Development of dna probe and immunological reagent for human tumor-related antigen

Description: TECHNICAL FIELD [Detailed description of the invention]

【0001】

This application is a partial continuation of US application SN.603,804 filed October 25, 1990, the content of which is incorporated herein by reference.

【0002】

Background of the Invention

Throughout this application, various publications are referenced in parenthesized Arabic numerals. A complete citation of these publications will be found at the end of the specification, just before the claims. The disclosures of these publications are hereby incorporated by reference in order to more fully describe the state of the art known to those of skill in the art at the time the claimed invention was made. Will be incorporated into.

【0003】

Studies over the past few years are based on several hypotheses.

【0004】

(1) Human cancers appear as a result of genetic alterations in cellular genetic traits. These genetic changes may include direct changes in the structure and / or expression of a particular gene that regulates the expression of the transformed state. Genetic trait markers and potential inducers of the tumor cell phenotype include activation and / or deletion or inactivation of certain oncogene elements (1,2).

【0005】

(2) Tumor phenotypes are often surface expression of novel tumor-associated antigen (TAA) subsets that are specific to tumors of different histological types and can be expressed differently in patients of the same tumor histological type. Characterized by. The use of specific cytokines can increase the level of expression of specific TAA, resulting in improved diagnostic improvement, visualization and ultimately enhanced monoclonal antibody (MoAb) based on the treatment of cancer. (3-5).

【0006】

(3) Certain TAA subsets expressed in certain tumors may not be recognized by the patient's own immune system, resulting in the patient being incapable of increasing an effective immune response and destroying tumor lesions. Become. This problem is caused by external application of MoAb (particularly human MoAb; primate MoAb or chimeric murine MoAb), increased immune potential of patients with cytokines and expression vector immunostimulants and / or their own tumor cells. Can be solved by denatured and acting as a vaccine.

【0007】

(4) In many cases, genetic elements, including tumor genotypes, can code specific cell surface TAAs. Therefore, both the tumor genotype and its corresponding TAA subset can be transferred together to a suitable recipient by DNA transfer techniques.

【0008】

Studies that directly test many of the above hypotheses have been conducted over the past few years, and many are now accepted as valid. One of the early trials included studies of the genetic and immunological basis of human prostate cancer differentiation.

【0009】

Prostate cancer is currently the most common cancer in adult men and is the leading cause of cancer death in men over the age of 55 (6). As human lifespan increases, the development of prostate cancer differentiation and the acquisition of the ability of prostate cancer cells to metastasize (especially to bone) remains unresolved (7). Attempts to show constant changes in the expression of known oncogenes in prostate cancer (8-10) have been unsuccessful. Similarly, conventional attempts to indicate dominant focus formation or tumor-inducing genes in either prostate cancer or prostate cancer cell lines by transducing calcium-mediated DNA have resulted in limited success (). 9). Analysis of the transformation focus that emerged in NIH-3T3 cells after transduction of DNA isolated from prostate cancer showed the presence of an activated Ki-ras oncogene (9).

【0010】

These results suggest that oncogene activation in human prostate cancer, at least the activation detected by the DNA transduction assay in NIH-3T3, is not a frequent event in prostate cancer. However, it utilizes a DNA cotransfection procedure in nude mice using high molecular weight human prostate cancer DNA (derived from the LNCaP cell line) and a dominant acting bacterial antigen resistance gene, followed by tumor induction. By doing so, the tumor gene phenotype was successfully transplanted into the rat embryonic fibroblast lineage, CREF (14). These presumed prostate cancer transformants not only do not form focus in CREF cells in in vitro, but also NIH-3T3 It cannot be detected by similar transduction / tumorigenesis attempted in cells. Tumor-derived CREF transducants containing human repeat (Alu) sequences and common molecular weight Alu fragments have been identified in both primary and independent secondary tumor-derived CREF transducers.

【0011】

In addition, nude mouse tumor-derived LNCaP DNA transduced CREF cells were also found to express the human tumor-related antigen (TAA) used to generate both polyclonal and monoclonal antibodies that react with the surface of LNCaP cells. .. These observations support the following hypothesis: (A) LNCaP cells contain dominant tumor-inducing genes that can be transferred and expressed in CREF but not in NIH-3T3 cells; (B) The presence of a common Alu fragment in the primary and secondary transfectants is the isolation and cloning of this tumor-induced genetic component by the physical binding of genes transferred from LNCaP cells to CREF cells to this sequence. And (C) monoclonal antibodies that specifically interact with LNCaP cells (and do not interact with other human tumors, including melanoma, lung cancer, normal human skin fibroblasts and CREF cells). The presence of TAA on the surface of primary and secondary tumor-derived CREF transfectants, which can be used in the production of, is between the expression of the tumor-induced prostate cancer gene and the transformed phenotype of LNCaP cells on this presumption. Further suggests a potential relationship.

【0012】

In summary, common methods have been developed to identify genes and produce immunological reagents that code TAA of human origin. By using CREF / transduction / monoclonal antibody technology, we can transfer genes intervening or related to human lung cancer and glial blastoma polymorphism, and antigens expressed in human lung cancer and other cancers. Achieved the development of both recognition polyclonal and MoAb. CREF / transduction / monoclonal antibody technology is also used to identify defined, transduced, and expressed surface molecules that contain 170,000 molecular weight cloned human (P-glycoprotein) complex drug resistance genes. Clone mdr-1 was transduced into CREF and human tumor (lung cancer and glial blastoma) cells and selected for corhitin resistance, indicating the presence and expression of the mdr-1 gene by Southern and Northern analysis, respectively. These transduced CREF cells were then used to generate MoAbs that react with P-glycoproteins expressed on transduced human lungs expressing the MDR phenotype and glial blastoma cells.

【0013】

[Summary of Invention]

The present invention provides a general method for identifying genes and producing immunological reagents that code cell surface antigens of human origin.

【0014】

Specifically, the present invention provides a method for preparing a hybridoma cell line that produces an antibody that specifically recognizes and binds to a cell surface antigen associated with human tumor cells. This method co-transfects (a) established non-human non-tumor-forming cell lines with DNA isolated from human tumor cells and DNA encoding selectable or identifiable traits ( b) Select transfection cells expressing selectable or identifiable traits, (c) recover the transfect cells so selected, and (d) appropriately harvest the transfect cells so recovered. Injected into a first murine host, (e) the resulting first murine host was maintained for an effective period of time during which the injected transfected cells were induced to form tumors within the first murine host. f) The resulting tumor was isolated from a first murine host, (g) tumor cells were obtained from the tumor so isolated, and (h) the tumor cells thus obtained were established as described above. The anti-serum produced against the non-human non-tumor-forming cell line was immobilized, (i) the anti-serum immobilized cells were injected into a suitable second host, and (j) the obtained second host. To identify the host that produces serum that reacts with human tumor cells, (k) remove the spleen from the second host so identified, and (l) remove the spleen in this way. Includes preparing a hybridoma from and (m) recovering from it a hybridoma cell line that produces an antibody that specifically recognizes and binds to cell surface antigens.

【0015】

The present invention also provides a method for producing a monoclonal antibody that specifically recognizes and binds to a cell surface antigen associated with human tumor cells. This method comprises producing a hybridoma according to the method described above and recovering a monoclonal antibody from the hybridoma thus produced.

【0016】

The present invention further provides a method for preparing a polyclonal antibody that specifically recognizes and binds to a cell surface antigen associated with human tumor cells. This method co-transfects (a) an established non-human non-tumor-forming cell line with DNA isolated from human tumor cells and a host encoding a selectable or identifiable trait. b) Select transfection cells expressing selectable or identifiable traits, (c) recover the transfecting cells so selected, and (d) appropriately harvest the transfecting cells so recovered. Injected into a first murine host, (e) the resulting first murine host was maintained for an effective period of time during which the infused transfected cells were induced to form a tumor within the first murine host. f) The resulting tumor was isolated from a first murine host, (g) tumor cells were obtained from the tumor so isolated, and (h) the tumor cells thus obtained were established as described above. The anti-sera generated against the non-human non-tumor-forming cell line was immobilized, (i) the anti-sero-immobilized cells were injected into a suitable second host, and (j) the obtained second host was obtained. Screening to identify the host that produces the serum that reacts with human tumor cells, and (k) recover the polyclonal antibody from the second host so identified.

【0017】

In addition, the present invention is a method of diagnosing a tumor condition in a subject, wherein the antibody specifically recognizes and binds to a cell surface antigen related to the tumor condition in a sample from the subject. Provided a method comprising contacting with any of the above antibodies labeled with a detectable marker to detect the presence of an antibody bound to the antigen and thereby diagnosing the condition of the tumor under possible conditions. To do.

【0018】

This method is also a method of treating a tumor condition, in which the human tumor cells associated with the tumor condition are labeled with the therapeutic agent under conditions such that the therapeutic agent selectively inhibits the growth of the human tumor cells. Provided are methods comprising contacting with any of the above antibodies.

【0019】

The present invention is further a method of forming an image of a human tumor cell, wherein the antibody specifically recognizes and binds to a cell surface antigen associated with the tumor cell in the human tumor cell for which the image is to be formed. Provided is a method of contacting with any of the above antibodies labeled with an image-forming agent and detecting the image-forming agent bound to them to form an image of tumor cells under possible conditions.

【0020】

The invention also provides a method of preparing DNA that encodes a cell surface antigen associated with human tumor cells. This method co-transfects (a) CREF-trans 6 cell lines with DNA isolated from human tumor cells and DNA encoding selectable or identifiable traits ( b) Select transfection cells expressing selectable or identifiable traits, (c) recover the transfect cells so selected, and (d) collect the transfect cells so recovered. Was injected into a suitable first murine host, and (e) the resulting first murine host was maintained for an effective period of time during which the injected transfected cells were induced to form tumors within the first murine host. And (f) isolate the resulting tumor from the first murine host, (g) obtain tumor cells from the tumor so isolated, and (h) from the tumor cells thus obtained. Includes recovering DNA that codes cell surface antigens associated with human tumor cells.

【0021】

Finally, the present invention is a method for diagnosing a tumor condition in a subject, in which a sample from the subject is labeled with a detectable marker under conditions where the DNA probe can hybridize with the DNA associated with the tumor condition. Provided is a method for diagnosing a tumor condition by contacting with a DNA probe that has been used to detect the presence of hybridized DNA.

【0022】

[Detailed description of the invention]

The present invention provides a general method for identifying genes and producing immunological reagents that code cell surface antigens of human origin.

