG CGG CAA CAA
CCC CTA CTA CG-3' (SEQ ID NO:22) and RHV 5'-CGT AGT AGG GGT TGT TGC
CGC TGT TGA TGC TGG CCA CC-3' (SEQ ID NO:23) (mutagenesis site is
underlined) via Quik change II Site-Directed Kit. The mutagenesis
variant was sequenced.
Results
[0152] The primary sequence of antibody rF1 contained one potential
asparagine deamidation site in light chain CDR2: NNGNN (SEQ ID
NO:24). Stress testing revealed an increase of deamidation in
stressed samples at N53 (numbering according to Kabat, 1991).
Site-directed mutagenesis was performed to stabilize the rF1 Mab by
N (AAC, (SEQ ID NO:25)) to 53S (AGC, (SEQ ID NO:26)) substitution
in heavy chain CDR2. Sequencing of the mutagenesis variant
confirmed an N53S mutation.
Example 5
Development of Cys Variants for Antibody-Drug Conjugate (ADC)
Applications
[0153] The rF1 pRK plasmids underwent a series of site-directed
mutagenesis with oligos of rF1pRK.LC(205/210)VCF (468075) 5'-GGG
CCT GAG CTC GCC CTG CAC AAA GAG CTT CAA CAG-3' (SEQ ID NO:27) and
rF1pRK.LC(205/210)VCR (468076) 5'-CTG TTG AAG CTC TTT GTG CAG GGC
GAG CTC AGG CCC-3' (SEQ ID NO:28) (Cys mutant is underlined) to
generate the light chain linkers of rF1 thioMab, and
rF1pRK.HCN53S.A121CF (468464) 5'-CTG GTC ACA GTC TCG AGT TGC AGC
ACC AAG GGC CCA TC-3' (SEQ ID NO:29) and rF1pRK.HCN53S.Al21CR
(468465) 5'-GAT GGG CCC TTG GTG CTG CAA CTC GAG ACT GTG ACC AG-3'
(SEQ ID NO:30) (Cys mutant is underlined) to make the heavy chain
linkers of rF1 thioMab via the Stratagene's Site-Directed method.
Light chain V205C and heavy chain A114C were confirmed by
sequencing of the variants (FIG. 9).
REFERENCES
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[0155] Greenberg J. W. et al. Infection and Immunity 64, 3318-3325
(1996)
[0156] Jaleco A. C. et al. Blood 94, 2637-2646 (1999)
[0157] Kabat et al. Sequences of Proteins of Immunological
interest, 5.sup.th Ed. Public Health Service, National Institute of
Health, Bethesda, Md. (1991)
[0158] Keller R. et al. Infect Immun 60, 3664-3672 (1992)
[0159] Kwakkenbos M et al. Nat Med accepted for publication
(2009)
[0160] Moran et al. NEMJ 355, 666-674 (2006)
[0161] Polotsky V. Y. et al. Infection and Immunity 64, 380-383
(1996)
[0162] Scheeren F. A. et al. Nat Immunol 6, 303-313 (2005)
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[0164] Weidenmaier C. et al. Nat Rev Microbiol 6, 276-287
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[0165] Kwakkenbos M et al. Nat Med. 16(1):123-8 (2010)
Sequence CWU 1
1
6415PRTArtificial SequenceSynthetic construct 1Arg Phe Ala Met Ser1
5 216PRTArtificial SequenceSynthetic construct 2Ser Ile Asn Asn Gly
Asn Asn Pro Tyr Tyr Ala Arg Ser Val Gln Tyr1 5 10 15
312PRTArtificial SequenceSynthetic construct 3Asp His Pro Ser Ser
Gly Trp Pro Thr Phe Asp Ser1 5 10 411PRTArtificial
SequenceSynthetic construct 4Arg Ala Ser Glu Asn Val Gly Asp Trp
Leu Ala1 5 10 57PRTArtificial SequenceSynthetic construct 5Lys Thr
Ser Ile Leu Glu Ser1 5 69PRTArtificial SequenceSynthetic construct
6Gln His Tyr Xaa Arg Phe Pro Tyr Thr1 5 7120PRTArtificial
SequenceSynthetic construct 7Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Leu Ser Arg Phe 20 25 30 Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile
Asn Asn Gly Asn Asn Pro Tyr Tyr Ala Arg Ser Val Gln 50 55 60 Tyr
Arg Phe Thr Val Ser Arg Asp Val Ser Gln Asn Thr Val Ser Leu65 70 75
80 Gln Met Asn Asn Leu Arg Ala Glu Asp Ser Ala Thr Tyr Phe Cys Ala
