WO1990014103A1 - Polypeptide-antibody conjugate for inhibiting cell adhesion - Google Patents

Abstract

A polypeptide-antibody conjugate is disclosed that contains an integrin-binding polypeptide operatively attached to an antibody molecule that immunoreacts with adhesitory cell surface antigens. Therapeutic compositions and methods using those compositions are also disclosed whereby the polypeptide-antibody conjugate can be used to inhibit cell adhesion and cell adhesion-dependent processes such as tumor growth and platelet aggregation.

Description

POLYPEPTIDE-ANTIBODY CONJUGATE FOR INHIBITING CELL ADHESION Description

Technical Field

The present invention relates to a polypeptide-antibody conjugate comprising an integrin- binding polypeptide operatively attached to an antibody molecule that immunoreacts with adhesitory cell surface antigens and thereby inhibits cell adhesion. Also contemplated is a therapeutic method wherein a polypeptide-antibody conjugate of the present invention is used to inhibit cell adhesion- dependent processes such as tumor growth and platelet aggregation.

Background

Cell adhesion is a critical process in a variety of cell-cell and cell-extracellular matrix interactions including tumor growth and platelet aggregation. Therefore, agents that inhibit cell adhesion are of therapeutic use at least to inhibit tumor growth or to inhibit platelet-mediated processes such as blood coagulation.

Cell adhesion generally involves an interaction between cell surface receptors and specific matrix proteins. Rouslahti et al.. Science 238:491-497 (1987). Of interest are those receptors that specifically bind to matrix proteins at sites that have as a part of their structure the tripeptide amino acid residue sequence Arg-Gly-Asp (RGD) . Such receptors are known in the art as RGD-directed adhesion receptors and include the fibronectin receptor (FNr) , the vitronectin receptor (VNr) , the platelet receptor known as GPIIb/IIIa and the collagen receptor. Recently, additional RGD-directed adhesion receptors were described on M21 human melanoma cells, Cheresh et al., J. Biol. Chem.. 262:1434-1437 (1987), and on human endothelial cells, designated ECr. Cheresh et al., Proc. Natl. Acad. Sci. USA. 84:6471- 6475 (1987).

RGD-containing polypeptides derived from matrix protein sequences have been shown to inhibit the ability of RGD-directed adhesion receptors to interact and adhere to a matrix protein-containing substrate. Pytela et al., Pro. Natl. Acad. Sci. USA. 82:5766-70 (1985); and Pytela et al; Science. 231:1559-62 (1986).

RGD-containing polypeptides have also been used to inhibit cell adhesion to a matrix protein- containing substrate in vitro. United States Patent NOS. 4,614,517, 4,517,686, 4,578,079, 4,683,291, 4,792,525; Pierschbacher et al., Nature, 309:30-33 (1984); Pierschbacher et al., Proc. Natl. Acad. Sci. USA. 81:5985-88 (1984); Hay an et al., J. Cell Biol.. 100:1948-54 (1985); Haverstick et al.. Blood. 66:946- 52 (1985); Ginsberg et al., J. Biol. Che .. 260:3931- 36 (1985); Ar ant et al., Proc. Natl. Acad. Sci. USA. 83:6751-55 (1986); and Ouaissi et al., Science. 234:603-7 ^"(1986) . One RGD-containing polypeptide was shown to inhibit murine melanoma cell adhesion and to also inhibit colonization of mouse lung tissue in vivo. Humphries et al, Science. 233:467-70 (1986).

A similar in vivo inhibition of lung tissue colonization was described using a polypeptide derived from the laminin matrix protein sequence. Iwamoto et al.. Science. 238:1132-1134 (1987). Both RGD- containing polypeptides and other cell adhesion inhibiting laminin-derived polypeptides were described that contained the pentapeptide amino acid sequence Tyr-Ile-Gly-Ser-Arg (YIGSR) , thus indicating that RGD- containing polypeptides are not the only candidates for inhibition of cell adhesion.

Inhibition of cell adhesion has also been demonstrated using monoclonal antibodies that immunoreact with a molecule present on the surface of cells that undergo cell adhesion. For example, cell adhesion-inhibiting antibodies were described that immunoreact with RGD-directed adhesion receptors such as GPIIb/IIIa, [Pytela et al.. Science. 231:1559-62 (1986); Charo et al., J. Biol. Chem.. 262:9935-38 (1987)], a 140 kilodalton fibronection specific receptor [Brown et al., Science. 228:1448-51 (19^'85)], the cell substrate attachment antigen (CSAT) [Horwitz et al., J.^' Cell Biol.. 101:2134-44 (1985)], and the human melanoma cell receptor [Cheresh et al., J. Biol. Chem.. 262:17703-11 (1987)].

Cell adhesion inhibiting-antibodies have also been described that immunoreact with adhesitory cell antigens where the antigens are not themselves RGD-directed adhesion receptors. For example, monoclonal antibodies that immunoreact with disialoganglioside GD2 or GD3 were shown to inhibit tumor cell adhesion to various matrix protein- containing substrates including collagen, vitronectin, laminin and fibronectin. Cheresh et al., J. Cell Biol.. 102:688-96 (1986).

In the medical arts, it is seen from the foregoing that RGD-containing polypeptides and other polypeptides derived from matrix protein primary sequences are useful for inhibiting cell adhesions, particularly adhesion of tumor cells or platelets. However, the systemic use of adhesion-inhibiting polypeptides is limited because it produces the side effect of systemically and indiscriminately inhibiting normal cellular adhesive events.

Brief Summary of the Invention

It has now been discovered that the cell adhesion-inhibiting effects of the polypeptides described herein can be directed (targeted) to particular tissues by means of their conjugation to specific antibodies so that the polypeptides can disrupt specific undesirable cell adhesion events without significantly effecting normal adhesive events.

The present invention provides an RGD- antibody comprising an antibody molecule operatively attached to an integrin-binding polypeptide. The antibody combining site of the RGD-antibody immunoreacts with an adhesitory cell surface antigen and the polypeptide has an amino acid residue sequence of about 5 to about 50 residues in length that includes a sequence having the formula: -RGD-; more preferably the formula: -GRGDSP-, and most preferably the formula: -CGGAGAGRGDSP-.

In addition, it is preferred that the antibody molecule of the contemplated RGD-antibody immunoreacts with an adhesitory cell surface antigen. Preferred adhesitory cell surface antigens are disialoganglioside GD2, disialoganglioside GD3, chondroitin sulfate proteoglycan, vitronectin receptor, endothelial cell receptor or collagen receptor. Preferably, the antibody molecule is produced by a hybridoma selected from the group consisting of 1418, 142A, 126, MB3.6, LM609 and LM142.

In preferred embodiments, a contemplated RGD-antibody immunoreacts with an antigen present on the surface of platelets or tumor cells. The present invention also contemplates a method for inhibiting the attachment of an adhesitory cell to an RGD-containing matrix comprising administering to a subject a therapeutically effective amount of an RGD-antibody of the present invention. Preferably, the adhesitory cell is a tumor cell or platelet and therefore the method contemplates the inhibition of tumor cell attachment or platelet aggregation, respectively.

.In another embodiment, the present invention contemplates a YIGSR-antibody comprising an antibody molecule that immunoreacts with an adhesitory cell surface antigen, operatively attached to a laminin receptor-binding polypeptide having an amino acid residue sequence of about 5 to about 50 residues in length that includes a sequence having the formula: - YIGSR-. More preferably the polypeptide has the formula:

YIGSR, CDPGYIGSR, or RGDSGYIGSR.

Further contemplated is a chimeric antibody that contains at least one hybrid protein molecule having an antibody combining site-forming fragment fused to at least one integrin-binding polypeptide, in which the hybrid protein molecule forms an antibody combining site that immunoreacts with a surface antigen of an adhesitory cell (adhesitory cell surface antigen) . In a preferred embodiment the integrin- binding polypeptide comprises an amino acid residue sequence of about 5 to about 50 residues in length that includes a sequence having the formula: -RGD-, and more preferably the formula: -GRGDSP-.

The present invention also contemplates a recombinant DNA molecule that encodes a hybrid protein molecule comprising: a) a first DNA segment encoding an antibody combining site-forming fragment, and b) a second DNA segment encoding an integrin-binding polypeptide that is operatively linked in phase to the first segment, wherein the recombinant DNA molecule is capable, when present in an appropriate expression vector, of expressing a hybrid protein molecule having an antibody combining site that immunoreacts with an adhesitory cell surface antigen.

Brief Description of the Drawings

Figure 1 is a composite figure comprising two panels (A and B) that graphically illustrate the effects after 20 minutes of a polypeptide-antibody conjugate on the attachment of M21 cells to microtiter wells, each well having been coated with either von illebrand factor (Panel A) or fibrinogen (Panel B) . ³H-leucine-labeled M21 cells were first immunoreacted with various indicated concentrations of either Mab 142A-conjugate (open boxes) or control activated Mab 142A (closed boxes) prior to reaction with the polypeptide. The antibody treated cells were then plated onto the coated microtiter wells and maintained for 20 minutes to allow the cells to adhere to the wells as described in Example 4. The results are expressed as the total number of cells bound, that number being a function of the cell-associated label, expressed in counts per minute (cpm xlO^*3) , bound to the matrix protein at each indicated Mab concentration.

