U.S. patent application number 17/537837 was filed with the patent office on 2022-06-02 for methods and compositions for reducing metastases.
The applicant listed for this patent is THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL. Invention is credited to Tyler GOODWIN, Leaf Huang, Rihe LIU, Lei MIAO.
Application Number | 20220168439 17/537837 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-02 |
United States Patent
Application |
20220168439 |
Kind Code |
A1 |
Huang; Leaf ; et
al. |
June 2, 2022 |
METHODS AND COMPOSITIONS FOR REDUCING METASTASES
Abstract
The subject matter described herein is directed to methods of
modifying the micro-environment of a target cell or The methods
comprise systemically administering to a subject a composition
comprising a vector, wherein the vector comprises a construct for
the expression of a trap in the target cell, wherein the trap is
expressed in the target cell thereby modifying the
micro-environment. Also described herein are methods of reducing
metastasis of a cancer comprising, systemically administering to a
subject suffering from the cancer, a composition comprising a
vector, wherein the vector comprises a construct for the expression
of a trap, wherein the trap is delivered to and then expressed in
tissue susceptible to metastasis, wherein metastasis of the cancer
to the tissue is reduced. Compositions for carrying out the methods
are also described.
Inventors: |
Huang; Leaf; (Durham,
NC) ; GOODWIN; Tyler; (Chapel Hill, NC) ; LIU;
Rihe; (Chapel Hill, NC) ; MIAO; Lei; (Chapel
Hill, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL |
Chapel Hill |
NC |
US |
|
|
Appl. No.: |
17/537837 |
Filed: |
November 30, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15763065 |
Mar 23, 2018 |
11219694 |
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PCT/US2016/051966 |
Sep 15, 2016 |
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17537837 |
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62232169 |
Sep 24, 2015 |
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International
Class: |
A61K 47/68 20060101
A61K047/68; A61K 47/65 20060101 A61K047/65; A61K 48/00 20060101
A61K048/00; C07K 14/47 20060101 C07K014/47; C07K 16/28 20060101
C07K016/28; C12N 15/62 20060101 C12N015/62 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0001] This invention was made with government support under Grant
Nos. CA151652, CA149387, CA157738, and DK100664 awarded by the
National Institutes of Health. The government has certain rights in
the invention.
Claims
1-145. (canceled)
146. A polypeptide capable of binding CXCL12, wherein said
polypeptide has at least 90% identity to a sequence selected from
the group consisting of SEQ ID NOs: 1, 2, 6, 7, 11, 12, and 16.
147. The polypeptide of claim 146, wherein said polypeptide has at
least 90% identity to a sequence selected from the group consisting
of SEQ ID NOs: 1, 2, 6, 7, 11, and 12; and wherein the polypeptide
comprises a VH region having three complementarity determining
regions (CDRs).
148. The polypeptide of claim 147, wherein the three CDRs are SEQ
ID NOs: 3, 4, and 5.
149. The polypeptide of claim 148, wherein the VH region has at
least 90% identity to SEQ ID NO: 2.
150. The polypeptide of claim 148, wherein the VH region is SEQ ID
NO: 2.
151. The polypeptide of claim 148, wherein the polypeptide has at
least 90% identity to SEQ ID NO: 1.
152. The polypeptide of claim 147, wherein the three CDRs are SEQ
ID NOs: 8, 9, and 10.
153. The polypeptide of claim 152, wherein the VH region has at
least 90% identity to SEQ ID NO: 7.
154. The polypeptide of claim 152, wherein the VH region is SEQ ID
NO: 7.
155. The polypeptide of claim 152, wherein the polypeptide has at
least 90% identity to SEQ ID NO: 6.
156. The polypeptide of claim 147, wherein the three CDRs are SEQ
ID NOs: 13, 14, and 15.
157. The polypeptide of claim 156, wherein the VH region has at
least 90% identity to SEQ ID NO: 12.
158. The polypeptide of claim 156, wherein the VH region is SEQ ID
NO: 12.
159. The polypeptide of claim 156, wherein the polypeptide has at
least 90% identity to SEQ ID NO: 11.
160. The polypeptide of claim 146, wherein said polypeptide has at
least 90% identity to SEQ ID NO: 16, and said polypeptide comprises
a) a V.sub.H region, wherein the V.sub.H region has at least 90%
identity to SEQ ID NO: 17, and wherein the V.sub.H region comprises
three complementarity determining regions (CDRs); and b) a V.sub.L
region, wherein the V.sub.L region has at least 90% identity to SEQ
ID NO: 18, and wherein the V.sub.L region comprises three
complementarity determining regions (CDRs).
161. The polypeptide of claim 160, wherein the three CDRs of the
V.sub.H region are SEQ ID NOS: 19, 20, and 21, respectively, and
the three CDRs of the V.sub.L region are SEQ ID NOS: 22, 23, and
24, respectively.
162. The polypeptide of claim 161, wherein the V.sub.L region is
SEQ ID NO: 18.
163. The polypeptide of claim 161, wherein the V.sub.H region is
SEQ ID NO: 17 and the V.sub.L region is SEQ ID NO: 18.
164. The polypeptide of claim 146, wherein the polypeptide does not
comprise a light chain or heavy chain constant region.
165. A nucleic acid encoding the polypeptide of claim 146.
Description
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
[0002] The sequence listing written in the file
482902_seqlisting.txt is 77,671 bytes, and was created on Sep. 14,
2016 and is hereby incorporated by reference.
FIELD OF THE INVENTION
[0003] The subject matter described herein is directed to
treatments that prevent or reduce the occurrence of metastatic
cancer by modifying cellular micro-environment factors in tissues
susceptible to metastases.
BACKGROUND
[0004] In treating cancer, early diagnosis and treatment before
metastasis is critical since once a cancer has metastasized, the
success rates of therapy are substantially lower. In particular,
colorectal cancer (CRC) is the third most prevalent cancer
diagnosed worldwide, leading to the third most cited deaths. In the
United States alone, approximately 143,460 patients are diagnosed,
resulting in 51,690 patient deaths yearly (American Cancer Society.
Cancer Facts and FIGS. 2012. Atlanta: American Cancer Society;
2012. p. 25-6). However, the cause of death is rarely due to the
primary colon cancer burden, in which local resection of the colon
where the primary cancer resides is quite efficient. Unfortunately,
the occurrence of liver metastasis is the leading cause of death in
CRC patients (American Cancer Society. Cancer Facts and FIGS. 2012.
Atlanta: American Cancer Society; 2012. p. 25-6).
[0005] At early stages of colorectal cancer detection, the
five-year survival rate is approximately 90%. Unfortunately, this
rate drops drastically to less than 12% survival once the liver
metastasis has occurred. Studies have also found that upon
diagnosis, 20% of patients have already developed liver metastasis,
with this number reaching up to 60-70% of patients having developed
metastatic lesions in the liver at time of death (Schima W, Kulinna
C, Langenberger H, et al. Liver metastases of colorectal cancer:
US, CT or MR? Cancer imaging. International Cancer Imaging Society.
2005; 5 (SpecNo A): S149-56).
[0006] Yet, treatments for diseases such as cancer, for which the
ultimate therapeutic goal is to kill the diseased cell or prevent
or inhibit its reproduction, include the administration of
cytotoxic drugs. Cytotoxic drugs include many chemotherapeutic
agents that are used in the treatment of cancers, including
alkylating agents, antimetabolites, and toxins. Most cytotoxic
drugs are non-selective, killing healthy cells as well as diseased
cells, which contributes to undesirable side effects when these
agents are delivered systemically. Thus, a need exists for
alternate therapies that do not rely on systemic administration of
toxic agents.
[0007] The subject matter herein addresses the shortcomings of
known therapies by modifying the micro-environment of tissues that
are susceptible to metastases. In doing so, metastasis is prevented
or reduced and the use of cytotoxic agents can be avoided.
BRIEF SUMMARY OF THE INVENTION
[0008] In an embodiment, the subject matter described herein is
directed to a method of modifying the micro-environment of a target
cell comprising, systemically administering to a subject a
composition comprising a vector, wherein the vector comprises a
construct for the expression of a trap, wherein the trap is
expressed in the target cell thereby modifying the
micro-environment.
[0009] In an embodiment, the subject matter described herein is
directed to a method of reducing metastasis of a cancer comprising,
systemically administering to a subject having cancer a composition
comprising a vector, wherein the vector comprises a construct for
the expression of a trap, wherein the trap is expressed in tissue
susceptible to metastasis thereby modifying the micro-environment
of the tissue and reducing metastasis of the cancer to the
tissue.
[0010] In an embodiment, the subject matter described herein is
directed to a method of treating cancer in a patient comprising,
administering to the patient a composition comprising a nucleic
acid sequence that encodes a polypeptide capable of binding CXCL12,
wherein the polypeptide comprises a signaling peptide for desired
extracellular or intracellular localization and an affinity or trap
region that interacts with CXCL12 and disrupts its interaction with
its endogenous receptor(s), and wherein said polypeptide is
transiently expressed.
[0011] In another embodiment, the subject matter described herein
is directed to a method further comprising administering a second
composition comprising a nucleic acid encoding a polypeptide
capable of binding PD-L1, wherein the polypeptide comprises a
signaling peptide for desired extracellular or intracellular
localization and an affinity or trap region that interacts with
PD-L1 and disrupts its interaction with its endogenous receptor(s),
and wherein said polypeptide is transiently expressed.
[0012] In another embodiment, a method further comprising
administering a second composition comprising a nucleic acid
encoding a polypeptide capable of binding PD-1, wherein the
polypeptide comprises a signaling peptide for desired extracellular
or intracellular localization and an affinity or trap region that
interacts with PD-1 and disrupts its interaction with its
endogenous receptor(s), and wherein said polypeptide is transiently
expressed.
[0013] These and additional embodiments are fully disclosed
herein.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 depicts the endogenous protein structures of the
chemokine CXCL12 used to establish a trap against wild-type CXCL12
with C-terminal biotin as target for positive selection. (A) The
Structure of CXCL12 mutant with CXCR4-interacting with the
N-terminus motif deleted for negative selection. (B) The structures
of wild-type CCL2 and CCL5 and their non-receptor-binding mutants
used for positive and negative selections. (C) Schematics of
homodimeric, heterodimeric, and bispecific chemo/cytokine traps
which can be used for single or combination therapies. (D) The
trivalent PD-L1 trap as well as the self-assembly process of the
trivalent trap. Furthermore, the binding kinetics of the trivalent
PD-L1 trap with PD-L1 using Octet is displayed along with the
plasmid map of the PD-L1 trap used for gene delivery based on the
nanoparticle system.
[0015] FIG. 2 depicts development and effect of engineered CXCL12
trap protein on CT-26 FL3 cellular migration and invasion. (A) The
plasmid vector map of pCXCL12 Trap DNA sequence. The coding
sequences of the CXCL12-binding VH and VL domains were used for
assembly of the trap gene. The final sequence for the CXCL12 trap
codes for a signaling peptide, VH domain, a flexible linker, VL
domain, E tag, and His(6.times.) tag, respectively. The complete
cDNA was cloned into pCDNA3.1 between Nhe I and Xho I sites and the
accuracy was confirmed by DNA sequencing. The binding affinity
between CXCL12 trap and CXCL12 by using Bio-Layer Interferometry.
CXCL12 was immobilized on the AR2G biosensor and different
concentrations of CXCL12 trap were used to measure the binding
kinetics, in which the CXCL12 trap was found to have a Kd=4 nM. (B)
The engineered CXCL12 trap was found to have one-half maximal
inhibition [ND50] against biological active CXCL12 (100 ng/ml) at a
concentration of approximately 120 nM. Analysis of CT-26 FL3 cell
migration stimulated with CXCL12 (100 ng/ml; 10 nM) in the presence
or absence of CXCL12 trap (2, 4, 8, or 12 .mu.g/ml; 60, 120, 240,
or 360 nM respectively) or positive control CXCL12 Ab (1, 2 or 4
.mu.g/ml; 6, 12, or 24 nM respectively). (C) Analysis of CT-26 FL3
cell invasion after stimulation with CXCL12 (100 ng/ml; 10 nM) in
the presence or absence of CXCL12 trap (4 or 12 .mu.g/ml; 120 or
360 nM) or positive control CXCL12 Ab (4 .mu.g/ml; 24 nM). Data was
expressed as mean.+-.s.d., calculated from samples ran in
triplicate and as a percentage of untreated (no CXCL12 or treatment
protein) control. *p<0.05, **p<0.01, compared to CXCL12 (100
ng/ml, 10 nM) stimulate cells (without protein trap or Ab
treatment) control. NS, not significant.
[0016] FIG. 3 depicts the LCP nanoparticle characterization by TEM
and DLS. (A) LCP core containing pDNA/mc-CR8C peptide (B) Final
galactose-LCP containing pDNA/mc-CR8C, with negative uranyl acetate
stain (C) Dynamic light scattering (DLS) analysis of final
galactose-LCP containing pDNA/mc-CR8C, yielding 45 nanometers in
diameter and Zeta potential of +10.0. Number, volume, and intensity
weighted size distribution illustrates two particle distribution.
The smaller population (.about.45 nm) is the desired LCP particles,
and the larger population (.about.350 nm) is due to excess DOTAP
and cholesterol which form liposomes following thin-film hydration
yielding a Z-average of -236.+-.32 nm; n=6. (D) The stability of
the LCP over time in a 10% serum solution was measured through DLS.
The LCP was suspended in 10% serum solution and incubated at
37.degree. C. The z-average was recorded over 24 hours in order to
observe any protein/LCP aggregation indicated by increased
z-average. Data were expressed as mean.+-.s.d., calculated from
samples ran in triplicate. The z-average (.about.30 nm) is
consistent over the 24 hours, yielding no significant increase in
the z-average, indicating minimal formation of protein/LCP
aggregates. (E) The vector map of the pCXCL12 plasmid encapsulated
into the LCP. (F) The DNA sequence of the pCXCL12 gene.
[0017] FIG. 4 depicts the pharmacokinetic and organ biodistribution
analysis of galactose-LCP-pCXCL12 Trap/mcCR8C with .sup.177Lu
incorporated into the LCP core. Approximately 250,000 counts were
administered into the mice by tail vein injection. (A) Blood
samples collected via tail vein cut were collected, weighed, and
measured for radioactive counts to determine the percentage of
injected dose (% ID) remaining in circulation. A biphase
distribution is observed, yielding T.sub.1/2.alpha. and
T.sub.1/2.beta. of 20 min and 1,054 min, respectively. (B) LCP
biodistribution/organ accumulation was measured at 16 hours after
tail vain injection (a time when blood radiation counts reached
background signal). Approximately 40-50% of the injected dose per
gram of tissue was found to accumulate in the liver. Data were
expressed as mean.+-.s.d., calculated from samples ran in
triplicate.
[0018] FIG. 5 depicts the biological trapping of endogenous CXCL12
and its role on immune cell recruitment as well as the transient
and liver specific expression of the pCXCL12 trap. (A) Endogenous
CXCL12 expression in paraffin-embedded sections of liver tissues
from BALB/c mouse models of colorectal cancer sacrificed 10 days
after the final treatment injection and control healthy livers
[healthy (No CRC)]. Immunofluorescent stain for CXCL12 (red), along
with DAPI nuclear stain (blue). Five groups were studied, including
healthy (No CRC), untreated (PBS), pGFP LCP control (10 .mu.g every
other day.times.3), pTrap LCP (10 .mu.g), pTrap LCP (10 .mu.g every
other day.times.3). All data were expressed as mean.+-.s.d.,
calculated from samples run in triplicate and reported as
fluorescent intensity. N.S. denotes no significance, N.D. denotes
under detection limit. The p-values of individual groups compared
to corresponding untreated control are displayed in graphs. Scale
bar: 250 .mu.m. (B) Additional sections were stained to determine
the recruitment of immune cells to the liver, including
immunosuppressive anti-inflammatory MDSCs [CD11b+(Green)/GR1+(Red)]
and T.sub.reg [CD4+(Green)/Foxp3+(Red)] as well as the CD8+ T cell
population (Green). Four groups were studied, including healthy (No
CRC), untreated (Tumor), untreated (Stroma), and pTrap LCP (10
.mu.g every other day.times.3). Trichrome staining is also shown to
distinguish normal and diseased liver. White arrows indicate
metastatic lesions. All data were expressed as mean.+-.s.d.,
calculated from samples run in triplicate and reported as
fluorescent intensity. N.S. denotes no significance, N.D. denotes
under detection limit. The p-values of individual groups compared
to corresponding untreated control are displayed in graphs. Scale
bar: 250 .mu.m demonstrates the transient liver-specific expression
of pGFP and engineered pCXCL12 trap. (C) Microscopy analysis of GFP
expression in major LCP-accumulating organs. The liver sections
demonstrate transient expression for at least 4 days after final
injection (10 .mu.g every other day.times.3. Scale bar: 250 .mu.m.
Data were expressed as mean.+-.s.d., calculated from at least
triplicated samples and reported as a fluorescent intensity
quantified by Image J software. N.S. denotes no significance, N.D.
denotes under detection limit, p-values represent significance to
untreated sample. Scale bar 250 .mu.m. (D) His(6.times.)-tag ELISA
and (E) Western blot analysis were conducted to determine the organ
distribution/expression of the pCXCL12 trap in all major
LCP-accumulating organs and serum. Doses were escalated from 2.0,
10.0, or 20.0 .mu.g pDNA administered via tail vein. (F) Western
blot analysis of organs show CXCL12 trap expression through use of
His(6.times.) mAb. The expression is transient and only lasts for
at least 4 days and no longer than 8 days after the final injection
(10 .mu.g every other day.times.3). Total protein concentrations
were determined by BCA and 50 .mu.g of total protein was loaded per
well/lane. Trap protein was detected at 28.6 kDa, as confirmed by a
protein standard ladder, consistent with the theoretical value.
GAPDH was used as a loading control, except in the serum samples,
where GAPDH is not present. Data were expressed as mean.+-.s.d.,
calculated from samples run in triplicate and shown as a fold
increase compared to untreated control. N.S. denotes no
significance, N.D. denotes under detection limit. The p-values of
groups compared to corresponding untreated control are displayed in
graphs.
[0019] FIG. 6 depicts Decreased incidence of liver metastasis after
pCXCL12 Trap LCP treatment. (A) Mice were inoculated with
2.times.10.sup.6 CT-26(FL3) RFP/Luc cells into the cecum wall.
Treatment schedule is shown above. Treatment, 10 .mu.g (0.5 mg/kg)
pDNA, was administered through the tail vein IV on days 10, 12, and
14. Groups included PBS (untreated; n=7) and pGFP LCP (10 .mu.g
every other day.times.3; n=6), as well as pCXCL12 Trap LCP (10
.mu.g every other day.times.3; n=7). Progression of overall tumor
mass was followed by administration of 200 .mu.l luciferin (10
mg/ml) IP. Luciferase bioluminescent imaging was recorded 10 min
after administration of luciferin. Whole mouse and liver tumor
burden were recorded. All data were expressed as mean.+-.s.d., and
reported as bioluminescent intensity. N.S. denotes no significance,
N.D. denotes under detection limit. The p-values of individual
groups compared to corresponding untreated control are displayed in
graph. (B) Total organ tumor burden of untreated (n=3) and
therapeutic pCXCL12 Trap LCP (n=4) groups. Quantification of tumor
burden in organs was performed with IVIS/Kodak software. All data
were expressed as mean.+-.s.d., and reported as bioluminescent
intensity. N.S. denotes no significance, N.D. denotes under
detection limit. The p-values of individual groups compared to
corresponding untreated control are displayed in graph. (C)
Paraffin-embedded liver sections were stained with trichrome. Large
tumor burden (indicated by black arrows) and cirrhosis/fibrosis
(blue stain, collagen) are clearly seen in the PBS (untreated) and
pGFP LCP treatment groups. The pCXCL12 Trap LCP treated livers have
normal healthy liver morphology and no detectable metastatic
burden. Scale bar is 250 .mu.m. Collagen quantification in liver
section was recorded. All data were expressed as mean s.d. N.S.
denotes no significance, N.D. denotes under detection limit. The
p-values of individual groups compared to corresponding untreated
control are displayed in graph.
[0020] FIG. 7 depicts a decreased incidence of liver metastasis and
enhanced T cell killing after pCXCL12 trap LCP therapy. Mice were
inoculated with 2.times.10.sup.6 CT-26(FL3) RFP/Luc cells into the
cecum wall. Treatment, 10 .mu.g (0.5 mg/kg) pDNA, was administered
through tail vein IV on days 10, 12, and 14. Groups included PBS
(untreated; n=5) and pCXCL12 Trap LCP (10 .mu.g every other
day.times.3; n=5) with either anti-Lyt2.2 or isotype IgG control
administrated on days 8 and 10 IP (400 .mu.g, 20 mg/kg).
Inoculation and treatment schedule/dose and liver tumor mass on day
21 are shown above. Mice were administered 200 .mu.l (10 mg/ml)
luciferin IP. After 5 min, mice were euthanized and livers were
extracted, rinsed in PBS, and placed in a solution of luciferin (1
mg/ml). The bioluminescent images were recorded using IVIS kinetic
with Kodak camera. Quantification of tumor burden in the liver was
performed with IVIS/Kodak software. Data were expressed as
log-transformed mean, normalized.+-.s.e. N.S. denotes no
significance, N.D. denotes under detection limit. The p-values of
individual groups compared to corresponding untreated control are
displayed in graph. ROI=region of interest.
[0021] FIG. 8 depicts a decreased incidence of 4T1 (Breast Cancer)
liver metastasis and increased survival after pCXCL12 Trap LCP
treatment. (A) The figure shows the inoculation and treatment
schedule and doses, as well as bioluminescent signal detection and
tumor burden quantification 7 days after inoculation. Treatment
groups included PBS (untreated; n=5), pGFP LCP/anti-CD8 (n=5),
pTrap LCP/anti-CD8 (n=5), pTrap LCP/Isotype IgG (n=5). Data were
expressed as log-transformed mean, normalized.+-.s.e. N.S. denotes
no significance, N.D. denotes under detection limit. The p-values
of individual groups compared to corresponding untreated control
are displayed in graph. ROI=region of interest. (B) Flow cytometry
analysis of tumor burden and quantification on day 10 (n=3 per
group). Gating consists of GFP positive tumor cells (P3) versus
non-GFP positive cells (P4) Data were expressed as mean, normalized
s.d. N.S. denotes no significance, N.D. denotes under detection
limit. The p-values of individual groups compared to corresponding
untreated control are displayed in graph. (C) Kaplan-Meier survival
curve including all 4 treatment groups (n=5 per group). Survival
was determined by evaluating mouse weight, activity, and quality of
life. N.S. denotes no significance, N.D. denotes under detection
limit. The p-values of individual groups compared to corresponding
untreated control are displayed in graph.
[0022] FIG. 9 depicts a comparison of therapeutic strategies for
reducing incidence of colorectal cancer (HT-29) liver metastasis.
(A) The timeline at the top shows the inoculation and treatment
schedule and dosing for the HT-29. Treatments were administered
every other day on days 0-16, through tail vein IV. Treatment
groups included PBS (untreated; n=5), pGFP LCP (10 .mu.g, 0.5 mg/kg
pDNA; n=5), pTrap LCP (10 .mu.g, 0.5 mg/kg pDNA; n=5), free CXCL12
trap protein (10 .mu.g, 0.5 mg/kg protein; n=5), and AMD3100 (100
.mu.g, 5.0 mg/kg; n=5). (B) Tumor burden analysis and
quantification on day 36 (n=5 per group). Liver metastasis burden
was quantified by resection and weighing of tumor nodules (in mg).
Image of liver from each treatment group with metastatic burden
shown, white arrows indicate metastatic lesion. Survival was
determined by evaluating mouse weight, activity, and quality of
life. Data were expressed as individual data points with
mean.+-.s.d. N.S. denotes no significance, N.D. denotes under
detection limit. The p-values of individual groups compared to
corresponding untreated control are displayed in graph.
[0023] FIG. 10 depicts the reduction in toxicity via LCP delivery.
(A) ALT, AST, creatinine, and BUN measurements and blood leukocyte
cell counts 24 hours after final treatment with PBS (untreated), 10
.mu.g pGFP LCP every other day.times.3, 10 .mu.g pCXCL12 Trap LCP
every other day.times.3, or free CXCL12 trap protein (20 .mu.g
every other day.times.3), in which mice were sacrificed on days 1,
7, and 14 after final administration. All data were expressed as
mean s.d. from samples run in triplicate. N.S. denotes no
significance, N.D. denotes under detection limit. The p-values of
individual groups compared to corresponding untreated control are
displayed in graph. (B) Trichrome histology sections of different
organs 24 hours after final treatment with PBS (untreated), 10
.mu.g pGFP LCP every other day.times.3, 10 .mu.g pCXCL12 Trap LCP
every other day.times.3, or free CXCL12 trap protein (20 .mu.g
every other day.times.3), in which mice were sacrificed on days 1,
7, and 14 after final administration. All trichrome histology
sections show no toxicity in any major organ including: heart,
lung, spleen, kidney, and liver. Scale bar=100 .mu.m.
[0024] FIG. 11 depicts the western blot analysis to evaluate
endogenous CXCR4 expression in mouse and human cancer cell lines.
Cells were cultured according to the conditions recommended by
ATCC, lysed, and normalized by BCA for accurate protein loading.
Each lane received 30 .mu.g of total protein. All samples were run
on same gel to ensure accurate exposure and relative expression.
Protein was identified at 42 kDa using a protein standard ladder.
Data were expressed as mean.+-.s.d., calculated from samples ran in
triplicate and reported as a relative intensity to the highest
intensity sample [CT-26(FL3) and HT-29] and normalized by GAPDH
intensity. N.S. denotes no significance, N.D. denotes under
detection limit, p-values represent significance to first cell line
in graph.
[0025] FIG. 12 depicts the endogenous CXCL12 in major organs of
mice (without CRC) and in the liver of a CRC mouse. Endogenous
CXCL12 expression in different organs from BALB/c mice. The images
show immunofluorescent staining against CXCL12 (red), along with
DAPI nuclear stain (blue). Data were expressed as mean.+-.s.d.,
calculated from at least triplicated samples and reported as a
fluorescent intensity. N.S. denotes no significance, N.D. denotes
under detection limit, p-values represent significance to liver
sample. Scale bar 250 .mu.m.
[0026] FIG. 13 depicts the total mouse tumor burden on day 24 after
cecal inoculation. Mice were inoculated with 2.times.10.sup.6 CT-26
F3 RFP/Luc cells into the cecum wall. Treatment consisting of 10
.mu.g pDNA was administered through tail vein IV on days 10, 12,
and 14. Groups included PBS (untreated; n=7), pGFP DNA LCP (10
.mu.g every other day.times.3; n=6), and pCXCL12 trap LCP (10 .mu.g
every other day.times.3; n=7). Progression of tumor mass was
followed by administration of 200 .mu.l luciferin (10 mg/ml) IP.
Luciferase bioluminescent imaging was performed 10 min after
administration of luciferin.
[0027] FIG. 14 depicts (A) TEM image of LPD NP (vector for
encapsulating plasmid). (B) Biodistribution of DiI-labeled LPD NP
(24 h post injection) in mice bearing KPC orthotopic tumor. (C)
Fluorescence images of DiI distribution in liver and tumor (white
numbers indicate % cells taken up DiI in the organ). Two daily
doses of GFP LPD NPs were intravenous injected into mice bearing
tumors. The GFP expression in liver and tumor are shown (green
numbers). Phalloidin labelled cellular actin. Results suggest that
though liver is the major organ taken up NPs, plasmid expression is
mainly in the tumor (n=3). (D) GFP expression in different cell
populations within tumor. The % of GFP positive cells in each cell
population was quantified (white numbers). .alpha.SMA positive
fibroblasts and RFP positive tumor cells are major GFP producing
cells within the tumor microenvironment. (E) Transient expression
of His-tag labeled trap plasmid were quantified by His-tag ELISA.
The expression of trap was transient within one week. And again,
tumor is the major producing organs. Compared to trap protein, the
plasmid delivery prolonged trap expression in tumor (n=4).
[0028] FIG. 15 depicts the tumor growth inhibition and host
survival. (A) Dosing schedule of different treatments on mice
bearing KPC allograft are shown in the upper panel. IVIS images of
KPC tumor after different treatments (n=5-7) are shown in the lower
panel. (B) Tumor inhibition curve of KPC (n=6-10). (C) The survival
proportions of the treated groups. Data show mean.+-.SD, n=5-8.
(D&E). The end time point tumor weight of mice bearing KPC with
low dose plasmid treatment (30 .mu.g/mice, 4 times, (D) and high
dose plasmid treatment (50 .mu.g/mice, 4 times, E). n=4. *
p<0.05, ** p<0.01, *** p<0.001. The statistical analyses
were calculated by comparison with the control group if not
specifically mentioned.
[0029] FIG. 16 depicts the long-term metastasis study. (A)
Metastasis of KPC cells in major organs 1 month after different
treatments (n=4-5). Liver, lung and spleen are major organs for KPC
metastasis. (B) H&E staining shown the histology of tumor
metastasis in the major organs of the PBS control group. Metastasis
was significantly inhibited when mice were treatment with combo
trap NP. Blue circles and arrows indicate metastatic tumor growth
in lung, spleen and liver. Bars in B represent 100 .mu.m.
[0030] FIG. 17 depicts the IFN-.gamma. ELISpot assay of splenocytes
from mice bearing orthotopic KPC pancreatic cancer with different
treatment. (A) Spleens were harvested from tumor bearing animals.
Splenocytes were re-stimulated with extracts from normal
splenocytes (control), KPC cells or KPC cells transduced with RFP
and Luciferase markers. Cells secreted IFN-.gamma. were stained
with anti-IFN-.gamma. antibody. Results are quantified and shown in
the right panel. ns: not significant, *** p<0.001. n=4. B.
Spleens were harvested from tumor bearing animals with different
treatments: PBS, CXCL12 trap NP, PDL1 trap NP and Combo trap NP.
Splenocytes were re-stimulated with extracts from KPC RFP/Luc.
Cells secreted IFN-.gamma. were stained with anti-IFN-.gamma.
antibody. No significant differences were found among different
treatments (n=4).
[0031] FIG. 18 depicts the combo trap NP facilitate T cells
infiltration into tumor microenvironment. (A) Tissue sections from
KPC allografts with different treatments were stained for CD3
(green), p53 (red), and DAPI (blue) and then analyzed by IF
microscopy. Adjacent H&E stainings show the stroma architecture
of the regions. Yellow dotted lines demonstrate the edge of tumor
cells' invasion into normal pancreas. Orange-rectangle areas are
zoomed in for better visualization. Tumor regions are also
presented in lower magnification. Scale bars indicate 400 .mu.m.
(B) The percentage of CD3+ cells within tumor regions were
quantified with image J of 5 representative images from each
treatment. (C) Single-cell suspensions of KPC allograft tumors
(within the tumor regions) after different treatments (n=5) were
stained with antibodies for CD3 and CD8. The percentage of
CD3.sup.+CD8 cells are quantified by flow cytometry. * p<0.05.
** p<0.01. D and E. Mice bearing KPC98027 tumors were pretreated
with 3 daily injections of CD8 mAb (300 .mu.g/mice) to deplete the
CD8+ T-cells in the mice. Isotype mAb were used as control. The
efficacy of combo trap NP in mice with or without CD8 depletion
were compared by imaging (D) and quantified (E).
[0032] FIG. 19 depicts the changes of tumor-infiltrating immune
cells in tumor microenvironment. The KPC murine tumor bearing mice
were divided into four groups and treated with either PBS, CXCL12
trap/Ctrl NP, PDL1 trap/Ctrl NP or Combo trap NP. At the end of
treatment, mice were euthanized and tumor tissues were collected
for (A) immunostaining evaluation and (B) flow cytometry assay: the
first panel shows the MDSC (yellow); the second panel shows the
Treg cells (yellow) and the third panel shows the macrophages
(red). Numbers showing in white indicate the average % of each cell
type in the tumor. Bars in A represent 200 .mu.m. * p<0.05; **
p<0.01. The statistical analyses were calculated by comparison
with the untreated group if not specifically mentioned.
[0033] FIG. 20 depicts the changing of CXCL12 and PD-L1 coverage
after trap plasmid treatment. Both fluorescence image (A) and
quantification (B) are presented (n=5). Bars in A represent 200
.mu.m. The statistical analyses were calculated by comparison with
the control group if not specifically mentioned. All data show
mean.+-.SEM (n=4), *p<0.05; ** p<0.01, ***p<0.001.
[0034] FIG. 21 depicts the changes of cytokines in tumor
microenvironment. Cytokine level were detected using quantitative
RT-PCR. The statistical analyses were calculated by comparison with
the control group if not specifically mentioned. All data show
mean.+-.SEM (n=4), *p<0.05; **p<0.01, ***p<0.001.
[0035] FIG. 22 depicts the tumor microenvironment changes after
various treatments. (A) The KPC bearing mice were divided into 4
groups and treated with either PBS, CXCL12 trap/Ctrl NP, PDL1
trap/Ctrl NP or Combo trap NP. At the end of treatment, mice were
euthanized and tumor tissues were harvested for double fluorescence
staining of CD31 (shown as green) and .alpha.SMA (fibroblast
staining, shown as red). Representative locations (yellow dotted
square) are zoomed in (yellow square). Blood vessels were
decompressed and normalized after CXCL12 trap or Combo trap
treatment. Yellow arrow indicates the normalized blood vessels.
Lower panel images are enlarged from the boxed areas in the
corresponding upper panel images. Bars in upper and lower panels
represents 500 and 100 .mu.m, respectively. (B) The % of .alpha.SMA
coverage, CD31 density and normalized blood vessels were quantified
using Image J from 5 representative images of each group. The
statistical analyses were calculated by comparison with the control
group if not specifically mentioned. All data show mean.+-.SD
(n=5), ** p<0.01, *** p<0.001.
[0036] FIG. 23 depicts the accumulation (A) and distribution (B) of
DiI labeled LPD NPs in different organs and tumors respectively,
from mice bearing KPC allografts treated with either PBS or combo
trap NPs.
[0037] FIG. 24 depicts the collagen coverage within tumors from
mice treated with PBS, CXCL12 trap/Ctrl NP, PDL1 trap/Ctrl NP and
Combo trap NP, respectively.
[0038] FIG. 25 depicts the H&E morphology of the KPC bearing
mice which were divided into 5 groups and treated with 4 doses of
PBS, Ctrl NP, CXCL12 trap/Ctrl NP, PD-L1 trap/Ctrl NP and Combo
trap NP every two days. At the end of the treatments, mice were
euthanized and the major organs were harvested for H&E
pathology staining. Blue rectangle highlights the liver and kidney
of PBS and Ctrl NP groups, indicating severe liver and kidney
toxicities. Cellular vacuolization, desquamated-degenerative cells
and focal necrosis (yellow arrows) were observed in these
organs.
DETAILED DESCRIPTION
[0039] As disclosed herein, improving the survival rate and
treatment of cancer patients rests in preventing or decreasing the
occurrence of metastases. In particular, colorectal cancer (CRC)
patients tend to develop liver metastases. Studies have shown that
the relationship between the chemokine receptor (CXCR4) expressed
on colon cancer cells and the chemokine ligand (CXCL12) secreted by
the hepatic stellate cells (HSC) plays a significant role in CRC
liver metastasis (Zeelenberg I, Ruuls-Van Stalle L, Roos E. The
Chemokine Receptor CXCR4 Is Required for Outgrowth of Colon
Carcinoma Micrometastases. Cancer Res., 63: 3833-3839, 2003). These
hepatic stellate cells are resident perisinusoidal cells which have
shown to produce high levels of endogenous CXCL12 for recruitment
of lymphocytes to areas of inflammation. Migration and invasion
studies have shown that in the presence of high levels of CXCL12,
colorectal cancer cells (CXCR4 positive) migrate and invade via the
CXCL12 concentration gradient. Further studies of human colorectal
cancer samples have also found that poor prognosis and a higher
rate of liver metastasis correlates with high levels of CXCR4
expression on the cancer cells (Zeelenberg I, Ruuls-Van Stalle L,
Roos E. The Chemokine Receptor CXCR4 Is Required for Outgrowth of
Colon Carcinoma Micrometastases. Cancer Res., 63: 3833-3839, 2003).
As described herein, a method of disrupting this CXCL12 gradient
(prophylactically) at the future site of metastasis (liver), can
decrease the occurrence of colorectal liver metastasis.
[0040] As described herein, the present methods avoid the problems
of known therapies. The treatment of animals with AMD3100, a small
molecule CXCR4 antagonist, has demonstrated that disrupting the
CXCL12/CXRC4 axis can decrease the occurrence of colorectal liver
metastasis (Matsusue R, Kubo H, Hisamori S, Okoshi K, Takagi H,
Hida K, Nakano K, Itami A, Kawada K, Nagayama S, Sakai Y. epatic
stellate cells promote liver metastasis of colon cancer cells by
the action of SDF-1/CXCR4 axis. Ann Surg Oncol. 16(9):2645-53,
2009). Subsequently, many CXCR4 antagonists have been developed.
However, the endogenous role of CXCL12 and CXCR4 in the immune
system is vital for normal homeostasis. Therefore, these
traditional treatments which include small molecule and protein
therapies come with systemic off-target toxicity concerns.
