WO1994012209A1

WO1994012209A1 - Malignant cell growth regulation by common acute lymphoblastic leukemia antigen

Info

Publication number: WO1994012209A1
Application number: PCT/US1992/010397
Authority: WO
Inventors: Margaret A. Shipp
Original assignee: Dana-Farber Cancer Institute, Inc.
Priority date: 1992-12-02
Filing date: 1992-12-02
Publication date: 1994-06-09

Abstract

A method is provided for treating cancer by administering preparations which increase the activity of the CD10 neutral endopeptidase. The methods of the invention also include inhibiting the growth of cells which are dependent on bombesin-like peptides, by administering CD10/NEP.

Description

MALIGNANT CELL GROWTH REGULATION BY COMMON

ACUTE LYMPHOBLASTIC LEUKEMIA ANTIGEN

Statement as to Federally Sponsored Research Work described herein was supported in part by National Institutes of Health Grants CA49232 and CA55095, and by National Institute on Drug Abuse Grant DA02243. The United States government, therefore, has certain rights in this invention.

The invention relates to the modulation of cell growth, particularly inhibition of malignant cell growth.

Background of the Invention

The common acute lymphoblastic leukemia antigen (CALLA, also referred to as CD10) has been shown to be a 100kd cell surface glycoprotein. It was initially

identified on human lymphoblastic leukemia cells (Ritz et al., Nature (London) 283:583-585, 1980). Later work showed that CD10 is also expressed by other lymphoid malignancies, such as lymphoblastic, Burkitts and nodular poorly

differentiated lymphocytic lymphomas, and by early lymphoid progenitors from fetal liver; fetal, pediatric and adult bone marrow; and fetal and pediatric thymus (Greaves et al., Blood 61:628-639, 1983; Hokland, J. Exp. Med. 152:114-129, 1983; Hokland et al., Blood 64:662-666, 1984; Hoffman-Fezer et al., Leuk . Res . 6:761-767, 1982; Neudorf et al., Leuk . Res . 8:173-179, 1984).

Molecular cloning and expression studies have shown that CD10 (CALLA) is the zinc metalloprotease, neutral endopeptidase 24.11 (NEP, "enkephalinase" (Shipp et al., Nature 347:394-396, 1990; Shipp et al., Proc. Natl . Acad.

Sci USA 86:297, 1989; LeTarte et al., J. Exp. Med. 168:1247, 1988). The three terms CALLA, CD10 and NEP are used

interchangeably herein. This cell membrane-associated enzyme cleaves peptide bonds on the amino side of hydrophobic amino acids and hydrolyzes a number of naturally occurring

peptides including the endogenous opioid pentapeptides Met- and Leu- enkephalin, substance P, neurotensin, oxytocin, bradykinin, angiotensins 1 and 2, atrial natriuretic factor, endothelin, and the chemotactic peptide fMet-Leu-Phe (Shipp et al., Proc. Natl . Acad. Sci USA 86:297, 1989). CD10/NEP, which is expressed on normal lymphoid progenitors, mature polymorphonuclear leukocytes, and a variety of

nonhematopoietic cell types (Greaves et al., Blood 61:628- 639, 1983; Cossman et al., J. Exp. Med. 157:1064-1069, 1983; Metzgar et al., J. Exp. Med. 154:1249-1254, 1981) functions in multiple organ systems to down-regulate induced responses to peptide hormones. For example, Met-enkephalin triggers inflammatory responses by inducing morphological changes, directed migration, and aggregation of CD10/NEP neutrophils; inhibition of CD10/NEP enzymatic activity reduces the amount of Met-enkephalin required for neutrophil activation by several orders of magnitude (Shipp et al., Nature 347:394- 396, 1990; Stephano et al., Proc. Natl . Acad. Sci USA

86:6307-6311, 1987). CD10/NEP, which is expressed at high levels in the lung (Johnson et al., Am. Rev. Resp. Dis.

132:564-568, 1985), similarly regulates responses to

substance P, the primary mediator of neurogenic inflammation of the respiratory tract (Lundberg et al., Proc. Natl . Acad. Sci USA 80:1120-1124, 1983). Stimulation of afferent nerves in the bronchial mucosa triggers substance P release;

resulting substance P-mediated responses include increased vascular permeability, neutrophil migration, bronchospasm, and cough (Lundberg et al., Proc. Natl . Acad. Sci USA

80:1120-1124, 1983). Inhibition of CD10/NEP dramatically increases the binding of substance P to bronchial membranes and the resulting physiological effects (Stimler-Gerard, J. Clin . Invest . 79:1819-1825, 1987; Kohrogi et al., J. Clin . Invest . 82:2063-2068, 1988; Umeno et al., J. Appl . Physiol . 66:2647-2652, 1989; Martins et al., J. Clin . Invest . 85:170- 176, 1990). Summary of the Invention

The present invention relates to a method of altering (reducing or enhancing) stimulation of cellular proliferation by cell-growth-enhancing peptides which are substrates of CD10/NEP, including in particular bombesin- like peptides (BLP). The present invention particularly relates to a method of inhibiting growth or proliferation of BLP-dependent malignant cells. In a preferred embodiment of the invention, a method is provided for treating a patient with cancer. The cancer may be one or more of many types, including carcinomas, sarcomas, and cancers derived from leukocytes or neuroendocrine cells. A particularly

preferred embodiment encompasses treatment of malignant pulmonary neuroendocrine cells (small cell carcinoma), the growth of which has been shown to be regulated in part by CD10/NEP. Small cell carcinoma is a type of pulmonary tumor characterized by small, undifferentiated cells, early metastasis, and poor clinical prognosis. A second

particularly important embodiment of the invention relates to the treatment of B and T cell cancers (leukemias and lymphomas), and of any epithelial cell cancer the growth of which is potentiated by a substrate for CD10. Other cancers which may be advantageously treated with the methods of the invention include those derived from lung, trachea,

bronchioles, bronchi, kidney, intestine, ovary, uterus, prostate, bone, connective tissue, leukemic cells and progenitor cells, spleen, nervous tissue, skin, muscle, liver, breast, esophagus, and spleen, among others. Proliferation of BLP-dependent malignant cells has now been shown to be inhibited by CD10/NEP, and, conversely, to be potentiated by CD10/NEP inhibition. In the present method, BLP-dependent malignant cell proliferation is reduced or inhibited (partially or totally) through the action of CD10/NEP on BPL-dependent malignant cells.

