JP2002527096A - Inhibitors of platelet activation and recruitment - Google Patents

Abstract

  (57) [Summary] The present invention provides soluble CD39 polypeptides and compositions, and methods of inhibiting platelet activation and recruitment in a mammal, comprising administering a soluble CD39 polypeptide.

Description

DETAILED DESCRIPTION OF THE INVENTION

REFERENCE TO RELATED APPLICATIONS This application is a pending provisional application Ser. No. 60 / 104,5, filed Oct. 16, 1998.
No. 85, No. 60 / 107,466, filed Nov. 6, 1998, and 1999.
No. 60 / 149,010 filed Aug. 13, 2016.

FIELD OF THE INVENTION The present invention relates to soluble CD39 compounds and compositions, their manufacture, and their use to inhibit platelet activation and recruitment in mammals.

[0003] BACKGROUND CD39 is based has been identified as a marker of mature B cells, B cells not terribly mature, Epstein - B cells were transformed with Barr virus, activated T cells, endothelial cells and It is a cell surface antigen that is also expressed on certain myeloid cells (Dorken et al., Leukocyte Typing IV; W. Knapp. B. Dorken, and W
R. Gilks, Eds; Oxford University Press, New York, NY; pp. 89-90, 1989.
). Monoclonal antibodies to CD39 induce homologous fusion of B cells, an activity that may be important in regulating immune function (Kansas and Tedder, J. et al.
Immunol. 147: 4094-4102, 1991). Molecular cloning and characterization of CD39 has shown that it is a unique cell surface molecule containing two potential transmembrane regions and a hydrophobic segment within the putative extracellular domain (Maliszewski et al., J. Biol.
Immunol. 153: 3574, 1994). The amino acid sequence of CD39 was reported to show some homology to guanosine diphosphatase from yeast (Maliszewski et a
l., ibid.).

In 1996, ATP diphosphohydrolase was cloned from potato tubers (Handa and Guidotti, Biochem. Biophys. Res. Commun. 218: 916, 199).
6). The amino acid sequences of this and some other NTPases showed a high degree of similarity, especially among several small "apyrase conserved regions" (ACRs). CD
39 shares the above conserved regions with soluble ATP-diphosphorylase from potato tubers, other apylases and related enzymes. It was subsequently reported that native CD39 and full-length recombinant CD39 had E-type ATP diphosphohydrolase (ATPase) activity (Marcus et al., J. Clin. Invest. 99:13
51, 1997); Kaczmarek et al., J. Biol. Chem. 271: 33116, 1996); Wang and
Guidotti, J. Biol. Chem. 271: 9898, 1996). ATPDase degrades nucleoside-3 and / or diphosphate but not monophosphate (Plesner,
Int. Rev. Cytol. 158: 141, 1995).

[0005] Vascular endothelial cells constitutively express cell surface ADPase (ecto-ATP diphosphohydrolase, apyrase, EC 3.6.1.5), which is at least three types that function in maintaining blood fluidity. Is one of the thromboregulatory systems (Marcus and Safie
r, FASEB J. 7: 516, 1983; Marcus et al., J. Clin. Invest. 88: 1690, 1991
). This ecto-ADPase, which belongs to the E-type ATPDase family, rapidly metabolizes ADP upon platelet release, stopping further platelet recruitment and aggregation.

[0006] Immunoprecipitation of detergent lysates of HUVECs with anti-CD39 mAbs completely captured cell-associated ADPase activity, suggesting that CD39 is the only ecto-ADPase on endothelial cells. (Marcus et al., J. Clin. Invest
99: 1351, 1997). In the same test, a CO expressing recombinant CD39 on the cell surface was used.
S cell transfectants completely inhibited ADP-induced platelet aggregation. Thus, CD39 plays a crucial role in thrombus control (Gayl
e et al., J. Clin. Invest., 101: 1851, 1998).

[0007] Excessive platelet activation (ie, stimulation by agonists) and supplementation results in platelet aggregation and vascular occlusion at the site of vascular injury in coronary, carotid, and peripheral arteries, but is a major component of cardiovascular medicine. Has become a major therapeutic challenge. Excessive activation and replacement of platelets can lead to stroke, unstable angina, myocardial infarction,
And restenosis following percutaneous coronary intervention, including angioplasty, atherectomy, stent placement and bypass surgery.

[0008] Glycoprotein IIb / IIIa antagonists such as the monoclonal antibody marketed as ReoPro® (Centcore)
) Is currently being developed to inhibit platelet aggregation in patients who have undergone percutaneous coronary intervention and in patients with acute coronary syndrome such as unstable angina or myocardial infarction. However, activation of the glycoprotein IIb / IIIa receptor is an event that occurs late in the platelet aggregation cascade.

There is a great need to identify additional therapeutic strategies and compositions to pharmacologically neutralize platelet reactivity (activation, recruitment, aggregation). In particular, there is a need to identify compounds and compositions that target early parts of the coagulation pathway, such as ADP-dependent platelet activation and recruitment. In fact, there is an urgent need to identify new strategies and compositions for treating stroke, the third leading cause of death in the United States. In cases of stroke, an advantageous therapeutic would be to reduce the burden of intravascular thrombi and the associated neurological deficits without increasing intracerebral hemorrhage.

[0010] soluble form of CD39 with an overview apyrase activity of invention constitute a novel approach to the prophylaxis and / or treatment of diseases. The present invention provides soluble CD39 polypeptides and nucleic acids, pharmaceutically acceptable carriers and compositions comprising the soluble CD39 polypeptides, and methods of making and using soluble CD39 polypeptides having apyrase activity.

The present invention relates to (a) having an amino acid sequence as shown in FIG. 1 (SEQ ID NO: 2), wherein the amino terminal is selected from the group consisting of amino acids 36 to 44, and the carboxy terminal is amino acids 471 to 471; A polypeptide selected from the group consisting of 478;
(B) a fragment of the polypeptide of (a), wherein the fragment has apyrase activity; (c) a variant of the polypeptide of (a) or (b),
A mutant polypeptide having apyrase activity; and (d) a fusion polypeptide comprising the polypeptide of (a), (b) or (c), wherein the fusion polypeptide has apyrase activity. The soluble CD39 polypeptide. The invention provides a composition comprising a pharmaceutically acceptable carrier and a soluble CD39 polypeptide.

[0012] The present invention is also directed to a nucleic acid encoding a soluble CD39 polypeptide. The present invention provides DNAs, vectors, recombinant cells and methods for producing a soluble CD39 polypeptide.

[0013] The invention further provides a soluble CD39 polypeptide in a mammal in need of such treatment as inhibiting platelet activation and recruitment, inhibiting angiogenesis, or degrading nucleoside tri- and / or diphosphate. Is intended for the use of The present invention provides a medicament for a mammal in need of such treatment as inhibiting platelet activation and platelet recruitment, inhibiting angiogenesis or degrading nucleoside tri- and / or diphosphate. And the use of soluble CD39 polypeptides to The above and other aspects of the present invention will become apparent with reference to the following figures, examples, and detailed description.

[0014]Detailed description of the invention The cDNA encoding the cell surface molecule CD39 was isolated, cloned and sequenced.
It was determined. The nucleic acid sequence and predicted amino acid sequence of this cDNA correspond to SEQ.
D NO: 1 and SEQ ID NO: 2. The present invention relates to amino- and
Soluble form of CD39 constructed by removing the carboxy-terminal domain
Provide a way to use state. Soluble CD39 physiologically converts ATP and ADP
Not only the ability of wild-type CD39 to metabolize at meaningful concentrations, but also platelet aggregation
Retains the ability to block and reverse ADP-induced platelet activation and recruitment
. The use of a soluble form of CD39 is advantageous because this plasmid from recombinant host cells is advantageous.
Facilitates purification of the polypeptide, and generally, the soluble polypeptide is membrane-bound
This is because it is more suitable for clinical administration. CD39 activates platelets and
Because the present invention inhibits recruitment and thus platelet aggregation, the present invention
Patent application title: METHODS AND COMPOSITIONS FOR INHIBITING THROMBOSIS IN A Mammalian Site
A method used to control the reactivity of hemostatic and thrombogenic processes by controlling the reactivity of And Methods for inhibiting and / or reversing platelet aggregation are provided.

A. Hemostasis is defined as the prevention of bleeding from damaged blood vessels and results from a series of physiological and biochemical events. At least three interacting biological systems are involved in hemostasis: vascular components (eg, subendothelial matrix), platelets, and plasma proteins (Marcus, AJ: Disorders of Hemostasis, Ratnoff and Forbes).
eds., WB Saunders, Philadelphia, 1996; pages 79-137; Marcus, AJ:
Platelet Activation, in: Atherosclerosis and Coronary Artery Disease, v
ol. 1, Fuster, Ross and Topol, eds., Lipincott-Raven, Philadelphia, 1996.
pages 607-637). Deficiencies in one or more of the above systems can cause bleeding disorders, and conversely, inappropriate activation of hemostasis can develop arterial or venous thrombosis.

When a blood vessel is damaged, it contracts, extruding vascular components such as collagen, von Willebrand factor, fibronectin, thrombospondin, laminin and microfibrils. Platelets adhere to these components and are activated, and a particularly effective agonist for platelet activation is collagen. Platelet-collagen contact initiates at least four physiological events: platelets release bioactive compounds; they express P-selectin on the cell surface (where they are neutrophils, monocytes and intervening adhesion of lymphocyte subsets); platelet eicosanoid pathway is activated (begins with the release of arachidonic acid to form a prostaglandin H _2); and a smooth disk shape of platelets to a sphere with a thorn And change dramatically.

The biologically active compounds released by platelets are numerous and multifunctional. This component group includes serotonin, ATP, ADP, calcium, adhesion proteins (fibrinogen, fibronectin, thrombospondin, vitronectin, von Willebrand factor), growth factors (platelet-derived growth factor, transforming growth factor- β, platelet factor 4) and aggregation factors (Factor V, high molecular weight kininogen, Factor XI, S protein and plasminogen activator inhibitor I (PA
II)). Some of the above compounds are responsible for further recruiting other cells, such as platelets and / or neutrophils and monocytes, to the site of activation, while others provide feedback mechanisms to down regulate excessive thrombus formation. Some are involved.

There are at least three distinct endothelial thromboregulatory systems: eicosanoids, including prostaglandins PGI ₁ and PGD ₂ ; endothelium-dependent relaxing factors (ED
RF / NO); and ectonucleotidase ATP-diphosphohydrolase (A
TPDase), which has both ADPase and ATPase activity. ADP is the most important platelet aggregation agonist present in platelet effluent, whereas collagen and thrombin are the major inducers of platelet secretion. Catabolism of ADP to AMP by ecto-ADPase prevents further recruitment of platelets to the site of hemostasis, reverses the agglutination and blocks subsequent thrombotic reactions.

Ecto-nucleotidase activity is measured by aggregometry, where the EDRF / NO effect and PGI ₂ production are blocked by hemoglobin and aspirin, respectively.
System demonstrated in vitro (Marcus and Safier, FASEB J. 7: 516; 1993
). In this system, loss of platelet stimulating activity in the supernatant correlates with ADP catabolism. Although ADPase activity has been identified in the membrane fraction of human endothelial cells, the enzyme activity detected by polyacrylamide gel electrophoresis showed both ATPase and ADPase (Marcus et al., Clin. Res. . 40: 226A (abstract), 1992).

B. Utility of the Invention Significant research activity has been directed to the discovery and characterization of platelet aggregation inhibitors because of the potential utility of such inhibitors in the treatment of occlusive vascular disorders. For example, WO 95/12412 discloses chimeric platelet-specific antibodies and methods of using them in the treatment of various thrombotic disorders. An exemplary description of the eagerness to develop this therapeutic and obtain approval for use as a human therapeutic (generic name: abciximab, brand name: ReoPro® (Leopro)) is provided by BS.
Coller, Circulation 92: 2373 (1995).

CD39 is an ecto-ADPase (apyrase) located on the surface of endothelial cells. This enzyme has a major role in maintaining blood fluidity, that is, maintaining platelets in a reference (stable) state. This is achieved by metabolizing adenosine diphosphate, a major platelet agonist, to adenosine monophosphate, which is not an agonist. Since ADP is the most important agonist of platelet aggregation and is present in platelet exudates, substances that catabolize ADP are useful in treating or preventing conditions involving inappropriate aggregation of platelets.

Examples of therapeutic uses of soluble CD39 and its compositions include coronary artery disease or injury after myocardial infarction, atherosclerosis, arteriosclerosis, preeclampsia, embolism, pulmonary ischemia, coronary ischemia Treatment of individuals suffering from platelet-related ischemic disorders, including blood and cerebral ischemia, reocclusion after thrombosis, coronary thrombosis, cerebral artery thrombosis, intracardiac thrombosis, peripheral arterial thrombosis, venous thrombosis, And thrombosis, including thrombosis and coagulation disorders associated with exposure to foreign matter or damaged tissue surfaces, including angioplasty, carotid endarterectomy, anastomosis of vascular grafts, and combination with implantation catheters or shunts Prevention of disability is included. Other examples where it may be useful to inhibit increased ADP release upon stimulation of elevated platelets include cases in individuals at high risk of thrombus formation or remodeling (severe arteriosclerosis), occlusion of blood vessels , Restenosis, stenosis and / or inhibition of restenosis. Platelet A
Individuals who would benefit from treatments involving inhibition of DP-induced aggregation include those at risk for progressive coronary artery disease and angioplasty (ie, balloon angioplasty,
Laser angioplasty, coronary atherectomy, and similar procedures). Inhibition of platelet aggregation can also occur in individuals undergoing surgery with a high risk of thrombus formation (ie, coronary bypass surgery, insertion of replacement valves or tubes, etc.), deep venous thrombosis (DVT), pulmonary embolism ( PE), transient ischemic attack (TIA)
) And other related conditions where arterial occlusion is a common basic feature. In addition, the ability of CD39 to block platelet activation and recruitment is useful in preventing stroke and treating patients experiencing stroke due to vascular obstruction. In particular, the methods, compounds and compositions of the present invention, during seizures, inhibit microvascular thrombus, improve cerebral blood flow after ischemia, reduce cerebral infarction volume and neurological deficits without inducing intracerebral hemorrhage. Has the ability to reduce. Soluble CD39 and compositions thereof according to the present invention may also be administered in other therapeutic settings where it may be useful to degrade nucleoside tri- and / or diphosphate. By way of example, soluble CD39 inhibits angiogenesis and / or prevents the survival benefits provided by ATP to tumor cells, or other mediation mediated by angiogenesis, such as ocular neovascularization. It can be used as an anti-neoplastic agent for treating a disease or condition.

[0023] CD39 polypeptides also have many non-therapeutic uses because they may be used in any application where soluble ATPase and / or ADPase activity is advantageous.
As an example, the soluble CD39 polypeptide is a Gepner-Puszkin (U.S. Pat.
378, 601) in a composition for preserving platelets. In another example, a soluble CD39 polypeptide is a Ronaghi e
(Science 281: 336, 1998) can be used in pyrophosphate-based DNA sequencing. As a further example, CD39 polypeptides may be used in screening for apyrase inhibitors.

C. Molecular cloning and structural characterization of the CD39 polypeptide CD39 has been described by Maliszewski et al. (J.
Immunol. 153: 3574, 1994). CD39, near the amino and carboxy termini, can help keep native proteins within the cell membrane.
Contains two putative transmembrane regions. The portion of the molecule that lies between the transmembrane regions is outside the cell. As used herein, the term “CD39 polypeptide” includes CD39, homologs of CD39, variants, fragments and derivatives of CD39, fusion polypeptides comprising CD39, and CD39 polypeptides. Soluble forms are included.

[0025] A soluble polypeptide is a polypeptide that can be secreted from the cell in which it is expressed. Secreted soluble polypeptide is identified by isolating intact cells expressing the desired polypeptide from the culture medium, for example by centrifugation, and assaying the medium (supernatant) for the presence of the desired polypeptide (and To distinguish it from its insoluble, membrane-bound counterpart. The presence of the desired polypeptide in the medium indicates that the polypeptide is secreted from the cell and is a soluble form of the polypeptide. The use of a soluble form of CD39 is advantageous for many applications. The soluble polypeptide is secreted from the cell, thereby facilitating purification of the polypeptide from the recombinant host cell. In addition, soluble polypeptides are generally more suitable for parenteral administration and many enzymatic approaches than membrane-bound forms.

Apyrase activity resides in the extracellular domain of CD39. Accordingly, CD39 polypeptides useful for applications requiring biological activity include an amino terminus selected from the group consisting of amino acids 36-44 of SEQ ID NO: 2, and SEQ ID NO: 2.
A soluble form of CD39 having a carboxy terminus selected from the group consisting of amino acids 471-478 of NO: 2 and exhibiting the biological activity of CD39 is included. Soluble CD39 polypeptides also include polypeptides that include a portion of one or both of the transmembrane regions, as long as the soluble CD39 polypeptide can be secreted from the cell and has the biological activity of CD39. In addition, soluble CD39 polypeptides include oligomeric or fusion polypeptides comprising the extracellular portion of CD39, and fragments of any of the above polypeptides that have biological activity.

The term “biological activity”, as used herein, includes apyrase enzyme activity, as well as the ex vivo and in vivo activities of CD39. While apyrase catalyzes the hydrolysis of nucleoside tri- and / or di-phosphates, certain apylases may exhibit different relative specificities for either nucleoside triphosphate or nucleoside diphosphate. The biological activity of the soluble form of CD39 can be quantified, for example, in an ectonucleotidase or apyrase assay (eg, an ATPase or ADPase assay) or an assay that measures inhibition of platelet aggregation.
While representative assays are disclosed herein, one of skill in the art will recognize that other similar types of assays may be used to measure biological activity.

[0028] Soluble CD39 polypeptides provided herein include native CD39 polypeptide variants (also referred to as analogs) that retain the biological activity of CD39.
There is. Such variants have substantial homology to native CD39,
Polypeptides having an amino acid sequence that differs from that of native CD39 due to one or more deletions, insertions or substitutions are included. Specific embodiments include, but are not limited to, deletions of 1 to 10 amino acids when compared to the native CD39 sequence,
CD39 polypeptides containing insertions or substitutions are included. The CD39 coding DNAs of the present invention include variants that differ from the native CD39 DNA sequence by one or more deletions, insertions or substitutions, but that encode biologically active polypeptides. Included as variants of the CD39 polypeptide are naturally occurring variants, such as allelic and alternatively spliced forms, as well as the amino acid sequence of the CD39 polypeptide or the nucleic acid encoding the CD39 polypeptide Is a variant constructed by modifying the nucleotide sequence of

In general, substitutions for one or more amino acids present in a native polypeptide should be made conservatively. Examples of conservative substitutions include amino acid substitutions outside the active domain and amino acids that do not alter the secondary and / or tertiary structure of CD39. Further examples include the substitution of one aliphatic residue such as Ile, Val, Leu or Ala for another, Lys and Arg;
Substitution of one polar residue for another, such as lu and Asp; or Gln and Asn. Other conservative substitutions, for example, replacing an entire region with a region having similar hydrophobic properties are also known in the art.

When a deletion or insertion strategy is employed, the potential effects of the deletion or insertion on biological activity must be considered. Subunits of the polypeptides of the invention can be constructed by deleting terminal or internal residues or sequences. Further guidance on the types of mutations that can be made is provided by comparing the sequence of CD39 with polypeptides of similar structure, and by performing structural analysis of the polypeptides of the invention.

