targeting of cancer gene therapy with antibodies or their genes against...

Gene Therapy and Molecular Biology Vol 9, page 107

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Gene Ther Mol Biol Vol 9, 107-112, 2005

Targeting of cancer gene therapy with antibodies ortheir genes against tumor-associated antigensReview Article

Masahide Kuroki1,2,*, Hirotomo Shibaguchi1, 2, Motomu Kuroki1, 2, TetsushiKinugasa1, Ken Hachimine1, Sotaro Enatsu1, Shin-ichi Maekawa1, Jian Huang1

and Jun Zhao2

1Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka, Japan2Fukuoka University Molecular Oncology Center, Fukuoka, Japan

__________________________________________________________________________________*Correspondence: Prof. Masahide Kuroki, Department of Biochemistry, Fukuoka University School of Medicine, 7-45-1 Jonan-ku,Fukuoka, 814-0180, Japan. Phone: + 81-92-801-1011 Ext. 3240; Fax: + 81-92-801-3600; E-mail: [email protected] words: Cancer gene therapy, viral vector, tumor-targeting, tumor-associated antigen

Abbreviations: adenoviral vectors, (adenovectors); carcinoembryonic antigen, (CEA); coxsackie-adenovirus rececptor, (CAR);epidermal growth factor receptor, (EGFR); epithelial cell adhesion molecule, (EpCAM); high-molecular weight melanoma-associatedantigen, (HMWMAA); inducible nitric oxide synthase, (iNOS); matrix metalloprotease, (MMP); murine leukemia virus, (MLV); nitricoxide, (NO); retroviral vectors, (retrovectors); arginine-glycine-aspartic acid, (RGD), single-chain diabody, (scDb); single-chain variablefragmented antibodies, (scFvs); severe combined immunodeficiency, (SCID); surface domain, (SU); tumor-associated antigens, (TAAs)

Received: 10 May 2005; Revised: 26 May 2005Accepted: 27 May 2005; electronically published: June 2005

SummaryGene therapy is expected to play a major role in future cancer treatment. Actually various therapeutic genes haveshown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient andtargeted gene transfer vectors. This overview summarizes the current use of anti-tumor-associated antigen (TAA)antibodies in cancer gene therapy. Current data suggest that antibodies or their genes against TAAs can be usedfor targeting viral vectors for cancer gene therapy.

I. IntroductionTumor-associated antigens (TAAs) are molecules

which occur in or on tumor cells and are either notdemonstrable or are significantly less abundant in normaltissues (Groen, 1987). There are many TAAs againstwhich monoclonal antibodies are now utilized forimmunotherapy of cancer (Kuroki et al, 2002).

The strategies of gene therapy for cancer can belargely categorized into either direct or indirect genetherapy (Kuroki et al, 2002). Direct gene therapy forcancer involves the insertion of a gene to tumor cells forthe direct killing or suppression of abnormal growth bygene products or their secondary products. The genes usedfor this strategy are suicide genes, functioning tumorsuppressor genes, anti-sense genes against knownoncogenes, or antiangiogenic genes (Kuroki et al, 2000).Indirect gene therapy involves the insertion of a gene thatmodifies or stimulates immunocytes to be more effectiveagainst tumor cells. Recent knowledge that T cell-recognized peptide epitopes are presented by HLA

molecules, and that the induction of immune responses isdependent on co-stimuli, has led to the development ofmore rational strategies (Roitt et al, 1998). The genes usedfor this strategy are cytokine genes, TAA genes,costimulatory molecule genes, or HLA class I genes,which are often inserted into tumor cells as well asimmunocytes (Kuroki et al, 2000).

