JP2009195236A - Compound for immunotherapy and diagnosis of breast cancer and method for their use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound and a method for the treatment and diagnosis of breast cancer. <P>SOLUTION: Provided are a polypeptide containing at least a portion of a breast tumor protein, a vaccine and a pharmaceutical composition for the immunotherapy of breast cancer containing the polypeptide, a polynucleotide molecule encoding the polypeptide, and a polynucleotide molecule for the preparation of the polypeptide. More particularly, provided are a polypeptide at least partly containing at least a portion of a protein preferentially expressed in the breast tumor tissue, and a polynucleotide molecule encoding the polypeptide. The polypeptide can be used in a vaccine and a pharmaceutical composition for breast cancer treatment. Such polypeptide and polynucleotide can be used for the immunodiagnosis of breast cancer. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

(Technical field)
The present invention relates generally to compositions and methods for the treatment and diagnosis of breast cancer. More particularly, the present invention relates to a polypeptide comprising at least a portion of a protein that is preferentially expressed in breast tumor tissue, and a polynucleotide molecule encoding such a polypeptide. Such polypeptides can be used in vaccines and pharmaceutical compositions for the treatment of breast cancer. In addition, such polypeptides and polynucleotides can be used in immunodiagnosis of breast cancer.

(Background of the Invention)
Breast cancer is a serious health problem for women in the United States and around the world. Although progress has been made in detecting and treating the disease, breast cancer remains the second leading cause of cancer death in women, affecting more than 180,000 women each year in the United States. . For women in North America, the chance of having a living breast cancer is currently 1 in 8.

  There are currently no vaccines or other generally superior methods for the prevention or treatment of breast cancer that are available. The response to the disease currently relies on a combination of early diagnosis (through routine breast screening procedures) and aggressive treatment, which includes surgery, radiation therapy, chemotherapy, and hormonal therapy One or more different treatments can be included. The course of treatment for a particular breast cancer is often selected based on various prognostic parameters, including analysis of specific tumor markers. See, for example, Porter-Jordan and Lipman, Breast Cancer 8: 73-100 (1994). However, the use of established markers often leads to difficult-to-interpret results and the high mortality observed in breast cancer patients indicates that improvements are needed in the treatment, diagnosis, and prevention of the disease. Show.

Porter-Jordan and Lippman, Breast Cancer 8: 73-100 (1994)

  Accordingly, there is a need in the art for improved methods of breast cancer treatment and diagnosis. The present invention fulfills these needs and provides other related advantages.

(Summary of the Invention)
The present invention provides compounds and methods for breast cancer immunotherapy. In one aspect, an isolated polypeptide is provided that includes at least an immunogenic portion of a breast tumor protein or a variant of that protein that differs only in conservative substitutions and / or modifications. Wherein the breast tumor protein comprises an amino acid sequence encoded by a polynucleotide molecule having a partial sequence selected from the group consisting of: (a) SEQ ID NOs: 3, 10, 17, 24, 45-52, and Nucleotide sequences shown in 55-67, 72, 73, and 89-94, (b) the complement of those nucleotide sequences, and (c) under moderate stringency conditions of (a) or (b) A sequence that hybridizes to a sequence.

  In a related aspect, an isolated polynucleotide molecule encoding the above polypeptide is provided. In certain embodiments, such polynucleotide molecules have the partial sequences provided in SEQ ID NOs: 3, 10, 17, 24, 45-52, and 55-67, 72, 73, and 89-94. The present invention further provides expression vectors comprising the polynucleotide molecules described above, and host cells transformed or transfected with such expression vectors. In a preferred embodiment, the host cell is E. coli. selected from the group consisting of E. coli, yeast, and mammalian cells.

  In another aspect, the present invention provides a fusion protein comprising the first and second polypeptides of the present invention, or a fusion protein comprising the polypeptide of the present invention and a known breast antigen.

  The invention also relates to a pharmaceutical composition comprising at least one of the above-mentioned polypeptides, or a polynucleotide molecule encoding such a polypeptide, and a physiologically acceptable carrier. Provided with a vaccine comprising a peptide or polynucleotide molecule in combination with a non-specific immune response enhancer. Pharmaceutical compositions and vaccines comprising one or more of the above fusion proteins are also provided.

  In a related aspect, a pharmaceutical composition for the treatment of breast cancer comprising at least one polypeptide and a physiologically acceptable carrier is provided. Wherein the polypeptide comprises an immunogenic portion of a breast tumor protein or variant thereof, wherein the breast tumor protein is encoded by a polynucleotide molecule having a partial sequence selected from the group consisting of: a) Nucleotide sequences shown in SEQ ID NOs: 1, 2, 4-9, 11-16, 18-23, 25-44, 53, 54, 68-71, and 74-88, (b) complementation of those nucleotides And (c) a sequence that hybridizes to the sequence of (a) or (b) under moderately stringent conditions. The invention also provides a vaccine for the treatment of breast cancer comprising such a polypeptide in combination with a non-specific immune response enhancer SEQ ID NO: 1, 2, 4-9, 11-16, 18-23, Provided with pharmaceutical compositions and vaccines comprising at least one polynucleotide molecule having the partial sequences provided in 25-44, 53, 54, 68-71, and 74-88.

  In yet another aspect, a method for inhibiting the development of breast cancer in a patient is provided, which comprises administering an effective amount of at least one of the above pharmaceutical compositions and / or vaccines.

  The invention also provides a method for immunodiagnosis of breast cancer, along with kits for use in such methods. In one particular aspect of the invention, a method for detecting breast cancer in a patient is provided, which includes: (a) a biological sample taken from a patient is one of the polypeptides of the invention. Contacting with a binding agent capable of binding; and (b) detecting a protein or polypeptide bound to the binding agent in the sample. In a preferred embodiment, the binding agent is an antibody, most preferably a monoclonal antibody.

  In a related aspect, a method is provided for monitoring the progression of breast cancer in a patient, comprising: (a) binding a biological sample taken from the patient to one of the above polypeptides. Obtaining, contacting with a binding agent; (b) determining the amount of protein or polypeptide that binds to the binding agent in the sample; (c) repeating steps (a) and (b); and A step of comparing the amounts of the polypeptides detected in (b) and (c).

  In a related aspect, the present invention uses antibodies, preferably monoclonal antibodies, that bind to the polypeptides of the present invention, and diagnostic kits containing such antibodies, and such antibodies to inhibit the development of breast cancer. Provide a method.

  The present invention further provides a method of detecting breast cancer, which comprises: (a) collecting a biological sample from a patient; (b) treating the sample with first and second in a polymerase chain reaction. In contact with the oligonucleotide primer, at least one of the oligonucleotide primers is specific for a DNA molecule encoding one of the above polypeptides; and (c) a first and second oligo Detecting in the sample a DNA sequence that amplifies in the presence of nucleotide primers. In a preferred embodiment, the at least one oligonucleotide primer comprises at least about 10 consecutive nucleotides of a DNA molecule having a subsequence selected from the group consisting of SEQ ID NOs: 1-94.

  In a further aspect, the invention provides a method for detecting breast cancer in a patient, comprising: (a) collecting a biological sample from the patient; (b) extracting the sample from the polypeptide described above. Contacting with an oligonucleotide probe specific for a polynucleotide molecule encoding one of: and (c) detecting a polynucleotide sequence that hybridizes to the oligonucleotide probe in the sample. Preferably, the oligonucleotide probe comprises at least about 15 consecutive nucleotides of a DNA molecule having a partial sequence selected from the group consisting of SEQ ID NOs: 1-94.

  In a related aspect, a diagnostic kit comprising the above-described oligonucleotide probe or primer is provided.

  These and other aspects of the invention will become apparent upon reference to the following detailed description. All references described herein are hereby incorporated by reference in their entirety, each as if individually incorporated.

FIGS. 1A and B show the lytic specific activity of the first and second B511S-specific CTL clones, respectively, which are autologous LCL transduced with B511 (black squares) or HLA-A3 (white squares). Was measured.

(Detailed description of the invention)
As noted above, the present invention generally relates to compositions and methods for immunotherapy and diagnosis of breast cancer. The composition of the invention is generally an isolated polypeptide comprising at least a portion of a breast tumor protein. Molecules (eg, antibodies or fragments thereof) that bind to a polypeptide of the invention are also included within the invention. Such molecules are referred to herein as “binding agents”.

  In particular, the present invention discloses a polypeptide comprising at least a portion of a human breast tumor protein, or a variant thereof. Wherein the breast tumor protein comprises an amino acid sequence encoded by a polynucleotide molecule comprising a sequence selected from the group consisting of: a nucleotide sequence set forth in SEQ ID NOs: 1-94; a complement of this nucleotide sequence; And variants thereof. The term “polypeptide” as used herein includes amino acid chains of any length, including full-length proteins, where amino acid residues are linked by covalent peptide bonds. Thus, a polypeptide comprising a portion of one of the above breast proteins may consist entirely of this portion, or this portion may be present within a larger polypeptide that includes additional sequences. . This additional sequence may be derived from a natural protein or heterologous, and such a sequence may be immunoreactive and / or antigenic.

