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Title:  Purposeful movement of human migratory cells away from an agent source

United States Patent:  6,448,054

Issued:  September 10, 2002

Inventors:  Poznansky; Mark C. (Charlestown, MA); Luster; Andrew T. (Wellesley, MA); Scadden; David T. (Weston, MA)

Assignee:  The General Hospital Corporation (Boston, MA)

Appl. No.:  546153

Filed:  April 7, 2000

Abstract

This invention relates to methods and compositions for modulating movement of eukaryotic cells with migratory capacity. More specifically, the invention relates to methods and compositions for modulating movement of cells of hematopoietic, neural, epithelial, or mesenchymal origin, in a specific site in a subject. The foregoing are useful, inter alia, in the treatment of conditions characterized by a need to modulate migratory-cell movement associated with specific sites in a subject. More specifically, specific sites include sites of inflammation and modulation of migratory-cell movement is movement away from an agent source, or repulsion. Other sites include tumor sites, sites of pathogenic infection, and germ cell bearing sites.

SUMMARY OF THE INVENTION

We describe herein the isolation of agents with migratory-cell repellant activity (hereinafter "fugetactic agents" and "fugetactic activity," and/or "chemo-fugetaxis"). We also describe the identification of previously isolated agents as fugetactic agents at defined concentrations. The invention provides pharmaceutical compositions containing the foregoing fugetactic agents, and various therapeutic and diagnostic methods utilizing the foregoing fugetactic agents. The invention also provides isolated fugetactic polypeptides and agents which bind such polypeptides, including antibodies. The foregoing can be used, inter alia, in the treatment of conditions characterized by a need to modulate migratory-cell movement in specific sites in a subject. Important such sites include inflammation sites. The invention also provides methods for identifying agents useful in the modulation of such fugetactic activity.

According to one aspect of the invention, a thymic stromal-cell isolate is provided that repels immune cells when contacted with immune cells, and does not contain an immune cell repelling concentration of SDF-1.alpha.. In certain embodiments, the immune cells that are repelled by the thymic stromal-cell isolate are mature T-cells. In some embodiments, the thymic stromal-cell isolate comprises a polypeptide, and it is the polypeptide that repels the immune cells. In certain embodiments, the thymic stromal-cell isolate mediates its repellant effects through a G-protein transduction pathway. In further embodiments, the thymic stromal-cell isolate is a supernatant, or fraction thereof, of thymic stromal-cells. In yet further embodiments, the thymic stromal-cell isolate is a substantially pure polypeptide. In some embodiments, the polypeptide is not genistein inhibited, and/or is wortmannin inhibited.

According to another aspect of the invention, a substantially pure organic agent that repels mature T-cells at effective concentrations but not at concentrations lower than effective concentrations, is provided. The agent is present at effective concentrations in the supernatant of confluent thymic stromal-cells, cultured under standard conditions for a period of at least one hour, and the culture conditions include a ratio of 107 cells/10 ml medium. The agent is also heat sensitive, protease sensitive, and it elutes-off an ion exchange column with 0.5M NaCl at pH 6.3, and is not SDF-1.alpha.. In an important embodiment, the agent is a polypeptide.

According to a further aspect of the invention, a pharmaceutical composition is provided. The pharmaceutical composition includes a thymic stromal-cell isolate according to any of the foregoing embodiments, in a pharmaceutically effective amount to repel immune cells, and a pharmaceutically acceptable carrier.

In a further aspect, the invention provides another pharmaceutical composition. The pharmaceutical composition includes a substantially pure organic agent that repels mature T-cells at effective concentrations but not at concentrations lower than effective concentrations, according to any of the foregoing embodiments, in a pharmaceutically effective amount to repel mature T-cells, and a pharmaceutically acceptable carrier. In some embodiments, the agent is not SDF-1.alpha.. In an important embodiment, the agent is a polypeptide.

