U.S. patent application number 14/750837 was filed with the patent office on 2016-02-25 for neisserial antigenic peptides.
This patent application is currently assigned to GLAXOSMITHKLINE BIOLOGICALS SA. The applicant listed for this patent is GLAXOSMITHKLINE BIOLOGICALS SA. Invention is credited to Cesira GALEOTTI, Guido GRANDI, Vega MASIGNANI, Marirosa MORA, Mariagrazia PIZZA, Rino RAPPUOLI, Giulio RATTI, Vincenzo SCARLATO, Maria SCARSELLI.
Application Number | 20160051658 14/750837 |
Document ID | / |
Family ID | 22586405 |
Filed Date | 2016-02-25 |
United States Patent
Application |
20160051658 |
Kind Code |
A1 |
GALEOTTI; Cesira ; et
al. |
February 25, 2016 |
NEISSERIAL ANTIGENIC PEPTIDES
Abstract
This invention provides, among other things, proteins,
polypeptides, and fragments thereof, derived from the bacteria
Neisseria meningitidis B. Also provided are nucleic acids encoding
for such proteins, polypeptides, and/or fragments, as well as
nucleic acids complementary thereto e.g., antisense nucleic acids).
Additionally, this invention provides antibodies which bind to the
proteins, polypeptides, and/or fragments. This invention further
provides expression vectors useful for making the proteins,
polypeptides, and/or fragments, as well as host cells transformed
with such vectors. This invention also provides compositions of the
proteins, polypeptides, fragments, and/or nucleic acids, for use as
vaccines, diagnostic reagents, immunogenic compositions, and the
like. Methods of making the compositions and methods of treatment
with the compositions are also provided. This invention also
provides methods of detecting the proteins, polypeptides,
fragments, and/or nucleic acids.
Inventors: |
GALEOTTI; Cesira; (Siena,
IT) ; GRANDI; Guido; (Siena, IT) ; MASIGNANI;
Vega; (Siena, IT) ; MORA; Marirosa; (Siena,
IT) ; PIZZA; Mariagrazia; (Siena, IT) ;
RAPPUOLI; Rino; (Siena, IT) ; RATTI; Giulio;
(Siena, IT) ; SCARLATO; Vincenzo; (Siena, IT)
; SCARSELLI; Maria; (Siena, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE BIOLOGICALS SA |
Rixensart |
|
BE |
|
|
Assignee: |
GLAXOSMITHKLINE BIOLOGICALS
SA
Rixensart
BE
|
Family ID: |
22586405 |
Appl. No.: |
14/750837 |
Filed: |
June 25, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13405213 |
Feb 24, 2012 |
9067987 |
|
|
14750837 |
|
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|
10111983 |
Feb 26, 2003 |
8734812 |
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PCT/IB2000/001661 |
Oct 30, 2000 |
|
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13405213 |
|
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60162616 |
Oct 29, 1999 |
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Current U.S.
Class: |
424/190.1 |
Current CPC
Class: |
A61K 39/00 20130101;
A61K 39/095 20130101; A61P 31/04 20180101; C07K 14/22 20130101;
A61K 2039/55505 20130101; A61P 31/00 20180101; A61K 48/00 20130101;
A61P 37/04 20180101 |
International
Class: |
A61K 39/095 20060101
A61K039/095 |
Claims
1. (canceled)
2. A composition comprising an adjuvant and an isolated polypeptide
including the amino acid sequence selected from SEQ ID NO: 11072
and SEQ ID NO: 24053.
3. The composition of claim 2, wherein the polypeptide does not
comprise the full length protein sequence of SEQ ID NO: 41367, SEQ
ID NO: 41369, or SEQ ID NO: 41371.
4. The composition of claim 2, wherein the amino acid sequence of
SEQ ID NO: 11072 is an antigenic fragment of SEQ ID NO: 41369; or
the amino acid sequence of SEQ ID NO: 24053 is an antigenic
fragment of SEQ ID NO: 41371.
5. The composition of claim 4, wherein the polypeptide does not
comprise the full length protein sequence of SEQ ID NO: 41367, SEQ
ID NO: 41369, or SEQ ID NO: 41371.
6. The composition of claim 2, further comprising a
pharmaceutically acceptable excipient or carrier.
7. The composition of claim 6, wherein the adjuvant comprises an
aluminum phosphate adjuvant.
8. The composition of claim 3, further comprising a
pharmaceutically acceptable excipient or carrier.
9. The composition of claim 8, wherein the adjuvant comprises an
aluminum phosphate adjuvant.
10. The composition of claim 4, further comprising a
pharmaceutically acceptable excipient or carrier.
11. The composition of claim 10, wherein the adjuvant comprises an
aluminum phosphate adjuvant.
12. The composition of claim 5, further comprising a
pharmaceutically acceptable excipient or carrier.
13. The composition of claim 12, wherein the adjuvant comprises an
aluminum phosphate adjuvant.
14. A method of inducing an immune response in a subject comprising
administering to the subject an immunologically effective dose of
the composition of claim 2.
15. The method of claim 14, wherein the polypeptide does not
comprise the full length protein sequence of SEQ ID NO: 41367, SEQ
ID NO: 41369, or SEQ ID NO: 41371.
16. The method of claim 14, wherein the amino acid sequence of SEQ
ID NO: 11072 is an antigenic fragment of SEQ ID NO: 41369; or the
amino acid sequence of SEQ ID NO: 24053 is an antigenic fragment of
SEQ ID NO: 41371.
17. The method of claim 16, wherein the polypeptide does not
comprise the full length protein sequence of SEQ ID NO: 41367, SEQ
ID NO: 41369, or SEQ ID NO: 41371.
18. The method of claim 14, wherein the composition further
comprises a pharmaceutically acceptable excipient or carrier.
19. The method of claim 18, wherein the adjuvant comprises an
aluminum phosphate adjuvant.
20. The method of claim 15, wherein the composition further
comprises a pharmaceutically acceptable excipient or carrier.
21. The method of claim 20, wherein the adjuvant comprises an
aluminum phosphate adjuvant.
22. The method of claim 16, wherein the composition further
comprises a pharmaceutically acceptable excipient or carrier.
23. The method of claim 22, wherein the adjuvant comprises an
aluminum phosphate adjuvant.
24. The method of claim 17, wherein the composition further
comprises a pharmaceutically acceptable excipient or carrier.
25. The method of claim 24, wherein the adjuvant comprises an
aluminum phosphate adjuvant.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 13/405,213, filed Feb. 24, 2012, now U.S. Pat.
No. 9,067,987, which is a Divisional of U.S. patent application
Ser. No. 10/111,983, claiming an international filing date of Oct.
30, 2000, now U.S. Pat. No. 8,734,812, which is the National Stage
of International Patent Application No. PCT/IB2000/001661, filed
Oct. 30, 2000, which claims the benefit of U.S. Provisional Patent
Application No. 60/162,616, filed Oct. 29, 1999, the disclosures of
which are herein incorporated by reference their entirety.
SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
303822000501SubSeqList.txt, date recorded: Jul. 30, 2015, size:
19,202 KB).
TECHNICAL FIELD
[0003] This invention relates to antigenic peptide sequences from
the bacteria Neisseria meningitidis and Neisseria gonorrhoea.
BACKGROUND ART
[0004] N. meningitidis is a non-motile, Gram-negative diplococcus
that is pathogenic in humans. Based on the organism's capsular
polysaccharide, 12 serogroups of N. meningitidis have been
identified. Group A is the pathogen most often implicated in
epidemic disease in sub-Saharan Africa. Serogroups B and C are
responsible for the vast majority of cases in the United States and
in most developed countries. Serogroups W135 and Y are responsible
for the rest of the cases in the United States and developed
countries.
[0005] The meningococcal vaccine currently in use is a tetravalent
polysaccharide vaccine composed of serogroups A, C, Y and W135.
Meningococcus B remains a problem, however. The polysaccharide
approach cannot be used because the menB capsular polysaccharide is
a polymer of .alpha.(2-8)-linked N-acetyl neuraminic acid that is
also present in mammalian tissue. One approach to a menB vaccine
uses mixtures of outer membrane proteins (OMPs). To overcome the
antigenic variability, multivalent vaccines containing up to nine
different porins have been constructed [e.g., Poolman J T (1992)
Development of a meningococcal vaccine. Infect. Agents Dis. 4:
13-28]. Additional proteins to be used in outer membrane vaccines
have been the opa and ope proteins, but none of these approaches
have been able to overcome the antigenic variability [e.g.,
Ala'Aldeen & Borriello (1996)]. The meningococcal
transferrin-binding proteins 1 and 2 are both surface exposed and
generate bactericidal antibodies capable of killing homologous and
heterologous strains. [Vaccine 14(1):49-53].
DISCLOSURE OF THE INVENTION
[0006] The invention provides fragments of the proteins disclosed
in international patent applications WO99/57280 and WO00/22430 (the
"International Applications"), wherein the fragments comprise at
least one antigenic determinant.
[0007] Thus, if the length of any particular protein sequence
disclosed in the International Applications is x amino acids, the
present invention provides fragments of at most x-1 amino acids of
that protein. The fragment may be shorter than this (e.g., x-2,
x-3, x-4, . . . ), and is preferably 100 amino acids or less (e.g.,
90 amino acids, 80 amino acids etc.). The fragment may be as short
as 3 amino acids, but is preferably longer (e.g., up to 5, 6, 7, 8,
9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, or 100 amino acids).
[0008] Preferred fragments comprise the meningococcal peptide
sequences disclosed in Table 1, or sub-sequences thereof. The
fragments may be longer than those given in Table 1 e.g., where a
fragment in Table 1 runs from amino acid residue p to residue q of
a protein, the invention also relates to fragments from residue
(p-1), (p-2), or (p-3) to residue (q+1), (q+2), or (q+3).
[0009] The invention also provides polypeptides that are homologous
(i.e., have sequence identity) to these fragments. Depending on the
particular fragment, the degree of sequence identity is preferably
greater than 50% (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more).
These homologous polypeptides include mutants and allelic variants
of the fragments. Identity between the two sequences is preferably
determined by the Smith-Waterman homology search algorithm as
implemented in the MPSRCH program (Oxford Molecular), using an
affine gap search with parameters gap open penalty=12 and gap
extension penalty=1.
[0010] The invention also provides proteins comprising one or more
of the above-defined fragments.
[0011] The invention is subject to the proviso that it does not
include within its scope proteins limited to any of the full length
protein sequences disclosed in the International Applications
(i.e., the even SEQ IDs: 2-3020 of WO99/57280 and the odd SEQ IDs:
963-1045 of WO00/22430).
[0012] The proteins of the invention can, of course, be prepared by
various means (e.g., recombinant expression, purification from cell
culture, chemical synthesis etc.) and in various forms (e.g.,
native, C-terminal and/or N-terminal fusions etc.). They are
preferably prepared in substantially pure form (i.e., substantially
free from other Neisserial or host cell proteins). Short proteins
are preferably produced using chemical peptide synthesis.
[0013] According to a further aspect, the invention provides
antibodies which recognise the fragments of the invention, with the
proviso that the invention does not include within its scope
antibodies which recognise any of the complete protein sequences in
the International Applications. The antibodies may be polyclonal or
monoclonal, and may be produced by any suitable means.
[0014] The invention also provides proteins comprising peptide
sequences recognised by these antibodies. These peptide sequences
will, of course, include fragments of the meningococcal proteins in
the International Applications, but will also include peptides that
mimic the antigenic structure of the meningococcal peptides when
bound to immunoglobulin.
[0015] According to a further aspect, the invention provides
nucleic acid encoding the fragments and proteins of the invention,
with the proviso that the invention does not include within its
scope nucleic acid encoding any of the full length protein
sequences in the International Applications. The nucleic acids may
be as short as 10 nucleotides, but are preferably longer (e.g., up
to 10, 12, 15, 18, 20, 25, 30, 35, 40, 50, 75, or 100
nucleotides).
[0016] In addition, the invention provides nucleic acid comprising
sequences homologous (i.e., having sequence identity) to these
sequences. The degree of sequence identity is preferably greater
than 50% (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more). Furthermore,
the invention provides nucleic acid which can hybridise to these
sequences, preferably under "high stringency" conditions (e.g.,
65.degree. C. in a 0.1.times.SSC, 0.5% SDS solution).
[0017] It should also be appreciated that the invention provides
nucleic acid comprising sequences complementary to those described
above (e.g., for antisense or probing purposes).
[0018] Nucleic acid according to the invention can, of course, be
prepared in many ways (e.g., by chemical synthesis, from genomic or
cDNA libraries, from the organism itself etc.) and can take various
forms (e.g., single stranded, double stranded, vectors, probes
etc.). In addition, the term "nucleic acid" includes DNA and RNA,
and also their analogues, such as those containing modified
backbones, and also peptide nucleic acids (PNA), etc.
[0019] According to a further aspect, the invention provides
vectors comprising nucleotide sequences of the invention (e.g.,
expression vectors) and host cells transformed with such
vectors.
[0020] According to a further aspect, the invention provides
compositions comprising protein, antibody, and/or nucleic acid
according to the invention. These compositions may be suitable as
vaccines, for instance, or as diagnostic reagents, or as
immunogenic compositions.
