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
1998-01-27
2003-10-14
Spector, Lorraine (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C435S252300, C435S069700, C435S320100, C536S023500
Reexamination Certificate
active
06632634
ABSTRACT:
This application relates to the preparation of decay is accelerating factor (hereinafter abbreviated as DAF) in recombinant cell culture. In particular, it is concerned with the large scale manufacture of DAF suitable for pharmaceutical or diagnostic use.
Antigenic cells targeted by the humoral immune response are lysed by a process called complement activation. This process consists of a series or cascade of proteolytic activities initiated by the binding of antibody with its antigen. The components that participate in complement activation are many and complex, although for the purposes herein the most important are C4b and C3b. In a key step in complement activation, these two proteins become covalently associated with the target cell surface and then serve as anchors for the assembly of C3 and C5 convertases, the amplifying enzymes of the cascade.
Complement activation must focus only on the target and must not occur on host cells. However, in the course of complement activation, large numbers of nascent C4b and C3b fragments are liberated into the fluid phase. Most react with water, but some by chance could bind to nearby host cells and lead to their damage. For this and possibly other reasons, the activities of bound, as well as free, C3b and C4b fragments are under strict control by a complex system of serum and membrane proteins.
Recent evidence (Medof. et al. 1982. “J. Exp. Med.” 156:1739; Medof, et al., 1984. “J. Exp. Med.” 159:1669) suggests that regulation of the activities of membrane-bound C4b and C3b is distinct from control of the fluid phase fragments. The functions of the former are controlled mainly by two membrane proteins: the C3b/C4b receptor (CR1) and DAF. CR1 dissociates C2 and factor B from C4b and C3b in C3 and C5 convertase complexes and promotes the cleavage of C3b (Medof, et al.,
J. Exp. Med.
156:1739 [1982]; Fearon, D. T.
Proc. Natl. Acad. Sci. USA:
76:5867 [1979]; Medicus, et al.,
Eur. J. Immunol.
13:465 [1983]; and Ross, et al.,
J. Immunol.
129:2051 [1982]) and C4b (Medof, et al.,
J. Exp. Med.
159:1669 [1984]; Iida et al.,
J. Exp.Med.
153:1138 [1981]) by the serum enzyme C3b/C4b inactivator (I). DAF has been shown also to enhance the decay dissociation of C2 and factor B from C3 convertases (Nicholson-Weller et al.,
J. Immunol.
129:205 [1982] and Pangburn, M. K. et al.,
J. Exp. Med.
157:1971 [1983]). The reason for the apparent redundancy in regulatory activities of the two membrane factors and their respective roles in convertase control has remained unclear. Abnormalities of CR1have been found in systemic lupus erythematosus (SLE) (Miyakawa, Y. et al.,
Lancet
2:493 [1981]; Iida, K. et al.,
J. Exp. Med.
155:1427 [1982]; Wilson, J. G. et al.,
N. Engl. J. Med.
307:981 [1982]; Taylor, R. P. et al.,
Arthritis Rheum.
26:736 [1983]), a condition associated with defective immune complex handling, and abnormalities of DAF have been found in paroxysmal nocturnal hemoglobinuria (PNH) (Pangburn, M. K. et al.,
J. Exp. Med.
157:1971 [1983]; Pangburn, J. K. et al.,
Proc. Natl. Acad. Sci.
80:5430 [1983]; Nicholson-Weller, A. et al.,
Proc. Natl. Acad. Sci.
80:5066 [1983]), a condition associated with heightened susceptibility of blood cells to lysis.
DAF was reported to have been purified as a single 70 Kd band on silver stained SDS-PAGE from a pooled extract of human erythrocyte stroma (Medof et al.,
J. Exp. Med.
160:1558 [1983]). The molecule was hydrophobic and tended to form multimers of greater than or equal to 150 Kd as determined by molecular sieve chromatography. Purified DAF could reassociate with red blood cells. Only a small number of DAF molecules (less than 100) had a significant effect on the hemolytic effect of activated complement. Medof et al. concluded that DAF can only function intrinsically within the cell membrane, and suggested that it offered the possibility of correcting in vitro the defect in the membranes of cells from patients with PNH.
