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Cancer Research 68, 9176, November 15, 2008. doi: 10.1158/0008-5472.CAN-08-1688
© 2008 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

"Active" Cancer Immunotherapy by Anti-Met Antibody Gene Transfer

Elisa Vigna1, Giovanni Pacchiana1, Massimiliano Mazzone2, Cristina Chiriaco1, Lara Fontani1, Cristina Basilico2, Selma Pennacchietti2 and Paolo M. Comoglio1,2

1 Laboratory for Gene Transfer and Therapy and 2 Division of Molecular Oncology, Institute for Cancer Research and Treatment (IRCC), University of Turin Medical School, Turin, Italy

Requests for reprints: Paolo M. Comoglio, Institute for Cancer Research and Treatment, University of Turin Medical School, Strada Provinciale 142, Turin, Italy. Phone: 39-11-993-3601; Fax: 39-11-993-3225; E-mail: paolo.comoglio{at}ircc.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Gene therapy provides a still poorly explored opportunity to treat cancer by "active" immunotherapy as it enables the transfer of genes encoding antibodies directed against specific oncogenic proteins. By a bidirectional lentiviral vector, we transferred the cDNA encoding the heavy and light chains of a monoclonal anti-Met antibody (DN-30) to epithelial cancer cells. In vitro, the transduced cells synthesized and secreted correctly assembled antibodies with the expected high affinity, inducing down-regulation of the Met receptor and strong inhibition of the invasive growth response. The inhibitory activity resulted (a) from the interference of the antibody with the Met receptor intracellular processing ("cell autonomous activity," in cis) and (b) from the antibody-induced cleavage of Met expressed at the cell surface ("bystander effect," in trans). The monoclonal antibody gene transferred into live animals by systemic administration or by local intratumor delivery resulted in substantial inhibition of tumor growth. These data provide proof of concept both for targeting the Met receptor and for a gene transfer–based immunotherapy strategy. [Cancer Res 2008;68(22):9176–83]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Due to their intrinsic specificity, monoclonal antibodies (mAb) represent one of the most promising avenues for the so-called "targeted therapy" of cancer when directed against specific oncogene products (1, 2). As "targeting drugs," mAbs may have features superior to small-molecule inhibitors (3). These include very high specificity, generally constant pharmacokinetic properties in different individuals, and the fact that they exert therapeutic effects through a wide spectrum of biological responses. mAbs, intefering with functional cell-surface molecules, such as receptors, can alter different intracellular pathways, thus interfering with the cancer cell biology. Moreover, through interaction with the body's immune system, mAbs can recruit effector functions for antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Clinical trials with mAbs have already provided encouraging results (4–6), but a number of limitations are currently still unresolved. These include (a) the complexity of the protocols needed to obtain a long-lasting high level of circulating antibody in recipients (e.g., repeated administrations); (b) the side effects of prolonged protein infusion; and (c) the neutralizing immune response arising in the host, especially on high doses of mAb injection. These and other phenomena hamper the success of the above classic, "passive" immunotherapy approach. Because mAbs are proteins encoded by specific genes, an alternative approach to cancer immunotherapy ("active immunotherapy") based on the delivery of mAb genes through a gene transfer technology has been proposed: Host cells may be genetically engineered to produce and release the therapeutic antibody (7).

The first key point for the success of "active" immunotherapy is the choice of the vehicle used for the direct in vivo gene transfer. Electroporation in muscles of plasmids carrying immunoglobulin genes has been explored (8, 9). The antibody is present in the serum of the experimental animals, but its expression is transient. The use of viral-derived vectors has proved to be more effective. An adenoviral vector has been tested, with limited success in terms of prolonged expression due to the lack of integration (10). Adeno-associated vectors were used to express in vivo an antibody with neutralizing activity against HIV-1 (11) and against vascular endothelial growth factor receptor 2 (VEGF-R2; ref. 12). In the first report, the vector, delivered by i.m. injection, resulted in a late long-term expression of the mAb. Heavy and light chain expression was obtained by two different promoters; the balanced expression of the two immunoglobulin chains was not measured. In the second study, a vector encoding an anti–VEGF-R2 antibody was delivered by tail vein injection. A single promoter drove transcription of an mRNA encoding a precursor protein containing both the heavy and light chains, separated by a sequence encoding a peptide (2A) of the foot and mouth disease virus. During translation, the 2A peptide is "self-cleaved," releasing the antibody chains. This elegant technology gave rise to a high and sustained level of circulating antibody. Thus far, the lentiviral vector has not been considered for the gene transfer–based immunotherapy although it fulfills the following recognized features: (a) stable integration into the chromatin of both dividing and non dividing cells (13), providing a wide range of target cells and a long term effect of delivery; (b) low genotoxicity on integration (14), providing a higher level of safety compared with other integrating vectors; and (c) the availability of a bidirectional promoter, driving high, sustained and coordinated expression of two different transgenes; this provides the tool of excellence to generate stoichiometric amounts of separate cDNA products (15). In the case of antibodies, the balanced expression of heavy and light chains prevents the assembly of aberrant molecules, interfering with the specificity.

