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62 Modification of rt-PA oligosaccharide structures to increase or decrease biological clearance
30 .a
6’1
.AnnrrrCLl+A.l ,-,, I,W,PL,V.ll”S%V~E ,jTR”,-T”RES rwulr u+nl Luo nrur _ArL-rn,,a ULI~~~LL~MKLUI albfic bLEARANCE A. TO INCREASE OR DECREASE BIOLOCTr"' F Hotchkiss, C. Refino, J. O'Connor, K. Tate, 3. Nakamura, C. Leonard, D. Powers, M. Mohler, J. McCabe. S. Anderson and M. Soellman. Genentech, In;,, -SoI-San Francisco, CA~64080
Recombinant L;;;r;Ad) i.s
tissue-type plasminogen activator a .glycoprftotpe~n that is rapidly complex contains 0ligosacch~rid~~~~6.S) at positions 184 and 448 and high mannose OS at position 117; these OS structures were modified by treatment with sodium periodate (PERIO), Endo H, or expression of rt-PA in a cell line that produces only high-mannose OS. An rt-PA mutant with a point mutation (Asn to Gln) at residue 117 was also produced in order to remove the high mannose OS. These four forms of rt-PA as well as q2$ontrol sample of rt-PA were labeled with I and The plasma injected as a bolus in rabbits.
63
'Ihe Pharmacokinetics and Circulatory Mataholism of a Lorq Half Life Mutant of rt-PA C.3. Refino, A.J. Hot&kiss, D.L. Higgens, M.A. Mohler, Depts. of Pharmacology and Cardiovascular &search, Genentech, Inc., So. San Prancisco, CA 94080, USA. Recanbinant tissue-type plasnincgen activator (rt-PA) is rapidly eliminated in humans and laboratory animals and it undergoes limited metabolism in the circulation. Previous studies in primates and rabbits have shown that endogenous protease inhibitors do not play an important role in its elimination. In the present sturdy, site specific mutagenesis was used to produce a mutant rt-PA in which the first 44 amino acids are missing (N44). The phazmacokinetics and circulatory metaholisn of this mutant were studied in rabbits aft755 I-N44 administration of a bolus i.v. dose of (5uCi/kg) and unlaballed N44 (0. m/kg). Blood samples were collected for 6 hours in the presence of the protease inhibitor, PPACK. RECOMBINANT tPA DELETION ANALOGUES: ACTIVITY, FIBRIN BINDING AND CLEARANCE STUDIES. M. Johannessen, V. Diness, K. Pingel, L.C. Petersen+, D.Rao, M. Insley and E. Mulvihill++ +)NOVO Research Institue, Bagsvaerd, Denmark and ++)ZymoGenetics, Seattle, WA 98105, USA. To examine the functions of structural domains of the tPA molecule, authentic rtPA (FGKlK2L) and a number of structural deletion mutants were constructed, expressed in mammalian cells, and the proteins purified. The tPA analogues studied includes l)deletion of the e idermal growth factor region (FKlKZL) 2yadditional - deletj.on of the finger region (KlK2L) and 3)deletion of also the kringle 1 region (K2L). In the fibrin plate and clot lysis assays the specific fibrinolytic activity of the analogues
timecourse of the TCA precipitable counts was fitted to a biexponential of elimination model. The elimination of PER10 rt-PA, Endo H-treated mutant were similar. with ~'~~~g%y"~~n~~~1~7 alpha an unchangkd t M~,~~_m~r~ed ~eois&?lbutio{of the eliminat# ~IILU Lne oeta pnase ana an increase in the area under the clearance curve. The rt-PA with all high-mannose OS was cleared more rapidly than control rt-PA. These results indicate that the high-mannose OS, which are removed or altered in every case, are a major factor in the clearance of rt-PA. Terminal galactose residues could also be expected to contribute to the clearance of rt-PA. However, the clearance of PER10 rt-PA was similar to that of Endo H-treated rt-PA and mutant rt-PA. Since periodate ~Zatm~!-217 would destroy terminal galactose and mannose residues, we conclude that the only significant OS-dependent clearance of rt-PA is through the high-mannose OS at position 117.
ware assayed for ICA Plasma fractiprjs precipitable I-N44, inmunoreactive N44 and axnplexes with protease inhibitors. B rates I-N44 of clearance for unlabelled N44 and ore 0.49+0.02 and 0.54_+0.01 ml/min/kg respectively ccmpared to a clearance rate for rt-PA of 12.4+_0.9. The initial volunes of distribution for the three forms of Et-PA were the saane, with a mean value of 53+6 ml/kg. Analyses by SDS gel electrophoresis of the plasma samples demonstratedlgat not more than I-N44 was bound 10% of the initial dose of to protease inhibitors at the 2 minute time point or any subsequent time during the 6 hours. lhese data suggest that despite the 20 fold decrease in the rate of clearance of N44 canpared to rt-PA, the formation of caaplexes of N44 with the fast acting tPA inhibitor, PA1 or, more significantly, with slower acting protease inhibitors such asd-2-macrcglobulin, is not a significant factor in its elimination frun the circulatory system.
was similar to that of tPA. In contrast, the activity of the analogues in a fibrin-stimulated chromogenic plasminogen activation assay was greatly reduced as compared to authentic tPA. Also the fibrin binding of all analogues was reduced. The results suggest that various plasminogen activation assays respond differently to changes in fibrin binding properties. Clearance studies were carried out in rats using 35S-labelled proteins and in rabbits using an immunosorbent activity assay. In both species the elimination half lives of the analogues were 5-10 times longer than for authentic tPA. Thus a prolonged half life was obtained by deletion of only the Gregion, however, this also reduced the fibrin affinity of tPA, possibly by affecting the spacing between remaining fibrin binding domains.
