Title: Compositions and methods for the treatment of sepsis
Abstract: The present invention relates to compositions and methods for the prevention and treatment of blood-borne and toxin mediated diseases, and in particular anti-C5a antibodies for the prevention and treatment of sepsis in humans as well as other animals. The present invention also relates to methods of generating anti-C5a antibodies employing C-terminal truncated C5a peptides.
Patent Number: 6,866,845 Issued on 03/15/2005 to Ward, et al.
| Inventors:
|
Ward; Peter A. (Ann Arbor, MI);
Huber-Lang; Markus (South Lyon, MI);
Sarma; Vidya (Ann Arbor, MI)
|
| Assignee:
|
The Regents of the University of Michigan (Ann Arbor, MI)
|
| Appl. No.:
|
651685 |
| Filed:
|
August 30, 2000 |
| Current U.S. Class: |
424/139.1; 530/387.9; 530/388.25; 530/389.3 |
| Intern'l Class: |
A61K 039//39.5; C07K 016//18 |
| Field of Search: |
424/139.1
530/387.9,388.25,389.3
|
References Cited [Referenced By]
U.S. Patent Documents
| 4357272 | Nov., 1982 | Polson.
| |
| 4686100 | Aug., 1987 | Raffin et al.
| |
| 5051448 | Sep., 1991 | Shashoua.
| |
| 5169862 | Dec., 1992 | Burke, Jr. et al.
| |
| 5192746 | Mar., 1993 | Lobl et al.
| |
| 5260203 | Nov., 1993 | Ladner et al.
| |
| 5340923 | Aug., 1994 | Carroll.
| |
| 5539085 | Jul., 1996 | Bischoff et al.
| |
| 5559103 | Sep., 1996 | Gaeta et al.
| |
| 5565332 | Oct., 1996 | Hoogenboom et al.
| |
| 5576423 | Nov., 1996 | Aversa et al.
| |
| 5585089 | Dec., 1996 | Queen et al.
| |
| 5658727 | Aug., 1997 | Barbas et al.
| |
| 5904922 | May., 1999 | Carroll.
| |
| Foreign Patent Documents |
| 0025949 | Apr., 1981 | EP.
| |
| 0245993 | Nov., 1987 | EP.
| |
| WO 96/39503 | Dec., 1996 | WO.
| |
Other References
Abaza et al. J. of Protein Chemistry, 11(5):433-444, 1992.*
Czermak et al. Nature Medicine, 5(7):788-792) (1999).*
Ames Rs et al., "Isolation of Neutralizing Anti-C5a Monoclonal Antibodies
from a Filamentous Phage Monovalent Fab Display Library," J. Immunol
152:4572-4581 (1994).
R.C. Bone, "The Pathogenesis of Sepsis," Ann. Intern. Med. 115:457-469
(1991).
E.S. Caplan and N. Hoyt, "Infection Surveillance and Control in the
Severely Traumatized Patient," Am. J. Med. 70:638-640 (1981).
Caruthers et al., "New chemical methods for synthesizing polynucleotides,"
Nuc. Acids Res. Symp. Ser. 7:215-233 (1980).
Chow and Kempe, "Synthesis of oligodeoxyribonucleotides on silica gel
support," Nuc. Acids Res. 9:2807-2817 (1981).
Crea and Horn, "Synthesis of oligonucleotides on cellulose bya
phosphotriester method," Nuc. Acids. Res. 9:2331 (1980).
Czermak BJ. et al., "Protective effects of a C5a blockade in sepsis,"
Nature Medicine 5:788-792 (1999).
Elzaim, et al., "Generation of Neutralizing Antipeptide Antibodies to the
Enzymatic Domain of Pseudomonas aeruginosa Exotoxin A." Infect. Immun.
May;66(5):2170-9 (1998).
Evans et al., "An engineered poliovirus chimaera elicits broadly reactive
HIV-1 neutralizing antibodies," Nature 339:385-388 (1989).
Gerard C. et al., "Amino Acid Sequence of the Anaphylatoxin from the fifth
Component of Porcine Complement," J. Biol. Chem. 255(10), 4710-4715,
(1980).
Gluzman, "SV40-Transformed Simian Cells Support the Replication of early
SV40 Mutants," Cell, 23:175-182 (1981).
Hatherhill JR et al., "Effects of Anti-C5a Antibodies on Human
Polymorphonuclear Leukocyte Function: Chemotaxis. Chemiluminescence, and
Lysosomal Enzyme Release" J of Biological Response Modifiers 8:614-624
(1989).
Hill, J.H. and Ward, P.A., "The Philogistic Role of C3 Leukotactic
Fragments in Myocardial Infarts of Rats," J. Exp. Med. 133:885-900 (1971).
Hochuli et al., "New Metal Chelate Adsorbent Selective for Proteins and
Peptides Containing Neighbouring Histidine Residues," J Chromatography
411:177-184 (1987).
A.M. Hoffman et al.,"Prognostic Variables for Survival of Neonatal Foals
Under Intensive Care," J. Vet. Int. Med. 6:89-95 (1992).
Huang et al.,"Vaccinia Virus Recombinants Expressing an 11-Kilodalton
.beta.-Galactosidase Fusion Protein Incorporate Active
.beta.-Galactosidase in Virus Particles," J. Virol. 62:3855-3861 (1988).
W.K. Joklik et al., (eds.), Zinsser Microbiology, 18th ed., Chapter 27
Streptococcus pneumoniae, p. 485-489, Appleton-Century-Crofts, Norwalk, CT
(1984).
Kettleborough, et al.,"Humanization of a mouse monoclonal antibody by
CDR-grafting: the importance of framework residues on loop conformation,"
Protein Engineering 4(7):773-783 (1991).
Kohl, J.,and Bitter-Suermann, D., Anaphylatoxins. Complement in health and
disease., Edited by Whaley, K., Loos. M., Weiler, J.M., Kluwer Academic
publishers, pp 299-324 (1993).
Kohler, G. and Milstein, C., "Continuous cultures of fused cells secrecting
antibody of predined specificity," Nature 256:495-497 (1975), Jun. 23,
2003.
Kola et al., "Epitope mapping of a C5a neutralizing mAb using a combined
approach of phage display synthetic peptides and site-directed
mutagenesis". Immunotechnology 1:115-126 (1996).
Kozbor and Roder, "The production of monoclonal antibodies from human
lymphocytes," Immunol. Today 4:72-79 (1983).