As described herein, an “immunogenic portion” of a human breast tumor protein is a portion that can elicit an immune response in a patient suffering from breast cancer and binds to an antibody present in serum from a breast cancer patient. To do. Such immunogenic moieties generally comprise at least about 5 amino acid residues, more preferably at least about 10 amino acid residues, and most preferably at least about 20 amino acid residues. Immunogenic portions of the proteins described herein can be identified in antibody binding assays. Such assays can be performed using any of a variety of means generally known to those skilled in the art, for example, as described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988. Can be done by. For example, the polypeptide can be immobilized on a solid support (as described below) and contacted with patient serum such that antibodies in the serum are bound to the immobilized polypeptide. Unbound serum can then be removed and bound antibody can be detected using, for example, ¹²⁵ I-labeled protein A. Alternatively, the polypeptides can be used to generate monoclonal and polyclonal antibodies for use in detecting polypeptides in the blood or other body fluids of breast cancer patients. Methods for preparing and identifying immunogenic portions of antigens of known sequence are well known in the art, and include those described by Paul, Fundamental Immunology, 3rd edition, Raven Press, 1993, 243-247. The methods summarized on the page are mentioned.

  The term “polynucleotide” as used herein refers to a single-stranded or double-stranded polymer of deoxyribonucleotide bases or ribonucleotide bases, and includes DNA molecules and corresponding RNA molecules (HnRNA and mRNA molecules, including both sense and antisense strands) and include cDNA, genomic DNA, and recombinant DNA, as well as fully or partially synthesized polynucleotides. HnRNA molecules contain introns and generally correspond to DNA molecules in a one-to-one manner. mRNA molecules correspond to HnRNA and DNA molecules, but exclude introns. The polynucleotide may be composed of the entire gene or any part thereof. An operable antisense polynucleotide may comprise a fragment of the corresponding polynucleotide, so the definition of “polynucleotide” includes all such operable antisense fragments.

  The compositions and methods of the present invention also include variants of the polypeptides and polynucleotides described above. A polypeptide “variant” as used herein is only in conservative substitutions and / or modifications such that the therapeutic, antigenic, and / or immunogenic properties of the polypeptide are retained. A polypeptide different from the described polypeptide. In a preferred embodiment, the modified polypeptide differs from the sequence identified by 5 amino acid substitutions, deletions or additions. Such variants generally modify one of the polypeptide sequences described above and, for example, the antigenicity of a polypeptide that has been modified using the representative procedures described herein. Can be identified by evaluating properties. The polypeptide variant preferably has at least about 70% identity, more preferably at least about 90% identity, and most preferably at least about 95% identity to the identified polypeptide. (Determined as described below).

  As used herein, a “conservative substitution” refers to a substitution in which an amino acid has been replaced with another amino acid that exhibits similar properties, so that one skilled in the art of peptide chemistry will not be substantially altered. Predict the secondary structure and hydrophobic hydrophilic index properties of peptides. In general, the following amino acid groups show conservative changes: (1) ala, pro, gly, glu, asp, gln, 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.

  Variants can also alternatively include other modifications, which include amino acid deletions or additions that have minimal effect on the antigenic properties, secondary structure and hydrophobic hydrophilicity index properties of the polypeptide. For example, a polypeptide can be linked to a signal (or leader) sequence at the N-terminus of the protein, which signal (or leader) sequence is directed to protein transport simultaneously or after translation. The polypeptide may also be linked to a linker or other sequence to facilitate synthesis, purification or identification of the polypeptide (eg, poly-His) or to increase the binding of the polypeptide to a solid support. Can be done. For example, the polypeptide can be bound to an immunoglobulin Fc region.

  Nucleotide “variants” are sequences that differ from the nucleotide sequences described in having one or more nucleotide deletions, substitutions, or additions. Such modifications can be readily introduced using standard mutagenesis techniques, such as oligonucleotide-directed site-directed mutagenesis, as taught by Adelman et al. (DNA 2: 183, 1983). . Nucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. The modified nucleotide sequence preferably has at least about 70% identity, more preferably at least about 80%, and most preferably at least about 90% identity (as described below) to the described sequence. Determined).

  Antigens provided by the present invention include variants encoded by DNA sequences that are substantially homologous to one or more DNA sequences described in detail herein. As used herein, “substantial homology” refers to a DNA sequence that can hybridize under moderately stringent conditions. Appropriate, moderately stringent conditions are pre-wash in a solution of 5 × SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); 50 ° C. to 65 ° C., 5 × SSC, one In the evening or cross-species homology events, hybridization at 45 ° C. with 0.5 × SSC; then 2 ×, 0.5 ×, and 0. 1% with 0.1% SDS. 2x SSC, 20 minutes at 65 ° C, each containing 2 washes. Such hybridizing DNA sequences are also within the scope of the present invention. Also included are nucleotide sequences that encode immunogenic polypeptides encoded by the hybridizing DNA sequences due to code degeneracy.

  Two nucleotide or polypeptide sequences are said to be “identical” if the sequences of nucleotides or amino acid residues in the two sequences are the same when aligned with the greatest correspondence, as described below. Comparison between two sequences is typically performed by comparing the sequences through a comparison window to identify and compare local regions of sequence similarity. As used herein, a “comparison window” refers to a segment of at least about 20 consecutive positions, usually 30 to about 75, more preferably 40 to about 50 consecutive positions. Here, the sequences can be compared against a reference sequence at the same number of consecutive positions after the two sequences are aligned as necessary.

好ましくは、「配列同一性のパーセント割合」は、少なくとも２０の位置の比較ウインドウによって、２つの最適にアラインされた配列を比較することによって決定される。ここで、比較ウインドウ中のポリヌクレオチド配列の部分は、２つの配列の最適なアラインメントについて参照配列（これは、付加または欠失を有さない）と比較して、２０％以下、通常は５〜１５％、または１０〜１２％の付加または欠失（すなわち、ギャップ）を含み得る。このパーセント割合は、位置の数（ここで、同一の核酸塩基またはアミノ酸残基が両方の配列で生じて、マッチした位置の数を得る）を決定し、参照配列中の位置の総数（すなわち、ウインドウサイズ）でマッチした位置の数を割り、そして結果に１００をかけて配列同一性のパーセント割合を得ることによって計算される。 Optimal alignment of sequences for comparison can be performed using the Megaalign program of Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) Using default parameters. This program includes several alignment schemes described in the following references:

Preferably, “percent sequence identity” is determined by comparing two optimally aligned sequences by a comparison window of at least 20 positions. Here, the portion of the polynucleotide sequence in the comparison window is no more than 20%, usually 5-5, compared to the reference sequence (which has no additions or deletions) for optimal alignment of the two sequences. It may contain 15%, or 10-12% additions or deletions (ie gaps). This percentage percentage determines the number of positions (where the same nucleobase or amino acid residue occurs in both sequences to obtain the number of matched positions) and the total number of positions in the reference sequence (ie, Calculated by dividing the number of matched positions by (window size) and multiplying the result by 100 to get the percent sequence identity.

  Alleles of the genes encoding the nucleotide sequences described herein are also within the scope of the invention. As used herein, an “allele” or “allelic sequence” is an alternative form of a gene that can result from at least one mutation in a nucleic acid sequence. Alleles can give rise to altered mRNAs or polypeptides that may or may not change in structure or function. Any given gene may have no allelic form or one or more allelic forms. The usual mutational changes that give rise to alleles are generally due to natural deletions, additions or substitutions of nucleotides. Each type of these changes may occur one or more times in a given sequence, either alone or in combination with the other.

  Alternatively, for breast tumor polypeptides having immunoreactive properties, variants are identified by altering the amino acid sequence of one of the aforementioned polypeptides and evaluating the immunoreactivity of the altered polypeptide Can be done. For breast tumor polypeptides useful for producing diagnostic binding agents, variants can be identified by evaluating the modified polypeptide for the ability to produce antibodies that detect the presence or absence of breast cancer. . Such modified sequences can be prepared and tested using, for example, representative procedures described herein.

  The breast tumor proteins of the invention and polynucleotide molecules encoding such proteins can be isolated from breast tumor tissue using any of a variety of methods well known in the art. A polynucleotide sequence corresponding to a gene (or portion thereof) encoding one of the breast tumor proteins of the invention is isolated from a breast tumor cDNA library using the subtraction technique described in detail below. Can be done. Examples of such DNA sequences are provided in SEQ ID NOs: 1-94. In this way, the resulting partial polynucleotide sequence can be used to design oligonucleotide primers for amplifying full-length polynucleotide sequences in polymerase chain reaction (PCR) using techniques well known in the art. (See, eg, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263, 1987; edited by Errich, PCR Technology, Stockton Press, NY, 1989). Once a polynucleotide sequence encoding the polypeptide is obtained, any of the above modifications may be performed using oligonucleotide-directed site-directed mutations as taught, for example, by Adelman et al. (DNA, 2: 183, 1983). It can be easily introduced using standard mutagenesis techniques such as induction.

  The breast tumor polypeptides disclosed herein can also be made by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, can be made using techniques well known to those skilled in the art. For example, such polypeptides can be obtained by using the Merrifield solid phase synthesis method in which amino acids are sequentially added to the extending amino acid chain (see, eg, Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963). ) And can be synthesized using any commercially available solid phase technique. Polypeptide automated synthesizers are commercially available from suppliers such as Perkin Elmer / Applied BioSystems Division (Foster City, Calif.) And can be operated according to the manufacturer's instructions.

  Alternatively, any of the above-described polypeptides can be produced recombinantly by inserting a polynucleotide sequence encoding the polypeptide into an expression vector and expressing the protein in a suitable host. Any of a variety of expression vectors known to those skilled in the art can be used to express the recombinant polypeptides of the invention. Expression can be achieved in any appropriate host cell transformed or transfected with an expression vector comprising a polynucleotide molecule encoding the recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cell used is E. coli. mammalian cell lines such as E. coli, yeast or CHO cells. A polynucleotide sequence expressed by this approach may encode a naturally occurring polypeptide, a portion of a naturally occurring polypeptide, or other variants thereof.

