U.S. patent application number 16/458275 was filed with the patent office on 2020-11-19 for cdkn2a companion diagnostic for bladder cancer interferon therapy.
This patent application is currently assigned to Trizell Limited. The applicant listed for this patent is Trizell Limited. Invention is credited to Colin P. DINNEY.
Application Number | 20200360499 16/458275 |
Document ID | / |
Family ID | 1000005037211 |
Filed Date | 2020-11-19 |
United States Patent
Application |
20200360499 |
Kind Code |
A1 |
DINNEY; Colin P. |
November 19, 2020 |
CDKN2a Companion Diagnostic for Bladder Cancer Interferon
Therapy
Abstract
A method of treating a human with bladder cancer that is not
responsive to treatment with bacillus Calmette-Guerin, comprising
measuring the human's level of CDKN2A expression, and then
instilling into the human an agent which induces interferon
expression, e.g., interferon polypeptide or a gene therapy vector
carrying an interferon transgene.
Inventors: |
DINNEY; Colin P.; (Houston,
TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Trizell Limited |
Chinnor |
|
GB |
|
|
Assignee: |
Trizell Limited
Chinnor
GB
|
Family ID: |
1000005037211 |
Appl. No.: |
16/458275 |
Filed: |
December 11, 2018 |
PCT Filed: |
December 11, 2018 |
PCT NO: |
PCT/US2018/064829 |
371 Date: |
July 1, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62597473 |
Dec 12, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
C12N 7/00 20130101; C12N 15/86 20130101; C12Q 1/6886 20130101; G01N
2800/7028 20130101; A61K 48/00 20130101; C07K 14/56 20130101; A61K
39/04 20130101; C12Q 2600/106 20130101; G01N 2800/00 20130101; C12N
2750/14143 20130101 |
International
Class: |
A61K 39/04 20060101
A61K039/04; C12Q 1/6886 20060101 C12Q001/6886; C07K 14/56 20060101
C07K014/56; C12N 7/00 20060101 C12N007/00; C12N 15/86 20060101
C12N015/86; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method of treating non-muscle invasive bladder cancer in a
human, comprising: a. identifying a human diagnosed with non-muscle
invasive bladder cancer, and then b. measuring the human's level of
CDKN2A expression, and then c. instilling into the lumen of the
bladder of said human interferon.
2. The method of claim 1, where the bladder cancer is
high-grade.
3. The method of claim 1, where the interferon is administered as a
non-replicating vector.
4. The method of claim 3, where said non-replicating vector
comprises a replication-deficient viral vector carrying an
interferon transgene.
5. The method of claim 4, where said replication-deficient viral
vector carrying an interferon transgene comprises nadofaragene
firadenovec.
8. The method of claim 1, where said measuring the human's level of
CDKN2A expression entails taking a bladder tissue sample and
measuring the level of CDKN2A expression in that tissue sample.
9. The method of claim 1, where said measuring the human's level of
CDKN2A expression entails taking a urine sample and measuring the
level of CDKN2A expression in it.
10. The method of claim 9, where said measuring the level of CDKN2A
expression comprises analyzing the exosomes in said urine
sample.
11. The method of claim 9, wherein said measuring the level of
CDKN2A expression comprises analyzing free DNA in said urine
sample.
12. The method of claim 1, further comprising the step of
administering to said human a checkpoint inhibitor after said step
of measuring the level of CDKN2A expression.
13. The method of claim 1, wherein said step of measuring the
human's level of CDKN2A expression comprises using fluorescent in
situ hybridization using a probe able to hybridize with a portion
of CDKN2A.
14. The method of claim 13 wherein said probe comprises a portion
which hybridizes with at least one exon contained in CDKN2A.
15. The method of claim 13 wherein said probe is about 40 bp in
length.
16. The method of claim 14 wherein said probe is about 40 bp in
length.
Description
RELATED APPLICATIONS
[0001] This application is a United States National Stage filing of
PCT/US2018/064829 filed 11 Dec. 2018, which in turn asserts
priority from United States provisional patent filing Ser. No.
62/597473 filed 12 Dec. 2017, the contents of which are here
incorporated by reference.
