THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology Inc

Vol. 263, No. 29, Issue of October 15, pp. 15064-15070,1388 Printed in U.S.A.

Relationship of Effective Molecular Size to Systemic Clearance in Rats of Recombinant Interleukin-2 Chemically Modified with Water-soluble Polymers*

(Received for publication, March 14, 1988)

Michael J. KnaufS, Dick P. Bell, Pam HirtzerQ, Zhen-Ping Luo, John D. Young, and Nandini V. Katregll From the Departments of SProcesslProduct Development and Pharmacology, Cetus Corporation, Emeryuille, California 94608

We have examined the effects of a variety of chemical modifications to recombinant human interleukin-2 (rIL-2) on its pharmacokinetic behavior in rats. Un- modified rIL-2 is cleared from plasma with half-lives of 3 and 44 min for the a and B phases. Modification of rIL-2 with monomethoxy polyethylene glycol or polyoxyethylated glycerol increased the half-lives as much as 20-fold, although the volume of distribution remained unchanged at 88 f: 13 ml/kg. The clearance rates correlated with the effective molecular size of the modified protein determined by size exclusion chromatography. Clearance decreased rapidly as the effective molecular size increased from 19.5 to 70 kDa, whereas above 70 kDa the clearance decreased more slowly. This abrupt change at 70 kDa may be related to the permeability threshold of the kidney glomerular membrane which retains proteins larger than albumin in the plasma. Using the relationship between clear- ance and effective molecular size, the clearance rates of mixtures of modified rIL-2 could be predicted based on their average effective molecular size. Since the effectiveness of rIL-2 therapy is likely to be related to its pharmacokinetic behavior, the ability to design a molecule with a predictable time course in plasma pro- vides a means to study this relationship.

Covalent attachment of monomethoxy polyethylene glycol (PEG)’ to a variety of enzymes prolonged their circulatory lifetimes in vivo (1-3). For example, when 18 PEG molecules were attached to superoxide dismutase, its half-life in rats increased from 5 min to 25 h (4). Similar changes in half-life were observed when recombinant interleukin-2 (rIL-2), a lymphokine currently being evaluated for anticancer activity in clinical trials (5-8), was modified with PEG (9).

The covalent attachment of PEG to rIL-2 also increased its potency in the murine Meth A sarcoma model (9). In that study, we compared the pharmacokinetic behavior of two forms of PEG-rIL-2, one more highly modified than the other. The more highly modified PEG-rIL-2 was cleared more slowly from the blood circulation of BALB/c mice. This observation

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Genencor Inc., 180 Kimball Way, S. San Fran- cisco, CA 94080.

7 To whom all correspondence should be addressed. The abbreviations used are: PEG, monmethoxy polyethylene

glycol; rIL-2, recombinant interleukin-2; POG, polyoxyethylated glyc- erol; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electro- phoresis; HPLC, high pressure liquid chromatography.

led us to construct a series of rIL-2 molecules modified with PEG of molecular weights from 350 to 20,000 and from one to five PEG molecules/rIL-2 molecule. This series was eval- uated for bioactivity and pharmacokinetic behavior in rats. We report here that both the degree of modification and the size of the attached PEG affect the in vivo clearance of the PEG-rIL-2 conjugate in a predictable manner.

MATERIALS AND METHODS

Preparation of Polymer Active Esters-The polymers used in this study were of two types, polyoxyethylated glycerol (POG) and mon- omethoxypolyethylene glycol. POG was custom synthesized by Pol- ysciences, Inc. (Warrington, PA). In the following structure for POG, n is approximately 38, and the nominal molecular weight is 5,000:

CHz-O-(CHz-CHzO)nH I

I CH-O-(CHz-CHzO)nH

CHz-O-(CHz-CHzO)nH

The structure of monomethoxy polyethylene glycol is CHaO-(CHz- CH,-O),H, where n is approximately 10 for PEG (350) and n is approximately 400 for PEG (20,000). PEG of molecular weights 10,000, 7,000, and 4,000 were custom synthesized by Union Carbide to contain less than 10% diol. PEG of molecular weights 5,000 and 350 was obtained from Aldrich.

