Peptide targeting of adenoviral vectors to augment tumor gene transfer

Adenovirus serotype 5 remains one of the most promising vectors for delivering genetic material to cancer cells for imaging or therapy, but optimization of these agents to selectively promote tumor cell infection is needed to further their clinical development. Peptide sequences that bind to specific cell surface receptors have been inserted into adenoviral capsid proteins to improve tumor targeting, often in the background of mutations designed to ablate normal ligand:receptor interactions and thereby reduce off target effects and toxicities in non-target tissues. Different tumor types also express highly variable complements of cell surface receptors, so a customized targeting strategy using a particular peptide in the context of specific adenoviral mutations may be needed to achieve optimal efficacy. To further investigate peptide targeting strategies in adenoviral vectors, we used a set of peptide motifs originally isolated using phage display technology that evince tumor specificity in vivo. To demonstrate their abilities as targeting motifs, we genetically incorporated these peptides into a surface loop of the fiber capsid protein to construct targeted adenovirus vectors. We then systematically evaluated the ability of these peptide targeted vectors to infect several tumor cell types, both in vitro and in vivo, in a variety of mutational backgrounds designed to reduce CAR and/or HSG-mediated binding. Results from this study support previous observations that peptide insertions in the HI loop of the fiber knob domain are generally ineffective when used in combination with HSG detargeting mutations. The evidence also suggests that this strategy can attenuate other fiber knob interactions, such as CAR-mediated binding, and reduce overall viral infectivity. The insertion of peptides into fiber proved more effective for targeting tumor cell types expressing low levels of CAR receptor, as this strategy can partially compensate for the very low infectivity of wild-type adenovirus in those cells. Nevertheless, the incorporation of relatively low affinity peptide ligands into the fiber knob, while effective in vitro, has only minimal targeting efficacy in vivo and highlights the importance of high affinity ligand:receptor interactions to achieve tumor targeting.     

Adenoviral gene therapy for renal cancer requires retargeting to alternative cellular receptors

Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies in humans. Therefore, the identification of new agents with better antitumor activity merits a high priority in the treatment of advanced RCC. In this regard, gene therapy with adenoviral (Ad) vectors is a promising new modality for cancer. However, a primary limiting factor for the use of Ad vectors for cancer gene therapy is their critical dependence on cellular expression of the primary Ad receptor, the coxsackie and adenovirus receptor (CAR), known to be down-regulated in many cancer types. Following the identification of CAR deficiency in RCC lines, we have found abundant membrane expression of alpha(v)beta 3 and alpha(v)beta 5 integrins and of the putative receptor to Ad serotype 3 (Ad3). As an alternative gene therapy approach for RCC that would circumvent CAR deficiency, we employed retargeting of replication-incompetent Ad vectors and replication-competent Ad viruses to alpha(v)beta 3 and alpha(v)beta 5 integrins and to the putative Ad3 receptor. These strategies to genetically alter Ad tropism were based on either the insertion of a cysteine-aspartate-cysteine-arginine-glycine-aspartate-cysteine-phenylalanine-cysteine (RGD) motif into the HI loop of the Ad fiber knob domain or on generation of a chimeric Ad fiber composed of adenovirus serotype 5 shaft/Ad3 knob. Both strategies proved highly efficient to circumvent CAR deficiency and enhance gene delivery into RCC cells. Furthermore, in the context of replication-competent Ad, tropism alteration resulted in distinct capacity of the retargeted viruses to infect, replicate, and lyse RCC models in vitro and in vivo. The retargeting strategies were particularly beneficial in the context of replication-competent Ad. These findings underscore the importance of CAR-independent cellular entry mechanisms in RCC and are highly consequential for the development of viral antitumor agents for RCC and other CAR-negative tumors.     

