Comparison of Recombinant Immunotoxins against LeY Antigen

tumors expressed the Le(Y) antigen. Objective antitumor activity was observed in 5 patients, 18 had stable disease, and 15 progressed. A complete remi...
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Bioconjugate Chem. 1998, 9, 736−743

Comparison of Recombinant Immunotoxins against LeY Antigen Expressing Tumor Cells: Influence of Affinity, Size, and Stability Tapan K. Bera and Ira Pastan* Laboratory of Molecular Biology, DBS, National Cancer Institute, National Institutes of Health, Building 37, Room 4E16, 37 Convent Drive MSC 4255, Bethesda, Maryland 20892-4255. Received March 6, 1998; Revised Manuscript Received August 11, 1998

Monoclonal antibody B3 (MAb B3) reacts with many epithelial cancers. It recognizes a carbohydrate antigen (Ley) which is expressed in a variety of solid tumors including breast and colon. We have used the Fab portion of MAb B3 and a portion of the constant domain of human IgG1 to make recombinant immunotoxins of different compositions. The toxin component employed is a truncated form of Pseudomonas exotoxin (PE38). The light chain or Fd of the antibody was cloned from hybridoma RNA and fused to PE38. Immunotoxin (IT) was then expressed in Escherichia coli as a fusion protein and refolded with either the Fd or the light chain. We have also made B3(Fab) immunotoxins of different sizes ranging 85-140 kDa, by introducing different portions of the constant domain of human IgG1 at the junction of Fd and PE38 fusion site. We compared the properties of the resulting immunotoxins with existing anti-Ley immunotoxins side by side. All recombinant Fabimmunotoxins made in this study were cytotoxic to antigen-positive cancer cell lines. However, in contrast to the B3(scFv) immunotoxin, the B3(Fab) immunotoxins are very stable, retaining 90% of their activity after 24 h of incubation in human serum albumin at 37 °C. A pharmacokinetics study with these immunotoxin molecules showed a longer survival in the circulation of mice compared to the smaller Fv immunotoxins. The smaller size of the Fab immunotoxins compared to B3Lys-PE38 and the increased T1/2 value compared to B3(scFv)-PE38 and B3(dsFv)-PE38 make these recombinant immunotoxins alternative therapeutic agents to treat Ley antigen positive cancers.

INTRODUCTION

Monoclonal antibodies (MAb) which recognize antigens on cancer cells are now being used for targeted therapy for the delivery of toxins, radioisotopes or drugs (Vitetta et al., 1987; Mulshine et al., 1991; Pastan et al., 1995; Brinkmann and Pastan, 1995). To produce an immunotoxin, the antibody is linked to a toxin made by bacteria or plants. Immunotoxins of this type are being developed for the treatment of cancer and other diseases including acquired immune deficiency syndrome (AIDS) and immunological disorders (Vitetta, 1990). In our laboratory we have used mutant forms of Pseudomonas exotoxin A (PE) to produce immunotoxins (Pastan and FitzGerald, 1991). PE is a three domain protein, each with a different function. Domain I is responsible for cell binding, domain II for the translocation across the cell membrane, and domain III for the ADP-ribosylation activity of the toxin (Hwang et al., 1987). Using recombinant DNA technology, we have deleted domain I of PE to make a 38 kDa fragment (PE38) that retains the full ADP-ribosylation activity of the toxin (Brinkmann et al., 1991; Pai et al., 1991). Immunotoxin B3Lys-PE38 (LMB1) is composed of MAb B3 chemically linked to PE38 (Pai et al., 1991). Singlechain Fv immunotoxins are recombinant fusion proteins composed of a cell-targeting moiety and the cytotoxic domain of the toxin molecule (Chaudhary et al., 1989). In B3(Fv)-PE38, the Fv portions of the monoclonal antibody B3 were cloned from the B3 hybridoma, fused to PE38 and expressed as a single molecule in Escheri* Corresponding author. Phone: (301) 496-4797. Fax: (301) 402-1344.

chia coli (Brinkmann et al., 1991). Using this approach, a variety of other recombinant single-chain immunotoxins have been made. B3(Fv)-PE38 and B3Lys-PE38 are currently being examined for the treatment of Leypositive solid tumors. A phase I study was conducted with B3Lys-PE38 immunotoxin for 38 patients with solid tumors who failed conventional therapy and whose tumors expressed the Le(Y) antigen. Objective antitumor activity was observed in 5 patients, 18 had stable disease, and 15 progressed. A complete remission was observed in a patient with metastatic breast cancer to supraclavicular nodes. A greater than 75% tumor reduction and resolution of all clinical symptoms lasting for more than 6 months were observed in a colon cancer patient with extensive retroperitoneal and cervical metastasis (Pai et al., 1996). Anti-Tac(Fv)-PE38 is currently being evaluated for the treatment of leukemia (Kreitman et al., 1992). Although single-chain immunotoxins are relatively easy to make, they have some limitations. One is that frequently these molecules are unstable, probably due to the fact that the hydrophobic residues in the Fv fragments which are covered by the constant region of the whole antibody get exposed, and as a result, they have a tendency to aggregate. This problem has been solved by generating an Fv molecule in which the VH and VL fragments are connected by a disulfide bond (Brinkmann et al., 1993; Reiter et al., 1994a-c). The disulfidestabilized Fv (dsFv) containing immunotoxins is very stable at 37 °C, effectively killing target cells and promoting tumor regressions. Depending on the antibody, the relative affinity of the dsFv molecule for antigen can be increased, unchanged, or reduced compared with the single chain Fv (Reiter et al., 1994a-c). Another

