U.S. patent application number 16/990036 was filed with the patent office on 2020-11-26 for combination treatment.
This patent application is currently assigned to Duke University. The applicant listed for this patent is Duke University. Invention is credited to Darell D. Bigner, Vidyalakshmi Chandramohan, Matthias Gromeier, Smita Nair.
Application Number | 20200368300 16/990036 |
Document ID | / |
Family ID | 1000005016299 |
Filed Date | 2020-11-26 |
United States Patent
Application |
20200368300 |
Kind Code |
A1 |
Bigner; Darell D. ; et
al. |
November 26, 2020 |
Combination Treatment
Abstract
Human clinical use of a chimeric poliovirus construct has
demonstrated excellent anti-tumor effect. Combination with immune
checkpoint inhibitors increases the anti-tumor effect. Tumors of
different types are susceptible to the combination treatment,
including but not limited to melanoma, glioglastoma, renal cell
carcinoma, prostate cancer, breast cancer, lung cancer,
medulloblastoma, and colorectal cancer.
Inventors: |
Bigner; Darell D.; (Mebane,
NC) ; Gromeier; Matthias; (Durham, NC) ; Nair;
Smita; (Durham, NC) ; Chandramohan; Vidyalakshmi;
(Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Duke University |
Durham |
NC |
US |
|
|
Assignee: |
Duke University
Durham
NC
|
Family ID: |
1000005016299 |
Appl. No.: |
16/990036 |
Filed: |
August 11, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15768147 |
Apr 13, 2018 |
10744170 |
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PCT/US16/57023 |
Oct 14, 2016 |
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16990036 |
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62361725 |
Jul 13, 2016 |
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62356831 |
Jun 30, 2016 |
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62242123 |
Oct 15, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/85 20130101;
A61K 39/39 20130101; A61K 9/127 20130101; A61K 39/13 20130101; C07K
2317/76 20130101; A61K 2039/505 20130101; C12N 2840/203 20130101;
A61K 35/768 20130101; C12N 2770/32621 20130101; A61K 2300/00
20130101; C07K 16/2818 20130101; A61P 35/00 20180101; C12N 7/00
20130101; Y02A 50/30 20180101; A61K 39/39541 20130101; C12N
2770/32632 20130101 |
International
Class: |
A61K 35/768 20060101
A61K035/768; A61K 39/13 20060101 A61K039/13; A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; C07K 16/28 20060101
C07K016/28; C12N 7/00 20060101 C12N007/00; C12N 15/85 20060101
C12N015/85; A61K 39/39 20060101 A61K039/39; A61K 9/127 20060101
A61K009/127 |
Goverment Interests
[0001] This invention was made using funds provided by the United
States government. The U.S. government retains certain rights
according to the terms of grants from the National Institutes of
Health R01 CA87537, P50 NS20023, P50 CA190991, R01 CA124756, and
R01 CA140510.
Claims
1. A method of treating a tumor in a patient, comprising:
administering to the patient a PVSRIPO chimeric poliovirus
construct comprising a Sabin type I strain of poliovirus with a
human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in
said poliovirus' 5' untranslated region between said poliovirus'
cloverleaf and said poliovirus' open reading frame; and
administering an anti-PD-1 antibody, whereby the patient is
treated.
2. A method of treating a tumor in a patient, comprising:
administering to the patient a PVSRIPO chimeric poliovirus
construct comprising a Sabin type I strain of poliovirus with a
human rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in
said poliovirus' 5' untranslated region between said poliovirus'
cloverleaf and said poliovirus' open reading frame; and
administering an anti-PD-L1 antibody to the patient, whereby the
patient is treated.
3. The method of claim 1 or 2 wherein the tumor is a
glioblastoma.
4. The method of claim 1 or 2 wherein the tumor is astrocytoma or
oligodendroglioma.
5. The method of claim 1 or 2 wherein the tumor is
astro-oligodendroglioma.
6. The method of claim 1 or 2 wherein the tumor is renal cell
carcinoma.
7. The method of claim 1 or 2 wherein the tumor is prostate
tumor.
8. The method of claim 1 or 2 wherein the tumor is bladder
tumor.
9. The method of claim 1 or 2 wherein the tumor is esophagus and/or
stomach tumor.
10. The method of claim 1 or 2 wherein the tumor is pancreas
tumor.
11. The method of claim 1 or 2 wherein the tumor is colorectal
tumor.
12. The method of claim 1 or 2 wherein the tumor is liver or gall
bladder tumor.
13. The method of claim 1 or 2 wherein the tumor is breast
tumor.
14. The method of claim 1 or 2 wherein the tumor is
medulloblastoma.
15. The method of claim 1 or 2 wherein the tumor is lung tumor.
16. The method of claim 1 or 2 wherein the tumor is head and neck
tumor.
17. The method of claim 1 or 2 wherein the tumor is melanoma.
18. The method of claim 1 or 2 wherein the tumor is sarcoma.
19. The method of claim 1 or 2 wherein the chimeric poliovirus
construct is administered by intracerebral infusion with convection
enhanced delivery.
