Biological activities of polyethylene-glycol immunoglobulin conjugates resistance to enzymatic degradation

Jonrnalofhnmunologk'al Methods, 152(1992) 177-19t|

177

© 1992 ElsevierScience Publishers B.V. All rights reserved {8}22-1759/92/$05AX)

JIM06370

Biological activities of polyethylene-glycol immunoglobulin conjugates Resistance to enzymatic degradation C h a r l o t t e C u n n i n g h a m - R u n d l e s , Z h o u Zhuo, Bernice Griffith and J a m e s K e e n a n Department of Medicine, Dicision of Clinical hnmmudogy, Mount Sinai Medical Center. New York City, NY 10020. USA

(Received 18 October 1991.revised received4 March 1992,accepted 4 March ITS2)

Serum lgG has been covalently bonded to polyethylene glycols of either 2000 or 8000 molecular weight to produce immunoglobulin conjugates with 4.4-27.2% of primary amines bonded to polyethylene glycol. Polyethylene glycol immunoglobulin conjugates retain the ability, comparable to native igG, to bind to a range of protein and microbial antigens, but have a reduced ability to bind to Fc receptors or to fix complement C3. When 6.8% or more of available primary amines are conjugated, lgG-PEG conjugates are impervious to trypsin, and at 14% or more conjugation, more resistant than native IgG to pepsin and chymotrypsin. We suggest that PEG-Ig conjugates may be useful for the oral treatment of various gastrointestinal diseases in which secretory humoral immunity is insufficient. Key words: IgG; IgG-polyethyleneglycolconjugate; Proteolyticdegradation; Enzyme

Introduction Covalent attachment of polyethylene glycol (PEG) to various proteins has been shown to reduce the antigenicity of these compounds, extend the half life, increase solubility, and variably alter the biologic activities of the modified proteins (Abuchowski et al., 1977a,b; Chen et al., 1981; Davis et al., 1981; Beauchamp et al., 1983). One of these modified compounds, PEG-adenosine deaminase, has been approved for clinical

Correspondence to: C. Cunningham-Rundles, Department of Medicine, Division of Clinical Immunology, Mount Sinai Medical Center, New York City, NY 10029, USA. Tel.: (212) 241-4014; Fax: (212) 348-7428. Abbreriations: PEG, polyethyleneglycol;PBS. phosphatebuffered saline; TPCK, tosyl-phenylchtomethyl ketone

use (Herschfieid et al., 1987); and at least one other, PEG-IL-2, is being investigated in clinical trials (Katre, 1990). In the current studies we have covalently bonded PEG to lgG isolated from serum to determine if these immunoglobulin molecules would retain a full spectrum of antigen binding capacities and be more impervious to proteolytic enzymes than native IgG. Our goal has been to determine if pooled human serum immunoglobulin, could be "protected" by covalent coupling with PEG to provide a polyclonal immunoglobulin substitute for secretory IgA, suitable for oral use. Previously, PEG modification of IgG has been performed to aid in separating antigen-antibody complexes (Ling and Mattiasson, 1983), or as an additive to solutions of intravenous immunoglobulin to discourage aggregation (Suzuki et al.,

178

Materials and methods

buffer, pH 8.5, with various concentrations of serum IgG, to provide molar ratios between 1:5 to !:1000. These solutions were then concentrated by lyophilization or by pressure dialysis in the Amicon cell. In other experiments PEG was activated with cyanuric chloride (Abuchowski et al., 1977a). To couple PEG to IgG, activated PEG was added to lgG in 0.1 M borate buffer at pH 9.2 to provide PEG: IgG molar ratios of 1:5 to 1:288. The mixture was incubated at 4°C for 1 h and then the unattached PEG was removed by pressure dialysis using 0.01 M phosphate buffer pH 7.3 as the dialyzing solution.

Pooled Imman serum imnumoglobalins

Et'aluation of modified primary amines

Serum immunoglobulin obtained by fractionation of pooled human plasma, fraction II (98% lgG), was obtained from Alpha Therapeutic Corporation (Los Angeles, CA). This preparation was dissolved in !1.01 M sodium phosphate buffer, pH 7.8, and then dialyzed against this buffer to remove residual salts. The final concentration of the immunoglobulin solution was determined spectrophotometrically using an extinction coefficient of 13.8 as E r~ for IgG (Putnum, 1975), and a Spectronic 601 (Fisher Scientific) spectrophotometer.

Samples of conjugates made with different molar ratios of IgG to PEG 2000 or PEG 8000 by the above two methods were then tested to determine the degree of conjugation by PEG by binding of an o-phthalaldehyde solution (Fluoraldehyde, Pierce, Rockford, IL) to available primary amines. PEG also binds to primary amines so the loss of fluorescence indicates the degree of PEG conjugation. IgG-PEG conjugates or unmodified IgG were diluted to provide 50, 25, 12.5 or 6.25 /xg/ml in PBS. To determine the IgG concentrations of these preparations, ultraviolet absorption, with spectrophotometric reading at 280 nm could be used since PEG has no absorbance at this wavelength. To 100/~1 aliquots of these solutions in Micro Fluor microtiter plates (DynaTech Laboratories, Alexandria, VA), 100 /.tl of fluoraldehyde was added. After 10 min the results were read in a Micro Floor Reader (DynaTech).

Iq84), or as a laboratory tool to diminish binding of immunoglobulin reagents to Fc receptors on cells (Anderson and "Fomasi, 1988). The ability of PEG modified lgG preparations to bind to a range of antigens has not been tested, although PEG modified IgG was found to bind ovalbumin (Anderson and Tomasi. 1988) and hepatitis B surface antigen (Suzuki et al., 1984); in contrast, in one study, the binding of anti-/32 microglobulin was destroyed by a PEG coupling procedure (Ling and Mattiasson, 1983).

