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Trispecific molecules fused with anti-CD19, anti-CD3 antibody domains and T-cell positive co-stimulatory molecule ligands and applications

A technology of co-stimulatory molecules and structural domains, applied in antibody medical components, antibodies, applications, etc., can solve problems such as storms, shock, and difficulty in dose control

Active Publication Date: 2021-01-15
CYTOCARES SHANGHAI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Trispecific molecules fused with anti-CD19, anti-CD3 antibody domains and T-cell positive co-stimulatory molecule ligands and applications
  • Trispecific molecules fused with anti-CD19, anti-CD3 antibody domains and T-cell positive co-stimulatory molecule ligands and applications
  • Trispecific molecules fused with anti-CD19, anti-CD3 antibody domains and T-cell positive co-stimulatory molecule ligands and applications

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Experimental program
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preparation example Construction

[0154] 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).

[0155] 6. The use of three functional molecules

[0156] 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.

[0157] 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 with CD19 re...

Embodiment 1

[0169] Embodiment 1: Construction of CD19-CD3-4-1BBL TsM_M and CD19-CD3-4-1BBL TsM_D eukaryotic expression vector

[0170] In the present invention, a fusion of 1) anti-lymphoma B cell surface human CD19 protein scFv domain, 2) anti-T cell surface human CD3 protein scFv domain and 3) T cell positive co-stimulatory molecule ligand 4-1BBL The TiTE trispecific molecule for the extracellular domain was named CD19-CD3-4-1BBL TsM.

[0171] 1. CD19-CD3-4-1BBL TsM_M and CD19-CD3-4-1BBL TsM_D construction scheme design

[0172] The specific construction scheme of CD19-CD3-4-1BBL TsM_M in monomeric form is as follows: the sequence of anti-CD19scFv, anti-CD3scFv and 4-1BBL extracellular region is connected by a linker (Linker), specifically, anti-CD19scFv and anti-CD3scFv are connected by The connecting fragment 1 (Linker 1) was connected, and the anti-CD3 scFv and the 4-1BBL extracellular region sequence were connected through the connecting fragment 2 (Linker 2).

[0173] The specifi...

Embodiment 2

[0213] Example 2: Expression and purification of CD19-CD3-4-1BBL TsM_M and CD19-CD3-4-1BBL TsM_D

[0214] 1. Expression of CD19-CD3-4-1BBL TsM_M and CD19-CD3-4-1BBL TsM_D

[0215] 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;

[0216] 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;

[0217] 1.3. Preparation of transfection complex: For each project (CD19-CD3-4-1BBL TsM_M and CD19-CD3-4-1BBLTsM_D), two centrifuge tubes / flasks should be prepared, take 20ml as an example, place them separately, and take the example The recombinant plasmid prepared in 1:

[0218] Add 600μl PBS and 20μg recombinant plasmid to tube ①, mix well;

[0219] Add 600μl PBS, 20ul FreeStyle to tube ② TM MAX Transfection Reagent (purchased from Thermo Fisher Scientific company), mixing;

[0220] 1.4. Add the...

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Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a three-specificity molecule fused with an anti-CD19 and anti-CD3 antibody structural domain and a T cell positive costimulatory molecule ligand and application of the three-specificity molecule. A first functional domain which can be combined to CD19, a second functional domain which can be combined to and activates T cell surface CD3 molecules and a ligand extracellular region structural region of T cell positive costimulatory molecules are fused to the same protein peptide to form trifunctionalmolecules, production is implemented by a eukaryotic cell expression system, an expressed product is single in structure, a purifying process is simple and convenient, the yield of protein is high, apreparation process and products are stable, and the three-specificity molecule is convenient to use; during adding independently, mediated T cells has excellent killing effect on CD19 positive targetcells; and operation steps such as virus mediated transgene, in-vitro T cell culture and retransfusion and the like are not involved, the three-specificity molecule is convenient to use and controllable in dosage, after the three-specificity molecule enters the body of a patient, the risk of causing excessive releasing of cell factors is small, and toxic and side effects during use of CAR-T are avoided.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a trispecific molecule fused with anti-CD19, anti-CD3 antibody domains and T cell positive co-stimulatory molecule ligands 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 surfac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00
CPCA61K39/00C07K16/2803C07K16/2809C07K16/2827C07K16/2875C07K16/2878C07K2317/31C07K2317/622C07K2317/73
Inventor 陈帅朱化星廖远平
Owner CYTOCARES SHANGHAI INC