Enhanced Stability in vitro and in vivo of Immunoconjugates Prepared

Bioconjugate Chem. 1994, 5, 248-256

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Enhanced Stability in Vitro and in Vivo of Immunoconjugates Prepared with 5-Methyl-2-iminothiolane Stephen F. Carroll,' Susan L. Bernhard,t Dane A. Goff,+Robert J. Bauer, Will Leach, and Ada H. C. Kung Departments of Biological Chemistry and Pharmacology/Toxicology, XOMA Corporation, 2910 Seventh Street, Berkeley, California 94710 . Received December 3, 1993"

Substituted 2-iminothiolanes (X2ITs) are new heterobifunctional crosslinking agents designed for the preparation of disulfide-linked conjugates with enhanced resistance to reduction. Based upon 2-IT substituted at the 4 and/or 5 position, these reagents appear to function by sterically protecting the conjugate disulfide bond from attack by thiolate nucleophiles. Here, we have used the X2ITs to prepare and evaluate a series of immunoconjugates (antibody-cytotoxin conjugates) between the murine monoclonal antibody 791/T36, which recognizes a 72-kDa surface antigen present on many human tumor cells, and RTA30, the naturally occurring 30-kDa glycoform of ricin A chain. The X2IT-linked conjugates were also compared to immunoconjugates prepared with N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) and 4- [(succinimidyloxy)carbonyll-a-methyl-cu-(2-pyridyldithio)toluene(SMPT), as well as with methyl- and dimethyl-substituted structural analogs of SPDP. In vitro, 791-(X2IT)TNB model compounds exhibited a 6000-fold range of stabilities. In contrast, the corresponding 791(X2IT)-RTA30 immunoconjugates were up to 20-fold more stable than conjugates made with unhindered linkages. These improvements resulted in immunoconjugates with prolonged serum half-lives in animals. Our data indicate that one of the crosslinking agents, 5-methyl-2-iminothiolane(MZIT), has optimal properties for the preparation of disulfide crosslinked immunoconjugates intended for therapeutic use in that (i) it is highly water soluble and reacts rapidly with protein amino groups at neutral pH, preserving the positive charge, (ii) it forms conjugateswith RTA30efficiently, and (iii) its conjugates exhibit enhanced disulfide bond stability in vitro and in vivo. The potential utility of M2IT and other X2ITs for the preparation of controlled release protein-drug conjugates is also discussed.

INTRODUCTION

Most of the RTA immunoconjugates prepared to date have utilized one of two crosslinking reagents, SPDP or Immunoconjugates (antibodies linked to cytotoxic pro2IT, to generate a disulfide bond linking antibody to teins) represent a specialized class of protein-protein cytotoxin. That a reducible bond is required for maximal conjugates designed for therapeutic use (for a review, see expression of cytotoxic activity has been demonstrated refs 1 and 2). As such, they typically are prepared by by numerous studies (1, 2, 9). However, many such covalently crosslinkingan antibody molecule to a cytotoxin conjugates are unstable in animals (10, II), where cleavage such as the A chain of ricin (RTA).' The antibody thus of the disulfide bond regenerates free antibody and serves to target the action of the cytotoxic component to cytotoxin. For immunoconjugate therapy this deconjucells bearing the target antigen. Once internalized, the gation has two important consequences. First, it reduces cytotoxin is released and then penetrates into the cytosol the effective concentration of circulating immunoconjuwhere it enzymically inactivates ribosomes, blocking gate, and as a result, larger clinical doses may be required. protein synthesis and causing cell death. This approach Second, the released antibody may remain in circulation for selective cellular elimination is currently being evalumuch longer than does conjugate, where it can compete ated clinically for the treatment of autoimmune disorders for antigen binding sites on target cells. Thus, the disulfide and cancer (2-8). bond linking antibodies to cytotoxin such as RTA must be sufficiently labile to facilitate intracellular cytotoxicity, + Current address: Chiron Corp., 4560 Horton Street, Embut it must also be sufficiently stable to survive admineryville, CA 94608. and delivery in vivo. * Current address: Gen Pharm Int., 297 N. Bernard0 Ave., istration To address these issues, several new crosslinking Mountain View, CA 94043. reagents have been prepared and tested for immunoconAbstract published in Advance A C S Abstracts, March 15, jugate preparation, and the in vitro and in vivo properties 1994. of such conjugates have been studied. Each of these Abbreviations: 2IT, 2-iminothiolane; 2-ME, 2-mercaptoreagents contains one (12,131 or two (14) methyl groups ethanol; 2TP, 2-thiopyridine; DTNB, dithionitrobenzoic acid; adjacent to the disulfide bond, and each has generated DTPO, 2,2'dithiobis(pyridineN-oxide);DTDP, 2,2'dithiodipyridine; GSH, reduced glutathione; HPSEC, high-performance conjugates with enhanced stability (12-15) and improved size-exclusion chromatography; MSPDP, the methyl-SPDP efficacy (15)in animals. Thus, hindering accesa of reducing analog N-succinimidyl 3-(2-pyridyldithio)butyrate;RTA, ricin agents to the antibody-cytotoxin linkage results in imtoxin A chain; RTABo,the 30-kDa glycoform of RTA; SAMBA, munoconjugates with improved i n vivo stability and the dimethyl-SPDP structural analog N-hydroxysuccinimidyl potency. 3-methyl-3-(acetylthio)butanoate;SMCC, succinimidyl 4-(NRecently, we described the synthesis and preliminary maleimidomethy1)cyclohexane-1-carboxylate;SMPT, 4-[(succharacterization of a new family of crosslinking reagents, cinimidyloxy)carbonyl]-a-methyl-a-(2-pyridyldithio)toluene; termed X2ITs (16),which are based upon 2-iminothiolane SPDP, N-succinimidyl 3-(2-pyridyldithio)propionate; TNB, (17). The X2ITs offer several advantages over other thionitrobenzoic acid; TPO, 2-thiopyridine N-oxide; XBIT, 2-iminothiolane substituted at the 4 and/or 5 position. crosslinking reagents: (i) they react with primary amines 1043- 18Q2/94/29Q5-Q248$Q4.5QlQ 0 1994 American Chemical Society

