EC-3, an inhibitor of .alpha.4.beta.1 and .alpha.4.beta.7 integrins Number:6,818,617 from the United States Patent and Trademark Office (PTO) owispatent This invention relates to the identification, purification, and characterization of a novel heterodimeric disintegrin, EC-3, from Echis carinatus viper venom. EC-3 inhibits .alpha.4 integrins in an RGD-independent manner. The invention further relates to methods of using EC-3, or a biologically active fragment or derivative thereof, to inhibit the interaction between cells expressing .alpha.4 integrins and cellular ligands.<H integrins, inhibitor, EC, 3, an, inhibit, 6818617.html, Patent, pto, 6818617

Title: EC-3, an inhibitor of .alpha.4.beta.1 and .alpha.4.beta.7 integrins

Abstract: This invention relates to the identification, purification, and characterization of a novel heterodimeric disintegrin, EC-3, from Echis carinatus viper venom. EC-3 inhibits .alpha.4 integrins in an RGD-independent manner. The invention further relates to methods of using EC-3, or a biologically active fragment or derivative thereof, to inhibit the interaction between cells expressing .alpha.4 integrins and cellular ligands.

Patent Number: 6,818,617 Issued on 11/16/2004 to Niewiarowski, et al.

Inventors: Niewiarowski; Stefan (Narbeth, PA); Marcinkiewicz; Cezary (Philadelphia, PA)
Assignee: Temple University- Of The Commonwealth System of Higher Education (Philadelphia, PA)

Appl. No.: 485323

Filed: February 7, 2000

PCT Filed: August 13, 1998

PCT NO: PCT/US98/16719

PCT PUB.NO.: WO99/13898

PCT PUB. Date: March 25, 1999

Current U.S. Class: 514/12 ; 514/13; 514/14; 514/15; 514/16; 514/17; 514/18; 530/324; 530/325; 530/326; 530/327; 530/328; 530/329; 530/330; 530/331; 530/344; 530/412; 530/415; 530/856

Field of Search: 514/12,13,14,15,16,17,18 530/324,325,326,327,328,329,330,331,344,412,415,856 424/183.1,173.1,184.1 435/7.1,7.8

References Cited [Referenced By]

U.S. Patent Documents


5318899	June 1994	Scarborough et al.
5510332	April 1996	Kogan et al.

Foreign Patent Documents


WO 96/00581	Jan., 1996	WO

Other References

Vanderslice et al. 1997. A Cyclic hexapeptide is a potent antagonist of alpha4 integrins. J. Immunology 158:1710-1718.* .
The Merck Index, Eighth Edition,. Edited by P. Stecher et al., Rahway, NJ, Merck & Co., Inc., 1968, p. 500. .
Siigur et al., cDNA Cloning and Deduced Amino Acid Sequence of Fibrinolytic Enzyme (Lebetase) from Vipera Lebetina Snake Venom. Biochemical and Biophysical Research Communications, 224(1): 229-336 (1996). .
Marchinkiewicz et al., "EC-3, A Novel MLD-Dependent Distintegrin From E. Carinatus Is A Potent Antagonist of .alpha.4 Integrins", Molecular Biology of the Cell Supp., vol. 8, Abstract 1682 (Nov. 1997). .
Marcinkiewicz et al., "Isolation and characterization of two novel dimeric disintegrins: EC-3 and EMF-10 inhibiting interaction of lymphoid cells with VCAM-1", Blood, 90(10: 59b Suppl. 1, Abstract 2966 (Nov. 15, 1997)..

Primary Examiner: Wax; Robert A.
Attorney, Agent or Firm: Drinker Biddle & Reath LLP

Government Interests

REFERENCE TO GOVERNMENT GRANT

The invention described herein was made, in part, in the course of work supported by the National Institutes of Health under Grant Nos. HL 45486 and HL 19055. The government has certain rights in the invention.

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 60/055,825, filed 15 Aug. 1997, and U.S. Provisional Application Ser. No. 60/055,957, filed 18 Aug. 1997.

Claims

We claim:

1. A substantially purified EC3 protein isolated from Echis carinatus venom, characterized by: (a) an apparent molecular mass of about 14,762 Da, as determined by electrospray ionization mass spectrometry; (b) elution from a C-18 high performance liquid chromatography column at about 40% acetonitrile; and (c) the ability to inhibit adhesion of Jurkat cells to vascular cell adhesion molecule-1.

2. A substantially purified EC-3A peptide isolated from EC-3 protein which has been reduced and alkylated, characterized by: (a) a molecular mass of about 8478 Da in its ethylpyridylated form, as determined by electrospray ionization mass spectrometry; (b) elation from a C-18 high performance liquid chromatography column at about 42% acetonitrile; and (c) the ability to inhibit adhesion of K562 cells to fibronectin.

3. A substantially purified EC-3B peptide isolated from EC-3 protein which has been reduced and alkylated with vinylpyridine, characterized by: (a) a molecular mass of about 7950 Da in its carboxymethylated form, as determined by electrospray ionization mass spectrometry; (b) elution from a C-18 high performance liquid chromatography column at about 46% acetonitrile; and (c) the ability to inhibit adhesion of Jurkat cells to vascular cell adhesion molecule-1.

4. The substantially purified EC-3A peptide of claim 2 comprising a sequence represented by SEQ ID NO:19 or a fragment characterized by the ability to inhibit adhesion of K562 cells to fibronectin thereof.

5. The substantially purified EC-3A peptide of claim 2 comprising a sequence represented by SEQ ID NO:2.

6. The substantially purified EC-3B peptide of claim 3 comprising a sequence represented by SEQ ID NO:20, or a fragment characterized by the ability to inhibit adhesion of Jurkat cells to vascular cell adhesion molecule-1 thereof.

7. The substantially purified EC-3B peptide of claim 3 comprising a sequence represented by SEQ ID NO:3.

8. The substantially purified EC-3 protein of claim 1 comprising two subunits, wherein one subunit comprises the sequence SEQ ID NO:19 or a fragment characterized by the ability to inhibit adhesion of K562 cells to fibronectin thereof and one subunit comprises the sequence SEQ ID NO:20 or a fragment characterized by the ability to inhibit adhesion of Jurkat cells to vascular cell adhesion molecule-1 thereof.

9. The substantially purified EC-3B peptide of claim 6, wherein the biologically active fragment comprises a peptide represented by an amino acid sequence X-Y-Met-Leu-Asp-Z, where X is H or a blocking group, Y is zero or more amino acids, and Z is OH or zero or more amino acids.

