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TP-3 Immunotoxins Improve Antitumor Activity in Mice with Osteosarcoma

Onda, Masanori MD, PHD*†; Bruland, Øyvind S MD; Pastan, Ira MD*

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Clinical Orthopaedics and Related Research: January 2005 - Volume 430 - Issue - p 142-148
doi: 10.1097/01.blo.0000137544.30200.b6
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Osteosarcoma is a highly malignant tumor that originates in bone with neoplastic mesenchymal cells that produce a primitive bone matrix. In 75% of the cases, the age of the patients is between 10 and 30 years.13 Metastatic tumors on the lung frequently occur by hematogenous spread. The reported 5-year survival rates of patients with osteosarcoma are in the range of 50-80%.1,8,16,24,36 The major current challenge in the treatment of osteosarcoma is to cure or prolong the survival of patients who do not respond to chemotherapy or who have metastatic tumors.14,32

Immunotoxins are therapeutic agents with a high specificity and unique mechanism of cell killing. Recombinant immunotoxins are chimeric proteins in which a targeting moiety is linked to a toxin. The targeting moiety selectively binds to tumor cells and enables the toxin to enter and kill the cell. Targeted therapeutics have the potential to be less toxic and more effective than conventional chemotherapeutic agents because the chemotherapeutic agents are toxic to all actively dividing cells, not just cancer cells. Also, the risk of a second malignant neoplasm associated with exposure to alkylating reagents should not occur with immunotoxins.24

Several tumor surface markers, including Met,12 ErbB2,26 and TP-1 and TP-3,7 have been reported to be expressed in osteosarcomas. These surface antigens should be useful as targets for specific immunotherapies. The TP-3 monoclonal antibody reacts with an 80-kd glycoprotein expressed on the surface of various sarcomas in humans, including osteosarcoma, malignant fibrous histiocytoma, and synovial sarcoma. Because of its low expression on healthy human tissues, it is an excellent target.6,15 The TP-3 monoclonal antibody has been used to stain frozen sections of osteosarcomas. The results show that all cells react with TP-3 monoclonal antibody, even though differences in expression levels exist.7 Therefore, an immunotoxin made with the TP-3 antibody is attractive as an alternative therapy for osteosarcoma. We previously prepared and evaluated a recombinant immunotoxin based on the TP-3 monoclonal antibody called TP-3(dsFv)-PE38,28 that selectively killed osteosarcoma cells with an IC50 of approximately 30 ng/mL in vitro. The PE38 is a truncated form of pseudomonas exotoxin A. Our goal in the current study was to increase the cytotoxic activity of the immunotoxin so that at the same dose, a better antitumor effect will be observed. One way to improve the activity of an immunotoxin is to increase its avidity by converting it into a bivalent molecule. Methods to do this were developed and used successfully when applied to immunotoxins reacting with erbB2 and mesothelin.2,4

This study addresses the following questions: Compared with the monovalent TP-3 immunotoxin, can the bivalent TP-3 immunotoxin be produced at a reasonable yield?; Is the cytotoxic activity in cell culture improved?; Is the binding activity improved?; and, Is the antitumor activity in severe combined immunodeficient (SCID) mice with human osteosarcoma xenografts improved?


The two human osteosarcoma cell lines used in this study are OHS and SaOS, which were reactive with TP-3 monoclonal antibody. The OHS cell line was established from a 13-year-old Caucasian boy’s femoral tumor at the Norwegian Radium Hospital (Oslo, Norway).11 It was maintained for several passages in Dulbecco’s Modified Eagle Medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, as described previously.28 The SaOS (CRL 7939) was purchased from the American Type Culture Collection (ATCC, Rockville, MD). The OHS-M1 is a subline of OHS, isolated from a tumor growing subcutaneously in SCID mice.29 To establish the OHS-M1 line, SCID mice were injected with 10 μg of antiasialo GM1 (Wako Pure Chemical Industries, Ltd, Osaka, Japan) intraperitoneally at the same time as the subcutaneous inoculation of OHS cells. These tumors grew to a mean volume of 0.1 cm3 by Day 20. After reaching a volume of 0.4 cm3, the tumors were harvested, cultured again, and the rapidly growing cells were cloned. The OHS-M1 clone produces subcutaneous tumors without treatment with antiasialo GM1. Human epidermoid carcinoma cell line (A431) and Burkitt’s lymphoma cell line (Raji) were used as antigen-negative cells in this study and were described previously.25,27 Mice were maintained in the National Cancer Institute Animal Facility under a protocol (LMB-015) approved by the National Institutes of Health.

