Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-05-01
2004-01-27
Chan, Christina (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S070100, C530S353000, C536S023500
Reexamination Certificate
active
06682911
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to the laminin 12, laminin subunit &ggr;3, and laminin subunit &bgr;1, and methods of making and using these molecules.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the discovery of a novel member of the laminin family, laminin 12. Accordingly, the present invention features a purified or isolated preparation or a recombinant preparation of laminin 12 which includes an &agr;2 subunit, a &bgr;1 subunit and a &ggr;3 subunit.
In a preferred embodiment, the &agr;2 subunit has at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human &agr;2 subunit, e.g., the human &agr;2 subunit of SEQ ID NO:7. The &agr;2 subunit can be identical to a human &agr;2 sequence, e.g., that of SEQ ID NO:7. In another embodiment, the &agr;2 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:8. In addition, the &agr;2 subunit can have substantially the same electrophoretic mobility as human &agr;2 subunit, e.g., it appears as a 205 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an &agr;2 subunit which is reactive with an &agr;2-specific antibody, e.g., an antibody which binds to the epitope recognized by mAb 5H2. &agr;2 specific antibodies can be made by methods known in the art.
Another preferred embodiment of the invention features a &bgr;1 subunit having at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human &bgr;1 subunit, e.g., the human &bgr;1 subunit of SEQ ID NO:9. Preferably, the &bgr;1 subunit has the identical amino acid sequence of human &bgr;1 subunit, e.g., that of SEQ ID NO:9. In another embodiment, the &bgr;1 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:10. In addition, the &bgr;1 subunit can have substantially the same electrophoretic mobility as human &bgr;1 subunit, e.g., it appears as a 185 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an &bgr;1 subunit which is reactive with an &bgr;1-specific antibody, e.g., an antibody which binds to the epitope recognized by mAb 545. &bgr;1-specific antibodies can be made by methods known in the art.
In yet another preferred embodiment, the &ggr;3 subunit of laminin 12 has at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human &ggr;3 subunit, e.g., the &ggr;3 subunit of SEQ ID NO:3. The &ggr;3 subunit can be identical to a naturally occurring human &ggr;3 subunit, e.g., that of SEQ ID NO:3. In another embodiment, the &ggr;3 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:4. In addition, the &ggr;3 subunit can have substantially the same electrophoretic mobility as human &ggr;3 subunit, e.g., it appears as a 170 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an &ggr;3 subunit which is reactive with an &ggr;3-specific antibody. &ggr;3-specific antibodies can be made by methods known in the art and taught herein.
In a preferred embodiment, the laminin 12 is a trimer which can be found in, or can be isolated from human placental chorionic villi. In another embodiment, the laminin 12 is expressed by a recombinant cell, e.g., a bacterial cell, a cultured cell (e.g., a cultured eukaryotic cell) or a cell of a non-human transgenic animal. Cultured cells can include CHO cells or SF8 cells. Expression of laminin 12 in a transgenic animal can be general or can be under the control of a tissue specific promoter. Preferably, one or more sequences which encode subunits of the laminin 12 trimer are expressed in a preferred cell-type by a tissue specific promoter, e.g., a milk specific promoter.
The present invention is also based, in part, on the discovery of a novel laminin subunit, &ggr;3. Accordingly, the invention features a recombinant or substantially pure or isolated preparation of a &ggr;3 polypeptide.
In a preferred embodiment, the &ggr;3 polypeptide has the following biological acitivities: 1) it promotes adhesion between tissue elements; 2) provides a site for insertion of nerves into the basement membrane. In other preferred embodiments: the &ggr;3 polypeptide includes an amino acid sequence with at least 60%, 80%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence from SEQ ID NO:3; the &ggr;3 polypeptide includes an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:3; the &ggr;3 polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the &ggr;3 polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:3; the &ggr;3 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring &ggr;3 subunit; the &ggr;3 polypeptide is a vertebrate, e.g., a mammalian, e.g. a primate, e.g., a human, &ggr;3 polypeptide.
In a preferred embodiment, the invention includes a &ggr;3 polypeptide encoded by a DNA insert of a plasmid deposited with ATCC as Accession No: 209357. In another embodiment, the &ggr;3 polypeptide is a polypeptide encoded by nucleotide sequences of the overlapping DNA inserts of more than one, preferably all seven of the plasmids deposited with ATCC as Accession No:209357.
In preferred embodiments: the &ggr;3 polypeptide is encoded by the nucleic acid in SEQ ID NO:4, or by a nucleic acid having at least about 85%, more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with the nucleic acid from SEQ ID NO:4.
In preferred embodiments, the &ggr;3 polypeptide includes a nidogen-binding domain. Generally, the nidogen-binding domain is at least 5 residues in length and preferably, has about 70, 80, 90, or 95% sequence identity with the nidogen-binding domain of the protein shown in SEQ ID NO: 3 (amino acid residues 750-755). In another embodiment, the &ggr;3 polypeptide includes at least 5, preferably 6 to 7, and most preferably 8 of the cysteins found in native &ggr;3 protein. In yet another embodiment of the invention features a &ggr;3 polypeptide that does not include or has an inactivated nidogen-binding domain which serves as an antagonist to &ggr;3 biological activities. Furthermore, a &ggr;3 polypeptide which has antagonist activity can have inactivated or excluded regions which comprise at least one cystein found in native &ggr;3 protein.
In a preferred embodiment, the &ggr;3 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO: 3. In other preferred embodiments, the &ggr;3 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10% of the residues from a sequence in SEQ ID NO: 3. Preferably, the differences are such that: the &ggr;3 polypeptide exhibits a &ggr;3 biological activity, e.g., the 73 polypeptide retains a biological activity of a naturally occurring &ggr;3 subunit.
In preferred embodiments the &ggr;3 polypeptide includes a &ggr;3 subunit sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In preferred embodiments, the &ggr;3 polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO: 3, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5′ to the genomic DNA which encodes a sequence from SEQ ID NO: 3.
In yet other preferred embodiments
Brunken William
Burgeson Robert E.
Champliaud Marie-France
Koch Manuel
Olson Pamela
Chan Christina
Fish & Richardson P.C.
Haddad Maher
The General Hospital Corporation
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