10. The substantially purified EC-3B peptide of claim 9, wherein the biologically active fragment comprises a peptide having from about 3 to about 20 amino acids.

11. The substantially purified EC-3B peptide of claim 9, wherein the biologically active fragment is represented by SEQ ID No: 16.

12. The substantially purified EC-3B peptide of claim 9, wherein the biologically active fragment is represented by SEQ ID No. 14.

13. A substantially purified echistatin polypeptide represented by SEQ ID NO: 9, in which the Arg-Gly-Asp residues at positions 24-26 are replaced by M et-Leu-Asp.

14. A method of isolating a peptide from a venom, wherein the peptide binds to an integrin of interest, comprising: (a) dissolving venom in a solvent, (b) centrifuging the dissolved venom to remove high molecular weight proteins, (c) fractionating the supernatant from step (b), (d) immobilizing the fractions from step (c) on a solid support, (e) adding detectably labeled cells that express the integrin of interest to the immobilized fractions, (f) detecting the number of cells bound to each immobilized fraction, and (g) isolating peptide from those fractions which showed enhanced cell binding in step(f).

15. A composition comprising a pharmaceutically acceptable carrier and the protein or peptide of any of claims 1-12, or a pharmaceutically acceptable salt thereof.

16. A method of inhibiting the binding of an .alpha.4 integrin to VCAM-1 comprising contacting a cell that expresses the .alpha.4 integrin with an effective amount of a protein or peptide according to one of claims 1-12, or a pharmaceutically acceptable salt thereof.

17. The method of claim 16 wherein the integrin is .alpha.4.beta.1 or .alpha.4.beta.7.

18. A method of inhibiting the binding of an .alpha.4.beta.7 integrin to MadCAM-1 comprising contacting a cell that expresses .alpha.4.beta.7 with an effective amount of a protein or peptide according to one of claims 1-12, or a pharmaceutically acceptable salt thereof.

19. A method of inhibiting the binding of an .alpha.4 integrin to CS-1 comprising contacting a cell that expresses the .alpha.4 integrin with an effective amount of a protein or peptide according to one of claims 1-12, or a pharmaceutically acceptable salt thereof.

20. A method of inhibiting the interaction between cells expressing an .alpha.4 integrin and VCAM-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition according to claim 15.

21. A method of inhibiting the interaction between cells expressing an .alpha.4 integrin and MadCAM-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition according to claim 15.

22. A method of inhibiting the interaction between cells expressing an .alpha.4 integrin and CS-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition according to claim 15.

23. A substantially purified EC-3A peptide characterized by: (a) a sequence having greater than 90% sequence identity with SEQ ID NO:2; and (b) the ability to inhibit adhesion of K562 cells to fibronectin.

24. A substantially purified EC-3B peptide characterized by: (a) a sequence having greater than 90% sequence identity with SEQ ID NO:3; and (b) the ability to inhibit adhesion of Jurkat cells to VCAM-1.

Description

FIELD OF THE INVENTION

This invention generally relates to methods and compositions for modulating cell adhesion and for inhibiting the interaction between integrins and their ligands. In particular, the invention relates to peptides that selectively inhibit .alpha.4 integrins.

BACKGROUND OF THE INVENTION

A. Integrins

Integrins are a family of cell surface proteins that mediate adhesion between cells (cell-cell adhesion) and between cells and extracellular matrix proteins (cell-ECM adhesion). Integrins are heterodimeric structures composed of noncovalently bound .alpha. and .beta. subunits. In humans there are at least 15 different .alpha. and eight different .beta. subunits, and these can combine to form proteins with diverse biological activities and ligand specificities.

The integrins play important roles in many diverse biological processes including platelet aggregation, tissue repair, angiogenesis, bone destruction, tumor invasion, inflammation, and immune reactions. The integrins are, therefore, important targets for therapeutic intervention in human disease.

Integrin .alpha.IIb.beta.3 (glycoprotein IIb/IIIa complex) binds fibrinogen on the platelet surface and mediates platelet aggregation. Integrin .alpha.v.beta.3 is predominantly expressed on endothelial cells and plays an important role in angiogenesis. Integrin .alpha.v.beta.3 is also expressed on osteoclasts and participates in bone destruction. Integrin .alpha.5.beta.1 is widely distributed on a variety of cells; it plays a critical role in cell adhesion to extracellular matrix as well as in the formation of tissues and organs during embryonic development. All three integrins .alpha.IIb.beta.3, .alpha.v.beta.3 and .alpha.5.beta.1 recognize RGD sequences in the adhesive ligands.

One example of the therapeutic targeting of integrins is the use of integrin inhibitors as antithrombotic agents. Peptides and peptidomimetics that block the adhesion of GPIIb/IIIa to fibrinogen can prolong bleeding times and prevent thrombotic occlusion in vivo. One group of naturally occurring peptides that inhibit platelet aggregation by interfering with fibrinogen binding to GPIIb/IIIa has been called the "disintegrins."

B. Disintegrins

The disintegrins are a family of low molecular weight cysteine-rich peptides that have been isolated from the venom of various snakes (reviewed in Niewiarowski et al., Seminars in Hematology 31(4):289-300 (1994)). Most disintegrins described to date contain an RGD motif. RGD is a recognition site for many integrins, and disintegrins inhibit fibrinogen binding to GPIIb/IIIa, as well as the binding of other ligands to RGD-dependant integrins on the surface of cells. Peptides modeled on the structure of disintegrins have potential clinical applications in the prevention and treatment of coronary thrombosis, stroke, and other vascular diseases.

The first disintegrin described in the literature, trigramin, was identified and characterized on the basis of its ability to block platelet aggregation and inhibit fibrinogen binding to .alpha.IIb.beta.3. Trigramin contains 72 amino acids including 12 cysteines, is linked by S-S bonds, and contains an RGD sequence. Subsequently several other RGD containing viper venom disintegrins of similar size were isolated.

A 49 amino acid disintegrin, called echistatin, has been isolated from the venom of Echis carinatus (Gan et al., J. Biol. Chem. 263:19827-32 (1988)). Like other disintegrins, echistatin contains an RGD sequence and inhibits GPIIb/IIIa binding of fibrinogen. Echistatin has been called a "promiscuous disintegrin" because it blocks .alpha.IIb.beta.3, .alpha.v.beta.3 and .alpha.5.beta.1 with similar potency.

NMR studies on RGD-containing disintegrins shows that the RGD sequence is located in a mobile loop joining two strands of .beta. sheet protruding from the protein core (reviewed in Niewiarowski et al., Semin. Hematol. 31:289-300 (1994)). The disulfide bonds around the RGD sequence maintain the hair-pin loop conformation in each peptide, and this conformation seems to be important for potency and selectivity.

