| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | October 31, 2000 |
| PATENT TITLE |
Use of chimeric vaccinia virus complement control proteins to inhibit complement |
| PATENT ABSTRACT |
Disclosed are chimeric proteins that are useful for inhibiting complement. The chimeric protein termed VCPFc is a fusion protein in which (i) an immunoglobulin Fc region is fused to (ii) a polypeptide that comprises a portion of a vaccinia virus complement control protein which binds complement components C4b and C3b, but not iC3b rosettes. This protein can be used in xenograft transplantation methods (e.g., by treating the donor mammal or organ) and in methods for treating complement-mediated disorders (e.g., inflammation) generally. In a second chimeric protein, a transmembrane anchoring domain is fused to a polypeptide that comprises a portion of a vaccinia virus complement control protein which binds complement components C4b and C3b, but not iC3b rosettes. The transmembrane anchoring domain can be, for example, short consensus regions 3 through 15 of human complement receptor 2 protein. Expression of the transmembrane-anchored fusion protein in a transgenic animal provides a well-suited organ donor for xenograft transplantation. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | November 30, 1998 |
| PATENT REFERENCES CITED |
McKenzie et al. "Regulation of Complement Activity by Vaccinia Virus Complement-Control Protein", The Journal of Infectious Diseases, 1992, vol. 166, pp. 1245-1250. Kroushus et al., "Complement Inhibition iwth an Anti-C5 Monoclonal Antibody Prevents Acute Cardiac Tissue Injury in an Ex Vivo Model of Pig-To-Human Xenotransplantation", Transplantation. Dec. 15, 1995, vol. 60, pp. 1194-1202. Hackl-Ostreicher et al., "Functional Activity of the Membrane-Associated Complement Inhibitor CD59 in a Pig-To-Human in vitro Model for Hyperacute Xenograft Rejection", Clin. Exp. Immunol., 1995, vol. 102, pp. 589-595. White et al., "Production of Pigs Transgenic for Human DAF to Overcome Complement-Mediated Hyperacute Xenograft Rejection in Man", Res. Immunol., Feb. 1996, vol. 147, pp. 88-94. Miller et al., Virology, 299: 126-133. 1997. |
| PATENT GOVERNMENT INTERESTS |
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made, at least in part, with funds from the Federal Government awarded through the National Institutes of Health (Grant HLB31331). |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A fusion protein that comprises (i) an immunoglobulin Fc region fused to (ii) a polypeptide that comprises a portion of a vaccinia virus complement control protein that binds complement components C4b and C3b. 2. The fusion protein of claim 1, wherein the polypeptide comprises short consensus regions 1 through 4 of vaccinia virus complement control protein. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention relates generically to the field of transplantation and rejection and specifically to a method for preventing rejection by transplant recipients and in improving the function of donor organs and tissues by inhibition of complement. BACKGROUND OF THE INVENTION Although the immune response is often perceived as beneficial, in certain circumstances the immune response to an antigen can actually be harmful to the animal in which the immune response occurs. Examples of situations where the immune response creates conditions where the animal is subject to serious pathologic sequelae are in such areas as graft versus host (GVH) rejection and host versus graft (HVG) rejection, and certain autoimmune diseases, such as lupus erythematosus, insulin-dependent diabetes mellitus, multiple sclerosis, myasthenia gravis, and rheumatoid arthritis. The utilization of organs from nonhuman donors is an appealing solution to the increasing shortage of organs available for clinical transplantation. Although xenotransplantation from primate donors has been performed with limited success clinically, the use of distantly related species, such as the pig, avoids ethical dilemmas, potential virus transmission, and limited availability associated with the use of primates as xenograft (Xg) donors. However, the use of organs from distantly related species for xenotransplantation has been hampered by the occurrence of hyperacute rejection (HAR), a process that leads to irreversible Xg damage and loss within