Method of making and binding CAIP polypeptides

Details Method of making and binding CAIP polypeptides Method of making and binding CAIP polypeptides

1995-06-07

2001-01-09

Spector, Lorraine (Department: 1646)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007240, C435S069100, C530S300000, C530S350000, C530S351000, C530S402000, C536S023100, C536S023500

Reexamination Certificate

active

06171800

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to CAIP gene, and other related genes, their products, and uses thereof.
SUMMARY OF THE INVENTION
The inventor has discovered several novel intracellular lymphocyte proteins. These proteins are termed, CAIP's, for “CD2 Associated Intracellular Proteins”. The CAIP's, thus discovered, can interact with the intracellular domain of CD2, posses a common central domain, and unique N-terminal regions.
Accordingly, the invention features, a recombinant polypeptide or substantially pure preparation of a peptide, the sequence of which includes, or is, the sequence of a CAIP polypeptide.
In preferred embodiments: the polypeptide has biological activity, e.g., it specifically binds a CD2 intracellular domain; the polypeptide includes an amino acid sequence at least 60%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; the polypeptide includes an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; the polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the 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:2, SEQ ID NO:4, or SEQ ID NO:6; the CAIP polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring CAIP For example, the CAIP polypeptide is an agonist or antagonist of CAIP-CD2 binding or of CAIP or CD2-mediated intracellular signaling. (Unless otherwise indicated, references to the amino acid sequences of SEQ ID NO:4 and SEQ ID NO:6 should be understood to exclude residues 1-5, which are vector sequences. Similarly, references to the nucleic acid sequences of SEQ ID NO:3 and SEQ ID NO:5 should be understood to exclude the bases which encode these 5 amino acid residues.)
In preferred embodiments: the CAIP polypeptide is encoded by the nucleic acid in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or by a nucleic acid having at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homology with the nucleic acid from SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.
In preferred embodiments, the CAIP polypeptide includes a central domain, and an N-terminal domain. Residues 86-553 of SEQ ID NO:2, residues 38-464 of SEQ ID NO:4, and residues 51-324 of SEQ ID NO:6, are exemplary central domains. Generally, a central domain is at least 50, 100, or 150 residues in length and is preferably 50, 60, 70, 80, 90, or 95% homologous with the central domains of the proteins shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
Residues 1-85 of SEQ ID NO:2, residues 6-37 of SEQ ID NO:4, and residues 6-50 of SEQ ID NO:6, are exemplary N-terminal domains. Generally, an N-terminal domain is at least 20, 30, 40, 50, or 60 residues in length and is preferably 50, 60, 70, 80, 90, or 95% homologous with the N-terminal region of the proteins shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
In a preferred embodiment, the subject CAIP polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, from a sequence in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. The differences, however, are such that: the CAIP polypeptide exhibits a CAIP biological activity, e.g., the CAIP polypeptide retains a biological activity of a naturally occurring CAIP, e.g., the CAIP polypeptide from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
In preferred embodiments the CAIP polypeptide includes a CAIP sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.
In preferred embodiments, the polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, 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:2, SEQ ID NO:4, or SEQ ID NO:6.
In yet other preferred embodiments, the CAIP polypeptide is a recombinant fusion protein having a first CAIP portion and a second polypeptide portion, e.g., a second polypeptide portion having an amino acid sequence unrelated to CAIP. The second polypeptide portion can be, e.g., any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.
In a preferred embodiment the CAIP polypeptide encodes amino acid residues 1-85; 86-553; 66-126; 177-236; or 347-406 from SEQ ID NO:2, 6-37; 38-464; 88-147; or 258-317 from SEQ ID NO:4, or 6-50; 51-324; 51-91; 142-201; or 312-324 from SEQ ID NO:6.
In preferred embodiments the CAIP polypeptide has antagonistic activity, and is capable of: inhibiting CD2/CAIP binding, inhibiting CD2 or CAIP mediated intracellular signaling, inhibiting lymphocyte proliferation, e.g., T cell proliferation, or inhibiting an immune disorder characterized by unwanted T cell proliferation, e.g., as in psoriasis or unwanted rejection of transplant tissue.
In a preferred embodiment, the CAIP polypeptide is a fragment of a naturally occurring CAIP which inhibits the CD2 mediated intracellular signaling.
Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events.
The invention includes an immunogen which includes a CAIP polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for said CAIP polypeptide, e.g., a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, e.g., a unique determinant, from a protein represented by SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
The present invention also includes an antibody preparation specifically reactive with an epitope of the CAIP immunogen or generally of a CAIP polypeptide, preferably an epitope which consists all or in part of residues from the N terminal domain, the central domain, an SH3 domain, or an SH3 binding domain.
In preferred embodiments the CAIP-like polypeptide, as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has a molecular weight of at least about 30 kDa, preferably at least about 40 kDa, more preferably at least about 50 kDa, as determined by SDS-PAGE.
In another aspect, the invention provides a substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a polypeptide, the amino acid sequence of which includes, or is, the sequence of a CAIP polypeptide. In preferred embodiments: the encoded polypeptide has biological activity, e.g., it specifically binds a CD2 intracellular domain; the encoded polypeptide includes an amino acid sequence at least 60%, 80%, 90%, 95%, 98%, or 99% homologous to an amino acid sequence from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; the encoded polypeptide includes an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; the encoded polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the encoded 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:2, SEQ ID NO:4, or SEQ ID NO:6; the encoded CAIP polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring CAIP. For example, the encoded CAIP polypeptide is an agonist or antagonist of the CAIP-CD2 binding or of CAIP or CD2-mediated intracellular signaling.
In preferred embodiments: the nucleic acid is or includes that of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; the nucleic acid is at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% homologous with a nucleic acid sequence from SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; the nucleic acid includes a fragment of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 at least 25, 50, 100, 200,

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