Actit'ation and lgG coupling of polyethylene glycol Polyethylene glycols (PEG) 2000 or 8000 (Sigma Chemical Co., St. Louis, MO) were activated in two ways. For the first, the method of Beauchamp et al. (1983) was used with the following changes: PEG was dissolved in dioxane at 37°C to provide a concentration of 50 mM and l,l'-carbonyidiimidazole (Sigma) was added to a final concentration of 500 raM. This solution was incubated at 37°C for 2 h with stirring to activate PEG. The mixture was then dialyzed extensively against distilled water at 4°C. The activated PEG 2000 or 8000 was then dialyzed against phosphate-buffered saline (PBS) in an Amicon ceU using a 76 mm Diaflo Ultrafiltration membrane (XM 50) (Amicon Corp., Beverly, MA). The solution containing activated PEG was then dialyzed against distilled water, lyophilized and stored desiccated. Activated PEG 2000 or 8000, was incubated at 4°C for 96 h in 0 01 mM sodium borate

Antibody binding capacity of PEG-IgG conjugates Antibody binding of PEG-IgG conjugates was determined by two sets of enzyme immunoassays. In the first, a screening ELISA was set up in which 100 p.I of 10 p.g/ml of various protein or microbial antigens in 0.1 M sodium-carbonate buffer pH 9.6, were used to coat wells of microtiter plates (Nunc, Maxisorb, Vanguard International, NY) The antigens used were egg albumin (3 × crystallized) (Sigma), bovine casein (Sigma), bovine immunoglobulin (Sigma), tetanus toxoid (Wyeth-Ayerst Laboratories, Philadelphia,

PAl, and mumps antigen (Merck Sharp and Dohme, West Point, PAl. In other experiments, a commercial ELISA kit was used to quantitate the amount of antibody to mumps, rubella, cytome~aluv!~us, herpes simplex, toxoplasmosis and her.es zoster antigens (Diamedix, Miami, FL). For beth systems, PEG-IgG conjugates or for c~mpa;ison, unmodified igG were diluted to supply e'ther 0.1 m g / m l for the first set or 0.157 m g / m l for the second set, in PBS-containing Tween-0.1%. 100 /M aliquots of these solutions were added to antigen-coated wells, previously well washed with normal saline containing Tween 0.1%. After 3 h at 37°C (or overnight at 4°C), wells were washed again and llX) p.I of goat anti-human IgG conjugate (Tago, Burlingame, CA) diluted 1/1000 in PBS-Tween was added to each well. These wells were reincubated at 37°C for 3 h, washed, and a substrate solution (nitrophenyi phosphate (NPP), l m g / m l in 0.1 M Tris-HCI containing also 0.001 M MgCI) was added. The resulting solution was then read in a Multiscan Titertek microtiter plate reader (Flow Laboratories, McLean, VA). Positive controls containing an established amount of binding for these antigens, and negative controls containing no binding activity, were included in each assay. For the kit ELISA, by reference to the absorbance at 405 nm produced by the positive control, the relative amount of antibody activity for test samples could be generated in ELISA U / m l . For all ELISA assays, tests were performed in triplicate, and on two separate occasions.

Binding of PEG conjugates to Fc receptors Heat aggregated human IgG and PEG conjugates were produced by heating l0 m g / m l solutions of each in PBS to 63°C for 30 min. After removing the largest (visible) aggregates by brief centrifugation (3000 rpm for 5 rain) the aggregates contained in the supernatants of these solutions were used to test for binding to human Fc receptors present on the macrophage cell line U937. These cells (previously found to bear FcRl, II and Ill, personal communication, Dr. K. Sperber) were maintained in medium containing RPMI 1640 (Gibco, Grand Island, NY) containing 10% fetal calf serum, 10 mM glutamine, peni-

cillin and streptomycin at 37°C in a 5% CO 2 incubator. 2(XI p.I of 3 x washed 5 × I,~)¢' cells/ml in PBS containing 2% BSA and 0.1% Tween 20 were incubated with 10 or 50 /.tl of aggregated IgG or PEG-IgG conjugates at 37°C for 30 min and then at 4°C for 30 min. Cells were then gently washed three times in 2% BSA/PBSTween, and 25/zl fluorescein conjugated F(ab') 2 goat anti-human lgG (Tago) was added to each tube. After incubation at 4°C for 45 min the cells were rewashed, and the degree of fluorescence for c'ach aliquot determined by flow cytometer ( F A C S / I V , Becton-Dickenson, Mountain View, CA.)

Complement actication To determine if PEG conjugates could fix complement component C3, two methods were used. In the first, heat aggregated conjugates were tested in comparimn to heat aggregated lgG for their ability to bind to microtiter plates coated with polyclonal anti-C3c or -C3d (Dako, Accurate Chem., Westbury, NY) antibodies. For this, microtiter plates were coated with rabbit anti-human C3c or -C3d, at 20 /zg/ml in O.I M sodium-carbonate buffer, pH 9.6, at 4°C for 16 h. 100 p,I of variously diluted aliquots of heat aggregated PEG-lgG conjugates or non-conjugated igG (providing I ng to l{X) p.g/ml IgG) were incubated with a standard amount of fresh normal serum (as a source of C3). After incubation at 37°C for 3 h, these immune complex mixtures were added to well- washed, anti-C3c or -C3d coated wells of microtiter plates. After another incubation at 37°C for 3 h, the wells were rewashed, and goat anti-human lgG-alkaline phosphatase conjugate was added. After the addition of the NPP solution, the resulting absorbance at 405 nm was subsequently determined as described above. Control coated wells contained PBS alone; the alkaline phosphatase conjugate was added as usual. In the second method, immune complexes were formed by taking advantage of the fact that pooled human immunoglobulin contains substantial antibody binding activity for a common antigen, bovine r casein. Immune complexes were formed by mixing PEG-!gG, or native IgG at 225/zg/ml, 22.5 p.g/ml or 2.25 p.g/ml with bovine r casein

