Antigenicity of Recombinant Proteins after Regioselective

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Bioconjugate Chem. 2004, 15, 458−466

Antigenicity of Recombinant Proteins after Regioselective Immobilization onto Polyanhydride-Based Copolymers Laure Allard,† Vale´rie Cheynet,† Guy Oriol,† Gaspard Gervasi,§ Emmanuelle Imbert-Laurenceau,† Bernard Mandrand,† Thierry Delair,† and Franç ois Mallet*,† Unite´ Mixte de Recherche 2714, CNRS-bioMe´rieux, IFR128 Biosciences Lyon-Gerland, ENS-Lyon, 46, alleé d’Italie, 69364 Lyon, France, and bioMe´rieux, Chemin de l’Orme, 69280 Marcy l’Etoile, France. Received August 22, 2003; Revised Manuscript Received February 13, 2004

We previously demonstrated that the introduction of a tag consisting of several contiguous lysines at the N- or C-terminus of a recombinant protein greatly improved the covalent grafting of the protein onto negatively charged maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer, under many different experimental conditions (Ladavie`re, C., et al. (1998) Bioconjugate Chem. 9, 655; Allard, L., et al. (2002) Biotechnol. Bioeng. 80, 341). The grafting efficiency was dependent on the charge and amine density of the tag, characteristics which were determined by the tag composition. The six lysine tag (Lys6) was found to be the most efficient (Allard, L., et al. (2001) Bioconjugate Chem. 12, 972). In the present work, the biological activity of Lys6-proteins covalently bound to polymer was investigated. N- or C-terminal Lys6-tagged HIV-1 p24 recombinant proteins (RK24H and RH24K) were grafted onto MAMVE, and the antigenicity each of the bioconjugates was evaluated using six monoclonal antibodies that recognized different epitopes distributed along the protein. We demonstrate that the position of the tag and the hydrolysis rate of the anhydride moieties of the polymer are the two main parameters involved in the conservation of the biological activity of the immobilized protein. We thus present a process which allows an efficient oriented immobilization of proteins onto copolymers with optimal biological activity that is suitable for the controlled production of active bioconjugates.

INTRODUCTION

Covalent immobilization of proteins onto polymers has found numerous applications in several fields, such as in chemotherapy to reduce proteolytic degradation (1) in gene therapy for targeted gene delivery (2) and in diagnostics to increase the stability of ELISA capture phase (3). However, immobilization may occur via a random attachment of the proteins to the support through several amino acid residues, leading to a partial loss of biological properties (1). Since protein covalent immobilization is a chemical alteration of the biomolecule, it is of great interest to make this reaction occur at a specific site of the protein that is not involved in the biorecognition process (4). The approach we developed consisted in introducing an oligoamino acid sequence (tag) at a defined position on the protein. The rationale for this was that an increase in the density of reactive groups at a particular position of the protein would direct the grafting and should enhance the efficiency of the immobilization reaction. The advantage in using amide bond formation to tether biomolecules to carriers is that the chemistry is simple, reproducible, and can sustain a diversity of experimental conditions which are prerequisites for the development of a generic grafting method. We have previously shown that addition of a hexalysine tag (Lys6) at the N- or C-terminal region of a p24 recombinant protein led to a dramatic increase in the yield of the tagged protein (more than 90% versus 30% for the nontagged p24 protein) * To whom correspondence should be addressed. E-mail: [email protected]. † Unite ´ Mixte de Recherche 2714, CNRS-bioMe´rieux. § bioMe ´ rieux, Chemin de l’Orme.

coupled to maleic anhydride-alt-methyl vinyl ether copolymer (MAMVE) (5, 6). The presence of the Lys6 peptide allowed the immobilization of up to 50 protein molecules per polymer chain. In addition, we have shown that the efficiency of the regioselective immobilization of the tagged protein onto the copolymer depended directly on the composition of the tag. The reactivity of the tag was determined by both the density of positive charges and by the number of reactive amino groups. The most efficient tag was found to be the Lys6 tag which contained both cationic groups and primary amine groups (7). Although such a tag allows quantitative efficient grafting, the localization of the tag should be carefully chosen to maintain the biological activity of the protein. We thus designed two recombinant proteins characterized by the N- or C-terminal position of the Lys6 tag, respectively termed RH24K and RK24H. The protein model used was the capsid p24 protein from HIV-1, which is well expressed in Escherichia coli, soluble, efficiently purified by immobilized metal affinity chromatography (IMAC) (8), and structurally characterized (9). The structure of the p24 allowed us to accurately locate domains involved in molecular recognition processes (such as epitopes and protease cleavage sites) and to evaluate the accessibility of internal lysines. The RH24K and RK24H recombinant proteins were grafted onto MAMVE, and the antigenicity of the bioconjugates was evaluated using monoclonal antibodies (mAbs). These mAbs were selected because they recognized different epitopes distributed along the protein, in the N-terminal domain, in the flexible interdomain peptide and in the C-terminal domain. The accessibility of the different epitopes on the bound proteins was

