CA2283628A1 - Chimeric adenoviral coat protein and methods of using same - Google Patents

Abstract

The present invention provides a chimeric adenoviral coat protein (particularly a chimeric adenovirus hexon protein). The chimeric adenovirus coat protein has a decreased ability or inability to be recognized by a neutralizing antibody directed against the corresponding wild-type adenovirus coat protein.

Description

WO 98/40509 PCT/~JS98/05033 CHIMERIC ADENOVIRAL COAT PROTEIN
AND METHODS OF USING SAME
TECHNICAL. FIELD OF THE INVENTION
The present invention relates to a chimeric adenoviral coat protein and a recombinant adenovirus comprising same. In particular, the invention provides a chimeric adenoviral hexon protein and a recombinant adenovirus comprising the chimeric adenoviral hexon protein. Such a recombinant adenovirus can be employed inter alia in gene therapy.
BACKGROUND OF THE INVENTION
In vivo gene therapy is a strategy in which nucleic acid, usually in the form of DNA, is administered to modify the genetic repertoire o' target cells for therapeutic purposes. This can be accor~p~ished efficiently using a recombina:~t aaenov~ru~ vector encoding a so-called "therapeutic gene". F, therapeutic gene is generally considered a gene that corrects or compensates for an underlying protein deficit or, alternately,. a gene that is capable of down-regulating a particular gene, or counteracting the negative effect, cf :ts encoded product, in a given disease state o.- ~>ynd:omc. :-:ecombinant adenoviral vectors have beer: used tc transfer one or more recombinant genes to diseased cells or tissues in need of treatment. As reviewed by Crystal, Science, 270, 904-410 (1995), such vectors are preferred over other vectors commonly employed for gene therapy (e. g., retroviral vectors) since adenoviral vectors can be produced in high titers (i.e., up to 1013 viral particles/ml), and they efficiently transfer genes to nonreplicating, as well as replicating, cells. Moreover, adenoviral vectors are additionally preferred based on their normal tropism for the respiratory epithelium in cases where the targeted tissue for somatic gene therapy is the lung, as well as for other reasons (see, e.g., Straus, In Adenoviruses, Plenan Press, New York, NY, 951-496 (1984)); Horwitz et al., In Virology, 2nd Ed., Fields et al., eds., Raven Press, New York, NY, 1679-1721 (1990); Berkner, BioTechniques, 6, 616 (1988); Chanock et al., JAMA, 195, 151 (1966); Haj-Ahmad et al., J. Virol., 57, 267 (1986);
and Ballay et al., EMBO, 4, 3861 (1985)).
There are 49 human adenoviral serotypes, categorized into 6 subgenera (A through F) based on nucleic acid comparisons, fiber protein characteristics, and biological properties (Crawford-Miksza et al., J. Virol., 70, 1836-1844 (1996)). The group C viruses (e.g., serotypes 2 and 5, or Ad2 and Ad5) are well characterized. It is these serotypes that currently are employed for gene transfer studies, including human gene therapy trials (see, e.g., Rosenfeld et al., Science, 252, 431-434 (1991); Rosenfeld et al., Cell, 68, 143-155 (1992); Zabner, Cell, 75, 207-216 (1993); Crystal et al., Nat. Gen., 8, 42-51 (1994);
Yei et al., Gene Therapy, 1, 192-200 (1994); Chen et al., Proc. Natl. Acad. Sci., 91, 3059-3057 (1994); Yang et al., Nat. Gen., 7, 362-369 (1994); Zabner et al., Nat. Gen., 6, 75-83 (1994)). Other groups and serotypes include, but are not limited to: group A (e. g., serotypes 12 and 31), group B (e. g., serotypes 3 and 7), group D (e. g., serotypes 8 and 30), group E (e. g., serotype 4) and group F (e. g., serotypes 40 and 41) (Horwitz et al., supra).
In terms of general structure, all adenoviruses examined to date are nonenveloped, regular icosahedrons of about 65 to 80 nanometers in diameter. Adenoviruses are comprised of linear, double-stranded DNA that is complexed with core proteins and surrounded by the adenoviral capsid. The capsid is comprised of 252 capsomeres, of which 240 are hexons and 12 are pentons. The hexon ...._ r. r ...._...

capsomere provides structure and form to the capsid (Pettersson, in The Adenoviruses, pp. 205-270, Ginsberg, ed., (Plenum Press, New York, NY, 1984)), and is a homotrimer of the hexon protein (Roberts et al., Science, 232, 1198-1151 (1986)). The penton comprises a penton base, which is bound to other hexon capsomeres, and a fiber, which is noncovalently bound to, and projects from, the penton base. The penton fiber protein comprises three identical polypeptides (i.e., polypeptide IV). The Ad2 penton base protein comprises five identical polypeptides (i.e., polypeptide III) of 571 amino acids each (Boudin et al., Virology, 92, 125-138 (1979)).
The adenoviruses provide an elegant and efficient means of transferring therapeutic genes into cells.
However, one problem encountered with the use of adenoviral vectors for gene transfer in vivo is the generation of antibodies to antigenic epitopes on adenoviral capsid proteins. If sufficient in titer, the antibodies can limit the ability of the vector to be used more than once as an effective gene transfer vehicle.
For instance, animal studies demonstrate that intravenous or local administration (e.g., to the lung, heart or peritoneum) of an adenoviral type 2 or 5 gene transfer vector can result in the production of antibodies directed against the vector which prevent expression from the same serotype vector administered 1 to 2 weeks later (see, e.g., Yei et al., su ra; Zabner (1999), supra;
Setoguchi et al., Am. J. Respir. Cell. Mol. Biol., 10, 369-377 (1994); Kass-Eisler et al., Gene Therapy, 1, 395-402 (1994); Kass-Eisler et al., Gene Therapy 3, 154-162 (1996)). This is a drawback in adenoviral-mediated gene therapy, since many uses of an adenoviral vector (e. g., for prolonged gene therapy) require repeat administration inasmuch as the vector does not stably integrate into the host cell genome. The mechanism by which antibodies directed against an adenovirus are able to prevent or reduce expression of an adenoviral-encoded gene is unclear. However, the phenomenon is loosely referred to as "neutralization", and the responsible antibodies are termed "neutralizing antibodies."
There are three capsid structures against which neutralizing antibodies potentially can be elicited:
fiber, penton, and hexon (Pettersson, supra). The hexon protein, and to a lesser extent the fiber protein, comprise the main antigenic determinants of the virus, and also determine the serotype specificity of the virus (Watson et al., J. Gen. Virol., 69, 525-535 (1988);
Wolfort et al., J. Virol., 62, 2321-2328 (1988); Wolfort et al., J. Virol., 56, 896-903 (1985); Crawford-Miksza et al., supra). Researchers have examined and compared the structure of these coat proteins of different adenoviral serotypes in an effort to define the regions of the proteins against which neutralizing antibodies are elicited.
The Ad2 hexon trimer is comprised of a pseudohexagonal base and a triangular top formed of three towers (Roberts et al., supra; Athappilly et al., J. Mol.
Biol., 242, 430-455 (1994)). The base pedestal consists of two tightly packed eight-stranded antiparallel beta barrels stabilized by an internal loop. The predominant regions in hexon protein against which neutralizing antibodies are directed appear to be in loops 1 and 2 (i.e., LI or 11, and LII or 12, respectively) in one of the three towers. For instance, Kinloch et al. (J. Biol.
Chem., 258, 6431-6436 (1984)) compared adenoviral hexon sequences and theorized that the serotype-specific antigenic determinants on hexon are located in amino acid residues 120 to 470 encompassing the 11 and 12 loops since type-specific sequence differences are mainly concentrated in this region. Toogood et al. (J. Gen.
T ~

Virol., 73, 1429-1435 (1992)) used peptides from this region to generate specific anti-loop antisera and confirmed that antibodies against residues 281-292 of 11 and against residues 441-455 of 12 were sufficient to neutralize infection. Also, Crompton et al. (J. Gen.
Virol., 75, 133-139 (1994)) modified these loops to accept neutralizing epitopes from polio virus, and demonstrated that infection with the resultant adenoviral vector generated neutralizing immunity against polio virus. More recently it was demonstrated that the hexon protein is composed of seven discrete hypervariable regions in loops and 1 and 2 (HVR1 to HVR7) which vary in length and sequence between adenoviral serotypes (Crawford-Miksza et al., supra).
Less is known regarding the regions of the fiber protein against which neutralizing antibodies potentially can be directed. However, much data is available on the structure of the fiber protein. The trimeric fiber protein consists of a tail, a shaft, and a knob (Devaux et al., J. Molec. Biol., 215, 567-588 (1990)). The fiber shaft region is comprised of repeating 15 amino acid motifs, which are believed to form two alternating beta strands and beta bends (Green et al., EMBO J., 2, 1357-1365 (1983)). The overall length of the fiber shaft region and the number of 15 amino acid repeats differ between adenoviral serotypes. The receptor binding domain of the fiber protein and sequences necessary for fiber trimerization are localized in the knob region encoded by roughly the last 200 amino acids of the protein (Henry et al., J. Virol., 68(8), 5239-5246 (1994)); Xia et al., Structure, 2(12), 1259-1270 (1994)). Furthermore, all adenovirus serotypes appear to possess a type of specific moiety located in the knob region (Toogood et al., supra.) Given the existence of these potential epitopes in hexon protein and fiber protein, it is understandable t.
.. . j. 1,,,°. ..r"~

CA 02283628 1999-09-lO,It' :'~'u'~' :~,~.+~I,3~~j;) v~:W~-~u.;~~ '~ -l21:\. \l)~:L:1';\-\11 Iv,W:IIL;yJt);5_n ,....1 . . 1~
~.. m. ~ v. . a .: .~
b that, in some cases, di~ficulties have been encountered using adenovirus as a vector for gene therapy.
Accordingly, recombinant adenoviral vectors capable o' escaping such r_eutral-zi:lg antibodies (in the ever.' t.hay are preexisting and hamper gene expression commanded by adenovirus in an initial dose;, and which would allow repeat. doses of ader_ov~.ral vec*~ors to be administered, would signiz~cantly advance current gene therapy methodology.
Thus, the present invention seeks to overcome at east some of the aforesaid prab'_e:ra of reco_mb~:~ant 'aderov~.ral none therapy. T_n particu'.ar, or.e aspect of the present invention provides a racomr~inant aderov-ir~~s ~ccmprising a chimeric coat protein that has a decreased ability or inability to be recognized by antibodies ;i.e., neutra_izi:~a antiDOdies) direr _ed agains t the corresponding wild-type adenovirus coat protein. These and ot:~er objects and advantages of Lhe present ir.venticr_, as ~aell as addit;on31 inventive features, wili be apparent i.rom the desc;ipz;o.~. cf the invention provided ~:erei:~.
BRIEF SU~RY OF THE INVENTION
The present invenL~or. provides a chimeric adoncvirus coat protein (part=cularly a chimer;c adenovirus hexon protein) comprising a nonnative amino acid sequence. The c'~imeric adenov:.rus coat prccein is not recognized by, cr has a decreased abi?ity to be recognized by, a neutralizing antibody directed against the corresponding wild-Lype (i.e., native) coat protein. ~'he chimeric adenovirus coat protein enables a vector (such as an adenovirus) comprising the corresponding protein to be ad:-~ir.:.stered repetitively, or to be administered following administration of an ad~encvir~ss vector comprising the corresponding wild-type coat protein. It also enables a AMENDED SHEET

vector (such as an adenovirus) comprising the chimeric protein to be administered and effect gene expression in the case where there are preexisting neutralizing antibodies directed against the wild-type adenovirus coat protein. The present invention also provides a vector, particularly an adenoviral vector, that comprises a chimeric adenovirus coat protein such as chimeric adenovirus hexon protein (and which optionally further comprises a chimeric adenovirus fiber and/or penton base protein), and methods of constructing and using such a vector.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a diagram of the method employed to construct the vector pAd70-100d1E3.fiber7.
Figure 2 is a partial restriction map of the vector pGBS.59-100(HSF:RGD).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides, among other things, a chimeric adenovirus coat protein. The chimeric adenovirus coat protein comprises a nonnative amino acid sequence, such that the chimeric adenovirus coat protein (or a vector comprising the chimeric adenovirus coat protein) has a decreased ability or inability to be recognized by antibodies (e. g., neutralizing antibodies) directed against the corresponding wild-type adenovirus coat protein.
Chimeric Adenovirus Coat Protein A "coat protein" according to the invention is either an adenoviral penton base protein, an adenoviral hexon protein, or an adenoviral fiber protein. Preferably a coat protein is a adenoviral hexon protein or an adenoviral fiber protein. Any one of the serotypes of human or nonhuman adenovirus can be used as the source of the coat protein, or its gene or coding sequence.
Optimally, however, the adenovirus coat protein is that of a Group B or C adenovirus and, preferably, is that of Adl, Ad2, Ad3, AdS, Ad6, Ad7, Adll, Adl2, Adl4, Adl6, Ad2l, Ad34, Ad35, Ad40, Ad4l, or Ad48.
The chimeric adenovirus coat protein (or a vector, such as adenoviral vector, comprising the chimeric adenovirus coat protein) has a decreased ability or an inability to be recognized by an antibody (e.g., a neutralizing antibody) directed against the corresponding wild-type adenovirus coat protein. A "neutralizing antibody" is an antibody that either is purified from or is present in serum. As used herein, an antibody can be a single antibody or a plurality of antibodies. An antibody is "neutralizing" if it inhibits infectivity of (i.e., cell entry) or gene expression commanded by an adenovirus comprising wild-type coat protein, or if it exerts a substantial deleterious effect on infectivity of or gene expression commanded by an adenovirus comprising wild-type coat protein, as compared, for instance, to any effect on any other adenoviral property.
An ability or inability of a chimeric coat protein to "be recognized by" (i.e., interact with) a neutralizing antibody directed against the wild-type adenovirus coat protein can be assessed by a variety of means known to those skilled in the art. For instance, the removal of one or more epitopes for a neutralizing antibody present in a wild-type adenovirus coat protein to generate a chimeric adenovirus coat protein will result in a decreased ability or inability of the chimeric coat protein to be recognized by the neutralizing antibody.
Also, such a decreased ability or inability to interact with a neutralizing antibody directed against wild-type coat protein can be demonstrated by means of a neutralization test (see, e.g., Toogood et al., supra;
Crawford-Miksza et al., su ra; Mastrangeli et al., Human Gene Therapy, 7, 79-87 (1996)), or as further described herein.
Generally, an "inability" of a chimeric adenovirus coat protein (or a vector comprising a chimeric adenovirus coat protein) to be recognized by a neutralizing antibody directed against wild-type adenovirus coat protein means that such an antibody does not interact with the chimeric coat protein, and/or exhibits no substantial deleterious effect on infectivity of or gene expression commanded by an adenovirus comprising wild-type coat protein, as compared, for instance, to any effect on any other adenoviral property.
A "decreased ability" to be recognized by neutralizing antibody directed against wild-type adenovirus coat protein refers to any decrease in the ability of the chimeric adenovirus coat protein (or a vector comprising the chimeric coat protein) to be recognized by an antibody directed against the corresponding wild-type adenovirus coat protein as compared to the wild-type adenovirus coat protein. When such ability/inability is assessed by means of a neutralization test in particular, preferably a "decreased ability" to be recognized by a neutralizing antibody directed against wild-type adenovirus coat protein is exhibited by from about a loo to about a 99~ increase in the ability of a recombinant adenovirus comprising the chimeric coat protein to cause a visible cytopathic effect (c.p.e.) in cells such as A599 cells or COS-1 cells (or other such cells appropriate for a neutralization assay) in the presence of the neutralizing antibody as compared to an adenovirus comprising the wild-type coat protein against which the neutralizing antibody is directed.

