A recombinant scFv-FasLext as a targeting cytotoxic agent against human Jurkat-Ras cancer
© Chan et al.; licensee BioMed Central Ltd. 2013
Received: 26 March 2012
Accepted: 22 February 2013
Published: 5 March 2013
Targeted therapy of human cancers is an attractive approach and has been investigated with limited success. We have developed novel cytotoxic agents for targeted therapy of human cancers based on the extracellular cytotoxicity domain of CD178 (FasL) and the specificity offered by single chain antibodies (scFv) against dominant human tumor Ag TAG-72 (cc49scFv) and TAL6 (L6scFv).
The cc49scFv-FasLext is highly effective in in vitro killing of human TAG-72+ Jurkat-Ras tumor cells with a 30,000 fold greater cytotoxicity as compared to soluble FasL (sFasL). On the other hand, L6scFv-FasLext only increased cytotoxicity 500-fold as compared with sFasL against TAL6+ HeLa cells in in vitro assays. The high specificity and strong cytotoxicity of cc49scFv-FasLext made it feasible to cure IP-implanted Jurkat-Ras tumors in SCID mice.
Our study demonstrated that scFv-FasLext with a strong cytotoxicity against sensitive human tumor targets may be useful as effective chemotherapeutic agents.
Targeted therapy of human cancers has been considered by many investigators and remains an attractive approach to avoid side effects of cancer treatment. Anti-CD95 (Fas) Ab that induces Fas+ tumor apoptosis in vitro has been investigated as a potential reagent for this purpose . One approach to increase the specificity of the killing is to link the FasL extracellular cytotoxic domain (FasLext) with a tumor Ag specific single chain Ab (scFv). Such fusion proteins may attract the binding of FasLext only to tumor cells and selectively induce apoptosis of cancer cells.
A study based on similar concept had been reported in a mouse tumor model . An scFv derived from anti-fibroblast activation protein (FAP) Ab was fused with FasLext. It was clearly demonstrated that such a fusion protein did not induce liver toxicity . Interestingly, local treatment with the fusion protein against Fas+ cancer cells that had been transfected with FAP prevented tumor growth in the skin whereas un-transfected tumor cells were marginally inhibited . Apparently, the FAP-transfected cancer cells continuously provided the source that allowed aggregate formation of the fusion protein such that strong cytotoxicity against the Fas+ cancer cells could be induced . In another related study, it was demonstrated that CTLA-4-FasLext recombinant protein did not display liver toxicity. It also did not express cytotoxicity against Fas+ target cells in vitro unless B7-bearing cells were added. Apparently, only through forming FasL aggregate on the B7+ cells could the toxicity be induced .
In contrast to the above studies, we generated two recombinant scFv-FasLext fusion proteins using scFv derived from cc49 and anti-TAL6 mAb, which target human tumor-associated glycoprotein (TAG-72) and tumor-associated Ag L6 (TAL6), respectively [4, 5]. As compared with soluble FasL (sFasL), L6scFv-FasLext was 500-fold more cytotoxic against TAL6+ HeLa cells and cc49scFv-FasLext was 30,000-fold more cytotoxic against TAG-72+ Jurkat lymphoma cells. The cc49- and L6- scFv-FasLext fusion proteins serve as important reciprocal controls, demonstrating the specificity and sensitivity of cytotoxicity against Jurkat and HeLa cells respectively in vitro. In animal models, cc49scFv-FasLext cured Jurkat-Ras (Jurkat cells transfected with human Ras oncogene) tumors implanted in SCID mice owing to increased sensitivity to the recombinant protein depending on TAG-72 and Fas expression as well as high-sensitivity of the tumor to cc49scFv-FasLext. Our study raises the possibility that certain human cancers may be highly sensitive to cc49scFv-FasLext such that they may be treated with the bioactive reagent.
