U.S. patent application number 11/694683 was filed with the patent office on 2007-08-30 for targeted vectors.
This patent application is currently assigned to Canji, Inc.. Invention is credited to Richard B. Murphy.
Application Number | 20070202524 11/694683 |
Document ID | / |
Family ID | 22573994 |
Filed Date | 2007-08-30 |
United States Patent
Application |
20070202524 |
Kind Code |
A1 |
Murphy; Richard B. |
August 30, 2007 |
Targeted Vectors
Abstract
This invention provides therapeutic and diagnostic agent
delivery vehicles, including viral vectors, that are complexed to a
targeting moiety by coordinate covalent linkages mediated by a
transition metal ion. The complex is typically formed with a
transition metal ion that is in a kinetically labile oxidation
state; after the complex is formed, the oxidation state of the
transition metal ion is changed to one that renders the complex
kinetically stable. The use of a coordinate covalent linkage to
attach the targeting moiety to the delivery vehicle provides
advantages such as the ability to readily attach a different
targeting moiety to a delivery vehicle without modifying the
delivery vehicle itself. This flexibility is achieved without
sacrificing stability of the complex.
Inventors: |
Murphy; Richard B.; (San
Diego, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Canji, Inc.
San Diego
CA
|
Family ID: |
22573994 |
Appl. No.: |
11/694683 |
Filed: |
March 30, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10646060 |
Aug 22, 2003 |
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11694683 |
Mar 30, 2007 |
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09687930 |
Oct 13, 2000 |
6635476 |
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10646060 |
Aug 22, 2003 |
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60159782 |
Oct 15, 1999 |
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Current U.S.
Class: |
435/6.1 ;
435/91.1 |
Current CPC
Class: |
C12N 2710/10345
20130101; C12N 15/86 20130101; C12N 2710/10343 20130101; A61K
47/6901 20170801; C12P 19/34 20130101; A61K 48/00 20130101; A61K
31/70 20130101; C12N 2810/859 20130101; C07K 16/00 20130101 |
Class at
Publication: |
435/006 ;
435/091.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 19/34 20060101 C12P019/34 |
Claims
1. A targeted complex of the formula: {(delivery
vehicle-CM)-TMI-(CM-targeting ligand)}; wherein CM is a chelating
moiety, TMI is a transition metal ion, and CM-targeting ligand is a
chelating moiety (CM) covalently linked to a targeting ligand.
2. The complex of claim 1, wherein the delivery vehicle is a virus
and the chelating moiety is a chelating peptide.
3. The complex of claim 2, wherein the virus lacks a native viral
ligand that binds to a native cellular receptor for the virus.
4. The complex of claim 2, wherein the virus is replication
competent.
5. The complex of claim 2, wherein the virus is replication
deficient.
6. The complex of claim 2, wherein the virus includes a
polynucleotide that encodes a p53 tumor suppressor polypeptide and
the targeting ligand is a antibody that binds to a tumor
antigen.
7. The complex of claim 2, wherein the virus is an adenovirus.
8. The complex of claim 7, wherein the viral coat protein is
selected from a fiber, a penton and a hexon.
9. The complex of claim 7, wherein the adenovirus is replication
competent.
10. The complex of claim 9, wherein the adenovirus is a wild-type
adenovirus.
11. The complex of claim 9, wherein the adenovirus is a selectively
replicating adenovirus.
12. The complex of claim 7, wherein the adenovirus is replication
deficient.
13. The complex of claim 12, wherein the genome of the adenovirus
comprises a partial or total deletion of the adenoviral E1
region.
14. The complex of claim 12, wherein the genome of the adenovirus
comprises a partial or total deletion of the protein IX-encoding
region.
15. The complex of claim 2, wherein the virus is selected from the
group consisting of a retrovirus, a vaccinia virus, a herpes virus,
an adeno-associated virus, a minute virus of mice (MVM), a human
immunodeficiency virus, a sindbis virus, an MoMLV, and a hepatitis
virus.
16. The complex of claim 1, wherein the delivery vehicle is
selected from the group consisting of a bacteriophage, a peptide
vector, a peptide-DNA aggregate, a liposome, a gas-filled
microsome, and an encapsulated macromolecule.
17. The complex of claim 1, wherein the targeting ligand is an
antibody.
18. The complex of claim 17, wherein the antibody is reactive with
a tumor antigen.
19. The complex of claim 17, wherein the antibody is selected from
the group consisting of Fab, Fab', Fab.sub.2' and Fv fragments.
20. The complex of claim 17, wherein the antibody is a human
antibody.
21. The complex of claim 17, wherein the antibody is a single chain
antibody.
22. The complex of claim 21, wherein the single chain antibody is
reactive with carcinoembryonic antigen.
23. The complex of claim 1, wherein the targeting ligand comprises
a conformationally constrained peptide.
24. The complex of claim 23, wherein the conformationally
constrained peptide comprises a portion of an adenoviral fiber
protein.
25. The complex of claim 1, wherein the CM is a chelating peptide
or an organic chelating agent.
26. The complex of claim 25, wherein the organic chelating agent is
selected from the group consisting of a bidentate, a tridentate, a
quadridentate ligand and a tripod ligand.
27. The complex of claim 26, wherein the organic chelating agent is
selected from the group consisting of iminodiacetic acid,
nitrilotriacetic acid, terpyridine, bipyridine,
triethylenetetraamine, and biethylenetriamine.
28. The complex of claim 1, wherein the delivery vehicle is a
liposome.
29. The complex of claim 1, wherein the delivery vehicle is a
paramyxovirus.
30. A viral vector complex that comprises a targeting ligand that
is attached to a surface polypeptide of a viral vector by a
coordinate covalent linkage mediated by a transition metal ion.
31. A method of producing a kinetically inert targeted delivery
vehicle complex, the method comprising: a) preparing a kinetically
labile transition metal complex by contacting a delivery vehicle-CM
and a CM-targeting ligand with a transition metal ion that is in a
kinetically labile oxidation state; and b) changing the oxidation
state of the metal ion to form the kinetically inert complex.
32. The method of claim 31, wherein the kinetically labile
transition metal complex is prepared by: a) contacting the
CM-targeting ligand with the transition metal ion in a reaction
vessel and allowing the transition metal ion to bind to the CM to
form a transition metal ion-CM-targeting ligand complex; b)
removing uncomplexed transition metal ion from the reaction vessel;
and c) contacting the transition metal ion-CM-targeting ligand
complex with the delivery vehicle-CM and allowing the transition
metal ion to bind to the CM to form the complex.
33. The method of claim 31, wherein the kinetically labile
transition metal complex is prepared by contacting the CM-targeting
ligand and the delivery vehicle-CM with the transition metal ion
simultaneously.
34. A method of delivering a therapeutic or diagnostic agent to a
target cell in an organism, the method comprising administering to
an organism a targeted complex of the formula: {(delivery
vehicle-CM)-TMI-(CM-targeting ligand)}; wherein delivery vehicle-CM
is a delivery vehicle that displays on its surface a polypeptide
that comprises a chelating moiety (CM), TMI is a transition metal
ion, and CM-targeting ligand is a chelating moiety (CM) covalently
linked to a targeting ligand that binds to the target cell.
35. The method of claim 34, wherein the delivery vehicle is a viral
vector and the chelating moiety is a chelating peptide (CP).
36. The viral vector of claim 35, wherein the viral vector is
selected from the group consisting of an adenovirus, a retrovirus,
a vaccinia virus, a herpes virus, an adeno-associated virus, a
minute virus of mice (MVM), a human immunodeficiency virus, a
sindbis virus, an MoMLV, and a hepatitis virus.
37. The viral vector of claim 35, wherein the viral vector is an
adenoviral vector and the surface polypeptide is a viral coat
protein selected from the group consisting of a penton base, a
hexon polypeptide, and a fiber polypeptide.
38. The method of claim 34, wherein the therapeutic agent is a gene
that encodes a therapeutic polypeptide.
39. The method of claim 38, wherein the gene encodes a polypeptide
selected from the group consisting of a tumor suppressor, an
antigenic polypeptide, a cytotoxic polypeptide, a cytostatic
polypeptide, a cytokine, a chemokine, a pharmaceutical protein, a
proapoptotic polypeptide, a prodrug-activating polypeptide, an
angiogenesis-inducing polypeptide, and an anti-angiogenic
polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of U.S. Provisional
Application No. 60/159,782, filed Oct. 15, 1999, which application
is incorporated by reference for all purposes.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Not Applicable.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention pertains to the field of targeting of gene
delivery systems (viral and non-viral) to particular cell and
tissue types.
[0005] 2. Background
[0006] The use of recombinant viral vectors for the delivery of
exogenous genes to mammalian cells is well established. See e.g.
Boulikas, T. in Gene Therapy and Molecular Biology Volume 1, pages
1-172 (Boulikas, Ed.) 1998, Gene Therapy Press, Palo Alto, Calif.
However, certain viral vectors commonly used in such instances,
such as adenoviruses, exhibit a broad tropism which permits
infection and expression of the exogenous gene in a variety of cell
types. While this can be useful in some instances, the treatment of
certain diseases is enhanced if the virus is able to be modified so
as "target" (i.e., to preferentially infect) only a limited type of
cell or tissue.
[0007] A variety of approaches to create targeted viruses have been
described in the literature. For example, cell targeting has been
achieved with adenovirus vectors by selective modification of the
viral genome knob and fiber coding sequences to achieve expression
of modified knob and fiber domains having specific interaction with
unique cell surface receptors. Examples of such modifications are
described in Wickham et al. (1997) J. Virol. 71(11):8221-8229
(incorporation of RGD peptides into adenoviral fiber proteins);
Arnberg et al. (1997) Virology 227:239-244 (modification of
adenoviral fiber genes to achieve tropism to the eye and genital
tract); Harris and Lemoine (1996) TIG 12(10):400-405; Stevenson et
al. (1997) J. Virol. 71(6):4782-4790; Michael et al. (1995) Gene
Therapy 2:660-668 (incorporation of gastrin releasing peptide
fragment into adenovirus fiber protein); and Ohno et al. (1997)
Nature Biotechnology 15:763-767 (incorporation of Protein A-IgG
binding domain into Sindbis virus).
[0008] However, the design of a functional chimeric protein for
targeting is not facile. For example, if one wishes to create a
chimeric adenoviral knob protein containing an targeting domain,
the recombinant knob protein must be able to (a) assemble properly
into the icosahedral viral structure and (b) also retain the
binding specificity of the targeting moiety. This may involve
significant and complex molecular modeling to incorporate the
targeting moiety into the appropriate region of the knob protein to
insure that the targeting moiety is on the surface of the knob
protein. Additionally, since the precise process for assembly of
the adenoviral particle is poorly understood it is possible that
insertion of a large targeting moiety will sufficiently interrupt
the three dimensional structure of the viral protein so that it
does not efficiently assemble into an infectious virion.