Viral vectors have been extensively used for cancergene therapy because of their relatively high efficacy ofgene transfer (El-Aneed, 2004). Retroviral vectors(retrovectors) and adenoviral vectors (adenovectors) areamong the most frequently chosen vector systems (Hunt etal, 2002). However, these vectors still have severalspecific problems regarding their pathogenicity(immunogenicity), their gene transfer efficacy, thestability and level of transgene expression, a limitation interms of the size of the inserted gene, and a limitation inspecifically targeting tumor cells, etc (El-Aneed, 2004;Hunt et al, 2002). Among them, the biggest problem isprobably the lack of tumor specificity of viral vectors used

Kuroki et al: Targeting of cancer gene therapy with antibody

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for gene transfer (Kuroki et al, 2000; Dachs et al, 1997). Inthis regard, genetically engineered single-chain variablefragmented antibodies (scFvs) or their genes to TAAshave recently been used for increasing tumor specificity ofviral vectors (Kuroki et al, 2003). This article provides abrief overview of the tumor targeting strategies ofretrovectors and adenovectors for cancer gene therapy byusing antibodies or their genes against TAAs.

II. Tumor targeting of retrovectorsRetrovectors remain an attractive option for clinical

gene delivery because integration of the vector genomeallows stable gene expression in the infected cell and itsprogeny (Hunt et al, 2002). The retrovectors used for mostclinical trials of gene therapy originate from a murineleukemia virus (MLV). Because viral coding regions aredeleted from the vector, viral proteins are not expressed inthe infected cells, avoiding stimulation of an inappropriateimmune response. Also, the host range of retrovectors isusually determined by the surface domain of the envelopeglycoprotein, which covers the viral capsid and binds to acell surface receptor. As retrovectors transduce onlydividing cells, they have been used to deliver therapeuticgenes to tumors in vivo, with surrounding normal tissuebeing largely refractory to transduction (Chowdhury et al,2004).

Recently, however, a patient with X-linked severecombined immunodeficiency (SCID), who received genetherapy using retrovirally transduced bone marrow cells,developed T cell leukemia caused by retrovectorintegration leading to insertional mutagenesis (Kohn et al,

2003). This highlights the need to target retroviral genedelivery specifically to tumors, if vectors or packagingcells are to be injected in vivo for cancer gene therapy.Most tumors induced by retrovectors involvehematopoietic cells transformed by insertionalmutagenesis. Thus, particular care is needed to avoidtransduction and potential transformation of these cells. Toestablish gene therapy as a feasible treatment of cancer,more emphasis is required on developing optimal genedelivery systems with a greater tumor tissue specificity.One of the efforts of tissue-specific targeting is based onattempts to engineer the normal retroviral envelope protein(Kuroki et al, 2003). Recent advances in the field ofgenetic engineering have led to development of a conceptfor target cell specificity by modifying the tropism of thenormal envelope, retroviral receptor-binding domain withan scFv antibody or a ligand that recognizes a TAA(Russell et al, 1993; Somia et al, 1995) or a specific cellsurface receptor (Kasahara et al, 1994). We focus here onthe strategy using anti-TAA scFv antibody genes forincreasing the tumor specificity of retrovectors. The majorantibody-recognized TAAs currently used as the targetsare carcinoembryonic antigen (CEA) (Chowdhury et al,2004; Konishi et al, 1998; Khare et al, 2001 and 2002) andhigh-molecular weight melanoma-associated antigen(HMWMAA) (Martin et al, 2002 and 2003).

In a recent study, we developed a novel bifunctionalMLV-based recombinant retrovector that displays achimeric envelope protein containing an scFv antibody toCEA and carries a suicide, inducible nitric oxide synthase(iNOS) gene in the genome (Figure 1) (Khare et al, 2001).

Figure 1. Specific targeting of retrovector carrying a suicide gene (the iNOS gene) to CEA-expressing tumor cells with a chimericenvelope protein containing an anti-CEA scFv antibody.


