Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-08-04
2003-10-07
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C530S350000, C530S370000, C530S378000
Reexamination Certificate
active
06630339
ABSTRACT:
TECHNICAL FIELD
The present invention relates to recombinant enzymes used in the conversion of type B erythrocytes to type O cells to render the cells useful for transfusion therapy. More specifically, the present invention provide novel recombinant galactosidases.
BACKGROUND OF THE INVENTION
The A, B, and H antigens are a clinically significant blood group (Landsteiner, 1901; Mollison et al, 1987). These antigens are terminal immunodominant monosaccharides on erythrocyte membrane glycoconjugates (Harmening, 1989). High densities of these epitopes are present on erythrocyte membranes and antibodies bound to these antigens readily fix complement (Economidou, et al, 1967; Romano and Mollison, 1987). Because these epitopes are ubiquitous in nature, immuno-potent and naturally occurring, complement fixing antibodies occur in individuals lacking these antigens, and transfusion of incompatible blood results in fatal hemolytic transfusion reactions (Fong et al, 1974; Schmidt, 1980).
Complex sugar chains in glycolipids and glycoproteins have often been implicated in the growth and development of eukaryotes (Watanabe et al., 1976). In particular, complex sugar chains play an important part in the recognition of self in the immune system (Mollison et al., 1987). Exoglycosidases are enzymes which can modify carbohydrate membrane epitopes, thereby modulating the immune response (Goldstein et al., 1982). The &agr;-D-galactosidase from Glycine is an enzyme that degrades the human blood group B epitope to the less immunogenic blood group H antigen also known as blood group O (Harpaz et al., 1977). &agr;-D-galactosidases [EC 3.2.1.22] are a common class of exoglycosidases. Although physical properties of these enzymes differ as a group, and the physiological significance of these enzymes are not clearly established, isozymes of &agr;-D-galactosidase are common to many plant species (Flowers et al, 1979; Corchete, et al 1987). Several investigators have studied &agr;-D-galactosidase from Coffea (Yatziv, 1971). There are reports that several isozymes exist for the Coffea &agr;-D-galactosidase enzyme (Courtois, 1966).
Modification of the A, B, and H antigens using exoglycosidases to hydrolyze the terminal immunodominant residue has previously been described (Tsuji et al, 1990; Levy & Aminoff, 1978; Yatziv & Flowers, 1971; Kubo, 1989). Hydrolysis of the terminal N-acetyl-&agr;-D-galactosamine by &agr;-N-acetyl-galactosaminidase (EC 3.2.1.49) converts blood type A
2
to blood type O, and similarly, hydrolysis of the terminal &agr;-D-galactose residue by &agr;-D-galactosidase (EC 3.2.1.22) converts blood type B to O (Yatziv & Flowers, 1971; Levy & Aminoff, 1978). An &agr;-D-galactosidase from
Coffea canephora
has been shown to effectively convert type B erythrocytes to type O erythrocytes (Harpaz, 1975). Because type O erythrocytes are generally universally transfusable, enzymatic deantigenation would have important medical applications.
Improvements of this technology could increase the compatible blood supply while reducing waste and risk of transfusion reactions. The primary impediments to seroconversion have been the large quantities of enzyme required for deantigenation, and washing the red cell concentrates to achieve the desired pH (Goldstein, 1989). Further, the reaction needs to take place at 24° C. Standard transfusion medicine protocol requires treating erythrocytes at or below 24° C. in order to decrease the possibility of bacterial contamination and maintain cell function and survival. Therefore, it is commercially important to isolate enzymes and develop buffer systems in which efficient seroconversion can occur at 24° C.
