WO1998017322A1

WO1998017322A1 - Enhanced delivery of adenoviral vectors to lung tissue

Info

Publication number: WO1998017322A1
Application number: PCT/US1997/019180
Authority: WO
Inventors: Michael A. Perricone; Judith A. St. George; Dianne D. Rees
Original assignee: Genzyme Corporation And President And Fellows Of Harvard College
Priority date: 1996-10-24
Filing date: 1997-10-23
Publication date: 1998-04-30
Also published as: AU4915897A

Abstract

Novel methods are provided that facilitate the binding of an adenoviral vector to epithelial cells in the lung, said methods comprising the steps of: (a) contacting said cells, at or prior to the time of administration of a therapeutic dose of said vector, with one or more proteinase inhibitors for a time sufficient to substantially protect the surface of said cells from one or more proteinases, and (b) administering to said lung, at or prior to the time of administration of said vector dose, one or more antibodies capable of binding specifically to human antibody present in said lung that is directed against a component of adenovirus. The methods thus facilitate effective contact of therapeutic doses of vector with lung epithelial cells. Therapeutic compositions are also described.

Description

Enhanced Delivery of Adenoviral Vectors to Lung Tissue

Background of the Invention

The present invention relates to methods and therapeutic compositions that facilitate treating diseases of the lung, such as by gene therapy. It is widely hoped that gene therapy will provide a long lasting and predictable form of therapy for certain disease states, and it is likely one of the few forms of therapy suitable for many inherited diseases.

Reported Developments

The transfer of a transgene to epithelial cells of the lung using a recombinant viral vector is currently being developed in order to correct inherited diseases such as cystic fibrosis (CF), and alpha-1-antitrypsin deficiency (see, for example, D. T. Curiel et al., Am. I. Respir. Cell Mol. Biol., 14, pp. 1-48, (1996) and J.M. Wilson et al., 1. Clin. Invest.. 96, pp. 2547-2554, (1995). The use of virus-derived vectors is intended to take advantage of the natural ability of a virus to enter the target cells of patients, where the therapeutic transgene built into the vector can be expressed. The lung is a particularly promising target for gene therapy as the target cells, the epithelial cells of the bronchi, bronchioles, and gas exchange regions, are accessible through the conducting airways.

Most attempts to use viral vectors for gene therapy have relied on retrovirus vectors, chiefly because of their ability to integrate mto the cellular genome. However, the disadvantages of retroviral vectors are becoming increasingly clear, including their tropism for dividing cells only, the possibility of insertional mutagenesis upon integration into the cell genome, decreased expression of the transgene over time, rapid mactivation by serum complement, and the possibility of generation of replication-competent retroviruses. See, for example, D. Jolly, et al., Cancer Gene Therapy. 1, pp. 51- 64, (1994) and C.P. Hodgson, et al, Bio Technology , 13, pp. 222-225 (1995).

Adenovirus is a nuclear DNA virus with a genome of about 36 kb, which has been well-characterized through studies m classical genetics and molecular biology (see, for example, M.S. Horwitz et al, "Adenoviπdae and Their Replication," in Virology. 2nd edition, B.N. Fields et al., eds., Raven Press, New York, 1990). In a simplified form, the genome is classified mto early (known as E1-E4) and late (known as L1-L5) transcπptional units, referring to the generation of two temporal classes of viral proteins. The demarcation between these events is viral DNA replication.

Adenovirus-based vectors offer several unique advantages, including tropism for both dividing and non-dividing cells, minimal pathogenic potential, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large therapeutic transgene inserts (K.L. Berkner, Curr. Top Micro Immunol. , 158, pp. 39-66, (1992); and D. Jolly, Cancer Gene Therapy. 1, pp. 51-64 (1994). The clonmg capacity of present adenovirus vectors is about 8 kb, resulting typically from: (1) the deletion of certain regions of the virus genome dispensable for virus growth, for example, E3; and (2) deletions of regions, such as El, whose function may be restored in trans, that is, from a packaging cell line such as the 293 cell, see F.L. Graham, Gen. Virol. . 36. pp. 59-72 (1977).

