Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, 1 Department of Biochemistry, Kyushu University School of Dentistry, Fukuoka 812-8582 and 2 Department of Preventive Dentistry and Kagoshima University Dental School, Kagoshima 890-8544, Japan
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Abstract |
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Keywords: chemical modification/immunological tolerance/monomethoxypolyethylene glycol/neutralizing antibody/protein pharmaceutics
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Introduction |
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mPEG proteins have also been reported to act as an effective tolerogen, i.e. injection of adult animals with a mPEG conjugate of a foreign protein renders them immunologically unresponsive to a subsequent injection of the native form (Lee and Sehon, 1977; Sehon, 1991
; Atassi et al., 1992
). In this respect, foreign proteins can be administered efficiently without evoking immune responses: the tolerogenic character enables us to make the best use of the activities of foreign proteins and should be advantageous in protein pharmaceutics. We have previously reported the effective tolerogenicity of mPEGtype II collagen and mPEGhen egg lysozyme (HEL) in both T-helper type 1 (Th1) and T helper type 2 (Th2) immune responses (Ito et al., 1997
, 1998
).
A large number of studies have been made on the usefulness of mPEG proteins; however, few reports ever have been intended to apply the immunotolerogenicity to protein pharmaceutics and, as a consequence, there is very little information available on the preparation of tolerogenic mPEG proteins. The main reason may be that the relationship between the degree of pegylation and the tolerogenicity has not been strictly addressed, despite the important association that has been suggested (Sehon, 1991).
To clarify this issue, we selected three distinct proteinsHEL, ovalbumin (OVA) and bovine gamma globulin (BGG)which were differentially conjugated with cyanuric chloride-activated mPEG, which has been used in many studies for evaluating the immunotolerogenicity of mPEG proteins. We found that the most tolerogenic conjugates possessed 1.52.0-fold the molecular weight (m.w.) of mPEG against that of protein. Higher or lower amounts were found to be less effective. The results suggest that it is necessary to introduce a constant amount of mPEG molecules into a protein in order to prepare tolerogenic conjugates and the value originally obtained in this study may provide practical information for the manufacture of mPEGprotein conjugates.
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Materials and methods |
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BALB/c mice were obtained from the Center of Biomedical Research, Kyushu University. The mice were immunized at age 812 weeks.
Antigens
Five times re-crystallized HEL was kindly donated by QP Co. (Tokyo, Japan), and five times re-crystallized OVA was purchased from Seikagaku Co. (Tokyo, Japan). BGG (No. G7516) was purchased from Sigma Chemical Co. (St Louis, MO). A purified protein derivative of Mycobacterium tuberculosis H37Ra (PPD) was purchased from Kainosu, Inc. (Tokyo, Japan).
Preparation of mPEG-conjugated proteins
mPEG (average mol. wt 5 kDa) was purchased from Aldrich Chemical Co. (Milwaukee, WI) and mPEG (average mol. wt 12 kDa) was kindly donated by Nippon Oil and Fats Co. (Tokyo, Japan). Cyanuric chloride, obtained from Sigma, was re-crystallized twice with benzene before use. Activated mPEG2, 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine, was synthesized according to Ono et al. (1991). Briefly, 30 g mPEG was dissolved in 150 ml of anhydrous benzene. The solution was refluxed at 80°C in the presence of 7 g molecular sieves 4A for 6 h. After cooling, 15 g zinc oxide and cyanuric chloride (a half equimolar amount of mPEG) were added. After refluxing at 80°C for 60 h, the resultant mixture was cooled to room temperature, diluted with 100 ml benzene and filtered. The filtrate was mixed with 250 ml anhydrous petroleum ether, and the precipitate was collected on a sintered glass filter, followed by re-dissolution in 100 ml anhydrous benzene and re-precipitated with petroleum ether. The product was applied to a Sephadex LH-60 column (3x130 cm) (Pharmacia, Uppsala, Sweden) and eluted with methanol. The absorbance at 254 nm of the eluate was monitored and the first peak was collected, followed by evaporation of the methanol. mPEGHEL, mPEGOVA and mPEGBGG were prepared as described (So et al., 1996a). Unreacted mPEG molecules and protein were removed by a Sephacryl S-100 column (Pharmacia, column size: 2.6x60 cm) equilibrated with 0.1 M NH4HCO3 and fractions of the mPEG proteins were exhaustively dialyzed against saline. The protein concentration was determined by amino acid analysis and the degree of mPEG introduction was determined by measuring the amount of free amino groups in a protein which are unreacted with activated mPEG2, using trinitrobenzenesulfonate (Habeeb, 1966
). mPEG24kDaHEL was prepared by conjugating HEL with activated mPEG2 (average mol. wt 24 kDa), and molecules in which one mPEG molecule had been introduced into the amino group of HEL was purified using a Sephacryl S-100 column and was used in this study.
