Bioconjugate Chem. 1994, 5,321-326
321
Mutations of Two Lysine Residues in the CDR Loops of a Recombinant Immunotoxin That Reduce Its Sensitivity to Chemical Derivatization Itai Benhar, Ulrich Brinkmann, Keith 0. Webber, and Ira Pastan' Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, Building 37, Room 4E16, Bethesda, Maryland 20892. Received January 18, 1994' ~
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B3(Fv)-PE38 is a recombinant single-chain immunotoxin in which the Fv region of monoclonal antibody B3 is connected to a truncated form of Pseudomonas exotoxin. It would be desirable to use the lysine residues of the molecule for chemical modification so that it can be derivatized with poly(ethy1ene glycol) to achieve reduced immunogenicity or with the Bolton-Hunter reagent for biodistribution studies. We found that derivatizing lysine residues of B3(Fv)-PE38 causes a marked loss of specific target cell cytotoxicity and/or immunoreactivity. Here we show that two lysine residues in the antibody-combining region of B3(Fv)-PE38 can be replaced with arginines, with only a small loss of cytotoxicity and no change in specificity. This mutant molecule is 3-fold more resistant to inactivation by derivatization with succinimidyl4-(N-maleimidomethyl)cyclohexanel-carboxylate (SMCC) or Bolton-Hunter reagent.
INTRODUCTION B3(Fv)-PE38 is a recombinant single-chain immunotoxin composed of the variable regions from the heavy and light chains of the B3 monoclonal antibody connected by a flexible peptide linker and joined to a truncated form of Pseudomonas exotoxin A (I). B3(Fv)-immunotoxins bind to a carcinoma-associated carbohydrate antigen present on many human breast, colon, gastric, lung, and other carcinomas and specifically kill carcinoma cells in vitro and cause complete regression of tumor xenografts in athymic mice. Thus, they are potential agents for cancer therapy in humans (2). To improve these immunotoxins for future therapeutic applications, we plan to modify B3(Fv)-PE38 by attaching poly(ethy1ene glycol) (PEG)' to reduce its immunogenicity and to prolong its survival in the circulation. Attachment of PEG commonly targets the a amino group of the lysines either directly, using activated PEG, or following chemical modification of the lysine with a heterobifunctional reagent. We have found that directly coupling PEG onto B3(Fv)-PE38, or modifying B3(Fv)-PE38 with the heterobifunctional reagent SMCC for subsequent PEGylation, resulted in the inactivation of the immunotoxin. We observed a similar inactivation when we attempted to label B3(Fv)-PE38 with radioactive Bolton-Hunter reagent for biodistribution assays. These results suggest that chemical modification of some of the lysine residues in B3(Fv)-PE38 with large molecules such as PEG (MW 6000) or even with small molecules such as SMCC or Bolton-Hunter reagent (MW 380) may be deleterious to one or more of its biological functions. There are 14 lysine residues in B3(Fv)-PE38; three of these are in the C-terminus of PE38, and 11are in the B3(Fv) portion of which two are in the binding region of B3(Fv). One is in CDRB of VH, and the other is in CDRB of VL. Both of these lysines are probably
* To whom all correspondence should be sent. Phone (301) 496-4797, fax (301) 402-1344. @
AbstractpublishedinAdvance ACSAbstracts, June 1,1994.