Genes that have been expressed to date using adenoviral vectors include p53 (Wills et al., Human Gene Therapy. 5, pp. 1079-188 (1994), dystrophin (Vincent et al., Nature Genetics , 5, pp. 130-134 (1993); erythropoietin (Descamps et al., Human Gene Therapy , 5, pp. 979-985 (1994); ornithine transcarbamylase (Perricaudet et al., Human Gene Therapy , 1, pp. 241-256, 1990); adenosme deaminase (Mitani et al., Human Gene Therapy ,5, pp. 941-948 (1994); mterleukm-2 (Haddada et al., Human Gene Therapy , 4, pp. 703-711 (1993); and d 1-antιtrypsin (Jaffe et al., Nature Genetics . 1, pp. 372-378 (1992).

The tropism of adenoviruses for cells of the respiratory tract has particular relevance to the use of adenoviral vectors in gene therapy for cystic fibrosis (CF), the most common autosomal recessive disease m Caucasians. Mutations in the gene for cystic fibrosis transmembrane conductance regulator protein (CFTR), a protein that regulates movements of ions (and therefore fluid) flow across the cell membrane of epithelial cells (including lung epithelial cells) are responsible for the CF disease state. Adenovirus vectors engineered to carry the CFTR gene have been developed (D. Rich et al., Human Gene Therapy. 4, pp. 461-476 (1993), and studies have shown the ability of such vectors to permit expression of the CFTR gene in nasal epitheha of CF patients (J. Zabner, et al., Cell. 75, pp. 207-216 (1993), the airway epitheha of cotton rats and primates (J. Zabner et al., Nature Genetics. 6, pp. 75-83 (1994), and the respiratory epithelium of CF patients (R.G. Crystal et al., Nature Genetics , 8, pp. 42-51 (1994). However, the environment of the cystic fibrosis-affected lung contams certam components that may act to dimmish the effectiveness of adenoviral vector-mediated gene transfer to the lung. One such component is sputum. Sputum is a viscous mucopurulent fluid found in the conductmg airways of the lungs. Sputum contams a number of components, some of which reflect the inflammatory stage of CF In addition to the ions and mucoplysacchaπdes found in normal airway secretions, sputum contams the products of inflammatory cells mcludmg proteases (see, for example, D.D. Rees et al., Am. T. Physiol.. 269, L657-665, 1995), and chemotactic cytokines, cellular debris, and microorganisms. Thus the sputum m the lung of a CF patient may present an additional banner, or otherwise adversely affect, a gene therapy treatment program based on adenoviral vectors. An additional component of the cystic fibrosis affected sputum is the titer of the patient s anti-adenovirus antibodies, a natural result of previous adenovirus infections

The present invention provides methods and therapeutic compositions designed to minimize the effects of the environment of the cystic fibrosis-affected lung on the efficacy of adenoviral vector-mediated CF gene therapy

Summary of the Invention

This present mvention provides methods to improve the administration to the lung of an adenoviral-based vector containing a transgene of therapeutic interest. In a preferred embodiment of the mvention the lung is affected by cystic fibrosis (CF) and the transgene of interest is that encoding the cystic fibrosis conductance regulator (CFTR) protem. According to the practice of the invention, it is recognized that the binding of adenoviral vectors to target lung epithelial cells is affected adversely by the presence in sputum of proteinases that alter the surface of said target cells, thus making less effective the binding of adenoviral vectors. It is recognized also that sputum in the CF-affected lung may contain antibodies directed against wild type adenovirus, which are capable of binding, for example, to surface components of adenovirus-based vectors, thereby affecting adversely the ability of said vectors to b nd and infect target cells.

Accordmgly, there is provided a method of facilitating the binding of an adenoviral vector to epithelial cells m the lung comprising the steps of-

(a) contacting said cells, at or pπor to the tune of administration of a therapeutic dose of said vector, with one or more proteinase inhibitors for a ime sufficient to substantially protect the surface of said cells from one or more proteinases, and

(b) administering to said lung, at or pπor to the time of admirustration of said vector dose , one or more antibodies capable of binding specifically to human antibody present n said lung that is directed against a component of adenovirus, thereby facilitating contact of said vector with target epithelial cells

Additional embodiments of the mvention are described according to the Detailed Descπption of the Invention which follows directly

Bπef Description of the Drawings

FIGURE 1 depicts the effect of CF sol concentration on expression of a transgene FIGURE 2 depicts the effect of neutrophil elastase on expression of a transgene FIGURE 3 depicts the effect of CF sol (treated with proteinase inhibitors) on expression of a transgene.