Bioconjugate Chem., Vol. 5, No. 3, 1994

Immunoconjugates Made with 2-Iminothiolanes

to form stable amidinium derivatives that retain the positive charge; (ii) inclusion of an aromatic disulfide (such as DTNB) in the reaction mixture both activates the newly exposed X2IT thiol and allows real-time spectrophotometric monitoring of the labeling reaction; and (iii) variation in the substituent at the 5-position (immediately adjacent to the linker thiol) alters the susceptibility of the resulting disulfide bond to reduction. For activated model compounds, these alterations in steric hindrance resulted in disulfide bonds that varied by over 4000-fold in their ability to be reduced by glutathione (16).The preparation and properties, both i n vitro and in vivo, of RTA immunoconjugates prepared with the X2IT reagents are the subject of this report. EXPERIMENTAL PROCEDURES

Materials. Solutions of DTNB (Sigma Chemical Co., St. Louis, MO) were prepared as described by Jocelyn (18). An ElmMlcm of 14.1 at 412 nm (19) was used to determine concentrations of the TNB anion. DTDP and DTPO were from Aldrich (Milwaukee, WI); the mM extinction coefficients (and wavelengths) used for 2TP (343 nm) and TPO (332 nm) were 7.06 and 4.16, respectively. Stock solutions of GSH (Sigma Chemical Co., St. Louis, MO) were prepared in PBS-EDTA (see below); prior to use, the concentration of free thiols was quantified by reaction with DTNB. DTT, 2-ME, Sephadex G25F, Phenyl-Sepharose (allfrom Sigma Chemical Co., St. Louis, MO), trisacryl GF-O5LS, and Ultrogel AcA44 (both from IBF Biotechnics, France) were purchased as indicated. All other reagents were of analytical grade. CrosslinkingReagents. 2IT, SMCC (both from Sigma ChemicalCo., St. Louis, MO),and SPDP (PierceChemical, Rockford, IL) were obtained from the indicated sources. The X2IT crosslinkingreagents,which have the structures shown in Table 1,were synthesized as described previously (16).Stock solutions were prepared in water; concentrations were determined spectrophotometrically by using appropriate extinction coefficients at 248 nm (16). In addition, three other crosslinkers were prepared for these studies. MSPDP and SMPT were synthesized according to Worrell et al. (12) and Thorpe et al. (13),respectively, with minor modifications. The structure of each linker was confirmed by lH NMR. N-Hydroxysuccinimidyl 3-methyl-3-(acetylthio)butanoate(SAMBA),a dimethylsubstituted structural analog of SPDP, was prepared as follows: 3-Methyl-3-(acetylthio)butanoicacid (ref 10,2.17 g, 12.3 mmol) in CHzClz (20 mL) was treated with N-hydroxysuccinimide (1.86 g, 16.2 mmol) and dicyclohexylcarbodiimide(3.34 g, 16.2 mmol) at room temperature for 66 h under N2. The reaction mixture was filtered, concentrated i n vacuo, and then subjected to flash chromatography on Si02 with elution in hexane/EtOAc (80/20, v/v, then 50/50). The desired ester was obtained as a pale yellow oil (2.78 g, 82% yield) that gave a white solid upon standing at room temperature: mp 63 "C; TLC (hexane/EtOAc, 80/20, v/v) Rf = 0.27; lH NMR (60 MHz, CDC13)3.23 (s, 2H), 2.80 ( 8 , 4H, NHS ester), 2.27 ( 8 , 3H, SAC), 1.60 (s, 6H). Preparation of Linker-Modified Antibody. The murine IgG2b monoclonal antibody 791/T36 (791, M,ca. 150000) was produced in an Accusyst hollow fiber bioreactor (Endotronics, Minneapolis, MN) and purified as described (20).The purified antibody was derivatized with each crosslinker so as to incorporate an average of 1-1.5 linkers per mol of antibody. For modification with SPDP, MSPDP, SAMBA, or SMCC, 791 antibody at 2 mg/mL in reaction buffer (0.1 M NaP04,0.1 M NaC1, pH 7.5) was first reacted with a 5- to 10-fold molar excess of

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crosslinker (previously dissolved in absolute ethanol). Following a 20-min incubation at 20 "C, excess reagent and reaction byproducts were removed by size-exclusion chromatography on a GF-05LS column equilibrated in reaction buffer at 4 "C. The number of crosslinkers introduced into the antibody was determined by spectrophotometric analysis following DTT-induced release of the 2TP leaving group (21). For some experiments, the 2TP leaving groups were replaced with TNB by mild reduction of the linker-modified antibody (0.1 mM DTT, 30 min, 25 "C), followed by reaction with 2 mM DTNB (30 min, 25 "C). The TNB-activated antibody was then purified by size-exclusion chromatography on a column of G5-05LS equilibrated in phosphate buffered saline containing 1mM EDTA, pH 7.4 (PBS-EDTA) and stored at 4 "C. The reaction of 21T and the X2ITs with 791 antibody was monitored spectrophotometrically as follows (16): 791 antibody (3 mg/mL; 20 pM) and DTNB (2.5 mM) in reaction buffer were equilibrated at 25 "C in a l-cm disposable cuvette and placed in a dual-beam spectrophotometer. An identical solution prepared without antibody was placed in the reference position. To initiate the reaction, X2IT (freshly dissolved in water) was rapidly added to each cuvette with mixing to a final concentration of 0.5 mM, and the absorbance at 412 nm was monitored. When the A412 reached a value of 0.28 (20 pM TNB, or 1 mol of TNB per mol of 7911, the reaction mixture was rapidly desalted on a l-cm X 20-cm column of Sephadex G25F equilibrated at 4 "C in PBS-EDTA. The excluded protein peak was pooled and stored at 4 "C. The number of linkers introduced per mole of antibody was determined spectrophotometrically as follows: The A2, of the linkeractivated protein was first measured. Then, following reaction with 2 mM DTT, released TNB was quantified at 412 nm. The corrected proteinA2, was calculated from the equation A,,(protein)