minutes to hours of transplantation. HAR is thought to be mediated by the binding of naturally occurring xenoreactive antibodies to the endothelium of the Xg, in particular, donor vascular endothelial cells, with subsequent activation of the classical pathway of complement (C). It has been shown that a predominate specificity of these antibodies is to the oligosaccharide moiety galactose(.alpha.1-3)galactose for primate recipients. Alternative C pathway activation also contributes to HAR in some species combinations. The complement cascade is activated following the binding of xenoreactive antibodies to donor tissue. This cascade leads to endothelial activation, thrombosis, intravascular coagulation, edema, and eventually loss of function of the transplanted organ. However, if xenoreactive natural antibodies are eliminated, the presence of complements is still adequate to mediate a rejection event, presumably via the alternative pathway. Complement-mediated cell lysis also plays a role in allograft rejection, and has therefore presented a hurdle in methods of allograft transplantation. Thus, complement-mediated tissue deterioration can cause dysfunction of donor organs and tissues both from human and non-human sources. In addition, complement activation causes the deterioration of blood products, such as platelets. Thus, the length of time that blood can be stored (e.g., for transfusions) is diminished by the activity of complement. Humans and microorganisms express complement inhibitors (CIs), which serve to inhibit complement-mediated attacks. CIs contain short consensus repeats (SCRs), which are 60-70 amino acid-long regions. The number of SCRs varies among CIs. For example, the human CI Complement Receptor 1 (CR1) has 30 SCRs, while the human CI Decay Accelerating Factor (DAF) has 4 SCRs. The binding specificity of the various CIs for the various complement factors also varies. For example, Complement Receptor 1 (CR1) in humans binds C3b, C3bi, and C4b, and functions via two mechanisms: Factor I cofactor activity and convertase decay acceleration. Another human CI, Decay Accelerating Factor (DAF) binds C3b and C4b, but only has convertase decay accelerating activity. Membrane Cofactor Protein (MCP), a different human CI binds C3b and C4b,but only has Factor I cofactor activity. SUMMARY OF THE INVENTION The invention provides a fusion protein that can be used to inhibit graft rejection and complement-mediated disorders generally. In particular, the invention provides a fusion protein in which (i) an immunoglobulin Fc region (e.g., IgG Fc) is fused to (ii) a polypeptide that includes a portion of a vaccinia virus complement control protein (VCP) that binds complement components C4b and C3b, but not iC3b rosettes. A preferred fusion protein includes short consensus regions 1 through 4 of VCP as the polypeptide that is fused to an immunoglobulin Fc region. The Fc portion of VCPFc confers stability to the fusion protein in vivo, thereby increasing the circulating half-life of the fusion protein, relative to that of VCP alone. The fusion protein of the invention, termed VCPFc, can be used in a method of allograft or xenograft transplantation, involving administering VCPFc to a donor mammal (e.g., a pig or human) to attain therapeutic levels of VCPFc by the time an organ is removed from the donor for transplantation. If desired, upon removing the organ prior to transplantation, the donor organ (e.g., a heart) can be treated (e.g., flushed) with VCPFc in a pharmaceutically acceptable carrier. The xenograft transplantation method can also include administering an immunosuppression agent to the xenograft recipient substantially contemporaneously with the transplant, for example. The invention also includes a method for inhibiting a complement-mediated disorder in a mammal generally. The method involves administering to the mammal an inhibition effective amount of a fusion protein that includes an immunoglobulin Fc region fused to a polypeptide that includes a portion of a vaccinia virus complement control protein that binds complement components C4b and C3b, but not iC3b rosettes. The VCPFc fusion protein also can be used in a method for inhibiting complement-mediated deterioration of a blood product by adding an inhibition-effective amount of VCPFc to a blood collection or storage unit