【0023】

In particular, the invention provides a method of preparing a hybridoma cell line that produces an antibody that specifically recognizes and binds to cell surface antigens associated with human tumor cells. This method co-transfects (a) established non-human non-tumor-forming cell lines with DNA isolated from human tumor cells and DNA encoding selectable or identifiable traits ( b) Select transfection cells expressing selectable or identifiable traits, (c) recover the transfect cells so selected, and (d) appropriately harvest the transfect cells so recovered. Injected into a first murine host, (e) the resulting first murine host was maintained for an effective period of time during which the injected transfected cells were induced to form tumors within the first murine host. f) The resulting tumor was isolated from a first murine host, (g) tumor cells were obtained from the tumor so isolated, and (h) the tumor cells thus obtained were established as described above. The anti-serum produced against the non-human non-tumor-forming cell line was immobilized, (i) the anti-serum immobilized cells were injected into a suitable second host, and (j) the obtained second host. To identify the host that produces serum that reacts with human tumor cells, (k) remove the spleen from the second host so identified, and (l) from the spleen thus removed. Includes preparing a hybridoma and (m) recovering from it a hybridoma cell line that produces an antibody that specifically recognizes and binds to cell surface antigens.

【0024】

Here, the established non-human non-tumor-forming cell line may be any non-human established cell line, exhibiting no transformation and tumorigenic phenotypes, and exogenous DNA consisting of both bound and unbound strands. Can be efficiently taken in and incorporated. In a preferred embodiment of the invention, the established non-human non-tumor-forming cell line is the CREF-trans 6 cell line, which is available at ATCC reception number CRL 10584, American Type Culture Collection, Rockville, Maryland. , 20852, deposited in USA. It was deposited satisfactorily in accordance with the requirements of the Budapest Convention on International Approval of Deposit of Microorganisms in Patent Procedures.

【0025】

Human tumor cells can be any benign or metastatic human tumor cells, derived from any human tumor cell lineage or any primary tumor (even a small amount of primary tumor). May be good. In one aspect of the invention, the human tumor cells are human prostate cancer cells derived from the cell line LNCaP. In another aspect of the invention, the human tumor cell is a human lung cancer cell derived from the cell line T47D. In another aspect of the invention, the human tumor cells are human colorectal cancer cells derived from the cell line SW 480. In another aspect of the invention, the human tumor cell is a human glioblastoma polymorph (stage IV astrocytoma) derived from the cell line GBM-18. In yet another aspect of the invention, the human tumor cell is a human glial blastoma polymorph (stage IV astrocytoma) derived from a primary tumor.

【0026】

Here, the DNA that codes the selectable or identifiable trait may be any DNA that codes the selectable or identifiable trait. In one aspect of the invention, the DNA that codes for selectable or identifiable traits is plasmid DNA that codes for resistance to antibiotics. In a preferred embodiment of the invention, the plasmid DNA comprises pSV2-neo and the antibiotic is G418.

【0027】

Here, a suitable second host can be a murine host or a non-human primate host.

【0028】

For the purposes of the present invention, the cell surface antigen associated with human tumor cells may be any cell surface antigen. Cell surface antigens include, but are not limited to, tumor-related antigens, growth factor receptors, surface-expressed antigens coded by viruses, antigens coded by cancer gene products, and surface epitopes. , Membrane proteins that mediate orthodox or abnormal complex drug resistance, antigens that mediate tumorigenetic phenotype, antigens that mediate metastatic phenotype, antigens that suppress tumorigenetic phenotype, antigens that suppress metastatic phenotype, T It can be an antigen recognized by a specific immunological effector cell such as a cell and an antigen recognized by a non-specific immunological effector cell such as a macrophage cell or a natural killer cell. In a preferred embodiment of the invention, the cell surface antigen is a tumor-related antigen.

【0029】

Hybridoma cell lines can be recovered using methods known to those of skill in the art. The invention also provides a hybridoma cell line produced according to the methods described above.

【0030】

It is also within the scope of the present invention that steps (a) to (g) can be repeated to obtain further tumor cells, that is, secondary transduced substances, tertiary transduced substances and the like.

【0031】

The present invention further provides a method for producing a monoclonal antibody that specifically recognizes and binds to a cell surface antigen associated with human tumor cells. The method comprises producing a hybridoma according to the method described above and recovering the monoclonal antibody from the hybridoma thus produced.

【0032】

For the purposes of the present invention, monoclonal antibodies can be recovered by methods known to those of skill in the art.

【0033】

The methods of the invention also provide monoclonal antibodies produced according to the methods described above.

【0034】

The present invention also provides a method for preparing a polyclonal antibody that specifically recognizes and binds to a cell surface antigen associated with human tumor cells. This method co-transfects (a) an established non-human non-tumor-forming cell line with DNA isolated from human tumor cells and a host encoding a selectable or identifiable trait. b) Select transfection cells expressing selectable or identifiable traits, (c) recover the transfecting cells so selected, and (d) appropriately harvest the transfecting cells so recovered. Injected into a first murine host, (e) the resulting first murine host was maintained for an effective period of time during which the infused transfected cells were induced to form a tumor within the first murine host. f) The resulting tumor was isolated from a first murine host, (g) tumor cells were obtained from the tumor so isolated, and (h) the tumor cells thus obtained were established as described above. The anti-sera generated against the non-human non-tumor-forming cell line was immobilized, (i) the anti-sero-immobilized cells were injected into a suitable second host, and (j) the obtained second host was obtained. Screening to identify the host that produces the serum that reacts with human tumor cells, and (k) recover the polyclonal antibody from the second host so identified.

【0035】

For the purposes of the present invention, polyclonal antibodies can be recovered by methods known to those skilled in the art.

【0036】

Here, the established non-human non-tumor-forming cell line may be any non-human established cell line, exhibiting no transformation and tumorigenic phenotypes, and exogenous DNA consisting of both bound and unbound strands. Can be efficiently taken in and incorporated. In a preferred embodiment of the invention, the established non-human non-tumor-forming cell line is the CREF-trans 6 cell line (ATCC reception number CRL 10584).

【0037】

Human tumor cells can be any benign or metastatic human tumor cells, derived from any human tumor cell lineage or any primary tumor (even a small amount of primary tumor). May be good. In one aspect of the invention, the human tumor cells are human prostate cancer cells derived from the cell line LNCaP. In another aspect of the invention, the human tumor cell is a human lung cancer cell derived from the cell line T47D. In another aspect of the invention, the human tumor cells are human colorectal cancer cells derived from the cell line SW 480. In another aspect of the invention, the human tumor cell is a human glioblastoma polymorph (stage IV astrocytoma) derived from the cell line GBM-18. In yet another aspect of the invention, the human tumor cell is a human glial blastoma polymorph (stage IV astrocytoma) derived from a primary tumor.

【0038】

The DNA that codes the selectable or identifiable trait can be any DNA that codes the selectable or identifiable trait. In one aspect of the invention, the DNA that codes for selectable or identifiable traits is plasmid DNA that codes for resistance to antibiotics. In a preferred embodiment of the invention, the plasmid DNA comprises pSV2-neo and the antibiotic is G418.

【0039】

Here, a suitable second host can be a murine host or a non-human primate host.

【0040】

For the purposes of the present invention, the cell surface antigen associated with human tumor cells may be any cell surface antigen. Cell surface antigens include, but are not limited to, tumor-related antigens, growth factor receptors, surface-expressed antigens coded by viruses, antigens coded by cancer gene products, and surface epitopes. , Membrane proteins that mediate orthodox or abnormal complex drug resistance, antigens that mediate tumorigenetic phenotype, antigens that mediate metastatic phenotype, antigens that suppress tumorigenetic phenotype, antigens that suppress metastatic phenotype, T It can be an antigen recognized by a specific immunological effector cell such as a cell and an antigen recognized by a non-specific immunological effector cell such as a macrophage cell or a natural killer cell. In a preferred embodiment of the invention, the cell surface antigen is a tumor-related antigen.

【0041】

It is also within the scope of the present invention that steps (a) to (g) can be repeated to obtain further tumor cells, that is, secondary transduced substances, tertiary transduced substances and the like.

【0042】

The present invention also provides a polyclonal antibody produced according to the above method.

【0043】

The invention also provides the monoclonal or polyclonal antibody labeled with a detectable marker. Here, detectable markers are known to those of skill in the art and can be, but are not limited to, enzymes, paramagnetic ions, biotins, phosphors, chromogenic groups, heavy metals or radioisotopes.

【0044】

The present invention is also a method for diagnosing a tumor condition in a subject, under conditions in which an antibody can specifically recognize and bind to a cell surface antigen associated with the tumor condition in a sample from the subject. The present invention provides a method for diagnosing a tumor state by contacting with any of the above antibodies labeled with a detectable marker and detecting the presence of the antibody bound to the antigen.

【0045】

The present invention also provides the above-mentioned monoclonal antibody or polyclonal antibody labeled with a therapeutic agent. Here, the therapeutic agent is well known to those of skill in the art and can be, but is not limited to, antibiotics, antiviral agents such as interferon, toxins, radioisotopes, or chemotherapeutic agents. ..

【0046】

This method is also a method of treating a tumor condition, in which the human tumor cells associated with the tumor condition are labeled with the therapeutic agent under conditions such that the therapeutic agent selectively inhibits the growth of the human tumor cells. Provided are methods comprising contacting with any of the above antibodies.

【0047】

The present invention also provides any of the above monoclonal or polyclonal antibodies labeled with an image-forming agent. Here, the image-forming agent is well known to those skilled in the art and can be a radioisotope, a dye or enzyme such as peroxidase or alkaline phosphate, a paramagnetic ion, or an element opaque to X-rays. It is not limited to.

【0048】

The present invention is also a method of forming an image of a human tumor cell, in which an antibody specifically recognizes and binds to a cell surface antigen associated with the tumor cell in the human tumor cell for which the image is to be formed. Provided is a method of contacting with any of the above antibodies labeled with an image-forming agent and detecting the image-forming agent bound to them to form an image of tumor cells under possible conditions.

【0049】

Here, the image forming method is any of many image forming methods known to those skilled in the art, for example, but not limited to, a method of visualizing the radiation emitted by a radioisotope. Can also be included.