The choice of the target antigen for the antibody is the second key point to achieve effective cancer immunotherapy. The ideal antigen should fulfill the following requirements: (a) be preferentially expressed by cancer versus normal cells (16); (b) be extracellular or present at the cell surface to allow antibody binding; and (c) be functionally involved in the onset and progression of malignancy. Molecules that meet most of the above listed features are growth factor receptors. For example, mAbs against the epidermal growth factor receptor (EGFR; HER-1) show therapeutic activity in epithelial tumors (17, 18), and one of them, cetuximab, has been approved for the treatment of colorectal (19) and head and neck carcinomas (5). A second example is trastuzumab, directed against the HER-2 receptor. This antibody, in combination with standard adjuvant chemotherapy, proved to be effective in the treatment of patients with mammary tumors overexpressing HER-2 (6).

Among the tyrosine kinase receptors, a growing clinical interest is focused on the product of the Met oncogene, the hepatocyte growth factor (HGF) receptor. Met drives a genetic program, known as "invasive growth," which, in pathologic conditions, promotes and sustains cell transformation and progression toward metastasis (reviewed in ref. 20). Recently, it has been shown that the mAb DN-30, an IgG2A directed against the extracellular moiety of the human Met receptor, acts as an inhibitor of tumor growth and metastasis through a mechanism of receptor "shedding" (21). In this article, we transfer DN-30 cDNAs by a bidirectional lentiviral vector to experimental animals to provide proof-of-concept for the therapeutic efficacy of anti-Met "active" immunotherapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell culture. A549, HTC-116, and DLD-1 cells were obtained from American Type Culture Collection. CoLo-741 cells were a gift from Dr. Venesio (Unit of Pathology, Institute for Cancer Research and Treatment, Candiolo, Turin, Italy). A2780 cells were from Sigma. All the above listed cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS; Sigma). MDA-MB-435 cells were obtained from the Georgetown University Tissue Culture Shared Resource (Washington, DC) and maintained in DMEM 10% FBS. U87-MG were obtained from Sigma-Tau and maintained in Iscove's modified Dulbecco's medium (IMDM), 10% FBS.

Lentiviral vectors. Vector stocks were produced by transient transfection of the bidirectional transfer plasmid, the packaging plasmids pMDLg/pRRE and pRSV.REV, and the vesicular stomatitis virus (VSV) envelope plasmid pMD2.VSV-G (15, 6.5, 2.5, and 3.5 µg, respectively, for 10-cm dishes) in 293T cells in the presence of 1 mmol/L Na butyric acid (Sigma) as described (22). When required, viral particles were purified and concentrated by ultracentrifugation as described (22). Determination of the viral p24 antigen concentration was done by HIV-1 p24 Core profile ELISA (Perkin-Elmer Life Science Life Science, Inc.). Cells were transduced in six-well plates (105 per well in 2 mL medium) using 25 ng/mL or the indicated amount of p24 gag equivalent particles in the presence of polybrene (Sigma) as described (22).

Cloning of DN-30 heavy and light chain sequences. We extracted total RNA from the hybridoma producing the DN-30 mAb (23) with RNAWiz (Ambion, Inc.) according to the manufacturer's instruction. Using this material as template, we performed reverse transcription-PCR (RT-PCR) using oligodT and murine leukemia virus reverse transcriptase (Promega Corp.). Then we applied a PCR strategy based on the use of degenerated oligonucleotides. To obtain the sequence of the light chain variable region (VL), we used the sense degenerated primer 5'-AGATCTSWGCTGACCCAGTCTCCA-3'and the antisense degenerated primer 5'-ACTAGTTTTGATYTCCARYTTKGTCCC-3. For the heavy chain variable region (VH), we used the sense degenerated primer 5'-CTGCAGSAGTCWGG-3' and the antisense primer 5'-CGTGGTCCCTTGGCCCCAG-3'. PCR amplifications were all done using the Proofstart DNA Polymerase from Qiagen. On analysis of the subfamily to which the two chains belonged,3 we designed new pairs of oligonucleotides able to amplify the entire cDNAs of the two antibody chains. For the light chain (L), we used the following oligonucleotides: sense, 5'-AAAACTGCAGCATCTAGTTCTCAGAGATGGAGAC-3', and antisense, 5'-GGACTAGTCTAACACTCATTCCTGTTGAAGC-3'; for the heavy chain (H), sense, 5'-TATACCCGGGCCACCATGGGATGGAGCTATATCATCC-3', and antisense, 5'-ATATGTCGACGCTAGCTTTACCCGGAGTCCGGGAGAAGC-3. These pairs of oligonucleotides contained the restriction sites to directly clone into the bidirectional lentiviral vector (15): PstI and XbaI for (L) and SmaI-SalI for (H). Details on the PCR amplifications are available on request. Into the sites NheI-SalI located at the 3'-terminus of (H), a sequence corresponding to a double repeated FLAG epitope (SDYKDDDDK) and a polyhistidine stretch (HHHHHHH) was added for mAb detection and purification.

