U.S. patent application number 10/115817 was filed with the patent office on 2002-12-19 for viral vectors having chimeric envelope proteins containing the igg-binding domain of protein a.
This patent application is currently assigned to New York University. Invention is credited to Meruelo, Daniel, Ohno, Kouichi.
Application Number | 20020192824 10/115817 |
Document ID | / |
Family ID | 25254855 |
Filed Date | 2002-12-19 |
United States Patent
Application |
20020192824 |
Kind Code |
A1 |
Meruelo, Daniel ; et
al. |
December 19, 2002 |
Viral vectors having chimeric envelope proteins containing the
IgG-binding domain of protein A
Abstract
The invention involves viral vectors that can be used to
tranduce a target cell, i.e. to introduce genetic material into the
cell. The targets of interest are eukaryotic cells and particularly
human cells. The transduction can be done in vivo or in vitro. More
particularly the invention concerns viral vectors that have
chimeric envelope proteins and contain the IgG-binding domain of
protein A. These vectors when used in conjunction with antibodies
targeting a particular cell are particularly useful for gene
therapy.
Inventors: |
Meruelo, Daniel;
(Scarborough, NY) ; Ohno, Kouichi; (Kagoshima,
JP) |
Correspondence
Address: |
DARBY & DARBY P.C.
805 Third Avenue
New York
NY
10022
US
|
Assignee: |
New York University
|
Family ID: |
25254855 |
Appl. No.: |
10/115817 |
Filed: |
March 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10115817 |
Mar 28, 2002 |
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08829558 |
Mar 28, 1997 |
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6432699 |
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Current U.S.
Class: |
435/456 ;
435/235.1 |
Current CPC
Class: |
C12N 2810/609 20130101;
C12N 2770/36152 20130101; C12N 2740/13022 20130101; C12N 2770/36143
20130101; C12N 2770/36145 20130101; C12N 2810/6018 20130101; A61K
48/00 20130101; C12N 7/00 20130101; C12N 2770/36122 20130101; C07K
14/005 20130101; C12N 15/86 20130101 |
Class at
Publication: |
435/456 ;
435/235.1 |
International
Class: |
C12N 015/86; C12N
007/00 |
Claims
What is claimed is:
1. A virus envelope protein modified by insertion of an IgG binding
domain of Protein A.
2. The virus envelope protein of claim 1, wherein the virus is an
Alphavirus.
3. The virus envelope protein of claim 2, wherein the Alphavirus is
a Sindbis virus and the envelope protein is an E2 protein.
4. The virus envelope protein of claim 1, wherein the Fc binding
domain of protein A is a ZZ domain.
5. The Sindbis virus E2 protein of claim 3, wherein the Fc binding
domain of protein A is a ZZ domain.
6. The Sindbis virus E2 protein of claim 3, wherein the Fc binding
domain of protein A is inserted between amino acid residues 71 and
74 of said E2 protein.
7. A complex for transducing a target cell comprising a viral
vector comprising a chimeric envelope protein containing an IgG
binding domain of Protein A sufficient to bind an Fc domain of an
antibody, wherein said envelope protein is a Sindbis E2 protein and
wherein said chimeric envelope protein alters the binding of said
E2 protein to its natural receptor; and an antibody directed
against a surface protein on said target cell.
8. The complex of claim 7, wherein the IgG binding domain of
protein A is a ZZ domain.
9. The complex of claim 7 wherein the IgG binding domain of protein
A is inserted between amino acid residues 71 and 74 of said E2
protein.
10. A viral vector comprising an Alphavirus envelope protein
modified by insertion of an IgG binding domain of protein A.
11. The viral vector of claim 10 wherein the Alphavirus is a
Sindbis virus and the envelope protein is an E2 protein.
12. The viral vector of claim 10 wherein the IgG binding domain of
protein A is a ZZ domain.
13. The Sindbis virus E2 protein of claim 11 wherein the IgG
binding domain of protein A is a ZZ domain.
14. The E2 protein of claim 11 wherein the IgG binding domain of
protein A is inserted between the amino acid residues 71 and 74 of
said E2 protein.
15. An isolated nucleic acid encoding a virus envelope protein
modified by insertion of an IgG binding domain of protein A.
16. The nucleic acid of claim 15 wherein the virus is an Alphavirus
and the envelope protein is an E2 protein.
17. The nucleic acid of claim 15 wherein the IgG binding domain of
protein A is a ZZ domain.
18. The nucleic acid of claim 17 wherein the IgG binding domain of
protein A is inserted between amino acid residues 71 and 74 of said
E2 protein.
Description
FIELD OF THE INVENTION
[0001] The invention involves viral vectors that can be used to
transduce a target cell, i.e., to introduce genetic material into
the cell. The targets of interest are eukaryotic cells and
particularly human cells. The transduction can be done in vivo or
in vitro. More particularly the invention concerns viral vectors
that have chimeric envelope proteins and contain the IgG-binding
domain of protein A. These vectors when used in conjunction with
antibodies targeting a particular cell are particularly useful for
gene therapy.
BACKGROUND OF THE INVENTION
[0002] A variety of viral based vectors have been employed to
transfer and to express a gene of interest into a eukaryotic target
cell. Recombinant DNA techniques are used to replace one or more of
the genes of the virus with the gene of interest operably linked to
a promoter that is functional in the target cell. The construct,
termed a viral vector, infects the target cell, using the
physiological infective "machinery" of the virus, and expresses the
gene of interest instead of the viral genes. Because not all the
genes of the virus are present in the vector, infection of the
target by the vector does not produce viral particles. Viruses that
have been used to infect human or mammalian target cells include
herpes virus, adenovirus, adeno-associated virus and derivatives of
leukemia-type retroviruses. Among the retroviruses of particular
interest in the transduction of cells of human origin are
constructs based on amphotropic retroviruses.
Use of Amphotropic and Ecotropic Retrovirus Vectors
[0003] Retroviruses are particularly well suited for transduction
of eukaryotic cells. The advantages of a vector based this type of
virus include its integration into the genome of the target cell so
that the progeny of the transduced cell express the gene of
interest. Secondly, there are well developed techniques to produce
a stock of infectious vector particles that do not cause the
production of viral particles in the transduced target cell.
Lastly, the production and purification of stocks vector particles
having titers of 10.sup.6 TCIU/ml can be accomplished.
[0004] One disadvantage of the use of retroviral vectors is that
there is presently no practical general, method whereby a
particular tissue or cell type of interest can be specifically
transduced. Previous efforts to this end have included surgical
procedures to limit to specific organs the physical distribution of
the viral vector particles (Ferry, N. et al., 1991, Proc. Natl.
Acad. Sci. 88:8377). Alternatively, practitioners have taken
advantage of the fact that type C retroviruses only infect dividing
cells. Thus, a population of cells, e.g., bone marrow cells, was
removed from a subject and cultured ex vivo in the presence of
growth factors specific for the specific target cell which, thus,
comprises most of dividing cells in the culture. See, e.g., Wilson,
J. M. et al., 1990, Proc. Natl. Acad. Sci. 87:439-47; Ohashi, T. et
al., 1992, Proc. Natl. Acad. Sci. 89:11332-36. After transduction
the dividing cells must be harvested and, for many purposes,
reimplanted into the subject. The technical difficulties of the ex
vivo culture technique combined with the unavailability of growth
factors of specific for some types of cells have limited the
application of this approach.
[0005] A second difficulty presented by the use retroviral based
vectors is that a recombination may occur between sequences of
vector and an endogenous retrovirus. Such recombination can give
rise to a replication competent virus that can cause the production
of infectious particles by the target cell. In contrast to herpes
virus or adenovirus infection, retroviral infections are not
necessarily self-limiting.
[0006] Notwithstanding these difficulties, retrovirus vectors based
on amphotropic murine leukemia retroviruses that infect human
cells, have been approved for use in human gene therapy of certain
diseases, for example adenosine deaminase and low density
lipoprotein receptor deficiencies and Gaucher's Disease. See, e.g.,
Miller A. D., 1992, Nature 357:455; Anderson, W. F., 1992, Science
256:808; and Crystal, R. G., 1995, Science 270:404-410.