Furthermore, to our knowledge no therapies targeting CXCL12 have
been developed or reported to reduce the occurrence of metastatic
lesions.
[0041] As disclosed herein, a unique anti-cancer strategy can be
accomplished, in which delivery of genes (encoding CXCL12 trap) to
the liver can alter the liver micro-environment, for example, the
protein factor levels therein. The methods described herein result
in local and transient modification of the micro-environment, thus
sparing other cells from undesirable toxicity. Delivery of such
genes can achieve reduced concentrations of factors such as CXCL12,
priming the liver to resist the migration/invasion of the
colorectal cancer cells. The association between CXCL12 and CXCR4
plays a crucial role in liver metastasis, in which over-expression
of CXCR4 is characteristic of highly metastatic human colorectal
liver metastasis lesions, as well as the high levels of CXCL12
expressed in the liver (Shan-shan Zhang, Zhi-peng Han, Ying-ying
Jing, et al., CD133+CXCR4+ colon cancer cells exhibit metastatic
potential and predict poor prognosis of patients. BMC Med. 10: 85,
2012).
[0042] The subject matter described herein is not limited to any
particular tissue or target cell. As disclosed herein, targeting
the micro-environment is a unique anti-cancer paradigm, in which
the metastatic lesions are not specifically targeted, but instead
the environment is primed to be unsuitable for the metastasis to
form or progress, ultimately allowing for decreased growth and
occurrence of metastasis. Through incorporation of a targeting
moiety, for example, the galactose targeting moiety for local liver
expression of the therapeutic protein, it is possible to target the
desired tissue with no expression found in off-target organs or
serum. Since this approach can be used in many tissues besides the
liver and with other traps besides CXCL12, other metastatic tissue,
such as breast, lung, lymph node, prostate, brain, pancreas, and
bone can be targeted.
[0043] Other micro-environment factors can play a role in
migration, invasion, and proliferation of cancer metastasis. These
factors become more pronounced in the organ of interest when
inflammation is induced. This inflammation can be associated with
many different environmental factors varying from secreted proteins
from the primary cancer to a patient's diet. Therefore, targeting
these factors is also contemplated in the subject matter described
herein. Accordingly, targeting factors in high metastatic tissue or
tissue that is susceptible to metastases is a promising therapy for
reducing the occurrence/progression of metastasis in many
organs.
[0044] The insufficient target specificity of most approaches in
treating diseases has limited the applications for successful
treatment of these diseases in the clinic. Furthermore, the
shortcomings of gene therapy, due to the numerous extracellular and
intracellular barriers have truly hampered clinical treatments of
many diseases. Therefore, in order to overcome these barriers we
have developed a vector for clinical applications with high
specificity, accumulation, and delivery into the nucleus of the
target cell. The subject matter described herein overcomes the
problems of prior therapies and can not only treat liver metastasis
and primary cancers, but numerous other liver diseases such as HBV,
fatty liver, liver cirrhosis and many others. Further, in addition
to liver diseases, through incorporation of different targeting
moieties, such as adenosine analogs, targeting highly expressed
Adenosine A2B receptors on lung epithelial cells, primes the vector
to accumulate and deliver traps, such as pDNA traps against CXCL12
or other micro-environment factors, that play a role in other
tissues susceptible to metastases. Accordingly, the methods and
compositions described herein can be used to prime the
micro-environment factors of numerous tissues known to have high
rates of metastasis such as the lung, lymph node, breast, bone, and
others.
[0045] Another type of recalcitrant cancer is pancreatic tumor,
which is known to be resistant to immunotherapy due to its strong
immune suppressive tumor microenvironment (TME). CXCL12 and PD-L1
are two molecules that control the suppressive TME. Fusion
proteins, also referred to herein as one type of trap, that bind
one of these two molecules with high affinity (Kd=4 nM and 16 pM,
respectively) were manufactured and tested for specific binding
with the target. Plasmid DNA encoding for each trap was formulated
in LPD nanoparticles and injected IV to mice bearing KPC orthotopic
pancreatic cancer. Expression of traps was mainly in the tumor and
secondarily in the liver. Combination trap therapy shrunk the tumor
and significantly prolonged the host survival by 57%. Either trap
alone only brought in a partial therapeutic effect. We also found
that CXCL12 trap allowed T-cell penetration into the tumor and
PD-L1 trap allowed the infiltrated T-cells to kill the tumor cells.
Combo trap therapy also significantly reduced metastasis of the
tumor cells to other organs. No toxicity was found in all major
organs including the liver and the kidney. Accordingly, combination
trap therapy significantly modified the suppressive TME to allow
the host immune system to kill the tumor cells.
[0046] The presently disclosed subject matter will now be described
more fully hereinafter. However, many modifications and other
embodiments of the presently disclosed subject matter set forth
herein will come to mind to one skilled in the art to which the
presently disclosed subject matter pertains having the benefit of
the teachings presented in the foregoing descriptions. Therefore,
it is to be understood that the presently disclosed subject matter
is not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. In other words, the
subject matter described herein covers all alternatives,
modifications, and equivalents. In the event that one or more of
the incorporated literature, patents, and similar materials differs
from or contradicts this application, including but not limited to
defined terms, term usage, described techniques, or the like, this
application controls. Unless otherwise defined, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in this field. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their
entirety.
I. DEFINITIONS
[0047] The term "microenvironment" refers to the target cell and
its adjacent milieu.
[0048] The term "construct" refers to an artificially constructed
segment of genetic material, such as a nucleic acid sequence, that
is to be expressed in a target tissue or cell. It can contain the
genetic insert, and any necessary promoters, etc. will also be
present in the vector.
[0049] The term "susceptible" refers to a tissue or cell that
because of its micro-environment certain types of cancers tend to
grow or metastasize in the tissue or cell. By way of a non-limiting
example, colorectal cancers tend to metastasize and grow in the
liver as a result of the micro-environment of liver cells. Other
cancers are known to metastasize to particular tissues and cells in
the body. In another non-limiting example, breast cancer tends to
metastasize in the liver, brain, and regional lymph nodes, and the
bone. Thus, these tissues and cells are susceptible as used
herein.
[0050] As used herein, "reducing metastasis" refers to the
inhibition or lessening of metastasis to susceptible tissues and
cells. Numerous ways of determining a reduction in metastasis can
be used. By way of a non-limiting example, subjects with a type of
cancer that is typically known to metastasize and would be expected
to metastasize who show little or no metastasis after treatment
will have shown a reduction in metastasis. In particular, the
subject matter described herein provides reducing metastasis of any
cancer to the liver.
[0051] As used herein, the term "trap" refers to an expression
product that binds, inhibits, or reduces the biological activity of
the target molecule in the micro-environment. The trap is delivered
by vectors, which can be viral, non-viral, synthetic, such as,
nanoparticles, and the like, each of which comprises the necessary
materials for subcloning and expression of the trap. The trap can
also be delivered by liposomes or living cells including monocytes
and stem cells. The inhibition can be measured by K.sub.d, for
example, or by showing a reduction in the activity of the target,
from 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80,
85, 90 or 95% or more. The trap is designed to work on a desired
target molecule, which in many instances is a trappable protein as
is known in the art in view of the subject matter described
herein.
[0052] The term "transient" refers to an effect that is
non-permanent.
[0053] The term "subject" refers to animals such as mammals,
including, but not limited to, primates (e.g., humans), cows,
sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like.
In certain embodiments, the subject is a human. In embodiments, the
subject has been diagnosed with a disease such as cancer or a liver
disease.
[0054] The terms "treat" and "treatment" refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) an undesired
physiological change or disorder, such as the development or spread
of cancer. For purposes of this disclosure, beneficial or desired
clinical results include, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented.
[0055] The phrase "therapeutically effective amount" means an
amount of the trap that (i) treats or prevents the particular
disease, condition, or disorder, (ii) attenuates, ameliorates, or
eliminates one or more symptoms of the particular disease,
condition, or disorder, or (iii) prevents or delays the onset of
one or more symptoms of the particular disease, condition, or
disorder described herein. In the case of cancer, the
therapeutically effective amount can reduce the number of cancer
cells; reduce the tumor size; inhibit (i.e., slow to some extent
and preferably stop) cancer cell infiltration into peripheral
organs; inhibit (i.e., slow to some extent and preferably stop)
tumor metastasis; inhibit, to some extent, tumor growth; and/or
relieve to some extent one or more of the symptoms associated with
the cancer.
[0056] The terms "cancer" refers to or describe the physiological
condition in mammals that is typically characterized by unregulated
cell growth. A "tumor" comprises one or more cancerous cells.
[0057] The term "pharmaceutically acceptable salts" denotes salts
which are not biologically or otherwise undesirable.
Pharmaceutically acceptable salts include both acid and base
addition salts. The phrase "pharmaceutically acceptable" indicates
that the substance or composition must be compatible chemically
and/or toxicologically, with the other ingredients comprising a
formulation, and/or the mammal being treated therewith.
[0058] As used herein, the term "deliver" refers to the transfer of
a substance or molecule (e.g., a polynucleotide) to a physiological
site, tissue, or cell. This encompasses delivery to the
intracellular portion of a cell or to the extracellular space.
Delivery of a polynucleotide into the intracellular portion of a
cell is also often referred to as "transfection."
[0059] As used herein, the term "intracellular" or
"intracellularly" has its ordinary meaning as understood in the
art. In general, the space inside of a cell, which is encircled by
a membrane, is defined as "intracellular" space. Similarly, as used
herein, the term "extracellular" or "extracellularly" has its
ordinary meaning as understood in the art. In general, the space
outside of the cell membrane is defined as "extracellular"
space.
[0060] A fibroblast is a cell that synthesizes extracellular matrix
and collagen, the structural framework (stroma) for animal tissues.
The main function of fibroblasts is to maintain the structural
integrity of connective tissues by continuously secreting
precursors of the extracellular matrix. In embodiments, fibroblasts
are a stroma target for the vectors described herein.
[0061] The following abbreviations may be used herein. Some
abbreviations are defined where they occur in the text of this
document. [0062] ALT Alanine Aminotransferase [0063] AST Aspartate
Aminotransferase [0064] BLI Bio-Layer Interferometry [0065] BUN
Blood Urea Nitrogen [0066] CMV Cytomegalovirus [0067] CRC
Colorectal Cancer [0068] DLS Dynamic Light Scattering [0069] DAPI
4',6-diamidino-2-phenylindole [0070] DOPA
1,2-dioleoyl-sn-glycero-3-phosphate [0071] DOTAP
1,2-dioleoyl-3-trimethylammonium-propane [0072] DSPE
1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine [0073] ELISA
Enzyme Linked Immunosorbent Assay [0074] GFP Green Fluorescent
Protein [0075] LCP Lipid Calcium Phosphate [0076] mc Mono-cyclic
[0077] NHS N-Hydroxysuccinimide [0078] PBS Phosphate Buffered
Saline [0079] PEG Polyethylene Glycol [0080] PK Pharmacokinetics
[0081] pTrap Galactose-PEG-LCP w/pCXCL12 trap/mc-CR8C [0082] TEM
Transmission Electron Microscopy
[0083] An immunoglobulin light or heavy chain variable region (also
referred to herein as a "light chain variable domain" ("VL domain")
or "heavy chain variable domain" ("VH domain"), respectively)
consists of a "framework" region interrupted by three
"complementarity determining regions" or "CDRs." The framework
regions serve to align the CDRs for specific binding to an epitope
of an antigen. The CDRs include the amino acid residues of an
antibody that are primarily responsible for antigen binding. From
amino-terminus to carboxyl-terminus, both VL and VH domains
comprise the following framework (FR) and CDR regions: FR1, CDR1,
FR2, CDR2, FR3, CDR3, and FR4. CDRs 1, 2, and 3 of a VL domain are
also referred to herein, respectively, as CDR-L1, CDR-L2, and
CDR-L3; CDRs 1, 2, and 3 of a VH domain are also referred to
herein, respectively, as CDR-H1, CDR-H2, and CDR-H3.
[0084] The assignment of amino acids to each VL and VH domain is in
accordance with any conventional definition of CDRs. Conventional
definitions include, the Kabat definition (Kabat, Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md., 1987 and 1991), The Chothia definition (Chothia
& Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature
342:878-883, 1989); a composite of Chothia Kabat CDR in which
CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition
used by Oxford Molecular's antibody modelling software; and, the
contact definition of Martin et al (bioinfo.org.uk/abs) (see Table
1). Kabat provides a widely used numbering convention (Kabat
numbering) in which corresponding residues between different heavy
chains or between different light chains are assigned the same
number. When an antibody is said to comprise CDRs by a certain
definition of CDRs (e.g., Kabat) that definition specifies the
minimum number of CDR residues present in the antibody (i.e., the
Kabat CDRs). It does not exclude that other residues falling within
another conventional CDR definition but outside the specified
definition are also present. For example, an antibody comprising
CDRs defined by Kabat includes among other possibilities, an
antibody in which the CDRs contain Kabat CDR residues and no other
CDR residues, and an antibody in which CDR H1 is a composite
Chothia-Kabat CDR H1 and other CDRs contain Kabat CDR residues and
no additional CDR residues based on other definitions.
TABLE-US-00001 TABLE 1 Conventional Definitions of CDRs Using Kabat
Numbering Composite of Chothia Loop Kabat Chothia & Kabat AbM
Contact L1 L24--L34 L24--L34 L24--L34 L24--L34 L30--L36 L2 L50--L56
L50--L56 L50--L56 L50--L56 L46--L55 L3 L89--L97 L89--L97 L89--L97
L89--L97 L89--L96 H1 H31--H35B H26--H32 . . . H34* H26--H35B*
H26--H35B H30--H35B H2 H50--H65 H52--H56 H50--H65 H50--H58 H47--H58
H3 H95--H102 H95--H102 H95--H102 H95--H102 H93--H101 *CDR-H1 by
Chothia can end at H32, H33, or H34 (depending on the length of the
loop). This is because the Kabat numbering scheme places insertions
of extra residues at 35A and 35B, whereas Chothia numbering places
them at 31A and 31B. If neither H35A nor H35B (Kabat numbering) is
present, the Chothia CDR-H1 loop ends at H32. If only H35A is
present, it ends at H33. If both H35A and H35B are present, it ends
at H34.
[0085] The term "epitope" refers to a site on an antigen to which
an antibody binds. An epitope can be formed from contiguous amino
acids or noncontiguous amino acids juxtaposed by tertiary folding
of one or more proteins. Epitopes formed from contiguous amino
acids (also known as linear epitopes) are typically retained on
exposure to denaturing solvents whereas epitopes formed by tertiary
folding (also known as conformational epitopes) are typically lost
on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation. Methods of determining
spatial conformation of epitopes include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See,
e.g., Epitope Mapping Protocols, in Methods in Molecular Biology,
Vol. 66, Glenn E. Morris, Ed. (1996). The epitope can be linear.
The epitope can also be a conformational epitope. It is to be noted
that the term "a" or "an" entity refers to one or more of that
entity; for example, "a cationic lipid" is understood to represent
one or more cationic lipids. As such, the terms "a" (or "an"), "one
or more," and "at least one" can be used interchangeably
herein.
[0086] Throughout this specification and the claims, the words
"comprise," "comprises," and "comprising" are used in a
non-exclusive sense, except where the context requires
otherwise.
[0087] As used herein, the term "about," when referring to a value
is meant to encompass variations of, in some embodiments .+-.50%,
in some embodiments .+-.20%, in some embodiments .+-.10%, in some
embodiments .+-.5%, in some embodiments .+-.1%, in some
embodiments.+-.0.5%, and in some embodiments.+-.0.1% from the
specified amount, as such variations are appropriate to perform the
disclosed methods or employ the disclosed compositions.
[0088] Additional definitions are set forth below.
II. METHODS
[0089] In an embodiment, the present subject matter is directed to
a method of modifying the micro-environment of a target cell
comprising, systemically administering to a subject a composition
comprising a vector, wherein the vector comprises a construct for
expression of a trap in the target cell, wherein the trap is
expressed in the target cell, wherein the micro-environment of the
target cell is modified.
[0090] As described herein, expression of the trap results in the
presence of an effective amount of the expressed trap to modify the
micro-environment of the target cell.
[0091] Modifying the micro-environment of a target cell comprises
reducing the amount of a target molecule that is normally present
in the micro-environment. As used herein, reducing the amount
refers to a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95% or more lowering of the amount of the target molecule as
compared to the amount in the absence of the trap.
[0092] Useful target molecules include proteins, such as enzymes,
chemokines, cytokines, protein factors, and combinations thereof.
Suitable targets include those in Table 2 below.
TABLE-US-00002 TABLE 2 CXC (alpha chemokines) CXCL1
GRO-.alpha./SCYB-1/MGSA/GRO-1/NAP-3 (MIP-2.alpha./KC) CXCR1, CXCR2
CXCL2 GRO-.beta./SCYB-2/GRO-2/MIp-2.alpha. (MIP-2.beta./KC) CXCR2
CXCL3 GRO-.gamma./SCYB-3/GRO-3/MIp-2.beta. (KC) CXCR2 CXCL4
PF-4/SCYB-4 Unknown CXCL5 ENA-78/SCYB-5 (LIX) CXCR2 CXCL6
GCP-2/SCYB-6 CXCR1, CXCR2 CXCL7
NAP-2/(SCYB-7/PBP/CTAP-III/.beta.-TG CXCR1, CXCR2 CXCL8
SCYB-8/GCP-1/NAP-1/MDNCF CXCR1, CXCR2 CXCL9 MIG/SCYB-9 CXCR3 CXCL10
IP-10/SCYB-10 CXCR3, KSHV-GPCR CXCL11
I-TAC/SCYB-11/.beta.-R1/H174/IP-9 CXCR3 CXCL12 SDF-1/SCYB-12/PBSF
CXCR4 CXCL13 BCA-1/SCYB-13 CXCR5 CXCL14 BRAK/SCYB-14/Bolekine
Unknown CXCL16 Small inducible cytokinc B6 CXCR6 C (gamma
chemokines) XCL1 Lymphotactin/SCYC1/SCM-1.alpha./Lympholactin
.alpha. XCR1 XCL2 SCM-1b/SCYC2/ymphotactin .beta. XCR1 CX.sub.3C
(delta chemokines) CX.sub.3CL1 Fractalkine/SCYD1 CX3CR1 CCL1 I-309
/SCYAI (TCA-3) CCR8 CCL2 MCP-1/SCYA2/MCAF/HC11 (JE) CCR2, CCR5,
CCR10 CCL3 MIP-1.alpha./SCYA3/LD78.alpha./SIS-.alpha. CCR1, CCR5
CCL4 MIP-1.beta./SCYA4/ACT-2/G-26/HC21/LAG-1/SIS-.gamma. CCR5,
CCR10 CCL5 RANTES/SCYA5/SIS-.delta. CCR1, CCR3, CCR5, CCR10 CCL7
MCP-3/SCYA7 CCRI, CCR2, CCR3 CCR5, CCL8 MCP-2/SCYA8/HC14 (MARC)
CCR2, CCR3, CCR5 CCR1, CCL11 Eotaxin/SCYA11 CCR3 CCL13
MCP-4/SCYA13/Ck .beta.10/NCC-1 CCR1, CCR2. CCR3, CCR5 CCL14
HCC-1/SCYA14/Ck .beta.1/MCIF/NCC-2/CC-1 CCR1 CCL15 MIP-1
.delta./SCYA15/Lkn-1/HCC-2/MIP-5/NCC-3/CC-2 CCR1, CCR3 CCL16
HCC-4/SCYA16/Ck .beta.12/LEC/LCC-1/NCC-4/ILINCK/LMC/Mtn-1 CCR1
CCL17 TARC/SCYA17 (ABCD-2) CCR4 CCL18
PARC/SCYA18/Ck.beta.7/DC-CK1/AMAC-1/MIP-4/DCtactin Unknown CCL19
MIP-3.beta./SCYA19/Ck.beta.11/ELC/Exodus-3 CCR7 CCL20
MIP-3.alpha./SCYA20/LARC/Exodus-1 CCR6 CCL21
6Ckine/SCYA21/Ck.beta.9/SLC/Exodus-2 CCR7 CCL22 MDC/SCYA22 (ABCD-1)
CCR4 CCL23 MPIF/SCYA23/Ck.beta.8/Ck.beta.8-1/MIP-3/MPIP-1 CCR1
CCL24 Eotaxin-2/SCYA24/Ck.beta.6/MPIF-2 CCR3 CCL25
TECK/SCYA25/Ck.beta.15 CCR9 CCL26
Eotaxin-3/SCYA26/MIP-4.alpha./TSC-1/IMA CCR3 CCL27
CTACK/SCYA27/ESkine/Skinkine CCR3, CCR2, CCR10 CCL28
CCL28/SCYA28/MEC CCR10, CCR3
[0093] A particularly useful target molecule is CXCL12. Other
Protein Factors include: EGF, Neuregulin, FGF, HGF, VEGF, VEGFR and
NRP-1, Ang1 and Ang2, PDGF (BB-homodimer) and PDGFR, TGF-.beta.,
endoglin and TGF-.beta. receptors, MCP-1, Histamine, Integrins
.alpha.2.beta.1, .alpha.V.beta.3, .alpha.V.beta.5, .alpha.V.beta.6,
.alpha.6.beta.4 and .alpha.5.beta.1, VE-cadherin and CD31, ephrin,
plasminogen activators, plasminogen activator inhibitor-1, eNOS and
COX-2, AC133, IDI/ID3, LOX, and HIF.
[0094] Inhibitory and Blocking Traps and their Targets: Inhibitory
traps for macromolecule targets include traps that can be protein
molecules that specifically bind and further inhibit or block the
biological functions of a target of interest. The targets of the
inhibitory or blocking traps can be cyto/chemokines and their
corresponding receptors, including but not limited to IL-1, IL-6,
IL-7, IL-8, IL-10, IL-15, IL-21 (and IL receptors), TNF-alpha (and
TNF-alpha receptor), TGF-beta (and TGF-beta receptor), CSF-1 (and
CSF-1 receptor), CXCR1 and its ligands (CXCL5, CXCL6, CXCL8), CXCR2
and its ligands (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7 and
CXCL8), CXCR3 and its ligands (CXCL4, CXCL9, CXCL10, CXCL11), CXCR4
and its ligand (CXCL12), CXCR5 and its ligand (CXCL13), CX3CR1 and
its ligand (CX3CL1), CCR1 and its ligands (CCL3, CCL4, CCL5, CCL6,
CCL7, CCL8, CCL13, CCL14, CCL15, CCL16, CCL23), CCR2 and its
ligands (CCL2, CCL5, CCL7, CCL8, CCL13, CCL16), CCR3 and its
ligands (CCL4, CCL5, CCL7, CCL11, CCL13, CCL15, CCL24, CCL26,
CCL28), CCR4 and its ligands (CCL17, CCL22), CCR5 and its ligands
(CCL3, CCL4, CCL5, CCL7, CCL14, CCL16), CCR6 and its ligand
(CCL20), CCR7 and its ligands (CCL19, CCL21), CCR9 and its ligand
(CCL25), CCR10 and its ligands (CCL2? and CCL28), ACKR3 and its
ligands (CCL11, CCL12), ACKR6 and its ligand (CCL18).
[0095] The targets of the inhibitory traps can be immune checkpoint
related proteins, including but not limited to CTLA-4, PD-1, PD-L1,
PD-L2, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (CD270 or TNFRSF14), BTLA
(CD272), GALS, TIGIT, A2aR, LAG-3, KIRs and MHC class I or II.
[0096] Targets also include those whose up- or downregulation by
inhibitory trapping will suppress the expression or reduce the
biological activity of IDO, TDO, arginase-1/2, adenosine receptors,
CD39, CD73, COX2, EP receptors, and iNOS that are involved in
catabolism (by IDO, TDO, ARG1/2) of amino acids (i.e. tryptophan,
arginine, cysteine, glutamine, and phenylalanine), generation of
adenosine (by CD39, CD73, and mediated through adenosine
receptors), prostaglandin E2 (by COX2), reactive oxygen species
(ROS) and reactive nitrogen species (RNS) (by iNOS), all resulting
in the immunosuppression of TME.
[0097] Inhibitory traps for small molecule metabolites: The targets
of the inhibitory traps can also be small molecules, including but
not limited to tryptophan metabolites (i.e. kynurenine that is
produced by IDO or TDO and signals through the aryl hydrocarbon
receptor), cAMP and adenosine, which play critical roles in the
inhibition of T cells, recruitment and/or expansion of
immunosuppressive cells and consequently the immunosuppressive
tumor microenvironment.
[0098] Stimulatory Traps and their Targets: The traps can also be
simulative, including those that agonistically act on immune
checkpoint targets CD28, ICOS (CD278), 4-1BB (CD137 or TNFRSF9),
OX40 (CD134 or TNFRSF4), GITR (CD357 or TNFRSF18), CD27 (TNFRSF7),
and CD40 (TNFRSF5). The stimulatory traps can also be those that
possess or mimic the agonistic effects of the ligands of the above
receptors, including but not limited to B7.1 (CD80), B7.2 (CD86),
B7-H5 (VISTA or Gi24), ICOSL (B7H2 or CD275), 4-1BBL (CD137L),
OX40L (CD252), GITRL, CD27L (CD70), and CD40L (CD154). The targets
for agonistic traps also include some toll-like receptors,
including but not limited to TLR4, TLR7, TLR8, and TLR9 that play
critical roles in the activation of T cells. Traps can be based on
antibody-like domains or fragments. It has been found that the
local and transient expression of the chemo/cytokine traps results
in desired biological activity and low toxicity. Most of the
target-binding biologics rely on a full-length monoclonal antibody
that has long half-life. Indeed, systemic and prolonged
administration of a CXCL12 trap can result in transient liver
damage and reduced white blood cell count, as demonstrated in our
in vivo data. In addition, due to the large size, complex
structure, and sophisticated post-translational modifications,
full-length antibodies have several intrinsic disadvantages in
serving as chemo/cytokine traps, including inefficiency in tissue
penetration to access TME, difficult to engineer and optimize the
target-trapping properties such as disruption of receptor binding,
high stability, bispecificity (if necessary), and Fc-induced side
effects. Our strategy to address the challenge is to develop novel
trapping molecules with desired features from protein libraries
based on small protein domains, including but not limited to the
immunoglobulin VH domain, immunoglobulin VL domain, a VH and VL
fusion protein, scFv, a peptide or protein derived from a binding
and/or framework region of an antibody, a non-immunoglobulin
target-binding domain such as a single domain antibody mimic based
on a non-immunoglobulin scaffold (such as an FN domain-based
monobody, Z domain-based affibody, DARPINs), singly and in any
combination.
[0099] Some known target-binding antibodies can be altered and
engineered to serve as the trap for the local and transient gene
delivery approach described in this work. To compete effectively
with the natural receptor(s), a trap should possess unusually high
binding specificity and affinity against the receptor-binding site,
a property that is more likely to achieve through directed protein
selection from a protein or antibody fragment library with a high
diversity at the surface loops or residues that could be utilized
in the interaction with a target of interest, using protein/peptide
display and selection technologies such as phage display, cell
surface display, mRNA display, DNA display, ribosome display that
are widely used in the in vitro protein selections.
[0100] As described herein, local and transient blockade of
signaling pathways mediated by certain key chemo/cytokines, e.g.,
CXCL12, a chemokine that has been implicated in playing a pivotal
role in the migration/invasion of CXCR4 positive colorectal cancer
cells to the liver, can significantly prevent CRC metastasis. We
first engineered a CXCL12 trap gene based on an anti-CXCL12
antibody sequences, by fusing a VH and a VL domain through a
protease-resistant flexible linker. To achieve efficient secretion
from liver hepatocytes after expression, a strong signal peptide
was incorporated at the N-terminus, whereas affinity tags were
introduced at the C-terminus to facilitate protein purification and
detection. The coding sequence of the resulting CXCL12 trap was
cloned into an expression vector pCDNA3.1 driven by a CMV promoter.
The resulting CXCL12 trap expressed in and purified from 293T cells
was found to have a K.sub.d of 4 nM with CXCL12 (FIG. 2A), whereas
its binding with CXCL1, CXCL8 and CXCL10 were not detectable. This
CXCL12 trap greatly suppressed the migration and invasion of CT-26
FL3 cells stimulated with CXCL12 (FIG. 2B-C). Local and transient
expression of this CXCL12 trap was tested using the gene delivery
system based on the lipid calcium phosphate (LCP) nanoparticle
(FIG. 5). As detailed below, three treatments with the pCXCL12-trap
pDNA formulated in LCP almost completely resolved any occurrence of
CT26-FL3 colorectal liver metastasis with no sign of cancer spread
to other organs (FIG. 6).
[0101] To generate a CXCL12 trap that has much higher potency, we
developed a CXCL12 trap based on the V.sub.H domain library. To
facilitate the in vitro protein selection, we expressed and
purified the wild-type recombinant CXCL12 containing a C-terminal
biotin tag (wtCXCL12-biotin, FIG. 1A), as well as a CXCL12 mutant
(.DELTA.CXCL12, FIG. 1A) in which the N-terminal 8 residues
(highlighted in cyan in FIG. 1A) that are implicated in
CXCR4-binding were deleted. wtCXCL12-biotin was used as target for
positive selection, whereas .DELTA.CXCL12 was used to remove
sequences that bind to non-desired sites through competitive
washing. Similar strategies were used to develop single domain
traps against other chemokines, as illustrated in FIG. 1B for CCL2
and CCL5.
[0102] In brief, displayed V.sub.H domain library pre-cleared with
a streptavidin-agarose column was incubated with an appropriate
amount of biotinylated wtCXCL12 in a binding buffer facilitating
the formation of disulfide bond. The mixture was incubated at room
temperature for 1 hr, followed by addition of pre-washed
streptavidin agarose beads to capture V.sub.H sequences. The
resulting beads were first washed with binding buffer to remove
nonspecifically bound sequences, followed by competitive washing
with large excess of .DELTA.CXCL12 to remove the VH sequences that
bound CXCL12 at sites away from the CXCR4-binding N-terminus. The
enriched pool was regenerated for a new round of selection.
Extensive competitive washing was performed to facilitate the
enrichment of sequences that bind to the N-terminal CXCL12 with
very slow off-rate. After five rounds of selection, we successfully
identified 6 VH sequences that tightly and specifically bind to
wtCXCL12, but not .DELTA.CXCL12. As shown in FIG. 1A, these
sequences have very slow off-rates, with a wtCXCL12-binding
affinity in the range of low nanomolars to picomolars.
[0103] Bivalent Traps with Synergistic Chemo/Cytokine Trapping
Effects
[0104] Chemokines exist as monomers and dimers under physiological
conditions, and compelling evidence suggests that both forms
regulate in vivo function. It was hypothesized that chemokine
dimerization perturbs the distribution of the conformational
substates, which in turn differentially affects the activation of
various downstream signaling pathways. CXCL1, CXCL12, CCR2, CCR5,
and IL-6 have all been reported to adopt dimeric or oligomeric
structures to interact with their pairing receptors.
[0105] In general, CXC chemokines dimerize using the first
.beta.-strand and .alpha.-helix forming a globular structure, with
the dimer interface located away from the receptor binding
N-terminal and N-loop regions. CC chemokines dimerize using their
N-loop residues and form an extended structure, and so their
dimerization and receptor binding domains overlap. CXCL12, for
example, forms under physiological conditions both monomer and
dimer, which possesses distinct effects on cell signaling and
function (Ray P 2012 PMID22142194). While monomeric CXCL12
preferentially activates CXCR4 signaling through G.alpha.i and Akt,
the dimeric form more effectively promotes recruitment of
.beta.-arrestin 2 to CXCR4 and chemotaxis of CXCR4-expressing
cancer cells. Significantly, the dimeric CXCL12 preferentially bind
to CXCR4 over CXCR7. These findings indicate that trapping dimeric
CXCL12 could more effectively block CXCR4-mediated signaling
pathways. Homodimeric or heterodimeric traps can be easily
generated through genetic fusion (FIG. 1C). To generate homodimeric
chemo/cytokine traps, each single domain trap can be genetically
fused with itself through a flexible linker as a recombinant fusion
protein. Similarly, heterodimeric traps that bind a chemokine of
interest at two unique sites can be generated using two traps that
bind to nonoverlapping sites with similar affinities.
[0106] Bispecific Traps that Block Signalings Mediated by Two
Different Chemo/Cytokines.
[0107] The redundancy of the chemokine network involved in tumor
metastasis and TME immunosuppression indicates that a trapping
therapy that acts on more than one signaling pathways should be
more effective. Bispecific traps that can simultaneously block the
signaling pathways mediated by two different chemo/cytokines can be
generated by genetically linking two traps with unique specificity
through a length tunable flexible linker (FIG. 1C).
[0108] Trivalent PD-L1 traps with very potent target-trapping
efficiency One of the most effective ways to develop a high quality
ligand that binds to a target of interest is by converting the
target-binding domain to its multivalent form, as observed in
almost all types of antibodies and numerous multimeric interactive
proteins. To minimize possible immunogenicity, highly stable
trimerization domains from abundant extracellular proteins in
mouse, human, or other organisms or trimerization domains based on
such proteins can be used for the generation of trivalent traps for
the local and transient gene delivery purpose. We chose to use a
trimerization domain from human CMP-1. The strong hydrophobic and
ionic interactions within the C-terminus of mouse or human CMP-1
result in a parallel, disulfide-linked, and rod-shaped trimeric
structure with high stability. We developed a robust technology
platform that allows for facile conversion of a target-binding
domain from endogenous proteins (such as PD-L1, PD-1) or an
affinity domain from protein selection into its trivalent form by
genetically fusing with the trimerization domain, resulting in
trivalent traps with high stability and significantly enhanced
avidity. Typically, trivalent traps that bind to a target of
interest with low nanomolar to picomolar binding affinities can be
easily generated from monomeric domains that are 1,000 times
weaker. The mouse sequence of this trimerization domain is highly
homologous to that of human CMP-1, making it easy to switch to the
human version if translational application is desired. Since the
trimeric trap is formed through self-assembly of three identical
monomers, it only requires a cDNA that codes for the monomer,
making the gene to be delivered much shorter and easier to deliver.
Using this strategy, we developed a potent PD-L1 trap by
genetically fusing the mouse or human extracellular domain of PD-1
that binds to PD-L1 with a stable trimerization domain that is very
abundant in mouse and human cartilages (FIG. 1D). The resulting
trivalent protein bound PD-L1 with a Kd at about 16 picomolar,
which is 10,000-higher than that between endogenous PD-1 and PD-L1
(FIG. 1D). Furthermore, in an immune competent KPC model of
pancreatic cancer, plasmid DNA encoding this trimeric trap (FIG.
15) efficiently induced tumor shrinkage after IV administration of
NPs, when used together with a trap against CXCL12.
[0109] In another embodiment, a trimer formed from three fusion
polypeptides, wherein each fusion polypeptide comprises a PD-1
extracellular domain, a flexible linker, and a trimerization
domain, said trimer capable of binding PD-L1, wherein the fusion
polypeptide is encoded by a nucleic acid sequence with at least
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence
identity to SEQ ID NO: 25.
[0110] In embodiments, useful traps include receptors. Cytokine
receptors exist in structurally related families and comprise
high-affinity molecular signaling complexes that facilitate
cytokine-mediated communication. Type I cytokine receptors have
certain conserved motifs in their extracellular amino-acid domain,
and lack an intrinsic protein tyrosine kinase activity. This family
includes receptors for IL2 (beta-subunit), IL3, IL4, IL5, IL6, IL7,
IL9, IL11, IL12, GM-CSF, G-CSF, Epo, LIF, CNTF, and also the
receptors for Thrombopoietin (TPO), Prolactin, and Growth hormone.
Type I cytokine receptor family is subdivided into three subsets on
the basis of the ability of family members to form complexes with
one of three different types of receptor signaling components
(gp130, common beta, and common gamma--the gamma-chain of the IL2
receptor).
[0111] Type II cytokine receptors are multimeric receptors composed
of heterologous subunits, and are receptors mainly for interferons.
This family includes receptors for IFN-alpha, IFN-beta, IFN-gamma,
IL10, IL22, and tissue factor. The extracellular domains of type II
cytokine receptors share structural similarities in their
ligand-binding domain. Several conserved intracellular sequence
motifs have been described, which probably function as binding
sites for the intracellular effector proteins JAK and STAT
proteins.
[0112] Chemokine receptors are G protein-coupled receptors with 7
transmembrane structure and couple to G-protein for signal
transduction. Chemokine receptors are divided into different
families: CC chemokine receptors, CXC chemokine receptors, CX3C
chemokine receptors, and XC chemokine receptor (XCR1).
[0113] Tumor necrosis factor receptor (TNFR) family members share a
cysteine-rich domain (CRD) formed of three disulfide bonds
surrounding a core motif of CXXCXXC creating an elongated molecule.
TNFR is associated with procaspases through adapter proteins (FADD,
TRADD, etc.) that can cleave other inactive procaspases and trigger
the caspase cascade, irreversibly committing the cell to
apoptosis.
[0114] TGF-beta receptors are single pass serine/threonine kinase
receptors. TGF-beta receptors include TGFBR1, TGFBR2, and TGFBR3
which can be distinguished by their structural and functional
properties.
[0115] Though the methods include systemic administration, the
methods are locally acting, in that, the effect on the
micro-environment is generally isolated in or around the target
cells. This can be accomplished by incorporating a targeting ligand
onto the vector as described elsewhere herein.
