WO2005023848A2

WO2005023848A2 - Adenoviral epitopes

Info

Publication number: WO2005023848A2
Application number: PCT/GB2004/003861
Authority: WO
Inventors: Niels Rudi Pedersen
Original assignee: Gardner, Rebecca, Katherine
Priority date: 2003-09-09
Filing date: 2004-09-09
Publication date: 2005-03-17
Also published as: WO2005023848A3; EP1664099A2

Abstract

The present invention provides a peptide comprising an adenoviral (Ad) penton base epitope for a human neutralising antibody; the epitope may be a fragment from within region 50-120, 191-233 or 310-408 of Ad5 or an equivalent region within another Ad serotype, or an immunologically equivalent variant of such a fragment; particularly preferred epitopes include GGRNSIRYSELA, TRVYLVDNKSTD, QTINLDDRSHWG, HYLKVGRQNGVL, FRLGFDPVTGLV, VTGLVMPGVYTN, SNSSGSGAEENS, DHAIRGDTFATR, DSTFTQYRSWYL, RRTCPYVYKALG; as well as the use of such peptides and corresponding nucleic acid molecules in therapy and formulations comprising these molecules, optionally together with an adenoviral vector.

Description

Adenoviral Epitopes

The present invention relates to Adenoviral epitopes, in particular to epitopes of the penton base (Pb) of Adenovirus (Ad) which are recognised by neutralising antibodies . Peptides incorporating these epitopes have utility in methods of treatment and evaluation/diagnosis of a patient. Currently, there are several ongoing clinical gene therapy trials with adenoviruses (Ads) as therapeutic agents . There are several reasons for such a wide-spread usage of Ad-derived vectors or viruses. (i) Ads are easy to grow to high titers and in a suitable clinical grade. (ii) Ads are biologically safe compared to other vectors such as retroviruses or lentiviruses since they are mostly responsible for benign infections in immunocompetent individuals, and their lack of genomic integration reduces the risk of genomic recombination and oncogenic transformation. (iii) Ads are able to transduce both dividing and nondividing cells and are particularly appropriate in clinical applications such as cancer therapy due to their transient expression. Despite these advantages, large numbers of the public acquire respiratory tract infections caused by various Ads, and host immune response due to the immunogenicity of Ad capsid components remains a major impediment to efficient Ad-mediated gene transfer and persistence of transgene expression. The relative inefficacy of Ad-mediated gene transfer has been attributed to the humoral response and to the presence of preexisting neutralizing antibodies .

Several research groups have shown Ad capsid epitopes in hexon and fiber proteins that were responsible for the immune response. However, the immunogenicity in humans and the characterization of neutralizing and non-neutralizing epitopes in the third major Ad capsid component, the Pb, has not been systemically studied.

It has now been shown that in sera from patients administered Ad were antibodies that recognise linear and conformational epitopes in the penton base protein of Adenovirus . The present inventors have further been able to identify the Pb antigenic epitopes and to evaluate which are epitopes for neutralising antibodies (NA) that can interfere with effective gene therapy transfer. It has also been shown that Pb antibodies make a significant contribution toward anti-Ad neutralisation.

According to one aspect of the present invention it is proposed to use one or more peptides which are able to mimic the Pb neutralising antibody epitopes in simultaneous administration with an Ad vector (e.g. a recombinant Ad vector in gene therapy) . These peptides can block the neutralising antibodies and help the Ad vector itself to escape the neutralising effect of the antibodies, thus enabling more effective gene delivery.

Thus, in one aspect the invention provides a peptide comprising an adenoviral penton base epitope for a (human) neutralising antibody. The 'epitope¹ either has the sequence of a naturally occurring penton base epitope for NA or is a related sequence which is immunologically equivalent. Preferably said epitope is from Ad5. Three major immunodominant domains have been identified and the epitope is preferably a fragment from within region 50-120, 191-233 (preferably 193-230) or

311-408 of Ad5 or an equivalent region within another Ad serotype, or an immunologically equivalent variant of such a fragment .

Ten particularly preferred epitopes of the invention are

^S0GGRNSIRYSELA^S1 "TRVYLVDNKSTD⁷⁸ ¹⁰⁹QTINLDDRSHWG¹²⁰

¹⁹¹HYLKVGRQNGVL²⁰¹

²¹⁵FRLGFDPVTGLV²²⁵

²²VTGLVMPGVYTN²³³ 3¹⁰SNSSGSGAEENS³²¹

³³⁵DHAIRGDTFATR³⁴⁷

³⁹⁷DSTFTQYRSWYL⁴⁰⁸

^54aRRTCPYVYKALG^5S9. A further group of suitable epitopes are immunologically equivalent variants or fragments of these 10 epitopes. The numbers correspond to the a ino acid in the Ad5 penton base protein. Preferred variants are the corresponding regions in other Ad serotypes, particularly Ad3. The epitope will preferably comprise at least 5 amino acids, e.g. at least 6 or 7 amino acids . Further suitable variants (which can be considered variants of fragments) include the hexamers of Table 2.

A preferred group of epitope fragments have an amino acid sequence selected from the group comprising

NSIRYSE, LGFDPVTGL, AIRGDTF, LDDRSHWG, SGSGAE, FTQYRS YL, YLVDNKS, LKVGRQ, PYVYK and GLVMPGV, any of the underlined sequences of Table 2, or immunologically equivalent variants or fragments thereof. Particularly preferred peptides are those which consist or comprise the following sequences NSIRYS, VTGL, SGAE, and FTQ.

