Modulators of complement function

Abstract

The invention relates generally to a modified human C3 protein containing a number of single amino acid changes in the and -chain of human C3 protein, designed to increase the affinity of the modified protein to factor B or Bb, to decrease the affinity of the modified protein to factor H, and to reduce the immunogenicity of the modified protein as compared to the native human C3 protein, a nucleotide sequence encoding the modified C3 protein, a plasmid or viral vector containing the nucleotide sequence for expression the modified C3 protein, and a host cell containing the plasmid or viral vector. We also present a polyethylene glycol covalently bound to the modified C3 protein for reducing immunogenicity and increasing plasma half-life of the modified C3 protein; a method for depleting complement in a patient by administering the modified C3 protein to the patient in an amount effective to deplete complement; a method of ameliorating effects caused by or disease or a method of ameliorating reperfusion injury in a patient by delivering the modified C3 protein.

Claims

1. A human C3 protein (SEQ ID NO:1) or an active fragment thereof (SEQ ID NO:1 without amino acid residues 650 through 726) modified to form a stable C3 convertase, with one or more amino acid replacements selected from the group consisting of: P1518S; S1550N; V1637D; N1642 S; and V1636S, wherein the position of the amino acid residue is based on the sequence of SEQ ID NO:1 numbering.

2. A human C3 protein (SEQ ID NO:1) or an active fragment thereof (SEQ ID NO:1 without amino acid residues 650 through 726) modified such that its affinity for complement factor H is lessened, thus increasing its half-life in vivo, wherein the modified C3 protein consists of one or more amino acid substitutions selected from the group consisting of: D733G; I734F; E738S; N739D; H897E; H898A; K1030T; T1033A; V1049T; Q1140Y; T1287R; H1291I; K1285P; and L1298G, wherein the position of the amino acid residue is based on the sequence of SEQ ID NO:1 numbering.

3. The modified C3 protein according to claim 1 with further sequence changes to decrease its affinity for complement factor H, wherein the sequence changes consist of one or more amino acid substitutions selected from the group consisting of: D733G; I734F; E738S; N739D; H897E; H898A; K1030T; T1033 A; V1049T; Q1140Y; T1287R; H1291I; K1285P; and L1298G.

4. The modified C3 protein according to claim 1 with polyethylene glycol covalently bound to the N-terminus, C-terminus, or a lysine residue on the modified protein, wherein the bound polyethylene glycol increases the half-life and stability of the modified protein in the blood when the modified C3 protein is injected parenterally, and reduces the immunogenicity of the modified protein, and wherein prior to the polyethylene glycol binding reaction, the active site of the modified protein is protected from alteration by pre-incubating with staphylococcal complement inhibitor protein (SCIN).

5. The modified C3 protein according to claim 1 with further sequence changes by adding 1 to 19 amino acids of a non-C3 signal peptide to the N-terminus of the modified protein.

6. The modified C3 protein according to claim 5, wherein the non-C3 signal peptide is a Drosophila BiP sequence or a mammalian signal peptide.

7. A composition comprising the modified C3 protein according to claim 1 or 2 and a pharmaceutically acceptable carrier.

8. A method for depleting complement in a patient by administering the modified C3 protein according to claim 1 to the patient in an amount effective to deplete complement.

9. The method according to claim 8, wherein the modified C3 protein is locally administered into an organ or subcutaneously administered into a cavity or a tissue.

10. The method according to claim 8, wherein the administration is a systemic administration, and the systemic administration is intravenous or intraperitoneal.

11. A method for avoiding or ameliorating reperfusion injury in a patient by delivering an effective amount of the modified C3 protein according to claim 1, sufficient to deplete complement and permit reperfusion in the patient.

12. The method according to claim 11, wherein the delivery step is a step of injecting the modified C3 protein into an artery, a local delivery or a systemic delivery.

13. The method according to claim 11, wherein the reperfusion is opening a blocked artery, or the reperfusion occurs in connection with transplantation of an organ.

14. The method according to claim 11, wherein the modified C3 protein has polyethylene glycol covalently bound to the N-terminus, C-terminus or a lysine residue of the modified protein, and wherein the bound polyethylene glycol increases the half-life and stability of the modified C3 protein in the blood after parenteral injection and decreases immunogenicity.

