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CD20-binding single domain antibody

A single-domain antibody, binding agent technology, applied in the direction of antibodies, antibody medical components, antibody mimetics/scaffolds, etc., can solve problems such as toxicity and side effects, rituximab treatment response, etc.

Active Publication Date: 2022-04-05
VIB VZW (BE) +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many patients do not respond to rituximab treatment
Additionally, responding patients often develop resistance to rituximab and eventually relapse
Additionally, rituximab appears to nonspecifically kill normal CD20-positive cells, resulting in significant toxicity and side effects

Method used

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  • CD20-binding single domain antibody
  • CD20-binding single domain antibody
  • CD20-binding single domain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0412] Example 1. Construction and evaluation of human CD20-specific VHH

[0413] Cloning of human CD20 gene

[0414] Human CD20 was amplified from Orfeome v5.1 collection (ID 11051 ) using forward primer 5'-GATAAGATCTCAGGCGGATCCACAACACCCAGAAATTCAG (O-7954) and reverse primer 5'-GGTTTTTCTCTAGATCAAGGAGAGCTGTCATTTTCTATTGG (O-7956). The amplified product was cut with BglII and Xbal and ligated into the mammalian expression vector pMet7. The plasmid was used for transient transfection of Hek293T cells and generation of CHO-K1 clones stably expressing human CD20.

[0415] Isolation of antigen-specific VHH

[0416] A VHH library was constructed and three consecutive rounds of panning (in solution) were performed on stably transfected CHO-K1 cells expressing human CD20. Parallel panning was performed on parental (untransfected CHO-K1 ) cells to serve as a negative control in order to calculate CD20-specific phage enrichment. Enrichment of antigen-specific phage was assessed aft...

Embodiment 2

[0447] Example 2. Construction and evaluation of mouse CD20-specific VHH

[0448] Cloning of mouse CD20 gene

[0449] Mouse CD20 was purchased from Imagenes, catalog number IRAVP968C1280D, and amplified with the forward primer 5'-gataagatctcaGGCGGATCCAGTGGACCTTTCCCAGCAGAGC (0-7962) and the reverse primer 5'-GGTTTTTCTCTAGATCAAGGAGCGATCTCATTTCCACTG (0-7964). The amplified product was cut with BglII and Xbal and ligated into the mammalian expression vector pMet7. The plasmid was used for transient transfection of Hek293T cells and generation of CHO-K1 clones stably expressing mouse CD20.

[0450] Isolation of antigen-specific VHH

[0451] The VHH library was constructed from peripheral blood lymphocytes (PBL). Specifically, total RNA from PBLs was used as template for first-strand cDNA synthesis with oligo(dT) primers. Using this cDNA, the VHH coding sequence was amplified by PCR, digested with PstI and NotI, and cloned into the PstI and NotI sites of the phagemid vector pM...

Embodiment 3

[0486] Example 3. Functional assessment of human CD20 and / or mouse CD20-specific VHHs

[0487] Demonstration of specific binding of VHHs by FACS

[0488] To determine the binding activity of human or mouse CD20-specific VHHs, FACS analysis was performed. Specifically, CHO-K1 cells stably expressing human or mouse CD20 and CHO-K1 cells were incubated with 5 μg / ml of VHH produced according to the present application. A FITC-labeled monoclonal anti-his antibody (Genscript, Cat. A01620) was used as a secondary stain. FACS analysis was performed on a FacsCalibur flow cytometer (Becton Dickinson). like Figure 8 As shown, all VHHs against human CD20 showed specific binding to human CD20. Similarly, all VHHs against mouse CD20 also showed specific binding to mouse CD20 ( Figure 9 ).

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Abstract

This application relates in part to agents that bind CD20 and their use as therapeutic agents. The present application also relates to a pharmaceutical composition comprising the CD20 binding agent and its use for treating various diseases.

Description

technical field [0001] The present invention relates, in part, to binding agents (eg, antibodies such as, but not limited to, VHHs) that bind CD20 and their use as therapeutic agents. Background technique [0002] CD20 is a transmembrane protein expressed on the surface of B lymphocytes. It is expressed during the development of B lymphocytes from the early pre-B stage until their final differentiation into plasma cells, at which point CD20 expression disappears. CD20 is also expressed on malignant B cells. Specifically, CD20 is expressed on more than 90% of B-cell non-Hodgkin's lymphoma (NHL) cells and more than 95% of B-type chronic lymphocytic leukemia (B-CLL) cells. [0003] Antibodies against CD20 have been used to treat leukemias and lymphomas of B-cell origin. Specifically, rituximab (Rituxan) is a chimeric mouse / human monoclonal antibody genetically engineered against CD20. Rituximab is currently approved for the treatment of relapsed or refractory follicular lym...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/00A61K39/395A61K31/00C07K14/715C07K16/28C07K16/30
CPCA61K38/00A61K39/3955C07K16/3061A61K2039/505C07K2317/33C07K2317/569C07K2319/00C07K14/56A61K45/06A61K31/704A61K33/243C07K16/2887A61P37/06A61P35/00A61K39/001124A61K2300/00C07K14/7156
Inventor J·塔威尼尔A·考威尔斯J·范德海登
Owner VIB VZW (BE)
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