HK1036016B - Inhibitors of complement activation - Google Patents

Description

Inhibitors of complement activation

Technical Field

The present invention relates to specific inhibitors of factor D, and the use of such inhibitors to inhibit complement system activation and to inhibit the alternative complement pathway.

Background

The complement system plays a central role in clearing immune complexes and immune responses to infectious agents, foreign antigens, virus-infected cells and tumor cells. However, complement is also involved in pathological inflammation and autoimmune diseases. Thus, inhibition of excessive or uncontrolled activation of the complement cascade may provide clinical benefit to patients with the disease or condition.

The complement system comprises two distinct activation pathways, termed the classical and alternative pathways (V.M.Holers, In Clinical Immunology: Principles and Practice, ed.R.R.Rich, Mosby Press; 1996, 363-391). The classical pathway is a calcium/magnesium dependent cascade, which is generally activated by the formation of an antigen-antibody complex. The alternative pathway is a magnesium-dependent cascade activated by C3 deposition and activation on specific susceptible surfaces (such as yeast, bacteria, and cell wall polysaccharides of specific biopolymers). Activation of the complement pathway produces biologically active fragments of complement proteins, such as C3a, C4a, and C5a anaphylatoxins and C5b-9 Membrane Attack Complex (MAC), that mediate inflammatory activities including activation of leukocyte chemotaxis, macrophages, neutrophils, platelets, mast cells, and endothelial cells, vascular permeability, cell lysis, and tissue injury.

Factor D is a highly specific serine protease required for the alternative pathway of complement activation. It cleaves factor B bound to C3B to produce the C3B/Bb enzyme, which is the active component of the alternative pathway C3/C5 convertase. Factor D may be a suitable target for inhibition because it is present at very low plasma concentrations in humans (1.8. mu.g/ml), and has been shown to be a restriction enzyme in the alternative pathway of complement activation (P.H.Lesavre and H.J.Muller-Eberhard.J.Exp.Med., 1978; 148: 1498. sup. 1510; J.E.Volanakis et al, New Eng.J.Med., 1985; 312: 395. sup. 401).

Down-regulation of complement activation has proven effective in the treatment of several diseases in animal models and in vitro studies, such as systemic lupus erythematosus and glomerulonephritis (Y.Wang et al, Proc.Natl.Acad.Sci.; 1996, 93: 8563-8568), rheumatoid arthritis (Y.Wang et al, Proc.Natl.Acad.Sci., 1995; 92: 8955-8959), cardiopulmonary bypass and hemolysis (C.S.Rinder, J.Clin.Invest. (1995; 96: 1564-1572), hyperacute rejection of organ Transplantation (T.J.Kroshus et al, Transplantation, 1995; 60: II 94-1202), cardiovascular embolism (J.W.Homeister et al, J.Immunol, 1993; 150: 1055-1064; H.F.Weisshu et al, Science, 1990: 149; AMBII et al, 175-149-151, 1992; adult respiratory syndrome, J.J.J.J.198, 1992; AMBII.151-151, J.J.J.J.J.J.1064-198, AMBII.J.J.J.H.H.H.175151, and H.J.J.. In addition, other inflammatory and autoimmune/immune complex diseases are also closely associated with complement activation (V.M. holers, ibid., B.P. Morgan. Eur.J.Clin. invest, 1994: 24: 219-.

Brief description of the invention

The invention includes inhibitors of factor D that bind to and block the functional activity of factor D in the alternative pathway of complement activation. Such inhibitors include antibody molecules as well as homologues, analogues and modified or derived forms thereof, including immunoglobulin fragments such as Fab, F (ab')₂And Fv. Small molecules including peptides, oligonucleotides, peptidomimetics, organic compounds capable of binding to factor D and blocking its functional activity are also encompassed by the invention.

A monoclonal antibody has been prepared which binds to factor D and blocks its complement activation function and is designated 166-32. The hybridoma producing this antibody was deposited with the American type culture Collection, 10801 University Blvd, Manassas, VA 20110-.

Brief Description of Drawings

FIG. 1 shows the binding of monoclonal antibodies (MAbs) against factor D to purified human factor D in an ELISA. The lines marked with filled circles represent MAb 166-11. The lines marked by filled triangles represent MAb 166-32. The lines marked with filled diamonds represent MAb 166-188. The lines marked by solid squares represent MAb 166-222. The reactivity of MAbs with factor D is shown on the Y-axis, which is expressed as Optical Density (OD) at 450nm, and the concentration of MAbs on the X-axis.

FIG. 2 shows the inhibition of Alternative Pathway (AP) hemolysis of unsensitized rabbit blood red cells (RBCs) by MAb166-32 in the presence of 10% human serum. The lines marked by solid squares represent MAbs 166-32. The solid circle marked lines represent the unrelated isotype matched control MAb G3-519, which is specific for the HIV envelope glycoprotein gp 120. As further described herein, the Y-axis represents% hemolysis inhibition. The X-axis indicates the concentration of MAbs.

FIG. 3 shows the inhibition of Alternative Pathway (AP) hemolysis of unsensitized rabbit blood red cells (RBCs) by MAb166-32 in the presence of 90% human serum. The lines marked by solid squares represent MAbs 166-32. The solid circle marked lines represent the unrelated isotype matched control MAb G3-519, which is specific for the HIV envelope glycoprotein gp 120. As further described herein, the Y-axis represents% hemolysis inhibition. The X-axis indicates the concentration of MAbs.

FIG. 4 shows that MAb166-32 is unable to inhibit Classical Pathway (CP) hemolysis of sensitized chicken RBCs, but the positive control anti-human C5 MAb137-76 is able to inhibit. The lines marked with filled circles represent MAbs 137-76. Lines marked with filled diamonds and filled squares represent MAb166-32 and negative control MAb G3-519, respectively. The Y-axis represents% hemolysis inhibition. The X-axis indicates the concentration of MAbs.

FIG. 5 shows the inhibitory effect of MAb166-32 on hemolysis of the Alternative Pathway (AP). Hemolysis can be enhanced by adding different concentrations of purified human factor D to human serum that has been cleared of its factor D using anti-factor D MAb 66-222 affinity chromatography. This assay was performed in the presence or absence of 0.3. mu.g/ml of the test MAbs. The solid square marked line represents no antibody. The lines marked with filled circles represent MAb 166-32. The line marked with filled triangles represents the irrelevant isotype-matched control MAb G3-519. The Y-axis represents% hemolysis inhibition. The X-axis represents the concentration of factor D.

FIG. 6 shows the inhibitory effect of MAb166-32 on factor-dependent EAC3b cell lysis. This alternative C3 convertase was assembled into EAC3B cells by incubation with factor B, factor P and factor D. MAb166-32 was added to incubation buffer at various concentrations to inhibit factor D activity. The lines marked by solid squares represent MAbs 166-32. The lines marked with filled circles represent MAb G3-519. The Y-axis represents% hemolysis inhibition. The X-axis represents the concentration of MAbs.

FIG. 7 shows that MAb166-32 inhibits the production of C3a from zymosan. Zymosan can activate the alternative complement pathway when present in human serum. The generation of C3a can be measured using an ELISA kit. The lines marked by solid squares represent MAbs 166-32. The solid circle-marked line represents the irrelevant isotype-matched control MAb G3-519. The Y-axis represents the% inhibition by C3 a. The X-axis represents the concentration of MAbs.

FIG. 8 shows that MAb166-32 inhibits the production of sC5b-9 from zymosan. Zymosan can activate the alternative complement pathway when present in human serum. The production of sC5b-9 can be measured using an ELISA kit. The lines marked by solid squares represent MAbs 166-32. The filled circle-marked line represents the irrelevant isotype-matched control MAb G3-519. The Y-axis represents the% inhibition of sC5b-9 production. The X-axis represents the concentration of MAbs.

FIG. 9 shows the inhibition of alternative pathway hemolysis of unsensitized rabbit RBCs by MAb166-32 and its Fab. The line marked with a filled circle represents MAb166-32 (whole IgG). The solid square marked lines represent the Fab of MAb 166-32. The Y-axis represents% hemolysis inhibition. The X-axis represents the concentration of MAbs.

FIG. 10 shows the inhibition of factor D in serum from different animal species by MAb166-32 on alternative pathway hemolysis of unsensitized rabbit RBCs. Lines marked with filled squares represent human serum. The lines marked with filled circles represent chimpanzee sera. Lines marked with filled triangles represent rhesus monkey serum. The filled inverted triangle-labeled line represents baboon serum. Lines marked with filled diamonds represent cynomlgus monkey serum. The line marked with empty circles represents sheep serum. Lines marked with empty triangles represent dog sera. The Y-axis represents% hemolysis inhibition. The X-axis represents the concentration of MAb 166-32.

FIG. 11 shows the reactivity of MAb166-32 with different baculovirus-expressed factor D ("FD") mutants and hybrids in ELISA. The line marked by a solid square represents the human factor D- - -FD/Hu. The line marked with a solid circle represents the Pig factor D- - -FD/Pig. Lines marked with filled triangles represent FD/Pighu. The solid inverted triangle marked line represents the fusion protein FD/Hupig. The line marked with filled diamonds represents the mutein FDNDA. The line marked by an empty circle represents the mutein FD/L. The line marked by an empty triangle represents the mutein FD/RH. The line marked with empty diamonds represents a blank without coating antigen. Recombinant proteins are described further herein.

FIG. 12 shows a schematic of the expression vector plasmid for chimera 166-32 Fab: (A) pSV2dhfrFd and (B) pSV2neo κ. The solid box represents the exon encoding the Fd or kappa genes. The diagonal line segments represent enhancer and promoter elements (E-P) derived from HCMV as shown below. The hollow cassette as its label represents dihydrofolate reductase (dhfr) and neo genes. The pSV2 plasmid contained DNA fragments from different sources: the pBR322 DNA (narrow line) contains the replication origin region (pBR ori) of the pBR322 DNA and the lactamase- - -ampicillin resistance gene (Amp); SV40 DNA is represented and indicated by broad diagonal lines and comprises the SV40 DNA replication origin region (SV40 ori), the early promoter (5 'for dhfr and neo genes), and the polyadenylation signal (3' for dhfr and neo genes). The polyadenylation signal (pA) from SV40 is also placed 3' to the Fd gene.

FIG. 13 shows inhibition of Alternative Pathway (AP) hemolysis in unsensitized rabbit RBCs. The line marked with filled squares represents mouse MAb 166-32. The filled circle marked line represents the chimera MAb 166-32. The line marked with solid triangles represents the isotype-matched negative control antibody G3-519. The Y-axis represents hemolysis inhibition (%). The X-axis represents the concentration of antibody.

FIG. 14 shows inhibition of Alternative Pathway (AP) hemolysis in unsensitized rabbit RBCs. The solid square-marked line represents the chimera 166-32 IgG. The solid circle marked line represents cFab/9 aa. Lines marked with filled triangles represent cFab. The Y-axis represents hemolysis inhibition (%). The X-axis represents protein concentration of IgG and Fab.

FIG. 15 shows the effect of anti-factor D MAb166-32 in treating hemodynamics of isolated rabbit hearts from human plasma infusion. Left Ventricular End Diastolic Pressure (LVEDP) is indicated by solid circles (MAb 166-32) and solid squares (MAb G3-519). Left ventricular pressure (LVDP) is represented by hollow circles (MAb 166-32) and hollow squares (MAb G3-519). MAb G3-519 was an unrelated isotype-matched control.

