NZ712211B2 - Thymidine kinase diagnostic assay for gene therapy applications - Google Patents
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- NZ712211B2 NZ712211B2 NZ712211A NZ71221114A NZ712211B2 NZ 712211 B2 NZ712211 B2 NZ 712211B2 NZ 712211 A NZ712211 A NZ 712211A NZ 71221114 A NZ71221114 A NZ 71221114A NZ 712211 B2 NZ712211 B2 NZ 712211B2
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Abstract
Nucleic acid sequences encoding improved Herpes Simplex Virus Thymidine Kinases (HSV-TK) are provided, including their use in diagnostic and therapeutic applications. The HSV-TK polynucleotide encodes a mutated form of HSV-TK comprising mutations at amino acid residues 32, 33, and 168, wherein the amino acid residues 32 and 33 are each independently mutated to an amino acid chosen from the group consisting of glycine, serine, glutamic acid, an acidic amino acid and cysteine, and wherein the mutated form of HSV-TK increases cell kill activity relative to a wild-type HSV-TK. Also provided are gene therapeutic systems, including viral and retroviral particles. mino acid residues 32 and 33 are each independently mutated to an amino acid chosen from the group consisting of glycine, serine, glutamic acid, an acidic amino acid and cysteine, and wherein the mutated form of HSV-TK increases cell kill activity relative to a wild-type HSV-TK. Also provided are gene therapeutic systems, including viral and retroviral particles.
Description
(12) Granted patent specificaon (19) NZ (11) 712211 (13) B2
(47) Publicaon date: 2021.12.24
(54) THYMIDINE KINASE DIAGNOSTIC ASSAY FOR GENE THERAPY APPLICATIONS
(51) Internaonal Patent Classificaon(s):
A01N 63/00 A61K 48/00 A61K 49/00 C12N 15/00
(22) Filing date: (73) Owner(s):
2014.03.14 GENVIVO, INC.
(23) Complete specificaon filing date: (74) t:
2014.03.14 PHILLIPS ORMONDE FITZPATRICK
(30) Internaonal Priority Data: (72) Inventor(s):
US 61/784,901 3.14 LEVY, John, P.
REED, Rebecca, A.
(86) Internaonal Applicaon No.: MCNULTY, Joseph
JOHNSON, Robert, G., Jr.
(87) Internaonal Publicaon number:
WO/2014/153205
(57) ct:
Nucleic acid sequences encoding improved Herpes Simplex Virus Thymidine Kinases (HSV-TK) are
ed, including their use in diagnosc and therapeuc applicaons. The HSV-TK polynucleode
encodes a d form of HSV-TK comprising mutaons at amino acid residues 32, 33, and
168, wherein the amino acid residues 32 and 33 are each independently mutated to an amino
acid chosen from the group consisng of glycine, serine, glutamic acid, an acidic amino acid and
cysteine, and wherein the mutated form of HSV-TK increases cell kill acvity relave to a wild-type
HSV-TK. Also provided are gene euc systems, including viral and retroviral parcles.
712211 B2
INE KINASE DIAGNOSTIC ASSAY FOR GENE THERAPY APPLICATIONS
CROSS-REFERENCE
This application claims the benefit of US. Provisional Application No. 61/784,901, filed
on March 14, 2013, which is incorporated herein by reference in its entirety.
This application is related to the following co-pending patent applications: application
Serial No. [not yet assigned], Attorney Docket No. 30863-722201, filed the same day herewith,
which application is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Proliferative diseases, such as cancer, pose a serious challenge to society. Cancerous
growths, including malignant cancerous growths, possess unique characteristics such as
uncontrollable cell proliferation resulting in, for example, unregulated growth of malignant
tissue, an ability to invade local and even remote s, lack of differentiation, lack of
able symptoms and most significantly, the lack of effective y and tion.
Cancer can develop in any tissue of any organ at any age. The etiology of cancer is not
clearly defined but mechanisms such as genetic susceptibility, chromosome breakage disorders,
viruses, environmental factors and immunologic ers have all been linked to a malignant cell
growth and transformation. Cancer encompasses a large category of medical conditions, affecting
millions of individuals worldwide. Cancer cells can arise in almost any organ and/or tissue of the
body. Worldwide, more than 10 million people are diagnosed with cancer every year and it is
estimated that this number will grow to 15 n new cases every year by 2020. Cancer causes
six million deaths every year or 12% of the deaths worldwide.
SUMMARY OF THE INVENTION
Provided herein are methods and compositions for identifying subjects or patients that
are capable of benefitting from gene therapy treatment. More specifically, ed herein are
methods and compositions for identifying ts or patients that express in ent
quantities a therapeutic protein included in a gene therapy agent. Preferably the therapeutic
protein is an enzyme, more specifically viral thymidine kinase or mutant viral thymidine .
Accordingly, provided herein are methods for identifying a patient capable of benefitting
from gene therapy treatment comprising administering a gene therapy retroviral particle
comprising an HSV-TK polynucleotide to the t; administering to the patient a substrate of
HSV-TK attached to a radioactive tracer; ing the relative amount and location of the
ctive signal present in the patient; and determining the location of lesions in the patient,
wherein patients with: radioactive signals above a n threshold, and location of the
radioactive signal correlating with lesions measured in step (d) of the patient, are identified as
capable of benefitting from gene therapy treatment.
In some embodiments, the substrate of HSV-TK is chosen from the group consisting of
FHBG (9-[4-fluoro(hydroxymethyl)butyl]guanine), FHPG (9~([3~fiuoro—l ~hydroxy—2—
propoxy]meihyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-
deoxy-2'-fluoro-l-B-D-arabinofi1ranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-
arabinofuranosyluracil), FMAU (fluoro-S-methyl-l-beta-D-arabinofuranosyluracil), FHOMP (6-
((l -fluorohydroxypropanyloxy)methyl)methylpryrimidine-2,4( l H,3H)—dione),
ganciclovir, valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled pyrimidine with 4-
hydroxy(hydroxymethyl)butyl side chain at N-l (HHG-S -FEP) or 5-(2-)hydroxyethyl)- and 5-
(3 -hydroxypropyl)-substituted pyrimidine derivatives bearing hydroxypropyl, acyclovir-,
ganciclovir- and penciclovir-like side chains. In yet other embodiments, the ate of HSV-
TK is FHBG (9-(4-fluoro(hydroxymethyl)butyl]guanine).
In still other embodiments, the radioactive tracer is 18F, 64Cu, 99mTe, 11C, 14C, 1241, 123I,
131I, 15O, 13N and/or . In other embodiments, the radioactive tracer is 18F.
In one ment, the HSV-TK substrate is [18F]FHBG (9-(4-18F-fluoro
(hydroxymethyl)butyl]guanine). In still other embodiments, the radioactive tracer signal is
measured using positron emission tomography (PET) ng.
In still other embodiments, the threshold level is at least above 2.0 SUV (standardized
uptake value) or at least 20% above ound on a PET scan, or n about 1.0 SUV and
about 3.0 SUV, or n about 20% to about 40% above background on a PET scan.
In some embodiments, the methods disclosed herein further comprises treating the
patient with the HSV-TK retroviral particle.
In still other embodiments, the viral nuclear localization sequence (NLS) of the encoded
HSV-TK polynucleotide is mutated. In yet other embodiments, the thymidine kinase
polynucleotide is mutated to include a nuclear export sequence (NES) at or near the amino
terminus of the expressed ine kinase protein. In one embodiment, the thymidine kinase
polynucleotide is d to increase substrate binding of the expressed thymidine kinase
protein. In another embodiment, the on is Al68H.
In still other embodiments, the methods disclosed herein further comprises mutating the
thymidine kinase polynucleotide to remove the viral nuclear zation sequence (NLS) and
include a nuclear export sequence (NES) at or near the amino terminus of the expressed
thymidine kinase protein. In some embodiments, the HSV-thymidine kinase polynucleotide is
SEQ ID NO: 18.
In one ment, the methods disclosed herein further comprises a targeting protein
expressed on the viral envelope. In some embodiments, the targeting protein binds to collagen,
laminin, fibronectin, elastin, aminoglycans, proteoglycans or RGD. In still other
embodiments, the targeting protein binds to collagen. In yet other embodiments, the targeting
protein is SEQ ID NO: 25.
Also provided herein are methods and compositions for identifying a t or subject in
need of treatment for lesions and capable of benefitting from gene therapy treatment: a)
administering a gene therapy retroviral particle comprising an HSV-TK cleotide and
transducing cells from the patient with the polynucleotide encoding HSV-thymidine ; b)
ng the cells with a substrate of HSV-TK attached to a radioactive tracer; c) measuring the
ve amount of radioactive signal present in target tissue; d) fying patients wherein the
level of radioactively-labelled HSV-TK substrate is above a threshold; e) determining the
location of lesions in the t; and f) treating said patient or t with the gene therapy
retroviral particle comprising an HSV-TK polynucleotide when the measured radioactive signal
in the patient is above a certain old, and the location of the measured radioactive signal
correlates with lesions measured in step (e) of the patient.
Provided herein are methods and compositions for measuring the enzymatic activity of a
transduced HSV-thymidine, the method comprising: a) administering a gene therapy retroviral
particle sing an HSV-TK polynucleotide and transducing cells from the t with the
polynucleotide encoding HSV-thymidine kinase; b) treating the cells with a substrate of HSV-
TK attached to a radioactive ; and c) measuring the relative amount of ctive signal
present in target tissue.
In on, provided herein are methods and compositions for determining the level of a
tracer signal in a subject or patient after administration of a gene therapy particle, and selecting
the subject or patient for treatment with the gene therapy particle when the level of the tracer
signal is above a set threshold. In some embodiments, the tracer is a radioactive, luminescent or
a cent signal. In some embodiments, the radioactive tracer element is 18F, 64Cu, 99mTe,
11C, 14C, 1241, 1231, 1311, 150’ 13N and/or 82RbCl.
In yet other embodiments, provided herein are methods and compositions for
determining the level of a radiotracer signal in a subject or patient after administration of a
thymidine kinase gene therapy construct, and selecting the subject or patient for treatment with
the gene therapy construct when the level of the tracer signal is above a set threshold. In some
embodiments, the tracer is a radioactive tracer. In other embodiments, the radioactive tracer
element is 18F, 64Cu, 99mTe, 11C, 14C, 124I, 123I, 131I, 15O, 13N and/or 82RbCl. In yet other
embodiments, the radioactive tracer element is coupled to a nucleoside or synthetic nucleoside
target to form a radioactive . In some embodiments, the nucleoside target is FHBG (9-[4-
fluoro(hydroxymethyl)butyl] guanine), FHPG (EMT[3~fluoro~ l roxva—
propoxflmethyi,jbguanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l -(2'-
deoxy-2'-fluoro-l-B-D-arabinofi1ranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-
arabinofuranosyluracil), FMAU (fluoro-S-methyl-l-beta-D-arabinofuranosyluracil), FHOMP (6-
((l -fluorohydroxypropanyloxy)methyl)methylpyrimidine-2,4( l H,3H)—dione),
ganciclovir, val-ganciclovir, acyclovir, val-aciVlovir, penciclovir, radiolabeled pyrimidine with
oxy(hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)— and
-(3 -hydroxypropyl)-substituted pyrimidine derivatives bearing hydroxypropyl, acyclovirganciclovir
and penciclovir-like side chains. Preferably the ctive target is [18F]FHBG (9-
(4-18F-fluoro[hydroxymethyl]butyl)guanine).
Also ed herein are methods comprising: (a) determining the level of [18F]FHBG
signal in a subject; and (b) selecting the subject for treatment with a composition wherein the
level of FHBG is above a threshold level. In some embodiments, the threshold level is at least
about 2.0 SUV (standardized uptake values) or at least 20% above background signal on a PET
scan.
Additionally provided herein is a method comprising: (a) determining the level of
[18F]FHBG signal in a t; (b) excluding the subject from treatment with a ition
wherein the level of FHBG in the t is greater than about 2.0 SUV or greater than about
% above background signal on a PET scan; and (c) stering to said subject an anti-
cancer agent.
In some embodiments, the ion provides a method for identifying a subject that is
susceptible to a cancer treatment, the method sing: a) identifying expression of
[18F]FHBG in the subject; b) treating the subject.
Provided herein is a method of ing HSV-TK-FHBG (9-[4-fluoro
(hydroxymethyl)butyl]guanine), FHPG (9»{I3ufiuomw l ~hydro:<y~2»propoxy]iriethylkiyguanine),
FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D-
arabinofuranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-arabinofi.1ranosyluracil),
FMAU (fluoro-S-methylbeta-D-arabinofuranosyluracil), FHOMP (6-((l-fluoro
hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l H,3H)—dione), ganciclovir, valganciclovir
, acyclovir, val-aciVlovir, penciclovir, radiolabeled pyrimidine with 4-hydroxy
(hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)- and 5-(3-
hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-like,
ganciclovir-like and penciclovir-like side chains-mediated bystander effect, the method
comprising: a) transducing cells with a polynucleotide encoding HSV-TK and a first fluorescent
WO 53205
protein; b) transducing the cells with a polynucleotide encoding a second or bioluminescent
protein that is optically discernible from the first fluorescent or bioluminescent protein; c)
treating the cells with an agent that becomes xic upon being phosphorylated by HSV-TK;
and d) measuring the relative amount of expression of the first fluorescent n and the
second fluorescent protein. In one embodiment, step d) comprises a Perkin Elmer Plate reader, a
fluorimeter; a fluorescent activated cell sorter (FACS); a cellometer; or a spectrophotometer. In
another embodiment, step d) comprises measuring fluorescent output of the second fluorescent
or inescent protein in vivo in the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel es of the invention are set forth with particularity in the appended
claims. A better understanding of the es and advantages of the present invention will be
obtained by reference to the following detailed description that sets forth illustrative
embodiments, in which the principles of the invention are utilized, and the anying
drawings of which:
Figure 1 illustrates the structure of 9-[4-[18F]Fluoro(hydroxymethyl)butyl]guanine
([18F]FHBG) and its mechanism of inhibition.
Figure 2 is whole body coronal images of [18F]FHBG biodistribution in a healthy human
subject at four distinct time periods after 4.53 mCi iv injection of the [18F]FHBG.
Figure 3 is a schematic for a Phase IA clinical trial.
Figure 4 is a schematic for a Phase IB clinical trial.
Figure 5 is a patient’s response to ent with HSV-TK in AAV retroviral vector
particle.
Figure 6 measuring the response of a patient to administration of HBG in PET
Scan (top panel), CT Scan e panel) and fiJsion of signals (bottom panel).
Figure 7 is a cent image of the biodistribution of the HSV-TK retroviral vector
particles in s.
Figure 8 is a comparison of coronal three hour images of 5 mm slices in rats
administered Reximmune Cl and C2. The tumor on the left expressed Reximmune-C2 and on
the right is Reximmune-Cl.
Figure 9 is a graphical representation of Figure 8, showing the averages of the mean
within tumors for one and three hour images. Error bars are standard error of the averages. B12
is the Reximmune-C2 expressing tumor and A9 the Reximmune-Cl expressing tumor. C6 is the
native cell line tumor.
DETAILED DESCRIPTION OF THE INVENTION
Provided herein are methods and compositions for identifying a patient susceptible to
treatment with a gene therapy delivery system. Also provided herein are methods and
compositions for identifying subjects or patients that are capable of benefitting from gene
therapy treatment. er, provided herein are methods and compositions for fying
subjects or patients that express in sufficient quantities a therapeutic protein included in a gene
therapy construct. The identification of subjects or ts that are capable of expressing
sufficient quantities of a therapeutic protein allows a practitioner to screen and identify patients
that can benefit from a particular gene therapy treatment. By doing so, patients and subjects are
identified at an early stage that are capable of delivering anti-cancer agents via gene therapy
particles to treat, for example, primary and metastatic lesions.
In some embodiments, anti-cancer agents expressed from gene therapy constructs
included in viral particles can be administered to patients by enous infusion. In yet other
embodiments, anti-cancer agents expressed from gene therapy constructs can be administered to
patients via inter-arterial infilsion. In yet other embodiments, the viral particles containing anti-
cancer agents can be stered intra-tumoral. In still other embodiments, anti-cancer agents
expressed from gene y constructs can be selectively transduced in vitro into target cells.
In yet other embodiments, anti-cancer agents expressed from gene therapy constructs can
be targeted to primary and metastatic lesions, y delivering a tumor-killing gene to y
and metastatic s while sparing normal cells and tissues. In some embodiments, the
ing of gene therapy constructs is ic. In yet other embodiments, the targeting of gene
therapy constructs is to a cell-surface or extracellular protein. In some embodiments, the cell-
surface or extracellular protein is collagen. In yet other embodiments, the targeting of gene
therapy constructs is to a c protein expressed by tumor cells. Such anti-cancer agents
provide a powerful tool that can specifically target cancer cells, thereby mitigating the ed
side-effects of other known cancer ies.
In some ments, the gene therapy uct is a retrovirus. Retroviruses typically
have three common open reading frames, gag, pol, and env, which encode the matrix, gag and
nucleocapsid structural ns, encode enzymes including reverse transcriptase, integrase and
protease, and encode envelope proteins and transmembrane fusogenic proteins, respectively.
Typically, retroviral vector particles are produced by packaging cell lines that provide the
necessary gag, pol, and env gene products in trans. (Miller, et al., Human Gene Therapy, Vol. 1,
pgs. 5-14 (1990)). This approach results in the production of retroviral vector particles which
transduce mammalian cells, but are incapable of filrther replication after they have integrated
into the genome of the cell.
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In some ments, the retrovirus comprises at least one therapeutic protein or
payload delivered by the gene therapy construct. In some embodiments, the therapeutic protein
or payload is an enzyme. In yet other embodiments, the therapeutic protein or payload is
thymidine kinase. In still other embodiments, the thymidine kinase is HSV (herpes simplex
virus) thymidine kinase. In yet other embodiments, the thymidine kinase is HSV (herpes
simplex virus) thymidine -l.
In some embodiments, the HSV-TK gene therapy construct is optimized with respect to
maximal gene expression and tumor kill activity both in vitro and in viva including cancer gene
therapy. In some embodiments, the HSV-TK gene is codon-optimized. In still other
ments, the HSV-TK gene therapy construct is targeted to a specific tumor cell or tissue.
In yet other embodiments, the HSV-TK gene therapy construct is targeted to a cell-surface
protein specifically expressed in tumor cells. In still other ments, the HSV-TK gene
therapy construct is targeted to a cell-surface protein expressed in tumor tissue or cells. In other
embodiments, the HSV-TK gene therapy construct is targeted to collagen.
When sed in vivo in cells, HSV-TK enzymatically cleaves a co-adminstered
nucleoside agent, such as ganciclovir, lovir, val-ganciclovir, acyclovir and val-aciclovir,
and subsequently transforms the inistered agent into a cytotoxic agent. Mammalian
thymidine kinases are insensitive to these co-administered agents. Sensitivity to the cytotoxic
agent is therefore only conferred upon tumor cells after expression of the HSV-TK gene.
Ganciclovir is converted by the resulting HSV-TK to the monophosphorylated product, which is
then converted to di- and triphosphates by host kinases, leading to cytotoxicity and tumor cell
death. Viral thymidine kinase therapy has been previously shown to have promise in the
ent of several cancers, including s, hepatoma and melanoma.
HSV-TK also selectively orylates the nucleoside analogue of, for example, 9-[4-
18F-fluoro(hydroxymethyl)butyl]guanine ([18F]FHBG) (FIG. I), which cleaves the radioactive
tracer 18F from the FHBG molecule. HSV-TK expression can therefore be closely monitored
with positron emission tomography (PET) scans.
Accordingly ed herein are methods and compositions for determining the level of
a tracer signal in a subject or patient after administration of a gene y vector, and selecting
the subject or patient for treatment with the gene therapy vector when the level of the tracer
signal is above a set threshold. In some ments, the tracer is a radioactive, luminescent or
a fluorescent signal. In some embodiments, the radioactive tracer element is 18F, 64Cu, 99mTe,
11C, 14C, 1241’ 1231’ 1311’ 150, 13N and/or 82RbCl.
In yet other embodiments, provided herein are methods and compositions for
determining the level of a radiotracer signal in a subject or patient after administration of a
thymidine kinase gene therapy vector, and selecting the subject or patient for treatment with the
gene therapy vector when the level of the tracer signal is above a set threshold. In some
embodiments, the tracer is a ctive tracer. In other embodiments, the radioactive tracer
t is 18F, 64Cu, 99mTe, 11C, 14C, 124I, 123 I, 131I, 15O, 13N and/or 82RbCl. In yet other
embodiments, the radioactive tracer element is coupled to a side or synthetic nucleoside
target to form a radioactive target. In some embodiments, the nucleoside target is FHBG (9-[4-
fluoro(hydroxymethyl)butyl]guanine), FHPG (9%[3 ~fluoro~ l y—Qw
propoxyknethyfi)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-
deoxy-2'-fluoro-l-B-D-arabinofi1ranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-
arabinofuranosyluracil), FMAU (fluoro-S-methyl-l-beta-D-arabinofuranosyluracil), FHOMP (6-
((l -fluorohydroxypropanyloxy)methyl)methylpryrimidine-2,4( l H,3H)—dione),
ganciclovir, valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled pyrimidine with 4-
hydroxy(hydroxymethyl)butyl side chain at N-l (HHG-S -FEP) or 5-(2-)hydroxyethyl)- and 5-
(3 xypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-
ganciclovir and penciclovir-like side chains. ably the radioactive target is [18F]FHBG (9-
(4-18F-fluoro[hydroxymethyl]butyl)guanine).
Also provided herein are methods comprising: (a) determining the level of [18F]FHBG
signal in a subject; and (b) selecting the subject for treatment with a gene therapy composition
wherein the level of [18F]FHBG is at least about 2.0 SUV or at least 20% above background on a
PET scan.
Additionally provided herein is a method comprising: (a) determining the level of
[18F]FHBG signal in a t; (b) including the subject with treatment with a composition
wherein the level of FHBG in the subject is greater than about 2.0 SUV on PET scan; and (c)
administering to said subject an anti-cancer agent.
In some embodiments, the invention es a method for identifying a subject that is
susceptible to a cancer treatment, the method comprising: a) identifying expression of
HBG in the t; b) treating the t.
Provided herein is a method of measuring HSV-TK-mediated FHBG (9-[4-fluoro
(hydroxymethyl)butyl]guanine), FHPG (9~([3wfluoron l nhydroxynzpropoxfimethybguanin c),
FGCV (fluoroganciclovir), FPCV penciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D-
arabinofuranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-arabinofi1ranosyluracil),
FMAU (fluoro-S-methylbeta-D-arabinofuranosyluracil), FHOMP (6-((l-fluoro
hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l H,3H)—dione), ganciclovir,
valganciclovir, vir, valacivlovir, penciclovir, radiolabeled pyrimidine with 4-hydroxy
(hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)- and 5-(3-
2014/029600
hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-,
ganciclovir- and penciclovir-like side chains-mediated bystander effect, the method comprising:
a) ucing cells with a polynucleotide encoding HSV-TK and a first fluorescent protein; b)
transducing the cells with a polynucleotide encoding a second fluorescent or bioluminescent
protein that is optically discernible from the first fluorescent or bioluminescent protein; c)
treating the cells with an agent that becomes cytotoxic upon being phosphorylated by HSV-TK;
and d) ing the ve amount of expression of the first fluorescent protein and the
second fluorescent protein. In one embodiment, step d) comprises a Perkin Elmer Plate reader, a
fluorimeter; a fluorescent activated cell sorter (FAC S); a cellometer; or a spectrophotometer. In
r embodiment, step d) comprises measuring cent output of the second fluorescent
or bioluminescent protein in vivo in the subject.
Also provided herein are methods for determining the level of [18F]FHBG signal in a
subject and selecting the subject for treatment with a gene therapy composition wherein the level
of [18F]FHBG is at least about 2.0 SUV or at least 20% above background on a PET scan.
DEFINITIONS
Unless defined ise, all technical and scientific terms used herein have the same
g as is commonly understood by one of skill in the art to which the invention(s) belong.
All patents, patent applications, published applications and publications, k sequences,
websites and other hed materials referred to throughout the entire disclosure herein, unless
noted otherwise, are incorporated by reference in their entirety. In the event that there are a
plurality of definitions for terms herein, those in this section prevail. Where reference is made to
a URL or other such identifier or address, it understood that such identifiers can change and
particular information on the intemet can come and go, but equivalent information can be found
by ing the intemet. Reference thereto evidences the availability and public dissemination
of such information.
As used herein, “nucleic acid” refers to a polynucleotide containing at least two
covalently linked nucleotide or nucleotide analog subunits. A nucleic acid is generally a
ibonucleic acid (DNA), a ribonucleic acid (RNA), or an analog of DNA or RNA.
Nucleotide analogs are commercially available and methods of preparing polynucleotides
containing such tide analogs are known (Lin et al. (1994) Nucl. Acids Res. 0-5234;
Jellinek et al. (1995) Biochemistry 34: 1 1363-1 1372; Pagratis et al. (1997) Nature hnol.
:68-73). The nucleic acid is generally single-stranded, double-stranded, or a mixture thereof.
For purposes herein, unless specified otherwise, the nucleic acid is double-stranded, or it is
apparent from the context.
As used herein, “DNA” is meant to include all types and sizes ofDNA molecules
including cDNA, plasmids and DNA including modified nucleotides and nucleotide analogs.
