U.S. patent application number 14/895551 was filed with the patent office on 2016-05-05 for compositions and methods for identification, assessment, prevention, and treatment of cancer using pd-l1 isoforms.
The applicant listed for this patent is DANA-FARBER CANCER INSTITUTE, INC.. Invention is credited to Peter Hammerman.
Application Number | 20160122829 14/895551 |
Document ID | / |
Family ID | 52008513 |
Filed Date | 2016-05-05 |
United States Patent
Application |
20160122829 |
Kind Code |
A1 |
Hammerman; Peter |
May 5, 2016 |
Compositions and Methods for Identification, Assessment,
Prevention, and Treatment of Cancer Using PD-L1 Isoforms
Abstract
The present invention relates to methods for identifying,
assessing, preventing, and treating cancer (e.g., head, neck,
and/or lung cancers in humans). A variety of PD-L1 isoforni
biomarkers are provided, wherein alterations in the copy number of
one or more of the biomarkers and/or alterations in the amount,
structure, and/or activity of one or more of the biomarkers is
associated with cancer status and indicates amenability to
treatment or prevention by modulating PD-1 and/or PD-L! levels.
Inventors: |
Hammerman; Peter; (Newton,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DANA-FARBER CANCER INSTITUTE, INC. |
Boston |
MA |
US |
|
|
Family ID: |
52008513 |
Appl. No.: |
14/895551 |
Filed: |
June 2, 2014 |
PCT Filed: |
June 2, 2014 |
PCT NO: |
PCT/US14/40504 |
371 Date: |
December 3, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61831894 |
Jun 6, 2013 |
|
|
|
Current U.S.
Class: |
514/44A ;
435/375; 435/6.11; 435/6.12; 435/7.23; 436/501; 506/16; 506/2;
506/9; 530/387.7; 536/24.31 |
Current CPC
Class: |
C12N 15/1138 20130101;
G01N 2333/4703 20130101; G01N 33/57492 20130101; C12Q 2600/156
20130101; C12Q 1/6886 20130101; G01N 2500/00 20130101; G01N 2500/04
20130101; G01N 2333/70596 20130101; G01N 33/57407 20130101; G01N
2333/70503 20130101; C12Q 2600/158 20130101; C12N 2320/30 20130101;
C12Q 2600/112 20130101; G01N 33/57423 20130101; G01N 2500/10
20130101; C12Q 2600/106 20130101; C12Q 2600/118 20130101; G01N
2800/50 20130101; G01N 2800/52 20130101; A61P 35/00 20180101; C12N
2310/11 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 15/113 20060101 C12N015/113; G01N 33/574 20060101
G01N033/574 |
Goverment Interests
STATEMENT OF RIGHTS
[0002] This invention was made with government support under Grant
NiH U24CA143867 awarded by the National Institutes of Health. The
U.S. government has certain rights in the invention. This statement
is included solely to comply with 37 C.F.R. .sctn.401.14(a)(f)(4)
and should not be taken as an assertion or admission that the
application discloses and/or claims only one invention.
Claims
1. A method of determining whether a subject is afflicted with a
head, neck, or lung cancer or at risk for developing a head, neck,
or lung cancer, the method comprising: a) obtaining a biological
sample from the subject; b) determining the copy number, level of
expression, or level of activity of one or more biomarkers listed
in Table 1 or a fragment thereof in the subject sample; c)
determining the copy number, level of expression, or level of
activity of the one or more biomarkers in a control; and d)
comparing the copy number, level of expression, or level of
activity of said one or more biomarkers detected in steps b) and
c); wherein a significant increase in the copy number, level of
expression, or level of activity of the one or more biomarkers in
the subject sample relative to the control copy number, level of
expression, or level of activity of the one or more biomarkers
indicates that the subject is afflicted with the head, neck, or
lung cancer or is at risk for developing the head, neck, or lung
cancer.
2. A method of determining whether a subject afflicted with a head,
neck, or lung cancer or at risk for developing a head, neck, or
lung cancer would benefit from modulating PD-1 and/or PD-L1 levels,
the method comprising: a) obtaining a biological sample from the
subject; b) determining the copy number, level of expression, or
level of activity of one or more biomarkers listed in Table 1 or a
fragment thereof in the subject sample; c) determining the copy
number, level of expression, or level of activity of the one or
more biomarkers in a control; and d) comparing the copy number,
level of expression, or level of activity of said one or more
biomarkers detected in steps b) and c); wherein a significant
increase in the copy number, level of expression, or level of
activity of the one or more biomarkers in the subject sample
relative to the control copy number, level of expression, or level
of activity of the one or more biomarkers indicates that the
subject afflicted with the head, neck, or lung cancer or at risk
for developing the head, neck, or lung cancer would benefit from
modulating PD-L1 and/or PD-L1 levels.
3. A method for monitoring the progression of a head, neck, or lung
cancer in a subject, the method comprising: a) detecting in a
subject sample at a first point in time the copy number, level of
expression, or level of activity of one or more biomarkers listed
in Table 1 or a fragment thereof; b) repeating step a) at a
subsequent point in time; and c) comparing the copy number, level
of expression, or level of activity of said one or more biomarkers
detected in steps a) and b) to monitor the progression of the head,
neck, or lung cancer.
4. The method of any one of claims 1-3, wherein the one or more
biomarkers comprises soluble PD-L1, optionally wherein the soluble
PD-L1 a) comprises an amino acid sequence of SEQ ID NO: 13 or 15 or
an amino acid sequence that is at least 80% identical to SEQ ID NO:
13 or 15; or b) comprises a nucleic acid molecule comprising a
nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:
13 or 15 or an amino acid sequence that is at least 80% identical
to SEQ ID NO: 13 or 15.
5. The method of claim 3, wherein an at least twenty percent
increase or an at least twenty percent decrease between the copy
number, level of expression, or level of activity of the one or
more biomarkers in the subject sample at a first point in time
relative to the copy number, level of expression, or level of
activity of the one or more biomarkers in the subject sample at a
subsequent point in time indicates progression of the head, neck,
or lung cancer; or wherein less than a twenty percent increase or
less than a twenty percent decrease between the copy number, level
of expression, or level of activity of the one or more biomarkers
in the subject sample at a first point in time relative to the copy
number, level of expression, or level of activity of the one or
more biomarkers in the subject sample at a subsequent point in time
indicates a lack of significant progression of the head, neck, or
lung cancer.
6. The method of claim 3, wherein between the first point in time
and the subsequent point in time, the subject has undergone
treatment to modulate PD-1 and/or PD-L1 levels.
7. A method for stratifying subjects afflicted with a head, neck,
or lung cancer according to predicted clinical outcome of treatment
with one or more modulators of PD-1 and/or PD-L1 levels, the method
comprising: a) determining the copy number, level of expression, or
level of activity of one or more biomarkers listed in Table 1 or a
fragment thereof in a subject sample; b) determining the copy
number, level of expression, or level of activity of the one or
more biomarkers in a control sample; and c) comparing the copy
number, level of expression, or level of activity of said one or
more biomarkers detected in steps a) and b); wherein a significant
modulation in the copy number, level of expression, or level of
activity of the one or more biomarkers in the subject sample
relative to the normal copy number, level of expression, or level
of activity of the one or more biomarkers in the control sample
predicts the clinical outcome of the patient to treatment with one
or more modulators of PD-1 and/or PD-L1 levels.
8. The method of claim 7, wherein the predicted clinical outcome is
(a) cellular growth, (b) cellular proliferation, or (c) survival
time resulting from treatment with one or more modulators of PD-1
and/or PD-L1 levels.
9. The method of claim 7, wherein the one or more biomarkers
comprises soluble PD-L1, optionally wherein the soluble PD-L1 a)
comprises an amino acid sequence of SEQ ID NO: 13 or 15 or an amino
acid sequence that is at least 80% identical to SEQ ID NO: 13 or
15; or b) comprises a nucleic acid molecule comprising a nucleic
acid sequence encoding an amino acid sequence of SEQ ID NO: 13 or
15 or an amino acid sequence that is at least 80% identical to SEQ
ID NO: 1.
10. The method of claim 7, wherein an at least twenty percent
increase or an at least twenty percent decrease between the copy
number, level of expression, or level of activity of the one or
more biomarkers in the subject sample compared to the control
sample predicts that the subject has a poor clinical outcome; or
wherein less than a twenty percent increase or less than a twenty
percent decrease between the copy number, level of expression, or
level of activity of the one or more biomarkers in the subject
sample compared to the control sample predicts that the subject has
a favorable clinical outcome.
11. The method of claim 7, further comprising treating the subject
with a therapeutic agent that specifically modulates the copy
number, level of expression, or level of activity of the one or
more biomarkers.
12. The method of claim 7, further comprising treating the subject
with one or more modulators of PD-1 and/or PD-L1 levels.
13. A method of determining the efficacy of a test compound for
inhibiting a head, neck, or lung cancer in a subject, the method
comprising: a) determining the copy number, level of expression, or
level of activity of one or more biomarkers listed in Table 1 or a
fragment thereof in a first sample obtained from the subject and
exposed to the test compound; b) determining the copy number, level
of expression, or level of activity of the one or more biomarkers
in a second sample obtained from the subject, wherein the second
sample is not exposed to the test compound, and c) comparing the
copy number, level of expression, or level of activity of the one
or more biomarkers in the first and second samples, wherein a
significantly modulated copy number, level of expression, or level
of activity of the biomarker, relative to the second sample, is an
indication that the test compound is efficacious for inhibiting the
head, neck, or lung cancer in the subject.
14. The method of claim 13, wherein the one or more biomarkers
comprises soluble PD-L1, optionally wherein the soluble PD-L1 a)
comprises an amino acid sequence of SEQ ID NO:13 or 15 or an amino
acid sequence that is at least 80% identical to SEQ ID NO: 13 or
15; or b) comprises a nucleic acid molecule comprising a nucleic
acid sequence encoding an amino acid sequence of SEQ ID NO: 13 or
15 or an amino acid sequence that is at least 80% identical to SEQ
ID NO: 1.
15. The method of claim 13, wherein the first and second samples
are portions of a single sample obtained from the subject or
portions of pooled samples obtained from the subject.
16. A method of determining the efficacy of a therapy for
inhibiting a head, neck, or lung cancer in a subject, the method
comprising: a) determining the copy number, level of expression, or
level of activity of one or more biomarkers listed in Table 1 or a
fragment thereof in a first sample obtained from the subject prior
to providing at least a portion of the therapy to the subject; b)
determining the copy number, level of expression, or level of
activity of the one or more biomarkers in a second sample obtained
from the subject following provision of the portion of the therapy;
and c) comparing the copy number, level of expression, or level of
activity of the one or more biomarkers in the first and second
samples, wherein a significantly decreased copy number, level of
expression, or level of activity of the one or more biomarkers in
the second sample, relative to the first sample, is an indication
that the therapy is efficacious for inhibiting the head, neck, or
lung cancer in the subject.
17. The method of claim 16, wherein the one or more biomarkers
comprises soluble PD-L1, optionally wherein the soluble PD-L1 a)
comprises an amino acid sequence of SEQ ID NO:13 or 15 or an amino
acid sequence that is at least 80% identical to SEQ ID NO: 13 or
15; or b) comprises a nucleic acid molecule comprising a nucleic
acid sequence encoding an amino acid sequence of SEQ ID NO: 13 or
15 or an amino acid sequence that is at least 80% identical to SEQ
ID NO: 1.
18. A method for identifying a compound which inhibits a head,
neck, or lung cancer, the method comprising: a) contacting one or
more biomarkers listed in Table 1 or a fragment thereof with a test
compound; and b) determining the effect of the test compound on the
copy number, level of expression, or level of activity of the one
or more biomarkers to thereby identify a compound which inhibits
the head, neck, or lung cancer.
19. The method of claim 18, wherein the one or more biomarkers
comprises soluble PD-L1, optionally wherein the soluble PD-L1 a)
comprises an amino acid sequence of SEQ ID NO: 13 or 15 or an amino
acid sequence that is at least 80% identical to SEQ ID NO: 13 or
15; or b) comprises a nucleic acid molecule comprising a nucleic
acid sequence encoding an amino acid sequence of SEQ ID NO: 13 or
15 or an amino acid sequence that is at least 80% identical to SEQ
ID NO: 1.
20. The method of claim 18, wherein the one or more biomarkers is
expressed on or in a cell.
21. The method of claim 20, wherein said cells are isolated from an
animal model of a head, neck, or lung cancer.
22. The method of claim 20, wherein said cells are from a subject
afflicted with a head, neck, or lung cancer.
23. A method for inhibiting a head, neck, or lung cancer, the
method comprising contacting a cell with an agent that modulates
the copy number, level of expression, or level of activity of one
or more biomarkers listed in Table 1 or a fragment thereof to
thereby inhibit the cancer.
24. The method of claim 23, wherein the one or more biomarkers
comprises soluble PD-L1, optionally wherein the soluble PD-L1 a)
comprises an amino acid sequence of SEQ ID NO: 13 or 15 or an amino
acid sequence that is at least 80% identical to SEQ ID NO: 13 or
15; or b) comprises a nucleic acid molecule comprising a nucleic
acid sequence encoding an amino acid sequence of SEQ ID NO: 13 or
15 or an amino acid sequence that is at least 80% identical to SEQ
ID NO: 1.
25. The method of claim 23, wherein the copy number, level of
expression, or level of activity of the one or more biomarkers is
downmodulated.
26. The method of claim 23, wherein the step of contacting occurs
in vivo, ex vivo, or in vitro.
27. The method of claim 23, further comprising contacting the cell
with an additional agent that inhibits the head, neck, or lung
cancer.
28. A method for treating a subject afflicted with a head, neck, or
lung cancer, the method comprising administering an agent that
downregulates the copy number, level of expression, or level of
activity of one or more biomarkers listed in Table 1 or a fragment
thereof such that the head, neck, or lung cancer is treated.
29. The method of claim 28, wherein the one or more biomarkers
comprises soluble PD-L1, optionally wherein the soluble PD-L1 a)
comprises an amino acid sequence of SEQ ID NO: 13 or 15 or an amino
acid sequence that is at least 80% identical to SEQ ID NO: 13 or
15; or b) comprises a nucleic acid molecule comprising a nucleic
acid sequence encoding an amino acid sequence of SEQ ID NO: 13 or
15 or an amino acid sequence that is at least 80% identical to SEQ
ID NO: 1.
30. The method of claim 28, further comprising administering one or
more additional agents that treats the cancer.
31. The method of claim 28, wherein the agent is one or more
modulators of PD-1 levels.
32. The method of claim 28, wherein the agent is one or more
modulators of PD-L1 levels.
33. A pharmaceutical composition comprising an antisense
polynucleotide that specifically binds to a polynucleotide of one
or more biomarkers listed in Table 1 or a fragment thereof useful
for treating a head, neck, or lung cancer in a pharmaceutically
acceptable carrier.
34. The pharmaceutical composition of claim 33, wherein the
antisense polynucleotide further comprises an expression
vector.
35. A method of using the pharmaceutical composition of claims 33
or 34 for treating a head, neck, or lung cancer in a subject.
36. A kit comprising an agent which selectively binds to one or
more biomarkers listed in Table 1 or a fragment thereof and
instructions for use.
37. The kid of claim 36, wherein the agent is selected from the
group consisting of polynucleotides and antibodies.
38. A biochip comprising a solid substrate, said substrate
comprising one or more probes capable of detecting one or more
biomarkers listed in Table 1 or a fragment thereof wherein each
probe is attached to the substrate at a spatially defined
address.
39. The biochip of claim 51, wherein the probes are complementary
to a genomic or transcribed polynucleotide associated with the one
or more biomarkers.
40. The pharmaceutical composition of claim 33, the kit of claim
36, or the biochip of claim 38, wherein the one or more biomarkers
comprises soluble PD-L1, optionally wherein the soluble PD-L1 a)
comprises an amino acid sequence of SEQ ID NO: 13 or 15 or an amino
acid sequence that is at least 80% identical to SEQ ID NO: 13 or
15; or b) comprises a nucleic acid molecule comprising a nucleic
acid sequence encoding an amino acid sequence of SEQ ID NO:13 or 15
or an amino acid sequence that is at least 80% identical to SEQ ID
NO: 1.
41. The method of any one of claims 1, 2, 3, 7, 13, 16, 18, 23, and
28, wherein the control is determined from a non-cancerous sample
from the subject or member of the same species to which the subject
belongs.
42. The method of any one of claims 1, 2, 3, 7, 13, 16, 18, 23, and
28, wherein the sample consists of or comprises body fluid, cells,
cell lines, histological slides, paraffin embedded tissue, fresh
frozen tissue, fresh tissue, biopsies, blood, plasma, serum, buccal
scrape, saliva, cerebrospinal fluid, urine, stool, mucus, or bone
marrow, obtained from the subject.
43. The method of claim 42, wherein the body fluid is selected from
group consisting of amniotic fluid, aqueous humor, bile, blood and
blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's
fluid or pre-ejaculatory fluid, chyle, chyme, stool, female
ejaculate, interstitial fluid, intracellular fluid, lymph, menses,
breast milk, mucus, pleural fluid, peritoneal fluid, pus, saliva,
sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal
lubrication, vitreous humor, and vomit.
44. The method of any one of claims 1, 2, 3, 7, 13, 16, 18, 23, and
28, wherein the copy number is assessed by microarray, quantitative
PCR (qPCR), high-throughput sequencing, comparative genomic
hybridization (CGH), or fluorescent in situ hybridization
(FISH).
45. The method of any one of claims 1, 2, 3, 7, 13, 16, 18, 23, and
28, wherein the expression level of the one or more biomarkers is
assessed by detecting the presence in the samples of a
polynucleotide molecule encoding the biomarker or a portion of said
polynucleotide molecule.
46. The method of claim 44, wherein the polynucleotide molecule is
a mRNA, cDNA, or functional variants or fragments thereof and,
optionally, wherein the step of detecting further comprises
amplifying the polynucleotide molecule.
47. The method of any one of claims 1, 2, 3, 7, 13, 16, 18, 23, and
28, wherein the expression level of the one or more biomarkers is
assessed by annealing a nucleic acid probe with the sample of the
polynucleotide encoding the one or more biomarkers or a portion of
said polynucleotide molecule under stringent hybridization
conditions.
48. The method of any one of claims 1, 2, 3, 7, 13, 16, 18, 23, and
28, wherein the expression level of the biomarker is assessed by
detecting the presence in the samples of a protein of the
biomarker, a polypeptide, or protein fragment thereof comprising
said protein.
49. The method of claim 48, wherein the presence of said protein,
polypeptide or protein fragment thereof is detected using a reagent
which specifically binds with said protein, polypeptide or protein
fragment thereof.
50. The method of claim 49, wherein the reagent is selected from
the group consisting of an antibody, an antibody derivative, and an
antibody fragment.
51. The method of any one of claims 1, 2, 3, 7, 13, 16, 18, 23, and
28, wherein the activity level of the biomarker is assessed by
determining the magnitude of modulation of the activity or
expression level of downstream targets of the one or more
biomarkers.
52. The method of any one of claims 1, 6, 7, 13, 16, 18, 23, and
28, wherein the agent or test compound modulates PD-1, PD-L1, and
or soluble PD-L1 levels.
53. The method of claim 52, wherein the agent or test compound
inhibits the expression and/or activity of soluble PD-L1,
optionally wherein the soluble PD-L1 a) comprises an amino acid
sequence of SEQ ID NO: 13 or 15 or an amino acid sequence that is
at least 80% identical to SEQ ID NO: 13 or 15; or b) comprises a
nucleic acid molecule comprising a nucleic acid sequence encoding
an amino acid sequence of SEQ ID NO: 13 or 15 or an amino acid
sequence that is at least 80% identical to SEQ ID NO: 1.
54. The method of claim 53, wherein the agent or test compound is
an antibody against soluble PD-L1, optionally wherein the soluble
PD-L1 comprises an amino acid sequence of SEQ ID NO: 13 or 15 or an
amino acid sequence that is at least 80% identical to SEQ ID NO:13
or 15.
55. The method of claim 52, wherein the agent or test compound is a
small molecule inhibitor of soluble PD-L1, optionally wherein the
soluble PD-L1 comprises an amino acid sequence of SEQ ID NO:13 or
15 or an amino acid sequence that is at least 80% identical to SEQ
ID NO: 13 or 15.
56. The method of claim 52, wherein the agent or test compound is
an anti-PD-L1 inhibitor selected from the group consisting of a
small molecule, antisense nucleic acid, interfering RNA, shRNA,
siRNA, aptamer, ribozyme, and dominant-negative protein binding
partner.
57. The method of any one of claims 1, 2, 3, 7, 13, 16, 18, 23, 28,
and 35, wherein the head or neck cancer is squamous cell carcinomas
of the head and neck (SCCHN) or associated with human
papillomavirus infection.
58. The method of any one of claims 1, 2, 3, 7, 13, 16, 18, 23, 28,
and 35, wherein the lung cancer is small-cell lung carcinoma (SCLC)
or non-small-cell lung carcinoma (NSCLC).
59. The method of any one of claims 1, 2, 3, 7, 13, 16, 18, 23, and
28, wherein the subject is a mammal.
60. The method of claim 59, wherein the mammal is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/831,894, filed on 6 Jun. 2013; the entire
contents of said application is incorporated herein in its entirety
by this reference.
BACKGROUND OF THE INVENTION
[0003] In order for T cells to respond to foreign proteins, two
signals must be provided by antigen-presenting cells (APCs) to
resting T lymphocytes (Jenkins, M. and Schwartz, R. (1987) J. Exp.
Med. 165:302-319; Mueller, D. L. et al. (1990) J. Immunol.
144:3701-3709). The first signal, which confers specificity to the
immune response, is transduced via the T cell receptor (TCR)
following recognition of foreign antigenic peptide presented in the
context of the major histocompatibility complex (MHC). The second
signal, termed costimulation, induces T cells to proliferate and
become functional (Lenschow et al. (1996) Annu. Rev. Immunol.
14:233). Costimulation is neither antigen-specific, nor MHC
restricted and is thought to be provided by one or more distinct
cell surface polypeptides expressed by APCs (Jenkins, M. K. et al.
(1988) J. Immunol. 140:3324-3330; Linsley, P. S. et al. (1991) J.
Exp. Med. 173:721-730; Gimmi, C. D., et al. 1991 Proc. Nat. Acad.
Sci. USA 88:6575-6579; Young. J. W. et al. (1992) J. Clin. Invest.
90:229-237; Koulova, L. et. al. (1991) J. Exp. Med. 173:759-762;
Reiser, H. et al. (1992) Proc. Natl. Acad. Sci. USA 89:271-275;
van-Seventer, G. A. et al. (1990) J. Immunol. 144:4579-4586;
LaSalle, J. M. et al. (1991) J. Immunol. 147:774-80; Dustin, M. I.
et al. (1989) J. Exp. Med. 169:503; Armitage, R. J. et al. (1992)
Nature 357:80-82; Liu, Y. et al. (1992) J. Exp. Med.
175:437-445).
[0004] The CD80 (B7-1) and CD86 (B7-2) proteins, expressed on APCs,
are critical costimulatory polypeptides (Freeman et al. (1991) J.
Exp. Med. 174:625; Freeman et al. (1989) J. Immunol. 143:2714;
Azuma et. al. (1993) Nature 366:76; Freeman et al. (1993) Science
262:909). B7-2 appears to play a predominant role during primary
immune responses, while B7-1, which is upregulated later in the
course of an immune response, may be important in prolonging
primary T cell responses or costimulating secondary T cell
responses (Bluestone (1995) Immunity 2:555).
[0005] One receptor to which B7-1 and B7-2 bind, CD28, is
constitutively expressed on resting T cells and increases in
expression after activation. After signaling through the T cell
receptor, ligation of CD28 and transduction of a costimulatory
signal induces T cells to proliferate and secrete IL-2 (Linsley, P.
S. et al. (1991) J. Exp. Med. 173:721-730; Gimmi, C. D. et al.
(1991) Proc. Natl. Acad. Sci. USA 88:6575-6579; June, C. H. et al.
(1990) Immunol. Today 11:211-6; Harding, F. A. et al. (1992) Nature
356:607-609). A second receptor, termed CTLA4 (CD152) is homologous
to CD28 but is not expressed on resting T cells and appears
following T cell activation (Brunet, J. F. et al. (1987) Nature
328:267-270). CTLA4 appears to be critical in negative regulation
of T cell responses (Waterhouse et al. (1995) Science 270:985).
Blockade of CTLA4 has been found to remove inhibitory signals,
while aggregation of CTLA4 has been found to provide inhibitory
signals that downregulate T cell responses (Allison and Krummel
(1995) Science 270:932). The B7 polypeptides have a higher affinity
for CTLA4 than for CD28 (Linsley, P. S. et al. (1991) J. Exp. Med.
174:561-569) and B7-1 and B7-2 have been round to bind to distinct
regions of the CTLA4 polypeptide and have different kinetics of
binding to CTLA4 (Linsley et al. (1994) Immunity 1:793). ICOS,
which is a polypeptide related to CD28 appears to be important in
IL-10 production (Hutloff et al. (1999) Nature 397:263. WO
98/38216), as has its ligand (Aicher A. et al. (200) J. Immunol.
164:4689-96; Mages H. W. et al. (2000) Eur. J. Immunol. 30:1040-7;
Brodie D. et al. (2000) Curr. Biol. 10:333-6; Ling V. et al. (2000)
J. Immunol. 164:1653-7; Yoshinaga S. K. et al. (1999) Nature
402:827-32). If T cells are only stimulated through the 1 cell
receptor, without receiving an additional costimulatory signal,
they become nonresponsive, anergic, or die, resulting in
downmodulation of the immune response.
[0006] The importance of the B7:CD28/CTLA4 costimulatory pathway
has been demonstrated in vitro and in several in vivo model
systems. Blockade of this costimulatory pathway results in the
development of antigen specific tolerance in murine and human
systems (Harding, F. A. et al. (1992) Nature 356:607-609; Lenschow,
D. J. et al. (1992) Science 257:789-792; Turka, L. A. et al. (1992)
Proc. Natl. Acad. Sci. USA 89:11102-11105; Gimmi, C. D. et al.
(1993) Proc. Natl. Acad. Sci. USA 90:6586-6590; Boussiotis, V. et
al. (1993) J. Exp. Med. 178:1753-1763). Conversely, expression of
B7 by B7 negative murine tumor cells induces T-cell mediated
specific immunity accompanied by tumor rejection and long lasting
protection to tumor challenge (Chen, L. et al. (1992) Cell
71:1093-1102; Townsend, S. E. and Allison. J. P. (1993) Science
259:368-370; Baskar, S. et al. (1993) Proc. Natl. Acad. Sci.
90:5687-5690).
[0007] Inhibitory receptors that bind to costimulatory polypeptides
have also been identified on immune cells. Activation of CTLA4, for
example, transmits a negative signal to a T cell. Engagement of
CTLA4 inhibits IL-2 production and can induce cell cycle arrest
(Krummel and Allison (1996) J. Exp. Med. 183:2533). In addition,
mice that lack CTLA4 develop lymphoproliferative disease (Tivol et
al. (1995) Immunity 3:541; Waterhouse et al. (1995) Science
270:985). The blockade of CTLA4 with antibodies may remove an
inhibitory signal, whereas aggregation of CTLA4 with antibody
transmits an inhibitory signal. Therefore, depending upon the
receptor to which a costimulatory polypeptide binds (i.e., a
costimulatory receptor such as CD28 or an inhibitory receptor such
as CTLA4), certain B7 polypeptides can promote T cell costimulation
or inhibition.
[0008] PD-1 has been identified as a receptor which binds to PD-L1
and PD-L2. PD-1 is a member of the immunoglobulin gene superfamily.
PD-1 (Ishida et al. (1992) EMBO J. 11:3887; Shinohara et al. (1994)
Genomics 23:704; U.S. Pat. No. 5,698,520) has an extracellular
region containing immunoglobulin superfamily domain, a
transmembrane domain, and an intracellular region including an
immunoreceptor tyrosine-based inhibitory motif (ITIM). These
features also define a larger family of polypeptides, called the
immunoinhibitory receptors, which also includes gp49B, PIR-B, and
the killer inhibitory receptors (KIRs) (Vivier and Dacron (1997)
Immunol. Today 18:286). It is often assumed that the tyrosyl
phosphorylated ITIM motif of these receptors interacts with
SH2-domain containing phosphatases, which leads to inhibitory
signals. A subset of these immunoinhibitory receptors bind to MHC
polypeptides, for example the KIRs, and CTLA4 bind to B7-1 and
B7-2. It has been proposed that there is a phylogenetic
relationship between the MHC and B7 genes (Henry et al. (1999)
Immunol. Today 20(6):285-8).
[0009] The nucleotide and amino acid sequence of PD-1 is published
in Ishida et al. (1992) EMBO J. 11:3887; Shinohara et al. (1994)
Genomics 23:704; and U.S. Pat. No. 5,698,520. PD-1 was previously
identified using a subtraction cloning based approach to select for
proteins involved in apoptotic cell death. Like CTLA4. PD-1 is
rapidly induced on the surface of T-cells in response to anti-CD3
(Agata et al. (1996) Int. Immunol. 8:765). In contrast to CTLA4,
however, PD-1 is also induced on the surface of B-cells (in
response to anti-IgM). PD-1 is also expressed on a subset of
thymocytes and myeloid cells (Agata et al. (1996) supra; Nishimura
et al. (1996) Int. Immunol. 8:773).
[0010] Two types of human PD-1 ligand polypeptides have been
identified. PD-1 ligand proteins comprise a signal sequence, and an
IgV domain, an IgC domain, a transmembrane domain, and a short
cytoplasmic tail. Both PD-L1 (Freeman et al. (2000) J. Exp. Med.
192:1027) and PD-L2 (Latchman et al. (2001) Nat. Immunol. 2:261)
are members of the B7 family of polypeptides. Both PD-L1 and PD-L2
are expressed in placenta, spleen, lymph nodes, thymus, and heart.
Only PD-L2 is expressed in pancreas, lung and liver while only
PD-L1 is expressed in fetal liver. Both PD-1 ligands are
upregulated on activated monocytes and dendritic cells. PD-L1 can
bind to either PD-1 or B7-1.
[0011] The fact that PD-1 binds to PD-L1 and PD-L2 places PD-1 in a
family of inhibitory receptors with CTLA4. While engagement of a
costimulatory receptor results in a costimulatory signal in an
immune cell, engagement of an inhibitory receptor, e.g., CTLA4 or
PD-1 (for example by crosslinking or by aggregation), leads to the
transmission of an inhibitory signal in an immune cell, resulting
in downmodulation of immune cell responses and/or in immune cell
anergy. While transmission of an inhibitory signal leads to
downmodulation in immune cell responses (and a resulting
downmodulation in the overall immune response), the prevention of
an inhibitory signal (e.g., by using a non-activating antibody
against PD-1) in immune cells leads to upmodulation of immune cell
responses (and a resulting upmodulation of an immune response).
[0012] Despite the fact that PD-1 ligands, such as PD-L1, are
usually membrane-bound polypeptides expressed on professional
immunological cells, it is known that such ligands can naturally be
expressed in a soluble form (i.e., lacking a cellular membrane
retention domain, such as a hydrophobic transmembrane domain) and
can be expressed by a variety of cells, such as cancer cells. For
example, Frigola et al. (2011) Clin. Cancer Res. 17:1915 describes
the association of a soluble form of PD-L1 with aggressive renal
cell carcinoma. Such soluble versions of PD-1 ligand (e.g., soluble
PD-L1) provide new biomarkers associated with the detection of
maladies and allow for a more non-invasive evaluation of cancer
status due to easier detection of the biomarker in a bodily fluid
as opposed to cancerous or pre-cancerous tissue. However, such
protein isoforms are thought to be cleavage products of
membrane-bound PD-L1, as opposed to products of alternative
splicing and transcript variants. Accordingly, there is a great
need to identify additional PD-1 ligand (e.g., soluble PD-L1)-based
biomarkers associated with deleterious conditions in subjects,
including the generation of diagnostic, prognostic, and therapeutic
agents to effectively control such disorders in subjects.
SUMMARY OF THE INVENTION
[0013] The present invention is based, at least in part, on the
discovery that PD-L1 isoforms, particularly those encoding soluble
forms of PD-L1, are overexpressed by specific cancers (e.g., head,
neck, and lung cancers) and maintain the ability to transmit
inhibitory signals to immune cells to thereby inhibit immune
responses (e.g., T cell activation, proliferation, and cytotoxic
function). Such PD-L1 isoforms and the encoded PD-L1 variants are
useful as biomarkers for the identification, assessment,
prevention, and/or treatment of such cancers.