Tolerance induction and immunization
Mice were given a single intraperitoneal (i.p.) injection of either a native protein or a mPEG protein 7 days prior to the challenge immunization on day 0. Control mice were injected with saline. The mice were challenge immunized intracutaneously in both hind foot pads with the native protein in a 0.1 ml emulsion of complete Freund's adjuvant (CFA, Difco Laboratories, Detroit, MI).
Measurement of immune responses in tolerized mice
Mice were killed 9 days after the immunization and draining lymph node cells were obtained. Cultures for the lymph node T cell proliferation assay were set up as previously described (Ito et al., 1998), and the proliferation was measured after 96 h using a colorimetric assay based on the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), as previously described (So et al., 1996a
). Sera were obtained from mice after the antigen administration and antigen-specific IgG titers were determined using enzyme-linked immunosorbent assay (ELISA), as described elsewhere (Ito et al., 1997
, 1998
).
Measurement of antibody binding capacity of mPEG conjugates
Antibody binding capacities of mPEGprotein conjugates were determined using competitive ELISA, as described elsewhere (So et al., 1997).
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Results |
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To clarify the effect of the extent of pegylation on the tolerogenic capacity of mPEG proteins, three different proteinsHEL, OVA, and BGGwere reacted with activated mPEG2 (average mol.wt 10 kDa). A series of mPEG proteins with differential mPEG contents were prepared. Each mPEG protein was i.p. administered 7 days prior to immunization with the unconjugated protein in the presence of CFA. Tolerogenic activities of the conjugates were evaluated by lymph node T cell proliferation in vitro, in response to respective native proteins and PPD (Figure 1, left) and by serum IgG levels to respective native proteins (Figure 1
, right).
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Similarly, native OVA did not tolerize antibody responses, and mPEGOVA, with a modification level of 36%, showed the optimum tolerogenicity (Figure 1, center). On the other hand, native BGG tolerized both T cell and antibody responses (Figure 1
, bottom) indicating the naturally tolerogenic character of
-globulins, as previously documented (Weigle, 1980
). However, as the extent of pegylation increased, the tolerogenic activity of mPEGBGG was augmented. We could not prepare a more heavily pegylated BGG than 33% because precipitation occurred during certain steps of the preparation.
The states of tolerance observed in this study were antigen-specific, as described elsewhere (Ito et al., 1997, 1998
); mPEG proteins were not toxic (So et al., 1996a
); and the covalent association of mPEG with the protein was essential for tolerogenicity (Ito et al., 1997
, 1998
). Hence, these results indicate that the extent of pegylation determines the tolerogenicity of mPEG proteins.
Amount of mPEG molecules in a tolerogenic mPEGprotein conjugate
We then searched for an underlying principle in tolerogenic conjugates, based on the results in Figure 1. The most tolerogenic activity of mPEGHEL was detected between 30 and 40% pegylation, as described above. As one HEL molecule possesses 7 amino groups, the tolerogenic conjugate could be calculated to be 2.12.8 mPEG molecules per one HEL molecule. Then, as the average molecular weight of the mPEG molecule was 10 kDa, we also estimated that 2128 kDa of mPEG per one HEL was introduced. Hence, the molecular weight ratio of mPEG to HEL was calculated to be 1.5 to 2.0 (Table I
). Similarly, in OVA and BGG, the most tolerogenic conjugates in this study were observed in the case of 36 and 33% pegylation, respectively, and the ratios were both 1.7. These findings suggest that the introduction of 1.52.0-fold the molecular weight of mPEG against that of protein results in optimal tolerogenicity.
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Reactivity of tolerogenic mPEGprotein conjugates to T cells and antibodies
Introduction of mPEG molecules into protein antigens results in a reduction in antigenicity against antigen-specific T cells and antibodies (Hershfield et al., 1991; So et al., 1996a
). However, if the pegylation nullifies the availability of T cell and B cell epitopes, mPEG proteins will be ignored by specific T and B cells and the tolerant state may not be established. To clarify the immunoreactivity of the tolerogenic mPEG protein, we measured their T cell stimulating capacity. As illustrated in Figure 2A
, HEL-specific T cell proliferative response of lymph node cells was reduced as pegylation increased, and the most tolerogenic derivative, 30% pegylated HEL, had a levelof T cell stimulating activity 1/400 that of native HEL. Similarly, in case of tolerogenic mPEGOVA (36%) and mPEG-BGG (33%) conjugates, T cell stimulating capacities were decreased at the level of 1/100 and 1/1000 that of their respective unconjugated forms (Figure 2B and C
).
