Carrier Design: New Generation of Polycationic Branched

Bioconjugate Chem. 1999, 10, 781−790

781

Carrier Design: New Generation of Polycationic Branched Polypeptides Containing OH Groups with Prolonged Blood Survival and Diminished in Vitro Cytotoxicity Ferenc Hudecz,*,† Malcolm V. Pimm,‡ E Ä va Rajnavo¨lgyi,§ Ga´bor Mezo _,† Angels Fabra,| Dezso _ Gaa´l,⊥ Attila L.Kovaćs,# Attila Horva´th,§ and Ma´ria Szekerke† Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Budapest 112, POB 32, H-1518, Budapest, Hungary, Cancer Research Laboratory, University of Nottingham, Nottingham NG7 2RD, U.K., Department of Immunology, Eo¨tvo¨s L. University, Go¨d, Hungary, Cancer Research Institute, Hospital Duran I Reynals, Barcelona, Spain, National Institute of Oncology, Budapest, Hungary, and Department of General Zoology, Eo¨tvo¨s L. University, Budapest, Hungary. Received February 10, 1999; Revised Manuscript Received May 12, 1999

For the construction of macromolecule-drug conjugates, it is important to provide rational basis to the selection of proper carrier. With respect to the importance of the side-chain structure and charge of the branched polypeptides in biological properties, we have prepared a new class of branched polypeptides with single or multiple hydroxyl groups and studied their solution conformation, in vitro cytotoxicity, biodistribution, and immunoreactivity. For comparative studies, polypeptides were designed to contain serine at various positions of the side chains, varying also the number. Ser was attached to the end of oligo(DL-Ala) side chains grafted to polylysine resulting polypeptides with the general formula poly[Lys(Seri-DL-Alam)], (SAK). Ser was also coupled directly to the polylysine backbone poly[Lys(Seri)] (SiK) and then elongated by polymerization of N-carboxy-DL-Ala anhydride resulting poly[Lys(DL-Alam-Seri)] (ASK). An additional polymer was also prepared, but instead of the oligo(DLAla) branches, oligo(DL-Ser) side chains were introduced (poly[Lys(DL-Serm)], SK). The presence of hydroxyl groups resulted in compounds with improved of water solubility. CD spectra of polypeptides showed significant differences correlating with the position and numbers of Ser residues in the side chains. Under physiological conditions, polycationic polypeptides assumed ordered secondary structure (SiK and LSK) or partially unordered conformation (SK, SAK, and ASK). Data of selected polymers demonstrate that these polycationic compounds are essentially nontoxic in vitro on normal rat liver or mouse spleen cells and have no cytostatic effect on mouse colorectal carcinoma C26 cells. The blood clearance and biodistribution of these derivatives were greatly dependent on the position and number of Ser residues in the branches and possess a rather extended blood survival in mice. Polypeptides were taken up predominantly by the liver and kidney (SiK, LSK, and ASK) or kidney and lung (SK and SAK). The best survival in the blood was found with SAK, representing the first polycationic branched polypeptide, which show extended blood clearance. The relative position of Ser residue had also a marked influence on the immunogenicity of polypeptides. The characteristics of the antibody response to polypeptide containing Ser at the end of the branches (SAK) or adjacent to the polylysine backbone (ASK) was also dependent on the genetic background of the mouse strains. We also found that these compounds have no effect on to the SRBC-specific humoral immune response, indicating the lack of nonspecific immunostimulatory potential. In conclusion, these studies suggest that synthetic branched polypeptides with Ser can be considered as candidates for constructing suitable conjugates for drug/epitope delivery. It is not only due to the presence of hydroxyl group to be used for oxime chemistry but also to their beneficial biological features.

INTRODUCTION

There is an increasing interest in the development of target-specific delivery systems for various drugs and isotopes as well as preparation of long-acting drugmacromolecule conjugates (1-3). Appropriate carrier molecules are required also for the construction of * To whom correspondence should be addressed. Fax: 36-12090602. Phone: 36-1-2090555. E-mail: [email protected]. † Hungarian Academy of Sciences. ‡ University of Nottingham. § Department of Immunology, Eo ¨ tvo¨s L. University. | Cancer Research Institute. ⊥ National Institute of Oncology. # Department of General Zoology, Eo ¨ tvo¨s L. University.

synthetic antigens for vaccination and/or for the production of conventional or monoclonal hapten specific antibodies (4, 5). In our laboratory different types of branched polypeptide have been synthesized to establish structurefunction relationship (6, 7). The synthetic branched polymeric polypeptides were characterized by their chemical (size, primary structure, and conformation) and biological (toxicity, pyrogenicity, biodegradation, immunoreactivity, and biodistribution) properties. Correlation has been demonstrated between the side-chain structure of polymers and some biological activities such as biodistribution (8) or in vitro cytotoxicity (9). Branched polypeptides were also used successfully as carriers for peptide epitopes of glycoprotein D of Herpes

10.1021/bc990015q CCC: $18.00 © 1999 American Chemical Society Published on Web 08/28/1999

782 Bioconjugate Chem., Vol. 10, No. 5, 1999

Hudecz et al.

Figure 1. Schematic presentation of branched polypeptides.

