Method for Diagnosing and Staging Cutaneous T-Cell Lymphoma Using Gene Expression in Blood Cells
Cutaneous T-cell lymphoma (CTCL) is a form of non-Hodgkin's lymphoma that affects the skin and involves malignant T-lymphocytes. There are about 20,000 patients with CTCL in North America and about 14,000 patients in Europe. The disease usually occurs in middle-aged adults and develops over an extended period of time. CTCL is difficult to diagnose, especially in its early stages, since the symptoms closely resemble those of more common skin ailments. If correctly diagnosed and treated early, CTCL patients can expect to survive for many years. However, the survival rate decreases as the disease progresses to more advanced stages. As a result, a major need exists for methods that can provide reliable early-stage diagnosis of CTCL.
To address this need, scientists at Wistar have identified gene expression profiles using complementary DNA (cDNA) microarray technology that can help in the diagnosis of patients with CTCL. By combining quantitative PCR (qPCR) and linear discriminant analysis, the scientists have adapted the cDNA microarray method and developed a reliable and practical diagnostic for CTCL using as few as 5 genes.
Additionally, expression patterns of a small number of genes (less than 10) can be predictive of patients that will undergo rapid advancement of the disease. Using this technology, gene expression patterns in samples from patients with Sezary Syndrome, an aggressive form of CTCL, were distinguishable from those in patients with more indolent stages of the disease. These changes may contribute significantly to the development and progression of CTCL and provide markers for the use of more aggressive forms of therapy than may otherwise be recommended.
The RA 27/3 strain of rubella virus was developed at The Wistar Institute. The seed stock from RA 27/3 has been successfully used worldwide since the 1970's as a vaccine for the prevention of rubella infection. This vaccine has been administered alone and as part of a combination vaccine (e.g. MMR; measles, mumps, rubella vaccine).
The Wistar Institute has maintained seed stocks of the RA/ 27/3 strain of rubella for use in production of vaccines. These vaccines have been licensed to regional public health agencies and to for-profit companies who are developing vaccines for local distribution, particularly in Asia and South America.
Rabies Vaccine - PM Seed Stock
The PM 1503 strain of rabies virus, developed at The Wistar Institute, has been successfully used worldwide to develop rabies vaccine for post-exposure treatment to prevent rabies infection of humans.
The Wistar Institute has seed stock of the PM 1503 strain of rabies for use in production of vaccines. The Wistar seed stocks were grown on Vero cells, however, they can be adapted to grow on human diploid cells. This rabies vaccine seed stock has been licensed to select regional public health agencies and to for-profit companies who are developing vaccines for local distribution in selected countries, particularly China and India.
Novel Influenza Vaccine
There is a critical unmet need for new influenza vaccines that are easily produced and that induce protective immunity which is broadly cross-reactive with current and future epidemic virus strains. The external M2 protein (M2e) is highly conserved among human influenza type A viruses and M2e-specific antibodies have the potential to provide broad protective immunity across influenza A strains. However, the natural M2 protein is poorly immunogenic so that individuals exposed to influenza virus or immunized with current vaccines do not develop these broadly-protective antibodies.
Wistar researchers have developed novel multiple antigenic peptide constructs (MAPs) that induce M2e-specific antibodies in vivo. These MAPs consist of M2e and helper T cell determinants that are covalently linked. Mice immunized intranasally with MAPs exhibited significant production of M2e-specific antibodies as well as decreased morbidity after influenza virus infection. These MAPs, administered independently or in conjunction with whole virus, may be useful for developing an effective broadly protective influenza vaccine.
Vaccine for Prevention of Rabies Infections in Humans
Researchers at Wistar have developed a new rabies vaccine that has the potential to produce the long-lasting immunity to rabies that is required for an effective prophylactic vaccine. This vaccine is derived from recombinant chimpanzee adenovirus (rAdC68) that contains the DNA sequence of the rabies glycoprotein.
In non-human primates, this adenovirus-based vaccine produces long-lasting immunity after just one intramuscular injection, even in the presence of pre-existing immunity to human adenovirus. In ongoing studies, primates immunized with one does of the rAdC68 rabies vaccine showed no decline in serum antibody levels after six months. Wistar is seeking a partner to develop this vaccine for prevention of human rabies infection, either as a single vaccine or in combination with the current human diploid cell rabies vaccine. The adenovirus-based vaccine is less expensive to produce, maintain, and distribute than the current human rabies vaccines. This new immunization approach may make it feasible and cost-effective to administer prophylactic rabies vaccines to children in areas where rabies still commonly infects dogs and other companion animals.