containing a blood product (e.g., whole blood or a solution containing any component(s) of blood), thereby inhibiting complement-mediated deterioration of the blood product. In particular, VCPFc is useful for inhibiting complement-mediated deterioration of a blood product that includes platelets. In another aspect, the invention provides a transgenic mammal (e.g., a pig), the genome of which includes a gene that encodes a fusion protein that inhibits rejection of an organ transplanted from the mammal to a recipient. Specifically, the fusion protein includes (i) a transmembrane anchoring domain fused to (ii) a polypeptide that includes a portion of a vaccinia virus complement control protein that binds complement components C4 b and C3b, but not iC3b rosettes. A preferred fusion protein includes (i) a polypeptide that includes the short consensus regions 3 through 15 of human complement receptor 2 protein, fused to (ii) a polypeptide that includes short consensus regions 1 through 4 of vaccinia virus complement control protein. Short consensus regions 3 through 15 of the human complement receptor 2 protein render this fusion protein membrane-bound, thus reducing concerns about clearance of the fusion protein from the circulation. Alternative methods that can be adapted to bind VCP to a cell membrane are described in U.S. Pat. No. 5,109,113, which is incorporated herein by reference. An organ(s) of this transgenic mammal can be transplanted to a recipient (e.g., a human) in a method of xenograft transplantation. Expression of the fusion protein in an organ derived from the transgenic mammal inhibits activation of complement and thereby inhibits rejection of the xenograft. If desired, an immunosuppression agent can be administered to the recipient substantially contemporaneously with the transplant. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a map of the expression construct pRelVCP1234. The immediate early CMV promoter of CDM8 drives transcription of the insert that encodes a VCP-CR2 chimeric receptor in which SCR-1 and SCR-2 of wild type human CR2 have been replaced with SCR-1 through -4 of wild type VCP. The unique PspAI and XhoI sites were also used to insert cassettes in which one or two SCR of VCP were deleted. FIGS. 2A-2C provide a schematic representation, recombinant protein expression levels, and C3b dimer binding-capacity of K562.wild type, K562.CR1, and six K562.VCP deletion mutants. FIG. 2A is a schematic representation of the relevant SCRs in various transfected cell lines. K562.CR1 expresses human CR1 with 30 SCRS. K562.RelVCP1234 encodes the four SCRs of VCP in place of SCR-1 and -2 of CR2. The remaining five deletion mutants have the relevant SCRs of VCP depicted in white in place of SCR-1 and -2 of CR2. FIG. 2B shows the results of flow cytometric analysis of K562 cells stably expressing CR1, VCP or one of five deletion mutants that have been indirectly stained with control antibody (thin solid line) or YZ1 anti-CR1 (thick solid line) or HB5 anti-SCR-3 or 4 of CR2 (thick solid line) followed by fluorescein-conjugated goat anti-mouse Ig. FIG. 2C shows the results of flow cytometric analysis of the C3b binding-capacity of K562 cells stably expressing CR1, VCP, or one of five deletion mutants. No ligand (thin solid line) or C3b-dimers (thin solid line) were stained using a fluorescein-conjugated goat anti-human C3 Ig. FIG. 3A-3C represents a flow cytometric analysis of mouse L cells expressing recombinant proteins. L cells.wild type (FIG. 3A), L cells.RelVCP1234 (FIG. 3B) and L cells.CR1 (FIG. 3C) indirectly stained with control anti-body (thin solid line) or either HB5 anti-SCR-3 and 4 of CR2 (thick solid line) for wild type and L cell.RelVCP1234 or YZ1 anti-CR1 for wild type and L cell.CR1 (thick solid line) followed by fluorescein-conjugated goat anti-mouse Ig. FIGS. 4A-4D represent function sorting of L cells.RelVCP1234 using flow cytometric analysis of HB5 expression levels after repeated exposures to heat inactivated or normal rabbit complement. FIGS. 4A and 4B show the levels of expression after 3 (FIG. 4A) or 7 (FIG. 4B) exposures to heat inactivated rabbit complement. FIGS. 4C-4D shows the level of expression after 3 (FIG. 4C) or 7 (FIG. 4C) exposures to normal rabbit complement. FIG. 5 illustrates the percent of L cells transfectants killed following exposure to