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TAA/CTLA-4/IL15 three-function fusion protein and application thereof

A CTLA-4, fusion protein technology, applied in the direction of peptide/protein components, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, etc., can solve the problem of limited response rate , drug resistance and other issues

Pending Publication Date: 2021-08-27
QUREBIO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] Existing CTLA-4 inhibitors still have common problems of limited response rate and drug resistance when single-agent treatment of solid tumors

Method used

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  • TAA/CTLA-4/IL15 three-function fusion protein and application thereof
  • TAA/CTLA-4/IL15 three-function fusion protein and application thereof
  • TAA/CTLA-4/IL15 three-function fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0241] Example 1: Cloning, expression and purification of three functional fusion proteins

[0242] Vector construction process

[0243] Construct a vector encoding an exemplary trifunctional fusion protein sequence (the trifunctional fusion protein sequence and design are shown in Table 1), and the plasmids contain DHFR and GS as selection markers, which can be used for stable strain screening; take CHO563880 as an example to illustrate the clone construction method : Construct QD3880 vector encoding ipilimumab VL-CL, see SEQ ID NO: 16 and ipilimumab VH-CH1-FC (Knob), Fc contains Knob mutation, namely T366W mutation, see SEQ ID NO: 17, the plasmid contains DHFR as a screening marker, can Used for stable strain screening; construction of CHO56 vector encoding light chain sequence of anti-tumor specific antigen antibody fusion IL15 is Codrituzumab VL-IL15 (L52C), see SEQ ID NO: 18, and variable heavy chain of anti-tumor specific antigen antibody Region fusion IL15Ra is Codrituzu...

Embodiment 2

[0259] Example 2: ELISA detection of trifunctional fusion protein binding to CTLA4 protein

[0260]Experimental method: Add His-tag antibody (GenScript, A00174-40) at 1 μg / ml, 100 μl per well into the ELISA plate, and incubate overnight at 4°C; after blocking with 5% non-fat milk for 1 hour at room temperature, add 0.1 μg / ml, 100 μl CTLA-4 protein (Sino Biological, 11159-H08H-B) per well, incubate at room temperature for 1 hour; each antibody to be tested was serially diluted with blocking solution, 100 μl was added to the well plate, and incubated at room temperature for 1 hour; HRP- goat anti human Fc (Abcam, ab97225) was diluted 1:10000, 100 μl per well was added to the ELISA plate, and incubated at room temperature for 1 hour; 100 μl was added to each well of TMB, and the color was developed for 5 minutes at room temperature in the dark, and 1M H2SO4 100 μl per well was added to stop the color development , Measure the absorbance at a wavelength of 450 nm in a microplate ...

Embodiment 3

[0261] Example 3: ELISA detection of trifunctional fusion protein binding to IL15 antibody

[0262] Method: Add goat anti human IL-15 antibody (R&D systems, AB-247-NA) at 5 μg / ml, 100 μl per well into the ELISA plate, incubate overnight at 4°C; block with 5% non-fat milk at room temperature for 1 hour , each antibody to be tested was diluted with blocking solution, 100 μl per well was added to the plate, and incubated at room temperature for 1 hour; HRP-goat anti human Fab (Abcam, ab87422) was diluted 1:10000, 100 μl was added to the ELISA plate, and incubated at room temperature 1 hour; 100 μl of TMB was added to each well, the color was developed for 10 minutes at room temperature in the dark, and 1M H 2 SO 4 100 μl per well was used to stop color development, and the absorbance value at a wavelength of 450 nm was measured with a microplate reader. The result is as Figure 10 shown. Experimental conclusion: CHO683880, CHO563880, CHO593880 and CHO653880 molecules all hav...

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Abstract

The invention provides a TAA / CTLA-4 / IL15 three-function fusion protein and an application thereof. The three-function fusion protein has the following forms: an anti-TAA antibody is an antibody, an antibody derivative or a polypeptide fragment combined with at least one antigen; the anti-CTLA-4 antibody is an antibody, an antibody derivative or a polypeptide fragment which is combined with CTLA-4; the compound is IL15 or IL5 / IL15Ra, or mutation, truncation and various derivatives of IL15 capable of being combined with IL15Ra, or mutation, truncation and various derivatives of IL15Ra capable of being combined with IL15, or a compound formed by the two; and a heterodimer formed by the FC. The three-function fusion protein can remove Treg cells, stimulate immune response to tumors and solve the problem of mismatching of light chains and heavy chains of a bispecific antibody at the same time.

Description

technical field [0001] The invention relates to a three-function fusion protein of tumor targeting TAA, anti-CTLA-4 antibody and T cell activation containing cytokine IL15, which belongs to the field of biotechnology. Background technique [0002] 1. Bispecific antibody [0003] Bispecific antibody BsAb, also known as bifunctional antibody, can simultaneously recognize and bind two different antigens and epitopes, and block two different signaling pathways to exert its effect. Compared with ordinary antibodies, BsAb has added a specific antigen-binding site, and has shown the following advantages in terms of treatment: [0004] Mediating the killing of immune cells on tumors: An important mechanism of action of bispecific antibodies is to mediate the killing of immune cells. Bispecific antibodies have two antigen-binding arms, one of which binds to the target antigen and the other binds to the target antigen on the effector cells. Labeled antigen binding, which can activat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/395A61P35/00A61P31/00A61P37/02A61K38/20
CPCA61K38/2086A61K39/39558A61K39/39566A61P31/00A61P35/00A61P37/02C07K14/5409C07K14/5443C07K16/28C07K16/2818C07K2319/30A61K2300/00
Inventor 屈向东潘琴
Owner QUREBIO LTD
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