* Abbreviations: CDR, complementaritydetermining region;
SMCC, succinimidyl4-(N-maleimidomethyl)cyclohexane-l-car-
boxylate; PE, Pseudomonas exotoxin A; PEG, poly(ethy1ene glycol); BES, [N,N-bis(2-hydroxymethyl)-2-amino]ethanesulfonic acid; BSA, bovine serum albumin. 1043-1802/94/2905-0321$04.50/0
exposed on the surface of the protein and therefore are good targets for chemical derivatization. Modification of these lysines in the binding region could account for the loss of binding and activity of BB(Fv)-immunotoxins either because the lysines directly contribute to antigen binding or because such a modified lysine interferes with antigen binding in another part of the binding cleft. Also, lysines in the toxin moiety of the immunotoxin, particularly near or a t the C-terminus, might cause reduced immunotoxin activity if they become modified. This paper evaluates these possibilities by making mutant immunotoxins in which the lysines in P E are mutated to glutamines and/or the Fv CDR lysines are mutated to arginines. EXPERIMENTAL PROCEDURES
Materials. Succinimidyl4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) was obtained from Pierce (Rockford, IL). Oligodeoxynucleotides were obtained from BioServe Biotechnologies (Laurel, MD). Other reagents were obtained from standard sources. Construction of Plasmids for Expression of B%(Fv)PE38 Derivatives. Plasmid pULI7 encodes the parental B3(Fv)-PE38 single-chain immunotoxin. The schematic structure of pULI7 and the amino acid sequence of B3(Fv)PE38 are described in Figure 1. Lysine codons that were mutated in this work are in boldface in Figure lB, with the replacing residues indicated below them. All the plasmids encoding B3(Fv)-PE38 derivatives were obtained either by site-specific mutagenesis according to Kunkel (3) using single-stranded pULI7 DNA as template or by subcloning (4). All the mutations were confirmed by DNA dideoxy sequencing (5). In plasmid pULI26, lysine codon 66 in VH CDR2 and lysine codon 190 in VL CDR2 were changed to arginine codons. The encoded protein is named B3(Fv)-PE38RR. In plasmid pULI14, PE38 lysine codons 575 and 591 were mutated to glutamine codons, and the 3' end lysine codon 598 was mutated to an amber (TAG) stop codon. The encoded protein is named B3(Fv)PE38QQA. Plasmid pITA27 carries the Fv CDR lysine to arginine mutation derived from pULI26 and the PE38 lysine to glutamine mutations and 3' end lysine-stop codon mutation derived from pULI14. The protein it encodes 0 1994 American Chemical Society
Benhar et al.
322 Bloconjugate Chem., Vol. 5, No. 4, 1994
is named B3(Fv)-PE38RRQQA. A scheme of the plasmids and their respective encoded proteins is given in Figure
A PUB
1c.
Expression and Purification of Recombinant Proteins. Expression plasmids encoding B3(Fv)-PE38 or its mutated derivatives were introduced into E . coli strain BL21 (XDE3) (6)by transformation, and the recombinant proteins were expressed as inclusion bodies as described ( I ) . The single-chain immunotoxins were obtained by solubilization and refolding of inclusion body protein as described (8). Properly refolded proteins were separated from contaminating proteins and aggregates by ionexchange chromatography on Mono Q (Pharmacia) followed by size exclusion chromatography on a TSK G3000SW (TosoHaas) column. Derivatization of Immunotoxins. Purified immunotoxins stored in phosphate-buffered saline were derivatized with a 30-fold molar excess of SMCC for 1 h a t 25 "C. Typically, 200 pg of protein was mixed with 30 pg of SMCC (Pierce) in a 1-mL reaction. Following derivatization, the immunotoxins were desalted, and aggregateswere removed by size exclusion chromatography on a TSK G3000SW (TosoHaas) column. Fractions containing monomeric immunotoxin were pooled and used for further analysis. To estimate the number of derivatized residues per toxin molecule, aliquots