Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Anti-cd16 binding molecules

A technology of binding molecules and binding fragments, which is applied in the direction of antibodies, anti-inflammatory agents, antibacterial drugs, etc.

Active Publication Date: 2009-11-18
AFFIMED GMBH
View PDF4 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there are no examples of molecules that specifically bind to FcγRIIIA expressed on NK cells but not to FcγRIIIB

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Anti-cd16 binding molecules
  • Anti-cd16 binding molecules
  • Anti-cd16 binding molecules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Example 1: Transient expression and purification of human CD16A-Fc fusion protein

[0130] HEK-293 cells were transiently transfected with the plasmid pCDM-CD16-Fc encoding human CD16A and human IgG1 Fc fragment recombinant protein to secrete soluble CD16A-Fc fusion protein (Mandelboim et al., 1999, supra).

[0131] Use full DMEM medium (with 10% heat-inactivated fetal calf serum (FCS), 2mM L-glutamine, 100 U / mL penicillin G sodium and 100 μg / mL streptomycin sulfate; all purchased from Invitrogen, Karlsruhe, Germany), at 37°C, certain humidity and 5% CO 2 Under the conditions of HEK-293 cells (ATCC deposit number CRL-1573) were cultured. Calcium phosphate-mediated transfection efficiencies for these cells are described below.

[0132] One day before transfection, 1.5×10 6 HEK-293 cells were inoculated in a 10 cm diameter cell culture dish with 10 mL of full DMEM medium. Take 10 μg of plasmid DNA with 500 μL of 250 mM CaCl 2 Mix well, then drop into 500μL 2x HEBS bu...

Embodiment 2

[0135] Example 2: Identification and isolation of CD16-A specific antibody

[0136] The CD16A-Fc fusion protein that obtains with above-mentioned similar method, is used for screening IgM derived scFv phage display library (D rsam etc., 1997FEBS Letts 414:7-13, Little etc., 1999J.Immunol.Methods 231:3-9 , Schwarz et al., FASEB J. 18:1704-6). Clones of this library contained the sequence between the scFv coding sequence and the phage M13pIII gene encoding a hexa-histidine tag, an amber stop codon and a c-myc epitope.

[0137] 500 μg of CD16A Fc fusion protein was biotinylated using the ECL protein biotinylation module (Amersham Pharmacia Biotech, Uppsala Sweden) and then used for three rounds of screening against a phage display library. Prior to screening of the library, sequential preabsorption using streptavidin-coated polystyrene and human IgG (Sigma-Aldrich, Taufkirchen, Germany) was performed in order to eliminate other potential molecules for these proteins. combinatio...

Embodiment 3

[0143] Example 3: Detection of scFv 50NI in human peripheral blood mononuclear cells and granulocytes stably transfected with CD16 subtype

[0144] To test whether 50NI scFv binds to native CD16A protein on the cell surface, we used flow cytometry to detect scFvs expressing either CD16A or CD16B in the cytoplasmic extracts of two different cell lines. The cell lines used for the test are: cell line BW / CD16A expressing CD16A (Mandelboim et al., 2001, Nature 409: 1055-1060); cell line 293-CD16 (stabilized with a plasmid encoding the NA-2 allele of the human CD16B subtype transfected HEK-293 cell line).

[0145] In order to verify whether 50NI scFv has the ability to bind CD16A expressed on the surface of natural killer cells (NK) and / or CD16B expressed on the surface of neutrophils (van de Winkeland Capel, 1993, supra; van Spriel et al., 2000, Immunol. Today 21 (8):391-397), which we detected by flow cytometry compared with freshly isolated pleomorphic cells containing 10-20% N...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention relates to binding molecules that specifically bind to the human Fc gamma receptor expressed on the surface of natural killer (NK) cells and macrophages (i.e. Fc Gamma RIIIA), and in particular binding molecules that specifically bind the A form Fc Gamma RIII but do not bind to the B form of Fc Gamma RIII, as well as to the use of such binding molecules in the diagnosis and treatment of disease. The invention further extends to polynucleotides encoding such binding molecules, host cells comprising such polynucleotides and methods of producing binding molecules of the invention using such host cells.

Description

technical field [0001] The present invention relates to binding molecules that can specifically bind to human FcγIII receptors (i.e. FcγRIIIA) expressed on the surface of natural killer (NK) cells and macrophages, and in particular, specifically bind to type A FcγRIII but not to type B A binding molecule to which FcγRIII binds, and applications of the binding molecule in disease diagnosis and treatment. The invention further relates to polynucleotides encoding such binding molecules, host cells comprising said polynucleotides and methods of using said host cells to produce binding molecules of the invention. Background technique [0002] CD16 (also known as FcγRIII), a receptor with low affinity for the Fc fragment of certain IgGs involved in antibody-dependent cellular cytotoxicity (ADCC), is the best characterized receptor responsible for eliciting NK cell response to Lysed membrane receptors of target cells (Mandelboim et al., 1999, PNAS 96:5640-5644). Human NK cells co...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K16/28A61K39/395A61K47/48
CPCC07K16/2878C07K2317/31C07K16/283C07K2317/34C07K2317/622C07K2317/92A61K47/48676C07K2317/732C07K2319/30A61K47/6879C07K16/18C07K2317/56C07K2317/565A61P29/00A61P31/00A61P31/04A61P35/00A61P37/00A61P37/02A61P37/08
Inventor 卡芮·霍夫曼瑟基·可普芮加欧斯蒂芬·旱斯·鸠其姆·克拉克马斯伐布芮斯·勒盖儿马威廉·利透乌娃·芮优其
Owner AFFIMED GMBH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com