CN1328469A - Use of parvovirus capsid particles to inhibit cell proliferation and migration - Google Patents

Abstract

The invention described herein relates to the discovery of methods and compositions for inhibiting the growth and/or migration of cells containing the P antigen, including but not limited to cells of hematopoietic origin and endothelial cells. More specifically, parvovirus capsid particles or fragments of parvovirus capsid proteins are used in the manufacture of medicaments for administration to a subject for inhibiting hematopoietic progenitor cell growth (e.g., prior to stem cell transplantation), endothelial cell growth (e.g., as an anti-tumorigenic treatment or to prevent restenosis or fibrotic hyperplasia following prosthesis implantation), or for preventing disorders involving abnormal proliferation of cells containing the P antigen (e.g., polycythemia vera).

Description

Use of parvovirus capsid particles to inhibit cell proliferation and migration

field of invention

The present invention relates to the discovery of methods and compositions for inhibiting cell growth and migration. More specifically, the B19 parvovirus capsid or B19 parvovirus capsid protein fragments are useful in the manufacture of medicaments that are administered to a subject to inhibit the growth of cells containing the P antigen, including but not limited to cells of hematopoietic origin and endothelial cells and/or migrate.

Background of the invention

Parvovirus B19 is a human pathogen that is associated with varying clinical symptoms in patients with immunocompromised or hemolytic anemia, ranging from moderate symptoms (erythema infectias) to more severe disease. Hydrops fetalis and intrauterine fetal death are well-known syndromes of B19 infection during pregnancy (Anderson and Young, Monographs in Virology, 20 (1997)). B19 parvovirus particles are icosahedral, 18-26 nm in diameter, and consist of 60 capsid proteins, about 95% of which are the major capsid protein (VP2), with a molecular weight of 58 kd. (Fields et al., Virology, vol. 2, 3 ed., Philadelphia, PA, Published by Lipponcott Raven Press, pp. 2202 (1996)), the coat constituting approximately 3-5% of the B19 parvovirus capsid The capsid protein is a minor capsid protein (VP1) with a molecular weight of 83 kd, VP1 differs from VP2 by an additional 227 amino acids at the amino terminus (ref. supra).

Parvovirus B19 is particularly susceptible to human erythrocyte and bone marrow cultures. For example, parvovirus B19 binds to human erythroid progenitor cells and inhibits hematopoietic colony formation by replicating in these cells. (Brown et al., Science, 262:114 (1993); Mortimer et al., Nature, 302:426 (1983)). Inhibition of hematopoietic cells has also been found in bone marrow samples from infected individuals, leading to transient anemia and, in rare cases, transient reductions in all types of blood cells. (Saunders et al., Br J Haematol, 63:407 (1986)). It was further found that parvovirus B19 causes myelosuppression in both natural and experimental human infections. (Anderson and Young, Monographs in Virology, 20 (1997)).

The cellular receptor for B19 parvovirus has been identified as erythrocyte glycoside or erythrocyte P antigen, a tetrahexoceramide. (Fields et al., Virology, Vol. 2, Ed. 3, Philadelphia, PA, Published by Lipponcott Raven Press, pp. 2204 (1996)). The P antigen is found in mature erythrocytes, erythroid progenitors, megakaryocytes, endothelial cells, renal cortex, placenta, fetal myocardium (von dem Brone et al., British Journal of Hematology, 63:35 (1986)) and in fetal liver. Erythrocytes (Morey and Flemming, British Journal of Hematology, 82:302 (1992)). Individuals congenitally deficient in P antigen are not susceptible to B19 parvovirus infection, and administration of excess P antigen or monoclonal antibodies against P antigen protects erythroid progenitor cells from B19 parvovirus infection. (References as above)

In addition, neutralizing antibodies that recognize several regions of the B19 parvovirus particle were produced. For example, monoclonal antibodies directed against epitopes of VP2, such as those found in amino acids 38-87, 253-272, 309-330, 328-344, 359-382, 449-468, and 491-515, and specific regions of VP1 can be Neutralization of B19 parvovirus (Fields et al., Virology, Vol. 2, Ed. 3, Philadelphia, PA, Lipponcott Raven Press, pp. 2207 (1996)).

A genetically engineered expression system for the production of B19 parvovirus antigens has also been developed (Kajigaya et al., Proc Natl Acad SciUSA, 86:7601 (1989); Kajigaya et al., Proc. Natl Acad. SciUSA, 88:4646 (1991).). The recombinant B19 parvovirus capsid produced in the baculovirus system is composed of VP1 and VP2 like the natural particles, and these capsid proteins self-assemble into virus-like particles (VLP). (Kajigaya et al., Proceedings of the National Academy of Sciences USA, 86:4646 (1991)). Electron microscopic analysis of the B19 parvovirus capsid revealed that the structure of the VLP is similar to that of plasma-derived virions (Kajigaya et al., Proc. Natl. Acad. Sci. USA 86:4646 (1991)). B19 VLP is currently being evaluated as a potential vaccine against B19 parvovirus infection, with preliminary results showing good neutralization without severe adverse effects. (Bostic et al., J. Infect. Dis., 179:619 (1999)). Many people are trying to administer B19 parvovirus to prevent B19 parvovirus infection, but no one has tried to exploit the properties of B19 parvovirus capsid, B19 capsid protein or fragments thereof to develop new drugs that inhibit cell proliferation or migration.

Brief description of the invention

In the invention described herein, the inventors disclose the discovery that the B19 parvovirus capsid, B19 parvovirus capsid protein or fragments thereof inhibit the growth and/or migration of cells containing the P antigen. Embodiments of the invention include medicaments comprising B19 parvovirus capsids, B19 capsid proteins or fragments thereof, which can be administered to a subject in need of an agent that inhibits cell growth and/or migration. Methods of treating diseases or conditions associated with proliferation or migration of hematopoietic or endothelial cells are also within the scope of the invention.

For example, one embodiment relates to the use of empty, non-infectious recombinant B19 parvovirus capsids, B19 capsid proteins or B19 capsid protein fragments for the production of a medicament for inhibiting the growth or migration of cells containing the P antigen. According to this use, the drug can be used to inhibit the growth of hematopoietic cells, the growth or migration of endothelial cells. Additionally, according to this use, the drug may also be used in the treatment of hematopoietic cell proliferation disorders, angiogenesis, tumorigenesis or ingrowth of endothelial cells into implanted prosthetic devices. Furthermore, according to this use, the drug can be used to treat a subject before stem cell transplantation, and the subject can be a fetus.

In another embodiment, a method of inhibiting the growth or migration of cells containing the P antigen is provided. The method comprises the steps of contacting cells with a capsid agent selected from the group consisting of B19 parvovirus capsids, B19 capsid proteins, and fragments thereof, and determining inhibition of cell growth or migration. In certain aspects, the cell can be a cell of hematopoietic origin or an endothelial cell.

The invention also includes a method of treating a subject prior to stem cell transplantation. The practice of the method comprises: identifying a subject in need of a capsid agent that inhibits the growth of hematopoietic cells, and providing to the subject in need an effective amount of a capsid selected from the group consisting of B19 parvovirus capsids, B19 capsid proteins, and fragments thereof agent. Likewise, another related embodiment relates to a method of treating a subject with a hematopoietic cell proliferative disorder, comprising the steps of: identifying a subject in need of a capsid agent that inhibits a hematopoietic cell proliferative disorder, and administering to the subject in need The latter provides an effective amount of a capsid agent selected from the group consisting of a B19 parvovirus capsid, a B19 capsid protein, and a fragment of a B19 capsid protein.

The invention also provides a method of inhibiting the growth of tissue into the implanted prosthesis. The method comprises the steps of: identifying a subject in need of a capsid agent that inhibits tissue growth into the implant prosthesis, and providing to the subject in need an effective amount of a B19 parvovirus capsid, a B19 capsid protein, and a B19 Capsid agents for capsid protein fragments. Another embodiment relates to a method of treating or preventing tumorigenesis, the method comprising the steps of: identifying a subject in need of a capsid agent that inhibits the growth of hematopoietic cells, and providing an effective amount of a drug selected from B19 to the subject in need. Capsid agent for parvovirus capsid, B19 capsid protein and B19 capsid protein fragment.

A kit containing a capsid agent is also an embodiment of the present invention, the kit comprising a capsid agent selected from the group consisting of B19 parvovirus capsid, B19 capsid protein and B19 capsid protein fragment, and for inhibiting Instructions for Dosage and Administration in Subjects of Growth of Hematopoietic Progenitor Cells, Inhibition of Endothelial Cell Growth, or Treatment of Hematopoietic Cell Proliferation.

Brief description of the drawings

Fig. 1 This figure shows the results of the colony formation test of human umbilical cord blood cells with different concentrations of B19 parvovirus capsid (VP1/2) in schematic form.

Figure 2 is a schematic diagram showing the results of colony formation assays on bone marrow cells of monkeys (baboons and macaques) using different concentrations of B19 parvovirus capsid (VP1/2).

Figure 3 is a schematic diagram showing the effects of different concentrations of B19 parvovirus capsid (BacVP1/2), B19 parvovirus capsid containing only VP2 (BacVP2 only) or control antigen (Bac control antigen) on human fetal liver cells The results of the colony formation assay.

Fig. 4 is a stick graph showing the results of the cell proliferation test in which human umbilical vein endothelial cells (HUVEC) were contacted with different concentrations of the control antigen (KYVTGIN) (SEQ ID No. 1). The x-axis represents the concentration of the control antigen gradually increasing (from left to right), 0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1.0 μg/ml, 10.0 μg/ml, and the y-axis represents the absorbance at 540 nm (A ₅₄₀ ).