Work by Goldstein et al., 1982, lead to the feasibility of large-scale enzymatic conversion of blood type B to O erythrocytes (Lenny et al, 1982, 1991). This group used Coffea &agr;-D-galactosidase in PCBS buffer to achieve deantigenation. These cells were transfused into individuals with anti-B antibodies and survived normally. The current problem with this application is that very high enzyme concentrations, about one to two grams of exoglycosidase per transfusable unit of red cells, are required for deantigenation (Lenny and Goldstein, 1991). The cost of this amount of enzyme is enormous and, without reduction, renders this technology impractical.
Data establishing the optimal ionic strength, pH, buffer species, or enzyme concentration for efficient deantigenation has not been published. It is presently unknown whether exoglycosidase activity can be modified to achieve more efficient hydrolysis of the B antigen in red cell concentrates.
U.S. Pat. No. 4,330,619, issued May 18, 1982; U.S. Pat. No. 4,427,777, issued Jan. 24, 1984; and U.S. Pat. No. 4,609,627, issued Sep. 2, 1986, all to Goldstein, relate to the enzymatic conversion of certain erythrocytes to type O erythrocytes. The above-mentioned U.S. Pat. Nos. 4,330,619 and 4,427,777 disclose the conversion of B-type antigen to H-type antigens by using &agr;-D-galactosidases from green coffee beans (
Coffea canephora
). The patent discloses the significant potential of such enzymes to be used in the conversion of type B erythrocytes to type O erythrocytes but does not provide a commercially feasible method. Additionally, other compounds such as tannins, present in &agr;-D-galactosidase enzyme extracts from plants such as Coffea beans can potentially inhibit or impair enzyme function which provides a further disadvantage for their commercial use (Goldstein et al, 1965).
It would also be useful to have additional exoglycosidases, particularly those active at neutral pH, that could be used in the deantigenation of blood group serotypes for transfusions. However, the screening procedures currently available to undertake a survey of procaryotic species that produce exoglycosidases active at neutral pHs against blood group epitopes (Tsuji et al., 1990; Aminoff & Furukawa, 1970; Levy & Aminoff, 1980) and to characterize the resulting cells are cumbersome, time consuming and expensive to run.
For example, quantitation of red cell membrane deantigenation has been accomplished by conventional hemagglutination assays. However, hemagglutination titers are not highly sensitive and are technically cumbersome. Furthermore, a 50% decrease in antibody concentration only correlates with a one-fold change in titer. Thus, it is difficult to vary a large number of parameters and detect subtle changes in deantigenation using this assay.
A sensitive, rapid assay that could be used in deantigenation studies on native red blood cells and could be used for screening culture banks or selecting bacterial mutants that constitutively express blood group specific enzymes would be very useful. It would also be useful if the assay could be used to characterize other blood group specific exoglycosidases as well as blood group A active &agr;-N-acetyl-galactosaminidases and blood group systems I and P which express terminal immunodominant saccharide epitopes.
Finally, it would be useful to have recombinant &agr;-D-galactosidases available from several sources so that deantigenation protocols can be optimized with a supply of purified enzyme for more efficient production of deantigenated red blood cells. The Glycine (soybean) &agr;-D-galactosidase is one such galactosidase for which would be useful to have a recombinant &agr;-D-galactosidase available. Additionally, the Phaseolus (pinto bean) &agr;-D-galactosidase is another galactosidase for which it would be useful to have a recombinant supply.
SUMMARY OF THE INVENTION AND ADVANTAGES
According to the present invention, a DNA (SEQ ID No.:2) and amino acid (SEQ ID No.:4) sequences of Glycine (soybean) &agr;-D-galactosidase are provided as well as a DNA sequence (SEQ ID No:5) and mature length amino acid sequence (SEQ ID No:7) of Phaseolus (pinto bean). The cDNA sequences and vectors containing these sequences allow the production of recombinant galactosidase enzymes which can be used in deantigenation protocols.
REFERENCES:
patent: 5633130 (1997-05-01), Smith et al.
patent: 95/07088 (1
Smith Daniel S.
Walker John C.
Graser Jennifer E.
Kohn & Associates PLLC
The Curators of the University of Missouri
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