Furthermore, whenever one wishes to change the targeting properties
of the adenovirus, it is necessary to reengineer the knob protein
taking into account all of the foregoing, which can be a lengthy
process. Moreover, the manipulation of the adenoviral genome to
obtain a gene that encodes the chimeric protein is a time consuming
process, due to the size and complexity of the adenoviral
genome.
[0009] In order to avoid these hurdles, other methods of cell
specific targeting rely on the conjugation of antibodies or
antibody fragments to the envelope proteins (see, e.g. Michael et
al. (1993) J. Biol. Chem. 268:6866-6869, Watkins et al. (1997) Gene
Therapy 4:1004-1012; Douglas et al. (1996) Nature Biotechnology 14:
1574-1578. This approach also has its limitations. First, in the
case of chemically conjugating the antibody (or antibody fragment)
to the surface of the virion, the linkage is generally achieved by
modification of amino acyl side chains in the antibody
(particularly through lysine residues). As it is difficult to
control the stoichiometry of this reaction, one can envision the
resulting virion being coated with antibodies in a variety of
orientations. As the binding specificity of the antibody is
contained in the variable regions, the random association of the
cross-linked antibody will result in many of the antibody variable
domains being "hidden" and thus ineffective. Accordingly, in order
to insure a sufficient number of exposed variable domains to
achieve efficient targeting, a significant excess of antibody must
be complexed to the virion. Additionally, the coating of the virion
with an excess of antibodies may interfere with internalization of
the virus in the target cell. For example, in the case of
adenoviruses, the interaction between the viral coat proteins and
the CAR receptor is believed to be an essential step in the
infectious process. If the viral coat proteins are obscured by an
excess of antibody proteins, one may expect that the efficiency of
binding to the CAR receptor and internalization would suffer. If
the virion is unable to infect the cell and exert its therapeutic
effect, it is questionable whether this targeting approach would
provide significant therapeutic benefit.
[0010] Alternative to the use of antibodies, others have complexed
targeting proteins to the surface of the virion. See, e.g. Nilson
et al. (1996) Gene Therapy 3:280-286 (conjugation of EGF to
retroviral proteins). However, this approach suffers many of the
same limitation as the use of antibodies, such as obscuring viral
coat proteins and potentially interfering with the infectious
mechanism.
[0011] In one attempt to avoid these problems, some groups have
used anti-knob or anti-fiber antibodies complexed to a targeting
moiety (see, e.g., U.S. Pat. No. 5,871,727). While this avoids the
problem of having a antibody-coated virion as discussed above, such
non-covalent complexes are in equilibrium with the free conjugated
antibody and virion species, i.e. {conjugated
antibody-virion}.revreaction.conjugated-antibody+virion. While the
affinity of the antibody for the knob may be high and the resulting
equilibrium constant of this reaction suggests the formation of a
"stable" complex, this does not indicate that the complex will be
kinetically stable in solution over a period of time. Additionally,
although a complex may be "stable" in a solution of limited volume,
upon introduction of the complex to a solution of essentially
infinite volume (e.g., the bloodstream of a mammal) the equilibrium
will be shifted in favor of dissociation of such a complex.
SUMMARY OF THE INVENTION
[0012] The present invention provides targeted complexes that are
useful for delivering molecules to a particular cell or tissue type
of interest. The invention provides targeted complexes of the
formula: {(delivery vehicle-CM)-TMI-(CM-targeting ligand)};
[0013] The delivery vehicle can be, for example, a peptide vector,
a peptide-DNA aggregate, a liposome, a gas-filled microsome, an
encapsulated macromolecule, and the like. In some embodiments, the
delivery vehicle is a viral vector. Particularly suitable viral
vectors include a retrovirus, a vaccinia virus, a herpes virus, an
adeno-associated virus, a minute virus of mice (MVM), a human
immunodeficiency virus, a sindbis virus, an MoMLV, and a hepatitis
virus.
[0014] "CM" is a chelating moiety, such as a chelating peptide or
an organic chelating agent. TMI is a transition metal ion.
CM-targeting ligand is a chelating moiety (CM) covalently linked to
a targeting ligand that can bind to a cell or tissue of
interest.
[0015] The invention also provides methods for producing a
kinetically inert targeted delivery vehicle complex. These methods
involve: a) preparing a kinetically labile transition metal complex
by contacting a delivery vehicle-CM and a CM-targeting ligand with
a transition metal ion that is in a kinetically labile oxidation
state; and b) changing the oxidation state of the metal ion to form
the kinetically inert complex
[0016] Also provided by the invention are methods of delivering a
therapeutic or diagnostic agent to a target cell in an organism.
These methods involve administering to an organism a targeted
complex of the formula: {(delivery vehicle-CM)-TMI-(CM-targeting
ligand)};
[0017] wherein delivery vehicle-CM is a delivery vehicle that
displays on its surface a polypeptide that comprises a chelating
moiety (CM), TMI is a transition metal ion, and CM-targeting ligand
is a chelating moiety (CM) covalently linked to a targeting ligand
that binds to the target cell.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 is an illustration of one embodiments of the
complexes of the present invention. The drawing provides is a
diagrammatic representation of a. complex wherein the virus is an
adenovirus is containing a modified knob domain containing a
chelating peptide and the targeting moiety is a single chain
antibody containing a chelating peptide chelating moiety.
[0019] FIG. 2 is an enhanced diagrammatic representation of the
linkage of the targeting moiety to the modified viral coat protein.
The central circular entity represents a transition metal ion. The
semi-circular structure shaded by cross-hatching represents the
chelating moiety which is covalently linked to the targeting
moiety. The semi-circular structure shaded with the dots represents
the viral coat protein which has been modified to contain a
chelating peptide.
DETAILED DESCRIPTION
[0020] The present invention provides viral vectors and other
delivery vehicles to which targeting ligands are attached by a
kinetically inert coordinate covalent linkage. The targeting
ligands allow the delivery vehicle to be targeted to a particular
cell or tissue type. The viral vectors, for example, display on
their virion surface a coat protein that has been modified to
include a chelating peptide. The targeting ligand is attached to a
chelating moiety (e.g., a chelating peptide or an organic chelating
agent), and a transition metal ion is employed to form a coordinate
covalent bond with the modified coat protein and the targeting
ligand. A coordinate covalent bond occurs when a given species
donates a lone electron pair to a vacant orbital in another
species.
[0021] The use of a coordinate covalent bond as a means to attach
the targeting ligand to the gene delivery system provides
significant advantages over previously available methods for
targeting vectors, which have significant limitations as previously
discussed. First, one need not reengineer a viral genome, for
example, to modify the gene that encodes the surface protein each
time one wishes to use a different targeting ligand. One simply
employs a different CM-targeting ligand to retarget the vector and
modify its tropism. Second, coordinate covalent complexes are
kinetically inert, resulting in a long-lasting targeted vector. In
contrast, attachment of targeting ligands by means of non-covalent
linkage, for example, antibodies that bind to viral coat proteins
is not kinetically inert.
[0022] It is essential that one appreciate the distinction between
a kinetically inert and a thermodynamically stable complex. This
distinction is discussed in detail in Anderson et al. (U.S. Pat.
No. 5,439,829 issued Aug. 8, 1995). Thermodynamic stability refers
to the thermodynamic tendency of a species to exist under
equilibrium conditions. A kinetically inert complex, on the other
hand, is one that is not labile, i.e., a particular complexed ion
is not able to readily engage in reactions that result in
replacement of one or more ligands in its coordination sphere by
others. For example, in an aqueous environment, unoccupied
coordination positions on a transition metal ion are occupied by
water. A chelating peptide or other chelating agent must displace
the water molecules to form a complex. When such reactions occur
rapidly, the reaction is termed "labile." However, where such
reactions occur very slowly or not at all, the complex is said to
be kinetically "inert." Kinetic lability or inertness, unlike
thermodynamic stability or instability, is thus related to the
reaction rate. A complex can be thermodynamically stable even
though the on/off reactions occur very rapidly (see, e.g., Advanced
Inorganic Chemistry, Cotton, F. A. and Wilkinson, G. (1972) 3rd ed.
Interscience Publishers, p. 652). Conversely, a complex can be
kinetically inert, and thus last for periods of time ranging from
days to years, even though the complex is thermodynamically
unstable (equilibrium lies in favor of dissociation) because the
rate of dissociation is low.
[0023] While the affinity of an antibody for a particular protein
may be high and the resulting equilibrium constant of this reaction
suggests the formation of a "stable" complex, this does not
indicate that the complex will be kinetically stable in solution
over a period of time. This presents a particularly serious
drawback when such non-covalent interactions are used to attach a
targeting ligand to a delivery system which is then introduced into
a biological system. The increased volume upon introduction of the
complex to an organism will result in an equilibrium constant
(K.sub.eq) favoring dissociation, since the blood volume is
essentially infinitely large in comparison to the administered
volume. Furthermore, the toxicity of the free components of the
complex may provide an additional degree of uncertainty in the use
of such complexes in mammalian systems. Since non-covalently linked
complexes will necessarily result in free species upon
administration to an organism, the toxicity of the free species in
addition to the complex would need to be evaluated. In human
beings, this would likely complicate the regulatory approval
process for such complexes as it would require additional
toxicology clinical studies. These problems are avoided by the
present invention, which uses a kinetically inert coordinate
covalent linkage to attach the targeting ligand to the viral coat
protein or other gene delivery system.
I. Targeted Complexes
[0024] Generally, the targeted complexes of the invention can be
represented by the formula: {(delivery
vehicle-CM)-TMI-(CM-targeting ligand)} (1) wherein delivery
vehicle-CM refers to a delivery vehicle that displays on its
surface a chelating moiety, TMI is a transition metal ion, and
CM-targeting ligand is a chelating moiety (CM) covalently linked to
a targeting ligand. In presently preferred embodiments, the
delivery vehicle is a viral vector, the chelating moiety is a
chelating peptide, and the polypeptide to which the chelating
peptide is attached is a viral coat protein.
[0025] A. Viral Vectors and Other Delivery Vehicles
[0026] The present invention provides complexes in which a viral
vector or other delivery vehicle is attached by a coordinate
covalent linkage to a targeting ligand. Such delivery vehicles
include, in addition to viral vectors, other molecules or carriers
that are useful for delivering an agent to a cell. Liposomes, for
example can be engineered to accept the coordinate covalently
linked targeting ligands, as can molecules that bind to nucleic
acids or other agents.
