Immunological Properties of Uricase Conjugated to Neutral Soluble

Bioconjugate Chem. 2001, 12, 515−522

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Immunological Properties of Uricase Conjugated to Neutral Soluble Polymers Paolo Caliceti,* Oddone Schiavon, and Francesco M. Veronese Department of Pharmaceutical Sciences, University of Padua, Via F. Marzolo, 5 Padova, Italy. Received September 25, 2000

For a comparative study of immunological properties of protein-polymer conjugates, uricase was modified with (a) poly(N-vinylpyrrolidone) 6000 Da, (b) poly(N-acriloylmorpholine) 6000 Da, (c) branched monomethoxypoly(ethylene glycol) 10000 Da, and (d) linear monomethoxypoly(ethylene glycol) 5000 Da. Spectroscopic studies performed by UV, fluorescence, and circular dichroism did not show any relevant difference in protein conformation among the native and the conjugates. Immunological studies showed that both uricase antigenicity and immunogenicity were altered by polymer conjugation to an extent that depended upon the polymer composition; in particular, monomethoxypoly(ethylene glycol) 10000 Da remarkably reduced the protein antigenicity, while unexpectedly, the poly(N-vinylpyrrolidone) derivative presented higher antigenicity than the native protein. In Balb/c mice, the native protein elicited a rapid and intense immunoresponse whereas all the conjugates induced a lower production of anti-native uricase antibodies. The rank order of immunogenicity was native uricase > uricase-poly(N-vinylpyrrolidone) g uricase-poly(N-acriloylmorpholine) > uricase-monomethoxypoly(ethylene glycol) 5000 Da > uricase-monomethoxypoly(ethylene glycol) 10000 Da. The four conjugates also induced anti polymer immunoresponse. Anti poly(N-vinylpyrrolidone) and anti poly(N-acriloylmorpholine) antibodies were generated from the first immunization while low levels of anti polymer antibodies were found with both poly(ethylene glycol) conjugates only after the second immunization.

INTRODUCTION

Polymer conjugation has so far been successfully used to enhance the therapeutic potential of many pharmacologically active proteins and peptides since it allows for the alteration of their physicochemical and biological properties improving permanence in circulation, stability, and solubility and reducing immunogenicity (1-3). Immunological properties of conjugates are of primary importance for their therapeutic application, but a number of items should be taken into consideration regarding both the conjugate components: polymer and protein (4). Although absence of immunogenicity is a general prerequisite for the polymers used in drug modification, some of them were shown to present inherent immunogenic character because of their repetitive structure or high molecular weight. Examples are polyamino acids, polyacryloil peptides, and polysaccharides (5, 6). Furthermore, in some cases, conjugation can provoke a dramatic change of polymer immunological properties. Low or nonimmunogenic macromolecules can in fact acquire haptogenic character by conjugation with immunogenic compounds. Poly(hydroxyethylstarch), poly(lysine), poly(ornithine), and dextrans, for example, were found to gain relevant antigenicity and immunogenicity after coupling with proteins and peptides (7-9). Also, poly(hydroxyethylacrylate), a polymer largely investigated for drug conjugation, was found to acquire unexpected immunogenicity after binding to oligopeptides, and anti poly(ethylene glycol) antibodies were generated using PEG* To whom correspondence should be addressed. Phone: 0039 049 8275695. Fax: 0039 049 8275366. E-mail: caliceti@ dsfarm.unipd.it.

superoxide dismutase and PEG-ovoalbumin although this polymer is nonimmunogenic when administered alone (10, 11). As far as the protein side is concerned, reduction of immungenicity often represents the target of conjugation when medical application of proteins entails the risk of severe immunoresponse. However, it is worth noting that although protein immunogenicity can be improved by modification with several macromolecules this result cannot be considered a general consequence of polymer coupling. Dextrans, for example, have been found to ameliorate the immunogenic behavior of many proteins, but paradoxically, in some cases they increase the protein immunogenicity as reported with bovine serum albumin or chicken serum albumin. (12, 13). Increased immunogenicity was also observed when polymer coupling induces partial modification of protein structure or dissociation into subunits that can be accompanied by introduction of neo-antigens (2). Another interesting immunological aspect of polymer binding is the possibility to obtain derivatives that modulate the immunoresponse toward allergens. In this regard, several studies demonstrated that polymer conjugation can switch immunogenic proteins into tolerogenic compounds, providing new therapeutic perspectives (14). These features indicate that conjugation can reflect in immunological properties of the derivatives, which can be quite different from those of the starting protein and polymer. These are often unpredictable since, as already reported in the literature, they depend on a number of parameters. In the framework of this research field, we carried out an immunological characterization of synthetic polymers

