U.S. patent application number 14/089824 was filed with the patent office on 2014-03-27 for organ transplant solutions and methods for transplanting organs.
This patent application is currently assigned to CHILDREN'S HOSPITAL MEDICAL CENTER. The applicant listed for this patent is Chrildren's Hospital Medical Center. Invention is credited to Prasad Devarajan, Jorg Kohl.
Application Number | 20140087357 14/089824 |
Document ID | / |
Family ID | 36925685 |
Filed Date | 2014-03-27 |
United States Patent
Application |
20140087357 |
Kind Code |
A1 |
Kohl; Jorg ; et al. |
March 27, 2014 |
Organ Transplant Solutions and Methods for Transplanting Organs
Abstract
A preservation solution for organs waiting to be transplanted is
disclosed; the method of using the solution in a transplantation
procedure is also disclosed. The preservation solutions comprise a
balanced isotonic aqueous solution comprising sodium, potassium,
calcium, magnesium and bicarbonate ions in a physiologically
acceptable amount, together with an effective amount of a mutein of
the C5a anaphylatoxin which is a C5a receptor antagonist wherein
the amino acid residue naturally occurring at sequence position 69
is mutated.
Inventors: |
Kohl; Jorg; (Cincinnati,
OH) ; Devarajan; Prasad; (Cincinnati, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chrildren's Hospital Medical Center |
Cincinnati |
OH |
US |
|
|
Assignee: |
CHILDREN'S HOSPITAL MEDICAL
CENTER
Cincinnati
OH
|
Family ID: |
36925685 |
Appl. No.: |
14/089824 |
Filed: |
November 26, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12239158 |
Sep 26, 2008 |
8617802 |
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14089824 |
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11368935 |
Mar 6, 2006 |
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12239158 |
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60660571 |
Mar 11, 2005 |
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Current U.S.
Class: |
435/1.2 |
Current CPC
Class: |
A01N 1/0226 20130101;
A01N 1/02 20130101 |
Class at
Publication: |
435/1.2 |
International
Class: |
A01N 1/02 20060101
A01N001/02 |
Claims
1.-20. (canceled)
21. A method of preservation, storage and reperfusion of an organ
intended for implantation, said method comprising perfusing said
organ with a solution comprising: (a) a balanced isotonic solution
comprising sodium, potassium, calcium, magnesium and bicarbonate
ions in a physiologically acceptable amount; (b) a safe and
effective amount of a mutein of the C5a anaphylatoxin which is a
C5a receptor antagonist wherein the amino acid residue naturally
occurring at sequence position 69 is mutated; and (c) water.
22. The method of preservation according to claim 21 wherein the
C5a mutein is present at from about 0.1 to about 10 .mu.M/liter of
the solution.
23. The method of preservation according to claim 22 wherein the
C5a mutein has an amino acid sequence selected from SEQ ID NO: 14
(C5a-(1-66, Cys27Ala-)A8B); SEQ ID NO: 15 (C5a-(1-66,
Cys27Ala)-A8B-Leu 70); SEQ ID NO: 16 (C5a-(1-66,
Cys27Ala)-Ab8-Cys71); SEQ ID NO: 17 (C5a-(1-66,
Cys27Ala)-A8B-Cys73); SEQ ID NO: 18 (C5a-(1-66,
Cys27Ala)-A8B-Leu70-Tyr73); SEQ ID NO: 19 (C5a-(1-66,
Cys27Ala)-A8B-Lys69-Ala70); SEQ ID NO: 20 (C5a-(1-66,
Cys27Arg)-A8B); SEQ ID NO: 21 (C5a-(1-66, Cys27Ala)-A8B-Del.71-73);
SEQ ID NO: 22 (C5a-(1-66, Cys-3, Gly-2, -1,Cys27Ala)-A8B); and SEQ
ID NO: 39 (C5a-(1-66, Cys27Ala)A5a).
24. A method of preservation of organs intended for implantation,
said method comprising perfusing the body of the dead organ donor,
prior to removal of the organs, with a solution comprising: (a) a
balanced isotonic solution comprising sodium, potassium, calcium,
magnesium and bicarbonate ions in a physiologically acceptable
amount; (b) a safe and effective amount of a mutein of the C5a
anaphylatoxin which is a C5a receptor antagonist wherein the amino
acid residue naturally occurring at sequence position 69 is
mutated; and (c) water.
25. The method of preservation according to claim 24 wherein the
C5a mutein has an amino acid sequence selected from SEQ ID NO: 14
(C5a-(1-66, Cys27Ala-)A8B); SEQ ID NO: 15 (C5a-(1-66,
Cys27Ala)-A8B-Leu 70); SEQ ID NO: 16 (C5a-(1-66,
Cys27Ala)-Ab8-Cys71); SEQ ID NO: 17 (C5a-(1-66,
Cys27Ala)-A8B-Cys73); SEQ ID NO: 18 (C5a-(1-66,
Cys27Ala)-A8B-Leu70-Tyr73); SEQ ID NO: 19 (C5a-(1-66,
Cys27Ala)-A8B-Lys69-Ala70); SEQ ID NO: 20 (C5a-(1-66,
Cys27Arg)-A8B); SEQ ID NO: 21 (C5a-(1-66, Cys27Ala)-A8B-Del.71-73);
SEQ ID NO: 22 (C5a-(1-66, Cys-3, Gly-2, -1,Cys27Ala)-A8B); and SEQ
ID NO: 39 (C5a-(1-66, Cys27Ala)A5a).
