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Bifunctional antibody for relieving immunosuppression in tumor immune microenvironment as well as application and preparation method of bifunctional antibody

A bifunctional antibody, immunosuppressive technology, applied in chemical instruments and methods, biochemical equipment and methods, antibodies, etc., can solve problems such as the disadvantage of tumor treatment effect, and achieve improved targeting and safety, efficient and safe selection. , the effect of enhancing affinity

Active Publication Date: 2021-10-15
拜盖特生物科技上海有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the contrary, in order to reduce the antibody gene, some studies compromised only the scFv structure of PD-1 / L1 antibody. Since scFv is usually much weaker than IgG antibody in blocking the combination of PD-1 and PD-L1, it may exist Significant disadvantage of tumor treatment effect

Method used

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  • Bifunctional antibody for relieving immunosuppression in tumor immune microenvironment as well as application and preparation method of bifunctional antibody
  • Bifunctional antibody for relieving immunosuppression in tumor immune microenvironment as well as application and preparation method of bifunctional antibody
  • Bifunctional antibody for relieving immunosuppression in tumor immune microenvironment as well as application and preparation method of bifunctional antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] This example constructs the recombinant PD-L1-huIgG1 Fc fusion protein, and its specific method is as follows:

[0050] 1. Synthesize the gene sequence of the 19th to 238th amino acid interval of PD-L1 and construct the expression vector of PD-L1-huIgG1 Fc fusion protein

[0051] The gene sequence from the 19th phenylalanine to the 238th arginine of human programmed death ligand-1 (PD-L1) (NCBI accession NP_054862) was synthesized by chemical synthesis, and the amino acid encoded by the gene sequence 序列为FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER(SEQID NO:1)。 Synthesize the gene sequence from the 100th proline to the 330th lysine of the human IgG1 heavy chain constant region (UniProtKB / Swiss-Protaccession No.P01857.1) by chemical synthesis, the amino acid sequence encoded by the gene sequence 为PKS...

Embodiment 2

[0055] This example provides an anti-human PD-L1 murine antibody, the preparation method of which is as follows:

[0056] 1. Immunization of animals

[0057] 2 mg / mL of the above recombinant PD-L1-huIgG1 Fc fusion protein was mixed and emulsified as an antigen with an equal volume of complete Freund's adjuvant (Sigma-Aldrich), and 10 6-week-old female Balb / c mice were subcutaneously immunized. After the initial immunization, a booster immunization was carried out every ten days, and a total of four subcutaneous immunizations were performed. In the fifth immunization, the spleen was directly immunized with MSLN-huIgG1Fc fusion protein.

[0058] 2. Serum titer detection

[0059] Before each booster immunization, 50 μL of blood was collected from the tail vein, and the cells were removed by centrifugation, and the serum was retained. Add recombinant PD-L1 50ng / well to the ELISA microwell plate, and coat overnight at 4°C. Wash three times with PBS, add 1% BSA / PBS, 200 μL / well, ...

Embodiment 3

[0115] Based on Example 2, this example provides a bifunctional antibody that relieves immunosuppression in the tumor immune microenvironment. The design and preparation process are as follows:

[0116] 1. Structural design of bifunctional antibody

[0117] Design a bifunctional scDb (single chain diabody) that is stabilized by a disulfide bond formed by the combination of the variable region of the anti-PD-L1 antibody and the variable region of the TGF-βs antibody (formation of a disulfide bond through the hinge region containing cysteine)- The CH3 structure (hereinafter referred to as bsDHC, bispecific scDb-Hinge-CH3), specifically consists of the light chain variable region of the PD-L1 antibody, the L1 short connecting peptide, the heavy chain variable region of the TGF-β antibody, the L2 long connecting peptide, Light chain variable region of TGF-β antibody, L3 short connecting peptide, heavy chain variable region of PD-L1 antibody, hinge domain of human IgG 1 that can ge...

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Abstract

The invention provides a bifunctional antibody for relieving immunosuppression in a tumor immune microenvironment as well as application and a preparation method of the bifunctional antibody. The bifunctional antibody is of a bifunctional scDb-CH3 structure with a stable disulfide bond formed by combining a PD-L1 antibody variable region and a TGF-[beta]s antibody variable region, and comprises the PD-L1 antibody variable region, the TGF-[beta]s antibody variable region, a hinge structural domain of human IgG 1 capable of generating the disulfide bond, a CH3 structural domain of human IgG1 and a connecting peptide. The bifunctional antibody provided by the invention is designed into a divalent form with a stable disulfide bond so as to enhance the affinity to a target spot, has a gene smaller than that of an IgG antibody, can block PD-L1 and TGF-beta signal channels at the same time, and can realize the effects of low dose and high drug effect; besides, the bifunctional antibody selects high-expression PD-L1 of tumors as a target spot, so that the bifunctional antibody has the effect of enriching in a tumor microenvironment in a targeted manner without blocking TGF-beta signals in a non-pathophysiological process, the targeting property and safety of drugs are improved, and more efficient and safe choices are provided for tumor treatment.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a bifunctional antibody capable of releasing immunosuppression in a tumor immune microenvironment and its application and preparation method. Background technique [0002] It is well known that the programmed cell death-1 receptor (programmed cell death-1, PD-1) / programmed cell death-ligand 1 (programmed cell death-ligand 1, PD-L1) immunosuppressive signaling channel will Inhibiting the expansion of T cells, reducing the function of effector T cells, causing immune tolerance, and promoting T cell apoptosis will seriously weaken the tumor killing effect of tumor infiltrating T cells. Many anti-PD-1 and PD-L1 antibody drugs that have been on the market have significant curative effects on a wide range of tumor indications. This is a huge advancement in the field of biopharmaceuticals, which has also prompted the industry to pay more attention to the release of immunosuppression in the tu...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/62C12N15/85C12N5/10C12N7/01A61K39/395A61K39/00A61K35/768A61P35/00
CPCC07K16/2827C07K16/22C07K14/7051C12N7/00C12N5/0636A61K39/39566A61K39/39558A61K39/3955A61K39/001111A61K39/00113A61K35/768A61P35/00C07K2317/31C07K2317/56C07K2319/30C12N2510/00A61K2039/507A61K2039/5158
Inventor 姜钰超程硕
Owner 拜盖特生物科技上海有限公司
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