Covalent Modifications of Antitetanus F(ab′)2 Fragments with Natural

Jul 17, 2008 - Phone: 33 (1) 42 86 20 51. Fax: 33 (1) 42 60 55 37. Email: [email protected]., † ... Cationization was shown to enhance ...
0 downloads 5 Views 1MB Size
Bioconjugate Chem. 2008, 19, 1543–1555

1543

Covalent Modifications of Antitetanus F(ab′)2 Fragments with Natural and Synthetic Polyamines and Their Effects on the Antibody Endocytosis in Cultured HL60 Cells Franc¸oise Herve´,*,† Nicolae Ghinea,‡ Philippe D’Athis,§ Pierre-Alain Carrupt,| and Jean-Michel Scherrmann⊥ CNRS, UPR2228, Universite´ Paris Descartes, UFR Biome´dicale, 45 rue des Saints-Pe`res, F-75270 Paris Cedex 06, France, INSERM, U841-EQ07, Universite´ Paris 12, Faculte´ de Me´decine, 8 rue du Ge´ne´ral Sarrail, F-94010 Cre´teil Cedex, France, Service de Biostatistique et Informatique Me´dicale, Centre Hospitalier Universitaire de Dijon, F-21034 Dijon Cedex, France, Unite´ de Pharmacochimie, Section des Sciences Pharmaceutiques, Universite´ de Gene`ve, Universite´ de Lausanne, Quai Ernest-Ansermet 30, CH-1211, Gene`ve 4, Switzerland, and INSERM, U705, CNRS, UMR7157, Universite´ Paris Diderot, Universite´ Paris Descartes, Hoˆpital Fernand Widal, 200 rue du Faubourg Saint-Denis, F-75475 Paris Cedex 10, France. Received February 3, 2008; Revised Manuscript Received May 26, 2008

For antibody therapeutics to succeed when intracellular target molecules are involved, a strategy must be applied to increase the delivery of antibodies into cells to reach their targets. Antibody cationization by chemical conjugation of a polyamine could be one such strategy. Both natural polyamines with increasing net charge valencies (putrescine, PUT; spermidine, SPD; and spermine, SPM) and a synthetic polyamine (hexamethylenediamine, HMD) can be used to cationize antibodies, but no comparison of the respective effects of these polyamines on intracellular delivery of antibodies has been performed yet. This study describes the covalent modification of antitetanus F(ab′)2 with these four polyamines using different reaction conditions, and compares the effects of these modifications on antibody interaction with cultured HL60 cells. The cationized antibodies retained g80% of the binding activity of the unmodified F(ab′)2 with regard to tetanus toxin, as measured by an antigen-binding capture enzyme immunoassay. This same method was used to quantify the amount of cell-associated F(ab′)2 following incubation with HL60 cells. Cationization was shown to enhance cell interaction of the F(ab′)2: the higher the number of coupled polyamine molecules, the greater the amount of antibody associated with the cells. Moreover, coupling the F(ab′)2 to the SPD and SPM polyamines had greater effect on cell interaction than coupling the F(ab′)2 to the PUT and HMD diamines. Internalization of the cationized antibodies by the HL60 cells was demonstrated by confocal microscopy. This technique also showed that SPD and SPM were more effective than PUT and HMD in terms of intracellular delivery of the F(ab′)2. It follows from all these results that electrostatic interaction involving charge density plays a predominant role in the endocytic transport mechanism of the F(ab′)2 modified with these polyamines. However, coupling the F(ab′)2 to SPM and SPD yielded the same maximum effects in terms of cell interaction, although coupling SPM was expected to increase the antibody net charge valency more than coupling SPD. This finding suggests that the effective global charge for the cell interaction and uptake of polyamine-modified antibodies does not simply correspond to the addition of the ionizable amine functions on the coupled polyamines, and that other factors may come into play.