of the SMCC-derivatized proteins were reduced with a 30-fold molar excess of 2-mercapto ethanol for 60 min a t 37 OC, which was followed by determination of free sulfhydryl using Ellman's reagent (Pierce) (9). Antigen Binding and Cytotoxicity of Recombinant Immunotoxins. Relative binding affinities of the immunotoxins were determined by competition against ['%I]labeled B3 IgG for binding to A431 adenocarcinoma cells a t 4 "C. Cells [in RPMI media (GIBCO) supplemented with 5% fetal calf serum] were plated a t 1X lo6 cells/well in 24-well plates on the day prior to assay. Cells were washed with RPMI, 1%BSA, 50 mM BES, pH 7.0 and then blocked with 5 % BSA in RPMI, 50 mM BES, pH 7.0 for 30 min. After the blocking solution was washed off, competitors and labeled tracer (0.01 pCi, 10 fmol labeled with monoiodo [12611-Bolton-Hunter reagent (New England Nuclear, Boston, MA) were added in a total volume of 100 pL and rocked gently for 2 h at 4 "C. Unbound tracer was removed by washing the cells twice with RPMI/ BSA/BES. Cells were lysed with 0.5% SDS in 10 mM Tris-HC1,l mM EDTA, pH 8.0, and the total lysate was counted in a Beckman Model 5500B y counter. The cytotoxic activities of B3(Fv)-PE38 and mutated derivatives were tested by determination of their ability to inhibit protein synthesis in cultured cells as described ( I ) . Immunoreactivity of [1251]-Labeled Immunotoxins. The immunoreactivity of [l26I1-labeledB3(Fv)-PE38 and B3(Fv)-PE38RRQQA were tested by binding to A431 adenocarcinoma cells a t 4 OC. Cells [in RPMI media (GIBCO) supplemented with 5% fetal calf serum] were plated a t 2 X lo4, 5 X lo4, 1 X 106, 2 X lo6, 5 X 106, 7.5 X lo6, and lo6cells/well in six-well plates on the day prior to assay. Cells were washed with RPMI, 1% BSA, 50 mM BES, pH 7.0 and then blocked with 5 % BSA in RPMI, 50 mM BES, pH 7.0 for 30 min. After the blocking solution was washed off, labeled immunotoxins (0.01 pCi, 3.3 fmol) labeled with monoiodo [l25II-Bolton-Hunter reagent (New England Nuclear, Boston, MA) according to the suppliers' recommendations were added in a total volume of 250 pL and rocked gently for 2 h a t 4 "C. Unbound immunotoxin was removed by washing the cells twice with RPMI/BSA/ BES. Cells were lysed with 0.5% SDS in 10 mM Tris-
PULI7
B MDVKLVESGG GLVQPGGSLK LSCATSGFTF Spxxp1xWVRQ TPEKRLEWA Y I S N D D S S A A R F T ISRDNARNTL YLQMSRLKSE DTAIYSCARL R
L A W G A W F m GQGTLVTVSS GGGGSGGGGS GGGGSDVLMT QSPLSLPVSL GDQASISC-
WYLQKP GQSPKLLIYK VSNRFSGVPD R RFSGSGSGTD FTLKISRVEA EDLGVYYC-FGS GTKLEIKASG
GPEGGSLAAL AARLSWNQVD FVRQGTGNDE NWTVERLLQA WRGFYIAGDP LTLAAPEAAG VIPSAIPTDP
Y
TAHQACHLPL ETFTRHRQPR GWEQLEQCGY QVIRNALASP GSGGDLGEAI REQPEQARLA AGAANGPADS GDALLERNYP TGAEFLGDGG HRQLEERGYV FVGYHGTFLE AAQSIVFGGV ALAYGYAQDQ EPDARGRIRN GALLRVWPR EVERLIGHPL PLRLDAITGP EEEGGRLETI RNVGGDLDPS SIPDKEQAIS ALPDYASQPG
Q
PVQRLVALYL LTLAAAESER DVSFSTRGTQ RARSQDLDAI SSLPGFYRTS LGWPLAERTV KPPREDLK Q A
C Plasmid
pULI14
pIT.427
construct
K R K R
K R K R
K..K...REDLK W(Fv)PE%RR Q..Q...REDL 63(Fv)PE38QQA Q..Q...REDL 53(Fv)PE38RRQQA
Figure 1. Expression of B3(Fv)-PE38. (A) The expression plasmid pULI7 was derived from PULI9 (IO)by replacing the 3' end sequence encoding the KDEL C-terminus with a sequence encoding the wild-type C-terminus of PE, REDLK. T7P is a T7 promoter, L is the (gly4ser)XB linker, C is the C3 connector, T is a transcription terminator, and F+ is a filamentous phage origin of replication. (B) Amino acid sequence of B3(Fv)-PE38. The Fv sequence is double spaced and the PE38 sequence is single spaced. The six CDRs are underlined. Lysines that were mutated in this study are in bold type with the replacing residue indicated below them. (C) Scheme showing the plasmids and their respective encoded B3(Fv)-PE38 derivatives used; shown are the positions of the mutated residues and the corresponding replacing residues.
