Releasable PEGylation of Mesothelin Targeted Immunotoxin SS1P

Bioconjugate Chem. 2007, 18, 773−784

773

Releasable PEGylation of Mesothelin Targeted Immunotoxin SS1P Achieves Single Dosage Complete Regression of a Human Carcinoma in Mice David Filpula,*,† Karen Yang,† Amartya Basu,† Raffit Hassan,‡ Laiman Xiang,‡ Zhenfan Zhang,† Maoliang Wang,† Qing-cheng Wang,‡ Mitchell Ho,‡ Richard Beers,‡ Hong Zhao,† Ping Peng,† John Zhou,† Xiguang Li,† Gerald Petti,† Ahsen Janjua,† Jun Liu,† Dechun Wu,† Deshan Yu,† Zhihua Zhang,† Clifford Longley,† David FitzGerald,‡ Robert J. Kreitman,‡ and Ira Pastan‡ Enzon Pharmaceuticals, Incorporated, 20 Kingsbridge Road, Piscataway, New Jersey 08854, and Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892. Received October 7, 2006; Revised Manuscript Received January 20, 2007

Recombinant immunotoxins exhibit targeting and cytotoxic functions needed for cell-specific destruction. However, antitumor efficacy, safety, and pharmacokinetics of these therapeutics might be improved by further macromolecular engineering. SS1P is a recombinant anti-mesothelin immunotoxin in clinical trials in patients with mesothelinexpressing tumors. We have modified this immunotoxin using several PEGylation strategies employing releasable linkages between the protein and the PEG polymers, and observed superior performance of these bioconjugates when compared to similar PEG derivatives bearing permanent linkages to the polymers. PEGylated derivatives displayed markedly diminished cytotoxicity on cultured mesothelin-overexpressing A431-K5 cells; however, the releasable PEGylated immunotoxins exhibited increased antitumor activity in A431-K5 xenografts in mice, with a diminished animal toxicity. Most significantly, complete tumor regressions were achievable with single dose administration of the bioconjugates but not the native immunotoxin. Pharmacokinetic analysis of the releasable PEGylated derivatives in mice demonstrated an over 80-fold expansion of the area under the curve exposure of bioactive protein when compared to native immunotoxin. A correlation in degree of derivatization, release kinetics, and polymer size with potency was observed in vivo, whereas in vitro cytotoxicity was not predictive of efficacy in animal models. The potent antitumor efficacy of the releasable PEGylated mesothelin-targeted immunotoxins was not exhibited by similar untargeted PEG immunotoxins in this model. Since the bioconjugates can also exhibit the attributes of passive targeting via enhanced permeability and retention, this is the first demonstration of a pivotal role of active targeting for immunotoxin bioconjugate efficacy.

INTRODUCTION Mesothelin is an attractive target for tumor-specific therapy due to its high expression on several cancers and limited expression on normal tissues. The 40 kDa glycosylphosphatidyl inositol-linked cell surface glycoprotein is overexpressed in pancreatic carcinoma, ovarian cancer, and malignant mesotheliomas, whereas normal pleura, pericardium, and peritoneum exhibit detectable mesothelin expression in the surrounding mesothelial cells (1-15). The biological function of mesothelin is not established; although it is not essential for normal mouse development (16-21), recent studies suggest that mesothelin * To whom correspondence should be addressed. Tel # 732-9804941; FAX # 732-885-2950; E-mail: [email protected]. † Enzon Pharmaceuticals, Inc. ‡ National Cancer Institute. 1 Abbreviations: ALD, aldehyde; AUC, area under the curve; B-SS1P, bicin-SS1P; Bicin, bis-N-2-hydroxyethylglycine, dsFv, disulfide stabilized Fv; Cmax, maximal concentration; D-SS1P, DGA2-SS1P; DGA2, diglycolic acid; ELISA, enzyme-linked immunosorbent assay; EPR, enhanced permeability and retention; HRP, horseradish peroxidase; i.v., intravenous; LD50, lethal dose with 50% mortality; mAb, monoclonal antibody; mPEG, monomethoxy-PEG; MRT, mean retention time; MW, molecular weight; NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline; PE, Pseudomonas exotoxin A; PEG, poly(ethylene glycol); PEG2, branched PEG; RP, reversed phase; rPEGylation, releasable PEGylation; RNL, reversible nitrogen linker; SC, succinimidyl carbonate; SE-HPLC, size exclusion high performance liquid chromatography; t1/2, serum half-life; Tmax, time of maximal concentration.

binds to the mucin glycoprotein CA125 and that this interaction mediates cell adhesion (22). An anti-mesothelin mAb1 was generated by immunizing mice with a mesothelin expression plasmid and library screening of splenic RNA variable domains using scFv phage display technology. Affinity maturation was conducted using siteselected mutagenesis and screening (6, 12-15, 23). The isolated SS1 Fv exhibited high affinity for mesothelin (Kd ) 0.7 nM) and was stabilized by a disulfide bond between the light and heavy variable domains of the Fv (24-26). The SS1 Fv was employed in the construction of an immunotoxin SS1P via genetic fusion to a truncated Pseudomonas exotoxin A (PE) (27-30). SS1P is highly cytotoxic to mesothelin-expressing cells in culture and demonstrates antitumor effects against the human epidermoid carcinoma cell line, A-431, that is stably transfected with a mesothelin cDNA expression vector (A431-K5) and subcutaneously implanted in mouse xenografts. SS1P [SS1(dsFv)PE38] has recently been investigated in clinical trials (1, 7, 9). Several recombinant immunotoxins composed of an antibody Fv targeting moiety and a PE cytotoxic moiety have been evaluated in animal models and clinical studies (31, 32). These immunotoxins have produced potent antitumor responses in human cancer xenografts in nude mice (33, 34), and major clinical responses to recombinant immunotoxins were observed in hematologic malignancies (35, 36). However, these chimeric proteins also exhibit side effects that limit the dose and frequency of administration. The PE domain of the immunotoxin may exhibit nonspecific binding to normal tissues, while

10.1021/bc060314x CCC: $37.00 © 2007 American Chemical Society Published on Web 03/09/2007

774 Bioconjugate Chem., Vol. 18, No. 3, 2007

neutralizing human anti-PE antibodies, and less frequently, antimouse Fv antibodies, are formed in patients treated with these therapeutics, resulting in either dose-limiting toxicity or diminished therapeutic potency. In addition, recombinant immunotoxins (∼63 kDa) do not exhibit prolonged blood residency, in contrast to mAbs, leading to increased distribution in both tumors and normal tissues, including kidneys and liver. Although a prolonged blood residency might be achieved via continuous infusion, it would be desirable to construct an immunotoxin with intrinsically improved pharmacokinetics, reduced toxicity, and diminished antigenicity/immunogenicity. PEGylation is a well-developed technology that has shown the capacity to address each of the above concerns. Bioconjugation of proteins with PEG via permanent linkages is employed in at least six marketed protein therapeutics (37). We previously reported an improvement in serum half-life, a reduction in immunogenicity and toxicity, and enhanced antitumor efficacy in a PEGylated chimeric toxin composed of transforming growth factor-R and PE (28). However, these positive attributes came at the expense of a marked loss of specific cytotoxic activity. Site-specific PEGylation of anti-Tac(Fv)-PE immunotoxin LMB-2 also improved antitumor activity and diminished toxicity and immunogenicity (29, 38). In this case, the engineered protein design of the bioconjugate employed a free thiol in the connector peptide of the immunotoxin. In practice, this approach may be limited by the inefficiency of protein refolding due to incorrectly disulfide-linked, misfolded protein impurities, and also by the inefficiency of conjugation and purification of the conjugates. It would be desirable to develop a bioconjugate strategy wherein no special polypeptide alterations are required and whereby nearly quantitative yields of bioconjugate from native protein are achieved. Designing the optimal immunotoxin through the combination of protein engineering and bioconjugation approaches is a challenging undertaking. Recombinant immunotoxins, such as SS1P, are multifunctional proteins that exhibit biological activity only after intracellular processing. Chemical modification strategies must consider the preservation of antigen-binding, translocation, and ADP-ribosylation functions, while enhancing composite pharmacological properties, including avoidance of rapid clearance from renal filtration, antibody or cellular clearance, or physical degradation. Although conventional PEGylation utilizes stable linkages between the polymers and the protein, recent research has investigated the potential of releasable PEGylation (rPEGylation), wherein the designed covalent linkages of the PEG strands are chemically unstable within a physiological environment and the attached polymers are detached via a controlled release mechanism (30, 39-44). This unconventional prodrug-based PEGylation approach might have an appropriate application in immunotoxin engineering. Immunotoxins are themselves biological prodrugs that are activated by a designed intracellular release mechanism. Further engineering of the immunotoxin as a double prodrug bioconjugate offers several hypothetical advantages. rPEGylation could provide multiple PEG attachments over the protein surface and allow optimized pharmacokinetics and biodistribution, with diminished immunogenicity and antigenicity. Although the rPEGylated immunotoxin may have severely compromised cytotoxic activity in its prodrug form, a designed incremental release in vivo of the unmodified immunotoxin protein from the attached polymers may provide a superior distribution of fully active protein at the target sites. Underlying this hypothesis is a requirement for a beneficial shift in the balance between the counteracting forces of clearance of the released protein and retention of the PEG modified protein. Furthermore, rPEGylated immunotoxins might accrue additional tumor targeting benefits from the enhanced permeability and retention (EPR) phenomenon (45, 46). In the first investigations

