EP1551446A1 - Marqueur neuroendocrinien du cancer et procede de production de ce marqueur - Google Patents

Marqueur neuroendocrinien du cancer et procede de production de ce marqueur

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Publication number
EP1551446A1
EP1551446A1 EP02744407A EP02744407A EP1551446A1 EP 1551446 A1 EP1551446 A1 EP 1551446A1 EP 02744407 A EP02744407 A EP 02744407A EP 02744407 A EP02744407 A EP 02744407A EP 1551446 A1 EP1551446 A1 EP 1551446A1
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EP
European Patent Office
Prior art keywords
nem
antibody
polypeptide
binding
cells
Prior art date
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EP02744407A
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German (de)
English (en)
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EP1551446A4 (fr
Inventor
Girish V. Shah
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University of Kansas Medical Center
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University of Kansas Medical Center
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Publication of EP1551446A1 publication Critical patent/EP1551446A1/fr
Publication of EP1551446A4 publication Critical patent/EP1551446A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates generally to markers for cancer diagnosis and treatment, and more specifically, to a novel neuroendocrine growth factor to diagnose and treat invasive and metastatic prostate cancer and other cancers that express this growth factor.
  • Prostatic carcinoma is the leading malignancy in human males in terms of incidence and the second leading cause of cancer deaths in men. While a majority of prostatic carcinomas remain dormant for a long period of time, a significant minority of them display rapid growth and invasive characteristics. The mechanisms responsible for the latent growth of tumors in a majority of cases and for rapid progression in a minority of cases have not been identified. It has been suggested that tumors with greater neuroendocrine cell populations may display autonomous growth, androgen-independence, and increased invasiveness.
  • PC prostatic carcinoma
  • LH-RH gonadotropin-releasing hormone
  • AJPC cells those cancer cells which require androgens for growth die when they are deprived of androgens during therapy, but those aggressive androgen-independent prostatic carcinoma cells ("AJPC cells") continue to proliferate even in the absence of androgens.
  • Two areas of prostate cancer biology currently under intense investigation include: (1) the cause of relapse of prostate carcinoma in patients who initially respond to androgen deprivation treatments; and (2) the early detection of prostatic carcinomas that will become metastatic. Once the relapse occurs, few treatment options are available and eventually the patient dies from the disease. Continued proliferation of clonally derived AIPC tumor cells is believed to be the underlying cause of the relapse, as metastatic tumors are frequently found to be androgen-independent.
  • AIPC cells are by definition independent from influence by androgens, there are neuroendocrine and other growth factors, which can stimulate their growth.
  • Tumor cells are known to express distinct markers at the very early stage of transformation. Several tumor markers have been identified and have been used to identify either the presence of malignancy and/or the severity of the malignancy. For example, prostate-specific antigen (PSA) has been identified and used for the diagnosis of prostate cancer.
  • PSA prostate-specific antigen
  • PSA has been used as a marker for PC for several years, current evidence suggests that serum PSA levels: (1) do not always correlate with progression of the disease; (2) are not expressed in a small but significant number of aggressive PCs which therefore are undetected by PSA screening; and (3) do not predict the clinical nature of the tumor, i.e., even if the tumor is detected early, PSA screening does not predict whether the tumor will be dormant and therefore relatively harmless, or will be aggressive requiring immediate clinical intervention.
  • PSA is used as a diagnostic marker for PC, neither PSA nor other serum markers can reliably identify the metastatic phenotype. Given the fact that not all prostate tumors secrete or express PSA, there is a critical need to develop other markers to identify metastatic phenotypes and PSA negative PC subtypes.
  • neuroendocrine factors such as bombesin and vasoactive intestinal polypeptide have been shown to influence the invasive behavior of prostate cancer cell lines.
  • Pindobind a serotonin Hla receptor antagonist, has been shown to inhibit the growth of prostate cancer cell lines.
  • foci of neuroendocrine differentiation have been demonstrated in between 47% and 100% of prostate cancers using a combination of sensitive argyrophil staining and immunocytochemistry. Thus, it is likely that neuroendocrine factors play an important role in prostate cancer.
  • the grade of the cancer determines the severity of the cancer and varies from low to high grades.
  • Low grade prostate tumor grows very slowly and often does not require any aggressive forms of therapy.
  • high-grade prostate tumor grows and metastasizes aggressively and requires aggressive therapeutic intervention. Determination of the grade of prostate tumor is crucial for choosing the right form of therapy.
  • Present methods of determination of the grade of prostate cancer involve examination of the architecture of the prostate tumor in sections of biopsy. However, this method is prone to error due to the fact that there is considerable inter-observer variation leading to wrong diagnosis of the grade of the tumor. Determination of the grade of the tumor based on a biochemical marker rather than the visual observation of the architecture of the tissue would provide a better method for identifying the grade of the tumor. This has led to major efforts at identifying new biochemical markers for prostate cancer.
  • the present invention relates to a novel neuroendocrine growth factor or marker ("NEM”) identified in cultured prostate cancer cells.
  • NEM neuroendocrine growth factor or marker
  • the present invention provides a method for preparing NEM and NEM receptor from tissue-cultured prostatic carcinoma cells and from prostatic tissues and by chemical methods of synthesis and purification through the use of recombinant DNA methods.
  • the present invention also provides a method for preparing the corresponding oligonucleotides, which are used as probes to detect NEM in mRNA in tissues by in-situ hybridization. Unknown metastases may be identified and prostatic carcinomas may be graded. As a result, metastatic prostatic cancer and PSA-negative cancers may be detected by measuring NEM immunoreactivity in tissues and in human fluids.
  • the present invention provides a method for making NEM antibodies, which are then therapeutically administered to inhibit the growth invasion of prostatic carcinoma and other cancers whose growth is influenced by NEM.
  • NEM and antibodies against NEM may employ radionuclides for detecting the presence of primary and secondary sites of prostate cancer.
  • NEM, antibodies against NEM or NEM receptor may target cell-killing radionuclides or other cytotoxic agents to cancer cells that selectively bind NEM to preferentially kill these cells.
  • NEM may be used as a prophylactic or therapeutic vaccine whereby these molecules, in conjunction with other immunostimulatory molecules, stimulate an immune response against cancer cells expressing these molecules to kill these cells.
  • NEM antibodies generated in the body by these procedures will neutralize NEM thereby providing therapeutic benefit.
  • FIG. 1 is a bar graph illustrating the effect of exogenously added prostatic NEM on DNA synthesis of an aggressive prostate cancer cell line, PC-3M.
  • FIG. 2 is a bar graph illustrating a dose-dependent relationship between the concentration of prostatic NEM and the invasiveness of PC-3M cells.
  • FIG. 3 is a bar graph illustrating a dose-dependent inhibition of growth of PC-3M cells by antibodies to prostatic NEM (dilution of 1 : 100).