[0021] The invention also provides nucleic acid, protein, or
antibody according to the invention for use as medicaments (e.g.,
as vaccines or as immunogenic compositions) or as diagnostic
reagents. It also provides the use of nucleic acid, protein, or
antibody according to the invention in the manufacture of: (i) a
medicament for treating or preventing infection due to Neisserial
bacteria; (ii) a diagnostic reagent for detecting the presence of
Neisserial bacteria or of antibodies raised against Neisserial
bacteria; and/or (iii) a reagent which can raise antibodies against
Neisserial bacteria. Said Neisserial bacteria may be any species or
strain (such as N. gonorrhoeae) but are preferably N. meningitidis,
especially strain A or strain B.
[0022] The invention also provides a method of treating a patient,
comprising administering to the patient a therapeutically effective
amount of nucleic acid, protein, and/or antibody according to the
invention.
[0023] According to further aspects, the invention provides various
processes, for example:
[0024] A process for producing proteins of the invention is
provided, comprising the step of culturing a host cell according to
the invention under conditions which induce protein expression;
[0025] A process for producing protein or nucleic acid of the
invention is provided, wherein the protein or nucleic acid is
synthesised in part or in whole using chemical means;
[0026] A process for detecting polynucleotides of the invention is
provided, comprising the steps of: (a) contacting a nucleic probe
according to the invention with a biological sample under
hybridizing conditions to form duplexes; and (b) detecting said
duplexes; and
[0027] A process for detecting proteins of the invention is
provided, comprising the steps of: (a) contacting an antibody
according to the invention with a biological sample under
conditions suitable for the formation of an antibody-antigen
complexes; and (b) detecting said complexes.
[0028] A summary of standard techniques and procedures which may be
employed in order to perform the invention (e.g., to utilise the
disclosed sequences for vaccination or diagnostic purposes)
follows. This summary is not a limitation on the invention but,
rather, gives examples which may be used, but which are not
required.
General
[0029] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature e.g., Sambrook Molecular Cloning; A Laboratory Manual,
Second Edition (1989); DNA Cloning, Volumes I and ii (D. N Glover
ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic
Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription and Translation (B. D. Hames & S. J. Higgins eds.
1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized
Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide
to Molecular Cloning (1984); the Methods in Enzymology series
(Academic Press, Inc.), especially volumes 154 & 155; Gene
Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos
eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds.
(1987), Immunochemical Methods in Cell and Molecular Biology
(Academic Press, London); Scopes, (1987) Protein Purification:
Principles and Practice, Second Edition (Springer-Verlag, N.Y.),
and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir
and C. C. Blackwell eds 1986).
[0030] Standard abbreviations for nucleotides and amino acids are
used in this specification.
[0031] All publications, patents, and patent applications cited
herein are incorporated in full by reference.
Definitions
[0032] A composition containing X is "substantially free of" Y when
at least 85% by weight of the total X+Y in the composition is X.
Preferably, X comprises at least about 90% by weight of the total
of X+Y in the composition, more preferably at least about 95% or
even 99% by weight.
[0033] The term "comprising" means "including" as well as
"consisting" e.g., a composition "comprising" X may consist
exclusively of X or may include something additional to X, such as
X+Y.
[0034] The term "antigenic determinant" includes B-cell epitopes
and T-cell epitopes.
[0035] The term "heterologous" refers to two biological components
that are not found together in nature. The components may be host
cells, genes, or regulatory regions, such as promoters. Although
the heterologous components are not found together in nature, they
can function together, as when a promoter heterologous to a gene is
operably linked to the gene. Another example is where a
meningococcal sequence is heterologous to a mouse host cell. A
further example would be two epitopes from the same or different
proteins which have been assembled in a single protein in an
arrangement not found in nature.
[0036] An "origin of replication" is a polynucleotide sequence that
initiates and regulates replication of polynucleotides, such as an
expression vector. The origin of replication behaves as an
autonomous unit of polynucleotide replication within a cell,
capable of replication under its own control. An origin of
replication may be needed for a vector to replicate in a particular
host cell. With certain origins of replication, an expression
vector can be reproduced at a high copy number in the presence of
the appropriate proteins within the cell. Examples of origins are
the autonomously replicating sequences, which are effective in
yeast; and the viral T-antigen, effective in COS-7 cells.
Expression Systems
[0037] The meningococcal nucleotide sequences can be expressed in a
variety of different expression systems; for example those used
with mammalian cells, baculoviruses, plants, bacteria, and
yeast.
i. Mammalian Systems
[0038] Mammalian expression systems are known in the art. A
mammalian promoter is any DNA sequence capable of binding mammalian
RNA polymerase and initiating the downstream (3') transcription of
a coding sequence (e.g., structural gene) into mRNA. A promoter
will have a transcription initiating region, which is usually
placed proximal to the 5' end of the coding sequence, and a TATA
box, usually located 25-30 base pairs (bp) upstream of the
transcription initiation site. The TATA box is thought to direct
RNA polymerase II to begin RNA synthesis at the correct site. A
mammalian promoter will also contain an upstream promoter element,
usually located within 100 to 200 bp upstream of the TATA box. An
upstream promoter element determines the rate at which
transcription is initiated and can act in either orientation
[Sambrook et al. (1989) "Expression of Cloned Genes in Mammalian
Cells." In Molecular Cloning: A Laboratory Manual, 2nd ed.].
[0039] Mammalian viral genes are often highly expressed and have a
broad host range; therefore sequences encoding mammalian viral
genes provide particularly useful promoter sequences. Examples
include the SV40 early promoter, mouse mammary tumor virus LTR
promoter, adenovirus major late promoter (Ad MLP), and herpes
simplex virus promoter. In addition, sequences derived from
non-viral genes, such as the murine metallotheionein gene, also
provide useful promoter sequences. Expression may be either
constitutive or regulated (inducible), depending on the promoter
can be induced with glucocorticoid in hormone-responsive cells.
[0040] The presence of an enhancer element (enhancer), combined
with the promoter elements described above, will usually increase
expression levels. An enhancer is a regulatory DNA sequence that
can stimulate transcription up to 1000-fold when linked to
homologous or heterologous promoters, with synthesis beginning at
the normal RNA start site. Enhancers are also active when they are
placed upstream or downstream from the transcription initiation
site, in either normal or flipped orientation, or at a distance of
more than 1000 nucleotides from the promoter [Maniatis et al.
(1987) Science 236:1237; Alberts et al. (1989) Molecular Biology of
the Cell, 2nd ed.]. Enhancer elements derived from viruses may be
particularly useful, because they usually have a broader host
range. Examples include the SV40 early gene enhancer [Dijkema et al
(1985) EMBO J. 4:761] and the enhancer/promoters derived from the
long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al.
(1982b) Proc. Natl. Acad. Sci. 79:6777] and from human
cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally,
some enhancers are regulatable and become active only in the
presence of an inducer, such as a hormone or metal ion
[Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et
al. (1987) Science 236:1237].
[0041] A DNA molecule may be expressed intracellularly in mammalian
cells. A promoter sequence may be directly linked with the DNA
molecule, in which case the first amino acid at the N-terminus of
the recombinant protein will always be a methionine, which is
encoded by the ATG start codon. If desired, the N-terminus may be
cleaved from the protein by in vitro incubation with cyanogen
bromide.
[0042] Alternatively, foreign proteins can also be secreted from
the cell into the growth media by creating chimeric DNA molecules
that encode a fusion protein comprised of a leader sequence
fragment that provides for secretion of the foreign protein in
mammalian cells. Preferably, there are processing sites encoded
between the leader fragment and the foreign gene that can be
cleaved either in vivo or in vitro. The leader sequence fragment
usually encodes a signal peptide comprised of hydrophobic amino
acids which direct the secretion of the protein from the cell. The
adenovirus triparite leader is an example of a leader sequence that
provides for secretion of a foreign protein in mammalian cells.
[0043] Usually, transcription termination and polyadenylation
sequences recognized by mammalian cells are regulatory regions
located 3' to the translation stop codon and thus, together with
the promoter elements, flank the coding sequence. The 3' terminus
of the mature mRNA is formed by site-specific post-transcriptional
cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349;
Proudfoot and Whitelaw (1988) "Termination and 3' end processing of
eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and
D. M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These
sequences direct the transcription of an mRNA which can be
translated into the polypeptide encoded by the DNA. Examples of
transcription terminater/polyadenylation signals include those
derived from SV40 [Sambrook et al (1989) "Expression of cloned
genes in cultured mammalian cells." In Molecular Cloning: A
Laboratory Manual].
[0044] Usually, the above described components, comprising a
promoter, polyadenylation signal, and transcription termination
sequence are put together into expression constructs. Enhancers,
introns with functional splice donor and acceptor sites, and leader
sequences may also be included in an expression construct, if
desired. Expression constructs are often maintained in a replicon,
such as an extrachromosomal element (e.g., plasmids) capable of
stable maintenance in a host, such as mammalian cells or bacteria.
Mammalian replication systems include those derived from animal
viruses, which require trans-acting factors to replicate. For
example, plasmids containing the replication systems of
papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or
polyomavirus, replicate to extremely high copy number in the
presence of the appropriate viral T antigen. Additional examples of
mammalian replicons include those derived from bovine
papillomavirus and Epstein-Barr virus. Additionally, the replicon
may have two replicaton systems, thus allowing it to be maintained,
for example, in mammalian cells for expression and in a prokaryotic
host for cloning and amplification. Examples of such
mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al.
(1989) Mol. Cell. Biol. 9:946] and pHEBO [Shimizu et al. (1986)
Mol. Cell. Biol. 6:1074].
[0045] The transformation procedure used depends upon the host to
be transformed. Methods for introduction of heterologous
polynucleotides into mammalian cells are known in the art and
include dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0046] Mammalian cell lines available as hosts for expression are
known in the art and include many immortalized cell lines available
from the American Type Culture Collection (ATCC), including but not
limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby
hamster kidney (BHK) cells, monkey kidney cells (COS), human
hepatocellular carcinoma cells (e.g., Hep G2), and a number of
other cell lines.
ii. Baculovirus Systems
[0047] The polynucleotide encoding the protein can also be inserted
into a suitable insect expression vector, and is operably linked to
the control elements within that vector. Vector construction
employs techniques which are known in the art. Generally, the
components of the expression system include a transfer vector,
usually a bacterial plasmid, which contains both a fragment of the
baculovirus genome, and a convenient restriction site for insertion
of the heterologous gene or genes to be expressed; a wild type
baculovirus with a sequence homologous to the baculovirus-specific
fragment in the transfer vector (this allows for the homologous
recombination of the heterologous gene in to the baculovirus
genome); and appropriate insect host cells and growth media.
[0048] After inserting the DNA sequence encoding the protein into
the transfer vector, the vector and the wild type viral genome are
transfected into an insect host cell where the vector and viral
genome are allowed to recombine. The packaged recombinant virus is
expressed and recombinant plaques are identified and purified.
Materials and methods for baculovirus/insect cell expression
systems are commercially available in kit form from, inter alia,
INVITROGEN, Carlsbab Calif. ("MaxBac" kit). These techniques are
generally known to those skilled in the art and fully described in
Summers and Smith, Texas Agricultural Experiment Station Bulletin
No. 1555 (1987) (hereinafter "Summers and Smith").
[0049] Prior to inserting the DNA sequence encoding the protein
into the baculovirus genome, the above described components,
comprising a promoter, leader (if desired), coding sequence of
interest, and transcription termination sequence, are usually
assembled into an intermediate transplacement construct (transfer
vector). This construct may contain a single gene and operably
linked regulatory elements; multiple genes, each with its owned set
of operably linked regulatory elements; or multiple genes,
regulated by the same set of regulatory elements. Intermediate
transplacement constructs are often maintained in a replicon, such
as an extrachromosomal element (e.g., plasmids) capable of stable
maintenance in a host, such as a bacterium. The replicon will have
a replication system, thus allowing it to be maintained in a
suitable host for cloning and amplification.
[0050] Currently, the most commonly used transfer vector for
introducing foreign genes into AcNPV is pAc373. Many other vectors,
known to those of skill in the art, have also been designed. These
include, for example, pVL985 (which alters the polyhedrin start
codon from ATG to ATT, and which introduces a BamHI cloning site 32
basepairs downstream from the ATT; see Luckow and Summers, Virology
(1989) 17:31.
[0051] The plasmid usually also contains the polyhedrin
polyadenylation signal (Miller et al. (1988) Ann. Rev. Microbiol.,
42:177) and a prokaryotic ampicillin-resistance (amp) gene and
origin of replication for selection and propagation in E. coli.
[0052] Baculovirus transfer vectors usually contain a baculovirus
promoter. A baculovirus promoter is any DNA sequence capable of
binding a baculovirus RNA polymerase and initiating the downstream
(5' to 3') transcription of a coding sequence (e.g., structural
gene) into mRNA. A promoter will have a transcription initiation
region which is usually placed proximal to the 5' end of the coding
sequence. This transcription initiation region usually includes an
RNA polymerase binding site and a transcription initiation site. A
baculovirus transfer vector may also have a second domain called an
enhancer, which, if present, is usually distal to the structural
gene. Expression may be either regulated or constitutive.