Filpula et al.

to examine the in vitro and in vivo performance of immunotoxins modified with rPEGylation, we have studied several rPEGylated bioconjugates composed of SS1P and designed rPEG polymers of varying release kinetics, polymer size, and degree of modification.

EXPERIMENTAL PROCEDURES Materials. PEG2-40 kDa-NHS was obtained from Nektar Therapeutics (Huntsville, AL). All other activated PEG polymers were supplied by Enzon Pharmaceuticals, Inc. (Piscataway, NJ). Hiload Superdex 75 or 200, Q Sepharose Fast Flow, and HiTrap Q-XL were supplied by GE Healthcare (Piscataway, NJ). Production of Recombinant SS1P. The subunits of SS1P were expressed in Escherichia coli BL21(λDE3) and purified as previously described for other immunotoxins (23, 25, 31). Active monomeric protein was purified from the refolding solution by ion exchange and size exclusion chromatography. LC-MS analysis confirmed the predicted 62 591 mass of SS1P. Linker Synthesis. The Bicin3, DGA2, and RNL-8a linkers were synthesized as previously described (39-41, 44). Preparation of Releasable PEG-SS1P Conjugates. In PEGylation reactions employing releasable linkers such as DGA2, RNL-8a, and Bicin3, SS1P at 1.5-2.5 mg/mL was PEGylated in 0.05-0.1 M sodium phosphate, pH 7.6, 25 °C, and purified by reported procedures (40, 44). With fast stirring without creating foam, the PEG powder at 10:1 to 50:1 reaction molar ratio of PEG to SS1P was added to the SS1P solution at 1 g/min. The reaction was continued at 25 °C for 1 h and quenched by adding glycine. Immediately after quenching, the solution pH was lowered to 6.5 with monobasic sodium phosphate and the conjugate was purified on a size exclusion column (Superdex 200 or 75 Hiload) equilibrated in 20 mM sodium phosphate, pH 6.5, 140 mM NaCl. Alternatively, the conjugate was purified on an anion exchange column (Q Sepharose Fast Flow) using either 10 mM sodium phosphate, pH 7.4, as the equilibration buffer, with 0.3 M NaCl in 10 mM sodium phosphate, pH 7.4, as the gradient elution buffer, or 10 mM Tris-HCl, pH 7.6, as the equilibration buffer, and 0.3 M NaCl in 10 mM Tris-HCl, pH 7.6, as the gradient elution buffer. The column fractions containing the peak were combined and dialyzed against PBS, pH 6.5. The sample was concentrated to about 1 mg/mL using Centriplus 30k (Millipore, MA) and passed through a sterile filter (Acrodisc Syringe Filter, 0.2 µm HT Tuffyn membrane, Pall, MI). The number of PEG polymers per SS1P molecule was initially estimated by SDS-PAGE prior to further analysis. The release of SS1P from releasable PEGSS1P was conducted by incubation in a PBS pH 8.5 buffer, at 37 °C or 25 °C. The purity of the product was analyzed on a TSK gel filtration column equilibrated in 50 mM sodium phosphate, pH 6.5, 150 mM NaCl (G4000SWXL, 7.8 × 30 cm, 8 µm, Tosoh Biosciences). The peak area was calculated at 220 nm. Since the conventional TNBS and fluorescamine assay methods for measurement of percent PEG modification require a basic pH which is not suitable for rPEG-SS1P, we used the ammonium ferrothiocyanate colorimetric method to analyze free and protein-coupled PEG (47). Preparation of Permanent PEG-SS1P Conjugates. For PEGylation reactions that employed permanent linkers such as SC and NHS, SS1P at 1.5-2.5 mg/mL was PEGylated in 0.05 M sodium phosphate, pH 6.0-9.0, at 25 °C and purified as previously described (29, 30, 37-41). With fast stirring without creating foam, the PEG powder at 3:1 to 15:1 reaction molar ratio of PEG to SS1P was added to SS1P solution at 1 g/min. The reaction was continued at 25 °C for 1-2 h for the pH 7.49.0 reactions and 4 h for pH 6.0-7.0 reactions. The reaction was quenched by adding glycine. The conjugate was purified by an anion exchange column (Q Fast Flow Sepharose) where

Bioconjugate Chem., Vol. 18, No. 3, 2007 775

Releasable PEGylation of Immunotoxin SS1P

the equilibration buffer was 10 mM Tris-HCl, pH 7.6, and the gradient elution buffer contained 0.3 M NaCl in 10 mM TrisHCl, pH 7.6. Alternatively, a size exclusion column (Superdex 200 or 75 Hiload) was used for purification with a pH 7.4 PBS equilibration buffer. The sample was concentrated to about 1 mg/mL using Centriplus 30k and passed through an Acrodisc sterile filter. Preparation of ALD-PEG-SS1P. For N-terminal selective PEGylation, SS1P at 2 mg/mL was incubated with aldehydePEG-20k (ALD-PEG-20k) at a 10:1 reaction molar ratio (PEG/ SS1P) in 100 mM sodium phosphate, pH 6.5, 25 °C, for 3 h. The reduction of Schiff base was completed in 15 mM sodium cyanoborohydride at 25 °C, 16 h. Mono-PEGylated and diPEGylated conjugates were isolated on HiTrap Q-XL at pH 7.5. SDS-PAGE analysis showed that the PEG attachment in mono PEG-SS1P was selectively located on the heavy chain (VH-PE) and the PEG in di-PEG-SS1P was found on both the heavy and light chains (VL) of SS1P. Cell Lines and Cytotoxicity Assay. A431-K5, a cell line established by transfection of an expressible cDNA encoding mesothelin into human epidermoid cancer A431 cells (American Type Culture Collection, Manassas, VA), was used in cytotoxicity assays in vitro and in vivo. A431-K5 has a stable and uniform expression of mesothelin (33 000 mesothelin sites per cell; Raffit Hassan, NCI, unpublished data), which is similar to that observed with tumor specimens obtained from patients (9, 10, 17, 18, 20). The specific cytotoxicity of SS1P and PEGylated SS1P compounds was assessed by protein synthesis inhibition assays in 96-well plates, by a modified nonradioactive assay (10, 34). Antitumor Activity of SS1P and PEGylated SS1P. The antitumor activity of SS1P and PEG-SS1P compounds was determined in nude mice bearing A431-K5 human cancer cells that express mesothelin. Starting on day 7, animals were treated with i.v. injections of each of the immunotoxin compounds diluted in 0.2 mL of phosphate buffered saline (PBS)/0.2% mouse serum albumin (MSA). Pharmacokinetics. BALB/c mice (6-8 week old females) were supplied by Sprague Dawley Harlan (Madison, WI). Mice (3/group) were injected i.v. with 100 µL per mouse of SS1P (0.125 or 0.4 mg/kg) or its PEGylated conjugates (0.5 or 1.5 mg/kg). Mice were terminally bled by cardiac puncture at 0.17, 0.5, 1, 3, 6, 24, 48, and 120 h. The concentrations of the compounds were analyzed by in vitro cytotoxicity with A431K5 cells, and data were modeled using WinNonlin software to determine pharmacokinetic parameters. Flow Cytometry. Flow cytometry studies with A431-K5 cells were used to analyze SS1P and PEG-SS1P compounds binding to mesothelin cell receptors. Cells were resuspended (106 cells/ mL) in D-MEM, 5% FBS. SS1P compounds (0.1, 1, 10, or 100 ng) in 100 µL of FACS medium were added to each tube and incubated for 30 min on ice, and 100 µL of mouse anti-PE-40 mAb (1 µg/mL) was added and incubated 30 min on ice. Phycoerythrin-conjugated goat anti-mouse polyclonal antibody (10 µg/mL; 100 µL) was added, and analysis was conducted on FACS Calibur (BD Biosciences, San Jose, CA). Surface Plasmon Resonance Analysis. Surface plasmon resonance techniques were performed using a Biacore X instrument (Biacore, Inc., Piscataway, NJ). Recombinant mesothelin-maltose binding protein (MBP) was prepared using the pMAL protein fusion and purification system (New England BioLabs, Beverly, MA). Anti-MBP mAb was immobilized on a CM5 chip and subjected to ligand stability analysis for six cycles with 500 nM mesothelin-MBP. For each kinetic experiment, equal amounts of mesothelin-MBP were allowed to be captured by the surface-immobilized anti-MBP mAb. The SS1P or PEG-SS1P proteins served as analytes. Over the stable mAb-