Furthermore, a decreased ability or inability of an adenovirus chimeric coat protein (or a vector comprising a chimeric adenovirus coat protein) to interact with a neutralizing antibody can be shown by a reduction of inhibition (from about loo to about 990) or no inhibition at all of cell infectivity by a recombinant vector (such as an adenoviral vector) containing the chimeric coat protein as compared to a recombinant vector containing the wild-type protein. Also, a decreased ability or inability of an adenovirus chimeric coat protein (or a vector comprising a chimeric adenovirus coat protein) to interact with a neutralizing antibody can be shown by a reduction of inhibition (from about loo to about 990) or no inhibition at all of gene expression commanded by a recombinant vector (such as an adenoviral vector) containing the chimeric coat protein as compared to a recombinant vector containing the wild-type coat protein.
These tests can be carried out when the recombinant adenovirus containing the chimeric coat protein is administered following the administration of an adenovirus containing the wild-type coat protein, or when the recombinant adenovirus is administered to a host that has never before encountered or internalized adenovirus (i.e., a 'naive" host). These methods are described, for instance, in the Examples which follow as well as in Mastrangeli et al., supra. Other means such as are known to those skilled in the art also can be employed.
The coat protein is "chimeric" in that it comprises a sequence of amino acid residues that is not typically found in the protein as isolated from, or identified in, wild-type adenovirus, which comprises the so-called native coat protein, or "wild-type coat protein". The chimeric coat protein thus comprises (or has) a "nonnative amino acid sequence". By "nonnative amino acid sequence" is meant any amino acid sequence (i.e., either component r . .. . rt residues or order thereof) that is not found in the native coat protein of a given serotype of adenovirus, and which preferably is introduced into the coat protein at the level of gene expression (i.e., by production of a nucleic acid sequence that encodes the nonnative amino acid sequence). Generally, the nonnative amino acid sequence can be obtained by deleting a portion of the amino acid sequence, deleting a portion of the amino acid sequence and replacing the deleted amino sequence with a so-called "spacer region", or introducing the spacer region into an unmodified coat protein. Preferably such manipulations result in a chimeric adenovirus coat protein according to the invention that is capable of carrying out the functions of the corresponding wild-type adenovirus coat protein (or, at least that when incorporated into an adenovirus, will allow appropriate virion formation and will not preclude adenoviral-mediated cell entry), and, optimally, that is not impeded in its proper folding.
Also, it is desirable that the manipulations do not result in the creation of new epitopes for differing antibodies, unless, of course, such epitopes do not interfere with use of an adenovirus containing the chimeric coat protein as a gene transfer vehicle in vivo.
In particular, a nonnative amino acid sequence according to the invention preferably comprises a deletion of a region of a wild-type adenovirus coat protein, particularly an adenovirus hexon or fiber protein.
Optimally the resultant nonnative amino acid sequence is such that one or more of the existing epitopes for neutralizing antibodies directed against the corresponding wild-type adenovirus coat protein have been rendered non-immunogenic. Desirably, the region deleted comprises from about 1 to about 750 amino acids, preferably from about 1 to about 500 amino acids, and optimally from about 1 to about 300 amino acids. It also is desirable that the region deleted comprises a smaller region less than about 200 amino acids, preferably less than about 100 amino acids, and optimally less than about 50 amino acids. The chimeric coat protein also desirably comprises a plurality of such deletions. Thus, according to the invention, the chimeric adenovirus coat protein comprises modification of one or more amino acids, and such modification is made in one or more regions.
In a preferred embodiment of the present invention, a nonnative amino acid sequence comprises a deletion of one or more regions of a wild-type adenovirus hexon protein, wherein preferably the hexon protein is the Ad2 hexon protein [SEQ ID N0:2] (which is encoded by the sequence of SEQ ID NO:1; GenBankO Data Bank Accession Number U20821), or the Ad5 hexon protein [SEQ ID N0:3] (GenBank~t Data Bank Accession Number M73260, which is encoded by the sequence of SEQ ID N0:4), or the Ad7 hexon protein (GenBankO Data Bank Accession Number x76551). Alternately, preferably the hexon protein is the protein sequence reported by Crawford-Miksza et al. (Ad2 hexon [SEQ ID N0:52], Ad5 hexon SEQ ID N0:54]). In particular, the sequences of Crawford-Miksza et al. differ over those reported in the GenBankO Data Bank in that the amino acid residue reported as the first in the Crawford-Miksza et al. sequences is not Met, and the Ad5 hexon sequence is reported as terminating with "Gln His" instead of with "Thr Thr". As employed herein, the numbering of adenovirus hexon amino acid residues corresponds to that in Crawford-Miksza et al.
Desirably the regions) of the deletion comprises an internal hexon protein sequence ("internal" meaning not at or near the C- or N-terminus of the protein; "near"
referring to a distance of 500 amino acids or less ), preferably a hypervariable region, e.g., as reported in Crawford-Miksza et al. In particular, optimally, the r internal region of the wild-type hexon protein that is deleted to generate the chimeric hexon protein comprises the entirety of Il loop, preferably from about residue 131 to about residue 331 of the Ad2 hexon protein [SEQ ID
N0:6] {which is encoded by the sequence of SEQ ID N0:5), or the corresponding region from another adenoviral serotype, e.g., particularly the corresponding region from Adl, Ad5 [SEQ ID N0:8] (which is encoded by the sequence of SEQ ID N0:7), Ad6, Ad7, Ad8, Adl2, Adl6, Ad40, Ad4l, Ad48, BAV3, or MAVl, especially as reported in Crawford-Miksza et al., supra.
Alternately, preferably the internal region of the wild-type hexon protein that is deleted to produce the chimeric hexon protein comprises one or more regions (e.g., smaller regions) of the 11 loop. Optimally the region deleted comprises a hypeavariable region.
Desirably the one or more regions c:f the 11 loop deleted are regions (i.e., hyperva~iab'.c~ ~eg:ons; selected from this group consisting of the HVF?1 region, ~he HVR2 region, the HVR3 region, the HVR4 region, the HVRS region, and the HVR6 region. Moreover, preferably the region of the wild-type protein that is deleted (or otherwise manipulated as described herein) occurs on the e~:te=na; surface of the hexon protein. Thus, HVR" E:V:~=;, HVRy, and HVRS -- each of which are externally locat~~i reaio.~.s o' the hexon protein -- are particularly preferred for deletion or modification.
The "HVR1 region" preferably comprises from about amino acid 137 to about amino acid 188 of the Ad2 hexon protein [SEQ ID N0:10] (which is encoded by the sequence of SEQ ID N0:9), or the corresponding region from another adenoviral serotype, e.g., particularly the corresponding region from Adl, Ad3, Ad5 [SEQ ID N0:12] (which is encoded by the sequence of SEQ ID N0:11), Ad6, Ad7, Ad8, Adll, Adl2, Adl4, Adl6, Ad2l, Ad34, Ad35, Ad40, Ad4l, Ad48, BAV3, or MAV1, especially as reported in Crawford-Miksza et al., supra.
The "HVR2 region" preferably comprises from about amino acid 194 to about amino acid 204 of the Ad2 hexon protein [SEQ ID N0:14] (which is encoded by the sequence of SEQ ID N0:13), or the corresponding region from another adenoviral serotype, e.g., particularly the corresponding region from Adl, Ad3, Ad5 [SEQ ID N0:16] (which is encoded by the sequence of SEQ ID N0:15), Ad6, Ad7, Ad8, Adll, Adl2, Adl4, Adl6, Ad2l, Ad39, Ad35, Ad40, Ad4l, Ad48, BAV3, or MAVl, especially as reported in Crawford-Miksza et al., supra.
The "HVR3 region" preferably comprises from about amino acid 222 to about amino acid 229 of the Ad2 hexon protein [SEQ ID N0:18] (which is encoded by the sequence of SEQ ID N0:17), or the corresponding region from another adenoviral serotype, e.g., particularly the corresponding region from Adl, Ad3, Ad5 [SEQ ID N0:20] (which is encoded by the sequence of SEQ ID N0:19), Ad6, Ad7, Ad8, Adll, Adl2, Adl4, Adl6, Ad2l, Ad34, Ad35, Ad40, Ad4l, Ad98, BAV3, or MAV1, especially as reported in Crawford-Miksza et al., s-upra.
The "HVR4 region" preferably comprises from about amino acid 258 to about amino acid 271 of the Ad2 hexon protein [SEQ ID N0:22] (which is encoded by the sequence of SEQ ID N0:21), or the corresponding region from another adenoviral serotype, e.g., particularly the corresponding region from Adl, Ad3, Ad5 [SEQ ID N0:24] (which is encoded by the sequence of SEQ ID N0:23), Ad6, Ad7, Ad8, Adll, Adl2, Adl4, Adl6, Ad2l, Ad34, Ad35, Ad40, Ad4l, Ad48, BAV3, or MAV1, especially as reported in Crawford-Miksza et al., supra.
The "HVRS region" preferably comprises from about amino acid 278 to about amino acid 294 of the Ad2 hexon protein [SEQ ID N0:26] (which is encoded by the sequence of SEQ ID N0:25), or the corresponding region from another adenoviral serotype, e.g., particularly the corresponding region from Adl, Ad3, Ad5 [SEQ ID N0:28] (which is encoded by the sequence of SEQ ID N0:27), Ad6, Ad7, Ad8, Adll, Adl2, Adl4, Adl6, Ad2l, Ad34, Ad35, Ad90, Ad4l, Ad48, BAV3, or MAVl, especially as reported in Crawford-Miksza et al., supra. In particular, preferably the deleted region comprises from about amino acid 297 to about amino acid 304 just outside of the HVR5 region of the Ad2 hexon protein [SEQ ID N0:30] (which is encoded by the sequence of SEQ ID N0:29), or the corresponding region from another adenoviral serotype, e.g., particularly the corresponding region from Adl, Ad3, Ad5 [SEQ ID N0:32] (which is encoded by the sequence of SEQ ID N0:31), Ad6, Ad7, Ad8, Adll, Adl2, Adl4, Adl6, Ad2l, Ad39, Ad35, Ad40, Ad4l, Ad98, BAV3, or MAV1, especially as reported in Crawford-Miksza et al., supra.
The "HVR6 region" preferably comprises from about amino acid 316 to about amino acid 327 of the Ad2 hexon protein [SEQ ID N0:34] (which is encoded by the sequence of SEQ ID N0:33), or the corresponding region from another adenoviral serotype, e.g., particularly the corresponding region from Adl, Ad3, Ad5 [SEQ ID N0:36] (which is encoded by the sequence of SEQ ID N0:35), Ad6, Ad7, Ad8, Adll, Adl2, Adl9, Adl6, Ad2l, Ad34, Ad35, Ad90, Ad4l, Ad48, BAV3, or MAV1, especially as reported in Crawford-Miksza et al., su ra.
In another preferred embodiment of the invention, the internal region of the wild-type hexon protein that is deleted to generate the chimeric hexon protein comprises the entirety of the 12 loop, preferably from about residue 423 to about residue 477 of the Ad2 hexon protein [SEQ ID
N0:38] (which is encoded by the sequence of SEQ ID N0:37), or the corresponding region from another adenoviral serotype, e.g., particularly the corresponding region from Adl, Ad3, Ad5 [SEQ ID N0:40] (which is encoded by the sequence of SEQ ID N0:39), Ad6, Ad7, Ad8, Adll, Adl2, Adl4, Adl6, Ad2l, Ad34, Ad35, Ad40, Ad9l, Ad48, BAV3, or MAV1, especially as reported in Crawford-Miksza et al., supra. Alternately, preferably the internal region of the wild-type hexon protein that is deleted to produce the chimeric hexon protein comprises one or more smaller regions (e.g., hypervariable regions) of the 12 loop. In particular, preferably the smaller region of the 12 loop comprises the HVR7 region.
The "HVR7 region" preferably comprises from about amino acid 433 to about amino acid 465 of the Ad2 hexon protein [SEQ ID N0:42] (which is encoded by the sequence of SEQ ID N0:41), or the corresponding region from another adenoviral serotype, e.g., particularly the corresponding region from Adl, Ad3, Ad5 [SEQ ID N0:44(which is encoded by the sequence of SEQ ID N0:4~;, Ad6, Ad7, Ad8, Adll, Adl2, Adl4, Adl6, Ad2l, Ad34, AdJS, Ad4C, Ad4l, Ad48, BAV3, or MAV1, especially as reported in Crawford-Miksza et al., supra. In particular, preferably the deleted region comprises from about amino acid 460 to about amino acid 466 of the HVR7 region (i.e., extending one base pair outside of this region) of the 11d2 hexon protein [SEQ ID
N0:46] (which is encoded by the seauen~e of SEQ ID N0:45), or the corresponding region from anothe~ adenoviral serotype, e.g., particularly the corresponding region from Adl, Ad3, Ad5 [SEQ ID N0:48] (which is encoded by the sequence of SEQ ID N0:47), Ad6, Ad7, Ad8, Adll, Adl2, Adl4, Adl6, Ad2l, Ad34, Ad35, Ad40, Ad4l, Ad48, BAV3, or MAVl, especially as reported in Crawford-Miksza et al., su ra.
Along the same lines, the chimeric adenovirus hexon protein desirably comprises deletions in one or both of the aforementioned regions, i.e., the hexon protein comprises deletions in one or both of the 11 and 12 loops, t which deletions can constitute the entirety of the loop(s), or can comprise deletions of one or more smaller regions (e.g., hypervariable regions) in one or both of the hexon loops. In particular, desirably the deleted regions) are selected from the group consisting of SEQ ID
N0:6, SEQ ID N0:8, SEQ ID NO:10, SEQ ID N0:12, SEQ ID
N0:14, SEQ ID N0:16, SEQ ID N0:18, SEQ ID N0:20, SEQ ID
N0:22, SEQ ID N0:29, SEQ ID N0:26, SEQ ID N0:28, SEQ ID
N0:30, SEQ ID N0:32, SEQ ID N0:34, SEQ ID N0:36, SEQ ID
N0:38, SEQ ID N0:40, SEQ ID N0:92, SEQ ID N0:44, SEQ ID
N0:46, and SEQ ID N0:48, and equivalents and conservative variations of SEQ ID N0:6, SEQ ID N0:8, SEQ ID NO:10, SEQ
ID N0:12, SEQ ID N0:14, SEQ ID N0:16, SEQ ID N0:18, SEQ ID
N0:20, SEQ ID N0:22, SEQ ID N0:24, SEQ ID N0:26, SEQ ID
N0:28, SEQ ID N0:30, SEQ ID N0:32, SEQ ID N0:39, SEQ ID
N0:36, SEQ ID N0:38, SEQ ID N0:90, SEQ ID N0:42, SEQ ID
N0:49, SEQ ID N0:46, and SEQ ID N0:48.
An "equivalent" is a naturally occurring variation of an amino acid or nucleic acid sequence, e.g., as are observed among different strains of adenovirus. A
conservative variation is a variation of an amino acid sequence that results in one or more conservative amino acid substitution(s). A "conservative amino acid substitution" is an amino acid substituted by an alternative amino acid of similar charge density, hydrophilicity/hydrophobicity, size, and/or configuration (e. g., basic, Arg and Lys; aliphatic Ala, Cys, Gly, Ile, Leu, Met and Val; aromatic, Phe, Tyr, Trp, and His;
hydrophilic, Glu, Gln, Asn, and Asp; hydroxyl, Ser and Thr) .
In another preferred embodiment, the nonnative amino acid sequence of the chimeric adenoviral coat protein (i.e., particularly a chimeric adenoviral fiber or hexon protein) comprises a deletion of one or more regions) of the wild-type adenovirus coat protein (particularly the 11 and/or 12 loops, and, most particularly, the HVR1, HVR2, HVR3, HVR4, HVRS, HVR6, and/or HVR7 regions of the wild-type adenovirus hexon protein) as previously described, and further comprises a replacement of the regions) with a spacer region preferably of from 1 to about 750 amino acids, especially of from about 1 to about 500 amino acids, and particularly of from about 1 to about 300 amino acids. It also is desirable that the region deleted and replaced comprises a smaller region less than about 200 amino acids, preferably less than about 100 amino acids, and optimally less than about 50 amino acids. The chimeric coat protein also desirably comprises a plurality of such replacements. Thus, according to the invention, the chimeric adenovirus coat protein comprises modification of one or more amino acids, and such modification is made in one or more regions which can be a smaller region. A spacer region of the aforementioned size also preferably simply can be inserted into one of the aforementioned regions (particularly into the I1 and/or 12 loop, or one or more of the aforementioned HVR1, HVR2, HVR3, HVR4, HVRS, HVR6, and HVR7 regions of the adenovirus hexon protein) in the absence of any deletion to render the resultant chimeric protein nonimmunogenic by, for instance, destroying the ability of a neutralizing antibody to interact with that particular site (e.g., by changing the spatial juxtaposition of critical amino acids with which the antibody interacts).
Optimally the spacer region comprises a nonconservative variation of the amino acid sequence of wild-type adenovirus coat protein (particularly wild-type adenovirus hexon protein) that comprises an epitope for a neutralizing antibody, and which may or may not be deleted upon the insertion of the spacer region. A
"nonconservative variation" is a variation of this amino acid sequence that does not result in the creation or ? t recreation in the chimeric adenovirus coat protein of the epitope for a neutralizing antibody directed against the wild-type adenovirus coat protein, and, in particular, is a variation of the spacer region that results in one or more nonconservative amino acid insertions) or substitutions) in this region. A "nonconservative amino acid substitution" is an amino acid substituted by an alternative amino acid of differing charge density, hydrophilicity/hydrophobicity, size, and/or configuration (e. g., a change of a basic amino acid for an acidic amino acid, a hydrophilic amino acid for a hydrophobic amino acid, and the like).
Desirably the spacer region does not interfere with the functionality of the chimeric adenovirus coat protein, particularly the chimeric adenovirus hexon or fiber protein, e.g., the ability of hexon protein to bind penton base protein or other hexon capsomeres, or the ability of penton fiber to bind penton base and/or to a cell surface receptor. Such functionality can be assessed by virus viability. Similarly, the absence of the creation or recreation of the epitope(s) for a neutralizing antibody directed against the wild-type coat (e. g., hexon and/or fiber) protein can be confirmed using techniques as described in the Examples which follow (e. g., by ensuring the antibody, which may be in a carrier fluid such as serum or other liquid, binds the wild-type adenovirus coat protein, but not the chimeric adenovirus coat protein).
Preferably the spacer region incorporated into the adenovirus coat protein (i.e., either as an insertion into the wild-type coat protein, or to replace one or more deleted regions) of the wild-type adenovirus coat protein) comprise a series of polar and/or charged amino acids (e.g., Lys, Arg, His, Glu, Asp, and the like), or amino acids with intermediate polarity (e. g., Gln, Asn, Thr, Ser, Met, and the like). In particular, desirably the spacer region comprises the sequence of SEQ ID N0:50 (which is encoded by the sequence of SEQ ID N0:49), and equivalents and conservative variations of SEQ ID N0:50.
Alternately, the spacer region can comprise any other sequence like the FLAG octapeptide sequence of SEQ ID
N0:50 that will not interfere with the functionality of the resultant chimeric protein.
In still yet another preferred embodiment, a region of a wild-type adenovirus coat protein (particularly an adenovirus hexon and/or fiber protein) is deleted and replaced with a spacer region comprising the corresponding coat protein region of another adenoviral serotype.
Preferably in this embodiment the spacer region is of a different adenoviral group. For instance, preferably a region of an Ad2 coat protein can be replaced with the corresponding region of an Ad5 or Ad7 coat protein (or any other serotype of adenovirus as described above), and vice versa. It also is preferable that such a spacer region comprising the coat protein region of another adenoviral serotype is simply inserted into the corresponding coat protein region of the chimeric coat protein. In this case, the likelihood of obtaining a chimeric hexon protein that is functional can be increased by making sure that the size of the hypervariable domain resulting from such insertion approximates the size of a known hypervariable domain. For instance, the HVR1 region of Ad90 is about 30 amino acids smaller than the HVR1 region of Ad2 (as well as other adenoviruses such as Ad5, Ad8, etc.). Thus, preferably a spacer region of about 30 amino acids can be incorporated into the Ad40 HVR1 region to produce a chimeric adenovirus hexon protein. In particular, desirably the region of Ad2 (or other adenovirus) that is not present in Ad40 (i.e., approximately amino acid residues 138 to 174), or a portion thereof, is introduced r r WO 98/40509 PCT/~JS98/05033 into Ad40 to produce the chimeric adenoviral hexon protein.
According to the invention, desirably the nonnative amino acid sequence of a chimeric coat protein comprises a plurality of such replacements or insertions. when the coat protein is incorporated into an adenoviral vector, preferably the entire coat protein of one adenoviral serotype can be substituted with the entire coat protein of another adenoviral serotype, as described further herein.
The region or regions of wild-type adenovirus hexon protein that are deleted and replaced by the spacer region, or into which the spacer region is inserted, can be any suitable regions) and desirably comprise one or more of the regions described above with respect to the hexon protein deletions. For instance, preferably the one or more regions into which the spacer region is inserted or which the spacer region replaces comprises the entirety of the 11 and/or 12 loop, or a sequence selected from the group consisting of SEQ ID N0:6, SEQ ID N0:8, SEQ ID
NO:10, SEQ ID N0:12, SEQ ID N0:14, SEQ ID N0:16, SEQ ID
N0:18, SEQ ID N0:20, SEQ ID N0:22, SEQ ID N0:24, SEQ ID
N0:26, SEQ ID N0:28, SEQ ID N0:30, SEQ ID N0:32, SEQ ID
N0:34, SEQ ID N0:36, SEQ ID N0:38, SEQ ID N0:90, SEQ ID
N0:42, SEQ ID N0:44, SEQ ID N0:46, and SEQ ID N0:48, and equivalents and conservative variations of SEQ ID N0:6, SEQ ID N0:8, SEQ ID NO:10, SEQ ID N0:12, SEQ ID N0:14, SEQ
ID N0:16, SEQ ID N0:18, SEQ ID N0:20, SEQ ID N0:22, SEQ ID
N0:24, SEQ ID N0:26, SEQ ID N0:28, SEQ ID N0:30, SEQ ID
N0:32, SEQ ID N0:34, SEQ ID N0:36, SEQ ID N0:38, SEQ ID
N0:40, SEQ ID N0:42, SEQ ID N0:44, SEQ ID N0:46, and SEQ
ID N0:48.
Similarly, the spacer region itself (i.e., both for insertion as well as replacement) preferably comprises the entirety of the 11 and/or 12 loop, or a sequence selected WO 98!40509 PCT/US98/05033 from the group consisting of SEQ ID N0:6, SEQ ID N0:8, SEQ
ID NO:10, SEQ ID N0:12, SEQ ID N0:14, SEQ ID N0:16, SEQ ID
N0:18, SEQ ID N0:20, SEQ ID N0:22, SEQ ID N0:24, SEQ ID
N0:26, SEQ ID N0:28, SEQ ID N0:30, SEQ ID N0:32, SEQ ID
N0:34, SEQ ID N0:36, SEQ ID N0:38, SEQ ID N0:40, SEQ ID
N0:42, SEQ ID N0:44, SEQ ID N0:46, and SEQ ID N0:48, and equivalents and conservative variations of SEQ ID N0:6, SEQ ID N0:8, SEQ ID NO:10, SEQ ID N0:12, SEQ ID N0:14, SEQ
ID N0:16, SEQ ID N0:18, SEQ ID N0:20, SEQ ID N0:22, SEQ ID
N0:24, SEQ ID N0:26, SEQ ID N0:28, SEQ ID N0:30, SEQ ID
N0:32, SEQ ID N0:34, SEQ ID N0:36, SEQ ID N0:38, SEQ ID
N0:40, SEQ ID N0:42, SEQ ID N0:49, SEQ ID N0:46, and SEQ
ID N0:98.
The fiber protein also preferably is altered in a similar fashion as described for modification of hexon protein to escape antibodies directed in particular against wild-type adenovirus fiber protein. Fiber protein sequences and methods of modifying fiber protein are known to those skilled in the art (see, e.g., Xia et al., supra; Novelli et al., Virology, 185, 365-376 (1991)). The fiber manipulations can be carried out in the absence of, or along with, modifications to the adenovirus hexon protein. In particular, preferably the fiber protein can be replaced in its entirety, or in part, with sequences of a fiber protein from a different serotype of adenovirus. Also, preferably, deletions can be made of fiber sites that constitute an epitope for a neutralizing antibody, and/or insertions can be made at the site to destroy the ability of the protein to interact with the antibody.
Nucleic Acid Encoding The Chimeric Adenovirus Coat Protein Preferably the chimeric adenovirus coat protein (particularly the chimeric adenovirus hexon or fiber protein) comprises a nonnative amino acid sequence wherein ~.

