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A bifunctional molecule binding CD3 and CD28 and its application

A bifunctional molecule and molecular technology, applied in the field of biomedicine, can solve the problems of increasing the workload and production cost of recombinant antibody expression and purification, and achieve the effects of stable preparation process and product, simple purification process, and better amplification effect

Active Publication Date: 2021-08-10
CYTOCARES SHANGHAI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 optimized when it is actually applied to the activation and expansion of T cells in vitro. Relative ratio of two full-length antibodies

Method used

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  • A bifunctional molecule binding CD3 and CD28 and its application
  • A bifunctional molecule binding CD3 and CD28 and its application
  • A bifunctional molecule binding CD3 and CD28 and its application

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preparation example Construction

[0075] The method of the present invention, the method of the present invention, comprising: constructing an expression vector comprising a dual function molecular gene sequence, and then transforming the expression vector of a dual functional molecular gene sequence to a host cell, separating from expression products Dual function molecule. In a preferred case of the present invention, the expression vector uses PCDNA 3.1. The host cells use Chinese hamster ovary CE1L, CHO.

[0076] 6. Use of dual function molecules

[0077] The dual functional molecules of the present invention can be used to prepare T cells having an oxidant propogent. In a preferred embodiment of the present invention, the human peripheral blood mononuclear cell, PBMC is the experimental material, and the dual function molecule prepared by the present invention, and anti-CD3 / anti-CD28 monoclonal full-length antibody (Anti-CD3 / Anti-CD28) acts on human blood PBMC, cell culture, which acts on the same donor s...

Embodiment 1

[0084] Example 1 CD3-CD28BSAB_M and CD3-CD28BSAB_D EQUG Expression Vector Construction

[0085] In the present invention, a bispecific antibody having a T cell surface human CD3 and a CD28 protein is a target point is Nd3-CD28BSAB.

[0086] I. Design of CD3-CD28BSAB_M and CD3-CD28BSAB_D

[0087] The specific construct of the CD3-CD28BSAB_M form in the monomer is: Between anti-CD3SCFV and anti-CD28SCFV sequences (GGGGS) 3 Linker is connected.

[0088] The CD3-CD28BSAB_D specific construction scheme in the form of dimer is: anti-CD3SCFV and anti-CD28SCFV sequences are connected via the IGD hinge region as Linker.

[0089] In order to express bispecific antibodies in mammalia, a codon optimization expressed in mammal systems is carried out for anti-CD3SCFV, anti-CD28SCFV and IgD hinge region sequences.

[0090] Specifically, the nucleotide sequence of the heavy chain variable region of the anti-CD3SCFV is shown in SEQ ID NO.12, specifically:

[0091] GACATCAAGCTGCAGCAGAGCGGCGCCGAGCTG...

Embodiment 2

[0121] Example 2: Expression and Purification of CD3-CD28BSAb_m and CD3-CD28BSAb_D

[0122] I. Expression of CD3-CD28BSAB_M and CD3-CD28BSAB_D

[0123] 1.1.cho-S cells (purchased from thermo fisher scientific) 1 day before transfection, 1 day is 0.5 ~ 0.6 × 10 6 / ml;

[0124] 1.2. Cell density statistics on the day of transfection, when the density is 1 ~ 1.4 × 10 6 / ml, viability> 90%, can be used for plasmid transfection;

[0125] 1.3. Transfection complex preparation: Each item (CD3-CD28BSAB_M and CD3-CD28BSAB_D) need to prepare two centrifuge / culture bottles, with 20 mL as an example, separately, and to take advantage of the recombinant plasmid prepared in the example:

[0126] 600 μL of PBS, 20 μg of recombinant plasmid, mixed;

[0127] Add 600 μL PBS, 20 ul freeestyle TM Max Transfection Reagent (purchased from Thermo Fisher Scientific), mixed;

[0128] 1.4. The diluted transfection reagent was added to the diluted recombinant plasmid, mixed uniform, formulated into tran...

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Abstract

The invention belongs to the technical field of biomedicine, and specifically relates to a bifunctional molecule binding to CD3 and CD28 and an application thereof. The bifunctional molecule of the present invention fuses the first functional domain capable of binding and activating the CD3 molecule on the surface of T cells and the second functional domain capable of binding and activating the CD28 molecule on the surface of T cells to the same protein peptide chain to form a bifunctional molecule. Produced by nuclear cell expression system, the expression product has a single structure, the purification process is simple, the protein yield is high, the preparation process and the product are stable; compared with the combination of anti-CD3 and anti-CD28 full-length antibodies, the bifunctional molecule has a greater effect on the expansion of T cells in vitro. The enhancement effect is better, the amount of protein is less, and it is easy to use. It can be added directly in the form of a solution without optimizing the relative ratio of the two full-length antibodies.

Description

Technical field [0001] The present invention belongs to the field of biomedical technology, and more particularly to a dual function molecule and its application thereof in conjunction with CD3 and CD28. Background technique [0002] T lymphocytes (T LYMPHOCYTE) comes from thymus, tap cells. Mature T cells exist in the thymus dependent area of ​​peripheral immune organs, occupying core status in adaptive cellular immune responses, while also playing an important aid in adaptive antigen-induced body fluid immune responses. Depending on the functionality, T cells can be divided into cytotoxic T Lymphocyte, CTL, auxiliary T cell (Helper T cell, TH) and regulatory T cells (Regulatory T cells (TREG). Among them, CTL expression CD8 is the main effector of adaptive cellular immunity. Its main function is to specifically identify endogenous antigenic peptide / MHC I molecular composites on the surface of the target cells, and they can secrete Polypoporin after it is activated. Granzyme, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C12N15/62C12N15/13C12N15/63C12N5/0783
CPCC07K16/2809C07K16/2818C07K16/468C07K2317/53C07K2317/55C07K2317/622C07K2319/00C12N5/0636C12N2501/50C12N2501/515
Inventor 陈帅廖远平朱化星
Owner CYTOCARES SHANGHAI INC