[0053] Structural genes, abundantly transcribed at late times in a
viral infection cycle, provide particularly useful promoter
sequences. Examples include sequences derived from the gene
encoding the viral polyhedron protein, Friesen et al., (1986) "The
Regulation of Baculovirus Gene Expression," in: The Molecular
Biology of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127
839 and 155 476; and the gene encoding the p10 protein, Vlak et
al., (1988), J. Gen. Virol. 69:765.
[0054] DNA encoding suitable signal sequences can be derived from
genes for secreted insect or baculovirus proteins, such as the
baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409).
Alternatively, since the signals for mammalian cell
posttranslational modifications (such as signal peptide cleavage,
proteolytic cleavage, and phosphorylation) appear to be recognized
by insect cells, and the signals required for secretion and nuclear
accumulation also appear to be conserved between the invertebrate
cells and vertebrate cells, leaders of non-insect origin, such as
those derived from genes encoding human .gamma.-interferon, Maeda
et al., (1985), Nature 315:592; human gastrin-releasing peptide,
Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human
IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, 82:8404;
mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human
glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be
used to provide for secretion in insects.
[0055] A recombinant polypeptide or polyprotein may be expressed
intracellularly or, if it is expressed with the proper regulatory
sequences, it can be secreted. Good intracellular expression of
nonfused foreign proteins usually requires heterologous genes that
ideally have a short leader sequence containing suitable
translation initiation signals preceding an ATG start signal. If
desired, methionine at the N-terminus may be cleaved from the
mature protein by in vitro incubation with cyanogen bromide.
[0056] Alternatively, recombinant polyproteins or proteins which
are not naturally secreted can be secreted from the insect cell by
creating chimeric DNA molecules that encode a fusion protein
comprised of a leader sequence fragment that provides for secretion
of the foreign protein in insects. The leader sequence fragment
usually encodes a signal peptide comprised of hydrophobic amino
acids which direct the translocation of the protein into the
endoplasmic reticulum.
[0057] After insertion of the DNA sequence and/or the gene encoding
the expression product precursor of the protein, an insect cell
host is co-transformed with the heterologous DNA of the transfer
vector and the genomic DNA of wild type baculovirus--usually by
co-transfection. The promoter and transcription termination
sequence of the construct will usually comprise a 2-5 kb section of
the baculovirus genome. Methods for introducing heterologous DNA
into the desired site in the baculovirus virus are known in the
art. (See Summers and Smith supra; Ju et al. (1987); Smith et al.,
Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For
example, the insertion can be into a gene such as the polyhedrin
gene, by homologous double crossover recombination; insertion can
also be into a restriction enzyme site engineered into the desired
baculovirus gene. Miller et al., (1989), Bioessays 4:91.The DNA
sequence, when cloned in place of the polyhedrin gene in the
expression vector, is flanked both 5' and 3' by polyhedrin-specific
sequences and is positioned downstream of the polyhedrin
promoter.
[0058] The newly formed baculovirus expression vector is
subsequently packaged into an infectious recombinant baculovirus.
Homologous recombination occurs at low frequency (between about 1%
and about 5%); thus, the majority of the virus produced after
cotransfection is still wild-type virus. Therefore, a method is
necessary to identify recombinant viruses. An advantage of the
expression system is a visual screen allowing recombinant viruses
to be distinguished. The polyhedrin protein, which is produced by
the native virus, is produced at very high levels in the nuclei of
infected cells at late times after viral infection. Accumulated
polyhedrin protein forms occlusion bodies that also contain
embedded particles. These occlusion bodies, up to 15 .mu.m in size,
are highly refractile, giving them a bright shiny appearance that
is readily visualized under the light microscope. Cells infected
with recombinant viruses lack occlusion bodies. To distinguish
recombinant virus from wild-type virus, the transfection
supernatant is plagued onto a monolayer of insect cells by
techniques known to those skilled in the art. Namely, the plaques
are screened under the light microscope for the presence
(indicative of wild-type virus) or absence (indicative of
recombinant virus) of occlusion bodies. "Current Protocols in
Microbiology" Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990);
Summers and Smith, supra; Miller et al. (1989).
[0059] Recombinant baculovirus expression vectors have been
developed for infection into several insect cells. For example,
recombinant baculoviruses have been developed for, inter alia:
Aedes aegypti, Autographa californica, Bombyx mori, Drosophila
melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO
89/046699; Carbonell et al., (1985) J. Virol. 56:153; Wright (1986)
Nature 321:718; Smith et al., (1983) Mol. Cell. Biol. 3:2156; and
see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol.
25:225).
[0060] Cells and cell culture media are commercially available for
both direct and fusion expression of heterologous polypeptides in a
baculovirus/expression system; cell culture technology is generally
known to those skilled in the art. See, e.g., Summers and Smith
supra.
[0061] The modified insect cells may then be grown in an
appropriate nutrient medium, which allows for stable maintenance of
the plasmid(s) present in the modified insect host. Where the
expression product gene is under inducible control, the host may be
grown to high density, and expression induced. Alternatively, where
expression is constitutive, the product will be continuously
expressed into the medium and the nutrient medium must be
continuously circulated, while removing the product of interest and
augmenting depleted nutrients. The product may be purified by such
techniques as chromatography, e.g., HPLC, affinity chromatography,
ion exchange chromatography, etc.; electrophoresis; density
gradient centrifugation; solvent extraction, or the like. As
appropriate, the product may be further purified, as required, so
as to remove substantially any insect proteins which are also
secreted in the medium or result from lysis of insect cells, so as
to provide a product which is at least substantially free of host
debris, e.g., proteins, lipids and polysaccharides.
[0062] In order to obtain protein expression, recombinant host
cells derived from the transformants are incubated under conditions
which allow expression of the recombinant protein encoding
sequence. These conditions will vary, dependent upon the host cell
selected. However, the conditions are readily ascertainable to
those of ordinary skill in the art, based upon what is known in the
art.
iii. Plant Systems
[0063] There are many plant cell culture and whole plant genetic
expression systems known in the art. Exemplary plant cellular
genetic expression systems include those described in patents, such
as: U.S. Pat. No. 5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat.
No. 5,608,143. Additional examples of genetic expression in plant
cell culture has been described by Zenk, Phytochemistry
30:3861-3863 (1991). Descriptions of plant protein signal peptides
may be found in addition to the references described above in
Vaulcombe et al., Mol. Gen. Genet. 209:33-40 (1987); Chandler et
al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol.
Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356
(1987); Whittier et al., Nucleic Acids Research 15:2515-2535
(1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et
al., Gene 122:247-253 (1992). A description of the regulation of
plant gene expression by the phytohormone, gibberellic acid and
secreted enzymes induced by gibberellic acid can be found in R. L.
Jones and J. MacMillin, Gibberellins: in: Advanced Plant
Physiology., Malcolm B. Wilkins, ed., 1984 Pitman Publishing
Limited, London, pp. 21-52. References that describe other
metabolically-regulated genes: Sheen, Plant Cell,
2:1027-1038(1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel
and Hickey, Proc. Natl. Acad. Sci. 84:1337-1339 (1987)
[0064] Typically, using techniques known in the art, a desired
polynucleotide sequence is inserted into an expression cassette
comprising genetic regulatory elements designed for operation in
plants. The expression cassette is inserted into a desired
expression vector with companion sequences upstream and downstream
from the expression cassette suitable for expression in a plant
host. The companion sequences will be of plasmid or viral origin
and provide necessary characteristics to the vector to permit the
vectors to move DNA from an original cloning host, such as
bacteria, to the desired plant host. The basic bacterial/plant
vector construct will preferably provide a broad host range
prokaryote replication origin; a prokaryote selectable marker; and,
for Agrobacterium transformations, T DNA sequences for
Agrobacterium-mediated transfer to plant chromosomes. Where the
heterologous gene is not readily amenable to detection, the
construct will preferably also have a selectable marker gene
suitable for determining if a plant cell has been transformed. A
general review of suitable markers, for example for the members of
the grass family, is found in Wilmink and Dons, 1993, Plant Mol.
Biol. Reptr, 11(2):165-185.
[0065] Sequences suitable for permitting integration of the
heterologous sequence into the plant genome are also recommended.
These might include transposon sequences and the like for
homologous recombination as well as Ti sequences which permit
random insertion of a heterologous expression cassette into a plant
genome. Suitable prokaryote selectable markers include resistance
toward antibiotics such as ampicillin or tetracycline. Other DNA
sequences encoding additional functions may also be present in the
vector, as is known in the art.
[0066] The nucleic acid molecules of the subject invention may be
included into an expression cassette for expression of the
protein(s) of interest. Usually, there will be only one expression
cassette, although two or more are feasible. The recombinant
expression cassette will contain in addition to the heterologous
protein encoding sequence the following elements, a promoter
region, plant 5' untranslated sequences, initiation codon depending
upon whether or not the structural gene comes equipped with one,
and a transcription and translation termination sequence. Unique
restriction enzyme sites at the 5' and 3' ends of the cassette
allow for easy insertion into a pre-existing vector.
[0067] A heterologous coding sequence may be for any protein
relating to the present invention. The sequence encoding the
protein of interest will encode a signal peptide which allows
processing and translocation of the protein, as appropriate, and
will usually lack any sequence which might result in the binding of
the desired protein of the invention to a membrane. Since, for the
most part, the transcriptional initiation region will be for a gene
which is expressed and translocated during germination, by
employing the signal peptide which provides for translocation, one
may also provide for translocation of the protein of interest. In
this way, the protein(s) of interest will be translocated from the
cells in which they are expressed and may be efficiently harvested.
Typically secretion in seeds are across the aleurone or scutellar
epithelium layer into the endosperm of the seed. While it is not
required that the protein be secreted from the cells in which the
protein is produced, this facilitates the isolation and
purification of the recombinant protein.
[0068] Since the ultimate expression of the desired gene product
will be in a eucaryotic cell it is desirable to determine whether
any portion of the cloned gene contains sequences which will be
processed out as introns by the host's splicosome machinery. If so,
site-directed mutagenesis of the "intron" region may be conducted
to prevent losing a portion of the genetic message as a false
intron code, Reed and Maniatis, Cell 41:95-105, 1985.
[0069] The vector can be microinjected directly into plant cells by
use of micropipettes to mechanically transfer the recombinant DNA.
Crossway, Mol. Gen. Genet, 202:179-185, 1985. The genetic material
may also be transferred into the plant cell by using polyethylene
glycol, Krens, et al., Nature, 296, 72-74, 1982. Another method of
introduction of nucleic acid segments is high velocity ballistic
penetration by small particles with the nucleic acid either within
the matrix of small beads or particles, or on the surface, Klein,
et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991,
Planta, 185:330-336 teaching particle bombardment of barley
endosperm to create transgenic barley. Yet another method of
introduction would be fusion of protoplasts with other entities,
either minicells, cells, lysosomes or other fusible lipid-surfaced
bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863,
1982.
[0070] The vector may also be introduced into the plant cells by
electroporation. (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824,
1985). In this technique, plant protoplasts are electroporated in
the presence of plasmids containing the gene construct. Electrical
impulses of high field strength reversibly permeabilize
biomembranes allowing the introduction of the plasmids.
Electroporated plant protoplasts reform the cell wall, divide, and
form plant callus.
[0071] All plants from which protoplasts can be isolated and
cultured to give whole regenerated plants can be transformed by the
present invention so that whole plants are recovered which contain
the transferred gene. It is known that practically all plants can
be regenerated from cultured cells or tissues, including but not
limited to all major species of sugarcane, sugar beet, cotton,
fruit and other trees, legumes and vegetables. Some suitable plants
include, for example, species from the genera Fragaria, Lotus,
Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum,
Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus,
Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion,
Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium,
Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis,
Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio,
Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum,
Sorghum, and Datura.
[0072] Means for regeneration vary from species to species of
plants, but generally a suspension of transformed protoplasts
containing copies of the heterologous gene is first provided.
Callus tissue is formed and shoots may be induced from callus and
subsequently rooted. Alternatively, embryo formation can be induced
from the protoplast suspension. These embryos germinate as natural
embryos to form plants. The culture media will generally contain
various amino acids and hormones, such as auxin and cytokinins. It
is also advantageous to add glutamic acid and proline to the
medium, especially for such species as corn and alfalfa. Shoots and
roots normally develop simultaneously. Efficient regeneration will
depend on the medium, on the genotype, and on the history of the
culture. If these three variables are controlled, then regeneration
is fully reproducible and repeatable.
[0073] In some plant cell culture systems, the desired protein of
the invention may be excreted or alternatively, the protein may be
extracted from the whole plant. Where the desired protein of the
invention is secreted into the medium, it may be collected.
Alternatively, the embryos and embryoless-half seeds or other plant
tissue may be mechanically disrupted to release any secreted
protein between cells and tissues. The mixture may be suspended in
a buffer solution to retrieve soluble proteins. Conventional
protein isolation and purification methods will be then used to
purify the recombinant protein. Parameters of time, temperature pH,
oxygen, and volumes will be adjusted through routine methods to
optimize expression and recovery of heterologous protein.
iv. Bacterial Systems
[0074] Bacterial expression techniques are known in the art. A
bacterial promoter is any DNA sequence capable of binding bacterial
RNA polymerase and initiating the downstream (3') transcription of
a coding sequence (e.g., structural gene) into mRNA. A promoter
will have a transcription initiation region which is usually placed
proximal to the 5' end of the coding sequence. This transcription
initiation region usually includes an RNA polymerase binding site
and a transcription initiation site. A bacterial promoter may also
have a second domain called an operator, that may overlap an
adjacent RNA polymerase binding site at which RNA synthesis begins.