Immunologically equivalent variants will be able to generate or are immunologically reactive with neutralising antibodies and will have sequence similarity with one of the sequences listed above, suitable immunologically equivalent epitopes/peptides are listed in Table 2. Variants will typically have 3 or more, preferably 4 or more, e.g. 5 or more amino acids which are preferably identical to or possibly homologous with amino acids in the actual Pb sequence. Amino acid pairs which can be considered homologous include leucine and isoleucine; cysteine and serine; asparagine and glutamine; and aspartic acid and glutamic acid.

Fragments and variants of the 10 epitopes defined above will typically be at least 6 or 7 amino acids in length, preferably 8 or 9 amino acids in length, more preferably 10 or 11, most preferably 12 amino acids in length. Variants will typically have at least 50%, if not 60%, preferably at least 70 or 80%, more preferably at least 90% sequence homology with these naturally occurring Ad5 epitopes (or equivalent epitopes in other serotypes) . Sequence 'homology' allows for conservative substitution of individual amino acids, Arg for Lys, Phe for Tyr, Glu for Asp etc. Preferably variants will have at least 40%, if not 50%, more preferably at least 60 or 70%, most preferably at least 80% sequence identity with these naturally occurring Ad5 epitopes (or equivalent epitopes in other serotypes) .

A method of determining immunological equivalence can be derived from the Examples which describe how bacteriophage expressing putative epitopes may be generated and their binding reactivity with (e.g. immobilised) Pb antibodies assessed. Variants and fragments may be more or less able to block the NA than a named sequence and still be considered ' immunogenically equivalent'; typically fragments and variants will have at least 20%, preferably at least 35%, more preferably at least 50%, most preferably at least 65% of the immunogenic activity of a named epitope . Each peptide of the invention incorporating an epitope will typically be no more than 25, preferably no more than 20 amino acids in length, e.g. 15 to 10 amino acids, most preferably 12 amino acids. Preferred peptides for use in co-administration with an Ad vector will comprise 2 or more epitopes as defined above, e.g. two epitopes or more in tandem (these peptides may therefore exceed the preferred values given above and be of 30, 40 or even 50 amino acids in length) , or circularised peptides incorporating disulfide bridges . The peptides may further be modified in a conventional manner to increase half life e.g. by N- or C-terminal modification but will typically be administered with the Ad vector without such modifications .

The peptide may comprise no other amino acids apart from the epitope or epitopes .

A 'neutralising antibody' (NA) is one which inhibits the ability of an Ad to replicatively infect a cell, typically at the step of cell uptake or endocytosis.

In a further aspect the invention provides a pharmaceutical composition comprising a peptide comprising an adenoviral penton base epitope for a human neutralising antibody and a pharmaceutically acceptable carrier, diluent or adjuvant. The peptides of the invention may be administered together with an Ad vector in a combined preparation for gene delivery or the two components may be administered separately. Thus in one preferred embodiment the invention provides a pharmaceutical composition comprising a peptide comprising an adenoviral penton base epitope for a human neutralising antibody and an (recombinant) adenoviral vector. In an alternative embodiment, the invention provides a product containing (a) a peptide comprising an adenoviral penton base epitope for a human neutralising antibody, and (b) an adenoviral vector as a combined preparation for separate, simultaneous or sequential administration to a patient as part of gene therapy or cancer treatment .

An "adenoviral vector" being an adenovirus based particle which is capable of delivering genetic material, typically but not exclusively recombinant genetic material, into a cell.

Suitable therapeutic genes which may be introduced into a patient in an adenoviral vector are known in the art and the list of suitable candidates, particularly in cancer therapy, are increasing all the time.

In a further aspect, the invention provides a peptide comprising an adenoviral penton base epitope for a human neutralising antibody for use in therapy, in particular for use in gene or cancer therapy as a masking or blocking agent for a co-administered Ad vector.

Similarly, the invention provides a method of gene or cancer therapy using an Adenoviral vector wherein a peptide comprising an adenoviral penton base epitope for a human neutralising antibody is co-administered to the patient with said vector. As discussed above co-administration need not be, but may be, as part of a single product or 2 simultaneously administered products .

The peptides of the invention may be administered to a patient as a mini peptide or a mini gene encoding the peptide which is inserted in a plasmid or other vector, e.g. an Ad vector. Thus, nucleic acid molecules encoding the peptides constitute a further aspect of the invention and the various therapeutic methods or uses of the peptides described herein apply equally to nucleic acid molecules encoding the peptides and to expression vectors incorporating these regions of nucleic acid, e.g. plasmids or viral vectors.

The Adenoviral vector may be of any serotype . The sequences referred to herein are based on the conditionally replicative mutant adenovirus AddI1520. Epitopes in other Ad may vary slightly in position or sequence but equivalent epitopes can be readily identified by sequence comparison and tested according to the methods described in the Examples section herein. A preferred group of epitope fragments are NSIRYS, VTGL, SGAE, FTQ, AVTGID, HSGAEF and NFTQVG and immunologically equivalent variants thereof. As discussed above Ads are the cause of common upper respiratory infections, and thus most people have preexisting immunity. Preexisting immunity to Ad is an important issue in clinical trials with Ad as vectors because these vectors are often used in direct in vivo delivery, and one of the limiting factors to Ad-mediated gene transfer had been attributed to preexisting neutralizing antibodies. It has been described above how the peptides incorporating the epitopes for these NA may be used to provide an effective 'cover' for the

Adenoviral vector itself. An alternative application of the present invention is the modification of these epitopes. Site directed mutagenesis, for example, may be used to modify the Ad genome so that the produced capsid Pb protein incorporates one or more modified epitopes which are able to avoid the NA.