the alteration is made at the level of DNA. Thus, the invention preferably provides an isolated and purified nucleic acid encoding a chimeric adenovirus coat protein.
Desirably, the invention provides an isolated and purified nucleic acid encoding a chimeric adenovirus hexon protein as defined herein, wherein the nucleic acid sequence comprises a deletion of a region (or a plurality of such deletions) that encodes from about 1 to about 750 amino acids of the wild-type adenovirus coat protein, preferably from about 1 to about 500 amino acids, and optimally from about 1 to about 300 amino acids. It also is desirable that the region deleted comprises a smaller region that encodes less than about 200 amino acids, preferably less than about 100 amino acids, and optimally less than about 50 amino acids. In particular, optimally the deletion (e.g., of an adenoviral hexon protein) comprises the entirety of the 11 and/or 12 loop, or a sequence selected from the group consisting of SEQ ID N0:5, SEQ ID N0:7, SEQ
ID N0:9, SEQ ID N0:11, SEQ ID N0:13, SEQ ID N0:15, SEQ ID
N0:17, SEQ ID N0:19, SEQ ID N0:21, SEQ ID N0:23, SEQ ID
N0:25, SEQ ID N0:27, SEQ ID N0:29, SEQ ID N0:31, SEQ ID
N0:33, SEQ ID N0:35, SEQ ID N0:37, SEQ ID N0:39, SEQ ID
N0:41, SEQ ID N0:43, SEQ ID N0:45, and SEQ ID N0:47, or a sequence comprising the corresponding region from Adl, Ad3, Ad6, Ad7, Ad8, Adll, Adl2, Adl4, Adl6, Ad2l, Ad34, Ad35, Ad40, Ad4l, Ad98, BAV3, or MAV1, especially as reported in Crawford-Miksza et al., supra.
The invention also preferably provides an isolated and purified nucleic acid encoding a chimeric adenovirus hexon protein as defined herein, wherein the nucleic acid sequence comprises a deletion of one or more sequences selected from the group consisting of equivalents and conservatively modified variants of sequences that encode the entirety of the 11 and/or 12 loop, or SEQ ID N0:5, SEQ
ID N0:7, SEQ ID N0:9, SEQ ID N0:11, SEQ ID N0:13, SEQ ID

WO 98!40509 PCT/US98/05033 N0:15, SEQ ID N0:17, SEQ ID N0:19, SEQ ID N0:21, SEQ ID
N0:23, SEQ ID N0:25, SEQ ID N0:27, SEQ ID N0:29, SEQ ID
N0:31, SEQ ID N0:33, SEQ ID N0:35, SEQ ID N0:37, SEQ ID
N0:39, SEQ ID N0:41, SEQ ID N0:43, SEQ ID N0:45, and SEQ
ID N0:47, or a sequence comprising the corresponding region from Adl, Ad3, Ad6, Ad7, Ad8, Adll, Adl2, Adl9, Adl6, Ad2l, Ad34, Ad35, Ad40, Ad4l, Ad48, BAV3, or MAVl, especially as reported in Crawford-Miksza et al., su ra.
With respect to the nucleic acid sequence, an "equivalent" is a variation on the nucleic acid sequence such as can occur in different strains of adenovirus, and which either does or does not result in a variation at the amino acid level. Failure to result in variation at the amino acid level can be due, for instance, to degeneracy in the triplet code. A "conservatively modified variant"
is a variation on the nucleic acid sequence that results in one or more conservative amino acid substitutions. In comparison, a "nonconservatively modified variant" is a variation on the nucleic acid sequence that results in one or more nonconservative amino acid substitutions.
In another preferred embodiment, the invention provides an isolated and purified nucleic acid encoding a chimeric adenovirus coat protein wherein the nucleic acid sequence further comprises a replacement of the deleted region (or a plurality of such replacements) with a spacer nucleic acid region (i.e., the nucleic acid sequence that encodes the aforementioned "spacer region") that encodes from about 1 to about 750 amino acids of the wild-type adenovirus coat protein, preferably from about 1 to about 500 amino acids, and optimally from about 1 to about 300 amino acids. It also is desirable that the region deleted and replaced comprises a smaller region that encodes less than about 200 amino acids, preferably less than about 100 amino acids, and optimally less than about 50 amino acids.
r Preferably, the spacer nucleic acid region comprises a FLAG octapeptide-encoding sequence [SEQ ID N0:49], and equivalents and conservatively modified variants of SEQ ID
N0:49. Similarly, a spacer nucleic acid region can be employed that substitutes one or more coat protein encoding regions (particularly a hexon protein encoding region) of a particular adenoviral serotype with a coat protein encoding region (particularly a hexon protein encoding region) of another adenoviral serotype. Thus, preferably a spacer nucleic acid region present in a chimeric adenoviral hexon protein is selected from the group consisting of sequences that encode the entirety of the 11 and/or 12 loop, or SEQ ID N0:5, SEQ ID N0:7, SEQ ID
N0:9, SEQ ID NO:11, SEQ ID N0:13, SEQ ID N0:15, SEQ ID
N0:17, SEQ ID N0:19, SEQ ID N0:21, SEQ ID N0:23, SEQ ID
N0:25, SEQ ID N0:27, SEQ ID N0:29, SEQ ID N0:31, SEQ ID
N0:33, SEQ ID N0:35, SEQ ID N0:37, SEQ ID N0:39, SEQ ID
N0:91, SEQ ID N0:93, SEQ ID N0:45, and SEQ ID N0:47, or a sequence comprising the corresponding region from Adl, Ad3, Ad6, Ad7, Ad8, Adll, Adl2, Adl4, Adl6, Ad2l, Ad34, Ad35, Ad40, Ad4l, Ad48, BAV3, or MAV1, especially as reported in Crawford-Miksza et al., supra, and equivalents and conservatively modified variants of these sequences.
As described above with respect to the chimeric adenovirus coat protein, the spacer nucleic acid region (or a plurality thereof) simply can be incorporated into the coat protein in the absence of any deletions. These manipulations can be carried out so as to produce the above-described chimeric adenovirus coat protein.
The means of making such a chimeric adenoviral coat protein (i.e., by introducing conservative or nonconservative variations at either the level of DNA or protein) are known in the art, are described in the Examples which follow, and also can be accomplished by means of various commercially available kits and vectors (e. g., New England Biolabs, Inc., Beverly, MA; Clontech, Palo Alto, CA; Stratagene, LaJolla, CA, and the like). In particular, the ExSiteTM PCR-based site-directed mutagenesis kit and the ChameleonTM double-stranded site-directed mutagenesis kit by Stratagene can be employed for introducing such mutations. Moreover, the means of assessing such mutations (e.g., in terms of effect on ability not to be neutralized by antibodies directed against wild-type hexon protein) are described in the Examples herein.
Accordingly, the present invention provides a preferred means of making a chimeric adenoviral coat protein, particularly a chimeric adenoviral hexon protein, which comprises obtaining an adenoviral genome encoding the wild-type adenovirus coat protein (e. g., the wild-type adenovirus hexon protein), and deleting one or more regions) of the chimeric adenovirus coat protein (particularly the chimeric adenovirus hexon protein) comprising from about 1 to about 750 amino acids by modifying the corresponding nucleic acid coding sequence.
Similarly, the invention provides a method of making a chimeric adenovirus coat protein (particularly a chimeric adenovirus hexon protein) which comprises obtaining an adenoviral genome encoding the wild-type adenovirus coat protein, deleting one or more regions) of the adenovirus coat protein comprising from about 1 to about 750 amino acids by modifying the corresponding coding sequence, and replacing the deleted regions) with a spacer region comprising from about 1 to about 300 amino acids by introducing a nucleic acid region (i.e., a "spacer nucleic acid region") that codes for same. Alternately, the spacer region preferably is simply incorporated into the coat protein (particularly the hexon protein) in the absence of any deletion. Optimally the spacer nucleic acid region encodes a nonconservative variation of the amino acid sequence of the wild-type adenovirus coat protein. The size of the DNA used to replace the native coat protein coding sequence may be constrained, for example, by impeded folding of the coat protein or improper assembly of the coat protein into a complex (e. g., penton base/hexon complex) or virion. DNA encoding 150 amino acids or less is particularly preferred for insertion/replacement in the chimeric coat protein gene sequence, and DNA encoding 50 amino acids or less is even more preferred.
Briefly, the method of mutagenesis comprises deleting one or more regions of an adenovirus coat protein, and/or inserting into an adenovirus coat protein one or more regions with a differing amino acid sequence, particularly by manipulating the DNA sequence. Several methods are available for carrying out such manipulations of adenovirus coat protein DNA sequences; these methods further can be used in combination. The method of choice depends on factors known to those skilled in the art, e.g., the size of the DNA region to be manipulated. For instance, convenient restriction sites (which further can be introduced into a sequence) can be used to introduce or remove segments of DNA, or entire genes or coding sequences. Alternately, other methods of mutagenesis involve the hybridization of a mismatched oligonucleotide to a region of single-stranded target DNA, extending the primer, for instance, using T7 DNA polymerase or other such means to produce a double-stranded heteroduplex, and isolating the mutant strand that incorporates the mismatched oligonucleotide from the parental nonmutant strand for use as a template and in further manipulations.
The mutant strand can be separated from the parental strand using various selection means known to those skilled in the art (see, e.g., Kunkel et al., Methods Enzymol., 204, 125-139 (1991), as well as the underlying methodology employed in the ChameleonTM kit). Alternately, the parental strand can be selectively degraded, for instance, with use of enzymes that nick the nonmethylated strand of a hemi-methylated DNA molecule (e. g., HpaII, MspI, and Sau3AI), and by extending the mutant strand using 5-methyl-dCTP, which renders the strand resistant to cleavage by these enzymes. Along the same lines, an entirely PCR-based approach can be employed for making mutations (e. g., Kunkel, Proc. Natl. Acad. Sci., 82, 488-992 (1985); Costa et al., Nucleic Acids Res., 22, 2423 (1994)), for instance, such as the approach encompassed by the ExSiteTM kit. More generally, amino acid substitutions or deletions can be introduced during PCR by incorporating appropriate mismatches in one or both primers. Once the chimeric coat protein sequence has been produced, the nucleic acid fragment encoding the sequence further can be isolated, e.g., by PCR ar~plificatio~ using 5' and 3' primers, or through use of cor.ve:-Wen~ restriction sites.
Vector Comprising a Chimeric Hexon Protein A "vector" according to the invention is a vehicle for gene transfer as that term is understood by those skilled in the art, and includes viruses, p~'~asmids, and the like. A preferred vec-,.o:~ :s an aaenovirus, particularly a virus o' th~~ farr.ily Adcnc~viridae, and desirably of the genus Mastadenovirus (e.g., comprised of mammalian adenoviruses) or Aviadenovirus (e. g., comprised of avian adenoviruses). Such an adenovirus (or other viral vector) can be transferred by its own means of effecting cell entry (e. g., by receptor-mediated endocytosis), or can be transferred to a cell like a plasmid, i.e., in the form of its nucleic acid, for instance, by using liposomes to transfer the nucleic acid, or by microinjecting or transforming the DNA into the cell. The nucleic acid vectors that can be employed for r r gene transfer, particularly the adenoviral nucleic acid vectors, are referred to herein as "transfer vectors".
Such nucleic acid vectors also include intermediary plasmid vectors that are employed, e.g., in the construction of adenoviral vectors.
Desirably an adenoviral vector is a serotype group C
virus, preferably an Ad2 or Ad5 vector, although any other serotype adenoviral vector (e. g., group A including serotypes 12 and 31, group B including serotypes 3 and 7, group D including serotypes 8 and 30, group E including serotype 9, and group F including serotypes 90 and 41, and other Ad vectors previously described) can be employed.
An adenoviral vector employed for gene transfer can be replication competent. Alternately, an adenoviral vector can comprise genetic material with at least one modification therein, which renders the virus replication deficient. The modification to the adenoviral genome can include, but is not limited to, addition of a DNA segment, rearrangement of a DNA segment, deletion of a DNA segment, replacement of a DNA segment, or introduction of a DNA
lesion. A DNA segment can be as small as one nucleotide and as large as 36 kilobase pairs (i.e., the approximate size of the adenoviral genome) or, alternately, can equal the maximum amount which can be packaged into an adenoviral virion (i.e., about 38 kb). Preferred modifications to the group C adenoviral genome include modifications in the E1, E2, E3 and/or E4 regions.
Similarly, an adenoviral vector can be a cointegrate, i.e., a ligation of adenoviral sequences with other sequences, such as other virus sequences, particularly baculovirus sequences, or plasmid sequences, e.g., so as to comprise a prokaryotic or eukaryotic expression vector.
In terms of an adenoviral vector (particularly a replication deficient adenoviral vector), such a vector can comprise either complete capsids (i.e., including a WO 98!40509 PCT/US98/05033 viral genome such as an adenoviral genome) or empty capsids (i.e., in which a viral genome is lacking, or is degraded, e.g., by physical or chemical means). The capsid further can comprise nucleic acid linked to the surface by means known in the art (e. g., Curie! et al., Human Gene Therapy, 3, 147-159 (1992)) or can transfer non-linked nucleic acid, for instance, by adenoviral-mediated uptake of bystander nucleic acid (e.g., PCT
International Application WO 95/21259).
Along the same lines, since methods are available for transferring an adenovirus in the form of its nucleic acid sequence (i.e., DNA), a vector (i.e., a transfer vector) similarly can comprise DNA, in the absence of any associated protein such as capsid protein, and in the absence of any envelope lipid. Inasmuch as techniques are available for making a RNA copy of DNA (e. g., in vitro transcription), and inasmuch as RNA viruses also can be employed as vectors or transfer vectors, a transfer vector also can comprise RNA. Thus, according to the invention whereas a vector comprises (and, further, may encode) a chimeric adenoviral coat protein, a transfer vector typically encodes a chimeric adenoviral coat protein (particularly a chimeric adenoviral hexon and/or fiber protein).
Based on this, the invention provides an adenoviral vector that comprises a chimeric coat protein (particularly a chimeric hexon and/or fiber protein) according to the invention. Preferably such a vector comprises a chimeric coat protein (particularly a chimeric adenovirus hexon protein and/or chimeric adenovirus fiber protein) as described above. Alternately, preferably the vector lacks wild-type fiber protein, e.g., the vector encodes a truncated or non-functional fiber protein, or fails to translate fiber protein. Such fiber mutations and the means of introducing fiber mutations are known to those skilled in the art (see, e.g., Falgout et al., J.
Virol., 62, 622-625 (1988)).
Of course, the chimeric adenoviral coat proteins include coat proteins in which the native (i.e., wild-type) hexon and/or fiber protein of an adenoviral vector is replaced by a hexon and or fiber amino acid sequence of a different adenoviral serotype such that the resultant adenoviral vector has a decreased ability or inability to be recognized by neutralizing antibodies directed against the corresponding wild-type coat protein. This replacement can comprise the entirety of the hexon and/or fiber amino acid sequence, or only a portion, as described above. Both proteins can be manipulated (e.g., in a single adenovirus), or only a single chimeric adenovirus coat protein can be employed, with the remaining coat proteins being wild-type.
A vector according to the invention (including a transfer vector) preferably comprises additional sequences and mutations, e.g., some that can occur within the coat protein coding sequence itself. In particular, a vector according to the invention further preferably comprises a nucleic acid encoding a passenger gene or passenger coding sequence. A "nucleic acid" is a polynucleotide (i.e., DNA
or RNA). A "gene" is any nucleic acid sequence coding for a protein or an RNA molecule. Whereas a gene comprises coding sequences plus any non-coding sequences, a "coding sequence" does not include any non-coding (e. g., regulatory) DNA. A "passenger gene" or 'passenger coding sequence" is any gene which is not typically present in and is subcloned into a vector (e. g., a transfer vector) according to the present invention, and which upon introduction into a host cell is accompanied by a discernible change in the intracellular environment (e. g., by an increased level of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide or protein, or by an altered rate of production or degradation thereof). A
"gene product" is either an as yet untranslated RNA
molecule transcribed from a given gene or coding sequence (e. g., mRNA or antisense RNA) or the polypeptide chain (i.e., protein or peptide) translated from the mRNA
molecule transcribed from the given gene or coding sequence. A gene or coding sequence is "recombinant" if the sequence of bases along the molecule has been altered from the sequence in which the gene or coding sequence is typically found in nature, or if the sequence of bases is not typically found in nature. According to this invention, a gene or coding sequence can be naturally occurring or wholly or partially synthetically made, can comprise genomic or complementary DNA (cDNA) sequences, and can be provided in the form of either DNA or RNA.
Non-coding sequences or regulatory sequences include promoter sequences. A "promoter" is a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis. "Enhancers" are cis-acting elements of DNA
that stimulate or inhibit transcription of adjacent genes.
An enhancer that inhibits transcription is also termed a "silencer". Enhancers differ from DNA-binding sites for sequence-specific DNA binding proteins found only in the promoter (which also are termed "promoter elements") in that enhancers can function in either orientation, and over distances of up to several kilobase pairs, even from a position downstream of a transcribed region. According to the invention, a coding sequence is "operably linked"
to a promoter (e.g., when both the coding sequence and the promoter constitute a passenger gene) when the promoter is capable of directing transcription of that coding sequence.
Accordingly, a "passenger gene" can be any gene, and desirably either is a therapeutic gene or a reporter gene.
Preferably a passenger gene is capable of being expressed t in a cell in which the vector has been internalized. For instance, the passenger gene can comprise a reporter gene, or a nucleic acid sequence which encodes a protein that can be detected in a cell in some fashion. The passenger gene also can comprise a therapeutic gene, for instance, a therapeutic gene which exerts its effect at the level of RNA or protein. Similarly, a protein encoded by a transferred therapeutic gene can be employed in the treatment of an inherited disease, such as, e.g., the cystic fibrosis transmembrane conductance regulator cDNA
for the treatment of cystic fibrosis. The protein encoded by the therapeutic gene can exert its therapeutic effect by resulting in cell killing. For instance, expression of the gene in itself may lead to cell killing, as with expression of the diphtheria toxin A gene, or the expression of the gene may render cells selectively sensitive to the killing action of certain drugs, e.g., expression of the HSV thymidine kinase gene renders cells sensitive to antiviral compounds including acyclovir, gancyclovir and FIAU (1-(2-deoxy-2-fluoro-b-D-arabinofuranosil)-5-iodouracil). Moreover, the therapeutic gene can exert its effect at the level of RNA, for instance, by encoding an antisense message or ribozyme, by affecting splicing or 3' processing (e. g., polyadenylation), or by encoding a protein which acts by affecting the level of expression of another gene within the cell (i.e., where gene expression is broadly considered to include all steps from initiation of transcription through production of a processed protein), perhaps, among other things, by mediating an altered rate of mRNA accumulation, an alteration of mRNA transport, and/or a change in post-transcriptional regulation.
Accordingly, the use of the term "therapeutic gene" is intended to encompass these and any other embodiments of that which is more commonly referred to as gene therapy and is known to those of skill in the art. Similarly, the recombinant adenovirus can be used for gene therapy or to study the effects of expression of the gene (e.g., a reporter gene) in a given cell or tissue in vitro or in vivo, or for diagnostic purposes.
Also, a passenger coding sequence can be employed in the vector. Such a coding sequence can be employed for a variety of purposes even though a functional gene product may not be translated from the vector sequence. For instance, the coding sequence can be used as a substrate for a recombination reaction, e.g., to recombine the sequence with the host cell genome or a vector resident in the cell. The coding sequence also can be an "anticoding sequence," e.g., as appropriate for antisense approaches.
Other means of using the coding sequence will be known to one skilled in the art.
The present invention. thus p-cvides recombinant adenoviruses comprising a chimeri-~ nexon protein and/or a chimeric fiber protein, and which p~eterably additionally comprise a passenger gene o~ genes capable of being expressed in a particular cell. The recombinant adenoviruses can be generated by use of a vector, specifically, a transfer vector, and preferably a viral (especially an adenovirali o~ plasrr.id t:ansfer vector, in accordance with the presen~ inven~io:~. Such a transfer vector preferably comprises a chimeric adenoviral hexon and/or fiber gene sequence as previously described.
Similarly, the means of constructing such a transfer vector are known to those skilled in the art. For instance, a chimeric adenovirus coat protein gene sequence can simply be ligated into the vector using convenient restriction sites. Alternately, a wild-type adenovirus gene sequence can be mutagenized to create the chimeric coat protein sequence following its subcloning into a vector. Similarly, a chimeric coat protein gene sequence can be moved via standard molecular genetic techniques from a transfer vector into baculovirus or a suitable prokaryotic or eukaryotic expression vector (e. g., a viral or plasmid vector) for expression and evaluation of penton base binding, and other biochemical characteristics.
Accordingly, the present invention also provides recombinant baculoviral and prokaryotic and eukaryotic expression vectors comprising an aforementioned chimeric adenoviral coat protein gene sequence, which, along with the nucleic acid form of the adenoviral vector (i.e., an adenoviral transfer vector) are "transfer vectors" as defined herein. By moving the chimeric gene from an adenoviral vector to baculovirus or a prokaryotic or eukaryotic expression vector, high protein expression is achievable (approximately 5-50°, of the total protein being the chimeric protein).
Similarly, adenoviral vectors (e. g., virions or virus particles) are produced using transfer vectors. For instance, an adenoviral vector comprising a chimeric coat protein according to the invention can be constructed by introducing into a cell, e.g., a 293 cell, a vector comprising sequences from the adenoviral left arm, and a vector comprising sequences from the adenoviral right arm, wherein there is a region of overlap between the sequences. As described in the Examples which follow, this methodology results in recombination between the sequences, generating a vector that comprises a portion of each of the vectors, particularly the region comprising the chimeric coat protein sequences.
The present invention thus preferably also provides a method of constructing an adenoviral vector that has a decreased ability or inability to be recognized by a neutralizing antibody directed against wild-type adenovirus hexon protein and/or fiber protein. This method comprises replacing a coat protein of the vector (i.e., a wild-type adenovirus hexon and/or fiber protein) with the corresponding chimeric adenovirus coat protein according to the invention to produce a recombinant adenoviral vector.
The coat protein chimera-containing particles are produced in standard cell lines, e.g., those currently used for adenoviral vectors. Deletion mutants lacking the fiber gene, or possessing shortened versions of the fiber protein, similarly can be employed in vector construction, e.g., H2d1802, H2d1807, H2d11021 (Falgout et al., su ra), as can other fiber mutants. The fiberless particles have been shown to be stable and capable of binding and infecting cells (Falgout et al., su ra).
Illustrative Uses and Benefits The present invention provides a chimeric coat protein that has a decreased ability or inability to be recognized by a neutralizing antibody directed against the corresponding wild-type coat protein, as well as vectors (including transfer vectors) comprising same. The chimeric coat protein (such as a chimeric hexon and/or fiber protein) has multiple uses, e.g., as a tool for studies in vitro of capsid structure and assembly, and capsomere binding to other proteins.
A vector (e.g., a transfer vector) comprising a chimeric coat protein can be used in strain generation, for instance, in generation of recombinant strains of adenovirus. Similarly, such a vector, particularly an adenoviral vector, can be used in gene therapy.
Specifically, a vector of the present invention can be used to treat any one of a number of diseases by delivering to targeted cells corrective DNA, i.e., DNA
encoding a function that is either absent or impaired, or a discrete killing agent, e.g., DNA encoding a cytotoxin that, for instance, is active only intracellularly.
T.