As used herein, “nucleotides” include nucleoside mono-, di-, and triphosphates.
Nucleotides also include modified nucleotides, such as, but are not limited to, phosphorothioate
nucleotides and deazapurine nucleotides and other nucleotide analogs.
The term “polynucleotide” as used herein means a polymeric form of nucleotide of any
length, and includes ribonucleotides and deoxyribonucleotides. Such term also includes single-
and double-stranded DNA, as well as single-and double-stranded RNA. The term also includes
modified polynucleotides such as methylated or capped polynucleotides.
As used , the term “subject” refers to animals, , insects, and birds into which
the large DNA molecules are introduced. Included are higher organisms, such as mammals and
birds, including humans, primates, rodents, , pigs, rabbits, goats, sheep, mice, rats, guinea
pigs, cats, dogs, horses, chicken and others.
As used herein, “administering to a subject” is a procedure by which one or more
delivery agents and/or large nucleic acid molecules, together or separately, are introduced into or
applied onto a subject such that target cells which are present in the subject are eventually
contacted with the agent and/or the large nucleic acid molecules.
As used , “delivery vector” or “delivery vehicle” or “therapeutic vector” or
“therapeutic system” refers to both viral and non-viral particles that harbor and ort
exogenous c acid molecules to a target cell or tissue. Viral vehicles include, but are not
limited to, iruses, adenoviruses, lentiviral viruses, herpes viruses and adeno-associated
viruses. Non-viral vehicles include, but are not d to, microparticles, nanoparticles,
virosomes and liposomes. “Targeted,” as used herein, refers to the use of ligands that are
ated with the delivery vehicle and target the e to a cell or tissue. Ligands include,
but are not limited to, antibodies, receptors and collagen-binding domains.
As used herein, “delivery,” which is used interchangeably with “transduction,” refers to
the s by which exogenous nucleic acid les are transferred into a cell such that they
are located inside the cell. Delivery of c acids is a distinct process from expression of
c acids.
As used herein, a “multiple cloning site (MCS)” is a nucleic acid region in a plasmid that
contains multiple restriction enzyme sites, any of which can be used in conjunction with
standard recombinant technology to digest the vector. “Restriction enzyme digestion” refers to
catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific
locations in a nucleic acid molecule. Many of these ction enzymes are commercially
ble. Use of such enzymes is widely tood by those of skill in the art. Frequently, a
vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable
exogenous sequences to be ligated to the vector.
As used herein, n of replication” (often termed “ori”), is a specific nucleic acid
sequence at which replication is initiated. Alternatively an autonomously replicating sequence
(ARS) can be ed if the host cell is yeast.
As used herein, “selectable or screenable markers” confer an identifiable change to a cell
permitting easy identification of cells ning an sion vector. Generally, a selectable
marker is one that confers a property that allows for selection. A positive selectable marker is
one in which the ce of the marker allows for its selection, while a negative selectable
marker is one in which its presence ts its selection. An example of a positive selectable
marker is a drug resistance .
Usually the inclusion of a drug selection marker aids in the cloning and fication of
transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin,
DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers
conferring a phenotype that allows for the discrimination of transformants based on the
implementation of conditions, other types of markers including screenable markers such as GFP,
whose basis is colorimetric analysis, are also contemplated. In some embodiments, screenable
enzymes such as herpes simplex Virus thymidine kinase (tk) or chloramphenicol
acetyltransferase (CAT) are utilized. One of skill in the art would also know how to employ
immunologic markers, possibly in conjunction with FACS analysis. The marker used is not
believed to be important, so long as it is e of being expressed simultaneously with the
c acid encoding a gene product. Further examples of selectable and screenable markers are
well known to one of skill in the art.
As used herein, “expression” refers to the process by which nucleic acid is ated into
peptides or is transcribed into RNA, which, for example, can be translated into peptides,
polypeptides or proteins. If the nucleic acid is derived from genomic DNA, expression includes,
if an appropriate eukaryotic host cell or organism is ed, splicing of the mRNA. For
heterologous nucleic acid to be expressed in a host cell, it must initially be delivered into the cell
and then, once in the cell, ultimately reside in the nucleus.
As used , a “therapeutic course” refers to the periodic or timed administration of
the vectors disclosed herein within a defined period of time. Such a period of time is at least one
day, at least two days, at least three days, at least five days, at least one week, at least two
weeks, at least three weeks, at least one month, at least two months, or at least six months.
Administration could also take place in a chronic , z'.e., for an undefined period of time.
The periodic or timed administration includes once a day, twice a day, three times a day or other
set timed administration.
As used herein, the terms “co-administration, 3, nistered in combination with” and
their grammatical equivalents or the like are meant to encompass stration of the selected
therapeutic agents to a single patient, and are intended to include treatment regimens in which
the agents are administered by the same or different route of administration or at the same or
different times. In some embodiments, a therapeutic agent as disclosed in the present application
will be co-administered with other agents. These terms ass administration of two or
more agents to an animal so that both agents and/or their metabolites are present in the animal at
the same time. They include simultaneous administration in separate compositions,
administration at different times in separate compositions, and/or administration in a
composition in which both agents are present. Thus, in some embodiments, a therapeutic agent
and the other agent(s) are administered in a single composition. In some embodiments, a
therapeutic agent and the other agent(s) are admixed in the composition. In further
embodiments, a therapeutic agent and the other agent(s) are administered at separate times in
separate doses.
The term “host cell” denotes, for e, microorganisms, yeast cells, insect cells, and
mammalian cells, that can be, or have been, used as recipients for multiple constructs for
producing a delivery vector. The term includes the progeny of the original cell which has been
transfected. Thus, a “host cell” as used herein generally refers to a cell which has been
ected with an exogenous DNA sequence. It is understood that the progeny of a single
parental cell may not necessarily be tely identical in logy or in genomic or total
DNA complement as the al parent, due to natural, accidental, or deliberate on.
As used herein, “genetic therapy” involves the transfer of heterologous DNA to the
certain cells, target cells, of a , particularly a human, with a disorder or conditions for
which therapy or diagnosis is sought. The DNA is uced into the selected target cells in a
manner such that the heterologous DNA is expressed and a therapeutic product d thereby
is produced. In some embodiments, the heterologous DNA, directly or ctly, mediates
expression ofDNA that encodes the eutic product. In some embodiments, the
heterologous DNA encodes a product, such as a peptide or RNA that mediates, directly or
indirectly, expression of a therapeutic t. In some embodiments, genetic therapy is used to
deliver a nucleic acid encoding a gene product to replace a defective gene or supplement a gene
product produced by the mammal or the cell in which it is introduced. In some embodiments, the
introduced c acid encodes a therapeutic compound, such as a growth factor or inhibitor
thereof, or a tumor necrosis factor or inhibitor thereof, such as a receptor therefore, that is not
generally produced in the mammalian host or that is not produced in therapeutically effective
amounts or at a therapeutically useful time. In some embodiments, the logous DNA
encoding the therapeutic product is modified prior to introduction into the cells of the afflicted
host in order to enhance or otherwise alter the product or expression thereof.
As used herein, “heterologous nucleic acid sequence” is generally DNA that encodes
RNA and proteins that are not ly produced in vivo by the cell in which it is sed or
that mediates or encodes ors that alter expression of endogenous DNA by affecting
transcription, translation, or other regulatable biochemical processes. Any DNA that one of skill
in the art would recognize or er as heterologous or n to the cell in which it is
sed is herein encompassed by heterologous DNA. Examples of heterologous DNA
include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein
that confers drug resistance, DNA that encodes therapeutically effective substances, such as
anti-cancer agents, enzymes and hormones, and DNA that encodes other types of ns, such
as antibodies. In some embodiments, antibodies that are encoded by heterologous DNA is
secreted or expressed on the e of the cell in which the heterologous DNA has been
introduced.
As used herein, the term “thymidine kinase mutant” refers to not only the specific protein
described herein (as well as the nucleic acid sequences which encode these ns), but
derivatives thereof which may e various structural forms of the primary protein which
retain biological actiVity.
As used herein, “unmutated thymidine kinase” refers to a native or wild-type thymidine
kinase polypeptide sequence.
As used herein, “suicide gene” refers to a nucleic acid encoding a product, n the
product causes cell death by itself or in the present of other compounds.
As used herein, the term “mutated” or “replaced by another nucleotide” means a
nucleotide at a certain position is replaced at that position by a nucleotide other than that which
occurs in the unmutated or previously mutated sequence. That is, in some instances, specific
modifications may be made in different nucleotides. In some embodiments, the replacements
are made such that the relevant splice donor and/or acceptor sites are no longer present in a
gene.
As used herein, a “polar amino acid” refers to amino acid residues Asp(N), Cys (C), Gln
(Q), Gly (G), Ser (S), Thr (T) or Tyr (Y).
As used herein, a olar amino acid” refers to amino acid residues Ala (A), Ile (I),
Leu (L), Met (M), Phe (F), Pro (P), Trp (W), or Val (V).
As used herein, a “basic amino acid” refers to amino acid es Arg (R), His (H), or
Lys (K).
As used herein, an c amino acid” refers to amino acid residues Asp (D) or Glu (E).
GENE THERAPY
Gene therapy involves the transfer of heterologous DNA to certain cells of a mammal,
particularly a human, with a disorder or conditions for which therapy or diagnosis is sought. The
DNA is introduced into the selected target cells in a manner such that the logous DNA is
expressed and a therapeutic product encoded thereby is produced.
In some embodiments, the heterologous DNA, directly or indirectly, es
expression ofDNA that encodes the therapeutic t. In some embodiments, the
heterologous DNA encodes a product, such as a peptide or RNA that mediates, directly or
indirectly, expression of a therapeutic product. In some embodiments, the introduced nucleic
acid encodes a therapeutic compound, such as a grth factor or inhibitor f, or a tumor
necrosis factor or inhibitor thereof, such as a receptor therefore, that is not generally produced in
the mammalian host or that is not produced in therapeutically effective s or at a
therapeutically useful time.
Non-viral and viral methods have been used to deliver heterologous therapeutic DNA
into the cell, including viral vector particles derived from retrovirus, irus, adeno-
associated viral particles, herpes virus les, vaccinia virus, lentivirus, pox virus, Semliki
virus and pseudotyped viruses.
Accordingly, provided herein are viral constructs for gene transfer to cells either in
viva, ex vivo or in vitro for gene therapy. Such viral vector les include, but are not limited
to retroviral vector particles, adenoviral vector particles, adeno-associated virus particles, herpes
virus particles, pseudotyped viruses, lentiviral vector particles, pox virus vector particles,
vaccinia virus vector particles and ral vectors. ably, the viral vector particle is a
retroviral vector le.
RETROVIRAL CONSTRUCTS
In some embodiments, the vector particle employed for gene therapy use is a retroviral
vector particle. In still other embodiments, the retroviral vector particle is derived from Moloney
Murine Leukemia Virus and is of the LN series of vectors, such as those hereinabove mentioned,
and described r in Bender, et al., J. Virol., Vol. 61, pgs. 1639-1649 (1987) and Miller, et
al., Biotechniques, Vol. 7, pgs 980-990 (1989). Such vectors, have a portion of the packaging
signal derived from a mouse sarcoma virus, and a mutated gag initiation codon. The term
ed" as used herein means that the gag initiation codon has been deleted or altered such
that the gag protein or fragments or truncations thereof, are not expressed.
In some ments, the retroviral vector particle includes a modified envelope,
including at least one polynucleotide encoding at least one heterologous polypeptide to be
expressed in a desired cell. The heterologous polypeptide may, in one embodiment, be a
eutic agent. The therapeutic agent is thymidine kinase, more preferably HSV-TK.
In still other embodiments, therapeutic agents e, but are not limited to, grth
factors such as, for example, epidermal grth factor (EGF), vascular elial growth factor
(VEGF), erythropoietin, G-CSF, GM-CSF, TGFu, TGF-B, and fibroblast growth factor,
cytokines, including, but not limited to, interleukins and tumor necrosis s. Other
therapeutic agents include, but are not limited to, anticoagulants, latelet agents, antiinflammatory
agents, tumor suppressor proteins, clotting factors, including Factor VII, Factor
VIII and Factor IX, protein S, n C, antithrombin III and von Willebrand Factor.
In some embodiments, the polynucleotide encoding the eutic agent is under the
control of a suitable promoter. Suitable promoters which may be employed include, but are not
limited to, the retroviral LTR; the SV40 promoter; the cytomegalovirus (CMV) promoter; the
Rous Sarcoma Virus (RSV) promoter; the histone promoter; the polIII promoter, the B-actin
promoter; inducible ers, such as the MMTV promoter, the metallothionein promoter; heat
shock promoters; adenovirus promoters; the albumin promoter; the ApoAI promoter; B l 9
parvovirus promoters; human globin promoters; viral thymidine kinase promoters, such as the
Herpes Simplex Virus thymidine kinase promoter; retroviral LTRs; human growth hormone
promoters, and the MxIFN inducible promoter. The promoter also may be the native promoter
which controls the polynucleotide encoding the therapeutic agent.
The polynucleotides encoding the modified envelope polypeptide and the therapeutic
agent may be placed into an appropriate vector by genetic engineering techniques known to
those skilled in the art. When the modified vector is a retroviral vector particle, the
polynucleotides encoding the modified envelope polypeptide and the therapeutic agent are
placed into an appropriate iral plasmid vector.
The retroviral plasmid vector es one on more promoters. Suitable promoters
which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter;
and the human cytomegalovirus (CMV) er bed in Miller, et al., hniques, Vol.
7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic
cellular promoters including, but not limited to, the e, pol III, and B-actin promoters).
Other viral promoters which may be employed include, but are not limited to, adenovirus
promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter
will be apparent to those d in the art from the teachings ned herein.
WO 53205
In one embodiment, the retroviral plasmid vector, which includes a cleotide
encoding the modified envelope and a polynucleotide encoding a therapeutic agent, is employed
to transduce a packaging cell line to form a producer cell line, which will generate ious
retroviral vector particles. In one embodiment, the packaging cell line is a "pre-packaging" cell
line which includes polynucleotides encoding the gag and pol retroviral proteins, but not the
pe, or env, n. Such cell lines, upon transduction with the retroviral plasmid vector,
generates infectious iral les including the d, or chimeric, envelope and a
polynucleotide encoding the therapeutic agent. The vector may transduce the ing cells
through any means known in the art. Such means include, but are not limited to, electroporation,
and use of liposomes, such as hereinabove described, and CaPO4 precipitation. Such producer
cells generate infectious iral vector particles which include the modified envelope, the
wild-type retroviral envelope, a polynucleotide encoding the modified, or chimeric, envelope,
and a polynucleotide encoding a therapeutic agent.
In another embodiment, there is provided a ing cell which es a nucleic
acid sequence encoding a modified chimeric envelope in accordance with the invention, and
which may further include nucleic acid sequences encoding the gag and pol proteins. A producer
cell for generating viral particles which includes a modified envelope in accordance with the
invention is produced by introducing into such packaging cell either a retroviral vector particle
or a retroviral plasmid vector, in each case including a polynucleotide encoding a therapeutic
agent. The producer cell line thus generates infectious retroviral particles including the modified
chimeric envelope and the polynucleotide encoding the therapeutic agent.
TARGETED RETROVIRAL VECTOR RY
In some embodiments, ed herein are vector particles having a modified viral
surface protein, such as, for example, a modified viral envelope polypeptide, for targeting the
vector particle to an extracellular matrix component. The viral surface protein is modified to
include a targeting polypeptide including a binding region which binds to, an ellular
matrix ent.
In some ments, the targeting polypeptide is ed between two consecutively
numbered amino acid residues of the native (i.e., unmodified) receptor binding region of the
retroviral envelope. In yet other embodiments, amino acid residues of the receptor binding
region may be removed and replaced with the targeting polypeptide.
As an alternative to modifying the receptor binding region, or in addition to the modified
or binding region, the retroviral particles may have modifications in other regions of the
envelope protein such that other regions of the envelope may include the targeting polypeptide,
such as, for example, the secretory signal or "leader" sequence, the hinge region, or the body
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2014/029600
portion. Such modifications may include deletions or substitutions of amino acid residues in the
retroviral envelope wherein amino acid residues from s other than the receptor binding
region of the envelope are removed and replaced with the targeting polypeptide, or the targeting
polypeptide is placed between consecutively numbered amino acid residues of regions other
than the receptor binding region of the viral envelope.
In another alternative embodiment, the retroviral envelope, prior to modification thereof
to include the targeting polypeptide which binds to the extracellular matrix component, may be
an envelope which includes regions of different tropisms. For example, the retroviral envelope
may be a y Murine Leukemia Virus envelope which includes a gp70 protein having an
ecotropic portion and an amphotropic and/or xenotropic portion.
In general, the targeting polypeptide includes a binding region which binds to an
extracellular matrix component, including, but not limited to, collagen (including collagen Type
I and collagen Type IV), laminin, f1bronectin, elastin, aminoglycans, proteoglycans, and
ces which bind to fibronectin, such as arginine-glycine-aspartic acid, or RGD, sequences.
Binding regions which may be included in the targeting polypeptide include, but are not limited
to, polypeptide domains which are functional domains within von rand Factor or
derivatives thereof, wherein such polypeptide domains bind to collagen. In one embodiment, the
binding region is a polypeptide having the following structural formula: Trp-Arg-Glu-Pro-Ser—
t-Ala-Leu-Ser. (SEQ ID NO: 25).
In addition to the binding region, the targeting ptide may fiarther include linker
sequences of one or more amino acid residues, placed at the N-terminal and/or C-terminal of the
binding region, whereby such s increase rotational flexibility and/or minimize steric
hindrance of the modified envelope polypeptide.
HSV-TK
Thymidine kinase is a salvage pathway enzyme which phosphorylates l nucleoside
substrates as well as nucleoside analogues. Generally, ine kinase is used therapeutically
by administration of a nucleoside analogue such as ganciclovir or vir to a cell expressing
ine kinase, wherein the ine kinase phosphorylates the nucleoside analogue,
creating a toxic t capable of killing the cell.
Polynucleotide sequences encoding ous thymidine kinase as used herein may be
prepared from a wide variety of thymidine kinases. In some embodiments, the thymidine kinase
mutant is derived from Herpesvz’rz’dae thymidine kinase including, for example, both e
herpes viruses, and non-primate herpes viruses such as avian herpes viruses. Representative
examples of suitable herpes viruses include, for example, Herpes Simplex Virus (HSV) Type 1,
Herpes Simplex Virus Type 2, Varicella zoster Virus, marmoset herpes virus, feline herpes virus
type 1, pseudorabies virus, equine herpes virus type 1, bovine herpes virus type 1, turkey herpes
virus, Marek's disease virus, herpes virus saimir and Epstein-Barr virus.
IMPROVEMENTS TO TK GENE
Disclosed herein, in some embodiments, is a polynucleotide sequence ng HSV-
TK. In some embodiments, the polynucleotide sequence encodes a wild-type HSV-TK amino
acid sequence. In some embodiments, the polynucleotide ce s a mutated HSV-TK
amino acid ce.
Exemplary procedures that may be used in preparation of an optimized polynucleotide
ce ed herein include, but are not limited to: codon optimization; correction of
splice sites, removal of yrimidine tracts and excess GC content; addition of single Kozak
sequence, removal of unwanted Kozak sequences; inclusion of restriction sites for ning
into retroviral or other vectors; removal of nuclear localization sequences or addition of nuclear
export sequences; addition of mutation sequences; addition of double stop codon sequences;
addition of tags, s and fiJsion sequences; ation of sequence file for submission to
gene synthesis company; subcloning of synthesized gene into retroviral vectors; inclusion of
fluorescent protein genes into retroviral vectors; inclusion of selectable marker genes into
iral vectors; preparation of Maxiprep plasmid DNA; transfection of retroviral er or
other cells; lab, pilot or GMP scale production of retrovirus; transduction of target cells with
retrovirus; GCV or analogus pro-drug mediated cell kill assay; Hypoxanthine/Aminopterin/
Thymidine (HAT) selection assay; selectable marker drug selection procedure to produce
retroviral uced cell lines; fluorescent microscopy and photography to detect and document
retroviral transduced target cells; quantitative fluorescent detection of retroviral transduced
target cells; Western protein expression assay; other procedures and assays as needed for HSV-
TK analysis; or a combination thereof. Protocols for such methods are described herein, are
cially available or are described in the public literature and databases.
In some embodiments, described herein is a method of obtaining an improved HSV-TK
sequence. In some embodiments, the method comprises: a) correction and/or removal of splice
sites; and/or b) adjustment to a single Kozak ce. ally, in some embodiments, the
method fithher comprises inclusion of restriction sites for sub-cloning of the HSV-TK sequence.
Optionally, or in addition, in some embodiments, the method further comprises removal of
nuclear localization sequences.
Provided herein is a polynucleotide sequence encoding a mutated form of viral
thymidine kinase from human simplex virus (HSV-TK), wherein the encoded HSV-TK is
mutated at amino acid residue 25, 26, 32, 33, 167, 168, or a combination thereof, wherein the
polynucleotide sequence is mutated compared to a cleotide sequence of SEQ ID NO: 1 or
3. In such sequences, 1,2, 3,4, 5, 6, 7, 8, 9, 10, ll, 12, 13, or 14 mutations may be made.
ations may be conservative or non-conservative mutations. A mutation may be
made such that the encoded amino acid is modified to a polar, non-polar, basic or acidic amino
acid.
Provided herein is a polynucleotide sequence encoding a mutated form of thymidine
kinase from human x virus (HSV-TK), wherein the encoded HSV-TK es a nuclear
export sequence. Provided herein is a polynucleotide sequence encoding a mutated form of
thymidine kinase from human simplex virus (HSV-TK), where the encoded HSV-TK is
improved in on compared to wild-type HSV-TK and comprises A168H dmNES (Q
system = QMV enhancer properly fused to LTR promoter regions), where NES refers to a
r export sequence. In one embodiment, a mutant HSV-TKA168HdmNES is a mutant
HSV-TK gene for inclusion in Reximmune-C2. In one embodiment, the NES is derived from
MAP Kinase Kinase (MAPKK). In yet another embodiment, the polynucleotide sequence for
NES is CTGCAGAAAAAGCTGGAAGAGCTGGAACTGGATGGC (SEQ ID NO: 23). In
other embodiments, the NES polypeptide sequence is LQKKLEELELDG (SEQ ID NO: 24).
In some embodiments, disclosed herein are mutations to a polynucleotide ce
encoding Human x Virus Thymidine Kinase (HSV-TK) wherein mutations are not made
to the polypeptide sequence of wildtype HSV-TK.
Nucleotide positions are referred to by reference to a position in SEQ ID NO: 1
ype (wt) HSVl-TK tide sequence) or SEQ ID NO: 3 (HSV-TK in Reximmune-C
HSV-TK; SR39 mutant and 26S Mutation of the HSV-TK nuclear localization signal
(NLS)).
] In one embodiment, a Sac I-Kpn I ction sites bounding the clonable double
stranded oligonucleotides of the mutant HSV-TK SR39 mutant region is provided. See, for
example, SEQ ID NOS: 6 and 7, where the Sac I and Kpn I sites are shown on the left and right,
respectively. Bold, underlining illustrates the sites where mutations may be made. SEQ ID
NOS: 8 and 9 illustrate an exemplary sequence after cutting with Sac I and Kpn I. Exemplary
forward and reverse primers that may be used to make the mutations are shown as SEQ ID NOS:
and 11.
Exemplary optimized HSV-TK polynucleotide sequences are provided, for example, as
SEQ ID NOS: 12-22.
However, when such references are made, the invention is not intended to be limited to
the exact sequence as set out in SEQ ID NO: 1 or 3, but includes variants and derivatives
thereof Thus, identification of nucleotide ons in other thymidine kinase sequences are
contemplated (z'.e., identification of tides at positions which the skilled person would
consider to correspond to positions recited in SEQ ID NO: 1 or 3).
In some embodiments, nucleotides are replaced by taking note of the genetic code such
that a codon is changed to a different codon which codes for the same amino acid residue. In
some embodiments, nucleotides are replaced within coding regions of a HSV-TK encoding
nucleic acid sequence, yet the nucleic acid sequence maintains wild type HSV-TK protein
expression.
In such embodiments, 5/21 codons contain “C or G” in third position (24%); 0/21
codons contain “C” in third position (0 %); 5/21 codons contain “G” in third position (24%); and
16/21 codons n “A or T” in third position (76%).
In yet other ments, 16/21 codons contain “C or G” in third on (76%);
11/21 codons n “C” in third position (52 %); 5/21 codons contain “G” in third position
(24%); and 5/21 codons contain “A or T” in third position (24%).
In some embodiments, the following rare codons are not used or are avoided in the
coding region of a polynucleotide encoding HSV-TK, or a variant thereof: GCG for alanine;
CGA or CGT for arginine; TTA or CTA for leucine; CCG for proline; TCG for serine; ACG for
threonine; and GTA for valine.
In some embodiments, altering codons as described herein results in about 2%, about
%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 55%, about 60%, about 70%, about 75%, about 80%, about 85%, about
90%, about 95%, or greater percentage increase in ty.
In some embodiments, disclosed herein is a nucleic acid ce ng a
ine kinase wherein at least one nucleotide corresponding to a splice donor site is replaced
by another nucleotide. In fiarther embodiments, the nucleotides of the splice acceptor sites are
not altered. In some embodiments, at least one nucleotide corresponding to a splice acceptor site
is replaced by another nucleotide.
