Method for making heteromultimeric polypeptides
First Claim
1. A method of preparing a heteromultimer comprising a first polypeptide and a second polypeptide which meet at an interface which has been engineered to promote heteromultimer formation, wherein the interface of the first polypeptide comprises a protuberance which is positionable in a cavity in the interface of the second polypeptide the method comprising the steps of:
- (a) culturing a host cell comprising nucleic acid encoding the first polypeptide and second polypeptide including the interfaces thereof, wherein the nucleic acid encoding the interface of the first polypeptide has been altered from nucleic acid encoding the original interface of the first polypeptide to encode the protuberance or the nucleic acid encoding the interface of the second polypeptide has been altered from nucleic acid encoding the original interface of the second polypeptide to encode the cavity or both, and wherein the culturing is such that the first polypeptide and second polypeptide are expressed, wherein the ratio of heteromultimer;
homomultimer which forms is greater than that for a multimer which lacks the protuberance and/or cavity; and
(b) recovering the heteromultimer from the host cell culture.
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Abstract
The invention relates to a method of preparing heteromultimeric polypeptides such as bispecific antibodies, bispecific immunoadhesins and antibody-immunoadhesin chimeras. The invention also relates to the heteromultimers prepared using the method. Generally, the method involves introducing a protuberance at the interface of a first polypeptide and a corresponding cavity in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heteromultimer formation and hinder homomultimer formation. "Protuberances" are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). The protuberance and cavity can be made by synthetic means such as altering the nucleic acid encoding the polypeptides or by peptide synthesis.
741 Citations
41 Claims
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1. A method of preparing a heteromultimer comprising a first polypeptide and a second polypeptide which meet at an interface which has been engineered to promote heteromultimer formation, wherein the interface of the first polypeptide comprises a protuberance which is positionable in a cavity in the interface of the second polypeptide the method comprising the steps of:
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(a) culturing a host cell comprising nucleic acid encoding the first polypeptide and second polypeptide including the interfaces thereof, wherein the nucleic acid encoding the interface of the first polypeptide has been altered from nucleic acid encoding the original interface of the first polypeptide to encode the protuberance or the nucleic acid encoding the interface of the second polypeptide has been altered from nucleic acid encoding the original interface of the second polypeptide to encode the cavity or both, and wherein the culturing is such that the first polypeptide and second polypeptide are expressed, wherein the ratio of heteromultimer;
homomultimer which forms is greater than that for a multimer which lacks the protuberance and/or cavity; and(b) recovering the heteromultimer from the host cell culture. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)
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31. A method of preparing a heteromultimer which comprises a first polypeptide and a second polypeptide which meet at an interface which has been engineered to promote heteromultimer formation the method comprising the steps of:
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(a) preparing the first polypeptide and second polypeptide including the interfaces thereof, wherein the interface of the first polypeptide comprises a protuberance which is positionable in a cavity in the interface of the second polypeptide, and wherein the amino acid sequence of an original interface has been altered so as to introduce the protuberance into the interface of the first polypeptide or the amino acid sequence of an original interface has been altered so as to introduce the cavity into the interface of the second polypeptide, or both, and (b) allowing the heteromultimer to form, wherein the ratio of heteromultimer;
homomultimer which forms is greater than that for a multimer which lacks the protuberance and/or cavity. - View Dependent Claims (32, 33)
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34. Isolated nucleic acid encoding a heteromultimer comprising a first polypeptide and a second polypeptide which meet at an interface, wherein the interface of the first polypeptide comprises a protuberance which is positionable in a cavity in the interface of the second polypeptide, and wherein nucleic acid encoding the interface of the first polypeptide has been altered from nucleic acid encoding the original interface of the first polypeptide to encode the protuberance or the nucleic acid encoding the interface of the second polypeptide has been altered from nucleic acid encoding the original interface of the second polypeptide to encode the cavity, or both, such that a greater ratio of hetermultimer:
- homomultimer forms than that for a multimer having a nonengineered interface.
- View Dependent Claims (36, 37, 38, 39, 40, 41)
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35. A composition comprising two or more recombinant nucleic acids encoding a heteromultimer comprising a first polypeptide and a second polypeptide which meet at an interface, wherein the interface of the first polypeptide comprises a protuberance which is positionable in a cavity in the interface of the second polypeptide, and wherein nucleic acid encoding the interface of the first polypeptide has been altered from nucleic acid encoding the original interface of the first polypeptide to encode the protuberance or the nucleic add encoding the interface of the second polypeptide has been altered from nucleic acid encoding the original interface of the second polypeptide to encode the cavity, or both, such that a greater ratio of hetermultimer:
- homomultimer forms than that for a multimer having a nonengineered interface.
Specification