HC1,l mM EDTA, pH 8.0, and the total lysate was counted in a Beckman Model 5500B y counter. RESULTS Plasmid Construction and Production of Mutated Derivatives of B3(Fv)-PE38. The parental plasmid, pULI7 (Figure lA), encodes B3(Fv)-PE38 (Figure lB), a single-chain immunotoxin composed of the B3 heavy-chain variable domain linked via a (Gly4Ser)a peptide linker to the B3 light-chain variable domain, which is fused through a C3 connector to PE38, a truncated form of Pseudomonas exotoxin. B3(Fv)-PE38 is almost identical to B3(Fv)PE38KDEL (I),except that the carboxyl terminus ends with the REDLK (the wild-type Pseudomonas exotoxin carboxyl terminus) instead of the mutant KDEL sequence. I t has a molecular weight of 63 kDaand contains 598 amino acids. Other plasmids used in this study are all pULI7 derivatives (Figure 1C). In pULI26, VH CDR2 lysine codon 66 (Figure lB, line 2, in boldface) and VL CDR2 lysine codon 190 (Figure lB, line 4, in boldface) were mutated to arginine codons. Arginine was chosen as the replacing amino acid in order to maintain the charge of the molecule. In pULI14, PE38 lysine codons 575 and 591 (Figure 1B bottom line in boldface) were mutated to glutamine codons, and the 3' end lysine codon 598 (codon 613 in native PE) was mutated to an amber (TAG) stop codon. This
Bioconlugete Chem., Vol. 5, No. 4, 1994
Lys Arg Mutations in B3(Fv)-PE38 CDRs
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ng/ml Figure 2. Cytotoxicity of B3(Fv)-PE38 and ita mutants toward two B3 antigen-positive human carcinoma cell lines: (A) A431, (B) MCF7. Assays were performed as described in the Experimental Procedures. combination of carboxyl-terminal replacements was previously shown by Chaudhary et al. (7)to preserve the cytotoxic activity of Pseudomonas exotoxin. Plasmid pITA27 carries the mutations of both pULI26 and pULI14 and thus has no lysine in the PE portion and the two CDR lysines of the Fv mutated to arginines. Cultures of BL21(XDE3) (6) transformed with each plasmid were used to produce immunotoxins. Following IPTG induction, the overproduced proteins accumulated in inclusion bodies. These were isolated, and the recombinant protein was solubilized, reduced, and refolded as previously described (8). Active immunotoxins were recovered from the refolded proteins by ion exchange and size exclusion chromatography, as described in the Experimental Procedures. Specific Binding and Specific Cytotoxicity of B3(Fv)-PE38 and B3(Fv)-PE38RR. The cytotoxic activity of B3(Fv)-PE38 and of its mutated derivatives was measured by incubation of various human carcinoma cell lines with serial dilutions of the immunotoxin in PBS containing 0.2 % BSA and measuring the incorporation of I3H1leucine as previously described (10). As shown in Figure 2, B3(Fv)-PE38 has an IC50 of 1.0 ng/mL on A431 cells and 1.2 ng/mL on MCF7 cells. Both are B3 antigen expressing cells. The mutant B3(Fv)-PE38RR had the same cytotoxic activity on these cells. B3(Fv)-PE38QQA and B3(Fv)-PE38RRQQA appear to have slightly lower cytotoxicities than B3(Fv)-PE38. B3(Fv)-PE38QQA and B3(Fv)-PE38RRQQA have cytotoxicities similar to each other. To test whether mutating CDR residues caused a change in specificity, the same assays were done on additional cell lines. As shown in Figure 3 and Table 1,both B3(Fv)PE38 and B3(Fv)-PE38RR had the same spectrum of recognition and cytotoxicity against the cell lines used. These cell lines differ in their level of B3 antigen expression (11). This result indicates that the antigen-binding
---(I
*
A431
MCF7
*- LNCaP
---t
L929 OVCar 3
4- HUT102 KB3-I
Figure 3. Specific cytotoxicity of B3(Fv)-PE38 and B3(Fv)PE38RR towards different cell lines (see Table 1).The cytotoxicity of B3(Fv)-PE38 (A) or B3(Fv)-PE38RR (B) was tested on B3 antigen strongly positive (A431, MCF7), weakly positive (LNCaP), or negative (OVCAR3, KB3-1, HUTlOQ,and L929) cell lines. Table 1. Cytotoxicity of Recombinant B3(Fv) Immunotoxins toward Various Cell Lines