[0116] The methods are also transient. That is, the effect of the
method lasts for about three (3) days or less. In embodiments, the
effect lasts for about 20 hours or less. In embodiments, the effect
lasts for about 16 hours or less. In embodiments, the effect lasts
for about 12 hours or less. In embodiments, the effect lasts for
about 10 hours or less. In embodiments, the effect lasts for about
8 hours or less. In embodiments, the effect lasts for about 6 hours
or less. In embodiments, the effect lasts for about 4 hours or
less. In embodiments, the effect lasts for about 3 hours or less.
In embodiments, the effect lasts for about 3 hours or less. In
embodiments, the effect lasts for about 1 hour or less.
[0117] In another embodiment, the present subject matter is
directed to a method of reducing metastasis of a cancer comprising,
systemically administering to a subject suffering from the cancer,
a composition comprising a vector, wherein the vector comprises a
trap, wherein the trap is delivered to and then expressed in and
released out of the tissue susceptible to metastasis, wherein
metastasis of the cancer to the tissue is reduced.
[0118] In this embodiment, the methods include all the variables
described above.
[0119] Additionally, the methods are particularly useful in cancers
as described elsewhere herein.
[0120] The reduction of metastasis can be from a total inhibition,
i.e., undetectable, up to a level of metastasis that is lower than
expected given the type and aggressiveness of the cancer and/or
tumor. Such types and tumors are known to those of skill in the
art. Data showing metastasis that is lower than a control or
comparator also evidence the methods described herein. The
reduction can be from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95% or more.
[0121] In another embodiment, the present subject matter is
directed to a method of treating cancer comprising, systemically
administering to a subject suffering from the cancer, a composition
comprising a vector, wherein the vector comprises a trap.
[0122] In another embodiment, the present subject matter is
directed to a method of treating cancer comprising, systemically
administering to a subject suffering from the cancer, a
combination, wherein the combination comprises at least two
vectors, wherein one of the vectors comprises a trap for a
cytokine/chemokine, and another vector comprises a trap for a
target associated with the cancer. In a particular aspect, the
combination comprises a trap for a CXCL12 chemokine and a trap for
PD-L1, and the cancer is pancreatic cancers, such as, pancreatic
ductal adenocarcinoma. In another aspect, the combination comprises
a trap for a CXCL12 chemokine and a trap for PD-1, and the cancer
is pancreatic cancers, such as, pancreatic ductal adenocarcinoma.
In another embodiment, the combinations include CXCL12 trap with
PD-1 trap, CXCL12 trap with PD-L1 trap, CXCL12 trap with PD-L2
trap, CXCL12 trap with agonistic CD27 trap, CXCL12 trap with
agonistic CD28 trap, CXCL12 trap with agonistic ICOS trap, CXCL12
trap with agonistic CD40 trap, CXCL12 trap with agonistic OX40
trap, CXCL12 trap with agonistic CD137 trap, CXCL12 trap with IDO
trap, CXCL12 trap with TDO trap, CXCL12 trap with ARG1 trap, CXCL12
trap with NOS trap, CXCL12 trap with TGF-beta trap, CXCL12 trap
with B7-H3 trap, CXCL12 trap with B7-H4 trap, CXCL12 trap with
CTLA4 trap, CXCL12 trap with HVEM trap, CXCL12 trap with BTLA trap,
CXCL12 trap with LAG3 trap, CXCL12 trap with KIR trap, CXCL12 trap
with TIM3 trap, CXCL12 trap with GALS trap, CXCL12 trap with A2aR
trap, PD-1 or PD-L1 trap with CXCR1 trap, PD-1 or PD-L1 trap with
CXCR2 trap, PD-1 or PD-L1 trap with CXCR4 trap, PD-1 or PD-L1 trap
with CXCR5 trap, PD-1 or PD-L1 trap with CXCR7 trap, PD-1 or PD-L1
trap with CCR2 trap, PD-1 or PD-L1 trap with CCR4 trap, PD-1 or
PD-L1 trap with CCR5 trap, PD-1 or PD-L1 trap with CCR7 trap, PD-1
or PD-L1 trap with CCR9 trap, PD-1 or PD-L1 trap with CXCL1 trap,
PD-1 or PD-L1 trap with CXCL8 trap, PD-1 or PD-L1 trap with CXCL10
trap, PD-1 or PD-L1 trap with CCL2 trap, PD-1 or PD-L1 trap with
CCL5 trap, PD-1 or PD-L1 trap with CCL22 trap, PD-1 or PD-L1 trap
with IL-6 trap, PD-1 or PD-L1 trap with IL-10 trap, PD-1 or PD-L1
trap with TGF-beta trap, PD-1 or PD-L1 trap with CSF1 trap, PD-1 or
PD-L1 trap with B7-H3 trap, PD-1 or PD-L1 trap with B7-H4 trap,
PD-1 or PD-L1 trap with CTLA4 trap, PD-1 or PD-L1 trap with HVEM
trap, PD-1 or PD-L1 trap with BTLA trap, PD-1 or PD-L1 trap with
LAG3 trap, PD-1 or PD-L1 trap with KIR trap, PD-1 or PD-L1 trap
with TIM3 trap, PD-1 or PD-L1 trap with GALS trap, PD-1 or PD-L1
trap with A2aR trap, PD-1 or PD-L1 trap with CCR4 trap, PD-1 or
PD-L1 trap with IDO-1 trap, PD-1 or PD-L1 trap with TDO trap, PD-1
or PD-L1 trap with ARG1 trap, PD-1 or PD-L1 trap with NOS trap,
PD-1 or PD-L1 trap with PI3K trap, PD-1 or PD-LL trap with
agonistic CD27 trap, PD-1 or PD-L1 trap with agonistic CD28 trap,
PD-1 or PD-L1 trap with agonistic ICOS trap, PD-1 or PD-L1 trap
with agonistic CD40 trap, PD-1 or PD-L1 trap with agonistic OX40
trap, and PD-1 or PD-L1 trap with agonistic CD137 trap.
[0123] In another embodiment, the CXCL12 trap comprises a VH region
from an anti-human CXCL12 antibody. In another embodiment, the
CXCL12 trap comprises a VL region from an anti-human CXCL12
antibody. In another embodiment, the CXCL12 trap comprises a fusion
protein comprising a VH and VL region from an anti-human CXCL12
antibody. In another embodiment, the CXCL12 trap comprises a
non-immunoglobulin domain that mimics antibodies, including but not
limited to FN domain-based monobody, Z domain-based affibody, and
DARPINs.
[0124] In another embodiment, the human CXCL12 is set forth in
GenBank Accession No. AAH39893 (SEQ ID NO:64) or GenBank Accession
No. AAV49999 (SEQ ID NO:65). In another embodiment, the nucleic
acid sequence of human CXCL12 is set forth in GenBank Accession No.
AY802782 (SEQ ID NO:66).
[0125] In another embodiment, the CXCL12 trap comprises a VH
region, wherein said VH region has at least at least 90%, 95%, 96%,
97%, 98% or 99% identity to to a sequence selected from the group
consisting of SEQ ID NOs: 2, 7, 12, and 17.
[0126] In another embodiment, the CXCL12 trap comprises a VH
region, wherein said VH region has at least at least 90%, 95%, 96%,
97%, 98% or 99% identity to SEQ ID NO:17 and a VL region, wherein
said VL region has at least at least 90%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID NO:18.
[0127] In another embodiment, the CXCL12 trap consists essentially
of a VH region, wherein the VH region has at least 90% identity to
a corresponding VH region of SEQ ID NO: 17. In another embodiment,
the CXCL12 trap consists essentially of a VL region, wherein the VL
region has at least 90% identity to a corresponding VL region of
SEQ ID NO: 18.
[0128] In another embodiment, the CXCL12 trap comprises a VH region
having three complementarity determining regions (CDRs) wherein the
three CDRs are (a) SEQ ID NOS: 3-5, respectively; (b) the three
CDRs are SEQ ID NOS: 8-10, respectively; (c) the three CDRs are SEQ
ID NOS: 13-15, respectively; or (d) the three CDRs are SEQ ID NOS:
19-21, respectively and a VL region having three complementarity
determining regions (CDRs), wherein the CDRs are SEQ ID NOS: 22,
23, and 24, respectively.
[0129] In another embodiment, a polypeptide capable of binding
CXCL12 encoded by a nucleic acid sequence having at least 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:
63.
[0130] Pancreatic ductal adenocarcinoma is a deadly disease that
kills 330,000 people globally each year (American Cancer Society.
Cancer Facts and Figures 2012. Atlanta: American Cancer Society;
2012. p. 25-6). The five-year survival rate is only about 12%. The
disease is known to be resistant to chemo and radiation therapies.
It is also resistant to the check-point inhibitors. Greater than
90% PDAC is KRas mutated and most also contain additional mutations
in the p53 gene. A genetically modified mouse model that contains
both the KRas and p53 mutations, i.e. the KPC mice, spontaneously
develops PDAC which closely mimic the clinical disease has been
developed. We have used a cell line derived from KPC tumor, called
KPC98027, which was inoculated orthotopically in the tails of the
pancreas in the syngeneic C57BL6 mice. The tumor cell line was
stably transduced with luciferase and red fluorescence protein
using a lentivirus vector.
[0131] Since the tumor is resistant to immunotherapy including the
check-point inhibitors, we hypothesized that suppressive immune TME
can be modified by locally expressing trap proteins that target key
molecules in the tumor. From the work of Feig et al, CXCL12 seems
to be a key chemokine that does not allow T-cells to infiltrate the
tumor (Feig et al., Targeting CXCL12 from FAP-expressing
carcinoma-associated fibroblasts synergizes with anti-PD-L1
immunotherapy in pancreatic cancer, PNAS, 2013 Dec. 10;
110(50):20212-7). A CXCL12 trap expressed locally in the tumor
should alleviate the problem. KPC tumor over-expresses PD-L1, which
is a check-point in the immune system. Over-expression of PD-L1 in
the tumor cells will result in the killing of T-cells via the
PD-1/PD-L1 axis interaction. Thus, we decided to deliver both traps
to the tumor via gene therapy. The well-established
Lipid-Protamine-DNA (LPD) nanoparticle (NP) was used to deliver
plasmid DNA encoding the trap to the tumor.
[0132] The combination can be synergistic. The term "synergistic"
as used herein refers to a therapeutic combination which is more
effective than the additive effects of the two or more single
therapeutic agents. A determination of a synergistic interaction
between, e.g., a cytokine trap and PD-L1 trap, can be based on the
results obtained from the assays described herein. For example, the
in vivo or in vitro methods disclosed herein. The results of these
assays can be analyzed using the Chou and Talalay combination
method and Dose-Effect Analysis with CalcuSyn software in order to
obtain a Combination Index (Chou and Talalay, 1984, Adv. Enzyme
Regul. 22:27-55). The combinations provided can be evaluated in one
or more assay systems, and the data can be analyzed utilizing a
standard program for quantifying synergism, additivism, and
antagonism among anticancer agents. An example program is that
described by Chou and Talalay, in New Avenues in Developmental
Cancer Chemotherapy, Academic Press, 1987, Chapter 2. Combination
Index values less than 0.8 indicate synergy, values greater than
1.2 indicate antagonism and values between 0.8 to 1.2 indicate
additive effects. The combination therapy may provide "synergy" and
prove "synergistic", i.e., the effect achieved when the active
agents used together is greater than the sum of the effects that
results from using the compounds separately. A synergistic effect
may be attained when the active agents are: (1) co-formulated and
administered or delivered simultaneously in a combined, unit dosage
formulation; (2) delivered by alternation or in parallel as
separate formulations; or (3) by some other regimen. When delivered
in alternation therapy, a synergistic effect may be attained when
the compounds are administered or delivered sequentially, e.g., by
different injections in separate syringes. In general, during
alternation therapy, an effective dosage of each active agent is
administered sequentially, i.e., serially, whereas in combination
therapy, effective dosages of two or more active agents are
administered together. Combination effects can be evaluated using
both the BLISS independence model and the highest single agent
(HSA) model (Lehar et al., Molecular Systems Biology, 3:80 (2007)).
BLISS scores quantify degree of potentiation from single agents and
a positive BLISS score (greater than 0) suggests greater than
simple additivity. A cumulative positive BLISS score greater than
250 is considered strong synergy observed within the concentration
ranges tested. An HSA score (greater than 0) suggests a combination
effect greater than the maximum of the single agent responses at
corresponding concentrations.
[0133] Specific embodiments described herein include:
[0134] 1. A method of modifying the micro-environment of a target
cell comprising, systemically administering to a subject a
composition comprising a vector, wherein the vector comprises a
construct for expression of a trap in the target cell, wherein the
trap is expressed in the target cell, wherein the micro-environment
of the target cell is modified.
[0135] 2. The method of embodiment 1, wherein the modifying the
micro-environment comprises reducing the amount of a target
molecule in the micro-environment.
[0136] 3. The method of embodiments 1-2, where the target molecule
is selected from the group consisting of a protein, a protein
factor, a chemokine, and a cytokine, or combinations thereof.
[0137] 4. The method of embodiments 1-3, wherein the target
molecule is a chemokine.
[0138] 5. The method of embodiments 1-4, wherein the chemokine is
CXCL12.
[0139] 6. The method of embodiments 1-5, wherein the construct
comprises a polynucleotide of interest.
[0140] 7. The method of embodiments 1-6, wherein the trap is a
CXCL12 trap.
[0141] 8. The method of embodiments 1-7, wherein the target cell is
an organ cell.
[0142] 9. The method of embodiments 1-8, wherein the cell is
selected from the group consisting of liver, lung, brain, and
breast.
[0143] 10. The method of embodiments 1-9, wherein the expression of
the trap is transient.
[0144] 11. The method of embodiments 1-10, wherein the modifying of
the micro-environment is transient.
[0145] 12. A method of reducing metastasis of a cancer comprising,
systemically administering to a subject having the cancer, a
composition comprising a vector, wherein the vector comprises a
construct for the expression of a trap, wherein the trap is
expressed in a tissue susceptible to metastasis, wherein metastasis
of the cancer to the tissue is reduced.
[0146] 13. The method of embodiment 12, wherein the cancer is a
solid cancer.
[0147] 14. The method of embodiments 12-13, wherein the cancer is
selected from the group consisting of lung, lymph node, breast,
bone, and colorectal cancer.
[0148] 15. The method of embodiments 12-14, wherein the cancer is
CRC and the tissue is liver tissue.
[0149] 16. The method of embodiments 12-15, wherein the construct
comprises a polynucleotide of interest.
[0150] 17. The method of embodiments 12-16, wherein the trap is a
CXCL12 trap.
[0151] The present methods overcome insufficient target specificity
of most approaches in treating diseases. Furthermore, the
shortcomings of gene therapy, due to the numerous extracellular and
intracellular barriers has truly hampered clinical treatments of
many diseases. Therefore, in order to overcome these barriers
disclosed herein is a vector that can find use in clinical
applications with high specificity, accumulation, and delivery into
target cells, such as, the nucleus of the hepatocytes of the liver.
In an embodiment, this vector yields a highly reproducible
non-viral vector capable of nuclear delivery of pDNA. The LCP
vector described herein provides the ability to incorporate a CMV
promoter, extracellular signaling peptide, trap protein, targeting
moieties, and nuclear penetrating peptides readily.
[0152] The vector can be a liposome. Liposomes are self-assembling,
substantially spherical vesicles comprising a lipid bilayer that
encircles a core, which can be aqueous, wherein the lipid bilayer
comprises amphipathic lipids having hydrophilic headgroups and
hydrophobic tails, in which the hydrophilic headgroups of the
amphipathic lipid molecules are oriented toward the core or
surrounding solution, while the hydrophobic tails orient toward the
interior of the bilayer. The lipid bilayer structure thereby
comprises two opposing monolayers that are referred to as the
"inner leaflet" and the "outer leaflet," wherein the hydrophobic
tails are shielded from contact with the surrounding medium. The
"inner leaflet" is the monolayer wherein the hydrophilic head
groups are oriented toward the core of the liposome. The "outer
leaflet" is the monolayer comprising amphipathic lipids, wherein
the hydrophilic head groups are oriented towards the outer surface
of the liposome. Liposomes typically have a diameter ranging from
about 25 nm to about 1 .mu.m. (see, e.g., Shah (ed.) (1998)
Micelles, Microemulsions, and Monolayers: Science and Technology,
Marcel Dekker; Janoff (ed.) (1998) Liposomes: Rational Design,
Marcel Dekker). The term "liposome" encompasses both multilamellar
liposomes comprised of anywhere from two to hundreds of concentric
lipid bilayers alternating with layers of an aqueous phase and
unilamellar vesicles that are comprised of a single lipid
bilayer.
[0153] Methods for making liposomes (LCP and LDP types) are well
known in the art, e.g., PCT/US2010/044209, herein incorporated by
reference in its entirety. A review of methodologies of liposome
preparation may be found in Liposome Technology (CFC Press NY
1984); Liposomes by Ostro (Marcel Dekker, 1987); Lichtenberg and
Barenholz (1988) Methods Biochem Anal. 33:337-462 and U.S. Pat. No.
5,283,185, each of which are herein incorporated by reference in
its entirety. For example, cationic lipids and optionally co-lipids
can be emulsified by the use of a homogenizer, lyophilized, and
melted to obtain multilamellar liposomes. Alternatively,
unilamellar liposomes can be produced by the reverse phase
evaporation method (Szoka and Papahadjopoulos (1978) Proc. Natl.
Acad. Sci. USA 75:4194-4198, which is herein incorporated by
reference in its entirety). In some embodiments, the liposomes are
produced using thin film hydration (Bangham et al. (1965) J. Mol.
Biol. 13:238-252, which is herein incorporated by reference in its
entirety). In certain embodiments, the liposome formulation can be
briefly sonicated and incubated at 50.degree. C. for a short period
of time (e.g., about 10 minutes) prior to sizing (see Templeton et
al. (1997) Nature Biotechnology 15:647-652, which is herein
incorporated by reference in its entirety).
[0154] In some embodiments, a targeted liposome or a PEGylated
liposome is made as described elsewhere herein, wherein the methods
further comprise a post-insertion step following the preparation of
the liposome or following the production of the liposome, wherein a
lipid-targeting ligand conjugate or a PEGylated lipid is
post-inserted into the liposome. Liposomes comprising a
lipid-targeting ligand conjugate or a lipid-PEG conjugate can be
prepared following techniques known in the art, including but not
limited to those presented herein (see Experimental section; Ishida
et al. (1999) FEBS Lett. 460:129-133; Perouzel et al. (2003)
Bioconjug. Chem. 14:884-898, which is herein incorporated by
reference in its entirety). The post-insertion step can comprise
mixing the liposomes with the lipid-targeting ligand conjugate or a
lipid-PEG conjugate and incubating the particles at about
50.degree. C. to about 60.degree. C. for a brief period of time
(e.g., about 5 minutes, about 10 minutes). In some embodiments, the
liposomes are incubated with a lipid-PEG conjugate or a
lipid-PEG-targeting ligand conjugate at a concentration of about 5
to about 20 mol %, including but not limited to about 5 mol %,
about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about
10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, about 14
mol %, about 15 mol %, about 16 mol %, about 17 mol %, about 18 mol
%, about 19 mol %, and about 20 mol %, to form a stealth delivery
system. In some of these embodiments, the concentration of the
lipid-PEG conjugate is about 10 mol %. The polyethylene glycol
moiety of the lipid-PEG conjugate can have a molecular weight
ranging from about 100 to about 20,000 g/mol, including but not
limited to about 100 g/mol, about 200 g/mol, about 300 g/mol, about
400 g/mol, about 500 g/mol, about 600 g/mol, about 700 g/mol, about
800 g/mol, about 900 g/mol, about 1000 g/mol, about 5000 g/mol,
about 10,000 g/mol, about 15,000 g/mol, and about 20,000 g/mol. In
certain embodiments, the lipid-PEG conjugate comprises a PEG
molecule having a molecular weight of about 2000 g/mol. In some
embodiments, the lipid-PEG conjugate comprises DSPE-PEG.sub.2000.
Lipid-PEG-targeting ligand conjugates can also be post-inserted
into liposomes using the above described post-insertion
methods.
[0155] In an embodiment, the liposome contains a vector encoding a
CXCL12 trap and PD-1 trap fusion protein linked via a cleavable 2A
peptide, which allows for administering just one composition
instead of two for the CXCL12/PD-L1 combination trap therapy.
[0156] Incorporation of the targeting galactose moiety through
conjugation to DSPE-PEG allows for active uptake in the hepatocytes
via asialoglycoprotein receptor which is highly expressed on the
liver hepatocytes. The use of DOTAP and the acid sensitive calcium
phosphate core allows for endosomal escape of the condensed
pDNA/mc-CR8C structure, which is released into the cytoplasm.
Furthermore, condensation of the pDNA with the membrane penetrating
cationic mc-CR8C peptide allows for improved nuclear uptake and
release. The incorporation of the CMV promoter within the pDNA
allows for high liver expression.
[0157] Through incorporation of these parts as described herein an
intelligently designed vector which yields high therapeutic levels
of expression in the desired targeted cell types is provided. This
pTrap LCP vector provides significant decrease the occurrence of
colorectal liver metastasis (80%), as well as significantly
decrease the tumor burden found within the liver (10 fold).
Increased levels of the CXCL12 trap, as well as decreased levels of
free CXCL12 protein was found in the liver in a dose dependent
manner, as well as a reduction in the recruitment of immune cells
(CD8+), demonstrating a biologically specific effect of pTrap LCP
treatment.
[0158] Thus, it shown herein that delivery of pDNA in a
Galactose-LCP vector shows no signs of off-target effects, with
minimal to no immune response following three injections QOD. In
these studies the His-tag incorporated onto the C-terminal end of
the pCXCL12 trap was necessary to determine expression levels
through western blot and ELISA assays. However, for further
clinical applications the His-tag may not be needed. This would aid
in circumventing any immune response such as induction of
neutralizing antibodies.
[0159] Furthermore, the ability to have transient expression of
this small CXCL12 trap (.about.28 kD) lasting no longer than 8
days, allows clinicians the ability to tightly control/monitor the
level and time of expression in order to limit the immune response
while still achieving therapeutic efficacy.
[0160] Disclosed herein is the affinity and production of the
engineered CXCL12 trap (protein) through Bio-Layer Interferometry
(BLI) as well as in vitro suppression of migration and invasion
(FIG. 2). The engineered CXCL12 trap was found to have a Kd=4 nM
through BLI analysis (FIG. 2A). Furthermore, producing one-half
maximal inhibition [ND50] against biological active CXCL12 (100
ng/ml) in vitro at a concentration of approximately 120 nM (FIG.
2B). It has also been reported that treatment of CT-26 cells with
the endogenous CXCL12 chemokine yields upregulation of the
migration/invasion/proliferation pathways. Therefore, we
investigated the ability of our CXCL12 trap and a commercially
available CXCL12 anti-body (Ab) to suppress the migration and
invasion of CT-26 FL3 cells stimulated with endogenous CXCL12.
(FIGS. 2B and 2C). These in vitro experiments demonstrate the
CXCL12 trap's ability to decrease the migration and invasion of
CT-26 FL3 cells stimulated with CXCL12 (100.0 ng/ml) yielding
complete suppression at 8.0 .mu.g/ml and 12.0 .mu.g/ml respectively
(FIGS. 2B and 2C). Commercially available antibody (ND50 of 2-4
ug/ml; 12-24 nM) was also used as a control.
[0161] A useful in vivo pDNA dose (0.5 mg/kg per single injection)
is substantially lower than doses previously shown to have in vivo
expression. Furthermore, this is the first instance where a
therapeutic amount of pDNA has been successfully delivered to the
liver via non-viral vectors other than through the use of an
invasive hydrodynamic injection which results in liver injury and
is not clinically applicable. Such a delivery is attributable to
the use of the intelligently designed LCP vector.
[0162] In 2013 Hu et al., first reported the use of this vector to
elicit high levels of luciferase expression in the liver (Hu, Y.,
et al., A Highly Efficient Synthetic Vector: Nonhydrodynamic
Delivery of DNA to Hepatocyte Nuclei in Vivo. ACS Nano, 2013. 7(6):
p. 5376-5384). Yet, this level of expression is the highest
obtained via non-viral vectors, only behind hydrodynamic injection
techniques. Hu et al. found that delivery of Cy-3 labelled pDNA via
this Gal-LCP vector preferentially accumulated in the nuclei of
hepatocytes 6 h post intravenous tail vein injection (Hu, Y., et
al., A Highly Efficient Synthetic Vector: Nonhydrodynamic Delivery
of DNA to Hepatocyte Nuclei in Vivo. ACS Nano, 2013. 7(6): p.
5376-5384). Organ distribution of radiolabeled Gal-LCP-pDNA/mcCR8C
vector demonstrated prominent uptake in the mouse liver.
Furthermore, through delivering a CXCL12 trap to the liver we
clearly demonstrate that the therapeutic effect was through
decreasing free CXCL12 found in the liver microenvironment. (FIG.
5A). This therapy yielded a decrease in liver tumor burden, which
subsequently decreased the liver inflammation compared to the
untreated diseased liver.
[0163] The untreated groups produced higher levels of CXCL12 and
further aided in liver metastasis accumulation. Therefore,
prophylactic treatment of colorectal patients with this
Gal-LCP-pDNA/mcCR8C vector can help decrease inflammation in the
liver, which plays a critical role in the livers CXCL12 expression
and liver metastasis progression.
[0164] Through the use of this vector, a number of applications and
therapies can be practiced, such as, the treatment of liver
diseases. It is shown herein that this vector has the ability to
deliver high levels of therapeutic pDNA to the hepatocytes of the
liver. Therefore, the ability to not only treat liver metastasis
and primary cancers, but numerous other liver diseases such as HBV,
fatty liver, liver cirrhosis and many others are provided herein.
In addition to liver diseases, the incorporation of different
targeting moieties, such as adenosine analogs, targeting highly
expressed Adenosine A2B receptors on lung epithelial cells, will
prime this vector to accumulate and deliver pDNA traps against
CXCL12 or other microenvironment factors known to play a role in
other highly metastatic tissues. Such a strategy can provide
modification to the micro-environment factors of numerous tissues
known to have high rates of metastasis such as the lung, lymph
node, breast, and bone.
III. COMPOSITIONS
[0165] Compositions are provided that are suitable for systemic
administration.
[0166] Compositions described herein comprise vectors. As used
herein, a vector includes viral vectors, non-viral vectors,
synthetic vectors and the like. Reference is made to U.S. Pub.
Appl. Nos. 2012-0201872; 2011-0117026; and 2011-0117141, each of
which is herein incorporated by reference in its entirety. Vectors
also include liposome vectors or living cell vectors such as
monocytes or stem cells.
[0167] Delivery Vectors
[0168] Suitable methods for delivering the trap of the invention
include viral vectors and non-viral vectors, such as plasmid
vectors, liposome vectors, or living cell vectors such as monocytes
or stem cells.
[0169] The term "vector," as used herein, refers to a nucleic acid
construct designed for transfer between different host cells. An
"expression vector" or "gene therapy vector" refers to a vector
that has the ability to incorporate and express heterologous DNA
fragments in a foreign cell. A cloning or expression vector may
comprise additional elements, for example, the expression vector
may contain an organ-specific promoter for the expression of the
trap gene, and contain signaling sequences for desired
extracellular or intracellular localization. The expression vector
may have two replication systems, thus allowing it to be maintained
in two organisms, for example in human cells for expression and in
a prokaryotic host for cloning and amplification. The term vector
may also be used to describe a recombinant virus, e.g., a virus
modified to contain the coding sequence for a therapeutic compound
or factor. As used herein, a vector may be of viral or non-viral
origin or liposomes or cells such as monocytes or stem cells.
[0170] The terms "virus," "viral particle," "vector particle,"
"viral vector particle," and "virion" are used interchangeably and
are to be understood broadly as meaning infectious viral particles
that are formed when, e.g., a viral vector of the invention is
transduced into an appropriate cell or cell line. Viral particles
according to the invention may be utilized for the purpose of
transferring DNA into cells either in vitro or in vivo. The terms
"vector," "polynucleotide vector," "polynucleotide vector
construct," "nucleic acid vector construct," and "vector construct"
are used interchangeably herein to mean any nucleic acid construct
for gene transfer, as understood by one skilled in the art.
[0171] As used herein, the term "viral vector" is used according to
its art-recognized meaning. It refers to a nucleic acid vector
construct that includes at least one element of viral origin and
may be packaged into a viral vector particle. The vector and/or
particle may be utilized for the purpose of transferring DNA, RNA
or other nucleic acids into cells either in vitro or in vivo.
Numerous forms of viral vectors are known in the art, the sources
of which include, but are not limited to, adenoviruses,
retroviruses, and adeno-associated viruses (AAV).
[0172] The present invention contemplates the use of any vector for
introduction of the trap of interest into mammalian cells.
Exemplary vectors include but are not limited to, viral and
non-viral vectors, such as retroviruses (including lentiviruses),
adenovirus (Ad) vectors including replication competent,
replication deficient and gutless forms thereof, adeno-associated
virus (AAV) vectors, simian virus 40 (SV-40) vectors, bovine
papilloma virus vectors, Epstein-Barr virus vectors, herpes virus
vectors, vaccinia virus vectors, Moloney murine leukemia virus
vectors, Harvey murine sarcoma virus vectors, murine mammary tumor
virus vectors, Rous sarcoma virus vectors and nonviral plasmid
vectors. In one embodiment, the vector is a viral vector. Viruses
can efficiently transduce cells and introduce their own DNA into a
host cell. In generating recombinant viral vectors, non-essential
genes are replaced with a gene or coding sequence for a
heterologous (or non-native) protein.
[0173] In constructing viral vectors, non-essential genes are
replaced with one or more genes encoding one or more therapeutic
compounds or factors. Typically, the vector comprises an origin of
replication and the vector may or may not also comprise a "marker"
or "selectable marker" function by which the vector can be
identified and selected. While any selectable marker can be used,
selectable markers for use in such expression vectors are generally
known in the art and the choice of the proper selectable marker
will depend on the host cell. Examples of selectable marker genes
which encode proteins that confer resistance to antibiotics or
other toxins include ampicillin, methotrexate, tetracycline,
neomycin (Southern et al., J., J Mol Appl Genet. 1982; 1(4):327-41
(1982)), mycophenolic acid (Mulligan et al., Science 209:1422-7
(1980)), puromycin, zeomycin, hygromycin (Sugden et al., Mol Cell
Biol. 5(2):410-3 (1985)) or G418.
[0174] Reference to a vector or other DNA sequences as
"recombinant" merely acknowledges the operable linkage of DNA
sequences which are not typically operably linked as isolated from
or found in nature. Regulatory (expression/control) sequences are
operatively linked to a nucleic acid coding sequence when the
expression/control sequences regulate the transcription and, as
appropriate, translation of the nucleic acid sequence. Thus
expression/control sequences can include promoters, enhancers,
transcription terminators, a start codon (i.e., ATG) in front of
the coding sequined, splicing signal for introns and stop
codons.
[0175] Adenovirus gene therapy vectors are known to exhibit strong
expression in vitro, excellent titer, and the ability to transduce
dividing and non-dividing cells in vivo (Hitt et al., Adv in Virus
Res 55:479-505 (2000)). When used in vivo these vectors lead to
strong but transient gene expression due to immune responses
elicited to the vector backbone. The recombinant Ad vectors for use
in the instant invention comprise: (1) a packaging site enabling
the vector to be incorporated into replication-defective Ad
virions; and (2) a polynucleotide of interest, such as a
polynucleotide encoding the trap of interest. Other elements
necessary or helpful for incorporation into infectious virions,
include the 5' and 3' Ad ITRs, the E2 and E3 genes, etc.
[0176] Replication-defective Ad virions encapsulating the
recombinant Ad vectors of the instant invention are made by
standard techniques known in the art using Ad packaging cells and
packaging technology. Examples of these methods may be found, for
example, in U.S. Pat. No. 5,872,005, incorporated herein by
reference in its entirety. A polynucleotide of interest is commonly
inserted into adenovirus in the deleted E1A, E1B or E3 region of
the virus genome. Preferred adenoviral vectors for use in
practicing the invention do not express one or more wild-type Ad
gene products, e.g., E1a, E1b, E2, E3, E4. Preferred embodiments
are virions that are typically used together with packaging cell
lines that complement the functions of E1, E2A, E4 and optionally
the E3 gene regions. See, e.g. U.S. Pat. Nos. 5,872,005, 5,994,106,
6,133,028 and 6,127,175, expressly incorporated by reference herein
in their entirety. Adenovirus vectors are purified and formulated
using standard techniques known in the art.
[0177] Recombinant AAV vectors are characterized in that they are
capable of directing the expression and the production of the
selected transgenic products in targeted cells. Thus, the
recombinant vectors comprise at least all of the sequences of AAV
essential for encapsidation and the physical structures for
infection of target cells.
[0178] Recombinant AAV (rAAV) virions for use in practicing the
present invention may be produced using standard methodology, known
to those of skill in the art and are constructed such that they
include, as operatively linked components in the direction of
transcription, control sequences including transcriptional
initiation and termination sequences, and the coding sequence for a
trap of interest. These components are bounded on the 5' and 3' end
by functional AAV ITR sequences. By "functional AAV ITR sequences"
is meant that the ITR sequences function as intended for the
rescue, replication and packaging of the AAV virion. Hence, AAV
ITRs for use in the vectors of the invention need not have a
wild-type nucleotide sequence, and may be altered by the insertion,
deletion or substitution of nucleotides or the AAV ITRs may be
derived from any of several AAV serotypes. An AAV vector is a
vector derived from an adeno-associated virus serotype, including
without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6,
AAV-7, AAV-8, etc. Preferred AAV vectors have the wild type REP and
CAP genes deleted in whole or part, but retain functional flanking
ITR sequences.
[0179] Typically, an AAV expression vector is introduced into a
producer cell, followed by introduction of an AAV helper construct,
where the helper construct includes AAV coding regions capable of
being expressed in the producer cell and which complement AAV
helper functions absent in the AAV vector. The helper construct may
be designed to down regulate the expression of the large REP
proteins (Rep78 and Rep68), typically by mutating the start codon
following p5 from ATG to ACG, as described in U.S. Pat. No.
6,548,286, expressly incorporated by reference herein. This is
followed by introduction of helper virus and/or additional vectors
into the producer cell, wherein the helper virus and/or additional
vectors provide accessory functions capable of supporting efficient
rAAV virus production. The producer cells are then cultured to
produce rAAV. These steps are carried out using standard
methodology. Replication-defective AAV virions encapsulating the
recombinant AAV vectors of the instant invention are made by
standard techniques known in the art using AAV packaging cells and
packaging technology. Examples of these methods may be found, for
example, in U.S. Pat. Nos. 5,436,146; 5,753,500, 6,040,183,
6,093,570 and 6,548,286, expressly incorporated by reference herein
in their entirety. Further compositions and methods for packaging
are described in Wang et al. (US 2002/0168342), also incorporated
by reference herein in its entirety, and include those techniques
within the knowledge of those of skill in the art.
[0180] Approximately 40 serotypes of AAV are currently known,
however, new serotypes and variants of existing serotypes are still
being identified today and are considered within the scope of the
present invention. See Gao et al (2002), PNAS 99(18):11854-6; Gao
et al (2003), PNAS 100(10):6081-6; Bossis and Chiorini (2003), J.
Virol. 77(12):6799-810). Different AAV serotypes are used to
optimize transduction of particular target cells or to target
specific cell types within a particular target tissue. The use of
different AAV serotypes may facilitate targeting of malignant
tissue. AAV serotypes including 1, 2, 4, 5 and 6 have been shown to
transduce brain tissue. See, e.g., Davidson et al (2000), PNAS
97(7)3428-32; Passini et al (2003), J. Virol 77(12):7034-40).
Particular AAV serotypes may more efficiently target and/or
replicate in target tissue or cells. A single self-complementary
AAV vector can be used in practicing the invention in order to
increase transduction efficiency and result in faster onset of
transgene expression (McCarty et al., Gene Ther. 2001 August;
8(16):1248-54).
[0181] Retroviral vectors are a common tool for gene delivery
(Miller, 1992, Nature 357: 455-460). Retroviral vectors and more
particularly lentiviral vectors may be used in practicing the
present invention. Retroviral vectors have been tested and found to
be suitable delivery vehicles for the stable introduction of a
variety of genes of interest into the genomic DNA of a broad range
of target cells. The ability of retroviral vectors to deliver
unrearranged, single copy transgenes into cells makes retroviral
vectors well suited for transferring genes into cells. Further,
retroviruses enter host cells by the binding of retroviral envelope
glycoproteins to specific cell surface receptors on the host cells.
Consequently, pseudotyped retroviral vectors in which the encoded
native envelope protein is replaced by a heterologous envelope
protein that has a different cellular specificity than the native
envelope protein (e.g., binds to a different cell-surface receptor
as compared to the native envelope protein) may also find utility
in practicing the present invention. The ability to direct the
delivery of retroviral vectors encoding a transgene to a specific
type of target cells is highly desirable for gene therapy
applications.
[0182] The present invention provides retroviral vectors which
include e.g., retroviral transfer vectors comprising one or more
polynucleotides of interest and retroviral packaging vectors
comprising one or more packaging elements. In particular, the
present invention provides pseudotyped retroviral vectors encoding
a heterologous or functionally modified envelope protein for
producing pseudotyped retrovirus.
