Antibody-directed therapy for human hepatocellular carcinoma

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Antibody-Directed Therapy for Human Hepatocellular Carcinoma LEONHARD MOHR,* ANDY YEUNG,‡ COSTICA ALOMAN,§ DANE WITTRUP,‡ and JACK R. WANDS§ *Department of Medicine II, University Hospital Freiburg, Freiburg, Germany; ‡Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts; and §The Liver Research Center, Rhode Island Hospital and Brown Medical School, Providence, Rhode Island

The goals of our research are to develop high-affinity and high-stability antibodies and fragments thereof for targeting tumor-specific antigens in an attempt to develop new therapeutic agents for human hepatocellular carcinoma (HCC). Tumor-associated antigens are excellent targets for drug and gene delivery, and offer the advantage of high cellular specificity. We have explored the use of a monoclonal antibody (Mab) AF-20 raised against a human hepatoma cell line (FOCUS) as a model system. This antibody binds to a 180-kDa homodimeric cell surface glycoprotein with high affinity. The antigen is uniformly expressed in HCC-derived cell lines and human tumors, including those with distant metastasis. There is minimal expression in nontumor tissues, and none detectable in normal liver. Because the AF-20 antigen antibody interactions on the cell surface is rapidly internalized at 37°C, there is an opportunity to deliver cytotoxic payloads to tumor cells. In addition, high-affinity single-chain monoclonal antibody fragments (scFv) have been created using a novel yeast display system. Drug conjugates with AF-20 monoclonal antibodies have been prepared for gene targeting of HCC both in vitro and in vivo using preclinical animal model systems. These studies show that it is possible to generate high-affinity intact scFv antibody fragments that will allow specific tumor targeting of adenoviruses containing suicide genes, chemotherapeutic agents such as methotrexate, and cytotoxic peptides to produce antitumor effects. Therefore, specific antibody targeting of antitumor agents to HCC cells has the potential for therapeutic application in this devastating disease.

or most patients with advanced hepatocellular carcinoma (HCC), treatment options are limited, resulting in a dismal prognosis. Novel strategies such as gene therapy, in which nucleic acids encoding for specific therapeutic genes are used as anti-tumor agents, are therefore urgently required. For gene therapy, delivery systems (vectors) are used to introduce DNA constructs into living cells, in which the therapeutic gene products are expressed. For the treatment of malignant tumors, the reintroduction of tumor suppressor genes and the

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blockage of oncogene expression are approaches for correcting the underlying molecular mechanisms of malignant transformation. The expression of suicide genes renders tumor cells and surrounding bystander cells sensitive to chemotherapeutic agents. Other gene products (eg, cytokines or chemokines) enhance tumor immunogenicity. The expression of genes, which inhibit tumor angiogenesis, is an additional therapeutic approach targeting the tumor vasculature, which is essential for the support of rapidly growing tumors. However, for gene therapy to become an accepted alternative for the treatment of HCC, further improvement of antitumor efficiency and tumor cell specificity is essential. In this respect, targeting gene therapy vectors to HCC cells by the use of specific monoclonal antibodies may result both in enhanced uptake of vectors and reduced toxicity, because targeted vectors will not damage normal cells not expressing the antigen. Thus, despite remarkable advances in the development of nucleic acid– based reagents and, in particular, ribozymes, antisense DNA and RNA, siRNA, and DNA vaccine constructs, there is always the problem of cellspecific delivery of therapeutic compounds to the liver. Therefore, gene therapy of HCC, has been hampered by the lack of tumor cell-specific delivery systems. In this review, we will comment on ongoing research in our laboratories as it relates to a HCC-specific delivery system. We will not address all of the approaches that are presently in use to develop cell-specific delivery systems, but rather will focus on an antigen–antibody interaction that shows promise in delivering drugs, toxins, and adenoviral constructs expressing suicide genes specifically to HCC both in vitro and in vivo. Abbreviations used in this paper: AFP, ␣-fetoprotein; CAR, Coxsackie and adenovirus receptor; HA, hemagglutinin; HCC, hepatocellular carcinoma; Mab, monoclonal antibody; scFv, single-chain monoclonal antibody fragments. © 2004 by the American Gastroenterological Association 0016-5085/04/$30.00 doi:10.1053/j.gastro.2004.09.037

