U.S. patent application number 17/115219 was filed with the patent office on 2021-07-08 for combination of topoisomerase-i inhibitors with immunotherapy in the treatment of cancer.
The applicant listed for this patent is Board of Regents, The University of Texas System. Invention is credited to Rodabe AMARIA, Patrick HWU, Rina M. MBOFUNG, Jodi A. MCKENZIE.
Application Number | 20210205293 17/115219 |
Document ID | / |
Family ID | 1000005462491 |
Filed Date | 2021-07-08 |
United States Patent
Application |
20210205293 |
Kind Code |
A1 |
HWU; Patrick ; et
al. |
July 8, 2021 |
COMBINATION OF TOPOISOMERASE-I INHIBITORS WITH IMMUNOTHERAPY IN THE
TREATMENT OF CANCER
Abstract
The present disclosure relates to compositions and methods for
treating cancer, more specifically to methods and compositions
comprising a Topoisomerase I inhibitor and an .alpha.-PD-L1
antibody
Inventors: |
HWU; Patrick; (Houston,
TX) ; MCKENZIE; Jodi A.; (Houston, TX) ;
MBOFUNG; Rina M.; (Houston, TX) ; AMARIA; Rodabe;
(Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents, The University of Texas System |
Austin |
TX |
US |
|
|
Family ID: |
1000005462491 |
Appl. No.: |
17/115219 |
Filed: |
December 8, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15760995 |
Mar 16, 2018 |
10894044 |
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PCT/US2016/052303 |
Sep 16, 2016 |
|
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17115219 |
|
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62219548 |
Sep 16, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2878 20130101;
A61K 2039/507 20130101; A61K 9/127 20130101; A61K 2300/00 20130101;
A61K 31/4745 20130101; A61P 35/00 20180101; A61K 9/0019 20130101;
A61K 9/1271 20130101; A61K 39/3955 20130101; C07K 16/2827 20130101;
A61K 2039/545 20130101; C07K 2317/76 20130101; A61K 2039/505
20130101; C07K 16/2818 20130101 |
International
Class: |
A61K 31/4745 20060101
A61K031/4745; A61K 9/00 20060101 A61K009/00; A61K 39/395 20060101
A61K039/395; C07K 16/28 20060101 C07K016/28; A61K 9/127 20060101
A61K009/127; A61P 35/00 20060101 A61P035/00 |
Claims
1.-11. (canceled)
12. The A method of treatment of cancer in a host in need thereof,
comprising administering to the host a combination of liposomal
irinotecan and pembrolizumab for the treatment of cancer, in an
amount and in a schedule of administration that is therapeutically
synergistic in the treatment of said cancer.
13. The method according to claim 12, wherein said schedule
comprises administering to a human host during a 28-day treatment
cycle: a total of 80 mg/m.sup.2 liposomal irinotecan (free base)
followed by the administration of 2 mg/kg pembrolizumab, once every
two weeks for two weeks; and repeating said 28-day treatment cycle
until a progression or an unacceptable toxicity is observed.
14. The method according to claim 12, wherein said schedule
comprises administering to a human host during a treatment cycle: a
total of 43, 50, 70 or 80 mg/m.sup.2 liposomal irinotecan (free
base) once every two weeks for two weeks and administration of 2
mg/kg pembrolizumab once every three weeks; and repeating said
treatment cycle until a progression or an unacceptable toxicity is
observed.
15. The method according to claim 12, wherein said schedule
comprises administering to a human host during a treatment cycle: a
total of 80 mg/m.sup.2 liposomal irinotecan (free base) once every
two weeks for two weeks and administration of 2 mg/kg pembrolizumab
once every three weeks; and repeating said treatment cycle until a
progression or an unacceptable toxicity is observed.
16. The method according to claim 12, wherein the cancer is
selected from the group consisting of melanoma, NSCLC and RCC.
17. The method according to claim 16, wherein the cancer is
melanoma.
18. The method according to claim 12, wherein the liposomal
irinotecan comprises liposomes having a unilamellar lipid bilayer
vesicle, approximately 110 nm in diameter, which encapsulates an
aqueous space containing irinotecan in a gelated or precipitated
state as the sucrose octasulfate salt; wherein the vesicle is
composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 6.81
mg/mL, cholesterol 2.22 mg/mL, and methoxy-terminated polyethylene
glycol (MW 2000)-distearoylphosphatidyl ethanolamine
(MPEG-2000-DSPE) 0.12 mg/mL. MM-398.
19. The method according to claim 12 wherein no other
antineoplastic agent is administered for the treatment of the
cancer.
Description
[0001] This application is a division of U.S. application Ser. No.
15/760,995, filed Mar. 16, 2018, which is a national stage entry
under 35 U.S.C. .sctn. 371 of International Application No.
PCT/US2016/052303, filed Sep. 16, 2016, which claims the benefit of
priority of U.S. Provisional Application No. 62/219,548, filed Sep.
16, 2015, the disclosure of which is hereby incorporated by
reference as if written herein in its entirety.
TECHNICAL FIELD
[0002] The present disclosure relates to compositions and methods
for treating cancer, more specifically to methods and compositions
comprising a Topoisomerase I inhibitor and an .alpha.-PD-L1 or
.alpha.-PD-1 antibody.
BACKGROUND
[0003] Generally, cancer results from the deregulation of the
normal processes that control cell division, differentiation, and
apoptotic cell death and is characterized by the proliferation of
malignant cells which have the potential for unlimited growth,
local expansion and systemic metastasis. Deregulation of normal
processes include abnormalities in signal transduction pathways and
response to factors which differ from those found in normal
cells.
[0004] Topoisomerases are a family of DNA enzymes, which are
involved in unwinding DNA and relieving torsional strain during
replication and transcription. Topoisomerases are nuclear enzymes
that control the changes in DNA structure by catalyzing the
breaking and rejoining of the phosphodiester backbone of DNA
strands during the normal cell cycle. These enzymes allow DNA to
relax by forming enzyme-bridged strand breaks that act as transient
gates or pivotal points for the passage of other DNA strands.
Topoisomerase-inhibiting drugs appear to interfere with this
breakage-reunion reaction of DNA topoisomerases, which ultimately
leads to cell death. Topoisomerase-inhibiting drugs have been found
to be effective for inhibiting cancer cell proliferation.
[0005] In addition to preventing proliferation of tumor cells
themselves, stimulating the patient's own immune response to target
tumor cells is another option for cancer therapy and many studies
have demonstrated effectiveness of immunotherapy using tumor
antigens to induce the immune response. PD-L1 (Programmed Cell
Death Ligand-1) binds PD-1 (Programmed Cell Death Protein 1) and
thus both play a role in the regulation of the immune system
functions including immunity and self-tolerance. PD-L1 is expressed
in tumors, and it appears that upregulation of PD-L1 may allow
cancers to evade the host immune system. Thus, interfering with the
inhibitory signal through the PD-L1:PD-1 pathway is a therapeutic
option for enhancing anti-tumor immunity. Antibodies blocking
activation of the programmed cell death 1 (PD-1) receptor have been
found to be effective for strengthening immune cells to target
cancer cells, however, long lasting responses are only observed in
a small subset of immunotherapy-treated patients.
[0006] Melanoma is a highly aggressive form of skin cancer, whose
rates of morbidity and mortality are continuously increasing. The
development of immunotherapeutic agents like anti-PD-L1 and
anti-CTLA4 antibodies has resulted in fundamental advances in the
treatment of melanoma. However, long lasting responses are only
observed in a small subset of immunotherapy-treated melanoma
patients. This shortfall highlights the need for a better
understanding of the molecular mechanisms that govern tumor
sensitivity or resistance to immunotherapy.
[0007] Despite these advances, there remains a need for improved
methods and compositions for treating cancer. This disclosure
relates to combining therapeutic approaches for inhibiting
proliferation of tumor cells and enhancing anti-tumor immunity. For
example, observed clinical responses to oncology immune-therapy
have been heterogeneous and limited in some patients due to a
variety of factors including, for example, patients having immune
sterile tumors, higher mutational loads, intra- and inter-tumoral
variabilities due to genetic and epigenetic differences between
patient cancers, and other still unknown mechanisms believed to
mediate responses or resistance to immune-therapy in the field of
oncology. As a result, immune-therapy has had limited clinical
benefit in some patients due to an inability to accurately predict
response to immuno-therapy. There remains a need to make tumors
more immunogenic and increase the efficacy of immune-therapy in
oncology treatment.
SUMMARY
[0008] In some embodiments, inventors have discovered that treating
tumor cells with certain bioactive compounds may enhance the
sensitivity of the patient-derived tumor cells to T-cell mediated
cytotoxicity, thereby providing novel combinatorial drug therapies
to improve the efficacy of cancer immunotherapy. For example, the
inventors herein disclose a synergistic effect between Top1
inhibitors and immune-based therapies in the treatment of cancer.
The invention is based in part on the discovery that treatment of
melanoma tumor cells with a Top1 inhibitor prior to exposure to
autologous T cells, produced a synergistic increase in tumor cell
death, as measured by intracellular staining of activated caspase
3, and computed using CalcuSyn.
[0009] In one embodiment, a screening approach is disclosed for
assaying T-cell mediated cytotoxicity. In another embodiment,
certain topoisomerase I inhibitors are identified as enhancers of T
cell mediated immune-therapy, including therapeutic combinations
that can provide a synergistic improvement of CTL-mediated killing
in vitro and enhanced anti-tumor response using a combination of
liposomal irinotecan (e.g., MM-398) and anti-PD-L1 or anti-PD-1
antibody in vivo. In another embodiment, the role of a p53
regulatory gene is identified as playing an essential role in the
enhanced response to T cell mediated killing, including
topoisomerase 1 inhibition resulting in upregulation of Teap, Teap
overexpression observed to recapitulate the relevant phenotype and
the observation that knockdown of Teap impedes the relevant
phenotype.
[0010] Autologous patient-derived tumor cell lines and tumor
infiltrating lymphocytes (TILs) were utilized in an in vitro
activated caspase 3-based high-throughput screen, to identify
compounds that increase the sensitivity of melanoma cells to T-cell
mediated cytotoxicity. The screen consisted of a library of 850
bioactive compounds. One group of compounds that was most able to
enhance T-cell killing of melanoma cells was topoisomerase I (Top1)
inhibitors including: topotecan, and irinotecan. Also disclosed
herein is an in vivo model, where a better anti-tumor effect was
observed in tumor- bearing mice treated with an antibody against
the co-inhibitory molecule Programmed Death Ligand 1 (PD-L1) in
combination with a nanoparticle liposomal formulation of
irinotecan, than in cohorts treated with either antibody or drug
alone. These findings relate to synergism between Top1 inhibitors
and immune-based therapies in the treatment of melanoma.
[0011] Genomic and proteomic changes elicited by inhibition of Top1
are now being investigated to identify the molecular factors that
mediate the effect of Top1 inhibitors on T cell-mediated killing of
melanoma. Our goal is to identify molecular changes mediated by
Top1 inhibition in melanoma tumor cells, and/or the tumor
microenvironment, can relieves immunosuppression and potentiates
the activity of cytotoxic T cell-based immunotherapy.
[0012] Provided is a method for killing cancer cells in a
biological sample comprising contacting the biological sample with
an effective amount of a Topoisomerase I inhibitor and an
.alpha.-PD-L1 antibody.
[0013] Provided is a method for inhibiting the growth of cancer
cells in a biological sample comprising contacting the biological
sample with an effective amount of a Topoisomerase I inhibitor and
an .alpha.-PD-L1 antibody.
[0014] Provided is a method for treating a cancer in a subject in
need thereof, comprising the step of administering to the subject
an effective amount of a Topoisomerase I inhibitor and an
.alpha.-PD-L1 antibody.
[0015] Provided is a method of treating cancer comprising the
administration of a therapeutically effective amount of an
.alpha.-PDL-1 antibody and a topoisomerase I inhibitor. In one
aspect, methods of treating cancer can include administering to a
patient in need thereof a therapeutically effective amount of the
.alpha.-PDL-1 antibody followed by the topoisomerase I inhibitor.
In another aspect, the topoisomerase I inhibitor is a liposomal
irinotecan formulation such as MM-398.
[0016] Provided is a composition comprising an effective amount of
a Topoisomerase I inhibitor and an .alpha.-PD-L1 antibody.
[0017] Provided is a composition comprising an effective amount of
a Topoisomerase I inhibitor and an .alpha.-PD-L1 antibody for use
in treating cancer.
[0018] Provided is a use of a composition as recited in claim 23
for the manufacture of a medicament to treat cancer.
[0019] Provided is a kit for treating a cancer in a subject in need
thereof, comprising: a) a Topoisomerase I inhibitor and an
.alpha.-PD-L1 antibody; and b) written instructions for
administering to the subject an effective amount of a Topoisomerase
I inhibitor and an .alpha.-PD-L1 antibody to treat the cancer.
[0020] Provided is a method for killing cancer cells in a
biological sample comprising contacting the biological sample with
an effective amount of a Topoisomerase I inhibitor and an
.alpha.-PD-1 antibody.
[0021] Provided is a method for inhibiting the growth of cancer
cells in a biological sample comprising contacting the biological
sample with an effective amount of a Topoisomerase I inhibitor and
an .alpha.-PD-1 antibody.
[0022] Provided is a method for treating a cancer in a subject in
need thereof, comprising the step of administering to the subject
an effective amount of a Topoisomerase I inhibitor and an
.alpha.-PD-1 antibody.
[0023] Provided is a method of treating cancer comprising the
administration of a therapeutically effective amount of an
.alpha.-PD-1 antibody and a topoisomerase I inhibitor. In one
aspect, methods of treating cancer can include administering to a
patient in need thereof a therapeutically effective amount of the
.alpha.-PD-1 antibody followed by the topoisomerase I inhibitor. In
another aspect, the topoisomerase I inhibitor is a liposomal
irinotecan formulation such as MM-398.
[0024] Provided is a composition comprising an effective amount of
a Topoisomerase I inhibitor and an .alpha.-PD-1 antibody.
[0025] Provided is a composition comprising an effective amount of
a Topoisomerase I inhibitor and an .alpha.-PD-1 antibody for use in
treating cancer.
[0026] Provided is a use of a composition as recited in claim 23
for the manufacture of a medicament to treat cancer.
[0027] Provided is a kit for treating a cancer in a subject in need
thereof, comprising: a) a Topoisomerase I inhibitor and an
.alpha.-PD-1 antibody; and b) written instructions for
administering to the subject an effective amount of a Topoisomerase
I inhibitor and an .alpha.-PD-1 antibody to treat the cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1A depicts the FACS analysis to determine tumor cells,
stained with the cell tracker dye DDAO, which are also positive for
activated caspase 3. FIG. 1B depicts a schematic for obtaining
data.
