Trifunctional molecule combining CD19, CD3 and T-cell negative costimulatory molecules and application of trifunctional molecule

A co-stimulatory molecule and three-function technology, applied in the field of biomedicine, can solve the problems of high experimental conditions, difficulty in dose control, cumbersome steps, etc., to improve the activation effect, avoid T cell incapacity and death, and complicate the preparation process Effect

Active Publication Date: 2018-07-10
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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  • Trifunctional molecule combining CD19, CD3 and T-cell negative costimulatory molecules and application of trifunctional molecule
  • Trifunctional molecule combining CD19, CD3 and T-cell negative costimulatory molecules and application of trifunctional molecule
  • Trifunctional molecule combining CD19, CD3 and T-cell negative costimulatory molecules and application of trifunctional molecule

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preparation example Construction

[0144]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).

[0145] 6. The use of three functional molecules

[0146] 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.

[0147] In a preferred embodiment of the present invention, it is found through experiments that the three functional molecules of the present invention all have in vitro binding activity to CD19, CD3 ...

Embodiment 1

[0159] Example 1: Construction of CD19-CD3-PD-1 TsAb_M and CD19-CD3-PD-1 TsAb_D eukaryotic expression vectors

[0160] In the present invention, the TiTE trispecific antibody targeting the human CD19 protein on the surface of lymphoma B cells, the human CD3 on the surface of T cells and the negative co-stimulatory molecule PD-1 protein on T cells is named CD19-CD3-PD- 1 TsAb.

[0161] 1. CD19-CD3-PD-1 TsAb_M and CD19-CD3-PD-1 TsAb_D construction scheme design

[0162] The specific construction scheme of CD19-CD3-PD-1 TsAb_M in monomeric form is as follows: the sequences of anti-CD19 scFv, anti-CD3 scFv and anti-PD-1 scFv are connected through a linker (Linker), specifically, anti-CD19 scFv and anti-CD3 scFv The sequences of the anti-CD3 scFv and anti-PD-1 scFv are connected by the linker 2 (Linker 2).

[0163] The specific construction scheme of CD19-CD3-PD-1 TsAb_D in dimer form is as follows: the sequences of anti-CD19 scFv, anti-CD3 scFv and anti-PD-1 scFv are connected t...

Embodiment 2

[0205] Example 2: Expression and purification of CD19-CD3-PD-1 TsAb_M and CD19-CD3-PD-1 TsAb_D

[0206] 1. Expression of CD19-CD3-PD-1 TsAb_M and CD19-CD3-PD-1 TsAb_D

[0207] 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;

[0208] 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;

[0209] 1.3. Preparation of transfection complex: For each item (CD19-CD3-PD-1 TsAb_M and CD19-CD3-PD-1 TsAb_D), two centrifuge tubes / flasks should be prepared, take 20ml as an example, place them separately, and take the implementation The recombinant plasmid prepared in Example 1:

[0210] Add 600μl PBS and 20μg recombinant plasmid to tube ①, mix well;

[0211] Add 600μl PBS, 20ul FreeStyle to tube ② TM MAX Transfection Reagent (purchased from Thermo Fisher Scientific company), mixing;

[0212] 1.4. ...

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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a trifunctional molecule combining CD19, CD3 and T-cell negative costimulatory molecules and an application of the trifunctional molecule. The trifunctional molecule structurally comprises a first functional domain, a second functional domain and a third functional domain, wherein the first functionaldomain can combine CD19, the second functional domain can combine and activate CD3, and the third functional domain can combine and block the T-cell negative costimulatory molecules. The trifunctional molecule has obvious advantages in terms of preparation process and practical application, the functions of activated T-cells are further improved while targeting of the T-cells for CD19 positive cells is achieved, the killing effect of the mediated T-cells on the CD19 positive target cells in individual addition is superior to that of CD19 / CD3 BiTE bispecific antibodies, and use convenience ofthe trifunctional molecule is superior to that of a targeted CD19 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 T cell negative co-stimulatory molecules and an application thereof. 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 act...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00
CPCA61K39/00C07K16/2803C07K16/2809C07K16/2818C07K2317/31C07K2317/622C07K2317/73
Inventor 陈帅朱化星廖远平
Owner CYTOCARES SHANGHAI INC
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