[0027] In some embodiments, the complexes include a viral vector to
which targeting ligands are attached. The term "virus" is used in
its conventional sense to refer to any of the obligate
intracellular parasites having no protein-synthesizing or
energy-generating mechanism and generally refers to any of the
enveloped or non-enveloped animal viruses commonly employed to
deliver exogenous transgenes to mammalian cells. The viruses
possess virally encoded viral coat proteins. The viruses useful in
the practice of the present invention include recombinantly
modified enveloped or non-enveloped DNA and RNA viruses. In
presently preferred embodiments, the viruses are selected from
baculoviridiae, parvoviridiae, picomoviridiae, herpesviridiae,
poxviridae, or adenoviridiae. Chimeric viral vectors which exploit
advantageous elements of each of the parent vector properties (See
e.g., Feng et al. (1997) Nature Biotechnology 15:866-870) can also
be employed in the practice of the present invention.
[0028] Viral vector systems useful in the practice of the instant
invention include, for example, naturally occurring or recombinant
viral vector systems. For example, viral vectors can be derived
from the genome of human or bovine adenoviruses, vaccinia virus,
herpes virus, adeno-associated virus (see, e.g., Xiao et al., Brain
Res. 756:76-83 (1997), minute virus of mice (MVM), HIV, sindbis
virus, and retroviruses (including but not limited to Rous sarcoma
virus), and MoMLV, hepatitis B virus (see, e.g., Ji et al., J.
Viral Hepat. 4:167-173 (1997). Typically, genes of interest are
inserted into such vectors to allow packaging of the gene
construct, typically with accompanying viral DNA, followed by
infection of a sensitive host cell and expression of the gene of
interest. One example of a preferred recombinant viral vector is
the adenoviral vector delivery system which has a deletion of the
protein IX gene (see, International Patent Application WO 95/11984,
which is herein incorporated by reference in its entirety for all
purposes).
[0029] In some instances it may be advantageous to use vectors
derived from a different species from that which is to be treated
in order to avoid the preexisting immune response. For example,
equine herpes virus vectors for human gene therapy are described in
WO98/27216 published Aug. 5, 1998. The vectors are described as
useful for the treatment of humans as the equine virus is not
pathogenic to humans. Similarly, ovine adenoviral vectors may be
used in human gene therapy as they are claimed to avoid the
antibodies against the human adenoviral vectors. Such vectors are
described in WO 97/06826 published Apr. 10, 1997.
[0030] The virus can be replication competent (e.g., completely
wild-type or essentially wild-type such as Ad dl309 or Ad dl520),
conditionally replicating (designed to replicate under certain
conditions) or replication deficient (substantially incapable of
replication in the absence of a cell line capable of complementing
the deleted functions). Alternatively, the viral genome can possess
certain modifications to the viral genome to enhance certain
desirable properties such as tissue selectivity. For example,
deletions in the E1 a region of adenovirus result in preferential
replication and improved replication in tumor cells. The viral
genome can also modified to include therapeutic transgenes (as more
fully described below). The virus can possess certain modifications
to make it "selectively replicating," i.e. that it replicates
preferentially in certain cell types or phenotypic cell states,
e.g., cancerous. For example, a tumor or tissue specific promoter
element can be used to drive expression of early viral genes
resulting in a virus which preferentially replicates only in
certain cell types. Alternatively, one can employ a
pathway-selective promoter active in a normal cell to drive
expression of a repressor of viral replication. For example, a
conditionally replicating adenoviral vector can be created by the
use of a promoter active in the presence of endogenous p53 to drive
expression of the E2F-Rb fusion protein (a potent inhibitor of the
E2 adenoviral promoter). In such instances, where there is a defect
in the p53 pathway such that active p53 is not present (e.g., a
tumor cell), the repressor of viral replication is not expressed
and the virus will replicate. However, where p53 is present (e.g.
normal cells) the repressor of viral replication is expressed and
viral replication is prevented. Selectively replicating adenoviral
vectors that replicate preferentially in rapidly dividing cells are
described in International Patent Application No. WO1999US0021451
(Publ. No. WO 022136) entitled "Recombinant E1A Deleted Adenoviral
Vectors." These vectors contain modifications to the E1 a coding
sequence so as to produce E1 a gene products that are deficient in
binding to one or more E1 a p300 protein family members and one or
more Rb protein family members, but retain the transactivating
function of the E1 a CR3 domain. Selectively replicating viruses
are also described in International Patent Application No.
WO1999US0021452 (Publ. No. WO 022137), which is entitled
"Selectively Replicating Viral Vectors." These viral vectors
replicate in cells that have a defective pathway (e.g., a p53 or
TGF-beta pathway), but not in cells with an active pathway.
[0031] Additionally, the viral vector may be replication deficient
or defective in that it possesses certain modifications to the
viral genome so as to essentially deprive the virus of its ability
to replicate in cells that are not capable to complementing the
deleted adenoviral functions. For example, recombinant adenoviral
vectors possessing a deletion of E1 gene functions are essentially
unable to replicate except in cell lines that have been engineered
to complement E1 functions, such as 293 cells, PERC.6 cells or
A549-E1 cells. Such replication defective vectors have been used
effectively to deliver therapeutic transgenes, such as the p53
tumor suppressor gene. Replication defective viral vectors are
preferably derived from adenovirus serotypes 2 or 5 and possess
deletions or mutations in the E1 region rendering one or more early
genes inoperative so as to attenuate the replication of the virus
in non-complementing cells. Additional deletions in the
non-essential E3 region are also permissible to increase the
packaging capacity of such vectors. Replication defective
adenoviral vectors may also contain mutations or deletions so as to
substantially eliminate protein IX function. Particularly preferred
adenoviral vectors are described in Gregory et al., U.S. Pat. No.
5,932,210, issued Aug. 3, 1999. Alternatively, where large DNA
inserts are desired to achieve the therapeutic effect in the target
cell, a "gutted" or minimal viral vector system can be employed.
Such vectors are well known in the art and a review of this
technology is provided in Morsy and Caskey, Molecular Medicine
Today, January 1999 pp. 18-24; Zhang, et al. (WO98/54345A1
published Dec. 3, 1998); and Kochanek et al. (1996) Proc. Nat'l.
Acad. Sci. USA 93: 5731-5736.
[0032] In a presently preferred embodiment of the invention, the
virus is an adenovirus. The use of adenoviral vectors for the
delivery of exogenous transgenes are well known in the art. See,
e.g., Zhang, W-W. (1999) Cancer Gene Therapy 6:113-138. The term
"adenovirus" refers collectively to animal adenoviruses of the
genus mastadenovirus including, but not limited to, human, bovine,
ovine, equine, canine, porcine, murine and simian adenovirus
subgenera. In particular, human adenoviruses include the A-F
subgenera as well as the individual serotypes thereof the
individual serotypes and A-F subgenera including but not limited to
human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Ad11A
and Ad 11P), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91. The bovine
adenoviruses useful in the invention include, but are not limited
to, bovine adenovirus types 1,2,3,4,7, and 10. Canine adenoviruses,
as used herein, includes but is not limited to canine types 1
(strains CLL, Glaxo, RI261, Utrect, Toronto 26-61) and 2. Equine
adenoviruses of interest include, but are not limited to, equine
types 1 and 2 and porcine adenoviruses of interest include, for
example, porcine types 3 and 4. In a presently preferred practice
of the invention, the virus is an adenovirus of serotype 2 or
5.
[0033] Adenoviral polypeptides into which one can incorporate a
chelating peptide include, for example, the fiber protein (see,
e.g., U.S. Pat. Nos. 5,846,789, 5,770,442, 5,543,328 and
5,756,086), the penton base protein (see, e.g., U.S. Pat. Nos.
5,559,099, 5,731,190 and 5,712,136), and the hexon protein (see,
e.g., U.S. Pat. No. 5,922,315).
[0034] Retroviral vectors can also be targeted using the coordinate
covalent complexes of the present invention. The envelope protein
of retroviral vectors is modified to include a chelating peptide.
The retroviral gene that encodes the env polypeptide is modified so
that a fusion between a chelating peptide and all or part of the
env polypeptide is expressed. Modifications of retroviral
env-encoding genes are described in, for example, U.S. Pat. Nos.
5,869,331. U.S. Pat. No. 5,736,387 describes the use of chimeric
targeting proteins that include a ligand (e.g., a cytokine analog)
that is capable of binding to a cytokine receptor to target
retroviral vectors to cells that display the cognate cytokine
receptor. Viral vectors having a chimeric envelope protein that
binds to cell surface receptors are described in, for example, U.S.
Pat. No. 5,985,655. The present invention allows such targeting
schemes to be accomplished without having to modify the viral
genome for each different targeting moiety.
[0035] Other suitable viral vectors include paramyxovirus, such as
simian virus 5 (SV5), a common and non-pathogenic RNA virus. Two
viral glycoproteins are found in the envelope of SV5: the HN
protein which functions in attachment to host cell receptors, and
the F protein which fuses the virion envelope with the target cell
plasma membrane. U.S. Pat. No. 5,962,275 describes the engineering
of SV5 to encode a foreign protein in place of the normal viral
attachment protein HN. Virions containing the foreign membrane
protein in the viral envelope are specific to cells expressing the
ligand that is complementary to the virion-associated foreign
protein or glycoprotein. The present invention provides a means to
make such chimeric envelope proteins without having to alter the
viral genome each time a different targeting moiety is used.
Instead, the viral genome is modified to express at least the
virion-bound portion of the HN protein fused to a chelating moiety.
No additional changes to the viral genome are then required to
substitute one targeting moiety for another. One simply expresses
the desired targeting moiety, linked to a chelating moiety, and
attaches it to the generic virion.
[0036] Bacteriophage are another delivery system to which the
present invention is applicable. Targeted bacteriophage vectors are
described in, for example, U.S. Pat. No. 6,054,312.
[0037] In some presently preferred embodiments, the viral vector is
modified so as to reduce or eliminate the native tropicity of the
virus. For example, the interaction of the a native viral envelope
protein with a cell surface receptor is often highly specific and
determines cell-type specificity of a particular virus (Weiss et
al. (1985) RNA tumor viruses, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.). Therefore, by engineering the chelating
peptide so that the portion of the env polypeptide that confers
cell specificity is disrupted or eliminated, one can obtain a
targeted viral complex that is not only has enhanced affinity for
the cell or tissue type that is recognized by the targeting ligand,
but also has reduced or eliminated affinity for the natural target
cell. Similarly, infection of adenoviruses into susceptible cells
involves the binding of the adenovirus fiber protein (in
particular, the C-terminal knob domain) to the coxsackievirus and
adenovirus receptor (CAR), which serves as the primary cellular
receptor. The subsequent internalization of the virion involves
Arg-Gly-Asp (RGD) sequences in the penton base, which interact with
the secondary host cell receptors, integrins .alpha..sub.v.beta.3
and .alpha..sub.v.beta..sub.5. Thus, by disrupting either or both
of the fiber protein and the penton base, one can eliminate the
native tropicity of the adenoviral vector (see, e.g., Douglas et
al. (1999) Nature Biotechnology, 17: 470-475; U.S. Pat. No.