15. A method for ameliorating effects caused by a disease comprising delivering an amount of the modified C3 protein according to claim 1 effective to deplete complement, wherein the disease is selected from the group consisting of: Neuromyelitis Optica, Multiple Sclerosis, Myasthenia Gravis, Rheumatoid arthritis, wet and dry Age-Related Macular Degeneration, Hemoglobin Urinary Syndrome, Paroxysmal Nocturnal Hemoglobinemia, Inflammatory Bowel Disease, Crohn's Disease, Alzheimer's disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis, Familial Mediterranean Fever, Dengue Fever, Cardiac or any Reperfusion Injury, Gout, Dense Deposit Disease, C3 Glomerulonephritis, Neuropathic Pain, and Inflammatory Pain.

16. A composition comprising the modified C3 protein according to claim 3 and a pharmaceutically acceptable carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A describes the amino acid sequence of human ProC3 (SEQ ID NO:1).

(2) FIG. 1B is a continuation of the amino acid sequence of human ProC3 (SEQ ID NO:1).

(3) FIG. 2A describes the nucleotide sequence of human ProC3 (SEQ ID NO:2).

(4) FIG. 2B is a continuation of the nucleotide sequence of human ProC3 (SEQ ID NO:2).

(5) FIG. 2C is a continuation of the nucleotide sequence of human ProC3 (SEQ ID NO:2).

(6) FIG. 2D is a continuation of the nucleotide sequence of human ProC3 (SEQ ID NO:2).

(7) FIG. 2E is a continuation of the nucleotide sequence of human ProC3 (SEQ ID NO:2).

(8) FIG. 3 shows expression of modified C3 in Baculovirus-infected Sf9 cells. Western blot showing expression of sC3 in the Baculovirus insect expression system. Different amounts of supernatant from HighFive cells infected with recombinant Baculovirus are shown on the left three lanes. As a positive control, purified C3b was run on the three lanes on the right. C3b consists of 2 chains, the -chain and the -chain. There are three bands immune-reacting bands in the expressed protein, the -chain, the -chain, and protein that has not been processed (pro-sC3). The -chain is the -chain from which the 77 amino acid C3a has been removed. The presence of the single-chain form of C3 shows that the protease responsible for the maturation of pro-C3 to C3 is only present in the Baculovirus supernatants in very limited amounts.

DETAIL DESCRIPTIONS OF THE INVENTION

(9) Previous studies on human C3/CVF hybrid proteins have shown it is possible to prepare C3 proteins with substitutions of CVF sequences that have many of the properties of CVF, including formation of a stable convertase, reduced factor H affinity, and low immunogenicity (12). Combining these studies with the available crystal structures of C3b, CVF, C3b complexed with factor Bb, CVF in a complex with factor B, C3b bound to factor H (SCRs 1 to 4), and C3b bound to factor H (SCRs 19 and 20) (8, 13, 14) has suggested which individual amino acid substitutions may allow the formation of more stable convertases and which amino acid substitutions may reduce the affinity of C3b for factor H, thereby increasing the stability of modified C3 proteins in vivo. Additionally, computer programs online, such as the Immune Epitope Database from National Institute of Allergy and Infectious Disease (www.iedb.org/home_v3.php) are able to predict which amino acid sequences within a protein increase the immunogenicity of the protein.

Example 1: Production of Modified Human C3 Proteins

(10) There are numerous amino acid substitutions in human C3 that should produce a modified human C3 that would have a higher affinity for factor B, thus forming a more stable convertase, a lower affinity for factor H, thus increasing the stability of the modified C3 in vivo, and will decrease the immunogenicity of the modified C3 protein.

(11) It has been well documented in the complement literature that Cobra Venom Factor (CVF) forms a more physiochemically stable C3 convertase (CVF,Bb) than does human C3b (6), with a half-life of dissociation of 7 hours at 37 C., versus 1.5 minutes for the C3b-containing convertase. Crystal structures have been derived for CVF,B, and C3b,Bb, and interactions between CVF or C3 and factor B (or Bb, respectively) have been determined from the crystal structures. A comparison of the ionic and non-ionic interactions were used to identify the amino acid residues of the C3 protein that interact with factor B or Bb, in which possible amino acid substitutions would result in a more stable C3 convertase. What follows is a list of some of the mutations: P1518N, Q, H, S, T, or -hydroxy-norvaline; S1550N, D, E, Q, V, I, L, or L-Glu--hydrazide; V1637N, T, S, E, Q, D, or mono-4-fluoroglutamic Acid; N1642I, E, D, Q, T, S, or -hydroxy-norvaline; G1519I, T, S, V, L, A, N, Q, H, D, E, or L-Glu--hydrazide; A1543Q, S, T, V, L, D, E, N, I, or L-threo--hydroxyl-aspartic acid; I1544G, K, W, V, 3-fluoro-valine, L-t-butyl-glycine, L-threonine, or L-allo-threonine; E1545D, N, Q, S, T, 5,5,5-trifluoro-leucine, or -t-butyl-alanine; Q1546S, T, M, A, D, E, N, mono-4-fluoro-glutamic acid, or 4,4-difluoro-glutamic acid; T1547A, V, L, I, G, W, 3-fluoro-valine, L-t-butyl-glycine; V1555T, S, D, N, Q, E, or L-Glu--hydrazide; Q1556L, I, M, V, G, P, thiazolidine-2-carboxylic acid, thiazolidine-4-carboxylic acid, 3,4-dehydro-proline, or L-azetidine-2-carboxylic acid; V1557S, T, N, Q, D, E, R, H, K, or L-canavanine; Q1559L, R, H, K, N, Q, S-2-aminoethylcysteine, or dehydrolysine; E1633L, M, I, Y, W, F, 3-fluoro-L-tyrosine, or 3-nitro-L-tyrosine; V1636T, S, D, E, or -hydroxynorvaline; A1630Q, N, T, S, D, E, or L-Glu--hydrazide.