10.1021/bc034146+ CCC: $27.50 © 2004 American Chemical Society Published on Web 05/04/2004

Biological Activity of Covalently Immobilized Proteins

Bioconjugate Chem., Vol. 15, No. 3, 2004 459

Figure 1. Chemical and antigenic features of the recombinant proteins derived from the HIV-1 p24 capsid protein. (A) Features of the three recombinant proteins, including the Lys6 (9) and His6 (9) tags, the main conserved domain (0), and specific AA residues. For each recombinant protein are indicated the length in AA and the theoretical and experimental molecular masses (MM) obtained using Mac Vector Version 6.5.3 and mass spectrometry (LC/ESI/MS), respectively. (B) Amino acid (AA) sequence of the native p24 protein from isolate HXB2. AA 3 (valine) to 224 (proline) represent the sequence conserved in all recombinant proteins. Lysine residues (K) are indicated in bold letters and epitopes recognized by 15F8, 1G5, 1B2, 23A5, 3D8, and 13B5 monoclonal antibodies (mAb) underlined. (C) In the context of a front and back RH24 3D structure, location of the N- and C-terminal ends of the protein and of the six epitopes and the 10 conserved lysines (red-squared). (D) Principle of the competitive ELISA test: (a) the RH24 protein was adsorbed on the microtiter plate, (b) various amounts of protein-polymer bioconjugate were incubated with the relevant mAb and then (c) loaded onto the RH24 capture phase; after a washing step which eliminated the bioconjugate-mAb complex, (d) the RH24 bound mAb was detected by adding an anti-mouse IgG peroxidase conjugate and (e) incubated with a colorimetric substrate; (f) curves representing the OD as a function of the bioconjugate concentrations were plotted, a rapid decrease indicating an efficient competition (black), a slow (dotted gray) or no decrease indicating an inefficient competition, which reflected an optimal or poor accessibility of the epitope, respectively.

evaluated using a competition ELISA test. The assay showed the recognition pattern of the bioconjugate epitopes was altered according to the N- or C-position of the tag. This was due to the involvement in the grafting reaction (in addition to the tag-driven grafting) of an internal lysine with spatial location close to the tag. We demonstrated that the reaction of internal lysine with the copolymer was avoided by modulating the reactivity of the latter, via controlled hydrolysis of some anhydride moieties. The location of the lysines involved in the covalent grafting of the protein onto the copolymer was determined by proteolytic digestion of the bioconjugates followed by MALDI-TOF mass spectrometry analysis. Thus, in addition to the previously demonstrated importance of the tag composition in the grafting reaction efficiency (7), all these results showed that the two main parameters involved in the conservation of the biological activity of the immobilized protein were the tag position and the number of reactive groups of the copolymer. EXPERIMENTAL PROCEDURES

Isolation and Characterization of Tagged Recombinant Proteins. The p24 gene was cloned in pMR (10)

and pMK81 and pMK83 expression vectors (6). Three HIV-1 p24 recombinant proteins were then obtained from E. coli using a procedure derived from Cheynet et al. (8). Each of the proteins present a His6 tag (H) at their Nor C-terminal end, allowing a one-step purification using IMAC, and two of the proteins display an additional Nor C-terminal Lys6 tag which allows efficient coupling of the protein to the copolymer (6, 7). The proteins were termed RH24, RH24K, and RK24H for recombinant protein with respectively no Lys6 tag, a C-terminal tag, and a N-terminal Lys6 tag (Figure 1A). Briefly, E. coli strain XL1 was transformed with the expression plasmids. The extracts of soluble proteins were purified by IMAC on FPLC (A ¨ kta explorer; Amersham Pharmacia Biotech) using a Zn-NTA-activated gel. Collected fractions were analyzed by SDS-PAGE, and the most concentrated fraction was dialyzed against phosphate buffer 50 mM, pH 7.8. Mass spectrometry (LC-ESI-MS) analysis was performed on a single quadrupole API 100 mass spectrometer, 140B pumps, and a 785A detector (Perkin-Elmer). Reversed-phase liquid chromatography was performed on a C4 column (Vydac ref 214PT5115, 5 µm particle size).

