Diagnostics/Analytical Methods/Drug Delivery/Mouse Models

Diagnostics

Use of JAG2 Expression in Diagnosis of Plasma Cell Disorders

U.S. Patent: Pending (Allowed)
Application Number: 10/837,722

Summary: The invention is based on the finding that plasma cell disorders such as Multiple Myeloma and Monoclonal Gammopathy of Unknown Significance are characterized by an increase in the expression of JAG2. Accordingly, the invention provides a method for diagnosis of plasma cell disorders by detecting the expression or overexpression of JAG2. The expression or overexpression of JAG2 may be detected as increased mRNA transcripts or increased protein.

Detail: The invention provides compositions and methods for detection of plasma cell disorders. The method is based on the observations that Jagged 2 (JAG2) expression is increased in MM cell lines as well as in primary tumors obtained from patients diagnosed with plasma cell disorders including MM or MGUS, but not with patients with other hematologic neoplasms or non-hematologic neoplasms. A method is provided to determine the expression of JAG2 in bone marrow samples obtained from patients. The expression of JAG2 may be evaluated by determining the levels of JAG2 mRNA or the levels of JAG2 protein. Methods for determination of levels of JAG2 mRNA include, but are not limited to, Northern blotting, oligonucleotide hybridization, PCR based techniques and in situ hybridization techniques. Methods for determination of JAG2 protein levels include, but are not limited to, immunoassays such as enzyme linked immunosorbent assays (ELISAs), immunofluorescence based techniques and FACS based techniques. Compositions are also provided for determination of JAG2 mRNA or protein. The compositions include primers useful for PCR amplification of reverse transcribed mRNA and antibodies including polyclonal and monoclonal antibodies for the detection of JAG2 protein.

Prognostic Significance of Molecular Genetic Aberrations on Chromosome Segment 11P15.5 in Non-Small-Cell Lung Cancer

Patent(s): 7,351,530
U.S. Patent Issued: April 1, 2008

Summary: The invention provides a method for detection of loss of heterozygosity (LOH) at human chromosome segment 11p15.5. The invention also provides a method for prognosis in an individual with NSCLC. The method comprises detecting LOH in the 11p15.5 region. The presence of LOH is indicative of poor prognosis in the NSCLC patient. This is useful for treatment decisions following surgical resection.

Detail: The invention provides a method for detection of loss of heterozygosity (LOH) at human chromosome segment 11p15.5 in a tumor sample from a patient with non-small-cell lung cancer (NSCLC), and a method for prognosis of the patient's disease progression. The prognosis is useful for determining the course of treatment following surgical resection of tumor.

Method for Identifying Altered Vitamin D Metabolism

Patent(s): Pending
U.S. Patent Application Number: 12/506,025

Summary: A method is provided for identifying an individual as having altered vitamin D metabolism comprising analyzing a biological sample from the individual for the presence of CYP24 SNPs and/or aberrantly spliced CYP24 mRNA. The presence of the SNPs and/or the aberrantly spliced CYP24 mRNA indicates that the individual has altered vitamin D metabolism. Also provided are methods for customizing dosages of biologically active vitamin D compounds for individuals who are determined to have altered vitamin D metabolism.

Detail: The invention provides a method for identifying an individual as likely having altered vitamin D metabolism. The method comprises obtaining a biological sample from the individual and determining the presence of certain CYP24 single nucleotide polymor- phisms (SNPs) and/or aberrantly spliced CYP24 mRNA, and/or correctly spliced CYP24 mRNA in the absence of calcitriol, wherein the presence of the SNPs and/or aberrantly spliced CYP24 and/or correctly spliced CYP24 mRNA in the absence of calcitriol is indicative that the individual is likely to have altered vitamin D metabolism. Also provided is a method for customizing dosing of calcitriol or calcitriol precursors, or a vitamin D analog compound that does not generate as much of a calcemic response as calcitriol. The method comprises obtaining a biological sample from the individual, identifying the pres- ence of CYP24 SNPs and/or aberrantly spliced CYP24 mRNA and/or correctly spliced CYP24 mRNA in the absence of calcitriol, and based upon such identification, prescrib- ing a lower or higher dose of calcitriol or calcitriol precursors.

