Oncolytic adenovirus retargeted to Delta-EGFR induces selective antiglioma activity.

The fact that glioblastomas, which are one of the most devastating cancers, frequently express the Delta-EGFR (epithelial growth factor receptor) also called mutant variant III of EGFR (EGFRvIII) suggests that this cancer cell-specific receptor might serve as an ideal target for cancer therapy. To assess its potential as such a target, we constructed an oncolytic adenovirus with Retargeted Infectivity Via EGFR (Delta-24-RIVER) on the backbone of Delta-24. This new oncolytic adenovirus targets, as Delta-24 does, the disrupted Rb pathway in cancer cells; in addition, this adenovirus has also been retargeted through the abrogation of CAR binding (Y477A mutation in adenoviral fiber protein) and insertion of an EGFRvIII-specific binding peptide in the HI loop of the fiber protein. As compared with Delta-24, Delta-24-RIVER induced EGFRvIII-selective cytotoxicity in U-87 MG isogenic cell lines and in tetracycline-inducible EGFRVIII expressing U-251 MG cells. Accordingly, by tittering the viral progeny and examining fiber protein expression in the above cells, we showed that the replication of this new construct also correlated with EGFRvIII expression. Consistently, immunohistochemistry staining of the adenoviral capsid protein hexon in the virus-treated tumors revealed that the virus replicated more efficiently in EGFRvIII-expressing U-87 MG.DeltaEGFR xenografts than in the tumors grown from U-87 MG cells. Importantly, treatment with Delta-24-RIVER prolonged the survival of animals with intracranial xenografts derived from U-87 MG.DeltaEGFR cells. Therefore, our results constitute the first proof of the direct targeting of a cancer-specific receptor using an oncolytic adenovirus.

Combining high selectivity of replication with fiber chimerism for effective adenoviral oncolysis of CAR-negative melanoma cells.

Oncolytic adenoviruses constitute a new and promising tool for cancer treatment that has been rapidly translated into clinical trials. However, minimal or absent expression of the adenovirus serotype 5 (Ad5) receptor CAR (coxsackievirus and adenovirus receptor) on cancer cells represents a major limitation for Ad5-based oncolysis. Here, we report on the resistance of CAR-negative primary melanoma cells to cell killing by wild-type Ad5 (Ad5wt) even after high titer infection, thus underlining the need for tropism-modification of oncolytic adenoviruses. We engineered a new generation of oncolytic adenoviruses that exhibit both efficient target cell infection by swapping Ad5 fiber domains with those of Ad serotype 3, which binds to a receptor distinct from CAR, and targeted virus replication. Fiber chimerism resulted in efficient cytopathicity to primary melanoma cells, which was at least 10(4)-fold increased relative to Ad5wt. Since viral infectivity mediated by such modified viral capsids was not cell type-specific, it was pivotal to carefully restrict adenoviral replication to target cells. Towards this end, we replaced both E1A and E4 promoters of fiber chimeric viruses by tyrosinase enhancer/promoter constructs. The resulting viruses showed melanoma-specific expression of E1A and E4 and combined efficient virus replication and cell killing in melanoma cell lines and primary melanoma cells with a remarkable specificity profile that implements strong attenuation in nonmelanoma cells, including normal fibroblasts and keratinocytes.

Bioluminescence imaging reveals a significant role for complement in liver transduction following intravenous delivery of adenovirus.

