WO1994021813A1 - Monoclonal antibodies specific for fibroblast growth factor receptors, immunotoxins, and use thereof - Google Patents

Abstract

Monoclonal antibodies (MAbs) that specifically bind to at least one subtype of human FGF high affinity receptors on intact human cells, but that do not bind to human T-lymphocytes or significantly to proliferating human smooth muscle cells are provided. The monoclonal antibodies can be advantageously radiolabeled or coupled to a drug. The monoclonal antibodies are useful as diagnostic reagents in vitro and in vivo. Immunotoxins prepared by coupling these MAbs to a cytotoxic agent, preferably a ribosome-inactivating protein, e.g. saporin are also provided. By administering an effective dose of such immunotoxins, the population of certain cells such as tumor cells which express this subtype of high affinity FGF receptor can be selectively reduced. Hybridoma cell lines that produce the antibodies are provided. One hybridoma cell line that produces monoclonal antibodies designated 11A8 has been deposited with the ATCC under Accession No. HB-11303.

Description

MONOCLONAL ANTIBODIES SPECIFIC FOR FIBROBLAST GROWTH FACTOR RECEPTORS , IMMUNOTOXINS, AND USE THEREOF.

This invention was made with Government support under Grant AM- 1881 1 awarded by the National Institutes of Health (DHHS). The Government has certain rights in this invention. 5 This application generally relates to monoclonal antibodies (MAbs) and to compositions, containing such MAbs, that inhibit cell proliferation, more specifically to MAbs to specific fibroblast growth factor receptors, immunotoxins containing the MAbs, to pharmaceutical compositions containing the immunotoxins and to methods using the immunotoxins and 10 the MAbs.

Background of the Invention During the last twenty-five years, a great deal of attention has been directed towards the identification and characterization of factors that stimulate the growth, proliferation and differentiation of specific cell types. 15 Numerous growth factors and families of growth factors that share structural and functional features have been identified. Many of these factors have multifunctional activities and affect a wide spectrum of cell types.

One family of growth factors that has a broad spectrum of activities 20 is the fibroblast growth factor (FGF) family. This family of proteins includes FGFs designated FGF-1 through FGF-9 (or acidic FGF (aFGF), basic FGF (bFGF), int-2, hst-1 /K-FGF, FGF-5, FGF-6/Hst-2, keratinocyte growth factor ^* (KGF), FGF-8 and FGF-9, respectively).

Acidic and basic FGF, which were the first members of the FGF 25 family that were characterized, are about 55% identical at the amino acid level and are highly conserved among species. Basic FGF has a molecular weight of approximately 16 kD, is acidic and temperature sensitive and has

< a high isoelectric point. Acidic FGF has an acidic isoelectric point. The other members of the FGF family have subsequently been identified on the basis of amino acid sequence homologies with aFGF and bFGF and common physical and biological properties, including the ability to bind to one or more FGF receptors. Basic FGF, int-2, hst-1 /K-FGF, FGF-5, hst- 2/FGF-6 and FGF-8 are oncogenes. For example, bFGF is expressed in melanomas, int-2 is expressed in mammary tumor virus and hst-1 /K-FGF is expressed in angiogenic tumors. Acidic FGF, bFGF, KGF and FGF-9 are expressed in normal cells and tissues.

FGFs exhibit a mitogenic effect on a wide variety of mesenchymal, endocrine and neural cells. They are also important in differentiation and development. Of particular interest is their stimulatory effect on collateral vascularization and angiogenesis. Such effects have stimulated considerable interest in FGFs as therapeutic agents, for example, as pharmaceuticals for wound healing, neovascularization, nerve regeneration and cartilage repair. In addition to potentially useful proliferative effects, FGF-induced mitogenic stimulation may, in some instances, be detrimental. For example, cell proliferation and angiogenesis are an integral aspect of tumor growth. Members of the FGF family, including bFGF, are thought to play a pathophysiological role, for example, in tumor development, rheumatoid arthritis, proliferative diabetic retinopathies and other complications of diabetes.

In addition to potentially useful proliferative effects, basic FGF- induced mitogenic stimulation may, in some instances, be detrimental. _, For example, cell proliferation and angiogenesis are an integral aspect of tumor growth, and basic FGF is thought to play a pathophysiological role, for example, in tumor development, rheumatoid arthritis, proliferative diabetic retinopathies and other complications of diabetes. bFGF has also gained widespread interest by virtue of its angiogenic activity and its production by numerous tumor cells.

The effects of FGFs are mediated by high affinity receptor tyrosine kinases on the cell surface membranes or FGF-responsive cells (see, e.g.. Imamura et aL (1988) Biochem. Biophvs. Res. Comm.:155:583-590: Huang et aL (1986) J. Biol. Chem.:261 :9568-9571 , which are incorporated herein by reference). Lower affinity receptors also play a role in mediating FGF activities. Certain of these receptor proteins appear to be single chain polypeptides with molecular weights ranging from 1 10 to 150 kD, depending on cell type. Some of these proteins bind basic FGF with high affinity (Kd = 10-80 pM) and are referred to as high-affinity FGF receptors (FGFR); often numbers of such receptors range from 2000 to 80,000 per cell. These receptors can be purified from rat brain, using a combination of lectin and ligand affinity chromatography and are associated with tyrosine kinase activity, see Imamura et al., Biochem. Biophvs. Res. Comm.. 155:583-590 (1988) and Huang and Huang, J. Biol. Chem.. 261 :9568-9571 (1986), the disclosures of which are incorporated herein by reference. FGF receptors have recently been shown to be involved in disease states of cell proliferation. It has been shown that there is altered receptor mRNA expression in the proliferative disease Dupuytren's Contracture, and in proliferating smooth muscle cells after balloon injury and in *. Becker et al., Oncogene. 7: 2303-2313, 1992, have shown that FGF receptors are expressed in primary and metastatic melanomas.

The high affinity receptor proteins constitute a family of structurally related FGF receptors. Four FGF receptor genes have been identified (see, e^, Lee, P.L., et al., Science. 245: 57-60, 1989; Patstone, G., et al., Devel. Biol., 755: 107-123, 1993; Johnson, D.E., et al., Mol. Cell. Biol.. 10: 4728-4736, 1 990; Houssaint, E., et al.. P.N.A.S. USA. 87: 8180- 8184, 1 990; and Pasquale, E.B.. P.N.A.S. USA. 87: 5812-5816, 1990), and, at least two of these genes generate multiple mRNA transcripts via alternative splicing of the primary transcript. This splicing potentially creates a large number of different molecular forms which can interact with FGF family members, thereby permitting cells to respond to different FGF family members, Dionne, C.A., et al., EMBO J., 9: 2685-2692, 1990; Keegan, K., et al.. P.N.A.S. USA, 88: 1095-1099, 1991 ; and Miki, T., et al., P.N.A.S., USA, 89: 246-250, 1992. This genetic variation and alternative splicing result in a variety of sequences of receptors proteins that have differential affinities for the FGF family members. For example, alternative splicing of a single gene results in the receptor FGFR2, which has high affinity for acidic and basic FGFs but no detectable affinity for KGF, and the KGF receptor, which has high affinity for KGF but reduced affinity for basic FGF. Similarly, alternative splicing of FGFR1 produces variants that have about a 50-fold decreased the affinity for basic FGF, but unchanged acidic FGF binding, Werner, S., et al., Mol. Cell. Biol.. 12:82-88, 1992.