[0014] In one aspect, a method of determining whether a subject is
afflicted with a head, neck, or lung cancer or at risk for
developing a head, neck, or lung cancer is provided, wherein the
method comprises: a) obtaining a biological sample from the
subject; b) determining the copy number, level of expression, or
level of activity of one or more biomarkers listed in Table 1 or a
fragment thereof in the subject sample; c) determining the copy
number, level of expression, or level of activity of the one or
more biomarkers in a control; and d) comparing the copy number,
level of expression, or level of activity of said one or more
biomarkers detected in steps b) and c); wherein a significant
increase in the copy number, level of expression, or level of
activity of the one or more biomarkers in the subject sample
relative to the control copy number, level of expression, or level
of activity of the one or more biomarkers indicates that the
subject is afflicted with the head, neck, or lung cancer or is at
risk for developing the head, neck, or lung cancer.
[0015] In another aspect, a method of determining whether a subject
afflicted with a head, neck, or lung cancer or at risk for
developing a head, neck, or lung cancer would benefit from
modulating PD-1 and/or PD-L1 levels is provided, wherein the method
comprises: a) obtaining a biological sample from the subject: b)
determining the copy number, level of expression, or level of
activity of one or more biomarkers listed in Table 1 or a fragment
thereof in the subject sample; c) determining the copy number,
level of expression, or level of activity of the one or more
biomarkers in a control; and d) comparing the copy number, level of
expression, or level of activity of said one or more biomarkers
detected in steps b) and c); wherein a significant increase in the
copy number, level of expression, or level of activity of the one
or more biomarkers in the subject sample relative to the control
copy number, level of expression, or level of activity of the one
or more biomarkers indicates that the subject afflicted with the
head, neck, or lung cancer or at risk for developing the head,
neck, or lung cancer would benefit from modulating PD-L1 and/or
PD-L1 levels.
[0016] In still another aspect, a method for monitoring the
progression of a head, neck, or lung cancer in a subject is
provided, wherein the method comprises: a) detecting in a subject
sample at a first point in time the copy number, level of
expression, or level of activity of one or more biomarkers listed
in Table 1 or a fragment thereof; b) repeating step a) at a
subsequent point in time: and c) comparing the copy number, level
of expression, or level of activity of said one or more biomarkers
detected in steps a) and b) to monitor the progression of the head,
neck, or lung cancer, wherein the one or more biomarkers comprises
soluble PD-L1, optionally wherein the soluble PD-L1 a) comprises an
amino acid sequence of SEQ ID NO: 13 or 15 or an amino acid
sequence that is at least 80% identical to SEQ ID NO: 13 or 15; or
b) comprises a nucleic acid molecule comprising a nucleic acid
sequence encoding an amino acid sequence of SEQ ID NO: 13 or 15 or
an amino acid sequence that is at least 80% identical to SEQ ID NO:
13 or 15.
[0017] In one embodiment of any aspect of the present invention, an
at least twenty percent increase or an at least twenty percent
decrease between the copy number, level of expression, or level of
activity of the one or more biomarkers in the subject sample at a
first point in time relative to the copy number, level of
expression, or level of activity of the one or more biomarkers in
the subject sample at a subsequent point in time indicates
progression of the head, neck, or lung cancer; or wherein less than
a twenty percent increase or less than a twenty percent decrease
between the copy number, level of expression, or level of activity
of the one or more biomarkers in the subject sample at a first
point in time relative to the copy number, level of expression, or
level of activity of the one or more biomarkers in the subject
sample at a subsequent point in time indicates a lack of
significant progression of the head, neck, or lung cancer. In
another embodiment, the subject has undergone treatment to modulate
PD-1 and/or PD-L1 levels between the first point in time and the
subsequent point in time.
[0018] In yet another aspect, a method for stratifying subjects
afflicted with a head, neck, or lung cancer according to predicted
clinical outcome of treatment with one or more modulators of PD-1
and/or PD-L1 levels is provided, wherein the method comprises: a)
determining the copy number, level of expression, or level of
activity of one or more biomarkers listed in Table 1 or a fragment
thereof in a subject sample; b) determining the copy number, level
of expression, or level of activity of the one or more biomarkers
in a control sample; and c) comparing the copy number, level of
expression, or level of activity of said one or more biomarkers
detected in steps a) and b); wherein a significant modulation in
the copy number, level of expression, or level of activity of the
one or more biomarkers in the subject sample relative to the normal
copy number, level of expression, or level of activity of the one
or more biomarkers in the control sample predicts the clinical
outcome of the patient to treatment with one or more modulators of
PD-1 and/or PD-L levels. In one embodiment, the predicted clinical
outcome is (a) cellular growth, (b) cellular proliferation, or (c)
survival time resulting from treatment with one or more modulators
of PD-1 and/or PD-L1 levels. In another embodiment, the one or more
biomarkers comprises soluble PD-L1, optionally wherein the soluble
PD-L1 a) comprises an amino acid sequence of SEQ ID NO: 13 or 15 or
an amino acid sequence that is at least 80% identical to SEQ ID NO:
13 or 15; or b) comprises a nucleic acid molecule comprising a
nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:
13 or 15 or an amino acid sequence that is at least 80% identical
to SEQ ID NO: 1. In still another embodiment, an at least twenty
percent increase or an at least twenty percent decrease between the
copy number, level of expression, or level of activity of the one
or more biomarkers in the subject sample compared to the control
sample predicts that the subject has a poor clinical outcome; or
wherein less than a twenty percent increase or less than a twenty
percent decrease between the copy number, level of expression, or
level of activity of the one or more biomarkers in the subject
sample compared to the control sample predicts that the subject has
a favorable clinical outcome. In yet another embodiment, the method
further comprises treating the subject with a therapeutic agent
that specifically modulates the copy number, level of expression,
or level of activity of the one or more biomarkers. In another
embodiment, the method further comprises treating the subject with
one or more modulators of PD-1 and/or PD-L1 levels.
[0019] In another aspect, a method of determining the efficacy of a
test compound for inhibiting a head, neck, or lung cancer in a
subject is provided, wherein the method comprises: a) determining
the copy number, level of expression, or level of activity of one
or more biomarkers listed in Table 1 or a fragment thereof in a
first sample obtained from the subject and exposed to the test
compound; b) determining the copy number, level of expression, or
level of activity of the one or more biomarkers in a second sample
obtained from the subject, wherein the second sample is not exposed
to the test compound, and c) comparing the copy number, level of
expression, or level of activity of the one or more biomarkers in
the first and second samples, wherein a significantly modulated
copy number, level of expression, or level of activity of the
biomarker, relative to the second sample, is an indication that the
test compound is efficacious for inhibiting the head, neck, or lung
cancer in the subject. In one embodiment, the one or more
biomarkers comprises soluble PD-L1, optionally wherein the soluble
PD-L1 a) comprises an amino acid sequence of SEQ ID NO: 13 or 15 or
an amino acid sequence that is at least 80% identical to SEQ ID NO:
13 or 15; or b) comprises a nucleic acid molecule comprising a
nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:
13 or 15 or an amino acid sequence that is at least 80% identical
to SEQ ID NO: 1. In another embodiment, the first and second
samples are portions of a single sample obtained from the subject
or portions of pooled samples obtained from the subject.
[0020] In still another aspect, a method of determining the
efficacy of a therapy for inhibiting a head, neck, or lung cancer
in a subject is provided, wherein the method comprises: a)
determining the copy number, level of expression, or level of
activity of one or more biomarkers listed in Table 1 or a fragment
thereof in a first sample obtained from the subject prior to
providing at least a portion of the therapy to the subject-b)
determining the copy number, level of expression, or level of
activity of the one or more biomarkers in a second sample obtained
from the subject following provision of the portion of the therapy;
and c) comparing the copy number, level of expression, or level of
activity of the one or more biomarkers in the first and second
samples, wherein a significantly decreased copy number, level of
expression, or level of activity of the one or more biomarkers in
the second sample, relative to the first sample, is an indication
that the therapy as efficacious for inhibiting the head, neck, or
lung cancer in the subject. In one embodiment, the one or more
biomarkers comprises soluble PD-L1, optionally wherein the soluble
PD-L1 a) comprises an amino acid sequence of SEQ ID NO:13 or 15 or
an amino acid sequence that is at least 80% identical to SEQ ID
NO:13 or 15; or b) comprises a nucleic acid molecule comprising a
nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:
13 or 15 or an amino acid sequence that is at least 80% identical
to SEQ ID NO: 1.
[0021] In yet another aspect, a method for identifying a compound
which inhibits a head, neck, or lung cancer is presented, wherein
the method comprises: a) contacting one or more biomarkers listed
in Table 1 or a fragment thereof with a test compound; and b)
determining the effect of the test compound on the copy number,
level of expression, or level of activity of the one or more
biomarkers to thereby identify a compound which inhibits the head,
neck, or lung cancer. In one embodiment, the one or more biomarkers
comprises soluble PD-L1, optionally wherein the soluble PD-L1 a)
comprises an amino acid sequence of SEQ ID NO: 13 or 15 or an amino
acid sequence that is at least 80% identical to SEQ ID NO:13 or 15;
or b) comprises a nucleic acid molecule comprising a nucleic acid
sequence encoding an amino acid sequence of SEQ ID NO: 13 or 15 or
an amino acid sequence that is at least 80% identical to SEQ ID NO:
1. In another embodiment, the one or more biomarkers is expressed
on or in a cell (e.g., cells isolated from an animal model of a
head, neck, or lung cancer or cells from a subject afflicted with a
head, neck, or lung cancer).
[0022] In another aspect, a method for inhibiting a head, neck, or
lung cancer is provided, wherein the method comprises contacting a
cell with an agent that modulates the copy number, level of
expression, or level of activity of one or more biomarkers listed
in Table 1 or a fragment thereof to thereby inhibit the cancer. In
one embodiment, the one or more biomarkers comprises soluble PD-L1,
optionally wherein the soluble PD-L1 a) comprises an amino acid
sequence of SEQ ID NO: 13 or 15 or an amino acid sequence that is
at least 80% identical to SEQ ID NO: 13 or 15; or b) comprises a
nucleic acid molecule comprising a nucleic acid sequence encoding
an amino acid sequence of SEQ ID NO: 13 or 15 or an amino acid
sequence that is at least 80%/i identical to SEQ ID NO: 1. In
another embodiment, the copy number, level of expression, or level
of activity of the one or more biomarkers is downmodulated. In
still another embodiment, the step of contacting occurs in vivo, e
vivo, or in vitro. In yet another embodiment, the method further
comprises contacting the cell with an additional agent that
inhibits the head, neck, or lung cancer.
[0023] In still another aspect, a method for treating a subject
afflicted with a head, neck, or lung cancer is provided, wherein
the method comprises administering an agent that downregulates the
copy number, level of expression, or level of activity of one or
more biomarkers listed in Table 1 or a fragment thereof such that
the head, neck, or lung cancer is treated. In one embodiment, the
one or more biomarkers comprises soluble PD-L1, optionally wherein
the soluble PD-L1 a) comprises an amino acid sequence of SEQ ID
NO:13 or 15 or an amino acid sequence that is at least 80%
identical to SEQ ID NO: 13 or 15; or b) comprises a nucleic acid
molecule comprising a nucleic acid sequence encoding an amino acid
sequence of SEQ ID NO: 13 or 15 or an amino acid sequence that is
at least 80% identical to SEQ ID NO:1. In another embodiment, the
method further comprises administering one or more additional
agents that treats the cancer. In still another embodiment, the
agent is one or more modulators of PD-1 levels and/or one or more
modulators of PD-L1 levels.
[0024] In yet another aspect, a pharmaceutical composition is
provided comprising an antisense polynucleotide that specifically
binds to a polynucleotide of one or more biomarkers listed in Table
1 or a fragment thereof useful for treating a head, neck, or lung
cancer in a pharmaceutically acceptable carrier. In one embodiment,
the antisense polynucleotide further comprises an expression
vector. In another embodiment, a method of using such
pharmaceutical composition for treating a head, neck, or lung
cancer in a subject is provided.
[0025] In another aspect, a kit comprising an agent which
selectively binds to one or more biomarkers listed in Table 1 or a
fragment thereof and instructions for use. In one embodiment, the
agent is selected from the group consisting of polynucleotides and
antibodies.
[0026] In still another aspect, a biochip is provided comprising a
solid substrate, said substrate comprising one or more probes
capable of detecting one or more biomarkers listed in Table 1 or a
fragment thereof wherein each probe is attached to the substrate at
a spatially defined address. In one embodiment, the probes are
complementary to a genomic or transcribed polynucleotide associated
with the one or more biomarkers.
[0027] Certain embodiments can apply to one, more than one, or all
aspects of the present invention. For example, in one embodiment of
any aspect of the present invention, the one or more biomarkers
comprises soluble PD-L1, optionally wherein the soluble PD-L1 a)
comprises an amino acid sequence of SEQ ID NO:13 or 15 or an amino
acid sequence that is at least 80% identical to SEQ ID NO:13 or 15;
or b) comprises a nucleic acid molecule comprising a nucleic acid
sequence encoding an amino acid sequence of SEQ ID NO: 13 or 15 or
an amino acid sequence that is at least 80% identical to SEQ ID NO:
1. In another embodiment, the control is determined from a
non-cancerous sample from the subject or member of the same species
to which the subject belongs. In still another embodiment, the
sample consists of or comprises body fluid, cells, cell lines,
histological slides, paraffin embedded tissue, fresh frozen tissue,
fresh tissue, biopsies, blood, plasma, serum, buccal scrape,
saliva, cerebrospinal fluid, urine, stool, mucus, or bone marrow,
obtained from the subject. In yet another embodiment, the body
fluid is selected from group consisting of amniotic fluid, aqueous
humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen
and earwax, cowper's fluid or pre-ejaculatory fluid, chyle, chyme,
stool, female ejaculate, interstitial fluid, intracellular fluid,
lymph, menses, breast milk, mucus, pleural fluid, peritoneal fluid,
pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears,
urine, vaginal lubrication, vitreous humor, and vomit. In another
embodiment, the copy number is assessed by microarray, quantitative
PCR (qPCR), high-throughput sequencing, comparative genomic
hybridization (CGH), or fluorescent in situ hybridization (FISH).
In still another embodiment, the expression level of the one or
more biomarkers is assessed by detecting the presence in the
samples of a polynucleotide molecule encoding the biomarker or a
portion of said polynucleotide molecule (e.g., a mRNA, cDNA, or
functional variants or fragments thereof and, optionally, wherein
the step of detecting further comprises amplifying the
polynucleotide molecule). In yet another embodiment, the expression
level of the one or more biomarkers is assessed by annealing a
nucleic acid probe with the sample of the polynucleotide encoding
the one or more biomarkers or a portion of said polynucleotide
molecule under stringent hybridization conditions.
[0028] In some embodiments, the expression level of the biomarker
is assessed by detecting the presence in the samples of a protein
of the biomarker, a polypeptide, or protein fragment thereof
comprising said protein, such as by using a reagent which
specifically binds with said protein, polypeptide or protein
fragment thereof (e.g., an antibody, an antibody derivative, and an
antibody fragment). In still another embodiment, the activity level
of the biomarker is assessed by determining the magnitude of
modulation of the activity or expression level of downstream
targets of the one or more biomarkers. In yet another embodiment,
the agent or test compound modulates PD-1, PD-L1, and or soluble
PD-L1 levels (e.g., compound inhibits the expression and/or
activity of soluble PD-L1, optionally wherein the soluble PD-L1 a)
comprises an amino acid sequence of SEQ ID NO: 13 or 15 or an amino
acid sequence that is at least 80% identical to SEQ ID NO: 13 or
15; or b) comprises a nucleic acid molecule comprising a nucleic
acid sequence encoding an amino acid sequence of SEQ ID NO: 13 or
15 or an amino acid sequence that is at least 80% identical to SEQ
ID NO: 1.
[0029] Examples of such agents include a) an antibody against
soluble PD-L1, optionally wherein the soluble PD-L1 comprises an
amino acid sequence of SEQ ID NO: 13 or 15 or an amino acid
sequence that is at least 80% identical to SEQ ID NO: 13 or 15; b)
a small molecule inhibitor of soluble PD-L1, optionally wherein the
soluble PD-L1 comprises an amino acid sequence of SEQ ID NO:13 or
15 or an amino acid sequence that is at least 80% identical to SEQ
ID NO: 13 or 15; and c) an anti-PD-L1 inhibitor selected from the
group consisting of a small molecule, antisense nucleic acid,
interfering RNA, shRNA, siRNA, aptamer, ribozyme, and
dominant-negative protein binding partner.
[0030] In some embodiments, the head or neck cancer is squamous
cell carcinomas of the head and neck (SCCHN) or associated with
human papillomavirus infection. In another embodiment, the lung
cancer is small-cell lung carcinoma (SCLC) or non-small-cell lung
carcinoma (NSCLC). In still another embodiment, the subject is a
mammal. In yet another embodiment, the mammal is a human.
BRIEF DESCRIPTION OF FIGURES
[0031] FIGS. 1-2 show a schematic of a mutational landscape of head
and neck cancers from The Cancer Genome Atlas (TCGA; FIG. 1)
project or an independent cohort of HPV- (left half) and HPV+
(right half) head and neck cancers (FIG. 2).
[0032] FIG. 3 shows a representation of sites of HPV integration in
the host genome in head and neck cancers analyzed as part of the
TCGA project described in FIG. 1.
[0033] FIGS. 4 and 5 show sequencing reads for the CD274 (PD-L1)
gene in a tumor (CV-5433) with a detected HPV integration in PD-L1.
FIG. 5 shows a zoomed in view of FIG. 4 according to a log
scale.
[0034] FIG. 6 shows a consolidation of the sequencing read data of
FIGS. 4 and 5 into a schematic showing the HPV integration within
the PD-L1 gene of tumor CV-5433.
[0035] FIG. 7 shows the predicted protein product following HPV
integration in tumor CV-5433.
[0036] FIG. 8 shows expression of each exon of full-length,
membrane-bound PD-L1 on a log scale from the CV-5433 sample and
demonstrates a dramatic drop in exons following exon 4 which is the
site of HPV integration.
[0037] FIGS. 9-10 show transcript variants of PD-L1 expressed by
head, neck, and lung cancers.
[0038] FIGS. 11A-11J show expression of short PD-L1 forms among
various cancers from the TCGA (The Cancer Genome Atlas)
database.
[0039] FIG. 12 shows expression of short PD-L1 forms among various
cancers from the Cancer Cell Line Encyclopedia database.
[0040] FIGS. 13-14 show Western blot results 293T cells transfected
with expression vectors encoding the full-length or short PD-L1
forms.
[0041] FIG. 15 shows the protein expression of short PD-L1 forms
from various cell lines.
[0042] FIG. 16 shows T cell viability in response to exposure to
short PD-L1 forms.
[0043] FIG. 17-18 shows that the short form of PD-L1 can
differentially kill proliferating T cells.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention is based, at least in part, on the
novel discovery of gene profiles useful for distinguishing among
cancer subtypes (e.g., head, neck, and/or lung cancers) and for
predicting the clinical outcome of such cancer subtypes to
therapeutic regimens, particularly to modulators of PD-1 and/or
PD-L1. Thus, agents such as miRNAs, miRNA analogues, small
molecules, RNA interference, aptamer, peptides, peptidomimetics,
antibodies that specifically bind to one or more biomarkers of the
invention (e.g., biomarkers listed in Table 1) and fragments
thereof can be used to identify, diagnose, prognose, assess,
prevent, and treat cancers (e.g., head, neck, and/or lung cancers)
or other conditions that would benefit from modulating immune
responses.
I. DEFINITIONS
[0045] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0046] The term "allogeneic" refers to deriving from, originating
in, or being members of the same species, where the members are
genetically related or genetically unrelated but genetically
similar. An "allogeneic transplant" refers to transfer of cells or
organs from a donor to a recipient, where the recipient is the same
species as the donor. The term "mismatched allogeneic" refers to
deriving from, originating in, or being members of the same species
having non-identical major histocompatability complex (MHC)
antigens (i.e., proteins) as typically determined by standard
assays used in the art, such as serological or molecular analysis
of a defined number of MHC antigens. A "partial mismatch" refers to
partial match of the MHC antigens tested between members, typically
between a donor and recipient. For instance, a "half mismatch"
refers to 50% of the MHC antigens tested as showing different MHC
antigen type between two members. A "full" or "complete" mismatch
refers to all MHC antigens tested as being different between two
members. These terms contrast with the term "xenogeneic," which
refers to deriving from, originating in, or being members of
different species, e.g., human and rodent, human and swine, human
and chimpanzee, etc. A "xenogeneic transplant" refers to transfer
of cells or organs from a donor to a recipient where the recipient
is a species different from that of the donor. The term "syngeneic"
refers to deriving from, originating in, or being members of the
same species that are genetically identical, particularly with
respect to antigens or immunological reactions. These include
identical twins having matching MHC types. Thus, a "syngeneic
transplant" refers to transfer of cells or organs from a donor to a
recipient who is genetically identical to the donor.
[0047] The term "altered amount" of a marker or "altered level" of
a marker refers to increased or decreased copy number of the marker
and/or increased or decreased expression level of a particular
marker gene or genes in a cancer sample, as compared to the
expression level or copy number of the marker in a control sample.
The term "altered amount" of a marker also includes an increased or
decreased protein level of a marker in a sample, e.g., a cancer
sample, as compared to the protein level of the marker in a normal,
control sample.
[0048] The "amount" of a marker, e.g., expression or copy number of
a marker or minimal common region (MCR), or protein level of a
marker, in a subject is "significantly" higher or lower than the
normal amount of a marker, if the amount of the marker is greater
or less, respectively, than the normal level by an amount greater
than the standard error of the assay employed to assess amount, and
preferably at least twice, and more preferably three, four, five,
ten or more times that amount. Alternately, the amount of the
marker in the subject can be considered "significantly" higher or
lower than the normal amount if the amount is at least about two,
and preferably at least about three, four, or five times, higher or
lower, respectively, than the normal amount of the marker. In some
embodiments, the amount of the marker in the subject can be
considered "significantly" higher or lower than the normal amount
if the amount is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or
more, higher or lower, respectively, than the normal amount of the
marker.
[0049] The term "altered level of expression" of a marker refers to
an expression level or copy number of a marker in a test sample
e.g., a sample derived from a subject suffering from cancer, that
is greater or less than the standard error of the assay employed to
assess expression or copy number, and is preferably at least twice,
and more preferably three, four, five or ten or more times the
expression level or copy number of the marker or chromosomal region
in a control sample (e.g., sample from a healthy subject not having
the associated disease) and preferably, the average expression
level or copy number of the marker or chromosomal region in several
control samples. The altered level of expression is greater or less
than the standard error of the assay employed to assess expression
or copy number, and is preferably at least twice, and more
preferably three, four, five or ten or more times the expression
level or copy number of the marker in a control sample (e.g.,
sample from a healthy subject not having the associated disease)
and preferably, the average expression level or copy number of the
marker in several control samples.
[0050] The term "altered activity" of a marker refers to an
activity of a marker which is increased or decreased in a disease
state, e.g., in a cancer sample, as compared to the activity of the
marker in a normal, control sample. Altered activity of a marker
may be the result of, for example, altered expression of the
marker, altered protein level of the marker, altered structure of
the marker, or, e.g., an altered interaction with other proteins
involved in the same or different pathway as the marker, or altered
interaction with transcriptional activators or inhibitors. For
example, the term "PD-1 ligand (e.g., soluble PD-L1) activity"
includes the ability of a PD-1 ligand (e.g., soluble PD-L1)
polypeptide to bind its natural receptor(s) (e.g., PD-1 or B7-1),
the ability to modulate immune cell costimulatory or inhibitory
signals, and the ability to modulate the immune response. With
respect to PD-1, the term "activity" includes the ability of a PD-1
polypeptide to modulate an inhibitory signal in an activated immune
cell, e.g., by engaging a natural PD-1 ligand (e.g., soluble PD-L1)
on an antigen presenting cell. PD-1 transmits an inhibitory signal
to an immune cell in a manner similar to CTLA4. Modulation of an
inhibitory signal in an immune cell results in modulation of
proliferation of, and/or cytokine secretion by, an immune cell.
Thus, the term "PD-1 activity" includes the ability of a PD-1
polypeptide to bind its natural ligand(s), the ability to modulate
immune cell costimulatory or inhibitory signals, and the ability to
modulate the immune response.
[0051] The term "altered structure" of a marker refers to the
presence of mutations or allelic variants within the marker gene or
maker protein, e.g., mutations which affect expression or activity
of the marker, as compared to the normal or wild-type gene or
protein. For example, mutations include, but are not limited to
substitutions, deletions, or addition mutations. Mutations may be
present in the coding or non-coding region of the marker.
[0052] The term "altered subcellular localization" of a marker
refers to the mislocalization of the marker within a cell relative
to the normal localization within the cell e.g., within a healthy
and/or wild-type cell. An indication of normal localization of the
marker can be determined through an analysis of subcellular
localization motifs known in the field that are harbored by marker
polypeptides.
[0053] Unless otherwise specified herein, the terms "antibody" and
"antibodies" broadly encompass naturally-occurring forms of
antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies
such as single-chain antibodies, chimeric and humanized antibodies
and multi-specific antibodies, as well as fragments and derivatives
of all of the foregoing, which fragments and derivatives have at
least an antigenic binding site. Antibody derivatives may comprise
a protein or chemical moiety conjugated to an antibody.
[0054] The term "antibody" as used herein also includes an
"antigen-binding portion" of an antibody (or simply "antibody
portion"). The term "antigen-binding portion", as used herein,
refers to one or more fragments of an antibody that retain the
ability to specifically bind to an antigen. It has been shown that
the antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al., (1989) Nature 341:544-546), which consists
of a VH domain; and (vi) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent polypeptides (known as
single chain Fv (scFv); see e.g., Bird et al. (1988) Science
242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883; and Osbounm et al. 1998, Nature Biotechnology 16:
778). Such single chain antibodies are also intended to be
encompassed within the term "antigen-binding portion" of an
antibody. Any VH and VL sequences of specific scFv can be linked to
human immunoglobulin constant region cDNA or genomic sequences, in
order to generate expression vectors encoding complete IgG
polypeptidcs or other isotypes. VH and VL can also be used in the
generation of Fab, Fv or other fragments of immunoglobulins using
either protein chemistry or recombinant DNA technology. Other forms
of single chain antibodies, such as diabodies are also encompassed.
Diabodies are bivalent, bispecific antibodies in which VH and VL
domains are expressed on a single polypeptide chain, but using a
linker that is too short to allow for pairing between the two
domains on the same chain, thereby forcing the domains to pair with
complementary domains of another chain and creating two antigen
binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl.
Acad. Sci. USA 90:6444-6448; Poljak. R. J., et al. (1994) Structure
2:1121-1123).
[0055] Still further, an antibody or antigen-binding portion
thereof may be part of larger immunoadhesion polypeptides, formed
by covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. Examples of
such immunoadhesion polypeptides include use of the streptavidin
core region to make a tetrameric scFv polypeptide (Kipriyanov, S.
M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use
of a cysteine residue, a marker peptide and a C-terminal
polyhistidine tag to make bivalent and biotinylated scFv
polypeptides (Kipriyanov. S. M., et al. (1994) Mol. Immunol.
31:1047-1058). Antibody portions, such as Fab and F(ab').sub.2
fragments, can be prepared from whole antibodies using conventional
techniques, such as papain or pepsin digestion, respectively, of
whole antibodies. Moreover, antibodies, antibody portions and
immunoadhesion polypeptides can be obtained using standard
recombinant DNA techniques, as described herein.
[0056] Antibodies may be polyclonal or monoclonal; xenogeneic,
allogeneic, or syngeneic; or modified forms thereof (e.g.,
humanized, chimeric, etc.). Antibodies may also be fully human. The
terms "monoclonal antibodies" and "monoclonal antibody
composition", as used herein, refer to a population of antibody
polypeptides that contain only one species of an antigen binding
site capable of immunoreacting with a particular epitope of an
antigen, whereas the term "polyclonal antibodies" and "polyclonal
antibody composition" refer to a population of antibody
polypeptides that contain multiple species of antigen binding sites
capable of interacting with a particular antigen. A monoclonal
antibody composition typically displays a single binding affinity
for a particular antigen with which it immunoreacts.
[0057] The term "antisense" nucleic acid polypeptide comprises a
nucleotide sequence which is complementary to a "sense" nucleic
acid encoding a protein, e.g., complementary to the coding strand
of a double-stranded cDNA polypeptide, complementary to an mRNA
sequence or complementary to the coding strand of a gene.
Accordingly, an antisense nucleic acid polypeptide can hydrogen
bond to a sense nucleic acid polypeptide.
[0058] The term "autologous" refers to deriving from or originating
in the same subject or patient. An "autologous transplant" refers
to the harvesting and reinfusion or transplant of a subject's own
cells or organs. Exclusive or supplemental use of autologous cells
can eliminate or reduce many adverse effects of administration of
the cells back to the host, particular graft versus host
reaction.
[0059] The term "biochip" refers to a solid substrate comprising an
attached probe or plurality of probes of the invention, wherein the
probe(s) comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, 100, 150, 200 or more probes. The probes
may be capable of hybridizing to a target sequence under stringent
hybridization conditions. The probes may be attached at spatially
defined address on the substrate. More than one probe per target
sequence may be used, with either overlapping probes or probes to
different sections of a particular target sequence. The probes may
be capable of hybridizing to target sequences associated with a
single disorder. The probes may be attached to the biochip in a
wide variety of ways, as will be appreciated by those in the art.
The probes may either be synthesized first, with subsequent
attachment to the biochip, or may be directly synthesized on the
biochip. The solid substrate may be a material that may be modified
to contain discrete individual sites appropriate for the attachment
or association of the probes and is amenable to at least one
detection method. Representative examples of substrates include
glass and modified or functionalized glass, plastics (including
acrylics, polystyrene and copolymers of styrene and other
materials, polypropylene, polyethylene, polybutylene,
polyurethanes, TeflonJ, etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses and
plastics. The substrates may allow optical detection without
appreciably fluorescing. The substrate may be planar, although
other configurations of substrates may be used as well. For
example, probes may be placed on the inside surface of a tube, for
flow-through sample analysis to minimize sample volume. Similarly,
the substrate may be flexible, such as a flexible foam, including
closed cell foams made of particular plastics. The biochip and the
probe may be derivatized with chemical functional groups for
subsequent attachment of the two. For example, the biochip may be
derivatized with a chemical functional group including, but not
limited to, amino groups, carboxyl groups, oxo groups or thiol
groups. Using these functional groups, the probes may be attached
using functional groups on the probes either directly or indirectly
using a linker. The probes may be attached to the solid support by
either the 5' terminus, 3' terminus, or via an internal nucleotide.
The probe may also be attached to the solid support non-covalently.
For example, biotinylated oligonucleotides can be made, which may
bind to surfaces covalently coated with streptavidin, resulting in
attachment. Alternatively, probes may be synthesized on the surface
using techniques such as photopolymerization and
photolithography.
[0060] The term "body fluid" refers to fluids that are excreted or
secreted from the body as well as fluids that are normally not
(e.g., amniotic fluid, aqueous humor, bile, blood and blood plasma,
cerebrospinal fluid, cerumen and earwax, cowper's fluid or
pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate,
interstitial fluid, intracellular fluid, lymph, menses, breast
milk, mucus, pleural fluid, peritoneal fluid, pus, saliva, sebum,
semen, serum, sweat, synovial fluid, tears, urine, vaginal
lubrication, vitreous humor, vomit).