20. The method of claim 1 or 2 wherein prior to administering, the
method comprises the step of testing the tumor to ascertain that it
expresses NECL5.
21. The method of claim 1 or 2 wherein the tumor expresses
NECL5.
22. The method of claim 1 or 2 wherein the chimeric poliovirus
construct is administered directly to the tumor.
23. The method of claim 1 or 2 wherein the antibody is administered
within 30 days of administering the chimeric poliovirus
construct.
24. The method of claim 1 or 2 wherein the antibody is administered
within 7 days of administering the chimeric poliovirus
construct.
25. The method of claim 1 or claim 2, wherein administration of the
antibody is repeated.
26. The method of claim 1 wherein the antibody is
pembrolizumab.
27. The method of claim 1 wherein the antibody is nivolumab.
28. A kit in a divided container for treating a tumor that
expresses NECL5 (nectin-like protein 5), comprising: a PVSRIPO
chimeric poliovirus construct comprising a Sabin type I strain of
poliovirus with a human rhinovirus 2 (HRV2) internal ribosome entry
site (IRES) in said poliovirus' 5' untranslated region between said
poliovirus' cloverleaf and said poliovirus' open reading frame; and
an anti-PD-1 antibody, wherein the chimeric poliovirus construct
and the antibody are in distinct vessels of the divided
container.
29. The kit of claim 28 wherein the antibody is pembrolizumab.
30. The kit of claim 28 wherein the antibody is nivolumab.
31. A kit in a divided container for treating a tumor that
expresses NECL5 (nectin-like protein 5), comprising: a PVSRIPO
chimeric poliovirus construct which comprises a Sabin type I strain
of poliovirus with a human rhinovirus 2 (HRV2) internal ribosome
entry site (IRES) in said poliovirus' 5' untranslated region
between said poliovirus' cloverleaf and said poliovirus' open
reading frame; and an anti-PD-L1 antibody, wherein the chimeric
poliovirus construct and the antibody are in distinct vessels of
the divided container.
32. A composition comprising a PVSRIPO chimeric poliovirus
construct and an anti-PD-1 antibody; wherein the chimeric
poliovirus construct comprises a Sabin type I strain of poliovirus
with a human rhinovirus 2 (HRV2) internal ribosome entry site
(IRES) in said poliovirus' 5' untranslated region between said
poliovirus' cloverleaf and said poliovirus' open reading frame.
33. The composition of claim 32 wherein the antibody is
pembrolizumab.
34. The composition of claim 32 wherein the antibody is
nivolumab.
35. A composition comprising a PVSRIPO chimeric poliovirus
construct and an anti-PD-L1 antibody; wherein the chimeric
poliovirus construct comprises a Sabin type I strain of poliovirus
with a human rhinovirus 2 (HRV2) internal ribosome entry site
(IRES) in said poliovirus' 5' untranslated region between said
poliovirus' cloverleaf and said poliovirus' open reading frame.
Description
TECHNICAL FIELD OF THE INVENTION
[0002] This invention is related to the area of anti-tumor therapy.
In particular, it relates to oncolytic virus anti-tumor
therapy.
BACKGROUND OF THE INVENTION
[0003] PVS-RIPO is an oncolytic poliovirus (PV) recombinant. It
consists of the live attenuated type 1 (Sabin) PV vaccine
containing a foreign internal ribosomal entry site (IRES) of human
rhinovirus type 2 (HRV2). The IRES is a cis-acting genetic element
located in the 5' untranslated region of the PV genome, mediating
viral, m.sup.7G-cap-independent translation. The virus has shown
exciting signs of efficacy in humans. Nonetheless there is a
continuing need in the art to identify and develop anti-cancer
treatments that are more effective and that are effective for more
humans, particularly for patients with brain tumors.
SUMMARY OF THE INVENTION
[0004] According to one aspect of the invention a method of
treating a tumor in a patient is provided. A chimeric poliovirus
construct is administered to the patient. The construct comprises a
Sabin type I strain of poliovirus with a human rhinovirus 2 (HRV2)
internal ribosome entry site (IRES) in said poliovirus' 5'
untranslated region between said poliovirus' cloverleaf and said
poliovirus' open reading frame. An immune checkpoint inhibitor is
also administered to the patient, either at the same time or within
about 30 days.
[0005] According to another aspect of the invention a kit is
provided for treating a tumor. The kit comprises a chimeric
poliovirus construct comprising a Sabin type I strain of poliovirus
with a human rhinovirus 2 (HRV2) internal ribosome entry site
(IRES) in said poliovirus' 5' untranslated region between said
poliovirus' cloverleaf and said poliovirus' open reading frame; and
an immune checkpoint inhibitor.
[0006] According to a further aspect of the invention, provided is
a combination of a chimeric poliovirus construct and an immune
checkpoint inhibitor for use as a medicament, or for use in
treating a tumor, wherein the chimeric poliovirus construct
comprises a Sabin type I strain of poliovirus with a human
rhinovirus 2 (HRV2) internal ribosome entry site (IRES) in said
poliovirus' 5' untranslated region between said poliovirus'
cloverleaf and said poliovirus' open reading frame; and an immune
checkpoint inhibitor.