  • FIG.4 is a bar graph illustrating a dose-dependent inhibition of invasiveness of PC- 3M cells by anti-NEM anti-serum.
  • FIG. 5 is a series of immunohistochemistry photographs of prostatic cancer tissue samples using antibodies to prostatic NEM, and illustrating the degree of malignancy correlating with an amount of NEM expression.
  • FIG. 5a is prostate cancer tissue at a premalignant stage (PEST);
  • FIG. 5b is of a moderate grade tumor; and
  • FIG. 5c is of an aggressive, high-grade tumor.
  • FIG. 6 is a series of photographs of prostate epithelium tissue samples following in- situ hybridization using prostatic mRNA to illustrate the expression of NEM mRNA in: (a)
  • BPH 1 BPH 1 ; (b) PIN 2; (c) low grade tumor 1 ; (d) moderate grade tumor 2; (e) high grade tumor 2; and (f) very aggressive tumor 1.
  • the NEM mRNA is expressed only by blue stained cells (primarily basal cells).
  • FIG. 7 is a series of photographs of tissue samples following in-situ hybridization using prostatic NEM mRNA illustrating that the metastases are prostatic in origin in the liver
  • FIG. 8 is a graph illustrating the detection of prostatic NEM in correlation with PSA.
  • FIG. 9 is a schematic illustrating the mechanism of action pathway of an NEM-based cancer therapy as compared to an untreated cancer pathway.
  • FIG. 10 is a schematic of the mechanism of EM-mediated proliferation and invasion of prostate cancer cells.
  • FIG. 11 is a schematic of NEM-based cell-targeted radiation therapy.
  • FIG. 12 is a series of photomicrographs depicting NEM selectively binding to prostate cancer cells.
  • FIG. 13 is series of photomicrographs of immunohisto-chemical detection of NEM in prostate cancer tissue sections, and detection of NEM in secondary sites of prostate cancer.
  • FIG. 14 is a series of photomicrographs of the expression of NEM in BPH and prostate cancer tissue sections. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
  • the present invention involves a novel neuroendocrine growth factor or marker (“NEM”) identified in cultured prostate cancer cells.
  • NEM neuroendocrine growth factor or marker
  • NEM is also produced by other cancers like small cell lung carcinoma, intestinal cancer, uterine cancer, certain types of breast cancer, among other cancers. This demonstrates that NEM also would have a role in the progression of these cancers.
  • One mechanism by which NEM induces the growth of cancer cells is by binding to its receptor (NEM receptor) present on cancer cells which then induces growtli and invasion. Blocking the interaction of NEM with its receptor by using an antibody directed against NEM or its receptor would inhibit the growth and invasion of these cancer cells thereby providing therapeutic benefits.
  • a significantly higher number of binding sites for this novel NEM is present on membranes of prostatic cancer cells than on those of benign prostatic hypertrophy (BPH), a common benign enlargement of the prostate in older men.
  • BPH benign prostatic hypertrophy
  • the ability to differentiate BPH from malignancy is highly important but cannot be effectively accomplished using PSA screening, the most commonly used method for detecting the presence of prostate cancer clinically.
  • PSA screening is overcome by screening patient blood for NEM which will identify metastatic prostate cancer, distinguish it from BPH, and can be used in excised tissues to identify the type of cancer and grade its degree of malignancy.
  • inhibition of NEM in turn decreases prostatic cancer growth, invasion and metastasis.
  • NEM antagonism offers new modes of therapy for prostate cancer.
  • NEM is a good biochemical marker for prostate cancer.
  • a mechanism of the proliferation and invasion of prostate cancer cells identified by Applicant relates to NEM's stimulation of a receptor on prostate cancer cells.
  • Figs. 9A-C illustrate NEM induced proliferation of prostate cancer cells.
  • prostate cancer cells secrete NEM.
  • NEM binds to NEM receptors on prostate cancer cells (Fig. 9B), in a lock and key manner, thereby stimulating cell division and invasion.
  • Prostate cancer cells then divide and invade as depicted in Fig. 9C.
  • Figure 10 illustrates the proposed mechanism of NEM-mediated proliferation and invasion of prostate cancer cells.
  • Figure 10A depicts a normal prostate cell that produces very little or no NEM and possesses very little or no NEM receptors.
  • Figure 10B depicts a prostate cancer cell that produces and secretes NEM. The cell also expresses
  • NEM receptors on the surface of the cell as shown by the Y-shaped molecule in Fig. 10B.
  • NEM receptors are specific proteins to which NEM must bind in order to mediate its action.
  • NEM binds to NEM receptors on prostate cancer cells, as depicted in Fig. 10C, thereby stimulating cell division and invasion. Prostate cancer cells then divide and invade, as shown in Fig. 10D.
  • NEM is prepared by first subcloning the complementary DNA in a vector.
  • the plasmid containing NEM cDNA is then transfected into prostate cancer cell line PC-3M cells, and after incubation and culturing, the expressed protein is obtained by affinity chromatography. Standard automated chemical synthesis and purification can also make the protein.
  • Polyclonal antibodies to NEM are prepared by injecting rabbits with antigen and removing a blood sample after the immune response has occurred.
  • the immunoglobulin fraction of the serum is purified and used as the antiserum.
  • More specific monoclonal antibodies are also prepared which can recognize a single antigenic determinant.
  • inventive polyclonal and monoclonal antibodies and the novel probes are utilized in the detection of NEM in tissues and body fluids and grading of invasive and metastatic prostatic cancers and other cancers that exhibit enhanced production of NEM as well as those cancers which are PSA negative.
  • This is successfully accomplished using immunohistochemistry, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, EIA, fluoroimmunoassay, and chemiluminescence assay techniques, or other techniques used for the detection of peptides.
  • cDNA for NEM is cloned into pGEM-T plasmids (Promega) and linearized with Sac II to make antisense strands and with Pstl to make sense strand.
  • the transcription reaction and digoxigenin 11-UTP (Boehringer Mannheim, Indianapolis, IN) labeling are performed with either SP6 (antisense) or T7 (sense) RNA polymerase provided in the riboprobe labeling kit (Promega).
  • the labeled probes are digested with deoxyribonuclease (Boehringer), extracted with pheno-chloroform, and purified with TE Microselect-D G-50 spin columns (5 Prime-3Prime, Inc., Boulder, CO).
  • the inventive antisense RNA (or nucleotide) probes against NEM can be used for detecting the presence of NEM RNA in prostatic and other cancer tissues that express NEM in order to determine the grade of the cancer.
  • Primers based on the DNA sequence can also be used to determine the presence of disseminated prostatic and other cancer cells producing NEM to monitor the presence of metastasis.
  • the inventive NEM (or NEM receptor) antiserum and monoclonal antibodies inhibit the invasiveness of the disease and, thus, offer new forms of cancer therapy.
  • the polyclonal and monoclonal antibodies of the present invention are conjugated to chemotherapeutic agents and/or radionuclides to treat prostate cancer or other cancers that produce NEM or whose growth is enhanced by NEM.