Larrick JW et al., "Characterization of Murine Monoclonal Antibodies That
Recognize Neutralizing Epitopes on Human C5a" Infection and Immunity
55:1867-1872 (1987).
G.W. Machiedo et al.,"Patterns of Mortality in a Surgical Intensive Care
Unit," Surg. Gyn & Obstet. 152:757-759 (1981).
Mandecki W, et al.,"Chemical synthesis of a gene encoding the human
complement fragment C5a and its expression in Escherichia coli," Proc Natl
Acad Sci U S A. 82(11):3543-7 (1985).
Matteucci and Caruthers,"The Synthesis of Oligodeoxypyrimidines on a
Polymer Support," Tetrahedron Lett 21:719-722 (1980).
M. Meek et al.,"The Baltimore Sepsis Scale: Measurement of Sepsis in
Patients with Burns Using a New Scoring System," J. Burn Care Rehab.
12:564-568 (1991).
Mohr M. et al., "Effects of anti-C5a monoclonal antibodies on oxygen use in
a porcine model of severe sepsis," Eur J Clin Invest 28:227-234 (1998).
D.D. Morris et al.,"Endotoxemia in neonatal calves given antiserum to a
mutant Escherichia coli (J-5)," Am. J. Vet. Res. 47:2554-2565 (1986).
Mulligan, M.S. et al.,"Requirement and Role of C5a in Acute Lung
Inflammatory Injury in Rats," J. Clin. Invest. 98:503-512 (1996).
Nardelli et al.,"A Chemically Defined Synthetic Vaccine Model for HIV-1,"
J. Immunol. 148:914-920 (1992).
R.L. Nichols in Decision Making in Surgical Sepsis, B.C. Decker, Inc.,
Philadelphia, "Classification of Surgical Wounds and Nonoperative Factors
Influencing Surgical Wound Infections," pp. 20-21 (1991).
Nilsson et al., "Affinity Fusion Strategies for Detection, Purification,
and Immobilization of Recombinant Proteins," Protein Expression and
Purification 11:1-16 (1997).
Olson, L.M., et al.,"The Role of C5 in Septic Lung Injury," Ann. Surg.
202:771-776 (1985).
Park K.W. et al., "Attenuation of Endothelium-Dependent Dilation of Pig
Pulmonary Arterioles After Cardiopulmonary Bypass Is Prevented by
Monoclonal Antibody to Complement C5a," Anesth Analg 89:42-48 (1999).
Posnett et al.,"A Novel Method for Producing Anti-peptide Antibodies," JBC
263:1719-1725 (1988).
Protein Accession No. A57689/ GI "2118416".
Protein Accession No. P01032/ GI "116605".
Protein Accession No. P12082/ GI "116604".
Roberge et al.,"A Strategy for a Convergent Synthesis of N-Linked
Glycopeptides on a Solid Support," Science 269:202-204 (1995).
Rothermel et al., Nucleotide and corrected amino acid sequence of the
functional recombinant rat anaphylatoxin C5a, Biochim. Biophys. Acta
1351:9-12, (1997).
Schlienger et al.,"Human Immunodeficieny Virus Type 1 Major Neutralizing
Determinant Exposed on Hepatitis B Surface Antigen Particles is Highly
Immunogenic in Primates," J. Virol. 66:2570-2576 (1992).
K.A. Schulman et al.,"Cost-effectiveness of HA-1A Monoclonal Antibody for
Gram-Negative Sepsis," JAMA 266:3466-3471 (1991).
J.M. Slack and I.S. Snyder, Bacteria and Human Disease, pp. 128-133,
Yearbook Medical Publishers (1978).
Solomkin, et al.,"Neutrophil dysfunction in sepsis. II. Evidence for the
role of complement activation products in cellular deactivation," Surgery
90:319-327, (1981).
Stahel, et al.,"TNF-.alpha.-Mediated Expression of the Receptor for
Anaphylatoxin C5a on Neurons in Experimental Listeria
Meninggoencephalitis," J. Immunol. 159(2):861-9 (1997).
Stevens JH. et al., "Effects of Anti-C5a Antibodies on the Adult
Respiratory Distress Syndrome in Septic Primates," J Clin Invest
77:1812-1816 (1986).
Van Epps, et al.,"Relationship of C5a Receptor Modulation to the Functional
Responsivesness of Human Polymorphonuclear Leukocytes to C5a," J. Immunol.
150:246-252 (1993).
Ward, P.A. & Becker, E.L.,"The Deactivation of Rabbit Neutrophils by
Chemotactic Factor and the Nature of the Activatable Esterase." J. Exp.
Med. 127:693-709 (1968).
Wilson, et al.,"The Structure of an Antigenic Determinant in a Protein,"
Cell 37:767-778 (1984).
S.M. Wolff, "Monoclonal Antibodies and the Treatment of Gram-Negative
Bacteremia and Shock," New Eng. J. Med. 324:486-488 (1991).
G.P. Youmans et al., Biological and Clinical Basis of Infectious Diseases,
3d ed., "Central Nervous System Infection: General Consideration," pp.
552-568, W.B. Saunders Co., (1985).
Zarbock, J., et al.,"A proton nuclear magnetic resonance study of the
conformation of bovine anaphylatoxin C5a in solution," FEBS Lett. 238(2),
289-294 (1988).
Zhong G. et al., "Production, specificity, and functionality of monoclonal
antibodies to specific peptide-major histocompatibility complex class II
complexes formed by processing of exogenous protein" Proc. Natl Acad Sci
USA 94:13856-13861 (1997).
|
Primary Examiner: Chan; Christina
Assistant Examiner: Vander Vegt; F. Pierre
Attorney, Agent or Firm: Medlen & Carroll, LLP
Goverment Interests
This invention was made with Government support under the National
Institutes of Health (NIH) awarded by contract GM29507 and HL31963. The
government has certain rights in this invention.
Parent Case Text
This is a Continuation-In-Part of copending application Ser. No. 09/387,671
filed on Aug. 31, 1999.
Claims
What is claimed is:
1. A method for the treatment of sepsis in a human comprising:
a) providing;
i) a human presenting symptoms of sepsis, and
ii) a therapeutic composition comprising antibody specific for SEQ ID NO:5,
wherein said composition is not reactive with the C-terminal region of C5a
peptide; and
b) administering said therapeutic composition to said human under
conditions such that at least one symptoms is reduced.
2. The method of claim 1, wherein said human presents the symptoms of
sepsis for a period in the range of approximately six to twelve hours
prior to the administration of said therapeutic composition.
