U.S. patent application number 15/155450 was filed with the patent office on 2016-12-15 for novel oprf/i fusion proteins, their preparation and use.
This patent application is currently assigned to Valneva Austria GmbH. The applicant listed for this patent is Valneva Austria GmbH. Invention is credited to ROBERT SCHLEGL.
Application Number | 20160362480 15/155450 |
Document ID | / |
Family ID | 45895497 |
Filed Date | 2016-12-15 |
United States Patent
Application |
20160362480 |
Kind Code |
A1 |
SCHLEGL; ROBERT |
December 15, 2016 |
NOVEL OPRF/I FUSION PROTEINS, THEIR PREPARATION AND USE
Abstract
The present invention relates to a novel trimeric OprF/I fusion
protein comprising a portion of the Pseudomonas aeruginosa outer
membrane protein F which is fused with its carboxy terminal end to
a portion of the amino terminal end of the Pseudomonas aeruginosa
out membrane protein I, wherein said portion of the Pseudomonas
aeruginosa outer membrane protein F comprises the amino acids
190-342 of SEQ ID NO: 1 and wherein said portion of the Pseudomonas
aeruginosa outer membrane protein I comprises the amino acids 21-83
of SEQ ID NO: 2, and further to a novel Opr F/I fusion protein
which contains a disulphide bond pattern, preferably selected from
the group consisting of (a) Cys18-Cys27-bond, (b) Cys18-Cys27-bond
and Cys33-Cys47-bond, and (c) Cys18-Cys47 and Cys27-Cys33-bond, and
to immunogenic variants thereof having at least 85% identity to the
amino acid sequence of SEQ ID NO: 3. The present invention also
relates to a novel method for producing said OprF/I fusion proteins
and to their use for the preparation of a pharmaceutical
composition and for the preparation of antibodies or antibody
derivatives which specifically bind said novel OprF/I fusion
proteins.
Inventors: |
SCHLEGL; ROBERT;
(Siegenfeld, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Valneva Austria GmbH |
Vienna |
|
AT |
|
|
Assignee: |
Valneva Austria GmbH
Vienna
AT
|
Family ID: |
45895497 |
Appl. No.: |
15/155450 |
Filed: |
May 16, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14005579 |
Oct 16, 2013 |
9359412 |
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PCT/EP2012/054783 |
Mar 19, 2012 |
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15155450 |
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61454075 |
Mar 18, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/02 20130101;
C07K 2319/00 20130101; C07K 14/21 20130101; C07K 2319/21 20130101;
C07K 16/1214 20130101; A61K 39/104 20130101 |
International
Class: |
C07K 16/12 20060101
C07K016/12; C07K 14/21 20060101 C07K014/21 |
Claims
1.-12. (canceled)
13. An antibody or antibody derivative which specifically binds a
protein complex comprising three OprF/I fusion proteins of SEQ ID
NO: 4 or an immunogenic variant thereof having at least 85%
identity to the amino acid sequence of SEQ ID NO: 4.
14. An antibody or antibody derivative which selectively binds a
protein complex comprising three OprF/I fusion proteins of SEQ ID
NO: 4 or an immunogenic variant thereof having at least 85%
identity to the amino acid sequence of SEQ ID NO: 4.
15. A pharmaceutical composition comprising the antibody or
antibody derivative according to claim 13 and one or more
pharmaceutically acceptable excipients.
16. A pharmaceutical composition comprising the antibody or
antibody derivative according to claim 14 and one or more
pharmaceutically acceptable excipients.
17. The antibody or antibody derivative of claim 13, wherein the
OprF/I fusion proteins are selected from the group consisting of
(a) the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys27-bond (SEQ ID NO: 9), (b) the OprF/I fusion protein of
SEQ ID NO: 4 with a Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID
NO: 10), (c) the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11), and
immunogenic variants thereof having at least 85%, preferably 90%,
in particular 95% identity to the amino acid sequence of SEQ ID NO:
4, and the same disulphide bond pattern as specified in (a), (b) or
(c).
18. The antibody or antibody derivative of claim 17, wherein the
sum of a) the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys27-bond (SEQ ID NO: 9), b) the OprF/I fusion protein of
SEQ ID NO: 4 with a Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID
NO: 10), and c) the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11) is equal or
greater than 75%.
19. The antibody or antibody derivative of claim 14, wherein the
OprF/I fusion proteins are selected from the group consisting of
(a) the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys27-bond (SEQ ID NO: 9), (b) the OprF/I fusion protein of
SEQ ID NO: 4 with a Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID
NO: 10), (c) the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11), and
immunogenic variants thereof having at least 85% identity to the
amino acid sequence of SEQ ID NO: 4, and the same disulphide bond
pattern as specified in either (a), (b) or (c).
20. The antibody or antibody derivative of claim 19, wherein the
sum of a) the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys27-bond (SEQ ID NO: 9), b) the OprF/I fusion protein of
SEQ ID NO: 4 with a Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID
NO: 10), and c) the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11) is equal or
greater than 75%.
21. The antibody or antibody derivative of claim 13, wherein the
OprF/I fusion proteins are the OprF/I fusion protein of SEQ ID NO:
4 with a Cys18-Cys27-bond (SEQ ID NO: 9) or an immunogenic variant
thereof having at least 85%, preferably 90%, in particular 95%
identity to the amino acid sequence of SEQ ID NO: 4, and the same
disulphide bond pattern as specified in SEQ ID NO: 9.
22. The antibody or antibody derivative of claim 13, wherein the
OprF/I fusion proteins are the OprF/I fusion protein of SEQ ID NO:
4 with a Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10) or
an immunogenic variant thereof having at least 85%, preferably 90%,
in particular 95% identity to the amino acid sequence of SEQ ID NO:
4, and the same disulphide bond pattern as specified in SEQ ID NO:
10.
23. The antibody or antibody derivative of claim 13, wherein the
OprF/I fusion proteins are the OprF/I fusion protein of SEQ ID NO:
4 with a Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11) or
an immunogenic variant thereof having at least 85%, preferably 90%,
in particular 95% identity to the amino acid sequence of SEQ ID NO:
4, and the same disulphide bond pattern as specified in SEQ ID NO:
11.
24. The antibody or antibody derivative of claim 14, wherein the
OprF/I fusion proteins are the OprF/I fusion protein of SEQ ID NO:
4 with a Cys18-Cys27-bond (SEQ ID NO: 9) or an immunogenic variant
thereof having at least 85%, preferably 90%, in particular 95%
identity to the amino acid sequence of SEQ ID NO: 4, and the same
disulphide bond pattern as specified in SEQ ID NO: 9.
25. The antibody or antibody derivative of claim 14, wherein the
OprF/I fusion proteins are the OprF/I fusion protein of SEQ ID NO:
4 with a Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10) or
an immunogenic variant thereof having at least 85%, preferably 90%,
in particular 95% identity to the amino acid sequence of SEQ ID NO:
4, and the same disulphide bond pattern as specified in SEQ ID NO:
10.
26. The antibody or antibody derivative of claim 14, wherein the
OprF/I fusion proteins are the OprF/I fusion protein of SEQ ID NO:
4 with a Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11) or
an immunogenic variant thereof having at least 85%, preferably 90%,
in particular 95% identity to the amino acid sequence of SEQ ID NO:
4, and the same disulphide bond pattern as specified in SEQ ID NO:
11.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/005,579, filed Oct. 16, 2013, now U.S. Pat.
No. 9,359,412, which is a national stage filing under 35 U.S.C.
.sctn.371 of international application PCT/EP2012/054783, filed
Mar. 19, 2012, which was published under PCT Article 21(2) in
English, and claims the benefit under 35 U.S.C. .sctn.119(e) of
U.S. provisional application Ser. No. 61/454,075, filed Mar. 18,
2011, the disclosures of which are incorporated by reference herein
in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel trimeric OprF/I
fusion protein comprising a portion of the Pseudomonas aeruginosa
outer membrane protein F which is fused with its carboxy terminal
end to a portion of the amino terminal end of the Pseudomonas
aeruginosa outer membrane protein I, wherein said portion of the
Pseudomonas aeruginosa outer membrane protein F comprises the amino
acids 190-342 of SEQ ID NO: 1 and wherein said portion of the
Pseudomonas aeruginosa outer membrane protein I comprises the amino
acids 21-83 of SEQ ID NO: 2, and further to a novel OprF/I fusion
protein which contains a disulphide bond pattern, preferably
selected from the group consisting of (a) Cys18-Cys27-bond, (b)
Cys18-Cys27-bond and Cys33-Cys47-bond, and (c) Cys18-Cys47 and
Cys27-Cys33-bond, and to immunogenic variants thereof having at
least 85% identity to the amino acid sequence of SEQ ID NO: 3. The
present invention also relates to a novel method for producing said
OprF/I fusion proteins and to their use for the preparation of a
pharmaceutical composition and for the preparation of antibodies or
antibody derivatives which specifically bind said novel OprF/I
fusion proteins.
BACKGROUND OF THE INVENTION
[0003] Nosocomial infections are infections that are a result of
treatment in a hospital or a healthcare service unit. Infections
are considered nosocomial if they first appear 48 hours or more
after hospital admission or within 30 days after discharge. This
type of infection is also known as a hospital-acquired infection
(or, in generic terms, healthcare-associated infection). In the
United States, the Center for Disease Control and Prevention
estimates that roughly 1.7 million hospital-associated infections,
from all types of microorganism, including bacteria, combined,
cause or contribute to 99,000 deaths each year. In Europe, where
hospital surveys have been conducted, the category of Gram-negative
infections are estimated to account for two-thirds of the 25,000
deaths each year. Nosocomial infections can cause severe pneumonia
and infections of the urinary tract, bloodstream and other parts of
the body. Many types are difficult to attack with antibiotics, and
antibiotic resistance is spreading to Gram-negative bacteria that
can infect people outside the hospital.
[0004] In Gram-negative bacteria, lipopolysaccharides (LPS) and
outer-membrane proteins are the major antigenic parts of the
bacterial envelope. LPS based vaccines have been extensively
studied in the 1970s (Priebe G & Pier G., Vaccines for
Pseudomonas aeruginosa 2003. New Bacterial vaccines, edited by
Elfis R W, Brodeur B. 260-82). Parke Davis produced a vaccine
Pseudogen from LPS of 7 different serogroups. Some activity was
observed with Pseudogen in non-randomized trials in cancer and burn
patients but not in cystic fibrosis (CF) and leukemia patients.
Being LPS based Pseudogen was very toxic and therefore not
registered (Priebe, supra). Using two different versions of
recombinant fusion proteins of Opr's F and I, von Specht and
colleagues have shown that active immunization can protect
neutropenic mice and passive immunization can protect SCID mice,
both against a challenge dose 1000-fold above the LD50 (von Specht,
B U et al., Protection of immunocompromised mice against lethal
infection with Pseudomonas aeruginosa by active or passive
immunization with recombinant Pseudomonas aeruginosa outer membrane
protein F and Outer membrane protein I fusion proteins. Infect
Immun 1995; 63(5):1855-1862; Knapp B et al., A recombinant hybrid
outer membrane protein for vaccination against Pseudomonas
aeruginosa. Vaccine 1999; 17(13-14):1663-1666). Said fusion protein
was then tested for safety and immunogenicity in healthy volunteers
reaching high levels of specific serum antibodies. To achieve an
enhanced mucosal immunogenicity in cystic fibrosis an emulgel
formulation of said fusion protein was developed and tested for
safety and immunogenicity in healthy volunteers and lung impaired
patients. However, the serum antibody response was comparatively
low. A systemic i. m. booster has enhanced serum antibody response
as compared to solely mucosal vaccination schedule.
[0005] An outer membrane protein preparation composed of 4
different strains of Pseudomonas aeruginosa with a molecular weight
range of 10-100 kDa was developed as a vaccine in Korea. The
vaccine contained minimal amounts of polysaccharide and was tested
in a double-blind, placebo-controlled trial in burn patients (Jang
II et al., Human immune response to a Pseudomonas aeruginosa outer
membrane protein vaccine. Vaccine 1999; 17(2): 158-68). Antibody
levels to the vaccine antigens rose by 2.3-fold in the placebo
group (19 patients) and 4.9 fold in the vaccine group (76 patients)
(Kim D K et al., Comparison of two immunization schedules for a
Pseudomonas aeruginosa outer membrane proteins vaccine in burn
patients. Vaccine 2001; 19(9-10):1274-83). Priebe and Pier
criticized the study because the follow-up of patients in the trial
was incomplete, analysis was not by intention-to-treat, and there
were no data regarding clinical outcomes (Priebe, supra. A similar
Opr vaccine was tested in Russia 10 years earlier (Stanislaysky E S
et al., Clinico-immunological trials of Pseudomonas aeruginosa
vaccine. Vaccine 1991; 9(7):491-4). Pseudomonas aeruginosa vaccine
(PV) containing predominantly cell-wall protein protective antigens
was tested for safety and immunogenicity by immunization of 119
volunteers. The PV vaccine was well tolerated. A high level of
specific antibodies persisted for the 5-month period of
observation. The antibody titers increased in 94-97% of volunteers
and moreover in 45.6% the antibody titers (the number of ELISA
units) increased 2.5-3-fold and more. Anti-Pseudomonas aeruginosa
plasma was used for the treatment of 46 patients with severe forms
of Pseudomonas aeruginosa infection (40 adults and six infants aged
up to 2 years) and 87% of the patients recovered. There have been
no follow-up studies with the PV vaccine after 1991.
[0006] Hospital-acquired infections are one of the major causes of
death and serious illness worldwide, resulting in an annual cost
burden of more than USD 20 billion in the developed world. In the
United States and Europe about 6 million patients become infected
annually resulting in 140,000 deaths per year. The incidence of
nosocomial infections is steadily increasing due to increasing
medical interventions and antibiotic resistance. Thus, minimizing
risk of mortality through hospital acquired infections by e.g.
vaccination of burn victims and fibrosis patients, ICU patients and
ventilated ICU patients is and is expected to become even more so a
major unmet medical need in said patients.