cytotoxicity ICwa ng/mL B~(Fv)- B~(Fv)cell lineb B3 antigen source expression PE38 PE38RR epidermoid carcinoma A431 +++ 0.6 0.9 MCF7 breast carcinoma +++ 2.0 2.9 LnCap prostate carcinoma +8.0 17.0 OVCAR3 ovarian carcinoma 24.0 70.0 KB 3-1 cervical carcinoma >lo00 >lo00 HUT102 T-cell leukemia >lo00 >lo00 L929 mouse fibroblast >lo00 >lo00 Cytotoxicity data are given as ICs0 values, the concentration of immunotoxin that causes a 50% inhibition of protein synthesis following ita incubation on the cells for 16 h. Expression level estimation of the B3antigen is based on immunofluorescence: +++, strong; +, weak, -, not detected. b All the cell lines except L929 are of human origin. specificity of the B3(Fv) was not altered by mutating the CDR lysines to arginines. The specific antigen binding of B3(Fv) immunotoxins was further analyzed by determination of the binding affinity of B3(Fv)-PE38 or BB(Fv)PE38RR to the B3 antigen by a competition assay in which increasing concentrations of either immunotoxin were used to compete out binding of iodinated B3 IgG to A431 cells a t 4 "C. As shown in Figure 4A, both B3(Fv)-PE38 and B3(Fv)-PE38RR competed for the binding of ['26II-B3 antibody to A431 cells by 50% a t about 1.1 MM. This result implies that the antigen-binding affinities of the two Fv parts of the two immunotoxins are similar and that antigen binding is not impaired by the lysine to arginine CDR mutations.
Benhar et al.
A M
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T.....-
B3(Fv)PE38 B3(Fv)PE38 SMCC
B3(Fv)PE38RR
derivatives. B3(Fv)-PE38, B3(Fv)-PE38RR, and B3(Fv)PE38RRQQA were derivatized with a 30-fold molar excess of SMCC, as described in the Experimental Procedures. Following derivatization, both unmodified and modified proteins were subjected to size exclusion chromatography to isolate the monomer fraction. Since improperly refolded B3(Fv) immunomonomers obtained toxinshave a strongtendency to aggregate (3, by size exclusion are predominantly of the correct active conformation. Lane 1: B3(Fv)-PE38. Lane 2: B3(Fv)-PE38SMCC. Lane 3: B3(Fv)-PE38RR. Lane 4 B3(Fv)-PE38RRSMCC. Lane 5: B3(Fv)-PE38RRQQA. Lane 6: B3(Fv)PE38RRQQA-SMCC.
+ B3(Fv)PE38RR SMCC
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Figure 4. Inhibition of B3 IgG binding to antigen positive A431
cells by competing immunotoxins. Iodinated B3 IgG was bound to cells in the presence of varying concentrations of competitors as described in the Experimental Procedures. (A) Competition with B3(Fv)-PE38 or B3(Fv)-PE38RR. (B) Competition with unmodified or SMCC-modifiedB3(Fv)-PE38. (C) Competition with unmodified or SMCC-modified B3(Fv)-PE38RR.
Activities of SMCC DerivatizedImmunotoxins. To determine the effect of modifying lysine residues and other amino groups on activity, the B3(Fv)-PE38 immunotoxins were derivatized with a 30-fold molar excess of SMCC as described in the Experimental Procedures. This ratio was chosen to achieve a 2-3 SMCC to 1 lysine ratio because using a lower ratio (Le., 10-15-fold) resulted in a low level of derivatization and lower inactivation of the immunotoxins. For example, using &fold SMCC over protein to modify B3(Fv)-PE38 resulted in derivatization of two to three lysines and 3-fold loss in cytotoxicity. The mutant B3(Fv)-PE38RR was not inactivated at all a t that level (data not shown). Thus, 6-10 residues per protein molecule were modified when a 30-fold molar excess of SMCC over protein was used (data not shown). Derivatization with SMCC resulted in a moderate amount of aggregation (5-10 % of the protein). Such aggregatesform when B3(Fv)-PE38 immunotoxins are incubated at 37 "C in buffers of low ionic strength. These were removed by size exclusion chromatography. Fractions containing monomeric proteins were pooled and 'used for further analyses. The recovery of monomer was 80-90% of the input protein. As shown in Figure 5, nonreducing SDSPAGE shows that pure monomers were obtained. Samples from-SMCC-derivatized proteins were tested for cytotoxic activity as described above. Underivatized proteins were subjected to the same treatment, excluding the SMCC, and were tested in parallel. The results are presented in Fi'gure 6. Derivatized B3(Fv)-PE38 and B3(Fv)-PE38QQA retained 8-10 % of their cytotoxic
v
m m
Immunotoxin
Figure 6. Effect of SMCC derivatization on the cytotoxicity of B3(Fv)-PE38 and its mutated derivatives. Immunotoxins were treated with a 30-fold molar excess of SMCC and purified as described in Experimental Procedures. The cytotoxicitiesof the derivatized immunotoxins were compared to their respective activities in the unmodified form.
