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[Cancer Research 66, 2271-2278, February 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Targeting of the Receptor Protein Tyrosine Phosphatase ß with a Monoclonal Antibody Delays Tumor Growth in a Glioblastoma Model

Erik D. Foehr, Gustavo Lorente, Jane Kuo, Rosie Ram, Karoly Nikolich and Roman Urfer

AGY Therapeutics, Inc., South San Francisco, California

Requests for reprints: Erik D. Foehr, AGY Therapeutics, Drug Discovery, 270 East Grand Avenue, South San Francisco, CA 94080. Phone: 650-228-1173; Fax: 650-615-4544; E-mail: efoehr{at}agyinc.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The receptor protein tyrosine phosphatase ß (RPTPß) is a functional biomarker for several solid tumor types. RPTPß expression is largely restricted to the central nervous system and overexpressed primarily in astrocytic tumors. RPTPß is known to facilitate tumor cell adhesion and migration through interactions with extracellular matrix components and the growth factor pleiotrophin. Here, we show that RPTPß is expressed in a variety of solid tumor types with low expression in normal tissue. To assess RPTPß as a potential target for treatment of glioblastoma and other cancers, antibodies directed to RPTPß have been developed and profiled in vitro and in vivo. The recombinant extracellular domain of human short RPTPß was used to immunize mice and generate monoclonal antibodies that selectively recognize RPTPß and bind to the antigen with low nanomolar affinities. Moreover, these antibodies recognized the target on living tumor cells as measured by flow cytometry. These antibodies killed glioma cells in vitro when coupled to the cytotoxin saporin either directly or via a secondary antibody. Finally, in vivo studies showed that an anti-RPTPß immunotoxin (7E4B11-SAP) could significantly delay human U87 glioma tumors in a mouse xenograft model. Unconjugated 7E4B11 provides a modest but statistically significant tumor growth delay when delivered systemically in mice bearing U87 glioma tumors. (Cancer Res 2006; 66(4): 2271-8)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There are currently >500 new oncology drugs in clinical trials. This reflects the huge medical need and intense competition for innovative drugs to novel targets. Cell surface receptors are prime targets of therapy due to the fact that they often play vital roles in tumor biology and are amenable to both small molecule and monoclonal antibody (mAb) drugs. Receptors that are overexpressed or display abnormal signaling often distinguish a tumor cell from normal cells. However, the pharmacologic targeting of receptors is often complicated by the reality that some normal tissues express the cancer target and cause unwanted organ uptake or dangerous side effects. Therefore, the search for a uniquely tumor-specific target goes on. Despite this hurdle, there are several instances of successful strategies to target receptor tyrosine kinases (RTK), these include Herceptin (Genentech, South San Francisco, CA) and Cetuximab (Imclone Systems, New York, NY), antibodies to the epidermal growth factor receptor (EGFR) family of RTKs (1). Few examples exist of the successful targeting of receptor protein tyrosine phosphatases (RPTP) by either small molecule or antibody-based therapeutics.

One such promising RPTP target is RPTPß, a protein that is overexpressed in astroctyomas (2–4). In astroctyomas, increased expression levels of RPTPß correlate with a poor clinical prognosis (3). RPTPß has a shown role in the migration and adhesion of tumor cells, and one of its primary ligands, pleiotrophin, is known to enhance tumor growth and angiogenesis (2, 5–7). Until recently, very little was known regarding the intracellular signaling of RPTPß. Reports indicate that pleiotrophin can bind and inactivate RPTPß and thereby lead to increased tyrosine phosphorylation of ß-catenin and Fyn (8, 9). The extracellular domain of RPTPß has three characterized domains: a carbonic anhydrase, fibronectin III, and glycine-serine rich region (10–13). These regions differentially interact with various cell adhesion molecules, such as NgCAM, Contactin, and tenascin C, to facilitate cell attachment, repulsion, and signaling (14, 15). Several splice variants of RPTPß have been described; these include the soluble matrix protein Phosphacan and long and short receptor forms (10, 11, 16). The short receptor form lacks the 806-amino-acid, glycine-rich domain in the extracellular portion of the receptor. This truncation produces a receptor with a unique junction and a modified ligand-binding profile (4, 17). Both long and short RPTPß are expressed in glioma tumor tissue (4).