According to another aspect of the invention, an isolated binding polypeptide which binds selectively to a thymic stromal-cell isolate or agent in the isolate with fugetactic activity according to any of the foregoing embodiments, is also provided. In certain embodiments, the isolated binding polypeptide is an antibody or an antibody fragment such as a Fab fragment, a F(ab)2 fragment, or a fragment including a CDR3 region selective for the thymic stromal-cell isolate or agent in the isolate with fugetactic activity.

According to a further aspect, the invention provides another isolated binding polypeptide which binds selectively to a substantially pure organic agent that repels mature T-cells according to any of the foregoing embodiments. In certain embodiments, the isolated binding polypeptide is an antibody or an antibody fragment such as a Fab fragment, a F(ab)2 fragment, or a fragment including a CDR3 region selective for the thymic stromal-cell isolate or agent in the isolate with fugetactic activity.

According to another aspect of the invention, another pharmaceutical composition is provided. This pharmaceutical composition includes an isolated binding polypeptide according to any of the foregoing embodiments which binds selectively to: (i) a thymic stromal-cell isolate, or (ii) a substantially pure organic agent that repels mature T-cells, or (iii) SDF-1.alpha., (according to any of the foregoing embodiments), in a pharmaceutically effective amount to inhibit repulsion of immune cells and/or T cells by the thymic stromal-cell isolate, the substantially pure organic agent that repels mature T-cells, and/or SDF-1.alpha., respectively, and a pharmaceutically acceptable carrier.

According to the invention a method for isolating a stromal-cell derived fugetactic agent is provided. The method involves preparing a culture of stromal-cells, isolating a supernatant suspected of containing a fugetactic agent from the culture of stromal-cells, fractionating the supernatant into a plurality of fractions, contacting a fraction from the plurality of fractions with a cell with migratory capacity, measuring movement of the cell with migratory capacity and determining whether the movement of the cell with migratory capacity is movement away from the fraction, wherein movement of the cell with migratory capacity away from the fraction is indicative of the presence of a fugetactic agent in the fraction. In certain embodiments, the stromal-cells are thymic stromal-cells and the cell with migratory capacity is a hematopoietic cell.

In certain embodiments, the stromal-cells are isolated from an immune-privileged site/tissue. In preferred embodiments, the cell with migratory capacity is a hematopoietic cell.

In any of the foregoing embodiments, the fraction from the plurality of fractions is undiluted or concentrated.

According to another aspect of the invention, a method of inhibiting migration of immune cells to a specific site in a subject is provided. The method involves locally administering to a specific site in a subject in need of such treatment a fugetactic agent in an amount effective to inhibit migration of immune cells to the specific site in a subject. In certain embodiments the specific site is a site of inflammation. In other embodiments, when the specific site is the site of inflammation, the method further comprises co-administering a non-fugetactic agent that inhibits migration of immune cells to the site of inflammation in the subject. In certain embodiments, the non-fugetactic agent includes an anti-inflammatory agent and/or an immunosuppressant.

In certain embodiments, the subject has an autoimmune disease. In preferred embodiments, the autoimmune disease includes rheumatoid arthritis, uveitis, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, Crohn's disease, Guillain-Barre syndrome, psoriasis, thyroiditis, Graves' disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, systemic lupus erythematosus. In further embodiments, the subject has multiple sclerosis, an abscess, a transplant, an implant, atherosclerosis, and/or myocarditis.

In any of the foregoing aspects and embodiments of the invention, the fugetactic agent is a CXCR-4 ligand. In certain embodiments, CXCR-4 ligands include, but are not limited to, HIV-1IIIB gp120, small molecules T134 and MD3010, and/or T22 ([Tyr5,12,Lys7]-polyphemusin II). In some embodiments, the fugetactic agent is SDF-1.alpha. at a concentration higher than about 1 .mu.g/ml, HIV-1IIIB gp120 at a concentration higher than about 200 ng/ml, non-diluted thymic stromal-cell-derived medium, concentrated thymic stromal-cell-derived medium, or thymic stromal-cell-derived polypeptide factor. In certain embodiments, the thymic stromal-cell-derived polypeptide factor mediates its chemorepellant effects through a G-protein transduction pathway. In other embodiments, the thymic stromal-cell-derived polypeptide factor is not genistein inhibited. In further embodiments, the thymic stromal-cell-derived polypeptide factor is wortmannin inhibited.