ELISA. DN-30RF quantification in cell culture supernatants or in mouse sera: Pure Fc-Met (R&D Systems) was adsorbed to 96-well ELISA plates overnight at 4°C. After saturation with 0.5% bovine serum albumin for 1 h at 37°C, different dilutions of the sample and increasing concentrations (0–100 ng/mL) of DN-30RF purified by immobilized-metal affinity chromatography, as described in ref. 24, were bound overnight at 4°C. Bound antibodies were revealed by a first incubation with anti-Flag M2 biotin conjugates (Sigma) followed by incubation with streptavidin-horseradish peroxidase (HRP; Amersham). Enzymatic reaction was developed in the presence of tetramethylbenzidine (Sigma) as substrate. Colorimetric assay was revealed by ELx-800 (BioTek Instrument, Inc.). Sample concentration values were obtained by interpolation of the sample absorbance values on the linear part of the curve obtained by dilutions of the standard.

Measurement of the mAb affinity for Met antigen: Pure extracellular portion of the Met receptor (100 ng/well), obtained as described in ref. 25, was absorbed on a 96-well ELISA plate as described above. Increasing concentrations (from 0 to 17 nmol/L) of pure DN-30 obtained by purification on Sepharose-protein A beads of the corresponding ascites and pure DN-30RF (obtained as described above) were absorbed on the plate as above. Bound antibodies were revealed by incubation with antimouse immunoglobulin HRP (Amersham) followed by enzymatic reaction as described above. Data were analyzed using Prism software (GraphPad Software).

Western blot analysis for DN-30 expression. At least 72 h after infection, transduced cells, 80% confluent, were incubated with medium without serum; after 48 h, supernatant containing DN-30RF was collected and cells were lysed with Laemmli buffer. Seventy-five microliters of culture supernatants or 50 µg of total proteins were subjected to SDS-PAGE under reducing conditions. Proteins were processed for Western blotting according to standard methods. Detection was done with antimouse immunoglobulin-HRP (Amersham) and the enhanced chemiluminescence detection system (Amersham).

Immunoprecipitation and Western blot. Immunoprecipitation assay was done as described in ref. 26. Antibodies used for immunoprecipitation were antihuman Met [DN-30, DO-24 (23), and DQ-13 (27)], anti–E-cadherin, and anti–phosphotyrosine PY20 (Transduction Laboratories). Separation on SDS-PAGE and transfer onto Hybond-C Extra membranes (Amersham) were done following standard methods. Primary antibodies for Western blot detection were antihuman Met, DL-21 mAb that recognizes a domain located in the extracellular portion of HGF receptor β chain and C-12 polyclonal antibody that recognizes the COOH-terminal tail of the HGF receptor (Santa Cruz Biotechnologies); anti–phospho-AKT (Ser473); and anti-AKT polyclonal antibodies (Cell Signaling Technology). Antimouse IgG, antirabbit IgG, and streptavidin conjugated with HRP were from Amersham.

Cell-surface protein labeling with biotin. Cells were subjected to surface biotinylation analysis using ECL Surface Biotinylation Module kit (Amersham Biosciences) according to the manufacturer's instructions. Cell extraction, immunoprecipitation, and Western blot were done as described above.

Stimulation of Met tyrosine phosphorylation by HGF. Subconfluent cell monolayers were washed twice with PBS and incubated with serum-free medium for 48 h. In 1 mL of spent medium, we added 100 ng/mL of recombinant HGF (R&D Systems) and cells were incubated for 10 min at 37°C. Then cells were immediately lysed as described in ref. 26.

In vitro biological assays. For proliferation assays, cells were seeded in 96-well Costar dishes. U87-MG cells were infected for 8 h with 60 ng of p24 gag equivalent lentiviral particles/mL. Eighteen hours after infection, cells were seeded in medium plus 10% FCS (1,000/0.1 mL/well). The amount of cells was evaluated every 24 h using ATPlite 1 step (Perkin-Elmer) according to the manufacturer's instruction. Signal was measured with DTX 880 multimode detector (Beckman Coulter). Each experimental point was the average of six repeated samples.