PEG (350) was derivatized to the N-hydroxysuccinimidyl-PEG carboxylate as described by Briickmann and Morr (10). The other polymers were derivatized to the N-hydroxysuccinimidyl glutamate esters as previously described (11,12), except for PEG (5,000), which was converted to the N-hydroxysuccinimidyl succinate by the same method.

Covalent Attachment of Polymers to rZL-2-hcombinant des-Ala Ser125 interleukin-2 was produced in Escherichia coli and purified as previously described (13,14). The concentration of rIL-2 and modified rIL-2 was calculated from its absorbance at 280 nm by using an e280

of 1.2 X 10‘ M” cm”. The conjugation of POG to rIL-2 (1 mg/ml) was in 0.01 M sodium borate, pH 9, containing 0.1% sodium dodecyl sulfate (SDS). At 22 “C, solid POG-ester glutarate-N-hydroxysucci- nimide was added in 2-fold molar excess to rIL-2. The solution was stirred for 45 min, then the POG-ester addition was repeated. After 45 min, the pH of the solution was adjusted to 5. We modified rIL-2 with PEG of various sizes by using the method described previously (9). The degree of modification was varied by the use of different mole ratios of PEG-ester to rIL-2. With PEG (350)-ester, a 30-fold molar excess of ester to protein was used. When PEG (4,000) and PEG (7,000) esters were used, the ratios were 5-fold ester to rIL-2. With PEG (10,000) and PEG (20,000) esters, the mole ratio of ester to rIL-2 used was 3.5. 35S-Labeled rIL-2 was purified as described previously ( X ) , after expression in E. coli grown with 35S-labeled sulfate. The purified protein had 35S-labeled methionines and cys- teines. Radiolabeled PEG-rIL-2 was obtained with 36S-rIL-2 and PEG (5000) using the conditions described above, with a 20-fold mole excess of the PEG-ester.

Purification and Fractionation of POG-rZL-2and PEG-rZL-2”PEG (350)-rIL-2 was purified from the reaction mixture by desalting on a

15064

rIL-2 Systemic Clearance Modulated by Chemical Modification 15065

45 X 1 cm Sephadex G-25 (Pharmacia LKB Biotechnology Inc.) column in 10 mM sodium phosphate, pH 7.5. PEG (5,000)-rIL-2 was purified from the reaction mixture by Sephadex G-25 chromatography to remove SDS, followed by hydrophobic interaction chromatography on a Bio-Rad TSK-phenyl-5-PW column (7.5 X 0.75 cm) maintained at 4-8 "C. The proteins were eluted by using a decreasing salt gradient as previously described (9). A final Sephadex G-25 column was used to remove residual SDS. The other PEG-rIL-2 and POG-rIL-2 species were purified from the crude reaction mixtures by Sephacryl S-200 (Pharmacia LKB Biotechnology Inc.), 100 X 2.5 cm, chromatography in 0.05 M sodium acetate, pH 5, with 0.1% SDS. Fractions were further purified by using Sephadex G-25 chromatography as described above. The extent of rIL-2 modification in each reaction mixture was monitored by observing the increase in its molecular size, using a Bio-Rad Bio-Si1 TSK-250 column in 0.005 M sodium phosphate, 0.15 M sodium chloride, 0.1% SDS, pH 7.0.

We used PEG with a molecular weight of 4000 to obtain PEG-rIL- 2 species with one, two, or three PEG molecules attached to rIL-2. In this reaction, a 5-fold molar excess of PEG ester was dissolved in 2.5 ml of 50 mM sodium acetate, pH 5.5, in 0.1% SDS. The ester solution was added to 5 mg/ml rIL-2 in 50 mM sodium borate, pH 9, and 0.1% SDS. The reaction was allowed to proceed for 30-40 min at room temperature. The reaction was terminated with 200 gl of 10% acetic acid. The conjugated protein was next chromatographed on a 2.5 X 100 cm S-200 column equilibrated in 50 mM sodium acetate, pH 5.5, in 0.1% SDS, at a flow rate of 0.5 ml/min. Fractions (2 ml) were collected, and the composition of each fraction was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Three frac- tions containing greater than 95% pure conjugates of 1-, 2-, or 3- PEG-rIL-2 were next desalted with 10 mM sodium phosphate, pH 7.5, by using an Amicon Centricon unit. Mixtures of the PEG-rIL-2 species containing variation in the degree of modification were ob- tained by pooling the appropriate fractions from the S-200 chroma- tography.