[0183] The core sequence of the retroviral vectors of the present
invention may be readily derived from a wide variety of
retroviruses, including for example, B, C, and D type retroviruses
as well as spumaviruses and lentiviruses (see RNA Tumor Viruses,
Second Edition, Cold Spring Harbor Laboratory, 1985). An example of
a retrovirus suitable for use in the compositions and methods of
the present invention includes, but is not limited to, lentivirus.
Other retroviruses suitable for use in the compositions and methods
of the present invention include, but are not limited to, Avian
Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus,
Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus,
Reticuloendotheliosis virus and Rous Sarcoma Virus. Particularly
preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley
and Rowe, J. Virol. 19:19-25, 1976), Abelson (ATCC No. VR-999),
Friend (ATCC No. VR-245), Graffi, Gross (ATCC No. VR-590), Kirsten,
Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998), and Moloney
Murine Leukemia Virus (ATCC No. VR-190). Such retroviruses may be
readily obtained from depositories or collections such as the
American Type Culture Collection ("ATCC"; Rockville, Md.), or
isolated from known sources using commonly available
techniques.
[0184] Preferably, a retroviral vector sequence of the present
invention is derived from a lentivirus. A preferred lentivirus is a
human immunodeficiency virus, e.g., type 1 or 2 (i.e., HIV-1 or
HIV-2, wherein HIV-1 was formerly called lymphadenopathy associated
virus 3 (HTLV-IE) and acquired immune deficiency syndrome
(AIDS)-related virus (ARV)), or another virus related to HIV-1 or
HIV-2 that has been identified and associated with AIDS or
AIDS-like disease. Other lentivirus vectors include, a sheep
Visna/maedi virus, a feline immunodeficiency virus (FIV), a bovine
lentivirus, simian immunodeficiency virus (SIV), an equine
infectious anemia virus (EIAV), and a caprine
arthritis-encephalitis virus (CAEV).
[0185] The various genera and strains of retroviruses suitable for
use in the compositions and methods are well known in the art (see,
e.g., Fields Virology, Third Edition, edited by B. N. Fields et
al., Lippincott-Raven Publishers (1996), see e.g., Chapter 58,
Retroviridae: The Viruses and Their Replication, Classification,
pages 1768-1771, including Table 1, incorporated herein by
reference).
[0186] The invention is applicable to a variety of retroviral
systems, and those skilled in the art will appreciate the common
elements shared across differing groups of retroviruses. All
retroviruses share the features of enveloped virions with surface
projections and containing one molecule of linear, positive-sense
single stranded RNA, a genome consisting of a dimer, and the common
proteins gag, pol and env.
[0187] Lentiviruses share several structural virion proteins in
common, including the envelope glycoproteins SU (gp120) and TM
(gp41), which are encoded by the env gene; CA (p24), MA (p117) and
NC (p7-11), which are encoded by the gag gene; and RT, PR and IN
encoded by the pol gene. HIV-1 and HIV-2 contain accessory and
other proteins involved in regulation of synthesis and processing
virus RNA and other replicative functions. The accessory proteins,
encoded by the vif, vpr, vpu/vpx, and nef genes, can be omitted (or
inactivated) from the recombinant system. In addition, tat and rev
can be omitted or inactivated, e.g., by mutation or deletion.
[0188] First generation lentiviral vector packaging systems provide
separate packaging constructs for gag/pol and env, and typically
employ a heterologous or functionally modified envelope protein for
safety reasons. In second generation lentiviral vector systems, the
accessory genes, vif, vpr, vpu and nef, are deleted or inactivated.
Third generation lentiviral vector systems are those from which the
tat gene has been deleted or otherwise inactivated (e.g., via
mutation).
[0189] Compensation for the regulation of transcription normally
provided by tat can be provided by the use of a strong constitutive
promoter, such as the human cytomegalovirus immediate early
(HCMV-IE) enhancer/promoter. Other promoters/enhancers can be
selected based on strength of constitutive promoter activity,
specificity for target tissue (e.g., liver-specific promoter), or
other factors relating to desired control over expression, as is
understood in the art. For example, in some embodiments, it is
desirable to employ an inducible promoter such as tet to achieve
controlled expression. The gene encoding rev is preferably provided
on a separate expression construct, such that a typical third
generation lentiviral vector system will involve four plasmids: one
each for gagpol, rev, envelope and the transfer vector. Regardless
of the generation of packaging system employed, gag and poi can be
provided on a single construct or on separate constructs.
[0190] Synthetic Non-Viral Delivery Agents
[0191] Synthetic non-viral agents that are capable of promoting the
transfer and expression of a polynucleotide of interest are also
suitable for use in the methods of the invention. Such agents
include, but are not limited to, cationic lipids and polymers.
Non-viral delivery agents that are cationic lipids bind to
polyanionic DNA. Following endocytosis, the nucleic acid must
escape from the delivery agent as well as the endosomal compartment
so that the genetic material is incorporated within the new host.
See Feigner, P. L. Nonviral Strategies for Gene Therapy Sci. Am.
1997, 276, 102-106; Feigner, P. L.; Gadek, T. R.; Holm, M.; Roman,
R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringgold, G. M.;
Danielsen, M. Lipofectin: A highly efficient, lipid mediated
DNA-transfection procedure Proc. Natl. Acad. Sci. USA 1987, 84,
7413-7417; Feigner, P. L.; Kumar, R.; Basava, C.; Border, R. C.;
Hwang-Feigner, J. In; Vical, Inc. San Diego, Calif.: U.S. Pat. No.
5,264,618, 1993; Feigner, J. H.; Kumar, R.; Sridhar, C. N.;
Wheeler, C. J.; Tsai, Y. J.; Border, R.; Ramsey, P.; Martin, M.;
Feigner, P. L. Enhanced Gene Delivery and Mechanism Studies with a
Novel Series of Cationic Formulations J. Biol. Chem. 1994, 269,
2550-2561; Freidmann, T. Sci. Am. 1997, 276, 96-101; Behr, J. P.
Gene Transfer with Synthetic Cationic Amphiphiles: Prospects for
Gene Delivery Bioconjugate Chem. 1994, 5, 382-389; Cotton, M.;
Wagner, B. Non-viral Approaches to Gene Therapy Curr. Op. Biotech.
1993, 4, 705-710; Miller, A. D. Cationic Liposomes for Gene Therapy
Angew. Chem. Int. 1998, 37, 1768-1785; Scherman, D.; Bessodes, M.;
Cameron, B.; Herscovici, J.; Hofland, H.; Pitard, B.; Soubrier, F.;
Wils, P.; Crouzet, J. Application of Lipids and Plasmid Design for
Gene Delivery to Mammalian Cells Curr. Op. Biotech. 1989, 9, 480;
Lasic, D. D. In Surfactants in Cosmetics; 2nd ed.; Rieger, M. M.,
Rhein, L. D., Eds.; Marcel Dekker, Inc.: New York, 1997; Vol. 68,
pp 263-283; Rolland, A. P. From Genes to Gene Medicines: Recent
Advances in Nonviral Gene Delivery Crit. Rev. Ther. Drug 1998, 15,
143-198; de Lima, M. C. P.; Simoes, S.; Pires, P.; Faneca, H.;
Duzgunes, N. Cationic Lipid-DNA Complexes in Gene Delivery from
Biophysics to Biological Applications Adv. Drug. Del. Rev. 2001,
47, 277-294.
[0192] These synthetic non-viral delivery agents have two main
functions, to condense the DNA to be transfected and to promote its
cell-binding and passage across the plasma membrane, and where
appropriate, the two nuclear membranes. Due to its polyanionic
nature, DNA naturally has poor affinity for the plasma membrane of
cells, which is also polyanionic. Several groups have reported the
use of amphiphilic cationic lipid-nucleic acid complexes for in
vivo transfection both in animals and humans. Thus, synthetic
non-viral delivery agents have cationic or polycationic charges.
See Gao, X; Huang, L. Cationic Liposome-mediated Gene Transfer Gene
Therapy 1995, 2, 710-722; Zhu, N.; Liggott, D.; Liu, Y.; Debs, R.
Systemic Gene Expression After Intravenous DNA Delivery into Adult
Mice Science 1993, 261, 209-211; Thierry, A. R.; Lunardiiskandar,
Y.; Bryant, J. L.; Rabinovich, P.; Gallo, R. C.; Mahan, L. C.
Systemic Gene-Therapy-Biodistribution and Long-Term Expression of a
Transgene in Mice Proc. Nat. Acad. Sci. 1995, 92, 9742-9746.
Cationic amphiphilic compounds that possess both cationic and
hydrophobic domains have been previously used for delivery of
genetic information. In fact, this class of compounds is widely
used for intracellular delivery of genes. Such cationic compounds
can form cationic liposomes, which are the most popular synthetic
non-viral delivery agent for gene transfection studies.
[0193] The cationic liposomes serve two functions. First, it
protects the DNA from degradation. Second, it increases the amount
of DNA entering the cell. Such liposomes have proven useful in both
in vitro and in vivo studies. Safinya, C. R. describes the
structure of the cationic amphiphile-DNA complex. See Radler, J.
O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275,
810-814; Templeton, N. S.; Lasic, D. D.; Frederik, P. M.; Strey, H.
H.; Roberts, D. D.; Pavlakis, G. N. Nature Biotech. 1997, 15,
647-652; Koltover, I.; Salditt, T.; Radler, J. O.; Safinya, C. R.
Science 1998, 281, 78-81; and Koltover, I.; Salditt, T.; Safinya,
C. R. Biophys. J. 1999, 77, 915-924. Many of these systems for gene
delivery in vitro and in vivo are reviewed in recent articles. See
Remy, J.; Sirlin, C.; Vierling, P.; Behr, J. Bioconj. Chem. 1994,
5, 647-654; Crystal, R. G. Science 1995, 270, 404-410; Blaese, X.;
et. al. Cancer Gene Ther. 1995, 2, 291-297; and Behr, J. P. and
Gao, X cited above. Unlike viral vectors, the lipid-nucleic acid
complexes can be used to transfer expression cassettes of
essentially unlimited size. Because these synthetic delivery
systems lack proteins, they may evoke fewer immunogenic and
inflammatory responses.
[0194] Behr discloses numerous amphiphiles including
dioctadecylamidologlycylspermine ("DOGS") for gene delivery. This
material is commercially available as TRANSFECTAM.TM.. Vigneron
describes guanidinium-cholesterol cationic lipids for transfection
of eukaryotic cells. Felgner discloses use of positively-charged
synthetic cationic lipids including
N-1-(2,3-dioleyloxy)propyl-chloride ("DOTMA"), to form lipid/DNA
complexes suitable for transfections. Byk describes cationic lipids
where the cationic portion of the amphiphile is either linear,
branched, or globular for gene transfection. Blessing and coworkers
describe a cationic synthetic vector based on spermine. Safinya
describes cationic lipids containing a poly(ethylene glycol)
segment for gene delivery. Bessodes and coworkers describe a
cationic lipid containing glycosidic linker for gene delivery. Ren
and Liu describe cationic lipids based on 1,2,4-butanetriol. Tang
and Scherman describe a cationic lipid that contains a disulfide
linkage for gene delivery. Vierling describes highly fluorinated
cationic amphiphiles as gene carrier and delivery systems. Jacopin
describes a cation amphiphile for gene delivery that contains a
targeting ligand. Wang and coworkers describe carnitine based
cationic esters for gene delivery. Zhu describes the use of a
cationic lipid, N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium
chloride for the intravenous delivery of DNA. See Behr, J. P.;
Demeneix, B.; Loeffler, J. P.; Perez-Mutul, J. Efficeint Gene
Transfer into Mammalian Primary Endocrine Cells with Lipopolyamine
Coated DNA Proc. Nat. Acad. Sci. 1989, 86, 6982-6986; Vigneron, J.
P.; Oudrhiri, N.; Fauquet, M.; Vergely, L.; Bradley, J. C.;
Basseville, M.; Lehn, P.; Lehn, J. M. Proc. Nat. Acad. Sci. 1996,
93, 9682-9686; Byk, G.; B Dubertret, C.; Escriou, V.; Frederic, M.;
Jaslin, G.; Rangara, R.; Pitard, B.; Wils, P.; Schwartz, B.;
Scherman, D. J. Med. Chem. 1998, 41, 224-235; Blessing, T.; Remy,
J. S.; Behr, J. P. J. Am. Chem. Soc. 1998, 120, 8519-8520;
Blessing, T.; Remy, J. S.; Behr, J. P. Proc. Nat. Acad. Sci. 1998,
95, 1427-1431; Schulze, U.; Schmidt, H.; Safinya, C. R. Bioconj.
Chem. 1999, 10, 548-552; Bessodes, M.; Dubertret, C.; Jaslin, G.;
Scherman, D. Bioorg. Med. Chem. Left. 2000, 10, 1393-1395;
Herscovici, J.; Egron, M. J.; Quenot, A.; Leclercq, F.;
Leforestier, N.; Mignet, N.; Wetzer, B.; Scherman, D. Org. Lett.
2001; Ren, T.; Liu, D. Tetrahedron Lett. 1999, 40, 7621-7625; Tang,
F.; Hughes, J. A. Biochem. Biophys. Res. Commun. 1998, 242,
141-145; Tang, F.; Hughes, J. A. Bioconjugate Chem. 1999, 10,
791-796; Wetzer, B.; Byk, G.; Frederic, M.; Airiau, M.; Blanche,
F.; Pitard, B.; Scherman, D. Biochemical J. 2001, 356, 747-756;
Vierling, P.; Santaella, C.; Greiner, J. J. Fluorine Chem. 2001,
107, 337-354; Jacopin, J.; Hofland, H.; Scherman, D.; Herscovici,
J. J. Biomed. Chem. Lett. 2001, 11, 419-422; and Wang, J.; Guo, X.;
Xu, Y.; Barron, L.; Szoka, F. C. J. Med. Chem. 1998, 41,
2207-2215.
[0195] In U.S. Pat. No. 5,283,185 to Epand et al., the inventors
describe additional examples of amphiphiles including a cationic
cholesterol synthetic vector, termed "DC-chol". The inventors
describe, in U.S. Pat. No. 5,264,618, more cationic compounds that
facilitate transport of biologically active molecules into cells.
U.S. Pat. Nos. 6,169,078 and 6,153,434 to Hughes et al. disclose a
cationic lipid that contains a disulfide bond for gene delivery.
U.S. Pat. No. 5,334,761 to Gebeyehu et al. describes additional
cationic amphiphiles suitable for intracellular delivery of
biologically active molecules. U.S. Pat. No. 6,110,490 to Thierry
describes additional cationic lipids for gene delivery. U.S. Pat.
No. 6,056,938 to Unger, et al. discloses cationic lipid compounds
that contain at least two cationic groups.
[0196] Polymeric systems for gene delivery are known in the art. In
Han's review, he discussed most of the common cationic polymer
systems including PLL, poly(L-lysine); PEI, polyethyleneimine;
pDMEAMA, poly(2-dimethylamino)ethyl-methacrylate; PLGA,
poly(D,L-lactide-co-glycolide) and PVP (polyvinylpyrrolidone). See
Garnett, M. C. Crit. Rev. Ther. Drug Carrier Sys. 1999, 16,
147-207; Han, S.; Mahato, R. I.; Sung, Y. K.; Kim, S. W. Molecular
Therapy 2000, 2, 302-317; Zauner, W.; Ogris, M.; Wagner, E. Adv.
Drug. Del. Rev. 1998, 30, 97-113; Kabanov, A. V.; Kabanov, V. A.
Bioconj. Chem. 1995, 6, 7-20; Lynn, D. M.; Anderson, D. G.; Putman,
D.; Langer, R. J. Am. Chem. Soc. 2001, 123, 8155-8156; Boussif, O.;
Lezoualc'h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.;
Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. USA 1995, 92,
7297-7301; Choi, J. S.; Joo, D. K.; Kim, C. H.; Kim, K.; Park, J.
S. J. Am. Chem. Soc. 2000, 122, 474-480; Putnam, D.; Langer, R.
Macromolecules 1999, 32, 3658-3662; Gonzalez, M. F.; Ruseckaite, R.
A.; Cuadrado, T. R. Journal of Applied Polymer Science 1999, 71,
1223-1230; Tang, M. X.; Redemann, C. T.; Szoka, F. C. In Vitro Gene
Delivery by Degraded Polyamidoamine Dendrimers Bioconjugate Chem.
1996, 7, 703-714; Kukowska-latallo, J. F.; Bielinska, A. U.;
Johnson, J.; Spinder, R.; Tomalia, D. A.; Baker, J. R. Proc. Nat.
Acad. Sci. 1996, 93, 4897-4902; and Lim, Y.; Kim, S.; Lee, Y.; Lee,
W.; Yang, T.; Lee, M.; Suh, M.; Park, J. J. Am. Chem. Soc. 2001,
123, 2460-2461.
[0197] Some representative examples of cationic polymers under
investigation are described below. For example, poly(beta-amino
esters) have been explored and shown to condense plasmid DNA into
soluble DNA/polymer particles for gene delivery. To accelerate the
discovery of synthetic transfection vectors parallel synthesis and
screening of a cationic polymer library was reported by Langer.
Wolfert describes cationic vectors for gene therapy formed by
self-assembly of DNA with synthetic block cationic co-polymers.
Haensler and Szoka describe the use of cationic dendrimer polymers
(polyamidoamine (PAMAM) dendrimers) for gene delivery. Wang
describes a cationic polyphosphoester for gene delivery. Putnam
describes a cationic polymer containing imidazole for the delivery
of DNA. See Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000, 122,
10761-10768; Wolfert, M. A.; Schacht, E. H.; Toncheva, V.; Ulbrich,
K.; Nazarova, O.; Seymour, L. W. Hum. Gene Ther. 1996, 7,
2123-2133; Haensler, J.; Szoka, F. Bioconj. Chem. 1993, 4, 372; and
Wang, J.; Mao, H. Q.; Leong, K W. J. Am. Chem. Soc. 2001; Putnam,
D.; Gentry, C. A.; Pack, D. W.; Langer, R. Proc. Nat. Acad. Sci.
2001, 98, 1200-1205.
[0198] A number of patents are also known that describe cationic
polymers for gene delivery. For example, U.S. Pat. No. 5,629,184 to
Goldenberg et al. describes cationic copolymers of vinylamine and
vinyl alcohol for the delivery of oligonucleotides. U.S. Pat. No.
5,714,166 to Tomalia, et al, discloses dendritic
cationic-amine-terminated polymers for gene delivery. U.S. Pat. No.
5,919,442 to Yin et al. describes cationic hyper comb-branched
polymer conjugates for gene delivery. U.S. Pat. No. 5,948,878 to
Burgess et al. describes additional cationic polymers for nucleic
acid transfection and bioactive agent delivery. U.S. Pat. No.
6,177,274 to Park et al. discloses a compound for targeted gene
delivery that consists of polyethylene glycol (PEG) grafted
poly(L-lysine) (PLL) and a targeting moiety, wherein at least one
free amino function of the PLL is substituted with the targeting
moiety, and the grafted PLL contains at least 50% unsubstituted
free amino function groups. U.S. Pat. No. 6,210,717 to Choi et al.
describes a biodegradable, mixed polymeric micelle used to deliver
a selected nucleic acid into a targeted host cell that contains an
amphiphilic polyester-polycation copolymer and an amphiphilic
polyester-sugar copolymer. U.S. Pat. No. 6,267,987 to Park et al.
discloses a positively charged poly[alpha-(omega-aminoalkyl)
glycolic acid] for the delivery of a bioactive agent via tissue and
cellular uptake. U.S. Pat. No. 6,200,956 to Scherman et al.
describes a pharmaceutical composition useful for transfecting a
nucleic acid containing a cationic polypeptide.
[0199] Nanoparticle delivery systems suitable for use in delivering
the traps described herein are disclosed in PCT/US2010/044209.
[0200] Targeting Ligands
[0201] The compositions can further comprise a targeting ligand
that is physically associated with the vector.
[0202] By "targeting ligand" is intended a molecule that targets
the vector or a physically associated molecule to a targeted cell
or tissue. Targeting ligands can include, but are not limited to,
small molecules, peptides, lipids, sugars, oligonucleotides,
hormones, vitamins, antigens, antibodies or fragments thereof,
specific membrane-receptor ligands, ligands capable of reacting
with an anti-ligand, fusogenic peptides, nuclear localization
peptides, or a combination of such compounds. Non-limiting examples
of targeting ligands include asialoglycoprotein, insulin, low
density lipoprotein (LDL), folate, benzamide derivatives, and
monoclonal and polyclonal antibodies directed against cell surface
molecules. In some embodiments, the small molecule comprises a
benzamide derivative. In some of these embodiments, the benzamide
derivative comprises anisamide.
[0203] By "targeted cell" is intended the cell to which a targeting
ligand recruits a physically associated molecule. The targeting
ligand can interact with one or more constituents of a target cell.
The targeted cell can be any cell type or at any developmental
stage, exhibiting various phenotypes, and can be in various
pathological states (i.e., abnormal and normal states). For
example, the targeting ligand can associate with normal, abnormal,
and/or unique constituents on a microbe (i.e., a prokaryotic cell
(bacteria), viruses, fungi, protozoa or parasites) or on a
eukaryotic cell (e.g., epithelial cells, muscle cells, nerve cells,
sensory cells, cancerous cells, secretory cells, malignant cells,
erythroid and lymphoid cells, stem cells). Thus, the targeting
ligand can associate with a constitutient on a target cell which is
a disease-associated antigen including, for example,
tumor-associated antigens and autoimmune disease-associated
antigens. Such disease-associated antigens include, for example,
growth factor receptors, cell cycle regulators, angiogenic factors,
and signaling factors.
[0204] In some embodiments, the targeting ligand interacts with a
cell surface protein on the targeted cell. In some of these
embodiments, the expression level of the cell surface protein that
is capable of binding to the targeting ligand is higher in the
targeted cell relative to other cells. For example, cancer cells
overexpress certain cell surface molecules, such as the HER2
receptor (breast cancer) or the sigma receptor. In certain
embodiments wherein the targeting ligand comprises a benzamide
derivative, such as anisamide, the targeting ligand targets the
associated molecule to sigma-receptor overexpressing cells, which
can include, but is not limited to, cancer cells such as small- and
non-small-cell lung carcinoma, renal carcinoma, colon carcinoma,
sarcoma, breast cancer, melanoma, glioblastoma, neuroblastoma, and
prostate cancer (Aydar, Palmer, and Djamgoz (2004) Cancer Res.
64:5029-5035).
[0205] The terms "cancer" or "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. As used herein, "cancer cells" or
"tumor cells" refer to the cells that are characterized by this
unregulated cell growth. The term "cancer" encompasses all types of
cancers, including, but not limited to, all forms of carcinomas,
melanomas, sarcomas, lymphomas and leukemias, including without
limitation, bladder carcinoma, brain tumors, breast cancer,
cervical cancer, colorectal cancer, esophageal cancer, endometrial
cancer, hepatocellular carcinoma, laryngeal cancer, lung cancer,
osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer,
renal carcinoma and thyroid cancer.
[0206] The targeted cell is one that is susceptible to metastases
of a distant cancer. As such, the targeted cell can be in any
organ. In a particular embodiment, the targeted cell is a liver
cell.
[0207] The targeting ligand can be physically associated a vector.
As used herein, the term "physically associated" refers to either a
covalent or non-covalent interaction between two molecules. As used
herein, the term "covalent bond" or "covalent interaction" refers
to a chemical bond, wherein a pair of electrons is shared between
two atoms. Two molecules are said to be chemically bound to one
another when the molecules have at least one chemical bond between
atoms that make up the molecules. One chemical bond between two
molecules is therefore comprised of the sharing of one pair of
electrons between an atom in one molecule with an atom in another
molecule. For example, a targeting ligand can be covalently bound
to a lipid of the invention through one of the nitrogen atoms or
one of the R groups of the cationic lipids. A "conjugate" refers to
the complex of molecules that are covalently bound to one another.
For example, the complex of a lipid covalently bound to a targeting
ligand can be referred to as a lipid-targeting ligand
conjugate.
[0208] Alternatively, the targeting ligand can be non-covalently
bound to the lipids of formula (I) or active derivatives thereof.
"Non-covalent bonds" or "non-covalent interactions" do not involve
the sharing of pairs of electrons, but rather involve more
dispersed variations of electromagnetic interactions, and can
include hydrogen bonding, ionic interactions, Van der Waals
interactions, and hydrophobic bonds. Such lipid-targeting ligand
conjugates can be readily obtained according to techniques widely
described in the literature.
[0209] Polynucleotide of Interest
[0210] The term "polynucleotide" is intended to encompass a
singular nucleic acid, as well as plural nucleic acids, and refers
to a nucleic acid molecule or construct, e.g., messenger RNA
(mRNA), plasmid DNA (pDNA), or short interfering RNA (siRNA). A
polynucleotide can be single-stranded or double-stranded, linear or
circular. A polynucleotide can comprise a conventional
phosphodiester bond or a non-conventional bond (e.g., an amide
bond, such as found in peptide nucleic acids (PNA)). The term
"nucleic acid" refers to any one or more nucleic acid segments,
e.g., DNA or RNA fragments, present in a polynucleotide. The term
"polynucleotide" can refer to an isolated nucleic acid or
polynucleotide, wherein by "isolated" nucleic acid or
polynucleotide is intended a nucleic acid molecule, DNA or RNA,
that has been removed from its native environment. Examples of an
isolated polynucleotide include recombinant polynucleotides
maintained in heterologous host cells or purified (partially or
substantially) polynucleotides in solution. Isolated
polynucleotides or nucleic acids according to the present invention
further include such molecules produced synthetically. Isolated
polynucleotides also can include isolated expression vectors,
expression constructs, or populations thereof. "Polynucleotide"
also can refer to amplified products of itself, as in a polymerase
chain reaction. The "polynucleotide" can contain modified nucleic
acids, such as phosphorothioate, phosphate, ring atom modified
derivatives, and the like. The "polynucleotide" can be a naturally
occurring polynucleotide (i.e., one existing in nature without
human intervention), or a recombinant polynucleotide (i.e., one
existing only with human intervention). While the terms
"polynucleotide" and "oligonucleotide" both refer to a polymer of
nucleotides, as used herein, an oligonucleotide is typically less
than 100 nucleotides in length.
[0211] As used herein, the term "polynucleotide of interest" refers
to a polynucleotide that is to be delivered to a cell to elicit a
desired effect in the cell (e.g., a therapeutic effect, a change in
gene expression). A polynucleotide of interest can be of any length
and can include, but is not limited to a polynucleotide comprising
a coding sequence for a polypeptide of interest. In certain
embodiments, when the polynucleotide is expressed or introduced
into a cell, the polynucleotide of interest or polypeptide encoded
thereby has therapeutic activity.
[0212] i. Polynucleotides Encoding Polypeptides
[0213] In some embodiments, the polynucleotide delivery systems
comprise a polynucleotide comprising a coding sequence for a
polypeptide of interest.
[0214] For the purposes of the present invention, a "coding
sequence for a polypeptide of interest" or "coding region for a
polypeptide of interest" refers to the polynucleotide sequence that
encodes that polypeptide. As used herein, the terms "encoding" or
"encoded" when used in the context of a specified nucleic acid mean
that the nucleic acid comprises the requisite information to direct
translation of the nucleotide sequence into a specified
polypeptide. The information by which a polypeptide is encoded is
specified by the use of codons. The "coding region" or "coding
sequence" is the portion of the nucleic acid that consists of
codons that can be translated into amino acids. Although a "stop
codon" or "translational termination codon" (TAG, TGA, or TAA) is
not translated into an amino acid, it can be considered to be part
of a coding region. Likewise, a transcription initiation codon
(ATG) may or may not be considered to be part of a coding region.
Any sequences flanking the coding region, however, for example,
promoters, ribosome binding sites, transcriptional terminators,
introns, and the like, are not considered to be part of the coding
region. In some embodiments, however, while not considered part of
the coding region per se, these regulatory sequences and any other
regulatory sequence, particularly signal sequences or sequences
encoding a peptide tag, may be part of the polynucleotide sequence
encoding the polypeptide of interest. Thus, a polynucleotide
sequence encoding a polypeptide of interest comprises the coding
sequence and optionally any sequences flanking the coding region
that contribute to expression, secretion, and/or isolation of the
polypeptide of interest.
[0215] The term "expression" has its meaning as understood in the
art and refers to the process of converting genetic information
encoded in a gene or a coding sequence into RNA (e.g., mRNA, rRNA,
tRNA, or snRNA) through "transcription" of a polynucleotide (e.g.,
via the enzymatic action of an RNA polymerase), and for
polypeptide-encoding polynucleotides, into a polypeptide through
"translation" of mRNA. Thus, an "expression product" is, in
general, an RNA transcribed from the gene (e.g., either pre- or
post-processing) or polynucleotide or a polypeptide encoded by an
RNA transcribed from the gene (e.g., either pre- or
post-modification).
[0216] As used herein, the term "polypeptide" or "protein" is
intended to encompass a singular "polypeptide" as well as plural
"polypeptides," and refers to a molecule composed of monomers
(amino acids) linearly linked by amide bonds (also known as peptide
bonds). The term "polypeptide" refers to any chain or chains of two
or more amino acids, and does not refer to a specific length of the
product. Thus, peptides, dipeptides, tripeptides, oligopeptides,
"protein," "amino acid chain," or any other term used to refer to a
chain or chains of two or more amino acids, are included within the
definition of "polypeptide," and the term "polypeptide" can be used
instead of, or interchangeably with any of these terms.
[0217] The term "polypeptide of interest" refers to a polypeptide
that is to be delivered to a cell or is encoded by a polynucleotide
that is to be delivered to a cell to elicit a desired effect in the
cell (e.g., a therapeutic effect). The polypeptide of interest can
be of any species and of any size.
[0218] Extensive sequence information required for molecular
genetics and genetic engineering techniques is widely publicly
available. Access to complete nucleotide sequences of mammalian, as
well as human, genes, cDNA sequences, amino acid sequences and
genomes can be obtained from GenBank at the website
www.ncbi.nlm.nih.gov/Entrez. Additional information can also be
obtained from GeneCards, an electronic encyclopedia integrating
information about genes and their products and biomedical
applications from the Weizmann Institute of Science Genome and
Bioinformatics (bioinformatics.weizmann.ac.il/cards), nucleotide
sequence information can be also obtained from the EMBL Nucleotide
Sequence Database (www.ebi.ac.uk/embl) or the DNA Databank or Japan
(DDBJ, www.ddbi.nig.ac.jp). Additional sites for information on
amino acid sequences include Georgetown's protein information
resource website (www.pir.georgetown.edu) and Swiss-Prot
(au.expasy.org/sprot/sprot-top.html).
[0219] As discussed above, the compositions of the invention can
comprise genetic material, such as a polynucleotide of interest,
e.g., pDNA (plasmid DNA), which when transcribed produces a trap.
In such embodiments, the genetic material can be part of an
expression cassette. In addition, polynucleotides comprise a coding
sequence found in an expression cassette.
[0220] The terms "introduction" or "introduce" when referring to a
polynucleotide refers to the presentation of the polynucleotide to
a cell in such a manner that the polynucleotide gains access to the
intracellular region of the cell.
[0221] The expression cassette comprises one or more regulatory
sequences, selected on the basis of the cells to be used for
expression, operably linked to a polypeptide of interest. "Operably
linked" is intended to mean that the nucleotide sequence of
interest (i.e., a coding sequence for a polypeptide of interest) is
linked to the regulatory sequence(s) in a manner that allows for
expression of the nucleotide sequence (e.g., in an in vitro
transcription/translation system or in a cell when the expression
cassette or vector is introduced into a cell). "Regulatory
sequences" include promoters, enhancers, and other expression
control elements (e.g., polyadenylation signals). See, for example,
Goeddel (1990) in Gene Expression Technology: Methods in Enzymology
185 (Academic Press, San Diego, Calif.). Regulatory sequences
include those that direct constitutive expression of a nucleotide
sequence in many types of host cells and those that direct
expression of the nucleotide sequence only in certain host cells
(e.g., tissue-specific regulatory sequences). It will be
appreciated by those skilled in the art that the design of the
expression cassette can depend on such factors as the choice of the
host cell to be transformed, the level of expression of the
silencing element or polypeptide of interest desired, and the like.
Such expression cassettes typically include one or more
appropriately positioned sites for restriction enzymes, to
facilitate introduction of the nucleic acid into a vector.
[0222] It will further be appreciated that appropriate promoter
and/or regulatory elements can readily be selected to allow
expression of the relevant transcription units/silencing elements
in the cell of interest. In certain embodiments, the promoter
utilized to direct intracellular expression of a silencing element
is a promoter for RNA polymerase III (Pol III). References
discussing various Pol III promoters, include, for example, Yu et
al. (2002) Proc. Natl. Acad. Sci. 99(9), 6047-6052; Sui et al.
(2002) Proc. Natl. Acad. Sci. 99(8), 5515-5520 (2002); Paddison et
al. (2002) Genes and Dev. 16, 948-958; Brummelkamp et al. (2002)
Science 296, 550-553; Miyagashi (2002) Biotech. 20, 497-500; Paul
et al. (2002) Nat. Biotech. 20, 505-508; Tuschl et al. (2002) Nat.
Biotech. 20, 446-448. According to other embodiments, a promoter
for RNA polymerase I, e.g., a tRNA promoter, can be used. See
McCown et al. (2003) Virology 313(2):514-24; Kawasaki (2003)
Nucleic Acids Res. 31 (2):700-7. In some embodiments in which the
polynucleotide comprises a coding sequence for a polypeptide of
interest, a promoter for RNA polymerase II can be used.
[0223] The regulatory sequences can also be provided by viral
regulatory elements. For example, commonly used promoters are
derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian
Virus 40. For other suitable expression systems for both
prokaryotic and eukaryotic cells, see Chapters 16 and 17 of
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See,
Goeddel (1990) in Gene Expression Technology: Methods in Enzymology
185 (Academic Press, San Diego, Calif.).
[0224] In vitro transcription can be performed using a variety of
available systems including the T7, SP6, and T3 promoter/polymerase
systems (e.g., those available commercially from Promega, Clontech,
New England Biolabs, and the like). Vectors including the T7, SP6,
or T3 promoter are well known in the art and can readily be
modified to direct transcription of silencing elements.
[0225] PEGylation
[0226] PEGylation of the vector enhances the circulatory half-life
of the delivery system by reducing clearance of the vector by the
reticuloendothelial (RES) system. While not being bound by any
particular theory or mechanism of action, it is believed that a
PEGylated vector can evade the RES system by sterically blocking
the opsonization of the particles (Owens and Peppas (2006) Int J
Pharm 307:93-102). In order to provide enough steric hindrance to
avoid opsonization, the exterior surface of the vector must be
completely covered by PEG molecules in the "brush" configuration.
At low surface coverage, the PEG chains will typically have a
"mushroom" configuration, wherein the PEG molecules will be located
closer to the surface of the lipid vehicle. In the "brush"
configuration, the PEG molecules are extended further away from the
particle surface, enhancing the steric hindrance effect. However,
over-crowdedness of PEG on the surface may decrease the mobility of
the polymer chains and thus decrease the steric hindrance effect
(Owens and Peppas (2006) Int J Pharm 307:93-102). The conformation
of PEG depends upon the surface density and the molecular mass of
the PEG on the surface of the vector. The controlling factor is the
distance between the PEG chains on the vehicle surface (D) relative
to their Flory dimension, R.sub.F, which is defined as aN.sup.3/5,
wherein a is the persistence length of the monomer, and N is the
number of monomer units in the PEG (Nicholas et al. (2000) Biochim
Biophys Acta 1463:167-178). Three regimes can be defined: (1) when
D>2 R.sub.F (interdigitated mushrooms); (2) when D<2 R.sub.F
(mushrooms); and (3) when D<R.sub.F (brushes) (Nicholas et
al.).
[0227] Pharmaceutical Compositions
[0228] The lipids and delivery systems of the invention are useful
in mammalian tissue culture systems, in animal studies, and for
therapeutic purposes. The cytotoxic cationic lipids of formula (I),
and delivery systems comprising a cationic lipid of formula (I),
wherein the cationic lipids of formula (I) have cytotoxic activity,
delivery systems comprising a cationic lipid of formula (I),
wherein the bioactive compound has therapeutic activity, and
delivery systems comprising a cytotoxic cationic lipid of formula
(I) and a bioactive compound with therapeutic activity can be used
in therapeutic applications. The presently disclosed subject matter
therefore provides pharmaceutical compositions comprising cytotoxic
cationic lipids of formula (I) or delivery systems comprising
cationic lipids of formula (I).
[0229] The presently disclosed compositions can be formulated for
delivery, i.e., administering to the subject, by any available
route including, but not limited, to parenteral (e.g.,
intravenous), intradermal, subcutaneous, oral, nasal, bronchial,
ophthalmic, transdermal (topical), transmucosal, rectal, and
vaginal routes. In some embodiments, the route of delivery is
intravenous, parenteral, transmucosal, nasal, bronchial, vaginal,
and oral.