[0061] The terms "cancer" or "tumor" or "hyperproliferative
disorder" refer to the presence of cells possessing characteristics
typical of cancer-causing cells, such as uncontrolled
proliferation, immortality, metastatic potential, rapid growth and
proliferation rate, and certain characteristic morphological
features. Cancer cells are often in the form of a tumor, but such
cells may exist alone within an animal, or may be a non-tumorigenic
cancer cell, such as a leukemia cell. Cancers include, but are not
limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's
macroglobulinemia, the heavy chain diseases, such as, for example,
alpha chain disease, gamma chain disease, and mu chain disease,
benign monoclonal gammopathy, and inununocytic amyloidosis,
melanomas, breast cancer, lung cancer, bronchus cancer, colorectal
cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian
cancer, urinary bladder cancer, brain or central nervous system
cancer, peripheral nervous system cancer, esophageal cancer,
cervical cancer, uterine or endometrial cancer, cancer of the oral
cavity or pharynx, liver cancer, kidney cancer, testicular cancer,
biliary tract cancer, small bowel or appendix cancer, salivary
gland cancer, thyroid gland cancer, adrenal gland cancer,
osteosarcoma, chondrosarcoma, cancer of hematological tissues, and
the like. Other non-limiting examples of types of cancers
applicable to the methods encompassed by the present invention
include human sarcomas and carcinomas, e.g., fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
colorectal cancer, pancreatic cancer, breast cancer, ovarian
cancer, prostate cancer, squamous cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland
carcinoma, papillary carcinoma, papillary adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma,
renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer,
choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,
cervical cancer, bone cancer, brain tumor, testicular cancer, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and
acute myelocytic leukemia (myeloblastic, promyelocytic,
myelomonocytic, monocytic and erythroleukemia); chronic leukemia
(chronic myelocytic (granulocytic) leukemia and chronic lymphocytic
leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease. In some embodiments,
the cancer whose phenotype is determined by the method of the
invention is an epithelial cancer such as, but not limited to,
bladder cancer, breast cancer, cervical cancer, colon cancer,
gynecologic cancers, renal cancer, laryngeal cancer, lung cancer,
oral cancer, head and neck cancer, ovarian cancer, pancreatic
cancer, prostate cancer, or skin cancer. In other embodiments, the
cancer is breast cancer, prostate cancer, lung cancer, or colon
cancer. In still other embodiments, the epithelial cancer is
non-small-cell lung cancer, nonpapillary renal cell carcinoma,
cervical carcinoma, ovarian carcinoma (e.g., serous ovarian
carcinoma), or breast carcinoma. The epithelial cancers may be
characterized in various other ways including, but not limited to,
serous, endometrioid, mucinous, clear cell, brenner, or
undifferentiated. In some embodiments, the present invention is
used in the treatment, diagnosis, and/or prognosis of lymphoma or
its subtypes, including, but not limited to, lymphocyte-rich
classical Hodgkin lymphoma, mixed cellularity classical Hodgkin
lymphoma, lymphocyte-depleted classical Hodgkin lymphoma, nodular
sclerosis classical Hodgkin lymphoma, anaplastic large cell
lymphoma, diffuse large B-cell lymphomas, MLL.RTM. pre B-cell ALL)
based upon analysis of markers described herein.
[0062] The term "classifying" includes "to associate" or "to
categorize" a sample with a disease state. In certain instances,
"classifying" is based on statistical evidence, empirical evidence,
or both. In certain embodiments, the methods and systems of
classifying use of a so-called training set of samples having known
disease states. Once established, the training data set serves as a
basis, model, or template against which the features of an unknown
sample are compared, in order to classify the unknown disease state
of the sample. In certain instances, classifying the sample is akin
to diagnosing the disease state of the sample. In certain other
instances, classifying the sample is akin to differentiating the
disease state of the sample from another disease state.
[0063] The term "coding region" refers to regions of a nucleotide
sequence comprising codons which are translated into amino acid
residues, whereas the term "noncoding region" refers to regions of
a nucleotide sequence that are not translated into amino acids
(e.g., 5' and 3' untranslated regions).
[0064] The term "complementary" refers to the broad concept of
sequence complementarity between regions of two nucleic acid
strands or between two regions of the same nucleic acid strand. It
is known that an adenine residue of a first nucleic acid region is
capable of forming specific hydrogen bonds ("base pairing") with a
residue of a second nucleic acid region which is antiparallel to
the first region if the residue is thymine or uracil. Similarly, it
is known that a cytosine residue of a first nucleic acid strand is
capable of base pairing with a residue of a second nucleic acid
strand which is antiparallel to the first strand if the residue is
guanine. A first region of a nucleic acid is complementary to a
second region of the same or a different nucleic acid if, when the
two regions are arranged in an antiparallel fashion, at least one
nucleotide residue of the first region is capable of base pairing
with a residue of the second region. Preferably, the first region
comprises a first portion and the second region comprises a second
portion, whereby, when the first and second portions are arranged
in an antiparallel fashion, at least about 50%, and preferably at
least about 75%, at least about 90%, or at least about 95% of the
nucleotide residues of the first portion are capable of base
pairing with nucleotide residues in the second portion. More
preferably, all nucleotide residues of the first portion are
capable of base pairing with nucleotide residues in the second
portion.
[0065] The term "control" refers to any reference standard suitable
to provide a comparison to the expression products in the test
sample. In one embodiment, the control comprises obtaining a
"control sample" from which expression product levels are detected
and compared to the expression product levels from the test sample.
Such a control sample may comprise any suitable sample, including
but not limited to a sample from a control cancer patient (can be
stored sample or previous sample measurement) with a known outcome;
normal tissue or cells isolated from a subject, such as a normal
patient or the cancer patient, cultured primary cells/tissues
isolated from a subject such as a normal subject or the cancer
patient, adjacent normal cells/tissues obtained from the same organ
or body location of the cancer patient, a tissue or cell sample
isolated from a normal subject, or a primary cells/tissues obtained
from a depository. In another preferred embodiment, the control may
comprise a reference standard expression product level from any
suitable source, including but not limited to housekeeping genes,
an expression product level range from normal tissue (or other
previously analyzed control sample), a previously determined
expression product level range within a test sample from a group of
patients, or a set of patients with a certain outcome (for example,
survival for one, two, three, four years, etc.) or receiving a
certain treatment. It will be understood by those of skill in the
art that such control samples and reference standard expression
product levels can be used in combination as controls in the
methods of the present invention. In one embodiment, the control
may comprise normal or non-cancerous cell/tissue sample. In another
preferred embodiment, the control may comprise an expression level
for a set of patients, such as a set of cancer patients, or for a
set of cancer patients receiving a certain treatment, or for a set
of patients with one outcome versus another outcome. In the former
case, the specific expression product level of each patient can be
assigned to a percentile level of expression, or expressed as
either higher or lower than the mean or average of the reference
standard expression level. In another preferred embodiment, the
control may comprise normal cells, cells from patients treated with
combination chemotherapy and cells from patients having benign
cancer. In another embodiment, the control may also comprise a
measured value for example, average level of expression of a
particular gene in a population compared to the level of expression
of a housekeeping gene in the same population. Such a population
may comprise normal subjects, cancer patients who have not
undergone any treatment (i.e., treatment naive), cancer patients
undergoing therapy, or patients having benign cancer. In another
preferred embodiment, the control comprises a ratio transformation
of expression product levels, including but not limited to
determining a ratio of expression product levels of two genes in
the test sample and comparing it to any suitable ratio of the same
two genes in a reference standard; determining expression product
levels of the two or more genes in the test sample and determining
a difference in expression product levels in any suitable control;
and determining expression product levels of the two or more genes
in the test sample, normalizing their expression to expression of
housekeeping genes in the test sample, and comparing to any
suitable control. In particularly preferred embodiments, the
control comprises a control sample which is of the same lineage
and/or type as the test sample. In another embodiment, the control
may comprise expression product levels grouped as percentiles
within or based on a set of patient samples, such as all patients
with cancer. In one embodiment a control expression product level
is established wherein higher or lower levels of expression product
relative to, for instance, a particular percentile, are used as the
basis for predicting outcome. In another preferred embodiment, a
control expression product level is established using expression
product levels from cancer control patients with a known outcome,
and the expression product levels from the test sample are compared
to the control expression product level as the basis for predicting
outcome. As demonstrated by the data below, the methods of the
invention are not limited to use of a specific cut-point in
comparing the level of expression product in the test sample to the
control.
[0066] As used herein, the term "costimulate" with reference to
activated immune cells includes the ability of a costimulatory
molecule to provide a second, non-activating receptor mediated
signal (a "costimulatory signal") that induces proliferation or
effector function. For example, a costimulatory signal can result
in cytokine secretion, e.g., in a T cell that has received a T
cell-receptor-mediated signal. Immune cells that have received a
cell-receptor mediated signal, e.g., via an activating receptor are
referred to herein as "activated immune cells."
[0067] The term "diagnosing cancer" includes the use of the
methods, systems, and code of the present invention to determine
the presence or absence of a cancer or subtype thereof in an
individual. The term also includes methods, systems, and code for
assessing the level of disease activity in an individual.
[0068] As used herein, the term "diagnostic marker" includes
markers described herein which are useful in the diagnosis of
cancer, e.g., over- or under-activity, emergence, expression,
growth, remission, recurrence or resistance of tumors before,
during or after therapy. The predictive functions of the marker may
be confirmed by, e.g., (1) increased or decreased copy number
(e.g., by FISH, FISH plus SKY, single-molecule sequencing, e.g., as
described in the art at least at J. Biotechnol., 86:289-301, or
qPCR), overexpression or underexpression (e.g., by ISH, Northern
Blot, or qPCR), increased or decreased protein level (e.g., by
IHC), or increased or decreased activity (determined by, for
example, modulation of a pathway in which the marker is involved),
e.g., in more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,
14%, 15%, 20%, 25%, or more of human cancers types or cancer
samples; (2) its presence or absence in a biological sample, e.g.,
a sample containing tissue, whole blood, serum, plasma, buccal
scrape, saliva, cerebrospinal fluid, urine, stool, or bone marrow,
from a subject, e.g., a human, afflicted with cancer; (3) its
presence or absence in clinical subset of subjects with cancer
(e.g., those responding to a particular therapy or those developing
resistance). Diagnostic markers also include "surrogate markers,"
e.g., markers which are indirect markers of cancer progression.
Such diagnostic markers may be useful to identify populations of
subjects amenable to treatment with modulators of PD-1 and/or PD-L1
levels and to thereby treat such stratified patient
populations.
[0069] A molecule is "fixed" or "affixed" to a substrate if it is
covalently or non-covalently associated with the substrate such the
substrate can be rinsed with a fluid (e.g., standard saline
citrate, pH 7.4) without a substantial fraction of the molecule
dissociating from the substrate.
[0070] The term "gene expression data" or "gene expression level"
as used herein refers to information regarding the relative or
absolute level of expression of a gene or set of genes in a cell or
group of cells. The level of expression of a gene may be determined
based on the level of RNA, such as mRNA, encoded by the gene.
Alternatively, the level of expression may be determined based on
the level of a polypeptide or fragment thereof encoded by the gene.
Gene expression data may be acquired for an individual cell, or for
a group of cells such as a tumor or biopsy sample. Gene expression
data and gene expression levels can be stored on computer readable
media, e.g., the computer readable medium used in conjunction with
a microarray or chip reading device. Such gene expression data can
be manipulated to generate gene expression signatures.
[0071] The term "gene expression signature" or "signature" as used
herein refers to a group of coordinately expressed genes. The genes
making up this signature may be expressed in a specific cell
lineage, stage of differentiation, or during a particular
biological response. The genes can reflect biological aspects of
the tumors in which they are expressed, such as the cell of origin
of the cancer, the nature of the non-malignant cells in the biopsy,
and the oncogenic mechanisms responsible for the cancer.
[0072] The term "head cancer" and "neck cancer" refer to cancers
arising from head or neck region or tissue, respectively. In
general, it is a group of cancers originating from the upper
aerodigestive tract, including the lip, oral cavity, nasal cavity,
paranasal sinuses, salivary glands, pharynx, and larynx.
[0073] Cancers of the head and neck are further identified by the
area in which they begin: cancers of the oral cavity, cancers of
the salivary glands, cancer of the paranasal sinuses and nasal
cavity, cancers of the pharynx and cancers of the larynx. The term
"oral cavity" includes the lips, the pharynx, the tongue, the gums
(gingiva), the lining inside the checks and lips (buccal mucosa),
the bottom (floor) of the mouth under the tongue, the bony top of
the mouth (hard palate), the soft palate and the small area behind
the wisdom teeth (retromolar area). The salivary glands include the
glands under the tongue lower jaw (submandibular and sublingual),
in front of the ears (parotid gland), as well as in other parts of
the upper digestive tract-minor salivary glands. The term
"paranasal sinuses" refers to the small hollow spaces in the bones
of the head surrounding the nose. The term "nasal cavity" refers to
the hollow space inside the nose. The term "pharynx" refers to the
hollow tube that starts behind the nose and leads to the esophagus
and the trachea. The pharynx has three parts: "nasopharynx," the
upper part of the pharynx, behind the nose; "oropharynx," the
middle part of the pharynx, which includes the soft palate, the
base of the tongue and the tonsils, and "hypopharynx," the lower
part of the pharynx. The term "larynx" is also known as the
voicebox, and is the passageway formed by cartilage below the
pharynx in the neck. It contains the vocal cords and the
epiglottis. Thus, the term "oral cancer" encompasses all
malignancies that originate in the oral tissues, in particular to
cancers located in any part of the mouth, including the lips, gum
tissue (gingival), tongue, cheek lining (buccal mucosa) and the
soft or hard palate, and floor of the mouth, or in the pharynx, the
top part of the throat. Sometimes, squamous cancer cells are also
found in the lymph nodes of the upper neck.
[0074] Most head and neck cancers begin in the squamous cells that
line the structures found in the head and neck and are therefore
termed squamous cell carcinomas (SCCHN). Because of this, head and
neck cancers are often referred to as squamous cell carcinomas.
Some head and neck cancers begin in other types of cells. For
example, cancers originating from glandular cells are called
adenocarcinomas. With approximately 500,000 new cases annually,
squamous cell carcinomas of the head and neck, the vast majority of
which arise in the oral cavity, represent the sixth most common
cancers in the world. This disease results in nearly about 11,000
deaths each year in the United States alone. The five-year survival
rate after diagnosis for HNSCC remains considerably low
(approximately 50%). This poor prognosis of squamous cell carcinoma
patients is likely due to the fact that most patients are diagnosed
at advanced disease stages, and often fail to respond to available
treatment options. As used herein a "squamous cell carcinoma" is a
cancer arising, at least in part, from a squamous cell population
and/or containing, at least in part, a squamous cell population
including, without limitation, cancers of the cervix; penis; head
and neck, including, without limitation cancers of the oral cavity,
salivary glands, paranasal sinuses and nasal cavity, pharynx and
larynx; lung; esophageal; skin other than melanoma; vulva and
bladder.
[0075] The term "homologous" as used herein, refers to nucleotide
sequence similarity between two regions of the same nucleic acid
strand or between regions of two different nucleic acid strands.
When a nucleotide residue position in both regions is occupied by
the same nucleotide residue, then the regions are homologous at
that position. A first region is homologous to a second region if
at least one nucleotide residue position of each region is occupied
by the same residue. Homology between two regions is expressed in
terms of the proportion of nucleotide residue positions of the two
regions that are occupied by the same nucleotide residue. By way of
example, a region having the nucleotide sequence 5'-ATTGCC-3' and a
region having the nucleotide sequence 5'-TATGGC-3' share 50%
homology. Preferably, the first region comprises a first portion
and the second region comprises a second portion, whereby, at least
about 50%, and preferably at least about 75%, at least about 90%,
or at least about 95% of the nucleotide residue positions of each
of the portions are occupied by the same nucleotide residue. More
preferably, all nucleotide residue positions of each of the
portions are occupied by the same nucleotide residue.
[0076] The term "host cell" is intended to refer to a cell into
which a nucleic acid of the invention, such as a recombinant
expression vector of the invention, has been introduced. The terms
"host cell" and "recombinant host cell" are used interchangeably
herein. It should be understood that such terms refer not only to
the particular subject cell but to the progeny or potential progeny
of such a cell. Because certain modifications may occur in
succeeding generations due to either mutation or environmental
influences, such progeny may not, in fact, be identical to the
parent cell, but are still included within the scope of the term as
used herein.
[0077] The term "humanized antibody," as used herein, is intended
to include antibodies made by a non-human cell having variable and
constant regions which have been altered to more closely resemble
antibodies that would be made by a human cell, for example, by
altering the non-human antibody amino acid sequence to incorporate
amino acids found in human germline immunoglobulin sequences.
Humanized antibodies may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo), for example in the CDRs. The term "humanized
antibody", as used herein, also includes antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework
sequences.
[0078] As used herein, the term "immune cell" refers to cells that
play a role in the immune response. Immune cells are of
hematopoietic origin, and include lymphocytes, such as B cells and
T cells; natural killer cells; myeloid cells, such as monocytes,
macrophages, eosinophils, mast cells, basophils, and
granulocytes.
[0079] As used herein, the term "immune response" includes T cell
mediated and/or B cell mediated immune responses. Exemplary immune
responses include T cell responses, e.g., cytokine production and
cellular cytotoxicity. In addition, the term immune response
includes immune responses that are indirectly effected by T cell
activation, e.g., antibody production (humoral responses) and
activation of cytokine responsive cells, e.g., macrophages.
[0080] As used herein, the term "inhibit" includes the decrease,
limitation, or blockage, of, for example a particular action,
function, or interaction. For example, cancer is "inhibited" if at
least one symptom of the cancer, such as hyperproliferative growth,
is alleviated, terminated, slowed, or prevented. As used herein,
cancer is also "inhibited" if recurrence or metastasis of the
cancer is reduced, slowed, delayed, or prevented.
[0081] As used herein, the term "inhibitory signal" refers to a
signal transmitted via an inhibitory receptor (e.g., CTLA4 or PD-1)
for a polypeptide on a immune cell. Such a signal antagonizes a
signal via an activating receptor (e.g., via a TCR, CD3, BCR, or Fc
polypeptide) and can result in, e.g., inhibition of second
messenger generation; an inhibition of proliferation; an inhibition
of effector function in the immune cell, e.g., reduced
phagocytosis, reduced antibody production, reduced cellular
cytotoxicity, the failure of the immune cell to produce mediators,
(such as cytokines (e.g., IL-2) and/or mediators of allergic
responses); or the development of anergy.
[0082] As used herein, the term "interaction," when referring to an
interaction between two molecules, refers to the physical contact
(e.g., binding) of the molecules with one another. Generally, such
an interaction results in an activity (which produces a biological
effect) of one or both of said molecules. The activity may be a
direct activity of one or both of the molecules. Alternatively, one
or both molecules in the interaction may be prevented from binding
their ligand, and thus be held inactive with respect to ligand
binding activity (e.g., binding its ligand and triggering or
inhibiting an immune response). To inhibit such an interaction
results in the disruption of the activity of one or more molecules
involved in the interaction. To enhance such an interaction is to
prolong or increase the likelihood of said physical contact, and
prolong or increase the likelihood of said activity.
[0083] An "isolated antibody." as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities. Moreover, an isolated
antibody may be substantially free of other cellular material
and/or chemicals.
[0084] As used herein, an "isolated protein" refers to a protein
that is substantially free of other proteins, cellular material,
separation medium, and culture medium when isolated from cells or
produced by recombinant DNA techniques, or chemical precursors or
other chemicals when chemically synthesized. An "isolated" or
"purified" protein or biologically active portion thereof is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the antibody,
polypeptide, peptide or fusion protein is derived, or substantially
free from chemical precursors or other chemicals when chemically
synthesized. The language "substantially free of cellular material"
includes preparations, in which compositions of the invention are
separated from cellular components of the cells from which they are
isolated or recombinantly produced. In one embodiment, the language
"substantially free of cellular material" includes preparations of
having less than about 30%, 20%, 10, or 5% (by dry weight) of
cellular material. When an antibody, polypeptide, peptide or fusion
protein or fragment thereof, e.g., a biologically active fragment
thereof, is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
protein preparation.
[0085] A "kit" is any manufacture (e.g., a package or container)
comprising at least one reagent, e.g., a probe, for specifically
detecting or modulating the expression of a marker of the
invention. The kit may be promoted, distributed, or sold as a unit
for performing the methods of the present invention.
[0086] As used herein, the term "lung cancer" refers to the
collection of cancers affecting lung tissue. Non-small cell lung
cancer (NSCLC) represents approximately 87% of all lung cancers.
The remaining 13% of all lung cancers are small cell lung cancers,
although mixed-cell lung cancers do occur. Because small cell lung
cancer is rare and rapidly fatal, the opportunity for early
detection is small. There are three main types of NSCLC: squamous
cell carcinoma, large cell carcinoma, and adenocarcinoma.
Adenocarcinoma is the most common form of lung cancer (30%-40% and
reported to be as high as 50%) and is the lung cancer most
frequently found in both smokers and non-smokers. Squamous cell
carcinoma accounts for 25-30% of all lung cancers and is generally
found in a proximal bronchus. Early stage NSCLC tends to be
localized, and if detected early it can often be treated by surgery
with a favorable outcome and improved survival. Other treatment
options include radiation treatment, drug therapy, and a
combination of these methods. NSCLC is staged by the size of the
tumor and its presence in other tissues including lymph nodes. In
the occult stage, cancer cells are found in sputum samples or
lavage samples and no tumor is detectable in the lungs. In stage 0,
only the innermost lining of the lungs exhibit cancer cells and the
tumor has not grown through the lining. In stage IA, the cancer is
considered invasive and has grown deep into the lung tissue but the
tumor is less than 3 cm across. In this stage, the tumor is not
found in the bronchus or lymph nodes. In stage IB, the tumor is
either larger than 3 cm across or has grown into the bronchus or
pleura, but has not grown into the lymph nodes. In stage IIA, the
tumor is more than 3 cm across and has grown into the lymph nodes.
In stage IIB, the tumor has either been found in the lymph nodes
and is greater than 3 cm across or grown into the bronchus or
pleura, or the cancer is not in the lymph nodes but is found in the
chest wall, diaphragm, pleura, bronchus, or tissue that surrounds
the heart. In stage IIIA, cancer cells are found in the lymph nodes
near the lung and bronchi and in those between the lungs but on the
side of the chest where the tumor is located. In stage IIIB, cancer
cells are located on the opposite side of the chest from the tumor
and in the neck. Other organs near the lungs may also have cancer
cells and multiple tumors may be found in one lobe of the lungs. In
stage IV, tumors are found in more than one lobe of the same lung
or both lungs and cancer cells are found in other parts of the
body. Current methods of diagnosis for lung cancer include testing
sputum for cancerous cells, chest x-ray, fiber optic evaluation of
airways, and low dose spiral computed tomography (CT). Sputum
cytology has a very low sensitivity. Chest X-ray is also relatively
insensitive, requiring lesions to be greater than 1 cm in size to
be visible. Bronchoscopy requires that the tumor is visible inside
airways accessible to the bronchoscope. The most widely recognized
diagnostic method is CT, but in common with X-ray, the use of CT
involves ionizing radiation, which itself can cause cancer. CT also
has significant limitations: the scans require a high level of
technical skill to interpret and many of the observed abnormalities
are not in fact lung cancer and substantial healthcare costs are
incurred in following up CT findings. The most common incidental
finding is a benign lung nodule. Lung nodules are relatively round
lesions, or areas of abnormal tissue, located within the lung and
may vary in size. Lung nodules may be benign or cancerous, but most
are benign. If a nodule is below 4 mm the prevalence is only 1.5%,
if 4-8 mm the prevalence is approximately 6%, and if above 20 mm
the incidence is approximately 20%. F or small and medium-sized
nodules, the patient is advised to undergo a repeat scan within
three months to a year. For many large nodules, the patient
receives a biopsy (which is invasive and may lead to complications)
even though most of these are benign.
[0087] A "marker" or "biomarker" includes a nucleic acid or
polypeptide whose altered level of expression in a tissue or cell
from its expression level in a control (e.g., normal or healthy
tissue or cell) is associated with a disease state, such as a
cancer or subtype thereof (e.g., head, neck, and/or lung cancers).
A "marker nucleic acid" is a nucleic acid (e.g., mRNA, cDNA, mature
miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding
site, or a variant thereof and other classes of small RNAs known to
a skilled artisan) encoded by or corresponding to a marker of the
invention. Such marker nucleic acids include DNA (e.g., cDNA)
comprising the entire or a partial sequence of any of the nucleic
acid sequences set forth in Table 1 and the Examples or the
complement of such a sequence. The marker nucleic acids also
include RNA comprising the entire or a partial sequence of any of
the nucleic acid sequences set forth in the Sequence Listing or the
complement of such a sequence, wherein all thymidine residues are
replaced with uridine residues. A "marker protein" includes a
protein encoded by or corresponding to a marker of the invention. A
marker protein comprises the entire or a partial sequence of any of
the sequences set forth in Table 1 and the Examples or the
Examples. The terms "protein" and "polypeptide" are used
interchangeably. In some embodiments, specific combinations of
biomarkers are preferred. For example, a combination or subgroup of
one or more of the biomarkers selected from the group shown in
Table.
[0088] As used herein, the term "modulate" includes up-regulation
and down-regulation, e.g., enhancing or inhibiting a response.
[0089] The "normal" or "control" level of expression of a marker is
the level of expression of the marker in cells of a subject, e.g.,
a human patient, not afflicted with a cancer. An "over-expression"
or "significantly higher level of expression" of a marker refers to
an expression level in a test sample that is greater than the
standard error of the assay employed to assess expression, and is
preferably at least twice, and more preferably 2.1, 2.2, 2.3, 2.4,
2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,
8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times
or more higher than the expression activity or level of the marker
in a control sample (e.g., sample from a healthy subject not having
the marker associated disease) and preferably, the average
expression level of the marker in several control samples. A
"significantly lower level of expression" of a marker refers to an
expression level in a test sample that is at least twice, and more
preferably 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4,
4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20 times or more lower than the expression
level of the marker in a control sample (e.g., sample from a
healthy subject not having the marker associated disease) and
preferably, the average expression level of the marker in several
control samples.
[0090] The term "pre-malignant lesions" as described herein refers
to a lesion that, while not cancerous, has potential for becoming
cancerous. It also includes the term "pre-malignant disorders" or
"potentially malignant disorders." In particular this refers to a
benign, morphologically and/or histologically altered tissue that
has a greater than normal risk of malignant transformation, and a
disease or a patient's habit that does not necessarily alter the
clinical appearance of local tissue but is associated with a
greater than normal risk of precancerous lesion or cancer
development in that tissue (leukoplakia, erythroplakia,
erytroleukoplakia lichen planus (lichenoid reaction) and any lesion
or an area which histological examination showed atypia of cells or
dysplasia.
[0091] The term "probe" refers to any molecule which is capable of
selectively binding to a specifically intended target molecule, for
example, a nucleotide transcript or protein encoded by or
corresponding to a marker. Probes can be either synthesized by one
skilled in the art, or derived from appropriate biological
preparations. For purposes of detection of the target molecule,
probes may be specifically designed to be labeled, as described
herein. Examples of molecules that can be utilized as probes
include, but are not limited to, RNA, DNA, proteins, antibodies,
and organic molecules.
[0092] The term "prognosis" includes a prediction of the probable
course and outcome of cancer or the likelihood of recovery from the
disease. In some embodiments, the use of statistical algorithms
provides a prognosis of cancer in an individual. For example, the
prognosis can be surgery, development of a clinical subtype of
cancer (e.g., head, neck, and/or lung cancers), development of one
or more clinical factors, development of intestinal cancer, or
recovery from the disease.
[0093] The term "response to cancer therapy" or "outcome of cancer
therapy" relates to any response of the hyperproliferative disorder
(e.g., cancer) to a cancer therapy, preferably to a change in tumor
mass and/or volume after initiation of neoadjuvant or adjuvant
chemotherapy. Hyperproliferative disorder response may be assessed,
for example for efficacy or in a neoadjuvant or adjuvant situation,
where the size of a tumor after systemic intervention can be
compared to the initial size and dimensions as measured by CT, PET,
mammogram, ultrasound or palpation. Response may also be assessed
by caliper measurement or pathological examination of the tumor
after biopsy or surgical resection for solid cancers. Responses may
be recorded in a quantitative fashion like percentage change in
tumor volume or in a qualitative fashion like "pathological
complete response" (pCR), "clinical complete remission" (cCR),
"clinical partial remission" (cPR), "clinical stable disease"
(cSD), "clinical progressive disease" (cPD) or other qualitative
criteria. Assessment of hyperproliferative disorder response may be
done early after the onset of neoadjuvant or adjuvant therapy,
e.g., after a few hours, days, weeks or preferably after a few
months. A typical endpoint for response assessment is upon
termination of neoadjuvant chemotherapy or upon surgical removal of
residual tumor cells and/or the tumor bed. This is typically three
months after initiation of neoadjuvant therapy. In some
embodiments, clinical efficacy of the therapeutic treatments
described herein may be determined by measuring the clinical
benefit rate (CBR). The clinical benefit rate is measured by
determining the sum of the percentage of patients who are in
complete remission (CR), the number of patients who are in partial
remission (PR) and the number of patients having stable disease
(SD) at a time point at least 6 months out from the end of therapy.
The shorthand for this formula is CBR=CR+PR+SD over 6 months. In
some embodiments, the CBR for a particular cancer therapeutic
regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, or more. Additional criteria for evaluating the
response to cancer therapies are related to "survival," which
includes all of the following: survival until mortality, also known
as overall survival (wherein said mortality may be either
irrespective of cause or tumor related); "recurrence-free survival"
(wherein the term recurrence shall include both localized and
distant recurrence); metastasis free survival; disease free
survival (wherein the term disease shall include cancer and
diseases associated therewith). The length of said survival may be
calculated by reference to a defined start point (e.g., time of
diagnosis or start of treatment) and end point (e.g., death,
recurrence or metastasis). In addition, criteria for efficacy of
treatment can be expanded to include response to chemotherapy,
probability of survival, probability of metastasis within a given
time period, and probability of tumor recurrence. For example, in
order to determine appropriate threshold values, a particular
cancer therapeutic regimen can be administered to a population of
subjects and the outcome can be correlated to copy number, level of
expression, level of activity, etc. of one or more biomarkers
listed in Table 1 and the Examples or the Examples that were
determined prior to administration of any cancer therapy. The
outcome measurement may be pathologic response to therapy given in
the neoadjuvant setting. Alternatively, outcome measures, such as
overall survival and disease-free survival can be monitored over a
period of time for subjects following cancer therapy for whom the
measurement values are known. In certain embodiments, the same
doses of cancer therapeutic agents are administered to each
subject. In related embodiments, the doses administered are
standard doses known in the art for cancer therapeutic agents. The
period of time for which subjects are monitored can vary. For
example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker
threshold values that correlate to outcome of a cancer therapy can
be determined using methods such as those described in the Examples
section. Outcomes can also be measured in terms of a "hazard ratio"
(the ratio of death rates for one patient group to another;
provides likelihood of death at a certain time point), "overall
survival" (OS), and/or "progression free survival." In certain
embodiments, the prognosis comprises likelihood of overall survival
rate at 1 year, 2 years, 3 years, 4 years, or any other suitable
time point. The significance associated with the prognosis of poor
outcome in all aspects of the present invention is measured by
techniques known in the art. For example, significance may be
measured with calculation of odds ratio. In a further embodiment,
the significance is measured by a percentage. In one embodiment, a
significant risk of poor outcome is measured as odds ratio of 0.8
or less or at least about 1.2, including by not limited to: 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2.0, 2.5, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0 and
40.0. In a further embodiment, a significant increase or reduction
in risk is at least about 20%, including but not limited to about
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% and 98%. In a further embodiment, a significant increase
in risk is at least about 50%. Thus, the present invention further
provides methods for making a treatment decision for a cancer
patient, comprising carrying out the methods for prognosing a
cancer patient according to the different aspects and embodiments
of the present invention, and then weighing the results in light of
other known clinical and pathological risk factors, in determining
a course of treatment for the cancer patient. For example, a cancer
patient that is shown by the methods of the invention to have an
increased risk of poor outcome by combination chemotherapy
treatment can be treated with more aggressive therapies, including
but not limited to radiation therapy, peripheral blood stem cell
transplant, bone marrow transplant, or novel or experimental
therapies under clinical investigation.
