Preparation and Characterization of Anti-Tenascin Monoclonal

Currently, we are evaluating the therapeutic potential of directly labeled monoclonal antibody (mAb) 81C6, reactive with the extracellular matrix prot...
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Bioconjugate Chem. 1999, 10, 867−876

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Preparation and Characterization of Anti-Tenascin Monoclonal Antibody-Streptavidin Conjugates for Pretargeting Applications Catherine F. Foulon,* Darell D. Bigner, and Michael R. Zalutsky Departments of Radiology and Pathology, Duke University Medical Center, DUMC 3808, Durham, North Carolina 27710. Received April 12, 1999; Revised Manuscript Received June 17, 1999

Radioimmunopretargeting is based on the separate injection of a modified mAb and the radionuclide and most frequently exploits the very high avidity of biotin for streptavidin (SA). Currently, we are evaluating the therapeutic potential of directly labeled monoclonal antibody (mAb) 81C6, reactive with the extracellular matrix protein tenascin, in surgically created glioma resection cavity patients. To be able to investigate pretargeting in this setting, the synthesis of 81C6 mAb-SA conjugates was required. In the current study, we have evaluated five methods for preparing both murine 81C6 (m81C6) and human/mouse chimeric 81C6 (c81C6) SA conjugates with regard to yield, biotin-binding capacity, immunoreactivity, and molecular weight. The 81C6 mAb and SA were coupled by covalent interaction between sulfhydryl groups generated on the mAb via N-succinimidyl-S-acetylthioacetate, dithiothreitol or 2-iminothiolane (2IT), and maleimido-derivatized SA, prepared via sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-succinimidyl-3-(2-pyridyldithio)-propionate. A noncovalent approach involving reaction of a biotinylated mAb, prepared using biotin caproate, and SA also was studied. The evaluation criteria were yield of mAb-SA 215 kDa monomer, as well as conjugate biotin-binding capacity and immunoreactive fraction. The optimal procedure involved activation of m81C6 or c81C6 with 30 equiv of 2IT and reaction of SA with 10 equiv of SMCC and yielded a conjugate with excellent biotin-binding capacity and immunoreactivity. The (125I-labeled m81C6)-2IT-SMCC-SA was stable and did not lose biotin-binding capacity after a 72 h incubation in human glioma cyst fluid in vitro. Although the conjugate was stable in murine serum in vivo, its biotin-binding capacity declined rapidly, consistent with high endogenous biotin levels in the mouse. After injection of the radioiodinated conjugate into athymic mice with subcutaneous D-54 MG human glioma xenografts, high tumor uptake (36.0 ( 10.7% ID/g at 3 days) and excellent tumor:normal tissue ratios were observed.

INTRODUCTION

One of the most significant limitations of monoclonal antibodies (mAbs)1 for radioimmunotherapeutic application is that their large molecular mass impedes homogeneous tumor uptake and clearance from normal tissues. To circumvent this problem, pretargeting strategies have been pursued in which the mAb and the radiolabel are injected separately. In such a protocol, a modified mAb conjugate is injected, and once optimal tumor-to-normal tissue ratios have been achieved (usually, within 2-4 days), a low molecular mass radiolabeled ligand that exhibits high-affinity binding to the conjugate is administered (1). Most pretargeting investigations have attempted to exploit the high-affinity binding of biotin, a 244 Da vitamin, for 65 kDa avidin or 54 kDa streptavidin (SA). * To whom correspondence should be addressed. Phone: (919) 684-7705. Fax: (919) 684-7122. E-mail: [email protected]. 1 Abbreviations: mAb, monoclonal antibody; SA, streptavidin; m81C6, murine 81C6, c81C6, human/mouse chimeric 81C6; IBA, biotinyl-3-iodoanilide; IBB, (3-iodobenzoyl)norbiotinamide; BnLCsNHS, biotinamidocaproic acid 3-sulfo-N-hydroxysuccinimide; BSA, bovine serum albumin; SATA, N-succinimidyl-Sacetylthioacetate; DTT, dithiothreitol; 2IT, 2-iminothiolane; SMCC, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane1-carboxylate; SPDP, N-succinimidyl-3-(2-pyridyldithio)-propionate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography; ITLC, instant thin-layer chromatography.