According to one aspect of the invention, a HepG2-cell isolate is provided that repels immune cells when contacted with immune cells. HepG2 is a cell line derived from human hepatocarcinoma (liver cancer). In certain embodiments, the immune cells that are repelled by the HepG2-cell isolate are mature T-cells. In some embodiments, the HepG2-cell isolate is a supernatant, or fraction thereof, of HepG2 cells. In further embodiments, the HepG2-cell isolate comprises a polypeptide, and it is the polypeptide that repels the immune cells. In yet further embodiments, the HepG2-cell isolate is a substantially pure polypeptide. A preferred such substantially pure polypeptide present in a HepG2-cell isolate is the polypeptide previously identified as inter-.alpha.-trypsin inhibitor heavy-chain II precursor (ITI H2) (SEQ ID NO: 1, GenBank Acc. No. P19823), or fragments thereof. In further embodiments, the substantially pure polypeptide present in a HepG2-cell isolate is the polypeptide having an amino acid sequence according to any one of GenBank Acc. Nos.: IYHU2, NP-- 034712, NP-- 002207, Q61703, P97279, O02668, CAA72308 (Y11545), BAA13939 (D89286), S54354, CAA49842(X70392), AAA60558(M18193), CAA30160(X07173), 1409219A, or AAA59195(M33033)(all members of the ITI H2 family of polypeptides), or fragments thereof.

According to another aspect of the invention, a substantially pure organic agent that repels mature T-cells at effective concentrations but not at concentrations lower than effective concentrations, is provided. The agent is present at effective concentrations in the supernatant of confluent HepG2 cells, cultured under standard conditions for a period of at least one hour, and the culture conditions include a ratio of 107 cells/10 ml medium. The agent is also heat sensitive, protease sensitive, and it elutes-off an ion exchange heparin column with 0.3M NaCl at pH 7.0. In an important embodiment, the agent is a polypeptide. In one embodiment, the polypeptide has a molecular weight of about 110 kD on a silver-stained SDS-PAGE gel. A preferred polypeptide is the polypeptide previously identified as inter-.alpha.-trypsin inhibitor heavy-chain II precursor (ITI H2) (SEQ ID NO: 1, GenBank Acc. No. P19823), or fragments thereof.

According to a further aspect of the invention, a pharmaceutical composition is provided. The pharmaceutical composition includes a HepG2-cell isolate according to any of the foregoing embodiments, in a pharmaceutically effective amount to repel immune cells, and a pharmaceutically acceptable carrier.

In a further aspect, the invention provides another pharmaceutical composition. The pharmaceutical composition includes a substantially pure organic agent that repels mature T-cells at effective concentrations but not at concentrations lower than effective concentrations, according to any of the foregoing embodiments relating to HepG2-cell isolates, in a pharmaceutically effective amount to repel mature T-cells, and a pharmaceutically acceptable carrier. In an important embodiment, the agent is a polypeptide.

According to another aspect of the invention, an isolated binding polypeptide which binds selectively to a HepG2-cell isolate or agent in the HepG2-cell isolate with fugetactic activity according to any of the foregoing embodiments, is also provided. In certain embodiments, the isolated binding polypeptide is an antibody or an antibody fragment such as a Fab fragment, a F(ab)2 fragment, or a fragment including a CDR3region selective for the HepG2-cell isolate or agent in the HepG2-cell isolate with fugetactic activity.

According to a further aspect, the invention provides another isolated binding polypeptide which binds selectively to a substantially pure organic agent that repels mature T-cells according to any of the foregoing embodiments relating to HepG2-cell isolates. In certain embodiments, the isolated binding polypeptide is an antibody or an antibody fragment such as a Fab fragment, a F(ab)2 fragment, or a fragment including a CDR3region selective for the HepG2-cell isolate or agent in the HepG2-cell isolate with fugetactic activity.