Figure 5 is a bar graph showing the results of cell proliferation assays in which different concentrations of B19 parvovirus capsids containing only VP1 were exposed to human umbilical vein endothelial cells (HUVEC). The x-axis represents increasing concentrations of B19 parvovirus capsid (VP1) (from left to right), 0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1.0 μg/ml, 10.0 μg/ml, and the y-axis represents 540 nm Absorbance at (A ₅₄₀ ).

Fig. 6 This graph shows the results of cell proliferation assays in which human umbilical vein endothelial cells (HUVEC) were exposed to different concentrations of B19 parvovirus capsids (VP1/2) in the form of bar graphs. The x-axis represents the increasing concentration of B19 parvovirus capsid (VP1/2) (from left to right), 0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1.0 μg/ml, 10.0 μg/ml, the y-axis Represents the absorbance at 540 nm (A ₅₄₀ ).

Fig. 7 This graph shows the results of the cell proliferation assay of human umbilical vein endothelial cells (HUVEC) with different concentrations of B19 parvovirus capsid (VP2) in the form of bar graphs. The x-axis represents increasing concentrations of B19 parvovirus capsid (VP2) (from left to right), 0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1.0 μg/ml, 10.0 μg/ml, and the y-axis represents 540 nm Absorbance at (A ₅₄₀ ).

Fig. 8 This figure shows the B19 parvovirus capsid with different concentrations (VP1/2), the B19 parvovirus capsid containing only VP1 (VP1 only), and the B19 parvovirus capsid containing only VP2 (VP2 only) in the form of a bar graph. ) or control antigen contact with human umbilical vein endothelial cells (HUVEC) cell migration test results.

Detailed description of the invention

In the invention described herein, the inventors disclose the discovery that the B19 parvovirus capsid, the B19 parvovirus capsid protein or a fragment thereof inhibits the growth and/or migration of cells containing the P antigen. Through colony formation assays, the inventors demonstrated that the B19 parvovirus capsid composed of VP1 and VP2 or only VP2 can inhibit several different types of hematopoietic cells (including human fetal liver cells, human umbilical cord blood cells, growth of adult bone marrow cells). In addition, the inventors found that the B19 parvovirus capsid inhibits the growth of bone marrow cells obtained from baboons and macaques. Further, the inventors found that the B19 parvovirus coat Capsid inhibits hematopoietic cell growth through interactions involving the P antigen. In addition, the inventors found that the B19 parvovirus capsid was internalized in cells containing the P antigen by incubation of the B19 parvovirus capsid with cells containing the P antigen followed by immunolabeling.

The inventors also found that the B19 parvovirus capsid inhibits the proliferation and migration of endothelial cells. Endothelial cell proliferation assays were performed by exposure of human umbilical vein endothelial cells (HUVEC) to fibroblast growth factor in the presence of B19 parvovirus capsid. Cell proliferation was monitored by crystal violet staining, which demonstrated that the B19 parvovirus capsid effectively reduced endothelial cell proliferation. Using the Boyden chamber assay, the inventors further demonstrated that the B19 parvovirus capsid inhibits the migration of HUVECs.

Several embodiments of the invention relate to the production of modified B19 parvovirus capsids. The inventors have disclosed a number of methods for producing B19 parvovirus capsids containing less than 5% VP1 and B19 parvovirus capsids containing only VP2. The inventors further teach methods of producing fragments of the B19 capsid protein and peptidomimetics similar to these peptides that can be used to inhibit the growth and/or migration of P antigen-containing cells. The length of the peptide fragment in the present invention is at least 3 amino acids, and can be as much as 780 amino acids, and can contain conservative amino acid substitutions. Additionally, the B19 parvovirus capsid, B19 capsid protein, or B19 capsid protein fragment can be modified by introducing substituent groups that are not native to the B19 capsid protein, by introducing mutations, or by creating fusion proteins. Derivatized or synthetic B19 capsid proteins are also an embodiment. The inventors also teach methods of designing and producing B19 parvovirus capsids, B19 capsid proteins, or fragments thereof, that elicit a minor immune response in a subject for use in long-term treatment regimens. Furthermore, the inventors describe the construction of various B19 capsid-based therapeutic methods, including the following information: sequence information, mutation or modification sites, information on the performance of functional analysis and information including disease indications, clinical evaluation, etc. Class treatment information.

Other embodiments of the invention include the preparation of multimer displays of B19 parvovirus capsids, B19 capsid proteins or fragments thereof, by means of supports (which can be beads, resins, plastic discs, preferably medical devices) , such as stent, valve or other prosthesis) and B19 parvovirus capsid, B19 capsid protein or its fragments are connected. These multimeric displays may provide potent agents for inhibiting the proliferation and/or migration of P antigen-containing cells (eg, restenosis after transplantation). The inventors also teach the preparation of a number of different medicaments and medical devices comprising the B19 parvovirus capsid, B19 capsid protein or fragments thereof. These drugs can be compatible with other additives, carriers or excipients for different routes of administration.

Methods of treatment and prophylaxis are also within the scope of the invention. In several embodiments, the inventors teach several methods of inhibiting P antigen-containing cells, including but not limited to cells of hematopoietic origin and endothelial cells, by administering a drug comprising a B19 parvovirus capsid, B19 capsid protein, or fragments thereof. Methods of proliferation and/or migration. In one embodiment, a method for inhibiting hematopoietic cells in a subject prior to intrauterine stem cell transplantation is provided. In another related aspect, the inventors teach methods of suppressing hematopoiesis in a subject prior to postnatal stem cell transplantation (eg, a new approach to non-myeloblastic therapy). Other methods of the invention include methods of inhibiting cell proliferation in a subject having a hematopoietic proliferative disorder, such as polycythemia vera. Still further, embodiments include methods of preventing angiogenesis, tumorigenesis or cancer, and methods of making medical devices, such as stents or valves, preventing fibrosis or restenosis or delaying endothelial cell ingrowth after heart valve surgery. A kit comprising a B19 parvovirus capsid, a B19 capsid protein or a fragment of a B19 capsid protein is also an embodiment of the present invention. In the following section, the inventors describe experiments that provide evidence that the B19 parvovirus capsid inhibits the growth of hematopoietic cells. B19 parvovirus capsid inhibits growth of hematopoietic cells

In a first set of experiments, the inventors found that recombinant B19 parvovirus capsids could be used to inhibit the growth of hematopoietic cells, as demonstrated by reduced colony formation of fresh human fetal liver cells, umbilical cord blood cells, and bone marrow cells. These tests are described below.

In order to obtain fetal liver tissue, fetuses with a gestational age of 6-12 weeks were obtained through legal abortion, and the subjects volunteered to donate fetal tissue. Gestational age is estimated based on specific anatomical landmarks and menstrual dates are given. Vacuum aspiration for abortion. Mince the fetal liver under aseptic conditions, place it in a sterile test tube containing RMPI 1640, pass it through a ethylene sieve to separate the cells and form a single-cell suspension. Then the nucleated cells were washed three times, counted, and diluted with culture medium.

The colony formation assay was performed using a commercial kit - "Stem Cell CFU Kit" (New York, USA, Life Technologies, GIBCO BRL). The kit provides a semi-solid support that mimics the intercellular matrix produced by stromal cells. Other components included in the kit are: Iscove's Modified Dulbecco's Medium, Modified Fetal Calf Serum, Methylcellulose, 2-Mercaptoethanol, Conditioned Medium, and Erythropoietin. The colonies formed were identified as BFU-E (burst-forming unit of erythroid cells) with densely packed hemoglobinated cells, CFU-GM (colony-forming unit of granulocytes, macrophages) with arrangement of non-hemoglobinated cells, and CFU-GEMM (colony forming units of granulocytes, erythroid cells, macrophages, megakaryocytes) of cells, small peripheral blood cells, and large peripheral blood cells.

Empty capsid particles of recombinant B19 parvovirus (Kajigaya et al., Proceedings of the National Academy of Sciences USA, 88:4646 (1991)), a gift from MedImmune (Gaithersburg, MD, USA), were cultured in recombinant baculovirus insect cells (Spodoptera frugiperda) prepared in an expression system. (Kajigaya et al., Proceedings of the National Academy of Sciences USA, 88:4646 (1991)). Dilute the capsid with buffer (20mM Tris, 0.5M NaCl, pH8.5), add each 30μl dilution to 100μl medium containing 25×10 ³ cells (50×10 ³ postnatal cells), and incubate at 4°C 1 hour. The mixture was then transferred to an incubation plate, and culture medium was added to each well to a final volume of 0.5 ml, incubated for 11 days in a humidified atmosphere of 5% CO ₂ , and then analyzed for BFU-E, CFU-E and CFU-E in the colony formation assay. GEMM-derived colony formation was scored.

In an 11-day colony formation assay, the inventors found that the B19 parvovirus capsid inhibited the growth of hematopoietic cells, as demonstrated by reduced colony formation in fresh human fetal liver cells, umbilical cord blood cells, and adult bone marrow cells. That is, when human fetal liver cells, umbilical cord blood cells, and adult bone marrow cells were incubated with B19 parvovirus capsids, BFU-E (erythroid burst colony-forming unit), CFU-GM (granulocyte, macrophage colony-forming unit) and CFU-GEMM (granulocyte, erythroid, macrophage, megakaryocyte colony-forming unit) colony formation decreased (see Table 1).

Table 1 Fetal liver cell colony-forming unit assay ^* Number of colonies (% medium control) Dilution of B19 parvovirus capsid (μg/ml) BFU-E CFU-E CFU-GEMM 70.0 twenty two% 14% 31% 0.7 39% 54% 63% 0.007 79% 95% 94% Medium (=100%), counted 95 37 16 ^* Cells were incubated with B19 capsids prior to 11 days of culture.

