Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • 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
  • C07K14/4727—Mucins, e.g. human intestinal mucin
• • 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
  • A61K39/0005—Vertebrate antigens
  • A61K39/0011—Cancer antigens
  • A61K39/001169—Tumor associated carbohydrates
  • A61K39/00117—Mucins, e.g. MUC-1
• • 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
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • 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
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55516—Proteins; Peptides

Definitions

• the invention relates to a mucin peptide with immunoenhancing properties, pharmaceutical compositions containing the peptide, and methods of treating or preventing cancer.
• the MUC1 gene is expressed in normal epithelium and several types of human cancers and at very high levels in breast tumors (1).
• a major product of this gene is a polymorphic type 1 transmembrane molecule consisting of a large, heavily glycosylated extracellular domain, a transmembrane domain, and a 72 amino acid cytoplasmic tail (2).
• the polymorphism mainly derives from variations in the numbers of a 20 amino acid tandem repeat unit present in the extracellular domain.
• a secreted MUC1 isoform (MUCl/sec) (3) has also been found, that includes a sequence of intron 2, terminates prematurely at a stop codon within this intron, and is therefore devoid ofthe transmembrane domain.
• MUC1 and fragments and derivatives thereof have been widely studied for possible use in anticancer vaccines and cancer therapy.
• U.S. Pat. No. 6,344,203 discloses peptides that mimic MUC1 and bind to IB4 lectin and anti-Gal.alpha.(l,3)Gal antibodies, for use in a cancer vaccine.
• the peptides are conjugated to carrier proteins such as tetanus toxoid, diphtheria toxoid or oxidised KLH in order to stimulate T cell help.
• U.S. Pat. No. 5,827,666 discloses a synthetic MUC1 peptide comprising at least two 20-amino acid tandem repeats of MUC1 that is capable of attaining native conformation in the absence of glycosylation.
• the synthetic peptide is indicated to be useful, inter alia, in a vaccine that also includes an amino acid sequence for an epitope.
• U.S. Pat. No. 5,989,552 discloses a conjugate of a MUC1 polypeptide or tandem repeats thereof and oxidized mannan, for use in immunotherapy.
• U.S. Pat. No. 6,080,725 discloses saponin analog adjuvants that may be utilized with MUCl and peptide fragments thereof as tumor-associated antigens in a vaccine.
• MUC1 has also been described and used as a cancer cell determinant or marker, as disclosed, for example, in published U.S. Patent applications 20020009759, 20020012931, 20020022235, and 20010051351.
• a peptide including the amino acid sequence VSIGLSFPMLP (SEQ ID NO:l), and analogs and derivatives thereof, demonstrates iimmmomodulatory activity in vivo.
• Such peptides and the nucleic acids (e.g.,, RNA, DNA) encoding them may be used as adjuvants to stimulate or otherwise enhance an immune response in a subject, animal or human, as assayed by humoral or cellular criteria known in the art.
• the enhancement of antibody production, secretion of their soluble immune mediators (e.g., cytokines), antigen presentation, effector cell production or functions (e.g., cytotoxicity), other l ⁇ iown measures of immune response, or a combination thereof may be used to show efficacy.
• the peptides or nucleic acids may be used as vaccine adjuvants, to enhance in vivo immune responses, to induce in vivo and/or in vitro the production of cytokines, as a mitogen of one or more cell subpopulations, or to enhance cellular cytoxicity. It is one object of the invention to provide a novel molecule for immunomodulation and potentiation.
• the peptide may be incorporated into a vaccine formulation with the antigen (e.g., a tumor antigen, or one derived from an infectious agent such as a bacterium, virus, protozoan, fungus, mold or yeast) or conjugated to the antigen.
• Nucleic acid may be incorporated into a formulation for genetic vaccination with the antigen and/or an expression vector encoding the antigen; or a nucleotide sequence encoding VSIGLSFPMLP (SEQ ID NO:l), or an analog or derivative thereof, may be included in an expression vector ligated to or distinct from a region encoding an antigen.
• compositions may be incorporated into cellular, acellular, fractionated or purified, recombinant, protein based, or "naked DNA" vaccines.