UNITED STATES GOVERNMENT FUNDING/INTEREST
[0002] None
BACKGROUND
[0003] Non-muscle-invasive bladder cancer (NMIBC) represents the
most common disease state for patients with newly diagnosed bladder
cancer. Those with high-grade (HG) tumors are at significant risk
for both recurrence and progression. Bacillus Calmette-Guerin (BCG)
represents the current preferred management. Nonetheless,
approximately 30% of patients will not respond to BCG; among those
who demonstrate an initial response, more than 50% will experience
recurrence and progression during long-term follow-up.
[0004] The optimal management of patients with persistent or
recurrent tumor after BCG remains controversial. Although radical
cystectomy provides cancer eradication, many patients are elderly,
have significant co-morbidities with an attendant diminished
performance status, and often are unwilling to undergo radical
extirpative surgery. Non-extirpative treatment options are
available, but studies to date have included relatively small
patient numbers and used varied definitions of treatment success.
Indeed, the US Food and Drug Administration (FDA) and genitourinary
oncology community agree that scant progress has been made in the
management of this disease since the initial approval of BCG. Thus
an effective alternative to radical cystectomy for patients with
disease recurrence after BCG treatment remains an important unmet
clinical need.
[0005] Several agents have been evaluated as second-line treatment
after BCG; however, none (to date) have provided robust and durable
responses. Valrubicin (Valstar; Endo Pharmaceuticals, Malvern,
Pa.), the only agent currently approved by the FDA for the
treatment of BCG-refractory CIS, provided a complete response rate
of 18% at 6 months and a 1-year disease-free survival rate of
approximately 10%. Promising results from early-phase trials have
been reported for intravesical taxane and gemcitabine. Joudi et al.
reported the final results from a national multicenter phase II
trial of BCG plus IFN.alpha.-2b and noted that 45% of patients with
BCG failure were free from recurrence at 2 years. However, only 44%
were treated for an HG recurrence, and 61% received only one prior
course of BCG. A recent retrospective analysis of BCG and
IFN.alpha.-2b reported a 38.6% RFS at 12 months. Again, many of
these patients (20 of 44) received only one prior course of BCG,
and 16 patients experienced relapse after 12 months. Overall, the
limited number of patients studied in previous trials, as well as
the modest RFS with treatment despite a less stringently defined
eligibility, illustrates the unmet need for effective and
evidence-based second-line therapy for patients with
BCG-unresponsive disease that improves disease-specific patient
outcomes and avoids cystectomy.
[0006] Recombinant intravesical interferon alfa-2b protein
(IFN.alpha.-2b; Intron A; Merck, Kenilworth, N.J.) demonstrated
promising initial clinical results in NMIBC.
[0007] Intravesical IFN.alpha.-2b gene delivery offers a novel
approach and increases the duration of exposure to IFN.alpha.-2b.
Recombinant adenovirus (rAd)-IFN.alpha.-2b is a
replication-deficient adenovirus-based gene transfer vector that
encodes the human IFN.alpha.-2b gene. Syn3, a polyamide surfactant,
is incorporated into the drug formulation (ADSTILADRIN.RTM.
rAd-IFN.alpha./Syn3.TM., FKD Therapies Oy, Kuopio, Finland) to
enhance adenoviral transduction of the bladder lining. Dramatic
enrichment of rAd-IFN.alpha. gene transfer and expression has been
shown with Syn3.TM. in both normal urothelium and human urothelial
carcinoma that grows in mice. rAd-IFN.alpha.-2b gene therapy mimics
the physiologic events associated with viral infection, which
results in local rather than systemic IFN.alpha.-2b production and
subsequent tumor regression.
[0008] We performed a phase I dose-ascending study of
rAd-IFN.alpha./Syn3 for patients with BCG-refractory and relapsing
NMIBC. First-generation replication-deficient serotype 5 adenovirus
vector, which expressed human interferon alfa-2b (IFN.alpha.-2b)
cDNA under a cytomegalovirus promoter, was produced under good
manufacturing practice conditions in 293 cells, as previously
described, with slight modifications of the process. It was tested
to be free of endotoxin, microbiologic contaminants, and other
impurities. The structure of the vector was verified by sequencing.
