WO2003090675A2

WO2003090675A2 - Methods and compositions useful for treating prostate cancer

Info

Publication number: WO2003090675A2
Application number: PCT/US2003/012392
Authority: WO
Inventors: Richard A. Lerner; Alan Saven
Original assignee: The Scripps Research Institute
Priority date: 2002-04-25
Filing date: 2003-04-21
Publication date: 2003-11-06
Also published as: AU2003237093A1; AU2003237093A8; WO2003090675A3

Abstract

Immunopeptides are provided that are composed of a prostate specific antigen (PSA) coupled to an autoimmune tag, or a prostate specific polypeptide (PSP) coupled to an autoimmune tag. Pharmaceutical compositions containing the immunopeptides are provided. Methods to treat prostate cancer are also provided. Antibodies to the immunopeptides are provided.

Description

Methods and Compositions Useful for Treating Prostate Cancer

Field of the Invention The invention generally relates to methods and compositions useful for treating prostate cancer. More specifically, the invention provides compositions and methods useful for overcoming self-tolerance and inducing an autoimmune response against prostate specific antigen (PSA) or a prostate specific polypeptide (PSP). Even more specifically, the invention relates to methods to induce autoimmunity against PSA or a PSP without inducing deleterious autoimmunity against cells residing throughout the body of a patient being treated for prostate cancer.

Background of the Invention Prostate cancer kills approximately 40,000 Americans each year. Most of these deaths occur in patients whose disease has become resistant to androgenic manipulation. Currently, there is no therapy shown to improve survival in patients with hormone refractory prostate cancer (HRPC). However, recent phase II studies have demonstrated combinations of agents with significant anti-tumor activity.

The most promising results to date have been achieved with a combination of estramustine and a taxane. In numerous phase II studies, these combinations have a consistent biochemical response rate of 50-70%, and corresponding objective response rates of 30-50%. Kreis et al., Br. J. Urol., 79, 196-202 (1997); Colleoni et al., American Journal of Clinical Oncology. 20(4). 383-6 (1997); Hudes et al., Seminars in Oncology. 22(5) Suppl 12. 41-5 (1995); Pienta et al., Journal of Clinical Oncology. 12(10), 2005-12 (1994); Kreis et al., Annals of Oncology. 10, 33-38 (1999); Petrylak et al., J. Clin. Oncol.. 17, 958- 967 (1999). A median survival of 23 months has been reported in a phase I study using the combination of estramustine and docetaxel. Petrylak et al. (1999). This doubling of the historic median survival has led to a large phase III trial comparing that combination to the FDA-approved combination of mitoxantrone and prednisone.

An approach to cancer therapy based on PSA has been explored. Sanda et al., Urology, 53, 260-266 (1999) performed a phase I study of a vaccinia virus modified to express the entire PSA gene. Anti-PSA IgG and IgM antibodies were demonstrated in 5 of 6 patients. Toxicity was limited to mild constitutional symptoms. A second study by Eder et al., Clin. Cancer Res., 6, 1632-1638 (2000) employed a vaccinia virus expressing PSA-3, a 9 mer subunit of PSA. Three immunizations were given for weeks apart combined with GM-CSF. One patient developed anti-PSA antibodies, and 5 of 7 patients demonstrated a two fold increase in PAS-3 peptide-specific cytolytic T lymphocyte (CTL) response in vitro. Stabilization of PSA levels was noted in six patients.

Despite significant advances in the systemic treatment of advanced, hormone-refractory prostate cancer, cytotoxic therapy alone appears unlikely to have a significant impact on the natural history of this disease. Furthermore, treatment methods involving expression of PSA are subject to tolerance mechanisms which cause a failure to produce an immune response to the PSA by the patient. Therefore, additional methods and compositions to treat prostate cancer are needed. Preferably, these methods and compositions advance treatment through immune system interaction.