The native sequence of full length CD39 is shown in FIG. 1 (SEQ ID NO: 2).
In one preferred embodiment, the CD39 variant comprises a native CD shown in the sequence listing.
39 amino acid sequences that are at least about 70% identical to the 39 amino acid sequences;
9 are at least about 80% identical to the amino acid sequence. In some more preferred embodiments, the CD39 variant comprises a native CD shown in the sequence listing.
39 is at least about 90% identical to the amino acid sequence of SEQ ID NO: 39; in some more preferred embodiments, the CD39 variants are native C
The amino acid sequence is at least about 95% identical to the amino acid sequence of D39.
In some most preferred embodiments, the CD39 variant is at least about 98% identical in amino acid sequence to the amino acid sequence of native CD39 set forth in the sequence listing; in some most preferred embodiments, the CD39 variant is native in the sequence listing. The amino acid sequence is at least about 99% identical to the amino acid sequence of CD39. Identity ratios can be determined visually for both polypeptides and nucleic acids. The identity ratio can be determined using the Needleman and Wunsch (J. Mol. Biol. 48: 443, 1970) juxtaposition method modified by Smith and Waterman (Adv. Appl. Math 2: 482, 1981). Preferably, the identity ratio is determined by a computer program, such as the Genetics Computer Group (GCG; Madison, WI;
Devereux et al., Nucl. Acids Res. 12: 387, 1984). GAP computer program, version 10. Determined using x. The preferred default variables for the GAP program are: (1) a single comparison matrix for nucleotides (including values of 1 for identity and 0 for non-identity), and Gribskov and Burgess for amino acids (Nucl. Acids Re.
s. 14: 6745, 1986), a weighted comparison matrix (Schwartz and Dayhoff, eds., A
tlas of Protein Sequence and Structure, National Biomedical Research Fou
ndation, pp. 353-358, 1979); (2) a penalty of 30 (amino acids) or 50 (nucleotides) for each gap, and an additional 1 for each symbol in each gap (
(Amino acid) or 3 (nucleotide) penalty; (3) no penalty for terminal gaps; and (4) no maximum penalty for long gaps. Those skilled in the art of sequence comparison techniques may use other programs. For the fragment of CD39, the identity ratio is calculated based on the portion of CD39 present in the fragment.

The primary amino acid structure of soluble CD39 is modified to create CD39 derivatives by forming covalent or aggregated conjugates with other chemical components such as glycosyl groups, lipids, phosphates, acetyl groups, etc. obtain. Covalent derivatives of CD39 are produced by linking a specific functional group to the amino acid side chain of CD39 or the N-terminus or C-terminus of a CD39 polypeptide, or the extracellular domain thereof. CD3
The 9 derivative also binds to various insoluble structures, including cyanogen bromide activated agarose structures, or similar agarose structures, or is adsorbed (with or without glutaraldehyde cross-linking) on polyolefin surfaces. Also included are CD39 polypeptides.

Soluble CD39 fusion polypeptides within the scope of the present invention include N-terminal or C-terminal.
Covalent or aggregate conjugates of CD39 or a fragment thereof with other polypeptides, such as by synthesis in a recombinant culture, such as terminal fusion, are included. One class of fusion polypeptides is discussed below in connection with soluble CD39 oligomers. As another example, a fusion polypeptide may comprise a signal peptide at the N-terminal or C-terminal region of a CD39 polypeptide (which may comprise a signal sequence, signal,
(Referred to variously as a leader peptide, leader sequence, or leader), which translates the polypeptide from a synthesis site, either simultaneously with or after translation, to a site inside or outside the cell membrane or cell wall. Instruct to move (
For example, the Saccharomyces α-factor leader; some leader sequences are discussed in the Examples below). Signal peptide that promotes extracellular secretion
It is particularly advantageous to fuse to the N-terminus of the 39 polypeptide. In this case, the signal peptide is typically cleaved when soluble CD39 is secreted from the cell.

In a particularly preferred embodiment, one or more amino acids are added to the N-terminus of the soluble CD39 polypeptide to enhance the expression level and / or stability of the CD39 polypeptide. The one or more amino acids include an Ala residue,
Another CD39 family member (eg, CD39L2, CD39L3, CD39
A fragment from the N-terminus of L4) or another polypeptide such as IL-2;
And other peptides that can take a stable secondary structure and that are naturally occurring or designed based on structural predictions.

In a most preferred embodiment, the soluble CD39 polypeptide comprises (a) a soluble CD3
A signal peptide that promotes extracellular secretion from the cells of cell No. 9 (the signal peptide is cleaved simultaneously with secretion), (b) 1 attached to the N-terminus of the soluble CD39 polypeptide to improve expression level and / or stability It is initially synthesized as a fusion polypeptide comprising one or more amino acids and (c) a biologically active CD39 fragment.

[0036] CD39 fusion polypeptides can also include polypeptides that have been added to provide novel multifunctional components. In addition, a soluble CD39-containing fusion polypeptide can include a peptide that is added to facilitate purification and identification of soluble CD39. Such peptides include, for example, poly-His, or US Pat.
1,912 and Hopp et al., Bio / Technology 6: 1204, 1988. One such peptide is FLAG®
Peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
(SEQ ID NO: 10), which is highly antigenic and reversibly binds to a particular monoclonal antibody to provide an epitope, allowing rapid assay and easy purification of the expressed polypeptide. . The mouse hybridoma, designated 4E11, is disclosed in US Pat. No. 5,011, (incorporated herein by reference).
No. 912, FLAG in the presence of certain divalent metal cations.
Produces a monoclonal antibody that binds to the peptide. This 4E hybridoma cell line has accession number no. American T as HB 9259
Deposited with the type Culture Collection. FLAG (
A monoclonal antibody that binds to the ® peptide is available from Eastman Kodak (Scientific Imaging Systems Division, New Haven, CT).

Another particularly useful class of fusion polypeptides includes those that allow the localization or enrichment of CD39 at sites of platelet activation and recruitment. Such a fusion polypeptide comprises a moiety that specifically binds to activated platelets and a CD3
9 and can be produced by using recombinant DNA technology or by using standard techniques for conjugation of polypeptides. For example, WO95
/ 12412 discloses platelet-specific chimeric antibodies and methods of using them in the treatment of various thrombotic disorders. The above antibodies, or other platelet-specific antibodies (eg, P
-Antibodies against selectin / CD62) are useful for forming fusion polypeptides with CD39. Further, it is possible to produce humanized or single-chain antibodies based on such platelet-specific antibodies.

Counterstructure molecules (molecules that specifically bind to polypeptides expressed on the cell surface of activated platelets) and fragments thereof that bind to platelets also specifically bind to activated platelets It is useful to form a fusion polypeptide that will Representative pair structures include P-selectin / CD62
(Proc. Natl. Acad. Sci. USA 91: 739).
0, 1994; Sammar et al., Int. Immunol. 6: 1027, 1994; Lenter et al., J. C.
ell Biol. 125: 471, 1994).

[0039] The invention includes oligomers containing the CD39 polypeptide. CD39
Oligomers can be in the form of multimers, covalently or non-covalently linked, including dimers, trimers, or higher oligomers. Oligomers can be linked by disulfide bonds formed between cysteine residues on various CD39 polypeptides. Aui, the oligomer is to build a Fc region of an immunoglobulin molecule such as a fusion polypeptide with human IgG ₁ of CD39,
It may be formed by producing a CD39 / Fc fusion polypeptide. The term "Fc polypeptide" as used herein includes native and mutein forms of the polypeptide derived from the Fc region of an antibody. Truncated forms of such polypeptides that contain a hinge region that promotes dimerization are also included. PCT Application No. WO93 / 10151 described (which are incorporated herein by reference), a suitable Fc polypeptide is a single chain extending from the N-terminal hinge region of the Fc region of human IgG ₁ antibody to native C-terminus Is a polypeptide. Another useful Fc polypeptide is described in US Pat. No. 5,457,035 and Baum.
Fc mutein described in et al., (EMBO J. 13: 3992-4001, 1994). The amino acid sequence of this mutein is described in WO 93/10151 except that the amino acid at position 19 changes from Leu to Ala, the amino acid at position 20 changes from Leu to Glu, and the amino acid at position 22 changes from Gly to Ala. Identical to that of the native Fc sequence shown. This mutein shows reduced affinity for the Fc receptor.
CD39 / Fc fusion polypeptides can associate in the same manner as the heavy chains of an antibody molecule form divalent CD39. If a fusion polypeptide is produced together with the H chain and L chain of the antibody, it is possible to form a CD39 oligomer having as many as four CD39 extracellular regions.

In one aspect of the invention, oligomers comprising multiple CD39 polypeptides are linked by covalent or non-covalent interactions between the peptide moieties fused to the CD39 polypeptide. Such a peptide moiety is a peptide linker (spacer) or a peptide having the property of promoting oligomerization. Certain polypeptides derived from leucine zippers and antibodies are among the peptides that can promote oligomerization of polypeptides.

The invention includes fusion polypeptides with or without spacer amino acid linking groups. For example, two soluble CD39 domains are described in US Pat.
The linker can be linked using a linker sequence such as (Gly) ₄ Ser (Gly) ₅ Ser described in US Pat. No. 3,627. Other linker sequences include, for example, GlyAl
aGlyGlyAlaGlySer (Gly) ₅ Ser, (Gly ₄ Ser) ₂ ,
(GlyThrPro) ₃ and (Gly ₄ Ser) ₃ Gly ₄ SerGly ₅ Ser
Is included. Alternatively, CD39 may be linked to other polypeptides (non-CD39) with or without the spacer amino acid linking group. As shown in Example 9, using a ThrSerSer or ThrSerSerGly linker,
IL2 residues can be fused to soluble CD39. For the expression of soluble CD39, the inventors have determined that the 12 amino acids from the N-terminus of mature human IL2 are solCD3
A surprising and unexpected finding was that fusion to the 9 coding region resulted in high levels of expression and activity in the supernatant of transfected cells. Accordingly, a particularly preferred embodiment of the present invention provides a soluble CD having the amino acid sequence SEQ ID NO: 6.
39 polypeptides and amino acid sequences SEQ ID NO: 5, such as SEQ ID NO: 5
There is a nucleic acid encoding a soluble CD39 polypeptide having ID NO: 6.

The invention further includes soluble CD39 polypeptides with or without associated native mode glycosylation. CD39 expressed in yeast or mammalian expression systems (eg, COS-7 cells) depends on the choice of expression system,
It may be similar or significantly different in native molecular weight and glycosylation mode from the native CD39 polypeptide. Expression of a CD39 polypeptide in a bacterial expression system such as E. coli results in a molecule that is not glycosylated.

Different host cells process the polypeptides differently, resulting in a mixture of heterogeneous polypeptides having various N- or C-termini. Expression of a CD39 polypeptide in a bacterial expression system such as E. coli generally provides a homogeneous polypeptide preparation. Polypeptides can be processed differently by eukaryotic cells, resulting in diverse N- and C-termini, resulting in heterogeneous polypeptide preparations. The invention includes polypeptides having various N- or C-termini produced by eukaryotic host cells. In one aspect of the CD39 polypeptides of the invention, the amino and carboxy termini may differ by about 5 amino acids from those disclosed herein.

Those skilled in the art will appreciate that the location at which the signal peptide is cleaved may be different from that predicted by the computer program, depending on factors such as the type of host cell utilized to express the recombinant soluble CD39 polypeptide. It will also be appreciated that it can change. Thus, a polypeptide preparation according to the invention may include a mixture of polypeptide molecules having various N-terminal amino acids resulting from cleavage of the signal peptide at one or more sites.

D. Nucleic acids The present invention relates to full-length nucleic acid molecules encoding soluble CD39, and SEQ.
Also included are isolated fragments and oligonucleotides derived from the nucleotide sequence of ID NO: 1. Such a nucleic acid sequence has SEQ ID NO: 1.
178-1494 or a fragment thereof, and SEQ ID N
O: The DNA and / or RNA sequences that hybridize under moderate stringency conditions to the nucleotide sequence of 1 or its complement, and which encode a polypeptide of the invention or a fragment thereof, may be included.

Nucleic acid sequence encoding a soluble CD39 polypeptide having an altered glycosylation site, a deleted or substituted Cys residue, or a modified proteolytic cleavage site, a subunit of a CD39 polypeptide or other Peptide and CD39
A nucleic acid sequence encoding a CD39 allele variant of CD39, a mammalian CD39 homolog, and a nucleic acid sequence encoding a CD39 polypeptide derived from an alternative mRNA construct, or having been substituted or added Nucleic acid sequences encoding peptides having a specific amino acid are examples of nucleic acid sequences according to the present invention.

Due to the degeneracy of the genetic code, there can be considerable variation in nucleotide sequences encoding identical amino acid sequences. Included as embodiments of the present invention are moderately stringent conditions (eg, 5X SSC, 0.5% SDS, 1.0 mM EDTA (p
H8) pre-wash solution and 50 ° C., 5 × SSC, overnight hybridization conditions) under conditions degenerate to a sequence capable of hybridizing to a DNA sequence encoding soluble CD39, and a sequence encoding soluble CD39. There are other arrays. One of skill in the art can determine additional combinations of salts and temperatures that make up moderate hybridization stringency. Higher stringency conditions include higher temperatures for hybridization and post-hybridization washes,
And / or lower salt concentrations.

In a preferred embodiment, the CD39 DNA encodes a polypeptide whose amino acid sequence is at least about 70% or at least about 80% identical to the amino acid sequence of a native CD39 polypeptide set forth in SEQ ID NO: 1. Things. In a more preferred embodiment, the encoded variant of CD39 is CD3
The amino acid sequence is at least about 90% or at least about 95% identical to the native form of 9; in the most preferred embodiment, the encoded variant of CD39 has at least about 98 amino acid sequences relative to the native form of CD39. % Or at least about 99% identical. For DNA encoding a fragment of CD39, the identity ratio for the fragment is based on the identity ratio to the corresponding portion of full-length CD39.

Mutations can be introduced into nucleic acids by synthesizing oligonucleotides containing the mutated sequence and flanking a restriction site that allows ligation to the native sequence fragment. After the ligation reaction, the resulting reconstructed sequence encodes a variant having the desired amino acid insertion, substitution or deletion.

Alternatively, site-directed mutagenesis directed to oligonucleotides may be used to provide altered genes having particular codons altered by the required substitutions, deletions or insertions. Representative methods for producing the variants shown above are Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37: 73, 1985).
; Craik (BioTechniques, January 1985, 12-19; Smith et al. (Genetic Engin
eering: Principles and Methods, Plenum Press, 1981); U.S. Patent Nos. 4,518,584 and 4,737,462 disclose suitable techniques,
Incorporated herein by reference.

The well-known polymerase chain reaction (PCR) method can also be used to produce and amplify a DNA sequence encoding a desired polypeptide or fragment thereof. DNA
Oligonucleotides that define the desired termini of the fragment are 5 ′ and 3 ′
Used as a primer. The oligonucleotide may additionally contain a recognition site for a restriction endonuclease to facilitate insertion of the amplified DNA fragment into an expression vector. For PCR technology, see Saiki et al., Sci.
ence 239: 487, 1988; Recombinant DNA Methodology, Wu et al., eds., Acade
mic Press, Inc., San Diego, 1989, pp. 189-196; PCR Protocols: A Guide to
Methods and Applications, Innis et al., Eds., Academic Press, Inc., 199
0 is described.

Various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues,
Alternatively, a DNA sequence encoding a CD39 polypeptide comprising a sequence not required for biological activity can be prepared. For example, the use of a yeast expression system allows for the expression of N-glycosylation while preventing homogenous, low carbohydrate variants.
Glycosylation sites may be modified. N-glycosylation sites in eukaryotic polypeptides are characterized by a triad of amino acids, Asn-XY, where X is an amino acid other than Pro and Y is Ser or Thr. Appropriate modifications to the nucleotide sequence encoding the triad result in substitutions, additions or deletions that prevent carbohydrate attachment to the Asn side chain.

In another example, the sequence encoding a Cys residue is altered such that the Cys residue is deleted or replaced with another amino acid, resulting in incorrect intermolecular disulfide bridge formation upon regeneration. Can be prevented. Thus, a Cys residue can be substituted with another amino acid or deleted without affecting the tertiary structure or disulfide bond formation of the polypeptide.

Other approaches to mutagenesis include modifying sequences encoding dibasic amino acid residues to enhance expression in yeast systems where KEX2 protease activity is present. Other variants are made by modifying adjacent dibasic amino acid residues to enhance expression in yeast systems where KEX2 protease activity is present. EP 212,914 discloses the use of site-directed mutagenesis to inactivate the processing site of KEX2 protease in a polypeptide. The processing site of KEX2 protease is A
It is inactivated by deleting, adding or substituting residues to alter the rg-Arg, Arg-Lys and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Similar modifications can be made to sequences encoding sites that are recognized and cleaved by other proteolytic enzymes. Subunits of a CD39 polypeptide can be constructed by deleting sequences encoding terminal or internal residues, or sequences that are not required for biological activity. Sequences encoding fusion polypeptides as shown below can be constructed by linking sequences encoding additional amino acid residues to a sequence of the invention without affecting biological activity.

Mutations in the nucleotide sequence that are constructed for the expression of soluble CD39 must, of course, preserve the reading frame phase of the coding sequence and preferably hybridize to translation of the receptor mRNA. It does not create complementary regions that can produce secondary structure of the mRNA, such as loops or hairpins that can adversely affect it. Although the mutation site may be predetermined, it is not necessary to predetermine the type of mutation itself. For example, to select for the optimal properties of a mutant at a particular site, random mutagenesis may be performed at the target codon and the expressed mutant polypeptide screened for the desired activity. .

Not all mutations in the nucleotide sequence encoding the CD39 polypeptide are expressed in the final product, for example, nucleotide substitutions are primarily expressed in transcribed mRNAs avoiding secondary structural loops. To increase (
(See EPA 75,444A, incorporated herein by reference.)
, Or codons that are more easily translated by the chosen host, such as E. coli.
To provide a well-known E. coli preference codon for expression.

In the genome, the CD39 polypeptide is encoded by a multi-exon gene.
The present invention further provides alternative mRNA constructs that can result from various post-transcriptional mRNA splicing events and hybridize under moderate stringency conditions to the cDNAs disclosed herein. included. CD39 polypeptides according to the invention include allelic variations of the sequence set forth in SEQ ID NO: 1, as well as sequences encoding CD39 polypeptides that include additional amino acids relative to the sequence of SEQ ID NO: 1.

The isolated nucleic acid sequences of the present invention are not associated with nucleic acid sequences encoding other proteinaceous substances and have few other substances found in living cells, such as proteins, lipids or carbohydrates. One of skill in the art can produce a vector for expression of a soluble CD39 polypeptide.

E. Recombinant Expression System The present invention also provides recombinant cloning and expression vectors containing CD39 DNA, and host cells containing the recombinant vectors. cDNA D
An expression vector comprising NA can be used to produce a soluble CD39 polypeptide encoded by this DNA. An expression vector carrying the DNA sequence of the recombinant CD39 can be introduced into a substantially homogeneous culture of a suitable host microorganism or mammalian cell line.
For example, it is introduced by transfection or transformation. A transformed host cell is a cell that has been transformed or transfected with a nucleotide sequence encoding a CD39 polypeptide and expresses the CD39 polypeptide.
The CD39 polypeptide to be expressed resides inside the host cell and / or is secreted into the culture supernatant, depending on the nature of the host cell and the gene construct inserted into the host cell. One skilled in the art will appreciate that the method of purifying the expressed CD39 will vary depending on factors such as the type of host cell utilized.

[0060] Any suitable expression system may be utilized. Soluble CD39 by recombinant DNA technology
A recombinant expression vector for expressing a DNA fragment derived from a mammalian, microbial, viral or insect gene operably linked to a synthetic or cDNA-derived DNA fragment encoding a CD39 polypeptide. Alternatively, a DNA sequence of CD39 comprising a translation regulatory nucleotide sequence is included.