Antibody targeting of RPTPß is a promising approach for the treatment of cancer. RPTPß tissue expression is largely central nervous system (CNS) and developmentally restricted (12, 16, 18). It has a shown role in tumor biology and is amenable to therapeutic targeting. The present study describes the generation and characterization of murine antibodies to RPTPß. The expression profile of RPTPß in both normal and tumor tissue (other than brain tumors) is described. We also detail the efficacy of anti-RPTPß antibodies in both cell-based and in vivo tumor studies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. Human U87 (American Type Culture Collection, Manassas, VA) cells were cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and 100 units/mL penicillin/streptomycin. Mouse mAbs against the COOH terminus of RPTPß were obtained from BD Transduction Labs (Franklin Lakes, NJ). Direct immunotoxin conjugates MAB-ZAP, IgG-SAP, DAT-SAP, 7A9B4-SAP, and 7E4B11-SAP were purchased (or created in the case of 7A9B4-SAP and 7E4B11-SAP) from Advanced Targeting Systems (San Diego, CA).

Mouse mAbs. Custom mouse mAbs were generated to the extracellular domain of recombinant human RPTPß short form. The extracellular domain for short RPTPß (residues 26-774), including the unique splice junction, was expressed with a COOH terminus 6x His tag using baculovirus. The protein was purified from the media with two chromatography steps: immobilized metal affinity (Ni2+-NTA FF, Qiagen, Valencia, CA) followed by anion exchange (Q Sepharose FF, GE Healthcare, Pittsburgh, PA; ref. 4). Mouse hybridomas were generated by Anaspec (San Jose, CA) using BALB/c mice with the protein immunogen in Freund's adjuvant. Spleen cells from immunized mice were fused with a mouse myeloma cell line. Supernatants were screened by ELISA using the RPTPß protein immunogen and isotyped, and RPTPß-specific IgG producing hybridomas were expanded, subcloned, and cultured. Promising hybridoma lines were grown, and the antibodies were purified from culture media using Protein A/G Montage Purification columns (Millipore, Billerica, MA).

Immunohistochemistry. Tissue MicroArray slides were used to study the expression RPTPß. Slides were placed on a heat block at 45°C for 4 to 6 hours and dewaxed using EZ-DeWax solution (Biogenex, San Ramon, CA). Slides were then placed in a bath containing Target Retrieval Solution (Innogenex) and simmered for 15 minutes in a microwave. Slides were stained with either the COOH terminus RPTPß antibody (Transduction Labs) or the custom made RPTPß mouse mAbs (Anaspec). The slides were processed using anti-mouse immunohistochemistry kits with 3,3'-diaminobenzidine colorimetric end-point detection and hematoxylin counterstain (Innogenex).

ELISA. To determine the specificity of antibody containing hybridoma supernatants and purified antibody preparations, 96-well Maxisorp plates (Nalgene, Rochester, NY) were coated in 0.5 mmol/L sodium bicarbonate solution containing 1 µg/mL antigen for 1 hour at room temperature. The plates were then washed with PBS and blocked with bovine serum albumin for 1 hour. Antibodies were added to triplicate wells for 1 hour and then washed thrice with PBS containing 0.1% Tween 20 to remove unbound or nonspecific proteins. The anti-mouse IgG-horseradish peroxidase detection antibody was then added for 1 hour before the final washes in PBS containing 0.1% Tween 20. The immune complex was incubated briefly with TMB substrate (Sigma-Aldrich, St. Louis, MO) and stopped with 0.1 N HCl, and absorbance was detected at 490 nm on a plate reader. Triplicate experiments were done, and error bars represent SDs.