5,885,808). The disruption of proteins involved in native viral
tropism can be as an intended consequence of the introduction of
the chelating peptide, or can be accomplished by other
manipulations of the viral genome. Parvoviral vectors are another
example of viral vectors that can be targeted using the modified
coat protein-chelating peptide complexed to a targeting ligand.
[0038] The invention also provides complexes in which a
conformationally restrained non-native amino acid sequence is
attached to a surface-displayed chelating moiety. Conformationally
constrained peptides are generally more effective in targeting
delivery to specific cells and/or tissues than unconstrained
peptides. U.S. Pat. No. 6,057,155 describes the use of such
conformationally-restrained, or "constrained" amino acid sequences
in a chimeric adenovirus fiber protein. The ability of the chimeric
fiber protein to bind to the cell and/or mediate cell entry is
increased, e.g., relative to the wild-type protein. According to
U.S. Pat. No. 6,057,155, the conformational constraint can be
achieved by placing a nonnative amino acid sequence in an exposed
loop of the chimeric fiber protein, or, through. the placement of
the sequence in another location and creation of a loop-like
structure comprising the nonnative amino acid sequence at that
site. The present invention facilitates making the chimeric fiber
protein by eliminating the need to alter the viral genome in order
to introduce the nonnative amino acid sequence. Rather, a
polypeptide that includes a chelating moiety and the nonnative
amino acid sequence and associated loop structure is made by, for
example, recombinant expression. This polypeptide is then attached
to a viral vector that displays a corresponding chelating moiety
through a transition metal ion.
[0039] The invention also provides methods for reducing or
eliminating the ability of a viral vector to be recognized by an
antibody that could otherwise neutralize the vector. Neutralizing
antibodies can, for example, inhibit entry of a vector into a cell,
or inhibit vector-mediated gene expression. Therefore, by modifying
coat proteins of the viral vector, one can reduce the
susceptibility of the vector to neutralization. U.S. Pat. No.
6,127,525 describes modifying a viral coat protein to decrease or
eliminate the ability of a neutralizing antibody to interact with
an adenoviral vector. These coat protein modifications can include,
for example, introducing non-native amino acids into the coat
protein. For example, a portion of the coat protein amino acid
sequence can be removed and replaced with a "spacer" amino acid
sequence, or simply by introducing a "spacer" sequence to an
unmodified naturally occurring coat protein. For example, the
deletion of one or more hypervariable regions (e.g., the I1 loop
and/or I2 loops) of the adenoviral hexon protein can result in
reduced sensitivity to neutralizing antibodies. Prior to the
instant invention, such modifications required altering the gene
that encodes the respective coat protein (e.g., for adenovirus:
penton base, hexon, or fiber protein). Through use of the
invention, however, one can simply attach an appropriately modified
extracellular region of the coat protein to a chelating moiety that
is displayed on the surface of the virion using a transition metal
ion. Thus, one can readily construct viral vectors that are
appropriate for avoidance of different neutralizing antibodies
without having to modify the viral genome. A chelating
moiety-modified extracellular domain molecule is constructed (e.g.,
by recombinant expression) for the particular application and
attached to the generic viral vector that displays a cell-surface
chelating moiety.
[0040] Attachment of a targeting moiety by means of a coordinate
covalent linkage according to the invention is useful not only for
viral vectors, but also for other delivery vehicles. For example,
one can attach a targeting ligand to a liposome using a coordinate
covalent linkage. The liposomes used in these embodiments of the
invention carry a chelating moiety on their surface. The chelating
moiety can be, for example, a chelating peptide that is present on
a polypeptide that is displayed on the surface of the liposome
membrane. Alternatively chelating peptides or other chelating
moieties can be attached chemically to lipids that comprise the
liposome membrane. The use of coordinate covalent linkages for
attaching a targeting ligand to a liposome is advantageous because
only one liposome structure need be developed; once such structures
having chelating moieties are made, it is a simple matter to attach
a desired targeting ligand. It is not necessary to reengineer a
liposome-anchored polypeptide or other anchoring moiety for each of
the targeting moieties that are of interest.
[0041] Coordinate covalent linkages are also useful for attaching
targeting moieties to other vehicles for delivering nucleic acids
or other compounds. For example, one can use these linkages to
attach a targeting moiety to a polycation, which is in turn
complexed with a nucleic acid that is to be targeted to a
particular cell or tissue (see, e.g., U.S. Pat. Nos. 5,874,297,
5,166,320, and 5,635,383). For example, gene constructs or other
agents can be conjugated to a cell receptor ligand for facilitated
uptake (e.g., invagination of coated pits and internalization of
the endosome; see, e.g., Wu et al. (1988) J. Biol. Chem.
263:14621-14624; WO 92/06180; U.S. Pat. No. 5,871,727) through a
coordinate covalent linkage. Again, the use of coordinate covalent
attachment simplifies the attachment of the targeting ligand
molecules to the delivery vehicle.
[0042] Other suitable delivery systems include, but are not limited
to, an HVJ (Sendai virus)-liposome gene delivery system (see, e.g.,
Kaneda et al., Ann. N.Y. Acad. Sci. 811:299-308 (1997)); a "peptide
vector" (see, e.g., Vidal et al., CR Acad. Sci III 32:279-287
(1997)); a peptide-DNA aggregate (see, e.g., Niidome et al., J.
Biol. Chem. 272:15307-15312 (1997)); lipidic vector systems (see,
e.g., Lee et al., Crit Rev Ther Drug Carrier Syst. 14:173-206
(1997)); polymer coated liposomes (Marin et al., U.S. Pat. No.
5,213,804, issued May 25, 1993; Woodle et al., U.S. Pat. No.
5,013,556, issued May 7, 1991); cationic liposomes (Epand et al.,
U.S. Pat. No. 5,283,185, issued Feb. 1, 1994; Jessee, J. A., U.S.
Pat. No. 5,578,475, issued Nov. 26, 1996; Rose et al, U.S. Pat. No.
5,279,833, issued Jan. 18, 1994; Gebeyehu et al., U.S. Pat. No.
5,334,761, issued Aug. 2, 1994); gas filled microspheres (Unger et
al., U.S. Pat. No. 5,542,935, issued Aug. 6, 1996), encapsulated
macromolecules (Low et al. U.S. Pat. No. 5,108,921, issued Apr. 28,
1992; Curiel et al., U.S. Pat. No. 5,521,291, issued May 28, 1996;
Groman et al., U.S. Pat. No. 5,554,386, issued Sep. 10, 1996; Wu et
al., U.S. Pat. No. 5,166,320, issued Nov. 24, 1992). In each case,
the transition metal ion-mediated chelation methods of the
invention can be used to attach a targeting moiety to the delivery
vector.
[0043] In order to mask the immunogenic effects of the delivery
system, especially viral vectors, it may be desirable to
additionally complex agents such as polyethylene glycol (PEG) to
the surface of the delivery system to minimize immunological
clearance of the complex. Preferred PEG-ylation protocols are
described in Frances et al. (1998) Int. J. Hematology 68:1-18 and
commercialized by PolyMASC Pharmaceuticals PLC (London UK) as the
"lipoMASC" and "viraMASC" technologies (www.polymasc.com).
[0044] B. Chelating Moiety (CM);
[0045] The delivery vehicles used in the targeted complexes of the
present invention include a polypeptide or other molecule that is
displayed on the surface of the delivery vehicle molecule, to which
a chelating moiety is attached. The term "chelating moiety"
(abbreviated herein as CM) refers collectively to chelating
peptides and organic chelating agents. For example, a viral vector
can have a coat protein that has been modified to include a
chelating peptide. Another chelating moiety is attached to the
targeting ligand. The targeting ligand is attached to the delivery
vehicle by means of a transition metal ion that forms a coordinate
covalent bond between the CM attached to the surface-displayed
molecule on the delivery vehicle and the CM attached to the
targeting ligand. The CM attached to the delivery vehicle can be
the same as, or different than, the CM that is attached to the
targeting ligand.
[0046] 1. Chelating Peptide (CP)
[0047] The term "chelating peptide" (abbreviated "CP") refers to a
peptide sequence that is capable of chelating a transition metal
ion as described in Smith et al. (U.S. Pat. No. 4,569,794 issued
Feb. 11, 1986) and Anderson et al. (U.S. Pat. No. 5,439,829 issued
Aug. 8, 1995) the entire teachings of which are herein incorporated
by reference. Generally, the chelating peptide is incorporated into
the viral coat protein or other delivery vehicle polypeptide by
modifying the viral coat protein coding sequence. The chelating
peptide is incorporated into the delivery vehicle component at a
location that will ensure its exposure on the delivery vehicle
surface. The chelating peptide can be appended to the amino or
carboxy terminus of the protein or can be incorporated internally
into the delivery vehicle protein in an surface-exposed domain of
the protein.
[0048] Examples of an adenovirus in which the knob protein has been
modified to contain a metal chelating peptide are known in the art.
For example, Douglas et al. describe a recombinant adenovirus in
which a poly-His metal chelating peptide has been incorporated into
the carboxy terminal domain of the adenoviral fiber protein (Nature
Biotechnology (1999) 17: 470-475). The penton and hexon
polypeptides are also suitable adenovirus coat proteins for
introduction of the chelating peptide. Apart from the insertion of
the metal chelating peptide in the coat protein, the remainder of
the viral genome can be wild-type or can be modified through
conventional recombinant DNA techniques to possess specific
properties.
[0049] Chelating peptides that are useful in the targeted vectors
of the invention include, for example, a polyhistidine sequence.
Generally, at least two histidine residues are required to obtain
binding to a transition metal ion; the use of additional adjacent
histidines increases the binding affinity. Typically, six adjacent
histidines are used, although one can use more or less than six.
Suitable polyhistidine peptides are described in, for example,
Anderson et al. (U.S. Pat. No. 5,439,829, issued Aug. 8, 1995),
Doebli et al. (U.S. Pat. No. 5,284,993, issued Feb. 8, 1994) and
Doebli et al. (U.S. Pat. No. 5,310,663, issued May 10, 1994).
[0050] In presently preferred embodiments, a nucleotide sequence
that encodes a chelating peptide is incorporated into a gene that
encodes a polypeptide that is displayed on the surface of a
delivery vehicle, and/or the peptidyl targeting ligand. This
typically involves constructing a fusion gene in which a nucleic
acid that codes for the polypeptide is linked, in reading frame, to
a nucleic acid that codes for the chelating peptide. In regard to
coat proteins of a virus, the nucleic acid encoding the chelating
peptide is preferably placed at a location in the surface
polypeptide gene that does not disrupt the ability of the fusion
protein obtained to be displayed on the surface of the delivery
vehicle. Where the targeting ligand is an antibody, the chelating
peptide-encoding nucleic acid can be placed at or near the region
of the antibody gene that encodes the carboxyl terminus of either
the light chain or the heavy chain, or both.