26. The method of preservation according to claim 25 wherein the
C5a mutein is present at from about 0.1 to about 10 .mu.M/liter of
the solution.
27. The method of claim 21, wherein, in the mutein, the amino acid
residue at sequence position 69 is replaced by leucine or a
positively charged amino acid residue.
28. The method of claim 21, wherein, in the mutein, the amino acid
residue at sequence position 67 is mutated.
29. The method of claim 21, wherein, in the mutein, at least one of
the amino acid residues at sequence positions 70 to 74 of the
natural amino sequence is mutated or at least one of the amino acid
residues at said sequence positions 70 to 74 is deleted.
30. The method of claim 21, wherein, in the mutein, the positively
charged amino acid residue at sequence position 69 is Arg or
Lys.
31. The method of claim 21, wherein the mutein comprises at
sequence position 67 an aromatic amino acid selected from the group
consisting of Phe, Trp and Tyr.
32. The method of claim 21, wherein the mutein comprises a
hydrophobic amino acid residue at sequence position 70.
33. The method of claim 21, wherein the mutein comprises Leu or Ala
at sequence position 70.
34. The method of claim 21, wherein the mutein comprises Ser at
sequence position 70.
35. The method of claim 1, wherein the mutein comprises a terminal
sequence selected from the group consisting of SEQ ID NO: 1; SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 7, and SEQ ID NO: 8.
36. The method of claim 21, wherein the mutein further comprises
Arg at sequence position 27.
37. The method of claim 21, wherein the mutein has an amino acid
sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID
NO: 10 SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14;
SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18.
38. The method of claim 21, wherein the mutein is fused to a
protein or a peptide tag with the proviso that the fusion proteins
of the mutein A8B with Jun/Fos alone or with Jun/Fos and the minor
coat protein (pIII) of the filamentous M13 phage mutein A8B,
wherein the Jun/Fos moiety is fused to the N-terminus of the mutein
A8B, as well as the mutein A8B having a hexahistidine tag directly
fused to the N-terminus are excluded.
39. The method of claim 21, wherein the mutein is conjugated to a
protein moiety via a suitable peptidic or non-peptidic linker which
enhances the in vivo half-life of the mutein.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is related to and claims priority from U.S.
Provisional Patent Application No. 60/660,571, Kohl et al., filed
Mar. 11, 2005, incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to organ preservation
solutions and to methods for transplanting organs. More
particularly, this invention relates to preservation solutions for
perfusing and storing an organ while awaiting implantation and to
methods of using the preservation solution during transplantation
of the organ.
BACKGROUND OF THE INVENTION
[0003] A great deal of research progress has been made over the
years in understanding cellular mechanisms, as well as developing
new transplantation techniques, for keeping organs viable not only
during storage but also after reperfusion of these organs (e.g.,
minimization of ischemia/reperfusion injury). As a result, organ
transplantation has become an established and effective technique.
A significant factor limiting clinical application of organ
transplantation is decrease in viability of the organ after removal
from the donor.
[0004] Generally, the two most frequently used methods for
preserving organs after removal from the donor are simple
hypothermic storage and continuous pulsatile perfusion. With simple
hypothermic storage, the organ is removed from the donor and cooled
rapidly. This is usually achieved by a combination of cooling and
short periods of perfusion to drop the organ temperature as quickly
as possible to a temperature between 0.degree. C. and 4.degree. C.,
where it may be held for up to about six hours. While cold storage
enables organs to be transplanted, the time during which the organ
is viable is short. Cold storage decreases the rate at which
intracellular enzymes, essential cellular components necessary for
organ viability, degrade but does not stop metabolism entirely.
[0005] The second method of organ preservation which has undergone
extensive investigation, continuous pulsatile perfusion, utilizes
the following elements: (1) pulsatile flow, (2) hypothermia, (3)
membrane oxygenation, and (4) a perfusate containing both albumin
and lipids. Although being more technically complex and costly,
continuous pulsatile perfusion provides significantly longer
viability of the organ when compared to simple hypothermia.
[0006] Preserving organs at between 0.degree. C. and 4.degree. C.
can result in damage to the organ during storage and upon
reperfusion with a warm reperfusion solution. Injury to the organ
occurs through damage to epithelial and endothelial cells. Although
some of the solutions of the prior art have been useful to extend
the storage time of donor organs and lessen injury to the organ
upon reperfusion, cell injury still does occur frequently. It is
desirable to extend the viable organ life and improve the quality
of the transplanted organ. For example, using preservation
solutions of the prior art, kidneys that have been in cold storage
beyond 48 hours frequently cannot be used and must be discarded.
Extending organ viability allows sufficient time for compatibility
testing of the donor and recipient, and increased organ
availability. It is also desirable to minimize damage to the organ
upon reperfusion. Ischemia-reperfusion injury to transplanted
organs preserved in solutions of the prior art commonly results in
delayed graft function, and predisposes the graft to acute and
chronic rejection.