As shown in Table 1, the concentration of B19 parvovirus capsid particles to inhibit hematopoietic cells can be as small as 0.007 μg/ml, and the growth of hematopoietic cells can be significantly inhibited at 70 μg/ml.

Recombinant papillomavirus capsids (cottontail rabbit papillomavirus and human papillomavirus type 6) were used as controls in the colony formation assay. These capsids are structurally similar to the B19 parvovirus capsid but do not interact with the P antigen. Recombinant human papillomavirus capsid (HPV6) and cottontail rabbit papillomavirus capsid (CRPV) were kindly provided by Dr. J. Dillner from Karolinska Institutet, Stockholm, Sweden. Results B19 parvovirus capsids inhibited the growth of hematopoietic cells, while papillomavirus capsids (test range 0.01-100 μg/ml) had no effect on colony formation.

In a second set of experiments, the inventors found that incubation of B19 capsids with anti-B19 monoclonal antibodies or B19 parvovirus IgG-positive human serum prior to adding the B19 capsid mixture to cells restored the ability of hematopoietic cell colonies to form . Anti-B19 parvovirus monoclonal antibody (MAB8292), an IgG family antibody, was purchased from Cehmicon AB in Malmo, Sweden. B19 IgG-positive (parvovirus B19 IgM-negative) sera were obtained from two asymptomatic individuals. In the neutralization assay, B19 parvovirus capsids were first incubated with anti-B19 monoclonal antibody before adding the mixture to fetal liver cells. Approximately 25 μl of anti-B19 parvovirus monoclonal antibody (MAB8292) was incubated with 25 μl of B19 parvovirus capsids for 2 hours at 4°C, and then the mixture was added to the cells, as described above for 11 days for the neutralized capsid/cell mixture. Colony formation assay.

Although a relatively high concentration of B19 parvovirus capsid was used (7 μg/ml, compared with the values in Table 1), anti-B19 monoclonal antibodies as low as 0.02 μg/ml reduced B19 parvovirus capsid inhibition of fetal liver cells The ability to grow, the anti-B19 monoclonal antibody concentration of 20.0 μg/ml can completely block the inhibition of BFU-E colony formation, and significantly reduce the impact on CFU-GM and CFU-GEMM colony formation (see Table 2).

Table 2 Neutralization assay using anti-B19 parvovirus monoclonal antibody ^* Number of colonies (% medium control) Parvovirus B19 capsid (7 μg/ml) + dilute solution of anti-parvovirus B19 monoclonal antibody (Error! Invalid link.) BFU-E CFU-GM CFU-GEMM 20.0 >100% 74% 67% 2.0 69% 35% 45% 0.2 52% twenty one% 19% 0.02 52% 30% twenty one% capsid only 43% 30% twenty one% Medium (=100%), counted 114 66 42 ^* Note: Cells were first incubated with corresponding reagents before 11-day culture.

Similarly, two batches of B19 parvovirus IgG positive sera were analyzed for their ability to neutralize the B19 parvovirus capsid. Approximately 25 μl of B19 parvovirus IgG-positive serum was incubated with 25 μl of B19 parvovirus capsid for 2 hours at 4°C, and then the mixture was added to fetal liver cells. Subsequently, the serum-neutralized B19 capsid/cell mixture was subjected to the previously described colony formation assay. As shown in Table 3, in the absence of serum, B19 parvovirus capsid (0.14 μg/m1) significantly inhibited the colony formation of fetal liver cells, while serum 1 at a dilution of 1:100 reduced B19 parvovirus capsid inhibited the growth of fetal liver cells Ability.

Table 3 Neutralization test with human B19 parvovirus IgG positive serum ^* Number of colonies (% medium control) B19 parvovirus capsid (0.14 μg/ml) + dilution of two human parvovirus B19 IgG positive sera BFU-E CFU-GM CFU-GEMM Serum 1, 1:10 70% 78% 90% Serum 1, 1:100 25% twenty three% 40% Serum 2, 1:10 48% 57% 57% Serum 2, 1:100 17% 27% 67% capsid only 18% 17% 63% Medium (=100%), counted 157 81 30 ^* Note: Cells were first incubated with corresponding reagents before 11-day culture.

Further evidence that the B19 parvovirus capsid inhibits the growth of hematopoietic cells was obtained in neutralization assays with monoclonal antibodies directed against the P antigen. Anti-P antigen monoclonal antibody (CLB-ery-2), a murine IgM antibody, was kindly provided by Dr. von dem Borne, Central Laboratory of the Dutch Red Cross Blood Transfusion Service, Amsterdam, The Netherlands (see von dem Borne et al., British Hematology Journal, 63:35 (1986)). In these experiments, approximately 2.5 x ¹⁰⁴ fetal hepatocytes were suspended in 100 μl of culture medium and then mixed with 25 μl of anti-P monoclonal antibody (CLB-ery-2) or 25 μl of anti-P ₁ (Seraclone) as a control monoclonal antibody Incubation. Cells and monoclonal antibodies were incubated for 1 hour at 4°C. Cell/antibody mixtures were washed twice with pre-chilled culture medium prior to addition of B19 parvovirus capsids, and colony formation assays were performed as previously described.

Consistent with data from previous experimental studies with natural virus particles (see Brown et al., Science, 262:114 (1993)), the inventors found that monoclonal antibodies against the P antigen were able to restore the presence of B19 parvovirus capsids. Growth capacity of incubated fresh fetal liver cells. As shown in Table 4, when the cells were incubated in the presence of CLB-ery-2, the inhibitory effect of the B19 parvovirus capsid was reduced by at least 25%. In contrast, anti-P1 monoclonal antibody (Seraclone) (Labdesign, Stockholm, Sweden) did not interact with the P antigen and had no effect on colony formation compared to the parvovirus B19 capsid control.

Table 4 Neutralization tests with anti-P or anti-P ₁ monoclonal antibodies ^* Number of colonies (% control medium) B19 parvovirus capsid (0.14 μg/ml) + anti-P antigen monoclonal antibody (titer) BFU-E CFU-GM CFU-GEMM 1:5 51% 39% 93% 1:500 twenty three% 10% 43% capsid only 18% 17% 63% Medium (=100%), counted 157 81 30 B19 parvovirus capsid (0.14 μg/ml) + anti-P ₁ antigen monoclonal antibody (μg/ml) 400.0 25% 20% 50% 4.0 17% twenty two% 47% capsid only 18% 17% 63% Medium (=100%), counted 157 81 30 ^* Note: Cells were first incubated with corresponding reagents before 11-day culture.

The inhibitory effect of B19 parvovirus capsids on colony formation was also tested using fresh stem cells isolated from umbilical cord blood and adult bone marrow samples. Colony formation assays were performed on cells isolated from cord blood and bone marrow in the presence of B19 parvovirus capsids as described above. Cord blood samples were obtained immediately after normal birth vaginal delivery, and adult bone marrow samples were obtained from healthy allogeneic donors. The fresh cell suspension was treated with heparin, diluted with 0.9% NaCl, and gradient centrifuged at 2000 rpm for 20 minutes on Lymphoprep (Nycomed, Palma, Oslo, Norway). The cells were carefully pipetted with a Pasteur pipette, washed 3 times with 0.9% NaCl, counted, diluted with culture medium, and prepared for colony formation test.

The ability of the B19 parvovirus capsid to inhibit cord blood and bone marrow-derived hematopoietic cells was comparable to that shown for fetal liver cells (see Table 5). For example, as shown in Figure 1, as parvovirus B19 capsid concentrations increased, the growth of cells from cord blood decreased. Further neutralization assays using cells from umbilical cord blood or bone marrow and B19 parvovirus capsids also showed similar results to assays with human fetal liver cells. Namely, incubation of B19 parvovirus capsids with an anti-B19 parvovirus monoclonal antibody (Mab8292) prior to exposure to cells from umbilical cord blood and bone marrow sources demonstrated a reduced ability to inhibit cell growth as evidenced by increased colony formation.

Table 5 Colony Formation Assays Using Umbilical Cord Blood and Adult Bone Marrow Cells ^* Number of colonies (% medium control) B19 parvovirus capsid (0.14μg/ml) umbilical cord blood cells BFU-E CFU-GM CFU-GEMM 7.0 10% 54% 43% 0.7 33% 62% 43% 0.07 49% 72% 50% 0.007 57% 67% 70% 0.0007 84% 79% 93% Medium (=100%), counted 134 39 30 bone marrow cells 7.0 18% 36% 6% 0.7 43% 45% 28% 0.07 63% 41% 44% 0.007 76% 80% 78% 0.0007 86% 77% 78% Medium (=100%), counted 134 39 30 ^* Note: Cells were first incubated with B19 parvovirus capsid (μg/ml) before 11-day culture.