  In general, regardless of the method of preparation, the polypeptides disclosed herein are prepared in isolated, substantially pure form (ie, the polypeptide is determined by amino acid composition and primary sequence analysis). If you are homogeneous) Preferably, the polypeptide is at least about 90% pure, more preferably at least about 95% pure, and most preferably at least about 99% pure. In certain preferred embodiments, described in further detail below, the substantially pure polypeptide is incorporated into a pharmaceutical composition or vaccine for use in one or more methods disclosed herein. It is done.

  In a related aspect, the present invention provides a fusion protein comprising the first and second polypeptides of the present invention, or alternatively a fusion protein comprising the polypeptide of the present invention and a known breast tumor antigen. Provided with the variant.

  The polynucleotide sequence encoding the fusion protein of the invention is constructed using known recombinant DNA techniques to assemble separate polynucleotide sequences encoding the first and second polynucleotides into an appropriate expression vector. The The 3 ′ end of the polynucleotide sequence encoding the first polypeptide contains two DNAs to a single fusion protein whose sequence reading frame retains the biological activity of both the first and second polypeptides. In combination to allow mRNA translation of the sequence, it is linked with or without a peptide linker to the 5 ′ end of the polynucleotide sequence encoding the second polypeptide.

  The peptide linker sequence can be used to separate the first and second polypeptides by a distance sufficient to ensure that each polypeptide is folded into its secondary and tertiary structure. Such peptide linker sequences are incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) ability to adopt a flexible extended conformation; (2) interact with functional epitopes on the first and second polypeptides. Inability to adopt a secondary structure that can act; and (3) deletion of hydrophobic or charged residues that can react with a functional epitope of the polypeptide. Preferred peptide linker sequences include Gly, Asn and Ser residues. Other near neutral amino acids such as Thr and Ala can also be used in the linker sequence. Amino acid sequences that can be usefully used as linkers are described in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8262, 1986; including the amino acid sequences disclosed in US Pat. No. 4,935,233 and US Pat. No. 4,751,180. The linker sequence can be from 1 to about 50 amino acids in length. The peptide sequence is not required if the first and second polypeptides have a nonessential N-terminal amino acid region that can be used to separate functional domains and prevent steric interference.

  The linked polynucleotide sequence is operably linked to appropriate transcriptional or translational regulatory elements. The regulatory elements responsible for the expression of the polynucleotide are located only 5 'to the polynucleotide sequence encoding the first polypeptide. Similarly, the stop codon required to terminate translation and transcription termination signals is present only 3 'to the polynucleotide sequence encoding the second polypeptide.

  Also provided is a fusion protein comprising a polypeptide of the invention with an unrelated immunogenic protein. Preferably, the immunogenic protein is capable of eliciting a recall response. Examples of such proteins include tetanus protein, tuberculosis protein and hepatitis protein (see, eg, Stoute et al., New Engl. J. Med., 336: 86-91 (1997)).

  A polypeptide of the invention comprising an immunogenic portion of a breast tumor protein can generally be used for breast cancer immunotherapy, where the polypeptide stimulates the patient's own immune response against breast tumor cells. . In a further aspect, the present invention relates to one or more immunoresponsive polypeptides (or one or more such polypeptides and / or encoded by a polynucleotide molecule having the sequence provided in SEQ ID NOs: 1-94). Or a fusion protein comprising a polynucleotide encoding such a polypeptide) for use in immunotherapy of breast cancer in a patient. As used herein, “patient” refers to any warm-blooded animal, preferably a human. The patient may be afflicted with a disease or may not have a detectable disease. Thus, the above-described immunoresponsive polypeptides (or fusion proteins or polynucleotide molecules encoding such polypeptides) can be used to treat breast cancer or used to inhibit breast cancer progression. Can be. The polypeptide can be administered either prior to or subsequent to surgical removal of the primary tumor and / or treatment by radiation therapy and administration of conventional chemotherapeutic drugs.

  In these aspects, the polypeptide or fusion protein is generally present in pharmaceutical compositions and / or vaccines. The pharmaceutical composition can include one or more polypeptides, each of which can each include one or more of the above-described sequences (or variants thereof), and a physiologically acceptable carrier. A vaccine can include one or more such polypeptides and a non-specific immune response enhancer, where the non-specific immune response enhancer can elicit or enhance an immune response against a foreign antigen. Examples of non-specific immune response enhancers include adjuvants, biodegradable microspheres (eg, polylactic galactide) and liposomes (in which the polypeptide is incorporated). Pharmaceutical compositions and vaccines can also be used to combine breast tumor antigens, either incorporated into a combination polypeptide (ie, a single polypeptide comprising multiple epitopes) or present in separate polypeptides. Other epitopes can be included.

  Alternatively, the polypeptide is produced in situ because the pharmaceutical composition or vaccine may comprise a polynucleotide encoding one or more of the polypeptides described above. In such pharmaceutical compositions and vaccines, the polynucleotide may be present in any of a variety of delivery systems known to those skilled in the art, including nucleic acid expression systems, bacterial and viral expression systems. A suitable nucleic acid expression system includes a polynucleotide sequence (eg, a suitable promoter) necessary for expression in a patient. Bacterial delivery systems involve the administration of bacteria (eg, Bacillus-Calmette-Guerrin) that express epitopes of breast tumor cell antigens that are present on the cell surface. In a preferred embodiment, the polynucleotide molecule can be introduced using a viral expression system (eg, urticaria or other poxviruses, retroviruses, or adenoviruses) that is non-pathogenic (deficient). May include the use of replicating competent cells. Suitable systems are described, for example, in Fisher-Hoch et al., PNAS 86: 317-321, 1989; Flexner et al., Ann. N. Y. Acad. Sci. 569: 86-103, 1989; Flexner et al., Vaccine 8: 17-21, 1990; US Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6: 616-627, 1988; Rosenfeld et al., Science 252: 431-434, 1991; Kolls et al., PNAS 91: 215-219, 1994; Kass-Eisler et al., PNAS 90: 11498-11502, 1993; Guzman et al., Circulation 88: 2838-2848, 1993; and Guz. man et al., Cir. Res 73: 1202-1207, 1993. Methods for incorporating polynucleotides into such expression systems are well known to those skilled in the art. Polynucleotides can also be “naked” (eg, published PCT application WO 90/11092, and Ulmer et al., Science 259: 1745-1749, 1993, review by Cohen, Science 259: 1691-1692. , 1993). Naked polynucleotide uptake can be increased by coating the polynucleotide onto a biodegradable bead, which is efficiently transported into the cell.

  The route and frequency of administration, as well as the dosage, will vary between individuals and may be similar to those currently used in immunotherapy of other diseases. In general, pharmaceutical compositions and vaccines can be administered by injection (eg, intradermal, intramuscular, intravenous or subcutaneous), intranasal (eg, by inhalation) or orally. 1 to 10 doses can be administered over 3-24 weeks. Preferably, 4 doses are administered at 3 month intervals, and thereafter booster administration can be performed periodically. Alternative protocols may be appropriate for each patient. An appropriate dose is the amount of a polypeptide or polynucleotide molecule that is effective to produce an immune response (cellular and / or humoral) against breast tumor cells in the treated patient. A suitable immune response is at least 10-50% above the basal (ie, untreated) level. In general, the amount of polypeptide present in a dose (or produced in situ by a dose of polynucleotide) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to About 1 mg, and preferably about 100 pg to about 1 μg. Suitable dosage sizes vary with patient size, but typically range from about 0.01 mL to about 5 mL.

  Although any suitable carrier known to those skilled in the art can be utilized in the pharmaceutical compositions of the invention, the type of carrier will vary depending on the mode of administration. For parenteral administration (eg, subcutaneous injection), the carrier preferably comprises water, saline, alcohol, lipids, waxes and / or buffers. For oral administration, any of the above carriers or solid carriers (eg, mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and / or magnesium carbonate) are utilized. Can be done. Biodegradable microspheres (eg, polylactic glycolide) can also be utilized as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are disclosed, for example, in US Pat. Nos. 4,897,268 and 5,075,109.

  Any of a variety of non-specific immune response enhancers can be utilized in the vaccines of the present invention. For example, an adjuvant can be included. Most adjuvants contain substances designed to protect the antigen from rapid catabolism (e.g., aluminum hydroxide or mineral oil, and non-specific immune responses such as lipid A, Bordella pertussis or Mycobacterium tuberculosis) Irritant). Such adjuvants are commercially available, for example, as Freund's incomplete and Freund's complete adjuvant (Difco Laboratories, Detroit, MI) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ).

  The polypeptides disclosed herein can also be used in adoptive immunotherapy for the treatment of cancer. Adoptive immunotherapy can also be broadly classified as either active or passive immunotherapy. In active immunotherapy, treatment relies on in vivo stimulation of the endogenous host immune system and responds to the tumor with the administration of immune response modifying agents (eg, tumor vaccines, bacterial adjuvants, and / or cytokines).

  In passive immunotherapy, treatment involves delivery of biological reagents (eg, effector cells or antibodies) with established tumor-immunoreactivity. This established tumor-immunoreactivity can mediate antitumor effects directly or indirectly and is not necessarily dependent on the intact host immune system. Examples of effector cells include T lymphocytes (eg, CD8 + cytotoxic T lymphocytes, CD4 + T helper, tumor infiltrating lymphocytes), killer cells (eg, natural killer cells, lymphokine activated killer cells), B cells, Or antigen-presenting cells (eg, dendritic cells and macrophages) that express the disclosed antigens. The polypeptides disclosed herein can also be used to produce antibodies or anti-idiotype antibodies (similar to US Pat. No. 4,918,164) for passive immunotherapy.