Characterization of Modified rZL-2-The extent of modification of rIL-2 by the different size polymer active esters was determined by SDS-PAGE with the Pharmacia Phast-gel system. The Pharmacia electrophoresis low molecular weight calibration kit was used for molecular weight determination. The modified species were also characterized by isoelectric focusing (Phast-Gel pH 3-10).

To characterize the effective size (hydrodynamic radius) of each modified species, two different gel-filtration columns were used. One system was the Du Pont Zorbax GF-250 (25 X 0.94 cm) and GF-450 (25 X 0.94 cm) HPLC columns in series. The second system was a fast protein liquid chromatography system with a Superose-12 column (Pharmacia LKB Biotechnology Inc.). Proteins were detected by UV absorbance, and polymers were detected by refractive index. For both systems the flow rate of eluting buffer, 0.01 M sodium phosphate, 0.1 M sodium sulfate, pH 7.0, was 1.0 mllmin. The polymer standards used for calibration were purified monomethoxy polyethylene glycols obtained from Alltech Associates. The Bio-Rad gel filtration stand- ards served as globular protein molecular weight markers. A Shi- madzu C-R 3A Chromatopac or a Maxima PC-based chromatography system was used to identify and integrate peaks.

In Vitro Biological Assays-The bioactivities of rIL-2 and the modified rIL-2 species were determined by using the standard short term proliferation response measured by [3H]thymidine incorporation into rIL-2-dependent HT2 cells (16, 17). A BRMP (Biological Re- sponse Modifier Program) standard was used for the rIL-2 bioassay. The specific activity of the standard was 13 ? 0.3 X lo6 BRMP units/ mg of rIL-2 protein.

Pharmacokinetics of rIL-2 and Modified rIL-2 in Rats-Male CD rats weighing between 200 and 250 g were obtained from Charles River Breeding Labs (Wilmington, MA). One day before administra- tion of the dose, rats were anesthetized with 80 mg/kg ketamine (Ketaset, Bristol Laboratories, Syracuse, NY) and 3.5 mg/kg xylazine (Rompum, Miles Laboratories, Shawnee, KS). A Silastic cannula was then placed in the right jugular vein according to the method of Harms and Ojeda (18). The cannula was tunneled subcutaneously and brought through the skin at the back of the neck, filled with heparinized saline, and closed with a wire stylet.

A dose of 5 mg/kg rIL-2 or 0.25 mg/kg modified rIL-2 was injected as a bolus into a tail vein, and samples of blood were obtained at selected times, up to 30 h. Blood was transferred to a heparinized capillary tube and centrifuged to obtain plasma. Plasma volume was determined by weight, diluted 3-fold in assay medium, and stored at -70 "C until assayed. Urine samples were obtained from selected rats given rIL-2 and PEG-rIL-2. Two mice/group were each injected with

either 10 gg (2.4 X lo6 cpm) of 35S-rIL-2 or PEG-35S-rIL-2 given as an intravenous bolus via a tail vein. Urine samples were collected a t 45 min and 7 h after injection for mice administered rIL-2 and at 45 min and 18 h after injection for mice administered PEG-rIL-2. Urine was analyzed by size exclusion chromatography, and radioactivity was measured in the fractions collected.