Figure 2 is a composite figure comprising two panels (A and B) that graphically illustrate the effects after 90 minutes of a polypeptide-antibody conjugate on the attachment of M21 cells to microtiter wells, each well having been coated with either von Willebrand factor (Panel A) or fibrinogen (Panel B) . Reagents and methods were as described for Figure 1, and data are reported as in Figure l.

Detailed Description of the Invention

A. Definitions

Amino Acid: All amino acid residues identified herein are the natural L-configuration. In keeping with standard polypeptide nomenclature, J. Biol. Chem. _f 243:3557-59 (1969), abbreviations for amino acid residues are as shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE

SYMBOL AMINO ACID

1-Letter 3-Letter

Y Tyr L-tyrosine

G Gly glycine

F Phe L-phenylalanine

M Met L-methionine

A Ala L-alanine

S Ser L-serine

I He L-isoleucine

L Leu L-leucine

T Thr L-threonine

V Val L-valine

P Pro L-proline

K Lys L-lysine

H His L-histidine

Q Gin L-glutamine

E Glu L-glutamic acid

W Trp L-tryptophan

R Arg L-arginine

D Asp L-aspartic acid N Asn L-asparagine

C Cys L-cysteine

It should be noted that all amino acid residue sequences are represented herein by formulae whose left to right orientation is in the conventional direction of amino-terminus to carboxyl-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a bond to a further sequence of one or more amino acid residues up to a total of about fifty residues in the polypeptide chain.

Polypeptide and Peptide: Polypeptide and peptide are terms used interchangeably herein to designate a linear series of no more than about 50 amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues.

Protein: Protein is a term used herein to designate a molecule having a primary structure comprised of a linear series of greater than 50 amino acid residues connected one to the other as in a polypeptide. Of course, both a protein and a polypeptide can further include intramolecular disulfide and other bonds that cause the protein or the polypeptide to assume a non-linear configuration when in solution.

B. Polypeptide-Antibody Conjugates 1. Background The polypeptide-antibody conjugate of the present invention comprises in its most broad embodiment an antibody molecule operatively attached to an integrin-binding polypeptide. The attached antibody molecule immunoreacts with an antigen present on the surface of an adhesitory cell, and the integrin-binding polypeptide inhibits cell adhesion. The terms adhesitory cell, integrin-binding polypeptide and cell adhesion are discussed and defined hereinafter. The combination of antibody molecule and polypeptide as disclosed herein produces a cell adhesion-inhibiting molecule because, upon immunoreaction of the antibody with an adhesitory cell surface antigen, the antibody situates the attached integrin-binding polypeptide into sufficient proximity with cell surface integrin molecules so that the polypeptide can interact with and be bound by (or bound to) -the integrin, thus acting as an antagonist and inhibiting the ability of the bound integrin to participate in adhesion processes.

In view of the utility of an integrin-binding polypeptide to inhibit cell adhesion, the combination of antibody molecule and polypeptide disclosed in the present invention provides the benefit of targeting the inhibitory effect to those cells that contain a particular adhesitory cell surface antigen. Without targeting, topical or systemic application of an integrin-binding polypeptide can indiscriminately inhibit adhesion of all cells exposed to the polypeptide, including adhesitory cells present in normal tissues. Therefore the benefit of localizing cell adhesion-inhibiting effects to specific adhesitory cells can be readily appreciated by one skilled in the art of cell adhesion. The phrase "cellular adhesion" refers to a process by which an adhesitory cell attaches (adheres) to another cell's surface or to a tissue substrate. The adhesion process occurs by a specific interaction between adhesitory cell surface protein receptors, called integrins, and matrix protein ligands that include the RGD-tripeptide as a part of their amino acid residue sequences. Thus, adhesitory cells, as used herein, are those cells that adhere to biological surfaces by means of the specific interaction (receptor/ligand complex formation) between an integrin and RGD-containing matrix proteins. RGD- containing matrix proteins are also referred to as adhesion protein ligands and include fibronectin, vitronectin, fibrinogen, von Willebrand factor, laminin, thrombospondin, osteopontin, collagens and the like.

An adhesitory cell is also characterized as any cell that contains on its surface one or more of the cell surface receptors known as integrins. Integrins are also referred to as RGD-directed adhesion receptors or cytoadhesins, and include vitronectin receptor, fibronectin receptor, collagen receptor, platelet glycoprotein GPIIb/IIIa, human endothelial cell receptor and the like. Exemplary adhesitory cells include platelets and numerous types of tumor cells including neuroblastoma, small cell carcinoma, adenocarcinomas of the lung, colon or breast, melanoma, astrocytoma, glioma, squamous cell carcinoma and the like. An adhesitory cell surface antigen refers to any antigen present on the surface of an adhesitory cell. "Antigen" is used herein to refer to any molecule that immunoreacts with and is bound by an antibody molecule to form an immunoreactant. Thus an adhesitory cell surface antigen can be any molecule on an adhesitory cell surface that immunoreacts with an antibody molecule while the antigen is present on the surface of the cell.

Exemplary adhesitory cell surface antigens include the disialogangliosides GD2 and GD3, tumor cell surface antigens, platelet surface antigens, chondroitin εulfate proteoglycans, cell surface receptors such as integrins, and the like. 2. Antibody Molecules Antibody molecules to be used as a component of the present invention include any antibody molecule that is capable of immunoreacting with an adhesitory cell surface antigen to form an immunoreaction product (antigen-antibody complex) . The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e., molecules that contain an antibody combining site or paratope.

An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab', F(ab')2 and F(v) .

Fab and F(ab')2 portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well known. See for example, U. S. Patent No. 4,342,566 to Theofilopolous and Dixon. Fab' antibody molecule portions are also well known and are produced from F(ab')2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide.

The term "antibody" in its various grammatical forms is used herein to refer to a composition containing a plurality of antibody molecules, e.g., an antiserum. An antibody containing intact antibody molecules is preferred. Particularly preferred is a monoclonal antibody.

The phrase "monoclonal antibody" or "Mab" in its various grammatical forms refers to an antibody containing only one species of antibody combining site capable of immunoreacting with a particular antigen and thus typically displays a single binding affinity for that antigen. A monoclonal antibody can therefore contain a bispecific antibody molecule having two antibody combining sites, each immunospecific for a different antigen.

A preferred monoclonal antibody is characterized as containing, within immunologically detectable limits, only one species of antibody combining site capable of immunologically binding (immunoreacting with) an adhesitory cell surface antigen.

Particularly preferred are monoclonal antibodies that immunoreact with disialogangliosides GD2 or GD3, vitronectin receptor, fibronectin receptor, human endothelial cell receptor, chondroitin sulfate proteoglycan or platelet glycoprotein GPIIb/IIIa. Table 1 lists monoclonal antibodies having immunospecificities useful in the present invention. Those Mabs are listed by the name utilized in a publication, by the reported American Type Tissue

Collection (ATCC) accession number of the hybridoma that produces the antibody and by the antigen indicated with which the Mab reportedly reacts. A citation to a discussion of each Mab and its immunospecificity is provided by the footnote to the listed antigen. Hybridomas producing monoclonal antibodies functionally equivalent to those in Table 1 can be made by techniques well known in the art including those described herein.

Table 1

Exemplary Monoclonal Antibodies That Immunoreact

With An Adhesitory Cell Surface Antigen

Mab ATTC No, Antigen

1418 HB 9118 disialoganglioside GD2¹

142A disialoganglioside GD2'

126 HB 8568 disialoganglioside GD2"

MB3.6 HB 8890 disialoganglioside GD3

11C64 disialoganglioside GD3"

R24 disialoganglioside GD3

9227 chondroitin sulfate proteoglycan⁷

LM609 HB 9537 vitronectin receptor

LM142 vitronectin receptor

HT29/26 HB 8247 colon cancer glycoprotein

T16 HB 8279 human bladder tumor glycoprotein gp48¹¹ 1116-NS-19-9 HB 8059 colorectal carcinoma monosialoganglioside¹²

CTHB-5 HB 135 complement C3d receptor¹³

O T 11 ^* CRL 8027 rosette receptors¹⁴

Anti-HLA¹⁵ - histocompatibility (HIA) antigens¹⁵

TM1 HB 169 leukocyte (leu-5) antigen¹⁶

C7 CRL 1691 low density lipoprotein receptor¹⁷

BBM HB 28 B-2 microglobulin¹⁸

¹ Cheresh et al.. Cancer Res.. 44:5112-18 (1986).

² Prepared as described in Example 1.

³ U.S. Patent No. 4,675,287.

⁴ Cheresh et al., Proc. Natl. Acad. Sci. USA.

82:5155-59 (1985).

⁵ Cheresh et al., J. Cell Biol.. 102:688-96 (1986).

⁶ U.S. Patent No. 4,507,391.

⁷ Bumol et al., Proc. Natl. Acad. Sci. USA. 79:1245-

49 (1982).

⁸ Cheresh et al., J. Biol. Chem. _P 262:17703-11

(1987) . 9 Cheresh et al., J. Biol. Chem. _f 262:17703-11 (1987) .