Affinity measurements/surface plasmon resonance. Biacore uses a sensor chip technology for monitoring interaction between two or more molecules in real time, without the use of labels. These studies were undertaken by Biacore contract services. The antibodies were captured on the sensor via anti-mouse IgG preimmobilized on the chip surface. The running buffer was HBSEP [50 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 3 mmol/L EDTA, and 0.005% Surfactant P-20], and the analysis temperature was 25°C. The recombinant human short RPTPß was injected, using an automated method, up to 10 minutes at flow rates ranging from 10 to 50 µL/min. Binding data was fit to a 1:1 binding model using Biacore software (BIAevaluation v 4.1) to obtain the kinetic and affinity constants.

Antibody-mediated internalization assay. U87 cells were plated at 3,000 per well (30,000/mL) onto black-walled 96-well plates (BD, Franklin Lakes, NJ) and incubated overnight at 37°C in 5% CO2. The cells were treated with 200 ng/well primary antibody prepared in Opti-MEM (Life Technologies). The antibody treatments include the RPTPß ectodomain antibodies and control IgG. The cells were processed and stained with anti-RPTPß endodomain mAb (Transduction Labs) and detected with anti-mouse Alexa 488 secondary antibody. The cells were imaged using an ArrayScan platform using Zeiss optics and Cellomics imaging algorithms. The punctuate nature of the staining indicates receptor-antibody internalization. Images representative of triplicate experiments are presented.

Colony formation in soft agar. To test the effect of 7E4B11 on colony formation, we used a cell transformation detection assay kit from Chemicon (Temecula, CA). Briefly, 24-well plates were prepared with 0.8% base agar in cell culture media (DMEM, 10% fetal bovine serum, 1% penicillin/streptomycin). U87 cells were harvested and mixed at 4,000 per well in 0.4% top agar solution in cell culture media. Medium containing 20 µg/mL IgG1 (Cymbus Biotechnology, Eastleigh, NH), 20 µg/mL EGFR-528 (Santa Cruz Biotechnology, Santa Cruz, CA; ref. 19), or 20 µg/mL 7E4B11 was added in triplicate to the appropriate wells. The cells were incubated for 21 days at 37°C in 5% CO2 with medium treatment changes every 3 to 4 days. At that time, colonies were stained and visualized using light microscopy. Triplicate experiments were done, and representative images were shown.

Immunotoxin-directed cell cytotoxicity. To evaluate if our RPTPß mouse antibodies can act as an immunotoxin, we tested indirect and direct immunotoxin-mediated cell cytotoxicity assays. For indirect immunotoxin assay, U87 cells were plated at 3,000 per well (30,000/mL) onto white-walled, 96-well plates (BD Falcon) and incubated overnight at 37°C in 5% CO2. The cells were treated with 200 ng/well primary antibody prepared in Opti-MEM (Life Technologies). The antibody treatments include the RPTPß antibodies as well as EGFR-528 (Santa Cruz Biotechnology; ref. 19), CD71 (Transduction Labs), and IgG1 (Cymbus Biotechnology). Half of the test wells (four of eight) were subsequently treated with saporin-conjugated secondary antibody (MAB-ZAP) at 200 ng/well. The cells were then incubated for 3 days 37°C in 5% CO2. At that time, the number of viable cells was assessed using the luminescence-based detection reagent Cell Titer Glo (Promega, Madison, WI) and read on a luminometer. For direct immunotoxin-mediated cell cytotoxicity assay, U87 cells were prepared the same as above but treated directly with the saporin toxin–conjugated antibodies IgG-SAP, DAT-SAP, 7A9B4-SAP, and 7E4B11-SAP (Advanced Targeting Systems). Triplicate experiments were done, and error bars represent SDs.

