La/single-stranded binding protein (La/SSB) chimera antigen modified NK cell, and preparation method and application thereof

A technology of NK cells and host cells, applied in genetically modified cells, cells modified by introducing foreign genetic material, blood/immune system cells, etc. disorder, etc.

Pending Publication Date: 2019-03-05
PERSONGEN BIOMEDICINESUZHOUCO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the etiology of some other autoimmune diseases (such as lupus erythematosus) is very complicated, and the immune system of patients with autoimmune diseases is disordered, with abnormal T cell function, Th cell control tolerance, B cell overreaction, self Immunopotential cells (PAL) are mostly in an unactivated or active closed state
Therefore, the treatment of autologous chimeric antigen receptor (CAR) T cells is very difficult and cannot achieve the ideal state, while allogeneic CAR-T cell transplantation has the risk of graft-versus-host reaction

Method used

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  • La/single-stranded binding protein (La/SSB) chimera antigen modified NK cell, and preparation method and application thereof
  • La/single-stranded binding protein (La/SSB) chimera antigen modified NK cell, and preparation method and application thereof
  • La/single-stranded binding protein (La/SSB) chimera antigen modified NK cell, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0203] Embodiment 1 Purification of LaA protein and LaA-BCR protein

[0204] 1.1 Induction and purification of LaA protein

[0205] To induce the expression of soluble LaA protein, the cDNA encoding LaA protein was subcloned into pET28a prokaryotic expression vector (LaA-pET28a) containing 6×HIS ( figure 1 B), 6×HIS tag is used for protein purification. Then the LaA-pET28a plasmid was transformed into E.coli BL21(DE3) strain for culture.

[0206] The LaA protein was induced and purified according to the following steps: Pick a single LaA-pET28a clone and culture it overnight at 37°C in 3 ml LB medium containing 100 μg / mL kanamycin. Then continue to cultivate in the fresh culture medium that contains 100 μ g / mL kanamycin according to the ratio dilution of 1:100, when bacterium early logarithmic phase (OD=0.6-0.8), add 0.5mMisopropylβ-D-1-thiogalactopyranoside ( IPTG, Sangon Biotech) at 37°C for 2.5-3 hours. Then LaA protein was purified by Ni-NTA agarose (GE Healthcare Life...

Embodiment 2

[0211] Example 2 Affinity Determination of LaA-BCR Protein and LaA Antigen

[0212] Purified LaA antigen added to 5×SDS-PAGE loading buffer (P0015L, Beyotime, Shanghai, China) and non-LaA protein as a negative control were used as samples, separated by 12% SDS-PAGE, and the proteins on SDS-PAGE were separated by Transfer to PDVF membrane (IPVH00010, Millipore, MA), block with 5% skimmed milk at room temperature for 1 hour (Guangming, Shanghai, China), apply purified LaA-BCR protein and incubate overnight at 4°C, and then apply 0.1% Tween- After washing three times with 20 PBST, the PVDF membrane was incubated with horseradish peroxidase-conjugated anti-mouse IgG (H+L) secondary antibody (A10677, Life Technologies, CA). After washing three times with PBST, the PDVF membrane was visualized by chemiluminescence by applying the enhanced chemiluminescence kit (WBKLS0500, Millipore Corporation, MA).

[0213] Purified LaA protein was coated on ELISA microplate overnight at 4°C. The...

Embodiment 3

[0215] Example 3 Construction, characteristics and functional verification of LaA-CAAR NK92MI cells

[0216] 3.1 Structure design and lentiviral transfection of LaA-CAAR

[0217] The molecular structure design of LaA-CAAR includes: a CD8 transmembrane signal peptide guides the expression of LaA-CAAR on the surface of NK92MI cells, the LaA antigen fragment is used as an extracellular fragment, F C Linking fragments, CD28 molecule (comprising transmembrane region), 4-1BB co-stimulatory factor and CD3ζ signal region are connected to form a fusion protein ( figure 2 A). The LaA-CAAR fusion protein contains: leader sequence, LaA peptide, hinge domain (FC), CD28 transmembrane domain TM, two co-stimulatory domains (CD28 and 4-1BB) and CD3ζ intracellular signaling domain.

[0218] The SP-LaA-Fc-CD28 / TM-4-1BB-CD3ζ fusion gene sequence was gene-synthesized, and then constructed into the pCDH-CMV-MCS-EF1-CopPuro (System Biosciences) lentiviral expression vector. Plate 293T cells, pac...

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PUM

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Abstract

The invention relates to an La / single-stranded binding protein (La / SSB) chimera antigen modified NK cell, and a preparation method and application thereof. Specifically, the invention provides the La / SSB chimera antigen modified fusion protein and the NK cell expressing the fusion protein, wherein the fusion protein has an optimized structure as shown in a formula I: X1-X2-L1-X3-X4-X4 (I), whereinthe elements are as described in the specification. Experimental results show that the fusion protein modified specific NK cell (such as LaA-CAARNK92MI cell) provided by the invention can be used fortargeted therapy of autoimmune diseases of positive LA / SSB autoantibodies, and has the advantages of good curative effect, small side effect, low production cost and the like.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to an anti-La / SSB chimera antigen-modified NK cell, its preparation method and application. Background technique [0002] Autoimmune diseases refer to the diseases caused by the body's immune response to self-antigens, resulting in damage to its own tissues. diseases of organs. Recently, people have increasingly realized that B cells play an increasingly extensive role in the development and development of autoimmune diseases, which provides exciting prospects for B cell-targeted therapy for autoimmune diseases. Among them, CD20-targeted B cell depletion experiments showed that 95% of pemphigus (PV) patients experienced short-term disease regression, but 81% of patients relapsed and developed fatal infection. After depletion of CD20-targeted B cells, autoantibody titers in patients' sera were significantly lower, suggesting that short-lived plasma cells are the so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N5/10A61K38/17A61P37/00
CPCA61K35/17C12N5/0646C07K14/47A61K38/00C07K2319/03C07K2319/02C12N2510/00
Inventor 杨林金泽明孟会敏
Owner PERSONGEN BIOMEDICINESUZHOUCO
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