The disintegrin eristostatin, originally described as a potent inhibitor of .alpha.IIb.beta.3, also inhibits human melanoma cell (MV3) metastases in immune deficient mice. It has been suggested that this effect is mediated by altering the function of an .alpha.4 integrin expressed on MV3 cells (Danen et al, Exp. Cell Res. 238:188-196 (1998)).

C. The .alpha.4 Integrins

The .alpha.4 integrins, .alpha.4.beta.1 and .alpha.4.beta.7, are expressed on leukocytes and lymphoid cells, and play a major role in inflammation and auto-immune diseases.

The .alpha.4.beta.1 integrin (which has also been called VLA4, very late activation antigen 4) mediates cell adhesion to vascular cell adhesion molecule-1 (VCAM-1), an adhesive molecule belonging to the IgG superfamily which is expressed on endothelial cells at sites of inflammation. The integrin .alpha.4.beta.1 also binds to alternatively spliced variants of fibronectin containing connecting segment 1 (CS-1).

The .alpha.4.beta.7 integrin binds to the gut homing receptor mucosa addressin cell adhesion molecule-1 (MadCAM-1) and to a lesser extent to CS-1 and VCAM-1.

Cytokine activated leukocytes express .alpha.4.beta.1 and .alpha.4.beta.7 integrins. Interaction of these integrins with VCAM-1 or MadCAM-1 (which are also up-regulated by cytokines) on endothelium mediates capillary infiltration by leukocytes, which can lead to tissue and organ destruction. Selectins and .beta.2 integrins also contribute to this process. Altevost et al. reported that the .alpha.4 subunit itself is a ligand for .alpha.4.beta.1 and .alpha.4.beta.7 integrins (J. Exp. Med. 182:345-55 (1995)), suggesting that .alpha.4 integrins may play a role in leukocyte communication during the immune response.

Activation and up-regulation of .alpha.4.beta.1 or .alpha.4.beta.7 on lymphocytes or macrophages is believed to play a significant role in the progression of many disease states, including insulin dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, arteriosclerosis, asthma, allergy, organ rejection, and restenosis of arteries after surgery or angioplasty. The .alpha.4 integrins are therefore targets for therapeutic intervention in a variety of inflammatory, auto-immune, and other diseases.

D. The .alpha.4 Integrins as Therapeutic Targets

There are several animal models of inflammatory and autoimmune diseases in which endothelial infiltration by lymphocytes and organ destruction are blocked by anti-.alpha.4 monoclonal antibodies. As an example, anti-.alpha.4 antibody inhibits lymphocyte infiltration of Langerhans islets in NOD mice, thus preventing development of spontaneous insulin dependent diabetes (Yang et al., Proc. Natl. Acad. Sci. USA 4 91:12604-08 (1994)). Anti-.alpha.4 monoclonal antibodies have also shown in vivo efficacy in animal models of asthma (Abraham et at., J. Clin. Invest. 93:776 (1994)), multiple sclerosis (Yednock et al., Nature 356:63 (1992)), inflammatory bowel disease (Podoisky et al., J. Clin. Invest. 92:372 (1993)), contact hypersensitivity (Chisholm et al., Eur. J. Immunol. 23:682 (1993)), and cardiac allograft rejection (Isobe et al., J. Immunol. 153:5810 (1994)).

There have been several attempts to isolate naturally occurring or to develop synthetic inhibitors of .alpha.4.beta.7 and .alpha.4.beta.1. Synthetic inhibitors that have been reported include cyclic RGD peptides and short peptides based on the sequences of MadCAM-1, VCAM-1, and CS-1. These peptides are typically active in vitro at the micromolar level.

Molossi et al. (J. Clin. Invest. 95:2601-10 (June 1995)) reported that blockade of .alpha.4.beta.1 (VLA-4) integrin binding to fibronectin with CS-1 peptide reduces accelerated coronary arteriopathy in rabbit cardiac allografts. The sequence of the CS-1 peptide was phenylacetic acid-Leu-Asp-Phe-d-Pro-amide.

Kogan et al. (WO 96100581) reported that cyclic peptides modeled after a portion of the CS-1 peptide inhibited the binding of .alpha.4.beta.1 integrin to VCAM-1 at concentrations of peptide less than about 10 .mu.M.

Kogan et al. (U.S. Pat. No. 5,510,332) reported that a peptide comprising the LDV domain of CS-1 peptide inhibited the binding of .alpha.4.beta.1 integrin to VCAM-1 with an IC.sub.50 of 30 .mu.M.

Shroff et al. (Bioorganic and Medicinal Chemistry Letters 6(21):2495-2500 (1996)) reported a series of peptides based on the N-terminal domain of MAdCAM-1 inhibited the binding of HUT78 cells activated with Mn.sup.++ to MAdCAM-1 with an IC.sub.50 of 5 to >1000 .mu.M. Cyclic peptides based on an N-terminal conserved motif in VCAM-1 have also been reported to inhibit VCAM/.alpha.4.beta.1 mediated leukocyte adhesion (Wang et al., Proc. Natl. Acad. Sci. USA 92:5714 (1995)).

Cyclic RGDS (SEQ ID NO:15) peptides have been reported as strong inhibitors of .alpha.4.beta.1 and .alpha.4.beta.7 integrins (Cardarelli et al., J. Biol. Chem. 269:18668-73 (1994); Yang et al. Eur. J. Immunol. 28:995-1004 (1998)).

Vanderslice et al. (J. Immunol. 158:1710-18 (1997)) recently reported that a cyclic peptide based on the LDV sequence of CS-1 inhibited .alpha.4.beta.1 dependent binding of lymphocytes to VCAM and CS-1 with an IC.sub.50 of 1-3 .mu.M.

There is a need for potent and specific inhibitors of .alpha.4 integrins, and for methods of specifically inhibiting the binding of .alpha.4 integrins to cellular ligands.

SUMMARY OF THE INVENTION

The present invention relates to the discovery of the EC-3 protein, a heterodimeric disintegrin that is an extremely potent antagonist of .alpha.4 integrins. EC-3 proteins and peptides inhibit adhesion of cells expressing .alpha.4 integrins in an RGD-independent manner.

The invention provides a substantially purified EC-3 protein, isolated from E. carinatus venom and characterized by: (a) an apparent molecular weight of about 14,762 Da, as determined by electrospray ionization mass spectrometry; (b) elution from a C-18 HPLC column at about 40% acetonitrile; and (c) the ability to inhibit adhesion of Jurkat cells to VCAM-1.