Summary of the Invention

The invention provides an immunopeptide composed of a prostate specific antigen (PSA) coupled to an autoimmune tag, or a prostate specific polypeptide (PSP) coupled to an autoimmune tag. Also provided are pharmaceutical compositions containing an immunopeptide of the invention. The invention also provides methods to treat prostate cancer by overcoming self- tolerance and inducing an autoimmune response against cells that express PSA or a PSP. Also provided by the invention are antibodies that bind to the immunopeptides of the invention.

An immunopeptide of the invention includes prostate specific antigen (PSA) or a prostate specific polypeptide (PSP) wherein each has been coupled to an autoimmune tag. Such an immunopeptide will overcome self-tolerance and produce an autoimmune response against cells that express PSA or a PSP when administered to a human without inducing deleterious autoimmune responses against tissues generally within the patient's body. Preferably the immunopeptide contains an autoimmune tag chosen from an acetyl or a picryl group. More preferably the autoimmune tag includes an acetyl group and a picryl group. Preferably the autoimmune tag is chosen from arsanilic acid or sulfanilic acid. More preferably the autoimmune tag includes arsanilic acid and sulfanilic acid. The invention provides pharmaceutical compositions that contain an immunopeptide of the invention. These pharmaceutical compositions may be administered to a human patient to overcome self-tolerance and produce an autoimmune response against PSA or a PSP produced by the patient. A pharmaceutical composition may be formulated to contain an immunopeptide, a pharmaceutically acceptable carrier and optional additional ingredients such as adjuvants, buffers, preservatives and the like. The pharmaceutical compositions may be formulated for use in a specific application by those of skill in the art. A pharmaceutical composition of the invention may also include other pharmaceutical agents. Such pharmaceutical agents are recognized in the official United States Pharmacopeia, official Homeopathic Pharmacopeia of the United States, official National Formulary of any supplement thereof.

The invention also provides a method to treat prostate cancer. The method involves administering an immunopeptide of the invention, preferably as a pharmaceutical composition of the invention, to a human such that self- tolerance is overcome and the human produces an autoimmune response to cells that express PSA or a PSP.

Antibodies that bind to the immunopeptides of the invention are also provided. Antibodies of the invention bind to the immunopeptides of the invention but do not bind to PSA or to a PSP lacking an autoimmune tag. Preferably the antibodies are polyclonal antibodies. More preferably the antibodies are monoclonal antibodies. The antibodies of the invention may be used to determine the concentration of an immunopeptide contained in a pharmaceutical composition or in a patient. The antibodies may also be used to determine and monitor the concentration of an immunopeptide administered to a patient in need thereof.

The invention also provides immunofragopeptides that are composed of a fragment of prostate specific antigen (PSA) coupled to an autoimmune tag, or a fragment of a prostate specific polypeptide (PSP) coupled to an autoimmune tag. More specifically, these immunofragopeptides include a fragment of PSA, a peptidomimetic of PSA, a fragment of a PSP, or a peptidomimetic of a PSP, wherein each is coupled to an autoimmune tag. Such an immunofragopeptide will overcome self-tolerance and cause an autoimmune cross-response against PSA, a PSP, or cells that express PSA or a PSP when administered to a human. Preferably the immunofragopeptide is conjugated to an autoimmune tag chosen from an acetyl or a picryl group. More preferably the autoimmune tag includes an acetyl group and a picryl group. Preferably the autoimmune tag is chosen from arsanilic acid or sulfanilic acid. More preferably the autoimmune tag includes arsanilic acid and sulfanilic acid. Preferably the autoimmune tag is coupled to a portion of the immunofragopeptide that does not have an amino acid sequence corresponding to that of PSA or a PSP. More preferably the autoimmune tag is coupled to a portion of the immunofragopeptide having an amino acid sequence corresponding to that of PSA or a PSP.

Pharmaceutical compositions containing the immunofragopeptides, methods to treat prostate cancer using the immunofragopeptides, and antibodies that bind to the immunofragopeptides are also included within the invention. A surprising aspect of the invention is that autoimmunity can be specifically induced against PSA, a PSP, or a cell that expresses PSA or a PSP, without causing deleterious effects due to other autoimmune reactions. This restricts the autoimmune response to prostate tissues and allows for the treatment of prostate cancer without the induction of a deleterious autoimmune response.