The operator permits negative regulated (inducible) transcription,
as a gene repressor protein may bind the operator and thereby
inhibit transcription of a specific gene. Constitutive expression
may occur in the absence of negative regulatory elements, such as
the operator. In addition, positive regulation may be achieved by a
gene activator protein binding sequence, which, if present is
usually proximal (5') to the RNA polymerase binding sequence. An
example of a gene activator protein is the catabolite activator
protein (CAP), which helps initiate transcription of the lac operon
in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev.
Genet. 18:173]. Regulated expression may therefore be either
positive or negative, thereby either enhancing or reducing
transcription.
[0075] Sequences encoding metabolic pathway enzymes provide
particularly useful promoter sequences. Examples include promoter
sequences derived from sugar metabolizing enzymes, such as
galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and
maltose. Additional examples include promoter sequences derived
from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al.
(1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids
Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 and
EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann
(1981) "The cloning of interferon and other mistakes." In
Interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake
et al. (1981) Nature 292:128] and T5 [U.S. Pat. No. 4,689,406]
promoter systems also provide useful promoter sequences.
[0076] In addition, synthetic promoters which do not occur in
nature also function as bacterial promoters. For example,
transcription activation sequences of one bacterial or
bacteriophage promoter may be joined with the operon sequences of
another bacterial or bacteriophage promoter, creating a synthetic
hybrid promoter [U.S. Pat. No. 4,551,433]. For example, the tac
promoter is a hybrid trp-lac promoter comprised of both trp
promoter and lac operon sequences that is regulated by the lac
repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983)
Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter
can include naturally occurring promoters of non-bacterial origin
that have the ability to bind bacterial RNA polymerase and initiate
transcription. A naturally occurring promoter of non-bacterial
origin can also be coupled with a compatible RNA polymerase to
produce high levels of expression of some genes in prokaryotes. The
bacteriophage T7 RNA polymerase/promoter system is an example of a
coupled promoter system [Studier et al. (1986) J. Mol. Biol.
189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074]. In
addition, a hybrid promoter can also be comprised of a
bacteriophage promoter and an E. coli operator region (EPO-A-0 267
851).
[0077] In addition to a functioning promoter sequence, an efficient
ribosome binding site is also useful for the expression of foreign
genes in prokaryotes. In E. coli, the ribosome binding site is
called the Shine-Dalgarno (SD) sequence and includes an initiation
codon (ATG) and a sequence 3-9 nucleotides in length located 3-11
nucleotides upstream of the initiation codon [Shine et al. (1975)
Nature 254:34]. The SD sequence is thought to promote binding of
mRNA to the ribosome by the pairing of bases between the SD
sequence and the 3' and of E. coli 16S rRNA [Steitz et al. (1979)
"Genetic signals and nucleotide sequences in messenger RNA." In
Biological Regulation and Development: Gene Expression (ed. R. F.
Goldberger)]. To express eukaryotic genes and prokaryotic genes
with weak ribosome-binding site [Sambrook et al. (1989) "Expression
of cloned genes in Escherichia coli." In Molecular Cloning: A
Laboratory Manual].
[0078] A DNA molecule may be expressed intracellularly. A promoter
sequence may be directly linked with the DNA molecule, in which
case the first amino acid at the N-terminus will always be a
methionine, which is encoded by the ATG start codon. If desired,
methionine at the N-terminus may be cleaved from the protein by in
vitro incubation with cyanogen bromide or by either in vivo on in
vitro incubation with a bacterial methionine N-terminal peptidase
(EPO-A-0 219 237).
[0079] Fusion proteins provide an alternative to direct expression.
Usually, a DNA sequence encoding the N-terminal portion of an
endogenous bacterial protein, or other stable protein, is fused to
the 5' end of heterologous coding sequences. Upon expression, this
construct will provide a fusion of the two amino acid sequences.
For example, the bacteriophage lambda cell gene can be linked at
the 5' terminus of a foreign gene and expressed in bacteria. The
resulting fusion protein preferably retains a site for a processing
enzyme (factor Xa) to cleave the bacteriophage protein from the
foreign gene [Nagai et al. (1984) Nature 309:810]. Fusion proteins
can also be made with sequences from the lacZ [Jia et al. (1987)
Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93; Makoff
et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EP-A-0 324 647]
genes. The DNA sequence at the junction of the two amino acid
sequences may or may not encode a cleavable site. Another example
is a ubiquitin fusion protein. Such a fusion protein is made with
the ubiquitin region that preferably retains a site for a
processing enzyme (e.g., ubiquitin specific processing-protease) to
cleave the ubiquitin from the foreign protein. Through this method,
native foreign protein can be isolated [Miller et al. (1989)
Bio/Technology 7:698].
[0080] Alternatively, foreign proteins can also be secreted from
the cell by creating chimeric DNA molecules that encode a fusion
protein comprised of a signal peptide sequence fragment that
provides for secretion of the foreign protein in bacteria [U.S.
Pat. No. 4,336,336]. The signal sequence fragment usually encodes a
signal peptide comprised of hydrophobic amino acids which direct
the secretion of the protein from the cell. The protein is either
secreted into the growth media (gram-positive bacteria) or into the
periplasmic space, located between the inner and outer membrane of
the cell (gram-negative bacteria). Preferably there are processing
sites, which can be cleaved either in vivo or in vitro encoded
between the signal peptide fragment and the foreign gene.
[0081] DNA encoding suitable signal sequences can be derived from
genes for secreted bacterial proteins, such as the E. coli outer
membrane protein gene (ompA) [Masui et al. (1983), in: Experimental
Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J.
3:2437] and the E. coli alkaline phosphatase signal sequence (phoA)
[Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212]. As an
additional example, the signal sequence of the alpha-amylase gene
from various Bacillus strains can be used to secrete heterologous
proteins from B. subtilis [Palva et al. (1982) Proc. Natl. Acad.
Sci. USA 79:5582; EP-A-0 244 042].
[0082] Usually, transcription termination sequences recognized by
bacteria are regulatory regions located 3' to the translation stop
codon, and thus together with the promoter flank the coding
sequence. These sequences direct the transcription of an mRNA which
can be translated into the polypeptide encoded by the DNA.
Transcription termination sequences frequently include DNA
sequences of about 50 nucleotides capable of forming stem loop
structures that aid in terminating transcription. Examples include
transcription termination sequences derived from genes with strong
promoters, such as the trp gene in E. coli as well as other
biosynthetic genes.
[0083] Usually, the above described components, comprising a
promoter, signal sequence (if desired), coding sequence of
interest, and transcription termination sequence, are put together
into expression constructs. Expression constructs are often
maintained in a replicon, such as an extrachromosomal element
(e.g., plasmids) capable of stable maintenance in a host, such as
bacteria. The replicon will have a replication system, thus
allowing it to be maintained in a prokaryotic host either for
expression or for cloning and amplification. In addition, a
replicon may be either a high or low copy number plasmid. A high
copy number plasmid will generally have a copy number ranging from
about 5 to about 200, and usually about 10 to about 150. A host
containing a high copy number plasmid will preferably contain at
least about 10, and more preferably at least about 20 plasmids.
Either a high or low copy number vector may be selected, depending
upon the effect of the vector and the foreign protein on the
host.
[0084] Alternatively, the expression constructs can be integrated
into the bacterial genome with an integrating vector. Integrating
vectors usually contain at least one sequence homologous to the
bacterial chromosome that allows the vector to integrate.
Integrations appear to result from recombinations between
homologous DNA in the vector and the bacterial chromosome. For
example, integrating vectors constructed with DNA from various
Bacillus strains integrate into the Bacillus chromosome (EP-A-0 127
328). Integrating vectors may also be comprised of bacteriophage or
transposon sequences.
[0085] Usually, extrachromosomal and integrating expression
constructs may contain selectable markers to allow for the
selection of bacterial strains that have been transformed.
Selectable markers can be expressed in the bacterial host and may
include genes which render bacteria resistant to drugs such as
ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin),
and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol.
32:469]. Selectable markers may also include biosynthetic genes,
such as those in the histidine, tryptophan, and leucine
biosynthetic pathways.
[0086] Alternatively, some of the above described components can be
put together in transformation vectors. Transformation vectors are
usually comprised of a selectable market that is either maintained
in a replicon or developed into an integrating vector, as described
above.
[0087] Expression and transformation vectors, either
extra-chromosomal replicons or integrating vectors, have been
developed for transformation into many bacteria. For example,
expression vectors have been developed for, inter alia, the
following bacteria: Bacillus subtilis [Palva et al. (1982) Proc.
Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO
84/04541], Escherichia coli [Shimatake et al. (1981) Nature
292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J.
Mol. Biol. 189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136
907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ.
Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988)
Appl. Environ. Microbiol. 54:655], Streptomyces lividans [U.S. Pat.
No. 4,745,056].
[0088] Methods of introducing exogenous DNA into bacterial hosts
are well-known in the art, and usually include either the
transformation of bacteria treated with CaCl.sub.2 or other agents,
such as divalent cations and DMSO. DNA can also be introduced into
bacterial cells by electroporation. Transformation procedures
usually vary with the bacterial species to be transformed. See
e.g., [Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et
al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and
EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc.
Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949,
Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci.
69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner
(1978) "An improved method for transformation of Escherichia coli
with ColE1-derived plasmids. In Genetic Engineering: Proceedings of
the International Symposium on Genetic Engineering (eds. H. W.
Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159;
Taketo (1988) Biochim. Biophys. Acta 949:318; Escherichia], [Chassy
et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler
et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al.
(1990) FEMS Microbiol. Lett. 66:203, Staphylococcus], [Barany et
al. (1980) J. Bacteriol. 144:698; Harlander (1987) "Transformation
of Streptococcus lactis by electroporation, in: Streptococcal
Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981)
Infect. Immun. 32:1295; Powell et al. (1988) Appl. Environ.
Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong.
Biotechnology 1:412, Streptococcus].
v. Yeast Expression
[0089] Yeast expression systems are also known to one of ordinary
skill in the art. A yeast promoter is any DNA sequence capable of
binding yeast RNA polymerase and initiating the downstream (3')
transcription of a coding sequence (e.g., structural gene) into
mRNA. A promoter will have a transcription initiation region which
is usually placed proximal to the 5' end of the coding sequence.
This transcription initiation region usually includes an RNA
polymerase binding site (the "TATA Box") and a transcription
initiation site. A yeast promoter may also have a second domain
called an upstream activator sequence (UAS), which, if present, is
usually distal to the structural gene. The UAS permits regulated
(inducible) expression. Constitutive expression occurs in the
absence of a UAS. Regulated expression may be either positive or
negative, thereby either enhancing or reducing transcription.
[0090] Yeast is a fermenting organism with an active metabolic
pathway, therefore sequences encoding enzymes in the metabolic
pathway provide particularly useful promoter sequences. Examples
include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase,
glucokinase, glucose-6-phosphate isomerase,
glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH),
hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and
pyruvate kinase (PyK) (EPO-A-0 329 203). The yeast PHO5 gene,
encoding acid phosphatase, also provides useful promoter sequences
[Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].
[0091] In addition, synthetic promoters which do not occur in
nature also function as yeast promoters. For example, UAS sequences
of one yeast promoter may be joined with the transcription
activation region of another yeast promoter, creating a synthetic
hybrid promoter. Examples of such hybrid promoters include the ADH
regulatory sequence linked to the GAP transcription activation
region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples of
hybrid promoters include promoters which consist of the regulatory
sequences of either the ADH2, GAL4, GAL10, OR PHO5 genes, combined
with the transcriptional activation region of a glycolytic enzyme
gene such as GAP or PyK (EP-A-0 164 556). Furthermore, a yeast
promoter can include naturally occurring promoters of non-yeast
origin that have the ability to bind yeast RNA polymerase and
initiate transcription. Examples of such promoters include, inter
alia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078;
Henikoff et al. (1981) Nature 283:835; Hollenberg et al. (1981)
Curr. Topics Microbiol. Immunol. 96:119; Hollenberg et al. (1979)
"The Expression of Bacterial Antibiotic Resistance Genes in the
Yeast Saccharomyces cerevisiae," in: Plasmids of Medical,
Environmental and Commercial Importance (eds. K. N. Timmis and A.
Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et
al. (1980) Curr. Genet. 2:109;].
[0092] A DNA molecule may be expressed intracellularly in yeast. A
promoter sequence may be directly linked with the DNA molecule, in
which case the first amino acid at the N-terminus of the
recombinant protein will always be a methionine, which is encoded
by the ATG start codon. If desired, methionine at the N-terminus
may be cleaved from the protein by in vitro incubation with
cyanogen bromide.