  The dominant method of procuring an appropriate number of T cells for adoptive immunotherapy is to proliferate immune T cells in vitro. Culture conditions for expanding single antigen-specific T cells to billions with retention of antigen recognition in vivo are well known in the art. These in vitro culture conditions typically utilize intermittent stimulation with antigen, often in the presence of cytokines such as IL-2 and non-dividing feeder cells. As described above, the immunoreactive polypeptides described herein rapidly expand antigen-specific T cell cultures to generate a sufficient number of cells for immunotherapy. Can be used to In particular, antigen presenting cells (eg, dendritic cells, macrophages or B cells) can be pulsed with immunoreactive polypeptides using standard techniques well known in the art, or It can be transfected with the nucleotide sequence (s). For example, antigen presenting cells can be transfected with a polynucleotide sequence, where the sequence includes a promoter region suitable for increasing expression, and part of a recombinant virus or other expression system. Can be expressed as In order for cultured T cells to be effective in therapy, the cultured T cells must be able to proliferate and be widely distributed and survive long-term in vivo. Studies have shown that cultured T cells can be induced to proliferate in vivo and to survive for a long time in substantial numbers by repeated stimulation with antigen supplemented with IL-2 (eg, See Chever, M., et al., “Therapeutic With Cultured T Cells: Principles Revised”, Immunological Reviews, 157: 177, 1997).

  The polypeptides disclosed herein can also be used to generate and / or isolate tumor-reactive T cells, which can then be administered to a patient. In one technique, antigen-specific T cell lines can be generated by immunizing in vivo with a short peptide corresponding to the immunogenic portion of the disclosed polypeptide. The resulting antigen-specific CD8 + CTL clone can be isolated from the patient, expanded using standard tissue culture techniques, and returned to the patient.

Alternatively, peptides corresponding to the immunogenic portion of the polypeptide can be obtained from autologous T cells as described, for example, in Chang et al. (Crit. Rev. Oncol. Hematol., 22 (3), 213, 1996). It can be used to generate tumor-reactive T cell subsets by selective stimulation and expansion in vitro and subsequently provide antigen-specific T cells that can be transferred to a patient. Cells of the immune system, such as T cells, are, for example, CellPro Incorporated's (Bothell, WA) CEPRATE ^™ system (US Pat. No. 5,240,856; US Pat. No. 5,215,926; WO 89/06280). Commercially available cell separation systems such as WO 91/16116 and WO 92/07243) can be isolated from the patient's peripheral blood. Isolated cells are stimulated with one or more of the immunoreactive polypeptides contained within a delivery vehicle (eg, macrosphere) to provide antigen-specific T cells. The tumor antigen specific T cell population is then expanded using standard techniques and the cells are administered back to the patient.

  In another embodiment, T cells and / or antibody receptors specific for the polypeptide can be cloned, expanded, and transferred to other vectors or effector cells for use in adoptive immunotherapy. .

  In a further embodiment, syngeneic or autologous dendritic cells can be pulsed with a peptide corresponding to at least an immunogenic portion of a polypeptide disclosed herein. The resulting antigen-specific dendritic cells can either be transferred to the patient or can be used to stimulate T cells to provide antigen-specific T cells that can be sequentially administered to the patient. It is. The use of peptide-pulsed dendritic cells to generate antigen-specific T cells, as well as the subsequent use of such antigen-specific T cells to eradicate tumors in a mouse model is described by Cheever et al. (Immunological Reviews 157: 177, 1997).

  In addition, vectors expressing the disclosed polynucleotides can be introduced into stem cells taken from a patient and can be clonal expanded in vitro for autotransplantation into the patient.

  The polypeptides of the present invention can also or alternatively be used to generate binding agents (eg, antibodies or fragments thereof) that can detect metastatic human breast tumors. The binding agents of the invention can generally be prepared using methods known to those skilled in the art, including the representative procedures described herein. A binding agent can distinguish between patients with and without breast cancer using the exemplary assays described herein. That is, antibodies or other binding agents produced against breast tumor proteins, or appropriate portions thereof, are present in the presence of primary or metastatic breast cancer in at least about 20% of patients affected by the disease. And a negative signal indicating the absence of disease in at least about 90% of individuals who do not have primary or metastatic breast cancer. Appropriate portions of such breast tumor proteins are primary or metastatic in substantially all (ie, at least about 80%, preferably at least about 90%) patients whose breast cancer is indicated using the full-length protein. The portion that can produce a binding agent that indicates the presence of sex breast cancer, as well as the absence of breast cancer in virtually all samples that are negative when tested with the full-length protein. Representative assays described below (eg, two-antibody sandwich assay) are generally used to assess the ability of binding agents to detect metastatic human breast tumors. obtain.

  The ability of a polypeptide prepared as described herein to produce an antibody capable of detecting a primary human breast metastasis or a metastatic human breast tumor generally has one or more antibodies against this polypeptide. Can be assessed by eliciting (eg, using the exemplary methods described herein) and by determining the ability of such antibodies to detect such tumors in a patient. . This determination can be made by assaying a biological sample from a patient with or without a primary human or metastatic breast tumor for the presence of a polypeptide that binds to the resulting antibody. Such a test assay can be performed, for example, using the representative procedure shown below. A polypeptide that produces an antibody capable of detecting at least 20% of primary or metastatic human breast tumors by such a procedure is for detecting primary or metastatic human breast tumors. It is considered useful for the assay. Polypeptide specific antibodies can be used alone or in combination to improve sensitivity.

  Polypeptides that can detect primary or metastatic human breast tumors can be used as markers for diagnosing breast cancer or for monitoring disease progression in a patient. In one embodiment, a patient's breast cancer can be diagnosed by evaluating a biological sample obtained from the patient for the level of one or more of the polypeptides above a predetermined cutoff value. As used herein, suitable “biological samples” include blood, serum and urine.

The level of one or more of the polypeptides can be assessed using any binding agent specific for the polypeptide. A “binding agent” in the context of the present invention is any agent (eg, compound or cell) that binds to a polypeptide as described above. As used herein, “binding” refers to the distance between two separate molecules (each molecule can be released (ie, in solution) or present on the surface of a cell or solid support). A non-covalent association in which a “complex” is formed. Such complexes can be free or immobilized on a support material (either covalently or non-covalently). Binding ability can generally be assessed by determining the binding constant for complex formation. This binding constant is the value obtained when the concentration of the complex is divided by the product of the concentrations of the components. In general, in the context of the present invention, two components are said to be “bound” if the binding constant for complex formation is greater than about 10 ³ L / mol. The binding constant can be determined using methods well known to those skilled in the art.

  Any agent that meets the above requirements can be a binder. For example, the binding agent can be a ribosome, RNA molecule or peptide with or without a peptide component. In a preferred embodiment, the binding partner is an antibody or a fragment thereof. Such an antibody can be a polyclonal antibody or a monoclonal antibody. Further, the antibody can be a single chain antibody, a chimeric antibody, a CDR-grafted antibody, or a humanized antibody. Antibodies can be prepared by the methods described herein and other methods well known to those skilled in the art.

  There are a variety of assay formats known to those of skill in the art for the use of binding partners to detect polypeptide markers in a sample. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In a preferred embodiment, the assay involves the use of a binding partner that binds to the polypeptide in the remainder of the sample and is immobilized on a solid support to remove the polypeptide therefrom. This bound polypeptide can then be detected using a second binding partner comprising a reporter group. Suitable second binding partners include antibodies that bind to the binding partner / polypeptide complex. Alternatively, a competitive assay can be utilized in which the polypeptide is labeled with a reporter group and allows binding to the immobilized binding partner after incubation of the sample with the binding partner. The degree to which the sample component inhibits the binding of the labeled polypeptide to the binding partner indicates the reactivity of the sample with the immobilized binding partner.

  The solid support can be any material known to those of skill in the art to which an antigen can be attached. For example, the solid support can be a test well of a microtiter plate, or a nitrocellulose membrane or other suitable membrane. Alternatively, the support can be a bead or disk (eg, glass, fiberglass, latex, or plastic material (eg, polystyrene or polyvinyl chloride)). The support can also be a magnetic particle or a fiber optical sensor (eg, as disclosed in US Pat. No. 5,359,681). The binder can be immobilized on the solid support using a variety of techniques known to those skilled in the art, which are well described in the patent and scientific literature. In the context of the present invention, the term “immobilization” refers to non-covalent association (eg adsorption) and covalent attachment (which is directly bound between an antigen and a functional group on the support). Or can be combined using a cross-linking agent). Immobilization by adsorption to a well of a microtiter plate or a membrane is preferred. In such cases, adsorption can be achieved by contacting the binder with a solid support in a suitable buffer at a suitable time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. Generally, contacting a well of a plastic microtiter plate (eg, polystyrene or polyvinyl chloride) with an amount of binder in the range of about 10 ng to about 10 μg, and preferably about 100 ng to about 1 μg, is appropriate. It is sufficient to immobilize the amount of binder.

  Covalent attachment of the binder to the solid support generally involves first reacting the support with a bifunctional reagent that reacts with both the support and a functional group (eg, a hydroxyl or amino group) on the binder. Can be achieved. For example, the binder can be covalently attached to a support having a suitable polymer that is coated by condensation of aldehyde groups on the support with benzoquinone or with an amine on the binding partner and an active hydrogen ( (See, for example, Pierce Immunotechnology Catalog and Handbook, 1991, A12-A13).

  In certain embodiments, the assay is a two antibody sandwich assay. The assay is performed by first contacting the antibody immobilized on a solid support (usually a well of a microtiter plate) with the sample and allowing the polypeptide in the sample to bind to the immobilized antibody. obtain. The unbound sample is then removed from the immobilized polypeptide-antibody complex and a second antibody (containing a reporter group) that can bind to a different site on the polypeptide is added. The amount of second antibody that remains bound to the solid support is then determined using a method appropriate for the particular reporter group.