181)

(Sigma) at a 2.5 : i molar ratio in 50/~1 PBS. PBS 0.1% Tween was added to each sample to bring the volume to 350 /~l, and 350 /~l of a fresh normal human serum (as a source of C3) was added which contained no K casein binding antibody, These solutions were mixed, incubated at 37°C for 1.5 h, and then I(X) t~l of these mixtures were added to microtiter plates coated with C3c or C3d antibody at 4°C overnight. After 16 II, the plate was washed, 10/xl of monoclonal antibody to K casein (Cunningham-Rundles and Feng, 1989) was added, and the incubation was continued for 3 h at 37°C. After washing, goat antimouse igG-alkaline phosphatase conjugate was added (1:1500 in PBS-Twecn), and the incubation continued for 3 h at 37°C. Then the plates were washed and developed with NPP as usual. Control coated wells contained PBS Tween alone, or the monoclonal anti-K casein antibody but no immune complexes; for both controls the alkaline phosphatase conjugate was added as usual.

Detection of PEG-IgG conjugates by a second antibody The various assays used to determine the biologic activities of the PEG-lgG conjugates in comparison to native IgG (described above) depend upon the detection of igG by a second, or sandwiching antibody conjugated to alkaline phosphatase or labeled with fluorescein. To be certain that these experiments were reliable, it was necessary to determine if the PEG-IgG conjugates could be detected to the same degree as native IgG. The PEG conjugation process could potentially have blocked the binding of these indicator antibodies. To ascertain this, two methods were used. In the first, PEG-IgG conjugates or native lgG were diluted to a concentration of 10 # g / m l (by absorbance at 280 rim) in 0.1 M sodium-carbonate buffer pH 9.8 and used to coat wells of microtiter plates at 37°C for 3 h. After washing, alkaline phosphatase goat anti-human lgG (Tago) was added to each well, and after further incubation at 37°C for 3 h, the wells were washed and NPP added. The plates were then read as usual. In the second assay, PEG-IgG conjugates and native lgG were diluted to the same concentration (0.5 m g / m l ) in PBS, and tested by standard

nephelometrie methods using a Beckman array protein system (Brea, CA) and a polyclonal goat anti-human IgG (Beckman), to determine the IgG concentration of each solution by reference to an IgG standard curve.

Enzymatic digestion of IgG and PEG-IgG conjugates Trypsin. Samples of IgG-PEG conjugates or unmodified IgG (4.0 m g / m l ) were incubated with trypsin (TPCK treated, 199 U / m g , Worthington, Free Hold, NJ) at an enzyme substrate ratio of 1 : 100 at 37°C for 3 h in 0.05 M Tris-HCi buffer, pH 8.0. After this, a four-fold excess of soybean trypsin inhibitor (Sigma) was added. Samples were frozen at - 7 0 ° C until analysis by HPLC. Chymotrypsin. Samples of native IgG and PEG-IgG conjugates (4.5 m g / m l in 0.2 M phosphate buffer pH 7.0) were also digested with chymotrypsin (Sigma Type 111, TPCK treated, 54 U / m g ) at an enzyme to substrate ratio of 1 : 200. These samples were incubated at room temperature for 8 h, and then the reaction was terminated by reducing the pH to 2.0 by 6 N HCi. Samples were then frozen at - 7 0 ° C prior to analysis by HPLC. Pepsin. IgG-PEG or unmodified lgG samples were incubated at 37°C at 2.5 m g / m i in 0.1 M sodium-acetate buffer, pH 4.5 with pepsin (Worthington Biochemical Corp., Free Hold, N J) at an enzyme substrate ratio of 1:200. The reaction was stopped at 6 h by the addition of solid Tris base to bring the pH to 8.0. The samples were frozen at - 7 0 ° C until analysis.

High pressure liquid chromatography High pressure liquid chromatography was performed to judge the size of PEG conjugates before and after enzymatic digestion. For this the FPLC system (Pharmacia-LKB, Piscataway, N J) was used, equipped with a Superose 12 Column. Fractions of native IgG, PEG conjugated immunoglobulins, or enzymatically digested native or PEG conjugated immunoglobulins were loaded onto the column, equilibrated and run in 0.05 M sodium-phosphate buffer pH 7.3. The flow rate was set at 0.3 ml/min, and fractions of 0.5 ml were collected.

TABLE ! PEG-CONJUGATES PRODUCED Preparation

PEG molecular weight

Conjugation method

Coupling ratio

v/r fluore~ence

% amines coupled

A B C D E F50 FI00 FIOIXI J K L !

8,1101) 8°000 8,IXX} 2,0~XI

Carbonyl diimidazole Carbonyl diimidazole Carbonyl diimidazole Carbonyl diimidazole Carbonyl diimidazole Carbonyl diimidazole Carbonyl diimidazole Carbonyl diimidazole Cyanuric chloride Cyanuric chloride Cyanuric chloride Cyanuric chloride

I : Ill I : IIX1 I : I.IX)O 1:5

95.6 93.2 72.8 91.3

4.4 6.8 27.2 8.7

I : 10 1:50 I : I(HI

86.9 86.0

13. I 14.11

84.9 83.9 96.1 93.6 92.7 ND

15.1 16.1 3.1 6.4 7.3 ND

2,1100 2,000

2,01XI 2,(100 2,11110 2.1X10 2,000 2,111X)

I : I,II00 1:5 I : Ill I : 50 I : 288

Results

coupling the o-phthaldehyde reagent to available primary amines; since activated PEG binds to