INTRODUCTION The potential of antibodies as diagnostic and therapeutic agents is limited, especially when intracellular target molecules are involved, because antibodies are poorly internalized by cells. Indeed, these hydrophilic molecules cannot cross the lipid matrix of the cell plasma membrane efficiently, and so cannot gain access to the cell interior or the different cellular compartments to reach the target molecules. The high adsorptive property of cationic proteins to anionic sites present on all cell surfaces has provided the basis for the development of cationization strategies for delivering proteins into cells (1, 2). Proteins can be cationized through the chemical * Corresponding author. Dr. Franc¸oise Herve´, CNRS UPR2228, Universite´ Paris Descartes, UFR Biome´dicale, 45 rue des Saints-Pe`res, F-75270 Paris Cedex 06, France. Phone: 33 (1) 42 86 20 51. Fax: 33 (1) 42 60 55 37. Email: [email protected]. † CNRS, UPR2228. ‡ INSERM, U841-EQ07. § Centre Hospitalier Universitaire de Dijon. | Universite´ de Gene`ve, Universite´ de Lausanne. ⊥ INSERM, U705, CNRS, UMR7157.

conversion of their surface carboxylic acid groups into basic moieties by covalent coupling of polyamines. Not only can the positively charged proteins thus formed bind to the various negatively charged plasma membrane components surrounding the cells, but the bound proteins are also internalized into cells in a receptor- and transporter-independent manner, by adsorptive-mediated endocytosis (AME1) (3). The ability of cationized antibodies or fragments to undergo enhanced cellular uptake (3–6) and exert intracellular activity (7, 8) has been demonstrated in different in vitro cell models. 1 Abbreviations: AME, adsorptive-mediated endocytosis; BSA, bovine serum albumin; C.V., coefficient of variation; EDC, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide; ELISA, enzyme-linked immunosorbent assay; HBSS, Hanks’ balanced saline solution; HBSS/BSA, HBSS containing 0.2% BSA; Hepes, N-2-hydroxyethylpiperazine-N′2-ethanesulfonic acid; HMD, hexamethylediamine; IEF, isolectric focusing; IgG, immunoglobulin G; Mes, 2-(N-morpholino)ethanesulfonic acid; MWCO, molecular weight cutoff; PBS, phosphatebuffered saline; pI, isolectric point; PUT, putrescine; RPMI 1640, Roswell Park Memorial Institute 1640 medium; S.D., standard deviation; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SPD, spermidine; SPM, spermine.

10.1021/bc800045x CCC: $40.75  2008 American Chemical Society Published on Web 07/17/2008

1544 Bioconjugate Chem., Vol. 19, No. 8, 2008

Hervé et al. 14

Figure 1. Structures of the synthetic diamine, hexamethylenediamine (HMD), and of the natural polyamines, putrescine (PUT), spermidine (SPD), and spermine (SPM). Due to coupling, each polyamine is found with one less primary amino group, i.e., one less positive charge, at neutral pH.

Moreover, it has also been shown that cationized antibodies can be transported across complex physiological barriers, such as the blood-brain barrier (1), indicating that cationization is a strategy able to increase both target cell endocytosis and transendothelial migration of antibodies. Antibody cationization has most generally been accomplished by coupling antibodies to the synthetic diamine molecule known as hexamethylenediamine (HMD) (Figure 1). In addition, a different type of protein cationization involving the covalent attachment of putrescine (PUT), spermidine (SPD), and spermine (SPM), a series of endogenously occurring polyamines with increasing net charge valencies (Figure 1), has also been described (9). The covalent modification of a variety of proteins with PUT, SPD, and/or SPM has been shown to facilitate the delivery of these proteins into the brain in vivo. More specifically, a remarkable increase in brain transfer was observed for PUT-modified immunoglobulin G (IgG) relative to the native protein (9). However, no comparison of the respective effects of PUT, SPD, and SPM on the cellular internalization of antibodies in vitro or their brain transfer in vivo has been performed yet. The same holds true for HMD in that it has yet to be shown whether synthetic polyamines are more, less, or equally effective as natural polyamines. Therefore, we chose to perform a study which included all of the aforementioned polyamines for antibody cationization in order to investigate these questions and to select the most effective polyamine in terms of intracellular delivery. Using polyclonal antitetanus F(ab′)2 fragments as an antibody model, the present study describes their covalent modification with HMD, PUT, SPD, and SPM and the effects of these modifications on antibody cell interaction and internalization in an in vitro model with HL60 cells. Optimization of the conditions of modification yielded two sets of F(ab′)2 with increasing net charge valencies: one set with an increasing number of coupled polyamine molecules, and the other with increasing charge densities associated with the polyamines after antibody modification. We then measured the antigen-binding activity of the modified F(ab′)2 samples, and quantitatively analyzed their interaction with the HL60 cells by an ELISA method which has been especially developed for these measurements (10). Finally, the distribution of these antibodies at the cellular level was visualized by confocal microscopy.