Gene delivery into malignant glioma by infectivity-enhanced adenovirus: in vivo versus in vitro models

Adenoviral (Ad) vectors demonstrate several attributes of potential utility for glioma gene therapy. Although Ad infection is limited in vitro by low expression levels of the coxsackie-adenoviral receptor (CAR), in vivo studies have shown the efficacy of Ad vectors as gene delivery vectors. To evaluate the in vivo utility of CAR-independent, infectivity-enhanced Ad vectors, we employed genetically modified Ad vectors in several experimental models of human gliomas. We used three capsid-modified Ad vectors: (1) a chimeric Ad vector with a human Ad backbone and a fiber knob of a canine Ad, (2) an Ad vector with a polylysine motif incorporated into the fiber gene, and (3) a double-modified Ad vector incorporating both an RGD4C peptide and the polylysine motif. These three modified Ad vectors target, respectively, the putative membrane receptor(s) of the canine Ad vector, heparan sulfate proteoglycans (HSPGs), and both integrins and HSPGs. Our in vitro studies indicated that these retargeting strategies all enhanced CAR-independent infectivity in both established and primary low-passage glioma cells. Enhancement of in vitro gene delivery by the capsid-modified vectors correlated inversely with the levels of cellular CAR expression. However, in vivo in orthotopic human glioma xenografts, the unmodified Ad vector was not inferior relative to the capsid-modified Ad vector. Although genetic strategies to circumvent CAR deficiency in glioma cells could reproducibly expand the cellular entry mechanisms of Ad vectors in cultured and primary glioma cells, these approaches were insufficient to confer in vivo significant infectivity enhancement over unmodified Ad vectors. Other factors, probably the extracellular matrix, stromal cells, and the three-dimensional tumor architecture, clearly play important roles in vivo and interfere with Ad-based gene delivery into glioma tumors.     

载体重靶向与肿瘤基因治疗

病毒载体的靶向性问题是肿瘤基因治疗中的重要研究热点,靶向病毒载体是提高肿瘤基因治疗安全性和有效性的重要途径。载体的靶向策略很多．载体重靶向就是其中重要的一种。本文就近年来在肿瘤基因治疗领域中关于载体重靶向问题的研究进展进行综述,并试图对载体重靶向的途径和研究策略进行归纳和总结。     

Fibroblast growth factor 2 retargeted adenovirus has redirected cellular tropism: evidence for reduced toxicity and enhanced antitumor activity in mice.

Adenovirus (Ad) have been used as vectors to deliver genes to a wide variety of tissues. Despite achieving high expression levels in vivo, Ad vectors display normal tissue toxicity, transient expression, and antivector immune responses that limit therapeutic potential. To circumvent these problems, several retargeting strategies to abrogate native tropism and redirect Ad uptake through defined receptors have been attempted. Despite success in cell culture, in vivo results have generally not shown sufficient selectivity for target tissues. We have previously identified (C. K. Goldman et al., Cancer Res., 57: 1447-1451, 1997) the fibroblast growth factor (FGF) ligand and receptor families as conferring sufficient specificity and binding affinity to be useful for targeting DNA in vivo. In the present studies, we retargeted Ad using basic FGF (FGF2) as a targeting ligand. Cellular uptake is redirected through high-affinity FGF receptors (FGFRs) and not the more ubiquitous lower-affinity Ad receptors. Initial in vitro experiments demonstrated a 10- to 100-fold increase in gene expression in numerous FGFR positive (FGFR+) cell lines using FGF2-Ad when compared with Ad. To determine whether increased selectivity could be detected in vivo, FGF2-Ad was administered i.v. to normal mice. FGF2-Ad demonstrates markedly decreased hepatic toxicity and liver transgene expression compared with Ad treatment. Importantly, FGF2-Ad encoding the herpes simplex virus thymidine kinase (TK) gene transduces Ad-resistant FGFR+ tumor cells both ex vivo and in vivo, which results in substantially enhanced survival (180-260%) when the prodrug ganciclovir is administered. Because FGFRs are up-regulated on many types of malignant or injured cells, this broadly useful method to redirect native Ad tropism and to increase the potency of gene expression may offer significant therapeutic advantages.     

Transductional targeting of adenovirus vectors for gene therapy

Cancer gene therapy approaches will derive considerable benefit from adenovirus (Ad) vectors capable of self-directed localization to neoplastic disease or immunomodulatory targets in vivo. The ablation of native Ad tropism coupled with active targeting modalities has demonstrated that innate gene delivery efficiency may be retained while circumventing Ad dependence on its primary cellular receptor, the coxsackie and Ad receptor. Herein, we describe advances in Ad targeting that are predicated on a fundamental understanding of vector/cell interplay. Further, we propose strategies by which existing paradigms, such as nanotechnology, may be combined with Ad vectors to form advanced delivery vehicles with multiple functions.     