Glycoprotein D Vaccine Adjuvant
Investigators at The Wistar Institute have developed a novel method for improving the immune response to vaccination by combining specific target antigens with a non-specific adjuvant. Wistar scientists have developed a new highly effective carrier protein adjuvant by fusing a Herpes simplex virus glycoprotein D (gD) sequence to the vaccine immunogen sequence. Glycoprotein D is a viral envelope protein that is normally expressed on the surface of cells infected with Herpes virus. The recombinant gD-antigen protein sequence preserves the structures responsible for interaction with an immune cell receptor, which is involved in controlling immune responses. The efficacy of recombinant gD as an adjuvant has been demonstrated with antigens from human papillomavirus (HPV), human immunodeficiency virus (HIV) and Influenza A virus.
In a mouse model of HPV-induced tumors, vaccination with a viral vector vaccine carrying HPV oncoproteins fused into the gD sequence induced strong immune response to the oncoproteins and resulted in significant tumor regression in mice with large tumor masses. Therefore, the gD adjuvant may be particularly useful for the development of a therapeutic HPV vaccine.
Unlike many carrier protein adjuvants that are targeted to intracellular compartments, the glycoprotein D-antigen proteins are expressed on the cell surface. This significantly reduces the likelihood of deleterious interaction with intracellular proteins such as those implicated in malignant transformation. The gD adjuvant may be applicable to multiple vaccine delivery methods. Vaccines in the form of naked DNA and viral vectors (adenovirus vectors and adeno-associated virus vectors) have been tested; testing of other vaccine delivery platforms are underway.
Vaccine to Treat Melanoma
Wistar scientists have developed novel peptides that mimic a BRAF mutation (BRAFV600E) which is expressed in a majority of melanomas. These mutated BRAF peptides stimulate T cell proliferation in melanoma patients who express HLA-2; this HLA type is expressed by about 50% of melanoma patients. Vaccines against BRAFV600E may induce both Class I and Class II-restricted lymphocyte responses and, as such, would be a useful tool for immunotherapy of melanoma.
In pre-clinical studies, 4 of 5 melanoma patients with BRAFV600E-positive lesions mounted a significant immune response (T cell proliferation) to stimulation with BRAFV600E peptide, thus demonstrating the feasibility of this immunotherapy in melanoma patients.
Method for Identifying Novel Cancer Antigens for Vaccine Development
A novel method for cloning MHC class II-dependent antigens from cancer cells has been developed for use in creating new cancer vaccines. Cancer antigens bound to MHC molecules on the surface of specialized antigen presenting cells (APCs) are recognized by CD4+ T-helper (Th) lymphocytes, which play a central role in providing protective immunity against cancer. New MHC class II-dependent cancer antigens therefore have significant promise as vaccine immunogens. They have previously been identified by artificially fusing libraries of cancer peptides with MHC molecules, expressing them in standard cell lines, and screening them for activation of CD4+ Th cells in culture. However, this method fails to recapitulate the complexity of antigen-MHC processing and presentation by APCs; requires prior knowledge of the MHC restriction element used by the Th cell and is very laborious. Wistar scientists have developed a phage method for expressing tumor cDNA libraries directly in APCs, without prior fusion to MHC. The APCs naturally process and present these tumor peptides on their surface, where they can be readily screened for CD4+ Th cell activation. The method has been used at Wistar to identify a novel Th cell antigen that is shared by melanomas and gliomas, and may be useful for the identification of other antigens for cancer vaccine development.
Novel Method for Inducing Tissue Repair
The healing of open wounds or replacement of lost tissue following injury, reconstructive surgery, transplantation or loss of blood supply requires the formation of new blood vessels as well as the growth of organized tissue. At present there are no reproducible procedures to speed up tissue growth, which requires formation of extracellular matrix and collagens by activated fibroblasts, and to induce new blood vessel formation. Recombinant tissue growth factors have been utilized for this purpose with limited success due to their short half-life in vivo, limited penetration into deeper tissue regions, and difficulty in application directly to naturally healing tissue.
Wistar scientists have constructed replication-defective recombinant adenoviruses which express platelet-derived growth factor B (PDGF-B), vascular endothelial growth factor (VEGF) as well as other growth factors. Clinical trials of these vectors to enhance wound healing in diabetic ulcers have been planned; additional applications of the technology, including the use of the vectors in tissue engineering are in development.