The method by which the plasmid was designed and constructed was described previously.2 Briefly, the plasmid pOND10-3 that encodes two Cys44 VH domains separated by a 15-amino acid linker (Gly4Ser)3, fused to PE38, was generated by polymerase chain reaction (PCR) using the pOND10-1 plasmid as the PCR template (Fig 1). The 5′ primer HNDST, 5′- CGT ACC CAT ATG GAG GTC CAG CTG CAA CAG TCT GGA GCT GAG CTG -3′ was used to provide the cloning site Nde I at the N-terminal of VH (the Nde I site underlined). The 3′ primer HGNDR, 5′- GAC TCG CAT ATG ACT TCC GCC ACC CCC TGA CCC ACC TCC GCC ACT ACC TCC GCC TCC TTT CAG CTC CAG CTT GGT CCC ACA -3′ was used to provide the 15-amino acid linker (Gly4-Ser)3 between two VH and the cloning site Nde I. Primers HNDST and HGNDR then were added to the 50-μL final reaction mixture and amplified for 25 cycles. High-fidelity polymerase mix (Roche Molecular Biochemicals, Indianapolis, IN) was used to avoid PCR errors. The resulting fragment was digested with Nde I restriction enzyme and cloned into the Nde I site of pOND 10-1, which codes for TP-3 Cys44 VH fused to PE38, a truncated form of pseudomonas exotoxin A. The Nde I site fuses the inserted fragment in-frame to the Cys44 VH fragment of TP-3 and the truncated toxin.20 The vector contains the T7 promoter for expression in the Escherichia coli BL21(λDE3) expression system.35 The plasmid pOND 10-2 encodes the VL domain of TP-3 Fv and contains a Cys100 mutation. All expression plasmids were confirmed by DNA sequencing on an ABI 373A sequencer using the dideoxy chain terminator-sequencing kit.

Fig 1.:
A-C. The schematics show the (A) monovalent and the (B) bivalent disulfide-stabilized Fv immunotoxin, and the (C) plasmid maps. The parental plasmid pOND 10-1, Ser44 of the TP-3 VH, is mutated to Cys in the framework region. The expression plasmid pOND 10-3 encodes two TP-3 VH domains that are fused in-frame to the PE38 toxin. Both VH domains have a Ser44 to Cys mutation and are held together by a 15-amino acid (Gly4Ser)3 linker. The expression plasmid pOND 10-2 encodes for the VL domain of TP-3, which has a Ala100 to Cys mutation.

For production of recombinant protein, the components of TP-3(dsFv)2-PE38 and TP-3(dsFv)-PE38 were expressed in Escherichia coli BL21(λDE3) and accumulated as inclusion-body proteins, as described for other recombinant immunotoxins.4,9 Escherichia coli BL21 (λDE3) cells containing the plasmids pOND10-3 and pOND10-2 for expression of the two components of TP-3(dsFv)2-PE38 were grown and induced with isopropyl-β-D-thiogalactopyranoside (IPTG) separately. The fusion proteins accumulated in intracellular inclusion bodies. To generate protein with a native conformation, we solubilized each type of inclusion body in guanidine hydrochloride solution, reduced with dithioerythritol (DTE), and refolded by dilution in a refolding buffer containing arginine to prevent aggregation, and oxidized and reduced glutathione to facilitate redox shuffling. Active monomeric protein was purified from the refolding solution by ion exchange and size-exclusion chromatography to near homogeneity, as described by Buchner et al.9 Protein concentration was determined by Bradford assay (Coomassie Plus, Pierce, Rockford, IL).