For anchorage-independent growth assays, cells were seeded in 24-well Costar dishes in medium 2% FCS plus 0.5% soft agar (SeaPlaque agarose, BMA; 1,000/0.5 mL/well). When added, the concentration of HGF was 80 ng/mL. Fresh medium +/– HGF was replaced every 3 d. For U87-MG, medium used was 5% FCS plus 0.6% soft agar. Grown colonies were finally visualized by tetrazolium staining after 14 d of culture. Each point was carried out at least in triplicate.

The invasion assay was done in Transwell chambers (Corning). The polycarbonate filters (8 µm pore size) were coated with 10 µg/well of Matrigel basement membrane (Collaborative Research). Cells (5 x 104) were seeded on the upper side of the filters and incubated in medium + 2% FBS, and 80 ng/mL HGF (R&D Systems) was added to the bottom wells of the chambers. After 24 h, cells on the upper side of the filters were mechanically removed. Cells migrated to the lower side were fixed and stained with crystal violet. Every point was in duplicate.

Systemic delivery of lentiviral vectors. Immunodeficient nu–/– female mice on Swiss CD-1 background (7 wk of age) were injected with the lentiviral vectors encoding DN-30RF or PBS (0.3 mL) in the tail vein. Starting from the 2nd week after vector delivery, mice were bled periodically and DN-30RF serum concentrations were estimated by ELISA. At the 8th week after vector delivery, mice were injected s.c. into the right posterior flank with HCT-116 cells (3 x 106 per mouse in 0.2 mL IMDM). Tumor size was evaluated periodically with a caliper. Tumor volume was calculated using the formula V = 2/3{pi}x2y, where x is the minor tumor axis and y is the major tumor axis. At the end of the experiments (42nd day from cells injection), mice were euthanized and tumors were extracted, weighed, embedded in paraffin, and processed for histology. Immunohistochemical analysis for the evaluation of DN-30RF presence in the tumors was done on 5-µm paraffin-embedded tumor sections with an anti-Flag M2 antibody (Sigma, 1:200). Sections were counterstained with Mayer's hematoxylin (Sigma).

Direct injection of lentiviral vectors into preformed tumors. Tumor cells (3 x 106 HCT-116 cells per mouse, 2 x 106 U87-MG cells per mouse in 0.2 mL of IMDM) were injected s.c. into the right posterior flank of 7-wk-old immunodeficient nu–/– female mice on Swiss CD-1 background. Tumor size was calculated as described above. HCT-116–derived tumors reached a size between 50 and 250 mm3 in 10 d. At this point (day 0), mice were randomly divided into two groups and high-titer lentiviral vector preparation (1 µg of gag p24 equivalents per mouse in 50 µL PBS) was administered intratumorally. One group received LV-DN-30RF; the other (control group) received lentiviral vectors (LV) expressing the enhanced green fluorescent protein (eGFP) under the control of the human phosphoglycerate kinase (PGK; ref. 22). A second delivery of vector particles (same amount, same procedure) was done at day 3. Tumor volume was measured every 3 to 4 d. At day 32, mice were euthanized. For U87-MG, after 10 d from injection of cells (day 0), we injected vector particles (2 µg of gag p24 equivalents per mouse in 50 µL of PBS, or PBS only as control) into masses <15 mm3. After 3 d, a second delivery was done. Tumor volume was measured with a caliper periodically and mice were scored tumor positive when masses were >20 mm3. Mice whose tumors were below this threshold were considered tumor-free. At day 71, mice were euthanized.

Statistical analysis. Statistical significance was determined using a two-tailed homoscedastic Student's t test (array 1, control group; array 2, experimental group). For all data analyzed, a significance threshold of P < 0.05 was assumed. In all figures, values are expressed as mean ± SD. The extent of a linear relationship between two data sets was evaluated by calculation of the Pearson product moment correlation coefficient, r.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Cloning the DN-30 cDNAs into a bidirectional lentiviral vector. Starting from total RNA of the corresponding hybridoma (23),we cloned the anti-Met mAb DN-30 cDNAs using RT-PCR based on a degenerated oligonucleotide strategy. The cDNAs of both heavy and light chains were introduced into a lentiviral vector containing a synthetic bidirectional promoter, driving high expression of the two separate cDNAs. The bidirectional promoter has been obtained joining a minimal promoter, derived from the human cytomegalovirus (CMV) virus, upstream and in the opposite orientation, to the human phosphoglycerate kinase ubiquitous promoter (15). Under the PGK promoter, in sense orientation relative to the vector long terminal repeat, we placed the c-DNA encoding the DN-30 heavy chain, whereas in antisense orientation, under the control of the minimal CMV promoter, we placed the DN-30 light chain (Fig. 1A ). Moreover, we tagged the DN-30 heavy chain by a 100-nucleotide-long sequence encoding a "Flag" and a histidine tail for purification purposes. This molecule was called DN-30 recombinant flag (DN-30RF).