Data Analysis-Plasma concentration data were corrected for di- lutions and analyzed for individual animals as well as for the arith- metic means of 3-5 animals/group. Preliminary pharmacokinetic parameter values for A and B intercepts and a and /3 rate constants were obtained by using the curve-stripping program, JANA (19). These parameter values, along with an estimate of the volume of the central compartment ( Vc), were used as a starting point for the nonlinear parameter estimation routine, PCNONLIN. Both programs were obtained from Statistical Consultants, Inc., Lexington, KY. The area under the curve (AUC) was calculated from the equation AUC = (A/a) + (B/p). Systemic clearance (Cl,) was then calculated from the equation C1. = dose/AUC. The volume of distribution at steady state (V,) was calculated from the equation V, = Vc((K12 + Kz,) / Kzl) , where Klz = a + P-KzI-K~o, KZI = (AP + Ba)/(A + B ) , and KIO = aP/Kn.

RESULTS

Covalent polymer-rIL-2 conjugates were prepared by the methods described. These conjugates included POG-rIL-2 and several PEG-rIL-2 species different in both degree of modifi- cation and size of the PEG attached. When the number of PEG or POG molecules attached to rIL-2 was fewer than four, the bioactivity of these conjugates was 12.6 & 1.2 X lo6 BRMP units/mg protein. The bioactivities of the PEG-rIL-2 species with four or more PEG molecules attached, ranged from 4.6 to 9.2 x lo6 BRMP units/mg of protein.

The SDS-PAGE characterization of the PEG-rIL-2 sam- ples is shown in Fig. 1. Fig. l a shows that increasing the number of PEG (4,000) molecules attached to rIL-2 (1-3) increases its size. However, attachment of a small size PEG (350) does not significantly alter the size of the conjugate as shown in Fig. l b in lane 3. PEG (20,000)-rIL-2 has the largest effective size of the modified rIL-2 molecules studied (Fig. lb, lane 4 ) . Reaction mixtures with varying degrees of modifica- tion were characterized either by SDS-PAGE or by isoelectric focusing. For example, PEG (7,000)-rIL-2 mixtures are shown in Fig. IC (lanes 2 and 3 ) and in Fig. 2 (lanes 3-5). PEG (350)- rIL-2 has five identifiable modifications (Fig. 2, lane 1 ) ; however, the effective size is not much greater than that of rIL-2.

Fig. 3 shows molecular exclusion chromatographic profiles, obtained by using GF 250/450 columns, of 1-, 2-, and 3-PEG (4000)-rIL-2. The polymer-rIL-2 conjugates produced broader peaks on the sizing column compared to the standards. The apex positions of these peaks were used to calculate the effective size (hydrodynamic radius) of each modified species. Sizing chromatography with a Superose-12 column gave sim- ilar profiles with comparable hydrodynamic radii. Unmodified rIL-2 chromatographed unreliably on the Zorbax GF 250/450 columns, unless 0.05% SDS was added to the eluting buffer. The molecular size of rIL-2 was more accurately determined using the Superose-12 column. Fig. 4 shows the size-exclusion profiles of PEG-rIL-2 samples containing a mixture of modi- fications. The relative areas obtained from these data were used to calculate the average size of each mixture.

The blood plasma curves for rIL-2 and a mixture of PEG (7000)-rIL-2 (Fig. IC, lane 2, and Fig. 2, lane 5 ) given intra- venously to rats are compared in Fig. 5. The a and @ half- lives of rIL-2 were 3 and 44 min, respectively. For PEG (7000)- rIL-2 these half-lives were increased to 48 and 310 min, and the systemic clearance rate decreased 16-fold from 1.15 to 0.07 ml/min. Blood plasma concentrations of rIL-2 bioactivity decreased 100-fold in less than 1 h for the unmodified protein,

15066 rIL-2 Systemic Clearance Modulated by Chemical Modification

FIG. 1. SDS-PAGE profiles of various PEG- and POG-modified rIL-2 species run under denaturing conditions. Unmarked lanes are stand- ards. a, 1-PEG (4,000)-rIL-2, 2-PEG (4,000)-rIL-2, and 3-PEG (4,000)-rIL-2. b, lane I , rIL-2; lane 2, PEG (10,000)- rIL-2; lane 3, PEG (350)-rIL-2; lane 4 , PEG (20,000)-rIL-2; lane 5, POG(5,OOO)- rIL-2. c, mixtures of PEG (7,000)-rIL-2 containing: 2-PEG-rIL-2 and 3-PEG rIL-2 (lane 2) ; unmodified rIL-2,l-PEG- rIL-2 and 2-PEG-rIL-2 (lane 3 ) .

