WO2001058947A1 - Complexes lipophiliniques utilises pour diagnostiquer et traiter un cancer - Google Patents

Complexes lipophiliniques utilises pour diagnostiquer et traiter un cancer Download PDF

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WO2001058947A1
WO2001058947A1 PCT/US2001/004439 US0104439W WO0158947A1 WO 2001058947 A1 WO2001058947 A1 WO 2001058947A1 US 0104439 W US0104439 W US 0104439W WO 0158947 A1 WO0158947 A1 WO 0158947A1
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Prior art keywords
antibody
lipophilin
complex
cancer
patient
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PCT/US2001/004439
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English (en)
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WO2001058947A9 (fr
Inventor
Darrick Carter
Thomas S. Vedvick
John Vallieve-Douglass
Raymond L. Houghton
Davin C. Dillon
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Corixa Corporation
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Priority to AU2001236911A priority Critical patent/AU2001236911A1/en
Publication of WO2001058947A1 publication Critical patent/WO2001058947A1/fr
Publication of WO2001058947A9 publication Critical patent/WO2001058947A9/fr

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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
    • C07K14/4721—Lipocortins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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 therapy and diagnosis of cancer, such as breast, prostate and ovarian cancer.
  • the invention is more specifically related to the use of lipophilin complexes for prevention and treatment of such cancers, as well as for cancer diagnosis and monitoring.
  • markers that permit an earlier and/or more accurate diagnosis and that may facilitate the selection of a course of treatment and monitoring of patients.
  • markers may further form the basis for more effective therapies for certain cancers.
  • immunotherapies employing such proteins, or the DNA encoding the proteins have the potential to substantially improve cancer treatment and survival.
  • Such therapies may involve the generation or enhancement of an immune response to an antigenic tumor marker.
  • the present invention provides compositions and methods for the diagnosis and therapy of cancers, such as breast, ovarian and prostate cancers.
  • the present invention provides isolated lipophilin complexes, as well as DNA molecules and constructs encoding such complexes, comprising a first lipophilin-like polypeptide linked to a second lipophilin-like polypeptide.
  • the first lipophilin-like polypeptide is a mammaglobin or mammaglobin- like polypeptide and/or the second lipophilin-like polypeptide is a lipophilin B or lipophilin B-like polypeptide.
  • the present invention provides pharmaceutical compositions comprising a complex as described above, in combination with a physiologically acceptable carrier.
  • vaccines are provided.
  • Such vaccines comprise a complex as described above, in combination with an immunostimulant.
  • the present invention provides antibodies, such as monoclonal antibodies, or antigen-binding fragments thereof, that bind to a complex as described above, wherein the antibodies or fragments thereof do not detectably bind to uncomplexed lipophilin, e.g., lipophilin A, lipophilin B or lipophilin C. Diagnostic kits comprising such antibodies and fragments thereof are also provided.
  • the present invention further provides pharmaceutical compositions that comprise: (a) an antibody or antigen-binding fragment thereof that specifically binds to a complex as described above; and (b) a physiologically acceptable carrier.
  • the present invention provides methods for inhibiting the development of cancer, such as breast, ovarian or prostate cancer, in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as recited above.
  • the patient may be afflicted with a cancer, in which case the methods provide treatment for the disease, or a patient considered at risk for such a disease may be treated prophylactically.
  • the present invention further provides, within other aspects, methods for removing tumor cells from a biological sample, comprising contacting a biological sample with T cells that specifically react with lipophilin complex, wherein the step of contacting is performed under conditions and for a time sufficient to permit the removal of cells expressing the complex from the sample.
  • methods for inhibiting the development of a cancer in a patient, comprising administering to a patient a biological sample treated as described above.
  • Methods are further provided, within other aspects, for stimulating and/or expanding T cells specific for a lipophilin complex, comprising contacting T cells with one or more of: (i) a lipophilin complex as described above; (ii) a polynucleotide encoding such a complex; and/or (iii) an antigen presenting cell that expresses such a complex; under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells.
  • Isolated T cell populations comprising T cells prepared as described above are also provided.
  • the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a T cell population as described above.
  • the present invention further provides methods for inhibiting the development of a cancer in a patient, comprising the steps of: (a) incubating CD4 + and/or CD8 + T cells isolated from a patient with one or more of: (i) a polypeptide comprising at least an immunogenic portion of a lipophilin complex; (ii) a polynucleotide encoding such a complex; and (iii) an antigen-presenting cell that expressed such a complex; and (b) administering to the patient an effective amount of the proliferated T cells, and thereby inhibiting the development of a cancer in the patient.
  • Proliferated cells may, but need not, be cloned prior to administration to the patient.
  • the present invention provides methods for determining the presence or absence of a cancer in a patient, comprising (a) contacting a biological sample obtained from a patient with an antibody or antigen-binding fragment thereof that specifically binds to a complex as described above; (b) detecting in the sample an amount of complex that binds to the antibody or fragment thereof; and (c) comparing the amount of complex with a predetermined cut-off value.
  • the antibody is a monoclonal antibody.
  • Step (b) may comprise, for example, a two-antibody sandwich assay.
  • the level of free components of the complex may also be assayed, and the ratio of complex to components determined.
  • step (c) involves comparing the ratio to a predetermined cut-off value.
  • the present invention also provides, within other aspects, methods for monitoring the progression of a cancer in a patient.
  • Such methods comprise the steps of: (a) contacting a biological sample obtained from a patient at a first point in time with an antibody or antigen-binding fragment thereof that specifically binds to a complex as described above; (b) detecting in the sample an amount of complex that binds to the antibody or fragment thereof; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of complex detected in step (c) with the amount detected in step (b).
  • the level of free components of the complex may also be assayed, and the change in the ratio of complex to components compared in step (d).
  • the present invention provides antibodies, such as monoclonal antibodies, that bind to a polypeptide complex as described above, as well as diagnostic kits comprising such antibodies. Diagnostic kits comprising one or more oligonucleotide probes or primers as described above are also provided.
  • Figure 1 is a diagram depicting a lipophilin complex comprising glycosylated mammaglobin and lipophilin B. Disulfide bonds link the two component proteins, which are released upon reduction of the complex.
  • Figures 2A and 2B are photographs illustrating the association of mammaglobin and lipophilin B by disulfide bonds.
  • Figure 2A depicts purified mammaglobin analyzed on a 4-20% SDS-PAGE gel stained with Glyco-Pro Glycoprotein detection kit (Sigma Chemical Co., St. Louis, MO). Lane 1 shows the complex in non-reducing SDS sample buffer, and lane 2 shows the complex with 10 mM DTT in the sample buffer.