[0029] FIG. 2A depicts the synergistic effect of Top 1 inhibitors
(TILs) on T-cell mediated killing of melanoma cells from patient
derived melanoma cell line 2338 by treatment with autologous TILs
at varying effector T cell to tumor cell (E:T) ratios for 3 hours,
as measured by percent activated caspase 3. FIG. 2B depicts the
synergistic effect of Top 1 inhibitors (TILs) on T-cell mediated
killing of melanoma cells from patient derived melanoma cell line
2400 by treatment with autologous TILs at varying effector T cell
to tumor cell (E:T) ratios for 3 hours, as measured by percent
activated caspase 3.
[0030] FIG. 3A is a normalized isobologram of the Combination Index
(CI) of combining SN38 with 2338 TIL. FIG. 3B is a normalized
isobologram of the Combination Index (CI) of combining SN38 with
2400 TIL.
[0031] FIG. 4A depicts the results of an in vivo experiment in
C57BL/6 mice inoculated sc with 5.times.10.sup.5 MC38/gp100 cells
and then treated with vehicle, SN38, .alpha.-PD-L1, or a
combination of SN38 and .alpha.-PD-L1 . FIG. 4B depicts the results
of an in vivo experiment in C57BL/6 mice inoculated sc with
5.times.10.sup.5 MC38/gp100 cells and then treated with free
irinotecan or MM-398. FIG. 4A shows that the combination of SN38
and .alpha.-PD-L1 trended better than SN38 or .alpha.-PD-L1 alone,
but not significantly. FIG. 4B shows that in vivo anti-tumor
response with MM-398 is significantly higher in comparison to free
irinotecan, and that efficacy increases with dose (wherein *
indicates P<0.0001). FIG. 4C depicts the results of an in vivo
experiments in C57BL/6 mice inoculated sc with 5.times.10.sup.5
MC38/gp100 cells and then treated with vehicle, SN38,
.alpha.-PD-L1, or a combination of SN38 and .alpha.-PD-L1. This is
a repeat of the experiment represented in FIG. 4A with the notable
change that treatment began 3 days after tumor inoculation (FIG.
4C) as opposed to 7 days after tumor inoculation (FIG. 4A). The
data represented in FIG. 4C were pulled from the experiment
described on FIG. 10. This shows the enhanced tumor control
observed in tumor-bearing mice treated with a combination of SN38
and anti-PD-L1 in comparison to the control group or to cohorts
treated with SN38 or anti-PD-L1 alone.
[0032] FIG. 5A is a graph of measured tumor volume over time after
administration of MM-398 liposomal irinotecan and the anti-PD-L1
antibody described in the Table 2 of Example 3 in a mouse xenograft
model, FIG. 5B is the corresponding plot of the survival curve
(FIG. 5B). FIG. 5C is a schematic of the experiment, designed to
determine the anti-tumor effect of combining MM-398 and anti-PD-L1
in our pre-clinical mouse model.
[0033] FIG. 6 shows gene expression changes in antigen processing
genes after Top1 inhibition. The heatmap in FIG. 6A represents the
differential expression of a subset of genes involved in antigen
presentation. The heatmap in FIG. 6B represents a subset of genes
differentially expressed after Top1 inhibition from microarray
analysis. In FIG. 6B, the leftmost side of the fold-change
spectrum, indicating downregulation, has been outlined to
distinguish it from upregulation, and the genes that were
downregulated in the array (APAF1 and USP15 in 2400 and 2549, and
EGR1 in 2549) have been outlined as well.
[0034] FIG. 7 shows Nano-liposomal irinotecan (nal-IRI),
MM-398.
[0035] FIG. 8A shows the formula for detecting the ComboScore
herein, and FIG. 8B is a scatter plot graph labeling selected data
points for certain Top1 inhibitor compounds. FIG. 8C is a scatter
plot graph showing the % caspase positive tumor cells exposed to
certain topoisomerase 1 inhibitor drugs plotted against % caspase
positive tumor cells exposed to a certain topoisomerase 1 inhibitor
drugs and T cells (Example 1).
[0036] FIG. 9A shows bar graphs showing the synergistic effect of
Top 1 inhibitors and autologous tumor infiltrating lymphocytes
(TILs) on T-cell mediated killing of melanoma cells from patient
derived melanoma cell line 2338 by treatment with treated with
autologous TILs at varying effector T cell to tumor cell (E:T)
ratios for 3 hours, as measured by percent activated caspase 3.
FIG. 9B shows bar graphs showing the synergistic effect of Top 1
inhibitors and autologous tumor infiltrating lymphocytes (TILs) on
T-cell mediated killing of melanoma cells from patient derived
melanoma cell line 2400 by treatment with treated with autologous
TILs at varying effector T cell to tumor cell (E:T) ratios for 3
hours, as measured by percent activated caspase 3. In each of FIGS.
9A and 9B, cells in the leftmost group of three bars was not
treated with a Top 1 inhibitor or TIL, the cells measured in the
second bar (from left) was treated only with the Top 1 inhibitor,
the cells measured in the third bar (from left) were treated with
TIL and the data for the bar on the far right was obtained from a
synergistic combination of TIL and the Top 1 inhibitor.
[0037] FIG. 10 is a graph of tumor volume over time in a xenograft
cancer model after administration of various immune modulatory
compounds with the Top1 inhibitor SN38. SN38 is the metabolite of
irinotecan.
[0038] FIG. 11A is a line graph from cancer xenograft models
obtained after administration of SN38 and/or anti-41BB. FIG. 11B is
a line graph from cancer xenograft models obtained after
administration of SN38 and/or anti-CTLA4. FIG. 11C is a line graph
from cancer xenograft models obtained after administration of SN38
and/or anti-0.times.40. FIG. 11D is a line graph from cancer
xenograft models obtained after administration of SN38 and/or anti
PD-L1 and anti CTLA4 antibodies.
[0039] FIG. 12 is a schematic of an animal model experiment to
determine the effect of MM-398 liposomal irinotecan and an
anti-PD-L1 antibody on different immune cell populations.
[0040] FIG. 13A is a graph showing CD8/gram measurements taken from
the animal model test of FIG. 12. FIG. 13B is a graph showing
CD8/gram measurements taken from the animal model test of FIG. 12.
FIG. 13C is a graph showing GranzA/gram measurements taken from the
animal model test of FIG. 12. FIG. 13D is a graph showing
GranzB/gram measurements taken from the animal model test of FIG.
12. FIG. 13E is a graph showing Mac/gram measurements taken from
the animal model test of FIG. 12.
[0041] FIG. 14 is a graph showing the change in TP531NP1 following
Top1 inhibition.
[0042] FIG. 15A is a graph showing measurements of relative mRNA
expression and overexpression in 2549 Teap. FIG. 15B is a graph
showing measurements of % caspase 3 positive in 2549 Teap.
[0043] FIG. 16A is a graph showing measurements of relative mRNA
expression and gene silencing in 2549 Teap KO. FIG. 16B is a graph
showing measurements of % caspase 3 positive in 2549 Teap KO.
[0044] FIG. 17A is a schematic for a first method of administering
a combination of MM-398 liposomal irinotecan and nivolumab to a
human in need thereof.
[0045] FIG. 17B is a schematic for a second method of administering
a combination of MM-398 liposomal irinotecan and nivolumab to a
human in need thereof.
[0046] FIG. 18 demonstrates that in vivo anti-tumor response and
survival are increased when nanoliposomal irinotecan (nal-IRI,
MM-398) is combined with .alpha.-PD1 antibody, including a plot of
tumor volume over time in a mouse xenograft model (FIG. 18B) and a
survival curve (FIG. 18C). The data was obtained from the
experiment described in the schematic of FIG. 18A.
DETAILED DESCRIPTION
Abbreviations and Definitions
[0047] To facilitate understanding of the disclosure, a number of
terms and abbreviations as used herein are defined below as
follows:
[0048] When introducing elements of the present disclosure or the
preferred embodiment(s) thereof, the articles "a", "an", "the" and
"said" are intended to mean that there are one or more of the
elements. The terms "comprising", "including" and "having" are
intended to be inclusive and mean that there may be additional
elements other than the listed elements.
[0049] The term "and/or" when used in a list of two or more items,
means that any one of the listed items can be employed by itself or
in combination with any one or more of the listed items. For
example, the expression "A and/or B" is intended to mean either or
both of A and B, i.e. A alone, B alone or A and B in combination.
The expression "A, B and/or C" is intended to mean A alone, B
alone, C alone, A and B in combination, A and C in combination, B
and C in combination or A, B, and C in combination.
[0050] The term "about," as used herein when referring to a
measurable value such as an amount of a compound, dose, time,
temperature, and the like, is meant to encompass variations of 20%,
10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.
[0051] Camptothecin is a drug used for the treatment of cancer, and
inhibits the DNA enzyme topoisomerase I. Its IUPAC name is
(S)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]
quinoline-3,14-(4H,12H)-dione.
[0052] The term "effective amount" as used herein means that the
amount of a Topoisomerase I inhibitor and an .alpha.-PD-L1 antibody
contained in the composition administered is of sufficient quantity
to achieve the intended purpose, such as, in this case, to kill
cancer cells in a biological sample, inhibit the growth of cancer
cells in a biological sample, or treat a cancer in a subject in
need thereof.
[0053] The term "humanized monoclonal antibodies" means that at
least a portion of the exposed amino acids in the framework regions
of the antibody (or fragment), which do not match with the
corresponding amino acids in the most homologous human
counterparts, are changed, such as by site directed mutagenesis of
the DNA encoding the antibody. The term "humanized monoclonal
antibody" also includes chimeric antibody wherein the light and
heavy variable regions of a monoclonal antibody generated by a
hybridoma from a non-human call line are each attached, via
recombinant technology, to one human light chain constant region
and at least one heavy chain constant region, respectively.
[0054] Irinotecan is a drug used for the treatment of cancer, and
inhibits the DNA enzyme topoisomerase I. Its IUPAC name is
(S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-
3,14-dioxo1H-pyrano[3',4':6,7]'-indolizino
[1,2-b]quinolin-9-yl-[1,4'bipiperidine]-1'-carboxylate.
[0055] Lamellarin D is a drug used for the treatment of cancer, and
inhibits the DNA enzyme topoisomerase I. Its IUPAC name is
3,11-Dihydroxy-14-(4-hydroxy-3-methoxyphenyl)-2,12-dimethoxy-6H-chromeno[-
4',3':4,5]pyrrolo[2,1-a]isoquinolin-6-one.
[0056] MM-398 is nano-liposomal irinotecan (nal-IRI), a liposomal
encapsulation of irinotecan (80,000 molecules/liposome) that is
engineered for stable encapsulation and prolonged circulation. The
AUC.sub.0-t of total irinotecan delivered by MM-398 in blood is
1,652 hr.mu.g/mL (120 mg/m.sup.2) and the AUC.sub.0-t of the active
metabolite, SN-38, is 476 hrng/mL. The T.sub.1/2 of total
irinotecan in blood is 21.2 h and of SN-38, 88.8 h. MM-398 is sold
under the trade name ONIVYDE.RTM.(irinotecan liposome injection)
(Merrimack Pharmaceuticals, Cambridge, Mass.).
[0057] Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody
against the programmed cell death receptor 1, and used in the
treatment of cancer.
[0058] Pembrolizumab is a human IgG4 anti-PD-1 monoclonal antibody
against the programmed cell death receptor 1, and used in the
treatment of cancer.
[0059] SN-38 is the active metabolite of irinotecan; it is 1000
times more active than irinotecan itself. In vitro cytotoxicity
assays show that the potency of SN-38 relative to rinotecan varies
from 2- to 2000-fold. Its IUPAC name is
(4S)-4,11-Diethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quin-
oline-3,14(4H,12H)-dione.
[0060] Anti-PD-L1 antibodies are known in the art and include the
mouse PD-L1-PE (clone 10F.9G2) which may be readily obtained from a
number of sources (e.g., Bio X Cell, 10 Technology Dr., Suite 2B,
West Lebanon, NH 03784-1671 USA). See also, Rodig N et al.,
"Endothelial expression of PD-L1 and PD-L2 down-regulates CD8+ T
cell activation and cytolysis," Eur J Immunol 2003; 33:3117-3126;
Brown JA et al., "Blockade of programmed death-1 ligands on
dendritic cells enhances T cell activation and cytokine
production," J Immunol. 2003 Feb. 1; 170(3):1257-66; and Drees J J
et al., "Soluble production of a biologically active single-chain
antibody against murine PD-L1 in Escherichia coli," Protein Expr
Purif, 2014 February; 94:60-6. Avelumab, atezolizumab, and
durvalumab are anti-PD-L1 antibodies under development.
[0061] Anti-PD-1 antibodies are known in the art and include
nivolumab and pembrolizumab.
[0062] The term topoisomerase I inhibitor refers to agents designed
to interfere with the action of topoisomerase enzyme I which
controls the changes in DNA structure by catalyzing the breaking
and rejoining of the phosphodiester backbone of DNA strands during
the normal cell cycle.
[0063] The term synergy refers to a phenomenon where treatment with
a combination of therapeutic agents manifests a therapeutically
superior outcome to the outcome achieved by each individual
constituent of the combination used at its optimum dose (T. H.
Corbett et al., 1982, Cancer Treatment Reports, 66, 1187). In this
context a therapeutically superior outcome is one in which the
patients either a) exhibit fewer incidences of adverse events while
receiving a therapeutic benefit that is equal to or greater than
that where individual constituents of the combination are each
administered as monotherapy at the same dose as in the combination,
or b) do not exhibit dose-limiting toxicities while receiving a
therapeutic benefit that is greater than that of treatment with
each individual constituent of the combination when each
constituent is administered in at the same doses in the
combination(s) as is administered as individual components. In
xenograft models, a combination, used at its maximum tolerated
dose, in which each of the constituents will be present at a dose
generally not exceeding its individual maximum tolerated dose,
manifests therapeutic synergy when decrease in tumor growth
achieved by administration of the combination is greater than the
value of the decrease in tumor growth of the best constituent when
the constituent is administered alone.
[0064] Thus, in combination, the components of such combinations
have an additive or superadditive effect on suppressing pancreatic
tumor growth, as compared to monotherapy. By "additive" is meant a
result that is greater in extent (e.g., in the degree of reduction
of tumor mitotic index or of tumor growth or in the degree of tumor
shrinkage or the frequency and/or duration of symptom-free or
symptom-reduced periods) than the best separate result achieved by
monotherapy with each individual component, while "superadditive"
is used to indicate a result that exceeds in extent the sum of such
separate results.
[0065] Topotecan is a drug used for the treatment of cancer, and
inhibits the DNA enzyme topoisomerase I. Its IUPAC name is
(S)-10-[(dimethylamino)methyl]-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]-
indolizino[1,2-b]quinoline-3,14(4H,12H)-dione
monohydrochloride.
[0066] An .alpha.-PD-L1 antibody is a monoclonal antibody that
works to activate the immune system by targeting Programmed cell
death ligand 1. An .alpha.-PD-1 antibody is a monoclonal antibody
that works to activate the immune system by targeting Programmed
cell death protein 1. Since PD-1 is the receptor for PD-L1,
interference with (e.g. inhibition of) either of these targets
(inhibition of the interaction between them) permits improved
immunologic targeting of cancer cells via immune checkpoint
blockade.
