A method for the preparation of protein-protein conjugates of predetermined composition

Journal of Immunological Methods, 24 (1978) 321--336

321

© Elsevier/North-Holland Biomedical Press

A METttOD FOR THE PREPARATION OF PROTEIN-PROTEIN CONJUGATES OF PREDETERMINED COMPOSITION

E.S. RECTOR, R.J. SCHWENK, K.S. TSE and A.H. SEHON {with the technical assistance of H. Chan) MRC Group for Allergy Research, Departments of Immunology and Medicine, Faculty of Medicine, The University of Manitoba. Winnipeg, Manitoba R3E OW3, Canada

(Received 23 January 1978, accepted 9 June 1978)

A novel procedure for the synthesis of well-defined protein-protein conjugates is described using ovalbumin (OA) and IgG as test proteins. This procedure involves the highly selective and rapid reaction of alkyl halide and sulfhydryl groups, which have been grafted, respectively, onto the proteins to be conjugated. Accordingly, iodoacetylated IgG, (ICtt2CO)nIgG , was prepared by the reaction of the e-amino groups of IgG with the N-hydroxysuccinimide ester of iodoacetic acid (NHIA), the degree of iodoacetylation (n) being proportional to the concentration of NHIA. OA was reacted with S-acetylmercaptosuccinic anhydride (SAMSA) under conditions yielding, on the average, a monosubstituted derivative. Following removal of the protective S-acetyl group, the resulting -SH derivative of OA was reacted with (ICH2CO)nIgG. The OAx-IgG conjugates so produced were characterized by gel filtration, specific radioactivity (using tritiated OA) and immunodiffusion. It was found that the average number of OA molecules coupled per IgG molecule could be controlled by varying the degree of iodoacetylation of IgG.

INTRODUCTION In this and o t h e r l a b o r a t o r i e s , it has been r e c e n t l y s h o w n t h a t c o n j u g a t e s o f small h a p t e n s with isologous IgG are c a p a b l e o f tolerizing i m m u n o c o m p e t e n t cells specific f o r t h e h a p t e n ( G o l a n a n d Borel, 1 9 7 1 ; Lee and S e h o n , 1976). F o r e x a m p l e , c o n j u g a t e s o f 2 , 4 - d i n i t r o p h e n y l ( D N P ) or b e n z y l penicilloyl g r o u p s with m o u s e IgG n o t o n l y i n h i b i t e d the i n d u c t i o n o f p r i m ary and s e c o n d a r y IgE a n t i b o d y r e s p o n s e s t o t h e r e s p e c t i v e h a p t e n s , b u t also a b r o g a t e d o n g o i n g IgE responses. T h e degree o f h a p t e n a t i o n o f t h e IgG was s h o w n to be i m p o r t a n t - - a low e p i t o p e d e n s i t y (1-4) was i n e f f e c t u a l w h e r e a s a high e p i t o p e d e n s i t y ( > 1 5 ) yielded an i m m u n o g e n r a t h e r t h a n a t o l e r o g e n . This s t u d y was u n d e r t a k e n with a view to e x p l o r i n g the feasibility o f e x t e n d ing this s y s t e m to the a b r o g a t i o n o f IgE a n t i b o d i e s t o c o m m o n allergens with c o n j u g a t e s o f IgG and allergenic p r o t e i n s o f well d e f i n e d c o m p o s i t i o n . In the p a s t m a n y p r o c e d u r e s have b e e n devised f o r c o v a l e n t l y c o u p l i n g p r o t e i n s to o n e a n o t h e r ( L i k h i t e and S e h o n , 1967). T h e s e p r o c e d u r e s utilized cross-linking agents such as g l u t a r a l d e h y d e , t o l u e n e - 2 , 4 - d i i s o c y a n a t e ,

322 carbodiimides and diazonium compounds. Most of these reagents function as cross-linking agents as a consequence of their ability to react with at least two amino acid residues found in proteins. A c o m m o n problem resulting from the reaction of two different proteins, e.g. P, and I)2, with such crosslinking reagents is the likelihood of producing many different reaction products. Depending on the particular circumstances, these reaction products may include a mixture of (1) intramolecularly cross-linked P~ and/or P2, (2) bimolecular conjugates P~--P2, PI--P1, P2--P2 and cross-linked aggregates of these and (3) combinations of (1) and (2). It is clear that the products from such conjugation reactions are in molecular terms complex, difficult to characterize and consequently difficult to reproduce. This paper describes the development of a conjugation procedure which can be used to produce tailor-made protein conjugates. The procedure, which utilizes the highly specific and rapid reaction of alkyl halides with sulfhydryl compounds results in the formation of thioether linkages. Such bonds are known to be very stable under physiological conditions and are, therefore, suitable for the covalent binding of protein molecules. The conjugation procedure reported in this article consists of 3 distinct reaction steps:

(1) The introduction of iodoacetyl groups into lgG This has been accomplished by the reaction of the e-amino groups of IgG with the N-hydroxysuccinimide ester of iodoacetic acid (NHIA): O

° II

© ~

° II

IgG-NH 2 ÷ ICH2C-O-N

~

IgG-NH-C-CH2I

* NO-N

O

O

(2) Introduction of a single sulfhydryl group into the allergenic protein (P~) This involves a two-stage preparation: (a) Reaction of P, with S-acetylmercaptosuccinic anhydride (SAMSA): 0 II