Receptor expression is altered by physical, chemical, and hormonal injury as well as in certain pathological conditions such as restenosis, tumors and selected proliferative diseases. Receptor messenger RNA and protein is expressed in melanoma cells, Becker, et al., Oncoqene, 7: 2303- 2313, 1992. The receptor message is not normally expressed in the pal¬ mar fascia, but is present in the proliferative hand disease Dupuytren's Contracture, Gonzales, A.-M., et al., Amer. J. Pathol., 141 : 661 -671 , 1992. Quiescent smooth muscle cells (SMCs) do not respond to bFGF, but proliferating SMCs, in a model of restenosis after balloon angioplasty, strongly respond to exogenous bFGF, Lindner, V., et al., Circ. Res.. 68: 106-1 13, 1991 , and Casscells, W., et al., P.N.A.S.. USA. £9: 71 59-7163, 1992. A potent cytotoxic agent was earlier developed, which inhibits proliferation of cells that express FGF receptors, by attachment of a ribosome-inactivating protein (RIP), e.g. saporin, to basic FGF to produce the conjugate bFGF-SAP, Lappi, et al., J. Cell. Phvsiol.. 147: 17-26, 1991 , and Lappi, et al., B.B.R.C, 160: 917-923, 1989. This agent has been used to inhibit smooth muscle cell after balloon injury in the rat, Casscells, W., et al., P.N.A.S., USA, 89: 71 59-7163, 1992, and Lindner, V., et al., Circ. Res., 78: 106-1 1 3, 1 991 , to inhibit proliferation of cells isolated from lesions of Dupuytren's Contracture, Lappi, et al., J. Hand Surq. [Am.], 17A: 324-332, 1992, and to inhibit in vivo tumor growth by tumor cells that present the FGF receptor, Beitz, et al., Cancer Res., 52: 227-230, 1992. While the high affinity of basic FGF for its receptor (5-50 pM) promises efficient binding of this ligand, there are some potential disadvantages to the use of this agent for such treatment. The heparin-binding activity of basic FGF could divert ■ RIP to compartments that do not offer bioavailability, or it co. ' be a source of non-specific toxicity by cellular internalization of cell-sυ ' ace proteoglycan. Because bFGF-SAP does not differentiate between t four known high affinity FGF receptors gene products and because there is a response in cells which express the low- affinity FGF receptor, the possibility of side-effects is increased. Moreover, the very fast removal (t_1/2 = 5 min) of basic FGF from systemic circulation and the similarly brief half-life of the conjugate basic FGF-saporin (FGF-SAP) is also a disadvantage.

Because bFGF does not appear to distinguish among subtypes of high affinity FGF receptors and because of the number of receptor variants that may exist and the correlation of receptor expression with certain disease states, injury and development, it would be useful to produce antibodies that specifically interact with subpopulations of FGF receptors. Such antibodies would not only serve as tools to elucidate important features of the receptors, development and disease states, but would also be clinically useful in diagnosis and treatment. Therefore, it is an object herein to provide monoclonal antibodies that specifically interact with specific subpopulations of FGF receptors and that thereby may be targeted to specific cell populations. It is also an object herein to provide methods for diagnosis and treatment using such antibodies. Summary of the Invention

Monoclonal antibodies have been created that recognize native human FGF receptors on intact cells that bear such FGF receptors. In addition, the antibodies appear to bind to only certain subpopulations of FGF receptors. The antibodies provided herein are promising as tools for characterizing the expression of FGF receptors that are produced on particular cells. They are also useful as a diagnostic reagents in assays for the detection of certain neoplasms, as generally disclosed in copending U.S. Application Serial No. 900,646, filed June 18, 1992, and Published International Application WO 94/00599 assigned to the assignee of this application. The monoclonal antibody may also be used to selectively label tumor cells in vivo for X-ray imaging. In particular, one such Mab 1 1A8 has been tested as an agent for targeting to tumor cells and has been found to provide the vehicle for a potent immunotoxin. To produce such antibodies, it was necessary to devise an immunization and selection protocol that permitted recognition of the native receptor rather than fragments or portions thereof. An immunization procedure was employed that used particular whole cells as the antigen, and appropriate monoclonal hybridomas were selected by using the extracellular domain (ECD) of the high affinity FGF receptor in the screening process and selecting for antibodies that internalize cytotoxic agents. As a result of this immunization and selection protocol, hybridomas were obtained that produce MAbs were that recognize native human FGF receptors on the surfaces of intact cells. These antibodies also recognize the ECD and immunoprecipitate FGF receptors as judged by Western blotting.

Methods for diagnosis of certain tumors are provided by reacting the antibodies with body fluids or tissues and detecting elevated levels of FGF receptors in the body fluids and tissues. Labeled Mabs are also provided. The labeled MAbs can be used to label FGF receptor-expressing cells in vivo and in vitro.

Immunotoxins containing the antibodies are also provided. Because of the specificity of the Mabs for intact native human FGF receptors, the immunotoxins can be targeted living melanoma cells and hepatomas that express the FGF receptor. It has now been found that the use of such immunotoxin in the form of an FGF receptor-specific MAb coupled to a cytotoxic agent is particularly advantageous because such an immunotoxin does not have an affinity for proteoglycan heparin, and thus compartmentalization and toxicity due to proteoglycan internalization are avoided. The half-life in plasma of antibodies is very much longer than that of a relatively short protein, such as basic FGF, and these saporin immunotoxins are considered to remain detectable in the serum for days, thereby allowing longer exposure of the cytotoxic agent to the proliferative cells being targeted after systemic administration.

The immunotoxins containing such the MAbs and a cytotoxic agent is useful in treating a variety of FGF-mediated pathophysiological conditions. In one embodiment, the cytotoxic agent is a ribosome- inactivating protein (RIP), such as, for example, saporin, although other cytotoxic agents can also be advantageously used. The cytotoxic agent can be attached to the MAb through a chemical bond, and the immunotoxin is designed and produced in such a way that the receptor-binding epitope of the MAb of the complex is unobstructed so as to permit recognition and binding to the FGF receptor. The immunotoxin can be used to treat FGF-mediated pathophysiological conditions by specifically targeting to cells having high affinity FGF receptors and inhibiting proliferation of or causing death of these cells. Such pathophysiological conditions include, for example, tumor development, Dupuytren's Contracture, certain complications of diabetes such as proliferative diabetic retinopathies, rheumatoid arthritis and similar diseases that are correlated with an alteration of FGF receptor expression. The treatment is effected by administering a therapeutically effective amount of the immunotoxin, for example, in a physiologically acceptable excipient. The immunotoxin can be used to target cytotoxic agents into cells having at least one certain subtype of high affinity FGF receptors in order to inhibit the proliferation of such cells, e.g. to thereby differentially target certain tumor cells.

