Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0006142 (breast cancer)
 160,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (+/-1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (beta-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.
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PMID:Characterization of engineered anti-p185HER-2 (scFv-CH3)2 antibody fragments (minibodies) for tumor targeting. 1518 22

The display of recombinant antibody fragments on the surface of filamentous phage mimicks B cells and is therefore a technology ideal to generate antibodies against any potential target antigen in vitro. In order to obtain tumor specific, high-affinity single chain antibody fragments (scFv), it has been speculated that lymph node tissue from cancer patients infiltrated with activated B cells must be a valuable source of antibody V-genes. The aim of this study was to generate a human scFv-phage library from lymph nodes of patients with breast cancer and to develop a stringent depletion and selection protocol in order to isolate specific single chain antibodies recognizing potentially new antigens in breast cancer. The amplification of the V-genes cloned from regional lymph node tissue and their assembly to single chain variable fragments was optimized in terms of library size and diversity. A large set of degenerated primers, annealing to all known V-gene families, was designed and used under optimized PCR conditions. The amplified V-genes were genetically fused in all possible combinations and cloned into a phagemid vector. Depletion and selection on mammary epithelial and primary breast carcinoma cell lines, respectively led to the isolation of a breast cancer cell line specific scFv (BCK-1 scFv) from this patient-derived scFv-phage display library as demonstrated in polyclonal and monoclonal ELISA, using immobilized cell membrane fractions of the indicated cell lines. A new recombinant breast cancer cell line specific antibody based on V-genes derived from reactive B-lymphocyte-infiltrated lymph nodes of patients with breast cancer was isolated via phage display, performing stringent depletion and selection protocols. We believe that this combination of antibody V-gene source and elaborated phage display depletion and selection strategy will be successful for the retrieval of numerous other recombinant, tumor specific antibody fragments.
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PMID:Construction of phage display libraries from reactive lymph nodes of breast carcinoma patients and selection for specifically binding human single chain Fv on cell lines. 1537 9

The tumor-associated antigen MUC1 is a cell surface mucin that is expressed on the apical surface of most glandular epithelial cells, including the ducts of the breast, ovary, pancrease, lung and colon. During malignancy, epithelial tissues regularly display elevated levels of MUC1 in a non-polar fashion and in an underglycosylated form, exposing cryptic peptide and carbohydrate epitopes. As such, MUC1 is regarded a potential target for immunotherapeutical intervention. Murine monoclonal H23 antibody specifically recognizes a MUC1 epitope on the surface of human breast cancer cells. We describe the cloning of the variable domains of H23 and their expression in (Escherichia coli) E. coli as maltose-binding protein-scFv (MBP-scFv) fusions. We humanized H23 and evaluated the binding properties of the murine and the humanized recombinant forms, which were similar in affinity and specificity, but lower in apparent affinity in comparison to the original monoclonal IgG. We mapped the epitope of humanized H23 by affinity-selecting a phage-displayed random peptide library on humanized H23 scFv-displaying bacteria. Our results show that humanized H23 binds an epitope corresponding to the MUC1 tandem repeat and an additional epitope not related to MUC1. These epitopes are competitive, bound with similar affinities and are recognized by the original murine H23 monoclonal antibody as well.
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PMID:Humanization and epitope mapping of the H23 anti-MUC1 monoclonal antibody reveals a dual epitope specificity. 1548 44

The successful application of cancer gene therapy has been hampered by the low efficiency of in vivo gene delivery by currently used replication-defective vectors. Accordingly, considerable efforts are now being directed toward development and use of vectors capable of replicating in cancer cells. However, for replicating retroviruses, insertion of additional reading frames into the viral genome often resulted in the generation of unstable viruses. Here, we report a novel concept for the generation of replication-competent murine leukemia virus (MLV) vectors capable of mediating the secretion of soluble therapeutic proteins from infected cells. As a proof of principle, we inserted transgene regions encoding either a single-chain variable region fragment (scFv), here, the laminin-specific L36-scFv, or the T-cell-specific 7A5-scFv, or the cytokine GM-CSF into the MLV envelope (env) gene after +1 codon of the envelope (Env) protein, followed by a sequence specifying a furin protease cleavage site. The resulting viruses, termed L36-furin-A, 7A5-furin-A and GMCSF-furin-Mo, respectively, infected a variety of human cell lines, including HMEC-1 (endothelial), A301 (lymphoid), MDA-MB231 and MDA-MB468 (breast cancer) and HT1080 (fibrosarcoma) cells. Western blot analysis of conditioned culture medium from HT1080 cells infected by replicating L36-furin A, as an example, revealed that more than 90% of the Env fusion protein molecules were indeed intracellularly cleaved. After 5 days of infection, up to 3-4 mug/ml of soluble L36-scFv accumulated in the supernatant of HT1080 cells. The eukaryotically produced L36-scFv and 7A5-scFv were able to recognize their native antigens with high avidity, as assessed by ELISA and flow cytometry. Furthermore, the replicating viruses were genetically stable for more than 12 cell passages. In conclusion, a new generation of replication-competent retroviral vectors capable of mediating long-term and efficient secretion of therapeutic proteins suitable for cancer therapy was generated.
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PMID:Replicating retroviral vectors mediating continuous production and secretion of therapeutic gene products from cancer cells. 1569 9