In some embodiments, disclosed herein is a nucleic acid sequence encoding a
thymidine kinase wherein at least one of the tides corresponding to splice donor site
nucleotides at positions 329 and 330 of a polynucleotide ce (e.g., SEQ ID NO: 1 or 3) is
replaced by another nucleotide. In some embodiments, both of the tides at positions 327
and 555 are replaced by other nucleotides. For example, on 327 may be mutated to an
amino acid residue selected from: G to A. Altemately, or in addition, position 555 may be
mutated to an amino acid e selected from: G to A. In one embodiment, the modified
HSV-TK has a polynucleotide sequence of SEQ ID NO: 18, in which HSV-TK was improved in
the following ways:
HSV-TK NESdmNLS Al68H, CO & SC
NES = nuclear export sequence from MAP Kinase Kinase (MAPKK)
dmNLS = double mutated HSV-TK Nuclear Localization Sequence
CO = codon optimized
SC = splice donor/acceptor site corrected at 327 and 555,
Underlined sequence
SEQ ID NO: 18
gtcaGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACT
GGATGGCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGC
CACAGCAACGGCAGCACCGCaCTGCGgCCaGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCG
AGATGCCCACCCTGCTGCGCGTGTACATCGACGGaCCaCACGGCATGGGCAAGACCAC
CACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATG
ACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCCAGCACCGCC
TGGACCAEGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATtACaAT
GGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCaCCaCACATCGGCGGCGAGGCCGGCAGC
AGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTTCGACCGgCACCCaATCGCaCACCTGCTGT
GCTACCCgGCaGCaCGCTACCTGATGGGCtcCATGACaCCaCAEGCCGTGCTGGCCTTCGTGGC
CCTGATCCCaCCaACaCTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCAC
CGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCA
TCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCG
CGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCaCCaCAGGGCGCCGAGCCaCAGAGCAAC
GCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTCACCCTGTTCCGgGCaCCaGAGCTGCTGG
CaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCG
CtCCATGCACGTGTTCATCCTGGACTACGACCAGtcaCCgGCCGGCTGCCGCGACGCCCTGCTG
CAGCTGACCAGCGGCATGGTGCAGACCCACGTGACaACaCCCGGCAGCATCCCaACaATCTGCG
ACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCT
Tgtca
In some embodiments, disclosed herein is a nucleic acid sequence encoding a
thymidine kinase wherein at least one of the nucleotides ponding to splice acceptor site
nucleotides at positions 554 and 555, or at least one of the nucleotides corresponding to splice
acceptor site nucleotides at positions 662 and 663, or at least one of the nucleotides
corresponding to splice acceptor sites at positions 541 and 542 of the Wild type sequence is
replaced by another nucleotide. For example, position 541 may be d to an amino acid
raflmflmmmmmeAPmMmmewmmWMMMmmmMMMWmmmhfid
from: G to A. Position 554 may be mutated to an amino acid residue selected from: G to A.
Position 555 may be mutated to an amino acid residue selected from: G to A. Position 662 may
be d to an amino acid residue selected from: G to A. Position 663 may be mutated to an
amino acid e selected from: G to A.
A Kozak sequence flanks the AUG start codon Within mRNA and influences the
ition of the start codon by eukaryotic ribosomes. In some embodiments, a polynucleotide
sequence encoding HSV-TK ses no more than one Kozak sequence. In some
embodiments, the Kozak sequence is upstream of the coding portion of the DNA ce. In
some embodiments, the Kozak sequence of a polynucleotide encoding HSV-TK is modified to
produce a Kozak sequence with a higher ncy of translation initiation in a mammalian cell.
In some embodiments, modification of the Kozak sequence does not produce an amino acid
substitution in the d HSV-TK polypeptide product. In some embodiments, modification
of the Kozak sequence s in at least one amino acid substitution in the encoded HSV-TK
polypeptide product. In one ment, the modified HSV-TK has a polynucleotide sequence
of SEQ ID NO: 18 or 22.
In some ments, the polynucleotide sequence encoding HSV-TK comprises at
least5,10,15,20,25,30,35,40,45,50,55,60,65,60,75,80,85,90,95,100(n1norecodon
substitutions. In some embodiments, the polynucleotide sequence encoding HSV-TK comprises
2H1east5,10,15,20,25,30,35,40,45,50,55,60,65,60,75,80,85,90,95,100(n1norecodon
substitutions, n the codon substitutions comprise the substitution of a codon having a
higher frequency of usage in a mammalian cell than the wild type codon at that position.
However, in some embodiments, less favored codons may be chosen for individual amino acids
depending upon the particular situation.
In some embodiments, the polynucleotide sequence encoding HSV-TK comprising at
least5,10,15,20,25,30,35,40,45,50,55,60,65,60,75,80,85,90,95,100(n1norecodon
subsfitufionshaslessflnu1about6596,6696,6796,6896,6996,7096,7196,7296,7396,7496,7596,
7696,7796,7896,7996,8096,8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,919&
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 or 3 wherein
the sequence identity is ined over the full length of the coding sequence using a global
alignment method. In some embodiments, the corresponding encoded polypeptide sequence has
atleast75 96,7696,7796,7896,7996,8096,8196,8296,8396,8496,8596,8696,8796,8896,8996,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-
TK amino acid ce, e.g., SEQ ID NO: 2 or 4.
In some embodiments, the polynucleotide sequence encoding HSV-TK comprises at
least5,10,15,20,25,30,35,40,45,50,55,60,65,60,75,80,85,90,95,100(n1norecodon
substitutions, n the codon substitutions comprise the substitution of a codon having the
highest ncy of usage in a mammalian cell for the wild type codon at that position. In some
embodiments, the corresponding encoded polypeptide sequence has at least 75 %, 76%, 77%,
7896,7996,8096,8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,9196,9296,939&
94%, 95%, 96%, 97%, 98%, 99%, or 100% ce identity to a HSV-TK amino acid
sequence, e.g., SEQ ID NO: 2 or 4.
In some embodiments, the polynucleotide sequence encoding HSV-TK comprises at
,10,l5,20,25,30,35,40,45,50,55,60,65,60,75,80,85,90,95,100(n1norecodon
substitutions, wherein the substituted codons have a frequency of usage greater than or equal to
aboutOJ,011,012,013,014,015,016,017,018,019,02,021,022,023,024,025,
7,028,029,03,031,032,033,034,035(ndnghmzhiynneentmdnnmnsthe
conespondnngencodedIXflypepfidesequencehasatleast7596,7696,7796,7896,7996,8096,
8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,9196,9296,9396,9496,9596,9696,
97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID
NO:2or4
In some embodiments, the polynucleotide sequence encoding HSV-TK comprises less
than about 45, 40, 35, 30, 25, 20 or fewer codons, wherein the codons have a frequency of usage
bssdmnaboutOl,011,012,013,014,015,016,017,018,019,02,02l,022,023,024
or 0.25. In some embodiments, the corresponding encoded polypeptide sequence has at least 75
96,7696,7796,7896,7996,8096,8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,9l96,
9296,9396,9496,9596,9696,9796,9896,9996,or10096sequenceidenfiuzu)aIISVCIliannno
acid sequence, e.g., SEQ ID NO: 2 or 4.
In some embodiments, the polynucleotide sequence encoding HSV-TK comprises at
least7896,7996,8096,8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,9196,9296,
93%, 94%, 95% or more of codons haVing a frequency of usage greater than or equal to about
01,011,012,013,014,015,016,017,018,019,02,02l,022,023,024,025,026,
0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, or higher. In some embodiments, the
conespondnngencodedIXflypepfidesequencehasatleast7596,7696,7796,7896,7996,8096,
8196,8296,8396,8496,8596,8696,8796,8896,8996,9096,9196,9296,9396,9496,9596,9696,
97%, 98%, 99%, or 100% sequence ty to a HSV-TK amino acid sequence, e.g., SEQ ID
NO:2or4
In some embodiments, the polynucleotide sequence encoding HSV-TK comprises at
least3596,3696,3796,3896,3996,4096,4196,4296,4396,4496,4596,4696,4796,4896,4996,
50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more of codons haVing
the highest ncy of usage in a ian cell. In some ments, the corresponding
encoded polypeptkk:sequenceliasatleast75 96,7696,7796,7896,7996,8096,8l96,8296,8396,
596,8696,8796,8896,8996,9096,9196,9296,9396,9496,9596,9696,9796,9896,9996,or
100% sequence identity to a HSV-TK amino acid sequence, e.g., SEQ ID NO: 2 or 4.
] In some embodiments, the polynucleotide sequence encoding HSV-TK comprises less
thmnabouu2l96,2096,l996,1896,l796,l696,l596,l496,l396,l296,1196,10960rlessof
codons haVing a frequency ofusage less than about 0.1, 0.1 l, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17,
0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24 or 0.25. In some embodiments, the polynuc1eotide
sequence comprises less than about21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%,
11%, 10% or less of codons having a frequency e less than about 0.1, 0.11, 0.12, 0.13,
0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24 or 0.25 in a mammalian cell. In
some embodiments, the corresponding d polypeptide sequence has at least 75 %, 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid
sequence, e.g., SEQ ID NO: 2 or 4.
In some embodiments, the polynuc1eotide sequence encoding HSV-TK comprises
codon tutions, wherein at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,
45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or more of the codons have been changed as ed to the wild type
sequence. In some embodiments, the polynuc1eotide sequence encoding HSV-TK comprises
codon substitutions, n at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,
40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more ofthe codons have been changed to a
codon haVing a higher frequency of usage in a ian cell as compared to the wild type
sequence. In some embodiments, the corresponding encoded polypeptide sequence has at least
75 %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence ty to a HSV-TK
amino acid sequence, e.g., SEQ ID NO: 2 or 4.
In some embodiments, the polynuc1eotide sequence encoding HSV-TK comprises
codon substitutions, wherein at least 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,
29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,
45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or more of the codons have been changed to a codon haVing the highest
frequency of usage in a mammalian cell as compared to the wild type sequence. In some
embodiments, the corresponding encoded polypeptide sequence has at least 75 %, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a HSV-TK amino acid
sequence, e.g., SEQ ID NO: 2 or 4.
The Viral thymidine kinase gene from the selected herpesvirus may be readily isolated
and d as described below, in order to construct nucleic acid les encoding a
thymidine kinase enzyme comprising one or more mutations which increases biological actiVity
of the thymidine kinase, as compared to unmutated wild-type thymidine kinase. The biological
activity of a thymidine kinase may be readily determined utilizing any of the assays known in
the art, including for example, determination of the rate of side analogue uptake or
determination of the rate of nucleoside or nucleoside analogue phosphorylation. In addition,
thymidine kinase mutants may be readily selected which are characterized by other biological
properties, such as thermostability and n stability.
In some embodiments, the polynucleotide sequence encoding HSV-TK is d to
remove or modify a predicted signal sequence. In some embodiments, the polynucleotide is
modified to remove or modify a nuclear localization sequence (NLS). In some embodiments,
the polynucleotide is modified to remove the nuclear localization sequence. In some
embodiments, the polynucleotide is modified to modify the NLS so that if no longer fianctions to
localize HSV-TK exclusively to the nucleus.
In some embodiments, a HSV-TK polypeptide sequence is mutated at amino acid
residues 167, 168, or both. In one example, the sequence is mutated at amino acid residue 167.
In another e, the sequence is mutated at amino acid e 168. In another example, the
sequence is mutated at amino acid residues 167 and 168. Amino acid residue 167 may be
mutated to serine or phenylalanine. Amino acid residue 168 may be mutated to ine, lysine,
cysteine, serine or phenylalanine. In some embodiments, a HSV-TK polypeptide sequence is
mutated at amino acid residues 25 and/or 26. In amino acid residues 25 and/or 26 may be
mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamate. In
some embodiments, the HSV-TK polypeptide sequence is mutated at amino acid residues 32
and/or 33. Amino acid residues 32 and/or 33 may be mutated to an amino acid chosen from the
group consisting of: glycine, serine, and glutamate. In some embodiments, the HSV-TK
ptide is mutated at amino acid residues 25, 26, 32, and/or 33. Amino acid residues 25, 26,
32, and/or 33, may be d to an amino acid chosen from the group ting of: glycine,
serine, and glutamate. Amino acid residue modifications may be made in comparison to a
polypeptide sequence of SEQ ID NOS: 2 or 4.
In accordance with the present ion, mutant thymidine kinase enzymes which are
encoded by the above-described nucleic acid molecules are provided, as well as vectors which
are capable of expressing such molecules. In some ments, expression vectors are
provided comprising a er operably linked to a nucleic acid molecule of the present
invention. In some embodiments, the vector is a Viral vector capable of directing the expression
of a nucleic acid molecule. Representative examples of such viral vectors e herpes
simplex viral vectors, iral vectors, adenovirus-associated viral vectors, pox vectors,
parvoviral vectors, virus vectors and iral vectors. In some embodiments, viral
vectors are provided which are capable of directing the expression of a nucleic acid molecule
which encodes a thymidine kinase enzyme comprising one or more ons, at least one of the
mutations encoding an amino acid substitution which increases a biological activity of
thymidine kinase, as ed to ted (z'.e. , wild-type) thymidine kinase.
In some ments, a nucleic acid molecule provided herein encodes a thymidine
kinase enzyme capable of phosphorylating a nucleoside analogue at a level at least 10% greater
than the level of phosphorylation of the nucleoside analogue by a ype thymidine kinase
enzyme. In some ments, the thymidine kinase enzyme is capable of orylating a
nucleoside analogue at a level at least 15%, at least 20%, at least 25%, at least 50%, at least
75%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500% greater than
the level of phosphorylation of the nucleoside analogue by a wild-type ine kinase
enzyme. Representative examples of suitable nucleoside analogues include gancyclovir,
acyclovir, famciclovir, buciclovir, penciclovir, valciclovir, trifluorothymidine, 1-[2-deoxy, 2-
fluoro, beta-D-arabino furanosyl]iodouracil, ara-A, araT 1-beta-D-arabinofuranoxyl thymine,
-ethyl-2'-deoxyuridine, 5-iodo-5'-amino-2, 5'-dideoxyuridine, idoxuridine, AZT, AIU,
dideoxycytidine and AraC. In some ments, the improved TK mutant lacks thymidine
kinase activity.
] In some embodiments, the KIII value thymidine kinase activity of a disclosed HSV-TK
mutant is at least 2.5 um. In some embodiments, the KIII value of a disclosed HSV-TK mutant is
at least 5 um, at least 10 um, at least 15 um, at least 20 um, at least 25 um, at least 30 um, at
least 40 um, at least 50 um, at least 60 um, at least 70 um, at least 80 um, at least 90 um, at least
100 um, at least 150 um, at least 200 um, at least 250 um, at least 300 um, at least 400 um, at
least 500 um, at least 600 um, at least 700 um, at least 800 um, at least 900 um, or at least 1000
um. In some embodiments, the percent KIn of a disclosed HSV-TK mutant compared to wild-
type HSV-TK is at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least
100%, at least 150%, at least 200%, at least 300%, or at least 500%.
Within one embodiment of the present invention, truncated derivatives of HSV-TK
mutants are provided. For example, site-directed mutagenesis may be readily performed in order
to delete the N—terminal 45 amino acids of a thymidine kinase mutant, thereby constructing a
truncated form of the mutant which retains its biological activity.
Mutations in tide sequences constructed for expression of derivatives of
ine kinase mutants should preserve the reading frame phase of the coding sequences.
Furthermore, the mutations will preferably not create complementary s that could
hybridize to produce secondary mRNA structures, such as loops or hairpins, which would
adversely affect translation of the or mRNA. Such derivatives may be readily constructed
using a wide variety of techniques, including those discussed above.
In some embodiments, a HSV-TK polypeptide ce is mutated at amino acid
residues 167, 168, or both. In one example, the sequence is mutated at amino acid residue 167.
In another example, the sequence is mutated at amino acid residue 168. In r example, the
sequence is mutated at amino acid residues 167 and 168. Amino acid residue 167 may be
mutated to serine or phenylalanine. Amino acid residue 168 may be d to histidine, lysine,
cysteine, serine or phenylalanine. In some embodiments, a HSV-TK polypeptide sequence is
mutated at amino acid residues 25 and/or 26. In amino acid residues 25 and/or 26 may be
mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamate. In
some embodiments, the HSV-TK polypeptide sequence is d at amino acid es 32
and/or 33. Amino acid residues 32 and/or 33 may be mutated to an amino acid chosen from the
group consisting of: glycine, serine, and glutamate. In some embodiments, the HSV-TK
polypeptide is mutated at amino acid residues 25, 26, 32, and/or 33. Amino acid residues 25, 26,
32, and/or 33, may be mutated to an amino acid chosen from the group consisting of: glycine,
serine, and glutamate. Amino acid residue modifications may be made in ison to a
polypeptide sequence of SEQ ID NOS: 2 or 4.
In accordance with the t invention, mutant thymidine kinase s which are
encoded by the above-described c acid molecules are provided, as well as vectors which
are capable of expressing such molecules. In some embodiments, expression vectors are
provided comprising a promoter operably linked to a nucleic acid molecule of the present
ion. In some embodiments, the vector is a viral vector capable of directing the expression
of a nucleic acid molecule. Representative examples of such Viral vectors include herpes
simplex Viral vectors, adenoviral vectors, adenovirus-associated Viral vectors, pox vectors,
parvoviral s, baculovirus vectors and retroviral vectors. In some embodiments, Viral
vectors are provided which are capable of directing the expression of a nucleic acid molecule
which s a thymidine kinase enzyme comprising one or more mutations, at least one of the
ons encoding an amino acid substitution which increases a biological activity of
thymidine kinase, as compared to unmutated (z'.e. , wild-type) thymidine kinase.
In some embodiments, a c acid molecule provided herein encodes a thymidine
kinase enzyme capable of phosphorylating a nucleoside analogue at a level at least 10% greater
than the level of phosphorylation of the nucleoside ue by a wild-type thymidine kinase
enzyme. In some ments, the thymidine kinase enzyme is capable of phosphorylating a
nucleoside analogue at a level at least 15%, at least 20%, at least 25%, at least 50%, at least
75%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500% greater than
the level of phosphorylation of the nucleoside analogue by a wild-type thymidine kinase
enzyme. Representative es of suitable nucleoside analogues include gancyclovir,
vir, lovir, buciclovir, penciclovir, valciclovir, trifluorothymidine, l-[2-deoxy, 2-
fluoro, beta-D-arabino furanosyl]iodouracil, ara-A, araT l-beta-D-arabinofuranoxyl thymine,
-ethyl-2'-deoxyuridine, 5-iodo-5'-amino-2, eoxyuridine, idoxuridine, AZT, AIU,
dideoxycytidine and AraC. In some embodiments, the improved TK mutant lacks thymidine
kinase activity.
Within one embodiment of the present invention, truncated derivatives of thymidine
kinase s are ed. For example, irected mutagenesis may be readily performed
in order to delete the N—terminal 45 amino acids of a ine kinase mutant, thereby
constructing a truncated form of the mutant which retains its biological activity.
Nhuafionsninudeofidesequencesconfirudedlbrexpmmsnniofdefiyafivesof
thymidine kinase mutants should preserve the g frame phase of the coding sequences.
Furthermore, the mutations will preferably not create complementary regions that could
hybridize to produce secondary mRNA structures, such as loops or hairpins, which would
adversely affect translation of the receptor mRNA. Such derivatives may be readily constructed
using a wide variety of ques, including those discussed above.
Using the methods described herein, the inventors determined that the majority of the
ates for zed HSV-TK genes appeared to be compatible with a retroviral expression
system and produce biologically useful retroviral titers.
Furthermore, the optimized HSV-TK genes which incorporated most of these
optimizations (SEQ ID NO: 18) exhibited ug GCV enzyme activity and selectivity for
their y to kill cancer cells following retroviral transduction delivery. The mutant HSV-TK
gene Al68H, which was codon optimized and splice corrected appeared to have the highest
GCV mediated cancer kill activity (SEQ ID NOs: l2, 16, 18, or 22). The same n of this
HSV-TK gene Al68H and mutated at amino acids 159-161 from LIF to IFL exhibited GCV
mediated cancer cell kill activity.
The mutant HSV-TK gene A167F (SEQ ID NOs: l3 ,l7, or 19) which was codon
optimized and splice corrected had very high GCV mediated cancer kill activity following
retroviral transduction delivery, but more surprisingly had NO thymidine kinase activity as
determined by expressing this gene following retroviral transduction delivery in 3T3 TK(-) cells
selected with HAT medium. To our knowledge, this is the most GCV selective HSV-TK
synthetic gene product for GCV activation which has no Thymidine activity ( HAT assay) ever
evaluated biologically.
] The double mutant HSV-TK gene Al67F + Al68H (SEQ ID NO: 14) unexpectedly
ablates both GCV and Thymidine enzyme activity by exhibiting very little GCV mediated
cancer kill activity and very little thymidine activity ( HAT assay),
The present inventors identified that it is possible to produce onal HSV-TK
fusions of genes such as bacterial ne deaminase, yeast cytosine deaminase, neomycin
phosphotransferase and include linker sequences and retain HSV-TK GCV mediated cancer cell
killing activity.
] In one embodiment, a codon optimized HSV-TK gene with GCV-mediated cancer
killing actiVity may be made which retains one or more nuclear localization sequences which is
not fused to one or more other therapeutic genes.
onal modifications to and/or evaluations of an optimized HSV-TK gene
described herein may include one or more of the following: removal ofknown nuclear
localization sequences within HSV-TK; increased pro-drug GCV enzyme actiVity and selectivity
for their y to kill cancer cells, evaluate the use of more tags, fusion proteins and linkers of
HSV-TK to other genes and ns, co-expression of HSV-TK optimized genes with other
optimized suicide and cancer killer genes in cancer cells, include optimized HSV-TK genes in a
Reximmune-C type iral vector system; production and testing of a Reximmune-C type
GMP product, or any combination thereof.
In one embodiment, a polynucleotide sequence described herein comprises a nuclear
export signal. For e, a polynucleotide sequence may comprise TKl68dmNES.
In another embodiment, a retroviral vector for use in the methods described herein
comprises one or more splice site modifications.
In another embodiment, a retroviral vector for use in the methods described herein
ses HSV-TK m (SEQ ID NO: 13).
In another embodiment, a retroviral vector for use in the methods described herein
comprises HSV-TK A168Hsm (SEQ ID NO: 12).
In another embodiment, a retroviral vector for use in the methods bed herein
comprises HSV-TK Al67de (SEQ ID NO: 17).
In another embodiment, a retroviral vector for use in the methods described herein
comprises HSV-TK Al68dm (SEQ ID NO: 16).
In another embodiment, a retroviral vector for use in the methods described herein
comprises HSV-TK A167de and an NES (SEQ ID NO: 19).
In another embodiment, a retroviral vector for use in the methods bed herein
comprises HSV-TK A168Hdm and an NES (SEQ ID NO: 18). In such an embodiment, the
sequence comprises HSV-TK A168H.
In another ment, a retroviral vector for use in the methods described herein
comprises a HSV-TK, n such vector ses an upgraded substrate binding domain and
a mNLS/NES set.
WO 53205
In another embodiment, a retroviral vector for use in the methods described herein
comprises a HSV-TK, wherein the vector comprises a selectable marker, a glowing gene and/or
one or more kill genes.
In another embodiment, a retroviral vector for use in the methods described herein
comprises at least two modifications.
Using the methods described herein, the inventors determined that the majority of the
optimized HSV-TK genes appeared to be compatible with a retroviral expression system and
produce ically useful retroviral titers.
The mutant HSV-TK gene A167F (SEQ ID NOs: l3 ,l7, or 19) which was codon
zed and splice corrected had very high GCV mediated cancer kill activity following
retroviral transduction delivery, but more singly had no thymidine kinase activity as
determined by expressing this gene ing retroviral uction delivery in 3T3 TK(-) cells
selected with HAT medium. This is highly GCV selective HSV-TK synthetic gene.
The double mutant HSV-TK gene Al67F + Al68H (SEQ ID NO: 14) exhibited very
little GCV mediated cancer kill activity and very little thymidine activity; thus, a proper double
mutant may have surprising null properties.
The present ors identified that it is possible to produce fianctional HSV-TK
fusions of genes such as bacterial cytosine deaminase, yeast cytosine deaminase, neomycin
phosphotransferase and include linker sequences and retain HSV-TK GCV mediated cancer cell
killing activity.
In one embodiment, a fully codon optimized HSV-TK gene with GCV-mediated
cancer killing activity may be made which retains one or more r localization sequences
which is not fused to one or more other therapeutic genes.
Additional modifications to and/or evaluations of an optimized HSV-TH gene
described herein may include one or more of the following: removal ofknown nuclear
localization sequences within HSV-TK; increased pro-drug GCV enzyme activity and selectivity
for their y to kill cancer cells, evaluate the use of more tags, fusion proteins and s of
HSV-TK to other genes and proteins, co-expression of HSV-TK optimized genes with other
optimized suicide and cancer killer genes in cancer cells, include optimized HSV-TK genes in a
Reximmune-C iral vector system; production and testing of a une-C GMP
product, or any combination thereof.