[0007] A storage solution for preserving organs which can be used
at temperatures from 0.degree. C. to 37.degree. C. is disclosed in
U.S. Pat. No. 5,145,771, Lemasters et al., issued Sep. 8, 1992. The
solution requires the use of a colloid, hydroxyethyl starch for
osmotic support against interstitial edema.
[0008] U.S. Pat. Nos. 4,879,283 and 4,798,824, Belzer et al.,
issued Nov. 7, 1989 and Jan. 17, 1989, respectively, relate to
organ preservation/storage solutions containing a specifically
defined synthetic hydroxyethyl starch in place of human serum
albumin. These patents cover the widely used organ preservation
solution commercially available under the trade name VIASPAN.TM.,
marketed by Barr Laboratories.
[0009] International Published Patent Application WO 03/078457,
published Sep. 25, 2003, describes muteins of the C5a
anaphylatoxin, which are taught to be C5a receptor antagonists. The
materials are taught to be useful for the treatment of C5a-mediated
disease or inflammatory conditions, such as asthma, adult
respiratory distress syndrome, ischemia/reperfusion injury, chronic
progressive pulmonary cystic fibrosis, and rheumatoid arthritis. It
is also taught that the C5a muteins can be used to treat patients
suffering from organ transplant rejection.
[0010] The present invention provides preservation solutions useful
for storing organs while awaiting implantation which extend the
vitality of the organ and reduce damage to organ cells. The present
invention also provides method for preserving organs which extend
the maximum life of the organ during transplantation.
SUMMARY OF THE INVENTION
[0011] In accordance with these objectives, there are disclosed
preservation solutions for use in the transplantation of organs,
and to methods for transplanting organs using those solutions,
which methods increase storage times and lessen injury to the
organs. The preservation solutions of the present invention
comprise: [0012] (a) a balanced isotonic solution comprising
sodium, potassium, calcium, magnesium and bicarbonate ions in a
physiologically acceptable amount; [0013] (b) a safe and effective
amount of a mutein of the C5a anaphylatoxin which is a C5a receptor
antagonist, wherein the amino acid residue naturally occurring at
sequence position 69 is mutated: and [0014] (c) water.
[0015] The present invention also relates to a method for
preservation, storage and reperfusion of organs intended for
implantation, said method comprising:
[0016] preserving or perfusing said organ with a solution
comprising: [0017] (a) a balanced isotonic solution comprising
sodium, potassium, calcium, magnesium and bicarbonate ions in a
physiologically acceptable amount; [0018] (b) a safe and effective
amount of a mutein of the C5a anaphylatoxin which is a C5a receptor
antagonist, wherein the amino acid residue naturally occurring at
sequence position 69 is mutated; and [0019] (c) water.
[0020] Finally, the present invention relates to a method of
preserving organs intended for implantation, said method comprising
perfusing the body of the dead organ donor, prior to removal of the
organs, with the preservation solution defined above.
[0021] The preservation solution in one embodiment includes at
least one antioxidant, such as dimethyl thiourea (DMTU), catalase
as a hydrogen peroxide scavenger, and apoferritin to decrease iron
content within the preservation solution. In addition, the
preservation solutions optionally may include hormones, such as
insulin and protaglandin, and antibiotics.
[0022] All patents and publications cited in this patent are fully
incorporated by reference herein.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention is directed to new preservation
solutions for storing and perfusing organs intended for
implantation in a patient requiring such implant. Suitable organs
on which the solutions of this invention may be used include, for
example, heart, liver, kidney, lungs, intestines, and pancreas.
[0024] The individual components of the present invention are all
nontoxic and have been found to be stable during storage.
[0025] The preservation solutions of the present invention are
based on a balanced isotonic solution including sodium, potassium,
calcium and magnesium ions, as well as glucose and sodium
bicarbonate, in a physiologically acceptable amount. Certain of
these types of solutions are well known, such as the one described
below, known as Krebs-Henseleit-bicarbonate solution, which has the
following composition:
TABLE-US-00001 TABLE 1 Concentration ranges in 1 Liter NaCl 85.0 mM
to 145 mM KCl 3.0 mM to 30 mM CaCl.sub.2 0.5 mM to 2.5 mM
KH.sub.2PO.sub.4 0.7 mM to 1.3 mM MgSO.sub.4 0.9 mM to 4.8 mM
NaHCO.sub.3 15.0 mM to 35 mM Glucose 1.0 mM to 50 mM
[0026] Isotonic preservation solutions are also described in U.S.
Pat. No. 5,693,462, Raymond, issued Dec. 2, 1997, incorporated
herein by reference.
[0027] The preservation solutions are designed to prevent or
inhibit various mechanisms which cause injury to the organ, and
thus should be a composition that performs one or more (and
preferably all) of the following functions: (1) prevents or
restricts intracellular acidosis, (2) prevents the expansion of
intracellular space, (3) prevents injury from oxygen-derived free
radicals, especially during reperfusion, (4) enables the
regeneration of high-energy phosphate compounds during reperfusion,
(5) sustains appropriate metabolic requirements and prevents the
rapid changes in intracellular Na.sup.+--H.sup.+--Ca.sup.++
following reperfusion.