Method of Producing a Stem Cell-Like Melanocyte
Wistar scientists have invented a method of producing a dedifferentiated melanocyte, a premelanoma stem cell-like cell, by activating specific intracellular targets and selecting for a stem cell-like melanocyte capable of proliferating under stem cell growth conditions. These dedifferentiated melanocytes increase expression of neural crest-related genes and exhibit a decrease in pigmentation-related genes, compared to untreated controls. Stem cell-like melanocytes are capable of further differentiation into various cell types, assuming neuronal, smooth muscle cell, oligodendrocyte, melanocyte, or chondrocyte phenotypes. Wistar scientists have defined an oncogenic role for Notch signaling in melanocytes and highlight the potential for Notch inhibition to be used as a therapeutic approach for the treatment of melanoma.
Small Molecule Inhibitors of MicroRNA miR21
Micro RNAs are non-coding RNAs that appear to regulate gene expression. Numerous microRNAs are implicated in various diseases, including cancer, and provide new targets for therapeutic intervention.
Researchers at The Wistar Institute have developed a high-throughput screening (HTS) assay to identify inhibitors of oncogenic microRNAs. Using chemical libraries and the HTS assay, Wistar scientists (in collaboration with Dr. Alex Dieters at North Carolina State University) were able to identify several compounds capable of reducing the expression of the microRNA miR-21. This assay system may be used to find activators or inhibitors of other microRNAs.
Using the results of the HTS assay, the researchers designed and synthesized group of small molecules that selectively inhibit the microRNA miR21. MiR-21 functions as an anti-apoptopic agent in cancer cells and elevated levels of miR-21 has been associated with breast, ovarian, lung, and brain cancers and with heart failure. These compounds may be particularly useful in combination with low doses of standard chemotherapeutic drugs. Additional studies to demonstrate the in vivo efficacy of these compounds are planned.
Cancer Therapeutics Based on Novel Cyclopamine Analogs
Researchers at The Wistar Institute and The University of Pennsylvania have developed novel cyclopamine analogs with high potential as anti-cancer agents. Cyclopamine is a plant-derived alkaloid that is an effective inhibitor of the sonic hedgehog-GLI (SHH) signaling pathway which has been implicated in the development of a variety of cancers, including lung, prostate, and breast cancer. Cyclopamine has been demonstrated to reduce cancer cell proliferation, and to promote apoptosis in vitro and in vivo. However, cyclopamine is difficult and expensive to synthesize, limiting its usefulness as a therapeutic agent. Wistar and Penn researchers have developed a simple method for synthesizing cyclopamine analogs. Several of these analogs have been demonstrated to be effective inhibitors of sonic hedgehog signaling in vitro.
Design of Novel Molecules That Regulate Telomerase
We used the 3-dimensional structure of the catalytic subunit of telomerase to identify novel target binding sites essential for telomerase ribonucleoprotein assembly and activity. Telomerase adds multiple identical repeats of DNA (telomeres) to the 3’-end of eukaryotic chromosomes thus providing the genomic stability required for cell survival. There is now clear evidence that links telomerase to both cancer and aging. For example 90% of human cancers show high levels of activity of this enzyme when such activity is absent in most healthy tissues. The absence of telomerase activity (in adults) in healthy tissues leads to loss of ~50-100 bases of telomeric DNA with every cell division. When telomeres reach a critically short length, cells enter a permanent state of dormancy to prevent genomic instability, a process known as senescence, the hallmark of aging. Our goal is to identify compounds that regulate telomerase function that can be used to combat cancer and age related diseases.
Design of Novel Molecules that Regulate Sirtuins
Researchers at The Wistar Institute have identified and characterized a novel target binding site involved in regulating the activity of Sir2 proteins (or Sirtuins, the class III family of histone deacetylases, silent information regulator 2). Sirtuins deacetylate numerous cell proteins, thereby stimulating diverse biological functions related to metabolism and cell growth. Their activity has been linked to increased lifespan and calorie-associated effects on longevity. Compounds that target Sirtuins show promise in the treatment of diabetes, and diseases of degeneration and aging. Wistar scientists have identified and characterized a new molecular binding target, the nicotinamide binding site, which is expected to enable the development of new classes of small molecule Sirtuin modifiers. Experimental studies have validated the role of this binding site in the regulation of Sirtuin activity. Small-molecule compounds that target this site can act either as inhibitors of Sirtuins, or as effective Sirtuin activators, influencing a range of biological functions including DNA regulation, programmed cell death, neuronal protection, insulin signaling, and fat mobilization.