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recently developed in our laboratory for protein conjugation: monofunctional poly(N-vinylpyrrolidone) (PVP) (15), poly(N-acryloilmorpholine) (PAcM) (16), and a branched form of poly(ethylene glycol) (PEG2) (17). These polymers were selected because they possess physicochemical properties similar to the most used linear PEG 5000 Da, namely solubility, chemical stability, monofunctionality in reaction, and absence of charge. However, these polymers were found to confer to proteins different physicochemical and biopharmaceutical properties (18). For the present comparative investigation, derivatives of uricase, an immunogenic protein of outstanding therapeutic value, bearing a close number of PVP, PAcM, PEG2, and PEG were prepared and spectroscopic studies and antigenic and immunogenic characterizations were carried out on the native enzyme and the four conjugates. EXPERIMENTAL SECTION

Uricase from Candida utilis was obtained from Toyobo (Osaka, Japan). [3H]Succinimidyl propionate was from Amersham International (Amersham, U.K.) and Instagel for radioactivity determinations from Canberra Packard (Groninghen, The Netherlands). The gel filtration Superose columns and the FPLC system were supplied by Pharmacia Biotech (Uppsala, Sweden); the ultrafiltration system was from Amicon Inc. (Beverly, MA). The alkaline phosphatase conjugated goat anti mouse IgG, IgM, and [IgG + IgM] were from Sigma (St. Louis, MO). All other reagents, of analytical grade, were purchased by Fluka (Buchs, Switzerland). Male Balb/c mice, weighing 24 ( 2 g and fed ad libitum, obtained from Charles River (Calco, Italy), were used for in vivo experiments. Animal treatments were performed according to institutional European guidelines. Polymer Preparation and Activation. Succynimidyl ester activated linear monomethoxypoly(ethylene glycol) MW 5000 Da (PEG 5000) and branched monomethoxypoly(ethylene glycol) MW 10000 Da (PEG 10000) were prepared as previously reported (17, 19). Carboxyterminating poly(N-vinylpirrolidone) MW 6000 Da (PVP) and poly(N-acryloilmorpholine) MW 6000 Da (PAcM) were synthesized, fractionated, and activated according to methods previously described (15, 16). Preparation of Protein-Polymer Conjugates. Uric acid (77 µmol) was dissolved in 1 mL of 0.2 M borate buffer, pH 8.0 containing 10 mg of uricase. Proper amounts of activated polymer (77 mg of PEG 5000, 270 mg of PEG 10000, 115 mg of PVP, or 140 mg of PAcM) were added to the protein solution under vigorous stirring. The reaction mixture was gently stirred for 1 h at room temperature, and 9 mL of 0.1 M borate buffer pH 8.0 was added. The solution was ultrafiltered with an Amicon system using a 10000 Da cut off membrane to reach a volume of 1 mL and the solution diluted with 5 mL of the same buffer and further ultrafiltered. The ultrafiltration procedure was repeated three times. The solution was finally loaded on a gel filtration Superose 12 preparative column that was eluted with 10 mM phosphate buffer, 0.15 M NaCl, pH 7.2 (PBS). The eluate was analyzed by OD at 280 nm for protein content, by iodine assay for polymer determination (20), and by enzyme activity (21). The protein-polymer conjugate peak was concentrated by ultrafiltration, and the protein concentration was determined by biuret assay (22). The degree of modification was estimated by a trinitrobenzensulfonic acid based colorimetric method (23). Preparation of Tritium-Labeled Derivatives. [3H]Succinimidylproprionate (10 µg, 519 mCi/mg) was added