460 Bioconjugate Chem., Vol. 15, No. 3, 2004

Solvent A was 0.1% (v/v) formic acid in water, and solvent B was 0.1% (v/v) formic acid in water/acetonitrile mixture (5:95 v/v). A gradient of 40 to 60% B was used. Theoretical molar masses were determined with the Mac Vector software (version 6.5.3) and compared with the experimental values obtained by mass spectrometry. p24 Epitope Mapping. To identify epitopes distributed along the p24 protein, we created a library of bacterial clones expressing peptide sequences of p24 (NovaTope System, Novagen). Briefly, the p24 gene was randomly cleaved in 50-150 bp fragments using DNase, and all fragments have been cloned in a plasmid designed for the inducible expression of small peptides as part of larger fusion proteins, i.e., p24 epitopes fused to phage T7 protein 10. Sixteen antibodies obtained by immunizing mice with whole HIV-1 virus extract (15F8, 1G5, 14D4, 1B2, 23A5, 3D10, 11D11, 9A4, 13H4, 11C10, 9B5) or RH24 recombinant protein (9A9, 7A1, 3D8, 4H3, 13B5) were used to screen the p24 epitope library by standard colony lift method. For each antibody, positive clones were analyzed by DNA sequencing and the epitope determined by aligning the overlapping sequences. Isolation and Characterization of Bioconjugates. The MAMVE alternated copolymer (molar mass ) 67 000 g/mol) was supplied by Polysciences Inc (Warrington, PA). The copolymer was dissolved to a final concentration of 1 g/L in either anhydrous dimethyl sulfoxide (DMSO from Aldrich) at 37 °C (named nonhydrolyzed polymer) or DMSO containing 10% (v/v) H2O and incubated for 48 h at 37 °C (named hydrolyzed polymer). A standard reaction consisted of 36 µg of protein (12.4 µM final concentration) and 5 µg of polymer (0.71 µM final concentration) in 100 µL of 50 mM phosphate buffer, pH 7.8. The reaction mixture was stirred for 3 h at 37 °C, and then the bioconjugates were stored at 4 °C. Bioconjugates were analyzed by size exclusion chromatography (SEC) using a Shodex Protein KW-803 (Waters), a Kontron HPLC 422 pump, a Kontron HPLC autosampler 465, and a Kontron UV diode array detector. Analyses were run in 0.1 M phosphate buffer, pH 6.8, containing 0.5% (w/v) sodium dodecyl sulfate (SDS) with a flow rate of 0.5 mL/min. Detection was achieved by measuring the absorbance at 280 nm (at the concentration used, the polymer had no absorption at 280 nm). The ratio of the peak area corresponding to the polymerbound proteins versus the sum of the two peaks corresponding to the unbound and bound proteins (i.e. the total amount of protein involved in the reaction) gave the coupling yield. Epitope Accessibility. The capacity of each antibody to recognize its cognate epitope was evaluated by ELISA competition. ELISA 96-well plates (Nunc Immuno Plate MaxiSorp surface) were coated overnight at room temperature with a 0.25 µg/mL solution of RH24 proteins in PBS buffer. After being coated, plates were saturated for 2 h at 37 °C with phosphate buffer saline (PBS) containing 1% dry milk. mAb and various amounts of proteinpolymer bioconjugate (ranging from 0.01 to 15 µg/mL) in PBS-0.2% dry milk were incubated for 2 h at 37 °C and then added to the RH24-coated microtiter-plates. After 2 h incubation at 37 °C, plates were washed three times with PBS-0.05% Tween 20. The mAb bound to the adsorbed antigen was detected by adding 100 µL of goat anti-mouse IgG (H+L) peroxidase conjugate (Jackson ImmunoResearch) (2 × 103 fold diluted in PBS dry milk) and incubating for 1 h at 37 °C. Plates were then washed and incubated for 10 min with O-phenylenediamine (OPD 30 mg) (Sanofi Pasteur) diluted in 10 mL substrate buffer

Allard et al.

0.03% H2O2 (Sanofi Pasteur) for 10 min in the dark at room temperature. OD values were measured at 492 nm. Amine Titration. Protein modification rate was evaluated using fluorescamine (Acros ref 19167-5000, FW 278,26) (11), a compound which reacts with primary amines leading to the formation of fluorescent products. A standard reaction consisted of 30 µL of fluorescamine solution (0.4 g/L in anhydrous DMSO) and 90 µL of recombinant protein solution (0.34 g/L in phosphate buffer 50 mM. pH 7.8). The reaction was allowed to proceed for 20 min in the dark, at room temperature, and the fluorescence emission intensity was measured at 477 nm for an excitation wavelength of 416 nm (Perkin-Elmer LS50 fluorimeter). The modification rate of the immobilized protein was determined as follows: 100 [fluorescence of the bound protein (assay) × 100/ fluorescence of the unbound protein]. Enzymatic Digestion of the RH24K-MAMVE67 Bioconjugates and Mass Spectrometry Analysis of the Fragments. RH24K immobilized onto nonhydrolyzed and hydrolyzed MAMVE67 copolymer was digested using Endoproteinase LysC (Roche ref 1 420 429) and protease V8 from Staphyloccocus aureus (Glu-C) (Roche ref 1 420 399) with an enzyme/protein ratio of 25/1 (w/ w). Prior to digestion, the bioconjugates were reduced in a solution of 3 M guanidine and 20 mM DTT for 1 h at 37 °C and alkylated in 45 mM iodoacetamide for 1 h at 37 °C. Bioconjugates were then dialyzed overnight at 4 °C against 50 mM ammonium bicarbonate (Sigma lot 10K0261). Free RH24 and RH24K proteins were digested under the same conditions. Digested bioconjugates were stored at 4 °C before molecular-mass analysis on a MALDI-TOF Biflex III (Bruker, Wissembourg, France) mass spectrometer. RESULTS AND DISCUSSION