a I b 9 4 K - 6 7 K - 4 4 K -

3 0 K -

20K-

1 4 . 4 K -

1 2 3 4 1 2 3 4 5

9 4 K - 6 7 K -

-3 PEG4K IL-2 4 4 K -

C 1 2 3

- 9 4 K - 6 7 K - 4 4 K

- 3 0 K - 2 0 K - 14.4K

I

-5.2

-’ 1 -4.6 I

1 2 3 4 5 FIG. 2. Isoelectric focusing of PEG-rIL-2 samples and un-

modified rIL-2, pH range 3-10. Lane 1 represents a highly modified PEG-rIL-2 sample obtained with PEG (350). Lane 2 is unmodified rIL-2. Lanes 3-5 are mixtures of PEG-rIL-2 obtained with PEG (7,000), containing mainly three lysines modified. Un- marked lanes are standards.

whereas with 2,3-PEG (7000)-rIL-2, this decrease was meas- ured after 24 h.

There was no IL-2 bioactivity found in any of the urine samples assayed. When 35S-labeled rIL-2 or 35S-PEG-rIL-2 was injected into mice, the urine samples had no IL-2 bioac- tivity at 45 min or at later time points. Some radioactivity was found in the urine 45 min after injection. Radioactivity

in the urine increased at the later time points (7 h for rIL-2; 18 h for PEG-rIL-2). These later urine samples were analyzed by size exclusion chromatography. Fig. 6 shows chromato- graphic sizing profile of urine samples from mice injected with the radiolabeled proteins. In both cases all the radioactivity sizes as low molecular weight species of about 1.3 kDa.

Table I compares the clearance rate of each rIL-2 species with its effective molecular size obtained by size exclusion chromatography. It is clear that the effective size of the polymer-rIL-2 conjugate dictates its plasma clearance; a larger size corresponds to a slower clearance rate. This effective size depends on both the degree of modification and the size of polymer used. Fig. 7 shows the change in clearance as the extent of modification is increased upon addition of 1-, 2-, or 3-PEG (4000) molecules to rIL-2. The effective molecular size increased from 40 to 66 to 104 kDa, and the systemic clearance decreased from 0.60 to 0.23 to 0.12 ml/min, respectively. Regardless of the nature of the polymer (PEG or POG) or the extent of modification, the hydrodynamic radius of the mod- ified species governs the plasma clearance of the rIL-2. Fig. 8 shows the similarity of the clearance curves for three modifi- cations of rIL-2 using 2-POG (5,000), 1-PEG (lO,OOO), and 3- PEG (4,000), which have similar molecular sizes of 72, 103, and 104 kDa.

The relationship between the systemic clearance rates of various modified rIL-2 species in rats and their effective molecular size (Table I) is shown in Fig. 9. This curve is composed of two slopes which intersect approximately at an effective molecular size of 70 kDa and a systemic clearance rate of 0.2 ml/min. The open circles represent mixtures of PEG-rIL-2 with weighted average effective sizes ranging from 46 to 164 kDa, Table 11. The systemic clearance rates pre- dicted by interpolation on the curve shown in Fig. 9 for these mixtures are compared to the observed rates in Table 11.

rIL-2 Systemic Clearance Modulated by Chemical Modification 15067

E d C

(u

m Q

C 0

m

cn 0

n U

r

c

2

b I I 1 I I

C I I 1 I 1

2 PEG 4K -IL-2

A

2 PEG n

16 1 8 2 0 2 2 2 4 26 MINUTES

FIG. 3. Size exclusion chromatography of 1-, 2-, or 3-PEG (4000)-rIL-2 using a Du Pont GF 260/450 HPLC column. The eluting buffer was 0.1 M sodium sulfate, 0.01 M sodium phosphate, pH 7.0, with a flow rate of 1 ml/min. Bio-Rad gel filtration standards were used to calibrate the system.