Existing methods for obtaining DAF are unsatisfactory for its commercial preparation. Red cells contain extremely small quantities of DAF. Furthermore, blood contains viruses and other biologically active components which pose a risk of adverse reactions in recipients or users.
Red blood cell DAF is limited to the native membrane bound form, including any naturally occurring alleles as may exist. Methods are needed for synthesizing amino acid and glycosylation variants which can function as DAF agonists or antagonists, or which will exhibit other desirable characteristics such as the absence of C-terminal lipid, resistance to proteases, or the ability to deliver DAF to the membranes of target cells.
Accordingly, it is an object herein to prepare DAF in commercial quantity from a therapeutically acceptable source.
It is a further object of obtain human DAF from a source that is completely uncontaminated with other human proteins.
It is an additional object to prepare amino acid sequence and glycosylation variants of DAF.
Other objects of this invention will be apparent from the specification as a whole.
SUMMARY
The objects of this invention are accomplished by expression of DAF in recombinant cell culture, a process that fundamentally comprises providing nucleic acid encoding DAP, transforming a host cell with the DAF-encoding nucleic acid, and culturing the cell in order to express DAF in the host cell culture.
The method of this invention enables the preparation of novel forms of DAF, including amino acid sequence variants and glycosylation variants. Amino acid sequence variants consist of deletions, substitutions and insertions of one or more DAF amino acid residues. DAF also is expressed in a form unaccompanied by the glycosylation associated with the native DAF (including unaccompanied by any glycosylation whatsoever), obtained as a product of expression of DAF in heterologous recombinant cell culture. DAF in any form as a component of a recombinant cell culture is novel.
Unexpectedly, I discovered during my studies of cell processing of DAF mRNA that the membrane-bound form of DAF (mDAF) is not the only form in which it is expressed in vivo. In fact, another form of DAF exists, called sDAF. This form is encoded by an mRNA species from which the last 3′ intron has not been spliced, resulting in an amino acid sequence C-terminal to residue 327 that is entirely different from that of MDAF. The novel C-terminus of sDAF is postulated to result in vivo in the secretion of the protein into the blood stream (where it may be biologically active) because the presence of the intron changes the reading frame of the last exon so as to eliminate the “signal” directing attachment of phosphatidylinositol (the membrane anchor for mDAF). This novel form of DAF was unappreciated until the pioneering work herein was accomplished, and it differs from MDAF in containing an antigenically distinct C-terminus. ODAF is useful in diagnosis of PNH since it is now possible to determine whether the condition in an individual results from a failure to express any of the DAF gene or a failure of post-translational processing to attach the phosphatidylinositol anchor.
Novel nucleic acids also are provided, including (1) cell free nucleic acid identified as encoding DAF, including genomic DNA, cDNA or RNA, (2) DNA encoding DAP free of an untranslated intervening sequence (introns) or flanking genomic DNA, and (3) nucleic acid encoding DAF which is free of nucleic acid encoding any other protein homologous to the source of the nucleic acid that encodes DAF. Also within the scope of this invention is nucleic acid which does not encode DAF but which is capable of hybridizing with nucleic acid encoding DAF.
Nucleic acid encoding DAF is useful in the expression of DAF in recombinant cell culture or for assaying test samples for the presence of DAF-encoding nucleic acid. Labelled DAF-encoding or hybridizing nucleic acid is provided for use i
Caras Ingrid W.
Davitz Michael A.
Martin, Jr. David W.
Nussenzweig Victor
Dreger Ginger R.
Genentech Inc.
Heller Ehrman White & McAuliffe LLP
Spector Lorraine
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