CD10/NEP function can be enhanced by administration of exogenous CD10/NEP, by administration of a therapeutic composition which increases CD10/NEP activity in tissues of the patient, by induction of the endogenous CD10/NEP gene, or by introduction of a vector encoding CD10/NEP (e.g., containing either the CD10/NEP gene or CD10/NEP cDNA) into a cell such that the CD10/NEP enzyme is expressed by the cell. By induction is meant increasing the rate of transcription or the stability of endogenous CD10/NEP mRNA such that more CD10/NEP is produced by the treated cell; this may be accomplished, e.g., by treating the cell with a compound that acts directly on the gene's regulatory DNA sequences, or which acts by signal transduction from the cell surface. In one embodiment, glucocorticoids are administered to the patient to increase endogenous CD10/NEP activity. The present method can be used to inhibit proliferation of BLP- dependent cells, including small-cell carcinoma of the lung, and can also be used as a part of therapeutic treatment for individuals having other medical conditions characterized by neoplastic growth of BLP-dependent cells.

In another embodiment of the invention, CD10/NEP is used to inhibit the proliferation of cells, either in a patient or in vitro, where the cells are stimulated to proliferate by any substrate of CD10/NEP. In addition to bombesin-like peptides which are herein disclosed to be substrates of CD10/NEP, there are likely to be other as yet unidentified substrates of CD10/NEP that are capable of inducing proliferation of normal and malignant cells.

Possible candidates include the known substrates of

CD10/NEP, e.g., the endogenous opioid pentapeptides such as Met- and Leu-enkephalin, as well as substance P,

neurotensin, oxytocin, bradykinin, angiotensins 1 and 2, atrial natriuretic factor, endothelin, and the chemotactic peptide fMet-Leu-Phe.

The methods of the invention also include

determining susceptibility of a patient to the development of cancer, by measuring the activity of CD10/NEP in cells of the patient. Suitable cells for the procedure might

include, for example, cells from the respiratory system, such as those present in sputum, as well as lymphocytes or cells from tissue biopsies.

A further object of the invention is to provide a method for increasing CD10/NEP activity in an animal, by exposing cells of the animal to isolated DNA encoding

CD10/NEP.

The treatment method of the invention may be carried out by administering to a patient with cancer a therapeutic composition containing an effective amount of CD10/NEP (or a composition which increases the level of expression and/or activity of endogenous CD10/NEP) and a pharmaceutically- acceptable carrier or diluent.

The term "patient" used herein is taken to mean any mammalian patient, as veil as any vertebrate animal used for research purposes. Patients specifically intended for treatment with the methods of the invention include humans, nonhuman primates, sheep, horses, cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice, as well as the organs and cells derived or originating from these hosts.

Purified recombinant CD10 may be produced using standard methods as are well known to those skilled in teh art, by expressing a plasmid containing cDNA encoding the CD10/NEP gene sequence, as disclosed in U.S. Patent

4,960,700, which is hereby incorporated by reference.

Surprisingly, active CD10/NEP has been found to be secreted into the cell culture medium by cells which express the CD10/NEP gene. The expressed CD10/NEP can be purified by standard methods, such as by affinity chromatography with anti-CD10/NEP antibodies such as the monoclonal antibody J5, which is available from Coulter, Inc.

Any appropriate, medically acceptable route of administration of CD10/NEP may be used, specifically

encompassing both administration via the gastrointestinal tract, and all routes of parenteral administration. Routes of parenteral administration include intravenous,

intraarterial, subcutaneous, intraperitoneal, intramuscular, intraventricular, intraepidural, or others, as well as nasal, ophthalmic, rectal, or topical. A preferred means of administration is by inhalation, such as by breathing a nebulized or aerosolized suspension of the compounds of the invention.

Compounds for use in practicing the invention may be formulated as sterile solutions with appropriate additions to make pharmaceutically acceptable preparations.

Components of the solution might include buffers, salts, preservatives, antioxidants, osmotic agents, fibrinolytic compounds, or complementary therapeutic agents. The

compounds may be incorporated into liposomes or other drug delivery systems for efficient delivery to the target site.

Other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims. Detailed Description

The drawings are first described.

Fig. 1 is a graphic representation showing that CD10/NEP inhibition increases thymidine incorporation of small cell carcinoma (SCCa) cell lines. Fig. 1a shows thymidine incorporation of SCCa cell lines (NCI H345 and H146) cultured with 10% FCS, no additives or GRP 1-27 (200 nM) in the presence (solid bars) or absence (open bars) of 10 μM phosphoramidon. Fig. 1b shows thymidine incorporation of SCCa cell line NCI H345 cultured with thiorphan (100 μM, stippled bar), captropril (100 μM, hatched bar) or no enzymatic inhibitors (open bar).

Fig. 2 is a graphic representation showing that SCCa colony formation in soft agar is potentiated by CD10/NEP inhibition. The numbers of SCCa colonies obtained when NCI H209 or NCI H146 cells were incubated without additives, with bombesin (50 nM) or with phosphoramidon (10 μM) are shown. Colony sizes (20-30 cells, open bars; 30-100 cells, stippled bars; or > 100 cells/colony, solid bars) are indicated.

Fig. 3 is a graphic representation showing that CD10/NEP inhibits thymidine incorporation by SCCa cell lines. Fig. 3a shows ³H-TdR incorporation following the addition of soluble CD10/NEP. Purified soluble CD10/NEP that was enzymatically active or boiled to destroy enzymatic activity was added at the indicated concentrations to unstimulated or GRP 14-27 stimulated NCI H345 cells.