Figure 16 is a representative representation of the left ventricular pressure (LVDP) generated by 2 antibody populations in an isolated rabbit cardiac study. The top panel represents hearts treated with the negative control antibody MAb G3-519, while the bottom panel represents hearts treated with MAb 166-32. Whereas hearts treated with MAb G3-519 failed to maintain LVDP after treatment with 4% human plasma, hearts treated with MAb166-32 maintained near baseline LVDP after 60 minutes of perfusion with 4% human plasma.

Figure 17 shows Bb concentrations in lymphatic effusions at selected time points during infusion of isolated rabbit hearts with 4% human plasma. The MAb166-32 treated heart samples (empty circles) contained significantly lower Bb than the MAb G3-519 treated hearts (filled squares), with p < 0.05.

FIG. 18 shows the alternative pathway hemolytic activity of plasma samples collected at different time points by the extracorporeal circulation system treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles).

FIG. 19 shows the C3a concentrations for plasma samples collected at different time points by extracorporeal circulation systems treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles).

FIG. 20 shows sC5b-9 concentrations for plasma samples collected at different time points from the extracorporeal circulation system treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles).

FIG. 21 shows the Bb concentrations of plasma samples collected at different time points by the extracorporeal circulation system treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles).

FIG. 22 shows the C4d concentrations for plasma samples collected at different time points by the extracorporeal circulation system treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles).

FIG. 23 shows the expression levels of CD11b on the surface of neutrophils collected at different time points by the extracorporeal circulation system treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles). The expression level of CD11b was expressed as Mean Fluorescence Intensity (MFI) measured by an immunocytometer analysis.

FIG. 24 shows the expression levels of CD62P on the surface of platelets collected at different time points from the extracorporeal circulation system treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles). The expression level of CD62P was expressed as Mean Fluorescence Intensity (MFI) measured by an immunocytometer analysis.

FIG. 25 shows neutrophil-specific Myeloperoxidase (MPO) concentrations in plasma samples collected at different time points by the extracorporeal circulation system treated with MAb166-32 (filled squares) or MAb G3-519 (filled circles).

Description of sequence listing

SEQ ID NO: 1 is the nucleotide sequence of human factor D.

SEO ID NO: 2 is the amino acid sequence of human factor D.

SEQ ID NO: 3 is the nucleotide sequence of porcine factor D.

SEQ ID NO: 4 is the amino acid sequence of porcine factor D.

SFQ ID NO: 5 is a primer for cloning MAb166-32 V.kappa.gene.

SEQ ID NO: 6 is an annealing linker (adaptor) used to clone the MAb166-32 V.kappa.gene as follows and used as a primer.

SEO ID NO: 7 is an annealing linker used to clone the MAb166-32 V.kappa.gene as follows.

SEQ ID NO: the MAb 166-32V clone was cloned as follows 8_H3' primer of gene.

SEQ ID NO: 9 for cloning MAb 166-32V_HPrimers for the gene.

SEQ ID NO: 10 for cloning MAb 166-32V_HPrimers for the gene.

SEQ ID NO: 11 is the 5' primer used for PCR amplification of the MAb166-32 Fd gene.

SEQ ID NO: 12 is the 3' primer used for PCR amplification of the MAb166-32 Fd gene.

SEQ ID NO: 13 is the 5' primer used for PCR amplification of the MAb166-32 Fd gene.

SEQ ID NO: 14 is the 3' primer used for PCR amplification of the MAb166-32 Fd gene.

SEQ ID NO: 15 is the other amino acid sequence used to add Fd to obtain recombinant Fab.

The invention has been accomplished and its use

A. Preparation of monoclonal antibodies (MAbs) against human factor D

In a particular embodiment of the invention, anti-factor D MAbs can be prepared by immunizing mice (e.g., mice, rats, hamsters, and guinea pigs) with the native factor D purified from human plasma or urine, or expressing recombinant factor D or fragments thereof in eukaryotic or prokaryotic systems. Other animals may also be used for immunization, such as non-human primates, transgenic mice expressing human immunoglobulins, and severe binding immunodeficiency (SCID) mice transplanted with human B lymphocytes. Hybridomas are prepared by conventional methods and obtained by fusing B lymphocytes from immunized animals with melanoma cells (e.g., Sp2/0 and NSO), as described in G.Kohler and C.Milstein (Nature, 1975: 256: 495-497). In addition, anti-factor D antibodies can also be obtained by screening recombinant single chain Fv or Fab libraries from human B lymphocytes in a phage display system. Specificity of MAbs for human factor D can be detected by ELISA, Western blotting, or other immunochemical techniques. The inhibitory activity of the antibody against complement activation can be evaluated using a hemolytic assay of non-sensitized rabbit or guinea pig Red Blood Cells (RBCs) for the alternative pathway and non-sensitized chicken or sheep RBCs for the classical pathway. Hybridomas in positive control wells were cloned with limiting dilutions. Purified antibodies were used in the above described assay to analyze specificity for human factor D.

If used to treat inflammation or autoimmunity in humans, anti-factor D antibodies are preferably used in the form of chimeric, deimmunized, humanized or human antibodies. Such antibodies may reduce immunity and thus avoid human anti-mouse antibody (HAMA) responses. The antibody is preferably IgG4, IgG2, or other genetically mutated IgG or IgM which does not enhance antibody-dependent cytotoxicity (S.M. Canfield and S.L. Morrison, J.exp. Med., 1991: 173: 1483-plus 1491) and complement-mediated cell lysis (Y.xu et al, J.biol chem., 1994: 269: 3468-plus 3474; V.L.Pulito et al, J.Immunol., 1996; 156: 2840-plus 2850).

Chimeric antibodies are prepared by recombinant techniques well known in the art and comprise animal variable regions and human constant regions. Humanized antibodies have a higher degree of human peptide sequence than chimeric antibodies. In humanized antibodies, only the Complement Determining Regions (CDRs) responsible for antigen binding and specificity are derived from an animal and contain the corresponding amino acid sequence of an animal antibody, while essentially all of the remainder of the molecule (except for a small portion of the framework region within the variable region in some instances) is derived from a human and corresponds to the amino acid sequence of a human antibody. See l.riechmann et al, Nature, 1988; 332: 323-327; winter, U.S. patent 5225539; queen et al, U.S. patent 5530101.

Deimmunized antibodies are antibodies that have removed T and B cell epitopes as described in International patent application PCT/GB 98/01473. It has no or reduced immunity when applied in vivo.

Human antibodies can be made by several methods, including the generation of fragments of human antibodies (VH, VL, Fv, Fd, Fab or F (ab')₂And these fragments can be used to construct fully human antibodies using techniques similar to those used to generate chimeric antibodies. Human antibodies can also be made in transgenic mice carrying the human immunoglobulin genome. Such mice are available from Abgenix, inc., Fremont, California, and Medarex, inc., anandale, New Jersey.

Single peptide chain binding molecules can also be constructed in which the heavy and light chain Fv regions are linked. Single chain antibodies ("ScFv") and methods for their construction are described in U.S. Pat. No. 4946778. Fab can also be constructed and expressed in a similar manner (M.J.Evans et al, J.Immunol meth, 1995; 184: 123-. All human antibodies, both whole and partial, are less immunogenic than the whole mouse MAbs, and fragments and single chain antibodies are less immunogenic. All these types of antibodies do not therefore elicit an immune or allergic response in comparison. At the same time, they are all more suitable for in vivo administration to humans than are intact animal antibodies, especially when repeated or prolonged administration is required. In addition, the smaller size of the antibody fragment may also facilitate tissue bioavailability, which may be important for better dose accumulation in acute diseases.

Based on the molecular structure of the variable region of the anti-factor D antibody, molecular models and rational molecular design can be used to prepare and screen small molecules that mimic the molecular structure of the antibody binding region and inhibit factor D activity. These small molecules may be peptides, peptidomimetics, oligonucleotides or organic compounds. Such mimetic molecules can be used as inhibitors of complement activation in inflammatory and autoimmune diseases. In addition, large-scale screening methods commonly used in the art can also be used to isolate appropriate small molecules from combinatorial compound libraries.

In one embodiment of the invention, a chimeric Fab having animal (mouse) variable regions and human constant regions is used for therapy because it is smaller than intact immunoglobulin and may be more tissue penetrating.

B. Use of anti-factor D molecules

The anti-factor D binding molecules, antibodies, and fragments of the invention may be administered to a patient in a suitable formulation by a variety of routes including, but not limited to, intravenous infusion, intravenous bolus injection, and peritoneal, dermal, intramuscular, subcutaneous, intranasal, bronchial, intravertebral, intracranial, and oral administration. Such administration allows them to bind to endogenous factor D and thereby inhibit the production of C3b, C3a, and C5a anaphylatoxins, as well as C5 b-9.

Estimated preferred doses of such antibodies and molecules are between 10 and 500 micrograms/ml serum. The actual dosage can be determined by measuring the optimum dosage according to the method commonly used in clinic, i.e. various dosages are given and the most effective dosage is determined.

Anti-factor D molecules can inhibit complement activation and/or the alternative complement pathway in vivo and the accompanying inflammation, such as macrophage, neutrophil, platelet and mast cell attraction and activation, edema and tissue damage. These inhibitors can be used to treat diseases or conditions mediated by excessive or uncontrolled activation of the complement system. They include, but are not limited to: (1) tissue damage due to ischemia reperfusion after cardiovascular embolism, aneurysm, stroke, hemorrhagic shock, impact damage, multiple organ failure, hypovolemic shock and intestinal ischemia; (2) inflammatory disorders such as burns, endotoxemia and septic shock, adult dyspnea, cardiopulmonary bypass, hemodialysis, anaphylactic shock, severe asthma, angioedema, Crohn's disease, sickle cell anemia, poststreptococcal glomerulonephritis and pancreatitis; (3) graft rejection such as hyperacute graft rejection; and (4) adverse drug reactions such as drug allergies, IL-2-induced vascular leakage, and radiocontrast media allergies. Autoimmune disorders include, but are not limited to: systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, Alzheimer's disease, and multiple sclerosis, which may also be treated with the inhibitors of the present invention.

Anti-factor D molecules may also be used in diagnostics to determine the presence or quantification of factor D in tissue or body fluid samples such as serum, plasma, urine or bone marrow. In this application, well known assay formats such as immunohistochemistry or ELISA, respectively, may be used. Such diagnostic tests can be used to determine whether a particular individual is deficient or overproducing factor D.

C. Animal model of therapeutic effectiveness of factor D inhibitors

The therapeutic activity of the factor D inhibitors described above in various diseases can be demonstrated using various animals available with various inflammatory and autoimmune diseases. The in vitro tests described in the examples below are sufficient to demonstrate their effectiveness.

Animal models comparable to various complement-associated clinical diseases in humans can also be used to demonstrate the in vivo effectiveness of factor D inhibitors. They include, but are not limited to: cardiovascular ischemia reperfusion injury (H.F. Weisman et al, Science, 1990; 249: 146-.

The following describes how to make and use the invention, and to demonstrate its utility.