MBP-mesothelin surface, different concentrations of SS1P or PEG-SS1P were examined. The data were analyzed using Biacore BiaEValuation software (version 3.0). Analysis of Anti-SS1P Antibodies in Human Sera by Competition ELISA. Microtiter wells were coated with 1 µg SS1P/50µL in 20 mM Tris-HCl, pH 8.0. Diluted human antisera, combined with serial dilutions of either SS1P or PEGSS1P, were added (100 µL total volume) and incubated at 25 °C for 1.5 h. The wells were washed and HRP conjugated rabbit anti-human Ig (1:40 000 dilution), 50 µL/well, was added and incubated at 25 °C for 45 min. Following washing, 100 µL of TMB-E substrate was added, and the plates were read at 450 nm.

RESULTS Preparation and Characterization of PEG-SS1P Compounds. Seven amine-reactive, NHS-activated PEG polymers were employed in the construction of PEGylated bioconjugates of the immunotoxin SS1P. As shown in Chart 1, four unconventional releasable linker designs were evaluated. The bis-N2-hydroxyethylglycine (Bicin3), diglycolic acid (DGA2), and reversible nitrogen linker (RNL-8a) linker chemistries have been recently described with regard to their mechanism of controlled release from PEGylated proteins (39-41, 44). The DGA2 (fast release) and RNL-8a (slow release) linkers are both aromatic designs wherein an initial ester cleavage triggers a classical 1,6benzyl elimination reaction which releases the original amine and results in linker degradation. In contrast, the Bicin3 linker employs a wholly aliphatic design. Following the hydrolysis of the exposed ester group of the linker, a cyclization event promotes linker self-immolation and release of the original amine (lysine) group. The Bicin3 linker was employed as a release linkage to either mono (linear) PEG or to branched (U) PEG in our studies such that examination of the influence of PEG geometry could also be included in our investigations. Release kinetics for either type of linker may be tailored by the inclusion of side chain groups strategically designed in the linker to provide either steric interference (for slower release) or anchimeric assistance (for faster release) to the degradation pathway, and we chose representative linkers from the two structural types that include a range of release kinetics. For example, Scheme 1 depicts the linkage chemistry of the Bicin3-mono version and its proposed mechanism of release. The NHS-activated PEG (1) is conjugated to SS1P protein to form the bioconjugate (2). Decapping is very rapid in vivo to produce 3, and the ester hydrolysis reaction proceeds at a rate determined by the molecular design of the linker side chains to produce 4. Cyclization occurs via participation of both hydroxyethyl side chains, and linker detachment releases the original lysine(s) of SS1P plus the morpholinolactone (5). DGA2 and RNL-8a linker characteristics and release mechanisms have also been described (40, 41). Protein bioconjugates with these rPEG polymers bonded to lysine residues are stable at pH 90% yields and provide the option of tailoring the number of PEG attachments through adjustments of reaction stoichiometry and purification protocols. Since both types of linkers have been successfully employed in reversible protein conjuga-


Filpula et al.

Chart 1. Structure of Activated PEG Polymers Composed Of Poly(ethylene glycol) Polymer, Releasable Linker, and N-Hydroxysuccinimide (NHS) Leaving Groupa

a Amide or carbamate linkages to primary amines in conjugates of SS1P are formed at the linker carbonyl group proximal to the NHS ester. The mechanisms of controlled release of authentic native protein from the linker and PEG have been described for both the aromatic-based DGA2 and RNL-8a linkers (40, 41) and the aliphatic Bicin3 linker (39, 44).

tion, further comparative studies in animal models may be instructive to evaluate these individual chemistries. We chose to focus our investigations on SS1P with an intermediate degree of rPEGylation, such that the number of PEG attachments and total PEG mass of most of the test compounds were comparable. In the design of the PEG-SS1P conjugates, a molecular mass mainly in the 40-60 kDa range was selected, since this strategy provided a practical compromise between optimizing the compound’s pharmacokinetics and ensuring release of the SS1P before systemic clearance of the intact bioconjugate. Bioconjugates of this size will not exhibit rapid renal clearance, due to their sufficient total polymer mass.

Therefore, the total numbers of PEG attachments were fewer for the large (40 kDa) PEG polymers than for smaller (12 kDa) PEG polymers. In addition, the use of branched linkers for PEG attachments allowed fewer total attachment sites for 12 kDa or 20 kDa linear PEGs, since the branched 40 kDa design tethers two 20 kDa linear PEGs to a common linker; similarly, the branched 24 kDa design contains two linear 12 kDa PEGs per linker. Multiple analytical methods were employed to characterize the composition of the multi-PEGylated SS1P compounds and confirmed batch consistency, as summarized in Table 1, using capillary electrophoresis, size-exclusion chromatography, chemical analysis of PEG, and SDS-PAGE. SS1P bioconjugates



Scheme 1. Example of the Chemistry of Conjugation and the Linker Decomposition in Vivo for the Bicin3-Mono Linkera

a Reaction of an activated PEG of the bicin linker type (1) with a protein amine forms the bioconjugate (2). In vivo, the acetyl group is rapidly lost, while the elimination reaction to release the PEG segment from (3) occurs at a controllable rate to produce (4). The final release of the original protein and morpholinolactone (5) is predicted to occur via a cyclization intermediate.

Figure 1. Comparison of release kinetics of four rPEG-SS1P compounds in PBS at pH 8.5, 25 °C: DGA2-12 kDa-PEG-SS1P ([), Bicin3-U-24 kDa-PEG-SS1P (9), Bicin3-mono-30 kDa-PEG-SS1P (2), and RNL-8a-12 kDa-PEG-SS1P (×). Release was monitored at 220 nm on SE chromatography on a TSK-gel, G4000SWXL column with a mobile phase of 50 mM sodium phosphate, pH 6.5, 140 mM NaCl, 0.5 mL/min. See Table 1 for release rates at pH 7.4.

with conventional permanent PEG polymers (SC-12k, ALD20k, PEG2-40k) (48) were further analyzed by MALDI-TOF, LC-MS, and reversed-phase chromatography. Cytotoxic Activity of PEG-SS1P. A modified nonradioactive cytotoxicity assay was used for the SS1P immunotoxin and its PEGylated counterparts. The specific cytotoxicity (IC50) of the immunotoxins was determined on A431-K5 cells, which highly express the target mesothelin surface protein (4, 23). As shown in Table 1, the in vitro potency of the SS1P immunotoxin was markedly compromised by the random PEGylation strategies used in this study. SS1P conjugates bearing three or more

permanent PEG attachments exhibited marginal or no activity in this bioassay. With conjugates derived from the site-selective PEG-aldehyde linkage, a single PEG attachment at one of the N-termini of the Fv subunits virtually eliminates in vitro bioactivity. Although releasable compounds such as DGA2-12 kDa-PEG-SS1P and Bicin3-24 kDa-PEG-SS1P, which also contain about 3-6 PEGs, retained some cytotoxic activity, this is attributed to the partial release of PEG during the incubation in cell culture. However, it is not uncommon for highly effective PEGylated protein therapeutics to exhibit fractional activity in vitro (37, 41, 48). Pharmacokinetics of PEG-SS1P. BALB/c mice were injected i.v. at 100 µL per mouse with SS1P (0.125 or 0.4 mg/ kg) or its PEGylated conjugates (0.5 or 1.5 mg/kg). Plasma concentration profiles of unmodified SS1P exhibited a rapid elimination with a half-life of about 26 min. In contrast, the plasma concentration profiles of DGA2-12 kDa-PEG and Bicin3-U-24 kDa-PEG bioconjugates demonstrated more complex elimination kinetics, as expected for these rPEG-SS1P compounds. As shown in Table 2, the plasma half-life, maximal concentration, and mean retention time of these two PEG-SS1P proteins were extended over 10-fold, while the time of maximal concentration was delayed from 30 min for SS1P to 2 h for the bioconjugates. Most significantly, the area under the plasma concentration curve (AUC) exposure of both rPEG-SS1P derivatives was increased about 80-fold compared to native SS1P. The PEG2-40 kDa-PEG-SS1P compound contained a permanent amide linkage of only one PEG per SS1P. In comparison to native SS1P, prolonged blood residency was achieved, although not to the extent exhibited by the first three rPEG-SS1P derivatives (Table 2). Animal Toxicity Studies. The nonspecific toxicity of the PEGylated immunotoxins in mice was examined by i.v.