[0007] It has recently been found (US provisional application with
application No. 61/426,760) that a vaccine of the above-described
hybrid fusion protein comprising the Pseudomonas aeruginosa outer
membrane protein I (Oprl or OMPI) which is fused with its amino
terminal end to the carboxy-terminal end of a carboxy-terminal
portion of the Pseudomonas aeruginosa outer membrane protein F
(OprF or OMPF) reduced the mortality rate in mechanically
ventilated intensive care patients significantly over alum as
placebo control. Mechanically ventilated intensive care patients
are at particular risk of acquiring severe and often
life-threatening forms of Pseudomonas aeruginosa or other
infections, such as Ventilator-Associated Pneumonia (VAP), sepsis
or soft tissue infection. Such infections also may affect burn
victims, severely burned victims, cancer and transplant patients
who are immunosuppressed, and cystic fibrosis patients, Intensive
Care Unit (ICU) patients or generally all hospitalized
patients.
[0008] Generally, the expression of soluble OprF/I fusion protein
in E. coli leads to the formation of non immunological aggregates
and misfolded variants. According to Worgall et al. (Worgall S et
al., Protection against P. aeruginosa with an adenovirus vector
containing an OprF epitope in The Capsid., J. of Clinical
Investigation, 2005, 115(5), 1281-1289) it is assumed that the
native OprF protein has one disulphide bridge from Cys200 to Cys209
of SEQ ID NO: 1 and two free cysteines at Cys215 and Cys229 of SEQ
ID NO: 1. In another publication (Rawling E G et al., Epitope
Mapping of the Pseudomonas aeruginosa Major Outer membrane Protein
OprF., Infection and Immunity, 1995, 63 (1), 38-42), however, two
disulphide bonds from Cys200 to Cys209 and from Cys215 to Cys229 of
SEQ ID NO:1 are proposed. It cannot be expected that the reported
disulphide bond pairing applies to the fusion protein OprF/I since
only amino acid No. 190 to amino acid No. 342 of SEQ ID NO: 1 from
the native OprF protein are expressed. Since native OprF is an
outer membrane protein and contains several transmembrane spans, it
is expected that folding in an aqueous environment differs from the
folded structure of the natively expressed protein located in a
membrane.
[0009] In addition, a pharmaceutical composition should be
homogenous and stable. Thus, both good manufacturing practice as
well as regulatory authority guidelines require that a dosage form
of a pharmaceutical or pharmaceutical combination should be in the
form of a homogeneous dispersion with respect to the active
substances. There is a concern in the field regarding aggregates
and a potential for immunogenicity (Leonard J. Schiff,
Biotechnology Products Derived from Mammalian Cell Lines: Impact of
Manufacturing Changes (2004) Regulatory Affairs Focus, October
2004, pages 29-31).
SUMMARY OF THE INVENTION
[0010] In accordance with the present invention, it has now
surprisingly been found that by a simple reduction and following
reoxidation under specific conditions an OprF/I fusion protein
variant could be recovered. This specific variant shows a disulfide
bond between Cys18 and Cys27 and two free cysteines at positions 33
and 47 (SEQ ID NO: 4) and a trimeric structure which has not been
shown before.
[0011] Thus, in accordance with the particular findings of the
present invention, there is provided: [0012] 1. An OprF/I fusion
protein comprising a portion of the Pseudomonas aeruginosa outer
membrane protein F which is fused with its carboxy terminal end to
a portion of the amino terminal end of the Pseudomonas aeruginosa
out membrane protein I, wherein said portion of the Pseudomonas
aeruginosa outer membrane protein F comprises the amino acids
190-342 of SEQ ID NO: 1 and wherein said portion of the Pseudomonas
aeruginosa outer membrane protein I comprises the amino acids 21-83
of SEQ ID NO: 2, and further wherein said fusion protein contains a
disulphide bond pattern, preferably selected from the group
consisting of (a) Cys18-Cys27-bond (SEQ ID NO: 9), (b)
Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10), and (c)
Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11), or an
immunogenic variant thereof having at least 85%, preferably 90%, in
particular 95% identity to the amino acid sequence of SEQ ID NO: 4
and the same disulphide bond pattern as specified. [0013] 2. A
trimeric OprF/I fusion protein comprising a portion of the
Pseudomonas aeruginosa outer membrane protein F which is fused with
its carboxy terminal end to a portion of the amino terminal end of
the Pseudomonas aeruginosa out membrane protein I, wherein said
portion of the Pseudomonas aeruginosa outer membrane protein F
comprises the amino acids 190-342 of SEQ ID NO: 1 and wherein said
portion of the Pseudomonas aeruginosa outer membrane protein I
comprises the amino acids 21-83 of SEQ ID NO: 2, or an immunogenic
variant thereof having at least 85%, preferably 90%, in particular
95% identity to the amino acid sequence of SEQ ID NO: 4. [0014] 3.
A method for producing the OprF/I fusion protein as herein
described, said method comprising the steps of [0015] (a) reducing
said OprF/I fusion protein with a reducing agent, preferably
dithiothreitol (DTT), dithioerythritol (DTE) or
.beta.-mercaptoethanol, and [0016] (b) oxidizing the reduced OprF/I
fusion protein with a redox agent, preferably the redox agent
glutathione disulfide/glutathione or the redox agent
cystine/cysteine, in the presence of a reducing agent, preferably
dithiothreitol (DTT), dithioerythritol (DTE) or
.beta.-mercaptoethanol. [0017] 4. A pharmaceutical composition, in
particular a vaccine, comprising said OprF/I hybrid. [0018] 5. An
antibody or antibody derivative which specifically binds said
OprF/I fusion protein. [0019] 6. A pharmaceutical composition
comprising said antibody or antibody derivative which specifically
binds said OprF/I fusion protein.
[0020] The invention will now be further illustrated below with the
aid of the Figures, Tables, Sequence Listings and Examples, without
being restricted hereto.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0021] The term "about" means a general error range of +/-5%.
[0022] The term "immunogenic variant" means a sequence variant of
the OprF/I fusion protein which shows in vivo immunogenicity, e.g.
in the BALB/c mouse model, e.g. have an ED50 value of 10 .mu.g of
lower, more preferably an ED50 value of 5 .mu.g or lower such as
e.g. 4 .mu.g or lower, 3 .mu.g or lower or 2 .mu.g or lower (see
example section).
[0023] The term "binding specificity" or "specifically bind(s)" as
used herein refers to the ability of an individual antibody
combining site to react with only one antigenic determinant. The
combining site of the antibody is located in the Fab portion of the
molecule and is constructed from the hypervariable regions of the
heavy and light chains. Binding affinity of an antibody is the
strength of the reaction between a single antigenic determinant and
a single combining site on the antibody. It is the sum of the
attractive and repulsive forces operating between the antigenic
determinant and the combining site of the antibody. Specific
binding between two entities means a binding with an equilibrium
constant (KA) of at least 1.times.10.sup.7 M.sup.-1, 10.sup.8
M.sup.-1, 10.sup.9 M.sup.-1, 10.sup.10 M.sup.-1, 10.sup.11
M.sup.-1, 10.sup.12 M.sup.-1, 10.sup.13 M.sup.1. The phrase
"specifically (or selectively) binds" to an antibody (e.g., an
OprF/I agent-binding antibody) refers to a binding reaction that is
determinative of the presence of an antigen (e.g., an OprF/I agent
such as a trimer of a mixture of SEQ ID NOs: 9 to 11) in e.g. a
heterogeneous population of proteins and other compounds. In
addition to the equilibrium constant (KA) noted above, an OprF/I
agent-binding antibody of the invention typically also has a
dissociation rate constant (Kd) of about 1.times.10.sup.-2
s.sup.-1, 1.times.10.sup.3 s.sup.-1, 1.times.10.sup.4 s.sup.-1,
1.times.10.sup.4 s.sup.-1, or lower, and binds to the OprF/I agent
such as a trimer of a mixture of SEQ ID NOs: 9 to 11 with an
affinity that is at least 2-fold, 5-fold, 10-fold, 20-fold,
50-fold, preferably 100-fold, more preferably 500-fold, or up to
1000-fold or more greater than its affinity for binding to a
non-specific antigen. The phrases "an antibody recognizing an
antigen" and "an antibody specific for an antigen" are used
interchangeably herein with the term "an antibody which binds
specifically to an antigen".
[0024] Specific Aspects of the Invention
[0025] According to the present invention the OprF/I fusion protein
contains parts of two outer membrane proteins of Pseudomonas
aeruginosa, OprF.sub.190-342 and OprI.sub.21-83, and preferably an
N-terminal tag which is in particular useful for the better
expression in a suitable host, e.g. E. coli, and/or purification of
said fusion protein. After expression, OprF/I exists as
heterogeneous mixture of misfolded forms (high and low molecular
weight aggregates) caused by disulfide scrambling as shown in FIG.
1. Surprisingly, during purification it has been found that after
reduction and reoxidation of the fusion protein, novel disulfide
bonds were created as shown in FIG. 8 resulting in a separable
product mixture of three main products (see FIGS. 7A and 7B, in
particular peaks 1, 2 and 3). Unexpectedly, the reoxidized fusion
protein or fusion protein mixture is stable and does not form
undesired aggregates. Moreover, it was unexpected that one of the
three main products corresponding to peak 1 shows the same
disulfide bond and two blocked cysteines (caused by covalent
reaction with redox-agent (e.g. cysteines) used for reoxidation) as
the native, not truncated OprF protein. However, not only this
specific fusion protein shows sufficient immunogenicity in vivo but
also the other two fusion protein variants corresponding to peaks 2
and 3 (see Table 3), which was indeed unexpected.
[0026] Therefore, one aspect of the present invention is directed
to said OprF/I fusion protein containing different disulphide bond
patterns. Preferably the disulfide bond pattern corresponds to a
single Cys18-Cys27-bond according to SEQ ID NO: 9. Another
preferred disulphide bond pattern corresponds either to a
Cys18-Cys27-bond and a Cys33-Cys47-bond according to SEQ ID NO: 10,
or to a Cys18-Cys47-bond and a Cys27-Cys33-bond according to SEQ ID
NO: 11.
[0027] The described OprF/I fusion protein variants can either
separately be isolated or as a mixture with or without further
protein components, in particular other fusion protein variants,
preferably obtained after the purification process described in the
present specification. In case of a mixture of the three main
variants (peaks 1-3; FIG. 8), the relative distribution of the
variants in the purified mixture analyzed by RP-HPLC are: about 15%
to about 18%, preferably about 16%, for the peak 1 variant; about
67% to about 62%, preferably about 66%, for the peak 2 variant; and
about 18% to about 20%, preferably about 18%, for the peak 3
variant (FIG. 9). In case of a mixture with further protein
components as e.g. shown in FIGS. 7A and 7B, the total relative
content or purity of all three main products (peaks 1-3) is at
least about 75%, preferably at least about 80% to about 90%, in
particular at least about 85%, e.g. 75% to 90% or 85% to 90%. The
relative distribution of the three main products in such mixture is
the same as described above for a mixture of only the three main
products. The specified values can be obtained e.g. by integration
of the peak areas obtained by RP-HPLC at 280 and 214 nm
[0028] The present invention also encompasses an immunogenic
variant of the described OprF/I fusion protein which has at least
85%, preferably 90%, in particular 95% identity to the amino acid
sequence of SEQ ID NO: 3 with the proviso that the specified
cysteine residues forming the disulphide bonds are maintained.
[0029] In view of the above explanations, a particularly preferred
embodiment of the present invention is a mixture, in particular a
complex, of OprF/I fusion proteins, each of the OprF/I fusion
proteins comprises a portion of the Pseudomonas aeruginosa outer
membrane protein F which is fused with its carboxy terminal end to
a portion of the amino terminal end of the Pseudomonas aeruginosa
out membrane protein I, wherein said portion of the Pseudomonas
aeruginosa outer membrane protein F comprises the amino acids
190-342 of SEQ ID NO: 1 and wherein said portion of the Pseudomonas
aeruginosa outer membrane protein I comprises the amino acids 21-83
of SEQ ID NO: 2, said mixture containing, in particular in the form
of a trimer,
[0030] (a) an OprF/I fusion protein having only a Cys18-Cys27-bond
(SEQ ID NO: 9),
[0031] (b) an OprF/I fusion protein having a Cys18-Cys27-bond and a
Cys33-Cys47-bond (SEQ ID NO: 10), and/or
[0032] (c) an OprF/I fusion protein having a Cys18-Cys47-bond and a
Cys27-Cys33-bond (SEQ ID NO: 11).
[0033] The amino acid numbering is according to the amino acid
sequence of SEQ ID NO: 4. The purity of said mixture is at least
about 75%, preferably at least about 80% to about 90%, in
particular at least about 85%, e.g. 75% to 90% or 85% to 90%
compared to the whole protein content of the mixture as preferably
measured by RP-HPLC.
[0034] As explained above, a particular advantage of the present
invention is that the OprF/I fusion protein does not form undesired
aggregates, in particular high molecular weight aggregates, but
preferably trimers. Interestingly, the OprF/I fusion protein
trimers have a rather elongated shape instead of a globular shape,
and a high hydrodynamic radius, in particular with a calculated
Stokes-radius of 5.6 nm. The trimer was stable in solution e.g.
under physiological conditions such as e.g. pH around 7 and room
temperature, i.e. no dissociation was monitored.
[0035] Therefore, another aspect of the present invention is a
trimeric OprF/I fusion protein comprising a portion of the
Pseudomonas aeruginosa outer membrane protein F which is fused with
its carboxy terminal end to a portion of the amino terminal end of
the Pseudomonas aeruginosa outer membrane protein I, wherein said
portion of the Pseudomonas aeruginosa outer membrane protein F
comprises the amino acids 190-342 of SEQ ID NO: 1 and wherein said
portion of the Pseudomonas aeruginosa outer membrane protein I
comprises the amino acids 21-83 of SEQ ID NO: 2, or an immunogenic
variant thereof having at least 85%, preferably 90%, in particular
95% identity to the amino acid sequence of SEQ ID NO: 3.
[0036] Preferably the trimeric OprF/I fusion protein possesses the
same disulfide bonds as explained above. In addition, the trimeric
OprF/I fusion protein(s) can be present in a mixture as also
explained above.
[0037] Another embodiment of the present invention concerns the
above-specified OprF/I fusion proteins which additionally contain a
N-terminal tag. Therefore, the present invention also concerns a
OprF/I fusion protein with 1-24 amino acids fused to its amino
terminal end. Preferably the N-terminal tag is selected from Met-,
Met-Ala-(His).sub.6- (SEQ ID NO: 5), Ala-(His).sub.6- (SEQ ID NO:
6),
Met-Lys-Lys-Thr-Ala-Ile-Ala-Ile-Ala-Val-Ala-Leu-Ala-Gly-Phe-Ala-Thr-Val-A-
la-Gln-Ala-(SEQ ID NO: 7),
Met-Lys-Leu-Lys-Asn-Thr-Leu-Gly-Val-Val-Ile-Gly-Ser-Leu-Val-Ala-Ala-Ser-A-
la-Met-Asn-Ala-Phe-Ala-(SEQ ID NO: 8), or any other N-terminal
sequence disclosed in Table 1 of Gabelsberger et al. (1997)
(Gabelsberger, J et al., A Hybrid Outer Membrane Protein Antigen
for Vaccination Against Pseudomonas aeruginosa, Behring Inst.
