The present invention relates to the discovery of methods and compositions for the inhibition of cell growth and migration. More specifically, B19 parvovirus capsids or fragments of B19 parvovirus capsid proteins are incorporated into medicaments that can be administered to a subject to inhibit the growth and/or migration of cells that have a receptor that interacts with a parvovirus B19 capsid or fragment thereof (e.g., a P antigen containing cell), including, but not limited to, cells of hematopoietic origin and endothelial cells.
The B19 parvovirus is a human pathogen that can be associated with various clinical conditions, ranging from mild symptoms (erythema infectiosum) to more serious diseases in persons who are immunocompromised or suffer from hemolytic anemias. Hydrops fetalis and intrauterine fetal death are well-known complications of B19 parvovirus infection during pregnancy. (Anderson and Young, Monographs in Virology, 20 (1997)). The B19 parvovirus particles have icosohedral symmetry, a diameter of 18 to 26 nm, and are composed of 60 capsid proteins, approximately 95% of which are major capsid proteins (VP2) that have a molecular weight of 58 kd. (Fields et al., Virology vol. 2, 3rd edition, Lipponcott-Raven Publishers, Philidelphia, Pa., p. 2202 (1996)). Approximately, 3-5% of the capsid proteins that compose a B19 parvovirus capsid are called minor capsid proteins (VP1), which have a molecular weight of 83 kd, and differ from VP2 by an additional 227 amino acids at the amino terminus. (Id.).
The B19 parvovirus is extraordinarily tropic for human erythroid cells and cultures of bone marrow. B19 parvovirus binds to human erythroid progenitor cells, for example, and inhibits hematopoietic colony formation by replicating in these cells. (Brown et al., Science, 262:114 (1993) and Mortimer et al., Nature, 302:426 (1983)). The suppression of hematopoietic cells has also been seen in bone marrow samples from infected individuals, resulting in transient anemia and, in rare case, transient pancytopenia. (Saunders et al., Br J Haematol, 63:407 (1986)). Further, B19 parvovirus is known to cause bone marrow suppression in natural and experimental human infections. (Anderson and Young, Monographs in Virology, 20 (1997)).
The cellular receptor for B19 parvovirus has been identified as globoside or erythrocyte P antigen, a textrahexoceramide. (Fields et al., Virology vol. 2, 3rd edition, Lipponcott-Raven Publishers, Philidelphia, Pa., p. 2204 (1996)). The P antigen is found on mature erythrocytes, erythroid progenitors, megakaryocytes, endothelium, kidney cortex, placenta, fetal myocardium (von dem Borne et al., Br J Hematol, 63:35 (1986)) and pronormoblasts from fetal liver. (Morey and Flemming, Br J Haematol, 82:302 (1992)). Individuals who genetically lack the P antigen are not susceptible to B19 parvovirus infection and administration of either excess P antigen or monoclonal antibodies directed to the P antigen can protect erythroid progenitors from infection with B19 parvovirus. (Id.).
Additionally, neutralizing antibodies that recognize several regions of the B19 parvovirus particle have been generated. For example, monoclonal antibodies directed to epitopes of VP2, such as found at amino acids 38-87, 253-272, 309-330, 328-344, 359-382, 449-468, and 491-515, and the unique region of VP1 can neutralize B19 parvovirus. (Fields et al., Virology vol. 2, 3rd edition, Lipponcott-Raven Publishers, Philidelphia, Pa., p. 2207 (1996)).
Genetically engineered expression systems for the production of B19 parvovirus antigens have also been developed. (Kajigaya et al., Proc Natl Acad Sci USA, 86:7601 (1989); Kajigaya et al., Proc Natl Acad Sci USA, 88:4646 (1991); Brown et al., J Virol, 65:2702 (1991)). Like the native particles, recombinant B19 parvovirus capsids, produced in a baculovirus system, are composed of both VP1 and VP2 and these capsid proteins self assemble to form virus-like particles (VLPs). (Kajigaya et al., Proc Natl Acad Sci USA, 88:4646 (1991)). Electron microscopic analyses of the B19 parvovirus capsids revealed that the VLPs are structurally similar to plasma-derived virions. (Kajigaya et al., Proc Natl Acad Sci USA, 88:4646 (1991)). B19 parvovirus VLPs are currently being evaluated as a potential vaccine against B19 parvovirus infection and preliminary results show a good neutralizing response without severe side effects. (Bostic et al, J. Infect. Dis., 179:619 (1999). While many are trying to prevent B19 parvovirus infection by administering B19 parvovirus capsids, none have sought to exploit the properties of the B19 parvovirus capsid, B19 parvovirus capsid proteins, or fragments thereof to develop novel medicaments that inhibit cell proliferation or migration.
It has been discovered that the B19 parvovirus capsid, B19 parvovirus capsid proteins, or fragments thereof inhibit the growth and/or migration of cells that have a receptor that interacts with a parvovirus B19 capsid or fragment thereof (e.g., a P antigen containing cell). The data presented herein demonstrate that the B19 parvovirus capsid, B19 parvovirus capsid proteins, and fragments thereof, inhibit hematopoiesis and endothelial cell growth and migration. Accordingly, the compositions described herein can be used to reduce the production of red blood cells, white blood cells, and blood platelets, as well as, inhibit the growth and migration of other cells having a receptor that interacts with a parvovirus B19 capsid or fragment thereof (e.g., a P antigen containing cell), such as endothelial cells. Because many diseases or maladies are associated with an overproduction of cells of hematopoietic origin and/or invasive growth or migration of cells of endothelial origin, the compositions and methods described herein have significant therapeutic and prophylactic utility.