[0028] While the preservation solution begins with the balanced
isotonic solution described above, there may be significant
differences in the final compositions. For example, the
preservation solution begins with the isotonic solution, wherein
the potassium concentration is maintained at preferably from about
3.0 mM to about 8.0 mM. Magnesium chloride may be used in place of
potassium chloride.
[0029] To the balanced isotonic solution is added a safe and
effective amount of the C5a mutein which is described in
International Published Patent Application WO 03/078457, published
Sep. 25, 2003, incorporated herein by reference. By "safe and
effective amount" is meant that amount of C5a mutein which provides
the beneficial preservation effect and minimization of ischemic
perfusion injury, but not so much as to cause damage to the organ
or desired side effects to the organ recipient. The C5a mutein is
preferably incorporated in the composition at a concentration of
from about 0.1 .mu.M to about 10 .mu.M. While not intending to be
bound by theory, it is believed that the C5a mutein binds to the
C5a receptor sites on the organ thereby reducing ischemic perfusion
injury and, as a result, reducing the potential for rejection of
the organ upon transplantation.
[0030] The mutein used in the present invention can be derived from
the natural C5a sequence of mammal and non-mammal species. It can,
for instance be of human, porcine, murine, bovine or rat origin. In
one embodiment, the mutein is a mutant protein of the human C5a
protein.
[0031] In one embodiment, the positively charged amino acid residue
at sequence position 69 of the C5a mutein is Arg or Lys.
[0032] In a further embodiment used in the invention, the mutein
comprises a hydrophobic amino acid residue at sequence position 67.
The aromatic hydrophobic amino acids Trp, Phe and Tyr are
particularly preferred as residues at sequence position 67.
[0033] Also preferred antagonists are muteins which comprise a
hydrophobic amino acid residue at one or more of the sequence
positions 70, 71 or 72. Such hydrophobic amino acid residues can be
selected independently from each other, they can be identical or
different. Preferred hydrophobic residues are Leu, Ile and Ala.
[0034] Such muteins preferably comprise, at sequence position 70,
an amino acid residue which is selected from Ala or Leu. Other
preferred muteins comprise Ser at sequence position 70.
[0035] A preferred amino acid at sequence position 71 is Leu. The
antagonistic mutein disclosed can also preferably comprise a Leu
residue at sequence position 72.
[0036] In a particularly preferred embodiment, the mutein comprises
Leu at all of the sequence positions 70, 71, and 72.
[0037] If present, i.e., not deleted, in the C5a mutant, the
sequence position 73 is preferably occupied by a Cys, Tyr, Arg or
Ser residue. In preferred embodiments the mutein has a length of
70, 71, 72 or 73 amino acid residues. In general, Arg, Cys, Tyr or
Ser are also preferred as C-terminal amino acid residues of a
truncated mutein, i.e., a mutein having 70, 71,72, or 73 amino acid
residues. An example of a mutein having a length of 70 amino acids
is the mutein C5a-(1-66, Cys27Ala)-FKRS-70 (cf. Table 1, SEQ ID NO:
21).
[0038] Further, muteins are also within the scope of the invention
in which the positively charged amino acid at position 69 is the
C-terminal (last) residue. Accordingly, such muteins can have a
length of 69 amino acids. However, it is also possible to introduce
deletions, for example, into the N-terminal region of the protein
so that an antagonistic protein used herein can comprise fewer than
69 amino acid residues. For clarity reasons it is noted once again
that such deletions can, of course, also be present in muteins of
the invention in which residues at sequence positions 70 to 74 are
not or only partly deleted.
[0039] The mutein used in the present invention preferably
comprises or has as C-terminal sequence a sequence selected from
the group consisting of 67-FKRSLLR-73 (cf. mutein ABB; SEQ ID NO:
14), 67-FKRLLLR-73 (cf. mutein A8B-Leu-70; SEQ ID NO: 15),
67-FKRSC-71 (cf. mutein Ab8-Cys71, SEQ ID NO: 16), 67-FKRSLLC-73
(cf. mutein Ab8-Cys73, SEQ ID NO: 17), 67-FKRLLLY-73 (cf. mutein
A8B-Leu70-Tyr73, SEQ ID NO: 18), 67-FKKALLR-73 (cf. mutein
A8B-Lys69Ala70; SEQ ID NO: 19), 67-FKRS-70 (cf. A8B-Del.71-73, SEQ
ID NO: 21) and 67-FKLLLLY-73 (cf. A5a, SEQ ID NO: 39). For the sake
of clarity, the numbering refers to the amino acid position of C5a,
i.e., 67-F means that phenylalanine is present as amino acid at
sequence position 67.
[0040] The mutein can further comprise an Arg residue at sequence
position 27, see, for example, mutein C5a-(1-66,Cys27Arg)-FKRSLLR
(A8B-Arg27, SEQ ID NO: 20). In fact, Arg at position 27 is found in
porcine and bovine C5a. In addition, muteins of human C5a with a
Cys27Arg replacement were selected from C5a mutant phage library
(Cain, S., et al. "Analysis of receptor/ligand interactions using
whole-molecule randomly-mutated ligand libraries," J. Immunol.