[0007] One approach to overcoming the limitations of using
amphotropic retrovirus vectors in human cells has been to mutate
the gene encoding the protein on the viral surface that determines
the specificity of infection of the virus, the gp70 protein. Using
recombinant DNA technology a "mutant" virus is constructed that has
had small regions of the gp70 sequence replaced by predetermined
sequences. The limits of this approach are set by the requirement
for knowledge of the sequence that will enable infection of the
target of interest. However, when this knowledge was available, the
anticipated alteration in viral specificity has been observed
(Valsesia-Wittmann, S., 1994, J. Virol. 68:4609-19).
[0008] Retrovirus vectors are the most efficient tools available
today to stably transduce genes into the genomes of vertebrate
cells. Murine leukemia retrovirus (MLV)-based vectors commonly used
for gene transfer are classified on the basis of their host range
as either ecotropic or amphotropic. Murine ecotropic virions can
only infect mouse or rat cells, but murine amphotropic viruses can
infect cells of most species, including human cells. Because of
their ability to infect such a broad spectrum of cells, a major
drawback to the use of amphotropic virus vectors is the fact that
these vectors lack target-cell specificity.
[0009] Several attempts to alter the host range of retroviruses
have been reported to date. Recently, direct modifications of the
envelope protein of murine leukemia virus (MLV) have been shown to
redirect the viral tropism. A recombinant virus containing a
fragment encoding a single Fv antibody chain at the N terminal
region of the MLV env gene has been shown to recognize the
corresponding epitopes and infect human cells (Russell, S. J. et
al., 1993, Nucleic Acids Res. 21:1081-1085; Somia, N. V. et al.,
1995, Proc. Natl. Acad. Sci. USA 92:7570-7574; Marin, M. et al.,
1996, J. Virol. 70:2957-2962). Kasahara et al. have made a chimeric
ecotropic virus containing an erythropoietin-envelope fusion
protein (Kasahara, N. et al., 1994, Science 266:1373-1376). This
chimeric virus has been shown to infect human cells bearing the
erythropoietin receptor. However, this type of approach suffers
from at least two limitations. First, each targetable vector must
be constructed de novo. It is unlikely that the incorporation of
different targeting elements in the envelope of the virus can
always be achieved with equal success and without reducing the
virus titers than can be obtained. Second, virions constructed to
directly bind to specific targets in human cells are intrinsically
unsafe, as wild-type recombinants could produce potentially harmful
effects patients treated with such vectors. By contrast, virions
constructed as outlined in this manuscript are uninfectious to
human cells in the absence of an accompanying targeting reagent,
such as a mAb, which is produced separately and only provided in
conjunction with the virus at a convenient time.
Known Viral Vector Complexes to Transduce Target Cells
[0010] An alternative to altering the specificity of binding of the
gp70 protein itself is to employ a second, novel structure that
binds or is bonded to both the viral particle and to the target
cell. In one example of this approach, lactose molecules were
covalently coupled, by a non-specific reaction, to the envelope
proteins of an ecotropic retrovirus, which does not normally infect
human cells. A human hepatocellular carcinoma that was known to
have receptors for lactose-containing proteins was found to be
susceptible to transduction by this vector complex, although the
integration of the transduced gene of interest in the target cell
chromosome was not directly demonstrated (Neda, H. et al., 1991, J.
Biol. Chem. 266:14143). No evidence of expression was observed in a
hepatocellular carcinoma that lacked the lactose specific receptor.
The method of Neda results in a variable number of binding sites
for the exposed acceptor on the target cell, attached to each
derivatized or bound envelope protein and, of course, is limited to
the case wherein the target cell has a lactose receptor.
[0011] Another approach to targeting is the use of adapter
molecules involved an adapter that was not covalently coupled to
the vector. The use of this type of adapter has been attempted by
Roux and his colleagues, who have published several reports that
relate to this strategy (Patent Publication FR 2,649,119 to
Piecheczyk, Jan. 4, 1991; Roux P. et al., 1989, Proc. Natl. Acad.
Sci. 86:9079-83; Etienne-Julan, M. et al., 1992, J. Gen. Virol.
73:3251-55). Roux and colleagues have constructed adapters from two
types of proteins, both typically antibodies, by biotinylating the
proteins and utilizing avidin or streptavidin tetramer, a protein
which binds four biotin molecules, to form aggregates of up to four
of the biotinylated proteins.
[0012] A better approach is described in U.S. Ser. No. 08/363,137,
filed Dec. 23, 1994, Meruelo et al., the contents of which are
hereby incorporated by reference into this patent application.
Meruelo et al. describe viral complexes and methods of use to
prepare pre-formed adaptors and linkers suitable for gen therapy.
They are particularly well-suited for retroviral systems.
Use of Sindbis Virus Vectors
[0013] Sindbis virus, a member of the Alphavirus genus, has
received considerable attention for use as virus-based expression
vectors. Many properties of alphavirus vectors make them a
desirable alternative to other virus-derived vector systems being
developed, including rapid engineering of expression constructs,
production of high-titered stocks of infectious particles,
infection of nondividing cells, and high levels of expression
(Strauss, J. H. et al., 1994, Microbiol. Rev. 58:491-562;
Liljestrom, P. et al., 1991, Biotechnology 9:1356-1361; Bredenbeek,
P. et al., 1992, Semin. Virol. 3:297-310; Xiong, C. et al., 1993,
Science 243:1188-1191). However, a major drawback to the use of
Sindbis virus vectors is the fact that these vectors lack
target-cell specificity. For mammalian cells, at least one Sindbis
virus receptor is a protein previously identified as the
high-affinity laminin receptor, whose wide distribution and highly
conserved nature may be in part responsible for the broad host
range of the virus (Strauss, J. H. et al. 1994; Wang, K. S. et al.,
1992, J. Virol. 66:4992-5001). It is desirable to alter the tropism
of the Sindbis virus vectors to permit gene delivery specifically
to certain target cell types. This will require both the ablation
of endogenous viral tropism and the introduction of novel tropism.
In the mature Sindbis virus virion, a plus-stranded viral genome
RNA is complexed with capsid protein C to form icosahedral
nucleocapsid that is surrounded by lipid bilayer in which two
integral membrane glycoproteins, E1 and E2 are embedded (Strauss,
J. H. et al., 1994). Although E1 and E2 form heterodimer that
functions as a unit, the E2 domain appears to be particularly
important for binding to cells. Monoclonal antibodies (mAbs)
capable of neutralizing virus infectivity are usually E2 specific,
and mutations in E2, rather than E1, are more often associated with
altered host range and virulence (Stanley, J. et al., 1985, J.
Virol. 56:110-119; Olmsted, R. A. et al., 1986, Virology
148:245-254; Polo, J. M. et al., 1988, J. Virol. 62:2124-2133;
Lustig, S. et al., 1988 J. Virol. 62:2329-2336). Recently, a
Sindbis virus mutant was identified which contained an insertion in
E2 and exhibited defective binding to mammalian cells. This mutant
is expected to be useful for development of targetable Sindbis
virus vectors (Dubuisson, J. et al., 1993, J. Virol.
67:3363-3374).
[0014] Grieve et al. (International Publication No. WO 94/17813
published Aug. 18, 1994, "Defective Sindbis Virus Vectors That
Express Toxoplasma Gondii P30 Antigens") report the use of
defective sindbis viral vectors to protect mammals from protozoan
parasites, helminth parasites, ectoparasites, fungi, bacteria and
viruses, the contents of which are hereby incorporated by
reference. Garoff et al. (International Publication No. WO 92/10578
published Jun. 25, 1992, "DNA Expression Systems Based On
Alphaviruses") describe the use of alphaviruses to express protein
sequences for immunization or protein production, the contents of
which are hereby incorporated by reference. Davis et al. (U.S. Pat.
No. 5,185,440 issued Feb. 9, 1993, entitled "cDNA Clone Coding For
Venezuelan Equine Encephalitis [(VEE)] Virus And Attenuating
Mutations Thereof) disclose cDNA encoding VEE and methods of
preparing attenuated Togaviruses, the contents of which are hereby
incorporated by reference. Huang et al. (U.S. Pat. No. 5,217,879
issued Jun. 8, 1993, entitled "Infectious Sindbis Virus Vectors")
describe infectious Sindbis virus vectors with heterologous
sequences inserted into the structural region of the genome, the
contents of which are hereby incorporated by reference.
Schlessinger et al. (U.S. Pat. No. 5,091,309 issued Feb. 25, 1992,
entitled "Sindbis Virus Vectors") describe RNA vectors based on the
Sindbis Defective Interfering (DI) particles with heterologous
sequences inserted, the contents of which are hereby incorporated
by reference. Dalemans et al. (International Publication No. WO
95/27069 published Oct. 12, 1995, "Alpha Virus RNA As Carrier For
Vaccines") report the medical use of alphaviruses, specifically the
Semliki Forest Virus, to delivery exogenous RNA encoding a
antigenic epitope or determinant, the contents of which are hereby
incorporated by reference. Dubensky et al., International
Publication No. WO 95/07994 published Mar. 23, 1995, "Recombinant
Alphavirus Vectors" describe recombinant retroviral alphavirus
vectors for delivery of heterologous genes to target cells, the
contents of which are hereby incorporated by reference. Sjoberg et
al., International Publication No. WO 95/31565 published Nov. 23,
1995, "Alphavirus Expression Vector" disclose vectors for enhanced
expression of heterologous sequences downstream from an alphavirus
base sequence, the contents of which are hereby incorporated by
reference. Liljestrom et al., International Publication No. WO
95/27044 published Oct. 12, 1995, "Alphavirus cDNA Vectors"
describe a cDNA construct that may be introduced and transcribed in
animal or human cells, the contents of which are hereby
incorporated by reference.
SUMMARY OF THE INVENTION
[0015] The invention concerns viral vectors and their use.
Specifically, the invention is concerned with viruses having a
protein on the viral particle surface that is a chimeric protein
comprising a viral envelope protein and an IgG-binding domain of
protein A. Because protein A binds to an Fc region of antibody,
these chimeric proteins enable one to use an antibody to target the
viral particle to a desired cell to which the antibody binds and
not to a cell to which the antibody does not bind.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1. A. Schematic representation of expression
constructs. p439 is the SV40-based expression vector including
wild-type Mo-MLV envelope gene. Plasmid p439-ZZ was constructed by
replacement of the Mo-MLV env gene with synthetic IgG-binding part
(ZZ) of protein A between unique restriction sites Bst EII and Bam
HI in p439 vector in the presence of compatible linker-spacer. See
Materials and Methods for details of construction. Abbreviations:
LTR, long terminal repeat ; SV40P, SV40 early enhancer/promoter; L,
leader sequence; SU, surface protein; TM, transmembrane protein;
ZZ, synthetic protein A; L/S, Linker-Spacer; p(A), polyadenylation
signal. B. Immunoblot analysis of lysates from COS-7 cells
transiently transfected with p439 and p439-ZZ. Lane 1 and 2 were
stained with a SU antiserum followed by HRP-conjugated rabbit
anti-goat IgG. Lane 3 and 4 were stained with HRP-conjugated rabbit
IgG for detection of protein A.
[0017] FIG. 2. A. Immunoblot analysis of virions produced by .psi.2
and .psi.2-ZZ10 packaging cells. Lane 1 and 2 were stained with a
SU antiserum followed by HRP-conjugated rabbit anti-goat IgG. Lane
3 and 4 were stained with HRP-conjugated rabbit IgG for detection
of protein A. B. ELISA for detection of IgG-binding activity of
chimeric virus produced by .psi.2-ZZ10 cells. Open circle, virions
from .psi.2; closed circle, virions from .psi.2-ZZ10. Results are
average of triplicate determinants.
[0018] FIG. 3. (A) Schematic strategy for retargeting an Sindbis
virus vector. A wild-type Sindbis virus (left) binds to mammalian
cells via its surface receptor which is known to be highly
conserved across species. A recombinant Sindbis virus displaying
IgG-binding domain of protein A (right) should permit binding to a
novel target molecule on the cell surface when used with a
corresponding monoclonal antibody (mAb). (B) Schematic
representation of recombinant helper constructs and a SinRep/LacZ
expression vector. DH-BB is a parental helper plasmid which
contains the genes for the structural proteins (capsid, E3, E2, 6K
and E1) required for packaging of the Sindbis viral genome.
DH-BB-Bst was constructed by introduction of a cloning site
(BstEII) into the E2 glycoprotein between amino acids 71 and 74.
The synthetic IgG-binding domain (ZZ) of protein A was inserted at
BstEII in the DH-BB-Bst helper plasmid and DH-BB-ZZ was obtained.
SinRep/LacZ, is a Sindbis virus-based expression vector which
contains the packaging signal, nonstructural protein genes for
replicating the RNA transcript and lacZ gene. Abbreviations:
P.sub.SG, Sindbis viral subgenomic promoter; C, capsid; nsP1-4,
nonstructural protein genes 1-4; ZZ, synthetic IgG-binding domain
of protein A; p(A), polyadenylation signal.
[0019] FIG. 4. Detection of Sindbis viral structural protein
components and a recombinant envelope. Cell lysates (A) from BHK
cells transfected with helper RNA and pellets of viral particles (B
and C) produced from these cells were subjected to SDS-PAGE
analysis. After transferring to a nitrocellulose filter, viral
proteins were stained with diluted anti-Sindbis virus mouse immune
ascitic fluid to detect all structural components (A and B) or with
HRP-conjugated goat anti-mouse IgG to detect protein A-envelope
chimeric protein (C). In each panel, lane 1, DH-BB; lane 2,
DH-BB-Bst; lane 3, DH-BB-ZZ.
[0020] FIG. 5. Infection of HeLa and HeLa-CD4.sup.+ cells with
recombinant Sindbis virus derived from DH-BB-ZZ helper RNA which is
transducing the bacterial lacZ gene. Viral supernatants (200 .mu.l)
were preincubated without or with anti-CD4 mAb (0.5 .mu.g/ml) at
room temperature for 1 hour, and added to each cells
(2.times.10.sup.5) in 6-well plates. After 1 hour incubation at
room temperature, cells were washed with PBS and incubated in
growth medium for 24 hours. Viral infection was evaluated by X-Gal
Staining.
[0021] FIG. 6. Antibody-dependent infectivities of recombinant
Sindbis virus particles on A431 and U87MG cells. Viral supernatants
(20 .mu.l for DH-BB, 500 .mu.l for DH-BB-ZZ) were preincubated
without or with anti-EGFR mAb (0.5 .mu.g/ml) at room temperature
for 1 hour, and added to cells (2.times.10.sup.5) in 6-well plates.
After 1 hour incubation at room temperature, cells were washed with
PBS and incubated in growth medium for 24 hours. Viral infection
was evaluated by X-Gal Staining.
[0022] FIG. 7. Antibody-dependent infectivities of recombinant
Sindbis virus particles on suspension cells Daudi and HL-60. Viral
supernatants (500 .mu.l) derived from DH-BB and DH-BB-ZZ
transfected BHK cells were preincubated without or with 0.5
.mu.g/ml of mAbs (anti-HLA-DR for Daudi and anti-CD33 for HL-60) at
room temperature for 1 hour, and added to cells (1.times.10.sup.6)
in 6-well plates. After 1 hour incubation at room temperature,
cells were washed with PBS and incubated in growth medium for 24
hours. Control shows uninfected cells. Viral infection was
evaluated by FACS-Gal analysis described in Experimental protocol.
Positive percent of infected cells were shown in each panel.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention provides for a means for modifying the
expression of genes in eukaryotic cells, such as mammalian cells or
avian cells, and, more particularly, of human cells for medical
practice and also of the cells of domesticated animals that are
valuable for agriculture and recreational purposes for veterinary
practice. The invention provides for the introduction and
expression of genetic material into the cells by means of a viral
vector complex. In the viral vector, some or all of the viral genes
have been replaced by a gene that is to be expressed in the
eukaryotic target cell. The essential viral genes that have been
removed from the vector are, in general, inserted into the genome
of the cell line that is used to produce stocks of the viral
particles. The producer cells lines thus complement the defects
that are present in the viral vector. In some embodiments, the only
viral gene contained in the genome of the vector is a gene that is
needed for the packaging of the vector genome into the viral
particles.