[0094] The term "resistance" refers to an acquired or natural
resistance of a cancer sample or a mammal to a cancer therapy
(i.e., being nonresponsive to or having reduced or limited response
to the therapeutic treatment), such as having a reduced response to
a therapeutic treatment by 25% or more, for example, 30%, 40%, 50%,
60%, 70%, 80%, or more, to 2-fold 3-fold, 4-fold, 5-fold, 10-fold,
15-fold, 20-fold or more. The reduction in response can be measured
by comparing with the same cancer sample or mammal before the
resistance is acquired, or by comparing with a different cancer
sample or a mammal who is known to have no resistance to the
therapeutic treatment. A typical acquired resistance to
chemotherapy is called "multidrug resistance." The multidrug
resistance can be mediated by P-glycoprotein or can be mediated by
other mechanisms, or it can occur when a mammal is infected with a
multi-drug-resistant microorganism or a combination of
microorganisms. The determination of resistance to a therapeutic
treatment is routine in the art and within the skill of an
ordinarily skilled clinician, for example, can be measured by cell
proliferative assays and cell death assays as described herein as
"sensitizing." In some embodiments, the term "reverses resistance"
means that the use of a second agent in combination with a primary
cancer therapy (e.g., chemotherapeutic or radiation therapy) is
able to produce a significant decrease in tumor volume at a level
of statistical significance (e.g., p<0.05) when compared to
tumor volume of untreated tumor in the circumstance where the
primary cancer therapy (e.g., chemotherapeutic or radiation
therapy) alone is unable to produce a statistically significant
decrease in tumor volume compared to tumor volume of untreated
tumor. This generally applies to tumor volume measurements made at
a time when the untreated tumor is growing log rhythmically.
[0095] The term "sample" used for detecting or determining the
presence or level of at least one biomarker is typically whole
blood, plasma, serum, saliva, urine, stool (e.g., feces), tears,
and any other bodily fluid (e.g., as described above under the
definition of "body fluids"), or a tissue sample (e.g., biopsy)
such as a small intestine, colon sample, or surgical resection
tissue. In certain instances, the method of the present invention
further comprises obtaining the sample from the individual prior to
detecting or determining the presence or level of at least one
marker in the sample.
[0096] The term "sensitize" means to alter cancer cells or tumor
cells in a way that allows for more effective treatment of the
associated cancer with a cancer therapy (e.g., chemotherapeutic or
radiation therapy. In some embodiments, normal cells are not
affected to an extent that causes the normal cells to be unduly
injured by the cancer therapy (e.g., chemotherapy or radiation
therapy). An increased sensitivity or a reduced sensitivity to a
therapeutic treatment is measured according to a known method in
the art for the particular treatment and methods described herein
below, including, but not limited to, cell proliferative assays
(Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res 1982; 42:
2159-2164), cell death assays (Weisenthal L M, Shoemaker R H,
Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94:
161-173; Weisenthal L M, Lippman M E, Cancer Treat Rep 1985; 69:
615-632; Weisenthal L M, In: Kaspers G J L, Pieters R, Twentyman P
R, Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia
and Lymphoma. Langhorne, P A: Harwood Academic Publishers. 1993:
415-432; Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82-90).
The sensitivity or resistance may also be measured in animal by
measuring the tumor size reduction over a period of time, for
example, 6 month for human and 4-6 weeks for mouse. A composition
or a method sensitizes response to a therapeutic treatment if the
increase in treatment sensitivity or the reduction in resistance is
25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to
2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 5-fold, 20-fold or more,
compared to treatment sensitivity or resistance in the absence of
such composition or method. The determination of sensitivity or
resistance to a therapeutic treatment is routine in the art and
within the skill of an ordinarily skilled clinician. It is to be
understood that any method described herein for enhancing the
efficacy of a cancer therapy can be equally applied to methods for
sensitizing hyperproliferative or otherwise cancerous cells (e.g.,
resistant cells) to the cancer therapy.
[0097] The term "synergistic effect" refers to the combined effect
of two or more anticancer agents or chemotherapy drugs can be
greater than the sum of the separate effects of the anticancer
agents or chemotherapy drugs alone.
[0098] The term "subject" refers to any healthy animal, mammal or
human, or any animal, mammal or human afflicted with a condition of
interest (e.g., cancer). The term "subject" is interchangeable with
"patient."
[0099] The language "substantially free of chemical precursors or
other chemicals" includes preparations of antibody, polypeptide,
peptide or fusion protein in which the protein is separated from
chemical precursors or other chemicals which are involved in the
synthesis of the protein. In one embodiment, the language
"substantially free of chemical precursors or other chemicals"
includes preparations of antibody, polypeptide, peptide or fusion
protein having less than about 30% (by dry weight) of chemical
precursors or non-antibody, polypeptide, peptide or fusion protein
chemicals, more preferably less than about 20% chemical precursors
or non-antibody, polypeptide, peptide or fusion protein chemicals,
still more preferably less than about 10% chemical precursors or
non-antibody, polypeptide, peptide or fusion protein chemicals, and
most preferably less than about 5% chemical precursors or
non-antibody, polypeptide, peptide or fusion protein chemicals.
[0100] The term "substantially pure cell population" refers to a
population of cells having a specified cell marker characteristic
and differentiation potential that is at least about 50%,
preferably at least about 75-80%, more preferably at least about
85-90%, and most preferably at least about 95% of the cells making
up the total cell population. Thus, a "substantially pure cell
population" refers to a population of cells that contain fewer than
about 50%, preferably fewer than about 20-25%, more preferably
fewer than about 10-15%, and most preferably fewer than about 5% of
cells that do not display a specified marker characteristic and
differentiation potential under designated assay conditions.
[0101] As used herein, the term "survival" includes all of the
following: survival until mortality, also known as overall survival
(wherein said mortality may be either irrespective of cause or
tumor related): "recurrence-free survival" (wherein the term
recurrence shall include both localized and distant recurrence);
metastasis free survival; disease free survival (wherein the term
disease shall include cancer and diseases associated therewith).
The length of said survival may be calculated by reference to a
defined start point (e.g., time of diagnosis or start of treatment)
and end point (e.g., death, recurrence or metastasis). In addition,
criteria for efficacy of treatment can be expanded to include
response to chemotherapy, probability of survival, probability of
metastasis within a given time period, and probability of tumor
recurrence.
[0102] A "transcribed polynucleotide" or "nucleotide transcript" is
a polynucleotide (e.g., an mRNA, hnRNA, cDNA, mature miRNA,
pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site,
or a variant thereof or an analog of such RNA or cDNA) which is
complementary to or homologous with all or a portion of a mature
mRNA made by transcription of a marker of the invention and normal
post-transcriptional processing (e.g., splicing), if any, of the
RNA transcript, and reverse transcription of the RNA
transcript.
[0103] As used herein, the term "vector" refers to a nucleic acid
capable of transporting another nucleic acid to which it has been
linked. One type of vector is a "plasmid", which refers to a
circular double stranded DNA loop into which additional DNA
segments may be ligated. Another type of vector is a viral vector,
wherein additional DNA segments may be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
or simply "expression vectors." In general, expression vectors of
utility in recombinant DNA techniques are often in the form of
plasmids. In the present specification, "plasmid" and "vector" may
be used interchangeably as the plasmid is the most commonly used
form of vector. However, the invention is intended to include such
other forms of expression vectors, such as viral vectors (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0104] An "underexpression" or "significantly lower level of
expression or copy number" of a marker refers to an expression
level or copy number in a test sample that is greater than the
standard error of the assay employed to assess expression or copy
number, but is preferably at least twice, and more preferably
three, four, five or ten or more times less than the expression
level or copy number of the marker in a control sample (e.g.,
sample from a healthy subject not afflicted with cancer) and
preferably, the average expression level or copy number of the
marker in several control samples.
[0105] As used herein, the term "unresponsiveness" includes
refractivity of immune cells to stimulation, e.g., stimulation via
an activating receptor or a cytokine. Unresponsiveness can occur,
e.g., because of exposure to immunosuppressants or exposure to high
doses of antigen. As used herein, the term "anergy" or "tolerance"
includes refractivity to activating receptor-mediated stimulation.
Such refractivity is generally antigen-specific and persists after
exposure to the tolerizing antigen has ceased. For example, anergy
in T cells (as opposed to unresponsiveness) is characterized by
lack of cytokine production, e.g., IL-2. T cell anergy occurs when
T cells are exposed to antigen and receive a first signal (a T cell
receptor or CD-3 mediated signal) in the absence of a second signal
(a costimulatory signal). Under these conditions, reexposure of the
cells to the same antigen (even if reexposure occurs in the
presence of a costimulatory polypeptide) results in failure to
produce cytokines and, thus, failure to proliferate. Anergic T
cells can, however, proliferate if cultured with cytokines (e.g.,
IL-2). For example, T cell anergy can also be observed by the lack
of IL-2 production by T lymphocytes as measured by ELISA or by a
proliferation assay using an indicator cell line. Alternatively, a
reporter gene construct can be used. For example, anergic T cells
fail to initiate IL-2 gene transcription induced by a heterologous
promoter under the control of the 5' IL-2 gene enhancer or by a
multimer of the API sequence that can be found within the enhancer
(Kang et al. (1992) Science 257:1134).
[0106] There is a known and definite correspondence between the
amino acid sequence of a particular protein and the nucleotide
sequences that can code for the protein, as defined by the genetic
code (shown below). Likewise, there is a known and definite
correspondence between the nucleotide sequence of a particular
nucleic acid and the amino acid sequence encoded by that nucleic
acid, as defined by the genetic code.
TABLE-US-00001 GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT
Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N)
AAC, AAT Aspartic acid (Asp, D) GAC, GAT Cysteine (Cys, C) TGC, TGT
Glutamic acid (Glu, E) GAA, GAG Glutamine (Gln, Q) CAA, CAG Glycine
(Gly, G) GGA, GGC, GGG, GGT Histidine (His, H) CAC, CAT Isoleucine
(Ile, I) ATA, ATC, ATT Laucine (Leu, L) CTA, CTC, CTG, CTT, TTA,
TTG Lysine (Lys, K) AAA, AAG Methionine (Met, M) ATG Phenylalanine
(Phe, F) TTC, TTT Proline (Pro, P) CCA, CCC, CCG, CCT Serine (Ser,
S) AGC, AGT, TCA, TCC, TCG, TCT Threonine (Thr, T) ACA, ACC, ACG,
ACT Tryptophan (Trp, W) TGG Tyrosine (Tyr, Y) TAC, TAT Valine (Val,
V) GTA, GTC, GTG, GTT Tarmination signal (end) TAA, TAG, TGA
[0107] An important and well known feature of the genetic code is
its redundancy, whereby, for most of the amino acids used to make
proteins, more than one coding nucleotide triplet may be employed
(illustrated above). Therefore, a number of different nucleotide
sequences may code for a given amino acid sequence. Such nucleotide
sequences are considered functionally equivalent since they result
in the production of the same amino acid sequence in all organisms
(although certain organisms may translate some sequences more
efficiently than they do others). Moreover, occasionally, a
methylated variant of a purine or pyrimidine may be found in a
given nucleotide sequence. Such methylations do not affect the
coding relationship between the trinucleotide codon and the
corresponding amino acid.
[0108] In view of the foregoing, the nucleotide sequence of a DNA
or RNA coding for a fusion protein or polypeptide of the invention
(or any portion thereof) can be used to derive the fusion protein
or polypeptide amino acid sequence, using the genetic code to
translate the DNA or RNA into an amino acid sequence. Likewise, for
a fusion protein or polypeptide amino acid sequence, corresponding
nucleotide sequences that can encode the fusion protein or
polypeptide can be deduced from the genetic code (which, because of
its redundancy, will produce multiple nucleic acid sequences for
any given amino acid sequence). Thus, description and/or disclosure
herein of a nucleotide sequence which encodes a fusion protein or
polypeptide should be considered to also include description and/or
disclosure of the amino acid sequence encoded by the nucleotide
sequence. Similarly, description and/or disclosure of a fusion
protein or polypeptide amino acid sequence herein should be
considered to also include description and/or disclosure of all
possible nucleotide sequences that can encode the amino acid
sequence.
[0109] Finally, nucleic acid and amino acid sequence information
for the loci and biomarkers of the present invention (e.g.,
biomarkers listed in Table 1 and the Examples) are well known in
the art and readily available on publicly available databases, such
as the National Center for Biotechnology Information (NCBI). For
example, exemplary nucleic acid and amino acid sequences derived
from publicly available sequence databases are provided below.
[0110] The nucleic acid and amino acid sequences of a
representative human PD-1 biomarker is available to the public at
the GenBank database under NM_005018.2 and NP_005009.2 (see also
Ishida et al. (1992) EMBO J. 11:3887; Shinohara et al. (1994)
Genomics 23:704; and U.S. Pat. No. 5,698,520). Nucleic acid and
polypeptide sequences of PD-1 orthologs in organisms other than
humans are well known and include, for example, monkey PD-1
(NM_001114358.1 and NP_001107830.1), mouse PD-1 (NM_0087998.2 and
NP_032824.1), rat PD-1 (NM_001106927.1 and NP_001100397.1), chicken
PD-1 (XM_422723.3 and XP_422723.2), cow PD-1 (NM_001083506.1 and
NP_001076975.1), and dog PD-1 (XM_543338.3 and XP_543338.3).
[0111] At least five transcript (i.e., splice) variants encoding
different human PD-L1 isoforms exist and are described herein.
PD-L1 proteins generally comprise a signal sequence, an IgV domain,
and an IgC domain. The sequence of human PD-L1 transcript variant 1
is the canonical sequence, all positional information described
with respect to the remaining isoforms are determined from this
sequence, and the sequences are available to the public at the
GenBank database under NM_014143.3 and NP_054862.1. In this
isoform, the signal sequence is shown from about amino acid 1 to
about amino acid 18, the IgV domain is shown from about amino acid
19 to about amino acid 134, the IgC domain is shown from about
amino acid 135 to about amino acid 227, the transmembrane domain is
shown from about amino acids 239 to about amino acid 259, and the
cytoplasmic domain is shown from about amino acid 260 to about
amino acid 290.
[0112] The sequences of human PD-L1 transcript variant 2 can be
found under NM_001267706.1 and NP_001254635.1 and the encoded
protein lacks an alternate in-frame excon in the 5' coding region
compared to variant 1 (i.e., missing amino acid residues 17-130) so
as to result in a shorter protein.
[0113] The sequences of human PD-L1 transcript variant 3 is
provided herein and encodes a naturally occurring B7-4 soluble
polypeptide, i.e., having a short hydrophilic domain and no
transmembrane domain. In this isoform, the signal sequence is shown
from about amino acid 1 to about amino acid 18, the IgV domain is
shown from about amino acid 19 to about amino acid 134, the IgC
domain of SEQ ID NO:2 is shown from about amino acid 135 to about
amino acid 227, and the hydrophilic tail is shown from about amino
acid 228 to about amino acid 245.
[0114] In addition, another soluble PD-L1 isoform exists having the
amino acid sequence shown herein. This fourth PD-L1 isoform differs
from that of the first PD-L1 isoform in that there is a K to D
substitution at amino acid position 178 and amino acid residues
179-290 are deleted.
[0115] Moreover, another soluble PD-L1 isoform exists having the
amino acid sequence of residues 1-227 encoded by transcript variant
1 and thereby only comprising a signal sequence, the IgV domain,
and the IgC domain.
[0116] In some embodiments, the soluble PD-L1 isoforms of the
present invention do not contain the signal sequence as such a
sequence is usually cleaved prior to secretion of the polypeptide
from the cell. In other embodiments, the soluble PD-L1 isoforms of
the presention invention consist of the IgV domain and the IgC
domain (i.e., the extracellular portion of the full-length,
membrane-bound PD-L1) and can further comprise heterologous
sequences, such as Fe domains, protein tags, conjugated
therapeutics, and the like. Such soluble PD-L1 isoforms can be
generated by alternative splicing in a number of ways well known to
the skilled artisan involving the elimination of exons 4, 5, and 6
of full-length, membrane-bound PD-L1 cDNA.
[0117] Nucleic acid and polypeptide sequences of PD-L1 orthologs in
organisms other than humans are well known and include, for
example, monkey PD-L1 (NM 001083889.1 and NP_001077358.1),
chimpanzee PD-L1 (XM_0011401705.2 and XP_001140705.1), mouse PD-L1
(NM 021893.3 and NP_068693.1), rat PD-L1 (NM_001191954.1 and
NP_001178883.1), chicken PD-L1 (XM_424811.3 and XP_424811.3), cow
PD-L1 (NM_001163412.1 and NP_001156884.1), and dog PD-L1
(XM_541302.3 and XP_541302.3).
[0118] Antibodies for the detection of PD-L1 and methods for making
them are known in the art.
TABLE-US-00002 TABLE 1 Nucleic Acid and Amnio Acid Sequences of the
Present Invention Human PD-1 cDNA Sequence SEQ ID NO: 1 1
atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg
61 ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt
ctccccagcc 121 ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct
gcagcttctc caacacatcg 181 gagagcttcg tgctaaactg gtaccgcatg
agccccagca accagacgga caagctggcc 241 gccttccccg aggaccgcag
ccagcccggc caggactgcc gcttccgtgt cacacaactg 301 cccaacgggc
gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 361
tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca
421 gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag
cccctcaccc 481 aggccagccg gccagttcca aaccctggtg gttggtgtcg
tgggcggcct gctgggcagc 541 ctggtgctgc tagtctgggt cctggccgtc
atctgctccc gggccgcacg agggacaata 601 ggagccaggc gcaccggcca
gcccctgaag gaggacccct cagccgtgcc tgtgttctct 661 gtggactatg
gggagctgga tttccagtgg cgagagaaga ccccggagcc ccccgtgccc 721
tgtgtccctg agcagacgga gtatgccacc attgtctttc ctagcggaat gggcacctca
781 tcccccgccc gcaggggctc agctgacggc cctcggagtg cccagccact
gaggcctgag 841 gatggacact gctcttggcc cctctga Human PD-1 Amino Acid
Sequence SEQ ID NO: 2 1 mqipqapwpv vwavlqlgwr pgwfldspdr pwnpptfspa
llvvtegdna tgtcsfsnts 61 esfvlnwyrm spsnqtdkla afpedrsqpg
qdcrfrvtql pngrdfhmsv vrarrndsgt 121 ylcgaislap kaqikrslra
elrvterrae vptahpspsp rpagqfqtlv vgvvggllgs 181 lvllwvwlav
icsraargti qarrtgqplk edpsavpvfs vdygeldfqw rektpeppvp 241
cvpeqteyat ivfpsgmgts sparrgsadg prsaqplrpe dghcswpl Mouse PD-1
cDNA Sequence SEQ ID NO: 3 1 atgtgggtcc ggcaggtacc ctggtcattc
acttgggctg tgctgcagtt gagctggcaa 61 tcagggtggc ttctagaggt
ccccaatggg ccctggaggt ccctcacctt ctacccagcc 121 tggctcacag
tgtcagaggg agcaaatgcc accttcacct gcagcttgtc caactggtcg 181
gaggatctta tgctgaactg gaaccgcctg agtcccagca accagactga aaaacaggcc
241 gccttctgta atggtttgag ccaacccgtc caggatgccc gcttccagat
catacagctg 301 cccaacaggc atgacttcca catgaacatc cttgacacac
ggcgcaatga cagtggcatc 361 tacctctgtg gggccatctc cctgcacccc
aaggcaaaaa tcgaggagag ccctggagca 421 gagctcgtgg taacagagag
aatcctggag acctcaacaa gatatcccag cccctcgccc 481 aaaccagaag
gccggtttca aggcatggtc attggtatca tgagtgccct agtgggtatc 541
cctgtattgc tgctgctggc ctgggcccta gctgtcttct gctcaacaag tatgtcagag
601 gccagaggag ctggaagcaa ggacgacact ctgaaggagg agccttcagc
agcacctgtc 661 cctagtgtgg cctatgagga gctggacttc cagggacgag
agaagacacc agagctccct 721 accgcctgtg tgcacacaga atatgccacc
attgtcttca ctgaagggct gggtgcctcg 781 gccatgggac gtaggggctc
agctgatggc ctgcagggtc ctcggcctcc aagacatgag 841 gatggacatt
gttcttggcc tctttga Mouse PD-1 Amino Acid Sequence SEQ ID NO: 4 1
mwvrqvpwsf twavlqlswq sgwllevpng pwrsltfypa wltvsegana tftcslsnws
61 edlmlnwnrl spsnqtekqa afcnglsqpv qdarfqilql pnrhdfhmnl
ldtrrndsgl 121 ylcgaislhp kakieespga elvvterile tstrypspsp
kpegrfqgmv igimsalvgi 181 pvllllawal avfcstsmse argagskddt
lkeepsaapv psvayeeldf qgrektpelp 241 tacvhteyat ivfteglgas
amgrrgsadg lqgprpprhe dghcswpl Human PD-L1 Variant cDNA Sequence
SEQ ID NO: 5 1 atgaggatat ttgctgtctt tatattcatg acctactggc
atttgctgaa cgcatttact 61 gtcacggttc ccaaggacct atatgtggta
gagtatggta gcaatatgac aattgaatgc 121 aaattcccag tagaaaaaca
attagacctg gctgcactaa ttgtctattg ggaaatggag 181 gataagaaca
ttattcaatt tgtgcatgga gaggaagacc tgaaggttca gcatagtagc 241
tacagacaga gggcccggct gttgaaggac cagctctccc tgggaaatgc tgcacttcag
301 atcacagatg tgaaattgca ggatgcaggg gtgtaccgct gcatgatcag
ctatggtggt 361 gccgactaca agcgaattac tgtgaaagtc aatgccccat
acaacaaaat caaccaaaga 421 attttggttg tggatccagt cacctctgaa
catgaactga catgtcaggc tgagggctac 481 cccaaggccg aagtcatctg
gacaagcagt gaccatcaag tcctgagtgg taagaccacc 541 accaccaatt
ccaagagaga ggagaagctt ttcaatgtga ccagcacact gagaatcaac 601
acaacaacta atgagatttt ctactgcact tttaggagat tagatcctga ggaaaaccat
661 acagctgaat tggtcatccc agaactacct ctggcacatc ctccaaatga
aaggactcac 721 ttggtaattc tgggagccat cttattatgc cttggtgtag
cactgacatt catcttccgt 781 ttaagaaaag ggagaatgat ggatgtgaaa
aaatgtggca tccaagatac aaactcaaag 841 aagcaaagtg atacacattt
ggaggagacg taa Human PD-L1 Isoform 1 Amino Acid Sequence SEQ ID NO:
6 1 mrifavfifm tywhllnaft vtvpkdlyvv eygsnmtiec kfpvekqldl
aalivyweme 61 dkniiqfvhg eedlkvqhss yrqrarllkd qlslgnaalq
itdvklqdag vyrcmisygg 121 adykritvkv napyniknqr ilvvdpvtse
heltcqaegy pkaeviwtss dhqvlsgktt 181 ttnskreekl fnvtstlrin
tttneifyct frrldpeenh taelvipelp lahppnerth 241 lvilgaillc
lgvaltfifr lrkgrmmdvk kcgiqdtnsk kqsdthleet Human PD-L1 Variant 2
cDNA Sequence SEQ ID NO: 7 1 atgaggatat ttgctgtctt tatattcatg
acctactggc atttgctgaa cgccccatac 61 aacaaaatca accaaagaat
tttggttgtg gatccagtca cctctgaaca tgaactgaca 121 tgtcaggctg
agggctaccc caaggccgaa gtcatctgga caagcagtga ccatcaagtc 181
ctgagtggta agaccaccac caccaattcc aagagagagg agaagctttt caatgtgacc
241 agcacactga gaatcaacac aacaactaat gagattttct actgcacttt
taggagatta 301 gatcctgagg aaaaccatac agctgaattg gtcatcccag
aactacctct ggcacatcct 361 ccaaatgaaa ggactcactt ggtaattctg
ggagccatct tattatgcct tggtgtagca 421 ctgacattca tcttccgttt
aagaaaaggg agaatgatgg atgtgaaaaa atgtggcatc 481 caagatacaa
actcaaagaa gcaaagtgat acacatttgg aggagacgta a Human PD-L1 Isoform 2
Amino Acid Sequence SEQ ID NO: 8 1 mrifavfifm tywhllnapy nkinqrilvv
tpvtsehelt cqaegypkae viwtssdhqv 61 lsgkttttns kreeklfnvt
stlrintttn eifyctfrrl dpeenhtael vipelplahp 121 pnerthlvil
gaillclgva ltfifrlrkg rmmdvkkcgi qdtnskkqsd thleet Human PD-L1
Isoform 3 cDNA Sequence SEQ ID NO: 9 gcttcccgag gctccgcacc
agccgcgctt ctgtccgcct gcagggcatt ccagaaag 58 atg agg ata ttt gct
gtc ttt ata ttc atg acc tac tgg cat ttg ctg 106 Met Arg Ile Phe Ala
Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 aac gca ttt
act gtc acg gtt ccc aag gac cta tat gtg gta gag tat 154 Asn Ala Phe
Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 ggt
agc aat atg aca att gaa tgc aaa ttc cca gta gaa aaa caa tta 202 Gly
Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40
45 gac ctg gct gca cta att gtc tat tgg gaa atg gag gat aag aac att
250 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile
50 55 60 att caa ttt gtg cat gga gag gaa gac ctg aag gtt cag cat
agt agc 298 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His
Ser Ser 65 70 75 80 tac aga cag agg gcc cgg ctg ttg aag gac cag ctc
tcc ctg gga aat 346 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu
Ser Leu Gly Asn 85 90 95 gct gca ctt cag atc aca gat gtg aaa ttg
cag gat gca ggg gtg tac 394 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu
Gln Asp Ala Gly Val Tyr 100 105 110 cgc tgc atg atc agc tat ggt ggt
gcc gac tac aag cga att act gtg 442 Arg Cys Met Ile Ser Tyr Gly Gly
Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 aaa gtc aat gcc cca tac
aac aaa atc aac caa aga att ttg gtt gtg 490 Lys Val Asn Ala Pro Tyr
Asn Lys Ile Asn Gln Arg Ile Leu Val Vla 130 135 140 gat cca gtc acc
tct gaa cat gaa ctg aca tgt cag gct gag ggc tac 538 Asp Pro Val Thr
Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 ccc
aag gcc gaa gtc atc tgg aca agc agt gac cat caa gtc ctg agt 586 Pro
Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170
175 ggt aag acc acc acc acc aat tcc aag aga gag gag aag ctt ttc att
634 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn
180 185 190 gtg acc agc aca ctg aga atc aac aca aca act aat gag att
ttc tac 682 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile
Phe Tyr 195 200 205 tgc act ttt agg aga tta gat cct gag gaa aac cat
aca gct gaa ttg 730 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His
Thr Ala Glu Leu 210 215 220 gtc atc cca ggt aat att ctg aat gtg tcc
att aaa ata tgt cta aca 778 Val Ile Pro Gly Asn Ile Leu Asn Val Ser
Ile Lys Ile Cys Leu Thr 225 230 235 240 ctg tcc cct agc acc
tagcatgatg tctgcctatc atagtcattc agtgattgtt 833 Leu Ser Pro Ser Thr
245 gaataaatga atgaatgaat aacactatgt ttacaaaata tatcctaatt
cctcacctcc 893 attcatccaa accatattgt tacttaataa acattcagca
gatatttatg gaataaaaaa 953 aaaaaaaaaa aaaaa 968 Human PD-L1 Isoform
3 Amino Acid Sequence SEQ ID NO: 10 Met Arg Ile Phe Ala Val Phe Ile
Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr
Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met
Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60
Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65
70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu
Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp
Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp
Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys
Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu
His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala
Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly
Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185
190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr
195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala
Glu Leu 210 215 220 Val Ile Pro Gly Asn Ile Leu Asn Val Ser Ile Lys
Ile Cys Leu Thr 225 230 235 240 Leu Ser Pro Ser Thr 245 Human PD-L1
Isoform 4 Amino Acid Sequence SEQ ID NO: 11 1
MRIFAVIFIMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMS 61
DKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGG 121
ADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGD Human
PD-L1 Isoform 5 cDNA Sequence SEQ ID NO: 12
ATGAGGATATTTGCTGTCTTTATATTCAATGACCTACTGGCATTTGCTGAACGCATTTACTGTCACGGT
TCCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAA
ACAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTCAATTTGTGCA
TGGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCT
CTCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTACCGCTGCAT
GATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGCCCCATACAACAAAATCAA
CCAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCC
CAAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGACCACCACCACCAATTC
CAAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTT
CTACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCATAA
Human PD-L1 Isoform 5 Amino Acid Sequence (Soluble 1) SEQ ID NO: 13
M R I F A V F I F M T Y W H L L N A F T V T V P K D L Y V V E Y G S
N M T I E C K F P V E K Q L D L A A L I V Y W E M E D K N I I Q F V
H G E E D L K V Q H S S Y R Q R A R L L K D Q L S L G N A A L Q I T
D V K L Q D A G V Y R C M I S Y G G A D Y K R I T V K V N A P Y N K
I N Q R
I L V V D P V T S E H E L T C Q A E G Y P K A E V I M T S S D H Q V
L S G K T T T T N S K R E E K L F N V T S T L R I N T T T N E I F Y
C T F R R L D P E E N H T A E L V I P Stop Human PD-L1 Isoform 6
cDNA Sequence SEQ ID NO: 14
ATGAGGATATTTGCTGTCTTTATATTCATGACCTACTGGCATTTGCTGAACGCATTTACTGTCACGGTT
CCCAAGGACCTATATGTGGTAGAGTATGGTAGCAATATGACAATTGAATGCAAATTCCCAGTAGAAAAA
CAATTAGACCTGGCTGCACTAATTGTCTATTGGGAAATGGAGGATAAGAACATTATTCAATTTGTGCAT
GGAGAGGAAGACCTGAAGGTTCAGCATAGTAGCTACAGACAGAGGGCCCGGCTGTTGAAGGACCAGCTC
TCCCTGGGAAATGCTGCACTTCAGATCACAGATGTGAAATTGCAGGATGCAGGGGTGTACCGCTGCATG
ATCAGCTATGGTGGTGCCGACTACAAGCGAATTACTGTGAAAGTCAATGCCCCATACAACAAAATCAAC
CAAAGAATTTTGGTTGTGGATCCAGTCACCTCTGAACATGAACTGACATGTCAGGCTGAGGGCTACCCC
AAGGCCGAAGTCATCTGGACAAGCAGTGACCATCAAGTCCTGAGTGGTAAGACCACCACCACCAATTCC
AAGAGAGAGGAGAAGCTTTTCAATGTGACCAGCACACTGAGAATCAACACAACAACTAATGAGATTTTC
TACTGCACTTTTAGGAGATTAGATCCTGAGGAAAACCATACAGCTGAATTGGTCATCCCAGGTAATATT
CTGAATGTGTCCATTAAAATATGTCTAACACTGTCCCCTAGCACCTAG Human PD-L1
Isoform 6 Amino Acid Sequence (Soluble 2) SEQ ID NO: 15 M R I F A V
F I F M T Y W H L L N A F T V T V P K D L Y V V E Y G S N M T I E C
K F P V E K Q L D L A A L I V Y W E M E D K N I I Q F V H G E E D L
K V Q H S S Y R Q R A R L L K D Q L S L G N A A L Q I T D V K L Q D
A G V Y R C M I S Y G G A D Y K R I T V K V N A P Y N K I N Q R I L
V V D P V T S E H E L T C Q A E G Y P K A E V I W T S S D H Q V L S
G K T T T T N S K R E E K L F N V T S T L R I N T T T N E I F Y C T
F R R L D P E E N H T A E L V I P G N I L N V S I K I C L T L S P S
T * Mouse PD-L1 cDNA Sequence SEQ ID NO: 16 1 atgaggatat ttgctggcat
tatattcaca gcctgctgtc acttgctacg ggcgtttact 61 atcacggctc
caaaggactt gtacgtggtg gagtatggca gcaacgtcac gatggagtgc 121
agattccctg tagaacggga gctggacctg cttgcgttag tggtgtactg ggaaaaggaa
181 gatgagcaag tgattcagtt tgtggcagga gaggaggacc ttaagcctca
gcacagcaac 241 ttcaggggga gagcctcgct gccaaaggac cagcttttga
agggaaatgc tgcccttcag 301 atcacagacg tcaagctgca ggacgcaggc
gtttactgct gcataatcag ctacggtggt 361 gcggactaca agcgaatcac
gctgaaagtc aatgccccat accgcaaaat caaccagaga 421 atttccgtgg
atccagccac ttctgagcat gaactaatat gtcaggccga gggttatcca 481
gaagctgagg taatctggac aaacagtgac caccaacccg tgagtgggaa gagaagtgtc
541 accacttccc ggacagaggg gatgcttctc aatgtgacca gcagtctgag
ggtcaacgcc 601 acagcgaatg atgttttcta ctgtacgttt tggagatcac
agccagggca aaaccacaca 661 gcggagctga tcatcccaga actgcctgca
acacatcctc cacagaacag gactcactgg 721 gtgcttctgg gatccatcct
gttgttcctc attgtagtgt ccacggtcct cctcttcttg 781 agaaaacaag
tgagaatgct agatgtggag aaatgtggcg ttgaagatac aagctcaaaa 841
aaccgaaatg atacacaatt cgaggagacg taa Mouse PD-L1 Amino Acid
Sequence SEQ ID NO: 17 1 mrifagiift acchllraft itapkdlyvv
eygsnvtmec rfpvereldl lalvvyweke 61 deqviqfvag eedlkpqhsn
frgraslpkd qllkgnaalq itdvklqdag vyccissygg 121 adykritlkv
napyrkinqr isvdpatseh elicqaegyp eaeviwtnsd hqpvsqkrsv 181
ttsrtegmll nvtsslrvna tandvfyctf wrsqpgqnht aeliipelpa thppqnrthw
241 vllgsillfl ivvstvllfl rkqvrmidve kcgvedtssk nrndqfeet
II. AGENTS AND COMPOSITIONS
[0119] Novel agents and compositions of the present invention are
provided herein and can be used for the diagnosis, prognosis,
prevention, and treatment of cancer (e.g., head, neck, and/or lung
cancers) and cancer subtypes thereof. Such agents and compositions
can detect and/or modulate, e.g., up- or down-regulate, expression
and/or activity of gene products or fragments thereof encoded by
biomarkers of the invention, including the biomarkers listed in
Table 1 and the Examples, Exemplary agents include antibodies,
small molecules, peptides, peptidomimetics, natural ligands, and
derivatives of natural ligands, that can either bind and/or
activate or inhibit protein biomarkers of the invention, including
the biomarkers listed in Table 1 and the Examples, or fragments
thereof, RNA interference, antisense, nucleic acid aptamers, etc.
that can downregulate the expression and/or activity of the
biomarkers of the invention, including the biomarkers listed in
Table 1 and the Examples, or fragments thereof.