Characterization of the p24 Recombinant Proteins. The three recombinant proteins, containing a His6 tag for IMAC purification, differed by the absence (RH24) or the presence at their N-terminal (RK24H) or Cterminal (RH24K) end of a Lys6 reactive tag for coupling onto copolymer (Figure 1A). All three recombinant proteins exhibited a high expression level representing up to 30% of the total amount of protein production by E. coli. Following purification by IMAC on a Zn-NTAactivated gel, the purity of the proteins was greater than 95% as determined on a Coomassie Blue stained SDS polyacrylamide gel, as described for other batches used in previous studies (5-8). Experimental determination of the molar masses was in accordance with the expected theoretical value for all recombinant proteins (Figure 1A). The amino acid sequence shared by all proteins is reported in Figure 1B. Ten lysine groups are almost evenly spaced along the nontagged RH24 protein macromolecule (residues 25, 30, 70, 131, and 140 in the N-terminal domain, residues 158, 170, 182, 199, 203 in the C-terminal domain. Residue numbering corresponds to the wild-type HIV-1 p24 HXB2 strain), and these lysine groups are conserved within all three recombinant proteins. NMR (12) and crystal structure data (9, 1314) demonstrated that, in the 3D structure, the 10 lysine groups were accessible for grafting (Figure 1C) although they displayed different reactivities (15). Characterization of the p24 RecombinantP(MAMVE) Bioconjugates. The RH24K and RK24H proteins were bound to MAMVE, and the coupling yields were determined by HPLC analysis. The coupling yields were close to 100%, irrespective of the N- or C-terminal



Figure 2. Influence of the Lys6 tag position on epitope accessibility on the bioconjugate. Competitive ELISA curves representing OD as a function of the bioconjugate concentration were obtained for the six mAbs used at an optimal concentration (see text) as indicated (in brackets). The two bioconjugates consisted of N- or C-terminal Lys6-tagged recombinant proteins, RK24H (0) and RH24K (9), respectively, grafted onto MAMVE copolymer. 15F8 and 1G5 mAbs targeted the N-terminal domain of p24, 1B2 the interdomain, and 23A5, 3D8, and 13B5 mAbs the C-terminal domain.

position of the Lys6 tag, as previously described (6), highlighting the robustness of the process. RK24H and RH24K proteins were bound to partially hydrolyzed MAMVE (48 h in DMSO, 10% H2O; see below), and the coupling yields were 100% and 94.4% and 90%, respectively. The 0% to 10% coupling yield reductions observed with these Lys6-tagged proteins grafted onto partially hydrolyzed versus nonhydrolyzed polymers were much lower than the 82% decrease previously observed with the RH24 nontagged counterpart in similar conditions (7), illustrating the high grafting efficiency of the Lys6 tag which compete efficiently with the water-induced hydrolysis during the coupling reaction (5, 7). Thus, the high coupling yields allowed use of the bioconjugates without subsequent purification. Although the tag position did not affect the yields, it could potentially be detrimental to the biological reactivity of the bioconjugate. We decided to use antigen (p24 epitopes)-antibody (monoclonal antibodies paratopes) recognition as a tool to analyze the biological reactivity of the immobilized proteins. P24 Epitope Mapping. All p24-derived proteins contained a principal conserved domain which was used to construct an epitope library in order to make an epitope map of the p24 protein. The epitope library was used to screen 16 anti-p24 monoclonal antibodies (mAb). These 16 antibodies covered seven epitope regions including six nonoverlapping epitopes. Six monoclonal antibodies were retained, 15F8, 1G5, 1B2, 23A5, 3D8, 13B5 (Figure 1B). Four out of the six identified epitopes, recognized by 15F8, 1G5, 1B2, and 23A5 mAb, were in agreement with previous identifications using peptide technologies (16, 17). One out of the six identified epitopes, recognized by 13B5 mAb, was in agreement with previous structural characterization determined during cocrystallization of RH24 and 13B5 Fab (9, 18). More precisely, 15F8 (residues 49-65) and 1G5 (77-82) mAb recognize epitopes located in the N-terminal domain, 1B2 (143-148) in the flexible interdomain peptide,

and the three remaining 23A5 (153-168), 3D8 (179195), and 13B5 (206-218) mAb in the C-terminal domain (Figure 1B,C). As those epitopes were evenly dispersed along the p24 protein, they were flanked or contained potentially reactive lysine groups (Figure 1B,C). Antigenicity of the Bioconjugates. Due to the homogeneous distribution and the close vicinity of epitopes and internal lysines (not belonging to the Lys6 tag), one can assume that, in the context of a protein coupled to a copolymer, any alteration of epitope-paratope interaction may reflect the involvement in the grafting reaction of residues other than those contained within the tag, leading to end-on (tag dependent) or side-on (tag unrelated) orientations as previously suggested (5). The bioreactivity of recombinant proteins immobilized onto copolymer was investigated in an ELISA competition test (Figure 1D), as the random adsorption of the bioconjugate onto a solid phase might have randomly hidden and/or damaged some of the recognition sites. We first determined the optimal concentration for each monoclonal antibody, using a classical ELISA test with RH24 as a coated antigen. Using a serial dilution of mAbs, we obtained OD as a function of mAb concentration curves and thus calculated the mAb concentration at the center of the linear part of the curve. This mAb concentration should be the most informative in competition assay as small variations in recognition efficiency should induce large signal variations. Determined optimal concentrations were 0.05 µg/mL, 2 ng/mL, 3 µg/mL, 0.12 µg/mL, 3 ng/mL and 0.25 ng/mL, for 15F8, 1G5, 1B2, 23A5, 3D8, 13B5 mAbs, respectively. The bioreactivity of RH24K and RK24H proteins immobilized onto MAMVE was investigated in an ELISA competition test with the six monoclonal antibodies. No influence of the position of the tag was observed for five out of the six mAbs. This was indicated by the exponential decrease of the OD signal as a function of the bioconjugate concentration (Figure 2). Conversely, the competition curves for 23A5 mAb showed that RH24K-