Suppression of Cell-Mediated Immunity by Down-regulation of Interleukin-12
The cytokine interleukin-12 (IL-12), which is produced during the immune response to a variety of stimuli, is critical for the development of cell-mediated immunity. Among its various functions in vivo, IL-12 is a potent inducer of interferon-gamma production by T lymphocytes and natural killer (NK) cells and is a co-factor in the mitogenic stimulation of these cell types. However, inappropriate cell-mediated immune responses, often accompanied by over-expression of IL-12, have been identified as having a key role in the development of a variety of autoimmune disorders, such as rheumatoid arthritis, multiple sclerosis, graft-versus-host disease, diabetes mellitus, systemic lupus erythematosus (lupus), and Crohn's disease. Reduction of IL-12 levels, for example by administration of antibodies to IL-12, has been effective in controlling autoimmune diseases in animal models.
Researchers at the Wistar Institute have determined that blockage of some complement receptors, including CD46 (the cellular receptor for measles virus) and CR3, can down-regulate production of IL-12 by monocytes. These findings support the well-known suppression of cell-mediated immunity following measles infection. Therefore, compounds that bind to these complement receptors, such as antibodies or other proteins, may be effective in reducing the production of IL-12 in autoimmune diseases and other conditions in which this cytokine is over-produced.
Therapeutic Applications of Interleukin-12 (IL-12)
IL-12, a cytokine that may be useful for treatment of certain diseases or for enhancing the immune response, was developed by researchers at The Wistar Institute. IL-12 is a heterodimeric protein, composed of a heavy chain (p40 subunit) and a light chain (p35 subunit) and was originally described as Natural Killer Stimulatory Factor (NKSF). Wistar is an owner of a series of U.S. and international patents on the IL-12 genes, IL-12 proteins (p35 and p40 subunits) and the uses of these compositions for treatment of a variety of conditions such as cancer and infection. Additionally, Wistar is an owner of a series of issued patents for the use of IL-12 as an adjuvant. These patents are available for license to companies developing products that incorporate IL-12.
BRAF35: The DNA-Binding Component of The BRCA2 Complex
Mutations of the human BRCA2 gene are associated with a very high risk of breast cancer, yet the majority of breast cancer patients do not carry any BRCA2 or BRCA1 mutations. Additionally the function of BRCA2 and its exact role in cell growth regulation have not yet been determined.
Wistar researches have determined that BRCA2 is part of a multiprotein complex and have identified a new protein, BRAF35 (BRCA2-Associated-Factor 35) that is the DNA-binding component of this complex. Similar to BRCA2, the expression of BRAF35 RNA is up-regulated in proliferating tissues. The BRCA2/BRAF35 complex is found associated with chromatin at the early stages of chromosome condensation. These findings suggest that the BRCA2/BRAF35 complex may have a key role in both DNA repair and cell cycle regulation. Thus BRAF35 and the BRCA2/BRAF35 complex may be useful targets for development of novel cancer therapeutics.
Bin1: A Novel Tumor Suppressor Protein
Bin1 is a novel gene product which offers utility as a diagnostic marker for malignant prostate cells. Bin1 (also known as amphiphysin II; amphiphysin-like protein, AMLP; and ALP) was identified through its ability to interact with and inhibit the oncogenic properties of the Myc oncoprotein and has features of a tumor suppressor protein. In preliminary clinical studies, monoclonal antibodies to Bin1 have been useful in the identification of malignant prostate cancer cells.
Bin1 structure suggests that it functions in a signal transduction pathway. This protein has features of a tumor suppressor that is widely lost in breast, prostate, and liver carcinoma. Detecting or manipulating Bin1 function in cells has potential applications for carcinoma and possibly other cancers involving Myc. Preliminary evidence suggests that Bin1 acts independently of p53. The Bin1 gene is located at a hotspot for deletion in metastatic prostate carcinoma and may be mutated in these and other tumor types, including cancers of breast, liver, and cervix.
Recent investigation of Bin1 indicates that it promotes cell differentiation (in the absence of activated Myc) and apoptosis (in its presence). Bin1 has several features of a tumor suppressor: it suppresses the oncogene activity of Myc, inhibits the growth of tumor cell lines, and has been implicated in cell differentiation and apoptosis. Thus, Bin1 is a molecule that is functionally deleted in cancer and that may have important roles in normal cell regulation.
Novel Mitotic Checkpoint Gene
Wistar researchers have identified a new mitotic checkpoint gene, chfr, that is expressed in normal tissues but is either absent or mutated in many cancer cell lines. When dividing cells that express the wild-type chfr are exposed to nocodazole, which inhibits microtubule formation, they become arrested at prophase, while cells that lack a functional chfr gene proceed through the cell cycle and division.
The restoration of normal chfr function may be a useful target for cancer therapy. Loss of chfr function in tumor cells may be indicative of cancers that are sensitive to chemotherapy.