Degree of conjugation of PEG to lgG

primary amines (the N terminus and iysine residues) by either method of activation, loss of f l u o r e s c e n c e is p r o p o r t i o n a l t o t h e a m o u n t o f

The degree of substitution of primary amines was determined by the fluorescence produced by TABLE I! ANTIBODY BINDING 1 Absorbance 4115nm ~' Antigens h Tetanus

Ovalbumin

Bovine Immunoglobulin

Casein

Mumps

1.493

0.713

11.713

0.725

I).318

1.5 l0 1.337 0.838

0.625 0.778 0.521

0.946 0.972 0.630

0.640 0.604 0.172

0.308 I).376 0.164

D E F-50 F- 100 F- 10110

I. 715 1.814 0.838 1.655 1.884

l).440 0.676 0.662 0.514 11.676

"~.476 0.648 I).785 11.518 0.775

11.631 0.572 0.592 0.606 0.509

I).268 I).283 0.455 0.318 0.389

J K L

1.706 0.820 0.830

11.777 11.76{1 0.750

I).764 0.7(10 I}.6511

11.746 0.5511 11.361

11.314 ND ND

I

0.146

0.112

0.128

0.139

ND

PBS

0.059

0.013

0.1147

0.038

0.(148

Native IgG PEG-IgG conjugates: A B C

" Data are expressed as, O.D. Units at Absorbance 41)5 nm. h Antigens used for coating microtiter plates at 10 p,g/ml, except fi)r mumps where whole vaccine was diluted to provide I(lO U/ml.

182 TABLE Ill ANTIBODY BINDING II '~ IgG Compound:

Antigens Rubella

PEG-IgG conjugates: A B C D E F50 FHI0 J K L

58.6 52.6 42.11 62.5 62. I 6 I. I 56.9 59. I 56.4 43.6

I

Negative control Pt~itive samples h contain:

ND

2.77 15

Varicella zosler

Herpes simplex I

Cytomegalo virus

108.5 110.7 97. I 121.5 122.0 I 10.7 ND 92.0 71.0 66.0

43.5 43.6 26.7 48.4 42.8 43.3 45.3 52.1 37.9 22.7

52.6 46.5 35.9 55.4 50. I 50.7 41.7 61.9 47.4 29.7

(1.6

1).6

4.5

1.8

(1.28

> 20

"ioxop|asma

48.9 7(I.1 46.(I 53.4 47.8 48.3 43.8 80.3 55.0 39. I 1.1 13.0

3.8

> 20

> 23

> 45

Data arc given in, ELISA index U per ml (EU/ml), where the known. E.U. of the positive eontrol+absorbance 405 nm of the positive control xabsorbance 41)5 nm of lest sample = EU/ml of test sample. All samples were tested at the same concentration. b Supplied in the kit used.

P E G coupled. Table 1 gives the coupling ratios used and the percentage of primary amines coupled using both procedures.

Antibody binding is preserced Antibody binding capacities of the P E G - I g G conjugates were c o m p a r e d to similar concentrations of lgG which had not been modified. Table II shows data for conjugates tested against the protein antigens, tetanus, chicken ovalbumin, m u m p s , bovine immunoglobulin and bovine casein. Overall, P E G - I g G conjugates A - L were as capable as native lgG of binding to the antigen coated plates although some conjugates had reduced capacities for binding individual antigens w h e n c o m p a r e d to native igG; for example F50 had reduced binding to tetanus toxoid, conjugates K and L, reduced binding to casein antigen, and conjugate C, greatly reduced binding for all antigens. Conjugate I had almost entirely lost the ability to bind to any of these antigen coated plates. Paradoxic results were also seen. For example, conjugates b o u n d less well to wells coated with tetanus toxoid and casein than conjugated D, but this conjugate b o u n d better than conjugate D to wells coated with ovalbumin and bovine

TABLE IV BINDING OF PEG-lg TO Fc RECEPTORS ON U937 MACROPHAGE CELL LINE Exper- Sample iments tested

Quadrant Percent of Percent percent control inhibition positive fluorescence

I

FITC-controla IgG PEG-IgG-A PEG-lgG-B PEG-IgG-C

5.17 96.00 81.00 69.00 68.00

100 84 72 45

0 16 28 55

FITC-control lgG PEG-IgG-D PEG-IgG-E

3.10 92.46 80.90 84.34

100 88 91

0 12 9

FITC-control 8.80 lgG 89.64 lgG-PEG-FI00 76.68 IgG-PEG-FI000 70.73

0 86 79

o 14 21

100 95 89 80

0 5 II 20

IV

FITC-control IgG PEG-lgG-J PEG-IgG-K PEG-IgG-L

4.3 90,16 85.77 79.83 72.06

a FITC Control = fluorescein isothiocyanate conjugated antibody directed to human lgG F(ab') 2 was added alone.

183 TABLE V COMPLEMENT FIXATION BY ANTIGEN-ANTIBODY COMPLEXES IgG compound"

Absorbance 405 nm

~/~of IgG control

% amines coupled

Native IgG PEG-IgG conjugates: A B C

0.270

l(g)

0.270

liX)

0.229 0.190

85 70

D FS0

0.234 0.127

8.7 13.1

FI(}O

0.097

56 47 36

J

0.097

49

3. I

PBS

0.(HI3

l)

1'4t 1A 1.2

0

[ 1.o 4.4 6.8 27.2 i

0,0 0.4

14.0

o.2~

~..~,~---

--..~_..~

0

. . . . . .

1

IO

" lgG and IgG-PEG conjugates tested at equal concentrations

lOO

lOOO

lOi~O

10oo0o

AGGREGATEDIMMUNOGLOBULIN I*g /m/

(22.5/.tg/ml); similar data were found for two other concentrations of igG tested, 225/zg/ml and 2.25 p.g/ml)

1.4 1.2

immunoglobulin. Tested by a commercial kit E L I S A , p r e p a r a t i o n s A - L were capable of binding to various microbial antigens in a s t a n d a r d kit E L I S A assay, with all p r e p a r a t i o n s found to contain positive a m o u n t s of antibody (Table liD.

