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Anti-CD3 antibody structural domain and T cell positive costimulatory molecular ligand fused bispecific molecule and application thereof

A technology of co-stimulatory molecules and structural domains, 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, simple purification process, and increased workload

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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  • Anti-CD3 antibody structural domain and T cell positive costimulatory molecular ligand fused bispecific molecule and application thereof
  • Anti-CD3 antibody structural domain and T cell positive costimulatory molecular ligand fused bispecific molecule and application thereof
  • Anti-CD3 antibody structural domain and T cell positive costimulatory molecular ligand fused bispecific molecule and application thereof

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preparation example Construction

[0142] 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).

[0143] 6. Uses of Bifunctional Molecules

[0144] 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 is proved by experiments that the bifunctional molecules in the form of monomers and dimers have in vitro binding activity with CD3 recombinant antigen and corresponding positive co-stimulatory molecular recombinant pro...

Embodiment 1

[0151] Embodiment 1: Construction of CD3-4-1BBL BsM_M and CD3-4-1BBL BsM_D eukaryotic expression vector

[0152] In the present invention, the bispecific molecule fused with the anti-T cell surface human CD3 protein scFv domain and T cell positive co-stimulatory molecule ligand 4-1BBL extracellular region domain is named CD3-4-1BBL BsM.

[0153] 1. CD3-4-1BBL BsM_M and CD3-4-1BBL BsM_D construction scheme design

[0154] The specific construction scheme of CD3-4-1BBL BsM_M in monomeric form is as follows: pass (GGGGS) between anti-CD3 scFv and 4-1BBL extracellular region sequence 3 Linker is connected.

[0155] The specific construction scheme of the CD3-4-1BBL BsM_D in dimer form is as follows: the anti-CD3 scFv and the 4-1BBL extracellular region sequence are connected through the IgD hinge region as a Linker.

[0156] In order to express the bispecific molecule in mammalian cells, codon optimization for expression in the mammalian system was carried out for the sequences ...

Embodiment 2

[0184] Example 2: Expression and purification of CD3-4-1BBL BsM_M and CD3-4-1BBL BsM_D

[0185] 1. Expression of CD3-4-1BBL BsM_M and CD3-4-1BBL BsM_D

[0186] 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;

[0187] 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;

[0188] 1.3. Preparation of transfection complex: For each item (CD3-4-1BBL BsM_M and CD3-4-1BBL BsM_D), two centrifuge tubes / bottles need to be prepared, take 20ml as an example, place them separately, and take the samples in Example 1 Prepare recombinant plasmids:

[0189] Add 600μl PBS and 20μg recombinant plasmid to tube ①, mix well;

[0190] Add 600μl PBS, 20ul FreeStyle to tube ② TM MAX Transfection Reagent (purchased from Thermo Fisher Scientific company), mixing;

[0191] 1.4. Add the diluted transfection reage...

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Abstract

The invention belongs to the technical field of biological medicines and particularly relates to an anti-CD3 antibody structural domain and T cell positive costimulatory molecular ligand fused bispecific molecule and application thereof. A first functional domain which can be combined with and activate T cell surface CD3 molecules and a ligand extracellular region structural domain of T cell positive costimulatory molecules are fused into a same protein peptide chain to form a difunctional molecule. A eukaryotic expression system is adopted for production, and structural singleness of expression products, simplicity and convenience in purification process, high protein yield and stability in preparation process and product are realized. Compared with anti-CD3 and anti-T cell positive costimulatory molecule full-length antibody combination, the difunctional molecule has advantages of excellent T cell in-vitro amplification effects, low protein consumption, simplicity and convenience inuse, availability for direct adding in a solution form and freeness of optimization of a relative proportion of two full-length antibodies.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a bispecific molecule fused with an anti-CD3 antibody domain and a T cell positive co-stimulatory molecule ligand 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 c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10
CPCC07K14/70521C07K14/70575C07K14/70578C07K16/2809C07K2319/00
Inventor 廖远平朱化星陈帅
Owner CYTOCARES SHANGHAI INC
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