Figure 1
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  		Figure 1. DN-30RF expression in epithelial cancer cell. A, scheme of the integrated form of the lentiviral vector expressing the DN-30 mAb. Gray boxes, vector backbone; white boxes, expression cassette (15). PminCMV, minimal core promoter elements from the human CMV joined upstream, and in opposite orientation, to the promoter of the human phosphoglycerate kinase gene, PGK. This promoter was driving divergent transcription of two RNAs, one encoding for the light chain and the other encoding for the heavy chain of the DN-30 mAb. Arrows, the expected transcripts. Flag Tag and His Tag, nucleotide sequences encoding, respectively, for the epitope recognized by the anti-FLAG antibody and a polyhistidine tail. B, DN-30RF expression in a representative cell line (MDA-MB-435) transduced with the indicated amount of LV-DN-30RF. Synthesized (right) and secreted (left) DN-30RF mAbs were revealed by Western blot probing filter with antimouse immunoglobulin.

 
The DN-30RF antibody is correctly synthesized, assembled, and secreted in vitro. We transduced a panel of cell lines derived from human carcinomas of lung, liver, ovary, or colon; a melanoma; and a glioblastoma with the above described lentiviral vector. The DN-30RF protein production was analyzed from cell lysates and cell culture supernatants. All the transduced cells produced the correctly assembled antibody that was secreted in the culture supernatant (Fig. 1B). The antibody production was stable (months) and in the range of 0.2 to 6 µg/mL (Table 1 ).


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  		Table 1. Secretion of recombinant Met antibody by human cancer cell lines

 
The DN-30RF antibody was functional and specific for the Met antigen, as assessed by immunoprecipitation (Fig. 2A ). The antibody detected both the precursor and the mature receptor form. The location of the epitope was assessed by immunoprecipitation of (a) the isolated extracellular domain of Met, including the {alpha} chain and the NH2-terminal segment of the β chain (amino acids 1–932); (b) the SEMA-PSI domains (amino acids 1–562); and (c) the PSI-IPTs domains (amino acids 1–25; 516–932). The epitope was located in the IPTs region of Met (Supplementary Fig. S1).


Figure 2
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  		Figure 2. Characterization of the recombinant DN-30 mAb obtained on gene transfer. A, Met immunoprecipitation from cell lysates on Sepharose-protein G beads preincubated with culture supernatants collected from cells transduced with LV-DN-30RF (DN30RF) or untransduced (CTRL) and from hybridomas producing anti-Met mAb (DN-30) or unrelated mAb (CTRL mAb). Selective immunoprecipitation was revealed by probing filter with anti-Met C12. p190Met, precursor form of the Met receptor; p145Met, mature form of the Met receptor. B, a 96-well plate coated with purified Decoy-Met molecules was incubated with increasing concentrations of purified DN-30 or DN-30RF. {blacksquare}, binding of the DN-30 antibody; {blacktriangleup}, binding of the DN-30RF antibody. O.D., absorbance; A.U., arbitrary units. Point, mean of triplicate values; bars, SD. C, shedding of the Met receptor induced by the DN-30RF mAb. HCT-116 cells were incubated for 24 h with purified DN-30RF mAb at the indicated concentration. Left, Met receptor amount (p190Met and p145Met) in total cell lysates; right, Met ectodomain (p80Met) in cell culture supernatants. Met receptor forms were revealed by probing filter with anti-Met DL-21.

 
The affinity of the DN-30RF antibody, produced by gene transfer, was compared with the affinity of the native parental antibody DN-30 (Fig. 2B). The two binding curves did not show significant differences, the Kd being 2.21 ± 0.11 x 10–9 and 1.43 ± 0.17 x 10–9 mol/L, respectively. The DN-30RF maintained also the biological properties of the native antibody, including the induction of Met receptor shedding from the cell surface (Fig. 2C).

DN-30RF gene transfer down-regulates Met receptor, impairing intracellular signaling. In cancer cells expressing the DN-30RF mAb, the amount of Met receptor exposed at the cell surface was substantially reduced, whereas the intracellular 190-kDa immature single-chain precursor accumulated (Fig. 3A ). Real-time PCR analysis showed that Met mRNA was unaffected (data not shown). It was concluded that accumulation of the precursor results from the interference of the antibody with the process of Met receptor maturation; the two molecules are in fact synthesized and stored in the same intracellular compartment. Processing and cell-surface expression of two unrelated membrane proteins, such as cadherins or {alpha}vβ3 integrin (Fig. 3A and data not shown), were not impaired, indicating a specific action of the antibody on its target rather than a general unspecific impairment of the RER-Golgi transition.