Detailed Description of the Invention Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. All U.S. patents and all publications mentioned herein are incorporated in their entirety by reference thereto. The term MAb 1 1 A8 should be understood to include useful binding fragments of the above monoclonal antibody which bind to FGFR, such as Fab, F(ab')₂, Fv fragments and so forth. The antibody fragments are obtained by conventional techniques. For example, useful binding fragments may be prepared by peptidase digestion of the antibody using papain or pepsin. The term is also intended to include all derivatives, issue, and offspring of the parent hybridoma that produce the monoclonal anti- human FGFR antibody produced by the parent, regardless of generations or karyotypic identity.

The antibodies provided herein are selected for their ability to specifically interact with an FGF receptor to be transported into the cell by virtue of its interaction with the FGF receptor.

As used herein, the term FGF receptor refers to any cell surface receptor that specifically interacts with an FGF polypeptide resulting in transport of the FGF into the cell by virtue of the interaction with the FGF receptor. Included among these are the receptors described in International Application No. WO 91 /00916, which is based on U.S. Patent Application Serial No.07/377,033; International Application No. WO 92/00999, which is based on U.S. Patent Application Serial No.07/549, 587; International Application No. WO 90/05522; and International Application No. WO 92/12948; see, also Imamura (1988) Biochem. Biophvs. Res. Comm. 155:583-590; Partanen et al. (1991 ) EMBO J. 10: 1347-1 354; and Moscatelli (1 987) J. Cell. Phvsiol. 131 : 1 23-130.

As used herein, FGF polypeptides include members of the FGF family of peptides, including FGF-1 through FGF-9 (see, e.g., Esch et al., Proc. Natl. Acad. Sci. USA, 82:6507-651 1 (1985); and Baird et al., Brit. Med. Bull., 45:438-452 (1989), chimeras or hybrids of any of FGF-1 through FGF-9, or FGFs that have deletions (see, e.g.. Published International Application No. WO 90/02800 and national stage applications thereof) or insertions of amino aids, as long as the resulting peptide or protein specifically interacts with an FGF receptor and is internalized by virtue of this interaction. For example, bFGF should be generally understood to refer to polypeptides having substantially the same amino acid sequences and receptor-targeting activity as that of bovine bFGF or human bFGF. It is understood that differences in amino acid sequences can occur among FGFs of different species as well as among FGFs from individual organisms or species.

The term FGF also refers to portions of an FGF that bind to FGF receptors and internalize linked proteins. For example, in Baird et al., Proc. Natl. Acad. Sci. USA, 85:2324-2328 (1988), which is incorporated herein by reference, there are examples of basic FGF peptide fragments that are reactive with FGF receptors. Immunotoxins of acidic fibroblast growth factor fused to several mutant forms of Pseudomonas exotoxin have been shown to be cytotoxic to a variety of tumor cell lines including hepatocellular, prostatic, colon, and breast carcinomas (Siegall et al., The FASEB Journal. 5:2843-2849, Oct., 1991 ). Purified preparations of basic FGF and acidic FGF are frequently observed to include several molecular forms of the mitogens which bind in varying degrees to the FGF receptors. It is also understood that differences in amino acid sequences can occur in FGF from different species, as well as between FGF from individual organs in an animal of a particular species. The amino acid sequence of an exemplary mammalian basic FGF derived from bovine pituitary tissue is provided in Esch et al., Proc. Natl. Acad. Sci. USA, 82:6507-651 1 (1 985).

As used herein, the term "basic FGF" refers to proteins or polypeptides having substantially the same amino acid sequence and mitogenic activity as that of the basic FGF described in Esch, supra. Moreover, DNA clones encoding human aFGF (Jaye et al., Science, 233:541 -545 (1986)) and DNA clones encoding bovine (Abraham et al., Science. 233:545-548 (1986), human (Abraham et al., EMBO J., 5:2523-2528 (1986); Abraham et al.. Quant. Biol., 51 :657-668 (1986)), and rat (Shimasaki et al., Biochem. Biophvs. Res. Commun. (1988); Kurokawa et al., Nucleic Acids Res.. 1 6:5201 (1988)) basic FGF have been cloned and sequenced; they predict the existence of proteins identical to those found by protein sequencing. FGF expresses mitogenic activity in a wide variety of normal diploid mesoderm-derived and neural crest-derived cells. A test of such "FGF mitogenic activity" is the ability to stimulate proliferation of cultured bovine aortic endothelial cells, as described in Gospodarowicz et al., J. Biol. Chem.. 257:12266-1 2278 (1982); Gospodarowicz et al., Proc. Natl. Acad. Sci. USA. 73:4120-4124 (1976).

As used herein, the term "FGF-mediated pathophysiological condition" refers to a deleterious condition characterized by or caused by proliferation of cells which are sensitive to basic FGF mitogenic stimulation and carry certain high affinity FGF receptor subtypes. Basic FGF-mediated pathophysiological conditions include, but are not limited to, tumors, rheumatoid arthritis, Dupuytren's Contracture and certain complications of diabetes such as proliferative retinopathy. Also included are types of breast cancer, ovarian cancer, prostate hyperplasia, prostate carcinoma, astrocytoma, glioblastoma and similar diseases that are linked to FGF receptor expression.

As used herein, the term cytotoxic agent broadly refers to a molecule capable of inhibiting cell function. The term includes agents that are only toxic when transported into the cell and also those whose toxic effect is mediated at the cell surface. A variety of well known cytotoxic agents can be effectively used including those which inhibit protein synthesis.

As used herein, to target a targeted agent, such as a cytotoxic agent, means to direct it to a cell that expresses a selected receptor by linking the agent to an antibody provided herein to produce an immunotoxin. Upon binding to the receptor the conjugate is internalized by the cell.

As used herein, the term biologically active, or reference to the biological activity of a cytotoxic agent or cytotoxicity of an immunotoxin, refers to the ability of such RIP or immunotoxin to inhibit protein synthesis by inactivation of ribosomes either in. vivo or in vitro or to inhibit the growth of kill cells upon internalization of the saporin-containing polypeptide by the cells. Such biological or cytotoxic activity may be assayed by any method known to those of skill in the art including, but not limited to, the In vitro assays that measure protein synthesis and ]n vivo assays that assess cytotoxicity by measuring the effect of a test compound on cell proliferation or on protein synthesis. Particularly preferred, however, are assays that assess cytotoxicity in targeted cells.

As used herein, FGF-mediated pathophysiological condition refers to a deleterious condition characterized by or caused by proliferation of cells that are sensitive to bFGF mitogenic stimulation. Basic FGF-mediated pathophysiological conditions include, but are not limited to, certain tumors, rheumatoid arthritis, restenosis, Dupuytren's Contracture and certain complications of diabetes, such as proliferative retinopathy.