More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are usually blocked. Any suitable blocking agent (eg, bovine serum albumin or Tween 20 ^™ (Sigma Chemical Co., St. Louis, MO)) is known to those skilled in the art. The immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind to the antibody. Prior to incubation, the sample can be diluted with an appropriate diluent, such as phosphate buffered saline (PBS). In general, a suitable contact time (ie, incubation time) is a time sufficient to detect the presence of a polypeptide in a sample obtained from an individual having breast cancer. Preferably, the contact time is sufficient to achieve a binding level at which an equilibrium between the bound and unbound polypeptides is achieved at least about 95%. One skilled in the art will recognize that by assaying the level of binding that occurs over a period of time, the time required to reach equilibrium can be readily determined. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound sample is then removed by washing the solid support with a suitable buffer (eg, PBS containing 0.1% Tween 20 ^™ ). A second antibody comprising a reporter group can then be added to the solid support. Preferred reporter groups include enzymes (eg, horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. Conjugation of the antibody to the reporter group can be accomplished using standard methods known to those skilled in the art.

  The second antibody is then incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound polypeptide. An appropriate amount of time can generally be determined by assaying the level of binding that occurs over a period of time. The unbound second antibody is then removed and the bound second antibody is detected using a reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopy can be used to detect dyes, luminescent groups and fluorescent groups. Vithion can be detected using avidin coupled to different reporter groups (generally radioactive or fluorescent groups or enzymes). Enzyme reporter groups can generally be detected by addition of a substrate (generally for a specified time) followed by spectroscopic or other analysis of the reaction product.

  In order to determine the presence or absence of breast cancer, the signal detected from the reporter group remaining bound to the solid support is generally compared to a signal corresponding to a predetermined cutoff value. In one preferred embodiment, this cutoff value is the average signal value obtained when the immobilized antibody is incubated with a sample from a patient without breast cancer. In general, samples that produce a signal that is 3 standard deviations above a given cutoff value are considered positive for breast cancer. In an alternative preferred embodiment, this cutoff value is determined by Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co. , 1985, pp. 106-7, using a receiver operator curve. Briefly, in this embodiment, this cutoff value is the true positive rate (ie sensitivity) and false positive rate (100% -specificity) corresponding to each possible cutoff value for a diagnostic test result. It can be determined from a paired plot. The cutoff value closest to the upper left hand corner on the plot (ie, the value surrounding the largest region) is the most accurate cutoff value, and positive for samples that yield a signal higher than the cutoff value determined by the method Can be considered. Alternatively, the cutoff value can be shifted to the left along the plot to minimize the false positive rate, or can be shifted to the right to minimize the false negative rate. In general, samples that produce a signal that is higher than the cut-off value determined by this method are considered positive for breast cancer.

  In related embodiments, the assay is performed in a flow-through test format or a strip test format (wherein the antibody is immobilized on a membrane such as nitrocellulose). In the flow-through test, the polypeptide in the sample binds to the immobilized antibody as the sample passes through the membrane. The second labeled antibody then binds to the antibody-polypeptide complex as the solution containing the second antibody flows through the membrane. Detection of the bound second antibody can then be performed as described above. In the strip test format, one end of the membrane to which the antibody is bound is immersed in a solution containing the sample. This sample travels along the membrane, through the region containing the second antibody, and to the region of immobilized antibody. The concentration of the second antibody in the area of immobilized antibody indicates the presence of breast cancer. Typically, the concentration of the second antibody at that site produces a pattern (eg, a line) that can be read visually. Not showing such a pattern indicates a negative result. In general, the amount of antibody immobilized on this membrane will be visual if the biological sample contains a level of polypeptide sufficient to produce a positive signal in a two-antibody sandwich assay in the format described above. Is selected to produce an identifiable pattern. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 500 ng. Such a test can typically be performed with a very small amount of biological sample.

  Of course, there are numerous other assay procedures that are suitable for use with the antigens or antibodies of the present invention. The above description is intended to be exemplary only.

  In another embodiment, the polypeptides described above can be used as markers for breast cancer progression. In this embodiment, assays such as those described above for the diagnosis of breast cancer can be performed over time and changes in the level of reactive polynucleotide (s) can be assessed. For example, the assay can be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter as needed. In general, breast cancer has progressed in patients whose level of polypeptide detected by the binding agent increases over time. In contrast, breast cancer is not progressing if the level of reactive polypeptide either remains constant or decreases with time.

  Antibodies for use in the above methods can be prepared by any of a variety of techniques known to those of skill in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogen containing an antigenic polypeptide is first injected into any of a wide variety of mammals (eg, mice, rats, rabbits, sheep, and goats). . In this step, the polypeptide of the present invention can be used as an immunogen without modification. Alternatively, particularly against relatively short polypeptides, an excellent immune response can be elicited when the polypeptide is coupled with a carrier protein (eg, 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 boosters) and the animals are bled regularly. Polyclonal antibodies specific for this polypeptide can then be purified from such antisera, for example, by affinity chromatography using the polypeptide bound to a suitable solid support.

  Monoclonal antibodies specific for the antigenic polypeptide of interest are described, for example, in Kohler and Milstein, Eur. J. et al. Immunol. 6: 511-519, 1976 and improvements thereto. Briefly, these methods involve the preparation of immortalized cell lines that can produce antibodies with the desired specificity (ie, reactivity with the polypeptide of interest). Such cell lines can be prepared, for example, from spleen cells obtained from animals immunized as described above. The spleen cells can then be immortalized by, for example, fusion with a myeloma cell fusion partner (preferably isogenic with the immunized animal). Various fusion techniques can be utilized. For example, spleen cells and myeloma cells can be combined with a nonionic detergent for several minutes and then plated at low density on selective media that supports hybrid cell growth but does not support myeloma cell growth. obtain. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After sufficient time (usually about 1-2 weeks), hybrid colonies are observed. Single colonies are selected and tested for binding activity against the polypeptide. Hybridomas with high reactivity and specificity are preferred.

  Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques such as injection of hybridoma cell lines into the peritoneal cavity of a suitable vertebrate host (eg, mouse) can be utilized to increase yield. The monoclonal antibody can then be collected from ascites or blood. Contaminants can be removed from the antibody by conventional techniques such as chromatography, gel filtration, sedimentation, and extraction. The polypeptides of the present invention can be used, for example, in a purification process in an affinity chromatography step.

The monoclonal antibodies of the invention can also be used as therapeutic agents to eliminate or eliminate breast cancer. The antibodies can be used on their own (eg, to prevent metastasis) or can be combined with one or more therapeutic agents. In this regard, suitable substances include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include ⁹⁰ Y, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At, and ²¹² Bi. Preferred drugs include methotrexate and pyrimidine and purine analogs. Preferable differentiation inducers include phorbol ester and butyric acid. Preferred toxins include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.

  The therapeutic agent can be coupled (eg, covalently coupled) to an appropriate monoclonal antibody either directly or indirectly (eg, via a linker group). When each has a substituent that can react with the other, a direct reaction between the substance and the antibody is possible. For example, nucleophilic groups such as amino or sulfhydryl groups react with carbonyl-containing groups such as anhydrides or acid halides, or on the other hand with alkyl groups having good leaving groups (eg halides). obtain.

  Alternatively, it may be desirable to couple the therapeutic agent and the antibody via a linker group. The linker group can function as a spacer that separates the antibody from the substance to avoid interfering with the binding ability. The linker group can also serve to increase the chemical reactivity of the substituents on the substance or antibody, thus increasing the coupling efficiency. Increased chemical reactivity may also facilitate the use of a substance (or otherwise impossible) or functional groups on the substance.

  A variety of bifunctional or multifunctional reagents, both homofunctional and heterofunctional (eg, as described in the catalog of Pierce Chemical Co., Rockford, IL) are utilized as linker groups. It will be apparent to those skilled in the art to obtain. Coupling can be done through, for example, an amino group, a carboxyl group, a sulfhydryl group, or an oxidized hydrocarbon residue. There are a number of references that describe such methodologies (eg, US Pat. No. 4,671,958 to Rodwell et al.).

  If the therapeutic agent is more effective in the absence of the antibody portion of the immune complex of the present invention, it is desirable to use a linker group that can be cleaved during or upon internalization into the cell. Let's go. A number of different cleavable linker groups have been described. Mechanisms for intracellular release of substances from these linker groups include reduction of disulfide bonds (eg, US Pat. No. 4,489,710 granted to Spitler), irradiation of photosensitive linkages (eg given to Senter et al.). US Pat. No. 4,625,014), induced amino acid side chain hydrolysis (U.S. Pat. No. 4,638,045 to Kohn et al.), Serum complement mediated hydrolysis. (US Pat. No. 4,671,958, granted to Rodwell et al.) And cleavage by acid-catalyzed hydrolysis (eg, US Pat. No. 4,569,789, granted to Blatter et al.).

  Binding of one or more substances to the antibody may be desired. In one embodiment, multiple molecules of the substance are bound to one antibody molecule. In another embodiment, more than one type of substance can be bound to one antibody molecule. Regardless of the particular embodiment, immune complexes having one or more substances can be prepared in a variety of ways. For example, one or more substances can be directly attached to the antibody molecule or linkers can be used that provide multiple sites for attachment. Alternatively, a carrier can be used.