• the tumor types that may be treated include, but are not limited to, sarcomas and carcinomas.
• a cancer such as leukemia, lymphoma or brain cancer may be treated.
• any antibody-based or other iimnunological treatment may be enhanced with the invention.
• the invention provides an efficacious and less toxic alternative to other adjuvants for enhancement of an immune response. It can be produced under conditions that satisfy standards for biologicals intended for medical or veterinary treatment, and is biocompatible with formulations used for therapy and prophylaxis.
• peptide refers to a sequence of naturally occurring amino acids.
• peptide is intended to refer to a sequence of less than 20 amino acid residues
• polypeptide to a sequence of 20 or more amino acid residues and as used herein is intended to encompass proteins as well.
• the peptide, polypeptide or protein of the invention comprises between 11 and 200 amino acid residues, more preferably between 11 and 100 residues, even more preferably between 11 and 50 residues, and most preferably 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 residues.
• isolated refers to material removed from its original environment (e.g.,, for naturally occurring substances, removed from their natural environment). Such material could be part of a vector or a composition of matter, or could be contained within a cell, if said vector, composition or cell is not the original environment of the material.
• the peptide of the invention has immunoenhancing properties and can be used in pharmaceutical compositions and vaccines.
• the term "analog”, as used herein, is intended to mean a sequence variant having the same or similar functional characteristics (i.e. producing an immunomodulatory effect). Such variants may have a single or several (2-3) substitutions of amino acid residues, preferably conservative substitutions, such that the variant retains the immunomodulatory characteristics ofthe "parent" compound.
• derivative is intended to mean a peptide, polypeptide or protein comprising sequence VSIGLSFPMLP (SEQ ID NO: 1) that has been modified by the addition of at least one additional group and that retains the immunomodulatory ability characteristic ofthe parent compound.
• the derivative comprises a KLH molecule to which multiple copies of the peptide of the invention that are conjugated.
• the derivative may also be an antigen to which the peptide, polypeptide or protein of the invention is attached.
• Other examples of derivatives include, but are not limited to, OVA and BSA conjugated to multiple copies of the peptide.
• the peptide should also be useful in its pure form.
• the peptide, polypeptide or protein ofthe present invention may be produced by conventional methods of chemical synthesis or by recombinant DNA techniques.
• a host microorganism may be transformed with a DNA fragment encoding the polypeptide and the polypeptide harvested from the culture.
• the host organism may be, for example, a bacterium, a yeast, a viral vector, or a mammalian cell, whereby the DNA fragment in question is integrated in the genome of the host organism or inserted into a suitable expression vector capable of replicating in the host organism.
• the DNA fragment is placed under the control of regions containing suitable transcription and translation signals. Methods for obtaining polypeptides by these means are familiar to persons skilled in the art.
• nucleic acid encoding the peptide, polypeptide or protein ofthe invention, or derivative or analog thereof.
• the nucleic acids of the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art, and may be cloned using techniques known in the art.
• the invention also includes a vector comprising the nucleic acid of the invention, and a host cell comprising the nucleic acid ofthe invention.
• the invention also includes an immunogenic pharmaceutical composition
• an immunogenic pharmaceutical composition comprising the peptide or polypeptide of the invention, or an analog or derivative thereof, and a pharmaceutically acceptable excipient or carrier.
• the pharmaceutical composition includes at least one antigen, preferably a tumor antigen.
• Tumor antigens include, for example, autologous tumor cells, allogeneic tumor cells, gangliosides, carcinoembryonic antigen, prostate specific antigen, melanoma associated antigens and p53 as well as other tumor antigens familiar to those of skill in the art.
• antigens that may be used in the invention include viral antigens, such as those derived from papillmaviruses, Hepatitis B and C, H V, and HTLN-1, and antigens derived from bacteria and other infectious microorganisms such as M. Tuberculosis.
• viral antigens such as those derived from papillmaviruses, Hepatitis B and C, H V, and HTLN-1
• antigens derived from bacteria and other infectious microorganisms such as M. Tuberculosis.
• the invention also includes a vaccine, and a method of inducing or enhancing an immunological response.