3. The method of claim 1, wherein said antibody is polyclonal.
4. The method of claim 1, wherein said antibody is monoclonal.
5. The method of claim 1, wherein said antibody is not reactive with
complement component C5.
Description
FIELD OF THE INVENTION
The present invention relates to compositions and methods for the
prevention and treatment of blood-borne and toxin-mediated diseases, and
in particular anti-C5a antibodies for the prevention and treatment of
sepsis in humans as well as other animals.
BACKGROUND OF THE INVENTION
Sepsis is a major cause of morbidity and mortality in humans and other
animals. It is estimated that 400,000-500,000 episodes of sepsis resulted
in 100,000-175,000 human deaths in the U.S. alone in 1991. Sepsis has
become the leading cause of death in intensive care units among patients
with non-traumatic illnesses. [G. W. Machiedo et al., Surg. Gyn. & Obstet.
152:757-759 (1981).] It is also the leading cause of death in young
livestock, affecting 7.5-29% of neonatal calves [D. D. Morris et al., Am.
J. Vet. Res. 47:2554-2565 (1986)], and is a common medical problem in
neonatal foals. [A. M. Hoffman et al., J. Vet. Int. Med. 6:89-95 (1992).]
Despite the major advances of the past several decades in the treatment of
serious infections, the incidence and mortality due to sepsis continues to
rise. [S. M. Wolff, New Eng. J. Med. 324:486-488 (1991).]
Sepsis is a systemic reaction characterized by arterial hypotension,
metabolic acidosis, decreased systemic vascular resistance, tachypnea and
organ dysfunction. Sepsis can result from septicemia (i.e., organisms,
their metabolic end-products or toxins in the blood stream), including
bacteremia (i.e., bacteria in the blood), as well as toxemia (i.e., toxins
in the blood), including endotoxemia (i.e., endotoxin in the blood). The
term "bacteremia" includes occult bacteremia observed in young febrile
children with no apparent foci of infection. The term "sepsis" also
encompasses fungemia (i.e., fingi in the blood), viremia (i.e., viruses or
virus particles in the blood), and parasitemia (i.e., helminthic or
protozoan parasites in the blood). Thus, septicemia and septic shock
(acute circulatory failure resulting from septicemia often associated with
multiple organ failure and a high mortality rate) may be caused by a
number of organisms.
The systemic invasion of microorganisms presents two distinct problems.
First, the growth of the microorganisms can directly damage tissues,
organs, and vascular function. Second, toxic components of the
microorganisms can lead to rapid systemic inflammatory responses that can
quickly damage vital organs and lead to circulatory collapse (i.e., septic
shock) and oftentimes, death.
There are three major types of sepsis characterized by the type of
infecting organism. Gram-negative sepsis is the most common and has a case
fatality rate of about 35%. The majority of these infections are caused by
Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.
Gram-positive pathogens such as the Staphylococci and Streptococci are the
second major cause of sepsis. The third major group includes fungi, with
fungal infections causing a relatively small percentage of sepsis cases,
but with a high mortality rate.
Many of these infections are acquired in a hospital setting and can result
from certain types of surgery (e.g., abdominal procedures), immune
suppression due to cancer or transplantation therapy, immune deficiency
diseases, and exposure through intravenous catheters. Sepsis is also
commonly caused by trauma, difficult newborn deliveries, and intestinal
torsion (especially in dogs and horses).
Many patients with septicemia or suspected septicemia exhibit a rapid
decline over a 2448 hour period. Thus, rapid methods of diagnosis and
treatment delivery are essential for effective patient care.
Unfortunately, a confirmed diagnosis as to the type of infection
traditionally requires microbiological analysis involving inoculation of
blood cultures, incubation for 18-24 hours, plating the causative organism
on solid media, another incubation period, and final identification 1-2
days later. Therefore, therapy must be initiated without any knowledge of
the type and species of the pathogen, and with no means of knowing the
extent of the infection.
It is widely believed that anti-endotoxin antibody treatment administered
after sepsis is established may yield little benefit because these
antibodies cannot reverse the inflammatory cascade initiated by endotoxin.
In addition, the high cost of each antibody could limit physicians' use of
a product where no clear benefit has been demonstrated. [K. A. Schulman et
al., JAMA 266:3466-3471 (1991).] Furthermore, these endotoxin antibodies
only target gram-negative sepsis, and no equivalent antibodies exist for
the array of gram-positive organisms and fungi.
Clearly, there is a great need for agents capable of preventing and
treating sepsis. It would be desirable if such agents could be
administered in a cost-effective fashion. Furthermore, approaches are
needed to combat all forms of sepsis.
SUMMARY OF THE INVENTION
The present invention relates to compositions and methods for the
prevention and treatment of blood-borne and toxin mediated diseases, and
in particular anti-C5a antibodies for the prevention and treatment of
sepsis in humans as well as other animals.
The present invention provides a composition comprising antibody specific
for complement component C5a peptide. In another embodiment, the
composition comprises antibody which is specific for complement component
C5a peptide, wherein the C5a peptide has a C-terminal region and an
N-terminal region, and the antibody is not reactive with the C-terminal
region. In further embodiments, the antibody is specific for the
N-terminal region of complement component C5a peptide. In an additional
embodiment, the antibody is also not reactive with complement component C5
protein.
It is not intended that the present invention be limited to antibodies
specific for C5a peptides from certain animals. In certain embodiments,
the antibody is specific for rat C5a peptide. In other embodiments, the
antibody is specific for bovine C5a peptide. In still other embodiments,
the antibody is specific for porcine C5a peptide. In a preferred
embodiment, the antibody is specific for human C5a peptide.
It is also not intended that the present invention be limited to antibodies
generated in a particular animal. A variety of animals are useful for
generating the antibodies of the present invention. In one embodiment, the
antibody is generated in an animal selected from a mouse, a rat, a horse,
a goat, a chicken, and a rabbit. In some embodiments, the antibodies are
collected from the blood of the animal. In other embodiments, the animal
generating the antibodies is a bird, and the antibodies are collected from
egg yolk.
It is not intended that the present invention be limited to the nature of
the antibodies, as a variety of antibody types are contemplated. In one
embodiment, the antibodies are monoclonal. In another embodiment, the
antibodies are humanized. In other embodiments, the antibodies are
chimaeric. In a preferred embodiment, the antibodies are polyclonal.