[0067] The term "subject" includes all mammals including humans,
and is equivalent to the terms "patient" and "host." Examples of
subjects include humans, cows, dogs, cats, goats, sheep, pigs, and
rabbits. Preferably, the subject is a human.
Methods
[0068] Provided is a method for killing cancer cells in a
biological sample comprising contacting the biological sample with
an effective amount of a Topoisomerase I inhibitor and an
.alpha.-PD-L1 or .alpha.-PD-1 antibody.
[0069] Provided is a method for inhibiting the growth of cancer
cells in a biological sample comprising contacting the biological
sample with an effective amount of a Topoisomerase I inhibitor and
an .alpha.-PD-L1 or .alpha.-PD-1 antibody.
[0070] Provided is a method for treating a cancer in a subject in
need thereof, comprising the step of administering to the subject
an effective amount of a Topoisomerase I inhibitor and an
.alpha.-PD-L1 or .alpha.-PD-1 antibody. In certain embodiments, the
.alpha.-PD-L1 or .alpha.-PD-1 antibody is a humanized monoclonal
antibody.
[0071] In certain embodiments, the subject is a human.
[0072] In certain embodiments, the cancer is chosen from skin
cancer, or a variant thereof.
[0073] In certain embodiments, administration of the Topoisomerase
I inhibitor and .alpha.-PD-L1 antibody is sequential.
[0074] In certain embodiments, administration of the Topoisomerase
I inhibitor occurs before administration of the .alpha.-PD-L1
antibody.
[0075] In certain embodiments, administration of the .alpha.-PD-L1
or .alpha.-PD-1 antibody occurs before administration of the
Topoisomerase I inhibitor.
[0076] In certain embodiments, administration of the .alpha.-PD-L1
or .alpha.-PD-1 antibody and Topoisomerase I inhibitor is
essentially simultaneous.
[0077] In certain embodiments, the .alpha.-PD-1 antibody is chosen
from nivolumab and pembrolizumab.
[0078] In certain embodiments, the Topoisomerase I inhibitor is
chosen from irinotecan, topotecan, camptothecin and lamellarin D.
In some embodiments, the method as recited in claim 12, wherein the
Topoisomerase I inhibitor is irinotecan. In some embodiments, the
irinotecan is provided in a composition comprising liposomes
(liposomal irinotecan).
[0079] In particular embodiments, the irinotecan is provided in a
composition comprising liposomes in an aqueous medium, the
liposomes having an interior aqueous space separated from the
aqueous medium by a membrane, the membrane comprising lipids, the
lipids comprising an uncharged lipid component and a neutral
phospholipid, with, entrapped inside the liposomes:
[0080] a. irinotecan and sucrose octasulfate, or
[0081] b. irinotecan and sucrose octasulfate and a substituted
ammonium compound, wherein, when administered into the bloodstream
of a mammal, said irinotecan has a half-release time from said
liposomes of at least 24 hours and the irinotecan entrapped inside
the liposomes is at a concentration that exceeds the irinotecan
concentration in the aqueous medium.
[0082] In particular embodiments, the liposomal irinotecan is
nano-liposomal irinotecan. In particular embodiments, the liposomal
irinotecan is MM-398 (ONIVYDE.RTM.).
[0083] In particular embodiments, the method comprises at least one
cycle, wherein the liposomal irinotecan is administered on day 1 of
a cycle at a dose of between about 60 and about 180 mg/m.sup.2,
except if the patient is homozygous for the UGT1A1*28 allele,
wherein the liposomal irinotecan is administered on day 1 of cycle
1 at a dose of between about 40 and about 120 mg/m.sup.2, wherein
the cycle is a period of 2 to 3 weeks. In particular embodiments,
the liposomal irinotecan is administered on day 1 of a cycle at a
dose of between about 90 and about 150 mg/m.sup.2, except if the
patient is homozygous for the UGT1A1*28 allele, wherein the
liposomal irinotecan is administered on day 1 of cycle 1 at a dose
of between about 60 and about 100 mg/m.sup.2. In particular
embodiments, the method comprises at least one cycle, wherein the
liposomal irinotecan is administered on day 1 of a cycle at a dose
of 120 mg/m.sup.2, except if the patient is homozygous for the
UGT1A1*28 allele, wherein the liposomal irinotecan is administered
on day 1 of cycle 1 at a dose of 80 mg/m.sup.2. In particular
embodiments, the cycle is a period of 2 weeks. In particular
embodiments, the cycle is a period of 3 weeks.
[0084] Also provided herein is a method of treatment of cancer in a
host in need thereof, comprising the step of administering to the
host an effective amount of a Topoisomerase I inhibitor and either
an .alpha.-PD-1 or .alpha.-PD-L 1 antibody. In certain embodiments,
the Topoisomerase I inhibitor and either .alpha.-PD-1 or
.alpha.-PD-L1 antibody are each administered in an amount and in a
schedule of administration that is therapeutically synergistic in
the treatment of said cancer. In certain embodiments, the method
comprises the step of administering to the host an effective amount
of a Topoisomerase I inhibitor and an .alpha.-PD-1 antibody. In
certain embodiments, the method comprises the step of administering
to the host an effective amount of a Topoisomerase I inhibitor and
an .alpha.-PD-L1 antibody.
[0085] In certain embodiments, the Topoisomerase I inhibitor is
irinotecan. In certain embodiments, the Topoisomerase I inhibitor
is liposomal irinotecan. In certain embodiments, the Topoisomerase
I inhibitor is MM-398.
[0086] In certain embodiments, the Topoisomerase I inhibitor and
either .alpha.-PD-1 or .alpha.-PD-L1 antibody are administered
every two to three weeks.
[0087] In certain embodiments, the .alpha.-PD-1 antibody is chosen
from nivolumab and pembrolizumab.
[0088] In certain embodiments, provided herein is are methods of
treatment of cancer in a host in need thereof comprising the
administration of a combination of liposomal irinotecan and
nivolumab, in an amount and in a schedule of administration that is
therapeutically synergistic in the treatment of said cancer.
[0089] In certain embodiments, said schedule comprises
administering to a human host during a 28-day treatment cycle: a
total of 50 mg/m.sup.2 liposomal irinotecan (free base) followed by
the administration of 3 mg/kg nivolumab, once every two weeks for
two weeks; and repeating said 28-day treatment cycle until a
progression or an unacceptable toxicity is observed.
[0090] In certain embodiments, said schedule comprises
administering to a human host during a 28-day treatment cycle: a
total of 43 mg/m.sup.2 liposomal irinotecan (free base) followed by
the administration of 3 mg/kg nivolumab, once every two weeks for
two weeks; and repeating said 28-day treatment cycle until a
progression or an unacceptable toxicity is observed.
[0091] In certain embodiments, said schedule comprises
administering to a human host during a 28-day treatment cycle: a
total of 70 mg/m.sup.2 liposomal irinotecan (free base) followed by
the administration of 3 mg/kg nivolumab, once every two weeks for
two weeks; and repeating said 28-day treatment cycle until a
progression or an unacceptable toxicity is observed.
[0092] In certain embodiments, said schedule comprises
administering to a human host during a 28-day treatment cycle: a
total of 80 mg/m.sup.2 liposomal irinotecan (free base) followed by
the administration of 3 mg/kg nivolumab, once every two weeks for
two weeks; and repeating said 28-day treatment cycle until a
progression or an unacceptable toxicity is observed.
[0093] In certain embodiments, the cancer is selected from the
group consisting of melanoma, pancreatic cancer, colorectal cancer,
Hodgkin's lymphoma, NSCLC and RCC. In certain embodiments, the
cancer is selected from the group consisting of melanoma, NSCLC and
RCC. In particular embodiments, for example, the cancer is
melanoma.
[0094] In certain embodiments, the liposomal irinotecan comprises
liposomes having a unilamellar lipid bilayer vesicle, approximately
110 nm in diameter, which encapsulates an aqueous space containing
irinotecan in a gelated or precipitated state as the sucrose
octasulfate salt; wherein the vesicle is composed of
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 6.81 mg/mL,
cholesterol 2.22 mg/mL, and methoxy-terminated polyethylene glycol
(MW 2000)-distearoylphosphatidyl ethanolamine (MPEG-2000-DSPE) 0.12
mg/mL.
[0095] In certain embodiments, each mL also contains
2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid (HEPES) as
a buffer 4.05 mg/mL and sodium chloride as an isotonicity reagent
8.42 mg/mL.
[0096] In certain embodiments, the host is human and is known not
to be homozygous for the UGT1A1*28 allele.
[0097] In certain embodiments, the combination of the
anti-neoplastic agent liposomal irinotecan and 3 mg/kg of the
anti-neoplastic agent nivolumab is administered to a human host
once every two weeks for a total of at least six weeks with each
administration of liposomal irinotecan comprising the
administration of a total of 43, 50, 70 or 80 mg/m.sup.2 liposomal
irinotecan (free base) followed by the administration of 3 mg/kg
nivolumab on the same day as the liposomal irinotecan, and no other
anti-neoplastic agents are administered during the six weeks.
[0098] In certain embodiments, provided herein is are methods of
treatment of cancer in a host in need thereof comprising the
administration of a combination of liposomal irinotecan and
pembrolizumab, in an amount and in a schedule of administration
that is therapeutically synergistic in the treatment of said
cancer.
[0099] In certain embodiments, said schedule comprises
administering to a human host during a 28-day treatment cycle: a
total of 80 mg/m.sup.2 liposomal irinotecan (free base) followed by
the administration of 2 mg/kg pembrolizumab, once every two weeks
for two weeks; and repeating said 28-day treatment cycle until a
progression or an unacceptable toxicity is observed.
[0100] In certain embodiments, said schedule comprises
administering to a human host during a treatment cycle: a total of
43, 50, 70 or 80 mg/m.sup.2 liposomal irinotecan (free base) once
every two weeks for two weeks and administration of 2 mg/kg
pembrolizumab once every three weeks; and repeating said treatment
cycle until a progression or an unacceptable toxicity is
observed.
[0101] In certain embodiments, said schedule comprises
administering to a human host during a treatment cycle: a total of
80 mg/m.sup.2 liposomal irinotecan (free base) once every two weeks
for two weeks and administration of 2 mg/kg pembrolizumab once
every three weeks; and repeating said treatment cycle until a
progression or an unacceptable toxicity is observed.
[0102] In certain embodiments, the cancer is selected from the
group consisting of melanoma, pancreatic cancer, colorectal cancer,
Hodgkin's lymphoma, NSCLC and RCC. In certain embodiments, the
cancer is selected from the group consisting of melanoma,
pancreatic cancer, NSCLC and RCC. In particular embodiments, for
example, the cancer is melanoma.
[0103] In certain embodiments, the cancer is melanoma.
[0104] In certain embodiments, the liposomal irinotecan comprises
liposomes having a unilamellar lipid bilayer vesicle, approximately
110 nm in diameter, which encapsulates an aqueous space containing
irinotecan in a gelated or precipitated state as the sucrose
octasulfate salt; wherein the vesicle is composed of
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 6.81 mg/mL,
cholesterol 2.22 mg/mL, and methoxy-terminated polyethylene glycol
(MW 2000)-distearoylphosphatidyl ethanolamine (MPEG-2000-DSPE) 0.12
mg/mL. MM-398
[0105] In particular embodiments of the above recited embodiments,
no other antineoplastic agent is administered for the treatment of
the cancer.
[0106] In certain embodiments, the method further comprises
administering another therapeutic agent.
[0107] In some embodiments, the therapeutic agent is chosen from a
taxane, inhibitor of bcr-abl, inhibitor of EGFR, DNA damaging
agent, and antimetabolite. In particular embodiments, the
therapeutic agent is chosen from aminoglutethimide, amsacrine,
anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin,
busulfan, campothecin, capecitabine, carboplatin, carmustine,
chlorambucil, chloroquine, cisplatin, cladribine, clodronate,
colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine,
dactinomycin, daunorubicin, demethoxyviridin, dichloroacetate,
dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin,
estradiol, estramustine, etoposide, everolimus, exemestane,
filgrastim, fludarabine, fludrocortisone, fluorouracil,
fluoxymesterone, flutamide, gemcitabine, genistein, goserelin,
hydroxyurea, idarubicin, ifosfamide, imatinib, interferon,
letrozole, leucovorin, leuprolide, levamisole, lomustine,
lonidamine, mechlorethamine, medroxyprogesterone, megestrol,
melphalan, mercaptopurine, mesna, metformin, methotrexate,
mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole,
octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin,
perifosine, plicamycin, porfimer, procarbazine, raltitrexed,
rituximab, sorafenib, streptozocin, sunitinib, suramin, tamoxifen,
temozolomide, temsirolimus, teniposide, testosterone, thioguanine,
thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin,
vinblastine, vincristine, vindesine, and vinorelbine.
[0108] In some embodiments, the method further comprises
administering non-chemical methods of cancer treatment. In
particular embodiments, the method further comprises administering
radiation therapy. In particular embodiments, the method further
comprises administering surgery, thermoablation, focused ultrasound
therapy, cryotherapy, or any combination thereof.
[0109] Also provided herein are embodiments equivalent to the
methods above, disclosing the corresponding uses of a combination
of liposomal irinotecan and nivolumab or liposomal irinotecan and
pembrolizumab.
Compositions
[0110] The present disclosure provides a composition comprising an
effective amount of a Topoisomerase I inhibitor and an
.alpha.-PD-L1 or .alpha.-PD-1 antibody.
[0111] In some embodiments, the .alpha.-PD-L1 antibody is a
humanized monoclonal antibody.
[0112] In some embodiments, the .alpha.-PD-L1 antibody is chosen
from nivolumab, and pembrolizumab.
[0113] In some embodiments, the Topoisomerase I inhibitor is chosen
from irinotecan, topotecan, camptothecin and lamellarin D. In
particular embodiments, the Topoisomerase I inhibitor is
irinotecan.
Kits
[0114] The present disclosure provides a kit for treating a cancer
in a subject in need thereof, comprising: [0115] a. Topoisomerase I
inhibitor and an .alpha.-PD-L1 or .alpha.-PD-1 antibody; and [0116]
b. written instructions for administering to the subject an
effective amount of a Topoisomerase I inhibitor and an
.alpha.-PD-L1 or a-PD-1 antibody to treat the cancer.
[0117] When the Topoisomerase I inhibitor and an .alpha.-PD-L1 or
.alpha.-PD-1 antibody are administered simultaneously, the kit may
contain the Topoisomerase I inhibitor and the .alpha.-PD-L1 or
.alpha.-PD-1 antibody in a single pharmaceutical composition or in
separate pharmaceutical compositions and packaged accordingly. When
the Topoisomerase I inhibitor and the .alpha.-PD-L1 or .alpha.-PD-1
antibody are not administered simultaneously, the kit will contain
Topoisomerase I inhibitor and the .alpha.-PD-L1 or .alpha.-PD-1
antibody in separate pharmaceutical compositions and packaged
accordingly.