DISCUSSION

We have shown that covalent attachment of the hydrophilic polymers PEG or POG to rIL-2 significantly affects its size. Molecular exclusion chromatography under nondenaturing conditions with either Superose-12 or Zorbax columns is an effective method to determine the molecular size (hydrody- namic radius) of proteins. The polymer-rIL-2 conjugates elute as broader peaks compared to the standards, most likely

C I I I I

e I I 1 1 I

0 10 20 30

MINUTES FIG. 4. Size exclusion chromatography of mixtures of PEG-

rIL-2 species on Pharmacia Superose-12 column. The eluting buffer was 0.1 M sodium sulfate, 0.01 M sodium phosphate, pH 7.0, with a flow rate of 1 ml/min. The numbers 0, 1, 2, or 3 indicate the number of PEG molecules attached/rIL-2 molecule. a, Bio-Rad gel filtration standards; b, mixture obtained from PEG (4000); c, mixture obtained from PEG (5000); d and e, mixture obtained from PEG (7000).

because of the inherent molecular weight distribution of the polymers themselves. rIL-2 chromatographed more reliably on the agarose-based Superose-12 sizing column. Because of its limited solubility, rIL-2 possibly interacts with the silica- based packing material of the Zorbax GF columns. However, with the polymer-rIL-2 conjugates, both systems gave similar results, perhaps, due to the enhanced solubility of these mod- ified proteins. Molecular exclusion chromatography has not been a very reliable method to determine the Stokes radii of large, asymmetric proteins when globular proteins were used

15068 1 oa

30

CI I- z 8 l a

n

0

U W Y

z I - 3 c c U z w 0

E 1 0 a VJ I a -1

0 P 0.3

w J c I 0 2

5

0.1

0.03

0.0 1

rIL-2 Systemic Clearance Modulated by Chemical Modification

2.3-PEG (7KbrlL-2

rlL-2

5 10 15 20 25

HOURS

FIG. 5. Semilogarithmic plot of mean blood plasma concen- trations of rIL-2 bioactivity, normalized at time 0 to 100% as a function of time showing the extensive change in plasma clearance achievable by modification with PEG. Groups of rats were given either 5 mg/kg rIL-2 or 0.25 mg/kg of a mixture of 1-, 2-, and 3-PEG (7000)-rIL-2 as a single intravenous bolus dose.

as standards (20-22). However, we believe this method is appropriate for comparing hydrodynamic radii within a set of modifications of a single protein such as rIL-2. This method of obtaining the hydrodynamic radii has allowed us to predict the systemic clearance rates of these molecules in rats.

Plasma clearance of unmodified rIL-2 is comparable to that predicted for small proteins which are cleared by glomerular filtration in the kidney (23, 24). The renal clearance rate of macromolecules relative to the glomerular filtration rate of inulin decreases with increasing effective molecular radius (24-26). An increase in the size (hydrodynamic radius) of rIL- 2 after its conjugation to PEG or POG, most likely due to hydration of the attached polymers, correlates with the de- crease in clearance rate of the protein from plasma.

This relationship between the systemic clearance rate of the rIL-2 species and hydrodynamic radius of the protein up to 70 kDa is consistent with the postulate that the kidney is the major organ for clearance of all these molecules (26). More direct evidence that the kidney is responsible for the changes observed was obtained from experiments with radiolabeled proteins and nephrectomized animals. Donohue and Rosen- berg (27) have reported a marked extension of rIL-2 half-life in mice following ligation of renal pedicles. We have observed

1200 r

-

6 0 0 - - -

0 -

FRACTION NO.

FIG. 6. Size exclusion chromatography of mouse urine on Bio-Rad TSK-250 HPLC column, run in phosphate-buffered saline. O"0 represents urine sample from mouse injected 36S- rIL-2, 7 h post-injection. C - -I represents mouse urine sample, 18 h post-injection of polyethylene gl~col-~~S-rIL-2. Gel filtration stand- ards proteins are marked. Elution positions of intact rIL-2 and PEG (5000)-rIL-2 are also indicated.