Thus in a further aspect, the present invention provides an adenovirus wherein one or more of the penton base epitopes for neutralising antibodies has been modified to reduce its reactivity with a corresponding neutralising antibody. A reduction in 'reactivity' includes a reduced ability to be recognised by NA and a reduced ability to generate NA; a reduction in immunogenicity. Alternatively, Pb capsomers may be swapped between Ad serotypes from different subgroups in order to improve the efficacy of Ad virions or Ad-derived vectors used as therapeutic agents in cancer or gene therapey. Thus in a further aspect the present invention provides a modified Adenovirus which incorporates one or more Pb capsomers from a non-native serotype. "Non-native" means that the capsomer is from a different serotype than the rest (or most of the rest) of the Ad.

Identification of these so called 'Neutralising Epitopes' within the Ad penton base provides further applications, in particular in predicting a patient's serum profile before Ad administration. Thus, the peptides can be incorporated in an ELISA test, for example, to check the presence and level of different penton base antibodies in the patient. This will give information as to the suitable dose of Ad vector which should be administered to the patient and also may be used to monitor the patient during the period of Ad administration, e.g. during the gene therapy trial. This is particularly useful as the type and quantity of NA varies significantly from patient to patient. Thus, in a further embodiment, the present invention provides a method of evaluating a patient ' s serum, which method comprises contacting a ( in vi tro) sample of the serum with one or more peptides comprising an adenoviral penton base epitope for a human neutralising antibody. Preferred peptides are discussed above. In order to obtain a useful profile relating to the different NA against the penton base, the sample is preferably contacted with three or more, preferably 5 or more peptides incorporating the neutralising epitopes . These methods may conveniently be carried out on a solid support, e.g. the peptides described above or antibodies thereto may be immobilised on a solid support. Thus in a further aspect, the invention provides a solid support having immobilised thereon one or more peptides comprising an adenoviral penton base epitope for a human neutralising antibody. Preferably the solid support will have immobilised thereon 3 or more, e.g. 5 or more peptides incorporating the neutralising epitopes. Methods of immobilising peptides on solid supports are well known in the art.

The peptides described above are of particular utility when Ad of serotype 5 is used as the adenoviral vector as the epitopes which have been characterised are from an Ad of this serotype . However the penton base sequence is highly conserved between Ad of the same subgroup and a 98.6% homology has been found between Ad2 and Ad5. The peptides of the invention may also be used with an Ad2 or Ad3 vector.

Despite utility in gene and anti-cancer therapies, Ad are responsible for many undesirable infections. The peptides described herein may also be used as a vaccine for preventing Ad infection. Thus in a further embodiment, the invention provides a vaccine comprising one or more peptides comprising an adenoviral penton base epitope for a human neutralising antibody. Typically the peptide would be used as an adjuvant, like tetanus toxoid or alum. Alternatively, the peptides, in the form of lipopeptides can be injected with recombinant Ad hexon proteins (which may be a complete hexon protein or an active fragment thereof, activity in this case being in the context of immunogenicity and the ability to act as an adjuvant) . In such an embodiment the C-terminus of the peptide is preferably modified to enhance immunogenicity e.g. by a palmitoyl-lysylamide group (as in J. Virology 76 [2000] , pp 127-135) . Thus the invention also provides a method of preventing or ameliorating natural Adenoviral infection in a patient which comprises administering to said patient a vaccine as described above.

A further application of the present invention is the use of the peptides of the invention in a method of haemofiltration or dialysis where the blood of the patient is taken out of the body and passed across a filter that contains these peptides. The peptides can chelate the NA and retain them in the filter, the blood can then be given back to the patient with much lower concentrations of NA. Thus in a further aspect, the invention provides a method of filtering blood to remove neutralising antibodies to epitopes of the adenoviral penton base, which method comprises contacting the blood outside the body with the peptides of the invention described above, preferably these peptides will be immobilised on a solid suport which filters the blood. The blood is then preferably returned to the patient . In a particularly preferred embodiment, the blood is passed through an affinity column which incorporates the peptides of the invention. An affinity column comprising the peptides of the invention is a further aspect of the present invention.

An alternative method of masking the neutralising ability of the NA is to use antibodies or antibody fragments directed against the neutralising antibodies. These antibody or Ab fragments can also attach to the NA so they cannot act on the adenovirus and as such can be considered immunoreactive with these NA. Methods of raising antibodies to known epitopes is well known in the art. Thus, the various therapeutic and diagnostic methods and uses discussed above which utilise the peptides of the invention apply, mutatis mutandis to the use of antibody or antibody fragments to the various epitopes described herein.

The invention will now be further described in the following Example and by reference to the Figures in which:

Ficfure 1 shows Western blot analysis of sera from patients 1, 2, 3 and 4 taken before (day 0 [DO]) or after (day 10 [D10] ) Addll520 administration. Aliquots of serum dilutions were reacted with membrane strips transferred with recombinant Ad Pb protein electrophoresed under^' native conditions (a) or a mixture of SDS-denatured hexon, Pb, and fiber proteins separated by conventional SDS-PAGE (b and c) . Control samples (lane 9 in panel a; lanes 9 to 11 in panel b; lanes 5 and 6 in panel c) consisted of strips that were reacted with rabbit anti-fiber (αFi) , anti-Pb (αPb) , or anti-whole Ad virion (αAd) antibodies, respectively. Ficrure 2 shows the virus NA activity of Pb antibodies from human sera. (a) Global effect of wholse sera; (b) global effect of Pb-specific antibodies; (c) effect of Pb-specific antibodies at the endocytotic step. In panel a, Ad5Luc3 recombinant was preincubated without (control, no antibody [con] ) or with total serum samples from patients 1 (PI) , 2 (P2) , 3 (P3) , or 4 (Pr) (using the DO sera of patients 1 and 2 and the D10 sera of patients 3 and 4) and then incubated with HeLa cells. In panel b, Ad5Luc3 was preincubated with or without isolated Pb antibodies. In panel c, isolated Pb antibodies were added to virus-cell monolayers after pre-attachment of Ad5Luc3 to HeLa cells at 4°C for 30 min. In the three sets of experiments (i.e., panels a to c) , virus infection was allowed to proceed for 18 h at 37°C, and the cells were processed for luciferase assays. The NA effect was indirectly assayed by the level of luciferase activity expressed as arbitrary units and then normalized to the percentage of the control samples .

Figure 3 demonstrates epitope mapping in Ad Pb protein. The Pb sequence is represented linearly. Under the Pb line are shown the positions of the epitopes defined in Table 2. The patient sera reacting with the different epitopes are indicated in parentheses. Above the Pb line are shown the major IDRs in humans, as defined in the present study, and in mice, after immunization with recombinant Pb (11) . The probability of immunogenicity (as evaluated by the antigenic index of Jameson-Wolf) is presented at the top of the figure .

Figure 4 gives graphical representation of phage-mediated depletion of Pb epitope-specific antibodies . Phages carrying the Pb epitopes indicated at the top of each panel were amplified and incubated with D10 serum samples from patients 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, and 16, as well as samples 18, 19, 20, and 21 from healthy donors. After removal of the phage-antibody complexes by centrifugation, the serum supernatants were assayed for their effect on Ad-mediated gene transfer by using the replication-competent Ad5Luc3 vector. Luciferase expression was compared in cells infected with Ad5Luc3 treated with depleted or nondepleted sera. Theoretically, values greater than 1.0 for the ratio of luciferase levels between depleted and nondepleted serum samples indicated a decrease in NA activity after phage depletion but, once the experimental errors are taken into account, only values of >1.3 were considered significant.

Example

MATERIALS AND METHODS

Patients and clinical trial.

A clinical study was performed with mutant Addll520 to assess its toxicity and efficacy in patients. The patients included in the studies had advanced primary or secondary liver cancer that had either failed to respond or was not suitable for conventional therapy, including surgery, chemotherapy, and radiotherapy (Habib, N. et al., 2002. Cancer Gene Ther. 9: 254-259 and Habib, N. et al., 2001, Hum. Gene Ther. 12: 219-226). Addll520 was given an intravenous injection in the arm vein (vena mediana cubiti) for the first dose (day 1) and then by direct intratumoral injection under ultrasound guidance on days 2, 15, 16, 29, and 30 (Habib, N., 2002. supra). All given doses were 1 ml in volume and contained 3 x 10¹¹ PFU of AddJ.1520, which had been found to be well tolerated in a previous phase 1 study (Habib, N. et al., 2001 supra) .

Viruses and mammalian cells.

The Ad mutant AddI1520 was donated by A.J. Berk

(Molecular Biology Institute, University of California at Los Angeles) . Addll520 carries a 827-bp deletion in the ElB-55-kDa protein coding region, in combination with a stop codon to ensure no expression of the 55-kDa gene product. Ad recombinant Ad5Luc3 (Mittal, S.K., et al. 1993. Virus Res. 28: 67-90), a kind gift from F. Graham (McMaster University, Hamilton, Ontario, Canada) , is a replication-competent Ad that carries the luciferase reporter gene under the control of the simian immunodeficiency virus early promoter inserted into the E3 region of the Ad5 genome . Ad5GFP vector (Adeno-CMV5-GFP) was obtained from Quantum Biotechnologies, Inc. (Montreal, Quebec, Canada) . HeLa and 293 cells were grown as monolayers in Dulbecco modified Eagle medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) , L-glutamine, and antibiotics.

Insect cells and baculoviruses .

Spodoptera frugiperda (Sf9) cells were maintained as monolayers at 28°C in Grace insect medium supplemented with 10% FCS and antibiotics. The recombinant baculoviruses expressing Ad2 capsid proteins hexon, Pb, and fiber have been described previously (Karayan, L. et al., 1997. J. Virol. 71: 8678-8689 and Molinier- Frenkel, V. et al . , 2002. J. Virol. 76: 127-135). Recombinant Ad capsid proteins were produced in Sf9 cells and purified as described in previous studies (Boulanger, P., and F. Puvion. 1973. Eur. J. Biochem. 39: 37-42 and Molinier-Frenkel, V. et al . , supra).

Gel electrophoresis and Western blotting.

Sodium dodecyl sulfate (SDS-denatured proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) in 10% acrylamide gels by using a discontinuous buffer system (Laemmli, U.K. 1970. Nature 227: 680-685). Native proteins were separated in 8% polyacrylamide gels in the same discontinuous buffer system but without SDS and twice the normal buffer concentration. Transfer of proteins onto nitrocellulose membranes (Hybond ECL; Amersham Biosciences) was carried out by using a semidry blotting system. Blots were blocked with skimmed milk in TBS-T (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Tween 20) and then successively reacted with patient sera, followed by the addition of phosphatase-labelled anti-human immunoglobulin M (IgM) and IgG conjugate (Sigma-Aldrich) .

Isolation of Pb antibody from serum samples.