Example 1: preparation of anti-factor D MAbs

8-12 week old male AIJ mice (Harlan, Houston, TX) were injected subcutaneously with 25 micrograms of factor D purified from human serum (Advanced Research Technologies, San Diego, Calif.) dissolved in Freund's complete adjuvant(Difco Laboratories, Detroit, Michigan) and dissolved in 200. mu.l Phosphate Buffered Saline (PBS) pH 7.4. The factor D preparation was > 95% pure as determined by Sodium Dithiobenzenesulfonate (SDS) -polyacrylamide gel electrophoresis (PAGE). This factor D was tested as described below and found to be biologically active in haemolysis. Mice were injected subcutaneously twice with 25 micrograms of human factor D in Freund's incomplete adjuvant at 2 week intervals. Mice were then given another intraperitoneal injection of 25 micrograms of the same antigen in PBS after 2 weeks and 3 days before killing. In each fusion, a single cell suspension was prepared from the spleen of each immunized mouse and used to fuse with Sp2/0 melanoma cells. 5X 10 in a culture broth containing 50% polyethylene glycol (M.W.1450) (Kodak, Rochester, NY) and 5% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.)⁸Sp2/0 and 5X 10⁸Spleen cells were fused. The cells were then adjusted to 1.5X 10 in lscove's medium (Gibco, Grand Island, N.Y.) supplemented with 10% calf serum, 100 units/ml penicillin, 100. mu.g/ml streptomycin, 0.1 mmole hypoxanthine, 0.4. mu. moles aminopterin and 16. mu. moles thymine⁵Concentration of splenocytes/200 microliters of suspension. 200 ml of the cell suspension was added to the wells of about 20 96-well micro-culture plates. After about 10 days, culture supernatants were removed for screening of those that could react with purified factor D in ELISA.

The wells of the Immulon 2(Dynatech Laboratories, Chantilly, Va.) micro-assay plate were coated at room temperature overnight by adding 50. mu.l of purified human factor (50. mu.mg/ml). Low concentrations of coating factor D allow for the screening of antibodies with high affinity. After beating the plate to remove the coating solution, 200 μ l of BLOTT0 (skim milk powder) in PBS was added to each well for 1 hour to block the non-specific sites. After 1 hour, the wells were washed with buffer PBST (PBS containing 0.05% Tween 20). 50 microliters of culture supernatant was collected from each fusion well, mixed with 50 microliters of BLOTTO, and added to each well of the micro-test plate. After 1 hour incubation, wells were washed with PBST. Goat anti-mouse IgG (Fc specificity) was then conjugated with horseradish peroxidase (HRP) diluted 1: 2000 with BLOTTO (Jackson Immunoresearch Laboratories, West G)Cave, PA) reaction to detect bound murine antibody. Peroxidase substrate solution containing 0.1% 3, 3, 5, 5 tetramethylbenzidine (Sigma, st. louis, MO) and 0.0003% hydrogen peroxide (Sigma) was added to each well and developed for 30 minutes. 50 microliter of 2M H was added₂SO₄To each well to stop the reaction. The reaction OD was read at 450nm using a BioTek ELISA counter (BioTek Instruments, Winooski, VM).

The culture supernatants of the positive control wells were then tested in 2 assays: i) the inhibition of alternative pathway hemolysis of unsensitized rabbit RBCs by pre-determined concentrations of human serum was tested as described below; and ii) the inhibition of zymosan-induced C3a production by treatment with human serum was examined as follows. Cells in positive wells were cloned by limiting dilution. The reactivity of the MAbs with factor D in ELISA was again tested. The selected hybridomas were cultured in spinner flasks, and the culture supernatants were collected and purified by protein a affinity column chromatography. ELISA assays were used to generate 4 MAbs that strongly respond to human factor D. These MAbs are designated 166-11, 166-32, 166-. Among these, MAb166-32 (IgG1) strongly inhibited the hemolysis of the unsensitized rabbit RBCs alternative pathway as described below.

Example 2: determination of kinetic constants of anti-factor D MAbs by surface plasmon resonance

The kinetic constants for binding of MAbs 166-11, 166-32, 166-188, and 166-22 to human factor D were determined using a BIAcore instrument (Pharmacia Biosensor AB, Uppsala, Sweden) using a measurement method based on surface plasmon resonance. All binding measurements were performed in HEPES-buffered saline (HBS) (10 mM HEPES, pH7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20) at 25 ℃. To measure the binding rate constant of factor D to MAbs, rabbit anti-mouse IgG (H + L) was immobilized to a CM5 detection plate (sensorchip) by amine coupling using N-hydroxysuccinimide and N-ethyl-N' - (3-diethylaminopropyl) carbodiimide. Each MAb is then captured first to the coated detector plateAnd re-injected with factor D at different concentrations. To measure the association constant (K)_assoc) Factor D was prepared at 5 dilution concentrations (2.5 nanomolar, 5 nanomolar, 10 nanomolar, 15 nanomolar and 20 nanomolar) as indicated by the manufacturer and injected into the wells at a rate of 5 μ l/min. To measure the dissociation rate constant (k)_dissoc) 100 nanomolar factor D was injected into the wells at a rate of 5 microliters/minute. The data in the form of sensing patterns can be analyzed using a data mosaicing procedure in the BIAcore system. Because MAb166-32 has an extremely fast K_assocAnother binding procedure may be used to measure its power rate constant because it has exceeded the confidence limit of the test procedure due to the limitations of the bulk input effect. Factor D was immobilized to the detection plate by amine coupling as described above, while different dilution concentrations of MAb166-32 (5 nM, 10 nM, 15 nM, 20 nM and 25 nM were used for K measurement_assocAnd 200 nanomolar for measuring K_dissoc) Flow to the detection plate at a rate of 5. mu.l/min. The data in the form of a sensing graph can be analyzed as described above. The kinetic constants for factor D binding to MAbs on BIAcore are listed in table 1 below. MAbs 166-32 and 166-222 have very high affinity for factor D, with equilibrium dissociation constant (K)_D) Less than 0.1 nanomolar.

TABLE 1 kinetic constants for factor D binding to MAbs on BIAcore

MAbs	K(×10MS)	K(×10S)	K(×100M)
				166-32	＞10	1.1	＜1

166-32	4.6	0.76	1.6
				166-188	8.75	2.1	2.4
166-11	8.0	1.0	1.24
				166-222	＞10	0.8	＜1

^aDuring the assay, factor D was used as the analyte and was flowed to a detection plate coated with anti-factor D MAb captured with rabbit anti-mouse IgG.

^bUsing MAb166-32 as the analyte, factor D was crosslinked to the detection plate by an amine coupling method.

^cK_DEquilibrium dissociation constant, ═ k_dissoc/k_assoc

Example 3: inhibition of complement activation hemolysis

To investigate the functional activity of anti-factor D MAbs in inhibiting complement activation in vitro, 2 hemolytic assays were used.

For the alternative pathway, unsensitized rabbit RBCs were washed 3 times with gelatin/sodium barbiturate buffered saline (GVB/Mg-EGTA) containing 2 mmole MgCl₂And 1.6 mmoles of EGTA. 10 millimoles of EGTA were used to inhibit the classical pathway (K.Whaley et al, in A.W.Dodds (Ed.), comment.A Practical approach.Oxford university Press, Oxford, 1997, pp.19-47). Washing the cells at 1.7X 10⁸Cells/ml were resuspended in the same buffer. In each well of a round bottom 96-well micro-assay plate, 50 microliters of normal human serum (20%) was mixed with 50 microliters of GVB/Mg-EGTA or serially diluted test MAb, and then 30 microliters of the washed rabbit RBCs suspension was added to the well containing the mixture. 50 ml of normal human serum (20%) was mixed with 80. mu.l of GVB/Mg-EGTA to give a serotoned background. Negative controls used isotype-matched anti-HIV-1 gp120MAb- - -G3-519. The final mixture was incubated at 37 ℃ for 30 minutes. The plate was then shaken in a microtest plate shakerShake for 15 seconds. The plates were then centrifuged at 300 Xg for 3 minutes. The supernatant (80 μ l) was collected and transferred to wells of a flat-bottomed 96-well micro-test plate to measure the OD at 405 nm. Percent inhibition of hemolysis was defined as 100 × [ (OD without MAb-serotonergic background OD) - (OD with MAb-serotonergic background OD)]/(OD without MAb-serotonergical background OD).

FIG. 2 shows data showing that MAb166-32 strongly inhibits alternative pathway hemolysis of unsensitized rabbit RBCs in the presence of 10% human serum in a dose-dependent manner, whereas the unrelated isotype-matched control (MAb G3-519) did not. MAb G3-519 is specific for the HIV envelope glycoprotein gp 120.

In an assay to detect the inhibitory activity of MAb166-32 in 90% human serum, frozen human serum was thawed and pre-treated with EGTA at a final concentration of 50 millimolar. 10 ml of serially diluted MAb166-32 or G3-519 were added to 90. mu.l of human serum treated with EGTA and two replicate wells were made in 96-well microtest plates and left for 15 minutes at room temperature. 30 ml of washed rabbit RBCs were added to each well. The plates were incubated at 37 ℃ for 30 minutes. The plates were then shaken on a plate shaker for 15 seconds and centrifuged at 300 Xg for 3 minutes. The supernatant (80. mu.l) was collected and transferred to a flat bottom 96-well micro-test plate to measure OD at 405 nm. Each plate had 2 wells containing 100 microliters of 90% human serum and 30 microliters of buffer as the background for developing serum, and 2 wells containing 100 microliters of 90% human serum in the absence of monoclonal antibody to lyse RBCs to represent complete lysis. FIG. 3 shows the data that MAb166-32 can strongly inhibit alternative pathway hemolysis of unsensitized rabbit RBCs in the presence of 90% human serum in a dose-dependent manner.

For the classical pathway, 0.5 millimole MgCl will be contained₂And 0.15 mmol of CaCl₂Chicken RBCs (5 × 107 cells/ml) in gelatin/barbituric sodium buffered saline (GVB + +) were sensitized with 8 μ g/ml of purified rabbit anti-chicken RBC immunoglobulin (Inter-Cell Technologies, Hopewell, NJ) at 4 ℃ for 15 minutes. Cells were then washed with GVB + +. The final concentration of human serum used was 2%.

FIG. 4 shows data that MAb166-32 and an unrelated isotype-matched control (G3-519) did not inhibit classical pathway hemolysis of sensitized chicken RBCs, but that a positive control, anti-human C5 MAb137-76, did. The data in FIGS. 2, 3 and 4 indicate that MAb166-32 specifically inhibits the alternative pathway of complement activation.

Example 4: specificity of MAb166-32 for factor D

As described below, 2 hemolytic assays were used to demonstrate the specificity of MAb166-32 for human factor D.

(1) Inhibition of factor D-dependent hemolysis assay using unsensitized rabbit RBCs

The human serum samples were first depleted of factor D by passing them through an affinity column packed with 3M emophaze Biosupport Medium (Pierce, Rockford, IL) conjugated with the anti-factor DMAb 166-222. The serum flow through was tested for the inability to activate alternative pathway hemolysis due to complete removal of factor D. The procedure of this experiment was similar to that described in example 3 above, except that each concentration of purified factor D was added to the factor D-depleted serum to reconstitute its hemolytic activity. In this case, hemolysis of rabbit RBCs is factor D dependent. Recombinant hemolytic activity showed a linear proportionality (from 0.01. mu.g/ml to 2. mu.g/ml) to the supplemented factor D concentration (FIG. 5). The data in FIG. 5 also show that 0.3. mu.g/ml of MAb166-32 completely inhibited hemolysis of unsensitized rabbit RBCs in the presence of 0.1. mu.g/ml of supplemented factor D, whereas negative control MAb G3-519 had no effect on factor D-dependent hemolysis. These data demonstrate that MAb166-32 is effective in inhibiting the biological activity of human factor D at a molar ratio of 1: 2 (MAb 166-32 vs. factor D). MAb166-32 is therefore a potent, high affinity antibody to factor D. Such antibodies have potential for use in the clinical treatment of diseases or conditions caused by the alternative complement pathway.