Animals, cancer cells, and immunochemical reagents
The immune compromised SCID mice on BALB/c background (CBySmn.CB17-Prkdc scid ) and Rag1 -/- mice in B6 background (B6.129S7-Rag1 tm1Mon /j) were obtained from the Jackson Laboratories, Bar Harbor, Maine, USA. Anti-TAG-72 (cc83) and anti-L6 mAb were provided by co-author Dr. S. Roffler. All human cancer cells with the exception of Jurkat-Ras were obtained either from ATCC or the tissue culture center of the University of Virginia. The Jurkat-Ras cell line was kindly provided by Dr. D. Faller at the Boston University School of Medicine. Experiments involving animals were conducted in accordance with the protocols approved by the Animal Care and Use Committee (ACUC) of the University of Virginia.
The plasmid p2c11-γ1-B7, containing 2c11scFv encoding anti-mouse CD3ε chain, pLHCX-cc49-hβG, a construct encoding cc49scFv, and pET22b-L6, a construct encoding L6scFv, were prepared by Liao et al and Chou et al [6, 7]. pBluescript II KS-hFasL, a plasmid containing the human FasL, was a gift from Dr. S. Nagata .
Construction of cc49scFv-FasLext and L6scFv-FasLext
The scFv-FasLext constructs were first assembled in a pHook-based vector. This involved first making 2c11scFv-FasLext from p2c11scFv-γ1-B7 construct by replacing the γ1-B7 portion with FasLext. Subsequently, 2c11scFv was replaced with cc49scFv and L6scFv.
To generate the pHook 2c11scFv-FasLext fusion protein construct, the γ1-B7 portion of p2c11-γ1-B7 was replaced with FasLext. The following primers were used to amplify FasLext from pBluescript II KS-hFasL: 5′- CGGACCAGGACAAGTACAACTACAAGAAAAAAAGGAGCTGAGG -3′ and 5′- TCTAGAATTCTCGAGTTAGAGCTTATATAAGCCGAAAAACGTCTG -3′ using Hotstart PFU Turbo (Stratagene), according to the manufacturer’s directions. Subsequently, T4 polynucleotide kinase (New England Biolabs) was used to blunt end the PCR product. Afterward, Xho I was used to digest the PCR product and the PCR mixture was electrophoresed. A band of about 460 bp was excised from the gel and purified using a QIAEX II gel extraction kit (Qiagen). In a separate reaction, pHook-scFv-γ1-B7 was digested with Sal I, filled in with Polymerase Klenow Fragment, cut with Xho I, and ligated with the FasLext fragment.
To generate cc49scFv-FasLext and L6scFv-FasLext, the 2c11scFv fragment was excised from pHook 2c11scFv-FasLext using Sfi I and Sal I restriction enzymes, and replaced with the PCR product coding for either the cc49scFv or L6scFv digested with Sfi I and Sal I. The primers used to amplify cc49scFv, L6scFv, and incorporate the Sfi I and Sal I restriction sites at either end were; cc49FP 5′-TGCCATGGCCCAGCCGGCCCAGGTTCAGTTGCAGCAGTCTGA-3′ and cc49RP 5′- TCCTGGTCCGTCGACTCCGCTTCCTCCGCTTCCTTTCAGCACCAGCTTGGTCCCAG-3′; L6FP: 5′- ATAGGTTGCCATGGCCCAGCCGGCCATTGTTCTCTCCCAGTCTCCAGCA -3′ and L6RP 5′- TCCTGGTCCGTCGACTCCGCTTCCTCCGCTTCCGGATGAGGAGACTGTGAGAGTGGT-3′.
To obtain higher expression levels, the cc49scFv-FasLext and L6scFv-FasLext expression cassettes were PCR amplified from the pHook based vectors using the following common primers, digested with Xho I and Not I restriction enzymes and cloned in the pBCMGSNeo vector at the Xho I and Not I sites. BCMGXhoI FP 5′- ATCGCCT CTAATCTCGAGATGGAGACAGACACACTCCTGCTA-3′ and BCMGNotI RP 5′- GGCCTCGACTCGCA TGCGGCCGCTTAGAGCTTATATAAGCC GAA-3′.