【0050】

In addition, the present invention provides a method of preparing DNA that encodes a cell surface antigen associated with human tumor cells. This method co-transfects (a) DNA isolated from human tumor cells and DNA encoding selectable or identifiable traits into a CREF-trans 6 cell line and (b) selectable or Transfect cells expressing identifiable traits are selected, (c) the transfect cells so selected are harvested, and (d) the transfect cells thus harvested are the appropriate first. Injected into a murine host, (e) the resulting first murine host was maintained for an effective period of time during which the infused transfected cells were induced to form tumors within the first murine host, and (f) obtained. The tumor was isolated from a first murine host, (g) tumor cells were obtained from the tumor so isolated, and (h) the tumor cells thus obtained were associated with human tumor cells. Includes the recovery of DNA that codes for cell surface antigens.

【0051】

It is also within the scope of the present invention that steps (a) to (g) can be repeated in order to obtain further tumor cells, i.e., secondary transducers, tertiary transducers, and the like.

【0052】

This method of preparing DNA is an improvement over the conventional method. This aspect of the invention is due to the use of newly cloned rat embryonic fibroblast lineages, especially the CREF-trans 6 cell line. This CREF-trans 6 cell line enables identification of genes that mediate tumor phenotypes and cell surface antigen expression using small amounts of tumor tissue. The CREF-trans 6 cell line also makes it possible to easily detect human repeats that act as genetic markers that describe the location of genes involved in the induction of tumor phenotype and cell surface expression. In addition, expression of many known oncogenes has not been observed in tumor cells derived from the CREF cell line (21). This affirms the potential use of this system for the identification of potentially novel classes of human oncogenes.

【0053】

Methods of recovering DNA from tumor cells thus obtained may include many isolation, purification and DNA cloning methods known to those of skill in the art. This method can be, but is not limited to, a polymerase chain reaction or a traditional phage cloning technique.

【0054】

Human tumor cells can be any benign or metastatic human tumor cells, derived from any human tumor cell lineage or any primary tumor (even a small amount of primary tumor). May be good. In one aspect of the invention, the human tumor cells are human prostate cancer cells derived from the cell line LNCaP. In another aspect of the invention, the human tumor cell is a human lung cancer cell derived from the cell line T47D. In another aspect of the invention, the human tumor cells are human colorectal cancer cells derived from the cell line SW 480. In another aspect of the invention, the human tumor cell is a human glioblastoma polymorph (stage IV astrocytoma) derived from the cell line GBM-18. In yet another aspect of the invention, the human tumor cell is a human glial blastoma polymorph (stage IV astrocytoma) derived from a primary tumor.

【0055】

The DNA that codes the selectable or identifiable trait can be any DNA that codes the selectable or identifiable trait. In one aspect of the invention, the DNA that codes for selectable or identifiable traits is plasmid DNA that codes for resistance to antibiotics. In a preferred embodiment of the invention, the plasmid DNA comprises pSV2-neo and the antibiotic is G418.

【0056】

For the purposes of the present invention, the cell surface antigen associated with human tumor cells may be any cell surface antigen. Cell surface antigens include, but are not limited to, tumor-related antigens, growth factor receptors, surface-expressed antigens coded by viruses, antigens coded by cancer gene products, and surface epitopes. , Membrane proteins that mediate orthodox or abnormal complex drug resistance, antigens that mediate tumorigenetic phenotype, antigens that mediate metastatic phenotype, antigens that suppress tumorigenetic phenotype, antigens that suppress metastatic phenotype, T It can be an antigen recognized by a specific immunological effector cell such as a cell and an antigen recognized by a non-specific immunological effector cell such as a macrophage cell or a natural killer cell. In a preferred embodiment of the invention, the cell surface antigen is a tumor-related antigen.

【0057】

The present invention also provides DNA prepared according to the above method.

【0058】

The present invention also provides a DNA probe capable of hybridizing with the above DNA. The invention further provides the DNA probe labeled with a detectable marker. Here, the detectable markers are well known to those of skill in the art and can be, but are not limited to, enzymes, paramagnetic ions, biotin, phosphors, chromogenic groups, heavy metals or radioisotopes. Absent.

【0059】

Finally, the present invention is a method for diagnosing a tumor condition in a subject, in which a sample from the subject is labeled with a detectable marker under conditions where the DNA probe can hybridize with the DNA associated with the tumor condition. Provided is a method for diagnosing a tumor condition by contacting with a DNA probe that has been used to detect the presence of hybridized DNA. The presence of a probe means detection of tumor status.

【0060】

The present invention will be further described in the following experimental details. This section is explained for the purpose of understanding the invention and is not intended to limit the invention described in the claims described below in any respect, and is construed as such. It shouldn't be.

【0061】

[Details of the experiment]

<Materials and methods> CREF-trans 6 cell line: The CREF-trans 6 cell line is a subclon of the CREF cell line, which is a specific clone of Fisher F2408 rat embryo cells (33). A CREF-trans 6 cell line was developed by plate culturing low density (50-100 cells / 6 cm plates) of CREF cells and isolating contiguous subclones from single cells. The subclones of these CREFs (identified from CREF-Trans 1 to 20) were tested for their morphological transformation potential following transfection with Ha-ras (T24) oncogenes. In addition, the ability of subclones transfected with the cloned neomycin resistance gene (pSV2neo) and selected for growth in G418-containing medium to form antibiotic-resistant colonies was also analyzed. CREF-Trans 6 is a specific subclon of CREF that is more sensitive to transformation by T24 than its parent or other CREF-Trans subclones and develops neomycin resistance.

【0062】

Other Cell Lines: The LN-CaP cell line was derived from human prostate cancer. The T47D cell line was derived from human breast carcinoma. GBM-18 was derived from human polymorphic glioblastoma (4th stage astrocytoma). It was also derived from a human polyneuroglioblastoma (4th stage polymorph) primitive tumor. The SW480 cell line was derived from human colorectal cancer. The MCF-7 cell line was derived from human breast cancer. The Colo38 cell line was derived from human melanoma. The HO-I cell line was derived from human melanoma. These cell lines are publicly available to those of skill in the art.

【0063】

Antiserum preparation: BALB / c female mice (8-10 weeks old) were hyperimmunized with CREF-Trans 6 cells. Mice were given (1) a single subcutaneous injection (SC) of scraped cells with a complete Freund's adjuvant (1: 1) on day 0; (2) incomplete Freund's on day 7. Subcutaneous injection of scraped cells (SC) once with adjuvant (1: 1); (3) Two intraperitoneal injections (ip) of scraped cells in a balanced salt solution of Hanks on days 14 and 21 .. The anti-CREF activity was tested by the Eliza method on sera collected by bleeding from the posterior eye of mice. CREF cells were (1) grown in a 96-well microtiter plate to near confluence; (2) cell-fixed in 3.7% formaldehyde PBS (solution) for 5 minutes at room temperature to 10%. Blockade cells with normal goat serum; (3) Tita for antiserum cells was measured at 37 ° C for 2 hours using a serial diluent; (4) Horseradish peroxidase-conjugated goat anti-mouse Ig second antibody ( Using GX MIg-HRP), the bond was detected at 37 ° C for 60 minutes; (5) A dye source was added in the presence of hydrogen peroxide solution, and the color change exhibited by the positive bond was observed with a spectrophotometer. Quantified. By adopting the above means, high titer serum (1: 3200 1: 6400) was obtained by day 21, and serum with titer 1: 100 was used to coat CREF-Trans 6 cells in the initial experiment. The second highest titer of serum was used in subsequent experiments.

【0064】

CREF-Trans 6 cell line cotransfection DNA transfection of receptor CREF cells was performed as described above, except that HMW-DNA was cleaved to form 6 to 25 kilobe-spare DNA fragments (22, 45, 47). This modification results in the effective transfer of certain cellular genes by calcium phosphate-mediated DNA transfection and tends to reduce the toxicity often encountered during HMW-DNA transfection (11). DNA is isolated from cell lines using the phenol extraction method (14,37). HMW-DNA was cleaved by repeated passage through a syringe with a 16-caliber (gauge) needle and sized by agarose gel electrophoresis (36). HMW-DNA was then mixed with pSV2-Neo DNA (HMW-DNA: Neo plasmid DNA 20-40 μg: 1 μg). 1 to 2 X of 20 to 40 μg of final DNA as a calcium phosphate precipitate⁶Was added to CREF-Trans cells (22, 45, 47). CREF-Trans cells were cultured with calcium phosphate DNA precipitate for 4 hours, and then excess DNA precipitate was removed. Cultures were briefly treated with glycerol (15 vol% glycerol PBS solution), quickly suspended in trypsin / versene, 5 x 10 per 10 cm plate.^6,、10^5,, And 2.5 x 10⁵Reprinted with.

【0065】

Selection of Transfected Cells Expressing Selectable or Recognitive Characteristics: Medium was replaced with medium containing G418 (500 μg / ml) 48 hours post-plate (36, 45). Change the selective medium twice a week, Neo^RColony can be detected within 7 to 14 days.

【0066】

Recovery of transfected cells and injection into mice: Once the culture of cultured cells from each plate has grown to an appropriate number, cells are pooled from each selected G418 selected plate.⁶Was subcutaneously injected into Nude mice.

【0067】

Tumor cell isolation and donation: When tumors develop in mice with an injection of transfected cells for a culture period of 8 weeks, the tumors are removed and reintroduced into the cell culture. Cells were removed by non-enzymatic separation or exfoliation by a rubber policeman from the plate. The resuspension was washed with Hanks BSS and resuspended with the minimum amount of Hanks BSS.

【0068】

Antiserum coverage of tumor cells: High titer anti-mouse-CREF-Trans 6 prior to injecting tumor cells from the tumor into mice to block the immune response of antigens normally expressed in CREF-Trans 6 cells. Covered with antiserum. Resuspended cells (1 to 2.5 x 10)⁶) Was mixed with MXCREF antiserum at a concentration of 1: 100 (antiserum: cells) and cultured at room temperature for 6 hours or at 4 ° C with shaking overnight.

【0069】

Injection of antiserum-coated cells into mice: BALB / c female mice (8-10 weeks old) with or without adjuvant_KeAntiserum-coated cells were repeatedly injected with ip for more than a month.

【0070】

Screening of serum-reactive hosts by malignant cells: blood and multicell cells of mice immunized and pooled as described above; for example, CREF-Trans 6, CREF 4-NMT (transfected with LNCaP DNA) , LNCaP (human prostate cancer cell line), SW480 (human colon-rectal cancer cell line), MCF-7 (human breast cancer cell line), HO-1 (human melanoma cell line), Colo38 human melano -Mass cell line), and human skin fibroblasts, were tested by the Eliza method for connectivity. MoAbs were produced by standard methods using animals confirmed to have serum reactive with appropriate human tumor cells.