• the vaccine comprises an immunomodulatory peptide or polypeptide of the invention, or an analog or derivative thereof, an antigen, and a pharmaceutically acceptable excipient or carrier, hi a particularly preferred embodiment, the antigen is a tumor antigen.
• the immunogenic pharmaceutical composition or vaccine of the invention may also include one or more additional adjuvants, such as BCG, KLH, IL-2, GM-CSF and cytoxan, which may be optionally linked to the immunomodulatory peptide or polypeptide of the invention, h one preferred embodiment, the immunomodulatory peptide ofthe invention is conjugated to KLH with malaimide-activated KLH.
• additional adjuvants such as BCG, KLH, IL-2, GM-CSF and cytoxan
• additional adjuvants such as BCG, KLH, IL-2, GM-CSF and cytoxan
• additional adjuvants such as BCG, KLH, IL-2, GM-CSF and cytoxan
• Safe and effective dosages of the pharmaceutical compositions and vaccines ofthe invention can be determined by persons of skill in the art without undue experimentation.
• a safe and effective dosage is considered to be one that can be administered to a subject to produce a beneficial effect on the subject's immune response without causing adverse effects that would be considered unacceptable by persons of skill in the art. It will be appreciated that any such adverse effects must be balanced against the benefits of the treatment of the invention, alternative available treatments, and other factors familiar to those of skill in the art.
• the invention provides a method for the prevention or treatment of tumors, particularly cancerous tumors.
• DA-3 mammary tumor cells or with DA-3 cells transfected with the neomycin vector only (DA-3/neo), or with the transmembrane MUCl isoform (DA-3/TM) or with the secreted MUC1 isoform (DA-3/sec).
• DA-3 cells transfected with the neomycin vector only (DA-3/neo), or with the transmembrane MUCl isoform (DA-3/TM) or with the secreted MUCl isoform (DA-3/sec).
• Figure 3 Growth of tumors in BALB/c (nuVnu ) mice that had been implanted with 10 untransfected DA-3 mammary tumor cells, or DA-3 cells transfected with the neomycin vector only (DA-3/neo), or with the transmembrane MUCl isoform (DA-3/TM) or with the secreted MUCl isoform (DA-3/sec).
• FIG. 4 Tumor growth in nude BALB/c mice after inoculation with 10 6 untransfected DA-3 mammary tumor cells or with DA-3 cells transfected with the neomycin vector only (DA-3/neo), or with the transmembrane MUCl isoform (DA-3/ TM) or with three separate transfectants ofthe secreted MUCl isoform (DA-3/sec).
• Figure 5 Effect of vaccination with DA-3 cells transfected with the secreted MUCl isoform (DA-3/sec) cells on the growth in BALB/c mice of a mixture of DA-3 cells transfected with the transmembrane MUCl isoform (DA-3/TM) and DA-3 sec cells.
• DA-3/sec secreted MUCl isoform
• Figure 6 Effect of vaccination with DA-3 cells transfected with the secreted MUCl isoform (DA-3/sec cells) on the growth in BALB/c mice of a mixture of DA-3 cells transfected with the neomycin vector only (DA-3/neo) and DA-3 sec cells.
• Figure 7. Effect of vaccination with DA-3 mammary tumor cells transfected with the secreted MUCl isoform (DA-3/sec cells) on the growth in BALB/c mice of a mixture of the RENCA renal cell carcinoma and DA-3/sec cells.
• Figure 8 Effect of vaccination with DA-3 mammary tumor cells transfected with the secreted MUCl isoform (DA-3/sec cells) on the growth in BALB/c mice of a mixture of K7 osteosarcoma cells and DA-3/sec mammary tumor cells.
• FIG. 9 Schematic representation of the cDNA sequences of the human transmembrane MUCl (MUCl/ TM) and the human secreted MUCl (MUCl/sec) isoforms.
• Figure 10 Peptide vaccination. Effect of vaccination with KLH-bound unique eleven amino acid peptide ofthe secreted human MUCl isoform on the growth of a mixture of KLH bound unique peptide with DA-3 cells transfected with either the neomycin vector alone (DA-3/neo) or with the transmembrane MUCl isoform (DA-3/TM).