Diseases that are candidates for such treatment include, but are not limited to, cancer, e.g., melanoma, glioma or lung cancers; genetic disorders, e.g., cystic fibrosis, hemophilia or muscular dystrophy; pathogenic infections, e.g., human immunodeficiency virus, tuberculosis or hepatitis; heart disease, e.g., preventing restenosis following angioplasty or promoting angiogenesis to reperfuse necrotic tissue; and autoimmune disorders, e.g., Crohn's disease, colitis or rheumatoid arthritis. In particular, gene therapy can be carried out in the treatment of diseases, disorders, or conditions that require repeat administration of the corrective DNA and/or the adenoviral vector, and thus for which current adenoviral-mediated approaches to gene therapy are less than optimal.
Moreover, such a vector, particularly an adenoviral vector, can be used to deliver material to a cell not as a method of gene therapy, but for diagnostic or research purposes. In particular, a vector comprising a chimeric adenovirus coat protein according to the invention can be employed to deliver a gene either in vitro or in vivo, for research and/or diagnostic purposes.
For instance, instead of transferring a so-called therapeutic gene, a reporter gene or some type of marker gene can be transferred instead. Marker genes and reporter genes are of use, for instance, in cell differentiation and cell fate studies, as well as potentially for diagnostic purposes. Moreover, a standard reporter gene such as a (3-galactosidase reporter gene, a gene encoding green fluorescent protein (GFP), or a ~3-glucuronidase gene can be used in vivo, e.g., as a means of assay in a living host, or, for instance, as a means of targeted cell ablation (see, e.g., Minden et al., BioTechniques, 20, 122-129 (1996); Youvan, Science, 268, 264 (1995); U.S. Patent 5,432,081; Deonarain et al., Br.
J. Cancer, 70, 786-794 (1994)).
Similarly, it may be desirable to transfer a gene to use a host essentially as a means of production in vivo of a particular protein. Along these lines, transgenic animals have been employed, for instance, for the production of recombinant polypeptides in the milk of transgenic bovine species (e. g., PCT International Application WO 93/25567). The use of an adenovirus according to the invention for gene transfer conducted for protein production in vivo further is advantageous in that such use should result in a reduced (if not absent) immune response as compared with the use of a wild-type adenovirus vector. Other "non-therapeutic" reasons for gene transfer include the study of human diseases using an animal model (e. g., use of transgenic mice and other transgenic animals including p53 tumor suppressor gene knockouts for tumorigenic studies, use of a transgenic model for impaired glucose tolerance and human Alzheimer's amyloid precursor protein models for the study of glucose metabolism and for the pathogenesis of Alzheimer's disease, respectively, etc.).
Furthermore, an adenoviral vector comprising a chimeric adenovirus coat protein and employed as described above is advantageous in that it can be isolated and purified by conventional means. For instance, it is likely that special cell lines will not need to be made in order to propagate adenoviruses comprising the chimeric coat proteins.
These aforementioned illustrative uses and recitation of benefits are by no means comprehensive, and it is intended that the present invention encompass such further uses which necessarily flow from, but are not explicitly recited, in the disclosure herein.

Means of Administration The vectors and transfer vectors of the present invention can be employed to contact cells either in vitro or in vivo. According to the invention "contacting"
comprises any means by which a vector is introduced intracellularly; the method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well known to those skilled in the art, and also are exemplified herein.
Accordingly, introduction can be effected, for instance, either in vitro (e. g., in an ex vivo type method of gene therapy or in tissue culture studies) or in vivo by methods that include, but are not limited to, electroporation, transformation, transduction, conjugation, triparental mating, (co-ltransfection, (co-)infection, high velocity bo~r.barame:~t with DNA-coated microprojectiles, incubation wilt. ~lciur~ phosphate-DNA
precipitate, direct microinjPct;en ir:to single cells, and the like. Similarly, the vectors can be introduced by means of membrane fusion using cationic lipids, e.g., liposomes. Such liposomes are commercially available (e. g., Lipofectin0, Lipo'e~~ac:inc~T", and the like, supplied by Life Technologies, Gibcc- 6f~I., GW tt:e:sburg, MD) .
Moreover, liposomes having ~r:cr~ase:t=ansfer capacity and/or reduced toxicity in vivo (see, e.g., PCT
International Application WO 95/21259 and references reviewed therein) can be employed in the present invention. Other methods also are available and are known to those skilled in the art.
According to the invention, a "host" encompasses any host into which a vector of the invention can be introduced, and thus encompasses an animal, including, but not limited to, an amphibian, bird, insect, reptile, or mammal. Optimally a host is a mammal, for instance, a rodent, primate (such as chimpanzee, monkey, ape, gorilla, orangutan, or gibbon), feline, canine, ungulate (such as ruminant or swine), as well as, in particular, a human.
Similarly, a "cell" encompasses any cell (or collection of cells) from a host into which an adenoviral vector can be introduced, e.g., preferably an epithelial cell. Any suitable organs or tissues or component cells can be targeted for vector delivery. Preferably, the organs/tissues/cells employed are of the circulatory system (e. g., heart, blood vessels or blood), respiratory system (e. g., nose, pharynx, larynx, trachea, bronchi, bronchioles, lungs), gastrointestinal system (e. g., mouth, pharynx, esophagus, stomach, intestines, salivary glands, pancreas, liver, gallbladder), urinary system (e. g., kidneys, ureters, urinary bladder, urethra), nervous system (e. g. brain and spinal cord, or special sense organs such as the eye) and integumentary system (e. g., skin). Even more preferably the cells being targeted are selected from the group consisting of heart, blood vessel, lung, liver, gallbladder, urinary bladder, and eye cells.
Thus, the present invention preferably also provides a method of genetically modifying a cell. This method preferably comprises contacting a cell with a vector comprising a chimeric adenovirus hexon protein and/or a chimeric adenovirus fiber protein, wherein desirably the vector is an adenovirus vector. The method preferably results in the production of a host cell comprising a vector according to the invention.
Moreover, the method of the invention of genetically modifying a cell can be employed in gene therapy, or for administration for diagnosis or study. The application of this method in vivo optimally comprises administering to a patient in need of gene therapy (e. g., a patient suffering from a disease, condition or disorder) a therapeutically effective amount of a recombinant adenovirus vector according to the invention. This method preferably can be employed as part of an ongoing gene therapy regimen, e.g., wherein the vector (e. g., a recombinant adenovirus vector) comprising the chimeric adenovirus coat protein is administered following (e.g., after from about 1 week to about 2 months) administration of a therapeutically effective amount of a vector comprising either the corresponding wild-type coat protein or a coat protein of a different adenoviral serotype. Alternately, the vector comprising the chimeric adenovirus coat protein can be employed as an initial attempt at gene delivery.
One skilled in the art will appreciate that suitable methods of administering a vector (particularly an adenoviral vector) of the present invention to an animal for purposes of gene therapy (see, for example, Rosenfeld et al. (1991), supra; Jaffe et al., Clin. Res., 39(2), 302A (1991); Rosenfeld et al., Clin. Res., 39(2), 311A
(1991a); Berkner, supra), chemotherapy, vaccination, diagnosis, and/or further study are available. Although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. For instance, local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration. Clinical trials regarding use of gene therapy vectors in vivo are ongoing. The methodology employed for such clinical trials as well as further technologies known to those skilled in the art can be used to administer the vector of the present invention for the purpose of research, diagnosis and/or gene therapy.