  • Gene therapy techniques and the preparation of an antagonist to prostatic NEM (or its receptor) can also be employed to treat the disease. 1. Preparation of Prostatic NEM
  • Amino acid SEQ ID NO: 1 was prepared by first subcloning the complementary DNA SEQ ID NO: 2 in pRC vector (Invitrogen, San Diego, CA).
  • the vector contains cytomegalovirus promoter upstream of the cloning site, and ensures high level expression of the cloned cDNA.
  • the pRC plasmid containing NEM cDNA is then transfected in prostate cancer cell line PC-3M cells using Lipofectamine (Life Technologies, Inc., Gaithersburg,
  • PC-3M cells were plated at a density of 15,000 cells per well in a six-well culture plate and transfected 24 hours later with either the vehicle plasmid or the plasmids carrying cDNAs. Aliquots containing 2 ⁇ g plasmid and 4 mg Lipofectamine in 1 ml serum- free, protein-free Dulbecco's Modified Eagle's medium (DMEM) were incubated for 45 minutes and added to culture wells. The transfection media was replaced with the complete medium 16 hours later. Two days later, the cells were cultured in selection medium (complete medium containing 400 ⁇ g/ml of G418).
  • DMEM Dulbecco's Modified Eagle's medium
  • NEM isolated by Applicant and the purified DNA are being deposited with the American Type Culture Collection, 10801 University Boulevard., Manassas, VA 20110.
  • SEQ ID NOs: 2-3 are alternative cDNA sequences derived from the NEM peptide based on Applicant's research and within reasonable margins for error.
  • Peptide SEQ ED NOs: 1, 4-6 are alternative sequences based on cDNA SEQ ID NOs: 2-3 depending on the reading frame employed to translate same. 2. Dose-Dependent Relationship of NEM on DNA Synthesis in PC-3M Cell Line
  • FIG. 1 illustrates the effect of exogenously added prostatic NEM on DNA synthesis of an aggressive prostate cancer (PC-3M) cell line.
  • PC-3M cells were seeded at a density of 10,000 cells per well and incubated overnight in the complete medium. The cells were then washed with and incubated in serum-free medium containing the agents and 0.5 ⁇ Ci of 3H-thymidine for 24 hours. At the end of incubation, the cells were washed three times with PBS-1 ⁇ M thymidine and the incorporated radioactivity was determined in TCA-precipitable fraction.
  • NEM stimulated 3H-thymidine incorporation in a dose-dependent fashion, and the increase in DNA synthesis produced by 10 nM or greater concentrations of NEM was significant.
  • NEM also stimulates the growth of certain non-prostate cancer cells like certain bone-derived cell lines (OVCA-5), thereby extending the use of NEM in other cancers that produce or are stimulated by NEM.
  • NEM as the antigen on the solid phase.
  • the cell lysate (50 ⁇ l) from NEM- PC-3M cells was coated on ELISA plates. This served as the solid antigen phase.
  • the anti- NEM rabbit serum was added to the wells at various concentrations (dilutions of 10-100) as the solid antigen phase.
  • the serum was then washed off with phosphate buffered saline and the solid phase was incubated with biotinylated Anti-rabbit gamma globulin. After incubation for 2 hours at room temperatures, the plates were washed and incubated with streptavidin-alkaline phosphatase (Sigma Chemical Co., St. Louis, MO). After thorough washing of the solid phase three times, the color reaction was initiated using BCIP-NBT substrate system (Bio-Rad, CA) according to the manufacturer's protocol. 4. Stimulation of Invasiveness of PC-3M Cells by NEM
  • FIG. 2 illustrates the effect of NEM on in vitro invasion of PC-3M cells.
  • the experiment used 3 well Matrigel invasion chambers (Becton Dickinson, Bedford, MA). Approximately 200,000 PC-3M cells were seeded in the upper chamber in RPM 1640 medium containing 0.1% BSA (Sigma, St. Louis), 4 mM L-glutamine, 100 IU/ml penicillin G and 100 ug/ml streptomycin.
  • the lower chamber medium contained chemoattractant medium consisting of 80% complete medium (RPMI 1640, 12% horse serum, 4% heat activated fetal calf serum, 4 mM L-giutamine, 100 IU/ml penicillin G and 100 mg/ml streptomycin).
  • Different concentrations of NEM or conditioned media from NEM-PC3-M cells that express NEM were added to the upper chamber.
  • Figure 2 demonstrates that NEM in a dose-dependent fashion significantly increases the invasiveness of PC-3M cells.
  • the cells demonstrated a 70% increase from OMto 10 "6 M NEM. All concentrations of NEM were significant in comparison to 0 CT for PC-3M. (P ⁇ 0.05).
  • NEM-induced DNA synthesis may influence proliferative activity of a prostate tumor and other cancer cells, then its immunoneutralization should reduce the growth. This hypothesis was tested by studying the rate of 3H-thymidine incorporation in PC-3M prostate cancer cells. The cells were treated either with non-immune antiserum or anti-NEM antiserum at a final dilution of 1 :50. The results presented in FIG. 3 show that anti-NEM antiserum significantly inhibited DNA synthesis in PC-3M cells.
  • FIG. 4 illustrates the effect of the anti-NEM antiserum on invasion of PC-3M cells.
  • the invasion assays were performed as described in conjunction with FIG. 3. Instead of stimulating PC-3M cells with NEM, the cells were treated with various concentrations anti- NEM serum. The parallel controls received equivalent amounts of non-immune rabbit serum. The results show that anti-NEM serum significantly reduced invasiveness of PC-3M prostate cancer cells and the decrease was significant at concentrations of 1 : 50 and 1:100. 7. Detection of Prostatic NEM Peptide in PC Biopsies
  • Figure 5 illustrates the detection of the peptide in PC biopsies (BPH-PIN-tumors of various grades). Paraffin-embedded sections were cut at 0.5 mm thicl ⁇ iess. Paraffin sections were air-dried for 30 minutes, oven-dried, deparaffinized and rehydrated. Slides were microwaved on "high” with ChemMate antigen retrieval solution (BioTek, Santa Barbara). As a control for nonspecific staining, some of the anti-NEM serum was preabsorbed with 10 ⁇ M NEM for one hour at 37°C prior to use. Non-absorbed and preabsorbed antisera were diluted at 1:70 in Antibody Dilution Buffer (BioTek).
  • Staining data were also assessed by image analysis on 35 mm Kodak Ektachrome slides of benign, PIN and invasive cancer acini. Slides were scanned by a Polaroid SprintScan 35 scanner at 337 dots/inch and saved as JPEG images using an Adobe Photoshop program. The staining of cytoplasm of 50 cells per image was assessed using an NIH Image program, and the mean staining for each image was calculated. The overall means ⁇ standard error were calculated for benign, PIN and invasive carcinoma acini and significance based on 2-tailed t-tests, assuming unequal variances.
