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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the present invention relates to novel methods of detecting and measuring levels of the tumor growth factor pleiotrophin (PTN). These measurements can be used to determine the presence of PTN-positive diseases, to determine the relative prognosis of the disease, to determine the efficacy of cytotoxic anticancer drugs, and for molecular targeting of PTN. More particularly, the present invention relates to immunoassays using anti-PTN antibodies to detect and measure levels of PTN in samples. The present invention also relates to kits for detecting and measuring PTN levels.
• PTN tumor growth factor pleiotrophin
• Tumor expansion and metastasis are dependent on growth factors produced by the tumor cells and/or by the stroma (Folkman J, N. Engl. J. Med. 333: 1757- 1763, 1995) and one would expect in particular that these growth factors, such as angiogenic factors that target sprouting blood vessels, can be shed into the circulation and hence may provide a direct measure of tumor progression.
• the tumor growth factor pleiotrophin belongs to a family of growth factors that includes midkine (MK) (Kadomatsu et al., Biochem. Biophys. Res. Commun., 3: 1312-1318, 1988). PTN and MK share 50% sequence homology (Laaroubi et al., Prog. Growth Factor Res., 6(1): 25-34, 1995). PTN is involved in growth and differentiation processes that are tightly regulated during development (Schulte et al., Tumor Angiogenesis, pp. 273-289, Oxford University Press, 1997), and it is a mitogen for fibroblasts (Fang et al., J. Biol.
• Knowing the levels of PTN in samples can play a significant role in diagnosing and prognosticating PTN-positive diseases, monitoring the efficacy of anti-PTN therapeutics, and detecting PTN inhibitors or stimulators. Accordingly, there is a need in the art for methods to detect and measure levels of pleiotrophin in samples and to diagnose pleiotrophin-positive diseases. There is also a need in the art for methods to monitor the effectiveness of therapeutic treatments for pleiotrophin-positive diseases and to test for agents or drugs that inhibit or stimulate pleiotrophin.
• It is a more specific object of the invention to provide a method utilizing immunoassays for detecting and measuring the levels of pleiotrophin in samples comprising incubating a sample of interest with an antibody directed against pleiotrophin and detecting the antibody-pleiotrophin antigen complex.
• It is another specific object of the invention to provide a method for diagnosing pleiotrophin-positive diseases comprising contacting a sample from a patient suspected of having a pleiotrophin-positive disease with antibodies which recognize pleiotrophin, detecting and measuring the presence or absence of antibody-pleiotrophin antigen complexes, and comparing the amount of pleiotrophin to a normal control, wherein an increase in the amount of pleiotrophin over the control indicates the presence of a pleiotrophin-positive disease.
• Figures 1A, IB and 1C illustrate the characterization of the PTN sandwich ELISA assay.
• the standard curve of 1 A displays the sensitivity of the PTN ELISA as ranging from 0.005 to 20 ng/ml.
• PTN were incubated in ELISA wells that had been preadsorbed without (-) or with (+) 4B7 mAb and BSA. Bound PTN was eluted with SDS-PAGE and Western- blotted with a goat anti-PTN antiserum (R&D).
• IB shows that the specificity of the ELISA for PTN.
• PTN lng/ml, MK 10 ng/ml, bFGF 10 ng/ml, or PTN 1 ng/ml plus MK 500 ng/ml were subjected to the ELISA assay. Results are from a representative experiment done in duplicate.
• 1C shows that serum components other than PTN do not interfere with the detection of PTN by ELISA.
• PTN concentrations ranging from 0.005 to 20 ng/ml were analyzed in the absence
• FIG. 2 illustrates xenograft tumor growth and serum levels of PTN in athymic nude mice over time.
• Subcutaneous tumor growth of 1205LU human melanoma xenografits (open circles) and serum levels of PTN (closed triangles) were determined in the nude mice.