As used herein, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.

As used herein, ED₆₀ refers to the concentration at which 50% of the cells are killed following a 72-hour incubation with an immunotoxin, such as 1 1A8-SAP.

As used herein, ID₅₀ refers to the concentration of immunotoxin required to inhibit protein synthesis in treated cells to 50% of the protein synthesis in the absence of the protein. Preparation of monoclonal antibodies

Mabs that specifically bind to FGF receptors and immunotoxins containing the MAbs, or fragments thereof that bind to an FGF receptor, coupled to a cytotoxic agent are provided. The immunotoxins and compositions containing the immunotoxins are effective for inhibiting growth and proliferation of cells having FGF receptors. The compositions can be used to counteract the mitogenic effects of basic FGF, where such an effect is deleterious, such as in tumor angiogenesis and proliferative complications of diabetes, such as proliferative retinopathies. An initial attempt was made to produce monoclonal antibodies to these receptors by using as antigen a 58 kDa fragment of FGF receptor type 1 (FGFR1 ) that included the extracellular domain of FGFR1 produced in a baculovirus expression system. The selected hybridoma secreted an antibody that was designated AB6. MAb AB6 is able to recognize the ECD and native receptor in Western blotting; however, it is unable to recognize native receptor protein on the cell surface of whole non-fixed cells or living cells. A similar attempt using a synthetic fragment of the receptor as an immunizing antigen also failed to result in selection of any hybridomas that produced antibodies the recognize the native FGFR on intact cells.

Subsequently, it was found that when mice were immunized using whole cells from the SK-HEP-1 cell line and antibodies from the resulting hybridomas were screened by ELISA using the extracellular domain, MAbs of interest were detected. To further screen the antibodies, a second stage of screening was performed testing the antibodies for cytotoxicity to cells in conditioned medium containing anti-mouse IgG bound to saporin. The antibody resulting in the most cytotoxicity was selected. This monoclonal antibody designated 1 1 A8 is able to recognize the ECD in Western blotting, is able to immunoprecipitate the native receptor from extracts of receptor-bearing cells, and most importantly demonstrably binds to receptors on intact living cells. The hybridoma cell line that producing MAb 1 1 A8 is on deposit with the ATCC under Accession No. HB-1 1303. Comparable MAbs that bind to this subtype or other subtypes of high affinity FGF receptor can be produced by repeating the protocol set forth hereinafter cells that express the particular FGF subtype.

The details of this work are set forth in the Examples included hereinafter; however, as a brief explanation, NS-0 cells were fused with a splenocyte from a mouse immunized with intact human hepatoma cells (SK- HEP-1 ). After screening with extracellular domain of the FGF receptor R1 and selecting for hybridomas that secret antibodies that internalize linked cytotoxic agents, a hybridoma secreting the antibody 1 1 A8 was selected. This monoclonal antibody recognizes at least one subtype of the human high affinity FGF receptor in Western blotting system and can immunoprecipitate FGF receptor from SK-HEP-1 cells and SK-MEL-28 cells. More significantly, immunofluorescent staining shows that this antibody stains cell surface receptors of SK-HEP-1 and SK-MEL-28 cell lines. Thus, this antibody, unlike other anti-FGF receptor antibodies previously produced, recognizes the human FGF receptor in its native form on the cell surface. Diagnostic Assays

As previously indicated, the receptor for FGF is differentially expressed jri vivo: for example, neoplastic tissues and cells after injury have elevated levels of FGF receptor when compared to the low to absent levels in non-malignant (quiescent) tissue. Thus, these significantly higher levels of expression of the receptor gene in select neoplasms can be used to confirm the presence of such neoplasms (see, Published International Application WO 94/00599). The higher levels of expression can be detected either by determining the presence of the receptor in cellular material, or by determining the presence of the receptor in body fluids as a result of sloughing from tumor cells or the like. The MAb 1 1 A8 can be used diagnostically in this respect.

The detection of a specific FGF receptor that is associated with malignant melanoma is particularly advantageous because it is increased in amounts when compared to normal or benign lesions. Furthermore, the expression of FGF has been directly implicated in the transformation from normal melanocytes to melanoma, see Halaban, R., et al., Oncoqene Res.. 3: 177-186 (1988). Assays using MAb 1 1 A8 for detecting malignant melanoma are one advantageous use; however, the MAbs can also be used to assay for numerous other neoplasms including, but not limited to, subtypes of breast cancer, ovarian cancer, prostate hyperplasia, prostate carcinoma, astrocytoma, glioblastoma and similar diseases that are linked to FGF receptor expression.

As indicated above, the assay allows the determination of the presence of a select neoplastic disease. A suitable sample is assayed for the presence of a product indicative of elevated levels of the FGF receptor protein of which there are numerous subtypes and isoforms of the cell surface protein receptor, each of which is potentially specific for a particular cell type, more highly expressed in malignant than in non- malignant tissue. The target FGFR to be assayed can be detected by any suitable immunological technique. Immunological techniques for assaying proteins are generally well known in the art and, accordingly, will not be described in detail. Immunological techniques that can be used for detecting FGFR in a fluid sample such as blood, serum or urine, for example, include competitive immunoassays that employ MAb 1 1A8 or a comparable MAb specific for this receptor. Such assays can also typically employ a labeled receptor as a second reagent which competes for the captive antibody with receptor present in the sample. The labeled receptor is typically conjugated with a moiety which permits direct or indirect detection. Among the useful labels are radioactive nuclides such as ³⁵S, enzymatic moieties that cause a color change, or luminescence-generating reactions with suitable substrates. Such enzymes include, for example, luciferase, horseradish peroxidase and alkaline phosphatase. Fluorescent labels also can be used. Alternatively, the labeled receptor can be conjugated with a moiety that is recognized by another reagent that permits detection. For example, the receptor can be bound to biotin which will bind to a detectable avidin derivative, e.g., avidin conjugated with an enzyme as described above.

Because the monoclonal antibodies are specific for native FGF receptors expressed on the surface of cells, labeled antibodies may be used administered to individuals as imaging agents. The labeled agents should preferentially bind to tumor cells that express the particular FGF receptor against which the antibody is prepared. Following administration, standard X-ray or other detecion methods may be used to detect or visualize such tumor cells. Preparation of immunotoxins

The MAbs are conjugated to a cytotoxic agent to produce an immunotoxin that can be used to target the cytotoxic agent specifically to cells that express specific FGF receptors.

An immunotoxin are made by conjugating the antibodies produced as described herein to a cytotoxic agent. The resulting immunotoxins toxic to cell lines that FGF receptors that were borne by the immunizing cells, but have little or no effect on cells that bear other subtypes of FGF receptors or that do not bear FGF receptors, such as T-lymphocytes.