(12) The crystal structures of factor H (CCPs 1-4 or CCPs 19-20) complexed with C3b have been derived (13, 14). The structures were used to map the ionic and non-ionic interactions between the two proteins. These interactions suggested amino acid substitutions on C3b that may reduce the strength of the interactions between the two proteins, either with substitutions that will eliminate ionic interactions, or that change the character of amino acids involved in non-ionic interactions between the two proteins by replacing polar residues with non-polar residues, or by making substitutions where a bulky residue is replaced by a small one (e.g. a tryptophane with an alanine or glycine). What follows is a list of the mutations: D733G, A, or V; I734F, W, or Y; E738S, or T; N739D, E, L, I, or V; H897D, E, T, S, G, A, V, L, or I; H898A, G, V, D, E, I, or L; K1030T, S, D, E, M, L, or I; T1033G, A, V, L, or I; V1049T, N, Q, D, or E; Q1140W, Y, F, M, S, T, D, or E; T1287R, K, H, N, or Q; H1291F, W, Y, I, L, S, T, D, or E; K1285P, V, A, G, F, D, or E; L1298G, A, or V.

(13) Potentially immunogenic regions of human C3 were determined using available internet programs, and substitutions designed by using similar amino acid residues that are less immunogenic but similar enough to the original residue to prevent loss of function. E176D, S, T, or L; V178A, I, L, M, or G; Q182N, S, T, I, or V; W183F, Y, M, or L; K184H, R, or Y; Y1173F, M, L, or I; A1174G, or V; Q1177N, S, or T; M1178L, I, or V; R1180H, K, T, S, Q, or N; K1182H, R, N, Q, T, or S; K1194R, Q, or N; N1197Q, S, or T; W1199F, Y, M, L, or I; K1204R, H, N, or Q; Y1207L, I, M, or V; V1232A, or G; R1233H, K, N, or Q; W1234F, Y, M, or L; E1237D; Q1238N, T, or S; R1239H, K, N, or Q; Y1240F, M, L, or I; W781F, Y, M, L, or V; I783L, M, V, or A; L784I, M, or V; M788L, I, V, or A; K791A, L, N, or Q; Y5F, W, L, I, or M; I7L, V, or A; S17T, N, or D; R14H, K, N, or Q; E1210N, S, or T; Y1214F, W, I, L, M, or V; L1216I, M, or A; Q1221N, T, or S; K1223R, H, N, or Q; F1252Y, W, L, I, or V; M1253L, I, or V; F1255Y, W, L, I, or V; Q1256N, or T; Y1261W, F, M, I, or L; K115H, R, Q, or N; I117L, V, or A; Y118F, W, L, M, or I; Y513F, W, L, M, or I; Y514F, W L, M, or I; L516I, V, or A; I517L, V, or A; S520T, D, or E.

(14) Stabilization of the protein could also be increased by the attachment of polyethylene glycol on the modified C3 protein.

(15) Since the preparation of many/most of the modified C3 proteins will involve producing coding sequences with several amino acid substitutions based on the nucleotide sequence (FIG. 2, SEQ ID NO:2) encoding the native human C3 protein (FIG. 1, SEQ ID NO:1), the most efficient method to produce these proteins would involve using synthesizing the entire coding sequence of the modified protein with the desired amino acid substitutions and the desired restriction enzyme recognition sequences at the 5 and 3 ends of the coding sequence.