[0118] In one embodiment the kit comprises: a first container
comprising the Topoisomerase I inhibitor in association with a
pharmaceutically acceptable adjuvant, diluent or carrier; and a
second container comprising the .alpha.-PD-L1 or .alpha.-PD-1
antibody in association with a pharmaceutically acceptable
adjuvant, diluent or carrier. The kit can also provides
instruction, such as dosage and administration instructions. Such
dosage and administration instructions can be of the kind that are
provided to a doctor, for example by a drug product label, or they
can be of the kind that are provided by a doctor, such as
instructions to a patient.
Formulation
[0119] The compositions of the present disclosure may be
administered in any way which is medically acceptable which may
depend on the condition or injury being treated. Possible
administration routes include injections, by parenteral routes such
as intramuscular, subcutaneous, intravenous, intraarterial,
intraperitoneal, intraarticular, intraepidural, intrathecal, or
others, as well as oral, nasal, ophthalmic, rectal, vaginal,
topical, or pulmonary, e.g., by inhalation. For the delivery of
liposomally drugs formulated according to the invention, to tumors
of the central nervous system, a slow, sustained intracranial
infusion of the liposomes directly into the tumor (a
convection-enhanced delivery, or CED) is of particular advantage.
See Saito, et al., Cancer Research, vol. 64, p. 2572-2579, 2004;
Mamot, et al., J. Neuro-Oncology, vol. 68, p. 1-9, 2004. The
compositions may also be directly applied to tissue surfaces.
Sustained release, pH dependent release, or other specific chemical
or environmental condition mediated release administration is also
specifically included in the invention, e.g., by such means as
depot injections, or erodible implants. Suitable compositions for
oral administration include solid formulations such as tablets,
lozenges and capsules, which can contain liquids, gels, or powders.
Liquid formulations can include solutions, syrups and suspensions,
which can be used in soft or hard capsules. Such formulations may
include a pharmaceutically acceptable carrier, for example, water,
ethanol, polyethylene glycol, cellulose, or an oil. The formulation
may also include one or more emulsifying agents and/or suspending
agents. Preparation of pharmaceutically acceptable formulations can
be accomplished according to methods known in the art.
Dosage and Administration
[0120] Compositions of the present disclosure may be administered
in a single dose or in multiple doses to achieve an effective
treatment objective. Typically the dosages for the liposome
pharmaceutical composition of the present invention are a
therapeutically effective dose in a range between about 0.005 and
about 500 mg of the therapeutic entity per kilogram of body weight,
most often, between about 0.1 and about 100 mg therapeutic
entity/kg of body weight.
[0121] An anti-PD-1 antibody is administered at a dosage amount of
from 2 mg/kg to 30 mg/kg every two to three weeks; suitably, from 3
mg/kg to 20 mg/kg every two to three weeks; suitably, 5 mg/kg to 10
mg/kg every two to three weeks; suitably, 6 mg/kg every two to
three weeks. In certain embodiments, anti-PD-1 antibody is
administered as above every two weeks. In certain embodiments,
anti-PD-1 antibody is administered as above every three weeks.
[0122] Typically, the liposome pharmaceutical compositions of the
present invention are prepared as a topical or an injectable,
either as a liquid solution or suspension. However, solid forms
suitable for solution in, or suspension in, liquid vehicles prior
to injection can also be prepared. The composition can also be
formulated into an enteric-coated tablet or gel capsule according
to known methods in the art.
[0123] The liposome composition of the present invention can be
administered in any way which is medically acceptable which may
depend on the condition or injury being treated. Possible
administration routes include injections, by parenteral routes such
as intramuscular, subcutaneous, intravenous, intraarterial,
intraperitoneal, intraarticular, intraepidural, intrathecal, or
others, as well as oral, nasal, ophthalmic, rectal, vaginal,
topical, or pulmonary, e.g., by inhalation. For the delivery of
liposomally drugs formulated according to the invention, to tumors
of the central nervous system, a slow, sustained intracranial
infusion of the liposomes directly into the tumor (a
convection-enhanced delivery, or CED) is of particular advantage.
See Saito, et al., Cancer Research, vol. 64, p. 2572-2579, 2004;
Mamot, et al., J. Neuro-Oncology, vol. 68, p. 1-9, 2004. The
compositions may also be directly applied to tissue surfaces.
Sustained release, pH dependent release, or other specific chemical
or environmental condition mediated release administration is also
specifically included in the invention, e.g., by such means as
depot injections, or erodible implants. . The quantity of liposome
pharmaceutical composition necessary to deliver a therapeutically
effective dose can be determined by routine in vitro and in vivo
methods, common in the art of drug testing. See, for example, D. B.
Budman, A. H. Calvert, E. K. Rowinsky (editors). Handbook of
Anticancer Drug Development, LWW, 2003. Therapeutically effective
dosages for various therapeutic entities are well known to those of
skill in the art; and according to the present invention a
therapeutic entity delivered via the pharmaceutical liposome
composition of the present invention provides at least the same, or
2-fold, 4-fold, or 10-fold higher activity than the activity
obtained by administering the same amount of the therapeutic entity
in its routine non-liposome formulation.
[0124] According to the present invention, a desired entity can be
loaded or entrapped into the liposomes by incubating the desired
entity with the liposomes of the present invention in an aqueous
medium at a suitable temperature, e.g., a temperature above the
component lipids' phase transition temperature during loading while
being reduced below the phase transition temperature after loading
the entity. The incubation time is usually based on the nature of
the component lipids, the entity to be loaded into the liposomes,
and the incubation temperature. Typically, the incubation times of
few minutes to several hours are sufficient. Because high
entrapment efficiencies of more than 85%, typically more than 90%,
are achieved, there is usually no need to remove unentrapped
entity. If there is such a need, however, the unentrapped entity
can be removed from the composition by various mean, such as, for
example, size exclusion chromatography, dialysis, ultrafiltration,
adsorption, or precipitation. It was unexpectedly found that
maintaining of the low ionic strength during the incubation of an
entity, such as, in particular, a camptothecin derivative or a
vinca alkaloid derivative, with the liposomes of the present
invention, followed by the increase in ionic strength at the end of
the incubation, results in higher loading efficiency, better
removal of unentrapped drug, and better liposome stability against
aggregation. Typically, the incubation is conducted, e.g., in an
aqueous solution, at the ionic strength of less than that
equivalent to 50 mM NaCl, or more preferably, less than that
equivalent to 30 mM NaCl. Following the incubation, a concentrated
salt, e.g., NaCl, solution may be added to raise the ionic strength
to higher than that of 50 mM NaCl, or more preferably, higher than
that of 100 mM NaCl. Without being bound by a theory, we
hypothesize that the increase of ionic strength aids dissociation
of the entity from the liposome membrane, leaving substantially all
entity encapsulated within the liposomal interior space.
[0125] In general, the entity-to-lipid ratio, e.g., drug load ratio
obtained upon loading an entity depends on the amount of the entity
entrapped inside the liposomes, the concentration of entrapped
substituted ammonium and/or polyanion, e.g., salt, the
physicochemical properties of the entrapped entity and the type of
counter-ion (anion), e.g., polyanion used. Because of high loading
efficiencies achieved in the compositions and/or by the methods of
the present invention, the entity-to-lipid ratio for the entity
entrapped in the liposomes is over 80%, over 90%, and typically
more than 95% of the entity-to-lipid ratio calculated on the basis
of the amount of the entity and the liposome lipid taken into the
loading process (the "input" ratio). Indeed, practically 100%
(quantitative) encapsulation is common. The entity-to lipid ratio
in the liposomes can be characterized in terms of weight ratio
(weight amount of the entity per weight or molar unit of the
liposome lipid) or molar ratio (moles of the entity per weight or
molar unit of the liposome lipid). One unit of the entity-to-lipid
ratio can be converted to other units by a routine calculation, as
exemplified below. The weight ratio of an entity in the liposomes
of the present invention is typically at least 0.05, 0.1, 0.2,
0.35, 0.5, or at least 0.65 mg of the entity per mg of lipid. In
terms of molar ratio, the entity-to-lipid ratio according to the
present invention is at least from about 0.02, to about 5,
preferably at least 0.1 to about 2, and more preferably, from about
0.15 to about 1.5 moles of the drug per mole of the liposome lipid.
In one embodiment, the entity-to-lipid ratio, e.g., drug load ratio
of camptothecin derivatives is at least 0.1, e.g., 0.1 mole of
camptothecin derivative per one mole of liposome lipid, and
preferably at least 0.2. In another embodiment, the entity-to-lipid
ratio, e.g., drug load is at least about 300 mg entity (e.g., vinca
alkaloid or a derivative thereof per mg of liposome-forming lipid.
In yet another embodiment, the entity-to-lipid ratio, e.g., drug
load is at least about 500 mg entity (e.g. camptothecin derivative
or camptothecin prodrug) per mg of liposome-forming lipid.
Surprisingly, the invention afforded stable and close to
quantitative liposomal encapsulation of a camptothecin derivative
drug, e.g., irinotecan, at the drug-to-lipid ratio of over 0.8 mmol
of the entity per 1 g of liposome lipid, over 1.3 mmol of entity
per 1 g of liposome lipid, and even at high as 1.7 mmol entity per
1 g liposome lipid (see Example 74).
[0126] If the liposome comprises a phospholipid, it is convenient
to express the entity content in the units of weight (mass) amount
of the drug per molar unit of the liposome phospholipid, e.g., mg
drug/mmol of phospholipid. However, a person skilled in the art
would appreciate that the drug content can be equivalently
expressed in a manner independent of the presence of phospholipids
in a liposome, and furthermore, can be equivalently expressed in
terms of a molar amount of the drug per unit (mass or molar) of the
liposome lipid content. For example, a liposome containing 3 molar
parts of distearoylphosphatidylcholine (DSPC, molecular weight
790), 2 molar parts of cholesterol (molecular weight 387), and
0.015 molar parts of poly(ethylene glycol)-derivatized
distearoylphosphatidylethanolamine (PEG-DSPE, molecular weight
2750), and containing a drug doxorubicin (molecular weight 543.5)
at the drug/lipid ratio of 150 mg/mmol phospholipid, the same drug
content can be equivalently expressed in terms of mg drug/mg total
lipid as follows:
[0127] (a) Calculate the molar amounts of liposome lipid components
normalized to the molar unit of liposome phospholipids (DSPC and
PEG-DSPE in this example) by dividing the molar quantity of a
component by the total of the molar quantities of the liposome
phospholipids:
DSPC 3/(3+0.015)=0.99502
Cholesterol 2/(3+0.015)=0.66335
PG-DSPE 0.015/(3+0.015)=0.00498
[0128] (b) Calculate the mass amount of total liposome lipid
corresponding to a unit molar amount of liposome phospholipid and
the components molecular weights:
[0129] Total lipid, mg/mmol
phospholipid=0.99502.times.790+0.66335.times.387+0.00498.times.2750=1056-
.48
[0130] (c) Calculate the mass amount of drug per mass unit of total
lipid by dividing the drug content expressed in mass units per
molar unit of phospholipid by the number obtained in step (b):
Doxorubicin, mg/mg total lipid=150/1056.48=0.14198.
[0131] (d) Calculate the molar amount of the drug per unit mass of
total lipid by dividing the number obtained in step (c) by the drug
molecular weight (in this case, 543.5):
Doxorubicin, mmol/g total lipid=0.14198/543.5.times.1000=0.261.
[0132] (e) Calculate the molar part of phospholipids in the
liposome lipid matrix:
[0133] Phospholipid molar part=(total moles of
phospholipids)/(total moles amount of
lipids)=(3+0.015)/(3+2+0.015)=0.6012.
[0134] (f) Calculate the molar ratio of doxorubicin to total
lipid.
[0135] Doxorubicin, mol/mol of total lipid=(Phospholipid molar
part).times.(Doxorubicin, g/mole phospholipid)/(Doxorubicin
molecular weight)=0.6012.times.150/543.5=0.166
[0136] Thus, the relationship between drug-to-lipid and
drug-to-phospholipid ratio expressed in various units is readily
established. As used herein, a `lipid` includes, without
limitation, any membrane-forming components of the liposome
membrane, such as, for example, polymers and/or detergents. See,
for example: U.S. Pat. No. 8,147,867 which is incorporated herein
by reference in its entirety for all purposes.
[0137] Unless otherwise indicated herein, the dose of a MM-398
irinotecan liposome is refers to the equivalent amount of
irinotecan hydrochloride trihydrate. For example, a 120 mg dose of
MM-398 irinotecan liposome contains an amount of irinotecan present
in 120 mg of irinotecan hydrochloride trihydrate. Converting a dose
based on irinotecan hydrochloride trihydrate to a dose based on
irinotecan free base is accomplished by substituting the molecular
weight of irinotecan hydrochloride trihydrate (677.19 g/mole) with
the molecular weight of irinotecan free base (586.68 g/mole), which
results in a conversion factor of 0.866.
[0138] In order that the disclosure described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting this disclosure in any
manner.
Biological Assays
[0139] Synergistic Effect of Top 1 Inhibitors on T-cell mediated
killing of Melanoma 2338 and 2400 Cells. The patient derived
melanoma cell lines 2338 and 2400 were treated with autologous
tumor infiltrating lymphocytes (TILs) at varying effector T cell to
tumor cell (E:T) ratios for 3 h. Cells were then stained for
activated caspase 3, to quantify apoptosis by flow cytometry. 2338
and 2400 cells were treated with the Top1 inhibitor SN38 for 24 h
using a concentration range of 0.125-1.0 uM. Cells were then
stained for activated caspase 3, or drug treated cells were washed
and then incubated with autologous TILs for 3 h.
[0140] Apoptosis was then quantified via a high throughput caspase
3-based cytotoxicity assay. Human melanoma cells were stained with
DDAO dye and either: (i) seeded for 24 h in 96 well plates with 1
uM of each of the 850 compounds in our screen or DMSO as a control,
(ii) seeded for 24h and then incubated with autologous T cells for
3 h, or (iii) seeded for 24 h with 1 uM compound, washed and then
incubated with autologous T cells for 3 h. Cells were then washed,
fixed, permeabilized and stained for activated caspase 3. Flow
cytometry was used to quantify staining as a measure of apoptosis.
Results are given in FIGS. 2A and 2B.
[0141] The data shown in FIGS. 2A and 2B were analyzed in Calcusyn
to compute the Combination Index (CI) of combining SN38 with 2338
and 2400 TILs. The CIs of 2338 and 2400 are represented in the
normalized isobolograms in FIGS. 3A and 3B respectively. Calcusyn
is based on the Chou-Talalay method of quantifying synergy where
synergism is CI<1 (points below the diagonal line), additive
effect is CI=1 (points on the diagonal line), and antagonism is
CI>1 (points above the diagonal line). See, e.g., Chou, T. C.,
"Drug combination studies and their synergy quantification using
the Chou-Talalay method," Cancer research 70, 440-446 (2010).