[0024] Specifically, the invention is directed to viral vectors for
transducing a target cell encoding a chimeric protein comprising an
envelope protein and an IgG-binding domain of protein A. In one
embodiment the envelope protein is a retroviral envelope protein.
An example of may be Moloney MLV envelope protein. In the envelope
protein is inserted the IgG binding domain of protein A. As used
herein, protein A may be a portion of native protein A or synthetic
protein having the Fc binding ability of native protein A. In one
embodiment it is inserted into the hypervariable region of
gp70.
[0025] In an alternative embodiment the envelope protein is an
alphavirus envelope protein. An example of an alphavirus may be a
Sindbis virus. For the Sindbis virus it is preferable to insert the
protein A into the E2 domain. The protein A is preferably inserted
so as to reduce or minimize the non-specific infectivity of the
Sindbis virus. One example of an insertion site is the position
between amino acids 71 and 74 of the E2 glycoprotein.
[0026] The construction of viral-based vectors suitable for the
general expression of genes in cells that are susceptible to
infection by the virus is described the following patent
publications: WO 89/05345 to Mulligan, R. C. and others, WO
92/07943 to Guild, B. C. and others concerning retroviral vectors;
WO 90/09441 and WO 92/07945 to Geller, A. I. and others concerning
herpes vectors; WO 94/08026 to Kahn, A. and others, and WO 94/10322
to Herz, J. and others concerning adeno virus vectors; U.S. Pat.
No. 5,354,678 to Lebkowski and U.S. Pat. No. 5,139,941 to Muzcyzka
concerning adeno-associated virus; and U.S. Pat. No. 5,217,879 to
Huang et al. and U.S. Pat. No. 5,091,309 to Schlesinger concerning
Sindbis viral vectors. Packaging systems for the production of
retroviral vectors have been described by Danos, O. et al., 1988,
Proc. Natl. Acad. Sci. 85:6460-64, and by Landau, N. R. et al.,
1992, J. Virol. 66:5110-13, the contents of the above are hereby
incorporated by reference.
[0027] The complexes described herein can be provided with a
variety of specificities. The application discloses methods of
constructing a complex comprising an antibody specific for an
acceptor on the target cell so that the vector complex are
internalized into the target cell after the vector complex is
bound. There are a large number of cell surface antigens suitable
for use as acceptors and for which antibodies are already
available. Such structures include, but are not limited to, the
class I and class II Major Histocompatibility Antigens; receptors
for a variety of cytokines and cell-type specific growth hormones,
brain derived neurotrophic factor (BDNF), ciliary neurotrophic
factor (CTNF), colony stimulating growth factors, endothelial
growth factors, epidermal growth factors, fibroblast growth
factors, glially derived neurotrophic factor, glial growth factors,
gro-beta/mip 2, hepatocyte growth factor, insulin-like growth
factor, interferons (.alpha.-IFN, .beta.-IFN, .gamma.-IFN,
consensus IFN), interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14), keratinocyte
growth factor, leukemia inhibitory factors, macrophage/monocyte
chemotactic activating factor, nerve growth factor, neutrophil
activating protein 2, platelet derived growth factor, stem cell
factor, transforming growth factor, tumor necrosis factors and
vascular endothial growth factor; cell adhesion molecules;
transport molecules for metabolites such as amino acids; the
antigen receptors of B- and T-lymphocytes; and receptors for
lipoproteins. The invention makes possible the specific infection
of a cell type by allowing the employ of differentiation antigens
as targets for the viral vector complex.
[0028] The invention is used to transduce a gene of interest into a
target cell. In practicing the preferred embodiment of the
invention, the viral vector and the antibody are preincubated prior
to contacting the target cell acceptor.
[0029] The practice of the invention can be performed by culturing
the target cells ex vivo. The cultured cells can be continued in
culture to produce the product encoded by the transduced gene.
Alternatively, the ex vivo transduced cell can be implanted into a
subject, which can be the host from which the cultured cells were
obtained.
[0030] In a yet further embodiment, the viral vector and
appropriate antibodies can be administered directly to the subject
thereby obviating the need for any ex vivo cell culture. The routes
of administration to the subject can be any route that results in
contact between the vector complex and the target cell. Thus for
example, intravenous administration is suitable for target cells in
the hepatic, splenic, renal cardiac and circulatory or
hematopoietic systems. The vector complex can also be administered
by catheterization of the artery or vein leading to the target
organ, thereby allowing the localized administration of the
complex. The complex can also be administered by inspiration when
the target cells are in the respiratory system.
[0031] Genes that can be transduced by the practice of the
invention include any gene that can be expressed in a eukaryotic
system. Illustrative examples of genes that can be expressed by use
of the present invention include glucocerebrosidase, adenosine
deaminase, and blood coagulation factors such as factor VIII and
factor IX.
[0032] The viral component of the vector complex can be based on
any virus, the particles of which are unable to bind or have been
modified to be unable to bind to cells of the same species as the
target cell. A non-limiting example of the virus are the murine
ecotropic leukemia retrovirus viruses, e.g., Moloney Leukemia Virus
or AKV. Alternatively, chemically modified viral particles can be
employed. In addition to ecotropic retroviruses, viruses that can
be employed to construct vectors according to this embodiment of
the invention include amphotropic retrovirus, herpes virus,
adenovirus and adeno-associated virus. In addition, the viral
component may be an alphavirus, such as a Sindbis Virus.
[0033] The viral vectors and viral complexes of the invention may
be used to treat a variety of disorders in man and animals. The
vectors based on the Sindbis virus are particularly well suited for
intracellular vaccination. That is, the viral complex carries with
it a gene of interest encoding a particular antigen. The viral
complex will be taken up into the cell and the gene of interest
encoding the antigen is will be expressed in the cellular
cytoplasm. By targeting the viral complex to desired cellular
target, the antigen will be expressed within the cell of
interest.
[0034] The complexes of this invention are also well suited for the
delivery of antisense sequences.
[0035] There are many examples of bacterial and viral diseases that
may be prevented or ameliorated by the methods described herein.
Specifically, the methods described herein may be used for the
following diseases: adenovirus, AIDS, antibiotic associated
diarrhea, bacterial pneumonia, bovine herpes virus (BHV-1),
chlamydia, croup, diphtheria, Clostridium difficile, cystitis,
cytomegovirus (CMV), gastritis, gonorrhea, heliobactor pyliori,
hepatitis A, hepatitis B, herpes virus, HSV-1, HSV-2, human
papilloma virus, influenza, legionnaires disease, Lyme disease,
malaria, multiple sclerosis, peptic ulcer, pertussis, psoriasis,
rabies, respiratory syncytial virus (RSV), rheumatoid arthritis,
rhinovirus, rotovirus, salmonella, Stomach cancer, strep throat,
tetanus or travelers diarrhea.
[0036] The embodiments of the invention are described in greater
detail hereinafter.
EXAMPLES
Example 1
[0037] In this example we describe the construction of a
recombinant ecotropic retrovirus displaying protein A-envelope
chimeric proteins. Protein A, a protein derived from Staphylococcus
aureus, has a strong affinity for the Fc region of various
mammalian IgGs (Surolia, A. et al., 1982, Trends Biochem. Sci.