Binding o f conjugates to Fc recepzors on cells

E

1.0

°°1 jo.,

T o test if the P E G - I g G conjugates could bind to Fc receptors, the conjugates were heat aggregated to maximize binding to the F c R I I and Ill ( A n d e r s o n , 1989) on U937. Binding was determined by fluoresceinated goat a n t i - h u m a n lgG immunoglobulin using a flow cytometer. T h e P E G - I g G conjugates did bind to U937, but with decreasing fluorescence being detected for im-

.Z,/" v

0.2

...............

0.0 1

10

IO0

1~

1OOO0

1 ~

AGG REGATED IMMUNOGLOEUUN pg/ml

2.0. 1C 1.6.

Fig. 1, Panels .4, B and C illustrate the abilities of PEG conjugates A, B and C (panel ,4), conjugates D and E (panel B), and conjugates F5I) and FI000 (panel C) exposed to normal human serum as a source of C3, to bind to microtiter plates coated with antibody to complement component C3c, as compared to native IgG. All immunoglobulin solutions were heat aggregated prior to exposure to normal human serum. The amount of immunoglobulin present in each sample is given in p.g/ml on the x axis. The relative amount of binding of IgG is shown on the 3' axis, was determined by absorbance at 405 urn. Results for binding to antibody C3d were similar.

~ ~

. 0.8.

,c 0.4.

O.Q " ' ~

.......

;;

. . . . . i~o . . . . i ~ o

.... ;~o

AGGREGATED IMMUNOGLOBUUN pgl/ml

"i~oo

munoglobulins bearing increasing amounts of PEG (Table IV). The relative degree of inhibition of cell surface binding by the PEG conjugation procedure is given.

Complement (C3) fixation by PEG-lgG conjugates Two methods of assessing complement fixation were used. In one, heat aggregated IgG-PEG conjugates, in comparison to heat aggregated lgG, were permitted to fix C3 from normal human serum. The resulting complexes were then incubated in wells of microtiter plates coated with anti-C3c or anti-C3d. Afterward, detection of

bound complexes was determined by an anti-human lgG-alkaline phosphatasc conjugate. As increasing amounts of PEG were bound to lgG fractions, reduced amounts of IgG were detected on coated microtiter plates. Fig. l shows results for PEG-lgG conjugates produced with PEG 8000, using the carbonyldiimidazole procedure. Conjugate A (4.4% coupled) bound less well than native IgG to the coated wells, while conjugates B (6.8% coupled with PEG 8000) and C (27.7% coupled) bound very poorly. Figs. 1B and 1C show results for conjugates produced using PEG 2000. Conjugates D (8.7%), E 03.1%), F50

2C

2A

IgG 150,

Is0,

t us u

m

e o

"

,;

"

~

"

~

"

do

2B

"

~

"

~

"

~o

A

i

so

'o

~'o

lOO,

lOO

"

~o

"

N

"

N

"

~oo t2 D

lm

"

N

"

N

"

F50

15o

so

lOO

10

20

40

50

60 " 70

00

10

20

30

40

50

60

Fig. 2. The relative susceptibilityof, PEG-IgG conjugatesto the enzymetrypsin,in comparison to native IgG, was determinedby digestingsamplesof these conjugatesand native lgG under the same conditions,and then analyzingthe molecularweightprofileof these samplesby HPLC. The panels A, B, C and D show the trypsin digestion results obtained for native lgG, and PEG-lgG conjugates A. B. FS0. Non-digestedIgG. and conjugates A, B. and FS0 eluted as a single, homogenouspeak with a maximum absorbance at fractions 25. 23. 23 and 23 respectively. Results for other conjugates, D, and FI00 shqw a similar lack of fragmentation(not shown).Standard ntolecularweight markers, IgM, igG, and BSA are shownby arrows (from the left to right).

185 (14.0%), a n d F1000 (16.1%) similarly lost t h e ability to bind to t h e s e coated plates as t h e perc e n t a g e of P E G b o u n d increased. C o n t r a s t i n g c o n j u g a t e B (6.8% conjugation) with c o n j u g a t e D (8.7% c o n j u g a t i o n with P E G 2000), however, s h o w s t h a t t h e larger m o l e c u l a r weight P E G a d d u c t a p p e a r s to p r o d u c e m o r e i m p a i r m e n t o f c o m p l e m e n t fixation at a slightly lower d e g r e e of conjugation. In a s e c o n d m e t h o d , n o h e a t a g g r e g a t i o n w a s n e e d e d since i m m u n e c o m p l e x e s were f o r m e d b e t w e e n l g G ( c o n t a i n i n g anti-K casein antibody) a n d , K casein antigen. A t t h r e e c o n c e n t r a t i o n s o f i m m u n o g l o b u l i n s tested, m o r e native IgG-Jc casein i m m u n e complex, could bind to microtiter plates t h a n could i m m u n e c o m p l e x e s f o r m e d using P E G - I g G (Table V). T h e b i n d i n g o f P E G - I g G c o n j u g a t e s r a n g e d f r o m 42.0 to 79.6% o f that f o u n d for native lgG. F o r c o n j u g a t e s A - C ( P E G 8000) or D - D F I 0 0 ( P E G 200), as m o r e a m i n e s were coupled, t h e less s u c h c o n j u g a t e s could bind. C o n j u g a t e s J ( P E G activated by cyanuric chloride) h a d few a m i n e s coupled, b u t still fixed c o m p l e m e n t poorly. H e r e , c o n j u g a t e s p r o d u c e d u s i n g e i t h e r P E G 8000 or P E G 2000 h a d r e d u c e d b i n d -

oWa [ lOO

o

.

o

10

,

20

30

.