【0071】

Monochrome- Production of monoclonal antibody: How human TAAs (50) which is utilized for the production of MoAbs, HLA antigen (51), the sprags were transformed with type 5 adenovirus - Dauri - (Sprague-Dawley) rat embryo cells ( 52), X-ray transformed C3H-10T 1/2 cells (53), neuroliblast (neu) oncogenes, expressed by MoAbs for the NIH-3T3 transfectant, as described above. Similar to the method. Spleen cells prepared from immunized mice are non-secretory in the presence of polyethylene glycol (PEG), with minor modifications to the Kahler and Milstein method. Hybridized with bone marrow cells NSI, and 24 hours after fusion, hybrid chloride was replateed in 96-well plate HAT medium to select hybrid cells. Collect the supernatant in the holes of the microtiter containing the proliferated hybrid clones and collect various target cells; For example, antibody activity against tertiary and secondary LN-CAP-CREF transfectants and normal CREF cells was tested. Early screening requires a small amount of target cells and does not consume time, so Terasaki Plate¹²⁵The I-Protein A binding assay (55) was used. Other assays, such as enzyme-bound immunoassays, can also be used. The initial screening below is a hybrid-ma culture supernatant that specifically reacts with the LN-CAP-CREF transfectant in a variety of target cells; For example, human prostate cancer cell line DU-145, PC-3, two human melanoma cell lines (Colo38 and HO-1), B lymphoblastic cell line (WIL-2), human skin fibroblasts, The specificity for CREF-Trans 6 cells, by other viruses (type 5 adenovirus, bovine fetal papyro-mavirus type 1 and Ha-ras), was retested. Hybriders that secrete antibodies specific to the human prostate cancer cell line are immediately subcloned at least 5 times with limited dilution. The final hybrid-macro-ne of interest, that is, those that produce antibodies that specifically react with human prostate cancer cell TAAs, are grown in in-vitro mass cultures or in vivo in pristane primed allogeneic mice. Proliferate as ascites. The antibody of interest is used to characterize the biochemical characteristics of prostate cancer-induced antigens expressed on transfected CREF cells or in human prostate cancer tumor cells.

【0072】

Identification of specific antigens by antibody use: MoAbs of interest are used to isolate specific antigens from human prostate cancer cells for biochemical analysis using the methods described above. First, a simple experiment is performed to determine the nature of the epitope-containing molecule recognized by MoAbs, namely whether it is a protein or a glycolipid. A radioimmunoprecipitation assay (56,57) is used to identify protein or glycolipid antigens.¹²⁵From cells labeled with I or³⁵S-Methionine or³Cell membrane detergent extracts from cells synthetically labeled with H-leucine are reacted with MoAbs bound to anti-mouse- (IgG + IgM) cephalos and the interaction between MoAbs and antigen is SDS-PAGE (56,57). Analyze by. To identify glycolipid antigens, lipid components isolated from human prostate cancer cells are analyzed by thin layer chromatography and the chromatograms are reacted with the corresponding MoAbs.

【0073】

The molecular weight and charge heterogeneity of the protein antigen is further analyzed by two-dimensional (2-D) gel electrophoresis as in the O ́ Farrell (58) method or Garrels modification method (59) described above. To do. Antigens isolated by immunoprecipitation with MoAbs are then subjected to one-dimensional isoelectric focusing of ampoline and urea-containing polyacrylamides. The gel is balanced by SDS-gel buffer and applied to SDS-PAGE gels. Prior to 2-D analysis, charge heterogeneity due to sialic acid is evaluated by removing sialic acid residues with neurominidase. Thus, 2-D analysis can simultaneously analyze isoelectric focusing (pI) and protein molecular weights.

【0074】

A Western blotting assay is performed to determine the stability of the protein antigen identified by MoAbs. In short, cell extracts denatured by SDS detergent are analyzed by SDS-PAGE in a reduced or non-reduced state, blotted on a nitrocellulosic membrane, and immunoperoxidase method (after treating the blot with MAb, then anti-antibody). MoAbs stained or blotted with MoAbs sorted with hydrogen peroxide and 3,3 ́-diaminobenzidein tetrahydrochloride) treated with mouse Ig antibody-binding peroxydase. Combine with¹²⁵Autoradiography is performed by binding the I-labeled staphylococcus aureus (S. aureus) protein A. These methods indicate whether (a) the epitope remains immunoreactive according to the treatment protocol and (b) whether the protein antigen is composed of subunits. In addition, these studies will show whether immunoblot assays can be applied to detect specific antigens in patient specimens.

【0075】

DNA Recovery from Tumor Cells: Tumor-derived tumor cells can be used to recover, identify, isolate, and clone at the molecular level using known methods. Southern blot analysis determines the presence of human DNA sequences in DNA extracted from tumor-derived cells that are progeny of the first, second, and third transfections of CREF-trans 6 cells. The second and third transfectants are usually less recovered, as if there was one band on the Southern blot probed with the human Alu DNA fragment, whereas in the primary or mouse. Fragments (Alu) containing human DNA are usually abundant in DNA digested and degraded by restriction enzymes derived from the cells to be transfected. Southern blot analysis revealed a 265 base spare Alu DNA sequence, namely Blur-8 (23) subcloned into a vector containing the SP 6 promoter [Racaniello (provided by V. Racaniell O)]. High specific activity³²It was used to produce P-labeled probe RNA. Due to the small size of the Blur-8 probe, it is not possible to detect a single copy of the Alu repeat integrated into the heterologous DNA. Contains only a single copy of Alu DNA V. It is known to contain a single copy of Alu DNA to determine if the Alu DNA sequence can be detected in transfected rat cells. DNA from the cell line provided by Racaniello (Columbia University) was used as the control for hybridization. If Southern blot analysis reveals the presence of inherited human Alu DNA containing restriction enzyme fragments in many second and third transfectants, human prostate cancer associated with tumorigenesis of CREF cells It is possible to isolate the gene. This includes the production of gene DNA libraries, the isolation of cloned DNA-containing Alu DNA sequences, and the biochemical activity of these cloned human DNAs by CREF cells cotransfected with pSV2-Neo DNA. This is possible by testing and determining whether the anti-G-418-transfectant is neoplastic in nude mice. MboI partial degradation of DNA extracted from CREF cells transfected with tumor-derived second or third LN-CAP DNA by MboI was used to construct the Lambda EMPL3 (BamHI site) library- (26). Library-Blur-8 Alu DNA Screened by a probe hybridization (27). DNA was extracted from recombinant lambda fage containing the Alu sequence, and cloned human DNA cotransfected pSV2-Neo DNA and CREF cells for its tumorigenicity, 10⁶Evaluate by recording the number of potentially tumorigenic anti-G-418- cells in nude mice after subcutaneous injection of cells. If the recombinant phase contains a tumor-inducing gene from LN-CAP prostate cancer cells, (a) the transcription rate of the newly isolated gene in the denuclearization analysis of the newly isolated gene. (b) Amount and multiplicity of intron homologous poly A + RNA by nozalen blot analysis; (c) S₁Intron / Exxon placement by analysis (28); and (d) Southern blot analysis to determine if the DNA placement of prostate oncogenes in LN-CaP cells is identical to the DNA placement in the normal human prostate cell genome; The smallest fragment of cloned DNA that can be mapped using restriction enzymes to transform CREF cells into the oncogene phenotype is LN- Used in CaP and normal prostate tissue. Genes involved in the oncogene phenotype of these prostate cancer cells by comparison of Southern blot analysis of normal, LN-CaP, primary, secondary, and tertiary LN-CaP transfected CREF cells You can check if there is amplification of.

【0076】

If there is a recombinant phase from secondary and tertiary transfectants containing Alu DNA and it does not transform lambda mice into CREF-trans 6 cells into a tumor state, then these pairs Transformation cells are used to screen cosmids (pJB8) (27) and lambda EMBL3 phase libraries, which are constructed by partial digestion of LN-CAP DNA with MboI and EcoRI. The DNA contained in the cells and cosmids that hybridize to the probe produced from the original transfectant DNA / lambda library is extensively mapped using restriction enzymes and homologous sequences in addition to the flanking DNA. DNA in the phage or cosmid that appears to contain is used to hybridize CREF-trans 6 cells and its potential for tumorigenesis is analyzed in nude mice. Cellular DNA sequences within the transfectant-producing phase or cosmid that test positive for the nude mouse tumorigenesis are determined by various restriction enzymes to determine the smallest DNA sequence involved in tumor-inducing activity. It is digested and decomposed.

【0077】

If human Alu DNA contains a sequence cloned within a single phase or cosmid constructed from second and third transfectants that transform CREF cells into a cancer phenotype. Without, normal human or LN-CAP DNA supplements the oncogene by supplying a polymerizable fragment from a cosmid library constructed by partial digestion degradation by SalI or EcoRI instead of lambda EMBL3 or MboI. -By recombination with vivo, it is possible to reconstruct an active oncogene. In recent years, this technique has been used in human nerves by transfecting a defined DNA portion of this gene with a DNA sequence selected from a normal human lambdalaribulary that hybridizes with a partial gene that encodes the human nerve growth factor receptor. It has been used to produce biochemical activity on growth factor receptors (24). As a result, the transfectant expresses a new growth factor receptor that exhibits NGF-binding activity (24). If the above approach is negative for the isolation of genes involved in canceration transformation of CREF cells, this approach would be useful.

【0078】

The gene involved in canceration of CREF-trans 6 cells cannot be isolated by using the gene clone and searching for this human gene associated with the human Alu sequence. As an alternative to this early approach, simply use cDNA that reflects the amount of heterologous mRNA or mRNA that is closely related to the tertiary transfectants of non-cancerous CREF cells and oncogene phenotypes. A gene isolation method can be taken by separating. The difference between CREF cell gene expression and neoplastic LN-CAP transfectant may be due to gene expression from human prostate cancer DNA. Based on this hypothesis, cloned tertiary transfectant-derived RNA was used to construct the cDNA / lambda gt-10 library-and these libraries were produced from CREF mRNA or CREF transfectant mRNA. To do³²Screen with a P-labeled cDNA probe.

【0079】

10 mg of poly A + RNA is 10⁶Sufficient to build a library containing an independent recombination page of. A λgt10-cDNA library of this size contains sequences with concentrations below 0.001% of total mRNA. The λgt10-cDNA library constructed from tertiary LN-CaP CREF cell transfectants is screened in a variety of ways. For example, comparative, competitive, and elimination screening (11-) to isolate cDNA clones that correspond to mRNAs that are specifically or highly expressed in CREF cell transfectants compared to CREF cells. 13, 29, 64).