The present invention also provides a method of producing polyclonal
antibody. In one embodiment, the method comprises, providing; an animal
and an immunogenic composition, wherein the composition comprises
C-terminal truncated C5a peptides; and immunizing the animal with the
immunogenic composition in order to generate antibodies. In some
embodiments, the immunogenic composition comprises adjuvant. In a further
embodiment, antibodies are collected from the animal.
It is not intended that the present invention be limited to antibodies
specific for C5a peptides from any particular animal. In certain
embodiments, the antibody is specific for rat C5a peptide. In other
embodiments, the antibody is specific for bovine C5a peptide. In still
other embodiments, the antibody is specific for porcine C5a peptide. In a
preferred embodiment, the antibody is specific for human C5a peptide.
It is not intended that the present invention be limited to particular
C-terminal truncated peptides. A variety of C-terminal truncated peptides
are contemplated. In one embodiment, the C-terminal truncated peptide
corresponds to the entire N-terminal region of C5a peptide. In another
embodiment, the C-terminal truncated peptide corresponds to the entire
N-terminal region of C5a peptide and a portion of the C-terminal region.
In another embodiment, the C-terminal truncated peptide is a fragment or
portion of the N-terminal region of C5a peptide. In another embodiment,
the C-terminal truncated C5a peptide is between approximately 5 and 50
amino acids in length. In some embodiments, the C-terminal truncated
peptide is approximately fifty amino acids in length. In other
embodiments, the C-terminal truncated peptide is approximately, five amino
acids in length. In preferred embodiments, the C-terminal truncated
peptides are 20 amino acids in length. In certain embodiments, the
C-terminal truncated peptides are selected from SEQ ID NOS:2, 4, 5, 14,
15, and 16.
The present invention also provides a method of treating a subject with the
antibodies of the present invention. In one embodiment, the method
comprises; providing; a subject, and a therapeutic composition comprising
an antibody specific for complement component C5a peptide, wherein the C5a
peptide has a C-terminal region and an N-terminal region, and wherein the
antibody is not reactive with the C-terminal region; and administering the
therapeutic composition to the subject. In another embodiment, the
antibody is specific for the N-terminal region of complement component C5a
peptide.
In one embodiment, the present invention provides a method comprising;
providing; a subject, and a therapeutic composition comprising an antibody
specific for complement component C5a peptide, wherein the C5a peptide has
a C-terminal region and an N-terminal region, and wherein the antibody is
not reactive with the C-terminal region; and administering the therapeutic
composition to the subject. In another embodiment, administering the
therapeutic composition reduces the binding of complement component C5a
peptide to one or more neutrophils of the subject. In a certain
embodiment, administering the therapeutic composition reduces bacteremia
in the subject. In yet another embodiment, administering the therapeutic
composition increases the H.sub.2 O.sub.2 production of neutrophils of the
subject. In a preferred embodiment, administering the therapeutic
composition reduces the symptoms of sepsis.
It is not intended that the therapeutic method of the present invention be
limited to particular subjects. A variety of subjects are contemplated. In
one embodiment the subject is selected from a pig, a rat, a cow, a horse,
and a human. In one embodiment, the therapeutic composition is
administered to a subject suffering from symptoms of sepsis. In another
embodiment, the therapeutic composition is administered prophylactically
to a subject at risk for sepsis, including new born humans and animals.
It is not intended that the therapeutic method of the present invention be
limited to certain modes of administration. A variety of modes of
administering the therapeutic composition are contemplated. In one
embodiment, the therapeutic composition is administered by a mode selected
from intravenously, intramuscularly, subcutaneously, intradermally,
intraperitoneally, intrapleurally, intrathecally, and topically.
It is not intended that the present invention be limited to a particular
therapeutic composition. A variety of compositions are contemplated. In
one embodiment the therapeutic composition comprises a soluble mixture of
anti-C5a antibodies. In another embodiment, the anti-C5a antibodies are
provided together with physiologically tolerable liquid, gels, solid
carriers, diluents, adjuvants or excipients, and combinations thereof. In
other embodiments, the therapeutic composition comprises anti-C5a
antibodies and other therapeutic agents (e.g. other immunoglobulins or
antibiotics).
The present invention also provides a method for screening C-terminal
truncated C5a peptides to identify immunogens for the production of
anti-C5a antibodies. In one embodiment, the method comprises, providing a
C-terminal truncated C5a peptide, modifying the amino acid sequence of
said C-terminal truncated C5a peptide, and screening said C-terminal
truncated C5a peptide to identify immunogens for the production of
anti-C5a antibodies. In one embodiment, the C-terminal truncated C5a
peptide which is provided is selected from SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In other
embodiments, the screening step involves a chemotaxis assay (See e.g.
Examples 7, 8 and 11). In a different embodiment, the screening step
involves a competitive binding assay (See e.g. Examples 10 and 11). In an
additional embodiment, the screening step involves administering the
C-terminal truncated peptides to septic animals (See e.g. Example 11).
In another embodiment, the present invention provides a method for sepsis
rescue, comprising: a) providing; i) a patient presenting symptoms of
sepsis, ii) a therapeutic composition comprising an antibody specific for
the complement component of a C5a peptide region, wherein said C5a peptide
region is some fraction of the complete C5a peptide; and b) administering
said therapeutic composition to said patient.
In a preferred embodiment, the C5a peptide region (recited in the sepsis
rescue method above) is selected from the group consisting of the peptides
defined by SEQ ID NO:2 and SEQ ID NO:75.
In another embodiment, the patient (recited in the sepsis rescue method
above) presents the symptoms of sepsis for a period in the range of
approximately six to twelve hours prior to the administration of said
therapeutic composition.
In another embodiment, the patient (recited in the sepsis rescue method
above) is selected from the group consisting of human, rat, cow, and pig.
In another embodiment, the antibody (recited in the sepsis rescue method
above) is polyclonal or monoclonal and are not reactive with complement
component C5.
DESCRIPTION OF THE FIGURES
FIG. 1 shows survival curves of septic rats with and without the
administration of anti-C5a antibodies.
FIG. 2 shows a graph demonstrating the ability of anti-C5a antibodies to
reduce bacteria blood of septic rats.
FIG. 3 shows a graph demonstrating the ability of anti-C5a antibodies to
reduce bacteria in the organs of septic rats.
FIG. 4 shows a graph demonstrating the ability of anti-C5a antibodies to
increase the level of H.sub.2 O.sub.2 production in neutrophils of septic
rats.
FIG. 5 shows a graph demonstrating the ability of synthetic peptides to
reduce human C5a-induced chemotaxis of neutrophils.
FIG. 6 shows the chemotactic activity of KLH-linked synthetic peptides of
human C5a peptide.