• Serum was collected from the orbital sinus of subgroups of the mice at different days. Mean ⁇ SE values are shown (n 18 tumors; 3 to 6 independent serum samplings at each time point).
• Inset Serum levels of PTN in mice with approximately 1 ,500 mm 3 subcutaneous human tumor cell xenografts of different origin. Tumors were grown from PTN-positive human breast cancer cells (MDA-MB 231) and melanoma (1205LU) as well as from PTN-negative, FGF-4-transfected MCF-7 breast cancer cells.
• Figure 3 illustrates the serum levels of PTN in healthy volunteers and patients with different gastrointestinal cancers.
• 3 A shows individual data points, and 3B shows the mean + SE of the different groups.
• Figure 4 illustrates the detection of PTN by immunohistochemistry of different gastrointestinal cancers in patients with elevated PTN serum levels (A) or with different serum levels of PTN (B).
• 4A shows the staining for PTN, a negative control without primary anti-PTN antibody (0), and staining for CEA as a positive control.
• 4B shows the staining for PTN in samples from patients with colon cancer and different serum levels of PTN as indicated above the respective panel. Note: the staining for PTN in the tumor cells and not the stroma.
• Figure 5 illustrates a comparison of pleiotrophin levels between healthy subjects and breast cancer patients.
• a method for detecting and measuring the presence of PTN comprises incubating a sample suspected of containing PTN with anti-PTN antibodies and determining the presence of PTN using techniques such as Western blots, immunoprecipitation, or Enzyme Linked Immunosorbent Assays (ELISAs).
• ELISAs Enzyme Linked Immunosorbent Assays
• PTN may be determined in a sample from any source.
• PTN is determined in a biological sample.
• Biological samples include, but are not limited to, blood, serum, urine, cerebrospinal fluid, cell culture supernatants, and tissue.
• PTN is detected using a sandwich ELISA that utilizes anti-PTN primary and secondary antibodies.
• a mouse monoclonal antibody (Mab) (4B7) is used as a primary antibody and a biotinylated affinity purified goat anti-PTN polyclonal secondary antibody is used to detect PTN bound to the mouse MAb.
• the secondary antibody may also be a monoclonal antibody, a mixture of monoclonal antibodies, a mixture of polyclonal antibodies, or a mixture of monoclonal and polyclonal antibodies.
• These secondary antibodies are preferably coupled to a detectable label, such as radiolabels, flourescent labels and enzymes.
• the label can be coupled to the secondary antibody by conventional methods known in the art. For example, chemical or physical bonding of the label to the antibody can be used.
• biotinylated goat anti-PTN polyclonal antibodies are preferred as the secondary antibody, it is also possible to use other polyclonal antibodies produced, for example, in rabbits, mice, and rats.
• antibodies attached to any reporter such as radiolabeled antibodies or antibodies directly conjugated to alkaline phosphatase (substrates include p-nitrophenyl phosphate (pNPP)), horseradish peroxidase (substrates include 5-aminosalicylic acid (5AS), 2-2' azino-di-(3-ethylbenzthiazoline sulfonate), o-dianisidine, o-phenylenediamine dihydrochloride (OPD), and 3,3'5,5'-tetramethylbenzidne (TMB)), ⁇ -galactosidase (substrates include o- nitrophenyl- ⁇ -D-galactopyranoside (oNPG) and p-
• pNPP p-nitrophenyl
• the present invention can be used to detect and diagnose PTN-positive diseases such as stomach cancer, breast cancer, prostate cancer, pancreatic cancer, and colon cancer.
• Patients having a PTN-positive disease have elevated levels of PTN when compared to PTN levels in healthy subjects.
• Measurements of PTN levels in accordance the with invention, can be used to detect PTN-positive diseases in samples from patients.
• the method for diagnosing a PTN-positive disease comprises contacting a sample from a patient suspected of having a PTN-positive disease with an antibody that recognize PTN, detecting the presence or absence of a complex formed between sample PTN and the antibody, and comparing the amount of PTN in the sample to PTN levels from normal controls.