Simplex virus (10), mucin 1 glycoprotein (11), and of M. tuberculosis proteins (12). Antitumor activity of daunomycin-EAK1 conjugate (13, 14) or Ac-[D-Trp1-3, D-Cpa2, D-Lys6, D-Ala10]-GnRH-AcEAK conjugate (15, 16) has shown very promising results. These studies have also demonstrated that the biological properties (e.g., epitoperelated immunogenicity or drug-related antitumor effect) and pharmacokinetics (e.g., blood survival, tissue distribution) of conjugates are greatly influenced by the chemical structure of the carrier. Several authors have reported the application of poly(ethyleneglycol) (PEG) or PEG-containing polymers as water soluble drug carriers (17, 18). It has been reported that PEG attached to proteins or synthetic polymers can increase their water solubility, blood survival, and/or lower the immunogenicity. In view of these observations published in the literature, we have designed and synthesized a new class of branched polypeptides with single or multiple hydroxyl groups (19). For comparative biodistribution, in vitro cytotoxicity and immunological studies, these polypeptides were designed to contain serine at various positions 1 Abbreviations: AXK, poly[Lys(DL-Ala -X )]; ASK, poly[Lysm i (DL-Ala6.5-Ser0.88)]; AK, poly[Lys(DL-Alam)]; AUC, area under the curve; CD, circular dicroism; DMSO, dimethyl sulfoxide; DPn, number average of the degree of polymerization; EAK, poly[Lys(Glui-DL-Alam)]; ESK, poly[Lys(Glu0.96-DL-Ser3)]; FCS, foetal calf serum; LAK, poly[Lys(Leui-DL-Alam)]; MTT 3-(4,5 dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; NCA, N-carboxyanhydride; PBS, phosphate-buffered saline; PEG, poly(ethyleneglycol); PFC, plaque forming cell; RPMI, Roswell Park Memorial Institute; SAK, poly[Lys(Ser0.96-DL-Ala3.1)]; SK, poly[Lys(DL-Ser3.1)]; SiK, poly[Lys(DL-Ser0.96)]; SRBC, sheep red blood cell; XAK, poly[Lys-(Xi-DL-Alam)].

of the side chains, varying also the number of the Ser residues (Figure 1). Serine was attached to the end of oligo(DL-Ala) side chains grafted to polylysine resulting polypeptides with the general formula poly[Lys(Seri-DLAlam)] (SAK). Ser was also coupled directly to the polylysine backbone poly[Lys(Seri)], (SiK) and then elongated by polymerization of N-carboxy-DL-Ala anhydrideresulting poly[Lys(DL-Alam-Seri)], (ASK). An additional polymer was also prepared, but instead of the above used oligo(DL-Ala) branches, oligo(DL-Ser) side chains were introduced (poly[Lys(DL-Serm)], SK) (19). The presence of hydroxyl functional groups could provide a new feature for oxime-based conjugation (20, 21) of branched polypeptides with appropriate biologically active molecules leading to compounds with improved water solubility. In this communication, we describe new branched polypeptides with a rather extended blood survival in mice. Data of selected polymers demonstrate that these compounds are essentially nontoxic in vitro on normal rat liver or mouse spleen cells and have no cytostatic features on mouse colorectal carcinoma C26 cells. The results of immunological experiments presented in this paper indicate that the position of Ser residue in the branches influences markedly the immunoreactivity of these polymers. EXPERIMENTAL SECTION

Abbreviations. Abbreviations for amino acids and their derivatives follow the revised recommendation of the IUPAC-IUB Committee on Biochemical Nomenclature, entitled “Nomenclature and Symbolism for Amino Acids and Peptides” (22). The nomenclature of branched polypeptides is used in accordance with the recommended nomenclature of graft polymers (23). For the sake of

Polypeptides with Blood Survival and Diminished Cytotoxicity

Bioconjugate Chem., Vol. 10, No. 5, 1999 783

Table 1. Characteristics of Branched Polypeptides amino acid compositionb polypeptide

codea

Lys

poly[Lys(Seri-DL-Alam)] poly[Lys(Seri)] poly[Lys(DL-Alam-Seri)] poly[Lys(DL-Serm)] poly[Lys(Leui-DL-Serm)] poly[Lys(Glui-DL-Serm)]

SAK SiK ASK SK LSK ESK

1.00 6.58 1.00 1.00 0.93 1.00 6.50 0.88 1.00 2.93 1.00 3.11 1.0 1.00 3.0 0.93

Ala

Ser

Xc

M h wd 191 200 58 600 186 700 107 300 149 300 142 700

a Code of branched polypeptides, based on one letter symbol of amino acids. b Determined by amino acid analysis after hydrolysis in 6 M HCl at 105 °C for 24 h. c X ) Leu or Glu. d Calculated from the number average degree of polymerization of polylysine (DPn ) 280) and from the amino acid composition of the branches.