Tumor xenografts. Female athymic nude mice were 11 to 12 weeks old on day 1 of the study. The U87 glioblastoma line used for this study was maintained in athymic nude mice. A tumor fragment (1 mm3) was implanted s.c. into the right flank of each test mouse. Tumors were monitored twice weekly and then daily as their volumes approached 120 to 160 mm3, at which time the treatment began. IgG-saporin and 7E4B11-saporin doses were prepared on each day of dosing by dilution with saline. Mice were sorted into eight groups with 10 mice per group. The antibody-treated groups received 15 and 30 µg/mouse, 2x/wk x 3 i.t. Each animal was euthanized when its neoplasm reached the predetermined end point size (1,5000 mm3). The log-rank test was employed to analyze the significant differences of TTE. These studies were done by Peidmont Research Center in compliance with the Guide for Care and Use of Laboratory Animals and accredited by Association for Assessmentand Accreditation of Laboratory Animal Care International.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RPTPß antigen expression and immunization. To provide the best source material for therapeutic antibody development, we subcloned the entire extracellular domain of short RPTPß into baculovirus expression vector. This construct has a polyhistidine tag introduced into the COOH terminus of the extracellular domain. The protein expressed from the baculovirus constructs was purified using conventional Nickel affinity column and other available methods to obtain the highest purity proteins (Fig. 1A). The short RPTPß antigen was tested for the ability to bind pleiotrophin and modulate cell adhesion (data not shown; ref. 4). Short RPTPß protein has a domain structure very similar to Phosphacan, an extracellular matrix proteoglycan with anti-adhesive properties. In fact, short RPTPß and Phosphacan have comparable anti-adhesive abilities when tested in cell-based assays (4). These studies indicate that the short RPTPß antigen used for immunization has retained structural and functional characteristics that may enhance the chances of generating functional antibodies.


Figure 1
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  		Figure 1. Antigen production and antibody selectivity. A, the protein encoding the extracellular domain of short RPTPß was purified from the media with two chromatography steps. Immobilized metal affinity (Ni2+-NTA FF) followed by anion exchange (Q Sepharose FF). Arrow, recombinant human short RPTPß in its purified form. This material was used to immunize mice and for screening purposes. B, a subset of RPTPß antibodies generated from the immunization of mice with short RPTPß were tested by ELISA for selectivity to either recombinant short RPTPß (black columns) or a nonspecific control protein (gray columns). An IgG1-negative control isotype was included as a reference. Bars, SD. C, flow cytometry analysis of RPTPß antibody binding to live U87 glioma cells. Human U87 glioma cells were stained with control IgG1, 1B9G4, 7A9B5, or 7E4B11 as indicated and detected with a fluorescent secondary antibody. Live cells were gated from dead cells and the fluorescent intensity of the cell population measured using flow cytometry. D, immunohistochemistry analysis of normal and tumor tissue for RPTPß expression. Normal colon (i) had modest expression in some duct cells, whereas colon adenocarcinoma (ii) was positive for RPTPß. Normal breast (iii) had low expression in some duct cells, whereas invasive ductal carcinoma of the breast stained intensely for RPTPß. Normal lung (v) did not stain for RPTPß, but lung adenocarcinoma displayed strong RPTPß immunoreactivity. Normal skin (vii) did not stain with RPTPß, but melanoma (viii) was immunoreactive. Representative of the pattern of staining multiple tissue sections using COOH terminus–specific RPTPß antibody (Transduction Labs).

 
Specificity and binding of RPTPß antibodies. Antibodies to RPTPß were screened by ELISA to determine specificity. In the ELISA experiment, a panel of purified antibodies was tested for binding to recombinant short RPTPß extracellular domain protein antigen (short RPTPß-ECD, black columns) or a unrelated but similarly expressed and purified control antigen, (NR-ECD, gray columns). The control protein used for this experiment is called GPR56 (20) and is a similarly glycosylated protein produced recombinately in a fashion nearly identical to RPTPß antigen. In this example, four of the five RPTPß antibodies tested gave a robust and specific signal for short RPTPß-ECD and did not recognize GPR56 (Fig. 1B). In addition, we tested the ability of the antibodies to cross react with rat chondroitin sulfate proteoglycan mix that includes Phosphacan. Phosphacan is essentially the extracellular domain of RPTPß. Our lead antibodies did not significantly cross react with rat Phosphacan, indicating species selectivity (data not shown). The hybridomas corresponding to three of the four positive antibodies were selected and used for further study.