6’1
.AnnrrrCLl+A.l ,-,, I,W,PL,V.ll”S%V~E ,jTR”,-T”RES rwulr u+nl Luo nrur _ArL-rn,,a ULI~~~LL~MKLUI albfic bLEARANCE A. TO INCREASE OR DECREASE BIOLOCTr"' F Hotchkiss, C. Refino, J. O'Connor, K. Tate, 3. Nakamura, C. Leonard, D. Powers, M. Mohler, J. McCabe. S. Anderson and M. Soellman. Genentech, In;,, -SoI-San Francisco, CA~64080
Recombinant L;;;r;Ad) i.s
tissue-type plasminogen activator a .glycoprftotpe~n that is rapidly complex contains 0ligosacch~rid~~~~6.S) at positions 184 and 448 and high mannose OS at position 117; these OS structures were modified by treatment with sodium periodate (PERIO), Endo H, or expression of rt-PA in a cell line that produces only high-mannose OS. An rt-PA mutant with a point mutation (Asn to Gln) at residue 117 was also produced in order to remove the high mannose OS. These four forms of rt-PA as well as q2$ontrol sample of rt-PA were labeled with I and The plasma injected as a bolus in rabbits.
63
'Ihe Pharmacokinetics and Circulatory Mataholism of a Lorq Half Life Mutant of rt-PA C.3. Refino, A.J. Hot&kiss, D.L. Higgens, M.A. Mohler, Depts. of Pharmacology and Cardiovascular &search, Genentech, Inc., So. San Prancisco, CA 94080, USA. Recanbinant tissue-type plasnincgen activator (rt-PA) is rapidly eliminated in humans and laboratory animals and it undergoes limited metabolism in the circulation. Previous studies in primates and rabbits have shown that endogenous protease inhibitors do not play an important role in its elimination. In the present sturdy, site specific mutagenesis was used to produce a mutant rt-PA in which the first 44 amino acids are missing (N44). The phazmacokinetics and circulatory metaholisn of this mutant were studied in rabbits aft755 I-N44 administration of a bolus i.v. dose of (5uCi/kg) and unlaballed N44 (0. m/kg). Blood samples were collected for 6 hours in the presence of the protease inhibitor, PPACK. RECOMBINANT tPA DELETION ANALOGUES: ACTIVITY, FIBRIN BINDING AND CLEARANCE STUDIES. M. Johannessen, V. Diness, K. Pingel, L.C. Petersen+, D.Rao, M. Insley and E. Mulvihill++ +)NOVO Research Institue, Bagsvaerd, Denmark and ++)ZymoGenetics, Seattle, WA 98105, USA. To examine the functions of structural domains of the tPA molecule, authentic rtPA (FGKlK2L) and a number of structural deletion mutants were constructed, expressed in mammalian cells, and the proteins purified. The tPA analogues studied includes l)deletion of the e idermal growth factor region (FKlKZL) 2yadditional - deletj.on of the finger region (KlK2L) and 3)deletion of also the kringle 1 region (K2L). In the fibrin plate and clot lysis assays the specific fibrinolytic activity of the analogues
timecourse of the TCA precipitable counts was fitted to a biexponential of elimination model. The elimination of PER10 rt-PA, Endo H-treated mutant were similar. with ~'~~~g%y"~~n~~~1~7 alpha an unchangkd t M~,~~_m~r~ed ~eois&?lbutio{of the eliminat# ~IILU Lne oeta pnase ana an increase in the area under the clearance curve. The rt-PA with all high-mannose OS was cleared more rapidly than control rt-PA. These results indicate that the high-mannose OS, which are removed or altered in every case, are a major factor in the clearance of rt-PA. Terminal galactose residues could also be expected to contribute to the clearance of rt-PA. However, the clearance of PER10 rt-PA was similar to that of Endo H-treated rt-PA and mutant rt-PA. Since periodate ~Zatm~!-217 would destroy terminal galactose and mannose residues, we conclude that the only significant OS-dependent clearance of rt-PA is through the high-mannose OS at position 117.
ware assayed for ICA Plasma fractiprjs precipitable I-N44, inmunoreactive N44 and axnplexes with protease inhibitors. B rates I-N44 of clearance for unlabelled N44 and ore 0.49+0.02 and 0.54_+0.01 ml/min/kg respectively ccmpared to a clearance rate for rt-PA of 12.4+_0.9. The initial volunes of distribution for the three forms of Et-PA were the saane, with a mean value of 53+6 ml/kg. Analyses by SDS gel electrophoresis of the plasma samples demonstratedlgat not more than I-N44 was bound 10% of the initial dose of to protease inhibitors at the 2 minute time point or any subsequent time during the 6 hours. lhese data suggest that despite the 20 fold decrease in the rate of clearance of N44 canpared to rt-PA, the formation of caaplexes of N44 with the fast acting tPA inhibitor, PA1 or, more significantly, with slower acting protease inhibitors such asd-2-macrcglobulin, is not a significant factor in its elimination frun the circulatory system.
was similar to that of tPA. In contrast, the activity of the analogues in a fibrin-stimulated chromogenic plasminogen activation assay was greatly reduced as compared to authentic tPA. Also the fibrin binding of all analogues was reduced. The results suggest that various plasminogen activation assays respond differently to changes in fibrin binding properties. Clearance studies were carried out in rats using 35S-labelled proteins and in rabbits using an immunosorbent activity assay. In both species the elimination half lives of the analogues were 5-10 times longer than for authentic tPA. Thus a prolonged half life was obtained by deletion of only the Gregion, however, this also reduced the fibrin affinity of tPA, possibly by affecting the spacing between remaining fibrin binding domains.