brevity, codes of branched polypeptides were constructed by us using the one-letter symbols of amino acids (Table 1). All amino acids are of the L configuration unless otherwise stated. Polypeptides. Branched polypeptides containing Ser residues were prepared in our laboratory by previously (19) published methods. Briefly, poly(L-Lys) was produced by the polymerization of NR-carboxy-N-(benzyloxycarbonyl)-lysine anhydride under conditions that allowed an average degree of polymerization of ∼300. After cleavage of the protecting groups with HBr in glacial acetic acid, poly[Lys(DL-Ala6.5)] (AK), or poly[Lys(Ser2.9)] (SK) were formed by grafting of short oligomeric DL-Ala or DL-Ser side chains onto the -amino groups of polylysine using the respective N-carboxy-DL-amino acid anhydride (NCA). The side chain of DL-Ser was protected by the benzyl group, which was removed with HBr/glacial acetic acid. For poly[Lys(Seri-DL-Alam)] (SAK), attachment of suitably protected and activated Ser residues to AK was carried out by the active ester method. Similarly, SK was reacted with Z-Leu-OPcp or with Z-Glu(OBzl)-OPcp to produce poly[Lys(Leui-DL-Serm)] (LSK) or poly[Lys(Glui-DL-Serm)] (ESK), respectively. Poly[Lys(Seri)] (SiK) was prepared by the attachment of protected and activated Ser residue to the -amino groups of polylysine followed by the removal of protecting groups, while poly[Lys(DL-Alam -Seri)] (ASK) was synthesized from SiK by polymerization of DL-Ala-NCA. After the deblocking, branched polypeptide samples were purified by extensive dialysis using Visking tubing followed by lyophilization. The removal of blocking groups was verified by UV spectroscopy (19). The average molar mass of the branched polypeptides was estimated from the number average degree of polymerization (DPn) of polylysine backbone (24) and from the amino acid composition of the side chains determined by quantitative amino acid analysis (Table 1). The synthesis of LSK and ESK is described in detail. Synthesis of Poly[Lys(Leui-DL-Serm)] and poly[Lys(Glui-DL-Serm)] (LSK and ESK). One and a half grams of DL-Ser(Bzl)-NCA (7.3 mmol) was dissolved in 3 mL dioxan, and it was added to 300 mg of polylysine‚ HBr (1.4 mmol) dissolved in 3 mL of deionized water and neutralized with 200 µL of triethylamine. The reaction mixture was stirred for 45 min and then allowed to stand for 24 h. The poly[Lys(DL-Ser(Bzl)m)] sample was dialyzed for 2 days and freeze-dried. The product was dried over P2O5 for 24 h, and then the Bzl-protecting groups were removed by 35% HBr/acetic acid (50 mL) in an overnight reaction at 4 °C. The product was precipitated by addition of dry ether (100 mL). The precipitate was washed with ether several times and dried over P2O5 and KOH. The poly[Lys(DL-Serm)] (SK) sample dissolved in deionized

Figure 2. CD spectra of branched polypeptides at pH 7.3 in PBS: SiK (s), SAK (- - -), ASK (-‚-‚), SK (-‚‚-‚‚), and LSK (O).

water was dialyzed in Visking tube (cutoff 8000-12000) against deionized water for 2 days and freeze-dried. A total of 115 mg (0.25 mmol) of SK was dissolved in 1 mL of deionized water, and then the solution was diluted with 5 mL of DMF. One and the half equivalent amount of Z-Leu-OPcp (193 mg; 0.375 mmol) or Z-Glu(OBzl)-OPcp (233 mg; 0.375 mmol) dissolved in 3 mL of DMF was added to the polymer solution. Equivalent amount of HOBt (61 mg, 20% water content) calculated according to the active ester derivatives was added in 1 mL of DMF to the reaction mixture. Finally, the pH of the solutions was adjusted to 7.5-8.0 with N-methylmorpholine. The mixture was stirred at RT for overnight, then the solvent was removed by evaporation. The remaining oil was solidified with ether, and the precipitate was washed several times with 10% DCM/ether, respectively. The dried product was suspended in 2 mL of abs. acetic acid and was treated with 20 mL of 35% HBr/acetic acid for overnight at 4 °C. The deprotected polymer samples were precipitated by addition of dry ether. The products were worked up as described above in case of SK polymer. Amino Acid Analysis. The amino acid composition of polypeptides was investigated by amino acid analysis using a Beckman (Fullerton, CA) model 6300 amino acid analyzer. Prior to analysis, samples were hydrolyzed in 6 M HCl in sealed and evacuated tubes at 110 °C for 24 h. Circular Dichroism Spectroscopy. Circular dichroism (CD) was measured with a Jobin-Yvon Mark VI dichrograph (Jobin-Yvon, Longjumean, France). CD spectra from 195 to 260 nm were recorded in a quartz cell with optical path of 0.02 cm at room temperature under constant nitrogen flush. The dichrograph was calibrated with D-(-)-pantoyllactone at 220 nm (25). CD spectra were recorded in PBS solution at pH 7.3, the concentrations of solutions being about 0.5 mg/mL (Figure 2.). The data of CD in the figure are expressed in terms of ∆, calculated for one Lys residue in the polypeptide backbone carrying an average side chain. The interpretation of CD spectra was based on the CD curves of polylysine (26), from which the branched polypeptides have been derived. The CD spectrum characterized by negative maxima at 208 and 221 nm of about the same intensity were considered helical, while the unordered structure is indicated with a strong negative maximum at 199 nm,