[0051] Similarly, when the CP-targeting ligand is created by
recombinant means, the nucleotide sequence encoding the chelating
peptide is incorporated into (or added to) the nucleotide sequence
encoding the targeting ligand. The chelating peptide should not
interfere with the ability of the targeting ligand to bind to the
target cell or tissue type.
[0052] Methods for constructing and expressing genes that encode
fusion proteins are well known to those of skill in the art.
Examples of these techniques and instructions sufficient to direct
persons of skill through many cloning exercises are found in Berger
and Kimmel, Guide to Molecular Cloning Techniques, Methods in
Enzymology 152 Academic Press, Inc., San Diego, Calif. (Berger);
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd
ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor
Press, N.Y., (Sambrook et al.); Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (1994 Supplement) (Ausubel); Cashion et al., U.S.
Pat. No. 5,017,478; and Carr, European Patent No. 0,246,864.
Alternatively, one can generate CP-targeting ligand species by
conventional chemical protein synthesis reactions. For example, an
isolated protein can be modified to incorporate a chelating peptide
by chemical linkage through the amino or carboxy termini, through
free sulfhydryl groups or free E-amino groups of Lysine or Arg.
[0053] 2. Organic Chelating Agent
[0054] The term "organic chelating agents" is used herein to refer
non-peptidyl bidentate, tridentate, quadridentate, tripod, and
macrocyclic ligands capable of chelating a transition metal ion.
Examples of such organic chelators include iminodiacetic acid,
nitrilotriacetic acid, terpyridine, bipyridine,
triethylenetetraamine, biethylene triamine and derivatives thereof.
Suitable chelating moieties are described in, for example, U.S.
Pat. No. 5,439,829.
[0055] C. Transition Metal Ion (TMI)
[0056] The term "transition metal ion" (abbreviated as TMI), as
described in Anderson et al., refers to a variety of metal ions
capable of forming coordinate complex between at least two
chelating moieties and possessing kinetically labile and
kinetically inert oxidation states. Octahedral complexes with
filled (d.sup.6) or half-filled(d.sup.3) levels such as Cr(III),
V(II), Mn(IV) and the low spin forms of Co(III), Fe(II), Ru(II),
Os(II), Rh(III), Ir(III), Pd(IV), and Pt(IV) tend to be extremely
inert and useful in the practice of the instant invention. Hanzik,
Robert P. in Inorganic Aspects of Biological and Organic Chemistry,
Academic Press, New York, 1976, p. 109. See also, Cotton, F. A. and
Wilkinson, G. supra. In the preferred practice of the invention the
metal ion is selected from the group comprising Te, Co, Cr, and Ru.
In the most preferred practice of the invention the metal ion is
Co. In the most preferred practice of the invention it is desirable
to proceed from Co(II), Cr(II), or Ru(III) to Co(III), Cr(III), or
Ru(II) respectively to form the inert complex. Producing the
necessary change in the oxidation state of the metal ion can be
achieved by a variety of redox reagents. For example, oxidizing
agents such as oxygen, hydrogen peroxide, and peracids can be used
in the practice of the invention. Examples of reducing agents
include, for example, thiols, potassium ferrocyanide, potassium
thiocyanate, sulfites, and sodium dithionite. These will be
prepared in aqueous solutions of appropriate concentrations.
[0057] In some instances, one may wish to incorporate a metal ion
which is readily detected by diagnostic testing equipment such as
x-ray or magnetic resonance imaging. In this manner, a clinician
can non-invasively follow the trafficking of the complex within an
organism. Additionally, certain heavy metals such as Te.sup.99
provide therapeutic (i.e., anti-tumor) effects and can be used to
complement the efficacy of the vector.
[0058] D. Targeting Ligand
[0059] The term "targeting ligand" refers to molecules that
interact with and bind to cell type surface ligands of particular
cells. Examples of such targeting moieties include antibodies
against cell surface proteins and ligands for cell surface
proteins. Examples of cell surface proteins include tumor antigens,
hormone receptors, G-protein coupled receptors, cytokine receptors,
and the like.
[0060] 1. Antibody
[0061] In some embodiments, the targeting ligand includes all or
part of an antibody that binds to the desired target tissue or
cell. The term "antibody" a term used to collectively describe
antibodies, fragments of antibodies (such as, but not limited to,
Fab, Fab', Fab.sub.2' and Fv fragments), chimeric, humanized, or
CDR-grafted antibodies capable of binding antigens of a similar
chain polypeptide binding molecules" as described in PCT
Application No. PCT/US 87/02208, International Publication No. WO
88/01649, International Publication Date: 10 Mar. 1988. Antibodies
can be monoclonal or polyclonal, but are preferably monoclonal. The
antibody can be derived from non-human sources (e.g., mice,
rabbits, goats) but when the complexes are being used in the
treatment of human beings, the antibody is preferably a "human"
antibody derived from non-human sources. Transgenic mice have been
developed which contain the entire human immunoglobulin gene
cluster and as such are capable of producing "human" antibodies.
Such technology and services are available from Abgenix, Inc., 7601
Dumbarton Circle, Fremont, Calif. 94555. As such antibodies are
derived from human genes, such antibodies are preferred as
targeting ligands due to a reduced potential immunogenicity to a
human host. Again, fragments of such human antibodies are
particularly preferred as targeting ligands. Single chain
antibodies modeled on such human antibodies are particularly
preferred as they can be prepared more economically in prokaryotic
culture procedures.
[0062] 2. Tumor Antigens
[0063] When the viral complex is being used to selectively target
tumor cells, it is preferred that the targeting ligand is reactive
with a tumor antigen. The term "tumor antigen" is used herein to
refer to proteins present only on tumor cells (tumor specific
antigens) as well as those present on normal cells but expressed
preferentially on tumor cells (tumor associated antigens). The term
tumor antigen includes, but is not limited to, alfa-fetoprotein
(AFP), C-reactive protein (CRP), cancer antigen-50 (CA-50), cancer
antigen-125 (CA-125) associated with ovarian cancer, cancer antigen
15-3 (CA15-3) associated with breast cancer, cancer antigen-19
(CA-19) and cancer antigen-242 associated with gastrointestinal
cancers, carcinoembryonic antigen (CEA), carcinoma associated
antigen (CAA), chromogranin A, epithelial mucin antigen (MC5),
human epithelium specific antigen (HEA), Lewis(a)antigen, melanoma
antigen, melanoma associated antigens 100, 25, and 150, mucin-like
carcinoma-associated antigen, multidrug resistance related protein
(MRPm6), multidrug resistance related protein (MRP41), Neu oncogene
protein (C-erbB-2), neuron specific enolase (NSE), P-glycoprotein
(mdr1 gene product), multidrug-resistance-related antigen, p170,
multidrug-resistance-related antigen, prostate specific antigen
(PSA), CD56, and NCAM. Antibodies which react with such tumor
antigens are commercially available or can be prepared through
conventional techniques used for the generation of antibodies.
[0064] 3. Ligands for Cell Surface Receptors/Proteins
[0065] Nearly every cell type in a tissue in a mammalian organism
possesses some unique cell surface receptor, e.g., G-protein
coupled receptors. Consequently, when targeting delivery of the
complex to a particular cell type, it is possible to incorporate
nearly any ligand for the cell surface receptor as a targeting
ligand into the complex. For example, peptidyl hormones can be used
a targeting moieties to target delivery to those cells which
possess receptors for such hormones. Chemokines and cytokines can
similarly be employed as targeting ligands to target delivery of
the complex to their target cells. A variety of technologies have
been developed to identify genes that are preferentially expressed
in certain cells or cell states and one of skill in the art can
employ such technology to identify ligands which are preferentially
or uniquely expressed on the target tissue of interest. When the
ligand is a non-peptidyl or non-protein ligand, it is preferred to
employ an organic chelating agent covalently linked to the ligand.
When the ligand is a protein or peptide, it is preferred that the
chelating agent is a chelating peptide. Again, the chelating
peptide can be incorporated at any convenient non-essential domain
of the ligand. The preparation of recombinant proteins comprising
chelating peptides is well known in the art and commercial vectors
are available to facilitate the recombinant production of proteins
incorporating chelating peptides such as the pBlueBacHis2 vector
commercially available from Invitrogen, San Diego, Calif.
[0066] 4. Other Ligands
[0067] Other suitable ligands include "totally synthetic affinity
reagents," which are described in U.S. Pat. Nos. 5,948,635,
5,852,167 and 5,844,076. Binding polypeptides obtained by directed
evolution, for example, as described in U.S. Pat. No. 5,837,500 can
also be used.
[0068] Nuclear localization sequences (NLS) can also be attached to
a vector using transition metal ion chelating methods of the
invention. NLS facilitate trafficking of proteins into a cell
nucleus. See, e.g., WO 96/41606 and U.S. Pat. No. 6,054,312.
II. Preparing the Targeted Complexes
[0069] The invention also provides methods of preparing kinetically
inert transition metal complexes between a chelating peptide that
is displayed on a delivery vehicle and a targeting ligand that is
attached to a chelating moiety. The methods involve:
[0070] a) preparing a kinetically labile transition metal complex
with a transition metal ion, the delivery vehicle-CM and the
CM-targeting ligand, and
[0071] b) changing the oxidation state of the metal ion to form the
kinetically inert complex.
[0072] The formation of the complex while the metal ion is in its
kinetically labile state and then converting the oxidation state to
form a kinetically inert complex is advantageous the rate of
complex formation with the transition metal ion in its inert state
would be very low. If it is desired to dissociate the targeting
ligand from the delivery vehicle, this can be accomplished simply
by contacting the complex with an appropriate redox reagent to
change the oxidation state back to the kinetically labile
state.
[0073] For embodiments in which the delivery vehicle is a viral
vector, the methods of the invention can involve preparing a
recombinant viral protein wherein the viral coat protein possesses
a chelating peptide. A recombinant targeting ligand that is
attached to a chelating moiety is also prepared. The viral coat
protein and the targeting ligand are then reacted with a transition
metal ion that is in a kinetically labile oxidation state. To make
the complex stable, the oxidation state of the transition metal ion
is changed to a kinetically inert oxidation state. The kinetically
inert complexes are then purified.
[0074] Each of the species to be complexed (i.e., the CM-delivery
vehicle and the CM-targeting ligand) can be prepared as described
above and isolated using conventional chromatographic techniques.
