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Bispecific antibodies and uses thereof

Inactive Publication Date: 2016-01-21
THE SCRIPPS RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a type of antibody that can connect to two different proteins simultaneously. This antibody can be used to target specific cells or molecules in the body, making it a potentially effective treatment for various diseases such as cancer. The antibody is made up of two parts: one part is a specific antibody that targets a specific protein, and the other part is a linker that connects the specific antibody to another antibody that targets a different protein. This allows for a more targeted and effective treatment. The patent also describes how the antibody can be designed to have improved properties such as better binding to specific proteins or increased effectiveness in treating disease.

Problems solved by technology

These current approaches may result in heterogeneous products and / or may require the development of antigen-specific ligands, rather than utilizing the diverse pool of existing selective, high affinity monoclonal antibodies.

Method used

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  • Bispecific antibodies and uses thereof
  • Bispecific antibodies and uses thereof
  • Bispecific antibodies and uses thereof

Examples

Experimental program
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Effect test

example 1

Expression of Anti-CD3 Fab Double Mutant in E. coli

[0234]UCHT1-Fab sequences were obtained from the literature and inserted into a pBAD vector behind the stII signal sequence. The sites for unnatural amino acid incorporation (light chain threonine 109 (LC-Thr109), light chain serine 202 (LC-Ser202), heavy chain alanine 123 (HC-A123), and heavy chain lysine 138 (HC-Lys138)) were quickchanged (QuikChange® Stratagene) to TAG amber nonsense codon. For the double mutant, two residues (LC-Ser202 and HC-Lys138) were mutated to TAG amber nonsense codon. FIG. 1 depicts a ribbon diagram of the UCHT1-Fab fragment. The pBAD vector was co-transformed with pULTRA-pAcF (a vector containing orthogonal M. jannaschii tRNA and aminoacyl-tRNA synthetase specific for pAcF) in DH10B cells. The cells were grown in LB media (2 L) supplemented with 100 mg / ml ampicillin, 25 mg / ml chloramphenicol, and 1 mM pAcF at 37° C. and 250 rpm. At OD600 0.8, cells were induced with 0.2% arabinose and moved to 30° C. fo...

example 2

Synthesis of Bispecific Antibodies Using Genetically Encoded Unnatural Amino Acids

[0235]Our strategy takes advantage of genetically encoded unnatural amino acids with orthogonal chemical reactivity relative to the canonical twenty amino acids to site-specifically modify antibody fragments. Specifically, we used an evolved tRNA / aminoacyl-tRNA synthetase pair to site-specifically incorporate p-acetylphenylalanine (pAcF, FIG. 2A) at defined sites in each of two Fab fragments in response to an amber nonsense codon. The mutant Fab fragments were then selectively coupled by stable oxime formation using the alkoxy-amine termini of two bifunctional linkers (FIG. 2B). In a second step, the two Fab-linker conjugates were linked to each other in order to obtain the heterodimer through a copper-free [3+2] Huisgen-cycloaddition (“Click” reaction) (FIG. 2C). This approach has a number of advantages over recombinant technologies and conventional coupling chemistries. For example, the use of bioort...

example 3

Synthesis of Anti-Her2 / Anti-CD3 Heterodimer

[0238]pAcPhe was substituted at HC-K138 in the anti-anti-CD3 antibody, UCHT1. This site is distal to the antigen binding site and, when conjugated to the LC-5202 mutant anti-HER2 Fab with the same polyethylene glycol linker used above, should be long and flexible enough to allow the resulting bispecific antibody to productively bind both a CD3 positive T-cell and the HER2 positive target cell simultaneously. UCHT1 Fab was expressed in E. coli by the same method as described for anti-HER2 Fab and in Example 2, and the cyclooctyne linker-modified anti-CD3 was prepared as described in Example 7 in >95% yield as confirmed by ESI-MS (FIG. 3E, 3J). The anti-CD3-Fab-cyclooctyne conjugate was then coupled to anti-HER2-Fab-azide conjugate as described in Example 7 in 70% percent yield (determined by SDS-PAGE and chromatographic separation) and purified from unreacted Fab monomers by Superdex-200 size exclusion column. FIG. 4A shows the SDS-PAGE of t...

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Abstract

Methods, compositions and uses are provided for bispecific antibodies comprising one or more unnatural amino acids. The bispecific antibodies may bind to two or more different receptors, co-receptors, antigens, or cell markers on one or more cells. The bispecific antibodies may be used to treat a disease or condition (e.g., cancer, autoimmune disease, pathogenic infection, inflammatory disease). The bispecific antibodies may be used to modulate (e.g., stimulate or suppress) an immune response.

Description

CROSS-REFERENCE[0001]This application claims the benefit of U.S. provisional application Ser. No. 61 / 783,426, filed Mar. 14, 2013 and U.S. provisional application Ser. No. 61 / 839,330, filed Jun. 25, 2013; which are incorporated by reference in their entirety.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH[0002]This invention was made with the support of the United States government under Contract number GM062159 by National Institutes of Health (NIH). The government has certain rights in the invention.FIELD OF THE INVENTION[0003]Described herein are immunoglobulin constructs comprising one or more unnatural amino acids, methods of making such constructs, pharmaceutical compositions and medicaments comprising such constructs, and methods of using such constructs and compositions to prevent, inhibit, and / or treat a disease or condition in a subject.BACKGROUND OF THE INVENTION[0004]Antibodies are natural proteins that the vertebrate immune system forms in response to foreign substances (a...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/46
CPCC07K16/468C07K16/2809C07K16/2863C07K2317/73C07K16/2887C07K16/2896C07K2317/31C07K16/2806C07K16/2803C07K16/2851C07K16/32A61K2039/505C07K2317/55C07K2317/33A61K47/542C07D249/04C07K2317/92A61P17/00A61P35/00A61P35/02Y02A50/30A61K39/3955A61K39/39558C07C275/16C07C275/24C07K16/3069C07K2317/94C07K2317/35C07K2317/52C07K2317/56
Inventor KIM, CHANHYUKAXUP, JUN Y.SCHULTZ, PETER G.YOUNG, TRAVISPINKERTON, STEPHANIEZHOU, QUANCAO, YU
Owner THE SCRIPPS RES INST
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