Examples of regulatory sequences include a transcription promoter, operator or enhancer, mR
Includes the NA ribosome binding site and appropriate sequences that control the initiation and termination of transcription and translation. Nucleotide sequences are operably linked when the regulatory sequence is operably related to the CD39 DNA sequence. Therefore, the nucleotide sequence of the promoter is such that the nucleotide sequence of this promoter is
If it controls transcription of the NA sequence, it is operably linked to the DNA sequence of the cDNA. Generally, the expression vector will incorporate an origin of replication that confers the ability to replicate in the desired host cell and a selection gene by which the transformed cells will be identified.

In addition, sequences encoding appropriate signal peptides (native or heterologous) can be incorporated into expression vectors. Signal peptide (secretory leader) DN
The A sequence can be fused in frame to the CD39 sequence, such that CD39 is initially translated as a fusion protein comprising this signal peptide. A signal peptide that functions in the intended host cell promotes extracellular secretion of the CD39 polypeptide. The signal peptide is cleaved from the CD39 polypeptide upon the secretion of soluble CD39 from the cell.

For signal peptides that can be utilized to produce soluble CD39, the native signal peptide can be replaced by a heterologous signal peptide or leader sequence, if desired. The choice of signal peptide or leader may depend on factors such as the type of host cell in which the recombinant polypeptide is produced. Illustratively, examples of heterologous signal peptides that function in mammalian host cells include the interleukin-7 (I) described in U.S. Patent No. 4,965,195.
L-7) signal sequence of interleukin-2 receptor described in Cosman et al., Nature 312: 768, 1984; interleukin-4 receptor signal peptide described in EP367,566; U.S. Pat. No. 4,968,6
No. 07 type I interleukin-1 receptor signal peptide; EP4
No. 60,846, including the type II interleukin-1 receptor signal peptide. For the expression of soluble CD39, we have found that using a leader containing a sequence derived from the human IL-2 polypeptide (SEQ ID NO: 9), high levels of ATP were found in the supernatants of transfected cells. A surprising and unexpected finding that ase activity occurs. Accordingly, a particularly preferred embodiment of the present invention is a nucleic acid encoding a soluble CD39 polypeptide having the amino acid sequence SEQ ID NO: 8.

[0064] Suitable host cells for expression of the CD39 polypeptide include prokaryotes, yeast or higher eukaryotic cells. Generally, preferred for use as host cells are mammalian or insect cells. Cloning and expression vectors suitable for use with bacterial, fungal, yeast and mammalian cell hosts are described, for example, in Pouwels et al. Cloning.
Vectors: A Laboratory Manual, Elsevier, New York, 1985. Cell-free translation systems can also be utilized to produce soluble CD39 polypeptides using RNA from the DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive microorganisms, such as E. coli or B.
acilli is included. Prokaryotic host cells suitable for transformation include, for example, E. coli, Ba.
cillus subtilis, Salmonella typhimurium and Pseudomonas, Streptomyces
And various species within the genus Staphylococcus. In prokaryotic host cells such as E. coli, the polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met can be cleaved from the expressed recombinant polypeptide.

[0066] Expression vectors used in prokaryotic host cells generally include one or more marker genes that are phenotypically selected. A phenotypically selected marker gene is, for example, a gene that encodes a protein that confers antibiotic resistance or provides autotrophic auxotrophy. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids, such as the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance, and thus provides a simple means to identify transformed cells. A suitable promoter and CD39 are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, WI, USA).

[0067] Promoter sequences commonly used in expression vectors for recombinant prokaryotic host cells include:
β-lactamase (penicillinase), lactose promoter system (Chang et a
l., Nature 275: 615, 1978; and Goeddel et al., Nature 281: 544, 1979)
, Tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Re
s. 8: 4057, 1980; and EP-A-36776) and the tac promoter (Mani
atis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laborat
ory, p. 412, 1982). In particular expression system useful prokaryotic host cell utilizes .lambda.P _L promoter and cI857ts thermolabile repressor sequence of the phage. the taken to a derivative of λP _L promoter, American Type Culture Collection
Plasmid pHUB2 (E. coli strain J
MB9, present in ATCC 37092) and pPLc28 (E. coli RP
1, existing in ATCC 53082).

The soluble CD39 is also preferably of the genus Saccharomyces (eg, S. cerevisi
It can also be expressed in yeast host cells from ae). Pichia or Kluyveromyces
Other yeast genera, such as Yeast vectors often contain an origin of replication sequence from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, a polyadenylation sequence, a transcription termination sequence, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, inter alia, metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073, 1980).
Or enolase, glyceraldehyde-triphosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose Other glycolytic enzymes such as isomerase and glucokinase (Hess et al., J. Adv. Enzyme Reg. 7:14
9, 1968; and Holland et al., Biochem. 17: 4900, 1978). Other vectors and promoters suitable for use in yeast expression are further described in Hitzeman, EPA-73,657. Other selection means are described in Russell et al. (J. Biol. Chem. 258: 2674, 1982) and Beier et al.
Nature 300: 724, 1982). A shuttle vector that is replicated in both yeast and E. coli is E.co.
li can be constructed by inserting the DNA sequence from pBR322 (Amp ^r gene and origin of replication) that is selected and replicated in li.

To direct the secretion of the recombinant polypeptide, the yeast α-factor leader sequence may be utilized. This α-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, for example, Kurjan et al., Cell 30: 933, 1982 and Bitte
Natl. Acad. Sci. USA 81: 5330, 1984. Other leader sequences suitable for promoting secretion of a recombinant polypeptide from a yeast host are known to those of skill in the art. The leader sequence may be modified to contain one or more restriction sites near its 3 'end. This promotes the fusion of the leader sequence to the structural gene.

[0070] Yeast transformation protocols are known to those skilled in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75: 1929, 1978. This Hinnen et al. Protocol selects for Trp ⁺ transformed cells in a selective medium, where the selective medium is 0.67% yeast nitrogen base, 0.5% casamino acid, 2% glucose, 10 μg / ml adenine and Contains 20 μg / ml of uracil.

[0071] Yeast host cells transformed with a vector containing the ADH2 promoter sequence can be grown to induce expression in "rich" media. An example of a rich medium is 1% supplemented with 80 μg / ml adenine and 80 μg / ml uracil.
It consists of yeast extract, 2% peptone, and 1% glucose. When glucose is completely consumed from the medium, derepression of the ADH2 promoter occurs.

[0072] Mammalian or insect host cell culture systems can also be used to express recombinant CD39 polypeptides. Baculovirus systems for producing heterologous polypeptides in insect cells are reviewed in Luckow and Summers, Bio / Technology 6:47, 1988. Cell lines of mammalian origin can also be used. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 165
1) (Gluzman et al., Cell 23: 175, 1981), L cells, C127 cells, 3T3
Cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, Hela cells, and BHK (ATCC CRL 10) cell lines, McMahan et al.
al. (EMBO J. 10: 2821, 1991) and includes a CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70). For the production of therapeutic polypeptides, it is particularly advantageous to use a mammalian host cell line adapted to grow in media that does not contain animal proteins. Soluble C
The use of such a cell line for the expression of D39 is described in Example 13.

An established method for introducing DNA into mammalian cells has been described (Ka
ufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15-69). L
ipofectamine (Gibco / BRL) or Lipofectami
Cells can be transfected using additional protocols using commercially available reagents such as ne-Plus (Felgner et al., Proc. Natl. Acad.
Sci. USA 84: 7413-7417, 1987). Further, Sambrook et al., Molecular Cloni
ng: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring Harbor Laboratory P
Mammalian cells can be transfected by electroporation using conventional methods, such as in Ress, 1989. Selection of stable transformed cells can be performed using methods known in the art, for example, resistance to cytotoxic drugs. Kaufman et al., Meth. In Enzymology 185: 487-511, 19
90 describes several selection schemes, such as dihydrofolate reductase (DHFR) resistance. A suitable host strain for DHFR selection may be the DHFR deficient CHO strain DX-B11 (Urlaub and Chasin, Proc. Natl. Acad.
Sci. USA 77: 4216-4220, 1980). Although a plasmid expressing the DHFR cDNA can be introduced into the DX-B11 strain, only cells containing this plasmid can grow on an appropriate selection medium. Other examples of selectable markers that can be incorporated into expression vectors include c418, which confers resistance to antibiotics such as G418 and hygromycin B.
DNA. Cells harboring this vector can be selected based on their resistance to these compounds.

The transcriptional and translational control sequences for mammalian cell expression vectors can be excised from the viral genome. Commonly used promoter and enhancer sequences are derived from polyoma virus, adenovirus 2, Simian virus 40 (SV40) and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, such as the SV40 origin of replication, early and late promoters, enhancers, splices, and polyadenylation sites are used to provide other genetic elements for expressing structural gene sequences in mammalian host cells. I can do it. The early and late promoters of the virus are particularly useful because both are readily available as fragments from the viral genome and may contain the viral origin of replication (Fiers et al., Nature 273: 113, 1978; Kaufman, IA US
Meth. In Enzymology, 1990). Smaller or larger SV40 fragments may also be used, as long as they include an approximately 250 bp sequence extending from the HindIII site to the BglI site located within the SV40 viral origin of replication.

Additional control sequences that have been shown to enhance the expression of heterologous genes from mammalian expression vectors include CHO cell-derived expression-enhancing sequence elements (EASE) (Morr
is et al., Animal Cell Technology, 1997, pp. 529-534);
TPL) and the VA gene RNA of adenovirus 2 (Gingeras et al., J. Bi
ol. Chem. 257: 13475-13491, 1982). An internal ribosome entry site (IRES) sequence at the viral origin allows the double cistronic mRNA to be efficiently translated (Oh and Sarnow, Current Opinion in Ge
netics and Development 3: 295-300, 1993; Ramesh et al., Nucleic Acids Re
search 24: 2697-2700, 1996). Heterologous DN as part of a double cistronic mRNA
Expression of A followed by expression of a selectable marker gene (eg, DHFR) has been shown to improve host transfection efficiency and heterologous cDNA expression (Ka
ufman, Meth. in Enzymology, 1990). Representative expression vectors utilizing dual cistronic mRNAs are pTR-DC / GFP described by Mosser et al., Biotechniques 22: 150-161, 1997, and Morris et al., Animal Cell Techno.
logy, 1997, pp. 529-534.

A useful high expression vector, pCAVNOT, is described in Mosley et al., Cell 59:33.
5-348, 1989. Other expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3: 280, 1983). A useful system for stable and high-level expression of mammalian cDNA in C127 mouse mammary epithelial cells is substantially the same as described in Cosman et al. (Mol.
mmunol. 23: 935, 1986). Cosman et al., Nature 312
: A useful high expression vector described by 768, 1984, PMLSV N1 /
N4 is stored as ATCC 39890. Additional useful mammalian expression vectors are described in EP-A-03675, which is incorporated herein by reference.
No. 66, and U.S. patent application Ser. No. 07 / 701,415, filed May 16, 1991. The vector may be from a retrovirus. Instead of the native signal sequence, the I signal described in U.S. Pat. No. 4,965,195
L-7 signal sequence; the signal sequence of the IL-2 receptor described in Cosman et al., Nature 312: 768, 1984; the IL-4 signal peptide described in EP 367,566; U.S. Pat. No. 968,607, a signal peptide of the type I IL-1 receptor; and a type II IL- described in EP 460,846.
A heterologous signal sequence can be added, such as a signal peptide for one receptor.

[0077] Another useful expression vector, pFLAG, may also be used. The FLAG® technology uses a low molecular weight (1 kD) hydrophilic FLAG® marker peptide as a pFLAG-1 ^™ Expression Vector (IBI Ko).
focusing on fusing to the N-terminus of the recombinant polypeptide expressed by Dak.

F. Purification of Soluble CD39 Polypeptide Soluble CD39 polypeptide can be produced by culturing the transformed host cell under the culture conditions necessary to express the CD39 polypeptide. Then
The resulting expressed polypeptide can be purified from the culture medium or cell extract. The supernatant derived from the cultured transformed host cells is purified by using a commercially available protein concentration filter such as A
Concentration may be achieved using a micon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate may be applied to a cation exchange matrix. Suitable cation exchangers include various insoluble matrices comprising sulfone or carboxymethyl groups, with sulfone groups being preferred. The matrix can be acrylamide, agarose, dextran, cellulose or other formats commonly used for protein purification. An anion exchange resin is then used, such as diethylaminoethyl (DEAE) or a matrix or substrate with quaternary amino groups, with quaternary amino groups being preferred. The matrix can be acrylamide, agarose, dextran, cellulose or other formats commonly used for protein purification. Additionally, media for gel filtration may be utilized to further purify the CD39 polypeptide with approximate molecular weight. Alternatively, some of the above steps may not be performed, or may be performed in the reverse order.

Reversed phase high performance liquid chromatography (RP-HPL) utilizing a hydrophobic RP-HPCL medium (eg, silica gel with pendant methyl or other aliphatic groups)
CD39 can be further purified using one or more steps of C). A substantially purified, homogenous polypeptide having the biological activity of CD39 can be eluted from a polyacrylamide gel and separated electrophoretically. Some or all of the above purification steps can be utilized in various combinations, containing less than about 1% by weight of protein contaminant residues from the manufacturing process, or otherwise,
A substantially purified, homogenous recombinant polypeptide is provided that is about 95% or more pure by gel electrophoresis.

To purify soluble CD39, affinity chromatography may be utilized. Example 12C describes the affinity purification of soluble CD39 from conditioned media. Further, CD39 was immunoprecipitated with a monoclonal antibody, the immunoprecipitate was electrophoresed on a polyacrylamide gel, a portion of the gel containing CD39 was excised, and CD39 was eluted from the excised portion of the gel. A small amount of CD39 is obtained.

In general, recombinant polypeptides produced in bacterial cultures are disrupted by host cells, centrifuged, extracted from cell pellets for insoluble polypeptides, or from supernatant for soluble polypeptides, and then extracted. Isolated by one or more steps of concentration, salting out, ion exchange, affinity purification, or size exclusion chromatography. Finally, RP-HPLC may be utilized for the final purification step. Microbial cells can be disrupted by conventional methods, including repeated freeze-thaw, sonication, mechanical disruption, or use of cell lysing agents.

[0082] Transformed yeast host cells can be used to express CD39 as a secreted polypeptide. This simplifies purification. Recombinant polypeptide secreted from yeast host cell fermentation is described in Urdal et al. (J. Chromatog. 296: 1
71, 1984). Urdal et a
l. describes two consecutive reverse-phase HPLC steps for purifying recombinant human IL-2 on a preparative HPLC column.

[0083] The desired purity of the soluble CD39 polypeptide will depend on the intended use of the polypeptide. For example, if the polypeptide is to be administered in vivo, relatively high purity is desired. Advantageously, the soluble CD39 polypeptide is an SDS
-Purified such that protein bands corresponding to other (non-CD39) polypeptides are not detected by polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, protein bands may be visualized by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography. One skilled in the art will recognize that due to different glycosylation, different post-translational processing, etc., a number of bands corresponding to the CD39 polypeptide may be present in SDS-PA
It will be seen that it can be visualized by GE.

G. Therapeutic compositions of CD39 polypeptides The present invention provides compositions comprising an effective amount of a soluble CD39 polypeptide in a pharmaceutically acceptable carrier. As used herein, "treatment"("th
erapy "," therapeutic "," treat ", and" treat "
The term "ment") generally includes not only treatment of an existing disease or condition, but also prophylaxis, i.e., prevention of a disease or condition.
) is also included. Thus, a therapeutic composition of a soluble CD39 polypeptide may need to be administered before, during, or after the onset of symptoms. For therapeutic use, the soluble CD39 polypeptide is administered to the patient to be treated in a manner appropriate for the indication. Thus, for example, a pharmaceutical composition of soluble CD39 administered to achieve a desired therapeutic effect may be provided by bolus injection, continuous infusion, sustained release from implants, etc., or other suitable techniques. Can be. Ideally, developing a stable form of CD39 or a closely related biologically active variant would allow its use in oral form, a preferred route of administration. CD
Because 39 is aspirin-insensitive, both therapeutic agents (CD39 composition and aspirin) can be used in combination for maximum benefit.

Typically, a soluble CD39 therapeutic agent can be administered in the form of a pharmaceutical composition comprising purified CD39 together with a physiologically acceptable carrier, including excipients or diluents. . Such carriers are non-toxic to patients at the dosages and concentrations employed. As described in the Examples below, administration of CD39 to a mouse or pig thrombus model has no observed toxic effects. In addition, CD39 2
The second dose does not cause immunogenic symptoms. Typically, the production of such compositions involves dissolving the soluble CD39 composition in a buffer, an antioxidant such as ascorbic acid, a low molecular weight (less than 10 residues) polypeptide, polypeptide, amino acid, glucose,
It involves complexing with carbohydrates, including sucrose or dextran, chelating agents such as EDTA, glutathione and other stabilizers, and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are examples of suitable diluents.

One type of sustained release technology that can be used when administering a soluble CD39 composition is a hydrogel material, such as a photopolymerized hydrogel (Sawhney et al., Macromol
ecules 26: 581; 1993). Prevent postoperative union formation (Hi
Ill-West et al., Obstet. Gynecol. 83:59, 1994), and preventing thrombosis and vascular occlusion following vascular injury (Hill-West et al., Proc. Natl. Acad. Sci. USA 91:
5697, 1994), similar hydrogels have been used. Polypeptides can be incorporated into such hydrogels to provide localized sustained release of the active ingredient (West and Hubbel, Reactive Polymers 25: 139, 1995; Hi
ll-West et al., J. Surg. Res. 58: 759; 1995). C incorporated into the hydrogel
Localized sustained release of D39 will be amplified by the long half-life of CD39. This is illustrated in the following example.

Thus, the soluble CD39 compositions described herein can also be incorporated into hydrogels for application to tissues where it is desired to locally inhibit hemostasis. . For example, hydrogels incorporating CD39 polypeptides can be applied to post-operative tissue to prevent or reduce post-operative fusion, or to prevent or reduce restenosis after angioplasty. , Using a catheter-based delivery system. Those of skill in the art will be able to formulate suitable hydrogels, for example, by applying standard pharmacokinetic testing methods as discussed in West and Hubbell, supra.

The effective amount may vary depending on the age, type and severity of the condition being treated, body weight, desired duration of treatment, mode of administration, and other parameters. Effective dosages are
Determined by a physician or other qualified medical professional. Typical dosages are between 0.
It is 01-100 mg / body weight (kg). In some embodiments, a single dose is sufficient, while in other embodiments, the soluble CD39 polypeptide is administered daily for one week or one month or more.

[0089] The biological effectiveness of a soluble CD39 polypeptide can be readily assessed. If the released platelet ADP is metabolized by the administered CD39 composition, an increase in bleeding time should be measured at a constant platelet count for a period of time after administration. This indicates that a therapeutic effect was probably obtained, as the above measures correlate with clinical improvement. Therapeutic effects may also be verified by testing ex vivo for platelet reactivity to ADP and other platelet agonists. The actual measurement of enzyme (apyrase) activity can also be performed after administration of soluble CD39. These methods of determining biological effectiveness are set forth in the Examples below.

H. Abbreviations used herein : ACR: apyrase conserved region; AG: affigel beads; ASA: acetylsalicylic acid; ATPDase: ATP diphosphohydrase; CHO: Chinese hamster ovary; CM: conditioned medium; DHFR: dihydrofolate reductase; FSBA: Fluorosulfonylbenzoyl-adenosine; HUVEC: Human umbilical vein endothelial cells; PRP: Platelet-rich plasma; PTCR: Percutaneous transluminal coronary angioplasty solCD39: Recombinant soluble human CD39; TBS: Tris-buffered saline

[0091]

【Example】

The following examples are intended to illustrate specific embodiments and do not limit the scope of the invention.