To measure binding of RPTPß antibodies to native epitopes, we used flow cytometry. Live human U87 glioma cells were incubated with the three lead candidate antibodies and detected using anti-mouse IgG-Alexa 488. Live cells were gated from dead cells (stained with propidium iodide) and measured for RPTPß-specific signal intensity (Fig. 1C). An IgG1 isotype control (top) was compared with the three lead candidate RPTPß antibodies 1B9G4, 7A9B5, and 7E4B11. All three lead candidate RPTPß antibodies bind native epitope with a similar profile as indicated by the shift in fluorescence intensity peak.

To determine the affinity of each lead candidate antibody for the RPTPß antigen, we undertook surface plasmon resonance measurements using BIAcore technology. Each of the antigens and antibodies were initially tested to determine the binding specificity, antibody capture levels, and chip surface regeneration. Once the proper binding conditions were achieved, a concentration series of each antigen was injected over their respective antibody surfaces using an automated method. Regeneration solution was used to remove the antigen from the amine coupled antibody surface or the antibody/antigen from the captured surface. Binding data were fit to a 1:1 binding model using BIAcore software (BIAevaluation v4.1) to obtain the kinetic and affinity constants (Table 1). All three lead candidate antibodies had similar low nmol/L affinities (KD = ~8 nmol/L).


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  		Table 1. Affinity of RPTPß antibodies

 
RPTPß expression in normal and tumor tissue. There are a few published reports of RPTPß overexpression in human carcinomas, including astrocytoma and melanoma, but little else is known about RPTPß expression in peripheral tissues and tumors (21, 22). We and others have studied the expression of RPTPß in high-grade astrocytomas (2–4). In our current work, we examined the expression of RPTPß in various tumor types (Table 2; Fig. 1D). Using an antibody to the COOH terminus of RPTPß, we surveyed several tumor types that revealed RPTPß overexpression (Table 2). Consistent with published reports, RPTPß is overexpressed in melanomas (21). Lung, colon, breast, and prostate carcinomas also stood out as having a significant number of tumor samples with RPTPß overexpression versus control tissue. Other tumor types, such as transitional carcinoma of the bladder and squamous cell carcinomas of the oral cavity and pharynx, were noteworthy. In addition, lymphoma also indicated up-regulation of RPTPß. A representative subset of the abovementioned cancers is presented in Fig. 1D, with matched normal controls. RPTPß is expressed most consistently in colon, breast, lung, and skin cancer. In some of these tissues, subsets of cells in the corresponding normal tissue (i.e., duct/mucosa cells) stained positive for RPTPß.


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  		Table 2. RPTPß expression in tumor tissue

 
RPTPß immunotoxin-mediated cell cytotoxicity. The critical criteria for selecting a lead antibody are the ability to bind native epitope, become internalized, and kill tumor cells. To show antibody-mediated receptor internalization, U87 cells were treated with anti-RPTPß ectodomain antibodies, and the internalization of the receptor-antibody complex was measured (Fig. 2A). U87 cells treated with anti-RPTPß antibody but not control IgG treated show a punctate staining pattern indicative of internalization.


Figure 2
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  		Figure 2. RPTPß antibody-mediated internalization and tumor cell killing. A, antibody-mediated internalization was shown by treating U87 glioma cells with control IgG (i) or RPTPß ectodomain antibodies (ii). The cells were then processed and stained for RPTPß using an antibody specific to the endodomain of RPTPß and detected with a fluorescence-conjugated secondary antibody. The punctate staining indicates internalized receptor antibody complex. B, for indirect immunotoxin experiments, U87 glioma cells are treated with RPTPß antibodies as well as positive control antibodies, anti-EGFR-528 and anti-CD71. The negative controls included media alone and an isotype control IgG1. Following primary antibody treatment, the cells are subsequently incubated with media (black columns) or a saporin toxin–conjugated anti-mouse antibody (gray columns). Bars, SD. C, for direct immunotoxin experiments, purified 7E4B11 and 7A9B5 antibodies were directly conjugated to saporin and evaluated in cell culture for the ability to kill glioma cells. In this experiment, glioma cells are treated with 7E4B11 and 7A9B5 immunotoxins along with control immunotoxins, nonspecific IgG-SAP (Neg. Ctrl), DAT-SAP (Pos. Ctrl), and vehicle. Bars, SD. D, U87 cells suspended in soft agar were treated with media containing IgG1 (20 µg/mL), 7E4B11 (20 µg/mL), or EGFR-528 (20 µg/mL). The cells were incubated for 21 days with medium treatment changes every 3 to 4 days. At that time, colonies were stained and imaged using light microscopy as shown here.