The therapeutic vectors may be administered alone or in conjunction with other
therapeutic treatments or active . Examples of other active agents that may be used
include, but are not d to, chemotherapeutic agents, anti-inflammatory agents, protease
inhibitors, such as HIV protease inhibitors, side analogs, such as AZT. In some
embodiments, the s of treatment fiarther comprise administering to the subject a
chemotherapeutic agent, a biologic agent, or radiotherapy prior to, contemporaneously with, or
subsequent to the administration of the therapeutic viral particles. One of skill in the art will
appreciate that the retroviral particles described herein may be administered either by the same
route as the one or more agents (6.g. the retroviral vector and the agent are both administered
intravenously) or by different routes (6.g. , the retroviral vector is administered intravenously and
the one or more agents are stered orally).
The dosage of the therapeutic viral particles lies preferably within a range of
circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary
within this range ing upon the dosage form ed and the route of administration
utilized. A therapeutically effective dose can be estimated initially from cell culture . A
dose may be formulated in animal models to achieve a circulating plasma concentration range
that includes the IC50 (z'.e., the concentration of the test compound which achieves a half-
maximal infection or a half-maximal tion) as determined in cell culture. Such information
can be used to more accurately determine useful doses in humans. Levels in plasma may be
measured, for example, by RT-qPCR or ddPCR methods.
An effective amount or eutically effective of the retroviral particles disclosed
herein to be administered to a subject in need of treatment may be determined in a variety of
ways. By way of example, the amount may be based on viral titer or efficacy in an animal
model. Alternatively the dosing regimes used in clinical trials may be used as general guidelines.
In some embodiments, the daily dose may be administered in a single dose or in
portions at various hours of the day. In some embodiments, a higher dosage may be required and
may be reduced over time when the optimal initial response is ed. In some embodiments,
treatment may be uous for days, weeks, or years, or may be at intervals with ening
rest periods. In some embodiments, the dosage is ed in accordance with other treatments
the individual may be receiving. However, the method of treatment is in no way limited to a
particular concentration or range of the retroviral particle and may be varied for each dual
being treated and for each derivative used.
Individualization of dosage may be required to e the maximum effect for a given
individual. In some embodiments, the dosage administered to an individual being treated varies
depending on the individual’s age, severity or stage of the disease and response to the course of
treatment. In some embodiments, clinical parameters for determining dosage include, but are not
d to, tumor size, alteration in the level of tumor markers used in al testing for
particular malignancies. In some embodiments, the treating physician determines the
therapeutically effective amount to be used for a given dual. In some embodiments, the
therapies disclosed herein are administered as often as necessary and for the period of time
judged necessary by the treating physician.
The therapeutic vectors, ing but not limited to the therapeutic iral particles
that are cally to the cell or system of interest, may be ically or ally (locally)
delivered to a subject in need of treatment. For example, the therapeutic vectors may be
systemically administered enously. Alternatively, the therapeutic s may also be
administered intra-arterially. The therapeutic vectors may also be administered topically,
intravenously, intra-arterially, intra-tumorally, intracolonically, intratracheally, intraperitoneally,
intranasally, intravascularly, intrathecally, intracranially, intramarrowly, intrapleurally,
intradermally, subcutaneously, intramuscularly, intraocularly, intraosseously and/or
intrasynovially or sterotactically. A combination of delivery modes may also be used, for
example, a patient may e the therapeutic vectors both systemically and regionally (locally)
to improve tumor responses with treatment of the therapeutic vectors.
In some embodiments, multiple therapeutic courses (e.g., first and second therapeutic
course) are administered to a subject in need of treatment. In some embodiments, the first
and/or second therapeutic course is administered intravenously. In other embodiments, the first
and/or second therapeutic course is administered via intra-arterial infusion, including but not
limited to infilsion through the hepatic artery, cerebral artery, coronary artery, pulmonary artery,
iliac artery, celiac trunk, gastric artery, splenic artery, renal artery, gonadal artery, subclavian
artery, vertebral artery, axilary artery, brachial artery, radial , ulnar , carotid artery,
femoral artery, or mesenteric artery and/or superior mesenteric artery. Intra-arterial
infusion may be accomplished using endovascular procedures, aneous procedures or open
surgical approaches. In some ments, the first and second therapeutic course may be
administered tially. In yet other embodiments, the first and second therapeutic course
may be administered simultaneously. In still other embodiments, the optional third therapeutic
course may be stered sequentially or simultaneously with the first and second therapeutic
courses.
In some embodiments, the therapeutic vectors disclosed herein may be administered in
conjunction with a sequential or concurrently administered therapeutic course(s) in high doses
on a cumulative basis. For example, in some embodiments, a patient in need thereofmay be
systemically administered, e.g., intravenously stered, with a first therapeutic course of at
least I x 109 TVP, at least I x 1010 TVP, at least I x 1011 TVP, at least I x 1012 TVP, at least I x
1013 TVP, at least I x 1014 TVP, at least I x 1015 TVP, at least I x 1016 TVP, at least I x 1017
TVP, at least I x 1018 TVP, at least I x 1019 TVP, at least I x 1020 TVP, at least I x 1021 TVP or
at least 1 x 1022 TVP delivery vector on a cumulative basis. The first therapeutic course may be
systemically administered. atively, the first therapeutic course may be administered in a
localized manner, e.g., intra-arterially, for example a patient in need thereofmay be
administered via intra-arterial infilsion with at least of at least 1 x 109 TVP, at least 1 x 1010
TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at
least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x
1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP delivery vector on
a cumulative basis.
In yet other embodiments, a subject in need thereofmay receive a ation, either
sequentially or concurrently, of systemic and intra-arterial infilsions administration of high doses
of delivery vector. For example, a patient in need fmay be first systemically administered
with at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012
TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015, at least 1 x 1016 TVP, at least
1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021
TVP or at least 1 x 1022 TVP delivery vector on a tive basis, ed by an onal
therapeutic course of intra-arterial infilsion, e.g., hepatic arterial infusion, administered delivery
vector of at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x 1011 TVP, at least 1 x
1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015 TVP, at least 1 x 1016
TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at least 1 x 1020 TVP, at
least 1 x 1021 TVP or at least 1 x 1022 TVP on a cumulative basis. In still another embodiment, a
patient in need thereofmay receive a combination of intra-arterial infilsion and ic
administration of delivery vector in high doses. For example, a patient in need thereofmay be
first be administered via intra-arterial infusion with at least of at least 1 x 109 TVP, at least 1 x
1010 TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014
TVP, at least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at
least 1 x 1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP delivery
vector on a cumulative basis, followed by an additional therapeutic course of systemically
administered delivery vector of at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at least 1 x
1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x 1015
TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019 TVP, at
least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP on a tive basis. The
therapeutic courses may also be administered simultaneously, z'.e., a therapeutic course of high
doses of delivery vector, for example, at least of at least 1 x 109 TVP, at least 1 x 1010 TVP, at
least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at least 1 x
1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x 1019
TVRatbwtlleoTVRatbwtlxINITVPormkmmlxlOnTVPddwmyvmmnona
2014/029600
cumulative basis, together with a therapeutic course of arterial infusion, e.g., hepatic
arterial infusion, administered delivery vector of at least of at least 1 x 109 TVP, at least 1 x 1010
TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at
least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x
1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP on a cumulative
basis.
In still other ments, a t in need thereofmay additionally receive, either
sequentially or concurrently with the first and second therapeutic courses, additional therapeutic
courses (e.g, third therapeutic course, fourth therapeutic course, fifth therapeutic course) of
cumulative dose of delivery vector, for example, at least of at least 1 x 109 TVP, at least 1 x 1010
TVP, at least 1 x 1011 TVP, at least 1 x 1012 TVP, at least 1 x 1013 TVP, at least 1 x 1014 TVP, at
least 1 x 1015 TVP, at least 1 x 1016 TVP, at least 1 x 1017 TVP, at least 1 x 1018 TVP, at least 1 x
1019 TVP, at least 1 x 1020 TVP, at least 1 x 1021 TVP or at least 1 x 1022 TVP delivery vector on
a cumulative basis.
In some embodiments, the subject in need of treatment is administered systemically
(e.g., intravenously) a dose of at least 1 x 1011 TVP, followed by the stration Via intra-
arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least 1 x 1011 TVP. In other
embodiments, the patient in need of treatment may be administered systemically (e.g.,
intravenously) a cumulative dose of at least 1 x 1012 TVP, followed by the administration Via
intra-arterial infilsion (e.g., c-arterial infusion) of a dose of at least 1 x 1012 TVP. In one
embodiment, the patient in need of treatment may be administered systemically (e.g.,
intravenously) a dose of at least 1 x 1013 TVP, followed by the administration Via intra-arterial
infusion (e.g., hepatic-arterial infusion) of a dose of at least 1 x 1013 TVP. In yet other
ments, the patient in need of treatment may be administered systemically (e.g.,
intravenously) a dose of at least 1 x 1014 TVP, concurrently with the administration Via intra-
arterial infusion (e.g., c-arterial infusion) of a dose of at least 1 x 1014 TVP. In still other
embodiments, the patient in need of treatment may be administered systemically (e.g.,
intravenously) a dose of at least 1 x 1015 TVP, together with the stration Via arterial
infusion (e.g., hepatic-arterial infusion) of a dose of at least 1 x 1015 TVP. In yet other
embodiments, the patient in need of treatment may be administered systemically (e.g.,
intravenously) a dose of at least 1 x 1016 TVP, concurrently with the administration Via intra-
arterial infusion (e.g., hepatic-arterial infusion) of a dose of at least 1 x 1016 TVP. In still other
embodiments, the patient in need of treatment may be administered systemically (e.g.,
enously) a dose of at least 1 x 10137TVP, together with the administration Via intra-arterial
infusion (e.g., hepatic-arterial infusion) of a dose of at least 1 x 1017 TVP.
A subject in need of treatment may also be administered, either systemically or
localized (for example intra-arterial on, such as hepatic arterial infusion) a therapeutic
course of delivery vector for a defined period of time. In some embodiments, the period of time
may be at least one day, at least two days, at least three days, at least four days, at least five
days, at least six days, at least seven days, at least one week, at least two weeks, at least three
weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least
eight weeks, at least 2 months, at least three months, at least four months, at least five months, at
least six , at least seven months, at least eight months, at least nine months, at least ten
months, at least eleven , at least one year, at least two years, at least three years, at least
four years, or at least five years. Administration could also take place in a chronic , z'.e.,
for an ed or indefinite period of time.
Administration of the therapeutic vector may also occur in a periodic manner, 6.g. at
least once a day, at least twice a day, at least three times a day, at least four times a day, at least
five times a day. Periodic administration of the delivery vector may be dependent upon the time
of delivery vector as well as the mode of administration. For example, parenteral administration
may take place only once a day over an extended period of time, whereas oral administration of
the delivery vector may take place more than once a day wherein administration of the delivery
vector takes place over a shorter period of time.
In one embodiment, the subject is d to rest 1 to 2 days between the first
therapeutic course and second eutic course. In some embodiments, the subject is allowed
to rest 2 to 4 days between the first therapeutic course and second therapeutic course. In other
ments, the subject is allowed to rest at least 2 days between the first and second
therapeutic course. In yet other embodiments, the subject is allowed to rest at least 4 days
between the first and second therapeutic course. In still other embodiments, the subject is
allowed to rest at least 6 days between the first and second therapeutic course. In some
embodiments, the subject is allowed to rest at least 1 week between the first and second
therapeutic course. In yet other embodiments, the subject is allowed to rest at least 2 weeks
between the first and second eutic course. In one embodiment, the subject is allowed to
rest at least one month between the first and second therapeutic . In some embodiments,
the subject is allowed to rest at least 1-7 days between the second therapeutic course and the
optional third therapeutic course. In yet other embodiments, the subject is allowed to rest at
least 1-2 weeks between the second therapeutic course and the optional third therapeutic course.
DIAGNOSING A PATIENT THAT IS TIBLE TO THYMIDINE KINASE GENE
THERAPYTREATMENT
Imaging tests, ing the use of radioactive tracers, contrast imaging technology and
other imaging technology can be used to identify ts that are susceptible to gene therapy
treatment, including thymidine kinase gene therapy treatment, and thus more likely to benefit
from such therapeutic measures.
In a red embodiment, positron emission tomography (PET) scans are used to
identify patients capable of ucing retroviral vector particles containing thymidine kinase
constructs for sion in vivo. A PET scan produces 3-dimensional images of fianctional
processes in the body by ing pairs of gamma rays emitted indirectly by ctive tracers
placed on a biological active molecule. PET scans detect energy emitted by positively charged
particles (positrons).
Patients administered a retroviral vector particle containing thymidine kinase
polynucleotide are co-administered a radiotracer agent capable of being d by expressed
thymidine kinase. An e is [18F]FHBG [18F]fiuoro
xymethyl)butyl]guanine), which is a high affinity substrate for HSV-TK enzyme, with
relative low affinity for mammalian TK enzymes. See Yaghoubi and Gambhir, Nat. Protocol
1:3069-75 (2006); Green et al., J. Nucl. Med. 45:1560-70 (2004). [18F]FHBG is phosphorylated
by HSVl-TK or HSVl-sr39TK, which is then trapped within cells expressing thymdine kinase
enzyme. Cleavage of this substrate in vivo in patients administered iral s containing
a thymidine kinase polynucleotide, including the mutated and/or optimized thymidine kinase
constructs bed herein, thus indicates efficient transduction of the retroviral vector particles
by the subjects and patients, and thus an initial of patient or subject tibility to thymidine
kinase-mediated gene therapy.
Alternatively, other methods for mearuing viral TK activity include chemical exchange
saturation transfer magnetic resonance imaging with 5-methyl-5,6-dihydrothymidine and related
compounds.
Accordingly, in some embodiments disclosed herein, provided are methods and
compositions for detecting thymidine kinase expression in patients administered a retroviral
viral particle containing a polynucleotide encoding a thymidine kinase protein. In some
ments, the thymidine kinase is derived from vz'rz'dae thymidine kinase. In some
embodiments, the thymidine kinase is HSV-TK. In other embodiments, the thymidine kinase is
HSV-TKl. In still other embodiments, the thymidine kinase is an optimized version of HSV-
In some embodiments, the HSV-TK gene is codon optimized for efficient expression
and/or transduction. In other embodiments, the amino us of the thymidine kinase is altered
to remove or ate the nuclear localization sequence (NLS) of the viral thymidine kinase
sequence. In other embodiments, the thymdine kinase nucleotide sequence es a nuclear
export sequence (NES) attached to the amino terminus. In some ments, the nuclear
export sequence is LQKKLEELELDG (SEQ ID NO: 24).
] In yet other embodiments, the thymidine kinase coding sequence is mutated to increase
substrate g of the expressed thymidine kinase n. In still other ments, the
thymidine kinase coding sequence es an Al 68H mutation.
Other examples of substrates targeted by thymidine kinase, including HSV-TK protein,
include: FHPG (9v(_[3~flum‘o~lhydro};y~2—pmpo><y]mctltyflguamine), FGCV (fluoroganciclovir),
FPCV (fluoropenciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D-arabinofi.1ranosyl)-5 -iodouracil),
FEAU (fluoroethyl- l -beta-D-arabinofuranosyluracil), FMAU (fluoromethylbeta-D-
arabinofuranosyluracil), FHOMP -fluorohydroxypropanyloxy)methyl)
pryrimidine-2,4(lH,3H)-dione), ganciclovir, valganciclovir, acyclovir, valacivlovir,
penciclovir, radiolabeled pyrimidine with 4-hydroxy-3 -(hydroxymethyl)butyl side chain at N-l
(HHGFEP) or 5-(2-)hydroxyethyl)- and 5-(3-hydroxypropyl)-substituted pyrimidine
derivatives bearing 2,3-dihydroxypropyl, acyclovir-ganciclovir and penciclovir-like side chains.
Examples of radiotracers that can be used to determine if a therapeutic protein, such as
thymidine kinase, is expressed in an individual treated with the thymidine kinase gene therapy
vectors described herein may also include 18F, 64Cu, 99mTe, 11C, 14C, 124I, I, 131I, 15O, 13N
and/or 82RbCl.
Clinical trials for 9—[438F—fluoro~.'§-{hydroxymethyDbutyl]guanine (FHBG) for PET
can be found on the following website: www.clinicaltrials.gov. Methods of measuring FHBG
with PET in clinical use can be found in clinical trials NCT0087l702, NCT00185 848 and
NCT01082926.
Briefly patients will receive a dose of therapeutic drug product on Day 1. On Day 3 to
6, preferably day 4, or at a time period after receiving the dose of therapeutic drug product
encoding a modified HSV-TK as disclosed herein, they will be infiJsed with [18F]FHBG
intravenously and imaged by PET scan 1-5 hours later, preferably 0.5, 1.0, 1.5, 2, 2.5, 3.0, 3.5
or 4.0 hours later or other riate time after administration for ng, for lation in
the tumor sites where HSV-l TK is shown to be expressed. Patients that show uptake of the
FHBG will be enrolled in the trial; those that do not will be excluded as disclosed herein. The
amount ofFHBG will be ined and based on previous studies. Additional protocols for
FHBG/PET may be found, for example, in references 15-39 below.
WO 53205
Accordingly provided herein is are methods and compositions for measuring a tagged
substrate of thymidine kinase, including HSV-TK, including FHBG (9-[4-fluoro
(hydroxymethyl)butyl]guanine), FHPG (gflffiufiuorow 1 ~hydro:<y~2»propoxy}iriethylkiyguanine),
FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D-
arabinofuranosyl)iodouracil), FEAU -S-ethyl-l-beta-D-arabinofi1ranosyluracil),
FMAU (fluoro-S-methylbeta-D-arabinofuranosyluracil), FHOMP -fluoro
hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l dione), ganciclovir,
valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled pyrimidine with 4-hydroxy
(hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)- and 5-(3-
hydroxypropyl)-substituted pyrimidine derivatives g 2,3-dihydroxypropyl, acyclovirganciclovir
and penciclovir-like side chains, the method comprising: a) transducing cells with a
polynucleotide encoding HSV-TK; b) treating the cells with a substrate of HSV-TK attached to
a radioactive tracer; and c) measuring the relative amount of radioactive signal present in target
tissue. In one embodiment, step c) comprises measuring the output of the radioactive tracer in
vivo in the subject using PET (positron emission tomography) scanning.
Also provided herein is are methods and compositions for identifying a patient or
subject capable of benefitting from gene y treatment, comprising measuring a tagged
substrate of thymidine kinase, including HSV-TK, including FHBG (9-[4-fluoro
(hydroxymethyl)butyl]guanine), FHPG (9ufi3~fluorou l uhydroxyulZmpropoxylmethyl)guanine),
FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D-
arabinofuranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-arabinofi1ranosyluracil),
FMAU (fluoro-S-methyl- 1 D-arabinofuranosyluracil), FHOMP (6-((l-fluoro
hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l H,3H)—dione), ganciclovir,
valganciclovir, acyclovir, valacivlovir, lovir, radiolabeled pyrimidine with 4-hydroxy
(hydroxymethyl)butyl side chain at N-l (HHG-S-FEP) or 5-(2-)hydroxyethyl)- and 5-(3-
hydroxypropyl)-substituted pyrimidine derivatives bearing hydroxypropyl, acyclovir-,
ganciclovir- and penciclovir-like side . In some embodiments, the method comprises: a)
administering a gene therapy retroviral particle comprising an HSV-TK polynucleotide and
transducing cells with the polynucleotide encoding HSV-thymidine kinase; b) treating the cells
with a substrate of HSV-TK attached to a radioactive tracer; c) measuring the relative amount of
radioactive signal present in target tissue; and d) identifying patients where the level of
radioactively-labelled HSV-TK ate is above a set threshold. In one ment, step c)
comprises measuring the output of the radioactive tracer in vivo in the subject using PET
(positron emission tomography) scanning. In some embodiments, patients e of benefitting
from a gene y protocol include patients or ts exhibiting a level above a set threshold
on a PET scan. In some embodiments, the level of ctive HSV-TK substrate is at least
about 2.0 SUV or at least 20% above background on a PET scan. In some embodiments, the
level of radioactive HSV-TK substrate is at least about 1.9 SUV or at least 20% above
background on a PET scan. In yet other embodiments, the level of radioactive HSV-TK
substrate is at least about 1.0 SUV, about 1.5 SUV, about 2.0 SUV or about 2.5 SUV or more, or
at least 10% above ound, at least 20% above background, at least 30% above background,
at least 40% above background or at least 50% above background or more on a PET scan.
In some embodiments, ed herein are methods and compositions for identifying a
patient or subject in need of treatment for benign or metastatic s and capable of tting
from gene therapy treatment. In some embodiments, the method for identifying patients capable
of benefitting from gene therapy for the treatment of benign or metastatic lesions include
ing a tagged substrate of thymidine kinase, including , including FHBG (9-[4-
fluoro(hydroxymethyl)butyl]guanine), FHPG (9~([3mfluoron 1 nhydroxyn2~
propoxyjmeihyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (1-(2'-
deoxy-2'-fluoroB-D-arabinofi1ranosyl)iodouracil), FEAU (fluoroethylbeta-D-
arabinofuranosyluracil), FMAU methylbeta-D-arabinofuranosyluracil), FHOMP (6-
((1 -fluorohydroxypropanyloxy)methyl)methylpryrimidine-2,4(1H,3H)—dione),
ganciclovir, valganciclovir, acyclovir, valacivlovir, lovir, radiolabeled pyrimidine with 4-
hydroxy(hydroxymethyl)butyl side chain at N-l (HHG-5 -FEP) or 5-(2-)hydroxyethyl)- and 5-
(3 -hydroxypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-,
ganciclovir- and penciclovir-like side chains after administration of the gene therapy treatment.
In some embodiments, the method comprises: a) administering a gene y retroviral particle
comprising an HSV-TK polynucleotide and transducing cells with the polynucleotide encoding
HSV-thymidine kinase; b) treating the cells with a substrate ofHSV-TK attached to a
radioactive tracer; c) measuring the relative amount of radioactive signal present in target tissue;
d) identifying patients where the level of ctively-labelled HSV-TK substrate is above a set
threshold; and e) treatment said patient or subject with the gene therapy retroviral particle. In
one embodiment, step c) comprises measuring the output of the ctive tracer in vivo in the
subject using PET (positron emission tomography) scanning. In some embodiments, patients
capable of benefitting from a gene therapy protocol include patients or subej cts exhibiting a
level above a set threshold on a PET scan. In some embodiments, the level of HBG signal
is at least about 2.0 SUV or at least 20% above background on a PET scan. In some
embodiments, the level of radioactive HSV-TK substrate is at least about 1.9 SUV or at least
% above background on a PET scan. In yet other embodiments, the level of ctive HSV-
TK substrate is at least about 1.0 SUV, about 1.5 SUV, about 2.0 SUV or about 2.5 SUV or
more, or at least 10% above background, at least 20% above background, at least 30% above
background, at least 40% above background or at least 50% above background or more on a
PET scan.
Also provided herein are methods sing: (a) determining the level of [18F]FHBG
signal in a subject; and (b) ing the subject for treatment with a composition wherein the
level of FHBG is at least about 2.0 SUV or at least 20% above background on a PET scan. In
some embodiments, the level of radioactive HSV-TK substrate is at least about 1.9 SUV or at
least 20% above background on a PET scan. In yet other embodiments, the level of radioactive
HSV-TK ate is at least about 1.0 SUV, about 1.5 SUV, about 2.0 SUV or about 2.5 SUV
or more, or at least 10% above background, at least 20% above background, at least 30% above
background, at least 40% above background or at least 50% above background or more on a
PET scan.
Additionally provided herein is a method comprising: (a) ining the level of
[18F]FHBG signal in a subject; (b) excluding the subject from treatment with a composition
n the level of FHBG in the subject is greater than about 2.0 SUV or at least above 20%
above background on a PET scan; and (c) administering to said subject an anti-cancer agent. In
some embodiments, the level of radioactive HSV-TK substrate is at least about 1.9 SUV or at
least 20% above background on a PET scan. In yet other embodiments, the level of radioactive
HSV-TK substrate is at least about 1.0 SUV, about 1.5 SUV, about 2.0 SUV or about 2.5 SUV
or more, or at least 10% above background, at least 20% above background, at least 30% above
background, at least 40% above background or at least 50% above background or more on a
PET scan.
In some embodiments, the invention provides a method for identifying a subject that is
susceptible to a cancer treatment, the method comprising: a) identifying expression of
[18F]FHBG in the subject; b) treating the subject.
Also provided herein are compositions and methods of ing -mediated
FHBG (9-[4-fluoro(hydroxymethyl)butyl]guanine), FHPG (9~([3~fiuoro—l oxy—2—
propoxy]methyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU -
deoxy-2'-fluoroB-D-arabinofi1ranosyl)iodouracil), FEAU (fluoroethylbeta-D-
arabinofuranosyluracil), FMAU (fluoromethylbeta-D-arabinofuranosyluracil), FHOMP (6-
((1 -fluorohydroxypropanyloxy)methyl)methylpryrimidine-2,4(1H,3H)—dione),
lovir, valganciclovir, acyclovir, valacivlovir, penciclovir, radiolabeled pyrimidine with 4-
hydroxy(hydroxymethyl)butyl side chain at N-l (HHG-5 -FEP) or 5-(2-)hydroxyethyl)- and 5-
(3 -hydroxypropyl)-substituted pyrimidine derivatives bearing hydroxypropyl, acyclovir-,
ganciclovir- and penciclovir-like side chains phosphorylation using a fluorescent imaging
system. In some embodiments, the method comprises: a) transducing cells with a polynucleotide
encoding HSV-TK and a first fluorescent protein; b) transducing the cells with a polynucleotide
ng a second fluorescent or bioluminescent protein that is optically discernible from the
first fluorescent or bioluminescent protein; c) treating the cells with an agent that becomes
cytotoxic upon being phosphorylated by ; and d) measuring the relative amount of
expression of the first fluorescent protein and the second fluorescent protein. In one
embodiment, step d) comprises a Perkin Elmer Plate reader, a eter; a fluorescent
activated cell sorter (FAC S); a cellometer; or a spectrophotometer. In another embodiment, step
d) comprises measuring fluorescent output of the second fluorescent or bioluminescent protein
in vivo in the subject using a fluorescent or inescent imaging system.
