The present invention relates to methods and compositions for the diagnosis and treatment of sepsis. The present invention also provides methods of providing a prognosis to a patient with sepsis. In particular, the present invention relates to compositions and methods for the detection of C5aR expression and the correlation of C5aR expression level with prognosis in sepsis.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for the diagnosis and treatment of sepsis. The present invention also provides methods of providing a prognosis to a patient with sepsis. In particular, the present invention relates to compositions and methods for the detection of C5aR expression and the correlation of C5aR expression level with prognosis in sepsis.

Accordingly, in some embodiments, the present invention provides a method of determining a prognosis, comprising providing a blood sample from a subject, wherein the blood sample comprises white blood cells (e.g., neutrophils), and wherein the subject is diagnosed with sepsis; and detecting the level of expression of C5aR on the white blood cells (e.g., neutrophils). In some embodiments, an increased level of expression of the C5aR on the neutrophils relative to a normal standard is indicative of an increased rate of survival of the subject. In other embodiments, a decreased level of expression of the C5aR on the neutrophils relative to a normal standard is indicative of a decreased rate of survival of the subject. In some embodiments, detecting the level of expression of C5aR on the neutrophils comprises exposing the blood sample to an anti-C5aR antibody. In some embodiments, the antibody is labeled (e.g., with a fluorescent label). In some embodiments, detecting the level of expression of C5aR on the neutrophils further comprises subjecting the blood sample to fluorescence activated cell sorting.

The present invention further provides a method of screening compounds, comprising providing a neutrophil, wherein the neutrophil expresses C5aR; and one or more test compounds; and contacting the neutrophil with the test compound; and detecting the level at which the neutrophil expresses the C5aR. In some embodiments, the neutrophil expresses more of the C5aR in the presence of the test compound than in the absence of the test compound. In some embodiments, detecting the level of expression of C5aR on the neutrophils comprises exposing the blood sample to an anti-C5aR antibody. In some embodiments, the antibody is labeled (e.g., with a fluorescent label). In certain embodiments, detecting the level of expression of C5aR on the neutrophils further comprises subjecting the blood sample to fluorescence activated cell sorting. In some embodiments, the cell is in a host. In certain embodiments, the host has been diagnosed with sepsis. In some embodiments, the host is a non-human animal (e.g., an animal model of sepsis). In some embodiments, the test compound is an anti-C5aR antibody.

The present invention additionally provides a kit for providing a prognosis to a subject diagnosed with sepsis, comprising a reagent for determining the level of C5aR expression on a neutrophil; and instructions for using the reagent for providing a prognosis to the subject. In some embodiments, the reagent is an anti-C5aR antibody. In some embodiments, the antibody is labeled with a fluorescent label. In some embodiments, the kit further comprises reagents for using fluorescence activated cell sorting to detect the antibody. In some embodiments, the kit further comprises a normal standard for C5aR expression. In some embodiments, the kit further comprises instructions for using the normal standard for quantitating the level of C5aR expression on neutrophils of the subject.

In still further embodiments, the present invention provides a method of treating sepsis, comprising providing a reagent capable of blocking a C5a receptor; and administering the reagent to a subject suffering from sepsis. In some preferred embodiments, the administering results in a decrease in symptoms of sepsis in the subject. In some embodiments, the reagent is a small molecule antagonist of the C5a receptor (e.g., including, but not limited to, F[OPdChaWR] and MeFKPdChaFR). In other embodiments, the reagent is an antibody specific for the C5a receptor (e.g., a monoclonal antibody).

DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for the diagnosis and treatment of sepsis. In particular, the present invention relates to compositions and methods for the detection of C5aR expression. The diagnostic methods of the present invention find use in the diagnosis of individuals at increased risk of developing sepsis, as well as methods of monitoring sepsis treatments. In other embodiments, the present invention provides methods of treating sepsis by blocking the C5aR.

I. C5a and C5aR in 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 macrophages 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)).

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.2O.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 and Becker, 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 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 et al., Ann. Surg. 202:771-776 (1985)).

C5aR content in various tissues (lung, liver, kidney and heart) is increased during the onset of sepsis, defined by up-regulation of C5aR (protein and mRNA) (J. Clin. Invest. 110:101-8, 2002). After binding of C5a to C5aR on neutrophils, the ligand/receptor complex is rapidly internalized and C5aR is ultimately recycled to the cell surface. This has been repeatedly demonstrated using in vitro experiments with human neutrophils.