Detailed Description of the Invention

The immune system responds to foreign antigens but is unresponsive (tolerant) to an individual's self antigens. Self-tolerance keeps an individual from mounting an unwanted autoimmune response against components of the individual's own body. Self- tolerance is maintained by selection processes that kill or block the maturation of potentially self-reactive lymphocytes and by mechanisms that inactivate self-reactive lymphocytes in peripheral tissues. One form of self-tolerance, called central tolerance, is due to death or inactivation of immature lymphocytes that specifically recognize self antigens in the generative lymphoid organs. Another form of self-tolerance, called peripheral T cell tolerance, is mainly due to inactivation (anergy) of mature T cells that recognize self antigens presented by costimulator-deficient, resting APCs, or death of T cells that are repeatedly stimulated by persistent self antigens. Abbas et al., Cellular and Molecular Immunology, W.B. Saunders Co., Philadelphia, (1997). Prostate specific antigen (PSA) is a serine protease secreted from epithelial cells lining prostatic ducts. While PSA is detectable in minute amounts in other organs, it is virtually specific for prostatic tissue. Ninety percent of all prostate tumors secrete PSA. This finding has led to the wide- spread use of serum PSA levels as a screening test for prostatic neoplasms. PSA levels may also be increased in benign prostatic hyperplasia, and with manipulation of the gland. After radical prostatectomy, PSA levels are undetectable. Any measurable PSA indicates the presence of residual tumor.

The invention provides a method to overcome self-tolerance to PSA in a human and to induce an autoimmune response against PSA and cells that express PSA. Also provided is a method to overcome self-tolerance to a PSP and induce an autoimmune response against a PSP and cells that express a PSP in a human. Through use of this method, a human's own immune system may be induced to attack and specifically eliminate cells that express PSA or a PSP, such as prostate cancer cells, and thereby reduce or eliminate cancerous prostate tissue. Deliberately inducing an autoimmune response against cells that express PSA or a PSP provides the positive effect of eliminating such cells by using the patient's own immune system.

A surprising advantage of the present invention is that a specific autoimmune response can be induced against cells that express PSA or a PSP without causing deleterious autoimmune responses against tissues generally found within the patient's body. Such autoimmune responses may be specific or non-specific. Furthermore, the present invention provides the opportunity to treat prostate cancer through the minimally invasive administration of an immunopeptide or pharmaceutical composition of the invention to a patient in need thereof. Thus, through use of this method, a minimal amount of an immunopeptide may be administered to a patient to overcome threshold self- tolerance and induce a desired autoimmune response that is specific to PSA, a PSP and cells that express PSA or a PSP.

The invention also provides immunopeptides and immunofragopeptides that are able to produce a specific autoimmune response against PSA, a PSP, or an immunofragopeptide when they are administered to a human. Such an immunopeptide or immunofragopeptide may be produced by coupling PSA, PSP, or an immunofragopeptide to an autoimmune tag. These immunopeptides and immunofragopeptides may be administered to a human in need thereof to treat prostate cancer by overcoming self-tolerance and inducing an autoimmune response against cells that express PSA or a PSP. These methods are thought to be particularly useful in patients who are at high risk of recurrence after radical prostatectomy, or in patients with more advanced disease.

Definitions

An "adjuvant" is generally defined as a substance that nonspecifically enhances the immune response to an antigen. A variety of adjuvants may be employed with the immunopeptides and immunofragopeptides of this invention. 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, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); 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. An "autoimmune tag" is a group that is used to create an immunopeptide or immunofragopeptide of the invention. For example, an autoimmune tag is coupled to PSA or to a PSP to create an immunopeptide. Administration of an immunopeptide to a human produces an immune response to PSA or to a PSP. Examples of autoimmune tags include, but are not limited to, arsanilic acid, sulfanilic acid, an acetyl group, a picryl group, or any combination thereof. Such autoimmune tags and methods to couple the autoimmune tags to form an immunopeptide or immunofragopeptide have been described. Weigle, J. Exp. Med., 116:913-928 (1962); Weigle, J. Exp. Med., 122:1049-1062 (1965); Weigle, J. Exp. Med., 121:289-308 (1965), and the references contained therein, all incorporated herein by reference.