Production of recombinant IFN.alpha.-2b was verified from each
production lot with immunologic methods. The excipient Syn3 is a
polyamide surfactant that enhances adenoviral gene transfer to the
bladder epithelium. Dose-dependent adenoviral gene transfer and
urine concentrations of IFN.alpha.-2b were confirmed. Of 14
patients treated with dose levels of rAd-IFN.alpha./Syn3.RTM. that
resulted in measurable urine IFN.alpha., six (43%) were free from
recurrence at 3 months and had no dose-limiting toxicity, and two
patients remained disease free at 29 and 39 months.
[0009] These provocative findings, predominantly at the two highest
doses, prompted us to pursue a phase II study, designed to evaluate
the efficacy and safety of intravesical rAd-IFN.alpha./Syn3.TM. for
patients with HG NMIBC refractory to, or with relapse after, BCG.
This randomized, open-label, parallel-arm study was conducted
across 13 centers in the United States between Nov. 5, 2012, and
Apr. 8, 2015. The protocol, administrative oversight, and accrual
timelines were designed and conducted by the Society of Urologic
Oncology Clinical Trials Consortium. The study protocol and
informed consent form were reviewed and approved by the respective
responsible site institutional review boards and biosafety
committees.
[0010] We assessed the efficacy and safety of recombinant
adenovirus interferon alfa with Syn3.TM. (rAd-IFN.alpha./Syn3.TM.),
a replication-deficient recombinant adenovirus gene transfer
vector, for patients with high-grade (HG) BCG-refractory or
relapsed NMIBC. In this open-label, multicenter (13 centers),
parallel-arm, phase II study, of 43 patients with HG BCG-refractory
or relapsed NMIBC received intravesical rAd-IFN.alpha./Syn3.TM.
(randomly assigned 1:1 to 1.times.1011 viral particles (vp)/mL or
3.times.1011 vp/mL). Patients who responded at months 3, 6, and 9
were retreated at months 4, 7, and 10. The primary end point was
12-month HG recurrence-free survival (RFS). All patients who
received at least one dose were included in efficacy and safety
analyses.
[0011] In our study, forty patients received
rAd-IFN.alpha./Syn3.TM. (1.times.1011 vp/mL, n=21; 3.times.1011
vp/mL, n=19) between Nov. 5, 2012, and Apr. 8, 2015. The trial was
designed to enroll 40 patients unable or unwilling to undergo
radical cystectomy, and there were two dosage groups of 20 patients
each. Eligible patients were 18 years or older and had HG
BCG-refractory or relapsed NMIBC, including papillary NMIBC alone
(Ta or T1), carcinoma in situ (CIS) alone, or a combination of CIS
and papillary disease. BCG-refractory disease was defined as the
inability to achieve a disease-free state at 6 months after
adequate induction BCG therapy with either maintenance or
reinduction at 3 months. "Adequate induction" was defined as a
minimum of five of six treatments, and "adequate maintenance" was
defined as a minimum of two of three treatments. BCG relapse was
defined as recurrence within 1 year after a complete response to
adequate BCG treatment (at least five and two instillations).
Patients were required to have undergone visually complete
resection of papillary lesions by transurethral resection of
bladder tumors. Patients could not have received intravesical
therapy within 3 months before beginning study treatment, with the
exception of cytotoxic agents when administered as a single
instillation immediately after a transurethral resection. All
participants who entered the study provided written or oral
informed consent.
[0012] Patients were assigned by computer-generated random
assignment, with a constrained 1:1 sequence, to receive either
low-dose (1.times.1011 viral particles [vp]/mL) or high-dose
(3.times.1011 vp/mL) rAd-IFN.alpha./Syn3. These doses were the most
promising observed in the phase I study. The total doses
administered were 7.5.times.1012 vp in the low-dose group and
2.25.times.1013 vp in the high-dose group. Treatment allocation was
performed centrally with a block size of two for all patients who
had successfully completed screening, with the constraint that the
first four patients at each site were balanced between cohorts.