10.1021/bc980028o Not subject to U.S. Copyright. Published 1998 by American Chemical Society Published on Web 10/20/1998

Immunotoxins against LeY Antigen

limitation for both scFv and dsFv-containing immunotoxins is that their half-lives in the circulation of mice and monkeys are short with a T1/2 ranging 20-60 min (Reiter et al., 1994c). In contrast, the half-life of the chemical conjugate of whole antibody is about 10 h in mice (Pai et al., 1991) and 9 h in humans (Pai et al., 1996). The plasma half-life of the immunotoxins constructed from the chemical conjugation between whole antibody and the toxin is mainly contributed by the antibody because the half-life of the toxin molecule is very short (Brinkmann and Pastan, 1995). The Fab fragments of antibodies composed of Fd and light chain linked by a disulfide bond are very stable. These have been used to make chemical conjugates with several different toxins and also recombinant immunotoxins (Reiter et al., 1997; Kreitman et al., 1994). In this study, we have made an Fab immunotoxin with MAb B3 and combined it with different portions of the constant domain of human IgG1 and PE38. Our goal was to determine if we could make immunotoxin molecules of different sizes containing one or two PE38 domains and to study the properties of these molecules. MATERIALS AND METHODS

Cloning of Fd and Light Chain of MAb B3. The Fd and light chain of MAb B3 were PCR-amplified from reverse transcribed hybridoma mRNA using primers designed from the published B3(Fv) sequences and from the sequences of the constant region of heavy and light chain. PCR primers used in this study are

T30

CAT ATG GAG GTG AAG CTG GTG GAA TCT

T31

TGA AGC TTC ACC ACA ATC CCT GGG CAC

T32

GG AAT TCA TTA ACA ATC CCT GGG CAC

T38

CAT ATG GAT GTT CTG ATG ACT CAA

T34

TGA AGC TTC ACA CTC ATT CCT GTT GAA GCT

T35

GG AAT TCA TTA ACA CTC ATT CCT GTT GAA GCT

T47

AGC CTC CAC CAA GGG CCC ATC G

T48

TCA TTT ACC CGG AGA CAG GGA G

T49

TCC ATC TCT TAA GCT TCA CCT GAG CTC CTG GGG GGA CCG

T50

CCC ACG GGT CCA AGC TTT GGC TTT GGA GAT GGT TTT CTC G

T51

AAC CTC TGT AAA GCT TCG CAG CCC CGA GAA CCA CAG

T52

CGG CCG TCG CAA AGC TTT ACC CGG AGA CAG GGA GAG

The 5′ primers (T30 and T38) used to amplify Fd or light-chain fragments were designed from B3(Fv) sequence. The 3′ primers (T31 and T32) for Fd fragment were designed from the nucleotide sequence of the constant region of heavy-chain subgroup III(A) to which B3 IgG belongs. Similarly, the 3′ primers (T34 and T35) for light chain were designed from the sequence of the constant region of κ chain. NdeI-HindIII or NdeI-EcoRI sites were introduced to clone the fragment into the expression vector to express either antibody-immunotoxin