Pa- NH2 +

0 11

//0

CH3C-S-~H --

0 II

~ - N H - C - C H -I

C\O

CH 2-~-.-C / ~O

S-C-CN

3

CH2COOH

(b) Generation of the sulfhydryl group: 0

0

II

II

Po- N H - C - C H - S - C CH 3 I CH2COOH

0

NH20 H ~--

II

Po- NH - C - CH - S H ; (PQ-SH) I C_2H2COOH

(3) Coupling of the mercapto derivative of P~ to the iodoacetylated IgG O II

I g G - N H - C,- C.H2I

+

O ii

Po-SH

m

IgG-NH-C-CH2-S

- Po + b.l

323 To verify the proposed reaction sequence, the low molecular weight hapten, 2,4-dinitrophenyl group (DNP), was coupled to mouse IgG. In subsequent experiments, ovalbumin (OA) was grafted onto IgG. MATERIALS AND METHODS

Chemicals All chemicals used were reagent grade. PBS--EDTA buffer consisted of 0.01 M sodium phosphate, 2% NaC1 and 1 mM EDTA, pH 7.5. Iodoacetic acid, N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC) and 1-fluoro-2,4-dinitrobenzene (DNFB) were obtained from Eastman Kodak, Rochester, NY, N-e-carbobenzoxy-L-lysine (N-e-CBZ-lysine) and S-acetylmercaptosuccinic anhydride {SAMSA) from Sigma Chemical Co., St. Louis, MO and 5 z: crystallized ovalbumin (OA) from ICN Pharmaceuticals Inc., Cleveland, OH. Dioxane was passed through neutral aluminium oxide (Mondray Ltd., Montreal, Canada) before use. Tritiated iodoacetic acid {206.6 Ci/mole) and Aquasol TM scintillation cocktail were obtained from New England Nuclear, Boston, MA. [methyl-3H]Acetic acid (551 Ci/mole) was purchased from Amersham/Searle, Arlington Heights, IL. Sephadex TM G-25, G-200 and Sepharose T M 6B were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden.

IgG Dog or mouse (A/HeJ) IgG was prepared from normal serum by precipitation with a m m o n i u m sulfate at 50% saturation and subsequent gel filtration of the precipitated whole ~,-globulins using Sephadex G-200; a molecular weight of 150,000 and ~2s0 •,,~ = 14 were assumed in all subsequent calculations.

Ovalbumin When required, a 4% solution centrifuged at 1 0 0 , 0 0 0 / g for 2 Beckman SW 50 rotor. The top use; a molecular weight of 45,000

of OA in PBS--EDTA was prepared and h at 4°C in 1.5 cm × 5 cm tubes using a half of the solution was stored at 4°C for and ~1%~2s°= 7.35 were assumed.

a-Dinitrophenyl-lysine (a-DNP-lysine) a-DNP-lysine was prepared by reaction of N-e-CBZ-lysine with DNFB, followed by removal of the e-CBZ protective group with HBr. Procedures similar to those described by Segal and Hurwitz {1976) were used.

a-DNP-e-(S-acetylmercaptosuccinyl)-lysine (a-DNP-e-SAMSA-lysine) a-DNP-lysine (0.32 mmole) was dissolved in 48 ml dioxane--water (2 : 1) and 0.64 mmole solid SAMSA was added gradually over a period of 1 h with constant stirring at room temperature. The pH of the solution was maintained at 7 by the dropwise addition of 1 M NaOH. Following extrac-

324 tion with ether, the aqueous solution was lyophilized to yield a yellow crystalline product.

a-DNP-e-mercaptosuccinyl-lysine (a-DNP-e-SH-tysine) The following reaction was pe r f or m e d under anaerobic conditions, a-DNPe-SAMSA-lysine (24 mg) was dissolved in 1 ml 0.10 N NaOH. The pH was raised and maintained between 11 and 11.5 using 0.10 N NaOH for 25 rain. The solution was adjusted to pH 9, diluted to 2 ml with water and stored at --70°C under N2.

NHS ester of [3H]iodoacetic acid ([3H]NHIA) [3H]iodoacetic acid (1 mCi, 0.9 rag) and unlabelled iodoacetic acid (12.6 mg) dissolved in a total volume of 2 ml anhydrous dioxane were mixed in a 3 ml glass vial equipped with a stirring bar. An equimolar a m o u n t {8.36 mg) o f NHS was added with stirring. DCC (16.42 mg) was added to the clear solution and the reaction mixture stirred at room t em perat ure for 1 h, during which time a white precipitate (dicyclohexyl urea) formed. After removal o f the precipitate by filtration, the dioxane solution was used as such.

Unlabelled NHIA Iodoacetic acid (3.9 mmoles) was added to 40 ml anhydrous dioxane containing 4 mmoles NHS and 4 mmoles DCC. After stirring at room temperature for 1 h, the solution was filtered, evaporated to dryness under vacuum and the p r o d u c t recrystallized from chl or oform , yielding a white crystalline p r o d u c t with m.p. 143--146°C. Mass spectrometric analysis* indicated a parent peak at 283 (theoretical = 283).

NtIS ester of [~H]acetic acid Equimolar quantities (0.05 mmole) of [3H]acetic acid (25 mCi), NHS and DCC were dissolved in 2 ml a n h y d r o u s dioxane and stirred at 20°C for 30 rain. The insoluble dicyclohexyl urea was removed by filtration and the dioxane solution stored frozen at --20°C.

Preparation of iodoacetylated IgG, (ICH2CO)nIgG (a) With [3H]iodoacetyl groups. Typically, [3H]NHIA (0.05 ml) was added to 4 mg IgG in 0.15 ml PBS-EDTA. After incubation at room temperature for 15 min, the solution was applied on a 0.8 cm ,~ 16 cm Sephadex G-25 column equilibrated with PBS--EDTA. The excluded fraction was assessed in terms of radioactivity and IgG cont ent , and the specific radioactivity (cpm/mg) determined. The degree of iodoacetylation, n, was subsequently calculated.