Thymidine incorporation of NCI H345 cells cultured with active CD10/NEP was compared to that of NCI H345 cells cultured with inactive (boiled) CD10/NEP, and the percent reduction in thymidine incorporation resulting from the addition of active CD10/NEP is displayed. Fig. 3b shows coculture of NCI H345 cells with CD10/NEP⁺ murine L cells. ³H-TdR incorporation (cpm x 10^-3) of murine-L-cell-vector- only transfectants (stippled bar), murine L-cell-vector-only transfectants + NCI H345 cells (solid above stippled bar), murine L cell CD10/NEP transfectants (hatched bar), and murine L cell CD10/NEP transfectants + NCI H345 cells (solid above hatched bar) are shown.

The present invention is based on the demonstration that BLP are CD10/NEP substrates and that CD10/NEP is involved in regulation of BLP-dependent cell growth, particularly in regulation of malignant cell growth. As described herein, it has been shown that malignant pulmonary neuroendocrine cells express low levels of the cell surface metalloendopeptidase CD10/NEP and that the growth of BLP- dependent SCCa cells is inhibited by CD10/NEP and

potentiated by CD10/NEP inhibition. CD10/NEP has been shown to hydrolyze BLP and to rapidly inactivate them by cleaving them at two sites within the seven amino acid conserved carboxyl terminus. As also described herein, exogenously added CD10/NEP has been shown to inhibit SCCa growth. Thus, CD10/NEP has been shown to be involved in the regulation of BLP-dependent cell growth and, particularly, in regulation of tumor cell proliferation. As a result, it is now

possible to regulate proliferation of BLP-dependent cells, especially BLP-dependent tumor cells, such as SCCa cells.

The present invention relates, therefore, to a method of enhancing or reducing BLP-dependent cell growth, and particularly to a method of reducing or inhibiting growth of malignant (or tumor) cell growth, such as SCCa growth or the growth of neoplastic leukemia cells. It also relates to compositions useful in the present method.

In the method disclosed herein for reducing

malignant cell growth, increased CD10/NEP activity

inactivates BLP expressed by the malignant cells, thereby leaving a lower level of BLP available to the cells. This results in decreased cellular proliferation compared to the proliferation observed in the presence of active BLP. BLP- dependent malignant cells are contacted with CD10/NEP in sufficient quantity to cleave, and thereby inactivate

(partially or totally) BLP which are essential growth factors for the cells, including those BLP expressed by the malignant cells themselves. Suitable tumors which may be treated with the method of the invention include carcinomas, adenocarcinomas, sarcomas, adenosarcomas, leukemias, and lymphomas. Specific tumor types which may be beneficially treated with these methods include uterine adenosarcoma and adenosarcoma, nephroblastoma, rhabdomyosarcoma,

neuroblastoma, Ewing's sarcoma, leiomyosarcoma, Triton tumor, fibrosarcoma, synovial sarcoma, giant cell tumor of tendon sheath, malignant fibrous histiocytoma, osteosarcoma, T cell acute lymphoblastic leukemia, Burkitt's lymphoma, follicular lymphoma, B cell lymphoblastic leukemia, and intestinal and mammary adenocarcinoma.

The CD10/NEP activity at a tumor site can be increased by a variety of means. For example, endogenous CD10/NEP activity can be enhanced (e.g., by treatment with glucocorticoids), exogenous (e.g., recombinant) CD10/NEP can be administered, or cells (such as genetically engineered cells) expressing CD10/NEP can be introduced into an

individual. Εxogenous CD10/NEP can be obtained from sources in which it occurs naturally or can be synthesized using known methods, such as by cells (e.g., bacterial, yeast, or mammalian cells) engineered to contain and express DNA or RNA encoding CD10/NEP. It is not necessary that the entire CD10/NEP molecule be used in the present method; a fragment of CD10/NEP possessing the BLP-inactivating activity of CD10/NEP can be used for this purpose. Such a fragment can be easily generated by standard genetic engineering methods, and tested for activity in, for example, the BLP-cleaving assay described below.

The dosage of purified CD10/NEP to be given to a patient will be from about 50 μg to about 50 g daily for an adult human patient. This dosage may also be administered more than once per day, and may be delivered continuously, as via implant or IV infusion. The amount (dose) of

CD10/NEP needed to inactivate endogenous BLP, thereby inhibiting tumor cell growth, will vary depending on the extent to which the malignant cells have grown, the size of the individual to whom it is given; the degree to which the dose can be targeted to the site of the tumor (e.g., by inhalation of the therapeutic for treatment of SSCa) as opposed to distributed throughout the blood and/or tissues of the patient; the half-life of the therapeutic in vivo; the route and speed of metabolism of administered CD10/NEP in a particular patient; and such other factors as are routinely taken into account by those of ordinary skill in the art of pharmacology when determining the optimal dosage regimen for a given pharmaceutical.

As described briefly in the next sections and in detail in the examples which follow, the cleavage sites of CD10/NEP substrates and the K_is of the substrates have been determined; immunohistochemical analysis of CD10/NEP was performed; enzymatic activity of CD10/NEP was analyzed; and the impact of CD10/NEP activity upon SCCa cell lines has been defined. As a result of the work described herein, it has been shown that CD10/NEP rapidly inactivates BLP family members by cleaving the peptides at two sites within the seven amino acid conserved carboxyl terminus required for biological activity, and that growth of BLP-dependent SCCa cell lines is potentiated by CD10/NEP inactivation and inhibited by CD10/NEP addition. These data suggest that an important mechanism for controlling cell growth is the catabolism of essential autocrine growth factors by cell surface enzymes such as CD10/NEP. CD10/NEP limits the proliferation of malignant pulmonary neuroendocrine cells which produce and respond to BLP. Therefore, in this context, the enzyme functions as a tumor suppressor, the loss of which may facilitate the development of small-cell carcinoma of the lung, as well as other BLP-stimulated tumors. This finding that CD10/NEP inactivates BLP and thereby inhibits the growth of BLP-sensitive cells suggests that normal proliferation of any BLP-stimulated cell type is modulated by the presence of CD10/NEP on the surface of the cells, and further indicates that increasing CD10/NEP activity in the vicinity of a BLP-stimulated cell, including a tumor cell, will inhibit proliferation of that cell.

