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
1995-05-16
2001-05-08
Low, Christopher S. F. (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S252300, C435S325000, C435S358000, C435S440000, C435S475000, C536S023400, C536S023500
Reexamination Certificate
active
06228620
ABSTRACT:
DESCRIPTION
1. Technical Field
This invention relates to protein complexes having Factor VIII:C activity, and to methods for producing said complexes by expression of suitable polynucleotide constructs. The protein complexes are useful in the treatment of classical (Type A) hemophilia.
2. Background of the Invention
Hemophilia A is an X-chromosome-linked inherited disease which afflicts 1-2 males per 10,000. The disease is caused by an absence or deficiency of Factor VIII:C. Factor VIII:C is a very large glycoprotein (native M
r
330 K−360 K), which is present in plasma at extremely low concentrations. It is a necessary element in the proteolytic cascade which converts soluble fibrinogen to insoluble fibrin, forming a clot to prevent blood loss from traumatized tissue. In the bloodstream, it is found in noncovalent association with Factor VIII:R (“von Willebrand factor”), which acts as a stabilizing carrier protein. Factor VIII:C is very susceptible to cleavage by thrombin, plasmin, protease C, and other serine proteases. It is generally isolated from plasma or plasma products as a series of related polypeptides ranging from M
r
160 K-40 K with predominant species of M
r
92 K and M
r
80 K-77 K. This complex pattern has made the analysis of the structure of active Factor VIII:C very difficult.
Factor VIII:C and the related polypeptides have been described by F. Rotblat et al,
Biochemistry
(1985) 24:4294-4300; G. A. Vehar et al,
Nature
(1984) 312:337-342; J. J. Toole et al,
Nature
(1984) 312:342-347; and M. A. Truett et al,
DNA
(1985) 4:333-349. E. Orr et al,
Molecular Genetics of Clotting Factors
, p. 54, s321, reported a “spacer” function for the heavily glycosylated region of Factor VIII:C. The sequence has been reported by J. J. Toole et al, supra; W. I. Wood et al,
Nature
(1984) 312:330-336; and M. A. Truett et al, supra. The full-length protein contains three repeats of one sequence (I), and two repeats of a second sequence (III). A third, heavily glycosylated sequence (II) is present between the second and third I repeats, and is apparently cleaved proteolytically to form the M
r
92 K and M
r
80 K polypeptides. The first two I repeats form the A domain, while the third I repeat and the two III repeats form the C domain. The II sequence forms the B domain. Thus, the full-length protein has the structure I
1
-I
2
-II-I
3
-III
1
-III
2
(A-B-C), while the M
r
92 K and M
r
80 K polypeptides (A and C) have the structures I
1
-I
2
and I
3
-III
1
-III
2
, respectively. C. Fulcher et al,
J Clin Invest
(1985) 76:117-124, suggested that based on antibody-epitope data with Factor VIII:C, both the M
r
92 K and the M
r
80 K polypeptides are necessary for Factor VIII:C function.
Factor VIII:C has historically been isolated from blood in a concentrated form for therapeutic treatment of hemophilia. However, concerns regarding transmission of HIV and other blood-borne diseases have stimulated activity to provide alternative supplies of Factor VIII:C. It is of substantial interest to be able to supply compositions having Factor VIII:C activity without concerns as to the transmission viral diseases associated with the native Factor VIII:C.
Although full-length recombinant human Factor VIII:C has been produced, it is difficult to purify and characterize, and it is unstable due to proteolysis. Efficient recombinant production of the full-length molecule for clinical use is doubtful at this time.
R. L. Burke et al,
J Biol Chem
(1986) 261:12574-78 disclosed the expression of an active Factor VIII:C complex from cells simultaneously transfected with polynucleotides encoding M
r
92 K and M
r
80 K polypeptides. The obtained protein demonstrated activity equal to that of cloned full-length Factor VIII:C expressed under similar conditions. O Nordfang et al,
J Biol Chem
(1988) 263:1115-18 disclosed the in vitro assembly of active Factor VIII:C complexes from separate preparations of M
r
92 K protein and M
r
80 K protein (FVIII-HC and -LC, respectively). Successful assembly required divalent metal ions (especially Mn
++
and Ca
++
) and thiols, but only a small amount of FVIII-HC could be complexed into active FVIII:C.