Analytical Methods

High Throughput Assay for Identification of Gene Expression Modifiers

U.S. Patent Number: 7,083,919
Date Issued: August 1, 2006

Summary: The invention provides a method for screening of a large number of compounds for their ability to modulate the expression of genes. The method uses gene trap technology and comprises the steps of transfecting a population of cells with a gene-trap vector, sorting cells according to their level of fluorescence, distributing sorted cells into pools and expanding the pools to obtain a sufficient number of cells representing each trapped gene to permit distinction of the effect of a test compound over controls, exposing the cells to the test compounds and identifying compounds which alter the fluorescence distribution pattern of cells using FACS analysis.

Detail: The invention provides a method for screening of a large number of compounds for their ability to modulate the expression of genes. The method uses gene trap technology and comprises the steps of transfecting a population of cells with a gene-trap vector comprising a polynucleotide sequence encoding a fluorescent reporter protein, subjecting the population of cells to FACS analysis to sort cells into windows based upon the level of fluorescence, pooling cells from each window, expanding the pooled cells to obtain a sufficient number of copies of cells containing each trapped gene so as to permit detection of altered expression, exposing a certain number of cells from each pool to a plurality of chemicals, subjecting exposed cells to FACS analysis, identifying pools in which the fluorescence pattern has been altered over control cells, and correlating the altered pattern of fluorescence to specific chemicals.

The invention further provides a method, wherein the trapped genes whose activity is altered upon exposure to test compounds are identified, cloned and sequenced. In the invention, methodologies are provided which improve the accuracy, stability and sensitivity of FACS screening thus allowing multiplex screening of chemical compounds. These methodologies are directed to reducing 1) the heterogeneity in the detection of fluorescence within a given cell during sorting that leads to inaccuracy in the determination of fluorescence; 2) heterogeneity in the detected fluorescence depending upon the stage of the cell cycle; and 3) cellular auto-fluorescence that reduces the sensitivity of fluorescence based assays. The accuracy is improved by increasing the residence time of each cell in the flow cytometer so as to reduce the heterogeneity in detection due to uneven illumination. The residence time is increased by reducing the cell stream velocity. The stability in improved by adjusting the parameters of sorting cells during FACS such that the variations due to cell cycling are minimized. The sensitivity is improved by suppressing the background by illuminating the sample with white light. Thus, an object of the invention is to overcome the limitation to the number of chemical compounds that can be screened for effects on populations of trapped genes in gene trap cellular libraries. Assessment of subsets of a gene trap cellular library for the effects of several chemical compounds simultaneously, i.e. multiplexing, allows for the enhancement in the number of genes .times. chemical compounds that can be assessed. A further object of the invention is to reduce the heterogeneity in fluorescence detection due to cell cycling.A further object of the present invention is to reduce the heterogeneity in fluorescence detection due to high-speed sorting during FACS analysis. A further objective is to suppress cellular auto-fluorescence. Suppression of auto-fluorescence increases the ability to measure low signal levels that result from genes expressed at minimal levels. In one embodiment, a method is provided for generating pools from each group (window in the fluorescence distribution patterns from FACS analysis) of cells in which approximately 500 trapped genes are represented where expression from these genes is detected using the fluorescent protein reporter enhanced green fluorescent protein and where expression of this reporter from the trapped genes varies by less than a factor of approximately three (the range of fluorescence separating two neighboring windows in FACS) and where less than approximately 0.02%-0.03% of the cells deviate from these parameters. In another embodiment, a population of cells is provided wherein cells maintain the desired properties for the assay period which may vary depending on the stability of the specific reporter gene used and whether up-regulated or down-regulated changes in gene expression are desired. In another embodiment, an assay is provided in which approximately 29,800 chemical compound interactions with specific genes were assessed through the application of approximately 2,000 chemical compounds, in groups of 10, to microtitre wells containing pools of approximately 5,000-10,000 cells windowed for low or intermediate levels of fluorescence and containing cells representing approximately 20 trapped genes and analysis of the effects of these compounds on fluorescence from as many as 6,000 cells per microtitre well. In another embodiment, an assay is provided in which approximately 875,000 chemical compound interactions with specific genes were assessed through the application of approximately 2,000 chemical compounds, in groups of 10, to microtitre wells containing pools of approximately 5,000-10,000 cells windowed for low or intermediate levels of fluorescence and containing cells representing approximately 500 trapped genes and analysis of the effects of these compounds on fluorescence from as many as 6,000 cells per microtitre well.