As described in Example 1, the cleavage sites of BLP, bombesin, gastrin-releasing peptide (GRP) and GRP 14- 27, were determined by incubation of these proteins with purified CD10/NEP enzyme, under conditions previously determined to be optimal for hydrolysis of known CD10/NEP substrates. HPLC analysis of the reaction products

identified two peptide bonds in the conserved 7 amino acid carboxyl terminus region as the CD10/NEP cleavage sites. Ki values for all proteins were determined as well (Example 1).

In other cellular systems in which CD10/NEP has been shown to have a physiological role, the cells that respond to a CD10/NEP peptide substrate express both the peptide receptor and the cell surface enzyme. Since BLP are (as disclosed herein) CD10/NEP substrates, and pulmonary since neuroendocrine cells produce and respond to BLP, these cells were evaluated for CD10/NEP expression using an anti-CD10 monoclonal antibody, J5 (see Example 2). Immunohistochemical staining of sections of fetal lung indicates that CD10/NEP is expressed by epithelial cells in both large and small airways.

Bombesin-like peptides are known to stimulate the growth of small cell carcinomas (SCCa) of the lung (Cuttita et al., Nature 316:823-826, 1985). Antibodies to BLP completely inhibit SCCa colony formation and tumorigenesis in nude mice, indicating that the bombesin autocrine loop is essential for the growth of these cells (Cuttita et al., Nature 316:823-826, 1985). Clinical studies suggest that BLP may also regulate SCCa growth in patients. The levels of circulating BLP detected with a sensitive

radioimmunoassay correlate with tumor burden in patients with SCCa of the lung and the levels of BLP detected in cerebrospinal fluid accurately predict leptomeningeal involvement with SCCa (Hoist et al., J. Clin . Oncol . 7:1831- 1838, 1989; Pederson et al., J. Clin . Oncol . 4:1620-1627, 1986).

Example 3 discusses enzymatic activity experiments performed on five SCCa cell lines, two CD10⁺ lines and one CD10- line. The cell lines were examined for GRP

transcription, cell surface CD10/NEP enzymatic activity, and CD10/NEP transcription. Reverse PCR products from all five SCCa cell lines contained CD10/NEP cDNA; all five SCCa lines also had detectable CD10/NEP enzymatic activity. Four of the five SCCa lines (NCF, H209, H345, H416 and H69) showed detectable GRP transcripts in the reverse PCR products;

these four were chosen for further analysis.

Culture of these SCCa cell lines in the presence of the CD10/NEP inhibitor phosphoramidon caused an increase in levels of thymidine incorporation compared to levels

observed in the controls, as did the presence of thiorphan, which inhibits both CD10/NEP and peptidyldipeptase. In contrast, incubation with captopril, which inhibits

peptidyldipeptase but is inactive against CD10/NEP, resulted in thymidine incorporation that was comparable to the controls (see Example 4). These experiments suggest that the potentiation of SCCa growth is a specific consequence of cell surface CD10/NEP inhibition.

Example 5 describes colony formation in soft agar and the effects of the CD10/NEP inhibitor phosphoramidon on the number and size of colonies of SCCa cells. SCCa colony formation increased significantly when phosphoramidon was added to cell cultures of NCI H209, H345, H69 and H146.

Further, addition of phosphoramidon reverses the inhibitory effect of the anti-bombesin monoclonal antibody 2A11 on SCCa colony formation, thus providing indirect evidence that CD10/NEP modulates BLP levels.

In additional experiments (Example 6), SCCa cell lines were cultured in the presence of specific

concentrations of purified soluble CD10/NEP (Jackson et al., J. Biol. Chem. 261:8649-8654, 1986) that was enzymatically active or previously boiled to destroy enzymatic activity, and ³H-thymidine incorporation by cells cultured with active CD10/NEP was compared to that of cells cultured with

inactive (boiled) CD10/NEP. The soluble active CD10/NEP inhibited thymidine incorporation in a dose-dependent manner. Also, co-culturing SCCa cells (NCI H345 cell line) with murine L cells transfected with pZipneo CD10/NEP led to inhibition of thymidine incorporation by the SCCa cells.

These results indicate that the incorporated vector

expressed active CD10/NEP, which in turn inhibited SCCa growth.

Example 7 provides an example of a protocol for treating a patient with small cell carcinoma using the methods of the invention. Example 8 details the use of CD10/NEP activity measurement for diagnosing susceptibility to cancer

development.

Example 9 teaches how to determine if a tumor is BLP-dependent.

The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way.

Example 1. Hydrolysis of BLP and Ki Determination

Bombesin, GRP and GRP 14-27 were obtained from

Peninsula Labs (San Carlos, CA) and CD10/NEP was purified as previously described (Jackson et al., J. Biol . Chem.

261:8649-8654, 1986).

The BLP, bombesin, GRP and GRP 14-27' were each incubated with purified CD10/NEP enzyme under conditions previously determined to be optimal for hydrolysis of known CD10/NEP substrates. Four hundred (400) pmole of peptide were incubated with 1 pmole of CD10/NEP in 100 λ of 20 mM MOPS, pH 6.5 at 37°C for 60 min. Reaction products were subsequently analyzed on a Hewlett Packard HPLC equipped with a 4.6 x 250 Browerlee RP300 column eluted at 1 ml/min with a 3%/min gradient of 0.1% aqueous trifluoracetic acid and 0.1% trifluoroacetic acid in acetonitrile.

HPLC analysis of the reaction products indicated that bombesin, GRP and GRP 14-27 were efficiently hydrolyzed by CD10/NEP. To identify these products, appropriate peptide peaks from each digest were collected and subjected to amino acid analysis using PLTC as previously described (Tarr, "Manual Edman Sequencing System", in Methods of

Protein Microcharacterization, Ed. J.E.Shively, The Humana Press,, Inc., Clifton, NJ, 1986).