There is an ever-growing interest in plant molecular farming as a system for producing valuable recombinant pharmaceutical molecules, such as single-chain variable fragments, on an industrial/agricultural scale and it appears that it is going to become a reality. Human epidermal growth factor receptor-2 (HER2) is an oncogene involved in abnormal cell growth in breast cancer and is considered for the development of new cancer therapies. We describe here the cloning and expression of a scFv-alpha HER2 that has been produced in Escherichia coli and in plants using both stable and transient systems in tobacco and Nicotiana benthamiana. Single-chain antibodies (ScFvs) extracted and purified from E. coli and plant tissues were tested for functionality and specificity by flow cytometry analysis on several cell lines and showed positive results when used on breast cancer slides coming from human frozen tissues. As a result, scFv-alpha HER2 represents a good opportunity for application and use in diagnosis and therapy.
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PMID:Expression of single-chain antibodies in transgenic plants. 1573 49

Breast cancer is the second most-common cause of cancer death in women in the United States. Although more than 60% of patients can now be cured by initial treatment, the rest, although perhaps receiving palliation with currently available therapy, will die of their disease. Early detection of micrometastasis and improved treatment strategies are needed. Monoclonal antibody (mAb)-based imaging and tumor targeted therapy holds the potential to impact these problems. The most significant results of systemically administered antibody-based radiopharmaceuticals for detection and targeted therapy (radioimmunotherapy [RIT]) of breast cancer give strong evidence that this potential can be realized. Interest in immunoimaging recently has focused on small mAb modules used with 18F, 64Cu, or 124I to detect minimal disease in breast cancer by positron emission tomography or single-photon emission computed tomography. Reported therapy trials in advanced breast cancer have yielded objective responses and minimal toxicity. These studies have spanned several radionuclides as well as several mAb, fragments and approaches, including dose intensification with bone marrow support; combined therapy with other modalities (ie, CM-RIT); biodegradable peptide linkers; and pretargeting. RIT evaluated in clinical breast cancer trials has delivered as much as 4000 cGy to metastatic breast cancer per therapy dose with marrow stem cell support. Preclinical studies have demonstrated further promising strategies for breast cancer. RIT studies must address the key issue: enhancing the therapeutic index (tumor effect verses most sensitive normal tissue (bone marrow) effect). Approaches now include newly engineered mAb, scFv modular constructs, blood clearance on demand, enhanced pretargeting, applications of both alpha and beta emitting radionuclides, and combination therapy using molecular triggers for therapeutic synergy. These strategies for detection and treatment of metastatic breast cancer should lead to notable clinical impact on management and cure of breast cancer.
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PMID:Radioimmunodetection and therapy of breast cancer. 1576 77

The aim of this study is to construct a phage display single-chain variable fragment (scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the library. The BALB/C mice were immunized with MCF-7 cells. Total RNA of spleens was isolated. The heavy-chain (VH) and light-chain variable region genes (VL) of the antibodies were amplified by RT-PCR and joined into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The assembled scFv fragments were cloned into the phagemids(pCANTAB5E) and the recombinant phagemids were used to transform competent E. coli TG1. The transformed TG1 cells were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The scFv fusion proteins were displayed on the surfaces of the recombinant phages. A phage display antibody library of repertoire of 1.2 x 10(6) clones was constructed. The specific antibodies against MCF-7 cells were enriched by 75 times after five rounds of affinity selection. Ten recombinant phages clones that exhibited specific binding to MCF-7 cells were identified. The specificity of those phage clones was analyzed by reactivity against HepG2 cells and Hela cells by ELISA. One of the selected phage clones against MCF-7cells was used to infect E. coli TOP10 to produce the soluble scFv antibodies after induction with IPTG. The strategy of construction and screening of antibody library directed against the whole tumor cells described in this report should be generally applicable to generate tumor cell-specific antibodies.
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PMID:[Construction of phage display antibody library to MCF-7 cells and screening of single-chain antibodies against breast cancer cells]. 1597 87