According to another aspect of the invention, another pharmaceutical composition is provided. This pharmaceutical composition includes an isolated binding polypeptide according to any of the foregoing embodiments which binds selectively to a HepG2-cell isolate (according to any of the foregoing embodiments), in a pharmaceutically effective amount to inhibit repulsion of immune cells and/or T cells by the HepG2-cell isolate, and a pharmaceutically acceptable carrier.

According to one aspect of the invention, a Kaposi's Sarcoma Herpes Virus (KSHV) infected-cell isolate is provided that repels immune cells when contacted with immune cells. In certain embodiments, the immune cells that are repelled by the KSHV infected-cell isolate are mature T-cells. In some embodiments, the KSHV infected-cell isolate is a supernatant, or fraction thereof, of KSHV infected-cells, preferably of chronically infected KSHV cells. Preferred chronically infected KSHV cells are cell lines BCBL-1 and VG-1. In further embodiments, the KSHV infected-cell isolate comprises a polypeptide, and it is the polypeptide that repels the immune cells. In yet further embodiments, the KSHV infected-cell isolate is a substantially pure polypeptide.

According to another aspect of the invention, a substantially pure organic agent that repels mature T-cells at effective concentrations but not at concentrations lower than effective concentrations, is provided. The agent is present at effective concentrations in the supernatant of confluent KSHV infected-cells, cultured under standard conditions for a period of at least one hour, and the culture conditions include a ratio of 107 cells/10 ml medium. The agent is also heat sensitive, and/or protease sensitive. In an important embodiment, the agent is a polypeptide.

According to a further aspect of the invention, a pharmaceutical composition is provided. The pharmaceutical composition includes a KSHV infected-cell isolate according to any of the foregoing embodiments, in a pharmaceutically effective amount to repel immune cells, and a pharmaceutically acceptable carrier.

In a further aspect, the invention provides another pharmaceutical composition. The pharmaceutical composition includes a substantially pure organic agent that repels mature T-cells at effective concentrations but not at concentrations lower than effective concentrations, according to any of the foregoing embodiments relating to KSHV infected-cell isolates, in a pharmaceutically effective amount to repel mature T-cells, and a pharmaceutically acceptable carrier. In an important embodiment, the agent is a polypeptide.

According to another aspect of the invention, an isolated binding polypeptide which binds selectively to a KSHV infected-cell isolate or agent in the KSHV infected-cell isolate with fugetactic activity according to any of the foregoing embodiments, is also provided. In certain embodiments, the isolated binding polypeptide is an antibody or an antibody fragment such as a Fab fragment, a F(ab)2 fragment, or a fragment including a CDR3 region selective for the KSHV infected-cell isolate or agent in the KSHV infected-cell isolate with fugetactic activity.

According to a further aspect, the invention provides another isolated binding polypeptide which binds selectively to a substantially pure organic agent that repels mature T-cells according to any of the foregoing embodiments relating to KSHV infected-cell isolates. In certain embodiments, the isolated binding polypeptide is an antibody or an antibody fragment such as a Fab fragment, a F(ab)2 fragment, or a fragment including a CDR3 region selective for the KSHV infected-cell isolate or agent in the KSHV infected-cell isolate with fugetactic activity.

According to another aspect of the invention, another pharmaceutical composition is provided. This pharmaceutical composition includes an isolated binding polypeptide according to any of the foregoing embodiments which binds selectively to a KSHV infected-cell isolate (according to any of the foregoing embodiments), in a pharmaceutically effective amount to inhibit repulsion of immune cells and/or T cells by the KSHV infected-cell isolate, and a pharmaceutically acceptable carrier.