Bioconjugate Chem., Vol. 9, No. 6, 1998 737

fusion protein (pTKB1.5.1 and pTKB3.1.1) or antibody fragment alone (pTKB2.1.1 and pTKB4.1.7), respectively. Isolation of Constant Region of Human IgG1 from Spleen cDNA. Polymerase-chain-reaction (PCR)ready human spleen cDNA was purchased from Clonetech (Palo Alto, CA), and the constant region of human IgG1 was isolated by PCR. Primers T47 and T48 were designed from the published sequences (Ellison, 1982) and were used to amplify the constant region of human IgG1. The amplified fragment was cloned into TA vector (Invitrogen, Carlsbad, CA), the sequence verified by DNA sequencing. The cDNA clone was then used as a template to amplify CH2, CH3, or CH2-CH3 region by primer pairs T49/T50, T51/T52 or T49/T52, respectively. Cloning of Various Constant Regions of Human IgG1 into Expression Vector pTKB1.5.1. Restriction site HindIII was generated by PCR on both sides of the fragment to be inserted between the Fd and the PE38 portions of plasmid pTKB1.5.1. To construct the plasmids expressing either CH2, CH3, or both CH2 and CH3 fragment, pTKB1.5.1 plasmid was linearized with HindIII and then ligated with HindIII restricted DNA fragments that contain either CH2, CH3, or both. The correct inframe insertion of the fragments in the resulting plasmids pTKB12.1.7, pTKB13.1.4, and pTKB15.1.3 were checked first by DNA sequencing and finally by determining the sizes of the fusion protein expressed in E. coli. Protein Expression and Purification. E. coli BL21 (λDE3) was transformed with different plasmids and the protein production was induced by adding 10 mM IPTG as described earlier (Brinkmann et al., 1991). All of the fusion proteins were accumulated as insoluble inclusion bodies (IBs) in E. coli. IBs were solubilized and refolded by the procedure described earlier (Buchner et al., 1992). Briefly, the purified IBs were dissolved in solubilization buffer (6 M guanidine-HCl, 0.1 M Tris-HCl, and 2 mM EDTA). After the protein concentration was determined, equimolar amounts of Fd or light chains and their PE38 fusion proteins were reduced by the addition of 0.3 M dithioerythritol (DTT) for 4 h at room temperature and then refolded for 48 h at 10 °C in a refolding buffer described earlier (Buchner et al., 1992). Properly folded protein was then chromatographically purified using first ion exchange (Q-Sepharose and MonoQ; Pharmacia) and finally size-exclusion chromatography (TSK G3000 SW, Toso HAAS) using the procedure described earlier (Buchner et al., 1992). Cytotoxicity Assay. The cytotoxicity of different immunotoxins was determined by measuring the inhibition of protein synthesis in different antigen-positive and -negative cancer cell lines by using a method described earlier (Chaudhary et al., 1989; Batra et al., 1990). All assays were performed in 96 well plates, each well containing 1.6 × 104 cells in 200 µL of RPMI medium containing either 10% (A431, LnCap, CRL1739, and Hut102) or 5% (MCF7) fetal bovine serum. Cytotoxicity assays were performed with different immunotoxins for 2 h, and then fresh medium was added to each well after removing the toxin. The incubation continued for 16 h before [3H]leucine was added. Assay of Blood Levels of B3(Fab)-PE38 Immunotoxin in Mice. Female BALB/c mice were injected in the tail vein with 10 µg of immunotoxin. Blood samples were drawn at various time intervals and the level of the active immunotoxin was measured by the cytotoxicity assay using A431 cells. The A431 cells were incubated with the diluted serum samples and their ability to inhibit protein synthesis was measured. A standard curve was made with each pure immunotoxin. The data

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were analyzed by an exponential curve fitting program RSTRIP (Version 5, MicroMath Scientific Software, Salt Lake City, UT). Stability Assay. The stability of B3(Fab)-PE38-1 and B3(Fab)-PE38-2 immunotoxin was determined by incubating the proteins at 10 µg/mL at 37 °C in human serum albumin. Active immunotoxin remaining at different times was determined by a cytotoxicity assay on A431 cells. Binding Assay. B3-antigen-positive A431 cells were used for a binding competition assay. B3 antibody, radioiodinated with Bolton-Hunter reagent (New England Nuclear, Boston, MA), was used as a tracer in the competition binding assay. Equal counts (50 000 cpm) of labeled B3 antibody with increasing amounts of competitors (cold B3 antibody or immunotoxin) were combined in RPMI, 1% BSA, 50 mM BES, pH 7, and 0.04% NaN3 and added to the cells after nonspecific sites on the cells were blocked. After 2 h of incubation with gentle rocking at 4 °C, unbound counts were washed off and the cells were lysed with 0.5% SDS. The bound radioactivity was assessed in a Beckman 5500B γ counter (Beckman Instruments, Fullerton, CA).

Bera and Pastan

RESULTS

Figure 1. Plasmids for expression of the components of B3 Fab immunotoxin. The plasmid pTKB2.1.1 and pTKB4.1.7 code for the Fd and light chain of the MAb B3, respectively. The expression plasmid pTKB1.5.1 encodes Fd-PE38 fusion protein and plasmid pTKB3.1.1 codes for the fusion protein of B3 light chain and PE38. The plasmids pTKB12.1.7, pTKB13.1.4, and pTKB15.1.3 were generated from the parental plasmid pTKB1.5.1 by introducing CH2, CH3, and CH2-CH3 fragment of human IgG1 between the Fd and PE38 and encode Fd-CH2PE38, Fd-CH3-PE38 and Fd-CH2-CH3-PE38 fusion protein, respectively.