Figure 3
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  		Figure 3. Analysis of Met expression/activation in cells transduced with LV-DN-30RF. A, total and surface Met receptor in transduced cells. HCT-116 cells transduced with LV-DN-30RF (DN30RF) or untransduced (WT) were surface labeled with biotin. Cell lysates were immunoprecipitated with anti-Met or anti–E-cadherin antibodies. Total Met was revealed with anti-Met C-12, whereas surface Met and surface E-cadherin were revealed by streptavidin-HRP. B, starved HCT-116 cells transduced with LV-DN-30RF (DN30RF) or untransduced (WT) were nonstimulated or stimulated with pure HGF. Immunoprecipitated Met was detected with anti-Met C-12 (right) and its phosphorylated amount was revealed by anti–P-Tyr antibody (left). C, phosphorylated AKT (P-AKT) molecules present in total lysates prepared from cells nontreated or treated with HGF were revealed by anti–phospho-AKT antibody (left); total AKT molecules were revealed with anti-AKT antibody (right).

 
In transduced cells, both the basal tyrosine phosphorylation of Met (precursor and the residual mature molecules) and the phosphorylation of downstream transducers such as AKT were not increased. The fact that Met and Met-dependent signals are not constitutively active indicates that a possible receptor dimerization induced by the intracellular antibody, if present, was ineffective. As expected, the response to HGF in DN-30RF–transduced cells was substantially impaired, as assessed by the measurement of Met receptor and AKT phosphorylation (Fig. 3B and C).

DN-30RF gene transfer inhibits Met-mediated biological responses. Growing the cells adherent on plastic dishes, the proliferation rate of the cell lines transduced with DN-30RF, compared with the wild-type cells, was unaffected (data not shown). A notable exception were U87-MG cells, derived from a human glioblastoma harboring a Met-HGF autocrine loop (28). These cells strongly slowed proliferation 2 days after gene transfer (Fig. 4A ), suggesting that the recombinant anti-Met antibody interferes with the autocrine loop of HGF. On the other hand, anchorage-independent growth (in soft agar), a property strictly related to the invasive growth phenotype, was impaired in all cell lines expressing the DN-30RF antibody (Table 1; Fig. 4B). Notably, this inhibition was observed not only in the case of colonies formed in response to exogenous HGF but also in colonies formed in the presence of serum alone (Supplementary Table S1). The inhibition was specific, as it was not observed in cells that do not express the Met receptor (Table 1). Invasion of an artificial basal membrane in a two-chamber transwell assay, another biological property strictly linked to the invasive growth phenotype, was strongly inhibited when DN-30RF–transduced cells were tested both in the absence and in the presence of HGF (Fig. 4C).


Figure 4
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  		Figure 4. Analysis of Met-mediated biological responses in LV-DN-30RF–transduced cells. A, proliferation rate of U87-MG cells expressing DN-30RF mAb. Cells infected with lentiviral vectors encoding for DN-30RF mAb ({square}) or eGFP ({blacklozenge}) or not infected ({blacksquare}). Representative of two independently done experiments. R.L.U., relative light units. B, anchorage-independent growth of U87-MG cells expressing DN-30RF mAb. DN-30RF, U87-MG cells transduced with LV-DN-30RF; WT, untransduced cells. Representative data of three independently done experiments. C, invasion through a reconstituted basal membrane of HCT-116 cells transduced with LV-DN-30RF (DN-30RF) or untransduced (WT) in a two-chamber transwell assay. Pictures show the amount of cells attached to the lower part of the filters. Similar data were obtained in two independently done experiments.

 
Systemic DN-30RF gene transfer inhibits growth of tumor xenografts. DN-30RF lentiviral particles were injected into the tail vein of athymic nude mice. By this route of delivery, the vector is primarily targeted to liver and spleen (29). Two weeks after injection, all the mice subjected to gene transfer (n = 16) showed measurable amounts of antibody in the serum. The level of circulating DN-30RF antibody was dose dependent on the amount of the vector injected, ranging from 144.6 ± 15.2 ng/mL, after administration of 16 µg of p24 equivalent vector particles per mouse, to 20.6 ± 4.4 ng/mL, after 8 µg of p24 equivalents; expression was stable and long-lasting when monitored up to 26 weeks (Fig. 5A ). The modest decrease in antibody concentration observed after the 14th week is compatible with the turnover of the liver Kupfer cells, the main hosts of the transgene (29).