  • Figure 2B depicts the same gel restained with silver stain. The arrow denotes the weakly staining mammaglobin. A new band is detected in the reduced lane (lane 2). The position of molecular weight markers is indicated on the left side of each figure.
  • Figures 3A and 3B illustrate the results of N-terminal sequencing of a lipophilin identified as being associated with mammaglobin. The first ten N-terminal residues were determined, as indicated.
  • FIG. 4 shows that mammaglobin is linked to lipophilin B via cysteine bridge from mammaglobin amino acid 4 to lipophilin B amino acid 67.
  • the 1621.6 ion matches the combined molecular weight of mammaglobin tryptic fragment 1-13 and lipophilin B tryptic fragment 67-69.
  • mammaglobin tryptic fragment 1- 13 MW 1316.5
  • lipophilin B tryptic fragment 67-69 MW 308.1
  • the present invention is generally directed to compositions and methods for the therapy and diagnosis of cancer, such as breast, ovarian and prostate cancer.
  • the compositions described herein generally include one or more lipophilin complexes (i.e., complexes comprising two different lipophilin-like polypeptides, linked by one or more disulfide bonds).
  • one lipophilin-like polypeptide is a mammaglobin or a mammaglobin-like polypeptide; more preferably the complex comprises a mammaglobin polypeptide and a lipophilin B polypeptide.
  • the present invention is based, in part, on the discovery that such complexes form in breast tumor cells, and may be of diagnostic, prognostic and therapeutic use.
  • the invention further provides antibodies, and antigen-binding fragments thereof, that specifically bind to lipophilin complexes. Such antibodies may be used within therapeutic and diagnostic methods, as described herein.
  • a lipophilin complex is an association of at least two different lipophilin-like polypeptides linked by disulfide bonds.
  • Lipophilin-like proteins are members of the uteroglobin superfamily of proteins, and include mammaglobin (SEQ ID NO:l), as well as lipophilin A, lipophilin B (SEQ ID NO:2) and lipophilin C (also known as mammaglobin B) (see Zhao et al., Biochem. Biophys. Res. Comm. 256:141- 155, 1999; Lehrer et al., FEBS Letters 432: 63-161, 1998).
  • Preferred complexes comprise a mammaglobin polypeptide, most preferably associated with (e.g., linked via disulfide bonds to) a lipophilin B polypeptide.
  • the mammaglobin polypeptide within such complexes is preferably glycosylated (as indicated in Figure 1).
  • two lipophilin-like polypeptides are said to form a complex if the polypeptides are linked.
  • the linkage between the two lipophilin-like polypeptides may vary, but will typically comprise one or more covalent linkages, and most typically will comprise one or more disulfide bonds.
  • Such disulfide linkages may be detected, for example, based on the presence of a higher molecular weight complex under non-reducing conditions, and the separation of the complex into lower molecular weight components under reducing conditions (e.g., using SDS-PAGE analysis as described herein).
  • a lipophilin-like polypeptide is a polypeptide that comprises: (i) a native lipophilin or mammaglobin protein, (ii) a portion of such a protein that is capable of forming a complex as described above, or (iii) a variant of such a protein that differs in one or more substitutions, deletions, additions and/or insertions, such that the ability of the variant to form a lipophilin complex is not substantially diminished.
  • a portion or other variant to associate with a lipophilin-like partner may be enhanced or unchanged, relative to the native lipophilin-like protein, or may be diminished by less than 50%, and preferably less than 20%, relative to the native lipophilin-like protein.
  • a mammaglobin polypeptide may comprise a full length native mammaglobin sequence, or a portion or other variant of such a sequence, provided that the ability of the polypeptide to associate with at least one other lipophilin-like protein (preferably lipophilin B) is not diminished, relative to the ability of a native mammaglobin.
  • a polypeptide is preferably glycosylated.
  • a lipophilin B polypeptide may comprise a full length native lipophilin B molecule, or a portion or other variant thereof that associates with at least one other lipophilin-like protein (preferably mammaglobin) to form a complex as described above.
  • Lipophilin-like protein variants may generally be identified by modifying a lipophilin-like protein sequence and evaluating the ability to form a complex. Preferred variants include those in which substitutions are made at no more than 20% of the residues in the native sequence.
  • a variant contains conservative substitutions.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
  • a variant may also, or alternatively, contain nonconservative changes.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
  • Polypeptides may further comprise sequences not normally present within a native lipophilin. Such sequences include signal (or leader) sequences at the N-terminal end of the polypeptide, which co-translationally or post-translationally direct transfer of the polypeptide.
  • a polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.
  • a polypeptide may be conjugated to an immunoglobulin Fc region.
  • Polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides may be readily prepared from DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells, such as mammalian and plant cells. Preferably, the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO.
  • Supernatants from suitable host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.
  • a suitable purification matrix such as an affinity matrix or an ion exchange resin.
  • Polypeptides having fewer than about 150 amino acids may also be generated by synthetic means using techniques well known to those of ordinary skill in the art.
  • such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 55:2149-2146, 1963.
  • Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc. (Foster City, CA), and may be operated according to the manufacturer's instructions.
  • lipophilin-like polypeptides as described herein are isolated.
  • An "isolated" polypeptide is one that is removed from its original environment.
  • a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system.
  • such polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
  • the present invention further provides agents, such as antibodies and antigen-binding fragments thereof, that specifically bind to a lipophilin complex.
  • an antibody, or antigen-binding fragment thereof is said to "specifically bind" to a complex if it reacts at a detectable level (within, for example, an ELIS A) with the complex.
  • Certain preferred antibodies are mammaglobin-specific (i.e., bind to mammaglobin, preferably in its glycosylated form, and do not detectably bind free lipophilin A, lipophilin B or lipophilin C under similar conditions).
  • Other antibodies that may be used within certain diagnostic methods provided herein specifically bind to lipophilin A, lipophilin B or lipophilin C.
  • Antibody binding refers to a noncovalent association between two separate molecules. The ability to bind may be evaluated by, for example, determining a binding constant for the association. The binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations, and may be determined using methods well known in the art. In general, an antibody is said to "bind" to a complex when the binding constant for non-covalent association with the complex exceeds about 10 L/mol. Binding agents may be further capable of differentiating between patients with and without a cancer, such as breast, ovarian or prostate cancer, using the representative assays provided herein.