Methods for Protein Interaction Determination

U.S. Patent Number: 7,323,313
Date Issued: January 29, 2008

Summary: The invention provides a method for identifying a plurality of pairs of interacting proteins and plasmids for use in the method. The pair of plasmids is adapted for use in a modified two hybrid system wherein wherein each plasmid comprises a recombinase recognition site. The method comprises the steps of providing cDNAs encoding test polypeptides, inserting the cDNAs into the first and second plasmids, recombining the first and second plasmids to obtain recombined plasmids, isolating and digesting the recombined plasmids, ligating the restriction fragments to a universal adapter to provide a pool of digested fragments flanked by a universal adapter, selecting and amplifying desired sequences, forming concatamers from the amplified sequences, and sequencing the concatamers to determine the nucleotide sequences encoding a plurality of pairs of interacting proteins.

Detail: The invention provides a method for identifying a plurality of pairs of interacting proteins and plasmids for use in the method. The invention provides a plasmid pair adapted for use in a modified two hybrid system wherein first plasmid comprises a coding sequence for a DNA binding domain of a transcription activator (the "DBD plasmid") and the second plasmid comprises a coding sequence for a transcription activation domain of a transcription activator (the "AD plasmid"), and each plasmid further comprises a recombinase recognition site. The method comprises the steps of providing cDNAs encoding test polypeptides, inserting the cDNAs into the first and second plasmids, recombining the first and second plasmids to obtain recombined plasmids, isolating and digesting the recombined plasmids, ligating the restriction fragments to a universal adapter to provide a pool of digested fragments flanked by a universal adapter, selecting and amplifying desired sequences, forming concatamers from the amplified sequences, and sequencing the concatamers to determine the nucleotide sequences encoding a plurality of pairs of interacting proteins.

Method for Predicting Susceptibility to Radiation Pneumonitis

Patent(s): Pending
U.S. Patent Application Number: 11/809,250

Summary: Provided is a method for determining whether an individual is likely to be susceptible to radiation pneumonitis from radiation therapy and for developing a treatment based on the determination of susceptibility. The method involves measuring SOD and GPX activity levels. A high SOD or low GPX activity, or a combination thereof, is indicative that the individual is likely to be susceptible to radiation pneumonitis.

Detail: The present invention provides a method for identifying an individual as likely to be susceptible to radiation pneumonitis. The method comprises obtaining a biological sample comprising red blood cells from the individual and determining the amount of glutathione peroxidase (GPX) activity and/or superoxide dismutase (SOD) activity in the sample. Low GPX or high SOD activity relative to a normal control is indicative that the individual is likely to be susceptible to radiation pneumonitis. The invention also takes advantage of the discovery that the ratio of GPX/SOD activity is a powerful predictor of susceptibility to radiation pneumonitis. In particular, a low GPX/SOD ratio relative to a normal control is indicative that the individual is likely to be susceptible to radiation pneumonitis. The invention also permits determination of a treatment regime for a cancer patient, since normal levels of GPX and SOD indicate that the individual is a candidate for aggressive radiation therapy, while those individuals identified as susceptible to radiation pneumonitis can be treated using smaller radiation dosages, more focused radiation, no RT, or other modifications to RT that are known to those skilled in the art.

Compositions and Methods for Identifying Agents that Alter Expression of Survivin

Patent(s): Issued
U.S. Patent Number: 7,569,221
Date Issued: August 4, 2009

Summary: Provided are compositions and methods for identifying agents that can modulate expression of the human survivin gene. The compositions include eukaryotic cells that contain a human survivin promoter sequence operably linked to a reporter gene. The method comprises determining whether a test agent can modulate transcription from a human survivin promoter sequence by adding a test agent to eukaryotic containing a human survivin promoter sequence operably linked to a reporter gene, measuring expression of the reporter gene and comparing expression of the reporter gene from to a control, wherein an increase or decrease of expression of the reporter gene relative to the control is indicative that the test agent can modulate transcription from a human survivin promoter. The method also comprises the use of SPlucTg mice for preclinical drug identification, including testing of candidate drug toxicity and efficacy.

Detail: The present invention provides eukaryotic cells comprising a human survivin promoter polynucleotide sequence operably linked to a reporter gene. The cells are useful for identifying agents that can modulate survivin gene transcription. In one embodiment, the cells are stably transfected cancer cells lines. In another embodiment, the invention provides a transgenic mouse comprising a germ line insertion of the survivin promoter in operable linkage with the reporter gene. Also provided are methods for identifying agents that can modulate survivin expression. The method comprises adding a test agent to cells comprising a human survivin promoter in operable linkage with a reporter gene. By comparison with a control, agents that can either increase or decrease transcription from the survivin promoter, as evidenced by increased or decreased expression of the reporter gene, can be identified. Thus, the invention is suitable for identifying agents that are expected to have therapeutic benefit for cancer patients, as well as for other disorders wherein it would be desirable to modulate the expression of survivin.