The invention also provides a substantially purified EC-3A peptide, isolated from EC-3 protein which has been reduced and alkylated, and characterized by: (a) a molecular mass of about 8478 Da in its ethylpyridylated form, as determined by electrospray ionization mass spectrometry; (b) elution from a C-18 HPLC column at about 42% acetonitrile; and (c) the ability to inhibit adhesion of K562 cells to fibronectin.

The invention further provides a substantially purified EC-3B peptide, isolated from EC-3 protein which has been reduced and alkylated with vinylpyridine, and characterized by: (a) a molecular mass of about 7950 Da in its carboxymethylated form, as determined by electrospray ionization mass spectrometry; (b) elution from a C-18 HPLC column at about 46% acetonitrile; and (c) the ability to inhibit adhesion of Jurkat cells to VCAM-1.

One preferred embodiment of the invention is a substantially purified EC-3A peptide comprising the sequence SEQ ID NO:19, or a biologically active fragment or derivative thereof. A more preferred embodiment is a substantially purified peptide comprising the sequence SEQ ID NO:2, or a biologically active fragment or derivative thereof.

Another preferred embodiment of the invention is a substantially purified EC-3B peptide comprising the sequence SEQ ID NO:20, or a biologically active fragment or derivative thereof. A more preferred embodiment is a substantially purified peptide comprising the sequence SEQ ID NO:3, or a biologically active fragment or derivative thereof.

A further embodiment of the invention is a substantially purified EC-3 protein comprising two subunits, wherein one subunit comprises the sequence SEQ ID NO:19 or a biologically active fragment or derivative thereof and one subunit comprises the sequence SEQ ID NO:20 or a biologically active fragment or derivative thereof.

The invention is also directed to a biologically active peptide fragment of EC-3B having the sequence X-Y-Met-Leu-Asp-Z, where X is H or a blocking group, Y is zero or more amino acids, and Z is OH or zero or more amino acids. In some preferred embodiments the biologically active peptide is from about 3 to about 20 amino acids. In a more preferred embodiment the peptide has the sequence SEQ ID NO:16. In another more preferred embodiment the peptide has the sequence SEQ ID NO:14.

Another aspect of the invention is a substantially purified nucleic acid encoding a protein or peptide according to the invention. One embodiment of the invention is a vector comprising a nucleic acid encoding a protein or peptide according to the invention. Another embodiment of the invention is a recombinant cell comprising a nucleic acid encoding a protein or peptide according to the invention.

The invention further provides an antibody which specifically binds to a protein or peptide according to the invention. The antibody may be a monoclonal antibody or a polyclonal antibody or an antibody fragment that is capable of binding antigen. One aspect of the invention is a hybridoma that produces a monoclonal antibody which specifically binds to a protein or peptide according to the invention.

Another aspect of the invention is a substantially purified echistatin polypeptide in which the Arg-Gly-Asp residues at positions 24-26 are replaced by Met-Leu-Asp, or a biologically active fragment or derivative thereof.

The invention further provides a method of isolating a peptide that binds to an integrin of interest from venom comprising: (a) dissolving venom in a solvent, (b) centrifuging the dissolved venom to remove high molecular weight proteins, (c) fractionating the supernatant from step (b), (d) immobilizing the fractions from step (c) on a solid support, (e) adding detectably labeled cells which express the integrin of interest to the immobilized fractions, (f) detecting the number of cells bound to each immobilized fraction, and (g) isolating peptide from those fractions which showed enhanced cell binding in step (f).

Another aspect of the invention is a composition comprising a pharmaceutically acceptable carrier and a protein, peptide, or nucleic acid according to the invention.

The invention encompasses a method of inhibiting the binding of an .alpha.4 integrin to VCAM-1 comprising contacting a cell that expresses the .alpha.4 integrin with an effective amount of a protein or peptide according to the invention. In preferred embodiments the integrin is .alpha.4.beta.1 or .alpha.4.beta.7.

The invention also encompasses a method of inhibiting the binding of .alpha.4.beta.7 integrin to MadCAM-1 comprising contacting a cell that expresses .alpha.4.beta.7 with an effective amount of a protein or peptide according to the invention.

The invention further encompasses a method of inhibiting the binding of an .alpha.4 integrin to CS-1 comprising contacting a cell that expresses the .alpha.4 integrin with an effective amount of a protein or peptide according to the invention.

One preferred embodiment of the invention is a method of inhibiting the interaction between cells expressing an .alpha.4 integrin and VCAM-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein or peptide according to the invention.

Another preferred embodiment of the invention is a method of inhibiting the interaction between cells expressing an .alpha.4 integrin and MadCAM-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein or peptide according to the invention.

A further preferred embodiment of the invention is a method of, inhibiting the interaction between cells expressing an .alpha.4 integrin and CS-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein or peptide according to the invention.

The invention is also directed to the use of a protein, peptide, or nucleic acid according to the invention for the preparation of a medicament for inhibiting the interaction between cells expressing an .alpha.4 integrin and its ligands.

Other aspects and advantages of the present invention are described in the drawings and in the following detailed description of the preferred embodiments thereof.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the HPLC fractionation of E. carinatus venom on a reverse phase C-18 HPLC column eluted with an acetonitrile gradient (dashed line). Fractions containing the disintegrin echistatin (EC1), the echicetin protein (which binds to GPIb/IX receptors on platelets, EC10) and EC3 are indicated.

FIG. 2 shows the effect of selected fractions obtained from Echis carinatus venom on adhesion of Jurkat cells to immobilized VCAM-1 (FIG. 2A) and K562 cells to immobilized fibronectin (FIG. 2B).

FIG. 3 shows the elution profile of reduced and alkylated EC-3 from a reverse phase HPLC column eluted with an acetonitrile gradient (dashed line). Fractions containing EC3A and EC3B are indicated.

FIG. 4 shows the amino acid sequences of EC-3 subunits EC-3A (SEQ ID NOs:19 and 2; FIG. 4A) and EC-3B (SEQ ID Nos:20 and 3; FIG. 4B), which were established by N-terminal sequencing of reduced and ethylpyridylethylated subunits and by sequencing of peptides isolated from digests with endoproteinase Lys-C. EC-3B was also digested by CNBr and two peptides were isolated.

FIG. 5 shows a comparison of the amino acid sequence of EC3A (SEQ ID NO:2), EC3B (SEQ ID NO:3), with two short disintegrins eristostatin (SEQ ID NO:8) and echistatin (SEQ ID NO:9), with two medium size disintegrins flavoridin (SEQ ID NO:10) and kistrin (SEQ ID NO:11), and with a disintegrin domain of Le3 (SEQ ID NO:7), a metallproteinase from Vipera lebetina venom. Boxes show the positions of conserved cysteine residues. The typical active site of the disintegrins (RGD) as well as the corresponding position in EC-3A(VGD), EC-3B (MLD), and Le3 (VGD) are underlined.