different dilutions of heat inactivated or normal rabbit complement. L cells.wild type, RelVCP1234, and CR1 were exposed to different concentration of complement or heat inactivated complement. The percent of cells killed were assessed using flow cytometric analysis for propidium iodide inclusion. This graph translated to CH.sub.50 of 30, 19, and 11 for L cells.wild type, L cells.CR1 and L cells.RelVCP1234, respectively, when using the equation log(y/1-y) versus serum dilution. y=(sample lysis-control lysis)/(100% lysis-control lysis). Control lysis=lysis at the same dilution of heat inactivated serum. Maximum killing=100%. FIGS. 6A-6B illustrate the percent killing of L cells.RelVCP1234 treated with different dilutions of normal rabbit complement and ratio of live cells expressing HB5 versus those live cells not expressing HB5. L cells.RelVCP1234 were exposed to different concentrations of complement. The percent of cells killed (FIG. 6A) and staining positive for HB5 (FIG. 6B) was assessed using flow cytometric analysis with double staining of the L cells.RelVCP 1234 for both propidium iodide and HB5 expression. FIG. 7A is a map of the pcVCPFc plasmid encoding the VCPFc fusion protein. The sequences of SCR-1 through 4 of VCP and mouse Fc were combined and inserted into pcDNA3 expression vector. FIG. 7B is a schematic diagram of the 4 SCRs of VCP attached to the hinge region of mouse Fc (IgG2a). FIG. 7C is an SDS-polyacrylamide reducing gel of purified VCPFc demonstrates the correct molecular size. FIGS. 8A-8E provide a function comparison of CR1 and VCP. EA (FIG. 8A) and EA bearing C3b (FIG. 8B) or C3bi (FIG. 8C) were characterized for the capacity to form rosettes with wild type L cells and with L cells expressing CR1 or VCP-CR2 (FIG. 8D). Characterization of erythrocyte intermediates (FIGS. 8A-8C) was performed by indirect immunofluorescence and flow cytometric analysis using monoclonal antibodies anti-C3d, anti-C3bi, and an isotype matched control. Anti-C3d recognizes both C3b and C3bi. Anti-C3bi is specific for a neoepitope on the C3bi fragment. Rosette formation was determined by coincubation of erythrocyte intermediates with wild type L cells, L cells bearing CR1, or L cells bearing VCP-CR2 for 45 minutes. At least 200 L cells were counted in each sample. A rosette was defined as an L cell bearing at least three erythrocytes. L cells bearing CR1 form rosettes with EAC3b and EAC3bi, whereas L cells bearing VCP-CR2 form rosettes only with EAC3b (FIG. 8D). VCPFc and soluble CR1 (100 .mu.g/ml) were compared for the capacity to block rosettes formed by L cells bearing VCP-CR2 and EAC3b (FIG. 8E). CTLA4Ig and CD44Fc served as negative controls. DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention involves application of a recombinant protein termed "VCPFc" in pretreating a donor and, if desired, a donor organ ex vivo before transplantation in order to prevent rejection of an allograft or xenograft (i.e., the transplanted organ(s) or tissue(s)). Although not bound by any particular mechanism, such graft rejection reactions are thought to be due to complement activation resulting from antibody binding and/or complement dysregulation. Immediately prior to transplantation, VCPFc is administered to a donor to attain therapeutic levels of VCPFc in the donor (and specifically in the donor organ) by the time the organ is removed from the donor for transplantation. Following removal of the organ, and prior to transplantation, the donor organ can be treated (e.g., flushed) with VCPFc in a pharmaceutically acceptable excipient. Typically, the organ recipient is treated prior and/or subsequent to transplantation with an immunosuppressive agent(s) that inhibits the host's antibody and cellular immune responses to the donor organ. For example, the invention includes a method for treating pig heart donors for transplantation of the hearts to human recipients. An illustrative treatment protocol follows: beginning one week prior to the transplantation, cyclophosphamide is administered to the recipient in order to reduce the potential for evoking an antibody response to the transplant. An immunosuppressive dose of cyclosporine or FK506 may be started shortly (e.g., 1-3 days) before transplantation to enhance graft acceptance. Immediately prior to transplantation, the donor is dosed with VCPFc to