Generation of cell lines expressing the fusion proteins
Transfection of plasmids was performed using lipofectamine (Invitrogen) or electroporation at 960 μF capacitance and voltages of 270 mV, 280 mV, or 290 mV using a BioRad Gene Pulser (BioRad). Ten million NIH-3T3 or BW5147 cells were utilized for each transfection. Immediately after transfection, cells were grown in DMEM (Invitrogen) containing 10% heat-inactivated FCS, vitamin solution (Cellgro), 100 mM sodium pyruvate solution, and penicillin-streptomycin-glutamine (Invitrogen). Two days later, 0.6 mg/ml of G418 (Invitrogen) was added to cultures and cells were allowed to adapt over a 2-week period. Cells were then transferred to fresh media containing 1 mg/ml of G418. Cell cultures were split as necessary, while maintaining G418 concentrations. After the mock transfected cells were dead, the mixtures were cultured for an additional 2 weeks in media containing G418. Subsequently, culture supernatants were collected and screened for cytotoxic activity against A20 cells. Cell populations whose supernatant expressed cytotoxicity were expanded for subsequent use in assays. Cells from wells with strongly cytotoxic supernatants were adapted to normal culture medium and expanded for further studies.
Flow cytometric analysis
Half a million cells were stained for Fas expression using PE-DX2 anti-Fas (Pharmingen) mAb, and with PE-mouse isotype control (Pharmingen). Expression analysis of L6 and TAG-72 was done using a two-step staining. L6 mAb, IgG2a isotype control (G155-178, Pharmingen), cc83 ascites fluid (a second generation mAb made against purified TAG-72) , and heat-inactivated BALB/c serum were used first, followed by washing and staining with fluorescent secondary Ab. For two-step staining using scFv-FasLext, 1 ml of respective transfected cell culture supernatant was added to each cell pellet. Cells were incubated on ice in the dark for 45 minutes. Cells were rinsed 2 times in PBS and supernatants were removed. Cells were then stained with FITC- or PE-goat anti-mouse IgG (Southern Biotech), PE-Alf 1.2 anti-FasL (Caltag), and PE-mouse IgG2a isotype control (Caltag) secondary Ab. Cells were incubated on ice in the dark for 45 minutes, rinsed 2 times with PBS, and then fixed in 2% paraformaldehyde. Samples were analyzed using CellQuest and a Becton-Dickenson flow cytometry machine.
40 μl of cell supernatants or media were loaded onto a 15% polyacrylamide gel using Laemmli buffer. A FasL recombinant protein corresponding to the human FasLext was used as a positive control (Santa Cruz Biotechnology) (data not shown). Proteins were resolved at 40 mA and transferred at 150 V for 75 minutes onto PVDF membranes using a BioRad MiniProtean II system (BioRad).
Membranes were then blotted with rabbit polyclonal anti-HA or rabbit polyclonal anti-FasL (C-20) (Santa Cruz Biotechnology) followed by HRP-conjugated donkey anti-rabbit Ab [HRP] (Amersham Biosciences). Bound HRP were exposed to ECL reagents (Amersham Biosciences) and results were recorded using X-OMAT AR film (Kodak).
Size exclusion analysis
Culture supernatants were fractionated using a Millipore molecular membrane filtration apparatus that separates molecules based on their sizes above or below 300 kDa. The volumes of both the “Flow-through” and “Retentate” fractions were brought to original volume with fresh culture medium and examined for cytotoxicity against A20 target cells.
Quantification of FasL
The scFv-FasLext concentration in supernatants was determined using a commercial kit for sFasL (Oncogene Science). The molecular mass of sFasL was assumed to be 26 kDa based on gene sequence data. The molecular mass of the fusion proteins was estimated to be 54 kDa. A standard curve was generated. Absorbance values of samples that fell within the linear range of the standard curve were used to determine the original sample concentrations.