FIG. 6 shows the effect of EC-3 on Jurkat cell adhesion to immobilized VCAM-1 in the presence (filled circles) or absence (open circles) of 1 mM Mn.sup.++.

FIG. 7 shows the effect of EC-3 on CHO (.alpha.4+, .alpha.5-) cell adhesion to immobilized VCAM-1.

FIG. 8 shows the effect of EC3 on the adhesion of CHO cells transfected with different integrins. Adhesion experiments were performed using Chinese hamster ovary (CHO) cells transfected with different human integrins. Open circles show the effect of EC-3 on the adhesion of CHO cells transfected with .alpha.4 to immobilized VCAM-1; closed circles: CHO cells transfected with .alpha.IIb.beta.3 to fibrinogen; open triangles: CHO cells transfected with .alpha.v.beta.3 to vitronectin; and closed triangles: non-transfected CHO cells expressing hamster .alpha.5.beta.1 to fibronectin. Error bars represent S.D. from three experiments.

FIG. 9 shows the effect of RGD peptides and HP2/1 on Jurkat cell-EC-3 interactions. FIG. 9A shows the effect of GRGDSP (SEQ ID NO:12), GRGESP (SEQ ID NO:13) and HP 2/1 monoclonal antibody on the adhesion of Jurkat cells to immobilized EC-3. FIG. 9B shows the effect of EC-3 on the binding HP 2/1 monoclonal antibody to cells expressing .alpha.4.beta.1 integrin. Error bars represent the S.D. from three experiments.

FIG. 10 shows the adhesion of CHO cells transfected with .alpha.4 and with .alpha.4 inactive G190A mutant to immobilized EC-3 and to immobilized HP2/1 antibody. The number of adhered cells is shown for: (a) CHO cells transfected with wild-type .alpha.4, (b) CHO cells transfected with G190A .alpha.4, (c) control CHO-K1 cells. The results with EC-3 are shown as open bars, and the results with HP2/1 are shown as closed bars. Error bars represent S.D. from three experiments.

FIG. 11 shows the effect of reduced and ethylpyridylethylated EC3A (open circles) and EC3B (closed circles) on adhesion of Jurkat cells to immobilized VCAM-1 (FIG. 11A) and K562 cells to immobilized fibronectin (FIG. 11B). Error bars indicate S.D. from three experiments.

FIG. 12 shows the effect of EC-3B derived peptides on Jurkat cell adhesion to immobilized VCAM-1. The inhibitory effect of MLDG peptide (SEQ ID NO:14) is shown by open circles, RGDS peptide (SEQ ID NO:15) by closed circles, cyclic *CKRAMLDGLNDYC* (SEQ ID NO:16) by open triangles and *CKRAMLAGLNDYC* (SEQ ID NO:17) by closed triangles. Error bars indicate S.D. from three experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery of a novel protein, EC-3, which is a potent inhibitor of .alpha.4.beta.1 and .alpha.4.beta.7 integrins. The EC-3 protein was purified from Echis carinatus suchoreki viper venom. EC-3 inhibits adhesion of cells expressing .alpha.4.beta.1 and .alpha.4.beta.7 integrins to natural ligands VCAM-1 and MadCAM-1 with IC.sub.50 =5-300 nM. Its blocking activity resembles that of some monoclonal antibodies but is 2 to 3 orders of magnitude higher than most synthetic antagonists. The EC-3 inhibitory effect is not RGD dependent but is competed by HP 2/4 and HP 2/1 monoclonal antibodies, which recognize epitopes on .alpha.4.

EC-3 comprises two types of subunits, EC-3A and EC-3B, which are covalently linked. The EC-3A and EC-3B subunits show a high degree of homology with other viper venom disintegrins, including the alignment of conserved cysteines. The conserved RGD motif of integrins is replaced, however, by VGD in EC-3A and by MLD in EC-3B.

A. Abbreviations and Short Forms

The following abbreviations and short forms are used in this specification.

"BSA" is bovine serum albumin.

"CHO cells" are Chinese hamster ovary cells.

"CMFDA" is 5-chloromethylfluorescein diacetate.

"CS-1" is connecting segment 1 of fibronectin.

"DBA" is direct binding assay.

"EC-3 protein" (also called EC3 protein) is a heterodimeric protein comprising an EC-3A (also called EC3A) subunit and an EC-3B (also called EC3B) subunit. The term "EC-3 peptide" (also called EC3 peptide) as used herein encompasses the EC-3 protein as well as the EC-3A and EC-3B subunits, and fragments thereof.

"ECM" is extracellular matrix.

"ep" is ethylpyridylethylated. The ethylpyridylethylated forms of EC-3A and EC-3B are designated "epEC3A" and "epEC3B."

"HBSS" is Hanks' balanced salt solution.

"HPLC" is high performance liquid chromatography.

"Ig" is immunoglobulin.

"IC.sub.50 " is the concentration of a biologically active agent such as a peptide, which inhibits 50% of the activity obtained in the absence of the agent.

"Mab" is monoclonal antibody.

"MadCAM-1" is mucosal addressin cell adhesion molecule-1, a member of the IgG protein superfamily having a mucin component. MadCAM-1 is a cellular ligand for the .alpha.4.beta.7 integrin.

"MLD" is the one letter designation of the amino acid sequence Met-Leu-Asp, which occurs in the EC-3 protein.

"NOD mouse" is a Non Obese Diabetic mouse.

"PBS" is phosphate buffered saline solution.

"RGD" is the one letter designation for the amino acid sequence Arg-Gly-Asp. RGD is a recognition site for many disintegrins. A related recognition site is "KGD" which is the one letter designation for Lys-Gly-Asp.

"TFA" is trifluoroacetic acid.

"VCAM-1" is vascular cell adhesion molecule-1, a member of the IgG protein superfamily which is a cellular ligand for the .alpha.4.beta.1) and .alpha.4.beta.7 integrins.

"VLA-4" is very late activation antigen 4.

B. Definitions

The following definitions, of terms used throughout the specification, are intended as an aid to understanding the scope and practice of the present invention.

A "peptide" is a compound comprised of amino acid residues covalently linked by peptide bonds. Amino acids have the following general structure: ##STR1##

Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group. Peptides comprising a large number of amino acids are sometimes called "polypeptides". The EC-3A and EC-3B subunits of EC-3 are peptides.