According to another aspect of the invention, a method for promoting migration of cells away from a specific site in a subject is provided. The method involves locally-administering to a specific site in a subject in need of such treatment a fugetactic agent in an amount effective to promote migration of cells away from the specific site in the subject. In some embodiments. the specific site is an inflammation site. In certain embodiments, the specific site is a germ cell-containing site. In further embodiments, the specific site is an area surrounding a tumor, but not the immediate area surrounding the tumor. In some important embodiments, the specific site is a transplanted tissue and/or an implant. Important fugetactic agents, cell-types, and so on, are as described above. In any of the foregoing embodiments, preferred cells are immune cells.

According to a further aspect of the invention, a method for distinguishing mature T-cells from immature T-cells in vitro is provided. The method involves contacting a fugetactic agent with a mixed population of mature and immature T-cells and measuring cell movement relative to the fugetactic agent, wherein movement of cells away from the fugetactic agent is indicative of mature T-cells.

According to another aspect of the invention, a method of screening for fugetactic agents is provided. The method involves contacting an agent suspected of being a fugetactic agent with a cell with migratory capacity, measuring movement of the cell with migratory capacity relative to the agent, and determining whether the movement of the cell with migratory capacity is movement away from the agent, wherein movement of the cell with migratory capacity away from the agent is indicative of the agent being a fugetactic agent. In certain embodiments, the cell with migratory capacity is a hematopoietic cell. In some embodiments, the cell with migratory capacity is a neural cell. In further embodiments, the cell with migratory capacity is an epithelial cell. In yet further embodiments, the cell with migratory capacity is a mesenchymal cell. In some embodiments, the cell with migratory capacity is an embryonic stem cell. In certain embodiments, the cell with migratory capacity is a germ cell. In another embodiment, the agent depicted of being a fugetactic agent is known to be a chemoattractant at a defined concentration and a fugetactic agent at a concentration higher than the defined concentration. In preferred embodiments such fugetactic agents include cytokines. In yet other embodiments, the agent suspected of being a fugetactic agent is an agent present in a biological fluid. In preferred embodiments, the biological fluid includes synovial fluid, cerebral spinal fluid, fellopian tube fluid, seminal fluid, ocular fluid, pericardial fluid, pleural fluid, inflammatory exudate and ascitic fluid. In other preferred embodiments, the agent suspected of being a fugetactic agent is an agent present in a tumor cell culture supernatant, tumor cell eluate and/or tumor cell lysate. In some embodiments, the tumor cells are HepG2 cells and the cell with migratory capacity is a hematopoietic cell. In further preferred embodiments, the agent suspected of being a fugetactic agent is an expression product of a cDNA library.

According to another aspect of the invention, a method of repelling immune cells from a material surface is provided. The method involves coating a material surface with an amount of a fugetactic agent effective to repel immune cells from the material surface. In certain embodiments, the material surface is part of an implant. The material comprising the implant may be synthetic material or organic tissue material. Important fugetactic agents, cell-types, and so on, are as described above.

According to another aspect of the invention, a method of screening for an agent that modulates migratory cell-specific fugetactic activity of a fugetactic agent is provided. The method involves forming a mixture comprising a cell with migratory capacity, a fugetactic agent and a candidate anti-fugetactic agent incubating the mixture under conditions which permit binding of the candidate anti-fugetactic agent to the fugetactic agent, determining the level of a test fugetactic activity of the fugetactic agent and the candidate anti-fugetactic agent on the cells with migratory capacity, and comparing the level of the test fugetactic activity to a control level of fugetactic activity determined in the absence of the candidate anti-fugetactic agent. In some embodiments, reduction in the test fugetactic activity level relative to the control level of fugetactic activity, indicates that the candidate anti-fugetactic agent is a lead compound for an anti-fugetactic agent which inhibits the fugetactic activity of the fugetactic agent. In certain other embodiments, an increase in the test fugetactic activity level relative to the control level of fugetactic activity, indicates that the candidate anti-fugetactic agent is a lead compound for an anti-fugetactic agent which increases the fugetactic activity of the fugetactic agent. In certain other embodiments, the fugetactic agent is SDF-1.alpha. at a concentration higher than about 1 .mu.g/ml, HIV-1IIIB gp120 at a concentration higher than about 200 ng/ml, non-diluted thymic stromal-cell-derived medium, concentrated thymic cell-derived medium or thymic stromal-cell-derived polypeptide factor, non-diluted HepG2-cell-derived medium, concentrated HepG2-cell-derived medium or HepG2-cell-derived polypeptide factor, inter-.alpha.-trypsin inhibitor heavy-chain II precursor (ITI H2), non-diluted Kaposi's Sarcoma Herpes Virus infected cell-derived medium, concentrated Kaposi's Sarcoma Herpes Virus infected cell-derived medium, or Kaposi's Sarcoma Herpes Virus infected cell-derived polypeptide factor.

