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Enhanced synthetic Notch receptor and application thereof

An enhanced, receptor-based technology, applied in applications, medical preparations containing active ingredients, receptors/cell surface antigens/cell surface determinants, etc., can solve problems such as error signals, achieve good safety, and expand applications Range, safety-enhancing effect

Pending Publication Date: 2020-06-12
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in our tests, it was found that the existing synNotch has a high level of ligand-independent activation (ligand-independent activation, LIA), which leads to its being able to be activated incorrectly without Ag activation, resulting in significant error signal

Method used

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  • Enhanced synthetic Notch receptor and application thereof
  • Enhanced synthetic Notch receptor and application thereof
  • Enhanced synthetic Notch receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1 Construction of synthetic Notch receptors and variants thereof, and recombinant expression vectors for the synthesis of Notch receptors and variants thereof

[0089] DNA sections were seamlessly ligated using ClonExpress (Vazyme #C115) and cloned into the P2Z plasmid backbone behind the PGK promoter. All constructs were verified by Sanger sequencing (Shenggong). The structures of all synthetic Notch receptors and their variants are summarized in Table 1.

[0090] Briefly, synthetic Notch receptors were constructed by fusing scFv (LaG16 or αCD19), Notch core regulatory domain (mN1c, EGF-mN1c, hN1c or Notch core regulatory domain with RAM sequence) and transcription factors (tTAA or GV2) and its variants. All synthetic Notch receptors and their variants contain an N-terminal CD8α signal peptide (MALPVTALLL PLALL LHAAR P) for membrane targeting and a Myc tag (EQKLI SEEDL) for determination of membrane surface expression.

[0091] Table 1. Synthetic Notch recep...

Embodiment 2

[0163] Example 2 Determination of the Ligand-Independent Activation Strength of Synthetic Notch Receptors

[0164] The schematic diagram of synNotch signal transduction induced by antigen (Ag) figure 1 shown. When cells expressing synNotch recognize specific antigens (antigens) presented on sender cells (sender, or target cells), sequential proteolytic cleavage occurs to release transcription factors (transcription factors, TFs) into the nucleus and induce their corresponding Transcription downstream of the promoter.

[0165] According to the method described in Example 1, we constructed a series of synthetic Notch receptors or variants thereof. The existing synNotch structure is formed by the sequential connection of scFv, NRR, TMD and TF, and does not contain the RAM domain. Plasmids for transient co-transfection assays such as figure 2 , to measure ligand-independent activation or misactivation of synNotch. Using the PGK promoter to drive the expression of synNotch, c...

Embodiment 3

[0171] Embodiment 3 inserting EGF can not reduce the LIA of synNotch

[0172] A previous study showed that insertion of an EGF repeat at the N-terminus of the Notch core regulatory domain reduced LIA.

[0173] On the basis of LaG16-mN1c-tTAA, we inserted an EGF repeat between LaG16 and NRR to construct LaG16-EGF-mN1c-tTAA. Both synNotchs were tested for LIA using transient co-transfection. Such as Figure 8 As shown in b, the median d2EGFP fluorescence intensity of each co-transfection was calculated and expressed in a histogram (mean ± SD); EGF (-) represents Figure 4 synNotch in a, while EGF(+) represents Figure 8 synNotch in a. The results showed that the LIA of LaG16-mN1c-tTAA and LaG16-EGF-mN1c-tTAA all increased significantly with the increase of synNotch transfection dose ( Figure 8 b). This experiment does not support the conclusions of previous studies, and we believe that the loss of the EGF domain is not responsible for the LIA or misactivation of synNotch....

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Abstract

The invention relates to an enhanced synthetic Notch receptor, which sequentially comprises: a, an extracellular recognition structural domain for specifically recognizing an antigen-related structure; b, a Notch core regulatory structural domain comprising a proteolytic cleavage site; c, an RAM structural domain; d, intracellular transcriptional modulating structural domain from an N terminal toa C terminal, wherein the length of the RAM structural domain is 5-100 amino acids, the RAM structural domain is an artificial sequence or is derived from a natural RAM structural domain, and the RAMstructural domain contains at least one or more diamino acid pairs randomly combined by hydrophobic amino acids. The enhanced synthetic Notch receptor provided by the invention has low ligand independent activation or error activation, and has good safety of clinical application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an enhanced synthetic Notch receptor, its coded nucleotide, recombinant expression vector and host cell, and application. Background technique [0002] The Notch signaling pathway is ubiquitous in organisms and is a highly conserved signaling pathway during evolution. It can mediate the interaction between cells, thereby regulating the development and differentiation of tissues and organs, and regulating the proliferation, differentiation and apoptosis of cells in the body. Death. [0003] Notch receptor is a type I transmembrane protein, which consists of Notch extracellular domain (Notch extracellular domain, NECD), Notch core regulatory domain (Notch core regulatory region, Nc), Notch intracellular domain (Notch intracellular domain, NICD )composition. The structure of NECD is very large, consisting of dozens of epidermal growth factor-like repeats (EGFs). NICD contains RBPJ-as...

Claims

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Application Information

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IPC IPC(8): C07K14/705C12N15/12A61K38/17A61P35/00A61P31/00A61P37/02
CPCC07K14/705A61P35/00A61P31/00A61P37/02A61K38/00
Inventor 蔡亮杨子杰
Owner FUDAN UNIV
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