A "conservative variant" or "conservative variation" is a polypeptide derived from the native protein by deletion (so-called truncation), or addition or subtraction of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion, addition or subtraction of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art. The PSA or PSP sequence may serve as a template to produce the amino acid sequence portion of an immunofragopeptide. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a prostate specific antigen can be prepared by mutagenesis of DNA encoding the polypeptides. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, Proc. Natl. Acad. Sci. USA. 82, 488 (1985); Kunkel et al., Methods in Enzvmol., 154:367 (1987); U. S. Patent No. 4,873,192; Walker and Gaastra, eds., Techniques in Molecular biology, MacMillan Publishing Company, New York (1983) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of PSA or a PSP may be found in the model of Dayhoff et al., Atlas of Protein Sequence and Structure. Natl. Biomed. Res. Found., Washington, CD. (1978), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, are preferred. Routine screening assays can be used to determine if an immunopeptide containing a PSA or PSP variant produces an autoimmune response when administered to a human. Examples of such screening assays are well known in the art and include enzyme linked immunosorbant assays, radioimmuno assays, and the like. Such assays have been described. Harlow et al., Antibodies: A Laboratory Manual, page 319 (Cold Spring Harbor Pub. 1988).

The terms "effective amount" and "therapeutically effective amount" are terms to identify an amount sufficient to obtain the desired physiological effect, e.g., treatment of a condition, disorder, disease and the like or reduction in symptoms of the condition, disorder, disease and the like. Such an effective amount of an immunopeptide or immunofragopeptide of the invention in the context of the disclosed methods is an amount that results in reducing, reversing, ameliorating or inhibiting prostate cancer.

An "immunopeptide" is prostate specific antigen (PSA) coupled to an autoimmune tag or prostate specific peptide (PSP) coupled to an autoimmune tag. An immunopeptide also includes PSA or PSP alleles that are coupled to an autoimmune tag. The nucleic acid and amino acid sequences for PSA or PSP and alleles thereof are available from the National Center for Biotechnology Information database and are provided in U.S. Patent No. 6,329,505.

An "immunofragopeptide" is a fragment of a PSP or PSA, a conservative variant of PSA or a PSP, a peptidomimetic of PSA or a PSP, wherein each has been coupled to an autoimmune tag. Examples of prostate specific polypeptides are described in U.S. Patent No. 6,329,505.

The term "peptidomimetic" or "peptide mimetic" describes a peptide analog, such as those commonly used in the pharmaceutical industry as non- peptide drugs, with properties analogous to those of the template peptide.

(Fauchere, J., Adv. Drug Res., 15: 29 (1986) and Evans et al., J. Med. Chem.. 30: 1229 (1987)). Peptidomimetics are structurally similar to PSA or a PSP used to create an immunopeptide of the invention, but have one or more peptide linkages optionally replaced by a linkage such as, — CH₂NH~, — CH₂S~, — CH₂~ CH₂-, -CH=CH- (cis and trans), -COCH₂~, -CH(OH)CH₂-, and -CH₂SO~, by methods known in the art. Advantages of peptide mimetics over natural polypeptide embodiments may include more economical production, greater chemical stability, altered specificity and enhanced pharmacological properties such as half-life, absorption, potency and efficacy. Accordingly, an immunopeptide of the invention may include a PSA or a PSP that is a peptidomimetic or a portion of PSA or a PSP that is a peptidomimetic.

The term "prostate specific antigen" or "PSA" includes a polypeptide that is encoded by the kallikrein 3 gene and alleles thereof. An example of a nucleic acid sequence corresponding to the human kallikrein 3 gene has Genebank accession number XM_031768.

