Genetically engineered red blood cells carrying anti-PD-1 single-chain antibody and preparation method thereof

A PD-1 and single-chain antibody technology, applied in the field of medicine and biology, can solve the problems of low utilization efficiency, side effects, high tumor immunity drugs, etc., and achieve the effect of wide circulation range, good biocompatibility, and large quantity

Active Publication Date: 2020-09-08
WEST LAKE BIOMEDICAL TECH HANGZHOU CO LTD
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Problems solved by technology

[0005] In view of the deficiencies in the above-mentioned existing technologies, the present invention provides genetically engineered red blood cells carrying anti-PD-1 single-chain antibodies, with the purpose of solving the problem that the delivery of anti-PD-1 monoclonal antibodies cannot be targeted to tumor tissues, and the utilization efficiency is low. And the technical problems that cause higher side effects of systemic tumor immune drugs

Method used

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  • Genetically engineered red blood cells carrying anti-PD-1 single-chain antibody and preparation method thereof
  • Genetically engineered red blood cells carrying anti-PD-1 single-chain antibody and preparation method thereof
  • Genetically engineered red blood cells carrying anti-PD-1 single-chain antibody and preparation method thereof

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Embodiment 1

[0062] 1. Construction of lentiviral vector containing anti-PD-1 scFv

[0063]Anti-PD-1 single-chain variable region fragment (Anti-PD-1 scFv) was designed, and the nucleotide sequence of Anti-PD-1 scFv is one or more of SEQ ID NO.1-SEQ ID NO.6 This combination was synthesized by conventional biosynthesis methods, and the vector was constructed by means of molecular cloning. The Anti-PD-1 scFv was constructed on the lentiviral vector pCDH-MCS-T2A-copGFP-MSCV to obtain the target sequence vector (MSCV- anti-PD-1 scFv) for viral packaging.

[0064] 2. Preparation of Lentivirus Concentrate

[0065] MSCV-anti-PD-1 scFv:pSPAX2:VSVG=2:1:1 ratio was used for lentivirus packaging and packaging. In this example, a 6-well plate was used, and the target sequence vector (MSCV-anti-PD -1 scFv): pSPAX2:VSVG=2 μg: 1 μg: 1 μg The plasmid was transfected into HEK 293T cells for virus packaging. The medium was changed 12 hours after transfection to remove calcium phosphate, the supernatant w...

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Abstract

Provided are a method for preparing genetically engineered red blood cells carrying an anti-PD-1 single-chain antibody, and genetically engineered red blood cells carrying the anti-PD-1 single-chain antibody. The preparation method comprises the following steps: constructing a target fragment sequence in a lentiviral expression vector; lentivirally packaging a target sequence vector to obtain a high-titer lentiviral concentrate; isolating and enriching Lin-CD34 cells from among peripheral blood mononuclear cells; inducing the Lin-CD34-cells to differentiate into red blood cells and performing amplification thereon; using the lentiviral concentrate to infect the Lin-CD34-cells; and obtaining mature anti-PD-1scFv-red blood cells by means of denucleating red blood cells. The present genetically engineered red blood cells carrying the anti-PD-1 single-chain antibody may perform the targeted delivery of an anti-PD-1 single-chain antibody to tumor tissue.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to a genetically engineered red blood cell carrying an anti-PD-1 single-chain antibody and a preparation method thereof. Background technique [0002] Immunotherapy has become one of the most powerful means of treating cancer. There are more and more drugs approved for immunotherapy, and more and more projects are in clinical and preclinical development. However, immunotherapy is a broad treatment strategy targeting the systemic immune system, so the key is still the regulation of the immune system. The main problems of current immunotherapy are side effects such as autoimmunity and non-specific inflammation. How to improve the therapeutic effect and control adverse reactions by improving the response rate of immunotherapy has become the key. How to more effectively deliver drugs for immunotherapy (such as: anti-PD-1, CTLA-4 monoclonal antibody, IL-2, TNF-α, etc.) in viv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867
CPCC07K16/2818C07K2317/622C12N5/0641C12N15/86C12N2510/02C12N2740/15043
Inventor 高晓飞黄彦杰李丹丹
Owner WEST LAKE BIOMEDICAL TECH HANGZHOU CO LTD
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