FIGURE 4 depicts the effects of anti-mouse Ig on adenoviral vector administration in mice.

Detailed Description of the Invention

The treatment of many chronic lung diseases, such as cystic fibrosis, by gene therapy, is expected to require repeat administrations of one or more vectors containing a therapeutic transgene, in many cases throughout the lifetime of the patient. The long term effectiveness of many viral vectors may be limited by the production in the patient of neutralizing concentrations of vector-specific antibodies. Thus, upon repeated readministration, newly added vector may be inactivated.

In order to overcome such effects, diverse solutions have been proposed. The broad use of immunosuppresants (see Engelhardt et al., Proc. Natl. Acad. Sci. USA. 91, pp. 6196-6200 (1994) has been described, as has the use of cytoablative agents, Dai et al., Proc. Natl. Acad. Sci. USA. 92, pp. 1401-1405 (1995).

Co-adminstration of interferon-gamma or interleukin-12 with recombinant adenovirus based (Ad-based) vectors has been shown to diminish the formation of Ad-specific neutralizing antibodies, and has thus allowed for re-adminsitration of Ad vector to the mouse airway (Yang et al., Nature Medicine. 1, pp. 890-893 (1995). However, there are and art-recognized disadvantages to such approaches.

Additionally , it is recognized that the disease-affected lung presents further challenges to successful viral vector administration. Λs aforementioned, components

E 25 that may act to dimmish the effectiveness of adenoviral vector-mediated gene transfer to the lung mcludmg proteases (see, for example, D.D. Rees et al., Am I Physiol.. 269, L657-665, 1995), chemotactic cytokines, cellular debris, and microorganisms, and numerous other components associated with disease states and resultant inflammation.

According to the practice of the present mvention, there are provided novel approaches to facilitate effective, and repeated, administration of adenovirus-based vectors to the lungs (preferably the epithelial cells thereof , and such cells within the small airways).

The present mvention involves, in part, the recognition that proteinases (proteases), that is, proteins which function to catalyze the breakdown of other proteins, have the capacity to damage the surface components of lung cells (such as airway epithelial cells) mcludmg giycoproteins or other proteins or protem-contammg molecules that may serve as receptors for adenovirus, or which are involved m translocating bound adenovirus mto the target cells. Thus, as a result of such interactions, and although affected lung cells may continue to function normally m the lung tissue such as for respiration, the capacity will have been substantially reduced to bmd or internalize therapeutically useful adenoviral vector.

It is recognized that the sputum of the CF-affected lung contains a wide variety of proteinases including , for example, neu rophil elastase, cathepsin G, trypsin, and proteinase 3 [purified samples of sputum denved protemases are m some cases available from commercial suppliers, for example, human sputum elastase (No. SE 563); protemase 3 (No. PN382); and trypsin (No. ST51), all from Elastm Products Company, Inc., Owensville , MO]. According to the practice of the mvention, gene therapy to the lung of a patient using transgenes carried by adenoviral vectors is conducted with additional programs of therapy, in order to preserve the integrity of cell surface components on the targeted lung cells, thereby to facilitate interaction of the vector with such target cells.

In the preferred practice of the mvention, therapeutic compositions comprising one or more protemase inhibitors (see, for example, the listing in Example 5) are administered either with or prior to doses of adenoviral gene therapy vector so as to preserve the surface mtegnty of target cells The selection of appropriate species of protemase inhibitor is determined by a number of factors that are appreciated in the art mcludmg acceptable toxicity, solubility or other capacity for formulation, such as m an aerosol, and a level of efficacy and chemical stability that permit an acceptable benefit. Preferred for administration in the practice of the mvention are protemase inhibitors that mactivate neutrophil elastase, such as, for example, alpha-1 antitrypsm Such protemase inhibitors may be formulated mto therapeutic compositions for nebulizers, powder inhalers, and via aerosolized solutions for delivery to the respiratory tract. The therapeutic protemase inhibitors useful m the practice of the mvention can, in general, also be formulated with excipients and may be lyophi zed and rehydrated prior to use

Although administration of such proteinase mhibitors provides benefit according to the practice of the invention, it is recognized that lung tissue of many patients is expected to have high titers of neutralizing antibodies against adenovirus. Alternatively, such neutralizing titers may arise as a result of repeated administration of the therapeutic adenoviral vector Accordmgly, the practice of the present invention also provides for administration to patients of therapeutic antibodies capable of inactivating the antibodies made by the patient that target the therapeutic vectors As aforementioned, the patient s reactive antibodies may have resulted from

R LE 25 a prior exposure to a related adenovirus or from repeated current exposure to the vector.