II. Diagnostic Applications

C5aR content in various tissues (lung, liver, kidney and heart) is increased during the onset of sepsis, defined by up-regulation of C5aR (protein and mRNA) (J. Clin. Invest. 110:101-8, 2002). Experiments conducted during the course of development of the present invention (See example 1) utilizing rat neutrophils from septic animals after cecal ligation/puncture (CLP) showed that blood neutrophils demonstrate a different pattern. The total amount of C5aR protein in and on blood neutrophils did not change during sepsis, nor did messenger RNA for C5aR. Experiments conducted during the course of development of the present invention demonstrated, however, that surface expression of C5aR on blood neutrophils significantly fell, starting as early as 4 hours after the onset of CLP-induced sepsis, reached a nadir at 24 hours, and slowly increased thereafter (FIG. 1, see Original Patent). The loss of C5aR on the neutrophil surface was due to internalization of C5aR triggered by contact with C5a in the blood. The ability of neutrophils from septic animals to respond chemotactically in vitro to C5a was depressed, inversely correlated with the number of C5aR on the surfaces of neutrophils (FIG. 2A, see Original Patent). The data show that neutrophils with higher numbers of C5aR during sepsis are associated with enhanced survival of the animals, while the opposite is true with neutrophils that have low numbers of C5aR. Another functional parameter is the ability of neutrophils to generate reactive oxygen species (ROS), which are required for bacterial killing by neutrophils. Experiments conducted during the course of development of the present invention demonstrated a positive correlation between the ability of neutrophils to produce ROS and higher C5aR levels on neutrophils (FIG. 2B, see Original Patent).

Further experiments conducted during the course of the present invention (See FIG. 12, see Original Patent) demonstrated a correlation between expression of C5aR on human neutrophils (PMNs) with sepsis. Flow cytometry was used to determine the level of expression of C5aR on human PMNs in healthy humans and humans with sepsis. The expression levels were increased in healthy humans.

Currently, there are no highly reliable single prognostic indicators for septic patients. The known laboratory methods to detect C5aR on blood neutrophils utilize '251-C5a binding, requiring the isolation of neutrophils, the processing of which can alter C5aR content. These procedures are very time consuming and also require larger volumes (40 ml or more) of blood samples.

Accordingly, in some embodiments, the present invention provides methods of characterizing (e.g., providing a prognosis) sepsis based on the level of C5aR expression on neutrophils. In other embodiments, the methods of the present invention are used in the diagnosis of sepsis (e.g., based on expression levels of C5aR on neutrophils). In some embodiments, the methods of the present invention are used in combination with other diagnostic methods useful in diagnosing sepsis. In still further embodiments, the methods of the present invention are used in monitoring the recovery of an individual diagnosed with sepsis.

The methods of the present invention involve common laboratory technologies (Flow Cytometry), and, in some embodiments, utilize antibody-based detection of C5aR on blood neutrophils (See Example 1 for a description of one embodiment of the assay). In some embodiments, a reference standard for C5aR content on blood neutrophils from normal humans is used for comparison. The diagnostic method of the present invention allows for the detection of C5aR on whole blood cells, eliminating the time consuming step of isolating neutrophils. This provides the advantages of requiring only a minimal amount of blood (as little as 100 .mu.l). In addition, the diagnostic method of the present invention is much more rapid than methods requiring the isolation of neutrophils, and, in some preferred embodiments, requires only one hour to perform.

In some embodiments, the level of C5aR expression is used to provide a prognosis to a patient suspected of having, or diagnosed with, sepsis. As described above, experiments conducted during the course of development of the present invention demonstrated that the level of C5aR expression on neutrophils correlated with an improved prognosis in sepsis. The appropriate course of treatment can then be chosen. For example, if a patient is found to have lower levels of C5aR expression, more aggressive treatment may be started earlier. Alternatively, in some embodiments, gene therapy or other pharmaceuticals may be used to increase the levels of C5aR expression.

III. Generating Antibodies to C5aR Peptides

The present invention contemplates monoclonal, polyclonal, and humanized antibodies to C5aR peptides and fragments thereof. 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 C5aR 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 C5aR or peptide fragments thereof.