• PTN-positive disease An increase in the amount of PTN in the sample in comparison to the control is indicative of the presence of a PTN-positive disease.
• Typical PTN levels in normal healthy subjects range from 0 to 100 pg/ml while abnormal PTN levels range from > 100 to 7000 pg/ml.
• the present invention is also applicable for detecting and diagnosing any PTN-positive disease including cancer, arthritis, multiple sclerosis, viral infections of the brain, hepatitis, and colitis.
• the effectiveness of treatment of PTN-positive diseases is monitored by measuring PTN levels in the patient post treatment and comparing the post-treatment PTN levels to initial PTN levels. A decrease in post-PTN levels indicates an effective treatment. PTN levels are measured in accordance with the invention as discussed above.
• Therapeutic treatments include, but are not limited to, surgical treatment, radiation treatment, chemical treatment, immunotherapy, gene therapy, antisense therapy or a combination thereof.
• an anti-PTN antibody can be used to treat PTN-positive diseases.
• a therapeutic amount of the PTN antibody is administered to a patient with a PTN-positive disease.
• the dose of the anti-PTN antibody to be administered can be determined by methods well known in the art.
• the antibody By binding to PTN protein, the antibody will prevent PTN from functioning as a tumor growth factor, thus, inhibiting tumor growth and angiogenesis.
• the efficacy of the treatment can be monitored in accordance with the procedure described above and
• Preferred antibodies to treat PTN-positive diseases include, but are not limited to, a monoclonal antibody, a mixture of monoclonal antibodies, polyclonal antibodies, a mixture of polyclonal antibodies, or a mixture of monoclonal and polyclonal antibodies. Additional preferred antibodies include anti-PTN antibodies produced, for example, in rabbits, mice, and rats. More preferably, a human anti-PTN antibody, a humanized anti-antibody, or an anti-PTN antibody produced by any method known in the art can be used.
• humanized antibody it is meant an antibody which is less immunogenic in humans. This is achieved by various methods known in the art, for example, one can produce a chimeric humanized antibody by grafting the non- human variable domains which retain antigen binding properties onto a human constant region. Additional methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. 81 : 6851-5 (1984); Morrison et al, Adv. Immunol. 44: 65-92 (1988); Verhoeyen et al, Science 239: 1534-1536 (1988); Padlan, Molec. Immun. 28: 489-
• the anti-PTN antibodies of the invention may be administered to a human or other animal in an amount sufficient to produce a therapeutic or prophylactic effect.
• Such antibodies of the invention can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
• the route of administration of the antibody (or fragment thereof) of the invention may be oral, parenteral, by inhalation or topical.
• parenteral as used herein includes intravenous, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Subcutaneous and intramuscular forms of parenteral administration are generally preferred.
• the daily parenteral and oral dosage regimens for employing compounds of the invention will generally be in the range of about 0.05 to 100, but preferably about 0.5 to 10, milligrams per kilogram body weight per day.
• the antibodies of the invention may also be administered by inhalation.
• inhalation is meant intranasal and oral inhalation administration.
• Appropriate dosage forms for such administration such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.
• the preferred dosage amount of a compound of the invention to be employed is generally within the range of about 10 to 100 milligrams.
• the antibodies of the invention may also be administered topically.
• topical administration is meant non-systemic administration and includes the application of an antibody (or fragment thereof) compound of the invention externally to the epidermis, to the buccal cavity, a instillation of such an antibody into the ear, eye and nose, where it does not significantly enter the blood stream.
• systemic administration is meant oral, intravenous, intraperitoneal and intramuscular administration.
• the amount of an antibody required for therapeutic or prophylactic effect will, of course, vary with the antibody chosen, the nature and severity of the PTN-positive disease being treated and the animal undergoing treatment, and is ultimately at the discretion of the physician.