FIG. 7 shows polyclonal rabbit anti-human C5a reactivity with regions of
human C5a peptide.
FIG. 8 shows the amino acid sequence of human C5a peptide (SEQ ID NO:77)
and various smaller portions of the human C5a peptide, specifically, amino
acids 1-20 (SEQ ID NO:4), amino acids 13-32 (SEQ ID NO:78), amino acids
21-40 (SEQ ID NO:79), amino acids 31-50 (SEQ ID NO:80), and amino acids
55-74 (SEQ ID NO:6).
FIG. 9 (SEQ ID NOS:4 to 6) shows a graph demonstrating the ability of
certain synthetic peptides to inhibit the binding of human C5a peptide to
human neutrophils.
FIG. 10 presents data demonstrating that PMA elicits a strong H.sub.2
O.sub.2 response, which is inhibitable by the C5a peptide, and to varying
degrees by peptides A, M, and C.
FIG. 11 projects the image of a Western Blot presents data showing that the
polyclonal anti-human C5a antibody was able to recognize both human and
rat C5a peptide.
FIGS. 12A-C present graphs demonstrating sepsis rescue via the
administration of C5a antibody to reverse sepsis symptoms (as compared to
control) at various timepoints following CLP (Cecal Ligation Puncture).
FIG. 13 (SEQ ID NO:3) projects the complete amino acid sequence of Human
C5a
FIG. 14 (SEQ ID NO:1) projects the complete amino acid sequence of Rat C5a
FIG. 15A (SEQ ID NO:5) projects the "M" fraction of Human C5a as defined by
amino acids 2140 (vis-a-vis the complete amino acid sequence of Human
C5a).
FIG. 15B (SEQ ID NO:81) presents a variant to the "M" fraction of Human C5a
(as compared to FIG. 15A) wherein the serine at amino acid 27 is
substituted for a cysteine.
FIG. 16 (SEQ ID NO:6) projects the "C" fraction of Human C5a as defined by
amino acids 55-74 (vis-a-vis the complete amino acid sequence of Human
C5a).
FIG. 17 (SEQ ID NO:2) projects the "M" fraction of Rat C5a as defined by
amino acids 17-36 (vis-a-vis the complete amino acid sequence of Rat C5a).
FIG. 18 (SEQ ID NO:75) projects the "C" fraction of Rat C5a as defined by
amino acids 58-77 (vis-a-vis the complete amino acid sequence of Rat C5a).
FIG. 19 (SEQ ID NO:82) projects the nucleic acid sequence of a Human C5a
analog set out in GenBank (NCBI gibbsq 170109).
DEFINITIONS
As used herein, the term "patient" includes members of the animal kingdom
including but not limited to human beings.
The phrase "symptoms of sepsis" refers to any symptoms characteristic of a
subject with sepsis including but not limited to, increased respiration,
increased heart rate, reduced arterial CO.sub.2 saturation, arterial
hypotension, metabolic acidosis, fever, decreased systemic vascular
resistance, tachypnea and organ dysfunction (as manefest by, but not
limited to, elevated transaminase, creatinine, and blood urea nitrogen).
Sepsis can result from septicemia (i.e., organisms, their metabolic
end-products or toxins in the blood stream), including bacteremia (ie.,
bacteria in the blood), as well as toxemia (i.e., toxins in the blood),
including endotoxemia (i.e., endotoxin in the blood). The term "sepsis"
also encompasses fungemia (i.e., fungi in the blood), viremia (ie.,
viruses or virus particles in the blood), and parasitemia (i.e.,
helminthic or protozoan parasites in the blood). Thus, phenotypes
associated with septicemia and septic shock (acute circulatory failure
resulting from septicemia often associated with multiple organ failure and
a high mortality rate) are symptoms of sepsis.
As used herein, the phrase "sepsis rescue" refers to the abatement of any
one of the symptoms of sepsis (as defined above) in a patient presenting
any one of the symptoms of sepsis wherein said septic patient is
subsequently administered a therapeutic composition comprising an antibody
specific for the complement component of a C5a peptide region, wherein
said C5a peptide region is some fraction of the complete C5a peptide.
The phrase "reduces the symptoms of sepsis" refers to a qualitative or
quantitative reduction in detectable symptoms, including but not limited
to a detectable impact on the rate of recovery from disease.
The phrase "at risk for sepsis" in reference to a subject is herein defined
as a subject predisposed to the development of sepsis by virtue of the
subject's medical status, including but not limited to such factors as
infection, trauma (e.g., abdominal perforation, such as by a gun shot
wound), surgery (e.g., intestinal surgery), and invasive procedures (e.g.,
placement of a catheter, etc.) and the like.
As used herein, the term "antigen" refers to any agent (e.g., any
substance, compound, molecule [including macromolecules], or other
moiety), that is recognized by an antibody, while the term "immunogen"
refers to any agent (e.g., any substance, compound, molecule [including
macromolecules], or other moiety) that can elicit an immunological
response in an individual. These terms may be used to refer to an
individual macromolecule or to a homogeneous or heterogeneous population
of antigenic macromolecules. It is intended that the term encompasses
protein and peptide molecules or at least one portion of a protein or
peptide molecule, which contains one or more epitopes. In many cases,
antigens are also immunogens, thus the term "antigen" is often used
interchangeably with the term "immunogen." The substance may then be used
as an antigen in an assay to detect the presence of appropriate antibodies
in the serum of the immunized animal.
The term "specific for" when used in reference to the interaction of an
antibody and a protein or peptide means that the interaction is dependent
upon the presence of a particular structure (i.e., the antigenic
determinant or epitope) on the protein; in other words the antibody is
recognizing and binding to a specific protein structure rather than to
proteins in general (i.e. non-specific or background binding).
The term "not reactive with" when used in reference to the potential
interaction of an antibody and a protein or peptide means that the
antibody does not recognize or bind specifically to that particular
protein (i.e. binding is at background levels).
The term "operably linked" refers to an arrangement of elements wherein the
components so described are configured so as to perform their usual
function. Thus, control sequences operably linked to a coding sequence are
capable of effecting the expression of the coding sequence. The control
sequences need not be contiguous with the coding sequence, so long as they
function to direct the expression.
As used herein, the phrase "anti-C5a antibody" refers to antibodies which
are specific for complement component C5a peptide, or portions thereof.