The activities of the immunotoxins were tested on TP-3 antigen-positive human osteosarcoma cell lines and antigen-negative human malignant cell lines. The specific cytotoxicity of TP-3 immunotoxins was assessed by protein synthesis inhibition (inhibition of incorporation of tritium-labeled leucine into cellular protein).5 Briefly, cells were placed in 96-well plates at the concentration of 2 × 104 cells per well, and incubated at 37°C overnight. Immunotoxin was diluted in phosphate-buffered saline (PBS)/0.2% bovine serum albumin (BSA) to the desired concentrations and added to the target cells in triplicate. The cells were incubated for 20 hours at 37°C, before the addition of 2 μCi of 3H-leucine per well and additional incubation for 2 hours at 37°C. Cells were frozen, thawed, and harvested onto glass fiber filter mats using an automated harvester. The radioactivity associated with the cells was counted in an automated scintillation counter. The activity of the molecule is defined by the IC50, the toxin concentration that reduced incorporation of radioactivity by 50% compared with cells that were not treated with toxin. Data from cytotoxicity assays are reported as the median of triplicate experiments, where each of the triplicate experiments usually was different from the mean by less than 10%. Data shown are representative of several individual experiments.

The binding affinity of TP-3 immunotoxins to the human osteosarcoma cell line, OHS-M1, was determined in a displacement assay.23 The TP-3 IgG was iodinated by Bolton-Hunter reagent (NEN Life Science Products, Boston, MA) and purified by gel filtration on a PD-10 column (Amersham Biosciences, Piscataway, NJ). The OHS-M1 cells were plated at 5 × 105 cells/0.5 mL in 24-well plates 24 hours before the assay. Cells were washed three times with ice-cold PBS containing BSA (2 mg/mL). Initial experiments determined that the binding of 125I-labeled TP-3 IgG (at specific activity of 0.35 μCi/μg) to OHS-M1 cells reached equilibrium at 2 hours. In the competition assays, various concentrations of unlabeled TP-3 IgG, TP-3(dsFv)-PE38, or TP-3(dsFv)2-PE38 were added to cells in the presence of a fixed concentration of labeled TP-3 IgG (1 nmol/L/well). Bound radioactivity was counted by an automated gamma counter.

Antitumor activities of TP-3(dsFv)2-PE38 and TP-3(dsFv)-PE38 were evaluated in SCID mice bearing OHS-M1 tumors. Tumor cells (2 × 106) were injected subcutaneously into SCID mice on Day 0 and produced tumors approximately 0.05 cm3 by Day 4 after tumor cell implantation. On Day 4, animals were treated with intravenous injections of bivalent TP-3(dsFv) 2-PE38 or monovalent TP-3(dsFv)-PE38 diluted in 0.2 mL of PBS-human serum albumin (HSA) (0.2%). Therapy was given on Days 4, 6, and 8. Control animals received a diluent of 0.2 mL of PBS-HSA (0.2%) on Days 4, 6, and 8. Each treatment group was comprised of five animals. Tumors were measured with a caliper every 2 or 3 days and the volume of the tumor was calculated using the formula: tumor volume (cm3) = length × (width)2 × 0.4.34

The results were analyzed statistically. For comparison between the two experimental groups, the Mann-Whitney U test was used. A p < 0.05 was considered statistically significant. Values are expressed as mean ± standard deviation.


There was a lower yield of properly folded active (dsFv)2-PE38 compared with the (dsFv)-PE38. The highly purified recombinant (dsFv)2 immunotoxin was recovered after the three-column purification steps (Fig 2).33 Approximately 1% of the refolded protein was obtained as the monomeric bivalent (dsFv)2-PE38 (1 mg of purified protein from 100 mg of inclusion bodies), compared with a yield of 15% for the monovalent TP-3(dsFv)-PE38. Purified immunotoxins were stored in aliquots at -80°C.

