Antibody-active agent conjugates and methods of use

a technology of active agents and conjugates, applied in the field of proteinactive agent conjugates, can solve the problems of antibody specificity loss, complex overall preparation process, and difficulty in obtaining the antigen specificity of antibodies,

Inactive Publication Date: 2014-06-12
LEGOCHEM BIOSCIENCES INC
View PDF2 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016]As described below, the present invention generally features protein-active agent conjugates and methods for making the protein-active agent conjugates. The invention also features methods for delivering the protein-active agent conjugate to a target cell in a subject, as well as methods for treating a subject in need of the active agent. The protein-active agent conjugates of the invention can be produced homogeneously and advantageously used for targeted treatment of a disease.

Problems solved by technology

Existing preparation methods, however, have some problems.
For example, the overall preparation process is complicated because the antibody-drug conjugates prepared by the existing preparation methods are not uniform (homogeneous).
Furthermore, when preparing antibody-drug conjugates by bonding lysine groups with a drug moiety, the electric charge of the lysine groups may be lost, thereby causing the antibody to lose its unique antigen specificity.
Likewise, the tertiary or quaternary structure of the antibody may not be maintained when preparing antibody-drug conjugates by reducing disulfide groups, thereby causing the antibody to be inactivated or become a non-specific antibody.
Although this method shows better result than the prior preparation methods in terms of toxicity, activity, and safety, this method still involves thiol-maleimide bonding and thus suffers from the diastereomer and instability problems associated with thiol-maleimide bonding.

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
  • Antibody-active agent conjugates and methods of use
  • Antibody-active agent conjugates and methods of use
  • Antibody-active agent conjugates and methods of use

Examples

Experimental program
Comparison scheme
Effect test

embodiment 1

[0115]A method for preparing a protein-active agent conjugate according to one embodiment of the invention comprises: (a) expressing a protein having an amino acid motif that can be recognized by an isoprenoid transferase; (b) enzymatically reacting, using the isoprenoid transferase, the expressed protein and at least one isosubstrate having a first functional group (FG1), thereby producing a functionalized protein; (c) attaching a second functional group (FG2) to an active agent, thereby producing a functionalized active agent; and (d) reacting the functionalized protein with the functionalized active agent, thereby producing the protein-active agent conjugate.

[0116]The term “protein” used herein is understood as two or more independently selected natural or non-natural amino acids joined by a covalent bond (e.g., a peptide bond). A peptide can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more natural or non-natural amino acids joined by peptide bo...

embodiment 2

[0156]A method for preparing a protein-active agent conjugate according to another embodiment comprises: (a) expressing a protein having an amino acid motif that can be recognized by an isoprenoid transferase; (b) attaching an isosubstrate of an isoprenoid transferase to an active agent; and (c) enzymatically reacting, using the isoprenoid transferase, the expressed protein with the active agent attached to the isosubstrate.

[0157]In this embodiment, once a protein having an amino acid motif that can be recognized by an isoprenoid transferase is expressed, the protein is reacted with an active agent attached to an isosubstrate of the isoprenoid transferase. In this case, thiol-maleimide conjugation may occur. However, even if thiol-maleimide conjugation occurs, the active agents are conjugated at the targeted positions only according to the present invention. Accordingly, a problem associated with the prior art that a non-homogeneous mixture is produced is avoided.

[0158]2. Protein-Ac...

example 1

Preparation of Ab(M)-CAAX

[0188]1-1. Construction, Expression, and Purification of Herceptin-CAAX

[0189]Modified Herceptin antibodies were generated using standard recombinant DNA technology and PCR cloning protocols with pNATABH::Herceptin HC plasmid or pNATABL::Herceptin LC plasmid. Recombinant plasmids were expressed in an HEK293E cell line by transient transfection. The antibodies were separated and purified by protein A column chromatography.

[0190]Construction of Herceptin-HC-GCVIM (“Herceptin-HC-GCVIM” disclosed as SEQ ID NO: 8) and Herceptin-LC-GCVIM (“Herceptin-LC-GCVIM” disclosed as SEQ ID NO: 11)

[0191]Modified Herceptin antibodies were generated using standard PCR cloning protocols. Generally, Herceptin-HC-GCVIM (“Herceptin-HC-GCVIM” disclosed as SEQ ID NO: 8) and Herceptin-LC-GCVIM (“Herceptin-LC-GCVIM” disclosed as SEQ ID NO: 11) plasmids were constructed by inserting a DNA sequence encoding a CAAX motif (e.g., GCVIM (SEQ ID NO: 1), G5CVIM (SEQ ID NO: 2), GCVIM (SEQ ID NO:...

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 invention provides protein-active agent conjugates having an amino acid motif that can be recognized by an isoprenoid transferase. The invention also provides compositions containing the conjugates. The invention further provides methods for using the conjugates to deliver the active agent to a target cell, as well as methods for using the conjugates to treat a subject in need thereof (e.g., a subject in need of the active agent).

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application is a Divisional of U.S. application Ser. No. 13 / 466,875, filed May 8, 2012, which claims priority to U.S. Provisional Application No. 61 / 483,698, filed May 8, 2011. The contents of these applications are incorporated herein by reference in their entirety.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 19, 2012, is named 87641305.txt and is 62,892 bytes in size.BACKGROUND[0003](a) Technical Field[0004]The present disclosure relates to a protein-active agent conjugate. The protein (e.g., an oligopeptide, a polypeptide, an antibody, or the like) has substrate specificity for a desired target, and the active agent (e.g., a drug, a toxin, a ligand, a detection probe, and the like) has a specific function or activity. The disclosure also relates to methods for pr...

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
Patent Type & Authority Applications(United States)
IPC IPC(8): A61K47/48
CPCC12Q1/48C07K16/32A61K47/65A61K47/6889A61K47/549A61K47/55A61K47/6803A61K47/6817A61K47/6849A61K47/6851A61K47/6855A61K47/6859A61K47/6811C07K16/2863A61P31/00A61P31/04A61P31/10A61P31/12A61P33/00A61P35/00A61P37/02A61K31/70A61K39/395A61K47/50A61K48/00
Inventor KIM, YONGZUPARK, TAEKYOWOO, SUNGHOLEE, HYANGSOOKKIM, SUNYOUNGCHO, JONGUNJUNG, DOOHWANKIM, YOUNGUNKWON, HYUNJINOH, KYUMANCHUNG, YUNSEOPARK, YUN-HEE
Owner LEGOCHEM BIOSCIENCES INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap