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Pd-1 variant having improved binding to pd-l1

A PD-L1 and PD-1 technology, applied in the direction of peptides containing affinity tags, fusion peptides, drug combinations, etc., can solve the problems of immunogenicity and other issues

Pending Publication Date: 2020-12-18
纽洛可遗传学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing PD-1 engineering is based on screening by yeast display, and there is a risk of immunogenicity due to glycan heterogeneity and various mutations

Method used

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  • Pd-1 variant having improved binding to pd-l1
  • Pd-1 variant having improved binding to pd-l1
  • Pd-1 variant having improved binding to pd-l1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1: Validation of N-linked glycosylation sites affecting binding to human PD-L1

[0101] To verify that the glycosylation of human PD-1 actually plays an important role in the binding affinity to PD-L1, each of the four glycosylation sites present in PD-1 was tested by deglycosylation Effect of glycosylation on binding affinity. Because PD-1 has four N-glycosylation sites, four genes (N25A, N34A, N50A, N92A) were made by replacing each asparagine at the four sites with alanine. Using the template gene (catalogue number: HG10377-M) and primers for PD-1 (CKJ#1, CKJ#2) and Vent polymerase to amplify by PCR encoding PD-1 outer membrane site (amino sequence L25-Q167) DNA fragments. The amplified DNA fragment was treated with BssHII and XbaI enzymes, and ligated into pMaz vector, which is a vector for expression in animal cells, which was treated with the same enzymes. Transformation into Jude1((F-mcrAΔ(mrr-hsdRMS-mcrBC) After 80 lacZΔM15 ΔlacX74 recA1 endA1 araD1...

Embodiment 2

[0105] Example 2: Cloning of Tetrameric Human PD-L1 (PD-L1-Streptavidin) for PD-1 Engineering

[0106] The cDNA of human PD-L1 gene was purchased from Sino Biotech (Cat. No.: HG10084-M), and the gene of PD-L1 outer membrane was amplified by PCR using primers (HW#1, HW#2) and Vent polymerase (Amino sequence F19-R238). Since the dissociation constant between wild-type PD-1 and wild-type PD-L1 is not good enough (equilibrium dissociation constant, KD = about 8.7 μM), tetramerization induction by efficient screening of aglycosylated PD-1 variants affinity effect. Tetramerization was induced by expressing streptavidin at the C-terminus of PD-L1, and a GS linker was inserted between streptavidin and PD-L1 to ensure the flexibility of each protein. After gene amplification using primers (HW#3, HW#4) and Vent polymerase (New England Biolab), assembly PCR was performed using the previously amplified PD-L1 gene and Vent polymerase. The prepared gene was treated with BssHII and XbaI r...

Embodiment 3

[0107] Example 3: Expression, purification and fluorescent labeling of tetrameric PD-L1-streptavidin in animal cells

[0108] The prepared vector for tetrameric PD-L1 expression was transfected into animal cells (HEK293F). After culturing the cells for 6 days, the cell culture was centrifuged at 6000 rpm for 20 minutes, and the supernatant was taken and filtered through a 0.22 μm filter. The filtered supernatant was bound to 1 mL of Ni-NTA resin (Qiagen) for 16 hours at 4°C. The bound resin was washed with 10 CV of 10 mM imidazole (Sigma) in PBS and then again with 10 CV of 20 mM imidazole in PBS. Finally, the protein was eluted and recovered with 250 mM imidazole in PBS. The purified PD-L1 tetramers were fluorescently labeled using the Alexa-488 labeling kit. The result of ELISA test is that fluorescently labeled tetrameric PD-L1 shows excellent binding affinity to PD-1 ( image 3 ).

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Abstract

The present invention relates to a PD-1 variant having improved binding to PD-L1. In addition, the present invention relates to a method for producing and a method for screening for the PD-1 variant.A PD-1 variant of the present invention effectively inhibits binding between wild-type PD-1 and PD-L1, and thus can be expected to have penetration power and an effect of cancer death by immunocytes or a treatment effect on infectious diseases, which are far greater than those of conventional therapeutic agents for immune checkpoint inhibition and, simultaneously, can minimize the possibility of immunogenicity occurrence and, further, can promote the convenience of developing biological pharmaceutical drugs through the implementation of aglycosylation.

Description

technical field [0001] The present disclosure relates to PD-1 variants having improved binding affinity to PD-L1 and thus effectively inhibiting the binding between wild-type PD-1 and PD-L1, and methods for preparing the same. Background technique [0002] Drugs for treating cancer are mainly classified into low-molecular-weight drugs and high-molecular-weight drugs. High-molecular-weight drugs with specificity are preferred over low-molecular-weight drugs that lack specificity and therefore have relatively more side effects. [0003] Recently, it has been reported that inhibiting the binding of immune checkpoint inhibitor proteins, especially PD-1 / PD-L1, is very effective in the treatment of cancer and has fewer side effects compared with other immune checkpoint inhibitor proteins (J. Naidoo et al (2015) Annals of Oncology, Lucia Gelao et al (2014) Toxins, Gorge K. Philips et al (2015) International Immunology). [0004] Big pharma companies such as Bristol-Myers Squibb a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C12N15/10A61P31/00A61P35/00A61P37/04A61K38/00
CPCA61K38/00A61P31/00A61P35/00A61P37/04C12N15/1034C12N15/1024C12N15/70C07K14/70596C07K14/4747C07K2319/02C07K2319/21C07K2319/30C07K2319/43C12N15/1055C07K14/70532C12N15/85
Inventor 郑相泽千光珍河志妍
Owner 纽洛可遗传学有限公司
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