[0093] Fusion proteins provide an alternative for yeast expression
systems, as well as in mammalian, baculovirus, and bacterial
expression systems. Usually, a DNA sequence encoding the N-terminal
portion of an endogenous yeast protein, or other stable protein, is
fused to the 5' end of heterologous coding sequences. Upon
expression, this construct will provide a fusion of the two amino
acid sequences. For example, the yeast or human superoxide
dismutase (SOD) gene, can be linked at the 5' terminus of a foreign
gene and expressed in yeast. The DNA sequence at the junction of
the two amino acid sequences may or may not encode a cleavable
site. See e.g., EP-A-0 196 056. Another example is a ubiquitin
fusion protein. Such a fusion protein is made with the ubiquitin
region that preferably retains a site for a processing enzyme
(e.g., ubiquitin-specific processing protease) to cleave the
ubiquitin from the foreign protein. Through this method, therefore,
native foreign protein can be isolated (e.g., WO88/024066).
[0094] Alternatively, foreign proteins can also be secreted from
the cell into the growth media by creating chimeric DNA molecules
that encode a fusion protein comprised of a leader sequence
fragment that provide for secretion in yeast of the foreign
protein. Preferably, there are processing sites encoded between the
leader fragment and the foreign gene that can be cleaved either in
vivo or in vitro. The leader sequence fragment usually encodes a
signal peptide comprised of hydrophobic amino acids which direct
the secretion of the protein from the cell.
[0095] DNA encoding suitable signal sequences can be derived from
genes for secreted yeast proteins, such as the yeast invertase gene
(EP-A-0 012 873; JPO. 62,096,086) and the A-factor gene (U.S. Pat.
No. 4,588,684). Alternatively, leaders of non-yeast origin, such as
an interferon leader, exist that also provide for secretion in
yeast (EP-A-0 060 057).
[0096] A preferred class of secretion leaders are those that employ
a fragment of the yeast alpha-factor gene, which contains both a
"pre" signal sequence, and a "pro" region. The types of
alpha-factor fragments that can be employed include the full-length
pre-pro alpha factor leader (about 83 amino acid residues) as well
as truncated alpha-factor leaders (usually about 25 to about 50
amino acid residues) (U.S. Pat. Nos. 4,546,083 and 4,870,008;
EP-A-0 324 274). Additional leaders employing an alpha-factor
leader fragment that provides for secretion include hybrid
alpha-factor leaders made with a presequence of a first yeast, but
a pro-region from a second yeast alphafactor. (e.g., see WO
89/02463.)
[0097] Usually, transcription termination sequences recognized by
yeast are regulatory regions located 3' to the translation stop
codon, and thus together with the promoter flank the coding
sequence. These sequences direct the transcription of an mRNA which
can be translated into the polypeptide encoded by the DNA. Examples
of transcription terminator sequence and other yeast-recognized
termination sequences, such as those coding for glycolytic
enzymes.
[0098] Usually, the above described components, comprising a
promoter, leader (if desired), coding sequence of interest, and
transcription termination sequence, are put together into
expression constructs. Expression constructs are often maintained
in a replicon, such as an extrachromosomal element (e.g., plasmids)
capable of stable maintenance in a host, such as yeast or bacteria.
The replicon may have two replication systems, thus allowing it to
be maintained, for example, in yeast for expression and in a
prokaryotic host for cloning and amplification. Examples of such
yeast-bacteria shuttle vectors include YEp24 [Botstein et al.
(1979) Gene 8:17-24], pC1/1 [Brake et al. (1984) PNAS USA
81:4642-4646], and YRp17 [Stinchcomb et al. (1982) J. Mol. Biol.
158:157]. In addition, a replicon may be either a high or low copy
number plasmid. A high copy number plasmid will generally have a
copy number ranging from about 5 to about 200, and usually about 10
to about 150. A host containing a high copy number plasmid will
preferably have at least about 10, and more preferably at least
about 20. Either a high or low copy number vector may be selected,
depending upon the effect of the vector and the foreign protein on
the host. See e.g., Brake et al., supra.
[0099] Alternatively, the expression constructs can be integrated
into the yeast genome with an integrating vector. Integrating
vectors usually contain at least one sequence homologous to a yeast
chromosome that allows the vector to integrate, and preferably
contain two homologous sequences flanking the expression construct.
Integrations appear to result from recombinations between
homologous DNA in the vector and the yeast chromosome [Orr-Weaver
et al. (1983) Methods in Enzymol. 101:228-245]. An integrating
vector may be directed to a specific locus in yeast by selecting
the appropriate homologous sequence for inclusion in the vector.
See Orr-Weaver et al., supra. One or more expression construct may
integrate, possibly affecting levels of recombinant protein
produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750].
The chromosomal sequences included in the vector can occur either
as a single segment in the vector, which results in the integration
of the entire vector, or two segments homologous to adjacent
segments in the chromosome and flanking the expression construct in
the vector, which can result in the stable integration of only the
expression construct.
[0100] Usually, extrachromosomal and integrating expression
constructs may contain selectable markers to allow for the
selection of yeast strains that have been transformed. Selectable
markers may include biosynthetic genes that can be expressed in the
yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418
resistance gene, which confer resistance in yeast cells to
tunicamycin and G418, respectively. In addition, a suitable
selectable marker may also provide yeast with the ability to grow
in the presence of toxic compounds, such as metal. For example, the
presence of CUP1 allows yeast to grow in the presence of copper
ions [Butt et al. (1987) Microbiol, Rev. 51:351].
[0101] Alternatively, some of the above described components can be
put together into transformation vectors. Transformation vectors
are usually comprised of a selectable marker that is either
maintained in a replicon or developed into an integrating vector,
as described above.
[0102] Expression and transformation vectors, either
extrachromosomal replicons or integrating vectors, have been
developed for transformation into many yeasts. For example,
expression vectors have been developed for, inter alia, the
following yeasts: Candida albicans [Kurtz, et al. (1986) Mol. Cell.
Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. Basic
Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J.
Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet.
202:302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol.
158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J.
Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology
8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic
Microbiol. 25:141], Pichia pastoris [Cregg, et al. (1985) Mol.
Cell. Biol. 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555],
Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl. Acad.
Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163],
Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706],
and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet.
10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].
[0103] Methods of introducing exogenous DNA into yeast hosts are
well-known in the art, and usually include either the
transformation of spheroplasts or of intact yeast cells treated
with alkali cations. Transformation procedures usually vary with
the yeast species to be transformed. See e.g., [Kurtz et al. (1986)
Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol.
25:141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol.
132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302;
Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De
Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et
al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al.
(1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic
Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 and 4,929,555; Pichia];
[Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75;1929; Ito et
al. (1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse
(1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al. (1985)
Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49;
Yarrowia].
Antibodies
[0104] As used herein, the term "antibody" refers to a polypeptide
or group of polypeptides composed of at least one antibody
combining site. An "antibody combining site" is the
three-dimensional binding space with an internal surface shape and
charge distribution complementary to the features of an epitope of
an antigen, which allows a binding of the antibody with the
antigen. "Antibody" includes, for example, vertebrate antibodies,
hybrid antibodies, chimeric antibodies, humanised antibodies,
altered antibodies, univalent antibodies, Fab proteins, and single
domain antibodies.
[0105] Antibodies against the proteins of the invention are useful
for affinity chromatography, immunoassays, and
distinguishing/identifying meningococcal proteins.
[0106] Antibodies to the proteins of the invention, both polyclonal
and monoclonal, may be prepared by conventional methods. In
general, the protein is first used to immunize a suitable animal,
preferably a mouse, rat, rabbit or goat. Rabbits and goats are
preferred for the preparation of polyclonal sera due to the volume
of serum obtainable, and the availability of labeled anti-rabbit
and anti-goat antibodies. Immunization is generally performed by
mixing or emulsifying the protein in saline, preferably in an
adjuvant such as Freund's complete adjuvant, and injecting the
mixture or emulsion parenterally (generally subcutaneously or
intramuscularly). A dose of 50-200 .mu.g/injection is typically
sufficient Immunization is generally boosted 2-6 weeks later with
one or more injections of the protein in saline, preferably using
Freund's incomplete adjuvant. One may alternatively generate
antibodies by in vitro immunization using methods known in the art,
which for the purposes of this invention is considered equivalent
to in vivo immunization. Polyclonal antisera is obtained by
bleeding the immunized animal into a glass or plastic container,
incubating the blood at 25.degree. C. for one hour, followed by
incubating at 4.degree. C. for 2-18 hours. The serum is recovered
by centrifugation (e.g., 1,000 g for 10 minutes). About 20-50 ml
per bleed may be obtained from rabbits.
[0107] Monoclonal antibodies are prepared using the standard method
of Kohler & Milstein [Nature (1975) 256:495-96], or a
modification thereof. Typically, a mouse or rat is immunized as
described above. However, rather than bleeding the animal to
extract serum, the spleen (and optionally several large lymph
nodes) is removed and dissociated into single cells. If desired,
the spleen cells may be screened (after removal of nonspecifically
adherent cells) by applying a cell suspension to a plate or well
coated with the protein antigen. B-cells expressing membrane-bound
immunoglobulin specific for the antigen bind to the plate, and are
not rinsed away with the rest of the suspension. Resulting B-cells,
or all dissociated spleen cells, are then induced to fuse with
myeloma cells to form hybridomas, and are cultured in a selective
medium (e.g., hypoxanthine, aminopterin, thymidine medium, "HAT").
The resulting hybridomas are plated by limiting dilution, and are
assayed for the production of antibodies which bind specifically to
the immunizing antigen (and which do not bind to unrelated
antigens). The selected MAb-secreting hybridomas are then cultured
either in vitro (e.g., in tissue culture bottles or hollow fiber
reactors), or in vivo (as ascites in mice).
[0108] If desired, the antibodies (whether polyclonal or
monoclonal) may be labeled using conventional techniques. Suitable
labels include fluorophores, chromophores, radioactive atoms
(particularly .sup.32P and .sup.125I), electron-dense reagents,
enzymes, and ligands having specific binding partners. Enzymes are
typically detected by their activity. For example, horseradish
peroxidase is usually detected by its ability to convert
3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment,
quantifiable with a spectrophotometer. "Specific binding partner"
refers to a protein capable of binding a ligand molecule with high
specificity, as for example in the case of an antigen and a
monoclonal antibody specific therefor. Other specific binding
partners include biotin and avidin or streptavidin, IgG and protein
A, and the numerous receptor-ligand couples known in the art. It
should be understood that the above description is not meant to
categorize the various labels into distinct classes, as the same
label may serve in several different modes. For example, .sup.125I
may serve as a radioactive label or as an electron-dense reagent.
HRP may serve as enzyme or as antigen for a MAb. Further, one may
combine various labels for desired effect. For example, MAbs and
avidin also require labels in the practice of this invention: thus,
one might label a MAb with biotin, and detect its presence with
avidin labeled with .sup.125I, or with an anti-biotin MAb labeled
with HRP. Other permutations and possibilities will be readily
apparent to those of ordinary skill in the art, and are considered
as equivalents within the scope of the invention.
Pharmaceutical Compositions
[0109] Pharmaceutical compositions can comprise either
polypeptides, antibodies, or nucleic acid of the invention. The
pharmaceutical compositions will comprise a therapeutically
effective amount of either polypeptides, antibodies, or
polynucleotides of the claimed invention.
[0110] The term "therapeutically effective amount" as used herein
refers to an amount of a therapeutic agent to treat, ameliorate, or
prevent a desired disease or condition, or to exhibit a detectable
therapeutic or preventative effect. The effect can be detected by,
for example, chemical markers or antigen levels. Therapeutic
effects also include reduction in physical symptoms, such as
decreased body temperature. The precise effective amount for a
subject will depend upon the subject's size and health, the nature
and extent of the condition, and the therapeutics or combination of
therapeutics selected for administration. Thus, it is not useful to
specify an exact effective amount in advance. However, the
effective amount for a given situation can be determined by routine
experimentation and is within the judgement of the clinician.
[0111] For purposes of the present invention, an effective dose
will be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about
10 mg/kg of the DNA constructs in the individual to which it is
administered.
[0112] A pharmaceutical composition can also contain a
pharmaceutically acceptable carrier. The term "pharmaceutically
acceptable carrier" refers to a carrier for administration of a
therapeutic agent, such as antibodies or a polypeptide, genes, and
other therapeutic agents. The term refers to any pharmaceutical
carrier that does not itself induce the production of antibodies
harmful to the individual receiving the composition, and which may
be administered without undue toxicity. Suitable carriers may be
large, slowly metabolized macromolecules such as proteins,
polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid copolymers, and inactive virus particles.
Such carriers are well known to those of ordinary skill in the
art.
[0113] Pharmaceutically acceptable salts can be used therein, for
example, mineral acid salts such as hydrochlorides, hydrobromides,
phosphates, sulfates, and the like; and the salts of organic acids
such as acetates, propionates, malonates, benzoates, and the like.
A thorough discussion of pharmaceutically acceptable excipients is
available in Remington's Pharmaceutical Sciences (Mack Pub. Co.,
N.J. 1991).
[0114] Pharmaceutically acceptable carriers in therapeutic
compositions may contain liquids such as water, saline, glycerol
and ethanol. Additionally, auxiliary substances, such as wetting or
emulsifying agents, pH buffering substances, and the like, may be
present in such vehicles. Typically, the therapeutic compositions
are prepared as injectables, either as liquid solutions or
suspensions; solid forms suitable for solution in, or suspension
in, liquid vehicles prior to injection may also be prepared.
Liposomes are included within the definition of a pharmaceutically
acceptable carrier.
Delivery Methods
[0115] Once formulated, the compositions of the invention can be
administered directly to the subject. The subjects to be treated
can be animals; in particular, human subjects can be treated.