Recombinant Pb protein was electrophoresed in a preparative SDS-PAGE gel and transferred onto a nitro-cellulose membrane. The protein band was then localized by Ponceau red staining and excised. Membrane strips with immobilized Pb protein were incubated with human sera overnight at 4°C. After several rinses with phosphate-buffered saline (PBS) , the antibodies were eluted with 5 mM glycine-HCl (pH 2.3) -500 mM NaCl-5% FCS for 30 s and immediately neutralized with 0.5 M Na₂HP0₄ (pH 9.5) to a final concentration of 50 mM. Two successive elutions were performed to maximize the antibody recovery.

Normalization of Pb antibody in serum samples and

Pb-specific antibody preparations.

The quantity of Pb antibodies eluted from the nitrocellulose membranes were determined in comparison to their corresponding original sera by using dot blot analysis . Aliquots of Pb antibody samples and of the patients ' sera from which Pb antibodies were isolated (at dilutions of 1:500, 1:1,000, 1:2,000, and 1:3,000) were spotted onto the same nitrocellulose membrane. The membrane was then reacted with peroxidase-conjugated anti-human IgG antibody and developed by chemiluminescence by using the SuperSignal substrate (Pierce) . The amounts of antibodies recovered from the patients ' sera were quantitatively determined by densitometric analysis of the dots by using the VersaDoc image analyzer and the Quantity One program (Bio-Rad) . Circulating anti-Ad antibodies.

(i) Anti-Ad antibody titer.

The whole anti-Ad antibody titers of patients' sera obtained at preinjection and 10 days after AddI1520 administration were determined by enzyme-linked immunosorbent assay (Virolab, Berkeley, Calif.) (Habib, N. 2002 supra) .

(ii) Neutralizing antibodies and virus NA assays.

Aliquots of Ad5GFP virus were preincubated with serial dilutions of heat-inactivated patient sera for

1 h at 37°C, the supernatants were removed and replaced with fresh culture medium. The cells were observed 48 h after infection for GFP expression. The NA titer was expressed as the dilution of serum that gave a 50% GFP expression compared to the control. Ad5Luc3 virus was used according to the protocol described above, except that the cells were harvested at 18 h after infection and the cell lysates were analyzed for luciferase activity by using luciferin substrate and a Lumat LB 9501 luminometer (Berthold, Germany) .

Epitope mapping of Pb antibodies by phage biomapping.

The filamentous phage hexapeptide library was a kind gift of G. Smith (University of Columbia, Columbia, Mo.) . Affinity selection of phages bound to immobilized Pb antibodies was carried out according to published protocols (Hong, S.S. et al., 2000, Viral Immunol. 13: 353-371; and Hong, S.S., and P. Boulanger. 1995. EMBO J. 14: 4714-4727; and Hong, S.S., 1997, EMBO J. 16:

2294-2306) . The phagotopes carried by the phages were identified by DNA sequencing of the piII gene of the phage .

Phage-mediated depletion of specific antibodies.

The principle of this method is based on the normal content of IgG in human serum (ca. 15 mg/ l) . Thus,

1 μg of total IgG molecules, and among them probably fewer than 10% would consist of Pb antibodies, and 1% of antibodies specific for a particular Pb epitope, i.e., 150 ng at most. Since the average molecular mass of an

IgG molecule is 150 kDa, incubation of 0.1 μl of serum in 100 μl of PBS (1:1,000 dilution, 6 x 10¹⁰ moles of IgG) with 10¹¹ phages (give pill proteins per phage, carrying one epitope each) would provide a 10-fold excess of epitope over their corresponding antibody molecules . Phage and serum were incubated overnight at

4°C with gentle mixing and then phage-antibody complexes were removed by centrifugation at 10,000 x g for 5 min. The supernatant was then assayed for virus NA as described above.

RESULTS

Characterization of circulating antibodies from patients administered AddI1520.

Human sera used in the present study were issued from 17 patients who underwent a clinical trial with the conditionally replicative Addll520. The serum samples were collected before (DO; pretreatment) and at 10 days after (D10) Ad administration and then assayed for their whole-antibody response against Ad5 virus and for their NA titer against Ad5. Their reactivity against the three major Ad capsid proteins - hexon, Pb, and fiber - was also analyzed. For comparison purposes, sera from four healthy blood donors were included in the present study (Table 1) .

TABLE 1. Characteristics of serum samples from healthy blood donors and from patients before or after administration of Addll520^a

(i) Ad5 antibodies and neutralizing antibodies.

The whole-antibody titer against Ad5, which was assayed for six patients (patients 3 to 8 , DO and D10 samples; Table 1), ranged between 128 to 1,024 in the pretreatment samples, whereas by 10 days after Ad administration there was an overall augmentation in antibody titer for all six patients: either 4-fold (patient 6), 16-fold (patient 4), 32-fold (patients 3, 5, and 7), or 64-fold (patient 8). The anti-Ad5 NA titers of the serum samples were analyzed, and 14 sera of 21 (67%) showed low titers of pre-existing NA (titers of <256) , whereas 7 sera (33%) had high titers of >1,024 (Table 1) . At 10 days after Ad treatment, there was an increase in NA titers for 12 patients (71%) , whereas there was no change for the five other patients . The increase in NA titers ranged from 2- to 16-fold, with one patient (patient 8) showing a 512-fold increase. These results indicated that pre-existing NA antibodies were low in most of the patients; however, after Ad administration their titers augmented rapidly and significantly.

(ii) Antibody reactivity profile against Ad capsid proteins.