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under stirring to 0.5 mL of 0.2 M borate buffer, pH 8.0, containing 5 mg of native uricase or equimolar amounts of its conjugates. The solution was gently stirred for 1 h at room temperature and then loaded on an analytical Superose 6 column eluted with PBS. The eluted fractions were analyzed for protein content by OD at 280 nm, enzyme activity, polymer content, and radioactivity using the Instagel cocktail. The radioactive peak corresponding to the [3H]-labeled uricase or its conjugates was collected and protein concentration and radioactivity were assessed. Spectroscopic Evaluations. The spectroscopic characterizations were carried out using solutions of 0.38 µM of native or polymer conjugated uricase in PBS. The circular dichroism spectra were recorded in the range of 200-250 nm while fluorescence spectra were determined in the range of 300-440 nm by excitation at 276 nm. ELISA Determinations. ELISA microplates were incubated overnight at 4 °C with 100 µL/well of coating solution (0.1 M NaHCO3, pH 9.5) containing 1 µg/mL of antigen. The wells were washed three times with 200 µL of PBS, 0.05% Tween, pH 7.2 (PBS-T) and then incubated for 2 h at room temperature with 200 µL of 0.1 M NaHCO3, pH 9.5, containing 50 µg/mL of bovine serum albumin. The wells, washed as described above, were incubated for 2 h at room temperature with 100 µL of serum samples serially diluted in PBS-T and, after further washing, for 2 h with 100 µL of alkaline phophatase conjugated goat anti mouse antibodies (anti IgG or anti IgM or anti [IgG + IgM]) properly diluted in PBS-T. The wells were washed as above and 100 µL of 1 M diethanolamine. MgCl2, pH 9.8, containing 1 mg/mL of p-nitrophenol phosphate (Sigma104). The enzymatic reaction was stopped after 1 h by addition of 50 µL of 3 N NaOH and the o.d. at 405 nm was determined. Antigen Coating Estimation. The amount of adsorbed protein on the well surface was estimated as follows: 200 µL of coating solutions containing serial dilutions (0.05-5 µg/mL) of tritium-labeled native protein or equimolar concentrations of its tritium-labeled conjugates in 0.1 M NaHCO3, pH 9.5, were incubated overnight at 4 °C in the ELISA wells. The solutions were completely removed and conserved, and the wells were washed three times with 200 µL of PBS-T. The coating and the washing solutions were pooled, lyophilized, and examined for radioactivity content. Antigenicity Determinations. Native uricase (10 µg) in 200 µL of PBS/complete Freund’s adjuvant (50/50) were intraperitoneously injected into seven male Balb/c mice on day 0. The animals were intraperitoneously boosted on days 7, 14, 21, and 28 with 10 µg of native uricase dissolved in 200 µL of PBS/incomplete Freund’s adjuvant (50/50). The blood was taken on day 35, pooled, and centrifuged, and the serum serially diluted in PBS-T was used for antigenicity determinations by ELISA. The assay was performed as described above using, for well coating, 0.2 µg/mL of native uricase or equimolar dilutions of uricase-PEG, uricase-PEG2, uricase-PVP, or uricase-PAcM. Immunogenicity Evaluations. Fifty male Balb/c mice were divided into five groups of 10 animals and immunized on days 0, 4, and 11 with 10 µg of native uricase (group 1) or equimolar amounts of uricase-PEG (group 2), uricase-PEG2 (group 3), uricase-PVP (group 4), and uricase-PAcM (group 5). The immunizing solutions were prepared by dissolution of the immunogen in 100 µL of PBS and 100 µL of Freund’s adjuvant (complete adjuvan was used in the first immunization, incomplete adjuvant was used for boosting). The solutions were

Immunological Properties of Polymer Conjugates

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Table 1. Reaction Conditions, Composition and Molecular Mass of Native Uricase, Uricase-PVP, Uricase-PAcM, Uricase-PEG, and Uricase-PEG2 mean no. of polymer molecules per molecular polymer/protein protein molecule mass molar ratio ((SDa) (KDa) native uricase uricase-PVP uricase-PAcM uricase-PEG uricase-PEG2

250/1 300/1 300/1 350/1

47 ( 2 43 ( 4 44 ( 3 40 ( 4

130 400 380 350 530

a (SD: the standard deviation was calculated on the basis of five experiments carried out to point out the conjugation reaction conditions.