In some embodiments, mice are immunized with a primary injection of C5aR 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 C5aR 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-C5aR antibodies. Suitable screening techniques include, but are not limited to, solid phase radioimmunoassay. A solid phase immunoadsorbent is prepared by coupling C5aR 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 labeled antibody against murine immunoglobulin. Label associated with the immunoadsorbent indicates the presence of hybridoma products reactive with C5aR peptides.

In preferred embodiments the monoclonal anti-C5aR antibodies are produced in large quantities by injecting anti-C5aR 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 there from. Alternatively, the antibodies are produced by culturing anti-C5aR antibody producing cells in vitro and isolating secreted monoclonal anti-C5aR 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-C5aR 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-C5aR polyclonal antibodies. Polyclonal antibodies can be prepared by immunizing an animal with a crude preparation of C5aR peptides, or purified C5aR 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.66:2170-9 (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; each of which is herein incorporated by reference).

The present invention also contemplates humanized antibodies (e.g., substantially non-immunogenic antibodies). Such antibodies are particularly useful in treating human subjects. Chimeric and `reshaped` humanized anti-C5aR 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; each of which is herein incorporated by reference). In one embodiment, humanized anti-C5aR chimeric antibodies 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.; each of which is herein incorporated by reference). The present invention also contemplates single polypeptide chain binding molecules which have binding specificity and affinity substantially similar to the binding specificity and affinity of the light and heavy chain aggregate variable region of an anti-C5aR antibody (see e.g. U.S. Pat. No. 5,260,203 to Ladner et al.; herein incorporated by reference).

IV. Drug Screening

In some embodiments, the detection methods of the present invention may be used to screen new therapeutics (e.g., treatments for sepsis). For example, in some embodiments, candidate compounds are contacted with neutrophils expressing low or high amounts of C5aR and the ability of the candidate compounds to increase the level of C5aR expression is evaluated (e.g., using the methods of the present invention). In some embodiments, candidate compounds are screened for their ability to improve the prognosis of patients with sepsis. In some embodiments, candidate compounds are small molecules. In other embodiments, candidate compounds are C5aR blocking agents (See below) such as C5aR antibodies or antagonists.

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckennann et al., J. Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the `one-bead one-compound` library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are preferred for use with peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909 [1993]; Erb et al., Proc. Nad. Acad. Sci. USA 91:11422 [1994]; Zuckermann et al., J. Med. Chem. 37:2678 [1994]; Cho et al., Science 261:1303 [1993]; Carrell et al., Angew. Chem. Int. Ed. Engl. 33.2059 [1994]; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061 [1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].

Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84 [1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores (U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids (Cull et al., Proc. Nad. Acad. Sci. USA 89:18651869 [1992]) or on phage (Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406 [1990]; Cwirla et al., Proc. NatI. Acad. Sci. 87:6378-6382 [1990]; Felici, J. Mol. Biol. 222:301 [1991]).

In other embodiments, candidate compounds are screened in animal models of sepsis (e.g., the CLP model disclosed herein). In some embodiments, candidate compounds identified as having activity in the in vitro drug screening methods described above are testing in animal models. Candidate compounds are analyzed in the animal model for their ability to increase survival in animals given experimental sepsis.

In other embodiments, the detection methods of the present invention are used to monitor the effectiveness of new or existing treatments for sepsis. Patients receiving treatment for sepsis are monitored on a regular basis for their levels of C5aR expression. Preferred treatments are those that increase the level of expression of C5aR.

V. Gene Therapy

The present invention also provides methods and compositions suitable for gene therapy to alter C5aR expression, production, or function. In some embodiments, it is contemplated that the gene therapy is performed by providing a subject with additional C5aR receptors on neutrophils to aid the prevention and/or treatment of sepsis. Subjects in need of such therapy may be identified by the methods described above (e.g., the diagnostic methods described above).

Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (See e.g., Miller and Rosman, BioTech., 7:980-990 [1992]). Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome of the replication defective viral vectors that are used within the scope of the present invention lack at least one region that is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (i.e., on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents.