Example 1 Assay for CD39 Expression This example illustrates the use of monoclonal antibodies in a FACS assay to analyze CD39 expression. B73 mAb, monoclonal anti-CD3
9 was derived from BALB / c mice immunized with the RPMI 1788 cell line (Rect
or et al., Immunology 55: 481, 1985), a mouse IgG1 characterized as CD39-specific by flow cytometry analysis and immunoprecipitation / SDS-PAGE. Monoclonal anti-CD39 is purified from ascites fluid by affinity chromatography using a protein A column, eluted with 0.05 M sodium citrate (pH 3), neutralized, and stored at 4 ° C. at a concentration of about 1 mg / ml.

The cells to be analyzed (eg, MP-1 cells, U937 cells, U937 cells stimulated with 5 ng / ml phorbol myristate acetate (PMA), or Daudi cells) were converted to phosphate buffered physiology containing 100 ng of human IgG1. Saline (PBS) 50
The cells were suspended in a volume of 10 ⁶ cells. The cells were then pelleted by centrifugation, suspended in PBS / azide containing the primary antibody (anti-CD39 or control antibody) and incubated (4 ° C, 30 minutes). The cells were then washed twice with PBS / azide, resuspended, incubated with a labeled second antibody, eg, goat anti-mouse immunoglobulin conjugated to phycoerythrin, and then washed again. The cells were analyzed by flow cytometry to quantify CD39 levels.

Example 2: Immunoselection of CD39-expressing cells This example describes a technique for panning (immunoselecting) cells expressing CD39. For selection of pan plates, purified anti-CD39 or control antibodies are diluted in phosphate buffered saline containing 0.1% heat-inactivated fetal calf serum (PBS / FCS). Anti-CD39 titers can be assayed to determine the most effective concentration of anti-CD39. Select plates are prepared by adding 3 ml of antibody solution or PBS / FCS alone to each plate. The plates are incubated for about 1 hour at room temperature, washed 5 times with PBS / FCS, and 3 ml of PBS / FCS containing 0.02% sodium azide is added to each plate.

The cells to be analyzed (eg, MP-1, U937 or Daudi cells) were prepared in PBS / 50 containing 5% goat serum, 5% rabbit serum and 100 μg / ml human IgG _1.
Suspend in 0 μM EDTA / 0.02% sodium azide (PEA) and add 2 × 1
0 ⁶ (cells) / ml of concentration was, the cell suspension 50 to the prepared selection plates
Add 0 μl. The selection plate is incubated with the cell suspension for about 2 hours at room temperature, and the plate is then incubated with PEA containing 10% FCS (PEA / FC
Wash three times with S) and three times with PEA. The plates are examined under a microscope and the relative number of cells bound to each plate is quantified.

Example 3 Construction of a cDNA Library This example illustrates the preparation of a CD39 library from a human B cell line called MP-1 for use in the expression cloning of human CD39.

This library construction technique is substantially equivalent to that described by Ausubel et al., Eds., Current Protocols In Molecular Biology, Vol. 1, 1987. Briefly, total RNA was extracted from a culture of MP-1 cells lysed with 8M guanidine hydrochloride using fractionated ethanol precipitation, and poly (A) ⁺ mRNA was isolated by oligo dT cellulose chromatography. Concentrate. Virtually Gubl
Double-stranded cDNA was prepared from the RNA template as described in er et al., Gene 25: 263, 1983. Primed with random hexanucleotides (pri
med) Poly (A) ⁺ mRNA was converted to an RNA-cDNA hybrid using reverse transcriptase. This RNA-cDNA hybrid was then converted to a double-stranded cDNA fragment using RNAse H with DNA polymerase I. The resulting double-stranded cDNA is blunt-ended with T4 DNA polymerase (
blunt-ended).

Using the adapter cloning method described in Haymerle et al., Nucleic Acids Res. 14: 8615, 1986, the 5 ′ end of the blunt-ended duplex is unkinased (ie, A (non-phosphorylated) BglII adapter was ligated. Under the above conditions, only the 24-mer oligonucleotide (upper strand) is covalently linked to the cDNA during the ligation reaction. Non-covalently attached adapters (including the complementary 20-mer oligonucleotide and unligated adapter described above) were removed by gel filtration chromatography at 65 ° C., leaving a non-self-complementary overhang of 24 nucleotides on the cDNA ends. Was.

The adapterd cDNA was converted to the adapterized pDC303 (E. coli).
(A mammalian expression vector that replicates within). pDC303 is pDC
201 (pML as previously described by Cosman et al., Nature 312: 768, 1984).
SV derivatives), assembled from SV40 and cytomegalovirus DNA, and along the direction of transcription from the replication origin, in turn, contains the following components: (1) origin of replication, including enhancer sequences and early / late promoters, 5171 SV of ~ 270th place
(2) cytomegalovirus promoter and enhancer region (Bo
nucleotides 671-63 from the sequence published by echart et al. (Cell 41: 521, 1985)); (3) positions 5779-6079 containing the first exon of the tripartite leader (TPL); Segments 7102-7171 and 9634-9693 containing part of the second and third exon, and XhoI;
Multiple cloning sites containing MCnI, SmaI and BglI sites (MC
Adenovirus-2 from S); (4) S from positions 4127-4100 and 2770-2533 containing polyadenylation and termination signals of early transcription.
V40 segment; (5) virus-associated RNA gene of pDC201, adenovirus-2 sequence from positions 10532-11156 of VAI and VAII; and (6) 4363-2486 containing ampicillin resistance gene and origin of replication
And the pBR322 sequence from positions 1094-375.

The MP-1 cDNA library in pDC303 was introduced into E. coli strain DH10B by electroporation. Plating the recombinant,
About 5,000 colonies were obtained per plate. These recombinants were pooled to give a stock of approximately 500,000 recombinants for screening. DNA was prepared from the transformed bacteria and isolated by cesium chloride centrifugation.

Example 4: cDNA molecule cloning of human CD39 This example illustrates the isolation of a DNA molecule encoding CD39 from the expression cloning library described in Example 3.

A. Round I: Transfection and immunoselection The isolated plasmid DNA was purified from COS-7 using DEAE-dextran and chloroquine treatment, essentially as described by McMahan et al., EMBO J. 10: 2821; 1991. The cells were transfected into a subconfluent layer.

COS-7 cells were cultured in a transfection / proliferation medium (10% (v / v) fetal calf serum, penicillin 50 U / ml, streptomycin 50 μg / ml).
Dulbecco's modified Eagle's medium containing 2 mM L-glutamine and 50 µg / ml gentamicin) and 10 ml of transfection / growth medium.
The cells were cultured in a / 10 cm dish to a density of about 1.5 × 10 ⁶ cells. The medium was removed from the adherent cells growing in a layer, to a confluence of about 70%, and 66.5 μm.
The medium was replaced with 10 ml of a complete medium containing M chloroquine. About 500 μl of DN is added to the cells.
A solution (D in transfection and growth medium containing 66.5 μM chloroquine)
5 μg NA, 0.5 mg / ml DEAE-dextran) was added and the mixture was incubated for about 5 hours at 37 ° C., 10% CO ₂ .

After the incubation, the medium was removed, and 5 ml of a transfection / growth medium containing 10% DMSO (dimethyl sulfoxide) was added over 2.5 to 20 minutes to bring the cells into a shock state. After shocking, this solution was replaced with 10 ml of fresh transfection and growth medium. 2-3 plates of cells from 12 plates
Propagated in culture for one day to allow for transient expression of the inserted DNA sequence. About 2
After 4 hours of growth, the cells were trypsinized and released from the plate. After an additional 1-2 days, CD39-expressing cells were selected by sorting, almost as described in Example 2. After 2 hours incubation at room temperature in mAb73 selection plates, unbound cells were released by gentle rinsing three times with PEA / FCS, then three times with PEA.

Cells not detached by rinsing expressed CD39. The CD39-expressing cells were lysed by adding 700 μl of lysis buffer containing sodium dodecyl sulfate (SDS) and incubated at room temperature for 20 minutes. Dissolve the solution of each dish with 5M Na
Each was transferred to a microcentrifuge tube containing 100 μl of Cl. The tube was capped, shaken about 20 times to mix thoroughly, and stored at 4 ° C. overnight. After overnight incubation at 4 ° C., high molecular weight DNA (residues) was removed by centrifugation and glycogen 2
μg was added to each supernatant. The supernatant was then washed twice with phenol / chloroform,
Extracted once with chloroform / isoamyl alcohol. The DNA was ethanol precipitated, washed with 80% ethanol and dried in vacuo. This purified DNA is
Was electroporated and cultured on an ampicillin plate. In this way, large-scale transformation was performed, and a total of about 48,000 colonies (sublibrary 1) were obtained. DNA was prepared from the colonies using CsCl, along with a frozen stock of the colonies.

B. Round II: DNA from electroporation and immunoselection sublibrary 1 was electroporated into COS cells (10 × 10 cm plate). Transfected COS cells were incubated, harvested and sorted essentially as described in Round I above. DNA was isolated substantially as described above and a sublibrary consisting of approximately 50,000 independent colonies (sublibrary 2) was prepared.

C. Round III: Electroporation and FACS Selection DNA from sublibrary 2 was electroporated into COS cells (10 × 10 cm plate). Transfected COS cells were incubated and harvested substantially as described in Round I. Harvested cells were analyzed by FACS substantially as described in Example 1 above. A small subpopulation of cells expressing CD39 was observed, which was sorted from the larger cell population and DNA was isolated from the sorted cells. A sub-library (sub-library 3) consisting of about 5,000 independent colonies was prepared. This D
NA was pooled into 10 pools of approximately 500 colonies each, and a frozen stock of isolated DNA and bacteria was prepared for each pool.

D. Round IV: Incubate cells from transfection and immunoselection sublibrary 3 on fibronectin-treated chamber slides (10 slides, 1 slide per pool, and 4 control slides) instead of 10 cm plates COS cells were transfected using DEAE-dextran and chloroquine treatment and incubated substantially as described in Round I above, except as noted. After two days of growth, cells were harvested as described above and analyzed not only by FACS as described in Example 1 above, but also by the slide dipping technique. For the slide dipping technique, slides were incubated with ¹²⁵ I-labeled mAb73 and fixed with glutaraldehyde. This result was determined by autoradiography in which cells containing silver particles were detected with a light microscope.

Two pools, each containing about 500 individual clones, were identified as potentially positive for CD39 production. This pool was titrated and cultured to obtain plates with an average of about 150 colonies each. A nitrocellulose filter for replication was prepared from each plate, then each plate was split into smaller pools of plasmid DNA.

E. Round V: Transfection and immunoselection COS-7 cells were treated with DNA from the small pool in DEA by the same method as above.
Transfected with E-dextran. The transfected cells were screened by slide dipping and FACS as described above. Two of the small pools contained colonies positive for CD39 as indicated by the presence of the expressed gene product binding to mAb73.

A total of 156 colonies were picked from the duplicate filters corresponding to one of the small positive pools and inoculated with the culture medium and grown overnight. After overnight growth, the culture was arranged in a 12 row, 13 column matrix format. The culture medium was pooled from each row and each column to prepare a culture medium subpool, a total of 24 subpools. This subpool is used to construct D for transfection and screening in the final round.
NA was prepared. The intersection of a positive row and a positive column indicated a potential positive colony. One potentially positive colony (ie, clone) was identified.

A streak plate was prepared from the positive clone (clone 1) and a mini-preparation of DNA (mi) from 9 individual colonies derived from the streak plate
nipreps). This DNA was digested with BglII and analyzed by SDS-PAGE. Nine of the nine individual clones from clone 1 were 1.8-2.
It contained the same insert of 0 Kb. 1 containing this 1.8-2.0 Kb insert
Two isolated clones were picked, inoculated into 10 ml of culture medium and grown overnight. DNA was prepared and sequenced by dideoxynucleotide sequencing. Clone 1
Is shown in SEQ ID NO: 1. pCD
A cloning vector containing the human CD39 sequence, designated as 39, was obtained under the Budapest Treaty on September 29, 1992 by the American Type Culture Collection,
Deposited with Rockville, MD (ATCC), and assigned deposit number 69077. A mouse homolog of CD39 was isolated by the interspecies hybridization method. The amino acid sequence of this mouse homolog is Maliszewski et al., J. Immunol.
153: 3574, 1994.

Example 5: Preparation of mAbs for CD39 This example describes the preparation of additional monoclonal antibodies to CD39, including antibodies to a region containing apyrase activity. For example, a purified CD39 fragment exhibiting apyrase activity, or a preparation of transfected cells expressing a CD39 polypeptide, is used as an immunogen in US Pat.
Monoclonal antibodies against CD39 are produced using conventional techniques as disclosed in US Pat. DNA encoding the CD39 fragment can also be used as an immunogen, for example, as described by Pardoll and Beckerleg (Immunity 3: 165, 1995). Such antibodies are useful in interfering with CD39-induced platelet aggregation, as a diagnostic or research component of CD39 or CD39 activity, and in the affinity purification of CD39.

To immunize rodents, a CD39 immunogen is adjuvanted (eg, complete or incomplete Freund's adjuvant, Alum, or Ribi adjuvant R700).
(Other adjuvants such as (Ribi, Hamilton, MT)) and into selected rodents, for example BALB / C mice or Lewis rats, from 10-100.
Inject subcutaneously in amounts in the range of μg. DNA was obtained intradermally (Raz et al., Proc. Natl. Ac
ad. Sci. USA 91: 9519, 1994) or intramuscularly (Wang et al., Proc. Natl. Acad.
Sci. USA 90: 4156, 1993), but a suitable diluent for DNA-based antigens has been found to be saline. After 10 days to 3 weeks, the immunized animals are boosted with an additional immunogen and then periodically boosted with an immunization schedule every 1 week, 2 weeks or 3 weeks.

Serum samples are collected periodically by retro-orbital bleeding or tail excision and include dot blot assays (antibody sandwich), ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, or other methods including FACS analysis. Tested by the appropriate assay. After detecting the appropriate antibody titer, positive animals are given an antigen intravenously in saline. Three to four days later, the animals are sacrificed, splenocytes harvested, and a mouse myeloma cell line (eg, NSI or preferably Ag8.653 [ATCC CRL 1580].
]). The hybridoma cell lines produced by this method are cultured in multiple microtiter plate selective media (eg, media containing hypoxanthine, aminopterin, and thymidine, or HAT), and the unfused cells, myeloma-myeloma hybrid And the proliferation of spleen cell-splenocyte hybrids.

The hybridoma clones thus produced are, for example, Engvall et a
l., Immunochem. 8: 871, 1971 and U.S. Pat. No. 4,703,004, and can be screened by ELISA for reactivity against CD39. Preferred screening techniques are described in Beckman et al., J. Immunol.
144: 4212, 1990. The positive clones are then injected intraperitoneally into syngeneic rodents to produce ascites containing high concentrations (> 1 mg / ml) of anti-CD39 monoclonal antibody. The resulting monoclonal antibodies can be purified by ammonium sulfate precipitation followed by gel exclusion chromatography. Or,
Affinity chromatography based on binding of the antibody to protein A or protein G can be utilized in the same way as affinity chromatography based on binding to CD39 polypeptide. Alternatively, another strategy is to use full-length C
Utilizing D39 to screen for antibodies that bind to CD39, for example by selecting for those that bind to a predefined epitope by screening with a CD39 fragment representing the predefined epitope.

[0117] Monoclonal antibodies can also be prepared by immunizing CD39 knockout mice with a CD39 immunogen, as described in Example 19D. In knockout mice, the entire CD39 sequence is considered "foreign", and this strategy allows antibodies that recognize epitopes shared across strain strains, including those that antagonize or agonize the biological activity of CD39. Produced.

Example 6: Physiological activity of CD39 This example demonstrates that ecto-A of endothelial cells where CD39 causes inhibition of platelet function.
Indicates that it is DPase. Human umbilical vein endothelial cells (HUVEC) constitutively inhibit platelet response to prothrombin stimulation by catabolism of exogenous platelet-derived ADP. This endothelial ecto-ADPase was identified as CD39 (
Marcus et al., J. Clin. Invest. 99: 1351, 1997). An anti-CD39 antibody immunoprecipitated ADPase activity from a preparation derived from endothelial cell membranes, and COS cells transfected with an expression vector comprising CD39 acquired ecto-ADPase activity, whereas a control vector transfected COS cells. The transfected COS cells did not gain it. Ecto-ADPase activity is determined by Marcus et al., J. Clin. In
vestigation 88: In a manner similar to that described in 1690, 1991, the activity of intact HUVEC monolayers or solubilized membranes was determined by conversion of ¹⁴ C-ADP to AMP by transfectant monolayers and membrane preparations. Greater or comparable.

HUVEC mRNA was analyzed by RT-PCR using a primer pair derived from the sequence of the activation antigen, weighted for the N-terminal part of human CD39 lymphoid cells. Lymphoid CD39 cDNA was used to directly compare the size of the PCR products. The data showed the identity of HUVEC and lymphoid CD39 in four fragments (about 1250 bp of 1850 bp of lymphoid CD39) spanning the part analyzed. Northern blot analysis was performed on CDs in HUVECs.
39 mRNA was shown to be expressed in the same band pattern as MP-1 cells from which CD39 was initially cloned.

Using confocal microscopy and fluorescence-activated cell sorting using mAb 73, H
It was determined whether the UVEC cells expressed CD39. The FACS protocol was substantially as described in Example 1. In confocal microscopy, cells grown on cover glasses (human umbilical vein endothelial cells or transfected COS-1 cells) were washed with PBS and fixed with 3% paraformaldehyde for 30 minutes at room temperature. 5
Autofluorescence was extinguished by treatment with 0 mM NH ₄ Cl for 10 minutes. Then 5% N
Cells were incubated in PBS containing GS (normal goat serum) + 0.1% Triton X-100 to prevent non-specific binding and permeabilize the cells. The cells were then incubated with an anti-CD39 antibody (5 μg / 5% NGS + 0.1% Triton X-10).
(PBS containing 0 ml) at room temperature for 1 hour. 5% NGS
After washing three times with PBS containing + 0.1% Triton X-100, a goat anti-mouse antibody (5 μg / ml, 10 mM YOYO (Molecule) labeled with Texas Red (Molecular Probes) was used for nucleic acid counterstaining.
ar Probes) were added for 1 hour at room temperature. 5% NGS + 0.1% Triton X-100, DABCO (1,4
Cells were washed three times with PBS supplemented with 100 mg / diaxabicyclo [2,2,2] octane) (Sigma) / 50% glycerol (ml). Next, Multi
probe 2001 Laser scanning confocal microscope (Molecular D)
cells were observed using the same method (dynamics). CD39 staining (Texas Red
) Was taken from the first contrast and a second contrast was taken from cell nucleus (YOYO) staining.

Both confocal microscopy and FACS experiments showed that HUVECs expressed CD39. The pattern of expression was similar to that observed in cells transfected with full length human CD39.