intraperitoneously injected, and blood samples were taken by retrobulbar bleeding at scheduled times. The blood samples were centrifuged for 2 min at 3500 rpm, and the serum was properly diluted in PBS for ELISA. Anti conjugate (whole construct) antibodies were assessed by ELISA using the corresponding uricase derivative for well coating. For the determination of anti native uricase antibodies the well coating was performed with native enzyme while for determination of anti-polymer antibodies, ribonuclease modified with PVP, PAcM, PEG2, and PEG was used. RESULTS AND DISCUSSION

To provide alternatives to monomethoxypoly(ethylene glycol) MW 5000 Da (PEG) for protein modification, we recently developed new soluble polymers: monofunctional poly(N-vinylpyrrolidone) MW 6000 (PVP), poly(Nacryloilmorpholine) MW 6000 Da (PAcM), and a branched form of monomethoxypoly(ethylene glycol) MW 10000 Da (PEG2). Previous studies showed that these macromolecules possess the prerequisites for medical application, and encouraging results were obtained in the conjugation of few protein models. Conjugation of these polymers endowed in fact proteins with improved biopharmaceutical and immunological properties (17, 18, 24, 25). Aiming to investigate closely the immunological aspects of PVP, PAcM, and PEG2 conjugation, we carried out a comparative study using uricase as protein model. Uricase conjugated to the most known linear PEG 5000 was used as a comparison model. PVP and PAcM with MW 6000 Da were selected because they have a molecular weight similar to PEG 5000, the polymer of choice for protein modification. PEG2 was considered in order to investigate the influence of the polymer molecular weight and shape on the immunological properties of the conjugate. Uricase was chosen because, despite its therapeutic value in treatment of uric acid overproduction, it presents a poor immunogenic character that greatly limits its medical use. For this reason, dextran and PEG derivatives of uricase with improved immunogenic and biopharmaceutical properties were already prepared (26, 27). Polymer Conjugation. Because of the comparative nature of this investigation, conjugates at a similar level of modification were prepared. To overcome the differences in reactivity of the polymer, which depend on both polymer activation degree and accessibility to the protein surface, different polymer/protein molar ratios were used in the conjugation reaction. Table 1 reports the reaction conditions, degree of modification, and molecular mass of the various uricase species. In all of the derivatives, an average of 44 protein

Figure 1. Circular dichroism spectra of native uricase (-) and uricase-PVP (---).

amino groups were modified by polymer. This yielded an higher molecular mass in the case of the PEG2 conjugate with respect to the other ones because of the higher molecular weight of this polymer that is about twice that of PVP, PAcM, and PEG. It is important to note that since PEG2 is a branched form of the linear PEG, the number of polymer chains bound to the protein surface corresponds to a double number with respect to the other single chain polymer conjugates. In all preparations, the addition of uric acid in the reaction mixture allowed for partial maintenance of the enzyme activity (20-80% of the unmodified uricase) probably because the substrate prevents the conjugation at the active site region (28). Structural Studies. Spectroscopic studies were carried out to investigate whether the polymer conjugation induced protein denaturation that would be reflected in increased protein antigenicity and immunogenicity. Native uricase and the four polymer conjugates displayed identical UV-vis and fluorescence spectra indicating that no macroscopic changes in protein structure occurred by polymer attachment. Also, no differences were found among the CD spectra of the various uricase species demonstrating that the secondary structure of the protein was not affected to a detectable degree by polymer conjugation. As an example, the CD spectra of native uricase and uricase-PVP are reported in Figure 1. The maintenance of the protein structure after polymer modification seems to indicate that the generation of new protein determinants, which could convey poor immunogenic properties to the conjugates, is unlikely. The spectroscopic data suggest also that the low enzyme activity of conjugates is not due to partial denaturation of uricase but most probably to masking of the active site by the polymer or to chemical modification of protein amino groups involved in the catalytic activity that takes place despite the use of uric acid. Antigenic Properties. The antigenicity of native and conjugated uricase was estimated by ELISA using mouse anti serum enriched with anti native uricase antibodies. In a preliminary study, the coating levels of the various enzyme forms on the ELISA microplate wells were