In addition, colony formation assays were carried out using hematopoietic cells obtained from the bone marrow of monkeys (baboons and macaques) in the presence of B19 parvovirus cells as described above. As shown in Figure 2, primate hematopoietic cell growth decreased as the B19 parvovirus capsid concentration increased. The results of this experiment not only demonstrate that primate hematopoietic cells contain the P antigen that interacts with the B19 parvovirus capsid, but also demonstrate that baboons and macaques are suitable for in vivo studies of the therapeutic and prophylactic embodiments of the present invention. In the following section, the inventors describe modified B19 parvovirus capsids and peptides that make up the B19 parvovirus capsid that can be used to inhibit the growth of hematopoietic cells. Modified B19 parvovirus capsids and peptides that make up the B19 parvovirus capsid inhibit hematopoietic cell growth

In this section, the inventors describe how to make modified B19 capsids containing different ratios of VP1 and VP2 proteins or VP2 only, which can be used in long-term therapeutic regimens to inhibit the growth of hematopoietic cells. In attempting to identify the region of the B19 parvovirus capsid involved in cell growth inhibition, the inventors found that upon binding to the P antigen, the capsid was internalized by fusion with cells containing the P antigen. In an experiment providing evidence of B19 parvovirus capsid internalization, the inventors incubated fetal liver cells with B19 parvovirus capsids, then fixed the capsid-treated cells on BioRad slides and treated them with anti-B19 monoclonal The cloned antibody (Mab8292) was labeled and detected with a fluorescent secondary antibody. Accordingly, fetal liver cells were washed twice with PBS solution to make a suspension with a concentration of 2×10 ⁶ /ml, and a part of the suspension was mixed with B19 natural capsid (0.35 μg capsid/ml cell suspension) at 37°C. Incubate for 1 hour. Then about 20 μl microdroplets of cell/capsid suspension (approximately 40,000 cells) were added to two BioRad slides, each with 10 wells, in two wells of each slide, no Cells treated with B19 capsid served as control. In the next step, the cells on one of the ^BioRacl® glass slides were permeabilized with saponin, which allowed the antibody to permeate, and subsequently, the primary anti-B19 monoclonal IgG antibody was added, bound and washed with PBS solution to remove unbound primary Antibody, then add secondary fluorescent anti-IgG antibody, after binding, wash with PBS to remove unbound secondary antibody. Analysis by ultraviolet light microscopy showed that cells permeated with saponin and then treated with B19 parvovirus capsid showed fluorescence both in the cell and on the cell membrane. In contrast, control cells not permeabilized with saponin showed fluorescence only on the cell surface. These results provide evidence that B19 parvovirus capsid-mediated inhibition of cell growth involves more protein sequences than those encoding the P antigen receptor.

While unmodified B19 parvovirus capsids may be included in embodiments of the invention, native B19 VLPs (i.e. capsids containing 95% VP2 and 5% VP1) can elicit an immune response and are not suitable for certain therapeutic applications (e.g. , for long-term treatment regimens). Someone constructed a modified B19 parvovirus capsid containing 25% VP1 and 75% VP2 in an attempt to develop a B19 parvovirus vaccine, but this modified VLP would cause enhanced neutralization in vivo (US Patent No. 5508186, Young et al.). This modified B19 parvovirus capsid is not suitable for long-term treatment regimens because the subject's immune response will quickly eliminate the VLP from the body and reduce the effective amount. Additionally, since antibodies against parvoviruses are close to 50% of the population, treatment regimens should favor capsid agents that elicit a smaller immune response. Since a unique region of VP1 plays an integral role in the immune response to B19 parvovirus (Fields et al., Virology, Vol. 2, ed. 3, Philadelphia, Pennsylvania, Lipponcott Raven Press, p. 2207 (1996)) , to produce modified capsids that contain less VP1 than native capsids, which are more effective in long-term treatment regimens. The following are some embodiments, which include VP1 containing VP1 and VP2 total amount less than or equal to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0% , 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7 %, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.8%, and 5.0% of B19 parvovirus capsids. Parvovirus B19 capsids (only VP1 and VP1/2) were provided by Klaus Heddman, University of Helsinki, Finland.

Furthermore, recombinant VP2 can spontaneously form a capsid structure similar to the VP1/VP2 structure without VP1 (US Patent No. 5508186, Young et al.). The VP2 capsid has only a small area of neutralization and is therefore a very effective therapy for use in long-term treatment regimens. Using the aforementioned colony formation assay method, the inventors confirmed that VP2 capsid can inhibit the growth of hematopoietic cells ( FIG. 3 ). Human fetal hepatocytes were incubated in the presence of VP2 capsids and then subjected to 11-day colony formation assays as previously described. The positive control of this experiment is natural B19 VLP, namely the B19 parvovirus capsid (VP1/2) containing 95% VP2 and 5% VP1.

The results in Fig. 3 show that the growth of hematopoietic cells is inhibited when the concentration of VP2 capsid is as low as 3 μg/ml, and its growth is significantly inhibited when the concentration is 30 μg/ml. Embodiments suitable for chronic treatment also include fragments of VP2 that are involved in the inhibition of cell growth (eg, the P antigen binding site or regions involved in fusion/internalization or both). Protein engineering techniques discussed in detail below, computer modeling, epitope mapping, and the 'capsid agent characterization assay' described here can be used to rapidly identify VP1, VP2 ( or both) peptides.

By 'capsid agent characterization assay' is meant an assay that analyzes the ability of a 'capsid agent' to inhibit the growth of cells containing the P antigen. Examples of capsid agents include, but are not limited to, B19 VLPs, or VP1/2, or VP1, or VP2 capsids, or modified or unmodified peptide fragments of VP1 or VP2 (or both), or peptide fragments with VP1 or VP2 ( or both) sequence, or a peptidomimetic like VP1 or VP2 or a region of either or both of these molecules. Examples of capsid agent characterization assays include, but are not limited to, colony formation assays, neutralization assays, protein binding or fusion assays, internalization assays, transcription or translation assays, assays to evaluate intracellular protein phosphorylation or calcium mobilization following exposure to capsid agents (US Pat. No. 5,508,186 to Young et al., cited herein by reference for some of the literature describing capsid agent characterization tests). In the next section, the inventors disclose the discovery that the B19 parvovirus capsid can inhibit another type of P antigen-containing cells, namely endothelial cells. B19 parvovirus capsid inhibits the growth of other P-antigen-expressing cells, including but not limited to endothelial cells

The first set of experimental results confirmed that the B19 parvovirus capsid and VP2 capsid effectively inhibited the growth of a large number of different P antigen-containing hematopoietic cells (including hematopoietic cells of different species). The inventors found in a second set of experiments that recombinant B19 parvovirus capsids inhibited the growth of another type of P antigen-containing cells. Specifically, the inventors discovered that the B19 parvovirus capsid inhibits the growth of endothelial cells, as manifested by a reduction in endothelial cell proliferation and migration. These experiments are described below.

In order to determine whether the B19 parvovirus capsid can inhibit the proliferation of endothelial cells, the following experiment was carried out. Primary human umbilical vein endothelial cells were seeded in 24-well plates, each well containing 1.5× ¹⁰⁴ cells, in 0.5% fetal bovine serum and Incubate with B19 parvovirus capsid in the presence of 10.0 ng/ml basic fibroblast growth factor. The next day different B19 capsid preparations (ie VP1/2 or VP1 or VP2 only capsids) were added to the wells and the cells were incubated with the capsids for an additional 72 hours. Cell proliferation was measured using a crystal violet staining assay. Accordingly, capsid-treated cells were washed with PBS, fixed with 3.7% formaldehyde, and incubated with crystal violet. Rinse well with distilled water to remove the dye. Crystal violet combined with cells can be dissolved in 10% acetic acid solution, and its absorption is measured at 540nm by ELISA plate analyzer.

The results of the endothelial cell proliferation assay are shown in Figures 4-7. In these figures, the x-axis represents the concentration of the control antigen KYVTGIN (SEQ ID No.1) representing increasing concentrations (Fig. 4), the concentration of B19 parvovirus capsid (only containing VP1) (Fig. 5), the concentration of B19 parvovirus capsid (Fig. VP1/2) (Figure 6), B19 parvovirus capsid concentration (only VP2) (Figure 7). Therefore, from left to right, the bar graphs represent absorbance at 540 nm at concentrations of 0 μg/ml, 0.01 μg/ml, 0.1 μg/ml, 1.0 μg/ml, and 10.0 μg/ml, respectively. The Y-axis represents the standard of absorbance at 540 nm. The standard deviation does not exceed 10%. As shown in Figure 7, VP2 capsid effectively inhibited the proliferation of endothelial cells at concentrations as low as 1.0 μg/ml, with significant inhibition observed at 10.0 μg/ml.

The effect of B19 parvovirus capsid preparations on cell migration was also determined. Migration assays were performed by a modified Boyden chamber assay (Neuroprobe, Inc). Basic fibroblast growth factor (40 ng/ml) was added to stimulate migration of human umbilical vein endothelial cells through type 1 collagen-coated 8 μm pore size microporous membranes. Cells were incubated with different B19 capsid agents (ie, VP1/2 or capsids containing only VP1 or VP2) before performing migration assays. For migration assays, Boyden chambers were incubated at 37°C, 10% CO ₂ for 4.5 hours, after which the filters were removed and fixed in 3.7% formaldehyde. Filters were stained overnight in Gil's hematoxylin to visualize cell migration. The number of migrating cells on the migrating side of the filter membrane under each high-magnification field of view was counted by counting stained cells (see Figure 8). As shown in Figure 8, VP2 capsid protein effectively inhibited endothelial cell migration at concentrations as low as 1 μg/ml. Furthermore, the inhibitory effect of VP2 capsid protein-mediated endothelial cell migration was significantly stronger than that observed for native capsids (VP1/2) or capsids containing only VP1.