Preferably, the CM-targeting ligand is purified to homogeneity
using CP-IMAC purification as described in Smith et al. (U.S. Pat.
No. 4,569,794) and the CM-virus purified in accordance with the
teaching of Shabram et al. (U.S. Pat. No. 5,837,520 issued Nov. 17,
1998, the entire teaching of which is herein incorporated by
reference). Alternatively the viral complex can be purified using
conventional CsC1 procedures.
[0075] The formation of a kinetically labile viral complex can be
accomplished by adding the metal ion to the CM-delivery vehicle or
the CM-targeting ligand independently, or both species can be
exposed to the metal ion in a single reaction vessel. However, in
order to maximize the yield and avoid the formation of homogenous
polymers of delivery vehicle or dimers of targeting ligand-CM
species, it is preferred that the metal ion be exposed to the
targeting ligand, excess metal removed, and the targeting ligand
containing the kinetically labile metal be exposed to the delivery
vehicle containing the modified viral coat protein. Adding the
metal to, for example, a viral vector first will likely result in
polymerization of the viral particles and precipitation.
[0076] The formation of the kinetically inert complex can be
achieved using a variety of oxidizing or reducing agents as
described above and will depend on the nature of the metal ion
involved. Care should be taken to use any particularly harsh
conditions which would result in denaturing of the targeting ligand
or CM-delivery vehicles.
[0077] The purification of the complexes can be accomplished using
conventional chromatographic techniques. Preferably, the
purification/isolation of the kinetically inert complexes should be
performed in the presence of imidazole or a similar agent capable
of competing with the formation of a kinetically labile
intermediate. This will facilitate the purification of only
kinetically inert complexes by disrupting kinetically labile
complexes, thus insuring a homogenous kinetically inert
complex.
III. Uses of the Targeted Complexes
[0078] The complexes of the present invention find use in a wide
variety of applications. Among these applications are the targeting
of therapeutic or diagnostic agents to particular cells or
tissues.
[0079] A. Therapeutic Applications
[0080] The complexes of the present invention are useful in the
treatment of a wide range of diseases in mammalian organisms. The
term "mammalian organism" includes, but is not limited to, humans,
pigs, horses, cattle, dogs, cats, and the like. In these
embodiments, a therapeutic agent is carried in, or attached to, a
viral vector, liposome, or other delivery vehicle, to which is
complexed the targeting ligand through a transition metal ion.
[0081] The methods and compositions of the present invention can be
used for the treatment of a variety of maladies common in mammalian
organisms. For example, the formulations and methods of the present
invention can be used for the treatment of a variety of mammalian
species suffering from such maladies including humans, pigs,
horses, cattle, dogs, cats. In a presently preferred practice of
the invention, the mammalian species is a human being.
[0082] For example, these complexes are useful in the treatment of
cancer wherein a viral or other vector targeted to a cancer cell is
designed to kill the infected cell, and can be designed to have a
by-stander effect so as to kill surrounding cancer cells. In such
instances the targeting ligand of the complex can be an antibody
against a tumor antigen or a ligand for a receptor preferentially
expressed on target tumor cells. A wide variety of tumor antigens
are well known in the art and antibodies to such antigens are
available from commercial sources such as BioDesign International
(105 York Street, Kennebunk, Me. 04043 USA). Generation of antibody
fragments of such intact antibodies are well known to those of
skill in the art. Additionally, such antibodies can be reengineered
to be chimeric, humanized, etc.
[0083] In a particularly preferred embodiment of the invention, the
targeting ligand is a single chain antibody directed against a
tumor antigen such as CEA. Such single chain antibodies against
tumor antigens are known in the art. For example, Anderson, et al.,
supra., describe the anti-CEA CHEL-13 single chain antibody
containing a chelating peptide chelating moiety.
[0084] 1. Gene Delivery
[0085] In some embodiments, the complexes of the present invention
are used to deliver nucleic acids, including, for example,
antisense nucleic acids, genes that encode therapeutic
polypeptides, and the like, to specific cells and/or tissues.
Nucleic acid delivery is useful for several applications, including
corrective gene replacement therapy for defective genes, nucleic
acid-mediated immunization, delivery of genes that encode
therapeutic polypeptides, and cancer therapy.
[0086] a. Expression systems.
[0087] The term "operably linked" refers to a linkage of
polynucleotide elements in a functional relationship. A nucleic
acid sequence is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the coding sequence.
Operably linked means that the nucleotide sequences being linked
are typically contiguous. However, as enhancers generally function
when separated from the promoter by several kilobases and intronic
sequences may be of variable lengths, some polynucleotide elements
may be operably linked but not directly flanked and may even
function in trans from a different allele or chromosome.
[0088] Expression of a nucleic acid, such as the production of a
polypeptide or an antisense nucleic acid, is desired for many
applications. Expression is typically accomplished by placing the
nucleic acid to be expressed in an "expression cassette," which is
a nucleic acid construct, generated recombinantly or synthetically,
that includes nucleic acid elements that are capable of effecting
expression of a structural gene in hosts compatible with such
sequences. Expression cassettes include at least promoters and
optionally, transcription termination signals. Typically, the
recombinant expression cassette includes a nucleic acid to be
transcribed (e.g., a nucleic acid encoding a desired polypeptide),
and a promoter. Additional factors necessary or helpful in
effecting expression may also be used as described herein. For
example, an expression cassette can also include nucleotide
sequences that encode a signal sequence that directs secretion of
an expressed protein from the host cell. Transcription termination
signals, enhancers, and other nucleic acid sequences that influence
gene expression, can also be included in an expression
cassette.
[0089] In order to effect expression of a nucleic acid of interest,
the nucleic acid is operably linked to a promoter sequence operable
in the mammal cell. Examples of promoters include, for example,
viral promoters endogenous to genome of a viral vector, or
promoters derived from other sources. The term "promoter" is used
in its conventional sense to refer to a nucleotide sequence at
which the initiation and rate of transcription of a coding sequence
is controlled. The promoter contains the site at which RNA
polymerase binds and also contains sites for the binding of
regulatory factors (such as repressors or transcription factors).
Promoters can be naturally occurring or synthetic. The promoters
can be endogenous to the virus or derived from other sources. The
promoter can be constitutively active, or temporally controlled
(temporal promoters), activated in response to external stimuli
(inducible), active in particular cell type or cell state
(selective) constitutive promoters, temporal viral promoters or
regulable promoters.
[0090] While the complexes of the present invention facilitate
targeting to particular cells, under certain circumstances
(particularly where the virus is designed to destroy the infected
cell) it may be desirable to further regulate the replication of a
replication competent virus or regulate the expression of the
nucleic acid. In the preferred practice of the invention, the
promoter is a selective promoter, i.e. promoters that are
preferentially active in selected cell types or cell states.
Examples of such selective promoters include tissue specific or
tumor specific promoters. Tissue specific and tumor specific
promoters are well known in the art and include promoters active
preferentially in smooth muscle (alpha-actin promoter), epidermal
specific (Polakowska et al. U.S. Pat. No. 5,643,746 issued Jul. 1,
1997) pancreas specific (Palmiter et al. (1987) Cell 50:435), liver
specific (Rovet et al. (1992) J. Biol. Chem. 267:20765; Lemaigne et
al. (1993) J. Biol. Chem. 268:19896; Nitsch et al. (1993) Mol.
Cell. Biol. 13:4494), stomach specific (Kovarik et al. (1993) J.
Biol. Chem. 268:9917), pituitary specific (Rhodes et al. (1993)
Genes Dev. 7:913), prostate specific (Henderson, U.S. Pat. No.
5,648,478, issued Jul. 15, 1997), etc. The term "selective
promoters" also includes promoters which have both tissue and tumor
cell specificity for example the alpha-fetoprotein promoter is both
liver specific and tumor specific replicating much more efficiently
in hepatocellular carcinoma cells than in either non-tumor or
non-liver cells.
[0091] The term "temporal promoters" refers to promoters which
drive transcription or the therapeutic transgene at a point later
in the viral cycle relative to the promoter controlling expression
of the pathway-responsive promoter. Examples of such temporally
regulated promoters include the adenovirus major late promoter
(MLP), other promoters such as E3. In the preferred practice of the
invention, the MLP promoter is employed. In the case of herpes
simplex virus genomes, the Latent Activated Promoters is an example
of such a temporally regulated promoter.
[0092] The term "inducible promoter" refers to promoters which
facilitate transcription of the therapeutic transgene preferable
(or solely) under certain conditions and/or in response to external
chemical or other stimuli. Examples of inducible promoters are
known in the scientific literature (see, e.g. Yoshida and Hamada
(1997) Biochem. Biophys. Res. Comm. 230:426-430; Iida et al. (1996)
J. Virol. 70(9):6054-6059; Hwang et al. (1997) J. Virol.
71(9):7128-7131; Lee et al. (1997) Mol. Cell. Biol.
17(9):5097-5105; and Dreher et al. (1997) J. Biol. Chem. 272(46);
29364-29371. Examples of radiation inducible promoters are
described in Manome et al. (1998) Human Gene Therapy
9:1409-1417).
[0093] b. Therapeutic Transgenes
[0094] The term "therapeutic transgene" refers to a nucleotide
sequence the expression of which in the target cell produces a
therapeutic effect. The term therapeutic transgene includes but is
not limited to tumor suppressor genes, antigenic genes, cytotoxic
genes, cytostatic genes, pro-drug activating genes, apoptotic
genes, pharmaceutical genes or anti-angiogenic genes. The vectors
of the present invention may be used to produce one or more
therapeutic transgenes, either in tandem through the use of IRES
elements or through independently regulated promoters.
[0095] 1) Tumor Suppressor Genes
[0096] The term "tumor suppressor gene" refers to a nucleotide
sequence, the expression of which in the target cell is capable of
suppressing the neoplastic phenotype and/or inducing apoptosis.
Examples of tumor suppressor genes useful in the practice of the
present invention include the p53 gene, the APC gene, the
DPC-4/Smad4 gene, the BRCA-1 gene, the BRCA-2 gene, the WT-1 gene,
the retinoblastoma gene (Lee et al. (1987) Nature 329:642), the
MMAC-1 gene, the adenomatous polyposis coli protein (Albertsen et
al., U.S. Pat. No. 5,783,666 issued Jul. 21, 1998), the deleted in
colon carcinoma (DCC) gene, the MMSC-2 gene, the NF-1 gene,
nasopharyngeal carcinoma tumor suppressor gene that maps at
chromosome 3p21.3 (Cheng et al. (1998) Proc. Nat'l. Acad. Sci. USA
95:3042-3047), the MTS1 gene, the CDK4 gene, the NF-1 gene, the NF2
gene, and the VHL gene.