[0120] In one embodiment, isolated nucleic acid molecules that
specifically hybridize with or encode one or more biomarkers listed
in Table 1 and the Examples or biologically active portions thereof
are presented. As used herein, the term "nucleic acid molecule" is
intended to include DNA molecules (i.e., cDNA or genomic DNA) and
RNA molecules (i.e., mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA. An "isolated" nucleic acid molecule is one
which is separated from other nucleic acid molecules which are
present in the natural source of the nucleic acid. Preferably, an
"isolated" nucleic acid is free of sequences which naturally flank
the nucleic acid (i.e., sequences located at the 5' and 3' ends of
the nucleic acid) in the genomic DNA of the organism from which the
nucleic acid is derived. For example, in various embodiments, the
isolated nucleic acid molecules corresponding to the one or more
biomarkers listed in Table 1 and the Examples can contain less than
about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide
sequences which naturally flank the nucleic acid molecule in
genomic DNA of the cell from which the nucleic acid is derived
(i.e., a head and neck or lung cancer cell). Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or chemical precursors or
other chemicals when chemically synthesized.
[0121] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having the nucleotide sequence of one or more
biomarkers listed in Table 1 and the Examples or a nucleotide
sequence which is at least about 50%, preferably at least about
60%, more preferably at least about 70%, yet more preferably at
least about 80%, still more preferably at least about 90%, and most
preferably at least about 95% or more (e.g., about 98%) homologous
to the nucleotide sequence of one or more biomarkers listed in
Table 1 and the Examples or a portion thereof (i.e., 100, 200, 300,
400, 450, 500, or more nucleotides), can be isolated using standard
molecular biology techniques and the sequence information provided
herein. For example, a human cDNA can be isolated from a human cell
line (from Stratagene, La Jolla, Calif., or Clontech, Palo Alto,
Calif.) using all or portion of the nucleic acid molecule, or
fragment thereof, as a hybridization probe and standard
hybridization techniques (i.e., as described in Sambrook. J.,
Fritsh, E. F., and Maniatis. T. Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N. Y. 1989). Moreover, a
nucleic acid molecule encompassing all or a portion of the
nucleotide sequence of one or more biomarkers listed in Table 1 and
the Examples or a nucleotide sequence which is at least about 50%,
preferably at least about 60%, more preferably at least about 70%,
yet more preferably at least about 80%, still more preferably at
least about 90%, and most preferably at least about 95% or more
homologous to the nucleotide sequence, or fragment thereof, can be
isolated by the polymerase chain reaction using oligonucleotide
primers designed based upon the sequence of the one or more
biomarkers listed in Table 1 and the Examples, or fragment thereof,
or the homologous nucleotide sequence. For example, mRNA can be
isolated from muscle cells (i.e., by the guanidinium-thiocyanate
extraction procedure of Chirgwin et al. (1979) Biochemistry 18:
5294-5299) and cDNA can be prepared using reverse transcriptase
(i.e., Moloney MLV reverse transcriptase, available from Gibco/BRL,
Bethesda, Md.; or AMV reverse transcriptase, available from
Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic
oligonucleotide primers for PCR amplification can be designed
according to well-known methods in the art. A nucleic acid of the
invention can be amplified using cDNA or, alternatively, genomic
DNA, as a template and appropriate oligonucleotide primers
according to standard PCR amplification techniques. The nucleic
acid so amplified can be cloned into an appropriate vector and
characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to the nucleotide sequence of one or
more biomarkers listed in Table 1 and the Examples can be prepared
by standard synthetic techniques, i.e., using an automated DNA
synthesizer.
[0122] Probes based on the nucleotide sequences of one or more
biomarkers listed in Table 1 and the Examples can be used to detect
transcripts or genomic sequences encoding the same or homologous
proteins. In preferred embodiments, the probe further comprises a
label group attached thereto, i.e., the label group can be a
radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells or tissue which express one or more
biomarkers listed in Table 1 and the Examples, such as by measuring
a level of nucleic acid in a sample of cells from a subject, i.e.,
detecting mRNA levels of one or more biomarkers listed in Table 1
and the Examples.
[0123] Nucleic acid molecules encoding proteins corresponding to
one or more biomarkers listed in Table 1 and the Examples from
different species are also contemplated. For example, rat or monkey
cDNA can be identified based on the nucleotide sequence of a human
and/or mouse sequence and such sequences are well known in the art.
In one embodiment, the nucleic acid molecule(s) of the invention
encodes a protein or portion thereof which includes an amino acid
sequence which is sufficiently homologous to an amino acid sequence
of one or more biomarkers listed in Table 1 and the Examples, such
that the protein or portion thereof modulates (e.g., enhance), one
or more of the following biological activities: a) binding to the
biomarker; b) modulating the copy number of the biomarker; c)
modulating the expression level of the biomarker; and d) modulating
the activity level of the biomarker.
[0124] As used herein, the language "sufficiently homologous"
refers to proteins or portions thereof which have amino acid
sequences which include a minimum number of identical or equivalent
(e.g., an amino acid residue which has a similar side chain as an
amino acid residue in one or more biomarkers listed in Table 1 and
the Examples, or fragment thereof) amino acid residues to an amino
acid sequence of the biomarker, or fragment thereof, such that the
protein or portion thereof modulates (e.g., enhance) one or more of
the following biological activities: a) binding to the biomarker;
b) modulating the copy number of the biomarker, c) modulating the
expression level of the biomarker; and d) modulating the activity
level of the biomarker.
[0125] In another embodiment, the protein is at least about 50%,
preferably at least about 60%, more preferably at least about 70%
75%, 80%, 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more homologous to the entire amino acid sequence of the biomarker,
or a fragment thereof.
[0126] Portions of proteins encoded by nucleic acid molecules of
the one or more biomarkers listed in Table 1 and the Examples are
preferably biologically active portions of the protein. As used
herein, the term "biologically active portion" of one or more
biomarkers listed in Table 1 and the Examples is intended to
include a portion, e.g., a domain/motif, that has one or more of
the biological activities of the full-length protein.
[0127] Standard binding assays, e.g., immunoprecipitations and
yeast two-hybrid assays, as described herein, or functional assays,
e.g., RNAi or overexpression experiments, can be performed to
determine the ability of the protein or a biologically active
fragment thereof to maintain a biological activity of the
full-length protein.
[0128] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence of the one or more
biomarkers listed in Table 1 and the Examples, or fragment thereof
due to degeneracy of the genetic code and thus encode the same
protein as that encoded by the nucleotide sequence, or fragment
thereof. In another embodiment, an isolated nucleic acid molecule
of the invention has a nucleotide sequence encoding a protein
having an amino acid sequence of one or more biomarkers listed in
Table 1 and the Examples, or fragment thereof, or a protein having
an amino acid sequence which is at least about 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous
to the amino acid sequence of the one or more biomarkers listed in
Table 1 and the Examples, or fragment thereof. In another
embodiment, a nucleic acid encoding a polypeptide consists of
nucleic acid sequence encoding a portion of a full-length fragment
of interest that is less than 195, 190, 185, 180, 175, 170, 165,
160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100,
95, 90, 85, 80, 75, or 70 amino acids in length.
[0129] It will be appreciated by those skilled in the art that DNA
sequence polymorphisms that lead to changes in the amino acid
sequences of the one or more biomarkers listed in Table 1 and the
Examples may exist within a population (e.g., a mammalian and/or
human population). Such genetic polymorphisms may exist among
individuals within a population due to natural allelic variation.
As used herein, the terms "gene" and "recombinant gene" refer to
nucleic acid molecules comprising an open reading frame encoding
one or more biomarkers listed in Table 1 and the Examples,
preferably a mammalian, e.g., human, protein. Such natural allelic
variations can typically result in 1-5% variance in the nucleotide
sequence of the one or more biomarkers listed in Table 1 and the
Examples. Any and all such nucleotide variations and resulting
amino acid polymorphisms in the one or more biomarkers listed in
Table 1 and the Examples that are the result of natural allelic
variation and that do not alter the functional activity of the one
or more biomarkers listed in Table 1 and the Examples are intended
to be within the scope of the invention. Moreover, nucleic acid
molecules encoding one or more biomarkers listed in Table 1 and the
Examples from other species.
[0130] In addition to naturally-occurring allelic variants of the
one or more biomarkers listed in Table 1 and the Examples sequence
that may exist in the population, the skilled artisan will further
appreciate that changes can be introduced by mutation into the
nucleotide sequence, or fragment thereof, thereby leading to
changes in the amino acid sequence of the encoded one or more
biomarkers listed in Table 1 and the Examples, without altering the
functional ability of the one or more biomarkers listed in Table 1
and the Examples. For example, nucleotide substitutions leading to
amino acid substitutions at "non-essential" amino acid residues can
be made in the sequence, or fragment thereof. A "non-essential"
amino acid residue is a residue that can be altered from the
wild-type sequence of the one or more biomarkers listed in Table 1
and the Examples without altering the activity of the one or more
biomarkers listed in Table 1 and the Examples, whereas an
"essential" amino acid residue is required for the activity of the
one or more biomarkers listed in Table 1 and the Examples. Other
amino acid residues, however, (e.g., those that are not conserved
or only semi-conserved between mouse and human) may not be
essential for activity and thus are likely to be amenable to
alteration without altering the activity of the one or more
biomarkers listed in Table 1 and the Examples.
[0131] The term "sequence identity or homology" refers to the
sequence similarity between two polypeptide molecules or between
two nucleic acid molecules. When a position in both of the two
compared sequences is occupied by the same base or amino acid
monomer subunit, e.g., if a position in each of two DNA molecules
is occupied by adenine, then the molecules are homologous or
sequence identical at that position. The percent of homology or
sequence identity between two sequences is a function of the number
of matching or homologous identical positions shared by the two
sequences divided by the number of positions compared .times.100.
For example, if 6 of 10, of the positions in two sequences are the
same then the two sequences are 60% homologous or have 60% sequence
identity. By way of example, the DNA sequences ATTGCC and TATGGC
share 50% homology or sequence identity. Generally, a comparison is
made when two sequences are aligned to give maximum homology.
Unless otherwise specified "loop out regions", e.g., those arising
from, from deletions or insertions in one of the sequences are
counted as mismatches.
[0132] The comparison of sequences and determination of percent
homology between two sequences can be accomplished using a
mathematical algorithm. Preferably, the alignment can be performed
using the Clustal Method. Multiple alignment parameters include GAP
Penalty=10, Gap Length Penalty=10. For DNA alignments, the pairwise
alignment parameters can be Htuple=2, Gap penalty=5, Window=4, and
Diagonal saved=4. For protein alignments, the pairwise alignment
parameters can be Ktuple=1, Gap penalty=3, Window=5, and Diagonals
Saved=5.
[0133] In a preferred embodiment, the percent identity between two
amino acid sequences is determined using the Needleman and Wunsch
(J. Mol. Biol. (48):444-453 (1970)) algorithm which has been
incorporated into the GAP program in the GCG software package
(available online), using either a Blossom 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment,
the percent identity between two nucleotide sequences is determined
using the GAP program in the GCG software package (available
online), using a NWSgapdna.CMP matrix and a gap weight of 40, 50,
60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In
another embodiment, the percent identity between two amino acid or
nucleotide sequences is determined using the algorithm of E. Meyers
and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated
into the ALIGN program (version 2.0) (available online), using a
PAM120 weight residue table, a gap length penalty of 12 and a gap
penalty of 4.
[0134] An isolated nucleic acid molecule encoding a protein
homologous to one or more biomarkers listed in Table 1 and the
Examples, or fragment thereof, can be created by introducing one or
more nucleotide substitutions, additions or deletions into the
nucleotide sequence, or fragment thereof, or a homologous
nucleotide sequence such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis. Preferably,
conservative amino acid substitutions are made at one or more
predicted non-essential amino acid residues. A "conservative amino
acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have
been defined in the art. These families include amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid, glutamic acid), uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine). Thus, a predicted
nonessential amino acid residue in one or more biomarkers listed in
Table 1 and the Examples is preferably replaced with another amino
acid residue from the same side chain family. Alternatively, in
another embodiment, mutations can be introduced randomly along all
or part of the coding sequence of the one or more biomarkers listed
in Table 1 and the Examples, such as by saturation mutagenesis, and
the resultant mutants can be screened for an activity described
herein to identify mutants that retain desired activity. Following
mutagenesis, the encoded protein can be expressed recombinantly
according to well-known methods in the art and the activity of the
protein can be determined using, for example, assays described
herein.
[0135] The levels of one or more biomarkers listed in Table 1 and
the Examples levels may be assessed by any of a wide variety of
well-known methods for detecting expression of a transcribed
molecule or protein. Non-limiting examples of such methods include
immunological methods for detection of proteins, protein
purification methods, protein function or activity assays, nucleic
acid hybridization methods, nucleic acid reverse transcription
methods, and nucleic acid amplification methods.
[0136] In preferred embodiments, the levels of one or more
biomarkers listed in Table 1 and the Examples levels are
ascertained by measuring gene transcript (e.g., mRNA), by a measure
of the quantity of translated protein, or by a measure of gene
product activity. Expression levels can be monitored in a variety
of ways, including by detecting mRNA levels, protein levels, or
protein activity, any of which can be measured using standard
techniques. Detection can involve quantification of the level of
gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme
activity), or, alternatively, can be a qualitative assessment of
the level of gene expression, in particular in comparison with a
control level. The type of level being detected will be clear from
the context.
[0137] In a particular embodiment, the mRNA expression level can be
determined both by in situ and by in vitro formats in a biological
sample using methods known in the art. The term "biological sample"
is intended to include tissues, cells, biological fluids and
isolates thereof, isolated from a subject, as well as tissues,
cells and fluids present within a subject. Many expression
detection methods use isolated RNA. For in vitro methods, any RNA
isolation technique that does not select against the isolation of
mRNA can be utilized for the purification of RNA from cells (see,
e.g., Ausubel et al., ed., Current Protocols in Molecular Biology,
John Wiley & Sons, New York 1987-1999). Additionally, large
numbers of tissue samples can readily be processed using techniques
well known to those of skill in the art, such as, for example, the
single-step RNA isolation process of Chomezynski (1989, U.S. Pat.
No. 4,843,155).
[0138] The isolated mRNA can be used in hybridization or
amplification assays that include, but are not limited to, Southern
or Northern analyses, polymerase chain reaction analyses and probe
arrays. One preferred diagnostic method for the detection of mRNA
levels involves contacting the isolated mRNA with a nucleic acid
molecule (probe) that can hybridize to the mRNA encoded by the gene
being detected. The nucleic acid probe can be, for example, a
full-length cDNA, or a portion thereof, such as an oligonucleotide
of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length
and sufficient to specifically hybridize under stringent conditions
to a mRNA or genomic DNA encoding one or more biomarkers listed in
Table 1 and the Examples. Other suitable probes for use in the
diagnostic assays of the invention are described herein.
Hybridization of an mRNA with the probe indicates that one or more
biomarkers listed in Table 1 and the Examples is being
expressed.
[0139] In one format, the mRNA is immobilized on a solid surface
and contacted with a probe, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probe(s) are immobilized on a solid surface and the mRNA is
contacted with the probe(s), for example, in a gene chip array,
e.g., an Affymetrix.TM. gene chip array. A skilled artisan can
readily adapt known mRNA detection methods for use in detecting the
level of the One or more biomarkers listed in Table 1 and the
Examples mRNA expression levels.
[0140] An alternative method for determining mRNA expression level
in a sample involves the process of nucleic acid amplification,
e.g., by RT-PCR (the experimental embodiment set forth in Mullis,
1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany,
1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self-sustained
sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), rolling
circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any
other nucleic acid amplification method, followed by the detection
of the amplified molecules using techniques well-known to those of
skill in the art. These detection schemes are especially useful for
the detection of nucleic acid molecules if such molecules are
present in very low numbers. As used herein, amplification primers
are defined as being a pair of nucleic acid molecules that can
anneal to 5' or 3' regions of a gene (plus and minus strands,
respectively, or vice-versa) and contain a short region in between.
In general, amplification primers are from about 10 to 30
nucleotides in length and flank a region from about 50 to 200
nucleotides in length. Under appropriate conditions and with
appropriate reagents, such primers permit the amplification of a
nucleic acid molecule comprising the nucleotide sequence flanked by
the primers.
[0141] For in situ methods, mRNA does not need to be isolated from
the cells prior to detection. In such methods, a cell or tissue
sample is prepared/processed using known histological methods. The
sample is then immobilized on a support, typically a glass slide,
and then contacted with a probe that can hybridize to the One or
more biomarkers listed in Table 1 and the Examples mRNA.
[0142] As an alternative to making determinations based on the
absolute expression level, determinations may be based on the
normalized expression level of one or more biomarkers listed in
Table 1 and the Examples. Expression levels are normalized by
correcting the absolute expression level by comparing its
expression to the expression of a non-biomarker gene, e.g., a
housekeeping gene that is constitutively expressed. Suitable genes
for normalization include housekeeping genes such as the actin
gene, or epithelial cell-specific genes. This normalization allows
the comparison of the expression level in one sample, e.g., a
subject sample, to another sample, e.g., a normal sample, or
between samples from different sources.
[0143] The level or activity of a protein corresponding to one or
more biomarkers listed in Table 1 and the Examples can also be
detected and/or quantified by detecting or quantifying the
expressed polypeptide. The polypeptide can be detected and
quantified by any of a number of means well known to those of skill
in the art. These may include analytic biochemical methods such as
electrophoresis, capillary electrophoresis, high performance liquid
chromatography (HPLC), thin layer chromatography (TLC),
hyperdiffusion chromatography, and the like, or various
immunological methods such as fluid or gel precipitin reactions,
immunodiffusion (single or double), immunoelectrophoresis,
radioimmunoassay (RIA), enzyme-linked immunosorbent assays
(ELISAs), immunofluorescent assays, Western blotting, and the like.
A skilled artisan can readily adapt known protein/antibody
detection methods for use in determining whether cells express the
biomarker of interest.
[0144] The present invention further provides soluble, purified
and/or isolated polypeptide forms of one or more biomarkers listed
in Table 1 and the Examples, or fragments thereof. In addition, it
is to be understood that any and all attributes of the polypeptides
described herein, such as percentage identities, polypeptide
lengths, polypeptide fragments, biological activities, antibodies,
etc. can be combined in any order or combination with respect to
any biomarker listed in Table 1 and the Examples and combinations
thereof
[0145] In one aspect, a polypeptide may comprise a full-length
amino acid sequence corresponding to one or more biomarkers listed
in Table 1 and the Examples or a full-length amino acid sequence
with 1 to about 20 conservative amino acid substitutions. An amino
acid sequence of any described herein can also be at least 50, 55,
60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
99.5% identical to the full-length sequence of one or more
biomarkers listed in Table 1 and the Examples, which is either
described herein, well known in the art, or a fragment thereof. In
another aspect, the present invention contemplates a composition
comprising an isolated polypeptide corresponding to one or more
biomarkers listed in Table 1 and the Examples polypeptide and less
than about 25%, or alternatively 15%, or alternatively 5%,
contaminating biological macromolecules or polypeptides.
[0146] The present invention further provides compositions related
to producing, detecting, characterizing, or modulating the level or
activity of such polypeptides, or fragment thereof, such as nucleic
acids, vectors, host cells, and the like. Such compositions may
serve as compounds that modulate the expression and/or activity of
one or more biomarkers listed in Table 1 and the Examples. For
example, anti-PD-L1 antibodies that may bind specifically to PD-L1
or soluble PD-L1 can be used to reduce soluble PD-L1 (i.e., both
forms of PD-L1 contain an extracellular domain typically targeted
by antibodies) and thereby a) stop the titration of such
therapeutic agents from binding to membrane-bound forms of PD-L1
and/or b) inhibit the inhibition of immunological responses
promoted by the soluble forms of PD-L1.
[0147] An isolated polypeptide or a fragment thereof (or a nucleic
acid encoding such a polypeptide) corresponding to one or more
biomarkers of the invention, including the biomarkers listed in
Table 1 and the Examples or fragments thereof, can be used as an
immunogen to generate antibodies that bind to said immunogen, using
standard techniques for polyclonal and monoclonal antibody
preparation according to well-known methods in the art. An
antigenic peptide comprises at least 8 amino acid residues and
encompasses an epitope present in the respective full length
molecule such that an antibody raised against the peptide forms a
specific immune complex with the respective full length molecule.
Preferably, the antigenic peptide comprises at least 10 amino acid
residues. In one embodiment such epitopes can be specific for a
given polypeptide molecule from one species, such as mouse or human
(i.e., an antigenic peptide that spans a region of the polypeptide
molecule that is not conserved across species is used as immunogen;
such non conserved residues can be determined using an alignment
such as that provided herein).
[0148] For example, a polypeptide immunogen typically is used to
prepare antibodies by immunizing a suitable subject (e.g., rabbit,
goat, mouse or other mammal) with the immunogen. An appropriate
immunogenic preparation can contain, for example, a recombinantly
expressed or chemically synthesized molecule or fragment thereof to
which the immune response is to be generated. The preparation can
further include an adjuvant, such as Freund's complete or
incomplete adjuvant, or similar immunostimulatory agent.
immunization of a suitable subject with an immunogenic preparation
induces a polyclonal antibody response to the antigenic peptide
contained therein.
[0149] Polyclonal antibodies can be prepared as described above by
immunizing a suitable subject with a polypeptide immunogen. The
polypeptide antibody titer in the immunized subject can be
monitored over time by standard techniques, such as with an enzyme
linked immunosorbent assay (ELISA) using immobilized polypeptide.
If desired, the antibody directed against the antigen can be
isolated from the mammal (e.g., from the blood) and further
purified by well-known techniques, such as protein A
chromatography, to obtain the IgG fraction. At an appropriate time
after immunization, e.g., when the antibody titers are highest,
antibody-producing cells can be obtained from the subject and used
to prepare monoclonal antibodies by standard techniques, such as
the hybridoma technique (originally described by Kohler and
Milstein (1975) Nature 256:495-497) (see also Brown et al. (1981)
J. Immunol. 127:539-46; Brown et al. (1980), J. Biol. Chem.
255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. 76:2927-31;
Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human
B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today
4:72), the EBV-hybridoma technique (Cole et al. (1985) Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or
trioma techniques. The technology for producing monoclonal antibody
hybridomas is well known (see generally Kenneth, R. H, in
Monoclonal Antibodies: A New Dimension In Biological Analyses,
Plenum Publishing Corp., New York, N.Y. (1980); Lerner, E. A.
(1981) Yale J. Biol. Med. 54:387-402; Gefter, M. L. et al. (1977)
Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line
(typically a myeloma) is fused to lymphocytes (typically
splenocytes) from a mammal immunized with an immunogen as described
above, and the culture supernatants of the resulting hybridoma
cells are screened to identify a hybridoma producing a monoclonal
antibody that binds to the polypeptide antigen, preferably
specifically.
[0150] Any of the many well-known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating a monoclonal antibody against one or more
biomarkers of the invention, including the biomarkers listed in
Table 1 and the Examples, or a fragment thereof (see, e.g., Galfre,
G. et al. (1977) Nature 266:550-52; Gefter et al. (1977) supra:
Lerner (1981) supra; Kenneth (1980) supra). Moreover, the ordinary
skilled worker will appreciate that there are many variations of
such methods which also would be useful. Typically, the immortal
cell line (e.g., a myeloma cell line) is derived from the same
mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines can be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from the American Type Culture Collection (ATCC),
Rockville, Md. Typically, HAT-sensitive mouse myeloma cells are
fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using
HAT medium, which kills unfused and unproductively fused myeloma
cells (unfused splenocytes die after several days because they are
not transformed). Hybridoma cells producing a monoclonal antibody
of the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind a given polypeptide, e.g.,
using a standard ELISA assay.
[0151] As an alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal specific for one of the above described
polypeptides can be identified and isolated by screening a
recombinant combinatorial immunoglobulin library (e.g., an antibody
phage display library) with the appropriate polypeptide to thereby
isolate immunoglobulin library members that bind the polypeptide.
Kits for generating and screening phage display libraries are
commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the Stratagene
SurfZAP.TM. Phage Display Kit, Catalog No. 240612). Additionally,
examples of methods and reagents particularly amenable for use in
generating and screening an antibody display library can be found
in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.
International Publication No. WO 92/18619; Dower et al.
International Publication No. WO 91/17271; Winter et al.
International Publication WO 92/20791; Markland et al.
International Publication No. WO 92/15679; Breitling et al.
International Publication WO 93/01288; McCafferty et al.
International Publication No. WO 92/01047; Garrard et al.
International Publication No. WO 92/09690; Ladner et al.
International Publication No. WO 90/02809; Fuchs et al. (1991)
Biotechnology (NY) 9:1369-1372; Hay et al. (1992) Hum. Antibod.
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992)
J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature
352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA
89:3576-3580; Garrard et al. (1991) Biotechnology (NY) 9:1373-1377;
Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et
al. (1991) Proc. Natl. Acad. &i. USA 88:7978-7982; and
McCaffcrty et al. (1990) Nature 348:552-554.
[0152] Additionally, recombinant polypeptide antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in Robinson et al. International Patent
Publication PCT/US86/02269; Akira et al. European Patent
Application 184.187; Taniguchi, M. European Patent Application
171,496; Morrison et al. European Patent Application 173,494;
Neuberger et al. PCT Application WO 86/01533; Cabilly et al. U.S.
Pat. No. 4,816,567; Cabilly et al. European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987)
J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood
et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl.
Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science
229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S.
Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525;
Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988)
J. Immunol. 141:4053-4060.
[0153] In addition, humanized antibodies can be made according to
standard protocols such as those disclosed in U.S. Pat. No.
5,565,332. In another embodiment, antibody chains or specific
binding pair members can be produced by recombination between
vectors comprising nucleic acid molecules encoding a fusion of a
polypeptide chain of a specific binding pair member and a component
of a replicable generic display package and vectors containing
nucleic acid molecules encoding a second polypeptide chain of a
single binding pair member using techniques known in the art, e.g.,
as described in U.S. Pat. Nos. 5,565,332, 5,871,907, or 5,733,743.
The use of intracellular antibodies to inhibit protein function in
a cell is also known in the art (see e.g., Carlson, J. R. (1988)
Mol. Cell. Biol. 8:2638-2646; Biocca, S. et al. (1990) EMBO J.
9:101-108; Werge, T. M. et al. (1990) FEBS Lett. 274:193-198;
Carlson, J. R. (1993) Proc. Natl. Acad. Sci. USA 490:7427-7428;
Marasco, W. A. et al. (1993) Proc. Natl. Acad. Sci. USA
90:7889-7893; Biocca, S. et al. (1994) Biotechnology (NY)
12:396-399; Chen. S-Y. et al. (1994) Hum. Gene Ther. 5:595-601;
Duan, L et at (1994) Proc. Natl. Acad. Sci. USA 91:5075-5079; Chen,
S-Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5932-5936; Beerli,
R. R. et al. (1994) J. Biol. Chem. 269:23931-23936; Beerli, R. R,
et al. (1994) Biochem. Biophys. Res. Commun. 204:666-672;
Mhashilkar, A. M. et al. (1995) EMBO J. 14:1542-1551; Richardson,
J. H. et al. (1995) Proc. Natl. Acad. Sci. USA 92:3137-3141; PCT
Publication No. WO 94/02610 by Marasco et al.; and PCT Publication
No. WO 95/03832 by Duan et al.).
[0154] Additionally, fully human antibodies could be made against
biomarkers of the invention, including the biomarkers listed in
Table 1 and the Examples, or fragments thereof. Fully human
antibodies can be made in mice that are transgenic for human
immunoglobulin genes, e.g., according to Hogan, et al.,
"Manipulating the Mouse Embryo: A Laboratory Manuel," Cold Spring
Harbor Laboratory. Briefly, transgenic mice are immunized with
purified immunogen. Spleen cells are harvested and fused to myeloma
cells to produce hybridomas. Hybridomas are selected based on their
ability to produce antibodies which bind to the immunogen. Fully
human antibodies would reduce the immunogenicity of such antibodies
in a human.
[0155] In one embodiment, an antibody for use in the instant
invention is a bispecific antibody. A bispecific antibody has
binding sites for two different antigens within a single antibody
polypeptide. Antigen binding may be simultaneous or sequential.
Triomas and hybrid hybridomas are two examples of cell lines that
can secrete bispecific antibodies. Examples of bispecific
antibodies produced by a hybrid hybridoma or a trioma are disclosed
in U.S. Pat. No. 4,474,893. Bispecific antibodies have been
constructed by chemical means (Staerz et al. (1985) Nature 314:628,
and Perez et al. (1985) Nature 316:354) and hybridoma technology
(Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453, and
Staerz and Bevan (1986) Immunol. Today 7:241). Bispecific
antibodies are also described in U.S. Pat. No. 5,959,084. Fragments
of bispecific antibodies are described in U.S. Pat. No.
5,798,229.
[0156] Bispecific agents can also be generated by making
heterohybridomas by fusing hybridomas or other cells making
different antibodies, followed by identification of clones
producing and co-assembling both antibodies. They can also be
generated by chemical or genetic conjugation of complete
immunoglobulin chains or portions thereof such as Fab and Fv
sequences. The antibody component can bind to a polypeptide or a
fragment thereof of one or more biomarkers of the invention,
including one or more biomarkers listed in Table 1 and the
Examples, or a fragment thereof. In one embodiment, the bispecific
antibody could specifically bind to both a polypeptide or a
fragment thereof and its natural binding partner(s) or a
fragment(s) thereof.
[0157] In another aspect of this invention, peptides or peptide
mimetics can be used to antagonize or promote the activity of one
or more biomarkers of the invention, including one or more
biomarkers listed in Table 1 and the Examples, or a fragment(s)
thereof. In one embodiment, variants of one or more biomarkers
listed in Table 1 and the Examples which function as a modulating
agent for the respective full length protein, can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, for antagonist activity. In one embodiment, a variegated
library of variants is generated by combinatorial mutagenesis at
the nucleic acid level and is encoded by a variegated gene library.
A variegated library of variants can be produced, for instance, by
enzymatically ligating a mixture of synthetic oligonucleotides into
gene sequences such that a degenerate set of potential polypeptide
sequences is expressible as individual polypeptides containing the
set of polypeptide sequences therein. There are a variety of
methods which can be used to produce libraries of polypeptide
variants from a degenerate oligonucleotide sequence. Chemical
synthesis of a degenerate gene sequence can be performed in an
automatic DNA synthesizer, and the synthetic gene then ligated into
an appropriate expression vector. Use of a degenerate set of genes
allows for the provision, in one mixture, of all of the sequences
encoding the desired set of potential polypeptide sequences.