The term "prostate specific polypeptide" or "PSP" includes a polypeptide that is specifically expressed by prostate cells. Such prostate specific polypeptides are exemplified by those described in U.S. Patent No. 6,329,505.

1. Immunopeptides

The invention provides immunopeptides able to induce an immune response to prostate specific antigen (PSA) when administered to a human. The invention also provides immunopeptides able to induce an immune response to a prostate specific polypeptide (PSP) when administered to a human. The immunopeptides of the invention include prostate specific antigen (PSA), and alleles thereof, coupled to an autoimmune tag. Furthermore, the immunopeptides of the invention include prostate specific polypeptides (PSP) and alleles thereof that are coupled to an autoimmune tag. Such prostate specific polypeptides are described in U.S. Patent No. 6,329,505. The immunofragopeptides of the invention include fragments, peptidomimetics and conservative variants of PSA or a PSP that are coupled to an autoimmune tag and that produce an immune response against PSA or the PSP when administered to a human. Induction of an immune response allows the immune system of the human to disable cells that express PSA or a PSP.

An autoimmune tag may be coupled to PSA or a PSP in a variety of art recognized ways to create an immunopeptide or an immunofragopeptide of the invention. Examples of such methods have been disclosed. Weigle, J. Exp. Med., 116:913-928 (1962); Weigle, J. Exp. Med.. 122:1049-1062 (1965);

Weigle, J. Exp. Med., 121:289-308 (1965). For example, diazonium derivatives of arsanilic and sulfanilic acid may be coupled to PSA or a PSP by first dissolving sulfanilic acid or arsanilic acid in a mixture of HC1 and NaNO₂. The tag solution is then added to a solution containing PSA or the PSP in phosphate buffer at pH 7.5 and 0°C. The pH is maintained at 7.5 to 7.8 by addition of NaOH. The non-coupled derivatives may be removed from the immunopeptide by passage through sephadex G-25. An acetyl group may be coupled to PSA or a PSP according to the method described by Olcott and Fraenkel-Conrat, Chem. Rev., 41 : 151 (1947). A picryl group may be coupled to PSA or a PSP according to the method described by Benacerraf and Gell, Immunology, 2:53 (1959). One skilled in the art recognizes that many methods may be used to couple an autoimmune tag to PSA or a PSP to create an immunopeptide or an immunofragopeptide of the invention.

PSA or a PSP used to create an immunopeptide or an immunofragopeptide of the invention can be obtained by a variety of methods known in the art. For example, PSA or a PSP can be purified from cells through use of conventional chromatography techniques. Examples of such techniques include, but are not limited to, ammonium sulfate precipitation, column chromatography, high pressure liquid chromatography, fast protein liquid chromatography, electrophoresis, and the like. Such methods are routine to those of skill in the art. PSA or a PSP may also be prepared through use of synthetic methods. Such methods are routine and have been described. Memfield, Science, 85:2149 (1963). Synthetic methods may also be used to produce PSA, a PSP, or a portion thereof that is a peptidomimetic. PSA or a PSP may also be prepared through use of recombinant techniques. For example, a gene encoding PSA or a PSP may be cloned into an expression vector and expressed in a cell into which the recombinant expression vector is introduced. Expression vectors include plasmids, viruses, artificial chromosomes, and the like. Methods to clone and express genes are well known in the art and have been described. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Such recombinant methods can be used to clone and express a nucleic acid sequence encoding PSA, a PSP, or a fragment or variant thereof as described herein.

An immunopeptide or immunofragopeptide of the invention will produce an immune response when administered to a human. The production of an immune response to the immunopeptide or immunofragopeptide can be determined through use of many methods known in the art. For example, production of antibodies that bind to the immunopeptides and immunofragopeptides of the invention can be used as an indicator of an immune response. Methods to screen for antibody production include, but are not limited to, antibody capture and antigen capture assays. Screening assays are well known and have been described. Harlow et al., Antibodies: A Laboratory Manual, page 319 (Cold Spring Harbor Pub. 1988).