• FIGS 11A and 11B Effect of KLH-bound unique amino acid peptide from the secreted human MUCl isoform on the growth of a mixture of KLH bound unique peptide with either RENCA tumor cells in BALB/c mice (11 A) or Lewis lung carcinoma on C57/BL6 mice (11B).
• the figure shows that efficacy of MUCl/sec peptide vaccination is not strain restricted.
• FIG. 12 Effect of administration of a mixture containing a lower number of challenge cells (DA-3/TM) with DA-3/sec cells on tumor growth.
• the figure shows that a lower number of challenge cells allows effective protection without vaccination.
• Figure 14 Effect of perform inhibitor concanamycin A on cytotoxicity of DA-3/sec splenic cells.
• Figure shows that perform mediates lysis of DA-3/sec tumor cells by activated splenocytes.
• Figure 15. Effect of anti-Fas antibody on the cytotoxicity of DA-3/sec activated splenocytes against DA-3/sec targets and positive controls.
• Figure shows that Fas/FasL does not play a role in DA-3/sec cytotoxicity.
• FIGS 17A and 17B Effect of MUCl/sec peptide on immune response of Dl- DMBA-3 tumor bearers in vitro (17A) and in vivo (17B).
• Figures show that MUCl/sec peptide stimulates the immune response of Dl-DMBA-3 tumor bearers.
• FIGS 18A and 18B Effect of normal BALB/c splenocytes with and without MUCl/sec peptide on lysis of YAC-1 (18A) and EL-4 (18B) target cells.
• FIGS 19A and 19B Effect of normal splenocytes from different mouse strains with and without MUCl/sec peptide on lysis of DA-3/sec tumor cells.
• Figure shows that normal splenocytes kill DA-3/sec tumor cells in the presence of MUCl/sec peptide.
• DA-3 cells a mammary tumor cell line that leads to metastatic lesions and death of BALB/c mice hosts, with the transmembrane MUCl isoform (DA-3/ TM), with the secretory form (DA-3/sec), or with the neomycin vector alone (DA- 3/neo).
• Transfection with the secretory form of MUCl renders the DA-3 cells incapable of growing in intact BALB/c mice, while they grow in nude BALB/c animals.
• Vaccination with DA-3/sec cells confers protection to challenge with the DA-3/TM or DA-3/neo cells and against two unrelated tumors also syngeneic to BALB/c mice. It appears that a unique
• mice and Tumors Intact BALB/c mice were maintained by brother-sister matings in our laboratory at the University of Miami School of Medicine. BALB/c nu+/nu+ were purchased from Taconic Labs.
• the DA-3 tumor cell line was derived from the in vivo Dl- DMBA-3 mammary tumor syngeneic to BALB/c mice (4). The DA-3 cells produce tumors in BALB/c mice and cause metastatic lesions in the lung.
• the cell line is grown in RPMI- 1640 media supplemented with 5% FCS (fetal calf serum), 100 U penicillin, 100 ⁇ g/ml streptomycin, 5 x 10 "5 M 2-Beta-mercaptoethanol (2-BME), 2 mM L-glutamine, 1% nonessential amino acids, 1% essential amino acids, and 1% sodium pyravate (all from GIBCO BRL, Gaithersburg, MD), and maintained by serial passage. These cells are free of endotoxin as ascertained by routine assays with Limulus amebocyte lysate (Pyrogent® plus; Whittaker M.A. Bioproducts, Inc., Walkersville, MD). Preparation of peptide fragments and antibodies.
• FCS fetal calf serum
• 2-BME 2-Beta-mercaptoethanol
• 2-BME 2-Beta-mercaptoethanol
• 2 mM L-glutamine 2 mM L-glutamine
• the monoclonal antibody designated H 3 was used to detect the tandem repeat sequence common to both MUCl/ TM and MUCl/sec.
• Antibody 1709 (Aves Labs, Tigard, OR) was prepared by immunizing chickens with the MUCl/sec specific peptide VSIGLSFMLP (SEQ ID NO:) conjugated to KLH with malaimide-activated keyhole limpet hemocyanin (KLH). Free peptide concentrations and conjugations of at least 150 peptide molecules per KLH molecule were utilized for immunization. Pre-immune IgY antibody was prepared from eggs collected prior to immunization.