[0097] 2) Antigenic Genes
[0098] The term "antigenic genes" refers to a nucleotide sequence,
the expression of which in the target cells results in the
production of a cell surface antigenic protein capable of
recognition by the immune system. Examples of antigenic genes
include carcinoembryonic antigen (CEA), p53 (as described in
Levine, A. PCT International Publication No. WO94/02167 published
Feb. 3, 1994). In order to facilitate immune recognition, the
antigenic gene may be fused to the MHC class I antigen.
[0099] 3) Cytotoxic Genes
[0100] The term "cytotoxic gene" refers to nucleotide sequence, the
expression of which in a cell produces a toxic effect. Examples of
such cytotoxic genes include nucleotide sequences encoding
Pseudomonas exotoxin, ricin toxin, diphtheria toxin, and the like.
Cytotoxic genes are generally employed in the situation where the
virus is designed to destroy the targeted cell and as such are
particularly preferred in the treatment of cancer. Given the nature
of the toxins produced by such genes, it is desirable to control
the expression of such genes. Consequently, when the virus is
designed to encode and express a cytotoxic gene, it is preferred
that the promoter be highly selective or able to be closely
regulated.
[0101] 4) Cytostatic Genes
[0102] The term "cytostatic gene" refers to nucleotide sequence,
the expression of which in a cell produces an arrest in the cell
cycle. Examples of such cytostatic genes include p21, the
retinoblastoma gene, the E2F-Rb fusion protein gene, genes encoding
cyclin dependent kinase inhibitors such as p16, p15, p18 and p19,
the growth arrest specific homeobox (GAX) gene as described in
Branellec et al. (PCT Publication WO97/16459 published May 9, 1997
and PCT Publication WO96/30385 published Oct. 3, 1996). Such genes
are generally employed where one does not wish to destroy the
targeted cell, but merely to prevent the hyperproliferation of such
cells. These genes are particularly useful in the treatment of
benign hyperproliferative diseases such as glaucoma surgery
failure, proliferative vitreoretinopathy. Other ocular diseases
associated with excessive angiogenesis such as age related
macular-degeneration, retinopathy of prematurity, and diabetic
retinopathy may also be treated with such cytostatic genes.
[0103] 5) Cytokine Genes
[0104] The term "cytokine gene" refers to a nucleotide sequence,
the expression of which in a cell produces a cytokine. Examples of
such cytokines include GM-CSF, the interleukins, especially IL-1,
IL-2, IL-4, IL-12, IL-10, IL-19, IL-20, interferons of the alpha,
beta and gamma subtypes especially interferon .alpha.-2b and
fusions such as interferon .alpha.-2.alpha.-1. In particular
disease states to be treated with cytokines, it is preferred that
the cytokine gene is closely regulated is a dose dependent fashion.
For example when using an interferon gene in a vector targeted to
liver cells, it is preferred that the promoter be able to be
closely regulated by an exogenous substance such as through the use
of the GeneSwitch.TM. regulatory system (GeneMedicine, Inc.
Woodlands, Tex.).
[0105] 6) Chemokine Genes
[0106] The term "chemokine gene" refers to a nucleotide sequence,
the expression of which in a cell produces a cytokine. The term
chemokine refers to a group of structurally related low-molecular
cytokines weight factors secreted by cells are structurally related
having mitogenic, chemotactic or inflammatory activities. They are
primarily cationic proteins of 70 to 100 amino acid residues that
share four conserved cysteine residues. These proteins can be
sorted into two groups based on the spacing of the two
amino-terminal cysteines. In the first group, the two cysteines are
separated by a single residue (C-x-C), while in the second group,
they are adjacent (C-C). Examples of member of the `C-x-C`
chemokines include but are not limited to platelet factor 4 (PF4),
platelet basic protein (PBP), interleukin-8 (IL-8), melanoma growth
stimulatory activity protein (MGSA), macrophage inflammatory
protein 2 (MIP-2), mouse Mig (m119), chicken 9E3 (or pCEF-4), pig
alveolar macrophage chemotactic factors I and I (AMCF-I and -II),
pre-B cell growth stimulating factor (PBSF),and IP10. Examples of
members of the `C-C` group include but are not limited to monocyte
chemotactic protein 1 (MCP-1), monocyte chemotactic protein 2
(MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte
chemotactic protein 4 (MCP-4), macrophage inflammatory protein 1
.alpha. (MIP-1-.alpha.), macrophage inflammatory protein 1 .beta.
(MIP-1-.beta.), macrophage inflammatory protein 1 .gamma.
(MIP-1-.gamma.), macrophage inflammatory protein 3-.alpha.
(MIP-3-.alpha., macrophage inflammatory protein 3 .beta.
(MIP-3-.beta.), chemokine (ELC), macrophage inflammatory protein 4
(MIP-4), macrophage inflammatory protein 5 (MIP-5), LD78 .beta.,
RANTES, SIS-epsilon (p500), thymus and activation-regulated
chemokine (TARC), eotaxin, I-309, human protein HCC-1/NCC-2, human
protein HCC-3, mouse protein C10.
[0107] 7) Pharmaceutical Protein Genes
[0108] The term "pharmaceutical protein gene" refers to nucleotide
sequence, the expression of which results in the production of
protein have pharmaceutically effect in the target cell. Examples
of such pharmaceutical genes include the proinsulin gene and
analogs (as described in PCT Intemational Patent Application No.
WO98/31397, growth hormone gene, dopamine, serotonin, epidermal
growth factor, GABA, ACTH, NGF, VEGF (to increase blood perfusion
to target tissue, induce angiogenesis, PCT publication WO98/32859
published Jul. 30, 1998), thrombospondin, etc.
[0109] 8) Proapoptotic Genes
[0110] The term "pro-apoptotic gene" refers to a nucleotide
sequence, the expression thereof results in the programmed cell
death of the cell. Such genes are particularly useful in the
destruction of the targeted cell for use in cancer therapy.
Examples of pro-apoptotic genes include p53, adenovirus E3-11.6K,
the adenovirus E4orf4 gene, p53 pathway genes, and genes encoding
the caspases.
[0111] 9) Pro-Drug Activating Genes
[0112] The term "pro-drug activating genes" refers to nucleotide
sequences, the expression of which, results in the production of
protein capable of converting a non-therapeutic compound into a
therapeutic compound, which renders the cell susceptible to killing
by external factors or causes a toxic condition in the cell. An
example of a prodrug activating gene is the cytosine deaminase
gene. Cytosine deaminase converts 5-fluorocytosine (5-FC) to
5-fluorouracil (5-FU), a potent antitumor agent. The lysis of the
tumor cell provides a localized burst of cytosine deaminase capable
of converting 5FC to 5FU at the localized point of the tumor
resulting in the killing of many surrounding tumor cells. This
results in the killing of a large number of tumor cells without the
necessity of infecting these cells with an adenovirus (the
so-called bystander effect"). Additionally, the thymidine kinase
(TK) gene (see e.g. Woo, et al. U.S. Pat. No. 5,631,236 issued May
20, 1997 and Freeman, et al. U.S. Pat. No. 5,601,818 issued Feb.
11, 1997) in which the cells expressing the TK gene product are
susceptible to selective killing by the administration of
gancyclovir can be employed.
[0113] 10) Anti-Angiogenic and Angiogenesis-Inducing Genes
[0114] The term "anti-angiogenic" genes refers to a nucleotide
sequence, the expression of which results in the extracellular
secretion of anti-angiogenic factors. Anti-angiogenesis factors
include angiostatin, inhibitors of vascular endothelial growth
factor (VEGF) such as Tie 2 (as described in Proc. Nat'1. Acad.
Sci. USA (1998) 95:8795-8800), endostatin.
[0115] Also of interest are angiogenesis-inducing genes that
encode, for example, vascular endothelial growth factor, and other
polypeptides that induce angiogenesis. Such genes are useful for
treating ischemia and other vascular disorders.
[0116] It will be readily apparent to those of skill in the art
that modifications and or deletions to the above referenced genes
so as to encode functional subfragments of the wild type protein
may be readily adapted for use in the practice of the present
invention. For example, the reference to the p53 gene includes not
only the wild type protein but also modified p53 proteins. Examples
of such modified p53 proteins include modifications to p53 to
increase nuclear retention, deletions such as the delta13-19 amino
acids to eliminate the calpain consensus cleavage site,
modifications to the oligomerization domains (as described in
Bracco et al. PCT published application WO97/0492 or U.S. Pat. No.
5,573,925).
[0117] Furthermore, the above therapeutic genes can be secreted
into the media or localized to particular intracellular locations
by inclusion of a targeting ligand such as a signal peptide or
nuclear localization signal (NLS). Also included in the definition
of therapeutic transgene are fusion proteins of the therapeutic
transgene with the herpes simplex virus type 1 (HSV-1) structural
protein, VP22. Fusion proteins containing the VP22 signal, when
synthesized in an infected cell, are exported out of the infected
cell and efficiently enter surrounding non-infected cells to a
diameter of approximately 16 cells wide. This system is
particularly useful in conjunction with transcriptionally active
proteins (e.g. p53) as the fusion proteins are efficiently
transported to the nuclei of the surrounding cells. See, e.g.,
Elliott, G. & O'Hare, P. (1997) Cell 88:223-233; Marshall, A.
& Castellino, A. (1997) Nature Biotechnology 15:205; O'Hare et
al. PCT publication WO97/05265 published Feb. 13, 1997. A similar
NLS derived from the HIV Tat protein is also described in Vives et
al. (1997) J. Biol. Chem. 272:16010-16017.
[0118] Additionally, it will be readily apparent to those of skill
in the art that a viral or other vector can be engineered to encode
more than one therapeutic transgene. The transgenes can be the same
(for example to increase the effective gene dosage) or different to
achieve complementary effects. Each transgene can be under control
of the same promoter (for example through the use of IRES elements)
or different promoters. In those situations where it is desirable
to produce a vector containing multiple transgenes, it is preferred
to use minimal vector systems. The construction of such minimal
vectors (also termed "gutted" or "gutless" vectors) are described
in Zhang, et al. International Publication No WO9854345A1 and Morsy
and Caskey (1999) Molecular Medicine Today, January 1999 issue, pp.
18-24.
[0119] 2. Other Therapeutic Agents
[0120] The terms "therapeutic agent", "therapeutic composition",
and "therapeutic substance" refer, without limitation, to any
composition that can be used to the benefit of a mammalian species.
Such agents may take the form of ions, small organic molecules,
peptides, proteins or polypeptides, oligonucleotides, and
oligosaccharides, for example.