  • Figure 6 illustrates the detection of the mRNA in PC biopsies (BPH, high grade PDSf- tumors of various grades).
  • BPH high grade PDSf- tumors of various grades.
  • the data clearly shows that while there is very little or no NEM expression in BPH tissues, it is clearly evident in high-grade PIN (prostatic, intraepithelial neoplasia) considered to be a pre-cancerous state.
  • the data also clearly shows that NEM expression increases proportionally with the grade of cancer (as determined by Gleason score).
  • Gleason score grade of cancer
  • Partial complementary DNA (cDNA) for prostatic NEM was cloned into pGEM-T plasmid (Promega, Madison, WI) was linearized with Sac II to make antisense strand and with Pstl to make sense strand. The transcription reaction and digoxigenin 11-UTP
  • FIG. 6A-F, 13A-E, and 14A-F demonstrate the expression of NEM mRNA in prostate epithelium.
  • Figure 13A depicts high-grade PIN, a precursor of invasive cancer;
  • Fig. 13B depicts moderate grade cancer;
  • Fig. 13C depicts high-grade cancer (left portion is cancer tissue and the right portion is normal); and
  • Fig. 13D depicts aggressive cancer.
  • Figure 13E depicts a section of a liver tissue showing a small prostate cancer secondary nodule. The cancer cells (in the middle) produce NEM (stained dark) while the surrounding liver tissue does not.
  • Figures 14A-F depict the expression of NEM in BPH and prostate cancer tissue sections: A) in BPH, no expression of NEM; B) in high-grade PIN (a pre-cancerous lesion), some moderate-grade cancer; C) a higher magnification of moderate-grade cancer wherein the cancer cells line the small glands; D) moderate-grade cancer with a large number of small glands; E) high-grade cancer; and F) aggressive cancer. Probes were also used to determine whether metastases were prostatic in origin in the liver, lymph node and tonsil (FIGS.7A-C). 10. Detection of NEM mRNA using RT-PCR
  • NEM mRNA was detected in prostate cancer specimens as well as cell lines using RT-PCR technique according to published procedures.
  • the primers used in this procedure were: agaacctgtgtgctggcta (forward) and catatactaccccggcta (reverse).
  • the total RNA from the specimens was extracted using a Quiagen RNA extraction kit (Quiagen, CA) according to the manufacturer's protocols, reverse transcribed using reverse transcriptase and amplified using the previously described primers pair.
  • the reaction mixture was then separated on 1% agarose gel, and the amplicon of approximate size of 350 bp was detected as predicted according to SEQ ID NO: 1 in prostrate cancer specimens and DU-145, MCF-7 and PC-3M cancer cell lines.
  • NEM mRNA was also detected by RT-PCR in certain breast cancer tissues demonstrating that NEM may be a marker for other cancers also, particularly the ones that show a high degree of differentiation into the neuroendocrine-type cells like small lung carcinoma, certain pancreatic cancers, renal cancer, adrenomedullary carcinoma etc. 11. Correlation of NEM Levels With PSA Levels
  • Figure 8 illustrates the detection of the NEM antigen in serum samples and the correlation with PSA.
  • Serum samples from patients with prostate cancer were obtained from the Department of Pathology and Laboratory Medicine at the University of Kansas Medical Center. Their PSA levels were determined at the Department of Pathology and they ranged from 13.2 - 2046 units.
  • the samples were then processed for NEM radioimmunoassay (RIA) by standard antigen-antibody reaction.
  • RIA NEM radioimmunoassay
  • the serum samples were processed (for the removal of macromolecules) by precipitating w/acetate-ethanol and the supernatant was passed through a 0.5 ml Sephadex 625 column. The fractions containing the peptide were used for assays.
  • the processed serum samples are incubated are incubated with anti- NEM serum for 24 hours at 4°C. I I25 -labeled NEM is then added (approximately 30,000 cpm). The samples are then incubated for an additional 24 hours. At the end of incubation, the samples are treated with 4V of chilled ethanol, centrifuged and pellet is counted for radioactivity. The values of NEM are derived by log-logit transformation of the data. The results are plotted as NEM levels (pg/ml) against PSA levels (units).
  • the NEM cDNA can be expressed in laboratory conditions or in vivo when the cDNA of the reported sequence or its antisense sequence or a ribozyme targeted against NEM mRNA or an aptamer sequence that binds NEM is cloned downstream of a promoter in a vector(s) which includes, but is not limited to, a viral vector or a plasmid vector under the control of a promoter where the promoters are chosen from a panel of eukaryotic promoters including, but not limited to, SV40 immediate early promoter, cytomegalovirus promoter, thymidine kinase promoter, Maloney murine leukemia virus long terminal repeat, Ul promoter, U6 promoter, tRNA promoter and VA promoter.
  • a vector(s) which includes, but is not limited to, a viral vector or a plasmid vector under the control of a promoter where the promoters are chosen from a panel of eukaryotic promoters
  • NEM cDNA or its antisense (when transcribed in the reverse direction), ribozyme or aptamer can be formulated in a carrier for delivery to the prostate cancer cells and can be administered to patients through intravenous, intramuscular, subcutaneous injections or direct injections into the tumor tissue. 13. NEM Vaccine
  • An NEM expression cassette can also be used for immunotherapy.
  • a patient can be freated for prostate cancer with an NEM vaccine.
  • NEM is first expressed in dentritic cells and then injected into the patient.
  • the NEM cDNA vaccine construct, which encodes for NEM can be directly inoculated into the host.
  • the expression cassette will be under the control of a strong promoter such as a cytomegalovirus promoter.
  • the transfected cells then become the production source of the NEM, and can sensitize immune cells against NEM.
  • the sensitized immune cells become capable of destroying NEM producing endogenous prostate cancer cells.
  • a similar approach using pCMV-PSA expression cassette has shown that a strong and persistent antibody response against PSA, a prostate protein, is observable for at least for six months after immunization.
  • Alternate approaches may include injecting NEM after conjugation with other protein or glycoproteins to enhance their immune recognition, i.e. immune co-stimulatory molecules, or injecting these conjugates using lipid formations.
  • the co-stimulatory molecules may include but are not limited to B7 and ICAM. Use of the above methods in addition to eliciting cellular immunity could also induce humoral immunity (production of NEM specific antibodies).
  • the antibody to NEM is produced when the NEM peptide of SEQ ID NOs: 1-6 or its precursor or fragments are conjugated to a large protein molecule by covalent linkage, mixed with an adjuvant and injected into either a rabbit, mouse or other host to generate an immune response.
  • the serum or the spleen of the injected animal is harvested.
  • the serum was found to contain immunoglobin molecules generated in response to NEM, such molecules being capable of recognizing various antigenic sites on NEM and, thus, are called polyclonal antibodies.
  • the serum is tested for the immune response whereas spleen cells are fused with tumor cells to generate hybridomas.