A "protein" is a polypeptide which plays a structural or functional role in a biological system. Proteins comprise one or more peptides. EC-3 is a protein.

"Fibrinogen" is a blood plasma glycoprotein, which is involved in platelet aggregation and fibrin formation.

"Integrins" are a family of heterodimeric cell surface proteins which mediate adhesion between cells and between cells and extracellular matrix proteins.

"Disintegrins" are a family of peptides isolated from snake venoms which inhibit the binding of various ligands to integrins.

"Echistatin" is a 49 amino acid disintegrin isolated from Echis carinatus venom.

"Homology" means similarity of sequence reflecting a common evolutionary origin. Peptides or proteins are said to have homology, or similarity, if a substantial number of their amino acids are either (1) identical, or (2) have a chemically similar R side chain. Nucleic acids are said to have homology if a substantial number of their nucleotides are identical.

"Substantial amino acid sequence homology" means an amino acid sequence homology greater than about 30 percent, preferably greater than about 60%, more preferably greater than about 80%, and most preferably greater than about 90 percent.

"Substantially purified peptide" or "substantially purified protein" means a peptide or protein which is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or peptides, nucleic acids, carbohydrates, lipids). "Substantially purified" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.

A "biologically active fragment" of an EC-3 peptide is a fragment derived from an EC-3 peptide which retains at least one biological activity of the EC-3 peptide.

A "biologically active derivative" of an EC-3 peptide is any analogue, variant, derivative, or mutant which is derived from an EC-3 peptide, which has substantial amino acid sequence homology with the EC-3 peptide, and which retains at least one biological property of the EC-3 peptide. Different variants of EC-3 peptides may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for EC-3 peptides, or may involve differential splicing or post-translational modification. The skilled artisan can produce derivatives having single or multiple amino acid substitutions, deletions, additions, or replacements. These derivatives may include, inter alia: (a) derivatives in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) derivatives in which one or more amino acids are added to an EC-3 peptide, (c) derivatives in which one or more of the amino acids includes a substituent group, and (d) derivatives in which the EC-3 peptide is fused with another peptide such as serum albumin. The techniques for obtaining these derivatives, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.

Biologically active fragments and biologically active derivatives of EC-3 peptides are intended to be included within the scope of this invention.

A "blocking group" is any group capable of blocking the N-terminal amino group of a peptide. A preferred blocking group is an alkyl group; a most preferred blocking group is an acetyl group.

A "nucleic acid" is a polymeric compound comprised of covalently linked subunits called nucleotides. Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA. The sequence of nucleotides that encodes a protein is called the sense sequence.

"Isolated nucleic acid" means a nucleic acid which is substantially free of those compounds that are normally associated therewith in its natural state. "Isolated" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.

The phrase "a nucleic acid which hybridizes at high stringency" means that the hybridized nucleic acids are able to withstand a washing under high stringency conditions. An example of high stringency washing conditions for DNA-DNA hybrids is 0.1.times.SSC, 0.5% SDS at 68.degree. C. Other conditions of high stringency washing are known to persons having ordinary skill in the art.

"Regulatory region" means a nucleic acid sequence which regulates the expression of a nucleic acid. A regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid. (a homologous region) or may include sequences of a different origin (responsible for expressing different proteins or even synthetic proteins). In particular, the sequences can be sequences of eukaryotic or viral genes or derived sequences which stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner. Regulatory regions include origins of replication, RNA splice sites, enhancers, transcriptional termination sequences, signal sequences which direct the polypeptide into the secretory pathways of the target cell, and promoters.

A regulatory region from a "heterologous source" is a regulatory region which is not naturally associated with the expressed nucleic acid. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences which do not occur in nature, but which are designed by one having ordinary skill in the art.

"Operatively linked to a regulatory region" means that the peptide or polypeptide coding region is connected to transcriptional and translational regulatory sequences in such a way as to permit polypeptide expression when the appropriate molecules (such as activator proteins and polymerases) are present in a cell or cell free system.

A "vector" is any means for the transfer of a nucleic acid according to the invention into a host cell. The term "vector" includes both viral and nonviral means for introducing the nucleic acid into a prokaryotic or eukaryotic cell in vitro, ex vivo, or in vivo. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers. Viral vectors include retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirus vectors. In addition to a nucleic acid according to the invention, a vector may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).

A "recombinant cell" is a cell which contains a nucleic acid which is not naturally present in the cell. "Recombinant cell" includes higher eukaryotic cells such as mammalian cells, lower eukaryotic cells such as yeast cells, prokaryotic cells, and archaebacterial cells.

"Antibody" as used herein includes monoclonal and polyclonal antibodies as well as fragments capable of binding antigen, including but not limited to Fab and F(ab).sub.2 fragments.

"Pharmaceutically acceptable carrier" includes diluents and fillers which are pharmaceutically acceptable for methods of administration, may be sterile, and may be aqueous or oleaginous suspensions formulated using suitable dispersing or wetting agents and suspending agents. The particular pharmaceutically acceptable carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the composition, the particular mode of administration, and standard pharmaceutical practice.

C. Proteins and Peptides

The present invention provides substantially purified EC-3 peptides, including the EC-3 protein, the EC-3A and EC-3B peptides, and biologically active fragments and derivatives of EC-3, EC-3A, and EC-3B. The invention also provides methods of isolation and peptides isolated from other venoms having biological activity similar to EC-3.

The peptides of the present invention may be recombinant peptides, natural peptides, or synthetic peptides. Each peptide is characterized by a reproducible single molecular weight and/or multiple set of molecular weights, chromatographic response and elution profile, amino acid composition and sequence, and biological activity.

The peptides of the present invention may be isolated from natural sources, such as viper venom, using the methods disclosed herein.

The peptides of the invention may also be chemically synthesized, using, for example, solid phase synthesis methods.

In conventional solution phase peptide synthesis, the peptide chain can be prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence. The use of various N-protecting groups, e.g., the carbobenzyloxy group of the t-butyloxycarbonyl group, various coupling reagents (e.g., dicyclohexylcarbodiimide or carbonyldimidazole, various active esters, e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide, and the various cleavage reagents, e.g., trifluoroacetic acid (TFA), HCl in dioxane, boron tris-(trifluoracetate) and cyanogen bromide, and reaction in solution with isolation and purification of intermediates is well-known classical peptide methodology. The preferred peptide synthesis method follows conventional Merrifield solid-phase procedures. See Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963) and Science 50: 178-85 (1965). Additional information about the solid phase synthesis procedure can be had by reference to the treatise by Steward and Young ("Solid Phase Peptide Synthesis," W. H. Freeman & Co., San Francisco, 1969, and the review chapter by Merrifield in Advances in Enzymology 32:221-296, F. F. Nold, Ed., Interscience Publishers, New York, 1969; and Erickson and Merifield, The Proteins 2:255 et seq. (ed. Neurath and Hill), Academic Press, New York, 1976. The synthesis of peptides by solution methods is described in Neurath et al., eds. (The Proteins, Vol. II, 3d Ed., Academic Press, NY (1976)).