The effect of complement on transgene expression was evaluated in vivo and in vitro using mice lacking complement components. Complement component 3 (C3) deficient mice (C3-/-) and appropriate wild-type controls were intravenously injected with a replication incompetent, luciferase-expressing normal Ad5 (Ad5Luc1), or fibritin-fiber Ad5 (Ad5FFLuc1). Repeated, noninvasive bioluminescence imaging was conducted over 35 days. Our data show for the first time that C3 facilitates both short- and long-term hepatic expression of luciferase following systemic delivery. C3-/- mice showed significantly less (P < 0.05) luciferase expression in their liver than treatment-matched wild-type mice when 2.3 x 10(9) (Ad5Luc1) and 4.0 x 10(9) (Ad5Luc1 or Ad5FFLuc1) viral particles (v.p.) were infused. The maximal difference in luciferase activity between C3-/- and wild-type mice was 99-fold difference at 3 days for the 2.3 x 10(9) v.p. dose (Ad5Luc1), 35-fold at 13 days for the 4.0 x 10(9) v.p. dose (Ad5Luc1), and 22-fold at 13 days for the 4.0 x 10(9) v.p. dose (Ad5FFLuc1). Preincubation of Ad5Luc1 with wild-type, C1q-/-, or factor B (FB) deficient mouse sera for 5 min significantly (P < 0.05) increased transduction of mouse liver cells, as compared to preincubation with C3-/- sera or PBS. These results suggest the classical or alternate complement pathway enhances Ad5-mediated liver transduction.

Approaches to utilize mesenchymal progenitor cells as cellular vehicles.

Mammalian cells represent a novel vector approach for gene delivery that overcomes major drawbacks of viral and nonviral vectors and couples cell therapy with gene delivery. A variety of cell types have been tested in this regard, confirming that the ideal cellular vector system for ex vivo gene therapy has to comply with stringent criteria and is yet to be found. Several properties of mesenchymal progenitor cells (MPCs), such as easy access and simple isolation and propagation procedures, make these cells attractive candidates as cellular vehicles. In the current work, we evaluated the potential utility of MPCs as cellular vectors with the intent to use them in the cancer therapy context. When conventional adenoviral (Ad) vectors were used for MPC transduction, the highest transduction efficiency of MPCs was 40%. We demonstrated that Ad primary-binding receptors were poorly expressed on MPCs, while the secondary Ad receptors and integrins presented in sufficient amounts. By employing Ad vectors with incorporated integrin-binding motifs (Ad5lucRGD), MPC transduction was augmented tenfold, achieving efficient genetic loading of MPCs with reporter and anticancer genes. MPCs expressing thymidine kinase were able to exert a bystander killing effect on the cancer cell line SKOV3ip1 in vitro. In addition, we found that MPCs were able to support Ad replication, and thus can be used as cell vectors to deliver oncolytic viruses. Our results show that MPCs can foster expression of suicide genes or support replication of adenoviruses as potential anticancer therapeutic payloads. These findings are consistent with the concept that MPCs possess key properties that ensure their employment as cellular vehicles and can be used to deliver either therapeutic genes or viruses to tumor sites.

The coxsackievirus and adenovirus receptor acts as a tumour suppressor in malignant glioma cells.

The coxsackievirus and adenovirus receptor (CAR) is a membrane glycoprotein with a cytoplasmic domain, a transmembrane domain and an extracellular region consisting of two immunoglobulin-like domains, an amino-terminal immunoglobulin variable (IgV)-related domain (D1), which is distal to the cell surface, and a proximal IgC2 domain (D2). The coxsackievirus and adenovirus receptor has been shown to exhibit tumour suppression activity in human bladder and prostate cancer cells. In the current paper, we demonstrate that CAR is a tumour suppressor in glioma cells and that the extracellular D2 domain is not required for this inhibitory effect. This finding provides a biological basis for the observation that expression of CAR is downregulated in malignant glioma cells. This suggests that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realise the full potential of adenoviral vectors for cancer gene therapy.

The therapeutic efficacy of adenoviral vectors for cancer gene therapy is limited by a low level of primary adenovirus receptors on tumour cells.