(2) Inhibition of the formation of alternative C3 convertase on EAC3b cells

FAC3b cells were encapsulated with human C3b (purchased from National Jewish Center of lmmunology and Respiratory medicine)ine, Denver, CO). In this assay, the alternative C3 convertase was assembled on the cell surface of EAC3B by the addition of factor B, factor P (properdin) and factor D. EAC3b cells (5X 10)⁸) With DGVB + + solution (50% barbiturate sodium buffered saline, pH7.2, containing 0.075 mmol of CaCl₂0.25 mmole MgCl₂0.1% gelatin, 2.5% (w/v) dextran, and 0.01% sodium azide) for 3 washes. The washed cells were suspended in 1.5 ml of DGVB⁺⁺Factor P (30 μ g) and factor B (20 μ g). The factor P and factor B concentrations were determined in advance to be excessive. 50 ml of the cell suspension was added to each well of a round bottom 96-well micro-assay plate. 50 microliters of a mixture of factor D (1.2 micrograms/ml) and serially diluted MAb166-32 or MAb G3-519 was then added to the wells containing the cells and incubated at 30 ℃ for 15 minutes. The concentration of factor D (1.2 μmg/ml) is in this case predicted to give a hemolysis higher than 90%. After incubation, cells were washed 2 times in GVB-EDTA medium (10 mM EDTA in gelatin/sodium barbiturate buffered saline). The cells were then suspended in 30. mu.l of GVB-EDTA medium. To initiate hemolysis, 100 μ l guinea pig serum (Sigma) (diluted 1: 10 in GVB-EDTA) was added to each well. The mixture was then incubated at 37 ℃ for 30 minutes. The micro-assay plates were then centrifuged at 300 Xg for 3 minutes. The supernatant was collected and the OD measured at 405 nm.

FIG. 6 shows the results of experiments in which MAb166-32 inhibited EAC3b cell lysis, whereas unrelated MAb G3-519 produced no inhibition. MAb166-32 inhibits factor D cleavage of factor B, thus avoiding the formation of C3 convertase on the cell surface of EAC 3B.

Example 5: inhibition of zymosan production C3a by complement activation by MAb166-32

To further determine the functional specificity of MAb166-32 for factor D, the effect of MAb on alternative complement pathway activation on zymosan (activated yeast particles) was examined. Zymosan A (from Saccharomyces cerevisiae, Sigma) (1 Mg/ml) was washed 3 times in GVB/Mg-EGTA and then suspended at 1 Mg/ml in the same medium. 25 microliters of different concentrations of MAb166-32 or G3-519 were mixed with 25 microliters of human serum (1: 5 diluted in GVB/Mg-EGTA) in a microtube and incubated for 15 minutes at room temperature. The control group contained no antibody, but culture medium and serum. After incubation, 50 microliters of the washed zymosan suspension was added to each tube and incubated at 37 ℃ for 30 minutes. The microtube was centrifuged at 2000 Xg for 5 minutes and the supernatant collected and mixed with an equal amount of sample stabilization solution (Quidel, San Diego, Calif.). The samples were frozen at-25 ℃ until analysis. The concentration of C3a and sC5b-9 in the sample was determined using a quantitative ELISA kit (Quidel) according to the manufacturer's instructions.

FIG. 7 shows that MAb166-32 inhibits the production of C3a self-complement-activated zymosan, whereas unrelated MAb G3-519 did not. These data indicate that MAb166-32 inhibits the C3 convertase production of factor D. Complete inhibition of factor D by MAb166-32 effectively blocks the production of the C3 convertase, as shown by its inability to produce C3 a. This will result in the inhibition of the C5 convertase in the subsequent step of the complement cascade, as evidenced by the inhibition of sC5b-9(MAC) formation (FIG. 8).

Example 6: inhibition of complement activation hemolysis by Fab of MAb166-32

To test whether the monovalent form of MAb166-32 inhibits the alternative complement pathway as effectively as the parent bivalent MAb166-32, Fab for MAb166-32 was prepared by papain digestion using a commercial kit (Pieree). The inhibitory activity of the Fab on alternative pathway hemolysis was then tested using unsensitised rabbit RBCs as described above.

Figure 9 shows experimental data, with both whole IgG and Fab showing similar ability to block alternative complement activation. Considering the two binding sites on each antibody molecule, these results indicate that the monovalent form of MAb166-32 is active, retaining the ability of antagonist D to resemble its parent diabody. This property is very important for considering the use of Fab or single-chain Fv as an alternative product. One advantage of using the latter monovalent form is that it may have better tissue permeability because of its small size. Given the activity of the Fab of MAb166-32, the binding epitope recognized by this MAb on factor D may be an important functional part.

Example 7: effect of MAb166-32 on alternative pathway hemolysis in sera of different species of animals

To study the cross-reactivity of MAb166-32 with factor D from different animal species, alternative pathway hemolysis analysis was performed using sera from different animal species. The CH50 values were first tested using fresh sera from different animal species (human, rhesus monkey, chimpanzee, baboon, cynomolgus monkey, sheep, dog, mouse, hamster, rat, rabbit, guinea pig and pig), with the CH50 value being defined as the dilution of serum that would achieve 50% hemolysis in unsensitized rabbit RBCs. The inhibitory activity of MAb166-32 on the same hemolytic activity (CH50) of each serum was then tested and compared.

FIG. 10 shows that MAb166-32 has strong inhibitory activity against human, rhesus, cynomolgus, baboon, and chimpanzee sera, and moderate inhibitory activity against sheep and dog sera. The antibody cannot inhibit serum of mice, hamsters, rats, rabbits, guinea pigs and pigs. These data indicate that MAb166-32 binds a common epitope of factor D in humans, rhesus monkeys, chimpanzees, baboons, cynomolgus monkeys, sheep, and dogs.

Example 8: construction of human factor D mutant for epitope mapping of MAb166-32

To delineate the binding epitope of human factor D recognized by MAb166-32, antibody reactivity with human factor D on Western blots was first examined. MAb166-32 did not react with SDS-denatured human factor D (reduced or non-reduced) immobilized on nitrocellulose membrane. This result indicates that MAb166-32 binds native, but not denatured, factor D.

Because MAb166-32 does not inhibit the hemolytic activity of mouse and porcine factor D as shown in example 7, MAb166-32 is most likely bound to the site of human factor D with high amino acid sequence differences from mouse and porcine factor D. In this regard, MAb166-32 binding epitope mapping was performed by substituting the corresponding amino acid residues of porcine factor D for the amino acid residues of human factor D to prepare various factor D mutants and hybrids, as described below.

(1) Construction of factor D mutants and heterozygotes

The human factor D gene fragment was obtained by Polymerase Chain Reaction (PCR) using human adipocyte cDNA (Clontech, San Francisco, Calif.) as a template and appropriate oligonucleotide primers. The amplified DNA fragment was restriction-cleaved with BamHI and EcoRI, and the cleavage product was inserted into BamHI and EcoRI sites of baculovirus transfer vector pVL1393(Pharmingen, San Diego, Calif.) to give wild-type pVL 1393-factor D/Hu. Human factor D was named factor D/Hu. The nucleotide sequence and deduced amino acid sequence of the mature human factor D protein are shown in SEQ ID NOS: 1 and 2(R.T. white et al, J.biol.chem., 1992: 267: 9210-9213; GenBank read number: M84526).

The cDNA clone of porcine factor D, pMon24909, was given as a gift by Nebrass university J.L.Miner (GenBank accession number: U29948). The BamHI-EcoRI fragment of pMon24909 was cloned into PVL1393 to give pVL 1393-factor D/pig. The porcine factor D was named factor D/pig. The nucleotide sequence and deduced amino acid sequence of the mature porcine factor D protein are shown in SEQ ID NOS: 3 and 4.

Three human factor D mutants were constructed using appropriate primers and overlapping PCR. Amino acid mutations are designed to replace amino acid based residues in human sequences with corresponding amino acid residues in porcine sequences, when the corresponding alignments of the amino acid sequences of human and porcine factor D are compared for homology. The first mutant was factor D/VDA, which had three amino acid mutations: V113E, D116E and a 118P. (this is a shorthand method for naming mutations, e.g.where V113E refers to the valine at amino acid group 113 of human factor D changed to the glutamic acid of porcine factor D). The second mutant is factor D/RH, which has two amino acid mutations: R156L and H159Y. The third mutant is factor D/L, which has a single amino acid mutation: L168M. The DNA sequences encoding these mutants were confirmed by DNA sequencing. After cleavage with an appropriate enzyme, the DNA fragment was inserted into BamHI and EcoRI sites of the baculovirus transfer vector pVL1393 to give pVL 1393-factor D/VDA, pVL 1393-factor D/RH and PVL 1393-factor D/L, respectively.

Two chimeric human-porcine factor D hybrids were also constructed by overlapping PCR with appropriate primers. The first hybrid is factor D/Hupig, which contains 52 amino acids from human factor D at the N-terminus, with the remaining amino acids from porcine factor D. The other hybrid is factor D/Pighu, which contains 52 amino acids from porcine factor D at the N-terminus, with the remaining amino acids from human factor D. The BamHI and EcoRI digested DNA fragment was inserted into BamHI and EcoRI sites of baculovirus transfer vector pVL1393 to give PVL 1393-factor D/Hupig and PVL 1393-factor D/Pighu.

(2) Expression of factor D mutants and hybrids

The steps of plasmid transfection, recombinant baculovirus preparation and recombinant factor D protein preparation in insect cell Sf9 were performed according to the manufacturer's instructions (baculovirus expression vector system, Pharmingen).

(3) Purification of factor D mutants and hybrids

Factor D mutants and hybrids from infected Sf9 cell culture supernatants were purified by affinity column chromatography using purified goat anti-human factor D polyclonal antibody (The Binding Site Limited, San Diego, CA). 3 ml of goat anti-human factor D antibody (13.2 mg/ml) was added to coupling buffer (0.1M borate and 0.75M Na)₂SO₄Ph9.0) and coupled with 4 ml Ultralink Biosupport Medium (Pierce) for 2 hours at room temperature. The micelles were first washed with 50 mM diethylamine (pH11.5) to saturate all remaining unreacted sites, and then washed with a solution containing 10 mM Tris, 0.15M NaCl, 5 mM EDTA, 1% Triton X-100, and 0.02% NaN₃Washing with a buffer solution (pH 8.0). The micelles were stored in buffer at 4 ℃.

Culture supernatants collected from 100 ml of each baculovirus mutant infected Sf9 cells in spin culture were passed through a goat anti-factor D affinity column, which had previously been pre-equilibrated with PBS to remove the preservation buffer. Bound factor D protein was eluted with 50 mM diethylamine, pH 11.5. The collected fractions were immediately neutralized to pH7.0 with 1M Hepes buffer. Residual salts were removed by buffer exchange with PBS using Millipore membrane ultrafiltration (m.w. cut-off: 3,000) (Millipore corp., Bedford, MA). Protein concentration was determined by BCA method (Pierce).