7:74-76). Native protein A has five homologous IgG-binding domains
(E, D, A, B and C) , and we have utilized the synthetic Z domain
which is based on the B domain of protein A (Nilsson, B. et al.,
1987, Protein Eng. 1:107-113). The development of retroviral
vectors that can bind IgGs (monoclonal antibodies) would have
important applications for specific gene delivery. Materials and
methods
Plasmids and Cell Line
[0038] A SV40-based plasmid, p439 (SV-E-MLV-env), which express
Moloney MLV (Mo-MLV) envelope protein (Landau, N. R. et al., 1992,
J. Virol. 66:5110-5113), was kindly provided Dr. Dan R. Littman,
New York University. pEZZ 18, which contains two synthetic Z
domains based on the B domain of protein A (Lowenadler, B. et al.,
1987, Gene 58:87-97) was purchased from Pharmacia Biotech, Uppsala,
Sweden. pZeoSV, which has Zeocin-resistant gene for selection, was
purchased from Invitrogen Co., San Diego, Calif. An ecotropic
retroviral packaging cell line .psi.2 (ATCC CRL9560) (Mann, R. et
al., 1983, Cell 33:153-159) and COS-7 cells (ATCC CRL1651) were
maintained in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (FBS).
[0039] 6.1.2. Construction of chimeric env gene
[0040] Two synthetic IgG-binding domain of protein A (ZZ) were
amplified by polymerase chain reaction (PCR) using PEZZ 18 as a
template. Primers used for PCR amplification are ZZ-5
(5'-CACGATGAGGTAACCGACAACAAATTCAAC-3'- ) (SEQ ID NO. 1), with Bst
EII site, and M13 (-40) sequencing primer (5'-GTTTTCCCAGTCACGAC-3')
(SEQ ID NO. 2) which locates downstream from the multiple cloning
sites of pEZZ vector. The resulting PCR products were digested with
Bst EII and Eco RI and replaced the Mo-MLV env gene between unique
restriction sites Bst EII (position 5923) and Bam HI (position
6537) of the p439 vector in the presence of compatible
oligonucleotides EB1 (5'-AATTCGGGAGGCGGTGGATCAGGTGGAGGCGGTTCAGG-3')
(SEQ ID NO. 3) and EB2 (5'-GATCCCTGAACCGCCTCCACCTGATCCACCGCCTCC-3')
(SEQ ID NO. 4) to act as a linker-spacer. Clones containing inserts
of proper size were sequenced to confirm that the correct reading
frames were maintained.
Cell transfection and virus production
[0041] The wild-type and protein A-gp70 chimeric envelope genes
were first transiently transfected into COS-7 cells.
2.times.10.sup.5 cells were seeded in 3.5 cm-diameter dishes and
transfected the next day with 2 .mu.g of plasmid with 10 .mu.l of
LipofectAmine reagent (Gibco-BRL, Gaithersburg, Md.). 72 h after
transfection, cells were collected and subjected to immunoblot
analysis. To create packaging cell lines expressing the recombinant
envelope, 5.times.10.sup.5 .psi.2 cells were transfected with 20
.mu.g of chimeric envelope plasmids and 1 .mu.g of pZeoSV by the
CaPO.sub.4 method (Stratagene, La Jolla, Calif.) (Mann, R. et al.,
1983). The medium was changed 16 hours later and transfected cells
were selected with 250 .mu.g/ml of Zeocin (Invitrogen Co., San
Diego, Calif.) After selection for 10 days, Zeocin-resistant cell
colonies were picked for expansion and screened by immunoblot
analysis and ELISA as described below.
Immunoblot assay
[0042] For monitoring of protein A-envelope chimeric protein
expression, transfected cells and viral samples were subjected to
immunoblot analysis. Virus samples were pelleted by
ultracentrifugation of the supernatants (10 ml) in an SW41 Beckmann
Rotor (25,000 rpm, 2 h, 4.degree. C.). Immunoblot analysis was
performed as described before (Marin, M. et al., 1996, J. Virol.
70:2957-2962) by using a goat antiserum against Rausher leukemia
virus SU protein (Quality Biotech Inc., Camden, N.J.) and
horseradish peroxidase-conjugated rabbit anti-goat IgG antibodies
(Pierce, Rockford, Ill.).
ELISA
[0043] ELISA was performed to detect chimeric virus carrying
protein A-envelope chimeric protein in the culture supernatants.
Briefly, pelleted viral particles from 10 ml culture supernatants
were resuspended in 400 .mu.l of phosphate buffered saline. 96-well
microtiter plates (Dynatech Laboratories, INC., Chantilly, Va.)
were coated with duplicate serial dilutions of viral samples for 2
h at RT followed by blocking with PBS containing 1% BSA and 0.05%
Tween 20. Then 0.1 .mu.g/ml of horseradish peroxidase-conjugated
rabbit anti-goat IgG antibodies (Pierce) was added to each well and
incubated for 2 h at RT. After washing with PBS containing 0.05%
Tween 20, the binding activity of each well was determined by using
o-Phenylenediamine (Pierce) as a substrate.
Results
[0044] Plasmid construction and transient expression in COS-7
cells
[0045] A modified Mo-MLV envelope expression vector, p439-ZZ, that
would express two synthetic IgG-binding domain of protein A was
generated (FIG. 1). The position of replacement in gp70 was
previously shown to allow the functional display of erythropoietin
(Kasahara, N. et al., 1994) and heregulin (Han, X. et al., 1995,
Proc. Natl. Acad. Sci. USA 92:9747-9751). The C-terminus of the
protein A gene is connected to a proline rich hypervariable region
of gp70 with the EB linker-spacer (SGGGGSGGGGS) (SEQ ID NO. 5) in
order to avoid interactions between the IgG-binding part of protein
A and the envelope protein segment of the recombinant fusion
protein. Expression is driven by the SV40 early enhancer/promoter
sequence and the 5' long terminal repeat (LTR). The plasmid
p-439-ZZ was deposited with the American Type Culture Collection
(ATCC) on Mar. 28, 1997.
[0046] To examine the expression of the recombinant envelope, we
transfected p439-ZZ expression plasmid into COS-7 cells. Lysates
from transfected and nontransfected cells were first analyzed for
envelope expression by using anti-Rauscher leukemia virus SU serum
which cross-reacts ecotropic (70 kDa) Mo-MLV SU protein. As
expected, the wild-type p439 plasmid expressed major protein bands
of gp70 and its precursor (80 kDa) (FIG. 1B, lane 2). The
recombinant p439-ZZ plasmid expressed immunoreactive proteins at 70
kDa corresponding to precursor protein of the recombinant envelope
suggesting that protein A-gp70 could be expressed in transfected
COS cells. The same lysates were used for detection of IgG-binding
activity using Horseradish peroxidase-conjugated rabbit anti-goat
IgG. As shown in FIG. 1B, lane 6, the protein A-gp70 chimeric
envelope precursor at 70 kDa expressed by p439-ZZ plasmid showed
IgG-binding activity. Stable expression of the chimeric protein
A-gp70 protein suggests that the protein A domain was properly
folded after translation.
[0047] Creation of packaging cell lines producing protein
A-envelope chimeric virus.
[0048] The chimeric envelope plasmid, p439-ZZ, and
Zeocin-resistance gene were cotransfected into .psi.2 packaging
cell line, which expresses gag, pol and env gene products of E-MLV.
After selection with Zeocin, subclones were isolated and screened
for protein A-gp70 expression by immunoblot analysis of whole cell
lysate using rabbit IgG. One subclone, designated .psi.2-ZZ10,
showed cytoplasmic IgG-binding activity and was chosen for further
characterization. To demonstrate the incorporation of the chimeric
envelope protein into virions, retroviral particles were purified
by sucrose density gradient centrifugation. The viral pellets were
then subjected to immunoblot analysis with anti-Rauscher leukemia
virus SU serum or rabbit anti-goat IgG. Major bands of 70 kDa,
which were derived from wild-type env gene of .psi.2 packaging
cells, could be detected in both virions from .psi.2 and
.psi.2-ZZ10 cells (FIG. 2A, lane 1 and 2). The band of 60 kDa,
which was estimated MW of protein A-gp70 chimeric protein, was also
detected in virions produced by .psi.2-ZZ10. However, less chimeric
envelope was found in virus pellet compared with wild-type
envelope. Virions produced by .psi.2-ZZ10 showed IgG-binding
activity at the band of 60 kDa whereas there was no IgG-binding
activity in that of untransfected .psi.2 cells (FIG. 2A, lane 3 and
4). The IgG-binding activity of chimeric virus was further
confirmed by ELISA. As shown in FIG. 2B, the protein A-envelope
chimeric virus produced by .psi.2-ZZ10 cells exhibited IgG-binding
activity in a concentration dependent manner compared with that of
untransfected .psi.2 cells. Taken together, these results
demonstrate that p439-ZZ produces recombinant retrovirus displaying
the IgG-binding domain in its envelope.
