Immune effector cell for chronic lymphocytic leukemia as well as preparation method and application thereof

A technology of immune effector cells and proteins, applied in the field of medical biology, can solve problems such as complexity, difficulty, and unsuitability for targeted therapy

Active Publication Date: 2020-08-07
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in view of the complexity of gene expression in vivo and various uncontrollable factors, it is very difficult to successfully select a suitable gene for CAR-based therapy. In a large amount of research work in this field, many tumor-specific antigens Found to be unsuitable for targeted

Method used

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  • Immune effector cell for chronic lymphocytic leukemia as well as preparation method and application thereof
  • Immune effector cell for chronic lymphocytic leukemia as well as preparation method and application thereof
  • Immune effector cell for chronic lymphocytic leukemia as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1, Construction of the Expression Vector of CD32b CAR Molecule

[0100] In this example, a lentiviral vector expressing CAR molecules is constructed, and the lentiviral vector pCDH is used as the backbone plasmid, and the structure of pCDH is as follows: figure 2 shown.

[0101] According to the difference of the CAR molecule scFv, the inventors constructed two kinds of CD32b CAR molecules, namely scFv1-BBZ and scFv2-BBZ: scFv1 and scFv2 are CD32b humanized antibodies. The molecular structure patterns of scFv1-BBZ and scFv2-BBZ are shown in figure 1 shown. The above CAR molecule was constructed into the lentiviral vector pCDH by conventional molecular cloning means.

[0102] Construction of scFv1-BBZ vector: First, scFv1-BBZ nucleic acid fragment was chemically synthesized into pUC57 vector (Beijing Huada Protein Biology Co., Ltd.), and scFv1-BBZ fragment (SEQ ID NO: 11) was obtained by XmaI / SalI double enzyme digestion. Wherein, scFv1 sequence such as SEQ ...

Embodiment 2

[0109] Example 2, Construction of CD32b CAR-T cells

[0110] In this example, the inventors constructed CD32b CAR-T cells.

[0111] Packaging of CAR lentivirus: First, use the MACHEREY-NAGEL endotoxin-free large-scale extraction plasmid kit to extract the lentiviral plasmids pCDH-scFv1-BBZ, pCDH-scFv2-BBZ and pCDH (without CD32b CAR control empty vector) constructed in Example 1 Plasmids) and lentiviral system auxiliary packaging plasmids Rev, VSV-G and pMDL. The day before transfection, 1×10 7 A 293T plate was placed in a T75 culture flask. 20 minutes before transfection, the medium of 293T cells was replaced with 5ml of serum-free DMEM medium. The plasmids were co-transfected into 293T cells using PEI transfection reagent, and the cell culture medium was replaced with 10 ml complete medium DMEM+10% FBS 6 hours after transfection. Cell supernatant was harvested 48 hours after transfection, and 10 ml of fresh complete medium was added. After 72 hours, the cell supernatant...

Embodiment 3

[0114] Example 3, Determination of CD32b CAR-T cell activity

[0115] In this example, the activity of CD32b CAR-T cells was measured.

[0116] 1. Identification and construction of CD32b CAR-T target cells

[0117] Human chronic lymphocytic leukemia Mec-1 cell line and Burkitt's lymphoma Raji cell line were used to detect the expression of CD32b on the two cell lines using CD32b-APC flow cytometry antibody.

[0118] The result is as Figure 5 As shown, the Raji cell line highly expresses the CD32b gene. Using a lentiviral vector encoding the GFP-Luciferase gene, the GFP-positive cells were sorted to construct a Raji cell line with a high expression of the GFP-Luciferase gene. CD32b and GFP-Luciferase expression levels in Raji cell lines see Figure 5 .

[0119] according to Figure 5 , Raji cell line is positive for CD32b expression.

[0120] 2. Cytokine secretion experiment

[0121] In a 96-well plate, scFv1-BBZ CAR-T cells or control pCDH T cells (effector cells) we...

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Abstract

The invention discloses a chimeric antigen receptor (CAR) targeting CD32b, an immune effector cell modified by the CAR, a preparation method of the immune effector cell and application of the immune effector cell in inhibition of chronic lymphocytic leukemia for the first time. The invention provides a novel treatment scheme for refractory chronic lymphocytic leukemia.

Description

technical field [0001] The invention belongs to the field of medical biology, and more specifically, the invention relates to an immune effector cell for chronic lymphocytic leukemia, its preparation method and application. Background technique [0002] In this field, adoptive immunotherapy based on immune effector cells has achieved certain effects in some tumors, and this immunotherapy method can overcome the above-mentioned defects of antibody therapy, but the curative effect in most tumors is still unsatisfactory[ Grupp SA, et al. Adaptive cellular therapy. Curr Top Microbiol Immunol., 2011; 344:149-72]. In recent years, based on the discovery that cytotoxic T lymphocytes (cytotoxic lymphocytes, CTLs) recognize target cells specifically depends on T cell receptors (T Cell Receptors, TCRs), the scFv of antibodies against tumor cell-associated antigens was combined with Intracellular signal activation motifs such as CD3ζ or FcεRIγ of T lymphocyte receptors are fused into ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10
CPCC07K14/7051C12N5/0636C12N5/0646A61K39/001111A61P35/02A61P35/00C07K2319/02C07K2319/03A61K2039/5156A61K2039/515C12N2510/02C07K2317/51C07K2317/515
Inventor 冯晓明王国玲孙晓蕾
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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