Figure 3. Influence of an internal lysine on epitope accessibility on the bioconjugate. Three bioconjugates were analyzed by competitive ELISA with the 23A5 mAb. Antibody and bioconjugate concentrations were 0.12 µg/mL and 1 µg/mL, respectively. The bioconjugates consisted of N-terminal, RK24H (black bar), or C-terminal, RH24K (empty bar), Lys6-tagged recombinant proteins, or a Lys 158 mutated form RH24K-K158A (grey bar), grafted onto MAMVE copolymer. Experiments were performed in triplicate. The indicated OD is the mean value ( two standard deviations.

MAMVE bioconjugate competes much less efficiently than the RK24H-MAMVE counterpart. This may suggest that in the context of a C-terminal Lys tag grafted protein, the 23A5 corresponding epitope is poorly recognized or not accessible. This epitope is located in the C-terminal domain of the p24 protein, as were the 3D8 and 13B5 epitopes which remained unaltered during the grafting reaction. The 23A5 epitope contained a very accessible primary amine on lysine 158, as determined by analysis of the 3D structure (SwissPdbViewer v3.6) and previously experimentally described (15). Another lysine (K170) was present within the 3D8 epitope but exhibited a lower accessibility than lysine 158, as determined from the 3D structure and previously observed experimentally (15). Overall, this suggested that the position of the tag might favor the reactivity (coupling) of the K158 intrinsic lysine, which was detrimental to the epitope accessibility. To demonstrate the involvement of this lysine in the covalent reaction, we designed a new p24 recombinant protein, derived from the C-terminal Lys6 tag protein RH24K, where the K158 lysine was replaced with alanine, a neutral amino acid that is unable to react covalently with the anhydride moieties of the polymer. The binding of this protein, termed RH24K-K158A, onto the 23A5 mAb was not affected by the single amino acid mutation within the epitope, in a direct ELISA test (data not shown). RH24K-K158A was coupled onto MAMVE (coupling yield 100%, 90% after partial hydrolysis), and the reactivity of the 23A5 mAb toward RK24H-MAMVE, RH24K-MAMVE, and RH24K-K158A-MAMVE bioconjugates was evaluated in the ELISA competition assay. The competition effect obtained with the RH24KK158A-MAMVE bioconjugate was approximately 10 times higher than the one produced by the original RH24K-MAMVE and was indeed similar to the inhibition produced by the N-terminal Lys6-tagged proteinpolymer bioconjugate, RK24H-MAMVE (Figure 3). This result clearly demonstrated that, in the context of a C-terminal Lys6 p24, lysine 158 was involved in the coupling reaction with the polymer. A simple way to avoid internal lysine reactivity would be to decrease the density of reactive groups on the polymer. Such a modulation of reactivity could be achieved by partial hydrolysis of the anhydride moieties of the MAMVE copolymer in DMSO containing 10% water for

Allard et al.