The bioactivity of CD39 is dependent on the ability of CD39-transfected COS cells to
Indicated by the ability to inhibit or completely reverse platelet aggregation by 0 μM ADP. COS cells transfected with CD39 were MP-1 cells or H
Like UVEC cells, this amount of ADP is metabolized to AMP within 3-4 minutes and rapidly added to platelet-rich plasma (substantially Marcus et al., Supra) when added to platelet-rich plasma. Was reversed. This activity occurred during the time frame of the process leading to platelet coalescence into the damaged subendothelial layer, ie, immediate ADP release, supplementation of additional platelets and formation of a hemostatic plug or thrombus. This time lapse is C
Parallel to platelet inhibition by D39 expressing cells, it was proportional to its ADPase activity. ADPase / CD39 activity was independent of the formation of other known thrombomodulators, nitrate or prostacyclin. The above results demonstrate the importance of ADPase / CD39 as a physiological, constitutively expressed endothelial cell thrombotic regulator.

Example 7 Phosphate Release Assay for ATPase Activity This example describes ATPase activity that can be used to track enzyme activity. Sample (concentrated CM or about 100 μl of purified polypeptide)
) With 10X assay buffer (200 mM HEPES, 1.2 M NaCl,
50 mM KCl, 15 mM CaCl ₂ , 15 mM MgCl ₂ and 3 mM A
TP) and mixed with sterile water to a final volume of 200 μl. Radiolabeled ATP (0.8 μCiγ [ ³² P] ATP; Amersham, Arlington Heights, IL) is added and the mixture is incubated at 37 ° C. for 20 minutes. A stop mix (0.5 ml of 20% activated carbon / 1M HCl) was added and the reaction was placed on ice for 1 hour.
Leave for 0 minutes. After centrifugation (14 K rpm for 10 minutes), the supernatant is assayed for free ³² P using a scintillation counter. Data are expressed as raw counts or total counts, or as picomoles of ATP degraded per minute.

Example 8: Binding Assay This example demonstrates the binding of CD39 polypeptide by biospecific interaction analysis (BIA) using a biosensor (a device that links a biological recognition mechanism to a detector or transducer). An assay for evaluating the binding to the CD39 antibody will be described. A representative biosensor is BIAcore ^™ (Fagerstam LG, Techniques in Protein Chemi) from Pharmacia Biosensor AB (Uppsala, Sweden).
stry II, ed. JJ Villafranca, Acad. Press, NY, 1991). BIAcore ^™ is a surface plasmon resonance (Kretschmann and Rad
ether, Z. Naturforschung, Teil. A 23: 2135, 1968) to monitor the interaction of two biological molecules. Suitable for characterization using BIAcore ^™ are affinity constants in the range of 10 ⁵ to 10 ¹⁰ M ⁻¹ , and 10 ³ to 10 ³
Pairs of molecules with binding rate constants in the range of 10 ⁶ M ⁻¹ s ⁻¹ .

A biosensor chip is coated with a CD39 antibody (eg, MAb73). The various CD39 constructs to be evaluated are then added at increasing concentrations. Between different constructs, for example, sodium hydroxide is added to regenerate the chip. The data obtained is analyzed to evaluate qualitatively or quantitatively the production of CD39 polypeptide. In a similar manner, other monoclonal antibodies or polypeptides that specifically bind to CD39 can be immobilized on a biosensor and the binding of various CD39 polypeptides can be evaluated.

Example 9 Transient Expression of Soluble CD39 Polypeptide This example describes the preparation of a construct for transient expression of a soluble CD39 polypeptide.

A. The reagent B73 mAb (mouse IgG1 recognizing human CD39) used was Guy Del.
Courtesy of Dr. espacese, University of Montreal, Quebec, Canada. FLAG® peptide (DYKDDDDK, SEQ ID
A mouse IgG1 that recognizes NO: 10) and mouse IgG1 were manufactured by Immunex. Immunoaffinity columns of Affigel 10 (Biorad, Hercules, CA) and CNBr-activated Sepharose 4B (Pharmacia Biotech, Piscataway, NJ) were prepared according to the manufacturer's instructions. Typically, coupling efficiencies in the range of 3-5 mg mAb / ml affinity gel slurry were obtained.

B. Construction of Soluble CD39 (solCD39) Expression Plasmid To produce a soluble molecule with the properties of CD39, the N-terminus and C-terminus of CD39 (see FIG. 2), including two transmembrane regions, were removed. To allow soluble CD39 to translocate to the medium, the amino terminus of the polypeptide was provided with a leader sequence allowing secretion.

The mammalian expression vector pDC206 (Kozlosky et al. Oncogene. 10
: 299, 1995), human IL2 (huIL2), human growth hormone (huIL).
GH) and mouse IL7 (muIL7)
A construct of 39 (solCD39) was produced.

A full length CD comprising nucleotides 178 to 1494 of SEQ ID NO: 1
The DNA sequence between the 39 putative transmembrane regions was amplified using PCR, replacing the C-terminal transmembrane coding region with a stop codon. This PCR was performed on a synthetic DNA fragment encoding an eight amino acid peptide tag (FLAG®).
The product was fused and ligated with the muIL7 leader (muIL7L) via the Spe1 and Bgl2 restriction sites into the plasmid pDC206 vector. This construct encoded solCD39 with a FLAG tag at the N-terminus.

An alternative leader was introduced by ligating this Spe1 / Bgl2 FLAG-solCD39 fragment to two different pDC206 plasmids. This leader comprises (1) human growth hormone (huGHL) and (2) human proinsulin / IL2 fusion polypeptide (huIL2L, Cullen, DNA. 7:
645, 1988). The coding region of the latter construct, shown in SEQ ID NOs: 25 and 26, contains a huIL2 leader (huIL2L, SEQ
ID NO: 25 nucleotides 1-72, amino acids 1-24), mature human I
The first 12 amino acids of L2 (nucleotides 73-108 of SEQ ID NO: 25, amino acids 25-36), a four amino acid linker (SEQ
D NO: 25 nucleotides 109-120, amino acids 37-40), FLA
G tag (nucleotides 121 to 144 of SEQ ID NO: 25, amino acid 4)
SolCD39 (nucleotide 1 of SEQ ID NO: 25)
45-1461, amino acids 49-487).

[0132] muIL7 leader, human growth hormone leader, and human proinsulin /
Constructs comprising the IL2 leader were each constructed with pIL7FlagSolCD3
9, named pGHLFlagSolCD39 and pIL2LFlagSolCD39.

Using each construct, the subconfluent layer of COS-1 cells was transiently purified using DEAE dextran followed by chloroquine as described in Cosman et al., Nature 312: 768, 1984. Was transfected. As a negative control, the CD40 ligand construct (pI12LCD40lig, Spriggs et al., J. Exp. Med. 176: 1543, 19)
92) was also transfected into COS-1 cells.

C. Preparation of conditioned medium from solCD39 transfectants Transfected COS-1 cells were incubated in DMEM-F12 medium supplemented with 5% FCS in 10 cm ² Petri dishes or 175 cm ² tissue culture flasks (37 ° C., 5% CO ₂ )did. Five days later, conditioned medium (CM) was collected from these cultures, and cells and debris were removed by centrifugation. YM-10 (10
The CM was concentrated 4-10 fold using a pressure stirred cell fitted with a (kD cutoff) membrane (Amicon, Danvers, MA).

D. AT in conditioned media derived from solCD39 transfectants
CM derived from Pase- active solCD39 transfectants (10-fold concentrated supernatant
At 0 μL), ATPase activity was assayed almost as described in Example 7, except that 10 × assay buffer contained 30 mM unlabeled ATP. Table 1 shows the results.

This transfection was repeated and CM (10, 20 and 30 μL, not concentrated) was assayed almost as described in Example 7. pIL2LFlagSo
lCD39 is pIL7FlagSolCD39 and pGHLFFagSolCD
This construct was chosen for the next study because it showed higher ATPase activity in COS-1 supernatant than 39. ATPase levels in CM from COS-1 cells transfected with pIL2LFlagSolCD39 were
It increased with culture time for at least 4 days after transfection.

[0137]

[Table 1]

[0138]

[Table 2]

E. Loss of immunoaffinity of solCD39 from COS-1CM To confirm that recombinant solCD39 is responsible for the ATPase activity observed in CM, CM from COS-1 transfectants was immunized prior to the enzyme assay. Incubated with affinity beads.

The pI cultured in DMEM / F12 supplemented with 5% FCS for 5 days
CM from COS-1 cells transfected with L2LFlagSolCD39
Was collected. 100 ml of shed Affigel beads (AG) conjugated to either chicken ovalbumin, anti-FLAG mAb, or anti-CD39 mAb per ml of CM
One aliquot was added. CM was bound for one or two cycles with one of the following: ovalbumin-bound AG, M2 mAb-bound AG, or B73 mAb-bound A
G. Each cycle included continuous gentle rocking of the slurry at 4 ° C. for 14 hours, followed by centrifugation to collect the supernatant for a subsequent binding cycle or ATPDase activity measurement.

As shown in FIG. 3, immunoprecipitation with anti-CD39 mAb-conjugated beads removed more than 80% of ATPase activity from CM. 9 by the adsorption step with the second antibody
More than 5% of ATPase activity was removed. Immunoprecipitation of anti-FLAG with beads coated with mAb (2 cycles) also resulted in a substantial loss of enzyme activity. Pre-incubation with control (ovalbumin-conjugated beads) for 2 rounds did not remove any ATPase activity from the supernatant.

F. Immunoprecipitation of Recombinant solCD39 To characterize the expression of recombinant solCD39 polypeptide, COS-1 cells were transformed with cell surface CD39 (pHuCD39, Marcus et al., J. Clin. Invest.
99: 1351, 1997), tagged soluble CD39 (pIL2LFlagSolCD3).
9) or 5% in a 10 cm ² dish, transfected with a mammalian expression vector encoding a soluble CD40 ligand (pIL-2L-CD40)
Grow in DMEM / F12 medium supplemented with FCS. 2 from transfection
One day later, the medium was replaced with Cys / Met-free medium and the cells were incubated at 37 ° C. for 1 hour. This medium was replaced with fresh Cys / Met-free medium supplemented with 5 μl of [ ³⁵ S] -Cys / Met (Amersham, Arlington Heights, IL), the newly synthesized polypeptide was labeled, and the cells were incubated at 37 ° C. The cells were cultured for 5 hours. CM from cells radiolabelled at the metabolic level was collected, purified by centrifugation and sterile filtration to remove cells and debris, and then stored at 4 ° C. until use.

For the radioimmunoprecipitation, 500 μl of ³⁵ S-labeled CM was added to 250 μl of 3% BSA /
After addition to Tris buffered saline (TBS), pH 7.7, 50 μl of 80% slurry of AG beads coated with mAb was added. If present, ³⁵ S-labeled C
M was incubated with ovalbumin-coated AG beads to remove non-specifically bound material and Ab-coated beads were added. After incubation at 4 ° C. for 14 hours, the beads were removed by centrifugation and washed three times with cold TBS.

For SDS-PAGE analysis, a 4x concentrated sample reduction buffer (250 mM Tris / HCl, pH 6.8, 8% (w / v) SDS, 40% (v / v) glycerol, 20% (v / v) ) 35 μl of 2-mercaptoethanol, 0.05% bromophenol blue dye) was added to each AG pellet, and the mixture was boiled for 5 minutes.
vex (San Diego, CA) loaded on polyacrylamide gel. The gel was electrophoresed at 25 mA, and Enhance (NEN Life Science) was used.
(Products, Boston, MA) and prepared for autoradiography by immersion in H ₂ O for 1 hour and then vacuum dried at 80 ° C. Gel is applied to Kodak (Rochester, NY) X-omatAR film.
After exposure for an hour, development was performed.

As shown in FIG. 4, the IL-2L-FLAGsolCD39 transfectant secreted an approximately 66 kD radiolabeled protein recognized by anti-CD39. This protein is an anti-CD39 from COS-1 cells transfected with a vector encoding full-length CD39 (including N-terminal and C-terminal hydrophobic regions) or a secreted protein, a vector encoding CD40 ligand. It was not detected in the immunoprecipitated CM. The anti-FLAG mAb is pIL-2LFa
A similar sized band was immunoprecipitated from the CM of the gsolCD39 transfectant, consistent with the presence of the FLAG® peptide on the recombinant solCD39. Pre-washing of the radiolabeled culture supernatant with anti-albumin-coated beads did not remove the 66 kD band, indicating specific binding to anti-CD39 and anti-FLAG. Beads coated with an unrelated isotype matched control antibody failed to immunoprecipitate a 66 kD band from solCD39 containing CM.

G. Production of an additional solCD39 fusion construct A DNA fragment comprising nucleotides 1-1488 of SEQ ID NO: 1 was produced using restriction enzymes, but the coding region was a second (most C-terminal) membrane. It is predicted to encode a fragment of CD39 lacking the transmembrane domain. Suitable linker oligonucleotides (SEQ ID NO: 14
And 15) to produce CD39 DNA, linker oligonucleotides, and regulatory elements that allow expression of the fusion polypeptides produced in mammalian cells, and to the Fc receptor (nucleotides 42-740 of SEQ ID NO: 16). Mutated human immunoglobulin Fc (SE) with reduced affinity
QID NOs: 16 and 17) were used in a three-way ligation of an expression vector comprising DNA encoding the region. This construct is called CD39 △ 2Fc, and when transfected into cells, amino acids 1-2 of SEQ ID NO: 1
And expressed a fusion polypeptide comprising amino acids 1-232 of SEQ ID NO: 17, which could be detected on the surface of transfected cells using anti-CD39 or anti-human IgG.

Using PCR technology, the first lacks the transmembrane region almost near the amino terminus, but lacks nucleotides 181-1488 (amino acids 39-4) of SEQ ID NO: 1.
DNA fragments were prepared from the CD39 DNA derived from CD39） 2Fc, including the CD39 DNA from 74) and the Fc mutein DNA from CD39 △ 2Fc (using the linkers shown in SEQ ID NOs: 18 and 19).

The resulting DNA was then ligated into an expression vector containing DNA encoding a mouse interleukin-7 leader sequence (SEQ ID NO: 20) ligated in the immediate vicinity of the CD39 coding sequence. This construct was named CD39 △ 1, △ 2Fc.

The DNA encoding the FLAG® peptide (SEQ ID NO: 10) and the codons corresponding to nucleotides 178, 179 and 180 of SEQ ID NO: 1 were isolated from the leader sequence and the CD39 coding sequence by CD39 △. 1, $ 2F
c construct to obtain a detectable tag for the amino terminus of the fusion polypeptide (using the linker shown in SEQ ID NOs: 21 and 22). This tagged construct was named FlagCD39 $ 1, $ 2Fc.

[0150] The Fc mutein sequence was removed and SEQ ID N, immediately downstream of the CD39 sequence.
O: the codon corresponding to nucleotides 1489 to 1494 of (SEQ ID
Another construct was made (using the linkers shown in NO: 23 and 24). This construct was named FlagCD39 △ 1, △ 2.

[0151] Each of the above constructs was transfected into mammalian cells, and protein levels on the surface, inside, or in the supernatant of the transfected cells were determined using antibodies to FLAG®, CD39 or human IgG. Assayed.

Example 10: To promote the expression of pIL2LSolCD39 and the establishment of stable production transfectants in activated CHO cells.
An untagged version of soluble human CD39 (solCD39) was constructed. pI
A similar fragment derived from solCD39 with the FLAG® tag of L2LFlagSolCD39 and the 523 bp Spe1 / Nde1 fragment containing the first 163 amino acids (aa) removed and a CAG-terminal FLAG® tag Replaced with In this way, the entire solCD39 coding region, retaining the HuIL2 leader and mature IL2 residues but lacking FLAG®, was reconstructed. This construct was named pIL2LSolCD39. The coding region of pIL2LSolCD39, shown in SEQ ID NO: 7, contains the huIL2 leader (huIL2L, nucleotides 1-72, amino acids 1-24 of SEQ ID NO: 7), the first 12 amino acids of mature human IL2 (SEQ ID NO: 7). NO: nucleotides 73-108 of amino acid 7, amino acids 25-
36) A three amino acid linker (nucleotide 10 of SEQ ID NO: 7)
9-117, amino acids 37-39), and solCD39 (SEQ ID NO:
: 7 nucleotides 118-1434, amino acids 40-478).

To determine if removal of the N-terminal FLAG® tag affected activity, COS-1 cells were treated with pIL2LFlagSolCD39 and pIL2.
Transfected with 2LSolCD39 and supernatants (sups) were collected after 5 days. Ixsups samples, 10, 20, and 30 μl were assayed for ATPase activity as described in Example 7. As shown in Table 3, activity was not affected by removal of the N-terminal FLAG® tag.

[0154]

[Table 3]

[0155] Example 11: production of additional solCD39 fusion construct A. Production and characterization of Trim1 and Trim2 mutants To characterize the effect of 12 mature human IL2 (huIL2) residues on solCD39 expression, two additional mutants of pIL2LSolCD39: pI
L2LTrim1 (“Trim1”) and pIL2LTrim2 (“Trim2
The huIL2 residue was removed during the construction of the nucleic acid sequence encoding ")).

The pIL2LTrim1 mutant has solCD39 except for the first four amino acids.
Hind3 from pIL2LSolCD39 containing the entire coding region
/ Bgl2 restriction fragment was constructed by purification. This fragment (containing the huIL2 leader and the first amino acid of mature huIL2)
Along with the synthetic oligo cassette, it was ligated to Sma1 / Bgl2 digested pDC206. Thus, the huIL2 leader was reintroduced and bound to solCD39 with intervening alanine residues.

The pIL2LTrim2 mutant is a Spe1 / derived from pIL2LSolCD39.
Using a Bgl2 fragment and a synthetic oligocassette containing the huIL2 leader and linker coding sequence (with the first codon changed to alanine),
Constructed in a similar way. In this way, the huIL2 reader is replaced with solCD3
Recovered with intervening Ala-Ser-Ser preceding 9

PIL2LSolCD39, pIL2LTrim1 and pIL2LTrim2
The N-terminal portion of the polypeptide is compared below. The predicted cleavage point is indicated by ★: pIL2LSolCD39 (SEQ ID NO: 11) MALWIDRMQLLSCIALSLALVTNS ★ APTSSSTKKT
QLts sT QNK. . . pIL2LTrim1 (SEQ ID NO: 12) MALWIDRMQLLSCIALSLALVTNS AT * QNK. . . pIL2LTrim2 (SEQ ID NO: 13) MALWIDRMQLLSCIALSLALVTNS as ★ sT QNK. . . The polypeptide encoded by the Trim1 construct has the sequence SEQ ID N
O: 27. Residues 26-464 are the soluble portion of CD39, with cleavage of the leader sequence between Ser24 and Ala25.

The expression of Trim1 and Trim2 constructs was determined using CO cultured on 10 cm plates.
Analyzed in S-1 cells. After 5 days incubation, (anti-CD39
1x supernatant was tested by ELISA and the phosphate release assay described in Example 7. As shown in Table 4, the specific activity of Trim1 and Trim2 (based on the concentration determined by ELISA) was equivalent to pIL2LFlagSolCD39. However, expression levels appear to have decreased 3-4 fold.

[0160]

[Table 4]

Also, COS-1 cells were transfected with pIL2LSolCD39, pIL2LTrim1 and pIL2LTrim2, and placed in a T175 flask (30 mL).
And cultured. Then, 5 days later, 1xsup was analyzed by ELISA. As shown in Table 5, the results of the ELISA also showed that the expression levels were reduced with the Trim1 and Trim2 mutants.

To further characterize the effect of human IL2 (huIL2) residues on the expression of solCD39, pIL2 was isolated using anti-CD39 coated sepharose.
The products of LSolCD39, pIL2LTrim1 and pIL2LTrim2 were purified. The N-terminal amino acid sequence was determined for each of the purified polypeptides. For solCD39, the N-terminus is APTSSSTKKT ... (SEQ I
D NO: 11 residues 25 to 34). In Trim1, the N-terminus is ATQ
NKALPEN ... (residues 25 to 34 of SEQ ID NO: 27).
In Trim2, the polypeptide had a heterogeneous N-terminus.