Method for Solid-Phase Amplification of DNA Using Multiarrays
Amplification of DNA is a common first step in nucleic acid diagnostics, particularly when testing for the presence or absence of an organism or a given nucleic acid sequence in a sample. For simplicity and sensitivity, DNA amplification assays have been developed wherein either the nucleic acid template or the primer is attached to a solid phase. These assays are particularly suitable for screening multiple samples in an automated system.
Wistar researchers have developed an improved assay for detecting the presence of a given test DNA in a sample, wherein the 5¹ and 3¹ primers for the test DNA are irreversibly bound to a solid phase and the DNA in the sample is reversibly bound to the solid phase. When these components are incubated under conditions that promote nucleic acid amplification, no signal is detectable unless the sample contains the test DNA. This assay may be useful for high throughput testing for pathogens (e.g. microorganisms or DNA viruses) or for detecting mutations in test samples, e.g. in multiarray systems.
Device and Method for Fractionation of Proteomes and Complex Biological Mixtures
Two-dimensional electrophoresis, which is widely used for quantitatively comparing changes in protein profiles (proteomes) of cells, is not capable of resolving all of the thousands of proteins in eukaryotic cell samples. While two-dimensional electrophoresis may resolve up to 2,000-3,000 individual proteins, most eukaryotic proteomes have over 10,000 protein components.
To address this limitation, Wistar researchers have developed a new device and method to separate proteomes and other complex mixtures into multiple discrete fractions. The subsequent fractions are then suitable for analysis by other analytical methods, such as two-dimensional electrophoresis or liquid chromatography/mass spectroscopy. Using this new device and method to pre-fractionate samples before analysis permits increase in overall protein loads (with a resulting increased sensitivity to proteins present in low concentrations), improved resolution of proteins or other components, and greater dynamic range compared to currently available pre-fractionation methods.
The new Wistar device is also useful for separation of components of other complex mixtures (e.g. nucleic acids, biological fluids) prior to analysis. The Wistar device may be used as a component of an automated system.
Stabilized Production of Ribozymes, iRNA, mRNA and Other Gene Products
Wistar researchers have developed a novel method to stabilize introns that offers the potential for stable production of ribozymes, iRNA, mRNA and related gene products. Using the Wistar technology, stabilized intron compositions are prepared with a sequence from the LAT intron of the HSV-1 virus. Polynucleotide encoding sequences are then ligated into a stabilized intron, resulting in a 105 -fold increase in stability. Unlike typical introns that rapidly degrade within seconds after excision, these stabilized introns have a half life of 24 hours (Thomas et al. 2002. J. Virol., 76, 532-540). The increased stability has been demonstrated in several models, indicating that the method is neither cell type- nor species-specific and can be applied to both eukaryotic and yeast expression systems.
These methods and compositions are useful for:
- Stabilizing a gene transcript to permit enhanced expression, and increased production, of a recombinant gene product.
- Production of vectors and gene products for research reagents, markers of gene production, and diagnostic and therapeutic compositions.
- Markers for determining whether a gene of interest is being transcribed, particularly for identification of genes transcribed only in a selected stage of the cell cycle.
RNA Editing Enzyme and Methods of Use Thereof
The ADAR (adenosine deaminase that acts on RNA) enzyme, previously known as DRADA, is involved in the RNA editing of glutamate-gated ion channels and the serotonin-2C receptor. This novel enzyme is implicated in central nervous system disorders characterized by abnormal neuronal transmission and intracellular signaling in mammalian brain such as stroke, schizophrenia, depression, and substance abuse.
A Method of Delivering Genes to the Central Nervous System
One of the limitations of gene therapy has been the lack of methods to effectively deliver genes to the central nervous system. Herpes simplex virus (HSV) is a neurotropic virus that naturally establishes latent infections in the peripheral nervous system and the central nervous system of humans. Wistar researchers have developed a novel method for using neurotropic HSV to deliver genes of therapeutic value. Additionally, the Wistar technology promotes long-term expression of the therapeutic genes, which is necessary for treatment of a genetic defect.
The Wistar technology has several advantages over other methods of delivering genes to the central nervous system (CNS). First, no helper virus is needed, and second, long-term expression of the gene is achieved using a naturally-occurring viral promoter. This technology has been used successfully in vivo in a mouse model. In this study, the gene encoding §-glucorinodase was delivered to the CNS and long-term expression (over 4 months) of the gene product was obtained.