[0013] rAd-IFN.alpha./Syn3.TM. in 75 mL was administered
intravesically through a urethral catheter, with a planned
retention time of 1 hour; an anti-cholinergic treatment was allowed
to relieve urinary urgency and permit adequate retention. Patients
without recurrence of HG disease at months 3, 6, and 9, as
evaluated by cytology, cystoscopy, and biopsy (if clinically
indicated) were then retreated at months 4, 7, and 10. At 12
months, a final efficacy evaluation was performed. This evaluation
included a protocol-mandated biopsy from the site of the index
tumor and at least five random biopsies, including the bladder
dome, trigone, right and left lateral wall, posterior wall, and
prostatic urethra in men with positive cytology or prior disease in
this region.
[0014] During the study, patients were contacted weekly by phone
for the first month after each treatment on days 7, 14 (of months 7
and 10 only), 21, and 28 (.+-.1 day) to provide information about
adverse events (AEs) and concomitant medication use. Assessments
for treatment failure were made between 14 and 7 days before
retreatment. Patients who were withdrawn from treatment before
study completion underwent a safety assessment at least 30 days
after last administration of the study drug. All patients are being
monitored in a 3-year long-term follow-up period to (1) determine
recurrence of HG disease in those patients with a complete response
and (2) to assess the long-term impact of treatment with
rAd-IFN.alpha./Syn3.TM.
[0015] The primary end point was freedom from HG disease recurrence
at 12 months, defined by a negative for cause or end of study
biopsy. Secondary end points included response to treatment,
defined as no evidence of recurrence of HG disease at 3, 6, and 9
months; incidence and time to cystectomy; and concentration of
IFN.alpha.-2b in the urine. Safety assessments included physical
examination, monitoring of vital signs, ECG, and standard clinical
chemistry, hematology, and urinalysis assessments (performed by
local laboratories). Safety end points include type, incidence,
relatedness, and severity of AEs and severe (.gtoreq.grade 3) AEs
(SAES), as assessed by National Cancer Institute Common Terminology
Criteria for Adverse Events (version 4.03).
[0016] We determined that a cohort of 20 patients would be
sufficient to give an 80% probability of rejection of a HG
recurrence-free survival (RFS) rate of 10% with an exact 5%
one-sided test when the true HG RFS rate was 35%. The operating
characteristics for this Fleming design were calculated exactly
with the binomial distribution described by A'Hern. The
hypothesis--that the response rate was equal to or less than the
reference rate-was rejected if five or more of the 20 patients
achieved HG RFS at 12 months. The proportion of patients who
achieved HG RFS at 3, 6, 9, and 12 months was reported for each
dose group, together with an exact 90% CI for the proportion. The
time to HG recurrence or death was summarized with the Kaplan-Meier
method. Analyses were performed with SASTM (version 9 or later; SAS
Institute, Cary, N.C.). Both the safety and efficacy (modified
intention-to-treat) analysis sets included all patients who
received at least one dose of rAd-IFN.alpha./Syn3.RTM.. A data
monitoring committee oversaw the study according to the data
monitoring plan. All analytical assays were developed and
validated. Samples were tested according to good laboratory
practices methods at Covance Laboratories Ltd (Harrogate, United
Kingdom).
[0017] Baseline patient characteristics are provided in Table
1:
TABLE-US-00001 TABLE 1 Baseline Patient Characteristics No. (%) by
rAd-IFN.alpha./Syn3 Dose Group 1 .times. 10.sup.11 3 .times.
10.sup.11 No. (%) vp/mL vp/mL Overall Characteristics (n = 21) (n =
19) (N = 40) Median (IQR) age, years 70 (67-74) 73 (62-81) 70.5
(64.5-77.5) Sex Male 19 (90) 14 (73.7) 33 (82.5) Female 2 (9.5) 5
(26.3) 7 (17.5) ECOG PS 0 16 (76.2) 18 (94.7) 34 (85.0) 1 5 (23.8)
1 (5.3) 6 (15.0) History of radiation therapy* 1 (4.8) 1 (5.3) 2
(5) BCG failure classification Relapsed 10 (47.6) 9 (47.4) 19
(47.5) Refractory 11 (52.4) 10 (52.6) 21 (52.5) No. of previous BCG
courses 1 1 1 2 2 10 12 22 .gtoreq.3.uparw. 10 6 16 Primary tumor
classification at enrollment CIS 12 (57.1) 9 (47.4) 21 (52.5) Ta 2
(9.5) 2 (10.5) 4 (10) Ta and CIS 3 (14.3) 1 (5.3) 4 (10) T1 2 (9.5)
4 (21.1) 6 (15) T1 and CIS 2 (9.5) 3 (15.8) 5 (12.5) Abbreviations:
BCG, bacillus Calmette-Guerin; CIS, carcinoma in situ; ECOP PS,
Eastern Cooperative Oncology Group performance status; IQR,
interquartile range; rAd-IFN.alpha./Syn3, recombinant adenovirus
interferon alpha protein/Syn3 (a nonreplicating recombinant
adenovirus gene transfer vector for patients with high-grade
BCG-refractory or relapsed non-muscle-invasive bladder cancer); Ta,
papillary urothelial carcinoma confined to the mucosa; T1,
micro-invasive urothelial carcinoma invasive into lamina propria
but not muscularis propria; vp, viral particles. *Radiology was 10
or more years before screening in each of these three patients; as
such, they were deemed eligible for study enrollment. .uparw.Range
of previous courses, 3 to 8.