= A,,(nonreduced)

- (0.33A4,,(reduced))

and the concentration of 791 antibody was determined by using anElmMlCm of 179at 280 nm (E1mg/mllcm = 1.2). The linkedantibody ratio was then calculated from the molar values of TNB and protein. Preparation and Purification of Immunoconjugates. Immunoconjugates containing SPDP-, SMPT-, and SMCC-activated 791 antibody and RTA30 (the naturally occurring 30-kDa glycoform of RTA) were prepared essentially as described (16,20).Briefly, linkeractivated antibody (1-2 mg/mL) in PBS-EDTA was reacted with a 5-fold molar excess of freshly reduced RTA~o.The disulfide exchange (SPDP, SMPT) or maleimide-based (SMCC) conjugation reactions proceeded for 16 h a t 4 "C. For SAMBA-activated 791 antibody, the conjugation reaction proceeded differently. The free thiol on RTA30 (5 mg/mL in reaction buffer) was first activated by reaction with 2 mM DTNB, and the RTA30-SS-TNB was purified by size-exclusion chromatography on Sephadex G25F. SAMBA-modified 791 antibody was then treated with 50 mM hydroxylamine (pH 7.5) for 30 min at 25 "C to remove the S-acetyl protecting group, and conjugation was initiated by the addition of RTA30-SSTNB (3-fold molar excess). Immunoconjugates were separated from excess RTAso and reaction byproducts by chromatography on a 1- X 50-cm column of Ultrogel AcA44 equilibrated at 4 "C in reaction buffer. The number of RTA30 molecules crosslinked to antibody was determined by densitometric analysis of samples following sodium dodecyl sulfate

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polyacrylamide gel electrophoresis in 5 % gels under nonreducing conditions (22)and Coomassieblue staining. The monoconjugate species (1RTA per 791 antibody) of selected immunoconjugates was purified by hydrophobic interaction chromatography on Phenyl-Sepharose (201, so as to remove residual free antibody and immunoconjugates containing multiple RTA30 moieties. Disulfide Bond Stability Assay. The susceptibility of 791-RTA30 immunoconjugates to reduction in vitro was evaluated in a high-performance size-exclusion chromatographic assay (HPSEC) which quantifies physical dissociation of the antibody-RTA3o conjugate2 (23). Immunoconjugates (0.23mg/mL in PBS-EDTA) were incubated at 37 "C for 30 min with increasing concentrations of reduced glutathione (0-10 mM). Upon completion, free thiols were quenched by the addition of excess iodoacetic acid (pH 7.5; final concentration, 50 mM), and aliquots were chromatographed on a BioSil TSK-250 column (BioRad Labs, Richmond, CA) equilibrated at 25 "C in 50 mM NaP04,lOO mM Na2S04, pH 6.8. The flow rate was 1.0 mL/min, the column effluent was monitored at 280 nm, and the amount of RTASO released was quantified by area integration. By comparison to samples incubated with 50 mM 2-ME (whichresulted in 100% deconjugation), plots were constructed correlating percent RTAsorelease with the concentration of glutathione in the incubation mixture. That concentration of glutathionewhich released 50% of the conjugated RTAN was termed the RC50. Cytotoxicity Assay. The cytotoxicities of 791-RTA30 immunoconjugates were determined using the 791T/M osteosarcoma cell line, which expresses the antigen recognized by 791 antibody (20). Cells (4 X 105/mL) were incubated in a humidified 5% COz incubator with increasing concentrations of immunoconjugates at 37 "C. After 42 h, 3H-thymidine (1 pCi/well) was added, and incubation was continued for an additional 18 h. Upon completion, cell-associated radioactivity was determined by liquid scintillation counting. The IC50was calculated as the concentration of immunoconjugate necessary to inhibit incorporation of radioactivity by 50% relative to untreated controls. These IC50values were corrected for the number of R T A ~ molecules o conjugated to antibody for each preparation by multiplying the conjugate IC50 by the RTA/Ab ratio. These normalized values compensate for slight variations in the number of cytotoxins per antibody in the different preparations and are expressed in terms of pM RTA30. Pharmacokinetic Studies. Pharmacokinetic studies of selected immunoconjugates were performed in 5-weekold male Sprague-Dawley rats (Simonsen Laboratories, Gilroy, CA) weighing an average of 149 g (range 122-175 g) at the initiation of dosing. All animals were delivered healthy to the XOMA animal care facility, where they were acclimated for at least 5 days prior to dosing, and were housed using standard NIH guidelines for husbandry procedures. Only purified monoconjugateswere used in these studies. Each monoconjugate was radiolabeled with 1251 by the Iodogen method (24) to a specific activity of 0.3-2 mCi/ mg and was injected intravenously (33-50 pg/kg) into 42 rats per study (three rats per timepoint). A t selected timepoints (0.05, 0.25, 0.5, 0.75, 2, 4, 8, 12, 18, 24, 36, 48, 72, and 96 h), blood samples were collected via the orbital sinus, and serum aliquots were counted in an LKB y Carroll,S. F., Goff, D., Reardan, D., and Trown, P. W. (1989) Abstractsfrom the fourth international conferenceon monoclonal antibody immunoconjugates for cancer, San Diego, CA, p 161.