[0007] These and other aspects which will be apparent to those of
skill in the art upon reading the specification provide the art
with new therapeutic regimens for treating cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 depicts experimental schema.
[0009] FIG. 2 shows results using four different tumor cell lines
representing, breast, melanoma, and prostate cancers. DCs were
seeded in dishes. Supernatant from onco-lysate was added to DC
cultures and incubated. Supernatant was then removed and DCs were
washed. DNase I-treated peripheral blood mononuclear cells (PBMCs)
were incubated at 37.degree. C. Non-adherent cells were harvested
and stimulated with DCs loaded with poliovirus-induced tumor lysate
at a responder cell to stimulator DC ratio of 10:1 in the presence
of IL-7 in CTL stimulation media. T cells were harvested on day
12-14, counted and used as effector T cells in a europium-release
CTL assay. Autologous DCs transfected with relevant and irrelevant
tumor antigen-encoding mRNA were used as control targets. For DC
control targets, mRNA-electroporated target cells were harvested,
washed to remove all traces of media and labeled with europium
(Eu). Alternatively, original target cells (Sum149, MDAMB231,
LNCaP, or DM6) were labeled with Eu. Ten thousand europium-labeled
targets (T) and serial dilutions of effector cells (E) at varying
E:T ratios were incubated in 96-well V-bottom plates. The plates
were centrifuged at for 3 minutes and incubated at 37.degree. C. 50
.mu.l of the supernatant was harvested and added to 150 .mu.l of
enhancement solution in 96-well flat-bottom plates and europium
release was measured by time resolved fluorescence using the
VICTOR3 Multilabel Counter (Perkin-Elmer). Specific cytotoxic
activity was determined using the formula: % specific
release=[(experimental release-spontaneous release)/(total
release-spontaneous release)].times.100. Spontaneous release of the
target cells was less than 25% of total release by detergent.
Spontaneous release of the target cells was determined by
incubating the target cells in medium without T cells. All assays
were done in triplicate, bars represent average % lysis and error
bars denote SEM.
[0010] FIGS. 3A-3D show results of in vivo testing in mouse tumor
model using CT2 A gliomas in C57B16 mice using a variety of
treatments including a combined poliovirus and checkpoint inhibitor
treatment analogous to the invention; both the mice and the CT2 A
cells express the human poliovirus receptor CD155. Results (tumor
volume over time) with the following experimental treatments are
shown in FIG. 3A: Group I (downward arrow): DMEM (vehicle to
control for virus)+IgG (to control for anti-PD1); Group II
(circle): single intra-tumoral injection of PVSRIPO+IgG; Group III
(upward arrow): single intra-tumoral injection of DMEM+anti-PD1;
Group IV (square): single intra-tumoral injection of PVSRIPO
("mRIPO")+anti-PD1. Anti-PD1 was given in three installments (days
3, 6, 9) by intraperitoneal injection. FIGS. 3B-3D show tumor
responses (tumor volume over time) in individual mice (each line a
different mouse) in the treatment groups II-IV.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The inventors have developed a combination therapy regimen
in which a viral construct and an immune checkpoint inhibitor are
administered to humans. Because the poliovirus is a potential
disease agent, extra precautions must be taken to ensure that
disease-causing agents are not introduced to the subjects. Using
good manufacturing procedures and purifications, a preparation was
made that was sufficiently pure to permit introduction into
humans.
[0012] Any technique for directly administering the viral
preparation to the tumor may be used. Direct administration does
not rely on the blood vasculature to access the tumor. The
preparation may be painted on the surface of the tumor, injected
into the tumor, instilled in or at the tumor site during surgery,
infused into the tumor via a catheter, etc. One particular
technique which may be used is convection enhanced delivery.
[0013] Immune checkpoint inhibitors which may be used according to
the invention are any that disrupt the inhibitory interaction of
cytotoxic T cells and tumor cells. These include but are not
limited to anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA4
antibody, anti-LAG-3 antibody, and/or anti-TIM-3 antibody. Approved
checkpoint inhibitors in the U.S. include ipimilumab,
pembrolizumab, and nivolumab. The inhibitor need not be an
antibody, but can be a small molecule or other polymer. If the
inhibitor is an antibody it can be a polyclonal, monoclonal,
fragment, single chain, or other antibody variant construct.
Inhibitors may target any immune checkpoint known in the art,
including but not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3,
B7-H4, BTLA, HVEM, TIM3, GALS, LAGS, VISTA, KIR, 2B4, CD160,
CGEN-15049, CHK 1, CHK2, A2aR, and the B-7 family of ligands.
Combinations of inhibitors for a single target immune checkpoint or
different inhibitors for different immune checkpoints may be used.
Additionally, CSF-1R blockade may be used in combination or as an
alternative to immune checkpoint inhibitor(s), to ensure generation
of potent and sustained immunity that effectively eliminates
distant metastases and recurrent tumors. Antibodies specific for
CSF-1R or drugs that inhibit or blockade CSF-1R may be used for
this purpose, including but not limited to imactuzumab and
AMG820.