Crude peptides may be purified using preparative HPLC. The amino terminus may be blocked according, for example, to the methods described by Yang et al. (FEBS Left. 272:61-64 (1990)).

Peptide synthesis includes both manual and automated techniques employing commercially available peptide synthesizers. Fragments and derivatives of EC-3 peptides may be prepared by chemical synthesis and biological activity can be tested using the methods disclosed herein.

Alternatively, the peptides of the invention may be prepared utilizing recombinant DNA technology, which comprises combining a nucleic acid encoding the peptide thereof in a suitable vector, inserting the resulting vector into a suitable host cell, recovering the peptide produced by the resulting host cell, and purifying the polypeptide recovered.

In some embodiments, the peptides of the present invention may be used in the form of a pharmaceutically acceptable salt.

Suitable acids which are capable of forming salts with the peptides include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like.

Suitable bases capable of forming salts with the peptides include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanol-amines (e.g., ethanolamine, diethanolamine and the like).

D. Nucleic Acids

The present invention provides substantially purified nucleic acids which encode peptides according to the invention.

The techniques of recombinant DNA technology are known to those of ordinary skill in the art. General methods for the cloning and expression of recombinant molecules are described in Maniatis (Molecular Cloning, Cold Spring Harbor Laboratories, 1982), and in Sambrook (Molecular Cloning, Cold Spring Harbor Laboratories, Second Ed., 1989), and in Ausubel (Current Protocols in Molecular Biology, Wiley and Sons, 1987), which are incorporated by reference.

Having the EC-3A and EC-3B amino acid sequences disclosed herein, one of skill in the art can isolate or prepare a gene that encodes the peptide of interest. Synthetic genes may be synthesized directly on a DNA synthesizer, or may be synthesized as complementary oligonucleotides which are ligated together to form the synthetic gene. Alternatively, the native gene encoding the peptides of the invention may be isolated from genomic or cDNA libraries. As an example, based upon the amino acid sequences disclosed herein, one of skill in the art can prepare suitable oligonucleotide probes and polymerase chain reaction (PCR) primers, which can be used to screen a cDNA library prepared from E. carinatus venom glands. Positive clones are purified and sequenced to confirm their identity.

The nucleic acids encoding EC-3 peptides may be operatively linked to one or more regulatory regions. Regulatory regions include promoters, polyadenylation signals, translation initiation signals (Kozak regions), termination codons, peptide cleavage sites, and enhancers. The regulatory sequences used must be functional within the cells of the vertebrate to be immunized. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art.

Promoters that may be used in the present invention include both constitutive promoters and regulated (inducible) promoters. The promoters may be prokaryotic or eukaryotic depending on the host. Among the prokaryotic (including bacteriophage) promoters useful for practice of this invention are lacI, lacZ, T3, T7, lambda P.sub.r, P.sub.l, and trp promoters. Among the eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g. MDR type, CFTR, factor VIII), tissue-specific promoters (e.g. actin promoter in smooth muscle cells), promoters which respond to a stimulus (e.g. steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, Ela, and MLP promoters. Tetracycline-regulated transcriptional modulators and CMV promoters are described in WO 96/01313, U.S. Pat. Nos. 5,168,062 and 5,385,839, the entire disclosures of which are incorporated herein by reference.

Examples of polyadenylation signals that can be used in the present invention include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals.

Fragments and derivatives of EC-3 peptides may be prepared using recombinant DNA technology. The biological activity of the fragments and derivatives can be assayed using the methods disclosed herein.

E. Antibodies

The present invention provides antibodies against EC-3 peptides. These antibodies may be monoclonal antibodies or polyclonal antibodies. The present invention includes chimeric, single chain, and humanized antibodies, as well as Fab fragments and the products of an Fab expression library.

Polyclonal antibodies may be generated against the intact protein or peptide, or against a fragment, derivative, or epitope of the protein or peptide. Antibodies may be obtained following the administration of the protein, polypeptide, fragment, derivative, or epitope to an animal, using the techniques and procedures known in the art.

Monoclonal antibodies may be prepared using the method of Mishell, B. B., et al., Selected Methods In Cellular Immunology, (W. H. Freeman, ed.) San Francisco (1980). Briefly, a peptide of the present invention is used to immunize spleen cells of Balb/C mice. The immunized spleen cells are fused with myeloma cells. Fused cells containing spleen and myeloma cell characteristics are isolated by growth in HAT medium, a medium which kills both parental cells, but allows the fused products to survive and grow.

Antibodies may be used to purify the peptides according to the invention, using immunoaffinity techniques which are well known by those of skill in the art.

F. Isolation of Related Peptides from Venoms

The present invention provides a method for isolating peptides that bind to an integrin of interest from the venom of snakes or other organisms. This method comprises the steps of:

(a) dissolving venom in a solvent,

(b) centrifuging the dissolved venom to remove high molecular weight proteins,

(c) fractionating the supernatant from step (b),

(d) immobilizing the fractions from step (c) on a solid support,

(e) adding detectably labeled cells which express the integrin of interest to the immobilized fractions,

(f) detecting the number of cells bound to each immobilized fraction, and

(g) isolating peptide from those fractions which showed enhanced cell binding in step (f).

This method may be used to isolate peptides from the venom of species including but not limited to vipers and other snakes including Agkistrodon acutus, Agkistrodon halys blomhoffi, Agkistrodon contortrix mokasen, Bitis arietans, Bitis caudalis, Bitis gabonica, Bitis g. rhinoceros, Bothrops asper, Bothrops alternata, Bothrops atrox, Bothrops cotiara, Bothrops jararaca, Bothrops newiedi, Bothrops medusa, Bothrops schlegli, Cerastes cerastes, Cerastes vipera, Crotalus adamanteus, C. atrox, C. basilicus, C. durissus totonatacus, C. h. horridus, C. m. molossus, C. ruber, C. scutalatus, C. v. cereberus, C. v. helleri, C. v. lutosus, C. v. oreganus, Echis carinatus sochurecki, Eristicophis macmahoni, Pseudocerastes persicus, Sistrurus m. barbouri, Sistrurus c. terqeminus, Trimeresurus flavoviridis, Trimeresurus gramineus, Vipera lebetina, Vipera ammondytes, Vipera palastinae, and Vipera r. russelli, leeches, ticks, and other organisms.