THYMIDINE KINASE STIC USES
In some embodiments, disclosed herein is a method of selecting a t for y,
or for excluding a patient from therapy. In one embodiment, the thymidine kinase gene therapy.
In other embodiments, the thymidine kinase is herpes simplex virus thymidine kinase (HSV-
TK). In yet other embodiments, the thymidine kinase is HSV-TKl.
As described herein, HBG and other HSV-TK labeled substrates may be used as
a marker for selection or exclusion of subjects for gene therapy. For example, cells sing
HSV-TK after administration of a iral vector particle comprising a cleotide
encoding HSV-TK will ively phosphorylate the nucleoside analogue 9-[4-fluoro
(hydroxymethyl) butyl]guanine ([18F]FHBG). See, e.g., Yaghoubi and Gambhir, Nat. Protocols
1:3069-75 (2006). [18F]FHBG imaging above a certain threshold can then be used to fy
HSV-TK positive cells and to select or exclude a t for gene therapy.
Accordingly, in one embodiment, a subject is administered a gene therapy
composition, wherein the gene therapy composition s an HSV-TK polypeptide. The
subject is stered a labeled nucleoside analog HSV-TK substrate after a predetermined
period of time, and monitored until background of the labeled substrate is reached in the subject.
The label activity is measured, and compared against a scan detecting lesions in the subject. If
the imaging activity: 1) is above a set old; and 2) correlates with the lesion location in the
subject, then the subject is a candidate for HSV-TK gene therapy.
In some embodiments, the nucleoside analog is FHBG (9-[4-fluoro
(hydroxymethyl)butyl]guanine), FHPG (9mfi3nfluorom l “hydroxywl«propoxyjmethyflguanine),
FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (l-(2'-deoxy-2'-fluoro-l-B-D-
arabinofuranosyl)iodouracil), FEAU (fluoro-S-ethyl-l-beta-D-arabinofi1ranosyluracil),
FMAU (fluoro-S-methylbeta-D-arabinofuranosyluracil), FHOMP (6-((l-fluoro
hydroxypropanyloxy)methyl)-5 -methylpryrimidine-2,4( l H,3H)—dione), ganciclovir,
valganciclovir, acyclovir, valacivlovir, lovir, radiolabeled pyrimidine with 4-hydroxy
(hydroxymethyl)butyl side chain at N-l (HHGFEP) or 5-(2-)hydroxyethyl)- and 5-(3-
hydroxypropyl)-substituted pyrimidine derivatives g 2,3-dihydroxypropyl, acyclovir-,
lovir- and penciclovir-like side chains. In some embodiments, the label is 18F, 64Cu,
99mTe, 11C, 14C, 124I, 123I, 131I, 15O, 13N and/or 82RbCl. ably, the labeled nucleoside analog
HSV-TK substrate is [18F]FHBG ((9-[4-18F-fluoro(hydroxymethyl)butyl]guanine).
In some embodiments, the nce pattern in a subject determines the length of time
delay for measuring background and determining, for example, [18F]FHBG imaging ty. In
humans, for example, [18F]FHBG ound rapidly decreases from most tissues outside the
lower abdomen, as seen in As seen, between 7.6 and 42.6 minutes after administration
of [18F]FHBG, levels are high in the liver, kidneys and bladder. This is in contrast to the heart
and lungs, for example, which show virtually no background [18F]FHBG signal after
administration. After 45.3 minutes to 80.3 minutes, [18F]FHBG levels have decreased
significantly in the liver and kidneys, with persistent high signal in the bladder. From 83.3
minutes to 155.6 minutes, levels in the liver and s and decreased even further, with
maintenance of high signal levels in the bladder. Accordingly, depending upon the organ and
subject individual, some time may be required for background levels to se in order to
measure HSV-TK gene expression. No time may be needed for measurement in organ systems
outside of the lower abdomen region, as seen in the heart and lungs. In these organ systems, a
sufficient threshold for gene therapy suitability may be, for example, at least above 1.0 SUV, at
least above 1.5 SUV, at least above 2.0 SUV, at least above 2.5 SUV, at least above 3.0 SUV, at
least above 3.5 SUV or at least above 4.0 SUV.
In contrast, some delay may be needed in order to image signals above background
levels in, for example, the liver and kidneys. See Yaghoubi et al. Nat. Protocols at vol. 1, p.
3073. Because of the background signals in these organ systems, a sufficient threshold for
determination of suitability for gene therapy treatment may be, for example, at least 10% above
background, at least 15% above background, at least 20% above background, at least 25% above
background, at least 30% above background, at least 35% above background, at least 40% above
background, at least 45% above background, at least 50% above background, at least 55% above
background, at least 60% above background, at least 65% above background, at least 70% above
background, at least 75% above background, at least 80% above ound, at least 85% above
ound, at least 90% above ound, at least 95% above background or at least 100% or
more above ound when measured after a predetermined amount of time. For example, as
seen in background signals in the liver are considerably less after at least 1 to 1-1/2
hours after administration of [18F]FHBG. Accordingly, [18F]FHBG signal measurements in the
liver should not be taken until after [18F]FHBG signal levels have decreased to ound,
approximately 1 to 1-1/2 hours, depending upon clearance rate in each individual subject.
In some organ systems, HSV-TK gene expression may not be measurable, for e,
in the bladder, where high background signal levels are maintained over time.
In other embodiments, a ratio of administered [18F]FHBG to measured [18F]FHBG
signal is ed to determine if a subject should be included or ed from a gene therapy
protocol. For example, if a subject that is ed with, for example, 500 MBq of [18F]FHBG
and exceeds the threshold of, for example, 50 MBq of [18F]FHBG signal, the subject is capable
of producing a therapeutically ive amount of phosphorylated lovir, or a derivative
thereof, from a construct described herein to be therapeutic, indicating that the subject may
respond in a gene therapy ion. In such an embodiment, the subject is a ate for
treatment with a gene therapy construct described herein.
In other embodiments, the subject is injected with 100-750 MBq or 100-600 MBq or
100-500 MBq or 200-500 MBq or 200-400 MBq, or 2.0 to 15.5 MBq/kg or 2.0 to 12.0 MBq/kg
or 2.0 to 10.0 MBq/kg or 2.0 to 7.5 MBq/kg of [18F]FHBG and s the threshold of, for
example, 10-100 MBq or 10-90 MBq or 10-80 MBq or 10-70 MBq or 10-60 MBq or 20-50
MBq or 20-40 MBq of [18F]FHBG signal. In some embodiments, the subjected is injection with
200-500 MBq of [18F]FHBG and exceeds the threshold of, for example, 20-50 MBq of
[18F]FHBG signal. In some embodiments, the ratio of [18F]FHBG signal injected to [18F]FHBG
signal measured is 2:1, 5:1, 10:1, 20:1 30:1, 40:1 or 50:1. In some embodiments, the ratio of
[18F]FHBG signal injected to [18F]FHBG signal measured is from about 2:1 to about 50:1, from
about 2:1 to about 40:1, from about 5:1 to about 30:1, from about 5:1 to about 20:1, from about
:1 to about 10:1 or about 10:1.
In another embodiment, if a subject produces ientphosphorylated FHBG to
generate a signal of greater than 2.0 SUV or at least 20% above background on PET scan, the
subject is likely to produce a therapeutically effective amount of TK from a construct described
herein and the t is likely respond in a gene therapy situation. In some embodiments, the
subject may be selected for combination therapy with another anti-cancer agent or treatment
described herein.
In other embodiments, the subject produces suff1cientphosphorylated FHBG to
generate a signal of greater than about 1.5 SUV, greater than about 2.0 SUV, greater than about
2.5 SUV, greater than about 3.0 SUV, r than about 4.0 SUV or greater than about 5.0
SUV. In yet other embodiments, the subject generates a signal of at least 10% above
ound, at least 20% above background, at least 30% above ound, at least 40% above
background or at least 50% or more above background.
CANCERS
Non-limiting examples of cancers can include: acute lymphoblastic leukemia, acute
myeloid leukemia, adrenocortical carcinoma, AIDS-related s, AIDS-related lymphoma,
anal cancer, appendix , astrocytomas, basal cell carcinoma, bile duct cancer, bladder
cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, al
astrocytoma/malignant , ependymoma, oblastoma, supratentorial primitive
neuroectodermal tumors, Visual pathway and hypothalamic glioma, breast cancer, bronchial
adenomas, Burkitt lymphoma, carcinoma ofunknown primary origin, central nervous system
lymphoma, cerebellar astrocytoma, cervical , childhood cancers, chronic lymphocytic
ia, chronic myelogenous leukemia, chronic roliferative disorders, colon cancer,
cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer,
ependymoma, esophageal , Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric
cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gliomas, hairy cell
leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, n lymphoma,
aryngeal cancer, intraocular ma, islet cell carcinoma, Kaposi sarcoma, kidney
cancer, laryngeal cancer, lip and oral caVity cancer, liposarcoma, liver , lung cancers, such
as non-small cell and small cell lung , lymphomas, leukemias, macroglobulinemia,
malignant fibrous histiocytoma of bone/osteosarcoma, oblastoma, melanomas,
mesothelioma, metastatic us neck cancer with occult primary, mouth cancer, multiple
endocrine neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity and
paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin ma,
non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous
histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor,
pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal caVity cancer,
yroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma,
pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia,
primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma,
renal pelVis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary
gland , sarcomas, skin cancers, skin carcinoma merkel cell, small intestine cancer, soft
tissue sarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer,
thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (gestational), cancers of
unkown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer,
Waldenstrom macroglobulinemia, and Wilms tumor.
In other embodiments, the s to be treated are chosen from the group consisting
of primary hepatocellular carcinoma, atic breast carcinoma to liver, metastatic pancreatic
cancer to liver, atic gastric cancer to liver, metastatic esophageal cancer to liver,
metastatic lung cancer to liver, metastatic melanoma to liver, metastatic ovarian carcinoma to
liver and metastatic kidney cancer to liver.
FORMULATIONS
ceutical compositions comprising a therapeutic vector can be formulated in any
conventional manner by mixing a ed amount of the therapeutic vector with one or more
physiologically acceptable rs or excipients. For example, the therapeutic vector may be
suspended in a carrier such as PBS (phosphate buffered saline). The active compounds can be
administered by any appropriate route, for example, , parenterally, intravenously,
intradermally, subcutaneously, or topically, in liquid, semi-liquid or solid form and are
formulated in a manner suitable for each route of administration.
In some embodiments, the therapeutic vector and physiologically acceptable salts and
solvates are formulated for administration by inhalation or insufflation (either through the mouth
or the nose) or for oral, buccal, parenteral or rectal administration. In some embodiments, for
administration by inhalation, the therapeutic vector is delivered in the form of an aerosol spray
presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
other le gas. In some embodiments, a pressurized aerosol dosage unit or a valve to deliver
a d amount. In some embodiments, capsules and cartridges (e.g., of gelatin) for use in an
inhaler or insufflator are formulated containing a powder mix of a eutic compound and a
suitable powder base such as lactose or starch.
In some embodiments, the pharmaceutical itions are formulated for oral
administration as tablets or capsules prepared by conventional means with pharmaceutically
acceptable excipients such as binding agents (e.g., atinized maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline
cellulose or calcium hydrogen phosphate); ants (e.g, magnesium te, talc or silica);
disintegrants (e.g, potato starch or sodium starch glycolate); or wetting agents (e.g., sodium
lauryl sulphate). In some embodiments, the tablets are coated by methods well known in the art.
In some embodiments, liquid preparations for oral administration are in the form of, for
example, solutions, syrups or suspensions, or they are ated as a dry product for
constitution with water or other suitable vehicle before use. In some embodiments, such liquid
preparations are prepared by tional means with pharmaceutically able additives
such as suspending agents (e.g., ol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous es (e.g., almond oil, oily
esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-
2014/029600
hydroxybenzoates or sorbic acid). In some embodiments, the preparations also contain buffer
salts, flavoring, coloring and sweetening agents as appropriate. In some embodiments,
pharmaceutical compositions are formulated oral administration to give controlled release of the
active compound. In some embodiments, the pharmaceutical compositions are formulated for
buccal in the form of tablets or lozenges formulated in conventional .
In some ments, the therapeutic vector is formulated for parenteral
administration by injection, e.g., by bolus injection, or continuous infusion. In some
embodiments, formulations for injection are in unit dosage form, e.g., in ampoules or in multi-
dose containers, with an added preservative. In some embodiments, the compositions are
ated as sions, solutions or emulsions in oily or aqueous vehicles. In some
embodiments, the formulations comprise formulatory agents such as ding, stabilizing
and/or dispersing agents. atively, in some embodiments, the active ingredient is in powder
lyophilized form for constitution with a suitable vehicle, e. g., sterile pyrogen-free water, before
use.
] In some embodiments, the therapeutic vector is formulated as a depot preparation. In
some embodiments, such long acting formulations are administered by implantation (for
example, subcutaneously, intramuscularly or ly into or in close proximity to a tumor) or by
intramuscular injection. Thus, for example, in some embodiments, the therapeutic compounds
are formulated with suitable polymeric or hydrophobic als (for example, as an emulsion in
an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for e, as a
sparingly soluble salt.
In some embodiments, the active agents are formulated for local or topical application,
such as for topical application to the skin and mucous membranes, such as in the eye, in the
form of gels, creams, and lotions and for application to the eye or for intracistemal or intraspinal
application. In some embodiments, such solutions, particularly those intended for ophthalmic
use, are formulated as 0.0l%-lO% isotonic solutions, pH about 5-9, with appropriate salts. In
some embodiments, the compounds are formulated as aerosols for topical application, such as by
inhalation.
The concentration of active compound in the drug composition will depend on
tion, inactivation and excretion rates of the active compound, the dosage schedule, and
amount administered as well as other factors known to those of skill in the art.
In some embodiments, the compositions are ted in a pack or dispenser device
which comprise one or more unit dosage forms containing the active ingredient. In some
embodiments, the pack may comprises metal or plastic foil, such as a r pack. In some
embodiments, the pack or dispenser device is accompanied by ctions for administration.
In some embodiments, the active agents are packaged as articles of cture
containing packaging al, an agent provided herein, and a label that indicates the disorder
for which the agent is provided.
ANIMAL MODELS
In some ments, the retroviral vector particles, hereinabove described are
administered to an animal in vivo as part of an animal model for the study of the effectiveness of
a gene therapy treatment. In some embodiments, the iral vector particles are administered
in varying doses to different animals of the same species. The animals then are evaluated for in
viva expression of the desired therapeutic or diagnostic agent. In some embodiments, from the
data obtained from such evaluations, a person of ordinary skill in the art determines the amount
of retroviral vector particles to be administered to a human t.
KITS
Also provided are kits or drug delivery systems comprising the compositions for use in
the methods described herein. All the essential materials and ts required for
administration of the retroviral particles disclosed herein may be assembled in a kit (6.g.
packaging cell construct or cell line, cytokine expression vector). The components of the kit
may be provided in a y of formulations as described above. The one or more therapeutic
retroviral particles may be formulated with one or more agents (e.g., a chemotherapeutic agent)
into a single pharmaceutically acceptable composition or separate pharmaceutically acceptable
compositions.
The components of these kits or drug delivery systems may also be ed in dried
or lyophilized forms. When reagents or components are provided as a dried form, titution
generally is by the addition of a suitable solvent, which may also be provided in another
container means.
Container means of the kits may generally include at least one vial, test tube, flask,
bottle, syringe and/or other container means, into which the at least one substance can be .
The kits disclosed herein may also comprise instructions regarding the dosage and or
stration information for the retroviral particle. Instructions can include instructions for
practicing any of the methods described herein including treatment methods. Instructions can
additionally e indications of a satisfactory clinical endpoint or any adverse symptoms that
may occur, or additional information required by regulatory agencies such as the Food and Drug
Administration for use on a human subject.
The ctions may be on “printed matter,” e.g., on paper or cardboard within or
affixed to the kit, or on a label affixed to the kit or packaging material, or attached to a vial or
tube containing a ent of the kit. Instructions may additionally be included on a computer
readable medium, such as a disk (floppy diskette or hard disk), optical CD such as CD- or DVD-
ROM/RAM, magnetic tape, electrical storage media such as RAM and ROM, IC tip and hybrids
of these such as magnetic/optical storage media.
In some embodiments, the kits or drug delivery systems include a means for containing
the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic
ners into which the desired vials are retained. Irrespective of the number or type of
ners, the kits may also comprise, or be ed with, an instrument for assisting with the
inj ection/administration or placement of the ultimate complex composition within the body of a
subject. Such an instrument may be an applicator, inhalant, syringe, pipette, forceps, measured
spoon, eye r or any such medically ed delivery vehicle.
Packages and kits can fiarther include a label specifying, for example, a t
description, mode of administration and/or indication of treatment. Packages provided herein
can include any of the compositions as bed herein. The package can further include a label
for treating one or more diseases and/or conditions.
The term “packaging material” refers to a physical structure housing the components of
the kit. The packaging material can maintain the components sterilely and can be made of
material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil,
ampules, etc). The label or packaging insert can include riate written instructions. Kits,
therefore, can additionally include labels or instructions for using the kit components in any
method described herein. A kit can include a compound in a pack, or dispenser together with
instructions for administering the compound in a method described herein.
EXAMPLES
In order that those in the art may be better able to practice the compositions and
s described herein, the following examples are provided for ration purposes.
Example 1: al Trial.
A dose escalation trial was conducted to evaluate the , pharmacokinetics, and
codynamics of Reximmune-C2 (Thymidine Kinase and GM-CSF Genes) in refractory
subjects with primary hepatocellular carcinoma or tumors metastatic to the liver.
Background and Rationale
Reximmune-C2 is comprised of a genetic delivery platform containing an internal
d that encodes for therapeutic ns of interest. The genetic delivery platform has been
dosed in over 280 subjects worldwide; approximately 270 subjects were d with the vector
containing dnGl as a payload (Rexin-G) and 16 subjects with thymidine kinase (vTK) and the
immune stimulator Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) as a d
(Reximmune-C). The genetic delivery platform is a highly engineered non-recombinant Mouse
Moloney Viral vector (MoMLV). Previously, a Phase 1 dose escalation trial was performed
investigating the combination of Rexin-G and Reximmune-C in subjects with refractory y
or metastatic solid tumors (Genevieve Trial). This ed Phase I clinical trial (entitled
Genevieve 2 Trial) is an extension of a trial undertaken investigating Reximmune-C2 alone —
without the Rexin-G — utilizing an improved form of thymidine kinase in a thymidine kinase
plus GM-CSF combination.
In the original Genevieve trial, sixteen t were recruited over 3 dose levels with
the mean exposure in the highest dose group being 8.0x 1010 cfus (# of pts = 7) and the longest
duration 6 cycles (range of cycles 3-6). For Part A of the study, treatment consisted of a
previously determined safe and effective (optimal) dose of G, and escalating doses of
Reximmune-C. Specifically, Rexin-G, 2 x 1011 cfu, on Days 1, 3, 5, 8, 10 and 12, Reximmune-
C, 1.0, 2.0 or 3.0 x 1010 Cfil on Day 3 (Dose Levels 1, II, 111 respectively), and valacyclovir at 1
gm p.o. three times a day on Days 6-19, as one cycle. For the Part B part of the study, subjects
who had no toxicity or in whom toxicity had resolved to Grade 1 or less could receive additional
cycles of therapy up to a total of 6 treatment cycles.
There were no dose-limiting toxicities at any dose level. ted adverse events were
reported for the 16 subjects in the study, but the number of events was low (in most cases 1 or 2
occurrences per preferred term), and most were Grade 1 or 2. Related non-serious adverse
events occurred in 2 subjects and both were Grade 2. Four subjects experienced serious adverse
events, all of which were deemed not related to the study drug.
The rationale for continuation of this Phase 1 trial is that: (l) ine kinase itself
could prove to be an effective ncer agent particularly in subjects whose tumors
trate a bystander effect; (2) administration of the genetic delivery rm to date to an
international group of subjects has demonstrated a very high degree of safety; and (3)
biodistribution in animals suggests a high biodistribution to the liver. Moreover, the addition of
GM-CSF could contribute to an logical effect and enhanced tumor cell kill h
tumor associated antigens h recruitment of the riate immune cells.
The biodistribution of the viral particles is t to the liver, followed by spleen, then
lung — this is the rationale for focusing initially on hepatocellular tumors where the dose
intensity should be the t. There is also a high clinical unmet need for effective anticancer
agents for these cancers.
It is understood that the embodiments disclosed herein are not limited to the particular
methods and components and other processes described as these may vary. It is also to be
understood that the terminology used herein is used for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the present invention. It must be
noted that as used herein and in the appended claims, the singular forms "a," "an," and "the"
include the plural reference unless the context clearly dictates ise. Thus, for example, a
reference to a "protein" is a reference to one or more proteins, and includes equivalents thereof
known to those skilled in the art and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this invention
s. Specific methods, devices, and materials are described, although any methods and
materials similar or equivalent to those described herein can be used in the practice or testing of
the present invention.
All publications cited herein are hereby incorporated by reference including all journal
articles, books, manuals, published patent applications, and issued patents. In addition, the
meaning of certain terms and s employed in the specification, examples, and appended
claims are provided. The definitions are not meant to be limiting in nature and serve to provide a
r understanding of certain aspects of the present invention.
Example 2: TK diagnostic assay for gene therapy ations.
Animal and human studies have usly shown the y of measuring vTK
expression by PET imaging using [18F]-FHBG. These imaging tools will be utilized as a
personalized surrogate test in accessing appropriate dosing and exposure and used in the IB
portion to determine which subjects have the best opportunity to benefit from the drug
candidates.
This clinical trial is divided into two phases: Phase IA in which Reximmune-C2 was
stered as a single intravenous dose on three out of five days and the presence of the HSV-
TK-m2 expression potentially monitored by HBG PET scanning after 3-8 days (Schematic
for Phase IA is illustrated in . Valganciclovir (the oral form of ganciclovir) dosing is
initiated on day 8 for 5 days irrespective of the PET scan s. An approximately one week
drug holiday s. Each cycle will be of three weeks duration.
There will be three patients in the first and subsequent cohorts until a patient
experiences Dose Limiting Toxicity (DLT) or two instances ofNCI-CTC Grade 2 toxicities
attributed to the study drug (except nausea/vomiting, fatigue, anorexia, alopecia, or anemia). If
there are no DLTs, patients will move to the next dose level. If there is a DLT, the cohort will
be expanded to 6 patients and the dose level will not be exceeded if 2 or more patients t
DLTs.
Once the Maximum Administered Dose (MAD) is reached, a modified Fibonacci
le will be followed starting with the cohort dose which had no DLTs and continuing until
dose-limiting toxicities are observed in two patients at a dose level. Once the Recommended
Phase 2 Dose (RP2D) is defined, 6-12 patients will be recruited.
Phase IB is designed to explore the activity of Reximmune-C2 in patients of a defined
tumor type and stage based on the Phase IA data and who are HBG scan positive day three
to six after one dose (RP2D) of Reximmune-C2. If the scan is positive, the patient is accepted
into the Phase IB ent phase of the protocol and the RP2D is given as three doses within 5
days, followed by 5 days of valganciclovir beginning on day 8 of that phase, followed by a one
week drug holiday. Each cycle is of three week duration. Patients who have a negative
[18F]FHBG PET scan after one single dose of Reximmune-C2 will be dosed with 5 days of
valganciclovir and will not continue in the study.
The patient DLT will be defined as the occurrence of any of the following events
which is attributed to une-C2 and occurring during the first cycle (3 weeks) of drug
administration:
Grade 4 neutropenia (i.e., te neutrophil count (ANC) < 500 cells/mm3) for 7 or
more consecutive days or febrile neutropenia (i.e., fever 2 38.50 C with an ANC < 1000
cells/mm3); Grade 4 ocytopenia (< 25,000 cells/mm3 or bleeding episode requiring
platelet transfusion); Grade 3 or greater nausea and/or vomiting despite the use of
adequate/maximal medical intervention and/or prophylaxis; Any Grade 3 or greater non-
hematological ty (except Grade 3 injection site reaction, alopecia, fatigue); Retreatment
delay of more than 3 weeks due to delayed recovery from a toxicity related to treatment with
Reximmune-C2; and Grade 3 or greater hypersensitivity reaction despite the riate use of
premedications (by Common ty Criteria defined as “symptomatic bronchospasm,
requiring parenteral medications(s), with or without urticaria; allergy-related edema-
dema”).
Reximmune-C2 is infused intravenously over 15-60 minutes (depending on the dose)
via an infusion pump. Reximmune-C2 is ed in 30 ml vials stored at -80 °Ci 10 0C.
In this Phase I trial, the safety, pharmacokinetics, and pharmacodynamics of escalating
doses of Reximmune-C2 will be investigated. The maximum tolerated dose will be identified
and a recommended Phase 2 dose will be defined for Reximmune C2. Any antitumor ty
and clinical responses to Reximmune-C2 treatment will be described.