[0163]

[Table 5]

B. Production and Characterization of Trim3 and Trim4 Mutants Nucleic acids encoding additional solCD39 variants were converted to pIL2LT Trim3 ("T
rim3 ") and pIL2LTrim4 (" Trim4 "), also constructed using a synthetic oligocassette strategy.

The N-terminal portions of the solCD39, Trim3 and Trim4 polypeptides are compared below. The predicted cleavage point is indicated by ★: pIL2LSolCD39 (SEQ ID NO: 11) MALWIDRMQLLSCIALSLALVTNS ★ APTSSST KK
TQLtssTQNK. . . pIL2LTrim3 (SEQ ID NO: 28) MALWIDRMQLLSCIALSLALVTNS ★ A KK
TQLtssTQNK. . . pIL2LTrim4 (SEQ ID NO: 29) MALWIDRMQLLSCIALSLALVTNS ST ★ KK
TQLtssTQNK. . . The polypeptide encoded by the Trim3 construct has the sequence SEQ ID N
O: 28. Residues 36-474 are the soluble portion of CD39 and the predicted cleavage of the leader sequence is between Ser24 and Ala25. The polypeptide encoded by the Trim4 construct has the sequence SEQ ID NO: 29.
Residues 35-473 are the soluble portion of CD39 and the predicted cleavage of the leader sequence is between Thr26 and Lys27.

Expressing the pIL2LTrim3 and pIL2LTrim4 polypeptides,
Purified using Sepharose coated with anti-CD39. For each of the polypeptides, determine the N-terminal amino acid sequence and specific activity.

C. Production and characterization of solCD39-L4 fusion polypeptides The CD39 gene family comprises at least four human members (CD39, C39
D39L2, CD39L3 and CD39L4) (Chadwi
ck and Frischauf, Genomics 50: 357, 1988). CD39-L4 is reported to be a secreted apyrase (Mulero et al., J. Biol. Chem. 274 (29): 2).
0064, 1999). Additional solCD39 variants are constructed by fusing the N-terminal sequence from CD39L2, CD39L3 or CD40L4 to the soluble portion of CD39. FIG. 2 shows the N-terminal amino acid sequences of human CD39 and human CD39L4.
4 side by side.

In one construct, CD39-L4-1, CD39-L4 amino acid residues 1-37
The nucleic acid encoding (Met1 to Ser37 of SEQ ID NO: 31) is C
Residues 38-476 of D39 (Thr38-Thr47 of SEQ ID NO: 2)
6) is fused to the nucleic acid encoding The polypeptide encoded by the CD39-L4-1 construct has the sequence of SEQ ID NO: 3. Residues 1-37 are from CD39-L4, residues 38-476 are the soluble portion of CD39, and the predicted site for cleavage of the leader sequence is between Ala20 and Val21.

In another construct, CD39-L4-2, CD39-L4 amino acid residue 1
-48 (Met1-Leu48 of SEQ ID NO: 31) are encoded by residues 49-476 of CD39 (Tyr49-Th of SEQ ID NO: 2).
r476). Another construct, CD39-L4
-3 means that the Cys residue at position 39 (Cys39) is another amino acid, preferably Ser
Is the same as CD39-L4-2 except that CD39-L4-
2 and the polypeptide encoded by the CD39-L4-3 construct have the sequence
It has the sequence of ID NO: 4. Residues 1-48 are from CD39-L4, residues 49-476 are the soluble portion of CD39, and the predicted site for cleavage of the leader sequence is between Ala20 and Val21.

Additional constructs include the N-terminal coding region of CD39-L4
It is constructed by fusing to the terminal coding region. After expression in recombinant cells, the N-terminal sequence, enzyme activity and platelet inhibitory activity are determined for each of the polypeptide products.

D. Production and characterization of IgkappaLsolCD39 Amino acids from IL-2 and a nucleic acid encoding an Igkappa leader sequence fused to solCD39 are also constructed. One such construct is Igcap
pa leader and CD39 soluble portion (residue Thr38 of SEQ ID NO: 2)
と し て encodes a polypeptide having 4 amino acids from IL-2 fused to the N-terminus of the polypeptide. Thus, the N of the encoded polypeptide
The end is 5'-METDTLLLWVLLLLVPGSTG ★ APTSQNK
ALPE. . . (Amino acids 1-32 of SEQ ID NO: 30), wherein amino acids Met1-Gly20 are Igkappa leaders and Ala21
Ser24 is derived from IL-2, and Thr25 to Glu32 is solCD39.
This is the beginning of the array. The predicted cleavage site is indicated by ★. Igkappa
The polypeptide encoded by the LsolCD39 construct has SEQ ID N
O: has an arrangement of 30. Residues 25-463 are the soluble portion of CD39 and the predicted cleavage of the leader sequence is between Gly20 and Ala21. After expression in recombinant cells, the N-terminal sequence, enzyme activity and platelet inhibitory activity are determined for each polypeptide product.

Example 12: Stably transfected cells secreting solCD39
Produce CHO cell lines expressing development solCD39 system, improving the production of recombinant solCD39 polypeptide.

A. Preparation of Constructs and Cell Lines for Stably Expressing Soluble CD39 Polypeptide A recombinant solCD39 sequence and a cDNA insert of solCD39 containing a non-FLAG® IL-2 leader were digested from the pIL2LSolCD39 plasmid by Xmal / BglII digestion. It was excised, inserted into 2A5Ib, an expression vector containing the DHFR gene, and optimized for a stable CHO cell line (Morris et al., Animal Cell Technology: From Vaccines to Gene).
tic Medicine, MJT Carrondo, B. Griffiths, and JLP Moreira
Kluwer Academic Publishers, Boston, 529-534, 1997).

The solCD39-2A5Ib plasmid was transferred to Lipo by the manufacturer's recommendations.
CHO cells were transfected using Fectamine (GIBCO BRL; Gaithersburg, MD). The CHO cell line, DX, used for this test
B-11 was already adapted to serum-free suspension culture conditions. The transfected cells are treated with peptone, glutamine, glucose, transferrin, lipid and I.
Grow in modified DMEM-F12 medium supplemented with GF-1 (insulin-like growth factor; used only when the culture is induced for protein expression).
After three days of growth, cells were transferred to a selective medium lacking hypoxanthine and thymidine. Stock cultures were grown in suspension at 37 ° C. and passaged every 2-3 days.
Induced cultures were grown in suspension at 31 ° C. with IGF-1 and sodium butyrate (1-2 mM). The cell density at the start of the induction culture is 1.5-2 × 10 ⁶
Cells / ml. The average induction period was 7 days, at which time CM was collected for later analysis.

B. ADPase and ATPase activities in solCD39-containing CM
After growth in TLC assay selection media, CM from CHO cell cultures was transformed with ATPase and A
It was analyzed for DPase activity. ADPase assay (Marcus et al., J.
Clin. Invest. 88: 1690, 1991) was mainly used in determining enzyme kinetics and pharmacokinetics. Test samples were assayed in assay buffer (15 mM Tris, 134 mM) containing 10 μM Ap5A (P ¹ , P ⁵ -di [adenosine-5 ′] pentaphosphate, 1 mM ouabain, 10 μM dipyridamole and 3 mM CaCl ₂ ).
mM NaCl, 5 mM glucose, pH 7.4), in a total volume of 50 μl,
50 μM [ ¹⁴ C] ADP (NEN Life Science Product)
s) (5 min, 37 ° C.). Place the reaction on ice for 1
0 μl of “stop solution” (160 mM disodium EDTA, pH 7, 17 mM
(ADP, 0.15 M NaCl) was added to prevent and stop further metabolism of ADP. Isobutanol / 1-pentanol / ethylene glycol monoethyl ether _{/ NH 4 OH / H 2 O} TL to use (90: 60: 180: 90 120)
C separated nucleotides, nucleosides and bases. Radiation activity TL
Quantification was performed by C scanning (RTLC multi-scanner; Packard, Meriden, CT). The results were calculated as the average of two to four measurements minus buffer blanks (typically less than 1% of total radioactivity). Data is 1 for 1 μl CM
Expressed as the ratio of ADP metabolized per minute or picomoles of metabolized ADP. One unit of activity is the amount of enzyme that degrades 1 μmol of ADP per minute at 37 ° C. The same assay was performed using ATP as a substrate to test the kinetics of ATPase activity of CD39.

As shown in Table 6, in the stably transfected CHO cells, any enzyme activity was 2 compared to the CM from the transfected COS-1 cells.
0-fold higher levels were secreted.

[0177]

[Table 6]

C. Sol CD39 from stably transfected CHO cells
30 ml of a 10-fold concentrated CM derived from affinity purified solCD39 secreting CHO cells was added to B73
It was added to the gel slurry of Sepharose 4B coated with mAb and mixed overnight at 4 ° C. The affinity matrix is centrifuged into a pellet and PBS
, And added to the plastic column. The specifically bound protein was eluted by adding 0.1 M triethylamine (pH 11.5). Fractions (1.2 ml each) are collected in a test tube containing 120 μl of a neutralization solution (1 M sodium phosphate, monobasic; pH 4.3), and analyzed by SDS-PAGE for protein content. Stained. AT as described in Example 7
Biological activity was quantified using a Pase assay, active peak fractions were pooled, the buffer was exchanged for PBS and concentrated 5-fold. Oxford Glycosystem
The N-linked sugar was removed from the purified protein using a kit from s (Rosedale, NY). This recombinant solCD39 was analyzed by SDS-PAGE.

A clear band of about 66 kD was present in the first eluted fraction, and a peak near the fifth fraction was observed by Coomassie blue staining. More than 90% of the proteins present were found as this major band. If overloaded on polyacrylamide gel,
Several low molecular weight contaminants have emerged, which are derived from the antibodies present on the column and appear to be independent of the protein loaded on the column.

The ATPDase activity of the affinity column fraction correlated with the intensity of the protein band on SDS-PAGE (FIGS. 5A and 5B). ATPDase activity is anti-CD
Few were detected in the flow-through fraction of the 39 columns, indicating that this affinity purification is an effective means of isolating biologically active recombinant CD39.
Treatment of the purified protein with N-glycanase for 18 hours to remove N-linked oligosaccharides resulted in the degradation of the broad band at 66 kD into a tighter protein band of about 52 kD. This is the expected molecular weight size for solCD39 (FIG. 5C).

The amount of total protein from 1 L of CM of CHO-solCD39 was about 2 mg. The production of solCD39 was scaled up to a 10 liter bioreactor. According to ELISA analysis, the produced conditioned medium contained about 50-100 μG /
ml of solCD39. Thus, it is expected that every 10 L bioreactor run will produce 500-1000 mg of recombinant polypeptide. CHO cell lines expressing additional solCD39 constructs were also produced,
Characterized.

Example 13: solCD39 in Veggie-CHO and CS-9 cells
In this example, CHO cells adapted to grow in suspension in animal protein free media (see Rasmussen et al., Cytotechnology, 28: 31, 1998), or IGF-1 Soluble CD39 is expressed in the clonal cell line CS-9 in the presence.

A Chinese hamster ovary cell line deficient in dihydrofolate reductase, DXB1
1-CHO is commonly used as a host cell to produce a recombinant polypeptide. DXB11-CHO was adapted to grow in suspension. Then
By adapting DXB11-CHO cells to grow in serum-free medium in the absence of exogenous growth factors such as transferrin and insulin-like growth factor (IGF-1), a serum-free host named Veggie-CHO Was produced. The latter adaptation gradually reduces the supplemented serum in the medium and lowers the levels of growth factors, IG
Achieved by replacing serum with F-1 and transferrin in a concentrated cell growth medium. Cells adapted to suspension and serum-free were detached from the above growth factors. The resulting Veggie-CHO cells maintain vigorous growth and high viability, as well as a DHFR-deficient phenotype, in a medium free of serum and animal-derived proteins. CS-9 cells were also derived from DXB11-CHO cells. Cells adapted to suspension and serum-free were adapted to grow in the absence of transferrin and individual clones were isolated. The CS-9 clone was selected for its stable recombinant protein expression.

Veggie-CHO cells and CS-9 cells are used as host cell lines to stably express high levels of soluble CD39 polypeptide using methods similar to those described in Example 12.

Example 14: Biochemical properties of affinity-purified solCD39 Purified solCD39 material was subjected to N-terminal amino acid sequencing and mass spectrometry. Quantitative amino acid analysis of the peak fractions (3-9) of the affinity column yielded the proportion of amino acid residues consistent with the calculated value of human CD39. pIL2LSo
The N-terminus of the ICD39 product had the following sequence: APTSSSTKKTQLtsssTQ. . . (Residues 25-41 of SEQ ID NO: 11) The first 12 residues represent the mature huIL2 residue; residues 13-15 (tss,
Lowercase letters are residues encoding the linker; residues 16, 17 and below (TQ ...
) Is solCD39.

Using the TLC assay described in Example 12, membrane-bound HUVEC Ect-A
The ADPase activity of DPase was measured using 100 mM bis-trispropane (Sigma,
(St. Louis, Mo.) at various pH values of the buffer. This was compared with the ADPase activity of purified solCD39 at the above pH. AT of CD39
Reaction kinetic constants for P and ADP metabolism were determined by measuring the initial reaction rate analyzed in the TLC system. 2.5-150 μM ADP or ATP were separately incubated with 2 × 10 ⁻⁹ M solCD39.

As shown in FIG. 6A, the optimal pH for the ATPase activity of the ecto-ADPase on the HUVEC surface and the recombinant solCD39 purified affinity was pH 8
〜8.5. This indicates that recombinant solCD39 is a native CD
39 / ecto-ATPase showed maximal activity under the same physiological conditions.

The initial rates of ATP and ADP metabolism by recombinant solCD39 are shown in FIG. 6B.
And determined the reaction kinetic constants. K for ADP_mAnd V_maxEach
5.9 μM and 72 pmol / min._mIs 2.1 μM,
V_maxWas determined to be 26 pmol / min. The assay is 2x10^-9M's so
Implemented with lCD39, 720 min^-1(ADP) and 260min^-1(ATP)
K_catI got As described above, the specificity constant, k_cat/ K_m(1.2 × 10⁸min ^-1 M^-1) Was identical for ADP and ATP. Purified recombinant solCD
The specific activity of 39 was 11 U / mg for ADP and 4 U / mg for ATP.
Was.

Example 15: Platelet inhibitory activity of solCD39 This example shows that affinity purified recombinant solCD39 is effective as an inhibitor of platelet activation and recruitment.

After obtaining informed consent from volunteers, citric acid-dextrose (38 mM citric acid; 75 mM sodium citrate;
Blood was collected via plastic tubing using 35 mM glucose). Where indicated, volunteers had taken 650 mg of acetylsalicylic acid (ASA) 18 hours prior to blood collection. First leukocyte centrifugation (200 g, 15
At 25 ° C.) to prepare a platelet-rich plasma (PRP), and the second centrifugation of PRP (90 g, 10 minutes) to remove residual red blood cells and white blood cells. It was maintained at room temperature stock suspension PRP under 5% CO ₂ / air.

A. Platelet aggregation test PRP containing 1.22 × 10 ⁸ platelets alone or solCD3
Agglutination measurement cuvette (Lumiaggregom)
pre; (Chrono-Log, Havertown, PA). TSG buffer (Marcus et al., J. Clin.
Invest. 88: 1690, 1991; Marcus et al., J. Clin. Invest. 99: 1351, 1997)
The total volume was adjusted to 300 μl using. After preincubation for 3 minutes,
A platelet agonist (ADP or collagen) is added at the specified concentration, and the
Minutes recorded. Where indicated, 10 μM indomethacin (Sigma, St. Louis, MO) was added to PRP to inhibit endogenous cyclooxygenase activity.

As shown in FIG. 7, the addition of 10 μM ADP to RPR alone resulted in complete, irreversible agglutination, whereas low concentrations of ADP resulted in partially irreversible agglutination. However, the presence of only 3.3 μg / ml of solCD39
Platelet aggregation induced by μM ADP stopped abruptly and the curve rapidly returned to baseline levels. Importantly, the degree of aggregation was reduced to levels below those observed with 1 μM ADP. Higher concentrations of solCD39 had a much stronger inhibitory effect, almost eliminating the initial aggregation burst induced by 10 μM ADP.

Platelet reactivity to 5 μM ADP was tested in PRP with and without the cyclooxygenase inhibitor indomethacin (10 μM) in the presence of CM containing solCD39 from COS-1 and CHO cells . As shown in FIG. 8A, indomethacin treatment partially reversed ADP-induced platelet aggregation in the absence of solCD39. In contrast, solCD39
CM was able to completely eliminate the platelet response to ADP with or without indomethacin treatment of PRP.

Inhibition of the platelet response by CD39 is shown in FIGS. 8B and 8C.
It is not limited to blocking the operation effect of P. Collagen, another important platelet agonist, was used at 1 μg / ml to induce platelet aggregation. The presence of solCD39 significantly reduced the response to collagen as compared to the control (FIG. 8B, upper curve). A similar effect of solCD39 was also observed with indomethacin treated PRP using collagen at 3.3 μg / ml (FIG. 8B, lower curve). As shown in FIG. 8C, the effect of solCD39 on collagen-induced aggregation was dose-dependent.

B. Inactivates solCD39 enzyme activity and inhibits platelet activation
To show that the ability of solCD39 to inhibit platelet activation and recruitment is due to the enzymatic activity of solCD39 and not to other properties,
Inhibits collagen-induced platelet activation (Colman et al., Blood 68: 565, 19
86), irreversibly binds to ATPDase found in several cell types (Se
vigny et al., Biochem. Biophys. Acta 1334: 73, 1997; Sevigny et al., Bio
chem. J., 312: 351, 1995) ATP analog FSBA (fluorosulfonylbenzoyl-adenosine) was reacted with solCD39.

SolCD39 (2 nmol) was added to a labeling buffer (100 mM Hepes, p
H7.4, 200 mM NaCl, 4 ml / vol% dimethylformamide (2 ml), 5 mM FSBA (Sigma Chemical Co.) 40 dissolved in ethanol
Combined with 0 μl and 1.52 ml of water. A mock-treated sample in which the FSBA solution was replaced with water was also prepared. After 90 minutes incubation at 37 ° C., the samples were
5,500 rpm in tricon-10 filter unit (Amicon)
The buffer was exchanged for PBS to remove unreacted substances. FSB
The effect of A-treated solCD39 on platelet reactivity is shown in FIG.

Induction of platelet activation by ADP (FIG. 9A) or collagen (FIG. 9B) was significantly inhibited by either purified solCD39 or mock FSBA-treated solCD39. In contrast, incubation with FSBA-treated solCD39 had no significant effect on platelet activation. Fake solCD39
Comparing the titers of FSBA-treated solCD39 with FSBA-treated solCD39 (FIG. 9C),
22.0 μg / ml of olCD39 was 0.88 μg / m of mock-treated solCD39
1 showed a similar aggregation profile. This indicates that 96% of the aggregation inhibitory activity of solCD39 was lost by FSBA treatment. FSBA processing solC
Analysis of the residual ADPase activity of D39 by a radio-TLC assay system showed that about 96% of the enzyme activity was blocked, indicating that the same proportion of ATPase activity was still lost in the phosphate release assay. Was.

C. First Embodiment Mutagenesis Assay Site-directed mutagenesis was used to alter selected amino acids of CD39 to identify amino acids involved in the biological activity of solCD39. Mutants were assayed for enzyme (ATPase and ADPase) and platelet inhibitory (dose-dependent inhibition of platelet aggregation) activity. In one mutant series, several residues in the conserved apyrase region were replaced with alanine.

