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Bifunctional molecule combining CD3 and T-cell negative costimulatory molecules and application of bifunctional molecule

A technology of bifunctional molecules and co-stimulatory molecules, applied in the field of biomedicine, can solve the problems of increasing the workload and production costs of recombinant antibody expression and purification, and achieve the effects of stable preparation process and products, single structure, and high protein yield

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, the combined use of two monoclonal full-length antibodies still has some shortcomings in specific applications, such as significantly increasing the workload and production costs of recombinant antibody expression and purification, and must be used in the actual application of T cell activation and expansion in vitro. Optimizing the Relative Ratio of Two Full-Length Antibodies

Method used

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preparation example Construction

[0140] The method for preparing the aforementioned bifunctional molecule of the present invention comprises: constructing an expression vector containing the gene sequence of the bifunctional molecule, then transforming the expression vector containing the gene sequence of the bifunctional molecule into a host cell to induce expression, and isolating the expression product to obtain the Bifunctional 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).

[0141] 6. Uses of Bifunctional Molecules

[0142] The bifunctional molecule of the invention can be used to prepare T cell expansion agent in vitro. In a preferred embodiment of the present invention, it has been proved by experiments that the bifunctional molecules in the form of monomers and dimers have in vitro binding activity to CD3 and corresponding T cell negative co-stimulatory molecule recombinant antigens, and...

Embodiment 1

[0149] Example 1: Construction of CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D eukaryotic expression vectors

[0150] In the present invention, the bispecific antibody targeting the human CD3 protein on the surface of T cells and the negative co-stimulatory molecule PD-1 protein on T cells is named CD3-PD-1 BsAb.

[0151] 1. CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D construction scheme design

[0152] The specific construction scheme of CD3-PD-1 BsAb_M in monomeric form is: passing between anti-CD3 scFv and anti-PD-1 scFv sequences (GGGGS) 3 Linker is connected.

[0153] The specific construction scheme of CD3-PD-1 BsAb_D in dimer form is as follows: the anti-CD3 scFv and anti-PD-1 scFv sequences are connected through the IgD hinge region as a Linker.

[0154] In order to express the bispecific antibody in mammalian cells, the sequences of anti-CD3 scFv, anti-PD-1 scFv and linker (Linker) were codon optimized for expression in mammalian system.

[0155] Specifically, the nucleotide sequen...

Embodiment 2

[0186] Example 2: Expression and purification of CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D

[0187] 1. Expression of CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D

[0188] 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;

[0189] 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;

[0190] 1.3. Preparation of transfection complex: For each project (CD3-PD-1 BsAb_M and CD3-PD-1 BsAb_D), two centrifuge tubes / flasks should be prepared. Take 20ml as an example and place them separately. Prepare recombinant plasmids:

[0191] Add 600μl PBS and 20μg recombinant plasmid to tube ①, mix well;

[0192] Add 600μl PBS, 20ul FreeStyle to tube ② TM MAX Transfection Reagent (purchased from Thermo Fisher Scientific company), mixing;

[0193] 1.4. Add the diluted transfection reagent to the diluted recombinant plasm...

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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a bifunctional molecule combining CD3 and T-cell negative costimulatory molecules and an application of the bifunctional molecule. A first functional domain capable of combining and activating a CD3 molecule on a T-cell surface and a second functional domain capable of combining and blocking the T-cell negative costimulatory molecule are fused into the same protein peptide chain to form the bifunctional molecule, eukaryotic expression system production is adopted, an expression product is single in structure, a purification process is simple and convenient, protein yield is high, and the preparation process and the product ate stable. Compared with CD3 and T-cell positive (negative) costimulatory molecule full-length antibody combination, the bifunctional molecule has better in-vitro expansion effects on T-cells, is simple and convenient to use and can be directly added in a solution form,protein dosage is less, and optimization of the relative proportion of two full-length antibodies is omitted.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a bifunctional molecule combined with negative costimulatory molecules of CD3 and T cells and an application thereof. Background technique [0002] T lymphocytes (T lymphocytes) are derived from the thymus (Thymus), so they are called T cells. Mature T cells exist in the thymus-dependent area of ​​peripheral immune organs, occupy a central position in the adaptive cellular immune response, and also play an important auxiliary role in the humoral immune response induced by thymus-dependent antigens. According to different functions, T cells can be divided into cytotoxic T cells (CTL), helper T cells (Helper T cells, Th) and regulatory T cells (Regulatory T cells, Treg). Among them, CTL expresses CD8 and is the main effector cell of adaptive cellular immunity. Its main function is to specifically recognize the endogenous antigen peptide / MHC class I molecular comple...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13C12N5/0783
CPCC07K16/2803C07K16/2809C07K16/2818C07K2317/31C07K2317/622C07K2317/74C12N5/0646C12N2501/51C12N2501/515C12N2501/599
Inventor 朱化星陈帅廖远平
Owner CYTOCARES SHANGHAI INC
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