* This analysis was performed in the Department of Chemistry, University of Manitoba.

325

(b) With unlabelled iodoacetyl groups. T o a stirred solution of 50 nag IgG in 2.45 ml PBS--EDTA, 1.89 mg NHIA in 0.05 ml dioxane was added in such a way as to avoid high local concent r a t i ons of dioxane. After stirring at room t e m p er atu r e for 5 rain the iodoacetylated IgG was passed through a Sephadex G-25 column, co nc e nt r at ed using negative pressure dialysis and stored at --20 ° C. Tritiated OA ([3H]OA) NHS ester of [3H]acetic acid in dioxane {0.06 ml) was added to a stirred solution of 50 mg OA in 1.25 ml PBS- -EDTA. After 5 min the reaction mixture was passed through a Sephadex G-25 column (1.5 cm ,' 70 cm) equilibrated with PBS---EDTA. The protein in the excluded fraction was concentrated and centrifuged at 100,000 × g to remove any aggregates which might have formed. The top two-thirds of the solution was stored at 4~C. In this particular case the specific radioactivity of the [3H]OA was 9.0 pCi/mg.

SAMSA derivative o f [3It]OA ([3tt]OA-SAMSA) The mercaptosuccinylation of [3H]OA was accomplished using previously described procedures (Klotz and Heiney, 1962; Marks, 1967) with some modifications. An aliquot containing 50 mg [3H]OA (1.25 ml PBS--EDTA) was placed in a 5 ml glass vial and stirred at room t em perat ure for 1 h. During this time and subsequently the vial was flushed with nitrogen. SAMSA (967 ,g, i.e. 5-fold molar excess) was added and the solution was stirred for an additional 1 h. The pH was maintained between 7.5 and 8.0 by the addition of 1 N NaOH. The [~H]OA-SAMSA was then isolated by gel filtration on Sephadex G-25 (PBS--EDTA), concentrated, and stored under N2 at --20°C.

Determination o f sulfhydryl content o f mercaptosuccinylated proteins Typically, 1 mg protein and a 20-fold molar excess of [ 3H]iodoacetic acid were stirred under a cons t a nt flow of N2 for 15 min. H y d r o x y l a m i n e (0.10 M in PBS--EDTA) was added to a final c o n c e n t r a t i o n of 0.01 M and the solution (0.20 ml) stirred in a sealed vial under N2 for 18 h. The labelled protein was separated from excess iodoacetic acid by gel filtration {0.8 cm ~ 16 cm Sephadex G-25) and its specific radioactivity was used to calculate the n u m b e r of -SH groups per protein molecule. Such determinations were found to compare favourably with those obtained using a spectrophotometric m e t h o d which involved the reaction of 2,2'-dithiodipyridine with thiols to yield 2-thiopyridone (Grassetti and Murray, 1967).

Conjugation o f a-DNP-e-SH lysine to iodoacetylated IgG Th e following reaction was p e r f o r m e d anaerobically. Typically, 30 mg (ICH:CO)12IgG in 4.2 ml PBS--EDTA was stirred under a stream of N2 for 30 min. To this solution 12 mg (1 ml) ~-DNP-c-SH lysine (10-fold molar excess with respect to -CH2I) was added and the reaction allowed to proceed at r o o m t e m p e r a t u r e overnight. The di ni trophenyl at ed IgG was separated

326

from free hapten by Sephadex G-25 gel filtration and the degree of haptenation (H), i.e. the number of DNP groups per IgG molecule, was calculated in a manner similar to that previously published (Eisen et al., 1954) using the formula: Hm

16.73 • A3~o A2~,, ~ (A.~,o/3.07) __.

where A = absorbance at the specified wavelength (nm). For a-DNP-lysine, the following constants were determined in this laboratory: E~6°= 12,550

and

A~6o

....". . . . 3.07 A2 ~o

Conjugation of [3H]OA-SAMSA to iodoacetylated IgG The conjugation was performed under anaerobic conditions at room temperature. [3H]OA-SAMSA (7 mg) and iodoacetylated IgG (5 mg), both in PBS--EDTA, were mixed and the solution made 0.01 M with respect to hydroxylamine. The reaction mixture (1.5 ml) was stirred for 20 h and passed through two 1.3 cm Sephadex G-200 columns arranged in series, equilibrated with PBS--EDTA (total length 176 cm). Fractions (1 ml) were collected and assessed for their 3H and protein contents. These two parameters allowed the calculation of the ratio of mercaptosuccinylated protein to IgG (the substitution ratio) in any particular fraction.

Determination of 3H radioactivity A Beckman LS-335 scintillation counter was used for 3H determinations. Typically, 0.05 ml samples were placed in 20 ml polyethylene scintillation vials (New England Nuclear) and dissolved in 5 ml Aquasol. Counting efficiencies ranged from 44 to 47.5%.