[0014] The immune checkpoint inhibitor may be administered at the
same time, before, or after the poliovirus. Typically the two
agents will be administered within 30, 28, 21, 14, 7, 4, 2, or 1
day(s) of each other. The agents may be given repeatedly, either
serially or in a cycle of first and second agents. It may be
advantageous but not necessary for the vaccine to be administered
prior to the checkpoint inhibitor. But the reverse order may also
be used. Priming of a cytotoxic T lymphocyte response by the viral
construct may take from about 5 to about 14 days. Administration of
the checkpoint inhibitor may beneficially be commenced during or
after the priming period.
[0015] Immune checkpoint inhibitors may be administered by any
appropriate means known in the art for the particular inhibitor.
These include intravenous, oral, intraperitoneal, sublingual,
intrathecal, intracavitary, intramuscularly, and subcutaneously.
Optionally, the immune checkpoint inhibitor may be administered in
combination with the poliovirus agent.
[0016] Any human tumor can be treated, including both pediatric and
adult tumors. The tumor may be in any organ, for example, brain,
prostate, breast, lung, colon, and rectum, Various types of tumors
may be treated, including, for example, glioblastoma,
medulloblastomas, carcinoma, adenocarcinoma, etc. Other examples of
tumors include, adrenocortical carcinoma, anal cancer, appendix
cancer, grade I (anaplastic) astrocytoma, grade II astrocytoma,
grade III astrocytoma, grade IV astrocytoma, atypical
teratoid/rhabdoid tumor of the central nervous system, basal cell
carcinoma, bladder cancer, breast sarcoma, bronchial cancer,
bronchoalveolar carcinoma, cervical cancer, craniopharyngioma,
endometrial cancer, endometrial uterine cancer, ependymoblastoma,
ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's
sarcoma, extracranial germ cell tumor, extragonadal germ cell
tumor, extrahepatic bile duct cancer, fibrous histiocytoma, gall
bladder cancer, gastric cancer, gastrointestinal carcinoid tumor,
gastrointestinal stromal tumor, gestational trophoblastic tumor,
gestational trophoblastic tumor, glioma, head and neck cancer,
hepatocellular cancer, Hilar cholangiocarcinoma, hypopharyngeal
cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma,
Langerhans cell histiocytosis, large-cell undifferentiated lung
carcinoma, laryngeal cancer, lip cancer, lung adenocarcinoma,
malignant fibrous histiocytoma, medulloepithelioma, melanoma,
Merkel cell carcinoma, mesothelioma, endocrine neoplasia, nasal
cavity cancer, nasopharyngeal cancer, neuroblastoma, oral cancer,
oropharyngeal cancer, osteosarcoma, ovarian clear cell carcinoma,
ovarian epithelial cancer, ovarian germ cell tumor, pancreatic
cancer, papillomatosis, paranasal sinus cancer, parathyroid cancer,
penile cancer, pharyngeal cancer, pineal parenchymal tumor,
pineoblastoma, pituitary tumor, pleuropulmonary blastoma, renal
cell cancer, respiratory tract cancer with chromosome 15 changes,
retinoblastoma, rhabdomyosarcoma, salivary gland cancer, small cell
lung cancer, small intestine cancer, soft tissue sarcoma, squamous
cell carcinoma, squamous non-small cell lung cancer, squamous neck
cancer, supratentorial primitive neuroectodermal tumor,
supratentorial primitive neuroectodermal tumor, testicular cancer,
throat cancer, thymic carcinoma, thymoma, thyroid cancer, cancer of
the renal pelvis, urethral cancer, uterine sarcoma, vaginal cancer,
vulvar cancer, and Wilms tumor.
[0017] Optionally, patients may be stratified on the basis of NECL5
(poliovirus receptor) expression. This can be assayed at the RNA or
protein level, using probes, primers, or antibodies, for example.
The NECL5 expression may guide the decision to treat or not treat
with the chimeric poliovirus of the present invention. The NECL5
expression may also be used to guide the aggressiveness of the
treatment, including the dose, frequency, and duration of
treatments. Antibodies to NECL5 (CD155) are commercially available
and may be used. NECL5 RNA expression can also be assayed. See
Hirota et al. Oncogene 24:2229-2235 (2005).
[0018] Treatment regimens may include, in addition to delivery of
the chimeric poliovirus construct and immune checkpoint inhibitor
combination, surgical removal of the tumor, surgical reduction of
the tumor, chemotherapy, biological therapy, radiotherapy. These
modalities are standard of care in many disease states, and the
patient need not be denied the standard of care. The chimeric
poliovirus and immune checkpoint inhibitor combination may be
administered before, during, or after the standard of care. The
chimeric poliovirus and immune checkpoint inhibitor combination may
be administered after failure of the standard of care. When a
combination is specified, it may be administered separately in time
as two separate agents within a single combination regimen.
Alternatively, the two (or more) agents may be administered in
admixture.