The results of the above experiments demonstrate that B19 parvovirus capsids, modified parvovirus B19 capsids and VP2 capsids can be produced and used to effectively inhibit the growth and/or migration of cells containing the P antigen, such as hematopoietic cells and endothelial cells. The above experiments also revealed that B19 capsid protein sequences that bind to the P antigen and/or participate in particle fusion or internalization may be involved in the inhibition of cell growth or cell migration. These embodiments are applicable to many therapeutic applications of the invention, and drugs containing fragments or synthetic molecules of the VP1 or VP2 proteins (or both) can be constructed for more efficient binding, fusion and internalization of cells containing the P antigen. In the next section, the inventors describe the production and characterization of further capsid agents that inhibit cell growth and migration. B19 capsid agents that inhibit the growth and migration of cells containing the P antigen

In this section, the inventors describe several techniques that can be used to produce, design and characterize capsid agents including but not limited to B19 parvovirus capsids, modified B19 parvovirus capsids, VP2 capsids and peptides or peptidomimetics containing sequences corresponding to VP1 or VP2 (or both). The structural genes of VP1 and VP2 have been fully sequenced and are available from the NCBL database under accession number U38506.1, or accession number AAB47788, or medline number 97081188, or see Erdman et al., J. General Virology, 77:2767 (1996 ), all references and sequences therein are incorporated herein by reference. VP1 or VP2 or fragments of both (or either) used in embodiments of the present invention correspond to sequences involved in the inhibition of cell growth and migration. The contemplated peptides of the present invention may comprise 3-780 amino acids of the VP1 and VP2 structural proteins, but must include at least some portion of the molecule involved in inhibiting the growth and/or migration of P antigen-containing cells. In other words, preferred embodiments of the invention may include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 of the VP1 and VP2 structural genes , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80 , 90 or 100 amino acids. Desirable embodiments may include at least 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 of the VP1 and VP2 structural proteins ,300,31 0,320,330,340,350,360,370,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, or 780 amino acids.

Peptides and peptide fragments or derivatives thereof involved in inhibiting the growth and migration of P antigen-containing cells include, but are not limited to, those regions found in native VP1 and VP2 structural genes. In addition, altered sequences are included in these capsid agents, wherein silent changes are produced by replacing residues in the sequence with functionally equivalent amino acid residues. Accordingly, one or more amino acid residues in the structural gene sequences of VP1 and VP2 can be replaced by other amino acids with similar polarity as functional equivalents, resulting in silent changes. An amino acid in the replacement sequence can be selected from other amino acids in the family to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Uncharged polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Aromatic amino acids include phenylalanine, tryptophan, and tyrosine. The aforementioned peptides are preferably analyzed in an assay to determine whether the fragment retains the ability to inhibit the growth and/or migration of P antigen-containing cells. The peptides used in the present invention may also be modified, for example, the peptides may contain substitutions that are unusual in peptides or substitutions that are common in peptides, but the substitutions occur in areas of the peptide that are not normal. For example, these peptides may eg be acetylated, acylated or aminated. Substituents for modifying peptides include, but are not limited to, H, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen, unsubstituted or substituted sulfonyl, 5-membered or 6-membered aliphatic or aromatic ring. In addition, VP1, VP2, or fragments of either (or both) can be derivatized such that the derivatized peptide contains amino acid sequences that affect the function and stability of the molecule. For example, peptides involved in the inhibition of growth and migration of cells containing the P antigen can be engineered to contain one or more cysteine residues to facilitate the formation of more stable derivatives via disulfide bonds (eg, U.S. Patent No. 4908773) . Computer graphics programs and the assays described herein are used to identify cysteine attachment sites that provide greater stability without affecting inhibition of growth or migration of P antigen-containing cells. (For example, Perry LJ and WetzelR, Science, 226:555-557 (1984); Pabo, C.O., Biochemistry (Biochemistry), 25:5987-5991 (1986); Bott, the people such as R., European patent application SEQID No. .130756, Perry LJ and Wetzel R, Biochemistry, 25:733-739 (1986);

Additional derivatives that are embodiments of the invention include peptide mimetics that resemble regions of VP1, VP2, or both. Synthetic peptides can be prepared using methods corresponding to the preparation of these molecules using conventional synthetic methods, using L-amino acids, D-amino acids, or mixtures of the two differently configured amino acids in varying ratios. Synthetic compounds that mimic the conformation and desired features of a specific peptide but avoid undesired features, such as elasticity (loss of conformation) and bond scission, are called "peptidomimetics" (for example, see Spatola AF , Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins (ed. Welstein B), Vol. 7, pp. 267-357, New York, Marcel Dekker (1983), which describes the enkephalins methenesulfide [ _CH2S ] bioisomers as amide substitutions in analogs; and Szelke et al., Eighth American Peptide Symposium, Peptides: Structure, Function, and Advances (Eds. Hruby and Rich) 579-582, Published by Pierce Chemical Co., Rockford, Illinois, this paper describes the leucine-valine peptide bond in an octapeptide derived from angiotensinogen 6-13 containing methamine [CH ₂ NH] and renin inhibitors of hydroxyethylene [CHOHCH ₂ ] bioisoligates). Various methods and techniques are known in the design and production of peptidomimetics, which can be chosen at will. (eg, Farmer PS, Drug Design (Ed., Ariens EJ) Vol. 10, pp. 119-143 (New York, London, Toronto, Sydney and San Francisco, Academic Press) (1980); Farmer et al., TIPS, 9/82, pp. 362-365; Verber et al., TINS, 9/85, pp. 393-396; Kaltenbronn et al., JMedChem, 33:838-845 (1990); Spatola AF, Amino Acids , Peptide and Protein Chemistry and Biochemistry, Volume VII, pp. 267-357, Chapter 5, "Peptide Backbone Modifications: Structure-Activity Analysis of Peptides Containing Amide Bond Substitutes. Constraints and Correlations of Configurations (Peptide Backbone Modifications: A Structure-Activity Analysis of Peptide Containing Amide Bond Surrogates. Conformational Constrains, and Relations)” (B Weisten, ed., New York: Marcell Dekker Publishing) (1983); Kemp DS, Peptide Mimicry and Templates for β-Sheet and α-Helical Nuclei in Peptides Method (Peptidomimetics and the Template Approach to Nucleation of beta-sheets and alpha-helice in Peptidea) Tibech, Vol. VIII, pp. 249-255 (1990). Additional techniques are available in U.S. Pat. Nos. 5,288,707; 5,552,534; 5,811,515; 5821231 and 5874529, incorporated herein by reference. Thus, as long as certain regions of the molecule are capable of inhibiting the growth or migration of cells containing the P antigen, the peptidomimetics of the present invention may have such a structure, at least in relation to the VP1 and VP2 structural proteins 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 ,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,390,400,410,420 ,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670 , 680, 690, 700, 710, 720, 730, 740, 750, 760, 770 or 780 amino acids are similar.

Conventional techniques of molecular biology, as described in US Patent No. 5,508,186, incorporated herein by reference, can be used to prepare various types of capsid agents. The term "capsid agent" may refer to fragments comprising VP1, VP2, VP1/2, or VP1 or VP2 or either or both in varying proportions, fusion proteins containing sequences corresponding to VP1 or VP2 or both, Modified or unmodified proteins or peptides or peptidomimetics corresponding to the VP1 and VP2 structural genes involved in the inhibition of growth and/or migration of P antigen-containing cells.

According to US Patent No. 5,508,186, the capsid agent can be produced by the following method. Plasmids containing full length VP1, VP2, or both were constructed. To construct plasmid pVP1/941, the cDNA encoding the VP1 gene of an almost full-length B19 parvovirus molecular clone was excised from pYT103c (Cotmore et al., Science, 226:1161 (1984); Ozawa et al., J. Virol., 62: 2884 (1988)), after digestion with restriction endonuclease Hind III (cutting at position 45 in the figure) and EcoRI (cutting at position 95 in the figure), the single-stranded end was filled in with mung bean nuclease. The obtained DNA fragment is inserted into the BamHI site of the baculovirus transformation vector pVL941 (processed into blunt end with DNA polymerase Klenow fragment), which is the Autographa californica polynuclear polyhedrosis virus (AcMNPV) that will delete the polyhedrin gene. ) gene cloned into the pUC8 plasmid (Summers et al., Tex. Agric. Exp. Stn. 1555 (1987)). The construction of pVP2/941 is to insert the PstI-EcoR digestion fragment of pYT103c into the BamHI site of pVL941 (Fig. A 20 nucleotide DNA fragment. Alternatively, the polymerase chain reaction (PCR) was used to clone the VP1 or VP2 gene or a portion of the full-length clone described by Erdman et al. (J. General Virology, 77:2767 (1996)), incorporated herein by reference. To facilitate cloning, primers can be designed to create convenient restriction sites as known in the art.

To produce recombinant baculoviruses, insect cells are transfected with recombinant plasmids encoding VP1, VP2, VP1/2 or fragments thereof. Accordingly, 8 μg of recombinant plasmid and 2 μg of wild-type AcMNPV were co-transfected into Sf9 cells by calcium phosphate-mediated precipitation. The Sf9 cell line (American Type Culture Collection, Rockville Md.) was obtained from the ovary (fall larvae) of Spodoptera frugiperda, and was cultured in 10% heat-inactivated fetal bovine serum, 2.5 μg/ml amphotericin, 50 μg/ml genta In 100% room air, 95% humidity , Cultured at 27°C. Progeny virus was harvested six days after transfection and plaques were regenerated on fresh Sf9 cells. Recombinant viruses are identified by the absence of macroscopic inclusion bodies in their nuclei (the inclusion body positive phenotype is the result of extensive polyhedrin protein synthesis). Recombinant virus can be subjected to 3 rounds of plaque purification prior to bulk VLP stock preparation and isolation or purification, specifically contemplated to contain 0.1%, 0.5%, 1%, 2%, 5%, 10%, 25% or more active ingredient (w/w) of the purified composition.