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Gene Transfer Into HCC Because cells take up nucleic acids very inefficiently, suitable vector systems are required to introduce nucleic acids. Viral and nonviral vector systems are being used for gene transfer techniques in vitro and in vivo. Because of their high level of efficiency in the transfer of nucleic acids, viral vectors have been used extensively for gene transfer in various animal models of HCC. In this respect, adenoviral vectors show several potential advantages, because they are highly hepatotropic and can transduce both dividing and nondividing cells. Furthermore, it is relatively easy to produce high titers of the vectors. However, immune responses against adenoviral proteins limit repeated administration in vivo, and intravenous delivery of high titers of adenoviral vectors may induce severe hepatic inflammation. Furthermore, because intravenously applied adenoviruses transduce predominantly normal hepatocytes and only to a much lesser extent HCC cells1,2 additional targeting strategies seem to be mandatory before their therapeutic use in humans. Nonviral vectors consist of a variety of mainly cationic lipids, which complex plasmid DNA and attach to the cell surface, thereby facilitating DNA uptake. Compared with viral vectors, the efficiency of gene transfer is relatively low, but they are less immunogenic than viral vectors and can carry large pieces of DNA.2 Vectors for gene therapy may be delivered into intrahepatic tumors by direct intratumoral injection or by intravascular administration. For gene therapy of multifocal HCC, the delivery of gene transfer vectors via the vascular route would be advantageous. In contrast to normal liver tissue, HCCs are mainly supplied by branches of the hepatic artery. Gene transfer into intrahepatic HCCs after intravenous administration has been analyzed in various murine HCC models. Intravenous application of adenoviral vectors resulted in only minimal transduction of tumor cells despite highly efficient gene transfer to normal hepatocytes,1,3 resulting in severe hepatic toxicity when suicide genes were expressed.4 Selective vector administration into the hepatic artery may enhance gene expression in intrahepatic tumors,5 which could further be improved by combination with vasoactive substances enhancing endothelial permeability. Alternatively, gene therapy vectors may be injected directly into the tumor tissue. This route of administration may be useful, because the technique of ultrasoundguided tumor injection is well established for the treatment of HCCs in clinical practice. Repeated intratumor injections of gene therapy vectors might enhance re-

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sponse rates for the treatment of intrahepatic tumors with suicide or immunomodulatory genes. However, transduction of a proportion of normal hepatocytes after direct adenovirus injection in murine HCCs was observed throughout the normal liver tissue,1 which may be caused by adenoviruses drained into the circulation during intratumoral injection. Severe hepatic toxicity was rarely observed after direct injection of HCCs, but hepatic failure was described after intratumoral injection of an adenovirus vector expressing HSV-tk followed by systemic ganciclovir administration in the woodchuck HCC model.6 Patients with HCC, many of whom suffer from cirrhosis and impaired liver function, would be at high risk if adenoviral vectors entering the circulation after intratumoral administration would transduce a proportion of hepatocytes. Both the direct intratumoral injection and the transarterial route of vector administration may be, therefore, feasible approaches for gene therapy of HCC, if the expression of therapeutic genes would be restricted to tumor cells by additional mechanisms of tumor cellspecific targeting. Targeting Gene Transfer Vectors to HCC The addition of tumor-specific ligands to gene therapy vectors such as monoclonal antibodies (Mabs) recognizing tumor-associated antigens may improve the selectivity of vector uptake by HCC cells. We have previously shown that AF-20 Mab raised against a human HCC cell line (FOCUS) binds to a 180-kd homodimeric cell surface glycoprotein expressed uniformly on all HCC cell lines examined and on other tumor cell lines as well (Figure 1). In primary HCC tumors, this antigen is highly expressed throughout the tumor and distant metastases, but there is minimal expression in nontumor tissue or adjacent normal liver.7 After binding of the AF-20 Mab to its antigen, the antigen–antibody complex is rapidly internalized at 37°C,8 and radiolabeled AF-20 Mab can be used for immunolocalization of subcutaneous HCC-derived tumors in nude mice.9 Therefore, this Mab may provide the opportunity to deliver gene therapy vectors specifically to HCC cells. Targeting of cationic liposomes with AF-20 Mab resulted in enhanced gene expression of reporter genes as compared with similar cationic liposomes targeted with a control Mab.10 However, there was still a low level of gene transfer by the control liposomes, which may have been mediated by the cationic charge of the large cationic liposomes used in these studies. To further improve targeting of nonviral vectors with AF-20 Mab, we constructed the novel vector system AF-20 cholesterylspermine by covalent coupling of AF-20 Mab to the

