Protein-active agent conjugates and method for preparing the same

Inactive Publication Date: 2012-12-06
LEGOCHEM BIOSCIENCES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0015]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

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 quat

Method used

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  • Protein-active agent conjugates and method for preparing the same
  • Protein-active agent conjugates and method for preparing the same
  • Protein-active agent conjugates and method for preparing the same

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Experimental program
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embodiment 1

[0114]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.

[0115]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

[0155]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.

[0156]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.

[0157]2. Protein-Ac...

example 1

Preparation of Ab(M)-CAAX

[0187]1-1. Construction, Expression, and Purification of Herceptin-CAAX

[0188]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.

[0189]Construction of Herceptin-HC-GCVIM and Herceptin-LC-GCVIM

[0190]Modified Herceptin antibodies were generated using standard PCR cloning protocols. Generally, Herceptin-HC-GCVIM and Herceptin-LC-GCVIM plasmids were constructed by inserting a DNA sequence encoding a CAAX motif (e.g., GCVIM, G5CVIM, GCVIM, G10CVIM, or G10CVLL), to the C-terminus of the heavy chain or light chain encoded in the pNATABH::Herceptin HC or pNATABH::Herceptin LC plasmid.

[0191]For example, a SacII recognition sequence is present at amino acid 172 in the C-terminus...

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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, as well as methods for making the conjugates and compositions. 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 claims the benefit of U.S. Provisional Application No. 61 / 483,698 filed May 8, 2011, the contents of which are incorporated herein by reference in their entirety.BACKGROUND[0002](a) Technical Field[0003]The present disclosure relates to a protein-active agent conjugate. The protein (e.g., an oligopeptide, a polypeptide, an antibody, or the like) has a 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 preparing the conjugate. The disclosure further relates to methods of using the conjugate to deliver an active agent to a target cell in a subject, as well as methods for treating a subject in need of the active agent (e.g., a subject having cancer).[0004](b) Background Art[0005]Methods for inhibiting growth of cancer cells by targeted delivery of anti-cancer agents h...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/00C12P21/00
CPCA61K47/48092A61K47/481A61K47/48338A61K47/48384A61K47/48438A61K47/48561A61K47/48584C12Q1/48C07K16/2863C07K16/32A61K47/48415A61K47/48569A61K47/65A61K47/6889A61K47/549A61K47/55A61K47/6803A61K47/6817A61K47/6849A61K47/6851A61K47/6855A61K47/6859A61K47/6811A61P31/00A61P31/04A61P31/10A61P31/12A61P33/00A61P35/00A61P37/02A61K31/70A61K39/395A61K47/50A61K48/00
Inventor KIM, YONGZUPARK, TAEKYOWOO, SUNGHOLEE, HYANGSOOKKIM, SUNYOUNGCHO, JONGUNJUNG, DOOHWANKIM, YOUNGUNKWON, HYUNJINOH, KYUMANCHUNG, YUNSEOPARK, YUN-HEE
Owner LEGOCHEM BIOSCIENCES INC
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