The primary goal of this study was to make recombinant immunotoxins of different sizes and compositions using the Fab fragment of MAb B3 and different portions of the human IgG1 constant region and to compare their cytotoxicity, pharmacokinetics, and binding affinity with the chemical conjugate B3-LysPE38 (Mr ≈ 195 kDa) and two other recombinant immunotoxins B3(Fv)-PE38 (Mr 63 kDa) and B3(dsFv)-PE38 (Mr 63 kDa) in which the Fv portion was fused to PE38. Plasmid Construction and Production of Recombinant Immunotoxin. The cDNA fragments encoding Fd and light chain of monoclonal antibody B3 were obtained by reverse transcriptase reaction of the mRNA from the B3 hybridoma cell line (Pastan et al., 1991), using the PCR primer pair as described in Materials and Methods. PCR-ready human spleen cDNA was purchased from Clonetech and used to isolate the constant region of human IgG1 using the primer pairs described in Materials and Methods. The CH2 and CH3 regions of the IgG1 were then isolated separately from the constant region using the specific primer pairs as described in Materials and Methods. Plasmid Construction. To produce the recombinant B3(Fab)-PE38 molecule, we constructed four expression plasmids, pTKB2.1.1, pTKB4.1.7, pTKB1.5.1, and pTKB3.1.1. (Figure 1). The first two plasmids were constructed to produce the Fd and light chain of MAb B3, respectively. The third and fourth plasmids were constructed to generate the Fd-PE38 and LC-PE38 fusion proteins. A polypeptide connector (C3, ASGGPE) was inserted between the antibody molecule and the toxin (Brinkmann et al., 1991). To produce recombinant immunotoxins of different sizes, we constructed three plasmids by introducing CH2, CH3, and CH2-CH3 fragments of human IgG1 into the plasmid pTKB1.5.1. Schematic pictures of the plasmids used in this study are shown in Figure 1. Protein Expression, Refolding, and Purification of Immunotoxins. Plasmids encoding different components of B3(Fab)-PE38 immunotoxins were expressed separately in E. coli BL21 (λDE3) cultures as described previously (Buchner et al., 1992). All recombinant proteins accumulated in large amounts as insoluble

intracellular inclusion bodies (IBs). IBs containing different components were used to produce different immunotoxins. Soluble recombinant immunotoxins were recovered using a purification scheme that included ion exchange and size-exclusion chromatography previously established for purification of single-chain immunotoxins (Buchner et al., 1992). The scheme we used to make different immunotoxin molecules is shown in Figure 2, with the summary of the expected molecular mass and the yield of each purified protein. B3(Fab)-PE38-1 was made by refolding Fd-PE38 and light chain. B3(Fab)-PE38-2 was made by refolding light chain-PE38 and B3 Fd. Both B3(Fab)-PE38-1 and B3(Fab)-PE38-2 could be produced as monomers with final yields from purification of about 2.5%. The purity of those molecules after purification is shown in Figure 3. B3(Fab)-PE38-3 did not refold into a soluble monomeric protein, perhaps because of the inhibition of the disulfide bond formation between Fd and light chain by the PE38 portion of each component. B3(Fab)-PE38-4 and B3(Fab)-PE38-6 also could be produced as pure proteins with final yields of about 2.5%. The purity of those molecules after refolding and subsequent purification is shown in Figure 3. B3(Fab)-PE38-5 and B3(Fab)-PE38-7 immunotoxins refolded very poorly. After size exclusion chromatography, the final yield for B3(Fab)-PE38-5 and B3(Fab)-PE38-7 was only about 0.1%. Specific Cytotoxicity of B3(Fab)-PE38 Recombinant Immunotoxin. PE38 is a truncated form of Pseudomonas exotoxin, and the fusion proteins of antibody fragments with PE38 are cytotoxic to cells that specifically bind and internalize the fusion protein. Since the degree of cytotoxicity to a given antigen depends primarily on binding affinity, cytotoxicity assays of immunotoxin fusion proteins can thus serve as an accurate test system for specificity as well as for the relative affinity of engineered antibody fragments (Brinkmann and Pastan, 1995). To test whether all of the six immunotoxins made retain not only the affinity but also specificity, we analyzed the activity of these recombinant immunotoxins using several B3 antigen-positive and

Immunotoxins against LeY Antigen

Bioconjugate Chem., Vol. 9, No. 6, 1998 739

Figure 2. Design of B3 Fab immunotoxin of different sizes. Linear composition of B3 Fab-PE38 immunotoxin of different sizes. The positions of the cystein residue forming the disulfied bond between Fd and light chain are Cys235 of Fd and Cys214 of light chain. The calculated molecular mass and the corresponding % yield of each immunotoxin is indicated.

-negative cancer cell lines. For convenience, protein synthesis inhibition was measured. This assay has shown a strong correlation with cell death (Brinkmann et al., 1991). The antigen-positive cell lines used for protein synthesis inhibition assays were A431, MCF-7, LnCAP, and CRL1739. All of the immunotoxins generated were very cytotoxic to B3 antigen positive cell lines but were nontoxic for the antigen negative HUT102 cell line. The IC50 values of individual immunotoxin molecules toward different cell lines are shown in Table 1. In 20 h cytotoxicity assays, both B3(Fab)-PE38-1 and B3(Fab)-PE38-2 were very cytotoxic for A431 cells (IC50, 0.6 ng/mL), CRL 1739 cells (IC50, 0.4 ng/mL), and MCF-7 cells (IC50, 1.0 ng/mL). All of the three cancer cell lines express large amounts of B3 antigen on the surface. The prostate cancer cell line LnCAP, which expresses a moderate amount of the B3 antigen, was also sensitive to these immunotoxin molecules with an IC50 of about 10 ng/mL. Except for B3(Fab)-PE38-5, the other three immunotoxins showed more or less similar IC50 values