The starting dose in this trial is based on: human clinical safety ence with the
related vector platform drug products Rexin-G and une-C and the results of the 21 day
rat GLP toxicology study for Reximmune-C2.
Objectives
] The primary objective of the study is to determine the maximum tolerated dose (MTD),
dose limiting toxicity (DLT), safety, and a recommended Phase 2 dose (RP2D) of Reximmune-
C2 stered over a three week cycle consisting of a series of three doses given
intraveneously within five days in week 1, ed by 5 daily doses of valganciclovir in week 2
in patients enrolled in this study who have been diagnosed with advanced primary or metastatic
tumors to the liver.
Secondary objectives include: (i) evaluation of the plasma cokinetics of
Reximmune-CZ; (ii) ment of the surrogate of HSV-TK-m2 n expression from
Reximmune-C2 via serial [18F]FHBG PET and/or SPECT imaging; (iii) description and
ment of any preliminary evidence of anti-tumor activity of Reximmune-CZ; and (iv) to
provide clinical research testing for antibodies to retrovector gp70 env, replication-competent
retrovirus in peripheral blood lymphocytes ; vector integration into genomic DNA of
PBLs, and circulating hGM-CSF protein.
Methods
Study Design: Parallel group, open label dose tion, three-center clinical trial.
Stratification: None.
Therapy: une-C2 will be administered as an intravenous infilsion to separate
patients. In Phase IA — investigating Reximmune-C2 - the dose will be escalated among cohorts
of patients until DLT is observed. At the RP2D, onal patients will be recruited. In Phase
IB patients will be pre-screened by [18F]FHBG PET for expression of the HSV-TK-m2. Those
that express HSV-TK-m2 will receive additional doses of Reximmune-C2. Patients will not be
pre-medicated unless hypersensitivity reactions occur.
Statistical Methods: Descriptive statistics will be used for statistical analysis.
Sample Size Determination: Precise sample size cannot be defined, as it is dependent
on the observed toxicity. For each schedule, cohorts of three to six subjects will be d at
each dose level until the MTD is defined. Once the MTD is identified, this dose level will be
expanded to a maximum of 12 patients who will be treated to better define the tolerability and
pharmacokinetics of the dose and schedule. It is expected that 45-70 subjects will be enrolled,
with 33 to 46 in the IA n.
Enrollment Criteria
Subjects must meet all of the following inclusion criteria to be eligible for
randomization into the study:
1. Diagnosis of histologically documented, ed stage, primary or metastatic adult
solid tumors in the liver that are refractory to standard therapy or for which no curative standard
therapy exists.
2. Evidence of radiographically measurable or evaluable disease.
3. All acute toxic effects of any prior radiotherapy, herapy, or surgical
ures must have resolved to National Cancer Institute (NCI) Common Toxicity Criteria
(CTC)(Version 4.0) Grade < 1.
4. Age must be > 18 years.
5. Last dose of antineoplastic therapy except for hormonal therapy must be > 21 days.
External beam radiotherapy must have been < 25% bone marrow-containing skeleton.
6. Patients may be Hepatitis B and C positive. (Patients may continue their antiviral
medications).
7. Patients may have intracranial metastases of any number if they have been brain
irradiated and stable for 6 weeks. Patients may be taking eizure medicines but must not be
on steroids.
8. Kamofsky performance status must be 2 70.
9. Life expectancy of at least 3 months.
10. Patients must be able to travel to St. Luke’s Medical Center for the PET scans.
ll. Required baseline tory data include:
Absolute neutrophil count
3 _ 9
2 mm [SI units 10 /L]
(ANC)
Platelets 2 75,000/mm3 [SI units 10%]
Hemoglobin 2 8.0 gm/dL [SI units ]
Serum Creatinine S 1.5 x laboratory upper limit of normal (L-ULN)
Bilirubin S 2.0 mg/dL
Alkaline phosphatase S 5 x L-ULN
AST, ALT S 5 x L-ULN
LDH S 5 x L-ULN
Pregnancy test (females of Negative within 7 days of ng Protocol
childbearing potential)
12. Signed informed consent indicating that they are aware of the neoplastic nature of
their disease and have been informed of the ures to be followed, the experimental nature
of the therapy, atives, potential benefits, side s, risks, and discomforts.
13. Willing and able to comply with scheduled visits, treatment plan, and laboratory
tests.
The presence of any of the following will exclude a subject from study enrollment
1. Concurrent therapy with any anticancer therapy including any other investigational
agent.
] 2. Known intracranial edema or a CVA within 6 weeks of screening.
3. Pregnant or breast-feeding women. Female subjects must agree to use effective
contraception, must be surgically sterile, or must be nopausal. Male ts must agree
to use effective contraception or be surgically sterile. The definition of effective contraception
will be based on the judgment of the Investigator or a designated associate. All at-risk female
subjects must have a negative pregnancy test within 7 days prior to the start of study treatment.
4. Clinically significant cardiac disease (New York Heart ation, Class III or IV).
5. Dementia or altered mental status that would prohibit informed consent.
6. Other severe, acute, or chronic medical or psychiatric condition or laboratory
abnormality that may increase the risk associated with study participation or study drug
administration or may interfere with the interpretation of study results and, in the judgment of
the Principal Investigator, would make the subject inappropriate for this study.
7. Known side effects to antivirals in the ganciclovir class.
8. Patients who are known to be HIV positive.
9. t must not be taking steroids at the time of screening.
Rationalefor the Starting Dose and Schedule
Reximmune-C has been dosed in 16 ts over a range of 1.0, 2.0 or 3.0 x1010 cfu
(Dose Levels 1, II, 111 respectively on day 3 of the . There were no dose-limiting toxicities
at any dose level. Unrelated adverse events were reported for the 16 patients in the study, but
the number of events was low (in most cases 1 or 2 occurrences per preferred term), and most
were Grade 1 or 2. Related ious adverse events occurred in 2 patients and both were
Grade 2. Four patients experienced serious e events, all of which were deemed not related
to the study drug. The trial was closed prior to ining the l dose and schedule of
Reximmune-C. In this trial, the new Genevieve-2 Trial, initial dosing will be based on the 21
day toxicology and the -ml study. Future dosing will proceed using total viral particles
(TVP) which is a more accurate measure of titer than cfu per mL.
The schedule is based on the rationale that Reximmune-C2 exposure will not transduce
all of the tumor cells. Therefore, patients will be dosed three times in a cycle over a period of 5
days.
The time between exposure to GDS and the expression of HSV-TK-m2 (and hGM-
CSF) is estimated to be 48 to 72 hours. Therefore, 72 hours after the third dose of Reximmune-
C2, valganciclovir will be initiated. The dose (which will be adjusted for renal on) will be
given at conventional antiviral dose levels. Due to the potential toxicity of valganciclovir and
the published observations that 5 days of ganciclovir should be sufficient to kill the majority of
cells ning HSV-TK-m2, 5 days of therapy was chosen. Due to the potential toxicity of
both Reximmune-C2 and valganciclovir, this will be followed by an approximately 9 day drug
holiday. The hGM-CSF may be at sufficient concentrations at the time of valganciclovir
addition to influence the presentation of any tumor associated antigens (TAAs) that may appear
during tumor cell apoptosis.
Plasma samples will be taken after the first and third doses in Cycle One and after the
first dose in Cycle Two for pharmacokinetics.
] As bution is primarily to the liver, toxicities will be carefillly monitored there and
e of the implications, the bone marrow.
This clinical protocol calls for the administration of Reximmune-C2 via intravenous
infusion to patients with advanced malignancies, either primary hepatocellular or tumors
metastatic to the liver. There will be two parts: Phase IA (dose tion 3 doses/week every
three weeks) and Phase IB (pre-screening after one dose of Reximmune-C2 and an HBG
scan). If the PET scan is positive, the patient will continue on study. If the PET scan is
negative, the patient will receive 5 days of valganciclovir and will not continue in the trial. For
Phase IA, dose tion will follow an accelerated titration design, incorporating three patients
per dose level until either one instance of DLT or two instances CTC Grade 2 toxicities
attributed to the study drug (except nausea/vomiting, fatigue, ia, alopecia or anemia) are
observed. Thereafter, dosing in the clinical protocol will follow a modified Fibonacci schedule
until dose-limiting toxicities are achieved.
Trial Design
This is a Phase 1, open-label, four center, scalating trial. The dose will be
increased until DLT is observed, and the MTD is defined.
Reximmune-C2 will be administered as an IV infusion over 15-60 minutes. It is
anticipated that 33-70 patients will be treated during the course of the study.
For Phase IA, the dose of Reximmune-C2 will be escalated from 6.0x1011 TVP. In the
accelerated dose escalation phase, cohorts of three patients will be enrolled at each dose level.
The dose escalation increment will be 100% until a DLT or two CTC Grade 2 or r
toxicities are observed. When the rated dose escalation ends, the dose escalation for a
new t in the standard dose escalation will follow a modified Fibonacci scheme (i.e., dose
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increments of 67%, 50%, 40%, 33% and 25%). A minimum of three patients per dose level will
be enrolled. For Phase 1B, the dose of Reximmune-C2 will be the RP2D. DLT will be assessed.
If a DLT is observed in 3 2 out of six patients at a dose level, there will be no further dose
escalation; this dose level will define the maximum administered dose (MAD).
The dose just below the MAD will be considered the MTD. Once the MTD is defined,
this dose level can be expanded to a maximum of twelve patients to fiarther characterize the
pharmacokinetic and pharmacodynamic parameters and suitability as a recommended dose for
Phase 2 clinical studies.
Treatment of Patients
Only qualified personnel who are familiar with procedures that minimize undue
exposure to themselves and to the environment should undertake the preparation, handling, and
safe disposal of rapeutic agents in an riate environment.
Reximmune C2 is a y Murine replication incompetent retrovector particle
containing the genes encoding for a HSV-TK-m2 and hGM-CSF. The drug t contains
DMEM (low glucose), RD-Retrovector Particles, L-glutamine, Sodium pyruvate, human serum
albumin, n-butyric acid, Pulmozyme®, magnesium and other excipients.
Drug t is available in one vial size: 30 mL type 1 clear glass vials with a 20 mm
finish (containing 25 mL of 31 .0x1010 TVP). The vials are closed with 20 mm Teflon coated
serum stoppers and 20 mm flip-off red flip tops.
] Reximmune-C2 will be administered intravenously by infusion pump over 15 s
up to a volume of 100 mL, from >100 mL to 200mL over 30 minutes, from >200 mL to 300 mL
over 45 minutes, and from >300 mL to 400 mL over 60 minutes. Volumes over 400 mL will be
administered at a rate determined by the Investigator and the Gleneagles Medical Monitor. Once
the MTD has been identified for the schedule, the time of administration may be changed, if
indicated (and as agreed between the Investigator and the Gleneagles Medical Monitor).
Valganciclovir is administered orally, and should be taken with food. Serum creatinine
or creatinine clearance levels should be monitored lly. Dosage adjustment is required
based on creatinine clearance as shown in the Table below. Valganciclovir dosing may begin on
day 7 to 9 of the cycle but must be given for 5 consecutive days.
Creatinine clearance can be calculated from serum creatinine by the following a:
For males = {(140 — age[years]) x (body weight [kg])}/ {(72) x (0.011 x serum
nine [micromol/L])}
For females = 0.85 x male value.
2014/029600
Table I. Valganciclovir Dosing for Renally Impaired Patients
Cr CL (ml/min) Dose Day 1 Dose Days 2 - 5
260 ml/min 900 mg (two 450 mg tablets) 900 mg (two 450 mg
bid tablets) qday
40-59 ml/min 450mg bid 450mg qday
-39 ml/min 450mg 450 mg Day 3 and Day 5
-24 ml/min 450mg 450 mg Day 4
The purpose of the Phase 1 study is to establish the MTD, DLT, safety and a RP2D of
the investigational agent. Toxic effects are thus the primary study endpoint and will be assessed
continuously. Response information will be obtained if patients have disease that can readily be
measured and re-assessed. These assessments will be made with every cycle. rmore, a
se must be noted between two examinations at least 6 weeks apart in order to be
documented as a confirmed response to therapy.
0 Evaluable for toxicity - All patients will be evaluable for toxicity if they
receive any study drug.
0 ble for se - All patients who have received at least a single cycle
of treatment and had tumor re-assessment will be considered evaluable for
response. In addition, those patients who develop early ssive disease
will also be considered evaluable for response. Patients on therapy for at
least two cycles of treatment will have their response evaluated.
] The determination of antitumor efficacy will be based on objective tumor ments
made according to the Immune-Related Response Criteria (irRC) system of evaluation and
treatment ons by the Investigator will be based on these assessments.
Given the presence of the GM-CSF transgene in Reximmune-C2 and the possibility of
an immune response contributing to the tumor effect, the Immune response Criteria will be
utilized for clinical response. The reasons for using The immune Response Criteria vs RECIST
1.1 are as follows: (1) the appearance of measurable anti-tumor ty may take longer for
immune therapies than for cytotoxic therapies; (2) responses to immune therapy occur after
conventional PD; (3) discontinuation of immune therapy may not be appropriate in some cases,
unless PD is confirmed (as is usually done for response); (4) allowance for “clinically
insufficient” PD (e.g. small new lesions in the presence of other responsive lesions) is
recommended; and (5) durable SD may represent antitumor activity.
The comparisons between RECIST 1.1 and the Immune-Related se Criteria are
listed below:
Table 11. Comparison ofWHO RECIST and Immune-Related se Criteria
WHO irRC
New measurable lesions Always represent PD Incorporated into tumor burden
(i.e., 2 5 X 5 mm)
New, nonmeasurable lesions Always represent PD Do not define progression (but
(i.e., < 5 X 5 mm) preclude irCR)
Non-index lesions Changes contribute to defining Contribute to defining irCR
BOR of CR, PR, SD, and PD (complete disappearance
CR earance of all lesions in Disappearance of all lesions in
two consecutive observations not two consecutive observations not
less than 4 wk apart less than 4 wk apart
PR 2 50% decrease in SPD of all 2 50% se in tumor burden
index lesions compared with compared with baseline in two
baseline in two ations at observations at least 4 wk apart
least 4 wk apart, in absence of
new lesions or unequivocal
progression of non-index lesions
SD 50% decrease in SPD compared 50% decrease in tumor burden
with baseline cannot be compared with baseline cannot be
established nor 25% increase established nor 25% increase
compared with nadir, in absence compared with nadir
ofnew lesions or unequivocal
progression of dex lesions
PD At least 25% increase in SPD At least 25% increase in tumor
compared with nadir and/or burden compared with nadir (at
unequivocal progression of non- any single time point) in two
indeX s and/or appearance consecutive observations at least
ofnew lesions (any any single 4 wk apart
time point)
Timing and Type of Assessments
All ne imaging-based tumor assessments are to be performed within 14 days
prior to the start of treatment. For the purposes of this study, all patients’ tumor ments
should be re-evaluated starting 9 weeks after initiation of treatment and every 6 weeks thereafter
(e. g., Week 9, Week 15, Week 21, etc.) for both Phase IA and Phase IB. All patients with
responding tumors (irCR or irPR) must have the response confirmed no less than 6 weeks after
the first documentation of response. All patients with tumor progression must have progression
confirmed no less than 6 weeks after the first documentation of progression.
The same method of assessment and the same que should be used to characterize
each identified and reported lesion at baseline and during follow-up. g-based evaluation
is preferred to evaluation by clinical examination when both methods have been used to assess
the antitumor effect of treatment. All measurements should be recorded in metric notation.
CT and CT/PET are the methods for tumor assessments. Conventional CT should be
performed with cuts of 10 mm or less in slice thickness contiguously. Spiral CT should be
performed using a 5 mm uous truction algorithm. This applies to the chest,
abdomen, and pelvis.
Chest CT will used for assessment of pulmonary lesions.
Clinical lesions will only be considered measurable when they are superficial (e. g.,
skin nodules, palpable lymph nodes). In the case of skin lesions, ntation by color
photography including a ruler to estimate the size of the lesion is recommended.
[18F]FHBG PET-CT scans will be obtained after the patient receives the first three
doses of ReXimmune-C2 (cycle 1) in Phase IA and after the screening dose of Reximmune-C2 in
Phase IB. In Phase IA additional [18F]FHBG PET-CT scans can be obtained in subsequent
cycles at the tion of the Investigator and with approval of the Medical Monitor.
Ultrasound should not be used to measure tumor lesions that are clinically not easily
accessible for objective response evaluation, e. g., visceral lesions. It is a possible alternative to
clinical measurements of superficial palpable nodes, SC lesions, and d nodules.
Ultrasound might also be useful to confirm the complete disappearance of superficial lesions
usually ed by clinical ation.
Endoscopy, laparoscopy, and radionuclide scan should not be used for se
ment.
All patients’ files and radiological images must be available for source verification and
may be submitted for extramural review for final assessment of mor ty.
ability of Tumor Lesions
At baseline, tumor lesions will be categorized by the igator as measurable or
non-measurable by the criteria as described below:
0 Measurable: Lesions that can be accurately measured in at least one
dimension (longest diameter to be recorded) as 2 20 mm with conventional
techniques or as 2 10 mm with spiral CT scan. Clinical s will only be
considered measurable when they are superficial (e. g., skin nodules, palpable
lymph nodes).
0 Non-Measurable: All other s, including small lesions (longest diameter
< 20 mm with conventional techniques or < 10 mm with spiral CT scan) and
bone lesions, leptomeningeal disease, ascites, pleural or pericardial effusions,
lymphangitis of the skin or lung, abdominal masses that are not ed
and followed by imaging techniques, cystic lesions, previously irradiated
lesions, and disease documented by indirect evidence only (e. g., by laboratory
tests such as alkaline phosphatase).
NOTE: Cytology and histology: If measurable disease is restricted to a solitary
lesion, its neoplastic nature should be confirmed by cytology/histology.
] Response to therapy may also be assessed by independent, central, radiologic blinded
review.
Recording Tumor Measurements
All measurable lesions up to a maximum of 10 lesions, representative of all involved
organs, should be identified as target lesions and measured and recorded at baseline and at the
stipulated intervals during treatment. Target lesions should be selected on the basis of their size
(lesion with the longest diameters) and their suitability for accurate repetitive measurements
(either by imaging techniques or clinically).
The longest diameter will be recorded for each target lesion. The sum of the longest
diameter for all target lesions will be calculated and ed as the baseline. The sum of the
longest diameters is to be used as reference to fiarther characterize the objective tumor response
of the measurable dimension of the disease during treatment. All measurements should be
recorded in metric notation in centimeters.
All other lesions (or sites of disease) should be fied as non-target lesions and
should also be recorded at baseline. Measurements are not required and these lesions should be
followed as “present” or “absent.”
Definitions of Tumor Response
Immune-Related se Criteria criteria will be followed for assessment of tumor
] Determination of Overall Response by Immune-Related Response Criteria
Target Lesions for Solid Tumors
0 Complete response (irCR) is defined as the disappearance of all
lesions (whether measurable or not, and no new lesions); confirmation
by a repeat, consecutive assessment no less than 6 weeks from the
date first documented.
0 l response (irPR) is defined as a > 50% decrease in tumor
burden relative to baseline ed by a consecutive assessment at
least 6 weeks after the first documentation.
0 Progressive disease (irPD) is defined as a > 25% increase in tumor
burden ve to nadir (minimum ed tumor burden) confirmed
by a repeat, consecutive assessment no less than 6 weeks from the
date first nted s recorded since the treatment started, or
the appearance of one or more new lesions.
0 Stable e (irSD) is defined as not meeting the ia for irCR or
irPR, in absence of irPD.
Non-Target Lesions for Solid Tumors
The cytological confirmation of the neoplastic origin of any effusion that s or
worsens during treatment when the measurable tumor has met criteria for response or irSD is
ory to differentiate between response or irSD and irPD.
Confirmation of Tumor Response
To be assigned a status of irPR or irCR, changes in tumor measurements in ts
with ding tumors must be confirmed by repeat studies that should be performed 3 6 weeks
after the criteria for response are first met. In the case of irSD, follow-up measurements must
have met the irSD criteria at least once after study entry at a minimum al of 6 weeks.
When both target and non-target lesions are present, individual assessments will be recorded
separately. The overall assessment of response will involve all ters as depicted in Table
111.
The best overall response is the best response ed from the start of the treatment
until disease progression/recurrence (taking as a reference for tumor progression the smallest
measurements recorded since the treatment started). The patient’s best response assignment will
depend on the achievement of both ement and confirmation criteria.
Patients will be defined as being not ble (NE) for response if there is no
post-randomization oncologic assessment. These patients will be counted as failures in the
analysis of tumor se data.
Clinical Efficacy Assessment: Performance Status.
Patients will be graded according to the Kamofsky performance status scale as
described in Table IV.
Table IV. Karnofsky Performance Status Criteria
Normal, no complaints, no evidence of e
Able to carry on normal activity, minor signs or symptoms of
disease
Normal activity with effort, some signs or ms of disease
Care for self. Unable to carry on normal activities or to do
active work
Requires occasional assistance, but is able to care for most of
his/her needs
50 Requlres erable ass1stance and frequent medlcal care. . . .
4O Disabled, requires special care and assistance
Severely disabled, hospitalization is indicated although death
not imminent
Hospitalization necessary, very sick, active supportive treatment
necessary
Moribund, fatal processes progressing rapidly
Death
-6l-
Tumor Marker Response
Method of ment
While not a fillly validated measure of efficacy in many malignancies, serial
determinations of tumor markers may allow evaluation of an easily performed, inexpensive,
quantitative, clinical tool as a potential additional means for following the course of the illness
during therapy.
A tumor marker se or increase will not be assessed as an ive measure of
e. In particular, a rising tumor marker value will not be considered in the definition of
tumor progression, but should prompt a repeat radiographic evaluation to document whether or
not radiographic tumor progression has occurred.
Calculated Endpoint Definitions
] Survival is defined as the time from date of first study drug treatment to date of death.
In the absence of confirmation of death, survival time will be ed at the last date of follow-
Tumor se rate is defined as the proportion of patients who have any evidence of
objective irCR or irPR.
TTP is defined as the time from treatment to first conf1rmed documentation of tumor
ssion or to death due to any cause. For patients who do not have objective evidence of
tumor progression and who are either d from study treatment or are given mor
treatment other than the study treatment, TTP will be censored. A tumor marker increase
meeting criteria for tumor marker progression does not constitute te objective evidence of
tumor progression. However, such a tumor marker increase should prompt a repeat raphic
evaluation to document whether or not objective tumor progression has occurred.
TTF is defined as the time from treatment to first conf1rmed documentation of tumor
progression, or to off-treatment date, or to death due to any cause, whichever comes first.
Patients who are still on treatment at the time of the analysis and patients who are removed from
therapy by their physicians during an objective response and who, at the off-treatment date, have
no evidence for objective tumor progression will not be considered to have experienced
treatment failure, unless the withdrawal is due to the occurrence of a medical event. For these
patients, TTF will be censored at the off-study date. Censoring for TTF will also be performed
in those patients who are given mor treatment, other than the study treatment, before the
first of ive tumor progression, off-study date, or death. A tumor marker increase meeting
criteria for tumor marker progression does not constitute adequate objective evidence of
treatment failure. However, such a tumor marker increase should prompt a repeat radiographic
evaluation to document whether or not objective tumor progression (and thus treatment failure)
has occurred.
Time to first definitive performance status worsening is the time from treatment until
the last time the performance status was no worse than at ne or to death, due to any cause,
in the absence of us documentation of definitive confimed performance status worsening.
For patients who do not have definitive mance status worsening and who are either
removed from study or are given mor treatment other than the study treatment, definitive
performance status worsening will be censored.
Time to first definitive weight loss is defined as the time from treatment until the last
time the percent weight decrease from baseline was < 5% or to death due to any cause in the
absence of us documentation of definitive weight loss. For patients who do not have
definitive weight loss and who are either removed from study or are given antitumor treatment
other than study treatment, definitive weight loss will be censored.
Additional evaluations of the data may include best objective response, confirmed and
unconfirmed objective response rate, duration of study ent, time to first ence ofnew
lesions, time to tumor response, stable disease at 24 weeks, and rate of progression free survival
at 24 weeks. Data may be evaluated by RECIST 1.1 criteria, if needed.
ent Administration Assessment
For both Phase IA and IB: dose intensity is defined as the total ycle times the
number ofweeks n start of treatment and last treatment plus 13 days.
Percent relative dose intensity is defined as the proportion of the actual dose intensity
divided by the planned dose intensity for that same period of time.