Pharmaceutically acceptable excipients also are well-known to those who are skilled in the art, and are readily available. The choice of excipient will be determined in part by the particular method used to administer the recombinant vector. Accordingly, there is a wide variety of suitable formulations for use in the context of the present invention. The following methods and excipients are merely exemplary and are in no way limiting.
Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
A vector of the present invention (including an adenoviral vector and a transfer vector), alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
t T

They may also be formulated as pharmaceuticals for non-pressured preparations such as in a nebulizer or an atomizer.
Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
Additionally, a vector of the present invention can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
The dose administered to an animal, particularly a human, in the context of the present invention will vary with the gene of interest, the composition employed, the method of administration, the particular site and organism undergoing administration, and the reason for the administration (e.g., gene therapy, diagnosis, means of producing a protein, further study, etc). Generally, the "effective amount" of the composition is such as to produce the desired effect in a host which can be monitored using several end-points known to those skilled in the art. For example, one desired effect might comprise effective nucleic acid transfer to a host cell.
Such transfer can be monitored in terms of a therapeutic effect (e. g., alleviation of some symptom associated with the disease or syndrome being treated), or by further evidence of the transferred gene or coding sequence or its expression within the host (e. g., using the polymerase chain reaction, Northern or Southern hybridizations, or transcription assays to detect the nucleic acid in host cells, or using immunoblot analysis, antibody-mediated detection, or particularized assays to detect protein or polypeptide encoded by the transferred nucleic acid, or impacted in level or function due to such transfer). One such particularized assay described in the Examples which follow includes an assay for expression of a chloramphenicol acetyl transferase reporter gene.
Generally, to ensure effective transfer of the vectors of the present invention, it is preferable that from about 1 to about 5,000 copies of the vector be employed per cell to be contacted, based on an approximate number of cells to be contacted in view of the given route of administration. It is even more preferable that from about 1 to about 300 plaque forming units (pfu) enter each cell. However, this is just a general guideline which by no means precludes use of a higher or lower amount of a component, as might be warranted in a particular application, either in vitro or in vivo. For example, the actual dose and schedule can vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts can vary in in vitro applications depending on the particular cell r r WO 98!40509 PCT1US98/05033 type utilized or the means by which the vector is transferred. One skilled in the art easily can make any necessary adjustments in accordance with the necessities of the particular situation.
The following examples further illustrate the present invention and, of course, should not be construed as in any way limiting its scope.
Example 1 This example describes experiments investigating adenoviral anti-vector neutralizing immunity.
To clarify the phenomenon of neutralizing immunity, an animal having circulating antibodies to one adenoviral vector type received intratracheal administration of another serotype adenoviral vector, and gene expression commanded by the second vector was monitored.
Specifically, either an Ad4 or Ad5 wild-type vector was administered to the lungs of Sprague-Dawley rats. Ten days later, an Ad5 reporter vector was administered to the lungs of the same animals. This reporter vector, which is referred to herein as the "pure 5" vector, comprises an E1-E3- type 5 adenoviral vector which expresses the chioramphenicol acetyl transferase (CAT) gene driven by the cytomegalovirus early/intermediate promoter/enhancer (CMV) (i.e., AdCMVCATgD described in Kass-Eisler et al., Proc. Natl. Acad. Sci., 15, 11498-11502 (1993)).
About twenty-four hours following administration of the "pure 5" vector, CAT activity was measured in homogenized lung tissue using a CAT assay as previously described (Kass Eisler et al. (1993), su ra). CAT
activity was monitored at various times thereafter up to 10 days following introduction of the "pure 5" vector.
CAT activity was determined relative to the "pure 5"
vector administered to naive animals (i.e., expression measured under this condition was considered 1000). The results of these studies are set out in Table I, and are further reported in Mastrangeli et al., Human Gene Therapy, l, 79-87 (1996) .
Table 1. Effect of (group anti-serotype E) neutralizing anti bodies on the ability of a "pure 5"

adenoviral vector to reporter transfer gene a to CAT

the lung Time (0 hours) Time (10 days) CAT Activity __ __ 0, -- pure 5 1000 Ad5 pure 5 Oo Ad4 pure 5 10510 These results confirm that in the presence of neutralizing antibodies elicited against on~:e adeneviragroup (e. g., against group E, serotype 41, it ~s possible to efficiently transfer and express a gene in vivo using an adenoviral vector derived from another group (e. g., derived from group C, serotype 5). Neutralizing immunity evoked against one serotype group does not protect against infection by another group o' ade~ov;ru~. These data support the paradigm of altern;tirc; adenoviral vectors derived from different subgro;:r~~ 3s a strategy to circumvent anti-adenoviral humoral immunity.
Example 2 The predominant epitopes that evoke neutralizing immunity are located on the fiber and hexon, but mainly on hexon. Based on this, the effect of switching the fiber protein was investigated. A vector was constructed that was identical to the "pure 5" vector except that the fiber gene was switched from a serotype 5, group C fiber to a r J

serotype 7, group B fiber. The resultant vector is referred to herein as the "5 base/? fiber" vector.
The Ad5/Ad7 fiber construct was generated as shown in Figure 1. An approximately 2.7 kb (Ad5 28689-31317 bp) fragment in pAd70-100 was replaced with a PacI linker (pAd70-100d1E3.Pac). A BamHI linker was inserted at a MunI site as indicated in Figure 2 to produce pAd70-100d1E3.Pac.Bam. A PCR-amplified PacI-BamHI fragment of approximately 1.1 kb containing the Ad7 fiber gene was inserted into pAd70-100d1E3.Pac.Bam to produce pAd70-100d1E3.fiber7.
In order to assess the ability of the Ad5 virus with Ad7 fiber to infect cells in vitro and in vivo, reporter gene assays were performed. A replication-defective recombinant adenoviral reporter vector designated AdCMV-CATNeo was used in the reporter gene assay. The reporter vector consists of the adenoviral origin of replication and viral packaging sequences, a combination of strong eukaryotic promoter (cytomegalovirus or CMV-1) and splicing elements, the bacterial chloramphenicol acetyl transferase (CAT) gene sequence, the mouse ~3ma~-globin poly(A) site, the neomycin gene sequence (Neo), and sufficient adenoviral DNA to allow for overlap recombination.
The reporter vector was used to generate AdCMV-CATNeo, AdCMV-CATNeo-dlE3 (AdCMV-CATNeo + pAd70-100d1E3) and AdCMV-CATNeo-dlE3-Fiber? (AdCMV-CATNeo + pAd70-1001E3.Fiber7) viruses. Each virus was grown in large scale, i.e., a one liter suspension of human embryonic kidney 293 cells, to yield virus at a concentration of 1012 particles/ml. A599 cells were infected with an estimated 100, 300 or 1,000 particles/cell of one of the three viruses. After 48 hours, the cells were harvested and lysates were prepared as described in Kass-Eisler et al.

(1993), supra. Using 50 ~l of each lysate, CAT assays were performed and acetylated chloramphenicol products were separated by thin layer chromatography using chloroform: methanol (95:5). The results of the assays confirm that each virus was able to infect cells and express gene products at appropriate levels. Accordingly, the virus in which the native fiber was replaced with a nonnative fiber could infect cells and express genes like the parental virus.
Following this study, adult Sprague-Dawley rats were infected with 108 viral particles by direct cardiac injection as described in Kass-Eisler et al. (1993), supra. Five days later, the rats were sacrificed, cardiac lysates were prepared, and CAT assays were performed. The amount of the CAT gene product produced was compared between the dlE3 and dlE3-Fiber? viruses. Results indicated that both viruses were able to infect cells in vivo. The replacement of the wild-type Ad5 fiber gene with that of Ad7 did not impair the ability of the virus to infect cells. Accordingly, the virus in which the native fiber was replaced with a nonnative fiber could also infect cells and express genes like the parental virus in vivo. These results support the utility of adenovirus with chimeric fiber in the context of gene therapy.
Example 3 This example describes the effect on neutralizing immunity of switching the fiber protein of an adenovirus from one serotype to another.
The "pure 5" and "5 base/? fiber" vectors described in the preceding Example were administered to Sprague-Dawley rats which either were naive or pre-immunized against wild-type Ad5. For these experiments, wild-type Ad5 or wild-type Ad7 (6 x 109 particles in phosphate r i buffered saline (PBS)) was administered intraperitoneally as a primary inoculation. Seventeen days later, serum samples were taken, and about 6 x 109 particles in about 50 ~l of PBS was injected. At about 120 hours following injection the animals were sacrificed, serum and heart tissue were harvested, and heart tissue was processed for CAT assays as previously described (Kass-Eisler et al.
(1993), supra). CAT assays also were performed on heart lysates of rat hearts infected with the "pure 5" vector or "5 base/7 fiber" vector alone.
Administration of either vector to naive animals resulted in comparable levels of CAT in heart tissue. In comparison, administration of either the "pure 5" vector or the "5 base/? fiber" vector to the animals that were pre-immunized against the "pure 5" vector resulted in a reduction of CAT levels by more than two orders of magnitude as compared with mock-infected controls. These and further results are reported in Gall et al., J.
Virol., 70, 2116-2163 (1996).
These results confirm that switching the fiber from that of adenoviral serotype 5 group C vector to that of an adenoviral serotype 7 group B vector by itself is insufficient to allow the vector to escape neutralizing antibodies generated against an adenoviral vector comprising Ad5 fiber. These results imply that antibodies against adenoviral structures other than fiber also are important in the process of neutralizing immunity.
Furthermore, whereas switching the fiber serotype to another serotype may be insufficient in and of itself to allow an adenovirus to escape immune detection, such switching when done in combination with removal of other epitopes may be desirable, for instance, to reduce an immune response .

Example 4 This example describes the construction of adenovirus vectors wherein the neutralizing immunity-evoking epitopes have been modified. In particular, this example describes vectors comprising chimeric adenoviral hexon protein, wherein the hexon neutralizing immunity-evoking epitopes are modified.
The results of the prior example indicate that it is possible to develop vectors for repeat administration in gene therapy from non-group C adenovirus, thus circumventing pre-existing neutralizing immunity. As another strategy, the dominant neutralizing immunity-evoking epitopes on existing group C vectors can be modified to render the vectors less susceptible (or "stealth") to the existing neutralizing immunity. For instance, adenoviral type 5-based E1- E3- CAT-expressing vectors can be constructed that have the same genetic composition as the "pure 5" and "5 base/? fiber" vectors described above, except for possessing a gene encoding a chimeric hexon that is not recognized by pre-existing anti-type 5 neutralizing immunity.
To derive the vectors, the chimeric hexon gene present in the "pure 5" parental vector can be modified, in particular, 11 and/or 12 can be altered. The hexon modifications that can be made on the "pure 5" CAT vector, or other adenoviral vector (such as any other adenoviral serotype vector), include, but are not limited to: (1) hexon with 11 deleted in its entirety; (2) hexon with 12 deleted in its entirety; (3) hexon with both I1 and 12 deleted; (4) hexon with any one or more of HVR1, HVR2, HVR3, HVR4, HVR5, HVR6, or HVR7, deleted; (5)-(8) hexon with a FLAG octamer epitope (i.e., Asp Tyr Lys Asp Asp Asp Asp Lys [SEQ ID N0:50]; Hopp et al., Biotechnology, 6, 1205-1210 (1988)) substituted for 11, 12, or both 11 and 12, or any one or more of HVR1, HVR2, HVR3, HVR4, HVRS, _. r. i HVR6 or HVR7; (9)-(12) hexon with a FLAG octamer epitope [SEQ ID N0:50] inserted into 11, 12, or both 11 and 12;
(13)-(16) hexon with comparable epitopes from Ad7 (group B) (GenBank~ Data Bank Accession Number x76551 for Ad7 hexon, and Number M73260 for Ad5 hexon) or Ad2, or any other adenoviral serotype, substituted for 11, 12, both 11 and 12, respectively, or for any one or more of HVRl, HVR2, HVR3, HVR4, HVR5, HVR6, or HVR7; (17)-(20) hexon with comparable epitopes from Ad7 (group B) (GenBankO Data Bank Accession Number x76551 for Ad7 hexon, and Number M73260 for Ad5 hexon) or Ad2, or any other adenoviral serotype, inserted into 11, 12, both 11 and 12, respectively, or any one or more of HVR1, HVR2, HVR3, HVR4, HVRS, HVR6, or HVR7; and (21) complete substitution of the hexon from Ad2 or another adenoviral serotype, for the Ad5 hexon. The use of the FLAG octamer epitope provides a sequence for incorporation in the chimeric hexon protein that is dif fere::~ f ror~. t:~e Ad5 hexon loop sequences, and also provides a posi~ive control using available specific anti-FLAG antibodies (Hopp et al., supra) .
These chimeric hexon proteins (and vectors containing them) can be made in seve:a~ steps. :'o modify the hexon in the "pure 5" vector, a w ral or plasmid vector can be constructed to contain th~= nexon type ~ coding sequence in a cassette that can be easily mod'_fied. The hexon is read off the 1 strand of the L3 transcription unit, i.e., map units 51.6 to 59.7, comprising a region of about 2.9 kb.
The two other transcripts that also are encoded by L3 --i.e., polypeptide VI and a 23 kDa protein -- do not overlap the hexon coding sequence. Moreover, there are no other coding sequences on the r strand that would be altered by the modification of the hexon coding sequence.
Thus, all the modifications of the type 5 hexon can be made using a "hexon 5 cassette" comprised of an approximate 6.7 kb SfiI-SfiI fragment of the "pure 5" CAT
vector. SfiI cuts Ad5 into 3 fragments, the center 6.7 kb fragment (i.e., comprising about 16,282 to 22,992 base pairs, as identified by agarose gel electrophoresis) of which contains all of the L3 region plus some overlap.
The "hexon 5 cassette" can be subcloned into a commercially available vector having restriction sites and the like making the vector easily manipulable in terms of modification and recovery of subcloned sequences. One such vector appropriate for subcloning is either the SK or KS version of the pBlueScript0 phagemid (Stratagene, LaJolla, CA).
The "hexon 5 cassette" can be mutagenized to generate site-specific mutations in the cloned DNA segment.
Several methods are available for carrying out site-specific mutagenesis. The 11 and 12 deletions, insertions, or replacements (or deletions, insertions, or replacements in HVRl, HVR2, HVR3, HVR4, HVR5, HVR6, or HVR7 regions contained therein) can be made by deleting the relevant sequences using restriction enzymes that cut uniquely within the vector inserts, or other similar means, e.g., by ligating in an end-polished, or otherwise modified, PCR product. Alternately, the hexon sequence contained in the hexon 5 cassette can be modified, e.g., using single-stranded mutagenesis in M13mp8 or some other convenient vector, and using appropriate oligonucleotides encompassing the flanking sequences for identification of plaques as described by Crompton et al., supra.
Alternately, a commercially available kit such as the ExSiteTM PCR-based site-directed mutagenesis kit and the ChameleonTM double-stranded site-directed mutagenesis kit by Stratagene can be used to introduce insertions, point mutations, or deletions into the chimeric hexon sequence without any need for subcloning into an M13, or other special vector.
t i Similarly, the FLAG octapeptide sequence (Hope et al., supra) can be introduced into the vectors (i.e., in the presence or absence of any deletion) by inserting the relevant 24 base pair sequence (GAY TAY AAR GAY GAY GAY
GAY AAR [SEQ ID N0:50), wherein Y is C or T/U, and R is A
or G)). The replacement of Ad5 hexon loop epitopes with comparable sequences of Ad7, Ad2, or any other adenoviral serotype, or an incorporation of these sequences in the absence of any deletion, can be accomplished by using unique restriction sites, or using one of the aforementioned means of mutagenesis. This usefully creates new serotypes of adenoviral vectors. For example, The replacement of the wildtype hexon protein of Ad5 with the chimeric Ad5 hexon comprising Ad7 hexon loops 1 and 2 gives rise to an adenoviral vector that is effectively neutralized by Ad7 neutralizing antibodies (i.e., neutralizing antibodies raised in response to Ad7 innoculation of a naive animal), but not by Ad5 neutralizing antibodies.
Moreover, both hypervariable loops 1 and 2 can be deleted from a serotype 5 or another serotype adenoviral vector. Adenoviral vectors and there genomes comprising these deletions are useful as a starting point to create other adenoviral vectors having loop replacements, as a tool for studying hexon structure-function relationships, and under some circumstances as a gene transfer vector with limited vulnerability to the adaptive immune system.
Example 5 This example describes the method of replacing the hexon protein of one serotype adenoviral vector with the hexon protein of another serotype adenoviral vector to generate a recombinant adenovirus. As representative of this method, the hexon protein of an Ad5 vector was replaced with the hexon protein of an Ad2 vector. This example also describes the method of incorporating the chimeric hexon proteins of the preceding Example into a vector to make a recombinant adenovirus.
Using standard molecular biology techniques, the Ad5 hexon gene open reading frame (ORF) was replaced with the Ad2 hexon gene ORF in such a fashion so as to maintain the proper Ad5 sequences upstream and downstream of the hexon gene. Adenoviral vectors comprising modified or chimeric hexon proteins can be constructed by homologous recombination using standard techniques and human embryonic kidney 293 cells (see, e.g., Rosenfeld et al.
(1991), supra; Rosenfeld et al. (1992), supra). For instance, map units 0 to 57.3 of dlAd5NCAT (Gall et al., supra) can be isolated by Bsu36I digestion, and map units 58.9 to 100 of dlAdSNCAT can be isolated by DrdI
digestion. These DNA fragments can be transfected into 293 cells along with pH5-2.
A neutralizing antibody directed against the parental vector can be employed to facilitate the generation of hexon replacement constructs. For example, when replacing the loop 1 and loop 2 regions of an Ad5 vector with Ad7 loop sequences, anti-Ad5 neutralizing polyclonal or monoclonal antibodies (directed against the loops 1 and 2 of Ad5 hexon) can be added to a the medium of cells in which the chimeric vector is being propagated. The presence of the Ad5 neutralizing antibodies substantially blocks the propagation of the undesired wildtype Ad5 vector(s), while the chimeric vector is unaffected.
Furthermore, the recombinant vectors comprising a chimeric hexon ORF can be generated by homologous recombination using a plasmid that carries a marker gene, such as Green Fluorescent Protein (GFP), adjacent to the chimeric or novel hexon ORF (e. g., between the fiber and hexon genes).
In this way, genomes that could harbor the chimeric hexon gene should also harbor the marker gene. The marker gene r t would then be expressed as a late protein, so that cells that potentially comprise the desired adenoviral genome can be easily identified.
Similarly, vectors (particularly adenoviral vectors) can be constructed that have the aforementioned hexon modifications, and which have further modifications, for instance, in the adenoviral fiber coding sequences. This can be accomplished by making the hexon modifications described above, and using different parental plasmids for homologous recombination, such as parental plasmids comprising mutations in fiber coding sequences. In particular, the "5 base/? fiber" vector can be employed as a starting vector for vector construction.
All of the viral vectors prepared according to this example can be plaque-purified, amplified, and further purified using standard methods (Rosenfeld et al. (1991), supra; Rosenfeld et al. (1992), su ra).
Example 6 This example describes a characterization of the activity in vitro and in vivo of the vectors described in the preceding Examples.
Each of the viruses prepared as described in the preceding Examples can be evaluated in vitro and in vivo using standard methods as previously described (e. g., Kass-Eisler et al., supra), and as set forth herein. In particular, for the in vitro studies, the various vectors along with control vectors (e.g., the "pure 5" and "5 base/? fiber" vectors, and the Ad5 wild-type vector) can be added to human lung carcinoma A599 cells alone, or in the presence of dilutions of serum from hosts infected with AdS, Ad7, "pure 5" CAT vector, or "5 base/? fiber"
CAT vector, or anti-FLAG epitope serum. The cells are then evaluated for CAT activity to determine the ability of antibodies present in the serum to block gene expression.
The in vivo studies can be carried out in Sprague-Dawley rats. The Sprague-Dawley rat as opposed to the mouse or cotton rat is preferred for these experiments since the rat is non-permissive, and the wild-type adenovirus cannot replicate in this host. Accordingly, immunizations can be carried out using wild-type viruses (e.g., wild-type Ad5 or Ad7), the "pure 5" CAT vector, and the "5 base/? fiber" CAT vector by intravenous administration (e.g., Kass-Eisler et al., supra). At various times ranging from about one to about four weeks later, the vector of interest can be administered intravenously or directly into the airways of the host.
Whereas intravenous administration allows an assessment of the "worst case scenario" (i.e., wherein the vector is in immediate contact with the circulating humoral immune system, and thus the strongest immune response is to be expected), introduction in the airways of the host allows an evaluation of a compartmentalized and mucosal humoral immune response .
CAT activity can be quantified as previously described in all the relevant organs, e.g., liver, heart, and lung for intravenous administration, and lung only for respiratory administration. Appropriate standards can be used to compensate for variations in organ expression of CAT activity (see e.g., Kass-Eisler et al., Gene Therapy, 2 395-402 (1994)). The in vitro and in vivo results can be compared and assessed using standard statistical methods.
All of the references cited herein, including the GenBank~ Data Bank sequence information, are hereby incorporated in their entireties by reference.
T _ r While this invention has been described with emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that the preferred embodiments can be varied. It is intended that the invention can be practiced otherwise than as specifically described herein.
Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: CORNELL RESEARCH FOUNDATION, INC.
(B) STREET: 20 Thornwood Drive, Suite 105 (C) CITY: Ithaca (D) STATE: New York (E) COUNTRY: US
(F) POSTAL CODE (ZIP): 14850 (G) TELEPHONE: 607-257-1081 (H) TELEFAX: 607-257-1015 (A) NAME: GENVEC, INC.
(B) STREET: 12111 Parklawn Drive (C) CITY: Rockville (D) STATE: Maryland (E) COUNTRY: US
(F) POSTAL CODE (ZIP): 20852 (G) TELEPHONE: 301-816-0396 (H) TELEFAX: 301-816-0085 (A) NAME: CRYSTAL, RONALD G.
(B) STREET: 13712 Canal Vista Court (C) CITY: Potomac {D) STATE: Maryland {E) COUNTRY: US
(F) POSTAL CODE (ZIP): 20854 (A) NAME: FALCK-PEDERSEN, ERIK
(B) STREET: 8719 Buena Vista Drive (C) CITY: Dobbs Ferry (D) STATE: New York (E) COUNTRY: US
(F) POSTAL CODE (ZIP): 10522 (A) NAME: GALL, JASON
(B) STREET: 920 E. 70th Street, #lOF
(C) CITY: New York (D) STATE: New York (E) COUNTRY: US
(F) POSTAL CODE (ZIP): 10021 (A) NAME: KOVESDI, IMRE
{B) STREET: 7713 Warbler Lane (C) CITY: Rockville (D) STATE: Maryland (E) COUNTRY: US
(F) POSTAL CODE (ZIP): 20855 (A) NAME: WICKHAM, THOMAS J.
(B) STREET: 2106 Hutchinson Grove Court (C) CITY: Falls Church (D) STATE: Virginia (E) COUNTRY: US
(F) POSTAL CODE (ZIP): 22043 (ii) TITLE OF INVENTION: CHIMERIC ADENOVIRAL COAT PROTEIN AND METHODS
OF USING SAME
(iii) NUMBER OF SEQUENCES: 56 (iv) COMPUTER READABLE FORM:
r T