• DA-3 cells Transfection of DA-3 cells.
• Stable transfectants expressing MUCl isoforms were generated by co-transfecting an expression plasmid harboring either MUCl/TM or the secreted form MUCl/sec cDNA with the neomycin plasmid (pSV2 neo) selection marker into DA-3 mouse mammary tumor cells.
• pSV2 neo neomycin plasmid
• Tumor Growth in vivo The parental DA-3 cells, the neomycin control and MUCl isoforms transfected cells were washed, counted, and 10 cells were injected subcutaneously (s.c.) in syngeneic intact or nude (nu + /nu + ) BALB/c mice. Tumor growth was monitored every 2-3 days and mice bearing a tumor of >10 mm as measured with a caliper were scored as tumor positive.
• mice were vaccinated two or three times two weeks apart with 10 DA-3/sec tumor cells in a physiological saline solution. Two weeks after the last vaccine administration the animals were challenged with DA-3 cells untransfected or transfected with neomycin vector alone or with the MUCl transmembrane isoform. In some experiments the tumors used to challenge were the renal cell carcinoma, RENCA, or the osteosarcoma K7. 10 6 tumor cells were used for each challenge mixed with 10 DA-3/sec cells.
• the unique 11 amino acid peptide present in the secreted MUCl isoform was synthesized by Aves Labs, hie. (Tigard, OR) and bound to KLH. Because the exact ratio of peptide to KLH varies with each synthesis; the preparations were normalized to deliver the same amount of peptide in each administration.
• the KLH peptide was used as a vaccine two weeks apart. Fifty ⁇ g of peptide was given to BALB/c mice with complete Freund's adjuvant for the first vaccination. Two weeks later 25 ⁇ g of the peptide was administered with incomplete Freund's adjuvant.
• mice were challenged with 10 DA-3/sec or DA-3/neo cells mixed with 25 ⁇ g of peptide with no ' adjuvant.
• unvaccinated mice were given the same tumor challenge as in the experimental groups to serve as controls. Tumor growth was monitored every two or three days and tumor size was measured by caliper.
• Example 1 DA-3 mammary tumor cells were transfected as described in Materials and Methods with either the transmembrane and secretory isoforms of the human MUCl. The success of the transfections was proven by staining the cells for the presence of the MUCl tandem repeat using the H 23 antibody specific for this sequence (5). The resulting cell lines were used in in vivo experiments to determine the incidence and time of tumor appearance in BALB/c mice. As seen in Table 1, implantation of DA-3, DA-3/neo and DA-3/TM mammary tumor cells into mice gave rise to palpable tumors of approximately the same size by seven days and by 15 days essentially all animals had sizable tumors.
• DA-3 cells transfected with the MUCl secreted form failed to cause tumor development.
• these animals remain tumor free even after more than 12 months after implantation, while animals with the other three types of DA-3 cells fail to survive past 100 days. It should be pointed out that no tumor growth was observed in mice that were implanted with the DA-3/sec tumor cells up to a concentration of 1 x 10 cells per inoculation.
• Figure 4 shows that the three separate DA-3/sec cell transfectants, i.e. DA-3/sec, DA-3/sec 11, and DA-3/sec 22, could all grow in vivo in BALB/c nude mice, although their growth kinetics in the immunodeficient mice were somewhat different.
• DA- 3/sec tumors that grew in nude BALB/c animals did not grow when implanted in immunologically intact BALB/c mice, but do grow when inoculated with other nude BALB/c animals (data not shown).
• the results of these experiments suggest that T cells, and to a lesser degree another type of non T cell effector, are involved in the control of growth of tumors transfected with the secreted form of MUC 1.
• DA-3 cells expressing no mucin were also included in the study.
• Experimental groups received two or three injections one week apart of 1 x 10 6 DA-3/sec cells prior to challenge with 1 x 10 6 cells DA-3/TM or DA-3/neo cells alone or mixed with DA-3/sec cells.