In other embodiments, the cell with migratory capacity is a hematopoietic cell, a neural cell, an epithelial cell, a mesenchymal cell, an embryonic stem cell, a cell involved in angiogenesis (blood vessel formation), or a germ cell.

According to yet another aspect of the invention, a method of enhancing an immune response in a subject having a condition that involves a specific site, is provided. The method involves locally administering to a specific site in a subject in need of such treatment an anti-fugetactic agent in an amount effective to inhibit immune cell-specific fugetactic activity at the specific site in the subject. In some embodiments, the specific site is a site of a pathogenic infection. In certain embodiments, the specific site is a germ cell-containing site. In further embodiments, the specific site is an area immediately surrounding a tumor. In certain embodiments, the anti-fugetactic agent is a cytokine binding agent. In other embodiments, the cytokine binding agent is an anti-cytokine antibody or a cytokine agonist. In a preferred embodiment the cytokine is SDF-1.alpha..

According to a further aspect of the invention, a method of inhibiting tumor cell metastasis in a subject, is provided. The method involves locally administering to a tumor site in a subject in need of such treatment an anti-fugetactic agent in an amount effective to inhibit metastasis of tumor cells from the tumor site in the subject. In certain embodiments, the anti-fugetactic agent is a cytokine binding agent. In other embodiments, the cytokine binding agent is an anti-cytokine antibody or a cytokine agonist. In a preferred embodiment the cytokine is SDF-1.alpha..

According to another aspect of the invention, a method of inhibiting endothelial cell migration to a tumor site in a subject, is provided. The method involves locally administering to an area surrounding a tumor site in a subject in need of such treatment a fugetactic agent in an amount effective to inhibit endothelial cell migration to the tumor site in the subject. In certain embodiments, the area surrounding the tumor site is not immediate to the tumor site. Important fugetactic agents are as described above.

According to a further aspect of the invention, a method of contraception in a subject, is provided. The method involves administering to a subject in need of such treatment, an anti-fugetactic agent in an amount effective to inhibit germ cell migration in the subject. In certain embodiments, the anti-fugetactic agent is a cytokine binding agent. In some embodiments, the cytokine binding agent is an anti-cytokine antibody or a cytokine agonist. In a preferred embodiment the cytokine is SDF-1.alpha..

According to another aspect of the invention, a method of treating infertility and premature labor, including premature delivery and impending miscarriage, is provided. The method involves administering to a subject in need of such treatment a fugetactic agent in an amount effective to inhibit immune cells from migrating close to a germ cell in the subject. In further embodiments, the administration is local to a germ cell-containing site of the subject.

In any of the foregoing aspects and embodiments of the invention where the fugetactic agent is identified as a polypeptide (e.g., SDF-1.alpha.., HIV-1IIIB gp120, ITI H2, etc.), fragments of such polypeptide useful according to the invention are only those with fugetactic activity. A person of ordinary skill in the art could easily determine which fragments have such activity by first creating deletions of the full length polypeptides using methods well known in the art, and testing such fragments for their fugetactic activity according to the teachings of the present invention.

Claim 1 of 11 Claims

We claim:

1. A method of repelling immune cells from a specific site in a subject, comprising:

locally administering to a specific site in a subject in need of such treatment a fugetactic agent in an amount effective to repel immune cells from the specific site in the subject.
 


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