(4) ELISA for factor D

The reactivity of MAb166-32 with different factor D mutants and hybrids was tested by ELISA assay. Different wells of a 96-well micro-assay plate were coated with proteins (factor D/Hu, factor D/pig, factor D/Hupig, factor D/Pighu, factor D/VDH, factor D/RH, and factor D/L) by adding each protein (0.5. mu.g/ml) to 100. mu.l of PBS solution. After overnight incubation at room temperature, wells were treated with PBSTB (PBST with 2% BSA) to saturate the remaining binding sites. The wells were washed with PBST. 100 ml of serially diluted MAb166-32 (1. mu.g/ml-0.5. mu.g/ml) was added to the wells and left at room temperature for 1 hour. The wells were then washed with PBST. Bound antibodies were detected by incubation with diluted HRP-goat anti-mouse igg (fc) (jackson immunoresearch) for 1 hour at room temperature. The peroxidase receptor solution was then added as described above to effect the color development reaction. OD at 450nm was determined using an ELISA reader.

FIG. 11 shows that MAb166-32 reacts with factor D/Hu, factor D/Pighu, and factor D/VDA, but not with factor D/pig, factor D/Hupig, factor D/RH, and factor D/L. The ELISA results showed that amino acid residues Arg156, His159 and Leu168 of human factor D are required for MAb166-32 binding. This is consistent with the fact that MAb166-32 does not bind to factor D/Hupig when the C-terminus of human factor D is replaced by the corresponding site in the pig. Amino acid residues Arg156, His159 and Leu168 are located in The so-called "methionine loop" which consists of The disulfide bond between Cys154 and Cys170 and a methionine at position 169 (j.e. volanakis et al, in The human comparative System in Health and Disease, j.e. volanakis and m.m.franks, eds., Marcel Dekker, 1998, pp.49-81). Structurally, a "methionine ring" is a tight type 1 β turn. Based on data from X-ray crystal diffraction pattern studies, it was found to be exposed on the surface of factor D molecules (S.V.L.Narayana et al, J.mol.biol., 1994, 235: 695-. However, the contribution of the "methionine ring" to receptor specificity and factor D catalytic activity has not been investigated (J.E.Volanakis et al, Protein Sci, 1996; 5: 553-one 564). This data demonstrates for the first time that the "methionine ring" plays an important role in factor D functional activity. MAb166-32 and its Fab, when bound to this region of factor D, effectively inhibit the catalytic activity of factor D.

Example 9: cloning of anti-factor D MAb166-32 variable region gene and construction and expression of chimera 166-32IgG and Fab thereof

To reduce the immunogenicity of MAb166-32 when used in humans, chimeras of MAb166-32 were prepared by substituting the mouse constant region with the constant region of human IgG 1. Two Fab chimeric forms of the antibody were also prepared with human corresponding regions replacing the mouse constant region. The cloning of the MAb166-32 variable region gene and the construction and expression of the chimeric 166-32 antibody and its Fab are described in detail below.

(1) Cloning of anti-factor D MAb variable region genes

Total RNA was isolated from hybridoma cells secreting anti-factor D MAb166-32 and isolated using RNAzol according to the manufacturer's instructions (Biotech, Houston, TX). The first strand of cDNA is synthesized from total RNA using oligo dT as a primer. PCR uses immunoglobulin constant (C) region derived 3' primers and derived from leader peptide or murine V_HOr a degenerate primer set of the first framework region of the V.kappa.gene as the 5' primer. Although the amplified DNA is designated V_HHowever, for V.kappa.DNA fragments of the expected length are not amplified. V_HAnd the vkappa gene were cloned by anchored PCR.

Chimeric anchored PCR can be performed as described by Chen and Platsucas (Scand. J. Immunol., 1992: 35: 539-549). In cloning the V.kappa.gene, a double-stranded cDNA was prepared using NotI-MAKI primer (5'-TGCGGCCGCTGTAGGTGCTGTCTTT-3' SEQ ID NO: 5). Annealing joint AD1 (5-GGAATTCACTCGTTATTCTCGGA-3' SEQ ID NO: 6) and AD2 (5'-TCCGAGAATAACGAGTG-3' SEQ ID NO: 7) ligated to the 5 'and 3' ends of the double-stranded cDNA. The 3' linker was removed by digestion with NotI. The digested product was used as a template in PCR with AD1 oligonucleotide as the 5 'primer and MAK2 (5'-CATTGAAAGCTTTGGGGTAGAAGTTGTTC-3'SEQ ID NO: 8) as the 3' primer. A DNA fragment of about 500bp was cloned into pUC 19. 12 clones were picked for further analysis. 7 clones were found to contain CDR3 sequences specific for the Sp2/O V signal and most likely misshapen k light chain signal from the fusion portion of the 166-32 hybridoma cell line. NotI-MAKI and MAK2 oligonucleotides derived from murine C_kRegions and are each at C_k182 bp and 84bp downstream of the first bp of the gene. Analysis of 3 clones by DNA sequencing resulted in sequences including a portion of murine C κ, all V κ and the leader peptide.

For clone V_HGene, double-stranded cDNA was prepared using NotI-MAG1 primer (5'-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3' SEQ ID NO: 9). Annealing adaptors AD1 and AD2 were ligated to the 5 'and 3' ends of the double stranded cDNA. The 3' end of the linker was removed by digestion with NotI. The digested product was used as a template in PCR with the primers AD1 oligonucleotide and MAG2 (5' -CGGTAAGCTTCACTGGCTCAGGGAAATA-31SEQ ID NO: 10). The DNA fragment of 500-600bp in length was cloned into pUC 19. NotI-MAG1 and MAG2 oligonucleotides were derived from the murine C.gamma.l region and were 180 and 93bp downstream of the first bp of the C.gamma.l gene, respectively. The 3 clones were analyzed by DNA sequencing to obtain sequences containing a portion of murine Cgamma.l, all of V_HAnd a leader peptide.

(2) Construction of expression vectors for chimeric 166-32IgG and Fab

Use of V in PCR_HAnd the V.kappa.gene as template to add Kozak sequence at the 5 'end and splice donor at the 3' end. After analyzing the sequence to confirm that there is no PCR error, V is added_HAnd inserting the V kappa gene into expression vector cassettes (cassettes) respectively containing human C gamma l and C kappa to obtain pSV2neoV_HHucgammal and pSV2 neoV-huckappa. Plasmid DNAs of CsC gradient-purified heavy and light chain vectors were electroporated into COS cells. After 48 hoursThe culture supernatant was assayed by ELISA for chimeric IgG at about 200 μmg/ml. Cells were collected and total RNA was prepared. First strand cDNA was synthesized from total RNA using oligo dT as a primer. Fd and kappa DNA fragments were prepared by PCR using this cDNA as a template. For the Fd gene, PCR was performed using (5'-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3' SEQ ID NO: 11) as a 5 'end primer and a 3' end primer derived from CH1 (5'-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3' SEQ ID NO: 12). The DNA sequence was confirmed to contain the entire V_HAnd the CH1 domain of human IgG 1. After digestion with an appropriate enzyme, the Fd DNA fragment was inserted into HindIII and BamHI restriction sites of the expression vector cassette pSV2dhfr-TUS to give pSV2dhfrFd (FIG. 12A).

For the kappa gene, PCR was performed using (5-AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3 ' SEQ ID NO: 13) as the 5 ' end primer and a C.kappa.derived 3 ' end primer (5'-CGGGATCCTTCTCCCTCTAACACTCT-3' SEQ ID NO: 14). The DNA sequence was shown to contain the entire V.kappa.and human C.kappa.regions. After digestion with appropriate restriction enzymes, the kappa DNA fragment was inserted into the HindIII and BamHI restriction sites of the expression vector cassette pSV2neo-TUS to give pSV2neo kappa (FIG. 126). Expression of the Fd and kappa genes can be driven by enhancers and promoters derived from HCMV. Since the Fd gene does not contain tryptophan residues involved in the interchain disulfide bond, the recombinant chimeric Fab contains a heavy and light chain that are non-covalently linked. This chimeric Fab is named cFab.

To obtain a recombinant Fab with disulfide bonds between the heavy and light chains, the Fd gene was expanded to include a sequence encoding another 9 amino acids from the hinge region of human IgG1 (EPKSCDKTH SEQID NO: 15). The BstEII-BamHI DNA fragment encoding 30 amino acids at the 3' end of the Fd gene was replaced with a DNA fragment encoding an expanded Fd. The extended Fd, which contained an additional 9 amino acids from the hinge region of human IgG1, was confirmed by DNA sequencing. The Fd/9aa gene was inserted into an expression vector cassette pSV2dhfr-TUS to obtain pSV2dhfrFd/9 aa. This chimeric Fab was designated cFab/9 aa.

(3) Expression of chimeras 166-32IgG and Fab

For obtaining secretory chimeras 166-32IgGCell line, NSO cells used pSV2neoV_HPurified plasmid DNAs of huC γ l and pSV2neoV-huC κ were transfected by electroporation. Transfected cells were selected in the presence of 0.7 mg/ml G418. Cells were cultured in 250-ml spinner flasks using serum-containing medium.

To obtain a cell line secreting chimera 166-32Fab, CHO cells were transfected with pSV2dhfrFd (or pSV2dhfrFd/9aa) and purified plasmid DNAs of pSV2neo κ by electroporation. Transfected cells were selected in the presence of G418 and methotrexate. The selected cell lines were amplified under conditions of increased methotrexate. Single cell subcloning of cells was performed by limiting dilution. The high-yielding single-cell subcloned cell lines were then cultured in 100-ml spinner culture using serum-containing medium.

(4) Purification of chimera 166-32IgG

100 ml of the spin culture supernatant was applied to ｃA 10 ml PROSEP-A column (Bioprocessing, Inc., Princeton, NJ). The column was washed with 10 bed volumes of PBS. Bound antibody was eluted with 50 mM citrate buffer, pH 3.0. An equal amount of 1M Hepes, pH8.0 was added to the purified antibody containing fraction to adjust the pH to 7.0. The residual salts were removed by buffer exchange with PBS and ultrafiltration using a Millipore membrane (M.W. cut-off: 3000). The protein concentration of the purified antibody was determined by the BCA method (Pierce).

(5) Purification of chimera 166-32Fab

The chimeric 166-32Fab can be purified by affinity chromatography using the anti-idiotypic MAb of mouse anti-MAb 166-32. This anti-idiotype MAb was designated MAb 172-25-3. It was prepared by immunizing mice with MAb166-32 conjugated to Keyhole Limpet Hemocyanin (KLH) and screening for specific antibodies that competitively bind to MAb166-32 with factor D.

The affinity chromatography matrix is prepared as follows: 25 mg of MAb 172-25-3 was mixed with 5 ml of dried azodone gel (UltraLink Biosupport Medium, Pierce) in buffer (0.1M Borate and 0.75M Na)₂SO₄pH9.0) and at room temperature for 2 hours. The remaining unreacted sites were then blocked with 1M ethanolamine (pH9.0) at room temperature for 2.5 hours. Then the colloidal particles were mixed with 10 mM Tris, 0.15M NaCl, 5 mM EDTA, 1% TritonX-100 and 0.02% NaN₃pH8.0) and stored at 4 ℃.

For purification, 100 ml of supernatant from a spun culture of cFab-or cFab/9aa CHO-producing cells was applied to an affinity column coupled to MAb 172-25-3. The column was washed thoroughly with PBS and bound Fab was eluted with 50 mmol diethylamine, ph 11.5. The residual salts were removed by buffer exchange as described above. The protein concentration of the purified Fab was determined by the BCA method (Pieree).