Figure 2. Antigenic profiles of native uricase (X), uricasePVP ([), uricase-PAcM (9), uricase-PEG ([), and uricasePEG2 (b). (SD was calculated on the basis of the values obtained with five experiments.

estimated using the following: [3H]uricase, [3H]uricasePVP, [3H]uricase-PAcM, [3H]uricase-PEG, and [3H]uricase-PEG2. This was essential for a correct comparison of the results obtained with the native and the polymer conjugated protein. Comparable protein adhesion was obtained with the various uricase species provided that the protein concentration of the coating solutions was 0.2 µg/mL. The antigenic profiles of native and polymer modified uricase are depicted in Figure 2. The different behavior obtained with the conjugates underlines the influence of the structural and physicochemical properties of the polymers on the protein antigenic character. PAcM, PEG, and PEG2 were found to reduce remarkably the uricase antigenicity. The reduction of uricase antigenicity obtained with both poly(ethylene glycol) derivatives confirms the known efficiency of these polymers in enhancing the antigenic character of this protein (29). PAcM also was found to reduce the protein antigenicity although at a lower extent with respect to the poly(ethylene glycol)s. Most probably, the reduction of protein antigenicity is due on one side to direct polymer masking of the protein epitopes and, on the other, to the steric hindrance of the polymer around the protein that prevents the antibody approach. Both of these events are strictly related to the physicochemical nature of the polymers that can be arranged on the protein surface in an extended or coiled conformation providing a different degree of hiding of the protein epitopes. Furthermore, these polymers possess a different capability to coordinate water molecules, which is reflected in different hydrodynamic volume of the conjugates. Therefore, the protein located in the hydrated polymeric core of the construct is differently accessible to the antibody interaction. To observe that the high efficiency of PEG2 in decreasing the uricase antigenicity with respect to the linear polymer is in agreement with the results obtained with other proteins (30). This can be ascribed to the higher molecular mass of polymer on the protein surface that efficiently enables it to mask the protein antigenic sites and reduces the approach of the antibodies to the protein structure as well as to prevent the proteolytic digestion (30). Unexpected results were instead obtained with the PVP derivative. In this case, the protein antigenicity is about 2-folds higher than the native enzyme one. This intriguing result is in contrast with the data obtained in our laboratory with superoxide dismutase and ribonuclease where PVP conjugation lowered the protein antigenicity. The different behavior observed with uricase with respect to ribonuclease and superoxide dismutase demonstrates that the antigenic performance of the

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Figure 3. Anti whole molecule immunogenic profiles. IgM + IgG levels toward native uricase, uricase-PVP, uricase-PAcM, uricase-PEG, and uricase-PEG2. V: immunization time. *: undetectable values.

conjugate is strictly related to the protein properties. In this regard we must remember that studies reported in the literature indicate that also PVP-conjugated D-Nacetyl hexosamidase A can strongly interact with the anti native protein antibodies (4). Conjugate Immunogenicity. The generation of antibodies against the native protein or the whole constructs was determined by ELISA using the various uricase forms for well coating and mouse serum obtained from the animals immunized with the corresponding conjugates. The immunological profiles depicted in Figure 3 indicate that the derivatives present a lower immunogenicity with respect to the native uricase, though at a different degree from one another. Uricase-PVP and uricasePAcM are the most immunogenic conjugates since they stimulate a high production of antibodies from the first immunization, and similar to the native enzyme, high antibody levels were maintained for the length of the experiment. On the other hand, the PEG and PEG2 derivatives present a remarkably lower immunogenicity with respect to native uricase. The very low amount of antibodies elicited by the PEG2 conjugate is worthy of note. Protein Immunogenicity. The anti native protein antibody levels obtained by animals immunized with the four conjugates were estimated and compared with the ones obtained with native uricase. The anti native protein [IgG + IgM] time courses depicted in Figure 4 A point out the high immunogenicity of native uricase. Anti uricase antibodies were found from the first immunization, and the maximal immunoresponse was obtained after animal boosting. The separate evaluation of IgM and IgG (Figure 4B,C) indicated that native uricase could elicit a rapid and intense IgM production since after the first immunization, and high IgM levels were maintained for 10 weeks while high amounts of anti native uricase IgG were elicited after the second immunization and maintained throughout the experiment. All the conjugates were found to stimulate a lower anti uricase immunorespone with respect to the native protein, although a different behavior was obtained with the four derivatives. PVP was the least effective in reducing the protein immunogenicity; anti uricase IgM were generated from the first immunization and IgG from the second one. PAcM was more effective than PVP in suppressing uricase immunogenicity. By treatment with