Filpula et al.

Table 1. Features and Analysis of PEGylated SS1P and rCD22 Bioconjugates compound

linkera

PEG mass on linker (kDa)

PEG # (AFTC/TNBS)b

PEG # (SEC/CE)

PEG # (PAGE)

SS1P release in vitro t1/2c(h)

IC50 (ng/mL)

flow cytd (% of SS1P)

SS1P DGA2-12k-SS1P Bicin3-U-24k-SS1P Bicin3-mono-30k-SS1P RNL-8a-12k-SS1P SC-12k-SS1P ALD-20k-SS1P PEG2-40k-SS1P DGA2-12k-RCD22 Bicin3-U-24k-RCD22

none R R R R P P P R R

none 12 24 30 12 12 20 40 12 24

none 4-5 2-3 3-5 4-5 5-6 1-2 1 4-5 2-3

none 4, 5, 6 2, 3, 4 4, 5 4, 5, 6 4, 5, 6 1 1 4, 5, 6 2, 3

none 4, 5, 6 3 4, 5 4, 5, 6 4, 5, 6 1 1 4, 5, 6 2, 3

n.a.f 125 90 140 >300 n.r.e n.r. n.r. 125 90

0.7 2.1 3.7 16.5 >100 >100 39 8.8 n.a. n.a.

100 33 39 11 6 19 23 63 n.a. n.a.

a R, releasable; P, permanent. b PEG # is the number of attached PEGs per SS1P determined by ammonium ferrothiocyanate (AFTC), trinitrobenzene sulfonic acid (TNBS), capillary electrophoresis (CE), size exclusion chromatography (SEC), and SDS-PAGE. c Half-life of SS1P release from PEG at pH 7.4, 25 °C, in PBS. d Flow cytometric analysis of % bound PEG-SS1P compound relative to unmodified SS1P on A431-K5 cells (native SS1P ) 100% bound). e n.r. ) no release. f n.a. ) not applicable.

Table 2. Pharmacokinetics of PEGylated SS1P Bioconjugates

compound SS1P DGA2-12k-SS1P Bicin3-U-24k-SS1P Bicin3-mono-30k-SS1P RNL-8a-12k-SS1P PEG2-40k-SS1P

AUCb t1/2 b(h) MRTb (min Tmaxb Cmaxb linkera in vivo (min) mg/mL) (min) (µg/mL) none R R R R P

0.44 4.8 4.8 5.0 4.4 2.5

0.64 4.7 5.2 6.5 5.5 3.6

0.36 28.6 31.3 18.8 0.77 2.0

30 120 120 180 60 10

3.4 51.5 47.0 20.9 1.1 8.7

a R, releasable; P, permanent. b Pharmacokinetic parameters are t , 1/2 biological half-life; MRT, mean residence time; AUC, area under the plasma concentration curve; Tmax, time of maximal concentration; Cmax, maximal concentration.

administration of 2.0, 3.0, 4.0, or 6.0 mg/kg. Almost all deaths occurred within 4 days of treatment. The enormous increase in drug exposure provided by the rPEGylated derivatives did not, however, correspond to an increase in animal toxicity. The LD50 of native SS1P was found to be about 1.0 mg/kg; in contrast, the PEG-SS1P compounds were much better tolerated. The LD50 values (mg SS1P protein/kg) for DGA2-12kDa-SS1P, Bicin3-U-24kDa-SS1P, and RNL-8a-12kDa-SS1P were 3.0, 3.0, and 4.0 mg/kg, respectively. For the antitumor studies, we employed one or two dose levels below the dose that produced sickness or death. Antitumor Activity of Permanent or rPEG-SS1P. Antitumor activity of SS1P and PEG-SS1P compounds was determined in nude mice bearing A431-K5 human cancer cells that express mesothelin. Cells (3 × 106) were injected s.c. into nude mice on day 0. Tumors of ∼140 mm3 in size developed in animals by day 7 after tumor implantation, after which animals were treated with i.v. injections of each of the immunotoxin compounds. In most experiments, therapy with native SS1P or with the PEG-SS1P compounds was given only once on day 7. In some experiments, animals received PEGSS1P twice on days 7 and 11 or SS1P three times on days 7, 9, and 11. The control groups received vehicle only. It was previously determined that SS1P inhibited tumor growth in a dose-dependent manner and that at least three doses of SS1P (0.5 mg/kg) were required to achieve regression in this mouse xenograft model (4, 23). Usually three independent studies were conducted with each of the PEGylated compounds using two different preparations of the SS1P conjugates. As shown in Figure 2A, DGA2-12 kDa-PEG-SS1P (DSS1P) was extremely active. A single dose of 2.0 mg/kg produced 90% tumor shrinkage on day 14 and 3/6 mice showed complete tumor regression. Mice receiving two doses of D-SS1P also showed a profound antitumor response with 4/8 mice showing complete response and an average tumor size regression of 90% (Table 3).

As shown in Figure 2B, Bicin3-U-24 kDa-PEG-SS1P (BSS1P) was also very active. Complete regression was achieved in 1/4 mice receiving 2.0 mg/kg and 3/4 mice receiving 3.0 mg/kg. In mice receiving two doses of 2.0 mg/kg, 4/8 mice showed complete regression of the tumors. Figure 2C shows the antitumor effects of the Bicin3-mono30 kDa-PEG-SS1P; it appeared to be somewhat less active than the first two compounds tested. At 3 mg/kg, a single dose caused a maximal decrease in tumor size of 68%, whereas two doses of 2.0 mg/kg caused an average decrease in size of 92% with 1/5 complete remission. The other compound with a slowly reversible linkage that showed significant antitumor activity in a small pilot study is RNL-8a-12 kDa-PEG-SS1P. However, as shown in Table 3, it is less active than the other compounds. Further antitumor investigations were carried out with permanent PEG derivatives, multi-PEGylated SC-12 kDa-PEGSS1P and mono-PEGylated PEG2-40 kDa-PEG-SS1P. Only the latter compound produced sustained regression with 1/4 of the mice showing complete regression at a dose of 3.0 mg/kg (Figure 3 and Table 3). PEGylated proteins have also been reported to accumulate in tumor xenografts via passive targeting by the EPR effect due to the leaky vasculature and deficient lymphatic drainage exhibited by tumors (45, 46). To investigate the capability of nontargeted rPEGylated immunotoxins to demonstrate antitumor effects, we generated analogous rPEGylated immunotoxins targeting CD22, which is not present on A431-K5 cells. These compounds showed no antitumor activity, confirming that the antitumor activities of the PEG-SS1P derivatives are specific. Immunogenicity and Immunoreactivity of PEG-SS1P. Since the immune response of patients to immunotoxins may limit their duration of efficacy, we assessed the immunoreactivity of PEG-SS1P compounds relative to unmodified SS1P. Figure 4A shows the results of sandwich ELISA analysis of SS1P and several PEG-SS1P derivatives, in which plates coated with a mAb reacting with PE38 were exposed to increasing amounts of each conjugate and the amount of immunotoxin bound was detected with a rabbit polyclonal antibody to PE38. Cross-reactivity of the PEG-SS1P compounds to the rabbit antiPE antibodies is diminished compared to native SS1P. Two independent preparations of SS1P exhibited equivalent strong immunoreactivity, while the two independent preparations of heavily PEGylated Bicin3-mono-30 kDa-PEG-SS1P demonstrated the weakest cross-reactivity. The N-terminally monoPEGylated ALD-20 kDa-PEG-SS1P was strongly reactive with the anti-PE antibodies as expected due to PEG attachment on the Fv portion of the Fv-PE toxin. Other rPEGylated derivatives were intermediate in signal. This suggests a possible strategy for evading the rapid drug clearance in patients due to existing neutralizing antibodies.