G. Methods of Treatment

The present invention provides methods of inhibiting the binding of .alpha.4.beta.1 or .alpha.4.beta.7 integrins to a ligand. A preferred embodiment is a method of inhibiting the interaction between cells expressing .alpha.4.beta.1 and/or .alpha.4.beta.7 integrins and a ligand, in a patient in need of such treatment, comprising administration of a therapeutically effective amount of a composition comprising one or more peptides according to the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. As an example, Non Obese Diabetic mice treated with EC-3 show reduced levels of lymphocyte infiltration in pancreatic islets (see Example 7, below).

Pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents, excipients, solvents, adjuvants, or vehicles, for parenteral injection, for intranasal or sublingual delivery, for oral administration, for rectal or topical administration or the like. The compositions are preferably sterile and nonpyrogenic. Examples of suitable carriers include but are not limited to water, saline, dextrose, mannitol, lactose, or other sugars, lecithin, albumin, sodium glutamate cystein hydrochloride, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyehtylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

The pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like). If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) may be used. The compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.

The compositions may be administered by any convenient route which will result in delivery to the blood stream in an amount effective for inhibiting .alpha.4.beta.1- and .alpha.4.beta.7-mediated adhesion, including orally, rectally, parenterally (intravenously, intramuscularly, intraarterially, or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray or aerosol. The compositions can also be delivered through a catheter for local delivery at a target site, or via a biodegradable polymer. The compositions may also be complexed to ligands, or antibodies, for targeted delivery of the compositions.

The compositions are most effectively administered parenterally, preferably intravenously. For intravenous administration, they may be dissolved in any appropriate intravenous delivery vehicle containing physiologically compatible substances, such as sodium chloride glycine, and the like, having a buffered pH compatible with physiologic conditions. Such intravenous delivery vehicles are known to those skilled in the art. In a preferred embodiment, the vehicle is a sterile saline solution. If the peptides are sufficiently small (e.g., less than about 8-10 amino acids) other preferred routes of administration are intranasal, sublingual, and the like.

The compositions according to the invention can be administered in any circumstance in which inhibition of .alpha.4.beta.1 and .alpha.4.beta.7 integrin function is desirable. Disease states which may be treated include but are not limited to diabetes, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, arteriosclerosis, asthma, allergy, autoimmune disorders, transplant rejection, and restenosis of the arteries following surgery or angioplasty. Because the .alpha.4 integrins are expressed on various cancer cells, including leukemia, melanomas, lymphomas, and sarcomas, inhibitors of .alpha.4 binding can also be useful in the treatment of some forms of cancer.

The amount of peptide administered depends upon the degree of integrin inhibition that is desired. Those skilled in the art will derive appropriate dosages and schedules of administration to suit the specific circumstances and needs of the patient. Typically, dosages are between about 0.001 mg/kg and about 100 mg/kg body weight. In some embodiments dosages are between about 0.01 mg/kg and about 10 mg/kg body weight. In some embodiments dosages are between about 0.05 mg/kg and about 5 mg/kg body weight.

EXAMPLES

The following examples illustrate the invention. These examples are illustrative only, and do not limit the scope of the invention.

Example 1

Materials and Methods Used in the Examples

A. Materials

Monoclonal antibodies HP2/1 (anti-.alpha.4 subunit of VLA-4) and SAM-1 (anti-.alpha.5 subunit of VLA-5) were purchased from Immunotech, Inc. (Westbrook, Me.). HP2/4 was received as a gift from Dr. Francisco Sanchez-Madrid (Madrid, Spain). The biological effects of HP2/1 and HP2/4 were identical. Highly purified human fibrinogen was gift from Dr. A. Budzynski (Temple University, Philadelphia Pa.), recombinant human VCAM-1 (Renz et al., J. Cell Biol. 125:1395-1406 (1994)) was a gift from Dr. M. Renz (Genentech, San Francisco, Calif.), human vitronectin and fibronectin were purchased from Calbiochem (La Jolla, Calif.) and Sigma (St. Louis, Mo.), respectively. GRGDSP and GRGESP peptides were purchased from Bachem (Torrance, Calif.). RGDS was purchased from Sigma. Highly purified MLDG peptide was kindly provided by Dr. Z. Huang (Thomas Jefferson University, Philadelphia, Pa.). Echistatin was isolated from Echis carinatus suchoreki venom as described by McLane et al. (Biochem. J. 301:429-436 (1994). Fluorescein isothiocynate (FITC)-conjugated goat anti-mouse IgG for flow cytometry was purchased from Jackson Immune Research (West Grove, Pa.).

A5 and VNRC3 cells, CHO cells transfected with human .alpha.IIb.beta.3 and .alpha.v.beta.3 integrins, respectively (O'Toole et al., Cell. Regul. 1:883-893 (1990)), were kindly provided by Dr. M. Ginsberg (Scripps Research Institute, La Jolla, Calif.). CHO cells with deleted .alpha.5 integrin (B2 cells) were kindly provided by Dr. R. Juliano (University of North Carolina, Chapel Hill, N.C.). CHO cells transfected with human .alpha.4 and its G190A mutant (Kamata et al, Biochem. J. 305:945-951 (1995)), and B2 cells transfected with human .alpha.4 (.alpha.4B2) were kindly provided by dr. Y. Takada (Scripps Research Institute). JY cells expressing .alpha.4.beta.7 were a gift of Dr. S. Burakoff (Dana-Farber Cancer Institute, Boston Mass.) K562 cells transfected with .alpha.6 integrin were a gift of Dr. A. Sonnenberg (Netherlands Cancer Institute, Amsterdam, Holland). Jurkat cells, K562 cells, and non transfected CHO-K1 cells were purchased from ATCC (Rockville, Md.).

B. Methods

Adhesion of cultured cells labeled with 5-chloromethylfluorescein diacetate (CMFDA) was performed as described earlier (Marcinkiewicz et al., Blood 90:1565-1575 (1997)). Briefly, ligands (such as EC-3, fibrinogen, vitronectin, fibronectin or VCAM-1) were immobilized on 96-well microtiter plate (Falcon, Pittsburgh, Pa.) in PBS buffer overnight at 4.degree. C. Wells were blocked by 1% BSA in HBSS buffer. Cells were labeled with fluorescein by incubation with 12.5 AM CMFDA in HBSS buffer containin