activity relative to their corresponding underivatized proteins. In contrast, B3(Fv)-PE38RR and B3(Fv)PE38RRQQA retained 30-40 % of their cytotoxic activity relative to their corresponding underivatized proteins. In another experiment, immunotoxins were incubated with a 30-fold molar excess of 2-mercaptoethanol following SMCC derivatization. This was done in order to block the reactive groups introduced by SMCC onto the immunotoxins, thereby preventing them from making nonspecific interactions with media components or cells. However, there was no difference in the activity of the SMCC-derivatized immunotoxins with or without the subsequent 2-mercaptoethanol treatment. SMCC Derivatization Causes a Loss of Antigen Binding, but Not Specificity. B3(Fv)-PE38 and B3(Fv)-PE38RR, both underivatized or derivatized with a 30-fold molar excess of SMCC as described above, were also tested for binding in competition assays. As shown in Figure 4B, SMCC derivatization caused a loss of more than 100-fold in antigen binding by B3(Fv)-PE38. However, as shown in Figure 4C, the loss in antigen binding
Bioconjugate Chem., Vol. 5, No. 4, 1994
Lys Arg Mutations in B3(Fv)-PE38 CDRs _....
670kD
158kD 44kD
17kD
1500
Fraction Y-
lZ5I-B3(Fv)PE38
No
l Z 5 I B3(Fv)PE38RRQQA
Figure 7. Profile of TSK G3000SW chromatography of BoltonHunter labeled immunotoxins. Labeled immunotoxins were mixed with gel-filtration molecular weight standards (Bio-Rad), whose sizes and elution positions are indicated above the chromatograms. of B3(Fv)-PE38RR was about 8-fold. SMCC-derivatized B3(Fv)-PE38RR was also tested for specificity by cytotoxic assays on cell lines which differ in their sensitivity to B3(Fv)-PE38 due to different levels (high to none) of the B3 antigen. B3(Fv)-PE38RR and SMCC-derivatized B3(Fv)-PE38RR had the same spectrum of activity on the various cell lines used (data not shown). Immunoreactivity of Bolton-Hunter Labeled Immunotoxins. To determine the effect of modifying B3(Fv)-PE38 lysine residues with the Bolton-Hunter reagent on immunoreactivity, B3(Fv)-PE38 and B3(Fv)PE38RRQQA were labeled with the Bolton-Hunter reagent as described in the Experimental Procedures. Like SMCC, Bolton-Hunter reagent targets amino groups. Aliquots of labeled immunotoxins were subjected to analytical gel filtration on a TSK G3000SW (TosoHaas) column in order to determine whether the treatment caused aggregation. As shown in Figure 7, both labeled proteins eluted from the column as a monomeric peak with a leading shoulder, indicating that little aggregation occurred. The immunoreactivities of [12511-B3(Fv)-PE38 and [125II-B3(Fv)-PE38RRQQA were tested by checking their respective binding to A431 cells a t 4 "C as described in the Experimental Procedures. The immunoreactivity of [1251]-B3(Fv)-PE38was 4-6 % , whereas that of [lz5I]B3(Fv)-PE38 RRQQA was 10-17%. For each labeled immunotoxin, the binding was maximal and similar for the three highest cell densities used, indicating that antigen was in excess over immunotoxin for those densities. Under similar radioiodination conditions, B3 IgG has 60-70 % immunoreactivity. Thus, B3(Fv)-PE38 is about 10-fold more sensitive to loss of immunoreactivity than the whole IgG, whereas B3(Fv)-PE38RRQQA is 3-4-fold more sensitive to loss of immunoreactivity than the whole IgG. Therefore, it is also about 3-fold less sensitive than the wild-type B3(Fv)-PE38 immunotoxin. DISCUSSION
In this study we have constructed a mutated form of the single-chain immunotoxin B3(Fv)-PE38 in which the only two lysines in the CDR regions were mutated to arginines. Additionally, we found that while the parental molecule is 90% inactivated by both treatment with the bifunctional reagent SMCC or by iodination with the Bolton-Hunter reagent, the mutated form is more resistant to such inactivation. We found that replacing the CDR lysines