[0230] Compositions can be formulated as a pharmaceutically
acceptable salt. The phrase "pharmaceutically acceptable salt(s),"
as used herein, means those salts of the presently disclosed
compounds that are safe and effective for use in a subject and that
possess the desired biological activity. Pharmaceutically
acceptable salts include salts of acidic or basic groups present in
compounds of the invention. Pharmaceutically acceptable acid
addition salts include, but are not limited to, hydrochloride,
hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate,
acid phosphate, borate, isonicotinate, acetate, lactate,
salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate,
succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,
saccharate, formate, benzoate, glutamate, methanesulfonate,
ethanesulfonate, benzensulfonate, p-toluenesulfonate, pamoate
(i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), mesylate
salts. Certain of the presently disclosed compounds can form
pharmaceutically acceptable salts with various amino acids.
Suitable base salts include, but are not limited to, aluminum,
calcium, lithium, magnesium, potassium, sodium, zinc, and
diethanolamine salts. For a review on pharmaceutically acceptable
salts see Berge et al. (1977) J. Pharm. Sci. 66:1-19, which is
incorporated herein by reference. The salts of the lipids described
herein can be prepared, for example, by reacting the appropriate
equivalent of the compound with the desired acid or base in
solution. After the reaction is complete, the salts are
crystallized from solution by the addition of an appropriate amount
of solvent in which the salt is insoluble.
[0231] As used herein the term "pharmaceutically acceptable
carrier" includes solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like, compatible with pharmaceutical or
cosmetic administration. Supplementary active compounds also can be
incorporated into the compositions.
[0232] As one of ordinary skill in the art would appreciate, a
presently disclosed pharmaceutical composition is formulated to be
compatible with its intended route of administration. Solutions or
suspensions used for parenteral (e.g., intravenous), intramuscular,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents, such as
benzyl alcohol or methyl parabens; antioxidants, such as ascorbic
acid or sodium bisulfite; chelating agents, such as
ethylenediaminetetraacetic acid; buffers, such as acetates,
citrates or phosphates; and agents for the adjustment of tonicity,
such as sodium chloride or dextrose. pH can be adjusted with acids
or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0233] Pharmaceutical compositions suitable for injectable use
typically include sterile aqueous solutions (where water soluble)
or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersions. For
intravenous administration, suitable carriers include physiological
saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany,
N.J.) or phosphate buffered saline (PBS). The composition should be
sterile and should be fluid to the extent that easy syringability
exists. In some embodiments, the pharmaceutical compositions are
stable under the conditions of manufacture and storage and should
be preserved against the contaminating action of microorganisms,
such as bacteria and fungi. In general, the relevant carrier can be
a solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyetheylene glycol, and the like), and suitable mixtures
thereof. The proper fluidity can be maintained, for example, by the
use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. In some embodiments, isotonic agents, for
example, sugars, polyalcohols, such as manitol or sorbitol, or
sodium chloride are included in the formulation. Prolonged
absorption of the injectable formulation can be brought about by
including in the formulation an agent that delays absorption, for
example, aluminum monostearate and gelatin.
[0234] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., cytotoxic cationic lipid
of formula (I) or a delivery system comprising a cationic lipid of
formula (I)) in the required amount in an appropriate solvent with
one or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. In certain embodiments,
solutions for injection are free of endotoxin. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In those
embodiments in which sterile powders are used for the preparation
of sterile injectable solutions, the solutions can be prepared by
vacuum drying and freeze-drying which yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0235] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions also can be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically or cosmetically compatible
binding agents, and/or adjuvant materials can be included as part
of the composition. The tablets, pills, capsules, troches, and the
like can contain any of the following ingredients, or compounds of
a similar nature: a binder, such as microcrystalline cellulose, gum
tragacanth or gelatin; an excipient, such as starch or lactose, a
disintegrating agent, such as alginic acid, Primogel, or corn
starch; a lubricant, such as magnesium stearate or Sterotes; a
glidant, such as colloidal silicon dioxide; a sweetening agent,
such as sucrose or saccharin; or a flavoring agent, such as
peppermint, methyl salicylate, or orange flavoring. Compositions
for oral delivery can advantageously incorporate agents to improve
stability within the gastrointestinal tract and/or to enhance
absorption.
[0236] For administration by inhalation, the presently disclosed
compositions can be delivered in the form of an aerosol spray from
a pressured container or dispenser which contains a suitable
propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Liquid aerosols, dry powders, and the like, also can be used.
[0237] Systemic administration of the presently disclosed
compositions also can be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate
to the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art, and include, for
example, for transmucosal administration, detergents, bile salts,
and fusidic acid derivatives. Transmucosal administration can be
accomplished through the use of nasal sprays or suppositories. For
transdermal administration, the active compounds are formulated
into ointments, salves, gels, or creams as generally known in the
art.
[0238] The compositions described herein also can be prepared in
the form of suppositories (e.g., with conventional suppository
bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
[0239] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical or cosmetic
carrier. The specification for the dosage unit forms of the
invention are dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of individuals. Guidance regarding dosing is provided elsewhere
herein.
[0240] As used herein, "therapeutic activity" when referring to the
compositions described herein is intended one that is able to
elicit a desired pharmacologic and/or physiologic effect when
administered to a subject in need thereof.
[0241] As used herein, the terms "treatment" or "prevention" refer
to obtaining a desired pharmacologic and/or physiologic effect. The
effect may be prophylactic in terms of completely or partially
preventing a particular infection or disease or sign or symptom
thereof and/or may be therapeutic in terms of a partial or complete
cure of an infection or disease and/or adverse effect attributable
to the infection or the disease. Accordingly, the method "prevents"
(i.e., delays or inhibits) and/or "reduces" (i.e., decreases,
slows, or ameliorates) the detrimental effects of a disease or
disorder in the subject receiving the compositions of the
invention. The subject may be any animal, including a mammal, such
as a human, and including, but by no means limited to, domestic
animals, such as feline or canine subjects, farm animals, such as
but not limited to bovine, equine, caprine, ovine, and porcine
subjects, wild animals (whether in the wild or in a zoological
garden), research animals, such as mice, rats, rabbits, goats,
sheep, pigs, dogs, cats, etc., avian species, such as chickens,
turkeys, songbirds, etc., i.e., for veterinary medical use.
[0242] Any type of unwanted condition or disease can be treated
therapeutically with the presently disclosed compositions. In some
embodiments, the disease or unwanted condition that is to be
treated is a cancer. As described elsewhere herein, the term
"cancer" encompasses any type of unregulated cellular growth and
includes all forms of cancer. In some embodiments, the cancer to be
treated is a colorectal cancer. Methods to detect the inhibition of
cancer growth or progression are known in the art and include, but
are not limited to, measuring the size of the primary tumor to
detect a reduction in its size, delayed appearance of secondary
tumors, slowed development of secondary tumors, decreased
occurrence of secondary tumors, and slowed or decreased severity of
secondary effects of disease.
[0243] It will be understood by one of skill in the art that the
administration of the compositions described herein can be used
alone or in conjunction with other therapeutic modalities,
including, but not limited to, surgical therapy, radiotherapy, or
treatment with any type of therapeutic agent, such as a drug. In
those embodiments in which the subject is afflicted with cancer,
the compositions described herein can be delivered in combination
with any chemotherapeutic agent well known in the art.
[0244] In some embodiments, the cytotoxic bioactive compound and
the compositions described herein can be administered
simultaneously to the subject, wherein the cytotoxic bioactive
compound and the compositions described herein are both present
within a single composition that is administered to the subject.
Alternatively, in other embodiments, the cytotoxic bioactive
compound and the compositions described herein are administered in
separate compositions sequentially. By "sequentially" is intended
that the two compositions are administered one after the other to
the subject, with two separate administrations of two distinct
compositions, wherein one composition comprises the cytotoxic
bioactive compound and the other composition comprises the
compositions described herein.
[0245] When administered to a subject in need thereof, the
compositions described herein can further comprise a targeting
ligand, as discussed elsewhere herein. In these embodiments, the
targeting ligand will target the physically associated ligand or
complex to a targeted cell or tissue within the subject. In some
embodiments, the targeted delivery system is cytotoxic. In certain
embodiments, the targeted cell or tissue will be diseased or
characterized by the unwanted condition.
[0246] Dosing
[0247] Delivery of a therapeutically effective amount of the
compositions described herein can be obtained via administration of
a pharmaceutical composition comprising a therapeutically effective
dose of this agent. By "therapeutically effective amount" or "dose"
is meant the concentration of the compositions described herein
that is sufficient to elicit the desired therapeutic effect.
[0248] As used herein, "effective amount" is an amount sufficient
to effect beneficial or desired clinical or biochemical results. An
effective amount can be administered one or more times.
[0249] The effective amount of the compositions described herein
will vary according to the weight, sex, age, and medical history of
the subject. Other factors which influence the effective amount can
include, but are not limited to, the severity of the subject's
condition, the disorder being treated, and, if desired, the
adjuvant therapeutic agent being administered along with the lipid
or lipid-comprising complex. Methods to determine efficacy and
dosage are known to those skilled in the art. See, for example,
Isselbacher et al. (1996) Harrison's Principles of Internal
Medicine 13 ed., 1814-1882, herein incorporated by reference.
[0250] The pharmaceutical composition can be administered at
various intervals and over different periods of time as required,
e.g., multiple times per day, daily, every other day, once a week
for between about 1 to 10 weeks, between 2 to 8 weeks, between
about 3 to 7 weeks, about 4, 5, or 6 weeks, and the like. The
skilled artisan will appreciate that certain factors can influence
the dosage and timing required to effectively treat a subject,
including but not limited to the severity of the disease, disorder,
or unwanted condition, previous treatments, the general health
and/or age of the subject, and other diseases or unwanted
conditions present. Generally, treatment of a subject can include a
single treatment or, in many cases, can include a series of
treatments.
[0251] It is to be understood that appropriate doses of the
compositions described herein depend upon its potency and can
optionally be tailored to the particular recipient, for example,
through administration of increasing doses until a preselected
desired response is achieved. It is understood that the specific
dose level for any particular animal subject can depend on a
variety of factors including the activity of the specific
compositions described herein employed, the age, body weight,
general health, gender, and diet of the subject, the time of
administration, the route of administration, the rate of excretion,
any drug combination, and the degree of expression or activity to
be modulated.
[0252] In another embodiment of the invention, a therapeutically
effective dose of the compositions described herein is administered
intermittently. By "intermittent administration" is intended
administration of a therapeutically effective dose of the
compositions described herein, followed by a time period of
discontinuance, which is then followed by another administration of
a therapeutically effective dose, and so forth. Administration of
the therapeutically effective dose can be achieved in a continuous
manner, as for example with a sustained-release formulation, or it
can be achieved according to a desired daily dosage regimen, as for
example with one, two, three, or more administrations per day. By
"time period of discontinuance" is intended a discontinuing of the
continuous sustained-released or daily administration of the
compositions described herein. The time period of discontinuance
may be longer or shorter than the period of continuous
sustained-release or daily administration. During the time period
of discontinuance, the level of the effect of the compositions
described herein in the relevant tissue is substantially below the
maximum level obtained during the treatment. In some embodiments,
the discontinuance period depends on the concentration of the
effective dose. The discontinuance period can be at least 2 days,
at least 4 days or at least 1 week. In other embodiments, the
period of discontinuance is at least 1 month, 2 months, 3 months, 4
months or greater. When a sustained-release formulation is used,
the discontinuance period must be extended to account for the
greater residence time of the compositions described herein at the
therapeutic site. Alternatively, the frequency of administration of
the effective dose of the sustained-release formulation can be
decreased accordingly. An intermittent schedule of administration
of the compositions described herein can continue until the desired
therapeutic effect, and ultimately treatment of the disease or
unwanted condition is achieved.
[0253] One of ordinary skill in the art upon review of the
presently disclosed subject matter would appreciate that the
presently disclosed compositions, including pharmaceutically
acceptable salts and pharmaceutical compositions thereof, can be
administered directly to a cell, a cell culture, a cell culture
medium, a tissue, a tissue culture, a tissue culture medium, and
the like. When referring to the compositions described herein, the
term "administering," and derivations thereof, comprises any method
that allows for the compound to contact a cell. The presently
disclosed compositions, or pharmaceutically acceptable salts or
pharmaceutical compositions thereof, can be administered to (or
contacted with) a cell or a tissue in vitro or ex vivo. The
presently disclosed compositions, or pharmaceutically acceptable
salts or pharmaceutical compositions thereof, also can be
administered to (or contacted with) a cell or a tissue in vivo by
administration to an individual subject, e.g., a patient, for
example, by systemic administration (e.g., intravenous,
intraperitoneal, intramuscular, subdermal, or intracranial
administration) or topical application, as described elsewhere
herein.
IV. ARTICLES OF MANUFACTURE
[0254] The article of manufacture can include a vial or other
container that contains a composition suitable for the present
method together with any carrier, either dried or in liquid form.
The article of manufacture further includes instructions in the
form of a label on the container and/or in the form of an insert
included in a box in which the container is packaged, for carrying
out the method of the invention. The instructions can also be
printed on the box in which the vial is packaged. The instructions
contain information such as sufficient dosage and administration
information so as to allow the subject or a worker in the field to
administer the pharmaceutical composition. It is anticipated that a
worker in the field encompasses any doctor, nurse, technician,
spouse, or other caregiver that might administer the composition.
The pharmaceutical composition can also be self-administered by the
subject.
[0255] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
[0256] The development and use of a reliable syngeneic orthotopic
colorectal liver metastasis animal model has allowed for further
investigation into the role of CXCL12 in driving colorectal liver
metastasis formation. This model, first reported by Zhang et al.,
involves CT-26 FL3 cells (2.0.times.10.sup.6) being inoculated into
the cecum wall and yielding a high occurrence of liver metastasis
(.about.90%) (Zhang, Y., et al., Development and Characterization
of a Reliable Mouse Model of Colorectal Cancer Metastasis to the
Liver. Clin Exp Metastasis, 30(7), 2013). Through the establishment
of a CT-26 FL3 (stably expressing RFP/Luc marker genes) cell line,
luciferase bioluminescent analysis was used to demonstrate that
intravenous (IV) injection of Galactose-PEG-LCP nanoparticles
delivering pDNA encoding a small engineered antibody binding domain
CXCL12/SDF-1-trap protein (28.6 kD), primes the liver to resist
metastatic lesions.
[0257] Materials and Methods
[0258] 1. Materials
[0259] 1,2-Distearoyl-sn-glycero-3-phosphatidyl
ethanolamine-N-[succinyl
(polyethyleneglycol)-2000]-N-hydroxysuccinimide
(DSPE-PEG2000-N-hydroxyl succinimide (NHS)) was purchased from NOF
Corporation (Tokyo, Japan). Radioactive .sup.177LuCl.sub.3 in 0.05
N HCl was purchased from PerkinElmer, Inc. and utilized immediately
upon receipt. DSPE-PEG2000-galactose was synthesized through the
conjugation of 10 eq. of 4-aminophenyl .beta.-d-galactopyranoside
and 1 eq. of DSPE-PEG2000-NHS in PBS buffer, followed by chloroform
extraction and dialysis against water using a MWCO 1000 dialysis
tube. All other lipids were purchased from Avanti Polar Lipids,
Inc. (Alabaster, Ala.). Peptides were purchased from Elim
Biopharmaceuticals, Inc. (Hayward, Calif.); monocyclic abbreviated
to mc. Hoechst nucleic acid stain 3342 was purchased from
ThermoFischer Scientific (Grand Island, N.Y.). Fluorescent Cy3 cDNA
labelling kit was acquired via (Mirus LabelIT kit, Mirus Bio,
Madison, Wis.). Luciferin was purchased from Promega Corporation
(Madison, Wis.). Plasmids encoding green fluorescence protein (GFP)
driven by the cytomegalovirus (CMV) promoter were custom prepared
by Bayou Biolabs (Harahan, L A). ELISA, IF, and IHC kits as well as
all antibodies including anti-His-tag, anti-CXCL12, and anti-CD8,
as well as secondary antibodies were purchases through Abcam
(Cambridge, Mass.). Invasion and Migration assay kits were
purchases through EMD Millipore, (Billerica, Mass.). All other
chemicals were obtained from Sigma-Aldrich (St. Louis, Mo.) and
used without further purification. Six-week-old BALB/c female mice
(.about.18 g each) were purchased from Charles River Laboratories
(Wilmington, Mass.).
[0260] 2. Methods
[0261] In Vitro Suppression of Migration and Invasion via CXCL12
Trap Protein: The engineered protein (CXCL12 trap) was tested to
determine its ability to suppress CT-26 FL3 migration and invasion.
The Chemotaxis 96-well Cell Migration and 24 well Cell Invasion
Assay (EMD Millipore, Billerica, Mass.) was used. Cells were
starved for 24 h and seeded on the trans-well plates at a density
of 0.5.times.10.sup.6 cells/ml in serum free medium. One group of
cells remained in serum free medium, while all other groups allowed
for the addition of the chemokine (chemoattractant) CXCL12 (100
ng/ml) in the feeder tray. Furthermore, three groups in which
CXCL12 was present, allowed for the addition of serum free medium
(no treatment), CXCL12 trap (2, 4, 8, 12 .mu.g/ml), or a
commercially available CXCL12 mAb (Abeam) (1, 2, 4 .mu.g/ml)
Incubation at 37.degree. C. in a 5% CO2 environment for 4 and 24 h
(Migration), and for 24 h (Invasion). Cells were dislodged,
collected, and lysed from the underside of the migration/invasion
plate. Lysis buffer was added along with luciferin (luciferase
assay solution), which is analyzed through bioluminescent plate
reader. Background wells were subtracted and quantification was
reported as relative to untreated (no CXCL12 chemoattractant).
[0262] Preparation and Characterization of LCP Loaded with DNA: LCP
was prepared using a modified protocol. Two separate microemulsions
(60 mL each) were prepared of Igepal 520 and cyclohexane (3:7 v/v)
and placed under stirring. A DNA (180 .mu.g) solution was prepared,
in which 1,800 .mu.L of 2.5 M CaCl.sub.2) was added. To this
solution, octaarginine peptide (mc-CR8C) was added at an N:P ratio
of 2:1 (.about.200 .mu.g) and immediately added to the
microemulsion. A Na.sub.2HPO.sub.4 solution (1,800 .mu.L, 50 mM)
was also prepared and added to the other microemulsion. Each
microemulsion was allowed to stir for 20 min. The microemulsion
containing Na.sub.2HPO.sub.4 was added to the microemulsion
containing the DNA/Peptide/CaCl.sub.2). This solution was allowed
to stir for 5 min before addition of 1,200 .mu.I, of 20 mM DOPA (in
CHCl.sub.3). After addition of DOPA the microemulsion was left to
stir an additional 30 min. An equal volume of 100% EtOH (120 ml)
was added to disrupt the emulsion. The mixture was transferred to
50 ml conical centrifuge tubes and centrifuged at 10,000 g for 20
min. After decanting the supernatant, the precipitate was washed
twice thereafter with 100% EtOH to remove traces of Igepal and/or
cyclohexane. The precipitate was then dried under N.sub.2, and
resuspended in CHCl.sub.3. This solution was centrifuged at 10,000
rpm for 5 min for the removal of large aggregates, and the
supernatant containing the LCP "cores" (DNA and peptide entrapped
within a calcium phosphate nanoprecipitate, supporting and
surrounded by a lipid monolayer of DOPA) was recovered.
[0263] To characterize DNA entrapment efficiency, cDNA was labeled
with Cy3 (Mirus LabelIT kit, Mirus Bio, Madison, Wis.) according to
manufacturer instructions. Such Cy3-DNA was formulated into the LCP
cores, after which recovery was assessed via fluorescence
spectrometry. Further studies used Hoescht nucleic acid stain to
confirm DNA entrapment efficiency in which pDNA/peptide was
encapsulated, cores were lysed in acetic acid buffer, peptide/DNA
was dissociated through addition of protease K, and Hoescht stain
was added and assessed via fluorescence spectrometry.
.sup.177Lu-labeled LCP cores were prepared as described above, in
which pDNA/peptide along with .sup.177LuCl.sub.3 was incorporated
into the CaCl.sub.2) solution of the calcium emulsion. Upon
co-precipitation of the two emulsions, .sup.177Lu-labeled LCP cores
were collected as described above, with centrifugation in
CHCl.sub.3 removing aggregates containing .sup.177Lu. The final LCP
cores encapsulated 80% of .sup.177Lu. Final Gal-LCP-pDNA/mc-CR8C
was produced through desiccation of a mixture of free lipids and
cores and rehydration via 5% aqueous sucrose solution. The ratio of
cores to outer leaflet lipids for optimal final particle
formulation was found to be 11 mg core: 600 .mu.l DOTAP (20 mM):
600 .mu.l Cholesterol (20 mM): 500 .mu.l DSPE-PEG2000 (20 mM).
Therein, 35 mol % DOTAP, 35 mol % cholesterol, and 30 mol %
DSPE-PEG2000 (or 25 mol % DSPE-PEG and 5 mol % DSPE-PEG-Gal) were
utilized as outer leaflet lipids. Zeta potential and particle size
of LCP were measured using a Malvern ZetaSizer Nano Series
(Westborough, Mass.). TEM images of LCP were acquired using a JEOL
100CX II TEM (JEOL, Japan).
[0264] Pharmacokinetics, Biodistribution, and cellular distribution
of Gal-LCP-pDNA/mc-CR8C: Pharmacokinetics and quantitative
biodistribution were determined via co-encapsulation of pDNA with
.sup.177Lu, as described above. Such methods have been utilized
previously to accurately determine LCP biodistribution. 8-week-old
BALB/c female mice (6 mice utilized for each group) were injected
individually (0.2 mL, balanced in osmolarity with the addition of
sucrose) with LCP at 0.5 mg pDNA/kg, corresponding to a dose of
1.times.10.sup.8 cpm/kg of .sup.177Lu. For pharmacokinetic
analysis, blood was recovered at various time points (0.5, 1, 2, 4,
8, 12, and 16 h) via tail-nick bleed. For biodistribution analysis,
16 h after the administration of LCP, the blood and major organs
were collected (6 mice utilized for each time point). Radioactivity
in the blood and tissues in both studies was measured using a
.gamma.-counter. Analysis was conducted under a two-compartment
model utilizing Phoenix WinNonlin (Version 6.3, Pharsight
Corporation; Mountain View, Calif.).
[0265] In Vivo Gene Dose Escalation and Expression Time:
Formulation of Galactose targeted LCPs containing pCXCL12 Trap DNA,
which contains a His-Tag at the C-terminal end were injected (0.2
mL, balanced in osmolarity with the addition of sucrose) into
8-week-old BALB/c female mice (0.1, 0.5, or 1 mg DNA/kg, 3 mice
utilized for each group) through the tail vein. Western Blot
analysis and quantification after tail vein IV administration of
increasing concentrations of pCXCL12 trap DNA LCP in order to
determine if the expression is dose dependent. Mice were sacked 24
h after administration. Liver, spleen, lungs, kidney, heart, and
blood were collected and homogenized in RIPA buffer. Total protein
concentration in the lysate was determined through a bicinchoninic
acid protein assay kit (BCA Protein Assay Kit, Pierce, Rockford,
Ill.). Subsequently, 50 .mu.g of total protein was loaded for
western analysis. The desired CXCL12 trap protein has a molecular
weight of 28.6 kD. GAPDH was used as a loading control. His
(6.times.)-tag mouse antibody was used as the primary antibody. The
expression of pCXCL12 trap was quantified as the relative HRP
intensity increase over PBS treated group.
[0266] His-tag ELISA kit was also used in which 5 .mu.g of total
protein was loaded for further expression analysis. The kit
provided standard proteins containing a His-tag to be used as a
standard calibration control. Therefore, quantification of protein
expression can be measured through ELISA analysis. Following the
dose escalation and expression studies, the 0.5 mg DNA/kg dose was
chosen for in vivo therapeutic studies.
[0267] Formulation of Galactose targeted LCPs containing pDNA
encoding pCXCL12 Trap, which contains a His-Tag at the C-terminal
end were injected (0.2 mL, balanced in osmolarity with the addition
of sucrose) into 8-week-old BALB/c female mice (0.5 mg
DNA/kg.times.3 QOD, 3 mice utilized for each group) through the
tail vein. Western Blot analysis and quantification after tail vein
IV administration of pCXCL12 trap DNA LCP in order to determine the
transient time of expression. Mice were sacked 1, 2, 4, or 8 days
after administration. Liver, spleen, lungs, kidney, heart, and
blood were collected and homogenized in RIPA buffer. Protein
content was measured using BCA. Subsequently, 50 .mu.g of total
protein was loaded for western analysis. All gels were loaded with
a specific organ and a standard liver sample in order to analyze
organ versus liver expression levels and maintain consistency in
quantification of organ and liver expression. The expression of
pCXCL12 trap was quantified as the relative HRP intensity increase
over PBS treated group.
[0268] Toxicity and Pathology Studies: Mice were treated with
pCXCL12 trap, pGFP, blank loaded LCP (0.5 mg DNA/kg.times.3, QOD)
(three mice utilized for each group). Furthermore, another
treatment group was administered free CXCL12 trap protein (1.0 mg
protein/kg.times.3, QOD). Mice were sacked 24 h post final tail
vein injection. Serum was obtained from the mice via cardiac
puncture and centrifugation. Hepatic and renal damage was assessed
by measuring the levels of AST, ALT and BUN in the serum samples.
Blood cell levels including, white blood cells, lymphocytes,
granulocytes, and monocytes were measured with whole blood
analysis. These measurements were quantified by the Animal Clinical
Chemistry and Gene Expression Laboratories at UNC Chapel Hill.
Further, the major organs of each mouse were collected, fixed, and
processed thereafter for trichrome staining. Images of tissue
sections were collected using a Nikon light microscope with
10.times. objective.
[0269] In Vivo Liver Metastasis Suppression: Mice were inoculated
with 2.times.10.sup.6 CT-26 FL3 RFP/Luc cells into the cecum wall.
Treatment of 10 .mu.g (pDNA) Gal-LCP-pCXCL12 trap/mc-CR8C on days
10, 12, and 14 was administered through tail vein IV (n=7). Control
groups included PBS/untreated (n=7) and Gal-LCP-GFP/mc-CR8C (n=6).
Progression of tumor mass was followed by administration of 200
.mu.l luciferin (10 mg/ml) IP Luciferase bioluminescent imaging was
recorded 10 min after administration of luciferin. Mouse tumor mass
on day 24 is shown above in bioluminescent image using IVIS with
Kodak camera. After 24 days, the mice were sacked and livers were
extracted. Quantification of tumor burden on livers was quantified
using image J software. Quantification is shown above in which
tumor burden was found to be reduced by over 85% compared to
control groups. Further analysis of other organs metastatic burden
post treatment were studied. Mouse metastasis was assessed on day
24 in which mice were treated with 200 .mu.l of Luciferin (10
mg/ml). Mice were imaged, sacked, and organs were then extracted
and placed in solution of luciferin (1 mg/ml) and imaged for
bioluminescence.
[0270] Statistical Analysis: Data were expressed as the
mean.+-.standard deviation (SD). Statistical analysis was performed
by the Students' t-test when only two value sets were compared, and
one-way analysis of variance (ANOVA) followed by Dunnett's test
when the data involved three or more groups. *, **, *** denotes
p<0.05, 0.01, and 0.001 respectively and was considered
significant and documented on figure or figure legend. In all
statistics the groups are compared against the untreated
control.
Example 1
[0271] Formulation of Galactose-LCP pDNA/mc-CR8C nanoparticles Hu
et al. first reported the formulation and delivery of the
Galactose-LCP with pDNA/mc-CR8C cargo to the liver (hepatocytes) of
mice (Hu, Y., et al., A Highly Efficient Synthetic Vector:
Nonhydrodynamic Delivery of DNA to Hepatocyte Nuclei in Vivo. ACS
Nano, 2013. 7(6): p. 5376-5384). As reported a reverse
micro-emulsion was used to prepare,
1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)-coated Calcium Phosphate
(CaP) nanoparticles (LCP "amorphous cores"). These cores can
encapsulate both DNA (60% efficiency) and the cationic peptides,
yielding a core size ranging from 15 to 25 nm in diameter. The
hollow core structure can be visualized under Transmission Electron
Microscopy (TEM) (FIG. 3A/B). Subsequently, the DOPA monolayer
surrounding the CaP core allows for the addition of the cationic
outer leaflet lipids (1,2-dioleoyl-3-trimethylammonium-propane
(DOTAP), helper lipid cholesterol, and
1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-[succinyl(polyethy-
lene glycol)-2000 (DSPE-PEG2000)) to assist in RES evasion,
producing sub-60 nm particles ("final" LCP, 40-60 nm in diameter,
shown in FIG. 3A-C which can easily penetrate hepatic sinusoidal
fenestrations.
Example 2
Determination of LCP Nanoparticle Size
[0272] The hydrodynamic diameter and the surface charge of the LCP
particle were found via dynamic light scattering to be
approximately 45 nm and 10 mV (FIG. 3C). Dynamic light scattering
indicated that LCP was narrowly dispersed around 45 nm in diameter,
with a positive zeta potential (around +10 mV) due to cationic
charge of DOTAP along with the cation shielding ability of
DSPE-PEG2000. The LCP and liposome mixture result in a z-average of
236.+-.32 nm; n=6. The solution was found to be stable in 10% fetal
bovine serum for at least 24 hours at 37.degree. C. in which no
significant increase in the z-average was observed. (FIG. 3D).
Example 3
pDNA Encapsulation Efficiency in LCP Cores
[0273] Determination of the pDNA loading of Galactose-LCP
pDNA/mc-CR8C was accomplished through lysing the cores in an acetic
acid buffer environment (pH=4). The DNA was dissociated from the
peptide through addition of a protease K solution. The addition of
Hoechst stain allowed for quantitative fluorescent reading to
determine DNA encapsulation efficiency. The DNA encapsulation
efficiency was found to the approximately 50-60%, which corresponds
closely to Hu et al. formulation (Hu, Y., et al., A Highly
Efficient Synthetic Vector: Nonhydrodynamic Delivery of DNA to
Hepatocyte Nuclei in Vivo. ACS Nano, 2013. 7(6): p. 5376-5384).
Example 4
Galactose-LCP-pDNA/Mc-CR8C Nanoparticle PK and Organ/Liver
Accumulation
[0274] Liver specificity, pharmacokinetics, and organ distribution
was determined through incorporation of Lu.sup.177 radioisotope
into the pDNA/mcCR8C LCP core. The Galactose-LCP pCXCL12
trap/mc-CR8C (pTrap LCP) particles containing the Lu.sup.177 were
injected through the tail vein into normal BALB/c mice. The PK and
organ distribution profile found that the galactose-LCP
nanoparticles exhibits a two-phase distribution with a
T.sup.1/2.alpha. and T.sup.1/2.beta. of 20 min and 1054 minutes
respectively, as well as approximately 50% of the LCP accumulating
in the liver 16 h post IV injection. (FIG. 4). Tail vein injection
of the pTrap LCP particles without Galactose targeting showed a
significant decrease in liver accumulation, approximately 10-15%
accumulation, which is comparable to the values Hu et al
reported.
Example 5
In Vivo Liver Expression Profile of Endogenous CXCL12
[0275] To validate the expression levels of endogenous CXCL12 in
the liver of diseased (colorectal liver metastasis model) BALB/C
mice, we extracted, formalin fixed, paraffin sectioned, and
assessed the amount of CXCL12 through immuno-fluorescent staining
with a primary CXCL12 antibody and fluorescently tagged (Alexa
Fluor 594) secondary antibody. We furthered assessed whether the
delivery of our pTrap LCP would yield decreased fluorescent signal
due to CXCL12 trapping as well as decreased inflammation due to
decreased metastatic lesions. Therefore, 10 days post final IV
administration of pTrap LCP (10 .mu.g pDNA QOD.times.3), we
collected the livers, formalin fixed the livers, paraffin sectioned
and used immune-fluorescent staining against CXCL12 (Red). Five
groups (four of which contained CRC) were studied, including
untreated (without CRC), untreated (PBS), Galactose-LCP
pGFP/mc-CR8C (pGFP LCP), pTrap LCP (10 .mu.g), pTrap LCP (10 .mu.g
QOD.times.3). Results are shown in FIG. 5a, in which the untreated
and pGFP had no significant difference in fluorescent intensity,
and had approximately a 5 to 6 fold increase in CXCL12 expression
compared to untreated liver from mice without CRC. However, both
pTrap LCP (10 .mu.g.times.1 and 10 .mu.g QOD.times.3) groups showed
a 2.5 and 5 fold decrease in fluorescent intensity respectively
compared to the untreated, ultimately reaching baseline levels of
CXCL12 found in untreated liver from mice without CRC. (p<0.05)
(FIG. 5A). Due to the decrease in CXCL12 found in the liver after
treatment of the pTrap LCP (10 .mu.g.times.1 and 10 .mu.g
QOD.times.3), we further stained sections to determine the effect
on the liver CD8 T-cell population (Green), MDSC, and T-regulatory
cells which is believed to be recruited by endogenous CXCL12. Four
groups were studied, including healthy (No CRC), untreated (Tumor),
untreated (Stroma), and pTrap LCP (10 .mu.g every other
day.times.3). Results are shown in FIG. 5B, in which pTrap LCP (10
.mu.g QOD.times.3) groups showed a decrease fluorescent intensity
compared to the untreated. (p<0.05) (FIG. 5B).
Example 6
In Vivo Organ Expression/Distribution of CXCL12-Trap Post pTrap LCP
Administration
[0276] Delivery of pTrap LCP via IV tail vein administration in
BALB/c mice yields nearly 50% of the injected dose accumulating in
the liver. (FIG. 4B). Hu et al. reported that the majority of LCP
is taken up and expressed in the hepatocytes. Hu et al. also showed
that decreased PEG density and the absence of galactose targeting
ligand, shifted the uptake preferentially into the Kupffer cells,
decreasing expression levels of the pDNA. Therefore, to insure
hepatocyte uptake and expression we mirror the PEG density (30%
mol. input) and galactose targeting ligand used in Hu et al,
formulation. We further investigated the preferential expression of
the pCXCL12 trap in the liver versus other organs/serum to insure
we have preferential liver specific expression of this CXCL12 trap.
In order to determine the organ expression of the pCXCL12 trap, we
have incorporated a His-tag at the C-terminus, which allows for
ELISA and western blot analysis via a His-Tag mAb. (FIGS. 5D, 5E,
and 5F). Mice were treated with increasing dose of pTrap LCP (0.1
mg/kg, 0.5 mg/kg, and 1.0 mg/kg). Through ELISA and Western blot
analysis we see after 24 h a dose dependent increase in liver
expression with no expression being found in other off-target
organs or serum (FIGS. 5D and 5E). Further studies in which pTrap
LCP (0.5 mg/kg QOD.times.3) was administered and the mice were
sacked on days 1, 2, 4, and 8. Organs were collected and analyzed
through western blot by using anti-His-tag mAb (FIG. 5F). These
results clearly demonstrate that the Galactose-LCP vector allows
for preferential transient expression in the liver, with minimal
expression in any other organ or serum. (FIGS. 5D, 5E, and 5F).
Subsequently, we report that the liver expression holds transient
properties in which expression is found to last up to 8 days post
final injection (10 .mu.g pDNA; 0.5 mg/kg QOD.times.3). (FIG.
5F).
[0277] The preferential expression of Green Fluorescent Protein
Plasmid (pGFP) and CXCL12 trap plasmid (pTrap) in the liver versus
other organs/serum is shown in FIG. 5A. Through fluorescent
microscopy analysis of organ sections on day 2, 4 and 8 after final
pGFP LCP injection, we were able to demonstrate transient
liver-specific expression lasting up to 4 days. No GFP signal was
found in any other major organ sections. Furthermore, expression of
GFP was found to be predominantly in the hepatocyte population
within the liver (FIG. 5A).
Example 7
Decreased Occurrence and Tumor Burden of Colorectal Liver
Metastasis in Mice Post pTrap LCP Administration
[0278] We examined the effect of pTrap LCP on the incidence and
metastasis burden found in mice. Mice were inoculated with
2.0.times.10.sup.6 CT-26 FL3 (RFP/Luc) cells orthotopically into
the cecum wall. In this series of experiments, treatments began 10
days post cecum inoculation. Three treatment groups were explored,
including PBS (untreated), a vector control consisting of pGFP LCP,
and pTrap LCP. Administration of the three treatment groups was
initiated on day 10 post inoculation, in which IV tail vein
injections (10 .mu.g pDNA) on day 10, 12, and 14. The total mouse
tumor burden was followed through IP administration of 200 .mu.l of
luciferin (10 mg/ml) followed by bioluminescent analysis. The whole
mouse tumor burden was recorded weekly, and used to sort mice into
treatment groups before day 10, whole mouse tumor burden on day 24
after inoculation is shown in FIG. 13. On day 24, 200 .mu.l of
luciferin (10 mg/ml) was administered IP, mice were sacked due to
heavy primary tumor burden in the cecum. Organs were collected and
rinsed in PBS before being placed in a diluted luciferin solution
(1 mg/ml). Livers and other organs were analyzed by bioluminescent
imaging to determine metastasis tumor burden. (FIGS. 6A and 6B).