Immunodiffusion and immunoelectrophoresis Immunodiffusion analysis was performed using 0.8% agarose in phosphate-buffered saline (PBS), pH 7.2. For immunoelectrophoresis, 1% Noble agar (Difco) was used in a buffer consisting of 0.03 M sodium barbitol, 0.0025 M calcium lactate pH 8.6. RESULTS

Iodoacetylation of IgG Initially experiments were designed to establish the conditions required for the controlled introduction of iodoacetyl groups into IgG. For this purpose, [3H]NHIA was prepared. Three parameters of the reaction were studied: (1) NHIA concentration. The degree of iodoacetylation of IgG, which designates the number of iodoacetyl groups grafted onto one IgG molecule,

327 26

~ 5 '3 ~g $ ,

"4

Fig. 1. Iodoacetylation of IgG. Mouse IgG was reacted with increasing amounts of [31t1NttIA as described in Materials and Methods. After 15 min, [3It]iodoacetyl-IgG was isolated from the reaction mixture by gel filtration and its specific radioactivity determined, e, IgG * [3H]NHIA;.., IgG + [3H]iodoacetic acid. w a s d e t e r m i n e d as a f u n c t i o n o f t h e a m o u n t o f N H I A a d d e d b y r e a c t i n g a fixed amount of IgG with increasing quantities of [aH]NHIA. It can be seen f r o m t h e r e s u l t s in Fig. 1 t h a t t h e n u m b e r o f i o d o a c e t y l g r o u p s c o u p l e d t o IgG was directly dependent on the amount of [3H]NHIA added, and varied b e t w e e n 1.2 a n d 1 7 . 4 g r o u p s p e r I g G m o l e c u l e . F u r t h e r m o r e , w h e n [ 3 H ] i o d o a c e t i c a c i d was u s e d in p l a c e o f [ 3 H ] N H I A , n o l a b e l l i n g o f I g G w a s apparent. (2) Reaction time. T h e k i n e t i c s o f t h e i o d o a c e t y l a t i o n r e a c t i o n was investigated by removing aliquots of a reaction mixture at different times, varying from 5 to 60 rain, and determining the extent of iodoacetylation.

TABLE 1 THE EFFECTS OF REACTION TIME AND DIOXANE CONCENTRATION ON THE DEGREE OF IODOACETYLATION OF IgG IgG (4 mg) was reacted with [3H]NHIA in a total volume of 0.20 ml and the reaction was terminated by gel filtration as described in Materials and Methods. Molar ratio of reactants (NHIA/IgG) .

.

.

.

.

.

.

.

Reaction time (rain) .

.

.

.

.

.

.

.

.

.

.

.

.

Dioxane concentration (%) .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

Degree of iodoacetylation .

.

.

.

.

.

.

.

.

(a)

20 20 20 20

5 15 30 60

25 25 25 25

10.5 10.4 10.4 10.4

(b)

5 5 5 5

5 5 5 5

25 10 4 2

3.5 3.0 2.9 2.9

.

.

.

.

.

.

.

.

.

328

The results (Table la) dem ons t r a t e d that the reaction was rapid and appeared essentially com pl e t e within 5 min. (3) Dioxane concentration. The a m o u n t of dioxane required during the reaction was also studied. Since NHIA appeared to be insoluble in water, it was necessary to add it as a dioxane solution. Due to the possible hydrolysis o f NIIIA by iI20 during iodoacetylation, different dioxane concentrations were tested. It was found that final concentrations of dioxane between 2~ and 25% did n o t significantly alter the degree of iodoacetylation (Table lb). In all subsequent experiments a final dioxane concent rat i on of 27~ was used in order to minimize protein denaturation.

Conjugation of DNP to lgG Initially, experiments were designed to assess the reactivity of iodoacetylated IgG with sulfhydryl compounds. One such experiment, which involved the conjugation of a-DNP-c-SH-lysine to iodoacetylated lgG, is described here. Tritiated (ICH2CO)12. ] IgG was prepared and characterized using I~I-I]NHIA. To 3 mg of this preparation, 1.2 mg of ~-DNP-c-SH-lysine (10-fold molar excess with respect to -CH2I) was added with stirring under N~ at 22°C. Aliquots (1 mg IgG) were removed at 0.75, 1.75 and 18 h later, passed through Sephadex G-25 and the e x t e n t of conjugation determined spectrophotometrically. The results are presented in Fig. 2. It can be seen that d i n i t r o p h e n y l a t i o n of lgG occurs rapidly under these conditions and

L9'.4 I!)

:):(2 t5 ~

"~9 10

~e a:

•

0

o~

4

:r~ ~ h O.b n~ pC) Z

z i

REACTION

lIME

:~;(]d:4S}

Fig. 2. D i n i t r o p h e n y l a t i o n o f [ 3 H ] i o d o a c e t y l - I g G . T r i t i a t e d ( I C H 2 C O ) 1 2 . 1 - I g G (3 mg) a n d ~-DNP-e-SH-lysine (1.2 mg) were m i x e d (0.64 ml) and stirred u n d e r N2 at 22°C. At t h e i n d i c a t e d times, a l i q u o t s were passed t h r o u g h S e p h a d e x G-25 a n d the degree of dinit r o p h e n y l a t i o n was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y , o, i o d o a c e t y l a t e d IgG + a-DNPc-Stt-lysine; m, u n m o d i f i e d IgG + ~-DNP-e-SH-lysine. Fig. 3. M e r c a p t o s u c c i n y l a t i o n o f OA. T o a 2.0 ml s o l u t i o n of OA (58 mg) in P B S - - E D T A , t h e a p p r o p r i a t e q u a n t i t y o f S A M S A dissolved in 0.05 ml d i o x a n e was a d d e d with stirring u n d e r N2. T h e pH was m a i n t a i n e d a t 8.5 w i t h 1 N N a O l ! for 45 m i n a n d t h e O A - - S A M S A isolated a n d c h a r a c t e r i z e d as d e s c r i b e d in Materials a n d M e t h o d s .

329 approaches its m axi m um value within 2 h, with little change thereafter. F u r t h e r m o r e , the m axi m um degree of di ni t rophenyl at i on achieved (DNPI~.7-1gG) was essentially equal to the degree of iodoacetylation of the IgG [(1CH2CO)12.1IgG]. Control experiments which involved mixing normal IgG with ~-DNP-e-SH-lysine did n o t result in any significant degree of dinit r o p h e n y l a t i o n (DNP0. ~0-IgG).