85 90 95 Lys Asp His Pro Ser Ser Gly Trp Pro Thr Phe Asp Ser Trp
Gly Pro 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
8110PRTArtificial SequenceSynthetic construct 8Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ala Leu Pro Ala Ser Val Gly1 5 10 15 Asp Arg Val
Ser Ile Thr Cys Arg Ala Ser Glu Asn Val Gly Asp Trp 20 25 30 Leu
Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40
45 Tyr Lys Thr Ser Ile Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Xaa
Arg Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys Arg Thr Val 100 105 110 9120PRTArtificial SequenceSynthetic
construct 9Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Leu Ser Arg Phe 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly
Arg Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Asn Asn Gly Asn Asn
Pro Tyr Tyr Ala Arg Ser Val Gln 50 55 60 Tyr Arg Phe Thr Val Ser
Arg Asp Val Ser Gln Asn Thr Val Ser Leu65 70 75 80 Gln Met Asn Asn
Leu Arg Ala Glu Asp Ser Ala Thr Tyr Phe Cys Ala 85 90 95 Lys Asp
His Pro Ser Ser Gly Trp Pro Thr Phe Asp Ser Trp Gly Pro 100 105 110
Gly Thr Leu Val Thr Val Ser Ser 115 120 10109PRTArtificial
SequenceSynthetic construct 10Asp Ile Gln Leu Thr Gln Ser Pro Ser
Ala Leu Pro Ala Ser Val Gly1 5 10 15 Asp Arg Val Ser Ile Thr Cys
Arg Ala Ser Glu Asn Val Gly Asp Trp 20 25 30 Leu Ala Trp Tyr Arg
Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Lys Thr
Ser Ile Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Xaa Arg Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Ala 100
105 11110PRTArtificial SequenceSynthetic construct 11Asp Ile Gln
Leu Thr Gln Ser Pro Ser Ala Leu Pro Ala Ser Val Gly1 5 10 15 Asp
Arg Val Ser Ile Thr Cys Arg Ala Ser Glu Asn Val Gly Asp Trp 20 25
30 Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile
35 40 45 Tyr Lys Thr Ser Ile Leu Glu Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His
Tyr Xaa Arg Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg Thr Val 100 105 110 1215DNAArtificial
SequenceSynthetic construct 12cgctttgcca tgagc 151348DNAArtificial
SequenceSynthetic construct 13tcgatcaata atgggaataa cccatactac
gcacggtcgg tacaatac 481436DNAArtificial SequenceSynthetic construct
14gatcacccta gtagtggctg gcccaccttt gactcc 361533DNAArtificial
SequenceSynthetic construct 15cgggccagtg aaaacgttgg tgactggttg gcc
331621DNAArtificial SequenceSynthetic construct 16aagacatcta
ttctagaaag t 211727DNAArtificial SequenceSynthetic construct
17caacactata tacgtttccc gtacact 271828DNAArtificial
SequenceSynthetic construct 18atggctgagg tgcagctggt ggagtctg
281961DNAArtificial SequenceSynthetic construct 19gaacacgctg
gggcccttgg tgctggcact cgagactgtg accagggtgc caggtcccca 60g
612030DNAArtificial SequenceSynthetic construct 20cggctcgacc
gatatccagc tgacccagag 302125DNAArtificial SequenceSynthetic
construct 21gatttccagc ttggtaccct ggccg 252238DNAArtificial
SequenceSynthetic construct 22ggtggccagc atcaacagcg gcaacaaccc
ctactacg 382338DNAArtificial SequenceSynthetic construct
23cgtagtaggg gttgttgccg ctgttgatgc tggccacc 38245PRTArtificial
SequenceSynthetic construct 24Asn Asn Gly Asn Asn1 5
253DNAArtificial SequenceSynthetic construct 25aac
3263DNAArtificial SequenceSynthetic construct 26agc
32736DNAArtificial SequenceSynthetic construct 27gggcctgagc
tcgccctgca caaagagctt caacag 362836DNAArtificial SequenceSynthetic
construct 28ctgttgaagc tctttgtgca gggcgagctc aggccc