The amino acid composition of the respective peptide fragments from each reaction mixture identified as CD10/NEP cleavage sites the Trp-Ala and His-Leu peptide bonds in the conserved 7 amino acid carboxyl terminus region required for BLP biologic activity (Table 1).

Bombesin GRP-(1-27), and GRP-(14-27) were hydrolyzed by CD10/NEP at the indicated alanine and leucine residues (1) within the conserved 7-amino acid C terminus required for biologic activity (boldface letters). The K_i values of bombesin, GRP, and GRP-(14-27) were determined by using the BLP as alternate substrate inhibitors of a reference peptide (dansyl-D-Ala-Gly- [NO₂]-Phe-Gly). Estisates for V_max based on the HPLC analyses and utilized to calculate K_cat values were 777.4, 22.8, and 388.6 nmol per min per mg of enzyme for bombesin. GRP and GRP-(14-27), respectively.

Although each BLP was efficiently hydrolyzed by CD10/NEP, GRP- (14-27) was inactivated by 10 times less enzyme than required to inactivate GRP or bombesin (data not shown). These results are of interest because GRP-(14-27) is as potent a mitogen as GRP-(1-27) at 10 times lower molar concentrations (Zacheray et al., Dev. Biol. 124:295-308, 1987), and a shorter GRP fragment [GRP-(18-27), neuromedin C (Spindle et al., Proc. Natl. Acad. Sci. 81:5699-5703, 1984; Spindle et al., Proc. Natl. Acad. Sci. 83:19-23, 1983;

Sausville et al., J. Biol. Chem. 261:2451-2457, 1986)], mediates pulmonary BLP effects. The K_i values of GRP-(14- 27), GRP-(1-27), and bombesin were determined by using these peptides as inhibitors of CD10/NEP hydrolysis of a reference peptide (dansyl-D-Ala-Gly-[NO₂ ]-Phe-Gly) (Cleland, Meth. Enzymol. 63:103-138, 1979) (Table 1). Estimates for K_cat for each BLP based on the HPLC analyses are also included in Table 1.

K_i values were obtained from Dixon plots using dansyl-D-Ala-Gly-[NO2]-Phe-Gly at a fixed concentration of 25 μM and an inhibitor range of 0-100 μM for GRP and GRP 14- 27, and 0-400 μM for bombesin. Data were fit to a weighted least squares program (Cleland, Methods in Enz . 63:103-138, 1979). K_i values were calculated from the relationship: Ki = KK_(obs) + Ki (1 + [S]/Km) where Ki_(obs) is the

experimentally observed value and [S] is the substrate concentration (25 μM). The observed 18 +3 - 117 + 2 μM Ki values (Table 1) are comparable to the 20-80 μM Kis of other peptides such as metenkephalin and leu-enkephalin that are efficiently hydrolyzed by CD10/NEP. Km was independently determined to be 87 μM.

Example 2. Immunohistoohemical Analysis of CD10/NEP

Immunohistochemical staining of serial sections of fetal lung was performed as previously described (Sunday, Meth . Neurosci . 5:123-136, 1991) using a rabbit anti- bombesin antiserum (Sunday, Meth . Neurosci . 5:123-136,

1991), an unreactive control monoclonal antibody, MOPC 21 (Sigma Chemical Co., St. Louis, MO), and the anti-CD10 MAb, J5 (Greaves et al., Blood 61:628-639, 1983; available from Coulter, Inc.). Colonies were photographed using a Zeiss Axiophot with a 20x objective. Fetal lung (12 weeks

gestation and 18 weeks gestation) was stained with the anti- CD10 monoclonal antibody, J5 preincubated with CD10/NEP, the unreactive control monoclonal antibody, MOPC 21, or the polyclonal rabbit anti-bombesin antiserum.

The results of these experiments showed that CD10/NEP is expressed by epithelial cells in both small and large airways in fetal lung. The specificity of J5

immunoreactivity for CD10/NEP was determined both by

examining the staining pattern of J5 monoclonal antibody preincubated (2 ml of 1:500 malignant ascites) with purified CD10/NEP (100 mg) prior to immunostaining and by comparing staining of the same tissue with an irrelevant control monoclonal antibody. Pulmonary neuroendocrine cells were identified in thin serial sections from fetal lung by location, morphology and expression of BLP. Neuroendocrine cells that were present in serial sections of the same fetal lung expressed CD10/NEP as did additional cells within the bronchial epithelial cell layer (Johnson et al., Am. Rev. Respir. Dis . 132:564-568, 1985). Similar results were obtained in adult lung.

Example 3. CD10/NEP Enzymatic Activity

Whole cell suspensions of the SCCa cell lines NCI- H345, H209, H146, H82, and H69, the CD10⁺ acute

lymphoblastic leukemia cell lines Nalm6 and Laz 221, and the CD10- T cell leukemia cell line J77 were evaluated for cell surface CD10/NEP enzymatic activity using a previously described sensitive fluorometric assay (Table 2) (Shipp et al., Proc. Natl . Acad. Sci . USA 86:297-301, 1989).

Values represent the mean of triplicate samples of

phosphoramidon-inhibitable CD10/NEP enzymatic activity.

SCCa cell lines were obtained from A. Gazdar, NCI, Bethesda, MD. Previous studies indicated that NCI H209 secretes high, H345 and H69 intermediate, H146 low, and H82 undetectable levels of BLP (Cuttita et al. Nature 316:823-826, 1985) and that NCI H345 expresses higher numbers of BLP receptors than the other SCCa cell lines (Moody et al., Life Sci . 37:105- 113, 1985). In previous studies, SCCa cell lines including NCI H345, H209, H146 and H69 responded to exogenous and autocrine BLP stimulation with increased thymidine

incorporation and colony formation in soft agar, and

increased tumor growth in a nude mouse model (Cuttita et al., Nature 316:823-826, 1985; Weber et al., J. Clin .