As used herein, the term "adjuvant" is defined as a substance known to
increase the immune response to other antigens when administered with
other antigens. If adjuvant is used, it is not intended that the present
invention be limited to any particular type of adjuvant--or that the same
adjuvant, once used, be used all the time. It is contemplated that
adjuvants may be used either separately or in combination. The present
invention contemplates all types of adjuvant, including but not limited to
agar beads, aluminum hydroxide or phosphate (alum), Incomplete Freund's
Adjuvant, as well as Quil A adjuvant commercially available from Accurate
Chemical and Scientific Corporation, Gerbu adjuvant also commercially
available (GmDP; C.C. Biotech Corp.), and bacterin (i.e., killed
preparations of bacterial cells).
As used herein, the term "N-terminal region of C5a peptide" refers to the
N-terminal 50% of the complement component C5a peptide.
As used herein, the term "C-terminal region of C5a peptide" refers to the
C-terminal 30% of the complement component C5a peptide.
As used herein, the term "wherein said antibody is not reactive with the
C-terminal of C5a region" refers to antibodies that do not recognize or
bind to the C-terminal 30% of the C5a peptide.
As used herein, the term "C-terminal truncated C5a peptides" refers to
peptides of varying lengths derived from the N-terminal 70% of the C5a
peptide, which do not include amino acid sequences from the C-terminal 30%
of the C5a peptide. Examples of these peptides include, but are not
limited to, SEQ ID NO:2 (from Rat C5a peptide), and SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16 (from Human C5a
peptide).
As used herein, the terms "C5a peptide", "C5a protein", and "complement
component C5a peptide" all refer to the complement component peptide in
animals which is cleaved from the amino terminus of complement component
C5 when the complement system is activated. Examples of animals with this
protein include, but are not limited to, mice, rats, cows, pigs, and
humans. This definition also includes peptides with synthetic sequences
which share substantial homology to naturally occurring C5a peptides. An
example of this type of sequence, includes, but is not limited to, the
sequence disclosed in Mandecki W, et al, Proc Natl Acad Sci USA. June;
82(11):3543-7(1985).
As used herein, the term "modifying the amino acid sequence" of said
C-terminal truncated C5a peptide refers to the addition, deletion, or
substitution of one or more amino acids to create a variant or modified
C-terminal truncated C5a peptide (See section II.b, below). Examples of
such variants or modified sequences are listed in Table 3 below.
A "variant" of a C5a peptide (or C-terminal truncated C5a peptide) is
defined as an amino acid sequence which differs by one or more amino acids
from the C5a peptide (or C-terminal truncated C5a peptide) sequence. The
variant may have "conservative" changes, wherein a substituted amino acid
has similar structural or chemical properties, e.g. replacement of leucine
with isoleucine. More rarely, a variant may have "nonconservative"
changes, e.g. replacement of glycine with a tryptophan. Similar minor
variations may also include amino acid deletions or insertions (i.e.,
additions), or both. A variant may be an epitope as short as four amino
acids in length, and as long as a modified full-lenth C5a peptide. More
preferably, a variant is greater than five amino acids in length, and less
than twenty-five amino acids in length. Variants may also contain a fusion
protein. In such cases, the variant may have more amino acids than the
natural, full-lenght C5a peptide.
DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for the
prevention treatment of blood-borne and toxin mediated diseases, and in
particular anti-C5a antibodies for the prevention and treatment of sepsis
caused by various types of organisms in humans as well as other animals.
It is contemplated that the present invention finds use in the treatment
of gram-negative and gram-positive sepsis. Although the invention may be
used for treatment of sepsis due to an individual organism, it may also be
used to treat sepsis caused by multiple organisms (e.g., sepsis and/or
bacteremia due to gram-negative and gram-positive organisms).
I. The C5a Peptide and Sepsis
The complement system is a complex group of proteins present in body fluids
that, working together with antibodies or other factors, plays an
important role as mediators of immune, allergic, immunochemical and
immunopathological reactions. Activation of the complement system can
result in a wide range of reactions such as lysis of various kinds of
cells, bacteria and protozoa, inactivation of viruses, and the direct
mediation of inflammatory processes. Through the hormone-like activity of
several of its components, the complement system can recruit and enlist
the participation of other humoral and cellular effector systems. These in
turn can induce directed migration of leukocytes, trigger histamine
release from mast cells, and stimulate the release of lysosomal
constituents from phagocytes.
The complement system consists of at least twenty distinct plasma proteins
capable of interacting with each other, with antibodies, and with cell
membranes. Many of these proteins, when activated, combine with still
others to form enzymes that cleave and activate still other proteins in
the system. The sequential activation of these proteins follows two main
pathways, the classical pathway and the alternative pathway. Both pathways
use a common terminal trunk that leads to cell lysis or virus
inactivation.
The classical pathway can be activated by antigen-antibody complexes,
aggregated immunoglobulins and non-immunological substances such as DNA
and trypsin-like enzymes. The classical pathway includes activation of C1,
C4, C2 and C3. These components can be grouped into two functional units:
C1 or recognition unit; and C4, C2 and C3 or activation unit. Five
additional components denominated C5, C6, C7, C8, and C9 define the
membrane attack unit forming the terminal trunk common to both pathways.
C5a peptide, also called anaphylatoxin, is a complement component peptide
which is cleaved from the amino terminus of component C5 when the
complement system is activated. C5a peptide has been shown to stimulate
contraction of smooth muscle, enhance vascular permeability, promote the
synthesis and release of other mediators including leukotrienes,
prostaglandins, platelet-activating factor, and histamine. In vivo, C5a
peptide results in the accumulation of polymorphonuclear leukocytes (PMN)
(i.e. neutrophils) and marcrophages at the site of inflammation, one of
the hallmark events of an acute inflammatory response. In vitro, C5a
peptide is a potent chemotaxin for leukocytes, most notably PMN and
macrophages, and it activates PMN causing them to release a variety of
hydrolytic enzymes and to generate oxygen radicals. These latter phenomena
are thought to be responsible not only for the killing of microorganisms
but for much of the tissue destruction that takes place in inflammatory
situations.
There is abundant evidence that in sepsis, complement activation,
production of cytokines, and unregulated inflammatory responses occurs. It
is well established in humans with sepsis that complement activation and
complement consumption have occurred, as defined by loss of whole
hemolytic activity of serum complement (CH50) and the presence of C5a
peptide in serum [Koehl, J., Bitter-Suermann, D., Anaphylatoxins.
Complement in health and disease., Edited by Whaley, K., Loos, M., Weiler,
J. M., Kluwer Academic publishers, pp 299-324, (1993), and Solomkin, et
al., Surgery 90:319-327, (1981)].