肿瘤靶向性腺病毒载体研究进展

近年来,腺病毒载体广泛应用于恶性肿瘤的靶向性基因治疗的研究。对传统腺病毒载体的改造策略主要包括:通过改造腺病毒的嗜性,构建感染靶向性腺病毒载体,如纤维嵌合型靶向性腺病毒载体和免疫靶向性腺病毒载体;在转录水平,控制外源基因的靶向表达或腺病毒基因组自身的靶向复制,即转录靶向性腺病毒载体和选择性裂解肿瘤细胞的条件复制型腺病毒载体。     

癌症靶向治疗的新趋势

靶向性是癌症治疗的关键所在,新的治疗方案必须在特异性作用于肿瘤的同时降低对正常细胞的损伤,目前已取得了许多成果:1)肿瘤的靶向基因-病毒治疗结合了基因治疗和病毒治疗的各自优势。所用的病毒载体具有特异性靶向肿瘤的作用并在肿瘤细胞中大量复制和高效表达抗癌基因,如果用2个特异性启动子严格控制病毒只在肿瘤中表达,并加上2个有协同效应的抗癌基因即可达到极好的抗癌效果。2)在抗体治疗方面,运用第1代腺病毒高效表达Her-ceptin或是Rituxan抗体基因已取得成功,它可以大大地降低目前昂贵的抗体治疗费用。3)RNA干扰技术可以沉默致病基因,选用质粒或是病毒做载体可以达到长期及高效的基因沉默效果。4)具有自我更新能力的肿瘤干细胞是肿瘤复发的关键。5)人们也逐渐意识到肿瘤的形成过程是癌细胞与基质细胞相互作用的结果,以肿瘤基质为靶标的药物也显示出了良好的效果。6)针对蛋白酪氨酸激酶或是抗凋亡蛋白而研制的小分子靶向药物也在肿瘤分子治疗上取得了成功。7)纳米技术正以其独特性而引起广泛关注。总之,在众多新型治疗方案研发的同时,我们必须认识到仅仅靠一种方法是难以成功根治肿瘤的,各种靶向治疗方案之间的相互结合将成为肿瘤治疗的主攻方向。     

Current developments in adenovirus-based cancer gene therapy

Adenovirus (Ad)-based cancer gene therapy is a promising, novel approach for treating cancer resistant to established treatment modalities. Unfortunately, the efficacy of nonreplicative first generation Ads was low and data from clinical trials were disappointing. To address this problem, conditionally replicating Ads have been constructed. Infection of tumor cells with conditionally replicating Ads results in tumor-specific replication, subsequent oncolysis and release of the virus progeny. Recently, it has been suggested that the low expression of the coxsackie-Ad receptor is the rate-limiting factor for infectivity with serotype 5 (Ad5). Unfortunately, coxsackie-Ad receptor expression is highly variable and often low on many tumor types. Consequently, molecular strategies have been applied for the development of coxsackie-Ad receptor-independent oncolytic Ads. This review describes recent developments of Ad-based cancer gene therapy, including novel engineering techniques of the Ad capsid for efficient tumor targeting, as well as targeting techniques, to restrict transgene expression to cancer cells.     

Transductional and transcriptional targeting of cancer cells using genetically engineered viral vectors

Gene delivery vectors, including adenovirus (Ad) and adeno-associated virus (AAV), are inefficient and non-selective for cancer due to low levels of viral receptors with high levels on other tissues, including liver. We tested Ads and AAVs with the SIGYPLP-targeting peptide inserted into virus capsids for transduction in a panel of cancer cells. Six of twelve lines (C8161, PC-3, G-CCM, MKN-45, LnCAP and A549) were transduced, independently of native viral tropism. Furthermore the candidate cancer gene therapy promoter FLT-1 was active in three of these six cell lines. This offers the potential for dual targeting of selected cancer cells.     

Gene transfer to carcinoma of the breast with fiber-modified adenoviral vectors in a tissue slice model system

Successful adenoviral (Ad) vector-mediated strategies for breast cancer gene therapy and virotherapy have heretofore been hindered by low transduction efficiency. This has recently been understood to result from a relative paucity of expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on primary tumor cells. To further investigate this issue, we evaluated the expression of CAR on breast cancer cell lines as well as primary breast cancer cells. With the exception of one patient sample, CAR expression was notably higher in the tumor cells from patients compared to CAR expression in the tumor cell lines. Furthermore, we explored CAR-independent targeting strategies to breast cancer tissue by exploring a panel of infectivity-enhanced Ad vectors, which contain CAR-independent targeting motifs for their utility in breast cancer gene therapy and virotherapy. These targeting motifs included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7), and were tested using the breast cancer tissue slice model, which is the most stringent substrate system available. Of all the tested tropism modified Ad vectors, Ad5/3 exhibited the highest transductional efficiency in breast cancer. These preclinical results suggest that Ad5/3 is the most useful modification to achieve higher clinical efficacy of breast cancer gene therapy and virotherapy.     

