Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1997-12-31
2001-01-09
Scheiner, Laurie (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000, C530S324000, C435S069700
Reexamination Certificate
active
06172190
ABSTRACT:
The invention relates to the field of programmed cell death.
BACKGROUND OF THE INVENTION
In multicellular organisms, homeostasis is maintained by balancing the rate of cell proliferation against the rate of cell death. Cell proliferation is influenced by numerous growth factors and the expression of proto-oncogenes, which typically encourage progression through the cell cycle. In contrast, numerous events, including the expression of tumor suppressor genes, can lead to an arrest of cellular proliferation.
In differentiated cells, a particular form of cell death called apoptosis is carried out when an internal suicide program is activated. This program can be initiated by a variety of external signals as well as signals that are generated within the cell in response to, for example, genetic damage. For many years, the magnitude of apoptotic cell death was not appreciated because the dying cells are quickly eliminated by phagocytes, without an inflammatory response.
The mechanisms that mediate apoptosis have been intensively studied. These mechanisms involve the activation of endogenous proteases, loss of mitochondrial function, and structural changes such as disruption of the cytoskeleton, cell shrinkage, membrane blebbing, and nuclear condensation due to degradation of DNA. The various signals that trigger apoptosis are thought to bring about these events by converging on a common cell death pathway that is regulated by the expression of genes that are highly conserved from worms, such as C. elegans, to humans. In fact, invertebrate model systems have been invaluable tools in identifying and characterizing the genes that control apoptosis. Through the study of invertebrates and more evolved animals, numerous genes that are associated with cell death have been identified, but the way in which their products interact to execute the apoptotic program is poorly understood.
Recently, several polypeptides were discovered which form a complex that transmits an apoptotic signal when the Fas/APO-1 receptor is bound (Boldin et al.,
Cell
85:803, 1996; Muzio et al.,
Cell
85:817, 1996). This receptor, also known as CD95, is present on the surface of a wide variety of cells (Boldin et al., supra; Muzio et al., supra). The Fas/APO-1 receptor and the TNF receptor (described below) are classified as members of the TNF
erve growth factor receptor family and both share a region of homology designated the “death domain” (Boldin et al., supra; Muzio et al., supra). The death domain of the Fas/APO-1 receptor interacts with FADD (Fas-associating protein with death domain, also known as MORT1) and RIP (receptor interacting protein), forming a complex that, when joined by Caspas-8, constitutes the Fas/APO-1 death-inducing signalling complex (Boldin et al., supra; Muzio et al., supra). The interaction between Fas/APO-1 and FADD is mediated by their respective C-terminal death domains (Chinnaiyan et al.,
Cell
81:505-512, 1995). Caspase-8 contains two N-terminal stretches of approximately 60 amino acids that are homologous to the DED of FADD (Muzio et al., supra). The remainder of Caspase-8 is highly homologous to the ICE/CED-3 family of cysteine proteases, which induce cell death if overexpressed. A number of forms of Caspase-8 have been described (Boldin et al., supra).
Caspase-8 may also be an important part of a second complex which is involved in cell death. This complex forms in association with the intracellular portion of the tumor necrosis factor (TNF) receptor (TNFR-1 or p55-R), and includes Caspase-8, TRADD (TNFR-1-associated death domain protein), and FADD/MORT1 (Boldin et al., supra; Muzio et al., supra).
SUMMARY OF THE INVENTION
The present invention relates to the discovery and characterization of two novel forms of Caspase-8. The first Caspase-8 of the invention, Caspase-8h, encodes a 220 amino acid polypeptide which includes two FADD death effector domains. The second caspase of the invention, Caspase-8i, encodes an 81 amino acid polypeptide having one FADD death effector domain.
The invention encompasses nucleic acid molecules encoding Caspase-8h and Caspase-8i, vectors containing these nucleic acid molecules, cells harboring recombined DNA encoding Caspase-8h and/or Caspase-8i, host fusion proteins which include Caspase-8h and/or Caspase-8i, transgenic animals which express Caspase-8h and/or Caspase-8i, recombinant knock-out animals which fail to express Caspase-8.
By “isolated nucleic acid molecule” is meant a nucleic acid molecule that is separated from either the 5′ or the 3′ coding sequence with which it is immediately contiguous in the naturally occurring genome of an organism. An isolated nucleic acid molecule is also referred to as “recombinant nucleic acid molecule.”
The nucleic acid molecules of the invention can be inserted into transcription and/or translation vectors, as described below, which will facilitate expression of the insert. The nucleic acid molecules and the polypeptides they encode can be used directly as diagnostic or therapeutic agents, or (in the case of a polypeptide) can be used to generate antibodies that, in turn, are therapeutically useful. Accordingly, expression vectors containing the nucleic acid of the invention, cells transfected with these vectors, the polypeptides expressed, and antibodies generated, against either the entire polypeptide or an antigenic fragment thereof, are among the preferred embodiments.
As used herein, the term “transfected cell” means any cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid encoding a polypeptide of the invention (e.g., a Caspase-8h polypeptide or a Caspase-8i polypeptide).
As used herein, both “protein” and “polypeptide” mean any chain of amino acid residues, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). The polypeptides of the invention are referred to as “substantially pure, ” meaning that they are at least 60% by weight (dry weight) the polypeptide of interest, e.g., a Caspase-8 polypeptide or a Caspase-8i polypeptide. Preferably, the polypeptide is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, the polypeptide of interest. Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The polypeptide can be a naturally occurring, synthetic, or a recombinant molecule consisting of a hybrid with one portion, for example, being encoded by all or part of the Caspase-8h or Caspase-8i gene, and a second portion being encoded by all or part of a second gene. For example, the AZB polypeptide may be fused to a hexa-histidine tag to facilitate purification of bacterially expressed protein, or to a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells.
The polypeptides of the invention can also be chemically synthesized, or they can be purified from tissues in which they are naturally expressed, according to standard biochemical methods of purification.
Also included in the invention are “functional polypeptides,” which possess one or more of the biological functions or activities of the caspase-8h or caspase-8i. These functions or activities are described in detail below and concern, primarily, inhibition of apoptosis and/or the ability to bind some or all of the proteins which normally bind to caspase-8h or caspas-8i. A functional polypeptide is also considered within the scope of the invention if it serves as an immunogen for production of antibodies that specifically bind to caspase-8h or caspase-8i. In many cases, functional polypeptides retain one or more domains present in the naturally-occurring form of the polypeptide. For example, a functional polypeptide may posses one or more FADD death effector domains. It is well within the abilities of skilled artisans to determine whether a polypeptide, regardless of size, retains the function activity of a polypeptide of the invention.
The functional polypeptides may contain
Hunter John J.
Shyjan Andrew W.
Wong Grace H. W.
Fish & Richardson P.C.
Millennium Pharmaceuticals Inc.
Parkin Jeffrey S.
Scheiner Laurie
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