attain therapeutic levels by the time of donor organ removal. Upon removal prior to transplantation, the donor organ is flushed with a solution containing VCPFc. Following transplantation by standard surgical techniques, the patient is maintained on routine immunosuppression using cyclosporine or FK506, cyclophosphamide and steroids plus VCPFc. Based on clinical signs and symptoms related to immune responsiveness, various of the immunosuppressants are reduced in dosage. The immunosuppressive agent used according to the method of the invention is an agent such as Cyclosporine A (CsA), however other agents that cause immune suppression, such as rapamycin, desoxyspergualine, and FK506 or functional equivalents of these compounds, can also be utilized. CsA is preferably administered by injection at an immunosuppressive dose. The duration of CsA treatment may range from about 2 to about 20 days. In a second aspect, the invention provides a method for inhibiting a complement-mediated disorder in a mammal, i.e., any condition in which complement activity is undesirably high. Examples of complement-mediated disorders include, but are not limited to, inflammation (including neurological inflammation), spinal cord injuries, arthritis, ischemia-induced reperfusion injuries, glomerulonephritis, encephalomyelitis, and burns. An inhibition effective amount of VCPFc is an amount that inhibits at least 20%, preferably 50%, and most preferably 90% of complement activity. If desired, an inhibition effective amount of VCPFc can be identified as an amount that ameliorates a sign(s) or symptom(s) of a complement-mediated disorder. Although a preferred embodiment of the invention involves transplantation of a pig heart into a human, it is understood that any organ can be transplanted. For example, other transplantable organs include cornea and kidney. Further, while the pig is the preferred donor, other donors may also be used. The human is the preferred recipient. As used herein, "substantially contemporaneously" refers to the time at which the immunosuppressant is administered to the recipient in relation to the time at which the organ is transplanted. For example, a heart transplant recipient may receive CsA for a short time prior to and immediately following the transplant for about 10-16 days, preferably about 14 days. In general, where transplant grafts are involved, the immunosuppressive agent can be administered from about 1 day to about 90 days before the transplant and until about 7 days to about 90 days after the transplant. Preferably, the immunosuppressive agent is administered from about 7 days to about 28 days before until about 7 days to about 28 days after. If desired, complement activity in the donor can be measured in the donor prior to transplantation of the organ. A "pre-assay", pre-existing, complement activity is determined by CH.sub.50, a standard procedure known to those of skill in the art (see for Example, Manual Clinical Immunology). Preferably, the fusion protein is expressed at a level sufficient to reduce complement activity to about 10% of the normal complement activity level in the donor. The invention also provides a transgenic mammal that is useful as a donor in organ transplantation methods. The transgenic mammal of the invention, or an ancestor of the mammal, is genetically engineered to encode a fusion protein that includes (i) a membrane anchoring domain (e.g., the short consensus regions 3 through 15 of human complement receptor 2 protein) fused to (ii) the short consensus regions 1 through 4 of vaccinia virus complement control protein. Conventional methods for producing transgenic mammals can be used to express a gene encoding the fusion protein as a transgene in a mammal (see, e.g., Elizabeth Hogan et al., Manipulation of the Mouse Embryo, 1994, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). The promoter of the construct could be the same as in the PiCR2 plasmid, i.e., CMV, or a different one. In all of the various aspects of the invention, the recipient can be treated prior or subsequent to transplantation with an immunosuppressive agent(s) that inhibits the host antibody and cellular immune responses to the donor organ. |
| PATENT EXAMPLES | available on request |
| PATENT PHOTOCOPY | available on request |
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