It is well established from in vitro studies that interaction of C5a
peptide with C5a receptor (C5aR) leads to phosphorylation of serine
residues of the receptor, followed by rapid internalization of the
receptor-ligand complex, dephosphorylation of the receptor and its
recycling back to the surface of the cell. All of this occurs fairly
rapidly. Furthermore, the maximal C5a-induced H.sub.2 O.sub.2 response of
the neutrophil requires that only a fraction of C5aR be occupied with
ligand [Van Epps, et al., J. Immunol. 150:246-252 (1993)]. Neutrophils
stimulated with C5a peptide become refractory ("deactivated") to further
stimulation with this peptide; following exposure to high doses of C5a
peptide, global deactivation to chemotactic peptides occurs [Ward, P. A. &
Becker, E. L., J. Exp. Med. 127:693-709 (1968)]. There is clinical
evidence that blood neutrophils from humans with early sepsis lose
functional responsiveness to C5a peptide and in the latter phases of
sepsis lose responsiveness to structurally different chemotaxins such as
the bacterial chemotactic factor [Solomkin, J. S., et al., Surgery
90:319-327 (1981)]. It has also been reported that C5 deficient mice
demonstrate somewhat prolonged survival times when sepsis is induced, but
ultimately all animals succumbed to the sepsis syndrome [Olson, L. M., et
al., Ann. Surg. 202:771-776 (1985)].
It is not necessary to the successful practice the present invention that
one understand the precise mechanism by which a therapeutic benefit is
achieved, nor is the present invention limited to any particular
mechanistic explanation. However, it is believed that sepsis results in
excessive production of C5a peptides, which leads to deactivation of
neutrophils, compromising the respiratory burst (H.sub.2 O.sub.2
production) of these cells and the closely linked bactericidal function,
which is dependent upon H.sub.2 O.sub.2 generation and participation of
myeloperoxidase. The anti-C5a antibodies of the present invention,
therefore, are believed to prevent the deactivation of neutrophils caused
by sepsis, thus preserving the bactercidial function of the neutrophils.
In this regard, the present invention contemplates antibodies specific for
complement component C5a peptides and methods of using these antibodies to
treat sepsis. In some embodiments, these antibodies are specific for
complement component C5a peptide, wherein said C5a peptide has a
C-terminal region and an N-terminal region, and wherein said antibody is
not reactive with said C-terminal region.
II. Generating Antibodies to C5a Peptides
a. Antibodies
The present invention contemplates monoclonal, polyclonal, and humanized
antibodies to C5a peptides. In some embodiments, the antibodies are
specific for complement component C5a peptide, wherein said C5a peptide
has a C-terminal region and an N-terminal region, and said antibody is not
reactive with said C-terminal region.
Monoclonal antibodies useful in this invention are obtained, for example,
by well known hybridoma methods. In one embodiment, an animal is immunized
with a preparation containing C-terminal truncated peptides. A fused cell
hybrid is then formed between antibody-producing cells from the immunized
animal and an immortalizing cell such as a myeloma. In one embodiment,
antibodies of the present invention are produced by murine hybridomas
formed by fusion of mouse myeloma or hybridoma which does not secrete
antibody with murine spleen cells which secrete antibodies obtained from
mice immunized against C-terminal truncated C5a peptides.
In some embodiments, mice are immunized with a primary injection of
C-terminal truncated C5a peptides, followed by a number of boosting
injections. During or after the immunization procedure, sera of the mice
may be screened to identify mice in which a substantial immune response to
the C-terminal truncated C5a peptides has been evoked. From the selected
mice, spleen cells are obtained and fusions are performed. Suitable fusion
techniques include, but are not limited to, the Sendai virus technique
[Kohler, G. and Milstein, C., Nature 256:495 (1975)] or the polyethylene
glycol method [Kennet, R. H., "Monoclonal Antibodies, Hybridoma--A New
Dimension in Biological Analysis," Plenum Press, NY (1980)].
The hybridomas are then screened for production of anti-C5a antibodies.
Suitable screening techniques include, but are not limited to, solid phase
radioimmunoassay. A solid phase immunoadsorbent is prepared by coupling
C5a peptides to an insoluble matrix. The immunoadsorbent is brought into
contact with culture supernatants of hybridomas. After a period of
incubation, the solid phase is separated from the supernatants, then
contacted with a labelled antibody against murine immunoglobulin. Label
associated with the immunoadsorbent indicates the presence of hybridoma
products reactive with C5a peptides.
In preferred embodiments the monoclonal anti-C5a antibodies are produced in
large quantities by injecting anti-C5a antibody producing hybridoma cells
into the peritoneal cavity of mice and, after an appropriate time,
harvesting acites fluid from the mice which yield a high titer of
homogenous antibody. The monoclonal antibodies are isolated therefrom.
Alternatively, the antibodies are produced by culturing anti-C5a antibody
producing cells in vitro and isolating secreted monoclonal anti-C5a
antibodies from the cell culture medium directly.
Another method of forming antibody-producing cells is by viral or oncogenic
transformation. For example, a B-lymphocyte which produces anti-C5a
specific antibody is infected and transformed with a virus, such as the
Epstein-Barr virus, to give an immortal antibody-producing cell [Kozbon
and Roder, Immunol. Today 4:72-79 (1983)].
The present invention also contemplates anti-C5a polyclonal antibodies.
Polyclonal antibodies can be prepared by immunizing an animal with a crude
preparation of C-terminal truncated C5a peptides, or purified C-terminal
truncated C5a peptides. The animal is maintained under conditions whereby
antibodies reactive with the components of the peptides are produced. [See
e.g. Elzaim, et al., Infect. Immun. May; 66(5):2170-9 (1998)]. Typically
the animal is "boosted" by additional immunizations to increase the
antibody titer. In one method, blood is collected from the animal upon
reaching a desired titer of antibodies. The serum containing the
polyclonal antibodies (antisera) is separated from the other blood
components. The polyclonal antibody-containing serum may be further
separated into fractions of particular types of antibodies (e.g. IgG or
IgM) or monospecific antibodies can be affinity purified from polyclonal
antibody containing serum. In another method, the immunized animal is a
bird. In this method antibodies (IgY) are collected from egg yolks. The
egg yolk is separated from the yolk lipid and non-antibody proteinaceous
matter, recovering the IgY anti-C5a antibodies in purified form (see e.g.
U.S. Pat. No. 4,357,272 to Polson and U.S. Pat. No. 5,904,922 to Carroll).