【0080】

To screen genes expressed in LN-CaP-transfected CREF cells rather than CREF-Trans 6 cells from the library, replicated nitrocellulos containing the appropriate recombinant phase tertiary CREF transfectant cDNA. Sfilter-generated from untransformed CREF-Trans 6 cells and tumor-induced LN-CaP-transfected CREF cells (64)³²Hybridize with a P-labeled cDNA probe. Nitrocellulos containing recombinant phase DNA molecules (CREF-Trans 6 cells transfected with cloned cDNAs from CREF-Trans 6 or LN-CaP DNA) as a second screening approach. -Sfilter- was prepared from tertiary transfectant CREF cells in the presence of hundreds of times more other CREF mRNA (11).³²Hybridize with a P-labeled cDNA probe. As a third approach, enriched with untransformed CREF cell sequences or tertiary CREF cell cDNA (12,13)³²There is an elimination screening method that hybridizes a recombinant phase-a nitrocellulose filter containing a tertiary transfectant DNA molecule-with a P-labeled cDNA probe. Using these methods, it is possible to compare with CREF cells and isolate a specific recombinant phase containing a gene sequence that is specifically expressed or overexpressed in the transfected CREF cells. ..

【0081】

The cDNA clones corresponding to mRNAs specifically expressed in tumor cells and tumor cells are homologous groups by cross-hybridization analysis and are mapped using restriction enzymes (29). To confirm that the cloned cDNA corresponds to human-specific mRNA, and to confirm that it is the gene that is involved in the carcinogenesis of CREF-Trans 6 cells and is involved in the regulation of prostate cancer. , CREF-Trans 6 cells, LN-CaP cells, CREF-Trans 6 cells transfected with LN-CaP DNA, as mRNA is present, size and quantitative characteristics of homologous mRNAs from these cells Is revealed from each homologous group using the largest cDNA insert.

【0082】

Nozalen blot analysis showed that these genes were (a) expressed at a low rate in normal prostate tissue or (b) in clinical tissue samples from prostate cancer that showed each stage of prostate cancer progression in humans. Show whether it is expressed or (c) expressed in prostate cells from normal and cancerous tissues derived from rats.

【0083】

Recombinant DNA containing cDNA corresponding to mRNA that is expressed differently is used as a probe for nozalene blot analysis of mRNA derived from cloned tertiary transfectants as well as mRNA derived from CREF-Trans 6 cells. used. If nozalen blot analysis identified gene expression only in the transfected cells, these cDNA clones would be tertiary to isolate and identify the gene sequences that control the expression of these genes. It is adopted as a probe for identifying cosmidophage from the gene library of transfectant DNA. Once one of the gene pairs has been isolated, the oncogene structure is S¹It is elucidated by the technique of nucleus mapping (29), restriction enzyme mapping (29), and DNA sequence (14).

【0084】

To confirm the biochemical action of the cloned cDNA, the cDNA is separated from the recombinant λgt10 DNA and inserted into the pCD expression vector- (15, 16). The pCD-cDNA recombinant plasmid is cloned into a λ-NMT bacterial opportunity containing the neomycin gene fused to the SV40 early gene transcription unit and is a potent promoter for transfectant and cDNA expression in mammalian cells. -Gives G418 resistance (15). λ-NMT-pCD-cDNA recombinant bacteriophage particles are used to effectively transfect CREF cells. G418-resistant strains are tested for transformation-related properties such as neoplasticity and changes in cell morphology, growth, and growth ability in agar-blended medium in nude mice.

【0085】

Identification of genes that control tumor-induced phenotypes in human prostate cell-derived CREF cells is as follows: (a) Whether similar genes are present and in other prostate cancer cell lines such as DU-145 and PC-3. In determining whether it is expressed; (b) in determining whether similar genes are present and / or in normal prostate, benign prostate enlargement and prostate cancer tissue corresponding to each stage of the same disease. (C) In determining whether a homologous gene is expressed in rat prostate cancer cells and whether expression of this gene may be converted in cells with different metastatic properties. In; (d) in identifying and qualifying special proteins involved in the expression of tumor-induced phenotypes in CREF cells; and (e) diagnosing specific stages of prostate cancer and treating prostate cancer It is useful in producing characteristic immunological drugs that are useful in investigating the course of treatment of patients undergoing treatment.

【0086】

Identification and Cloning of Tumor Inducing Genes Transferred from Human Prostate Cancer Cell Lines (LNCaP) to CREF Cells: An Attempt to Demonstrate Consistent Changes in Oncogene Expression in Primary and Established Human Prostate Cancer Cells Has not yet succeeded (8-10). However, defects in the expression of the retinoblastoma (RB) tumor suppressor gene (located on chromosome 13q14) were found in the DU145 human prostate cancer cell line (64) and in human prostate cancer specimens (65) at a rate of 2 out of 10. Was done. Special allelic defects on chromosomes 16q and 10q are involved in the pathology of human prostate cancer (66-68). The role of genetic alterations in RB and other speculative tumor suppressor genes (located on chromosomes 16q and 10q) in suppressing the behavior of specialty prostate cancers or providing insight into the course of a patient's prognosis is known. Not (64 --68) .. Further research is needed to determine whether changes in the tumor suppressor genes themselves induce prostate cancer, or whether these genetic changes are associated with unidentified oncogene activity. DNA transfection of high molecular weight DNA from prostate cancer cells to NIH 3T3 cells via calcium mediation has not been able to identify oncogenes that focus on and preferentially act on common tumor induction in this class of tumors ( 9). Elaborate single DNA transfection techniques allow the identification of additional oncogenes with weak transforming potential in histologically distinct human tumors (12, 13, 68-71). This method consists of co-transfection into mature cell lines with human tumor DNA and selectable antibiotic resistance genes, selection of antibiotic resistance and injection of resistant colonies into nude mice (12, 13,). 69-71). DNA of the cloned neomycin resistance gene (pSV2neo) and high molecular weight DNA derived from LNCaP cells were cotransfected into CREF or NIH 3T3 cells, and selection was made for resistance to G418, resulting in morphological transformation. It didn't become focal. However, injection of co-transfected CREF instead of co-transfected NIH3T3 cells into nude mice resulted in tumor induction in 3 sets of 4 sets of animals that received cells pooled from heterologous transfections. It was observed. As a result of cotransfection of CREF cells with pSV2neo and DNA derived from normal skin fibroblasts or DNA derived from salmon sperm, no tumor formation was observed. DNA from primary LNCaP-CREF tumor (1 ° transfectant) induced tumor phenotype after cotransfection into secondary CREF cells. After cotransfection into secondary CREF cells, screened antibiotic-resistant colonies (2 ° transfectants) are in No morphological transformation was shown in vitro. However, these antibiotic-resistant morphologically normal cultures showed tumor induction in 6 of 7 sets of animals pooled from heterologous transfections. Both 1 ° and 2 ° transfectants obtained from LNCaP-CREF induced tumors in nude mice containing human (Alu) repeat sequences not detected in CREF cells (these transfectants). Some of these are shown in Figure 1). Both 1 ° (CREF 4 NMT) and 2 ° transfectant (CREF 4-5 NMT; CREF 4-7 NMT) contain a clearly unique Alu fragment in addition to a common Alu fragment of approximately 7.5 kb. There was. This finding suggests that a putative tumor-inducing gene from human prostate cancer cells is located near this DNA sequence. As described below, 1 ° and 2 ° transfectants are used to isolate genes involved in the phenotype of tumor cells transferred from human prostate cancer cell line LNCaP to CREF.

【0087】

Gene cloning strategy used to isolate transduced tumor-inducing genes from LNCaP cells: Cloning in EMBL3 phage: The basic procedure used has been established to identify Alu human repeat sequences present in rodent animal cells transduced with human DNA and to clone these sequences (23, 70-72). ). In addition, recently this approach has co-transduced NIH 3T3 cells with pSV2neo DNA with cellular DNA to identify potentially novel oncogenes from patients with familial adenocarcinoma-like polyposis after tumorigenesis in nude mice. It is used in (71). As shown in Figure 1, studies on Southern blots show that LNCaP DNA-CREF transducers from both 1 ° and 2 ° tumors generate after cleavage at EcoRI, which hybridizes with the Blur 8 (Alu) probe. It is shown to contain a DNA fragment of approximately 7.5 kb (23). The following measures can be used to isolate genomic clones containing this putative LNCaP tumor-inducing gene. EcoRI digestion of 7-9 kb, positive when searched on Blur 8 DNA fragments are isolated and extracted from low melting point agarose gels by treating with phenol, phenol: chloroform and chloroform. This DNA fragment is ligated to an EcoRI (Stratagene, CA) cleaved bacteriophage EMBL3 arm (72). After ligation, the reaction mixture is packed using the Stratagene Packing Extract. The entire phage library is applied to LE392 host bacteria and plaque hybridization (73) on nitrocellulose paper using Blur 8 as a probe identifies Alu-positive plaques. The Alu probe was [α-32P] dCTP (Amersham, Inc.,) using the random oligonucleotide labeling method (76). Label with IL). Positive genomic clones are characterized by restriction enzyme mapping and Southern blot analysis (77,78). Positive clones are subcloned into pUC19 or M13 phage for sequencing and used as probes in Northern blot studies. Cytoplasmic RNA isolated from LNCaP, 1 ° and 2 ° tumor-derived LNCaP DNA-CREF transfectants and additional human prostate cancer cell lines (including DU145 and PC-3) were used for studies in Northern blots. Be done. Clones that are positive in these Northern blot analyzes and contain exons of the same size are further characterized by sequencing. Clones containing the appropriate exons are used to screen a cDNA library composed of LNCaP and 1 ° and 2 ° tumor-derived LNCaP DNA-CREF transducers to obtain full-length cDNA clones corresponding to the putative tumor-inducing genes.

【0088】

The following approaches can be used as a second approach using EMBL3 cloning to obtain putative tumor-inducing genes present in 1 ° and 2 ° tumor-derived LNCaP DNA-CREF transduction bodies (71, 74, 76). .. Secondary tumor-induced LNCaP DNA-CREF transformant DNA is partially digested with Sau3AI and then fractionated by sucrose gradient centrifugation. Purified fractions containing 20-30 kb fragments are ligated with a purified EMBL3 phage arm cleaved with BamHI. This recombinant phage library is screened with an Alu probe to identify positive clones. The cloned DNA is then digested with SalI and analyzed by Southern blot using the Alu probe and the probe obtained above.