In particular, the MAbs can been combined with a ribosome- inactivating protein (RIP), such as the type-1 RIP saporin-6 (SAP-6). SAP is a potent RIP, which is isolated from the seeds of the plant Saponaria officinalis (see Stirpe, et al., Biochem. J.. 216:617-625 (1983)). Other suitable RIPs include, but are not limited to, ricin, ricin A chain, gelonin, diphtheria toxin, diphtheria toxin A chain, trichosanthin, tritin, pokeweed antiviral protein (PAP), mirabilis antiviral protein (MAP), Dianthins 32 and 30, abrin, monordin, bryodin, and shiga. L. Barbieri et al., Cancer Surveys, 1, 489-520 (1982) and EPO published patent application No. 466,222 , provide lists of numerous RIPs and their sources, and a more extensive list is set forth in Stirpe, et al., Biotechnology. 10:405-412 (April 1992). Other cytotoxic agents that are considered to be functionally equivalent to the aforementioned RIPs include Pseudomonas exotoxin and metabolic inhibitors, which are known in this art, but they are not limited thereto. Therefore, the term RIPs is used in this application to broadly include such cytotoxins. For example, chimeric proteins composed of acidic fibroblast growth factor fused to several mutant forms of Pseudomonas exotoxin have proven to be cytotoxic to a variety of tumor cell lines, including hepatoceliular, prostatic, colon, and breast carcinomas (Siegall et al., The FASEB Journal. 5:2843-2849, Oct., 1991 ). The Pseudomonas toxin has also been shown to be effective at killing cells expressing epidermal growth factor receptors when fused to transforming growth factor type α as a chimeric protein. Chaudhary et al., PNAS, 84:4538-4542, (1 987). The cytotoxic agent may also be a drug, and examples of such drugs are anthracyclines, such as the daunomycins (including daunorubicin and doxorubicin), and methotrexate and its analogs. Others are also known to those skilled in the art.

An immunotoxin was synthesized by conjugating antibody 1 1 A8 to saporin. in vitro cytotoxicity assays have established the resulting immunotoxin is cytotoxic to endothelial cells, but it has no effect on cells that do not bear high affinity FGF receptors, e.g. T-lymphocytes, or that bear different subtypes of FGFR, including proliferating smooth muscle cells. Thus, the immunotoxin is a potent cell killing agent for SK-MEL-28 and SK-HEP-1 cells (ED₅₀ = 30 pM), while having no effect on normal human T-lymphocytes that do not express FGF receptors. Coupling of the Mabs to cytotoxic agents The MAbs can be conjugated to a protein cytotoxic agent by any suitable means known to those skilled in the art, such as through derivatization with a reactive sulfhydryl-containing moiety such as SPDP, or via a suitable cross linking agent, such as glutaraldehyde or carbodiimide. Methods for conjugating compounds to antibodies and antibody fragments are well-known in the art. Such methods may be found in U.S. Patents Nos. 4,220,450; 4,235,869; 3,935,074 and 3,996,345. In one embodiment, the cytotoxic agent is derivatized with a reactive sulfhydryl containing agent, such as N-succinimidyl-3(2-pyridyldithio)propionate. The MAb is then added to and mixed with the derivatized cytotoxic agent, and the resulting immunotoxin is separated from the unreacted products on a suitable column. Alternatively, the MAb can be conjugated to a drug, such as 14 bromo doxorubicin through the sugar moiety, as by the cis-aconitate method as described in Shen and Riser, B.B.R.C, 102: 1048 (1981 ). Alternatively, immunotoxins may be prepared by recombinant methods by providing DNA that encodes all or a receptor binding and internalization portion of MAb 1 1 A8 or other such antibody. Such methods as applied to conjugates of IL-2 or TGFσ are provided in Chaudhary et al., Proc. Natl. Acad. Sci. USA, 84:4538-4542 (1987) and Lorberman-Galski et al., Proc. Natl. Acad. Sci. USA. 85: 1922-1926 (1988). See also, Sambrook et al.. Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Laboratory (1989). Use of the immunotoxins

These MAb-cytotoxic agent immunotoxins can be used to target the cytotoxic agent to cells expressing certain high-affinity FGF receptors in order to cause cell death. The number of FGF receptors per cell has been found to have a direct relationship to the dose at which 50% of the cells are killed (the ED₅₀). Moreover, for cells with extremely high receptor numbers, for example, BHK cells, the ED₅₀ is identical to the affinity constant of basic FGF for its receptor (about 25 pM for BHK cells). This indicates that the presence of even such a large molecule as SAP as the cytotoxic agent does not reduce the ability of the MAb to bind to the desired receptors. Moreover, it is indicated that cells that express a large number of basic FGF receptors are particularly sensitive to the immunotoxin. In order to treat FGF-mediated pathophysiological conditions, a therapeutically effective amount of the MAb-cytotoxic agent immunotoxin is administered to a mammal in a physiologically acceptable excipient or pharmaceutical carrier. Examples of physiologically acceptable excipients include PBS and saline. ln therapeutic use for treating a mammalian host, for example an experimental animal host, affected by a tumor, the immunotoxins provided herein will be administered in an amount effective to inhibit the growth of the tumor, that is, a tumor growth-inhibiting amount will be in the range of about 0.1 to about 15 mg/kg of animal body weight/day. It is to be understood that the actual preferred dosage of immunotoxin for treating a tumor or other such FGF-mediated pathophysiological condition will vary widely depending upon the requirements of the animal being treated, the composition being used, and the route of administration. Many factors that modify the action of the anti-neoplastic agent will be taken into account by one skilled in the art to which this invention pertains including, for example, age, body weight and sex of the animal host; diet; time of administration; rate of excretion; condition of the host; severity of the disease; and the like. Administration may be carried out simultaneously or periodically within the maximum tolerated dose. Optimal administration (or application) rates for a given set of conditions may be readily ascertained by those skilled in the art using conventional dosage determination tests.

The following examples are intended to illustrate preferred methods of creating MAbs having the desired characteristics, immunotoxins incorporating such MAbs and methods for using the foregoing, but they should not be considered to limit the invention.

EXAMPLE 1 Materials and Methods T lymphocytes were obtained from the blood of normal healthy volunteers and were isolated by the different migration of cell types during centrifugation using Ficoll-Paque (Pharmacia, LKB,

Piscataway, NJ). The manufacturer's protocol for isolation of lymphocytes was followed, and the collected cells were washed three times before further processing. The human hepatoma cell line SK-HEP-1 was obtained from the American Type Culture Collection (Rockville, MD) and cultured in the HEPES-buffered Dulbecco's Modified Eagle's Medium with 4.5 g/l glucose and 10% fetal bovine serum. The human melanoma cell line (SK- MEL-28) was also obtained from the American Type culture collection (Rockville, MD) and cultured in the hepes buffered Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum. NS-0 myeloma cells are readily available, and they can be obtained from the University of London, Postgraduate Medical School. They were cultured in the RPMI-1640 with 10% fetal bovine serum. Primary cultures of normal human aortic endo- thelial cells and normal human aortic smooth muscle cells were obtained from Clonetics (San Diego, CA), and they were cultured with media and reagents provided by Clonetics. BALB/C female mice, 5-6 weeks of age, were purchased from Harlan Sprague-Dawley (Indianapolis, IN).