"Isolated"refers to a material that is separated from its original environment (eg, if the material is naturally occurring, the natural environment). For example, a protein naturally occurring in living cells is not isolated, but the same protein is isolated from some or all of the substances that coexist with it in a natural system. "Purification" does not require absolute purity, but a relative concept. For example, by Coomassie staining. After routine purification of the protein to electrophoretic purity, it is suitable for several assays despite the presence of impurities. Capsid agent characterization assays are preferably performed on isolated or purified capsid agents, including but not limited to those described in U.S. Pat. microscopy, immunoelectron microscopy, and the aforementioned capsid agent characterization analysis).

In some embodiments, particularly applications involving chronic administration of capsid agents, it is desirable to produce a drug that does not elicit a significant immune response in the subject. The general framework for producing capsid agents that do not elicit an immune response includes agent design, agent construction, analysis of the ability of the agent to inhibit cell growth and/or cell migration, and analysis of the immune response to the agent. The immunogenic regions of many B19 parvovirus capsids are known. These immunogenic regions can be deleted, mutagenized, or modified using conventional molecular biology techniques. The newly designed synthetic capsid proteins can use one or more capsid agents. Characterization assays (for example, colony formation assays and neutralization assays with sera from asymptomatic individuals) were performed. A number of methods can be used to identify the immunogenic region of the B19 parvovirus capsid and to produce non-immunogenic VLPs that inhibit cell growth and/or cell migration. The following example provides a possible method.

The design, production and analytical testing of expression constructs can be carried out as follows. This step can be repeated to generate several VLPs and capsid-containing drugs that differ in their ability to inhibit cell growth, cell migration, and elicit an immune response in a subject. Accordingly, through a method, primers are designed according to the published VP2 sequence, and the VP2 structural gene can be cloned from clinical isolates by PCR. The VP2 gene was then subcloned into Bluescript (Pharmacial) for mutagenesis and pVL1393 (Stratagene) for expression in Sf9 cells. Mutations corresponding to the VP2 immunogenic region (eg, amino acids 253-272, 309-330, 328-344, 359-382, 449-468, and 491-515) were introduced into the VP2 gene using the Amersham Sculptor in vitro mutagenesis kit. Those of ordinary skill in the art will appreciate that carboxy-terminal truncation, amino-terminal truncation, internal truncation and site-directed mutagenesis of the VP1 and VP2 structural proteins can be accomplished in several ways. It is preferred to generate several different clones containing one or more deletions as described above. Desired mutations were confirmed by sequencing, and the mutated genes were subcloned into pVL1393 for expression in Sf9 cells. Sf9 cells were transfected with Baculogold transfection kit (Pharmingen). Transfection was performed according to the instructions with the following modifications. Approximately 8 x 10 ⁸ Sf9 cells were transfected with 4 μg Baculogold DNA and 6 μg test DNA in 100 mM plates, harvested 6 days later and analyzed for VLP production.

Next, the cells are scraped and centrifuged at low speed. Cells were resuspended in 300ml lysis buffer (1M NaCl, 0.2M Tris, pH 7.6) and homogenized in a Falcon 1259 tube with a Polytron PT 1200 B with PT-OA1205/2-A (Brinkman) probe for 30 Second. Samples were centrifuged at 2500 rpm for 3 minutes to pellet debris and tubes were washed with an additional 150 ml of lysis buffer. The supernatant was collected into a 1.5 ml microcentrifuge tube and centrifuged in an Eppendorf microcentrifuge tube (Brinkman) for 5 minutes. The collected supernatant was stored at 4°C.

The isolated VLPs were then subjected to enzyme-linked immunoassay assay (ELISA) as follows. About 5 ml of the extract was diluted to 50 ml of 1% BSA in PBS (phosphate buffered saline, 20 mM NaPO ₄ , pH 7.0, 150 mM NaCl), and spread on a polystyrene plate. Incubate overnight at 4°C. Extracts were removed and polystyrene plates were blocked with 5% milk powder in PBS. All the following washing steps use 1% BSA in PBS. Polystyrene plates are incubated with primary antibody (eg, sera from asymptomatic subjects) for 1 hour at room temperature. After washing away unbound antibody, the polystyrene plate was incubated with secondary antibody for an additional 1 hr. Secondary antibody, peroxidase-labeled goat anti-mouse IgG (g) can be purchased from Kirkegaard & Perry Laboratories, Inc, and can be diluted to 10 ³ times with 1% BSA in PBS solution before use. After the last wash, an alkaline phosphorylase assay was performed and the absorbance was measured at 405 nm. The most successful capsid agents should be undetectable by this test method. That is, the desired mutant VP2 capsid should lose the antigenic determinant that can be recognized by the antibody present in the serum, so it cannot be detected by ELISA. By performing these assays with different batches of sera from different individuals, monoclonal antibodies that neutralize the inhibition of colony formation or cell migration, one skilled in the art can quickly identify the immunogenic regions of VP2 and the ones that best avoid immunogenicity. Response to mutant VP2 capsids.

Then, the capability of inhibiting cell growth and cell migration was analyzed by capsid agent characterization test for the mutant VP2 capsid that successfully avoided the above-mentioned ELISA detection. A "capsid agent profile" was obtained by evaluating the ability of each mutant VP2 capsid to inhibit cell growth and cell migration, reconciled with immunogenicity results from ELISA assays. A "capsid agent profile" may include a symbol or icon representing a mutant capsid protein or mutant VLP, sequence information (e.g., location of mutation or modification), description of capsid agent family (e.g., information on relationship to other capsid agents) ), application information (e.g., indication or therapeutic information, clinical or biotechnological use), functional information from capsid agent characterization assays (e.g., colony formation assays, neutralization assays, fusion/internalization assays, binding assays , phosphorylation test, cell migration test, proliferation test, immunogenicity test results including ELISA).

The capsid agent profile can be recorded on a computer readable medium, stored in a database on a hard disk, floppy disk, or internal memory, read by a search engine, and can be compared with each other or with a disease state or "disease state profile" (i.e., with disease, condition or information on applicable therapies). Investigators can use these capsid agent profiles and disease state profiles to rationally design drugs or perform biochemical assays, and physicians or clinicians can use them to select the appropriate drug composition based on the duration of treatment that balances inhibition of cell growth and migration with the effects of affected cells. The level of the immune response of the subjects.

In some embodiments, the capsid agent is placed on a support to produce a polymeric capsid agent. Monomer formulations (ie, formulations representing a single dispersed molecule, carrying only one binding domain) are sufficient to achieve the desired effect, whereas polymeric capsid formulations (ie, formulations representing multiple molecules, containing several domains) Usually produces a larger effect. It should be pointed out that "multimer" refers to more than one molecule on a support, for example, several individual VP2 molecules are linked on a support, which should be distinguished from "multimerization", which refers to Instead, multiple molecules are linked as a single compound to the support, for example, several VP2 molecules are linked to form a single compound molecule attached to the support. The polymeric form of the capsid agents described herein is advantageous in many biotechnological and clinical applications due to the higher affinity of the agent for P antigen-containing cells.

Multimeric capsid agents can be obtained by coupling proteins such as VP2 or fragments thereof to macromolecular supports. A support can also be called a carrier, resin, or any macromolecular structure used to attach or immobilize proteins. The macromolecular support can have a hydrophobic surface that interacts hydrophobically and non-covalently with regions of the capsid agent. The hydrophobic surface of the support may, for example, be a polymer such as plastic or any other polymer containing hydrophobic groups attached to polystyrene, polyethylene, polytetrafluoroethylene. Capsid agents can optionally be covalently bound to carriers including proteins and oligo/polysaccharides (eg, cellulose, starch, glycogen, chitosan, or hydrogenated Sepharose). In the latter embodiment, reactive groups in the capsid agent (such as hydroxyl groups or amino groups on the peptide) can be used to link with reactive groups on the carrier to form a covalent bond. Embodiments also include a support with a charged surface that can interact with the capsid agent. Additional embodiments relate to supports with other reactive groups that can be chemically activated to attach to capsid agents. For example, cyanogen bromide-activated matrices, epoxy-activated matrices, thio and thiopropyl gels, chains of nitrophenyl chloroformate and N-hydroxysuccinyl chloroformate, or ethylene oxide acrylic supports (SIGMA).

Further, the support may also include an inorganic carrier of silicon oxide (such as silica gel, zeolite, diatomaceous earth or ammoniated glass), and the capsid agent is formed by the hydroxyl, carboxyl or amino group on the peptide and the reactive group on the carrier. covalently linked. Thus, in the appropriate context, a "support" may be a reaction plate, test tube, catheter, stent, balloon, prosthesis, medical device, polystyrene beads, magnetic beads, nitrocellulose tape, membrane, Walls or pores of tiny particles (such as latex particles), sheep (or other animal) erythrocytes, Duracyte( ^R) artificial cells, etc. Inorganic carriers such as silicon oxides (such as silica gel, zeolite, diatomaceous earth, and ammoniated glass) form covalent bonds with the capsid agent through hydroxyl, carboxyl, amino groups and reactive groups on the carrier. Carriers for in vivo use (eg, for prophylactic, therapeutic use) are preferably physiologically non-toxic and non-immunogenic. Such carriers include, but are not limited to, poly-L-lysine, poly-D-, L-alanine, and ^Chromosorb® (Johns-Manville, Products, Denver Co)

In other embodiments, a linker of suitable length, such as lambda linker, biotin-avidin (or streptavidin) is inserted between the capsid agent and the support to increase elasticity to overcome the steric position on the support. resistance. Capsid agents containing linkers of different lengths were screened using the capsid agent characterization assay described to determine the appropriate length of linker to ensure optimal function.