Invest . 75:306-309, 1985; Carney et al., Cancer Res . 47:

821-825, 1987). GRP transcripts were analyzed in these SCCa cell lines by performing reverse PCR using oligonucleotide probes derived from the human GRP cDNA sequence (5' sense, bp 125-150, and 3' antisense, bp 492-497) (Spindel et al., Proc. Natl . Acad. Sci USA 81:5699-5703, 1984). Reverse PCR products from four of the five SCCa cell lines contained the appropriately sized 372 bp GRP cDNA fragment. The only cell line which did not have detectable GRP transcripts (NCI H82) was one that had been unresponsive to BLP in earlier

functional assays (Cuttita et al., Nature 316:823-826,

1985).

All five SCCa lines had low but detectable levels of CD10/NEP enzymatic activity (123-906 nmol per hr per 10⁶ cells) that were less than 3-30% of the levels of known CD10/NEP⁺ controls (Laz 221 and Nalm-6) (Table 2). In contrast, CD10/NEP- cells (such as J77) had virtually no detectable CD10/NEP enzymatic activity (<17 nmol per hr per 10⁶ cells). We also confirmed that the SCCa cell lines transcribed CD10/NEP by performing reverse PCR using

oligonucleotide probes derived from the human CD10/NEP sequence (SEQ ID NO:1; disclosed as Fig. 1 of U.S. Patent 4,960,700). The specific oligonucleotide sequences are specified by nucleotides 1185 to 1209 (5' sense) and 1571 to 1596 (3' antisense) of SEQ ID NO:l, respectively. [The same sequences are shown by Shipp et al., Proc. Natl. Acad. Sci. 85: 4819-4823, 1988: 5' sense (bp 1200-1224) and 3' antisense (bp 1586-1611)]. Reverse PCR products from each of the five SCCa cell lines contained the appropriately sized 411-bp CD10/NEP cDNA fragment.

Example 4. Analysis of SCCa Proliferation

To determine whether inhibition of cell surface CD10/NEP enzymatic activity affected growth, the SCCa cell lines were cultured for 48 h in serum-free Hites medium

(Cuttita et al., Nature 316:823-826, 1985) with no addition, 10% fetal calf serum, or GRP 1-27 (200 μM) in the presence or absence of phosphoramidon (10 μM), thiorphan (10 μM), or captopril (10 μM), and subsequently analyzed for ³H- thymidine incorporation. In representative experiments, shown in Fig. 1A, NCI H345 and H146 cells incubated with GRP incorporated, respectively, 834% and 50% more thymidine than unstimulated cells. Preincubation with the specific

CD10/NEP inhibitor phosphoramidon (N-[α-L- rhamnopyranosyloxyhydroxy-phosphinyl]-L-leucyl-L-tryptophan (Matsas et al., Biochem. J. 223:433-440, 1984) significantly increased thymidine incorporation by unstimulated SCCa and/or BLP-stimulated SCCa. For example, NCI H345 cells incubated with phosphoramidon alone incorporated 350% more thymidine than untreated cells (p = 0.01) and NCI H146 cells incubated with phosphoramidon incorporated 72% more

thymidine than untreated cells (p = 0.05). NCI H345 and H146 cells stimulated with exogenous GRP also incorporated more thymidine following addition of phosphoramidon.

To demonstrate that the phosphoramidon effects were a specific consequence of CD10/NEP inhibition, SCCa cell lines were cultured with one of two additional chemically related enzymatic inhibitors, thiorphan (N-[D,L-2-benzyl-3- mercaptopropionyl] glycine (Matsas et al. Biochem . J.

223:433-440, 1984)) or captopril (D-3-mercaptol-2- methylpropenyl-L-proline (Matsas et al. Biochea J. 223:433- 440, 1984)). Thiorphan inhibits both CD10/NEP and peptidyl dipeptidase, whereas captopril inhibits peptidyl dipeptidase but is inactive against CD10/NEP (Matsas et al. Biochem . J. 223:433-440, 1984). As indicated in Fig. 1b, SCCa cells (NCI H345) cultured with thiorphan (100 μM) had

significantly higher levels of thymidine incorporation than cells cultured with no enzymatic inhibitor (p <0.001), whereas cells cultured with captopril (100 μM) had similar levels of thymidine incorporation as cells cultured with no inhibiter (p = 0.145). That SCCa growth was potentiated by phosphoramidon or thiorphan but not captopril (Figs, la and 1b) suggests that these effects are a specific consequence of cell surface CD10/NEP inhibition. Values shown are the mean ± standard deviation of triplicate samples in a

representative experiment. The differences in ³H-TdR incorporation of NCI H345 or NCI H146 cells cultured in the presence or absence of enzymatic inhibitors were evaluated using a one-sided students t test. P values are as follows: NCI H345 + phosphoramidon, no addition, p = 0.011; + GRP 1- 27, p - 0.1; NCI H146 + phosphoramidon, no addition, p = 0.05; + bombesin p = 0.002; + GRP, p - 0.006; NCI H345, no addition, + thiorphan, p <0.001; ± captopril, p = 0.145.

Example 5. Colony Formation in Soft Agar

The most sensitive assay for analyzing the effects of BLP on SCCa cell growth is colony formation in soft agar (Cuttica et al., Nature 316:823-826, 1985; Carney et al., Cancer Res. 47:823-826, 1987). Colony formation by SCCa cells is markedly enhanced by the addition of exogenous BLP (Cuttica et al., Nature 316:823-826, 1985; Carney et al., Cancer Res. 47:823-826, 1987; Fig. 2) and completely inhibited by the addition of anti-bombesin monoclonal antibodies (Cuttica et al., Nature 316:823-826, 1985).