Table 1. Reaction of Substituted 2-Iminothiolanes with 791 Antibody. reaction rate4 linker substitution structure k X lo6 21T (none) 5.0

(")

+NH2

M2IT

5-methyl

"0hi2

4.0

Ph2IT

5-phenyl

+NH2

8.8

TB2IT

5-tert-butyl

cF -7 -cs) fNH2

7.4

C%

cH3

DMPIT

5-dimethyl

4.6

SPIT

5-spiro

4.6

R2IT

4,5-ring

5.6

a Rates of reaction of X2ITs (0.5 mM) with 791 antibody (20 pM) as monitoredby coupling the reaction with 2.5 mM DTNB in 0.1 M NaP04,O.l M NaCI, pH 7.5,and monitoringthe change in absorbance at 412 nm. First-order rate constants were determined from the linear slopes of plots for log [XBIT] against time.

counter. Serum samples from each timepoint were also analyzed by SDS-PAGE and autoradiography to determine the fraction of intact monoconjugate prior to pharmacokinetic analysis (13). Pharmacokinetic parameters were determined from a two compartmental analysis using the program PCNONLIN (Statistical Consultants, Inc., Lexington, KY). RESULTS

Reaction of X2ITs with Proteins. The structures of the X2IT crosslinking reagents are summarized in Table 1. Prior studies had shown that the reactivity of the X2ITs with the amino group of glycine was relatively unaffected by the X2IT ring substituent (16). We therefore examined the reactivity of the X2ITs with protein amino groups, in preparation for conjugate production. Each X2IT (0.5 mM) was incubated at pH 7.5 with the murine IgG2b monoclonalantibody 791 (20pM) in the presence of DTNB (2.5 mM), and changes in the absorbance at 412 nm were recorded. Following reaction of the X2ITs with the protein amino groups, DTNB undergoes disulfide exchange with the newly exposed X2IT thiol to yield a free TNB group (monitored at 412 nm) and an activated 791-(X2IT)-SSTNB molecule. This coupling of the reactions between protein modification and TNB production simplifies the analysis of the rate and extent of the reaction. As was found for the reaction with glycine (16),reaction rates for the X2ITs with 791 antibody followed first-order kinetics and varied less than 2-fold for the entire series of crosslinkers (Table 1). Similar reaction conditions were therefore employed for the preparation of protein conjugates utilizing each X2IT linker. Stability of Model Disulfides. The relative stability of X2IT model protein disulfides was assessed by measuring the rates of release of the TNB leaving group from 791-(X2IT)-SS-TNB molecules followingincubation with 200 pM reduced glutathione (GSH). For comparison, two additional control analogs were examined. 791-(SPDP)SS-TNB was prepared by derivatizing 791 antibody with the heterobifunctional crosslinking reagent SPDP and replacing the 2TP leaving group with TNB. A similar



251

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Figure 1. Glutathione-induced release of T N B from 791-TNB analogs. Samples of activated conjugates (791-(X)-SS-TNB, 10 pM)in PBS-EDTA were placed in a cuvette a t 25 "C, and at T = 0 "C, reduced glutathione was added to a final concentration of 200 pM.The release of TNB was monitored spectrophotometrically a t 412 nm for 500 s, and 2-ME was then added to a final concentration of 200 mM to determine maximal release of TNB. Results were normalized by quantifying percent maximal release, as determined by dividing the absorbance a t any timepoint by that obtained with 200 mM 2-ME and then multiplying the product by 100. Table 2. Relative Stabilities of TNB-Activated791 Antibody Analogs

analog 791-(2IT)-SS-TNB 791-(R2IT)-SS-TNB 791-(M2IT)-SS-TNB 791-(Ph2IT)-SS-TNB 791-(TB2IT)-S S-TNB 791-(DM2IT)-SS-TNB 791-(S2IT)-SS-TNB 791-(SPDP)-SS-TNB 791-(SMPT)-SS-TNB

TNB release ratea (k X 104) 235 21.7 18.2 11.5 4.5 0.039 0.036 102 14.7

stability increase relative to 21Tb SPDP" 1.0 0.4 10.8 4.7 12.9 5.6 20.4 8.9 52.2 22.7 6030 2620 6530 2830 2.3 16.0

1.0 6.9

a Reaction mixtures contained 791-X-SS-TNB (20 pM) and reduced glutathione (40-103 mM) and were incubated at 25 "C and monitored at 412 nm. Plots of log [791-X-SS-TNB] vs time were linear for all analogs except 791-(SMPT)-SS-TNB. Pseudo-firstorder reaction rates were calculated by computerized nonlinear curve fitting (GraF'it, version 2.0, Erithacus Software Ltd., Staines, U.K.). Relative increase in disulfide stability compared to the 21T analog. c Relative increase in disulfide stability compared to the SPDP analog.

procedure was used to prepare 791-(SMPT)-SS-TNB, which incorporates the methyl-hindered crosslinking reagent developed by Thorpe et al. (13). Figure 1indicates that the X2IT reagents create model protein disulfides which vary greatly in their susceptibility to reduction by GSH. However, each of the substituted X2ITs produced linkages that were significantly more stable than those produced by SPDP or 21T. At appropriate concentrations of reductant, pseudo-first-order rate constants for TNB release were calculated (Table 2). Relative to 2IT, the most stable linkages (DM2IT and SBIT) were more than 6000-foldmore resistant to reduction by GSH. The order from least to most stable was as follows: 21T < R2IT < M2IT < Ph2IT < TB2IT < DMBIT < SBIT. For the most stable analogs (those made with DMBIT and SBIT), prolonged incubation (30-60 min) with 200 mM 2-ME was required for complete release of TNB. In this assay, the stability of the SMPT analogwas intermediate between those of M2IT and Ph2IT.

4-

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Figure 2. Conjugate formation by 791-TNB analogs. Each 791(X2IT)-SS-TNB analog (1TNB per 791 antibody) was incubated with a &fold molar excess of freshly reduced RTA30 in PBSEDTA. The final concentration for both 791-(X2IT)-TNB and RTA30 was 1.6 mg/mL. After 16 h a t 4 "C, aliquots (10 pg) were analyzed by SDS-PAGE in a 5 % gel under nonreducing conditions. Upon completion, the gel was stained with Coomassie blue.