a weak negative maximum at 234 nm, and a weak positive maximum at 218 nm (7). In Vitro Cytotoxicity Assays. In vitro toxicity was evaluated by measuring the viability of isolated rat liver (method A) or mouse spleen (method B) cells after incubation with various amount of polypeptides. Method A. Isolated rat liver cells were prepared by the collagenase perfusion method of Seglen (27). Cells were incubated with varying amounts of polymers in rapidly shaking centrifuge tubes in suspension buffer (28) for 1 h at 37 °C. Controls were incubated with buffer alone. Viability of liver cells was measured by the trypan blue exclusion test. Three parallel samples were incubated for each polymer at each concentration, and 200-300 cells were counted for staining with trypan blue. Percentage viability was calculated from the mean of the three measurements (28). Method B. Spleen cells were prepared from spleens of six 10-11 week old 22-24 g female BDF1 mice and resuspended in RPMI medium (28). Cells were taken at 3 mL (6 × 107 cells/mL) and incubated at 37 °C for 1 or 4 h. Polypeptides dissolved in PBS were diluted with RPMI. Each dilution was added in 100 µL to triplicate culture tubes, the concentration stated in the Results being the final concentration in the test tube. Control culture tubes were treated with 100 µL of RPMI alone. Viability of spleen cells was measured by the trypan blue exclusion test. Three parallel experiments were carried out with each polypeptide at each concentration. Percentage viability was calculated from the mean of the three measurements. Values for cell samples are given as percentage of mean of control samples incubated without any compound. Cytostatic Assay. In vitro cytostatic properties were evaluated by measuring the viability of mouse colorectal carcinoma C26 cells after incubation with various amount of polypeptides. C26 cells were grown in tissue culture flasks at 37 °C in a humidified atmosphere in DMEM: Ham F12 medium and supplemented with 10% (v/v) heat inactivated foetal bovine serum (Biological Industries, Israel). Routine test showed the cells to be free of Mycoplasma. C26 cells in logarithmic growth were trypsinized, resuspended in medium, and adjusted to a concentration of 1.5 × 104 cell/mL. A total of 200 µL of cell suspension was plated in a 96-well-flat-bottom tissue culture grade microtiter plates (Costar, Cambridge MA) and incubated at 37 °C for 24 h. A total of 100 µL of polymer solutions in serum-free medium at different concentrations was added in quadruplicate. After incubation at 37 °C for 3 h, medium was removed by aspiration, the cells were washed twice, and 200 µL of fresh complete medium containing FCS was added. Incubation for an additional 72 h was followed by the viability measurements using MTT assay (29). The absorbance was measured at λ ) 540 nm using a microplate reader (Titertek Multiskan). Absorbance of treated wells was compared to the control wells corresponding to the metabolically active cells. Cytostasis was calculated as a measure of growth inhibition, expressed as percentage of controls and calculated by the formula (100 - absorbance of treated wells) x 100/ absorbance of control wells. Labeling with 125I. 125I labeling of polypeptides was carried out with N-succinimidyl 3-(4-hydroxy-phenyl)propionate prelabeled with 125I (Amersham International plc, Amersham, U.K.) as described before (3). The labeled polypeptides were purified of unreacted [125I]sodium iodide by passage through Sephadex G-25, elution being in PBS at pH 7.2, using prepacked PD-10 columns

Hudecz et al.

(Pharmacia, Milton Keynes, U.K.). Electrophoresis on native polyacrylamide gel with a continuous 8 to 25% gradient was applied to assess the low molecular weight labeled product content of the preparation. The labeling efficacy was 35-40%. Specific activities of the final products were ∼2 MBq/mg. Blood Clearance and Biodistribution Studies. All in vivo studies were carried out in adult (∼20 g) female BALB/c mice (Biomedical Services Unit, University of Nottingham) with appropriate licenses from the U.K. Home Office and with due consideration for animal welfare. Groups of mice (n ) 4) were injected intravenously via a tail vein 5-10 mg of labeled polypeptides in 0.2 mL of PBS. Serial blood samples (10 mL) were taken from the tail tip at 1, 10, and 30 min and at 1, 2, and 3 h after injection directly into microcapillary pipets (Drummond Microcaps, Drummond Scientific Co., Broomhall, PA). Blood clearance curves were constructed of the percent of the total initially injected count rates in the total blood volumes against time. The total intravascular blood volumes were calculated assuming the blood volume of the mice (in milliliters) to be 11.2% of the body weight (in grams). Areas under curves (AUC), as percent dose per hour, were calculated using the trapezoidal rule. At 4 h after injection, mice were killed, and weighed samples of blood, visceral organs, and residual carcass were assayed for radioactivity. Earlier comparative studies with 125I, 111In, and 51Cr labeled branched polypeptides showed no significant differences in radioactivity in tissue samples (31). On the basis of these data, we assumed that 125 I-labeled marker is associated with the polypeptide during the study period. Results of the tissue-distribution analysis were expressed as a percentage of the total initially injected count rate recovered per gram of tissue. Immunization. C57/Bl/6 (H-2b, Igh-1b), CBA (H-2k, Igh-1e), and BALB/c (H-2d, Igh-1a) mouse strains (LATI, Go¨do¨llo¨, Hungary) were used for immunization. One experimental group consisted of six 10-12-week old male mice. Twenty micrograms of synthetic polypeptide dissolved in sterile saline (50 µL, 2 mg/mL) was emulsified in complete Freund’s adjuvant (1:1, v/v) and injected sc into the backpaws and also ip. Serum samples were taken 7 and 14 days after antigen administration and titrated for IgM and IgG type antibodies and for IgG subclasses. Four weeks later the primed mice were boosted with the same amount of antigen emulsified in incomplete Freund’s adjuvant (1:1, v/v) and administered as before. Serum samples were collected 20 days after the injection. A third challenge was given ip in PBS on day 55, and blood samples were taken 14 days after antigen administration. Measurements of Antibody Titers by Solid-Phase Indirect Enzyme Immunoassay. Detection of the antigen-specific serum antibodies was performed by solidphase indirect enzyme immunoassay using antigencoated plates as described previously (9). SAK or ASK were coated on the surface of PVC plates (Propileń, Pećs, Hungary) at 5 µg/mL. After the washing and blocking procedures, various dilutions of the immune sera of mice were added. The bound antibodies were detected by horseradish-peroxidase-labeled anti-mouse IgM or IgG antibodies (Sigma, St. Louis, MO). Antibody titers were characterized by serum dilution corresponding to a fixed OD value (∼30%) chosen from the linear phase of the binding curve. For specificity studies, SAK or ASK were coated to the surface of the plates at graded concentrations (0.1-10 µg/mL) and a pretitrated dilution of pooled sera were added. For titration of IgG subclasses antibody