EXPERIMENTAL PROCEDURES Materials. The antitetanus toxoid horse polyclonal F(ab′)2 fragments (batch no U-5000-2) and the tetanus anatoxin were gifts from Aventis-Pasteur (Marcy l’Etoile, France). The HL60 (Human Caucasian promyelocytic leukemia) cell line was obtained from the European Collection of Animal Cell Cultures (Salisbury, Wiltshire, UK). Putrescine • 2HCl, spermidine • 3HCl, spermine • 4HCl, hexamethylenediamine, and 1-ethyl3-(3-dimethylaminopropyl)carbodiimide (EDC) were from Fluka (Saint-Quentin Fallavier, France). [2,3-3H-(N)]putrescine (1243.2 GBq/mmol; 185 MBq/mL) and [terminal methylenes-3H(N)]spermidine (919.5 GBq/mmol; 185 MBq/mL) were obtained from PerkinElmer Life Sciences (Boston, MA), and [1,4-

C]spermine (4.07 GBq/mmol; 1.85 MBq/mL) from GE Healthcare (Orsay, France). Alkaline phosphatase-labeled streptavidin, p-nitrophenyl phosphate, protamine base, and 2-(Nmorpholino)ethanesulfonic acid (Mes) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Rabbit biotin conjugated affinity purified antihorse IgG (F(ab′)2 specific) was from Rockland (Gilbertsville, PA). The following chemicals were obtained from Invitrogen (Cergy Pontoise, France): streptavidin conjugated to Alexa Fluor 488, toto-3 iodide, Roswell Park Memorial Institute 1640 medium (RPMI 1640), Hepes buffer solution 1 M, Hanks’ balanced saline solution (HBSS), and 7.5% (w/v) bovine serum albumin (BSA) solution. All other chemicals were of the purest grade available and purchased from VWR (Fontenay-sous-Bois, France) unless otherwise noted. Milli-Q Plus purified water (Millipore, Bedford, MA) was used throughout. Preparation of Polyamine-Modified Antitetanus F(ab′)2. The method used for antibody modification was similar to that described previously (10). The polyamines, HMD, PUT, SPD, and SPM, respectively, were covalently attached to carboxylic acid groups of the F(ab′)2 using EDC. A sample of the native F(ab′)2 diluted in 0.1 M Mes buffer with a pH of 4.5, 5.5, or 6.5 was added to the desired polyamine in water while stirring. This was followed by the addition of water-soluble EDC, and the modification reaction was allowed to proceed for 4 h at 4 °C in the dark. The concentrations of tested polyamine, EDC, and antibody in the EDC(+) mixture (50 mL) were 50 mM, 0.96 mg/mL, and 0.5 mg/mL, respectively. The reaction was quenched with the addition of an equal volume of 0.1 M sodium acetate buffer, pH 5.0. To remove the excess of polyamine and EDC, the product was first filtered on Omega-50 membrane filter with a MWCO of 50 000 (Pall/Gelman Sciences, Ann Arbor, MI), and then on Bio-Gel 10DG column (Bio-Rad, Marnes-laCoquette, France), and finally dialyzed against water with multiple changes for 60 h at 4 °C using Spectra/Por CE membrane with a MWCO of 25 000 (VWR). Concurrently, F(ab′)2 samples were treated under the same conditions as those described above, except that water was used in place of EDC. These EDC(-) F(ab′)2 samples were used as controls throughout this study. The cationized and the control antibody preparations were analyzed for protein content using the Bio-Rad DC Protein Assay kit with bovine IgG as a standard (Bio-Rad). The antibody solutions were then individually aliquoted and stored at -20 °C for subsequent use. These aliquots were stable over a oneyear storage period at this temperature. Estimation of the Polyamine Substitution Ratio of the F(ab′)2. The polyamine, PUT, SPD, or SPM, was added to two samples of the F(ab′)2 in 0.1 M Mes buffer with the desired pH, ranging from 4.5 to 6.5, and the samples spiked with known amounts of [3H]PUT, [3H]SPD, or [14C]SPM. Then, EDC was added to one F(ab′)2 sample (EDC(+)) and water to the other (EDC(-)). The concentrations of polyamine and F(ab′)2 in the EDC(+) and EDC(-) samples (each 5 mL), and the concentration of EDC in the EDC(+) sample were the same as those used above. The concentration of radioactive polyamine in each sample was 2 µM for [3H]PUT and for [3H]SPD and 10 µM for [14C]SPM. After incubation followed by the addition of 0.1 M sodium acetate buffer, pH 5.0 (5 mL), the EDC(+) and EDC(-) F(ab′)2 samples were individually filtered and dialyzed as described. Dialysis was stopped when no radioactivity was detected in the EDC(-) sample (generally after 48-60 h dialysis), indicating that all free polyamine had been removed from both samples. Radioactivity counting was performed using liquid β-scintillation spectrometry (Tri-Carb 1900-TR model, Packard Instruments, Les Ulis, France). The dialyzed EDC(+) F(ab′)2 sample was analyzed for radioactivity and protein