Discussion
[0049] In this study we have shown that protein A can be displayed
on the surface of murine ecotropic retroviral particles fused to
the native envelope protein. The protein A-gp70 chimeric protein
derived from p439-ZZ was correctly expressed and incorporated into
virions. Furthermore, IgG-binding activity was detected in virions
produced by .psi.2-ZZ10 cells. In this study the chimeric envelope
did not express as efficiently as that of wild type envelope in
virions produced by .psi.2-ZZ10 (FIG. 2A). We are currently trying
to increase the expression of protein A-gp70 protein by changing
the enhancer/promoter of the expression plasmid as well as
utilizing other packaging cell lines.
[0050] The use of antibody-antigen interactions as the basis for
targeting has a great advantage because a number of monoclonal
antibodies have been developed and investigated. Since the protein
A portion of the chimeric envelope binds to the Fc domain of the
antibody (Surolia, A. et al., 1982), it allows flexibility with
regard to the targeting elements, as any of a variety of mAbs can
be selected. It has been reported that the binding of
retrovirus-associated antibody fragments to the cell surface is
followed by membrane fusion between virus and target cells
(Etienne-Julan, M. et al., 1992, Roux, P. et al., 1989). The
protein A-envelope chimeric retrovirus displaying mAbs against cell
surface antigens should bind preferentially to target cells
expressing those antigens, and this may facilitate their
infection.
[0051] Furthermore, in principle, a similar approach may be used
with other viral vectors, such as adenovirus and Sindbis virus
vectors by inserting the synthetic IgG binding domain (ZZ) of
protein A. We also have constructed a recombinant Sindbis virus
vector with protein A-envelope and demonstrated its high efficient
cell-specific infection against variety of human cells, see Example
2. The protein A-envelope retroviral vector as described in this
example should also permit infection against specific cell types
once the expression of chimeric envelope successfully increased in
the virion. In conclusion, the novel cell targeting system which
utilizes protein A-mAb interaction for virus infection would have
broad applications for gene expression studies and therapy.
Example 2
[0052] In this example we describe the construction of a
recombinant Sindbis virus vector displaying protein A-envelope
chimeric proteins to redirect the viral tropism. Protein A (PA), a
protein derived from Staphylococcus aureus, has a strong affinity
for the Fc region of various mammalian IgGs (Surolia, A. et al.,
1982). In contrast to the targeted retroviral vectors described
above, the PA-envelope chimeric virus vector once successfully
generated needs no further modification to target distinct cells.
The targeting is achieved simply by changing the complementary mAb
(FIG. 3A). More importantly, we demonstrate that this chimeric
virus used in conjunction with mAbs can infect human cells and
transfer a test gene, bacterial .beta.-galactosidase with high
efficiency. The novel cell targeting system which utilizes PA-mAb
interaction for virus infection would have important applications
for gene expression studies and therapy.
Results
Construction of protein A-envelope Sindbis virus helper plasmid
[0053] To modify the Sindbis virus envelope protein, we have
utilized the DH-BB helper plasmid (FIG. 3B) which was constructed
by deletion of the region between BspMII and BamHI sites of the
full-length Sindbis virus cDNA clone (Bredenbeek, P. J. et al.,
1993, J. Virol. 67:6439-6446). When RNA from DH-BB is cotransfected
with recombinant RNA from the Sindbis virus expression vector (for
example, SinRep/LacZ, FIG. 3B), the structural proteins expressed
in trans, from the DH-BB RNA transcript allows packaging of the
recombinant RNA into virions. Since DH-BB does not contain a
packaging signal, it will not form a defective interfering particle
or be packaged with recombinant RNA. Two modified Sindbis virus
helper plasmids were constructed: DH-BB-Bst into which a BstEII
cloning site was inserted and DH-BB-ZZ into which two IgG-binding
domain of PA were inserted in the E2 region, were generated (FIG.
3B). Native protein A has five homologous IgG-binding domains (E,
D, A, B and C) , and we have utilized the synthetic Z domain which
is based on the B domain of protein A (Nilsson, B. et al., 1987).
The insertion position, between codons 71 and 74 amino acids in E2,
was chosen because mutations in this region were previously shown
to allow normal particle assembly and release block virus entry at
the level of attachment (Dubuisson, J. et al., 1993).
Expression and incorporation of chimeric envelopes into virions
[0054] After linearization of helper and SinRep/LacZ plasmids, in
vitro transcription was performed and the quality of RNA was
checked on agarose gels (data not shown). To examine the expression
of the recombinant envelope, recombinant helper RNA was
cotransfected with RNA from SinRep/LacZ plasmid into BHK cells by
electroporation. The transfection efficiency was usually nearly
100% under the procedure described in Experimental protocol below
(data not shown). Lysates from transfected cells were first
analyzed for expression of structural protein by using anti-Sindbis
virus immune ascitic fluid. As shown in FIG. 4A, DH-BB-Bst helper
RNA expressed a 50-55 kDa band of envelope (E1 and E2) and a 33 kDa
of capsid protein which is the same protein profile as the parental
virus produced by DH-BB. A band of 60 kDa corresponding to the E2
precursor PE2 was also detected. In the protein profile expressed
by DH-BB-ZZ RNA, a major band between 65-70 kDa, which is the
estimated MW of PA-E2 and PA-PE2 chimeric protein, was observed as
well as the 33 kDa capsid protein. These results suggest that the
mutants were correctly expressed and processed. A band of envelope
(E1) looks slightly shifted below in the lysate from DH-BB-ZZ
transfected cells due to the disappearance of E2 glycoprotein.
[0055] To demonstrate the incorporation of the chimeric envelope
protein into virions, viral pellets were subjected to immunoblot
analysis. As shown in FIG. 4B, virions produced by DH-BB and
DH-BB-Bst RNA contain capsid and envelope (E1 and E2) proteins
indicating that the mutation in DH-BB-Bst does not affect virus
assembly. The PA-E2 chimeric protein was also incorporated into
virions and exhibited IgG-binding activity which is not detected in
that of DH-BB and DH-BB-Bst (FIG. 4B and C). These results
demonstrate that DH-BB-ZZ produces recombinant Sindbis
pseudovirions displaying the IgG-binding domain in its envelope.
The protein band of E1, which was expressed in transfected cells
(FIG. 4A, lane 3) could not be detected in the virions produced by
DH-BB-ZZ RNA.
Infection with viruses carrying mutant envelopes
[0056] Infectivities of recombinant viruses against hamster and
human cells were determined by transfer of the Sindbis virus vector
(SinRep/LacZ) that can transduce bacterial .beta.-galactosidase
gene. As shown in Table 1, viruses derived from DH-BB and DH-BB-Bst
helper showed very high infectious titer (10.sup.8 LacZ CFU/ml)
against BHK cells whereas viruses produced by DH-BB-ZZ showed very
low infectivity (10.sup.3 LacZ CFU/ml) suggesting that the protein
A insertion into E2 blocked virus binding to host cells supporting
previous observations (Dubuisson, J. et al., 1993). The PA-envelope
virus also showed minimal titer against human HeLa-CD4.sup.+ cells
(10.sup.2 LacZ CFU/ml). When virions were preincubated with
anti-CD4 mAb, however, the protein A-envelope chimeric virus could
infect HeLa-CD4.sup.+ cells in a antibody dose-dependent manner
(Table 1). When the viral supernatant was preincubated with 0.5
.mu.g/ml mAb, an infectious titer was approximately 10.sup.5 LacZ
CFU/ml. The enhancement of infectivities by mAb was not observed
with that of DH-BB and DH-BB-Bst derived viruses. As shown in FIG.