Cytotoxicity assays against various target cells were conducted as previously described. Target cells (2×104), labeled with Na251CrO4 (New England Nuclear), were cultured with various doses of Jo2 mAb (BD Pharmingen) or culture supernatant in 0.2 ml in individual wells of a 96-well plate for 5 hours in a 37°C, 10% CO2 incubator [9, 10]. Supernatants were removed and radioactivity (cpm) was determined using an LKB-Wallac gamma counter (Perkin-Elmer). Background release was determined by culturing cells alone. Cytotoxicity is expressed as% specific 51Cr release, which is determined by the formula: 100% × (experimental release–background release)/(total cpm released by 0.5% NP − 40–background release). Assays were carried out in duplicate and the experiment was repeated three times. For adherent human cancer cell lines such as HeLa, LS-174 T, and several breast cancer cell lines, cells in 96-well plate were either treated overnight with IFN-γ (100 ng/ml) or left untreated followed by the addition of the cytotoxic proteins. The standard MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma) assays were conducted 18 hours after IFN-γ treatment . In some experiments, cytotoxicity assays were conducted in the presence of inhibitors such as Alf 1.2 anti-FasL, NOK-1 anti-FasL , cc83 anti-TAG-72  or anti-L6  mAb with isotype included as appropriate controls.
Treatment of cancer cells in vivo
We could not grow Jurkat cells in SCID and Rag1 -/- mice using (30-50) ×106 cells per IP injection even at 4 months after implantation. However, tumor growth was readily achieved by using Jurkat-Ras cells. This dose was used for the treatment studies with cc49scFv-FasLext. SCID mice implanted IP with Jurkat-Ras cells and not treated with the cc49scFv-FasLext fusion protein usually died within 5 weeks.
Two days after tumor implantation, scFv-FasLext containing supernatant (1 ml for cc49scFv-FasLext containing approximately 2 μg FasL equivalent, based on ELISA assay) was injected IP into the peritoneum every other day for as long as 4 weeks or until the mice died. In some experiments, the first injection of cc49scFv-FasLext was administered at 6 days after tumor implantation.
Derivation and immunological characterization of scFv-FasLext
Biochemical characterization of scFv-FasLext
We determined the molecular sizes of the cytotoxic components secreted by the scFv-FasLext transfected cells by molecular membrane filtration through a 300 kDa Millipore filter. As shown in Figure 1B, the large proportion of Fas-dependent cytotoxicity was present in the “Retentate” of the L6scFv-FasLext whereas the great majority of cytotoxicity was present in the “Flow-through” of cc49scFv-FasLext supernatant (Figure 1C). The result indicated that individual scFv-FasLext form different levels of multimeric structures. The cytotoxic activities in the respective fractions were verified by Western blotting to correspond to the scFv-FasLext fusion proteins (not shown). Western blotting analyses of the culture supernatants from both clones using polyclonal anti-HA for scFv and anti-C20 FasLext Ab demonstrated that the scFv-FasLext fusion proteins expressed the expected molecular size under denaturing and reducing conditions (Figure 1D).
Targeting cancer Ag specifically increases cytotoxicity against the Ag-bearing human cancer cells
In another series of experiments, we included A20 as a control for scFv-FasLext for Fas-mediated cytotoxicity without the tumor Ag influence because mouse A20 expresses only Fas but not the human TAG-72 nor TAL6. The culture supernatants from L6scFv-FasLext and cc49scFv-FasLext showed comparable cytotoxicity against the A20 target (Figure 3A). In addition, both scFv-FasLext were more cytotoxic to A20 than sFasL. HeLa cells (TAL6+TAG72-Fas+) were found to be 500-fold more sensitive to L6scFv-FasLext than sFasL (Figure 3B) whereas Jurkat cells (TAL6-TAG-72+Fas+) were 30,000 times more sensitive to cc49scFv-FasLext than sFasL (Figure 3C). In addition, L6scFv-FasLext was significantly more cytotoxic against HeLa cells as compared to cc49scFv-FasLext (Figure 3B). Conversly, cc49scFv-FasLext displayed far more enhanced cytotoxicity against Jurkat cells as compared to L6scFv-FasLext (Figure 3C). The results clearly demonstrated that the increase in Fas-mediated cytotoxicity of scFv-FasLext required targets bearing the cognate tumor Ag.