Multipotent Adult Stem Cells from Human Hair Follicles
Scientists at The Wistar Institute and the University of Pennsylvania have developed a novel strategy for isolating, growing, and differentiating broadly multipotent adult stem cells from human hair follicles. Adult stem cells, like embryonic stem cells, can differentiate into multiple types of function-dedicated daughter cells, such as nerve or muscle cells. However, most adult stem cells are restricted to producing cell types from only one tissue type (lineage). Researchers at Wistar, in collaboration with researchers at the University of Pennsylvania isolated a unique adult stem cell from human hair follicles, which is capable of producing daughter cells of multiple tissue lineages. The human hair follicle stem cell is a rare, but permanent, inhabitant of the skin follicular microenvironment, and exhibits features that are characteristic of highly-adaptable embryonic stem cells. This technology allows hair follicle stem cells to be collected, maintained in renewable culture, and stimulated to produce daughter cells of multiple classes, including functional skin cells, muscle cells, and nerve cells.
Monoclonal Antibodies to ADAR
Adenosine Deaminase Acting on RNA (ADAR) is a member of a gene family that is involved in site-selective RNA editing that changes adenosine residues to inosine. Three vertebrate ADAR gene family members, ADAR1, ADAR2,and ADAR3, have been identified. ADAR1 and ADAR2 form a stable enzymatically active homodimer complex and ADAR3is a monomeric, enzymatically inactive form.
anti-ADAR1 (made against dsRNA binding domain region)
Western Blot & Immunohistochemistry
References: Q. Wang et al. (2000) Requirement of the RNA Editing Deaminase ADAR1 Gene for Embryonic Erythropoiesis, Science Vol. 290, 1765-1768
Dan-Sung C. Cho et al. (2003) Requirement of Dimerization for RNA Editing Activity of Adenosine Deaminases Acting on RNA, The Journal of Biological Chemistry Vol. 278 No. 19, 17093-17102
anti-ADAR2 (made against N-terminal region)
Western Blot & Immunohistochemistry
References: Dan-Sung C. Cho et al. (2003) Requirement of Dimerization for RNA Editing Activity of Adenosine Deaminases Acting on RNA, The Journal of Biological Chemistry Vol. 278 No. 19, 17093-17102
anti-ADAR3 (made against N-terminal region)
References: Chun-Xia Chen et al. (2000) A third member of the RNA-specific adenosine deaminase gene family, ADAR3, contains both single- and double-stranded RNA binding domains, RNA Vol. 6, 755-767
Monoclonal Antibodies For Influenza Research
Characteristics: These antibodies specifically bind to the identified antigens on influenza viruses. All of the antibodies are useful for ELISA and some of the antibodies are also useful for Western blot. The specificity of the various antibodies that have been characterized is as shown:
2, 3, 5, 7-8, 11
Reacts with 1934-1957 strains
Later strains nd
H3 (Aichi/68, Mem/72, others nd)
NP (x-reacts with PR8 and Aichi)
NP (x-reacts with early H1N1 strains)
1. Yewdell, J.W., Frank, E., and Gerhard, W. J. Immunol. 126: 1814-1819, 1981.
2. Gerhard, W., Yewdell, J., Frankel, M., and Webster, R.G. Nature 290: 713-717, 1981.
3. Caton, A.J., Brownlee, G.G., Yewdell, J.W., and Gerhard, W. Cell 31: 417-427, 1982.
4. Jackson, D.C., Murray, J.M., White, D.O., and Gerhard, W. Infec. and Immunity 37: 912-918, 1982.
5. Staudt, L.M. and Gerhard, W. J. Exp. Med. 157: 687-704, 1983.
6. Nohinek et al. Virology 143: 651-656, 1985.
7. Kavaler J et al. J. Immunol. 145: 2312-2321, 1990.
8. Caton, A.J., Stark, S.E., Kavaler, J., Staudt, L.M., Schwartz, D., and Gerhard, W. J. Immunol. 147: 1675-1687, 1991.
9. Mozdzanowska et al. Virology 254: 138-146, 1999.
10. Zhang, M., Zharikova, D., Mozdzanowska, K., Otvos, L., and Gerhard, W. Mol. Immunol. 43: 2195-2206, 2006.
11. Mozdzanowska, K., Feng, J., Eld, M., Zharikova, D., Gerhard, W. Virology (in press), 2006.
Antibodies That Detect a Novel Tumor Supressor (BIN1) Implicated in Human Carcinomas and Apoptosis
Wistar researchers have developed polyclonal and monoclonal antibodies (PAb 99Pst and MAbs 99D through 99I) that bind to BIN1, a novel tumor suppressor protein that interacts with and inhibits the oncogenic activity of the MYC oncoprotein. BIN1 is functionally deleted in carcinomas of the breast, colon, lung, liver and cervix and has been implicated in programmed cell death (apoptosis). Apoptosis is an active process of physiological cell suicide that is critical to normal tissue homeostasis but that does not function properly in tumor cells. Work aimed at defining the genetic control of apoptosis and understanding its dysfunction in cancer has centered on the MYC oncoprotein, because it can promote either cell proliferation or apoptosis. Wistar scientists theorize that BIN1 may activate or facilitate apoptosis, thereby overcoming MYC's malignant growth activity. The antibodies generated by Wistar scientists are useful for research aimed at understanding the role of the BIN1 protein in the genetic events associated with apoptosis.