 
To show immunotoxin-mediated cell killing, glioma cells are treated with RPTPß antibodies as well as positive control antibodies (anti-EGFR-528 and anti-CD71), with known ability to bind, internalize, and kill target cells. The negative controls included an isotype control antibody and media alone. Following primary antibody treatment, the cells are incubated with a saporin toxin–conjugated anti-mouse IgG antibody (MAB-ZAP). If the primary antibody recognizes its target and gets internalized, then the toxin-antibody complex is delivered and kills the cells. Figure 2B shows that the lead RPTPß antibodies (1B9G4, 7A9B5, and 7E4B11), as well as the positive control antibodies, effectively kill tumor cells when coupled to the anti-mouse IgG immunotoxin. The isotype control antibody was unable to bind and internalize and thus could not kill these cells. Because 7A9B5 and 7E4B11 seemed to work slightly better than 1B9G4 and their corresponding hybridoma lines are more productive, they were selected for further evaluation.

Purified RPTPß-7E4B11 and RPTPß-7A9B5 antibodies were directly conjugated to saporin and evaluated in cell culture for the ability to kill glioma cells. In this experiment, glioma cells are treated with these RPTPß immunotoxins as well as control immunotoxins. The negative controls included an IgG control antibody (Neg. Ctrl) or media alone (Vehicle). The positive control DAT-SAP antibody (Pos. Ctrl) targets the dopamine transporter expressed on astrocytoma cells. If the immunotoxin recognizes its target and gets internalized, then the toxin payload is delivered and kills the cells. Figure 2C shows that the immunotoxins 7A9B5-SAP and 7E4B11-SAP, as well as the positive control antibody, effectively kill tumor cells when directly coupled to the toxin. The isotype control antibody was unable to bind and internalize and thus could not kill these cells.

RPTPß has been shown to play a role in tumor cell invasion, migration, and adhesion (2). However, to date, it is unclear if RPTPß activity promotes tumor growth. Using short interfering RNA to knock down RPTPß, we did not see an effect on U87 cell proliferation (data not shown). In addition, studies using pleiotrophin, one of RPTPß's ligands, failed to show a clear enhancement of cell proliferation (data not shown). In contrast, pleiotrophin does enhance colony formation in soft agar assays (5), supporting the notion that RPTPß may play a role in tumor growth in complex cellular systems. To better understand the role of RPTPß in tumor cell growth, we tested whether the lead RPTPß antibody, 7E4B11, could function as an unconjugated antibody to inhibit tumor cell growth. The 7E4B11 antibody (and other RPTPß antibodies) failed to inhibit U87 cell proliferation when simply grown in standard culture conditions over a number of days (data not shown). We then set out to test if 7E4B11 could inhibit or prevent colony formation in soft agar. U87 cells were seeded into soft agar, and then the cells were grown in the presence of test and control antibodies (Fig. 2D). Control IgG1 antibody did not inhibit colony formation in soft agar. In contrast, the positive control EGFR antibody and 7E4B11 had fewer and smaller colonies. Therefore, 7E4B11 is able to modulate RPTPß function and thereby inhibit U87 tumor formation in soft agar.

In vivo efficacy of 7E4B11 immunotoxin. As part of the in vivo proof-of-concept studies for RPTPß targeted immunotoxin, we undertook U87 tumor growth delay studies using the 7E4B11 antibody. These studies are designed to determine the ability of 7E4B11 to target and delay tumor growth and are summarized in Table 3. The immunotoxin consists of 7E4B11 directly linked to saporin (7E4B11-SAP).