【0089】

Polymerase Chain Reaction (PCR) Cloning: Isolation of human DNA sequences from 1 ° and 2 ° tumor-derived LNCaP DNA-CREF transducers using recombinant phage is a whole-genome library within EMBL3 (2-4). × 10⁵ The production of (including independent recombinant phage) and screening of this library using Alu as a probe are required. Recent studies have shown that the Alu PCR approach includes somatic hybrids that carry human chromosomal fragments in the rodent genome background, genomic DNA cloned into lambda phage, and yeast artificial chromosome (YAC) vectors. It has been shown that it can be used as a single method for the identification and purification of human DNA sequences from complex sources (79, 80). This approach is facilitated by using Alu-repeating consensus sequences that are reasonably conserved in the human genome (79). PCR primer consensus sequences that have proven to have special value in identifying human DNA sequences in somatic hybrid and YAC vectors are TC-65 and 517 (79). Primer TC-65 is located within the only 31 bp sequence of the second monomer of the primate Alu repeat (81). Primer 517 has the same 17 bp as TC-65 It recognizes the Alu sequence, but in the opposite direction. Based on a study by Nelson et al. (79), it is estimated that TC-65 identifies fragments of human DNA that produce every 500 kb, and primer 517 produces fragments every 1000 bp. If these consensus Alu sequences are present in 1 ° and 2 ° tumor-derived LNCaP DNA-CREF transduced bodies, the Alu PCR approach should be used to prepare a genomic library and screen this library for Alu-positive clones. It disappears. First, 1 μg of 1 ° and 2 ° tumor-derived LNCaP DNA-CREF transduced DNA, and PCR with TC-65 and 517 as described by Nelson et al. (79) are used. Modification of this approach by either the use of primers or reverse PCR (82), which allows bidirectional amplification from a single Alu repeat with two primers, is the amplification of most Alu sequences from regions or clones. To enable. If this PCR Alu methodology is successful, the sequences identified will be 1 ° and It can be used directly as a probe used to identify cDNA clones of interest in screening recombinant libraries derived from 2 ° tumor-derived LNCaP DNA-CREF transducers.

【0090】

Discriminatory screening (subtractive screening): Screening of subtracted libraries constructed from LNCaP DNA-CREF transfectants from 2 ° tumors totals 1 × 10⁶ It results in a greater enrichment of hybridization-positive phage clones than can be obtained from screening of individual recombinant phage cDNA libraries. First, a unidirectional cDNA expression library from CREF and 2 ° tumor-derived LNCaP DNA-CREF transfectant cells was prepared from 5 μg of poly A + RNA, followed by a Stratagene cDNA synthesis kit. Heavy-chain cDNA is prepared (83). The cDNAs are ligated into Lambda Zap's bacteriophage vector and packaged with packaging extracts. 1x10 in total⁶ The number of clones is counted. The subtractive library is made from the above-mentioned cDNA library by the method of Sargent and Dawid (84). 30 μg of LNCaP-DNA-CREF transfectant cDNA from a 2 ° tumor present in the lambda zap vector is cleaved with XhoI and RNA transcripts are produced by T3 RNA polymerase (85). This RNA transcript is converted to single-stranded cDNA by the Moloney murine leukemia virus reverse transcriptase upon addition of the XhoI linker primer. This single-stranded cDNA hybridizes to excess CREF cytoplasmic RNA at 60 ° C for 48 hours. Unhybridized cDNA is recovered by passing it through a hydroxyapatite column (86). Approximately two rounds of subtraction results in a 90% subtraction from 2 ° tumor-derived LNCaP DNA-CREF transfectant cDNA. This subtractive cDNA can be used as a probe, or is further processed into double strands with Klenow polymerase and ligated into a phage vector for packaging. The total number of colonies obtained is 5 ~ 10 × 10⁴ Is. This approach is voluntary and is only used if, for some reason, the identification of the putative LNCaP tumor-inducing gene using EMBL3 cloning or PCR-Alu methods is unsuccessful.

【0091】

Another approach that can be theoretically used to clone the LNCaP tumor-inducing gene from CREF transfectants combines both subtractive hybridization and PCR techniques (87). In this method, a large excess of driver DNA (CREF) is combined with tester DNA (LNCaP DNA-CREF transfectant from a 2 ° tumor containing the gene in question that is not present in the driver DNA) and the subtraction procedure. Removes common sequences and results in enrichment of the target DNA sequence (in the tester DNA sample). After separating the subtracted tester DNA from the driver DNA by avidin / biotin affinity chromatography, the single-stranded target DNA is amplified by PCR, which can be cloned as double-stranded. Using this new method, Wieland et al. (87) achieved a 100- to 700-fold enrichment of the target DNA sequence. By adopting this approach, the common sequences present in both CREF and 2 ° tumor-derived LNCaP DNA-CREF transfectant are removed, and 2 ° tumor-derived LNCaP DNA- It is possible to result in enrichment of Alu-linked sequences that are present only in CREF transfectants. These Alu-linked sequences can then be amplified using PCR and the gene can be cloned directly into the pUC18 vector for further analysis.

【0092】

Probing with Lambdazap expression vector and polyclonal antibody: Polyclonal antibody and monoclonal antibody that react with LNCaP cells are highly potent to coat 1 ° tumor-derived LLNCaP DNA-CREF transfectant prior to immunosensitization of mice. Developed by using valent CREF antiserum. Using this approach and rabbit immunosensitization, it reacts with 1 ° and 2 ° tumor-derived LNCaP DNA-CREF transfectants and antigens expressed on the surface of LNCaP cells, but not with CREF cells. Polyclonal antibody has been produced. LNCaP-DNA-CREF transfectants from 1 ° and 2 ° tumors and cDNA libraries from LNCaP cells were constructed in Lambdazap expression vectors and colonies were constructed as polyclonal antibodies (as described by Young and Davis (Young and Davis). 88) and then screened with (77) neutralized against E. coli extract. Approximately 1 x 10⁶ Screening of recombinant phage clones with polyclonal antibodies 3-4 times in a row to obtain positive clones. This cDNA is then subcloned and sequenced as described above (77,78). This approach is provided only as an alternative approach for identifying and cloning LNCaP tumor-inducing genes that have been introduced into CREF cells.

【0093】

Presumably, the methods outlined above are present in LNCaP cells and isolate and identify presumed prostate cartinoma tumor-inducing or tumor-related genes that are introduced into CREF cells via calcium-mediated DNA transduction. It seems that it will bring about.

【0094】

Organization and expression of transfected tumor-inducing genes derived from LNCaP cells in normal prostate, benign prostatic hypertrophy (BPH), prostatic cartinoma and metastatic tumors to bone: The methods described above are from LNCaP cells. Introduced into CREF cells, it results in the isolation of cDNA or gene clones (referred to as Prostatic Cartinoma Tumor Induction / Association (PCTI) Genes) that mediate the tumor expression type of transfected cells. Once identified, this clone is used to determine its potential role in the development of human prostate carcinoma. These studies include (a) analysis of its expression in normal human prostate, BPH and prostatic cultinoma cell lines (at different Gleason stages); (b) normal human prostate, BPH, (different Gleason). ) Analysis of its expression in primary tissues from benign prostatic hyperplasia and metastatic tumors to bone; and (c) whether or not the homologous gene is expressed in rat prostate cultinoma cells, and the expression of this gene is different. Includes determination of whether or not it changes intracellularly with a degree of tumorigenicity / metastatic potential.

【0095】

Expression of PCTI Genes in Human Prostate Cell Lines Representing Different Stages of Prostate Cartinoma Development: LNCaP-DNA Transfection Tumor-Derived CREF Cell-Derived PCTI Genes Organized in Normal Prostate, BPH and Prostate Cartinoma Cell Lines (8) And expression is measured by Southern (6,71,74,75) and Northern Blotting analysis (47,71) as described above. The organization and expression of the PCTI gene is also measured in additional normal cell lines and tumor-derived cell lines. These include normal human cutaneous fibroblasts, epidermal keratinocytes, melanocytes, breast epithelial cells and colonic epithelial cells; and from sarcomas, hematological malignancies, melanomas, central nervous system tumors and various carcinomas (breast cancer, colorectal cancer, etc.). Tumor-derived cell lines are included. Since rat prostate model systems, including the Danning rat dorsal prostate adenocartinoma system (7,89), have proven useful in determining the functional properties of the PCTI gene, these systems are also evaluated. .. The above studies provide information on the distribution of expression of the PCTI gene isolated from CREF cells from tumors transduced with LNCaP DNA.

【0096】

Expression of the PCTI gene in primary prostate tissue representing different stages of prostate cultinoma development: It is important to determine whether the PCTI gene is expressed in vivo in human prostate cultinoma cells. RNA is extracted from normal prostate, BPH, various Gleason-stage prostate carcinomas, and prostate carcinomas that have metastasized to the bone using the technique described above (90). These RNAs are analyzed by slot-blot (90) and Northern blotting (47) to measure the expression level of the PCTI gene. To normalize RNA expression and loading, the blot is reblotted with glyceraldehyde phosphate dehydrogenase (GAPDH).

【0097】

If the PCTI gene is expressed differently in primary prostate tissue, this gene is subcloned into a pGEM-4 or SK vector and frozen from normal prostate, BPH and prostate carcinoma tissues by in-situ hybridization (91). Expression of the PCTI gene in tissue sections is measured. By using rodent-human hybrid and Southern blotting analysis carrying specific human chromosomes, studies are also conducted to determine the chromosomal location of the PCTI gene in the human genome (71). ..

【0098】

Functional Study of PCTI Genes Derived from Human Prostate Cartinoma (LNCaP) Cells: The key question is whether isolated PCTI genes expressed in CREF cells can function as tumor-inducing genes, and / or Whether the gene can function as a tumor-inducing gene when expressed in normal human, rat and mouse prostate cells. The study also to determine whether the PCTI gene can alter the phenotype, as shown by the proliferation of human BPH and human, rat and mouse prostate carcinoma cells in agar and tumorigenesis in nude mice. It is also done. A useful model system for determining whether the PCTI gene can cooperate with other oncogenes such as ras and myc in the induction of prostate carcinoma is the mouse remodeling organ system described by Thomas et al. (92). In this system, expression of ras in normal mouse prostate cells results in dysplasia associated with angioplasty, myc induces prostate hyperplasia that would otherwise occur normally, and the combination of ras and myc Induces primary carcinoma (92).