Replication-defective adenoviral vectors are currently being employed as gene delivery vehicles for cancer gene therapy. To address the hypothesis that the therapeutic efficacy of adenoviral vectors is restricted by their inability to infect tumour cells expressing low levels of the primary cellular receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR), we have employed a pair of ovarian cancer cell lines differing only in the expression of a primary receptor for Ad5. This novel system thus allowed the direct evaluation of the relationship between the efficacy of an adenoviral vector and the primary receptor levels of the host cancer cell, without the confounding influence of other variable cellular factors. We demonstrate that a deficiency of the primary cellular receptor on the tumour cells restricts the efficacy of adenoviral vectors in two distinct cancer gene therapy approaches, TP53 gene replacement therapy and herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy. Moreover, we show that a deficiency of the primary receptor on the tumour cells limits the efficiency of adenovirus-mediated gene transfer in vivo. Since a number of studies have reported that primary cancer cells express only low levels of CAR, our results suggest that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realize the full potential of adenoviral vectors in the clinical setting.

Improved gene transfer efficiency to primary and established human pancreatic carcinoma target cells via epidermal growth factor receptor and integrin-targeted adenoviral vectors.

In this study we analyzed two ways of retargeting of Ad-vectors to human pancreatic carcinoma with the aim of enhancing the gene transfer efficiency. First, we analyzed the expression of the epidermal growth factor receptor (EGFR) on primary, as well as established pancreatic carcinoma cells by flow cytometry which revealed high expression levels of EGFR on the surface of these cells. We showed that EGFR-retargeted entry pathway using a bispecific fusion protein formed by a recombinant soluble form of truncated Coxsackie and Adenovirus Receptor (sCAR) genetically fused with human EGF (sCAR-EGF) redirects them to the EGFR leading to an enhanced gene transfer efficiency to pancreatic carcinoma cells. Since flow cytometry revealed absence of CAR expression, but the presence of at least one of both alphav integrins on the pancreatic carcinoma cells, a second way of targeting was investigated using a genetically modified Ad vector which has an RGD (Arg-Gly-Asp)-containing peptide inserted into the HI-loop of the fiber knob. This RGD targeted Ad (AdlucRGD) revealed efficient CAR-independent infection by allowing binding to cellular integrins resulting in a dramatic enhancement of gene transfer. These findings have direct relevance for Ad-vector based gene therapy strategies for pancreatic carcinoma.

Genetic targeting of an adenovirus vector via replacement of the fiber protein with the phage T4 fibritin.

The utility of adenovirus (Ad) vectors for gene therapy is restricted by their inability to selectively transduce disease-affected tissues. This limitation may be overcome by the derivation of vectors capable of interacting with receptors specifically expressed in the target tissue. Previous attempts to alter Ad tropism by genetic modification of the Ad fiber have had limited success due to structural conflicts between the fiber and the targeting ligand. Here we present a strategy to derive an Ad vector with enhanced targeting potential by a radical replacement of the fiber protein in the Ad capsid with a chimeric molecule containing a heterologous trimerization motif and a receptor-binding ligand. Our approach, which capitalized upon the overall structural similarity between the human Ad type 5 (Ad5) fiber and bacteriophage T4 fibritin proteins, has resulted in the generation of a genetically modified Ad5 incorporating chimeric fiber-fibritin proteins targeted to artificial receptor molecules. Gene transfer studies employing this novel viral vector have demonstrated its capacity to efficiently deliver a transgene payload to the target cells in a receptor-specific manner.

Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells.

Exploiting the lytic life cycle of viruses has gained recent attention as an anticancer strategy (oncolysis). To explore the utility of adenovirus (Ad)-mediated oncolysis for rhabdomyosarcoma (RMS), we tested RMS cell lines for Ad gene transduction and infection. RMS cells were variably transduced by Ad. Compared with control cells, RMS cells were less sensitive or even resistant to oncolysis by wild-type virus. RMS cells expressed the Ad internalization receptors, alpha(v) integrins, but had low or undetectable expression of the major attachment receptor, coxsackievirus-Ad receptor (CAR). Mutant Ads with ablated CAR binding exhibited only 5-20% of transgene expression in RMS cells seen with a wild-type vector, suggesting that residual or heterogeneous CAR expression mediated the little transduction that was detectable. Immunohistochemical analysis of archived clinical specimens showed little detectable CAR expression in five embryonal and eight alveolar RMS tumors. Stable transduction of the cDNA for CAR enabled both efficient Ad gene transfer and oncolysis for otherwise resistant RMS cells, suggesting that poor CAR expression is the limiting feature. Gene transfer to RMS cells was increased >2 logs using Ads engineered with modified fiber knobs containing either an integrin-binding RGD peptide or a polylysine peptide in the exposed HI loop. The RGD modification enabled increased oncolysis for RMS cells by a conditionally replicative Ad, Ad delta24RGD, harboring a retinoblastoma-binding mutation in the E1A gene. Thus, the development of replication-competent vectors targeted to cell surface receptors other than CAR is critical to advance the use of Ad for treating RMS.