binding was developed by biotin-labeled subclass specific antibodies (Southern Biotechnology Associates Inc., Birmingham, AL). Titers were calculated from four or five independent titrations and mean values (SD were given. Results of separate experiments were compared on the basis of reference sera used in all tests. Immunomodulatory activity. Sheep red blood cells (SRBC) as immunogen were injected at 108 cell/0.2 mL of sterile saline ip into 10-12 week old mice. Control mice were injected with 0.2 mL of saline. The branched polypeptides and levamisole as potential immunomodulators were used at 0.2 and 1.0 mg/kg of body weight and were administered simultaneously with SRBC. The hemolytic plaque-forming cell (PFC) assay, first described by Jerne and Nordin (32) and modified by Behling and Nowotny (33), was applied for the assessment of immunomodulatory ability of polypeptides. Spleens from each treatment group of five mice were obtained 4 days after immunization with SRBC and pooled. The washed spleen cells were resuspended in TC199 medium, filtered, and adjusted to concentrations of 1:10 and 1:100 (v/v) for plating. Two milliliters of a 0.7% Noble agar solution, containing 0.1 mL of 1% DEAE dextran, 0.1 mL of a 10% suspension of freshly washed SRBC, and 0.1 mL of any of the various spleen cell concentrates were carefully mixed at 45 °C and layered on plates covered with 4 mL of 1.4% solidified but prewarmed agar. The plates were incubated for 60 min at 37 °C, and 2.5 mL of guinea pig complement diluted with 1:10 (v/v) balanced salt solution was added. The plates were incubated for an additional 30 min. Hemolyzed plaques in the agar were counted in triplicate and analyzed blind. RESULTS AND DISCUSSION

In the present study, the development of a new series of branched polypeptides, which contain hydroxyl groups in their side chains, is reported. Ser was attached by HOBt-catalyzed active ester method to the N-terminals of oligo(DL-Ala) chains grafted to a polylysine backbone resulted poly[Lys(Seri-DL-Alam)] (SAK). Ser was coupled also directly to the -amino groups of polylysine followed by polymerization of DL-Ala NCA resulting oligo(DL-Ala) chain terminals. In this way, a reverse sequence was built up in the side chains corresponding to the poly[Lys(DLAlam Seri)] (ASK). The number of hydroxyl groups in the polymer was increased by oligo(DL-Ser) branches instead of oligo(DL-Ala) ones [poly[Lys-(DL- Serm)] (SK)] (Figure 1). Solution Conformation. Classification of solution conformations as an important property of branched polypeptides was carried out by CD spectroscopy. The CD curves of SiK, SK, SAK, ASK, and LSK in PBS at pH 7.3 are shown in Figure 2. In the 190-250 nm wavelength region, the circular dichroic spectra, originated from the optical activity of amide bonds, indicate the formation of a helical secondary structure for SiK and LSK, but only partially ordered conformation for SAK, ASK, and SK (Figure 2). It should be noted that the CD spectrum of polypeptide AK corresponds to a disordered conformation (35). The incorporation of hydroxyl groups in side chains of branched polypeptides resulted in slightly more ordered conformation than observed with oligo(DL-Ala) side chains. In line with the hydrophilic character of the Ser residue(s), the aggregation of polymers was not detectable neither in solution nor after storage in solid state. Branched polypeptides with a number of hydroxyl groups