Methods. 2000. pp 139-145, 245), incorporated herein by reference.
Muteins of C5a with only a Cys27Arg replacement are agonists of the
C5a receptor (Ibid).
[0041] Particularly preferred is a mutein of the human C5a
anaphylatoxin having or comprising the amino acid sequence of SEQ
ID NO: 14, i.e., C5a-(1-66, Cys27Ala-)A8B; SEQ ID NO: 15, i.e.,
C5a-(1-66, Cys27Ala)-A8B-Leu 70; SEQ ID NO: 16, i.e., C5a-(1-66,
Cys27Ala)-Ab8-Cys71; SEQ ID NO: 17, i.e., C5a-(1-66,
Cys27Ala)-A8B-Cys73; SEQ ID NO: 18; i.e., C5a-(1-66,
Cys27Ala)-A8B-Leu70-Tyr73); SEQ ID NO: 19, i.e., C5a-(1-66,
Cys27Ala)-A8B-Lys69-Ala70); SEQ ID NO: 20; i.e., C5a-(1-66,
Cys27Arg)-A8B; SEQ ID NO: 21, i.e., C5a-(1-66,
Cys27Ala)-A8B-Del.71-73); SEQ ID NO: 22, i.e., C5a-(1-66, Cys-3,
Gly-2, -1,Cys27Ala)-A8B; and SEQ ID NO: 39, i.e., C5a-(1-66,
Cys27Ala)A5a.
[0042] A mutant C5a antagonist of the present invention cannot only
be present as the isolated (recombinant) protein but it can also be
modified. In one embodiment, a mutein of the invention can be
dimerized either with the same or a different mutein to form a
homo- or heterodimer. For this purpose the mutein can comprise an
N-terminal linker sequence which is capable of dimerizing the C5a
mutein. One example of a preferred linker sequence linked to the
N-terminus comprises the sequence Cys-Gly-Gly which can be used for
spontaneous dimerization of the C5a mutein A8B in the course of the
recombinant production of the mutant protein (cf. the mutein
C5a-(1-66; Cys-3, Gly-2,-1; Cys27Ala)-A8B). Another example of such
a suitable linker is Cys-(Gly-Gly-Gly-Gly-Ser).sub.2.
[0043] If the mutein carries a cysteine as C-terminal residue (cf.
the muteins A8B-Cys71 and A8B-Cys73), the dimerization can also
occur by coupling of two muteins via these C-terminal cysteine
residues as described by Pellas et al., "Novel C5a receptor
antagonists regulate neutrophil functions in vitro and in vivo,"
Journal of Immunology, Jun. 1, 1998, pp 5616-5621, vol. 160, no.
11, incorporated herein by reference. The dimerization can also be
achieved by linking a nucleotide sequence encoding a mutein in an
appropriate reading frame with the nucleotide sequence coding for a
protein which forms a homodimer in its native fold. Subsequent
expression of the nucleic acid molecule yields a fusion protein
consisting of the dimerization module linked to the C5a mutant
polypeptide, which then dimerizes spontaneously. Examples of such
proteins which can be used as dimerization modules are alkaline
phosphatase, superoxide-dismutase or glutathione-S-transferase. The
use these proteins is in particular useful because the respective
functional fusion protein can readily be obtained by periplasmic
expression in bacterial expression systems such as E. coli. The use
of dimerization modules such as alkaline phosphatase or
superoxide-dismutase provides the further advantage that such a
fusion protein can easily be detected using a chromogenic reaction
which is catalyzed, e.g., by alkaline phosphatase. Suitable
chromogenic substrates for these enzymes, such as
5-bromo-4-chloro-3-indolylphosphate for alkaline phosphatase, are
well known to the person skilled in the art. Those fusion proteins
are therefore suitable as diagnostic reagents.
[0044] In accordance with the disclosure of the above paragraph,
the mutein of the invention is in a further embodiment linked to a
protein or a peptide tag, i.e., in which a fusion protein
containing the C5a mutein is also part of the invention. However,
the fusion proteins of the mutein A8B with Jun/Fos alone, and, with
Jun/Fos and the minor coat protein (pIII) of the filamentous M13
phage fused to the N-terminus of the mutein A8B, which are known
from Heller et al, "Selection of a C5a receptor antagonist from
phage libraries attenuating the inflammatory response in immune
complex disease and ischemia/reperfusion injury," Journal of
Immunology, Jul. 15, 1999, pp 985-994, vol. 163, no. 2, are
excluded from the invention. The same applies to the mutein A8B
that has a hexahistidine tag directly fused to the N-terminus,
because this polypeptide is known from Hennecke, Untersuchung zur
C5a-C5a Rezeptor-Interaktion ureter Verwendung des Phage-Displays,
PhD thesis, 1998, Medical School Hannover, Germany.