Methods for synthesizing degenerate oligonucleotides are known in
the art (see, e.g., Narang. S. A. (1983) Tetrahedron 39:3; Itakura
et al. (1984) Annu. Rev. Biochem. 53:323, Itakura et al. (1984)
Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
[0158] In addition, libraries of fragments of a polypeptide coding
sequence can be used to generate a variegated population of
polypeptide fragments for screening and subsequent selection of
variants of a given polypeptide. In one embodiment, a library of
coding sequence fragments can be generated by treating a double
stranded PCR fragment of a polypeptide coding sequence with a
nuclease under conditions wherein nicking occurs only about once
per polypeptide, denaturing the double stranded DNA, renaturing the
DNA to form double stranded DNA which can include sense/antisense
pairs from different nicked products, removing single stranded
portions from reformed duplexes by treatment with S1 nuclease, and
ligating the resulting fragment library into an expression vector.
By this method, an expression library can be derived which encodes
N-terminal, C-terminal and internal fragments of various sizes of
the polypeptide.
[0159] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of polypeptides. The most widely used techniques, which
are amenable to high through-put analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a technique
which enhances the frequency of functional mutants in the
libraries, can be used in combination with the screening assays to
identify variants of interest (Arkin and Youvan (1992) Proc. Natl.
Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Eng.
6(3):327-331). In one embodiment, cell based assays can be
exploited to analyze a variegated polypeptide library. For example,
a library of expression vectors can be transfected into a cell line
which ordinarily synthesizes one or more biomarkers of the
invention, including one or more biomarkers listed in Table 1 and
the Examples, or a fragment thereof. The transfected cells are then
cultured such that the full length polypeptide and a particular
mutant polypeptide are produced and the effect of expression of the
mutant on the full length polypeptide activity in cell supernatants
can be detected, e.g., by any of a number of functional assays.
Plasmid DNA can then be recovered from the cells which score for
inhibition, or alternatively, potentiation of full length
polypeptide activity, and the individual clones further
characterized.
[0160] Systematic substitution of one or more amino acids of a
polypeptide amino acid sequence with a D-amino acid of the same
type (e.g., D-lysine in place of L-lysine) can be used to generate
more stable peptides. In addition, constrained peptides comprising
a polypeptide amino acid sequence of interest or a substantially
identical sequence variation can be generated by methods known in
the art (Rizo and Gierasch (1992) Annu. Rev. Biochem. 61:387,
incorporated herein by reference); for example, by adding internal
cysteine residues capable of forming intramolecular disulfide
bridges which cyclize the peptide.
[0161] The amino acid sequences disclosed herein will enable those
of skill in the art to produce polypeptides corresponding peptide
sequences and sequence variants thereof. Such polypeptides can be
produced in prokaryotic or eukaryotic host cells by expression of
polynucleotides encoding the peptide sequence, frequently as part
of a larger polypeptide. Alternatively, such peptides can be
synthesized by chemical methods. Methods for expression of
heterologous proteins in recombinant hosts, chemical synthesis of
polypeptides, and in vitro translation are well known in the art
and are described further in Maniatis et al. Molecular Cloning: A
Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger
and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular
Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.;
Merrifield, J. (1969) J. Am. Chem. Soc. 91:501; Chaiken I. M.
(1981) CRC Crit. Rev. Biochem. 11: 255; Kaiser et al. (1989)
Science 243:187; Merrifield, B. (1986) Science 232:342; Kent, S. B.
H. (1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980)
Semisynthetic Proteins, Wiley Publishing, which are incorporated
herein by reference).
[0162] Peptides can be produced, typically by direct chemical
synthesis. Peptides can be produced as modified peptides, with
nonpeptide moieties attached by covalent linkage to the N-terminus
and/or C-terminus. In certain preferred embodiments, either the
carboxy-terminus or the amino-terminus, or both, are chemically
modified. The most common modifications of the terminal amino and
carboxyl groups are acetylation and amidation, respectively.
Amino-terminal modifications such as acylation (e.g., acetylation)
or alkylation (e.g., methylation) and
carboxy-terminal-modifications such as amidation, as well as other
terminal modifications, including cyclization, can be incorporated
into various embodiments of the invention. Certain amino-terminal
and/or carboxy-terminal modifications and/or peptide extensions to
the core sequence can provide advantageous physical, chemical,
biochemical, and pharmacological properties, such as: enhanced
stability, increased potency and/or efficacy, resistance to serum
proteases, desirable pharmacokinetic properties, and others.
Peptides disclosed herein can be used therapeutically to treat
disease, e.g., by altering costimulation in a patient.
[0163] Peptidomimetics (Fauchere, J. (1986) Adv. Drug Res. 15:29;
Veber and Freidinger (1985) TINS p. 392; and Evans et al. (1987) J.
Med. Chem. 30:1229, which are incorporated herein by reference) are
usually developed with the aid of computerized molecular modeling.
Peptide mimetics that are structurally similar to therapeutically
useful peptides can be used to produce an equivalent therapeutic or
prophylactic effect. Generally, peptidomimetics are structurally
similar to a paradigm polypeptide (i.e., a polypeptide that has a
biological or pharmacological activity), but have one or more
peptide linkages optionally replaced by a linkage selected from the
group consisting of: --CH2NH--, --CH2S--, --CH2-CH2-, --CH.dbd.CH--
(cis and trans), --COCH2-, --CH(OH)CH2-, and --CH2SO--, by methods
known in the art and further described in the following references:
Spatola, A. F. in "Chemistry and Biochemistry of Amino Acids,
Peptides, and Proteins" Weinstein, B., ed., Marcel Dekker, New
York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol.
1, Issue 3. "Peptide Backbone Modifications" (general review);
Morley, J. S. (1980) Trends Pharm. Sci. pp. 463-468 (general
review); Hudson, D. et al. (1979) Int. J. Pep. Prot. Rev.
14:177-185 (--CH2NH--, CH2CH2-); Spatola, A. F. et al. (1986) Life
Sci. 38:1243-1249 (--CH2-S); Hann, M. M. (1982) J. Chem. Soc.
Perkin Trans. 1. 307-314 (--CH--CH--, cis and trans); Almquist, R.
G. et al. (190) J. Med. Chem. 23:1392-1398 (--COCH2-);
Jennings-White, C. et al. (1982) Tetrahedron Lett. 23:2533
(--COCH2-); Szelke, M. et al. European Appln. EP 45665 (1982) CA:
97:39405 (1982)(--CH(OH)CH2-); Holladay, M. W. et al. (1983)
Tetrahedron Let. (1983) 24:4401-4404 (--C(OH)CH2-); and Hruby, V.
J. (1982) Life Sci. (1982) 31:189-199 (--CH2-S--); each of which is
incorporated herein by reference. A particularly preferred
non-peptide linkage is --CH2NH--. Such peptide mimetics may have
significant advantages over polypeptide embodiments, including, for
example: more economical production, greater chemical stability,
enhanced pharmacological properties (half-life, absorption,
potency, efficacy, etc.), altered specificity (e.g., a
broad-spectrum of biological activities), reduced antigenicity, and
others. Labeling of peptidomimetics usually involves covalent
attachment of one or more labels, directly or through a spacer
(e.g., an amide group), to non-interfering position(s) on the
peptidomimetic that are predicted by quantitative
structure-activity data and/or molecular modeling. Such
non-interfering positions generally are positions that do not form
direct contacts with the macropolypeptides(s) to which the
peptidomimetic binds to produce the therapeutic effect.
Derivitization (e.g., labeling) of peptidomimetics should not
substantially interfere with the desired biological or
pharmacological activity of the peptidomimetic.
[0164] Also encompassed by the present invention are small
molecules which can modulate (either enhance or inhibit)
interactions, e.g., between biomarkers listed in Table 1 and the
Examples and their natural binding partners, or inhibit activity.
The small molecules of the present invention can be obtained using
any of the numerous approaches in combinatorial library methods
known in the art, including: spatially addressable parallel solid
phase or solution phase libraries; synthetic library methods
requiring deconvolution; the `one-bead one-compound` library
method; and synthetic library methods using affinity chromatography
selection. (Lam, K. S. (1997) Anticancer Drug Des. 12:145).
[0165] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem.
37:1233.
[0166] Libraries of compounds can be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA
89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al.
(1990) Proc. Natl. Acad. Sci. USA 87:6378-6382); (Felici (1991) J.
Mol. Biol. 222:301-310); (Ladner supra.). Compounds can be screened
in cell based or non-cell based assays. Compounds can be screened
in pools (e.g., multiple compounds in each testing sample) or as
individual compounds.
[0167] The invention also relates to chimeric or fusion proteins of
the biomarkers of the invention, including the biomarkers listed in
Table 1 and the Examples, or fragments thereof. As used herein, a
"chimeric protein" or "fusion protein" comprises one or more
biomarkers of the invention, including one or more biomarkers
listed in Table 1 and the Examples, or a fragment thereof,
operatively linked to another polypeptide having an amino acid
sequence corresponding to a protein which is not substantially
homologous to the respective biomarker. In a preferred embodiment,
the fusion protein comprises at least one biologically active
portion of one or more biomarkers of the invention, including one
or more biomarkers listed in Table 1 and the Examples, or fragments
thereof. Within the fusion protein, the term "operatively linked"
is intended to indicate that the biomarker sequences and the
non-biomarker sequences are fused in-frame to each other in such a
way as to preserve functions exhibited when expressed independently
of the fusion. The "another" sequences can be fused to the
N-terminus or C-terminus of the biomarker sequences,
respectively.
[0168] Such a fusion protein can be produced by recombinant
expression of a nucleotide sequence encoding the first peptide and
a nucleotide sequence encoding the second peptide. The second
peptide may optionally correspond to a moiety that alters the
solubility, affinity, stability or valency of the first peptide,
for example, an immunoglobulin constant region. In another
preferred embodiment, the first peptide consists of a portion of a
biologically active molecule (e.g., the extracellular portion of
the polypeptide or the ligand binding portion). The second peptide
can include an immunoglobulin constant region, for example, a human
C.gamma.1 domain or C.gamma.4 domain (e.g., the hinge, CH2 and CH3
regions of human IgC.gamma. 1, or human IgC.gamma.4, see e.g.,
Capon et al. U.S. Pat. Nos. 5,116,964; 5,580,756; 5,844,095 and the
like, incorporated herein by reference). Such constant regions may
retain regions which mediate effector function (e.g., Fe receptor
binding) or may be altered to reduce effector function. A resulting
fusion protein may have altered solubility, binding affinity,
stability and/or valency (i.e., the number of binding sites
available per polypeptide) as compared to the independently
expressed first peptide, and may increase the efficiency of protein
purification. Fusion proteins and peptides produced by recombinant
techniques can be secreted and isolated from a mixture of cells and
medium containing the protein or peptide. Alternatively, the
protein or peptide can be retained cytoplasmically and the cells
harvested, lysed and the protein isolated. A cell culture typically
includes host cells, media and other byproducts. Suitable media for
cell culture are well known in the art. Protein and peptides can be
isolated from cell culture media, host cells, or both using
techniques known in the art for purifying proteins and peptides.
Techniques for transfecting host cells and purifying proteins and
peptides are known in the art.
[0169] Preferably, a fusion protein of the invention is produced by
standard recombinant DNA techniques. For example, DNA fragments
coding for the different polypeptide sequences are ligated together
in-frame in accordance with conventional techniques, for example
employing blunt-ended or stagger-ended termini for ligation,
restriction enzyme digestion to provide for appropriate termini,
filling-in of cohesive ends as appropriate, alkaline phosphatase
treatment to avoid undesirable joining, and enzymatic ligation. In
another embodiment, the fusion gene can be synthesized by
conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of gene fragments can be carried
out using anchor primers which give rise to complementary overhangs
between two consecutive gene fragments which can subsequently be
annealed and reamplified to generate a chimeric gene sequence (see,
for example, Current Protocols in Molecular Biology, eds. Ausubel
et al. John Wiley & Sons: 1992).
[0170] In another embodiment, the fusion protein contains a
heterologous signal sequence at its N-terminus. In certain host
cells (e.g., mammalian host cells), expression and/or secretion of
a polypeptide can be increased through use of a heterologous signal
sequence.
[0171] The fusion proteins of the invention can be used as
immunogens to produce antibodies in a subject. Such antibodies may
be used to purify the respective natural polypeptidcs from which
the fusion proteins were generated, or in screening assays to
identify polypeptides which inhibit the interactions between one or
more biomarkers polypeptide or a fragment thereof and its natural
binding partner(s) or a fragment(s) thereof.
[0172] Also provided herein are compositions comprising one or more
nucleic acids comprising or capable of expressing at least 1, 2, 3,
4, 5, 10, 20 or more small nucleic acids or antisense
oligonucleotides or derivatives thereof, wherein said small nucleic
acids or antisense oligonucleotides or derivatives thereof in a
cell specifically hybridize (e.g., bind) under cellular conditions,
with cellular nucleic acids (e.g., small non-coding RNAS such as
miRNAs, pre-miRNAs, pri-miRNAs, miRNA*, anti-miRNA, a miRNA binding
site, a variant and/or functional variant thereof, cellular mRNAs
or a fragments thereof). In one embodiment, expression of the small
nucleic acids or antisense oligonucleotides or derivatives thereof
in a cell can enhance or upregulate one or more biological
activities associated with the corresponding wild-type, naturally
occurring, or synthetic small nucleic acids. In another embodiment,
expression of the small nucleic acids or antisense oligonucleotides
or derivatives thereof in a cell can inhibit expression or
biological activity of cellular nucleic acids and/or proteins,
e.g., by inhibiting transcription, translation and/or small nucleic
acid processing of, for example, one or more biomarkers of the
invention, including one or more biomarkers listed in Table 1 and
the Examples, or fragment(s) thereof. In one embodiment, the small
nucleic acids or antisense oligonucleotides or derivatives thereof
are small RNAs (e.g., microRNAs) or complements of small RNAs. In
another embodiment, the small nucleic acids or antisense
oligonucleotides or derivatives thereof can be single or double
stranded and are at least six nucleotides in length and are less
than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50,
40, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, or 10
nucleotides in length. In another embodiment, a composition may
comprise a library of nucleic acids comprising or capable of
expressing small nucleic acids or antisense oligonucleotides or
derivatives thereof, or pools of said small nucleic acids or
antisense oligonucleotides or derivatives thereof. A pool of
nucleic acids may comprise about 2-5, 5-10, 10-20, 10-30 or more
nucleic acids comprising or capable of expressing small nucleic
acids or antisense oligonucleotides or derivatives thereof.
[0173] In one embodiment, binding may be by conventional base pair
complementarity, or, for example, in the case of binding to DNA
duplexes, through specific interactions in the major groove of the
double helix. In general, "antisense" refers to the range of
techniques generally employed in the art, and includes any process
that relies on specific binding to oligonucleotide sequences.
[0174] It is well known in the art that modifications can be made
to the sequence of a miRNA or a pre-miRNA without disrupting miRNA
activity. As used herein, the term "functional variant" of a miRNA
sequence refers to an oligonucleotide sequence that varies from the
natural miRNA sequence, but retains one or more functional
characteristics of the miRNA (e.g., cancer cell proliferation
inhibition, induction of cancer cell apoptosis, enhancement of
cancer cell susceptibility to chemotherapeutic agents, specific
miRNA target inhibition). In some embodiments, a functional variant
of a miRNA sequence retains all of the functional characteristics
of the miRNA. In certain embodiments, a functional variant of a
miRNA has a nucleobase sequence that is a least about 60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identical to the miRNA or precursor thereof over a region of
about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60), 65, 70, 75, 80,
85, 90, 95, 100 or more nucleobases, or that the functional variant
hybridizes to the complement of the miRNA or precursor thereof
under stringent hybridization conditions. Accordingly, in certain
embodiments the nucleobase sequence of a functional variant is
capable of hybridizing to one or more target sequences of the
miRNA.
[0175] miRNAs and their corresponding stem-loop sequences described
herein may be found in miRBase, an online searchable database of
miRNA sequences and annotation, found on the world wide web at
microrna.sanger.ac.uk. Entries in the miRBase Sequence database
represent a predicted hairpin portion of a miRNA transcript (the
stem-loop), with information on the location and sequence of the
mature miRNA sequence. The miRNA stem-loop sequences in the
database are not strictly precursor miRNAs (pre-miRNAs), and may in
some instances include the pre-miRNA and some flanking sequence
from the presumed primary transcript. The miRNA nucleobase
sequences described herein encompass any version of the miRNA,
including the sequences described in Release 10.0 of the miRBase
sequence database and sequences described in any earlier Release of
the miRBase sequence database. A sequence database release may
result in the re-naming of certain miRNAs. A sequence database
release may result in a variation of a mature miRNA sequence.
[0176] In some embodiments, miRNA sequences of the invention may be
associated with a second RNA sequence that may be located on the
same RNA molecule or on a separate RNA molecule as the miRNA
sequence. In such cases, the miRNA sequence may be referred to as
the active strand, while the second RNA sequence, which is at least
partially complementary to the miRNA sequence, may be referred to
as the complementary strand. The active and complementary strands
are hybridized to create a double-stranded RNA that is similar to a
naturally occurring miRNA precursor. The activity of a miRNA may be
optimized by maximizing uptake of the active strand and minimizing
uptake of the complementary strand by the miRNA protein complex
that regulates gene translation. This can be done through
modification and/or design of the complementary strand.
[0177] In some embodiments, the complementary strand is modified so
that a chemical group other than a phosphate or hydroxyl at its 5'
terminus. The presence of the 5' modification apparently eliminates
uptake of the complementary strand and subsequently favors uptake
of the active strand by the miRNA protein complex. The 5'
modification can be any of a variety of molecules known in the art,
including NH.sub.2, NHCOCH.sub.3, and biotin. In another
embodiment, the uptake of the complementary strand by the miRNA
pathway is reduced by incorporating nucleotides with sugar
modifications in the first 2-6 nucleotides of the complementary
strand. It should be noted that such sugar modifications can be
combined with the 5' terminal modifications described above to
further enhance miRNA activities.
[0178] In some embodiments, the complementary strand is designed so
that nucleotides in the 3' end of the complementary strand are not
complementary to the active strand. This results in double-strand
hybrid RNAs that are stable at the 3' end of the active strand but
relatively unstable at the 5' end of the active strand. This
difference in stability enhances the uptake of the active strand by
the miRNA pathway, while reducing uptake of the complementary
strand, thereby enhancing miRNA activity.
[0179] Small nucleic acid and/or antisense constructs of the
methods and compositions presented herein can be delivered, for
example, as an expression plasmid which, when transcribed in the
cell, produces RNA which is complementary to at least a unique
portion of cellular nucleic acids (e.g., small RNAs, mRNA, and/or
genomic DNA). Alternatively, the small nucleic acid molecules can
produce RNA which encodes mRNA, miRNA, pre-miRNA, pri-miRNA,
miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof.
For example, selection of plasmids suitable for expressing the
miRNAs, methods for inserting nucleic acid sequences into the
plasmid, and methods of delivering the recombinant plasmid to the
cells of interest are within the skill in the art. See, for
example, Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl
(2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002),
Science 296:550-553; Miyagishi et al. (2002), Nat. Biotechnol.
20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et
al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002),
Nat. Biotechnol. 20:505-508, the entire disclosures of which are
herein incorporated by reference.
[0180] Alternatively, small nucleic acids and/or antisense
constructs are oligonucleotide probes that are generated ex vivo
and which, when introduced into the cell, results in hybridization
with cellular nucleic acids. Such oligonucleotide probes are
preferably modified oligonucleotides that are resistant to
endogenous nucleases, e.g., exonucleases and/or endonucleases, and
are therefore stable in vivo. Exemplary nucleic acid molecules for
use as small nucleic acids and/or antisense oligonucleotides are
phosphoramidate, phosphothioate and methylphosphonate analogs of
DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).
Additionally, general approaches to constructing oligomers useful
in antisense therapy have been reviewed, for example, by Van der
Krol et al. (1988) BioTechniques 6:958-976; and Stein et al. (1988)
Cancer Res 48:2659-2668.
[0181] Antisense approaches may involve the design of
oligonucleotides (either DNA or RNA) that are complementary to
cellular nucleic acids (e.g., complementary to biomarkers listed in
Table 1 and the Examples). Absolute complementarity is not
required. In the case of double-stranded antisense nucleic acids, a
single strand of the duplex DNA may thus be tested, or triplex
formation may be assayed. The ability to hybridize will depend on
both the degree of complementarity and the length of the antisense
nucleic acid. Generally, the longer the hybridizing nucleic acid,
the more base mismatches with a nucleic acid (e.g., RNA) it may
contain and still form a stable duplex (or triplex, as the case may
be). One skilled in the art can ascertain a tolerable degree of
mismatch by use of standard procedures to determine the melting
point of the hybridized complex.
[0182] Oligonucleotides that are complementary to the 5' end of the
mRNA. e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have recently been shown to be
effective at inhibiting translation of mRNAs as well (Wagner, R.
(1994) Nature 372:333). Therefore, oligonucleotides complementary
to either the 5' or 3' untranslated, non-coding regions of genes
could be used in an antisense approach to inhibit translation of
endogenous mRNAs. Oligonucleotides complementary to the 5'
untranslated region of the mRNA may include the complement of the
AUG start codon. Antisense oligonucleotides complementary to mRNA
coding regions are less efficient inhibitors of translation but
could also be used in accordance with the methods and compositions
presented herein. Whether designed to hybridize to the 5',3' or
coding region of cellular mRNAs, small nucleic acids and/or
antisense nucleic acids should be at least six nucleotides in
length, and can be less than about 1000, 900), 800, 700, 600, 500,
400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17,
16, 15, or 10 nucleotides in length.
[0183] Regardless of the choice of target sequence, it is preferred
that in vitro studies are first performed to quantitate the ability
of the antisense oligonucleotide to inhibit gene expression. In one
embodiment these studies utilize controls that distinguish between
antisense gene inhibition and nonspecific biological effects of
oligonucleotides. In another embodiment these studies compare
levels of the target nucleic acid or protein with that of an
internal control nucleic acid or protein. Additionally, it is
envisioned that results obtained using the antisense
oligonucleotide are compared with those obtained using a control
oligonucleotide. It is preferred that the control oligonucleotide
is of approximately the same length as the test oligonucleotide and
that the nucleotide sequence of the oligonucleotide differs from
the antisense sequence no more than is necessary to prevent
specific hybridization to the target sequence.
[0184] Small nucleic acids and/or antisense oligonucleotides can be
DNA or RNA or chimeric mixtures or derivatives or modified versions
thereof, single-stranded or double-stranded. Small nucleic acids
and/or antisense oligonucleotides can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, hybridization, etc., and may
include other appended groups such as peptides (e.g., for targeting
host cell receptors), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al (1989) Proc. Natl. Acad.
Sci. U.S.A. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. 84:648-652; PCT Publication No. WO88/09810, published Dec. 15,
1988) or the blood-brain barrier (see, e.g., PCT Publication No.
WO89/10134, published Apr. 25, 1988), hybridization-triggered
cleavage agents. (See, e.g., Krol et al. (1988) BioTechniques
6:958-976) or intercalating agents. (See, e.g., Zon (1988). Pharm.
Res. 5:539-549). To this end, small nucleic acids and/or antisense
oligonucleotides may be conjugated to another molecule, e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, hybridization-triggered cleavage agent, etc.
[0185] Small nucleic acids and/or antisense oligonucleotides may
comprise at least one modified base moiety which is selected from
the group including but not limited to 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,
4-acetylcytosine, 5-(carboxyhydroxytiethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), S-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Small nucleic acids and/or antisense
oligonucleotides may also comprise at least one modified sugar
moiety selected from the group including but not limited to
arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0186] In certain embodiments, a compound comprises an
oligonucleotide (e.g., a miRNA or miRNA encoding oligonucleotide)
conjugated to one or more moieties which enhance the activity,
cellular distribution or cellular uptake of the resulting
oligonucleotide. In certain such embodiments, the moiety is a
cholesterol moiety (e.g., antagomirs) or a lipid moiety or liposome
conjugate. Additional moieties for conjugation include
carbohydrates, phospholipids, biotin, phenazine, folate,
phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,
coumarins, and dyes. In certain embodiments, a conjugate group is
attached directly to the oligonucleotide. In certain embodiments, a
conjugate group is attached to the oligonucleotide by a linking
moiety selected from amino, hydroxyl, carboxylic acid, thiol,
unsaturations (e.g., double or triple bonds),
8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
6-aminohexanoic acid (AHEX or AHA), substituted C1-C10 alkyl,
substituted or unsubstituted C2-C10 alkenyl, and substituted or
unsubstituted C2-C10 alkynyl. In certain such embodiments, a
substituent group is selected from hydroxyl, amino, alkoxy,
carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl,
aryl, alkenyl and alkynyl.
[0187] In certain such embodiments, the compound comprises the
oligonucleotide having one or more stabilizing groups that are
attached to one or both termini of the oligonucleotide to enhance
properties such as, for example, nuclease stability. Included in
stabilizing groups are cap structures. These terminal modifications
protect the oligonucleotide from exonuclease degradation, and can
help in delivery and/or localization within a cell. The cap can be
present at the 5'-terminus (5'-cap), or at the 3'-terminus
(3'-cap), or can be present on both termini. Cap structures
include, for example, inverted deoxy abasic caps.
[0188] Suitable cap structures include a 4',5'-methylene
nucleotide, a 1-(beta-D-erythrofuranosyl) nucleotide, a 4'-thio
nucleotide, a carbocyclic nucleotide, a 1,5-anhydrohexitol
nucleotide, an L-nucleotide, an alpha-nucleotide, a modified base
nucleotide, a phosphorodithioate linkage, a threo-pentofuranosyl
nucleotide, an acyclic 3',4'-seco nucleotide, an acyclic
3,4-dihydroxybutyl nucleotide, an acyclic 3,5-dihydroxypentyl
nucleotide, a 3'-3'-inverted nucleotide moiety, a 3'-3'-inverted
abasic moiety, a 3'-2'-inverted nucleotide moiety, a 3'-2'-inverted
abasic moiety, a 1,4-butanediol phosphate, a 3'-phosphoramidate, a
hexylphosphate, an aminohexyl phosphate, a 3'-phosphate, a
3'-phosphorothioate, a phosphorodithioate, a bridging
methylphosphonate moiety, and a non-bridging methylphosphonate
moiety 5'-amino-alkyl phosphate, a 1,3-diamino-2-propyl phosphate,
3-aminopropyl phosphate, a 6-aminohexyl phosphate, a
1,2-aminododecyl phosphate, a hydroxypropyl phosphate, a
5'-5'-inverted nucleotide moiety, a 5'-5'-inverted abasic moiety, a
5'-phosphoramidate, a 5'-phosphorothioate, a 5'-amino, a bridging
and/or non-bridging 5'-phosphoramidate, a phosphorothioate, and a
5'-mercapto moiety.
[0189] Small nucleic acids and/or antisense oligonucleotides can
also contain a neutral peptide-like backbone. Such molecules are
termed peptide nucleic acid (PNA)-oligomers and are described,
e.g., in Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. U.S.A.
93:14670 and in Eglom et al. (1993) Nature 365:566. One advantage
of PNA oligomers is their capability to bind to complementary DNA
essentially independently from the ionic strength of the medium due
to the neutral backbone of the DNA. In yet another embodiment,
small nucleic acids and/or antisense oligonucleotides comprises at
least one modified phosphate backbone selected from the group
consisting of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0190] In a further embodiment, small nucleic acids and/or
antisense oligonucleotides are .alpha.-anomeric oligonucleotides.
An .alpha.-anomeric oligonucleotide forms specific double-stranded
hybrids with complementary RNA in which, contrary to the usual
b-units, the strands run parallel to each other (Gautier et al.
(1987) Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a
2'-0-methylribonucleotide (Inoue et al. (1987) Nucl. Acids Res.
15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0191] Small nucleic acids and/or antisense oligonucleotides of the
methods and compositions presented herein may be synthesized by
standard methods known in the art, e.g., by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(1988) Nucl. Acids Res. 16:3209, methylphosphonate oligonucleotides
can be prepared by use of controlled pore glass polymer supports
(Sarin et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451),
etc. For example, an isolated miRNA can be chemically synthesized
or recombinantly produced using methods known in the art. In some
instances, miRNA are chemically synthesized using appropriately
protected ribonucleoside phosphoramidites and a conventional
DNA/RNA synthesizer. Commercial suppliers of synthetic RNA
molecules or synthesis reagents include, e.g., Proligo (Hamburg,
Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce
Chemical (part of Perbio Science, Rockford, Ill., USA), Glen
Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA),
Cruachem (Glasgow, UK), and Exiqon (Vedbaek, Denmark).
[0192] Small nucleic acids and/or antisense oligonucleotides can be
delivered to cells in vivo. A number of methods have been developed
for delivering small nucleic acids and/or antisense
oligonucleotides DNA or RNA to cells, e.g., antisense molecules can
be injected directly into the tissue site, or modified antisense
molecules, designed to target the desired cells (e.g., antisense
linked to peptides or antibodies that specifically bind receptors
or antigens expressed on the target cell surface) can be
administered systematically.
[0193] In one embodiment, small nucleic acids and/or antisense
oligonucleotides may comprise or be generated from double stranded
small interfering RNAs (siRNAs), in which sequences fully
complementary to cellular nucleic acids (e.g., mRNAs) sequences
mediate degradation or in which sequences incompletely
complementary to cellular nucleic acids (e.g., mRNAs) mediate
translational repression when expressed within cells. In another
embodiment, double stranded siRNAs can be processed into single
stranded antisense RNAs that bind single stranded cellular RNAs
(e.g., microRNAs) and inhibit their expression. RNA interference
(RNAi) is the process of sequence-specific, post-transcriptional
gene silencing in animals and plants, initiated by double-stranded
RNA (dsRNA) that is homologous in sequence to the silenced gene, in
vivo, long dsRNA is cleaved by ribonuclease III to generate 21- and
22-nucleotide siRNAs. It has been shown that 21-nucleotide siRNA
duplexes specifically suppress expression of endogenous and
heterologous genes in different mammalian cell lines, including
human embryonic kidney (293) and HeLa cells (Elbashir et al. (2001)
Nature 411:494-498). Accordingly, translation of a gene in a cell
can be inhibited by contacting the cell with short double stranded
RNAs having a length of about 15 to 30 nucleotides or of about 18
to 21 nucleotides or of about 19 to 21 nucleotides. Alternatively,
a vector encoding for such siRNAs or short hairpin RNAs (shRNAs)
that are metabolized into siRNAs can be introduced into a target
cell (see, e.g., McManus et al. (2002) RNA 8:842; Xia et al. (2002)
Nature Biotechnology 20:1006; and Brummelkamp et al. (2002) Science
296:550). Vectors that can be used are commercially available,
e.g., from OligoEngine under the name pSuper RNAi System.TM..
[0194] Ribozyme molecules designed to catalytically cleave cellular
mRNA transcripts can also be used to prevent translation of
cellular mRNAs and expression of cellular polypeptides, or both
(See, e.g., PCT International Publication WO90/11364, published
Oct. 4, 1990; Sarver et al. (1990) Science 247:1222-1225 and U.S.
Pat. No. 5,093,246). While ribozymes that cleave mRNA at site
specific recognition sequences can be used to destroy cellular
mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead
ribozymes cleave mRNAs at locations dictated by flanking regions
that form complementary base pairs with the target mRNA. The sole
requirement is that the target mRNA have the following sequence of
two bases: 5'-UG-3'. The construction and production of hammerhead
ribozymes is well known in the art and is described more fully in
Haseloff and Gerlach (1988) Nature 334:585-591. The ribozyme may be
engineered so that the cleavage recognition site is located near
the 5' end of cellular mRNAs; i.e. to increase efficiency and
minimize the intracellular accumulation of non-functional mRNA
transcripts.
[0195] The ribozymes of the methods and compositions presented
herein also include RNA endoribonucleases (hereinafter "Cech-type
ribozymes") such as the one which occurs naturally in Tetrahymena
thermophila (known as the IVS, or L-19 IVS RNA) and which has been
extensively described by Thomas Cech and collaborators (Zaug, et
al. (1984) Science 224:574-578; Zaug, et al. (1986) Science
231:470-475; Zaug, et al. (1986) Nature 324:429-433; published
International patent application No. WO88/04300 by University
Patents Inc.; Been, et al. (1986) Cell 47:207-216). The Cech-type
ribozymes have an eight base pair active site which hybridizes to a
target RNA sequence whereafter cleavage of the target RNA takes
place. The methods and compositions presented herein encompasses
those Cech-type ribozymes which target eight base-pair active site
sequences that are present in cellular genes.