2. Pharmaceutical compositions The invention provides pharmaceutical compositions that include an immunopeptide or an immunofragopeptide of the invention in combination with a pharmaceutically acceptable carrier. The invention also includes pharmaceutical compositions containing an immunopeptide or immunofragopeptide and another pharmaceutical agent known in the art. Such pharmaceutical agents are listed in the official United States Pharmacopeia, official Homeopathic Pharmacopeia of the United States, official National Formulary or any supplement thereof.

The pharmaceutical compositions of the invention may be prepared in many forms that include tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow-release formulations, such as shaped polymeric gels. An oral dosage form may be formulated such that an immunopeptide or immunofragopeptide of the invention is released into the intestine after passing through the stomach. Such formulations are described in U.S. Patent No. 6,306,434 and in the references contained therein. Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non- aqueous vehicles (which may include edible oils), or preservatives.

The immunopeptides and immunofragopeptides of the invention are preferably formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampules, prefilled syringes, small volume infusion containers or multi-dose containers with an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the immunopeptides or immunofragopeptides of the invention may be in powder form, obtained by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile saline, before use.

For topical administration to the epidermis, the immunopeptides and immunofragopeptides of the invention may be formulated as ointments, creams, lotions or patches for transdermal delivery. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.

Pharmaceutical compositions suitable for rectal administration are most preferably presented as unit dose suppositories. Suitable carriers include saline solution and other materials commonly used in the art. For administration by inhalation, the immunopeptides and immunofragopeptides of the invention are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, an immunopeptide or immunofragopeptide of the invention may take the form of a dry powder composition, for example, a powder mix of an immunopeptide and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator. For intra-nasal administration, the immunopeptides and immunofragopeptides of the invention may be administered via a liquid spray, such as via a plastic bottle atomizer.

Pharmaceutical compositions of the invention may also contain other ingredients such as flavorings, colorings, anti-microbial agents, or preservatives.

It will be appreciated that the amount of the present immunopeptides and immunofragopeptides, required for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient. Ultimately the attendant health care provider may determine proper dosage.

3. Methods to treat prostate cancer

The invention provides methods to treat prostate cancer. The methods involve administering an effective amount of an immunopeptide or immunofragopeptide to a patient in need thereof such that the patient overcomes self-tolerance and produces an autoimmune response to cells that express PSA or a PSP. Thereafter the immune system of the patient will disable cells that express PSA or a PSP. In this manner, patients suffering from prostate cancer may be treated. An immunopeptide or immunofragopeptide of the invention may be administered to a patient according to methods known in the art and described herein. The immunopeptides and immunofragopeptides of the invention may be administered to a patient alone or in a pharmaceutical composition. The immunopeptides and immunofragopeptides of the invention may also be administered to a patient in need thereof in conjunction with additional therapeutic agents.

The amount or route of administration of an immunopeptide or immunofragopeptide can be determined by the attending physician or other practitioner. Such amounts can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅o (the dose lethal to 50% of the population) and the ED₅o (the dose therapeutically effective in 50% of the population). The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized.

The dosage regimen and amount for treating patients with the immunopeptides or immunofragopeptides of the invention is selected in accordance with a variety of factors that include, for example, the age, weight and medical condition of the patient, the severity of the condition, and the route of administration. An ordinarily skilled physician can readily determine and prescribe an effective amount of an immunopeptide or immunofragopeptide to treat prostate cancer. In so proceeding, the physician can employ relatively low initial dosages and subsequently increase the dose until a maximum response is obtained.