Preparation of RTA30 Immunoconjugates. Antibody-RTA immunoconjugates are typically prepared by performing a disulfide-exchangereaction between the free -SH group of RTA and an activated linker disulfide present on the antibody. Because this exchange reaction (like the stability assay described above) is essentially a reductive cleavageof the activated linker-SS-TNB bond, variations might be expected in the efficiency with which the linkeractivated antibody is converted to immunoconjugate. Activated 791-(X2IT)-SS-TNB antibodies (1.0-1.3 linkers/Ab) were therefore individually reacted with a 5-fold molar excess of RTA30 for 16 h at 4 "C and then aliquots were analyzed by SDS-PAGE. The results (Figure 2) suggest an inverse correlation between disulfide bond stability and the efficiency of conjugation, as determined by either the disappearance of the free antibody band or by the appearance of higher molecular weight conjugate bands. Utilizing densitometry to quantify the conversion of antibody into immunoconjugate, we found that the efficiency of conjugation followed the order 21T > MBIT > R2IT = Ph2IT > TB2IT. Under identical conditions, SMPT-activated antibody was converted to immunoconjugate roughly as efficiently as was TBBIT-activated antibody (data not shown). No immunoconjugates were detected in reaction mixtures containing DM2IT- or S2IT-modified antibody, suggesting that RTA30 (like GSH and 2-ME) could not easily displace the TNB leaving group from these two linkers disubstituted at the 5 position (immediately adjacent to the disulfide bond). Similarly, little or no conjugation was detected with DM2IT- or S2IT-modified antibody even followingprolonged incubation with RTA3o for severalmonths at 4 O C or after increasingthe incubation temperature to 25 or 37 "C.Antibody activated by reaction with a dimethyl-substituted analog of SPDP (synthesized according to Worrell et al. (12)) was also incapable of making conjugateswhen incubated with an excessof RTA30 (data not shown). Conjugation Efficiency Is Influenced by the Leaving Group. Because the reactions of RTA30 with DM2IT, TNB- or S2IT-TNB-activated antibodies were not productive, the effect of alternate leaving groups on conjugation efficiency was investigated. Initially, 791-(SPDP)-SS-2TP was studied, together with SPDP linkages activated by two additional diary1 disulfides. As before, the 2TP leaving group,of SPDP was first removed by mild reduction, and the newly exposed linker thiol was then reacted with either DTNBor DTPO, thus generating 791(SPDP)-SS-TNB a n d 791-(SPDP)-SS-TPO, re-

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CH,

0

0

200

400

600

0

Time (seconds)

Figure 3. Glutathione-induced release of leaving groups from activated 791 antibody. Aliquota of 791-(SPDP)-SS-2TP were converted to the corresponding 791-(SPDP)-SS-TNB and 791(SPDP)-SS-TPO analogs by mild reduction and subsequent reaction with the corresponding diary1 disulfide (DTNB and DTPO, respectively). The activated 791 antibodies were isolated by size-exclusion chromatography, and glutathione-induced release of the leaving groups was monitored as described in the legend for Figure 1. The final concentration of GSH in these assays was 40 pM.

spectively. These compoundswere then evaluated for their susceptibility to reduction by GSH. As shown in Figure 3, the TPO derivative was most easily reduced, followed by TNB and then 2TP. On the basis of first-order rate constants, the release of TPO was &fold faster than TNB and 15-fold faster than 2TP. When these activated antibodieswere reacted with RTAm, conjugationefficiency was also highest for the TPO analog, followed again by TNB and then 2TP (data not shown). Essentially identical results were obtained with the M2IT-activated 791 antibody (791 reacted with M2IT in the presence of DTDP, DTNB, or DTPO); i.e., the TPO derivative was most easily reduced and was most efficiently conjugated with R T A ~ o (data not shown). In fact, the conjugation efficiency of 791-(M2IT)-SS-TPO exceeded 95%, even when only a 3-fold molar excess of RTABowas used for conjugation. As before, however, no immunoconjugates were detected following reaction of 791-(DM2IT)-SS-TPO or 791(S2IT)-SS-TPO with R T A ~ ounder any of the reaction conditions tested. Disulfide Bond Stability and Cytotoxicity of 791RTA30 Immunoconjugates in Vitro. The in vitro stabilities of 791-RTA30 immunoconjugates were analyzed directly by monitoring thiol-dependent release of RTA30. In addition to the conjugates described above, three additional immunoconjugateswere also prepared, purified, and tested. 791-(MSPDP)-SS-RTA30 (which incorporates a methyl-substituted analog of SPDP) and the thioether-linked conjugate 791-(SMCC)-CS-RTAm (which is not reducible) were prepared by standard reactions with linker-modified antibodies. The third conjugate was prepared in a effort to evaluate an immunoconjugate disubstituted at the carbon atom adjacent to the disulfide bond and, since conjugations with DM2IT- and S2ITlinked antibodies were unsuccessful, required alternate chemistries. An analog of SPDP was therefore prepared (SAMBA) which incorporated two methyl groups adjacent to the linker thiol and an S-acetyl protecting group instead of the usual 2TP moiety. Following reaction of the

SAMBA

@-NH

y

3

I1 -c - c H , - - C - - S S a I CH3

Figure 4. Linkage structures formed by the different croaslinking agents. For the X2ITs, only M2IT is shown as an example. Table 3. Stability and Cytotoxicity of 791-RTAao Immunotoxins in Vitro stability increase relative to ICE4 immunotoxin RCaa 21Tb SPDP' (pMRTAm) 791-(21T)-SS-RTAso 1.9 1.0 0.6 70.6 791-(R2IT)-SS-RTAm 11.8 6.2 3.7 65.8 791-(M2IT)-SS-RTAm 26.2 13.8 8.3 69.0 791-(Ph2IT)-SS-RTA30 16.5 8.7 5.2 94.6 791-(TB2IT)-SS-RTA30 9.5 5.0 3.0 93.6 79l-(SPDP)-SS-RTAso 791-(MSPDP)-SS-RTA30 791-(SAMBA)-SS-RTA30 791-(SMPT)-SS-RTAso 791-(SMCC)-CS-RTA30