Methods to produce such therapeutic antibodies are well known in the art (see, for example, E. Harlow and D. Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York , 1988) and include procedures to humanize monoclonal antibodies made in another animal or bacteria; or recovering fully human antibodies from hybridomas that were themselves derived from transgenic mice that include within their genome fully human antibody genes, while lacking those of the mouse. Such antibodies can then be administered in doses known to be acceptable in the art, or which are capable of being readily determined, for any of the methods of administration that can provide benefit to the lung, such as intravenously, intranasally, or by aerosol administration, and the like.

Examples

The following examples are representative of the practice of the invention.

Example 1 CF sol fraction inhibited the rransfection efficiency of Ad2/β gal-2

The effect of CF sol fraction on rransfection efficiency of an adenoviral vector, Ad2/β gal-2 , was investigated in a model system using Fisher rat thyroid (FRT) cells. The FRT cells were treated with serially diluted CF sol containing Ad2/β gal-2 (at 10 MOI) for 1 hour. Transfection efficiency was determined 48 hours after the start of infection by measuring β-galactosidase activity using a chemiluminescent substrate. The detailed procedure was as follows. Sputum was collected from adult patients having advanced CF and have been hospitalized for acute pulmonary exacerbation (Children's Hospital, Boston, MA). The patients were on antibiotic therapy and recombinant deoxyribonuclease (Pulrnozy e, Genentech, South San Francisco, CA). Sputum was separated into aqueous sol and gel fractions by ultracentπfugation at 50,000 X g for 90 min ( see D.D. Rees et al.. Am. J. Physiol. ,269, L195-202, 1995). Multiple samples of CF sol from different individuals were pooled for use in this study. Two pools of CF sol were used in this study, designated CF sol pool #4 and CF sol pool #7.

Fisher rat thyroid (FRT) epithelial cells were received from the Amencan Type Culture Collection (Rockville, MD). FRT cells were cultured using F12 (Ham's formulation) supplemented with 2% fetal bovine serum (FBS).

The recombinant adenoviral vector used in this study was adenovirus serotype 2, El-deleted and contained the £. coh Lac Z reporter gene driven by the cytomegalovirus early-immediate promoter, wherein the E4 region thereof has been replaced by only ORF6 of E4 (Ad2/βgal-2, see J. St. George et al. Gene Therapy, 1996). Ad2/βgal-2 was titered by microplate assay and activity was expressed in International Units (IU)/ml. T e activity of the Ad2/βgal-2 used in these studies was from 2 different gradients having titers of either 2.26 X 10⁶ IU/ml or 5.38 X 10° IU/ml.

To perform the rransfection assays underlying the dose response depicted in Figure 1, FRT cells were seeded mto the wells of 24- well or 96- ell plates at a density of 30,000 cells per cm² and grown for 24 hrs at 37°C and 5% Cθ2- Ad2/βgal-2 was diluted in either CF sol or OptiMEM (Gibco/BRL, Gaithersburg, MD) to 1, 10, or 100 MOI and incubated with the cells for 1 hr at 37°C and 5% CO2- The Ad2/βgal-2 was then aspirated and the wells washed twice with OptiMEM and then complete growth

10

SUBSTITUTE SHEET RULE 25) medium was added to the wells. The cells were grown for 48 hrs at 37°C and 5% CO2-

Transfection efficiency was determined by measuring b-galactosidase activity using the Galactolight Plus (Tropix Inc., Bedford, MA) chemiluminescent assay according to package instructions. Chemiluminescence was quantified using a BetaMax multiplate luminometer (Wallach,) and results are reported as luminometer counts per second (LCPS). To generate the dose-response (see Figure 1), cells were seeded as described above for a transfection assay. CF sol (pool #4) was then serially diluted in a 1:1 ratio in F12 and Ad2/^βgal-2 was added to each concentration of CF sol to a final adenoviral concentration of 10 MOI. The CF sol containing Ad2/βgal-2 was added to the cells and the cells incubated for 1 hr at 37°C and 5% C 2- Transfection efficiency was then determined as described directly above.

