Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-10-12
2003-12-30
Kunz, Gary (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S069100, C435S188000, C435S235100, C435S325000, C435S375000, C435S320100, C435S007200, C530S300000, C530S350000, C536S023100, C536S023500
Reexamination Certificate
active
06670135
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to semaphorin polypeptides, nucleic acids encoding such semaphorin polypeptides, processes for producing recombinant semaphorin polypeptides, pharmaceutical compositions containing such polypeptides relates and processes for treating disorders associated with semaphorin activity.
BACKGROUND OF THE INVENTION
The semaphorin gene family includes a large number of molecules that encode related transmembrane and secreted glycoproteins known to be neurologic regulators. The semaphorins are generally well conserved in their extracellular domains which are typically about 500 amino acids in length. Semaphorin family proteins have been observed in neuronal and nonneuronal tissue and have been studied largely for their role in neuronal growth cone guidance. For example, the secreted semaphorins known as collapsin-1 and Drosophila semaphorin II are selectively involved in repulsive growth cone guidance during development. Flies having semaphorin II genes that are mutated so that their function is reduced exhibit abnormal behavior characteristics.
Another semaphorin gene has been identified in several strains of poxvirus. This semaphorin is found in vaccinia virus (Copenhagen strain) and Ectromelia virus, and is encoded in an open reading frame (ORF) known as A39R. The A39R encoded protein has no transmembrane domain and no potential membrane linkage and is known to be a secreted protein. Variola virus ORF also contains sequences that share homology with the vaccinia virus ORF A39R at the nucleotide level and the amino acid level. Another viral semaphorin. AHV-sema, has been found in the Alcelaphine Herpesvirus (AHV).
Genes encoding mammalian (human, rat, and mouse) semaphorins have been identified, based upon their similarity to insect semaphorins. Functional studies of these semaphorins suggest that embryonic and adult neurons require a semaphorin to establish workable connections. Significantly, the fast response time of growth cone cultures to appropriate semaphorins suggests that semaphorin signaling involves a receptor-mediated signal transduction mechanism. Semaphorin ligands that are secreted into the extracellular milieu signal through receptor bearing cells in a located and systemic fashion. In order to further investigate the nature of cellular processes regulated by such local and systemic signaling, it would be beneficial to identify additional semaphorin receptors and ligands. Furthermore, because virus encoded semaphorins are produced by infected cells and are present in viruses that are lytic (poxviruses) and viruses that are not known to be neurotropic (AHV), it is unlikely that their primary function is to modify neurologic responses. It is more likely that the virus encoded semaphorins function to modify the immunologic response of the infected host and it is likely that mammalian homologous to virus encoded semaphorins function to modify the immunologic response. In view of the suggestion that viral semaphorins may function in the immune system as natural immunoregulators it would be beneficial to identify semaphorins that may be therapeutic agents for enhancing or diminishing the immune response.
SUMMARY OF THE INVENTION
The present invention pertains to novel semaphorins as isolated or homogeneous proteins. In particular, the present invention provides semaphorin polypeptides, that are homologous to the viral semaphorins A39R and AHV Sema. Within the scope of the present invention are DNAs encoding semaphorin polypeptides and expression vectors that include DNA encoding semaphorin polypeptides. The present invention also includes host cells that have been transfected or transformed with expression vectors that include DNA encoding a semaphorin polypeptide, and processes for producing semaphorin polypeptides by culturing such host cells under conditions conducive to expressing semaphorin polypeptides. The present invention further includes antibodies directed against semaphorin polypeptides.
Further within the scope of the present invention are processes for purifying or separating certain novel semaphorin polypeptides or cells that express novel semaphorins to which semaphorin receptor polypeptides bind. Such processes include binding at least one semaphorin receptor to a solid phase matrix and contacting a mixture containing a semaphorin polypeptide to which the semaphorin receptor binds, or a mixture of cells expressing the semaphorin with the bound semaphorin receptor, and then separating the contacting surface and the solution.
The present invention additionally provides processes for treating mammals afflicted with a disease that is ameliorated by the interaction of semaphorins and their receptors. Such processes involve activating immune cells that express receptors for the semaphorins of the present invention and include administering a therapeutically effective amount of semaphorin to a mammal afflicted with the disease. The therapeutically effective amount is sufficient to activate immune system cells that express semaphorin receptors. Such an activation results in the secretion of cytokines, the regulation of activation antigens or the migration of the cell to sites of immune activity.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel semaphorin polypeptides, DNA encoding semaphorin polypeptides and recombinant expression vectors that include DNA encoding semaphorin polypeptides. The present invention further provides methods for isolating semaphorin polypeptides and methods for producing recombinant semaphorin polypeptides by cultivating host cells transfected with the recombinant expression vectors under conditions appropriate for expressing semaphorin and recovering the expressed semaphorin polypeptide.
This invention additionally provides antibodies directed against semaphorin polypeptides.
Particular semaphorin embodiments of the present invention include polypeptides homologous to AHV sema. The native semaphorin polypeptide described herein was discovered using data base search and comparison techniques that resulted in the identification of at least one EST having some homology to viral semaphorins. As described in Example 1, PCR techniques were used to identify and clone the full viral semaphorin homologue. The human semaphorin of the present invention is found in placenta, testis, ovary, spleen, dendritic cells, and B cells. Semaphorin polypeptides of the present invention bind to the semaphorin receptor, designated VESPR (described in copending application S/N 08/958,598, incorporated herein by reference). Evidence suggests that the interaction between the semaphorins of the present invention and their receptors are associated with the immune suppression of mature dendritic cells. For this reason, the semaphorins of the present invention are designated DCSema.
Example 1 describes identifying a native DCSema of the present invention. The amino acid sequence of the identified native DCSema is disclosed in SEQ ID NO:2 and the DNA encoding the amino acid sequence is disclosed in SEQ ID NO:1. The amino acid sequence presented in SEQ ID NO:2 is a secreted soluble polypeptide, but may additionally exist as a membrane bound protein. The amino acid sequence of SEQ ID NO:2 has a predicted signal sequence that includes amino acids 1-44. Also encompassed within the present invention are soluble DCSema polypeptides that lack the signal sequence. An example of such a polypeptide is amino acids 45-666 of SEQ ID NO:2. The EST 151129 portion of SEQ ID NO:2 has a 28% identity to A39R and 44% identity to AHV sema both of which bind to VESPR, a semaphorin receptor described in copending patent application Ser. No. 08/958,598. A39R and AHV sema share 29% identity.
The terms “semaphorin polypeptide”, “human semaphorin homologue”, “DCSema” and homologous of AHV Sema encompass polypeptides having the amino acid sequence disclosed in SEQ ID NO:2, and proteins that are encoded by nucleic acids that contain the nucleic acid sequence of SEQ ID NO:1. In addition, those polypeptides that have a high
Basi Virmal S.
Fowler Kathleen
Henry Janis C.
Immunex Corporation
Kunz Gary
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