Mitt., 1997, 98, 302-314) namely the E. coli OmpT signal peptide or
the E. chrysanthemii PelB signal peptide. It is also possible that
a spacer, preferably a Ser-Thr-Gly-Ser-spacer (SEQ ID NO: 12),
between the tag and the N-terminus of the OprF/I fusion protein is
located. A particularly preferred OprF/I fusion protein contains an
Ala-(His).sub.6-N-terminus (SEQ ID NO: 6) because the fusion
protein can easily be purified by immobilized metal affinity
chelate chromatography as explained below.
[0038] In view of the above explanations, another particularly
preferred embodiment of the present invention is, therefore, a
mixture, in particular a complex, of OprF/I fusion proteins, each
of the OprF/I fusion proteins comprises a portion of the
Pseudomonas aeruginosa outer membrane protein F which is fused with
its carboxy terminal end to a portion of the amino terminal end of
the Pseudomonas aeruginosa out membrane protein I, wherein said
portion of the Pseudomonas aeruginosa outer membrane protein F
comprises the amino acids 190-342 of SEQ ID NO: 1 and wherein said
portion of the Pseudomonas aeruginosa outer membrane protein I
comprises the amino acids 21-83 of SEQ ID NO: 2, and each of the
OprF/I fusion proteins contains an Ala-(His).sub.6-N-terminus, said
mixture containing
[0039] (a) an OprF/I fusion protein having only a Cys18-Cys27-bond
(SEQ ID NO: 9),
[0040] (b) an OprF/I fusion protein having a Cys18-Cys27-bond and a
Cys33-Cys47-bond (SEQ ID NO: 10), and/or
[0041] (c) an OprF/I fusion protein having a Cys18-Cys47-bond and a
Cys27-Cys33-bond (SEQ ID NO: 11).
[0042] The amino acid numbering is according to the amino acid
sequence of SEQ ID NO: 4. The purity of said mixture is at least
about 75%, preferably at least about 80% to about 90%, in
particular at least about 85%, e.g. 75% to 90% or 85% to 90%
compared to the whole protein content of the mixture as preferably
measured by RP-HPLC as described above. Furthermore, the mixture
contains preferably dimers and in particular trimers of said OprF/I
fusion protein.
[0043] Another aspect of the present invention concerns a method
for producing the above-specified OprF/I fusion protein(s). The
preferred method according to the present inventions comprises the
steps of [0044] (a) reducing said OprF/I fusion protein(s) with a
reducing agent, preferably dithiothreitol (DTT), dithioerythritol
(DTE) or .beta.-mercaptoethanol, and [0045] (b) oxidizing the
reduced OprF/I fusion protein(s) with a redox agent, preferably the
redox agent glutathione disulfide/glutathione or the redox agent
cystine/cysteine, in the presence of a reducing agent, preferably
dithiothreitol (DTT), dithioerythritol (DTE) or
.beta.-mercaptoethanol.
[0046] The purpose of the reduction step is to break up all intra-
and intermolecular disulfide bonds of highly cross-linked disulfide
aggregates formed during expression in e.g. E. coli. Consequently,
the fully reduced protein elutes as a single peak from a RP-HPLC
column (see e.g. FIG. 2). The concentration of the reducing agent
is in particular from about 3 mM to about 10 mM, preferably from
about 3 mM to about 6 mM, e.g. about 5 mM. DTT is the most
preferred reducing agent because it is non-toxic. The reaction time
of the reduction step (a) is in particular from about 15 minutes to
about 2 hours, preferably from about 30 minutes to about 1 hour,
especially about 30 minutes, and/or the pH value is preferably from
about 7.0 to about 8.5, in particular about 8.0.
[0047] The reoxidation can be carried out with different redox
systems. The progress of reoxidation, i.e. the formation of
disulfide bonds can be monitored by RP-HPLC. Surprisingly it was
found that in the presence of reducing and oxidizing agent, in
particular at low concentrations, reshuffling of the disulfide
bonds resulted in essentially correct bond formation, i.e.
misfolded forms of high and low molecular weight aggregates as e.g.
shown in FIG. 1 were minimized and a stable solution of immunogenic
fusion proteins containing preferably dimers and in particular
trimers could be obtained (see e.g. FIGS. 2 and 3). The fusion
protein(s) are stable in aqueous solution at neutral pH in the
presence of a salt like NaCl, e.g. 0.15 M NaCl, e.g. the fusion
protein of SEQ ID NO: 4 in form as a trimer is stable for up to 24
months formulated in PBS at 2 to 8.degree. C. The most preferred
redox agent is cystine/cysteine and the most preferred reducing
agent in the reoxidation step is DTT. The preferred concentration
of the redox agent is from about 0.2 mM to about 4 mM, preferably
about 0.2 mM to about 1 mM, in particular about 0.2 mM to about 0.5
mM, and the concentration of the reducing agent is from about 0.5
mM to about 1.5 mM, preferably about 1 mM. The most preferred
reoxidation of the fusion protein(s) can be carried out in the
presence of 0.5 mM cystine and 1 mM DTT final concentrations. The
reaction temperature is in particular from about 18.degree. C. to
about 25.degree. C., preferably at about 20.degree. C. The reaction
time of the oxidation step (b) is in particular from about 1 hour
to about 20 hours, preferably from about 1 hour to about 6 hours,
especially from about 1.5 hours to about 2 hours, and/or the pH
value is preferably from about 7.5 to about 8.5, in particular
about 8.0. Generally, a protein concentration from about 0.2 mg/mL
to about 10 mg/mL, preferably from about 0.2 mg/mL to about 1
mg/mL, in particular from about 0.2 mg/mL to about 0.5 mg/mL,
especially at about 0.35 mg/mL is applicable.
[0048] Another preferred embodiment of the present invention
concerns the subsequent purification of the reoxidized fusion
protein(s) by an anion exchange chromatography, in particular
Diethylaminoethyl- (DEAE-), Diethyl-(2-hydroxypropyl)aminoethyl-
(QAE-) or Trimethylaminomethyl- (Q-) exchange chromatography,
preferably DEAE- and/or Q-exchange chromatography in order to
reduce e.g. the endotoxin content and the genomic DNA content.
These remaining impurities can bind to anion exchange media at
neutral to slightly basic pH even at higher conductivity, whereas
the fusion protein product(s) remain in the flow through. It is
most preferred to purify the reoxidized OprF/I fusion protein(s)
sequentially by DEAE- and Q-exchange chromatography, preferably by
DEAE Sepharose.RTM. and Q-Sepharose.RTM.-HP chromatography, because
the additional chromatography can separate between the various
forms of the fusion protein(s), e.g. peak 1, 2, 3, 4, 5, and high
molecular weight aggregates, and degradation by-products, e.g. a 7
kD fragment, which still may be present after the reoxidation and
the first chromatography purification step. Finally, the purified
OprF/I fusion protein(s) can be diafiltrated against a buffer
solution, in particular a formulation buffer, e.g. an isotonic
phosphate buffer saline solution (pH 7.4).
[0049] Generally, the above-described OprF/I fusion protein is
produced by fermentation, preferably by expression in a suitable
host, e.g. E. coli. Usually, the fusion protein is expressed
intracellularly in soluble form e.g. at 30.degree. C. and isolated
after cell lysis with e.g. lysis buffer containing e.g. high
concentrations of a salt, e.g. NaCl, in particular 0.5 M NaCl, and
low concentration of a diazole e.g. imidazole, and in particular
0.06 M imidazole. A preferred lysis buffer contains 0.1 M Tris (pH
7.4), 0.5 M NaCl and 0.06 M imidazole.
[0050] Thereafter it is preferred to purify the OprF/I fusion
protein by affinity chromatography prior to the above-described
reduction step. Preferred affinity chromatographies are
immunoaffinity or immobilized metal ion affinity chromatography, in
particular immobilized metal ion affinity chromatography which can
be used for capturing the His-tagged OprF/I fusion protein.
Chelating Sepharose.RTM. loaded with copper ions is most preferred.
Thereafter, desalting e.g. on Sephadex G50 or by
ultra/diafiltration using a 100 kDa cut-off membrane is further
preferred in order to reduce the content of low molecular weight
impurities, e.g. imidazole or copper. In addition, a buffer change
is conducted with this purification step. A preferred elution
buffer is 0.1 M Tris (pH 8.0) with 0.15M NaCl because this buffer
is also a preferred buffer for the following reduction and
reoxidation steps. An overview of the most preferred production and
purification process is shown in FIG. 6. In short, the process can
be summarized as follows: [0051] (a) fermenting a suitable host,
e.g. E. coli, expressing the described OprF/I fusion protein,
[0052] (b) lysing the host, [0053] (c) capturing the produced
OprF/I fusion protein by affinity chromatography, preferably by
IMAC, [0054] (d) desalting the eluted OprF/I fusion protein, [0055]
(e) reducing of the OprF/I fusion protein with a reducing agent,
[0056] (f) reoxidizing the reduced OprF/I fusion protein with a
redox agent in the presence of a reducing agent, [0057] (g)
purifying the reoxidized OprF/I fusion protein on anion exchange
chromatography, preferably on DEAE Sepharose.RTM., [0058] (h)
purifying the eluted OprF/I fusion protein on a further anion
exchange chromatography, preferably on Q-Sepharose.RTM., [0059] (i)
diafiltration the eluted OprF/I fusion protein into a formulation
buffer.
[0060] The formulation buffer is preferably an isotonic salt
solution buffer containing, e.g. KCl, NaCl and phosphate buffer (pH
7.4), as in particular specified under the section "Materials".
[0061] Consequently, the fusion protein(s) directly obtained by the
above-described methods is also a specific embodiment of the
present invention. Examples of such fusion protein(s) are also
described above and in the following examples.
[0062] Another aspect of the present invention is also a
pharmaceutical composition, in particular a vaccine, comprising the
described OprF/I fusion protein(s) or obtained by the
above-described method(s), and optionally at least one additive or
adjuvant, in particular aluminium hydroxide, which may serve as an
additional stabilizer. A typical formulation of the pharmaceutical
composition contains an isotonic phosphate buffer saline solution
(pH 7.4).
[0063] This preferred composition (SEQ ID NO:4 prepared according
to the method described herein and formulated in PBS) is stable up
to 24 months at about 2.degree. C. to about 8.degree. C.
[0064] Another aspect of the present inventions concerns an
antibody or antibody derivative which specifically binds the
above-specified OprF/I fusion protein(s) such as e.g. the trimer
comprising the herein specified OprF/I fusion protein(s). The
antibody is either polyclonal or monoclonal, preferably it is a
monoclonal antibody. The term "antibody derivative" is understood
as also meaning antigen-binding parts of the inventive antibody,
prepared by genetic engineering and optionally modified antibodies,
such as, for example, chimeric antibodies, humanized antibodies,
multifunctional antibodies, bi- or oligospecific antibodies,
single-stranded antibodies, F(ab) or F(ab).sub.2 fragments, which
are all well known for a person skilled in the art.
[0065] The invention includes isolated antibodies and binding
fragments thereof that selectively bind trimers of OprF/I fusion
proteins as described herein. As used herein with respect to the
binding of trimers of OprF/I fusion proteins by the antibodies and
binding fragments, "selectively binds" means that an antibody
(binding fragment thereof) preferentially binds to a trimer of
OprF/I fusion proteins (e.g., with greater avidity and/or binding
affinity) than to an OprF/I fusion protein monomer. In preferred
embodiments, the antibodies of the invention and binding fragments
thereof bind to a trimer of OprF/I fusion proteins with an avidity
and/or binding affinity that is 1.1-fold, 1.2-fold, 1.3-fold,
1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold,
3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,
20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold,
90-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold or more
than that exhibited by the antibody and binding fragments thereof
for an OprF/I fusion protein monomer. Preferably, the antibody
selectively binds trimers of OprF/I fusion proteins, and not OprF/I
fusion protein monomers, i.e., substantially exclusively binds to
trimers of OprF/I fusion proteins, or specifically binds trimers of
OprF/I fusion proteins without substantial binding to OprF/I fusion
protein monomers.
[0066] In some embodiment, the isolated antibodies or
antigen-binding fragments thereof bind to a trimer-specific
epitope. Generally, antibodies or antigen-binding fragments thereof
that bind to a trimer-specific epitope preferentially bind a trimer
of OprF/I fusion proteins rather than a OprF/I fusion protein
monomer. To determine if a selected antibody binds preferentially
(i.e., selectively and/or specifically) to a trimer of OprF/I
fusion proteins, each antibody can be tested in comparative assays
(e.g., a surface plasmon resonance (SPR) assay such as BiaCore or
immunoprecipitation followed by Western blotting) using trimers of
OprF/I fusion proteins and OprF/I fusion protein monomers. A
comparison of the results will indicate whether the antibodies bind
preferentially to the trimer or to the monomer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0067] FIG. 1 schematically depicts the reduction and controlled
reoxidation processes according to the present invention.
[0068] FIG. 2 shows the superimposition of RP-HPLC profiles of the
OprF/I fusion protein after expression and capturing on IMAC, after
reduction, and after reoxidation/purification.
[0069] FIG. 3 shows the superimposition of SEC profiles of the
OprF/I fusion protein after expression and capturing on IMAC, and
after reoxidation/purification.
[0070] FIG. 4 shows the RP-HPLC analysis of the reoxidized IMAC/G50
pool. Samples were analyzed after 300 minutes and 21 hours.
[0071] FIG. 5 shows the change in retention time during SEC
analysis of OprF/I fusion protein samples at pH 8.0 and pH 2.
[0072] FIG. 6 shows a flow scheme of an exemplary production and
purification process of the OprF/I fusion protein.
[0073] FIG. 7A shows preparative RP-HPLC elution profiles; and FIG.
7B shows analytical RP-HPLC elution profiles of an elected QSHP
fraction.
[0074] FIG. 8 shows the disulphide bond pattern of peaks P1, P2 and
P3 of the OprF/I fusion protein.