Monoclonal Antibodies Against BAP1 (BRCA1-Associated Proteins)
Wistar researchers have identified a novel protein, BAP1, which is a nuclear-localized, ubiquitin carboxyl-terminal hydrolase that binds to the wild-type RING finger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. Murine Bap1 and Brca1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3 and rearrangements, deletions and missense mutations of BAP1 have been found in lung carcinoma cell lines and in primary breast tumor samples. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified.
Diagnostic Antibodies Against: T-Cells, NK Cells, Receptors and Cytokines
monocytes (55,000 MW antigen)
granulocytes from precursors cells to mature polymorphs (145,000 MW glycoprotein and glycolipids) also reacting with colon-carcinoma and other tumors and with mouse embryonic antigen (SSEA1) similar to My1
Granulocytes from promyelocytes to polymorphs, monocytes and natural killer cells (similar to OK1, Mac1)
Helper T cells (similar to OKT4, Leu 3, T4)
cells (67,000 MW glycoprotein)
Cytotoxic/suppressor T cells, NK cells (33K)
Anti-glycoprotein IIIa on platelets
21K antigens on platelets and some lymphocytes, coded for by chromosome 12
Immune (gamma) interferon
Tumor necrosis factor
Tumor necrosis factor
Tumor necrosis factor
Monoclonal Antibody BR64 for the Identification of BRCA1
Analysis of the underlying genetic component of breast cancer localized the heritable component of this disease to a gene, the breast cancer susceptibility gene (BRCA1), located on chromosome 17 (at q21). As expected, the BRCA1 gene is mutated in the majority of heritable breast cancers and the mutations within the coding region occur throughout the gene with little apparent clustering. The majority of these mutations lead to shortened BRCA1 gene products, thus describing the need for a marker of the amino-terminus of the protein. Wistar scientists have isolated antibodies secreted by hybridomas derived from the immunization of mice with purified polypeptide representing a 100-amino acid segment of the amino terminus of the BRCA1 gene product. Hybridomas with restricted reactivities were selected, subcloned and the secreted antibodies have been tested for anti-BRCA1 reactivity in a variety of assays. The antibody demonstrates an extreme specificity to the BRCA1 gene product by immunoprecipitation analysis. The Wistar-developed antibody described herein provides an excellent tool for the identification of both the full-length and mutated (shortened) forms of BRCA1.
Monoclonal Antibodies Against Wilms' tumor (WT1)
Scientists at The Wistar Institute have created monoclonal antibodies that react with the Wilms' tumor (WT1) protein. These antibodies can be used diagnostically in a variety of malignancies that over-express the WT1 protein. In particular, they can be used for distinguishing mesotheliomas from other tumors or nonmalignant conditions that affect the pleura. In addition, the antibodies may be used to monitor disease activity in acute leukemias.
The wt1 gene was originally identified as a candidate tumor suppressor gene by researchers investigating the causes of Wilms' tumor, a childhood kidney tumor. The gene codes for the WT1 protein, which has the structural features of a DNA-binding transcription factor. The WT1 protein has been shown to be over-expressed in a variety of tumors.
Three monoclonal antibodies have been developed, designated 6F-H2, 6F-H7, 6F-H17, and the isotype for each is IgG1.
The hybridomas producing these antibodies resulted from the fusion of spleen cells from mice immunized with a peptide comprising the N-terminal 173 amino acids of the WT1 protein and cells of the myeloma fusion partner P3x63AG8/653. This N-terminal region of the WT1 protein has little sequence homology to any other known protein. The resulting antibodies do not cross react with other transcription factors containing the zinc finger motif. Consequently, these monoclonal antibodies are more specific than either polyclonal or monoclonal antibodies to WT1 that have been produced by other researchers.
Monoclonal Antibodies that Bind to Nerve Growth Factor Receptor (NGFR)(ME20.4 and ME82.11)
Nerve growth factor is important for regulation of development of sympathetic and sensory neurons and other neural crest-derived cell types. MAbs ME20.4 and ME82.11 bind to the low affinity NGFR of human origin. Using these MAbs, we have carefully defined the biochemical nature of the NGF receptor and its distribution among normal and transformed neural crest tissues.