An immunopeptide or immunofragopeptide is administered in unit dosage form, for example, containing about 0.05 mg to about 500 mg, preferably about 0.1 mg to about 250 mg, and more preferably about 1 mg to about 150 mg, of an immunopeptide or immunofragopeptide per unit dosage form. The desired dose can be presented in a single dose, or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself can be further divided, e.g., into a number of discrete loosely spaced administrations. An immunopeptide or immunofragopeptide can be administered in a variety of ways that include orally, sublingually, transdermally, or parenterally at dose levels of about 0.01 to about 150 mg/kg, preferably about 0.1 to about 50 mg/kg, and more preferably about 0.1 to about 10 mg/kg of body weight. The exact regimen for administration of the immunopeptides, immunofragopeptides and pharmaceutical compositions disclosed herein necessarily depends upon the needs of the individual subject being treated and the judgment of the attending practitioner or physician. In practice, the physician determines the actual dosing regimen which is most suitable for an individual patient, and the dosage varies with the age, weight, and response of the particular patient. The above dosages are exemplary, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of this invention. Treatment of prostate cancer with the immunopeptides, immunofragopeptides and pharmaceutical compositions disclosed herein may be assessed through use of many art recognized methods. These methods include monitoring PSA level, acid phosphatase level and tumor volume, with a decrease in level or volume indicating a decrease in prostate cancer progression.

4. Antibodies that bind the immunopeptides of the invention

The invention provides antibodies to the immunopeptides and immunofragopeptides of the invention. These antibodies are useful for determining the concentration of an immunopeptide or immunofragopeptide in a pharmaceutical composition. These antibodies are also useful for determining and monitoring the concentration of an immunopeptide or immunofragopeptide that has been administered to a patient.

The antibodies of the invention may be used to assess a regimin used to administer the immunopeptides, immunofragopeptides and pharmaceutical compositions disclosed herein. For example, the clearance rate of an intervenously administered immunopeptide or immunofragopeptide may be determined by drawing blood from the patient at multiple times following administration and using the antibody to detect the level of the immunopeptide or immunofragopeptide in the blood. This information may be used to determine a proper administration regimin for a particular patient.

Antibodies that bind to the immunopeptides and immunofragopeptides of the invention 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. In one technique, the immunopeptide or immunofragopeptide is injected into an animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animal is bled periodically. Polyclonal antibodies specific for the immunopeptide or immunofragopeptide may then be purified from such antisera by, for example, affinity chromatography using the immunopeptide or immunofragopeptide coupled to a suitable solid support.

A variety of techniques may be used to remove undesired antibodies that bind to PSA or a PSP that is not coupled to an autoimmune tag. These methods include passage of an antibody preparation over a column containing PSA or a PSP that is not coupled to an autoimmune tag and collection of the preparation after it exits the column. Such a preparation will lack undesired antibodies that bind to PSA or a PSP that is not coupled to an autoimmune tag. In another example, undesired antibodies may be removed from an antibody preparation by immunoprecipitation with PSA or a PSP that is not coupled to an autoimmune tag. Many additional methods are known in the art and may be used within the scope of the invention.

Monoclonal antibodies specific for an immunopeptide or immunofragopeptide of the invention 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 immunopeptide or immunofragopeptide 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. For example, 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 immunopeptide. Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, 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 immunopeptides and immunofragopeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.

Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, 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.

References

1. W. Kreis, D. R. Budman, A. Calabro, Unique synergism or antagonism of combinations of chemotherapeutic and hormonal agents in human prostate cancer cell lines. Br. J. Urol., 79, 196-202 (1997). 2. M. Colleoni et al., Phase II study of estramustine, oral etoposide, and vinorelbine in hormone-refractory prostate cancer. American Journal of Clinical Oncology, 20(4), 383-6 (1997).

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All publications, patents and patent applications cited herein are incorporated herein by reference. The foregoing specification has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, however, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

What is Claimed is:

1. An immunopeptide comprising prostate specific antigen (PSA) or an allele thereof coupled to at least one autoimmune tag.

2. The immunopeptide of claim 1, wherein the autoimmune tag is a diazonium derivative of arsanilic acid, a diazonium derivative of sulfanilic acid, an acetyl group or a picryl group.

3. The immunopeptide of claim 1, wherein the autoimmune tag is a diazonium derivative of arsanilic acid and a diazonium derivative of sulfanilic acid or an acetyl and a picryl group.

