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Anti-PD-L1 and 4-1BB bifunctional antibody and medical application thereof

A bifunctional antibody, PD-L1 technology, applied in non-active ingredients medical preparations, antibodies, anti-tumor drugs, etc., can solve the problems of drug resistance, weak anti-tumor effect, limited efficacy in tumor patients, etc. Toxic and side effects, the effect of improving immunostimulatory activity

Pending Publication Date: 2022-07-12
INNOLAKE BIOPHARMA (HANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with Urelumab, Utomilumab has a good safety profile, but its antitumor effect is relatively weak
[0004] In recent years, although monoclonal antibody drugs that block the interaction between PD-L1 and PD-1 have achieved encouraging curative effects in the clinical treatment of some cancer patients, they still face limited efficacy and drug resistance for most tumor patients. The problem

Method used

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  • Anti-PD-L1 and 4-1BB bifunctional antibody and medical application thereof
  • Anti-PD-L1 and 4-1BB bifunctional antibody and medical application thereof
  • Anti-PD-L1 and 4-1BB bifunctional antibody and medical application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1 Construction of PD-L1 / 4-1BB bifunctional antibody

[0102] A hu6F9B1A6 / hu10B2-scFv bifunctional antibody was constructed.

[0103] In the construction, the 4-1BB humanized antibody was designed as a single-chain scFv (see SEQ ID NO: 17 for the sequence) and linked to the C-terminus of the hu6F9B1A6 heavy chain through Linker (G4S) 2 (eg, figure 1 shown), co-transfected with the light chain vector into 293 cells. Shake flasks at 37°C for 6 days. The supernatant was collected by centrifugation and purified with Protein A. After purification, various assays were performed, including binding and functional activity.

Embodiment 2

[0104] Example 2 Determination of the binding effect of humanized PD-L1 / 4-1BB bifunctional antibody to PD-L1 and 4-1BB proteins by ELISA

[0105] 96-well ELISA plates (Corning, Cat No: 9018) were coated with PD-L1-ECD-His or 4-1BB-ECD-His, left at 4 degrees overnight, and washed 3 times with washing buffer (PBS+0.05% Tween20) Then, add blocking buffer (PBS+1% BSA (Sigma, Cat No: V90093)) and incubate for 1 hour; wash the 96-well plate 3 times; add serially diluted samples to be tested including positive control and negative control, and then incubate For 1 hour, wash 3 times; add 100 μL of 1:10000-fold diluted goat anti-mouse IgG secondary antibody (Thermo, Cat No: 31432) to each well, incubate for 1 hour at room temperature, and wash 3 times; add 100 μL TMB (Beijing Bioall) to each well Cyber, Cat No: ES-002) for 3 minutes, and then add 100 μL / well of stop solution (2N H 2 SO 4 ) to terminate the reaction, and the OD450nm signal of each sample was measured with a Tecan Spar...

Embodiment 3

[0107] Example 3 Determination of the binding effect of humanized PD-L1 / 4-1BB bifunctional antibody to 293T-PD-L1 and Jurkat-4-1BB cells by FACS method

[0108] Add 50 μL of serially diluted samples to be tested including positive and negative controls mixed with 50 μL of 293T-PD-L1 or Jurkat-4-1BB cells (1×10 5 cells / well) were added to a U-bottom 96-well plate and incubated for 1 hour, centrifuged and washed twice with FACS buffer (PBS+3% FCS), and 1:400-fold diluted PE-labeled goat anti-mouse secondary antibody (Biolegend, CatNo: 405307), incubated for 30 minutes, washed with FACS buffer, and detected the PE signal of 293T-PD-L1 cells by BD C6 flow cytometer.

[0109] The experimental results are as Figure 4 and Figure 5 As shown, the PD-L1 / 4-1BB bifunctional antibody maintained its binding activity to 293T-PD-L1 and Jurkat-4-1BB cells.

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Abstract

The invention relates to the technical field of antibodies, in particular to a bifunctional antibody for resisting PD-L1 and 4-1BB and medical application of the bifunctional antibody. The selected 4-1BB antibody needs to be supported by a cross-linking effect in the aspect of immunocompetence stimulation, the cross-linking effect is avoided, and the 4-1BB antibody has no activity, so that toxic and side effects on the liver are avoided. In a tumor environment, tumor cells have a relatively high PD-L1 expression level. According to the PD-L1 / 4-1BB bifunctional antibody, the PD-L1 antibody part is combined with PD-L1 on the surface of a tumor cell, so that a relatively strong cross-linking effect is provided for a 4-1BB antibody. Therefore, the immunostimulatory activity of the PD-L1 / 4-1BB bifunctional antibody can be specifically and remarkably improved in a tumor microenvironment, so that toxic and side effects of the 4-1BB antibody in the liver are avoided or reduced.

Description

technical field [0001] The present invention relates to the technical field of antibodies, in particular, to anti-PD-L1 and 4-1BB bifunctional antibodies and their medical uses. Background technique [0002] PD-L1 (programmed death ligand) is a 40 kDa I-type transmembrane protein whose receptor is programmed cell death protein 1 (PD-1, also known as CD279). PD-L1 consists of an IgV-like domain, an IgC-like domain, a transmembrane domain, and a short cytoplasmic domain peptide (Keir et al., (2008) Annu Rev Immunol 26:677-704; Lin et al. al., (2008) PNAS 105:3011-3016). By interacting with PD-1 and CD80, PD-L1 plays an important role in the regulation of the body's immune system, such as inhibiting T cell receptor (TCR)-mediated signaling of IL-2 production and T cell proliferation activation. Knockdown of the PD-L1 gene results in an upregulation of T cell responses, resulting in autoreactive T cells (Latchman et al., (2004) PNAS 101:10691-10696). Abnormal expression level...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13C12N15/85C12N5/10A61K39/395A61K47/68A61P35/00A61P35/02
CPCC07K16/2827C12N15/85C12N5/0686A61K47/6801A61P35/00A61P35/02C07K2317/565C07K2317/622C07K2317/51C07K2317/56C12N2510/00C12N2800/107A61K2039/505C07K2317/31
Inventor 邱均专陈均勇王振生孙锴孙键李忠良区日山
Owner INNOLAKE BIOPHARMA (HANGZHOU) CO LTD
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