10 U.S. Patent No. 4,579,827.

11 European Patent Application No. 84102517.4, publication No. 0 118 891, published September 19, 1984.

12 U.S. Patent No. 4,471,057.

13 Weis et al., Proc. Natl. Acad. Sci. USA. 81:881- 885 (1984).

H U.S. Patent No. 4,364,937.

¹⁵ ATTC Catalogue of Cell Lines and Hybridomas. 6th edition, 1988, at pg. 283 lists over 27 different Mabs that immunoreact with numerous HLA antigens.

16 Grumet et al., Human Immunol.. 6:63-73 (1983).

17 Goldstein et al.. Cell. 30:715-24 (1982).

18 Parham et al., Eur. J. Immunol. 9:536-45 (1979).

Antibodies or monoclonal antibodies that immunoreact with adhesitory cell surface antigens can be obtained from a variety of commercial vendors. Alternatively, antibodies can be prepared by immunization techniques well known in the art. The preparation of monoclonal antibodies and their subsequent purification sufficient for use in the present invention are well known. Typically, a monoclonal antibody is prepared from a hybridoma culture supernatant by separating the supernatant from cultured hybridoma cells to form a cell-free monoclonal antibody molecule-containing solution. Alternatively, a monoclonal antibody can be prepared from ascites by introducing a hybridoma cell, as by injection, into the peritoneal cavity of a mammal such as a mouse and later harvesting the resulting peritoneal exudate (ascites tumor fluid) from the mouse by well known techniques. See, for example, H. Zola, Monoclonal Antibodies: A Manual of Technicrues_r CRC Press, Inc. (1987). Additionally, a monoclonal antibody can be produced by recombinant DNA methodologies in which a monoclonal antibody molecule-encoding gene is cloned and manipulated into a suitable expression medium for production of a recombinantly produced antibody molecule. Monoclonal antibody production by recombinant methods is described in more detail below under Section D entitled "Chimeric Antibodies". Particularly preferred are the monoclonal antibody molecules 142A, 1418, 126, MB3.6, LM142, and LM609, as disclosed in more detail herein. Hybridomas that produce monoclonal antibody molecules 1418, 126, MB3.6 and LM609 were deposited pursuant to Budapest Treaty requirements with the American Type Tissue Collection (ATCC) , Rockville, MD, on June 4, 1986, May 28, 1984, August 16, 1985 and September 15, 1987, respectively, and were assigned the accession numbers HB 9118, HB 8568, HB 8890 and HB 9537, respectively. 3. Polypeptides An integrin-binding polypeptide useful in the present invention is a polypeptide having an amino acid residue sequence of about 5 to about 50 residues in length, and exhibiting the property of competitively inhibiting cellular adhesion. A useful polypeptide binds to an integrin expressed on the surface of an adhesitory cell. It also inhibits (antagonizes) cellular adhesion between an adhesitory cell and a surface that contains a matrix protein.

Many polypeptides are known to have such adhesion inhibiting properties. See, for example, Rouslahti et al.. Cell. 44:517-518 (1986);

Pierschbacher et al.. Nature. 309:30-33 (1984); Ouaissi et al.. Science, 234:603-607 (1986); Iwamoto et al.. Science. 238:1132-1134 (1987); Haverstick et al.. Blood. 66:946-952 (1985); and U.S. Pat. Nos. 4,578,079, 4,792,525, 4,614,517, 4,517,686 and

4,683,291. (The teachings of the art cited herein are incorporated by reference) .

Adhesion-inhibiting conditions for integrin- binding polypeptides are well known in the art. Exemplary conditions are described in Example 4 and in the teachings cited immediately above.

Typically, integrin-binding polypeptides contain an amino acid residue sequence that corresponds to a portion of the sequence of an adhesion protein. This polypeptide sequence is present in that portion of the adhesion protein that participates in contacting the integrin to which it binds when adhesion occurs. This contacting portion of the adhesion protein has been referred to as the cell recognition site. For many of the characterized adhesion proteins, the recognition site includes the amino acid tripeptide RGD. See, for example, Rouslahti et al.. Science. 238:491-497 (1987) .

The ability of an integrin-binding polypeptide to bind to an integrin, and to inhibit cellular adhesion, is quantified by means well known in the art. Exemplary means are set forth in detail in Examples 3 and 4. Further exemplary means can be found in the disclosures cited above that describe exemplary integrin-binding polypeptides. A preferred integrin-binding polypeptide has an amino acid residue sequence that includes residues represented by the formula: -RGD-. Exemplary polypeptides are represented by the formulas: VTGRGD,

GRGDS, and RGDSPASSKP. Preferably, the polypeptide sequence includes amino acid residues represented by the formula: -GRGDSP-. Exemplary polypeptides are represented by the formulas:

AVTGRGDSP,

GRGDSP, and CGGAGAGRGDSP. For a polypeptide having more residues than the above-recited included sequences, it is preferred that any additional residues be selected such that the entire resulting polypeptide has a sequence that corresponds to a portion of the amino acid residue sequence of fibronectin. The sequence of an 11.5 kilodalton fragment of fibronectin that contains the RGD sequence is described by Pierschbacher et al., J. Biol. Chem.. 257:9593-97 (1982).

Where a polypeptide-antibody conjugate contains the tripeptide -RGD- as a part of its integrin-binding polypeptide, it is referred to herein as an RGD- antibody.

An additional preferred integrin-binding polypeptide has an amino acid residue sequence that includes residues represented by the formula: -YIGSR-. For a polypeptide having more residues than this included sequence, it is preferred that any additional residues be selected such that the entire resulting polypeptide has a sequence that corresponds to a portion of the amino acid residue sequence of the Bl chain of laminin. The sequence of the laminin Bl chain is described by Sasaki et al., Proc. Natl. Acad. Sci. USA. 84:935-39 (1987).

Where a polypeptide-antibody conjugate contains the pentapeptide -YIGSR- as a part of its integrin- binding polypeptide, it is referred to herein as a YIGSR-antibody.

Exemplary polypeptides suitable for use in a YIGSR-antibody include polypeptides represented by the formulas

YIGSR, CDPGYIGSR, and RGDSGYIGSR. An integrin-binding polypeptide to be utilized in a polypeptide-antibody conjugate of the present invention can be synthesized by any suitable method known to those skilled in the polypeptide art, including recombinant DNA techniques. Thus, synthesis can be by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensations or cleavages, or by classical solution addition.

The polypeptides are preferably prepared using the solid-phase Merrifield-type synthesis for reasons of purity, freedom from undesired side products, ease of production and the like, such as that described by Merrifield, J. Am. Chem. Soc.. 85:2149 (1964), although other equivalent chemical syntheses known in the art can also be used, such as the syntheses of Houohten. Proc. Natl. Acad. Sci. USA. 82:5132 (1985). A summary of the many techniques available can also be found in J. M. Steward and J. D. Young, "Solid Phase Peptide Synthesis", W. H. Freeman Co., San Francisco, 1969; M. Bodanszky, et al., "Peptide Synthesis", John Wiley & Sons, Second Edition, 1976; and J. Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic Press (New York), 1983, for solid phase peptide synthesis, and E. Schroder and K. Kubke, "The Peptides", Vol. 1, Academic Press (New York), 1965, for classical solution synthesis.

4. Polypeptide to Antibody Attachment The polypeptide-antibody conjugate of the present invention contains an integrin-binding polypeptide that is operatively attached to a before- discussed antibody molecule that immunoreacts with an adhesitory cell surface antigen.

As used herein, the term "operatively attached" means that the polypeptide and the antibody molecule are physically associated by a linking means that does not significantly interfere with the ability of either of the linked groups to function as described herein. This ability is not significantly interfered with where the number of polypeptides operatively attached is limited so that each antibody molecule has linked to it from 1 to about 30 polypeptides. It is preferred that from about 8 to about 12 polypeptides are linked per antibody molecule, more preferably about 10 polypeptides per molecule.

Methods for controlling the number of polypeptides that are linked (conjugated) to an antibody molecule are well known in the art and typically depend upon the particular linkage chemistry utilized. Typically, those methods rely on controlling the ratio of polypeptide to antibody molecule in the linkage reaction mixture or the time allowed for the linkage reaction to occur, or both. An exemplary linkage method is described in Example 2.

Because antibody molecules are proteins themselves, the techniques of protein conjugation or coupling through activated functional groups is particularly applicable to operatively attached the polypeptide to the antibody molecule. See, for example, Aurameas, et al., Scand. J. Immunol.. Vol. 8 Suppl. 7:7-23 (1978); U.S. Pat. Nos. 4,493,795, and 4,671,950.

One or more additional amino acid residues can be added to the amino- or carboxyl-termini of the synthetic polypeptide to assist in binding the polypeptide to a carrier. Cysteine residues added at the amino- or carboxyl-termini of the synthetic polypeptide have been found to be particularly useful for forming linkages via disulfide bonds. However, other methods well known in the art for preparing conjugates can also be used. Exemplary additional linking procedures include the use of Michael addition reaction products, dialdehydes such as glutaraldehyde, Klipstein et al., J. Infect. Pis.. 147:318-326 (1983) and the like, or the use of carbodimide technology as in the use of water-soluble carbodimide to form amide links to the antibody molecule.