Following bioluminescent analysis, livers were rinsed in PBS, fixed
in formalin solution, sectioned, and trichrome stained for further
morphological analysis. (FIG. 6C). It is clear from the luciferase
(bioluminescent intensity) that the PBS and pGFP LCP have large
metastatic tumor burdens on the liver (FIGS. 6A and 6B), which
subsequently is causing cirrhosis and fibrotic tissue to become
more prominent (FIG. 6C). In contrast, mice treated with pTrap LCP
(10 .mu.g pDNA) three times (QOD) showed a significant (10 fold
reduction in liver metastasis burden) and approximately a 70-80%
decrease in the incidence of liver metastasis formation. The
fibrotic area detected via microscope analysis of the trichrome
stained liver sections was significantly less in specimens from
pGFP LCP mice than in control specimens (p<0.01) (FIG. 6C). This
is the first report to our knowledge that has successfully
expressed a therapeutic protein, via delivery of pDNA in a liver
specific non-viral vector, yielding therapeutic efficacy.
Furthermore, we observe that the metastasis is not only reduced in
burden and incidence, but the metastasis is not found to migrate
and invade other organs. (FIG. 6B).
Example 8
Cancer-Specific T Cells Enhance the Anti-Metastasis Efficacy of
pTrap LCP Therapy
[0279] The decreased MDSC and Treg populations in the liver after
pTrap LCP treatment, along with the presence of CD8+ lymphocytes,
implicate a shift from a pro-tumor (immunosuppressive) to
anti-tumor environment within the liver. Therefore, we examined the
cytotoxic T lymphocytes' (CTLs') ability to decrease the
establishment of metastasis in the liver after pTrap LCP therapy.
To investigate the pTrap LCPs' ability to enhance cancer-specific
CD8 T cell killing, we studied the anti-cancer efficacy of the
pTrap LCP in mice with a depleted CD8+ T cell population. We
followed a protocol similar to that reported by Harimoto et al., in
which .gtoreq.95% of the CD8+ T cell population was depleted after
two intraperitoneal injections of 400 m anti-Lyt2.2 (2.43; rat
IgG2b)(14). Mice were inoculated with CRC according to the
orthotopic syngeneic model described earlier, followed by T cell
depletion before treatment. In this series of experiments,
treatments began 10 days after cecum inoculation. The animals were
divided into three treatment groups: untreated (PBS), pTrap LCP
with anti-CD8 (Anti-Lyt2.2), and pTrap LCP with an antibody isotype
control (rat IgG2b isotype control). To maintain the depletion of
the CD8+ T cell population, an intraperitoneal injection of the
Anti-Lyt2.2 or isotype control IgG (400 jig) was administered on
day 8 and 10. Treatment was initiated on day 10 after inoculation,
with IV tail vein injection (10 .mu.g pDNA) on days 10, 12, and 14.
Mice were euthanized because of heavy primary tumor burden in the
cecum on day 21, and the liver tumor burden was determined through
bioluminescent imaging (FIG. 7). All mice treated with the PBS
developed large metastatic tumor lesions in the liver (FIG. 7). The
T cell-depleted mice treated with anti-Lyt2.2 followed by three
doses of pTrap LCP (10 .mu.g pDNA) showed similar liver tumor
burden to the untreated mice. In contrast, mice treated with the
isotype control IgG2b antibody followed by pTrap LCP showed a
5-fold reduction in liver metastasis burden and approximately 80%
decrease in the incidence of liver metastasis compared to untreated
animals. These results show that the presence of CTLs along with
reduction in CXCL12 decreases the risk of establishing metastatic
lesions in the liver.
Example 9
Reduced Metastatic Burden is Associated with Increased Survival in
Breast Cancer Liver Metastasis Model
[0280] We examined the effect of pTrap LCP on the median survival
and liver tumor burden in an aggressive mouse breast cancer liver
metastasis model. The breast cancer liver metastasis model consists
of the hemi-splenic implantation of a highly metastatic murine
breast cancer cell line 4T1. These studies modeled the clinical
standard of care, in which the primary tumor is resected and death
usually results from the metastatic burden. BALB/c mice were
inoculated with 1.0.times.106 (0.1 mL) of 4T1(GFP/Luc) cells into
one half of the spleen, which had been tied off and separated into
two halves before the tumor inoculation. The hemi-spleen that
received the cells was resected 10 min after inoculation to
decrease primary tumor growth. In this series of experiments,
treatments began on the day of inoculation because of the rapid
migration of cells to the liver, often within 5 min after
inoculation (15). We studied four treatment groups for the breast
cancer liver metastasis model: untreated (PBS), pGFP LCP with
anti-CD8 (10 .mu.g, 0.5 mg/kg pDNA and 400 .mu.g, 20 mg/kg
anti-Lyt2.2), pTrap LCP with anti-CD8 (10 .mu.s, 0.5 mg/kg pDNA and
400 .mu.g, 20 mg/kg anti-Lyt2.2), and pTrap LCP with isotype IgG
(10 .mu.g, 0.5 mg/kg pDNA and 400 .mu.g, 20 mg/kg Isotype IgG).
PBS, pGFP LCP, or pTrap LCP was administered IV via tail vein
injections every other day starting on day 0 and ending on day 6.
Administration of the anti-Lyt2.2 or Isotype IgG control involved
two IP injections on days 0 and 2. Tumor progression was monitored
by bioluminescent imaging (FIG. 8A). Mice were euthanized when one
of the following conditions applied: drastic weight gain or loss
greater than 10% within one week or clear signs of distress, such
as dehydration, inactivity, or shortness of breath/weak breathing.
Three mice from each group were euthanized 10 days after
inoculation, their organs were collected and rinsed in PBS, and the
livers were analyzed for tumor burden by flow cytometry analysis
(FIG. 8B). Mice that did not receive pTrap LCP treatment developed
large metastatic tumor lesions in the liver within the first week
after inoculation (FIGS. 8A and 8B). In contrast, mice treated with
pTrap LCP showed a reduction in liver metastasis burden and
decrease in the incidence of liver metastasis formation, as well as
an almost 2-fold increase in the median survival versus all other
treatment groups (14 vs 25 days) (FIG. 8C).
Example 10
Reducing the Establishment of Liver Metastasis by pTrap LCP, Trap
Protein, and CXCR4 Antagonist
[0281] We compared the efficacy of different therapeutic modalities
[anti-CXCL12 trap protein, CXCR4 small molecule antagonist
(AMD3100), and the pTrap LCP] in reducing the establishment of
liver metastasis using a human colorectal cancer cell line (HT-29)
in immunodeficient athymic mice. The human colorectal cancer liver
metastasis model was established according to the same hemi-splenic
implantation procedure as above, using colorectal cancer cell line
HT-29, which has high expression of CXCR4 (FIG. 11). In this series
of experiments, treatments again began on the day of inoculation
because of the rapid migration of cells to the liver within 5 min
after inoculation (15). The five treatment groups studied for the
colorectal cancer liver metastasis model were untreated (PBS), pGFP
LCP (10 .mu.g, 0.5 mg/kg pDNA), pTrap LCP (10 .mu.g, 0.5 mg/kg
pDNA), free CXCL12 trap protein (10 .mu.g, 0.5 mg/kg protein), and
AMD3100 (100 .mu.g, 5.0 mg/kg). The treatments were administered IV
by tail vein injection every other day, initiated on day 0 and
terminated on day 16 (FIG. 9A). Mice were euthanized on day 36,
their livers were collected and rinsed in PBS, and tumor nodules
were resected from the livers and weighed (FIG. 9B). Mice that did
not receive pTrap LCP or AMD3100 treatment developed numerous
metastatic tumor lesions in the liver (FIG. 9B). In contrast, mice
treated with pTrap LCP or AMD3100 showed a reduction in liver
metastasis burden and decreased incidence of liver metastasis
formation during the treatment compared to all other treatment
groups.
Example 11
Effects of pTrap LCP on Liver, Kidney, and Blood Function (Toxicity
Analysis)
[0282] Administration of pTrap LCP (10 .mu.g QOD.times.3) showed no
significant changes in ALT, AST, creatinine, or BUN levels, as well
as no sign of toxicity in analyzing trichrome histology sections of
any organ 24 h post final IV tail vein injection (FIGS. 10A and
10B). Further analysis of blood/immune cell levels showed no signs
of change compared to untreated mice (FIG. 10A). Toxicological
analysis was also confirmed in histological trichrome organ
sections in which all treatments showed normal tissue/cell
morphology. (FIG. 10B).
Example 12
Combination Therapy
[0283] Materials and Methods
[0284] 1,2-distearoryl-sn-glycero-3-phosphoethanolamine-N-[methoxy
(polyethyleneglycol-2000)] ammonium salt (DSPE-PEG) were purchased
from NOF (Ebisu Shibuya-ku, Tokyo). Dioleoyl phosphatydic acid
(DOPA) and 1,2-Dioleoyl-3-trimethylammonium-propane chloride salt
(DOTAP) were purchased from Avanti Polar Lipids (Alabaster, Ala.,
USA). Cholesterol and protamine were purchased from Sigma-Aldrich
(St. Louis, Mo., USA). All other chemicals were purchased from
Sigma-Aldrich if not specifically mentioned (St. Louis, Mo.,
USA).
[0285] CXCL12 trap gene construction: The coding sequences of the
CXCL12-binding VH and VL domains were used for assembly of the trap
gene. The final sequence for the CXCL12 trap codes for a signaling
peptide, VH domain, a flexible linker, VL domain, E tag, and
His(6.times.) tag, respectively. The complete cDNA was cloned into
pCDNA3.1 between NheI and XhoI sites and the accuracy was confirmed
by DNA sequencing.
[0286] PDL1 trap gene construction: The coding sequences of the
extracellular domain of mouse or human PD-1 and the trimerization
domain of mouse or human CMP1 were used for assembly of the PD-L1
trap gene. The final sequence for the PD-L1 trap codes for a
signaling peptide, the PD-L1 binding domain of PD-1, a flexible
linker, a trimerization domain, E tag, and His(6.times.) tag,
respectively. The complete cDNA was cloned into pCDNA3.1 between
NheI and XhoI sites and the accuracy was confirmed by DNA
sequencing.
[0287] Cell lines: Primary tumor cell line KPC98027 derived KPC
pancreatic ductal adenocarcinoma mouse model (LSL-Kras.sup.G12D/+;
LSL-Trp53.sup.R172II/+; Pdx-1-Cre, on C57Bl/6 background) were
provided by Dr. Serguei Kozlov (National Cancer Institute, Center
for Advanced Preclinical Research) and cultivated in Dulbecco's
Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)
supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1%
Penicillin/Streptomycin at 37.degree. C. and 5% CO2 in a humidified
atmosphere. Lentivirus transfection of cell lines was performed in
which KPC98027 cells were stably transfected with the vector
carrying the mCherry red fluorescent protein (RFP), firefly
luciferase (Luc), and the puromycin resistance gene. Stable
transfected KPC98027 cells (KPC98027 RFP/Luc) were selected in the
presence of puromycin.
[0288] Orthotopic allografting KPC model in mice: Sub-confluent
KPC98207 (with or without RFP/Luc) cells were harvested and washed
in phosphate buffered saline (PBS) just prior to implantation.
Orthotopic allografting KPC model was established by orthotopic
injection of 1.times.10.sup.6 cells into the tail of pancreas. In
brief, eight-week-old C57Bl/6 mice were anesthetized by IP
injection of ketamine/xylazine solution and placed in supine
position. A midline incision was made to exteriorize the spleen and
pancreas. Using an insulin-gage syringe, 1.times.10.sup.6 cells in
40 .mu.L were injected into the tail of pancreas. And the abdominal
wall and skin closed with 6-0 polyglycolic acid sutures. The
injection site was sealed with a tissue adhesive (3M, St. Paul,
Minn.) and sterilized with 70% alcohol to kill cancer cells that
may have leaked out.
[0289] Antibodies: Primary antibodies, fluorescent conjugated
primary and secondary used for immunostainings (IF) and flow
cytometry (flow cytr) were listed in Table 3 below.
TABLE-US-00003 TABLE 3 Antibodies Used in the Study Antibodies
Company Catalog Application Anti-.alpha.SMA Abcam Ab5694 IF
Anti-CD31 Abcam Ab28364 IF Anti-SDF1 (CXCL12) Abcam Ab9797 IF
Anti-PDL1 Abcam Ab80276 IF Ant CD8.alpha. BD 553031 flow cyt
(FITC-conjugated) Ant CD4 BD 561828 flow cyt (FITC-conjugated)
Anti-FOXP3 BD 560408 flow cyt (PE-conjugated) Anti-CD11b BD 553310
flow cyt (FITC-conjugated) Anti-Gr1 (Ly-6G and Ly- BD 553128 flow
cyt 6C) PharmingenTM (PE-conjugated) Anti-CD206 BD flow cyt
(PE-conjugated) Anti-CCR7 BD flow cyt (APC-conjugated) APC Rat
IgG2b, BD 553991 flow cyt .kappa. Isotype Control PharmingenTM
Anti-Rabbit IgG Cell Signaling 4414 IF, flow cyt (Alex Fluor .RTM.
647 Conjugate) Goat anti-rabbit IgG-HRP Santa Cruz Sc-2030 WB
Anti-RFP Invitrogen R10367 WB
[0290] Preparation and Characterization of LPD: LPD were prepared
through a stepwise self-assembly process based on a
well-established protocols in the art. Briefly, DOTAP and
cholesterol (1:1, mol/mol) were dissolved in chloroform, and the
solvent was removed. The lipid film was then hydrated with
distilled water to make the final concentration of 10 mmol/L
cholesterol and DOTAP. Then, the liposome was sequentially extruded
through 200 nm and 100 nm polycarbonate membranes (Millipore,
Mass.) to form 70-100 nm unilamellar liposomes. The LPD polyplex
cores were formulated by mixing 140 .mu.L of 36 .mu.g protamine in
5% glucose with equal volume of 50 .mu.g plasmid (either pcDNA 3.1
as a control plasmid, or plasmids encoding CXCL12 or PDL1 trap) in
5% glucose. The mixture was incubated at room temperature for 10
min and then 60 .mu.l cholesterol/DOTAP liposomes (10 mmol/L each)
were added. Post insertion of 15% DSPE-PEG was further performed at
60.degree. C. for 15 minutes. The size and surface charge of the
NPs were determined by a Malvern ZetaSizer Nano series
(Westborough, Mass.). TEM images were acquired where NPs were
negatively stained using a JEOL 100 CX II TEM (JEOL, Japan).
[0291] Biodistribution and cellular distribution of LPD NPs:
Approximately 0.1% of hydrophobic dye DiI was incorporated into
DOTAP liposomes to formulate the DiI-labeled LPD NPs. Twenty-four
hour after intravenously injection of the DiI-labeled LPD NPs, mice
were euthanized, major organs and tumors were collected. The
distribution of LPD NPs in major organs were quantitatively
visualized with IVIS Kinetics Optical System (Perkin Elmer, CA).
The excitation wavelength was set at 520 nm, while the emission
wavelength was set at 560 nm. Livers and tumors were further
sectioned by a cryostat (1-1/I Hacker Instruments & Industries,
Winnsboro, S.C.) to quantify the distribution of LPD NPs within the
tissues. Accumulation and distribution of NPs before or after Combo
trap LPD NPs treatment in tumors were further compared and
quantified (n=4).
[0292] Transient, local, and intra-tumoral cellular distribution of
trap protein: Formulation of LPD NPs encapsulated pCXCL12 trap DNA,
pPD-L1 trap DNA were injected (50 .mu.g plasmid/mice) intravenously
into mice bearing KPC98027 RFP/Luc allografts (Daily injection,
twice in total). Both pCXCL12 trap DNA and pPD-L1 trap DNA contain
His-Tag at the C-terminal end, which can be used as a tracker for
the expression of the trap protein. At day 1, 3, 5 post the final
injection, mice were sacrificed, major organs and tumors were
collected and homogenized in RIPA buffer. Total protein
concentration in the lysate was determined through a bicinchoninic
acid protein assay kit (BCA Protein Assay Kit, Pierce, Rockford,
Ill.). The transfection and expression efficiency of His-tag
protein in organs and tumors of different time points were
quantified using ELISA (Cell biolabs, INC., n=4). CXCL12 trap
protein was also directly intravenously injected into mice and
compared with the plasmid counterpart in biodistribution and
accumulation level at the time points monitored. Mice bearing
KPC98027 RFP/Luc were also given two doses daily injection of LPD
NPs encapsulating pGFP DNA. Three days after final injection, tumor
tissues were cyro-sectioned and processed with staining of
fibroblast marker .alpha.SMA, leucocyte marker CD45 and the
endothelial marker CD31. Tumor cells were pre-transfected with RFP.
GFP protein expression in different cell populations within the
tumor tissues were observed using a Nikon light microscope (Nikon
Corp., Tokyo). The % of GFP positive cells in each cell populations
were quantified using image J from 5 representative images from
each type of staining. Here's an example of the calculation:
% .times. .times. of .times. .times. CD .times. .times. 45 +
.times. GFP .times. .times. cells = % .times. .times. CD .times.
.times. 45 + .times. .times. GFP + .times. .times. Cells % .times.
.times. GFP + .times. .times. Cells ##EQU00001##
[0293] Tumor Growth Inhibition, Metastasis Suppression and Survival
Analysis:
[0294] Mice bearing KPC98027 RFP/Luc allografts were established as
mentioned above. Treatments were initiated on day 13. Mice were
then randomized into 6 group (n=5-7) as follows: Untreated group
(PBS), Ctrl LPD NP (encapsulated with pcDNA3.1 backbone), CXCL12
trap/Ctrl NPs, PD-L1 trap/Ctrl NP, Combo trap NP, and free combo
trap protein. Intravenous injections were performed every two days
for a total of 4 doses of 50 .mu.g per plasmid/mice. Tumor growth
was monitored using IVIS.RTM. Kinetics Optical System (Perkin
Elmer, CA) every five days. The increases of tumor volumes were
calculated as the radiance of the intensities and standardized with
the initial tumor volume (Vt/V0). Long term survival was also
monitored on mice bearing the KPC98027 RFP/Luc allografts with
different treatments (n=7, in each treatment groups). Mice were
monitored for over two months. Kaplan-Meier curves and Median
Survival were quantified and calculated using Image J. For the
study of metastasis, mice bearing tumors were treated with PBS
(n=5), CXCL12 trap NPs (n=4), PD-L1 trap NPs (n=4), and Combo traps
(n=5). One month after inoculation, mice were injected with 10
mg/mL luciferin and sacrificed. Major organs and tumors were then
extracted and placed in solution of luciferin (5 m/mL) and imaged
for bioluminescence. Major organs were then fixed and processed
with H&E staining to observe the pathology of tumor metastasis
in each organs.
[0295] ELISpot Assay for IFN-.gamma. Production: Re-stimulation of
spleen cells for mice bearing KPC98027 or KPC98027 RFP/Luc
allografts was performed as described previously. In brief, 13 days
after tumor inoculation, spleens in healthy mice, mice bearing
KPC98027, and KPC98027 RFP/Luc were harvested and separated into
single cell suspensions in a sterile condition. Following the
BD.TM. ELISPOT assay instructions, cells were seeded at
2.times.10.sup.5 per well in a capture antibody coated 96 well
plate. The single cell suspensions were then co-cultured with
either inactivated KPC98027, KPC98027 RFP/Luc cell lysates or
healthy mice spleen cell lysates at 37.degree. C. for 40 h. At the
due time, cells were removed by several washing steps. The
production of INF-.gamma. was measured by detection antibody
addition followed by enzyme conjugate magnification. Red dots
signals were developed with a BD ELISpot substrate set and
calculated manually.
[0296] Quantitative Real-time PCR (qPCR) Assay: Total RNA was
extracted from the tumor tissues using an RNeasy kit (Qiagen,
Valencia, Calif.). cDNA was reverse-transcribed using the
First-Strand Synthesis System for RT-PCR (Invitrogen, Grand Island,
N.Y.). One hundred ng of cDNA was amplified with the Taqman
Universal Probes Supermix system (Bio-rad, Hercules, Calif.). All
the mouse-specific primers for RT-PCR reactions are listed in Table
4 (Life Technologies, Grand Island, N.Y.). GAPDH was used as the
endogenous control. Reactions were conducted using the 7500
Real-Time PCR System and the data were analyzed with the 7500
Software.
TABLE-US-00004 TABLE 4 Primer Applied Biosystems/Ref Mouse
IFN-.gamma. Mm01178820_m1 Mouse IL12 .alpha. Mm00446190_m1 Mouse
TNF-.alpha. Mm00443260_g1 Mouse IL4 Mm00441242_m1 Mouse IL10
Mm00441242_m1 Mouse GAPDH Mm99999915_g1
[0297] Flow Cytometry Assay: Tumor-infiltrating immune lymphocytes
were analyzed by flow cytometry. In brief, tissues were harvested
and digested with collagenase A and DNAase at 37.degree. C. for
40-50 min. After red blood cell lysis, cells were dispersed with 1
mL of PBS. For intracellular cytokine staining, the cells from the
tissues were penetrated with penetration buffer (BD) following the
manufacturer's instructions. Different immune lymphocytes
(5.times.10.sup.6/mL) were stained with the fluorescein-conjugated
antibodies mentioned in the previous section.
[0298] Immunofluorescence Staining: After the deparaffinizing step,
antigen retrieval and permeabilization, tissue sections were
blocked in 1% bovine serum albumin (BSA) at room temperature for 1
h. Primary antibodies conjugated with fluorophores (BD, Franklin
Lakes, N.J.) were incubated overnight at 4.degree. C. and nuclei
were counterstained with DAPI containing mounting medium (Vector
Laboratories Inc., Burlingame, Calif.). All antibodies were diluted
according to the manufacturer's manual. Images were taken using
fluorescence microscopy (Nikon, Tokyo, Japan). Three randomly
selected microscopic fields were quantitatively analyzed using
Image J software.
[0299] TUNEL Assay: TUNEL assays were carried out using a DeadEnd
Fluorometric TUNEL System (Promega, Madison, Wis.) according to the
manufactures instructions. Cell nuclei that were fluorescently
stained with FITC (green) were defined as TUNEL-positive nuclei.
Slides were cover-slipped with 4,6-diaminidino-2-phenyl-indole
(DAPI) Vectashield (Vector laboratories, Burlingame, Calif.).
TUNEL-positive nuclei were monitored using fluorescence microscopy
(Nikon, Tokyo, Japan). Three randomly selected microscopic fields
were quantitatively analyzed using Image J.
[0300] H&E Morphology Evaluation and Blood Chemistry Analysis:
Four days after the final treatment of the tumor inhibition study,
mice with different treatments were all subjected to a toxicity
assay. Both whole blood and serum were collected. Whole blood
cellular components were counted and compared. Creatinine, blood
urea nitrogen (BUN), serum aspartate aminotransferase (AST) and
alanine aminotransferase (ALT) in the serum were assayed as
indicators of renal and liver function. Organs including the heart,
liver, spleen, lungs and kidneys were collected and fixed for
H&E staining by UNC histology facility to evaluate the
organ-specific toxicity.
[0301] Statistical Analysis: A two-tailed Student's t-test or a
one-way analysis of variance (ANOVA) were performed when comparing
two groups or larger than two groups, respectively. Statistical
analysis was performed using Prism 5.0 Software. Differences were
considered to be statistically significant if the P-value was less
than 0.05.
[0302] Transient and Local Distribution and Expression of pDNA
(Trap or GFP) within Tumor Microenvironment Post pDNA LPD
Administration
[0303] LPD preferentially deliver macromolecules, including plasmid
DNA, siRNA, mRNA to tumors for anticancer therapy. To prepare LPD,
plasmid DNA (pDNA) was condensed with cationic protamine to form a
slightly anionic complex core. The core was further coated with the
preformulated cationic liposomes (DOTAP, Cholesterol and DSPE-PEG).
TEM images confirm the size of LPD (.about.70 nm) and indicate its
spherical shape and homogenous distribution (FIG. 14A).
Approximately 0.1% of DiI was incorporated into the lipid membrane
of LPD as an in vivo tracker for evaluating the biodistribution of
DiI-labeled LPD.
[0304] Desmoplastic KPC pancreatic tumor model was generated from
orthotopic injection of the primary KPC98027 cells into the tail of
the pancreas. DiI-labeled LPD NPs were intravenously injected into
mice. Twenty-four hour after injection, NPs accumulation in major
organs were analyzed. Consistent with other NPs of similar size,
liver were the major organs taken up LPD NPs (FIG. 14B). Besides
liver, tumor is another major NP accumulation site (FIG. 14B).
Tissue cyrosection data suggest the scattered distribution of
DU-labeled NP over all the liver tissues, with more than 40% of
liver cells were labelled (FIG. 14C). In contrast, only less than
25% of tumor cells took up DiI NPs, and the distribution of NPs
within tumors were heterogeneous and uneven, mostly due to the high
interstitial fluidic pressure (IFP) and thick extracellular matrix
within pancreatic tumor microenvironment. The distribution of GFP
protein in liver and tumor were further compared as an indication
of the transfection efficiency of the LPD delivered plasmid (pGFP).
Despite the higher accumulation of NPs in liver, the expression of
GFP is extremely low in comparison to tumors (FIG. 14C). This can
be attributed to that, the Kupffer cells, which localized in
vicinity of blood vessels, nonspecifically phagocytosed the LPD
NPs. The transfection efficiency of plasmid in Kupffer cells are
relatively low. Therefore, our results demonstrate that LPD
encapsulating plasmid can be locally delivered and expressed within
the KPC pancreatic cancer.
[0305] Immunofluorescence staining was performed to determine the
LPD accumulation in various cell populations within the bulk tumor
mass (FIG. 14D). Stable transgene expression of RFP and
fluorophore-conjugated antibody against mouse .alpha.SMA, CD45 and
CD31 defined tumor cells, fibroblasts, leukocytes and endothelial
populations, respectively. Results show that tumor cells are one of
the major cell populations that take up NPs; more than 60% of the
tumor cells expressed GFP. In addition, more than .about.30% of
fibroblasts take up LPD four days post intravenous injection of LPD
pGFP (two doses, daily), accounting for .about.30% of the total
GFP-positive cells. In contrast, the expression of GFP in
leucocytes and endothelial are negligible, confirming that
fibroblasts are the major stroma off-target sites for NP
distribution and plasmid expression. Due to the adjacent
distribution of fibroblasts to tumor cells, fibroblasts' expression
of the secreted trap would benefit their neighboring effect to
tumor cells rather than as an off-targeting site that diminishes
the therapeutic concentration of drugs approaching tumor cells.
[0306] Subsequently, distribution and expression of the trap
protein (either PDL1 or CXCL12) was assessed through ELISA via the
targetable His-tag incorporated into the C-terminus of the trap
(FIG. 14E). The pure trap protein (CXCL12 trap) was also injected
and compared. After two daily doses of the trap plasmid NP and trap
protein, the mice were sacrificed on days 2, 4, 6, and demonstrated
of transient plasmid transfection and expression in the tumor (FIG.
14E) rather than other organs. In contrast, protein trap was
cleared rapidly, with significantly lower concentration in all the
organs at time monitored.
[0307] These results demonstrate that LPD vector allows for
preferential transient expression of trap plasmid within the
tumors, in particular, fibroblasts and tumor cells, with minimal
expression in any other organs. We report that the tumor expression
holds transient properties in which expression is found to last up
to 4 days post trap plasmid injection (FIG. 14E).
Combined Therapy with LPD NP Encapsulating pCXCL12 Trap DNA and LPD
NP Encapsulating pPD-L1 Trap DNA Improved Antitumor Response
Against KPC Allografts and Suppressed Metastasis
[0308] Previous study by Feig et al. suggested that inhibiting the
interaction of CXCL12 with CXCR4 uncovers the antitumor activity of
anti PD-L1 (Feig et al., Targeting CXCL12 from FAP-expressing
carcinoma-associated fibroblasts synergizes with anti-PD-L1
immunotherapy in pancreatic cancer, PNAS, 2013 Dec. 10; 110(50).
20212-7). Due to the local and transient expression feature of
plasmid delivered by LPD vector, plasmid encoding PD-L1 trap and
CXCL12 trap were encapsulated into LPD vector, separately, and
administered as combined regimens for KPC pancreatic cancer
treatment. KPC98027 RFP/Luc was orthotopically inoculated into the
tail of the pancreas. The dosing schedule of LPD NPs is presented
in FIG. 15A. PBS, LPD NPs encapsulating pcDNA3.1 backbone (Ctrl
NP), free combo trap proteins were set as controls. Tumor volume
correlated from the number of photons emitted from the tumor were
assessed, A) and quantified. B). Results demonstrated that both
CXCL12 trap NPs and PDL1 trap NPs monotherapy showed minimal
antitumor efficacy at low doses (FIG. 15D). Antitumor efficacy for
the monotherapy increased slightly but only partial effective while
increasing the dose (FIG. 15E). On the contrary, the combo trap NP
group significantly inhibited tumor growth (P<0.01) compared to
the PBS group. Tumor weight of the combo group decreased
dramatically both at low and high doses (FIG. 15E). Meanwhile, the
free combo trap protein only showed slight but not significant
anti-cancer effect, suggesting the advantages of using plasmid
rather than protein (FIGS. 15A and 15B). Further, in an overall
survival analysis after the final day of treatment, median survival
was enhanced in the combo trap NP therapy (63.5 days) as compared
to other treatment groups (40.5, 49, 47, 50 days for PBS, Ctrl NP,
CXCL12 trap NP and PDL1 trap NP groups, respectively; FIG. 15C),
conveying not only a potent therapeutic effect but also a
long-lasting overall response. This is consistent with the
observation by Feig et al. who had used a combination of a CXCR4
antagonist and anti-PD-L1 antibody to inhibit the KPC tumor growth
(Feig et al., Targeting CXCL12 from FAP-expressing
carcinoma-associated fibroblasts synergizes with anti-PD-L1
immunotherapy in pancreatic cancer, PNAS, 2013 Dec. 10;
110(50):20212-7). Data in FIG. 15 suggested that the combined
CXCL12 trap NPs with PD-L1 trap NPs indeed exhibited superior
antitumor efficacy in the desmoplastic KPC tumor-bearing mouse
model.
[0309] Further, metastasis of tumors in major organs were monitored
one month after the inoculation of KPC allografts. Consistent with
the patients bearing pancreatic ductal adenocarcinoma (PDAC), liver
and lung are the major metastasis sites for orthotopic KPC models
(FIG. 16A). Tumors were also observed in spleen and kidney due to
invasion within peritoneal cavity. Histology shows large nodules of
metastasis in the lung, spleen and liver of the control group (FIG.
16B) Monotherapy can slightly suppressed tumor metastasis, only the
combo therapy was able to significantly inhibit or even abrogate
metastasis (FIGS. 16A and 16B). Thus, it was apparent that the
combo trap NP strategy was capable of reducing tumor
metastasis.
Enhanced T Cells Infiltration into Tumor Microenvironment Explains
the Superior Anti-Tumor Effect of the Combo Trap NPs
[0310] Cancer cell specific T cell response was reported in KPC
model previously, and was further confirmed herein by the ELISpot
Assay (FIG. 17A). FIG. 17A shows INF-.gamma. ELISpot assay data
using splenocytes from tumor bearing animals. Extract from KPC
cells, with or without the transfected RFP/Luc markers, could
stimulate the splenocytes to secret IFN-.gamma., but not the
extracts from normal splenocytes. The data indicate that KPC tumor
could induce tumor specific T-cell response. The immune response
seen in the tumor bearing mice was not directed to the luciferase
or the red fluorescence markers, but to the yet-to-be identified
tumor associated antigens. However, the absence of any significant
increase in IFN-.gamma.-secreting CD8+ T cells from the spleens of
PD-L1 trap NPs and CXCL12 trap NPs (either mono- or combo-therapy)
treated mice indicates that the antitumor effect of combo trap NPs
was not accomplished by enhanced systemic priming of
cancer-specific CD8+ T cells (FIG. 17B). Since the ELISpot
activities were relatively weak (FIGS. 17A and 17B), a vaccine
which can boost the cancer-specific cytotoxic T-lymphocyte activity
would further enhance the therapeutic activity of the traps.
[0311] To determine if the immunotherapeutic effect was caused by
enhanced T-cell accumulation among cancer cells, the distribution
of T-cells (CD3.sup.+) in the pancreas was shown by
immunofluorescence (FIG. 18A). It was shown that T-cells were
mostly located in the border between tumor and normal pancreas
tissue in the PBS control. Small amounts of T-cells were found in
the tumor region, but they were located in the stroma area. The
pancreas from animals treated with PD-L1 trap showed some
penetration of T-cells into the tumor region, but the ones treated
with CXCL12 trap NP (with or without the PD-L1 trap NP) showed
extensive T-cell infiltration into the tumor region. The
localization of T-cells in the tumor region is quantified in FIG.
18B. The tumors were further collected and dispersed into single
cells. CD3.sup.+CD8.sup.+ cells were analyzed with flow cytometry
(FIG. 18C). Results, again, confirms that the CD8.sup.+ T-cells
were significantly increased in tumors of the combo trap NPs
treated mice. Thus, we conclude that, CXCL12, rather than PD-L1,
trap was the major factor that enhanced T-cell infiltration.
Further, the role of CD8.sup.+ T-cells in the combo trap NPs
therapy were evaluated by depleting CD8.sup.+ T-cells using
monoclonal antibody against CD8 (FIGS. 18D and 18E). It was shown
that combo trap NPs significantly slowed tumor growth, but not when
CD8 T cells were removed. Collectively, enhanced T-cells
infiltration into tumor microenvironment is one major reason
resulting in the superior anti-tumor effect of the combo trap
NPs.
Changes of Tumor-Infiltrating Immune Cells and Cytokine Levels in
Tumor Microenvironment
[0312] To further elucidate why the combo trap NP strategy could
efficiently improve T cell infiltration and accumulation around
tumor cells, we then evaluated the changes of the related distinct
myeloid subsets and cytokines in the tumor microenvironment after
different trap NP treatments, which, partake in a complicated
interplay network to mask CD8+ T-cell anti-tumor activity.
[0313] Since immunosuppressive subsets, such as regulatory T cells
(Tregs), myeloid-derived suppressor cells (MDSCs), and tumor
associated macrophages (TAMs) are the dominate myeloid infiltrates
within the desmoplastic PDAC models, we examine the accumulation of
these immune suppressive cells within the tumor microenvironments
by both flow cytometry and immunostaining of tumor sections. MDSC
were checked as the first regulatory subset. As shown in FIGS. 19A
and 19B, the percentages of MDSC in the trap only group (either
CXCL12 trap and PDL1 trap) and combination group were much lower
than the control groups. Since MDSC can establish immune tolerance
by induction of Treg development, the blockage of MDSC may lead to
inhibition of Treg. We therefore detected the percentage of Treg in
tumor tissues, as shown in FIGS. 19A and 19B. Consistent with the
trends of MDSC, the CXCL12 trap NP treated group and combination
group exhibited fewer Treg cells than the control groups. However,
PD-L1 trap NPs slightly increased T cell infiltration, which was
also observed by Feig. et al using PD-L1 check point inhibitor
(Feig et al., Targeting CXCL12 from FAP-expressing
carcinoma-associated fibroblasts synergizes with anti-PD-L1
immunotherapy in pancreatic cancer, PNAS, 2013 Dec. 10;
110(50):20212-7). This is most likely due to the fact that
PD-L1/PD1 interaction negatively regulates Treg proliferation and
activation by controlling STAT-5 phosphorylation. Macrophage, is
another important component of the suppressive tumor immune
microenvironment. As shown in FIG. 19A, both PD-L1 monotherapy and
combo therapy can significantly decreased the accumulated
macrophages, and efficiently turned the macrophages favorable to M1
state (FIG. 19B). Thus, there was a significant remodeling of the
suppressive TME by the traps in favor of therapy.
[0314] To correlate the observation of immune suppressive subsets
with the level of CXCL12 and PD-L1, we next test the neutralizing
efficiency of the intravenously delivered trap NPs (FIGS. 20A and
20B). It was shown that CXCL12 trap NPs, but not PD-L1 trap NPs can
efficiently neutralize the intratumoral secreted CXCL12, leading to
a substantial decrease of the protein detected by an anti-CXCL12
primary antibody, and subsequently inhibit MDSC and Treg
infiltration through CXCL12/CXCR4 mediated interaction. Whereas,
the overall PD-L1 level was not only diminished by applying PD-L1
trap NPs but also partially affected by CXCL12 trap treatment. This
is likely due to the fact that myeloid cells can induce the
expression of PD-L1 in tumor cells in an epidermal growth factor
receptor (EGFR)/mitogen-activated protein kinases (MAPK)-dependent
manner. Therefore, reduced recruitment of myeloid cells by CXCL12
decreased the level of PD-L1. Efficient depletion of MDSC and Treg
cells subsequently facilitate the infiltration of effector T cells
within the tumor microenvironment, explaining the superior
antitumor efficacy.
[0315] We then monitored the cytokine levels in the local tumor
tissue in order to see whether or not the combo group could reverse
the suppressive microenvironment as shown by cytokine levels (FIG.
21). IL-4 and IL-10 are known as Th-2 cytokines which are critical
for immunosuppression to promote cancer metastasis. Meanwhile,
IFN-.gamma., IL-12a and TNF-.alpha. (considered as Th-1 cytokines)
are the cytokines secreted by cytotoxic T cells that facilitate T
cell killing and fight against tumor progression. In the CXCL12
trap NP monotherapy group, though IL-12a and IFN-.gamma. increased
and IL-4 decreased substantially, IL-10 still increased suggesting
a slightly suppressive microenvironment. Similarly, in the PD-L1
trap NP group, despite the increased level of the overall Th1
cytokines, suppressive cytokines remain consistently high. However,
in the combination group, both IL-4 and IL-10 were significantly
decreased. Meanwhile, IL-12.alpha., TNF-.alpha. and IFN-.gamma.
were dramatically increased, indicating a M2 to M1 phonotype switch
to an immune-stimulating microenvironment. This would consequently
activate the recruitment of lymphocytes to act as scavengers,
facilitate tumor antigen presentation and result in an intensified
cytotoxic T cell mediated, tumor-specific killing.
