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Bispecific antibody capable of combining PD-L1 and TGF-beta of mouse as well as preparation method and application thereof

A bispecific antibody, PD-L1 technology, applied in chemical instruments and methods, antibodies, specific peptides, etc., can solve problems such as reducing stromal cell TGF-β activity, stimulating immune response, tumor regression, etc., to correct immune microbes. environment, enhanced tumor suppressor activity, and high expression

Pending Publication Date: 2021-05-28
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mouse breast cancer EMT-6 model simulates the phenotype of epithelial cancer, and the effect of blocking PD-L1 or TGF-β alone is not good, and the combined inhibition of TGF-β and PD-1 signaling reduces the TGF-β activity of stromal cells , promote T cell entry into the tumor, stimulate a strong immune response, and lead to tumor regression

Method used

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  • Bispecific antibody capable of combining PD-L1 and TGF-beta of mouse as well as preparation method and application thereof
  • Bispecific antibody capable of combining PD-L1 and TGF-beta of mouse as well as preparation method and application thereof
  • Bispecific antibody capable of combining PD-L1 and TGF-beta of mouse as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1 Construction of bispecific antibody YM101 binding to mouse PD-L1 and TGF-β:

[0101] In the present invention, a bispecific antibody binding to mouse PD-L1 and TGF-β is constructed by using the commonly used molecular cloning technology. The bispecific antibody contains four polypeptide chains and can bind to two antigens. Antigen A is PD- L1, antigen B is TGF-β; specifically, the DNA sequence of the coding gene of each chain corresponding to the diabody is cloned into the pcDNA3.1(-) eukaryotic expression vector (commercially available, purchased from Invitrogen, catalog number: V79520) middle. Wherein the obtained bispecific antibody includes figure 1 The structure shown, its specific sequence information is shown in Table 2 below.

[0102] Table 2 Sequence information corresponding to the double antibody

[0103]

[0104] Combining the above table 2 and figure 1 It can be seen that the bispecific antibody constructed in the present invention includes...

Embodiment 2

[0118] Example 2 Preparation, expression and purification of bispecific antibody YM101 binding to mouse PD-L1 and TGF-β:

[0119] Plasmids were extracted according to conventional plasmid extraction methods, and used to transfect 293 cells or CHO-S cells. The transfection reagent could be Lipofectamine2000 (Thermo fisher, Cat. No. 11668019). Transfected 293 cells or CHO cells were incubated at 37°C in 5% CO 2 Suspended and shaken culture in a shaker for 7 to 10 days. The supernatant was harvested by centrifugation at 3000 xg and filtered through a 0.22 μm filter. The bispecific antibody was purified by protein A affinity chromatography and cation exchange chromatography.

[0120]The concentration of purified diabody was determined by UV absorbance at 280 nm and the corresponding extinction coefficient for each protein. The purified protein was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular weight of the bispecific antibody was about 203KD.

[...

Embodiment 3

[0122] Example 3 Affinity detection of the PD-L1 end of the bispecific antibody YM101 that binds to mouse PD-L1 and TGF-β:

[0123] In this example, an enzyme-linked immunosorbent assay (ELISA) was used to detect the affinity between the bispecific antibody and PD-L1.

[0124] Among them, mouse PD-L1 (50010-M03H, Sino Biological) (200ng per well, 100ul per well) was coated on a polyacrylamide plate and kept at 4°C overnight. The plate was washed with PBS containing 0.05% Tween20, and blocked with PBS containing 3% BSA for 3 hours at 37°C. Then, add 100uL YM101 or other antibodies, and incubate at 37°C for 1 hour. Wash the plate, add 100uL HRP-labeled anti-hIgG secondary antibody (1:5000, A80-319P, Bethyl), and incubate at 37°C for 1 hour. The plate was washed, and 100 uL ELISA chromogenic substrate (555214, BD Biosciences) was added to each well. After reacting at room temperature for 15 minutes, 100 ul of 2 mol / L HCl was added to each well to stop the color development. A...

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Abstract

The invention discloses a bispecific antibody capable of combining PD-L1 and TGF-beta of a mouse as well as a preparation method and application thereof, and belongs to the technical fields of immunology and antibody engineering. The bispecific antibody comprises two long chains which are symmetrically distributed and form pairing and short chains which are positioned on the outer sides of the long chains and are respectively paired with the long chains, the amino acid sequence of each long chain is SEQ ID NO: 1 and comprises a polypeptide chain as shown in the following formula I, and the amino acid sequence of each short chain is SEQ ID NO: 2 and comprises a polypeptide chain as shown in the following formula II. Formula 1: VHa-CH1-X1-VLb-X2-CH2-CH3, and formula II: VLa-CL-X1-VHb. The bispecific antibody disclosed by the invention is specifically combined with PD-L1 and TGF-beta of a mouse with high affinity, and a good biological function is achieved by adjusting signal transduction pathways participated by various antigens.

Description

technical field [0001] The invention relates to a newly prepared bispecific antibody, which belongs to the technical field of immunology and antibody engineering, in particular to a bispecific antibody binding to mouse PD-L1 and TGF-β, its preparation method and application. Background technique [0002] Monoclonal antibodies are highly specific antibodies that only act on a single antigenic epitope and have been widely used in the treatment of many diseases, such as cancer, inflammatory diseases, autoimmune diseases and infectious diseases. However, when this therapeutic molecule is used alone, the therapeutic molecule cannot exhibit sufficient pharmacological effects. This may be due to the complexity of the disease. For example, cancer or inflammatory diseases often involve multiple disease-mediated molecular pathways and intersecting interactions between signaling pathways. In these cases, molecules targeting a single target do not provide optimal therapeutic efficacy....

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/85A61K39/395A61P35/00A61P37/00
CPCC07K16/2827C07K16/2863C12N15/85A61P35/00A61P37/00A61K2039/505C07K2317/31C07K2317/56C07K2317/52
Inventor 吴孔明易铭方丽娟严永祥周鹏飞
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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