It is preferred that the linking means be a covalent coupling between a cysteine terminus on the polypeptide and the epsilon amino group of a lysine residue present in the monoclonal antibody molecule. As is also well known in the art, it is often beneficial to bind a synthetic polypeptide to its antibody molecule by means of an intermediate, linking group. As noted above, glutaraldehyde is one such linking group. However, when cysteine is used, the intermediate linking group is preferably an m- maleimidobenxoyl N-hydroxy succinimide (MBS) or 4- (maleimidomethyl)-l-cyclohexane carboxylic acid N- hydroxysuccinimide ester(NHS) , as was used herein. Additionally, MBS can be first added to the antibody molecule by an ester-amide interchange reaction as disclosed by Liu et al., Biochem.. 80:690 (1979) . Thereafter, the addition can be followed by addition of a blocked mercapto group such as thiolacetic acid (CH₃C0SH) across the maleimido- double bond. After cleavage of the acyl blocking group, a disulfide bond is formed between the deblocked linking group mercaptan and the mercaptan of the added cysteine residue of the synthetic polypeptide. In addition, site directed coupling reactions can be carried out so that the attached polypeptide does not substantially interfere with the immunoreaction of the antibody molecule with the' adhesitory cell surface antigen. See, for example, Rodwell et al., Biotech.. 3:889-894 (1984).

The polypeptides utilized in this invention can contain additional residues at either terminus for the purpose of providing a "spacer" by which the integrin- binding polypeptide can be operatively attached to the -antibody. The use of a spacer will thereby extend the polypeptide further out from the site of linkage upon the antibody molecule than if the polypeptide were attached without a spacer.

Amino acid residue spacers are usually from 1 to about 50 residues in length, more often 2 to 10 residues and do not necessarily comprise sequences that correspond to the sequence of an adhesion protein.

A preferred spacer polypeptide contains the amino acid residue sequence CGGAGA operatively attached by its carboxyl-terminal alanine through a peptide bond to the amino terminal residue of the integrin-binding polypeptide. More preferably, the spacer polypeptide when attached to an integrin- binding polypeptide comprises the sequence CGGAGAGRGDSP.

A polypeptide utilized in the present invention can be connected together to form a polymer (synthetic multi er) comprising a plurality of polypeptide repeating units. Such a polymer typically has the advantage of increased exposure of the cell recognition site for the potential binding to integrins.

A polymer for use in this invention can be prepared by synthesizing a polypeptide as discussed before, and including a cysteine residue at both the amino- and carboxyl-termini to form a "diCys- terminated" polypeptide. After polypeptide synthesis, in a typical laboratory preparation, 10 mg of the diCys polypeptide (containing cysteine residues in un- oxidized form) are dissolved in 250 ml of 0.1 M ammonium bicarbonate buffer. The dissolved diCys- terminated polypeptide is then air oxidized by stirring the resulting solution gently for a period of about 18 hours in the air, or until there is no detectable free mercaptan by the Ell an Test. [See Ellman, Arch. Bioche . Biophvs.. 82:70-77 (1959).]

The polymer so prepared contains a plurality of the synthetic polypeptide repeating units that are randomly bonded together by cysteine residues. Such polymers typically contain their polypeptide repeating units bonded together in a head-to-tail manner as well as in head-to-head and tail-to-tail manners; i.e., the amino-termini of two polypeptide repeating units can be bonded together through a single cysteine residue as can two carboxyl-termini since the linking groups at both polypeptide termini are identical.

Alternatively, the polypeptides can be connected in numerous branched polymer configurations to provide a similar advantage as above for the polymer that contains linear repeating units.

In a further embodiment, it is contemplated that different integrin-binding polypeptides can be operatively attached to a single antibody molecule. For example there can be two or more different polypeptides that are independently linked as monomers, polymers or branched polymers to the antibody molecule by the same linkage means described above for .the polymers. In this embodiment each polymer is comprised of different integrin-binding polypeptide monomers.

C. Therapeutic Methods and Compositions The present invention contemplates a polypeptide-antibody conjugate and compositions containing said conjugate wherein said conjugate comprises an antibody molecule operatively attached to an integrin-binding polypeptide, said antibody molecule being capable of immunoreacting with an adhesitory cell surface antigen and said polypeptide being a polypeptide described herein having adhesion inhibiting properties.

Preferably, the polypeptide-antibody conjugate is an RGD-antibody in which said conjugate contains a polypeptide comprising an amino acid residue sequence of about 5 to about 50 residues in length that includes a sequence having the formula: -RGD-, and more preferably having the formula: -GRGDSP-.

In another embodiment the polypeptide-antibody conjugate is a YIGSR-antibody in which said conjugate contains a polypeptide comprising an amino acid residue sequence of 5 to about 50 residues in length that includes a sequence having the formula: -YIGSR-. The present invention further contemplates that a polypeptide-antibody conjugate of the present invention and compositions containing said conjugate can be used in a method to inhibit the attachment of adhesitory cells to an adhesion protein-containing surface.

The polypeptide-antibody conjugate of the present invention can be used in a pharmaceutically acceptable composition that, when administered to a subject in a therapeutically effective amount, is capable of inhibiting the ability of an integrin molecule, present on adhesitory cells, to bind to an RGD-containing matrix protein (adhesion protein) .

That inhibition of integrin is believed to result in the decreased capacity for attachment (adhesion) by cells containing an integrin molecule to other adhesion grotein-containing cell surfaces or tissue substrates in the body. Thus, in vivo administration of a polypeptide-antibody conjugate composition of the present invention can be used to inhibit cell adhesion in a specific manner depending upon the choice of antibody molecule to be included in the conjugate as disclosed further herein.

For example, the use of a polypeptide-antibody conjugate containing a monoclonal antibody molecule that immunoreacts with the disialoganglioside GD2, a molecule present on a variety of tumor cell types, inhibits tumor cell adhesion and tumor metastasis. This embodiment is effective to inhibit growth and metastasis of tumor cells because those events depend on tumor cell attachment, which attachment is specifically inhibited by the adhesion-inhibiting polypeptide present in the conjugate that is targeted to those tumor cells having GD2 as a cell surface antigen by means of the anti-GD2 antibody present in the conjugate.

The use of a polypeptide-antibody conjugate containing a monoclonal antibody that immunoreacts with a platelet surface antigen is also contemplated to inhibit platelet adhesion. It is preferred that the monoclonal antibody immunoreact with the platelet glycoprotein GPIIb/IIIa, a platelet aggregation- dependent molecule. This use is effective to inhibit blood coagulation because coagulation depends in part on the ability of platelets to aggregate by means of cell adhesion.

Other cell adhesion inhibitory uses of the claimed polypeptide-antibody conjugate will be apparent to one skilled in the cell adhesion arts and depend upon the selection of specific monoclonal antibodies.

It is therefore seen that a polypeptide-antibody conjugate of the present invention is useful in a variety of methods for inhibiting cell adhesion and for inhibiting the processes that are dependent upon a particular cell's ability to adhere to matrix proteins. Thus, the present invention contemplates a method for inhibiting the attachment of an adhesitory cell to an RGD-containing matrix comprising administering to a subject a therapeutically effective amount of a polypeptide-antibody conjugate described herein. Preferably the conjugate is an RGD-antibody or YIGSR-antibody.

Where the adhesitory cell is a tumor cell, the invention contemplates a method for inhibiting tumor cell adhesion and tumor metastasis. Where the adhesitory cell is a platelet, the invention contemplates a method for inhibiting platelet adhesion, platelet aggregation and platelet- dependent blood coagulation.

The preparation of therapeutic compositions which contain polypeptide-antibody conjugate molecules as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents which enhance the effectiveness of the active ingredient.

As used herein, the phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction, such as gastric upset, dizziness, and the like, when administered to a subject.

A polypeptide-antibody conjugate can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide-antibody conjugate molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The therapeutic polypeptide-antibody conjugate molecule-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active material calculated to produce the desire therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.

Therapeutic polypeptide-antibody conjugate- containing compositions can also be administered topically, as by means of a salve or ointment, or by application of a packet or patch containing the composition in a formulation suitable for diffusion of the active ingredient into the contacted tissue as is well known.

The polypeptide-antibody conjugate compositions administered typically contain 0.1-95% of active ingredient, preferable 25-70%.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to the treated, capacity of the subject's system to utilize the active ingredient without adverse side affects, and degree of inhibition of interaction desired between an integrin and an adhesive protein.

The phrase "therapeutically effective amount" when used herein refers to an amount of a polypeptide- antibody conjugate sufficient to measurably inhibit a particular cell's ability to adhere to an adhesion protein-containing substrate. Typically, that ability is measured in vitro by an adhesion assay, such as is described "in Example 4.

Measuring for a therapeutically effective amount by means of the in vitro adhesion assay depends on the type of cell to be treated, which in turn depends on the particular antibody molecule contained in the polypeptide-antibody conjugate. Thus, the cells to be utilized in the adhesion assay are of a cell type that contain an adhesitory cell surface antigen with which the antibody molecule immunoreacts, preferably the same cell type as is to be treated by the use of the contemplated composition, and more preferably the cells utilized are a population of cells obtained from the subject to be treated.