  • the fused cells that secrete the antibody to NEM are thus immortalized and each cell secretes an immunoglobin molecule that recognizes a single antigenic site of NEM.
  • the antibodies can also be obtained by phage display when the expression vector (described above in "Preparation of Vector for RNA Synthesis") is injected into an animal.
  • the antibody to NEM is generated when purified or synthetic NEM peptide (or its fragment, precursor or other modified form) is conjugated to key hole limpet hemocyanin by covalent linkage, mixed with an adjuvant and injected into either a rabbit, mouse or other host to generate an immune response (according to published procedure in Harlow, et al, ANTIBODIES: A LABORATORY MANUAL (1988) Cold Spring Harbor Laboratory, NY).
  • the serum of the animal is then obtained.
  • the serum contains immunoglobulin molecules generated in response to NEM injections which can recognize various antigenic sites on the NEM sequence.
  • the IgC function of this serum is used for diagnostic purposes such as immunohistochemistry (as demonstrated by FIG. 5), immunoassays (as depicted in FIG. 8), and is also used for the immunoneutralization of NEM
  • Purified NEM or a synthetic fragment of the peptide is conjugated to an immunogen like keyhole limpet hemocyanin or similar protein according to published procedures (Harlow, et al, supra).
  • the conjugated immunogen is injected into a mouse and monoclonal antibodies are prepared according to published procedures.
  • Several hybridoma clones secreting the murine monoclonal antibodies are isolated and the antibodies secreted by the cells are tested for their ability to bind to radioiodinated NEM. Generally, the hybridoma clones secreting antibodies with the highest affinity are selected. These antibodies are further tested for their ability to inhibit the growth and invasive properties of prostate cancer cells as described above.
  • the hybridoma culture is then scaled up in large bioreactors as is known in the art to produce large amounts of monoclonal antibodies.
  • the antibody that is secreted into the cell culture media is purified using standard chromatographic methods and is used for diagnostic purpose or administered to prostate cancer patients as an aqueous formulation intravenously. 15. NEM Antibody for the Treatment of Prostate Cancer
  • An ideal therapeutic agent against prostate cancer should minimally possess three qualities: (1) It should interfere with the growth of prostate cancer cells; (2) it should prevent the spread of prostate cancer cells; and (3) it should be minimally toxic to normal cells.
  • NEM selectively binds to NEM receptors on prostate cancer cells. Cells with NEM bound to them were stained dark brown (the wide band of cells on the right of Fig. 12 abutting normal cells on the left). NEM was conjugated to a detection tag (digoxigenin-alkaline phosphatase) in order to visualize its binding to cancer cells.
  • the digoxigenin tag may be replaced with a radioisotope including but not limited to In-111. NEM would direct the radioisotope to prostate cancer cells wherever they are present in the body.
  • the sites of accumulation of NEM-radioisotope can be detected using a gamma camera.
  • the binding of the antibodies to NEM prevents NEM's interaction with its specific receptors, thereby inhibiting its growth promoting and metastatic properties.
  • the therapeutic pathway that prevents prostate cancer cell division is depicted in Figs. 9D-F.
  • Prostate cancer cells secrete NEM as shown in Fig. 9D.
  • NEM antibodies bind to NEM and block NEM from binding to its receptor, depicted in Fig. 9E. As a result, NEM is neutralized by the antibody and prostate cancer cell division is prevented, shown in Fig. 9F.
  • antibodies directed against NEM could be used as an effective therapy for prostate cancer. These antibodies could be administered alone or in conjunction with one of the currently available therapies. This type of combination therapy is being increasingly used for the treatment of a variety of diseases.
  • Antibodies particularly humanized monoclonal antibodies, or human antibodies, are gaining acceptance as therapeutics designed to block the action of various bioactive molecules involved in disease processes, and specifically for the treatment of cancer.
  • An example is the use of antibodies against HER2, a receptor protein that is expressed in high amounts in breast cancer and which mediates the action of certain peptide growth factors that induce breast cancer growth. These antibodies have shown efficacy in the treatment of breast cancer in animal models and in humans. 16.
  • variable region that determines the specificity towards an antigen is restricted to a few amino acids and hence the new hybrid antibody would be of predominantly human nature thus preventing the formation of antibodies against it once they are injected into humans, hi addition, humanized antibodies have longer retention times in the body thereby allowing fewer doses of the antibody to the patient.
  • Several such "humanized" monoclonal antibodies are in clinical trials for various indications.
  • Example 1 - Treatment of Prostate Cancer In this embodiment, hybridoma-producing monoclonal antibodies against NEM are prepared as above. The cDNA encoding the heavy and light chain of the antibody is isolated from the selected hybridoma using standard recombinant DNA techniques.
  • the VH and VL domains of the murine antibody is fused at the DNA level to the human Ck and Cg domains (CHI, hinge, CH2 and CH3) of the human antibody again using standard recombinant DNA technology. Grafting loops of the VH and VL domains onto human V domain framework can compensate the loss of affinity sometimes observed with this procedure.
  • the chimeric humanized antibody gene is cloned into a mammalian plasmid vector with a drug selection marker (e.g., neomycin selection marker) and the plasmid transfected into a human myeloma cell line. Stable cell lines producing the chimeric humanized antibodies are selected using the drug selection marker. The recombinant antibody is tested for its cross reactivity with a drug selection marker (e.g., neomycin selection marker) and the plasmid transfected into a human myeloma cell line. Stable cell lines producing the chimeric humanized antibodies are selected using the drug selection marker. The
  • NEM NEM and its ability to inhibit the growth and invasion of prostate cancer cells.
  • Selected myeloma cells are scaled up as mentioned above and the purified antibody is used for the treatment of prostate cancer.
  • human B cells are grown in vitro and NEM is added to the cells in presence of certain ligands like CD40 and human T cell clones. Some of the human B cells immunized in vitro can undergo clonal selection and isotype switching. The clones of the B cells producing NEM antibodies with high affinity are selected. The cDNA encoding the antibody is isolated and transfected into human myeloma cells and stable cell lines producing the human antibody against NEM are selected, scaled up and the antibodies purified by methods mentioned above.
  • NEM or peptides derived from NEM
  • transgenic mice that carry humanV-genes.
  • These mice are defective for the expression of their endogenous (mouse) V genes.
  • the murine B cells that secrete human antibodies are then immortalized by fusing to human myeloma cells using known procedures.
  • the immortalized human myeloma cells producing the human antibodies are scaled up, purified from the cell culture medium, and used for the treatment of prostate cancer.
  • Example 4 Detection of Prostatic NEM in PC Biopsies Using Immunofluorescence
  • Prostatic NEM can be detected in PC biopsies by immunofluorescence.
  • the frozen PC specimens are sectioned to 5-10 ⁇ m size, fixed in Zamboni's fixative and incubated with
  • An aptamer is either an RNA, DNA or composed of other modified nucleosides that can bind sequences encoded by cDNA SEQ ID NOs: 2 and 3.
