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A kind of cd123 binding protein, car containing it and application thereof

A technology for binding proteins and variable regions, which is applied in the field of CAR and CD123 binding proteins, and can solve the problems of normal hematopoietic cytotoxicity and other issues

Active Publication Date: 2021-12-28
西安桑尼赛尔生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This CART cell has specific anti-AML ability in vivo and in vitro, but has certain toxicity to normal hematopoietic cells

Method used

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  • A kind of cd123 binding protein, car containing it and application thereof
  • A kind of cd123 binding protein, car containing it and application thereof
  • A kind of cd123 binding protein, car containing it and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0147] Example 1: Production and immunoassay of murine monoclonal antibodies

[0148] 1.1 Immune mice and serum affinity ELISA detection

[0149] In order to generate antibodies against CD123, in this example, CD123 protein (No. SNS001-002, purchased from ACRO, Beijing Baipsis Biotechnology Co., Ltd.) was used to immunize mice.

[0150] Three mice were immunized by subcutaneous injection, and after three rounds of immunization, serum samples were obtained by blood sampling through the tail vein. Affinity ELISA was performed on the serum of the third immunized mice.

[0151] Specific steps are as follows:

[0152] 1) Antigen coating: Use affinity Elisa plate, add SNS001-002 diluted with PBS to 2 μg / ml in 96-well semi-well microtiter plate, 30 μl per well, and coat overnight at 4 degrees.

[0153] 2) Wash the plate: wash the plate 3 times with PBST.

[0154] 3) Blocking: 5% PBSM, room temperature, 2 hours.

[0155] 4) Wash the plate: wash the plate 3 times with TBST.

[01...

Embodiment 2

[0271] Example 2: Construction and virus packaging of lentiviral plasmids containing chimeric antigen receptors

[0272] 2.1 Synthesis of Chimeric Antigen Receptor Nucleic Acid Sequence

[0273] In this embodiment, the arrangement order of the chimeric antigen receptor sequence is designed as follows: from the 5th end to the 3rd end: leading chain-scFv-CD8 hinge region-CD8 transmembrane region-4-1BB-CD3ζ.

[0274] Use the light chain variable region and heavy chain variable region sequences (VL, VH sequence numbers in Table 11) of the 18 kinds of positive clones screened in Example 1 and submit the following sequences at the same time, respectively numbered as 32716 clones (from the United States Hope City) light and heavy chain variable region sequence (from patent CN201480024929.6), leading chain region (SEQ ID NO:73), linker region (SEQ ID NO:75), CD8 hinge region (SEQ ID NO:77), CD8 transmembrane region (SEQ ID NO:79), 4-1BB co-stimulatory region (SEQ ID NO:81) and CD3ζ s...

Embodiment 3

[0297] Example 3: Construction and verification of stable expression cell lines of K562-CD123

[0298] First, NCBI (https: / / www.ncbi.nlm.nih.gov / ) searched for the CD123 protein sequence (XP_016884980.1), commissioned Nanjing GenScript to synthesize a lentiviral plasmid vector containing the protein gene, and transfected into competent For cell stb13, the plasmid was extracted according to Example 2.2. The extracted target plasmid and packaging plasmid were transfected into 293T cells according to a certain ratio, and the CD123 virus liquid was collected and filtered after 48 hours and 72 hours of transfection respectively, and referred to Example 2.4 Perform virus titration.

[0299] According to MOI=5:1, K562 cells (from Peking University Medical Center) were infected with CD123 virus solution. Puromycin selection was carried out 2 days after infection (the screening concentration was 5 μg / ml). One week after the selection, unstained K562 cells were used as control cells, a...

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Abstract

The invention discloses a CD123 binding protein, a CAR containing the same and an application thereof. The CD123 binding protein and the CAR comprise the sequences shown in the present invention. The anti-CD123 binding protein and the CAR of the present invention can reduce or avoid damage to normal cells while ensuring specific killing of cancer cells with high CD123 expression, and improve the safety of clinical treatment.

Description

technical field [0001] The present application relates to the field of biomedicine, in particular to a CD123 binding protein, a CAR containing the same, and applications thereof. Background technique [0002] 1. Current status of acute myeloid leukemia treatment: [0003] Acute myeloid leukemia (AML) is a malignant tumor of the hematological system, that is, a malignant disease of myeloid hematopoietic stem cells (HSCs) / progenitor cells. Features (Hope KJ, Jin L, Dick JE. Human acute myeloid leukemia stemcells. Arch Med Res, 2003, 34(6):507-514.). [0004] AML is the most common hematological malignancy in my country, with an annual incidence rate of 1.8 / 100,000, and the incidence of elderly people over 65 has increased to 17.2 / 100,000. Traditional treatment drugs have relatively low efficacy and strong side effects, and the five-year survival rate of patients is very low (about 10%) (Tamamyan G, Kadia T, Ravandi F, et al. Frontline treatment ofacute myeloid leukemia in ad...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62C12N15/867C12N7/01C12N5/10A61K47/68A61K38/07A61K31/537A61K39/00A61P35/00A61P35/02
CPCC07K16/2866C07K14/7051C12N15/86C12N7/00C12N5/0636C12N5/0646A61K47/6849A61K38/07A61K31/537A61K39/0011A61P35/00A61P35/02C07K2319/02C07K2319/03C07K2319/33C07K2317/622C07K2317/92C07K2317/565C12N2740/15021C12N2740/15043C12N2510/00
Inventor 彭作翰徐之艳张蕊刘佳玫李玏
Owner 西安桑尼赛尔生物医药有限公司
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