A therapeutically effective amount is an amount sufficient to cause a decrease in the cell's ability to adhere ^"in the in vitro adhesion assay by at least 10 percent, and preferably by about 50 percent. That amount can be expressed as an effective concentration as determined by the in vitro assay. Thus, a therapeutically effective amount can be expressed as that amount sufficient to deliver an effective concentration to the subject's blood. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of 0.1 to 10 milligrams, preferably one to several milligrams, of active ingredient per kilogram bodyweight of subject per day, and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to produce effective intravascular concentrations are contemplated, preferably intravascular concentrations of about ten nanomolar to about ten micromolar.

D. Chimeric Antibodies

The present invention also contemplates a chimeric antibody that is capable of immunoreacting with an adhesitory cell surface antigen and inhibiting integrin-mediated cell adhesion.

A "chimeric antibody" as used herein refers to a type of antibody molecule that contains the amino acid residue sequence of an integrin-binding polypeptide of the present invention as a part of the antibody molecule's primary structure, eg., amino acid residue sequence. As an antibody molecule, a chimeric antibody is comprised of heavy and light chains, at least one chain of which is a hybrid protein molecule having an antibody combining site-forming fragment that is fused to an integrin-binding polypeptide.

An antibody combining site-forming fragment as used herein refers to a linear series of amino acid residues that correspond to the variable region portion of either a heavy or light chain of an antibody molecule that contributes to the structure known as the antibody combining site.

A hybrid protein molecule refers herein to a molecule that contains a continuous length of amino acid residues comprising a first length corresponding to an antibody combining site-forming fragment fused to a second length corresponding to an integrin- binding polypeptide.

The term "fused" refers to the manner in which the fragment and the polypeptide are associated and is used to distinguish a chimeric antibody from a polypeptide-antibody conjugate in which the polypeptide is associated with the antibody molecule by operative attachment. For a hybrid protein molecule, the fusion refers to the peptide bond between adjacent amino acid residues, one adjacent residue being the carboxyl-terminal residue of the first length and the other adjacent residue being the amino-terminal residue of the second length. Although a chimeric antibody typically contains a single integrin-binding polypeptide component fused to each antibody combining site-forming fragment, it is not to be construed as so limited. For example, a hybrid protein can contain one antibody combining site-forming fragment that is fused to one or more integrin-binding polypeptides. Further, the included polypeptides can be polymers comprised of repeating or different integrin-binding polypeptides, or both.

In preferred embodiments, the integrin-binding polypeptide portion of a chimeric antibody is the same polypeptide as described herein as a component of the polypeptide-antibody conjugate.

A hybrid protein can additionally contain more than two lengths, such as where the first and second lengths are followed by a third length of amino acid residues in which the amino-terminal residual of the third length is fused to the carboxyl-terminal residue of the second length. The third length can have an amino acid sequence that corresponds to a second integrin-binding polypeptide, a portion of an antibody molecule or a portion of a third protein molecule unrelated to the first two.

In a preferred embodiment a third length has an amino acid sequence that corresponds to a portion of the Fc region of an antibody molecule. Typically the resulting hybrid protein in this embodiment has a structure .in which the integrin-binding polypeptide comprising the second length is inserted into an intact (native) heavy chain of an antibody molecule so as to interrupt the native heavy chain molecule and produce a first length that includes an antibody combining site-forming fragment and a third length that includes a portion of the Fc region.

From the foregoing it can be seen that the location of the interrupting second length within the antibody heavy chain amino acid residue sequence can vary along the length of the Fc portion of the native heavy chain. In preferred embodiments, the site- of the interruption by the second length will reside nearer to the carboxyl terminus than to the hinge region that makes the amino terminus of the Fc region. More preferably, however, the second length is fused to the carboxyl terminus of the heavy chain of an antibody molecule. The general structure of antibody molecules, and the terminology used herein to identify the heavy and light chains, the hinge region and the Fc region of antibody molecules (immunoglobulins) are well known. See, for example Paul et al., "Fundamental Immunology," fourth printing. Raven Press, N.Y. (1984), at pages 7 and 150.

The combination of an antibody combining site and an integrin-binding polypeptide in the chimeric antibody provides the same uses and benefits as described herein above for a polypeptide-antibody conjugate.

Therefore the present invention contemplates a chimeric antibody that contains at least one hybrid protein molecule having an antibody combining site- forming fragment fused to at least one integrin- binding polypeptide such that the hybrid protein molecule forms an antibody combining site that is capable of immunoreacting with an adhesitory cell surface antigen and thereby inhibiting cell adhesion. In preferred embodiments, a chimeric antibody contains a polypeptide having an amino acid residue sequence of about 5 to about 50 residues in length that includes a sequence having the formula: -RGD-, and more preferably includes residues represented by the formula: -GRGDSP-.

A preferred chimeric antibody additionally contains an antibody combining site-forming fragment portion of an antibody molecule whose primary amino acid residue sequence corresponds to the amino acid residue sequence of a monoclonal antibody as described herein. Exemplary monoclonal antibodies are shown in Table 1. A preferred chimeric antibody contains an antibody combining site-forming fragment whose amino acid residue sequence corresponds to the amino acid residue sequence present in the monoclonal antibody Mab 142A.

The preparation of chimeric antibodies of the present invention can be accomplished using methods well known that involve the cloning of immunoglobulin genes, the subsequent manipulation of the cloned genes to incorporate integrin-binding polypeptide encoding sequences, the introduction of the manipulated genes into a suitable expression vector and expression medium, and the production of the resulting hybrid protein molecules to form chimeric antibodies. The above-recited preparative steps are routine recombinant DNA cloning, manipulation and expression procedures, and are described in more detail herein below. The inventive aspect of a chimeric antibody does not reside in the techniques for preparing a chimeric antibody, but rather in the resulting manipulated gene, and it's expressed antibody molecule having a combination of integrin-binding polypeptide fused to an antibody combining site that immunoreacts with an adhesitory cell surface antigen.

The cloning of immunoglobulin protein-encoding genes, and their manipulation to form recombined DNA ^* molecules that encoded antibody combining sites fused to other heterologous protein fragments are generally known methods. Once prepared, these recombined DNA molecules are introduced into an expression medium that produces assembled antibody molecules that are capable of immunoreaction with the same specificity as the antibody produced by the antibody producing cell from which the immunoglobulin protein-encoding genes were isolated. See, for example Roberts et al.. Protein Engineering. 1:59-65 (1986) , Morrison, Science. 229:1202-07 (1985), U.S. Patent No. 4,474,893, and published patent application Nos. EP 0125023, EP 0239400 and WO 89/00999,

For the isolation of an immunoglobulin protein- encoding gene in preparation of a chimeric antibody, the gene can be obtained from any cell that produces an antibody molecule that immunoreacts with an adhesitory cell surface antigen. A preferred cell is a hybridoma cell. Hybridomas can be prepared as disclosed herein, or obtained from commercial sources. Exemplary hybridomas are listed in Table 1.

For examples of general recombinant DNA cloning methods, see Molecular Cloning. Maniatis et al.. Cold Spring Harbor Lab., N.Y., 1982; DNA Cloning. Glover, ed., IRL Press, McLean, VA (1985). For the genomic cloning and expression of immunoglobulin genes in lymphoid cells, see Neuberger et al.. Nature. 312:604- 8 (1984); Ochi, et al., Proc. Natl. Acad. Sci. USA. 80:6351-55 (1987); and Oi et al., Proc. Natl. Acad. Sci. USA. 80:825-29 (1983). For cloning of immunoglobulin genes from hybridoma cells, and their expression in Xenopus oocytes, see Roberts et al., Protein Engineering. 1:59-65 (1986), and see Wood et al.. Nature. 314:446-9 (1985) for their expression in yeast.

A DNA segment is included as a part of the above described manipulated recombinant DNA molecule and that segment has a nucleotide sequence that encodes at least a polypeptide whose amino acid residue sequence corresponds to the amino acid residue sequence of an integrin-binding polypeptide of the present invention. A DNA segment may further encode an antibody combining site-forming fragment. The nucleotide sequence of the DNA segment can correspond to the sequence of an antibody combining site-forming fragment of an immunoglobulin protein cloned as described above, or the sequence can correspond, for example, to known sequences. Exemplary antibody combining site-forming fragment-encoding nucleotide sequences are described by Kabat, et al. in "Sequences of Proteins of Immunological Interest", 4th Edition, National Institutes of Health, Bethesda, MD (1987) . The DNA segment that encodes an integrin-binding polypeptide, an antibody combining site-forming fragment, or both, can be synthesized by chemical techniques, as by, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Soc. _f 103:3185 (1981). Once prepared the segment is included in the recombinant DNA molecule that is being manipulated to encode and express the entire hybrid protein molecule.

The present invention therefore also contemplates a recombinant DNA molecule that encodes the hybrid protein disclosed herein and is useful for the preparation of a chimeric antibody of the present invention.