[0019] Kits may comprise, in a single divided or undivided
container, both the chimeric poliovirus construct PVSRIPO as well
as a checkpoint inhibitor. They two may be in separate vessels or
in a single vessel in admixture. Instructions for administration
may be included. Optionally, an antibody for testing NECL5
expression in the patient is a component of the kit.
[0020] Applicants have found that the clinical pharmaceutical
preparation of the chimeric poliovirus has admirable genetic
stability and homogeneity. This is particularly advantageous as the
poliovirus is known to be highly mutable both in culture and in
natural biological reservoirs. Any suitable assay for genetic
stability and homogeneity can be used. One assay for stability
involves testing for the inability to grow at 39.5 degrees C.
Another assay involves bulk sequencing. Yet another assay involves
testing for primate neurovirulence.
[0021] While applicants do not wish to be bound by any particular
mechanism of action, it is believed that multiple mechanisms may
contribute to the efficacy of the poliovirus construct. These
include lysis of cancer cells, recruitment of immune cells, and
specificity for cancer cells. Moreover, the virus is
neuro-attenuated.
[0022] The above disclosure generally describes the present
invention. All references disclosed herein are expressly
incorporated by reference. A more complete understanding can be
obtained by reference to the following specific examples, which are
provided herein for purposes of illustration only, and are not
intended to limit the scope of the invention.
Example 1
[0023] Animal tumor models. An IND-directed efficacy trial of
PVS-RIPO was conducted in the HTB-15 GBM xenograft model in athymic
mice. PVS-RIPO (from the clinical lot) was administered at the
`mouse-adjusted`, FDA-approved max. starting dose [the FDA-approved
max. starting dose (10e8 TCID) was adjusted for the reduced tumor
size in mice (to 6.7.times.10e6 TCID)]. Delivery mimicked the
intended clinical route, i.e., slow intratumoral infusion. Under
these conditions, PVS-RIPO induced complete tumor regress in all
animals after 15 days. While virus was recovered from treated
tumors until day 10, the levels were modest at best, indicating
that direct viral tumor cell killing alone cannot account for the
treatment effect.
[0024] Evidence from animal tumor models suggests that intratumoral
inoculation of PVS-RIPO causes direct virus-induced tumor cell
killing and elicits a powerful host immunologic response against
the infected/killed tumor (3, 7, 10). The response to virus
infusion is characterized by a strong, local inflammatory response,
leading to immune infiltration of the tumor. Eventually the slow
tissue response to PVS-RIPO infusion leads to the demise of the
tumor mass and its replacement by a scar.
Example 2
[0025] Clinical trials. IND no. 14,735 `Dose-finding and Safety
Study of PVSRIPO Against Recurrent Glioblastoma` was FDA-approved
on Jun. 19, 2011 and IRB-approved on Oct. 27, 2011. A phase I/II
clinical trial in patients with recurrent glioblastoma (GBM)
(NCT01491893) is currently enrolling patients.
[0026] Two human subjects have so far been treated with PVS-RIPO
per IRB-approved protocol. Preliminary findings from the first
subject are described in Example 3.
Example 3
[0027] Preliminary findings with first human subject. The patient
is a 21-year-old female nursing student diagnosed with a right
frontal GBM (WHO grade IV). She was first diagnosed in June 2011,
at the age of 20 years, following a history of severe headaches and
unsuccessful treatment for a suspected sinus infection. Brain
imaging was obtained on Jun. 17, 2011 and showed a large right
frontal mass, measuring .about.5.times.6 cm. She underwent a
subtotal resection of the right frontal mass on Jun. 22, 2011, with
pathology confirming GBM (WHO grade IV). Given the young age of the
patient, her excellent performance status and the subtotal tumor
resection, it was decided to treat her aggressively with a
combination of six weeks of radiation therapy with concurrent
Temodar chemotherapy at 75 mg/m.sup.2 by mouth daily and
bevacizumab (antiangiogenic agent) administered every 2 weeks. The
patient completed six weeks of treatment on Sep. 18, 2011. On Oct.
11, 2003, the patient initiated adjuvant therapy with monthly,
five-day Temodar chemotherapy in addition to bevacizumab 10 mg/kg
every two weeks.
[0028] On Apr. 16, 2012, the patient presented to clinic after
having experienced her first generalized seizure, which occurred in
her sleep. By that time, she had completed six months of the
combination of Temodar and bevacizumab. She had attributed the
seizure to increased stress at school, as she was completing a
degree to become a pediatric oncology nurse, despite her diagnosis
of GBM and ongoing chemotherapy treatment. The brain MRI obtained
on that day showed tumor recurrence, with a new nodular enhancement
along the medial aspect of the resection cavity (FIG. 12).
[0029] The patient was offered multiple treatment options, but
elected to pursue the PVS-RIPO clinical trial. Following her first
generalized seizure, she was initiated on Keppra, but forgot to
take it on occasion and because of this and the known tumor
recurrence, the patient experienced a second generalized seizure in
her sleep on May 6, 2012. She went back to her baseline neurologic
condition and was worked up to enroll on protocol.