[0018] The 12-month HG RFS rate was comparable between the two dose
groups, with 33.3% of patients (7 of 21; 90% CI, 16.8 to 53.6) in
the low-dose group and 36.8% (7 of 19; CI, 18.8 to 58.2) in the
high-dose group alive and free of HG disease at 12 months. Overall,
35.0% of patients (14 of 40; 90% CI, 22.6% to 49.2%) remained free
of HG recurrence at 12 months after the initiation of
rAd-IFN.alpha./Syn3 treatment. Off-schedule disease assessments did
not affect findings (Appendix, online only). The median time to HG
recurrence or death was 6.5 months (90% CI, 3.52 to 12.78 months);
the median time to HG recurrence was 3.52 months (90% CI, 3.02 to
12.78 months) for the low-dose group and was 11.73 months (90% CI,
5.88 months to not evaluable) for the high-dose group.
[0019] Fourteen patients (35.0%; 90% CI, 22.6% to 49.2%) remained
free of HG recurrence 12 months after initial treatment. Comparable
12-month HG RFS was noted for both doses. Of these 14 patients, two
experienced recurrence at 21 and 28 months, respectively, after
treatment initiation, and one died as a result of an upper tract
tumor at 17 months without a recurrence. rAd-IFN.alpha./Syn3 was
well tolerated; no grade four or five adverse events (AEs)
occurred, and no patient discontinued treatment because of an
adverse event. The most frequently reported drug-related AEs were
micturition urgency (n=16; 40%), dysuria (n=16; 40%), fatigue
(n=13; 32.5%), pollakiuria (n=11; 28%), and hematuria and nocturia
(n=10 each; 25%).
[0020] When patient subgroups and secondary end points were
considered in exploratory analyses, the 12-month HG RFS rates were
broadly similar for men and women, for younger and older patients,
for refractory or relapsed NMIBC, for CIS only or papillary tumors
and CIS, and for patients with Ta and T1 disease only.
TABLE-US-00002 TABLE 2 Incidence of HG RFS at 3, 6, 9 and 12 Months
rAd-IFN.alpha./Syn3 Dose Group 1 .times. 10.sup.11 vp/mL 3 .times.
10.sup.11 vp/mL Overall (n = 21) (n = 19) (N = 40) No. (%) No. (%)
No. (%) of 90% Cl of 90% Cl of 90% Cl Variable Patients (%)*
Patients (%)* Patients (%)* RFS at secondary end point analysis
time 3 months 10 (47.6) 28.6-67.2 13 (68.4) 47.0-85.3 23 (57.5)
43.3-70.8 6 months 8 (38.1) 20.6-58.3 9 (47.4) 27.4-68.0 17 (42.5)
29.2-56.7 9 months 8 (38.1) 20.6-58.3 9 (47.4) 27.4-68.0 17 (42.5)
29.2-56.7 12 months 7 (33.3) 16.8-53.8 7 (36.8) 18.8-58.2 14 (35.0)
22.6-49.2 HG recurrence-free subgroup at 12 months Refractory NMIBC
8 (38.1) 20.6-58.3 (n = 31) Relapsed NMIBC 6 (31.6) 14.7-53.0 (n =
19) CIS only 6 (28.6) 13.2-48.7 (n = 21) Papillary tumor 3 (33.3)
9.7-65.6 (n = 9) Ta + Ti disease only 5 (50.0) 22.2-77.8 (n = 10)
Serum antiadenoviral antibody Positive (n = 22) 10 (45.5) 27.1-64.7
Negative (n = 17) 4 (23.5) 8.5-46.1 Abbreviations: CIS, carcinoma
in situ; GG, high-grade; NMIBC, non-muscle-invasive bladder cancer;
rAd-IFN.alpha./Syn3, recombinant adenovirus interferon alpha
protein/Syn3 (a nonreplicating recombinant adenovirus gene transfer
vector for patients with high-grade bacillus
Calmette-Guerin-refractory or relapsed NMIBC); RFS, relapse-free
survival; Ta, papillary urothelial carcinoma confined to the
mucosa; Ti, micro invasive urothelial carcinoma invasive into
lamina propria but not muscularis propria; vp, viral particles. *Cl
is for the proportion of patients with HG RFS; 90% Cls are based on
the exact binomial method.