Preferably, the replication defective virus retains the sequences of its genome that are necessary for encapsidating the viral particles. DNA viral vectors include an attenuated or defective DNA viruses, including, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, that entirely or almost entirely lack viral genes, are preferred, as defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al., Mol. Cell. Neurosci., 2:320-330 [1991]), defective herpes virus vector lacking a glycoprotein L gene (See e.g., Patent Publication RD 371005 A), or other defective herpes virus vectors (See e.g., WO 94/21807; and WO 92/05263); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest., 90:626-630 [1992]; See also, La Salle et al., Science 259:988-990 [1993]); and a defective adeno-associated virus vector (Samulski et al., J. Virol., 61:3096-3101 [1987]; Samulski et al., J. Virol., 63:3822-3828 [1989]; and Lebkowski et al., Mol. Cell. Biol., 8:3988-3996 [1988]).

Preferably, for in vivo administration, an appropriate immunosuppressive treatment is employed in conjunction with the viral vector (e.g., adenovirus vector), to avoid immuno-deactivation of the viral vector and transfected cells. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-gamma (IFN-.gamma.), or anti-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors. In addition, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.

In a preferred embodiment, the vector is an adenovirus vector. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types. Various serotypes of adenovirus exist. Of these serotypes, preference is given, within the scope of the present invention, to type 2 or type 5 human adenoviruses (Ad 2 or Ad 5), or adenoviruses of animal origin (See e.g., WO94/26914). Those adenoviruses of animal origin that can be used within the scope of the present invention include, for example, adenoviruses of canine, bovine, murine (e.g., Mav1, Beard et al., Virol., 75-81 [1990]), ovine, porcine, avian, and simian (e.g., SAV) origin. Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800)).

Preferably, the replication defective adenoviral vectors of the invention comprise the ITRs, an encapsidation sequence and the nucleic acid of interest. Still more preferably, at least the E1 region of the adenoviral vector is non-functional. The deletion in the E1 region preferably extends from nucleotides 455 to 3329 in the sequence of the Ad5 adenovirus (PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3A fragment). Other regions may also be modified, in particular the E3 region (e.g., WO95/02697), the E2 region (e.g., WO94/28938), the E4 region (e.g., WO94/28152, WO94/12649 and WO95/02697), or in any of the late genes L1-L5.

In a preferred embodiment, the adenoviral vector has a deletion in the E1 region (Ad 1.0). Examples of E1-deleted adenoviruses are disclosed in EP 185,573, the contents of which are incorporated herein by reference. In another preferred embodiment, the adenoviral vector has a deletion in the E1 and E4 regions (Ad 3.0). Examples of E1/E4-deleted adenoviruses are disclosed in WO95/02697 and WO96/22378. In still another preferred embodiment, the adenoviral vector has a deletion in the E1 region into which the E4 region and the nucleic acid sequence are inserted.

The replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art (See e.g., Levrero et al., Gene 101:195 [1991]; EP 185 573; and Graham, EMBO J., 3:2917 [1984]). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid which carries, inter alia, the DNA sequence of interest. The homologous recombination is accomplished following co-transfection of the adenovirus and plasmid into an appropriate cell line. The cell line that is employed should preferably (i) be transformable by the elements to be used, and (ii) contain the sequences that are able to complement the part of the genome of the replication defective adenovirus, preferably in integrated form in order to avoid the risks of recombination. Examples of cell lines that may be used are the human embryonic kidney cell line 293 (Graham et al., J. Gen. Virol., 36:59 [1977]), which contains the left-hand portion of the genome of an Ad5 adenovirus (12%) integrated into its genome, and cell lines that are able to complement the E1 and E4 functions, as described in applications WO94/26914 and WO95/02697. Recombinant adenoviruses are recovered and purified using standard molecular biological techniques, that are well known to one of ordinary skill in the art.

The adeno-associated viruses (AAV) are DNA viruses of relatively small size that can integrate, in a stable and site-specific manner, into the genome of the cells that they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two essential regions that carry the encapsidation functions: the left-hand part of the genome, that contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, that contains the cap gene encoding the capsid proteins of the virus.

The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (See e.g., WO 91/18088; WO 93/09239; U.S. Pat. No. 4,797,368; U.S. Pat. No. 5,139,941; and EP 488 528, all of which are herein incorporated by reference). These publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferring the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). The replication defective recombinant AAVs according to the invention can be prepared by co-transfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line that is infected with a human helper virus (for example an adenovirus). The AAV recombinants that are produced are then purified by standard techniques.

