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Chimeric antigen receptor specifically binding to CD33 and application thereof

A specific, CDR-H3 technology, applied in the direction of application, antibody, cancer antigen components, etc., to achieve significant clinical value

Pending Publication Date: 2022-05-13
CHENGDU KELUN PRECISION BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, no single antigen has been shown to be unique to AML cells and leukemia stem cells

Method used

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  • Chimeric antigen receptor specifically binding to CD33 and application thereof
  • Chimeric antigen receptor specifically binding to CD33 and application thereof
  • Chimeric antigen receptor specifically binding to CD33 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0314] Example 1: Preparation of specific single-chain antibody (scFv) binding to human CD33

[0315] 1.1 Screening of CD33 scFv based on phage display

[0316] (1) Take 0.5ml of a fully human phage library and screen it with biotinylated CD33 and SV magnetic beads, and the screened products are tested for phage titration by plating.

[0317] (2) Mix 0.5ml of the product of the first round of panning with 0.5ml of M PBST, and carry out the second, third or fourth round of panning according to the above steps.

[0318] 1.2 ELISA detection of monoclonal phage

[0319] Inoculate a single colony from the phage panning product titer plate into a 96-deep well plate, and detect monoclonal phage by ELISA.

[0320] 1.3 CD33-scFv sequencing

[0321] The forward and reverse primers used for sequencing were: PKLT1F (SEQ ID NO: 81); PKLT1R: (SEQ ID NO: 82). The sequence results were analyzed using Sequcher software, and finally 11 clone sequences were obtained.

[0322] 1.4 Constructi...

Embodiment 2

[0340] Example 2: Construction of lentiviral plasmid and viral packaging

[0341]1) Overview of the construction of the lentiviral plasmid: the scFv sequence was synthesized after codon optimization and constructed into the Lenti-EF1a-AT-Free (manufactured by Suzhou Aikangde Co., Ltd.) vector, and a single clone was picked for cultivation and preservation, and the plasmid was extracted Sequencing is performed, and the bacterial liquid with correct sequencing is used to prepare lentiviral plasmids.

[0342] In detail: based on the scFv sequence obtained in the above examples, further construct the CAR lentiviral expression vector. Using the intracellular domain of CD137 and the ITAM region of CD3ζ as the activation signal, fuse with the above scFv, add signal peptide, CD8 hinge region, and CD8 transmembrane region at the same time, construct a chimeric antigen receptor expression vector, and subclone it into In the Lenti-EF1a-AT-Free (manufactured by Suzhou Aikond Co., Ltd.) v...

Embodiment 3

[0348] Example 3: Preparation of CAR-T cells

[0349] 3.1 Isolation and activation of primary T cells:

[0350] (1) Use lymphocyte separation medium to separate human PBMC cells, add totipotent nuclease according to the ratio of cell fluid:totipotent nuclease=10ml:1ul, place at 37°C, 5% CO 2 Cultivate for 3 hours in an incubator;

[0351] (2) Aspirate CD3 / CD28 magnetic beads according to the ratio of PBMC: magnetic beads = 3:1, wash with HDPBS (DPBS containing 2% human albumin), and resuspend in 100ul HDPBS in a cryopreservation tube.

[0352] (3) Collect the suspended cells in a cryopreservation tube, centrifuge at 500g for 5 minutes, add 1ml HDPBS to resuspend the cells, transfer the cell suspension to 100ul magnetic beads, mix gently, and incubate at room temperature for 30 minutes;

[0353] (4) Insert the incubated mixture into the magnetic pole, let it stand at room temperature for 2 minutes, keep the cryopreservation tube in the magnetic pole, invert it gently, and pou...

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Abstract

The invention relates to the field of cellular immunotherapy, in particular to an antibody specifically binding to CD33 or an antigen binding fragment thereof, and a chimeric antigen receptor (CAR) and a conjugate containing the antibody or the antigen binding fragment thereof. The invention also relates to sequences and expression vectors encoding such CARs, engineered immune cells comprising such CARs and methods of making such engineered immune cells. The invention also relates to uses and methods of these antibodies, conjugates, CARs and engineered immune cells such as T cells and NK cells for treating myeloid malignancies and malignant lymphomas such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS).

Description

technical field [0001] The present invention relates to the field of cellular immunotherapy, in particular, the present invention relates to an antibody specifically binding to CD33 or an antigen-binding fragment thereof and a chimeric antigen receptor (CAR) and a conjugate comprising the antibody or an antigen-binding fragment thereof. The present invention also relates to sequences and expression vectors encoding the CAR, engineered immune cells comprising the CAR and methods for preparing the engineered immune cells. The present invention also relates to the use of these antibodies, conjugates, CAR and engineered immune cells such as T cells and NK cells for the treatment of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS ) and other myeloid malignancies and malignant lymphomas and methods thereof. Background technique [0002] AML is a common hematologic malignancy and one of the most common malignant tumors in China. The inci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62C12N15/867C12N15/861C12N5/10A61K47/68A61K39/395A61K39/00A61P35/00A61P35/02
CPCC07K16/2803C07K14/7051C12N15/86A61K47/6803A61K39/0011A61P35/00A61P35/02C07K2317/622C07K2317/565C07K2317/92C07K2319/02C07K2319/03C07K2319/33C12N2740/15043C12N2710/10043C12N2740/10043A61K2039/505
Inventor 常建辉罗永华朱雁林宋婷玉余超恒王潇东田海军王晶翼
Owner CHENGDU KELUN PRECISION BIOTECHNOLOGY CO LTD
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