Single variable domain antibody targeting human programmed death ligand 1 (PD-L1) and derivative of single variable domain antibody

A PD-L1, domain antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. In order to achieve the effect of inhibiting tumor growth, flexible application form, and good biological activity

Pending Publication Date: 2021-10-22
MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, PD-1 / PD-L1 monoclonal antibodies have shown good therapeutic effects in the clinical treatment of various malignant tumors, but there are problems such as large dosage and low overall response rate.

Method used

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  • Single variable domain antibody targeting human programmed death ligand 1 (PD-L1) and derivative of single variable domain antibody
  • Single variable domain antibody targeting human programmed death ligand 1 (PD-L1) and derivative of single variable domain antibody
  • Single variable domain antibody targeting human programmed death ligand 1 (PD-L1) and derivative of single variable domain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Phage Display Camel Single Variable Domain Antibody Immune Library Construction

[0066] Antigens were used to immunize camels, peripheral blood mononuclear cells (PBMCs) were isolated and total RNA was extracted for reverse transcription, and the reverse transcription product was used as a template to amplify the variable domain of the heavy-chain antibody domain (variable domain of the heavy-chain) ofheavychain antibody, VHH) and connected to the phage display vector, electroporated into Escherichia coli TG1 competent cells, and camel immune library was constructed. Camels are immunized every two weeks, a total of 4 times. Each injection of 0.8mg PD-L1 extracellular region recombinant protein (self-expression and purification, gene sequence number: NP_054862.1, 19aa-238aa), supplemented with Freund's incomplete adjuvant (Sigma, Cat: F5506-10ml), take Subcutaneous multi-point injection. Two weeks after each immunization, 1 mL of serum was collected, and th...

Embodiment 2

[0067] Example 2: Anti-human PD-L1 specific single variable domain antibody screening

[0068] The constructed camel immune library was screened by solid-phase screening to obtain specific phage-displayed single variable domain antibodies.

[0069] (1) The original library is presented. The camel immune library was transferred to 2YT medium containing ampicillin and tetracycline, cultivated to the logarithmic growth phase, added M13 helper phage, and then added kanamycin, and presented overnight at a lower temperature. The culture supernatant was collected the next day, and the phages were concentrated by PEG precipitation to obtain high-titer antibody library display products for subsequent screening.

[0070] (2) Screening. Screening of specific antibodies was carried out by solid-phase method. Coat the specific antigen on the surface of the immune tube, block the immune tube and the antibody library with a blocking agent, add the antibody library to the immune tube and inc...

Embodiment 3

[0072] Example 3: Preliminary identification of anti-human PD-L1 specific single variable domain antibody

[0073] The five obtained single-domain VHH antibodies were induced and expressed in Escherichia coli TG1, and the induction conditions were 1 mM IPTG, 30°C, 150 rpm overnight culture. Bacterial samples with induced expression were sonicated and filtered, followed by affinity purification using a nickel column and ultrafiltration to obtain single-domain VHH antibodies. The inhibitory effect of the single domain on the binding of human PD-L1 to its receptor PD-1 was then detected by ELISA. Specifically, the fusion protein of human PD-1 extracellular region and human Fc (PD-1-hFc, PD-1 sequence number: NP_005009.2, 21aa-167aa) was coated at a concentration of 0.5 μg / mL overnight at 4°C. , blocked with 5% BSA for 60 min in a 37°C incubator. Single-domain VHH antibodies (concentrations of 50, 10, and 2 nM) were co-incubated with 1 μg / mL PD-L1-mFc, and reacted in a 37°C cons...

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Abstract

The invention provides a single variable domain antibody targeting human programmed death ligand 1 (PD-L1) and a derivative of the single variable domain antibody. PBMC (peripheral blood mononuclear cell) is extracted from a human PD-L1 immunized camel to construct a phage surface display VHH antibody library, an anti-human PD-L1 specific single variable domain antibody 2-2F2 is obtained through screening and identification, and a chimeric antibody chF2 and a humanized modified antibody hzF2 mutant are prepared on the basis of the anti-human PD-L1 specific single variable domain antibody 2-2F2. The affinity of the hzF2 mutant can reach or even exceed that of an initial single variable domain antibody 2-2F2, binding of PD-1 and PD-L1 can be blocked in vitro, and growth of tumors can be inhibited in a tumor-bearing mouse in-vivo test.

Description

[0001] This patent application claims priority to the Chinese invention patent application with application number CN 202010324761.8 filed on April 22, 2020, the entire contents of which are hereby incorporated by reference. technical field [0002] The present invention relates to the field of antibody medicine, in particular, the present invention relates to a single variable domain antibody targeting human programmed death ligand 1 (PD-L1), its derivative protein and its use in the preparation of medicines, especially in Use in the treatment and / or prevention, or diagnosis of PD-L1-related diseases such as tumors. Background technique [0003] PD-1 and its ligand PD-L1 are important targets of tumor immunity. PD-1 and PD-L1 are a pair of immunosuppressive molecules, which are important components of the immune system to prevent autoimmune hyperactivity. The activation of their pathways can inhibit tumor immune response and induce tumor-specific T cell apoptosis, which is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCC07K16/2827A61P35/00C07K2317/569C07K2317/56A61K2039/505C07K2317/94C07K2317/76C07K2317/92C07K2317/24C07K2317/22
Inventor 王双曾大地焦莎莎张畅王荣娟
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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