Figure 4. Anti native uricase immunogenic profiles: IgM + IgG(A), IgM (B), and IgG (C) immunoresponse after immunization with native uricase, uricase-PVP, uricase-PAcM, uricase-PEG, and uricase-PEG2. V: immunization time. *: undetectable values.

PAcM-uricase, anti protein IgM and IgG were detected only after the second immunization, and the reduction in immunoresponse was reduced to 2% of the native enzyme one. Even higher reduction in uricase immunogenicity was found with the linear PEG conjugate (below 1% of the native protein one). In this case, detectable amounts of anti native uricase IgM were determined only after the second immunization to disappear rapidly while

low amounts of IgG were generated after the third treatment. With the PEG2 derivative anti native protein IgM were not found, and negligible amounts of IgG were detected after the third immunization only. Similar to what reported for the conjugate immunogenicity, the different immunogenic profiles depicted in Figure 4 can be explained with the different ability of the polymer to mask the protein epitopes and to prevent


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Figure 5. Anti polymer immunogenic profiles: IgM + IgG(A), IgM (B), and IgG (C) immunoresponse after immunization with uricase-PVP, uricase-PAcM, uricase-PEG, and uricase-PEG2. V: immunization time. *: undetectable values.

the approach of the immunocompetent cells to the conjugate. Probably the unlike arrangement of the four polymers on the protein surface as well as their degree of hydration affect in a different way the uricase immunogenicity. Also in this case, the immunogenic behavior of uricasePVP is in contrast with the results previously obtained

in our laboratory with other proteins. Superoxide dismutase-PVP, for example, was found to be less immunogenic than the PEG derivative (24). However, the low effectiveness of PVP in reducing uricase immunogenicity could be related to the high immunogenic character of this protein as well as to a possible low in vivo stability of the protein induced by PVP attachment that could


induce the molecule dissociation into high immunogenic subunits. The rapid and intense production of anti native uricase IgM obtained with uricase-PVP indicates that a T cell indepenent mechanism may be involved in antibody response while with the other conjugates the immunoresponse appears to take place by a T cell dependent mechanism only. On the basis of such a hypothesis, we can think that PAcM, linear PEG, and PEG2 can convey to the conjugates a different capability to interact with macrophages and different stability toward the cell proteolytic processing. As far as the two poly(ethylene glycol) derivatives are concerned, the PEG2 conjugate presents an immunogenic character that is more convenient for therapeutic application as already observed in our laboratory (30). Polymer Immunogenicity. Interesting results were obtained regarding the ability of the conjugates to induce anti polymer immunoresponse. For this study, ELISA microplate wells were coated with ribonulcease (RNAse) extensively modified with the four polymers. To avoid misinterpretation of the results, the absence of antibody cross reactivity with native RNAse was previously verified. Figure 5 reports the anti-polymer immunogenic profiles obtained with the four uricase-polymer conjugates. Unexpectedly, all the conjugates were found to induce anti polymer immunoresponses although at different levels. The PVP and PAcM conjugates were found to induce a relevant production of IgM soon after the first immunization, suggesting a T cell independent mechanism antibody response (Figure 5B), while high levels of anti polymer IgG were estimated after the second treatment (Figure 5C). On the contrary, PEG and PEG2 derivatives gave rise to slight and delayed anti polymer antibody production. In particular, the immunogenic profiles reported in parts B and C of Figure 5 show that low levels of anti PEG IgM and IgG were found by immunization with uricase-PEG after the second and the thirrd boosting, respectively, while the PEG2 derivative stimulated only negligible amounts of anti polymer IgM and IgG These results indicate that despite the absence of inherent immunogenic character of the polymers used for the present investigation (7, 31, 32) their immunogenicity can significantly increase by linkage to proteins. In this regard, we must remember that conjugation to proteins generally induces a deep modification of polymer physicochemical properties that can be reflected in different immunological properties. Covalent binding of polymers to the protein surface achieves high molecular weight constructs that may result in partial loss of polymer flexibility and prolonged exposure to the biological environment due to their long permanence in circulation. These events could be the reason immunocompetent cells are stimulated at a greater extent to produce anti polymer antibodies. With regard to the molecular weight, it is worth noting that this parameter is of primary importance in dictating polymer immunogenicity. Immunogenic character of many polymers has been, in fact, demonstrated to increase as their molecular mass increases as, for instance, in the case of poly(N-vinylpyrrolidone)s of high molecular weights that are definitely immunogenic (32, 33). In the case of conjugates, the protein in the core of the construct behaves as an anchoring point for the polymer so that the conjugate could virtually acquire the properties of a high mol wt polymer. This may contribute