362938DNAArtificial SequenceSynthetic construct 29ctggtcacag
tctcgagttg cagcaccaag ggcccatc 383038DNAArtificial
SequenceSynthetic construct 30gatgggccct tggtgctgca actcgagact
gtgaccag 38315PRTArtificial SequenceSynthetic construct 31Leu Pro
Xaa Thr Gly1 5 324PRTArtificial SequenceSynthetic construct 32Ser
Asp Ser Asp1 335PRTArtificial SequenceSynthetic construct 33Ser Asp
Ser Asp Ser1 5 346PRTArtificial SequenceSynthetic construct 34Ser
Asp Ser Asp Ser Asp1 5 35360DNAArtificial SequenceSynthetic
construct 35gag gtg caa ctg ttg gag tcg ggg ggg ggc ttg gtg cag ccg
ggg ggg 48Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc
ctt agc cgc ttt 96Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Leu Ser Arg Phe 20 25 30 gcc atg agc tgg gtc cgc cag gct cca gga
agg gga ctg gaa tgg gtc 144Ala Met Ser Trp Val Arg Gln Ala Pro Gly
Arg Gly Leu Glu Trp Val 35 40 45 gca tcg atc aat aat ggg aat aac
cca tac tac gca cgg tcg gta caa 192Ala Ser Ile Asn Asn Gly Asn Asn
Pro Tyr Tyr Ala Arg Ser Val Gln 50 55 60 tac cgc ttc acc gtc tcc
cgg gac gtc tcc cag aac act gtg tct ctg 240Tyr Arg Phe Thr Val Ser
Arg Asp Val Ser Gln Asn Thr Val Ser Leu 65 70 75 80 cag atg aac aac
ctg aga gcc gaa gac tcg gcc aca tat ttc tgt gct 288Gln Met Asn Asn
Leu Arg Ala Glu Asp Ser Ala Thr Tyr Phe Cys Ala 85 90 95 aaa gat
cac cct agt agt ggc tgg ccc acc ttt gac tcc tgg ggc ccg 336Lys Asp
His Pro Ser Ser Gly Trp Pro Thr Phe Asp Ser Trp Gly Pro 100 105 110
gga acc ctg gtc acc gtc tcc tcg 360Gly Thr Leu Val Thr Val Ser Ser
115 120 36120PRTArtificial SequenceSynthetic construct 36Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Arg Phe 20
25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp
Val 35 40 45 Ala Ser Ile Asn Asn Gly Asn Asn Pro Tyr Tyr Ala Arg
Ser Val Gln 50 55 60 Tyr Arg Phe Thr Val Ser Arg Asp Val Ser Gln
Asn Thr Val Ser Leu65 70 75 80 Gln Met Asn Asn Leu Arg Ala Glu Asp
Ser Ala Thr Tyr Phe Cys Ala 85 90 95 Lys Asp His Pro Ser Ser Gly
Trp Pro Thr Phe Asp Ser Trp Gly Pro 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 3790DNAArtificial SequenceSynthetic construct
37gag gtg caa ctg ttg gag tcg ggg ggg ggc ttg gtg cag ccg ggg ggg
48Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ctt agc
90Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser 20 25 30
3830PRTArtificial SequenceSynthetic construct 38Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser 20 25 30
3942DNAArtificial SequenceSynthetic construct 39tgg gtc cgc cag gct
cca gga agg gga ctg gaa tgg gtc gca 42Trp Val Arg Gln Ala Pro Gly
Arg Gly Leu Glu Trp Val Ala 1 5 10 4014PRTArtificial
SequenceSynthetic construct 40Trp Val Arg Gln Ala Pro Gly Arg Gly
Leu Glu Trp Val Ala1 5 10 4196DNAArtificial SequenceSynthetic
construct 41cgc ttc acc gtc tcc cgg gac gtc tcc cag aac act gtg tct
ctg cag 48Arg Phe Thr Val Ser Arg Asp Val Ser Gln Asn Thr Val Ser
Leu Gln 1 5 10 15 atg aac aac ctg aga gcc gaa gac tcg gcc aca tat
ttc tgt gct aaa 96Met Asn Asn Leu Arg Ala Glu Asp Ser Ala Thr Tyr
Phe Cys Ala Lys 20 25 30 4232PRTArtificial SequenceSynthetic
construct 42Arg Phe Thr Val Ser Arg Asp Val Ser Gln Asn Thr Val Ser
Leu Gln1 5 10 15 Met Asn Asn Leu Arg Ala Glu Asp Ser Ala Thr Tyr
Phe Cys Ala Lys 20 25 30 4333DNAArtificial SequenceSynthetic
construct 43tgg ggc ccg gga acc ctg gtc acc gtc tcc tcg 33Trp Gly
Pro Gly Thr Leu Val Thr Val Ser Ser 1 5 10 4411PRTArtificial