[0116] Direct delivery of the compositions will generally be
accomplished by injection, either subcutaneously,
intraperitoneally, intravenously or intramuscularly or delivered to
the interstitial space of a tissue. The compositions can also be
administered into a lesion. Other modes of administration include
oral and pulmonary administration, suppositories, and transdermal
or transcutaneous applications (e.g., see WO98/20734), needles, and
gene guns or hyposprays. Dosage treatment may be a single dose
schedule or a multiple dose schedule.
Vaccines
[0117] Vaccines according to the invention may either be
prophylactic (i.e., to prevent infection) or therapeutic (i.e., to
treat disease after infection).
[0118] Such vaccines comprise immunising antigen(s), immunogen(s),
polypeptide(s), protein(s) or nucleic acid, usually in combination
with "pharmaceutically acceptable carriers," which include any
carrier that does not itself induce the production of antibodies
harmful to the individual receiving the composition. Suitable
carriers are typically large, slowly metabolized macromolecules
such as proteins, polysaccharides, polylactic acids, polyglycolic
acids, polymeric amino acids, amino acid copolymers, lipid
aggregates (such as oil droplets or liposomes), and inactive virus
particles. Such carriers are well known to those of ordinary skill
in the art. Additionally, these carriers may function as
immunostimulating agents ("adjuvants"). Furthermore, the antigen or
immunogen may be conjugated to a bacterial toxoid, such as a toxoid
from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.
[0119] Preferred adjuvants to enhance effectiveness of the
composition include, but are not limited to: (1) aluminum salts
(alum), such as aluminum hydroxide, aluminum phosphate, aluminum
sulfate, etc; (2) oil-in-water emulsion formulations (with or
without other specific immunostimulating agents such as muramyl
peptides (see below) or bacterial cell wall components), such as
for example (a) MF59.TM. (WO 90/14837; Chapter 10 in Vaccine
design: the subunit and adjuvant approach, eds. Powell &
Newman, Plenum Press 1995), containing 5% Squalene, 0.5% TWEEN.TM.
80, and 0.5% SPAN.TM. 85 (optionally containing various amounts of
MTP-PE (see below), although not required) formulated into
submicron particles using a microfluidizer such as Model 110Y
MICROFLUIDIZER.TM. (Microfluidics, Newton, Mass.), (b) SAF,
containing 10% Squalane, 0.4% TWEEN.TM. 80, 5% PLURONIC.TM. blocked
polymer L121, and thr-MDP (see below) either microfluidized into a
submicron emulsion or vortexed to generate a larger particle size
emulsion, and (c) Ribi.TM. adjuvant system (RAS), (Ribi Immunochem,
Hamilton, Mont.) containing 2% Squalene, 0.2% TWEEN.TM. 80, and one
or more bacterial cell wall components from the group consisting of
monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell
wall skeleton (CWS), preferably MPL+CWS (Detox.TM.); (3) saponin
adjuvants, such as Stimulon.TM. (Cambridge Bioscience, Worcester,
Mass.) may be used or particles generated therefrom such as ISCOMs
(immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA)
and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as
interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12,
etc.), interferons (e.g., gamma interferon), macrophage colony
stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and
(6) other substances that act as immunostimulating agents to
enhance the effectiveness of the composition. Alum and MF59.TM. are
preferred.
[0120] As mentioned above, muramyl peptides include, but are not
limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
[0121] The immunogenic compositions (e.g., the immunising
antigen/immunogen/polypeptide/protein/nucleic acid,
pharmaceutically acceptable carrier, and adjuvant) typically will
contain diluents, such as water, saline, glycerol, ethanol, etc.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, pH buffering substances, and the like, may be present in
such vehicles.
[0122] Typically, the immunogenic compositions are prepared as
injectables, either as liquid solutions or suspensions; solid forms
suitable for solution in, or suspension in, liquid vehicles prior
to injection may also be prepared. The preparation also may be
emulsified or encapsulated in liposomes for enhanced adjuvant
effect, as discussed above under pharmaceutically acceptable
carriers.
[0123] Immunogenic compositions used as vaccines comprise an
immunologically effective amount of the antigenic or immunogenic
polypeptides, as well as any other of the above-mentioned
components, as needed. By "immunologically effective amount", it is
meant that the administration of that amount to an individual,
either in a single dose or as part of a series, is effective for
treatment or prevention. This amount varies depending upon the
health and physical condition of the individual to be treated, the
taxonomic group of individual to be treated (e.g., nonhuman
primate, primate, etc.), the capacity of the individual's immune
system to synthesize antibodies, the degree of protection desired,
the formulation of the vaccine, the treating doctor's assessment of
the medical situation, and other relevant factors. It is expected
that the amount will fall in a relatively broad range that can be
determined through routine trials.
[0124] The immunogenic compositions are conventionally administered
parenterally, e.g., by injection, either subcutaneously,
intramuscularly, or transdermally/transcutaneously (e.g.,
WO98/20734). Additional formulations suitable for other modes of
administration include oral and pulmonary formulations,
suppositories, and transdermal applications. Dosage treatment may
be a single dose schedule or a multiple dose schedule. The vaccine
may be administered in conjunction with other immunoregulatory
agents.
[0125] As an alternative to protein-based vaccines, DNA vaccination
may be employed [e.g., Robinson & Torres (1997) Seminars in
Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol
15:617-648; see later herein].
Gene Delivery Vehicles
[0126] Gene therapy vehicles for delivery of constructs including a
coding sequence of a therapeutic of the invention, to be delivered
to the mammal for expression in the mammal, can be administered
either locally or systemically. These constructs can utilize viral
or non-viral vector approaches in in vivo or ex vivo modality.
Expression of such coding sequence can be induced using endogenous
mammalian or heterologous promoters. Expression of the coding
sequence in vivo can be either constitutive or regulated.
[0127] The invention includes gene delivery vehicles capable of
expressing the contemplated nucleic acid sequences. The gene
delivery vehicle is preferably a viral vector and, more preferably,
a retroviral, adenoviral, adeno-associated viral (AAV), herpes
viral, or alphavirus vector. The viral vector can also be an
astrovirus, coronavirus, orthomyxovirus, papovavirus,
paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus
viral vector. See generally, Jolly (1994) Cancer Gene Therapy
1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly
(1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature
Genetics 6:148-153.
[0128] Retroviral vectors are well known in the art and we
contemplate that any retroviral gene therapy vector is employable
in the invention, including B, C and D type retroviruses,
xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1
(see O'Neill (1985) J. Virol. 53:160) polytropic retroviruses e.g.,
MCF and MCF-MLV (see Kelly (1983) J. Virol. 45:291), spumaviruses
and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold
Spring Harbor Laboratory, 1985.
[0129] Portions of the retroviral gene therapy vector may be
derived from different retroviruses. For example, retrovector LTRs
may be derived from a Murine Sarcoma Virus, a tRNA binding site
from a Rous Sarcoma Virus, a packaging signal from a Murine
Leukemia Virus, and an origin of second strand synthesis from an
Avian Leukosis Virus.
[0130] These recombinant retroviral vectors may be used to generate
transduction competent retroviral vector particles by introducing
them into appropriate packaging cell lines (see U.S. Pat. No.
5,591,624). Retrovirus vectors can be constructed for site-specific
integration into host cell DNA by incorporation of a chimeric
integrase enzyme into the retroviral particle (see WO96/37626). It
is preferable that the recombinant viral vector is a replication
defective recombinant virus.
[0131] Packaging cell lines suitable for use with the
above-described retrovirus vectors are well known in the art, are
readily prepared (see WO95/30763 and WO92/05266), and can be used
to create producer cell lines (also termed vector cell lines or
"VCLs") for the production of recombinant vector particles.
Preferably, the packaging cell lines are made from human parent
cells (e.g., HT1080 cells) or mink parent cell lines, which
eliminates inactivation in human serum.
[0132] Preferred retroviruses for the construction of retroviral
gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia
Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus,
Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma
Virus. Particularly preferred Murine Leukemia Viruses include 4070A
and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC
No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol
VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No.
VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such
retroviruses may be obtained from depositories or collections such
as the American Type Culture Collection ("ATCC") in P.O. Box 1549
Manassas, Va. 20108 USA or isolated from known sources using
commonly available techniques.
[0133] Exemplary known retroviral gene therapy vectors employable
in this invention include those described in patent applications
GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349,
WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698,
WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825,
WO95/07994, U.S. Pat. No. 5,219,740, U.S. Pat. No. 4,405,712, U.S.
Pat. No. 4,861,719, U.S. Pat. No. 4,980,289, U.S. Pat. No.
4,777,127, U.S. Pat. No. 5,591,624. See also Vile (1993) Cancer Res
53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer
Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503;
Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane
(1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene
Therapy 1.
[0134] Human adenoviral gene therapy vectors are also known in the
art and employable in this invention. See, for example, Berkner
(1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431,
and WO93/07283, WO93/06223, and WO93/07282. Exemplary known
adenoviral gene therapy vectors employable in this invention
include those described in the above referenced documents and in
WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984,
WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320,
WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102,
WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241,
WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively,
administration of DNA linked to killed adenovirus as described in
Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed. The gene
delivery vehicles of the invention also include adenovirus
associated virus (AAV) vectors. Leading and preferred examples of
such vectors for use in this invention are the AAV-2 based vectors
disclosed in Srivastava, WO93/09239. Most preferred AAV vectors
comprise the two AAV inverted terminal repeats in which the native
D-sequences are modified by substitution of nucleotides, such that
at least 5 native nucleotides and up to 18 native nucleotides,
preferably at least 10 native nucleotides up to 18 native
nucleotides, most preferably 10 native nucleotides are retained and
the remaining nucleotides of the D-sequence are deleted or replaced
with non-native nucleotides. The native D-sequences of the AAV
inverted terminal repeats are sequences of 20 consecutive
nucleotides in each AAV inverted terminal repeat (i.e., there is
one sequence at each end) which are not involved in HP formation.
The non-native replacement nucleotide may be any nucleotide other
than the nucleotide found in the native D-sequence in the same
position. Other employable exemplary AAV vectors are pWP-19, pWN-1,
both of which are disclosed in Nahreini (1993) Gene 124:257-262.
Another example of such an AAV vector is psub201 (see Samulski
(1987) J. Virol. 61:3096). Another exemplary AAV vector is the
Double-D ITR vector. Construction of the Double-D ITR vector is
disclosed in U.S. Pat. No. 5,478,745. Still other vectors are those
disclosed in Carter U.S. Pat. No. 4,797,368 and Muzyczka U.S. Pat.
No. 5,139,941, Chartejee U.S. Pat. No. 5,474,935, and Kotin
WO94/288157. Yet a further example of an AAV vector employable in
this invention is SSV9AFABTKneo, which contains the AFP enhancer
and albumin promoter and directs expression predominantly in the
liver. Its structure and construction are disclosed in Su (1996)
Human Gene Therapy 7:463-470. Additional AAV gene therapy vectors
are described in U.S. Pat. Nos. 5,354,678, 5,173,414, 5,139,941,
and U.S. Pat. No. 5,252,479.
[0135] The gene therapy vectors of the invention also include
herpes vectors. Leading and preferred examples are herpes simplex
virus vectors containing a sequence encoding a thymidine kinase
polypeptide such as those disclosed in U.S. Pat. No. 5,288,641 and
EP0176170 (Roizman). Additional exemplary herpes simplex virus
vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar
Institute), pHSVlac described in Geller (1988) Science
241:1667-1669 and in WO90/09441 and WO92/07945, HSV Us3::pgC-lacZ
described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2
RH 105 and GAL4 described in EP 0453242 (Breakefield), and those
deposited with the ATCC as accession numbers ATCC VR-977 and ATCC
VR-260.
[0136] Also contemplated are alpha virus gene therapy vectors that
can be employed in this invention. Preferred alpha virus vectors
are Sindbis viruses vectors. Togaviruses, Semliki Forest virus
(ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross
River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine
encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC
VR-532), and those described in U.S. Pat. Nos. 5,091,309,
5,217,879, and WO92/10578. More particularly, those alpha virus
vectors described in U.S. Ser. No. 08/405,627, filed Mar. 15, 1995,
WO94/21792, WO92/10578, WO95/07994, U.S. Pat. No. 5,091,309 and
U.S. Pat. No. 5,217,879 are employable. Such alpha viruses may be
obtained from depositories or collections such as the ATCC in P.O.
Box 1549 Manassas, Va. 20108 USA or isolated from known sources
using commonly available techniques. Preferably, alphavirus vectors
with reduced cytotoxicity are used (see U.S. Ser. No.
08/679640).
[0137] DNA vector systems such as eukaryotic layered expression
systems are also useful for expressing the nucleic acids of the
invention. See WO95/07994 for a detailed description of eukaryotic
layered expression systems. Preferably, the eukaryotic layered
expression systems of the invention are derived from alphavirus
vectors and most preferably from Sindbis viral vectors.
