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Monomeric fc domains

a technology fc domains, applied in the field of monomeric fc domains, can solve the problems of long serum half-life, short half-live of many of these formats compared to full-size iggs, and risk of immunogenicity, and achieve the effect of reducing toxicity

Inactive Publication Date: 2017-12-28
INNATE PHARMA SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a new type of molecule that can be easily produced and contains a native CH3 domain. These molecules can be used to create bispecific antibodies that can target two different antigens. These antibodies have a unique structure that makes them effective at eliminating target cells while reducing the risk of harmful side effects. Overall, this patent provides a way to create highly targeted and effective treatments for diseases like cancer.

Problems solved by technology

However, many of these formats have very short half-lives compared to full-size IgG.
This biological system protects IgG from degradation and results in a long serum half-life.
However, introduction of mutations involves risk of immunogenicity when administered to humans.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Bispecific Monomeric Tandem CH3 Fc Polypeptides

Materials and Methods

[0174]Different constructs were made for use in the preparation of a bispecific Fc-polypeptide based on a scFv specific for human tumor antigen CD19 (anti-CD19 scFv) and a scFV specific for human activating receptor NKp46 on NK cells (anti-NKp46 scFv).

[0175]Shown below in Table 1 are the light chain and heavy chain variable regions amino acid sequences for the anti-CD19 component of the proteins.

TABLE 1Anti-CD19SequenceAnti-CD19-VK DNASEQ ID NO: 3Anti-CD19-VK amino acidSEQ ID NO: 4Anti-CD19-VH DNASEQ ID NO: 5Anti-CD19-VH amino acidSEQ ID NO: 6

[0176]Shown below in Table 2 are the light chain and heavy chain variable regions and scFv amino acid sequences for the anti-NKp46 component of the proteins.

TABLE 2Anti-NKp46scFV sequence (VHVK) / - stopNKp46-3 VH amino acidSEQ ID NO: 7NKp46-3 VL amino acidSEQ ID NO: 8NKp46-3 scFv amino acidSEQ ID NO: 9

Cloning and Production of the Recombinant Proteins

[0177]Coding...

example 2

NKp46 Binding Affinity by Bispecific Proteins by Surface Plasmon Resonance (SPR)

Biacore T100 General Procedure and Reagents

[0213]SPR measurements were performed on a Biacore T100 apparatus (Biacore GE Healthcare) at 25° C. In all Biacore experiments HBS-EP+ (Biacore GE Healthcare) and NaOH 10 mM served as running buffer and regeneration buffer respectively. Sensorgrams were analyzed with Biacore T100 Evaluation software. Protein-A was purchase from (GE Healthcare). Human NKp46 recombinant proteins were cloned, produced and purified at Innate Pharma.

Immobilization of Protein-A

[0214]Protein-A proteins were immobilized covalently to carboxyl groups in the dextran layer on a Sensor Chip CM5. The chip surface was activated with EDC / NHS (N-ethyl-N′-(3-dimethylaminopropyl) carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GE Healthcare)). Protein-A was diluted to 10 μg / ml in coupling buffer (10 mM acetate, pH 5.6) and injected until the appropriate immobilization level was reache...

example 3

NKp46 Mechanism of Action

[0219]NKp46×CD19 bispecific proteins having an arrangement according to the F3 format described in Example 1 were compared to rituximab (anti-CD20 ADCC inducing antibody), and a human IgG1 isotype control antibody for functional ability to direct CD16- / NKp46+ NK cell lines to lyse CD19-positive tumor target cells.

[0220]Briefly, the cytolytic activity of the CD16- / NKp46+ human NK cell line KHYG-1 was assessed in a classical 4-h 51Cr-release assay in U-bottom 96 well plates. Daudi or B221 cells were labelled with 51Cr (50 μCi (1.85 MBq) / 1×106 cells), then mixed with KHYG-1 at an effector / target ratio equal to 50:1, in the presence of test antibodies at dilution range starting from 10−7 mol / L with 1 / 5 dilution (n=8 concentrations)

[0221]After brief centrifugation and 4 hours of incubation at 37° C., 50 μL of supernatant were removed and transferred into a LumaPlate (Perkin Elmer Life Sciences, Boston, Mass.), and 51Cr release was measured with a TopCount NXT bet...

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Abstract

Monomeric Fc-domains are provided, as well as multimeric and single chain proteins comprising the monomeric Fc-domains. The proteins have utility in the treatment of disease. Included, inter glia, are multi specific polypeptides.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 62 / 099,634, filed Jan. 31, 2015; which is incorporated herein by reference in its entirety; including any drawings.REFERENCE TO THE SEQUENCE LISTING[0002]The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “NTECH2 PCT_ST25 txt”, created Dec. 31, 2015, which is 100 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0003]Monomeric Fc-domains are provided, as well as multimeric and single chain proteins comprising the monomeric Fc-domains. The proteins have utility in the treatment of disease. Included, inter alia, are multispecific polypeptides.BACKGROUND[0004]Antibody fragments such as Fab, Fv, scFv, VH and VHH, and more new antibody fragment formats are under development that hav...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/28G06K9/62G06T7/11G06T7/00G06T11/00C07K16/46G06K9/66
CPCC07K16/2803C07K16/468G06K9/6267G06K9/66G06T11/003G06T7/0012G06T2207/30096C07K2317/41C07K2317/526C07K2317/64C07K2317/71C07K2317/92G06T2207/10081G06T7/11C07K2317/31C07K2317/622C07K2317/524A61P29/00A61P31/00A61P35/00A61P37/00A61P37/06A61B6/032G06T2207/30024G06T2207/30061G06F18/24
Inventor GAUTHIER, LAURENT
Owner INNATE PHARMA SA