• a suitable topical dose of an antibody of the invention will generally be within the range of about 1 to 100 milligrams per kilogram body weight daily.
• the ability of agents or drugs of interest to inhibit or stimulate pleiotrophin production is determined.
• Drugs or agents are administered in vitro to cells, for example, neuronal or other brain cells expressing PTN.
• PTN levels are measured in accordance with the invention.
• the post-treatment PTN levels are compared to PTN levels of control cells that did not receive the agent or drug.
• a decrease in PTN in the cell culture medium compared to control cells indicates a PTN inhibitory agent or drug while an increase indicates a PTN stimulatory agent or drug.
• retinoids are stimulatory agents of PTN.
• the present invention can be used to monitor the effectiveness of blocking pleiotrophin production on a molecular level.
• an agent that destroys pleiotrophin mRNA or blocks the production of pleiotrophin in any manner can be administer to a patient.
• the levels of pleiotrophin can be measured in accordance with the invention to determine whether the agent and the amount of the agent as well as the time interval for administering the agent were effective or sufficient.
• Another embodiment of the invention involves a kit to detect the presence of PTN, such as PTN present in biological samples, to diagnose and prognosticate
• kits comprises an antibody directed against pleiotrophin and ancillary reagents for use in detected the presence of pleiotrophin.
• the kit contains any of: (1) a solid support, such as a microtiter plate, on which to bind a primary anti-PTN antibody; (2) a solution containing the primary antibody; (3) buffer solutions to block unbound sites on the solid support and to wash the solid support; (4) a solution containing the labeled secondary anti body; and (5) PTN protein for a control standard curve.
• PTN may be isolated and purified from samples by methods well known in the art such as affinity chromatography, immunoprecipitation, ammonium sulfate precipitation, ethanol precipitation, and anion or cation exchange chromatography. See Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor Press, New York, 1989, which is incorporated herein by reference, for additional isolation/purification methods.
• PTN is isolated by immunoassays utilizing anti-PTN antibodies which recognize epitopes of PTN.
• the antibodies may be polyclonal or monoclonal, preferably monoclonal.
• the anti-PTN antibodies are bound to a solid support.
• Materials that can be used as solid supports include, but are not limited to, polysaccharide based materials such as cellulose and dextran, silica, nylon, magnetic particles such as beads, and microtiter plates.
• the sample of interest is applied to an affinity chromatography column packed with, for example, Protein A or Protein
• Another embodiment of the invention includes detecting elevated levels of PTN by adding to a sample a first antibody that binds to PTN. Addition of a second antibody that has an affinity to the first causes the antibody-PTN-antibody complex to precipitate. The precipitated complex can be assayed to determine the amount of PTN present using methods well known in the art.
• EXAMPLE I ELISA Procedure The mouse monoclonal antibody (4B7) was diluted to 1 ⁇ g/ml in Tris- Buffered Saline (TBS; 50mM TrisHCl pH 7.5, 0.15 M NaCl). 100 ⁇ l aliquots of the diluted antibody were incubated in 96 well plates (Corning, New York, NY) at 4°C overnight. The wells were washed three times with TBST (TBS with 0.5% Tween 20). The remaining free binding sites in the wells were then blocked with 200 ⁇ l of blocking solution (TBST with 1% BSA) for 2 hours at 4°C and the wells were washed three times with TBST.
• TSS Tris- Buffered Saline
• Samples suspected of containing PTN were diluted in 2xTBST and 100 ⁇ l aliquots of the sample were added to the wells and incubated at room temperature for 1 hour. The wells were then washed three times with TBST and the second antibody (biotinylated, affinity-purified goat anti- human pleiotrophin IgG (R&D Systems, Minneapolis, MN)), was added at a concentration of 500 ng/ml and incubated at room temperature for 1 hour. After washing three times with TBST, 100 ⁇ l of streptavidin conjugated to alkaline phosphatase at a concentration of 50 ng/ml was added to each well and the plate was incubated for 1 hour at room temperature.