Mercaptosuccinylation o f OA It has previously been dem ons t r a t e d (Klotz and tteiney, 1962) that the degree o f protein mercaptosuccinylation using SAMSA was d e p e n d e n t on (a) the protein ano (b) the a m o u n t of SAMSA added to a constant a m o u n t of protein. The results presented in Fig. 3 describe the conditions whereby a low n u mb er of -SH groups could be attached to OA. It can be seen that the degree of mercaptosuccinylation of OA was directly proportional (r = 0.96) to the SAMSA/OA molar ratio. When a molar ratio of 5 was used, the population of OA molecules after deacetylation with NH2OH exhibited, on the average, 0.7 -SH groups/OA molecule. Although in t h e o r y one -SH group per molecule is required for conjugation, a degree of mercaptosuccinylation less than this was routinely used in order to favour the production of derivatives of OA having not more than one -SH group per OA molecule. Conjugation o f OA to IgG In order to d em ons t r a t e protein-protein coupling using the reaction p ro to co l described here, 3 reaction parameters were investigated. (1) Reaction time. [3H]OA-SAMSA and iodoacetylated IgG (molar ratio OA/IgG = 4.6) were mixed and the conjugation initiated by the addition of NH2OH. After stirring for either 3 h or 20 h under N2, the reaction solution was applied on Sephadex G-200 (1.3 cm :~' 176 cm). The elution profiles are shown in Fig. 4a and b. The salient features of Fig. 4a are: (a) The A2s0 profile consisted of two p r o m i n e n t peaks which were assigned to native IgG and OA, {corresponding to elution volumes of 133 and 179 ml, respectively). (b) A significant a m o u n t of ~tl-labelled protein was eluted between the void volume and the IgG peak indicating the presence of [3H]OA-IgG conjugates. The 3tI-profile in this region indicated the presence of at least two components. The first c o m p o n e n t (F~) eluted in the void volume was assigned a molecular weight ~ 4 0 0 , 0 0 0 . The OA/lgG ratio for this c o m p o n e n t was calculated as 1.1. On the basis of these two criteria, this c o m p o n e n t was assigned the composition o f (OA~-IgG)y, with y > 1. The second and major c o m p o n e n t eluted at fraction 121 with an apparent molecular weight of 215,000 and its OA/IgG ratio was calculated as 1.0; consequently, this c o m p o n e n t was designated OA~-IgG {theoretical M.W. = 195,000). (c) From the total a m o u n t of [31t]OA emerging within the elution volume of 100-* 135 ml, and taking an

330 IgG

Vc

OA,-I~S /

o.3-®

A 1.~

_

OA-O,~

tO

/

OA

i \

..-.... -

g 2 Lu

o '~,

_

0.1

x

o~® 02

0

~:

l 0

1.O

r

'

1OO

,~

120

",IC v O I UM~

16k; {n,}

180

•

200

Fig. 4. E|ution profiles of reaction products involving the conjugation of OA to IgG using reaction times of (a) 3 h and (b) 20 h. [3H1OA-SAMSA0.; (7 rag, 8.95 x 10 ~ cpm/mg) and (IClt2CO)lcjlgd (5 rag) were stirred at 22~C und~,r N 2 in the presence of 0.0l M

NIl:Oft (total reaction volume = 1.57 ml PBS--EDTA). A Sephadex G-200 column (1.3cm x 176 cm, PBS--EDTA) was used and 1 ml fractions collected. -- .... , A:s,; . . . . . . , 3H cpm/0.05 ml.

overall O A / I g G ratio o f 1, it w~s d e d u c e d t h a t 4 5 - - 5 0 ~ o f the IgG had been c o n j u g a t e d to OA during the 3 h r e a c t i o n time. Fig. 4 b illustrates the elution profiles o f c o n j u g a t e s o b t a i n e d w h e n the r e a c t i o n t i m e was increased f r o m 3 h to 20 h. T h e a b s o r b a n c e profile d e f i n e d t w o f r a c t i o n s o f high m o l e c u l a r weight d e s i g n a t e d as F1 and F2. It was concluded that, as in Fig. 4a, the m a i n c o n j u g a t i o n p r o d u c t s were (OA~-IgG),,, y > 1 (F1) and O A , - I g G (121 ml). T h e p r o m i n e n t IgG f r a c t i o n p r e s e n t a f t e r o n l y 3 h r e a c t i o n t i m e (Fig. 4a) was replaced by the m a j o r f r a c t i o n F2, which eluted with an a p p a r e n t m o l e c u l a r weight ranging b e t w e e n IgG and OA~-IgG. Since the a b s o r b a n c e and r a d i o a c t i v i t y profiles were n o t c o i n c i d e n t in F2, s o m e native IgG was likely present. Q u a n t i t a t i o n o f the total a m o u n t o f [~H]OA in F~ a n d F2 i n d i c a t e d t h a t the longer r e a c t i o n t i m e had resulted in a 1 . 5 - - 2 - f o l d increase in the yield o f lgG c o n j u g a t e s ; a c c o r d i n g l y , a 20 h r e a c t i o n t i m e was a d o p t e d in s u b s e q u e n t e x p e r i m e n t s . (2) Molar ratio o f reactants. In o r d e r to d e t e r m i n e the e x t e n t to which the s u b s t i t u t i o n r a t i o was d e p e n d e n t on the m o l a r ratio o f the r e a c t a n t s , experim e n t s were c o n d u c t e d in which increasing a m o u n t s o f m e r c a p t o s u c c i n y l a t e d OA were r e a c t e d with a fixed a m o u n t o f (ICHeCO)~olgG. T h e elution profiles f r o m t w o such e x p e r i m e n t s are p r e s e n t e d in Fig. 5a and b. It can be