[0142] In vivo anti-tumor response with the Top1 inhibitor nal-IRI
(MM-398) is significantly higher in comparison to free irinotecan.
In a first experiment, C57BL/6 mice were injected subcutaneously
with 5.times.10.sup.5 MC38/gp100 cells. Mice were treated with 40
mg/kg SN38 (3 times weekly intraperitoneally), 150 ug .alpha.-PD-L1
(mouse PD-L1-PE (clone 10F.9G2) obtained from Bio X Cell, 10
Technology Dr., Suite 2B, West Lebanon, NH 03784-1671 USA) (every 3
days intraperitoneally), or a combination of SN38 and
.alpha.-PD-L1. Control group received phosphate-buffered saline
(PBS) and Rat IgG2B control antibody. Mice were treated for 3
weeks. Results are shown in FIG. 4A, which shows that the
combination of SN38 and .alpha.-PD-L1 trended better than SN38 or
.alpha.-PD-L1 alone, but not significantly. In a second experiment,
C57BL/6 mice were injected subcutaneously with 5.times.10.sup.5
MC38/gp100 cells. Three days later when tumors were palpable, mice
were randomized into treatment groups (n=5). Beginning on day 3,
mice received nal-IRI (MM-398, intravenously), free irinotecan
(intraperitoneally), or PBS (intravenously) as the vehicle, once
weekly for 3 weeks. Results are shown in FIG. 4B, which
demonstrates that MM-398 was better at all doses than free
irinotecan, and was increasingly efficacious as the dose increased
(achieving significance at 40 mg/kg.
[0143] In vivo anti-tumor response and survival are increased when
nanoliposomal irinotecan, nal-IRI (MM-398) is combined with
.alpha.-PD-L1 antibody. In a first experiment, C57BL/6 mice were
injected s.c. with 5.times.10.sup.5 MC38/gp100 cells. Three days
later when tumors were palpable, mice were randomized into
treatment groups (n=5) receiving nal-IRI (40 mg/kg), .alpha.-PD-L1
antibody (mouse PD-L1-PE (clone 10F.9G2) obtained from Bio X Cell,
10 Technology Dr., Suite 2B, West Lebanon, N.H. 03784-1671 USA)
(150 ug/mouse), or both nal-IRI and .alpha.-PD-L1 antibody. Vehicle
control group received PBS and isotype-matched control antibody Rat
IgG2b (150 ug). Beginning on day 3, mice received once weekly doses
of nal-IRI and antibody was administered every 3 days. FIG. 5A
shows tumor volume up to day 21; FIG. 2B shows tumor survival data
for mice treated with MM-398 or .alpha.-PD-L1 antibody alone, or a
combination of both agents.
[0144] Gene expression changes in antigen processing genes after
Top1 inhibition. RNA was isolated from patient derived melanoma
cell lines treated with SN38 or DMSO as a control. The heatmap in
FIG. 6A represents the differential expression of a subset of genes
involved in antigen presentation. FIG. 6A is a subset of the data
of the microarray analysis that was performed on SN38-treated tumor
cells described in Example 5. This subset of the data focused on
the differential expression changes of genes involved in antigen
processing and presentation in tumor cells. Antigen processing and
presentation is a fundamental step in the cancer immunity cycle
that allows for the recognition of tumor cells by cytolytic T
cells. We observed significant upregulation in the expression of
MHC Class I (HLA-A, B, C) and in Beta-2-microglobulin (B2M) and the
transporter proteins TAP and TAP binding protein (TAPBP), all
crucial for the antigen processing and presentation pathway. This
data suggests that one way by which Top1 inhibitor-treatment of
melanoma tumor cells may improve T cell mediated killing is by
increasing antigen processing and presentation, which may allow for
increased recognition and targeting by T cells, and subsequent
greater induction of tumor cell killing.
[0145] Referring to FIGS. 6B and 14: the heatmap in FIG. 6B
represents a subset of genes differentially expressed after Top1
inhibition from microarray analysis. The data shown represents a
portion of the gene expression analysis which was described in
Example 5. This portion of the data focuses on the differential
expression of some genes related to p53 signaling. In particular,
we have chosen to focus on TP53INP1 (or Teap), which is a p53
regulatory gene shown to be involved in directing an apoptotic
response in tumor cells (Gironella et al., Natl Acad Sci USA 2007;
Tomasini et al., J Biol Chem 2001). We observed a significant
upregulation in the expression of Teap with SN38 treatment in
melanoma. This phenotype was also validated by quantitative real
time PCR (qRT-PCR) performed on a number of melanoma
patient-derived tumor cell lines treated with 2 different Top1
inhibitors (Top 1 inh. 1=SN38, Top1 inh. 2=Topotecan).
[0146] Unless otherwise indicated, the nano-liposomal irinotecan
material used where indicated by corresponding the data in the
Figures comprises irinotecan sucrose octasulfate encapsulated in a
liposome as depicted in FIG. 7. FIG. 7 shows Nano-liposomal
irinotecan (nal-IRI), MM-398. MM-398 irinotecan sucrose octasulfate
salt liposome injection may also be referred to as irinotecan HCl
liposome injection because irinotecan HCl (trihydrate) is the
active pharmaceutical ingredient that is used to load irinotecan
into liposomes containing triethylammonium sucrose octasulfate to
prepare MM-398 liposomes. This nomenclature may be used even though
the hydrochloride ion of the irinotecan HCl reacts with the
triethylammonium ion of the triethylammonium sucrose octasulfate to
yield triethylammonium chloride (triethylamine hydrochloride),
leaving irinotecan sucrose octasulfate salt as the entrapped
pharmaceutical agent within the MM-398 liposomes. Further details
about irinotecan liposomes are provided in the publication
WO2013/188586, filed Jun. 12, 2013 (incorporated by reference
herein in its entirety).
[0147] The liposomal irinotecan comprises liposomes having a
unilamellar lipid bilayer vesicle, approximately 110 nm in
diameter, which encapsulates an aqueous space containing irinotecan
in a gelated or precipitated state as the sucrose octasulfate salt;
wherein the vesicle is composed of
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 6.81 mg/mL,
cholesterol 2.22 mg/mL, and methoxy-terminated polyethylene glycol
(MW 2000)-distearoylphosphatidyl ethanolamine (MPEG-2000-DSPE) 0.12
mg/mL. Each mL can also contain
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) as
a buffer 4.05 mg/mL and sodium chloride as an isotonicity reagent
8.42 mg/mL.
[0148] As provided herein, irinotecan can be administered in a
stable liposomal formulation as irinotecan sucrose sulfate liposome
injection (otherwise termed "irinotecan sucrose octasulfate salt
liposome injection" or "irinotecan sucrosofate liposome
injection"), the formulation referred to herein as "MM-398" (also
known as PEP02, see U.S. Pat. No. 8,147,867). MM-398 may be
provided as a sterile, injectable parenteral liquid for intravenous
injection. The required amount of MM-398 may be diluted, e.g. in
500mL of 5% dextrose injection USP and infused over a 90 minute
period.
[0149] An MM-398 liposome is a unilamellar lipid bilayer vesicle of
approximately 80-140 nm in diameter that encapsulates an aqueous
space which contains irinotecan complexed in a gelated or
precipitated state as a salt with sucrose octasulfate. The lipid
membrane of the liposome is composed of phosphatidylcholine,
cholesterol, and a polyethyleneglycol-derivatized
phosphatidyl-ethanolamine in the amount of approximately one
polyethyleneglycol (PEG) molecule for 200 phospholipid
molecules.
[0150] This stable liposomal formulation of irinotecan has several
attributes that may provide an improved therapeutic index. The
controlled and sustained release improves activity of this
schedule-dependent drug by increasing duration of exposure of tumor
tissue to drug, an attribute that allows it to be present in a
higher proportion of cells during the S-phase of the cell cycle,
when DNA unwinding is required as a preliminary step in the DNA
replication process. The long circulating pharmacokinetics and high
intravascular drug retention in the liposomes can promote an
enhanced permeability and retention (EPR) effect. EPR allows for
deposition of the liposomes at sites, such as malignant tumors,
where the normal integrity of the vasculature (capillaries in
particular) is compromised resulting in leakage out of the
capillary lumen of particulates such as liposomes. EPR may thus
promote site-specific drug delivery of liposomes to solid tumors.
EPR of MM-398 may result in a subsequent depot effect, where
liposomes accumulate in tumor associated macrophages (TAMs), which
metabolize irinotecan, converting it locally to the substantially
more cytotoxic SN-38. This local bioactivation is believed to
result in reduced drug exposure at potential sites of toxicity and
increased exposure at cancer cells within the tumor.
[0151] Irinotecan is converted to SN-38 within the body upon
release from a MM-398 liposome. The metabolic transformation of
MM-398 to SN-38 (e.g. in plasma) includes two steps: (1) the
release of irinotecan from the liposome and (2) the conversion of
free irinotecan to SN-38. While not intending to be limited by
theory, it is believed that once irinotecan leaves the liposomes,
it is catabolized by the same metabolic pathways as conventional
(free) irinotecan. Therefore the genetic polymorphisms in humans
predictive for the toxicity and efficacy of irinotecan and those of
MM-398 can be considered similar. Nonetheless, in the MM-398
formulation compared to free irinotecan, the deficient genetic
polymorphisms may show less association with severe adverse events
and/or efficacy.
[0152] Liposomal irinotecan can be administered intravenously,
either alone or in combination with 5-fluorouracil (5-FU) and/or
leucovorin, prior to administration of an anti-PDL-1 antibody. In
one embodiment, liposomal irinotecan is administered (alone or in
combination with or prior to 5-FU and leucovorin) and prior to a
checkpoint inhibitory antibody (e.g., an antibody binding to
anti-PD 1). In another embodiment, the liposomal irinotecan is
administered as part of a treatment cycle comprising the
administration of a therapeutically effective dose of MM-398,
followed by administration of leucovorin and 5-FU as a series of
infusions over a total time period of about 48 hours. The liposomal
irinotecan treatment cycle can be followed by administration of the
checkpoint inhibitory antibody. For example, liposomal irinotecan
can be administered intravenously over 90 minutes, leucovorin can
be administered over 30 minutes, and 5-FU can be administered
intravenously over 46 hours. Leucovorin can administered
intravenously over 30 minutes, as a composition comprising about
200 mg/m.sup.2 of the active (1) form or as a composition
comprising 400 mg/m.sup.2 of the (l+d) racemic form. In various
embodiments the liposomal irinotecan is MM-398.
[0153] One method of treating cancer comprises the administration
of 60-120 mg/m.sup.2 of MM-398 liposomal irinotecan (i.e., a dose
of MM-398 containing the amount of irinotecan corresponding to
60-120 mg/m.sup.2 of irinotecan hydrochloride trihydrate) having a
half-life of at least about 24 hours, in combination with the
administration of 3 mg/kg of checkpoint inhibitor antibody that
binds to anti-PD1. For example, the MM-398 liposomal irinotecan can
be administered at a dose of 60, 80 or 120 mg/m.sup.2 every 2
weeks. The antibody can be nivolumab administered over 60 minutes
every 2 weeks. Optionally, the method further includes
administration of 5-fluorouracil (e.g., 2,400 mg/m.sup.2) and
leucovorin (e.g., 200 mg/m.sup.2 of the 1-form or 400 mg/m.sup.2 of
the l+d racemic form) in combination with the MM-398, and prior to
administration of the checkpoint inhibitor antibody. When
administered once every two weeks at 80 mg/m.sup.2 (hydrochloride
trihydrate basis, equivalent to 70 mg/m.sup.2 free base), MM-398
has the mean (+/- standard deviation) total irinotecan and total
SN-38 in Table 1 below.
TABLE-US-00001 TABLE 1 Total Irinotecan Total SN-38 C.sub.max
AUC.sub.0-.infin. t.sub.1/2 CL V.sub.d C.sub.max AUC.sub.0-.infin.
t.sub.1/2 Dose [.mu.g/mL] [h .mu.g/mL] [h] [L/h] [L] [ng/mL] [h
ng/mL] [h] (mg/m.sup.2) (n = 25) (n = 23) (n = 23) (n = 23) (n =
23) (n = 25) (n = 13) (n = 13) 70 37.2 1364 25.8 0.20 4.1 5.4 620
67.8 (8.8) (1048) (15.7) (0.17) (1.5) (3.4) (329) (44.5) C.sub.max:
Maximum plasma concentration AUC.sub.0-.infin.: Area under the
plasma concentration curve extrapolated to time infinity t.sub.1/2:
Terminal elimination half-life CL: Clearance V.sub.d: Volume of
distribution
[0154] In a particular example, a method of treating cancer
comprises administering by infusion to the patient in need thereof
once every three weeks (a) a liposomal irinotecan treatment cycle
comprising or consisting of a dose of 120 mg/m.sup.2 MM-398 over 90
minutes, followed by the leucovorin over 30 minutes, followed by
the 5-fluorouracil over 46 hours; followed by (b) a checkpoint
antibody treatment cycle comprising an antibody that binds to
anti-PD1 (e.g., 3 mg/kg of nivolumab administered over 60 minutes).
A therapeutically effective time period can be selected between
administration of the liposomal irinotecan treatment cycle and the
checkpoint antibody treatment cycle. When administered once every
three weeks at 120 mg/m.sup.2, MM-398 has an AUC.sub.0-1 of total
irinotecan in blood that is 1,652 hrug/ml (120 mg/m.sup.2) and
SN38, the active metabolite, is 476 hrng/ml, and T.sub.1/2 of total
irinotecan in blood is 21.2 h and SN38 is 88.8 h.
[0155] In another particular example, a method of treating cancer
comprises administering by infusion to the patient in need thereof
(a) a liposomal irinotecan treatment cycle comprising or consisting
of a dose of 80 mg/m.sup.2 MM-398 over 90 minutes, followed by the
leucovorin over 30 minutes, followed by the 5-fluorouracil over 46
hours; followed by (b) a checkpoint antibody treatment cycle
comprising an antibody that binds to anti-PD1 (e.g., 3 mg/kg of
nivolumab administered over 60 minutes). A therapeutically
effective time period can be selected between administration of the
liposomal irinotecan treatment cycle and the checkpoint antibody
treatment cycle.
[0156] In one particular example, a method of treating cancer
comprises administering by infusion to the patient in need thereof
(a) a liposomal irinotecan treatment cycle comprising or consisting
of a dose of 60 mg/m.sup.2 MM-398 over 90 minutes, followed by the
leucovorin over 30 minutes, followed by the 5-fluorouracil over 46
hours; followed by (b) a checkpoint antibody treatment cycle
comprising an antibody that binds to anti-PD1 (e.g., 3 mg/kg of
nivolumab administered over 60 minutes). A therapeutically
effective time period can be selected between administration of the
liposomal irinotecan treatment cycle and the checkpoint antibody
treatment cycle
[0157] One method of treating cancer comprises the administration
of 60-120 mg/m.sup.2 of liposomal irinotecan octasulfate
(containing an amount of irinotecan equivalent to 60-120 mg/m.sup.2
of irinotecan hydrochloride trihydrate) having an irinotecan
half-life of at least about 24 hours in combination with the
administration of 3 mg/kg of a checkpoint inhibitor antibody such
as nivolumab. For example, the MM-398 can be administered at a dose
of 60, 80 or 120 mg/m.sup.2 every 2 weeks. The nivolumab can be
administered over 60 minutes every 2 weeks.