  The carrier may hold this material in a variety of ways, including covalent bonds either directly or through a linker group. Suitable carriers include proteins such as albumin (eg, US Pat. No. 4,507,234, granted to Kato et al.), Peptides such as aminodextran and polysaccharides (eg, given to Shih et al., U.S. Pat. No. 4,699,784). The carrier may also carry the material by non-covalent bonding or by encapsulation (eg, in liposome vesicles (eg, US Pat. No. 4,429,008 and US Pat. No. 4,873,088)). Specific carriers for radionuclide materials include radiohalogenated small molecules and chelate compounds. For example, US Pat. No. 4,735,792 discloses representative radiation halogenated small molecules and their synthesis. Radionuclide chelates can be formed from chelating compounds that contain nitrogen and sulfur atoms as donor atoms for binding metal radionuclides, or metal oxide radionuclides. For example, US Pat. No. 4,673,562, granted to Davison et al. Discloses representative chelating compounds and their synthesis.

  Various routes of administration can be used for this antibody and immune complex. Typically, administration is intravenous, intramuscular, subcutaneous, or in a resected tumor bed. The exact dose of the antibody / immune complex will vary depending on the antibody used, the antigen density to the tumor, and the clearance rate of the antibody.

  The diagnostic reagents of the present invention can also include a polynucleotide sequence encoding one or more of the above polynucleotides, or one or more portions thereof. For example, at least two oligonucleotide primers can be utilized in a polymerase chain reaction (PCR) based assay to amplify breast cancer specific cDNA from a biological sample, wherein at least one oligonucleotide primer is Specific for the DNA molecule encoding the breast cancer protein of the present invention. The presence of the amplified cDNA is then detected using techniques well known in the art such as gel electrophoresis. Similarly, an oligonucleotide probe specific for a DNA molecule encoding a breast cancer protein of the invention can be used in a hybridization assay to detect the presence of a polypeptide of the invention in a biological sample.

  As used herein, the term “oligonucleotide primer / probe specific for a DNA molecule” refers to at least about 60%, preferably at least about 75%, and more preferably, for the DNA molecule in question. Means an oligonucleotide sequence having at least about 90% identity. Oligonucleotide primers and / or probes that can be usefully utilized in the diagnostic methods of the present invention preferably have at least about 10-40 nucleotides. In a preferred embodiment, the oligonucleotide primer contains at least about 10 consecutive nucleotides of a DNA molecule having a subsequence selected from SEQ ID NOs: 1-94. Preferably, the oligonucleotide probe for use in the diagnostic method of the present invention contains at least about 15 consecutive oligonucleotides of a DNA molecule having the partial sequence provided in SEQ ID NOs: 1-94. Techniques for both PCR-based assays and hybridization assays are well known in the art (see, eg, Mullis et al., Supra; Ehrlich, supra). Thus, primers or probes can be used to detect sequences specific for breast cancer in biological samples including blood, urine, and / or breast cancer tissue.

(Example)
The following examples are provided for purposes of illustration and are not intended to be limiting.

(Isolation and characterization of breast tumor polypeptides)
This example describes the isolation of a breast tumor polypeptide from a breast tumor cDNA library.

A human breast tumor cDNA expression library was prepared from 3 patients using a superscript plasmid system for cDNA synthesis and plasmid cloning kit (BRL Life Technologies, Gaithersburg, MD 20897) according to the manufacturer's protocol. Constructed from a pool of tumor poly A ⁺ RNA. Specifically, breast tumor tissue was homogenized with polytron (Kinematica, Switzerland) and total RNA was extracted with Trizol reagent (BRL Life Technologies) as directed by the manufacturer. Poly A ⁺ RNA was then purified using the Qiagen oligotex spin column mRNA purification kit (Qiagen, Santa Clara, CA 91355) according to the manufacturer's protocol. The first cDNA strand was synthesized using NotI / Oligo-dT18 primer. Double stranded cDNA was synthesized, ligated using EcoRI / BstX I adapter (Invitrogen, Carlsbad, Calif.) And cut with NotI. After size fractionation on a Chroma Spin-1000 column (Clontech, Palo Alto, CA 94303), this cDNA was ligated to pCDNA3.1 (Invitrogen, Carlsbad, CA) and ElectroMax E. by electroporation. E. coli DH10B cells (BRL Life Technologies) were transformed.

Using the same procedure, a normal human breast cDNA expression library was prepared from a pool of four normal breast tissue samples. This cDNA library is characterized by the number of independent colonies, the percentage of clones carrying the insert, determining the average insert size, and sequence analysis. The breast tumor library contained 1.14 × 10 ⁷ independent colonies, more than 90% of the colonies had obvious inserts, and the average insert size was 936 base pairs. The normal breast cDNA library contained 6 × 10 ⁶ independent colonies, 83% of the colonies had inserts, and the average insert size was 1015 base pairs. Sequencing analysis showed that both libraries contained good composite cDNA clones synthesized from mRNA with minimal contaminating sequences of rRNA and mitochondrial DNA.

Subtraction of the cDNA library using the breast tumor cDNA library and normal breast cDNA library described above (with some modifications) as described by Hara et al. (Blood, 84: 189-199, 1994). And executed. In detail, a breast tumor-specific subtracted cDNA library was prepared as follows. A normal breast cDNA library (70 μg) was digested with EcoRI, NotI and SfuI, and then a filling reaction was performed with DNA polymerase Klenow fragment. After phenol-chloroform extraction and ethanol precipitation, the DNA is dissolved in 100 μl H ₂ O, heat denatured and mixed with 100 μl (100 μg) photoprobe biotin (Vector Laboratories, Burlingame, Calif.). The resulting mixture was irradiated with a 270 W sunlamp on ice for 20 minutes. Additional photoprobe biotin (50 μl) was added and the biotinylation reaction was repeated. After 5 extractions with butanol, the DNA was ethanol precipitated and dissolved in 23 μl of H ₂ O to form driver DNA.

To form tracer DNA, 10 μg of a breast tumor cDNA library was cut with BamHI and XhoI, phenol chloroform extracted and passed through a Chroma spin-400 column (Clontech). After ethanol precipitation, this tracer DNA was dissolved in 5 μl of H ₂ O. Tracer DNA is mixed with 15 μl driver DNA and 20 μl 2 × hybridization buffer (1.5 M NaCl / 10 mM EDTA / 50 mM HEPES pH 7.5 / 0.2% sodium dodecyl sulfate), overlaid with mineral oil, and Completely heat denatured. The sample was immediately transferred to a 68 ° C. water bath and incubated for 20 hours (long term hybridization [LH]). The reaction mixture was then subjected to streptavidin treatment followed by phenol / chloroform extraction. This process was repeated three more times. The subtracted DNA was precipitated, dissolved in 12 μl H ₂ O, mixed with 8 μl driver DNA and 20 μl 2 × hybridization buffer and subjected to hybridization at 68 ° C. for 2 hours (short-term hybridization [ SH]). After removal of the biotinylated double-stranded DNA, the subtracted cDNA was ligated to the BamHI / XhoI site of chloramphenicol resistant pBCSK ⁺ (Strategene, La Jolla, CA 92037) and electroporated by ElectroMax E. coli. E. coli DH10B cells were transformed to produce a breast tumor-specific subtracted cDNA library.

  To analyze the subtracted cDNA library, plasmid DNA was prepared from 100 independent clones randomly selected from the subtracted breast tumor-specific library and the Perkin Elmer / Applied Biosystems Division's Automated Sequencer 373A model (Foster City, Characterized by DNA sequencing using (CA). 38 different cDNA clones were subtracted and found in a breast tumor specific cDNA library. The determined 3 'cDNA sequences for 14 of these clones are provided in SEQ ID NOs: 1-14. This corresponds to the 5 'cDNA sequences provided for SEQ ID NOs: 15-28, respectively. Single stranded (5 'or 3') cDNA sequences determined for the remaining clones are provided in SEQ ID NOs: 29-52. A comparison of these cDNA sequences with known sequences in the gene bank (GenBank) using the EMBL and GenBank database (Release 97) provides the sequences provided in SEQ ID NOs: 3, 10, 17, 24, and 45-52. It was revealed that there was no significant homology to. It has been found that the sequences provided in SEQ ID NOs: 1, 2, 4-9, 11-16, 18-23, 25-41, 43 and 44 show at least some homology to known human genes. The sequence of SEQ ID NO: 42 was found to show some homology to known yeast genes.

  Data was analyzed using Synteni, GEMTOOLS software. Twenty-one different cDNA clones were found to be overexpressed in breast tumors and expressed at low levels in all normal tissues tested. The partial cDNA sequences determined for these clones are provided in SEQ ID NOs: 53-73. A comparison of the gene bank sequences as described above with the sequences of SEQ ID NOs: 53, 54 and 68-71 showed some homology to the previously identified human genes. No significant homology was found in the sequences of SEQ ID NOs: 55-67, 72 (referred to as JJ 9434, 7117), and 73 (referred to as B535S).

  In further experiments, cDNA fragments analyzed by DNA microarray (microarray) were obtained from two subtraction libraries obtained by conventional subtraction as described above. In one example, the tester was derived from a primary breast tumor. In the second example, a metastatic breast tumor was used as a tester. The driver consists of a normal breast.

  CDNA fragments from these two libraries were presented as templates for DNA microarray analysis. The DNA chip was analyzed by hybridizing with a fluorescent probe derived from mRNA derived from both tumor tissue and normal tissue. Analysis of this data was accomplished by creating three groups from the probe set. The composition of these probe groups is called breast tumors / mets, normal non-breast tissue, and metastatic breast tumors. Two comparisons were performed using a modified Gemtool analysis. The first comparison was to identify templates with increased expression in breast tumors. The second was to identify templates that were not recovered in the first comparison that produced increased expression in metastatic breast tumors. Any level of increased expression (average of tumor expression relative to average of normal tissue expression) was set at about 2.2.