[0196] As in the antisense approach, the ribozymes can be composed
of modified oligonucleotides (e.g., for improved stability,
targeting, etc.). A preferred method of delivery involves using a
DNA construct "encoding" the ribozyme under the control of a strong
constitutive pol III or pol II promoter, so that transfected cells
will produce sufficient quantities of the ribozyme to destroy
endogenous cellular messages and inhibit translation. Because
ribozymes unlike antisense molecules, are catalytic, a lower
intracellular concentration is required for efficiency.
[0197] Nucleic acid molecules to be used in triple helix formation
for the inhibition of transcription of cellular genes are
preferably single stranded and composed of deoxyribonucleotides.
The base composition of these oligonucleotides should promote
triple helix formation via Hoogsteen base pairing rules, which
generally require sizable stretches of either purines or
pyrimidines to be present on one strand of a duplex. Nucleotide
sequences may be pyrimidine-based, which will result in TAT and CGC
triplets across the three associated strands of the resulting
triple helix. The pyrimidine-rich molecules provide base
complementarity to a purine-rich region of a single strand of the
duplex in a parallel orientation to that strand. In addition,
nucleic acid molecules may be chosen that are purine-rich, for
example, containing a stretch of G residues. These molecules will
form a triple helix with a DNA duplex that is rich in GC pairs, in
which the majority of the purine residues are located on a single
strand of the targeted duplex, resulting in CGC triplets across the
three strands in the triplex.
[0198] Alternatively, the potential sequences that can be targeted
for triple helix formation may be increased by creating a so called
"switchback" nucleic acid molecule. Switchback molecules are
synthesized in an alternating 5'-3', 3'-5' manner, such that they
base pair with first one strand of a duplex and then the other,
eliminating the necessity for a sizable stretch of either purines
or pyrimidines to be present on one strand of a duplex.
[0199] Small nucleic acids (e.g., miRNAs, pre-miRNAs, pri-miRNAs,
miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof),
antisense oligonucleotides, ribozymes, and triple helix molecules
of the methods and compositions presented herein may be prepared by
any method known in the art for the synthesis of DNA and RNA
molecules. These include techniques for chemically synthesizing
oligodeoxyribonucleotides and oligoribonucleotides well known in
the art such as for example solid phase phosphoramidite chemical
synthesis. Alternatively, RNA molecules may be generated by in
vitro and in vivo transcription of DNA sequences encoding the
antisense RNA molecule. Such DNA sequences may be incorporated into
a wide variety of vectors which incorporate suitable RNA polymerase
promoters such as the T7 or SP6 polymerase promoters.
Alternatively, antisense cDNA constructs that synthesize antisense
RNA constitutively or inducibly, depending on the promoter used,
can be introduced stably into cell lines.
[0200] Moreover, various well-known modifications to nucleic acid
molecules may be introduced as a means of increasing intracellular
stability and half-life. Possible modifications include but are not
limited to the addition of flanking sequences of ribonucleotides or
deoxyribonucleotides to the 5' and/or 3' ends of the molecule or
the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages within the oligodeoxyribonucleotide
backbone. One of skill in the art will readily understand that
polypeptides, small nucleic acids, and antisense oligonucleotides
can be further linked to another peptide or polypeptide (e.g., a
heterologous peptide), e.g., that serves as a means of protein
detection. Non-limiting examples of label peptide or polypeptide
moieties useful for detection in the invention include, without
limitation, suitable enzymes such as horseradish peroxidase,
alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
epitope tags, such as FLAG, MYC, HA, or HIS tags; fluorophores such
as green fluorescent protein; dyes; radioisotopes; digoxygenin;
biotin; antibodies; polymers: as well as others known in the art,
for example, in Principles of Fluorescence Spectroscopy, Joseph R.
Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999).
[0201] In addition to the agents described herein, additional
agents are particularly useful for upregulating or downregulating
immune responses according to the present invention. For example,
modulation of the interaction between PD-1 and PD-1 ligand (e.g.,
soluble PD-L1) (e.g., membrane-bound PD-L1 and/or soluble PD-L1),
or between PD-1 ligand (e.g., soluble PD-L1) (e.g., membrane-bound
PD-L1 and/or soluble PD-L1) and a B7 polypeptide, results in
modulation of the immune response. In general, in embodiments where
PD-L1 binds to a costimulatory receptor such as B7-1, upregulation
of PD-L1 activity results in upregulation of immune responses,
whereas downregulation of PD-L1 activity results in downregulation
of immune responses. In embodiments where PD-L1 binds to inhibitory
receptors such as PD-1, upregulation of PD-L1 activity results in
downregulation of immune responses, whereas downregulation of PD-L1
activity results in upregulation of immune responses. It is also
believed that soluble forms of PD-L1, whether naturally occurring
or cleavage products of membrane-bound PD-L, can still interact
with PD-L1 receptors, such as B7-1 or PD-L1, to modulate immune
responses as the membrane-bound version.
[0202] Non-limiting examples of how such agents can modify immune
responses include the observation that the interaction between a B7
polypeptide and a PD-1 ligand (e.g., soluble PD-L1) polypeptide
prevents PD-1 ligand (e.g., soluble PD-L1) from binding to PD-1
and, thus, inhibits delivery of an inhibitory immune signal. Thus,
in one embodiment, agents which block the interaction between PD-1
and PD-1 ligand (e.g., soluble PD-L1) can prevent inhibitory
signaling. In one embodiment, agents that block the binding of a B7
polypeptide to a PD-1 ligand (e.g., soluble PD-L1) polypeptide
allow PD-1 ligand (e.g., soluble PD-L1) to bind PD-1 and provide an
inhibitory signal to an immune cell. PD-1 ligand (e.g., soluble
PD-L1), by binding to a B7 polypeptide, also reduces the B7
polypeptide binding to the inhibitory receptor CTLA4. In one
embodiment, agents that block the binding of a B7 polypeptide to a
PD-1 ligand (e.g., soluble PD-L1) polypeptide allow the B7
polypeptide to bind CTLA4 and provide an inhibitory signal to an
immune cell. In another embodiment, PD-L1, by binding to a B7
polypeptide, also reduces the B7 polypeptide binding to the
costimulatory receptor CD28. Thus, in one embodiment, agents that
block the binding of a B7 polypeptide to a PD-1 ligand (e.g.,
soluble PD-L1) polypeptide allow the B7 polypeptide to bind CD28,
and provide a costimulatory signal to an immune cell.
[0203] For example, in one embodiment, agents that increase the
interaction between a PD-1 ligand (e.g., soluble PD-L1) and a B7
polypeptide can enhance an immune response while agents that
decrease the interaction between a PD-1 ligand (e.g., soluble
PD-L1) and a B7 polypeptide can reduce an immune response by
enhancing the interaction between the PD-1 ligand (e.g., soluble
PD-L1) and PD-1 and or the interaction between the B7 polypeptide
and CTLA4. In one embodiment, agents that modulate the interaction
between a PD-1 ligand (e.g., soluble PD-L1) and a B7 polypeptide do
not produce inhibition of the interaction between a PD-1 ligand
(e.g., soluble PD-L1) and PD-1 and/or between the B7 polypeptide
and CTLA4. In another embodiment, agents that increase a PD-1
ligand (e.g., soluble PD-L1) interaction with a B7 polypeptide,
also decrease the interaction between the PD-1 ligand (e.g.,
soluble PD-L1) and PD-1, and/or between the B7 polypeptide and
CTLA4. In yet another embodiment, agents that decrease the
interaction of a PD-1 ligand (e.g., soluble PD-L1) and a B7
polypeptide, enhance or increase the interaction between the PD-1
ligand (e.g., soluble PD-L1) and PD-1, and/or between the B7
polypeptide and CTLA4.
[0204] Exemplary agents for modulating (e.g., reducing) an immune
response include antibodies against PD-1, a PD-1 ligand (e.g.,
soluble PD-L1), or a B7 polypeptide which inhibit the interaction
of the PD-1 ligand (e.g., soluble PD-L1) with PD-1 or B7
polypeptide; bispecific antibodies that enhance PD-1 signaling,
such as bispecific antibodies against PD-1 and PD-L1; agents that
reduce the expression of inhibitory receptor-ligand interactions,
such as antisense nucleic acid molecules, triplex oligonucleotides,
or ribozymes targeting PD-1 and/or PD-L1; small molecules or
peptides which inhibit the interaction of the PD-1 ligand (e.g.,
soluble PD-L1) with the B7 polypeptide; and fusion proteins (e.g.,
the extracellular portion of the PD-1 ligand (e.g., soluble PD-L1)
or B7 polypeptide, fused to the Fe portion of an antibody) which
bind to the B7 polypeptide or PD-1 ligand (e.g., soluble PD-L1),
respectively, and prevent the interaction between the PD-1 ligand
(e.g., soluble PD-L1) and B7 polypeptide.
[0205] In another embodiment, agents that increase the interaction
between a PD-ligand (e.g., soluble PD-L1) and a B7 polypeptide,
decrease an immune response by decreasing the ability of the B7
polypeptide to bind to CD28. In yet another embodiment, agents that
decrease the interaction between a PD-1 ligand (e.g., soluble
PD-L1) and a B7 polypeptide can increase the immune response by
increasing the interaction between the B7 polypeptide and CD28.
[0206] Agents that modulate the interaction between a PD-1 ligand
(e.g., soluble PD-L1) and a PD-1 polypeptide can also be used to up
or down regulate the immune response. For example, agents that
increase the interaction between the PD-1 ligand (e.g., soluble
PD-L1) and PD-1 polypeptide can decrease an immune response while
agents that decrease the interaction between the PD-1 ligand (e.g.,
soluble PD-L1) and PD-1 polypeptide can increase an immune
response. Preferably, agents that modulate the interaction between
the PD-1 ligand (e.g., soluble PD-L1) and PD-1, do not modulate
(have no direct affect on) the interaction between the PD-1 ligand
(e.g., soluble PD-L1) and a B7 polypeptide. In another embodiment,
agents that increase the interaction between the PD-1 ligand (e.g.,
soluble PD-L1) and PD-1, decrease the interaction between the PD-1
ligand (e.g., soluble PD-L1) and the B7 polypeptide. In yet another
embodiment, agents that decrease the interaction between the PD-L1
ligand and PD-1 increase the interaction between the PD-1 ligand
(e.g., soluble PD-L1) and the B7 polypeptide. Exemplary agents for
modulating (e.g., enhancing) an immune response include antibodies
against PD-1 or a PD-1 ligand (e.g., soluble PD-L1) which block the
interaction between PD-1 and the PD-1 ligand (e.g., soluble PD-L1);
bispecific antibodies that enhance 87 signaling, such as bispecific
antibodies against PD-L1 and B7-1; multivalent antibodies against
such a target that ligate many such molecules together in order to
increase local concentrations and stimulate interactions; agents
that reduce the expression of costimulatory receptor-ligand
interactions, such as antisense nucleic acid molecules, triplex
oligonucleotides, or ribozymes targeting B7-1; small molecules or
peptides which block the interaction between PD-1 and the PD-1
ligand (e.g., soluble PD-L1); and fusion proteins (e.g., the
extracellular portion of a PD-1 ligand (e.g., soluble PD-L1) or
PD-1 polypeptide fused to the Fe portion of an antibody) which bind
to PD-1 or a PD-1 ligand (e.g., soluble PD-L1) and prevent the
interaction between the PD-1 ligand (e.g., soluble PD-L1) and
PD-1.
[0207] In some embodiments, agents useful for upregulating or
downregulating PD-1 and/or PD-L1 in particular are useful.
Combinations of any such agents are contemplated.
[0208] Exemplary agents for use in downmodulating PD-L1 (i.e.,
PD-L1 antagonists) include (for example): antisense molecules,
antibodies that recognize PD-L1, compounds that block interaction
of PD-L1 and one of its naturally occurring receptors on a immune
cell (e.g., soluble, monovalent PD-L1 molecules, and soluble forms
of B7-4 ligands or compounds identified in the subject screening
assays). In some embodiments, combinations of antibodies that
target either the membrane-bound PD-L1 form or the soluble PD-L1
form are useful for functionally inactivating both forms of PD-L1.
Exemplary agents for use in downmodulating PD-1 (i.e., PD-1
antagonists) include (for example): antisense molecules, antibodies
that bind to PD-1, but do not transduce an inhibitory signal to the
immune cell ("non-activating antibodies"), and soluble forms of
PD-1.
[0209] Exemplary agents for use in upmodulating PD-L1 (i.e., PD-L1
agonists) include (for example): nucleic acid molecules encoding
PD-L1 polypeptides, multivalent forms of PD-L1, compounds that
increase the expression of PD-L1, and cells that express PD-L1, and
the like. Exemplary agents for use in upmodulating PD-1 (i.e., PD-1
agonists) include (for example): antibodies that transmit an
inhibitory signal via PD-1, compounds that enhance the expression
of PD-1, nucleic acid molecules encoding PD-1, and forms of B7-4
that transduce a signal via PD-1.
[0210] The modulatory agents described herein (e.g., antibodies,
small molecules, peptides, fusion proteins, or small nucleic acids)
can be incorporated into pharmaceutical compositions and
administered to a subject in vivo. The compositions may contain a
single such molecule or agent or any combination of agents
described herein. Based on the genetic pathway analyses described
herein, it is believed that such combinations of agents is
especially effective in diagnosing, prognosing, preventing, and
treating cancer. Thus, "single active agents" described herein can
be combined with other pharmacologically active compounds ("second
active agents") known in the art according to the methods and
compositions provided herein. It is believed that certain
combinations work synergistically in the treatment of particular
types of cancer. Second active agents can be large molecules (e.g.,
proteins) or small molecules (e.g., synthetic inorganic,
organometallic, or organic molecules).
[0211] Examples of large molecule active agents include, but are
not limited to, hematopoietic growth factors, cytokines, and
monoclonal and polyclonal antibodies. Typical large molecule active
agents are biological molecules, such as naturally occurring or
artificially made proteins. Proteins that are particularly useful
in this invention include proteins that stimulate the survival
and/or proliferation of hematopoietic precursor cells and
immunologically active poietic cells in vi/ro or in vivo. Others
stimulate the division and differentiation of committed erythroid
progenitors in cells in vitro or in vivo. Particular proteins
include, but are not limited to: interleukins, such as IL-2
(including recombinant IL-I1 ("rIL2") and canarypox IL-2), IL-10,
IL-12, and IL-18; interferons, such as interferon alfa-2a,
interferon alfa-2b, interferon alpha-n1, interferon alpha-n3,
interferon beta-1a, and interferon gamma-1b; GM-CF and GM-CSF; and
EPO.
[0212] Particular proteins that can be used in the methods and
compositions provided herein include, but are not limited to:
filgrastim, which is sold in the United States under the trade name
Neupogen.RTM. (Amgen, Thousand Oaks, Calif.); sargramostim, which
is sold in the United States under the trade name Leukine.RTM.
(Immunex, Seattle. Wash.); and recombinant EPO, which is sold in
the United States under the trade name Epogen.RTM. (Amgen, Thousand
Oaks, Calif.). Recombinant and mutated forms of GM-CSF can be
prepared as described in U.S. Pat. Nos. 5,391,485; 5,393,870; and
5,229,496; all of which are incorporated herein by reference.
Recombinant and mutated forms of G-CSF can be prepared as described
in U.S. Pat. Nos. 4,810,643; 4,999,291; 5,528,823; and 5,580,755;
all of which are incorporated herein by reference.
[0213] Antibodies that can be used in combination form include
monoclonal and polyclonal antibodies. Examples of antibodies
include, but are not limited to, trastuzumab (Herceptin.RTM.),
rituximab (Rituxan.RTM.), bevacizumab (Avastin.RTM.), pertuzumab
(Omnitarg.RTM.), tositumomab (Bexxar.RTM.), edrecolomab
(Panorex.RTM.), and G250. Compounds of the invention can also be
combined with, or used in combination with, anti-TNF-.alpha.
antibodies. Large molecule active agents may be administered in the
form of anti-cancer vaccines. For example, vaccines that secrete,
or cause the secretion of, cytokines such as IL-2, G-CSF, and
GM-CSF can be used in the methods, pharmaceutical compositions, and
kits provided herein. See, e.g., Emens, L. A., et al., Curr.
Opinion Mol. Ther. 3(1):77-84 (2001).
[0214] Second active agents that are small molecules can also be
used to in combination as provided herein. Examples of small
molecule second active agents include, but are not limited to,
anti-cancer agents, antibiotics, immunosuppressive agents, and
steroids.
[0215] In some embodiments, well known "combination chemotherapy"
regimens can be used. In one embodiment, the combination
chemotherapy comprises a combination of two or more of
cyclophosphamide, hydroxydaunorubicin (also known as doxorubicin or
adriamycin), oncovorin (vincristine), and prednisone. In another
preferred embodiment, the combination chemotherapy comprises a
combination of cyclophsophamide, oncovorin, prednisone, and one or
more chemotherapeutics selected from the group consisting of
anthracycline, hydroxydaunorubicin, epirubicin, and
motixantrone.
[0216] Examples of other anti-cancer agents include, but are not
limited to: acivicin; aclarubicin; acodazole hydrochloride;
acronine; adozelesin; aldesleukin; altretamine; ambomycin;
ametantrone acetate; amsacrine; anastrozole; anthramycin;
asparaginase; asperlin; azacitidine; azetepa; azotomycin;
batimastat; benzodepa; bicalutamide; bisantrene hydrochloride;
bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar
sodium; bropirimine; busulfan; cactinomycin; calusterone;
caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor);
chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol
mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin;
daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine;
dezaguanine mesylate; diaziquone; docetaxel; doxorubicin;
doxorubicin hydrochloride; droloxifene; droloxifene citrate;
dromostanolone propionate; duazomycin; edatrexate; eflornithine
hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;
epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;
estramustine; estramustine phosphate sodium; etanidazole;
etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;
fazarabine; fenretinide; floxuridine; fludarabine phosphate;
fluorouracil; flurocitabine; fosquidone; fostriecin sodium;
gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin
hydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan;
irinotecan hydrochloride; lanreotide acetate; letrozole; luprolide
acetate; liarozole hydrochloride; lometrexol sodium; lomustine;
losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine
hydrochloride; megestrol acetate; melengestrol acetate; melphalan;
menogaril; mercaptopurine; methotrexate; methotrexate sodium;
metoprine; meturedepa; mitindomide; mitocarcin; mitocromin;
mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazole; nogalamycin;
ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;
pentamustine; peplomycin sulfate; perfosfamide; pipobroman;
piposulfan; piroxantrone hydrochloride; plicamycin; plomestane;
porfimer sodium; porfiromycin; prednimustine; procarbazine
hydrochloride puromycin; puromycin hydrochloride; pyrazofurin;
riboprine; safingol; safingol hydrochloride; semustine; simtrazene;
sparfosate sodium; sparsomycin; spirogermanium hydrochloride;
spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur
talisomycin; tecogalan sodium; taxotere; tegafur; teloxantrone
hydrochloride; temoporfin; teniposide; teroxirone; testolactone;
thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine;
toremifene citrate; trestolone acetate; triciribine phosphate;
trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole
hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin;
vinblastine sulfate; vincristine sulfate; vindesine; vindesine
sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine
sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine
sulfate; vorozole; zeniplatin; zinostatin; and zorubicin
hydrochloride.
[0217] Other anti-cancer drugs include, but are not limited to:
20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;
aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin;
ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine;
aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist
G; antarelix; anti-dorsalizing morphogenetic protein-1;
antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;
antisense oligonucleotides; aphidicolin glycinate; apoptosis gene
modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;
arginine deaminase; asulacrine; atamestane; atrimustine;
axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin;
azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL
antagonists; benzochlorins; benzoylstaurosporine; beta lactam
derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF
inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;
bisnafide; bistratene A; bizelesin; breflate; bropirimine;
budotitane; buthionine sulfoximine; calcipotriol; calphostin C;
camptothecin derivatives; capecitabine; carboxamide-amino-triazole;
carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived
inhibitor; carzelesin; casein kinase inhibitors (ICOS);
castanospermine; cecropin B; cetrorelix; chlorins;
chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue,
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cyclosporin A; cypemycin; cytarabine ocfosfate;
cytolytic factor; cytostatin; dacliximab; decitabine;
dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide;
dexrazoxane; dexverapamil; diaziquone; didemnin B; didox;
diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-;
dioxamycin; diphenyl spiromustine; docetaxel; docosanol;
dolasetron; doxifluridine; doxorubicin; droloxifene; dronabinol;
duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;
eflornithine; elemene; emitefur; epirubicin; epristende;
estramustine analogue; estrogen agonists; estrogen antagonists;
etanidazole; etoposide phosphate; exemestane; fadrozole;
fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;
flezelastine; fluasterone; fludarabine; fluorodaunorunicin
hydrochloride; forfenimex; formestane; fostriecin; fotemustine;
gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;
gelatinase inhibitors; gemcitabine; glutathione inhibitors;
hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;
ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;
ilomastat; imatinib (e.g., Gleevec.RTM.), imiquimod;
immunostimulant peptides; insulin-like growth factor-1 receptor
inhibitor, interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;
isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic
peptides; maitansine; mannostatin A; marimastat; masoprocol;
maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors;
menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF
inhibitor; mifepristone; miltefosine; mirimostim; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
Erbitux, human chorionic gonadotrophin; monophosphoryl lipid
A+myobacterium cell wall sk; mopidamol; mustard anticancer agent;
mycaperoxide B; mycobacterial cell wall extract; myriaporone;
N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;
naloxone+pentazocine; napavin; naphterpin; nartograstim;
nedaplatin; nemorubicin; neridronic acid; nilutamide; nisamycin;
nitric oxide modulators; nitroxide antioxidant; nitrullyn;
oblimersen (Genasense.RTM.); O6-benzylguanine; octreotide;
okicenone; oligonucleotides; onapristone; ondansetron; ondansetron;
oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;
oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel
derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;
panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;
peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;
perflubron; perfosfamide; perillyl alcohol; phenazinomycin;
phenylacetate; phosphatase inhibitors; picibanil; pilocarpine
hydrochloride; pararubicin; piritrexam; placetin A; placetin B;
plasminogen activator inhibitor; platinum complex; platinum
compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor, protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed ramosetron; ms farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rohitukine; romurtide; roquinimex;
rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A;
sargramostim; Sdi 1 mimetics; semustine; senescence derived
inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; sizofuran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; sonermin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin L; squalamine; stipiamide; stromelysin inhibitors;
sulfinosine; superactive vasoactive intestinal peptide antagonist;
suradista; suramin; swainsonine; tallimustine; tamoxifen
methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur;
tellurapyrylium; telomerase inhibitors; temoporfin; teniposide;
tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline;
thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin
receptor agonist; thymotrinan; thyroid stimulating hormone; tin
ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin;
toremifene; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride;
tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor; urokinase
receptor antagonists; vapreotide; variolin B; velaresol; veramine;
verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;
zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
[0218] Specific second active agents include, but are not limited
to, chlorambucil, fludarabine, dexamethasone (Decadron.RTM.),
hydrocortisone, methylprednisolone, cilostamide, doxorubicin
(Doxil.RTM.), forskolin, rituximab, cyclosporin A, cisplatin,
vincristine, PDE7 inhibitors such as BRL-50481 and IR-202, dual
PDE4/7 inhibitors such as IR-284, cilostazol, meribendan,
milrinone, vesnarionone, enoximone and pimobendan. Syk inhibitors
such as fostamatinib disodium (R406/R788), R343, R-112 and
Excellair.RTM. (ZaBeCor Pharmaceuticals, Bala Cynwyd, Pa.).
III. METHODS OF SELECTING AGENTS AND COMPOSITIONS
[0219] Another aspect of the invention relates to methods of
selecting agents (e.g., antibodies, fusion proteins, peptides,
small molecules, or small nucleic acids) which bind to upregulate,
downregulate, or modulate one or more biomarkers of the invention
listed in Table 1 and the Examples and/or a cancer (e.g., a head,
neck, or lung cancer). Such methods can use screening assays,
including cell based and non-cell based assays.
[0220] In one embodiment, the invention relates to assays for
screening candidate or test compounds which bind to or modulate the
expression or activity level of, one or more biomarkers of the
invention, including one or more biomarkers listed in Table 1 and
the Examples, or a fragment thereof. Such compounds include,
without limitation, antibodies, proteins, fusion proteins, nucleic
acid molecules, and small molecules.
[0221] In one embodiment, an assay is a cell-based assay,
comprising contacting a cell expressing one or more biomarkers of
the invention, including one or more biomarkers listed in Table 1
and the Examples, or a fragment thereof, with a test compound and
determining the ability of the test compound to modulate (e.g.,
stimulate or inhibit) the level of interaction between the
biomarker and its natural binding partners as measured by direct
binding or by measuring a parameter of cancer.
[0222] For example, in a direct binding assay, the biomarker
polypeptide, a binding partner polypeptide of the biomarker, or a
fragment(s) thereof, can be coupled with a radioisotope or
enzymatic label such that binding of the biomarker polypeptide or a
fragment thereof to its natural binding partner(s) or a fragment(s)
thereof can be determined by detecting the labeled molecule in a
complex. For example, the biomarker polypeptide, a binding partner
polypeptide of the biomarker, or a fragment(s) thereof, can be
labeled with .sup.125I, .sup.35S, .sup.14C, or .sup.3H, either
directly or indirectly, and the radioisotope detected by direct
counting of radioemmission or by scintillation counting.
Alternatively, the polypeptides of interest a can be enzymatically
labeled with, for example, horseradish peroxidase, alkaline
phosphatase, or luciferase, and the enzymatic label detected by
determination of conversion of an appropriate substrate to
product.
[0223] It is also within the scope of this invention to determine
the ability of a compound to modulate the interactions between one
or more biomarkers of the invention, including one or more
biomarkers listed in Table 1 and the Examples, or a fragment
thereof, and its natural binding partner(s) or a fragment(s)
thereof, without the labeling of any of the interactants (e.g.,
using a microphysiometer as described in McConnell, H. M. et al.
(1992) Science 257:1906-1912). As used herein, a "microphysiometer"
(e.g., Cytosensor) as an analytical instrument that measures the
rate at which a cell acidifies its environment using a
light-addressable potentiometric sensor (LAPS). Changes in this
acidification rate can be used as an indicator of the interaction
between compound and receptor.
[0224] In a preferred embodiment, determining the ability of the
blocking agents (e.g., antibodies, fusion proteins, peptides,
nucleic acid molecules, or small molecules) to antagonize the
interaction between a given set of polypeptides can be accomplished
by determining the activity of one or more members of the set of
interacting molecules. For example, the activity of one or more
biomarkers of the invention, including one or more biomarkers
listed in Table 1 and the Examples, or a fragment thereof, can be
determined by detecting induction of cytokine or chemokine
response, detecting catalytic/enzymatic activity of an appropriate
substrate, detecting the induction of a reporter gene (comprising a
target-responsive regulatory element operatively linked to a
nucleic acid encoding a detectable marker, e.g., chloramphenicol
acetyl transferase), or detecting a cellular response regulated by
the biomarker or a fragment thereof (e.g., modulations of
biological pathways identified herein, such as modulated
proliferation, apoptosis, cell cycle, and/or ligand-receptor
binding activity). Determining the ability of the blocking agent to
bind to or interact with said polypeptide can be accomplished by
measuring the ability of an agent to modulate immune responses, for
example, by detecting changes in type and amount of cytokine
secretion, changes in apoptosis or proliferation, changes in gene
expression or activity associated with cellular identity, or by
interfering with the ability of said polypeptide to bind to
antibodies that recognize a portion thereof.
[0225] In yet another embodiment, an assay of the present invention
is a cell-free assay in which one or more biomarkers of the
invention, including one or more biomarkers listed in Table 1 and
the Examples or a fragment thereof, e.g., a biologically active
fragment thereof, is contacted with a test compound, and the
ability of the test compound to bind to the polypeptide, or
biologically active portion thereof, is determined. Binding of the
test compound to the biomarker or a fragment thereof, can be
determined either directly or indirectly as described above.
Determining the ability of the biomarker or a fragment thereof to
bind to its natural binding partner(s) or a fragment(s) thereof can
also be accomplished using a technology such as real-time
Biomolecular Interaction Analysis (BIA) (Sjolander, S. and
Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al.
(1995) Curr. Opin. Struct. Biol. 5:699-705). As used herein, "BIA"
is a technology for studying biospecific interactions in real time,
without labeling any of the interactants (e.g., BIAcore). Changes
in the optical phenomenon of surface plasmon resonance (SPR) can be
used as an indication of real-time reactions between biological
polypeptides. One or more biomarkers polypeptide or a fragment
thereof can be immobilized on a BIAcore chip and multiple agents,
e.g., blocking antibodies, fusion proteins, peptides, or small
molecules, can be tested for binding to the immobilized biomarker
polypeptide or fragment thereof. An example of using the BIA
technology is described by Fitz et al. (1997) Oncogene 15:613.
[0226] The cell-free assays of the present invention are amenable
to use of both soluble and/or membrane-bound forms of proteins. In
the case of cell-free assays in which a membrane-bound form protein
is used it may be desirable to utilize a solubilizing agent such
that the membrane-bound form of the protein is maintained in
solution. Examples of such solubilizing agents include non-ionic
detergents such as n-octylglucoside, n-dodecylglucoside,
n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide. Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether).sub.n,
3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),
3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane
sulfonate.
[0227] In one or more embodiments of the above described assay
methods, it may be desirable to immobilize either the biomarker
polypeptide, the natural binding partner(s) polypeptide of the
biomarker, or fragments thereof, to facilitate separation of
complexed from uncomplexed forms of one or both of the proteins, as
well as to accommodate automation of the assay. Binding of a test
compound in the assay can be accomplished in any vessel suitable
for containing the reactants. Examples of such vessels include
microtiter plates, test tubes, and micro-centrifuge tubes. In one
embodiment, a fusion protein can be provided which adds a domain
that allows one or both of the proteins to be bound to a matrix.
For example, glutathione-S-transferase-base fusion proteins, can be
adsorbed onto glutathione Sepharose.RTM. beads (Sigma Chemical, St.
Louis, Mo.) or glutathione derivatized microtiter plates, which are
then combined with the test compound, and the mixture incubated
under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtiter plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of binding or activity determined
using standard techniques.
[0228] In an alternative embodiment, determining the ability of the
test compound to modulate the activity of one or more biomarkers of
the invention, including one or more biomarkers listed in Table 1
and the Examples, or a fragment thereof, or of natural binding
partner(s) thereof can be accomplished by determining the ability
of the test compound to modulate the expression or activity of a
gene, e.g., nucleic acid, or gene product, e.g., polypeptide, that
functions downstream of the interaction. For example, inflammation
(e.g., cytokine and chemokine) responses can be determined, the
activity of the interactor polypeptide on an appropriate target can
be determined, or the binding of the interactor to an appropriate
target can be determined as previously described.
[0229] In another embodiment, modulators of one or more biomarkers
of the invention, including one or more biomarkers listed in Table
1 and the Examples, or a fragment thereof, are identified in a
method wherein a cell is contacted with a candidate compound and
the expression or activity level of the biomarker is determined.
The level of expression of biomarker mRNA or polypeptide or
fragments thereof in the presence of the candidate compound is
compared to the level of expression of biomarker mRNA or
polypeptide or fragments thereof in the absence of the candidate
compound. The candidate compound can then be identified as a
modulator of biomarker expression based on this comparison. For
example, when expression of biomarker mRNA or polypeptide or
fragments thereof is greater (statistically significantly greater)
in the presence of the candidate compound than in its absence, the
candidate compound is identified as a stimulator of biomarker
expression. Alternatively, when expression of biomarker mRNA or
polypeptide or fragments thereof is reduced (statistically
significantly less) in the presence of the candidate compound than
in its absence, the candidate compound is identified as an
inhibitor of biomarker expression. The expression level of
biomarker mRNA or polypeptide or fragments thereof in the cells can
be determined by methods described herein for detecting biomarker
mRNA or polypeptide or fragments thereof.
[0230] In yet another aspect of the invention, a biomarker of the
invention, including one or more biomarkers listed in Table 1 and
the Examples, or a fragment thereof, can be used as "bait proteins"
in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat.
No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al.
(1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)
Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene
8:1693-1696; and Brent WO94/10300), to identify other polypeptides
which bind to or interact with the biomarker or fragments thereof
and are involved in activity of the biomarkers. Such
biomarker-binding proteins are also likely to be involved in the
propagation of signals by the biomarker polypeptides or biomarker
natural binding partner(s) as, for example, downstream elements of
one or more biomarkers-mediated signaling pathway.