Scatchard binding of SK-HEP-1 CELLS was performed according to the method of Moscatelli, D., J. Cell. Phvsiol.. 131 : 123-130, 1987. Iodinated basic FGF was suitably prepared using recombinant human basic FGF. SK-HEP-1 cells were incubated at 4°C in the presence of varying concentrations of ¹²⁵l-basic FGF for 2 hours in serum-free medium containing 0.1 5% gelatin. The medium was removed, cells were washed 3 times with PBS. The medium and the wash were used to determine unbound cpm. Cells were then washed with 2 M NaCI in 20 mM HEPES buffer, pH 7.2 to remove proteoglycan-bound basic FGF. The cells were then extracted with 0.5% Triton X-100. Wells were washed with 0.5% Triton X-100, and the Triton-extracted cells and Triton wash were used, to determine cpm bound. Cell number per well was 47,600. Graphic analysis and parameter determination were done using the MacLigand Scatchard Analysis program. Binding of iodinated FGF to cells was determined after washing with 2 M NaCI in order to eliminate low affinity proteoglycan- based receptor binding. The resulting Scatchard analysis (see Scatchard, G., Ann. N. Y. Acad. Sci.. 51 : 660-672, 1949) determined the presence of high affinity receptor binding for basic FGF with a Kd of 7.7 ± 0.4 pM and a total of 5400 receptors per cell. These data indicated that this SK-HEP-1 line would be a good choice for use as an immunogen for the present purpose.

Preparation of the hybridoma

The female BAIb/C mice were injected with 10⁷ SK-HEP-1 cells in 0.2 ml Dulbecco's PBS in the peritoneal cavity. The animals were boosted 14 and 28 days later with 10⁷ cells injected interperitoneally. The fusion was done 4 days after the final immunization. Spleen cells taken from an immunized mouse were fused with NS-0 cells using PEG-1 500. Hybridoma cells were selected in RPMI-1640 containing HAT and 0.005% 2- mecaptoethanol, followed by RPMI-1640 containing HAT.

The initial screening process employed the ECD of the FGF receptor R1 . An enzyme-linked immunosorbant assay was used for screening the hybridomas as described in Harlow, E. and Lane, D., Antibodies: a laboratory manual, p. 182. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1988. Briefly, 50 μ\ of the ECD of basic FGF receptor (100 ng/ml) were added to wells and incubated. AB6, an antibody raised using as antigen the ECD, was used as a positive control. Second antibody conjugated to horseradish peroxidase (Bio-Rad, 1 : 1000 dilution) and o- phenylenediamine were used to develop the color. The reaction was stopped with H₂S0₄, and optical density 492 nm was measured. After quantitation, only the value more than the positive control was considered a positive well, and these wells were cloned by limited dilution.

Because the initial screening ELISAs resulted in such a large number of positive hybridomas, a second antibody immunotoxin was used, Till, M., et al.. Cancer Res.. 48: 1 1 19-1 123, 1988, for further screening of the hybridomas. This technique utilizes an anti-mouse IgG-saporin conjugate as a second antibody. Saporin is a potent ribosome-inactivating protein from the plant Saponaria officinalis. First antibody, as conditioned medium in this case, is added to cells, and the antibody is allowed to bind. Then the second antibody immunotoxin is added, binds to the first antibody on the cell surface, and is internalized by "piggy-backing" on the first. Upon internalization, saporin inhibits protein synthesis. Thus the technique selects for an internalizing antibody and for an IgG antibody.

The hybridoma 1 1 A8 proved to be the most cytotoxic in this system, and this hybridoma has been cultured for more than six months and has retained its stability. Its production of antibody in conditioned medium is 25 mg/ml and in ascites fluid is 2.5 mg/ml.

Subtyping of the 1 1 A8 antibody to determine its subclass was done using a Mouse-Hybridoma-Subtyping kit enzyme Immunoassay (Boehringer Mannheim, Mannheim, Germany) following the manufacturer's instructions. It has been determined that the 1 1 A8 monoclonal antibody is an IgGI . The light chain is K and the heavy chain is y. Testing using the MAb 1 1A8

To carry out Western blotting of the extracellular domain of bFGF receptor-1 , iodination of antibody 1 1A8 was first performed using the chloramine T procedure as described in Harlow, E. and Lane, D., supra, p. 328. 50 ng of the extracellular domain of bFGF receptor-1 were electrophoresed on a 10% polyacrylamide gel containing sodium dodecyl sulphate (SDS-PAGE). The protein was transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in TRIS buffered saline (10 mM TRIS, 0.14 M NaCI pH 7.4) (TBS) overnight at room temperature. It was then incubated with 10-20 ml of iodinated 1 1.A8 antibody (1 μCi/10 ml 4% non-fat milk in TBS) for two hours at room temperature. The receptor was visualized by autoradiography using X-ray film detection method.

To carry out immunoprecipitation of bFGF receptor from SK-HEP-1 and SK-Mel-28 cells, these cells in 60 mm dishes were washed three times using PBS and scraoed from the plate with a rubber policeman into eppendorf tubes and pelleted by centrifugation. The cells were resuspended in 100 μl PBS, and sonicated two minutes. The sonicated cells were centrifuged, and the supernatant was added to 10 μg of 1 1 A8 or AB6 and incubated overnight at room temperature. The following morning, 100 μl of goat anti-mouse IgG agarose (Pharmacia, LKB, Piscataway, NJ) was added to the samples, incubated another four hours and centrifuged. After washing the pelleted beads with PBS and 0.5% P- 40 in PBS, the beads were added to 0.1 ml 2.5 x reducing sample buffer. After centrifugation, the total samples were run on a 7.5% polyacrylamide gel. The gel proteins were transferred onto a nitrocellulose membrane and incubated with 10 ml of 1 1 A8 (1 .5 //g/ml) antibody followed by rabbit anti- mouse IgG (1μg/ml). This was followed by incubation with 2 μCi of iodinated Protein A (ICN Biomedicals, Irvine, CA) in 10 ml PBS, and the results were visualized by autoradiography using X-ray film detection methods. The results showed the immunoprecipitation by antibody 1 1 A8 of FGFR from SK-HEP-1 hepatoma cells and from SK-Mel-28 melanoma cells. AB6 was used as positive control. Monclonal antibodies 1 1 A8 and AB6 immunoprecipitated receptors from the two cell lines that have the same molecular weight as native FGF receptor. The observed pattern of two bands around 100 kd and 150 kd was similar to bands reported by Partanen et al., supra, as being indicative of FGFR1 . Immunohistochemical staining of SK-HEP-1 cells is carried out using

MAb 1 1 A8 and MAb AB6, a monoclonal antibody raised using the ECD as the immunogen and for screening. AB6 recognizes the ECD and receptor from chick brain extracts, but apparently it is unable to recognize the receptor on the cell surface, as judged by binding experiments with whole cells.