  • antibodies or other binding agents that bind to a lipophilin complex will generate a signal (within at least one of the methods provided herein) indicating the presence of a cancer in at least about 20% of patients with the disease, and will generate a negative signal indicating the absence of the disease in at least about 90% of individuals without the cancer.
  • biological samples e.g., blood, sera, urine, sputum and/or tumor biopsies
  • a cancer as determined using standard clinical tests
  • a binding agent may be a ribosome, with or without a peptide component, and RNA molecule or a polypeptide.
  • a binding agent is an antibody or an antigen-binding fragment thereof.
  • Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.
  • an immunogen comprising the complex is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats).
  • the complexes of this invention may serve as the immunogen without modification.
  • a superior immune response may be elicited if the complex is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin.
  • the immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the complex may then be purified from such antisera by, for example, affinity chromatography using the complex coupled to a suitable solid support.
  • Monoclonal antibodies specific for a complex of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the complex of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed.
  • the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells.
  • a preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the complex. Hybridomas having high reactivity and specificity are preferred.
  • Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies.
  • various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood.
  • Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.
  • the complexes of this invention may be used in the purification process in, for example, an affinity chromatography step.
  • the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques.
  • immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments.
  • the Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns.
  • Monoclonal antibodies of the present invention may be coupled to one or more therapeutic agents.
  • Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof.
  • Preferred radionuclides include 90 Y, l23 I, 125 I, ,3 , I, 186 Re, 188 Re, 211 At, and 212 Bi.
  • Preferred drugs include methotrexate, and pyrimidine and purine analogs.
  • Preferred differentiation inducers include phorbol esters and butyric acid.
  • Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.
  • a therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group).
  • a direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other.
  • a nucleophilic group such as an amino or sulfhydryl group
  • on one may be capable of reacting with a carbonyl- containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.
  • a linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities.
  • a linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible. It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, IL), may be employed as the linker group.
  • Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues.
  • a linker group that is cleavable during or upon intemalization into a cell.
  • a number of different cleavable linker groups have been described.
  • the mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Patent No. 4,489,710, to Spitler), by irradiation of a photolabile bond (e.g., U.S. Patent No. 4,625,014, to Senter et al.), by hydrolysis of derivatized amino acid side chains (e.g., U.S.
  • Patent No. 4,638,045 to Kohn et al.
  • serum complement-mediated hydrolysis e.g., U.S. Patent No. 4,671,958, to Rodwell et al.
  • acid-catalyzed hydrolysis e.g., U.S. Patent No. 4,569,789, to Blattler et al.
  • immunoconjugates with more than one agent may be prepared in a variety of ways. For example, more than one agent may be coupled directly to an antibody molecule, or linkers that provide multiple sites for attachment can be used. Alternatively, a carrier can be used.
  • a carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group.
  • Suitable carriers include proteins such as albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.).
  • a carrier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Patent Nos. 4,429,008 and 4,873,088).
  • Carriers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds.
  • U.S. Patent No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis.
  • a radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
  • U.S. Patent No. 4,673,562 to Davison et al. discloses representative chelating compounds and their synthesis.
  • a variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration will be intravenous, intramuscular, subcutaneous or in the bed of a resected tumor. It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon the antibody used, the antigen density on the tumor, and the rate of clearance of the antibody.
  • Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for lipophilin complex.
  • T cells may generally be prepared in vitro or ex vivo, using standard procedures.
  • T cells may be isolated from bone marrow, peripheral blood, or a fraction of bone marrow or peripheral blood of a patient, using a commercially available cell separation system, such as the IsolexTM System, available from Nexell Therapeutics, Inc. (Irvine, CA; see also U.S. Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243).
  • T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures.
  • T cells may be stimulated with a lipophilin complex, or polynucleotides encoding a lipophilin complex and/or an antigen presenting cell (APC) that expresses such a complex.
  • APC antigen presenting cell
  • Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the complex.
  • the complex is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.
  • T cells are considered to be specific for a lipophilin complex if the T cells specifically proliferate, secrete cytokines or kill target cells coated with the complex or expressing a gene encoding some or all of the complex.
  • T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold, increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res. 54:1065-1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques.
  • T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA).
  • a lipophilin complex -100 ng/ml - 100 ⁇ g/ml, preferably 200 ng/ml - 25 ⁇ g/ml
  • contact with a lipophilin complex for about 3 - 7 days should result in at least a two fold increase in proliferation of the T cells.
  • T cells that have been activated in response to a lipophilin complex, polynucleotide or complex-expressing APC may be CD4 + , CD8 + or may comprise other T cell types.
  • Lipophilin complex- specific T cells may be expanded using standard techniques.
  • the T cells are derived from a patient, a related donor or an unrelated donor, and are administered to the patient following stimulation and expansion.
  • CD4 + or CD8 + T cells that proliferate in response to a lipophilin complex, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways. For example, the T cells can be re-exposed to a lipophilin complex, or a portion thereof corresponding to an immunogenic portion of such a complex, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a lipophilin complex. Alternatively, one or more T cells that proliferate in the presence of a lipophilin complex can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution.
  • the present invention concerns formulations of one or more of the compositions disclosed herein in pharmaceutically-acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy.
  • compositions as disclosed herein may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents.
  • agents such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents.
  • additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues.
  • the compositions may thus be delivered along with various other agents as required in the particular instance.
  • Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein.
  • Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation, as described below for the purposes of illustration..
  • compositions disclosed herein may be delivered via oral administration to an animal.
  • these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (see, for example, Mathiowitz et al., 1997; Hwang et al., 1998; U. S. Patent 5,641,515; U. S.
  • Tablets, troches, pills, capsules and the like may also contain any of a variety of additional components, for example, a binder, such as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder such as gum tragacanth, acacia, cornstarch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.
  • these formulations may contain at least about 0.1 % of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 60% or 70%) or more of the weight or volume of the total formulation.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically-effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
  • solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally will contain a preservative to prevent the growth of microorganisms.
  • Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U. S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., vegetable oils
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution for parenteral administration in an aqueous solution, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570- 1580).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations will of course preferably meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.
  • compositions disclosed herein are formulated in a neutral or salt form.
  • Illustrative pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be inco ⁇ orated into the compositions.
  • phrases "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described, e.g., in U. S. Patent 5,756,353 and U. S. Patent 5,804,212 (each specifically inco ⁇ orated herein by reference in its entirety).