(A) MEDIUMTYPE: Floppy disk (B) COMPUTER:IBMPC ompatible c (C) OPERATINGSYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE:PatentIn Release 0, #1.30(EPO) #1. Version (vi)PRIOR APPLICATION DATA:

(A) APPLICATION 8-816346 NUMBER:
US

(B) FILINGDATE: 13-MAR-1997 (2) INFORMATION SEQID N0:1:
FOR

(i)SEQUENCE ARACTERISTIC S:
CH

(A) LENGTH: 07 pairs 29 base (B) TYPE: nucleicacid (C) STRANDEDNESS:double (D) TOPOLOGY:linear (ii)MOLECULE PE:DNA(genomic) TY

(xi)SEQUENCE SCRIPTION: EQ D
DE S I NO:1:

ATG GCTACC CCT ATGATGCCG CAGTGG TCTTACATG CACA1'CTCG 98 TCG

Met AlaThr Pro MetMetPro GlnTrp SerTyrMet HisIle Ser Ser TCG

Gly GlnAsp Ala GluTyrLeu SerPro GlyLeuVal GlnPhe Ala Ser ACG

Arg AlaThr Glu TyrPheSer LeuAsn AsnLysPhe ArgAsn Pro Thr ACG

Thr ValAla Pro HisAspVal ThrThr AspArgSer GlnArg Leu Thr ATC

Thr LeuArg Phe ProValAsp ArgGlu AspThrAla TyrSer Tyr Ile ACC

Lys AlaArg Phe LeuAlaVal GlyAsp AsnArgVal LeuAsp Met Thr TTT

Ala SerThr Tyr AspIleArg GlyVal LeuAspArg GlyPro Thr Phe TCC

Phe LysPro Tyr GlyThrAla TyrAsn AlaLeuAla ProLys Gly Ser TGT

Ala ProAsn Ser GluTrpGlu GlnThr GluAspSer GlyArg Ala Cys GAA

Val AlaGlu Asp GluGluGlu AspGlu AspGluGlu GluGlu Glu Glu GCT

Glu GluGln Asn ArgAspGln AlaThr LysLysThr HisVal Tyr Ala GCCCAG GCTCCT TTGTC'I'GGA GAAACAATT ACA AGCGGGCTA CAA 576 AAA

AlaGln AlaPro LeuSerGly GluThrIle ThrLys SerGlyLeu Gln IleGly SerAsp AsnAlaGlu ThrGlnAla LysPro ValTyrAla Asp ProSer TyrGln ProGluPro GlnIleGly GluSer GlnTrpAsn Glu AlaAsp AlaAsn AlaAlaGly GlyArgVal LeuLys LysThrThr Pro ATGAAA CCATGC TATGGATCT TATGCCAGG CCTACA A.~1TCCTTTT GGT 768 MetLys ProCys TyrGlySer TyrAlaArg ProThr AsnProPhe Gly GGTCAA TCCGTT CTGGTTCCG GATGAAAAA GGGGTG CCTCT'1'CCA AAG 816 GlyGln SerVal LeuValPro AspGluLys GlyVal ProLeuPro Lys GTTGAC TTGCAA TT'CT1'CTCA AATACTACC TCTTTG AACGACCGG CAA 869 VslAsp LeuGln PhePheSer AsnThrThr SerLeu AsnAspArg Gln GlyAsn AlaThr LysProLys ValValLeu TyrSer GluAspVal Asn MetGlu ThrPro AspThrHis LeuSerTyr LysPro GlyLysGly Asp GluAsn SerLys AlaMetLeu GlyGlnGln SerMet ProAsnArg Pro AsnTyr IleAla PheArgAsp AsnPheIle GlyLeu MetTyrTyr Asn AGCACT GGCAAC ATGGGTGTT CT:GCTGGT CAGGCA TCGCAGCTA AAT 1104 tee:Thr GlyAsn MetGlyVal LeuAlaGly GlnAla SerGlnLeu Asn AlaVal ValAsp LeuGlnAsp ArgAsnThr GluLeu SerTyrGln Leu LeuLeu AspSer IleGlyAsp ArgThrArg TyrPhe SerMetTrp Asn GlnAla ValAsp SerTyrAsp ProAspVal ArgIle IleGluAsn His GlyThr G1uAsp GluLeuPro AsnTyrCys PhePro LeuGlyGly Ile r ACT

GlyValThr AspThrTyr GlnAla IleLysAla AsnGlyAsn GlySer GlyAspAsn GlyAspThr ThrTrp ThrLysAsp GluThrPhe AlaThr ArgAsnGlu IleGlyVal GlyAsn AsnPheAla MetGluIle AsnLeu 465 970 975 9g0 AsnAlaAsn LeuTrpArg AsnPhe LeuTyrSer AsnIleAla LeuTyr CTGCCAGAC AAGCTAAAA TACAAC CCCACCAAT GTGGAAATP.TCTGAC 1536 LeuProAsp LysLeuLys TyrAsn ProThrAsn ValGluIle SerAsp AsnProAsn ThrTyrAsp TyrMet AsnLysArg ValValAla ProGly LeuValAsp CysTyrIle AsnLeu GlyAlaArg TrpSerLeu AspTyr MetAspAsn ValAsnPro PheAsn HisHisArg AsnAlaGly LeuArg TATCGCTCC A1'GTTGTTG GGAAAC GGCCGCTAC GTGCCCTTT CACATT 172.8 TyrArgSer MetLeuLeu GlyAsn GlyArgTyr ValProPhe HisIle GlnValPro GlnLysPhe PheAla IleLysAsn LeuLeuLeu LeuPro GlySerTyr ThrTyrGlu TrpAsn PheArgLys AspValAsn MetVal LeuGlnSer SerLeuGly AsnAsp LeuArgVal AspGlyAla SerIle LysPheAsp SerIleCys LeuTyr AlaThrPhe PheProMet AlaHis AsnThrAla SerThrLeu GluAla MetLeuArg AsnAspThr AsnAsp GlnSerPhe AsnAspTyr LeuSer AlaAlaAsn MetLeuTyr ProIle ProAlaAsn AlaThrAsn ValPro IleSerIle ProSerArg AsnTrp AlaAla PheArgGly TrpAlaPhe ThrArg LeuLysThr LysGluThr ProSer LeuGlySer GlyTyrAsp ProTyr TyrThrTyr SerGlySer IlePro TyrLeuAsp GlyThrPhe TyrLeu AsnHisThr PheLysLys ValAla IleThrPhe AspSerSer ValSer TrpProGly AsnAspArg CTGCTT ACTCCCAAT GAGTTTGAG AT'rAAA CGCTCAGTT GACGGGGAG 2304 LeuLeu ThrProAsn GluPheGlu IleLys ArgSerVal AspGlyGlu GlyTyr AsnValAla GlnCysAsn MetThr hysAspTrp PheLeuVal GlnMet LeuAlaAsn TyrAsnIle GlyTyr GlnGlyPhe TyrIlePro GluSer TyrLysAsp ArgMetTyr SerPhe PheArgAsn PheGlnPro MetSer ArgGlnVal ValAspAsp ThrLys TyrLysGlu TyrGlnGln ValGly IleLeuHis GlnHisAsn AsnSer GlyPheVal GlyTyrLeu AlaPro ThrMetArg GluGlyGln AlaTyr ProAlaAsn ValProTyr ProLeu IleGlyLys ThrAlaVal AspSer IleThrGln LysLysPhe LeuCys AspArgThr LeuTrpArg IlePro PheSerSer AsnPheMet SerMet GlyAlaLeu ThrAspLeu GlyGln AsnLeuLeu TyrAlaAsn SerAla HisAlaLeu AspMetThr PheGlu ValAspPro MetAspGlu ProThr LeuLeuTyr ValLeuPhe GluVal PheAspVal ValArgVal t fi His Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTfi: 968 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (r,i) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser 1'yr Met His Ile Ser Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Va1 Leu Asp Met Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly Ala Pro Asn Ser Cys Glu Trp Glu Gln Thr Glu Asp Ser Gly Arg Ala Val Ala Glu Asp Glu Glu Glu Glu Asp Glu Asp Glu Glu Glu Glu Glu Glu Glu Gln Asn Ala Arg Asp Gln Ala Thr Lys Lys Thr His Val Tyr Ala Gln Ala Pro Leu Ser Gly Glu Thr Ile Thr Lys Ser Gly Leu Gln Ile Gly Ser Asp Asn Ala Glu Thr Gln Ala Lys Pro Val Tyr Ala Asp Pro Ser Tyr Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Asp Ala Asn Ala Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Pro Phe Gly Gly Gln Ser Val Leu Val Pro Asp Glu Lys Gly Val Pro Leu Pro Lys Val Asp Leu Gln Phe Phe Ser Asn Thr Thr Ser Leu Asn Asp Arg Gln Gly Asn Ala Thr Lys Pro Lys Val Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly Lys Gly Asp Glu Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Asn Gly Asn Gly Ser Gly Asp Asn Gly Asp Thr Thr Trp Thr Lys Asp Glu Thr Phe Ala Thr Arg Asn Glu I1e Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile r Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile Lys Phe Asp Ser Ile C.ys Leu Tyr Ala Thr Phe Phe Pro Mgt Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Fro Tyr Tyr Thr Tyr Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys Vai Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg 740 '745 750 Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro Glu Ser T'yr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Glu Tyr Gln Gln Val Gly Ile Leu His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val His Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2858 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ( ir. ) FEATURE
(A) NAME/KEY: misc_feature (B) LOCATION: 951, 952 (D) OTHER INFORMATION: /note="Xaa can be either Gln, His, or Thr"
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:3:

MetAla ThrProSer MetMetPro GlnTrp SerTyrMet HisIleSer GlyGln AspAlaSer GluTyrLeu SerPro GlyLeuVal GlnPheAla ArgAla ThrGluThr TyrPheSer LeuAsn AsnLysPhe ArgAsnPro ThrVal AlaProThr HisAspVal ThrThr AspArgSer GlnArgLeu ThrLeu ArgPheIle ProValAsp ArgGlu AspThrAla TyrSerTyr LysAla ArgPheThr LeuAlaVal GlyAsp AsnArgVal LeuAspMet AlaSer ThrTyrPhe AspIleArg GlyVal LeuAspArg GlyProThr PheLys ProTyrSer GlyThrAla TyrAsn AlaLeuAla ProLysGly AlaPro AsnProCys GluTrpAsp GluAla AlaThrAla LeuGluIle r AsnLeu GluGluGlu AspAspAsp AsnGluAsp GluVal AspGluGln AlaGlu GlnGlnLys ThrHisVal PheGlyGln AlaPro TyrSerGly IleAsn IleThrLys GluGlyIle GlnIleGly ValGlu GlyGlnThr ProLys TyrAlaAsp LysThrPhe GlnProGlu ProGln IleGlyGlu SerGln TrpTyrG1u ThrGluIle AsnHisAla AlaGly ArgValLeu LysLys ThrThrPro MetLysPro CysTyrGl.ySerTyr AlaLysPro ThrAsn GluAsnGly GlyGlnGly IleLeuVal LysGln GlnAsnGly LysLeu GluSerGln ValGluMet GlnPhePhe SerThr ThrGluAla ThrAla GlyAsnGly AspAsnLeu ThrProLys ValVal LeuTyrSer GluAsp ValAspIle GluThrPro AspThrHis IleSer TyrMetPro ThrIle LysGluGly AsnSerArg GluLeuMet GlyGln GlnSerMet ProAsn ArgProAsn TyrIleAla PheArgAsp AsnPhe IleGlyLeu MetTyr TyrAsnSer ThrGlyAsn MetGlyVal LeuAla GlyGlnAla SerGln LeuAsnAla ValValAsp LeuGlnAsp ArgAsn ThrGluLeu SerTyr GlnLeuLeu LeuAspSer IleGlyAsp ArgThr ArgTyrPhe SerMet TrpAsnGln AlaValAsp SerTyrAsp ProAsp ValArgIle AAT

IleGlu AsnHis GlyThrGlu AspGluLeu ProAsn TyrCysPhe Pro LeuGly GlyVal IleAsnThr GluThrLeu ThrLys ValLysPro Lys ThrGly GlnGlu AsnGlyTrp GluLysAsp AlaThr GluPheSer Asp LysAsn GluIle ArgValGly AsnAsnPhe AlaMet GluIleAsn Leu AsnAla AsnLeu TrpArgAsn PheLeuTyr SerAsn IleAlaLeu Tyr LeuPro AspLys LeuLysTyr SerProSer AsnVal LysIleSer Asp AsnPro AsnThr TyrAspTyr MetAsr:Lys ArgVal ValAlaPro Gly LeuVal AspCys TyrIleAsn LeuGlyAla ArgT'rpSerLeuAsp Tyr MetAsp AsnVal AsnProPhe AsnHisHis ArgAsn AlaGlyLeu Arg TyrArg SerMet LeuLeuGly AsnGlyArg TyrVal ProPheHis Ile GlnVal ProGln LysPhePhe AlaIleLys AsnLeu LeuLeuLeu Pro GlySer TyrThr TyrGluTrp AsnFheArg LysAsp ValAsnMet Val LeuGln SerSer LeuGlyAsn AspLeuArg ValAsp GlyAlaSer Ile LysPhe AspSer IleCysLeu TyrAlaThr PhePhe ProMetAla His AsnThr AlaSer ThrLeuGlu AlaMetLeu ArgAsn AspThrAsn Asp GlnSer PheAsn AspTyrLeu SerAlaAla AsnMet LeuTyrPro Ile ProAla AsnAlaThr AsnValPro IleSer IleProSer ArgAsnTrp AlaAla PheArgGly TrpAlaPhe ThrArg LeuLysThr LysGluThr ProSer LeuGlySer GlyTyrAsp ProTyr TyrThrTyr SerGlySer IlePro TyrLeuAsp GlyThrPhe TyrLeu AsnHisThr PheLysLys ValAla IleThrPhe AspSerSer ValSer TrpProGly AsnAspArg LeuLeu ThrProAsn GluPheGlu IleLys ArgSerVal AspGlyGlu GlyTyr AsnValAla GlnCysAsn MetThr LysAppTrp PheLeuVal CAAATG CTAGCTAAC TACAACATT GGCTAC CF,.G "' ': ATCCCA 2352 _;,r. AT
, GlnMet LeuAlaAsn TyrAsnIle GlyTyr Glr:;;:yEt;.e:'yrIlePro 770 775 79;.