Fig 2.:
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis shows recombinant TP-3 immunotoxins under reduced and nonreduced conditions. Lane 1=reduced TP-3(dsFv)2-PE38; Lane 2=reduced TP-3(dsFv)-PE38; Lane 3=blank; Lane 4=nonreduced TP-3(dsFv)2-PE38; Lane 5=nonreduced TP-3(dsFv)-PE38; M=molecular weight markers

The bivalent immunotoxin was more cytotoxic to antigen-positive cells than monovalent immunotoxin, and both immunotoxins were not cytotoxic to antigen-negative cells (Table 1). The IC50 value using OHS-M1 cells were 4 ± 1 ng/mL for bivalent immunotoxin and 30 ± 7 ng/mL for monovalent immunotoxin (Fig 3). Therefore, the bivalent immunotoxin is 7.5 times more cytotoxic than monovalent immunotoxin. Using OHS cells, the bivalent immunotoxin is eight times more active. Using SaOS cells, the bivalent type is six times more cytotoxic. Neither type of immunotoxin affected the antigen-negative A431 or Raji cell lines at more than 1000 ng/mL.

Table 1:
Cytotoxicity of TP-3(dsFv)2-PE38 and TP-3(dsFv)-PE38 on Different Cells
Fig 3.:
The comparison shows the cytotoxicity of TP-3(dsFv)2-PE38 (Δ) and TP-3(dsFv)-PE38 (o) using OHS-M1 cells. The error bars show standard deviation.

The binding of the TP-3 immunotoxin was specific, because bound 125I-TP-3 immunoglobulin (IgG) was displaced from OHS-M1 cells by increasing concentrations of TP-3 IgG (Fig 4). It was not displaced by increasing concentrations of TP-1 IgG, which recognizes a different epitope from TP-3 on the same antigen (Fig 4). Binding to OHS-M1 cells was reduced by 50% at 30 nmol/L for TP-3(dsFv)2-PE38, and 350 nmol/L for TP-3(dsFv)-PE38.

Fig 4.:
Displacement of 125I-TP-3 with TP-1 IgG (□), TP-3(dsFv)2-PE38 (▴), and TP-3(dsFv)-PE38 (▪) using the human osteosarcoma cell line, OHS-M1 is shown. Triplicate sample values were averaged and the standard deviation was calculated for each data point. The error bars show standard deviation.

The antitumor activity of the bivalent TP-3 immunotoxin was approximately twofold better than the monovalent TP-3 immunotoxin. The antitumor activity of bivalent TP-3 immunotoxin was estimated using human osteosarcoma xenograft growing in SCID mice. The OHS-M1 cells were injected into mice subcutaneously on Day 0. On Day 4 after injection of tumor cells, the tumors had grown to 0.05 cm3. The animals then received intravenous injections of either 23 pmol per mouse (0.1 mg/kg) or 46 pmol per mouse (0.2 mg/kg) of TP-3(dsFv)2-PE38, or 46 pmol per mouse (0.143 mg/kg) or 92 pmol per mouse (0.285 mg/kg) of TP-3(dsFv)-PE38 on Days 4, 6, and 8. Animals in the control group that were treated with 0.2% HSA-PBS had large tumors develop and were sacrificed on Day 21, when the tumors had grown to more than 0.4 cm3. The tumors for the TP-3(dsFv)2-PE38 and TP-3(dsFv)-PE38 groups decreased to less than 0.05 cm3 after three injections of immunotoxin. Animals treated with 23 pmol per mouse with three injections of TP-3(dsFv)2-PE38 had tumors that decreased to 0.15 ± 0.16 cm3 by Day 25 (Fig 5A), and animals treated with 46 pmol per mouse with three injections of TP-3(dsFv)2-PE38 had tumors that decreased to less than 0.05 cm3 by Day 25 (0.01 ± 0.02 cm3). The difference in tumor size between these two groups is significant (p = 0.03). In contrast, mice treated with monovalent immunotoxin at 46 pmol per mouse did not show significant tumor regression (0.19 ± 0.09 cm3 by Day 25)(Fig 5B). When TP-3(dsFv)-PE38 was injected three times at 92 pmol per mouse, the tumors decreased to less than 0.05 cm3 by Day 25 (0.02 ± 0.02 cm3). The difference of the tumor size between these two groups was significant (p = 0.008). These results showed that a dose of monovalent TP-3 immunotoxin less than 0.05 cm3 leads to a tumor two times larger than that of the corresponding bivalent immunotoxin. Intravenous administration of TP-3(dsFv)-PE38 or TP-3(dsFv)2-PE38 at the doses previously described did not produce significant systemic toxicity.