325
with arginine residues did not result in any significant changes in antigen binding, specificity, or specific cytotoxicity of the mutated immunotoxin. Chemical conjugation is a well-established method of producing antibody-toxin conjugates (12 and references cited therein) or for conjugation of other macromolecules to antibodies or to their fragments (13-15).In some cases, chemical modification has led to loss of antigen binding by the modified antibody (16,17).Chemical conjugation was used to prepare the first-generation immunotoxin B3PE38, in which the whole B3 IgG which contains lysine residues was conjugated to PE38. This conjugate binds almost as well as the underivatized antibody to target cells and is very active (18). Radioiodination of proteins for use in a wide variety of basic and clinical investigations is another example of chemical modification of proteins (19,20).We have routinely labeled the B3 IgG tQbe used as a tracer in our antigen-binding competition assays, with retention of about 60-70% of the immunoreactivity.2 It is thus evident that the whole antibody and PE38 are relatively resistant to chemical modification. B3(Fv)PE38, however, contains only the Fv part of the IgG, so it is more likely that a functionally important lysine will be modified following chemical derivatization with a possible loss of activity. Because general chemical modification is a-priori not usually position-specific (21), emphasis should be placed on modifying proteins on a predetermined position using site-directed mutagenesis. We have found that the single-chain immunotoxin B3(Fv)PE38 can be site-specifically modified on cysteine residues that replace surface-exposed residues in either domain I1 or 111of PE38.3 Among residues replaced and derivatized without activity loss were lysine 575 and lysine 591. In this study they were replaced with glutamine because previous data indicated that such replacements would not impair the immunotoxins' cytotoxicity (7). Also it has been shown that the C-terminal lysine of Pseudomonas exotoxin can be deleted or replaced with arginine without significant activity loss, but replacement with other amino acids reduces activity (7). Therefore, chemical modification of the C-terminal lysine could interfere with the toxin's activity. The similar sensitivity of B3(Fv)-PE38 and B3(Fv)-PE38QQA to inactivation following chemical derivatization on the one hand, and the similar resistance of B3(Fv)-PE38RR and B3(Fv)-PE38RRQQA on the other hand, implies that loss of activity does not result from chemical modification of the PE38 part of B3(Fv)PE38. The same results further suggest that the immunotoxins' sensitivity to such inactivation resides mainly in one or both of its CDR lysines. The modification of framework lysines in the Fv or of the amino terminal a-amino group may also interfere with the formation of the optimal configuration for antigen binding or, indirectly, with the antigen binding itself, as implied by the residual loss of activity of B3(Fv)-PE38RR and B3(Fv)PE38RRQQA following SMCC treatment or radioiodination. However, most of the interference with antigen binding following chemical derivatization of B3(Fv)-PE38 results from the fact that either the CDR lysines are directly involved in antigen binding or the bound reagent sterically hinders a binding interaction between a neighboring CDR residue and the B3 antigen. The fact that both lysines can be mutated to arginines without a significant activity loss suggests that the latter is the more likely explanation. Both lysine 65 in VH CDR2 (Kabat No. 64) (22) and lysine 190 in VL CDR2 (Kabat No. 50) 2
Benhar and Webber, 1993 (unpublished data). Benhar et. al., 1993 (manuscript in preparation).