[0021] Interestingly, of the 14 patients who were recurrence free
at 12 months, 10 (71%) of the 14 had an anti-adenovirus antibody
response (defined as four times the pre-dose titer), compared with
11 (24%) of 25 who experienced recurrence.
[0022] In long-term follow-up, seven patients (18%) who withdrew
from the study because of HG disease recurrence within the 12-month
study period died at a median of 16 months (range, 2 to 26 months)
after the withdrawal date. There is no indication that these deaths
were treatment related. The cause of death was unknown in four
patients, whereas two died as a result of progressive bladder
cancer and one died as a result of liver failure unrelated to
treatment 17 months after withdrawal from the study. The four
patients for whom the cause of death is unknown were being observed
locally after they completed their end-of-study evaluation.
Fourteen patients (35%) who experienced an HG recurrence within the
first year underwent a radical cystectomy at a median of 9 months
(range, 4 to 28 months) from day 1 of month 1.
[0023] Patients are being monitored for 3 years to collect
long-term follow-up data. Of the 14 patients who remained disease
free at 12 months, additional follow-up data are being collected
for 11; 3 withdrew from the study. Nine of these 11 patients are
alive, and eight remained disease-free during a period of 15 to
more than 36 months. Two patients experienced HG recurrence at 21
and 28 months, respectively, from the start of treatment. One of
these patients who experienced progression to muscle invasion
underwent a radical cystectomy 31 months after the initiation of
treatment and later died at 41 months. The other, who experienced
recurrence at 21 months, remained alive and free from distant
recurrence at 36 months. One patient free from bladder recurrence
at 12 months died as a result of an upper tract tumor at 17
months.
TABLE-US-00003 TABLE 3 Durability of HG RFS Since Start of
Treatment With rAd-IFN.alpha./Syn3 .RTM. Duration of Bladder Time
of Last Stage at Dose HG RFS Since Follow-Up from Status at Last
Entry Group Day 1 (months) Day1 (months) Follow-Up Ta/CIS High 21
47 Recurrence of HGD Died at 38 months Ta Low 28 41 Recurrence of
28 months Cystectomy at 31 months Died at 41 months CIS Low 15 15
CR Withdrew Ta/CIS Low 30 36 Recurrence of HGD Ta High 16 16 CR
Withdrew T1/CIS Low 35 37 CR T1 Low 30 50 CR T1 High 36 36 CR CIS
High 38 39 CR CIS High 34 37 CR CIS High 27 27 CR CIS Low 34 37 CR
T1/CIS Low 17 17 Died of upper tract recurrence Ta High 13 13 CR
Withdrew NOTE. Duration of HG RFS represent the number of months
from day 1 that a complete response within the bladder has been
documented based on yearly report. Three patients withdrew from the
study after the 1-month end-of-study evaluation. Two patients had
recurrence of HGD at 21 and 28 months from day 1. One of these
patients underwent a cystectomy but later died. One patient died of
an upper tract tumor with a bladder recurrence. Abbreviations: CIS,
carcinoma in situ; CR, complete response; HG, high-grade; HGD,
high-grade disease; rAd-IFN.alpha./Syn3, recombinant adenovirus
interferon alpha protein/Syn3 .RTM. (a nonreplicating recombinant
adenovirus gene transfer vector for patients with HG bacillus
Calmette-Guerin-refractory or relapsed non-muscle-invasive-bladder
cancer); RFS, relapse-free survival; Ta, papillary urothelial
carcinoma confined to the mucosa; T1, micro-invasive urothelial
carcinoma invasive into lamina propria but not muscularis
propria.