Thus in one embodiment the present invention contemplates a recombinant DNA molecule that encodes a hybrid protein molecule comprising a first DNA segment encoding an antibody combining site-forming fragment operatively linked in phase to a second DNA segment encoding an integrin-binding polypeptide, such that the recombinant DNA molecule is capable, when present in an appropriate expression vector, of expressing a hybrid protein molecule having an antibody combining site that immunoreacts with an adhesitory cell surface antigen. A DNA segment, as is generally understood and used herein, refers to a molecule comprising a linear stretch of nucleotides wherein the nucleotides are present in a sequence that encodes, through the genetic code, a molecule comprising a linear sequence of amino acid residues that is referred to as a protein, a protein fragment or a polypeptide.

A particularly useful DNA segment is characterized as including a DNA sequence that encodes (1) an antibody combining site-forming fragment, or (2) a DNA sequence that encodes an integrin-binding polypeptide.

A recombinant DNA molecule, as used herein, refers to a molecule that contains two or more DNA segments operataively linked to produce a third DNA segment. Thus a recombinant DNA molecule (rDNA) is a hybrid DNA molecule comprising at least two nucleotide sequences (DNA segments) not normally found in nature.

The phrase "operatively linked" indicates that the two joined DNA segments are connected by a typical phosphodiester bond normally found between adjacent nucleotide residues in a DNA sequence. Where the two linked DNA segments each encode an amino acid residue sequence of interest, the term further indicates that the linkage maintains the reading frame of the joined DNA segments so that the two encoded amino acid residue sequences can be translated from the hybrid DNA molecule in phase as a single larger hybrid protein.

A recombinant DNA molecule of the present invention contains at least a DNA segment that encodes an antibody combining site-forming fragment operatively linked in phase to a DNA segment that encodes an integrin-binding polypeptide to form a hybrid protein encoding DNA segment. Typical recombinant DNA molecules also contain a vector operatively linked to the hybrid protein encoding DNA segment.

As used herein, the term "vector" refers to a DNA molecule capable of autonomous replication in a cell and to which another DNA segment can be operatively linked so as to bring about replication of the attached segment. Vectors capable of directing the expression of a hybrid protein-encoding DNA segment are referred to herein as "expression vectors". The recombinant DNA molecule having a hybrid protein-encoding DNA segment operatively linked to an expression vector-containing DNA segment provides a system for expressing a hybrid protein product in a medium compatible with the included expression vector.

The choice of vector to which a recombinant DNA molecule of the present invention is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing and expressing recombinant DNA molecules. However, a vector contemplated by the present invention is at least capable of directing the replication, and preferably also expression, of the hybrid protein- encoding DNA segment included in recombinant DNA molecule to which it is operatively linked.

In preferred embodiments, a vector contemplated by the present invention includes a procaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell, transformed therewith. Such replicons are well known in the art. In addition, those embodiment that include a procaryotic replicon also include a gene whose expression confers drug resistance to a bacterial host transformed therewith. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline.

Those vectors that include a procaryotic replicon can also include a procaryotic promoter capable of directing the expression (transcription and translation) of the hybrid protein-encoding DNA segment in a bacterial host cell, such as E. coli. transformed therewith. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically .provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Typical of such plasmid vectors are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA) and pPL and pKK223 available from Pharmacia (Piscataway, NJ) .

Expression vectors compatible with eucaryotic cells, preferably those compatible with vertebrate cells, can also be used to form the recombinant DNA molecules .of the present invention. Eucaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia) , pBPV-l/pML2d (International Biotechnologies, Inc., New Haven, CT) and pTDTl (ATCC, #31255).

In preferred embodiments, the eucaryotic cell expression vectors used to construct the recombinant DNA molecules of the present invention include a selection marker that is effective in an eucaryotic cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neo ycin phosphotransferase (neo) gene. Southern et al., J. Mol. APPI. Genet.. 1:327-341 (1982).

The use of retroviral expression vectors to form the rDNAs of the present invention is also contemplated. As used herein, the term "retroviral expression vector" refers to a DNA molecule that includes a promoter sequence derived from the long terminal repeat (LTR) region of a retrovirus genome. In preferred embodiments, the expression vector is typically a retroviral expression vector that is preferably replication-incompetent in eucaryotic cells. The construction and use of retroviral vectors has been described by Sorge et al., Mol. Cell. Biol.. 4:1730-37 (1984). Also contemplated by the present invention are RNA equivalents of the above described recombinant DNA molecules.

EXAMPLES

The following examples are intended to illustrate, but not limit, the present invention. 1. Preparation of Monoclonal Antibodies The hybridoma cell line 1418, on deposit with the ATCC as HB 9118, produces a monoclonal antibody (Mab) that immunoreacts with the disaloginglioside GD2. The -hybridoma cell line 142A was isolated as one of several isotype switch variants from a culture of hybridoma 1418 using the floursecence activated cell aorting (FACS) techniques described by Kipps et al., J. EXP. Med.. 161:1-17 (1985). Hybridoma 142A produces a monoclonal antibody that also immunoreacts with GD2. Hybridoma 1418 and the FACS-isolated hybridomas were then characterized to determine the isotype of their produced antibodies using the following isotyping ELISA assay. Fifty microliter volumes (1:1000 dilutions in

PBS) of rabbit anti-mouse IgGl, IgG2a, IgG2b, IgG3 or IgM (Southern Biotech Associates, Birmingham, AL) were plated per well of a flat-bottom polyvinyl chloride microliter plate (Dynatech, Alexandria, VA) The plates were then maintained overnight at 37"C in a drying oven. The dried plates were stored at 4*C until use. Prior to the ELISA assay, dried plates were rehydrated by two washes of two minutes each with wash buffer (10 millimolar [mM] PBS, pH 7.4, containing 0.1 percent polyoxyethylene [20] sorbitan monolaurate [Tween 20] and 0.02 percent Thimerosal [sodium ethylmercurithiosalicylate; Sigma, St. Louis, MO]).

Culture supernatant from FACS-isolated hybridomas were diluted 1:2 in wash buffer, containing 0.1% BSA (bovine serum albumin), added at fifty microliters per well and maintained at 4"C for 1 hour. After 2 wash buffer rinses, fifty microliters (ul) of horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Biorad Laboratories, Richmond, CA) diluted 1:1000 were added to each well and maintained at 4°C for 1 hour.

The substrate used to assay bound peroxidase activity was prepared just prior to use and consisted of 400 micrograms/ml o-phenylenediamine (Sigma, St.

Louis, MO) in 80 mM citrate-phosphate buffer, pH 6.0, containing 0.23 percent H₂0₂. After two final wash buffer rinses, 50 ul of substrate solution was added to each well and color was allowed to develop for 15 minutes in the dark. Color development was stopped by adding 25 ul of 4 Normal (N) H₂S0₄ to each well, and the optical density at 492 nanometers (nm) was measured with Multiskan ELISA Plate reader (Bio-Tek Instruments Inc. , Burlington, VA) . Isotype designations were assigned to those hybridoma-produced culture supernatants that generated an optical density at 492 nm that was at least 10 times the value obtained for a control non-reacting antibody. By this assay, Mab 1418 was verified to be an IgG3 isotype and Mab 142A was identified to be an IgG2a isotype.

The monoclonal antibodies Mab 142A were prepared by introducing a culture of hybridoma 142A cells into the peritoneal cavity of a Balbc/byj mouse (Scripps Clinic Vivarium, La Jolla, CA) and later harvesting the resulting peritoneal exudate (ascites tumor fluid) from the mouse by well known techniques. Mab 142A antibody molecules present in the recovered ascites fluid were further isolated by affinity chromatography using protein-A Sepharose (Bio-Rad Laboratories, Richmond, CA) at a ratio of 1.0 milliliters (ml) ascites fluid per ml packed Sepharose beads according to the methods recommended by the manufacturer with the exception that the elution buffer was adjusted to pH 5.0 before use. The Sepharose eluant was collected in fractions and the protein content of the fractions was determined using the BCA Protein Assay Reagent available from Pierce Chemical Co. (Rockford, IL) . Peak protein-containing fractings were pooled to form purified Mab 142A.

In addition to Mab 142A, the following Mabs were used to demonstrate the present invention, listed here with their respective isotypes and sources denoted in parentheses: Mab 9227 (IgG2a, Dr. D. Cheresh; Scripps Clinic and Research Foundation, La Jolla, CA, hereinafter Scripps) , directed against chondroitin sulfate proteoglycan of human melanoma cells [Cheresh et al., J. Cell. Biol.. 102:688-696 (1986); Harper et al., J. Immunol. 132:2096-2104 (1984); and Bumol et al., Proc. Natl. Acad. Sci. USA. 79:1245-1249 (1982)]; and Mab KS14, a control antibody that does not immunoreact with M21 melanoma cells but does immunoreact with UCLA-P3 cells.

Additional purified monoclonal antibodies were prepared by the same method as described above for Mab 142A except that hybridomas 9227 and KS14 were utilized to form purified Mab 9227 and purified Mab KS14, respectively.