4. An immunopeptide comprising a portion of prostate specific antigen (PSA) coupled to an autoimmune tag.

5. The immunopeptide of claim 4, wherein administration of the immunopeptide to a human causes the human to produce antibodies against prostate specific antigen (PSA).

6. The immunopeptide of claim 4, wherein the autoimmune tag is a diazonium derivative of arsanilic acid or a diazonium derivative of sulfanilic acid.

7. The immunopeptide of claim 4, wherein the autoimmune tag is a diazonium derivative of arsanilic acid and a diazonium derivative of sulfanilic acid.

8. A pharmaceutical composition comprising the immunopeptide of claim 1 and a pharmaceutically acceptable carrier.

9. A pharmaceutical composition comprising the immunopeptide of claim 4 and a pharmaceutically acceptable carrier.

10. A method to treat prostate cancer comprising administering to a patient in need thereof an effective amount of an immunopeptide of claim 1.

11. A method to treat prostate cancer comprising administering to a patient in need thereof an effective amount of the pharmaceutical composition of claim 8.

12. The method of claim 10, wherein the immunopeptide is administered in conjunction with an adjuvant.

13. The method of claim 10, wherein a therapeutic agent and the immunopeptide are administered to the patient.

14. A method to treat prostate cancer comprising administering to a patient in need thereof an effective amount of a composition of claim 4.

15. An antibody to an immunopeptide of claim 1.

16. The antibody of claim 15, wherein the antibody is a single-chain antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, or a recombinant antibody.

17. An antibody to an immunopeptide of claim 4.

18. The antibody of claim 17, wherein the antibody is a single-chain antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, or a recombinant antibody.

19. An immunopeptide comprising a prostate specific polypeptide (PSP) or an allele thereof coupled to at least one autoimmune tag.

20. The immunopeptide of claim 19, wherein the autoimmune tag is a diazonium derivative of arsanilic acid, a diazonium derivative of sulfanilic acid, an acetyl group or a picryl group.

21. The immunopeptide of claim 19, wherein the autoimmune tag is a diazonium derivative of arsanilic acid and a diazonium derivative of sulfanilic acid or an acetyl and a picryl group.

22. A pharmaceutical composition comprising the immunopeptide of claim 19 and a pharmaceutically acceptable carrier.

23. A method to treat prostate cancer comprising administering to a patient in need thereof an effective amount of an immunopeptide of claim 19.

24. The method of claim 23, wherein the immunopeptide is administered in conjunction with an adjuvant.

25. The method of claim 23, wherein a therapeutic agent and the immunopeptide are administered to the patient.

26. An antibody to an immunopeptide of claim 19.

27. The antibody of claim 26, wherein the antibody is a single-chain antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, or a recombinant antibody.

28. An immunofragopeptide comprising a fragment of PSA, a fragment of a PSP, a conservative variant of PSA, a conservative variant of a PSP, a peptidomimetic of PSA, or a peptidomimetic of a PSP, wherein each has been coupled to an autoimmune tag.

29. The immunopeptide of claim 28, wherein the autoimmune tag is selected from a diazonium derivative of arsanilic acid, a diazonium derivative of sulfanilic acid, an acetyl group or a picryl group.

30. The immunopeptide of claim 28, wherein the autoimmune tag is a diazonium derivative of arsanilic acid and a diazonium derivative of sulfanilic acid or an acetyl and a picryl group.

31. A pharmaceutical composition comprising the immunofragopeptide of claim 28 and a pharmaceutically acceptable carrier.

32. A method to treat prostate cancer comprising administering to a patient in need thereof an effective amount of an immunofragopeptide of claim 28.

33. The method of claim 32, wherein the immunofragopeptide is administered in conjunction with an adjuvant.

34. The method of claim 32, wherein a therapeutic agent and the immunofragopeptide are administered to the patient.

35. An antibody to an immunofragopeptide of claim 28.

36. The antibody of claim 35, wherein the antibody is a single-chain antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, or a recombinant antibody.