40

3OO

3B E

250-

[

2OO 150 100

o 50.

< 0 10

20

30

40

50

60

3C 4O0

TABLE VI BINDING OF A SECOND ANTIBODY TO PEG-lgG CONJUGATES IgG compound

Native IgG PEG-igG conjugates: A B C D E FI00 FI000 J K L

% of control igG bound to ELISA plate ;'

% of control IgG detected in solution b

100

100

100 87 63 100 100 93 86 100 96 95

95 100 39 95 100 100 100

E

o

200.

100-

o "

10

• ~L~

"

~

" ~

• ~

" ~

Fig. 3. Similar to Fig. 2. the molecular weight profile of native IgG was compared to PEG-lgG conjugates E. and FS0 by HPLC after each sample had been digested with chymotrypsin under the same conditions. Conjugate D shows a pattern similar to conjugate E (not shown)• Non-digested conjugates eluted as a single peak with a maximum absorbance at fraction 23: molecular weight markers are as shown in Fig. 2.

I(X)

100 100

a Percent of control lgG solution bound to wells of microtiter plates: all solutions at 10 ~.g/ml concentration. b Percent of control IgG solution detected by nephelometer: all solutions were tested at 0.5 mg/ml.

ing activity, b u t with P E G 2000-1gG c o n j u g a t e s b e i n g s o m e w h a t m o r e impaired t h a n P E G 8000 conjugates.

186

Binding of a second antibody to PEG conjugates Since in the above experiments the binding of a sandwiching antibody to PEG-IgG conjugates was used to determine the biologic activities of these conjugates, experiments were needed to determine the relative binding of this second antibody to PEG conjugates in comparison to native lgG. Table V1 shows the result for binding of alkaline phosphatase labeled goat anti-human lgG to PEG-IgG conjugates or native IgG coated onto wells of microtiter plates, and the binding of an unlabeled goat anti-human lgG for detection by a nephelometer. Some degree of impairment of binding of these second antibodies was found for conjugates B, C, and FI000 when these conjugates were used to coat wells of microtiter plates. However, when the same conjugates were tested in solution, the second antibody detected all conjugates well, except for the most heavily PEG conjugated immunoglobulin, conjugatL C (27.7% of available amines coupled).

Enzyme digestion of PEG-IgG conjugates A main goal of these experiments was to determine if binding of PEG to IgG would render lgG resistant to intestinal enzymes. To test this, solutions of IgG or various PEG-IgG conjugates were exposed to trypsin, chymotrypsin or pepsin; the degree of fragmentation of IgG was then assessed by HPLC. Fig. 2 shows the results for trypsin digestion of native IgG as compared to PEG-IgG conjugates. While IgG was fragmented into two major peaks, conjugate A (4.4% conjugation) and conjugate B (6.8% lysine conjugation) displayed less cleavage after this procedure. Conjugate D (8.7% conjugated) remained almost totally intact in this analysis (not shown), and conjugates F50 (14% conjugated) and F100 (15.0% conjugated) (the latter not shown) were not di-

2o0 IgG 250 •

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Fig. 4. Similarto Figs. 2 and 3. the molecularweight profile of native lgG was compared to. PEG-lgG conjugates FI00 and (2, by HPLC after each sample had been digested with pepsin under the same condilions. Molecularweight markers are as shown in Fig. 2. Non-digestedconjugates FI00 and C eluted as a single peak with maximum absorbance at fractions 23, and 20 respectively.

so

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FRACTION NUMBERS

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187 gested. Thus PEG conjugation produced IgG compounds impervious to trypsin. Fig. 3 shows results for chymotrypsin digestion of PEG conjugates in comparison to native lgG. This enzyme fragmented IgG into several fragments; PEG-IgG conjugates with 14% or more PEG conjugation were less susceptible to the actions of this enzyme although some fragmentation was still seen. Examples shown are conjugates E (13.1%) and FS0 (14%) shown in Fig. 3. Although PEG-IgG conjugates were partially digested by pepsin, these compounds remained more intact than native IgG after incubation with this enzyme. For example, Fig. 4 shows data for native IgG, conjugates C and FI00 after 6 h of exposures to pepsin. In comparison to native IgG, conjugates C and FI00 produced about 50% of the F(ab') 2 fragment of IgG as did native lgG. . For other less modified conjugates, less protection against pepsin was observed. Chromatographic analyses of PEG-compounds also revealed that the more heavily substituted IgG conjugates eluted earlier from Superose than the less heavily substituted compounds, or native IgG. These observations are noted in the legends of Figs. 2-4, which point out the different elution positions of these conjugates.

Discussion

Polyethylene glycol conjugation of various biologically active proteins has previously been shown to increase the half life, reduce the immunogenicity, and variably preserve the biological functions of the protein modified (Abuchowski et al., 1977a,b; Davis et al., 1981; Katre, 1990). In these analyses we show that modification of the available primary amines of human serum immunoglobulin IgG, up to 27%, when using a mild procedure, can produce an immunoglobulincapable of binding proteins or viruses using either PEG 2000 or PEG 8000. Using the carbonyldiimidazole procedure and a coupling ratio up to 1 : 100 (producing a conjugate with 6.8% of available iysines conjugated), binding of PEG-IgG to various antigens was equivalent to that of native lgG. However, using this procedure and a coupling, ratio of 1:1000, some losses of antibody

