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Anti-CD47 antibodies and methods of use thereof

A technology of antibodies and monoclonal antibodies, applied in the fields of antibodies, antibody medical components, chemical instruments and methods, etc., can solve problems such as reduced efficacy and poor pharmacokinetics

Active Publication Date: 2015-12-02
NOVIMMUNE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The widespread expression of CD47 in healthy tissues raises questions about the safety and efficacy of treatments: first, targeting CD47 with neutralizing monoclonal antibodies (Mabs) can affect healthy tissues, leading to preclinical studies such as those in mice and cynomolgus monkeys. Severe toxicity shown in (Willingham SB, et al., Proc Natl Acad Sci USA. 2012 Apr24;109(17):6662-7; WeiskopfK, et al., Engineered SIRPα Variants as Immunotherapeutic Adjuvants to Anticancer Antibodies, Science. 2013 Jul5;341(6141):88-91)
Second, even though severe toxicity could be avoided or mitigated by using alternative forms (WeiskopfK, et al., Science. 2013 Jul 5;341(6141):88-91), widespread expression of CD47 still results in CD47 binding via target-mediated drug distribution Rapid elimination of the molecule, resulting in poor pharmacokinetics and reduced potency

Method used

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  • Anti-CD47 antibodies and methods of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0603] Example 1: Cloning, expression and purification of human CD47

[0604] clone. The sequence corresponding to the extracellular domain of human CD47 (hCD47) was amplified from human cDNA by polymerase chain reaction (PCR) using specific oligonucleotides. Amplification products were gel purified and cloned into the pEAK8 mammalian expression vector (EdgeBiosystems, Gaithersburg, MD). The vector was further modified to introduce Avitag™ (Avidity, Denver CO) and a hexa-histidine tag at the C-terminus, which allows single-site biotinylation of the protein and purification by IMAC (Immobilized Metal Ion Affinity Chromatography). Constructs were verified by DNA sequencing.

[0605] Express. The plasmid is then transfected into mammalian cells using a liposome-based transfection reagent such as TransIT-LT1 (Mirus, Madison, WI). The transfection step requires only a small amount of DNA and cells, typically 2x10 5 cells and 2 μg plasmid DNA / well, and transfections were perfo...

Embodiment 2

[0608] Example 2: Cloning, expression and purification of human CD19

[0609] clone. The sequence corresponding to the extracellular domain of human CD19 (hCD19) was amplified from human cDNA by polymerase chain reaction (PCR) using specific oligonucleotides. Amplification products were gel purified and cloned into the pEAK8 mammalian expression vector (EdgeBiosystems, Gaithersburg, MD). The vector was further modified to introduce Avitag™ (Avidity, Denver CO) and a hexa-histidine tag at the C-terminus, which allows single-site biotinylation of the protein and purification by IMAC (Immobilized Metal Ion Affinity Chromatography). Constructs were verified by DNA sequencing.

[0610] expression and purification. Expression, purification and biotinylation of soluble hCD19 were performed as described in Example 1.

Embodiment 3

[0611] Example 3: Phage display selection using a human scFv library containing immobilized variable heavy chains

[0612] General procedures for the construction and manipulation of human scFv libraries displayed on M13 phage are described in Vaughan et al., (Nat. Biotech. 1996, 14:309-314), which is hereby incorporated by reference in its entirety. Libraries used for selection and screening encoded scFvs all sharing the same VH domain and differing only in the VL domain. Methods for producing immobilized VH libraries and their use for identifying and assembling bispecific antibodies are described in US2012 / 0184716 and WO2012 / 023053, each of which is incorporated herein by reference in its entirety. Procedures for identifying scFvs that bind hCD19 or hCD47 are described below.

[0613] Liquid phase selection. An aliquot of the scFv phage library (10 12 Pfu) were blocked with PBS containing 3% (w / v) skim milk for one hour at room temperature in a rotary mixer. Blocked pha...

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Abstract

The invention provides anti-CD47 antibodies and methods of use thereof. The invention relates to monoclonal and / or monovalent antibodies that bind CD47. The invention relates to monoclonal and / or monovalent antibodies that bind CD19. The invention also relates to novel bispecific monoclonal antibodies carrying a different specificity for each binding site of the immunoglobulin molecule, where one of the binding sites is specific for CD47. The invention also relates to novel bispecific monoclonal antibodies carrying a different specificity for each binding site of the immunoglobulin molecule, where one of the binding sites is specific for CD19.

Description

[0001] related application [0002] This application claims the benefit of U.S. Provisional Application No. 61 / 732452, filed December 3, 2012; U.S. Provisional Application No. 61 / 816788, filed Apr. 28, 2013; U.S. Provisional Application No. 61 / 816788, filed Apr. 7, 2013 No. 61 / 863106; U.S. Provisional Application No. 61 / 881523, filed September 24, 2013; and U.S. Provisional Application No. 61 / 898710, filed November 1, 2013; each of which is incorporated by reference in its entirety Incorporated into this article. field of invention [0003] The present invention relates to monoclonal and / or monovalent antibodies that bind CD47. The present invention relates to monoclonal and / or monovalent antibodies that bind CD19. The present invention also relates to novel bispecific monoclonal antibodies having different specificities for each binding site of an immunoglobulin molecule, wherein one of said binding sites is specific for CD47. The present invention also relates to novel bi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/00
CPCC07K16/468A61K2039/505C07K16/2803C07K16/2896C07K16/461C07K2317/30C07K2317/31C07K2317/565C07K2317/622C07K2317/76C07K2317/92A61P35/00
Inventor K.马斯特纳克N.费希尔F.卢梭E.海利M.科斯科-维尔博瓦
Owner NOVIMMUNE
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