to increase immunogenic character of the polymer after protein conjugation. However, this relationship between polymer immunogenicity and molecular weight is not a general rule since data reported in the literature indicate that poly(ethylene glycol) immunogenicity decreases as the molecular weight increases. This is in agreement with the lower anti polymer immunoresponse obtained with the PEG2 derivative relative to the PEG one (7). CONCLUSIONS

The data reported in this paper demonstrate that the four polymers investigated here, PVP, PAcM linear and branched PEG, can improve the immunogenic character of uricase. However, despite their apparently similar physicochemical properties, they convey different immunological properties to the conjugates. In particular, our data demonstrate that the immunogenic properties of the conjugates are strictly related to the protein structure. This is in agreement with that reported in the literature and explains the different results obtained in our laboratory with other protein models modified with the same polymers. In the case of uricase, the high protein immunogenicity is probably the reason for the low efficiency of PVP and PAcM in preventing the anti native protein immmunoresponse. Furthermore, this protein can confer to the polymers haptogenic character allowing for stimulation of anti polymer antibody production. On the other hand, our results underline the relevance of the polymer choice in fabrication of conjugates with suitable immunogenic properties. Polymer flexibility, extended or coiled conformation, hydrophilic/hydrophobic balance, water coordination, and arrangement on the protein surface, together with molecular weight and chemical structure, are, in fact, all parameters that dictate the immunological properties of the conjugate. In conclusion, from the investigation reported here we can say that, although neutral amphiphilic polymers seem to possess suitable immunological properties on their own for protein modification, the immunogenic performance of conjugates is not easily predictable because of the complexity of the immunoresponse process. Accurate immunological investigations are therefore required in the development of any new derivative, and both protein and polymer immunogenicity before and after conjugation must be thoroughly investigated. LITERATURE CITED (1) Poly(ethylene glycol): chemistry and biological applications (1997) (J. Milton Harris and Samuel Zilpsky, Eds.) American Chemical Society, Washington, DC. (2) Delgado, C., Francis, G. E., and Fisher, D. (1992) The uses and properties of PEG-linked proteins. Crit. Rev. Ther. Drugs Carrier Syst. 9, 249-304. (3) Kartre, K. (1993) The conjugation of proteins with poly(ethylene glycol) and other polymers. Adv. Drug Deliv. Rev. 10, 91-114. (4) Davis, F. F., Kazo, G. M., Nucci, M. L., and Abuchovski, A. Reduction of immunogenicity and extension of circulating half-life of peptides and proteins. In Peptide and Protein Drug Delivery, (V. H. L. Lee, Ed.) pp 831-864, Marcel Dekker Inc., New York. (5) Borras Cuesta, F., Fedon, Y., and Petit Camurdan, A. (1988) Enhancement of peptide immungenicity by linear polymerization. Eur. J. Immunol. 18, 199-202. (6) Jackson, D. C., O’Brien-Simpson, N., Ede, N. J., and Brown, L. E. (1997) Free radical induced polymerization of synthetic peptides into polymeric immunogens. Vaccine 15, 1697-1705. (7) Richter, A. W., and de Bedler, A. N. (1976) Antibodies against hydroxyethylstarch produced by immunization with

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Immunological Properties of Uricase Conjugated to Neutral Soluble

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