binding were observed. In some cases, individual conjugates appeared to bind some antigens better than others (examples being conjugates D and L, both containing PEG 2000). Whether the method of PEG activation can alter some, but not other, antibody specificities, is unknown. The use of the cyanuric chloride method produced some losses of antibody binding capacity at coupling ratios as low as 1:50, and at a ratio of 1:288, antibody binding activity was lost when this procedure was used. This is in agreement with Ling et al. (1983) who found total loss of antibody binding activity using cyanuric chloride, at a coupling ratio of 1 : 1000. The central observation of these experiments, which could be a basis for the potential use of PEG-lgG conjugates for oral reconstitution of secretory immunity, is that PEG-IgG conjugates are rendered relatively resistant to enzymatic cleavage, particularly to trypsin. Since trypsin cleaves at peptide bonds contributed by iysine or arginine residues (Bennett, 1967) and PEG binds predominantly to lysines, the lack of susceptibility of PEG-lg conjugates to trypsin digestion is understandable. IgG contains approximately 90 mol of lysine/moi IgG (Habeeb, 1966). Blockage of some of these (as for PEG-IgG conjugates B, C, D, E, F50 or F100) is apparently sufficient to inhibit almost completely the activities of trypsin. Presumably, only a proportion of the lysine residues of IgG are actually available to trypsin digestion when dissolved in a solution in the absence of denaturants. PEG-IgG conjugates were also rendered less susceptible to the actions of chymotr~sin and pepsin, especially for the more highly substituted conjugates. Chymotrypsin has specificity for peptide bonds adjacent to tryptophan, phenylalanine, and tyrosine residues (Bennett, 1967); pepsin cleaves at defined regions in the hinge of IgG (Edelman and Marchalonis, 1967). Presumably steric hindrance by the bonded PEG residues accounts for the increased resistance of conjugates to these enzymes. The most abundant proteolytie enzyme present in the small intestinal tract is trypsin, followed by ebymotrypsin; both are secreted by the pancreas. Pepsin is also present, but in the alkaline environment of the intestinal tract, it is less active

188 (Brooks, 1985). The exact amounts of trypsin and chymotrypsin which might be available in the intestinal tract are difficult to determine, but using the Lundh test, an established measure of pancreatic secretion of trypsin, the average secretion of trypsin in normal subjects after a test meal is 7-38 U / I (Mottaleb, 1973). The amount of trypsin supplied in these experiments was at least 1000 times this range (7.96 U/ml), thus the stability of conjugates to this enzyme is impressive. As expected, chromatographic analyses of PEG-IgG conjugates showed more rapid elutions as compared to native lgG; these observations have previously been interpreted as confirming the presence of PEG on the outer surface of the IgG molecule, which would increase the effective radius (Suzuki et al., 1984). Since PEG conjugation thus changes the elution position of lgG conjugates and enzymatically cleaved fragments on chromotography, an accurate estimation of true molecular weights for these conjugates or fragments, by reference to the molecular weight standards, is probably not possible. In these analyses we also investigated if the PEG-IgG conjugates could fix complement component C3, or bind to cell surface Fc receptors. All conjugates appeared to have a variably reduced ability to fix complement C3, with conjugates B, C, F100 and F1000 having very little activity when heat aggregated and permitted to bind to C3. These data agree with those of Suzuki et al. (1984) who found that heat aggregated PEG-igG conjugates (formed by activitating PEG in the cyanuric chloride) fixed complement less readily than native IgG. However, as Suzuki et al. point out, IgG-PEG conjugates may not form aggregates well when heated, and by size-exclusion chromatography, such complexes are more heterogeneous in size than those containing native IgG. In our studies we used an additional method for testing complement fixation by PEGIgG complexes, which also indicated overall impairment of C3 fixation by the more highly substituted conjugates. While the two tests of complement fixation gave similar results, specific correlations for results of individual conjugates tested by these methods could not be made. In our experiments, heat aggregated complexes formed with the more highly substituted

PEG conjugates also bound less well (only 55%) to cell surface Fc receptors. These results could be due to PEG binding to the Fc portion of lgG, preventing attachment to Fc receptors, or to the differences between heat aggregated PEG-IgG complexes and native lgG complexes. In a previous work, using antigen-antibody-complexes, Fc receptor binding of antigen-antibody complexes on murine cells was totally inhibited when 20% or more of available lysines on the antibody were coupled to PEG 5000 (Anderson and Tomasi, 1988). The differences in our experimental results may be due to the methods of complex formation, PEG-activation, or the differences in the types of cells used. We also investigated if the PEG-IgG conjugates could be detected less well by a second antibody since binding of the heterologous antisera can be diminished by PEG conjugation (Abuchowski et al 1977a,b). We investigated this in two ways: by binding conjugates to wells of polystyrene microtiter plates and detecting IgG by alkaline phosphatase-labeled goat anti-human IgG, and by testing binding of conjugates to a second antibody in solution with detection by nephelometer. The more highly substituted conjugates B, C, and F1000 were less well detected on microtiter plates, but all conjugates, except conjugate C, were detected similarly by nephelometer. Thus it appears that the more highly substituted conjugates may either bind less well to polystyrene or are less well detected when fixed to polystyrene, but that except for conjugate C, all are capable of being identified by a polyclonal heterologous antiserum when tested in solution. Thus our conclusions (derived from experiments in which PEG-lgG was not fixed to polystyrene plates) seem valid. Since serum lgA fixes complement C3 only by the alternative pathway (Russell et ai., 1989), and binds to Fc receptors present mostly on granulocytes and monocytes (cells not normally found in the gastrointestinal lumen, Deha et al., 1989), the relative loss of complement fixation and cell surface binding by PEG-IgG conjugates may be desirable. The role of secretory IgA in the gastrointestinal tract has previously been likened to that of 'a 'discreet housekeeper' (Conley and Delacroix, 1987); antigen binding is efficient but since