5, the protein A-envelope chimeric virus with anti-CD4 mAb could
not infect HeLa cells which do not express CD4 on its surface
indicating that the infection is dependent on both an antibody and
a corresponding antigen. These data demonstrate that the PA-E2
chimeric envelope derived from DH-BB-ZZ helper RNA can redirect
Sindbis virus infection via a new receptor/antigen in the presence
of recognizing antibody.
[0057] Next, we determined whether PA-E2 displaying virus particles
were capable of infection against various human cell lines
expressing specific antigens on their surface. For adherent cells,
epidermoid carcinoma cell line A431 and glioblastoma cell line
U87MG, both overexpressing epidermal growth factor receptors
(EGFR), were used. As expected, viruses with PA-envelope could
infect these cells efficiently only when virions were preincubated
with anti-EGFR mAb (FIG. 6). Infectious titers of the recombinant
virus with mAb (0.5 .mu.g/ml) against A431 and U87MG cells were
approximately 10.sup.4 LacZ CFU/ml. Again, minimal infectivities
(10.sup.2 LacZ CFU/ml) were seen on these cells when infected
without mAb. We next used two human suspension cell lines,
Burkitt's lymphoma cells, Daudi, and promyelocytic leukemia cells,
HL-60. In this experiment infected cells were detected by FACS-Gal
analysis. Typical FACS results of infectivity are presented in FIG.
7. In contrast to the data with adherent cells (FIG. 6), the
wild-type virus particles derived from DH-BB helper RNA have very
low infectivities against Daudi and HL-60 cells. However, the
PA-envelope virus preincubated with corresponding mAbs (anti-HLA-DR
for Daudi and anti-CD33 for HL-60) could infect these cells with
very high efficiency, and the positive percent of infected cells
were more than 90% in both cell lines. Infection by the protein
A-envelope virus of these cells was not observed in the absence of
mAb.
Discussion
[0058] In this invention we describe the construction of a
recombinant Sindbis virus vector displaying protein A-envelope
chimeric proteins on the viral surface. The synthetic IgG-binding
domain of protein A (ZZ) at the position between 71 and 74 amino
acids of the E2 glycoprotein; this site has been shown to block
Sindbis virus binding to host cells (Dubuisson, J. et al., 1993).
The PA-E2 chimeric protein was correctly expressed and incorporated
into Sindbis virions and exhibited IgG-binding activity as shown in
FIG. 4B and C. In this experiment, however, the incorporation of E1
glycoprotein into virions could not be detected (FIG. 4C, lane 3)
although it is expressed in transfected cells (FIG. 4A, lane 3).
Insertion of the IgG-binding domain produces structural change of
recombinant E2 chimeric protein that inhibits its interaction with
E1 to form a heterodimer. The interaction between E1 and PA-E2
protein is not fully understood. This result also indicates that
Sindbis virus assembly may occur without incorporation of the E1
glycoprotein. This observation may provide insight into mechanism
of Sindbis virus assembly.
[0059] The PA-envelope chimeric Sindbis virus vector showed minimal
infectivities against BHK and other human cell lines. However, when
used in conjunction with mAbs which react with cell surface
antigens, the PA-envelope chimeric virus was able to transfer the
LacZ gene into human cell lines with high efficiency. The new
tropism of the recombinant virus depends on antigen-antibody
interaction since the PA-envelope virus could not infect targeted
cells without mAb and corresponding antigen on cell surface (FIG.
5). Taken together, the PA-E2 chimeric envelope derived from
DH-BB-ZZ helper RNA can redirect Sindbis virus infection with high
efficiency by antigen-antibody interaction.
[0060] Several retrovirus and adenovirus-based cell-targeting
vectors have been developed recently (Russell, S. J. et al., 1993;
Somia, N. V. et al., 1995; Marin, M. et al., 1996; Douglas, J. T.
et al., 1996, Nature, Biotechnology 14:1574-1578). The novel
cell-targeting system developed in this study has some advantages
compared with these retroviral and adenoviral retargeting vectors.
In this approach it is not necessary to construct each targetable
vector de novo. It is unlikely that the incorporation of different
targeting elements in the envelope of the virus can always be
achieved with equal success and without reducing the virus titers
that could be obtained. Since the protein A portion of the chimeric
envelope binds to the Fc domain of the antibody (Surolia, A. et
al., 1982), it allows flexibility with regards to the targeting
elements, as any of a variety of mAbs can be selected. In addition,
replication occurs entirely in the cytoplasm of the infected cells
as an RNA molecule, without a DNA intermediate (Strauss, J. H. et
al., 1994). This is in contrast to retrovirus vectors, which must
enter the nucleus and integrate into the host genome for initiation
of vector activity. Thus, retrovirus-derived vectors have
applications for long-term expression of foreign proteins, while
alphavirus vectors are useful primarily for transient high-level
expression. Furthermore, although adenovirus vectors can express
high levels of foreign proteins, these systems are far more complex
than alphaviruses and express many highly antigenic virus-specific
gene products including structural proteins (Rosenfeld, M. A. et
al., 1991, Science 252:431-434). In contrast, current alphavirus
vectors express only the four viral replicase proteins
(nonstructural proteins nsP1 through nsP4) required for RNA
amplification in the transduced cells.
[0061] There are several issue which have to be considered in
working with Sindbis vectors. First, Sindbis virus infection of
vertebrate cells usually results in cell death by apoptosis
(Levine, B. et al. 1993, Nature 361:739-742), with the notable
exception of neuronal cells in which a persistent infection may be
established (Levine, B. et al. 1992, J. Virol. 66:6429-6435).
Although this cytotoxicity may be suitable for gene therapy for
cancer, long-term or inducible expression vectors would have
broader application. It has been reported that the transformation
of cells with the cellular oncogene bcl-2 led to a cell line in
which Sindbis virus no longer induces apoptosis and instead
establishes a persistent infection (Levine, B. et al., 1993;
Levine, B. et al., 1996, Proc. Natl. Acad. Sci. USA 93:4810-4815,
the contents of which are hereby incorporated by reference into the
present application). bcl-2 may be used to construct a long-term
Sindbis virus expression vector that overcomes the problems of
apoptosis. The bcl-2 vector would be particularly well suited to
create a master packaging cell line also expressing the both
chimeric Sindbis envelop protein and a heterologous gene of
interest under the control a Sindbis promotor. Second, the
recombinant Sindbis virus vector developed in this invention may
have low infectivities even in the absence of antibody.
Accordingly, there might be other sites in E2 or E1 which are
involved in receptor binding (Strauss, J. H. et al., 1994).
Furthermore, different receptors have been identified on chicken
embryo fibroblast (Wang, K. S. et al., Virology 181:694-702) and
mouse neuronal cells (Ubol, S. et al., 1991, J. Virol.
65:6913-6921), suggesting that the Sindbis virus can utilize more
than one receptor. For safety reason, it is desirable to develop
improved recombinant Sindbis virus vector which do not infect any
mammalian cells when not used with mAbs.
[0062] This invention represents the first demonstration of the
retargeting of a Sindbis virus vector by a novel utilization of the
protein A-antibody interaction. A similar approach may be used with
other viral vectors, such as retrovirus and adenovirus vectors by
inserting the synthetic IgG binding domain (ZZ) of protein A. The
virus-based vectors displaying protein A-envelope could be very
useful and have a broad applicability for gene transfer study and
for the gene therapy field.
Experimental protocol
[0063] Cell lines. Baby hamster kidney (BHK) cells were obtained
from Invitrogen Co., San Diego, Calif., and maintained in minimum
essential medium alpha-modification (.alpha.MEM, JRH Biosciences,
Lenexa, Kans.) supplemented with 5% fetal bovine serum (FBS, Gemini
Bio-Products, Inc., Calabasas, Calif.). A human epidermoid
carcinoma cell line A431 (ATCC CRL1555), a human epitheloid
carcinoma cell line HeLa (ATCC CRL2) and a human glioblastoma cell
line U87MG (ATCC HTB14) were grown as monolayers in Dulbecco's
modified Eagle's medium (DMEM; GIBCO-BRL, Gaithersburg, Md.)
supplemented with 10% FBS. HeLa CD4.sup.+ Clone 1022 (NIH AIDS
Research and Reference Reagent Program), which express CD4 on their
surface and a human Burkitt's lymphoma cell line Daudi (ATCC
CCL213), (ATCC CRL1582) was maintained in RPMI 1640 (JRH
Bioscience) supplemented with 10% FBS. HL-60, promyelocytic
leukemia cell line (ATCC CCL240), was maintained in RPMI 1640
supplemented with 20% FBS.