In other embodiments, the multimeric supports discussed above are combined with multimerized capsid agents to produce "multimerized-multimeric supports." Embodiments of multimerizing capsid agents are obtained by creating expression constructs comprising (eg linked together) two or more nucleotide sequences encoding VP2 or fragments thereof. The expressed fusion protein is one embodiment of a multimerized capsid agent, which is then attached to a support. A support containing multiples of such multimerization agents is referred to as a multimerization-multimer support. In some embodiments, linkers and spacers between the domains that make up the multimerization formulation can be bound to the support, preferably spacer linkers can be determined by capsid agent characterization assays.

In some embodiments, the capsid is placed on a prosthetic device to be implanted in a subject. With various types of prostheses such as stents and valves, a certain amount of tissue ingrowth is required to stabilize the implant. However, as a result of injury to the surrounding tissue during transplantation, cellular proliferation is greatly exacerbated, which can cause fibrotic hyperplasia or restenosis, which over time can compress the stent or alter the position of the valve. Prior art devices attempt to solve this problem by using radioactivity, but the success of the treatment and the possibility of exposing the body to the radioactive material released from the device makes this approach unpopular. Similarly, techniques like balloon revascularization often suffer from restenosis due to endothelial cell infiltration.

Attaching capsids to medical prostheses such as stents, valves or using porous catheters (such as balloon catheters used in angiogenesis) to deliver capsids, endothelial cell migration, proliferation, fibrotic hyperplasia, tissue Ingrowth and restenosis can be effectively inhibited. Furthermore, tissue ingrowth can be delayed by using capsid agents that are cleared by the immune system within a certain period of time after resolution of inflammation associated with the therapeutic process. Using the methods described above, capsid agents can be bound to various prostheses such as stents or valves through hydrophobic interactions or covalent linkages. Furthermore, catheters used in the prior art can be adapted to deliver capsid agents to the site of revascularization. Through the analysis of the profile of the capsid agent, the doctor can select the appropriate capsid agent-coated prosthesis for transplantation or the appropriate capsid agent according to the required cell inhibition time or tissue ingrowth delay time. Topical delivery of capsid agents by other means is also contemplated. For example, the growth of vascular endothelial cells can be inhibited by implanting a sustained-release composition adjacent to a stent, graft, valve, or other prosthesis or by delivering the drug to the site using an infusion pump or other suitable device. In addition to formulations that can be coated into medical devices and delivered as catheters, the capsid agents described herein can be formulated into medicines for the treatment and prevention of human diseases and conditions associated with P antigen-containing cell proliferation or migration. The following section discusses the many ways in which capsids can be formulated into pharmaceuticals and appropriate dosages determined. Production and dosing of prophylactic and therapeutic preparations

The capsid agents (eg, VP1, VP1/2, VP2 or fragments thereof) of the invention are useful in the treatment of subjects as a prophylactic measure for diseases and conditions, or in the treatment of subjects already infected with diseases. These pharmacologically active compounds are formulated according to the traditional methods of Galenic Pharmacy for administration to subjects, eg mammals including humans. Modified and unmodified active ingredients can be added to pharmaceutical formulations. Furthermore, methods for the production of therapeutic agents or drugs that deliver the pharmacologically active compounds of the present invention via several routes are also within the scope of the present invention, for example, in embodiments that are not limited to the use of DNA, RNA, and viral vectors that encode capsids . Nucleic acids encoding capsids can be administered alone or in combination with other active ingredients.

The compounds of the present invention may be used in admixture with conventional excipients, i.e. pharmaceutically acceptable for parenteral, enteral (e.g., oral) or topical application, without prejudice to the pharmacologically active compounds of the present invention Functional inorganic or organic carrier substances. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohols, acacia, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (such as lactose), amylose or starch, stearic acid Magnesium acid, talc, silicic acid, viscous paraffin, sesame oil, fatty acid monoglycerides, fatty acid diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. Many more suitable excipients are reported in the following literature, Remmington's Pharmaceutical Sciences, 15th Edition, Easton: Mack Press, 1405-1412, 1461-148 (1975); National Drug Collection, American Pharmaceutical Association (1975). Pharmaceutical preparations can be sterilized and, if desired, combined with active substances such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, flavoring agents and/or fragrance substances, etc. The compound does not produce ancillary reagent mixing that impairs the reaction.

The effective amount and method of administration of a particular pharmaceutical formulation will vary depending on the particular subject, the type of disease, the extent of the disease and other factors well known in the art. The efficacy and toxicity of these compounds can be determined according to standard pharmacological procedures in cell culture and animal assays, eg, ED50 (50% therapeutically effective dose). The aforementioned macaques or baboons are suitable experimental models. The results of cell culture assays and experimental animal studies were used to determine a range of dosage for use in humans. The dosage range of such compounds lies preferably within a range of circulating concentrations that include the ED50 without toxicity. The dosage within this range will vary with the type of capsid, the dosage form used, the sensitivity of the subject and the mode of administration.

Depending on the route of administration, normal doses may range from about 1 [mu]g to 100,000 [mu]g, up to a total dose of about 10 g. Suitable doses include 250 μg, 500 μg, 1 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1 g, 1.1 g , 1.2g, 1.3g, 1.4g, 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g and 10g. Additionally, in embodiments for topical administration, the capsid agent may be present in high concentrations. Molar concentrations of capsid agents may be employed in some embodiments. Desired concentrations for epidermal administration and/or coating of medical devices are from 100 [mu]M to 800 mM. Preferred concentrations for these embodiments are from 500 μM to 500 mM, for example, preferred concentrations for epidermal administration and/or coating of medical devices include 500 μM, 550 μM, 600 μM, 650 μM, 700 μM, 750 μM, 800 μM, 850 μM, 900 μM, 950 μM, 1 mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 300mM, 20 325mM, 350mM, 375mM, 400mM, 425mM, 450mM, 475mM and 500mM.

In some embodiments, doses of capsid agents preferably result in tissue or blood concentrations (or both) of about 0.1 [mu]M to 500 mM. Desired doses produce tissue or blood concentrations (or both) of about 1 [mu]M to 800 [mu]M. Preferably the dose produces a tissue or blood concentration (or both) greater than about 10 μM to about 500 μM, e.g., an amount of capsid agent is preferably required to achieve a tissue or blood concentration (or both) of 10 μM, 15 μM, 20 μM, 25 μM, 30 μM , 35μM, 40μM, 45μM, 50μM, 55μM, 60μM, 65μM, 70μM, 75μM, 80μM, 85μM, 90μM, 95μM, 100μM, 110μM, 120μM, 130μM, 140μM, 150μM, 160μM, 170μM, 180μM, 22000μM, , 240 μM, 250 μM, 260 μM, 280 μM, 300 μM, 320 μM, 340 μM, 360 μM, 380 μM, 400 μM, 420 μM, 440 μM, 460 μM, 480 μM, and 500 μM. Doses that produce tissue concentrations greater than 800 [mu]M, although not preferred, may be used in some embodiments of the invention. In order to maintain a stable tissue concentration similar to blood levels, capsid agents can also be infused continuously.

The precise dosage will be determined by the individual physician on a case-by-case basis. Dosage and dosage regimens will be adjusted in order to provide adequate levels of the active ingredient and maintain the desired effect. Other factors should also be considered, including the subject's condition, age and weight; diet, time and frequency of administration, drug combination, sensitivity of response, and tolerance/response to treatment. The short-acting active pharmaceutical composition is administered once a day, while the long-acting active pharmaceutical composition is administered every 2, 3, 4 days, every week or every 2 weeks. Depending on the half-life and clearance rate of the particular formulation, the pharmaceutical composition of the present invention is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times per day.

Routes of administration of the present invention include, but are not limited to, transdermal, parenteral, gastrointestinal, transbronchial, and alveolar. Transdermal administration is accomplished through creams, lotions, gels, etc. that allow the pharmaceutically active compound to penetrate the skin. Parenteral routes of administration include, but are not limited to, electrical or direct injection, such as direct injection into a central vein, intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection. Gastrointestinal routes of administration include, but are not limited to, oral and rectal administration. Bronchial and alveolar administration includes, but is not limited to, oral or nasal inhalation.

Compositions of the present invention containing pharmacologically active compounds suitable for transdermal administration include, but are not limited to, pharmaceutically acceptable suspensions that can be applied directly to the skin or incorporated into a protective carrier of a transdermal device (transdermal tablet). , oils, creams and ointments. Examples of suitable creams, ointments, etc. can be found in the Physician's Manual. Examples of such suitable transdermal devices are described in US Patent No. 4,818,540, Chinen et al., issued April 4, 1989, incorporated herein by reference.

Compositions of the present invention containing pharmacologically active compounds suitable for parenteral administration include, but are not limited to, pharmaceutically acceptable sterile isotonic solutions. These solutions include, but are not limited to, saline and phosphate buffered saline for central venous, intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection.

Compositions of the present invention containing pharmacologically active compounds suitable for bronchial and alveolar administration include, but are not limited to, various aerosol formulations for inhalation. Devices suitable for bronchial and alveolar administration are also embodiments of the invention, such devices include, but are not limited to nebulizers and vaporizers. The many forms of nebulizers and vaporizers currently in use are readily adapted to deliver the compositions of the present invention containing pharmacologically active compounds.

Compositions of the present invention containing pharmacologically active compounds suitable for gastrointestinal administration include, but are not limited to, pharmaceutically acceptable powders, pills and liquids for oral administration and suppositories for rectal administration. Because of the ease of application, gastrointestinal administration, especially oral administration, is a preferred embodiment. Once obtained, the medicament containing the capsid agent can be administered to a subject in need of treatment or prevention of a disease associated with the proliferation or migration of P antigen-containing cells.