Reference: Ross, et al., Characterization of nerve growth factor receptor inneural crest tumors using monoclonal antibodies, Proc. Natl. Acad. Sci. 81: 6681-6685, 1984.
Monoclonal Antibodies Against bFGF
Wistar scientists have developed two MAbs (designated 8 and 11) against bFGF (basic fibroblast growth factor), which is a major mitogen for cells of various embryological origin. It is important for development, tissue regeneration, cancer, and inflammatory diseases. Its detection is highest in activated tissues. In certain cancer such as melanomas, bFGF is an autocrine growth factor. Neutralization of its activity leads to cessation of growth.
Reference: Mark Nesbit, Heike KE Nesbit, Jean Bennett, Thomas Andl, Mei-Yu Hsu, Emma Dejesus, Michelle McBrian, Abha R. Gupta, Stephen L. Eck, Meenhard Herlyn (1999) "Basic fibroblast growth factor induces a transformed phenotype in normal human melanocytes" Oncogene Vol 18, 6469-6476.
Monoclonal Antibody A32 Against MEL-CAM/MUC18
Cell surface melanoma-associated antigens can mediate cell-cell or cell-substrate adhesion, signal transduction, proteolysis, or immune recognition and play a key role in determining invasive and metastatic competence of the tumor cells. MAb A32 binds to endothelial and melanoma cells and detects a 113 Kd cell surface adhesion receptor of the immunoglobulin supergene family. The Mel-CAM/MUC18 antigen is a cell surface molecule involved in heterotypic adhesion. The ligand for Mel-CAM/MUC18 has not yet been identified. Its characterization will be important for endothelial cell adhesion during inflammation.
Monoclonal Antibody 452 Against Aminopeptidase N (CD13)
Anti-Human Tenascin Monoclonal Antibodies 300-1, 300-3, 300-2, 302-1, 302-9
Monoclonal Antibody Against Melanotransferrin (ME D63)
Monoclonal Antibody SAP for the Detection of the ß3 Subunitof the Vitronectin Receptor
Monoclonal Antibody to Phosphorylated p53
The gene for the tumor suppressor protein p53 encodes a nuclear phosphoprotein that is altered by mutation or deletion in about 50% of human tumors. When single point mutations modify p53 structure and function, post-translational modifications of mutated p53 molecules are expected to interfere even more strongly with its function of controlling cell growth and division. The most prominent post-translational modification of p53 is phosphorylation at one or more of many sites along the protein. Wistar researchers developed a double-phosphorylated peptide that is phosphorylated at the Ser378 and Ser392 sites of human p53. This peptide was then used to generate a highly sensitive monoclonal antibody (mAb p53-18) to p53 protein. This antibody can be used to distinguish the phosphorylated from the non-phosphorylated forms of p53. Since sera from cancer patients preferentially label the double-phosphorylated p53 peptide, the mAb p53-18 may be useful in developing cancer diagnostics.
Monoclonal Antibody for Delineating CD8+ T Cell Population Subsets (C1.7)
CD8+ T cells have been traditionally classified as cytotoxic lymphocytes. However, a number of studies have suggested that the CD8+ T cell population is in fact functionally heterogeneous. For example, research has demonstrated that cytotoxic or regulatory (suppressor) activities and an ability to release distinct cytokine profiles distinguish subsets within the CD8+ T cell population. Specifically, research has classified CD8+ T cells as Type I (producing IFN, GMCSF, and TNF) or Type II (producing IL-4, IL-10, GMCSF, and TNF).
Antibody: Wistar scientists have developed a monoclonal antibody designated C1.7 that recognizes a novel surface marker (p38) expressed on all NK cells and 50% of peripheral CD8+ T cells. The C1.7+ CD8+ T Cell subset has been shown to have higher cytotoxic activity and predominantly release a type I cytokine profile. The C1.7- subset has a lower cytotoxic activity and some of the cells may release a type II cytokine profile. On NK cells, C1.7 is a signaling molecule that when crosslinked, induces cytotoxic activity and release of cytokines.
Monoclonal Antibodies That Bind to Cell Surface Antigens on Human Embryonal Carcinoma Cells
Wistar researchers have developed monoclonal antibodies against five cell surface markers that are useful in the identification and characterization of human embryonic stem cells and monitoring their differentiation. Investigation of the function and expression of developmentally regulated cell surface antigens may yield clues to the control of cell differentiation, not only during embryogenesis but also during oncogenesis. These monoclonal antibodies bind to cell surface antigens SSEA-1 (stage-specific mouse embryonic antigen), SSEA-3, SSEA-4, TRA-1-60R, and TRA-1-81.