2. Coupling of Polypeptide to Monoclonal Antibody

A three ml solution containing 14.4 milligrams (mg) of purified Mab 142A in 100 millimolar (mM) phosphate buffered saline (PBS; pH 7.8) was prepared as described in Example 1. About 25 microliters (ul) of a solution of NHS [75 mM 4-(maleimidomethyl)-l- cyclohexane carboxilic acid N-hydroxysuccinimide ester in N-methylpyrolidone] was admixed with the prepared Mab 142A-containing solution and the admixture was maintained while stirring for 3 hours at room temperature to form an activated Mab 142A-containing solution. The activated Mab 142A- containing solution was then applied to a Sepharose G-25 column having a bed volume of 1.0 mis and pre-equilibrated with 10 mis, and the resulting eluant was monitored for protein content and collected in fractions. Peak fractions containing protein were pooled to form activated Mab 142A.

A synthetic polypeptide having the amino acid sequence Cys-Gly-Gly-Ala-Gly-Ala-Gly-Arg-Gly-Asp-Ser- Pro (CGGAGAGRGDSP) was provided by Dr. M.D.

Pierschbacher (La Jolla Cancer Research Foundation, La Jolla, CA) after having been synthesized by Peninsula Laboratories (San Carlos, CA) . Ten mg of the polypeptide was admixed as a solid to the above- prepared activated Mab 142A and the admixture was maintained while stirring for 3 hours at room temperature to form a reaction admixture. The reaction admixture was then applied to a Sephadex G-25 column having a bed volume of 1.0 is that was pre-equilibrated with 10 mis and the resulting eluant was monitored for protein as before and peak fractions containing protein were collected to form a polypeptide Mab 142A conjugate (Mab 142A-conjugate) .

Polypeptide-Mab conjugates were also prepared by the above procedure using purified Mab 9227 or control Mab KS14 prepared as described in Example 1, in place of purified Mab 142A to form Mab 9227-conjugate and control Mab-conjugate, respectively.

Polypeptide-Mab conjugates can also be prepared by the above methods of Examples 1 and 2 using the monoclonal antibodies produced by hybridoma 1418, 126, MB3.6, 9227, LM142or LM609.

3. Monoclonal Antibody Conjugate Binding To Cultured Cells M21 human melanoma cells were kindly provided by

Dr. D.L. Morton (University of California at Los Angeles, hereinafter*UCLA) and grown as a suspension culture in growth medium [RPM1 1640 containing 2mM L- glutamine and 50 mg/ml gentamicin sulfate; all from GIBCO Laboratories (Grand Island, NY) ] containing 10 percent fetal calf serum (FCS) at 37'C in 7.5% Cθ₂/92.5% air. UCLA-P3 human lung adenocarcinoma cells were provided by Dr. Martin (UCLA) and grown as a suspension culture as above for M21 cells. Cultured M21 cells were washed twice in normal saline and plated in a flat-bottom polyvinyl microtiter plate (Dynatech, Chantilly, VA) at 5 x 10⁴ cells per well in 50 ul in growth medium containing 10% FCS. Thereafter the plates were maintained at 37"C for about 12-18 hours to dry the plates and to form dried M21 plates. Dried UCLA-P3 plates were similarly prepared using cultured UCLA-P3 cells.

Dried plates were rehydrated by two washes each comprising first the addition of wash buffer [lOmM PBS, pH 7.4, 0.1% Tween 20 (polyoxyethylenesorbitan monolaurate) 0.02% thimerosal (sodium ethylmercurithiosalicylate) ] , then the maintenance of the plates at room temperature for two minutes, and finally the removal of the liquid by inverting the plates.

Purified Mabs prepared in Example 1 and Mab- conjugates prepared in Example 2 each were then serially diluted to the final concentrations indicated in Table 2 using diluent buffer [1.10 (v/v) in wash buffer containing 0.1% BSA (bovine serum albumin)] and 50 ul thereof was admixed into each well of the rehydrated plates to form immunoreaction admixtures. The plates were maintained for one hour at 4*C on a gyroshaker, emptied of their liquid contents by inversion and shaking, and then washed twice as described above to isolate solid-phase bound immunoreaction products. Afterwards, 50 ul of a solution containing peroxidase-1inked, goat anti-mouse Ig (Tago, Burlingame, CA) diluted 1:1000 (v/v) in diluent were admixed into each well of the plates and maintained for 1 hour at 4'C to allow formation of labeled immunoreaction products. The plates were again emptied by inversion and shaking, washed twice as described above, and 50 ul of freshly prepared chromogenic substrate solution [400 ug/ml O-phenylene diamine (Sigma Chemical Co., St. Louis, MO), 80 mM citrate-phosphate buffer, pH 5.0, 0.012% (v/v) H₂0₂ (Eastman Kodak, Rochester, NY) ] was admixed into each well, The plates were then maintained in the dark for 30 minutes after which time 15 ul of 4N H₂Sθ₄ was admixed into each well. The optical absorbance at 492 nm (A;₉₂) of the resulting solutions in each well was then measured with a Multiscan ELISA plate reader (Flow Laboratories, McLean, VA) . Results of studies of binding of purified Mab

("Free") and Mab-conjugate ("Conjugate") by immunoreaction with adhesitory cell surface antigens present on M21 and UCLA-P3 cells are shown in Table 2, below.

Table 2 Binding of Polypeptide Monoclonal Antibody Conjugates

To Cells

A. M21 Cell Free" Conjugates

[Mab]^c 9227 142A Control 9227 142A Control

5.0 1.171 1.995 0.147 1.088 1.687 0.994

1.0 1.146 1.602 0.114 0.985 1.758 0.951

0.5 1.142 1.590 0.071 1.023 1.786 0.963

0.1 1.101 1.402 0.032 0.929 1.471 0.554

0.05 0.510 0.105 0 0.408 0.078 0

0.005 0.261 0.015 0 0.121 0.017 0

0 0 0 0 0 0 0

B. UCLA-P3 Cells

Free" Conjugates¹³

[Mab]^c 9227 142A Control 9227 142A Control

5.0 .0.090 0.256 1.754 0.234 0.345 1.858

1.0 0.093 0.116 2.192 0.122 0.174 2.076

0.5 0.077 0.149 2.127 0.126 0.155 1.996

0.1 0.022 0.081 1.815 0.070 0.085 2.047

0.05 0.030 0.085 0.241 0.001 0.114 0.335

0.005 0.056 0.042 0.103 0.050 0.070 0.148

0 0.050 0.037 0.021 0.013 0.034 0.017

"Free" designates the use of indicated purified Mab's (Mab 9227, Mab 142A or Control Mab) prepared as described in Example 1 and being free of polypeptide conjugate.

"Conjugate" designates the use of the same Mab's as indicated for "Free" except that the Mab's have been operatively attached (conjugated) to polypeptide CGGAGAGRGDSP as described in Example 2.

"[Mab]" indicates the final protein concentration of the serially diluted Mab (free) or Mab-conjugate (conjugate) preparation.

Table 2 shows that both free and conjugated Mab 9277 were able to immunoreact with chondroitin sulfate proteoglycan present on M21 cells, as were the Mab

142A preparations able to immunoreact with ganglioside GD2 on those same cells. _ For both of these Mabs, the immunoreaction was only slightly decreased when using Mab-conjugate as compared to free Mab. Similarly, both free and conjugated control Mab KS14 were able to immunoreact with the target antigen present on UCLA-P3 cells. These data indicate that the operative attachment of an integrin-binding polypeptide to an antibody molecule by the disclosed linking methods does not significantly interfere with the Mab- conjugates ability to immunoreact with its target antigen when present on adhesitory cell surfaces.^"

Control Mab KS14 that does not immunoreact with M21 cells was observed to bind to M21 cells upon polypeptide conjugation. Because this increased binding was observed only after formation of a Mab- conjugate it is believed that the binding results from an interaction between the RGD-containing polypeptide present on the Mab-conjugate and the RGD-directed adhesion receptors present on the M21 cells.

It is not known why the Mab 9227 or Mab 142A conjugates do not also show an increase in binding to UCLA-P3 by the mechanism proposed above for Mab KS14 on M21 cells. However, UCLA-P3 cells can have significantly fewer RGD-directed adhesion receptors than M21 cells as measured by the assay.

4. Monoclonal Antibody Conjugates Inhibit

Cell Adhesion In Vitro The following cell adhesion assay was performed to characterize the ability of monoclonal antibody (Mab) conjugates to inhibit cell adhesion.

The M21 cells were grown in suspension culture in RPMI 1640 "growth medium containing 10% fetal calf serum (FCS) at 37"C with 7.5% C0₂/92.5% air. These cells were metabolically labeled in leucine-free growth media containing 50 microcurie (uCi)/ml ³H leucine (ICN, Irvine, CA) for 72 hours at 37"C. Thereafter labeled cells were washed by centrifugation in growth medium containing 1% FCS to remove unincorporated radiolabel forming ³H-leucine-labeled M21 cells.

Approximately 5 x 10^{3 3}H-leucine labeled M21 cells were resuspended in 100 ul growth medium containing various concentrations of a Mab conjugate prepared as described in Example 2 to form an immunoreaction admixture. As controls, labeled cells were resuspended in growth medium alone, or a control antibody was used. The control Mab is known as activated Mab and was prepared as described in Examp^'le 2.

The admixture was then maintained for 1 hour at 4'C to allow formation of Mab-M21 cell immunoreaction products. The cells were then washed by two cycles of first a low speed centrifugation at 400 x g followed by resuspension of the resulting pellet using growth medium containing 1% FCS. After the wash the cells were resuspended in growth medium to form antibody treated M21 cells.