[0064] Monoclonal antibodies (mAbs).
[0065] A murine mAb of IgG2a type against the human epidermal
growth factor receptor (EGFR) was obtained from Upstate
Biotechnology (Lake Placid, N.Y.). Anti-HLA-DR (mouse IgG2a),
anti-CD4 (mouse IgG1) and anti-CD33 (mouse IgG1) were purchased
from Becton Dickinson (San Jose, Calif.).
[0066] Plasmids.
[0067] A helper plasmid DH-BB (Invitrogen Co., FIG. 1B)
(Bredenbeek, P. J. et al., 1993) which contains the genes for the
structural proteins (capsid, E3, E2, 6K and E1) required for
packaging of the Sindbis viral genome was used for construction of
the recombinant envelope gene. A Sindbis virus-based expression
vector SinRep/LacZ (Invitrogen Co., FIG. 3B) (Bredenbeek, P. J. et
al., 1993) contains the packaging signal, nonstructural protein
genes 1-4 (nsP1-4) for replicating the RNA transcript and the lacZ
gene. Plasmid pEZZ 18, which contains two synthetic Z domains based
on the B domain of protein A (Lowenadler, B. et al., 1987), was
purchased from Pharmacia Biotech, Uppsala, Sweden. The phagemid
pALTER-1 vector (Promega Co. Madison, Wis.) was used to introduce
the BstEII site in E2 region of DH-BB plasmid by oligo-directed
site-specific mutagenesis.
[0068] Construction of the recombinant Sindbis virus structural
gene.
[0069] Altered Sites in vitro Mutagenesis System (Promega Co.) was
used to introduce a specific restriction site into the E2 region of
Sindbis virus structural gene. First, a BssHII site was introduced
between XbaI and HindIII sites of the pALTER-1 vector by using two
compatible oligonucleotides 5'-CTAGAGCGCGCAAA-3' and
5'-AGCTTTTGCGCGCT-3' (SEQ ID NOS. 6-7). A fragment between SacI and
BssHII of the DH-BB plasmid containing the E2 region of structural
gene was cloned into the pALTER-1 vector. A single-stranded
template of the recombinant pALTER-1 vector was prepared by
infection of helper phage M13KO7. A mutagenic oligonucleotide
(5'-ATGTCGCTTAAGCAGGTAACCACCGTTAAAGAAGGC-3') (SEQ ID NO. 8) which
introduces a BstEII cloning site between codons 71 and 74 amino
acids in E2 polypeptides and an ampicillin repair oligonucleotide
(5'-GTTGCCATTGCTGCAGGCATCGTGGTG-3') (SEQ ID NO. 9) were annealed to
the single-stranded template, followed by synthesis of the mutant
strand with T4 DNA polymerase. After transformation into E. coli,
mutants were selected in the presence of ampicillin and screened by
direct sequencing of the plasmid DNA. The SacI-BssHII region of
original DH-BB plasmid was replaced with the mutated fragment and
the DH-BB-Bst plasmid was obtained (FIG. 3B). A region of protein A
(ZZ) containing two synthetic IgG-binding domain was amplified by
the polymerase chain reaction (PCR) using pEZZ 18 as a template.
Primers used for PCR amplification are ZZ-5
(5'-CACGATGAGGTAACCGACAACAAATTCAAC-3') and ZZ-3
(5'-GGTCGAGGTTACCGGATCCCC- GGGTACCGA-3') (SEQ ID NOS. 10-11) both
encoding unique BstEII sites. The resulting PCR products were
digested with BstEII and inserted into predigested DH-BB-Bst
plasmid at the BstEII site. Clones containing inserts of proper
size and orientation were sequenced to confirm that the correct
reading frames were maintained and the DH-BB-ZZ plasmid was
obtained (FIG. 3B). The plasmid p-DH-BB-ZZ was deposited with the
American Type Culture Collection (ATCC) on Mar. 28, 1997.
[0070] In vitro transcription and transfection for recombinant
virus production.
[0071] Plasmids for in vitro transcription were prepared by use of
Qiagen (Chatsworth, Calif.) columns. All helper plasmids (DH-BB,
DH-BB-Bst and DH-BB-ZZ) and SinRep/LacZ plasmid were linearized by
XhoI restriction enzyme digestion and purified by phenol/chloroform
extraction followed by ethanol precipitation. Transcription
reactions were carried out by using InvitroScript Cap Kit
(Invitrogen Co.) to produce large quantities of capped mRNA
transcript from the SP6 promoter. For cotransfections of helper and
SinRep/LacZ RNA into BHK cells, electroporations were performed as
described before (Liljestrom, P. et al., 1991, Biotechnology
9:1356-1361). Electroporated cells were transferred to 10 ml of
.alpha.MEM containing 5% FCS and incubated for 12 hours. Cells were
then washed with PBS and incubated in 10 ml of Opti-MEM I medium
(GIBCO-BRL) without FCS. After 24 hours, culture supernatants were
harvested and aliquots were stored at -80.degree. C.
[0072] Immunoblot assay. Cells were lysed in 20 mM Tris-HCl buffer
(pH 8.0) containing 1% Triton X, 0.15 M NaCl, 1 mM
phenylmethylsulfonyl fluoride, 1 mM EDTA and 10% glycerol 24 hour
after transfection. Cell extracts were then sonicated and mixed
with electrophoresis loading buffer (125 mM Tris-HCl, pH 6.8, 10 mM
.beta.-mercaptoethanol, 2% SDS, 10% glycerol and 0.01% bromphenol
blue). Virus samples were pelleted by ultracentrifugation of the
supernatants (10 ml) in an SW41 Beckmann Rotor (35,000 rpm, 2 h,
4.degree. C.) and resuspended in electrophoresis loading buffer.
Cell extracts and viral samples were subjected to immunoblot
analysis as described before (Marin, M. et al., 1996) by using
anti-Sindbis virus mouse immune ascitic fluid (ATCC VR-1248) and
horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG
antibodies (Pierce, Rockford, Ill.).
[0073] Infection assays.
[0074] Infectivity of recombinant chimeric viruses to BHK and human
cell lines was determined by transfer of the Sindbis virus vector
(SinRep/LacZ) that can transduce the bacterial .beta.-galactosidase
gene (Bredenbeek, P. J. et al., 1993). Viral supernatant dilutions
were incubated with or without monoclonal antibodies at room
temperature for 1 hour, then added to adherent (2.times.10.sup.5)
and suspension (1.times.10.sup.6) cells in 6-well plates. After 1
hour incubation at room temperature, cells were washed with PBS and
incubated in growth medium for 24 hours. Viral infection was
evaluated by X-Gal Staining and FACS-Gal as described below and
titers were estimated in LacZ CFU per milliliter.
[0075] X-Gal Staining and FACS-Gal Assay.
[0076] For X-gal staining, commercial protocol was followed.
Briefly, cells were fixed in PBS containing 0.5% glutaraldehyde for
15 min followed by washing with PBS three times. Then cells were
stained with PBS containing 1 mg/ml X-gal, 5 mM potassium
ferricyanide, 5 mM potassium ferrocyanide and 1 mM MgSO.sub.4 at
37.degree. C. for 2 hours. The FACS-Gal assays were performed as
described previously (Fiering, S. N. et al., 1991, Cytometry
12:291-301).
[0077] The present invention is not to be limited in scope by the
specific embodiments described which were intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components were within the
scope of the invention. Indeed, various modifications of the
invention, in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
DEPOSIT OF MICROORGANISMS
[0078] The following organisms were deposited with the American
Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville,
Md. 20852 on Mar. 28, 1997.
1 Strain Designation Containing Accession No. p-439-ZZ Expression
plasmid p-DH-BB-ZZ Expression plasmid
[0079]
Sequence CWU 1
1
* * * * *