  • the delivery of drugs using intranasal microparticle resins Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U. S. Patent 5,725,871, specifically inco ⁇ orated herein by reference in its entirety) are also well-known in the pharmaceutical arts.
  • transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is described in U. S. Patent 5,780,045 (specifically inco ⁇ orated herein by reference in its entirety).
  • compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • liposomes are generally known to those of skill in the art (see for example, Couvreur et al, 1977; Couvreur, 1988; Lasic, 1998; which describes the use of liposomes and nanocapsules in targeted therapy for intracellular bacterial infections and diseases).
  • Liposomes have been developed with improved serum stability and circulation half-times (see, for example, Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U.S. Patent 5,741,516, specifically inco ⁇ orated herein by reference in its entirety). Further, various methods of liposome and liposome like preparations as potential drug carriers have been reviewed (Takakura, 1998; Chandran et al, 1997; Margalit, 1995; U.S.
  • Patent 5,567,434 U.S. Patent 5,552,157; U.S. Patent 5,565,213; U.S. Patent 5,738,868 and U.S. Patent 5,795,587, each specifically inco ⁇ orated herein by reference in its entirety).
  • Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al, 1990; Muller et al, 1990). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs (Heath and Martin, 1986; Heath et al, 1986; Balazsovits et al, 1989; Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et al, 1987), enzymes (Imaizumi et al, 1990a; Imaizumi et al.
  • liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the peptide compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, i.e. in the aqueous spaces and within the bilayer itself, respectively. Moreover, the drug-bearing liposomes may be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation.
  • Antibodies may be used to bind to the liposome surface and to direct the antibody and its drug contents to specific antigenic receptors located on a particular cell-type surface.
  • Carbohydrate determinants may also be used as recognition sites as they have potential in directing liposomes to particular cell types. Usually, it is contemplated that intravenous injection of liposomal preparations would be used, but other routes of administration are also conceivable.
  • the invention provides for pharmaceutically-acceptable nanocapsule formulations of the compositions of the present invention.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al, 1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987).
  • ultrafine particles sized around 0.1 ⁇ m
  • biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention.
  • Such particles are easily made, as described, for example, by Couvreur et al, 1980; 1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry et al, 1995 and U. S. Patent 5,145,684, specifically inco ⁇ orated herein by reference in its entirety.
  • the pharmaceutical compositions of the invention comprise immunogenic compositions, particularly vaccine compositions.
  • such compositions will comprise one or more polynucleotide and/or polypeptide lipophilin complex compositions of the present invention in combination with an immunostimulant.
  • An immunostimulant may be any substance that enhances or potentiates an immune response (antibody and/or cell- mediated) to an exogenous antigen.
  • compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive.
  • one or more immunogenic portions of other tumor antigens may be present, either inco ⁇ orated into a fusion polypeptide or as a separate compound, within the composition or vaccine.
  • Illustrative vaccines may contain DNA encoding one or more of the lipophilin polypeptides as described above, such that some or all of the complex is generated in situ.
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 75:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal).
  • Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette- Guerri ⁇ ) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope.
  • the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus.
  • vaccinia or other pox virus, retrovirus, or adenovirus e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • Suitable systems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 56:317-321, 1989; Flexner et al., Ann. NY. Acad. Sci.
  • the DNA encoding some or all of the lipophilin complex may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993.
  • the uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
  • a vaccine may comprise both a polynucleotide and a polypeptide component. Such vaccines may provide for an enhanced immune response.
  • a vaccine may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides provided herein.
  • Such salts may be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).
  • compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration.
  • parenteral administration such as subcutaneous injection
  • the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • Biodegradable microspheres may also be employed as carriers for the pharmaceutical compositions of this invention.
  • Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,81 1,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.
  • Modified hepatitis B core protein carrier systems are also suitable, such as those described in WO/99 40934, and references cited therein, all inco ⁇ orated herein by reference.
  • One may also employ a carrier comprising the particulate-protein complexes described in U.S. Patent No. 5,928,647, which are capable of inducing a class I- restricted cytotoxic T lymphocyte responses in a host.
  • compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose or dextrans
  • mannitol proteins
  • proteins polypeptides or amino acids
  • proteins e.glycine
  • antioxidants e.g., mannitol
  • immunostimulants may be employed in the vaccines of this invention.
  • an adjuvant may be included.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
  • Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
  • Cytokines such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
  • the adjuvant composition is preferably designed to induce an immune response predominantly of the Thl type.
  • High levels of Thl-type cytokines e.g., IFN- ⁇ , TNF , IL-2 and IL-12
  • Th2-type cytokines e.g., IL-4, IL-5, IL-6 and IL-10
  • a patient will support an immune response that includes Thl- and Th2- type responses.
  • Thl-type cytokines will increase to a greater extent than the level of Th2-type cytokines.
  • the levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.
  • Preferred adjuvants for use in eliciting a predominantly Thl-type response include, for example, a combination of monophosphoryl lipid A, preferably 3- de-O-acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt.
  • MPL adjuvants are available from Corixa Co ⁇ oration (Seattle, WA; see US Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).
  • CpG-containing oligonucleotides in which the CpG dinucleotide is unmethylated also induce a predominantly Thl response.
  • oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996.
  • Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, MA), which may be used alone or in combination with other adjuvants.
  • an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739.
  • Other preferred formulations comprise an oil-in-water emulsion and tocopherol.
  • a particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
  • compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel (composed of polysaccharides, for example) that effects a slow release of compound following administration).
  • sustained release formulations i.e., a formulation such as a capsule, sponge or gel (composed of polysaccharides, for example) that effects a slow release of compound following administration.
  • Such formulations may generally be prepared using well known technology (see, e.g., Coombes et al., Vaccine 74:1429-1438, 1996) and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
  • Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane.
  • Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release.
  • Such carriers include microparticles of poly(lactide-co- glycolide), polyacrylate, latex, starch, cellulose, dextran and the like.
  • Other delayed- release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as a phospholipid (see e.g., U.S. Patent No.
  • delivery vehicles may be employed within pharmaceutical compositions and vaccines to facilitate production of an antigen-specific immune response that targets tumor cells.
  • delivery vehicles will comprise antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs.
  • APCs antigen presenting cells
  • Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and/or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype).
  • APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
  • Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:501-529, 1999).
  • dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate naive T cell responses.
  • Dendritic cells may, of course, be engineered to express specific cell- surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention.
  • secreted vesicles antigen-loaded dendritic cells called exosomes
  • exosomes antigen-loaded dendritic cells
  • Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid.
  • dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF ⁇ to cultures of monocytes harvested from peripheral blood.
  • CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF ⁇ , CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.
  • Dendritic cells are conveniently categorized as “immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which co ⁇ elates with the high expression of Fc ⁇ receptor and mannose receptor.
  • the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and cosfimulatory molecules (e.g., CD40, CD80, CD86 and 4- IBB).
  • cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and cosfimulatory molecules (e.g., CD40, CD80, CD86 and 4- IBB).
  • APCs may generally be transfected with a polynucleotide encoding a lipophilin complex (or portion or other variant thereof) such that the complex, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition or vaccine comprising such transfected cells may then be used for therapeutic pu ⁇ oses, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo.
  • In vivo and ex vivo transfection of dendritic cells may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.
  • Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the lipophilin complex, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).
  • the polypeptide Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule).
  • an immunological partner that provides T cell help e.g., a carrier molecule.
  • a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.
  • Vaccines and pharmaceutical compositions may be presented in unit- dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve sterility of the formulation until use.
  • formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles.
  • a vaccine or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
  • compositions described herein may be used for immunotherapy of cancer, such as breast, ovarian or prostate cancer.
  • pharmaceutical compositions and vaccines are typically administered to a patient in order to elicit an immune response directed against the lipophilin complex, or portion thereof, contained within the pharmaceutical compositions.
  • a "patient" refers to any warm-blooded animal, preferably a human.
  • a patient may or may not be afflicted with cancer.
  • the above pharmaceutical compositions and vaccines may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer.
  • a cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor.
  • Pharmaceutical compositions and vaccines may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs.
  • immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immune response-modifying agents (such as tumor vaccines, bacterial adjuvants and/or cytokines).
  • immune response-modifying agents such as tumor vaccines, bacterial adjuvants and/or cytokines.
  • immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established tumor-immune reactivity (such as antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system.
  • agents with established tumor-immune reactivity such as antibodies
  • the complexes provided herein may be used to generate antibodies or anti- idiotypic antibodies (as described above and in U.S. Patent No. 4,918,164) for passive immunotherapy.
  • Such antibodies may be of particular benefit in the treatment of tumors with steroid-dependence, replacing the current steroid-related treatments with a less harsh, more directed form of treatment.
  • compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally.
  • injection e.g., intracutaneous, intramuscular, intravenous or subcutaneous
  • intranasally e.g., by aspiration
  • between 1 and 10 doses may be administered over a 52 week period.
  • 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter.
  • Alternate protocols may be appropriate for individual patients.
  • a suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above the basal (i.e., untreated) level.
  • Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine- dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro.
  • Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non- vaccinated patients.
  • the amount of each complex present in a dose ranges from about 25 ⁇ g to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
  • an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit.
  • a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients.
  • Increases in preexisting immune responses to a lipophilin complex may co ⁇ elate with an improved clinical outcome.
  • immune responses may be evaluated using assays, which may be performed using samples obtained from a patient before and after treatment.
  • therapies may be used alone, or in combination with other anti-cancer therapies.
  • pretreatment with steroids may increase the effectiveness of anti-complex treatment as described herein.
  • the effect of existing cancer drugs may be enhanced by concu ⁇ ent treatment with anti-complex immunotherapy.
  • lipophilin complexes may associate with steroids and may participate in the regulation of growth of steroid-dependent cancers.
  • Therapies directed at hormone-dependent growth regulation, such as Tamoxifen may be affected by complex formation.
  • the therapeutic methods provided herein may decrease the amount of complex present, thereby facilitating treatment using such existing therapies.
  • Mammaglobin protein is expressed as a small polypeptide of 93 amino acids in length with a predicted molecular weight of 10.5 kDa and a putative cleavage site at amino acid 19 (Watson (1996)). Mammaglobin shares some homology with lipophilin C (also known as mammaglobin B), having 52% identity on the amino acid level ( Lehrer, R.I., Xu, G., Abduragimov, A., Dinh, N.N., Qu, X.D., Martin, D., and Glasgow, B.J.
  • the lipophilin complexes described herein have utility in detection and diagnostic methods for patients having or suspected of having cancer, particularly breast, ovarian or prostate cancer.
  • a cancer may be detected in a patient based on the presence of one or more lipophilin complexes described herein, or antibodies directed thereto, in a biological sample obtained from the patient.
  • such complexes may be used as markers to indicate the presence or absence of a cancer such as breast, ovarian or prostate cancer.
  • the presence of such a complex, or antibody thereto, at a level that is higher, for example at least three fold higher, in tumor tissue than in normal tissue is indicative of a cancer.
  • the presence of such a complex may further provide information useful in the selection of therapeutic options.
  • the presence of lipophilin complexes may be indicative of tumor cell refractoriness to steroid mimic treatment.
  • the type of cancer, as well as stage information may be derived from the type and composition of complexes found in a tumor.
  • the presence or absence of a cancer in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of complex that binds to the binding agent; and (c) comparing the level of complex with a predetermined cut-off value.
  • the level of complex and the levels of free complex components are assayed.
  • a ratio of complex to free components i.e., binding constant
  • This ratio may be indicative of the presence, or the severity, of a cancer.
  • the assay involves the use of binding agent immobilized on a solid support to bind to and remove the complex from the remainder of the sample.
  • the bound complex may then be detected using a detection reagent that contains a reporter group and specifically binds to the bound complex.
  • detection reagents may comprise, for example, an antibody that specifically binds to the complex or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin.
  • a competitive assay may be used, in which a complex is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled complex to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent.
  • the solid support may be any material known to those of ordinary skill in the art to which the binding agent may be attached.
  • the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
  • the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride.
  • the support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.
  • the binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature.
  • immobilization refers to both noncovalent association, such as adso ⁇ tion, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adso ⁇ tion to a well in a microtiter plate or to a membrane is prefe ⁇ ed. In such cases, adso ⁇ tion may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day.
  • contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 ⁇ g, and preferably about 100 ng to about 1 ⁇ g, is sufficient to immobilize an adequate amount of binding agent.
  • a plastic microtiter plate such as polystyrene or polyvinylchloride
  • Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
  • a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
  • the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
  • the assay is a two-antibody sandwich assay.
  • This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that complex within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized complex/antibody and a detection reagent (preferably a second antibody capable of binding to a different site on the complex) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.
  • one antibody specifically binds glycosylated mammaglobin, and the other specifically binds a lipophilin protein, such as lipophilin B.