癌症靶向治疗的新趋势

靶向性是癌症治疗的关键所在，新的治疗方案必须在特异性作用于肿瘤的同时降低对正常细胞的损伤，目前已取得了许多成果：1）肿瘤的靶向基因一病毒治疗结合了基因治疗和病毒治疗的各自优势。所用的病毒载体具有特异性靶向肿瘤的作用并在肿瘤细胞中大量复制和高效表达抗癌基因，如果用2个特异性启动子严格控制病毒只在肿瘤中表达，并加上2个有协同效应的抗癌基因即可达到极好的抗癌效果。2）在抗体治疗方面，运用第1代腺病毒高效表达Herceptin或是Rituxan抗体基因已取得成功，它可以大大地降低目前昂贵的抗体治疗费用。3）RNA干扰技术可以沉默致病基因，选用质粒或是病毒做载体可以达到长期及高效的基因沉默效果。4）具有自我更新能力的肿瘤干细胞是肿瘤复发的关键。5）人们也逐渐意识到肿瘤的形成过程是癌细胞与基质细胞相互作用的结果，以肿瘤基质为靶标的药物也显示出了良好的效果。6）针对蛋白酪氨酸激酶或是抗凋亡蛋白而研制的小分子靶向药物也在肿瘤分子治疗上取得了成功。7）纳米技术正以其独特性而引起广泛关注。总之，在众多新型治疗方案研发的同时，我们必须认识到仅仅靠一种方法是难以成功根治肿瘤的，各种靶向治疗方案之间的相互结合将成为肿瘤治疗的主攻方向。     

Retargeting of adenoviral infection to melanoma: combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scDb) adapter that binds to fiber knob and HMWMAA

Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus-mediated gene transfer to melanoma cells. This strategy combines genetic ablation of native adenoviral tropism with redirected viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single-chain diabody, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma-associated antigen (HMWMAA). This antigen is widely and specifically expressed on the surface of melanoma cells and its expression is associated with tumor development and progression. Our results showed specific and strong binding of the anti-HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection experiments, we demonstrated i) substantially (>50-fold) reduced infectivity of capsid mutant adenoviruses, ii) restored (up to 367-fold increase), CAR-independent and HMWMAA-mediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of transgene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wild-type fibers. We confirmed the utility of this targeting strategy with human primary melanoma cells that represent clinically relevant substrates. These experiments established that the retargeting strategy mediates up to 54-fold increased adenoviral gene transfer to CAR-negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA-targeted adenoviral vector lacking native tropism exhibits both enhanced specificity and augmented infectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma.     

Gene transfer to cervical cancer with fiber-modified adenoviruses

Successful adenoviral (Ad) vector-mediated strategies for cancer gene therapy mandate gene-delivery systems that are capable of achieving efficient gene delivery in vivo. In many cancer types, in vivo gene-transfer efficiency remains limited due to the low or highly variable expression of the primary Ad receptor, the coxsackie Ad receptor (CAR). In this study, we evaluated the expression of CAR on cervical cancer cells as well as CAR-independent targeting strategies to integrins (Ad5.RGD), heparan sulfate proteoglycans (Ad5.pK7) or both (Ad5.RGD.pK7). We used a panel of established cervical cancer cell lines and primary cervical cancer cells isolated from patients to quantify the expression of CAR mRNA and to evaluate the gene-transfer efficiency of fiber-modified Ads. Of the fiber-modified vectors, Ad5.pK7 and Ad5.RGD.pK7 displayed significantly enhanced gene-transfer efficiency in vitro. Gene-delivery efficiency in vivo was evaluated using an s.c. cervical cancer mouse model. Ad5.RGD.pK7 significantly improves tumor targeting in vivo, resulting in a significantly improved tumor/liver ratio in mice. Our results suggest that the double-modified Ad5.RGD.pk7 vector enhances gene transfer to clinically relevant cervical cancer substrates, while the infectivity of nontarget cells in the mouse is not increased and comparable to Ad5. The fiber-modified virus described here can help achieve higher clinical efficacy of cervical cancer gene therapy.     