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The MLV-based retrovector used here is ecotropic, andoriginally infects only murine cells. CEA is expressed by anumber of tumors of epithelial origin, most notablycolorectal carcinoma. The iNOS gene product yields nitricoxide (NO), which directly induces autocytotoxicity andcytolysis of by-stander cells. An anti-CEA scFv antibodygene derived from the mouse hybridoma F11-39 wasgenetically inserted between the sixth and seventh aminoacid of the ecotropic envelope. The resultant bifunctionalretrovector, GPEscFv-env/iNOS, showed a specificdelivery of the iNOS gene to human CEA-expressingtumor cells (MKN-45 gastric carcinoma cells) and directlyand efficiently killed the infected CEA-expressing tumorcells by the induction of apoptosis without any additionaldrugs. The targeted vector was able to produce tumorsuppression in a SCID mouse xenograft model with a 70%reduction in tumor weight (Khare et al, 2002).

In a previous study, Martin et al, (1999) describedretrovectors targeted to HMWMAA, which is expressed inmore than 90% of human melanomas. The chimericenvelope surface domain (SU) contained an scFvrecognizing HMWMAA followed by a proline linker anda matrix metalloprotease (MMP) cleavage site. Theproline linker prevented binding of the chimeric SU to itsPit-2 receptor. However, when these vectors bound toHMWMAA, they were then cleaved by cell surfaceMMPs, revealing the amphotropic 4070A (MLV-A)backbone that mediated transduction via the Pit-2 receptor.The targeted vector (LMH2/ProMMP) infectedHMWMAA-positive cells when injected into a nudemouse xenograft model (Martin et al, 2002). Recently,they also reported a new retrovector targeted to CEA usingthe MFE23 scFv antibody against CEA (Chowdhury et al,2004). The envelope MFE23/ProMMP was constructed bylinking MFE23 to the amino terminus of MLV-A SUusing a proline-rich spacer followed by a cleavage site forMMPs. Retrovectors incorporated the MFE23/ProMMPenvelope as efficiently as the unmodified MLV-Aenvelope, in contrast to the relatively poor incorporation ofmany chimeric envelopes (Martin et al, 1999), and couldspecifically transduce CEA-positive cells or tumors withhigh efficiency (Chowdhury et al, 2004).

Taken together, these results suggest that a tumor-specific therapeutic effect could be achieved by using thescFv-chimeric retroviral envelope protein model to deliversuicide genes in vivo and this approach could also beapplied to other TAAs expressing on cancer cells.

III. Tumor targeting of adenovectorsAdenovectors are also promising reagents for clinical

gene delivery because of their superior in vivo genetransfer efficiency on a wide spectrum of cell types andtheir low risk of mutagenesis. Adenovectors, likeadenoviruses, do not have an envelope and their majorcapsid components are hexon, penton (or penton base),and knobbed fiber (fiber and fiber knob). Adenoviralinfection is mediated by binding of the knob region,located at the carboxy terminus of the fiber, to itscorresponding receptor, which is the coxsackie-adenovirusrececptor (CAR) (Bauerschmitz et al, 2002). Binding isfollowed by interaction between cellular integrins and an

arginine-glycine-aspartic acid (RGD) motif located at thepenton base. Infection is not dependent on cell cyclephase; therefore, both cycling and non-dividing cells areinfected, and adenoviral DNA is not integrated into thehost genome. Nevertheless, the limited duration of geneexpression may render adenovectors less desirable for thegene therapy of hereditary diseases where long-termexpression is needed, but it is adequate for cancer genetherapy approaches where the primary purpose is to killthe target cells (Bauerschmitz et al, 2002).