Table 3. Antitumor Effects of PEGylated SS1P Bioconjugates

Figure 2. Antitumor effects of rPEG-SS1P and SS1P on A431-K5 solid tumors. Cells (3 × 106) were injected s.c. into nude mice on day 0. Starting on day 7 (tumor volume ) ∼140 mm3), animals were treated with i.v. injections of each of the immunotoxin compounds at the indicated doses. Therapy with native SS1P or PEG-SS1P was given once on day 7. In an additional group, therapy with the PEG-SS1P compounds was given on days 7 and 11 (arrows). Control mice received vehicle only. All PEG-SS1P compounds are compared to SS1P (0.6 mg/kg × 1) administration and tumor volumes (mm3) are shown (mean ( SD). (A) DGA2-12 kDa-PEG-SS1P (1.0 mg/kg × 2, or 2.0 mg/kg × 1). (B) Bicin3-U-24 kDa-PEG-SS1P (2.0 mg/kg × 1, 2.0 mg/kg × 2, or 3.0 mg/kg × 1). (C) Bicin3-mono-30 kDa-PEG-SS1P (2.0 mg/kg × 2).

To assess the immunogenicity of the PEG-SS1P compounds, BALB/c mice were immunized i.v. once per week with SS1P (2.5 µg/mouse) or PEG-SS1P (10 µg/mouse) for four weeks. Higher doses of unmodified SS1P could not be administered due to toxicity of the native toxin. Blood samples were collected every 7 days before subsequent immunization. The specific IgG and IgM levels were determined by capture ELISA. At day 28, IgM antibody levels were similar for SS1P and two rPEGylated derivatives; IgG antibody levels were also similar at day 28 for these compounds (data not shown). The mouse antisera were also investigated in cytotoxicity assays, and both the anti-SS1P and anti-PEG-SS1P antisera were determined to contain neutralizing antibodies. Although immunogenicity in mice does

compound

dose

DGA2-12k-SS1P DGA2-12k-SS1P Bicin3-U-24k-SS1P Bicin3-U-24k-SS1P Bicin3-U-24k-SS1P Bicin3-mono-30k-SS1P Bicin3-mono-30k-SS1P RNL-8a-12k-SS1P RNL-8a-12k-SS1P SC-12k-SS1P SC-12k-SS1P PEG2-40k-SS1P PEG2-40k-SS1P positive control: SS1P negative control: no SS1P

1.0 mg/kg × 2 2.0 mg/kg × 1 2.0 mg/kg × 1 2.0 mg/kg × 2 3.0 mg/kg × 1 2.0 mg/kg × 2 3.0 mg/kg × 1 2.0 mg/kg × 1 4.0 mg/kg × 1 1.0 mg/kg × 1 2.0 mg/kg × 1 2.0 mg/kg × 1 3.0 mg/kg × 1 0.6 mg/kg × 1 none

day of % best decrease mice/ response in size CR group 13 13 13 13 11 13 13 14 14 4 5 12 14 9 none

92% 92% 78% 92% 92% 68% 68% 73% 89% 51% 38% 64% 86% 32% none

4/8 3/6 1/4 4/8 3/4 1/5 0/2 0/2 1/2 0/2 0/2 0/4 1/4 0/5 0/20

8 6 4 8 4 5 2 2 2 2 2 4 4 5 20

not predict human immunogenicity, these data do suggest that an immune response is not precluded in the current rPEGSS1P compounds. The great increase in blood residency time of the PEG-SS1P compounds in mice might provide an increased opportunity for development of an immune response. To assess the cross-reactivity of PEG-SS1P and native SS1P to human antibodies versus SS1P, antisera were collected from two patients undergoing SS1P therapy and analyzed in competition ELISA as shown in Figure 4B. Human antisera were mixed with dilutions of either SS1P or PEG-SS1P and then added to SS1P coated plates. The detection reagent was rabbit anti-human IgG (HRP conjugate). The permanent or rPEG-SS1P compounds demonstrated cross-reactivity to the anti-SS1P human antibodies, although the PEG-SS1P compounds exhibited a one- to two-log reduced binding efficiency in this competition immunoassay when compared to native SS1P. These data support the hypothesis that PEG-SS1P may be less prone to rapid clearance in patients with existing antibodies to the SS1P protein. Target Binding and Uptake of PEG-SS1P. To assess the bioactivity of the PEG-SS1P derivatives in (1) mesothelin binding, (2) cell surface mesothelin binding, and (3) cell uptake and processing, we performed several studies. For the DGA212 kDa-PEG-SS1P (D-SS1P) and Bicin3-U-24 kDa-PEG-SS1P (B-SS1P), a determination of KD, kon, and koff were performed

Figure 3. Antitumor effects of PEGylated SS1P with a nonreleasable linker on A431-K5 solid tumors. Cells (3 × 106) were injected s.c. into nude mice on day 0. Starting on day 7, animals were treated with i.v. injections of the immunotoxin compounds (arrow). Therapy with native SS1P (0.6 mg/kg × 1) was given once on day 7; therapy with the PEG2-40 kDa-PEG-SS1P compound was given once at day 7 (2.0 mg/kg or 3.0 mg/kg). Control mice received vehicle only. Tumor volumes (mm3) are shown (mean ( SD).


Filpula et al.

Figure 4. Immunoreactivity of PEG-SS1P. (A) Antigenicity of PEG-SS1P compounds was evaluated by sandwich ELISA. The ELISA plate was coated with mouse monoclonal anti-PE antibody. Different concentrations of SS1P or PEG-SS1P conjugate were added, and rabbit polyclonal anti-PE antiserum (1:40 000 dilution) was added as the primary antibody reagent, while HRP conjugated goat anti-rabbit IgG (1:5000 dilution) and TMB were the secondary reagents. Absorbance was read at 450 nm. The 12 compounds are numbered: (1) native SS1P, lot #1; (2) native SS1P, lot #2; (3) Bicin3-24 kDa-PEG-SS1P; (4) SC-12 kDa-PEG-SS1P; (5) Bicin3-mono-30 kDa-PEG-SS1P, lot #1; (6) Bicin3-mono-30 kDa-PEGSS1P, lot #2; (7) PEG2-40 kDa-PEG-SS1P; (8) hydrazide-12 kDa-PEG-SS1P; (9) RNL-8a-12 kDa-PEG-SS1P; (10) ALD-mono-20 kDa-PEGSS1P; (11) ALD-di-20 kDa-PEG-SS1P; and (12) DGA2-12 kDa-PEG-SS1P. (B) (left) Cross-reactivity of human anti-SS1P serum to PEG-SS1P (patient #1). Plates were coated with 1 µg SS1P/50 µL per well. Human antiserum (1:40 000 dilution) versus SS1P was mixed with serial dilutions of SS1P or PEG-SS1P compounds (based on µg protein) and added to the wells (25 °C, 1.5 h). Following incubation and washing, HRP conjugated rabbit anti-human Ig was added to each well. Following incubation and washing, 100 µL TMB-E substrate were added, and the plates were read immediately at 450 nm. Native SS1P ([), DGA2-12 kDa-PEG-SS1P (9), PEG2-40 kDa-PEG-SS1P (2), and SC-12 kDa-PEG-SS1P (/) were included in the analysis. (right) Cross-reactivity of human anti-SS1P serum to PEG-SS1P (patient #2, 1:4000 dilution).