  • a frozen section of human prostate cancer specimen was probed with anti-NEM IgG. The antibody was removed, washed and incubated with anti-rabbit IgG-FITC. The presence of NEM-immunopositive cells along the acinar regions was observed.
  • Serum prostatic NEM can be detected using many known techniques such as radioimmunoassays, fluorescence immunoassays, ELISA, EIA and the like and as generally described in Rose, et al, MANUAL OF CLINICAL LABORATORY IMMUNOLOGY (1997).
  • Radioactive isotopes can be easily incorporated into NEM and can be easily detected.
  • a radioimmunoassay is a competitive immunoassay where a fixed number of antibody sites are made to react with two sets of antigens (i.e., NEM and NEM*R-NEM incorporated with a radioisotope).
  • NEM is usually present in variable amounts either in serum samples (unknown amounts) or in standard solutions (where known amounts of NEM are added in variable amounts in series of dilutions).
  • the radioisotope incorporated into NEM is generally either I 125 , 3H or other molecule.
  • the method for conducting NEM RIA comprises: (1) preparing NEM samples; (2) incubating the samples with NEM antiserum; (3) adding NEM*R; (4) terminating the reaction and precipitating the NEM*R-As-NEM complexes; and (5) measuring radioactivity and calculation. 1. Preparation of NEM Samples
  • the unknown serum NEM samples (e.g., patient serum) needs to be partially purified. Since NEM is a small peptide molecule, NEM*R can conjugate with various other protein molecules in serum giving rise to very high non-specific binding.
  • the serum samples are precipitated with ethanol acetate, centrifuged and the supernatant is passed through a
  • the NEM*R (approximately 25,000 cpm) is added to each tube and the incubation is continued.
  • One or more of the polyclonal antibody, monoclonal antibody, single chain antibody, phage-display antibody, in vitro evolved antibody, and aptamer is conjugated to one or more molecules including, but not limited to, biotin, avidin, fluorescein, rhodamine, alkaline phosphates, horseradish peroxidase and radionuclides such that the conjugate retains its ability to bind prostatic NEM. This conjugate is incubated with serum and the unbound conjugates are removed and the amount of conjugate bound to the NEM is calculated.
  • Example 8-Sandwich ELISA Sandwich ELISA is an improvement of ELISA that enables one to use unprocessed serum samples for the NEM assay. This eliminates the need for purification of serum samples for the assay and thus improves reliability of the results and simplifies the assay procedure.
  • ELISA plates are coated with the antibody that recognizes one antigenic site of the antigen, which in this case is NEM.
  • Untreated serum samples are added on to wells, and the coated antibody will bind with NEM in the serum samples.
  • NEM is retained because of its binding to the coated antibody.
  • a second primary antibody that recognizes another antigenic site of the NEM will react with NEM in proportion to the concentration of NEM.
  • the second antibody is conjugated with a reporter enzyme such as alkaline phosphatase. Therefore, the amount of the antigen can be measured by the color produced on addition of the substrate.
  • FIA Fluoroimmunoassay
  • Example 10-Chemiluminescence Assay In the chemiluminescence assay, either NEM or a second antibody is tagged to chemiluminescent compounds such as luciferase or green-fluorescent protein. The light is measured in a luminometer. A standard curve from NEM concentrations is generated and NEM concentrations of unknown samples are derived from this curve.
  • Example 11 - Detection of Urine Prostatic NEM Prostate cancer patients have been shown to release various prostatic products in urine. Since prostatic tumor cells produce NEM, it may also be released into urine. NEM in urine samples can be analyzed using RIA, ELISA, fluoroimmunoassay or chemiluminescence assay as described above. This process could be used to screen for prostate cancer.
  • NEM tagged with a dye or a radiochemical can be injected intravenously into a patient and can be tracked to the prostate by imaging devices.
  • the radiochemical-NEM conjugate can selectively bind and destroy cancer cells, and can also be used as noninvasive treatment procedure.
  • the prostate cancer Once the prostate cancer has spread to secondary locations, the only treatment currently available for the patient is androgen ablation therapy. However, the rumor becomes insensitive to this therapy after about 12 to 18 months leaving the patient with hardly any treatment choice. However, if one can detect the location of these secondary tumors, they could be surgically removed if such loci are limited in number.
  • a targeting molecule akin to a homing devise that can seek out the tumor. Examples of such molecules are antibodies that specifically bind to a protein that is specific for a particular type of cancer. These molecules are conjugated to a radioisotope like Tc-99.
  • the conjugate once injected into the patient seeks out the tumor as a result of the inherent affinity of the targeting molecule for proteins uniquely present on the cancer cells.
  • the location of concentration of the targeting molecule-radioisotope conjugate can be identified with a radioactive scanner such as a gamma scanner.
  • a radioactive scanner such as a gamma scanner.
  • NEM may be suitably positioned to fill this void due.
  • NEM is conjugated to Technetium 99m (Tc99), although several other isotopes are equally useful for tumor imaging.
  • Tc99 Technetium 99m
  • Several procedures for conjugating radioisotope to proteins and peptides are published and several use chelation reagents.
  • the conjugate is injected into the cancer patients. After several hours or days scintigraphs are acquired by gamma cameras. Scintigraphy is routinely used with different isotopes for the detection of tumors or other lesions. The scintigraphs reveal the site of accumulation of NEM-Tc99 conjugate, which would indicate the location and extent of primary and secondary prostate cancer.
  • Example 13-D-Peptides Blockers of NEM NEM binds to its receptors on prostate cancer cells with high affinity (low nM) and initiates biological response.
  • Current pharmacological approaches serve to identify the binding region and biological response regions of a bioactive peptide.
  • NEM fragments of various sizes are synthesized and used for receptor binding studies as well as for in vitro bioassay such as growth of prostate cancer cells. The smallest fragment that retains the receptor binding ability but fails to generate biological response could serve as a competitive antagonist of NEM. Further modifications in the primary structure of the molecule that would increase stability of the peptide, but would not affect its receptor-binding capacity, would lead to the development of antagonists.
  • an antagonist could provide a therapeutic approach for the treatment of PC, because when administered, the antagonist will occupy NEM receptors but will not generate biological response. In addition, it will prevent the action of locally produced NEM (by blocking its receptors).
  • Molecules that can block the ability of NEM to bind to its receptors could prevent the growth inducing and spread of prostate cancer cells.
  • mirror image phage display selection one can select D-peptides that bind to proteins or peptides (e.g. NEM) with high affinity (T.N. Schumacher et. al, SCIENCE, 271:1854 (1996)) and prevent its interaction with its receptor.
  • D-peptides unlike the natural L-peptides, are not degraded by the peptidases in the body.
  • the technique involves the display of random peptide sequences on a phage filament protein (usually pill of Ml 3).
  • the phage library is then panned against the target peptide that is chemically synthesized as its D-enantiomer.