  In the first comparison to identify overexpression in breast tumors, two novel gene sequences (hereinafter referred to as B534S and B538S (SEQ ID NO: 89, 90)) and previously identified genes And six sequences (SEQ ID NOs: 74-79) showing some degree of homology. In addition, in a second comparison to identify elevated expression in metastatic breast tumors, five novel sequences were identified, hereinafter referred to as B535S (overexpressed in this and the above analysis), It is referred to as B542S, B543S, P501S and B541S (SEQ ID NOs: 73 and 91 to 94). Nine genes (SEQ ID NOs: 80 to 88) showing a certain degree of homology with known genes were also identified. It was found that clones B534S and B538S (SEQ ID NOs: 89 and 90) were overexpressed in both breast and metastatic breast tumors.

(Production of human CD8 + cytotoxic T cells recognizing antigen-presenting cells expressing breast tumor antigen)
This example illustrates the production of T cells that recognize target cells that also express the B511S antigen, also known as 1016-F8 (SEQ ID NO: 56). Human CD8 + T cells were primed in vitro to the B511S gene product using dendritic cells infected with recombinant vaccinia virus engineered to express B511S as follows (Yee et al., Journal of Immunology ( 1996) 157 (9): 4079-4086). Dendritic cells (DC) were produced from peripheral blood derived monocytes by differentiation for 5 days in the presence of 50 μg / ml GMCSF and 30 μg / ml IL-4. DCs were collected, plated in wells of 24-well plates at a density of 2 × 10 ⁵ cells / well, and infected for 12 hours with B511S expressing vaccinia at a multiplicity of infection of 5. DCs were then matured overnight by addition of 3 μg / ml CD40-ligand and UV irradiated at 100 μW for 10 minutes. CD8 + T cells were isolated using magnetic beads, and priming cultures were separated into individual wells (typically 24 wells) using 7 × 10 ⁵ CD8 + T cells and 1 × 10 ⁶ irradiated CD8 depleted PBMC. On the first day, 10 ng / ml IL-7 was added to the culture. Cultures were restimulated every 7-10 days with autologous primary fibroblasts transduced with retrovirus using B511S and costimulatory molecule B7.1. 15 I. of the culture. U. Of IL-2 was added on day 1. After four such stimulation cycles, the interferon gamma Elispot assay (see Lalvani et al. J. Experimental Medicine (1997) 186: 859-965) was used to identify autologous fibroblasts transduced with B511S. CD8 + cultures were tested for the ability of CD8 + to recognize automatically. Briefly, T cells from individual microcultures are added to 96-well Elispot plates containing autologous fibroblasts transduced to express either B511S or the negative control antigen EGFP, and 37 Incubated overnight at 0C; wells also contained 10 ng / ml IL-12. It was confirmed that the culture specifically produced interferon gamma only in response to fibroblasts transduced with B511S; such strains were further expanded and also retrovirally transduced with B511S. Cloning was performed by limiting dilution with autologous B-LCL. It was confirmed that cell lines and clones can specifically recognize autologous B-LCL transduced with B511, but not autologous B-LCL transduced with control antigen EGFP or HLA-A3. An example demonstrating the ability of a human CTL cell line derived from such experiments to specifically recognize and lyse B51115 expressing the target is shown in FIG.

(Synthesis of polypeptides)
Polypeptides were synthesized on a Perkin Elmer / Applied Biosystems Division peptide synthesizer 430A using FMOC chemistry with HPTU (O-benzotriazole-N, N, N ′, N′-tetramethyluronium hexafluorophosphate) activation. Can be synthesized. The Gly-Cys-Gly sequence can be attached to the amino terminus of a peptide and provide a method of conjugation that binds to an immobilized surface or peptide label. Cleavage of the peptide from the solid support can be carried out using the following cleavage mixture: trifluoroacetic acid: ethanedithiol: thioanisole: water: phenol (40: 1: 2: 2: 3). After 2 hours cleavage, the peptide could be precipitated in chilled methyl-t-butyl-ethanol. The peptide pellet could then be dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized prior to purification by C18 reverse phase HPLC. A gradient of 0% to 60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) could be used for peptide elution. After lyophilization of the pure fractions, the peptides could be characterized using electrospray or other types of mass spectrometry and by amino acid analysis.

  From the foregoing, it will be understood that although particular embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. The

Claims (52)