The present invention also contemplates humanized antibodies (i.e.
substantially non-immunogenic antibodies). Such antibodies are
particularly useful in treating human subjects. Chimeric and `reshaped`
humanized anti-C5a antibodies may be produced according to techniques
known in the art (see e.g. U.S. Pat. No. 5,585,089 to Queen et al., and
Kettleborough, et al., Protein Engineering, vol. 4, no. 7, pp 773-783,
1991). In one embodiment, humanized anti-C5a chimeric antiboides are
produced using a combinatorial approach (see e.g. U.S. Pat. No. 5,565,332
to Hoogenboom et al. and U.S. Pat. No. 5,658,727 to Barbas et al.). The
present invention also contemplates single polypeptide chain binding
molecules which have binding specificity and affinity subtantially similar
to the binding specificity and affinity of the light and heavy chain
aggregate variable region of an anti-C5a antibody (see e.g. U.S. Pat. No.
5,260,203 to Ladner et al.).
b. C5a Peptide Immunogens
The present invention provides various C5a peptide immunogens. For example,
the C5a peptides can be from various animals (e.g. human, rat, pig, and
cow). The amino acid sequence of these C5a peptides are described in the
literature [see Rothermel et al., Biochim. Biophys. Acta 1351 (1-2), 9-12,
(1997){rat}; Babkina, I. N., et al., Bioorg Khim, May; 21(5):359-64,
(1995) (human); Gerard, C. et al., J. Biol. Chem. 255(10), 4710-4715,
(1980){pig}; and Zarbock, J., et al., FEBS Lett. 238(2), 289-294,
(1988)(cow)]. The C5a immunogen may be the full length C5a peptide, or
various peptides derived from the full length C5a peptide. In particular
embodiments, the peptides are C-terminal truncated peptides (e.g. SEQ ID
NOS:2, 4, 5, 14, 15 and 16). Representative sequences are listed in Table
1. Representative human and rat DNA sequences which are used to generate
various C-terminal truncated C5a peptides are listed in Table 2, along
with the full human and full rat C5a DNA sequences. Modifications of these
sequences (i.e. longer/shorter sequence, from various regions) are
contemplated by the present invention. Generation of these various
C-terminal truncated C5a peptide immunogens are described below.
Variants of the C-terminal truncated C5a peptides are contemplated as
useful immunogens (See e.g. Table 3). For example, it is contemplated that
an isolated replacement of a leucine with an isoleucine or valine, an
aspartate with a glutamate, a threonine with a serine, or a similar
replacement of an amino acid with a structurally related amino acid (i.e.,
conservative mutations) will not have a major effect on the biological
activity of the resulting molecule. Conservative replacements are those
that take place within a family of amino acids that are related in their
side chains. Genetically encoded amino acids can be divided into four
families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine,
histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine,
asparagine, glutamine, cysteine, serine, threonine, tyrosine).
Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly
as aromatic amino acids. In similar fashion, the amino acid repertoire can
be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine,
arginine histidine), (3) aliphatic (glycine, alanine, valine, leucine,
isoleucine, serine, threonine), with serine and threonine optionally be
grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine,
tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6)
sulfur-containing (cysteine and methionine) (See e.g., Stryer ed.,
Biochemistry, 2nd ed,, WH Freeman and Co. [1981]).
Thus, in certain embodiments, modifications of the C-terminal truncated C5a
peptides are contemplated by the present invention. Similar minor
variations may also include amino acid deletions or insertions (i.e.
additions), or both. Guidance in determining which and how many amino acid
residues may be substituted, inserted or deleted without abolishing
biological or immunological activity may be found using computer programs
well known in the art, for example, DNAStar software or GCG (Univ. of
Wisconsin).
Whether a change in the amino acid sequence of a C-terminal truncated C5a
peptide results in a useful immunogen for producing the anti-C5a
antibodies of the present invention can be readily determined. One method
involves screening the C-terminal truncated C5a peptides for the ability
to inhibit the chemotaxis of neutrophils. Useful immunogens are identified
by the ability to induce chemotaxis (See e.g. Examples 7, 8 and 11).
Another indication of a useful immunogen is the ability of the C-terminal
truncated C5a peptide to inhibit chemotaxis when combined with C5a peptide
(See e.g. Examples 7, 8, and 11). Another method involves screening the
C-terminal truncated C5a peptides for the ability to antagonize the
binding of labelled C5a peptides to neutrophils in a competitive assay
(See e.g. Examples 10 and 11). Yet another method involves administering
the C-terminal truncated C5a peptides to CLP sepsis induced rats, and
monitoring their response over a given time period. Useful immunogens are
identified by the ability to reduce the symptoms of sepsis, and/or
increase survival times of the rats (See e.g. Example 11).
The C-terminal truncated C5a peptides employed in the present invention may
also comprise a fusion partner. Examples of fusion partners include
Protein A, ABP, GST, poly histidine, HA, KLH, and MBP. Other fusion
partners are well known in the art. (See Nilsson et al., Prot. Expr.
Purif., 11(1):1-16 [1997]). The fusion partner may serve various
functions, including, but not limited to, enhancement of the solubility of
the C-terminal truncated C5a peptides, as well as providing an "affinity
tag" to allow the purification of the recombinant fusion C-terminal
truncated C5a peptide from the host cell or culture supernatant, or both.
If desired, the exogenous protein fragment may be removed from the peptide
of interest prior to immunization by a variety of enzymatic or chemical
means known in the art.
In some embodiments, nucleic acid sequences corresponding to these various
C-terminal truncated C5a peptides (e.g., SEQ ID NOS:10, 11, 13, 17, 18,
and 19) are used to generate recombinant DNA molecules that direct the
expression of the C-terminal truncated C5a peptides in appropriate host
cells, which are then purified and used as immunogens to generate the
antibodies of the present invention. These DNA sequences may be included
in any one of a variety of expression vectors for expressing C-terminal
truncated C5a peptides in various hosts. Examples of vectors include, but
are not limited to, chromosomal, nonchromosomal and synthetic DNA
sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of plasmids
and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and
pseudorabies, and the like. Any vector may be used as long as it is
replicable and viable in the host.
Large numbers of suitable vectors are known to those of skill in the art,
and are commercially available. Such vectors include, but are not limited
to, the following: 1) Bacterial--pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10,
phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A
(Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); and
2) Eukaryotic--pWLNEO, pSV2CAT, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV,
pMSG, pSVL (Pharmacia). In g