SCCa colony forming assays were performed as previously described using 5 x 10⁴ cells per 30 mm well in serum-free Hites medium (Cuttita et al., Nature 316:823-826, 1985) containing no additives, 10% fetal calf serum,

phosphoramidon (10 μM) or bombesin (50 nM). In NCI H416 colony forming assays, 0.1% BSA was added to the top agar to enhance cloning efficiency as previously described. To prepare the wells for counting of colonies, agar samples were dehydrated, methanol fixed and stained with Gill's hematoxylin solution. As indicated in Fig. 2, SCCa colony formation was significantly increased when cells were plated with the CD10/NEP inhibitor phosphoramidon. In a

representative experiment, NCI H209 cells plated without additives formed 34 colonies of 30-100 cells each and one colony of >100 cells each per well, whereas NCI H209 cells plated with phosphoramidon formed 114 colonies of 30-100 cells each and 41 colonies of >100 cells each per well (p = 0.002, p < 0.001, respectively; p = 0.137 for 20-30 cell colonies). There was also increased colony formation by each of the other SCCa cell lines (NCI H345, H69 and H146) when plated in the presence of phosphoramidon. In a

representative experiment, inhibition of NCI H146 CD10/NEP increased the number of 20-30 cell and 30-100 cell colonies by 4.6x (p = 0.005) and 4.3x (p = 0.003), respectively. Of note, more NCI H209 and H146 SCCa colonies were formed when cell surface CD10/NEP was inhibited than when exogenous BLP was added to the SCCa cells. Values shown are the mean ± standard deviation of samples in a representative

experiment. The differences in NCI H209 and NCI H146 colony formation in the presence or absence of phosphoramidon were evaluated using a one-sided students t test. Indirect evidence that CD10/NEP functions by

modulating BLP levels was obtained by analyzing the number of SCCa colonies formed in the presence or absence of an antibombesin monoclonal antibody (2A11), or by treatment with a bombesin receptor antagonist. As previously reported (Cuttita et al., Nature 316:823-826, 1985), 2A11 inhibits SCCa colony formation and exogenously added bombesin

reverses the inhibitory effect of 2All or SCCa colony formation. Phosphoramidon similarly reverses the inhibitory effect of 2All on SCCa colony formation.

Indirect evidence that CD10/NEP functions primarily by modulating BLP levels was obtained by treating SCCa cells with phosphoramidon in the presence or absence of the bombesin receptor antagonist [¹³ψ¹⁴, CH₂NH]bombesin (Coy et al., J. Biol. Chem. 264:14691-14697, 1989). As shown in Table 3, this bombesin receptor antagonist reverses the increase in NCI H345 SCCa colony formation resulting from the addition of either phosphoramidon or bombesin.

+

Number of colonies (>20 cells per colony) obtained when NCI H209 cells were incubated without addition, with [^{1 3}

¹⁴, CH₂NH]bombesin at 50 nM, bombesin at 50 nM, bombesin + [¹

¹⁴, CH₂NH] bonbesin, phosphor amidon at 50 nM, or phosphor amidon + [¹

¹⁴, CH ₂NH] -bombesin are shown as are

percentage changes in number of colonies formed.

Example 6. Exogenous CD10/NEP Addition and Co-Culture

Experiments

Since BLP are CD10/NEP substrates and SCCa secrete the autocrine growth factor and express low levels of the cell surface enzyme, the effect of exogenously added

CD10/NEP on SCCa cell growth was evaluated.

Purified soluble CD10/NEP (Bensch et al., Cancer 22:1163-1172, 1968; Jackson et al., J. Biol. Chem. 261:8649- 8654, 1986) that was (a) enzymatically active or (b) boiled to destroy enzymatic activity was added at the indicated concentrations to unstimulated or GRP 14-27 stimulated NCI H345 cells. Thymidine incorporation of NCI H345 cells cultured with active CD10/NEP was compared to that of NCI H345 cells cultured with inactive (boiled) CD10/NEP (onesided Student's t test) and the percent reduction in thymidine incorporation resulting from the addition of active CD10/NEP (1 μg - 5239 nmol/hr enzymatic activity) displayed. Actual values for ³H-TdR incorporation of NCI H345 cultured with the indicated concentration of active or boiled enzyme (active/boiled x 10^-4) are as follows: No additives 1 μg (4.67 ± 1.09/8.13 ± 0.6, p = 0.004); 0.5 μg (5.69 ± 0.4/7.04 ± 0.5, p = 0.01); 0.1 μg (5.55 ± 0.15/6.47 ± 0.83, p not done); 0.01 μg (7.3 ± .35/6.93 ± 0.62, p not done); GRP 14-27 1 μg (5.51 + 0.23/10.22 ±0.26, p <0.001); 0.5 μg (6.69 ± 0.49/9.74 ± 0.71 p - 0.001); 0.1 μg (7.15 ± 0.57/9.49 ± 0.13, p not done); 0.01 μg (9.06 ± 1.15/9.01 ± 0.92, p not done). Values are the mean ± standard deviation of triplicate samples in a representative experiment. This illustrates that soluble active CD10/NEP enzyme inhibits thymidine incorporation of unstimulated or BLP-stimulated SCCa cells in a dose-dependent manner.

Since CD10/NEP normally functions as a membrane- bound cell surface endopeptidase, the next step was to determine the effects of cell surface CD10/NEP enzyme on SCCa growth. It was not possible to transfect SCCa directly with CD10/NEP. For this reason, cells of the SCCa cell line NCI H345 were co-cultured with murine L cells transfected with CD10/NEP. Murine L cells were transfected with pZipneo (Pfeifer et al., Proc. Natl . Acad. Sci . USA 86:10075-10079, 1989) or pZipneo CD10/NEP, selected for G418 resistance, and analyzed for CD10/NEP enzymatic activity. Irradiated L cells were plated at 20,000/well in 96 well plates, and NCI H345 cells (10,000/well) were added to the L cell monolayer 24 hr later in serum-free Hites medium supplemented with ³H- TdR. Thymidine incorporation was determined 16 hr

thereafter. As L cells are thymidine kinase negative, the transfected adherent L cells themselves incorporated low levels of exogenous thymidine (Fig. 3b). L cells transfected with vector alone had virtually undetectable CD10/NEP enzymatic activity (<25 nmol/10⁶ cells/hr) whereas L cells transfected with CD10/NEP had 1360 nmoles/hr/10⁶ cells of cell surface enzyme. In a representative

experiment, NCI H345 cells co-cultured with the CD10/NEP⁺ L cell transfectants incorporated 60% less thymidine than NCI H345 cells co-cultured with vector-only L cell transfectants (p = 0.018) Fig. 3b). Values are the mean ± standard deviation of triplicate samples in a representative

experiment. The differences in the ³H-TdR incorporation of NCI H345 cells cultured in the presence of L cells

transfected with vector only versus incorporation by H345 cells cultured in the presence of L cells transfected with CD10/NEP were determined using a one-sided Student's t test. Example 7. Treatment of SSCa with CD10/NEP