The patients ' sera were assayed for IgM and IgG subclass antibodies against native or denatured hexon, Pb, and fiber proteins. However, the reactivity of IgM antibodies were found to be very low and negligible compared to IgG antibodies (data not shown) , and thus only IgG reactivity is described here. The antibody reactivity profile of the patients ' sera are summarized in Table 1. In the samples taken before Ad injection, 13 samples (62%) had hexon antibodies, 17 samples (81%) had Pb antibodies, and 17 samples (81%) had fiber antibodies. At 10 days after virus injection, most of the patients ' sera showed either an increase in reactivity or acquired antibodies to hexon (4 of 17 patients [24%]), Pb (8 of 17 patients [45%]), or fiber (7 of 17 patients [41%]) proteins.

In the majority of the samples, hexon antibodies were detected mainly against the native protein and very few were detected against SDS-denatured hexon, implying that most of the antibodies were directed against SDS-sensitive, conformational epitopes on the protein. In contrast, fiber and Pb antibodies were detected against both the native and the SDS-denatured proteins, suggesting that these antibodies recognized both linear and conformational-dependent epitopes. Taken together, these observations suggested that among the three capsid components of the virus, antibodies to both Pb and fiber appeared to be better induced compared to antibodies to hexon.

There was no clear correlation between the NA titers and antibody reactivity against the capsid proteins. However, it is noteworthy that, for two serum samples (sera 15 and 17) that had uniquely hexon antibodies, the NA titers were low.

Isolation of Pb-specific antibodies.

We selected sera from two patients who had preexisting Pb antibodies prior to the Ad treatment (DO sera of patients 1 and 2) and from two other patients with newly acquired Pb antibodies (D10 sera of patients

3 and 4) (refer to Table 1 and Fig. la and b; patients 1, 2, 3, and 4) for further studies. Antibodies recognizing the Ad Pb protein were isolated by adsorption of the patients ' sera on electrophoretically purified Pb protein immobilized on a nitrocellulose membrane. The adsorbed antibodies were then acid eluted from the membrane, immediately neutralized, and analyzed for their recovery and reactivity.

To ensure their functionality and monospecificity against Pb proteins, the isolated antibodies were tested for their reactivity against Pb protein by Western blot analysis (Fig. lc) . The positive reactivity of all four different Pb antibodies suggested that the brief acid treatment during the elution process was apparently not detrimental to their antigen-binding capacity and to their functionality as antibodies. Their nonreactivity against fiber protein that was present on the same blot showed that the antibodies recovered were specific toward Pb proteins (Fig. lc) . The amounts of IgG antibodies recovered were determined by dot blotting, followed by quantitative densitometric analysis. The Pb antibodies recovered from serum samples 1, 2, 3, and 4 contained 1/2,500, 1/2,000, 1/1,428, and 1/1,666 of the total IgG content of their original sera, respectively.

Neutralizing activity of isolated Pb antibodies.

The Pb of the Ad virion has been shown to react with cell surface integrin molecules which subsequently result in the internalization of the virion. Pb antibodies could therefore have a neutralizing effect on Ad infection by interfering with the step of virion endocytosis and internalization. We therefore tested whether the isolated Pb antibodies had any neutralizing activity against Ad virus: first, in the course of a normal viral infection, and, second, at the step of virus internalization. The Ad5Luc3 virus used was a replication-competent virus that carried the luciferase reporter gene in its deleted E3 region (Mittal, S.K., et al., 1993, Virus Res. 28: 67-90). Ad5Luc3 was preincubated for 1 h at 37°C with (i) mock, serum, (ii) total serum, or (iii) the corresponding isolated Pb antibodies . The total sera were diluted accordingly to normalize their IgG content to that of their corresponding Pb antibody preparations . After the preincubation period, the virus-antibody samples were added to HeLa cells, and infection was allowed to occur for 18 h at 37°C. At 18 h post-infection, the cells were harvested and assayed for luciferase activity as a measure of Ad infection efficiency. The four serum samples from patients 1, 2, 3, and 4 blocked Ad-mediated gene delivery by 65, 83, 96, and 98%, respectively (Fig. 2a) . Their corresponding Pb antibodies inhibited Ad-mediated gene transfer by 59, 56, 77, and 86%, respectively (Fig. 2b) . This result suggested that a significant proportion of the Ad Na activity from the serum samples could be attributed to Pb antibodies .

We next addressed the question as to whether virus NA by the Pb antibodies occurred at the step of virus internalization. The entry of Ad into cells is a two-step process that first involves virus attachment on the cell surface, followed by endocytosis and internalization of the virus. The virus attachment step can occur at 4°C, but virus internalization can only occur at 37°C. To study the effect of Pb antibodies at the level of virus internalization, Ad5Luc3 virus was first allowed to attach to HeLa cells at 4°C for 30 in. Unattached viruses were then removed, Pb antibodies were added to the virus-cell monolayer for another 30 min at

4°C, and the cells were warmed to 37°C for virus internalization to occur. The infection was then allowed to proceed for 18 h at 37°C, and the cells were collected and assayed for luciferase activity. As shown in Fig. 2c, the presence of Pb antibodies at the step of virus internalization significantly reduced Ad-mediated gene delivery. The percent inhibitions observed were 47, 57, 55, and 57% for the Pb antibodies isolated from the serum samples of patients 1, 2, 3, and 4, respectively.

Mapping of the Pb epitopes by phage biopanning of Pb antibodies .