  • the phage that binds to the peptide is isolated, amplified in a bacterial host and panned again against the target. After several rounds of selection, the phages that display peptides with high affinity are selected.
  • the sequence of the peptide can be deduced the gene that encodes the filament protein- peptide sequence.
  • An enantiomer of the peptide (mirror image), which is the D form of the peptide, would bind to the L-form of the target peptide.
  • NEM was synthesized as a D-peptide using conventional peptide synthesis techniques and purified by chromatography.
  • NEM was bound to a plastic petri dish and panned against a phage display library (M13) having 108 individual members.
  • M13 phage display library
  • Such libraries are available from commercial sources (e.g., New England Biolabs, MA).
  • the unbound phage was then washed off and the phage bound to NEM was eluted from the petri dish using mild acid.
  • the eluted phage was then grown in an E.coli host.
  • the phage was again panned against a fresh petri dish coated with NEM. The cycle of binding and elution was repeated 7-8 times until no more enrichment was achieved.
  • the peptide sequence from several isolated phages was determined by PCR sequencing.
  • the deduced peptide sequences were then synthesized as D- peptides and tested for their ability to block NEM-induced growth and invasion in a prostate cancer cell line. Several candidates were tested for their ability to stop tumor growth and invasion in nude mice implanted with human prostate cancer cells. The active sequence was then tested for its toxicity properties in appropriate animal models and then formulated in an aqueous media and administered to prostate cancer patients in clinical trials. All variants, homologues, mutated variants, alternatively spliced gene variants, pre- mRNA and mRNA molecules of SEQ ID NOs: 2-3 were identified using known techniques. Promoters driving the expression of SEQ ID NOs: 2-3 were also identified. Detection of SEQ ID NO:2
  • the expression of the NEM gene can be detected in prostate cancer cells by various techniques such as:
  • RT-PCR Reverse Transcriptase-Polymerase Chain Reaction
  • NEM cDNA probe The probe to detect NEM RNA can be prepared by incorporation of radiochemicals using P 32 or S 35 isotopes, by introducing fluorescence or chemiluminescence tags into the cDNA molecule, or by developing antibodies to the incorporated nucleotide. These antibodies are usually conjugated to gold or reporter enzymes such as alkaline phosphatase or horseradish peroxidase. 4. Ribozyme Amplification. Example 14-Treatment of PC Using Gene Therapy
  • the factors that suppress NEM gene expression can be used or administered to inhibit endogenous NEM gene expression.
  • An alternative approach is the targeted synthesis of these modulators in PC cells accomplished by producing recombinant retroviruses such as adenovirus driven by prostate-specific promoters such as PSA and contain cDNAs for NEM gene suppressors in their expression cassette. When injected at the site of tumor, the virus invades the tumor site, enters into cells and incorporates into their genome. Because PSA promoter drives its expression, its expression will be selectively induced in prostate cells and other cells such as blood cells, macrophages or cells of connective tissues.
  • NEM gene suppressors Because of the high efficiency of the viral genome, its expression will be high and will produce large amounts of NEM gene suppressors in the tumor area. These suppressors then act on NEM secretors to attenuate NEM synthesis by tumor cells. This approach could provide a clean, targeted and effective treatment for advanced PC.
  • Antisense molecules are gaining importance as tools for specifically inactivating mRNA that code for proteins that are involved in disease processes.
  • Antisense molecules are oligonucleotides that are complementary to a target mRNA.
  • the most commonly used antisense molecules have a DNA backbone, which is stabilized against degradation by endonucleases and exonucleases present in the body by chemical substitution of the phosphate backbone. It is believed that the primary mechanism of antisense action is through endogenous RNase H-mediated cleavage of the RNA at the region of the DNA-RNA hybrid.
  • Improved antisense molecules have a short (7-nucleotide) stretch of DNA flanked by sequences with 2'-substitution.
  • the entire molecule is usually modified in the phosphate backbone to enhance stability. These molecules are more specific and have less toxicity.
  • the backbone substitution of choice is phosphorothioate and the 2' modification includes methyl, ethyl, propyl, methoxyethoxy and allyl.
  • ribozymes are molecules that can cleave mRNA. Ribozymes can be designed to cleave specific mRNA by introducing sequences that are complementary to the target mRNA. This would allow the ribozymes to specifically bind to the target mRNA and inactivate it. Ribozymes can be chemically synthesized and administered like a conventional drug or can be expressed in appropriate tissues using viral vectors. In the former case, the molecules are stabilized against nuclease degradation by introducing appropriate chemical substitutions in the molecule.
  • Triplex molecules bind to the major grove of the DNA usually in the purine rich region and prevent the transcription of the mRNA coding for the protein of interest.
  • Several 21 nucleotide antisense molecules are synthesized using a standard DNA synthesizer. These molecules are composed of a 7-nucleotide DNA sequence in the middle and two 7 nucleotides 2' O-methyl RNA sequences flanking the central DNA portion. The entire molecule is modified in the phosphate backbone with sulfur substitution (phosphorothioate backbone). Forty sequences complementary to different regions of NEM mRNA are synthesized and purified by reverse phase HPLC method.
  • the antisense molecules are added to PC-3M cells growing in culture and the ability of the antisense to block growth and invasion and its ability to reduce NEM mRNA are monitored.
  • the more active and least toxic (as measured by cellular toxicity assays) sequences are scaled up and tested in nude mice baring prostate tumor to test the efficacy of the antisense molecule to stop the tumor growth and spread.
  • the best candidate is selected, scaled up and administered as an intravenous infusion in prostate cancer patients to determine the efficacy of these molecules in preventing the growth and spread of prostate cancer.
  • Example 16-Preparation of Prostatic NEM Antagonist-Antineoplastic Conjugates Compositions to treat prostate cancer are produced using known techniques wherein
  • NEM or NEM antibodies are conjugated to chemotherapeutic agents such as Paclitaxel, Cladribine, Pentostatin, Fludarabine, Carboplain, Isofamide, Octreotide acetate, Mitoxantrone, Streptozocin, Stoposide, Flutamie, Leuprolide, Tamoxifen, Bleomycin, Doxrubicin, Cisplatin, dacabazine, Daunorubicin, Nitrosoureas, Mithramycin, Cytarabine, Procarbazine, Hydrozyurea, Mitomycin C, Vinca alkaloids, Mitotane, Cyclophosphamide,
  • chemotherapeutic agents such as Paclitaxel, Cladribine, Pentostatin, Fludarabine, Carboplain, Isofamide, Octreotide acetate, Mitoxantrone, Streptozocin, Stoposide, Flutamie, Leuprolide, Tamoxifen, Bleo
  • Progestins 5-Flurouracil, Actinomycin D, 6-mercaptopurine, and the like. These compositions are made into a pharmaceutically acceptable form for human administration.