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Figure 1. Properties of AF-20 Mab. The AF-20 antigen is synthesized as a 90-kd monomer and is assembled on the cell surface as a 180-kd homodimer linked by disulfide bonds (upper panels). The antigen is highly expressed on HCC tumor cells (lower right panel) by immunohistochemical staining. The AF-20 antibody–antigen complex is internalized from the cell surface at 37°C, as shown (left lower panel).

cationic amphophile cholesteryl-spermine.11 This nonviral gene transfer system resulted in highly specific and efficient gene transfer to human HCC cells with only minimal gene expression in control cells not expressing the AF-20 antigen. Furthermore, a similar construct in which a control Mab (C7-57) was conjugated to cholesteryl-spermine showed virtually no detectable gene transfer to both AF-20 Ag–positive HCC cells and AF-20 Ag–negative control cells. Therefore, attachment of DNA-binding molecules such as cholesteryl-spermine directly to AF-20 Mab, rather than targeting highly cationic liposomes with Mabs, generated a specific and effective nonviral vector for gene delivery to HCC. These findings showed for the first time the high level of specificity and efficiency achieved by targeting a gene transfer vector to HCC cells by AF-20 Mab. However, nonviral vector systems are currently still limited by their relatively low efficiency of gene transfer as compared with viral vectors. Therefore, we analyzed the potential of the AF-20 Mab to target recombinant adenoviral vectors using a bifunctional Fab-antibody conjugate that was generated by chemical cross-linking a Fab fragment derived from the antihexon Mab 2Hx-2 recognizing the adenoviral hexon protein to the AF-20 Mab. We examined the effects of the 2Hx-2–AF20 conjugate on directing adenovirus-mediated gene delivery to HCC cells under conditions in which the natural Coxsackie and adenovirus receptor (CAR) on the cell surface was preoccupied by excess of fiber knob protein.12 After blockage of CAR with an excess of fiber knob protein adenoviral vectors preincubated with the 2Hx-2–AF20 conjugate

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transduced the majority of cells. This effect could be inhibited by an excess of free AF-20 Mab but not by control Mab. This study, therefore, provided evidence that targeting of adenovirus vectors by AF-20 Mab could result in specific adenovirus uptake by HCC cells expressing the AF-20 antigen resulting in high levels of gene expression. Because under in vivo conditions complete blockade of the CAR is not possible, additional mechanisms must be involved to result in the generation of adenoviral vectors, which are only able to transduce AF-20 antigen-expressing HCC cells. For example, genetic alterations of the adenoviral fiber protein to replace the natural binding site of the fiber to the CAR with the specific binding domain of AF-20 Mab might result in highly efficient and tumor-selective adenoviral vectors for gene therapy of HCC. These vectors should no longer be capable of infecting cells via the natural receptor, therefore limiting the potential side effects of systemic adenoviral application.

Conjugation of AF-20 Mab to Methotrexate HCC is often quite resistant to chemotherapeutic agents because of high expression of the multidrugresistant genes that pump the drugs quickly out of the tumor cells. To determine whether a chemotherapeutic agent would be more effective after internalization, we performed experiments with FOCUS HCC cells with a methotrexate AF-20 antibody conjugate. Preliminary studies showed that methotrexate in concentrations that would be in the high micromolar range, and therefore lethal in vivo, had no effect on the viability of FOCUS HCC cells in vitro. However, as shown in Figure 2, a conjugation of methotrexate directly to the AF-20 monoclonal antibody showed that increasing concentrations in the low micromolar range had a substantial effect on cell viability, particularly at day 6 after administration. Thus, by presenting a totally ineffective chemotherapeutic agent in a different way, these highly resistant HCC cells seemed susceptible to the cytotoxic action of methotrexate when the antigen–antibody complex was internalized. Such studies offer hope that other chemotherapeutic agents, with perhaps a greater therapeutic index, might be delivered by a similar fashion through the intact or antibody fragments of AF-20 Mab. Development of Single-Chain Antibody Fragments of AF-20 Mab It became advantageous to determine whether high-affinity single-chain antibody fragments (scFv) of AF-20 could be generated for potential therapeutic purposes. The rationale for this approach was that the small-