Platelet inhibitory activity generally correlated with enzyme activity. The E174 mutant (residues counted as in FIG. 1) completely lacked enzymatic activity and had no effect on platelet reactivity. Also, the S218A mutant retained less than 10% ADPase activity and about 10% platelet aggregation activity. Thus, glutamic acid 174 and serine 218 are considered to be important for both the CD39 enzyme and platelet inhibitory activities.

Additional CD39 mutant forms were expressed and assayed for enzyme and platelet inhibitory activity, mutants with increased or decreased activity, and ATPase or ADP
Mutants that preferentially catalyze the ase reaction are identified.

Example 16: Persistent Balb / c mice of solCD39 after in vivo administration to mice (6-8 weeks old; maintained under special sterile conditions; Jackson L)
(laboratory, Bar Harbor, ME) was injected intravenously with 100 μl of recombinant solCD39 (50 μg) dissolved in sterile saline (0.9% NaCl). No apparent problems were observed in the animals after the injection. At various time points after injection (5, 10, 30 minutes, 1, 2, 4, 8, 24 hours), a pair of mice were bled by cardiac puncture and euthanized. Serum was prepared from each blood sample and stored frozen until assay. Biologically active C present in serum samples
D39 was measured in ATPase and ADPase activity. This data was fitted using Deltagraph (Deltapoint, Monterrey, CA). The best fit was derived from the use of double exponential decay. When specified, the specificity of the enzyme activity was determined by incubating the serum sample with beads coated with anti-CD39 mAb and removing CD39 before testing for ATPase activity.

As shown in FIG. 10, data showing the best fit to the biphasic exponential curve was obtained.
The amount of ATPase activity derived from 25 μg / ml of solCD39 in mouse serum is shown for comparison. t _1/2 α (distribution phase) was 59 minutes in the ATPase assay, A
The DP assay calculated 43 minutes. In this layer, about 55-65% of the apyrase activity had disappeared from the circulation. The disappearance phase was about _1/2 hour t _1/2 β in both assays. Prior washing of the 10 min, 2 h and 24 h time points samples with beads coated with anti-CD39-mAB completely abolished serum ATPase / ADPase activity. The above data also indicates that this assay was performed using recombinant human solC
This shows that D39 is specifically detected.

Example 17: Pilot dose setting trial using Yorkshire / Hampshire pigs
Sol to a Yorkshire / Hampshire pig developed as a pig model of test thrombus
CD39 was administered. After intravenous injection, CD39 persisted in the circulation and was able to inhibit platelet aggregation and recruitment for as long as one week after injection. This is in sharp contrast to other therapeutic agents used for platelet inhibition, where the inhibition time is very short.

Ten pigs were randomly assigned to receive a low (72 μg / kg), medium (221 μg / kg) or high (72 μg / kg) dose of solCD39. Aspirin was administered orally every day. The placebo subject was aspirin. Control saline and solCD39 were administered as a single bolus. The time from this administration was measured. Blood samples were collected via an external jugular vein catheter. Bleeding time was measured at baseline and at 60 minutes in placebo control pigs and pigs receiving solCD39. At specific time intervals of dose, ADP-induced platelet aggregation was measured. The serum concentration of solCD39 at a certain time was measured using an ELISA assay.

Administration of solCD39 was well tolerated. It also did not induce anemia or thrombocytopenia, and importantly, no adverse effects such as hypotension, plasmacytopenia or bleeding could be observed with the second administration of solCD39.

A. Effect of solCD39 on bleeding time Bleeding time is an absolute measure of platelet function. As shown in FIG.
lCD39 induced prolonged bleeding time. This indicated that a therapeutic effect was obtained through mild interference with platelet function. This slight increase in bleeding time was similar to that obtained with aspirin administration. This means
Indicates that the bleeding defect is minor.

B. Effect of Aspirin and solCD39 on Platelet Aggregation FIG. 12A shows the effect of aspirin on baseline and on day 5 of aspirin. FIG. 12B shows the effect of high-dose CD39 on baseline and platelet aggregation on day 7. FIG. 13 plots the peak height of the platelet aggregation curve for each of the three solCD39 doses. The platelet aggregation data is also compared by plotting the relative area of the platelet curve for each of the three solCD39 doses. A dose of 670 μg / kg inhibited more than 90% of ADP-induced platelet aggregation. This inhibitory effect is long lasting (high doses of solCD3
There was a 30% inhibition in 2 weeks (after 9 doses). The above experiments show that solCD39 exhibits a potent and long lasting antiplatelet effect and that the effects are superior to those obtained using aspirin.

C. Persistence of solCD39 in Serum FIG. 14 shows the persistence of solCD39 in porcine serum as determined by ELISA. Distribution and clearance half-lives were determined using a biphasic curve fitting method. t _1/2 α (distribution phase) was calculated to be 29 minutes. Elimination phase had a t _1/2 beta of about 51 hours. The biological activity of solCD39 (ADPase activity) also showed a long elimination half-life approaching 5 to 7 days, and could be detected more than 2 weeks after administration. During this time, the hematological parameters did not change, and there was no evidence of bleeding when the bleeding time tripled.

D. Percutaneous Transluminal Coronary Angioplasty (PTCA) Test Pigs were labeled with ¹¹¹ indium and ¹²⁵ iodine, respectively, which injected piglets with platelets and fibrinogen. Twelve pigs were given a sedative, anesthetized and given intravenous so
They were randomly assigned to receive either ICD39 (670 μg / kg) plus heparin and ASA, or intravenous saline placebo plus heparin and ASA. ASA was administered to all pigs orally at least 3 days prior to coronary angioplasty and heparin (100 U / kg) was administered at the time of angioplasty. Platelets and fibrin, CD3, inserted into the external jugular vein line 1-3 days prior to angiogenesis
9 or saline was administered to facilitate blood aspiration. Labeled platelets and fibrinogen were administered about 18 hours prior to balloon injury. An oversized balloon was used to injure the coronary artery. First, the coronary guide catheter was advanced into the ascending artery. The oversized balloon is then advanced into the coronary vessels, for a total of 6
Inflated at 8 atm. The balloon was then deflated and withdrawn. The average ratio of balloon size to blood vessel size was 1.32 in the placebo group and 1.32 in the CD39 group.
29.

Thirty minutes after administration, platelet aggregation and bleeding time were measured. Balloon damage and sol
Pigs were sacrificed 24 hours after CD39 administration and labeled platelet ( ¹¹¹ indium) and fibrin ( ¹²⁵ iodine) deposition was measured on damaged coronary artery sections. Table 7 shows the results. CD39 administration was well tolerated without bleeding or hemodynamic complications. In addition, no bleeding was observed during PTCA or after removal of the sheath, and there was no significant difference in hematocrit or platelet count between each group.

The above results show that administration of solCD39 results in significant inhibition of platelet aggregation and prolonged bleeding time, and a tendency to inhibit platelet and fibrin deposition following balloon injury in animals. The results also suggest that CD39 has a very low risk of inducing bleeding.

[0212]

[Table 7]

After the radioactivity collapsed, the damaged coronary arteries stained with toluidine blue were examined histologically to further characterize the extent of thrombus formation. The degree of histological damage in coronal sections was assessed by blinded observers. The rating was on a scale of 1 to 4, with 4 being the most severe injury (severity for medial and internal elastic lamina detachment, medial separation and bleeding). An overall damage score was obtained by summing the three individual scores. The median damage scores for the placebo and CD39 groups were 2.5 and 2.2, respectively; the medial segregation scores for the placebo and CD39 groups were 2.0 and 1.6, respectively; the bleeding degree for the placebo and CD39 groups was 2 respectively. 0.3 and 2.5. The overall damage score for the placebo and CD39 groups was 6.6 and 6.2, respectively. These in vivo results correlate well with the results reported herein obtained in vitro and ex vivo.

Example 18: Soluble CD39 was obtained from aspirin and abciximab.
) Provides additive platelet aggregation inhibition . Add soluble CD39 to platelet-rich serum from patients receiving placebo, aspirin, clopidogrel, ticlopidine or abciximab ex vivo
Studies were performed to evaluate the additive inhibition of platelet aggregation. Each group contained 3-6 patients. The clopidogrel, ticlopidine and abciximab groups also received aspirin. The reference platelet aggregation of each group when responding to the platelet agonist ADP, collagen, or thrombin receptor activating peptide (TRAP _1-6 ) was measured. Then, solCD39 (10 μg / ml or 100 μg / ml) was added,
The additive inhibition of platelet activation (relative to criteria responding to platelet agonists) was measured in each of the five groups. Table 8 shows the results.

[0215]

[Table 8]

SolCD39 at a concentration of 10 μg / ml, ADP, collagen and TRA
P-mediated platelet aggregation was synergistically inhibited in patients with aspirin (p <0.0
01), this effect was independent of clopidogrel or ticlopidine. Abciximab alone prevented platelet aggregation by ADP and collagen, but CD3
9 provided a synergistic inhibition of platelet aggregation induced by TRAP (p <0.
007). solCD39, at a concentration of 100 μg / ml, provided increased inhibition of platelet aggregation for all agonists. Since collagen and TRAP induce platelet aggregation through a mechanism other than ADP release and recruitment, collagen and TRAP
The ability of CD39 to inhibit AP-mediated platelet aggregation is due to CD3 as an antithrombotic agent
9 suggests further versatility.

Example 19: Soluble CD39 is found in wild-type and reconstituted CD39-deficient mice.
Inhibits thrombosis and inhibits ischemic brain damage . The above examples suggested that soluble CD39 inhibits the amplification of ADP-mediated platelet recruitment in distal microvessels, thereby reducing post-stroke thrombosis. The following experiments illustrate the use of CD39 in a microvascular thrombosis (mouse ischemic stroke) model. solCD39 inhibited microvascular thrombosis and provided cerebral protection during stroke. A notable feature of solCD39 treatment is the lower incidence of intracerebral hemorrhage compared to that reported for other antithrombotic agents.

A. Materials and Methods C57BL / 6J mice (6-8 weeks old) were purchased from Jackson Laborato
ries (Bar Harbor, ME). Untreated mouse, sol
Mice treated with CD39 (4 mg / kg), aspirin (5 mg / kg) or phosphate buffered saline were anesthetized, heparinized (10 U / g) and blood was collected via cardiac puncture. 80 μL of 3.8% sodium citrate per 1 ml of blood
Was added. Samples from 6-8 mice were pooled and platelet rich plasma (PRP) was prepared by centrifugation. This PRP contains 400 to 1 μL.
700 × 10 ³ platelets were included. All experiments were completed within 2 hours of blood collection. PRP (200 μL) was added to an agglutination measurement cuvette (Lumiggg).
regometer; Chromo-Log, Havertown, PA) in Tris-buffered saline (15 mM NaCl, 5 mM glucose, p.
H7.4) Preincubate with 100 μL for 3 min at 37 ° C. and add platelet agonists ADP, collagen or sodium arachidonic acid at specified final concentrations. The agglutination was recorded for 2-4 minutes and expressed as the area under the curve (width at height x 1/2 height).

The effect of soluble CD39 was demonstrated on a mouse seizure injury model (Choudh
ri, TF, et al., J. Clin. Invest. 102: 1301-1310 (1998); Connolly, E. S.
., Jr., et al., J. Clin. Invest. 97: 209-216 (1996); and Connolly, ES
, Jr., et al., Neurosurg. 38 (3): 523-532 (1996)). Anesthetized mice were maintained at 37 ± 2 ° C. during surgery and for 90 minutes after surgery. A midline incision was made in the neck and the right carotid artery was exposed. The middle cerebral artery was occluded by advancing 13 mm 6-0 nylon suture, heat treated at the tip, to the external carotid stump by arteriotomy. The external carotid artery was cauterized to ensure hemostasis and arterial blood flow was reestablished. Occlusion of the carotid artery did not exceed 3 minutes. After 45 minutes, the occluded suture was removed and local cautery was applied again to prevent bleeding at the site of the artery resection. Surgical staples were used to suture the wound.

Doppler measurement of cerebral cortical blood flow, neurological score (Huang, Z. et al., Science
265: 183-185 (1994)), calculation of infarct volume, measurement of cerebral thrombus using ¹¹¹ In-labeled platelets (Choudhri, TF, et al., J. Clin. Invest. 102: 1301-1310 (1998)
) And Naka, Y., et al., Circ. Res. 76: 900-906 (1995)), detection of fibrin in the brain (Choudhri, TF, et al., J. Clin. Invest. 102: 1301-1310). (1998)
), And measurement of intracerebral hemorrhage (Choudhri, TF, et al., J. Clin. Invest. 102: 1
301-1310 (1998) and Choudhri, TF, et al., Stroke 28: 2296-2302 (1997)
) Was measured as described previously. The results are shown below.

B. Soluble CD39 blocks ex vivo aggregation of mouse platelets . Platelet-rich plasma was obtained from mice 1 hour after injection with saline (vehicle), soluble CD39, or aspirin. Ex vivo platelet aggregation was tested to determine the relative potency of solCD39 compared to aspirin, which could improve outcome after a transient ischemic attack. Platelets from control and aspirin-treated mice aggregated strongly after stimulation with ADP (FIG. 15A) or collagen (FIG. 15B).

Soluble CD39 blocked platelet aggregation in the presence of ADP and attenuated aggregation in the presence of collagen and arachidonic acid. In contrast, aspirin treatment only blocked the platelet response to arachidonic acid (FIG. 15C). solCD
Platelets from mice pre-treated with 39 initially showed aggregation in the presence of arachidate, but soon disaggregated and returned to a stable state before a complete reaction occurred (FIG. 15C).

C. Soluble CD39 is effective even when added at the peak of the agglutination reaction . ADP (5 μM) was added to mouse platelets in vitro to induce agglutination. At the peak of the agglutination reaction, solCD39 (2.5 μg / ml or 1.25 μg / ml
ml) was added. solCD39 immediately reversed the agglutination reaction, as shown in FIG. This result indicates that solCD39, even when added at the peak of the agglutination reaction, can reverse this reaction and return platelets to a stable state. This result may reflect the fact that ADP is dominant in the release from aggregated platelets at the peak of agglutination. Soluble CD39 metabolizes this ADP almost instantaneously to the biologically inactive compound AMP, resulting in a rapid drop in the aggregation curve on the right side of FIG.

D. Soluble CD39 reduces sequelae of seizures . Soluble CD39 injected intravenously has shown therapeutic utility in stroke. Soluble CD39 inhibited platelet accumulation in the ipsilateral brain hemisphere after seizure induction, as shown in FIG. Similarly, solCD39 reduced fibrin accumulation levels in the ipsilateral hemisphere (as opposed to contralateral) as measured by Western blot analysis using a fibrin-specific antibody (FIG. 18B).

The ability of solCD39 to reduce thrombosis, as measured by reduced platelet and fibrin deposition, resulted in improved post-ischemic cerebral perfusion 24 hours after seizure induction, as shown in FIG. 18A. . In contrast, administration of aspirin at clinically relevant doses (inhibiting the ex vivo response of platelets to arachidate) did not improve cerebral blood flow after ischemia (FIG. 18A). ).

Pre-operatively administered solCD39 reduced cerebral infarct volume in a dose-dependent manner, as measured by digital histology (FIG. 18B). In contrast, although aspirin tended to reduce cerebral infarct volume, this effect was not statistically significant. Administration of soluble CD39 3 hours before or 3 hours after the seizure reduced both neurological deficit score (FIG. 18C) and mortality (FIG. 18D).

Effect of soluble CD39 and aspirin on the development of post-stroke intracerebral hemorrhage.
Shown in Aspirin significantly increased cerebral hemorrhage (as measured spectroscopically), whereas there was no significant increase in cerebral hemorrhage at any of the solCD39 tested. At the above doses, solCD39 inhibited platelet and fibrin accumulation and promoted post-ischemic blood flow, as shown in FIGS. 17A, 17B and 18A. FIG. 19 shows a covariate plot of cerebral infarct volume and cerebral hemorrhage for each treatment, showing that aspirin cannot reduce infarct volume or prevent cerebral hemorrhage as much as solCD39. In summary, at the doses tested in the mouse seizure model, solCD39 provided protection without inducing bleeding problems often associated with antithrombotic therapy.

E. CD39 deficient mice can be reconstituted with soluble CD39 . As shown in FIG. 20A, apyrase conserved regions 2 to 4 (Handa, M. & Guidotti
, G., Biochem. Biophys. Res.Commun. 218: 916-923 (1996); Wang, TF &
Guidotti, G., J. Biol. Chem. 271: 9898-9901 (1996); Maliszewski, CR e.
t al., J. Immunol. 153: 3574-3583 (1994); and Schoenborn, MA, et al.
, Cytogen Cell Gen. 81 (3-4): 287-280 (1998)).
Using a gene targeting vector in which PGKneo cassette was replaced
− / − Mice were produced. 4.1 kb SpeI-Bgl containing exons 4-6
The II fragment was replaced with the PGKneo cassette and this gene targeting vector was introduced into 129-derived ES cells. Cells were selected on G418 and ganciclovir. As shown in FIG. 20B, nine CD39 disrupted ES clones, as identified by Southern blot genomic analysis of BglII digested DNA, were injected into the blastula and the resulting chimeras were bred to C57BL / 6, CD39 +/− heterozygotes were produced. CD39 − / − mice produced at the expected Mendelian frequency from the CD39 +/− intercross. CD used in the experiments described below
39 − / − mice represent random C57BL / 6 × 129 hybrids.

Heterozygous CD39 − / − mice were outwardly normal and did not show a prominent phenotype in the calm state. Hematological profiles, including red blood cell parameters, platelet counts, white blood cell counts and specific morphology, and coagulation screening were normal. As shown below, unless challenged by experimental seizures, CD39 deficient mice did not show a prothrombinic phenotype. Under such conditions, solCD39
Administration resolved this defect and restored normal blood fluidity. Bleeding times in CD39 − / − mice were normal, indicating that normal blood flow in calm animals was not dependent on endogenous CD39 expression. As shown in FIG. 21, CD39 − / − animals showed a significant increase in bleeding time following administration of aspirin or increasing amounts of solCD39, similar to that seen in normal mice. . CD39 − / − mice exposed to cerebral ischemia exhibit reduced blood flow after reperfusion compared to genetically matched control animals (FIG. 22A), and endogenous CD39 contributes to maintaining hemostasis during vascular injury episodes I showed you. Sol to CD39-/-mouse
Upon administration of CD39 (8 mg / kg), these mice were "reconstituted" as shown by a post-ischemic blood flow similar to untreated controls. CD39 − / − mice showed increased cerebral infarct volume after seizure induction compared to genotype matched control animals (FIG. 22B). CD39 − / − mice “reconstituted” with solCD39 had a markedly reduced infarct volume, indicating a protective effect of solCD39. Other parameters (neuronal deficiency score-total mortality, and intracerebral hemorrhage) did not change between groups (FIG.
2C, D, E).

The above results indicate that CD39 inhibits microvascular thrombosis without inducing intracerebral hemorrhage in a mouse seizure model, resulting in a cerebral protective effect. Soluble CD3
9 reduced platelet deposition, fibrin deposition, and cerebral infarct volume. Soluble CD
No. 39 reduced infarct volume and restored blood flow after ischemia even when administered 3 hours after seizure induction. This result is important because the average patient who has experienced a seizure is brought to the emergency room approximately three hours after the first event. 3
Being able to treat patients with solCD39 even after hours provides an advantage over many other drugs designed to inhibit platelet activity.