Endocytosis of Polyamine-Modified Antitetanus F(ab′)2

content to determine its polyamine substitution ratio (i.e., the number of coupled polyamine molecules per antibody molecule). Characterization of the Polyamine-Modified F(ab′)2 by Electrophoresis. The control F(ab′)2 and the polyaminemodified F(ab′)2 samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using gels with an acrylamide monomer concentration of either 12% or 8% T and 2.67% C, and a Tris-glycine buffer system (11). Protein samples were solubilized in SDS under reducing conditions with 2-mercaptoethanol, and under nonreducing conditions. This step was followed by electrophoresis and staining with Coomassie Blue R-250. Gels were run at constant voltage with the Mini-Gel apparatus (Bio-Rad), according to the manufacturer’s instructions. Protein standards used were the Precision Plus protein standards from Bio-Rad or the high molecular weight calibration kit from GE Healthcare. Isoelectric focusing (IEF) was performed as described previously (10), using ampholine polyacrylamide gel plates of 1mm thickness, with acrylamide monomer concentration of 5% T and 3% C and 2.2% carrier ampholytes pH 3.5-9.5 (GE Healthcare). IEF standards from GE Healthcare were used containing 11 proteins with isoelectric points in the range of 3.5-9.3. The proteins were visualized with Coomassie Blue R-250. Determination of the Antigen-Binding Activity of the Antitetanus F(ab′)2. An antigen-binding capture enzyme-linked immunosorbent assay (ELISA) for the antitetanus F(ab′)2 (10) was performed on preparations of control and polyaminemodified antitetanus F(ab′)2 to determine their binding activity. The protocol followed was the same as that reported in ref (10). Briefly, the method used tetanus anatoxin coated on microtiter plates as capture antigen (0.5 µg/well) to bind the F(ab′)2, with the amount of antibody binding being quantified using, first, a rabbit biotinylated antihorse F(ab′)2 (60 ng/well), and then a streptavidin-alkaline phosphatase conjugate (0.15 µg/well). Substrate for making the color reaction was p-nitrophenyl phosphate (100 µg/well). A solution of HBSS supplemented with 0.2% (w/v) BSA (HBSS/BSA) was used as assay diluent for the F(ab′)2 samples. Each dilution of samples (over the concentration range 2.5-25 ng/mL for both the control and polyamine-modified F(ab′)2) was assayed in duplicate. Blanks (HBSS/BSA) were included in each assay. The color was developed for 60 min in the dark at room temperature and the enzyme reaction stopped with 1 M NaOH (100 µL/well). Plates were read on a Bio-Tek ELX 800 microplate reader (Winooski, VT) at a wavelength of 405 nm. Linear regression curves were determined by plotting the concentrations of the control or polyamine-modified F(ab′)2 versus the absorbance according to the following equation: y) a×x+b (1) where y is the absorbance at 405 nm, x the antibody concentration in ng/mL, and a and b are the slope and intercept of the curve, respectively. HL60 Cell Assays and Measurement of Cell-Associated F(ab′)2. HL60 cells were grown in suspension cultures as reported (6), using a RPMI 1640 medium with 10% fetal calf serum, and maintained in a logarithmic growth phase at a maximum density of ∼1.5 × 106 cells/mL by subculturing every 3 days. For the assays with the polyamine-modified and control F(ab′)2, the HL60 cells were harvested from cultures by centrifugation at 200 × g (5 min, room temperature), washed three times with RPMI 1640 containing 10 mM Hepes buffer (pH 7.0) and 0.2% (w/v) BSA, and suspended to a density of ∼2 × 106 cells/mL. The cell suspension was divided between two sample sets of polypropylene centrifuge tubes (1 mL/tube): one set was incubated at 4 °C and the other at 37 °C. To each tube was then added 1 mL of dilutions of either the polyaminemodified or the control F(ab′)2 prepared in RPMI 1640/10 mM