SK-HEP-1 cells were grown in tissue culture chamber slides (Tissue- Tek) (5 million cells per ml). Cells were fixed in 1 % formaldehyde in PBS containing 2% glucose and 0.02% sodium azide. Certain cells were permeabilized by methanol treatment for 10 minutes. Prior to primary incubation, non-specific binding was blocked with 3% normal goat serum (30 mins). The primary antibodies were diluted in PBS containing 5% BSA, and cells were incubated for one hour. After washing, cells were incubated with goat anti-mouse IgG conjugated to fiuorescein (Tago) for one hour. Slides were rinsed and cover slips applied. Cells were analyzed with epifluorescent microscope. All incubations and washes were done with ice- cold solutions.

The results are as follows: for permeabilized SK-HEP-1 cells treated with 1 1 A8, immunofluorescence was evident in the cytosol and nuclei, with some cells only staining in the nucleus. The immunofluorescence is similar to that for cells treated with AB6. For non-permeabilized SK-HEP-1 cells treated with 1 1 A8, the fluorescent staining is associated only with the plasma membrane; a pattern observed for surface proteins and receptors. Some cells exhibit the typical halo-like appearance seen for receptor- staining. For permeabilized SK-HEP-1 cells treated with AB6, FGF receptor staining in the cytosol and nuclei of many cells was evident. Some cells show staining only in the nucleus. With respect to non-permeabilized SK- HEP-1 cells treated with AB6, no immunofluorescence was observed, indicating that is antibody cannot stain cell-surface FGF receptors. In SK-HEP-1 cells permeabilized by methanol treatment, after binding with MAb 1 1 A8 and a second antibody labeled with fiuorescein, the cytoplasm and nucleus of many cells show the presence of FGF receptor; however, in some cells only the nucleus was positive. When the same cells are again treated with MAb 1 1 A8 but not permeabilized, there was staining of the cell surface that appears as a ring around the cells; this staining is typical of staining of a celi-surface-bound receptor. The immunofluorescence from staining of permeabilized cells with AB6 shows a distribution that is quite similar to that of 1 1 A8, i.e. immunofluorescence is observed in the cytoplasm and nuclei, and some cells show only staining of the nucleus. Thus, these antibodies are indistinguishable in staining the interior of cells. The nonpermeabilized cells, however, show absolutely no staining, indicating, in agreement with previous data, that AB6 cannot recognize FGF receptor when expressed on the cell surface, illustrating a major difference between the two antibodies. In addition, SK-MEL-28 cells were also stained in a similar manner with similar results, though somewhat lesser immunofluorescence intensity. These findings, in conjunction with the specificity in Western blotting, demonstrate that antibody 1 1 A8 recognizes FGF receptor on the cell surface.

EXAMPLE 2 Preparation of the immunotoxin. An immunotoxin was prepared by conjugation of MAb 1 1A8 with saporin. The 1 1 A8 monoclonal antibody was purified by ammonium sulfate precipitation and Affi-Gel Protein A agarose column (Bio Rad) according to the manufacturer's protocol. The purity of the antibody was checked by a 7.5% PhastGel (Pharmacia) with commasie blue stain. The resulting protein was dialyzed against 0.1 M sodium phosphate, 0.1 M sodium chloride, pH 7.5.

Saporin was isolated from the seeds of the plant Saponaria officinalis and purified as described in Lappi et al., Biochem. Biophvs. Res. Commun., 129: 934-942, 1985. Protein concentrations were determined using the BCA protein assay (Pierce, Rockford, IL). A six-fold molar excess of N- succinimidyl-3(pyridildithio)propionate (SPDP) (Pharmacia, Piscataway, NJ) was used to derivatize the antibody according to the manufacturer's instructions. The range of derivatization for saporin was 0.9-1 .1 and for antibody 1 1 A8 was 3.5 to 4. After reducing saporin-SPDP with 0.1 M dithiothreitol, the mixture was passed over Sephadex G25 (Pharmacia) and equilibrated in 10 mM acetic acid, 0.14 M sodium chloride, pH 5.0. A sufficient amount of thiolated saporin was pooled and then added to the SPDP-modified 1 1 A8 antibody (molar excess of thiolated saporin:deriva- tized 1 1A8 = 3:1 ). The mixture was vortexed at room temperature, incubated on a platform agitator for two hours and left overnight at 4°C. The reaction mixture was dialyzed against 0.02 M sodium phosphate, pH 7.0 and applied to a Protein G affinity column (Pharmacia) according to the manufacturer's instructions. A fraction was obtained which contained only antibody-conjugated saporin and free antibody. The peak pool was dialyzed against borate buffered saline (BBS). The dialysate was loaded onto an AffiGel 10 anti-saporin column, thoroughly washed, and the column was eluted with 0.1 M acetic acid. After fractions were analyzed on a 7.5% PhastGel, a pool was made, and it was dialyzed against Dulbecco's PBS. After purification by anti-saporin affinity gel column and Protein G affinity column, no free saporin or free antibody is detected by sodium dodecylsulphate gel electrophoresis.

EXAMPLE 3 Cγtotoxictγ of the Immunotoxin Cell lines which express FGF receptors were treated with the MAb 1 1 A8-saporin immunotoxin, or with saporin alone, or with antibody MAb 1 1 A8 alone, and cytotoxicity was measured using four different assay protocols.

To carry out a thymidine-incorporation inhibition assay, cells in 100 μl of growth media were added to the wells of a 96-well plate at concentrations ranging from 1000 to 5000 cells per well. The number of cells per well plated varies depending on the doubling time of the cells being tested. The following morning, 100 l of samples, diluted in grow,th media, were added in triplicate, and the plates were returned to the incubator for 72 hours. 50 μl of media containing 0.2 μCi [³H]- thymidine (New England Nuclear, Dupont, Wilmington, DE) was added to each well, and the plate was then returned to the incubator for 3 to 6 hours. The plates were frozen at -80°C overnight. The plates were then thawed, and the well contents were collected with a Skatron ceil harvester onto glass fiber filter sheets. The radioactivity incorporated therein was determined by liquid scintillation techniques, and results are expressed as a percent of the count per minute in untreated control wells.

To carry out a MTT cell titer 96 cytotoxicity assay (Promega, Madison WI), the manufacturer's instructions were followed. Briefly, late in the day, cells we^f= plated in 96-well plates at 1000 to 5000 cells per well in 90 μl media. * following morning, 10 μl of samples were added to the wells in triplicaiw and incubated at 37°C. After 72 hours, 1 5 μl of MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added to each well, and the plate was returned to the incubator for four hours. Solubilization solution (100 μl) was added, and after the tetrazolium salt crystals had dissolved, the optical density 560 was read. Results are expressed as percent of the OD 560 of untreated control wells.

A protein synthesis inhibition assay was performed as described by Lappi et al., J. Cell. Phvsiol.. 147: 17-26, 1991 , with the exception that cells were incubated for 72 hours (instead of 48) with the various agents before counting.