  An isolated polypeptide, wherein the polypeptide comprises an immunogenic portion of a breast protein, or a variant of the protein that differs only in conservative substitutions and / or modifications, wherein the protein comprises a polynucleotide Comprising the amino acid sequence encoded by the molecule, the polynucleotide molecule is listed below: (a) SEQ ID NOs: 3, 10, 17, 24, 45-52, 56-67, 72, 73, and 89-94 A sequence selected from the group consisting of: (b) a complement of the nucleotide sequence; and (c) a sequence that hybridizes to the sequence of (a) or (b) under moderately stringent conditions. An isolated polypeptide comprising   An isolated polynucleotide molecule comprising a nucleotide sequence encoding the polypeptide of claim 1.   An isolated polynucleotide molecule comprising the sequence provided in SEQ ID NOs: 3, 10, 17, 24, 45-52, 56-67, 72, 73, and 89-94.   An expression vector comprising the polynucleotide molecule of any one of claims 2 and 3.   A host cell transformed with the expression vector according to claim 4.   The host cell is E. coli. 6. A host cell according to claim 5, which is selected from the group consisting of E. coli, yeast and mammalian cell lines. 2. The polypeptide of claim 1, and an isolated polypeptide, wherein the polypeptide comprises an immunogenic portion of a breast protein, or a variant of the protein that differs only in conservative substitutions and / or modifications. Wherein the protein comprises an amino acid sequence encoded by a polynucleotide molecule, the polynucleotide molecule comprising: (a) a nucleotide sequence of SEQ ID NO: 55; (b) a complement of the nucleotide sequence; c) a poly selected from the group consisting of the above isolated polypeptides comprising a sequence selected from the group consisting of sequences hybridizing to the sequence of (a) or (b) under moderately stringent conditions A pharmaceutical composition comprising a peptide and a physiologically acceptable carrier. 2. The polypeptide of claim 1, and an isolated polypeptide, wherein the polypeptide comprises an immunogenic portion of a breast protein, or a variant of the protein that differs only in conservative substitutions and / or modifications. Wherein the protein comprises an amino acid sequence encoded by a polynucleotide molecule, the polynucleotide molecule comprising: (a) a nucleotide sequence of SEQ ID NO: 55; (b) a complement of the nucleotide sequence; c) a poly selected from the group consisting of the above isolated polypeptides comprising a sequence selected from the group consisting of sequences hybridizing to the sequence of (a) or (b) under moderately stringent conditions A vaccine comprising a peptide and a non-specific immune response enhancer.   9. A vaccine according to claim 8, wherein the non-specific immune response enhancer is an adjuvant. (I) the polynucleotide molecule of any one of claims 2 and 3,
(Ii) an isolated polynucleotide molecule encoding an isolated polypeptide, wherein the polypeptide is an immunogen of a breast protein or a variant of the protein that differs only in conservative substitutions and / or modifications Wherein the protein comprises an amino acid sequence encoded by a polynucleotide molecule, the polynucleotide molecule comprising: (a) a nucleotide sequence of SEQ ID NO: 55; (b) a complement of the nucleotide sequence And (c) the polynucleotide molecule comprising a sequence selected from the group consisting of sequences that hybridize to the sequence of (a) or (b) under moderately stringent conditions; and (iii) SEQ ID NO: A polynucleotide selected from the group consisting of isolated polynucleotide molecules comprising the sequence provided in 55. A vaccine comprising a renucleotide molecule and a non-specific immune response enhancer.   11. The vaccine of claim 10, wherein the non-specific immune response enhancer is an adjuvant.   A pharmaceutical composition for the treatment of breast cancer, the composition comprising a polypeptide and a physiologically acceptable carrier, wherein the polypeptide comprises an immunogenic portion of a breast protein, wherein the The protein comprises an amino acid sequence encoded by a polynucleotide molecule, the polynucleotide molecule comprising: (a) SEQ ID NO: 1, 2, 4-9, 11-16, 18-23, 25-44, 53, Nucleotide sequences listed in 54, 68-71, and 74-88; (b) the complement of the nucleotide sequence; and (c) the sequence of (a) or (b) under moderately stringent conditions A pharmaceutical composition comprising a sequence selected from the group consisting of: a hybridizing sequence.   A vaccine for the treatment of breast cancer, the vaccine comprising a polypeptide and a non-specific immune response enhancer, wherein the polypeptide comprises an immunogenic portion of a breast protein, wherein the protein comprises a polynucleotide Comprising the amino acid sequence encoded by the molecule, said polynucleotide molecule: (a) SEQ ID NO: 1, 2, 4-9, 11-16, 18-23, 25-44, 53, 54, 68-71 And (b) the complement of the nucleotide sequence; and (c) a sequence that hybridizes to the sequence of (a) or (b) under moderately stringent conditions; A vaccine comprising a sequence selected from the group consisting of:   14. A vaccine according to claim 13, wherein the non-specific immune response enhancer is an adjuvant.   A vaccine for the treatment of breast cancer, said vaccine comprising a polynucleotide molecule and a non-specific immune response enhancer, said polynucleotide molecule comprising: (a) SEQ ID NO: 1, 2, 4-9, 11 -16, 18-23, 25-44, 53, 54, 68-71, and 74-88; (b) the complement of the nucleotide sequence; and (c) moderately stringent A vaccine comprising a sequence selected from the group consisting of a sequence that hybridizes to the sequence of (a) or (b) under conditions.   16. The vaccine of claim 15, wherein the non-specific immune response enhancer is an adjuvant.   13. A pharmaceutical composition according to claim 7 or 12, for use in the manufacture of a medicament for inhibiting the development of breast cancer in a patient.   16. A vaccine according to any one of claims 8, 10, 13, or 15 for use in the manufacture of a medicament for inhibiting the development of breast cancer in a patient.   A fusion protein comprising at least one polypeptide according to claim 1.   20. A pharmaceutical composition comprising the fusion protein of claim 19 and a physiologically acceptable carrier. 20. The fusion protein of claim 19, and an isolated polypeptide, wherein the polypeptide comprises an immunogenic portion of a breast protein, or a variant of the protein that differs only in conservative substitutions and / or modifications. Wherein the protein comprises an amino acid sequence encoded by a polynucleotide molecule, the polynucleotide molecule comprising: (a) a nucleotide sequence of SEQ ID NO: 55; (b) a complement of the nucleotide sequence; c) from a fusion protein comprising at least one of the above isolated polypeptides comprising a sequence selected from the group consisting of sequences that hybridize to the sequence of (a) or (b) under moderately stringent conditions A fusion protein selected from the group consisting of:
A vaccine comprising a non-specific immune response enhancer.   The vaccine of claim 21, wherein the non-specific immune response enhancer is an adjuvant.   21. A pharmaceutical composition according to claim 20 for use in the manufacture of a medicament for inhibiting the development of breast cancer in a patient.   23. A vaccine according to claim 21 for use in the manufacture of a medicament for inhibiting the development of breast cancer in a patient. A method for detecting breast cancer in a patient comprising the following steps:
(A) contacting a biological sample from a patient with a binding agent capable of binding to the polypeptide, the polypeptide comprising an immunogenic portion of a breast protein, wherein the protein Comprising the amino acid sequence encoded by the nucleotide molecule, the polynucleotide molecule under the nucleotide sequences listed in SEQ ID NOs: 1-54, 56-94, the complement of the nucleotide sequence, and moderately stringent conditions Comprising a sequence selected from the group consisting of sequences that hybridize to the sequences provided in SEQ ID NOs: 1-54, 56-94; and (b) a protein or polypeptide that binds to the binding agent in a sample And thereby detecting breast cancer in the patient.   26. The method of claim 25, wherein the binding agent is a monoclonal antibody.   27. The method of claim 26, wherein the binding agent is a polyclonal antibody. A method for monitoring the progression of breast cancer in a patient comprising the following steps:
(A) contacting a biological sample from a patient with a binding agent capable of binding to the polypeptide, the polypeptide comprising an immunogenic portion of a breast protein, wherein the protein Comprising the amino acid sequence encoded by the nucleotide molecule, the polynucleotide molecule under the nucleotide sequences listed in SEQ ID NOs: 1-54, 56-94, the complement of the nucleotide sequence, and moderately stringent conditions Comprising a sequence selected from the group consisting of sequences that hybridize to the sequences provided in SEQ ID NOs: 1-54, 56-94;
(B) determining the amount of protein or polypeptide that binds to the binding agent in the sample;
(C) repeating steps (a) and (b); and (d) comparing the amount of polypeptide detected in steps (b) and (c) to monitor the progression of breast cancer in the patient. Process,
Including the method.   A monoclonal antibody that binds to a polypeptide, wherein the polypeptide comprises an immunogenic portion of a breast protein, or a variant of the protein that differs only in conservative substitutions and / or modifications, wherein the protein Comprising the amino acid sequence encoded by the nucleotide molecule, which polynucleotide molecule is listed below: (a) SEQ ID NOs: 3, 10, 17, 24, 45-52, 56-67, 72, 73, and 89-94 Selected from the group consisting of: (b) the complement of the nucleotide sequence; and (c) a sequence that hybridizes to the sequence of (a) or (b) under moderately stringent conditions. A monoclonal antibody comprising a sequence.   30. The monoclonal antibody of claim 29 for use in the manufacture of a medicament for inhibiting the development of breast cancer in a patient.   32. The monoclonal antibody of claim 30, wherein the monoclonal antibody is conjugated with a therapeutic agent. A method for detecting breast cancer in a patient comprising the following steps:
(A) contacting a biological sample from a patient with at least two oligonucleotide primers in a polymerase chain reaction, wherein at least one of said oligonucleotides comprises an immunogenic portion of a breast protein Wherein the protein comprises an amino acid sequence encoded by the polynucleotide molecule, wherein the polynucleotide molecule is a nucleotide listed in SEQ ID NOs: 1-54, 56-94. Comprising a sequence selected from the group consisting of a sequence, a complement of the nucleotide sequence, and a sequence that hybridizes to the sequences of SEQ ID NOs: 1-54, 56-94 under moderately stringent conditions; b) Presence of the oligonucleotide primer in the sample Detecting a polynucleotide sequence to be amplified below, thereby detecting breast cancer;
Including the method.   35. The method of claim 32, wherein at least one of the oligonucleotide primers comprises at least about 10 contiguous nucleotides of a polynucleotide molecule comprising a sequence selected from SEQ ID NOs: 1-54, 56-94. Diagnostic kit with the following:
30. A kit comprising: (a) one or more monoclonal antibodies of claim 29; and (b) a detection reagent. Diagnostic kit with the following:
(A) one or more monoclonal antibodies that bind to a polypeptide encoded by the polynucleotide molecule, wherein the polynucleotide molecule is SEQ ID NO: 1, 2, 4-9, 11-16, 18-23, 25; A nucleotide sequence selected from the group consisting of -44, 53, 54, 68-71, and 74-88, the complement of the sequence, and SEQ ID NO: 1, 2, 4-9 under moderately stringent conditions A monoclonal antibody comprising a sequence selected from the group consisting of a sequence that hybridizes to a sequence of 11-16, 18-23, 25-44, 53, 54, 68-71 or 74-88; and (b) detection reagent,
Including a kit.   36. A kit according to claim 34 or 35, wherein the monoclonal antibody is immobilized on a solid support.   37. The kit of claim 36, wherein the solid support comprises nitrocellulose, latex, or plastic material.   36. The kit of claim 34 or 35, wherein the detection reagent comprises a reporter group conjugated to a binding agent.   40. The kit of claim 38, wherein the binding agent is selected from the group consisting of anti-immunoglobulin, protein G, protein A, and lectin.   40. The kit of claim 38, wherein the reporter group is selected from the group consisting of a radioisotope, a fluorescent group, a luminescent group, an enzyme, biotin, and dye particles.   A diagnostic kit comprising at least two oligonucleotide primers, wherein at least one of the oligonucleotide primers is specific for a polynucleotide molecule encoding a polypeptide comprising an immunogenic portion of a breast protein, the protein Comprises the amino acid sequence encoded by the polynucleotide molecule, the polynucleotide molecule comprising the nucleotide sequence listed in SEQ ID NOs: 1-54, 56-94, the complement of the nucleotide sequence, and moderate stringency conditions A diagnostic kit comprising a sequence selected from the group consisting of sequences that hybridize to the sequences of SEQ ID NOs: 1-54, 56-94 below.   42. The diagnostic kit of claim 41, wherein at least one of the oligonucleotide primers comprises at least about 10 contiguous nucleotides of a polynucleotide molecule comprising a sequence selected from SEQ ID NOs: 1-54, 56-94. A method for detecting breast cancer in a patient comprising the following steps:
(A) obtaining a biological sample from the patient;
(B) contacting the biological sample with an oligonucleotide probe specific for a polynucleotide molecule encoding the polypeptide, the polypeptide comprising an immunogenic portion of a breast protein, Comprises an amino acid sequence encoded by a polynucleotide molecule, the polynucleotide molecule comprising the nucleotide sequence listed in SEQ ID NOs: 1-54, 56-94, the complement of the nucleotide sequence, and moderately stringent A sequence selected from the group consisting of sequences that hybridize to the sequences of SEQ ID NOs: 1-54, 56-94 under conditions; and (c) a polyhybrid that hybridizes to the oligonucleotide probe in the sample Detecting a nucleotide sequence and thereby detecting breast cancer in said patient ,
Including the method.   44. The method of claim 43, wherein the oligonucleotide probe comprises at least about 15 contiguous nucleotides of a polynucleotide molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-54, 56-94.   A diagnostic kit comprising an oligonucleotide probe specific for a polynucleotide molecule encoding a polypeptide, the polypeptide comprising an immunogenic portion of a breast protein, wherein the protein is encoded by the polynucleotide molecule Comprising a nucleotide sequence listed in SEQ ID NOs: 1-54, 56-94, the complement of the nucleotide sequence, and SEQ ID NOs: 1-54 under moderately stringent conditions, A diagnostic kit comprising a sequence selected from the group consisting of sequences that hybridize to sequences 56-94.   46. The diagnostic kit of claim 45, wherein the oligonucleotide probe comprises at least about 15 contiguous nucleotides of a polynucleotide molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1-54, 56-94.   2. Peripheral blood cells from a patient for use in the manufacture of a medicament for treating breast cancer in a patient, incubated in the presence of at least one polypeptide according to claim 1 such that T cells proliferate.   48. The peripheral blood cell of claim 47, wherein the T cell is repeated one or more times.   A composition for the treatment of breast cancer in a patient comprising T cells expanded in the presence of the polypeptide of claim 1 in combination with a pharmaceutically acceptable carrier.   Antigen presenting cells for use in the manufacture of a medicament for treating breast cancer in a patient, incubated in the presence of at least one polypeptide according to claim 1.   51. The cell of claim 50, wherein the antigen presenting cell is selected from the group consisting of dendritic cells and macrophage cells.   A composition for the treatment of breast cancer in a patient comprising antigen presenting cells incubated in the presence of the polypeptide of claim 1 in combination with a pharmaceutically acceptable carrier.

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