[0024] Our results showed that rAd-IFN.alpha./Syn3.TM. was well
tolerated. It demonstrated promising efficacy for patients with HG
NMIBC after BCG therapy who were unable or unwilling to undergo
radical cystectomy.
[0025] While potentially promising, however, these data show
several failings in our treatment plan. First, treatment was
ineffective in the majority of patients: fully 65% of patients
failed to achieve a 12-month HG RFS by intention-to-treat analysis
of all patients dosed. Likewise, the 12-month RFS in heavily
pretreated patients was 31%. Notably, responses were durable: the
majority remained disease-free for close to 24 months. We noted
that for patients with any element of CIS, 70% failed to achieve a
durable complete response. Indeed, for patients with papillary
disease only at study entry, only 50% achieved RFS.
[0026] To address these failings, we have pursued two approaches.
First, we have designed and are currently pursuing a larger trial
involving significantly more patients and a high-dose of
rAd-IFN.alpha./Syn3.TM.. This trial may provide evidence that a
higher dose of rAd-IFN.alpha./Syn3.TM. is effective where a lower
dose was not merely less effective, but entirely ineffective.
[0027] Second, we collected tissue samples from patients involved
in the above-discussed Phase II completed trial. These samples
enable us to analyze gene expression for each patient. This enables
us to compare gene expression in patients who responded to
treatment against gene expression in patients who did not
respond.
[0028] Gene expression analysis may be done using tumor biopsy
samples.
[0029] Alternatively, for bladder cancer, urine samples if properly
preserved may contain exosomes which enable analysis of oncogene
expression. Exosomes are small (30-100 nm) endocytic, cell-derived
vesicles. They are secreted by most human cell types, including
cancer cells, and they may be absorbed via endocytosis into
recipient cells. Exosomes can contain functional bio-molecules such
as dsDNA, and may be found in human blood and urine. Exosomal DNA
("exoDNA") represents the entire genome and, where the exosome is
produced from a tumor cell, reflects the mutational status of the
tumor cell. ExoDNA in tumor-derived exosomes found in a urine
sample taken from a bladder cancer patient thus provides a
non-invasive circulating biomarker useful for the sensitive
detection of cancer and a more accurate determination of potential
responsiveness to interferon-based therapies.
[0030] ExoDNA includes both single-stranded and double-stranded
DNA. ExoDNA is found encapsulated in the interior of the exosome
membrane and bound to the exterior of the exosome membrane. In
internal exoDNA, dsDNA predominates. Typically, exoDNA encapsulated
in the interior of the exosome membrane ranges from 0.1-2.5 kb,
while exoDNA bound to the exosome membrane exterior is >2.5 kb.
Much of the exoDNA associated with tumor exosomes is
double-stranded DNA bound to the exterior of the exosome membrane.
ExoDNA, which includes dsDNA, can provide an unusually accurate
diagnostic tool because it provides a complete sample of the
complete genome in readily-assayable form in the urine of a patient
with bladder cancer.
[0031] Alternatively, one can assay micro vesicles. Micro vesicles
are another type of extracellular vesicle, between 50 and 1,000
nanometers (nm) in diameter, found in many types of body fluids as
well as the interstitial space between cells. Micro vesicles are
made from fragments of plasma membrane. They are thus distinct from
exosomes, which are smaller and generated intra-cellularly. In
contrast to exosomes, which do not contain mitochondrial DNA, micro
vesicles derived from astrocytes and glioblastoma include
mitochondrial DNA. Micro vesicles appear equivalent to exosomes for
the purposes of this invention.
[0032] For a human patient diagnosed with bladder cancer, one may
collect exosomes by collecting a urine sample. Sample storage
techniques able to preserve DNA and RNA are known in the art.
Similarly, isolation of exosomes from urine may be done using
conventional separation techniques.