3.2 11.8 32.9 5.7 nde

1.7 6.2 17.3 3.0

1.0 3.7 10.4 1.8

89.1 72.2 89.6 76.1 9949.5

The concentration of reduced glutathione, in mM, that releases 50% of the RTA3o from the immunotoxin. b Obtained by dividing the RCm for each conjugate by 1.9, the value for 791-(2IT)-SSRTA~o.Obtained by dividing the RCw value for each conjugate by 3.2, the value for 791-(SPDP)-SS-RTAm. The concentration of immunotoxin that inhibited protein synthesis in 791T/M cells by 50%. The data are expressed in terms of RTAso equivalents. The RTA/Ab ratios varied between 1.1and 1.5. Not determined. The amount of RTA~o released by reducing agents did not exceed 10%.

SAMBA NHS ester with antibody amino groups, a free -SH group was exposed on the linker by treatment with hydroxylamine. Thiol-activated RTA30-SS-TNB was then added, and conjugation occurred via disulfide exchange, thus producing 79l-(SAMBA)-SS-RTA30. The linkage structures of these and other immunoconjugates are shown in Figure 4 (note that the linkage made by SAMBA is identical to that which would have been made by dimethyl-SPDP). Following preparation and purification, the stability of the disulfide bond linking antibody and RTA30 to reduction with GSH i n vitro was then examined. The calculated RCmvalues(the concentration of GSH causing 50% release of RTA30) for all immunoconjugates are shown in Table 3. On the basis of these analyses, the dimethyl-substituted SAMBA conjugate was the most stable, followed closely by conjugates made with M2IT and Ph2IT. However, unlike the 791-TNB protein-leaving group compounds (which exhibited stabilities over an 6000-fold range), the


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0

791-21T-RTA3,

v

791-SPDP-RTA30

'

C

2

003

1

c

1

40

2

u

001 0 003

20

0 001 0 0003

0 10

20

30

40

0

50

Time (hours)

Figure 5. Stability of 791-RTA30 immunoconjugates in rats. 1251-labeled immunoconjugatemonoconjugates were injected iu into rats as described in the Experimental Procedures. As a function of time thereafter, serum samples were collected and

analyzed by SDS-PAGE and autoradiography.The percentage of radioactivity in serum associated with intactimmunoconjugate at each timepoint was then quantified by densitometry.

increases in stability for the series of 791-RTA30 proteinprotein conjugates varied by less than a factor of 20. In all cases, the 2IT-linked forms were the least stable. The cytotoxic activity of each 791 immunoconjugate was accessed against 791T/M cells in our standard 60-h assay (20). The IC50 values calculated for all conjugates are presented in Table 4. Despite the 20-fold range of stabilities in the i n vitro disulfide stability assay, each of the conjugates was highly cytotoxic and of similar potency for antigen-bearing target cells. Not unexpectedly, the thioether-linked conjugate (791-(SMCC)-CS-RTA30) was much less cytotoxic (ca. 100-fold)than the disulfide-linked conjugates. Pharmacokinetics of 791-RTA30 immunoconjugates. On the basis of the results of the in vitro stability studies,the two most stable (linked via SAMBA and M2IT) and the two least stable (linked via SPDP and 21T) 791R T A ~ immunoconjugates o were selected for pharmacokinetic studies in rats. In order to simplify the interpretation of these studies, the monoconjugate species (1RTA per antibody) of each immunoconjugate form was purified by hydrophobic interaction chromatography. Each monoconjugate was then radiolabeled with 1251 and injected iu into groups of rats. As a function of time thereafter, serum samples were collected and radioactivities were determined. Rat serum aliquots were also analyzed for the presence of intact immunoconjugate by SDS-PAGE, autoradiography, and densitometry. These data were used to identify the fraction of radioactivity at each time point that was associated only with the intact monoconjugates (the remaining bands were primarily free 791 antibody). Figure 5 shows that the fraction of serum-associated radioactivity found in the immunoconjugate band varied greatly with the different conjugates. As was found in vitro, the 2ITand SPDP-linked immunoconjugates appeared to deconjugate more quickly i n vivo, with the fraction of counts present in the immunoconjugate bands decreasing to 50 % within ca. 8 h. Similarly, the SAMBA- and M2IT-linked conjugates, which were significantly more stable in vitro, appeared to deconjugate more slowly in vivo. For these two conjugates,more than 50 % of the radioactivity present

12

24

36

48

60

72

84

96

Time after injection (hours)

Figure 6. Pharmacokinetic serum elimination curves for 791-

RTAsOimmunoconjugates.125I-labeledimmunoconjugateswere injected iu into rats as described in the ExperimentalProcedures, and serum sampleswere collected as a function of time thereafter. The percent injected dose corresponding to intact immunoconjugate was determined by y counting and correction,based upon the data in Figure 5. Results are expressed as concentration of the monoconjugates (in percent of dose per mL of serum) vs time. in the serum samples was still associated with intact immunoconjugate a t 30 h. The percentages of intact immunoconjugates at each time point, together with the total serum radioactivities, were then used to construct pharmacokinetic serum elimination curves for each of the immunoconjugates. As shown in Figure 6, two sets of clearance curves were obtained. The two conjugates most stable in vitro and in vivo (prepared with SAMBA and MBIT) were eliminated from the serum more slowly. Similarly, the two immunoconjugates least stable in vitro and in vivo (prepared with 21T and SPDP) were cleared more rapidly. The pharmacokinetic parameters calculated from these data (Table 4) indicate that the conjugate serum mean residence times (sMRT)and AUCs were both increased by enhancing the stability of the linker. The elimination rate constants (CLc/Vc) of the SAMBA- and M2IT-linked immunoconjugates (0.098 and 0.077 h-l, respectively) were decreased more than 2-fold relative to the 21T- and SPDP-linked immunoconjugates (0.20 h-l). DISCUSSION