Although many different approaches have been described (2-4), theoretical calculations predict that administration of mAb-SA conjugates, followed by a radiolabeled biotin derivative, should provide optimal tumor targeting (5). Indeed, some of the most promising clinical results to date have been achieved with pretargeting protocols that utilize biotin as the radionuclide carrier (6, 7). Monoclonal antibodies directed against tenascin, an extracellular matrix protein found on gliomas, melanomas, and breast carcinomas (8-10), might be useful for pretargeting because they do not internalize after antigen binding. Currently, we are investigating the therapeutic potential of the 131I-labeled anti-tenascin mAb m81C6 administered into surgically created glioma resection cavities and via the intrathecal route in patients with neoplastic meningitis (11, 12). Encouraging results have been obtained in many patients with these directly labeled mAbs; however, pretargeting approaches using 81C6-SA conjugates and radiolabeled biotin molecules might offer significant advantages. Recent results suggest that it might be possible to deliver high radiation doses to intracranial malignancies using pretargeting, even when reagents are administered intravenously (7). In addition, by labeling biotin instead of the mAb, radiotherapy with promising short half-life radionuclides, such as the 7.2 h half-life R-emitter 211At, should be facilitated. Prior to initiating clinical investigations, methodologies for the preparation, purification, and characterization of reagents with optimal properties for pretargeting must

10.1021/bc990040w CCC: $18.00 © 1999 American Chemical Society Published on Web 08/03/1999

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be developed. We have recently described the synthesis of radiohalogenated biotin conjugates including (3-[125/131I]iodobenzoyl)norbiotinamide (IBB) and its 211At-labeled analogue and documented their resistance to degradation by biotinidase (13, 14). In the current study, we have investigated several methods for preparing anti-tenascin 81C6-SA conjugates and evaluated them with regard to yield, biotin-binding capacity, immunoreactivity and molecular mass. Approaches involving the noncovalent interaction between biotinylated 81C6 (via sulfo-NHSBnLC) and SA, as well as covalent binding between SA and thiolated mAb, were investigated. Variables that were studied included SA maleimido-activation (SMCC and SPDP), mAb thiolation (reduction of existing disulfide bonds via DTT, addition of sulfhydryl groups using 2IT or SATA), and mAb (m81C6 and c81C6). Our results indicate that using covalent approaches, a 215 kDa mAb-SA monomer, consisting of one SA molecule and one mAb molecule, can be prepared in reasonable yield and with excellent biotin and antigen binding capacities. Furthermore, (125I-labeled m81C6)-2IT-SMCC-SA exhibited excellent in vitro stability in human glioma tumor cyst fluid and a tissue distribution in athymic mice with subcutaneous D-54 MG human glioma xenografts that compares favorable with that of directly radioiodinated m81C6. MATERIALS AND METHODS

Materials. Murine 81C6 (m81C6) is an IgG2b that binds to domain 12 of alternatively spliced fibronectin type III repeats of tenascin (8, 15). Details concerning the generation and characterization of human/mouse chimeric 81C6 (c81C6), consisting of human IgG2 constant regions and m81C6 variable regions, can be found in a previous publication (16). Both mAbs were purified using Protein A Sepharose and an ion-exchange method compatible with mAb isoelectric point (17). Streptavidin, 2-IT, iodoacetamide, and BnLCsNHS were purchased from Sigma Chemical Co. (St. Louis, MO); all other reagents were purchased from Pierce (Rockford, IL). Radioiodine (Na125I or Na131I) in 0.1 N NaOH was obtained from Dupont-NEN (Boston, MA). The radioiodinated biotin conjugates [131I]IBA and [131/125I]IBB were prepared as described elsewhere (13, 18). General. For spin column purification, Sephadex G-25 (Pharmacia, Pistacaway, NJ) was reconstituted in PBS containing 0.05 M EDTA and added to a 3 mL syringe fitted with a cotton ball. The column was conditioned with 200 µL of 0.1% BSA and washed with 3 mL of the above buffer. The column was spun at 1000 rpm for 3 min, the sample (68% for m81C6 and >79% for c81C6, values comparable to those for native mAbs. In addition, SDSPAGE indicated the presence of a single ∼215 kDa peak capable of biotin binding. The exception was the reaction run at a 30:1 DTT:mAb molar ratio where a 75 kDa

species that did not bind biotin, as well as multiple 150180 kDa species that did bind biotin, was observed. On the basis of ease of synthesis, yield, and binding properties of the resultant conjugate, the method selected for further study involved activation of mAb with 30 equiv of 2IT followed by reaction with SMCC-SA (prepared at 10:1 molar ratio). Representative size-exclusion HPLC chromatograms obtained using this procedure with m81C6

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Figure 2. Size-exclusion TSK 3000 profile (UV absorbance at 280 nm) of (A) monomeric mAb-SA, mAb, and SA standards, and (B-F) conjugate reaction mixtures prepared at 10:1 reagent:mAb and reagent:SA molar ratios. Peak at approximately 25 min corresponds to iodoacetamide.