【0099】

DNA transformation and expression studies: First, the PCTI gene is inserted into a suitable mammalian expression vector system containing the neomycin resistance gene (and a replication-deficient retroviral vector containing the neomycin resistance gene) and transduced (or retro) into CREF cells. In the case of a virus, it was infected). If these transduced cells expressing the PCTI gene induce a tumor, the tumor is resected and the presence of the appropriate gene is analyzed (Southern analysis) (14) and its expression is analyzed (Northern analysis). (47). It is also possible to determine whether or not a tumor-derived CREF cell expressing the PCTI gene expresses TAAs recognized by the MoAbs developed above on its cell surface. Positive results support the assumption that the cloned gene is a tumor-inducing gene element officially introduced from LNCaP cells into CREF cells.

【0100】

Next, the expression of the PCTI gene in normal mouse, rat and human prostate cells is transformed to a partially (immortal) or complete (anchorage independence and tumorigenicity in nude mice) transformation phenotype. Whether or not to induce is decided. These studies transduced the PCTI gene into appropriate target cells and G414 It is done by selecting for resistance and isolating individual clones (93). These clones were then analyzed biologically (1) and molecularly (14,47,93) to see if they contained and expressed the PCTI gene, and they had an acquired phenotypic alteration. Whether or not it is determined. The protocol (1,95) described above is also used to determine the tumorigenic performance and metastatic potential of these transformed prostate cells in nude mice. Similar studies are performed on human BPH cells to determine if the PCTI gene can advance these cells to tumorigenic and / or metastatic states. By expressing the PCTI gene in Danning rat prostate adenocarcinoma cells and human prostate carcinoma cells, we will further explore the role of the PCTI gene in the regulation of tumorigenicity and metastasis of transformed cells in nude mice. It is possible. Experiments have also been described by Thompson et al. (92) to determine whether the PTCI gene induces carcinoma in reconstituted mouse prostate gland by itself or in combination with ras or myc. It is done in this way. These studies described above provide valuable information on the presumed functional role of the PTCI gene in the development of human prostate carcinoma.

【0101】

Characteristics of Monoclonal Antibodies (MoAbs) Developed Against Tumor-Derived CREF Cells Transduced with LNCaP / DNA: Neoplastic Expressions Are Novel, Specific for Tumors of Different Histological Types Often characterized by surface expression of tumor-associated antigen (TAA) subsets. In some cases, genetic elements that induce tumorigenic phenotypes can also encode these surface TAAs. Tumors transduced with LNCaP DNA to generate MoAbs that react with LNCaP cells because of the direct relationship between the PCTI gene transduced into CREF cells and the expression of the prostate cartinoma-expressing form Suggested by the availability of derived CREF cells. Tumor-derived CREF cells transduced with LNCaP DNA are coated with a highly specific CREF polyclonal antiserum and injected into mice. Serum from these animals contains polyclonal antibodies that react with LNCaP cells, and spleens from these animals are LNCaP and PC3. It was used to generate MoAbs that specifically react with human prostate carcinoma cells, including. These same MoAbs do not cross-react with DNA-transformed CREF or CREF cells from other histologically different human tumors and are normal human skin fibroblasts, melanoma, breast cancer, glial blast carcinoma. Does not respond to form or colon-rectal carcinoma. MoAbs that react with LNCaP but do not cross-react with the types of cells listed above or PC3 cells have also been isolated using the approach described above.

【0102】

In previous studies, NIH 3T3 cells transduced with DNA from acute lymphocytic leukemia (ALL) (96) or pancreatic adenocartinoma cell line (HPAF) (97) were morphologically transformed. Formed focus. These morphologically transformed NIH3T3 cells were then used to develop MoAbs that do not react with NIH3T3 cells but bind to epitopes on human tissue cell membranes that are histologically identical. For NIH3T3-derived MoAbs transduced / transformed with ALL-DNA, NIH3T3 cells transduced / transformed with ALL-DNA, as well as fresh human leukemia cell lines, cultured leukemia cell lines, and T cell lines. And reactivity with normal human hematopoietic cells (96) was observed. In the case of NIH3T3-derived MoAbs transduced / transformed with HPAF-DNA, reactivity with the HPAF cell line and another 6 human pancreatic adenocartinoma cell lines was found. In contrast, HPAF-DNA transduced / transformed NIH3T3 The derived MoAbs are lymphoblast tumor cell line (myeloid (K562 and HL-60), T cell line (CEM, MOLT and RESKW3), B cell line (SB, Daudi, WIL-2 and CALLA), melanoma cell line). , Showed no cross-reactivity with prostate cultinoma cell line or normal cell line (skin fibroblasts and normal pancreas). Using MoAbs generated for CREF cells transfected with tumor-derived LNCaP DNA. These experiments and studies described above suggest that (a) human tumor-related antigens can be introduced and expressed in heterologous mouse and rat cells; (b) traits. The introduced cells can be used to generate MoAbs that exhibit specific and limited reactivity with human tissues of different tissue types. For tumor-derived LNCaP-DNA-transfected CREF cells. The experiments described below are planned to further characterize the generated MoAbs as a possible means for the diagnosis and ultimately treatment of prostatic cultinoma.

【0103】

This developed MoAbs that react with LNCaP cells is characterized as previously described (98). These studies included formalin-fixed normal human cell lines (prostatic epithelium, colonic epithelium, breast epithelium, etc.) and tumor-derived human cell lines (BPH, prostatic carcinoma, metastatic prostate carcinoma from bone, colonic rectal carcinoma, breast cancer). Etc.), which includes extensive analysis by ELISA on the reactivity of MoAbs with. To determine (a)-(d) below, (98-100) additional studies are performed as described above: (a) the antigenic epitope that reacts with the MoAbs is cultured. Whether or not it is shed in the fluid; (b) Expression of MoAbs by FACS analysis in live permeable normal prostate cells, BPH cells and prostate cultinoma cells; (c) PAGE and Western blot analysis were used. , Molecular weight of epitope recognized by MoAbs; (d) Various enzymes (fucosidase, β-glucosidase, β- The biochemical properties of the antigen recognized by MoAbs using galactosidase, mixed glycosidase, periodic acid, mannosidase, neuraminidase and protease). MoAbs are also compared to prostate-specific antigen (PA) and prostatic acid phosphatase (PAP) MoAbs for identification of prostate cell types and additional cell types. PA and PAP are not useful as early markers of prostate cancer, but are useful for monitoring disease recurrence and therapeutic response. Therefore, it is determined how MoAbs are compared to these established immunological reagents and whether the epitopes recognized by the MoAbs are present in normal prostate, BPH and / or prostate carcinoma cells. That is interesting.

【0104】

Responsiveness spectrum of MoAbs developed for tumor-derived CREF cells transfected with LNCaP / DNA to human tissues: MoAbs developed for tumor-derived CREF cells transfected with LNCaP / DNA are patients It is important to determine whether the antigenic epitope on the prostate cultinoma tissue obtained from the tissue can be recognized. Therefore, to see if MoAbs can identify prostate cells in frozen and formalin-fixed / paraffin-embedded tissue from bone sections containing normal prostate, BPH, benign prostatic hyperplasia and prostate cultinoma metastases. Research is done. If MoAbs can detect the missing antigenic epitopes, then urine, semen secretions and blood samples from patients with prostate carcinoma (of different degrees of Gleason) and patients with prostate carcinoma that has metastasized to the bone will have these antigens. Is determined whether or not it contains. Similarly, if a good correlation is observed between the expression of antigenic epitopes recognized by MoAbs and prostate carcinoma, then the MoAbs have a broad spectrum of additional normal and tumor cells (different tissue origins). It is possible to determine whether or not it can react with (the thing). PAs and PAPs are used as suitable control reagents for all these studies. These studies are to determine whether the developed MoAbs may be useful in diagnosing prostate cancer in humans and whether they offer some selective advantage over PA and PAP / MoAbs. Is necessary for.

【0105】

Biological studies to address the potential function of TAAs, recognized by MoAbs developed for tumor-derived CREF cells transduced with LNCaP DNA: Currently introduced from LNCaP cells into CREF cells The role of the TCTI gene is unknown. Whether LNCaP cells and tumor-derived CREF cells transduced with LNCaP DNA are coated with MoAbs to alter proliferation in monolayers and / or agar suspensions to initiate addressing to this problem. Is determined (94). MoAb-coated cells are also evaluated for changes in tumorigenesis and proliferation after transplantation into nude mice (97). Suitable controls for the above experiments are (a) PA and PAP-coated LNCaP cells and LNCaP-DNA transduced tumor-derived CREF cells, (b). Similar studies will be conducted on LNCaP cells and tumor-derived CREF cells transduced with LNCaP / DNA that have been incubated with non-reactive antibodies such as high molecular weight melanoma-related antibodies that do not bind to these cells. If any MoAbs suppresses tumorigenesis of LNCaP cells in nude mice, this study is extended to include an assessment of the effects of these MoAbs on additional prostate carcinoma that reacts with the MoAbs. Although negative results are not beneficial, suppression of fixation-dependent growth and / or tumorigenesis by MoAbs in prostate carcinoma cells is associated with the PCTI gene-encoded TAAa and the transformed state in human prostate carcinoma cells. Gives strong evidence that there is. In addition, if a positive result is obtained, the identified MoAbs can be demonstrated to be useful after being combined with a toxin or high-energy radionuclide for the treatment of prostate cancer (102).

