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TGF-Beta receptor II isoform, fusion peptide, methods of treatment and methods in vitro

An isotype, RII-SE technology, used in fusion peptides, hybrid peptides, gene therapy, etc., to solve problems such as signal transduction disruption

Pending Publication Date: 2020-05-05
国家科学技术委员会 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This mutant based on the receptor's isoform A is able to bind TGF-β1 but has disrupted signaling due to the absence of the serine / threonine kinase domain

Method used

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  • TGF-Beta receptor II isoform, fusion peptide, methods of treatment and methods in vitro
  • TGF-Beta receptor II isoform, fusion peptide, methods of treatment and methods in vitro
  • TGF-Beta receptor II isoform, fusion peptide, methods of treatment and methods in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1: Isolation, cloning and sequencing of TβRII-SE isoforms

[0116]Human adipose-derived mesenchymal stromal cells (hASCs) were obtained from 20 g of subcutaneous fat according to the protocol described by Zuk et al. and 1% L-glutamine in DMEM. Epstein Barr virus immortalized lymphoblasts were generated from peripheral blood mononuclear cells as described in "Protocols in Immunology" and cultured in RPMI medium. Human A459 (lung adenocarcinoma), HT1080 (fibrosarcoma), Caco-2 (colorectal cancer), Hep 3B (hepatocellular carcinoma), Jurkat (acute lymphoblastic leukemia), HEK293 (human embryonic kidney) and 293T cell lines. Place cells in humidified 5% CO 2 Cultured at 37°C in an incubator.

[0117] Purification of different leukocyte subsets

[0118] Granulocytes, lymphocytes and monocytes were isolated from heparinized peripheral blood by Ficoll-Paque™ PLUS (GE Healthcare Bio-Sciences AB) gradient centrifugation. After centrifugation, two fractions were obtai...

Embodiment 2

[0121] Example 2: Cloning of codon-optimized (co)TβRII-SE / Fc isotype fusion constructs

[0122] The TβRII-SE coding sequence containing the AgeI site was codon-optimized, the stop codon was deleted, and the Kozak sequence (Epoch Biolabs Inc. Texas, USA) was included. Use specific oligonucleotides as primers (forward: 5'AGA TCT GAC AAA ACT CAC ACA TGC 3' (SEQ ID No. 8) and reverse: 5'GAT ATC TTT ACCCGG AGA CAG G 3' (SEQ ID No.9)), the human IgG1 Fc coding sequence was obtained from total blood mRNA by RT-PCR, and the primers contained BglII site (forward primer) and EcoRV (reverse primer) to allow integration with TβRII-SE and Lentiviral vector in-frame fusion. The 951 bp AgeI / EcoRV fusion construct (coTβRII-SE / Fc) comprised a 258 bp coTβRII-SE fused in-frame to a 693 bp human IgG1-Fc.

Embodiment 3

[0123] Embodiment 3: lentiviral vector

[0124] The cDNAs encoding these three human TβRII isoforms were cloned into the pRRLsin18.cPPT.WPRE lentiviral vector, resulting in transfer vectors pRRLsin18.cPPT.CMV-TβRII-SE.ireseGFP.WPRE, pRRLsin18.cPPT.CMV-TβRII-DN .ireseGFP.WPRE and pRRLsin18.cPPT.CMV-coTβRII-SE / Fc.ireseGFP.WPRE. As previously described (R.A. Dewey et al., Experimental Hematology 34:1163-1171, 2006), by combining the transfer vector with an envelope plasmid (pCMV-VSVG), a packaging plasmid (pMDLg / pRRE) and a Rev plasmid (pRSV-REV) Transient transfection into the 293T cell line produced vesicular stomatitis virus G-pseudotyped lentivirus (VSV-G). Supernatants were collected every 12 hours for 48 hours and frozen in aliquots. Virus titers were determined by transduction of A549 cells (produced 10 7 infection particles). The pRRLsin18.cPPT.CMV-eGFP.WPRE lentiviral vector was used as a control.

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Abstract

The invention relates to a TGF-Beta receptor II isoform, fusion peptide, methods of treatment and methods in vitro. ATGF-p receptor II isoform, fusion peptide, methods of treatment and methods in vitro An isoform of the TGF beta receptor II comprising a sequence of about of 80 amino acids and lacking a transmembrane domain. A fusion peptide is provided comprising an isoform of the TGF beta II receptor fused to a ligand, wherein a vector comprising the fusion peptide is used to treat cancer and / or hepatic fibrosis. A method of treating hepatic fibrosis or cancer diseases, comprising the step ofadministering to a mammal in need thereof a vector bearing a polynucleotide sequence set forth in SEQ ID No.2. A fusion peptide is provided comprising an isoform of the TGF beta II receptor fused toa ligand, wherein a vector comprising the fusion peptide is used to treat cancer and / or hepatic fibrosis. An antibody binding the soluble isoform of the TGF beta II receptor is provided. The antibodybinds the amino acid sequence shown in SEQ ID No. 12 and is used in in vitro methods.

Description

technical field [0001] The present invention relates to isoforms, methods and uses of TGF-beta receptor II encoding polynucleotides, vectors, cells, transforming peptides and fusion peptides. More specifically, it involves an isoform of TGF-beta receptor II, which comprises a sequence of approximately 80 amino acids and lacks a transmembrane domain. Said isoform comprises the amino acid sequence of SEQ ID No.12. The isoform may have the amino acid sequence shown in SEQ ID No. 2 or a sequence with at least 85% sequence identity to the sequence shown in SEQ ID No. 2. Background technique [0002] Transforming growth factor-β (TGF-β) is abundant in the bone matrix and has been shown to regulate osteoblast and osteoclast activity in vitro and in vivo. Human adipose-derived mesenchymal stromal cells (ASCs) are precursors of osteoblasts, adipocytes and chondrocytes. Therefore, initial studies focused on cytokines (such as Tgf-β1), osteoprotegerin (OPG), and hepatocyte growth fa...

Claims

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Application Information

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IPC IPC(8): C12N15/867C07K14/71C07K19/00G01N33/68A61K48/00A61K38/18A61P1/16A61P35/00
CPCC12N15/86C07K14/71C07K16/22G01N33/6872A61K48/005A61K38/1841A61P1/16A61P35/00C12N2740/15043G01N2333/71C07K2319/30
Inventor A·洛莫A·B·拉科拉M·A·普雷塞格尔M·S·贝尔托利奥R·A·德维P·D·瓦兹奎斯A·N·基萨里T·M·罗德里格斯B·J·维拉索·扎莫拉
Owner 国家科学技术委员会
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