Methods for Analysis of PDEF and Survivin as Interconnected Cancer Biomarkers and Targets for Personalized Medicine

Patent(s): Pending
U.S. Patent Application Number: 12/186,981

Summary: Provided are methods for determining whether an individual is a candidate to receive treatment with a DNA methylation inhibitor. The method can be performed a biologi- cal sample of cancerous tissue of the individual. Determining that PDEF expression is absent or low and survivin expression is present identifies the individual as a candidate to receive a treatment with a DNA methylation inhibitor. The method also includes communicating the result of identifying an individual as a candidate for receiving a DNA methylation inhibitor to a health care provider.

Detail: The present invention provides a method for determining whether an individual diagnosed with cancer is a candidate for treatment with a DNA methylation inhibitor. The method comprises obtaining a biological sample of cancerous tissue from the individual and determining the expression of PDEF and survivin from the sample. A determination that PDEF expression is absent or low relative to a control and that the sample is positive for survivin expression indicates that the individual is a candidate for receiving a DNA methylation inhibitor. A determination that PDEF expression is normal or high and survivin expression is absent indicates that the individual is a not a candidate for receiving the DNA methylation inhibitor. A determination that PDEF expression is absent or low and that survivin expression is absent is also indicative that the individ- ual is a not a candidate for receiving the DNA methylation inhibitor. Determining that PDEF expression is normal or high and detecting survivin expression is likewise indicative that the individual is a not a candidate for receiving the DNA methylation inhibitor. Identification of an individual as a candidate for receiving a DNA methylation inhibitor is considered to also be indicative that the individual is a candidate for receiving a histone deacetylase inhibitor in combination with the DNA methylation inhibitor. The invention includes communicating the result of identifying an individual as a candidate for receiving a DNA methylation inhibitor to a health care provider.

Drug Delivery

Method for Nasal Application of a Medicinal Substance

U.S. Patent Number: 6,534,040
Date Issued: March 25, 2003

Summary: Compositions that are a chemical combination of porphyrins, chlorins, bacteriochlorins, and related tetra-pyrrolic compounds with radioactive elements such as Technetium.sup.99, Gadolinium, Indium.sup.111 and radioactive iodine. When the element can form cations, the compound is usually a chelate with the porphyrin or chlorin structure. When the element forms anions, the compound is usually a direct chemical combination of the radioactive element into the porphyrin or chlorin structure. The invention further includes the method of using the compounds of the invention for diagnostic imaging of hyperproliferative tissue such as tumors and new blood vessel growth as is associated with the wet form of age related macular degeneration and methods of making the compounds. Compounds for MRI contrast imaging of the invention are usually Tc.sup.99, In.sup.111 or Gd(III) complexes of compounds of the formula: ##STR1##

Detail: The invention includes compositions that are chemical combination of porphyrins and chlorins and related tetra-pyrrolic compounds with radioactive elements such as Technetium.sup.99, Gadolinium, Indium.sup.111 and radioactive iodine. When the element can form cations, the compound is usually a chelate with the porphyrin or chlorin structure. When the element forms anions, the compound is usually a direct chemical combination of the radioactive element into the porphyrin or chlorin structure.

Examples of porphyrin and chlorin structures that can form compounds with radioactive elements, when modified in accordance with the invention, are for example described in U.S. Pat. Nos. 5,756,541; 5,028,621; 4,866,168; 4,649,151; 5,438,071; 5,198,460; 5,002,962; 5,093,349; 5,171,741; 5,173,504; 4,968,715; 5,314,905; 5,459,159; 5,770,730; 5,864,035; 5,190,966; and 5,952,366 all of which are incorporated by reference as background art. The invention further includes the method of using the compounds of the invention for diagnostic imaging of hyperproliferative tissue such as tumors and new blood vessel growth as is associated with the wet form of age related macular degeneration. Unexpectedly, porphyrins and chlorins, as above described, upon injection, carry the element into cells of hyperproliferative tissue and dramatically enhance the signal produced by tumor tissue in MR imaging.

