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Bispecific antibody targeting CD96 as well as preparation method and application of bispecific antibody

A bispecific antibody and construction method technology, applied in the field of biopharmaceuticals, can solve problems such as heavy chain and light chain mismatch, inability to remove by-products, etc., to reduce drug resistance, reduce construction difficulty, and reduce by-product production Effect

Active Publication Date: 2021-06-15
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the two heavy chains and light chains of a classic bispecific antibody are expressed in host cells at the same time, there will be heavy and light chain mismatches and by-products that cannot be removed, and how to construct a stable Bispecific antibodies have also become a challenge, and there is currently no literature disclosing the use of CD96 in combination with other antibodies

Method used

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  • Bispecific antibody targeting CD96 as well as preparation method and application of bispecific antibody
  • Bispecific antibody targeting CD96 as well as preparation method and application of bispecific antibody
  • Bispecific antibody targeting CD96 as well as preparation method and application of bispecific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Construction and Production of CD96 ScFv-Holes-PD1 ScFv-Knobes and CD96 ScFv-(G4S)4-PD1 ScFv-Fc

[0069] (1) Construction and production of CD96 ScFv-Holes-PD1 ScFv-Knobes

[0070] T366W, S354C, S228P, R409K mutations were introduced into PD1 ScFv-Fc, and T366S, L368A, Y407V, Y349C, S228P, R409K mutations were introduced into CD96 ScFv-Fc to construct anti-human CD96 ScFv-holes lentiviral expression plasmids and anti-human CD96 ScFv-holes respectively. The lentiviral expression plasmids of human PD1 ScFv-knobes were packaged into lentiviruses respectively, then infected CHO cells at the same multiplicity of infection and screened for single clones to obtain the target cell line, cultured the target cell line and collected the culture supernatant, passed Protein A The target protein was purified by affinity chromatography column (nanomicro), desalted by molecular exclusion chromatography column Superdex 200pg (GE), and the purity was detected by SDS-PAGE to obt...

Embodiment 2

[0077] Example 2 Size identification of CD96 ScFv-Holes-PD1 ScFv-Knobes and CD96 ScFv-(G4S)4-PD1 ScFv-Fc antibodies

[0078] The purified antibody in Example 1 was verified using SDS-PAGE (the verification result is as follows: image 3 Shown), the position of the band is found to be correct, the theoretical size of CD96 ScFv-Holes-PD1 ScFv-Knobes is 103.79kDa, and its monomers are 51.49kDa and 52.32kDa respectively.

[0079] The theoretical size of the CD96 ScFv-(G4S)4-PD1 ScFv-Fc monomer is 79.40kDa, and the purified antibody was verified by SDS-PAGE (the verification result is as follows image 3 shown), and found that the position of the band was correct, and then determined by molecular exclusion (measurement results such as Figure 4 shown), the bispecific antibody was of high purity (>95%) and formed dimers.

Embodiment 3

[0080] The biological activity verification of embodiment 3 antibody

[0081] (1) Biological activity of monoclonal antibody

[0082] 1) The combination of antibody and antigen

[0083] The CHO-PD1 cell line was constructed exogenously, and the expression of the target antigen and the binding of the antibody to the target antigen were detected by flow cytometry. It was proved that the PD1 antibody can specifically recognize the PD1 antigen molecules on the exogenously constructed cell line CHO-PD1 and T cells. The 293T-CD96 cell line was constructed exogenously, and the expression of the target antigen and the binding of the antibody to the target antigen were detected by flow cytometry. It was proved that the CD96 antibody could specifically recognize the CD96 antigen molecules on the exogenously constructed cell line 293T-CD96 and T cells.

[0084] like Figure 5 and Image 6 As shown, the expression rates of target antigens of exogenously constructed CHO-PD1 and 293T-CD9...

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Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to a bispecific antibody targeting CD96 as well as a preparation method and application of the bispecific antibody. The bispecific antibody contains an anti-human CD96ScFv mutant, an anti-human PD1ScFv mutant and an IgG4Fc mutant, and the configuration of the bispecific antibody comprises CD96ScFv holes-PD1ScFv knobes and CD96ScFv-(G4S)4-PD1ScFv-Fc. The CD96 and PD-1 are combined for the first time, the CD96-PD1 bispecific antibody is constructed for the first time, the bispecific antibody has high affinity with the PD1 and the CD96, the CD96 is highly expressed in activated T cells and also highly expressed in activated NK cells, the expression quantity is increased along with the activation time, and the drug resistance problem caused by the single antibody of PD1 in the treatment process can be greatly reduced through the addition of the CD96.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a bispecific antibody targeting CD96 and its preparation method and application. [0002] technical background [0003] Currently, immune checkpoint blockers are used in the immunotherapy of various tumors. PD1 acts as an immune checkpoint on the surface of T cells. When it interacts with the ligands PD-L1 and PD-L2 on the surface of tumor cells, it initiates downstream signaling pathways and inhibits T cell activation. Antibodies are used to block PD1 and its ligands The interaction of PD1 has been proved to be effective in the clinical treatment of various malignant tumors, but due to the single antibody of PD1, drug resistance will occur during the treatment. Therefore, blocking PD1 alone cannot meet clinical expectations, and the development of bispecific antibodies for tumor treatment has become an urgent need. Due to the simultaneous targeting of two antigens ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/867C12N5/10A61K35/17A61P35/00
CPCC07K16/2896C07K16/2818C12N15/86C12N5/0636A61K35/17A61P35/00C07K2317/31C07K2319/30Y02A50/30
Inventor 马佳兵赵文旭洪娟徐艳敏沈俊杰幺瑞娜
Owner CHONGQING PRECISION BIOTECH CO LTD
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