(6) SDS-PAGE of SDS-PAGE chimeras 166-32IgG, cFab and cFab/9aa

Purified chimeras 166-32IgG, cFab and cFab/9aa were analyzed for purity and molecular weight by SDS-PAGE. The protein was treated with a sample buffer with or without mercaptoethanol. The samples were then electrophoresed in prestained gel (12.5%) (Amersham Pharmacia Biotech, Uppsala, Sweden) using a PhastSystem (Amersham Pharmacia Biotech) together with prestained molecular weight standards (low molecular weight range) (BIO-RAD Laboratories, Hercules, Calif.). The gel was then stained in Coomassie Brilliant blue (BIO-RAD) for 5 minutes and destained in an aqueous solution containing 40% methanol and 10% acetic acid.

SDS-PAGE results of CFab, cFab/9aa and chimeric IgG under non-reducing and reducing conditions showed that chimeric IgG had a 150kD protein band and two Heavy (HC) and Light (LC) protein bands of approximately 50kD and 29kD, respectively. As expected, the cFab/9aa had only 1 protein band of approximately 40kD under non-reducing conditions, indicating that the heavy and light chains are linked by interchain disulfide bonds. On the other hand, the cFab has 2 protein bands under non-reducing conditions, indicating that the heavy and light chains are not linked by interchain disulfide bonds.

(7) Determination of the Activity of chimeras 166-32IgG, cFab and cFab/9aa

The activity of the chimeras 166-32IgG, cFab and cFab/9aa was determined as described above using the alternative complement hemolytic assay. FIG. 13 shows that murine and chimeric forms of MAb166-32 have the same potency as inhibitor D. FIG. 14 shows that the cFab and cFab/9aa have substantially the same potency for inhibiting factor D. More importantly, both forms of chimeric Fab are as potent as chimeric IgG, taking into account the two binding sites per IgG molecule. Taken together, these results demonstrate that the chimeras IgG, cFab and cFab/9aa retain the potency of the parent murine MAb 166-32.

Example 10: protection of complement-mediated tissue injury by MAb166-32 in an in vitro model of rabbit Heart perfused with human plasma

Activation of the complement system can cause hyperacute graft rejection. This may be the result of the following process: binding of antibodies capable of fixing complement, direct activation of complement at the surface of foreign cells via the alternative pathway, and/or inability of foreign organs to regulate complement (J.L. Platt et al, Transplantation, 1991; 52: 937-947). Complement activation can be via either the predominant classical or alternative pathway, depending on the particular species-to-species interaction, although sometimes both pathways can operate simultaneously (T.Takabashi et al, Immunol Res., 1997; 16: 273-297). Previous studies have shown that hyperacute rejection can occur via the alternative activation pathway in the absence of anti-donor antibodies (P.S. Johnston et al, transplantation. Proc., 1991; 23: 877-879).

To demonstrate the importance of the alternative complement pathway for tissue damage, MAb166-32 against factor D was tested using an in vitro model in which isolated rabbit hearts were perfused with diluted human plasma. Previous studies in this model have shown that rabbit myocardium may be damaged by the alternative complement activation pathway. (M.R. Gralinski et al, Immunopharmacology, 1996: 34: 79-88). (1) Rabbit hearts perfused by Langendorff:

male New Zealand white rabbits (2.2-2.4 kg) were killed by cervical spondylolysis. Rapid removal of heart, cannulation to pass throughAnd (5) arterial perfusion. Perfusion medium contained a circulating volume (250 ml) of modified Krebs-Henseleit (K-H) buffer (pH7.4, 37 ℃) administered at a constant rate of 20-25 ml/min. The composition of the buffer broth (mmol/L) was: NaCl, 117; KCl, 4.0; CaCl₂.H₂O，2.4；MgCl₂.6H₂O，1.2；NaHCO₃，25；KH₂PO₄1.1; glucose, 5.0; monosodium L-glutamate, 5.0; 2.0 parts of sodium pyruvate; and BSA, 0.25% (w/v). The K-H buffer passed through a porous "lung" consisting of Silastic Laboratory GradeTubin (Dow Corning, Midland, MI) 55.49 meters in length, 1.47 mm in inside diameter and 1.96 mm in outside diameter. Membranous "lungs" were exposed continuously to 95% O₂/5％CO₂Next, the partial pressure of oxygen in the perfusion culture was set to 500 mm Hg. Throughout the experiment, the heart was pulsed by electrodes attached to the right pulmonary artery. Pulsatile stimulation (3Hz, 4msec duration) was delivered by a laboratory square wave generator (Grass SD-5, Quincy, MA). The pulmonary artery was cannulated with polyethylene tubing to facilitate collection of pulmonary artery infusate, which represents coronary venous return. The external and internal lumens of the vena cava and the pulmonary veins are ligated to prevent the perfusate from flowing out of the severed tube. A left ventricular tube, thermistor probe and rubber ball are inserted through the left artery and positioned in the left ventricle. The fluid filled rubber balloon was hard piped to a pressure transmitter to measure the systolic and the final diastolic pressure of the left ventricle. Left ventricular levelled pressure is defined as the difference between the systolic and the final diastolic pressure of the left ventricle. The interventricular balloon was dilated with distilled water to reach the left end diastolic pressure at the starting baseline of 5 mm hg. Coronary perfusion pressure was measured with a pressure transmitter connected to the side arm of the cardiac catheter. All hemodynamics were continuously monitored using a multichannel counter (grasss Polygraph 79D, Quincy, MA). The isolated heart was maintained at 37 ℃ throughout the experiment by placing the heart in a double glass chamber that was tempered and perfused with culture medium by a heater and conveyor.

(2) Antibody treatment:

two treatment groups were used to determine the ability of anti-factor D MAb166-32 to inhibit complement activation in isolated rabbit hearts perfused with human plasma. Group 1: an isotype matched negative control consisting of hearts perfused with 4% human plasma in the presence of 0.3 μ G/ml MAb G3-519 specific for HIV-1 gp120 (n ═ 6). Group 2: the treatment group consisted of hearts perfused with 4% human plasma in the presence of 0.3 μ g/ml MAb166-32 (n ═ 6). Human plasma can be isolated from freshly collected whole blood and stored at-80 ℃ until use. This percentage of human plasma is used because it can severely disrupt myocardial function in a reasonable length of time and allows for an assessment of the effectiveness of the treatment. High concentrations of human plasma in this system can cause rapid contraction of the heart, while the effectiveness of the drug at low concentrations is difficult to analyze. Preliminary studies have determined that 0.3 μ g/ml is the lowest effective concentration at which the heart can be protected from the effects of complement activation. All hearts were equilibrated in a Langendorff apparatus for 10-15 minutes before any antibody was added to the perfusion medium. After 10 minutes of antibody addition, 4% human plasma was added to the perfusion medium (250 ml, recirculated). Hemodynamic changes included Coronary Perfusion Pressure (CPP), Left Ventricular Systolic Pressure (LVSP), Left Ventricular End Diastolic Pressure (LVEDP) and left ventricular pressure (LVDP) were recorded before antibody addition (baseline), before 4% human plasma addition, and every 10 minutes thereafter for 60 minutes.

MAb166-32 (0.3. mu.g/ml) reduced the increase in Coronary Perfusion Pressure (CPP) when exposed to 4% human plasma compared to hearts treated with MAb G3-519 (0.3. mu.g/ml). An increase in CPP shows coronary vascular resistance, which is often associated with myocardial tissue injury. Isolated rabbit hearts perfused with MAb166-32 maintained Left Ventricular End Diastolic Pressure (LVEDP), which is the inverse of the results obtained with MAb G3-519 (fig. 15). The latter group of hearts exposed to 4% human plasma increased LVEDP, indicating that the ventricles were not relaxed at diastole (fig. 15). MAb166-32 also reduced the increase in left ventricular pressure (LVDP) after exposure to diluted human plasma compared to hearts treated with MAb G3-519 (FIG. 15).

Figure 16 depicts representative recordings of cardiac function before perfusion and after 10, 30 and 60 minutes in the presence of 4% human plasma. After 10 minutes, rabbit hearts treated with MAb G3-519 had a marked progressive increase in LVEDP and decrease in LVDP, and within the next 50 minutes, ventricular function progressively deteriorated. On the other hand, hearts treated with MAb166-32 maintained ventricular function after 60 minutes.

In summary, hemodynamics data indicate that anti-factor D MAb166-32 protects isolated rabbit hearts from human complement-mediated damage, as demonstrated by the ability to maintain complete myocardial function following human plasma challenge.

(3) ELISA for complement Bb:

factor D catalyzes the cleavage of factor B in the bound state, producing Ba and Bb fragments. The concentration of Bb present can be used as an indicator of factor D activity. The concentration of the active ingredient Bb in the lymph fluid collected from isolated rabbit hearts was measured using a commercial ELISA kit (Quidel). This assay measures activation of the human complement system using mabs against human complement Bb during perfusion of rabbit hearts in the presence of human plasma. Lymphatic effusions lymph fluid may be collected from the apex of the heart, snap-frozen in liquid nitrogen, and stored at-70 ℃ until analysis. The flow rate of lymphatic outflow was recorded and indicated to normalize Bb concentrations.

After 10 min of perfusion with 4% human plasma and at each time point, the hearts treated with MAb166-32 had significantly (p < 0.05) lower concentrations of Bb in lymphatic exudates than the hearts treated with MAb G3-519 (fig. 17). The reduced production of complement activation product Bb in rabbit hearts treated with MAb166-32 demonstrates the inhibitor activity of this antibody against factor D.

(4) Immunohistochemical localization of C5b-9 deposits:

after completion of the above procedure, the heart was removed from the Langendorff apparatus, cut into transverse sections, and frozen in liquid nitrogen. The apex and artery tissue were discarded. Sections were embedded IN o.c.t. compound embedding medium (Miles, inc., Ekhart, IN), cut to 3 microns, and placed on poly-L-valine coated slides. After washing with Phosphate Buffered Saline (PBS), sections were incubated with 4% paraformaldehyde in PBS at room temperature. Heart sections were washed with PBS and incubated with 1% BSA for 15 minutes to reduce nonspecific staining. After washing with PBS, the sections were incubated with a 1: 1000 dilution of murine anti-human C5b-9MAb (Quidel) for 1 hour at room temperature. Sections were washed again with PBS and incubated with goat anti-mouse FITC conjugated antibody (Sigma, 1: 320 dilution) for 1 hour at room temperature. After final washing with PBS, sections were fixed with Fluorocount-G (Electron Microscopy Sciences, Forr Washington, Pa.) and coverslipped. Controls included sections not treated with primary antibody and sections with isotype-matched murine antibody IgG1(Sigma) in place of anti-C5 b-9 MAb.

Human MAC (or C5b-9) deposition in cardiac sections of hearts treated with MAb166-32 and MAb G3-519 was detected using immunofluorescence staining. Hearts treated with MAb166-32 showed reduced MAC deposition compared to MAb G3-519 treated hearts.

In summary, data from in vitro rabbit heart studies demonstrate the efficacy of MAb166-32 in preventing cardiac tissue damage by inhibiting the alternative complement pathway. Inhibition of complement activation has been shown to prolong graft survival (s.c. makrides, Pharmacological rev., 1998, 50: 59-87). Thus, MAb166-32 has potential for use as a therapeutic agent to protect grafts from human plasma damage.