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Discussion |
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Surface modification of a protein with mPEGs reduces the antibody binding capacity and the T cell stimulating capacity of the protein, as shown in Figures 2 and 3. The reduced T cell stimulating capacity is caused by the depressed generation of immunogenic peptides in antigen presenting cells due to their lowered susceptibility to processing enzymes (So et al., 1996a
), and the reduced recognition of pegylated proteins by antibodies is attributed to epitope shielding by the introduced mPEG molecules (Hershfield et al., 1991
). Thus, as the extent of pegylation increased, the heavily pegylated protein is ignored by antigen-specific T and B cells and tolerogenicity may decrease. On the contrary, as the extent of pegylation increased, the blood half-life of proteins is extended, owing to a reduced intracellular uptake, proteolysis and renal filtration (Francis et al., 1992
). Concentration of the circulating antigen is a key parameter controling tolerance states (Goodnow et al., 1989
; Cibotti et al., 1992
). Therefore, the extent of pegylation may positively regulate the tolerogenicity of the mPEG protein. These opposing factors in tolerance induction, i.e. immunoreactivity and in vivo half-life, negate each other and this may explain why a regular amount of mPEG molecules is required to obtain optimal tolerogenicity in the mPEGprotein conjugate, and why higher or lower ratios were less effective in inducing tolerance (Figure 1
).
Another important finding is that the mildly pegylated molecules possessed the adjuvanticity in antibody responses, i.e. mPEGHEL (14%) and mPEGOVA (17%), as demonstrated in Figure 1. A similar result was obtained when HEL was mildly pegylated with a low molecular weight of mPEG (data not shown). Brumeanu et al. (1995) reported that mildly pegylated IgG was more potent in inducing in vivo cellular and humoral immune responses and able to elicit immune response without requirement for adjuvant. Hence, it must be noted that the process of mild pegylation of proteins may have a potential to enhance the antibody response against the protein moiety and one would be careful to prepare therapeutic mPEG proteins with consideration of the extent of pegylation.
We previously evaluated the surface area of a mPEG molecule introduced onto HEL and estimated that one mPEG (average mol. wt 5 kDa) covered about 15% of the HEL surface (So et al., 1996b). If this result can be applied simply to the tolerogenic mPEGHEL in this study, 6080% of the surface of HEL should be covered by mPEG molecules. The shielding effect of mPEG effects leads to depression of antigenic peptides for T cells by reducing the susceptibility of mPEG proteins against processing enzymes (Figure 2
; So et al., 1996a). Recent studies demonstrate that inadequate antigenic stimuli lead specific T cells to the tolerant states (Kersh et al., 1998
). Thus, the generation of depressed antigenic peptides by the shielding effect of mPEG might be a possible mechanism for the tolerance in mPEG proteins.
Recent developments in genetic engineering make it possible to create more functional sites for pegylation, such as cysteine or lysine residues, with site-directed mutagenesis (Hershfield et al., 1991; Kuan et al., 1994
). With the aid of structural data from X-ray crystallography or NMR spectrometry, we can select the appropriate pegylation sites unrelated to the desired functions of proteins. The combining of this structural information with our results allows the preparation of potent mPEG protein drugs possessing both activity and immunotolerogenicity. Hence, our results may help to tolerize undesired antibody responses against drugs, e.g. interferons (Itri et al., 1989
) and adenoviral vectors for gene therapy (Yeh et al., 1996
).
In conclusion, this report is apparently the first to show the critical correlation between the molecular weight ratio (mPEG/protein) and the immunotolerogenicity in mPEG proteins. Protein pegylation is now a powerful strategy in the enhancement of protein activity in vivo. The information that the tolerogenic mPEG protein is conjugated 1.52.0-fold the molecular weight of mPEGs against that of protein may provide a practical index for manufacturing tolerogenic mPEG proteins and will increase further the usefulness of mPEG proteins in therapeutics.
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Acknowledgments |
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Notes |
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References |
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Received March 29, 1999; revised April 23, 1999; accepted April 28, 1999.