To be effective therapeutically, immunoconjugates made with cytotoxins such as RTA must possess two apparently conflicting properties; a labile disulfide bond linking the antibody and cytotoxin appears necessary for expression of full cytotoxic activity (1,2,9), but that disulfide bond must remain sufficiently stable i n vivo to allow efficient delivery to target cells. Other studies have shown that reductive deconjugation of antibody-RTA immunoconjugates can occur in animals (10,111 and that enhancing the stability of the linkage between antibody and cytotoxin improves immunoconjugate survival (12-15) and potency (15). The improved linkers used in these studies have had one (12,13,15) or two (14) methyl groups introduced immediately adjacent to the disulfide bond, suggesting that sterically hindering access to the antibody-cytotoxin linkage provides a simple means to enhance immunoconjugate stability and efficacy i n vivo. In an effort to more critically evaluate factors affecting immunoconjugate preparation and potency, we recently

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Carroll et al.


Table 4. Summary of Pharmacokinetic Parameters from a Two-Compartmental Analysis after Intravenous Administration of 791-RTAso Immunotoxins into Male Sprague-Dawley Rats

Tip a Tip P AUC CLC vss

VC sMRTR bMRTb

h h

% d0seimL.h mL/h mL mL h h

2.3 h 0.36 6.5 f 0.40 31.1 f 1.26 3.2 f 0.13 20.1 f 1.09 15.7 f 0.92 4.9 f 0.29 6.2 f 0.17

2.4 h 0.26 7.5 f 0.24 34.3 i 1.27 2.9 f 0.11 19.8 f 1.01 14.5 f 0.78 5.0 f 0.27 6.8 f 0.17

3.2 h 0.57 23.2 f 1.58 60.7 h 2.08 1.6 h 0.06 42.9 h 2.41 20.9 i 1.27 12.7 i 0.82 26.0 f 1.16

3.2 f 0.50 21.5 i 1.55 76.8 f 2.93 1.3 f 0.05 28.2 f 1.77 13.3 f 0.88 10.2 f 0.70 21.7 h 0.98

a Serum mean residence time (sMRT): The average time a molecule (the drug) resides in serum. Serum MRT is also the time it takes to clear approximately 63% of the drug from the serum. *Whole body mean residence time (bMRT): The average time a molecule (the drug) resides in the body, assuming drug is eliminated only from the central compartment. Body MRT is also the time it takes to clear approximately 63% of the drug from the body.

developed a new series of heterobifunctional crosslinking reagents, which we termed X2ITs (16). Based upon the structure of 2IT, a variety of analogs with substituents a t the 4 and/or 5 position were synthesized, which, following reaction with an amino group and DTNB, places each substituent immediately adjacent to the linker-SS-TNB disulfide bond. For a series of model low molecular weight compounds (NHZ-(X~IT)-SS-TNB),stability to reduction by GSH varied over 4000-fold; whereas linkages made with the parental 21T were the least stable, those produced by DM2IT and S2IT (both disubstituted on the carbon adjacent to the disulfide bond) were very difficult to reduce. Overall, the stability of the series increased with increasing bulk (HC methyl < tert-butyl < dimethyl), suggesting that the X2IT substituents function by sterically protecting the disulfide bond from attack by thiolate nucleophiles. The present studies have investigated the utility of the X2ITs to prepare protein-protein conjugates suitable for in vivo use. As was true for their reaction with glycine (16),the X2ITs reacted rapidly and with equal rates with 791 antibody, producing disulfide-activated 791-(X2IT)SS-TNB analogs. And, like the small model analogs examined previously, the thiol-activated protein analogs also varied greatly (more than 6000-fold) in their susceptibility to reduction, with the DM2IT and S2IT analogs again being the most stable. In fact, activated disulfide bonds prepared with these two linkers required incubation with 200 mM 2-ME for more than 30 min to achieve maximal release of the TNB leaving groups. Although the overall magnitudes of the TNB release rates for the protein analogs studied here are similar to those reported earlier for the small model analogs (161, differences were noted between the two systems. For example, most of the protein-X2IT-TNB analogs exhibited glutathione-induced TNB release rates nearly 4-fold slower than those measured for the corresponding NH2X2IT-TNB compounds. Not surprisingly, this result suggests that the enviroment of the disulfide bond in the protein-TNB conjugates, or its accessibility to glutathione, differs from that in the NH2-TNB compounds. The ability of the 791-(X2IT)-SS-TNB analogs to create immunoconjugate conjugates via disulfide-exchange reactions with R T A ~ owas also markedly influenced by the nature of the X2IT substituent. Consistent with their relative ease of reduction, immunoconjugates were most easily produced with the parental 791-(2IT)-SS-TNB analog, whereas no conjugates were detected with either the disubstituted DM2IT or S2IT analogs. Even when a more efficient leaving group (TPO) was used in the conjugation reactions, no conversion of the DM2IT- or S2IT-activated antibodies to immunoconjugates were observed. High levels of conversion to immunoconjugate