Other Embodiments
[0158] The detailed description set-forth above is provided to aid
those skilled in the art in practicing the present disclosure.
However, the disclosure described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed
because these embodiments are intended as illustration of several
aspects of the disclosure. Any equivalent embodiments are intended
to be within the scope of this disclosure. Indeed, various
modifications of the disclosure in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description, which do not depart from the spirit
or scope of the present inventive discovery. Such modifications are
also intended to fall within the scope of the appended claims.
[0159] Also provided are embodiments wherein any of embodiment
above may be combined with any one or more of these embodiments,
provided the combination is not mutually exclusive. As used herein,
two embodiments are "mutually exclusive" when one is defined to be
something which cannot overlap with the other.
[0160] In some embodiments, a method of killing cancer cells in a
biological sample can comprise contacting the biological sample
with an effective amount of a Topoisomerase I inhibitor and an
.alpha.-PD-L1 antibody. These or other embodiments can be
characterized by one or more of the following, alone or in any
combination: [0161] the .alpha.-PD-L1 or .alpha.-PD-1 antibody can
be a humanized monoclonal antibody; [0162] the subject can be a
human; [0163] the cancer can be chosen from skin cancer, pancreatic
cancer, or a variant thereof; [0164] the administration of the
Topoisomerase I inhibitor and .alpha.-PD-L1 or .alpha.-PD-1
antibody can be sequential; [0165] the administration of the
Topoisomerase I inhibitor occurs before administration of the
.alpha.-PD-L1 or .alpha.-PD-1 antibody; [0166] the administration
of the .alpha.-PD-L1 or .alpha.-PD-1 antibody can occur before
administration of the Topoisomerase I inhibitor, or the
administration of the .alpha.-PD-L1 or .alpha.-PD-1 antibody and
Topoisomerase I inhibitor can be essentially simultaneous; [0167]
the .alpha.-PD-L1 antibody can be chosen from nivolumab, and
pembrolizumab; [0168] the Topoisomerase I inhibitor is chosen from
irinotecan, topotecan, camptothecin and lamellarin D, or liposomal
formulations thereof, or preferably the Topoisomerase I inhibitor
is a liposomal irinotecan, or the irinotecan is provided in a
composition comprising liposomes in an aqueous medium, the
liposomes having an interior aqueous space separated from the
aqueous medium by a membrane, the membrane comprising lipids, the
lipids comprising an uncharged lipid component and a neutral
phospholipid, with, entrapped inside the liposomes: irinotecan and
sucrose octasulfate, or irinotecan and sucrose octasulfate and a
substituted ammonium compound, wherein, when administered into the
bloodstream of a mammal, said irinotecan has a half-release time
from said liposomes of at least 24 hours and the irinotecan
entrapped inside the liposomes is at a concentration that exceeds
the irinotecan concentration in the aqueous medium; [0169] the
method comprises at least one cycle, wherein the liposomal
irinotecan is administered on day 1 of a cycle at a dose of between
about 60 and about 180 mg/m.sup.2, except if the patient is
homozygous for the UGT1A1*28 allele, wherein the liposomal
irinotecan is administered on day 1 of cycle 1 at a dose of between
about 40 and about 120 mg/m.sup.2, wherein the cycle is a period of
2 to 3 weeks; [0170] the topoisomerase I inhibitor is liposomal
irinotecan administered on day 1 of a cycle at a dose of between
about 90 and about 150 mg/m.sup.2, except if the patient is
homozygous for the UGT1A1*28 allele, wherein the liposomal
irinotecan is administered on day 1 of cycle 1 at a dose of between
about 60 and about 100 mg/m.sup.2. [0171] the method comprises at
least one cycle, wherein the liposomal irinotecan is administered
on day 1 of a cycle at a dose of 120 mg/m.sup.2, except if the
patient is homozygous for the UGT1A1*28 allele, wherein the
liposomal irinotecan is administered on day 1 of cycle 1 at a dose
of 80 mg/m.sup.2; [0172] the cycle is a period of 2 weeks; [0173]
the cycle is a period of 3 weeks; [0174] the method further
comprises administering another therapeutic agent, the therapeutic
agent is optionally chosen from a taxane, inhibitor of bcr-abl,
inhibitor of EGFR, DNA damaging agent, and antimetabolite thereof,
or the therapeutic agent is chosen from aminoglutethimide,
amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin,
buserelin, busulfan, campothecin, capecitabine, carboplatin,
carmustine, chlorambucil, chloroquine, cisplatin, cladribine,
clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine,
dacarbazine, dactinomycin, daunorubicin, demethoxyviridin,
dichloroacetate, dienestrol, diethylstilbestrol, docetaxel,
doxorubicin, epirubicin, estradiol, estramustine, etoposide,
everolimus, exemestane, filgrastim, fludarabine, fludrocortisone,
fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein,
goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib,
interferon, letrozole, leucovorin, leuprolide, levamisole,
lomustine, lonidamine, mechlorethamine, medroxyprogesterone,
megestrol, melphalan, mercaptopurine, mesna, metformin,
methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide,
nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate,
pentostatin, perifosine, plicamycin, porfimer, procarbazine,
raltitrexed, rituximab, sorafenib, streptozocin, sunitinib,
suramin, tamoxifen, temozolomide, temsirolimus, teniposide,
testosterone, thioguanine, thiotepa, titanocene dichloride,
topotecan, trastuzumab, tretinoin, vinblastine, vincristine,
vindesine, and vinorelbine; [0175] the method further comprises
administering non-chemical methods of cancer treatment; [0176] the
method further comprises administering radiation therapy; and/or
[0177] the method further comprises administering surgery,
thermoablation, focused ultrasound therapy, cryotherapy, or any
combination thereof.
[0178] In some embodiments, provided is a composition comprising an
effective amount of a Topoisomerase I inhibitor and an
.alpha.-PD-L1 or .alpha.-PD-1 antibody, useful in human therapy.
The composition can comprise an effective amount of a Topoisomerase
I inhibitor and an .alpha.-PD-L1 antibody for use in treating
cancer. In some embodiments, use of a composition can be for the
manufacture of a medicament to treat cancer.
[0179] In some embodiments, provided is a kit for treating a cancer
in a subject in need thereof, comprising: a Topoisomerase I
inhibitor and an .alpha.-PD-L1 antibody; and written instructions
for administering to the subject an effective amount of a
Topoisomerase I inhibitor and an .alpha.-PD-L1 antibody to treat
the cancer.
[0180] In some embodiments, provided is a method of treating cancer
comprises administering to a patient in need thereof a
therapeutically effective amount of a MM398 irinotecan liposome in
combination with the administration of a therapeutically effective
amount of a PD-L1blocking antibody. The MM398 irinotecan liposome
can be administered in a dose providing an amount of irinotecan
equivalent to 60-120 mg/m.sup.2 of irinotecan hydrochloride
trihydrate. The MM-398 irinotecan liposome can be administered in a
dose providing an amount of irinotecan equivalent to 60 mg/m.sup.2
of irinotecan hydrochloride trihydrate. The MM-398 irinotecan
liposome can be administered in a dose providing an amount of
irinotecan equivalent to 80 mg/m.sup.2 of irinotecan hydrochloride
trihydrate. The MM-398 irinotecan liposome can be administered in a
dose providing an amount of irinotecan equivalent to 120 mg/m.sup.2
of irinotecan hydrochloride trihydrate. The MM-398 liposome can be
administered as an infusion over 90 minutes. The administration of
the MM-398 irinotecan liposome can be followed by the additional
administration of leucovorin and 5-fluorouracil. The leucovorin can
be administered as 200 mg/m.sup.2 of the (1) form of leucovorin.
The leucovorin can be administered as 400 mg/m.sup.2 of the (l+d)
racemic form of leucovorin. The 5-fluorouracil can be administered
as a dose of 1,800-2,400 mg/m.sup.2. The MM-398 can be administered
at a dose of 60 mg/m.sup.2 and the 5-fluorouracil is administered
as a dose of 1,800 mg/m.sup.2. The MM-398 can be administered at a
dose of 80 mg/m.sup.2 and the 5-fluorouracil is administered as a
dose of 2,400 mg/m.sup.2. The therapeutically effective amount of a
PD-L1blocking antibody can be administered after the MM-398. The
PD-L1 blocking antibody can be nivolumab. The PD-L1 blocking
antibody can be administered at a dose of 3mg/kg. The PD-L1
blocking antibody can be administered by infusion over 60 minutes.
The PD-L1 blocking antibody can be administered every 2 weeks. The
MM-398 irinotecan liposome can be administered following the
administration of a therapeutically effective amount of a PD-L1
blocking antibody. The cancer can be melanoma. The cancer can be
metastatic melanoma. The patient can be previously been treated
with nivolumab prior to the administration of the MM-398 irinotecan
liposome.
EXAMPLES
Example 1: High Throughput Cytotoxicity Assay
[0181] In one embodiment, a screening approach is disclosed for
assaying T-cell mediated cytotoxicity. Human melanoma cancer cell
lines (BRAF/NRAS/CKIT/NF1 wild type) were incubated with 1
micromolar concentration of various test compounds for 24 hours.
The human melanoma cells were stained with the cell tracker dye
DDAO (APC channel) and either: (i) seeded for 24 h in 96 well
plates with 1 uM of each of the 850 compounds in our screen or DMSO
as a control, (ii) seeded for 24 h and then incubated with
autologous T cells for 3 h, or (iii) seeded for 24 h with 1 uM
compound, washed and then incubated with autologous T cells for 3
h. Cells were then washed, fixed, permeabilized and stained with a
PE-conjugated antibody for activated caspase 3. Flow cytometry was
used to quantify staining as a measure of apoptosis. Cells in the
indicated gate would be positive for both DDAO (APC) and activated
caspase 3 (PE), and were quantified as a percentage of the total
number of DDAO-positive tumor cells.
[0182] The flow cytometry analysis of intracellular staining for
activated caspase 3 is shown in FIG. 1A. FIG. 1A depicts data
obtained from a flow cytometry T cell cytotoxicity assay for high
throughput screen. FIG. 1B depicts the methodology of the Flow
cytometry based T cell cytotoxicity assay for high throughput
screen. The dot plots for gating and flow cytometric analysis are
depicted on the right. Briefly, patient derived melanoma tumor
cells (stained with a far-red cell tracker dye), are incubated with
reactive autologous T cells, followed by intracellular staining for
active caspase 3. The level of cytotoxicity is measured by the
percentage of active caspase 3 positive tumor cells (PE-conjugated
caspase 3 antibody).
Example 2: Topoisomerase 1 Inhibitor Enhances T Cell Mediated Tumor
Killing In Vitro in Patient Derived Melanoma Cell lines 2338 and
2400
[0183] In another embodiment, certain topoisomerase I inhibitors
are identified as enhancers of T cell mediated immune-therapy,
including therapeutic combinations that can provide a synergistic
improvement of CTL-mediated killing in vitro. Studies were
conducted with additional patient-derived melanoma cell lines with
NRAS or BRAF mutations, which also showed enhanced T cell mediated
tumor killing.
[0184] Building on the observation from the high throughput assay
where Top1 inhibitors were identified as hits, we further assessed
the effect of Top1 inhibitors on T cell mediated killing of a
number of melanoma patient-derived cell lines in vitro. In the
experiments shown here, melanoma patient-derived cell lines 2338
and 2400, were treated with SN38, the active metabolite of the Top1
inhibitor irinotecan, for 24 h at a concentration of 1 uM. DMSO was
used as a solvent control. Drug-treated cells were then processed
as outlined in the cytotoxicity assay. Briefly, SN38-treated cells
were then processed for flow cytometry analysis, or incubated with
2338 or 2400 autologous TILs for 3 h at an effector to target cell
ratio (E:T) of 4:1 for 2338 and 10:1 for 2400. Flow cytometry
analysis for activated caspase 3 was used to quantify the apoptotic
effect of Top1 inhibitor or TIL alone, as well as the combination
of Top1 inhibitor and TIL. The normalized isobolograms shown depict
the CI for the combined effect of SN38 and TIL on apoptosis in
melanoma tumor cells. CalcuSyn was used to compute the combination
indices (CI) for the effect of SN38 and TIL. CI less than 1
indicate synergy between the 2 agents. CI greater than 1 would
indicate antagonism, while CI equal to 1 indicate an additive
effect. (Note: the data shown here is a subset of the data shown in
FIGS. 2A, 2B and 3A of the patent application draft which shows the
full experiment conducted with a concentration range of SN38 from
0.125-1 uM, and E:T ratios of 1-4:1 (2338) and 1-10:1 (2400).
[0185] FIGS. 2A, 2B, 9A, 9B, 3A and 3B depict the synergistic
effect of Top 1 inhibitors (TILs) on T-cell mediated killing of
melanoma cells from patient derived melanoma cell lines 2338 (FIG.
2A, top) and 2400 (FIG. 2B, bottom) by treatment with treated with
autologous TILs at varying effector T cell to tumor cell (E:T)
ratios for 3 hours, as measured by percent activated caspase 3. In
each of FIGS. 2A or 9A and 2B or 9B, cells in the leftmost group of
three bars was not treated with a TIL. In each of the rightmost
four groups of three bars, cells were treated at the given
concentrations of TIL and no effector T-cells (right), effector T
cells in a 2:1 ration with tumor cells, or effector T cells in a
4:1 ratio with tumor cells. The patient derived melanoma cell lines
2338 (NRAS Q61R) and 2400 (BRAF V600E) were treated with autologous
TILs at varying effector T cell to tumor cell (E:T) ratios for 3
hours. Cells were then stained for activated caspase 3, to quantify
apoptosis by flow cytometry. The 2338 and 2400 cells were treated
with the Top1 inhibitor SN38 for 24 hours using a concentration
range of 0.125 to 1.0 micromolar. Cells were then stained for
activated caspase 3, or drug treated cells were washed and then
incubated with autologous TILs for 3 hours. Apoptosis was then
quantified as described.