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  		Table 3. Treatment response summary for 7E4B11-SAP study

 
U87 tumors were engrafted in Matrigel s.c., and treatments began when the tumors reached 140 mm3. The activities of 7E4B11-SAP were compared with that of vehicle (PBS) and nonspecific IgG1-SAP delivered i.t. Mice were euthanized when tumors reached the end-point volume (1,500 mm3). Nontargeted IgG1-SAP treatments at 15 µg/dose produced only 2% tumor growth delay (TGD). However, at 30 µg/dose, IgG1-SAP produced 24% TGD. At 15 and 30 µg/dose, 7E4B11-SAP produced 25% and 73% TGD, respectively, and were highly significant (P < 0.001; Fig. 3A). All tumors reached the end-point volume by day 44. The median time to end point (TTE) of PBS-treated mice was 18.6 days. Whereas the 7E4B11-SAP (30 µg/dose) treated mice had a TTE of 32.1 days (Fig. 3B). These results show that 7E4B11-SAP has significant antitumor activity.


Figure 3
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  		Figure 3. A, effect of 7E4B11-SAP on tumor growth delay in established U87 xenografts. Mice received i.t. injections twice a week for 2 weeks once tumors had reached a mean tumor volume of 130 mm3. The immunotoxins 7E4B11-SAP (30 µg/dose) and IgG-SAP (30 µg/dose) were tested alongside the vehicle control (PBS) for tumor growth delay of established tumors. Points, mean tumor volume versus time; bars, SD. B, points, mean time to end point (growth of tumor to 1,500 mm3). C, examination of normal human tissue for 7E4B11 reactivity: (i) astrocytoma-positive control, (ii) colon mucosa, (iii) colon muscularis mucosa, (iv) kidney cortex renal tubular, (v) kidney cortex glomerulus, (vi) kidney medulla renal tubular, (vii) liver hepatocytes, (viii) liver portal area, (ix) small intestine myenteric plexus and musculars propria, (x) small intestine villi, (xi) stomach muscularis propria, (xii) stomach muscosa, (xiii) astrocytoma IgG1-negative control. Magnification, x40. Representative of replicate tissue samples from different subjects, in a study conducted by LifeSpan. Additional normal human tissue is also presented in Supplementary Fig. S1.

 
We then set out to determine the antitumor activity of unconjugated 7E4B11 in the human U87 glioblastoma xenograft model. Nude mice received i.p. injections of 7E4B11, control IgG, or PBS twice weekly for 2 weeks. All treatments began on day 1 in groups of 10 mice bearing well-established (130 mm3) U87 glioblastomas; i.p. 7E4B11 (20 µg/dose) treatment produced 27% tumor growth delay (P < 0.05) relative to the vehicle, whereas the IgG treatment group did not show statistically significant antitumor activity. In addition, 7E4B11 treatment yielded two 60-day survivors with partial regression responses. The 7E4B11 unconjugated antibody was well tolerated, and no toxic deaths were recorded (data not shown).

The cross-reactivity of 7E4B11 to peripheral tissue, especially those tissues involved in absorption, secretion, and toxicity, is important to establish. Especially, if an antibody toxin conjugate is to be generated. To this end, we studied, colon, kidney, liver, small intestine, and stomach tissues for cross-reaction to 7E4B11 (Fig. 3C). No cross-reactivity was seen in these tissues. The astrocytoma-positive control displays extensive staining, whereas the isotype-negative control IgG did not stain.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Astrocytomas are the most common and often deadly form of brain tumor. RPTPß expression in astrocytomas has been described and correlates with increased malignancy (3). One approach for the treatment of astrocytoma is the delivery of therapeutic antibodies i.c. This avoids the difficulties in penetrating the blood-brain barrier and may be an effective way to target and destroy tumor cells that remain after surgical resection. RPTPß has many hallmark features of a functional tumor biomarker. RPTPß seems to be very CNS specific. This suggests the opportunity to target peripheral tumors for destruction while avoiding toxicity to normal tissue. In the case of astrocytoma/glioblastoma, RPTPß is overexpressed and could be an excellent biomarker and therapeutic target. Others and we have shown the overexpression of both long and short RPTPß isoforms in astrocytoma using functional genomics and immunohistochemistry (2–4). The role of short and long RPTPß in cell adhesion and migration suggests an important function in tumor cell biology (4). Therefore, the development of RPTPß antibodies may prove useful for the treatment of astrocytoma and other cancers. RPTPß seems to be nonessential to normal tissues, as shown by the lack of phenotype of the knockout mouse (23). These mice are normal except for sensitivity to EAE (murine multiple sclerosis model) and have impaired remyelination following insult (24). In contrast, tumor cells seem to require RPTPß for adhesion, migration, and growth (2, 4, 25). These observations would suggest that RPTPß can be safely targeted by therapeutic antibodies.