Example 2: Expression of Modified Human C3 Proteins

(16) Modified C3 proteins will be expressed in either Drosophila S2 cells, in Baculovirus-infected Sf9 (FIG. 3) or HighFive cells, or, for final production in a mammalian protein expression system, such as in COS7 or CHO cells. For production in Drosophila S2 cells, the Drosophila BiP signal sequence will be used so that the expressed proteins will be exported to the media. Plasmids containing DNA coding for the modified C3 proteins described above will be cloned into one of the Drosophila S2 expression plasmids (pMT/BiP-V5-HisA, . . . HisB, or . . . HisC). Use of these plasmids allows expression of the proteins with the Drosophila BiP signal sequence, thus ensuring high expression and export in S2 cells. pMT/BiP-V5-HisA, B, or C plasmids containing the coding sequence of a modified C3 protein will be transfected into Drosophila S2 cells using the calcium phosphate method of Chen and Okayama (15). S2 cells were transfected with a mixture of expression plasmid and pCoBlast, using a ratio of 19:1 (w:w). Following transfection, cells containing both plasmids were selected using blasticidin (25 g/ml). For expression, 1-liter cultures of transfected cells were grown in serum-free medium (High Five plus L-glutamine), in the absence of blasticidin. When the cells reached a density of 510.sup.6 cells/ml, production of the recombinant proteins was induced by the addition of CuSO.sub.4 to a final concentration of 25 M. Cultures were allowed to express recombinant proteins for 4-5 days. Hybrid proteins were then purified from the media by a combination of ANX, Sephacryl H-300, and CM-FF chromatography.

(17) For Baculovirus-infected insect cell production of modified C3 proteins, the coding sequences will be cloned into the Baculovirus co-transfection plasmid pBacPAK8. The C3-coding sequence containing plasmid will then be co-transfected into insect cells (Sf9 or HighFive), using a lipotransfection reagent (16). The cells will be allowed to grow for 4-5 days at 27 C. The cell supernatant will be centrifuged to remove cell debris, and approximately 0.5 ml of the P0 virus stock will be used to infect Sf9 or HighFive cells growing in log phase. Cells will be removed by centrifugation. This is the P1 virus stock. This stock will be used to infect Sf9 cells at an MOI of 0.1, to create the P2 virus stock, which needs to be filtered through 0.2 micron filters to ensure the removal of all cellular debris. This is repeated to prepare the P3 virus stock, which should have a titer of >510.sup.8 pfu/ml. This stock is used for protein expression.

(18) For protein expression in Baculovirus-infected cells, cultures in growing in log phase will be infected the P3 supernatant at an MOI of 3. The culture will be harvested at a time that had been previously determined, and the cells separated from the supernatant by centrifugation.

(19) For expression of modified C3 proteins in mammalian cells, coding sequences for modified C3 proteins will be cloned into pSecTag2A, B, or C, depending on the restriction sites at either end of the modified C3 coding sequence. Since these vectors contain the mouse IgK secretion signal, the coding sequence insert will not contain the human C3 signal sequence (17). Coding sequences will be cut with the appropriate restriction enzymes, and ligated into the appropriate pSecTag2/Hygro vector cut with the same enzymes. Following ligation, plasmids will be transformed into E. coli DH5F, and plated out on LB plates containing 100 g/ml Ampicillin. Plasmids will be isolated, and correct clones determined by restriction mapping and sequencing. Plasmids will be transfected into COST cells using the Lipofectamine transfection protocol. Cells containing the Hygromycin B resistance gene will be selected by culturing the cells in media containing varying concentrations of Hygromycin B. Expression of the modified C3 will be determined by PAGE on cell supernatants, and detection using goat anti-human C3 antibodies.

Example 3: Activity Measurements of Modified Human C3 Proteins

(20) The purified modified human C3 proteins will be subjected to a number of functional analyses as follows.

(21) Complement Depletion

(22) This assay measures the ability of a protein to deplete complement in human (or other) serum. The assay was done in two steps. In the first step, the protein of interest will be diluted to the desired concentrations in buffer, usually by serial dilution (typically from less than a nanogram/microliter up to approximately 320 ng/microliter or 3.2 g in the 10 microliters used in the assay). Then, a 1 L aliquot of the diluted protein will be mixed with 9 L undiluted serum. The mixture will be incubated at 37 C. for 3 hours, allowing the protein to exhaustively activate and thus deplete C3 and factor B in the serum. Then, to measure the amount of complement activity left, the serum will be diluted and mixed with antibody-sensitized sheep erythrocytes, which are easily lysed by complement when it is present in serum. This reaction will be allowed to proceed for 30 minutes, and will be stopped by diluting the mixture in cold buffer. The cells will be centrifuged and the lysed cells quantified by measuring the hemoglobin released (12).