[0030] A follow-up MRI was obtained on May 9, 2012 (FIG. 13),
before the patient underwent infusion of PVS-RIPO on May 11, 2012
with the FDA-approved max. starting dose (10e8) by the intended
clinical delivery method (convection-enhanced, intratumoral
infusion of 3 mL of virus suspension containing the contrast
Gd-DTPA over 6 hrs; see Example 4) and experienced no neurologic or
other complications related to this.
[0031] An MRI obtained immediately after completion of the infusion
documents the distribution of the infusate (FIG. 14).
[0032] Our research team followed up on the patient on a weekly
basis and she was seen in clinic two weeks post infusion, at which
time she denied any new neurologic symptoms, seizure recurrence,
fatigue, shortness of breath or weakness. She again was evaluated
in clinic on Jun. 7, 2012 and her physical and neurological
conditions remained normal. The brain MRI obtained at that visit
showed stability of the disease (FIG. 15).
[0033] The patient was seen in clinic on Jul. 9, 2012. Once more,
she denied any new neurologic symptoms, including the absence of
any recurrent seizure activity since the seizure observed on May 6,
2012, prior to PVS-RIPO infusion. She also reported that her mood
was good, that she was content with her progress in nursing school,
feeling that she is able to focus in school much better since after
her infusion. She was also excited by her move with two roommates
and by the fact that she is able to exercise regularly. Her brain
MRI obtained on that day showed a slightly increased mass effect
and minimal increase in superior linear enhancement, concerning for
progression of disease (FIG. 16).
[0034] In view of worrisome radiographic changes with no clinical
worsening, we decided to obtain an 18-FDG PET scan. The 18-FDG PET
scan demonstrated hypometabolic activity in the area of concern on
the MRI, suggestive of a necrotic process (treatment response
effect; FIG. 17). The PET scan from 07/09 suggests the absence of
viable tumor. After discussion with the patient and her mother, it
was decided to continue to follow the patient from a clinical and
radiographic standpoint.
[0035] In check-ups on 8/27 and 10/22 the patient denied any new
neurologic symptoms, including the absence of any seizure activity
since the seizure on May 6, 2012 (prior to PVS-RIPO infusion). The
patient reports improved cognitive/memory function, motor function
(exercise). As of 10/26, the patient is neurologically normal.
[0036] Because of the favorable radiographic presentation at 08/27,
a PET scan was not ordered. The patient was re-scanned on 10/22 and
there was a quantifiable radiographic response.
[0037] An MRI/PET overlay demonstrates the absence of signal from
the general area of the tumor recurrence.
Example 4
[0038] Convection infusion. Preoperatively the BrainLab iPlan Flow
system is used to plan catheter trajectories based on predicted
distributions using information obtained from a preoperative
MRI.
[0039] This invention uses one mM of gadolinium as a surrogate
tracer to identify the distribution of the poliovirus. This could
be used for other drug infusions as well. The gadolinium is
co-infused with the drug and various MRI sequences are used to
quantify the distribution.
[0040] The entire volume of the agent to be delivered will be
pre-loaded into a syringe by the investigational pharmacist and
connected to the catheter under sterile conditions in the operating
room or the NICU just prior to beginning of infusion. Due to the
complexity of scheduling all of the necessary components for the
infusion (operating room time, pharmacy time, and radiology
appointments), a +1 day window has been built in to the study for
the study drug infusion. This means that the infusion is allowed to
start the following day after the biopsy/catheter placement. This
will still be considered "day 0" in regards to the protocol and the
timing of the subsequent events. At the time of virus injection,
emergency drugs, including epinephrine and diphenhydramine will be
available and the neurologic status, oxygen saturation, and cardiac
rhythm will be monitored. Drug infusion will occur in the
Neuro-Surgical Intensive Care Unit (NSCU) so that all other
emergency facilities will be available. Patients will be treated
with a prophylactic antibiotic such as nafcillin, a
second-generation cephalosporin or vancomycin starting with the
induction of anesthesia for the catheter placement.
[0041] Based on our own experience, previously published reports
(19) and IRB- and FDA-approved trials using similar infusion
techniques (IRB #4774-03-4R0), patients will be infused at a rate
of 500 .mu.L/hr. A Medfusion 3500 infusion pump will be
pre-programmed to a delivery rate of 500 .mu.L/hr. The agent (which
will be in a total volume of 10 mL to account for `dead-space` of
3.3723 mL in the infusion system) will be loaded in a 20 mL syringe
into the syringe pump at the initial onset to avoid any
interruptions in the infusion. The total amount of the inoculum
delivered to the patient will be 3 mL. The catheter itself (30 cm
length, 1 mm interior diameter) cannot be preloaded with virus
suspension. Therefore, the initial .about.250 .mu.L of infusion
will be preservative-free salinein the `dead-space` of the
indwelling catheter. To account for this, the infusion pump will be
programmed for delivery of 3.250 mL. The infusion will be performed
using a Medfusion 3500 (Medex, Inc., Duluth, Ga.) syringe infusion
pump. The virus injection procedure will be completed within 6.5
hrs. The catheter will be removed immediately following the
delivery of PVSRIPO.