GAGAGC TACAAGGAC CGCATGTAC TCC'."".'C':':F, F,l,_T CAGCCC 2400 T _:F, T
C

GluSer TyrLysAsp ArgMetTyr Serfete-E~!~.F,r:a.',:_.,:~neGlnPro 785 790 '79'- 800 ATGAGC CGTCAGGTG GTGGATGAT ACTAAA TACAAGGF,CTACCAACAG 2498 MetSer ArgGlnVal ValAspAsp ThrLys TyrLysAsp TyrGlnGln GTGGGC ATCCTACAC CAACACAAC AAr':'~'T,:1G:~.TT.TGTT'C;_;~TACCTT 2996 ValGly IleLeuHis GlnHisAsn A:,:~:'er~lyE;u:~'.'mlGlyTyrLeu 820 8~'-.': =a0 GCCCCC ACCATGCGC GAAGGACAG GC"I 'T C' iv.''. ACCTAT 254 u" .~': . 4 ,, AlaPro ThrMetArg GluGlyGln A;sTyr E~r~>hl F,:~:,theYroTyr ProLeu IleGlyLys ThrAlaVal AspSer IleThrGln LysLysPhe LeuCys AspArgThr LeuTrpArg IlePro PheSerSer AsnPheMet SerMet GlyAlaLeu ThrAspLeu GlyGln AsnLeuLeu TyrAlaAsn SerAla HisAlaLeu AspMetThr PheGlu ValAspPro MetAspGlu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val His Arg Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Xaa Xaa (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 952 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE
(A) NAME/KEY: misc_feature (B) LOCATION: 951,952 (D) OTHER INFORMATION: /note= "Xaa can be either Gln, His, or Thr"
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:9:

MetAla ThrProSer MetMetPro GlnTrp SerTyrMet HisIleSer GlyGln AspAlaSer GluTyrLeu SerPro GlyLeuVal GlnPheAla ArgAla ThrGluThr TyrPheSer LeuAsn AsnLysPhe ArgAsnPro ThrVal AlaProThr HisAspVal ThrThr AspArgSer GlnArgLeu ThrLeu ArgPheIle ProValAsp ArgGlu AspThrAla TyrSerTyr LysAla ArgPheThr LeuAlaVal GlyAsp AsnArgVal LeuAspMet AlaSer ThrTyrPhe AspIleArg GlyVal LeuAspArg GlyProThr PheLys ProTyrSer GlyThrAla TyrAsn AlaLeuAla ProLysGly AlaPro AsnProCys GluTrpAsp GluAla AlaThrAla LeuGluIle AsnLeu GluGluGlu AspAspAsp AsnGlu AspGluVal AspGluGln AlaGlu GlnGlnLys ThrHisVal PheGly GlnAlaPro TyrSerGly IleAsn IleThrLys GluGlyIle GlnIle GlyValGlu GlyGlnThr i Pro Lys Tyr Ala Asp Lys Thr Phe Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Tyr Glu Thr Glu Ile Asn His Ala Ala Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Lys Pro Thr Asn Glu Asn Gly Gly Gln Gly Ile Leu Val Lys Gln Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Thr Glu Ala Thr Ala Gly Asn Gly Asp Asn Leu Thr Pro Lys Val Val Leu Tyr Ser Glu Asp Val Asp Ile Glu Thr Pro Asp Thr His Ile Ser Tyr Met Pro Thr Ile Lys Glu Gly Asn Ser Arg Glu Leu Met Gly Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly Gly Val Ile Asn Thr Glu Thr Leu Thr Lys Val Lys Pro Lys Thr Gly Gln Glu Asn Gly Trp Glu Lys Asp Ala Thr Glu Phe Ser Asp Lys Asn Glu Ile Arg Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Ser Pro Ser Asn Val Lys Ile 5er Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arq Leu Lys T.hr Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro ~w?: 7y- ':.r Ty- Scr Gly Ser 690 695 70C:
Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu A_~n :!__ :t.. F'he Lys Lys 705 710 7:5 720 Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Se: Val Asp Gly Glu Gly Tyr Asn Val A1a Gln Cys Asn M~~' '."r._ 1.';, r~~: ..-f f~t:~~ Leu Val 755 760 'IC_ Gln Met Leu Ala Asn Tyr Asn Ile G;y "'~;r ~~1.-~ G_y E~h~~~ Tyr I';e Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp Tyr Gln Gln Val Gly Ile Leu His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Phe Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln-Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn SerAla HisAlaLeuAsp MetThr PheGluVal AspProMet AspGlu ProThr LeuLeuTyrVal LeuPhe GluValPhe AspValVal ArgVal HisArg ProHisArgGly ValIle GluThrVal TyrLeuArg ThrPro PheSer AlaGlyAsnAla XaaXaa (2)INFORMATION FORSEQ ID
N0:5:

(i) SEQUENCE ARACTERISTICS:
CH

(A) :

base pairs (B) nucleicacid TYPE:

(C) double STRANDEDNESS:

(D) linear TOPOLOGY:

(ii)MOLECUL E DNA(genomic ) TYPE:

(xi)SEQUENCE D
DESCRIPTION: N0:5:
SEQ
I

SerCys GluTrpGluGln ThrGlu AspSerGly ArgAlaVal AlaGlu AspGlu GluGluGluAsp GluAsp GluGluGlu GluGluGlu GluGln AsnAla ArgAspGlnAla ThrLys LysThrHis ValTyrAla GlnAla ProLeu SerGlyGluThr IleThr LysSerGly LeuGlnIle GlySer AspAsn AlaGluThrGln AlaLys ProValTyr AlaAspPro SerTyr GlnPro GluProGlnIle GlyGlu SerGlnTrp AsnGluAla AspAla AsnAla AlaGlyGlyArg ValLeu LysLysThr ThrProMet LysPro CysTyr GlySerTyrAla ArgPro ThrAsnPro PheGlyGly GlnSer GAT GGG AAG

ValLeu Val Pro GluLys ValPro LeuPro LysValAsp Leu Asp Gly AAT TCT

GlnPhe Phe Ser ThrThr LeuAsn AspArg GlnGlyAsn Ala Asn Ser GTG TAC

ThrLys Pro Lys ValLeu SerGlu AspVal AsnMetGlu Thr Val Tyr CTG AAA

ProAsp Thr His SerTyr ProGly LysGly AspGluAsn Ser Leu Lys GGT TCT

LysAla Met Leu GlnGln Met Gly Ser (2)INFORMATION SEQID N0:6:
FOR

(i) SEQUENCE CS:
CHARACTERISTI

(A) LENGTH: acids 201 amino (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE peptide TYPE:

(x.i)SEQUENCE SEQID
DESCRIPTION: N0:6:

Ser Cys Glu Trp Glu Gln Thr Glu Asp Ser Gly Arg Ala Val Ala Glu Asp Glu Glu Glu Glu Asp Glu Asp Glu Glu Glu Glu Glu Glu Glu Gln Asn Ala Arg Rsp Gln Ala Thr Lys Lys Thr His Val Tyr Ala Gln Ala Pro Leu Ser Gly Glu Thr Ile Thr Lys Ser Gly Leu Gln Ile Gly Ser Asp Asn Ala Glu Thr Gln Ala Lys Pro Val Tyr Ala Asp Pro Ser Tyr Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Asp Ala Asn Ala Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Pro Phe Gly Gly Gin Ser Val Leu Val Pro Asp Glu Lys Gly Val Pro Leu Pro Lys Val Asp Leu Gln Phe Phe Ser Asn Thr Thr Ser Leu Asn Asp Arg Gln Gly Asn Ala Thr Lys Pro Lys Val Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly Lys Gly Asp Glu Asn Ser LysAla MetLeuGly GlnGlnSer Met (2)INFORMATION FOR SEQID
N0:7:

(i) SEQUENCE
CHARACTERISTICS:

(A) :

base pairs (B) nucleic acid TYPE:

(C) double STRANDEDNESS:

(D) linear TOPOLOGY:

(ii)MOLECUL E DNA(genomic ) TYPE:

(xi)SEQUENCE D
DESCRIPTION: N0:7:
SEQ
I

ProCys GluTrpAsp GluAlaAla ThrAlaLeu GluIleAsn LeuGlu GluGlu AspAspAsp AsnGluAsp GluValAsp GluGlnAla GluGln GlnLys ThrHisVal PheGlyGln AlaProTyr SerGlyIle AsnIle ThrLys GluGlyIle GlnIleGly ValGluGly GlnThrPro LysTyr AlaAsp LysThrPhe GlnProGlu ProGlnIle GlyGluSer GlnTrp TyrGlu ThrGluIle AsnHisAla AlaGlyArg ValLeuLys LysThr ThrPro MetLysPro CysTyrGly SerTyrAla LysProThr AsnGlu AsnGly GlyGlnGly IleLeuVal LysGlnGln AsnGlyLys LeuGlu SerGln ValGluMet GlnPhePhe SerThrThr GluAlaThr AlaGly AsnGly AspAsnLeu ThrProLys ValValLeu TyrSerGlu AspVal AspIle GluThrPro AspThrHis IleSerTyr MetProThr IleLys Glu Gly Asn Ser Arg Glu Leu Met Gly Gln Gin Ser Met (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 189 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Pro Cys Glu Trp Asp Glu Ala Ala Thr Ala Leu Glu Ile Asn Leu Glu Glu Glu Asp Asp Asp Asn Glu Asp Glu Val Asp Glu Gln Ala Glu Gln Gln Lys Thr His Val Phe Gly G1n Ala Pro Tyr Ser Gly Ile Asn Ile Thr Lys Glu Gly Ile Gln Ile Gly Val Glu Gly Gln Thr Pro Lys Tyr Ala Asp Lys Thr Phe Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Tyr Glu Thr Glu Ile Asn His Ala Ala Gly Arg Val Leu Lys Lys Thr Thr Pro Met hys Pro Cys Tyr Gly Ser Tyr Ala Lys Pro Thr Asn Glu Asn Gly Gly Gln Gly Ile Leu Val Lys Gln Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Thr Glu Ala Thr Ala Gly Asn Gly Asp Asn Leu Thr Pro Lys Val Val Leu Tyr Ser Glu Asp Val Asp Ile Glu Thr Pro Asp Thr Eiis Ile Ser Tyr Met Pro Thr Ile Lys Glu Gly Asn Ser Arg Glu Leu Met Gly Gln Gln Ser Met (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 153 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:

Thr Glu Asp Ser Gly Arg Ala Val Ala Glu Asp Glu Glu Glu Glu Asp Glu Asp Glu Glu Glu Glu Glu Glu Glu Gln Asn Ala Arg Asp Gln Ala Thr Lys Lys Thr His Val Tyr Ala Gln Ala Pro Leu Ser Gly Glu 'rhr Ile Thr Lys (2) INFORMATION FOR SEQ ID NO:10:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Thr Glu Asp Ser Gly Arg Ala Val Ala Glu Asp Glu Glu Glu Glu Asp Glu Asp Glu Glu Glu Glu Glu Glu Glu Gln Asn Ala Arg Asp Gln Ala Thr Lys Lys Thr His Val Tyr Ala Gln Ala Pro Leu Ser Gly Glu Thr Ile Thr Lys (2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 135 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Ala Ala Thr Ala Leu Glu Ile Asn Leu Glu Glu Glu Asp Asp Asp Asn Glu Asp Glu Val Asp Glu Gln Ala Glu Gln Gln Lys Thr His Val Phe Gly Gln Ala Pro Tyr Ser Gly Ile Asn Ile Thr Lys Glu (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 95 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi)SEQUENCE DESCRIPTION: SEQ ID N0:12:

AlaAla Thr Ala Leu Glu Ile Asn Leu Glu Asp Asp Asp Asn Glu Glu GluAsp Glu Val Asp Glu Gln Ala Glu Gln Thr His Val Phe Gln Lys GlyGln Ala Pro Tyr Ser Gly Ile Asn Ile Glu Thr Lys (2)INFORMATION
FOR
SEQ
ID
N0:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (xi)SEQUENCE DESCRIPTION: SEQ ID N0:13:

GTA

SerAsp Asn Ala Glu Thr Gln Ala Lys Pro Val (2)INFORMATION
FOR
SEQ
ID
N0:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi)SEQUENCE DESCRIPTION: SEQ ID N0:14:

SerAsp Asn Ala Glu Thr Gln Ala Lys Pro Val (2)INFORMATION
FOR
SEQ
ID
N0:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (xi)SEQUENCE DESCRIPTION: SEQ ID N0:15:

ValGlu Gly Gln Thr Pro Lys (2)INFORMATION FOR SEQ ID N0:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: N0:16:
SEQ ID

ValGlu Gly Gln Thr Pro Lys (2)INFORMATION FOR SEQ ID N0:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: N0:17:
SEQ ID

AACGAA GCT GAT GCT AAT GCG GCA 2q AsnGlu Ala Asp Ala Asn Ala Ala (2)INFORMATION FOR SEQ ID N0:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (x.i) SEQUENCE DESCRIPTION: N0:18:
SEQ ID

AsnGlu Ala Asp Ala Asn Ala Ala (2)INFORMATION FOR SEQ ID N0:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: N0:19:
SEQ ID

TyrGlu Thr Glu Ile Asn His Ala (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Tyr Glu Thr Glu Ile Asn His Ala (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:

Ser Val Leu Val Pro Rsp Glu Lys Gly Val Pro Leu Pro Lys (2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Ser Val Leu Val Pro Asp Glu Lys Gly Val Pro Leu Pro Lys (2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:

Gly Ile Leu Val Lys Gln Gln Asn Gly Lys Leu Glu Ser Gln (2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
Gly Ile Leu Val Lys Gln Gln Asn Gly Lys Leu Glu Ser Gln (2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:

Ser Asn Thr Thr Ser Leu Asn Asp Arg Gln Gly Asn Ala Thr Lys Pro Lys (2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (F3) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Ser Asn Thr Thr Ser Leu Asn Asp Arg Gln Gly Asn Ala Thr Lys Pro Lys (2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:

Ser Thr Thr Glu Ala Thr Ala Gly Asn Gly Asp Asn Leu Thr Pro Lys (2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii} MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Ser Thr Thr Glu Ala Thr Ala Gly Asn Gly Asp Asn Leu Thr Pro Lys (2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:

Leu Tyr Ser Glu Asp Val Asn Met (2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Leu Tyr Ser Glu Asp Val Asn Met (2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:

Leu Tyr Ser Glu Asp Val Asp Ile (2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Leu Tyr Ser Glu Asp Val Asp Ile (2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:

Gly Lys Gly Asp Glu Asn Ser Lys Ala Met Leu Gly (2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
Gly Lys Gly Asp Glu Asn Ser Lys Ala Met Leu Gly (2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:

Thr Ile Lys Glu Gly Asn Ser Arg Glu Leu Met G1y (2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:

Thr Ile Lys Glu Gly Asn Ser Arg Glu Leu Met Gly (2)INFORMATION FOR SEQ ID N0:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 165 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (xi)SEQUENCE DESCRIPTION: SEQ
ID N0:37:

ATT GGG CAA

AsnTyr Cys Phe Pro Leu Gly Gly Val Thr Asp Thr Tyr Ile Giy Gln TCA GGC ACA

AlaIle Lys Ala Asn Gly Asn Gly Asp Asn Gly Asp Thr Ser Gly Thr TGGACA AAA GAT GAA ACT TTT GCA AAT GAA AT'A GGA GTG 144 ACA CGT GGT

TrpThr Lys Asp Glu Thr Phe Ala Asn Glu Ile Gly Val Thr Arg Gly AsnAsn Phe Ala Met Glu Ile (2)INFORMATION FOR SEQ ID N0:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (}:i)SEQUENCE DESCRIPTION: SEQ ~':_in:
I:; ":

AsnTyr Cys Phe Pro Leu Gly Gly ,... :r.: r:_: .::r =:~ ~.~.~ 1'yr Gln 1 5 1 ~ .5 AiaI Lys Ala Asn Gly Asn Gly F,= ~~ F,::r. ~ : y le Scar ;.. y A<~~; Thr Thr TrpThr Lys Asp Glu Thr Phe Ala Asn Glu Ile Gl.y Val Thr Arg Gly AsnAsn Phe Ala Met Glu Ile (2)INFORMATION
FOR
SEQ
ID
N0:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 153 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) . ..

(xi)SEQUENCE DESCRIPTION: ID
SEQ N0:39:

GGT GTG ACT

AsnTyr Cys Phe Pro Leu Gly IleAsn Thr Glu LeuThr Gly Val Thr CAG GAA AAA

LysVal Lys Pro Lys Thr Gly AsnGly Trp Glu AspAla Gln Glu Lys GAA ATA AAT

ThrGlu Phe Ser Asp Lys Asn ArgVal Gly Asn PheAla Glu Ile Asn MetGlu Ile (2.)INFORMATION
FOR
SEQ
ID
N0:90:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 51 amino s acid (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (xi)SEQUENCE DESCRIPTION: ID
SEQ N0:90:

AsnTyr Cys Phe Pro Leu Gly IleAsn Thr Glu LeuThr Gly Val Thr LysVal Lys Pro Lys Thr Gly AsnGly Trp Glu AspAla Gln Glu Lys ThrGlu Phe Ser Asp Lys Asn ArgVal Gly Asn PheAla Glu Ile Asn MetGlu Ile (2)INFORMATION
FOR
SEQ
ID
N0:41:

{i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (xi)SEQUENCE DESCRIPTION: ID
SEQ N0:91:

ATT AAG GGC

ValThr Asp Thr Tyr Gln Ala AlaAsn Gly Asn SerGly Ile Lys Gly GATAAT 5q AspAsn (2)INFORMATION
FOR
SEQ
ID
N0:42:

(i) SEQUENCE CHARACTERISTICS:

{A) LENGTH: 18 amino s acid (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Asn Gly Asn Gly Ser Gly Asp Asn (2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:

Asn Thr Glu Thr Leu Thr Lys Val Lys Pro Lys Thr Gly Gln Glu Asn Gly Trp Glu Lys Asp Ala Thr Glu Phe Ser Asp Lys Asn (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
Asn Thr Glu Thr Leu Thr Lys Val Lys Pro Lys Thr Gly Gln Glu Asn Gly Trp Glu Lys Asp Ala Thr Glu Phe Ser Asp Lys Asn (2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:

Thr Phe Ala Thr Arg Asn Glu T i (2)INFORMATION FOR SEQ ID N0:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: N0:46:
SEQ ID

ThrPhe Ala Thr Arg Asn Glu (2)INFORMATION FOR SEQ ID N0:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: N0:97:
SEQ ID

ThrGlu Phe Ser Asp Lys Asn Glu (2)INFORMATION FOR SEQ ID N0:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: N0:98:
SEQ ID

ThrGlu Phe Ser Asp Lys Asn Glu (2)INFORMATION FOR SEQ ID N0:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other acid nucleic (xi) SEQUENCE DESCRIPTION: N0:49:
SEQ ID

AspTyr Lys Asp Asp Asp Asp Lys (2)INFORMATION FOR SEQ ID N0:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
Asp Tyr Lys Asp Asp Asp Asp Lys (2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2907 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:

TAC

AlaThr ProSerMet MetProGln TrpSer TyrMetHisIle Ser GlyGlnAsp AlaSerGlu TyrLeuSer ProGly LeuValGlnPhe Ala ArgAlaThr GluThrTyr PheSerLeu AsnAsn LysPheArgAsn Pro ThrValAla ProThrHis AspValThr ThrAsp ArgSerGlnArg Leu ThrLeuArg PheIlePro ValAspArg GluAsp ThrAlaTyrSer Tyr LysAlaArg PheThrLeu AlaValGly AspAsn ArgValLeuAsp Met AlaSerThr TyrPheAsp IleArgGly ValLeu AspArgGlyPro Thr PheLysPro TyrSerGly ThrAlaTyr AsnAla LeuAlaProLys Gly AlaProAsn SerCysGlu TrpGluGln ThrGlu AspSerGlyArg Ala ValAlaGlu AspGluGlu GluGluAsp GluAsp GluGluGluGlu Glu GluGluGlnAsn AlaArg AspGlnAla ThrLysLys ThrHisVal Tyr AlaGlnAlaPro LeuSer GlyGluThr IleThrLys SerGlyLeu Gln IleGlySerAsp AsnAla GluThrGln AlaLysPro ValTyrAla Asp ProSerTyrGln ProGlu ProGlnIle GlyGluSer GlnTrpAsn Glu AlaAspAlaAsn AlaAla GlyG7.yArg ValLeuLys LysThrThr Pro MethysProCys TyrGly SerTyrAla ArgProThr AsnProPhe Gly GlyGlnSerVal LeuVal ProAspGlu LysGlyVal ProLeuPro Lys ValAspLeuGln PhePhe SerAsnThr ThrSerLeu AsnAspArg Gln GGCAATGCTACT AAACCA AAAGTGGTT TTGTACAG'rGAAGATGTA AAT 912 GlyAsnAlaThr LysPro LysValVal LeuTyrSer GluAspVal Asn MetGluThrPro AspThr HisLeuSer TyrLysPro GlyLysGly Asp GluAsnSerLys AlaMet LeuGlyGln GlnSerMet ProAsnArg Pro AsnTyrIleAla PheArg AspAsnPhe IleGlyLeu MetTyrTyr Asn SerThrGlyAsn MetGly ValLeuAla GlyGlnAla SerGlnLeu Asn AlaValValAsp LeuGln AspArgAsn ThrGluLeu SerTyrGln Leu LeuLeuAspSer IleGly AspArgThr ArgTyrPhe SerMetTrp Asn GlnAlaValAsp SerTyr AspProAsp ValArgIle IleGluAsn His GlyThrGlu AspGlu LeuProAsn TyrCysPhe ProLeu GlyGlyIle GlyValThr AspThr TyrGlnAla IleLysAla AsnGly AsnGlySer GlyAspAsn GlyAsp ThrThrTrp ThrLysAsp GluThr PheAlaThr ArgAsnGlu IleGly ValGlyAsn AsnPheAla MetGlu IleAsnLeu AsnAlaAsn LeuTrp ArgAsnPhe LeuTyrSer AsnIle AlaLeuTyr 4g0 485 990 995 LeuProAsp LysLeu LysTyrAsn ProThrAsn ValGlu IleSerAsp AsnProAsn ThrTyr AspTyrMet AsnLysArg ValVal T~laProGly CTTGTAGAC TGCTAC ATTAACCTT GGGGCGC~_'TGG'."CT~TGGACTAC 1632 LeuValAsp CysTyr IleAsnLeu G:iyAlaA:~ ".r,>per L.euAspTyr 530 53 5 S4(~

ATGGr'1CAAC GTTAAT CCCTTTAAC CF::CACC~;'F,F,:'~_"~~a CTCCGT 1680 ~'_' MetAspAsn ValAsn ProPheAsn Hipt:i:;A: A~:;ia:~~';yLeuArg ~;

TyrArgSer MetLeu LeuGlyAsn GlyArgTyr ValPro PheHisIle, CAGGTGCCC CAAAAG TTTTTTGCC AT".'F,F,i~Fv,~'CTCCT;'CTCCTGCCA 1776 GlnValPro GlnLys PhePheAla IleaLy::h:;-.:.<~,:~,~w.:LeuLeuPro 580 ',~'~ 590 GGCTCATAT ACATAT GAATGGAAC '.".f,';i~A~C,i,:GT".i,i~CATGGTT 1824 , -GlySerTyr ThrTyr GluTrpAsn Pt.Ark:.y:;Any:Vul AnnMe~Val LeuGlnSer SerLeu GlyAsnAsp LeuArgVal AspGly AlaSerIle LysPheAsp SerIle CysLeuTyr AlaThrPhe PhePro MetAlaHis AsnThrAla SerThr LeuGluAla MetLeuArg AsnAsp ThrAsnAsp GlnSerPhe AsnAsp TyrLeuSer AlaAlaAsn MetLeu TyrProIle T

ProAla AsnAlaThr AsnValPro IleSerIle ProSerArg AsnTrp AlaAla PheArgGly TrpAlaPhe ThrArgLeu LysThrLys GluThr ProSer LeuGlySer GlyTyrAsp ProTyrTyr ThrTyrSer GlySer IlePro TyrLeuAsp GlyThrPhe TyrLeuAsn HisThrPhe LysLys ValAla IleThrPhe AspSerSer ValSerTrp ProGlyAsn AspArg LeuLeu ThrProAsn GluPheGlu IleLysArg SerValAsp GlyGlu GGCTAC AACGTAGCT CAGTGCAAC ATGACCAAG GACTGGTTC CTGGTG 2352.

GlyTyr AsnValAla GlnCysAsn MetThrLys AspTrpPhe LeuVal GlnMet LeuAlaAsn TyrAsnIle GlyTyrGln GlyPheTyr IlePro GluSer TyrLysAsp ArgMetTyr SerPhePhe ArgAsnPhe GlnPro MetSer ArgGlnVal ValAspAsp ThrLysTyr LysGluTyr GlnGln ValGly IleLeuHis GlnHisAsn AsnSerGly PheValGly TyrLeu AlaPro ThrMetArg GluGlyGln AlaTyrPro AlaAsnVal ProTyr ProLeu IleGlyLys ThrAlaVal AspSerIle ThrGlnLys LysPhe LeuCys AspArgThr LeuTrpArg IleProPhe SerSerAsn PheMet SerMet GlyAlaLeu ThrAspLeu GlyGlnAsn LeuLeuTyr AlaAsn SerAla HisAlaLeu AspMetThr PheGluVal AspProMet AspGlu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val His Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr (2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 967 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly Ala Pro Asn Ser Cys Glu Trp Glu Gln Thr Glu Asp Ser Gly Arg Ala Val Ala Glu Asp Glu Glu Glu Glu Asp Glu Asp Glu Glu Glu Glu Glu Glu Glu Gln Asn Ala Arg Asp Gln Ala Thr Lys Lys Thr His Val Tyr Ala Gln Ala Pro Leu Ser Gly Glu Thr Ile Thr Lys Ser Gly Leu Gln Ile Gly Ser Asp Asn Ala Glu Thr Gln Ala Lys Pro Val Tyr Ala Asp Pro Ser Tyr Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala r WO 98!40509 PCT/US98/05033 Asp Ala Asn Ala Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Pro Phe Gly Gly Gln Ser Val Leu Val Pro Asp Glu Lys Gly Val Pro Leu Pro Lys Val Asp Leu Gln Phe Phe Ser Asn Thr Thr Ser Leu Asn Asp Arg Gln Gly Asn Ala Thr Lys Pro Lys Val Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly Lys Gly Asp Glu Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr I1e Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Rsn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Rla Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Asn Gly Asn Gly Ser Gly Asp Asn Gly Rsp Thr Thr Trp Thr Lys Asp Glu Thr Phe Ala Thr Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly A1a Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Al.a Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Glu Tyr Gln Gln Val Gly Ile Leu His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser r MetGlyAla LeuThrAsp LeuGlyGln AsnLeu LeuTyrAla AsnSer AlaHisAla LeuAspMet ThrPheGlu ValAsp ProMetAsp GluPro ThrLeuLeu TyrValLeu PheGluVal PheAsp ValValArg ValHis GlnProHis ArgGlyVal IleGluThr ValTyr LeuArgThr ProPhe SerAlaGly AsnAlaThr Thr (2)INFORMATION FORSEQ ID
N0:53:

(i)SEQUENCE ARACTERISTICS:
CH

(A) : pairs base (B) nucleic acid TYPE:

(C) double STRANDEDNESS:

(D) linear TOPOLOGY:

(ii)MOLECULE DNA(genomic) TYPE:

(~:i)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:53:

AlaThr ProSerMet MetProGln TrpSe: ':yrMetHis IleSer ;?

GGCCAGGAC GCCTCGGAG TACCTGAGC C~:'~~~ ''TC;:,:CF,GTTTGCC 96 ;

GlyGlnAsp AlaSerGlu TyrLeuSe: P.-c~C;_ L.<:V' G::iPheAla y ~: l 20 ~'.. 30 ArgAlaThr GluThrTyr PheSerLeu AsnAsn LysPheArg AsnPro ACGGTGGCG CCTACGCAC GACGTGACC ACRGA_~CGGTCCCAG CGTTTG 192 ThrValAla ProThrHis AspValT';rT.rF~~p.y-~~.~:Gln ArgLeu 50 55 r~;

ACGCTGCGG TTCATCCCT GTGGACCGT G.y~'.:;a".'~,~(;~'"'t,'.TCGTAC 29 :' _; 0 ThrLeuArg PheIlePro ValAsh.Rrg G: i,~E~': F,:':'yrSerTyr a ~: a 65 70 'J, LysAlaArg PheThrLeu AlaValGly AspAsn ArgValLeu AspMet AlaSerThr TyrPheAsp IleArgGly ValLeu AspArgGly ProThr PheLysPro TyrSerGly ThrAlaTyr AsnAla LeuAlaPro LysGly AlaProAsn ProCysGlu TrpAspGlu AlaAla ThrAlaLeu GluIle Asn Leu Glu Glu Glu Asp Asp Asp Asn Glu Asp Glu Val Asp Glu Gln AlaGlu GlnGlnLys ThrHis ValPheGly GlnAlaPro TyrSerGly IleAsn IleThrLys GluGly IleGlnIle GlyValGlu GlyGlnThr ProLys TyrAlaAsp LysThr PheGlnPro GluProGln IleGlyGlu SerGln TrpTyrGlu ThrGlu IleAsnHis AlaAlaGly ArgValLeu LysLys ThrThrPro MetLys ProCysTyr GlySerTyr AlaLysPro 1'hrAsn GluAsnGly GlyGln GlyIleLeu ValLysGln GlnAsnGly LysLeu GluSerGln ValGlu MetGlnPhe PheSerThr ThrGluAla ThrAla GlyAsnGly AspAsn LeuThrPro LysValVal LeuTyrSer GluAsp ValAspIle GluThr ProAspThr HisIleSer TyrMetPro ThrIle LysGluGly AsnSer ArgGluLeu MetGlyGln GlnSerMet ProAsn ArgProAsn TyrIle AlaPheArg AspAsnPhe IleGlyLeu MetTyr TyrAsnSer ThrGly AsnMetGly ValLeuAla GlyGlnAla SerGln LeuAsnAla ValVal AspLeuGln AspArgAsn ThrGluLeu SerTyr GlnLeuLeu LeuAsp SerIleGly AspArgThr ArgTyrPhe SerMet TrpAsnGln AlaVal AspSerTyr AspProAsp ValArgIle AAT

r Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro LeuGlyGlyVal IleAsn ThrGluThr LeuThrLys ValLysPro Lys ThrGlyGlnGlu AsnGly TrpGluLys AspAlaThr GluPheSer Asp LysAsnGluIle ArgVal GlyAsnAsn PheAlaMet GluIleAsn Leu AsnAlaAsnLeu TrpArg AsnPheLeu TyrSerAsn IleAlaLeu Tyr 465 970 q75 LeuProAspLys LeuLys TyrSerPro SerAsnVal LysIleSer Asp AsnProAsnThr TyrAsp TyrMetAsn LysArgVal ValAlaPro Gly LeuValAspCys TyrIle AsnLeuGly AlaArgTrp SerLeuAsp Tyr MetAspAsnVal AsnPro PheAsnHis HisArgAsn AlaGlyLeu Arg TyrArgSerMet LeuLeu GlyAsnGly ArgTyrVal ProPheHis Ile GlnValProGln LysPhe PheAlaIle LysAsnLeu LeuLeuLeu Pro GlySerTyrThr TyrGlu TrpAsnPhe ArgLysAsp ValAsnMet Val LeuGlnSerSer LeuGly AsnAspLeu ArgValAsp GlyAlaSer Ile LysPheAspSer IleCys LeuTyrAla ThrPhePhe ProMetAla His AsnThrAlaSer ThrLeu GluAlaMet LeuArgAsn AspThrAsn Asp GlnSerPheAsn AspTyr LeuSerAla AlaAsnMet LeuTyrPro Ile ProAlaAsn AlaThrAsn ValProIle SerIlePro SerArg AsnTrp AlaAlaPhe ArgGlyTrp AlaPheThr ArgLeuLys ThrLys GluThr ProSerLeu GlySerGly TyrAspPro TyrTyrThr TyrSer GlySer IleProTyr LeuAspGly ThrPheTyr LeuAsnHis ThrPhe LysLys ValAlaIle ThrPheAsp SerSerVal SerTrpPro GlyAsn AspArg LeuLeuThr ProAsnGlu PheGluIle LysArgSer ValAsp GlyGlu GlyTyrAsn ValAlaGln CysAsnMet ThrLysAsp TrpPhe LeuVal GlnMetLeu AlaAsnTyr AsnIleGly TyrGlnGly PheTyr IlePro GluSerTyr LysAspArg MetTyrSer PhePheArg AsnPhe GlnPro MetSerArg GlnValVal AspAspThr LysTyrLys AspTyr GlnGln ValGlyIle LeuHisGln HisAsnAsn SerGlyPhe ValGly TyrLeu AlaProThr MetArgGlu GlyGlnAla TyrProAla AsnPhe ProTyr ProLeuIle GlyLysThr AlaValAsp SerIleThr GlnLys LysPhe LeuCysAsp ArgThrLeu TrpArgIle ProPheSer SerAsn PheMet SerMetGly AlaLeuThr AspLeuGly GlnAsnLeu LeuTyr AlaAsn SerAlaHis AlaLeuAsp MetThrPhe GluValAsp ProMet AspGlu f Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val His Arg Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Gln His (2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 951 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
Ala Thr Pro Ser Met Met Pro Gln 1'rp Ser 1'yr Met His Ile Ser Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly Ala Pro Asn Pro Cys Glu Trp Asp Glu Ala Ala Thr Ala Leu Glu Ile Asn Leu Glu Glu Glu Asp Asp Asp Asn Glu Asp Glu Val Asp Glu Gln Ala Glu Gln Gln Lys Thr His Val Phe Gly Gln Ala Pro Tyr Ser Gly Ile Asn Ile Thr Lys Glu Gly Ile Gln Ile Gly Val Glu Gly Gln Thr Pro Lys Tyr Ala Asp Lys Thr Phe Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Tyr Glu Thr Glu Ile Asn His Ala Ala Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Lys Pro Thr Asn Glu Asn Gly Gly Gln Gly Ile Leu Val Lys Gln Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Thr Glu Ala Thr Ala Gly Asn Gly Asp Asn Leu Thr Pro Lys Val Val Leu Tyr Ser Glu Asp Val Asp Ile Glu Thr Pro Asp Thr His Ile Ser Tyr Met Pro Thr Ile Lys Glu Gly Asn Ser Arg Glu Leu Met G1y Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu Asn H3is Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly Gly Val Ile Asn Thr Glu Thr Leu Thr Lys Val Lys Pro Lys Thr Gly Gln Glu Asn Gly Trp Glu Lys Asp Ala Thr Glu Phe Ser Asp Lys Asn Glu Ile Arg Val G1y Asn Asn Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Ser Pro Ser Asn Val Lys Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln t Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr 'I'yr Thr Tyr Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp Tyr Gln Gln Val Gly Ile Leu His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Phe Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val His Arg Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Gln His (2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 base pairs {B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
GAA CTC GGA GG'r GGA GGT GGA ACT AGT TTT GGA CGC GGA GAC ATT CGC 98 Glu Leu Gly Gly Gly Gly Gly Thr Ser Phe Gly Arg Gly Asp Ile Arg Asn (2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
Glu Leu Gly Gly Gly Gly Gly Thr Ser Phe Gly Arg Gly Asp Ile Arg Asn r T

Claims (19)

WHAT IS CLAIMED IS:

1. A chimeric adenovirus hexon protein comprising a deletion, insertion, or a replacement of a region of from about 1 to about 750 amino acids of a wild-type adenovirus hexon protein, wherein paid chimeric adenovirus hexon protein has an inability or decreased ability to be recognized by a neutralizing antibody directed against the wild-type adenovirus coat protein.

2. The chimeric adenovirus hexon protein of claim 1, comprising a plurality of deletions, insertions, and/or replacements.

3. The chimeric adenovirus hexon protein of claim 1 or 2, wherein said region deleted or replaced comprises a hypervariable region in either the 11 loop or the 12 loop.

4. The chimeric adenovirus hexon protein of claim 3, wherein said hypervariable region is selected from the group consisting of HVR1, HVR2, HVR3, HVR4, HVR5, HVR6, and HVR7.

5. The chimeric adenovirus hexon protein of any of claims 1-4, comprising a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:10, SEQ
ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO; 18, SEQ ID
NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID
NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, and SEQ ID NO:48.

6. The chimeric adenovirus hexon protein of any of claims 1-5, comprising the sequence of SEQ ID NO:50.

7. The chimeric adenovirus heron protein of any of claims 1-6, comprising an amino acid sequence of a hexon protein of another serotype of adenovirus.

8. An isolated or purified nucleic acid that encodes the chimeric adenovirus hexon protein of any of claims 1-7.

9. The isolated or purified nucleic acid of claim 8 comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID N0:45, and SEQ ID NO:47.

10. The isolated or purified nucleic acid of claim 2 or 9, comprising SEQ ID NO:49.

11. An adenoviral vector that comprises the chimeric adenovirus hexon protein of any of claims 1-7.

12. The adenoviral vector of claim 11, further comprising a nonnative fiber protein.

13. The adenoviral vector of claim 12, wherein said nonnative fiber protein is derived from an adenoviral serotype other than said adenoviral vector.

14. The adenoviral vector of any of claims 12-13, wherein said nonnative fiber protein comprises a deletion, insertion, or a replacement of a region of from about 2 to about 750 amino acids of a wild-type adenovirus fiber protein.

15. A method of genetically modifying a cell which comprises contacting said cell with the adenoviral vector of any of claims 11-14.

16. A host cell that comprises the chimeric adenovirus hexon protein of any of claims 1-7.

17. A host cell that comprises the nucleic acid of any of claims 8-10.

18. A host cell that comprises the vector of any of claims 11-14.

19. A method of constructing an adenoviral vector that has a decreased ability or inability to be recognized by a neutralizing antibody directed against wild-type adenovirus hexon protein, which method comprises obtaining an adenoviral vector comprising a wild-type adenovirus hexon protein and replacing said wild-type adenovirus hexon protein with the chimeric adenovirus hexon protein of any of claims 1-7.