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CD123 binding protein, CAR containing CD123 binding protein and application thereof

A binding protein, H12Y technology, applied in the field of CAR, CD123 binding protein, can solve the problem of normal hematopoietic cell toxicity

Active Publication Date: 2020-12-15
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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  • CD123 binding protein, CAR containing CD123 binding protein and application thereof
  • CD123 binding protein, CAR containing CD123 binding protein and application thereof
  • CD123 binding protein, CAR containing CD123 binding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0148] 1.1 Immune mice and serum affinity ELISA detection

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

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

[0151] Specific steps are as follows:

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

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

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

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

[0156]...

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, and 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 cell...

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Abstract

The invention discloses a CD123 binding protein, a CAR containing the CD123 binding protein and application thereof. The CD123 binding protein and the CAR comprise sequences as shown in the sequence listing. An anti-CD123 binding protein and the CAR disclosed by the invention can achieve specific killing effects on cancer cells with high CD123 expression under the premise of reducing or avoiding damage on normal cells; and thus, safety of clinical treatment can be improved.

Description

technical field [0001] This application relates to the field of biomedicine, in particular to a CD123-binding protein, a CAR containing it 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 blood system, that is, a malignant disease of myeloid hematopoietic stem cells (HSCs) / progenitor cells, mainly characterized by abnormal proliferation of primitive and immature myeloid cells in bone marrow and peripheral blood. Characteristics (Hope KJ, Jin L, Dick JE. Human acute myeloid leukemia stemcells. Arch Med Res, 2003, 34(6): 507-514.). [0004] In my country, AML is the most common malignant blood disease, with an annual incidence rate of 1.8 / 100,000 people, and the incidence rate of the elderly over 65 years old has increased to 17.2 / 100,000 people. Traditional treatment drugs have relatively low efficacy and strong side effects, and the five-year surv...

Claims

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Application Information

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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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