[0042] The infusion catheter (PIC 030) and infusion tubing (PIT
400) will be supplied by Sophysa, Inc. (Crown Point, Ind.). The
Infusion Catheter Kit is a 30 cm clear, open-ended catheter (1.0 mm
ID/2.0 mm OD) with 1 cm markings for 20 cm. The catheter comes with
a 30 cm stainless steel stylet, a barbed female luer lock with cap
and a stainless steel trocar. The Infusion Tubing Kit consists of a
3-way stopcock connector with air filter, 4 m of microbore tubing
with antisiphon valve, a red, vented cap and a white luer lock cap.
The catheter products are packaged sterile and non-pyrogenic and
are intended for single (one-time) use only. The infusion will be
performed using a Medfusion 3500 (Medex, Inc. Duluth, Ga.) syringe
infusion pump.
Example 5--Results
[0043] The mechanism of immune checkpoint inhibitors is to release
cytotoxic T cell function from events instigated by tumors that
block their effector functions. Tumors engage a system of naturally
existing `brakes` that control cytotoxic T cells. To the tumor,
this has the advantage of limiting the potential for the immune
system to attack tumors that express mutant proteins and,
therefore, represent a foreign signature. Immune checkpoint
inhibitors reverse this tumor mechanism and release immune
function.
[0044] We have found that PVSRIPO elicits an immune response that
relies on cytotoxic T cells (CTL) attacking tumors. Thus
combination of PVSRIPO with checkpoint inhibitors enhances the
therapeutic effect. As shown below, PVSRIPO, indeed, works to treat
tumors by inducing CTL responses.
[0045] We infected melanoma, breast, brain tumor, prostate cancer
cells with PVSRIPO and collected the supernatant from dying/dead
cells. The supernatant from the infected tumor cells was used to
expose dendritic cells (a population of immune cells that is
responsible for communicating with CTLs and coordinating their
activation) from human subjects. As a consequence, the dendritic
cells exhibited powerful signs of pro-inflammatory activation
(i.e., the virus infection of the tumor cells produced soluble
factors that promote the CTL activation functions of dendritic
cells).
[0046] The activated dendritic cells were then co-cultivated with T
cells (including CTLs) from the same human subject that donated the
dendritic cells. The co-cultured T cells (including CTLs) were then
co-cultivated with uninfected tumor cells from the same lines used
for the infection step.
[0047] We observed high-level cytotoxicity of the activated CTLs
against the tumor cells. See FIG. 2.
[0048] This experiment, in vitro, exemplifies what we believe
happens in patients: virus infection elicits a series of events
that ultimately leads to the generation of a CTL response against
the tumor. This series of events can be enhanced synergistically
with immune checkpoint inhibitors.
[0049] One of the natural existing `brakes` on T cell function
(immune checkpoints) is the PD1-PD-L1 link. Dendritic cells in
tumor often are induced to express PD-L1, which then binds to PD1
on T cells to inhibit activation of the T cells.
[0050] We have demonstrated that dendritic cells exposed to
PVSRIPO/PVSRIPO-tumor lysate increase PD-L1 expression. PD-1 or
PD-L1 inhibitors, paradigmatic checkpoint inhibitors, prevent this
effect and increase CTL activation by PVSRIPO oncolysis.
Example 6--Methods
[0051] Confluent 10 cm dishes of Sum149, MDAMB231, LNCaP, or DM6
cells were infected with mock (DMEM) or PVSRIPO (MOI 0.1) in AIMV
medium for 48 hours. Supernatants were collected and cell debris
was removed by centrifugation. Frozen PBMCs were thawed, washed in
PBS and resuspended at 2.times.10.sup.8 cells in 30 ml AIM-V media
in T-150 tissue culture flasks (3). Cells were incubated for 1 h at
37.degree. C. The non-adherent cells were harvested by rocking the
flask from side to side to dislodge them. The adherent cells were
replenished with 30 ml AIM-V supplemented with 800 U/ml human
GM-CSF and 500 U/ml human IL-4, then incubated at 37.degree. C. DCs
were harvested on day 6, by collecting all non-adherent cells,
followed by a cold PBS wash. Cells that were still adherent were
dissociated with cell dissociation buffer. DCs were washed in AIMV
medium, counted and seeded in 35 mm dishes at 1.times.10.sup.6
cells per dish. Supernatant from onco-lysate was added to DC
cultures and incubated for 24 hours. Supernatant was then removed
and DCs were washed in AIMV medium. PBMCs were thawed and
resuspended in PBS and treated with DNase I at 200 U/ml for 20 min
at 37.degree. C. DNase I-treated PBMCs were incubated for 1 h at
37.degree. C., Non-adherent cells were harvested and stimulated
with DCs loaded with poliovirus-induced tumor lysate at a responder
cell to stimulator DC ratio of 10:1 in the presence of 25 ng/ml
IL-7. All stimulations were done in RPMI 1640 with 10% FCS, 2 mM