DISCLOSURE OF THE INVENTION
We have now invented an improved method for expressing recombinant protein complexes with high stability and Factor VIII:C activity. The M
r
92 K polypeptide (FVIII-HC) and the M
r
80 K polypeptide (FVIII-LC) are expressed as two separate polypeptides, under the control of separate promoters, within the same host cell. Each polypeptide is preferably expressed using a signal sequence which directs export to the extracellular space with cleavage of the signal sequence. FVIII-HC is preferably expressed as a fusion protein having a C-terminal extension. The extension comprises a polypeptide sequence homologous to the B domain N-terminal sequence (which may allow cleavage by thrombin), a polypeptide spacer of 3 to 100 amino acids, and a sequence homologous to the C-terminal B domain sequence. The C-terminal extension of FVIII-HC results in a higher yield of active polypeptide upon expression in eukaryotic host cells. FVIII-LC is preferably expressed as an LC polypeptide using a signal peptide. The FVIII-LC polypeptide is processed and secreted efficiently with the correct N-terminal amino acid residue, and correct glycosylation. Cotransfection with polynucleotides encoding FVIII-HC and FVIII-LC in a suitable host cell provides recombinant protein complexes having Factor VIII:C activity in high yield.
MODES OF CARRYING OUT THE INVENTION
A. Definitions
The term “polynucleotide” as used herein refers to a sequence of DNA or RNA, which may be single or double-stranded (ss or ds), or a DNA-RNA heteroduplex. In most cases herein, polynucleotide will refer to dsDNA.
The term “signal peptide” as used herein refers to a peptide sequence which is recognized and acted upon by signal peptidase during expression of the polypeptide. Signal peptides encode peptide sites for signal peptidase cleavage, and cause the attached polypeptide to be transported into the secretion pathway leading to the extracellular medium.
The term “A domain” refers to that portion of human Factor VIII:C which constitutes the M
r
92 K protein subunit. The A domain contains from about 740 to about 760 amino acids, and is found at the N-terminus of the native human Factor VIII:C. The A domain polypeptide will extend from amino acid 10, usually amino acid 1, to at least about amino acid 620, usually at least about amino acid 675, usually at least about amino acid 710, ±10 amino acids, particularly ±1 amino acid, an may extend to amino acid 740, ±10 amino acids, more usually at least about amino acid 740. The polypeptide will include at least about 85% of the A domain (Wood et al, supra), more usually at least about 90% and may optionally include a portion of the N-terminus of the B domain, up to 110% (a portion of the B domain, more usually not including more than about 105% typically not exceeding about amino acid 1405. Of particular interest is an N-terminal chain having the entire sequence to the thrombolytic cleavage site at Arg
740
-Ser
741
.
The term “B domain” refers to that portion of native human Factor VIII:C which is generally removed by intracellular cleavage, and which is heavily glycosylated when expressed in mammalian cells such as COS7 and CHO. The B domain contains an N-terminal sequence, which allows cleavage of the A domain from the B domain by thrombin. The B domain also has a C-terminal processing site which allows cleavage of the C domain from the A-B precursor by an enzyme located in the Golgi apparatus of the mammalian cell. The sequences of the N-terminal and C-terminal sequences are set forth in the Examples below. The complexes of the invention which lack “a substantial portion of the B domain” lack all of the B domain, or essentially all of the B domain except for the N-terminal and C-terminal sequences.
The term “C domain” refers to that portion of native human Factor VIII:C which constitutes the
Burke Rae Lyn
Chapman Barbara
Mikkelson Jan Moller
Rasmussen Mirella Ezban
Blackburn Robert P.
Bugaisky Gabriele E.
Chiron Corporation
Guth Joseph H.
Low Christopher S. F.
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