and c81C6 are illustrated in Figure 4. Even though the mAbs had different elution times on this column (m81C6, 15.5 min; c81C6, 16.5 min), with both mAbs, the predominant peak in the reaction mixture eluted at 12 min, corresponding to the desired mAb-SA conjugate. In Vivo Stability of (125I-Labeled m81C6)-2ITSMCC-SA in Normal Mice. The in vivo stability of (125Ilabeled m81C6)-2IT-SMCC-SA was investigated after injection of the conjugate to normal mice. Figure 5 presents an SDS-PAGE autoradiograph comparing the molecular mass profile of the conjugate to those present in murine serum samples obtained 2-48 h after injection. The only radioactive band observed in serum had a

molecular mass of ∼215 kDa with no evidence for the generation of lower molecular mass labeled catabolites. The biotin-binding capacity of the mAb-SA conjugate in serum, measured using [131I]IBA, was 92.1 ( 3.1% at 2 h, 32.4 ( 4.0% at 6 h, 11.5 ( 0.5% at 24 h, and 16.6 ( 1.1% at 24 h. In Vivo Distribution of (125I-Labeled m81C6)-2ITSMCC-SA. Murine 81C6 was labeled with 125I, thiolated using 2IT, and reacted with SMCC-SA. Table 2 summarizes the tissue distribution of this conjugate in athymic mice bearing subcutaneous D-54 MG human glioma xenografts. The radioiodinated mAb-SA conjugate exhibited high tumor uptake, reaching a maximum

Streptavidin-Anti-Tenascin mAb Conjugates

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Table 1. Coupling Yields, Biotin-Binding Capacities, and Immunoreactive Fractions for Anti-Tenascin 81C6 mAb-Streptavidin Conjugates

mAb

mAb activationa

SA activationb

yield (%)c

biotin binding (%)d

IRF (%)e

m81C6 m81C6 c81C6 m81C6 m81C6 m81C6 m81C6 m81C6 m81C6 c81C6

BnLC (10:1) DTT DTT (10:1) DTT (30:1) SATA (10:1) SATA (30:1) 2IT (10:1) 2IT (10:1) 2IT (30:1) 2IT (30:1)

none SMCC SMCC SMCC SMCC SMCC SPDP SMCC SMCC SMCC

13 5 4 4 9 15 13 8 28 31

79 96 87 95 95 95 99 96 96 79

49 71 83 68 69 79 86 76 73 74

a Reagent used for mAb modification (reagent:mAb molar ratio). Reagent used for SA modification (reagent:SA molar raito). c Yield of 215 kDa product calculated based on initial mAb concentration. d Percentage of labeled biotin counts bound in vitro. e Immunoreative fraction. b

of 36.04 ( 10.69% ID/g at 3 days. Deiodination of the conjugate was reflected by the fact that about 3% of the injected dose was taken up by the thyroid at all three time points. Three days after injection, tumor-to-blood ratios were 3.6:1, while tumor-to-normal organ ratios were greater than 10:1, demonstrating that this SA coupling method did not compromise the tumor localizing capacity and normal tissue clearance of m81C6. In Vitro Stability of (125I-Labeled m81C6)-2ITSMCC-SA in Tumor Cyst Fluid. No evidence for degradation of the conjugate was observed on SDSPAGE autoradiographs during the 72 h incubation period. Incubation in glioma resection cyst fluid had no effect on the biotin-binding capacity of the conjugate. The percentage of [131I]IBA bound decreased only slightly, from 98.8% after 2 h to 94.3% after 72 h. For comparison, after incubation of SA in cyst fluid, [131I]IBA binding ranged 98.9-94.7% from 2 to 72 h. As a control, the binding of [131I]IBA to cyst fluid alone was measured and, as expected, was less than 1-5% during the 72 h incubation period. DISCUSSION

Our ongoing radioimmunotherapy trials for the treatment of brain tumors involve direct administration of radiolabeled anti-tenascin mAbs into surgically created glioma resection cavities (12). As one of our secondgeneration strategies, we envision investigating a twostep pretargeted approach in which the 81C6-SA conjugate would be injected into the cavity in order to saturate tenascin-binding sites on the tumor. The radiolabeled biotin derivative would be delivered about 2 days later, also into the resection cavity. By associating the radiolabel with a small molecule instead of the mAb, we hope to increase the diffusion of the labeled moiety into the tumor and extend the zone of effective treatment beyond the regions adjacent to the cavity interface. With this goal in mind, we have previously designed a series of biotin conjugates derivatives, including [131I]IBA, [131I]IBB, and their 211At-labeled analogues (13, 14, 18, 21) with varying degrees of resistance to biotinidase degradation. Herein, we have attempted to optimize the production of the other reagent needed for pretargeting, the anti-tenascin mAb-SA conjugate. Some of the methods that were investigated have been described in the literature; however, evaluation of their relative merit is complicated by the fact that different mAbs, reagent amounts, reaction times, and conjugate isolation methods