In an hypothetical example illustrating the

therapeutic method of the invention, an adult male patient with a history of smoking a pack of cigarettes daily for the last ten years presents with a 2.5 cm nodule at the

periphery of the right inferior lobe, as shown by computed tomography. A percutaneous transthoracic aspiration needle biopsy of the lesion indicates that the nodule is a small cell carcinoma. The patient, who weighs 150 pounds (68 kg) is given 50 mg CD10/NEP per day for 14 days, administered in sterile normal saline as a nebulized inhalant preparation. Changes in the size of the tumor can monitored by computed tomography scan.

Example 8. Diagnosis of Cancer Susceptibility

In a hypothetical example illustrating the

diagnostic method of the invention, a patient with a history of smoking, and a family history of lung cancer, is

evaluated for risk of developing pulmonary neoplasms. A sputum sample from the patient is collected, and bronchial epithelial cells derived from the sputum sample are analyzed for CD10/NEP activity using an immunoperoxidase assay (as described above in Example 3). The patient's cells are found to express CD10/NEP activity of 500 nmol per hour per lθ⁵ cells. Since normal bronachial epithelial cells

typically express CD10/NEP activity of 2000 or more nmol per hour per 10⁵ cells, the patient expresses an abnormally low activity of CD10/NEP, and is at incresed risk for developing lung cancer.

Example 9. Characterisation of Tumor as BLP- Responsive

The following is a hypothetical example illustrating how the BLP-depence of a given tumor would be determined. A metastatic tumor is removed from a patient with small cell carcinoma. To determine whether the tumor expresses

bombesin-like peptide receptors and, therefore, is likely to respond with increased proliferation to bombesin

stimulation, a simple radioimmunoassay is performed to evaluate the binding of iodinated bombesin to a putative BLP receptor-positive tumor cell. The tumor cells show greatly increased ¹³¹I binding as compared to control cells which are known not to respond to BLP. The patient is

subsequently treated with CD10/NEP, to which she responds with tumor regression and remission.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the

invention described herein. Such equivalents are intended to be encompassed by the following claims.

What is claimed is:

Claims

CLAIMS 1. A method of treating a patient with cancer comprising administering an effective amount of therapeutic composition comprising CD10/NEP.

2. The method of claim 1, wherein the therapeutic composition is administered parenterally.

3. The method of claim 2, wherein the therapeutic composition is administered as an inhalant aerosolized preparation.

4. The method of claim 1, wherein the cancer is a carcinoma.

5. The method of claim 4, wherein the cancer is a pulmonary carcinoma.

6. The method of claim 4, wherein the cancer is a small cell carcinoma.

7. The method of claim 4, wherein the cancer is derived from neuroendocrine cells.

8. The method of claim 1, wherein the cancer comprises neoplastic leukocytes.

9. The method of claim 1, wherein the cancer is a sarcoma.

10. The method of claim 1, wherein the cancer is uterine adenosarcoma and adenosarcoma, nephroblastoma, rhabdomyosarcoma, neuroblastoma, Ewing's sarcoma,

leiomyosarcoma, Triton tumor, fibrosarcoma, synovial sarcoma, giant cell tumor of tendon sheath, malignant fibrous histiocytoma, osteosarcoma, T cell acute

lymphoblastic leukemia, Burkitt's lymphoma, follicular lymphoma, B cell lymphoblastic leukemia, and intestinal and mammary adenocarcinoma.

11. The method of claim 1, wherein the patient is human.

12. The method of claim 1, wherein the cancer comprises cells the growth of which is stimulated by a substrate for CD10.

13. The method of claim 12, wherein said substrate is a bombesin-like peptide.

14. A method of treating a patient with cancer, comprising administering a therapeutic composition which increases CD10/NEP activity in said patient.

15. The method of claim 14, wherein said CD10/NEP activity is increased by induction of an endogenous CD10/NEP gene.

16. The method of claim 14, wherein CD10/NEP

activity is increased in cells of said cancer.

17. The method of claim 14, wherein said therapeutic composition comprises a glucocorticoid.

18. The method of claim 14, wherein said cancer cells are pulmonary carcinoma cells.

19. A method for increasing CD10/NEP activity in the respiratory system of a patient, comprising administering to a patient a therapeutic composition comprising CD10/NEP.

20. A method for determining susceptibility of a patient to cancer, comprising measuring the activity of CD10/NEP in a sample from said patient.

21. The method of claim 20, wherein the sample comprises cells from the respiratory system of said patient.

22. The method of claim 21, wherein the sample comprises sputum.

23. A method for increasing CD10/NEP activity in an animal, comprising exposing cells of said animal to isolated DNA encoding CD10/NEP.

24. A method of inhibiting proliferation of a cell the proliferation of which is stimulated by a substrate for CD10/NEP, which method comprises increasing the amount of CD10 activity to which said cells are exposed.

25. The method of claim 24, wherein the CD10/NEP activity is increased by contacting said cells with

exogenous CD10/NEP.

26. The method of claim 24, wherein CD10/NEP activity is increased by contacting said cells with isolated DNA encoding CD10/NEP.

27. The method of claim 24, wherein the BLP- dependent cells are carcinoma cells.

28. The method of claim 24, wherein said substrate is a BLP.