48 h at 37 °C (7). The RH24K protein was therefore grafted onto nonhydrolyzed and partially hydrolyzed (MAMVE.hy) polymers, and the antigenic determinants reactivity of the two bioconjugates was investigated in the ELISA competition test with the six monoclonal antibodies. First, the competition curves for 23A5 mAb showed that the RH24K-MAMVE.hy bioconjugate competed much more efficiently than the RH24K-MAMVE counterpart (Figure 4A). This indicated that, in the context of a less reactive polymer, the 23A5 corresponding epitope was efficiently recognized and accessible onto the bioconjugate, in a similar way to that obtained for the RH24K-K158A-MAMVE and RK24H-MAMVE bioconjugates. These three different situations are likely to avoid the involvement of lysine 158 in the coupling reaction. It was also found that 1G5, 1B2, 3D8, and 13B5 mAbs behaved similarly, independently of the hydrolysis or nonhydrolysis status of the copolymer, as illustrated by 3D8 mAb in Figure 4A. Finally, the 15F8 mAb presented an opposing situation to the 23A5 mAb, the less competitive bioconjugate being the RH24KMAMVE.hy, from 1 µg/mL bioconjugate concentrations (Figure 4A). In addition, independently of the C-terminal or N-terminal position of the Lys6 tag on the protein, the 15F8 corresponding epitope was less efficiently recognized following immobilization of the protein onto the partially hydrolyzed copolymer than on the nonhydrolyzed form, again from 1 µg/mL bioconjugate concentrations (data not shown). To understand why the epitope accessibility of the 15F8 mAb was lowered after grafting onto partially hydrolyzed copolymer, we compared the reactivity of 23A5, 3D8, and 15F8 mAbs in an ELISA competition assay using the unbound RH24 protein as a competitor. Classical competition curves were obtained for 23A5 and 3D8 mAbs, although they illustrated different efficiencies, probably due to different affinities, 4.1 × 108 M-1 and 3.6 × 109 M-1, respectively (Figure 4B). Conversely, an inverted bell shape curve was obtained with 15F8 antibody which had an affinity similar to the 23A5 antibody (2.9 × 108 M-1). Antigen concentrations from 1 µg/mL induced, intriguingly, a signal increase (Figure 4B). Overall, this strongly suggested that the 15F8 mAb recognized an epitope which was more efficiently displayed on the RH24K-MAMVE bioconjugate, probably as a result from several points of attachment of the protein on to the nonhydrolyzed polymer. This is in agreement with previous observations showing that a modification of the lysine residues of a recombinant p24 protein by a maleic anhydride monomer increased the affinity of a murine mAb (15) that recognized an epitope sharing eight residues with 15F8 mAb related epitope. Conversely, this epitope would be partially hidden either on bioconjugate defined by a tagrestricted attachment (hydrolyzed polymers) or at high concentration of noncoupled protein. These features correspond to a cryptic epitope. Indeed, (i) the 15F8 mAb was obtained by immunization with viral lysate, (ii) it recognized an epitope located within a pocket formed by the dimerization of two p24 N-terminal domains (19), (iii) the N-terminal composition of all p24 recombinant proteins is altered in comparison with the native p24 form, which may affect both N-terminal domain structure and self-association properties (12, 19, 20), and finally (iv) the p24 surface plasticity allows N-N and N-C intermolecular interfaces (9, 13, 14, 19). Identification of the Lysines Involved in the Coupling Reaction. The main results from the antigenic analysis were (i) that depending on the position of the polylysine tag, intrinsic lysines could react with



Figure 4. Influence of the hydrolysis of the polymer on epitope accessibility on the bioconjugate. (A) Competitive ELISA curves representing OD as a function of the bioconjugate concentration were obtained for the mAbs used at an optimal concentration. The two bioconjugates consisted of C-terminal Lys6-tagged recombinant protein RH24K, grafted onto nonhydrolyzed (9) or hydrolyzed (0) MAMVE copolymer. Hydrolysis of the polymer improved inhibition efficiency for the 23A5 mAb, had no effect for the 3D8 mAb (and 1G5, 1B2, and 13B5 mAbs, see text), and reduced inhibition efficiency for the 15F8 mAb. (B) Competitive ELISA curves representing OD as a function of the RH24 concentration were obtained for all mAbs. Classical inhibition curves were observed for 23A5 (b), 3D8 (b) (and 1G5, 1B2, and 13B5 mAbs), and an inverted bell shape curve was obtained for 15F8 (O) mAb. Table 1. Quantitation of Free Amino Groups of the Unbound and Polymer-Bound Recombinant Proteinsa RH24K tested

productb

unbound proteins proteins grafted onto MAMVE proteins grafted onto hydrolyzed MAMVE

RH24-K158A

OD477 nm

modificationc

747 369 593

50.6 20.6

RK24H

OD477nm

modificationc

OD477nm

modificationc

806 475 713

41.1 11.5

863 609 750

29.5 13

a The presence of free amino groups was measured by detecting fluorescent products generated by the chemical reaction of fluorescamine with primary amines of the tested product (OD477nm). b Tested product consisted of unbound proteins (RH24K, RH24K-K158A, RK24H) or proteins grafted onto nonhydrolyzed or partially hydrolyzed MAMVE copolymers. c The percentage of modification of the bound protein is defined as 100 - (fluorescence of bound protein × 100/fluorescence of unbound protein).

anhydride groups of MAMVE and (ii) that this involvement of internal lysines could be impeached by a decrease in the density of reactive groups of MAMVE via a controlled hydrolysis. To strengthen these conclusions, two additional methods were used to perform both quantitative and qualitative analyses. A rough estimate of the number of lysines participating in the reaction was indirectly evaluated using fluorescamine, a compound which reacts specifically with primary amines to produce fluorescent products. Amine titers of three free p24-derived recombinant proteins and of their corresponding polymer bioconjugates were compared, and the percentages of protein modification were calculated (Table 1). The mean modification ratio of nonhydrolyzed polymer-based bioconjugates (40.4 ( 10.6) was 2.3 to 3.6 times higher than that obtained with the partially hydrolyzed ones (15.0 ( 4.9). Thus, irrespective of the recombinant protein tested, these results confirmed that the reduction of the amount of reactive groups onto the polymer reduced alterations of the grafted protein, as a consequence of a lowered involvement of intrinsic lysines in the grafting process. Nevertheless, the higher modification ratio in RH24K-MAMVE conjugates as compared to RK24H-MAMVE ones suggested that the C-terminal position of the tag might favor the involvement of intrinsic lysines, as confirmed by the RH24KK158A mutant (Table 1). To identify lysine residues which were involved in the coupling reaction and the direct incidence of the polymer partial hydrolysis on avoiding internal lysine reaction, we performed a comparative enzymatic digestion of the unbound C-terminally tagged RH24K protein and of the nonhydrolyzed and hydrolyzed polymer-based bioconjugates. Two different proteolytic enzymes were used, and