We have recently described the in vivo properties of an iodinated anti-p185HER2 engineered antibody fragment [minibody (scFv-C(H)3)2; 80 kDa], made from the internalizing 10H8 monoclonal antibody. Although the 10H8 minibody showed excellent binding to the target in vitro, only modest tumor uptake [5.6 +/- 1.7% injected dose per gram (ID/g) of tissue] was achieved in nude mice bearing MCF7/HER2 breast cancer tumors. Here, in an attempt to improve targeting, the 10H8 minibody was conjugated to 1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA), radiometal labeled, and evaluated in vivo. The tumor uptake of 111In-DOTA 10H8 minibody was 5.7 +/- 0.1% ID/g, similar to the radioiodinated 10H8 minibody. However, in addition to the expected liver clearance, the kidneys had unexpectedly high activity (34.0 +/- 4.0% ID/g). A minibody derived from a second anti-p185(HER2) antibody (trastuzumab; hu4D5v8) was also made. Tumor uptakes, evaluated by quantitative microPET using 64Cu-DOTA hu4D5v8 minibody, were 4.2 +/- 0.5% ID/g. Furthermore, in non-tumor-bearing mice, 111In-DOTA hu4D5v8 minibody exhibited similar elevated uptake in the kidneys (28.4 +/- 6.5% ID/g). Immunohistochemical staining of kidneys from non-tumor-bearing mice showed strong specific staining of the proximal tubules, and Western blot analysis of kidney lysate confirmed the presence of cross-reactive antigen. To further improve tumor uptake and normal tissue distribution, a larger hu4D5v8 fragment [(scFv-C(H)2-C(H)3)2; 105 kDa] was made, engineered to exhibit rapid clearance kinetics. This fragment, when evaluated by microPET, exhibited improved tumor targeting (12.2 +/- 2.4% ID/g) and reduced kidney uptake (13.1 +/- 1.5% ID/g). Thus, by manipulating the size and format of anti-p185(HER2) antibody fragments, the kidney activity was reduced and high or low expression of p185HER2 in xenografts could be distinguished by microPET imaging.
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PMID:Optimizing radiolabeled engineered anti-p185HER2 antibody fragments for in vivo imaging. 1599 69

Breast cancer is a leading cause of cancer-related deaths in women worldwide. Although tumorectomy, radiotherapy, chemotherapy and hormone replacement therapy have been used for the treatment of breast cancer, there is no effective therapy for patients with invasive and metastatic breast cancer. Immunotherapy may be proved effective in treating patients with advanced breast cancer. Breast cancer immunotherapy includes antibody based immunotherapy, cancer vaccine immunotherapy, adoptive T cell transfer immunotherapy and T cell receptor gene transfer immunotherapy. Antibody based immunotherapy such as the monoclonal antibody against HER-2/neu (trastuzumab) is successfully used in the treatment of breast cancer patients with over-expressed HER-2/neu, however, HER-2/neu is over-expressed only in 25-30% of breast cancer patients. Cancer vaccine immunotherapy is a promising method to treat cancer patients. Cancer vaccines can be used to induce specific anti-tumor immunity in breast cancer patients, but cannot induce objective tumor regression. Adoptive T cell transfer immunotherapy is an effective method in the treatment of melanoma patients. Recent advances in anti-tumor T cell generation ex vivo and limited clinical trial data have made the feasibility of adoptive T cell transfer immunotherapy in the treatment of breast cancer patients. T cell receptor gene transfer can redirect the specificity of T cells. Chimeric receptor, scFv(anti-HER-2/neu)/zeta receptor, was successfully used to redirect cytotoxic T lymphocyte hybridoma cells to obtain anti-HER-2/neu positive tumor cells, suggesting the feasibility of treatment of breast cancer patients with T cell receptor gene transfer immunotherapy. Clinical trials will approve that immunotherapy is an effective method to cure breast cancer disease in the near future.
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PMID:Breast cancer immunotherapy. 1622 67

We used atomic force microscopy to measure the binding forces between Mucin1 (MUC1) peptide and a single-chain variable fragment (scFv) antibody selected from a scFv library screened against MUC1. This binding interaction is central to the design of molecules used for targeted delivery of radioimmunotherapeutic agents for prostate and breast cancer treatment. Our experiments separated the specific binding interaction from nonspecific interactions by tethering the antibody and MUC1 molecules to the atomic force microscope tip and sample surface with flexible polymer spacers. Rupture force magnitude and elastic characteristics of the spacers allowed identification of the rupture events corresponding to different numbers of interacting proteins. We used dynamic force spectroscopy to estimate the intermolecular potential widths and equivalent thermodynamic off rates for monovalent, bivalent, and trivalent interactions. Measured interaction potential parameters agree with the results of molecular docking simulation. Our results demonstrate that an increase of the interaction valency leads to a precipitous decline in the dissociation rate. Binding forces measured for monovalent and multivalent interactions match the predictions of a Markovian model for the strength of multiple uncorrelated bonds in a parallel configuration. Our approach is promising for comparison of the specific effects of molecular modifications as well as for determination of the best configuration of antibody-based multivalent targeting agents.
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PMID:Dynamic force spectroscopy of parallel individual Mucin1-antibody bonds. 1626 47


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