In order to determine the epitopes recognized by the Pb antibodies in the Pb sequence, we used the technique of phage biopanning with a phage-displayed hexapeptide library. Bacteriophages bound to immobilized Pb antibodies were selected, enriched, and sequenced after three rounds of adsorption and/or elution. The amino acid sequences (or phagotopes) thus obtained were compared to the Pb protein sequence. As shown in Table 2, the different phagotopes were tentatively aligned with correspding regions of the Pb sequence according to amino acid and peptide motif identity or homology. A sequence alignment could be proposed with a reasonable degree of probability for the majority of the phagotopes isolated, since there were at least three to four contiguous amino acids out of six that were identical or had a strong homology with corresponding residues in Pb .

Ten individual different antigenic epitopes within the Pb protein sequence could be identified. They were separated into three different classes (Table 2) . Class I included epitopes unique to DO samples containing preexisting Pb antibodies. Class II epitopes were unique to D10 samples recovered from patients' sera after Ad administration (newly acquired Pb antibodies) .

Class III epitopes were common to both types of Pb antibodies, since they were found in samples from patients both before and after Ad treatment. TABLE 2. Phagotypes isolated by phage biopanning of Pb antibodies

a The phagotopes recovered from immobilized Pb-specific Pb antibodies (Ab) were grouped according to conserved residues and peptide motifs and then aligned with homologous regions of the Ad Pb sequence (underlines, with residue numbers as indicated) . Homologous or identical residues at similar locations are in boldface. Patient numbers correspond to the patient identification numbers in Table 1. Note that motif LDD-WGV likely corresponded to a discontinuous epitope.

Most of the epitopes corresponded to regions of high antigenic index, according to the predictive method of Jameson-Wolf (Fig. 3) . This was the case for the three major epitopes of the first class, mapped to positions ^S3NSIRYSE⁵⁹, ²¹⁷LGFDPVTGL²²⁵, AND ³³⁸AIRGDTF³⁴⁴ in the Pb sequence. Interestingly, one of the epitopes contained the tripeptide sequence, RGD (underlined) , which is responsible for interacting with cell surface integrins . Likewise, the three epitopes of class III that mapped within the regions ¹¹³LDDRSHWG¹²¹, ³¹³SGSGAE³¹⁸, AND ⁴⁰⁰FTQYRSWYL⁴⁰⁸ showed a high probability of accessibility and antigenicity (Fig. 3) . In the class II epitopes, ⁷⁰YLVDNKS^7S, ¹⁹³LKVGRQ¹⁹⁸, AND ^S53PYVYK^SS6 corresponded to regions of high antigenic index; however, the epitope ²²⁴GLVMPGV²³⁰ had a relatively low probability of antigenicity.

Phage-mediated depletion of RGD- and other epitope-directed Pb antibodies.

In order to determine the contribution of anti-RGD antibody in Ad vector NA, the unique phage carrying the epitope RGDVTF was amplified and incubated with 17 different serum samples which had Pb antibodies and moderate to high Ad NA titers (D10 samples from patients 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, and 16 and samples 18, 19, 20, and 21 from healthy donors) . After removal of the phage (RGD) -antibody complexes by centrifugation, the serum supernatants were tested for Ad NA, which were indirectly assayed by their effect on gene transduction by using the Ad5Luc3 vector. As a control for nonspecific depletion of antibodies, the phage library carrying random peptides was used. As shown in Fig. 4a, depletion of RGD-antibodies resulted in an approximately 1.5-fold increase in Ad-mediated gene transfer for only three serum samples, whereas there was no significant change for the 14 other samples, suggesting that RGD antibodies were present in negligible amounts, if any, in these serum samples.

We performed the same antibody depletion assays by using phages carrying the epitopes NVRYSW (Fig. 4b) , AVTGID (Fig. 4c), HSGAEF (Fig. 4d) , NFTQVG (Fig. 4e) , and LDDSYS (Fig. 4f) , representing the different class I and class III epitopes mapped on the Pb protein. The class II epitopes YLVDNKS, LKVGRQ, GLVMPGV, and PYVYK were not included in the present study since we were not able to resuscitate the phages carrying these epitopes. The strongest effect observed after antibody depletion with the different phages was in the order NVRYSW, AVTGID, HSGAEF, NFTQVG, RGDVTF, and LDDSYS, based on the fold increase in Ad-mediated gene transfer efficiency and the number of serum samples that induced a significant increase in luciferase expression. These data implied (i) that the RGD antibodies were poorly neutralizing and (ii) that the motifs identified on Pb, i.e., ^S3NSIRYS^S8, ²²²VTGL²²⁵, ³¹⁵SGAE³¹⁸, and ⁴⁰¹FTQ⁴⁰³, which were homologous to the phage sequences NVRYSW, AVTGID, HSGAEF, and NFTQVG, respectively, represented potential major epitopes of neutralizing antibodies.

Claims

1. A peptide comprising an adenoviral (Ad) penton base epitope for a human neutralising antibody.

2. The peptide of claim 1 wherein the epitope is a fragment from within region 50-120, 191-233 or 310-408 of Ad5 or an equivalent region within another Ad serotype, or an immunologically equivalent variant of such a fragment.

3. The peptide of claim 1 wherein the epitope is selected from the following group of Ad5 epitopes or an equivalent region within another Ad serotype, or is an immunologically equivalent variant or fragment of one of these epitopes :

^S0GGRNSIRYSELA⁶¹ "TRVYLVDNKSTD⁷⁸ 1⁰⁹QTINLDDRSHWG¹²⁰

¹⁹¹HYLKVGRQNGVL²⁰¹ 2¹⁵FRLGFDPVTGLV²²⁶ 2²²VTGLVMPGVYTN²³³ 3¹⁰SNSSGSGAEENS³²¹ 3³⁶DHAIRGDTFATR³⁴⁷

³⁹⁷DSTFTQYRSWYL⁴⁰⁸ S⁴⁸RRTCPYVYKALG⁵⁵⁹.