Treatment to increase sensitivity of TAL6+Fas+ cells to L6scFv-FasLext
IFN- γ treatment enhances the sensitivity of cancer cells to L6scFv- FasL ext mediated apoptosis
% Specific killing of 104cells/well by L6scFv-FasLext(μl/well)
Treatment of TAG-72+Fas+ human cancer cells in SCID mice with scFv-FasLext
Highly cytotoxic cc49scFv-FasL ext eradicated Jurkat-Ras cancer in SCID mice a
Cells injected on day 0
Treatment begins on
# mice treated
# tumor-free mice at the end of the treatmentb
(40-50)×106 Jk-Ras Control
6 at 4-6 wk later
(40-50)×106 Jk-Ras Test
0 at 4-6 wk later
20×106 Jk-Ras Control
4 at 4-6 wk later
20×106 Jk-Ras Test
1 at 4-6 wk later
The present study suggests that using scFv-FasLext fusion protein as a therapeutic agent against human cancers is feasible only if the target tumor is extremely sensitive to the scFv-FasLext –mediated cytotoxicity, such as the case for cc49scFv-FasLext mediated cytotoxicity against Jurkat cells. However, other than Jurkat-Ras, we have not found a second commonly used human cancer cell line that displays such sensitivity to cc49scFv-FasLext and can be cured in an animal model. In the vast repertoire of tumor diversity in human cancers, highly sensitive tumors like Jurkat cells may exist and as such may be sensitive to cc49scFv-FasLext. In this respect, various cancer cells present in repositories that express Fas and TAG-72 should be examined for their sensitivity to cc49scFv-FasLext. Even if a small fraction of the TAG-72+ human tumors were found to be nearly as sensitive as Jurkat, it would be significant that the cc49scFv-FasLext could completely eradicate the cancer cells. Moreover, our study also raises the possibility in using scFv-FasLext in combination therapy in which tumor cell sensitivity to scFv-FasLext can be enhanced by commonly used cancer treatment protocols.
Intuitively, it is difficult to understand how such a dramatic increase in cytotoxicity against Jurkat cells was observed when cc49scFv was fused with FasLext. A 30,000-fold increase in cytotoxicity is not easily comprehended simply by adding a co-operative ligand to aid for binding. Several possibilities may explain this observation. First, only Jurkat among many cancers displayed such a high susceptibility, suggesting that high sensitivity to FasL-mediated apoptosis is a property characteristic of the Jurkat target. A recent study has shown that Fas-mediated apoptosis in Jurkat cells triggers rapid release of ATP via pannexin1 channels, leading to an ATP-dependent purinergic receptor-mediated death . Higher order structures formed due to aggregation of scFv component , so as to form scFv dimers/trimers of FasLext trimers could have enhance the cytotoxicity of FasLext moiety, as evidenced by higher cytotoxicity of both cc49 and L6 -scFv-FasLext on A20, HeLa and Jurkat cells compared to sFasL (Figure 3). Previous studies have shown that controlled cross-linking of sFasL trimers increased the cytotoxicity by over a 1000-fold as compared to uncross-linked sFasL, a property which is also applicable to soluble TRAIL and other members of the TNF super-family ligands . Second, The unique multimeric structure of cc49scFv-FasLext (each Fas binding unit contains 3 TAG-72 binding sites) may allow more efficient binding to Jurkat TAG-72 Ag to outcompete anti-TAG-72 mAb. This will also add to the sensitivity of Jurkat to cc49scFv-FasLext cytotoxicity and resistance to cc83-mediated inhibition. Third, several TAG-72+ tumors increase their TAG-72 expression (10-100 folds) after implanting in athymic mice further leading to increased sensitivity of Jurkat-Ras tumors to cc49scFv-FasLextin vivo. Fourth, the great majority of TAG-72 has a molecular size of ≥106. Although TAG-72 has been well established as the target Ag for cc49, high TAG-72 or TAG-72-related target sites that are reactive to cc49scFv-FasLext may be present on Jurkat since the reactive epitopes on Jurkat cells have not been characterized. These sites may be revealed or modified by the post-translational modifications of the TAG-72 .