328 Bloconlugate Chem., Vol. 5, No. 4, 1994
(22) are on the framework-CDR boundary and are conserved in murine immunoglobulin genes belonging to the same structural group (22). This suggests that they may not be directly involved in antigen binding but rather have a structural role so that they can be replaced with arginine residues that have properties similar to the original lysines. LITERATURE CITED (1) 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, 86168620. (2) Pastan, I., Pai, L. H., Brinkmann, U., and FitzGerald, D. J. (1993)Recombinanttoxins: New therapeutic agents for cancer. Annals N . Y. Acad. Sci. (in press). (3) Kunkel, T. A. (1985) Rapid and efficient site-directed mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. U.S.A. 82,488-492. (4) Sambrook,J.,Fritch, E. F., and Maniatis, T. (1989)Molecular cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. ( 5 ) Sanger, F., Nicklen, S., and Coulsen, A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467. (6) Studier, F. W., and Moffat, B. A. (1986)Use of bacteriophage T7 polymerase to direct selective expression of cloned gene. J.Mol. Biol. 189, 113-130. (7) Chaudhary, V. K., Jinno, Y., FitzGerald, D. J., and Pastan, I. (1990)Pseudomonas exotoxin contains a specific sequence at the carboxylterminus that is required for cytotoxicity.Proc. Natl. Acad. Sei. U.S.A.87, 308-312. (8)Buchner, J., Pastan, I., and Brinkmann, U. (1992)A method to increase the yield of properly folded recombinant fusion proteins, e.g., single-chain immunotoxins from renaturations of bacterial inclusion bodies. Anal. Biochem. 205, 267-270. (9) Ellman, G. L. (1958)A colorimetric method for determining low concentrations of mercaptans. Arch. Biochem. Biophys. 74, 443-450. (10) Jinno, Y., Chaudhary, V. K., Kondo, T., Adhya, S., FitzGerald, D. J., and Pastan, I. (1988) Mutational analysis of domain I of Pseudomonas exotoxin: Mutations in domain I of Pseudomonas exotoxin that reduce cell binding and animal toxicity. J. Biol. Chem. 263, 13203-13207.
Benhar et
al.
(11) Brinkmann, U., Reiter, Y., Jung, S.-H., Lee, B., and Pastan,
I. (1993) A recombinant immunotoxin containing a disulfidestabilized Fv fragment (dsFv). Proc. Natl. Acad. Sci. U.S.A. 90,7538-7542. (12) Cumber, A. J., Forrester, J. A., Foxwell, B. M. J., Ross, W. C. J., and Thorpe, P. E. (1985)Preparation of antibody-toxin conjugates. Methods Enzymol. 112, 207-225. (13) Mearea,C. F.,and Goodwin,D. A. (1984)Linkingradiometals to proteins with bifunctionalchelatingagents. J.Protein Chem. 3, 215-228.
(14) Byers, V., and Baldwin, R. (1988) Therapeutic strategies with monoclonal antibodies and immunoconjugates. Immunology 65, 329-335. (15) Lyons, A., King, D. J., Owens, R. J., Yarranton, G. T., Millican,A., Whittle, N. G., and Adair,J. R. (1990)Site-specific attachment to recombinant antibodies via introduced surface cysteine residues. Protein Eng. 3, 703-708. (16) Lindmo, T., Boven, E., Cuttitta, F. Fedorko, J., and Bunn, P. A., Jr. (1984)Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J.Immunol. Methods 72, 77-89. (17) Rodwell, J. D., Alvarez, V. L., Lee, C., Lopez, A. D., Goers, J. W., King, H. D., Powsner, H. J., and McKearn, T. J. (1986) Site-specificcovalent modification of monoclonal antibodies: in vitro and in vivo evaluations. Proc. Natl. Acad. Sci U.S.A. 83,2632-2636. (18) Pai, L. H., Batra, J. K., FitzGerald, D. J., Willingham, M. C., and Pastan, I. (1991)Anti-tumor activitiesof immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin. Proc. Natl. Acad. Sci. U.S.A.88,33583362. (19) Langone, J. J. (1989) Radioiodination by use of the BoltonHunter and related reagents. Methods Enzymol. 70,221-247 (20) Vaidyanathan, G.,Affleck,D. J., and Zalutsky,M. R. (1993). Radioicdination of proteins using N-Succinimidyl4-Hydroxy3-iodobenzoate. (21) Smith, R. A .G., Dewdney, J. M., Fears, R., and Foste, G. (1993) Chemical derivatization of therapeutic proteins. TIBTECH 11,397-403. (22) Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman,K S., and Foeller, C. (1991) Sequences of proteins of immunological interest, 5th ed., U.S. Department of Health and Human Services,Public Health Service,National Institutes of Health, Bethesda, MD.