However, adenovectors should also possess criticalproperties required for the development of efficient andtargeted gene transfer vectors for the successful clinicaltranslation of cancer gene therapy (Nettelbeck et al, 2004).These include a highly evolved gene transfer mechanism,the stability of virus particles and the ease of virusproduction at high titers. The necessity of suchimprovement is predicated by the observation that CAR iswidely expressed on normal tissues resulting innonspecific susceptibility to adenoviral infection. Inaddition, reduced or absent expression of CAR has beenreported for various tumor types, indicating resistance toadenoviral infection by tumor cells in situ. Theseconsiderations of adenoviral biology are paralleled by theobservation of limited efficacy and vector-related toxicityin preclinical and clinical adenoviral gene therapy studies.Therefore, the development of tropism-modified, tumor-targeted adenovectors is a key endeavor in current genetherapy approach. To this end, the native tropism ofadenoviruses needs to be ablated and a new, tumor-specific tropism needs to be engineered into viral particles(Nettelbeck et al, 2004). The trial has been performed inseveral ways: a) fusion protein of soluble CAR (sCAR)and targeting-receptor ligand (Dmitriev et al, 2000), b)fusion protein of anti-fiber knob antibody and targeting-receptor ligand (Watkins et al, 1997), c) bispecificantibody to fiber knob and TAA (or cell receptor) (Haismaet al, 1999; Nettelbeck et al, 2001), d) fusion protein ofsCAR and scFv antibody to TAA (Kashentseva et al,2002), and e) immunoglobulin-binding domain insertedfiber-knob protein (Volpers et al, 2003), etc. Here wefocus on the last three strategies that have been utilizinganti-TAA (or anti-cell receptor antibodies) or their genesfor increasing tumor specificity of adenovectors. Theantibody-recognized TAAs (or cell receptors) used as thetargets are HMWMAA (Nettelbeck et al, 2004), epithelialcell adhesion molecule (EpCAM) (Haisma et al, 1999;Heideman et al, 2001), epidermal growth factor receptor(EGFR) (Volpers et al, 2003; Haisma et al, 2000), HER-2(Her-2/neu or c-erbB-2) (Kashentseva et al, 2002), CD-40(Korokhov et al, 2003), CD-70 (Israel et al, 2001), andCD-105 (Nettelbeck et al, 2001), etc.

In previous studies, Haisma et al (1999) andHeideman et al (2001) demonstrated tumor-specific genetransfer via an adenovector targeted to the pan-carcinomaantigen EpCAM. An anti-fiber knob Fab’ antibodyconjugated to an anti-EpCAM Fab’ antibody was createdthat targets the adenovirus to the EpCAM antigen presenton tumor cells. The EpCAM antigen was chosen as thetarget because this antigen is highly expressed on a varietyof adenocarcinomas of different origin such as breast,


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ovary, colon and lung. In these studies, the EpCAM-targeted adenovector was shown to specifically infectcancer cell lines of different origin expressing EpCAM.Gene transfer was blocked by excess anti-EpCAMantibody and dramatically reduced in EpCAM negativecell lines, thus showing the specificity of the EpCAM-targeted adenovector. Importantly, infection with targetedadenovector was independent of CAR, which is the naturalreceptor for adenovirus binding, since blocking of CARwith recombinant fiber knob did not affect infection withtargeted adenovirus. Apart from the cancer cell lines, theefficacy of targeted viral infection was studied in freshlyisolated primary human colon cancer cells. As coloncancer predominantly metastasizes to liver, and adenovirushas a high tropism for hepatocytes, they determined if theEpCAM-targeted adenovector showed reduced infectivityof human liver cells. The bispecific antibody couldsuccessfully mediate gene transfer to primary human coloncancer cells, whereas it almost completely abolishedinfection of liver cells. Thus, chemically preparedbispecific antibodies are versatile tools, but the productionand purification of the conjugates poses problems ofheterogeneity and is time consuming. More recently,Nettelbeck et al, (2004) reported retargeting of adenoviralinfection to melanoma by combining genetic ablation ofnative tropism with a recombinant bispecific single-chaindiabody (scDb) adapter that binds to fiber knob andHMWMAA. This strategy combines genetic ablation ofnative adenoviral tropism with redirected viral binding tomelanoma cells via a bispecific adapter molecule, abacterially expressed single-chain diabody, scDb MelAd,that binds to both the adenoviral fiber knob and toHMWMAA. The results showed specific and strongbinding of the bispecific adapter scDb MelAd tomelanoma cells. In adenoviral infection experiments, they

demonstrated a) substantially reduced infectivity of capsidmutant adenoviruses, b) restored, CAR-independent andHMWMAA-mediated infectivity of these mutant virusesby scDb MelAd specifically in melanoma cells, and c)higher levels of transgene expression in melanoma cells byfiber mutant virus complexed with scDb MelAd, relativeto a vector with wild-type fibers. Hence, the HMWMAA-targeted adenovector lacking native tropism exhibits bothenhanced specificity and augmented infectivity of genetransfer to melanoma cells, suggesting that it is feasible touse this vector to improve gene therapy for malignantmelanoma.