Example 20: Soluble CD39 enhances survival in a mouse ischemia model . C57BL / 6 mice were anesthetized, ventilated, their thorax opened, and both hilars were surgically exposed. After intravenous administration of either saline or soluble CD39 (8 mg / kg), the left hilum was clamped with cross forceps for 1 hour. The cross-forceps were removed during 3 hours of reperfusion, and then the cross-forceps were applied to the right hilum during the 30 minute observation period. The mice had to survive the function of the left lung after ischemia as this last operation effectively removed the normal lungs from the circulation. The results are shown in the form of a Kaplan-Meier survival plot in FIG. Mice to which physiological saline was administered (n = 6)
All died before the time of 30 minutes, whereas solCD39 treated mice (n =
3) survived for 30 minutes in all cases.

The permanent effects of soluble CD39 have also been shown to be clinically useful in reducing atherosclerotic complications, such as myocardial infarction, stroke and peripheral vascular obstruction. In patients suffering from the above conditions, activated platelets are abundant in their circulation, and such activated platelets lower the response threshold to ADP stimulation. Soluble CD39 depletes ADP from the activated platelet fluid phase and reverses its prothrombin profile.

The relevant disclosures of the references cited herein are hereby specifically incorporated by reference. The embodiments shown above are not exhaustive and do not limit the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements in light of the above teachings, and such modifications and changes fall within the scope of the invention.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the predicted amino acid sequence of human CD39 (SEQ ID NO: 2).
Is shown. The predicted amino acid sequence contains 6 potential N-linked glycosylation sites (double underline) and 11 cysteine residues (bold). The two predicted transmembrane regions are underlined (single line).

FIG. 2 shows the domain structure of full-length and engineered soluble forms of CD39. The location of the transmembrane region near the amino- and carboxy termini, the centrally located hydrophobic sequence, and the portion containing the four putative apyrase conserved regions (ACRs) are indicated. Cysteine residues are denoted as "C". Soluble CD39 is FLAG (
(R) peptide and lacks two transmembrane regions, containing a novel leader sequence.

FIG. 3 shows that after one (1 ×) or two (2 ×) adsorption, COS-1 conditioned medium (C
(M) shows that the soluble (sol) CD39 is immunoaffinity-depleted. Samples were assayed for ATPase activity as described in Example 7. Data are expressed as picomoles of ATP degraded per minute.

FIG. 4 shows immunoprecipitation of solCD39 from COS-1 CM. Lane 2
Indicates a substance specifically bound to the antibody-coated beads. Lane 1 shows material pre-incubated with ovalbumin-coated beads to remove non-specific binding material prior to addition of Ab-coated beads. The mobility of the molecular weight standard is in kilodaltons (
kDa).

FIG. 5 shows immunoaffinity purification and characterization of soluble CD39 (solCD39). FIG. 5A shows the fraction of the immunoaffinity column analyzed by SDS-PAGE, FIG. 5B shows the enzymatic activity of the fraction, and FIG. 5C shows before (lane 1) and after (lane 2) N-glycanase treatment. 3) shows solCD39 purified in (1).

FIG. 6A shows HUVEC membrane ecto-ADPase (●) and recombinant solCD3.
9 (black square) shows the optimal pH profile. FIG. 6B shows purified solCD3.
9 shows Edie Hofstadt lots for the metabolic rate of ATP (●) or ADP (solid squares) at various concentrations and concentrations using 9 (6.5 ng).

FIG. 7 shows AD in platelet-rich plasma from donors receiving aspirin.
4 shows that P-induced platelet response is inhibited by purified solCD39. The response to increasing concentrations of ADP is shown in FIG. 7A. The effect of increasing the amount of purified solCD39 on the platelet aggregation reaction to 10 μM ADP is shown in FIG. 7B. Arrows indicate addition of agonist. Data is expressed as relative light transmission over time (4 minutes).

FIG. 8 shows a comparison of platelet reactivity modulated by various agonists and inhibitors. The effect of CM derived from solCD39 expressing cells on platelet aggregation induced by 5 μM ADP (FIG. 8A) and collagen (FIG. 8B)
Comparisons were made in μM indomethacin treated PRP. In FIG. 8B, the upper sample contains 1 μg / ml of collagen and the lower (indomethacin-treated) sample contains 3.
3 μg / ml was used. FIG. 8C shows the inhibition of collagen-induced aggregation when increasing the amount of solCD39 in PRP from aspirin-fed donors.
Arrows indicate addition of agonist. Data is expressed as relative light transmission over time (4 minutes).

FIG. 9 shows the effect of FSBA-treated solCD39 on platelet reactivity. FIG. 9A shows purified so on ASA-treated PRP after addition of 10 μM ADP.
The effects of lCD39, FSBA-treated solCD39, and mock-treated solCD39 (4.4 μg / ml each) are shown. FIG. 9B shows FSBA-treated solCD39 and mock-treated s for ASA-treated PRP after addition of 3.3 μg / ml collagen.
The effect of olCD39 (22 μg / ml each) is shown. FIG. 9C shows mock-treated solCD39 (0.88 / ml) versus FSBA-treated solCD39 (22 μg / ml).
-2.2 [mu] g / ml). ASA-treated PRP with 10 μM ADP
Stimulated. Arrows indicate addition of agonist. Data is expressed as relative light transmission over time.

FIG. 10 shows a pharmacokinetic analysis of solCD39 in mice. ATPase assay (solid square) or ADPase assay (radioactive phosphate release)
In (), CD39 in the serum was measured. Activity is expressed as picomoles of nucleotides degraded per minute. The dashed line indicates that solCD39 was 2 in mouse serum.
5 shows ATPase activity at 5 μg / ml. Using a biphasic curve fitting method, the distribution (t _1/2 α = 59 min (ATP); 43 min (ADP)) half-life of the clearance (t _1/2 β = 40 h (ATP and ADP)) It was determined.

FIG. 11. 0 and 60 of pigs treated with low, medium or high doses of solCD39.
Shows the bleeding time in minutes.

FIG. 12 shows the effect of aspirin on piglet platelet aggregation at baseline and 5 days after intravenous administration (FIG. 12A), and the effect of high-dose solCD39 on platelet aggregation and baseline at day 7 (FIG. 12). 12B).

FIG. 13 shows low, medium, and high doses of solCD with respect to time after bolus administration.
9 shows the inhibition of porcine platelet aggregation by 39.

FIG. 14 shows CD in porcine serum with time after low, medium or high dose administration.
39 indicates the concentration. Biphasic curve fitting was used to determine the half-life of the distribution (t1 _/ 2α = 29 minutes) and clearance (t1 _/ 2β = 51 hours).

FIG. 15 shows ex vivo aggregation of mouse platelets. After administration of vehicle (saline), soluble CD39 (4 mg / kg) or aspirin (5 mg / kg), 10
μM ADP (FIG. 15A), collagen 2.5 μg / ml (FIG. 15B) or 0.1 μg / ml.
Platelets were stimulated with 1 mM sodium arachidonate (FIG. 15C). Soluble CD
Aggregation curves returned to baseline after agonist stimulation by 39 treatment, whereas aspirin treatment produced such patterns only when arachidonic acid was the agonist.

FIG. 16 shows the reversal of ADP-induced agglutination in mouse platelets when solCD39 was added at the peak of the agglutination.

FIG. 17 shows inhibition of platelet (n = 20, FIG. 17A) and fibrin (n = 3, FIG. 17B) deposition after seizure induction in mice pre-treated with soluble CD39 (8 mg / kg). “Fibrin” is a positive control, “Ipsilateral” is ipsilateral (ie, ischemic hemisphere), and “Contralateral” is a non-ischemic hemisphere.

FIG. 18 shows vehicle (n = 23) versus seizure results induced in mice.
, Soluble CD39 (n = 67) and aspirin (n = 27).
FIG. 18A shows cerebral blood flow, 18B shows cerebral infarct volume, 18C shows neurological deficit score (higher scores indicate more severe deficit (Connolly, ES, Jr.,
et al., Neurosurg. 38 (3): 523-532 (1996)), 18D indicates mortality,
E indicates intracerebral hemorrhage. ^* p <0.05, Δp <0.01, Δp <0.001.

FIG. 19 shows a covariate plot of cerebral infarct volume versus intracerebral hemorrhage. Vehicle (saline), aspirin (ASA, 5 mg / kg before seizure), soluble CD39 (
4 and 8 mg / kg before seizure) and soluble CD39 (8 mg / kg 3 hours after seizure induction in mice).

FIG. 20A shows the construct used to produce CD39 − / − by homologous recombination. Labeled restriction sites were BglII (B), SpeI (S) and Asp
718 (A). FIG. 20B shows ES with the disrupted CD39 allele.
2 shows a Southern blot of the genome used to identify clones.

FIG. 21 shows control (n = 15), aspirin treatment (5 mg / kg, n = 10),
solCD39 treatment (4,8 and 20 mg / kg, n = 25) and solCD3
The bleeding time in 9 − / − mice (n = 10) is shown. ( ^* P <0.05, ‡ p
<0.01, Δp <0.001).

FIG. 22 shows control (C57BL / 6J × 129 / JF1) mice (n = 6), C
D39 − / − mice (n = 5), and CD39 “reconstructed” with solCD39
7 shows comparison of seizure results in / − (n = 6). FIG. 22A shows cerebral blood flow,
2B indicates brain infarct volume, 22C indicates nerve score, 22D indicates mortality, and 22E indicates intracerebral hemorrhage. ^* p <0.05, Δp <0.01, Δp <0.00
One.

FIG. 23 shows solCD39 in a stringent pulmonary ischemia-reperfusion model.
2 is a Kaplan-Meier plot, showing that the results in improved survival. FIG. 24 shows the alignment of the N-terminal amino acid sequences of human CD39 and human CD39-L4.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/19 C12N 9/14 4H045 5/10 15/00 ZNAA C12P 21/02 A61K 37/02 // C12N 9/14 C12N 5/00 B (31) Priority claim number 60 / 149,010 (32) Priority date August 13, 1999 (August 13, 1999) (33) Priority claim country United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG) , ZW ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN , CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK , SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Gail, Richard Bee, The Third Woodinville, Washington 98072, United States of America , One Hundred and Forty-Nine Venue Northeast 17833 (72) Inventor Price, Virginia El. 98102, Washington, United States of America, Seattle, Boyer Avenue East 2617 (72) Inventor Gimpel, Steven D. 98115, Washington, United States of America, Twenty Six Avenue Northeast 6842 F-term (reference) 4B024 AA01 AA15 BA31 CA04 CA12 DA02 GA14 HA15 4B050 CC03 DD11 EE10 LL01 4B064 AG31 CA19 DA01 4B065 AA94Y AB01 BA03 CA24 CA44 4C084 AA02 AA07 BA01 BA08 BA22 BA42 A53 CA53 AA20 AA30 BA10 CA42 DA50 DA86 DA89 EA24 FA74 GA26

Claims (35)

[Claims] 1. (a) a group having the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2), wherein the amino terminus is selected from the group consisting of amino acids 36 to 44, and the carboxy terminus is composed of amino acids 471 to 478; (B) a fragment of the polypeptide of (a), wherein the fragment has apyrase activity; (c) a variant of the polypeptide of (a) or (b), (D) a fusion polypeptide comprising the polypeptide of (a), (b) or (c), wherein the fusion polypeptide has apyrase activity; and A soluble CD39 polypeptide. 2. A polypeptide having a sequence consisting of amino acids 38 to 476 or 39 to 476 of SEQ ID NO: 2; (b) an amino acid relative to amino acids 36 to 478 of SEQ ID NO: 2 or a fragment thereof. A variant polypeptide having at least 70% sequence identity, said variant polypeptide having apyrase activity; (c) having at least 80 amino acid sequence relative to amino acids 36-478 of SEQ ID NO: 2 or a fragment thereof. A variant polypeptide having apyrase activity; (d) the amino acid sequence is at least 90% identical to amino acids 36-478 of SEQ ID NO: 2 or a fragment thereof. A variant polypeptide, wherein the variant has apyrase activity (E) a variant polypeptide having an amino acid sequence at least 95% identical to amino acids 36 to 478 of SEQ ID NO: 2 or a fragment thereof, wherein the variant polypeptide has apyrase activity; (f) C.) A variant polypeptide having an amino acid sequence at least 98% identical to amino acids 36-478 of SEQ ID NO: 2 or a fragment thereof, said variant polypeptide having apyrase activity; and A variant polypeptide having an amino acid sequence at least 99% identical to amino acids 36-478 of NO: 2 or a fragment thereof, wherein the variant polypeptide is selected from the group consisting of: a variant polypeptide having apyrase activity. 2. The soluble CD39 polypeptide according to 1. 3. A polypeptide having the structure XY, wherein Y is the soluble CD39 polypeptide according to claim 1, wherein X is an Ala residue and a peptide capable of forming a stable secondary structure. The polypeptide, selected from: 4. The polypeptide of claim 3, wherein X is a peptide fragment derived from the amino-terminal portion of mature IL-2, CD39-L2, CD39-L3 or CD39-L4. 5. A polypeptide having the structure ABC, wherein A is 0-20 amino acids derived from the amino-terminal portion of mature IL-2 and B is 0-
The polypeptide of claim 15, wherein the polypeptide is a linker of 15 amino acids and C is the soluble CD39 polypeptide of claim 1. 6. (a) SEQ ID NO: 6, SEQ ID NO: 27
Amino acids 25-464 of SEQ ID NO: 28;
(B) (a) amino acids 27 to 473 of SEQ ID NO: 29, amino acids 21 to 476 of SEQ ID NO: 3, amino acids 21 to 476 of SEQ ID NO: 4, or amino acids 21 to 463 of SEQ ID NO: 30; (C) a fragment of the polypeptide having apyrase activity; (c) a mutant of the polypeptide of (a) or (b), wherein the mutant has apyrase activity; and (d) ( A soluble CD39 polypeptide comprising the polypeptide of (a), (b) or (c), wherein the soluble CD39 polypeptide is selected from the group consisting of the fusion polypeptides having apyrase activity. (A) a mutant polypeptide having an amino acid sequence at least 70% identical to the soluble CD39 polypeptide of claim 6, wherein said mutant polypeptide has apyrase activity; (d) A) a mutant polypeptide having an amino acid sequence at least 80% identical to the soluble CD39 polypeptide of claim 6, wherein the mutant polypeptide has apyrase activity; (e) the mutant polypeptide of claim 6; A mutant polypeptide having an amino acid sequence at least 90% identical to a soluble CD39 polypeptide, said mutant polypeptide having apyrase activity; (f) a mutant CD39 polypeptide according to claim 6; A variant polypeptide having at least 95% amino acid identity and having apyrase activity (G) a mutant polypeptide having an amino acid sequence at least 98% identical to the soluble CD39 polypeptide of claim 6, wherein the mutant polypeptide has apyrase activity; And (h) a mutant polypeptide having an amino acid sequence at least 99% identical to the soluble CD39 polypeptide of claim 6, wherein the mutant polypeptide is selected from the group consisting of the mutant polypeptides having apyrase activity. , Polypeptide. 8. Amino acids 25 to 464 of SEQ ID NO: 6, SEQ ID NO: 27, amino acids 25 to 474 of SEQ ID NO: 28, amino acids 27 to 473 of SEQ ID NO: 29, and SEQ ID NO: 3. 7. The soluble CD39 polypeptide of claim 6, having an amino acid sequence selected from the group consisting of amino acids 21-476, amino acids 21-476 of SEQ ID NO: 4, and amino acids 21-463 of SEQ ID NO: 30. . 9. An isolated nucleic acid encoding a polypeptide according to claim 1. 10. The nucleic acid according to claim 9, wherein said nucleic acid is DNA. 11. (a) SEQ ID NO: 5; (b) a DNA sequence encoding the polypeptide encoded by SEQ ID NO: 5 due to the degeneracy of the genetic code; (c) moderately stringent A DNA sequence that hybridizes to SEQ ID NO: 5 under conditions; (d) a DNA sequence whose sequence is at least 70% identical to SEQ ID NO: 5 or a fragment thereof; (e) SEQ ID NO: 5 or a fragment thereof. A DNA sequence that is at least 80% identical to the fragment; (f) a DNA sequence that is at least 90% identical to SEQ ID NO: 5 or a fragment thereof; (g) a SEQ ID NO: 5 or sequence thereof A DNA sequence whose sequence is at least 95% identical to the fragment; (h) SEQ ID NO: 5 or a fragment thereof. A sequence selected from the group consisting of: a DNA sequence that is at least 98% identical to a sequence of said fragment; and (i) a DNA sequence that is at least 99% identical to SEQ ID NO: 5 or a fragment thereof. The DNA according to claim 10. 12. The DNA of claim 10, further comprising a leader peptide operably linked to the N-terminus of the polypeptide, wherein the leader peptide promotes extracellular secretion of the polypeptide. 13. The DNA of claim 12, wherein the leader peptide comprises all or part of a leader derived from IL-2, proinsulin, human growth hormone (huGH), IL-7 or Igkappa. 14. The leader peptide comprises the sequence SEQ ID NO: 9.
The DNA according to claim 13. 15. (a) SEQ ID NO: 7; (b) due to the degeneracy of the genetic code, a DNA sequence encoding the polypeptide encoded by SEQ ID NO: 7; (c) moderately stringent A DNA sequence that hybridizes under conditions to SEQ ID NO: 7; (d) a DNA sequence that is at least 70% identical to SEQ ID NO: 7 or a fragment thereof; (e) SEQ ID NO: 7 or its A DNA sequence that is at least 80% identical to the fragment; (f) a DNA sequence that is at least 90% identical to SEQ ID NO: 7 or a fragment thereof; (g) a SEQ ID NO: 7 or sequence thereof A DNA sequence whose sequence is at least 95% identical to the fragment; (h) SEQ ID NO: 7 or a fragment thereof. A sequence selected from the group consisting of: a DNA sequence that is at least 98% identical to a sequence of said sequence; and (i) a DNA sequence that is at least 99% identical to SEQ ID NO: 7 or a fragment thereof. The DNA according to claim 12. 16. A vector comprising the nucleic acid according to claim 9. 17. The vector according to claim 16, wherein said vector is an expression vector. 18. The vector according to claim 17, wherein said vector is a eukaryotic expression vector. 19. A eukaryotic expression vector comprising the sequence SEQ ID NO: 5. 20. A eukaryotic expression vector comprising the sequence SEQ ID NO: 7. 21. A recombinant cell comprising the nucleic acid according to claim 9. 22. The cell of claim 21, wherein said cell is a prokaryotic cell. 23. The cell of claim 21, wherein said cell is a eukaryotic cell. 24. The cell according to claim 23, wherein the cell is a COS cell or a CHO cell.
The cell according to 1. 25. The cell of claim 24, wherein said cell is a CHO cell adapted to grow in suspension and in the absence of serum. 26. A recombinant CHO cell comprising the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7. 27. A soluble CD39 polypeptide comprising culturing the recombinant cell of claim 21 under conditions allowing expression of the CD39 polypeptide and recovering the CD39 polypeptide from the culture. How to manufacture. 28. The method of claim 27, wherein the recombinant cell is a eukaryotic cell. 29. The method of claim 27, wherein the recombinant cells are CHO cells adapted to grow in suspension and in the absence of serum. 30. A polypeptide produced by the method according to claim 27. 31. A polypeptide produced by the method of claim 29. 32. A composition comprising a pharmaceutically acceptable carrier and the polypeptide according to one of claims 1 to 8. 33. A composition comprising a pharmaceutically acceptable carrier and the polypeptide of claim 30. 34. A composition comprising a pharmaceutically acceptable carrier and the polypeptide of claim 31. 35. A method of inhibiting angiogenesis in a mammal in need of such a treatment that inhibits angiogenesis, comprising administering a therapeutic amount of a soluble CD39 polypeptide.