[0075] FIG. 9 shows the RP-HPLC peak pattern of purified OprF/I
drug substance mixture.
TABLE-US-00001 [0076] SEQUENCES SEQ ID NO: 1 (full length Opr F) 1
MKLKNTLGVV IGSLVAASAM NAFAQGQNSV EIEAFGKRYF TDSVRNMKNA DLYGGSIGYF
61 LTDDVELALS YGEYHDVRGT YETGNKKVHG NLTSLDAIYH FGTPGVGLRP
YVSAGLAHQN 121 ITNINSDSQG RQQMTMANIG AGLKYYFTEN FFAKASLDGQ
YGLEKRDNGH QGEWMAGLGV 181 GFNFGGSKAA PAPEPVADVC SDSDNDGVCD
NVDKCPDTPA NVTVDANGCP AVAEVVRVQL 241 DVKFDFDKSK VKENSYADIK
NLADFMKQYP STSTTVEGHT DSVGTDAYNQ KLSERRANAV 301 RDVLVNEYGV
EGGRVNAVGY GESRPVADNA TAEGRAINRR VEAEVEAEAK SEQ ID NO: 2 (precursor
Opr I) 1 MNNVLKFSAL ALAAVLATGC SSHSKETEAR LTATEDAAAR AQARADEAYR
KADEALGAAQ 61 KAQQTADEAN ERALRMLEKA SRK SEQ ID NO: 3 (OprF/I with
N-tag plus Met) 1 MAHHHHHHAP APEPVADVCS DSDNDGVCDN VDKCPDTPAN
VTVDANGCPA VAEVVRVQLD 61 VKFDFDKSKV KENSYADIKN LADFMKQYPS
TSTTVEGHTD SVGTDAYNQK LSERRANAVR 121 DVLVNEYGVE GGRVNAVGYG
ESRPVADNAT AEGRAINRRV ESSHSKETEA RLTATEDAAA 181 RAQARADEAY
RKADEALGAA QKAQQTADEA NERALRMLEK ASRK SEQ ID NO: 4 (OprF/I with
N-tag without Met) 1 AHHHHHHAPA PEPVADVCSD SDNDGVCDNV DKCPDTPANV
TVDANGCPAV AEVVRVQLDV 61 KFDFDKSKVK ENSYADIKNL ADFMKQYPST
STTVEGHTDS VGTDAYNQKL SERRANAVRD 121 VLVNEYGVEG GRVNAVGYGE
SRPVADNATA EGRAINRRVE SSHSKETEAR LTATEDAAAR 181 AQARADEAYR
KADEALGAAQ KAQQTADEAN ERALRMLEKA SRK SEQ ID NO: 5 (N-tag plus Met)
1 MAHHHHHH SEQ ID NO: 6 (N-tag without Met) 1 AHHHHHH SEQ ID NO: 7
(OmpA signal peptide E. coli) 1 MKKTAIAIAV ALAGFATVAQ A SEQ ID NO:
8 (OprF signal peptide P. aeruginosa) 1 MKLKNTLGVV IGSLVAASAM AAFA
SEQ ID NO: 9 (OprF/I with Cys18-Cys27-bond) Disulfide bond between
Cys18 (underlined) and Cys27 (underlined) 1 AHHHHHHAPA PEPVADVCSD
SDNDGVCDNV DKCPDTPANV TVDANGCPAV AEVVRVQLDV 61 KFDFDKSKVK
ENSYADIKNL ADFMKQYPST STTVEGHTDS VGTDAYNQKL SERRANAVRD 121
VLVNEYGVEG GRVNAVGYGE SRPVADNATA EGRAINRRVE SSHSKETEAR LTATEDAAAR
181 AQARADEAYR KADEALGAAQ KAQQTADEAN ERALRMLEKA SRK SEQ ID NO: 10
(OprF/I with Cys18-Cys27-bond and a Cys33-Cys47-bond) Disulfide
bond between Cys18-Cys27 (both underlined) and Cys33-Cys47 (both
italic) 1 AHHHHHHAPA PEPVADVCSD SDNDGVCDNV DKCPDTPANV TVDANGCPAV
AEVVRVQLDV 61 KFDFDKSKVK ENSYADIKNL ADFMKQYPST STTVEGHTDS
VGTDAYNQKL SERRANAVRD 121 VLVNEYGVEG GRVNAVGYGE SRPVADNATA
EGRAINRRVE SSHSKETEAR LTATEDAAAR 181 AQARADEAYR KADEALGAAQ
KAQQTADEAN ERALRMLEKA SRK SEQ ID NO: 11 (OprF/I with
Cys18-Cys47-bond and a Cys27-Cys33-bond) Disulfide bond between
Cys18-Cys47 (both underlined) and Cys27-Cys33 (both italic) 1
AHHHHHHAPA PEPVADVCSD SDNDGVCDNV DKCPDTPANV TVDANGCPAV AEVVRVQLDV
61 KFDFDKSKVK ENSYADIKNL ADFMKQYPST STTVEGHTDS VGTDAYNQKL
SERRANAVRD 121 VLVNEYGVEG GRVNAVGYGE SRPVADNATA EGRAINRRVE
SSHSKETEAR LTATEDAAAR 181 AQARADEAYR KADEALGAAQ KAQQTADEAN
ERALRMLEKA SRK SEQ ID NO: 12 (spacer) 1 STGS
EXPERIMENTAL PART OF THE INVENTION
Abbreviations
TABLE-US-00002 [0077] Abbreviation Explanation AUC Analytical
ultracentrifugation CV Column volume DTT Dithiothreitol DV
Diafiltration volumes DS Drug substance ED50 Reverse of the
dilution of the samples resulting in 50% seroconversion rate EGT
Eurogentec gDNA Genomic DNA GMT Geometric mean titer GSH Reduced
glutathione GSSG Oxidized glutathione HCP Host cell protein HPLC
High performance liquid chromatography ICLL Intercell IMAC
Immobilized metal affinity chelate chromatography MALDI-ToF Matrix
assisted Lased Desorption Ionization Mass Spectrometry-Time of
Flight MALS Multi Angle Light Scattering .beta.-ME
Beta-mercaptoethanol PAGE Polyacrylamide gel Electrophoresis QSHP
Q-Sepharose HP RP Reversed phase RT Room temperature (about
20.degree. C.) SCD Sedimentation Coefficient Distributions SEC Size
exclusion chromatography UF/DF Ultrafiltration/Diafiltration
[0078] Materials
[0079] NaOH (Riedel-de Haen), NaCl (Riedel-de Haen),
Tris(hydroxymethyl)aminomethane (Merck KGaA, Darmstadt), L-Cystine
(Aldrich), DTT (Sigma), HCl (Merck KGaA), Q-Sepharose.RTM. HP (GE
Healthcare), DEAE-Sepharose.RTM. FF (GE Healthcare). All other
materials were of analytical grade if not otherwise stated.
[0080] Formulation buffer: Dulbecco's 1.times.PBS pH 7.4 (H15-002),
lx concentrate (g/L)
TABLE-US-00003 KCl 0.2 g/L KH.sub.2PO.sub.4 0.2 g/L NaCl 8.0 g/L
Na.sub.2HPO.sub.4 anhydrous 1.15 g/L
[0081] General Methods
[0082] Analytical RP-HPLC
[0083] Analytical RP-HPLC analysis of samples was performed on a
Jupiter C4 column (4.6 mm.times.150 mm, 300 A, 5 .mu.m, Phenomenex)
connected to a Dionex Ultimate 3000 HPLC system. Solvent A was
water containing 0.1% TFA, solvent B was acetonitrile containing
0.1% TFA. Separation of peaks was performed by linear gradient
elution from 27% B to 37% B in 13 min at a flow rate of 1 mL/min.
The column temperature was set to 40.degree. C. Peak detection was
performed at 214 nm and 280 nm.
[0084] For downstream development work an estimation of the
specific OprF/I content in IMAC/G50 was necessary to calculate step
yields. OprF/I content was determined by RP-HPLC. The HPLC system
was calibrated with purified, native (unreduced) OprF/I working
standard. The protein content of the working standard was
determined by UV 280 nm measurement based on a calculated
theoretical extinction coefficient for a 1 mg/mL solution of
.epsilon..sub.0.1%=0.373. Prior to analysis of IMAC/G50 pools by
RP-HPLC, an aliquot was fully reduced by addition of DTT or
.beta.-mercaptoethanol (100 mM final concentration) to split up the
various aggregated and misfolded (most probably disulfide
scrambled) OprF/I variants. The samples were incubated at room
temperature for 30 minutes and analyzed by RP-HPLC. After
reduction, OprF/I eluted as a single peak compared to the untreated
IMAC/G50 pool. The content of reduced OprF/I after IMAC/G50 was
calculated by integration of the peak area.
[0085] All other samples (e.g. reoxidized OprF/I, fractions from
QS-HP etc.) were directly injected without further treatment and
the OprF/I concentration was calculated.
[0086] Reoxidized samples can be immediately analyzed by RP-HPLC or
formation of disulfide bonds can be quenched by acidification to pH
2-3 (.about.20 .mu.L 6% HCl per 1 ml reoxidation solution) and
stored at 2-8.degree. C. for subsequent analysis.
[0087] Semi-Preparative RP-HPLC
[0088] Semi-Preparative RP-HPLC was used for isolation of
individual peaks detected by analytical RP-HPLC. Purification was
done on a Jupiter C4 column (10 mm.times.250 mm, 300 A, 5 .mu.m,
Phenomenex) connected to an Akta Purifier chromatography system.
The stationary phase at preparative scale was the same as the one
used at analytical scale. Solvent A was water containing 0.1% TFA,
solvent B was 80% acetonitrile in water containing 0.1% TFA. Sample
volume was 2 to 4 mL (total protein load <2 mg). Separation of
peaks was performed by linear gradient elution from 35% B to 40% B
over 8 column volumes at a flow rate of 2.5 mL/min. The column
temperature was set to 40.degree. C. Peak detection was done at 280
and 214 nm Fractions of 0.8 mL were collected and the pH was
adjusted to pH.about.7 by addition of 0.25 mL 0.1 M sodium
phosphate buffer, pH 7.0. Higher quantities (.about.0.5 to 2 mg) of
P1 to 4 were prepared by several preparative purification runs.
After pooling of the desired fractions containing the individual
peaks, samples were concentrated approximately 5 times using a 5
kDa ultracentrifugation device (Millipore). Concentrated pools were
desalted by PD10 columns (GE Healthcare) and the buffer was
exchanged against final drug product formulation buffer (1/10 PBS
diluted with 0.9% NaCl, pH .about.7). Final samples containing the
isolated OprF/I variants were analyzed for purity and content by
RP-HPLC and SEC-HPLC. The relative purity determined by RP-HPLC was
at least 90%. Samples were stored at -20.degree. C. until further
analysis.
[0089] SDS-PAGE
[0090] SDS-PAGE was done on 4-12% NuPAGE gels (Invitrogen) using
MES running buffer. Samples were mixed with LDS sampling buffer
under reducing or non-reducing conditions and incubated for 5 min
at 70.degree. C. if not otherwise stated. Staining was done with
colloidal Commassie or silver stain (Heukeshoven).
[0091] Western Blot Analysis
[0092] Western blotting was done with antibodies anti OprF/I 944/5
D5 epitope (1:20000 diluted) and 966/363 E3 epitope (1:10000
diluted).
[0093] pH and Conductivity Measurement
[0094] For determination of pH and conductivity of samples and
buffers a WTW 720 system was used. Conductivity was measured using
the linear temperature compensation mode at 25.degree. C.
[0095] Endotoxin Measurement
[0096] Endotoxin measurement was done with a chromogenic LAL-assay
(Cambrex). Selected samples were also measured in an external
certified laboratory with a conventional gel clot assay (Limulus
Amoebocyte Lysate test).
[0097] Host Cell Protein Measurement (HCP)
[0098] For quantification of HCPs, a generic E. coli HCP ELISA kit
(Cygnus Technologies, Inc.) was used.
[0099] Peptide-Mass Fingerprint and Disulphide Mapping
[0100] Purified fractions obtained from preparative RPC were
analyzed by LC-MS/MS. Samples were digested with AspN or trypsin
without reduction or after reduction and alkylation.
[0101] MALDI-ToF Mass Spectrometry
[0102] MALDI-ToF analysis was performed on a Voyager STR 4069
system (Applied Biosystems). Sinapinic acid dissolved in 0.1%
TFA/30% AcN was used as sample matrix. DS samples were diluted
five-fold with sample matrix and 2 .mu.l were placed on the target.
A delayed extraction mode and positive polarity was used. The
system was externally calibrated with BSA (Mass calibration kit,
Applied Biosystems). For internal calibration Myoglobin (Sigma
M-0630, average Mr 16951.5) was spiked into DS samples at a
concentration of approximately 100 .mu.g/mL. The mass accuracy for
internal calibration can be estimated with approximately .+-.0.3%
(e.g. 24100.+-.72 Da), for external calibration .+-.0.6% (e.g.
24100.+-.145 Da).
[0103] Native PAGE
[0104] The NativePAGE.TM. Novex.RTM. Bis-Tris Gel system is a near
neutral pH, pre-cast polyacrylamide mini gel system to perform
native (non-denaturing) electrophoresis. Native PAGE of OprF/I
fusion protein samples was done on NativePAGE 4-16% Bis-Tris gels
(Invitrogen) according to the manufacturers instruction. Sample
buffer was 50 mM BisTris, 50 mM NaCl, 16 mM HCl, 10% w/v Glycerol,
0.001% Ponceau S, pH 7.2. Running buffer was 50 mM BisTris, 50 mM
Tricine, pH 6.8. Cathode buffer was running buffer including 0.02%
Coomassie G-250.
[0105] N-Terminal Sequencing
[0106] N-terminal sequencing was carried out using an Applied
Biosystems 494HT machine and the method of N-terminal Edman
sequencing, where the N-terminal amino acid of the protein was
sequentially removed chemically and identified by HPLC. The protein
was first immobilized inside the sequencing instrument by either
blotting it onto a PVDF membrane or adsorbing it onto a biobrene
treated glass fibre filter. Subsequently the bound protein reacted
with the Edman reagent, (phenylisothiocyanate, PITC) at high pH.