the generated fragments were analyzed by MALDI-TOF mass spectrometry. The three substrates were digested by Staphylococcus aureus protease V8, which cleaved on the C-terminal side of glutamic and aspartic acids residues in order to produce fragments containing lysines, and endoproteinase Lys-C which cleaved on the C-terminal side of lysines in order to directly check the involvement of an intrinsic lysine in the coupling reaction. Mass spectrometry analysis of peptides from protease V8 digestion of RH24K, RH24K-MAMVE, and RH24K-MAMVE-hydrolyzed allowed identification of three out of eight fragments with specific features (Table 2). First, the isolation of a 2363.2 kDa fragment, obtained exclusively after digestion of the free RH24K protein and identified as the tag-containing C-terminal portion of the protein, confirmed the direct involvement of the hexalysine tag in the grafting reaction. Second, the 1758.8 kDa fragment, absent exclusively from the RH24K-MAMVE bioconjugate and identified as the N-terminal part of the protein, suggested that the N-terminal amino group was involved in the coupling reaction onto a nonhydrolyzed polymer, but that such an involvement was avoided by partial hydrolysis of the polymer. Third, the detection of a 3291.7 kDa fragment, obtained after digestion of RH24K and RH24K-MAMVEhydrolyzed but not RH24K-MAMVE and corresponding to the S1-E29 region, showed that the primary amino group of internal lysine 25 was involved in the grafting reaction onto the nonhydrolyzed polymer. This parasitic coupling could be avoided by partial hydrolysis of the polymer, as described above. Mass spectrometry analysis of peptides from endoproteinase Lys-C digestion of all three substrates was indeed more informative concerning the involvement of


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Table 2. Identification of Proteolysis Fragments of RH24K-Derived Bioconjugates by MALDI-TOF Analysis protease V8

Lys-C

MMa

deduced sequences

amino acids position

cont

604.3 677.3 1758.8 2363.2 2755.3 2779.5 3291.7 3665.8 111.7 1358.7 1633.8 2032.9 2073.1 4232.0

TINEEc KAFSPE MRGSHHHHHHGSVDE MMTACQGVGGPGKKKKKKSVDE GATPQDLNTMLNTVGGHQAAMQMLKE TLLVQNANPDCKTILKALGPAATLEEc SMVQNIQGQMVHQAISPRTLNAWVKVVEEc WDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQE RWIILGLNK TLRAEQASQEVK EPFRDYVDRFYK NWMTETLLVQNANPDCK IVRMYSPTSILDIRQGPK AFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLK

72-76 30-35 N-term(H6) (K6)C-term 46-71 188-213 1-29 80-113 132-140 171-182 159-170 183-199 141-158 30-70

X X X X X X X X X X X X X X

RH24K bioconjugatesb MAMVE MAMVE.hy X X O O X X O X O X O O O O

X X X O X X X X X X X X X X

a Molar mass (Da) of fragments observed in MALDI-TOF analysis and resulting from the digestion of the bioconjugates by Staphylococcus aureus V8 protease and endoproteinase Lys-C. b Bioconjugates consisted of free RH24K protein as a control (cont) or RH24K protein grafted onto nonhydrolyzed MAMVE (MAMVE) or hydrolyzed MAMVE (MAMVE.hy) copolymers; X and O indicated that the fragment was detected or not detected, respectively. c Partial proteolysis.

Figure 5. Relative analytical sensitivity of protein-polymer bioconjugate. (A) RH24 (0) or RK24H-MAMVE (9) antigens were coated onto microtiter plates (1 µg/mL), and anti-p24 antibodies were detected in direct ELISA, using serial dilutions of serum from one HIV-1 seropositive patient ranging from 1/500 to 1/1 665 000. OD as a function of the serum dilution is plotted. The cutoff value (mean + three standard deviations) was determined with pooled OD obtained from three control sera, from HIV-1 seronegative individuals, tested in the same conditions as in the HIV-1 assay. OD values corresponding to 5×, 10×, and 20× the cutoff value are indicated. (B) The relative increase in sensitivity was calculated for six HIV-1 positive sera tested as indicated above. For each serum, the indicated data corresponded to the mean ( two standard deviations of the increase calculated at 5×, 10×, and 20× the cutoff value. The overall mean increase is indicated by a horizontal lane.