Liver has been shown to be a target with lethal outcome when mice were treated with anti-Fas mAb Jo2 . This lethal effect was originally attributed to the toxicity of hepatocytes. We demonstrated that FcRII knockout mice are resistant to Jo2 treatment . In addition, anti-Fas mAb lacking FcRII binding activity also failed to induce lethality . Moreover, sFasL was extremely ineffective in inducing lethality due to its inability to form aggregates on the target cells that lacked FcRII. Our study suggested that FcRII-expressing sinusoid endothelial cells in the liver are the real target for FcRII-dependent, Jo2-mediated lethality, which cannot be induced with scFv-FasLext. The scFv-FasLext lacks FcRII binding activity and bears a ligand for tumor target Ag that selectively enhances binding to the cancer targets with minimal non-specific cytotoxicity against Fas-expressing normal cells that lack scFv targeted Ag.
There are potentially several ways to enhance the use of scFv-FasLext for therapy. First, any existing protocols for cancer therapies are likely to make the tumor target cells more sensitive toward the cytotoxicity of the scFv-FasLext. Second, increase in Fas expression levels on target cells are often observed under various in vivo conditions. Amongst them, activation of cells and induction by cytokines such as IFN-γ almost always enhances Fas expression levels. Others have claimed that IFN-α or IFN-γ could induce higher expression of cancer Ag [19, 20]. Our studies on many tumor cells cultured in the presence of IFN-γ only enhanced the cytotoxicity expression by 3-fold in a single in vitro treatment within 36 hours (pretreatment and cytotoxicity times), a level that may be helpful but not very effective. Additional studies are needed to determine if modulation of TAG-72 and TAL6 may be achieved to help the therapeutic effect of the cc49scFv-FasLext and L6scFv-FasLext, respectively.
Our studies demonstrate the reciprocal specificity of cc49scFv-FasLext and L6scFv-FasLext reagents and strongly suggest that targeted therapy of human cancers with FasLext based reagents is feasible, provided that the target antigen is expressed on the cancer cells and that the tumors are highly susceptible to FasLext. Hematopoietic cancers are attractive targets due to their likelihood of FasLext sensitivity. The possibility of the use of interferons for modulation of tumor antigen expression and increase in susceptibility to L6scFv-FasLext -mediated cytotoxicity offers additional strategies. The eradication of Jurkat-Ras tumors with cc49scFv-FasLext without hepatic toxicity warrants the screening of human hematopoietic cancer repositories for TAG-72 and Fas expression and their subsequent curability with this reagent.
Derek V Chan in partial fulfillment of Ph.D thesis (2001-2004).
This work is supported in part by NIH grants AR-051203, DE-017579, AI-36938 and ES10244 (STJ), “Institutional Start-up funds” from University of Virginia Department of Medicine (RS) and NSC grants NSC-99-2320-B001-011-MY3 (SRR). Dr. Shyr-Te Ju, PhD, Professor of Medicine and Microbiology at the University of Virginia School of Medicine, died at the age of 65 in Charlottesville, VA, USA on September 25, 2012 after a long battle with lung cancer. He was a member of many federal and non-federal review panels, served on several editorial boards and made numerous contributions to immunology research, having published 140 peer-reviewed papers, including one on Fas ligand biology, which was cited more than 1,200 times. Dr. Ju was a conscientious and compassionate human being with an unmatched enthusiasm for applied medical research.
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