Efficient gene transduction by RGD-fiber modified recombinant adenovirus into dendritic cells.

Dendritic cells (DC) are important antigen-presenting cells in the development of an anti-tumor T cell response. To extend the range of current immuno / gene therapies, we tested luciferase-expressing RGD-adenovirus (Ad) (Ad5lucRGD)-mediated transduction into DC. Phenotypically characterized DC were generated from peripheral blood CD14(+) cells by incubation with granulocyte-macrophage colony-stimulating factor, interleukin-4 and tumor necrosis factor alpha. On the 7th day of culture, the cells became mature DC with a CD1a(+), CD11c(+), CD80(+), CD83(+), CD86(+), human leukocyte antigen (HLA)-DR(+), CD14- phenotype. The expression of alpha( v)beta(3) integrin was enhanced on day 3 and returned to the basal level on day 7. We then compared the transduction efficiency of an Ad5lucRGD system to that using conventional Ad, in cells harvested on days 1, 3 and 7 of culture. Luciferase activity was negligible in AdCMVLuc, but remarkable in cells processed with Ad5lucRGD. Activity was maximal in cells that had been cultured for 3 days. Recombinant Ad5 fiber knob protein blocked AdCMVLuc- and Ad5lucRGD-mediated gene transduction by 90% and 20%, respectively. Surface markers and cytokine production were not affected by Ad5lucRGD-mediated transduction.

A conditionally replicative adenovirus with enhanced infectivity shows improved oncolytic potency.

The absence or the presence of low levels of the Coxsackievirus and adenovirus receptor (CAR) on several tumor types might limit the efficacy of recently proposed tumor-specific or conditionally replicative adenoviruses (CRAds). To address this issue, we used a genetic modification of the fiber knob in the context of an E1A-defective CRAd to allow CAR-independent target cell infection as a means to enhance oncolytic potency. Such infectivity-enhanced CRAd showed higher replication, more efficient infection, and lysis of tumor cells in vitro. Of note, the improved antitumor effect of the fiber-modified CRAd could be demonstrated in vivo. We conclude that the combination of genomic modification to achieve tumor-selective replication and capsid modification to enhance infectivity yields more potent oncolytic adenoviruses for use in cancer treatment.

Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid.

Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.

Ectodomain of coxsackievirus and adenovirus receptor genetically fused to epidermal growth factor mediates adenovirus targeting to epidermal growth factor receptor-positive cells.

Human adenovirus (Ad) is extensively used for a variety of gene therapy applications. However, the utility of Ad vectors is limited due to the low efficiency of Ad-mediated gene transfer to target cells expressing marginal levels of the Ad fiber receptor. Therefore, the present generation of Ad vectors could potentially be improved by modification of Ad tropism to target the virus to specific organs and tissues. The fact that coxsackievirus and adenovirus receptor (CAR) does not play any role in virus internalization, but functions merely as the virus attachment site, suggests that the extracellular part of CAR might be utilized to block the receptor recognition site on the Ad fiber knob domain. We proposed to design bispecific fusion proteins formed by a recombinant soluble form of truncated CAR (sCAR) and a targeting ligand. In this study, we derived sCAR genetically fused with human epidermal growth factor (EGF) and investigated its ability to target Ad infection to the EGF receptor (EGFR) overexpressed on cancer cell lines. We have demonstrated that sCAR-EGF protein is capable of binding to Ad virions and directing them to EGFR, thereby achieving targeted delivery of reporter gene. These results show that sCAR-EGF protein possesses the ability to effectively retarget Ad via a non-CAR pathway, with enhancement of gene transfer efficiency.