Bioconjugate Chem., Vol. 19, No. 8, 2008 1545

Hepes/0.2% (w/v) BSA. Dilutions of each antibody were made up to give F(ab′)2 concentrations of 5, 12.5, 25, or 50 µg/mL. Each antibody level was tested in duplicate. The tubes were then incubated for 120 min at 4 or 37 °C. Kinetic experiments using SPD-F(ab′)2 showed that cellular antibody concentrations reached steady state at 120 min of incubation at 37 °C. The kinetic results were similar to those obtained with PUT-F(ab′)2 in a previous study (10). After incubation, the cells were immediately centrifuged at 200 × g (5 min, 4 °C) and washed three times with ice-cold HBSS to remove the unbound antibody. Cells were then suspended in 1 mL of ice-cold HBSS and finally lysed on ice by ultrasound sonication for 10 s. Blank cell lysates from HL60 cells incubated without antibodies were prepared as stated above. Each cell lysate was then separated into two parts, the first part for the cellular protein assay and the second for the measurement of cell-associated F(ab′)2. The term cell-associated F(ab′)2 includes the surface-bound antibody plus the internalized antibody, if any. Protein determination was performed in duplicate as described above, using BSA (BioRad) as a standard. The concentrations of the antitetanus F(ab′)2 in total cell lysates were determined by ELISA. Prior to this, the total lysates were supplemented with BSA (0.2%, final w/v), and dilutions ranging from 1/2 to 1/50 were then prepared in HBSS also containing 0.2% BSA. The dilutions were assayed by ELISA concurrently with dilutions of a F(ab′)2 standard, i.e., the same F(ab′)2 as that used for the assays with the HL60 cells. Standards were prepared in HBSS/BSA over the concentration range 2.5-25 ng/mL for both the control and the polyamine-modified F(ab′)2. Blanks (HBSS/BSA) were included in each assay. The plates were then processed as described above. A linear regression curve was calculated for the standard and used to compute antibody levels in cell lysates. The amount of cell-associated F(ab′)2 was normalized to milligrams of cell proteins. Inhibition Assays. HL60 cells (∼2 × 106 cells in RPMI 1640/10 mM Hepes/0.2% (w/v) BSA) were incubated for 120 min at 37 °C with 50 µg/mL of control or SPD-modified F(ab′)2 and different concentrations of inhibitor, SPD, or protamine base. SPD was used in the concentration range 1.25-1250 µg/ mL, and protamine in the range 0.205-1025 µg/mL. After incubation, the cells were centrifuged and washed with ice-cold HBSS to remove the excess unbound antibody and inhibitor. The cells in each tube were then suspended in 1 mL HBSS and lysed using ultrasound sonication. Each cell lysate was analyzed for protein content and its antitetanus F(ab′)2 level as already described. The amount of cell-associated F(ab′)2 was normalized to milligrams of cell proteins. The relationship between the amount of cell-associated F(ab′)2 (C) and the concentration of inhibitor (It) was modeled with the following equation C ⁄ Cmax ) IC50 ⁄ (IC50 + It)