Example 11: inhibition of complement activation and inflammatory response by MAb166-32 in an extracorporeal circulation model of cardiopulmonary bypass

Patients undergoing cardiopulmonary bypass (CPB) often exhibit a systemic inflammatory response. Clinically, these reactions can be manifested as postoperative leukocytosis, fever, and extravascular fluid accumulation, which can lead to prolonged recovery and occasionally severe organ dysfunction (J.K. Kirklin et al, J.Thorac.Cardiovasc.Surg., 1983; 86: 845-. This inflammatory response involves changes in body fluids and cells, which results in tissue damage and disruption of hemostatic function. Complement activation has been considered to be the leading cause of systemic inflammatory responses (P.Haslam et al, Anaesthesia, 1980; 25: 22-26; A.Salama et al, N.Eng.J.Med., 1980; 318: 408-414; J.Steinberg et al, J.Thorac.Cardiovasc.Surg., 1993; 106: 1008-1016). Complement activation is responsible for the surface interaction of blood with the CPB extracorporeal circulation machinery (D.Royston, J.Cardiotac.Vasc.Anest, 1997; 11: 341-354). Proinflammatory substances are produced following activation of the complement system and include anaphylatoxins C3a and C5a, opsonin C3b, and membrane attack complex C5 b-9. C5a has been shown to upregulate CD11b (integrin) and CD18 (integrin) in the MAC-1 complex in polymorphonuclear PMN (predominantly neutrophils) in vitro (M.P.Fletcher et al, am.J.physiol., 1993; 265: H1750-H1761) and induce PMN release of lysosomal enzymes. C5b-9 induced the expression of P-selectin (CD62P) on platelets (T.VViedmer et al, Blood, 1991; 78: 2880-2886), and both C5a and C5b-9 induced the expression of P-selectin on the surface of endothelial cells (K.E.Foreman et al, J.Clin.invest., 1994; 94: 1147-1155). C3a and C5a stimulate chemotaxis of human mast cells (K.Hartmann et al, Blood, 1997; 89: 2868-2870) and trigger histamine release (Y.Kubota, J.Dermatol., 1992; 19: 19-26), which induces vascular permeability (T.J.Williams, Agents Actions, 1983; 13: 451-455).

In vitro recirculation of whole blood in the extracorporeal shunt circulation system has been widely used as a model for activation of leukocytes in CPB (J.Kappelamyer et al, Circuit. Res., 1993; 72: 1075-. The effectiveness of MAb166-32 against factor D in inhibiting cell and complement activation in human whole blood was studied using this model of CPB extracorporeal circulation.

(1) Preparation of extracorporeal circulation system:

the extracorporeal circulation system is assembled using the following components: hollow fiber pediatric membrane oxygen generator with an embedded heat exchanger (D901 LILLPUT 1; didco, mirandola (mo), Italy), pediatric venous reservoir with an embedded open heart filter (D752 venous reservoir; didco), a perfusion tubing set (soda Biomedical, inc., Irvine, CA), and multi-stream rotary pump (Stockert Instruments GmbH, Munich, Germany). plasma-Lyte 148 fluid (Baxter Healthcare corp., Deerfield, IL) was injected into the oxygen generator and circulator for start-up. The starting solution was warmed to 32 ℃ by a cold-hot apparatus (Sarns; 3M Health Care, Ann Arbor, MI) and circulated at 500 ml/min, maintaining the aeration flow at 0.25 l/min with 100% oxygen. After addition of blood to the circulatory system, the ventilation flow was changed to oxygen (95%) and CO₂(5%) of mixed gas. Continuous monitoring of pH, PCO throughout the recycle period₂、PO₂And perfusate temperature. Sodium bicarbonate was added as needed to maintain the pH at 7.25-7.40.

(2) Operation and sampling of the extracorporeal circulation system:

450 ml of blood was drawn from healthy, non-dosed volunteers within 5-10 minutes into a transfer bag (Haemo-Pak; Chartermed, Inc., Lakewood, N.J.) containing porcine heparin (5 units/ml, final concentration; Elkins-Sinn, Cherry Hill, N.J.) and anti-factor D MAb166-32 or an isotype matched negative control MAb G3-519 (18. mu.g/ml; final concentration). This concentration of antibody corresponds to about 1.5 times the molar concentration of factor D in blood. Before blood was added to the extracorporeal circulation system, a blood sample was taken from the bag as a "pre-circulation" sample, named "-10 min sample". Blood is then added to the reservoir via the activated channel. While the priming fluid was pumped to the oxygen generator distal outlet, a final circulation volume of 600 ml was produced, with a final hematocrit of 25-28%. Circulating the blood and the starting solution together to complete mixing within 3 minutes; the baseline sample was withdrawn and designated time 0. To simulate the general course of a low body temperature surgery, the circulating fluid was cooled to 27 ℃ for 70 minutes and then warmed to 37 ℃ for 50 minutes (120 minutes total recirculation).

During recirculation, blood samples were also drawn at 10, 25, 40, 55, 70, 80 and 120 minutes. Plasma samples were immediately prepared by centrifugation at 2000 Xg and 4 ℃. Subpackaging for bypass route hemolysis analysis and neutrophil-specific myeloperoxidase analysis, freezing the subpackaged samples in dry ice, and storing at-80 ℃. Aliquots of samples for ELISA measurements of complement C3a, C4d, sC5b-9, and Bb were immediately mixed with an equal volume of sample stabilization medium (Quidel), frozen on dry ice, and stored at-80 ℃. Whole blood samples were also collected for immunostaining of activated cell surface markers CD11b and CD62P on neutrophils and platelets. To prevent subsequent complement activation of the whole blood sample during staining, 10 microliters of 1M EDTA was added to each ml of whole blood to a final concentration of 10 millimoles.

(3) Alternative pathway hemolysis assay:

the alternative complement activity in plasma samples at different time points was measured using rabbit red blood cells in MAb166-32 treated and MAb G3-519 treated cycles as described above. 50 microliters of each sample (20%) was added to 30 microliters of rabbit red blood cells (1.7X 10)⁸Cells/ml) was mixed with 50 μ l GVB/Mg-EGTA buffer before. After incubation at 37 ℃ for 30 minutes, the supernatant was collected. The OD at 405nm was read using an ELISA plate counter.

FIG. 18 shows that alternative complement activity in the circulatory system treated with MAb166-32 is completely inhibited by this antibody, whereas MAb G3-519 had no effect on complement activity when used in the equivalent circulatory system. These results indicate that MAb166-32 is a potent inhibitor of the alternative complement pathway. MAb166-32 completely inhibited alternative complement activity even at a molar ratio of only 1.5: 1 (MAb: factor D).

(4) Analysis of complement activation products:

in addition to the hemolysis assay described above, plasma samples from both extracorporeal circulation systems were also tested for levels of C3a, sC5b-9, Bb, and C4 d. These materials can be quantified using a commercial ELISA kit (Quidel) according to the manufacturer's instructions. Like C5a, sC5b-9 is another marker of C5 convertase activity in the complement cascade. C5a and sC5b-9 are both products of C5 cleaved by C5 convertase. Complement Bb is a marker specific for the alternative complement pathway, whereas C4d is a marker specific for the classical pathway.

FIGS. 19 and 20 show that MAbs 166-32 were effective at inhibiting C3a and sC5b-9 production, respectively, but that isotype-matched negative control MAb G3-519 was not able to inhibit their production. The specificity and potency of MAb166-32 are further illustrated in FIGS. 21 and 22. Bb produced by the alternative complement pathway is completely inhibited by MAb166-32, while Bb levels in the G3-519 cycle increase with time during the recirculation process. Interestingly, the levels of C4d in the MAb166-32 and G3-519 cycles did not change significantly over time. The latter results regarding Bb and C4d levels strongly indicate that complement activation in the extracorporeal circulation system is mainly mediated by the alternative pathway.

Taken together, these results indicate that MAb166-32 is a potent inhibitor of the alternative complement pathway. Inhibition of factor D may terminate complement activation in subsequent steps of the cascade, as may be evidenced by a reduction in the formation of C3a and sC5 b-9.

(5) Analysis of neutrophil and platelet activation:

activation of neutrophils and platelets can be quantified by measuring cell surface expression levels of CD11b and CD62P on neutrophils and platelets, respectively. For CD11b labeling of neutrophils, 100 microliters of blood collected from the circulation system was immediately incubated in a microfuge tube at room temperature for 10 minutes with 20 microliters of rhodoxanthin (PE) -anti-CD 11b antibody (clone D12, Becton Dickinson, San Jose, CA). 1.4 ml of FACS lysate (Becton Dickinson) was added for 10 minutes at room temperature to lyse the erythrocytes and fix the leukocytes. The microcentrifuge tube was centrifuged at 300 Xg for 5 minutes. The supernatant was aspirated and the cells were suspended in PBS for washing. The microcentrifuge tube was centrifuged once more, the supernatant aspirated, and the cells were suspended in 0.5 ml of 1% paraformaldehyde overnight before analysis using an EPIC-XL flow cytometer (Coulter corp. For double labeling to identify the neutrophil population at the same time, 5 microliters of Fluorescein Isothiocyanate (FITC) -anti-CD 15 antibody (clone MMA, Becton Dickinson) was added to incubate with the PE-anti-CD 11b antibody.

For CD62P labeling of platelets, 40 microliters of blood collected from the circulatory system was immediately incubated with 20 microliters of PE-anti-CD 62P antibody (clone aci.2, becton dickinson) in a microfuge tube at room temperature for 10 minutes. The mixture was treated with FACS lysate as described above. The microcentrifuge tube was centrifuged at 2000 Xg for 5 minutes. Platelets were washed with PBS, fixed with 1% paraformaldehyde, and analyzed as described above. For double labeling to identify platelet populations simultaneously, 5 microliters of FITC-anti-CD 42a antibody was added to incubate with PE-anti-CD 62P antibody.

For flow cytometry, PMNs (containing predominantly neutrophils) and platelet populations were identified using a movable gate based on antero-lateral scatter parameters and specific staining with FITC-anti-CD 15 antibody and FITC-anti-CD 42a antibody, respectively. Background staining gates were set using isotype matched labeled antibodies. The expression intensities of CD11b and CD62P are expressed as Mean Fluorescence Intensity (MFI).

FIG. 23 shows that neutrophils from MAb166-32 treated extracorporeal circulation showed significantly lower CD11b expression than neutrophils from MAb G3-519 treated circulation. These data, along with the other data described above, suggest that inhibition of activation of the alternative complement pathway by MAb166-32 prevents neutrophil activation.

Similarly, FIG. 24 shows that platelets from the extracorporeal circulation system treated with MAb166-32 exhibit significantly lower expression of CD62P than platelets from the circulation system treated with MAb G3-519. Again, these data, along with the other data above, show that inhibition of activation of the alternative complement pathway by MAb166-32 prevents platelet activation.

(6) Analysis of neutrophil-specific Myeloperoxidase (MPO)

The degree of neutrophil activation can also be measured using a commercial ELISA kit (R & DSystems, inc., Minneapolis, MN) to determine the amount of neutrophil-specific Myeloperoxidase (MPO) in plasma samples from the extracorporeal circulation system. MPO is stored predominantly in neutrophils' primordium (aniline-philic blue cells). It is released when neutrophils degranulate due to activation. MPO is therefore a soluble marker of neutrophil activation. The analysis may be performed according to the manufacturer's instructions. Briefly, samples were incubated in the wells of a microplate, which had been coated with a first MAb against MPO. The MPO-MAB complex was labeled with a biotin-conjugated polyclonal antibody prepared from goat MPO-antiserum. The final step of this assay is based on a biotin-avidin coupling in which avidin has been covalently linked to alkaline phosphatase. After addition of the substrate 4-nitrophenyl-phosphate (pNPP), the amount of MPO in each sample was determined enzymatically by reading the OD at 405.