by Biacore analysis. The high-affinity SS1P (Kd ) 0.7 nM) demonstrated diminished affinity in the D-SS1P (Kd ) 39.5 nM) and B-SS1P (Kd ) 8.1 nM) derivatives. In agreement with prior studies on PEGylated Fv compounds (49, 50), this was largely the consequence of a reduction in association rate. For SS1P, kon ) 1.1 × 106 M-1 s-1, while the D-SS1P and B-SS1P derivatives have kon rates of 6.3 × 103 M-1 s-1 and 6.4 × 104 M-1 s-1, respectively. Dissociation rates (koff) were relatively conserved among the compounds, SS1P (8.3 × 10-4 s-1), D-SS1P (2.5 × 10-4 s-1), and B-SS1P (5.1 × 10-4 s-1). It is noteworthy that on-rates of a binding molecule are concentration-dependent, whereas off-rates are not. Hence, the attached PEG strands may in effect diminish the dynamic

concentration of binding competent molecules available for association with their target. Flow cytometry investigations were conducted with the PEG-SS1P compounds to evaluate their cell surface binding to A431-K5 cells. SS1P and PEG-SS1P compounds at equimolarity were incubated with the cells, and mouse anti-PE mAb was added, followed by phycoerythrin-conjugated goat antimouse polyclonal antibody. A shift toward low fluorescence intensity indicates reduced binding of PEG-SS1P to the cells. These flow cytometry results (Figure 5) correlate with the determined IC50 values from the cytotoxicity analysis (see Table 1). For example, SS1P demonstrated maximal cell binding and maximal cytotoxicity (IC50 ) 0.7 ng/mL) in these analyses,



and differential interference contrast (DIC) microscopy (data not shown) to investigate the subcellular localization of this fluorescein-labeled PEG-SS1P compound after incubation with A431-K5 cells in vitro. Figure 6 shows phase contrast, fluorescence, and merged microscopic analysis of cellular uptake and subcellular localization. The images revealed an efficient uptake of the compound by A431-K5 cells and localization mainly in the endosomal/lysosomal compartment in the perinuclear area of the cytoplasm, probably as a consequence of the interference of the dye moiety in correct protein processing and trafficking. Control cells (A431), which do not exhibit cell surface expression of mesothelin, demonstrated no cellular uptake of the fluorescein-labeled PEG-SS1P compound. Therefore, as expected, SS1P bioconjugates must maintain at least two general biological functions for efficacy, both cell binding and intracellular processing.

DISCUSSION

Figure 5. Flow cytometry analysis following PEG-SS1P binding to A431-K5 cells. In flow cytometry studies, A431-K5 cells were incubated with PEG-SS1P compounds prior to addition of mouse antiPE mAb and phycoerythrin-conjugated goat anti-mouse polyclonal antibody for analysis on FACS Calibur. A shift toward low fluorescence intensity indicates reduced binding of PEG-SS1P to the cells. Cell populations are shown (upper graph): (1) cells without anti-mouse Ab fluorescence labeling, (2) without SS1P, with labeled anti-mouse Ab, (3) with di-PEGylated ALD-20 kDa-PEG-SS1P, (4) with monoPEGylated ALD-20 kDa-PEG-SS1P, (5) with mono-PEGylated NHS40 kDa-PEG-SS1P, (6) with mono-PEGylated SC-20 kDa-PEG-SS1P, and (7) with native SS1P; or (lower graph): (1) as above, (2) as above, (3) with DGA2-12 kDa-PEG-SS1P, after 16 h in vitro PEG release, (4) with DGA2-12 kDa-PEG-SS1P, before PEG release, (5) with DGA2-12 kDa-PEG-SS1P, after 4 h in vitro PEG release, and (6) with native SS1P.

whereas the mono-PEGylated ALD-PEG-SS1P (IC50 ) 39 ng/mL) and di-PEGylated ALD-PEG-SS1P (IC50 ) 310 ng/ mL) demonstrated decreasing cell binding with PEG attachments. Two independent preparations of PEG2-40 kDa-SS1P compounds were intermediate in IC50 and in cell binding in this comparison. As shown in Figure 5 (lower graph), SS1P exhibited maximal binding, while DGA2-12 kDa-PEG-SS1P (IC50 ) 2.1 ng/mL) showed reduced cell binding. Upon 4 h of in vitro pH 8.5 hydrolysis of the PEG linkage and release of native SS1P, both the cytotoxicity (IC50 ) 0.93 ng/mL) and the degree of cell binding increase together. However, following prolonged hydrolysis at pH 8.5, the immunotoxin partially denatures as revealed by both a decrease in cytotoxicity (IC50 ) 30.2) and diminished cell binding by flow cytometry. Overall activity is a function of the efficiency of intracellular processing of the PEG-SS1P compounds as well. To investigate the immunotoxin processing of PEG-SS1P from binding through subcellular localization, we used a fluorescein-linked 5 kDa NHS-PEG, which forms a permanent amide linkage in conjugation with SS1P. Mono-PEGylated conjugates were purified and characterized. Although the fluorescein-PEG conjugated SS1P exhibited stronger cell binding by flow cytometry than any of the rPEG-SS1P compounds (data not shown), it demonstrated essentially no in vitro cytotoxicity (IC50 >1000 ng/mL). We used fluorescence microscopy (Figure 6)

We describe macromolecular engineering of recombinant immunotoxin SS1P through employment of rPEGylation. Four rPEGylated designs were investigated wherein three to five large PEG polymers were linked to SS1P via releasable linkers. The rPEG-SS1P compounds exhibited prolonged blood residency time and greatly expanded AUC therapeutic exposure while reducing the nonspecific toxicity of the immunotoxin. A major achievement of this program was a substantial improvement in antitumor efficacy demonstrated by the rPEG-SS1P compounds. For the first time, complete remissions of established mesothelin-expressing tumor xenografts were observed in mice with single-dose administration of rPEG-SS1P conjugates. The significant increase in a therapeutic window for rPEG-SS1P may offer clinical advantages for the use of immunotoxins in cancer patients. Bioconjugation of proteins with PEG via permanent linkages is a well-developed technology employed in marketed PEGylated protein therapeutics and numerous experimental compounds (37, 48). PEG attachments to proteins have significant benefits in prolonging the in vivo activity of proteins by impeding the rapid clearance of proteins from renal filtration, antibody or cellular recognition, or physical degradation. Although an extensive surface modification of proteins, especially foreign proteins, by multiple PEG strands is often preferred in development of PEGylated protein drugs, this strategy can be limited by the reduction or elimination of biological activity of the PEG protein. Recent methods in PEGylation technology have focused on designing a composite bioconjugate using both protein engineering and improved chemical linkages (13, 36, 42). Site-specific attachments of PEG polymer strands may afford better retention of bioactivity, while minimizing the heterogeneity of the conjugates. However, some unwanted polymer-derived effects may still be encountered either in formulation or in pharmacological outcomes. Therefore, our recent research has examined the potential development of rPEGylation, wherein covalent attachments of strands of PEG polymers are shed via a controlled release mechanism derived from a degradable linkage at the attachment site on the protein surface (39, 40, 44). The immunotoxin SS1P must maintain target binding, protein translocation/processing, and enzymatic ADP-ribosylating activities to be an effective therapeutic. These requirements challenge conventional PEGylation approaches. First, random permanent attachment of one large polymer (or a few small polymers) to the protein will likely compromise one or more of these functions while providing suboptimal surface shielding from antibodies. Second, permanent site-specific attachments of PEG will also reduce biological activity and may drastically reduce yields during manufacturing. In contrast, employment


Filpula et al.

Figure 6. A431-K5 cellular internalization studies with 20 nM fluorescein labeled 5 kDa-PEG-SS1P in vitro (16 h). Phase contrast, fluorescence, and merged microscopic images are shown with 10 µm scale bar (lower right) with fluorescein in green and the nucleus stained blue with DAPI.

of rPEGylation may allow widespread modification of the protein while actually simplifying purification, since the heavily PEGylated proteins are readily purified from native protein. While such formulations of SS1P are heterogeneous, the end product is homogeneous and corresponds to the original unmodified SS1P, which is regenerated in vivo. Although this strategy does add complexity to the analysis of in vivo biodistribution, pharmacokinetics, and pharmacodynamics, the overall benefits in increased antitumor efficacy and diminished toxicity, suggested by our present work, support rPEGylation as an important new platform technology for immunotoxins and related therapeutics. Further studies on the biodistribution of the PEG-SS1P compounds may provide insight into the differential deposition of the immunotoxin in tumors and major organs. However, the design of these labeled conjugates may be more complex than the recently reported biodistribution study of 111In-labeled SS1P, which also used a conjugation method for the radioisotope complex (51). Immunogenicity is a central concern in the development and drug monitoring of protein therapeutics, including PEGylated proteins. Binding antibodies and neutralizing antibodies are commonly encountered. Clinical experience with SS1P has revealed the eventual development of neutralizing antibodies in most patients. It is uncertain whether our current rPEGSS1P can completely overcome this problem. Immunized mice did develop neutralizing antibodies against SS1P following prolonged administration of rPEG-SS1P compounds. While these animal results are not predictive of outcomes in patients, it may be challenging to completely abolish the immune response to the SS1P conjugates in humans. In this context, however, two PEGylated nonhuman protein drugs, ADAGEN and ONCASPAR, have been administered to patients successfully over multiple cycles, despite the occurrence of antibody responses in several of these patients. Either animal or human antisera against SS1P bind much less efficiently to the PEGSS1P compounds, suggesting that PEG-SS1P compounds will have a lower in vivo clearance in patients as a result of binding or neutralizing antibodies. We also utilized multiple physical and functional assays to evaluate the composition, stability, and activities of the rPEGSS1P conjugates. From flow cytometry and cytotoxicity investigations, we established a correlation between the efficiency of target binding by the SS1P derivatives and their in vitro IC50 values. However, using fluorescence and DIC microscopy, we also demonstrated that uptake alone of a fluorescein labeled PEG-SS1P conjugate does not guarantee correct intracellular processing. In this instance, it is proposed that the PEG-dye moiety, while permitting internalization, interferes with the biological release mechanism of the toxin. We further observed that in vitro binding or activity assays are inadequate to predict efficacy in vivo of the rPEG-SS1P, since the controlled release