Embodiments of the invention include medicaments comprising, consisting essentially of, or consisting of B19 parvovirus capsid, B19 parvovirus capsid proteins, or fragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5), which can be administered to subjects in need of an agent that inhibits cell growth and/or migration. The B19 parvovirus capsid proteins and fragments thereof, preferably VP2 capsid proteins and fragments thereof, can be prepared synthetically, using peptide chemistry or genetic engineering, or can be made by cleaving B19 parvovirus capsids, desirably VP2 capsids, with various proteases, for example an endoprotease, which cleaves at a lysine or arginine residue.
The methods of the invention include approaches to manufacture medicaments that can be used to inhibit the growth or migration of cells that have a receptor that interacts with a parvovirus B19 capsid or fragment thereof (e.g., a P antigen containing cell). By one approach, for example, an active ingredient consisting of, consisting essentially of, or comprising empty, noninfectious, recombinant B19 parvovirus capsids, B19 parvovirus capsid proteins, or fragments of B19 parvovirus capsid proteins, preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5), are incorporated into a medicament with or without a pharmaceutically acceptable carrier or support. The amount of active ingredient can be varied depending on the potency of the inhibition needed and the length of treatment period desired from a single dose.
The medicaments described herein are preferably used to inhibit hematopoietic cell growth, endothelial cell growth, or endothelial cell migration. The medicaments described herein are used, for example, to treat hematological proliferative disorders, angiogenesis, tumorigenesis, or endothelial cell ingrowth into an implanted prosthetic device. Further, the medicaments described herein can be provided to a subject, including a fetus, prior to stem cell transplantation. Accordingly, methods of prevention and/or treatment of diseases or conditions associated with hematopoietic or endothelial cell proliferation or migration are aspects of the invention and many of the embodied methods are characterized in that they involve providing a subject at risk of having such a disease or a subject that is afflicted with such a disease with a therapeutically effective amount of a B19 parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2 capsid proteins, or fragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5).
In other embodiments, methods to prevent or treat Polycytemia Vera are provided, wherein a subject at risk for Polycytemia Vera or a subject afflicted with Polycytemia Vera is identified and said subject is provided a therapeutically effective amount of a capsid agent selected from the group consisting of B19 parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2 capsid proteins, and fragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5). The capsid agents, described above, can be provided to said subject neat or as part of a medicament containing other materials including, but not limited to, pharmaceutically acceptable supports or carriers. In a similar fashion, other hematopoietic proliferative disorders can be treated or prevented. That is, a subject in need of a capsid agent that inhibits a hematopoietic proliferative disorder is identified and said subject is provided an effective amount of capsid agent selected from the group consisting of B19 parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2 capsid proteins, and fragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5). In some embodiments, the progress or effectiveness of treatment is monitored or measured (e.g., analysis of red blood cell hematocrit).
A method of treating a subject prior to stem cell transplantation is also provided, wherein a subject in need of stem cell transplantation is identified and said subject is provided an effective amount of capsid agent selected from the group consisting of B19 parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2 capsid proteins, and fragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5).
In more embodiments, methods of inhibiting the growth or migration of a cell having a receptor that interacts with a parvovirus B19 capsid or fragment thereof (e.g., a P antigen containing cell) are provided. By one approach, for example, a cell is contacted with a capsid agent selected from the group consisting of B19 parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2 capsid proteins, and fragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5), and the inhibition of cell growth or cell migration is monitored or measured. In some embodiments, the cell is of hematopoietic origin or an endothelial cell.
Methods of inhibiting tissue ingrowth into an implanted prosthesis are also provided. These approaches involve identifying a subject in need of a capsid agent that inhibits tissue ingrowth into an implanted prosthesis and providing said subject an effective amount of capsid agent selected from the group consisting of B19 parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2 capsid proteins, or fragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5). Approaches to manufacture ingrowth inhibiting implantable prosthesis are also provided, wherein a capsid agent selected from the group consisting of a B19 parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2 capsid proteins, and fragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5) is joined to said implantable prosthesis. Embodiments also include implantable prosthesis, e.g., stents, valves, artificial joints, having said capsid agents.
More embodiments involve methods of treating or preventing tumorigenesis, wherein a subject in need of a capsid agent that inhibits hematopoietic and/or endothelial cell growth is identified and said subject is provided an effective amount of capsid agent selected from the group consisting of B19 parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2 capsid proteins, and fragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5).
Still more embodiments concern kits having a capsid agent selected from the group consisting B19 parvovirus capsid, B19 parvovirus capsid proteins, preferably VP2 capsid proteins, and fragments thereof, preferably the tripeptide (QQY) and a 10-mer peptide (NKGTQQYTDQ SEQ. ID. NO. 5), and instructions for dosage and administration to a subject for hematopoietic progenitor cell growth inhibition, hematopoietic progenitor cell growth inhibition, endothelial cell growth inhibition or treatment of a hematological proliferative disease. These kits may also have a device to remove blood from a subject and/or a devices to measure blood hematocrit or endothelial cell growth.