FIG. 25 shows that MAb166-32 processes circulating systems at significantly lower MPO levels than MAbG3-519 circulating systems. These results are consistent with CD11b expression in the immunofluorescent counting study described above.

In summary, data on complement, neutrophils and platelets support the argument that effective inhibition of activation of the alternative complement pathway in the extracorporeal circulation by anti-factor D MAb166-32 prevents the formation of inflammatory substances C3a, C5a and sC5b-9 and thus reduces neutrophil and platelet activation. It is predicted that MAb166-32, and fragments, homologues, analogues and small molecule portions thereof are effective in preventing or reducing clinical inflammatory responses due to CPB.

Example 12: study of the Effect of MAb166-32 in dog models with ischemia and perfusion injury

A study was designed to examine whether MAb166-32 could protect myocardial tissue from injury in ischemia-perfused dogs, although it was recognized from the outset that dogs may not be an ideal animal model for studying the utility of MAb 166-32. In hemolytic assays, MAb166-32 was at least 10-fold less able to neutralize dog factor D than human factor D (see example 7). Because there is then only a limited amount of MAb166-32, MAb166-32 is administered to the heart via the coronary vessels. We expect that antibody concentrations of at least 60 μ g/ml can be accumulated in coronary blood to completely inhibit dog factor D in the heart. The calculated dose was 3.15 mg/kg/perfusion for a total of 6 infusions. MAb G3-519 served as an isotype-matched control.

Briefly, experimental beagle dogs were anesthetized. A thoracotomy was performed in the fourth intercostal left position to expose the heart. The proximal left circumflex coronary artery was isolated, ligated for 90 minutes to induce ischemia, and reperfused for 6 hours. Antibodies were administered 6 times 30 min before ischemia, 10 min before perfusion, and 75, 150, 225, 300 min during perfusion, respectively. Radioactive microspheres were injected at different time points to measure regional blood flow. At the end of the experiment, the heart was perfused with an evan blue dye and a triphenized four file to measure the area at risk and infarct size, respectively. Coronary lymph and blood were collected from the jugular vein before ischemia and at the end of the experiment. These samples were used to measure the concentration of injected antibody and the alternative pathway hemolytic activity.

The results show that the highest achievable concentration of MAb166-32 in coronary lymph is approximately 30 μ g/ml, which is well below the concentration required to completely inhibit dog factor D in the coronary circulation. Antibodies were also detected in systemic circulation, indicating that the injected antibodies spread out of the heart. Hemolytic assay data showed no decrease in alternative complement pathway activity; this is consistent with the fact that antibody concentrations are low. Therefore, the effect of MAb166-32 in the perfusion experiment cannot be concluded from these experiments in dogs.

The above description, terms, expressions and examples are illustrative only and not restrictive. The invention includes all equivalents of the foregoing embodiments, whether known or unknown. The present invention is limited only by the claims of the following patent and is not limited by any statement or any other source in any other part of this document.

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Bill N.C.Sun

Cecily R.Y.Sun

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Arg Asp Ser Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Gly Gly

180 185 190

gtg gcc gag gga gtg gtt acc tca ggc tcc cga gtc tgc ggc aac cgc 624

Val Ala Glu Gly Val Val Thr Ser Gly Ser Arg Val Cys Gly Asn Arg

195 200 205

aag aaa ccc ggc atc tac acg cgc ttg gcg agc tac gtg gcc tgg atc 672

Lys Lys Pro Gly Ile Tyr Thr Arg Leu Ala Ser Tyr Val Ala Trp Ile

210 215 220

gac gga gtg atg gct gac agc gca gcc gcc tagtaggaat tc 714

Asp Gly Val Met Ala Asp Ser Ala Ala Ala

225 230

<210>4

<211>233

<212>PRT

<213> pig

<400>4

Ile Leu Gly Gly Gln Glu Ala Lys Ser His Glu Arg Pro Tyr Met Ala

1 5 10 15

Ser Val Gln Val Asn Gly Lys His Val Cys Gly Gly Phe Leu Val Ser

20 25 30

Glu Gln Trp Val Leu Ser Ala Ala His Cys Leu Glu Asp Val Ala Glu

35 40 45

Gly Lys Leu Gln Val Leu Leu Gly Ala His Ser Leu Ser Gln Pro Glu

50 55 60

Pro Ser Lys Arg Leu Tyr Asp Val Leu Arg Ala Val Pro His Pro Asp

65 70 75 80

Ser Gln Pro Asp Thr Ile Asp His Asp Leu Leu Leu Leu Lys Leu Ser

85 90 95

Glu Lys Ala Glu Leu Gly Pro Ala Val Gln Pro Leu Ala Trp Gln Arg

100 105 110

Glu Asp His Glu Val Pro Ala Gly Thr Leu Cys Asp Val Ala Gly Trp

115 120 125

Gly Val Val Ser His Thr Gly Arg Arg Pro Asp Arg Leu Gln His Leu

130 135 140

Leu Leu Pro Val Leu Asp Arg Thr Thr Cys Asn Leu Arg Thr Tyr His

145 150 155 160

Asp Gly Thr Ile Thr Glu Arg Met Met Cys Ala Glu Ser Asn Arg Arg

165 170 175

Asp Ser Cys Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Gly Gly Val

180 185 190

Ala Glu Gly Val Val Thr Ser Gly Ser Arg Val Cys Gly Asn Arg Lys

195 200 205

Lys Pro Gly Ile Tyr Thr Arg Leu Ala Ser Tyr Val Ala Trp Ile Asp

210 215 220

Gly Val Met Ala Asp Ser Ala Ala Ala

225 230

<210>5

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>5

tgcggccgct gtaggtgctg tcttt 25

<210>6

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>6

ggaattcact cgttattctc gga 23

<210>7

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>7

tccgagaata acgagtg 17

<210>8

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>8

cattgaaagc tttggggtag aagttgttc 29

<210>9

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>9

cgcggccgca gctgctcaga gtgtaga 27

<210>10

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>10

cggtaagctt cactggctca gggaaata 28

<210>11

<211>37

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>11

aagaagcttg ccgccaccat ggattggctg tggaact 37

<210>12

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>12

cgggatcctc aaactttctt gtccaccttg g 31

<210>13

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>13

aagaaagctt gccgccacca tgttctcact agctct 36

<210>14

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> primer

<400>14

cgggatcctt ctccctctaa cactct 26

<210>15

<211>9

<212>PRT

<213> human

<400>15

Glu Pro Lys Ser Cys Asp Lys Thr His

1 5

Claims (33)

1. An antibody or binding fragment thereof that binds to a region between amino acid residue numbers Cys154 and Cys170 and containing Cys154 and Cys170 in human factor D and inhibits complement activation, the binding fragment being Fab, F (ab')₂Fv or single chain Fv fragments.

2. The antibody or binding fragment thereof of claim 1, which does not bind human factor D in the absence of amino acid residues Arg156, His159 and Leu 168.

3. The antibody or binding fragment thereof of claim 1, wherein the antibody is a chimeric, deimmunized, humanized, deimmunized or human antibody.

4. The antibody or binding fragment thereof according to claim 1, wherein the antibody is monoclonal antibody 166-32 produced by the hybridoma deposited with the american type culture collection with accession number HB-12476.

5. A hybridoma that produces monoclonal antibody 166-32, deposited with the American type culture Collection with accession number HB-12476.

6. A chimeric form of the antibody as defined in claim 3, which comprises the mouse variable region and the human constant region of monoclonal antibody 166-32 produced by the hybridoma deposited with the american type culture collection with accession number HB-12476.

7. A chimeric form of the antibody as defined in claim 3, which comprises the mouse variable region and the human constant region of the Fab fragment of monoclonal antibody 166-32 produced by the hybridoma deposited with the american type culture collection with accession number HB-12476.

8. A cell line that produces the antibody or binding fragment thereof of any one of claims 1-4 and 6-7.

9. A pharmaceutical composition comprising the antibody of any one of claims 1-3 and 6-7 and optionally a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 9 for use in the treatment of a disease or condition mediated by excessive or uncontrolled activation of the complement system.

11. The pharmaceutical composition of claim 9 or 10, wherein the antibody is administered in vivo or in vitro.

12. The pharmaceutical composition of any one of claims 9 or 10, wherein the antibody is administered to a human.

13. The pharmaceutical composition of claim 12, wherein the human has undergone cardiopulmonary bypass surgery.

14. The pharmaceutical composition of claim 11, wherein the medicament is for the treatment of post-cardiovascular embolization ischemia reperfusion, aneurysm, stroke, hemorrhagic shock, stroke injury, multiple organ failure, hypovolemic shock, or intestinal ischemia.

15. The pharmaceutical composition of claim 11, wherein the medicament is for treating an inflammatory disorder.

16. The pharmaceutical composition of claim 15, wherein the medicament is for the treatment of burn, endotoxemia, septic shock, adult dyspnea, cardiopulmonary bypass, hemodialysis, anaphylactic shock, severe asthma, angioedema, crohn's disease, sickle cell anemia, poststreptococcal glomerulonephritis, or pancreatitis.

17. The pharmaceutical composition of claim 11, wherein the medicament is for treating an adverse drug response.

18. The pharmaceutical composition of claim 17, wherein the adverse drug response is caused by drug allergy, IL-2 induced vascular leak or radiocontrast media allergy.

19. The pharmaceutical composition of claim 11, wherein the medicament is for treating an autoimmune abnormality.

20. The pharmaceutical composition of claim 19, wherein the autoimmune disorder is selected from the group consisting of systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, alzheimer's disease, and multiple sclerosis.

21. The pharmaceutical composition of claim 11, wherein the medicament is for treating a disease or condition associated with transplant rejection.

22. Use of an antibody of any one of claims 1-3 and 6-7 in the manufacture of a medicament for treating a disease or condition mediated by excessive or uncontrolled activation of the complement system.

23. The use of claim 22, wherein the antibody is administered in vivo or in vitro.

24. The use of claim 22 or 23, wherein the antibody is administered to a human.

25. The use of claim 24, wherein the human is undergoing cardiopulmonary bypass surgery.

26. The use of claim 23, wherein the medicament is for the treatment of post-cardiovascular embolization ischemia reperfusion, aneurysm, stroke, hemorrhagic shock, impact injury, multiple organ failure, hypovolemic shock, or intestinal ischemia.

27. The use of claim 23, wherein the medicament is for treating an inflammatory disorder.

28. The use of claim 27, wherein the medicament is for the treatment of burns, endotoxemia, septic shock, adult dyspnea, cardiopulmonary bypass, hemodialysis, anaphylactic shock, severe asthma, angioedema, crohn's disease, sickle cell anemia, poststreptococcal glomerulonephritis, or pancreatitis.

29. The use of claim 23, wherein the medicament is for treating an adverse drug response.

30. The use of claim 29, wherein the adverse drug response is caused by drug allergy, IL-2 induced vascular leak or radiocontrast media allergy.

31. The use of claim 23, wherein the medicament is for the treatment of an autoimmune abnormality.

32. The use of claim 31, wherein the autoimmune disorder is selected from the group consisting of systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, alzheimer's disease, and multiple sclerosis.

33. The use of claim 23, wherein the medicament is for treating a disease or condition associated with transplant rejection.