[0186] Although we observed a good treatment effect with the
combination of SN38 and anti-PD-L1, we wanted to find a Top1
inhibitor with more favorable chemical properties (e.g.: stability,
solubility, ease of use of in vivo studies), to use in combination
with anti-PD-L. We therefore tested the anti-tumor activity of
MM-398, a nano-liposomal formulation of irinotecan (nal-IRI), and
compared it to the anti-tumor activity of Free Irinotecan to
determine if this would be a suitable Top1 inhibitor to be used for
further pre-clinical testing in our model system. C57BL/6 mice were
inoculated with 500K mc38/gp100 tumor cells. 3 days later when
tumors were palpable, mice were randomized into 1 of 5 experimental
groups: (i) PBS-200 ul ip once a week, (ii) Free Irinotecan-50
mg/kg ip once a week, or MM-398 at (iii) 10 mg/kg, (iv) 20 mg/kg,
or (v) 40 mg/kg iv once a week for 4 doses. From this experiment,
we chose to proceed with using an MM-398 dose of 20 or 40 mg/kg for
in vivo assessment in combination studies.
[0187] Using the software program CalcuSyn, we determined that
Topoisomerase 1 (Top1) inhibition synergistically improves ability
of T cells to kill tumor cells. The data shown in FIGS. 3A and 3B
were analyzed in CalcuSyn to compute the Combination Index (CI) of
combining SN38 with 2338 and 2400 TILs. The CI are represented in
the normalized isobologram above. CalcuSyn is based on the
Chou-Talalay method of quantifying synergy where synergism is
CI<1 (points below the line), additive effect is CI=1 (points on
the line), and antagonism is CI>1 (points above the line). FIGS.
3A and 3B depict the combination Index of the Top1 inhibitor SN38
and T cell cytotoxicity. The data shown in FIGS. 2A and 2B were
analyzed in Calcusyn to compute the Combination index (CI) of
combining SN38 with 2338 and 2400 TILs. The CI are represented in
the normalized isobologram above. Calcusyn is based on the
Chou-Talalay method of quantifying synergy where synergism is
CI<1 (points below the red line), additive effect is CI=1
(points on the red line), and antagonism is CI>1 (points above
the red line).
[0188] The level of cytotoxicity induced by the drug alone in
comparison to the combination of the drug and T cells was evaluated
and used to compute a comboscore to identify hits from the screen.
FIG. 8A is the formula developed to calculate Comboscores, which
was used as an analytical tool to initially narrow down the number
of hit compounds from the HTPS.
[0189] The Tableau plot displays data obtained from the previously
described high throughput cytotoxicity assay performed on the
patient-derived melanoma cell line 2549, with its autologous
2549TILs (tumor infiltrating lymphocytes or T cells). The apoptosis
induced by the drug alone (indicated as percentage of caspase 3
positive tumor cells on the y axis) is graphed versus the apoptosis
induced by the combination of the T cells and the drug (indicated
as the percentage of caspase 3 positive tumor cells on the x axis).
Hits from the screen were identified based on the computed
comboscore, which takes into account the level of killing induced
by the combination of drug and T cells in comparison to the level
of killing induced by either single agent. Drugs that improved T
cell killing would generate a high comboscore (>1.5) and drugs
that had no effect or a negative effect on T cell killing would
generate a low comboscore (<1). The 3 Top1 inhibitors identified
as hits from the screen are indicated as Top1 inhibitor 1, 2 and 3,
and are: camptothecin, topotecan, and irinotecan, and were shown to
increase T cell mediated killing of melanoma tumor cells.
[0190] FIG. 8C is a scatter plot showing that topoisomerase 1
inhibitors can enhance T cell mediated killing of melanoma cancer
cells. Referring to FIG. 8B, topotecan, irinotecan and
camptothecin, all inhibitors of Top1 were determined to have high
Comboscores, indicating that pre-treatment with these drugs caused
more melanoma cells to be killed by T cells, than if they were only
exposed to T cells or drug alone. Referring to the scatter plot
graph of FIG. 8C, the data points show the Comboscore calculated
according to the formula in FIG. 8A, ranging from low (0.5)
indicating low to minimal T cell killing to high (1.5) indicating
high T cell killing. The observed hits were minimally cytotoxic
alone and showed synergistic T cell-mediated killing of tumor
cells. Camptothecin-derived inhibitors of topoisomerase 1 were
identified as top compounds from the screen.
Example 3: Combination of SN38 and Different Immune Modulatory
Antibodies In Vivo
[0191] In another embodiment, certain topoisomerase I inhibitors
are identified as enhancers of T cell mediated immune-therapy,
including therapeutic combinations that can provide a synergistic
improvement of CTL-mediated killing in vitro. A series of animal
model xenograft tests were performed to demonstrate the anti-tumor
activity of combinations of Top1 inhibitors and a variety of immune
modulatory antibodies.
[0192] Having demonstrated in vitro that SN38 could enhance T cell
mediated killing of tumor cells, we next investigated the effect of
SN38 on the anti-tumor response to different T cell based
immunotherapies using a pre-clinical mouse model. In this
experiment, C57BL/6 mice were inoculated with 500K mc38/gp100 cells
sub-cutaneously. 3 days after tumor inoculation, mice were
randomized and treated with: (i) vehicle+isotype-matched control
antibody (IgG 2B-clone LTF-2), (ii) SN38-40mg/kg ip 3 times per
week, (iii) anti-PD-L1-150 ug ip every 3 days (clone 10F.9G2), (iv)
anti-41BB-350 ug every 3 days (clone LOB12.3), (v) anti-CTLA4-100
ug every 3 days (clone 9H10), (vi) anti-OX40-250 ug every 3 days
(clone OX-86), combination of SN38 and (vii) anti-PD-L1, (viii)
anti-41BB, (ix) anti-CTLA4, (x) anti-OX40, (xi) anti-PD-L1 and
--CTLA4, or a combination of (xii) anti-PD-L1 and anti-CTLA4. The
data shows tumor volume over time. FIG. 10 is a graph showing the
tumor volume measured over about three weeks, after treatment with
the Top1 inhibitor SN38, an anti-anti PD-L1 antibody, an
anti-alpho-CTLA4 antibody, anti-anti ox40 antibody, an anti-41BB
antibody, and various combinations thereof.
[0193] The sequences of the antibodies are provided in the Table 2
below.
TABLE-US-00002 TABLE 2 Antibody Antibody Sequence (literature
reference) .alpha.-PD-L1 10F.9G2, Bioxcell .alpha. -41BB LOB12.3,
Bioxcell .alpha. -CTLA4 9H10, Bioxcell .alpha. -OX40 OX-86,
Bioxcell
[0194] FIGS. 11A-11D are graphs of measured tumor volume over time
after administration of certain combinations of the Top1 inhibitor
SN38 and various antibodies against anti-41BB, anti-CTLA4,
anti-0X40 and anti-PD-L1 (as described in the Table 2 of Example 3
herein). The data represented in FIGS. 11A-11D were pulled out of
the experiment described on FIG. 10, to show the tumor volume over
time of the different combination groups in comparison to the
single agent and control-treated groups. As shown, no increase in
tumor control was observed in tumor-bearing mice treated with a
combination of SN38 and anti-41BB, or anti-CTLA4, or anti-OX40, in
comparison to tumor-bearing mice treated with SN38 alone or the
mentioned immunotherapy alone.
[0195] Having demonstrated in vitro that SN38 could enhance T cell
mediated killing of tumor cells, we next investigated the effect of
SN38 on the anti-tumor response to different T cell based
immunotherapy using a pre-clinical mouse model. In this experiment,
C57BL/6 mice were inoculated with 500K mc38/gp100 cells
sub-cutaneously. 7 days after tumor inoculation, mice were
randomized and treated with: (i) vehicle+isotype-matched control
antibody (IgG 2B-clone LTF-2), (ii) SN38-40 mg/kg ip 3 times per
week, (iii) anti-PD-L1-150 ug (clone 10F.9G2), or (iv) combination
of SN38 and anti-PD-L1. The data shows tumor volume over time. From
this experiment, we saw no overall significance in the difference
in tumor control between the different treatment groups, suggesting
that further optimizations were required. Mc38/gp100 is an
aggressive tumor model, which grows rapidly and is prone to
ulceration. We determined that randomization and treatment can be
performed 3 days after tumor inoculation instead of the delayed
time point of 7 days after inoculation. Therefore, for proceeding
in vivo experiments where tumor volume and survival are monitored,
randomization and treatment are started 3 days after tumor
inoculation when tumors are first palpable. This allows us to have
a therapeutic window in which to work and determine the anti-tumor
effect of the each agent singly or in combination. FIG. 4A is a
graph of the measured tumor volume over time after administration
of certain combinations of the Top1 inhibitor SN38 and the
anti-PD-L1 antibody described in the Table 2 in Example 3
herein.
[0196] Increased tumor control in animal xenograft models was
observed when liposomal irinotecan was administered in combination
with certain immune modulatory antibodies. FIG. 4B is a graph of
the measured tumor volume over time after administration of various
concentrations of liposomal irinotecan (MM-398).
[0197] FIG. 5C is a schematic of the mouse xenograft experiment
performed to obtain the data in FIGS. 5A and 5B. C57BL/6 mice were
inoculated with 500K mc38/gp100 tumor cells. Three days later,
tumor-bearing mice were randomized into 1 of 4 experimental groups:
(i) vehicle, (ii) MM-398 (Top1 Inh.)--40 mg/kg iv once a week,
(iii) anti-PD-L1-150 ug ip every 3 days, (iv) MM-398 and anti-PD-L1
(Top1 Inh+anti-PD-L1). The top panel depicts the treatment
schedule. Shown below that are tumor volume over time, and a
Kaplan-Meier curve for survival. The data shows that the
combination of MM-398 and anti-PD-L1 produced increased anti-tumor
activity over MM-398 or anti-PD-L1 alone. This also translated into
significantly increased survival of tumor-bearing mice treated with
the combination of MM-398 and anti-PD-L1 in comparison to cohorts
treated with either single agent. Data from the experiment in FIG.
5 demonstrates that in vivo anti-tumor response and survival are
increased when nanoliposomal irinotecan (nal-IRI, MM-398) is
combined with .alpha.-PD-L1 antibody, including a plot of tumor
volume over time in a mouse xenograft model (FIG. 5A) and a
survival curve (FIG. 5B). The data was obtained from the experiment
described in the schematic of FIG. 5C.
[0198] FIG. 5A is a graph of measured tumor volume over time after
administration of MM-398 liposomal irinotecan and the anti-PD-L1
antibody described in the Table 2 of Example 3. FIG. 5 demonstrates
that in vivo anti-tumor response and survival are increased when
nanoliposomal irinotecan (nal-IRI, MM-398) is combined with a-PD-L1
antibody. FIG. 5A shows tumor volume up to day 21 (* indicates
P<0.0001). C57BL/6 mice were injected s.c. with 5.times.10.sup.5
MC38/gp100 cells. Three days later, when tumors were palpable, mice
were randomized into treatment groups (n=5) receiving the Top1
inhibitor MM-398 irinotecan liposome (40 mg/kg), anti-PD-L1
antibody (150 micrograms/mouse), or both MM-398 irinotecan liposome
and the anti-PD-L1 antibody. Vehicle control group received PBS and
isotype-matched control antibody Rat IgG2b (150 micrograms).
Beginning on day 3, mice received once weekly doses of MM-398
irinotecan liposome and antibody was administered every 3 days.
Shown here is tumor volume up to day 21, with P<0.0001. FIG. 5A
shows in vivo anti-tumor response and FIG. 5B shows survival, both
increased when a MM-398 liposomal irinotecan Top1 inhibitor is
combined with an anti-PDL-1 antibody. FIG. 5B shows the percent
survival over time of mice treated as in FIG. 5A; the rightmost
stepwise curve in 5B represents the combination of MM-398 with
.alpha.-PD-L1; moving to the left, the stepwise curves represent
MM-398, .alpha.-PD-L1, and vehicle, respectively (* indicates
P<0.0174). The tumor survival data for mice treated with MM-398
liposomal irinotecan or anti-PD-L1 antibody alone, or in
combination, is shown in FIG. 5B, having P<0.0174.
[0199] The data in FIG. 18 was obtained from an experiment designed
to determine the anti-tumor effect of combining MM-398 and anti-PD1
in our pre-clinical mouse model. PD1 is the receptor for PD-L1 and
forms the second part of this T cell checkpoint barrier that we can
interrogate therapeutically. Therefore we wanted to see if we could
see a similar increase in the anti-tumor effect with the
combination of MM-398 and anti-PD1 as we observed with the
combination of MM-398 and anti-PD-L1. C57BL/6 mice were inoculated
with 500K mc38/gp100 tumor cells. Three days later, tumor-bearing
mice were randomized into 1 of 4 experimental groups: (i) vehicle,
(ii) MM-398 (Top1 Inh.)--20 mg/kg iv once a week, (iii)
anti-PD1-200 ug (clone 29F.1A12) ip every 3 days, (iv) MM-398 and
anti-PD1 (Top1 Inh+anti-PD-L1). The top panel depicts the treatment
schedule. Shown below that are tumor volume over time, and a
Kaplan-Meier curve for survival. The data shows that the
combination of MM-398 and anti-PD1 produced increased anti-tumor
activity over MM-398 or anti-PD1 alone. The added survival benefit
of the combination of MM-398 and anti-PD1 was not as extensive as
the added survival benefit observed in the combination of MM-398
and anti-PD-L1.
[0200] Data in FIG. 18 demonstrates that in vivo anti-tumor
response and survival are increased when nanoliposomal irinotecan
(nal-IRI, MM-398) is combined with .alpha.-PD1 antibody. FIG. 18A
is a schematic of the mouse xenograft experiment performed to
obtain the data in FIGS. 18B and 18C. FIG. 18B is a graph of
measured tumor volume over time after administration of MM-398
liposomal irinotecan and the anti-PD1 antibody described in the
Table 2 of Example 3. FIG. 18B shows tumor volume up to day 21 (**
indicates P<0.01). C57BL/6 mice were injected s.c. with
5.times.10.sup.5 MC38/gp100 cells. Three days later, when tumors
were palpable, mice were randomized into treatment groups (n=5)
receiving the Top1 inhibitor MM-398 irinotecan liposome (20 mg/kg),
anti-PD1 antibody (200 micrograms/mouse), or both MM-398 irinotecan
liposome and the anti-PD1 antibody. Vehicle control group received
PBS and isotype-matched control antibody Rat IgG2b (200
micrograms). Beginning on day 3, mice received once weekly doses of
MM-398 irinotecan liposome and antibody was administered every 3
days. Shown here is tumor volume up to day 21, with P<0.01. FIG.
18B shows in vivo anti-tumor response and FIG. 18C shows survival,
both increased when a MM-398 liposomal irinotecan Top1 inhibitor is
combined with an anti-PD-1 antibody. FIG. 18B shows the percent
survival over time of mice treated as in FIG. 18A; the bottom curve
in 18B represents the combination of MM-398 with .alpha.-PD1;
moving upward, the stepwise curves represent MM-398, .alpha.-PD1,
and vehicle, respectively (* indicates P<0.0273). The tumor
survival data for mice treated with MM-398 liposomal irinotecan or
anti-PD1 antibody alone, or in combination, is shown in FIG. 18C,
having P<0.0273.