Temperature Controlled Content Release from Liposomes

U.S. Patent Number: 6,964,778
Date Issued: November 15, 2005

Summary: The invention provides a liposomal composition for targeted delivery of drugs. The composition comprises large unilamellar vesicles (LUV) encapsulating poloxamers and one or more delivery agents. The composition and concentration of the poloxamer inside the LUVs is such that upon heating to temperatures above the critical micellar temperature of the poloxamer, the LUVs becomes leaky causing release of the encapsulated drug. The invention also provides a method for delivery of agents to targeted sites and a method for preparing the LUVs suitable for use according to the method described herein.

Detail: The invention provides a liposomal composition for targeted delivery of drugs. The composition comprises large unilamellar vesicles (LUV) encapsulating poloxamers and one or more agents for delivery. Poloxamers do not significantly associate with the liposome bilayer at temperatures below their critical micellar temperature (CMT). However, above CMT, poloxamers partition into the LUV bilayer, causing defects in the bilayer leading to its eventual disruption. Therefore, in one embodiment, the poloxamer has a CMT around the physiological temperature. The concentration of the poloxamer inside the LUVs is such that upon incorporation of the poloxamer into the LUVs, the LUVs becomes leaky causing release of the encapsulated drug. In one embodiment, the poloxamer Pluronic F127 (M.W. .about.12,600, PEO.sub.98 --PPO.sub.67 --PEO.sub.98) was used because of its high molecular weight and desired hydrophilic-hydrophobic balance (HLB), both factors reducing the detergent-like toxicity, and a CMT around the physiological temperature.

The invention also provides a method for delivery of agents to targeted sites. The method comprises (1) preparing large unilamellar vesicles encapsulating the agent and poloxamer molecules, wherein the concentration of the poloxamer is such that upon incorporation of the poloxamer molecules into the LUV membrane at a temperature above the CMT, the membrane becomes leaky causing the release of the encapsulated Delivery Agent; (2) administration of the LUVs to the individual; and (3) increasing the temperature of the target site to effect release of agent from the LUVs. As an example, the release of tracer molecules of different molecular weights, from liposomes of different lipid compositions is described. The invention further provides a method for preparing the LUVs suitable for use according to the method described herein. The method of preparation comprises the steps of forming multilamellar vesicles (MLV)s in the presence of one or more agents and poloxamer molecules, preparing LUVs therefrom such that the poloxamer and the drug are encapsulated within the LUVs. Unencapsulated i.e., free agent and poloxamer molecules are then separated from the LUVs. The poloxamer is selected so that it has a CMT around the physiological temperature. The concentration of the poloxamer inside the LUV is such that upon incorporation into the LUV membrane, the LUV becomes leaky.

Temperature Sensitive Control of Liposome-Cell Adhesion

U.S. Patent Number: 6,991,805
Date Issued: January 31, 2006

Summary: The invention provides a liposomal composition for targeted delivery of drugs. The composition comprises poloxamer molecules and liposomes encapsulating one or more delivery agents. At above the critical micellar temperature of the poloxamer, a fraction of the poloxamer molecules form micelles and another fraction becomes incorporated into the liposome surface, thereby inhibiting their adhesion to cells. At a temperature below the critical micellar temperature, the poloxamer molecules dissociate into monomers allowing the liposomes to adhere to adjacent cells and effecting retention of the liposomes in the surrounding tissue. A method is provided for delivery of agents to target site comprising administering the composition to an individual and cooling the target site to cause retention of the liposomes at or near the target site.

Detail: The invention provides a method for targeted delivery of agents comprising the steps of providing a mixture of poloxamer molecules, and liposomes encapsulating the delivery agent; heating the mixture to above the critical micellar temperature (CMT) for the poloxamer, so as to allow a fraction of the poloxamer molecules to form micelles and another fraction of the poloxamer molecules to become incorporated into the liposomes; administering the heated mixture to an individual; and cooling the target site to below the CMT so as to cause the poloxamer molecules forming the micelles and incorporated into the liposomes to dissociate into monomers thereby exposing the liposomal adhesion sites causing the liposomes to be retained at or near the target site.

The invention also provides a liposomal composition for targeted delivery of agents comprising poloxamer molecules and liposome vesicles encapsulating one or more agents for delivery, wherein upon heating of the composition to above the CMT, a fraction of the poloxamer molecules form micelles and another fraction of the poloxamer molecules become incorporated into the liposomes resulting in stealthing of the liposomes such that the liposomal-cell adhesion is reduced, and wherein upon cooling the mixture at the target site to below the CMT of the poloxamer, the poloxamer molecules become dissociated into monomers to expose cell-adhesion sites thereby causing retention of the liposomes at or near the target site. In one embodiment, the poloxamer Pluronic F127 (M.W..about.12,600, PEO.sub.98- PPO.sub.67- PEO.sub.98) is used.