ranging 1-3 ng/mL for the A431 and MCF cell lines (Table 1). B3(Fab)-PE38-5 was about 10-fold more cytotoxic to all of the B3 antigen positive cell lines tested, compared to B3(Fab)-PE38-4, B3(Fab)-PE38-6, and B3(Fab)-PE38-7 molecules. The adult T-cell leukemia cell line HUT102, which does not express B3 antigen, was not sensitive to any of the above immunotoxin molecules but very sensitive to anti-Tac(scFv)-PE38 immunotoxin directed to IL2 receptor (Chaudhary et al., 1989). B3(Fv)-PE38 immunotoxin is very unstable at 37 °C, however, the molecule is very cytotoxic to B3 antigenpositive cancer cell line. B3(Fv)-PE38 loses much of its activity after 2 h at 37 °C (Reiter et al., 1994c), even so its IC50 value is similar to the IC50 value of B3(dsFv)PE38 in a 20 h cytotoxicity assay. This is because B3(Fv)-PE38 has a higher affinity for the LeY antigen than B3(dsFv)-PE38 (Reiter et al., 1994c). In this study, we determined the cytotoxicity of B3 (Fab) immunotoxin at 37 °C for 2 h and compared the values with B3(Fv)-PE38 and B3(dsFv)-PE38.

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Bera and Pastan Table 1. Specific Cytotoxicity of B3(Fab)-Immunotoxins cell lines, B3 antigen A431 MCF7 LnCap CRL1739 (+++) (+++) (+) (+)

immunotoxins LMB1 LMB7 B3dsFv B3Fab1 B3Fab2 B3Fab4 B3Fab5 B3Fab6 B3Fab7 anti-Tac(scFv)PE38

2.0 0.5 2.5 0.6 0.55 1.0 0.1 1.8 1.5

1.2 2.0 5.5 0.9 1.0 1.0 0.15 3.5 2.5

2.8 20 60 11 10 18 2.5 150 45

1.5 0.32 4.5 0.4 0.4

HUT102 (-) >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 0.05

Table 2. Specific Cytotoxicity of B3(Fab)-Immunotoxins; 2 h Assaya cell lines, B3 antigen immunotoxins

A431 (+++)

MCF7 (+++)

LnCap (+)

CRL1739 (+)

HUT102 (-)

LMB1 LMB7 B3dsFv B3Fab1 B3Fab2

5.2 0.4 40 4.5 5.0

10 3.5 7.5 5.5 5.5

5.0 35 275 42 60

2.5 0.8 30 6.5 4.5

>1000 >1000 >1000 >1000 >1000

a Cytotoxicity assays were performed by measuring incorporation of tritiated leucine into cellular proteins as described in the Materials and Methods section. IC50 is the concentration that caused 50% inhibition of protein synthesis after incubation with immunotoxin. Antigen expression (+++); very high (+); present (-); absent.

Table 3. Stability of B3(Fab) Immunotoxins in Human Serum Albumin at 37 °Ca % activity remaining time (h)

B3dsFv

B3Fab-1

B3Fab-2

B3Fab4

B3Fab6

0 2 6 24

100 96 80 75

100 93 90 82

100 91 91 91

100 100 90 90

100 100 100 87

a Different immunotoxins at a concentration of 10 µg/mL were incubated with 0.2% human serum albumin for the times shown at 37 °C and then assayed for cytotoxic activity on A431 cells.

Figure 3. SDS-PAGE analysis of purified recombinant B3 Fab-PE38 immunotoxin. Coomassie blue stained SDS PAGE of (A) reduced, and (B) nonreduced samples of B3(Fab)-PE38 immunotoxin; lane 1, B3(Fab)-PE38-6; lane 2, B3(Fab)-PE384; lane 3, B3(Fab)-PE38-2; lane 4, B3(Fab)-PE38-1.

In 2 h cytotoxicity assays, the immunotoxins were washed away after 2 h of incubation, the cells were incubated for additional 16 h, and protein synthesis inhibition was measured after 18 h as described in Materials and Methods. The IC50 values of individual immunotoxin molecules for different cell lines in 2 h. cytotoxicity assays are summarized in Table 2. Both B3(Fab)-PE38-1 and B3(Fab)-PE38-2 molecules were cytotoxic to all B3 antigen-positive cell lines tested. They were less active than B3(Fv)-PE38 but more active than B3(dsFv)-PE38. This difference in activity can be explained by the fact that the affinity of the B3(Fab)-PE38 immunotoxin is higher than the B3(dsFv)-PE38 but lower than B3(Fv)-PE38, and therefore in 2 h, the binding of the B3(Fab)-PE38 immunotoxin to the target cells was