In another embodiment, the gene can be introduced in a retroviral vector (e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764, 4,980,289 and 5,124,263; Mann et al., Cell 33:153 [1983]; Markowitz et al., J. Virol., 62:1120 [1988]; PCT/US95/14575; EP 453242; EP178220; Bernstein et al. Genet. Eng., 7:235 [1985]; McCormick, BioTechnol., 3:689 [1985]; WO 95/07358; and Kuo et al., Blood 82:845 [1993]; each of which is herein incorporated by reference). The retroviruses are integrating viruses that infect dividing cells. The retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env). In recombinant retroviral vectors, the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest. These vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("murine Moloney leukemia virus" MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Defective retroviral vectors are also disclosed in WO 95/02697; herein incorporated by reference).

In general, in order to construct recombinant retroviruses containing a nucleic acid sequence, a plasmid is constructed that contains the LTRs, the encapsidation sequence and the coding sequence. This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions that are deficient in the plasmid. In general, the packaging cell lines are thus able to express the gag, pol and env genes. Such packaging cell lines have been described in the prior art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719, herein incorporated by reference), the PsiCRIP cell line (See, WO 90/02806; herein incorporated by reference), and the GP+envAm-12 cell line (See, WO 89/07150; herein incorporated by reference). In addition, the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences that may include a part of the gag gene (Bender et al., J. Virol., 61:1639 [1987]). Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.

Alternatively, the vector can be introduced in vivo by lipofection. For the past decade, there has been increasing use of liposomes for encapsulation and transfection of nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et. al., Proc. Natl. Acad. Sci. USA 84:7413-7417 [1987]; See also, Mackey, et al., Proc. Natl. Acad. Sci. USA 85:8027-8031 [1988]; Ulmer et al., Science 259:1745-1748 [1993]). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Felgner and Ringold, Science 337:387-388 [1989]). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127, each of which is herein incorporated by reference.

Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931; herein incorporated by reference), peptides derived from DNA binding proteins (e.g., WO96/25508; herein incorporated by reference), or a cationic polymer (e.g., WO95/21931; herein incorporated by reference).

It is also possible to introduce the vector in vivo as a naked DNA plasmid. Methods for formulating and administering naked DNA to mammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and 5,589,466, both of which are herein incorporated by reference.

DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, including but not limited to transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (See e.g., Wu et al., J. Biol. Chem., 267:963-967 [1992]; Wu and Wu, J. Biol. Chem., 263:14621-14624 [1988]; and Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 [1991]). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., Hum. Gene Ther., 3:147-154 [1992]; and Wu and Wu, J. Biol. Chem., 262:4429-4432 [1987]).

VI. Treatment of Sepsis

The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated, based on the experiments disclosed herein (See e.g., Examples 2 and 3), that blockade of the C5a receptor (C5aR) results in a beneficial effect in the outcome of sepsis. Accordingly, in some embodiments, the present invention provides methods of treating sepsis by blocking the C5aR receptor (e.g., with a C5aR antibody or antagonist).

The CLP mouse model was used to investigate the effect of C5aR blockage (See Examples 2 and 3). Blockade of C5aR by C5aR antagonists resulted in improved survival compared to control animals. The activity of the C5aR antagonist was also confirmed in vitro by chemotaxis experiments, showing significantly reduced chemotactic responses of mouse neutrophils to mouse C5a when the cells were pre-incubated with the C5aR antagonist. In addition, blockade of C5aR by this antagonist resulted in significantly reduced lung injury in a model of immune complex induced lung injury as measured by leakage of .sup.125I-labeled bovine serum albumin (BSA). Administration of an antibody against C5aR resulted in increased survival compared to the group of animals injected with irrelevant IgG.

Thus, experiments conducted during the course of development of the present invention demonstrated that survival in sepsis in rodents can be significantly improved by blockade of C5aR. Accordingly, in some embodiments, the present invention provides methods of treating or preventing sepsis and associated organ damage by blocking C5aR. C5aR may be blocked using any suitable blocking agent, including, but not limited to, specific antagonists (e.g., small molecule antagonists) or specific antibodies directed against C5aR.

Accordingly, in some embodiments, C5aR blocking therapy is used to treat patients at high risk of developing sepsis (e.g., ICU patients after trauma or laparotomy). In other embodiments, patients judged to be in the early phases of a developing a septic syndrome are treated with C5aR blocking reagents to lower the harmful effects of C5a triggered by the increased amount of C5aR in organs in the early onset of sepsis. In yet other embodiments, C5aR blocking reagents are used in patients with fully developed septic syndrome to prevent further harmful organ effects induced by C5a. The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that C5aR blockade prevents patients from multi-organ damage caused by harmful effects of C5a linked to increased C5aR expression in lung, liver, kidney and heart.