ABBREVIATIONS
ALT Alanine aminotransferase
ANC Absolute neutrophil count
AST Aspartate aminotransferase
AUC Area under the plasma concentration-time curve
BSA Body surface area (mg/m2)
CL Systemic plasma clearance
Cmax Peak plasma concentration
CR te response
CRF Case report form
CT Computerized tomography
CTC Common Toxicity Criteria
DLT Dose Limiting Toxicities
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EOI End of infusion
FDA Food and Drug Administration
G-CSF Granulocyte-colony stimulating factor (filgrastim, Neupogen®)
GCP Good clinical practice
GM-CSF Granulocyte-macrophage colony-stimulating factor (sargramostim,
Leukine®)
HIV Human Immunodeficiency Virus
HR Hazard ratio
IEC Independent Ethics Committee
i.p. Intraperitoneal
IRB utional Review Board
IV Intravenous, intravenously
LD10 or Dose that is lethal to 10% or 50% of animals
Lactate dehydrogenase
VIaximum Administered Dose
VIagnetic resonance imaging
VIaximum tolerated dose
\Iational Cancer Institute
\Iot evaluable for tumor response
\10 Observed Adverse Effect Level
\Ion-complete response
\Ion-progressive disease
Peripheral Blood Mononuclear Cells
Propylene Glycol: hor® EL: Ethanol
Progressive disease
Partial response
SAER-S Serious Adverse Event Report-Study
Subcutaneous, aneously
Stable disease
Dose that is severely toxic to 10% of s
Time to Progression
Time to e
Half-life
Time ofmaximum plasma concentration
V SS Steady state volume of distribution
While preferred embodiments have been shown and described herein, it will be
obvious to those skilled in the art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions will now occur to those skilled in the art
without departing from the disclosed embodiments. It should be understood that various
alternatives to the embodiments described herein may be employed in practicing the
embodiments. It is intended that the following claims define the scope of the embodiments and
that methods and structures within the scope of these claims and their equivalents be covered
thereby.
nces
1. Lentivirus-based 2-transfected atic cancer cells for deep in vivo
imaging of metastatic disease. Yu Z, Zhou J, Hoffman RM., Methods Mol Biol. 2012;872:69-
83.doi:lO.l007/978617792_5.
2. Color-coded real-time subcellular fluorescence imaging of the interaction between
cancer and host cells in live mice.Yamauchi K, Tome Y, Yamamoto N, Hayashi K, Kimura H,
Tsuchiya H, Tomita K, Bouvet M, Hoffman RM.Anticancer Res. 2012 Jan;32(l):39-43.
3. Lentivirus-based DsRedtransfected pancreatic cancer cells for deep in vivo
imaging of metastatic disease.Zhou J, Yu Z, Zhao S, Hu L, Zheng J, Yang D, Bouvet M,
Hoffman RM.J Surg Res. 2009 Nov;lS7(l):63-70. doi: lO.lOl6/j.jss.2008.08.027. Epub 2008
Oct 9.
4. Fluorescent LYVE-l antibody to image dynamically lymphatic trafficking of cancer
cells in vivo.McElroy M, i K, Garmy-Susini B, Kaushal S, Vamer JA, Moossa AR,
n RM, Bouvet M.J Surg Res. 2009 Jan;15 l(l):68-73. doi: lO.lOl6/j.jss.2007. .
Epub 2008 Jan 18.
] 5. Lentiviral reporter constructs for fluorescence tracking of the temporospatial pattern
of Smad3 signaling.Stuelten CH, Kamaraju AK, Wakefield LM, Roberts techniques.
2007 Sep;43(3):289-90, 292, 294.
6. lular imaging in the live mouse.Hoffman RM, Yang M.Nat Protoc.
2006;1(2):775-82.
7. In vivo color-coded imaging of the interaction of colon cancer cells and splenocytes
in the ion of liver metastases.Bouvet M, Tsuji K, Yang M, Jiang P, Moossa AR, Hoffman
RM.Cancer Res. 2006 Dec l;66(23): 1 1293-7.
8. Dual-color imaging of r-cytoplasmic dynamics, ity, and proliferation of
cancer cells in the portal vein area.Tsuji K, Yamauchi K, Yang M, Jiang P, Bouvet M, Endo H,
Kanai Y, Yamashita K, Moossa AR, Hoffman RM.Cancer Res. 2006 Jan l;66(l):303-6.
] 9. FL-CTL assay: fluorolysometric ination of cell-mediated cytotoxicity using
green fluorescent protein and red fluorescent protein expressing target cells.Chen K, Chen L,
Zhao P, Marrero L, Keoshkerian E, Ramsay A, Cui Y.J Immunol Methods. 2005 May;300(1-
2): 100-14.
10. Murine leukemia virus (MLV) replication monitored with fluorescent
proteins.Sliva K, Erlwein O, Bittner A, Schnierle BS.Virol J. 2004 Dec 20;1 :14.
11. Real-time whole-body imaging of an orthotopic metastatic te cancer model
expressing red fluorescent n.Yang M, Jiang P, Yamamoto N, Li L, Geller J, Moossa AR,
Hoffman state. 2005 Mar 1;62(4):374-9.
12. Cellular dynamics visualized in live cells in vitro and in vivo by differential dual-
color nuclear-cytoplasmic fluorescent-protein expression.Yamamoto N, Jiang P, Yang M, Xu M,
Yamauchi K, Tsuchiya H, Tomita K, Wahl GM, Moossa AR, Hoffman RM.Cancer Res. 2004
Jun 15;64(12):4251-6.
13. In vivo imaging with fluorescent ns: the new cell biology.Hoffman RM.Acta
Histochem. 2004;106(2):77-87.
14. Real-time imaging of individual fluorescent-protein color-coded metastatic
colonies in vz'v0.Yamamoto N, Yang M, Jiang P, Xu M, Tsuchiya H, Tomita K, Moossa AR,
Hoffman RM.Clin Exp asis. 0(7):633-8.
15. Yaghoubi S, Barrio JR, m M, Iyer M, Namavari M, Satyamurthy N,
Goldman R, Herschman HR, Phelps ME, Gambhir SS. Human pharmacokinetic and dosimetry
studies of [(18)F]FHBG: a reporter probe for imaging herpes simplex virus type-1 thymidine
kinase reporter gene expression. J Nucl Med. 2001 Aug;42(8):1225-34.
16. Pafieda A, Collantes M, Beattie SG, Otano I, Snapper J, Timmermans E, Guembe
L, Petry H, Lanciego JL, Benito A, Prieto J, Rodriguez-Pena MS, Pefiuelas I, Gonzalez-
nolaza G. Adeno-associated virus liver transduction efficiency measured by in vivo
HBG positron emission tomography imaging in rodents and nonhuman primates. Hum
Gene Ther. 2011 Aug;22(8):999-1009. doi: 10.1089/hum.2010.190. Epub 2011 Apr 6.
17. Johnson M, Karanikolas BD, an SJ, Powell R, Black ME, Wu HM, Czemin
J, Huang SC, Wu L. ion of variant HSV1-tk gene expression to determine the sensitivity of
HBG PET imaging in a prostate tumor. J Nucl Med. 2009 May;50(5):757-64. doi:
.2967/jnumed.108.058438. Epub 2009 Apr 16.
18. Pefiuelas I, Mazzolini G, Boan JF, Sangro B, Marti-Climent J, Ruiz M, Ruiz J,
Satyamurthy N, Qian C, Barrio JR, Phelps ME, Richter JA, Gambhir SS, Prieto J. Positron
on Tomography Imaging of Adenoviral-Mediated Transgene Expression in Liver Cancer
Patients Gastro (2005)128: 1787.
19. Sangro B, Mazzolini G, Ruiz M, Ruiz J, Quiroga J, Herrero I, Qian C, Benito A,
Larrache J, Olagiie C, Boan J, Pefiuelas I, Sadaba B, Prieto J. A phase I clinical trial of
thymidine kinase-based gene therapy in advanced hepatocellular carcinoma Can. Gene Ther.
(2010) 17: 837—843.
20. Willmann JK, Paulmurugan R, Rodriguez-Porcel M, Stein W, Brinton TJ,
Connolly AJ, Nielsen CH, Lutz AM, Lyons J, Ikeno F, Suzuki Y, Rosenberg J, Chen IY, Wu JC,
Yeung AC, Yock P, Robbins RC, Gambhir SS. g gene sion in human
mesenchymal stem cells: from small to large animals. Radiology. 2009 Jul;252(1):117-27. doi:
.1148/radiol.2513081616. Epub 2009 Apr 14. PubMed PMID: 19366903; PubMed Central
PMCID: PMC2702468.
21. Yaghoubi SS, Jensen MC, Satyamurthy N, Budhiraja S, Paik D, Czemin J,
Gambhir SS. Noninvasive detection of therapeutic cytolytic T cells with [18F]FHBG PET in a
patient with glioma. Nat Clin Pract Oncol. 2009 Jan;6(1):53-8. doi: 10.1038/ncponc1278. Epub
2008 Nov 18. PubMed PMID: 19015650; PubMed Central PMCID: PMC3526373.
22. Roelants V, Labar D, de Meester C, Havaux X, Tabilio A, Gambhir SS, Di Ianni
M, Bol A, Bertrand L, Vanoverschelde JL. Comparison between adenoviral and retroviral
s for the transduction of the thymidine kinase PET er gene in rat mesenchymal stem
cells. J Nucl Med. 2008 Nov;49(11):1836-44. doi: 10.2967/jnumed.108.052175. Erratum in: J
Nucl Med. 2009 Jan;50(1):17. PubMed
PMID: 18984872.
23. Lee SW, Padmanabhan P, Ray P, Gambhir SS, Doyle T, Contag C, Goodman SB,
Biswal S. Stem cell-mediated accelerated bone g observed with in vivo molecular and
small animal imaging technologies in a model of skeletal injury. J Orthop Res. 2009
Mar;27(3):295-302. doi: 10.1002/jor.20736. PubMed PMID: 18752273.
24. Chin FT, Namavari M, Levi J, Subbarayan M, Ray P, Chen X, Gambhir SS.
Semiautomated radiosynthesis and biological evaluation of [18F]FEAU: a novel PET imaging
agent for HSVl-tk/sr39tk er gene expression. Mol Imaging Biol. 2008 Mar-Apr;10(2):82-
91. Epub 2007 Dec 22. PubMed PMID: 18157580.
25. Yaghoubi SS, Gambhir SS. PET g of herpes simplex virus type 1 thymidine
kinase (HSVl-tk) or mutant HSVl-sr39tk reporter gene expression in mice and humans using
HBG. Nat Protoc. (6):3069-75. PubMed PMID: 17406570.
26. Deroose CM, De A, Loening AM, Chow PL, Ray P, Chatziioannou AF, r
SS. Multimodality imaging of tumor xenografts and metastases in mice with combined small-
animal PET, small-animal CT, and bioluminescence g. J Nucl Med. 2007 (2):295-
303. PubMed PMID: 17268028; PubMed Central PMCID: PMC3263830.
27. Kim SJ, Doudet DJ, Studenov AR, Nian C, Ruth TJ, Gambhir SS, sh CH.
Quantitative micro positron emission tomography (PET) imaging for the in vivo ination
of pancreatic islet graft survival. Nat Med. 2006 Dec;12(12):1423 -8. Epub 2006 Dec 3. PubMed
PMID: 17143277.
28. Yaghoubi SS, Couto MA, Chen CC, Polavaram L, Cui G, Sen L, Gambhir SS.
Preclinical safety evaluation of [18F]FHBG: a PET reporter probe for imaging herpes simplex
virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr3 9tk's expression. J Nucl Med.
2006 Apr;47(4):706-15. PubMed PMID: 16595506.
29. Xiong Z, Cheng Z, Zhang X, Patel M, Wu JC, Gambhir SS, Chen X. Imaging
chemically d adenovirus for targeting tumors expressing integrin alphavbeta3 in living
mice with mutant herpes simplex virus type 1 ine kinase PET reporter gene. J Nucl Med.
2006 Jan;47(1):130-9. PubMed PMID: 97.
30. Shu CJ, Guo S, Kim YJ, Shelly SM, Nijagal A, Ray P, Gambhir SS, Radu CG,
Witte ON. Visualization of a primary anti-tumor immune se by positron emission
tomography. Proc Natl Acad Sci U S A. 2005 Nov 29;102(48):17412-7. Epub 2005 Nov 17.
PubMed PMID: 16293690; PubMed Central PMCID: PMC1283986.
31. Sen L, Gambhir SS, Furukawa H, Stout DB, Linh Lam A, Laks H, Cui G.
Noninvasive imaging of ex vivo intracoronarily delivered nonviral therapeutic transgene
expression in heart. Mol Ther. 2005 Jul;12(1):49-57. PubMed PMID: 15963920.
32. Yaghoubi SS, Barrio JR, Namavari M, urthy N, Phelps ME, man
HR, Gambhir SS. Imaging progress of herpes simplex virus type 1 thymidine kinase suicide
gene therapy in living ts with positron emission tomography. Cancer Gene Ther. 2005
Mar;12(3):329-39. PubMed PMID: 15592447.
33. Miyagawa M, Anton M, Haubner R, Simoes MV, Stadele C, Erhardt W, Reder S,
Lehner T, Wagner B, Noll S, Noll B, Grote M, Gambhir SS, Gansbacher B, Schwaiger M,
Bengel FM. PET of cardiac transgene expression: comparison of 2 approaches based on
herpesviral thymidine kinase reporter gene. J Nucl Med. 2004 Nov;45(11):1917-23. PubMed
PMID: 15534063.
34. Green LA, Nguyen K, Berenji B, Iyer M, Bauer E, Barrio JR, Namavari M,
Satyamurthy N, Gambhir SS. A tracer kinetic model for [18F]FHBG for tating herpes
x virus type 1 thymidine kinase reporter gene sion in living animals using PET. J
Nucl Med. 2004 Sep;45(9):1560-70. PubMed PMID: 15347725.
35. Wu JC, Chen IY, Wang Y, Tseng JR, Chhabra A, Salek M, Min JJ, Fishbein MC,
Crystal R, Gambhir SS. Molecular imaging of the kinetics of ar endothelial growth factor
2014/029600
gene expression in ic myocardium. Circulation. 2004 Aug 10;110(6):685-91. PubMed
PMID: 15302807.
36. Su H, Forbes A, Gambhir SS, Braun J. tation of cell number by a positron
emission tomography reporter gene strategy. Mol Imaging Biol. 2004 May-Jun;6(3): 139-48.
PubMed PMID: 15193248.
37. Chen IY, Wu JC, Min JJ, Sundaresan G, Lewis X, Liang Q, Herschman HR,
Gambhir SS. Micro-positron emission tomography imaging of cardiac gene expression in rats
using bicistronic adenoviral vector-mediated gene ry. Circulation. 2004 Mar
23;109(11):1415-20. Epub 2004 Mar 8. PubMed PMID: 15007006.
38. Sundaresan G, Paulmurugan R, Berger F, Stiles B, Nagayama Y, Wu H, Gambhir
SS. MicroPET g of Cre-loxP-mediated conditional activation of a herpes simplex virus
type 1 thymidine kinase reporter gene. Gene Ther. 2004 Apr;l l(7):609-l8. PubMed PMID:
14724687.
39. Green LA, Yap CS, Nguyen K, Barrio JR, Namavari M, Satyamurthy N, Phelps
ME, Sandgren EP, Herschman HR, Gambhir SS. Indirect monitoring of endogenous gene
expression by positron emission aphy (PET) imaging of reporter gene expression in
transgenic mice. Mol Imaging Biol. 2002 Jan;4(1):7l-8l. PubMed PMID: 50.
40. Ghosh P. Reproducible quantification in PET-CT: Clinical relevance and
technological approaches. White Paper Siemens ary 2012).
41. Shankar L.K. et al. Consensus recommendations for the use of 18F-FDG as an
indicator of therapeutic response in National Cancer Institute trials. J. Nucl. Med. 2006 June;
47: 1059-66.
42. Bar-Shir A. et al. Transforming thymidine into a ic resonance imaging
probe for gene expression. J. Am. Chem. Soc. 2013 Jan; 17-24.
43. Muller U. et al. Synthesis and inical evaluation of a new C-6 alkylated
pyrimidine derivative as a PET imaging agent for HSVl-tk gene expression. Am. J. Nucl. Med.
Mol. Imaging 2013; 3:71-84.
44. Mescic A. et al. C-5 hydroxyethyl and hydroxypropyl acyclonucleosides as
substrates for thymidine kinase of Herpes simplex virus type 1 (HSV-l TK): Syntheses and
biological evaluation. Molecules 2013; 18:5104-24.
VV()2014/153205
SE UENCES
SEQ ID NO: 1: wild type HSVl-TK nucleotide ce
a:ggcttcgtaccccggccatcaacacgcg SC egchchaccaggctgcgcgt :ctcgcggcc
a:agcaaccgacg':acggcg:tgcgccc:cgccggcagcaagaagccacggaag':ccgcccgga
gcagaaaatgcccacgctaetgcggg “a,a eagacggtccccacgggatggggaaaaccacc
accacgcaachc egg vggcccvggg “cgcgcgacga:atcgtctacgtacccgagccgatga
cttactggcgggtgctgggggc :tccgagacaatcgcgaacatctacaccacacaacaccgcc:
cgaccaggg ,gagaLa ggggacgcggcgg egg ,aa egacaagcgcccagataacaatg
ggca,gch ,aLgccg ,gaccgacgcchLchgc “CC ,Ca,a ecgggggggaggc:gggagc
cacatgccccgcccccggccc:caccctcatc:tcgaccgcca :cccatcgccgccctcctg:g
C:acccggccgcgcgg ,ach eanggcagcatgaccccccaggccg ,chggch ,chggcc
Ceca ,cccgccgacct:gcccggcaccaacatcgtgc :tggggccc eccggaggacagacaca
:cgaccgcc:ggccaaacgccagcgccccggcgagcggc:ggaccngcLaLchggc ,gcgaL
:cgccgcgt:tacgggc:acttgccaaLacgg egcgg ea “cvgcagvgcggcgggtcg'199099
gaggactggggacagc ecggggacggccgtgccgccccaggg':gccgagccccagagcaacg
cacgacccca ,cggggacachLa ,Laccc eg ,Lchggcccccgagttgctggc
ccccaacggcgacc eg ,aacg,gLLLgcc ,gggccttggacg':cttggccaaacgcc:ccgt
tccatgcachcL ea ,chgga ,Lacgaccaa:cgcccgccggctgccgggacgccctgctgc
aacttacctccggga egg eccagacccacgtcaccacccccggc :ccataccgacgatatgcga
cctggcgcgcacg ,gcccgggagatgggggaggctaactga
SEQ ID NO: 2: wild type HSVl-TK amino acid sequence
MASYPGHQ {ASAEiDQAARSRGHSN {{TALR?RRQQ fiAT fiVRP fiQKMPTLL<VYn DGPHGMG {TT
TTQLLVALGS { "VY
V?: ?MTYWQV. AN YTTQ {LDQGZ SAGDAAVVMTSAQ: TMG ?YAVTDAVLAP I
G {A}
b QYLMGSMTPQAVPAFVAL" N"VPGAPP 13R {PAK {Q ?G
I R<VYG
LLAWTVQYLQCGGSW {Ll. DWGQLSGTAVPPQGAEPQSNAGPRP { GDTPETPE{A' fiPPA?NG3L
YWVFAWALDVLA<RLQ
S HVF: LDYDQS ?AGCaDALLQLTSGMVQTHVTTPGS PT CDPA{TEAR fiMG fiAW
SEQ ID NO: 3: HSV-TK in une-C HSV-TK; SR 39 mutant and R25G-R26S
Mutation of NLS
a:ggcctcgtaccccggccatcaacacgcg SC accaggctgcgcgt :ctcgcggcc
atagcaacggatccacggcg:tgcgccc:cgccggcagcaagaagccacggaag':ccgcccgga
gcagaaaatgcccacgctaetgcggg “a,a eagacggtccccacgggatggggaaaaccacc
accacgcaactgctggtggccc eggg “cgcgcgacga:atcgtctacgtacccgagccgatga
ggcgggtgctgggggc :tccgagacaatcgcgaacatctacaccacacaacaccgcc:
cgaccaggg ,gagaLa ecggccggggacgcggcgg egg ,aa egacaagcgcccagataacaatg
ggca,gch g ,gaccgacgcch,chgc “CC ,Ca,a gggaggCZgggagC
cacatgccccgcccccggccc:caccatCttcctcgaccgcca ,cccachchLcaLchg eg
C:acccggccgcgcgg ,ach ,anggcagcatgaccccccaggccg ,chggch ,chggcc
Ceca ,cccgccgacct:gcccggcaccaacatcgtgc :tggggccc eccggaggacagacaca
:cgaccgcc:ggccaaacgccagcgccccggcgagcggc:ggaccngcLaLchggc ,gcgaL
:cgccgcgt:tacgggctacttgccaaLacgngcgg ea chgcagvgcggcgggtcg'199099
gaggactggggacagcsLscggggacggccgtgccgccccagggtgccgagccccagagcaacg
cgggcccacgacccca,a,cggggacacgttat2taccctgtttcgggcccccgagttgctggc
cggcgacc,g,a,aacg,gLLLgcc,gggccttggacg:cttggccaaacgcc:ccgt
tccatgcachcLSea,chgga,Lacgaccaa:cgcccgccggctgccgggacgccctgctgc
aacttacctccgggaegg,ccagacccacgtcaccacccccggc:ccataccgacgatatgcga
cctggcgcgcacg,SSgcccgggagatgggggaggctaactga
SEQ ID NO: 4 (amino acid sequence encoded by SEQ ID NO: 3)
MASYPGHQHASAEDQAA<S{GHSNGSTALR?RRQQfiATfiV{?fiQ<M?TLLRVYIDGPHGMGKTT
TTQLLVALGSRDD VYV?%?MTYWQVPGASHT AN YTTQi<PDQGfi SAGDAAVVMTSAQIT
mLu'r'G) ?YAVTDAVPA?{ GGHAGSS{A???ALT ELDR{? AF PCY?AARYLMGSMTPQAVLAFVA
"?PTLPGTW"VLGAL?fiD{H D<PA<RQ<?G%{LDLAMPAA {{VYGLLAWTVRYLQCGGSW?