higher than the B3(dsFv)-PE38 and lower than the B3 (Fv)-PE38 (Reiter et al., 1994c). Stability of B3(Fab)-PE38 Immunotoxin. The recombinant Fv or Fab fragments of antibody are widely used in diagnosis and therapy of cancer in the form of radiolabeled or toxin fusion protein. For such applications, it is important that these therapeutic molecules are stable at 37 °C in human circulation so they will retain activity for a long time after injection into patients. Therefore, we examined the stability of B3(Fab)-PE381, B3(Fab)-PE38-2, B3(Fab)-PE38-4, and B3(Fab)-PE38-6 by incubating the purified proteins at 37 °C. The immunotoxins, at a concentration of 10 µg/mL, were incubated at 37 °C for different periods of time in phosphate-buffered saline containing 0.2% human serum albumin. The remaining activity of the incubated immunotoxins was determined by protein synthesis inhibition assays using B3 antigen positive A431 cell lines. The data in Table 3 shows that all of the molecules were very stable at 37 °C retaining 80-90% of initial activity for at least 24 h when incubated in human serum albumin. Pharmacokinetics of B3(Fab)-PE38 Recombinant Immunotoxin. We next determined the pharmacokinetics of B3(Fab)-PE38-1 and B3(Fab)-PE38-4 immunotoxin in mice. Each immunotoxin was injected i.v. with

Immunotoxins against LeY Antigen

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Figure 4. Blood levels of B3(Fab)-PE38 immunotoxin in mice. Female BALB/c mice were injected i.v. with 10 µg of B3(Fab)PE38-1 or B3(Fab)-PE38-4. The level of immunotoxin in blood samples drawn at different times was measured by protein synthesis inhibition assay on A431 cells as described in Materials and Methods. Results are average of three animals for each time point. Open circle, blood levels of B3(Fab)-PE38-1; Close circle, blood levels of B3(Fab)-PE38-4. Table 4. Relative Binding Affinity (IC50) of B3 (Fab)-Immunotoxinsa analyte

tracer

IC50 (nM)

cell line

B3 IgG B3-Lys-PE38 B3 Fab-PE38-1 B3 Fab-PE38-2

B3 IgG B3 IgG B3 IgG B3 IgG

85 2500 3000 3500

A431 A431 A431 A431

a The affinties of different immunotoxins were determined by competition binding analyses to cells expressing B3 (Ley) antigen. The antigen positive cell line used was A431. The relative binding affinities were calculated from the concentration of competitor which caused 50% inhibition of the binding of 125I-B3 IgG to the Ley-expressing cell line.

a single dose of 10 µg administered to a series of mice. Blood was drawn at different time points after the injection and assayed for immunotoxin activity by measuring its ability to inhibit protein synthesis on A431 cells as described in Materials and Methods. In Figure 4 the plasma clearance curve of B3(Fab)-PE38-1 and B3(Fab)PE38-4 immunotoxin was shown. The T1/2 for B3(Fab)PE38-1 and B3(Fab)-PE38-4 molecules was found to be 104 and 121 min, respectively. Relative Binding of B3(Fab)-PE38 Immunotoxin. The relative binding affinities of B3(Fab)-PE38-1 and B3(Fab)-PE38-2 immunotoxins were determined by competition binding assay on B3 antigen expressing A431 cells. Various amounts of B3(Fab)-PE38-1, B3(Fab)-PE38-2, B3Lys-PE38, or B3 IgG were used to compete for the binding of radiolabeled B3 IgG to the B3 antigen positive A431 cells. Table 4 summarizes the relative binding of B3(Fab)-PE38 immunotoxins. B3(Fab)-PE38-1, B3(Fab)PE38-2, and B3Lys-PE38 are equipotent competitors with 125I-labeled B3 IgG for antigen sites on cultured A431 cells. The IC50 was between 2 and 5 µM. Both B3(Fab)-PE38-1 and B3(Fab)-PE38-2 competed approximately 30-fold less well than the whole B3 IgG antibody (IC50 ≈ 85 nM). Since B3(Fab)-PE38-1 and B3(Fab)PE38-2 are both monovalent, a decrease in apparent binding affinity is to be expected. DISCUSSION

In this study, we have made recombinant immunotoxins of different sizes and compositions using the Fab fragment of the B3 antibody, a truncated version of Pseudomonas exotoxin (PE38), and different portions of the human IgG1 constant region. We have successfully made recombinant immunotoxins with molecular masses of 86 000, 96 000, and 134 000 kDa.