[0033] Alternatively, one may employ fluorescent in situ
hybridization (FISH). FISH is a molecular cytogenetic technique
that uses fluorescent probes that bind to specific target parts of
the chromosome, but when adequately stringent hybridization
conditions are used, does so with a high degree of sequence
complementarity. FISH was developed in the early 1980s. See e.g.,
Langer-Safer, P. R. et al., Immunological Method For Mapping Genes
On Drosophila Polytene Chromosomes, 79 Proceedings of the National
Academy of Sciences 4381 (1982). FISH can detect and localize the
presence or absence of specific DNA sequences on chromosomes or in
DNA fragments such as free DNA found in urine.
[0034] To use FISH in our diagnostic method, one constructs a probe
able to hybridize with a portion of CDKN2A, and preferably with one
or more of the eight exons contained in CDKN2A. The probe must be
large enough to hybridize specifically with its target but not so
large as to require non-stingent hybridization conditions, which
conditions may lead to formation of false-positive hybridization to
an inappropriate target sequence. We prefer a probe of about 40 bp,
but longer or shorter may be used depending on the specific CDKN2A
mutation sought. Once made, the probe is tagged directly with
fluorophores, with targets for antibodies or with biotin. Tagging
can be done in various ways taught in the art, such as nick
translation or Polymerase Chain Reaction using tagged
nucleotides.
[0035] To find out whether and where the fluorescent probe is bound
to the target DNA in e.g., a urine sample, one may use fluorescence
microscopy. FISH can also be used to detect and localize specific
RNA targets (e.g., mRNA) in cells, circulating tumor cells, and
tissue samples.
[0036] As an alternative to sequencing exoDNA in a urine sample,
one may isolate free DNA from the urine sample and sequence that
free DNA.
[0037] Analysis of exoDNA reveals a critical difference in the
populations of bladder cancer patients who respond to interferon
therapy and those who do not. Patients who respond to treatment
have a different level of expression of CDKN2A than do patients who
do not respond to treatment. These different levels in expression
appear to arise from deletions in the gene, or a loss of
heterozygosity in the gene.
[0038] Our insight can improve the safety and efficacy of rAd-IFN
gene therapy because it enables the attending physician to limit
treatment to those patients most likely to respond. Further, our
insight can similarly improve the safety and efficacy of other
instilled treatments which induce interferon expression, e.g., BCG
vaccine. Similarly, our insight can improve the effectiveness of
cancer "checkpoint inhibitors." Checkpoint inhibitors are thought
to be ineffective in cancer patients with CDKN2A deletions.
Identifying such patients enables the artisan to combine interferon
therapy with checkpoint inhibitor therapy in such patients, using
the interferon to overcome the inhibition of the checkpoint
inhibitor.
[0039] We thus here intend this patent to cover a method of
treating bladder cancer, comprising diagnosing bladder cancer in a
human, measuring the human's level of CDKN2A expression, and then
instilling into the human an agent which induces interferon
expression.
[0040] We similarly intend this patent to cover that method of
treating bladder cancer, where the bladder cancer is
high-grade.
[0041] We similarly intend this patent to cover that method of
treating bladder cancer, where the agent is a non-replicating agent
such as rAd-IFN or mycobacterium cell wall extract.
[0042] We similarly intend this patent to cover that method of
treating bladder cancer, where the agent is a replicating agent
such as BCG vaccine.
[0043] We similarly intend this patent to cover that method of
treating bladder cancer, where measuring the human's level of
CDKN2A expression entails taking a bladder tissue sample and
measuring the level of CDKN2A expression in that tissue sample.
Alternatively, we intend this patent to cover that method of
treating bladder cancer, where measuring the human's level of
CDKN2A expression entails taking a urine sample, and measuring the
level of CDKN2A expression in it, preferably by analyzing the
exosomes in it, but also by analyzing e.g., free DNA in it.
[0044] We similarly intend this patent to cover that method of
treating bladder cancer, where the human also gets treated with a
cancer checkpoint inhibitor.
[0045] We similarly intend our patent to cover a companion
diagnostic test able to measure the level of CDKN2A expression in a
bladder tissue sample.
[0046] Further embodiments and variations will be readily apparent
to the artisan after reviewing this disclosure. We thus intend our
appended legal claims to encompass such embodiments.
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