[0121] B. Diagnostic Applications
[0122] The complexes of the invention also find use in diagnostic
and labeling applications. A coordinate covalent linkage mediated
by a metal ion joins a targeting moiety to a detectable label. The
label can be present on a viral or other vector, on a liposome, or
can be attached to a molecule that includes a label. Upon
administration to an organism, or to a population of cells, the
targeting moiety will mediate attachment of the label to the
targeted cells or tissues. One can then detect the presence of the
label to determine which cells and/or tissues have the moiety to
which the targeting ligand is directed. Also, as previously
discussed, a heavy metal visualizable through conventional
diagnostic procedures can be employed, providing the ability to
follow the targeted therapeutic complex through the organism
non-invasively and thus providing both therapeutic and diagnostic
value.
[0123] Detectable labels can be primary labels (where the label
comprises an element that is detected directly or that produces a
directly detectable element) or secondary labels (where the
detected label binds to a primary label, as is common in
immunological labeling). An introduction to labels, labeling
procedures and detection of labels is found in Polak and Van
Noorden (1997) Introduction to Immunocytochemistry, 2nd ed.,
Springer Verlag, NY and in Haugland (1996) Handbook of Fluorescent
Probes and Research Chemicals, a combined handbook and catalogue
published by Molecular Probes, Inc., Eugene, Oreg. Primary and
secondary labels can include undetected elements as well as
detected elements. Useful primary and secondary labels in the
present invention can include spectral labels such as fluorescent
dyes (e.g., fluorescein and derivatives such as fluorescein
isothiocyanate (FITC) and Oregon Green", rhodamine and derivatives
(e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.),
digoxigenin, biotin, phycoerythrin, AMCA, CyDyes", and the like),
radiolabels (e.g., 3H, 125I, 35S, 14C, 32P, 33P, etc.), enzymes
(e.g., horse radish peroxidase, alkaline phosphatase etc.),
spectral colorimetric labels such as colloidal gold or colored
glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)
beads. The label may be coupled directly or indirectly to a
component of the detection assay (e.g., the detection reagent)
according to methods well known in the art. As indicated above, a
wide variety of labels may be used, with the choice of label
depending on sensitivity required, ease of conjugation with the
compound, stability requirements, available instrumentation, and
disposal provisions.
[0124] Preferred labels include those that use: 1)
chemiluminescence (using horseradish peroxidase or luciferase) with
substrates that produce photons as breakdown products as described
above) with kits being available, e.g., from Molecular Probes,
Amersharn, Boehringer-Mannheim, and Life Technologies/Gibco BRL; 2)
color production (using both horseradish peroxidase and/or alkaline
phosphatase with substrates that produce a colored precipitate
[kits available from Life Technologies/Gibco BRL, and
Boehringer-Mannheim]); 3) hemifluorescence using, e.g., alkaline
phosphatase and the substrate AttoPhos [Amersham] or other
substrates that produce fluorescent products, 4) fluorescence
(e.g., using Cy-5 [Amersharn]), fluorescein, and other fluorescent
tags]; 5) radioactivity. Other methods for labeling and detection
will be readily apparent to one skilled in the art.
[0125] Preferred enzymes that can be conjugated to targeting
ligands using the coordinate covalent linkages of the invention
include, e.g., luciferase, and horse radish peroxidase. The
chemiluminescent substrate for luciferase is luciferin. Embodiments
of alkaline phosphatase substrates include p-nitrophenyl phosphate
(pNPP), which is detected with a spectrophotometer;
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium
(BCIP/NBT) and fast red/napthol AS-TR phosphate, which are detected
visually; and 4-methoxy-4-(3-phosphonophenyl)
spiro[1,2-dioxetane-3,2'-adamantane], which is detected with a
luminometer. Embodiments of horse radish peroxidase substrates
include 2,2'azino-bis(3-ethylbenzthiazoline-6 sulfonic acid)
(ABTS), 5-aminosalicylic acid (5AS), o-dianisidine, and
o-phenylenediamine (OPD), which are detected with a
spectrophotometer; and 3,3,5,5'-tetramethylbenzidine (TMB),
3,3'diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), and
4-chloro-1-naphthol (4C1N), which are detected visually. Other
suitable substrates are known to those skilled in the art.
[0126] In general, a detector which monitors a particular label is
used to detect the label. Typical detectors include
spectrophotometers, phototubes and photodiodes, microscopes, x-ray,
magnetic resonance imaging (MRI), scintillation counters, cameras,
film and the like, as well as combinations thereof. Examples of
suitable detectors are widely available from a variety of
commercial sources known to persons of skill. Commonly, an optical
image of a substrate comprising bound labeling moieties is
digitized for subsequent computer analysis.
[0127] C. Other Uses
[0128] The targeted vectors of the invention are also useful to
introduce a gene into a host for in vivo production of a protein
encoded by the gene. For example, transgenic bovines and goats are
used for production of proteins in milk (see, e.g., WO 93/25567).
The vectors are also useful for making "knockout" animals that are
useful for the study of human diseases and other purposes.
IV. Formulations and Treatment Regimes
[0129] The complexes prepared above can be formulated for
administration to a mammalian organism in accordance with
techniques well known in the art. The complexes can be administered
in conventional solutions such as sterile saline and can
incorporate one or more carriers of agents to preserve the
stability and sterility of the solution. The formulations can also
include carrier molecules conventionally used in the formulation of
pharmaceutical agents. The term "carriers" refers to compounds
commonly used on the formulation of pharmaceutical compounds used
to enhance stability, sterility and deliverability of the
therapeutic compound. When the viral, non-viral or protein delivery
system is formulated as a solution or suspension, the delivery
system is in an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers can be used, e.g., water, buffered
water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like.
[0130] These compositions can be sterilized by conventional, well
known sterilization techniques, or can be sterile filtered. The
resulting aqueous solutions can be packaged for use as is, or
lyophilized, the lyophilized preparation being combined with a
sterile solution prior to administration. The compositions can
contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, calcium chloride, sorbitan
monolaurate, triethanolamine oleate, etc.
[0131] The formulations can also include delivery enhancing agents
to increase uptake of the targeted complexes into the target cells.
The terms "delivery enhancers" or "delivery enhancing agents" are
used interchangeably herein and includes agents that facilitate the
transfer of the nucleic acid or protein molecule to the target
cell. Examples of such delivery enhancing agents detergents,
alcohols, glycols, surfactants, bile salts, heparin antagonists,
cyclooxygenase inhibitors, hypertonic salt solutions, and acetates.
Suitable alcohols include for example the aliphatic alcohols such
as ethanol, N-propanol, isopropanol, butyl alcohol, acetyl alcohol.
Glycols include glycerine, propyleneglycol, polyethyleneglycol and
other low molecular weight glycols such as glycerol and
thioglycerol. Acetates such as acetic acid, gluconic acid, and
sodium acetate are further examples of delivery-enhancing agents.
Hypertonic salt solutions like 1M NaCl are also examples of
delivery-enhancing agents. Bile salts such as taurocholate, sodium
tauro-deoxycholate, deoxycholate, chenodesoxycholate, glycocholic
acid, glycochenodeoxycholic acid and other astringents such as
silver nitrate can be used. Heparin-antagonists like quaternary
amines such as protamine sulfate can also be used. Anionic,
cationic, zwitterionic, and nonionic detergents can also be
employed to enhance gene transfer. Exemplary detergents include but
are not limited to taurocholate, deoxycholate, taurodeoxycholate,
cetylpyridium, benalkonium chloride, Zwittergent 3-14 detergent,
CHAPS (3-[(3-Cholamidopropyl) dimethylammnoniol]-1-propanesulfonate
hydrate), Big CHAP, Deoxy Big CHAP, Triton-X-100 detergent, C12E8,
Octyl-B-D-Glucopyranoside, PLURONIC-F68 detergent, Tween 20
detergent, and TWEEN 80 detergent (CalBiochem Biochemicals).
Particularly preferred delivery enhancing reagents are derivatives
of particular impurities that are found in some preparations of Big
CHAP; these derivatives are described in PCT Application No.
US98/14241 (published Jan. 21, 1999 as WO99/02191).
[0132] The formulations of the invention are typically administered
to enhance transfer of an agent to a cell. The cell can be provided
as part of a tissue, such as an epithelial membrane, or as an
isolated cell, such as in tissue culture. The cell can be provided
in vivo, ex vivo, or in vitro. The formulations containing delivery
enhancing compounds and modulating agents can be introduced into
the tissue of interest in vivo or ex vivo by a variety of methods.
In some embodiments of the invention, the modulating agent is
introduced to cells by such methods as microinjection, calcium
phosphate precipitation, liposome fusion, or biolistics. In further
embodiments, the therapeutic agent is taken up directly by the
tissue of interest.
[0133] In some embodiments of the invention, the targeted complexes
of the invention are administered ex vivo to cells or tissues
explanted from a patient, then returned to the patient. Examples of
ex vivo administration of therapeutic gene constructs include
Arteaga et al., Cancer Research 56(5): 1098-1103 (1996); Nolta et
al. Proc. Nat'l. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al.,
Seminars in Oncology 23 (1):46-65 (1996); Raper et al., Annals of
Surgery 223(2):116-26 (1996); Dalesandro et al., J. Thorac. Cardi.
Surg., 11(2):416-22 (1996); and Makarov et al., Proc. Nat'l. Acad.
Sci. USA 93(1):402-6 (1996).
[0134] It will be appreciated by those of skill in the art that the
particular dosage of a given complex will depend on a variety of
factors. The targeted complexes of the present invention provide an
advantage over their non-targeted counterparts in that a lower
dosage can achieve an equivalent therapeutic or diagnostic effect.
However, this does not necessarily mean that a reduced dosage will
be indicated in all cases. For example, in oncology applications,
administration of the maximum tolerated dose of the therapeutic
agent is generally accepted as the preferred dosage. Clinical
trials in human beings have indicated that a dose of
2.5.times.10.sup.13 adenoviral particles administered for 5
consecutive days for three courses of therapy is well tolerated
(Nielsen et al. (1998) Hum Gene Ther. 9: 681-94). Consequently,
viral doses of this magnitude would be suitable for therapeutic
applications. For oncology applications the therapeutic agent may
also be combined with other treatment regimens such as radiation,
etc.
[0135] In non-oncology therapeutic applications and diagnostic
applications, a more limited dose would be preferred. Again, the
precise nature of the dose will depend on the type of delivery
vehicle, the therapeutic or diagnostic effect sought, the degree of
control of transgene, expression in addition to more common factors
such as the patient's age, weight, sex, physical condition, etc.
However, the determination of appropriate dose is a matter of
routine experimentation to those of skill in the art. Dose
escalation trials in mammalian species generally are initially
carried out in small animal species such as swine, eventually in
primates. Phase I clinical trials in human beings also include such
dose escalation and toxicity assessments. Although such experiments
are time-consuming, the skill necessary to achieve the clinically
relevant dosage range is a matter of routine experimentation.
[0136] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference for all purposes.
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