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Three-function molecule combining CD19, CD3 and CD28 and application of three-function molecule

A three-function, molecular technology, applied in the field of biomedicine, can solve the problems of high experimental conditions, difficulty in dose control, cumbersome steps, etc., and achieve the effects of improving the activation effect, avoiding T cell incapacity and death, and complicating the preparation process

Active Publication Date: 2017-04-26
CYTOCARES SHANGHAI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, CAR-T technology itself also has some shortcomings: first, the technology relies on virus transfection to genetically modify T cells, the steps are cumbersome, and the requirements for experimental conditions are high; Compared with antibody drugs, it is more difficult to control the dose of CAR-T cells reinfused into the patient; in addition, the sharp increase in the number of CAR-T cells after entering the patient's body can lead to a cytokine storm (Cytokine storm), resulting in excess in a short period of time Cytokines, which cause side effects such as high fever, low pressure, shock and even death

Method used

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  • Three-function molecule combining CD19, CD3 and CD28 and application of three-function molecule
  • Three-function molecule combining CD19, CD3 and CD28 and application of three-function molecule
  • Three-function molecule combining CD19, CD3 and CD28 and application of three-function molecule

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preparation example Construction

[0089] The method for preparing the aforementioned trifunctional molecule of the present invention comprises: constructing an expression vector containing the gene sequence of the trifunctional molecule, then transforming the expression vector containing the gene sequence of the trifunctional molecule into a host cell to induce expression, and isolating the expression product to obtain the the three functional molecules described above. In a preferred case of the present invention, the expression vector uses pcDNA3.1. The host cell is Chinese hamster ovary cell (CHO).

[0090] 6. The use of three functional molecules

[0091] The trifunctional molecule of the present invention can be used for tumor treatment drugs. The tumor is a tumor whose cell surface is positive for CD19.

[0092] In a preferred embodiment of the present invention, human peripheral blood mononuclear cells (PBMC) are used as experimental materials, and the above-mentioned trifunctional molecules in the f...

Embodiment 1

[0104] Example 1 Construction of CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D eukaryotic expression vectors

[0105] In the present invention, the TiTE trispecific antibody targeting human CD19 protein on the surface of lymphoma B cells and human CD3 and CD28 proteins on the surface of T cells is named CD19-CD3-CD28TsAb.

[0106] 1. CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D construction scheme design

[0107] The specific construction scheme of CD19-CD3-CD28TsAb_M in monomeric form is as follows: the sequences of anti-CD19scFv, anti-CD3scFv and anti-CD28scFv are connected through a linker (Linker), specifically, anti-CD19scFv and anti-CD3scFv are connected through a linker 1 (Linker 1) The anti-CD3scFv and anti-CD28scFv sequences were connected by linker 2 (Linker 2).

[0108] The specific construction scheme of the CD19-CD3-CD28TsAb_D in dimer form is as follows: the sequences of anti-CD19scFv, anti-CD3scFv and anti-CD28scFv are connected through a linker (Linker), specific...

Embodiment 2

[0152] Example 2 Expression and purification of CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D 1. Expression of CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D

[0153] 1.1. The passage density of CHO-S cells (purchased from Thermo Fisher Scientific) 1 day before transfection was 0.5-0.6×10 6 / ml;

[0154] 1.2. Count the cell density on the day of transfection, when the density is 1~1.4×10 6 / ml, when the activity is >90%, it can be used for plasmid transfection;

[0155] 1.3. Preparation of transfection complex: For each project (CD19-CD3-CD28TsAb_M and CD19-CD3-CD28TsAb_D), two centrifuge tubes / flasks should be prepared, taking 20ml as an example, place them separately, and take the recombinant prepared in Example 1 Plasmid:

[0156] Add 600μl PBS and 20μg recombinant plasmid to tube ①, mix well;

[0157] Add 600μl PBS, 20ul FreeStyle to tube ② TM MAX Transfection Reagent (purchased from Thermo Fisher Scientific company), mixing;

[0158] 1.4. Add the diluted transfection re...

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Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a three-function molecule combining CD19, CD3 and CD28 and application of the three-function molecule. The three-function molecule capable of identifying CD19, CD3 and CD28 simultaneously is constructed through a genetic engineering method and an antibody engineering method. In the aspects of a preparation technology and practical application, the molecule has the obvious advantages that the effect of activated T cells is further improved while the targeting property on CD19 positive cells is given to the T cells; when only CD19 is added, the bonding and killing effects of the CD19-mediated T cells on CD19 positive target cells are superior to those of an anti-CD19 / anti-CD3 BiTE bispecific antibody, and the using convenience is superior to that of a CD19-targetted CAR-T technology.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a trifunctional molecule combined with CD19, CD3 and CD28 and its application. Background technique [0002] Human CD19 antigen is a 95kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily. In addition to being expressed on the surface of normal B lymphocytes, CD19 is also highly expressed in B cell malignant tumors. Therefore, anti-CD19 monoclonal full-length antibodies have been Developed and applied to the treatment of acute / chronic lymphocytic leukemia and B-cell lymphoma (Wang K et al., Experimental Hematology & Oncology, 1:36-42, 2012). Given that anti-CD19 monoclonal antibodies cannot effectively recruit cytotoxic T lymphocytes (CTL), this type of CD3 / CD8 double-positive T cells can specifically recognize the antigen peptide / MHC class I molecule complex on the surface of the target cell, and activate itself After release of perforin (Pe...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00
CPCA61K2039/505C07K16/468C07K2317/622
Inventor 陈帅朱化星廖远平
Owner CYTOCARES SHANGHAI INC
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