Mouse Models

Murine Model for Human Carcinoma

U.S. Patent Number: 5,986,170
Date Issued: November 16, 1999

Summary: A murine model for human ovarian and prostate cancer is provided. The model comprises an immunodeficient mouse containing human primary ovarian or prostate carcinoma tissue implanted within the gonadal fat pad. Methods of using the murine model, for example, to grow tumors and to screen treatments for ovarian and/or prostate cancer are also provided.

Detail: Briefly stated, the invention provides murine models for breast, ovarian and prostate carcinomas. In one aspect, an immunodeficient mouse containing human primary breast, ovarian or prostate carcinoma tissue is provided, wherein the carcinoma tissue is implanted within a gonadal fat pad of the immunodeficient mouse. Within related aspects, the present invention provides methods for generating a murine model for breast, ovarian or prostate cancer. In one embodiment, the method comprises implanting a specimen of a human breast, ovarian or prostate carcinoma within the gonadal fat pad of an immunodeficient mouse. In a related embodiment, the method further comprises removing a portion of the implanted carcinoma specimen and subsequently implanting the portion within a gonadal fat pad of a second immunodeficient mouse. In yet another aspect, methods are provided for growing human breast, ovarian or prostate carcinoma tissue, comprising: (a) implanting a specimen of a human breast, ovarian or prostate carcinoma within a gonadal fat pad of an immunodeficient mouse; and (b) allowing the carcinoma to grow within the immunodeficient mouse. In further aspects, the invention provides methods for evaluating the effectiveness of a breast, ovarian or prostate cancer therapy, comprising: (a) implanting a specimen of a human breast, ovarian or prostate carcinoma within a gonadal fat pad of an immunodeficient mouse; (b) exposing the immunodeficient mouse to a candidate therapy; and (c) determining a change in size of the implanted specimen, the extent of tumor cell death and/or the level of metastatic spread in the mouse, and therefrom determining the effectiveness of the therapy. In another aspect, methods are provided for producing tumor-reactive human cytolytic T lymphocytes, comprising: (a) implanting a specimen of a human carcinoma containing human T lymphocytes within a gonadal fat pad of an immunodeficient mouse, wherein the carcinoma is a breast, ovarian or prostate carcinoma; (b) allowing the human T lymphocytes to grow within the immunodeficient mouse; (c) isolating a human cytolytic T lymphocyte from the specimen-bearing immunodeficient mouse; and (d) evaluating the lymphocyte for the ability to kill carcinoma cells, and therefrom identifying a tumor-reactive lymphocyte. In a related aspect, methods are provided for generating an anti-tumor antiserum, comprising: (a) implanting a specimen of a human carcinoma containing human B cells within a gonadal fat pad of an immunodeficient mouse, wherein the carcinoma is a breast, ovarian or prostate carcinoma; (b) allowing the human B cells to grow and produce human anti-tumor antibodies within the immunodeficient mouse; (c) isolating an antiserum from the immunodeficient mouse; and (d) evaluating the antiserum for the ability to bind to carcinoma cells, and therefrom identifying an anti-tumor antiserum. In yet another related aspect, methods are provided for generating an anti-tumor monoclonal antibody, comprising: (a) implanting a specimen of a human carcinoma containing human B cells within a gonadal fat pad of an immunodeficient mouse, wherein the carcinoma is a breast, ovarian or prostate carcinoma; (b) allowing the human B cells to grow and produce human antitumor antibodies within the immunodeficient mouse; (c) generating a monoclonal antibody from the isolated B cells; and (d) evaluating the monoclonal antibody for the ability to bind to carcinoma cells, and therefrom identifying an anti-tumor monoclonal antibody. Within other related aspects, the present invention provides tumor-reactive human cytolytic T lymphocytes, anti-tumor antisera, and monoclonal antibodies produced by the methods described above. In yet another aspect, the invention provides methods for treating cancer in a patient, wherein the cancer is a breast, ovarian or prostate cancer. In one embodiment, the method comprises administering to a patient a tumor-reactive cytolytic T lymphocyte as described above. In another embodiment, the method comprises administering to a patient an anti-tumor monoclonal antibody as described above. These and other aspects of the invention will become apparent upon reference to the following detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.