Figure 5
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  		Figure 5. DN-30RF expression and tumor growth in mice subjected to LV-DN-30RF gene transfer by systemic administration. A, kinetics of DN-30RF expression in sera from athymic nude mice subjected to LV-mediated antibody gene transfer (16 µg of gag p24 equivalents per mouse). {blacklozenge}, mice not injected with HCT-116 tumorigenic cells (n = 8 before 8th week, n = 3 after the 8th week); {square}, mice (n = 5) s.c. injected with HCT-116 cells (at the 8th week after vector delivery; arrow). The amount of circulating DN-30RF was measured by ELISA done on mouse sera. Points, mean obtained from each group; bars, SD. Mice injected with PBS were completely negative (data not shown). B, growth rate of tumors obtained on s.c. injection of HCT-116 cells into the flank of athymic nude mice. {blacksquare}, mice not subjected to LV-DN-30RF administration (n = 4); {square}, mice subjected to LV-DN-30RF administration (n = 5). Points, mean tumor volume; bars, SD. C, analysis of DN-30RF presence in the tumor masses by immunohistochemistry. Serial slices of the paraffin-embedded tumors were stained with anti-Flag M2 antibody. Representative images obtained by the analysis done on a tumor developed in a mouse subjected (right) or not (left) to LV-DN-30RF administration.

 
Athymic nude mice expressing the DN-30RF gene and controls were transplanted with HCT-116 human colon cancer cells (Fig. 5B). Tumors were monitored for 5 weeks, showing a slower growth in transduced mice (n = 5) and 50% mass reduction (P = 0.02) with respect to controls (n = 4). Interestingly, the concentration of circulating DN-30RF, which was otherwise stable, decreased dramatically. This suggested depletion of the antibody from the serum as a consequence of adsorption to the growing tumor where the antigen is expressed (Fig. 5A). The flagged recombinant antibody was indeed detected bound on the surface of tumor cells by immunohistochemical staining (Fig. 5C).

Local DN-30RF gene delivery inhibits tumor growth. Athymic nude mice were xenografted by s.c. injection of HCT-116 colon carcinoma cells. Once the explants reached a detectable size (50–250 mm3), lentiviral particles carrying DN-30RF, or eGFP cDNA as control, were directly injected into tumors. Tumors injected with LV-DN-30RF (n = 6) were found to grow slower compared with controls (n = 6, Fig. 6A ). At the end of the experiment (32 days after vector injection), a 40% reduction in tumor mass was observed (P = 0.002). In all mice subjected to gene transfer, the recombinant antibody was present in the serum at the end of the experiment; notably, the concentration was inversely proportional to tumor weight (Pearson coefficient, r = –0.79). Again, this was interpreted as adsorbtion of the antibody by the tumor.


Figure 6
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  		Figure 6. Tumor growth in mice subjected to LV-DN-30RF gene transfer by in situ administration. A, growth of HCT-116–derived tumor in athymic nude mice. Lentiviral particles were injected into the tumors (1 µg of gag p24 equivalents per mouse two times separated by 3 d). {blacksquare}, mice subjected to LV-eGFP administration (n = 6); {square}, mice subjected to LV-DN-30RF administration (n = 6). Points, mean tumor volume; bars, SD. B, tumor incidence in nude mice xenografted with U87-MG cells. LV-DN-30RF particles were delivered into the site of cell injection (2 µg of gag p24 equivalents per mouse two times separated by 3 d). {blacksquare}, mice injected with vehicle (n = 5); {square}, mice subjected to LV-DN-30RF administration (n = 5).

 
When LV-DN-30RF was injected into xenografts of U87-MG glioblastoma cells harboring the HGF-Met autocrine loop, the therapeutic effect was stronger. Four of five mice receiving the local injection of the vector did not develop tumor masses. Untreated mice transplanted with the same number of glioma cells developed tumors with high incidence (Fig. 6B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
In this article, we further strengthen the concept that targeting the Met receptor may be a successful strategy for cancer therapy. Met receptor, generating promitogenic, antiapopotoptic, and proinvasive signals, is one of the key molecules driving a genetic program, known as "invasive growth," which, when inappropriately activated in cancer cells, fosters the transformed and metastatic phenotypes (30). Overexpression of the Met oncogene is a frequent event in a wide spectrum of solid tumors and is often correlated with poor prognosis (reviewed in ref. 31). In most cases, overexpression is a transcriptional response of the cancer cell to the unfavorable microenvironment and it is secondary to hypoxia (32). In fewer patients, Met overexpression is the result of gene amplification, found in primary carcinomas (reviewed in ref. 31), in metastasis (33), and in cancers resistant to EGFR inhibitors (34). Taken together, these reports show that, in a large number of cases, cancer cells have a higher amount of Met receptor compared with normal cells. This feature highlights the Met receptor as a potential good target antigen for antibody-based immunotherapy. Other tumors show Met receptor activation by concomitant expression of HGF, the Met receptor ligand (reviewed in ref. 31). This condition is appropriate for the therapeutic use of an anti-Met antibody able to block the autocrine loop.