[0138] Other viral vectors suitable for use in the present
invention include those derived from poliovirus, for example ATCC
VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin
(1973) J. Biol. Standardization 1:115; rhinovirus, for example ATCC
VR-1110 and those described in Arnold (1990) J Cell Biochem L401;
pox viruses such as canary pox virus or vaccinia virus, for example
ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch
(1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci
569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat. No. 4,603,112 and
U.S. Pat. No. 4,769,330 and WO89/01973; SV40 virus, for example
ATCC VR-305 and those described in Mulligan (1979) Nature 277:108
and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example
ATCC VR-797 and recombinant influenza viruses made employing
reverse genetics techniques as described in U.S. Pat. No. 5,166,057
and in Enami (1990) Proc Natl Acad Sci 87:3802-3805; Enami &
Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110,
(see also McMichael (1983) NEJ Med 309:13, and Yap (1978) Nature
273:238 and Nature (1979) 277:108); human immunodeficiency virus as
described in EP-0386882 and in Buchschacher (1992) J. Virol.
66:2731; measles virus, for example ATCC VR-67 and VR-1247 and
those described in EP-0440219; Aura virus, for example ATCC VR-368;
Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou
virus, for example ATCC VR-922; Chikungunya virus, for example ATCC
VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924;
Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach
virus, for example ATCC VR-927; Mayaro virus, for example ATCC
VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244;
Ndumu virus, for example ATCC VR-371; Pixuna virus, for example
ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC
VR-925; Triniti virus, for example ATCC VR-469; Una virus, for
example ATCC VR-374; Whataroa virus, for example ATCC VR-926;
Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern
encephalitis virus, for example ATCC VR-65 and ATCC VR-1242;
Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251,
ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC
VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med
121:190.
[0139] Delivery of the compositions of this invention into cells is
not limited to the above mentioned viral vectors. Other delivery
methods and media may be employed such as, for example, nucleic
acid expression vectors, polycationic condensed DNA linked or
unlinked to killed adenovirus alone, for example see U.S. Ser. No.
08/366,787, filed Dec. 30, 1994 and Curiel (1992) Hum Gene Ther
3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem
264:16985-16987, eucaryotic cell delivery vehicles cells, for
example see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S.
Ser. No. 08/404,796, deposition of photopolymerized hydrogel
materials, hand-held gene transfer particle gun, as described in
U.S. Pat. No. 5,149,655, ionizing radiation as described in U.S.
Pat. No. 5,206,152 and in WO92/11033, nucleic charge neutralization
or fusion with cell membranes. Additional approaches are described
in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994)
Proc Natl Acad Sci 91:1581-1585.
[0140] Particle mediated gene transfer may be employed, for example
see U.S. Ser. No. 60/023,867. Briefly, the sequence can be inserted
into conventional vectors that contain conventional control
sequences for high level expression, and then incubated with
synthetic gene transfer molecules such as polymeric DNA-binding
cations like polylysine, protamine, and albumin, linked to cell
targeting ligands such as asialoorosomucoid, as described in Wu
& Wu (1987) J. Biol. Chem. 262:4429-4432, insulin as described
in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as
described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or
transferrin.
[0141] Naked DNA may also be employed. Exemplary naked DNA
introduction methods are described in WO 90/11092 and U.S. Pat. No.
5,580,859. Uptake efficiency may be improved using biodegradable
latex beads. DNA coated latex beads are efficiently transported
into cells after endocytosis initiation by the beads. The method
may be improved further by treatment of the beads to increase
hydrophobicity and thereby facilitate disruption of the endosome
and release of the DNA into the cytoplasm.
[0142] Liposomes that can act as gene delivery vehicles are
described in U.S. Pat. No. 5,422,120, WO95/13796, WO94/23697,
WO91/14445 and EP-524,968. As described in U.S. Ser. No.
60/023,867, on non-viral delivery, the nucleic acid sequences
encoding a polypeptide can be inserted into conventional vectors
that contain conventional control sequences for high level
expression, and then be incubated with synthetic gene transfer
molecules such as polymeric DNA-binding cations like polylysine,
protamine, and albumin, linked to cell targeting ligands such as
asialoorosomucoid, insulin, galactose, lactose, or transferrin.
Other delivery systems include the use of liposomes to encapsulate
DNA comprising the gene under the control of a variety of
tissue-specific or ubiquitously-active promoters. Further non-viral
delivery suitable for use includes mechanical delivery systems such
as the approach described in Woffendin et al (1994) Proc. Natl.
Acad. Sci. USA 91(24):11581-11585. Moreover, the coding sequence
and the product of expression of such can be delivered through
deposition of photopolymerized hydrogel materials. Other
conventional methods for gene delivery that can be used for
delivery of the coding sequence include, for example, use of
hand-held gene transfer particle gun, as described in U.S. Pat. No.
5,149,655; use of ionizing radiation for activating transferred
gene, as described in U.S. Pat. No. 5,206,152 and WO92/11033
[0143] Exemplary liposome and polycationic gene delivery vehicles
are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915; in
WO 95/13796; WO94/23697; and WO91/14445; in EP-0524968; and in
Stryer, Biochemistry, pages 236-240 (1975) W. H. Freeman, San
Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979)
Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119;
Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem
176:420.
[0144] A polynucleotide composition can comprise therapeutically
effective amount of a gene therapy vehicle, as the term is defined
above. For purposes of the present invention, an effective dose
will be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about
10 mg/kg of the DNA constructs in the individual to which it is
administered.
Delivery Methods
[0145] Once formulated, the polynucleotide compositions of the
invention can be administered (1) directly to the subject; (2)
delivered ex vivo, to cells derived from the subject; or (3) in
vitro for expression of recombinant proteins. The subjects to be
treated can be mammals or birds. Also, human subjects can be
treated.
[0146] Direct delivery of the compositions will generally be
accomplished by injection, either subcutaneously,
intraperitoneally, intravenously or intramuscularly or delivered to
the interstitial space of a tissue. The compositions can also be
administered into a lesion. Other modes of administration include
oral and pulmonary administration, suppositories, and transdermal
or transcutaneous applications (e.g., see WO98/20734), needles, and
gene guns or hyposprays. Dosage treatment may be a single dose
schedule or a multiple dose schedule.
[0147] Methods for the ex vivo delivery and reimplantation of
transformed cells into a subject are known in the art and described
in e.g., WO93/14778. Examples of cells useful in ex vivo
applications include, for example, stem cells, particularly
hematopoetic, lymph cells, macrophages, dendritic cells, or tumor
cells.
[0148] Generally, delivery of nucleic acids for both ex vivo and in
vitro applications can be accomplished by the following procedures,
for example, dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei, all
well known in the art.
Polynucleotide and Polypeptide Pharmaceutical Compositions
[0149] In addition to the pharmaceutically acceptable carriers and
salts described above, the following additional agents can be used
with polynucleotide and/or polypeptide compositions.
A. Polypeptides
[0150] One example are polypeptides which include, without
limitation: asioloorosomucoid (ASOR); transferrin;
asialoglycoproteins; antibodies; antibody fragments; ferritin;
interleukins; interferons, granulocyte, macrophage colony
stimulating factor (GM-CSF), granulocyte colony stimulating factor
(G-CSF), macrophage colony stimulating factor (M-CSF), stem cell
factor and erythropoietin. Viral antigens, such as envelope
proteins, can also be used. Also, proteins from other invasive
organisms, such as the 17 amino acid peptide from the
circumsporozoite protein of plasmodium falciparum known as RII.
B. Hormones, Vitamins, etc.
[0151] Other groups that can be included are, for example:
hormones, steroids, androgens, estrogens, thyroid hormone, or
vitamins, folic acid.
C. Polyalkylenes, Polysaccharides, etc.
[0152] Also, polyalkylene glycol can be included with the desired
polynucleotides/polypeptides. In a preferred embodiment, the
polyalkylene glycol is polyethlylene glycol. In addition, mono-,
di-, or polysaccharides can be included. In a preferred embodiment
of this aspect, the polysaccharide is dextran or DEAE-dextran.
Also, chitosan and poly(lactide-co-glycolide)
D. Lipids, and Liposomes
[0153] The desired polynucleotide/polypeptide can also be
encapsulated in lipids or packaged in liposomes prior to delivery
to the subject or to cells derived therefrom.
[0154] Lipid encapsulation is generally accomplished using
liposomes which are able to stably bind or entrap and retain
nucleic acid. The ratio of condensed polynucleotide to lipid
preparation can vary but will generally be around 1:1 (mg
DNA:micromoles lipid), or more of lipid. For a review of the use of
liposomes as carriers for delivery of nucleic acids, see, Hug and
Sleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger
(1983) Meth. Enzymol. 101:512-527.
[0155] Liposomal preparations for use in the present invention
include cationic (positively charged), anionic (negatively charged)
and neutral preparations. Cationic liposomes have been shown to
mediate intracellular delivery of plasmid DNA (Felgner (1987) Proc.
Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl.
Acad. Sci. USA 86:6077-6081); and purified transcription factors
(Debs (1990) J. Biol. Chem. 265:10189-10192), in functional
form.
[0156] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are available under the trademark LIPOFECTIN.TM., from GIBCO BRL,
Grand Island, N.Y. (See, also, Felgner supra). Other commercially
available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boerhinger). Other cationic liposomes can be prepared from readily
available materials using techniques well known in the art. See,
e.g., Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198;
WO90/11092 for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
[0157] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Alabaster, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0158] The liposomes can comprise multilammelar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs). The various liposome-nucleic acid complexes are prepared
using methods known in the art. See e.g., Straubinger (1983) Meth.
Immunol. 101:512-527; Szoka (1978) Proc. Natl. Acad. Sci. USA
75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta
394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976)
Biochim. Biophys. Acta 443:629; Ostro (1977) Biochem. Biophys. Res.
Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA 76:3348);
Enoch & Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145;
Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka &
Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and
Schaefer-Ridder (1982) Science 215:166.
E. Lipoproteins
[0159] In addition, lipoproteins can be included with the
polynucleotide/polypeptide to be delivered. Examples of
lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL,
and VLDL. Mutants, fragments, or fusions of these proteins can also
be used. Also, modifications of naturally occurring lipoproteins
can be used, such as acetylated LDL. These lipoproteins can target
the delivery of polynucleotides to cells expressing lipoprotein
receptors. Preferably, if lipoproteins are including with the
polynucleotide to be delivered, no other targeting ligand is
included in the composition.
[0160] Naturally occurring lipoproteins comprise a lipid and a
protein portion. The protein portion are known as apoproteins. At
the present, apoproteins A, B, C, D, and E have been isolated and
identified. At least two of these contain several proteins,
designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.
[0161] A lipoprotein can comprise more than one apoprotein. For
example, naturally occurring chylomicrons comprises of A, B, C, and
E, over time these lipoproteins lose A and acquire C and E
apoproteins. VLDL comprises A, B, C, and E apoproteins, LDL
comprises apoprotein B; and HDL comprises apoproteins A, C, and
E.
[0162] The amino acid of these apoproteins are known and are
described in, for example, Breslow (1985) Annu Rev. Biochem 54:699;
Law (1986) Adv. Exp Med. Biol. 151:162; Chen (1986) J Biol Chem
261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann
(1984) Hum Genet 65:232.
[0163] Lipoproteins contain a variety of lipids including,
triglycerides, cholesterol (free and esters), and phospholipids.
The composition of the lipids varies in naturally occurring
lipoproteins. For example, chylomicrons comprise mainly
triglycerides. A more detailed description of the lipid content of
naturally occurring lipoproteins can be found, for example, in
Meth. Enzymol. 128 (1986). The composition of the lipids are chosen
to aid in conformation of the apoprotein for receptor binding
activity. The composition of lipids can also be chosen to
facilitate hydrophobic interaction and association with the
polynucleotide binding molecule.
[0164] Naturally occurring lipoproteins can be isolated from serum
by ultracentrifugation, for instance. Such methods are described in
Meth. Enzymol. (supra); Pitas (1980) J. Biochem. 255:5454-5460 and
Mahey (1979) J Clin. Invest 64:743-750. Lipoproteins can also be
produced by in vitro or recombinant methods by expression of the
apoprotein genes in a desired host cell. See, for example, Atkinson
(1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim
Biophys Acta 30: 443. Lipoproteins can also be purchased from
commercial suppliers, such as Biomedical Techniologies, Inc.,
Stoughton, Mass., USA. Further description of lipoproteins can be
found in Zuckermann et al. WO98/06437.
F. Polycationic Agents
[0165] Polycationic agents can be included, with or without
lipoprotein, in a composition with the desired
polynucleotide/polypeptide to be delivered.
[0166] Polycationic agents, typically, exhibit a net positive
charge at physiological relevant pH and are capable of neutralizing
the electrical charge of nucleic acids to facilitate delivery to a
desired location. These agents have both in vitro, ex vivo, and in
vivo applications. Polycationic agents can be used to deliver
nucleic acids to a living subject either intramuscularly,
subcutaneously, etc.
[0167] The following are examples of useful polypeptides as
polycationic agents: polylysine, polyarginine, polyornithine, and
protamine. Other examples include histones, protamines, human serum
albumin, DNA binding proteins, non-histone chromosomal proteins,
coat proteins from DNA viruses, such as X174, transcriptional
factors also contain domains that bind DNA and therefore may be
useful as nucleic aid condensing agents. Briefly, transcriptional
factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF,
Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains
that bind DNA sequences.
[0168] Organic polycationic agents include: spermine, spermidine,
and purtrescine.
[0169] The dimensions and of the physical properties of a
polycationic agent can be extrapolated from the list above, to
construct other polypeptide polycationic agents or to produce
synthetic polycationic agents.