189 complement fixation and binding to cells is restricted, little local inflammation or other immune activation is triggered. Secretory immune deficiency is not an uncommon disorder, since it can arise from several different physiologic, congenital, or pathologic mechanisms, Infants normally have secretory IgA deficiency since IgA is produced in only very low amounts for the first few months of life (Mellander et al., 1984). Patients with selective lgA deficiency, or common variable immunodeficiency also have secretory lgA deficiency (Cunningham-Rundles et al., 1979; Cunningham-Rundies and Carr, 1986). In addition to these causes, chemotherapy and radiation therapy may deplete IgA producing plasma cells, resulting in secondary secretory IgA deficiency. Possibly the best described instance of acquired, prolonged secretory IgA deficiency is in the bone marrow transplant recipients (Beschorner et al., 1981; Izutsu et al., 1983). To attempt the reconstitution of secretory immunity in such patients, human milk, especially h u m a n colostrum, might provide the ideal form of immunoglobulin for oral use. However there are major difficulties in the procurement, sterilization, and standardization of this substance, in addition there is the possibility that banked breast milk could transmit virus infections. Commercially available immunoglobulins derived from human serum could surmount these difficulties, and in some studies oral use of serum immunoglobulin concentrates have been tried with clinical benefits (Blum et al., 1981; Barnes et al., 1982; Copeland et al., 1988; Tuschka et ai., 1988; Eibl et al., 1989). In some cases the survival of a portion of the orally administered antibody has been documented. Most studies have used serum IgG but in one, a concentrate containing serum IgA and lgG was used (Eibl et ai., 1988). Although the use of serum IgA could conceivably afford a somewhat different spectrum of anti-microbial antibody activity, as opposed to serum lgG, the susceptibility of monomeric serum lgA to proteolytic attack, is very little different from that of serum IgG (Brown et al, 1970; Lindh, 1975). In the current studies, serum IgG has been covalently bonded to polyethylene glycol, in order to produce conjugates retaining antibody binding activity, but which resist enzymatic cleavage.

Polyethylene glycol is biologically inert, nonimmunogenic and inexpensive. It is a common additive to foods and drugs and a frequent component of proprietary formulations such as cough remedies. Various molecular sizes of polyethylene glycols have been used in orally administered tests of gastrointestinal permeability (Olaison et al., 1989), and as treatments for constipation (Adorsky et al., 1989). It is possible that PEG-IgG conjugates could be useful for the oral treatment of gastrointestinal immune deficiency states.

Acknowledgements We gratefully acknowledge the secretarial skills of Mrs. Lydia Lopez and thank Dr. K. Sperber for the gift of U937 cells, Alpha Therapeutics for the immunoglobulin fractions and Dr. Jay Unkeless for the use of his microfluorometer.

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Hershfield, M.S., Buckley, R.H., Greenberg, M.L., Milton, A.L., Schiff, R., Harem, C., Kurtzbcrg, J., Markert, M.L., Kobayashi, R,H., Kobayashi. A.L. and Abuehowski, A. (1987) Treatment of adenosine deaminase deficiency with polyethylene glycol-modified adenosine deaminase. New. Engl. J. Med. 316, 589-596. izutsu, K.T., Sullivan, K.M., Schubert, M.M., Truelove, E.L., Schulman, H.M., Sale, G.E., Morton, T.H., Rice, J.C,, Witherspoon, R.P., Storb, R. and Thomas, E.D. (1983) Disordered salivary immunoglohulin secretion and sodium transport in human chronic graft-vs-host disease. Transplantation 35, 441-450. Katrc, N.V. (1990) lmmunogenicity of recombinant IL-2 modified by covalent attachment of polyethylene glycol. J. lmmunol. 144, 209-213. Lindh, E. (1975) Increased resistance of immunoglobulin A dimers to proteolyt;,~ degradation after binding of secretory component. J. lmmunol. 114, 284-286. Ling, T.G.I. and Mattiasson, B. (1983) A general study of the binding and separation in partition affinity legand assay. Immunoassay of/32 microglobulin. J. lmmunol. Methods 59, 327-337. Losonsky, G.A., Johnson, J.P., Winkelstein, J.A. and Yolken, R.H. (1985) Oral administration of human serum immunogtobnlin in immunodeficient patients with viral gastroenteritis. J. Clin. Invest. 2362-2367. Mellander, L., Carlsson, B. and Hanson, L.A. (1984) Appearance of secretory IgM and IgA antibodies to Escherichia coil in saliva during early infancy and childhood. J. Pediair. 104, 564-568. Mottaleb, A., Kapp, F., Noguera, E.C.A, Kellock, T.D., Wiggins, H.S., and Waller, S.L. (1973) The Lundh test in the diagnosis of pancreatic disease: A review of five years experience. Gut 14, 835-841. Olaison, G., Sjodahl, R., Leandersson, P. and Tagesson, C. (1989) Abnormal intestinal permeability pattern in colonic Crohn's disease. Absorption of low molecular weight polyethylene glycols after oral or colonic load. Scand. J. Gastroenterol. 24, 571-576. Putnum, F.W. (Ed.) (1975) The Plasma Proteins. Academic Press, New York, pp. 62. Russell, M.W. and Mansa, B. (1989) Complement fixing properties of human lgA antibodies. Alternative pathway complement activation by plastic-bound, but not specific antigen bound, IgA. Scand. J. Immunol. 30, 175-183. Suzuki, T., Kanbara, N., Tomono, T., Hayashi, N. and Shinohara, I. (1984) Physicochemical and biological properties of polyethylene glycol-coupled immunoglobulin G. Biochim. Biophys. Acta 248-255. Tutschka, P.J. (1988) Infectious and immunodeficiency in bone marrow transplan~.ation. Pediatr. Infect. Dis. J. 7, 522-529. Underdown, B.J. and Dorrington, KJ. (1974) Studies on the shuctural and conformational basis for the relative resistance of serum and secretory immunoglobulin A to proteolysis. J. lmmunol. 112, 949-959.