【0106】

<Results and discussion> CREF cells co-transduced with DNA from the human prostate carcinoma cell line, LNCaP and pSV2neo induce tumors in nude mice. Tumor-derived CREF cells at 1 ° (first transduction) and 2 ° (second transduction) contain human Alu sequences. All tumors are approximately 7.5 In addition to the common Alu fragment of kb, it contains a seemingly unique Alu fragment (Fig. 1). This finding suggests that presumed tumor-inducing genes from these human prostate carcinoma cells are located adjacent to this DNA sequence. Various strategies can be used to isolate genes that mediate tumor cell phenotype using 1 ° and 2 ° tumors derived from nude mice transduced with human prostate DNA transduced CREF cells. .. One approach is to prepare a genomic DNA library within EMBL3 phage using DNA corresponding to the Alu-positive signal found in 1 ° and 2 ° tumor-derived CREF transfectants. Alu-positive clones can then be identified, subcloned and Northern blotting to determine the presence of exons. Subclones that hybridize to RNA of the same size on Northern blots are further characterized by sequencing. These can be used as probes for screening genomic libraries or cDNA / LNCaP libraries. The second approach is to use Alu-specific probes to identify and amplify DNA flanking these sequences in 1 ° and 2 ° tumor-derived CREF trans-6 transfectants by polymerase chain reaction (PCR). Is. These amplified DNA sequences are then cloned directly into the expression vector system and tested for tumor inducibility in nude mice. The third approach is CREF / NMT4 cells and CREF- Creating a subtractive library from trance 6 cells. Yet another approach can be used to mediate tumorigenesis within CREF cells and to identify and clone genes within LNCaP cells that can be involved in human prostate carcinoma development. These include partial digestion of LNCaP DNA with SauIIIAI, selection of 30-35 kb DNA, and production of genomic libraries within genomic neo expression vector systems. These DNAs are then transduced into neo-resistant CREF cells and the pooled cell population for tumorigenesis is injected into nude mice. It is highly possible that the above strategies can be used to successfully identify and clone a presumed tumor-inducing gene derived from LNCaP cells. In addition, the approach outlined above has also been used to identify and clone other tumor-inducible genes from other specific human malignancies transduced and expressed in CREEF-trans 6 cells. obtain. Once identified, this putative tumor-inducible gene from LNCaP (and other identified putative tumor-inducible genes obtained from LNCaP and other tumors) is then the normal, human prostate, benign prostate. It can be used as a probe to measure its organization and expression in prostatic cartinoma of hypertrophy and different Gleason stages. The potential role of this tumor-inducing gene from LNCaP cells in mediating metastasis of prostate carcinoma cells can also be addressed. It can also be used to identify and clone other tumor-inducible genes from certain other specific human malignancies transduced and expressed in trans 6 cells. Once identified, this putative tumor-inducible gene from LNCaP (and other identified putative tumor-inducible genes obtained from LNCaP and other tumors) is then the normal, human prostate, benign prostate. It can be used as a probe to measure its organization and expression in prostatic cartinoma of hypertrophy and different Gleason stages. The potential role of this tumor-inducing gene from LNCaP cells in mediating metastasis of prostate carcinoma cells can also be addressed. It can also be used to identify and clone other tumor-inducible genes from certain other specific human malignancies transduced and expressed in trans 6 cells. Once identified, this putative tumor-inducible gene from LNCaP (and other identified putative tumor-inducible genes obtained from LNCaP and other tumors) is then the normal, human prostate, benign prostate. It can be used as a probe to measure its organization and expression in prostatic cartinoma of hypertrophy and different Gleason stages. The potential role of this tumor-inducing gene from LNCaP cells in mediating metastasis of prostate carcinoma cells can also be addressed.

【0107】

As shown above, neoplastic phenotypes are often characterized by surface expression of novel tumor-associated antigen (TAA) subsets specific for different histological types of tumors. In some cases, genetic elements that induce tumorigenic expression can also encode these specific cell surface TAAs. In many cases, TAAs have proven to be suitable targets for MoAb-based therapies that result in direct suppression of tumor growth. Possible relationships between induction of tumor cell phenotype and expression of specific TAAa have been directly tested using LNCaP-transduced tumor-derived CREF-trans 6 cells (CREF · 4, NMT). There is. CREF-A antigen expressed on CREF-4 NMT cells and LNCaP cells by injecting mice with CREF-4 NMT cells coated with a highly specific activity polyclonal antibody generated against trans 6 cells. Both reactive polyclonal and monoclonal antibodies were successfully produced (Table 1 and Figures 2, 3 and 4).

【0108】

table 1 Mouse antiserum Cell line origin CREF CREF-NMT CREF-Trans 6 rat fetal Fibroblast +- CREF-NMT 4 LNCaP-transduced Tumor-derived CREF ++ LNCaP human prostate Carcinoma-+ SW480 Human colorectal rectum Carcinoma-+ MCF-7 human breast cancer -+ Colo38 Human Melanoma --- CREF-trans 6 cells performed an immune response against CREF-trans 6 cells and CREF 4 NMT cells, but did not respond to cells of human origin. In contrast, CREF-4 NMT cells coated with CREF-trans 6 identify antigens on CREF 4 NMT cells, LNCaP cells, human colon-rectal cultinoma cells (SW480) and human breast cancer cells (MCF-7). Antigens on CREF-trans 6 cells, human skin fibroblasts (WI-38) or human melanoma cells (HO-1) produced an unidentified polyclonal antibody response (Table 1 and Figure 2). Further characterization by titration analysis of the polyclonal antibody produced by injecting mice with CREF-trans 6 coated CREF 4NMT cells is shown in FIG. As you can see here, anti-CREF 4 NMT polyclonal antibodies can bind to the human prostate carcinoma cell line, breast cancer cell line and colorectal carcinoma cell line. MoAbs were then generated from animals hyperimmunized with CREF 4 NMT coated with high titer CREF high serum by standard methods. As shown in FIG. 4, these MoAbs (MoAb 1.5.6 and MoAb 5.3.1.3) have good reactivity with LNCaP and other human cultinoma. In contrast, these MoAbs do not bind to HO-1 cells, WI-38 cells or CREF cells. These results show that the gene introduced from LNCaP DNA into CREF-trans 6 cells is expressed in the transduced cells, and these show MoAbs that react with the original cells from which the human tumor DNA was isolated. It clearly shows that it can be used to generate.

【0109】

The CREF-trans 6 system was tested for expression of tumor-inducible genes from other human tumor cell lines as well as primary human tumor DNA samples. Human breast cancer cell line (T47D), human glioblastoma polymorph (stage IV astrocytoma) cell line (GBM-18), and primary tumor-derived primary human glioblastoma polymorph (stage IV astrocytoma) Tumor) The human tumorigenesis expression form from DNA was successfully introduced. In the case of the CREF-T47D transfectant, as expected, Alu sequences different in size from those found in CREF 4 NMT cells were detected. In addition, by using this antibody coating technique with highly specific CREF antiserum, it binds to T47D cells as well as MCF-7 cells, but SW480 cells, LNCaP cells, HO-1 cells or CREF cells. MoAb (4.2.6) was generated that did not bind to (Fig. 5). The second MoAb (5.1.4) produced by exposing animals to CREF-T47D cells coated with CREF-trans 6 antiserum reacts with LNCaP cells as well as T47D cells and MCF-7 cells. These results are MoAb It is suggested that the epitope recognized by 5.1.4 is expressed in breast cancer cells and prostate carcinoma but not in SW480 or HO-1 cells to the same extent. Further analysis of these MoAbs with multiple cell types (as well as MoAbs 1.5.6 and 5.3.1.3 generated for CREF4 NMT cells) and analysis of resected tumor tissue reveals the specificity of these MoAbs. And its diagnostic usefulness will be determined.

【0110】

In summary, common methods have been developed to identify genes encoding TAA of human origin and to produce immunological reagents. The ability to generate both polyclonal and MoAbs that bind to human prostate carcinoma cells suggested an association between the tumor-inducing gene introduced into CREF cells by transfection and the encoded human gene product (TAAs). .. In addition, by using CREF / transduction / monoclonal antibody technology, genes that mediate or are associated with HITO breast cancer and gliablastoma polymorphism are introduced, and antigens that are expressed in human breast cancer and other carcinomas. Recognizing polyclonal and MoAbs have been developed. This CREF / transduction / monoclonal antibody technology is also available for 170,000 cloned humans. It was also used to identify defined transduced and expressed surface molecules, including molecular weight (P-glycoprotein) multidrug resistance genes. CREF and human tumor (breast cancer and glial blastoma) cells were transduced with the cloned mdr-1 gene and selected for corhitin resistance, and Southern and Northern analysis showed the presence and expression of the mdr-1 gene, respectively. Shown. These transduced CREF cells were then used to generate MoAbs that react with P-glycoprotein expressed in transduced human breast cancer cells and glial blastoma cells expressing the MDR phenotype. It was.

【0111】

[Reference]

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[Simple explanation of drawings]

[Figure 1]

Southern blot analysis. No. 1 obtained from nude mice following injection of CREF-trans 6 cells transduced with high molecular weight DNA, pSV2-neo and LNCap DNA (CREF-4 NMT) from CREF-trans 6 (represented by CREF in Figure 1). Two independent second tumors obtained after injecting one tumor and CREF-trans 6 cells into nude mice with pSV2-neo and CREF 4 NMT DNA. Southern blots were examined with a 300 bp nick-translated 32P labeled Alu probe.

[Figure 2]

CREF 4 NMT-immobilization and CREF-trans 6 (represented by CREF in Figure 2) immobilized antisera against LNCaP. Animals immunized with CREF-trans 6 cells derived from nude mouse tumors transduced with LNCaP DNA (CREF-4 NMT) express an immune response against the original LNCaP cells. Estimated from LNCaP cells CREF-trans 6 cells lacking human TAA do not produce an immune response against LNCaP cells.

[Fig. 3]

Binding of CREF-4 NMT polyclonal antiserum to human cancer cells. Polyclonal antibodies generated against high titer CREF-trans 6 cells (represented by CREF cells in Figure 3) transduced with LNCaP DNA (CREF-4 NMT) are the human prostate, lung and colorectal cancer cell lines. Combine to. Conversely, like CREF-4 NMT, polyclonal antibodies generated against CREF-trans 6 cells immobilized with high titer CREF-trans 6 antiserum do not bind to human cancer-derived cell lines.

[Fig. 4]

Binding of CREF-4 NMT MoAb to human tumor cell lines and CREF-trans 6. CREF-4 NMT cells treated with MoAb prepared from animals immunized with high titer CREF-trans 6 antiserum show good specificity for LNCaP and other human cancers. Conversely, the same MoAb does not bind to HO-I human melanoma, WI-38 human skin fibroblasts or CREF-trans 6 cells (represented by CREF in Figure 4).

[Fig. 5]

Binding of CREF-T47D MoAb to human cell lines and CREF-trans 6 cells. MoAbs generated for CREF-trans 6 cells transduced with nude mouse tumor-derived DNA show good specificity for human lung cancer cells. MoAb 5.1.4 also shows some binding to human prostate cancer cells. MoAb does not bind to HO-I human melanoma cells or non-transduced CREF-trans 6 cells (represented by CREF in Figure 5).

[Fig. 6]

Binding of MoAb 5.1.4 (CREF-T47D) ascites to humans and CREF-trans 6 cell lines (represented by CREF in Figure 6). In nude mouse tumor-derived CREF-trans 6 cells transfected with human lung cancer DNA from T47D cells, which are immobilized with high titer CREF-trans 6 antisera prior to injection into animals. MoAb 5.1.4 for immunized animals continues to show good specificity for human lung cancer cells after subcloning and ascites formation.