Results are depicted in Figure 1 where β-galactosidase activity is expressed as luminometer counts per second (LCPS) per well. The substantial effect on β- galactosidase activity is apparent for CF sol derived from both pools used, Nos 4 and 7. For example, CF sol pool #4, when diluted to 50% in F12 (Ham's) medium, suppressed the transfection efficiency to about 90% of the control value as determined by β- galactosidase activity. The rransfection efficiency was suppressed by CF sol pool #4 to 50% of the maximum (ID50) when diluted to 12%.

Exampl 2 Preincubation of Ad2/β gal-2 with CF sol did not inhibit the capacity of the vector to infect cells.

Ad2/bgal-2 (1.13 X 10⁸ IU) was incubated in 45 ul of undiluted CF sol (pool #4) or PBS at 37°C for 15, 30, and 60 mm. The adenovirus was then diluted in F12 to 100, 10, or 1 MOI and the efficiency of rransfection was tested using the transfection assay described above. The pre-incubation of Ad2/bgal-2 in undiluted CF sol pπor to dilution (to 1, 10, or 100 MOI) and subsequent cell infection did not suppress the transfection efficiency of FRT cells at any MOI (Table 1) To dilute the adenovirus solution after incubation with CF sol for subsequent cell infection, at least a 1 100 dilution was performed This resulted in the infection of cells in the presence of, at most, 1% CF sol solution during the infection. Based on the dose-response values (see Figure 1), this amount of CF sol remaining with the adenovirus during the subsequent cell transfection should not suppress infection. In fact, the values for b-galactosidase activity tended to be higher for the CF sol group than for the PBS control group The differences between the PBS and CF sol groups were not, however, significant

TABLE 1

EFFECT OF Ad2/^βGal2 PRE-INCUBATED WITH CF SOL ON TRANSFECTION EFFICIENCY

Ad2/βgal-2 was pre-incubated in CF sol or PBS pπor to dilution and infection of FRT cells at 1, 10, and 100 MOI. Transfection efficiency was determined 48 hours after the start of infection by measuring β-galactosidase activity

Example 3 Lability of factor in CF sol responsible for transfection suppressive effect Usmg the above descπbed ^β-galactosidase assay, the heat stability of CF sol component responsible for transfection supression was assessed. CF sol (pool #4 or pool #7) was mcubated at 95°C for 30 minutes, and then centπfuged at high speed in a microcentπfuge for 5 minutes. The transfection efficiency of heat-treated CF sol was compared with untreated CF sol, and it was determmed that CF sol did not supress adenovirus transfection efficiency when heat treated at 95 °C for 30 mmutes pπor to use.

Example 4 Purified neutrophil elastase inhibits transfection efficiency of Ad2/β gal-2 a dose dependent manner

The effect of neutrophil elastase on transfection efficiency was determmed according to the following protocol. Ad2/βgal-2 was diluted to 10 MOI in OptiMEM containing either neutrophil elastase (Elasπn Products Co., Inc., Owensville, MO) or cathepsm G (Athens Research and Technology, Athens, GA). Neutrophil elastase was serially diluted to final concentration range of 20 uM to 20 pM and cathepsm G was serially diluted to a final concentration range of 4 U/ml to 0.06 U/ml. FRT cells were mcubated with the protemase & Ad2/βgal-2 mixture at 37°C and 5% CO2 for 1 hr. The protemase & Ad2/βgal-2 mixture was then aspirated and the wells washed 2 times with OptiMEM and the cells cultured for 48 hrs in complete medium. Transfection efficiency was determmed by measuring b-galactosidase activity usmg the Galactolight Plus (Tropix Inc., Bedford, MA) cherniluminescent assay, as above, and accordmg to package instructions.

Figure 2 demonstrates the substantial effect of punfied neutrophil elastase (at 20 to 0.3 μM) on transfection efficiency usmg FRT cells. In contrast, purified cathepsm G,

13

5 another component of CF sputum, appeared to have no effect on the transfection efficiency of Ad2/βgal-2.