Incorporation of a laminin-derived peptide (SIKVAV) on polymer-modified adenovirus permits tumor-specific targeting via alpha6-integrins

Effective gene therapy for disseminated metastatic cancer is currently impossible because of poor delivery of vector to target sites. Modification of viral vectors to target advanced cancer has long been a challenge. In this study, we aimed to redirect adenovirus tropism to infect prostate cancer cells via alpha6beta1 integrins, whose expression is upregulated during prostate cancer progression. To ablate normal mechanisms of infection and provide a framework for attachment of targeting ligands, viruses were non-genetically modified with pHPMA-ONp polymer. Addition of polymer-coated virus to prostate cells showed significantly reduced transgene expression compared with unmodified virus. To restore infectivity, an alpha6-integrin binding peptide (-SIKVAV-) derived from laminin was incorporated onto the surface of the polymer-coated viruses. Photon correlation spectroscopic analysis revealed a small increase in the mean diameter of the particles following retargeting. Addition of -SIKVAV- peptide restored virus infectivity of PC-3 cells in a ligand concentration-dependent manner that was significantly improved following removal of unincorporated polymer and peptide. Competition assays using cells preincubated with Ad5 fiber protein or free -SIKVAV- peptide confirmed that entry of retargeted viruses was mediated via the incorporated ligand. Application of retargeted viruses to a panel of human cell lines revealed varying levels of transduction efficiency. Flow cytometric analysis of cells using anti-alpha6 integrin and anti-beta1 integrin antibodies demonstrated that for prostate cells, greater transduction efficiency correlated with higher levels of expression of both integrin subunits. Furthermore with the exception of LNCaP cells, increased alpha6beta1 integrin expression correlated with advanced disease. Intravenous administration of retargeted viruses to tumor-bearing mice resulted in slower plasma clearance and greatly reduced liver tropism, and hence toxicity compared with unmodified virus, while maintaining reporter gene expression in the tumor. The data suggest that YESIKVAVS-retargeted viruses have potential for systemic delivery for the treatment of metastatic disease.     

Soluble coxsackievirus adenovirus receptor is a putative inhibitor of adenoviral gene transfer in the tumor milieu.

PURPOSE: Several barriers that collectively restrict gene delivery by viral vectors in vivo have been described. Previously, we identified soluble chondroitin sulfate-proteoglycans/glycosaminoglycans in malignant pleural effusions (MPEs) as inhibitors of retroviral vector transduction. Soluble components of MPE also inhibited adenoviral (Ad) gene transfer, and the factors were characteristically filterable, titrable, stable at 56 degrees C, and blocked the binding of Ad to target cells. Depleting immunoglobulin from MPE, partially reversed the block to Ad transduction, instigating a search for additional factors that bound Ad in MPE. EXPERIMENTAL DESIGN: Vector-protein interactions were identified after the resolution of MPE-components by SDS-PAGE. Viral overlays and immunoblots delineated significant interactions, and the potential relevance of those interactions was tested in transduction efficiency bioassays. RESULTS: Immunoglobulin is the predominant factor inhibiting Ad gene transfer in MPE. Albumin also interacted with Ad, although at predicted serum concentrations, it did not effect Ad transduction efficiency in vitro. Soluble coxsackievirus-Ad receptor (sCAR) was then identified in MPE. In a survey of 18 MPE, the mean concentration of sCAR was variable and estimated to be 3.51 +/- 5.02 ng/ml by ELISA. The impact of sCAR on transduction efficiency in this milieu was next assessed. Whereas immunodepletion of sCAR from MPE by affinity chromatography resulted in enhanced gene transfer within MPE, the inhibition of adenoviral gene transfer was not evident when the predicted concentrations of recombinant sCAR were added into the transduction medium. CONCLUSIONS: These studies indicate that, in addition to anti-Ad antibodies, other specific and nonspecific factors interact with viral vectors and may impair gene transfer in the tumor milieu. The presence of sCAR in MPE puts forward the notion that in certain contexts (e.g., within the extracellular matrix of solid tumors) the concentrations of secreted (or shed) CAR may be high enough to effectively compete with Ad gene delivery.