After this reaction, the resulting compound was cleaved off the
protein using anhydrous acid. The coupling and cleavage process was
repeated for as many times as required. Usually 15 to 20 amino
acids ("amino acids" herein also referred to as "aa") could be
analyzed. The cleaved products were converted to their stable
phenylthiohydantoins, PTH, with aqueous acid, and then analyzed
using the on-board HPLC. Identification of the amino acids was
achieved by comparing elution times compared to a standard mixture.
Data from the HPLC was collected on a computer for visual calling
of the sequence.
[0107] Alkylation of Thiolgroups
[0108] Free thiol groups in proteins can be detected by alkylation
using iodoacetamide, which reacts selectively with free thiol
groups of cysteines to produce carboxamidomethyl cysteine. If free
thiol groups are present, these would be covalently blocked
resulting in a mass increase of 57 Da per attached iodacetamide
molecule.
[0109] 47 mg iodoacetamide were dissolved in 1 mL 1 M Tris-HCl, pH
8.0 (0.2 M iodoacetamide solution). 200 .mu.L each of purified peak
1, 2 and 3 (protein concentration approximately 200 .mu.g/mL) were
mixed with 20 .mu.L of iodoacetamide stock solution (final
iodoacetamide concentration .about.0.02M). The OprF/I fusion
protein sample (protein concentration approximately 1 mg/mL) was 3
fold diluted with PBS to a final concentration of approximately 330
.mu.g/mL. 30 .mu.L iodacetamide stock solution were added to 300
.mu.L diluted DS. In another experiment the sample was reduced with
5 mM DTT (20 min) before dilution and alkylation. All samples were
incubated at room temperature in the dark for 30 min followed by
LC-MS analysis.
[0110] Static Light Scattering Analysis
[0111] The chromatographic system consisted of an HPLC system from
Dionex including an Ultimate 3000 pump and degasser, an Ultimate
3000 autosampler and an Ultimate 3000 column compartment. Column
and chromatographic conditions were the same as described for
SEC-HPLC. All solvents were filtered through a 0.1 .mu.m Supor
Membrane filter (Pall VacuCap 60). An injection volume of 100 .mu.L
was used for all samples if not stated otherwise.
[0112] Chromatographic detectors included a Dionex Ultimate 3000
photodiode array detector set to 214 nm and 280 nm, a Shodex RI-101
refractive index detector and a DAWN TREOS MALS (multi angle light
scattering) detector (Wyatt Technology Corporation), which was used
in on-line mode. Chromatographic data collection and analysis was
performed using the Chromeleon software package (vers. 6.80,
Dionex). Experimental collection and data analysis of the
MALS-signals were performed with the ASTRA software package
(version 5.3.2.13, Wyatt Technology). Using this software it was
possible to collect and subsequently analyze the light scattering
signals (3 MALS angles) along with the UV-, and RI-signals.
[0113] Analytical Ultracentrifugation (AUC)
[0114] All experiments were performed with a BeckmanCoulter XL-I
Analytical Ultracentrifuge at 50.000 rpm and 25.degree. C. Samples
were placed in sapphire-capped two-sector titanium centerpieces of
12 mm optical path length. 390 .mu.L of solution and solvent were
placed in the sample and reference sectors, respectively.
Sedimentation traces were detected by recording local differences
in refractive index (interference optics). The samples were
analyzed with a ten-fold dilution or without further dilution.
Diffusion-corrected Sedimentation Coefficient Distributions (SCD)
were calculated using the finite element approach proposed by P.
Schuck, NIH (Peter Schuck et al., Biopolymers, Vol 54, Issue 5,
pages 328-341, October 2000). The frictional ratio f/f0 was treated
as a fitting variable. The density and viscosity of the buffer
(phosphate buffered saline, PBS) as well as the partial specific
volume (v) of the proteins were calculated from composition with
Sednterp. These values were used when calculating the respective
SCD.
[0115] Analysis of OprF/I Fusion Protein Samples Including
Aluminium Hydroxide by RP-HPLC
[0116] Aliquots (0.25 ml) of formulated OprF/I fusion protein were
centrifuged at 16000.times.g for 10 minutes at 20.degree. C. to
separate the aluminium hydroxide sediment from the supernatant. The
clear supernatant was removed and used for analysis of unbound
fusion protein by RP-HPLC. The remaining pellet was resuspended in
0.25 ml of 0.1% TFA in water (pH .about.2). Samples were incubated
at RT for 2 h, followed by 10 minutes centrifugation at 16.000 g at
room temperature to spin down the Aluminium particles. The clear
supernatant was used for analysis by RP-HPLC (TFA desorption).
[0117] Specific Methods and Results
[0118] Expression and Recovery of OprF/I Fusion Protein
[0119] OprF/I is a fusion protein of the pseudomonas outer membrane
porin proteins OprF and OprI. It is expressed as a 224 aa hybrid
protein containing a His.sub.6-tag at its N-terminus. The
N-terminal Met is cleaved off after expression in E. coli. The
primary structure of the expressed protein (including the
N-terminal methionine) is shown in SEQ ID NO: 3.
[0120] The molecular weight of the native protein has been
calculated as 24118.2 Da (full reduced protein, no N-terminal
methionine). The pI has been calculated as 5.3.
[0121] The protein of the present examples is a fusion protein of
outer membrane protein F and I containing a N-terminal histidine
tag (His tag). The protein was expressed in E. coli
XL1-Blue/pTrc-Kan-OprF/I_His strain. The OprF/I-His protein was
expressed intracellularly in soluble form at 30.degree. C.
[0122] Cell Lysis
[0123] OprF/I may be degraded by bacterial proteases, in particular
when lysis buffer without high concentration of NaCl and imidazole
was used. Therefore, cells were resuspended in cold lysis buffer
(1:5 dilution of cell paste in buffer) consisting of 0.1 M Tris, pH
7.4, 0.5 M NaCl, 0.06 M imidazole. Addition of 0.5 M NaCl
particularly inhibited proteolytic degradation of the molecule in
the lysate. Resuspension and subsequent homogenization (2 cycles at
800 bar) was done at cold room temperature and the lysate was
placed on ice immediately. Higher temperatures may lead to product
degradation or higher protease activity.
[0124] IMAC-Copper Capture Step
[0125] Chelating Sepharose FF (loaded with copper ions) was used
for capturing the His-tagged OprF/I. After loading the lysate,
elution was performed with different concentrations of imidazole:
0.07 M, 0.325 M and 0.5 M imidazole. OprF/I containing fractions
elute at 0.325 M imidazole as two separate peaks. Analytical data
showed that RP-HPLC elution profile contained several peaks. If the
same samples were analyzed under reduced conditions (addition of
DTT or .beta.-ME) only one major peak was observed. The various
peaks in the untreated sample were most probably disulfide
scrambled variants and aggregates of the native molecule.
[0126] An exemplary purification run was done with 992 g cell paste
that is equivalent to 8.59 L of fermentation broth. After the IMAC
purification and desalting on Sephadex.RTM. G50 (see below) the
total amount of OprF/I was approximately 1600 mg which is
equivalent to 186 mg OprF/I per liter fermentation broth.
[0127] Desalting on Sephadex G50
[0128] This step reduced the content of low molecular weight
impurities (e.g. imidazole, copper, etc.) and a buffer exchange was
conducted. The loading volume was approximately 20% of the column
volume. As elution buffer 0.1M Tris-HCl, 0.15M NaCl, pH 8.0 was
used. It was the same buffer used for reduction and reoxidation.
Alternatively, this step was also replaced by UF/DF with a 100K
cut-off membrane.
[0129] Reduction
[0130] After the IMAC/G50 steps, OprF/I exists as heterogeneous
mixture of misfolded forms (high and low molecular weight
aggregates) caused by disulfide scrambling as schematically
depicted in FIG. 1. Reduction of disulfide bonds was done with 5 mM
DTT to break up all intra- and intermolecular disulfide bonds. The
fully reduced protein elutes as a single peak according to RP-HPLC
data. DTT can be substituted by .beta.-ME. Since DTT is not stable
over a longer period of time in aqueous solution, an aliquot of a
freshly prepared DTT solution (1 M in water, used within 1 hour) is
added to the IMAC/G50 pool under gentle stirring (5 mL of 1 M DTT
stock solution per liter IMAC/G50 pool). The pool is incubated at
room temperature for 30 minutes without stirring. Samples can be
analyzed by RP-HPLC to monitor the progress of reduction.
[0131] Reoxidation
[0132] For optimization of the reoxidation conditions, different
redox systems (GSSG/GSH, cystamine/cysteamine, cystine/cysteine)
were tested out in presence of low concentration of DTT (1 mM) to
allow correct reshuffling of the disulfide bond. The progress of
reoxidation (formation of disulfide bonds) can be monitored by
RP-HPLC after various time intervals since the folding variants
have different retention times. Reoxidation with
cystamine/cysteamine was unsuccessful under the tested conditions.
In a first set of experiments, GSSG and GSH were tested out as
reoxidation agents. The reduced IMAC/G50 pool in 5 mM DTT was
diluted 5-fold into 0.1 M Tris-HCl, 0.15 M NaCl pH 8.0 containing
GSSG (0-4 mM) under gentle stirring. DTT reacts with GSSG and forms
GSH, GSSG and reduced/oxidized DTT. The final reoxidation
conditions tested out covered a broad range of different ratios of
GSH, GSSG and DTT. Aliquots of the samples were also quenched with
HCl after various time intervals and analyzed by RP-HPLC. At
increasing GSSG concentration peak 1 increases and peak 2
decreases. Formation of peak 1 occurs very early in the reoxidation
process and remains constant over time. The total recovery for
peaks 1+2 was estimated to be .about.60% starting from the
completely reduced protein (100%), the recovery of all detected
peaks was approximately 90% compared to the starting material.
[0133] In a second set of experiments, cystine and cysteine were
tested out as reoxidation agents. The reduced IMAC/G50 pool (5 mM
DTT) was diluted 5-fold into 0.1 M Tris-HCl, 0.15 M NaCl pH 8.0
containing various concentration of cystine (0-3 mM) and cysteine
(0-3 mM). The final DTT concentration was 1 mM. Please note that
the 0.2 M stock solution of cystine was prepared in 0.5 M NaOH.
Samples were analyzed after 300 min and over night incubation at
room temperature. No difference in RP-HPLC peak pattern for each
individual experiment between the two time points was observed
except for the sample containing 1 mM DTT and no cystine. The
protein was still reduced after 5 h, after over night incubation
peak 2 appeared. Depending on the final cystine and cysteine
concentration, different ratios of peak 1 and peak 2 were detected.
RP-HPLC profiles showed that peak 1 concentration was sufficiently
low in presence of 0.5 mM cystine.
[0134] After preliminary studies of the various redox systems, it
was decided to use cystine as the oxidizing agent. During scale-up
of the production process for GMP production the concentration was
further lowered to 0.375 mM cystine. Representative RP-HPLC and SEC
elution profiles prior and after reduction/reoxidation of IMAC/G50
pool are shown in FIG. 2 and FIG. 3. After reoxidation in presence
of 0.5 mM cystine, the elution profiles observed by RP-HPLC and SEC
were much more homogeneous compared to the "untreated" IMAC/G50
pool. The various peaks, present in the IMAC pool before reduction,
shift to one major peak under reducing conditions. After
reoxidation, one major peak (named as peak 2 in FIG. 4) is observed
with a different retention time compared to the reduced protein.
Peak 2 should represent the correctly folded OprF/I. Peak 2 is
surrounded by three smaller peaks (peak 1, peak 3 and peak 4 in
FIG. 4) that should be folding variants. Peaks eluting at
approximately 13.17 and 13.81 min, named as peak 5 and peak 6 in
FIG. 4, are other folding variants (disulfide cross-linked
aggregates according to MS data).
[0135] Further characterization of peak 1 by LC-MS showed an
increase in molecular weight of 240 Da compared to peak 2. This
mass shift was most probably caused by covalent attachment of two
molecules cysteine. Free cysteine was formed by the reaction of DTT
with cystine, which resulted in 2 molecules cysteine. It was
further discovered that peak 1 increases while peak 2 decreases at
increasing concentration of oxidizing agent (GSSG or cystine).
[0136] Evaluation of the main peak after reoxidation by SEC shows
that the protein does not exist as a monomer. The SEC column was
calibrated with reference proteins (BioRad's size exclusion
standard) ranging from 1.35 to 670 kDa. The retention time of the
main peak (.about.25 min) corresponds to a calculated theoretical
mass of .about.180 kDa under the assumption of a globular shape and
no unspecific interactions with the stationary phase. It was
observed that this defined multimeric state was formed preferential
under the process and formulation conditions applied and seemed to
be stable in aqueous solution at neutral pH in presence of NaCl. At
pH 7 to 8 the OprF/I fusion protein elutes as a multimer
corresponding to 180 kDa, whereas in the acidified sample (pH
.about.2) the peak shifts to higher retention time (.about.28 min)
corresponding to approximately 55 kDa (see FIG. 5). This change in
retention time could be caused by dissociation of the multimer at
low pH.
[0137] Purification by DEAE Sepharose FF
[0138] Additional purification of the OprF/I containing process
stream by anion exchange chromatography after reoxidation was
tested out to reduce the content of remaining endotoxins and gDNA.
These remaining impurities would bind to anion exchange media at
neutral to slightly basic pH even at higher conductivity, whereas
the product should remain in the flow through. DEAE Sepharose was
tested out and found to have good properties to remove endotoxins
without any major product losses by binding of OprF/I onto the
resin.
[0139] Purification by Q-Sepharose HP (QSHP)
[0140] After reoxidation and DEAE flow through chromatography, the
protein solution was further purified by Q-Sepharose HP.
Purification by QSHP resulted in an endotoxin concentration of
.about.2 EU/mg in the main pool, which was within an acceptable low
level.
[0141] Ultrafiltration/Diafiltration
[0142] Finally, the QS-HP pool was diafiltrated against formulation
buffer (1.times.PBS buffer pH 7.4, Dulbecco, without Ca, Mg). A 10
kDa or 30 kDa regenerated cellulose membrane (Amicon Ultra 15
centrifugal filter device, Millipore), was used. OprF/I was
detected in the permeate of the 30 kDa membrane. Therefore, a 10
kDa membrane was used for final UF/DF into formulation buffer
resulting in a step yield of >95%. The pool was adjusted to a
final protein concentration of 1 mg/ml based on UV measurement.
[0143] An overview of the whole production and purification process
is shown in FIG. 6. An overall yield of about 34% to about 40% of
purified OprF/I fusion protein was achieved.