Advanced generation adenoviral vectors possess augmented gene transfer efficiency based upon coxsackie adenovirus receptor-independent cellular entry capacity.

Adenoviral (Ad) vectors have been widely used in the context of cancer gene therapy approaches. Their utility in these contexts, however, has frequently been limited by tumor cell resistance to Ad infection. The basis of this resistance has been defined recently as resulting from a deficiency of the primary adenovirus receptor, coxsackie adenovirus receptor. As a means to circumvent this limitation, a variety of tropism modification strategies have allowed coxsackie adenovirus receptor-independent gene delivery via the Ad vector. These advanced generation adenovirus vectors exhibit enhanced infectivity, which can allow direct therapeutic gain. Such vectors may allow improvements in efficacy in the context of ongoing human clinical gene therapy approaches for cancer.

Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector.

In vivo cancer gene therapy approaches for squamous cell carcinoma of the head and neck (SCCHN) based on adenoviral vector-mediated gene delivery have been limited by the suboptimal efficacy of gene transfer to tumor cells. We hypothesized that this issue was due to deficiency of the primary adenoviral receptor, the coxsackie-adenovirus receptor (CAR), on the tumor targets. Studies of CAR levels on SCCHN cell lines confirmed that their relative refractoriness to the adenoviral vector was based on this deficiency. To circumvent this deficiency, we applied an adenoviral vector targeted to a tumor cell marker characteristic of SCCHN. In this regard, integrins of the alpha2beta1 and alpha3beta1 class are frequently overexpressed in SCCHN. Furthermore, these integrins recognize the RGD peptide motif. On this basis, we applied an adenoviral vector genetically modified to contain such a peptide within the HI loop of the fiber protein as a means to alter viral tropism. Studies confirmed that the CAR-independent gene delivery achieved via this strategy allowed enhanced gene transfer efficiencies to SCCHN tumor cells. Importantly, this strategy could achieve preferential augmentation of gene transfer in tumor cells compared with normal cells. The ability to achieve enhanced and specific gene transfer to tumor cells via adenoviral vectors has important implications for gene therapy strategies for SCCHN and for other neoplasms in general.

Reduction of ischemia-reperfusion injury of the liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic Bcl-2 gene.

OBJECTIVE: To examine the possibility of reducing ischemia-reperfusion injury (I/R injury) to the mouse liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic human Bcl-2 gene. SUMMARY BACKGROUND DATA: Ischemia-reperfusion injury has been demonstrated in a number of clinically relevant diseases such as myocardial infarction, cerebrovascular disease, sepsis, peripheral vascular disease, and organ transplantation. In this regard, apoptosis plays a central role. METHODS: Normal C57BL/6 mice were used. An adenovirus (deltaE1) vector containing the human Bcl-2 gene was developed in the authors' laboratory. An adenovirus vector encoding an irrelevant gene (beta-galactosidase, AdCMVLacZ) was used as a control. Taking advantage of the hepatotropic properties of adenovirus vectors, gene transfer was performed with 1 x 10(9) plaque-forming units by intravenous tail injection, 48 hours before the ischemic injury. Ischemic-reperfusion injury was induced by temporal and segmental occlusion of hepatic blood flow. Aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase activity was measured using standard assays. Liver biopsies were obtained before and 6 hours after I/R injury for morphologic assessment, and apoptosis was determined in situ with a histochemical assay. RESULTS: The expression of AdCMVhBcl-2 vector was confirmed by reverse transcription-polymerase chain reaction and functionally validated in apoptotic studies in endothelial cells. Expression of the Bcl-2 gene protects against I/R injury, as shown by a significant decrease in transaminases (p < 0.05) and necrosis and apoptosis (p < 0.001), and permanent survival (p < 0.0001), compared with sham-operated animals and animals treated with AdCMVLacZ. CONCLUSIONS: Genetic modification of the liver to induce cytoprotection has potential applications to prevent I/R injury to the liver in surgical interventions, including liver transplantation.