It is noted that normal human bronchial epithelial (NHBE) cells can also be used for this procedure. Such cells were purchased from Clonetics Corporation (San Diego, CA). The cells were cultured usmg the Bronchial Epithelial Basal Medium supplemented with the bullet kit (BEGM) supplied with the cells. The NHBE cells were then used at passage number 2.

Example 5 The pre-treatment of CF sol with a panel of proteinase inhibitors does not neutralize its suppressive effect on adenoviral vector-mediated cell transfection.

CF sol (pool #4 or #7) was pre-incubated with different proteinase inhibitors prior to use in the cell transfection assay: 4 mg/ml of -l-antitrypsin (Sigma, St. Louis, MO); 1 mM of E-64 (Boehringer Mannheim, Indianapolis, IN); 1 μg/ml of Pepstatin A (Boehπnger Mannheim, Indianapolis, IN); and 1 mM 1,10-phenanthroline (Sigma, St. Louis, MO). The incubations were conducted at room temperature for 30 minutes, although times of 1 hour and temperatures of about 30 C° are also representative of those that can be used.

NHBE cells (see Example 4) were then transfected in the presence of CF sol (or CF sol pre-rreated with proteinase inhibitor) using Ad2/βgal-2 (at 10 MOI). Transfection efficiency was then determined 48 hours after the start of infection by measurmg β-galactosidase activity (as above) reported as percentage of the PBS control (see Figure 3). It is demonstrated that pre-incubation with the proteinase inhibitors was ineffective to restore transfection activity reduced by the presence of CF sol. Without bemg limited as to theory, it is believed that natural antibody titers in the CF 98/17322

sol directed against wild type adenovirus to which the donor(s) had been previously exposed was sufficiently high to mask, in this expenment, the beneficial effects of contacting the CF sol with protemase inhibitors.

Exa ple 6 Detection of Antibody Ti r*;

The titer of antibodies (IgG and IgA) against Ad2 m CF sol pools #4 and #7 were determmed using an enzyme-linked immunoassay (ELISA). Neutralizmg activity of CF sol pools #4 and #7 were determmed usmg a cytopathic effect reduction (CPER) assay.

The cytopathic effect reduction (CPER) assay was used to determine the neutralizmg activity of CF sol. CF sol was seπally diluted m wells contammg 293 cells and Ad2/CFTR-2 (33,625 IU) was added to each well. Cells were grown for 4 days until the cytopathic effect of the adenovirus infection was complete. The neutralizmg capacity of CF sol on adenovirus mfection was determmed from the CPER assay and expressed as neutralizmg mfectious units/μl (nlU/μl). Although antibody titer was determmed, the assay did not provide direct evidence that the antι-Ad2 antibody m CF sol was responsible for the neutralizmg effect

TABLE 2 ANΗ-ADENOVIRUS ANTIBODY ANALYSIS IN CF SOL (POOLS #4 AND #7)

Example 7 Antibody directed against mouse Ig enhanced transfection in mouse lung

The following procedure was used to demonstrate that anti-adenovirus neutralizing antibodies found in the mouse lung could be inactivated such that adenoviral vector delivery to the mouse lung could be sustained.

Mouse strain B10.A5R (Taconic, Germantown, NY) was used. The animals were divided into 5 groups, as shown in Table 3 below. Ad2/CFTR-5 [an Ad2 based vector having a CMV promoter, a CFTR encoding gene placed into the deleted Ela/ Elb region, a BGH poly A tail, a protein IX gene moved 3' of the CFTR gene and an E4 region replaced only with E4 ORF6 thereof, see U. S. Serial No. 08/540,077, and published international patent application PCT/US96/03818] was used to stimulate anti-Ad antibodies in the test animals. Blood samples taken from the eye on the 30th, 40th, and if necessary 50th days, and were used to determine whether the test animal had acquired a sufficient titer of neutralizing antibody (defined where neutralizing capability was still present at a serum dilution of 25,000 fold). On the indicated day (Table 3) the mice were infected intranasally with Ad2/β gal-4 vector (similar to above-defined Ad2/β gal-2 but having the full E4 region), in order to determine whether administration of this subsequent vector would lead to transgere expression (as measured by β-gal detection). Anti-mouse IgA (from a commercial source and anti-mouse IgG (also from a commercial source) were administered intranasally as indicated in Table 3. The concentration of these antibody solutions used was about 1 mg/ml. Test animal groups 3, 4, and 5 also represent controls as described in Table 3.