[0144] Characterization of the Purified OprF/I Fusion Protein
[0145] Preparative Isolation of OprF/I Fusion Protein Variants
[0146] Selected side fractions from QSHP chromatography steps were
used for preparative isolation. A typical preparative elution
profile and nomination of peaks detected is shown in FIGS. 7A and
7B. All combined fractions containing the individual peaks were
analyzed by SDS-PAGE and Western blot under reducing and
non-reducing conditions. Under reducing conditions all bands had
similar migration properties compared to an OprF/I standard. Under
non-reducing conditions, the content of multimeric OprF/I variants
detected at approximately 60 kDa (calibrated against the molecular
weight marker) increased for Peak C, D, 5 and 6. All bands were
also detected by western blot analysis using monoclonal anti OprF/I
antibodies. These results indicate that all peaks detected by
RP-HPLC are product related. This finding was also confirmed by
peptide-mass fingerprint analysis of the individual fractions. In
final DS only P1, 2, 3, 4 and 5 can be detected by RPC. The other
peaks, A, B, C, D and 6, could be separated by preparative
chromatography on Q-Sepharose HP from the main fractions. During
Q-Sepharose HP chromatography a small peak eluted before the main
peak. This fraction contained a higher concentration of an OprF/I
degradation product (denoted as 7 kDa peak) as detected by
analytical RP-HPLC and MALDI-ToF. This peak was also shown to be a
product related fragment consisting of a 15.5 kDa and 7.2 kDa
OprF/I fragment.
[0147] Analytical Characterization of OprF/I Fusion Protein
Variants
[0148] The purified OprF/I fusion protein consists of different
forms of the molecule as shown by RP-HPLC (see FIG. 4). Five peaks
could be detected by RP-HPLC. Peak 2 (P2) was the most prominent
peak with a relative content of 50 to 55%, surrounded by peak 1
(P1), peak 3 (P3) and Peak 4 (P4). Peak 5 (P5) was well separated
from the other peaks eluting at a slightly higher retention time.
The relative peak content is summarized in Table 1. After reduction
of the sample with .beta.-ME or DTT, the elution profile changes.
One major peak eluted and the individual variants exhibited the
same chromatographic retention time. Based on these results P1 to
P4 are regarded as folding variants caused by differences in
disulphide bonding.
TABLE-US-00004 TABLE 1 Peak Sample 1 Sample 2 Sample 3 Sample 4 1
19% 14% 13% 11% 2 50% 55% 54% 60% 3 18% 17% 19% 14% 4 9% 9% 9% 9%
Sum of Peaks 87% 86% 86% 85% 1, 2 and 3 Note: Reoxidation of sample
1 was done in presence of 0.5 mM cystine; samples 2, 3 and 4 were
reoxidized in presence of 0.375 mM cystine. The slightly higher
cystine concentration resulted in minor increase in peak 1 content
for sample 1.
[0149] MALDI-ToF Analysis
[0150] For MALDI-ToF analysis the system was calibrated externally
against BSA. For internal calibration Myoglobin was used. All four
samples showed similar mass spectra. The main signal was from
native OprF/I monomer followed by OprF/I dimer and trimer peaks.
Table 2 summarizes molecular mass obtained after internal
calibration. Deviation from the expected molecular mass was within
the experimental error (.+-.0.3%). Mass peaks at 24 kDa, 48 kDa and
72 kDa were detected, showing the presence of the monomeric,
dimeric and trimeric OprF/I fusion proteins.
TABLE-US-00005 TABLE 2 Deviation from theoretical mass (Da)* (rel.
% deviation from Peak Analyzed mass (Da) theoretical MW) Monomer
Sample 1 24096 -20 (-0.08) Sample 2 24053 -63 (-0.26) Sample 3
24097 -19 (-0.08) Sample 4 24045 -71 (-0.30) Dimer Sample 1 48408
+176 (+0.36) Sample 2 48104 -128 (-0.27) Sample 3 48239 -7 (-0.01)
Sample 4 48031 -201 (-0.42) Trimer Sample 1 72379 +31 (+0.04)
Sample 2 72105 -243 (-0.34) Sample 3 72135 -213 (-0.30) Sample 4
72250 -98 (-0.14) *theoretical mass: monomer 24114 Da under the
assumption of two disulfide bonds, dimer 48228, trimer 72342
[0151] Native PAGE
[0152] Native PAGE of OprF/I fusion protein samples under
non-reducing and reducing conditions were carried out as explained
above. Band intensities after Commassie blue staining were
evaluated by densitometry. Under native conditions one OprF/I main
band was detected in the range of approximately 180 kDa with a
relative intensity of approximately 94 to 97%. Under reducing
conditions the apparent molecular size was determined as 206 kDa.
The apparent molecular weight is in good correlation with SEC-HPLC
data, but different from SEC-MALS and AUC results where OprF/I mass
was in the range of 80 kDa (trimer). The separation mechanism for
native PAGE is the same as for native SEC, separation properties
strongly depend on the shape of the protein complex when it passes
through the gel. This result confirms that OprF/I has a rather
elongated shape with a high hydrodynamic radius.
[0153] N-Terminal Sequencing
[0154] The first 13 or 15 amino acids of two different samples were
analyzed. No differences between the theoretical and detected amino
acid sequence were found. The sequencing results confirmed that the
N-terminal Met was completely cleaved off during expression.
[0155] Alkylation of Thiolgroups
[0156] The results of the alkylation of the thiogroups of a OprF/I
fusion protein sample showed a mass increase after alkylation of
+226 Da corresponding to 4 attached molecules of iodacetamide
(theoretical mass increase +228 Da; mass increase of +57 Da per
attached iodacetamide molecule). This result was expected since the
reduced protein contains 4 free cysteine residues. All other
samples did not show an increase in mass. Based on these results
peak P1 of the RP-HPLC (FIG. 4) could be considered as a twofold
cysteinylated variant containing one additional disulphide bond.
Peaks P2 and P3 were considered as variants containing two
disulphide bonds.
[0157] Static Light Scattering (SEC/MALS)
[0158] SEC with refractive index/UV detection at 280 nm was
combined with light scattering for protein characterization and
molecular weight detection. As the molar mass was constant over the
cross section of the main peak eluting between 23 to 26 min, a
defined monodisperse molecule species eluted. For the main peak a
molecular mass in the range of approx. 80 to 86 kDa was detected.
The cumulative mass fraction was in the range of 94 to 98% (species
1).
[0159] The high molecular weight fraction (species 2) eluting
between 20 to 22 min showed a molecular mass in the range of 140 to
190 kDa. Due to the low Rayleigh signal intensity for high
molecular weight fraction the molecular mass determined exhibited a
higher degree of variation. The cumulative mass fraction of species
2 was in the range of 0.5 to 1% at a range between 120 to 200
kDa.
[0160] These results exhibit that OprF/I exists as a trimer
(species 1) and that only a small portion of the protein forms
aggregates of higher molecular mass (species 2).
[0161] The results obtained by SEC-MALS are also in good
correlation with AUC results (see below). Results obtained by
SEC/UV detection and native PAGE indicated higher molecular masses
for the OprF/I fusion protein in the range of 180 kDa. Results
obtained by SEC and native PAGE are based on the assumption of a
globular protein shape, whereas the protein shape does not
influence static light scattering or AUC data. Based on the results
from the different methods that were applied, it was concluded that
the OprF/I trimer does not exist in a globular shape but exhibits a
large hydrodynamic radius.
[0162] Analytical Ultracentrifugation (AUC)
[0163] Sedimentation velocity profiles were recorded and
deconvoluted with SedFit software to yield the sedimentation
coefficient values of the sample components. The resulting
calculated sedimentation coefficient and molecular mass for the
individual species 1 (OprF/I fusion protein main peak) and species
2 (aggregates) were determined. The sedimentation coefficient
values for the dominant component species 1 agree rather well for
all samples studied. This indicates that no significant differences
exist between the different samples examined. The molar mass of the
main component species 1 differs within experimental variation for
this parameter. It generally indicates a trimer of the OprF/I
fusion protein. The molar masses of the monomer and trimer, as
calculated from the sequence, are 24.1 kDa and 72.3 kDa,
respectively.
[0164] No dissociation of this trimer occurred over the
concentration range examined. The Stokes-radius for the trimer was
calculated to be 5.6 nm. The Stokes-radius for a globular protein
of the expected trimer mass is 2.8 nm. This indicates a highly
asymmetrical and/or hydrated molecule. Species 2 appeared as a
distinct peak at varying sedimentation coefficients. This indicates
that species 2 corresponds to a component with a distinct
stoichiometry (hexamer, nonamer, etc.), as opposed to unspecific
aggregation. These data are in very good correlation to the
SEC-MALS results showing that the native OprF/I fusion protein
exists as a trimer, but are significantly different from the
calculated molecular mass obtained by SEC and native PAGE
(overestimation of mass due to non-globular shape). The primary and
most reliable parameter from a sedimentation velocity experiment is
the sedimentation coefficient itself. For the calculation of the
SCD, a single frictional coefficient was assumed to apply for all
sedimentation coefficients calculated. It was optimized in a
fitting step. The frictional coefficient is necessary for the
transformation of the SCD to a molar mass distribution (MMD). In
the present study the signal for sedimentation coefficients <2 S
only appeared at a ten-fold dilution. The possibility can be ruled
that this peak corresponds to a putative monomer of OprF/I out
because species 1 did not change. In conclusion, OprF/I is present
in solution as a trimeric molecule. No dissociation occurred over
the range of concentrations examined.
[0165] Disulfide Mapping
[0166] Disulphide Bond Mapping Using Nano-MS/MS Analysis
[0167] The aim of this study was to identify the differences in the
disulphide bridge pattern between peaks 1, 2 and 3. The individual
peaks were isolated and enriched. The primary sequence contains 4
cystein residues at position 18 (C1), 27 (C2), 33 (C3) and 47 (C4)
(see SEQ ID NO: 3). It was concluded from the data of the intact
molecular weight determination by on-line LC/ES-MS that peak 1 has
one disulphide bridge and two cysteinylations, and peaks 2 and 3
have two disulphide bridges. The tryptic digest of all three peaks
produced the peptide fragment 1 to 55, which contains all four
cysteins of the protein. The observed masses for this fragment in
the three peaks confirmed the assignment from the intact MW
analysis. The peptide fragment 1 to 55 from all three peaks were
collected and subdigested with AspN and analysed by LC-MS. Based on
the interpretation of the raw data the structures according to FIG.
8 were derived for the predominant component in the three different
peaks.
[0168] These findings were confirmed by reduction and MS/MS
experiments of selected signals from the AspN subdigest. In
addition to the disulphide bridge pattern deamidation was observed
in the three different peaks. In the tryptic peptide 120 to 132,
the Asn 124 is probably partly deamidated. In different peptides,
deamidation of Asn 45 was observed as well.
[0169] Influence of Temperature on Stability
[0170] SDS-PAGE gels (reducing and non-reducing conditions) were
run for OprF/I fusion protein samples incubated at different
temperatures over 10 days. Relative content of OprF/I fusion
protein main band in reduced gels was calculated by densitometric
evaluation of the gels by normalization of band intensities to
2-8.degree. C. samples (reference). No degradation or changes in
band pattern were observed for samples stored at -80.degree. C.,
-20.degree. C., 2-8.degree. C. and RT (20.degree. C.) over the
storage period of 10 days.
[0171] Influence of pH on Stability
[0172] OprF/I fusion protein samples were incubated at different pH
values at pH 1.98 to pH 11.1 and analyzed by RP-HPLC and SEC-HPLC.
The main peak of the OprF/I fusion protein, which corresponds to
the non-covalent trimer, was constant with approximately 90% at pH
5.9 to 11.1 over the storage period of at least 23 days at
2-8.degree. C. The trimer reversibly dissociated at low pH (pH
2).
[0173] Aluminium Hydroxide as Additive/Adjuvant
[0174] RP-HPLC results showed that the OprF/I fusion protein could
further be stabilized at pH 4.88 by binding onto aluminium
hydroxide and could be desorbed at high recoveries.
[0175] Immunogenicity of Different OprF/I Fusion Protein Fractions
(BALB/c Mouse Model)
[0176] Five BALB/c mice per group received 1 ml of different OprF/I
fusion protein fractions (peaks 1, 2 and 3 of obtained from
semi-preparative RP-HPLC fractions) and of the unfractionated
OprF/I fusion protein (DS) i.p. at days 0 and 14. At day 21 the
blood of the mice was tested for specific antibodies and the values
(GMT [.mu.g/ml]+SD) determined at specific doses (.mu.g protein).
The results are summarized in Table 3.
TABLE-US-00006 TABLE 3 dose Peak 1 Peak 2 Peak 3 DS 31.6 29.36
40.75 49.53 83.54 10 15.58 4.59 24.63 31.04 3.16 0.09 0.03 0.24
0.70 1 0.01 0.01 0.01 0.05 0.316 0.01 0.01 0.01 0.01
[0177] It was concluded that all fractions as well as the
unfractionated OprF/I fusion protein induced specific antibodies.
The ED50 value for the peak 2 fraction has additionally been
determined as 5.6 .mu.g (unfractionated OprF/I fusion protein: 1.8
.mu.g).
CONCLUSIONS
[0178] 1. The OprF/I fusion protein can be produced and purified
without cross-linked disulfide aggregates in an over all yield up
to 40% starting with the IMAC-Cu capture step (i.e. SEQ ID NO: 4 in
the form of a trimer wherein trimer content of more than about 90%
according to SEC and an aggregate content of less than 1%). [0179]
2. The OprF/I fusion protein (SEQ ID NO: 4) produced in different
production lots is very consistent. [0180] 3. The OprF/I fusion
protein (SEQ ID NO: 4) exists as a trimer under physiological
conditions with a mean molecular mass of approximately 80 kDa and a
relative content of 94 to 98%. [0181] 4. The OprF/I fusion protein
(SEQ ID NO: 4) produced according to the present invention can be
separated in several variants by RP-HPLC (see FIGS. 4 and 8) Peak 1
(P1) is a two-fold cysteinylated adduct at position 33 (C3) and 47
(C4) containing a disulphide bond between position 18 (C1) and 27
(C2) (see also SEQ ID NO: 4). Peak 2 (P2) is a variant containing
two disulphide bridges at positions 18 (C1)-27 (C2) and 33 (C3)-47
(C4). Peak 3 (P3) is a further variant containing 2 disulphide
bridges at positions 18 (C1)-48 (C4) and 27 (C2)-33 (C3). [0182] 5.