(2)

where Cmax is the maximum amount of cell-associated antibody measured in the absence of inhibitor, and IC50 is the theoretical concentration of inhibitor producing a 50% decrease in Cmax. IC50 was estimated with its standard deviation by nonlinear regression analysis using Micro-Pharm software (12). Confocal Laser Microscopy. The polyamine-modified and control F(ab′)2 (each used at a concentration of 50 µg/mL) were incubated with HL60 cells at either 4 or 37 °C, as described above. Following incubation, the cells were centrifuged and washed with ice-cold HBSS to remove the unbound antibody. The cells (∼2 × 106 cells/tube) were then fixed with 1 mL of 4% (w/v) paraformaldehyde in 10 mM phosphate buffer/138 mM NaCl/2.7 mM KCl, pH 7.4, (PBS) for 60 min. All subsequent steps were performed at room temperature, and PBS was used as washing buffer between each step. After washing,

1546 Bioconjugate Chem., Vol. 19, No. 8, 2008

the cells were suspended in 0.4 mL PBS and then immobilized on the glass surface of Lab-Tek II chambers (Nunc, Roskilde, Denmark) previously coated with 0.1% (w/v) poly(L-lysine) (Sigma-Aldrich) in water. The immobilized cells were permeabilized with 0.2% (v/v) Triton X-100 in PBS for 10 min, and the free aldehyde groups then blocked with 0.8% (w/v) glycine in PBS for 30 min. Thereafter, the cells were successively incubated with 2% (v/v) rabbit serum (Sigma-Aldrich) in PBS for 30 min, rabbit biotinylated antihorse F(ab′)2 (8 µg/mL in PBS containing 2% (v/v) rabbit serum) for 60 min in the dark, and finally with a streptavidin-Alexa Fluor 488 conjugate (2 µg/mL in PBS containing 1% (w/v) BSA) for 30 min in the dark. The biotinylated secondary antibody was the same antibody as that used in ELISA. For specific nuclear labeling, the cells were incubated with toto-3 (1 µg/mL in PBS) for 10 min in the dark. The internalization of antibodies was examined using a confocal microscope (Zeiss LSM 510) and a 63/1.4× oil immersion objective. Confocal optical sections were obtained through the nuclear plane of the cells. As controls, we used HL60 cells which were incubated without antitetanus F(ab′)2 at either 4 or 37 °C. These cells were then treated in exactly the same way as those incubated with antibodies. Statistical Analysis. Data analyzed with the Triomphe software (13) are presented as mean ( standard deviation (S.D.). Coefficient of variation (C.V. ) S.D./mean) was used to evaluate variation between measures. To study the variation in antigen binding activity between the different F(ab′)2 preparations, we used a two-way analysis of variance (14) to compare mean values of antibody binding, and an analysis of covariance (15) to compare the slopes of the titration curves of the F(ab′)2. The analysis of variance studied the effects of the reaction pH on the F(ab′)2 cell interaction, as well as the effects of cationization at the different pH values, for each temperature and each antibody. It also studied the variation in amount of cellassociated antibody between the different F(ab′)2, and between temperatures for each antibody. Statistical significance was set at the p < 0.05 level.

RESULTS Polyamine Modification of the Antitetanus F(ab′)2. The antitetanus F(ab′)2 was covalently modified with PUT, SPD, SPM, or HMD, by activating carboxylic acid groups to the reactive ester with water-soluble carbodiimide (EDC), and then by reacting with each polyamine as the nucleophilic reagent (16). A large excess of the polyamines was used in the reaction to ensure that only one primary amine group of the molecules would couple with one carboxylic acid group on the antibody, and thus avoid intermolecular cross-linking and the formation of F(ab′)2 polymers (2). Following the reaction, the excess of polyamine was removed from the polyamine-F(ab′)2 conjugates using a three-step purification procedure. The conditions for the carbodiimide-mediated amidation of the F(ab′)2 carboxylic acid groups were optimized as follows. Because the initial phase of the reaction depends on the ionization of carboxylic acid, preliminary experiments using PUT and [3H]PUT were conducted to adjust the ionization of the F(ab′)2 carboxylic acid groups with pH. As illustrated in Figure 2, reactions at different pH values decreasing from 7.0 to 4.5 demonstrated increasing degrees of modification of the F(ab′)2 which reached a maximum value of ∼3 molecules of PUT coupled per antibody molecule at pH 4.5. Decreasing the pH to values below 4.5 did not further increase the amount of coupling and led to the risk of antibody denaturation. Another variable that can influence the extent of cationization of a protein is the molar excess of polyamine relative to the carboxyl groups (17). Therefore, experiments were conducted where the F(ab′)2 was modified with PUT and [3H]PUT at pH 4.5, using different

Hervé et al.