were also observed with analogs such as 791-(M2IT)-SSTNB, and efficienciesapproaching 100%could be achieved with the corresponding TPO-activated analog. Thus, increasing the intrinsic stability of the activated protein disulfide had a detrimental effect on conjugate formation and yield, but these negative effects could (for some linkers) be reversed by appropriate selection of the leaving group. Since immunoconjugates were not detected with the disubstituted X2IT-activated analogs, we prepared an immunoconjugate containing a new dimethyl-substituted analog of SPDP (SAMBA) and compared its properties to those of conjugates prepared with the remaining X2IT series, as well as to conjugates containing two other sterically hindered heterobifunctional crosslinkers, MSPDP (12) and SMPT (13). In animal models, immunoconjugates containing SMPT have exhibited improved potency, which has been attributed to their improved stability and prolonged serum half-lives (15). For each of the disulfide-linked 791-RTA30 immunoconjugates, cytotoxicity for antigen-bearing cells was relatively unaffected by the linker substituents, whereas the activity of a nonreducible thioether-linked conjugate was decreased 100-fold. From these data we conclude that, as assayed herein, the linker substituents we have studied have little impact on the in vitro potency of the resulting immunoconjugates. In contrast to the range of stabilitiesobtained with model 791-TNB analogs, the stability of the different 791-RTA30 immunoconjugates varied by less than 20-fold in vitro. Thus, although the 791-(SAMBA)-SS-TNB analog was 2000-fold more resistant to reduction in vitro than was 791-(SPDP)-SS-TNB, the corresponding R T A ~ immuo noconjugates (791-(SAMBA)-SS-RTA30 and 791-(SPDP)-SS-RTAso) varied by only a factor of 10. Moreover, there was little difference between the relative stabilities of the SAMBA-linked and M2IT-linked conjugates (10.4fold and 8.3-fold, respectively),whereas both the methylsubstituted SPDP conjugates (MSPDP and SMPT) appeared somewhat less stable. Clearly, adding a second methyl group immediately adjacent to the disulfide bond enhances the stability of model antibody-TNB compounds by more than 460-fold, whereas the corresponding antibody-RTA30 immunoconjugates were much less affected (1.3-fold). Similar trends were observed in vivo, where the M2ITlinked and SAMBA-linked conjugates exhibited comparable stabilities and their serum elimination profiles were essentially equivalent. Moreover, both conjugates were more stable and persisted longer than the corresponding conjugates made with 21T and SPDP. Interestingly, the D. Fishwild and A. Kung, unpublished data. S.F. Carroll et al., unpublished data.


serum elimination curves for the M2IT- and SAMBAlinked immunoconjugates closely approached that of nonreducible thioether-linked conjugates,2 and the improvements (relative to the unhindered linkers) were comparable to those reported for MSPDP (12) or SMPT (13, 151, as well as those reported for the dimethylsubstituted reagent sNHS-ATMBA (14). Thus, enhancing immunoconjugate disulfide bond stability beyond the level generated with M2IT would apparently provide little additional therapeutic benefit. Presumably, the differential magnitudes of stability noted with the antibodyRTA~oconjugates reflect structural constraints imposed upon the linkages within protein-protein conjugates that are not present in the corresponding protein-TNB analogs. Taken together, our results suggest that increasing the steric bulk of the X2IT substituent adjacent to the disulfide bond beyond that of a single methyl group offers little improvement in the in vitro and in vivo stability of proteinprotein conjugates and that the stabilities of proteinprotein conjugates (but not protein-TNB conjugates) to reduction in vitro are good indicators of stability in vivo. As a result of these evaluations, the M2IT crosslinker appears to possess many properties optimal for the preparation and therapeutic use of antibody-RTA immunoconjugates. Indeed, preliminary studies in animal models suggest that M2IT-linked immunoconjugates are more potent than conjugates prepared with SPDP.3 Moreover, although the present studies were conducted with a single murine IgG2b monoclonal antibody, essentially equivalent results have been obtained with other murine (IgG1, IgG2a) and mouse/human chimeric antibodies and antibody fragments (25) as well as with other cytotoxins (26) and ribosome-inactivating proteins.4 It is noteworthy, however, that the therapeutic utility of MBIT (or the other X2ITs) is not limited to the preparation of protein-protein conjugates. For example, given the 6000-fold differences in stabilities noted with the antibody-TNB analogs, it should be possible to prepare protein-drug conjugates that exhibit a range of stabilities in vivo. Such conjugates may find use in the controlled, time-dependent release of active drug following systemic delivery or in the preparation of timed-release matrices for implantation. ACKNOWLEDGMENT

We are grateful to Eddie Bautista, Charlotte Chang, Maria Fang, Bill Smith, and Will Leach for excellent assistance in the preparation and evaluation of conjugates and to Carroll Hess for assisting in manuscript preparation. LITERATURE CITED (1) Ramakrishnan, S., Fryxell, D., Mohonraj, D., Olson, M., and Li, B.-Y. (1992) Cytotoxic conjugates containing translational inhibitory proteins. Ann. Rev. Pharmacol. Toxicol. 32, 579621. (2) Vitetta, E. S., Thorpe, P. E., and Uhr, J. W. (1993) Immunotoxins: magic bullets or misguided missiles? Immunol. Today 14, 252-259. (3) Byers, V. S., Henslee, P. J., Kernan, N. A., Blazar, B. R., Gingrich, R., Phillips, G. L., LeMaistre, C. F., Gilliland, G., Antin, J. H., Martin, P., et al. (1990) Use of antipan T-lymphocyte ricin A chain immunotoxin in steroid-resistant graftversus-host disease. Blood 75, 1426-1432. (4) Strand, V., Lipsky, P., Cannon, G., Calabrese, L., Wiesenhutter, C., Cohen, S., Olsen, N., Lee, M., Lorenz, T., Nelson, B., et al. (1993) Effects of administration of an anti-CD5 Plus immunoconjugate in rheumatoid arthritis. Arthritis Rheum. 36, 620-630. (5) Wacholtz, M. C., and Lipsky, P. E. (1992)Treatment of lupus nephritis with CD5 Plus, and immunoconjugate on an anti-


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Carroll et al. (26) Better, M., Bernhard, S. L., Lei, S.-P.,Fishwild, D. M., and Carroll, S.F. (1992) Activity of recombinant mitogillin and mitogillin immunoconjugates. J. Biol. Chem. 267, 1671216718.

Enhanced Stability in vitro and in vivo of Immunoconjugates Prepared

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