described in this paper represent a new class of polycationic biodegradable polymers (36) with significantly increased water solubility. Polycationic polypeptide, LSK, assumed essentially ordered (helical) secondary structure (Figure 2). Effect of Polypeptides on the Viability of Isolated Liver and Spleen Cells. In vitro cytotoxicity against isolated rat liver and mouse spleen cells was investigated and compared with data obtained earlier using the same cellular assays (28). Polypeptides at various concentrations (1.5-50.0 µg/mL) were incubated with rat liver cells or with mouse spleen cells, and the percentage of cell viability was determined (Figure 3, panels A and B). As reported previously, poly(L-Lys) proved to be rather toxic to the liver cells in the above concentration range (28). The present set of data indicate that the attachment of a single Ser residue to the -amino group of polylysine resulted in an essentially nontoxic polypeptide, SiK. Cell toxicity of AK for spleen or liver cells was also reported earlier at c ) 50 µg/mL concentration (28). In contrast, treatment with SK containing oligo(DL-Ser) instead of oligo(DL-Ala) did not reduce the viability of liver or spleen cells (Figure 3, panels A and B). ASK on spleen cells and LSK on liver cells were marginally toxic (5% decrease), while in case of other polypeptides, the number of viable liver or mouse cells even after 4 h incubation period was almost identical to the controls. Effect on Growth of C26 Cells. Estimation of cytotoxicity was extended to the evaluation of the growth of C26 mouse colorectal carcinoma cells in the presence of various amounts of polycationic polymers. Cytostatic effect of polymers was determined by MTT assay (29). Results are demonstrated in Figure 3C. As described previously, poly[L-Lys] was shown to retard the growth of various tumor cells (9, 34). The treatment of C26 cells with poly[L-Lys] resulted in a similar effect. Elongation at the -amino groups by oligo(DL-Ala) branches in AK reduced the cytostatic effect by more then 1 order of magnitude. Similar effect was observed with ASK. Interestingly, SiK the backbone of ASK exhibit greatly reduced cytostatic effect as compared to poly[L-Lys], but the extension of Ser with oligo(DL-Ala) resulted in an elevated growth (Figure 3C). In contrast to ASK, much diminished cytostatic effect could be demonstrated with polypeptide SAK containing one Ser residue attached to oligo(DL-Ala) side chains. Poly[Lys(DL-Serm)], comprising only oligo(DL-Ser) branches exhibited no cytostatic effect in the concentration range studied. Our data summarized in Figure 3 clearly suggest that there is a correlation between the number and location of Ser residues and cell killing activity of branched polypeptides. The incorporation of hydroxyl amino acid(s) could reduce markedly the in vitro cytotoxic effect of polycationic polypeptides. Biodistribution of Polymeric Polypeptides. 125Ilabeled polypeptides were prepared by introduction of N-succinimidyl 3-(4-hydroxyphenyl)propionate containing 125 I onto the R-amino groups of the side-chain terminals as described before (30). The blood clearance profile and tissue distribution of polypeptides after iv administration are depicted in Figure 4 and Table 2. We found no significant difference in the area under the curve (AUC 0-4 h) for polypeptides prepared with either single Ser residue (SiK) adjacent to the polylysine backbone or its oligo(DL-Ala) elongated version (ASK) (Figure 4A). Percentage of the total injected dose 4 h after injection was somewhat elevated for SiK (38.3 ( 3.1% injected dose vs 27.8 ( 3.6% injected dose for ASK). Besides an increased kidney uptake of SiK, the tissue distribution profiles of


Hudecz et al.

Figure 3. Effect of branched polypeptides on viability of isolated rat liver cells (A), of isolated mouse spleen cells (B) or on C26 carcinoma cell growth (C).

Figure 4. Blood clearance curves of 125I labeled branched polypeptides. (A) SiK, SK, AK, SAK, ASK; (B) LSK, LAK, ESK, EAK. Four mice/group. The areas under these time: activity curves are shown in Table 2. Table 2. Biodistribution Data of Branched Polypeptides code

AUC; 0-4 h (% dose h ( SD)

percent of the total injected dose at 4 h (% dose ( SD)

blood

SAK ASK SK SiK LSK ESK

177.5 ( 7.6 58.7 ( 6.3 97.1 ( 2.6 66.3 ( 4.0 95.8 ( 1.6 161.8 ( 3.0

32.9 ( 4.2 34.6 ( 3.7 27.8 ( 3.6 38.3 ( 3.1 47.7 ( 2.7 41.4 ( 1.2

11.0 ( 0.8 2.6 ( 0.1 3.7 ( 0.3 3.3 ( 0.3 6.0 ( 0.3 13.9 ( 0.7

these polymers were similar (Table 2.), both compounds being taken up predominantly by kidney and liver. The presence of multiple Ser residues in the branches (SK) resulted in significantly increased blood survival (Figure 4A) with an AUC of 97.1% dose/h (Table 2). Interestingly, branched polypeptide with oligo(DL-Ala) branches (AK) in accord with earlier published data was cleared rapidly from circulation with an AUC of only 17.8% dose/h (8).

percent of the total injected dose (( SD)/gram of spleen kidney liver lung 1.8 ( 0.3 3.1 ( 0.5 1.0 ( 0.2 2.7 ( 1.4 5.7 ( 0.7 4.0 ( 0.3

5.9 ( 0.9 4.6 ( 0.5 3.2 ( 0.2 13.0 ( 1.9 19.5 ( 1.5 11.8 ( 0.7

2.3 ( 0.3 8.7 ( 1.1 1.5 ( 0.1 9.3 ( 1.1 14.5 ( 0.7 11.5 ( 0.5

3.9 ( 0.4 1.4 ( 0.3 3.0 ( 1.0 3.1 ( 1.2 3.8 ( 0.4 4.8 ( 0.3

carcass 1.4 ( 0.2 0.9 ( 0.3 1.2 ( 0.1 0.9 ( 1.2 1.0 ( 0.8 1.2 ( 0.1

Elongation of the side chains of AK by a single Ser residue led to a dramatic increase in blood survival (AUC of 177.5% dose/h). The major sites of retention of 125Ilabeled SAK at the 4 h dissection time were the kidney with about 6% of the injected dose/g and the lung with about 4% (Table 2). As described earlier, the branched polypeptide containing Leu at the end of the oligo(DL-Ala) branches (LAK)



Figure 5. Magnitude of the IgG type memory antibody responses C57/BL/6, CBA and BALB/c mice to synthetic branched polypeptides SAK (A) or ASK (B). Table 3. Antibody Responses Induced by Branched Polypeptides in Inbred Mouse Strains SAK

ASK

strain

IgM

IgG (primary)

IgG (memory)

IgM

IgG (primary)

IgG (memory)

C57/Bl/6 CBA BALB/c

457 ( 86a 583 ( 47 667 ( 51

111 ( 18 2210 ( 459 1122 ( 8

107 ( 4 >20 000 1459 ( 5

247 ( 42 222 ( 47 3119 ( 190

143 ( 6 147 ( 6 10 321 ( 1651

125 ( 7 145 ( 10 19 688 ( 2473

a Antibody titer ( SD. Pooled serum samples taken at different points of time were titrated using solid-phase indirect immunoassay as described in the Experimental Section. Mean values ( SD of three parallel titrations are given.