Figure 2. The effect of pH on the EDC-mediated polyamine modification of the antitetanus F(ab′)2. The average number of PUT molecules coupled per F(ab′)2 molecule (b) estimated at each pH value is shown along with its S.D., and is the result of five independent experiments. Table 1. Polyamine Substitution Ratio of the Antitetanus F(ab′)2a polyamine PUT PUT PUT SPD SPM HMD

pH of the reaction 6.5 5.5 4.5 4.5 4.5 4.5

cationized antibody

substitution ratio

PUT-F(ab′)2 PUT-F(ab′)2 PUT-F(ab′)2 SPD-F(ab′)2 SPM-F(ab′)2 HMD-F(ab′)2

1.11 2.29 2.98 2.68 2.89 nd b

( ( ( ( (

0.16 0.22 0.12 0.11 0.19

a The results are presented according to the polyamine and pH used in the modification reaction. The substitution ratio of the F(ab′)2 corresponds to the number of polyamine molecules coupled per antibody molecule. Mean values are shown in all cases along with their S.D., and are the results of five independent experiments. b nd, not determined.

molar ratios of polyamine over antibody. The amount of coupling was shown to increase almost linearly from 0.02 to 3.2 molecules of PUT per F(ab′)2 molecule when the molar ratio of polyamine over antibody increased from 100 to 10 000. The use of PUT/F(ab′)2 ratios higher than 10 000 resulted in a contamination of the antibody conjugate by free polyamine which could not be removed by two successive filtrations on Omega-50 membrane filter and Bio-Gel 10DG column, followed by dialysis, even when dialysis was continued for more than 7 days. On the basis of these results, the antitetanus F(ab′)2 was modified with PUT at pH 6.5, 5.5, and 4.5. Table 1 shows that these modifications resulted in substitution ratios of roughly 1, 2, and 3 coupled PUT molecules per antibody molecule, yielding a set of antibodies with increasing positive charge valencies due to the number of coupled polyamine molecules. Using the same maximized reaction conditions as for PUT, the F(ab′)2 was modified with either HMD, SPD, or SPM, resulting in a second set of antibodies with different charge valencies due to the different number of corresponding protonated amino groups left after coupling (Figure 1). The substitution ratios determined for the F(ab′)2 modified with SPD and SPM at pH 4.5 and using a molar polyamine/antibody ratio of 10 000 were both close to 3 (Table 1). The coupling was reproducible in all cases (C.V. 0.500). Moreover, these activities were very similar to that determined for the native antitetanus F(ab′)2 in a previous study (10), also using HBSS/BSA as assay diluent. Quantification of Antitetanus F(ab′)2 in HL60 Cell Lysates by ELISA. The quantitative analysis of cationized and control F(ab′)2 which were associated to the HL60 cells following incubation at 4 and 37 °C, respectively, was performed with an ELISA method (10) using total cell lysates. Thus, all cell-associated F(ab′)2, including surface-bound and internalized antibodies, were measured. Because this method is an antigenbinding capture ELISA, the antibodies measured are necessarily active antibodies. The results for the PUT-F(ab′)2 and the corresponding control F(ab′)2 are presented in Figure 6, and those for the HMD-, SPM-, and SPD-F(ab′)2 and their respective controls in Figure 7.

1548 Bioconjugate Chem., Vol. 19, No. 8, 2008

Hervé et al.

Figure 4. Analysis of polyamine-modified antitetanus F(ab′)2 by IEF. (a) Coomassie Blue stain of polyacrylamide gel IEF of PUT-F(ab′)2 modified at pH 4.5 (lane 1) along with the corresponding control F(ab′)2 (lane 4), and PUT-F(ab′)2 modified at pH 5.5 (lane 2) and pH 6.5 (lane 3), and the native F(ab′)2 (lane 5). (b) Coomassie Blue stain of polyacrylamide gel IEF of HMD-F(ab′)2 (lane 1), SPD-F(ab′)2 (lane 2), SPM-F(ab′)2 (lane 3) along with the corresponding control F(ab′)2 (lane 4), and the native F(ab′)2 (lane 5). Twenty micrograms of each F(ab′)2 were applied to the gel in (a), and 25 µg in (b), along with pI standards (lanes 6 in (a) and (b)). The pIs of the standards are shown on the right-hand border.