(23) Factor B Activation Assay

(24) This is an assay to measure the ability of a modified protein to activate factor B and form a C3/C5 convertase. The convertase formation will be measured as a function of the cleavage of factor B into Bb and Ba. In the assay, purified modified C3 proteins will be incubated with a three-fold molar excess of factor B and catalytic amounts of factor D (all highly purified) in the presence of magnesium at 37 C. At various times, aliquots of the reaction will be withdrawn, and the reaction stopped by adding EDTA, which chelates the magnesium. The reaction products will be run on a non-reducing SDS-polyacrylamide gel, which will be stained for proteins with Coomassie Blue. The amount of Factor B converted will be quantified by scanning the gel into a specialized computer program and measuring the amount of protein in the factor B and Bb bands. The results of this assay are dependent on both the rate of factor B activation and the stability of the resulting convertase. Since there is an excess of factor B in the reaction, a very rapid production of Ba and Bb would indicate an unstable convertase (12).

(25) C3 Convertase Activity Assay

(26) This assay measures the activity of C3/C5 convertases containing modified proteins to activate human C3, by cleaving off the C3a peptide. To perform this assay, convertases will be formed as described above, and the reaction stopped by the addition of EDTA. The convertase will then be mixed with human C3, and the reaction incubated at 37 C. At the indicated times, aliquots will be removed, and the reaction stopped by mixing with gel loading buffer containing SDS and -mercaptoethanol. The SDS denatures the proteins, and the -mercaptoethanol reduces the disulfide bonds between cysteines in the proteins. After electrophoresis under reducing conditions, the gel will be stained with Coomassie Blue dye, and the relative amounts of the C3a-chain and the C3a-chain will be quantified as described above. Care is taken to use the same amount of convertase in each reaction. The results of this assay are dependent both on the activity of the modified C3-containing convertase and its stability, as an active but unstable convertase will rapidly cleave C3, but will stop as the convertase dissociates (12).

(27) Assay for Cleavage of Modified C3 Proteins by Factors H and I

(28) Modified C3 proteins will incubated with purified human Factor H and Factor I at 37 C. for several hours. The reactions are analyzed by subsequent 7% (w/v) SDS polyacrylamide gel electrophoresis under reducing conditions. Factor H binding and I activity is determined by the reduction in the strength of the 105 kDa -chain band, and appearance of bands with a molecular weight of 37 and 40 kDa (12).

(29) Assays for Immunogenicity.

(30) Various methods can be used to analyze immunogenicity, including but not limited to, skin tests, testing the modified C3 protein in transgenic animals which have been genetically engineered to have human immune systems, in vitro methods, including RIA tests using serum generated in such transgenic animals, radioimmunoprecipitation assays, ELISA assays, electrochemiluminescence, and Surface Plasmon Resonance. In addition, mouse, rat or guinea pig analogs of some proteins can be constructed, using either mouse, rat or guinea pig C3 sequences. These can be injected into the appropriate animal, and serum is collected and analyzed for the production of antibodies against the modified proteins.

(31) This method measures the stability of the modified C3 protein in plasma in different ways. However, it is to be understood that one or all of the methods can be used as well as any other methods known to one of skill in the art.

(32) The first method measures the stability in serum in vitro. Rabbit serum is isolated and separated from whole blood. Aliquots of different concentrations of the modified C3 proteins that have been biotinylated are added to the serum and allowed to incubate. Aliquots of the serum are removed at various time intervals, and the amount of modified C3 that persists is identified in an ELISA assay using a monoclonal antibody which is specific to human C3. This is one example of animal serum that can be used. The choice of serum will depend on the cross-reactivity of the human C3 antibodies with C3 of that species.

(33) A second method allows the determination of modified C3 in any animal. Modified C3 proteins are biotinylated, and injected into an animal. At times, blood is withdrawn from the animal, and serum separated from the blood. The amount of biotinylated protein can be measured by ELISA, using biotin antibodies (or streptavidin), and the activity of the modified protein measured by measuring the amount of C3 remaining in the serum, using the second part of the complement depletion assay to determine remaining complement activity.

(34) All illustrations of the drawings are for the purpose of describing selected versions of the present invention and are not intended to limit the scope of the present invention.

(35) Although the invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention as hereinafter claimed.