  • complex may be captured with one antibody (e.g., anti-lipophilin B) and detected using the other antibody (e.g., anti-mammaglobin).
  • an appropriate contact time is a period of time that is sufficient to detect the presence of complex within a sample obtained from an individual with breast, ovarian or prostate cancer.
  • the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound complex.
  • a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound complex.
  • the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.
  • Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1 % Tween 20TM.
  • the second antibody which contains a reporter group, may then be added to the solid support.
  • the detection reagent is then incubated with the immobilized antibody/complex for an amount of time sufficient to detect the bound complex.
  • An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time.
  • Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group.
  • the method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
  • the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that co ⁇ esponds to a predetermined cutoff value.
  • the cut-off value for the detection of a cancer is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without the cancer.
  • a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for the cancer.
  • the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specif ⁇ city) that co ⁇ espond to each possible cut-off value for the diagnostic test result.
  • true positive rates i.e., sensitivity
  • false positive rates (100%-specif ⁇ city
  • the cut-off value on the plot that is the closest to the upper left-hand corner is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive.
  • the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate.
  • a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for a cancer.
  • a similar process may be used to establish a cut-off value for the ratio of complex to free components.
  • ⁇ For certain embodiments (e.g., sandwich assays), quantitative measurements of antigen may be obtained.
  • a standard curve may be generated. Signals obtained for antigen levels in particular samples may then be compared to the standard curve, to allow quantitation.
  • the cut-off value within such assays may be an amount of complex indicative of the presence of breast, ovarian or prostate cancer.
  • the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose.
  • a membrane such as nitrocellulose.
  • a second, labeled binding agent then binds to the binding agent/complex as a solution containing the second binding agent flows through the membrane.
  • the detection of bound second binding agent may then be performed as described above.
  • the strip test format one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent.
  • Concentration of second binding agent at the area of immobilized antibody indicates the presence of a cancer.
  • concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result.
  • the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of complex that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above.
  • Prefe ⁇ ed binding agents for use in such assays are antibodies and antigen-binding fragments thereof.
  • the amount of antibody immobilized on the membrane ranges from about 25 ng to about l ⁇ g, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.
  • Antibodies as provided herein may further be used to facilitate cell identification and sorting in vitro, permitting the selection of cells expressing a lipophilin complex (or varying levels of lipophilin complex).
  • antibodies for use in such methods are linked to a detectable marker. Suitable markers are well known in the art and include radionuclides, luminescent groups, fluorescent groups, enzymes, dyes, constant immunoglobulin domains and biotin.
  • an antibody linked to a fluorescent marker such as fluorescein, is contacted with the cells, which are then analyzed by fluorescence activated cell sorting (FACS).
  • the above complexes may be used as markers for the progression of cancer.
  • assays as described above for the diagnosis of a cancer may be performed over time, and the change in the level of reactive complex(es) evaluated.
  • the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed.
  • a cancer is progressing in those patients in whom the level of complex detected by the binding agent increases over time.
  • the cancer is not progressing when the level of reactive complex either remains constant or decreases with time.
  • the ratio of complex to the product of the free components may be monitored over time to evaluate cancer progression.
  • Certain in vivo diagnostic assays may be performed directly on a tumor.
  • One such assay involves contacting tumor cells with a binding agent.
  • the bound binding agent may then be detected directly or indirectly via a reporter group.
  • binding agents may also be used in histological applications.
  • assays as described herein may be combined with assays to detect other tumor-associated antigens. It will be apparent that binding agents specific for different proteins may be combined within a single assay. The selection of tumor protein markers may be based on routine experiments to determine combinations that results in optimal sensitivity. Alternatively, pretreatment with steroids may increase the sensitivity of a complex-based diagnostic method.
  • kits for use within any of the above diagnostic methods.
  • Such kits typically comprise two or more components necessary for performing a diagnostic assay.
  • Components may be compounds, reagents, containers and/or equipment.
  • one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a lipophilin complex.
  • Such antibodies or fragments may be provided attached to a support material, as described above.
  • One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay.
  • Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.
  • Prefe ⁇ ed kits are those designed for use within sandwich assays.
  • kits comprise two or more components for use within such assays.
  • a kit may comprise standards for use in preparing a standard curve.
  • Such a kit may comprise one or both antibodies for use within the assay (i.e., the capture antibody and/or signal antibody), with or without additional reagents for use in detecting complex binding.
  • a kit comprises an anti-mammaglobin antibody (or fragment thereof) and an anti-Lipophilin B antibody (or fragment thereof).
  • the invention also relates to therapeutics for targeting the immunosuppressive and anti-inflammatory properties of mammaglobin/lipophilin complexes.
  • Some proteins in the uteroglobin family have been reported to be immunomodulatory molecules.
  • the unique expression pattern found for mammaglobin-like molecules and lipophilins A, B and C and related molecules and their physical properties make it likely that these molecules may have anti-inflammatory or immunosuppressive effects. These effects may be utilized by tumors to circumvent recognition by the immune system and explain the overexpression of these molecules in a number of tumors.
  • these molecules themselves may be manufactured for use in human subjects as immunomodulators, and antibodies targeting these molecules and complexes could be used to unmask tumors and make them accessible to the immune system.
  • lane 1 shows the complex in non-reducing SDS sample buffer
  • lane 2 shows the complex with 10 mM DTT in the sample buffer.
  • N-terminal sequencing ( Figures 3A-3B) of an equivalent gel blotted onto PVDF membrane revealed the presence of Lipoprotein B in the band in lane 1 , while no such sequence was detected in the band shown in lane 2.
  • Figure 2B depicts the same gel restained with silver stain.
  • the a ⁇ ow denotes the weakly staining mammaglobin.
  • a new band was detected in the reduced lane (lane 2), and was confirmed to be lipophilin B by N-terminal sequencing.
  • Tris[hydroxymethyl]aminomethane (Tris), sodium chloride, ammonium acetate, phosphate buffered saline (PBS), glycine, methanol, silver nitrate, and sodium thiosulfate were obtained from Sigma (St. Louis, MO). Acetonitrile was from Mallinckrodt Laboratory Chemicals (Phillipsburg, NJ).
  • MDA-MB-415 cells (ATCC #HTB-128) cells were initially grown in DMEM (Life Technologies Inc., Rockville, MD) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT). Once the cells were confluent, the serum containing media was removed; the cells were rinsed with PBS and low serum media was added.
  • the low serum media consisted of a mix of 50% SFX-CHO (Hyclone, Logan, UT) supplemented with 2mM GlutaMAXTM (Life Technologies Inc., Rockville, MD) and 50% Opti-MEM® l(Life Technologies Inc., Rockville, MD).