[0045] A fusion protein of the invention can comprise any suitable
fusion partner, e.g., alkaline phosphatase or the green fluorescent
protein (GFP) as long as the fusion partner does not interfere with
the antagonistic properties of the mutein disclosed here and
converts the mutein into an agonist when given to a patient, for
example. A fusion partner appropriate for therapeutic purpose is a
protein such as albumin which can enhance the in vivo (circulation)
half-life of a mutein of the invention. The fusion partner can be
fused to the N-terminus of the C5a mutein. Likewise, any peptide
tag can be fused to the N-terminus of the mutein as long as its
antagonistic property is maintained. Examples of suitable affinity
tags are the STREP-TAG.RTM. which has specific binding affinity for
streptavidin or mutants thereof as STREP-TACTIN.RTM. (see U.S. Pat.
No. 5,506,121, Skerra et al., issued Apr. 9, 1996, and U.S. Pat.
No. 6,022,951, Sano et al. issued Feb. 8, 2000, both incorporated
by reference herein), the Flag-tag or the myc-tag, all of which can
be used for purification of the mutein by affinity
chromatography.
[0046] It should, however, be noted that in the event of, e.g.,
inventive C5a muteins conjugated or fused to a partner that confers
agonistic properties, the antagonistic muteins can be readily
generated/released from its (fusion) partner by treatment such as
limited proteolysis or cleavage, for example enzymatic or chemical
cleavage, of a (peptide) bond which links the C5a mutein to the
(fusion) partner. Accordingly, it is also within the scope of the
present invention, to use a fusion partner, for example, for
improved purification of the mutein, for example, even if this
fusion partner confers an agonistic activity as long as this
activity can be eliminated before (and thus the antagonistic
activity of the inventive mutein is generated) the muteins is used,
for instance, in a desired therapeutic application. It is also
possible to use a mutein the antagonistic activity of which is
reduced by the (fusion) partner but not completely abolished. In
this case, it is thus not necessary to deliberate the mutein of the
invention by cleavage from its (fusion) partner. Rather, the fusion
protein or the conjugate as explained in the following can be used
in a desired application.
[0047] The mutein used in the present invention can also be
conjugated to a protein or a different chemical (macromolecular)
moiety via a suitable peptidic or non-peptidic linker that can be
attached to any suitable residue within the primary sequence of the
mutein. A protein can, for instance, be conjugated with the C5a
mutein using solvent exposed .alpha.-amino groups of lysine
residues and glutaraldehyde as linker. Another suitable coupling
chemistry is amine-amine crosslinking using bis(succinimidylesters)
of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) as described in
Haugland, R. Handbook of Fluorescent Probes and Research Chemicals,
6th Ed. 1996, Molecular Probes, Eugene, Oreg., on page 96,
incorporated herein by reference. Any protein can be coupled to the
C5a mutein, depending on the desired application. For example, a
conjugate with streptavidin, horseradish peroxidase or green
fluorescent protein might be used as a diagnostic reagent or
research tool for visualizing a C5a receptor on the surface or
within different compartments of a cell.
[0048] In a preferred embodiment a mutein of the invention is
conjugated to a moiety which enhances the in vivo half-life of the
mutein. Such a conjugate is particularly useful when a present C5a
receptor antagonist is used for the treatment of the organ
recipient where a long term presence of the antagonist within the
graft is desired. Suitable moieties are proteins such as human
serum albumin. It is also possible to use anon-protein
(macromolecular) moiety such as polyethylene glycol.
[0049] The C5a muteins including conjugates or fusion proteins
thereof are useful in the treatment and/or prevention or
prophylaxis of a variety of injurious conditions or diseases in
which the complement system, and more particularly C5a and the C5a
receptor, are involved. They are therapeutically very suitable when
administered to any mammal such as cats, dogs, monkeys, rabbits,
mice, rats and, of course, especially humans that face a high risk
of C5a-mediated tissue destruction and death. In general, the
conditions or diseases are usually those such as inflammatory
disorders where C5a is generated proteolytically in serum or
tissue.
[0050] The preservation solution also preferably includes
pharmaceutically-acceptable adjunct materials, such as ethylene
diaminetetraacetic acid (EDTA) in an amount from 0.5 .mu.M to about
1.5 .mu.M as a chelating agent (or other chelating agents known in
the art may also be used). It has also been found desirable to add
from 10 .eta.M to about 100 .eta.M of caprylic acid which helps the
solution to bypass blocked fatty acid utilization and from 10
.mu.g/L to 100 .mu.g/L of apoferritin which serves to eliminate
iron (Fe.sup.++) which causes breakdown of the cells.
Desferrioxamine may also be used to chelate the iron.
Dichloroacetic acid may be employed to reduce lactate.
[0051] Suitable pharmaceutically acceptable antioxidants include,
but are not limited to, allopurinol, glutathione, beta-carotene,
catalase, superoxide dismutase, dimethyl thiourea (DMTU), diphenyl
phenylene diamine (DPPD), mannitol or cyanidanol in an amount
effective to inhibit the generation of oxygen-derived free
radicals. The antioxidants are present generally in an amount from
about 1 .eta.M to 10 .eta.M. Antibiotics may be added for
transplantable organs, but are not generally added during acute
studies.
