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    • 4. 发明申请
    • METHOD FOR THE GENERATION OF T CELL HYBRIDOMA FOR CLONING AND ANALYSIS OF RECEPTORS USING ENGINEERED MEASLES VIRUS FUSOGENIC GLYCOPROTEINS
    • 产生T细胞杂交瘤克隆的方法和使用工程化的病毒的融合蛋白
    • WO2018033544A1
    • 2018-02-22
    • PCT/EP2017/070684
    • 2017-08-15
    • MAX-DELBRÜCK-CENTRUM FÜR MOLEKULARE MEDIZIN IN DER HELMHOLTZ-GEMEINSCHAFT
    • BUNSE, MarioEDES, InanUCKERT, WolfgangBUCHHOLZ, Christian J.SCHNEIDER, Irene
    • C12N5/0783C07K14/12
    • C12N5/163C07K14/12C07K14/705C07K2319/33C12N2510/04
    • The invention relates to the field of immunology and immunotherapy, in particular, to adoptive T cell therapy of cancer utilizing T cell receptor (TCR)-engineered T cells. The invention provides novel methods and tools for identification and cloning of TCR, which are also applicable for identification of other receptors such as B cell receptors or antibodies. The invention provides immortalized cell lines able to induce subset specific (e.g. CD4 + or CD8 + specific) hybridization during co-culture, e.g., with primary lymphocytes of mice or men. The immortalized cells are engineered to express two mutated glycoproteins derived from the paramyxovirus family, namely hemagglutinin (H) and fusion (F) or a derivative thereof. H is a chimeric protein not able to bind to its natural ligands, fused to a targeting ligand capable of specifically binding to a specific cell surface antigen such as a lymphocytic subset-marking ligand (e.g. CD4 or CD8); fusion (F) mediates the fusion of cellular membranes of the immortalized cell line and cells, e.g., primary lymphocytes, H has bound to. The methods of the invention comprise preparing a hybridoma cell expressing a receptor, comprising steps of co-culturing a cell expressing a specific cell surface antigen and said receptor with the specific immortalized cells of the invention. The invention also relates to the hybridoma cells obtainable by said method.
    • 本发明涉及免疫学和免疫疗法领域,具体涉及利用T细胞受体(TCR) - 工程化T细胞的癌症的过继性T细胞治疗。 本发明提供了鉴定和克隆TCR的新方法和工具,其也可用于鉴定其他受体如B细胞受体或抗体。 本发明提供了能够在共培养过程中,例如与小鼠或男性的原代淋巴细胞共同培养时诱导子集特异性(例如CD4 + / CD8 +特异性)杂交的永生化细胞系。 工程化永生化细胞以表达衍生自副粘病毒科的两种突变糖蛋白,即血凝素(H)和融合蛋白(F)或其衍生物。 H是不能与其天然配体结合的嵌合蛋白,与能够特异性结合特定细胞表面抗原如淋巴细胞亚群标记配体(例如CD4或CD8)的靶向配体融合; 融合体(F)介导永生化细胞系和细胞(例如H已结合的初级淋巴细胞)的细胞膜融合。 本发明的方法包括制备表达受体的杂交瘤细胞,其包括将表达特定细胞表面抗原的细胞和所述受体共同培养于本发明的特定永生化细胞的步骤。 本发明还涉及通过所述方法可获得的杂交瘤细胞。
    • 10. 发明申请
    • METHOD FOR NUCLEIC ACID DETECTION BY OLIGO HYBRIDIZATION AND PCR-BASED AMPLIFICATION
    • WO2022084528A1
    • 2022-04-28
    • PCT/EP2021/079393
    • 2021-10-22
    • MAX-DELBRÜCK-CENTRUM FÜR MOLEKULARE MEDIZIN IN DER HELMHOLTZ-GEMEINSCHAFT
    • POMBO, AnaSPARKS, Thomas
    • C12Q1/6813C12Q1/6841
    • The present invention relates to the field of nucleic acid sequencing at the single cell level, e.g., single-cell RNA sequencing (scRNA-seq). In particular, the invention provides a method of detecting nucleic acid in a fixated or non-fixated nucleic acid-containing compartment such as a eukaryotic cell or nucleus thereof, by hybridizing a plurality of single-stranded (ss)DNA oligonucleotide probes to complementary nucleic acid molecules within said compartment; removing ssDNA oligonucleotide probes from the compartment that have not specifically hybridized to nucleic acid; and identifying the ssDNA oligonucleotide probes specifically hybridized to nucleic acid molecules within said compartment by sequencing or amplification, thereby determining the corresponding nucleic acids present in said compartment. The method does not require a step of sequential ssDNA probe hybridization to the same target nucleic acid as a means for increased specificity or sensitivity, and preferably further does not require steps of RNA isolation and cDNA generation. The method of the invention has the potential to detect substantially every known and/or unknown nucleic acid species, in particular RNA, e.g., protein-encoding mRNAs as well as non-coding RNAs. The method further enables spatial mapping of detected nucleic acids, wherein the compartment is sectioned or dissociated into a single cell suspension prior to probe hybridization to obtain a collection of fractions and thus nucleic acid molecules are separated from each other depending on their localization or which cell type they belonged to. Spatial mapping of detected nucleic acids may be combined with the detection of at least one DNA locus, at least one protein, or with the analysis of chromatin condensation. The method of the invention is designated oligo-seq.