GTAVP?QGAE?QSNAG?{?{ GDTLETLE{A?flLLA?WGDLYNVFAWALDVLAKRLQ
{VFILDYDQS?AGC?DALLQLTSG VQTiVTTPGS ?T CDLARTEARH GfiAN
SEQ ID NO: 5: HSV-TK Sites to mutate are in bold, underlining (HSV-TK nuclear
localization sequence, RR, and Substrate Binding Domain, LIF and AAL
atggcctcgtaccccggccatcaacacgcgtCtgcgttcgaccaggctgcgcgttctcgc 60
M A S Y P G H Q T A S A F D Q A A R S R
ggccatagcaaccgacgtacggcg:tgcgccc:cgccggcagcaagaagccacggaagtc
G H S N 3 3 T A L R P 3 3 Q Q E A T E V
cgcccggagcagaaaatgcccacgctactgcgggtttatatagacggtccccacgggatg
R P E Q K M P T L L R V Y I D G P H G M
gggaaaaccaccaCcacgcaactgctggtggccctgggttcgcgcgacgatatcgtctac
G K T T T T Q L L V A L G S R D D I V Y
gtacccgagccgatgacttactggcgggtgctgggggcttccgagacaatcgcgaacatc
V P E P M T Y W R V L G A S E T I A N
acacaacaccgcctcgaccagggtgagatatcggccggggacgcggcggtggta
Y T T Q H R L D Q G d S A G D A A V V
atgacaagcgcccagataacaatgggcatgccttatgccgtgaccgacgccgttctggct
M T S A Q I T M G M P Y A V T D A V L A
cctcatatcgggggggaggctgggagctcacatgccccgcccccggccctcaccctcatc
P H I G G E A G S S T A P P P A L T E l
EEggaccgccatcccatcgccgccctcctgtgctacccggccgcgcggtaccttatgggc
F D R H P I A A E L C Y P A A R Y L M G
agcatgaccccccaggccgtgctggcgttcgtggccctcatcccgccgaccttgcccggc
WO 53205
S M T P Q A V L A F V A L I P P T L P G
accaacatcgtgcttggggcccttccggaggacagacacatcgaccgcctggccaaacgc
T N I V L G A L P Lti D R H I D R L A K R
CagcgccccggcgagcggctggaccngcLaLchgchgcgaLchccgcgtttacggg
Q R P G E R L D L A M L A A I R R V Y G
ctacttgccaaLacgngcgg,aLCLgcagLgcggcgggtcgtggcgggaggactgggga
L L A N T V R Y L Q C G G S W R m D W G
cagctttcggggacggccgtgccgccccagggtgccgagccccagagcaacgcgggccca
Q L S G T A V P P Q G A E P Q S N A G P
cgaccccatatcggggacacg,La,LLacchgLLchggcccccgagttgctggccccc
R P H I G D T L F T L F R A P m L L A P
aacggcgachgLaLaachgLLLgccnggccttggacgtCttggccaaacgcctccgt
N G D L Y N V F A W A L D V L A K R L R
tccatgcacgtctttatcctggattacgaccaatcgcccgccggctgccgggacgccctg
1020
S M H V F I L D Y D Q S P A G C R D A L
ctgcaacttacctccgggatggtccagacccacgtcaccacccccggc:ccataccgacg
1080
L Q L T S G M V Q T T V T T P G S I P T
atatgcgacctggcgcgcacgtttgcccgggagatgggggaggctaactga
C D L A R T F A R E M G E A N *
SEQ ID NOS: 6 and 7: Sac I-Kpn I (SR39) mutant region
GAGCTCACATGCCCCGCCCCCGGCCCTCACCéTCETCETCGACCGCCATCCCATCGCC-
ETCGAGTGTACGGGGCGGGGGCCGGGAGTGGEAGéAGEAGCTGGCGGTAGGGTAGCGG-
Sac "
—EEC§T§CTGTGCTACCCGGCCGCGCGGTACC (SfiQ 3 NO: 6)
—§§GEAEGACACGATGGGCCGGCGCGCCATGG (SfiQ 3 NO: 7)
Kpn "
IIII: :IIII Gm-
IIIIIIIIIII AG” ' GAGCTC
IIIIIIIIIII
SEQ ID NOS: 8 and 9: Sac I-Kpn I (SR39) mutant region (cut)
CACATGCCCCGCCCCCGGCCCTCACCéTCETCETCGACCGCCATCCCATCGCCEECéTE
TCGAGTGTACGGGGCGGGGGCCGGGAGTGGEAGéAGEAGCTGGCGGTAGGGTAGCGGéé
Sac I (cut)
CTGTGCTACCCGGCCGCGCGGTAC (SfiQ 3 NO: 8)
GEAEGACACGATGGGCCGGC (SfiQ 3 NO: 9)
Kpn 2(Cut)
GGTACC IIIIIIIIIII GTAC - 3’
IIIIIIIIIII
SEQ ID NOS: 10 and 11: Primers
SR39sackpn F1
’CACATGCCCCGCCCCCGGCCCTCACCéTCETCETCGACCGCCATCCCATCGCCEECéTECTG
TGCTACCCGGCCGCGCGGTAC 3’ (SfiQ 3 NO: 10)
ckpn R1
’CGCGCGGCCGGGTAGCACAGCATGAAGGCGATGGGATGGCGGTCGAEGAéGAEGGTGAGGGC
CGGGGGCGGGGCATGTGAGCT 3’ (SfiQ 3 NO: 11)
SEQ ID NO: 12 Gene #3 mHSV-TK CO A168H(LIF...AHL): Length:1185
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC
CCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC
CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC
GACCGGCACCCAATCGCACACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA
CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT
CGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG
CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC
GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC
ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC
ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG
CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC
ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA
ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG
CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA
SEQ ID NO: 13 Gene #4 mHSV-TK CO TK A167F(LIF...FAL): Length:1185
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC
CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC
CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC
CACCCAATCTTCGCACTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA
CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT
GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG
CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC
GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC
ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC
ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG
ACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC
CGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA
ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG
CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA
SEQ ID NO: 14 Gene #5 mHSV-TK CO dual mutant A167F-A168H (LIF...FHL):
Length:1185
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC
CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC
CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC
GACCGGCACCCAATCTTCCACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA
CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT
GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG
CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC
GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC
ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC
ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG
CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC
ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA
ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG
CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA
SEQ ID NO: 15 Gene #6 mHSV-TK CO MB-IFL A168H(IFL...AHL): Length:1185
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC
CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCACGGCGC
CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCATCTTCCTG
GACCGGCACCCAATCGCACACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA
CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT
GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG
CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC
GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC
ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC
ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG
CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC
ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA
ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG
CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA
SEQ ID NO: 16 Gene #1 HSV-TK A168H dmNLS CO SC: Length:1185
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC
CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCAGGATCT
GAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC
CACCCAATCGCACACCTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA
CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT
GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG
CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC
GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC
ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC
ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG
CCCTGGACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC
ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA
ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG
CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA
SEQ ID NO: 17 Gene #2 HSV-TK A167F dmNLS CO SC: Length:1185
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC
CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCACTGCGGCCAGGATCT
CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGACCACACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATTACAATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
ACCACACATCGGCGGCGAGGCCGGCAGCAGCCACGCACCACCACCAGCACTGACCCTGATCTTC
GACCGGCACCCAATCTTCGCACTGCTGTGCTACCCGGCAGCACGCTACCTGATGGGCTCCATGA
CACCACAAGCCGTGCTGGCCTTCGTGGCCCTGATCCCACCAACACTGCCCGGCACCAACATCGT
GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG
CGCCTGGACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC
TGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC
ACCACAGGGCGCCGAGCCACAGAGCAACGCCGGACCACGACCACACATCGGCGACACCCTGTTC
ACCCTGTTCCGGGCACCAGAGCTGCTGGCACCAAACGGCGACCTGTACAACGTGTTCGCCTGGG
ACGTGCTGGCCAAGCGCCTGCGCTCCATGCACGTGTTCATCCTGGACTACGACCAGTC
ACCGGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACA
ACACCCGGCAGCATCCCAACAATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG
CCAACTAATAGGGATCCCTCGAGAAGCTTGTCA
SEQ ID NO: 18 Gene #3 HSV-TK A168H NESdmNLS CO SC: Length:1221
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACT
GGATGGCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGC
CACAGCAACGGCAGCACCGCACTGCGGCCAGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCG
AGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCGACGGACCACACGGCATGGGCAAGACCAC
CACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATG
ACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCCAGCACCGCC
TGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATTACAAT
GGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCACCACACATCGGCGGCGAGGCCGGCAGC
AGCCACGCACCACCACCAGCACTGACCCTGATCTTCGACCGGCACCCAATCGCACACCTGCTGT
GCTACCCGGCAGCACGCTACCTGATGGGCTCCATGACACCACAAGCCGTGCTGGCCTTCGTGGC
CCTGATCCCACCAACACTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCAC
ATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCA
TCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCG
CGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCACCACAGGGCGCCGAGCCACAGAGCAAC
GCCGGACCACGACCACACATCGGCGACACCCTGTTCACCCTGTTCCGGGCACCAGAGCTGCTGG
CACCAAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCG
GCACGTGTTCATCCTGGACTACGACCAGTCACCGGCCGGCTGCCGCGACGCCCTGCTG
CAGCTGACCAGCGGCATGGTGCAGACCCACGTGACAACACCCGGCAGCATCCCAACAATCTGCG
CCCGCACCTTCGCCCGCGAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCT
TGTCA
SEQ ID NO: 19 Gene #4 HSV-TK A167F NESdmNLS CO SC: Length:1221
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCCCTGCAGAAAAAGCTGGAAGAGCTGGAACT
GGATGGCAGCTACCCCGGCCACCAGCACGCCAGCGCCTTCGACCAGGCCGCCCGCAGCCGCGGC
CACAGCAACGGCAGCACCGCACTGCGGCCAGGATCTCAGCAGGAGGCCACCGAGGTGCGCCCCG
AGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCGACGGACCACACGGCATGGGCAAGACCAC
CACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGACGACATCGTGTACGTGCCCGAGCCCATG
ACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATCGCCAACATCTACACCACCCAGCACCGCC
TGGACCAAGGCGAGATCAGCGCCGGCGACGCCGCCGTGGTGATGACCAGCGCCCAGATTACAAT
GGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGCACCACACATCGGCGGCGAGGCCGGCAGC
AGCCACGCACCACCACCAGCACTGACCCTGATCTTCGACCGGCACCCAATCTTCGCACTGCTGT
GCTACCCGGCAGCACGCTACCTGATGGGCTCCATGACACCACAAGCCGTGCTGGCCTTCGTGGC
CCTGATCCCACCAACACTGCCCGGCACCAACATCGTGCTGGGCGCCCTGCCCGAGGACCGCCAC
ATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAGCGCCTGGACCTGGCCATGCTGGCCGCCA
TCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGCGCTACCTGCAGTGCGGCGGCAGCTGGCG
CGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCCACCACAGGGCGCCGAGCCACAGAGCAAC
GCCGGACCACGACCACACATCGGCGACACCCTGTTCACCCTGTTCCGGGCACCAGAGCTGCTGG
CACCAAACGGCGACCTGTACAACGTGTTCGCCTGGGCCCTGGACGTGCTGGCCAAGCGCCTGCG
CTCCATGCACGTGTTCATCCTGGACTACGACCAGTCACCGGCCGGCTGCCGCGACGCCCTGCTG
CAGCTGACCAGCGGCATGGTGCAGACCCACGTGACAACACCCGGCAGCATCCCAACAATCTGCG
ACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGGCCAACTAATAGGGATCCCTCGAGAAGCT
TGTCA
SEQ ID NO: 20 Gene #5 HSV-TK A168H NESdmNLS JCO SC: Length:1221
GGCCGCACCGGTACGCGTCCACCATGGCTCTGCAGAAAAAGCTGGAAGAGCTGGAACT
GGATGGCTCTTATCCTGGACATCAGCATGCTTCTGCTTTTGATCAGGCTGCCAGATCTAGAGGA
CATTCTAATGGCAGCACAGCACTGCGGCCAGGATCTCAGCAGGAAGCTACAGAAGTGAGACCTG
AACAGAAAATGCCTACACTGCTGAGAGTGTATATTGATGGACCACATGGAATGGGAAAAACAAC
CACAACCCAGCTGCTGGTGGCTCTCGGATCTAGAGATGATATTGTGTATGTGCCTGAACCTATG
ACATATTGGAGAGTGCTGGGAGCTTCTGAAACAATTGCTAATATCTATACAACACAGCATAGAC
TGGATCAAGGAGAAATTTCTGCCGGAGATGCTGCCGTGGTGATGACATCTGCTCAGATTACAAT
GCCTTATGCTGTGACAGATGCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGC
TCTCATGCACCACCACCAGCACTGACACTGATTTTTGATCGGCATCCAATTGCACATCTGCTGT
GTTATCCGGCAGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGC
TCTGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGATAGACAT
ATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGCCATGCTGGCTGCCA
TTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCTGCAGTGTGGAGGCTCTTGGAG
AGAGGATTGGGGACAGCTGTCTGGCACAGCTGTGCCACCACAGGGAGCCGAACCACAGAGCAAT
GCTGGACCACGACCACATATCGGAGACACACTGTTTACACTGTTTCGGGCACCAGAACTGCTGG
CACCAAATGGAGACCTGTACAACGTGTTTGCCTGGGCTCTGGATGTGCTGGCTAAACGGCTGAG
ATCTATGCATGTGTTTATCCTGGACTATGATCAGTCACCGGCCGGATGTCGCGATGCCCTGCTG
CAGCTGACATCTGGGATGGTGCAGACACATGTGACAACACCTGGATCTATCCCAACAATCTGTG
ATCTGGCTAGAACATTCGCTAGGGAGATGGGAGAGGCCAACTAATGAGGATCCCTCGAGAAGCT
TGTCA
SEQ ID NO: 21 Gene #6 HSV-TK A167F LS JCO SC: Length:1221
GTCAGCGGCCGCACCGGTACGCGTCCACCATGGCTCTGCAGAAAAAGCTGGAAGAGCTGGAACT
GGATGGCTCTTATCCTGGACATCAGCATGCTTCTGCTTTTGATCAGGCTGCCAGATCTAGAGGA
CATTCTAATGGCAGCACAGCACTGCGGCCAGGATCTCAGCAGGAAGCTACAGAAGTGAGACCTG
AACAGAAAATGCCTACACTGCTGAGAGTGTATATTGATGGACCACATGGAATGGGAAAAACAAC
CACAACCCAGCTGCTGGTGGCTCTCGGATCTAGAGATGATATTGTGTATGTGCCTGAACCTATG
ACATATTGGAGAGTGCTGGGAGCTTCTGAAACAATTGCTAATATCTATACAACACAGCATAGAC
TGGATCAAGGAGAAATTTCTGCCGGAGATGCTGCCGTGGTGATGACATCTGCTCAGATTACAAT
GGGAATGCCTTATGCTGTGACAGATGCTGTGCTGGCACCACATATTGGAGGCGAAGCTGGAAGC
TCTCATGCACCACCACCAGCACTGACACTGATTTTTGATCGGCATCCAATTTTCGCACTGCTGT
GTTATCCGGCAGCAAGATATCTGATGGGAAGCATGACACCACAAGCCGTGCTGGCTTTTGTGGC
TCTGATTCCACCAACACTGCCTGGAACAAACATCGTGCTGGGAGCTCTGCCTGAAGATAGACAT
ATCGATCGGCTGGCCAAACGGCAGAGACCTGGAGAACGGCTGGATCTGGCCATGCTGGCTGCCA
TTCGGAGAGTGTATGGCCTGCTGGCTAACACAGTGAGATATCTGCAGTGTGGAGGCTCTTGGAG
TTGGGGACAGCTGTCTGGCACAGCTGTGCCACCACAGGGAGCCGAACCACAGAGCAAT
GCTGGACCACGACCACATATCGGAGACACACTGTTTACACTGTTTCGGGCACCAGAACTGCTGG
CACCAAATGGAGACCTGTACAACGTGTTTGCCTGGGCTCTGGATGTGCTGGCTAAACGGCTGAG
ATCTATGCATGTGTTTATCCTGGACTATGATCAGTCACCGGCCGGATGTCGCGATGCCCTGCTG
CAGCTGACATCTGGGATGGTGCAGACACATGTGACAACACCTGGATCTATCCCAACAATCTGTG
ATCTGGCTAGAACATTCGCTAGGGAGATGGGAGAGGCCAACTAATGAGGATCCCTCGAGAAGCT
TGTCA
SEQ ID NO: 22
HSV-TK dmNLS Al68H, CO & SC
dmNLS = double mutated Nuclear Localization Sequence
CO = codon optimized
SC = splice corrected at 327 and 555
Kozak Sequence, Underlined
gtcaGCGGCCGCACCGGTACGCGTCCACCATGGCCAGCTACCCCGGCCACCAGCACGCCAGCGC
CTTCGACCAGGCCGCCCGCAGCCGCGGCCACAGCAACGGCAGCACCGCaCTGCGgCCaGGATCT
CAGCAGGAGGCCACCGAGGTGCGCCCCGAGCAGAAGATGCCCACCCTGCTGCGCGTGTACATCG
ACGGaCCaCACGGCATGGGCAAGACCACCACCACCCAGCTGCTGGTGGCCCTGGGCAGCCGCGA
CGACATCGTGTACGTGCCCGAGCCCATGACCTACTGGCGCGTGCTGGGCGCCAGCGAGACCATC
GCCAACATCTACACCACCCAGCACCGCCTGGACCAaGGCGAGATCAGCGCCGGCGACGCCGCCG
TGGTGATGACCAGCGCCCAGATtACaATGGGCATGCCCTACGCCGTGACCGACGCCGTGCTGGC
aCCaCACATCGGCGGCGAGGCCGGCAGCAGCCACGCaCCaCCaCCaGCaCTGACCCTGATCTTC
GACCGgCACCCaATCGCaCACCTGCTGTGCTACCCgGCaGCaCGCTACCTGATGGGCtccATGA
CaCCaCAaGCCGTGCTGGCCTTCGTGGCCCTGATCCCaCCaACaCTGCCCGGCACCAACATCGT
GCTGGGCGCCCTGCCCGAGGACCGCCACATCGACCGCCTGGCCAAGCGCCAGCGCCCCGGCGAG
GACCTGGCCATGCTGGCCGCCATCCGCCGCGTGTACGGCCTGCTGGCCAACACCGTGC
GCTACCTGCAGTGCGGCGGCAGCTGGCGCGAGGACTGGGGCCAGCTGAGCGGCACCGCCGTGCC
aCCaCAGGGCGCCGAGCCaCAGAGCAACGCCGGaCCaCGaCCaCACATCGGCGACACCCTGTTC
ACCCTGTTCCGgGCaCCaGAGCTGCTGGCaCCaAACGGCGACCTGTACAACGTGTTCGCCTGGG
CCCTGGACGTGCTGGCCAAGCGCCTGCGCtccATGCACGTGTTCATCCTGGACTACGACCAGtc
aCCgGCCGGCTGCCGCGACGCCCTGCTGCAGCTGACCAGCGGCATGGTGCAGACCCACGTGACa
ACaCCCGGCAGCATCCCaACaATCTGCGACCTGGCCCGCACCTTCGCCCGCGAGATGGGCGAGG
CCAACTAATAGGGATCCCTCGAGAAGCTTgtca
SEQ ID NO: 23 — MAP Kinase Kinase Nuclear Export Polynucleotide Sequence
CTGCAGAAAAAGCTGGAAGAGCTGGAACTGGATGGC
SEQ ID NO: 24 - MAP Kinase Kinase r Export Polypeptide ce
LQKKLfifiLfiLDG
SEQ ID NO: 25 — Targeting Moiety
WREPSF ALS
Claims (43)
1. Use of a retroviral particle sing an HSV-TK polynucleotide encoding a mutated form of HSV-TK comprising a nuclear export sequence (NES) in the manufacture of a diagnostic for identifying a patient with lesions capable of benefitting from gene therapy, n the identifying comprises: a) administering the retroviral particle comprising the HSV-TK polynucleotide comprising the NES to the patient, wherein the HSV-TK polynucleotide encodes a d form of HSV-TK comprising ons at amino acid residues 32, 33, and 168 of the viral r localization sequence (NLS), wherein the amino acid residues 32 and 33 are each independently mutated to an amino acid chosen from the group consisting of glycine, serine, glutamic acid, an acidic amino acid, and cysteine, and wherein the mutated form of HSV-TK increases cell kill activity relative to a wild-type HSV-TK; b) administering to the patient a substrate of HSV-TK attached to a radioactive tracer; c) ing the ve amount and location of the radioactive signal present in the patient; and d) determining the on of lesions in the patient, wherein patients with: ctive signals above a certain threshold, and location of the radioactive signal correlating with lesions measured in step c) of the patient, are identified as capable of tting from gene therapy treatment.
2. The use of claim 1, wherein the ate of HSV-TK is chosen from the group consisting of FHBG (9-[4-fluoro(hydroxymethyl)butyl]guanine), FHPG (9-([3-fluoro- oxy propoxy]methyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (1-(2'- deoxy-2'-fluoro13-D-arabinofuranosyl)iodouracil), FEAU (fluoroethyl-l-beta-D- arabinofuranosyluracil), FMAU (fluoromethyl-l-beta-D-arabinofuranosyluracil), FHOMP (6-((1 - fluorohydroxypropanyloxy)methyl)methylpyrimidine-2,4(1H,3H)-dione), ganciclovir, valganciclovir, acyclovir, valacyclovir, penciclovir, radiolabeled pyrimidine with 4-hydroxy (hydroxymethyl)butyl side chain at N-1 (HHGFEP) or 5-(2-)hydroxyethyl)-and 5-(3- hydroxypropy1)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains.
3. The use of claim 1 or claim 2, wherein the substrate of HSV-TK is FHBG (9-[4-fluoro (hydroxymethyl)butyl]guanine).
4. The use of any one of claims 1-3, wherein the radioactive tracer is 18F, 64Cu, 99mTe, 11C, 14C, 124I, 123I, 131I, 15O, 13N and/or 82RbCl.
5. The use of any one of claims 1-4, wherein the radioactive tracer is 18F.
6. The use of claim 1, wherein the HSV-TK substrate is [18F]FHBG 18F-fluoro (hydroxymethyl)butyl]guanine).
7. The use of any one of claims 1-6, n the ctive signal is measured using positron emission tomography (PET) scanning.
8. The use of claim 7, wherein the threshold level is at least above 2.0 SUV (standardized uptake value) or at least 20% above background when the radioactive signal is measured using PET scanning.
9. The use of claim 7, n the threshold level is between 1.0 SUV and 3.0 SUV, or between 20% to 40% above background when the radioactive signal is measured using PET scanning.
10. The use of any one of claims 1-9, wherein the mutation is A168H.
11. The use of any one of claims 1-10, wherein the nuclear export sequence (NES) is located at or near the amino terminus of the expressed d form of HSV-TK.
12. The use of claim 1, wherein the HSV-TK polynucleotide is SEQ ID NO: 18.
13. The use of claim 12, wherein the retroviral particle further comprises a polynucleotide encoding for a targeting protein expressed on the viral envelope.
14. The use of claim 13, wherein the targeting protein binds to collagen, laminin, fibronectin, elastin, glycosaminoglycans, proteoglycans or RGD.
15. The use of claim 14, wherein the targeting protein binds to collagen.
16. The use of claim 15, wherein the ing protein is SEQ ID NO: 25.
17. Use of a retroviral particle comprising an HSV-TK polynucleotide encoding a mutated form of HSV-TK comprising a NES in the manufacture of a medicament for treating a patient in need of treatment for lesions capable of tting from gene therapy treatment, wherein the patient is identified by the following steps a)-e): a) administering the retroviral particle comprising the HSV-TK polynucleotide comprising the NES to the patient, and transducing cells from the patient with the polynucleotide encoding HSV-TK, wherein the HSV-TK polynucleotide s a d form of HSV-TK sing mutations at amino acid residues 32, 33, and 168, wherein the amino acid residues 32 and 33 are each independently mutated to an amino acid chosen from the group ting of glycine, serine, glutamic acid, an acidic amino acid and ne, and wherein the mutated form of HSV-TK increases cell kill activity relative to a wild-type HSV-TK; b) treating the cells with a substrate of HSV-TK attached to a radioactive tracer; c) measuring the relative amount and on of radioactive signal present in target tissue; d) identifying if a level of radioactive signal is above a threshold; e) determining the location of lesions in the patient; and f) re-administering the patient with the retroviral particle comprising the HSV-TK polynucleotide of a) if: (i) the measured radioactive signal in the patient is above the threshold; and (ii) the location of the ed radioactive signal correlates with the location of the lesions measured in e).
18. The use of claim 17, wherein the substrate of HSV-TK is chosen from the group consisting of FHBG fluoro(hydroxymethyl)butyl]guanine), FHPG (9-([3-fluoro- l—hydroxy propoxy]methyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (1-(2'- 2'-fluoro(3-D-arabinofuranosyl)iodouracil), FEAU (fluoroethyl-l-beta-D- arabinofuranosyluracil), FMAU (fluoromethyl-l-beta-D-arabinofuranosyluracil), FHOMP (6-((1 - fluorohydroxypropanyloxy)methyl)methylpyrimidine-2,4(1H,3H)-dione), ganciclovir, valganciclovir, acyclovir, clovir, lovir, radiolabeled pyrimidine with 4-hydroxy (hydroxymethyl)butyl side chain at N-1 (HHGFEP) or 5-(2-)hydroxyethyl)-and 5-(3- ypropyl)-substituted pyrimidine derivatives bearing 2,3-dihydroxypropyl, acyclovir-, ganciclovir- and penciclovir-like side chains.
19. The use of claim 17, wherein the substrate of HSV-TK is FHBG (9-[4-fluoro (hydroxymethyl)butyl]guanine).
20. The use of any one of claims 17 to 19, wherein the radioactive tracer is 18F, 64Cu, 99mTe, 11C, 14C, 124I, 123I, 131I, 15O, 13N and/or 82RbCl.
21. The use of any one of claims 17 to 20, n the radioactive tracer is 18F.
22. The use of claim 21, wherein the HSV-TK substrate is [18F]FHBG (9-[4-18F-fluoro (hydroxymethyl)butyl]guanine).
23. The use of any one of claims 17 to 22, n the radioactive signal is measured using positron emission tomography (PET) scanning.
24. The use of claim 23, wherein the threshold level is at least above 2.0 SUV (standardized uptake value) or at least 20% above background when the radioactive signal is ed using the PET scan.
25. The use of claim 23, wherein the threshold level is between 1.0 SUV and 3.0 SUV when the radioactive signal is measured using the PET scan.
26. The use of claim 24 or claim 25, wherein the gene therapy retroviral particle comprises a second therapeutic polynucleotide.
27. The use of any one of claims 17 to 26, wherein the mutation is A168H.
28. The use of any one of claims 17 to 27, wherein the NES is located at or near the amino terminus of the expressed mutated form of .
29. The use of claim 17, wherein the HSV-TK polynucleotide is SEQ ID NO: 18.
30. The use of claim 29, wherein the retroviral particle further comprises a polynucleotide encoding for a ing protein expressed on the viral envelope.
31. The use of claim 30, wherein the targeting protein binds to collagen, laminin, fibronectin, elastin, glycosaminoglycans, proteoglycans, or RGD.
32. The use of claim 31, wherein the targeting protein binds to collagen.
33. The use of claim 32, wherein the targeting protein is SEQ ID NO: 25.
34. The use of any one of claims 1 to 9, wherein the d form of HSV-TK r comprises mutations at amino acid residues 25 or 26, wherein the amino acid residues correspond to positions 25 and 26 of SEQ ID NO: 2.
35. The use of claim 34, wherein amino acid es 25 or 26 are mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamic acid.
36. The use of any one of claims 1 to 9, or 34, wherein amino acid residue 168 is d to an amino acid selected from the group consisting of: histidine, lysine, cysteine, serine, and phenylalanine.
37. The use of any one of claims 17 to 26, wherein the d form of HSV-TK further comprises mutations at amino acid residues 25 or 26, wherein the amino acid residues correspond to positions 25 and 26 of SEQ ID NO: 2.
38. The use of claim 37, wherein amino acid es 25 or 26 are mutated to an amino acid chosen from the group consisting of: glycine, serine, and glutamic acid.
39. The use of any one of claims 17 to 26, wherein amino acid residue 168 is mutated to an amino acid selected from the group consisting of: histidine, lysine, cysteine, serine, and phenylalanine.
40. The use of claim 34, wherein the mutation is A168H.
41. The use of claim 34 or claim 40, n the nuclear export sequence (NES) is located at or near the amino terminus of the expressed d form of HSV-TK.
42. The use of claim 37, wherein the mutation is A168H.
43. The use of claim 37 or claim 42, wherein the nuclear export sequence (NES) is located at or near the amino terminus of the expressed mutated form of HSV-TK.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361784901P | 2013-03-14 | 2013-03-14 | |
US61/784,901 | 2013-03-14 | ||
PCT/US2014/029600 WO2014153205A1 (en) | 2013-03-14 | 2014-03-14 | Thymidine kinase diagnostic assay for gene therapy applications |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ712211A NZ712211A (en) | 2021-08-27 |
NZ712211B2 true NZ712211B2 (en) | 2021-11-30 |
Family
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