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Bispecific antibody and application thereof

A bispecific antibody, binding activity technology, applied in the field of tumor therapy and molecular immunology, can solve the problems of low binding activity and low tumor cell killing activity

Active Publication Date: 2020-01-10
L&L BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the low binding activity of the TIM-3 antibody part in the bispecific antibody and its corresponding independent intact antibody to human TIM-3 and the activation of PBMC (NK) cell killing in the prior art. Defects such as low activity of tumor cells, providing a bispecific antibody and its use

Method used

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  • Bispecific antibody and application thereof
  • Bispecific antibody and application thereof
  • Bispecific antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Cloning, expression and purification of embodiment 1 antigen and antibody

[0063] Human TIM-3, PD-1, PD-L1 extracellular region-human IgG1 Fc fusion protein and -his tag protein used in the present invention are obtained by cloning, expression and purification of the present invention. Parts were purchased from the following different companies: TIM-3-his (Cat. No.: TM3-H5229), TIM-3-hFc (Cat. No.: TM3-H5258) were purchased from Beijing Baipusaisi Biotechnology Co., Ltd.; TIM-3-his- hFc was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., item number: 10390-H03H.

[0064] Antibodies used in the present invention include recombinant antibodies, bispecific antibodies, Tim-3 positive control antibody ABTIM3 (the sequence is from the heavy chain shown in SEQ ID NO:34 in US20150218274A1, and the light chain shown in SEQ ID NO:22) Chain), referred to as positive control or positive antibody or control antibody in the following examples. PD-1 antibody Nivo (Nivo...

Embodiment 2

[0079] Example 2 Anti-TIM-3 Antibody Binding ELISA Experiment

[0080] Dilute goat-anti-hFc (Jackson, 109-005-008) to a concentration of 1 μg / ml with PBS buffer at pH 7.4, and add to a 96-well microtiter plate (Corning, CLS3590-100EA) at a volume of 50 μl / well , placed in a 37°C incubator for 2 hours. (Or directly use the antigen TIM-3, 0.5 μg / ml to coat the plate and directly add the antibody to be tested). After discarding the liquid, add 200 μl / well of 5% skimmed milk (bright skimmed milk powder) blocking solution diluted with PBS, incubate at 37°C for 2.5 hours or place at 4°C overnight (16-18 hours) for blocking. Discard the blocking solution, and wash the plate 5 times with PBST buffer (PBS with pH 7.4 containing 0.05% tweeen-20), then add 50 μl / well, 0.5 μg / ml TIM-3-hFc (Example 1), set Incubate in a 37°C incubator for 2 hours. After incubation, wash the plate 6 times with PBST, add 50 μl / well supernatant (containing detection antibody) or different concentrations of...

Embodiment 3

[0081] Example 3 Anti-human TIM-3 antibody human PBMC tumor cell killing activity test (anti-human TIM-3 antibody activates human PBMC tumor cell killing activity test)

[0082] Human NK cells come from human peripheral blood mononuclear cells (PBMC), and PBMC comes from peripheral blood donated by healthy people, and is separated and extracted by the present invention. Divide PBMCs by 2.5 × 10 5 cells / well, K562 (ATCC catalog number: CCL-243 TM Agent: Shanghai Suer Biotechnology Co., Ltd.) Press 5×10 4 Cells / well were plated in 96-well plates (Corning 3599). The hybridoma supernatant antibody or purified antibody to be tested was added to a 96-well cell plate, incubated in a 37°C incubator for 6 hours, and then detected with an LDH detection kit (Shanghai Tongren Biotechnology Co., Ltd., catalog number: CK12) according to the instructions , read the 490nm absorbance value (OD) with MMLTISKAN Go microplate reader, calculate the percentage change of the release amount of LDH...

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Abstract

The invention discloses a bispecific antibody and an application thereof. Wherein the bispecific antibody comprises a first protein functional area and a second protein functional area; wherein the first protein functional area is the protein functional area targeting TIM-3; the TIM-3 full-length antibody of the targeting TIM-3 corresponding to the first protein functional area has weak binding activity with macaca TIM-3, has strong binding activity with marmoset and human TIM-3, and can activate the killing effect of human NK cells on tumor cells. The bispecific antibody not only can retain the activity of activating PBMC (NK) to kill tumor cells of the single TIM-3 antibody, but also can retain the original activity of the other protein functional area, and can achieve the activity equivalent to or better than the activity of activating T lymphocytes by combining two molecules (synergistic effect). The bispecific antibody (e.g., LB141) of the present invention has a more excellent effect in therapy than PD-1 antibody treatment alone or combined use of the TIM-3 antibody and the PD-1 antibody.

Description

technical field [0001] The invention belongs to the fields of tumor therapy and molecular immunology, and specifically relates to a bispecific antibody comprising two protein functional regions, wherein one protein functional region is a protein functional region targeting TIM-3. Background technique [0002] Tumor immunotherapy is a major breakthrough in the field of tumor treatment in recent years. In recent years, the research on the immune checkpoint PD-1 / PD-L1 has been extremely hot. In the past less than a year, 5 PD-1 antibody drugs have been launched in China. Among immune checkpoints other than PD-1 / PD-L1, T cell immunoglobulin domain and mucin domain 3 (TIM-3) is an immune system that may cooperate with PD-1 / PD-L1 to regulate T cell activity. One of the checkpoint molecules. TIM-3 is a transmembrane receptor protein expressed on IFN-γ-secreting Th1 (T helper 1) CD4+ cells and cytotoxic CD8+ T (Tc1) cells. In addition to the expression of Th1 and Tc1 that secrete...

Claims

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Application Information

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IPC IPC(8): C07K16/46A61K39/395A61P35/00A61P35/02G01N33/68
CPCC07K16/2803C07K16/2818A61P35/00A61P35/02C07K2317/31C07K2317/24C07K2317/92C07K2317/565C07K2317/76C07K2317/73C07K2317/94A61K39/39541A61K47/00A61K47/22A61K47/26A61K2039/505C07K2317/622G01N33/68G01N2333/70503G01N2333/70521
Inventor 刘佳建
Owner L&L BIOPHARMA CO LTD
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