The OprF/I fusion protein (SEQ ID NO: 4) is stable from -80.degree.
C. to +20.degree. C. over a period of 10 days, and at pH 5.9 to
11.1 over a period of 23 days at 2-8.degree. C. Furthermore (data
not shown), the OprF/I fusion protein (SEQ ID NO: 4) is stable up
to 24 months at 2 to 8.degree. C. in PBS. At pH 4.88 the OprF/I
fusion protein can be further stabilized by binding onto aluminium
hydroxide. [0183] 6. All three variants (peaks 1, 2 and 3) as well
as the unfractionated OprF/I fusion protein induced specific
antibodies after vaccination of BALB/c mice.
[0184] Preferred Aspects [0185] 1. An OprF/I fusion protein
comprising a portion of the Pseudomonas aeruginosa outer membrane
protein F which is fused with its carboxy terminal end to a portion
of the amino terminal end of the Pseudomonas aeruginosa outer
membrane protein I, wherein said portion of the Pseudomonas
aeruginosa outer membrane protein F comprises the amino acids
190-342 of SEQ ID NO: 1 and wherein said portion of the Pseudomonas
aeruginosa outer membrane protein I comprises the amino acids 21-83
of SEQ ID NO: 2, and further wherein said fusion protein contains a
disulphide bond pattern, preferably selected from the group
consisting of (a) Cys18-Cys27-bond (SEQ ID NO: 9), (b)
Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10), and (c)
Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11), or an
immunogenic variant thereof having at least 85%, preferably 90%, in
particular 95% identity to the amino acid sequence of SEQ ID NO: 4,
and the same disulphide bond pattern as specified. [0186] 2. The
OprF/I fusion protein according to aspect 1, wherein said fusion
protein is trimeric. [0187] 3. The OprF/I fusion protein according
to aspect 1 or 2, wherein said fusion protein further contains 1-24
amino acids fused to its amino terminal end, preferably selected
from the group consisting of Met-, Met-Ala-(His).sub.6- (SEQ ID NO:
5), Ala-(His).sub.6- (SEQ ID NO: 6),
Met-Lys-Lys-Thr-Ala-Ile-Ala-Ile-Ala-Val-Ala-Leu-Ala-Gly-Phe-Ala-Thr-Val-A-
la-Gln-Ala-(SEQ ID NO: 7),
Met-Lys-Leu-Lys-Asn-Thr-Leu-Gly-Val-Val-Ile-Gly-Ser-Leu-Val-Ala-Ala-Ser-A-
la-Met-Asn-Ala-Phe-Ala-(SEQ ID NO: 8), in particular
Ala-(His).sub.6- (SEQ ID NO: 6). [0188] 4. An OprF/I fusion protein
mixture or complex containing or consisting essentially of three
OprF/I fusion proteins according to aspect 1 or 3, in particular in
the form of a trimer. [0189] 5. The OprF/I fusion protein mixture
or complex according to aspect 4, said mixture or complex
containing or consisting essentially of [0190] (a) an OprF/I fusion
protein having only a Cys18-Cys27-bond (SEQ ID NO: 9), [0191] (b)
an OprF/I fusion protein having a Cys18-Cys27-bond and a
Cys33-Cys47-bond (SEQ ID NO: 10), and/or [0192] (c) an OprF/I
fusion protein having a Cys18-Cys47-bond and a Cys27-Cys33-bond
(SEQ ID NO: 11). [0193] 6. The OprF/I fusion protein mixture or
complex according to aspect 5, wherein the relative distribution of
the components are for component (a) about 15% to about 18%,
preferably about 16%; for component (b) about 67% to about 62%,
preferably about 66%; and for component (c) about 18% to about 20%,
preferably about 18%. [0194] 7. The OprF/I fusion protein mixture
or complex according to aspect 5 or 6, wherein the total relative
content of all components (a) to (c) compared to the total protein
content is at least 75%, preferably at least about 80% to about
90%, in particular at least about 85%. [0195] 8. The OprF/I fusion
protein mixture or complex according to any of aspects 5-7, wherein
each of the OprF/I fusion proteins contains an
Ala-(His).sub.6-N-terminus, said mixture containing or consisting
essentially of, in particular in the form of a trimer, [0196] (a)
an OprF/I fusion protein having only a Cys18-Cys27-bond (SEQ ID NO:
9), [0197] (b) an OprF/I fusion protein having a Cys18-Cys27-bond
and a Cys33-Cys47-bond (SEQ ID NO: 10), and/or [0198] (c) an OprF/I
fusion protein having a Cys18-Cys47-bond and a Cys27-Cys33-bond
(SEQ ID NO: 11). [0199] 9. A trimeric OprF/I fusion protein
comprising a portion of the Pseudomonas aeruginosa outer membrane
protein F which is fused with its carboxy terminal end to a portion
of the amino terminal end of the Pseudomonas aeruginosa out
membrane protein I, wherein said portion of the Pseudomonas
aeruginosa outer membrane protein F comprises the amino acids
190-342 of SEQ ID NO: 1 and wherein said portion of the Pseudomonas
aeruginosa outer membrane protein I comprises the amino acids 21-83
of SEQ ID NO: 2, or an immunogenic variant thereof having at least
85%, preferably 90%, in particular 95% identity to the amino acid
sequence of SEQ ID NO: 3. [0200] 10. The trimeric OprF/I fusion
protein according to aspect 9, wherein said fusion protein further
contains 1-24 amino acids fused to its amino terminal end,
preferably selected from the group consisting of Met-,
Met-Ala-(His).sub.6- (SEQ ID NO: 5), Ala-(His).sub.6- (SEQ ID NO:
6),
Met-Lys-Lys-Thr-Ala-Ile-Ala-Ile-Ala-Val-Ala-Leu-Ala-Gly-Phe-Ala-Thr-Val-A-
la-Gln-Ala-(SEQ ID NO: 7),
Met-Lys-Leu-Lys-Asn-Thr-Leu-Gly-Val-Val-Ile-Gly-Ser-Leu-Val-Ala-Ala-Ser-A-
la-Met-Asn-Ala-Phe-Ala-(SEQ ID NO: 8), in particular
Ala-(His).sub.6- (SEQ ID NO: 6). [0201] 11. A method for producing
the OprF/I fusion protein according to any of aspects 1-10, said
method comprising the steps of [0202] (a) reducing said OprF/I
fusion protein with a reducing agent, preferably dithiothreitol
(DTT), dithioerythritol (DTE) or .beta.-mercaptoethanol, and [0203]
(b) oxidizing the reduced OprF/I fusion protein with a redox agent,
preferably the redox agent glutathione disulfide/glutathione or the
redox agent cystine/cysteine, in the presence of a reducing agent,
preferably dithiothreitol (DTT), dithioerythritol (DTE) or
.beta.-mercaptoethanol. [0204] 12. The method according to aspect
11, wherein in step (a) the concentration of the reducing agent is
from about 3 mM to about 10 mM, preferably from about 3 mM to about
6 mM. [0205] 13. The method according to aspect 11 or 12, wherein
in step (b) the concentration of the redox agent is from about 0.2
mM to about 4 mM, preferably about 0.2 mM to about 1 mM, in
particular about 0.2 mM to about 0.5 mM, and the concentration of
the reducing agent is from about 0.5 mM to about 1.5 mM, preferably
about 1 mM. [0206] 14. The method according to any of the aspects
11 to 13, wherein the reaction temperature is from about 18.degree.
C. to about 25.degree. C., preferably at about 20.degree. C. [0207]
15. The method according to any of the aspects 11 to 14, wherein
the reaction time of the reduction step (a) is from about 15
minutes to about 2 hours, preferably from about 30 minutes to about
1 hour, in particular about 30 minutes, and/or the pH value is from
about 7.0 to about 8.5, in particular about 8.0. [0208] 16. The
method according to any of the aspects 11 to 15, wherein the
reaction time of the oxidation step (b) is from about 1 hour to
about 20 hours, preferably from about 1 hour to about 6 hours, in
particular from about 1.5 hours to about 2 hours, and/or the pH
value is from about 7.5 to about 8.5, in particular about 8.0.
[0209] 17. The method according to any of the aspects 11 to 16,
wherein the reoxidized OprF/I fusion protein is further purified by
an anion exchange chromatography, preferably Diethylaminoethyl-
(DEAE-), Diethyl-(2-hydroxypropyl)aminoethyl- (QAE-) or
Trimethylaminomethyl- (Q-) exchange chromatography, preferably
DEAE- and/or Q-exchange chromatography, in particular wherein the
reoxidized OprF/I-fusion protein is sequentially purified by DEAE-
and Q-exchange chromatography, preferably by DEAE Sepharose.RTM.
and Q-Sepharose.RTM. chromatography. [0210] 18. The method
according to any of the aspects 11 to 17, wherein prior to the
reduction of the OprF/I fusion protein, the OprF/I fusion protein
is purified by affinity chromatography, preferably by
immunoaffinity or immobilized metal ion affinity chromatography, in
particular by immobilized metal ion affinity chromatography. [0211]
19. A pharmaceutical composition, in particular a vaccine,
comprising the OprF/I fusion protein according to any of the
aspects 1 to 10 or obtained by the method according to any of the
aspects 11 to 17, and optionally at least one additive or adjuvant,
in particular aluminium hydroxide, preferably formulated in an
isotonic phosphate buffer saline solution (pH 7.4). [0212] 20. An
antibody or antibody derivative which specifically binds the OprF/I
fusion protein according to any of the aspects 1 to 10 or obtained
by the method according to any of the aspects 11 to 17. [0213] 21.
A protein complex comprising three OprF/I fusion proteins of SEQ ID
NO: 4 or an immunogenic variant thereof having at least 85%,
preferably 90%, in particular 95% identity to the amino acid
sequence of SEQ ID NO: 4. [0214] 22. A protein complex consisting
at least 80%, preferably 85%, more preferably 90% of three OprF/I
fusion proteins of SEQ ID NO: 4 or an immunogenic variant thereof
having at least 85%, preferably 90%, in particular 95% identity to
the amino acid sequence of SEQ ID NO: 4. [0215] 23. Complex of
aspect 21 or 22, wherein the OprF/I fusion proteins are selected
from the group consisting of [0216] (a) the OprF/I fusion protein
of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQ ID NO: 9), and [0217]
(b) the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10), and [0218]
(c) the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11), [0219] or an
immunogenic variant thereof having at least 85%, preferably 90%, in
particular 95% identity to the amino acid sequence of SEQ ID NO: 4,
and the same disulphide bond pattern as specified in (a), (b) or
(c). [0220] 24. Complex of aspect 23, wherein the complex consists
of a) about 15% to about 18% of the OprF/I fusion protein of SEQ ID
NO: 4 with a Cys18-Cys27-bond (SEQ ID NO: 9), b) about 62% to 67%
of the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10), and c) about
18% to about 20% the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11). [0221] 25.
Complex of aspect 23, wherein the sum of a) the OprF/I fusion
protein of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQ ID NO: 9), b)
the OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond
and Cys33-Cys47-bond (SEQ ID NO: 10), and c) the OprF/I fusion
protein of SEQ ID NO: 4 with a Cys18-Cys47-bond and
Cys27-Cys33-bond (SEQ ID NO: 11) is equal or greater than 75%.
[0222] 26. Complex of aspect 21 or 22, wherein the OprF/I fusion
proteins are selected from the group consisting of [0223] (a) the
OprF/I fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond (SEQ
ID NO: 9), or [0224] (b) the OprF/I fusion protein of SEQ ID NO: 4
with a Cys18-Cys27-bond and Cys33-Cys47-bond (SEQ ID NO: 10), or
[0225] (c) the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys47-bond and Cys27-Cys33-bond (SEQ ID NO: 11), [0226] or an
immunogenic variant thereof having at least 85%, preferably 90%, in
particular 95% identity to the amino acid sequence of SEQ ID NO: 4,
and the same disulphide bond pattern as specified in (a), (b) or
(c). [0227] 27. Complex of aspect 21 or 22, wherein the OprF/I
fusion proteins is the OprF/I fusion protein of SEQ ID NO: 4 with a
Cys18-Cys27-bond (SEQ ID NO: 9) or an immunogenic variant thereof
having at least 85%, preferably 90%, in particular 95% identity to
the amino acid sequence of SEQ ID NO: 4, and the same disulphide
bond pattern as specified in SEQ ID NO: 9. [0228] 28. Complex of
aspect 21 or 22, wherein the OprF/I fusion protein is the OprF/I
fusion protein of SEQ ID NO: 4 with a Cys18-Cys27-bond and
Cys33-Cys47-bond (SEQ ID NO: 10) or an immunogenic variant thereof
having at least 85%, preferably 90%, in particular 95% identity to
the amino acid sequence of SEQ ID NO: 4, and the same disulphide
bond pattern as specified in SEQ ID NO: 10. [0229] 29. Complex of
aspect 21 or 22, wherein the OprF/I fusion protein is the OprF/I
fusion protein of SEQ ID NO: 4 with a Cys18-Cys47-bond and
Cys27-Cys33-bond (SEQ ID NO: 11) or an immunogenic variant thereof
having at least 85%, preferably 90%, in particular 95% identity to
the amino acid sequence of SEQ ID NO: 4, and the same disulphide
bond pattern as specified in SEQ ID NO: 11. [0230] 30. A
pharmaceutical composition, in particular a vaccine, comprising the
protein complex according to any of the aspects 21 to 29 or the
protein complex obtained by the method according to any of the
aspects 11 to 17, and optionally at least one additive or adjuvant,
in particular aluminium hydroxide, preferably formulated in an
isotonic phosphate buffer saline solution (pH 7.4). [0231] 31. An
antibody or antibody derivative which specifically binds the
protein complex according to any of the aspects 21 to 29 or the
protein complex obtained by the method according to any of the
aspects 11 to 17. [0232] 32. The antibody or antibody derivative of
aspect 31, wherein said antibody or antibody derivative selectively
binds to the protein complex according to any of the aspects 21 to
29 or the protein complex obtained by the method according to any
of the aspects 11 to 17. [0233] 33. The antibody or antibody
derivative of aspect 31, wherein said antibody or antibody
derivative binds to a) the protein complex according to any of the
aspects 21 to 29 or b) the protein complex obtained by the method
according to any of the aspects 11 to 17 but does not bind to a
monomer of the OprF/I fusion protein according to aspects 1 to 3.