TABLE I Effect of modification of rIL-2 on pharmacokinetic Darameters

rIL-2 No. PEG/ species CY

v," Effective r1L-2 aluha beta size

rnin rnllrnin rnl /kg kDa rIL-2 0 3 44 1.15 100 19.5 PEG (350) 5 6 57 0.97 89 21 PEG (4,000) 1 5 44 0.60 93 40 PEG (4,000) 2 14 162 0.23 95 66 POG (5,000) 2 21 238 0.14 88 72 PEG (10,000) 1 12 263 0.12 80 103 PEG (4,000) 3 32 292 0.12 86 104

PEG (5,000) >5 19 409 0.05 106 280 PEG (20,000) 2 32 256 0.11 91 326

PEG (5,000) 4-5 61 370 0.05 60 208

' Systemic clearance. * Volume of distribution at steady state.

that the clearance rate of PEG (5000)-rIL-2 mixture (from Table II), 46% of which sized below 70 kDa, decreased in nephrectomized rats when compared to normal rats. The kidney is therefore responsible for a major portion of the plasma clearance of both rIL-2 and PEG-rIL-2.

Our data with 35S-labeled PEG-rIL-2 and rIL-2 in mice is in agreement with earlier results using 35S-rIL-2 (15) in which only small radioactive fragments, but no detectable rIL-2 bioactivity, were excreted in the urine. Since rIL-2 is cleared by the kidney, but not excreted intact in the urine, the mostly likely mechanism for its clearance is metabolism, followed by excretion of the fragments in the urine.

The rapid decrease in systemic clearance rate of PEG-rIL- 2 as the effective molecular size increases from 21 to approx- imately 70 kDa, is most likely due to a progressive exclusion of the protein from glomerular filtration, and therefore from its site of metabolism in the cells lining the proximal tubule (15,23), or other sites in the kidney. This conclusion is partly based on the abrupt change in the slope of the curve around 70 kDa, which corresponds to the molecular weight of serum albumin, a protein which is predominantly excluded from filtration by the kidney glomerular basement membrane and

rIL-2 Systemic Clearance Modulated by Chemical Modification 15069 100,000

0 2 POG SK -rlL-2

100,000

10,ooc

=. 1,ooa E 2

2

a Q m

. c c 0

100

l a

l

a Ib

\ \ 2 P E G ~ K -rlL-2 \

1 PEG 4K -rlL-2

7

\

1 1 I I I

5 10 15 20 25

HOURS

FIG. 7. Semilogarithmic plot of mean blood plasma concen- trations of rIL-2 bioactivity as a function of time showing the effect of adding 1-, 2-, or 3-PEG molecules to rIL-2 on the shape of the curve. Groups of rats were given single intravenous bolus doses of 1-, 2-, or 3-PEG (4000)-rIL-2 at 0.25 mg/kg.

appears in the urine in only small concentrations (25). Since above the molecular size of 70 kDa the systemic clearance of rIL-2 does not drop to zero, a clearance mechanism which does not include the kidney is implied. Even in nephrecto- mized rats the rIL-2 is cleared, although at a much slower rate.

Glomerular selectivity is based not only on molecular size, but also on shape and charge of the molecule (25, 26). Our unpublished study using circular dichroism analysis shows that the structures of the polymers themselves are random in nature and that modification of rIL-2 with PEG or POG does not alter its shape. The charge of the rIL-2 is affected by modification with PEG or POG. The polymers are covalently attached to lysine side chains of the protein, thereby progres- sively decreasing its isoelectric point (PI) upon modification, Fig. 2 (9). Hence the net negative charge of the protein is increased after its modification. PEG (350)-rIL-2 has 5 lysines modified but its effective molecular size is not much larger than rIL-2. The clearance rate of this PEG (350)-rIL-2 reflects its size and not its charge. In addition, our data with 1-PEG (10,000)-rIL-2, 2-POG (5,000)-rIL-2, and 3-PEG (4,00O)-rIL- 2 clearly show that regardless of the number of lysines modi- fied and the differences in net charge, these three modified proteins show similar plasma clearance rates, reflecting their similar effective molecular radius. The volumes of distribution of rIL-2 and the modified proteins are similar, suggesting that the pattern of distribution in the tissue for the modified and unmodified molecule is not greatly changed.