An advanced generation of adenoviral vectors selectively enhances gene transfer for ovarian cancer gene therapy approaches.

OBJECTIVE: We hypothesized that incorporation of an integrin binding Arg-Gly-Asp (RGD)-containing peptide to the HI loop of the adenovirus fiber knob would allow enhanced, coxsackie-adenovirus receptor-independent gene transfer by modified Ad vector in the context of ovarian cancer. METHODS: Ovarian cancer cell lines, primary ovarian cancer cells, primary tumor explants, and mesothelial tissue were transfected with luciferase encoding adenovirus (AdCMVLuc) or a genetically modified adenovirus (Ad5lucRGD) which contained an RGD motif within the HI loop of the knob. The luciferase activity was measured and the transduction efficiencies of both viruses were compared. RESULTS: In all established ovarian cell lines and primary tumor cell samples there was dramatically augmented gene transfer observed with the Ad5lucRGD compared to AdCMVLuc. The enhanced gene transfer in ovarian cancer cell lines ranged from 2.5- to 471.6-fold, in ascites samples from 26.1- to 64.0-fold, and in tumor explants from 1.6- to 11.1-fold. Although gene transfer to normal mesothelial tissue was slightly augmented by RGD retargeting, the level of gene transfer was much lower than that seen in ovarian cancer cells. CONCLUSION: This study demonstrates that genetically altered adenoviruses with modified tropism are capable of more efficient gene transfer in the context of ovarian cancer. The higher level of transfer with respect to peritoneal mesothelium can be exploited to enhance the therapeutic index of interventions using adenoviral vectors. Studies are warranted, therefore, to determine the in vivo utility of this targeted vector approach in the context of gene therapeutic strategies for cancer of the ovary.

Adenovirus-mediated gene expression in vivo is enhanced by the antiapoptotic bcl-2 gene.

An adenovirus vector encoding the human Bcl-2 gene (hBcl-2) was derived. In vivo expression of hBcl-2 in murine livers enhanced and prolonged adenovirus-mediated gene expression. Furthermore, in the hBcl-2-treated group a significant reduction in the apoptosis induced by the adenovirus vector was observed. Thus, the cytoprotection of the vector-infected cells with antiapoptotic genes appears promising for successful in vivo gene therapy.

A system for the propagation of adenoviral vectors with genetically modified receptor specificities.

The development of genetically modified adenovirus (Ad) vectors with specificity for a single cell type will require both the introduction of novel tropism determinants and the ablation of endogenous tropism. Consequently, it will not be possible to exploit the native cellular entry pathway in the propagation of these targeted Ad vectors. Based on the concept that Ad enters cells by a two-step process in which a primary receptor serves as a high affinity binding site for the Ad fiber knob, with subsequent internalization mediated by alpha v integrins, we designed two artificial primary receptors. The extracellular domain of one of these synthetic receptors was derived from a single-chain antibody (sFv) with specificity for Ad5 knob, while the second receptor consisted of an icosapeptide identified by biopanning a phage display library against Ad5 knob. Expression of either of these artificial virus-binding receptors in fiber receptor-negative cells possessing alpha v integrins conferred susceptibility to Ad infection. We then created a novel mechanism for cell binding by genetically modifying both the vector and the target cell. In this approach, six histidine (His) residues were incorporated at the C-terminal of the Ad fiber protein. The resultant Ad vector was able to infect nonpermissive cells displaying the cognate artificial receptor, containing an anti-His sFv. This strategy, comprising a genetically engineered Ad virion and a modified cell line, should be useful in the propagation of targeted Ad vectors that lack the ability to bind the native fiber receptor.