L-glutamine, 20 mM HEPES, 1 mM sodium pyruvate, 0.1 mM MEM
non-essential amino acids, 100 IU/ml penicillin, 100 .mu.g/ml
streptomycin and 5.times.10.sup.-5 M -mercaptoethanol (CTL
stimulation medium). The responder T-cell concentration was
2.times.10.sup.6 cells/ml. IL-2 was added at 100 U/ml on day 3 and
every 4-5 days for 12-14 days. T cells were maintained at
1-2.times.10.sup.6 cells/ml in CTL stimulation medium. T cells were
harvested on day 12-14, counted and used as effector T cells in a
europium-release CTL assay. Autologous DCs transfected with tumor
antigen-encoding mRNA were used as targets as controls. For DC
target controls, mRNA-electroporated target cells (as designated in
FIG. 2) were harvested, washed to remove all traces of media and
labeled with europium (Eu). Alternatively original target cells
(Sum149, MDAMB231, LNCaP, or DM6) were labeled with Eu. The
Eu-labeling buffer (1 ml per target) contained 1 ml HEPES buffer
(50 mM HEPES, 93 mM NaCl, 5 mM KCl, 2 mM MgCl.sub.2, pH 7.4), 10
.mu.l Eu (10 mM EuCl.sub.3.6H.sub.2O in 0.01 N HCl), 5 .mu.l DTPA
(100 mM diethylenetriamine pentaacetate in HEPES buffer) and 4
.mu.l DS (1% dextran-sulfate) (4) 5.times.10.sup.6 target cells
were resuspended in 1 ml of the europium-labeling buffer very
gently and incubated on ice for 20 minutes. 30 .mu.l of CaCl2
solution (100 mM) was then added to the labeled cells, mixed and
the cells were incubated for another 5 minutes on ice. 30 ml of
Repair buffer (HEPES buffer with 10 mM glucose, 2 mM CaCl.sub.2)
was added to the cells and the cells were centrifuged at 1000 rpm
for 10 minutes. Cells were counted and 5.times.10.sup.6 cells were
washed 4 times with Repair buffer. After the final wash the cells
were resuspended in CTL stimulation medium without
penicillin-streptomycin at 10.sup.5 cells/ml. Ten thousand
europium-labeled targets (T) and serial dilutions of effector cells
(E) at varying E:T ratios were incubated in 200 .mu.l of CTL
stimulation medium with no penicillin-streptomycin in 96-well
V-bottom plates. The plates were centrifuged at 500.times.g for 3
minutes and incubated at 37.degree. C. for 4 hours. 50 .mu.l of the
supernatant was harvested and added to 150 .mu.l of enhancement
solution (Wallac, Perkin-Elmer) in 96-well flat-bottom plates and
europium release was measured by time resolved fluorescence using
the VICTOR3 Multilabel Counter (Perkin-Elmer). Specific cytotoxic
activity was determined using the formula: % specific
release=[(experimental release-spontaneous release)/(total
release-spontaneous release)].times.100. Spontaneous release of the
target cells was less than 25% of total release by detergent.
Spontaneous release of the target cells was determined by
incubating the target cells in medium without T cells. All assays
were done in triplicate, bars represent average % lysis and error
bars denote SEM.
Example 7
[0052] PVSRIPO antitumor efficacy may be aided by the virus'
ability to elicit strongly immunogenic type 1 interferon (IFN)
responses in infected tumor cells and in infected
antigen-presenting cells (dendritic cells, macrophages, microglia).
However, although type 1 IFN responses are highly desirable as
mediators of immunotherapy, they also engage known immune
checkpoints that can dampen the anti-neoplastic immune response
elicited by PVSRIPO, e.g., PD-L1. Therefore, to maximize PVSRIPO
immunotherapy, combination with immune checkpoint blockade may be
indicated. This is evident in assays in immune-competent, syngeneic
glioma tumor models (e.g., CT2 A). See Martinez-Murillo et al.,
Histol. Histopathol. 12:1309-26 (2007).
[0053] We implanted subcutaneous CT2 A gliomas in C57B16 mice
transgenic for the poliovirus receptor CD155. The CT2 A cells used
to initiate tumors were previously transduced with CD155 (to enable
PVSRIPO infection analogous to human cells). Four groups of
tumor-bearing animals (n=10) were treated as follows: Group I: DMEM
(vehicle to control for virus)+IgG (to control for anti-PD1); Group
II: single intra-tumoral injection of PVSRIPO+IgG; Group III:
single intra-tumoral injection of DMEM+anti-PD1; Group IV: single
intra-tumoral injection of PVSRIPO+anti-PD1. Anti-PD1 was given in
three installments (days 3, 6, 9) by intraperitoneal injection.
Results are shown in FIG. 3; the top panel shows the group results
and the bottom panels shows results for individual mice.
[0054] Both PVSRIPO and anti-PD1 had significant anti-tumor effects
individually (top panel). The combination of the two agents had
added therapeutic effects, suggesting mechanistic synergy (top
panel). Importantly, durable tumor remission (indicated by
flat-lining of the tumor response curves at very low tumor volumes)
was only achieved with the combination treatment.
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