The peculiar role of Met in tumor development prompted a variety of attempts to inhibit Met functions (35). The drug studies include (a) small molecules endowed with tyrosine kinase inhibitory activity; (b) short hairpin RNAs inhibiting Met transcription; (c) engineered antagonists, such as receptor "decoys," which titre out the ligand (HGF) and impair full-size receptor homodimerization; and (d) ligand competitors, which bind, but do not activate, the receptor. As far as the immunotherapy approach is concerned, antibodies against HGF, the Met receptor ligand, have been successfully tested (36–39). Antibodies against the Met receptor have also been investigated, but most of them displayed agonist activity (23, 40). One strategy to reach a pure antagonist has been to convert, by molecular engineering, a divalent agonistic antibody to a monovalent (one-arm) form (41). The anti-Met DN-30 antibody induces receptor shedding, reducing the number of receptors at the cell surface and generating a soluble 130-kDa ectodomain ("decoy"; ref. 21). The latter competes for ligand binding, forms heterodimeric complexes with the intact transmembrane receptors, and inhibits homodimerization and tyrosine kinase activation (25).

The data reported in this article show that the in vitro and in vivo efficacies of the DN-30 antibody vary in different cells types. In cells in which Met is expressed at physiologic levels and displays regulated enzymatic activity, antibody-mediated interference resulted in reduced cell growth only under stressful conditions (lack of anchorage) and slowed down tumor growth in nude mice. In contrast, in cells where Met activity is constitutive (as in the case of HGF autocrine loop), the antibody impairs viability in standard cell cultures and dramatically blocks tumor development in xenografts. Together, these results reinforce the notion that the therapeutic efficacy of tyrosine kinase inhibitors is directly correlated with the extent of steady-state activation of the targeted enzyme. Due to the nature of DN-30, which specifically recognizes the human form of the Met receptor (21), our experiments measured only the antibody effect on tumor cells. What remains to be investigated is if the targeting of Met expressed by the cells populating the tumor microenvironment will give rise to a stronger therapeutic efficacy.

In this article, we show that the "active" administration of the DN-30 antibody by gene transfer, as an alternative to the "passive" infusion of the protein, resulted in production of sustained levels of antibody followed by a detectable therapeutic response. We investigated two routes for gene transfer: systemic delivery by tail vein injection or local delivery by injection of the vector into the tumor. In both cases, the lentiviral vector was stably integrated into the host.

When administered systemically, DN-30RF antibody is produced in a variety of tissues due to the ubiquitous promoter. The concentration of the circulating antibody, in the range of 100 to 200 ng/mL, gives measurable therapeutic responses. The minimal concentration for therapeutic efficacy of a given antibody is unpredictable. In a previous report, high levels (1 mg/mL) of circulating anti–VEGF-R2 antibody were observed on gene transfer (12), but the requirement of such a high concentration was not investigated. On the other hand, an unnecessary high amount of the antibody produced in a chronic way could generate detrimental effects, such as the development of anti-idiotypic antibody responses in immunocompetent hosts (42), and/or the appearance of side effects due to the biological activity of the antibody in sites other than the tumor. This has been dramatically observed in the case of immunotherapy against the EGFR (43).

After direct transfer into the tumor mass due to the ubiquitous promoter, both stroma and tumor cells are targeted. The antibody synthesized by cells harboring the tumor is active both in cis and in trans. In the first instance, the antibody interferes with the biosynthesis and processing of the nascent Met receptor into the endoplasmic reticulum and Golgi apparatus, as shown by the intracellular accumulation of the uncleaved precursor. This event results in reduction of the mature receptor at the cell surface and, ultimately, in growth inhibition. On the other hand, because transduction of the tumor cells does not occur in the whole population, the unaffected cells are selected and ultimately should prevail in the population. However, the antibody secreted by the stroma and by the transduced cancer cells, working in trans, generates a "bystander" effect, slowing down the growth of the overall tumor (i.e., transduced and not transduced cells). Obviously, the cis effect, as well as the constant release in trans of the antibody into the tumor site, cannot be achieved by conventional passive immunotherapy and could be considered an advantage of gene transfer. In this regard, it is also evident that the side effects of massive doses of therapeutic antibodies, administered as a "bolus" by conventional routes, are minimized.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
P.M. Comoglio: Consultant, research agreement with the University of Turin, and patent #WO2007090807, Metheresis Translational Research SA. The other authors disclosed no potential conflicts of interest.


   Acknowledgments
 
Grant support: Associazione Italiana per la Ricerca sul Cancro grants (E. Vigna and P.M. Comoglio) and institutional grants from Fondazione CRT and Compagnia di San Paolo.

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.

We thank L. Trusolino for advice and discussion, and R. Albano and M. Galluzzo for invaluable technical help.


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

Current address for M. Mazzone: Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.

3 htttp://www.ebi.ac.uk/imgt Back

Received 5/ 5/08. Revised 7/ 4/08. Accepted 8/16/08.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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