On the other hand, Kashentseva et al, (2002) haveproposed the use of the sCAR ectodomain fused with aligand to block CAR-dependent native tropism and tosimultaneously achieve infection through a novel receptoroverexpressed in target tumor cells. To conferadenovector-targeting capability on cancer cellsexpressing the HER-2 oncogene, they engineered abispecific adapter protein, sCARfC6.5, that consisted ofsCAR, phage T4 fibritin polypeptide, and the C6.5 scFvantibody against HER-2 oncoprotein. They demonstratedthat the sCARfC6.5 protein binds to cellular HER-2oncoprotein and mediates efficient adenovector targetingvia a CAR-independent pathway. Targeted adenovector,complexed with sCARfC6.5 adapter protein, providedsignificant enhancement of gene transfer compared withadenovector alone and untargeted adenovector complexedwith sCAR control protein. Thus, the use of recombinanttrimeric sCAR-scFv adapter proteins may augmentadenovector potency for targeting cancer cell types.

In addition, Volpers et al, (2003) developed a novelmodified adenovector that displays a synthetic IgG-binding domain in the capsid and carries a reporter lacZgene (Figure 2).

Figure 2. Specific targeting of adenovector carrying a reporter gene (the lacZ gene) to EGFR-expressing tumor cells with a chimericfiber-knob protein containing an immunoglobulin-binding domain (Z33).


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A synthetic 33-amino-acid IgG-binding domain (Z33),derived from staphylococcal protein A, was inserted intothe adenovirus fiber protein. The fiber retained the abilityto assemble into trimers, bound IgG with high affinity, andwas incorporated into vector particles. The transductionefficiency of the Z33-modified adenoector in humanEGFR-expressing tumor cells (A431 epidermoidcarcinoma cells) was strongly and dose-dependentlyenhanced by combination with an EGFR-specificmonoclonal antibody. The antibody-mediated increase incellular transduction was abolished in the presence ofcompeting protein A. More recently, Henning et al, (2005)constructed two kinds of adenovirus 5 vectors carryingknobless fibers with antibody-binding domains fromStaphylococcal protein A or from Streptococcal protein G,respectively. Both adenovectors bound their specific Igisotypes with the expected affinity. They transducedhuman carcinoma cells independently of the CARpathway, via cell surface receptors targeted by specificmonoclonal antibodies, that is, EGFR expressed on A549,HT29 and SW1116, HER-2/neu on SK-OV-3 and SK-BR-3, CA242 antigen on HT29 and SW1116, and prostate-specific membrane antigen on HEK-293 cells,respectively. Thus, the antibody-binding adenovector alsoholds promise for directed gene transfer to a wide varietyof cell types by simply changing the target-specificantibody.

IV. ConclusionCancer gene therapy is one of the main applications

of gene therapy. In the past decade, both viral and non-viral vectors have been developed and evaluated fordelivering therapeutic genes that can eliminate tumor cells.In the last few years, numerous modifications to thedelivery systems have been made to optimize thetransfection efficacy. Among them, the strategies to targetviral vectors to tumor tissues by modifying the tropismswith antibodies or their genes against TAAs are verypromising from a practical point of view.

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First row from left to right: Jian Huang, Ken Hachimine, Hirotomo Shibaguchi, Masahide Kuroki

Second row from left to right: Motomu Kuroki, Shin-ichi Maekawa, Jun Zhao, Tetsushi Kinugasa, Soutaro Enatsu

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