Normal T-lymphocytes were isolated from normal blood as described above. Isolated normal T-lymphocytes (2 x 10⁵ cells/well) were incubated with immunotoxin and control samples at 37°C in 5% C0₂ humidified atmosphere for two hours in RPMI-1640 media (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum. At the end of this incubation, the 96-well plates were centrifuged. The cells were washed twice, and the media was replaced with media containing 1 5% fetal bovine serum and 10 μg/ml of the T-cell mitogen phytohemagglutinin (PHA-P, Difco, Detroit, Ml). The plates were returned to the incubator for 72 hours, and mitogen- induced DNA synthesis was determined using the above-described assay for measuring tritiated thymidine incorporation.

The results show that the immunotoxin is potently cytotoxic to these cells SK-MEL-28 and SK-HEP-1 cells, which express FGF receptors. The ED₅₀ for both cell types is approximately 30 pM as measured by inhibition of thymidine incorporation. There is one-thousand-fold increase in cyto^¬ toxicity by conjugation of saporin to the antibody; saporin alone has an ED₅₀ of 30 nM to these cell. Only the cytotoxicity of SAP alone to SK- MEL-28 cells is shown; however, the data for SK-HEP cells is similar. SAP conjugated to AB6 had no inhibitory effect on thymidine incorporation by either of these cell types at any level tested (10 nM to 0.01 nM). These data agree with the immunofluorescence data which shows that AB6 is unable to bind to the cell surface. Determination of the effect of 1 1 A8- SAP on thymidine incorporation by T-lymphocytes showed that that is no significant effect of the immunotoxin on human T- lymphocytes, which have not been reported to express any high affinity FGF receptor. OKT1 - SAP, a well-characterized immunotoxin, Siena, S., et al., Blood. 72: 756- 765, 1 988, which is specifically cytotoxic to T-lymphocytes, was used as a positive control. It is concluded that 1 1 A8-SAP immunotoxin is specific for cells that bear a high affinity FGF receptor. Other cytotoxicity experiments indicate that the immunotoxin has no substantial affinity for FGF receptors expressed on quiescent or proliferating smooth muscle cells.

EXAMPLE 4 Effects of immunotoxins on Dupuytren's cells Cells obtained from surgical removal of tissue from the hand of adult patients diagnosed as having Dupuytren's Contracture, a malady effecting movement of the hand, are placed in primary culture. Such cells are estimated to have between 10,000 and 15,000 basic FGF receptors per cell. The cells are grown overnight in a 24-well tissue culture dish at a concentration of 10,000 cells per well in HEPES-buffered DMEM with 10% FCS. The next morning, the media is removed and replaced with media containing concentrations of the MAb 1 1 A8-SAP immunotoxin ranging from 10^"8 to 10^"12 molar. Controls include wells treated with media only, and wells treated with similar concentrations of MAb 1 1 A8 alone, saporin alone, and MAb 1 1 A8 and saporin together but not conjugated. The cells are returned to the incubator for 72 hours. At the end of this incubation, the cells are washed, removed with trypsin and counted on a Coulter cell counter. The number of cells in the media controls is compared with the number of cells in the treated wells. These cell killing assays show that Dupuytren's cells are sensitive to this immunotoxin.

Pseudomonas exotoxin (PE) is substituted for saporin in the protocol of Example 2, derivatized with SPDP and conjugated with MAb 1 1 A8. The MAb 1 1 A8-PE immunotoxin is employed as described in the above Examples, and similar results are obtained. Deposit of biological material

A hybridoma cell line producing the MAb 1 1 A8 has been deposited on March 17, 1993, with the American Type Culture Collection (ATCC), Rockville, MD under ATCC Accession No. HB-1 1303. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for purposes of patent procedure. Substantially purified MAbs are readily obtained from such a hybridoma cell line in culture by one having the ordinary skill in this art and are generally accepted as having at least about 90% homogeneity of product. This deposit is provided merely as convenience for those of skill in the art, and it is not an admission that a deposit is necessary. A license may be required to make, use, or sell the deposited material, and no such license is hereby granted.

The disclosures of all patents, pending patent applications and literature references cited herein are incorporated by reference. Although the invention has been described with reference to the presently-preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

WHAT IS CLAIMED IS:

1 . Monoclonal antibodies (MAbs) that specifically bind to at least one subtype of native human fibroblast growth factor (FGF) receptor on the surface of intact human cells, but do not bind to human T-lymphocytes, and that, upon binding, are internalized.

2. Monoclonal antibodies that specifically bind to a fibroblast growth factor (FGF) receptor expressed on the surface of human endothelial cells but that do not, bind to fibroblast growth factors expressed on the surface of proliferating human smooth muscle cells, wherein binding is assessed by the ability of the antibody to internalize a linked cytotoxic agent into the receptor bearing cells.

3. Monoclonal antibodies that are produced by a hybridoma cell line having all of the identifying characteristics of the hybridoma cell line 1 1 A8 that is deposited under ATCC Accession No. HB-1 1303.

4. An immunotoxin, comprising a monoclonal antibody of Claim 1 or Claim 2 and a cytotoxic agent.

5. The immunotoxin of Claim 4, wherein the cytotoxic agent is a ribosome-inactivating protein.

6. The immunotoxin of Claim 4, wherein the cytotoxic agent is saporin.

7. A pharmaceutical composition, comprising immunotoxins of any of Claims 4-6 and a pharmaceutical carrier.

8. A method for selectively reducing a population of tumor cells, comprising: treating the cells with an effective amount of immunotoxin of any of claims 4-6.

9. A method of treating an FGF-mediated pathophysiological condition, comprising, administering a therapeutically effective amount of immunotoxin of any of Claims 4-6.

10. The method of Claim 9, wherein the cytotoxic agent is a ribosome- inactivating protein.

1 1 . The method of Claim 10, wherein the cytotoxic agent is saporin.

12. The MAbs of Claim 1 or Claim 2 coupled to a radioisotope label.

13. An immunotoxin, comprising a monoclonal antibody of Claim 1 and claim 2 and a drug.

14. A hybridoma cell line that produces a monoclonal antibody that binds to at least one subtype of human fibroblast growth factor (FGF) high affinity receptors on human intact endothelial cells, but does not bind to FGF receptors present on proliferating human smooth muscle cells, wherein binding is assessed by the ability of the antibody to internalize a linked cytotoxic agent into the FGF receptor bearing cells.

1 5. A process for produces a hybridoma cell line that produces a monoclonal antibody that binds to at least one subtype of human fibroblast growth factor (FGF) high affinity receptors on human intact cells, comprising:

(a) immunizing an animal with intact cells that bear FGF receptors;

(b) preparing hybridomas from the spleen cells from the immunized animals;

(c) selecting hybridomas that secrete antibodies that bind to the extracellular domain of an FGF receptor and that internalize anti-isotype antibody bound to a cytotoxic agent into cells that bear FGF receptors, wherein the anti-isotype antibody is specific for an isotype of the immunized animal, whereby the selected hybridoma produces antibodies that bind native human FGF receptors on intact cells.