[0052] The transplantation method of the present invention is to
preserve and store the organ with the preservation solution and
reperfuse with preservation solution prior to implantation. In a
preferred method, the surgeon removes the organ and connects it to
a perfusion apparatus comprising tubing and pumps. The preservation
solution is then perfused through the organ while gassed with
oxygen and carbon dioxide while it is awaiting implantation into a
patient. A perfusion rate of from about 25 to about 150 mL/hour,
preferably about 50 mL/hour, at 1.degree. C. has been found to be
effective. This organ perfusion can occur at either a constant flow
or pressure. In a variation of this method, after the donor has
died, but before the organs have been removed from the donor, the
donor's body can be perfused with the preservation solution of the
present invention in order to provide the beneficial effects of the
solution to the organs at the earliest possible time. Once the
organs are removed, they are then perfused with the preservation
solution as described above.
[0053] The preservation solution can be used at all temperatures
ranging from 0.degree. C. to normal body temperature, 37.degree. C.
At temperatures of from about 12.degree. C. to about 37.degree. C.
the solution is particularly effective.
[0054] The following example is provided to further illustrate the
present invention and is not intended to be construed as limiting
the invention in any manner.
EXAMPLE
[0055] A liter of preservation solution having the following
composition is prepared.
TABLE-US-00002 TABLE 2 Component Concentration NaCl 118 mM KCl 30
mM CaCl.sub.2 1.75 mM KH.sub.2PO.sub.4 1.2 mM MgSO.sub.4 1.2 mM
NaHCO.sub.3 25 mM Glucose 11 mM Adenosine 10 .mu.M EDTA 1.0 mM DMA
1.0 .mu.M Heparin 1000 units Distilled, deionized water q.s.
[0056] To this solution is added the C5a mutein in sterile water or
other sterile injectable medium to reach a final concentration in
the preservation solution of from about 0.1 to about 10 .mu.M. The
C5a mutein used has the following structure: SEQ ID NO: 21
(C5a-(1-66,Cys27Ala)-A8B-Del. 71-73).
[0057] The preservation solution defined above is perfused through
a kidney or a heart, at a rate of about 50 mL per hour, after
removal from the donor and prior to implantation. Similar results
are obtained when the specific C5a mutein component utilized is
replaced, in whole or in part, with the following:
SEQ ID NO: 14 (C5a-(1-66, Cys27Ala)-A8B);
SEQ ID NO: 15 (C5A-(1-66, Cys27Ala)-A8B-Leu 70);
[0058] SEQ ID NO: 16 (C5a-(1-66, Cys27Ala)-Ab8-Cys71); SEQ ID NO:
17 (C5a-(1-66, Cys27Ala)-A8B-Cys73); SEQ ID NO: 18 (C5a-(1-66,
Cys27Ala)-A8B-Leu70-Tyr73); SEQ ID NO: 19 (C5a-(1-66,
Cys27Ala)-A813-Lys69-Ala70); SEQ ID NO: 20 (C5a-(1-66,
Cys27Arg)-A8B); SEQ ID NO: 22 (C5a-(1-66,
Cys-3,Gly-2,-1,Cys27Ala)-A8B); and SEQ ID NO: 39 (C5a-(1-66,
Cys27Ala)A5a).
[0059] The present invention has been described in detail and with
particular reference to the preferred embodiments. Those skilled in
the art will appreciate that changes can be made without departing
from the spirit and scope thereof. Accordingly, the present
invention is to be defined by the following claims, with
equivalents of the claims to be included therein.
Sequence CWU 1
1
1917PRTArtificial SequenceC5aR 1Phe Lys Arg Ser Leu Leu Arg1 5
27PRTArtificial SequenceC5aR 2Phe Lys Arg Leu Leu Leu Arg1 5
35PRTArtificial SequenceC5aR 3Phe Lys Arg Ser Cys1 5
47PRTArtificial SequenceC5aR 4Phe Lys Arg Ser Leu Leu Cys1 5
57PRTArtificial SequenceC5aR 5Phe Lys Arg Leu Leu Leu Tyr1 5
67PRTArtificial SequenceC5aR 6Phe Lys Lys Ala Leu Leu Arg1 5
74PRTArtificial SequenceC5aR 7Phe Lys Arg Ser1 87PRTArtificial
SequenceC5aR 8Phe Lys Leu Leu Leu Leu Arg1 5 973PRTHomo
Sapiens(C5a-(1-66,Cys27Ala)-A8B 9Thr Leu Gln Lys Lys Ile Glu Glu
Ile Ala Ala Lys Tyr Lys His Ser1 5 10 15 Val Val Lys Lys Cys Cys
Tyr Asp Gly Ala Ala Val Asn Asn Asp Glu 20 25 30 Thr Cys Glu Gln
Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile 35 40 45 Lys Ala
Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu Arg Ala Asn 50 55 60
Ile Ser Phe Lys Arg Ser Leu Leu Arg65 70 1073PRTHomo
Sapiens(C5A-(1-66,Cys27Ala)-A8B-Leu70 10Thr Leu Gln Lys Lys Ile Glu
Glu Ile Ala Ala Lys Tyr Lys His Ser1 5 10 15 Val Val Lys Lys Cys
Cys Tyr Asp Gly Ala Ala Val Asn Asn Asp Glu 20 25 30 Thr Cys Glu
Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile 35 40 45 Lys
Ala Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu Arg Ala Asn 50 55
60 Ile Ser Phe Lys Arg Leu Leu Leu Arg65 70 1171PRTHomo
Sapiens(C5a(1-66,Cys27Ala)-A8B-Cys71) 11Thr Leu Gln Lys Lys Ile Glu
Glu Ile Ala Ala Lys Tyr Lys His Ser1 5 10 15 Val Val Lys Lys Cys
Cys Tyr Asp Gly Ala Ala Val Asn Asn Asp Glu 20 25 30 Thr Cys Glu
Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile 35 40 45 Lys
Ala Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu Arg Ala Asn 50 55
60 Ile Ser Phe Lys Arg Ser Cys65 70 1273PRTHomo
Sapiens(C5a-(1-66,Cys27Ala)-A8B-Cys73) 12Thr Leu Gln Lys Lys Ile
Glu Glu Ile Ala Ala Lys Tyr Lys His Ser1 5 10 15 Val Val Lys Lys
Cys Cys Tyr Asp Gly Ala Ala Val Asn Asn Asp Glu 20 25 30 Thr Cys
Glu Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile 35 40 45
Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu Arg Ala Asn 50
55 60 Ile Ser Phe Lys Arg Ser Leu Leu Cys65 70 1373PRTHomo
Sapiens(C5a(1-66, Cys27Ala)-A8B-Leu70-Tyr73) 13Thr Leu Gln Lys Lys
Ile Glu Glu Ile Ala Ala Lys Tyr Lys His Ser1 5 10 15 Val Val Lys
Lys Cys Cys Tyr Asp Gly Ala Ala Val Asn Asn Asp Glu 20 25 30 Thr
Cys Glu Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile 35 40
45 Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu Arg Ala Asn
50 55 60 Ile Ser Phe Lys Arg Leu Leu Leu Tyr65 70 1473PRTHomo
Sapiens(C5a-(1-66, Cys27Ala)-A8B-Lys69-Ala70) 14Thr Leu Gln Lys Lys
Ile Glu Glu Ile Ala Ala Lys Tyr Lys His Ser1 5 10 15 Val Val Lys
Lys Cys Cys Tyr Asp Gly Ala Ala Val Asn Asn Asp Glu 20 25 30 Thr
Cys Glu Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile 35 40
45 Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu Arg Ala Asn
50 55 60 Ile Ser Phe Lys Lys Ala Leu Leu Arg65 70 1573PRTHomo
Sapiens(C5a(1-66,Cys27Arg)-A8b) 15Thr Leu Gln Lys Lys Ile Glu Glu
Ile Ala Ala Lys Tyr Lys His Ser1 5 10 15 Val Val Lys Lys Cys Cys
Tyr Asp Gly Ala Arg Val Asn Asn Asp Glu 20 25 30 Thr Cys Glu Gln
Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile 35 40 45 Lys Ala
Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu Arg Ala Asn 50 55 60
Ile Ser Phe Lys Arg Ser Leu Leu Arg65 70 1670PRTHomo
Sapiens(C5a-(1-66,Cys27Ala)-A8B-Del 71-73 16Thr Leu Gln Lys Lys Ile
Glu Glu Ile Ala Ala Lys Tyr Lys His Ser1 5 10 15 Val Val Lys Lys
Cys Cys Tyr Asp Gly Ala Ala Val Asn Asn Asp Glu 20 25 30 Thr Cys
Glu Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile 35 40 45
Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu Arg Ala Asn 50
55 60 Ile Ser Phe Lys Arg Ser65 70 1776PRTHomo
Sapiens(C5a-(1-66,Cys-3,Gly-2,-1,Cys27Ala)-A8B) 17Cys Gly Gly Thr
Leu Gln Lys Lys Ile Glu Glu Ile Ala Ala Lys Tyr1 5 10 15 Lys His
Ser Val Val Lys Lys Cys Cys Tyr Asp Gly Ala Arg Val Asn 20 25 30
Asn Asp Glu Thr Cys Glu Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro 35
40 45 Arg Cys Ile Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser Gln
Leu 50 55 60 Arg Ala Asn Ile Ser Phe Lys Arg Ser Leu Leu Arg65 70
75 1873PRTHomo Sapiens(C5a-(1-66,Cys27Ala)A5a) 18Thr Leu Gln Lys
Lys Ile Glu Glu Ile Ala Ala Lys Tyr Lys His Ser1 5 10 15 Val Val
Lys Lys Cys Cys Tyr Asp Gly Ala Arg Val Asn Asn Asp Glu 20 25 30
Thr Cys Glu Gln Arg Ala Ala Arg Ile Ser Leu Gly Pro Arg Cys Ile 35
40 45 Lys Ala Phe Thr Glu Cys Cys Val Val Ala Ser Gln Leu Arg Ala
Asn 50 55 60 Ile Ser Phe Lys Leu Leu Leu Leu Arg65 70
1911PRTArtificial SequenceLinker 19Cys Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser1 5 10
* * * * *