  • Insulin- Transfe ⁇ in-Selenium-A (Life Technologies Inc., Rockville, MD) was added for a final concentration of lx. The cells were grown in this media for a period of several weeks, with weekly applications of fresh media. Supernatants were collected and pooled for subsequent mammaglobin isolation.
  • the lyophilized powder was redisolved in phosphate buffered saline and run over a column containing Affi-Gel Blue (Bio-Rad, Hercules, CA). The unbound fractions contained most of the mammaglobin protein and was used for further studies.
  • SDS-PAGE was performed according to the method of Laemmli (Laemmli, U.K. (1970) Nature 227, 680-685). Samples were diluted 1 :1 in Laemmli Sample Buffer (Invitrogen, Carlsbad, CA) and boiled for 5 minutes. Proteins were then loaded on 4-20% acrylamide gradient gels (Bio-Rad, Hercules, CA) and run at 250V constant for 30 minutes.
  • Membranes were probed with 1 ug / ml of a monoclonal antibody to mammaglobin overnight and washed again as above.
  • An horseradish peroxidase-conjugated goat anti rabbit IgG was used as a secondary antibody, developed with ECL solution (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and visualized using scientific imaging film (Kodak, Rochester, NY).
  • N-terminal Sequencing Amino terminal sequence data were obtained from purified proteins which were dried directly onto TFA treated glass fiber filters (Perkin Elmer/ Applied Biosystems Division) or from samples which were separated on SDS-PAGE and electroblotted onto Sequi-Blot PVDF membranes according to the method of Matsudaira (Matsudaira, P. (1987) J Biol Chem 262, 10035-10038). The membranes were stained with the Glyco-Pro glycoprotein detection kit (Sigma, St Louis, MO) to visualize the protein bands that were excised with a clean razor. Traditional Edman degradation sequence analysis was performed using a Perkin Elmer /Applied Biosystems Division Procise 494 Protein Sequencer.
  • Quantitative Real-time PCR The specificity and sensitivity of the different genes was determined using quantitative PCR analysis. Breast metastases, primary breast tumors, benign breast disorders and normal breast tissue along with other normal tissues and tumors were tested in quantitative ("Real time") PCR. This was performed either on the ABI 7700 Prism or on a Gene Amp® 5700 sequence detection system (PE Biosystems, Foster City, CA). The 7700 system uses a forward and a reverse primer in combination with a specific probe with a 5 'fluorescent reporter dye at one end and a 3' quencher dye at the other end (TaqmanTM).
  • the 5700 system uses SYBR® green, a fluorescent dye, that only binds to double stranded DNA, and the same forward and reverse primers as the 7700 instrument. Matching primers and fluorescent probes were designed for each of the genes according to the primer express program (PE Biosystems, Foster City, CA). The primers used for mammaglobin detection were:
  • Mamm 2f 5'-TGCCATAGATGAATTGAAGGAATG-3' (SEQ ID NO:3) ; and Mamm 2r: 5'-TGTCATATATTAATTGCATAAACACCTCA-3' (SEQ ID NO:4).
  • the primers were:
  • LipoBf 5'-TGCCCCTCCGGAAGCT-3' (SEQ ID NO:5); and LipoBr: 5'-CGTTTCTGAAGGGACATCTGATC-3' (SEQ ID NO:6).
  • Primers and probes so produced were used in the universal thermal cycling program in real-time PCR. They were titrated to determine the optimal concentrations using a checkerboard approach. A pool of cDNA from target tumors was used in this optimization process. The reaction was performed in 25 ⁇ l volumes. The final probe concentration in all cases was 160nM. dATP, dCTP and dGTP were at 0.2mM and dUTP at 0.4mM. Amplitaq gold and Amperase UNG (PE Biosystems, Foster City, CA) were used at 0.625 units and 0.25 units per reaction. MgCl 2 was at a final concentration of 5mM.
  • This mix also contained 3mM MgCl 2 , 0.25units of AmpErase UNG, 0.625 units of Amplitaq gold, 0.08%> glycerol, 0.05% gelatin, 0.0001% Tween 20 and ImM dNTP mix. In both formats, 40 cycles of amplification were performed.
  • a standard curve is generated for each run using a plasmid containing the gene of interest. Standard curves were generated using the Ct values determined in the real-time PCR which were related to the initial cDNA concentration used in the assay.
  • Mammaglobin-lipophilin B was purified to greater than 90% purity as assayed by N-terminal sequencing of the purified liquid pool after reverse phase chromatography. Due to the poor staining of the complex by conventional means this was the best measure to use to assay purity. To verify that there was no significant contaminant with a blocked N-terminus, a blot was stained by Coomassie Brilliant Blue and the only visible other band sequenced. This band was not N-terminally blocked and was revealed to be serum albumin left from the cell culture medium.
  • Mammaglobin glycosylation Purified mammaglobin stained easily by a carbohydrate specific stain. Fine tuning of the acetonitrile gradient used to purify the complex revealed a sequence of peaks cumulating on one major final peak. These peaks were identified all as having a major mammaglobin component by Western blotting (data not shown) and are inte ⁇ reted as differentially glycosylated forms of mammaglobin with the predominant form being the final product of glycosylation.
  • Mammaglobin - lipophilin B association Mammaglobin and lipophilin B copurified through numerous different biochemical purification steps. They also co- migrated on SDS-PAGE gels under non-reducing conditions. When blotted on PVDF membranes and sequenced, the diffuse band which contained mammaglobin by Western blotting also contained equimolar amounts of lipophilin B. This association of mammaglobin and lipophilin B can be broken by pre-treatment with reducing agents such as lOmM dithiothreitol indicating an association by disulfide linkage. Prior to reduction, the center of the mammaglobin band was at about 25 kD on SDS-PAGE gels.
  • N-terminal sequences determined are consistent with mammaglobin and lipophilin B being processed at their respective predicted cleavage sites, between amino acids 19 and 20 for mammaglobin; and amino acids 21 and 22 for lipophilin B.
  • mammaglobin a diagnostic marker for breast cancer
  • lipophilin B in its association with mammaglobin may be a good serological marker for breast cancer.
  • the significant association found in breast tumors using a rank co ⁇ elation coefficient which assumes nothing about distribution of values or the linearity of the association, indicates a significant paralleling of message expression for both mammaglobin and lipophilin B.