• unvaccinated animals were challenged with either DA-3/neo or DA-3/TM cells.
• Animals receiving two injections of unmixed DA-3 sec (10 6 cells) caused only a short delay in the appearance of tumors when challenged with DA-3/TM cells alone, i.e.
• mice that had received three injections of 10 6 DA-3/sec cells before challenge unexpectedly retarded the growth of not only DA-3/TM cells but also DA-3/neo cells compared to the unvaccinated control groups.
• the DA-3/TM tumor cells used for challenge were mixed with the DA-3/sec cells at the time of implantation ( Figure 5), there was not only a retardation in the time of tumor appearance compared to the control groups, but there was an actual substantial protection resulting in lack of growth of the DA-3/TM tumor cells in 50%> ofthe vaccinated mice.
• a schematic drawing of the cDNAs from the MUCl/TM and MUCl/SEC isoforms is shown in Figure 9.
• the cDNAs are depicted from their 5' termini at the left of the figure.
• the tandem repeat array is depicted by the barred region and the regions coding for the signal peptide, transmembrane domain, and cytoplasmic domain are indicated by SP, TM, and CYT respectively and have the same shadings in the two forms.
• the amino acid sequence at the terminal end ofthe MUCl/sec isoform is VSIGLSFPMLP (3).
• a direct ELISA utilizing this reagent was developed to detect the peptide in the supematants of DA-3/sec cultures. Briefly, 50 ⁇ l of coating buffer containing 0.2 - 10.0 ⁇ g of protein/ml or tissue culture supematants from semm free cultures were plated overnight at 4°C. Blocking buffer was added for 1 hour at 37° C, plates washed 3 times, primary antibody incubated for 1 hour or overnight; wash 3 times; add secondary antibody, if required, for 1 hour, wash 3 times; add visualization solution; incubate and read. A positive control of purified peptide at known concentrations was employed in all assays. All cultures tested were positive for this molecule (data not shown).
• Example 7 Experiments to • evaluate the possible beneficial effects of vaccination with this peptide were performed (Figure 10).
• Control mice that received injections with DA-3/TM or DA-3/neo cells (10 6 cells) resulted in 100%> tumor growth whether in the presence or absence of adjuvant alone.
• Experimental mice received two injections eight days apart with KLH-bound unique peptide in the presence of adjuvant. Eight days after the second injection the animals were challenged with a mixture of KLH bound peptide and tumor cells. Challenge with the peptide mixed with DA-3/TM cells resulted in tumor growth in only 7 out of 12 mice, with an additional animal developing a tumor at 4 weeks.
• Example 9 In order to determine whether a lower number of challenge cells mixed with DA-3 sec cells will obviate the necessity of previous exposure to the latter in order to provide protection against tumor growth, 5 x 10 5 DA-3/TM cells were mixed with an equal amount of DA-3/sec cells and administered to mice. As shown in Figure 12, this treatment provided 60% protection against tumor development without any previous vaccination.
• cytotoxicity assays against 51 Cr labeled cells were performed using splenic cells from mice that received an inoculation of DA-3/sec tumor cells two weeks prior to obtaining the cells.
• 10 x 10 6 splenocytes were cultured for 5 days in the presence of 2 x 10 5 - 2 x 10 6 mitomycin-C (MMC) treated DA-3/sec cells.
• MMC mitomycin-C
• DA-3/sec primed splenocytes have low levels of cytotoxicity against DA-3/TM, DA- 3/neo and DA-3 targets.
• Cells ofthe immune system have multiple mechanisms of killing, which include Fas - Fas ligand interactions (7) and perforin-granzyme mediated killing (8)
• the DA-3/sec primed splenocytes were treated with anti-Fas antibody or Concanamycin A (CMA), an inhibitor of vacuole acidification through blockage of H + ATPase that blocks perforin- mediated killing (9).
• CMA effectively blocked killing of DA-3/sec targets in a dose- dependent manner ( Figure 14), while no effect on cytotoxicity was observed with the addition of the anti-Fas antibody ( Figure 15).