The present invention is not limited to a particular C5aR blockage agent. Any suitable agent may be utilized. For example, in some embodiments, an antibody against C5aR is utilized. Is some embodiments, the antibody is humanized or fully human (See e.g., above section describing antibodies).

In other embodiments, the blocking agent is a C5aR antagonist (e.g., a small molecule antagonist). In some embodiments, the antagonist is F[OPdChaWR] (Wong et al., (1998) J. Med. Chem. 41,3417-3425; herein incorporated by reference). In other embodiments, the hexapeptide MeFKPdChaFR (Mollison et al., (1992) FASEB J 6,A2058; Drapeau et al., (11993) Biochem. Pharmacol. 45,1289-1299; each of which is herein incorporated by reference) or variants thereof (Konteatis et al., (1994) J. Immunol. 153,4200-4205; herein incorporated by reference) are utilized as antagonists. Additional antagonists may be identified using the drug screening methods disclosed herein, or other suitable methods.

The present invention is not limited to the treatment of sepsis with C5aR blockage. Any disease states associated with increased C5aR are contemplated for treatment with C5aR blockage. For example, in some embodiments, blockade of C5aR is used as preventative or acute therapy for organ inflammatory diseases such as autoimmune disorders, glomerulonephritis, ischemic injury of the control nervous system or heart, and adult respiratory distress syndrome (ARDS).

VII. Pharmaceutical Compositions Containing C5aR or Effectors Thereof

The present invention further provides pharmaceutical compositions which may comprise all or portions of C5aR inhibitors or antagonists of C5aR bioactivity, including antibodies, alone or in combination with at least one other agent, such as a stabilizing compound, and may be administered, for example, in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.

Pharmaceutical compositions can be administered to the patient intravenously in a pharmaceutically acceptable carrier such as physiological saline. Standard methods for intracellular delivery of peptides can be used (e.g., delivery via liposome). Such methods are well known to those of ordinary skill in the art. The formulations of this invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal. Therapeutic administration of a polypeptide intracellularly can also be accomplished using gene therapy as described herein.

As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and interaction with other drugs being concurrently administered.

Accordingly, in some embodiments of the present invention, pharmaceutical compositions can be administered to a patient alone, or in combination with other nucleotide sequences, drugs or hormones or in pharmaceutical compositions where it is mixed with excipient(s) or other pharmaceutically acceptable carriers. In one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert. In another embodiment of the present invention, polynucleotide sequences or amino acid sequences may be administered alone to individuals subject to or suffering from a disease (e.g., sepsis).

Depending on the condition being treated, these pharmaceutical compositions may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton Pa.). Suitable routes may, for example, include oral or transmucosal administration; as well as parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.

For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. For tissue or cellular administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

In other embodiments, the pharmaceutical compositions of the present invention can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral or nasal ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. For example, an effective amount of a pharmaceutical composition may be that amount that prevents or decreases symptoms of sepsis. Determination of effective amounts is well within the capability of those skilled in the art, especially in light of the disclosure provided herein.

In addition to the active ingredients these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known (e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes).

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, etc; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, (i.e., dosage).

Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

Compositions comprising a compound of the invention formulated in a pharmaceutical acceptable carrier may be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Conditions indicated on the label may include treatment of condition related to sepsis.

The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.

For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Then, preferably, dosage can be formulated in animal models (particularly murine models) to achieve a desirable circulating concentration range that adjusts drug levels.

A therapeutically effective dose refers to that amount of drug that ameliorates symptoms of the disease state. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED.sub.50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and additional animal studies can be used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state; age, weight, and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature (See, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212, each of which are herein incorporated by reference).
 

Claim 1 of 2 Claims

1. A method of treating sepsis in a subject suffering from sepsis, wherein said sepsis is selected from the group consisting of sepsis due to gram-positive bacteremia and sepsis due to gram-negative bacteremia comprising (a) providing a reagent capable of blocking C5a receptor, wherein said reagent is a monoclonal antibody that specifically binds to said C5a receptor; and (b) administering said reagent to said subject, wherein said subject's survival is prolonged.

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