Because both long and short RPTPß are expressed in astrocytomas and facilitate tumor cell adhesion and migration, we developed antibodies to the ectodomain of RPTPß and characterized them for binding and efficacy in tumor cell killing. The entire ectodomain of short RPTPß was used to immunize mice and generate antibodies. Therefore, a better characterization of the antibody-binding sites and understanding of ligand-receptor interactions will be undertaken. We show that 7E4B11 and several other RPTPß antibodies have low nanomolar affinity. This seems to be adequate for in vivo efficacy. However, higher-affinity interactions could facilitate increased uptake of immunotoxin or enhance the presumed effects on signal transduction (altered adhesion, invasion, and growth).

The RPTPß antibodies described here are selective, have reasonable affinities, bind native surface RPTPß, and get internalized (either actively or passively). RPTPß is probably continuously recycled (much like other receptors) and thereby could passively deliver the immunotoxin, alternatively, antibodies to RPTPß may actively facilitate internalization. Active internalization could result from the formation of specific receptor conformations resulting from antibody-receptor interactions. Interestingly, RPTPß normally has constitutive phosphatase activity, and when it binds pleiotrophin, RPTPß becomes inactive (8). Therefore, a receptor conformation that is in the off state (lacks phosphatase activity) may be the one rapidly internalized. This is in contrast to the models of antibody-mediated internalization suggested for receptor tyrosine kinases. Some EGFR antibodies bind and activate this receptor and thereby trigger internalization (1).

RPTPß seems to be important for maximum tumor growth in situations of contact independence or in complex cellular environments. RPTPß binds to matrix proteins, such as NCAMs and tenascin (15–17). These interaction may be necessary for tumor growth or survival (resistance to anoikis) in in vivo settings. The complex nature of RPTPß biology is consistent with the studies of pleiotrophin and the protumor role it plays. Wherein, pleiotrophin promotes colony formation and in vivo tumor growth but lacks direct cell proliferative activity in cell culture models (5, 7). In our hand, the RPTPß antibody-7E4B11 seems to slow tumor growth in soft agar and tumor growth in vivo.

The ability of RPTPß antibodies to target and kill U87 tumors provides proof of concept for the development of better RPTPß immunotoxins. Currently, several small molecular peptide and protein toxins are being evaluated for efficacy as antibody conjugates (26, 27). Ideally, the antibody-toxin conjugate should be selective to tumor cells and have a linker that is cleaved inside the cell and a toxin that is potent. These characteristics ensure that only tumors get killed and normal tissues are spared. Saporin is a ribosomal inhibiting protein toxin and, although useful for research studies, has not proved itself for clinical application, due to its size and immunogenecity (28). Immunotoxins must not be immunogenic (as is often the case for protein toxins) and should retain the desired characteristics of the antibody (stability, affinity, selectivity, and function). Systemic delivery of an immunotoxin is risky if the antibody is not sufficiently targeted or if the toxin has undesired side effects. A "naked," unconjugated antibody is ideal, but many of the same hurdles still need to be overcome (29). The naked antibody must be highly tumor selective and able to modulate its target such that tumor cells die and normal cells are not affected. In many cases, this can be a "tall order" and unwanted side effects due to dysregulation of signaling pathways can be just as devastating as toxin-mediated side effects. This is because many antibody targets play important roles in normal cellular functions. In many ways, RPTPß is an ideal target (tumor-selective expression, functional relevance) and is nonessential for normal tissues. The further study of RPTPß and its antibodies may facilitate development of therapeutics for the treatment of cancer.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 4/ 7/05. Revised 11/23/05. Accepted 12/ 9/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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