were used. In addition, to the best of our knowledge, the use of SATA and SPDP for the preparation of mAb-SA conjugates has not been reported. The simplest method for preparing mAb-SA conjugates is by reaction between a biotinylated mAb and SA, thereby exploiting the SA-biotin reaction itself to form the conjugate. This approach is attractive because several activated biotin agents are commercially available, and it can be used for generating the conjugate in vivo by preinjection of the biotinylated mAb, followed by intravenous injection of SA (22, 23). Furthermore, biotinylation results in negligible structural modification of the protein with retention of immunoreactivity (24). On the other hand, a limitation of this approach, when used in vitro, is that no form of quenching is available, and polymerization and cross linkage can take place, resulting in high molecular mass proteins with compromised binding properties and tissue distribution. The results that we obtained with m81C6-BnLC-SA were consistent with the formation of higher molecular mass species than the desired 215 kDa mAb1-SA1 conjugate. The immunoreactive fraction of m81C6-BnLCSA was less than 50%, considerably lower than that observed with other conjugate formation methods. In addition, this conjugation method makes use of the highest affinity SA biotin-binding site in the conjugation process and decreases the binding capacity of the conjugate for subsequently administered radiolabeled biotin. For these reasons, we decided to focus our efforts on covalent methods for coupling mAb and SA. Covalently linked mAb-SA have been generated using classical cross-linking chemistry involving the high reactivity of sulfhydryl residues with maleimido groups. Typically, free thiols are generated on one protein by direct reduction of existing disulfide bridges using mercaptoethylamine (MEA) or DTT, or by addition of sulfhydryl groups on the lysyl residues with 2IT or SATA. Maleimido groups are substituted on the lysyl residues of the other protein using sulfo-SMCC or SPDP. Several of the possible combinations have been applied to the coupling of mAb and SA including mAb-SMCC-2IT-SA (25, 26), mAb-2IT-SMCC-SA (27), mAb-MEA-SMCC-SA (25, 27), and mAb-DTT-SMCC-SA (28). In addition, sitespecific biotinylation of thiolated mAbs also has been described (29, 30). Our goals were to identify a covalent approach for producing anti-tenascin mAb-SA conjugates with high biotin-binding capacity and immunoreactivity, and good yield of the 215 kDa mAb-SA monomer. We decided to focus on this molecule because it is the smallest product containing both intact mAb and SA, and thus should diffuse more rapidly through the tumor than larger conjugates. Only limited information is available concerning the molecular mass profiles of mAb-SA conjugates. After reaction of a disulfide-reduced mAb with SMCC-SA, FPLC analysis indicated the formation of a single ∼210 kDa species capable of binding biotin (25). However, in a subsequent study using similar reaction conditions, the molecular mass of the mAb-SA conjugate was determined to be about 350 kDa by size-exclusion HPLC (31). SA-mAb conjugates in the 440-600 kDa range also have been reported (32). Initially, we attempted covalent synthesis of mAb-SA conjugates using the approach described by NeoRx Corporation (28). This procedure involves thiolation of the mAb with 10 equiv of DTT, activation of SA with 10 equiv of SMCC, and reaction of the proteins at an equimolar ratio. Although biologically active 81C6 mAbSA conjugates were obtained, yields of the 215 kDa

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Figure 3. SDS-PAGE of size-exclusion-HPLC-purified m81C6-SA; silver stain on left, autoradiograph after exposure of reaction mixture to [125I]IBB on right, of the same gel. Lane 1, molecular mass standards; lane 2, murine mAb; lane 3, SATA:mAb 10:1; lane 4, SATA:mAb 30:1, lane 5, 2IT:mAb 10:1, lane 6, 2IT:mAb 30:1; lane 7, DTT:mAb 10:1; lane 8, DTT:mAb 30:1; lane 9, streptavidin.

Figure 4. Size-exclusion TSK 3000 profile (UV absorbance at 280 nm) of (A) murine and (B) chimeric 81C6-streptavidin conjugate prepared at 30:1 2IT:mAb molar ratio.

monomer were less than 5% in our hands. Interestingly, the c81C6 reaction mixture contained higher levels of unreacted SA and mAb while, with m81C6, prominent peaks in the 170-200 kDa and