331 OA,- IgG VoOAi:~G I IgG

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120

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200

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Fig. 5. Conjugation of OA to IgG. Five mg (ICH2CO)101gG were reacted with [3H]OASAMSA0. 8 in the presence of 0.01 M NH2OH (total volume = 1.10 ml). Following 20 h incubation at 22°C under N2, the reaction solutions were applied to Sephadex G-200 (1.5 cm × 172 cm, PBS--EDTA), and 1.5 ml fractions collected, a: 14.5 mg [3H]OASAMSA (9.62× l0 s cpm/mg); OA/IgG=9.7. b: 21.0 mg [3H]OA-SAMSA (1.05× 106 cpm/mg); OA/IgG = 14.0. - - , A2s0; . . . . . . ,3H cpm/0.05 ml.

seen t h a t based on the a b s o r b a n c e profiles, t h e O A - I g G c o n j u g a t e s were resolved i n t o t w o m a i n f r a c t i o n s , FI and F~. F r a c t i o n F~ eluted with t h e void v o l u m e o f t h e c o l u m n {M.W. ~> 4 0 0 , 0 0 0 ) , w h e r e a s F2, w h i c h a c c o u n t e d f o r t h e b u l k o f the c o n j u g a t e d material, eluted with an a p p a r e n t m o l e c u l a r w e i g h t o f 1 9 5 , 0 0 0 (OAL0-IgG). A l t h o u g h F2 a p p e a r e d t o consist p r e d o m i n a n t l y o f OA~-IgG, m o l e c u l a r h e t e r o g e n e i t y was e v i d e n t w i t h i n this f r a c t i o n since t h e a b s o r b a n c e a n d r a d i o a c t i v i t y profiles w e r e n o t c o i n c i d e n t . T h e subs t i t u t i o n ratios calculated a t 3 d i f f e r e n t p o i n t s across F2 varied f r o m 1.3 to 2.1 (Fig. 5a) and 1.3--2.4 {Fig. 5b); t h e r e f o r e , it was c o n c l u d e d t h a t F~ was a m i x t u r e o f OA~-IgG and OA2-IgG. T h e r a d i o a c t i v i t y profile in this region p e a k e d at 132 ml, w h i c h i n d i c a t e d the e l u t i o n o f a m o l e c u l a r species with an a p p a r e n t m o l e c u l a r weight, based on gel filtration, o f 2 3 8 , 0 0 0 ( t h e o r e t i c a l OA2-IgG = 240,000}. F r o m these results it was c o n c l u d e d t h a t t h e maximum s u b s t i t u t i o n r a t i o which c o u l d be o b t a i n e d u n d e r these c o n j u g a t i o n conditions was 2, and t h a t this value c o u l d n o t be significantly increased b y increasing the m o l a r r a t i o o f r e a c t a n t s f r o m 9.7 (Fig. 5a) t o 14.0 {Fig. 5b). T h e s e results are s u m m a r i z e d in T a b l e 2. It was evident, t h e r e f o r e , t h a t o f .the 10 i o d o a c e t y l g r o u p s p r e s e n t on the IgG m o l e c u l e , o n l y t w o w e r e a p p a r e n t l y available for r e a c t i o n w i t h m e r c a p t o s u c c i n y l a t e d OA. (3) Degree of IgG iodoacetylation. Since t h e results in the p r e v i o u s s e c t i o n

332 TABLE 2

MAXIMUM SUBSTITUTION RATIO OF OA x-IgG Figure

,tb 5a 51) 6

Molar ratio of reactants (OA/IgG)

Degree of iodoacetylation of Ig(;

Elution volume (ml)

Gel filtration

Specific radioactivity

,t.6 9.7 1,1.0 9.7

10 10 10 20

1 21 1 32 t 32 11 2

1.4 2.0 2.0 5.3

1.0 2.1 2.4 3.7

Substitution ratio (x) a

a Substitution ratio determinect either in terms of the elution volume on gel filtration or by specific radioactivity at the elution volumes indicated.

suggested t h a t o n l y a very limited n u m b e r o f i o d o a c e t y l g r o u p s were available for r e a c t i o n , the degree o f lgG i o d o a c e t y l a t i o n was increased in o r d e r to increase the n u m b e r of such reactiw~ g r o u p s and t h e r e b y increase the subs t i t u t i o n ratio. A c c o r d i n g l y , the n u m b e r o f i o d o a c e t y l g r o u p s was increased f r o m 10 to 20 per lgG m o l e c u l e and this p r e p a r a t i o n r e a c t e d with m e r c a p t o s u c c i n y l a t e d OA. S u b s e q u e n t l y , the reaction m i x t u r e was applied to a S e p h a r o s e - 6 B c o l u m n . T h e r e s u l t a n t elution profiles are p r e s e n t e d in Fig. 6. It can be seen t h a t the c o n j u g a t e d material eluted p r i m a r i l y as a single fraction F2, with an a p p a r e n t m o l e c u l a r weight o f 3 9 0 , 0 0 0 , i.e. OAr,.:~-IgG (basc
Immunodiffusion and immunoelectrophoretic analysis of OA-IgG conjugates F r o m the analysis o f the i m m u n o d i f f u s i o n p a t t e r n s o b s e r v e d by reacting an O A - I g G c o n j u g a t e ( O A 3 . s - l g G ) with r a b b i t a n t i b o d i e s to OA and IgG, which are illustrated in Fig. 8a, it m a y be c o n c l u d e d t h a t the c o n j u g a t e consisted o f a m o l e c u l a r species which possessed antigenic d e t e r m i n a n t s of