As shown in Figure 6, effective adminsitration of the Ad2/βgal-4 vector was possible following anti-Ig treatment, with the mean in each case indicated bv a dark diamond.

Table 3

Example 8 Additional Controls

Additional controls were performed to rule out alternate explanations for some of the data observed (A) Short term cytptoxicity

A short term cvtotoxicity assav was performed to confirm that cells remained viable on contact with CF sol Briefly, FRT, A549, HeLa, and NHBE cells were seeded mto 96-well plates at 30,000 cells per cm² and grown for 24 hrs (48 hrs for NHBE cells) at 37°C and 5% Cθ2 CF sol was seπallv diluted in a 1.1 ratio with either F12 (for FRT cells), DMEM (for A549 and HeLa cells), or BEBM (for NHBE cells) and then added to the cells usmg triplicate wells The cells were mcubated with the CF sol for up to 2 hrs at 37°C and 5% CO2 The cells were tested for viability at 2 hrs usmg trypan blue (Sigma, St Louis, MO)

CF sol had no short-term cytotoxic effect on any of the cell Imes tested as determmed by the trypan blue exclusion assay. Cell viability was greater than 95% for all cell lines tested (A549, FRT, HeLa, and NHBE) when the cells were mcubated with CF sol (50% in either PBS, F12, or OptiMEM) for 3 hrs at 37°C and 5% CO2 In all cases, viability was not significantly less than the viability of untreated cells. CF sol did, however, cause all of the cell Imes to round up. The amount of time it took for 50% of the cells to round-up (partially detach from the plastic support) varied between cell Imes. The time for 50% of the cells to round for HeLa, A549, FRT, and NHBE cell Imes was 30 mm, 45 mm, 2 hrs, and 2.5 hrs, respectively. Furthermore, there was no cell rounding or detachment m either the FRT and NHBE cell cultures after 1 hr of treatment with CF sol. As a result, the FRT and NHBE cells were used for the transfection studies.

(B) Effect of pH change from contact with CF sol

CF sol is somewhat acidic, and the following controls were performed to confirm that pH change was not contributing substantially to the phenomena herein described. Ad2/βgal-2 (1.13 X 10⁸ IU) was mcubated in 45 ul of PBS (pH range from 6.2 to 7.0) at room temperature or 37°C for 60 mm. The adenovirus was then diluted in F12 to 100, 10, or 1 MOI and the efficiency of transfection was determmed by measurmg b-galactosidase activity as descπbed above.

Ad2/βgal-2 (1.13 X 10⁸ IU) was also diluted to 10 MOI m PBS ranging from 5.9 - 7.2) and mcubated with FRT cells for 1 hr at 37°C and 5% CO2. The PBS was replaced with complete medium and the cells grown for 48 hrs. Efficiency of transfection was determmed by measurmg ^β-galactosidase activity as described above There was no significant suppression of the transfection efficiency of Ad2/βgal-2 in a low pH buffer (5.9 - 6.8) compared with the control group (pH 7.2). Values for b- galactosidase activity were 6,479 ± 1,003 and 5,945 ± 543 LCPS/x ug protein for pH 5.9 and pH 7.2, respectively. Similarly, the pre-mcubation of Ad2/βgal-2 in low pH buffer prior to dilution (to 1, 10, or 100 MOI) and subsequent cell infection did not suppress the transfection efficiency of FRT cells at any MOI. The dilution of the adenovirus into culture medium prior to cell infection effectively neutralized the pH.

(C) Adenovirus binding assays

Additional assays were performed to confirm that effects that appeared to indicate failure of vector to bmd to the target cells were not due to another cause, such as, that the viral vector bound but was not endocytosed, or that following endocytosis the vector could not escape from the endosome.

