Coiled coil and/or tether containing protein complexes and uses thereof

a protein complex and tether technology, applied in the field of new engineered proteins and multispecific protein complexes, can solve the problems of elusive multispecific antibody building technologies, and low yield of heterodimer formation

Inactive Publication Date: 2012-11-29
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0003]The present invention provides novel protein complexes and methods of creating and manufacturing protein complexes. In one aspect, the invention involves a coiled coil domain that is linked to an Fc CH component, which coiled coil domain may or may not be cleavable from the Fc containing protein if desired. In another aspect, the invention involves a protein comprising a tether and an Fc CH component complex, which tether may or may not be cleavable from the protein. In another aspect, the invention involves a protein comprising a coiled coil, a tether and an Fc CH component, optimally able to form a protein complex, which tether and / or coiled coil may or may not be cleavable from the protein depending on the desired effect. In another aspect, the invention provides a process of preparing the protein comprising a tether, wherein the tether is cleaved by a host cell or cleaved by a chemical or enzymatic reaction in vitro. In another aspect, the invention involves a protein comprising a coiled coil, a tether and an Fc CH component, optimally able to form a protein complex, which tether and / or coiled coil are cleavable from the protein by a host cell that expresses the protein and overexpresses enzymes capable of cleaving the tether and / or coiled coil from the protein.

Problems solved by technology

Finding technologies for building multispecific antibodies that are useful and scalable for commercial and therapeutic purposes has been elusive.
Many methods have been tried, but nearly all suffer significant drawbacks such as being poorly soluble; inexpressible in mammalian cells, demonstrating low yield of heterodimer formation, technically challenging to manufacture, immunogenic, short half-life in vivo, unstable among other problems (e.g., Hollinger et al., (1993) PNAS 90:6444-6448; U.S. Pat. Nos. 5,932,448; 6,833,441; 5,591,828; 7,129,330; 7,507,796; Fischer et al., (2007) Pathobiology 74:3-14; Booy (2006) Arch. Immunol. Ther. Exp. 54:85-101; Cao et al (2003) 55:171-197; and Marvin et al., (2006) Current Opinion in Drug Discovery & Development 9(2):184-193.

Method used

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  • Coiled coil and/or tether containing protein complexes and uses thereof
  • Coiled coil and/or tether containing protein complexes and uses thereof
  • Coiled coil and/or tether containing protein complexes and uses thereof

Examples

Experimental program
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Effect test

example 1

Construction of Vectors for the Expression of Coiled Coil Containing Antibodies

[0368]The coiled coil heterodimerization domains described herein can be linked to a constant chain (e.g., the C-terminus of the HC) of any antibody. Numerous antibody sequences that can be used to construct coiled coil containing antibodies are known in the art and techniques required to manipulate DNA sequences are also well known in the art. An exemplary method for constructing coiled coil containing antibodies is described below.

[0369]The HC backbone for the generation of antibodies containing a coiled coil was constructed as follows. Sense and anti-sense oligonucleotides were designed and synthesized to encode either the ACID.p1 (GGSAQLEKELQALEKENAQLEWELQALEKELAQGAT; SEQ ID NO:33) or BASE.p1 (GGSAQLKKKLQALKKKNAQLKWKLQALKKKLAQGAT; SEQ ID NO:34) coiled coil domain sequence with 5′ AscI and 3′ XbaI overhangs. The oligonucleotides were annealed, phosphorylated, and ligated into a digested and dephosphory...

example 2

Purification of Coiled Coil Containing Antibodies

[0375]An exemplary schema that can be used to purify coiled coil containing antibodies is shown below.

[0376]In particular, antibodies were purified from conditioned media using mAbSure Select resin from GE Healthcare (Sweden) overnight at 4° C. The column was washed with two column volumes (CV) of PBS (phosphate buffered saline), followed by 10 CV of PBS+0.1% Triton X114 detergent, followed by 10 CV potassium phosphate buffer. The columns were eluted with 10 mM Acetic Acid (pH 2.9) and immediately diluted with Arginine (100 mM final concentration) and Tris (200 mM final concentration), pH 8.0. Coiled coils were removed from antibodies upon treatment with a 1:500 (weight:weight) ratio of Lys-C endopeptidase (Wako Pure Chemical Laboratories) at 37° C. for 1-5 hours. Cleaved samples were loaded back over an mAbSure resin column to separate cleaved coiled-coils from antibodies and eluted as above. Antibody concentrations were adjusted to ...

example 3

Cleavage of Coiled Coil Containing Antibodies

[0381]The various coiled coil containing antibodies were subjected to cleavage experiments to show that the coiled coil (and tether, if present) could be cleaved from the antibody sequence while leaving the antibody sequence intact. In particular, FIGS. 13A and B show that the coiled coil was cleaved from an exemplary α-FcεR1 / α-FcγR2b antibody using Lys-C endopeptidase and that the antibody remained intact. The theoretical mass for the antibody with the coiled coil is within the margin of error of the mass experimentally observed by mass spectrometry. Similary, the theoretical mass for the engineered antibody without the coiled coil is within the margin of error of that experimentally observed by mass spectrometry showing that Lys-C cleaved the coiled coil from the antibody.

[0382]Mass spectrometry results also demonstrated that Lys-C endopeptidase did not cleave the LC or HC of the exemplary α-FcER1 / α-FcγR2b antibody (FIGS. 14A and B). In...

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Abstract

The invention provides engineered protein complexes constructed using a coiled coil and/or a tether and methods for making, using, and purifying such complexes, such as multispecific antibodies or other multispecific Fc containing complexes.

Description

FIELD OF THE INVENTION[0001]This invention relates to novel engineered proteins, multispecific protein complexes, including multispecific antibodies, methods of constructing them and producing them. This invention also relates to the new application of technologies useful in obtaining the multispecific protein complexes.BACKGROUND OF THE INVENTION[0002]Finding technologies for building multispecific antibodies that are useful and scalable for commercial and therapeutic purposes has been elusive. Many methods have been tried, but nearly all suffer significant drawbacks such as being poorly soluble; inexpressible in mammalian cells, demonstrating low yield of heterodimer formation, technically challenging to manufacture, immunogenic, short half-life in vivo, unstable among other problems (e.g., Hollinger et al., (1993) PNAS 90:6444-6448; U.S. Pat. Nos. 5,932,448; 6,833,441; 5,591,828; 7,129,330; 7,507,796; Fischer et al., (2007) Pathobiology 74:3-14; Booy (2006) Arch. Immunol. Ther. E...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/00C07K19/00C07K16/46C12P21/02
CPCC07K16/283C07K16/2863C07K16/32C07K16/468C07K2319/73C07K2317/73C07K2317/92C07K2319/00C07K2317/53C07K2317/31C07K2317/51
Inventor CHRISTENSEN, ERIN H.EATON, DAN L.VENDEL, ANDREW C.WRANIK, BERND
Owner F HOFFMANN LA ROCHE & CO AG
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