Changes of the Tumor Vessel and Tumor Associated Fibroblast
[0316] Tumor associated fibroblasts (TAFs) and angiogenesis impede
the infiltration of cytotoxic T lymphocytes to the tumor tissue.
The effect of trap NPs on TAFs was investigated by staining for
.alpha.-smooth muscle actin (.alpha.SMA), a marker of TAFs, and
CD31, a marker for the vasculature. The density and mean
florescence were detected by fluorescence microscopy. Five
microscopic fields were randomly selected for analysis. As shown in
FIG. 22A, the density of CD31 in both mono- and combo-groups were
lower than that of the control group. The combo group, in
particular, demonstrates a substantial blood vessel normalization
(FIG. 22B). The blood vessel was decompressed significantly, and
subsequently increased NP perfusion and extended distribution after
multiple combo trap treatments (FIG. 23). The normalized blood
vessel is a result of released IFP, which mostly due to decreased
stroma and cell density.
[0317] Therefore, we next evaluate the density of fibroblasts. It
was shown that the combo trap NP group exhibited the lowest density
of .alpha.SMA. Interestingly, we found that only CXCL12 trap, but
not PD-L1 trap results in the decreasing of .alpha.SMA in both the
monotherapy and combo therapy (FIGS. 22A and 22B). Consistently, we
noted that collagen, one of the major extracellular matrix secreted
by fibroblasts were decreased dramatically in both CXCL12 trap NP
treated group and combo trap NPs (FIG. 24). Therefore, we conclude
that CXCL12 trap NPs not only increased T-cell infiltration,
uncovered the antitumor efficacy of PD-L1 trap by tuning the
suppressive immune microenvironment, but also by depleting
fibroblasts and collagen content. Since fibroblasts are considered
as major source of CXCL12 in KPC tumor microenvironment, a
CXCR4-mediated autocrine loop may explain the decreasing of
fibroblasts and remodeling of the stroma.
Toxicity Evaluation for the Different Treatments and Blood
Chemistry Analysis
[0318] The results of the toxicological pathology evaluation
demonstrated that there were not any noticeable morphological
changes in the heart, liver, spleen, lungs and kidneys for
monotherapy and combo trap NPs (FIG. 25). However, cellular
vacuolization, desquamated-degenerative cells and focal necrosis
were detected in liver and renal tissues of mice in PBS and Ctrl NP
groups, suggesting severe liver and kidney damages, which were most
likely due to the burden of tumors. Consistently, the serum
biochemical value analysis demonstrated that liver (AST and ALT) or
kidney (creatinine and BUN) toxicity caused by tumor progression in
these two groups but not the combo trap NPs treated mice (Table 6).
In addition, the whole blood cell counts (Table 5) remain constant
within normal ranges for all the groups, suggesting no systemic
anemia or inflammation occurred after treatments.
TABLE-US-00005 TABLE 5 Whole cell counts of mice treated with
different groups Sample# WBC LYMF GRAN MONO HCT RBC HGB PLT Health
5.8 .+-. 0.1 3.8 .+-. 0.6 1.1 .+-. 0.3 0.8 .+-. 0.3 46.2 .+-. 3.1
9.8 .+-. 1.0 14.9 .+-. 1.3 1036.0 .+-. 92.7 PBS 5.5 .+-. 0.7 3.7
.+-. 0.4 1.3 .+-. 0.2 0.6 .+-. 0.1 42.7 .+-. 0.8 9.5 .+-. 0.2 14.0
.+-. 0.4 1159.5 .+-. 34.7 Protein trap 6.3 .+-. 0.2 4.0 .+-. 0.6
1.6 .+-. 0.4 0.7 .+-. 0.1 46.8 .+-. 0.3 10.2 .+-. 0.1 14.9 .+-. 0.3
1220.5 .+-. 46.7 Combo trap NP 5.8 .+-. 1.2 3.1 .+-. 0.7 2.1 .+-.
0.3 0.6 .+-. 0.2 41.7 .+-. 0.7 9.2 .+-. 0.2 13.6 .+-. 0.3 1182.3
.+-. 25.8 Ctrl NP 5.8 .+-. 1.2 1.4 .+-. 0.2 1.8 .+-. 0.9 0.6 .+-.
0.3 38.1 .+-. 1.9 8.6 .+-. 0.3 12.5 .+-. 0.6 951.0 .+-. 13.1 *
Numbers in bold indicate that the value is over the normal
range
TABLE-US-00006 TABLE 6 Serum biochemical value analysis Creatinine
Sample# BUN mg/dL mg/dL AST U/L ALT U/L Health 22.0 .+-. 2.5 0.2
.+-. 0.0 186.7 .+-. 30.2 24.7 .+-. 10.6 PBS 24.0 .+-. 4.8 0.4 .+-.
0.1 360.0 .+-. 58.8 68.0 .+-. 3.6 Protein trap 33.0 + 2.5 0.2 .+-.
0.0 134.0 .+-. 8.2 21.0 .+-. 8.9 Combo trap NP 26.0 .+-. 2.5 0.2
.+-. 0.0 173.3 .+-. 23.1 28.0 .+-. 8.6 Ctrl NP 30.0 .+-. 0.2 0.4
.+-. 0.0 406.0 .+-. 124.1 74.0 .+-. 4.8 * Numbers in bold indicate
that the value is over the normal range
[0319] All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0320] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
661175PRTArtificial sequenceCXCL12-Trap-hSDA-1 1Met Lys Trp Val Thr
Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Gly Ser
Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val 20 25 30Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Lys 35 40 45Val Ser
Ala Lys Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60Leu
Glu Trp Val Ser Ser Ile Asn Asn Arg Asp Gly Ser Thr Tyr Tyr65 70 75
80Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
85 90 95Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala 100 105 110Val Tyr Tyr Cys Ala Gly Arg Arg Arg Arg Thr Ala Asn
Phe Arg Tyr 115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly Gly Ser Ala Ala Ala
Gly Ala Pro Val Pro Tyr Pro145 150 155 160Asp Pro Leu Glu Pro Arg
Gly Gly Ser His His His His His His 165 170 1752119PRTArtificial
sequenceVH domain of CXCL12-Trap-hSDA-1 2Gln Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Val Lys Val Ser Ala Lys 20 25 30Asn Met Ala Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile
Asn Asn Arg Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Gly Arg Arg Arg Arg Thr Ala Asn Phe Arg Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 115310PRTArtificial
sequenceCDR-H1 of CXCL12-Trap-hSDA-1 3Gly Val Lys Val Ser Ala Lys
Asn Met Ala1 5 10417PRTArtificial sequenceCDR-H2 of
CXCL12-Trap-hSDA-1 4Ser Ile Asn Asn Arg Asp Gly Ser Thr Tyr Tyr Ala
Asp Ser Val Lys1 5 10 15Gly510PRTArtificial sequenceCDR-H3 of
CXCL12-Trap-hSDA-1 5Arg Arg Arg Arg Thr Ala Asn Phe Arg Tyr1 5
106176PRTArtificial sequenceCXCL12-Trap-hSDA-2 6Met Lys Trp Val Thr
Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Gly Ser
Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val 20 25 30Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys 35 40 45Ile Asn
Asn Lys Val Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60Leu
Glu Trp Val Ser Thr Ile Gln Lys Arg Gly Gly Ser Thr Tyr Tyr65 70 75
80Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
85 90 95Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala 100 105 110Val Tyr Tyr Cys Ala Arg Glu Ser Ala Arg Thr Ala Asp
Lys Leu Gly 115 120 125Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Gly Ser Ala Ala
Ala Gly Ala Pro Val Pro Tyr145 150 155 160Pro Asp Pro Leu Glu Pro
Arg Gly Gly Ser His His His His His His 165 170
1757120PRTArtificial sequenceVH domain of CXCL12-Trap-hSDA-2 7Gln
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Lys Ile Asn Asn Lys
20 25 30Val Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Thr Ile Gln Lys Arg Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ser Ala Arg Thr Ala Asp Lys
Leu Gly Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
115 120810PRTArtificial sequenceCDR1-H1 of CXCL12-Trap-hSDA-2 8Gly
Phe Lys Ile Asn Asn Lys Val Met Ala1 5 10917PRTArtificial
sequenceCDR1-H2 of CXCL12-Trap-hSDA-2 9Thr Ile Gln Lys Arg Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly1011PRTArtificial
sequenceCDR3-H3 of CXCL12-Trap-hSDA-2 10Glu Ser Ala Arg Thr Ala Asp
Lys Leu Gly Tyr1 5 1011177PRTArtificial sequenceCXCL12-Trap-hSDA-3
11Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1
5 10 15Tyr Ser Gly Ser Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val 20 25 30Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Asp Ser 35 40 45Phe Thr Thr Lys Asn Met Ala Trp Val Arg Gln Ala Pro
Gly Lys Gly 50 55 60Leu Glu Trp Val Ser Ala Ile Ser Lys Arg Ser Gly
Ser Thr Tyr Tyr65 70 75 80Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys 85 90 95Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala 100 105 110Val Tyr Tyr Cys Ala Gly Leu
Thr Gln Arg His Gly His Ala Lys Leu 115 120 125Lys Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 130 135 140Gly Ser Gly
Gly Gly Gly Gly Ser Ala Ala Ala Gly Ala Pro Val Pro145 150 155
160Tyr Pro Asp Pro Leu Glu Pro Arg Gly Gly Ser His His His His His
165 170 175His12121PRTArtificial sequenceVH domain of
CXCL12-Trap-hSDA-3 12Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Asp Ser Phe Thr Thr Lys 20 25 30Asn Met Ala Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Lys Arg Ser Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Gly Leu Thr
Gln Arg His Gly His Ala Lys Leu Lys Tyr Trp Gly 100 105 110Gln Gly
Thr Leu Val Thr Val Ser Ser 115 1201310PRTArtificial sequenceCDR-H1
of CXCL12-Trap-hSDA-3 13Gly Asp Ser Phe Thr Thr Lys Asn Met Ala1 5
101417PRTArtificial sequenceCDR-H2 of CXCL12-Trap-hSDA-3 14Ala Ile
Ser Lys Arg Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly1512PRTArtificial sequenceCDR-H3 of CXCL12-Trap-hSDA-3 15Leu
Thr Gln Arg His Gly His Ala Lys Leu Lys Tyr1 5 1016288PRTArtificial
sequenceCXCL12-Trap-VH/VL-4 16Met Lys Trp Val Thr Phe Ile Ser Leu
Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Gly Ser Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val 20 25 30Gln Pro Gly Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Ser 35 40 45Leu Thr Val Tyr Ser Val
His Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60Leu Glu Trp Val Gly
Ala Leu Trp Gly Ser Gly Gly Thr Glu Tyr Asn65 70 75 80Ser Asn Leu
Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn 85 90 95Thr Val
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105
110Tyr Tyr Cys Ala Arg Asp Gln Gly Leu Asn Tyr Gly Ser Leu Phe Asp
115 120 125Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
Ile Gln Met Thr145 150 155 160Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly Asp Arg Val Thr Ile 165 170 175Thr Cys Arg Ala Ser Glu Ser
Ile Ser Tyr Ser Leu Ser Trp Tyr Gln 180 185 190Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile Tyr Asn Ala Val Lys 195 200 205Leu Glu Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 210 215 220Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr225 230
235 240Tyr Tyr Cys Lys Gln Tyr Trp Asn Thr Pro Phe Thr Phe Gly Gln
Gly 245 250 255Thr Lys Val Glu Ile Lys Arg Ala Ala Ala Gly Ala Pro
Val Pro Tyr 260 265 270Pro Asp Pro Leu Glu Pro Arg Gly Gly Ser His
His His His His His 275 280 28517120PRTArtificial sequenceVH domain
of CXCL12-Trap-VH/VL-4 17Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Ser Leu Thr Val Tyr 20 25 30Ser Val His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Leu Trp Gly Ser Gly
Gly Thr Glu Tyr Asn Ser Asn Leu Lys 50 55 60Ser Arg Phe Thr Ile Ser
Arg Asp Thr Ser Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Asp Gln
Gly Leu Asn Tyr Gly Ser Leu Phe Asp Tyr Trp Gly Gln 100 105 110Gly
Thr Leu Val Thr Val Ser Ser 115 12018108PRTArtificial sequenceVL
domain of CXCL12-Trap-VH/VL-4 18Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Glu Ser Ile Ser Tyr Ser 20 25 30Leu Ser Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asn Ala Val Lys Leu
Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Lys Gln Tyr Trp Asn Thr Pro Phe 85 90 95Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 1051910PRTArtificial
sequenceCDR-H1 of CXCL12-Trap-VH/VL-4 19Gly Phe Ser Leu Thr Val Tyr
Ser Val His1 5 102016PRTArtificial sequenceCDR-H2 of
CXCL12-Trap-VH/VL-4 20Ala Leu Trp Gly Ser Gly Gly Thr Glu Tyr Asn
Ser Asn Leu Lys Ser1 5 10 152112PRTArtificial sequenceCDR-H3 of
CXCL12-Trap-VH/VL-4 21Asp Gln Gly Leu Asn Tyr Gly Ser Leu Phe Asp
Tyr1 5 102211PRTArtificial sequenceCDR-L1 of CXCL12-Trap-VH/VL-4
22Arg Ala Ser Glu Ser Ile Ser Tyr Ser Leu Ser1 5 10237PRTArtificial
sequenceCDR-L2 of CXCL12-Trap-VH/VL-4 23Asn Ala Val Lys Leu Glu
Ser1 5249PRTArtificial sequenceCDR-L3 of CXCL12-Trap-VH/VL-4 24Lys
Gln Tyr Trp Asn Thr Pro Phe Thr1 525690DNAArtificial sequencePD-L1
Trap 25atgaaatggg tcacctttat cagcctgctg ttcctgttca gcagcgccta
cagcggatcc 60ggtccgccta cctttagtcc ggcactgctg gttgttaccg aaggtgataa
tgcaaccttt 120acatgcagct ttagcaatac cagcgaaagc tttgttctga
attggtatcg tatgagcccg 180agcaatcaga ccgataaact ggcagcattt
ccggaagatc gtagccagcc tggtcaggat 240agccgttttc gtgttaccca
gctgccgaat ggtcgtgatt ttcatatgag cgttgttcgt 300gcacgtcgta
atgatagcgg cacctatctg tgtggtgcaa ttagcctggc accgaaagca
360cagattaaag aaagcctgcg tgcagaactg cgtgtgaccg aacgtcgtgc
agaaggcccg 420caaccgcaac cgaaaccgca gccgaaaccg gaaccggaac
cgcaaccgca aggcggttct 480gaggaagacc cctgtgcctg tgagtccata
ctgaaatttg aggccaaggt ggagggtctg 540ctgcaggccc tgaccaggaa
gctggaagct gtgagcgggc ggctggctgt cctggagaac 600agaatcatcg
cggccgctgg cgcccctgtg ccttatcctg atcccctgga acctagaggc
660ggcagccacc accaccatca ccactgatga 69026228PRTArtificial
sequencePD-L1 Trap 26Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe
Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Gly Ser Gly Pro Pro Thr Phe Ser
Pro Ala Leu Leu Val Val 20 25 30Thr Glu Gly Asp Asn Ala Thr Phe Thr
Cys Ser Phe Ser Asn Thr Ser 35 40 45Glu Ser Phe Val Leu Asn Trp Tyr
Arg Met Ser Pro Ser Asn Gln Thr 50 55 60Asp Lys Leu Ala Ala Phe Pro
Glu Asp Arg Ser Gln Pro Gly Gln Asp65 70 75 80Ser Arg Phe Arg Val
Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met 85 90 95Ser Val Val Arg
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly 100 105 110Ala Ile
Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala 115 120
125Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Gly Pro Gln Pro Gln Pro
130 135 140Lys Pro Gln Pro Lys Pro Glu Pro Glu Pro Gln Pro Gln Gly
Gly Ser145 150 155 160Glu Glu Asp Pro Cys Ala Cys Glu Ser Ile Leu
Lys Phe Glu Ala Lys 165 170 175Val Glu Gly Leu Leu Gln Ala Leu Thr
Arg Lys Leu Glu Ala Val Ser 180 185 190Gly Arg Leu Ala Val Leu Glu
Asn Arg Ile Ile Ala Ala Ala Gly Ala 195 200 205Pro Val Pro Tyr Pro
Asp Pro Leu Glu Pro Arg Gly Gly Ser His His 210 215 220His His His
His22527117PRTHomo sapiens 27Pro Pro Thr Phe Ser Pro Ala Leu Leu
Val Val Thr Glu Gly Asp Asn1 5 10 15Ala Thr Phe Thr Cys Ser Phe Ser
Asn Thr Ser Glu Ser Phe Val Leu 20 25 30Asn Trp Tyr Arg Met Ser Pro
Ser Asn Gln Thr Asp Lys Leu Ala Ala 35 40 45Phe Pro Glu Asp Arg Ser
Gln Pro Gly Gln Asp Ser Arg Phe Arg Val 50 55 60Thr Gln Leu Pro Asn
Gly Arg Asp Phe His Met Ser Val Val Arg Ala65 70 75 80Arg Arg Asn
Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala 85 90 95Pro Lys
Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr 100 105
110Glu Arg Arg Ala Glu 11528130PRTHomo sapiens 28Pro Gly Trp Phe
Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr1 5 10 15Phe Ser Pro
Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe 20 25 30Thr Cys
Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr 35 40 45Arg
Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu 50 55
60Asp Arg Ser Gln Pro Gly Gln Asp Ser Arg Phe Arg Val Thr Gln Leu65
70 75 80Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg
Asn 85 90 95Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro
Lys Ala 100 105 110Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
Thr Glu Arg Arg 115 120 125Ala Glu 13029143PRTHomo sapiens 29Leu
Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala1 5 10
15Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe
20 25 30Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser
Pro 35 40 45Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg
Ser Gln 50 55 60Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro
Asn Gly Arg65 70 75 80Asp Phe His Met Ser Val Val Arg Ala Arg Arg
Asn Asp Ser Gly Thr 85 90 95Tyr Leu
Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu 100 105
110Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro
115 120 125Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe
Gln 130 135 14030117PRTMus musculus 30Ser Leu Thr Phe Tyr Pro Ala
Trp Leu Thr Val Ser Glu Gly Ala Asn1 5 10 15Ala Thr Phe Thr Cys Ser
Leu Ser Asn Trp Ser Glu Asp Leu Met Leu 20 25 30Asn Trp Asn Arg Leu
Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala Ala 35 40 45Phe Cys Asn Gly
Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln Ile 50 55 60Ile Gln Leu
Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp Thr65 70 75 80Arg
Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu His 85 90
95Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val Thr
100 105 110Glu Arg Ile Leu Glu 11531147PRTMus musculus 31Ser Gly
Trp Leu Leu Glu Val Pro Asn Gly Pro Trp Arg Ser Leu Thr1 5 10 15Phe
Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala Asn Ala Thr Phe 20 25
30Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met Leu Asn Trp Asn
35 40 45Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala Ala Phe Cys
Asn 50 55 60Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln Ile Ile
Gln Leu65 70 75 80Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp
Thr Arg Arg Asn 85 90 95Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser
Leu His Pro Lys Ala 100 105 110Lys Ile Glu Glu Ser Pro Gly Ala Glu
Leu Val Val Thr Glu Arg Ile 115 120 125Leu Glu Thr Ser Thr Arg Tyr
Pro Ser Pro Ser Pro Lys Pro Glu Gly 130 135 140Arg Phe
Gln14532143PRTMus musculus 32Leu Glu Val Pro Asn Gly Pro Trp Arg
Ser Leu Thr Phe Tyr Pro Ala1 5 10 15Trp Leu Thr Val Ser Glu Gly Ala
Asn Ala Thr Phe Thr Cys Ser Leu 20 25 30Ser Asn Trp Ser Glu Asp Leu
Met Leu Asn Trp Asn Arg Leu Ser Pro 35 40 45Ser Asn Gln Thr Glu Lys
Gln Ala Ala Phe Cys Asn Gly Leu Ser Gln 50 55 60Pro Val Gln Asp Ala
Arg Phe Gln Ile Ile Gln Leu Pro Asn Arg His65 70 75 80Asp Phe His
Met Asn Ile Leu Asp Thr Arg Arg Asn Asp Ser Gly Ile 85 90 95Tyr Leu
Cys Gly Ala Ile Ser Leu His Pro Lys Ala Lys Ile Glu Glu 100 105
110Ser Pro Gly Ala Glu Leu Val Val Thr Glu Arg Ile Leu Glu Thr Ser
115 120 125Thr Arg Tyr Pro Ser Pro Ser Pro Lys Pro Glu Gly Arg Phe
Gln 130 135 14033117PRTRattus norvegicus 33Pro Leu Thr Phe Ser Pro
Thr Trp Leu Thr Val Ser Glu Gly Ala Asn1 5 10 15Ala Thr Phe Thr Cys
Ser Phe Ser Asn Trp Ser Glu Asp Leu Lys Leu 20 25 30Asn Trp Tyr Arg
Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala Ala 35 40 45Phe Cys Asn
Gly Tyr Ser Gln Pro Val Arg Asp Ala Arg Phe Gln Ile 50 55 60Val Gln
Leu Pro Asn Gly His Asp Phe His Met Asn Ile Leu Asp Ala65 70 75
80Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu Pro
85 90 95Pro Lys Ala Gln Ile Lys Glu Ser Pro Gly Ala Glu Leu Val Val
Thr 100 105 110Glu Arg Ile Leu Glu 11534143PRTRattus norvegicus
34Leu Glu Val Leu Asn Lys Pro Trp Arg Pro Leu Thr Phe Ser Pro Thr1
5 10 15Trp Leu Thr Val Ser Glu Gly Ala Asn Ala Thr Phe Thr Cys Ser
Phe 20 25 30Ser Asn Trp Ser Glu Asp Leu Lys Leu Asn Trp Tyr Arg Leu
Ser Pro 35 40 45Ser Asn Gln Thr Glu Lys Gln Ala Ala Phe Cys Asn Gly
Tyr Ser Gln 50 55 60Pro Val Arg Asp Ala Arg Phe Gln Ile Val Gln Leu
Pro Asn Gly His65 70 75 80Asp Phe His Met Asn Ile Leu Asp Ala Arg
Arg Asn Asp Ser Gly Ile 85 90 95Tyr Leu Cys Gly Ala Ile Ser Leu Pro
Pro Lys Ala Gln Ile Lys Glu 100 105 110Ser Pro Gly Ala Glu Leu Val
Val Thr Glu Arg Ile Leu Glu Thr Pro 115 120 125Thr Arg Tyr Pro Arg
Pro Ser Pro Lys Pro Glu Gly Gln Phe Gln 130 135 14035220PRTHomo
sapiens 35Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr
Gly Ser1 5 10 15Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln
Leu Asp Leu 20 25 30Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys
Asn Ile Ile Gln 35 40 45Phe Val His Gly Glu Glu Asp Leu Lys Val Gln
His Ser Ser Tyr Arg 50 55 60Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu
Ser Leu Gly Asn Ala Ala65 70 75 80Leu Gln Ile Thr Asp Val Lys Leu
Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95Met Ile Ser Tyr Gly Gly Ala
Asp Tyr Lys Arg Ile Thr Val Lys Val 100 105 110Asn Ala Pro Tyr Asn
Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro 115 120 125Val Thr Ser
Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 130 135 140Ala
Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys145 150
155 160Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val
Thr 165 170 175Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe
Tyr Cys Thr 180 185 190Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr
Ala Glu Leu Val Ile 195 200 205Pro Glu Leu Pro Leu Ala His Pro Pro
Asn Glu Arg 210 215 22036200PRTHomo sapiens 36Leu Phe Thr Val Thr
Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly1 5 10 15Ser Asn Val Thr
Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn 20 25 30Leu Gly Ala
Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser 35 40 45Pro His
Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly 50 55 60Lys
Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln65 70 75
80Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu
85 90 95Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile
Leu 100 105 110Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln
Ala Thr Gly 115 120 125Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val
Ser Val Pro Ala Asn 130 135 140Thr Ser His Ser Arg Thr Pro Glu Gly
Leu Tyr Gln Val Thr Ser Val145 150 155 160Leu Arg Leu Lys Pro Pro
Pro Gly Arg Asn Phe Ser Cys Val Phe Trp 165 170 175Asn Thr His Val
Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser 180 185 190Gln Met
Glu Pro Arg Thr His Pro 195 20037219PRTMus musculus 37Phe Thr Ile
Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser1 5 10 15Asn Val
Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu Asp Leu 20 25 30Leu
Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val Ile Gln 35 40
45Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn Phe Arg
50 55 60Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn Ala
Ala65 70 75 80Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val
Tyr Cys Cys 85 90 95Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile
Thr Leu Lys Val 100 105 110Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg
Ile Ser Val Asp Pro Ala 115 120 125Thr Ser Glu His Glu Leu Ile Cys
Gln Ala Glu Gly Tyr Pro Glu Ala 130 135 140Glu Val Ile Trp Thr Asn
Ser Asp His Gln Pro Val Ser Gly Lys Arg145 150 155 160Ser Val Thr
Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val Thr Ser 165 170 175Ser
Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys Thr Phe 180 185
190Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile Ile Pro
195 200 205Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg 210
21538200PRTMus musculus 38Leu Phe Thr Val Thr Ala Pro Lys Glu Val
Tyr Thr Val Asp Val Gly1 5 10 15Ser Ser Val Ser Leu Glu Cys Asp Phe
Asp Arg Arg Glu Cys Thr Glu 20 25 30Leu Glu Gly Ile Arg Ala Ser Leu
Gln Lys Val Glu Asn Asp Thr Ser 35 40 45Leu Gln Ser Glu Arg Ala Thr
Leu Leu Glu Glu Gln Leu Pro Leu Gly 50 55 60Lys Ala Leu Phe His Ile
Pro Ser Val Gln Val Arg Asp Ser Gly Gln65 70 75 80Tyr Arg Cys Leu
Val Ile Cys Gly Ala Ala Trp Asp Tyr Lys Tyr Leu 85 90 95Thr Val Lys
Val Lys Ala Ser Tyr Met Arg Ile Asp Thr Arg Ile Leu 100 105 110Glu
Val Pro Gly Thr Gly Glu Val Gln Leu Thr Cys Gln Ala Arg Gly 115 120
125Tyr Pro Leu Ala Glu Val Ser Trp Gln Asn Val Ser Val Pro Ala Asn
130 135 140Thr Ser His Ile Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr
Ser Val145 150 155 160Leu Arg Leu Lys Pro Gln Pro Ser Arg Asn Phe
Ser Cys Met Phe Trp 165 170 175Asn Ala His Met Lys Glu Leu Thr Ser
Ala Ile Ile Asp Pro Leu Ser 180 185 190Arg Met Glu Pro Lys Val Pro
Arg 195 20039219PRTRattus norvegicus 39Phe Thr Ile Thr Ala Pro Lys
Asp Leu Tyr Val Val Glu Tyr Gly Ser1 5 10 15Asn Val Thr Met Glu Cys
Arg Phe Pro Val Glu Gln Lys Leu Asp Leu 20 25 30Leu Ala Leu Val Val
Tyr Trp Glu Lys Glu Asp Lys Glu Val Ile Gln 35 40 45Phe Val Glu Gly
Glu Glu Asp Leu Lys Pro Gln His Ser Ser Phe Arg 50 55 60Gly Arg Ala
Phe Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn Ala Val65 70 75 80Leu
Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Cys Cys 85 90
95Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu Lys Val
100 105 110Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Met Asp
Pro Ala 115 120 125Thr Ser Glu His Glu Leu Met Cys Gln Ala Glu Gly
Tyr Pro Glu Ala 130 135 140Glu Val Ile Trp Thr Asn Ser Asp His Gln
Ser Leu Ser Gly Glu Thr145 150 155 160Thr Val Thr Thr Ser Gln Thr
Glu Glu Lys Leu Leu Asn Val Thr Ser 165 170 175Val Leu Arg Val Asn
Ala Thr Ala Asn Asp Val Phe His Cys Thr Phe 180 185 190Trp Arg Val
His Ser Gly Glu Asn His Thr Ala Glu Leu Ile Ile Pro 195 200 205Glu
Leu Pro Val Pro Arg Leu Pro His Asn Arg 210 21540200PRTRattus
norvegicus 40Leu Phe Thr Val Thr Ala Pro Lys Glu Val Tyr Thr Val
Asp Phe Gly1 5 10 15Ser Ser Val Ser Leu Glu Cys Asp Phe Asp Arg Arg
Glu Cys Thr Glu 20 25 30Leu Glu Gly Ile Arg Ala Ser Leu Gln Lys Val
Glu Asn Asp Thr Ser 35 40 45Ser Gln Ser Gln Arg Ala Thr Leu Leu Glu
Glu Leu Leu Pro Leu Gly 50 55 60Lys Ala Ser Phe His Ile Pro Ser Val
Gln Val Arg Asp Ser Gly Gln65 70 75 80Tyr Arg Cys Leu Val Ile Cys
Gly Ala Ala Trp Asp Tyr Lys Tyr Leu 85 90 95Thr Val Lys Val Lys Ala
Ser Tyr Val Arg Ile Asp Thr Gly Ile Leu 100 105 110Glu Val Pro Gly
Thr Gly Glu Val Gln Leu Ile Cys Gln Ala Arg Gly 115 120 125Tyr Pro
Leu Ala Glu Val Ser Trp Gln Asn Val Ser Val Pro Ala Asn 130 135
140Thr Ser His Ile Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser
Val145 150 155 160Leu Arg Leu Lys Pro Gln Pro Asn Arg Asn Phe Ser
Cys Met Phe Trp 165 170 175Asn Ala His Met Lys Glu Leu Thr Ser Ala
Ile Ile Asp Pro Leu Ser 180 185 190Trp Met Glu Pro Lys Val Pro Arg
195 2004143PRTHomo sapiens 41Glu Glu Asp Pro Cys Ala Cys Glu Ser
Leu Val Lys Phe Gln Ala Lys1 5 10 15Val Glu Gly Leu Leu Gln Ala Leu
Thr Arg Lys Leu Glu Ala Val Ser 20 25 30Lys Arg Leu Ala Ile Leu Glu
Asn Thr Val Val 35 404243PRTHomo sapiens 42Glu Glu Asp Pro Cys Ala
Cys Glu Ser Leu Val Thr Phe Gln Ala Lys1 5 10 15Val Glu Gly Leu Leu
Gln Ala Leu Thr Arg Lys Leu Glu Ala Val Ser 20 25 30Lys Arg Leu Ala
Ile Leu Glu Asn Thr Val Val 35 404343PRTHomo sapiens 43Glu Glu Asp
Pro Cys Ala Cys Glu Ser Leu Val Lys Phe Gln Ala Lys1 5 10 15Val Glu
Gly Leu Leu Gln Ala Leu Thr Ser Pro Leu Glu Ala Val Ser 20 25 30Lys
Arg Leu Ala Ile Leu Glu Asn Thr Val Val 35 404446PRTHomo sapiens
44Leu Arg Ser Pro Cys Glu Cys Glu Ser Leu Val Glu Phe Gln Gly Arg1
5 10 15Thr Leu Gly Ala Leu Glu Ser Leu Thr Leu Asn Leu Ala Gln Leu
Thr 20 25 30Ala Arg Leu Glu Asp Leu Glu Asn Gln Leu Ala Asn Gln Lys
35 40 454544PRTHomo sapiens 45His Asp Gln Cys Lys Cys Glu Asn Leu
Ile Met Phe Gln Asn Leu Ala1 5 10 15Asn Glu Glu Val Arg Lys Leu Thr
Gln Arg Leu Glu Glu Met Thr Gln 20 25 30Arg Met Glu Ala Leu Glu Asn
Arg Leu Arg Tyr Arg 35 404645PRTHomo sapiens 46Thr Glu Asp Ala Cys
Gly Cys Glu Ala Thr Leu Ala Phe Gln Asp Lys1 5 10 15Val Ser Ser Tyr
Leu Gln Arg Leu Asn Thr Lys Leu Asp Asp Ile Leu 20 25 30Glu Lys Leu
Lys Ile Asn Glu Tyr Gly Gln Ile His Arg 35 40 454743PRTMus musculus
47Glu Glu Asp Pro Cys Ala Cys Glu Ser Ile Leu Lys Phe Glu Ala Lys1
5 10 15Val Glu Gly Leu Leu Gln Ala Leu Thr Arg Lys Leu Glu Ala Val
Ser 20 25 30Gly Arg Leu Ala Val Leu Glu Asn Arg Ile Ile 35
404846PRTMus musculus 48Leu Arg Ser Pro Cys Glu Cys Glu Ser Leu Val
Glu Phe Gln Gly Arg1 5 10 15Thr Leu Gly Ala Leu Glu Ser Leu Thr Gln
Asn Leu Ala Arg Leu Thr 20 25 30Glu Arg Leu Glu Glu Leu Glu Asn Gln
Leu Ala Ser Arg Lys 35 40 454945PRTMus musculus 49Thr Gln Asp Gln
Cys Lys Cys Glu Asn Leu Ile Gln Phe Gln Asn Leu1 5 10 15Ala Asn Glu
Glu Val Arg Lys Leu Thr Gln Arg Leu Glu Glu Met Thr 20 25 30Gln Arg
Met Glu Ala Leu Glu Asn Arg Leu Arg Tyr Arg 35 40 455043PRTMus
musculus 50Asp Gln Cys Lys Cys Glu Asn Leu Ile Leu Phe Gln Asn Val