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Figure 2. Effect of methotrexate on HCC viability when conjugated to AF-20 Mab. Note the concentration effect and the delayed response of tumor cells to the conjugate. Methotrexate alone even at high concentrations had no effect on the visibility of tumor cells.

sized scFvAF-20 (⬃25 kd) would allow for optimal tissue penetration. The rapid renal clearance of such fragments would also potentially reduce toxicity. Furthermore, this reagent might be useful for imaging HCC as well. Finally, another major advantage may be to create potential therapeutic molecules by generating fusion proteins with bacterial or plant toxins via recombinant DNA technology, or to synthesize bivalent linker systems to deliver adenoviral vectors containing tumor suicide genes as well as direct conjugation of cytotoxic agents for cell-specific delivery. Protein Engineering by Yeast Surface Display Protein engineering has been used extensively to modify the functions and properties of antibodies.13 Examples of protein engineering include reducing the size of an antibody from IgG format to scFv, modifying the valency of an antibody, changing the affinity of the Fc receptor, and more frequently, improving the affinity and stability of an antibody. Directed evolution involves displaying a diverse array of antibody mutants on a genetic package14 and then selecting favorable mutants from the pool. Such packages, which link the phenotype to its genotype, include ribosome,15–17 bacteriophage,18 –20 bacteria,21,22 and yeast.23,24 We have developed a yeast surface display approach for protein-directed evolution. One of the advantages of using yeast for displaying protein is that as a eukaryote, yeast contains similar protein-processing machinery to that of a mammalian cell. Thus, yeast is more likely than prokaryotes to correctly express and display mammalian surface or secreted proteins, in

this case antibodies. Yeast surface display involves presenting the antibody of interest through fusion with a 2-unit cell wall glycoprotein called a-agglutinin, whose original function is to mediate cell– cell adhesion between haploid cells23 (Figure 3). A-agglutinin consists of 2 distinct domains, Aga1p and Aga2p, connected by 2 disulfide bonds. Aga1p subunit anchors the assembly to the cell wall via a ␤-glucan covalent linkage. The antibody is fused to the C-terminus of Aga2p, where the native A-agglutinin binding activity localizes. The antibody in the display construct is flanked by 2 epitope tags, with hemagglutinin (HA) and c-myc on the N- and C-terminus of the antibody, respectively. Expression of the fulllength antibody on the cell surface can be simply confirmed by detecting the presence of a c-myc tag on the cell surface. Additionally, because of the intrinsic quality control of the endoplasmic reticulum, the presence of a c-myc tag on the cell surface strongly implies that the surface antibody is folded properly.25 Antibody engineering using yeast surface display involves mutagenizing the antibody gene, displaying the library of mutant antibodies on the yeast surface, and subsequently screening for the desired mutants. As shown in Figure 3, multiple methods have been used to mutagenize an antibody gene: random mutagenesis,26 –29 hot-spot site-directed mutagenesis,30,31 targeted mutagenesis of complementary determined region residues,32–34 DNA shuffling,29,35–37 complementary determined region shuffling,38 – 41 and chain shuffling.38,39

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Figure 3. Schematic of yeast surface display. The antibody of interest (scFv) is displayed on the yeast surface through fusion to Aga1–Aga2 protein. Two epitope tags, HA and c-myc, were fused to the N-terminus and C-terminus of the antibody, respectively. The displayed antibody was able to bind ligands on the yeast surface, and the presence of epitope tags can be detected using commercially available antibodies. The mutagenesis approach to generate high-affinity scFv is shown (right).