Figure 6. The distribution of different IgG subclasses during the memory antibody response of BALB/c mice immunized with ASK. The relative levels of specific antibody isotypes was determined as described in the Experimental Section.

was cleared rapidly from circulation (8). In contrast, LSK with oligo(DL-Ser) showed a longer blood survival (Figure 4B) with an AUC of 95.8% dose/h and the whole body retention was the highest value observed among polypeptides with Ser residue (47.7% dose/h) (Table 2). The blood clearance curve and AUC of amphoteric [125I]EAK (8) was virtually identical to that of the labeled polypeptide ESK with oligo(DL-Ser) in the branches (Figure 4B, Table 2). It should be noted that the amphoteric ESK displayed greater blood survival than its polycationic counterpart LSK (Figure 4B). There was also a different pattern of tissue biodistribution, with reduced levels in the lung (3.8% for LSK vs 4.8% for ESK) but higher levels in spleen and liver (19.5% and 14.5% for LSK vs 11.8% and 11.5% for ESK) (Table 2.) The results of this comparative study suggest that the survival of polycationic polypeptides is prolonged, and their tissue distribution is altered by the incorporation

of Ser residue into the side chains. The substitution of Ala residues by Ser ones (AK vs SK, LAK vs LSK) elevated significantly the blood circulation time. Interestingly enough, the relative position of Ser in the branches influenced markedly the biodistribution of the polycationic polypeptides. The most favorable blood clearance profile and tissue distribution were obtained with SAK in which Ser is situated at the end of the side chain. For the evaluation of these findings, it should be borne in mind that this is the first branched polypeptide reported in the literature which is capable to circulate in the blood for an extended period of time despite its polycationic feature. This observation has to be taken in consideration for the design of future conjugates. Immunoreactivity of branched polypeptides. The immunoreactivity of two representatives of polycationic Ser-containing polypeptides was characterized by their immunogenicity and immunomodulatory properties. In the first set of experiments SAK and ASK were used as immunogen and their capability of provoking antibody responses was investigated. In another assay we studied the effect of these polypeptides on sheep red blood cell (SRBC) antigen-induced immune responses. Antibody Response. The immune response induced by the branched polypeptides was studied in three inbred mouse strains of different H-2 haplotype and Igh-1 allotype (37). The qualitative and quantitative features of the antibody response of polycationic polypeptides were characterized by IgM- and IgG-type antibody levels (Figure 5., Table 3), IgG isotype distribution (Figure 6), and fine specificity of antibodies (Figure 7) produced during the primary and memory response of BALB/c, CBA, and C57/Bl/6 mice. The magnitude of the IgM and IgG type antibody response was measured at different points of time. As summarized in Table 3 and in Figure 5, SAK and ASK induced negligible level of IgM or IgG type antibodies in C57/Bl/6 mice even after the secondary antigen challenge. After repeated immunization of BALB/c mice with SAK or CBA mice with ASK, the antibody titers were rela-


Hudecz et al.

Figure 7. Cross reactivity pattern of antibodies induced by ASK in BALB/c mice (A) and by SAK in CBA mice (B). Mean antibody titers (SD measured in the serum of immunized mice taken 14 days after antigen challenge on ASK- or SAK-coated plates.

tively low. Increased levels of IgG-type antibodies could be only detected after the third injection of ASK to BALB/c and of SAK to CBA mice (Figure 5). These findings indicate that the immunogenicity of branched polypeptides is highly dependent on the responsiveness of the inbred mouse strain (e.g., SAK is capable to elicit antibody response in CBA, but this compound is nonimmunogenic in C57/Bl/6 and poor immunogen in BALB/ c) as well as on the amino acid sequence of the branches (e.g., SAK is not immunogenic, while ASK induced antibody response in BALB/c). The elevated level of ASKand SAK-specific IgG isotypes in memory response of BALB/c and CBA mice of different MHC class II haplotypes (H-2d vs H-2k) point to a helper T-cell-dependent antibody response against these antigens (Figure 6). Fine specificity of the polyclonal IgG type antibodies after hyperimmunization with ASK in BALB/c or with SAK in CBA was also studied (Figure 7). We found that the ASK-induced antibodies of BALB/c mice react with ASK but do not recognize SAK adsorbed to the solid phase (Figure 7A). Similarly, antibodies raised by SAK in CBA mice react with the immunizing antigen, but do not bind to ASK (Figure 7B). The recognition pattern of ASK- and SAK-specific antibodies demonstrate the lack of significant cross-reactivity between the two analogues, indicating that their antigenic determinants are different. Immunomodulatory Effect. In a separate experiment the ability of SAK and ASK of modulating the ongoing immune response of BDF1 mice against sheep red blood cells (SRBC) was tested by the hemolytic direct PFC assay. On the basis of earlier data obtained from branched polypeptide studies in the same assay (28), the immunomodulatory activity of these polypeptides was studied at two concentrations (0.2 mg/kg and 1.0 mg/kg). The results are presented in Figure 8. Polypeptides administered in aqueous solution simultaneously with primary SRBC immunization have no pronounced effect on PFC levels expressed as adjuvant index. Small but concentration-independent enhancement could be detected in PFC formation after administration of ASK (AI ) 1.2 ( 0.1) Treatment with levamisol used as positive control increased PFC formation (AI ) 3.2 ( 0.4). These data indicated that the polypeptides had apparently no effect on the SRBC specific immune responses under the conditions studied. CONCLUSION