Ala Asn1 5 10 15Glu Glu Val Arg Lys Leu Thr Gln Arg Leu Glu Glu Met
Thr Gln Arg 20 25 30Met Glu Ala Leu Glu Asn Arg Leu Lys Tyr Arg 35
405144PRTMus musculus 51Glu Asp Ala Cys Gly Cys Gly Ala Thr Leu Ala
Phe Gln Glu Lys Val1
5 10 15Ser Ser His Leu Gln Lys Leu Asn Thr Lys Leu Asp Asn Ile Leu
Lys 20 25 30Lys Leu Lys Val Thr Glu Tyr Gly Gln Val His Arg 35
405243PRTCeratotherium simum 52Glu Glu Asp Pro Cys Ala Cys Glu Ser
Ile Val Lys Phe Gln Ala Lys1 5 10 15Val Glu Gly Leu Leu Gln Ala Leu
Thr Arg Lys Leu Glu Ala Val Ser 20 25 30Lys Arg Leu Ala Val Leu Glu
Asn Arg Ile Val 35 405343PRTNannospalax galili 53Glu Glu Asp Pro
Cys Ala Cys Glu Ser Ile Val Arg Phe Glu Ala Lys1 5 10 15Val Glu Gly
Leu Leu Gln Ala Leu Thr Arg Lys Leu Glu Ala Val Ser 20 25 30Lys Arg
Leu Ala Val Leu Glu Asn Arg Ile Val 35 405443PRTBison bison 54Glu
Glu Asp Pro Cys Ala Cys Glu Ser Ile Val Lys Phe Gln Thr Lys1 5 10
15Val Glu Gly Leu Leu Gln Ala Leu Thr Arg Lys Leu Glu Ala Val Ser
20 25 30Lys Arg Leu Ala Val Leu Glu Asn Arg Ile Val 35
405543PRTRattus norvegicus 55Glu Glu Asp Pro Cys Ala Cys Glu Ser
Ile Val Arg Phe Glu Ala Lys1 5 10 15Val Glu Gly Leu Leu Gln Asp Leu
Thr Arg Lys Leu Glu Ala Val Ser 20 25 30Lys Arg Leu Ala Val Leu Glu
Asn Arg Val Ile 35 405646PRTRattus norvegicus 56Glu Thr Gln Asp Gln
Cys Lys Cys Glu Asn Leu Ile Gln Phe Gln Asn1 5 10 15Leu Ala Asn Glu
Glu Val Arg Lys Leu Thr Gln Arg Leu Glu Glu Met 20 25 30Thr Gln Arg
Met Glu Ala Leu Glu Asn Arg Leu Arg Tyr Arg 35 40 455726PRTRattus
norvegicus 57Glu Asp Ala Cys Ser Cys Gly Ala Thr Leu Ala Phe Gln
Glu Lys Val1 5 10 15Ser Ser His Leu Gln Lys Leu Asn Thr Thr 20
255811PRTArtificial sequencelinker sequenceREPEAT(1)..(11)linker
sequence can be repeated n times, wherein n=1-20 58Gly Pro Gln Pro
Gln Pro Lys Pro Gln Pro Lys1 5 10595PRTArtificial sequencelinker
sequenceREPEAT(1)..(5)linker sequence can be repeated n times,
wherein n=1-20 59Gly Gly Gly Gly Ser1 56015PRTHomo sapiens 60Thr
Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser Pro1 5 10
156116PRTMus musculus 61Glu Phe Pro Lys Pro Ser Thr Pro Pro Gly Ser
Ser Gly Gly Ala Pro1 5 10 156216PRTCamelus dromedarius 62Pro Gln
Pro Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu1 5 10
15631543DNAArtificial SequenceCXCL12 trap 63gttgacattg attattgact
agttattaat agtaatcaat tacggggtca ttagttcata 60gcccatatat ggagttccgc
gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120ccaacgaccc
ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
180ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac
ttggcagtac 240atcaagtgta tcatatgcca agtacgcccc ctattgacgt
caatgacggt aaatggcccg 300cctggcatta tgcccagtac atgaccttat
gggactttcc tacttggcag tacatctacg 360tattagtcat cgctattacc
atggtgatgc ggttttggca gtacatcaat gggcgtggat 420agcggtttga
ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
480tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
ccattgacgc 540aaatgggcgg taggcgtgta cggtgggagg tctatataag
cagagctctc tggctaacta 600gagaacccac tgcttactgg cttatcgaaa
ttaatacgac tcactatagg gagacccaag 660ctggctagcc accatgaaat
gggtcacctt tatcagcctg ctgttcctgt tcagcagcgc 720ctacagcgga
tccgaggtgc agctggtgga atctggcgga ggactggtgc agcctggcgg
780ctctctgaga ctgtcttgtg ccgccagcgg cttcagcctg accgtgtact
ctgtgcactg 840ggtgcgccag gccccaggca aaggactgga atgggtggga
gccctgtggg gctctggcgg 900aaccgagtac aacagcaacc tgaagtcccg
gttcaccatc agccgggaca ccagcaagaa 960caccgtgtac ctgcagatga
acagcctgcg ggccgaggac accgccgtgt actattgcgc 1020cagagatcag
ggcctgaact acggcagcct gttcgactat tggggccagg gcacactcgt
1080gaccgtgtct agcggaggcg gaggaagtgg cggaggggga tctggcggcg
gaggcagcga 1140tattcagatg acccagtccc ccagcagcct gagcgcctct
gtgggcgaca gagtgaccat 1200cacctgtcgg gccagcgaga gcatcagcta
cagcctgtcc tggtatcagc agaagcccgg 1260caaggccccc aagctgctga
tctacaacgc cgtgaagctg gaaagcggcg tgcccagcag 1320attttccggc
agcggctctg gcaccgactt caccctgacc atcagctccc tgcagcccga
1380ggacttcgcc acctactact gcaagcagta ctggaacacc cccttcacct
tcggacaggg 1440caccaaggtg gaaatcaaga gagcggccgc tggcgcccct
gtgccttatc ctgatcccct 1500ggaacctaga ggcggcagcc accaccacca
tcaccactga tga 15436489PRTHomo sapiens 64Met Asn Ala Lys Val Val
Val Val Leu Val Leu Val Leu Thr Ala Leu1 5 10 15Cys Leu Ser Asp Gly
Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys 20 25 30Arg Phe Phe Glu
Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys 35 40 45Ile Leu Asn
Thr Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys 50 55 60Asn Asn
Asn Arg Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln65 70 75
80Glu Tyr Leu Glu Lys Ala Leu Asn Lys 856593PRTHomo sapiens 65Met
Asn Ala Lys Val Val Val Val Leu Val Leu Val Leu Thr Ala Leu1 5 10
15Cys Leu Ser Asp Gly Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys
20 25 30Arg Phe Phe Glu Ser His Val Ala Arg Ala Asn Val Lys His Leu
Lys 35 40 45Ile Leu Asn Thr Pro Asn Cys Ala Leu Gln Ile Val Ala Arg
Leu Lys 50 55 60Asn Asn Asn Arg Gln Val Cys Ile Asp Pro Lys Leu Lys
Trp Ile Gln65 70 75 80Glu Tyr Leu Glu Lys Ala Leu Asn Lys Arg Phe
Lys Met 85 906618818DNAHomo sapiens 66ctttcaggct tctgggacag
atcctaggtc cagctgccca cctgattttg tggcaaagaa 60aaaaagaaga agaagaagcg
agaggcgatg gcgtttgctt tggccagatt taagggcaag 120cgaggctgcg
cgcggctccc gcagggtcgg atctccgagc tccagggcgc ccctccaccc
180gggtgtagat ttcccgcgga ccccttcgcc ctcccgggtt tcatcagctg
cgcaggaatg 240gagctggcca gagctctggg agcggggagg gaggcgccgc
caccagaggg cgccggagcc 300ccagcgctgc ggccggtgag cggccaggct
ccccggccca gcccagcaaa ggcctgggga 360cgcccgacgg ctgccttctt
cactggacct cactgccttc agttctttaa gagcagggcc 420aagtcagtgg
ggttcccggc tccaagccca gtgcccaggg tgggtgggtg ggtgggtggg
480tgggtggatg gatggatgga tggatggatg gagtgccggc ccacagccat
ctaacggcca 540aagtggtttt ggaaaaaaaa tgcacagaag acacctactc
ccaccagcgg agttccggag 600ccctcgcagc ctcctgttga ccgctcccgc
ctaatgcagc cgctgaccgc ccactccccg 660acggccagga ctccccaggg
acagggacgt gtccccaggg caggcccctg gatggacgcg 720gcgactgacc
ccacttcgct ggacgctgtg ctgggaagga cacagagagg tggctggggc
780agcctgcggt cacaaagcga ggcccaaagg ggcgctctcc tcacccccac
gcctcctggg 840tgccgacctg caccctccct tcgccaccgg actggggcca
tctgggatgt ctcgggggta 900tccggagggc taagcaccgc cgagggacgg
ctccgtggga agagttttct ggacccagaa 960ggcagacgcc agtagtactg
tcctaggagt cggaggtcgg ggtgggggag ttctcagctc 1020tttgggtcgc
acggagcttt tcttgggtaa ggcagtaagt acttaggttt aaaggactta
1080cttacagcta ccatttattg agtactgtct gcttgtcaga cacgatgcag
agaatttcgc 1140ggcgtggggc gggtctcatt gaatctcccg tcccactccg
cggggtgggc ctgtgattag 1200ctcatttcac cattgagagg tcggaagtac
aaaggctaca ttcgctttta ctgagagccg 1260ccggcgcctt ctgctttgtt
tgtacaggcg aggaaactga ggctcggctg gtggcgccgt 1320gggcttggag
tccgagccac gctgactgca aagacgggtc tcattcccgc agatcgagct
1380ctgccggcgg ctgcgccgca agccgggcag gtggcgagct tgagccccca
cgcacagaaa 1440gcaggacccc ctcggctgcc ttgggccgcc accgccagca
ggccctccgc ccgggactaa 1500cttgtttgct tttcattggt tctcattcag
ttcccgccat cgaaaggccc cgtcccgcag 1560ctttccacgc gcgccccact
ttacgcctaa ggtcctcagt ctctccagtg gggccctgtc 1620acagggacaa
taagcggccc tccagccggc gtcgctcagg ctgcggacct cactgcagac
1680cgggccagcg gtgcggggcc cagcggagcc tgagaaggtc aaaggccgga
gcgcactgcg 1740cctcgggagc acagagggag cggaggaggg gcgaagggga
tgggtggggg gtgccgccga 1800gggagtcgcg cgtcagagac cccggccacg
gccagcactc ggctccgggc ccgcccctca 1860ccgcgcgccc cgccccgccc
cgcctggctc tcccctctaa agcgcccggc gcgcgcctcc 1920caccgccgca
ctttcactct ccgtcagccg cattgcccgc tcggcgtccg gcccccgacc
1980cgcgctcgtc cgcccgcccg cccgcccgcc cgcgccatga acgccaaggt
cgtggtcgtg 2040ctggtcctcg tgctgaccgc gctctgcctc agcgacggta
agtgcgctcg gcggggaggc 2100cctggcgagg cggccctggc gaggctctgg
gcttcggctc ccgcccggcg cagagccgcg 2160gctcctctgc ctgcgcccgc
agtttgcgcc gcccgacaca gtgggggtgg aggcagctcg 2220cttagctggg
cgctctggtg cggtgtcgcg ttcaaagccc tacttttgcg ccgagggttt
2280gtgacctgca gagagcgacc gcctgacctc aagctggctg cagagcgagg
tcagccggga 2340caactgggca ggaacgcccc aagagagcgt tcgggactgg
cgcgggaagg cgcgagggtg 2400gggagccgcg gaccccagac ccctgcctgc
ccaccctcca cccactgcct tcccgctgcg 2460ggctcggttt ccacaggcga
atgggctccg aggtcctgct gcgcacagtg ctggaggtcg 2520ggcggtgcat
ggcgagagcg cgctttgcaa agttcgcctc atgccgcgac ttgaggctgt
2580gaggtccgat gcagccgcgc agcctccgct ccctgcgaag ccgatcctct
ccccacaccc 2640tcgcggggct cttggccagg cgcgggcggg ctgcgcggcg
cagctgtcgg gcttgtgcca 2700cccgccaggc cgcgcttctg cacagctccg
cggtccgcag cgacgcggac ctgggcgcgc 2760gtccagccgc tgtcttcctg
tctcttctcg ggttcacctg agagaggccc agggaccctc 2820cgagcctcac
acgccccctc cccagctccc caaacacacc tgcagacagg ctcttttgtc
2880accagctggc caagcgcttt gccccagaat gagagctgca gcaaagttca
atctccaagg 2940ccctgggcca ctgggaaccg tggtgtcccg tttcactggg
agagggcttt tttcttttct 3000ttcgctgaag agaaactcgc tgcgctgtca
caggacaccg atgcaaacca aaataaactg 3060ttgcctctga acacacaaaa
caaaaccctg tggctcaggc cgctgagctt ccttggctgt 3120gaagtttcct
gcaaagagtt gaaggacact ctgagtgctc ttgcagccgc gggctgtgca
3180ggcccagcgc gcctcgagcc cttggctggg ttggtgcaga cgtggccccg
gcgtcctagc 3240tggcttggac tgtgcgctgg tgtgagcagg accagcgcag
ctgagtcggg ctggagaagc 3300tgggcagcct ggggtcgtgg cggactgcct
aagtgttaag gacacagact gaaggcaatt 3360gggaatgttc gtcgttgatg
aaaagtccct gaggctgtgt ctgcacactg accccccccc 3420ccccccgcaa
gtcatggggc atgtttctag tttttattgc tggatattct tctgttcctg
3480taaggtctct ggtttgggca gttattgact gattggtggg atgagcgcgg
aggtgtcaca 3540gtgcgccgct ctgggaaatg gtgctgcccc ctccccggag
ggctggagag aggattggaa 3600tgaggaacct cggtctttga gttatgggtg
caactgacct gggggaagca cagagacgtg 3660gggttcatcc cagactcctc
ctggcctggg aagggtgaca tggccctcag agttgcccca 3720ttggcagcca
agtaaccagg gcaggagcct gggctccagg cagatctgct ccttacacac
3780atcgccagaa tgttttgttt tgttttgttt tcccctcacc tcacagtgcc
tatgtgcccc 3840ctatccccac acttagtcct caagctgcac tacagagaga
gcttggccag gggtcctgcc 3900cctcccccat tggctgactt ccgtcaggca
gctgggccct tgggtttccc gggcttaaac 3960tcagggtggg cccccttccc
aagcctgctt ggaggggcca gctgggctcc tgtctctgca 4020ttagactgga
gggctttctg aaaactcctg gagccaagct cacccggtcc caaatccggg
4080gtaccatcag agccagtcag gactcttgcc ggtacacgga tttacatcca
gaacggttgg 4140gaaagttagt cttactgaca aaaagctgga ggaaggagca
catgaggtac acacgagacc 4200atgttctttt ggtaagcaca cctgggggct
ctgtcacttt aaaaagacct gttgaaattc 4260gctgcttgac aggcgggcct
taggtcctgg gagtagaagc aagaattgct tttgaaggaa 4320atggataatc
ccagggggtg gagggggaga ggagagagag cctgtgtgtt gagctctgaa
4380taggctctga tttctggcct catcaccact gcttggttgg ggggccagag
gagcttctct 4440cctctgggcg ctggagagtc acagccccac ccatcccagc
gggggcggct gcagcacttg 4500cccttggctg catatcttca agctcactgt
tgccgggagg gggcttgtgc ctcactaccg 4560gcctcctatt ctcctgctcc
acccccaacc caccccctgc ctcagcttgg aagggcccaa 4620aagaaatcca
gggaaacctg gaggcacaac actcaggagc gtttagctcc tcagttaccc
4680aggctgcagg ccccggccct gggggtggct ttcccaggtg atctggttac
agtgcagtgc 4740tgacttcccc tgcagggtcc cagtgcagct gctggaagct
ggccccagag gtgcctttct 4800ggagttagat caggtgtaac tgtgaaggct
gctttaggtc tctccagtgc catgagtcac 4860cagcaccctg atgcttggct
ttttcctggt tttcagggag ttgcatttgg tcctggggac 4920aaggaagtgg
ggggacaagt ggtccacttt tcctgcaact tggtggctga atacacaatg
4980caattcctag tccacatcat ccctcaattg attgcagtct gtagaaaaaa
taaaataaaa 5040tgagcccctt actcaagatt tcctcctact tccccagcat
tctttcccaa aggtgcagaa 5100tttcagaacc acagaggtct ctcagaactt
tgtaaacagg caaacttaaa aaaaaagaaa 5160aaagaaaaaa aatctctgga
ccaaatattc ttagacatat atttaatctc tgatgaaagg 5220atccacaagt
tcaaataatt tggggtatta agtggggctt ggataaaatc ttcaaggaaa
5280aaagaaaaac aaaaagcaca cacaaatccc agccctccag ggcttgcaat
cctatattta 5340aagggtgagc agtgggttct gcaggagccc cttgctaatt
tacactaatg agtgtcaatt 5400atggcatttt gtaaattggt gattttgcaa
agatcttaat acaattcctg aggctacagc 5460atccctgcct aggcaatgaa
aaccacattt aactccagct ccactaatct ctaagccttg 5520gggaaagtgc
tgggcagaag gtgggtcctt ggcctgctct caggggactc acaggtagga
5580agccagccag gattcttttg tgccttccag agctttaggt gaatgtgagg
aggggtctct 5640ggggtggggg agggcagcat tcctgagtac tactgtgctt
cctggctaga ggcctgtgcc 5700agaggagaga ggccagggag cctggagcct
cttgtccccc tttcttttcc ctgagtaggg 5760agatctttct cagttgtccc
ttgaggaatt ttttttacaa agagtaactg gaaatttcag 5820taccttggac
tgaaaccgtt atctactata attactacct ttatcttgag acactgagca
5880ggtggcttct aatttaatct cccccctcat ctaggctccg gctctcccca
ggcctgttgg 5940gctccttgcc ctgtagaagc tgagcctgag ctcagccagc
actcactgtg ttgcctgagg 6000agatggcctt tcccaacata caccagtaac
ctggaactta ccccagtgat gccgtctctt 6060gatccttaat cgctgcacca
actgctgcag cccagacaca cattaccaaa atctggcagg 6120gtagtaaagt
gccttgttgt ccttacctct tctagggaag cccgtcagcc tgagctacag
6180atgcccatgc cgattcttcg aaagccatgt tgccagagcc aacgtcaagc
atctcaaaat 6240tctcaacact ccaaactgtg cccttcagat tgtgtaagtc
ttgaaattga acatcatcta 6300acgaacatag ttgcatctaa cgaacatagt
aacatctaac ccgagtctta ttaaaggtgt 6360agaggtgaca agccaagtgt
ccaaccttga acttggcata attagtggca gctattctta 6420tcatgaggac
ctttagtatg ccaccaggca ttaattttaa aagccgctct tggcctgggc
6480accctgctgt gctcgtagta gttctctctt ctttccaaac ctgtgttctt
cagcacagca 6540cagctggatg ctggagaaac tatgggcttc accaaggttt
tggctgaagc ctagtaaatt 6600agccagccct tagctaataa cagtgataac
tggcctgcca gtttcgggga tctgatgccc 6660tacattaact cacaagaccc
aaatgcagtt taagtcctct ttaaccattg ggtgaaactt 6720caacattttc
agctcacttg cctgtatttt ctcaagtaca acatatgagg ttttggtgtt
6780atttggtgtg gatggcagcc actaacattt gattttattc atttgggaaa
caaagaccaa 6840aatcacacat ccacaagaca ggaagatctc agctgggtac
agaagaccgg gagaaatgcg 6900ggaagaaagc catgtgcttg aaagccctct
catgctcaag aggtcctgtg tctgaacacg 6960gggaagcgag ggctcaggga
gtctcctgga gaggatttga ttgctcagaa gttctagtgc 7020taaaaatggg
gtggggagac agcagttgga accaaagcta caactttccc aagacttttc
7080cacccaacac caaatgtttt tacttttgat tttagaaaac tgtttttagt
tttgagttaa 7140ccctttactg ccttgaatgt gtttgttatt tacagtatgt
tttagggccc tagaaaatga 7200atgttttttc agtactttgc ctacaacatg
atactttatc tcttgtgaag agtattttaa 7260agatgctcaa tttagctagc
ttggacctgc tttacagcac tgatcatgct ggtttactgg 7320cctggaaatg
caccttgaag ttgaagatgc caaatacaat tctcaggtct gagcagacac
7380taaattaaca aagaataggc cccagatttg gaccccaatg tgggttgctc
cgagccacac 7440ccatctctgc acccctcttc acctaatgga caggctgctt
gaagactccc tgggtgcccc 7500tgaaccactg ttctggggct tagctgtcct
caagaccctt aagtacttgg gctgcatgag 7560attacctggc accaaagggc
tttctggcaa gagatccaga gagatgaaat caggccttcc 7620ccaggagttt
gcatgtaggg aagcttttaa gagtgtgaac ttttaaaaaa caaatgagac
7680caagaaacat tcagtgaact tgtaaaatct ttctagaaac tattttctgg
taagccacct 7740gcactgcccc tcccctccct ggctggtcac cctcccggtc
agaccgcggt gacttctggc 7800aggcctcctt cctccaggct tcctcatgcc
catccttaag tcggccctat atctatcaag 7860atgacccatc caaataaacc
tctgctctgt tgctctccat tctcccctcc ccaggtgcct 7920agagcatcct
gtcgtcccct tcctcagttt cctctgtatg ctttgatgac ccccctggcg
7980tatttgctcc tgattttccc ttcccgttta tttactctcc aagctcctac
ttatccttca 8040aaggcagctc aaatgctccc ttacatgaat tcccctgcac
tgccccggaa agacttctgt 8100tcacatttcc atgtctctgc aatggaactt
cctgcactgc cagcgctgag ctttccccag 8160cacaccgcga gcacctctgg
agcctgagcg taccaagtgg agcacggggt gccagcgagg 8220gccctagatg
ggtgttgggg gagcggatgc acacgttgca gccgcgcctt cctcctgtgc
8280agccgcacac tgttgccatt ctgttttcac agagcccggc tgaagaacaa
caacagacaa 8340gtgtgcattg acccgaagct aaagtggatt caggagtacc
tggagaaagc tttaaacaag 8400taagcacaac agccaaaaag gactttccgc
tagacccact cgaggaaaac taaaaccttg 8460tgagagatga aagggcaaag
acgtggggga gagggggcct taaccatgag gaccaggtgt 8520gtgtgtgggg
tgggcacatt gatctgggat cgggcctgag gtttgccagc atttagaccc
8580tgcatttata gcatacggta tgatattgca gcttatattc atccatgccc
tgtacctgtg 8640cacgttggaa cttttattac tggggttttt ctaagaaaga
aattgtatta tcaacagcat 8700tttcaagcag ttagttcctt catgatcatc
acaatcatca tcattctcat tctcattttt 8760taaatcaacg agtacttcaa
gatctgaatt tggcttgttt ggagcatctc ctctgctccc 8820ctggggagtc
tgggcacagt caggtggtgg cttaacaggg agctggaaaa agtgtccttt
8880cttcagacac tgaggctccc gcagcagcgc ccctcccaag aggaaggcct
ctgtggcact 8940cagataccga ctggggctgg gcgccgccac tgccttcacc
tcctctttca acctcagtga 9000ttggctctgt gggctccatg tagaagccac
tattactggg actgtgctca gagacccctc 9060tcccagctat tcctactctc
tccccgactc cgagagcatg cttaatcttg cttctgcttc 9120tcatttctgt
agcctgatca gcgccgcacc agccgggaag agggtgattg ctggggctcg
9180tgccctgcat ccctctcctc ccagggcctg ccccacagct cgggccctct
gtgagatccg 9240tctttggcct cctccagaat ggagctggcc ctctcctggg
gatgtgtaat ggtccccctg 9300cttacccgca aaagacaagt ctttacagaa
tcaaatgcaa ttttaaatct gagagctcgc 9360tttgagtgac tgggttttgt
gattgcctct gaagcctatg tatgccatgg aggcactaac 9420aaactctgag
gtttccgaaa tcagaagcga aaaaatcagt gaataaacca tcatcttgcc
9480actaccccct cctgaagcca cagcagggtt tcaggttcca atcagaactg
ttggcaaggt 9540gacatttcca tgcataaatg cgatccacag aaggtcctgg
tggtatttgt aactttttgc 9600aaggcatttt tttatatata tttttgtgca
catttttttt tacgtttctt tagaaaacaa 9660atgtatttca aaatatattt
atagtcgaac aattcatata tttgaagtgg agccatatga 9720atgtcagtag
tttatacttc tctattatct caaactactg gcaatttgta aagaaatata
9780tatgatatat aaatgtgatt gcagcttttc aatgttagcc acagtgtatt
ttttcacttg 9840tactaaaatt
gtatcaaatg tgacattata tgcactagca ataaaatgct aattgtttca
9900tggtataaac gtcctactgt atgtgggaat ttatttacct gaaataaaat
tcattagttg 9960ttagtgatgg agcttataga cgtttctggt ttatatagtt
aagcctgcct gcagtcaggt 10020gtctgagacc ccttctcaca gcccatgtgt
gacagtgtat gggcttttct cacgagcaga 10080ttagatctgc agctcaagtt
tttggatctt tttttttttt ttttaacccg attgaaatag 10140cagtgctggt
tttctgaaga ataatatttg actcactaat tcgtcttccc tccctcctcc
10200tccttggttc tcctaacttc cccatgtaat ccccagagac tcaaccctag
taatatcaac 10260cttttacatt ttcccatgta aaaatcccat gactccaggc
catggttaat atgaagcttt 10320cacagggaca ggtggcctca ccccataaat
cattaaatac cattcagctt gaatcatttt 10380aatgtgacag tcacgagcca
gttgctctaa taaaattctg ctaaccagct ctctcccttg 10440ctctccagaa
caatcgcatt cattcccagg agtgttcaca ggcgtctaga aaggggaagg
10500tgggaccact gccttttttc tcctcttctg aatggcagtc tgaacctggg
gcctgcagcc 10560tccaaaacgt tattcaattt gaaatgcaga catttcttag
agaaagctaa gagttcagcc 10620ctgcattgag aagaagaaaa agtcccatga
gagcagggca gggtggggtg acaaggacca 10680cgatggccag cctggccctg
ggtgtaccct gagatgtgga ttccttgtcc ccagtgggaa 10740tcaggttcag
gttggcagat tggcagtcgc tgtaacccat tcacaaaggt gttctaggag
10800cccattgaac atttgtttga atggaaggaa atgtccaatt tattttaatg
aattactgat 10860aaacagtgct ttgaccaggg gcctccaggc ctgagactga
aggcacagtt taaccagtac 10920acgggccagt gttaaatctt atgcagactg
agacgaactt ttgtttattt ccacattatt 10980aatggttcta ctaattttat
ctaagggggc gcagagaaga aaaagtgggg aaaaaagaaa 11040agataggaaa
aaagaagcga cagaagaaga gaaaggctgc ccagaaaagg aaaaactagt
11100tatctgccac ctcgagatgg accacagttc acttgctctc ggcgctttgt
aaatttgctc 11160gatcctcctc caggacagac ccccatgcag actgggcagg
ggctcagact tccgtggggg 11220agcagtgctt tgctgccctg ccagccacac
cggcttctgt atttatgtgc tttttaaggc 11280ccttgttggt ctgctaagtt
atgaagaaag tagttgtgca gagactgggg cgggggtctg 11340tgacgcggag
cctgtgtgct caggactctg tccagaatag cctgggagct ccaggaatgc
11400ccaggttgct gagcccccca gcccgccctc cacttcctct cttagaaggc
ctgcgctcac 11460tggggagctc accagctcca cacacttgca gtctgcattc
ctgtggagac caggctgtgg 11520ccacctggcc agtgtgcagg gcaacctgct
agcccagcag aggtggcctc gccaggaagg 11580gggtgtgcca cccctctgtg
gccctcagga aaggtgaaaa ctgactgacc cttaggaatg 11640ggccctggct
ttttccaaat acccattgcc tttccctcca gcactctgcc acctgggaag
11700gggcttcttc agcccctcct tcctgggttg tgggagtggc tttatcccca
agcccgggtg 11760ggtttctgca aactgtgtgg ggtgagcccg agggaatgct
gcattctcag agagcagatg 11820tggaaacact tctcagggag ctcctctttg
ggcaattctc tttagagtct ttaaacgggt 11880cccccacgtg gaggacagat
gtgctatgga actttgcaag ggtcctgaat ccctggggat 11940cccagcctgt
cccctcccgc ctcctgcgtg atggcgtgtg ctccagctgc agggcacagc
12000tgcctctgtc ctcattcatg ggaaatacct tatctgccta aagcaaatac
cacttaagct 12060ctaggaactt tcctgcaccc tccttccctg cccatgtaga
tgtatgtgtg agattttttg 12120gcaagtttct ctaatctgga ccgggaggat
ggaagagcaa ggaccccatt tcagtagtgc 12180tcggaaaaag gatgcgttga
atttctcagc ccttctccac ctcacataaa cacccgccca 12240ccctgcaccc
ggatcctggg tcataatttt aataaatgca gaaagagaaa gtggttggag
12300gatggagcac atggaattca ggagaaaacc cacaaagacc cctgcatgtc
agacacaccc 12360tgtcccggag cgtggtgtcc ccttgagctt taatgagctc
cctgtgatca cagccatgcc 12420ttctcctcgt tggggaggtg tcctaggatg
cttcagccaa agacctttgt ttcccgctgc 12480tatctctttt acctggacaa
ctctcctggc ccacgttcct cttgccagca ctgggggtca 12540caggcctgag
ccctgggtac aggggtgccc tagtcttctg ccctccccac ctcttaaggc
12600acagagctgt tgggtgggct gcctggggct gccatccttc ccgtggaagc
cagtagccac 12660tctagtccat gggactcttg acaaaagcgc cccgagaggg
caaacctgtg cccccatact 12720cgcctgcatt cttcggactc cacatgcagc
agggctttgt gcctggggag gggtggccag 12780tctgtcctgg tcagtatgaa
aagctgttgg ccccctaggg acagagggcc cagctaaggc 12840tgcctgagga
tacaaactgc ttgctatccc actcctgggg agcagggtct gcagggactg
12900agagtgggtc ccaccttgag aacgcatgca aggtccgtcc tgtcttgatg
tcttgatgtg 12960actgtatgtg ccctgggggc tcactgtggt ttacaagtgg
cttgtgaagc tcctgggagc 13020aggtggtaca cccagtgctg aagacagggt
cgccgtggaa gagcgaagag cctgaccggg 13080attcctggtg ggttgaaact
aggaagtgct cacaccagtc agagccaaat gaggggtgcg 13140ctatggtcac
tgctctgtcc agcatgcgtt cctcctggga ggtcctggcc acctgtgcac
13200ccacccctgt gccacctcca gcagtcccac ctggggccac ctacggtggc
atggcccctg 13260gctgagaggc cccgagggcg aagggttact ggaagccacg
aaagtgcctc ttgggacagc 13320cgaggccagg atgcagggca gcagcatcct
gagcctcagc cccacgccgg tgccgggtaa 13380gcagtgtgcc ctgtccccgt
cgtatgacca ctctgatggg cctctctgtg ccttcgtgcg 13440tctgccacgc
ccagtgcttg ccacgtgtct gtcctctgct ttctgccatc catgggtccc
13500tccgcttcag cctggctgcg tctcgcactc ccctcccgtc tgttgtcgca
gggcctctga 13560agggagatgc atggccaagg tggcaacttg gaagtaggga
ttggccccag ggcctccgcg 13620caggccgctg tcctgctgga gctggctggg
tgtgggggga acctgcctta atggtgtttc 13680cctctgttct tgtcaacagg
aggttcaaga tgtgagaggg tcagacgcct gaggaaccct 13740tacagtagga
gcccagctct gaaaccagtg ttagggaagg gcctgccaca gcctcccctg
13800ccagggcagg gccccaggca ttgccaaggg ctttgttttg cacactttgc
catattttca 13860ccatttgatt atgtagcaaa atacatgaca tttatttttc
atttagtttg attattcagt 13920gtcactggcg acacgtagca gcttagacta
aggccattat tgtacttgcc ttattagagt 13980gtctttccac ggagccactc
ctctgactca gggctcctgg gttttgtatt ctctgagctg 14040tgcaggtggg
gagactgggc tgagggagcc tggccccatg gtcagcccta gggtggagag
14100ccaccaagag ggacgcctgg gggtgccagg accagtcaac ctgggcaaag
cctagtgaag 14160gcttctctct gtgggatggg atggtggagg gccacatggg
aggctcaccc ccttctccat 14220ccacatggga gccgggtctg cctcttctgg
gagggcagca gggctaccct gagctgaggc 14280agcagtgtga ggccagggca
gagtgagacc cagccctcat cccgagcacc tccacatcct 14340ccacgttctg
ctcatcattc tctgtctcat ccatcatcat gtgtgtccac gactgtctcc
14400atggccccgc aaaaggactc tcaggaccaa agctttcatg taaactgtgc
accaagcagg 14460aaatgaaaat gtcttgtgtt acctgaaaac actgtgcaca
tctgtgtctt gtttggaata 14520ttgtccattg tccaatccta tgtttttgtt
caaagccagc gtcctcctct gtgaccaatg 14580tcttgatgca tgcactgttc
cccctgtgca gccgctgagc gaggagatgc tccttgggcc 14640ctttgagtgc
agtcctgatc agagccgtgg tcctttgggg tgaactacct tggttccccc
14700actgatcaca aaaacatggt gggtccatgg gcagagccca agggaattcg
gtgtgcacca 14760gggttgaccc cagaggattg ctgccccatc agtgctccct
cacatgtcag taccttcaaa 14820ctagggccaa gcccagcact gcttgaggaa
aacaagcatt cacaacttgt ttttggtttt 14880taaaacccag tccacaaaat
aaccaatcct ggacatgaag attctttccc aattcacatc 14940taacctcatc
ttcttcacca tttggcaatg ccatcatctc ctgccttcct cctgggccct
15000ctctgctctg cgtgtcacct gtgcttcggg cccttcccac aggacatttc
tctaagagaa 15060caatgtgcta tgtgaagagt aagtcaacct gcctgacatt
tggagtgttc cccttccact 15120gagggcagtc gatagagctg tattaagcca
cttaaaatgt tcacttttga caaaggcaag 15180cacttgtggg tttttgtttt
gtttttcatt cagtcttacg aatacttttg ccctttgatt 15240aaagactcca
gttaaaaaaa attttaatga agaaagtgga aaacaaggaa gtcaaagcaa
15300ggaaactatg taacatgtag gaagtaggaa gtaaattata gtgatgtaat
cttgaattgt 15360aactgttctt gaatttaata atctgtaggg taattagtaa
catgtgttaa gtattttcat 15420aagtatttca aattggagct tcatggcaga
aggcaaaccc atcaacaaaa attgtccctt 15480aaacaaaaat taaaatcctc
aatccagcta tgttatattg aaaaaataga gcctgaggga 15540tctttactag
ttataaagat acagaactct ttcaaaacct tttgaaatta acctctcact
15600ataccagtat aattgagttt tcagtggggc agtcattatc caggtaatcc
aagatatttt 15660aaaatctgtc acgtagaact tggatgtacc tgcccccaat
ccatgaacca agaccattga 15720attcttggtt gaggaaacaa acatgaccct
aaatcttgac tacagtcagg aaaggaatca 15780tttctatttc tcctccatgg
gagaaaatag ataagagtag aaactgcagg gaaaattatt 15840tgcataacaa
ttcctctact aacaatcagc tccttcctgg agactgccca gctaaagcaa
15900tatgcattta aatacagtct tccatttgca agggaaaagt ctcttgtaat
ccgaatctct 15960ttttgctttc gaactgctag tcaagtgcgt ccacgagctg
tttactaggg atccctcatc 16020tgtccctccg ggacctggtg ctgcctctac
ctgacactcc cttgggctcc ctgtaacctc 16080ttcagaggcc ctcgctgcca
gctctgtatc aggacccaga ggaaggggcc agaggctcgt 16140tgactggctg
tgtgttggga ttgagtctgt gccacgtgtt tgtgctgtgg tgtgtccccc
16200tctgtccagg cactgagata ccagcgagga ggctccagag ggcactctgc
ttgttattag 16260agattacctc ctgagaaaaa aggttccgct tggagcagag
gggctgaata gcagaaggtt 16320gcacctcccc caaccttaga tgttctaagt
ctttccattg gatctcattg gacccttcca 16380tggtgtgatc gtctgactgg
tgttatcacc gtgggctccc tgactgggag ttgatcgcct 16440ttcccaggtg
ctacaccctt ttccagctgg atgagaattt gagtgctctg atccctctac
16500agagcttccc tgactcattc tgaaggagcc ccattcctgg gaaatattcc
ctagaaactt 16560ccaaatcccc taagcagacc actgataaaa ccatgtagaa
aatttgttat tttgcaacct 16620cgctggactc tcagtctctg agcagtgaat
gattcagtgt taaatgtgat gaatactgta 16680ttttgtattg tttcaattgc
atctcccaga taatgtgaaa atggtccagg agaaggccaa 16740ttcctatacg
cagcgtgctt taaaaaataa ataagaaaca actctttgag aaacaacaat
16800ttctactttg aagtcatacc aatgaaaaaa tgtatatgca cttataattt
tcctaataaa 16860gttctgtact caaatgtagc caccaacagt ttgaaattag
tgttactact tggaattttc 16920tggacgtgct ttttttcccc acaaacccaa
aactgagggt tgtgtaatcc tggctacagt 16980ggttcatgga aaacagggca
ttgtaatcat gctaatcaca gctgagaatt ctggaggcat 17040acttgcctct
tctggacagc tgatccttag cagggaaagg tgctcccttt tctctgagtg
17100tctttgattt tgctagaatt gcttctgaaa ggccagcctg tcctccactc
cacaagggat 17160ggttttgtgc aagggtctca ggtatttctt gacagctgtg
aggagggaag cttccactct 17220gcccttctca gtcctgggag aatgcaacag
aggtctggcc ctgatggtaa ttgctgagga 17280ccagcactct gtgtgtttcc
tgcactgcac agagaccttg ctggaagcca gacacgattc 17340tctcacctag
caatggctag ccagcactgt cctggacatg gggcatagga gatgagccca
17400gcagacaggt tctggtcctc acagaggtca gcccttaaca ctctctagca
cattctctgt 17460catccacagt caactttgtt agaaaggaga gtcagcacaa
aagttccgaa tgtctgagtc 17520agattgtccg gtcctccaag ccctcttccc
cattggtcca gatgtgatca catcatccca 17580tgccaaatct tcccatgact
taatgggatg gccgtcatgt gggtgctaag ctgcagatgg 17640actggggaag
gaagaagggt tagagcaggc cgggaagggc agcgtggtgt caggggacct
17700gtgcaggtgt gaagatcagt agactggggc ttgcgtccca ttcactctct
gctccctctg 17760tgatttgaga tgcatccctt gatctctctg agttccagtc
tcccagctgc aacactgaag 17820cttattactg ccaacggtca agaggattag
actgggagag tgtgtggaag aagctagctt 17880ttgagggaca ggacaaagcc
cgcccctcca ccctgaaagt tccccatttt gctccctctc 17940tccacctcct
gtttcctgag gtccacagag ctagaataag acccacatct gcctctgctg
18000agccccttaa caaagctcag agaccaaggg agcaggaagt gacctggaag
ctgagcctgc 18060ccctcagcta aaggatgggg agaagtcact gggtcgcatc
ccctgtatct ccggcacacc 18120acactgctat cctagcactg ggaatcgaca
atgggccaac ccttcaaaat gacctggcat 18180cagaaagaaa gcctgagccc
tggcatgctt gcattctccc ctcatttact tggccaacaa 18240cagctgtgat
gcctctcctg caccgcagga gtgtggggag caagacacac tcactctaat
18300tccatgaagc ctgcagtctg gcggctgccg tgtgagaagt gggtattttc
atagattgtg 18360gtgcattcag gcatcaggga agcgtaaaac agaaaagacc
cctaacttag gcatcagtaa 18420ggatttgctt aaaaagcgct ggtgaaggtg
agacctgaga aaagtggatt ctttggggag 18480gtggtctact gagcccatga
gggctggcat ggagagccca ggtggtatcc tcctgtgaat 18540tggaaagggg
gcttattcta gaagccggag gtgtagctca cacagggagt ctggcatggg
18600gggcacaggg cactgggttt cagcttcctt attcccatca cttgtgggag
gcacaaacac 18660agctgagaaa gctggacccc cacggtgatg gggtcccctc
agcacggctg attgcatgga 18720gtggacactg gatgcatatt gcaggccgat
ctgattttct gtcacaggag tacaaggctg 18780tgattcaaca gcatcgggct
ttgctccctt tctctgtg 18818
* * * * *
References