Example 4: Profile of Immune Response to Administration of
Liposomal Irinotecan in Combination with Anti-PD-L1 Antibody
[0201] In another embodiment, certain topoisomerase I inhibitors
are identified as enhancers of T cell mediated immune-therapy,
including enhanced anti-tumor response using a combination of
liposomal irinotecan (e.g., MM-398) and anti-PD-L1 antibody in
vivo.
[0202] FIG. 12 is a schematic of a mouse xenograft experiment
including the administration of MC38 colon cancer cell which have
been transduced to express the melanoma antigen gp100, followed by
administration of liposomal irinotecan and an anti-PD-L1 antibody.
The diagram in FIG. 12 outlines our experimental design for
exploring the effect of MM-398 or anti-PD-L1 alone or in
combination on different immune cell subsets in the tumor
microenvironment. C57BL/6 mice were inoculated with 500K mc38/gp100
tumor cells. 7 days later, mice were randomized into 1 of 4
experimental groups: (i) vehicle, (ii) MM-398-40 mg/kg iv once a
week, (iii) anti-PD-L1-150 ug ip every 3 days, (iv) MM-398 and
anti-PD-L1. Tumors were harvested on day 18 (post tumor
inoculation), and subjected to flow cytometry analysis for: CD8 T
cells and their effector function, regulatory T cells, and myeloid
derived macrophages.
[0203] Tumors from the experiment described in FIG. 12 were
dissociated and analyzed by flow cytometry analysis for effector
and regulatory T cells. FIGS. 13A-13B are graphs obtained from the
following experiment. CD8 T cells were identified based on the
following criteria: CD3+, CD8+. Regulatory T cells are defined as
CD3+, CD4+, CD25+, and FoxP3+. We observed that while the MM-398
alone group exhibited no increase in the CD8 T cell population,
both the anti-PD-L1 alone and the combination group of MM-398 and
anti-PD-L1 exhibited an increase in the number of CD8 T cells per
gram of tumor in comparison to the control group. The same is true
for the ratio of CD8 T cells to regulatory T cells (CD8/Treg),
which is higher in the anti-PD-L1 group and the combination group.
It would seem that this increase in the CD8 T cell number and the
ratio of the CD8 T cells to regulatory cells is being driven more
by the effect of the anti-PD-L1 antibody.
[0204] Data in FIGS. 13C and 13D was obtained as follows. The
effector activity of CD8 T cells was assessed by looking at the
expression levels of granzyme A and B, which are functional enzymes
produced by T cells which deliver cytolytic signals to target tumor
cells. Effector activity was quantified based on flow cytometry
analysis of: CD3+, CD8+, and GzA/GzB+. As shown in the FIGS. 13C
and 13D, there is a significant increase in the level of granzymes,
particularly in granzyme B which is the predominant effector
molecule for CD8 T cells. The data shows that the highest amount of
cytolytic activity was detected in CD8 T cells in the setting of
the combination of MM-398 and anti-PD-L1. Further experiments are
required to more comprehensively understand the effect of MM-398 on
the tumor and the tumor microenvironment that would be permissive
for increased T cell cytolytic activity.
[0205] Data in FIG. 13E was obtained as follows. In addition to
looking at different T cell populations, we also assessed the
effect of MM-398, anti-PD-L1, and the combination of the two on the
myeloid derived macrophages defined by: CD11b+, F4/80+ cells. The
data in FIG. 13E shows a general trend towards an increase in the
number of macrophages detected in the single agent treated groups
and also in the combination treatment group, with the highest
number of macrophages per gram of tumor detected in the combination
setting. The formulation of MM-398 lends itself to uptake by
macrophages and it was unsurprising to us to note this observation
of increased tumor associated macrophages.
Example 5: Top1 Inhibition Resulting in Upregulation of Tumor
Protein 53-Induced Nuclear Protein 1 (Teap)
[0206] In order to better understand the tumor molecular mechanisms
involved in Top1 inhibitor-enhancement of T cell mediated killing,
we performed gene expression analysis of SN38-treated melanoma cell
lines (DMSO-treatment of the same melanoma cell lines served as
controls). For this analysis, 4 melanoma cell lines were chosen (A:
2338, B: 2400, C: 2549, and D: 2559) and treated for 24 h with 1 uM
SN38 before being harvested for microarray analysis using the
Illumina HumanHT-12 v4 Expression BeadChip array. The data
collected was pathway analysis performed using Ingenuity Pathway
Analysis (IPA) to determine what signaling pathways and cell master
regulators are differentially regulated in SN38-treated cells in
comparison to DMSO-treated cells. Pathways and regulators are
ranked based on the Log2 fold change and on the activation score
respectively. The data indicated a significant and highly ranked
activation of the p53 signaling pathway in our Top1-inhibitor
treated tumor cells.
[0207] IPA analysis of the differential activation of the p53
signaling pathway in our 5N38-treated melanoma tumor cells also
indicated that based on the gene expression changes of the factors
involved in the p53 signaling pathway, there was a significant
activation of the cell death pathway in these cells and a
repression in proliferative and survival signals. These
computations based on the gene expression changes are indicative of
an increased apoptotic response in Top1 inhibitor-treated tumor
cells. This is important for our studies which seek to understand
how Top1 inhibitors can modulate tumor cells to make them more
susceptible to additional death signals from T cells.
[0208] In some embodiments, the discovery of synergy between
Topoisomerase 1 inhibition and checkpoint blockade provides novel
methods of treating cancer comprising the administration of a Top1
inhibitor (e.g., liposomal irinotecan) with a checkpoint inhibitor
compound. In this embodiment, the role of a p53 regulatory gene is
identified as playing an essential role in the enhanced response to
T cell mediated killing, including topoisomerase 1 inhibition
resulting in upregulation of Tumor protein 53-induced nuclear
protein 1 ("Teap"), Teap overexpression observed to recapitulate
the relevant phenotype and the observation that knockdown of Teap
impedes the relevant phenotype. Microarray analysis suggested that
p53 inducible nuclear protein 1 (TP53INP1) levels increase in
response to Top1 inhibition. In Example 5, the inventors
investigated whether TP53INP1 (Teap) can act as an apoptotic sensor
and lower the apoptotic threshold in the tumor cells through
activation of a TP53 regulated apoptotic pathway, thereby making
them more sensitive to T cell induced cell death, in addition to
whether Top1 inhibition can increase effector T cells and increase
the ratio of effector to regulatory T cells.
[0209] The p53 pathway is highly activated following the inhibition
of Top1. The Top1 inhibition results in activation of the cell
death pathway and repression of proliferation and survival
signaling. The induction of p53 pathway can be activated by p73 in
the absence of p53. Teap induces apoptosis in response to cell
stress, including the regulation of stress response genes like p21,
bax or md.sup.m2. Teap can also regulate autophagy via interactions
with LC3 and regulation of ATGS and beclin-1 activity.
[0210] FIG. 6B is a gene expression "heat map" for various genes
expressed in three cell lines (2338, 2400 and 2549). The data shown
in FIG. 6B represents a portion of the gene expression analysis
which was described above. This portion of the data focuses on the
differential expression of some genes related to p53 signaling. In
particular, we have chosen to focus on TP53INP1 (or Teap), which is
a p53 regulatory gene shown to be involved in directing an
apoptotic response in tumor cells (Gironella et al., Natl Acad Sci
USA 2007; Tomasini et al., J Biol Chem 2001). We observed a
significant upregulation in the expression of Teap with SN38
treatment in melanoma. This phenotype was also validated by
quantitative real time PCR (qRT-PCR) performed on a number of
melanoma patient-derived tumor cell lines treated with 2 different
Top1 inhibitors (Top1 inh. 1=SN38, Top1 inh. 2=Topotecan).
[0211] FIG. 14 is a graph showing the comparative change in
TP53INP1 in response to a first Top1 inhibitor and a second Top1
inhibitor.
[0212] Overexpression of Teap increases T cell mediated killing in
vitro. Given the significant increase observed in the expression
level of TP53INP1 in Top1 inhibitor-treated tumor cells, we next
investigated the functional relevance of this change to T cell
mediated killing using our in vitro cytotoxicity assay. We used a
lentivirus system to overexpress GFP-tagged Teap in melanoma tumor
cells (overexpression of GFP was used as a control). We validated
the overexpression of Teap in the tumor cells by qRT-PCR. We
incubated GFP or Teap overexpressing 2549 melanoma cells with 2549
autologous T cells to determine what effect overexpression of Teap
would have on T cell mediated killing of the tumor cells. We
observed increased T cell killing of 2549 tumor cells overexpres
sing Teap in comparison to control GFP-overexpressing 2549 cells.
This observation recapitulated what we observed with treatment of
melanoma cells with Top1 inhibitors; which resulted in increased
expression of Teap as well as increased T cell mediated killing of
tumor cells. FIGS. 15A and 15B are graphs showing relative mRNA
expression of Teap normalized to GAPDH (FIG. 15A) and % Caspase 3
positive (FIG. 15B).
[0213] Silencing Teap impedes T cell mediated killing in Top1
inhibitor treated tumor cells. We then asked the complementary
question of the necessity of Teap for Top1 inhibitor enhancement of
T cell mediated killing of melanoma tumor cells. We addressed this
question by using lentiviral shRNAs to silence the expression of
Teap in melanoma tumor cells (shRNAs targeting luciferase were used
as a control). We validated the knockdown of Teap expression by
qRT-PCR. We then asked whether or not Top1 inhibitor-treatment
would result in increased T cell mediated killing of melanoma tumor
cells if the expression of Teap was silenced. As is shown,
silencing of Teap in melanoma cells impeded the capacity of Top1
inhibitor treatment to enhance T cell mediated killing of tumor
cells. However, TEAP silencing did not impede the caspase
activation in tumor cells by TOP1 inhibition or TIL co-incubation
alone. This indicates that Teap is necessary for the enhancement
observed in T cell mediated killing of Top1 inhibitor-treated tumor
cells. FIGS. 16A is a graph showing relative mRNA expression
normalized to GAPDH for 2549 and % Caspase 3 positive (FIG. 16B)
for a Teap knockout melanoma cell line.
Example 6: Combination Top1/Immunomodulatory Therapy for the
Treatment of Human Cancer
[0214] In another embodiment, methods of treating humans diagnosed
with cancer such as melanoma comprise administration of a
topoisomerase 1 inhibitor (e.g., MM-398 liposomal irinotecan) in
combination with an anti-PD1 antibody (e.g., nivolumab).
[0215] FIGS. 17A and 17B are schematic diagrams of exemplary
methods of treating a human with a combination therapy of MM-398
liposomal irinotecan and the anti-PD1 therapy nivolumab. The method
of FIG. 17A is useful, for example, to determine the recommended
Phase II dose of an anti-PD-1 antibody (e.g. nivolumab) and a
liposomal irinotecan (e.g., MM-398) in a combination therapy,
including determination of pharmacokinetics of the combination
therapy. The method of FIG. 17B is useful, for example, to
determine the overall response rate of an anti-PD-1 antibody (e.g.
nivolumab) and a liposomal irinotecan (e.g., MM-398) in a
combination therapy in a patient who is refractory to prior anti
PD-1 antibody therapy, including the determination of progression
free survival and overall survival, and evaluation of the safety
profile of the combination therapy. One or both methods in FIGS.
17A and 17B can further include one or both of the following: (a)
assessing pre and post treatment biopsy and blood samples for
biomarker analysis; including assessment of immunologic and
molecular markers in patients with metastatic melanoma enrolled on
with combination therapy, including specifically an assessment of
TP53NP1 (Teap) which has been identified as a target of interest
based on pre-clinical studies, and/or (b) immunological markers to
be analyzed include CD4, CD8, CD25, FoxP3 to monitor circulating
effector and regulatory T cells. The expression of tumor intrinsic
factors such as TP53INP1 and pro-apoptotic molecules can also be
monitored during either or both methods of FIGS. 17A and 17B. The
methods can be practiced in medically appropriate patients.
Preferably, the patients have one, multiple or all of the following
characteristics: Age >18, ECOG 0-; Measurable disease by RECIST
1.1; tumor amenable to serial biopsy that is not counted as
measurable disease; adequate organ/marrow function; and/or
treatment refractory to anti PD-1 or anti PD-L1 based therapy. Also
preferably, patients with active autoimmune diseases with
requirement for chronic steroid replacement (>10 mg
prednisone/equivalents) are excluded from treatment. In addition
patients with prior CNS metastases can be allowed provided that
disease is treated and stable at least 4 weeks prior to
treatment.
[0216] The methods of treatment include treating the human patient
with at least one of dose level 1, -1, 2 or 3 from the Table 3
below given once every 14 days intravenously (in a 28-day treatment
cycle), corresponding to specific doses of MM-398 liposomal
irinotecan (dose based on free base of irinotecan) and
nivolumab.
TABLE-US-00003 TABLE 3 Dose Level MM-398 (mg/m.sup.2) Nivolumab
(mg/kg) -1 43 3 1 (starting dose) 50 3 2 70 3 3 80 3
[0217] Preferably, the MM-398 liposomal irinotecan is administered
prior to the nivolumab. Preferably, the methods of treatment are
used to treat human patients diagnosed with a form of cancer that
is FDA approved for nivolumab. Nivolumab is currently FDA-approved
in melanoma, non-small cell lung cancer (NSCLC), Renal Cell Cancer
(RCC), and Hodgkin lymphoma. A Bayesian design can be used for a
phase 1 study using the method of treatment in FIG. 17A. A phase II
study using the method of treatment in FIG. 17B can have a target
of 20% overall response rate (ORR) (e.g., estimate a total of 50
patients treated to target of 20% ORR with one-sided significance
level of 5% and power of 75%).
[0218] The protocol above could be altered in several ways to
assess efficacy and proper dosing. Pembrolizumab could be used in
place of nivolumab, to be tested in combination with liposomal
irinotecan. Pembrolizumab is typically dosed at 2 mg/kg every 3
weeks, so the protocols shown in FIGS. 17A and 17B could be
modified such that each cycle would be three weeks long instead of
two. Preferably, the methods of treatment are used to treat human
patients diagnosed with a form of cancer that is FDA approved for
pembrolizumab. Pembrolizumab is currently FDA-approved in melanoma.
Alternatively, an anti-PD-L1 antibody could be used in place of an
anti-PD-1 antibody.
Other Embodiments
[0219] The detailed description set-forth above is provided to aid
those skilled in the art in practicing the present disclosure.
However, the disclosure described and claimed herein is not to be
limited in scope by the specific embodiments herein disclosed
because these embodiments are intended as illustration of several
aspects of the disclosure. Any equivalent embodiments are intended
to be within the scope of this disclosure. Indeed, various
modifications of the disclosure in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description, which do not depart from the spirit
or scope of the present inventive discovery. Such modifications are
also intended to fall within the scope of the appended claims.
[0220] All references cited in this specification are hereby
incorporated by reference. The discussion of the references herein
is intended merely to summarize the assertions made by their
authors and no admission is made that any reference constitutes
prior art relevant to patentability. Applicant reserves the right
to challenge the accuracy and pertinence of the cited
references.
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