B3(Fab)-PE38-1 and B3(Fab)-PE38-2, both have molecular masses of 86 000 kDa. In the case of B3(Fab)PE38-1, the Fd fragment of monoclonal antibody B3 was fused to PE38 and disulfide bonded with B3 light chain. The Fd fragment of B3 antibody, in B3(Fab)-PE38-2, was linked with light chain-PE38 fusion molecules by a disulfide bond. Both recombinant immunotoxin molecules refolded well and could be purified as active monomers. There is no difference between B3(Fab)PE38-1 and B3(Fab)-PE38-2 molecules with regard to cytotoxicity toward antigen positive cancer cell lines, stability of the molecule, and total yield after refolding and subsequent purification steps. In a previous report (Choe et al., 1994), the B3(Fab) was made from the fusion of the Fv fragment of MAb B3 and the constant region of Fd and the VL of MAb MAK33 (Buckel et al., 1987). To make the immunotoxin, the resulting Fd or VL chain was fused to PE38M and then refolded with the corresponding VL or Fd chain. In PE38M, two of the lysine residues in domain III were mutated to glutamine and lysine 613 was deleted (Choe et al., 1994). In this study, we have used the natural constant region of the Fd and the VL of MAb B3 to make the Fab. These fragments were isolated using mRNA from the B3 hybridoma. Although the C κ chain sequence for antibody MAK33 is identical to that of B3 antibody, there is an alanine (Kabat position 113) instead of serine at the FR4 region of the VH domain of the B3 antibody. Interestingly, the immunotoxins made using the natural B3 Fab fragment were about 4-5-fold more active than the previously made Fab immunotoxin on B3 antigenpositive cell line. At the same time, the final yield of the properly folded immunotoxin was severalfold less than the previous report. Whether the mutations in the PE38M domain and the serine to alanine substitution at the FR4 region of the heavy chain have any effect on the yield and the activity of the immunotoxin molecules are not clear. The components used to prepare B3(Fab)-PE38-3 were B3(Fd)-PE38 and the light-chain PE38 fusion protein. This molecule aggregated during the refolding procedure and purified monomer could not be produced. Western blot analysis of the material obtained after the ionexchange chromatography purification step indicated the presence of nondisulfide bonded individual components in the mixture (data not shown). It is possible that the two PE38 portions interacted and prevented association of Fd with VL. It is possible that the C3 linker used to fuse Fd or light chain to the PE38 molecule, which is only six amino acids long, is too short and caused the PE38 domains to interact and prevent folding and disulfide bond formation between Fd and the light chain. This view is supported by the fact that the monomeric form of B3(Fab)-PE38-6 could be produced with a yield of 2.5%. In B3(Fab)-PE38-6, the CH2 fragment of the human IgG1 was introduced between the Fd and PE38 and probably served as a flexible linker and thus prevented destructive interaction of the PE38 portions of the Fd and light chain. For B3(Fab)-PE38-5 and B3(Fab)-PE38-7, we were not able to make highly purified monomeric molecules and the final yield was very low (0.1%). Both molecules contain the CH3 domain of human IgG1. In B3(Fab)PE38-5, the CH3 domain was inserted between Fd and PE38. The B3(Fab)-PE38-7 molecule has the CH2 and CH3 domains of human IgG1. The CH3 portion of the IgG1 has a tendency to form a homodimer (Ridgway et al., 1996), and probably, that region is responsible for making noncovalent dimer with those immunotoxin molecules. B3(Fab)-PE38-4, which contained a CH2

742 Bioconjugate Chem., Vol. 9, No. 6, 1998

Bera and Pastan

Table 5. Summary of the Properties of Recombinant Immunotoxins against Ley Antigen immunotoxin

size (kDa)

IC50 on A431 cells (ng/mL)

binding (µM)

stability

T1/2 in circulation of mice

ref

B3Lys-PE38 B3Fab-PE38-6 B3Fab-PE38-4 B3Fab-PE38-2 B3Fab-PE38-1 B3scFv-PE38 B3dsFv-PE38

190 134 96 86 86 65 65

2.0 1.8 1.0 0.55 0.6 0.5 2.5

2.5 NDa ND 3.5 3.0 1.3 32

Stable Stable Stable Stable Stable Unstable Stable

10 h ND 2h ND 2h 30 min 30 min

present study, Pai (1991) present study present study present study present study present study, Reiter (1994c) present study, Reiter (1994c)

a

ND; not determined.