several internal lysine residues in the coupling reaction onto nonhydrolyzed polymer (Table 2). Six clearly identified peptide fragments were generated after digestion of the RH24K protein. Five peptides were not detected after digestion of the bioconjugate based on a nonhydrolyzed polymer. This suggested an involvement of lysines 70 (Nterminal domain), 140 (flexible inter domain peptide), 158, 170, and 199 (C-terminal domain) in the formation of amide bonds with the copolymer. These results were in agreement with the antibody binding analysis showing the involvement of lysine158 (23A5 mAb analyses) and suggested the multipoint attachment process (15F8 mAb analyses) in grafting conditions with nonhydrolyzed polymer. Conversely, peptide T171-K182 was also observed after digestion of the bioconjugate based on a nonhydrolyzed polymer. It contained lysine K182 which according to the 3D structure (SwissPdbViewer v3.6) was poorly accessible and hence did not contribute to the grafting reaction. Finally, the six peptides obtained with the unbound protein were all identified after digestion of the RH24K-MAMVE-hydrolyzed bioconjugate, confirming the partial hydrolysis as a powerfull process to avoid the side-on grafting by intrinsic lysines.

CONCLUSION

We had previously demonstrated that the introduction of a six-lysine tag into a recombinant protein drastically improved the immobilization of the protein onto a copolymer, with the nature of the buffer and the nature, size, and reactivity of the polymer being minor parameters with respect to the control of the covalent immobilization (5, 7). The present work demonstrated that the two main parameters involved in the conservation of the biological activity of the immobilized protein were the tag position and the number of reactive groups on the copolymer. This demonstration was achieved using bioconjugates derived from coupling of RK24H or RH24K, two recombinant proteins containing a N- or C-terminal six-lysine tag characterized by positive charges and primary amines, onto MAMVE, a copolymer bearing negative charges and anhydride moieties. The alteration of the recognition by a specific monoclonal antibody of a bioconjugate consisting of a Cterminal Lys6-tagged p24 protein grafted onto MAMVE reflected a loss of accessibility of the corresponding epitope. The local protein conformational alteration was due to the involvement, in the grafting reaction, of an internal lysine with a spatial location close to the tag.


Such an alteration could be avoided by reducing the reactivity of the polymer, a parameter controlled by the partial hydrolysis of the anhydride moieties. These observations were confirmed by another molecular recognition process, i.e., the site specific digestion of the bioconjugates by protease V8 and endoproteinase Lys-C followed by mass spectrometry analysis. This technique highlighted the close relationships between the tag position, the position and accessibility of internal lysines, and the density of reactive anhydride groups of the copolymer. So we have developed a process to engineer bioconjugates by using judiciously tagged recombinant proteins and polymers with a modulated reactivity. The interest of this methodology was illustrated by the using of RK24H grafted onto hydrolyzed MAMVE as a capture antigen in an ELISA test format. Serial dilutions (up to 2 × 106) of sera from six HIV-1 seropositive patients were used as sources of antibodies to compare the immunoreactivity of RK24H-MAMVE with the RH24 antigen classically used in immunoassay (21). A significant signal increase was observed with the bioconjugate as compared to the standard assay using RH24 (Figure 5). More precisely, the mean increase of analytical sensitivity was 7.2 ( 2.7, the minimal and maximal increase being 3.9 and 11.4, respectively. The evaluation of this bioconjugate with a panel of sera from 107 HIV-1 seropositive individuals at various stages of HIV infection, including two seroconversion panels, confirmed that such a proteincopolymer bioconjugate allowed the detection of antibody titers lower than the adsorbed protein (6). Thus, based on this antigen(s)/antibody(s) model illustrating the local modifications due to the immobilization process and how to control/avoid them, we propose a versatile technology of oriented covalent immobilization of biologically active recombinant protein onto MAMVE polymer. The isolation of biologically active bioconjugates would be based on three main parameters consisting of introducing a tag of a defined composition (six lysines), in a predefined position (N- or C-terminal) with respect to the predicted or determined protein active site(s) and/ or the intrinsic lysine content and/or accessibility, and using hydrolysis to control the density of reactive groups onto the polymer if the protein functional requirements imposed the tag position. To validate this concept with other proteins such as antigens, enzymes, or cell-binding ligands, a set of expression vectors was developed (6). More generally, it will be of interest to evaluate if the physicochemical parameters identified as critical in this protein-polymer system (refs 5-7, this study) would lead to a generic process of immobilization onto other negatively charged supports compatible with amide bond formation. ACKNOWLEDGMENT

The authors are grateful to Franç ois Penin and Christophe Geourjon for helpful discussions, and Jennifer Burgess et Saint John Skilton for critical reading of the manuscript. We would like to thank Marcelle Sauzon for mass spectrometry analysis. L.A. is thankful to Fondation Me´rieux for financial support. LITERATURE CITED (1) Monfardini, A., and Veronese, F. M. (1998) Stabilization of substances in circulation. Bioconjugate Chem 9, 418-450. (2) Varga, C. M., Wickham, T. J., and Lauffenburger, D. A. (2000) Receptor-mediated targeting of gene delivery vectors:

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Antigenicity of Recombinant Proteins after Regioselective

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