Fig 5.:
A-B. Antitumor activity of (A) bivalent and (B) monovalent recombinant immunotoxins [TP-3(dsFv)-based immunotoxins] in SCID mice bearing OHS-M1 tumors are shown. Groups of five animals were injected with 2.5 × 106 OHS-M1 cells and treated intravenously in the bivalent group on Days 4, 6, and 8 with 23 pmol per mouse (□) or 46 pmol/mouse (o) of TP-3 immunotoxins. (B) In the monovalent group on Days 4, 6, and 8 the mice received injections of 46 pmol per mouse (o), or 92 pmol per mouse (Δ) of TP-3(dsFv)-PE38. Mice in the control groups received only diluent (○). The error bars show standard deviation. IV = intravenous injection; *Fig 5A, p = 0.03; *Fig 5B, p = 0.008


This study addressed four important issues concerning the preclinical evaluation of improved TP-3(dsFv)2-PE38 that are relevant to its development for clinical use: (1) the yield of this agent; (2) determination of its cytotoxic activity on several osteosarcoma cell lines; (3) determination of its binding activity to the antigen on the surface of cells that react with monoclonal antibody TP-3; and (4) determination of its antitumor activity.

There are several limitations in the current study. The cell line is different from the spontaneous tumor in the human body. During establishment of the cell line, the characteristics of the tumor cells often change. Because cell lines usually keep most of the properties of the original tumor, we used several cell lines as models of human osteosarcomas. Also, the mouse xenograft tumor model is different from spontaneous human tumors. The animal tumor xenografts grow rapidly and are composed of a monoclonal population of cells. However, human tumors grow slowly and are composed of a mosaic of cells. The tumor xenograft model, however, often is used to estimate preclinical antitumor activity in vivo.

Several approaches have been described to generate recombinant bivalent Fvs. These include mini-antibodies, diabodies, and disulfide-linked fragments.17-19,22,31 Our approach to make bivalent molecules consists of linking each to the cognate VL by a disulfide bond and connecting the two VHs by a 15-amino acid linker (Fig 1). Because the dsFv is extremely stable,33 the resulting molecule is very stable under physiologic conditions. This approach, to make bivalent immunotoxins targeting erb-B2 and mesothelin with improved cytotoxicity in vitro and antitumor activities in mice, was described previously.2-4 In the current study, bivalent TP-3 immunotoxin was developed and tested the antitumor activity in vivo.

To develop novel targeted therapies to combat osteosarcoma, we made an immunotoxin with the Fv of TP-3 monoclonal antibody that was cytotoxic to osteosarcoma cells.28 The TP-3 antibody reacts with 100% of osteosarcoma frozen tissue sections.7 The avidity of this immunotoxin was increased by producing a bivalent immunotoxin, which was tested in cell culture and in mice. The yield of TP-3(dsFv)2-PE38 was only 1%. This is similar to the yield of anti-ErbB2(dsFv)2-PE38. Adding one component of Fv may result in a decreased yield of the immunotoxin. The bivalent TP-3(dsFv) immunotoxin showed a sixfold to eightfold improved cytotoxicity in vitro (Table 1; Fig 3) compared with the corresponding monovalent TP-3(dsFv) immunotoxin. The bivalent immunotoxin has an approximately twofold higher affinity than the monovalent molecule. These results show that an increase in affinity helps to improve the cytotoxicity in vitro, as reported previously.2,4