[0234] 34. A pharmaceutical composition comprising the antibody or
antibody derivative according to aspects 32 or 33, and optionally
pharmaceutically acceptable excipients. [0235] 35. The antibody or
antibody derivative according to aspects 32 or 33 for use as a
medicament, preferably for use in the reduction of mortality.
Sequence CWU 1
1
121350PRTPseudomonas aeruginosa 1Met Lys Leu Lys Asn Thr Leu Gly
Val Val Ile Gly Ser Leu Val Ala 1 5 10 15 Ala Ser Ala Met Asn Ala
Phe Ala Gln Gly Gln Asn Ser Val Glu Ile 20 25 30 Glu Ala Phe Gly
Lys Arg Tyr Phe Thr Asp Ser Val Arg Asn Met Lys 35 40 45 Asn Ala
Asp Leu Tyr Gly Gly Ser Ile Gly Tyr Phe Leu Thr Asp Asp 50 55 60
Val Glu Leu Ala Leu Ser Tyr Gly Glu Tyr His Asp Val Arg Gly Thr 65
70 75 80 Tyr Glu Thr Gly Asn Lys Lys Val His Gly Asn Leu Thr Ser
Leu Asp 85 90 95 Ala Ile Tyr His Phe Gly Thr Pro Gly Val Gly Leu
Arg Pro Tyr Val 100 105 110 Ser Ala Gly Leu Ala His Gln Asn Ile Thr
Asn Ile Asn Ser Asp Ser 115 120 125 Gln Gly Arg Gln Gln Met Thr Met
Ala Asn Ile Gly Ala Gly Leu Lys 130 135 140 Tyr Tyr Phe Thr Glu Asn
Phe Phe Ala Lys Ala Ser Leu Asp Gly Gln 145 150 155 160 Tyr Gly Leu
Glu Lys Arg Asp Asn Gly His Gln Gly Glu Trp Met Ala 165 170 175 Gly
Leu Gly Val Gly Phe Asn Phe Gly Gly Ser Lys Ala Ala Pro Ala 180 185
190 Pro Glu Pro Val Ala Asp Val Cys Ser Asp Ser Asp Asn Asp Gly Val
195 200 205 Cys Asp Asn Val Asp Lys Cys Pro Asp Thr Pro Ala Asn Val
Thr Val 210 215 220 Asp Ala Asn Gly Cys Pro Ala Val Ala Glu Val Val
Arg Val Gln Leu 225 230 235 240 Asp Val Lys Phe Asp Phe Asp Lys Ser
Lys Val Lys Glu Asn Ser Tyr 245 250 255 Ala Asp Ile Lys Asn Leu Ala
Asp Phe Met Lys Gln Tyr Pro Ser Thr 260 265 270 Ser Thr Thr Val Glu
Gly His Thr Asp Ser Val Gly Thr Asp Ala Tyr 275 280 285 Asn Gln Lys
Leu Ser Glu Arg Arg Ala Asn Ala Val Arg Asp Val Leu 290 295 300 Val
Asn Glu Tyr Gly Val Glu Gly Gly Arg Val Asn Ala Val Gly Tyr 305 310
315 320 Gly Glu Ser Arg Pro Val Ala Asp Asn Ala Thr Ala Glu Gly Arg
Ala 325 330 335 Ile Asn Arg Arg Val Glu Ala Glu Val Glu Ala Glu Ala
Lys 340 345 350 283PRTPseudomonas aeruginosa 2Met Asn Asn Val Leu
Lys Phe Ser Ala Leu Ala Leu Ala Ala Val Leu 1 5 10 15 Ala Thr Gly
Cys Ser Ser His Ser Lys Glu Thr Glu Ala Arg Leu Thr 20 25 30 Ala
Thr Glu Asp Ala Ala Ala Arg Ala Gln Ala Arg Ala Asp Glu Ala 35 40
45 Tyr Arg Lys Ala Asp Glu Ala Leu Gly Ala Ala Gln Lys Ala Gln Gln
50 55 60 Thr Ala Asp Glu Ala Asn Glu Arg Ala Leu Arg Met Leu Glu
Lys Ala 65 70 75 80 Ser Arg Lys 3224PRTArtificial SequenceOprF/I
fusion protein with N-tag plus Met 3Met Ala His His His His His His
Ala Pro Ala Pro Glu Pro Val Ala 1 5 10 15 Asp Val Cys Ser Asp Ser
Asp Asn Asp Gly Val Cys Asp Asn Val Asp 20 25 30 Lys Cys Pro Asp
Thr Pro Ala Asn Val Thr Val Asp Ala Asn Gly Cys 35 40 45 Pro Ala
Val Ala Glu Val Val Arg Val Gln Leu Asp Val Lys Phe Asp 50 55 60
Phe Asp Lys Ser Lys Val Lys Glu Asn Ser Tyr Ala Asp Ile Lys Asn 65
70 75 80 Leu Ala Asp Phe Met Lys Gln Tyr Pro Ser Thr Ser Thr Thr
Val Glu 85 90 95 Gly His Thr Asp Ser Val Gly Thr Asp Ala Tyr Asn
Gln Lys Leu Ser 100 105 110 Glu Arg Arg Ala Asn Ala Val Arg Asp Val
Leu Val Asn Glu Tyr Gly 115 120 125 Val Glu Gly Gly Arg Val Asn Ala
Val Gly Tyr Gly Glu Ser Arg Pro 130 135 140 Val Ala Asp Asn Ala Thr
Ala Glu Gly Arg Ala Ile Asn Arg Arg Val 145 150 155 160 Glu Ser Ser
His Ser Lys Glu Thr Glu Ala Arg Leu Thr Ala Thr Glu 165 170 175 Asp
Ala Ala Ala Arg Ala Gln Ala Arg Ala Asp Glu Ala Tyr Arg Lys 180 185
190 Ala Asp Glu Ala Leu Gly Ala Ala Gln Lys Ala Gln Gln Thr Ala Asp
195 200 205 Glu Ala Asn Glu Arg Ala Leu Arg Met Leu Glu Lys Ala Ser
Arg Lys 210 215 220 4223PRTArtificial SequenceOprF/I fusion protein
with N-tag without Met 4Ala His His His His His His Ala Pro Ala Pro
Glu Pro Val Ala Asp 1 5 10 15 Val Cys Ser Asp Ser Asp Asn Asp Gly
Val Cys Asp Asn Val Asp Lys 20 25 30 Cys Pro Asp Thr Pro Ala Asn
Val Thr Val Asp Ala Asn Gly Cys Pro 35 40 45 Ala Val Ala Glu Val
Val Arg Val Gln Leu Asp Val Lys Phe Asp Phe 50 55 60 Asp Lys Ser
Lys Val Lys Glu Asn Ser Tyr Ala Asp Ile Lys Asn Leu 65 70 75 80 Ala
Asp Phe Met Lys Gln Tyr Pro Ser Thr Ser Thr Thr Val Glu Gly 85 90
95 His Thr Asp Ser Val Gly Thr Asp Ala Tyr Asn Gln Lys Leu Ser Glu
100 105 110 Arg Arg Ala Asn Ala Val Arg Asp Val Leu Val Asn Glu Tyr
Gly Val 115 120 125 Glu Gly Gly Arg Val Asn Ala Val Gly Tyr Gly Glu
Ser Arg Pro Val 130 135 140 Ala Asp Asn Ala Thr Ala Glu Gly Arg Ala
Ile Asn Arg Arg Val Glu 145 150 155 160 Ser Ser His Ser Lys Glu Thr
Glu Ala Arg Leu Thr Ala Thr Glu Asp 165 170 175 Ala Ala Ala Arg Ala
Gln Ala Arg Ala Asp Glu Ala Tyr Arg Lys Ala 180 185 190 Asp Glu Ala
Leu Gly Ala Ala Gln Lys Ala Gln Gln Thr Ala Asp Glu 195 200 205 Ala
Asn Glu Arg Ala Leu Arg Met Leu Glu Lys Ala Ser Arg Lys 210 215 220
58PRTArtificial SequenceN-tag plus Met 5Met Ala His His His His His
His 1 5 67PRTArtificial SequenceN-tag without Met 6Ala His His His
His His His 1 5 721PRTEscherichia coli 7Met Lys Lys Thr Ala Ile Ala
Ile Ala Val Ala Leu Ala Gly Phe Ala 1 5 10 15 Thr Val Ala Gln Ala
20 824PRTPseudomonas aeruginosa 8Met Lys Leu Lys Asn Thr Leu Gly
Val Val Ile Gly Ser Leu Val Ala 1 5 10 15 Ala Ser Ala Met Ala Ala
Phe Ala 20 9223PRTArtificial SequenceOprF/I fusion protein with
N-tag without Met 9Ala His His His His His His Ala Pro Ala Pro Glu
Pro Val Ala Asp 1 5 10 15 Val Cys Ser Asp Ser Asp Asn Asp Gly Val
Cys Asp Asn Val Asp Lys 20 25 30 Cys Pro Asp Thr Pro Ala Asn Val
Thr Val Asp Ala Asn Gly Cys Pro 35 40 45 Ala Val Ala Glu Val Val
Arg Val Gln Leu Asp Val Lys Phe Asp Phe 50 55 60 Asp Lys Ser Lys
Val Lys Glu Asn Ser Tyr Ala Asp Ile Lys Asn Leu 65 70 75 80 Ala Asp
Phe Met Lys Gln Tyr Pro Ser Thr Ser Thr Thr Val Glu Gly 85 90 95
His Thr Asp Ser Val Gly Thr Asp Ala Tyr Asn Gln Lys Leu Ser Glu 100
105 110 Arg Arg Ala Asn Ala Val Arg Asp Val Leu Val Asn Glu Tyr Gly
Val 115 120 125 Glu Gly Gly Arg Val Asn Ala Val Gly Tyr Gly Glu Ser
Arg Pro Val 130 135 140 Ala Asp Asn Ala Thr Ala Glu Gly Arg Ala Ile
Asn Arg Arg Val Glu 145 150 155 160 Ser Ser His Ser Lys Glu Thr Glu
Ala Arg Leu Thr Ala Thr Glu Asp 165 170 175 Ala Ala Ala Arg Ala Gln
Ala Arg Ala Asp Glu Ala Tyr Arg Lys Ala 180 185 190 Asp Glu Ala Leu
Gly Ala Ala Gln Lys Ala Gln Gln Thr Ala Asp Glu 195 200 205 Ala Asn
Glu Arg Ala Leu Arg Met Leu Glu Lys Ala Ser Arg Lys 210 215 220
10223PRTArtificial SequenceOprF/I fusion protein with N-tag without
Met 10Ala His His His His His His Ala Pro Ala Pro Glu Pro Val Ala
Asp 1 5 10 15 Val Cys Ser Asp Ser Asp Asn Asp Gly Val Cys Asp Asn
Val Asp Lys 20 25 30 Cys Pro Asp Thr Pro Ala Asn Val Thr Val Asp
Ala Asn Gly Cys Pro 35 40 45 Ala Val Ala Glu Val Val Arg Val Gln
Leu Asp Val Lys Phe Asp Phe 50 55 60 Asp Lys Ser Lys Val Lys Glu
Asn Ser Tyr Ala Asp Ile Lys Asn Leu 65 70 75 80 Ala Asp Phe Met Lys
Gln Tyr Pro Ser Thr Ser Thr Thr Val Glu Gly 85 90 95 His Thr Asp
Ser Val Gly Thr Asp Ala Tyr Asn Gln Lys Leu Ser Glu 100 105 110 Arg
Arg Ala Asn Ala Val Arg Asp Val Leu Val Asn Glu Tyr Gly Val 115 120
125 Glu Gly Gly Arg Val Asn Ala Val Gly Tyr Gly Glu Ser Arg Pro Val
130 135 140 Ala Asp Asn Ala Thr Ala Glu Gly Arg Ala Ile Asn Arg Arg
Val Glu 145 150 155 160 Ser Ser His Ser Lys Glu Thr Glu Ala Arg Leu
Thr Ala Thr Glu Asp 165 170 175 Ala Ala Ala Arg Ala Gln Ala Arg Ala
Asp Glu Ala Tyr Arg Lys Ala 180 185 190 Asp Glu Ala Leu Gly Ala Ala
Gln Lys Ala Gln Gln Thr Ala Asp Glu 195 200 205 Ala Asn Glu Arg Ala
Leu Arg Met Leu Glu Lys Ala Ser Arg Lys 210 215 220
11223PRTArtificial SequenceOprF/I fusion protein with N-tag without
Met 11Ala His His His His His His Ala Pro Ala Pro Glu Pro Val Ala
Asp 1 5 10 15 Val Cys Ser Asp Ser Asp Asn Asp Gly Val Cys Asp Asn
Val Asp Lys 20 25 30 Cys Pro Asp Thr Pro Ala Asn Val Thr Val Asp
Ala Asn Gly Cys Pro 35 40 45 Ala Val Ala Glu Val Val Arg Val Gln
Leu Asp Val Lys Phe Asp Phe 50 55 60 Asp Lys Ser Lys Val Lys Glu
Asn Ser Tyr Ala Asp Ile Lys Asn Leu 65 70 75 80 Ala Asp Phe Met Lys
Gln Tyr Pro Ser Thr Ser Thr Thr Val Glu Gly 85 90 95 His Thr Asp
Ser Val Gly Thr Asp Ala Tyr Asn Gln Lys Leu Ser Glu 100 105 110 Arg
Arg Ala Asn Ala Val Arg Asp Val Leu Val Asn Glu Tyr Gly Val 115 120
125 Glu Gly Gly Arg Val Asn Ala Val Gly Tyr Gly Glu Ser Arg Pro Val
130 135 140 Ala Asp Asn Ala Thr Ala Glu Gly Arg Ala Ile Asn Arg Arg
Val Glu 145 150 155 160 Ser Ser His Ser Lys Glu Thr Glu Ala Arg Leu
Thr Ala Thr Glu Asp 165 170 175 Ala Ala Ala Arg Ala Gln Ala Arg Ala
Asp Glu Ala Tyr Arg Lys Ala 180 185 190 Asp Glu Ala Leu Gly Ala Ala
Gln Lys Ala Gln Gln Thr Ala Asp Glu 195 200 205 Ala Asn Glu Arg Ala
Leu Arg Met Leu Glu Lys Ala Ser Arg Lys 210 215 220
124PRTArtificial sequencesynthetic polypeptide 12Ser Thr Gly Ser
1
* * * * *