Figure 5. Titration curves measured by ELISA for the PUT-F(ab′)2 modified at pH 6.5 (b), pH 5.5 (O), and pH 4.5 (9), along with the control F(ab′)2 (0) corresponding to the latter. All antibodies were assayed at concentrations of 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, and 25 ng/mL (duplicate measurements per level). Each titration curve was determined from the data of five different ELISA experiments. The experimental points correspond to mean values which are shown along with their S.D.. The solid lines are computer-estimated values. The equations of the titration curves for the PUT-F(ab′)2 modified at pH 6.5 and 5.5 are y ) -0.0913x + 0.107 and y ) -0.0860x + 0.090, respectively. The coefficients of correlation (r) of these curves were 0.9985 and 0.9976. The equations of the curves for the PUT-F(ab′)2 modified at pH 4.5 and the corresponding control are y ) -0.0729x + 0.099 (r ) 0.9977) and y ) -0.0925x + 0.097 (r ) 0.9988). x is the concentration of the F(ab′)2 in ng/mL, and y is the absorbance. The titration results for the SPD-, SPM-, and HMD-F(ab′)2 modified at pH 4.5 were not significantly different from those obtained with the PUTF(ab′)2 modified at pH 4.5. No significant differences were found between the various control samples.

The amounts of cell-associated F(ab′)2 were greater at 37 than at 4 °C for the cationized and the control antibodies (p < 0.001). The amounts also increased as the initial antibody concentration was increased in the incubation medium (p < 0.001), the effect of concentration being greater at 37 °C than at 4 °C (p < 0.001 for the temperature-concentration interaction). No F(ab′)2 was measured in the blank cell samples (no antibody present).

Effect of the Degree of Antibody Modification on Cell Interaction. As shown in Figure 6, the amounts of PUT-F(ab′)2 associated with the cells increased as the number of coupled PUT molecules increased. The amounts measured at 4 or 37 °C for the F(ab′)2 coupled with three PUT molecules (the antibody modified at pH 4.5) were approximately 1.5 times higher, on average, than that measured for the F(ab′)2 coupled with two PUT molecules (the antibody modified at pH 5.5) and 2 times higher than that coupled with one PUT molecule (the antibody modified at pH 6.5). All the differences observed were significant (p < 0.05). The cell-associated amounts of all the PUT-F(ab′)2 were significantly higher than those of the corresponding control F(ab′)2 at each temperature and antibody level tested (p < 0.05). When the pH of the modification reaction decreased from 6.5 to 4.5, the variation between the cationized and control antibodies was accentuated. Small, albeit statistically significant, differences in cell-associated amounts were observed between the various control F(ab′)2 at both 4 and 37 °C (p < 0.05 for each temperature). These differences increased as the pH decreased. This suggests that the accessibility and/or relative distribution of amino acid side-chain groups on the antibody surface may be slightly changed by the pH of the reaction, thus mildly affecting the interaction of the control antibodies with the cells. Nonetheless, this was seen to have minimal effect on the IEF profiles of the control F(ab′)2 and none on their antigenbinding activities relative to the native antibody. Effect of the Charge Valency of F(ab′)2 Coupled Polyamine. The amounts of cell-associated antibody measured for the F(ab′)2 modified with HMD, SPD, and SPM at pH 4.5 are shown in Figure 7. These antibodies had substitution ratios of approximately 3 coupled polyamine molecules per antibody molecule, which were similar to that of the PUT-F(ab′)2 modified at pH 4.5 (Table 1). Since 1 protonated amino group is left on a HMD or PUT molecule after coupling, and 2 and 3 of these groups are left on SPD and SPM molecules, respectively (Figure 1), the expected number of positive charges associated with these polyamines after coupling should increase in the order of HMD or PUT < SPD < SPM, with a corresponding increase in the antibody net charge valency of +3, +6, and +9, respectively. At all antibody concentrations tested, both at 4 and at 37 °C, the amounts of cell-associated HMD-F(ab′)2 (Figure 7) were similar to those measured for the PUT-F(ab′)2 modified at pH 4.5 (C.V.