  • homologous proteins found in the rat prostatic binding protein complex are also know to be attached to each other ( Claessens, F., Rushmere, N.K., Davies, P., Celis, L., Peeters, B., and Rombauts, W.A. (1990) Mol Cell Endocrinol 74, 203-212).
  • C1-C3 heterodimer there are two heterodimers: the C1-C3 heterodimer; and the C2- C3 heterodimer.
  • Cl and C2 are homologous to Lipophilins A and B, while C3 is homologous to Mammaglobin and Lipophilin C (also known as Mammaglobin B).
  • pairwise sequence alignment of these different proteins sorts them into two groups: mammaglobin-like molecules, and lipophilin-like molecules.
  • mammaglobin-like molecules For each mammaglobin-like molecule, a dimerization partner has been detected, except for mammaglobin itself (Lehrer, R.I., Xu, G., Abduragimov, A., Dinh, N.N., Qu, X.D., Martin, D., and Glasgow, B.J. (1998) FEBS Lett 432, 163-167; Claessens, F., Rushmere, N.K., Davies, P., Celis, L., Peeters, B., and Rombauts, W.A. (1990) Mol Cell Endocrinol 74, 203-212).
  • mammaglobin-like C3 is complexed to lipophilin-like Cl and C2, and in rat lacrimal gland the C3 component is disulfide bonded to another as yet unidentified 10 kD polypeptide which is distinct from either Cl or C2 ( Vercaeren, I., Vanaken, H., Devos, A., Peeters, B., Verhoeven, G., and Heyns, W. (1996) Endocrinology 137, 4713-4720).
  • Vercaeren, I., Vanaken, H., Devos, A., Peeters, B., Verhoeven, G., and Heyns, W. (1996) Endocrinology 137, 4713-4720).
  • C3 message is highly regulated by androgens both in the prostate and in the lacrimal gland. If this is also true in humans, then up-regulation of the messages in breast cancer tissues may be an indicator of steroid responsiveness for tumors expressing the mammaglobin-lipophilin B complex.
  • Non-fat dried milk 25 g pH to 9.6
  • the buffers were allowed to come to room temperature. Plates were coated the night before and incubated overnight at 4°C. 50 ⁇ l per well of antigen was used, diluted in coating buffer. (The plate can be coated the same day at 37°C for 1 hour.) Recombinants were coated at 200 ng/well, Peptides at 1 ug/well. Plates were aspirated, and 250 ⁇ l of Blocking Buffer was added to each well and incubated for 2 hours. A 1 :100 dilution of serum was made in serum diluent using 50ul/well. Plates were washed at 350 ⁇ l per well and washed 6 times.
  • Mass spec data indicate that mammaglobin purified from MB415 cells is covalently linked to lipophilin B via a cysteine bridge from mammaglobin amino acid 4 to lipophilin amino acid 67.
  • the results of the mass spec analysis are shown in Figure 4.
  • the 1621.6 ion matches the combined molecular weight of mammaglobin tryptic fragment 1-13 and lipophilin B tryptic fragment 67-69.
  • mammaglobin tryptic fragment 1-13 MW 1316.5
  • lipophilin fragment 67-69 MW 308.1
  • Mammaglobin lipophilin B complex purified from MB415 cells was subjected to digestion with trypsin. Fragments were analyzed by mass spectrometry (MS/MS). In addition to the predicted tryptic fragments, two fragments at +14 Daltons were identified. MS/MS data from th mammaglobin tryptic fragment of amino acids 1- 13 indecated that amino acids 8, 9 or 10 were modified in such a manner as to cause a +14 Dalton shift in the predicted molecular weight of the tryptic fragment. Similar analysis of lipophilin B tryptic fragment comprised of amino acids 54-62 indicated that a similar polymo ⁇ hism occu ⁇ ed between amino acids 54 and 59.
  • polymo ⁇ hisms in mammaglobin and lipophilin may be used in diagnostic and prognostic applications.
  • Specific therapeutics and drugs may be targeted to these polymo ⁇ hisms and, consequently, may find utility in the development of vaccines.
  • the polynucleotides encoding the mammaglobin reference molecule and the above-mentioned variants are included in SEQ ID NO: 18-26, and the amino acid sequences encoded by these are included in SEQ ID NO:28-34 (see also SEQ ID NO:l for the mammaglobin reference protein).
  • the polynucleotides encoding the lipophilin B reference molecule and variants are included in SEQ ID NO:7-17, and the amino acid sequences are included in SEQ ID NO:35-42 (see also SEQ ID NO:2 for the lipophilin B reference protein). Note that not all of the polynucleotide variants result in an amino acid change in the encoded protein.
  • Mammaglobin cDNA was amplified from 8 primary breast tumors, 10 metastatic breast tumors, and a single normal breast tissue sample. These were then subcloned and sequenced as described in Example 5 to determine the extent of sequence variants that exist.
  • the sequence variants identified are summarized in Table 4. The DNA sequences are disclosed in SEQ ID NOs:43-50, and 55 and the amino acid sequence of those variants resulting in amino acid changes are disclosed in SEQ ID NOs:51-54.

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Abstract

L'invention concerne des compositions et des méthodes permettant de diagnostiquer et de traiter le cancer du sein, des ovaires, et de la prostate. Ces compositions peuvent comprendre au moins un complexe lipophilinique qui renferme au moins deux polypeptides différents du type lipophiline liés par des liaisons disulfidiques. Ces compositions peuvent s'utiliser pour diagnostiquer et traiter le cancer du sein, des ovaires, et de la prostate. L'invention concerne également des méthodes de diagnostic permettant de détecter la présence de ces complexes, ou anticorps chez un patient.
PCT/US2001/004439 2000-02-11 2001-02-08 Complexes lipophiliniques utilises pour diagnostiquer et traiter un cancer WO2001058947A1 (fr)

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WO1999014230A1 (fr) * 1997-09-18 1999-03-25 Washington University La mammaglobine, proteine secretee du cancer du sein specifique de la glande mammaire

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999014230A1 (fr) * 1997-09-18 1999-03-25 Washington University La mammaglobine, proteine secretee du cancer du sein specifique de la glande mammaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
C.ZHAO ET AL.: "Lipophilins: Human Peptides Homologous to Rat Prostatein", BIOCHEM.BIOPHYS.RES.COMMUN., vol. 256, 1999, pages 147 - 55, XP002170548 *
R.I.LEHRER ET AL.: "Lipophilin, a novel heterodimeric protein of human tears", FEBS LET., vol. 432, 1998, pages 163 - 7, XP000999229 *

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