Ad2/bgal-2 was labeled with [3$S] by growing in the presence of 1 mCi of [35s]-L- methiomne (NEG-009T; DuPont NEN, Boston, MA) per roller bottle of 293 cells. [35s]-L metfuonine-labeled Ad2/bgal-2 ([35s]-Ad2/bgal-2) was purified on a cesium chloride gradient and dialyzed extensively at 4°C with 3 changes of PBS contammg 1% sucrose. The [35s]-Ad2/bgal-2 was stored m 5% sucrose in PBS at -80°C until used.

NHBE cells were seeded mto the wells of 96-well plates at a density' of 30,000 cells per cm2 and grown for 48 hrs at 37°C and 5% Cθ2 The plates were placed on ice and the wells washed 2 times with ice-cold PBS. The last PBS wash was aspirated and [ ⁵S]-Ad2/bgal-2 (serially diluted in either OptiMEM or CF sol in a range of 1 X 10⁸ - 1 X lO^ particles per well) was added to the wells. The cells were mcubated with β^S}- Ad2/bgal-2 at 4°C for 90 mm. The adenovirus was then aspirated and the wells washed 3 times with PBS. The cells were then lysed with 50 ul of lysis buffer () and the lysate centrifuged at high speed in a microcen rifuge for 5 min. The amount of [35s]- Ad2/bgal-2 bound to the cells was determined from an aliquot of the lysate supernatant by using liquid scintillation spectrophotometry. Results are reported as percentage of control.

£121 Statistical Methods

During the course of the experiments described herein, statistical tests for significance were performed between two groups using the independent t-test or between multiple groups using one-way analysis of variance (ANOVA) and were considered to be significantly different at P < 0.05 with the Tukey HSD test for post-hoc comparisons of mean differences. All statistics were done using Systat Version 5.2 (Systat, Inc., Evanston, IL) for the Macintosh. Values are reported as mean ± standard deviation (sd) in the test and as error bars in the graphs unless otherwise noted.

Claims

Claim

1. A method of facilitating the binding of an adenoviral vector to epithelial cells in the lung comprising the steps of :

(a) contacting said cells, at or prior to the time of administration of said vector, with one or more proteinase inhibitors for a time sufficient to substantially protect the surface of said cells from one or more proteinase, and

(b) administering to said lung, at or prior to the time of administration of said vector dose, one or more antibodies capable of binding specifically to human antibody present in said lung that is directed against a component of said adenoviral vector, thereby facilitating contact of said vector with target epithelial cells.

2. The method of Claim 1. in which the proteinase is neutrophil elastase.

3. The method of Claim 1, in which the proteinase inhibitor is alpha- 1 antitrypsin.

4. The method of Claim 1, in which the proteinase inhibitor is pepstatin.

5. The method of Claim 1 , in which the proteinase inhibitor is 1.10-phenanthroline.

6. The method of Claim 1. in which the component of the adenoviral vector is the fiber protein.

7. The method of Claim 1. in which the component of the adenoviral vector is the hexon protein.

8. The method of Claim 1, in which the component of the adenoviral vector is the penton protein.

9. The method of Claim 1. in which the component of the adenoviral vector is a transgene.

10. A method of facilitating the binding of an adenoviral vector to epithelial cells in the lung comprising contacting said cells, at or prior to the time of administration of said vector, with one or more proteinase inhibitors for a time sufficient to substantially protect the surface of said cells from one or more proteinases.

1 1. The method of Claim 10. in which the proteinase is neutrophil elastase.

12. The method of Claim 10. in which the proteinase inhibitor is alpha- 1 antitrypsin.

13. The method of Claim 10. in which the proteinase inhibitor is pepstatin.

14. The method of Claim 10, in which the proteinase inhibitor is 1,10-phenanthroline.

15. A method of facilitating the binding of an adenoviral vector to epithelial cells in the lung comprising administering to said lung, at or prior to the time of administration of said adenoviral vector, one or more antibodies capable of binding specifically to human antibody present in said lung that is directed against a component of said adenoviral vector; thereby facilitating contact of said vector with target epithelial cells.

16. The method of Claim 15, in which the component of the adenoviral vector is the fiber protein.

17. The method of Claim 15, in which the component of the adenoviral vector is the hexon protein.

18. The method of Claim 15. in which the component of the adenoviral vector is the penton protein.

19. The method of Claim 15, in which the component of the adenoviral vector is a transgene.