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Anti-cd123 Nanobody and Its Application

A nanobody and chimeric antigen receptor technology, applied in the field of biomedicine, can solve problems such as failure to achieve ideals

Active Publication Date: 2022-07-29
HUADAO SHANGHAI BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although TKIs provide different treatment options for leukemia, they are still in the development stage, and more treatment strategies are needed clinically
[0004] The concept of Chimeric antigen receptor T cells (CAR-T) appeared as early as 1989, but it has not been able to achieve the desired effect in clinical trials.

Method used

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  • Anti-cd123 Nanobody and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Construction, panning and ELISA preliminary screening of phage nanobody library

[0073] (1) Construction of phage nanobody library

[0074] In this example, the extracellular region of CD123 antigen was used to immunize Bactrian camels, and after ELISA was used to verify the titer, 200 mL of peripheral blood was extracted; lymphocytes were sorted from peripheral blood to obtain peripheral blood mononuclear lymphocyte precipitation, and RNA extraction was performed;

[0075] Using the extracted RNA as a template, The first-strand cDNA was synthesized by reverse transcriptase, and then the VHH gene was amplified by nested PCR; the amplified VHH gene was inserted into the pMECS phage display vector, and TG1 competent cells were electrotransformed. Cultures were evenly spread on LB / AMPGLU plates;

[0076] After the bacteria grow out, collect the bacterial moss, add 1 / 3 volume of 50% glycerol, mix and distribute, and store at -80 °C, that is, the storage capacity...

Embodiment 2

[0089] Example 2 FACS screening of candidate clones

[0090] In this example, the cells were cultured according to the standard cell culture protocol:

[0091] Use trypsin to digest the cells to prepare a CD123-positive or CD123-negative cell suspension, centrifuge at 300g for 5 min to remove the culture medium, and resuspend the cells in Flow Buffer to a cell concentration of 2×10 6 pcs / mL;

[0092] Add 2 x 10 to each well of a V-bottom 96-well plate 5 Cells were centrifuged at 300 g for 5 min to remove the supernatant, and the crude VHH antibody extract was added to resuspend the cells, and incubated at 4°C for 1 h;

[0093] Centrifuge at 300 g for 5 min to remove the supernatant, resuspend the cells in Flow Buffer, add 100 μL of APC anti-his antibody (2 μg / mL) diluted in Flow Buffer, and incubate at 4°C for 1 h;

[0094] After washing the cells three times with Flow Buffer, resuspend the cells in 200 μL of Flow Buffer for flow cytometry.

Embodiment 3

[0095] Example 3 Expression, purification and affinity determination of VHH-mIgG2a Fc Nanobody

[0096] In order to further identify the screened antibodies, this example constructs a VHH-expressing vector C-4pCP.Stuffer-mCg2a-FC (with a mouse Fc tag), and the steps are as follows:

[0097] PCR amplification of the anti-CD123 heavy chain variable region encoding gene, wherein the upstream primer of CD123-9 (SEQ ID NO: 6) is HD-F, the downstream primer is HD-B12-R2, CD123-12 (SEQ ID NO: 6) 7) The upstream primer is HD-F, and the downstream primer is HD-CD-12-R. The sequence is shown in Table 2. The PCR reaction system is shown in Table 3. The reaction conditions are pre-denaturation at 95°C for 1min, and denaturation at 95°C for 10s. , annealing at 55°C for 10s, extension at 72°C for 10s, 30 cycles, extension at 72°C for 5 min, and storage at 4°C.

[0098] Table 2

[0099]

[0100] table 3

[0101]

[0102] The empty vector was digested with enzyme at 37°C for 6 hours....

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Abstract

The present invention provides an anti-CD123 nanobody and application thereof, the nanobody comprises a heavy chain variable region; the heavy chain variable region comprises CDR1 shown in SEQ ID NO: 1 and CDR2 shown in SEQ ID NO: 2 , and CDR3 shown in SEQ ID NO:3. The antibodies screened from the camel VHH immune library of the present invention have the CDR regions shown in SEQ ID NOs: 1 to 3, and the nanobodies formed by combining with different framework regions FR have strong affinity with CD123, and the chimeric antigen receptor constructed by this , Chimeric antigen receptor immune cells have significant cytotoxicity to CD123 positive cells, and have broad application prospects in the field of tumor therapy.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to an anti-CD123 nanobody and an application thereof. Background technique [0002] More than 30 years ago, the "3+7" regimen (3 days of daunorubicin chemotherapy combined with 7 days of cytarabine chemotherapy) resulted in remission in about 60% of AML patients and became the standard induction regimen for acute leukemia in children and adults . In the 1990s, clinical experts began to focus on post-remission treatment options and their benefits, and many studies were conducted, including high-dose cytarabine chemotherapy or hematopoietic stem cell transplantation (HSCT). Although remission rates and overall survival rates for childhood acute leukemia have now reached greater than 90% and 60%, respectively, existing treatment options are still limited to anthracyclines, nucleoside analogs, and intensive post-remission therapy. In order to improve the prognosis of patients with A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C07K19/00C12N5/10A61K35/17A61P35/02
CPCC07K16/2866C07K14/7051C12N5/0634A61K35/17A61P35/02C07K2317/565C07K2317/569C07K2319/02C07K2319/03C12N2510/00
Inventor 狄升蒙侯莉石磊刘芳茅健余学军
Owner HUADAO SHANGHAI BIOPHARMA CO LTD
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