333

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Fig. 6. C o n j u g a t i o n o f OA to IgG. Five mg (ICII2CO)2(}IgG were reacted with 14.5 mg [ 3 H ] O A - S A M S A 0 . 6 (7.36 x 10 s c p m / m g ) in the p r e s e n c e o f 0.01 M Ntt2OH (total v o l u m e = 1.10 ml). F o l l o w i n g 20 h i n c u b a t i o n at 22°C u n d e r N2, the s o l u t i o n was applied to Sepharose-6B (1.5 c m x 110 cm, P B S - - E D T A ) , and 1.5 ml fractions collected. A2~{}; . . . . . . , 3ti c p m / 0 . 0 5 ml. Fig. 7. R e l a t i o n s h i p b e t w e e n the conjugate s u b s t i t u t i o n ratio x and the degree o f iodoa c e t y l a t i o n o f IgG, n. M e r c a p t o s u c c i n y l a t e d OA and i o d o a c e t y l a t e d IgG were reacted at 22°C for 20 h (molar ratio o f reactants OA/IgG = 9.7) and the resulting OAx-IgG conjugates c h a r a c t e r i z e d by gel filtration. The r e l a t i o n s h i p b e t w e e n x and n was d e f i n e d as: x = 0 . 4 3 n -- 1.35 (correlation c o e f f i c i e n t r = 0.91).

O

Fig. 8. a: i m m u n o d i f f u s i o n analysis o f OAa.5-IgG conjugate. C e n t e r well: OA-IgG conjugate; wells 1, 4: rabbit anti-IgG 7-globulins; wells 2, 5: rabbit anti-OA 7-globulins; well 3: r a b b i t anti-OA + r a b b i t anti-IgG 7-globulins; well 6: 0.9% saline, b: i m m u n o e l e c t r o p h o r e t i c analysis o f OAa.5-IgG conjugate. T o p well: OA; c e n t e r well: OA-IgG conjugate; b o t t o m well: IgG. The h o r i z o n t a l t r o u g h s were filled with rabbit anti-OA ~-globulins ( t o p ) and rabbit anti-IgG ")'-globulins ( b o t t o m ) .

334 both OA and IgG. Moreover, as would be expected and as illustrated by the immunoelectrophoresis patterns shown in Fig. 8b, the electrophoretic migration of the conjugate was slower than that of OA but faster than that of IgG. DISCUSSION lgE responses to haptenic determinants were suppressed by conjugates of haptens with isologous "),-globulins in mice (l,ee and Sehon, 1975, 1976; Borel et al. 1976b), rats (Pan et al., 1976) and dogs (Tse et al., 1978}. The ability of such hapten-lgG conjugates to induce tolerance appears to be related to the subclass of lgG used and to depend on the integrity of the Fc region, thereby suggesting the; importance of the Fc portion of the molecule (Borel et al., 1976a; Pan et al., 1976). Furthermore, conjugates with high epitope densities (i.e. 15) do not function as tolerogens, presumably due to profound structural alterations caused by the high degree of substitution, which may involve rendering the Fc region of tile lgG inaccessible to the Fc receptor on the i m m u n o c o m p e t e n t cell which is to be tolerized (l,ee and Sehon, 1976). In order to extend this system to the induction of tolerance to protein antigens, a conjugation procedure was required which was able to produce well-defined conjugates of small peptides or proteins and IgG. The ability to cross-link proteins using bifunctional reagents has been known for some time (Likhite mid Sehon, 1967), however, most of these reagents react with groups which are usually present in large numbers on both proteins to be coupled (e.g. amino, tyrosyl) and, therefore, the reaction leads to a mixture.' of ill-defined intermolecular conjugates as well as to intramolecularly crosslinked products. Hence, this study was undertaken with a view to incorporating, on both the antigen and IgG, a limited number of reactive groups not normally found on these proteins. This limited number would provide the basis for a controlled conjugation reaction. The coupling procedure described here utilizes the well-known reaction of alkyl halides and sulfhydryl compounds. This reaction has been used previously for the attachment of small haptenic determinants to proteins. For example, the iodinated hapten p-(p-iodoacetylaminobenzeneazo) hippuric acid was conjugated to thiolated human serum albumin (Marks, 1967). Similarly, steroids such as progesterone and testosterone haw.' been coupled to bovine serum albumin by reaction of their 6-13-bromide derivatives with thiolated BSA (Pang and Johnson, 1974). For the synthesis of protein-IgG conjugates described here, iodoaeetylated IgG was reacted with thiolated protein. It was decided to introduce the iodoacetyl groups into IgG since (a) IgG does not contain free sulfhydryl residues and (b) some antigens or allergens may have free sulfhydryl groups intrinsic to their structure and, therefore, could be reacted with iodoacetylated IgG directly. As shown in Fig. 1, iodoacetyl groups were incorporated into IgG with NtIIA. Under the conditions used, the extent of iodoacetylation, as deter-