For the construction of macromolecule-drug conjugates, it is important to provide rational basis for the selection of a proper carrier. With respect to the impor-

Figure 8. Dose-dependent effect of branched polypeptide ASK (dark bars) and SAK (light bars) on the PFC immune response to SRBC in BDF1 mice. Polypeptides and SRBC were applied simultaneously, ip, in saline.

tance of the side-chain structure and charge of the branched polypeptides in various chemical and biological properties (6, 7), we have studied the conformation, the immunoreactivity, the biodistribution, and in vitro cytotoxicity of a new set of branched polypeptides containing Ser residue. We found that the water solubility of these polycationic compounds with single or multiple OH group(s) was higher than of those containing only amino groups at the end of the branches. CD spectra of polypeptides showed significant differences correlating with the position and numbers of Ser residues in the side chains (19). Under physiological conditions, polycationic polypeptides assumed ordered (helical) secondary structure (SiK and LSK) or partially unordered conformation (SK, SAK, and ASK). It should be noted that in case of Ala-containing polypeptides an expressed tendency could be demonstrated in the formation of more ordered structure as compared to the respective serine analogues (SK vs AK and LSK vs LAK) (7). Consequently, the incorporation of even a single Ser could have an effect on the solution conformation by promoting its transition toward unordered secondary structure. The data summarized in this report indicate that these new compounds under in vitro conditions are essentially nontoxic on rat liver or mouse spleen cells up to a concentration of 50 µg/mL. The blood clearance, biodistribution, and immunogenicity of these derivatives were dependent on the position


and number of Ser residues in the branches. The polypeptides investigated were taken up predominantly by the liver and kidney (SiK, LSK, and ASK) or kidney and lung (SK and SAK). The best survival in the blood was found with SAK, representing the first polycationic branched polypeptide which show extended blood clearance similar to various proteins or the amphotheric polypeptide EAK (8, 37). The investigation of the immunostimulatory capacity of the new analogues indicate that these compounds have no effect on to the SRBC specific humoral immune response in the dose interval investigated. In contrast to the high pKa value of the -NH2 groups of polylysine, branched polypeptides, including compounds described in this paper, possess R-NH2 groups with lower pKa. The introduction of Ser residue(s) does not change the number of amino functional group in the polypeptides; therefore, the reduced cytotoxicity and prolonged blood circulation observed could mainly be attributed to the presence of OH groups, which might iniciate a further acidic shift in the pKa of the free R-NH2 groups positioned at the end of the branches. In conclusion, these studies have indicated that synthetic branched polypeptides with Ser can be considered as potential candidates for constructing suitable conjugates for drug/epitope delivery. It is not only due to the presence of hydroxyl functional group to be used for oxime chemistry, but also to their beneficial biological features. Data summarized here indicate that the incorporation a single Ser residue preferably at the N-terminal position of the side chain provide optimal biological properties. No additional improvements were observed in case of polypeptides possessing oligo(DL-Ser) branches. These polypeptides are essentially nontoxic, nonimmunomodulatory under conditions studied and can be present in the circulation for a much longer period of time than previously described polycationic polymers. ACKNOWLEDGMENT

These studies were supported by grants from the Hungarian Research Fund, (OTKA T 014964), from the Ministry of Health (ETT 115/1996), from PECO-COST SPIDER program (CIPA 4031), and from the Association for International Cancer Research (UK). M.V.P. is supported by the Cancer Research Campaign, London, UK. LITERATURE CITED (1) Duncan, R. (1992) Drug-polymer conjugates; potential for improved chemotherepy. Anti-Cancer Drugs 3, 153-156. (2) Maeda, H., Seymour, L. W., and Miyamoto, Y. (1992) Conjugates of anticancer agents and polymers: advantages of macromolecular therapeutics in vivo. Bioconjugate Chem. 3, 351-362. (3) Takakura, Y., and Hashida, M. (1995) Macromolecular drug carrier systems in cancer chemotherapy: macromolecular prodrugs. Crit. Rev. Oncol./Haematol. 18, 207-231. (4) Del Giudice, G. (1992) New carriers and adjuvants in the development of vaccines. Curr. Opin. Immunol. 4, 454-459. (5) Hudecz, F., and To´th, G. K. (1994) Synthetic peptide constructs to increase the immunogenicity of B-cell epitopes. In Synthetic peptides in the search for B-and T-cell epitopes (E. Rajnavo¨lgyi, Ed.) pp 97-119, R. G. Landes Company, Austin. (6) Hudecz, F., Votavova, H., Gaa´l, D., Sponar, J., Kajta´r, J., Blaha, K., and Szekerke, M. (1985) Branched polypeptides with a poly(L-lysine) backbone: synthesis, conformation and immunomodulation. In Polymeric Materials in Medication (Ch. G. Gebelein and Ch. E. Carraher, Eds.) pp 265-289, Plenum Press, New York.

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Carrier Design: New Generation of Polycationic Branched

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