• DA-3 cells transfected with the human MUCl secreted form but not the transmembrane form, stimulate effector cells to respond against DA-3/sec cells both in vivo and in vitro.
• a synthetic peptide was made corresponding to this peptide (termed “immunoenhancing peptide", or IEP), which when used in vaccination protocols, has been found to have protective effects against the DA3, D A3, neo, DA3-TM, K7 osteosarcoma, RENCA, and Lewis lung carcinoma cells.
• this peptide to stimulate immune cells in a NK cell assay was tested. This assay was chosen based on the observation that the DA-3/sec tumor was suppressed during the initial stages of growth in the nude mice, suggesting that an innate immune component may be involved in the immediate reaction against DA-3/sec. Therefore, to test for immunomodulatory activity of the immunoenhancing peptide (IEP), BALB/c splenocytes were pretreated for 30-45 minutes with 10 ⁇ g IEP and subsequently, chromium-labeled YAC-1 or EL-4 targets were added in a 4-hour assay as previously described (11).
• IEP immunoenhancing peptide
• the data disclosed herein indicate that transfection of a novel secreted isoform ofthe MUCl gene (MUCl/sec) prevents the growth of an aggressive, immunogenic established tumor cell line in BALB/c mice. When used in vaccination protocols, it can afford protection against syngeneic tumors in a non-specific manner. A unique eleven amino acid peptide present in the MUCl/sec molecule is involved in the protective effects and when used in vaccination protocols it is capable of acting effectively against various tumor cell lines. The data also suggest that cytotoxicity by effector cells of the innate and/or adaptive immune system is involved in the observed results.
• this peptide Since the effects of this peptide are not restricted to a given type of tumor or mouse strain, it should be possible to use it alone and/or in combination with other immunomodulatory molecules against a wide variety of tumors. No toxicity has been detected in the animals injected with the peptide.
• the broad spectrum of action of this IEP may permit the use of this agent not only against tumors, but also against viral or bacterial diseases such as HIV, hepatitis B and C, and other microbial infections (e.g., tuberculosis), where an enhancement of the immune responses may have positive effects in the exposed individuals.
• Ceriani RL, Thompson KE, Peterson JA, and Abrahams S Surface differentiation antigens of human mammary epithelial cells carried on the human milk fat globule. Proc. Natl. Acad. Sci. (Wash) 74: 582-587, 1977.
• Herberman RB, Nunn ME, and Lavrin DH Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. I. Distribution of reactivity and specificity. Int. J. Cancer 16: 216-223, 1975. 11. Paul R, Padmanabhan R, and Lopez DM: Characterization of the effector cells mediating an "innocent bystander" cytotoxicity reaction induced by a syngeneic mammary adenocarcinoma in mice. Cancer Res. 44: 4480-4486, 1984.

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• 2002
  • 2002-04-02 JP JP2002576876A patent/JP4223813B2/ja not_active Expired - Fee Related
  • 2002-04-02 CN CNA028077784A patent/CN1525864A/zh active Pending
  • 2002-04-02 DE DE60223232T patent/DE60223232T2/de not_active Expired - Fee Related
  • 2002-04-02 NZ NZ528234A patent/NZ528234A/en unknown
  • 2002-04-02 CA CA002443045A patent/CA2443045A1/en not_active Abandoned
  • 2002-04-02 US US10/113,800 patent/US7285282B2/en not_active Expired - Fee Related
  • 2002-04-02 WO PCT/US2002/010155 patent/WO2002078607A2/en not_active Ceased
  • 2002-04-02 KR KR10-2003-7012901A patent/KR20030092044A/ko not_active Withdrawn
  • 2002-04-02 AT AT02757916T patent/ATE376836T1/de not_active IP Right Cessation
  • 2002-04-02 EP EP02757916A patent/EP1372691B1/en not_active Expired - Lifetime
  • 2002-04-02 BR BR0208771-5A patent/BR0208771A/pt not_active IP Right Cessation
• 2004
  • 2004-08-04 US US10/910,612 patent/US20050095675A1/en not_active Abandoned
• 2005
  • 2005-04-05 JP JP2005109271A patent/JP2005247861A/ja not_active Withdrawn

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