The relationship between clearance and size does allow us to predict the clearance parameters of modified rIL-2 after determining its hydrodynamic radius by molecular exclusion

10 5 10 15 20 25 30

HOURS

FIG. 8. Semilogarithmic plot of mean blood plasma concen- trations of rIL-2 bioactivity as a function of time showing the similarity between 3 modified rIL-2 species with apparent molecular sizes between 72 and 104 kDa. Polymer-rIL-2 con- jugates were given as an intravenous bolus dose of 0.25 mg/kg.

0.1 - n I -1 0 30 100 300 1000

EFFECTIVE MOLECULAR WEIGHT x

FIG. 9. Relationship between systemic clearance rate in rats and effective molecular size of rIL-2 modified with PEG or POG. Solid circles represent modified rIL-2 from Table I and open circles represent mixtures from Table 11. The line is the best fit to the solid circles by nonlinear least squares regression analysis.

chromatography. Thus, our ability to design the in vivo clear- ance parameters of rIL-2 by attachment of appropriate poly- mers will allow us to better define the therapeutic use of rIL- 2.

15070 rIL-2 Systemic Clearance Modulated by Chemical Modification

TABLE I1 D. W., Thurman, G. B., and Oldham, R. K. (1987) N. Engl. J .

effective molecular size of PEG-rIL-2 mixtures 8. Mageed, A. A., Findley, H. W., Jr., Franco, C., Singhapakdi, S., Alvarado, C., Chan, W. C., and Ragab, A. H. (1987) Cancer 60,

Prediction of systemic clearance rate (CU in rats based on average of Med. 316,898-905

PEG-rlL-2 PEG/rIL- Size of Weighted 2913-2918 Mixture ~

2 each average CL 9. Katre, N. V., Knauf, M. J., and Laird, W. J. (1987) Proc. Natl. No. % size Predicted Observed Acad. Sci. U. S. A. 8 4 , 1487-1491

kDa kDa rnllrnin 10. Briickman, A. F., and Morr, M. (1981) Makromol. Chem. 182,

11. Zalipsky, S., Gilon, C., and Zilkha, A. (1983) Eur. Polym. J . 19,

0'35 12. Anderson, G. W., Zimmerman, J. E., and Callahan, F. M. (1964) J. Am. Chem. SOC. 86,1839-1842

13. Kato, K., Yamada, T., Kawahara, K., Onda, H., Asano, T., Sugino, H., and Kakinuma, A. (1985) Biochem. Biophys. Res. Commun. 130,692-699

14. Koths, K., Thomson, J., Kunitani, M., Wilson, K., and Hanisch, W. (February 11,1986) US. Patent 4,569,790

15. Koths, K., and Halenbeck, R. (1985) Cellular and Molecular Biology of Lymphokines (Sorg, C., and Schimple, A., eds) pp. 779-783, Academic, New York

0'07 0'07 16. Gillis, S., Ferm, M. M., Ou, W., and Smith, K. A. (1978) J. Immunol. 120,2027-2032

PEG (4000) 0 11 19.5 1379-1384

1 61 40 2 28 66 46 0.45

1 42 55 2 54 100 78 0.15 0.15

1 44 64 2 51 123 92 0.12 0.12

PEG (7000) 0 0 -

1 3.5 64 2 34.5 125 3 62 190 164

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Acknowledgments-We thank the Cetus Bioassay Group for the 17. Watson, J. (1979) J. Med. 1 5 0 9 l5lo-l5l9 IL-2 cell proliferation assays, and Sharon Nilson for the graphics. 18. Harms, p. G.9 and Ojeda, s. R. (1974) J. APPl. PhYSiOl. 36, 391-

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