instead of a CH3 domain and is the same size as B3(Fab)-PE38-5, can be prepared on good yield as a monomer. These data support our interpretation that CH3 causes dimerization. Cytotoxicity of B3(Fab)-PE38 Recombinant Immunotoxins. All of the six B3(Fab)-PE38 immunotoxins were tested for their cytotoxicity on several B3 antigenpositive cell lines and one negative cancer cell line. The molecules were very cytotoxic to B3 antigen-positive cell lines and nontoxic to the antigen-negative cell line. In 20 h cytotoxicity assays, both B3(Fab)-PE38-1 and B3(Fab)-PE38-2 gave IC50 values very similar to B3(Fv)PE38, which were lower than the values for B3Lys-PE38 and B3(dsFv)-PE38 for most of the B3 antigen-positive cell lines tested (Table 1). But in 2 h cytotoxicity assays, the IC50 values for B3(Fab)-PE38-1 and B3(Fab)-PE38-2 were closer to B3Lys-PE38, which were higher than the values for B3(Fv)-PE38 (Table 2). The IC50 values for B3(dsFv)-PE38 in 2 h cytotoxicity assays were much higher than the values for B3(Fab)-PE381 and B3(Fab)PE38-2 molecules for all of the B3 antigen-positive cell lines. The cytotoxicity data for the B3(Fab)-PE38 immunotoxins in 2 and 20 h assays are directly correlated with their relative binding affinity to B3 antigen and also the stability of the molecule at 37 °C. B3(Fv)-PE38 is very unstable and loses much of its activity by 2 h. Nevertheless, it has a very low IC50 as the result of its higher affinity to B3 antigen and there is no difference in IC50 values between 2 and 20 h assay. B3(dsFv)-PE38, on the other hand, had very poor IC50 in a 2 h assay because of its poor binding to B3 antigen; but due to its extreme stability at 37 °C, it can kill the B3 antigenpositive cells as efficiently as B3(Fv)-PE38 in 20 h assays. B3(Fab) immunotoxins are also very stable at 37 °C, and their binding affinity is much better than B3(dsFv)-PE38. As a result, B3(Fab) immunotoxins had the same IC50 values as B3(Fv)-PE38 in 20 h cytotoxicity assays and much better IC50 than B3(dsFv)-PE38 in 2 h assays. Except B3(Fab)-PE38-5, the other three immunotoxins had similar IC50 values which are closer to the values of B3Lys-PE38. B3(Fab)-PE38-5 was about 10-fold more cytotoxic to all B3 antigen-positive cell lines tested, compared to B3(Fab)-PE38-4, B3(Fab)-PE38-6, and B3(Fab)-PE38-7. Increased cytotoxicity of B3(Fab)-PE38-5 immunotoxin to the B3 antigen-positive cells could be due to its dimer formation and activity as a bivalent molecule. Surprisingly, B3(Fab)-PE38-6 which contains two PE38 molecules did not show increased cytotoxic activity to B3 antigen-positive cells. Stability Compensates for Loss of Affinity. The stability of immunotoxin, as for all other therapeutic drugs, is an important factor. B3(Fv)-PE38 is very unstable when incubated at 37 °C and loses most of its activity within 2 h. We determined the stability of B3(Fab)-PE38 immunotoxins by incubating the molecules at 37 °C. All molecules are very stable at least up to 24 h at 37 °C as are B3(dsFv)-PE38 molecules reported

earlier (Reiter et al., 1994c). The relative binding affinity of B3(Fab)-PE38 molecule was determined by radioactive binding and competition assay. Both B3(Fab)-PE38-1 and B3(Fab)-PE38-4 immunotoxins competed approximately 30-fold less than the whole B3 IgG antibody (IC50 ≈ 85 nM). The lower affinity of these molecules toward B3 antigen compared to B3(Fv)-PE38 is compensated by the fact that all B3(Fab)-immunotoxins are very stable at 37 °C. As a result, the cytotoxic effect of those molecules is very similar to B3(Fv)-PE38 immunotoxin. Increased Half-Life of B3(Fab)-PE38 Immunotoxin. Both B3(Fv)-PE38 and B3(dsFv)-PE38 have very short half-lives in mouse serum. The rate of elimination of these molecules from the mouse blood is very similar, with a T1/2 of 20-23 min (Reiter et al., 1994c). On the other hand, the chemical conjugate of the B3 antibody with PE38 (LMB-1) has a longer survival time in mouse blood with a T1/2 of about 10 h (Pai et al., 1991). We determined the pharmacokinetics of B3(Fab)-PE38-1 and B3(Fab)-PE38-4 immunotoxins in mice. The T1/2 for B3(Fab)-PE38-1 and B3(Fab)-PE38-4 molecules was found to be 104 and 121 min, respectively, which are much higher than B3(Fv)-PE38 and B3(dsFv)-PE38 (Reiter et al., 1994c) but lower than B3Lys-PE38 (Pai et al., 1991). The increased T1/2 values of B3(Fab)-PE38-1 and B3 (Fab)-PE38-4 immunotoxin molecules compared to B3(Fv)-PE38 and B3(dsFv)-PE38 could be due to their increased size or to the CH1 and CL portion of the molecules or both. The CH2 domain insertion in B3(Fab)-PE38-4 molecule does not have any significant effect on T1/2 value of the molecule in mouse blood. Conclusion. We have made B3 immunotoxins of different compositions and compare their properties with other existing anti Ley immunotoxins. The side by side comparison of the properties of these molecules is summarized in Table 5. We have successfully made recombinant immunotoxin molecules of sizes 86 000, 96 000, and 134 000 kDa. All molecules are very cytotoxic to B3 antigen-positive cancer cell lines. We are able to increase the retention time of the immunotoxin molecule in mouse blood by introducing the constant region of human IgG1. We believe that the increased T1/2 value compared to B3(Fv)-PE38 and smaller size compared to B3 lys-PE38 will make these immunotoxin molecules potential alternative therapeutic agents to treat cancer patients with Ley antigen positive cancer soon. ACKNOWLEDGMENT

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