335 mined by the incorporation of ~H, was shown to be dependent on the a m o u n t of [3H]NHIA added. Since the ~H label did not constitute the actual reactive moiety (-I), it was important to demonstrate a correlation between 3H incorporation and the number of iodide groups present. This was accomplished by reaction of the iodo groups with a-DNP-e-SH-lysine. As shown in Fig. 2 a direct correlation was obtained between the number of iodide groups calcula~d to be grafted onto the IgG molecule and the number of DNP residues attached to IgG. This result demonstrated that essentially "all the iodo groups incorporated into IgG were (a) intact and (b) available for reaction with the low molecular weight thiolated hapten. Bromoacetyl derivatives of DNP hapten have been used for affinity labelling of the antibody combining sites of anti-DNP antibodies (tlaimovich et al., 1970). This type of labelling involved reaction of the bromoacetyl group with either the nucleophilic tyrosyl (-O-) or lysyl (-NH2) residues at a plt close to the pK of these residues (pH 9). Conceivably similar reactions could occur internally within the iodoacetylated IgG preparations described here, resulting in extensive inter- and intramolecular cross-linking of the IgG. However, at the pH used here (pH 7.5) for the preparation and conjugation of iodoacetylated IgG, the corresponding tyrosyl (-OH) and lysyl (-NH3 ~) residues are not sufficiently nucleophilic to displace the halide residue.. Moreover, in the presence of sulfhydryl groups (pK ~ 7) at pH 7.5, the reaction of alkyl halides with other amino acid residues can normally be considered insignificant (Means and Feeney, 1971}. Consequently, cross-linking of iodoacetylated IgG does not occur to any significant degree under the conditions described in this report. The introduction of sulfhydryl groups into proteins using SAMSA was first demonstrated by Klotz and Heiney (Klotz and Heiney, 1959, 1962). The extent of thiolation was shown to be related to the ratio of SAMSA to -NH: groups of the protein. As shown in Fig. 3, the number of -Stl groups introduced into OA could be varied in this manner. The procedure could be used to produce thiolated OA with an average value of 1 or less than 1 -SH group per OA molecule. The importance of rendering the tigand molecule (i.e. allergenic molecule) chemically monofunctional by the introduction of only 1 -Stl group was demonstrated in Figs. 4, 5 and 6 by the generation of high molecular weight (OAx-IgG)y (y > 1) conjugates. The formation of these conjugates was likely due to the reaction of OA molecules bearing two or more -SH groups. In general, the yield of such conjugates was relatively low and would probably be further reduced if lower degrees of OA mercaptosuccinylation were used. The results in 'Fable 2 and Fig. 7 demonstrate that the maximum substitution ratios obtained in the OA-lgG conjugates were consistently lower than the degree of IgG iodoacetylation. In fact, only 25--40% of the iodide groups were reactive with mercaptosuccinylated OA. Since virtually all the iodoacetyl groups were reactive with the small thiolated DNP hapten {Fig. 2), sterie hindrance is judged to be an important factor when coupling

336 a m u c h larger molecule, such as OA, to IgG. The possibility o f e x t e n d i n g the i o d o a c e t y l groups from the IgG m o l e c u l e and t h e r e b y increasing their accessibility is presently being considered. The biological activity o f IgG c o n j u g a t e s is u n d e r active study. Preliminary e x p e r i m e n t s indicate t h a t DNP-IgG c o n j u g a t e s prepared by the m e t h o d described here can specifically inhibit IgE anti-DNP responses in mice, thus d e m o n s t r a t i n g the applicability o f this p r o c e d u r e to the p r e p a r a t i o n of tolerogenic h a p t e n - l g G conjugates. This system is being e x t e n d e d to include the synthesis o f tailor-made c o n j u g a t e s o f distinct c o m p o s i t i o n consisting o f varying n u m b e r s o f allergenic molecules c o u p l e d per lgG molecule. ACKNOWLEDGEMENT This w o r k was s u p p o r t e d by grants f r o m the Medical Research Council of Canada. REFERENCES Borel, Y., D.T. Golan, L. Kilham and H. Borel, 1976a, J. Immunol. 116, 854. Borel, Y., L. Kilham, N. Hyslop and It. Borel, 1976b, Nature 261, 50. Eisen, H.N., M.E. Carsten and S. Belman, 1954, J. Immunol. 73, 296. Golan, D.T. and Y. Borel, 1971, J. Exp. Med. 134, 1046. Grassetti, D.R. and J.F. Murray, Jr., 1967, Arch. Biochem. Biophys. 119, .tl. Haimovich, J., D. Givol and H.N. Eisen, 1970, Proc. Natl. Acad. Sci. U.S.A. 67, 1656. Klotz, I.M. and R.E. Iteiney, 1959, J. Amer. Chem. Soc. 81,3802. Klotz, I.M. and R.E. lteiney, 1962, Arch. Biochem. Biophys. 96, 605. Lee, W.Y. and A.It. Sehon, 1975, J. Immunol. 114, 829. Lee, W.Y. and A.H. Sehon, 1976, J. Immunol. 117,927. Likhite, V. and A.tt. Sehon, 1967, in: Methods in Immunology and Immunochemistry, Vol. 1, eds. C.A. Williams and M.W. Chase (Academic Press, New York) p. 150. Marks, R., 1967, in : Methods in Immunology and Immunochemistry, Vol. 1, eds. C.A. Williams and M.W. Chase (Academic Press, New York) p. 126. Means, G.E. and R.E. Feeney, 1971, Chemical Modification of Proteins (Holden-Day, San Francisco, CA) p. 105. Pan, D., W.Y. Lee, A.H. Sehon and It. Bazin, 1976, Specific suppression of reaginic antibodies in rats. 60th Annual Meeting of the Federation of the American Society of Experimental Biologists, Anaheim, CA (Abstract). Pang, C.N. and D.C. Johnson, 1974, Steroids 23, 203. Segal, D.M. and E. Hurwitz, 1976, Biochemistry 15, 5253. Tse, K.S., W. Kepron, and A.H. Sehon, 1978, J. Allergy Clin. Immunol. 61, 303.