Monovalent and bivalent TP-3(dsFv)-PE38 showed strong antitumor activities in a mouse xenograft model of human osteosarcoma, the bivalent molecule being the most active. The bivalent immunotoxin improved antitumor activity, with an approximately twofold difference between monovalent and bivalent immunotoxins. Because tumors are composed of a capsule and interstitial spaces, the ability of an immunotoxin to penetrate the tumor is important for its antitumor activity. Smaller molecules penetrate tumor tissue more rapidly than larger molecules.37 Because the molecular weight of the bivalent immunotoxin is 86 kd, the penetration of a monovalent immunotoxin should be better than a bivalent immunotoxin. However, because the bivalent immunotoxin has better cytotoxic activity than the monovalent immunotoxin, the combined effect of tumor penetration and increased binding ability leads to a small improvement of antitumor activity in vivo. These results confirm that improvement of avidity produces a small but significant increase in cytotoxic activity in vitro, as described previously.3,4 In our subcutaneous xenograft model, complete regression of tumors occurred after injecting the immunotoxins. The cytotoxic effect of drugs on osteosarcoma tumors is described as a percent of necrosis based on pathologic evaluation of the specimen.16 Pathologic examination of larger tumors, in which complete regression may not occur, will be done to evaluate the amount of tumor necrosis that occurs.

Ek et al10 used intact TP-3 IgG chemically coupled to pokeweed antiviral protein (PAP), a plant hemitoxin, to study its antitumor effect in a SCID mouse xenograft model of human osteosarcoma. They injected 1 mg/kg TP-3-PAP for 3 days. The TP-3-PAP treatment did not show rapid reduction of tumor volume. However, the tumors in the TP-3(dsFv)-PE38 and TP-3(dsFv)2-PE38 groups decreased to less than 0.05 cm3 after three injections of 0.2-0.3 mg/kg, although the control animals treated with 0.2% HSA-PBS had large tumors develop. The difference in antitumor activities in the SCID mouse xenograft model of human osteosarcoma may be influenced by the differences in tumor penetrating ability, because of the difference in the size of molecules.21,37 Our uniform-sized recombinant immunotoxins are 62 kd for TP-3(dsFv)-PE38 and 86 kd for TP-3(dsFv)2-PE38, respectively. However, TP-3-PAP is a mixture of molecules with a molecular weight ranging from 150-240 kd. Furthermore, a direct comparison is difficult, because we used an OHS-M1 cell, a subline of OHS cells.

Bruland et al7 reported that all osteosarcoma tissue sections were stained with TP-3 antibody. However, the expression level of the TP-3 antigen can vary among different tumors. For patients who have low expression of the TP-3 antigen, another antigen might be used as an immunotoxin target. Recently, a new immunotoxin, 8H9(Fv)-PE38, was shown to have good antitumor activity in a human osteosarcoma model.29 Using combination therapy with both immunotoxins may improve the antitumor response against the osteosarcoma.

We have generated two antiosteosarcoma immunotoxins, TP-3(dsFv)2-PE38 and TP-3(dsFv)-PE38. Both molecules have good antitumor activities in a mouse xenograft model, with the bivalent molecule being more active. The major side effect of immunotoxin is liver toxicity, based on our Phase I clinical studies.30 We plan to measure the nonspecific toxicity of these immunotoxins in primates and to do pharmacokinetic studies. Because of its potent antitumor activity, TP-3(dsFv)-PE38 merits additional investigation for possible treatment of osteosarcomas in humans.


We thank Anna Mazzuca for expert editorial assistance and Maria Gallo for careful reading of the manuscript.


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