4. The peptide of claim 3 wherein the epitope is selected from the group comprising:

NSIRYSE, LGFDPVTGL, AIRGDTF, LDDRSHWG, SGSGAE, FTQYRSWYL, YLVDNKS, LKVGRQ, PYVYK and GLVMPGV, GRNSIRYSEL, TINLDDRSHWG, RLGFDPVTGLV, DHAIRGDTFA, NNSGSGAEEN, VYLVDNKST, STFTQYRSWYL, LKVGRQNG, TGLVMPGV, CPYVYKALGI, NSIRYS, VTGL, SGAE, and FTQ, or immunologically equivalent variants and fragments thereof .

5. The peptide of any preceding claim which is no more than 25 amino acids in length.

6. A pharmaceutical composition comprising a peptide as defined in any one of claims 1 to 5 and a pharmaceutically acceptable carrier, diluent or adjuvant .

7. A pharmaceutical composition comprising a peptide as defined in any one of claims 1 to 5 and an adenoviral vector.

8. A product containing (a) a peptide as defined in any one of claims 1 to 5, and (b) -an adenoviral vector as a combined preparation for separate, simultaneous or sequential administration to a patient as part of a gene therapy or cancer treatment.

9. A peptide as claimed in any one of claims 1 to 5 for use in therapy.

10. A method of gene or cancer therapy involving administration to a patient of an adenoviral vector, wherein a peptide as claimed in any one of claims 1 to 5 is also administered to said patient as part of the therapeutic regimen.

11. A method as claimed in claim 10 wherein said peptide is administered simultaneously with said adenoviral vector.

12. A nucleic acid molecule encoding a peptide as claimed in any one of claims 1 to 5.

13. A pharmaceutical composition comprising a nucleic acid molecule as claimed in claim 12 and a pharmaceutically acceptable carrier, diluent or adjuvant .

14. A pharmaceutical composition comprising a nucleic acid molecule as claimed in claim 12 and an adenoviral vector.

15. A product containing (a) a nucleic acid molecule as claimed in claim 12, and (b) an adenoviral vector as a combined preparation for separate, simultaneous or sequential administration to a patient as part of a gene therapy or cancer treatment .

16. An adenovirus wherein one or more of its penton base epitopes for a neutralising antibody has been modified to reduce its immunogenicity.

17. An adenovirus as claimed in claim 16 wherein the modified epitope is found within region 50-120, 191-233 or 310-408 of Ad5 or an equivalent region within another Ad serotype .

18. An adenovirus as claimed in claim 16 wherein the modified epitope is selected from the following group of Ad5 epitopes or an equivalent region within another Ad serotype :

S⁰GGRNSIRYSELA⁶¹

"TRVYLVDNKSTD⁷⁸

¹⁰⁹QTINLDDRSHWG¹²⁰

¹⁹¹HYLKVGRQNGVL²⁰¹

²¹⁵FRLGFDPVTGLV²²⁶ 2²²VTGLVMPGVYTN²³³

³¹⁰SNSSGSGAEENS³²¹

³³⁶DHAIRGDTFATR³⁴⁷ ³⁹⁷DSTFTQYRSWYL⁴⁰⁸ 5⁴⁸RRTCPYVYKALG⁵⁵⁹.

19. A modified Adenovirus which incorporates one or more Pb capsomers from a non-native serotype .

20. A method of evaluating a patient's serum, which method comprises contacting, in vi tro, a sample of the serum with one or more peptides as defined in any one of claims 1 to 5.

21. A method as claimed in claim 20 wherein the sample is contacted with at least three different epitopes as defined in any one of claims 1 to 5.

22. A solid support having immobilised thereon one or more peptides as defined in any one of claims 1 to 5.

23. A solid support as claimed in claim 22 having immobilised thereon at least three different epitopes as defined in any one of claims 1 to 5.

24. A vaccine formulation comprising as an adjuvant a peptide as defined in any one of claims 1 to 5.

25. A product containing (a) a peptide as claimed in any one of claims 1 to 5, and (b) an Ad hexon protein as a combined preparation for separate, simultaneous or sequential administration to a patient as a vaccine.

26. A product as claimed in claim 25 wherein the peptide of component (a) is in the form of a lipopeptide.

27. A product as claimed in claim 25 or 26 wherein the peptide of component (a) is conjugated at the C-terminus with palmitoyl-lysylamide .

28. A method of filtering blood to remove neutralising antibodies to epitopes of the adenoviral penton base, which method comprises contacting the blood outside the body with one or more peptides as claimed in any one of claims 1 to 5.

29. An antibody or antibody fragment which is immunoreactive with a neutralising antibody which itself has as an epitope therefor an epitope as defined in any one of claims 1 to 5, for use in therapy.

30. A method of gene or cancer therapy involving administration to a patient of an adenoviral vector, wherein an antibody or antibody fragment as defined in claim 29 is also administered to said patient as part of the therapeutic regimen.

31. A method of evaluating a patient's serum, which method comprises contacting, in vi tro, a sample of the serum with one or more antibody or antibody fragments as defined in claim 29.

32. A method of filtering blood to remove neutralising , antibodies to epitopes of the adenoviral penton base, which method comprises contacting the blood outside the body with one or more antibodies or antibody fragments as defined in claim 29.