Also included within the context of the invention are coatings for therapeutic devices such as prostheses, implants and instruments. Coatings suitable for the therapeutic device may be provided by gels or powders containing the shell agent, or by a polymeric coating in which the shell is suspended. Polymeric materials suitable for coating should be physiologically acceptable and capable of diffusing a therapeutically effective dose of the shell. Suitable polymers include, but are not limited to, polyurethane, polymethacrylate, polyamide, polyester, polyethylene, polypropylene, polystyrene, polytetrafluoroethylene, polyvinyl chloride, cellulose acetate, Silicone resin elastic materials, collagen and silk, etc. Such coatings are described, for example, in US Patent No. 4,612,337, Fox et al., issued September 16, 1986, incorporated herein by reference. In the following section, the inventors disclose several methods of treating diseases associated with the proliferation or migration of P antigen-containing cells which involve providing a medicament comprising a capsid. Treatment and Prevention

In several aspects of the invention, capsid agents, especially medicaments comprising capsid agents, are provided to a subject in need of treatment or prevention of a disease or condition associated with abnormal cell proliferation and/or cell migration. Methods of formulating a medicament that inhibits the growth and/or cell migration of P antigen-containing cells including, but not limited to, hematopoietic cells and endothelial cells are embodiments of the invention. That is, embodiments of the invention include the use of a capsid-containing medicament for inhibiting the growth and/or migration of P-antigen-containing cells, such as hematopoietic cells and endothelial cells.

In one embodiment, capsid agents may be used to suppress hematopoietic cells in a recipient subject prior to intrauterine stem cell transplantation. In a previous study of the tissue distribution of stem cells in the human fetus, it was estimated that transplantation of 5 x ¹⁰⁷ cells to the fetus during the second trimester yielded a donor-recipient ratio of approximately 1:1000-1: 10000. Such a low ratio does not provide a margin for the transplanted cells to compete with the recipient's own stem cells. (Westren et al., Am J Obstet Gynecol, 176:49 (1996)). To increase this rate and the success of stem cell transplantation, capsid agents can be given prior to transplantation to suppress the autologous stem cell population and thereby promote transplantation. In addition, treatment of donor stem cells with anti-P antigen monoclonal antibodies before transplantation can protect them from inhibition by capsid agents, resulting in better results. Therefore, one embodiment includes providing the patient with a capsid-containing medicament prior to stem cell transplantation. This method of treatment may be performed by identifying a subject in need of intrauterine stem cell transplantation and then providing the subject with a therapeutically effective amount of a capsid agent that inhibits the growth of hematopoietic cells.

Another similar aspect of the invention is a method of modulating non-severe myelosuppression prior to postnatal stem cell transplantation. Recently, approaches to the modulation of non-severe myelosuppression have received considerable attention because these regimens are less toxic to patients than standard regimens using high-dose chemotherapy. (Giral et al., Blood, 89:4531 (1997); Slavin et al., Blood, 91:756 (1998)). However, achieving complete donor hematopoietic chimerism in non-severe myelosuppressive therapy has not been very successful with existing techniques. By administering capsid agents prior to postnatal stem cell transplantation, the ratio of donor cells to recipient cells can be favorably altered to achieve donor hematopoietic cell chimerism without radiation. Accordingly, a method of non-severe myelosuppressive modulation can be achieved by identifying a subject in need of non-severe myelosuppressive modulation prior to postnatal stem cell transplantation, and then administering to the subject a therapeutically effective amount of a capsid agent.

Another aspect of the invention is a method of treating a subject suffering from a blood proliferative disorder (eg, polycythemia vera). Polycythemia vera (PVC) is a blood disorder caused by the uncontrolled proliferation of red blood cells in the bone marrow. Cells of other lineages (leukocytes and platelets) are also involved, but do not cause symptoms of similar severity. The disease occurs in middle-aged or elderly people (median age at diagnosis is 60 years), and the incidence in Sweden is 1.5 cases per 100,000 people. At present, there is no specific drug treatment, and the current measures are to relieve the symptoms of this slowly developing disease. The median survival time without treatment is very short. Young individuals, with optimal treatment, can live up to 20 years with a comparable quality of life.

By administering the capsid agent to patients suffering from PVC, the proliferation of hematopoietic cells can be inhibited, which is an effective treatment for this fatal disease. Accordingly, a method of treating PVC may be achieved by identifying a subject in need of treatment for PVC and then administering to the subject a therapeutically effective amount of a capsid agent. Since long-term treatment regimens are considered, the capsid agent used should preferably be one that elicits a minor immune response.

Yet another aspect of the invention is a method of treating a patient to inhibit the growth of endothelial cells. As noted above, a subject may experience undesired growth of endothelial cells, for example, following surgical injury, ie following implantation of a valve, stent or other repair or angioplasty. In addition, tumor formation and metastasis require endothelial cell growth and cell migration. Embodiments of the invention therefore include agents that inhibit cancer, and more precisely, angiogenesis and cell migration associated with metastasis.

Angiogenesis is the formation of new capillaries through the process of sprouting from existing blood vessels. Angiogenesis occurs during development and under many physiological and pathological conditions and is required for tissue growth, wound healing, female reproductive function, and a step in pathological processes such as hemangioma formation and eyeball neovascularization. However, much of the long-standing interest in angiogenesis stems from the discovery that solid tumors must undergo angiogenesis in order to grow beyond a critical volume. That is, tumors must recruit endothelial cells from the surrounding stroma to form their own endogenous microcirculation.

Administering capsid agents to cancer patients can inhibit the proliferation and migration of endothelial cells, so tumorigenesis and migration can be prevented. Accordingly, a method of inhibiting angiogenesis, tumorigenesis or cancer can be achieved by identifying a subject in need of inhibition of angiogenesis, tumorigenesis or cancer, and then administering to the patient a therapeutically effective amount of a capsid agent. Since long-term treatment regimens are a concern, the capsid agent used should preferably be one that elicits a minor immune response.

Additional embodiments of the invention include a kit comprising a capsid agent and written instructions for dosing and administration for inhibiting hematopoietic progenitor cells, the dose for inhibiting the growth of hematopoietic progenitor cells in said patient (such as a fetus) prior to stem cell transplantation and instructions for use, dosage and instructions for inhibiting the growth of endothelial cells in patients, and/or instructions for dosage and instructions for inhibiting hematopoietic proliferative disorders of P antigen-positive cells (such as hyperplasia vera) in patients.

While the invention has been described based on the embodiments and examples, it should be clear that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All documents cited herein are incorporated by reference.

SEQUENCE LISTING <110> Broliden, Kristina

Westgren, Magnue <120> Use of parvovirus capsid particles to inhibit cell proliferation and migration <130> TRIPEP.019A<150>SE 9,804,022-3<151>1998-11-28<160>1<170> FastSEQ for Windows Version 4.0<210>1<211>7<212>PRT<213>Artificial sequence<220><223>Peptide<400>1Lys Tyr Val Thr Gly Ile Asn1 5

Claims (12)

1. empty, noninfective reorganization B19 parvovirus capsid, B19 capsid protein or B19 capsid protein fragment are used to produce the purposes of the medicine that suppresses to contain antigenic cell growth of P or migration.

2. purposes according to claim 1, medicine wherein are to suppress the medicine of hematopoietic cell growth, endothelial cell growth or endothelial cell migration.

3. purposes according to claim 1, medicine wherein are the disorder of treatment blood hypertrophy, angiogenesis, tumor generation or the endotheliocyte medicines to the prosthetic appliance growth of implanting.

4. purposes according to claim 1, medicine wherein are the medicines of before the stem cell transplantation experimenter being treated.

5. purposes according to claim 4, experimenter wherein is a fetus.

6. an inhibition contains the method for the growth or the migration of the antigenic cell of P, comprise the following steps: to be selected from segmental a kind of capsid agent of B19 parvovirus capsid, B19 capsid protein and B19 capsid protein and described cells contacting, and measure inhibition this cell growth or cell migration.

7. method according to claim 6, cell wherein are hemopoietic source cell or endotheliocyte.

One kind before stem cell transplantation the treatment experimenter method, comprise the following steps: to identify the experimenter of the capsid agent that needs the growth of inhibition hematopoietic cell, the experimenter for these needs provides the capsid agent that is selected from B19 parvovirus capsid, B19 capsid protein and the segmental effective dose of B19 capsid protein then.

9. method for the treatment of the experimenter of hemopoietic hypertrophy disorder, comprise the following steps: to identify the experimenter of the capsid agent that needs the disorder of inhibition hemopoietic hypertrophy, the experimenter for these needs provides the capsid agent that is selected from B19 parvovirus capsid, B19 capsid protein and the segmental effective dose of B19 capsid protein then.

10. one kind is suppressed to organize the method for growing in the prosthese of implanting, comprising the following steps: to identify needs to suppress the experimenter of tissue to the capsid agent of the prosthese growth of implanting, and the experimenter for these needs provides the capsid agent that is selected from B19 parvovirus capsid, B19 capsid protein and the segmental effective dose of B19 capsid protein then.

11. the method that treatment or prophylaxis of tumours take place, comprise the following steps: to identify the experimenter of the capsid agent that needs the growth of inhibition hematopoietic cell, the experimenter for these needs provides the capsid agent that is selected from B19 parvovirus capsid, B19 capsid protein and the segmental effective dose of B19 capsid protein then.

12. test kit, it comprises and is selected from B19 parvovirus capsid, B19 capsid protein and the segmental capsid agent of B19 capsid protein, and be used for hemopoietic progenitor cell growth inhibited, endothelial cell growth suppresses or treatment hemopoietic hypertrophy is used explanation with dosage to the experimenter.

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