Protein variants having modified immunogenicity

Abstract

The present invention relates to a method of selecting a protein variant having modified immunogenicity as compared to the parent protein comprising the steps obtaining antibody binding peptide sequences, using the sequences to localise epitope sequences on the 3-dimensional structure of parent protein, defining an epitope area including amino acids situated within 5 Å from the epitope amino acids constituting the epitope sequence, changing one or more of the amino acids defining the epitope area of the parent protein by genetical engineering mutations of a DNA sequence encoding the parent protein, introducing the mutated DNA sequence into a suitable host, culturing said host and expressing the protein variant, and evaluating the immunogenicity of the protein variant using the parent protein as reference. The invention further relates to the protein variant and use thereof, as well as to a method for producing said protein variant.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a 35 U.S.C. 371 national application of PCT / DK01 / 00293 filed Apr. 30, 2001 and claims, under 35 U.S.C. 119, priority or the benefit of Danish application nos. PA 2000 00707 and PA 2001 00327 filed Apr. 28, 2000 and Feb. 28, 2001, respectively, and U.S. application Ser. Nos. 60 / 203,345 and 60 / 277,817 filed May 10, 2000 and Mar. 21, 2001, respectively, the contents of which are fully incorporated herein by reference.FIELD OF INVENTION [0002] The present invention relates to a method of selecting a protein variant having modified immunogenicity as compared to the parent protein, to the protein variant and use thereof, as well as to a method for producing said protein variant. BACKGROUND OF THE INVENTION [0003] An increasing number of proteins, including enzymes, are being produced industrially, for use in various industries, housekeeping and medicine. Being proteins they are likely to stimulate an immunological response...

AI Technical Summary

Benefits of technology

[0044] A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. “Homologous recombination” refers to the insertion of a foreign DNA sequence of a vector in a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination. Nucleic Acid Sequence

[0053] The term “control sequences” is defined herein to include all components which are necessary or advantageous for expression of the coding sequence of the nucleic acid sequence. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0086] The vectors of the present invention may be integrated into the host cell genome when introduced into a host cell. For integration, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination. These nucleic acid sequences may be any sequence that is homologous with a target sequence in the genome of the host cell, and, furthermore, may be non-encoding or encoding sequences.

Problems solved by technology

This may result in a reduced importance of such an epitope, maybe converting it from a high affinity to a low affinity epitope, or maybe even result in epitope loss, i.e. that the epitope cannot sufficiently bind an antibody to elicit an immunogenic response.

2) reduce the potential of commercial proteins to cross-react with environmental allergens and hence cause allergic reactions in people sensitized to the environmental allergens (or vice versa).

All of these methods, however, only leads to identification of linear epitopes, not to identification of ‘structural’ or ‘discontinuous’ epitopes, which are found on the 3-dimensional surface of protein molecules and which comprise amino acids from several discrete sites of the primary sequence of the protein.

The major drawbacks of this approach are the ‘trial and error’ character, which makes it a lengthy and expensive process, and the lack of general information on the epitope patterns.

Further, their method cannot be used in the absence of such crystallographic data for antigen-antibody complexes, which are very cumbersome, sometimes impossible, to obtain—especially since one would need a separate crystal structure for each epitope to be changed.

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