of the active drug is not fully revealed in vitro. rPEG-SS1P may acquire full bioactivity in vivo following programmed release. An intrinsic feature of recombinant immunotoxins is targetspecific delivery. In juxtaposition to active targeting, the phenomenon of passive targeting of drugs via the EPR effect has been shown to offer significant drug accumulation in tumors (45, 46). PEG modification is one common approach to exploiting the EPR effect (45). Therefore, we wished to determine the extent to which an analogous nontargeted immunotoxin would produce antitumor responses in the mesothelin-expressing xenografts. An rPEGylated dsFv-PE immunotoxin, which binds CD22, was prepared with equivalent linkers and PEG attachments and investigated in the A431-K5 xenograft model. No antitumor effects were observed. These results suggest a pivotal role of active targeting via mesothelin binding and internalization for the antitumor effects of the PEGSS1P bioconjugates. In summary, rPEGylation of mesothelin-targeted immunotoxin SS1P greatly improves antitumor efficacy while decreasing nonspecific toxicity. The further co-development of protein engineering and bioconjugate technologies may provide superior immunotoxins with tailored pharmacological properties.

ACKNOWLEDGMENT This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. We thank Steve Youngster, Diane Chang, Qing Dong, and Michelle Boro for performance of SE-HPLC, RP-HPLC, and endotoxin analysis. We are grateful to Mary Mehlig and Virna Borowski for performance of mouse PK and immunogenicity studies. We are also grateful to Jack Lipman and Ivan Horak for critical review of the manuscript.

LITERATURE CITED (1) Hassan, R., Bera, T., and Pastan, I. (2004) Mesothelin: a new target for immunotherapy. Clin. Cancer Res. 10, 3937-3942. (2) Sato, N., Hassan, R., Axworthy, D. B., Wong, K. J., Yu, S., Theodore, L. J., Lin, Y., Park, L., Brechbiel, M. W., Pastan, I., Paik, C. H., and Carrasquillo, J. A. (2005) Pretargeted radioimmunotherapy of mesothelin-expressing cancer using a tetravalent single-chain Fvstreptavidin fusion protein. J. Nucl. Med. 46, 1201-1209. (3) Fan, D., Yano, S., Shinohara, H., Solorzano, C., Van Arsdall, M., Bucana, C. D., Pathak, S., Kruzel, E., Herbst, R. S., Onn, A., Roach, J. S., Onda, M., Wang, Q. C., Pastan, I., and Fidler, I. J. (2002) Targeted therapy against human lung cancer in nude mice by highaffinity recombinant antimesothelin single-chain immunotoxin. Mol. Cancer Ther. 1, 595-600. (4) Hassan, R., Lerner, M. R., Benbrook, D., Lightfoot, S. A., Brackett, D. J., Wang, Q. C., and Pastan, I. (2002) Antitumor activity of SS(dsFv)PE38 and SS1(dsFv)PE38, recombinant antimesothelin im-

Releasable PEGylation of Immunotoxin SS1P munotoxins against human gynecologic cancers grown in organotypic culture in vitro. Clin. Cancer Res. 8, 3520-3526. (5) Hassan, R., Wu, C., Brechbiel, M. W., Margulies, I., Kreitman, R. J., and Pastan, I. (1999) 111Indium-labeled monoclonal antibody K1: biodistribution study in nude mice bearing a human carcinoma xenograft expressing mesothelin. Int. J. Cancer 80, 559-563. (6) Chowdhury, P. S., Viner, J. L., Beers, R., and Pastan, I. (1998) Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity. Proc. Natl. Acad. Sci. U.S.A. 95, 669-674. (7) Ho, M., Hassan, R., Zhang, J., Wang, Q. C., Onda, M., Bera, T., and Pastan, I. (2005) Humoral immune response to mesothelin in mesothelioma and ovarian cancer patients. Clin. Cancer Res. 11, 3814-3820. (8) Bera, T. K., Williams-Gould, J., Beers, R., Chowdhury, P., and Pastan, I. (2001) Bivalent disulfide-stabilized fragment variable immunotoxin directed against mesotheliomas and ovarian cancer. Mol. Cancer Ther. 1, 79-84. (9) Hassan, R., Kreitman, R. J., Pastan, I., and Willingham, M. C. (2005) Localization of mesothelin in epithelial ovarian cancer. Appl. Immunohistochem. Mol. Morphol. 13, 243-247. (10) Li, Q., Verschraegen, C. F., Mendoza, J., and Hassan, R. (2004) Cytotoxic activity of the recombinant anti-mesothelin immunotoxin, SS1(dsFv)PE38, towards tumor cell lines established from ascites of patients with peritoneal mesotheliomas. Anticancer Res. 24, 13271335. (11) Hassan, R., Viner, J. L., Wang, Q. C., Margulies, I., Kreitman, R. J., and Pastan, I. (2000) Anti-tumor activity of K1-LysPE38QQR, an immunotoxin targeting mesothelin, a cell-surface antigen overexpressed in ovarian cancer and malignant mesothelioma. J. Immunother. 23, 473-479. (12) Brinkmann, U., Webber, K., Di Carlo, A., Beers, R., Chowdhury, P., Chang, K., Chaudhary, V., Gallo, M., and Pastan, I. (1997) Cloning and expression of the recombinant Fab fragment of monoclonal antibody K1 that reacts with mesothelin present on mesotheliomas and ovarian cancers. Int. J. Cancer 71, 638-644. (13) Chowdhury, P. S., Chang, K., and Pastan, I. (1997) Isolation of anti-mesothelin antibodies from a phage display library. Mol. Immunol. 34, 9-20. (14) Chang, K., and Pastan, I. (1996) Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc. Natl. Acad. Sci. U.S.A. 93, 136-40. (15) Chang, K., and Pastan, I. (1994) Molecular cloning and expression of a cDNA encoding a protein detected by the K1 antibody from an ovarian carcinoma (OVCAR-3) cell line. Int. J. Cancer 57, 90-97. (16) Bera, T. K., and Pastan, I. (2000) Mesothelin is not required for normal mouse development or reproduction. Mol. Cell Biol. 20, 2902-2906. (17) Muminova, Z. E., Strong, T. V., and Shaw, D. R. (2004) Characterization of human mesothelin transcripts in ovarian and pancreatic cancer. BMC Cancer 4, 19. (18) Robinson, B. W., Creaney, J., Lake, R., Nowak, A., Musk, A. W., de Klerk, N., Winzell, P., Hellstrom, K. E., and Hellstrom, I. (2003) Mesothelin-family proteins and diagnosis of mesothelioma. Lancet 362, 1612-1616. (19) Ordonez, N. G. (2003) Application of mesothelin immunostaining in tumor diagnosis. Am. J. Surg. Pathol. 27, 1418-1428. (20) Argani, P., Iacobuzio-Donahue, C., Ryu, B., Rosty, C., Goggins, M., Wilentz, R. E., Murugesan, S. R., Leach, S. D., Jaffee, E., Yeo, C. J., Cameron, J. L., Kern, S. E., and Hruban, R. H. (2001) Mesothelin is overexpressed in the vast majority of ductal adenocarcinomas of the pancreas: identification of a new pancreatic cancer marker by serial analysis of gene expression (SAGE). Clin. Cancer Res. 7, 3862-3868. (21) Onda, M., Willingham, M., Nagata, S., Bera, T. K., Beers, R., Ho, M., Hassan, R., Kreitman, R. J., and Pastan, I. (2005) New monoclonal antibodies to mesothelin useful for immunohistochemistry, fluorescence-activated cell sorting, Western blotting, and ELISA. Clin. Cancer Res. 11, 5840-5846. (22) Rump, A., Morikawa, Y., Tanaka, M., Minami, S., Umesaki, N., Takeuchi, M., and Miyajima, A. (2004) Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion. J. Biol. Chem. 279, 9190-9198.