SequenceSynthetic construct 44Trp Gly Pro Gly Thr Leu Val Thr Val
Ser Ser1 5 10 45330DNAArtificial SequenceSynthetic construct 45gac
atc cag ttg acc cag tct cct tcc gcc ctg cct gca tct gtg gga 48Asp
Ile Gln Leu Thr Gln Ser Pro Ser Ala Leu Pro Ala Ser Val Gly 1 5 10
15 gac aga gtc agc atc act tgt cgg gcc agt gaa aac gtt ggt gac tgg
96Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Glu Asn Val Gly Asp Trp
20 25 30 ttg gcc tgg tat cgg cag aaa ccg ggg aaa gcc cct aat ctt
ctc atc 144Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Asn Leu
Leu Ile 35 40 45 tat aag aca tct att cta gaa agt ggg gtc cca tca
agg ttc agc ggc 192Tyr Lys Thr Ser Ile Leu Glu Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aca gaa ttc act ctc acc
atc agc agc ctg cag cct 240Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 gat gat ttt gca act tat tac tgt
caa cac tat ata cgt ttc ccg tac 288Asp Asp Phe Ala Thr Tyr Tyr Cys
Gln His Tyr Ile Arg Phe Pro Tyr 85 90 95 act ttt ggc cag ggg acc
aag ctg gag atc aaa cga act gtg 330Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg Thr Val 100 105 110 46110PRTArtificial
SequenceSynthetic construct 46Asp Ile Gln Leu Thr Gln Ser Pro Ser
Ala Leu Pro Ala Ser Val Gly1 5 10 15 Asp Arg Val Ser Ile Thr Cys
Arg Ala Ser Glu Asn Val Gly Asp Trp 20 25 30 Leu Ala Trp Tyr Arg
Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Lys Thr
Ser Ile Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Ile Arg Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
100 105 110 4769DNAArtificial SequenceSynthetic construct 47gac atc
cag ttg acc cag tct cct tcc gcc ctg cct gca tct gtg gga 48Asp Ile
Gln Leu Thr Gln Ser Pro Ser Ala Leu Pro Ala Ser Val Gly 1 5 10 15
gac aga gtc agc atc act tgt 69Asp Arg Val Ser Ile Thr Cys 20
4823PRTArtificial SequenceSynthetic construct 48Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ala Leu Pro Ala Ser Val Gly1 5 10 15 Asp Arg Val
Ser Ile Thr Cys 20 4945DNAArtificial SequenceSynthetic construct
49tgg tat cgg cag aaa ccg ggg aaa gcc cct aat ctt ctc atc tat 45Trp
Tyr Arg Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile Tyr 1 5 10 15
5015PRTArtificial SequenceSynthetic construct 50Trp Tyr Arg Gln Lys
Pro Gly Lys Ala Pro Asn Leu Leu Ile Tyr1 5 10 15 5196DNAArtificial
SequenceSynthetic construct 51ggg gtc cca tca agg ttc agc ggc agt
ggg tct ggg aca gaa ttc act 48Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr 1 5 10 15 ctc acc atc agc agc ctg cag
cct gat gat ttt gca act tat tac tgt 96Leu Thr Ile Ser Ser Leu Gln
Pro Asp Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 5232PRTArtificial
SequenceSynthetic construct 52Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Thr1 5 10 15 Leu Thr Ile Ser Ser Leu Gln
Pro Asp Asp Phe Ala Thr Tyr Tyr Cys 20 25 30 5339DNAArtificial
SequenceSynthetic construct 53ttt ggc cag ggg acc aag ctg gag atc
aaa cga act gtg 39Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr
Val 1 5 10 5413PRTArtificial SequenceSynthetic construct 54Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val1 5 10
55230PRTArtificial SequenceSynthetic construct 55Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Arg Phe 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35
40 45 Ala Ser Ile Asn Asn Gly Asn Asn Pro Tyr Tyr Ala Arg Ser Val
Gln 50 55 60 Tyr Arg Phe Thr Val Ser Arg Asp Val Ser Gln Asn Thr
Val Ser Leu65 70 75 80 Gln Met Asn Asn Leu Arg Ala Glu Asp Ser Ala