The wells of polystyrene microtiter plates (96- well; Flow Laboratories, McLean, VA) were coated with matrix proteins (adhesion substrates) by admixing solutions of PBS (pH 7.2) containing 5 ug/ml of an adhesion substrate to a well. The wells were maintained overnight at 25"C to allow adsorption of the proteins onto the wells. Matrix proteins used included fibrinogen, provided by Dr. E. Plow (Research Institute of Scripps Clinic, La Jolla, CA hereinafter RISC); von Willebrand factor, provided by Drs. Z. Ruggeri and T. Zimmerman (RISC) ; and human fibronectin, provided by Dr. M. Pierschbacher (La Jolla Cancer Research Foundation, La Jolla, CA, hereinafter LCRF) .

Fifty ul of growth medium containing 5 x 10³ antibody treated M21 cells were admixed in an adhesion substrate-coated well to form an adhesion-reaction admixture. The admixtures were maintained at 37*C in a humidified incubator for 20 minutes to allow cell adhesion to occur, at which point the plates were inverted to removed growth medium and non-adhered cells. A duplicate set of plates were similarly prepared and maintained for an adhesion period of 90 minutes. All wells were washed twice with 150 ml PBS (pH 7.2) to assure removal of unattached cells. The remaining attached cells were harvested by adding 100 ul of Trypsin/EDTA (IX; Gibco Laboratories, Grand

Island, NY) to each well, incubating the cells at 37"C for 30 minutes and then removing the cells and collecting them on glass fiber filters using a Skatron automated cell harvester (Skatron Instruments, Sterling, VA) according to instructions provided by the manufacturer. The fiber filter disks were place into vials containing 3 ml of liquid scintillation cocktail and the amount of radioactive label present determined as counts per minute in a liquid scintillation counter. Results were expressed as the total number of cells (count per minute) that adhered

(bound) to the matrix protein at the designated concentrations of added Mab-conjugate or control Mab.

As shown in Figures 1 and 2, M21 cell adhesion was inhibited by Mab 142A conjugate during either a 20 minute (Figure 1) or 90 minute (Figure 2) adhesion period, when using either von Willebrand factor (vWF) for fibrinogen (Fb) as the adhesion substrate. Inhibition was observed at concentration less than 2 ug Mab conjugate per ml. Adhesion inhibiting potency (IC₅₀) can be expressed as the concentration of Mab conjugate sufficient to effect a 50% decreased in adhesion when compared to the adhesion obtained with no added Mab conjugate. Thus, the IC₅₀ for Mab 142A conjugate during a 20 min adhesion time was 4 ug/ml for adhesion to vWF and 0.5 ug/ml for adhesion to Fb. Similarly,, the IC₅₀ for the conjugate during a 90 minute adhesion time was 15 ug/ml for adhesion to vWF and 0.5 ug/ml for adhesion to Fb. Using the in vitro cell adhesion assay, essentially as described above, soluble polypeptide CGGAGAGRGDSP from Example 2 was added at various concentration to the cell suspensions in place of the Mab 142 conjugate. The results of those adhesion measurements is expressed as adhesion inhibiting potency (IC₅₀) in Table 3, below.

Table 3

EFFECTS OF SOLUBLE OR MAB 142A-CONJUGATED

CGGAGAGRGDSP ON M21 CELL ATTACHMENT TO FIBRINOGEN, VON WILLEBRAND FACTOR OR FIBRONECTIN^*3

SOLUBLE MAB 142A

TIME PEPTIDE fuM) CONJUGATE fuM)

VWF

20 min. 10 0.24

90 min. 55 0.91

Fibrinogen

2200 mmiinn.. NN//TT^cc N/T 90 min. 2 .7 0. 03

Fibronectin

20 min. 106 >18 90 min. >500 >18

^a Length of time cells were maintained for adhesion to occur. ^b Effects on cell attachment is expressed as a icromolar (uM) concentration of polypeptide present in solution or on the conjugate sufficient to cause a 50% decrease in adhesion when compared to adhesion with no added polypeptide. ^c N/T indicates not tested.

Table 3 shows that at each adhesion time, 20 or 90 minutes, and for all adhesion substrated tested, there was a significant reduction in the concentration of polypeptide necessary to inhibit cell adhesion when the polypeptide was conjugated.

Thus although it is known that RGD-containing polypeptides and other adhesion protein derived polypeptides have the ability to inhibit cell adhesion, the data in Table 3 shows that the adhesion inhibition potency of these polypeptides was improved by conjugation to an antibody that immunoreacts with an adhesitory cell surface antigen.

The foregoing specification, including the specific embodiments and examples, it intended to be illustrative of the present invention and is not to be taken as limiting. Numerous other variations and modifications can be effected without departing from the true spirit and scope of the present invention.

Claims

What is Claimed is:

1. An RGD-antibody comprising an antibody molecule that is capable of immunoreacting with an adhesitory cell surface antigen, operatively attached to an integrin-binding polypeptide comprising an amino acid residue sequence of about 5 to about 50 residues in length that includes a sequence having the formula: - RGD-.

2. The RGD-antibody of claim 1 wherein said polypeptide includes a sequence having the formula: -GRGDSP-.

3. The RGD-antibody of claim 2 wherein said " polypeptide has the formula: -CGGAGAGRGDSP-.

4. The RGD-antibody of claim 1 wherein said antibody molecule is capable of immunoreacting with an adhesitory cell surface antigen selected from the group consisting of disialoganglioside GD2, disialoganglioside GD3, chondroitin sulfate proteoglycan, vitronectin receptor, and endothelial cell receptor.

5. The RGD-antibody of claim 1 wherein said antibody molecule is selected from the group of monoclonal antibodies consisting of 1418, 142A, 126, MB3.6, LM609, and LM142.

6. The RGD-antibody of claim 1 wherein said surface antigen is an antigen present on a cell selected from the group consisting of platelets and tumor cells.

7. The RGD-antibody of claim 1 wherein said antibody molecule contains from 2 to about 30 polypeptides operatively attached to said antibody molecule.

8. The RGD-antibody of claim 7 wherein said antibody molecule contains about 10 polypeptides.

9. The RGD-antibody of claim 1 wherein said antibody molecule is operatively attached by means of polypeptide spacer having a length of 2 to about 50 amino acid residues.

10. The RGD-antibody of claim 9 wherein said polypeptide spacer consists essentially of a polypeptide having the amino acid sequence CGGAGA wherein the terminal alanine residue is operatively attached by a peptide bond to the terminal glycine residue of the integrin-binding polypeptide GRGDSP.

11. An RGD-antibody comprising an antibody molecule that is capable of immunoreacting with an adhesitory cell surface antigen, is selected from the group of monoclonal antibodies consisting of 1418, 142A, 126, MB3.6, 11C64, R24, LM609 and LM142, and is operatively attached to an integrin-binding polypeptide having the amino acid residue sequence CGGAGAGRGDSP, wherein said polypeptide is attached to said antibody molecule in a ratio of about 10 polypeptides per antibody molecule by means of a NHS linkage molecule.

12. A method for inhibiting the attachment of an adhesitory cell to an RGD-containing matrix comprising administering to a subject a therapeutically effective amount of an RGD-antibody according to claim 1.

13. The method of claim 12 wherein said effective amount is an amount sufficient to produce an intravascular concentration of RGD-antibody of ten nanomolar to ten micromolar.

14. The method of claim 12 wherein said effective amount is in the range of 0.1 to 10 milligrams of RGD- antibody per kilogram bodyweight of subject per day.

15. The method of claim 12 wherein said adhesitory cell is a platelet.

16. The method of claim 12 wherein said adhesitory cell is a tumor cell.

17. A YIGSR-antibody comprising an antibody molecule that is capable of immunoreacting with an adhesitory cell surface antigen, operatively attached to a laminin receptor-binding polypeptide comprising an amino acid residue sequence of about 5 to about 50 residues in length that includes a sequence having the formula: -YIGSR-.

18. The YIGSR- antibody of claim 17 wherein said polypeptide has the formula:

YIGSR, CDPGYIGSR, or RGDSGYIGSR.

19. A chimeric antibody that contains at least one hybrid protein molecule having an antibody combining site-forming fragment fused to at least one integrin- binding polypeptide, said hybrid protein molecule forming an antibody combining site that immunoreacts with an adhesitory cell surface antigen.

20. The chimeric antibody of claim 19 wherein said integrin-binding polypeptide comprises an amino acid residue sequence of about 5 to about 50 residues in length that includes a sequence having the formula: RGD-.

21. The chimeric antibody of claim 20 wherein said polypeptide includes a sequence having the formula: -GRGDSP-.

22. A recombinant DNA molecule that encodes a hybrid protein molecule comprising: a) a first DNA segment encoding an antibody combining site-forming fragment, and b). a second DNA segment encoding an integrin- binding polypeptide that is operatively linked in phase to the first segment, wherein said recombinant DNA molecule is capable, when present in an appropriate expression vector, of expressing a hybrid protein molecule having an antibody combining site that immunoreacts with an adhesitory cell surface antigen.