  • Example 17-Preparation of Prostatic NEM Antagonist-Radionuclide Conjugates Compositions to treat prostate cancer are produced using known techniques wherein the above-described NEM immunogens are conjugated to radionuclides such as Ac-225, Ac- 227, Au-198, B-ll, Bi-212, Bi-213, Br-77, Cf-252, Co-60, Cs-137, Cu-67, Ir-192, Os-194, Pb-203, Pb-212, Pd-103, Pd-109, Ra-223, Ra-226, Re-186, Re-188, Rh-105, Sc-47, Si-28, Sm-145, Sr-89, Sr-90, Ta-182, Tb-149, Th-228, Th-229, W-188, Y-88, Y-90, Y-91,
  • compositions are made into a pharmaceutically acceptable form for human administration.
  • Fig. 11 such cell-targeted therapy is employed to selectively kill cancer cells.
  • Figure 11 A depicts a prostate cancer cell.
  • An NEM-radioisotope conjugate binds to NEM receptors present on the cancer cell surface, shown in Fig. 1 IB.
  • the radioisotope emits radiation in close proximity to the cancer cell.
  • Figure 11 C illustrates that the prostate cancer cells are then killed by radiation.
  • Figure 1 ID illustrates the specificity of the NEM-radioisotope preventing the NEM-radioisotope from binding to non-prostate cells present in other organs.
  • This cancer treatment composition is a combination of the above wherein a chemotherapeutic agent and a radionuclide are conjugated to an NEM immunogen and then administered to the patient.
  • Example 19 - NEM Receptor Antagonist or Blockers of NEM Receptor NEM binding to NEM receptor is necessary for eliciting the proliferative activity of NEM as evidenced by the growth and invasion inhibiting activity of NEM antisera. Furthermore, the data shows that NEM receptor is selectively expressed in cancer cells and is not detectable in normal cells (Fig. 13). Blocking the binding of NEM to its receptor or use of an antagonist of NEM receptor will result in the growth inhibition of these cells, thereby leading to therapeutic benefits. In order to identify a molecule that possesses this property, an in vitro binding assay and a growth assay may be used.
  • Plasma membranes from PC-3M or PC3 cells are isolated by standard techniques and held in suspension in small aliquots at 4 °C in tubes or 96-well microtiter plates.
  • NEM is radiolabeled, preferably using radioactive iodine, or fluorescent labeled, purified, and used to bind to NEM receptor on the plasma membrane. It has been demonstrated that radiolabeled NEM binds to its receptor on an isolated plasma membrane from PC3-M cells and competes with unlabeled NEM.
  • Target molecules may be used to compete with radiolabeled or fluorescent labeled NEM for receptor binding to identify molecules that bind to NEM receptor and thereby prevent NEM from binding to NEM receptor. The inhibition of binding of labeled NEM to NEM receptor is monitored by a radiation counter or a fluorescence scanning detector.
  • a second assay involves using PC-3M or similar cells and monitoring the ability of target molecules to block NEM-induced growth of PC-3M, PC-3 or LNCaP cells. Growth of the cells is monitored by 3 H-thymidine incorporation as described above.
  • a library of NEM fragments are synthesized either specifically or randomly and screened for their ability to prevent binding of NEM to NEM receptor.
  • the members of this library of CT-like molecules are used for receptor biding studies in an automated or semi-automated fashion as well as in an in vitro bioassay (inhibition of growth of PC cells by NEM).
  • the smallest molecules from the library that inhibit the binding of NEM to its NEM receptor at the lowest concentration are selected for further testing.
  • chemical modifications to the molecule that would increase the stability or solubility of the molecule without affecting its receptor binding properties would lead to the development of therapeutic molecules for prostate cancer. The ideal candidates would then be screened for any toxic properties in animals prior to administration to patients.
  • a library of fragments of NEM receptor are chemically synthesized or are expressed as recombinant proteins and are purified. These fragments are incubated with radiolabled NEM and the ability of NEM to bind to these receptor fragments is determined by isolating the complex with anti-NEM antibody or by size exclusion chromatography. These molecules are then tested for their ability to inhibit binding of NEM to its receptor in isolated plasma membrane preparations from PC-3M cells (see above) or in preventing the NEM-induced growth of PC-3 M, PC3 or LnCaP cells (see above).
  • binding affinity of these soluble receptors are enhanced, if necessary, by introducing a variety of chemical modifications and further testing in the in vitro assays to see if the binding affinity has been enhanced. Predicting which modifications increase the binding of these molecules to NEM involves a mix of logical design and trial and error method. The stability and solubility of the identified molecules are tested and are enhanced, if needed, through further chemical modifications. A variety of modifications, as are generally known in the art and incorporated herein by reference, may increase the solubility and stability in blood of peptides and peptide mimetics and one or more of these methods can be used for this purpose. Finally, the soluble NEM receptor is tested for its ability to block growth of PC in animal models and its potential toxicity determined in animals prior to administration to human patients for testing efficacy.
  • a large library of combinatorial organic compounds are screened using the above assays to identify a molecule that binds to NEM receptor and interrupt binding of NEM to NEM receptor.
  • the candidates that have the maximal effect on inhibiting the binding of NEM to its receptor, as well as inhibit the proliferation of PC- 3M or similar cells, are further tested in prostate cancer animal models for their efficacy in blocking or inhibiting the growth of prostate cancer in these animal models. Its potential toxicity is determined in animals and then tested in humans patients.
  • Methods of producing a large library of combinatorially created organic compounds are generally known in the art and incorporated herein by reference.
  • This embodiment of the invention is also useful for treating other cancers with significant neuroendocrine differentiation.

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Abstract

La présente invention concerne un nouveau facteur de croissance ou marqueur neuroendocrinien ("NEM") identifié dans des cultures de cellules cancéreuses, ainsi que des conjugués de NEM et d'un agent de liaison pouvant inhiber la liaison du NEM à son récepteur.
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Title
CHIEN J. ET AL.: "Calcitonin is a prostate epithelium-derived growth stimulatory peptide" MOLECULAR AND CELLULAR ENDOCRINOLOGY, vol. 181, no. 1-2, 5 July 2001 (2001-07-05), pages 69-79, XP002332581 ISSN: 0303-7207 *
DATABASE EMBL [Online] 6 February 2002 (2002-02-06), "Rattus norvegicus clone CH230-226B10, *** SEQUENCING IN PROGRESS ***, 3 unordered pieces." XP002332537 retrieved from EBI Database accession no. AC109091 *
DATABASE EMBL [Online] 6 October 1999 (1999-10-06), "Cloning vector pGEM-URA3, complete sequence." XP002332538 retrieved from EBI Database accession no. AF173954 *
DATABASE EMBL [Online] 729 nt 20 July 2001 (2001-07-20), "EST008 Subtracted larval fat body library, bacteria-induced genes Manduca sexta cDNA clone 008 3' similar to coatomer alpha subunit, mRNA sequence." XP002332539 retrieved from EBI Database accession no. BI262743 *
See also references of WO03105892A1 *

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