Display of AF-20 scFv on the Yeast Surface The genes of the mouse AF-20 IgG variable regions were cloned from the AF-20 secreting hybridoma cells. The AF-20 scFv was constructed in the configuration of VH-linker-VL, with four repeats of G4S (glycineglycine-glycine-glycine-serine) being the middle linker. The AF-20 scFv was ligated into the display plasmid, which contained both the HA and the c-myc tags, and subsequently, the plasmid was transformed into yeast. The transformed yeast cells were then induced to display the AF-20 scFv on the cell surface. After confirming that the AF-20 scFv was well expressed on yeast surfaces, binding of these AF-20 scFv-displaying yeast cells to the FOCUS cells was determined. These experiments indicated that the AF-20 scFv displaying on the yeast surface was functional and responsible for the binding of the yeast cells to the FOCUS cells.

Secretion of Soluble AF-20 scFv in Yeast The AF-20 scFv gene was then ligated into a yeast secretion plasmid. Similar to the display plasmid, 2 epitope tags flanking the scFv were constructed along with the scFv, with a FLAG tag at the N-terminus and a 6xHIS tag at the C-terminus of the scFv. The epitope tags were later used for both scFv detection and purification. AF-20 scFv was expressed in Saccharomyces cerevisiae, using strain YVH10. AF-20 scFv was purified from the yeast supernatant using affinity chromatography. One of the potentially useful properties of the AF-20 antigen is that once AF-20 IgG binds, the antigen– antibody complexes are internalized readily into tumor cells.8 AF-20 scFv was shown to internalize into the FOCUS cell in a similar manner as AF-20 IgG. Incubating at 4°C, the AF-20 scFv, which was indicated by the green fluorescence, primarily resided on the outer member of the cells (Figure 4). On the other hand, when being incubated at 37°C, AF-20 scFv mainly clustered in

Figure 4. Internalization of the AF-20 scFv into the FOCUS tumor cells.

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the middle of the cells, indicating the internalization of Af-20 scFv into the cells. In conclusion, the AF-20 scFv was constructed, and its function in the format of both soluble scFv and yeast-displaying scFv was confirmed. Because of its smaller size, AF-20 scFv should permit better tumor penetration than AF-20 IgG. Thus, the AF-20 scFv may have potential application in tumor targeting. In addition, because the gene of AF-20 IgG is available, protein engineering techniques can be applied to construct antibodies with different sizes and avidity,42,43 and to create immunotoxins by fusion with different toxins, enzymes, and viruses for targeted therapy.44 – 47

Transcriptional Control of Therapeutic Genes Tumor specificity of gene therapy may also be provided by the use of so-called tumor-specific promoters. Because ␣-fetoprotein (AFP) is highly expressed in the majority of HCCs, transcriptional control of therapeutic genes by the AFP-promoter results in tumorspecific gene expression.48 As vitro and in vivo gene therapy of experimental HCCs using various vector systems and therapeutic genes under the control of the human AFP-promoter showed tumor-specific transgene expression in AFP-positive tumor cells and showed therapeutic efficiency.49,50 However, in comparison with strong viral promoters such as the cytomegalovirus promoter, the transcriptional activity of the AFP promoter is relatively low, thus limiting the potential therapeutic efficiency. This might be improved by the use of a mutated AFP promoter with stronger transcriptional activity51 or the construction of hybrid promoters containing regulatory elements of the AFP-promoter fused to strong promoter elements. These approaches may combine the tumor specificity of the AFP-promoter with enhanced levels of gene expression. As an alternative approach, 2 separate adenoviral vectors were constructed, 1 carrying the gene encoding for Cre recombinase under the control of the AFP promoter and a vector containing a potent gene expression unit activated by Cre.52

Conclusions In the future, the combination of targeting gene vectors to HCC cells by Mabs that bind to tumorassociated antigens such as AF-20 with transcriptional control of gene expression may result in the development of vector systems combining the high levels of efficiency and safety needed for human gene therapy of HCC.

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Address requests for reprints to: Jack R. Wands, MD, Brown University, The Liver Research Center, 55 Claverick Street, 4th Floor, Providence, Rhode Island 02903. e-mail: [email protected]; fax: (401) 444-2939. Supported by grants AA-02666, AA-02169, AA-11431, AA-12908, and CA-35711 from the National Institutes of Health, and BPEC from the National Science Foundation.