[0170] Synthetic polycationic agents which are useful include, for
example, DEAE-dextran, POLYBRENE.TM., LIPOFECTIN.TM., and
LIPOFECTAMINE.TM. are monomers that form polycationic complexes
when combined with polynucleotides/polypeptides.
Immunodiagnostic Assays
[0171] Meningogoccal antigens of the invention can be used in
immunoassays to detect antibody levels (or, conversely,
anti-meningococcal antibodies can be used to detect antigen
levels). Immunoassays based on well defined, recombinant antigens
can be developed to replace invasive diagnostics methods.
Antibodies to meningococcal proteins within biological samples,
including for example, blood or serum samples, can be detected.
Design of the immunoassays is subject to a great deal of variation,
and a variety of these are known in the art. Protocols for the
immunoassay may be based, for example, upon competition, or direct
reaction, or sandwich type assays. Protocols may also, for example,
use solid supports, or may be by immunoprecipitation. Most assays
involve the use of labeled antibody or polypeptide; the labels may
be, for example, fluorescent, chemiluminescent, radioactive, or dye
molecules. Assays which amplify the signals from the probe are also
known; examples of which are assays which utilize biotin and
avidin, and enzyme-labeled and mediated immunoassays, such as ELISA
assays.
[0172] Kits suitable for immunodiagnosis and containing the
appropriate labeled reagents are constructed by packaging the
appropriate materials, including the compositions of the invention,
in suitable containers, along with the remaining reagents and
materials (for example, suitable buffers, salt solutions, etc.)
required for the conduct of the assay, as well as suitable set of
assay instructions.
Nucleic Acid Hybridisation
[0173] "Hybridization" refers to the association of two nucleic
acid sequences to one another by hydrogen bonding. Typically, one
sequence will be fixed to a solid support and the other will be
free in solution. Then, the two sequences will be placed in contact
with one another under conditions that favor hydrogen bonding.
Factors that affect this bonding include: the type and volume of
solvent; reaction temperature; time of hybridization; agitation;
agents to block the non-specific attachment of the liquid phase
sequence to the solid support (Denhardt's reagent or BLOTTO);
concentration of the sequences; use of compounds to increase the
rate of association of sequences (dextran sulfate or polyethylene
glycol); and the stringency of the washing conditions following
hybridization. See Sambrook et al. [supra] Volume 2, chapter 9,
pages 9.47 to 9.57.
[0174] "Stringency" refers to conditions in a hybridization
reaction that favor association of very similar sequences over
sequences that differ. For example, the combination of temperature
and salt concentration should be chosen that is approximately 120
to 200.degree. C. below the calculated Tm of the hybrid under
study. The temperature and salt conditions can often be determined
empirically in preliminary experiments in which samples of genomic
DNA immobilized on filters are hybridized to the sequence of
interest and then washed under conditions of different
stringencies. See Sambrook et al. at page 9.50.
[0175] Variables to consider when performing, for example, a
Southern blot are (1) the complexity of the DNA being blotted and
(2) the homology between the probe and the sequences being
detected. The total amount of the fragment(s) to be studied can
vary a magnitude of 10, from 0.1 to 1 .mu.g for a plasmid or phage
digest to 10.sup.-9 to 10.sup.-8 g for a single copy gene in a
highly complex eukaryotic genome. For lower complexity
polynucleotides, substantially shorter blotting, hybridization, and
exposure times, a smaller amount of starting polynucleotides, and
lower specific activity of probes can be used. For example, a
single-copy yeast gene can be detected with an exposure time of
only 1 hour starting with 1 .mu.g of yeast DNA, blotting for two
hours, and hybridizing for 4-8 hours with a probe of 10.sup.8
cpm/.mu.g. For a single-copy mammalian gene a conservative approach
would start with 10 .mu.g of DNA, blot overnight, and hybridize
overnight in the presence of 10% dextran sulfate using a probe of
greater than 10.sup.8 cpm/.mu.g, resulting in an exposure time of
.about.24 hours.
[0176] Several factors can affect the melting temperature (Tm) of a
DNA-DNA hybrid between the probe and the fragment of interest, and
consequently, the appropriate conditions for hybridization and
washing. In many cases the probe is not 100% homologous to the
fragment. Other commonly encountered variables include the length
and total G+C content of the hybridizing sequences and the ionic
strength and formamide content of the hybridization buffer. The
effects of all of these factors can be approximated by a single
equation:
Tm=81+16.6(log.sub.10Ci)+0.4[%(G+C)]-0.6(% formamide)-600/n-1.5(%
mismatch).
where Ci is the salt concentration (monovalent ions) and n is the
length of the hybrid in base pairs (slightly modified from Meinkoth
& Wahl (1984) Anal. Biochem. 138: 267-284).
[0177] In designing a hybridization experiment, some factors
affecting nucleic acid hybridization can be conveniently altered.
The temperature of the hybridization and washes and the salt
concentration during the washes are the simplest to adjust. As the
temperature of the hybridization increases (i.e., stringency), it
becomes less likely for hybridization to occur between strands that
are nonhomologous, and as a result, background decreases. If the
radiolabeled probe is not completely homologous with the
immobilized fragment (as is frequently the case in gene family and
interspecies hybridization experiments), the hybridization
temperature must be reduced, and background will increase. The
temperature of the washes affects the intensity of the hybridizing
band and the degree of background in a similar manner. The
stringency of the washes is also increased with decreasing salt
concentrations.
[0178] In general, convenient hybridization temperatures in the
presence of 50% formamide are 42.degree. C. for a probe with is 95%
to 100% homologous to the target fragment, 37.degree. C. for 90% to
95% homology, and 32.degree. C. for 85% to 90% homology. For lower
homologies, formamide content should be lowered and temperature
adjusted accordingly, using the equation above. If the homology
between the probe and the target fragment are not known, the
simplest approach is to start with both hybridization and wash
conditions which are nonstringent. If non-specific bands or high
background are observed after autoradiography, the filter can be
washed at high stringency and reexposed. If the time required for
exposure makes this approach impractical, several hybridization
and/or washing stringencies should be tested in parallel.
Nucleic Acid Probe Assays
[0179] Methods such as PCR, branched DNA probe assays, or blotting
techniques utilizing nucleic acid probes according to the invention
can determine the presence of cDNA or mRNA. A probe is said to
"hybridize" with a sequence of the invention if it can form a
duplex or double stranded complex, which is stable enough to be
detected.
[0180] The nucleic acid probes will hybridize to the meningococcal
nucleotide sequences of the invention (including both sense and
antisense strands). Though many different nucleotide sequences will
encode the amino acid sequence, the native meningococcal sequence
is preferred because it is the actual sequence present in cells.
mRNA represents a coding sequence and so a probe should be
complementary to the coding sequence; single-stranded cDNA is
complementary to mRNA, and so a cDNA probe should be complementary
to the non-coding sequence.
[0181] The probe sequence need not be identical to the
meningococcal sequence (or its complement)--some variation in the
sequence and length can lead to increased assay sensitivity if the
nucleic acid probe can form a duplex with target nucleotides, which
can be detected. Also, the nucleic acid probe can include
additional nucleotides to stabilize the formed duplex. Additional
meningococcal sequence may also be helpful as a label to detect the
formed duplex. For example, a non-complementary nucleotide sequence
may be attached to the 5' end of the probe, with the remainder of
the probe sequence being complementary to a meningococcal sequence.
Alternatively, non-complementary bases or longer sequences can be
interspersed into the probe, provided that the probe sequence has
sufficient complementarity with the a meningococcal sequence in
order to hybridize therewith and thereby form a duplex which can be
detected.
[0182] The exact length and sequence of the probe will depend on
the hybridization conditions, such as temperature, salt condition
and the like. For example, for diagnostic applications, depending
on the complexity of the analyte sequence, the nucleic acid probe
typically contains at least 10-20 nucleotides, preferably 15-25,
and more preferably at least 30 nucleotides, although it may be
shorter than this. Short primers generally require cooler
temperatures to form sufficiently stable hybrid complexes with the
template.
[0183] Probes may be produced by synthetic procedures, such as the
triester method of Matteucci et al. [J. Am. Chem. Soc. (1981)
103:3185], or according to Urdea et al. [Proc. Natl. Acad. Sci. USA
(1983) 80: 7461], or using commercially available automated
oligonucleotide synthesizers.
[0184] The chemical nature of the probe can be selected according
to preference. For certain applications, DNA or RNA are
appropriate. For other applications, modifications may be
incorporated e.g., backbone modifications, such as
phosphorothioates or methylphosphonates, can be used to increase in
vivo half-life, alter RNA affinity, increase nuclease resistance
etc. [e.g., see Agrawal & Iyer (1995) Curr Opin Biotechnol
6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues such as
peptide nucleic acids may also be used [e.g., see Corey (1997)
TIBTECH 15:224-229; Buchardt et al. (1993) TIBTECH 11:384-386].
[0185] Alternatively, the polymerase chain reaction (PCR) is
another well-known means for detecting small amounts of target
nucleic acids. The assay is described in: Mullis et al. [Meth.
Enzymol. (1987) 155: 335-350]; U.S. Pat. Nos. 4,683,195 and
4,683,202. Two "primer" nucleotides hybridize with the target
nucleic acids and are used to prime the reaction. The primers can
comprise sequence that does not hybridize to the sequence of the
amplification target (or its complement) to aid with duplex
stability or, for example, to incorporate a convenient restriction
site. Typically, such sequence will flank the desired meningococcal
sequence.
[0186] A thermostable polymerase creates copies of target nucleic
acids from the primers using the original target nucleic acids as a
template. After a threshold amount of target nucleic acids are
generated by the polymerase, they can be detected by more
traditional methods, such as Southern blots. When using the
Southern blot method, the labelled probe will hybridize to the
meningococcal sequence (or its complement).
[0187] Also, mRNA or cDNA can be detected by traditional blotting
techniques described in Sambrook et al [supra]. mRNA, or cDNA
generated from mRNA using a polymerase enzyme, can be purified and
separated using gel electrophoresis. The nucleic acids on the gel
are then blotted onto a solid support, such as nitrocellulose. The
solid support is exposed to a labelled probe and then washed to
remove any unhybridized probe. Next, the duplexes containing the
labeled probe are detected. Typically, the probe is labelled with a
radioactive moiety.
MODES FOR CARRYING OUT THE INVENTION
Preferred Fragments
[0188] The protein sequences disclosed in the International
Applications have been, inter alia, subjected to computer analysis
to predict antigenic peptide fragments within the full-length
proteins. Three algorithms have been used in this analysis: [0189]
AMPHI This program has been used to predict T-cell epitopes [Gao et
al. (1989) J. Immunol. 143:3007; Roberts et al. (1996) AIDS Res Hum
Retrovir 12:593; Quakyi et al. (1992) Scand J Immunol suppl. 11:9]
and is available in the Protean package of DNASTAR, Inc. (1228
South Park Street, Madison, Wis. 53715 USA). [0190] ANTIGENIC INDEX
as disclosed by Jameson & Wolf (1988) The antigenic index: a
novel algorithm for predicting antigenic determinants. CABIOS,
4:181:186 [0191] HYDROPHILICITY as disclosed by Hopp & Woods
(1981) Prediction of protein antigenic determinants from amino acid
sequences. PNAS, 78:3824-3828
[0192] The three algorithms often identify the same fragments. Such
multiply-identified fragments are particularly preferred. The
algorithms often identify overlapping fragments (e.g., for antigen
"013", AMPHI identifies aa 42-46, and Antigenic Index identifies aa
39-45). The invention explicitly includes fragments resulting from
a combination of these overlapping fragments (e.g., the fragment
from residue 39 to residue 46, in the case of "013"). Fragments
separated by a single amino acid are also often identified (e.g.,
for "018-2", antigenic index identifies aa 19-23 and 25-41). The
invention also includes fragments spanning the two extremes of such
"adjacent" fragments (e.g., 19-41 for "081-2"). The Example
provides preferred antigenic fragments of the proteins disclosed in
the International Applications.
Example 1
Preferred Antigenic Protein Fragments
[0193] The following amino acid sequences in Table 1 are identified
by titles indicating the number assigned to the particular open
reading frame (ORF), consistent with those designated in the
International Applications. The titles are of the following form:
[no prefix, g, or a] [#], where "no prefix" means a sequence from
N. meningitidis serotype B, "a" means a sequence from N.
meningitidis serotype A, and "g" means a sequence from N.
gonorrhoeae; and "#" means the number assigned to that open reading
frame (ORF). For example, "127" refers to an N. meningitidis B
amino acid sequence, ORF number 127. The presence of a suffix "-1"
or "-2" to these titles indicates an additional sequence found for
that particular ORF. Thus, for example, "a12-2" refers to an N.
meningitidis A amino acid sequence, ORF number 12, which is another
sequence found for ORF 12 in addition to the originally designated
ORF 12 and ORF 12-1. Each amino acid sequence is preceded by the
beginning amino acid position number and followed by the ending
amino acid position number.
TABLE-US-00001 Lengthy table referenced here
US20160051658A1-20160225-T00001 Please refer to the end of the
specification for access instructions.
[0194] It will be understood that the invention is described above
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160051658A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160051658A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160051658A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References