Thr Tyr Phe Cys Ala 85 90 95 Lys Asp His Pro Ser Ser Gly Trp Pro
Thr Phe Asp Ser Trp Gly Pro 100 105 110 Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165
170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro225 230
56230PRTArtificial SequenceSynthetic construct 56Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Arg Phe 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35
40 45 Ala Ser Ile Asn Ser Gly Asn Asn Pro Tyr Tyr Ala Arg Ser Val
Gln 50 55 60 Tyr Arg Phe Thr Val Ser Arg Asp Val Ser Gln Asn Thr
Val Ser Leu65 70 75 80 Gln Met Asn Asn Leu Arg Ala Glu Asp Ser Ala
Thr Tyr Phe Cys Ala 85 90 95 Lys Asp His Pro Ser Ser Gly Trp Pro
Thr Phe Asp Ser Trp Gly Pro 100 105 110 Gly Thr Leu Val Thr Val Ser
Ser Cys Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165
170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro225 230
57213PRTArtificial SequenceSynthetic construct 57Asp Ile Gln Leu
Thr Gln Ser Pro Ser Ala Leu Pro Ala Ser Val Gly1 5 10 15 Asp Arg
Val Ser Ile Thr Cys Arg Ala Ser Glu Asn Val Gly Asp Trp 20 25 30
Leu Ala Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35
40 45 Tyr Lys Thr Ser Ile Leu Glu Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr
Met Arg Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys Arg Ala Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165
170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 58214PRTArtificial
SequenceSynthetic construct 58Asp Ile Gln Leu Thr Gln Ser Pro Ser
Ala Leu Pro Ala Ser Val Gly1 5 10 15 Asp Arg Val Ser Ile Thr Cys
Arg Ala Ser Glu Asn Val Gly Asp Trp 20 25 30 Leu Ala Trp Tyr Arg
Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Lys Thr
Ser Ile Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln His Tyr Met Arg Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160 Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Cys Thr Lys Ser 195 200
205 Phe Asn Arg Gly Glu Cys 210 5913PRTArtificial SequenceSynthetic
construct 59Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val1 5
10 6016PRTArtificial SequenceSynthetic construct 60Ser Ile Asn Ser
Gly Asn Asn Pro Tyr Tyr Ala Arg Ser Val Gln Tyr1 5 10 15
6130PRTArtificial SequenceSynthetic construct 61Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser 20 25 30
62120PRTArtificial SequenceSynthetic construct 62Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Arg Phe 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35
40 45 Ala Ser Ile Asn Ser Gly Asn Asn Pro Tyr Tyr Ala Arg Ser Val
Gln 50 55 60 Tyr Arg Phe Thr Val Ser Arg Asp Val Ser Gln Asn Thr
Val Ser Leu65 70 75 80 Gln Met Asn Asn Leu Arg Ala Glu Asp Ser Ala
Thr Tyr Phe Cys Ala 85 90 95 Lys Asp His Pro Ser Ser Gly Trp Pro
Thr Phe Asp Ser Trp Gly Pro 100 105 110 Gly Thr Leu Val Thr Val Ser
Ser 115 120 63110PRTArtificial SequenceSynthetic construct 63Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro 100 105 110 64104PRTArtificial
SequenceSynthetic construct 64Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys1 5 10 15 Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg 20 25 30 Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 35 40 45 Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 50 55 60 Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys65 70 75
80 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
85 90 95 Lys Ser Phe Asn Arg Gly Glu Cys 100
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