Bioconjugate Chem., Vol. 18, No. 3, 2007 783 (23) Chowdhury, P. S., and Pastan, I. (1999) Improving antibody affinity by mimicking somatic hypermutation in vitro. Nat. Biotechnol. 17, 568-572. (24) Brinkmann, U., Reiter, Y., Jung, S. H., Lee, B., and Pastan, I. (1993) A recombinant immunotoxin containing a disulfide-stabilized Fv fragment. Proc. Natl. Acad. Sci. U.S.A. 90, 7538-7542. (25) Reiter, Y., Brinkmann, U., Kreitman, R. J., Jung, S. H., Lee, B., and Pastan, I. (1994) Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions. Biochemistry 33, 5451-5459. (26) Reiter, Y., Brinkmann, U., Lee, B., and Pastan, I. (1996) Engineering antibody Fv fragments for cancer detection and therapy: disulfide-stabilized Fv fragments. Nat. Biotechnol. 14, 1239-1245. (27) Allured, V. S., Collier, R. J., Carroll, S. F., and McKay, D. B. (1986) Structure of exotoxin A of Pseudomonas aeruginosa at 3.0Ångstrom resolution. Proc. Natl. Acad. Sci. U.S.A. 83, 1320-1324. (28) Wang, Q. C., Pai, L. H., Debinski, W., FitzGerald, D. J., and Pastan, I. (1993) Polyethylene glycol-modified chimeric toxin composed of transforming growth factor R and Pseudomonas exotoxin. Cancer Res. 53, 4588-4594. (29) Tsutsumi, Y., Onda, M., Nagata, S., Lee, B., Kreitman, R. J., and Pastan, I. (2000) Site-specific chemical modification with polyethylene glycol of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB2) improves antitumor activity and reduces animal toxicity and immunogenicity. Proc. Natl. Acad. Sci. U.S.A. 97, 8548-8553. (30) Roberts, M. J., and Harris, J. M. (1998) Attachment of degradable poly(ethylene glycol) to proteins has the potential to increase therapeutic efficacy. J. Pharm. Sci. 87, 1440-1445. (31) Brinkmann, U., Pai, L. H., FitzGerald, D. J., Willingham, M., and Pastan, I. (1991) B3(Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice. Proc. Natl. Acad. Sci. U.S.A. 88, 8616-8620. (32) Pastan, I., and FitzGerald, D. (1991) Recombinant toxins for cancer treatment. Science 254, 1173-1177. (33) Bang, S., Nagata, S., Onda, M., Kreitman, R. J., and Pastan, I. (2005) HA22 (R490A) is a recombinant immunotoxin with increased antitumor activity without an increase in animal toxicity. Clin. Cancer Res. 11, 1545-1550. (34) Kreitman, R. J., and Pastan, I. (1998) Accumulation of a recombinant immunotoxin in a tumor in ViVo: fewer than 1000 molecules per cell are sufficient for complete responses. Cancer Res. 58, 968-975. (35) Kreitman, R. J., Wilson, W. H., Robbins, D., Margulies, I., StetlerStevenson, M., Waldmann, T. A., and Pastan, I. (1999) Responses in refractory hairy cell leukemia to a recombinant immunotoxin. Blood 94, 3340-3348. (36) Kreitman, R. J., Wilson, W. H., Bergeron, K., Raggio, M., StetlerStevenson, M., FitzGerald, D. J., and Pastan, I. (2001) Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapyresistant hairy-cell leukemia. N. Engl. J. Med. 345, 241-247. (37) Harris, J. M., and Chess, R. B. (2003) Effect of PEGylation on pharmaceuticals. Nat. ReV. Drug DiscoVery 2, 214-221. (38) Benhar, I., Wang, Q. C., FitzGerald, D., and Pastan, I. (1994) Pseudomonas exotoxin A mutants: replacement of surface-exposed residues in domain III with cysteine residues that can be modified with polyethylene glycol in a site-specific manner. J. Biol. Chem. 269, 13398-404. (39) Zhao, H., Yang, K., Martinez, A., Basu, A., Chintala, R., Liu, H. C., Janjua, A., Wang, M., and Filpula, D. (2006) Linear and branched bicin linkers for releasable PEGylation of macromolecules: controlled release in vivo and in vitro from mono- and multi-PEGylated proteins. Bioconjugate Chem. 17, 341-351. (40) Greenwald, R. B., Yang, K., Zhao, H., Conover, C. D., Lee, S., and Filpula, D. (2003) Controlled release of proteins from their poly(ethylene glycol) conjugates: drug delivery systems employing 1,6elimination. Bioconjugate Chem. 14, 395-403. (41) Greenwald, R. B., Choe, Y. H., McGuire, J., and Conover, C. D. (2003) Effective drug delivery by PEGylated drug conjugates. AdV. Drug DeliVery ReV. 55, 217-250. (42) Zalipsky, S., Qazen, M., Walker, J. A., II, Mullah, N., Quinn, Y. P., and Huang, S. K. (1999) New detachable poly(ethylene glycol) conjugates: cysteine-cleavable lipopolymers regenerating natural phospholipid, diacyl phosphatidylethanolamine. Bioconjugate Chem. 10, 703-707.

784 Bioconjugate Chem., Vol. 18, No. 3, 2007 (43) Tsubery, H., Mironchik, M., Fridkin, M., and Shechter, Y. (2004) Prolonging the action of protein and peptide drugs by a novel approach of reversible polyethylene glycol modification. J. Biol. Chem. 279, 38118-38124. (44) Greenwald, R. B., Zhao, H., Yang, K., Reddy, P., and Martinez, A. (2004) A new aliphatic amino prodrug system for the delivery of small molecules and proteins utilizing novel PEG derivatives. J. Med. Chem. 47, 726-734. (45) Yu, D., Peng, P., Dharap, S. S., Wang, Y., Mehlig, M., Chandna, P., Zhao, H., Filpula, D., Yang, K., Borowski, V., Borchard, G., Zhang, Z., and Minko, T. (2005) Antitumor activity of poly(ethylene glycol)-camptothecin conjugate: the inhibition of tumor growth in vivo. J. Controlled Release 110, 90-102. (46) Maeda, H., Sawa, T., and Konno, T. (2001) Mechanism of tumortargeted delivery of macromolecular drugs, including the EPR effect in solid tumor and clinical overview of the prototype polymeric drug SMANCS. J. Controlled Release 74, 47-61. (47) Li, S., Yang, Z., Sun, X., Tan, Y., Yagi, S., and Hoffman, R. M. (2003) A simultaneous colorimetric assay of free and protein-coupled polyethylene glycol. Anal. Biochem. 313, 335-337.

Filpula et al. (48) Roberts, M. J., Bentley, M. D., and Harris, J. M. (2002) Chemistry for peptide and protein PEGylation. AdV. Drug DeliVery ReV. 54, 459-476. (49) Yang, K., Basu, A., Wang, M., Chintala, R., Hsieh, M. C., Liu, S., Hua, J., Zhang, Z., Zhou, J., Li, M., Phyu, H., Petti, G., Mendez, M., Janjua, H., Peng, P., Longley, C., Borowski, V., Mehlig, M., and Filpula, D. (2003) Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation. Protein Eng. 16, 761-770. (50) Kubetzko, S., Sarkar, C. A., and Pluckthun, A. (2005) Protein PEGylation decreases observed target association rates via a dual blocking mechanism. Mol. Pharmacol. 68, 14391454. (51) Zhang, Y., Xiang, L., Hassan, R., Paik, C. H., Carrasquillo, J. A., Jang, B. S., Le, N., Ho, M., and Pastan, I. (2006) Synergistic antitumor activity of taxol and immunotoxin SS1P in tumor-bearing mice. Clin. Cancer Res. 12, 4695-4701. BC060314X

Releasable PEGylation of Mesothelin Targeted Immunotoxin SS1P

Recommend Documents