• the second antibody biotinylated, affinity-purified goat anti- human pleiotrophin IgG (R&D Systems, Minneapolis, MN)
• microtiter plate was then washed three times with TBST and incubated with 100 ⁇ l p-nitrophenyl phosphate (PNPP) substrate (Pierce) in the dark at 4 °C for 18 hours or at room temperature for 2 hours. Absorbance was measured using a microtiter plate reader at 405 nm.
• PNPP p-nitrophenyl phosphate
• Figure 1A shows that the minimum detectable concentration of PTN ranges from 5 to 10 pg/ml.
• the intra-assay coefficient of variation (CV) was 1.67% and the inter-assay coefficient of variation was 2.10%.
• PTN with a 500-fold excess of human MK did not alter the signal generated by PTN alone ( Figure IB).
• the PTN ELISA is highly specific for PTN and not MK or bFGF. Since secreted mouse and human PTN protein are identical at the amino acid level (Bohlen et al., Prog Growth Factor Res., 3: 143-157, 1991), the assay cross-reacts with the murine PTN protein.
• the serum of athymic nude mice contained PTN levels below the detection limit of the ELISA.
• EXAMPLE II Detection of PTN in Human Tumor Cell Line Supernatants Using the procedure outlined in Example I, human tumor cell lines of different origin were screened for the presence of PTN in the culture medium. The
• PTN ELISA detected 6.3 ⁇ 0.1 and 6.9 ⁇ 0.2 ng of PTN per ml of conditioned medium of subconfluent 1205LU melanoma and MDA-MB 231 breast cancer cells, respectively. Both cell lines had previously been shown to express PTN mRNA and PTN protein. The cell lines were cultivated as reported earlier (Fang et al., J. Biol. Chem., 267: 25889-25897, 1992; Czubayko et al., Proc. Natl. Acad.
• mice with Human Xenograft Tumors Tumor xenografts grown from PTN-positive human tumor cells (MDA-MB 231 breast cancer and 1205LU melanoma) were established and monitored in athymic nude mice as described in Czubayko et al., Proc. Natl. Acad. Sci., 93: 14753-14758, 1996). MCF-7 human breast cancer cells transfected with FGF-4 (MCF-7/FGF-4) served as a negative control because these cells do not produce PTN (Fang et al, J. Biol. Chem., 267: 25889-25897, 1992). Serum samples from mice bearing tumors of at least 1 ,500 mm 3 were analyzed according to Example
• Figure 5 shows the comparison of PTN levels between healthy subjects and breast cancer patients. As in the example above, PTN levels in breast cancer patients are significantly elevated over the healthy control subjects.
• Cryostat prepared sections of tumors were acetone-fixed and incubated in a 1 : 100 dilution of H 2 0 2 to block endogenous peroxidases. After washing in PBS, the samples were incubated with the primary anti-PTN antibody used in the ELISA (goat anti-human PTN; R&D) diluted to 5 ⁇ g/ml (1:20) in 1% BSA in PBS at room temperature for 2 hours or at 4°C overnight. After washing three times with PBS, the secondary antibody (peroxidase-coupled rabbit anti-goat IgG; Dianova, Hamburg, Germany) was added in 10% ABO human serum and incubated for 1 hour at room temperature. After a further wash in PBS, peroxidase activity was revealed by staining with DAB as a substrate (Vector Lab, Burlingame). The sample was then washed twice in water, dried, and covered.
• monitoring of PTN serum levels in patients could be a useful measure of residual tumor burden and/or recurrence of tumors after therapy. This is supported by the drop in PTN serum levels after successful tumor removal and the lack thereof with residual tumor mass.
• drugs targeting PTN for therapeutic purposes e.g. synthetic ribozymes
• monitoring PTN serum levels is a very attractive way of assessing pharmacologic efficacy of the particular therapeutic molecule or its mode of delivery.

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