[0231] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for one or more
biomarkers polypeptide is fused to a gene encoding the DNA binding
domain of a known transcription factor (e.g., GAL-4). In the other
construct, a DNA sequence, from a library of DNA sequences, that
encodes an unidentified polypeptide ("prey" or "sample") is fused
to a gene that codes for the activation domain of the known
transcription factor. If the "bait" and the "prey" polypeptidcs are
able to interact, in vivo, forming one or more biomarkers-dependent
complex, the DNA-binding and activation domains of the
transcription factor are brought into close proximity. This
proximity allows transcription of a reporter gene (e.g., LacZ)
which is operably linked to a transcriptional regulatory site
responsive to the transcription factor. Expression of the reporter
gene can be detected and cell colonies containing the functional
transcription factor can be isolated and used to obtain the cloned
gene which encodes the polypeptide which interacts with one or more
biomarkers polypeptide of the invention, including one or more
biomarkers listed in Table 1 and the Examples or a fragment
thereof.
[0232] In another aspect, the invention pertains to a combination
of two or more of the assays described herein. For example, a
modulating agent can be identified using a cell-based or a
cell-free assay, and the ability of the agent to modulate the
activity of one or more biomarkers polypeptide or a fragment
thereof can be confirmed in vivo, e.g., in an animal such as an
animal model for cellular transformation and/or tumorigenesis.
[0233] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in an appropriate animal model. For example, an
agent identified as described herein can be used in an animal model
to determine the efficacy, toxicity, or side effects of treatment
with such an agent. Alternatively, an agent identified as described
herein can be used in an animal model to determine the mechanism of
action of such an agent. Furthermore, this invention pertains to
uses of novel agents identified by the above-described screening
assays for treatments as described herein.
III. USES AND METHODS OF THE INVENTION
[0234] The biomarkers of the invention described herein, including
the biomarkers listed in Table 1 and the Examples or fragments
thereof, can be used in one or more of the following methods: a)
screening assays; b) predictive medicine (e.g., diagnostic assays,
prognostic assays, and monitoring of clinical trials); and c)
methods of treatment (e.g., therapeutic and prophylactic, e.g., by
up- or down-modulating the copy number, level of expression, and/or
level of activity of the one or more biomarkers).
[0235] The biomarkers described herein or agents that modulate the
expression and/or activity of such biomarkers can be used, for
example, to (a) express one or more biomarkers of the invention,
including one or more biomarkers listed in Table 1 and the Examples
or a fragment thereof (e.g., via a recombinant expression vector in
a host cell in gene therapy applications or synthetic nucleic acid
molecule), (b) detect biomarker mRNA or a fragment thereof (e.g.,
in a biological sample) or a genetic alteration in one or more
biomarkers gene, and/or (c) modulate biomarker activity, as
described further below. The biomarkers or modulatory agents
thereof can be used to treat conditions or disorders characterized
by insufficient or excessive production of one or more biomarkers
polypeptide or fragment thereof or production of biomarker
polypeptide inhibitors. In addition, the biomarker polypeptides or
fragments thereof can be used to screen for naturally occurring
biomarker binding partner(s), to screen for drugs or compounds
which modulate biomarker activity, as well as to treat conditions
or disorders characterized by insufficient or excessive production
of biomarker polypeptide or a fragment thereof or production of
biomarker polypeptide forms which have decreased, aberrant or
unwanted activity compared to biomarker wild-type polypeptides or
fragments thereof (e.g., cancers, including head, neck, and/or lung
cancers).
[0236] A. Screening Assays
[0237] In one aspect, the present invention relates to a method for
preventing in a subject, a disease or condition associated with an
unwanted, more than desirable, or less than desirable, expression
and/or activity of one or more biomarkers described herein.
Subjects at risk for a disease that would benefit from treatment
with the claimed agents or methods can be identified, for example,
by any one or combination of diagnostic or prognostic assays known
in the art and described herein (see, for example, agents and
assays described in III. Methods of Selecting Agents and
Compositions).
[0238] B. Predictive Medicine
[0239] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
and monitoring of clinical trials are used for prognostic
(predictive) purposes to thereby treat an individual
prophylactically. Accordingly, one aspect of the present invention
relates to diagnostic assays for determining the expression and/or
activity level of biomarkers of the invention, including biomarkers
listed in Table 1 and the Examples or fragments thereof, in the
context of a biological sample (e.g., blood, serum, cells, or
tissue) to thereby determine whether an individual is afflicted
with a disease or disorder, or is at risk of developing a disorder,
associated with aberrant or unwanted biomarker expression or
activity. The present invention also provides for prognostic (or
predictive) assays for determining whether an individual is at risk
of developing a disorder associated with biomarker polypeptide,
nucleic acid expression or activity. For example, mutations in one
or more biomarkers gene can be assayed in a biological sample.
[0240] Such assays can be used for prognostic or predictive purpose
to thereby prophylactically treat an individual prior to the onset
of a disorder characterized by or associated with biomarker
polypeptide, nucleic acid expression or activity.
[0241] Another aspect of the invention pertains to monitoring the
influence of agents (e.g., drugs, compounds, and small nucleic
acid-based molecules) on the expression or activity of biomarkers
of the invention, including biomarkers listed in Table 1 and the
Examples, or fragments thereof, in clinical trials. These and other
agents are described in further detail in the following
sections.
[0242] 1. Diagnostic Assays
[0243] The present invention provides, in part, methods, systems,
and code for accurately classifying whether a biological sample is
associated with a cancer or a clinical subtype thereof (e.g., head,
neck, and/or lung cancers). In some embodiments, the present
invention is useful for classifying a sample (e.g., from a subject)
as a cancer sample using a statistical algorithm and/or empirical
data (e.g., the presence or level of one or biomarkers described
herein).
[0244] An exemplary method for detecting the level of expression or
activity of one or more biomarkers of the invention, including one
or more biomarkers listed in Table 1 and the Examples or fragments
thereof, and thus useful for classifying whether a sample is
associated with cancer or a clinical subtype thereof (e.g., head,
neck, and/or lung cancers), involves obtaining a biological sample
from a test subject and contacting the biological sample with a
compound or an agent capable of detecting the biomarker (e.g.,
polypeptide or nucleic acid that encodes the biomarker or fragments
thereof) such that the level of expression or activity of the
biomarker is detected in the biological sample. In some
embodiments, the presence or level of at least one, two, three,
four, five, six, seven, eight, nine, ten, fifty, hundree, or more
biomarkers of the invention are determined in the individual's
sample. In certain instances, the statistical algorithm is a single
learning statistical classifier system. Exemplary statistical
analyses are presented in the Examples and can be used in certain
embodiments. In other embodiments, a single learning statistical
classifier system can be used to classify a sample as a cancer
sample, a cancer subtype sample, or a non-cancer sample based upon
a prediction or probability value and the presence or level of one
or more biomarkers described herein. The use of a single learning
statistical classifier system typically classifies the sample as a
cancer sample with a sensitivity, specificity, positive predictive
value, negative predictive value, and/or overall accuracy of at
least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%.
[0245] Other suitable statistical algorithms are well known to
those of skill in the art. For example, learning statistical
classifier systems include a machine learning algorithmic technique
capable of adapting to complex data sets (e.g., panel of markers of
interest) and making decisions based upon such data sets. In some
embodiments, a single learning statistical classifier system such
as a classification tree (e.g., random forest) is used. In other
embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
learning statistical classifier systems are used, preferably in
tandem. Examples of learning statistical classifier systems
include, but are not limited to, those using inductive learning
(e.g., decision/classification trees such as random forests,
classification and regression trees (C&RT), boosted trees,
etc.), Probably Approximately Correct (PAC) learning, connectionist
learning (e.g., neural networks (NN), artificial neural networks
(ANN), neuro fuzzy networks (NFN), network structures, perceptrons
such as multi-layer perceptrons, multi-layer feed-forward networks,
applications of neural networks, Bayesian learning in belief
networks, etc.), reinforcement learning (e.g., passive learning in
a known environment such as naive learning, adaptive dynamic
learning, and temporal difference learning, passive learning in an
unknown environment, active learning in an unknown environment,
learning action-value functions, applications of reinforcement
learning, etc.), and genetic algorithms and evolutionary
programming. Other learning statistical classifier systems include
support vector machines (e.g., Kernel methods), multivariate
adaptive regression splines (MARS), Levenberg-Marquardt algorithms,
Gauss-Newton algorithms, mixtures of Gaussians, gradient descent
algorithms, and learning vector quantization (LVQ). In certain
embodiments, the method of the present invention further comprises
sending the cancer classification results to a clinician, e.g., an
oncologist or hematologist.
[0246] In another embodiment, the method of the present invention
further provides a diagnosis in the form of a probability that the
individual has a cancer or a clinical subtype thereof. For example,
the individual can have about a 0%, 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
greater probability of having cancer or a clinical subtype thereof.
In yet another embodiment, the method of the present invention
further provides a prognosis of cancer in the individual. For
example, the prognosis can be surgery, development of a clinical
subtype of the cancer (e.g., subtype of leukemia), development of
one or more symptoms, development of malignant cancer, or recovery
from the disease. In some instances, the method of classifying a
sample as a cancer sample is further based on the symptoms (e.g.,
clinical factors) of the individual from which the sample is
obtained. The symptoms or group of symptoms can be, for example,
those associated with the IPI. In some embodiments, the diagnosis
of an individual as having cancer or a clinical subtype thereof is
followed by administering to the individual a therapeutically
effective amount of a drug useful for treating one or more symptoms
associated with cancer or the cancer.
[0247] In some embodiments, an agent for detecting biomarker mRNA,
genomic DNA, or fragments thereof is a labeled nucleic acid probe
capable of hybridizing to biomarker mRNA, genomic DNA, or fragments
thereof. The nucleic acid probe can be, for example, full-length
biomarker nucleic acid, or a portion thereof, such as an
oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides
in length and sufficient to specifically hybridize under stringent
conditions well known to a skilled artisan to biomarker mRNA or
genomic DNA. Other suitable probes for use in the diagnostic assays
of the invention are described herein. In some embodiments, the
nucleic acid probe is designed to detect transcript variants (i.e.,
different splice forms) of a gene.
[0248] A preferred agent for detecting one or more biomarkers
listed in Table 1 and the Examples or a fragment thereof is an
antibody capable of binding to the biomarker, preferably an
antibody with a detectable label. Antibodies can be polyclonal, or
more preferably, monoclonal. An intact antibody, or a fragment
thereof (e.g., Fab or F(ab')2) can be used. The term "labeled",
with regard to the probe or antibody, is intended to encompass
direct labeling of the probe or antibody by coupling (i.e.,
physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with
fluorescently labeled streptavidin. The term "biological sample" is
intended to include tissues, cells, and biological fluids isolated
from a subject, as well as tissues, cells, and fluids present
within a subject. That is, the detection method of the invention
can be used to detect biomarker mRNA, polypeptide, genomic DNA, or
fragments thereof, in a biological sample in vitro as well as in
vive. For example, in vitro techniques for detection of biomarker
mRNA or a fragment thereof include Northern hybridizations and in
situ hybridizations. In vitro techniques for detection of biomarker
polypeptide include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations and immunofluorescence. In
vitro techniques for detection of biomarker genomic DNA or a
fragment thereof include Southern hybridizations. Furthermore, in
vivo techniques for detection of one or more biomarkers polypeptide
or a fragment thereof include introducing into a subject a labeled
anti-biomarker antibody. For example, the antibody can be labeled
with a radioactive marker whose presence and location in a subject
can be detected by standard imaging techniques.
[0249] In one embodiment, the biological sample contains
polypeptide molecules from the test subject. Alternatively, the
biological sample can contain mRNA molecules from the test subject
or genomic DNA molecules from the test subject. A preferred
biological sample is a hematological tissue (e.g., a sample
comprising blood, plasma, B cell, bone marrow, etc.) sample
isolated by conventional means from a subject.
[0250] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting
polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA,
pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a
variant thereof, genomic DNA, or fragments thereof of one or more
biomarkers listed in Table 1 and the Examples such that the
presence of biomarker polypeptide, mRNA, genomic DNA, or fragments
thereof, is detected in the biological sample, and comparing the
presence of biomarker polypeptide, mRNA, cDNA, small RNAs, mature
miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding
site, or a variant thereof, genomic DNA, or fragments thereof in
the control sample with the presence of biomarker polypeptide,
mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*,
anti-miRNA, or a miRNA binding site, or a variant thereof, genomic
DNA, or fragments thereof in the test sample.
[0251] The invention also encompasses kits for detecting the
presence of a polypeptide, mRNA, cDNA, small RNAs, mature miRNA,
pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site,
or a variant thereof, genomic DNA, or fragments thereof, of one or
more biomarkers listed in Table 1 and the Examples in a biological
sample. For example, the kit can comprise a labeled compound or
agent capable of detecting one or more biomarkers polypeptide,
mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*,
anti-miRNA, or a miRNA binding site, or a variant thereof, genomic
DNA, or fragments thereof, in a biological sample; means for
determining the amount of the biomarker polypeptide, mRNA, cDNA,
small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA,
or a miRNA binding site, or a variant thereof, genomic DNA, or
fragments thereof, in the sample: and means for comparing the
amount of the biomarker polypeptide, mRNA, cDNA, small RNAs, mature
miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding
site, or a variant thereof, genomic DNA, or fragments thereof, in
the sample with a standard. The compound or agent can be packaged
in a suitable container. The kit can further comprise instructions
for using the kit to detect the biomarker polypeptide, mRNA, cDNA,
small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA,
or a miRNA binding site, or a variant thereof, genomic DNA, or
fragments thereof.
[0252] In some embodiments, therapies tailored to treat stratified
patient populations based on the described diagnostic assays are
further administered.
[0253] 2. Prognostic Assays
[0254] The diagnostic methods described herein can furthermore be
utilized to identify subjects having or at risk of developing a
disease or disorder associated with aberrant expression or activity
of one or more biomarkers of the invention, including one or more
biomarkers listed in Table 1 and the Examples, or a fragment
thereof. As used herein, the term "aberrant" includes biomarker
expression or activity levels which deviates from the normal
expression or activity in a control.
[0255] The assays described herein, such as the preceding
diagnostic assays or the following assays, can be utilized to
identify a subject having or at risk of developing a disorder
associated with a misregulation of biomarker activity or
expression, such as in a cancer (e.g., head, neck, and/or lung
cancers). Alternatively, the prognostic assays can be used to
identify a subject having or at risk for developing a disorder
associated with a misregulation of biomarker activity or
expression. Thus, the present invention provides a method for
identifying and/or classifying a disease associated with aberrant
expression or activity of one or more biomarkers of the invention,
including one or more biomarkers listed in Table 1 and the
Examples, or a fragment thereof. Furthermore, the prognostic assays
described herein can be used to determine whether a subject can be
administered an agent (e.g., an agonist, antagonist,
peptidomimetic, polypeptide, peptide, nucleic acid, small molecule,
or other drug candidate) to treat a disease or disorder associated
with aberrant biomarker expression or activity. For example, such
methods can be used to determine whether a subject can be
effectively treated with an agent for a cancer (e.g., head, neck,
and/or lung cancers). Thus, the present invention provides methods
for determining whether a subject can be effectively treated with
an agent for a disease associated with aberrant biomarker
expression or activity in which a test sample is obtained and
biomarker polypeptide or nucleic acid expression or activity is
detected (e.g., wherein a significant increase or decrease in
biomarker polypeptide or nucleic acid expression or activity
relative to a control is diagnostic for a subject that can be
administered the agent to treat a disorder associated with aberrant
biomarker expression or activity). In some embodiments, significant
increase or decrease in biomarker expression or activity comprises
at least 2 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4,
4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20 times or more higher or lower,
respectively, than the expression activity or level of the marker
in a control sample.
[0256] The methods of the invention can also be used to detect
genetic alterations in one or more biomarkers of the invention,
including one or more biomarkers listed in Table 1 and the Examples
or a fragment thereof, thereby determining if a subject with the
altered biomarker is at risk for cancer (e.g., head, neck, and/or
lung cancers) characterized by aberrant biomarker activity or
expression levels. In preferred embodiments, the methods include
detecting, in a sample of cells from the subject, the presence or
absence of a genetic alteration characterized by at least one
alteration affecting the integrity of a gene encoding one or more
biomarkers polypeptide, or the mis-expression of the biomarker
(e.g., mutations and/or splice variants). For example, such genetic
alterations can be detected by ascertaining the existence of at
least one of 1) a deletion of one or more nucleotides from one or
more biomarkers gene, 2) an addition of one or more nucleotides to
one or more biomarkers gene, 3) a substitution of one or more
nucleotides of one or more biomarkers gene, 4) a chromosomal
rearrangement of one or more biomarkers gene, 5) an alteration in
the level of a messenger RNA transcript of one or more biomarkers
gene, 6) aberrant modification of one or more biomarkers gene, such
as of the methylation pattern of the genomic DNA, 7) the presence
of a non-wild type splicing pattern of a messenger RNA transcript
of one or more biomarkers gene, 8) a non-wild type level of one or
more biomarkers polypeptide, 9) allelic loss of one or more
biomarkers gene, and 10) inappropriate post-translational
modification of one or more biomarkers polypeptide. As described
herein, there are a large number of assays known in the art which
can be used for detecting alterations in one or more biomarkers
gene. A preferred biological sample is a tissue or serum sample
isolated by conventional means from a subject.
[0257] In certain embodiments, detection of the alteration involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364),
the latter of which can be particularly useful for detecting point
mutations in one or more biomarkers gene (see Abravaya et al.
(1995) Nucleic Acids Res. 23:675-682). This method can include the
steps of collecting a sample of cells from a subject, isolating
nucleic acid (e.g., genomic DNA, mRNA, cDNA, small RNA, mature
miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding
site, or a variant thereof) from the cells of the sample,
contacting the nucleic acid sample with one or more primers which
specifically hybridize to one or more biomarkers gene of the
invention, including the biomarker genes listed in Table 1 and the
Examples, or fragments thereof, under conditions such that
hybridization and amplification of the biomarker gene (if present)
occurs, and detecting the presence or absence of an amplification
product, or detecting the size of the amplification product and
comparing the length to a control sample. It is anticipated that
PCR and/or LCR may be desirable to use as a preliminary
amplification step in conjunction with any of the techniques used
for detecting mutations described herein.
[0258] Alternative amplification methods include: self-sustained
sequence replication (Guatelli, J. C. et al. (1990) Proc. Natl.
Acad. Sci. USA 87:1874-1878), transcriptional amplification system
(Kwoh. D. Y. et al. (1989) Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi, P. M, et al. (1988)
Bio-Technology 6:1197), or any other nucleic acid amplification
method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art. These detection
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers.
[0259] In an alternative embodiment, mutations in one or more
biomarkers gene of the invention, including one or more biomarkers
listed in Table 1 and the Examples, or a fragment thereof, from a
sample cell can be identified by alterations in restriction enzyme
cleavage patterns. For example, sample and control DNA is isolated,
amplified (optionally), digested with one or more restriction
endonucleases, and fragment length sizes are determined by gel
electrophoresis and compared. Differences in fragment length sizes
between sample and control DNA indicates mutations in the sample
DNA. Moreover, the use of sequence specific ribozymes (see, for
example, U.S. Pat. No. 5,498,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[0260] In other embodiments, genetic mutations in one or more
biomarkers gene of the invention, including a gene listed in Table
1 and the Examples, or a fragment thereof, can be identified by
hybridizing a sample and control nucleic acids, e.g., DNA, RNA,
mRNA, small RNA, cDNA, mature miRNA, pre-miRNA, pri-miRNA, miRNA*,
anti-miRNA, or a miRNA binding site, or a variant thereof, to high
density arrays containing hundreds or thousands of oligonucleotide
probes (Cronin, M. T. et al. (1996) Hum. Mutat. 7:244-255; Kozal.
M. J. et al. (1996) Nat. Med. 2:753-759). For example, genetic
mutations in one or more biomarkers can be identified in two
dimensional arrays containing light-generated DNA probes as
described in Cronin et al. (1996) supra. Briefly, a first
hybridization array of probes can be used to scan through long
stretches of DNA in a sample and control to identify base changes
between the sequences by making linear arrays of sequential,
overlapping probes. This step allows the identification of point
mutations. This step is followed by a second hybridization array
that allows the characterization of specific mutations by using
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene.
[0261] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence one or
more biomarkers gene of the invention, including a gene listed in
Table 1 and the Examples, or a fragment thereof, and detect
mutations by comparing the sequence of the sample biomarker gene
with the corresponding wild-type (control) sequence. Examples of
sequencing reactions include those based on techniques developed by
Maxam and Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:560 or
Sanger (1977) Proc. Natl. Acad Sci. USA 74:5463. It is also
contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
(Naeve, C. W. (1995) Biotechniques 19:448-53), including sequencing
by mass spectrometry (see, e.g., PCT International Publication No.
WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and
Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
[0262] Other methods for detecting mutations in one or more
biomarkers gene of the invention, including a gene listed in Table
1 and the Examples, or fragments thereof, include methods in which
protection from cleavage agents is used to detect mismatched bases
in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science
230:1242). In general, the art technique of "mismatch cleavage"
starts by providing heteroduplexes formed by hybridizing (labeled)
RNA or DNA containing the wild-type sequence with potentially
mutant RNA or DNA obtained from a tissue sample. The
double-stranded duplexes are treated with an agent which cleaves
single-stranded regions of the duplex such as which will exist due
to base pair mismatches between the control and sample strands. For
instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA
hybrids treated with S1 nuclease to enzymatically digest the
mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA
duplexes can be treated with hydroxylamine or osmium tetroxide and
with piperidine in order to digest mismatched regions. After
digestion of the mismatched regions, the resulting material is then
separated by size on denaturing polyacrylamide gels to determine
the site of mutation. Sec, for example, Cotton et al. (1988) Proc.
Natl. Acad. Sci. USA 85:4397 and Saleeba et al. (1992) Methods
Enzymol. 217:286-295. In a preferred embodiment, the control DNA or
RNA can be labeled for detection.
[0263] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in
biomarker genes of the invention, including genes listed in Table 1
and the Examples, or fragments thereof, obtained from samples of
cells. For example, the mutY enzyme of E. coli cleaves A at G/A
mismatches and the thymidine DNA glycosylase from HeLa cells
cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis
15:1657-1662). The duplex is treated with a DNA mismatch repair
enzyme, and the cleavage products, if any, can be detected from
electrophoresis protocols or the like. See, for example, U.S. Pat.
No. 5,459,039.
[0264] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in biomarker genes of
the invention, including genes listed in Table 1 and the Examples,
or fragments thereof. For example, single strand conformation
polymorphism (SSCP) may be used to detect differences in
electrophoretic mobility between mutant and wild type nucleic acids
(Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766; see also
Cotton (1993) Mutat. Res. 285:125-144 and Hayashi (1992) Genet.
Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample
and control nucleic acids will be denatured and allowed to
renature. The secondary structure of single-stranded nucleic acids
varies according to sequence, the resulting alteration in
electrophoretic mobility enables the detection of even a single
base change. The DNA fragments may be labeled or detected with
labeled probes. The sensitivity of the assay may be enhanced by
using RNA (rather than DNA), in which the secondary structure is
more sensitive to a change in sequence. In a preferred embodiment,
the subject method utilizes heteroduplex analysis to separate
double stranded heteroduplex molecules on the basis of changes in
electrophorectic mobility (Keen et al. (1991) Trends Genet.
7:5).
[0265] In yet another embodiment the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to ensure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys.
Chem. 265: 12753).
[0266] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324:163; Saiki
et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA or a number of different mutations when the oligonucleotides
are attached to the hybridizing membrane and hybridized with
labeled target DNA. In some embodiments, the hybridization
reactions can occur using biochips, microarrays, etc., or other
array technology that are well known in the art.
[0267] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner (1993) Tibtech 11:238). In
addition it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88:189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0268] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving one or more biomarkers of the invention,
including one or more biomarkers listed in Table 1 and the
Examples, or fragments thereof.
[0269] 3. Monitoring of Effects During Clinical Trials
[0270] Monitoring the influence of agents (e.g., drugs) on the
expression or activity of one or more biomarkers of the invention,
including one or more biomarkers listed in Table 1 and the
Examples, or a fragment thereof (e.g., the modulation of a cancer
state) can be applied not only in basic drug screening, but also in
clinical trials. For example, the effectiveness of an agent
determined by a screening assay as described herein to increase
expression and/or activity of one or more biomarkers of the
invention, including one or more biomarkers listed in Table 1 and
the Examples or a fragment thereof, can be monitored in clinical
trials of subjects exhibiting decreased expression and/or activity
of one or more biomarkers of the invention, including one or more
biomarkers of the invention, including one or more biomarkers
listed in Table 1 and the Examples, or a fragment thereof, relative
to a control reference. Alternatively, the effectiveness of an
agent determined by a screening assay to decrease expression and/or
activity of one or more biomarkers of the invention, including one
or more biomarkers listed in Table 1 and the Examples, or a
fragment thereof, can be monitored in clinical trials of subjects
exhibiting decreased expression and/or activity of the biomarker of
the invention, including one or more biomarkers listed in Table 1
and the Examples or a fragment thereof relative to a control
reference. In such clinical trials, the expression and/or activity
of the biomarker can be used as a "read out" or marker of the
phenotype of a particular cell.
[0271] In some embodiments, the present invention provides a method
for monitoring the effectiveness of treatment of a subject with an
agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide,
peptide, nucleic acid, small molecule, or other drug candidate
identified by the screening assays described herein) including the
steps of (i) obtaining a pre-administration sample from a subject
prior to administration of the agent; (ii) detecting the level of
expression and/or activity of one or more biomarkers of the
invention, including one or more biomarkers listed in Table 1 and
the Examples or fragments thereof in the preadministration sample;
(iii) obtaining one or more post-administration samples from the
subject; (iv) detecting the level of expression or activity of the
biomarker in the post-administration samples; (v) comparing the
level of expression or activity of the biomarker or fragments
thereof in the pre-administration sample with the that of the
biomarker in the post administration sample or samples; and (vi)
altering the administration of the agent to the subject
accordingly. For example, increased administration of the agent may
be desirable to increase the expression or activity of one or more
biomarkers to higher levels than detected (e.g., to increase the
effectiveness of the agent.) Alternatively, decreased
administration of the agent may be desirable to decrease expression
or activity of the biomarker to lower levels than detected (e.g.,
to decrease the effectiveness of the agent). According to such an
embodiment, biomarker expression or activity may be used as an
indicator of the effectiveness of an agent, even in the absence of
an observable phenotypic response.
[0272] D. Methods of Treatment
[0273] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder characterized by insufficient or
excessive production of biomarkers of the invention, including
biomarkers listed in Table 1 and the Examples or fragments thereof,
which have aberrant expression or activity compared to a control.
Moreover, agents of the invention described herein can be used to
detect and isolate the biomarkers or fragments thereof, regulate
the bioavailability of the biomarkers or fragments thereof, and
modulate biomarker expression levels or activity.
[0274] 1. Prophylactic Methods
[0275] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant expression or activity of one or more biomarkers of the
invention, including one or more biomarkers listed in Table 1 and
the Examples or a fragment thereof, by administering to the subject
an agent which modulates biomarker expression or at least one
activity of the biomarker. Subjects at risk for a disease or
disorder which is caused or contributed to by aberrant biomarker
expression or activity can be identified by, for example, any or a
combination of diagnostic or prognostic assays as described herein.
Administration of a prophylactic agent can occur prior to the
manifestation of symptoms characteristic of the biomarker
expression or activity aberrancy, such that a disease or disorder
is prevented or, alternatively, delayed in its progression.
[0276] 2. Therapeutic Methods
[0277] Another aspect of the invention pertains to methods of
modulating the expression or activity or interaction with natural
binding partner(s) of one or more biomarkers of the invention,
including one or more biomarkers listed in Table 1 and the Examples
or fragments thereof, for therapeutic purposes. The biomarkers of
the invention have been demonstrated to correlate with cancer
(e.g., head, neck, and/or lung cancers). Accordingly, the activity
and/or expression of the biomarker, as well as the interaction
between one or more biomarkers or a fragment thereof and its
natural binding partner(s) or a fragment(s) thereof can be
modulated in order to modulate the immune response.
[0278] Modulatory methods of the invention involve contacting a
cell with one or more biomarkers of the invention, including one or
more biomarkers of the invention, including one or more biomarkers
listed in Table 1 and the Examples or a fragment thereof or agent
that modulates one or more of the activities of biomarker activity
associated with the cell. In some embodiments, the biomarkers are
or encode secreted molecules such that contacting a cell with one
or more biomarkers of the invention or agent that modulates one or
more of the activities of biomarker activity is unnecessary and
contact with a bodily fluid (e.g., blood, serum, lung pleural
fluid, etc.) is sufficient. An agent that modulates biomarker
activity can be an agent as described herein, such as a nucleic
acid or a polypeptide, a naturally-occurring binding partner of the
biomarker, an antibody against the biomarker, a combination of
antibodies against the biomarker and antibodies against other
immune related targets, one or more biomarkers agonist or
antagonist, a peptidomimetic of one or more biomarkers agonist or
antagonist, one or more biomarkers peptidomimetic, other small
molecule, or small RNA directed against or a mimic of one or more
biomarkers nucleic acid gene expression product.
[0279] An agent that modulates the expression of one or more
biomarkers of the invention, including one or more biomarkers of
the invention, including one or more biomarkers listed in Table 1
and the Examples or a fragment thereof is, e.g., an antisense
nucleic acid molecule, RNAi molecule, shRNA, mature miRNA,
pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site,
or a variant thereof, or other small RNA molecule, triplex
oligonucleotide, ribozyme, or recombinant vector for expression of
one or more biomarkers polypeptide. For example, an oligonucleotide
complementary to the area around one or more biomarkers polypeptide
translation initiation site can be synthesized. One or more
antisense oligonucleotides can be added to cell media, typically at
200 .mu.g/ml, or administered to a patient to prevent the synthesis
of one or more biomarkers polypeptide. The antisense
oligonucleotide is taken up by cells and hybridizes to one or more
biomarkers mRNA to prevent translation. Alternatively, an
oligonucleotide which binds double-stranded DNA to form a triplex
construct to prevent DNA unwinding and transcription can be used.
As a result of either, synthesis of biomarker polypeptide is
blocked. When biomarker expression is modulated, preferably, such
modulation occurs by a means other than by knocking out the
biomarker gene.
[0280] Agents which modulate expression, by virtue of the fact that
they control the amount of biomarker in a cell, also modulate the
total amount of biomarker activity in a cell.
[0281] In one embodiment, the agent stimulates one or more
activities of one or more biomarkers of the invention, including
one or more biomarkers listed in Table 1 and the Examples or a
fragment thereof. Examples of such stimulatory agents include
active biomarker polypeptide or a fragment thereof and a nucleic
acid molecule encoding the biomarker or a fragment thereof that has
been introduced into the cell (e.g., cDNA, mRNA, shRNAs, siRNAs,
small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA,
or a miRNA binding site, or a variant thereof, or other
functionally equivalent molecule known to a skilled artisan). In
another embodiment, the agent inhibits one or more biomarker
activities. In one embodiment, the agent inhibits or enhances the
interaction of the biomarker with its natural binding partner(s).
Examples of such inhibitory agents include antisense nucleic acid
molecules, anti-biomarker antibodies, biomarker inhibitors, and
compounds identified in the screening assays described herein.
[0282] These modulatory methods can be performed in vitro (e.g., by
contacting the cell with the agent) or, alternatively, by
contacting an agent with cells in vivo (e.g., by administering the
agent to a subject). As such, the present invention provides
methods of treating an individual afflicted with a condition or
disorder that would benefit from up- or down-modulation of one or
more biomarkers of the invention listed in Table 1 and the Examples
or a fragment thereof, e.g., a disorder characterized by unwanted,
insufficient, or aberrant expression or activity of the biomarker
or fragments thereof. In one embodiment, the method involves
administering an agent (e.g., an agent identified by a screening
assay described herein), or combination of agents that modulates
(e.g., upregulates or downregulates) biomarker expression or
activity. In another embodiment, the method involves administering
one or more biomarkers polypeptide or nucleic acid molecule as
therapy to compensate for reduced, aberrant, or unwanted biomarker
expression or activity.
[0283] Stimulation of biomarker activity is desirable in situations
in which the biomarker is abnormally downregulated and/or in which
increased biomarker activity is likely to have a beneficial effect.
Likewise, inhibition of biomarker activity is desirable in
situations in which biomarker is abnormally upregulated and/or in
which decreased biomarker activity is likely to have a beneficial
effect.
[0284] In addition, these modulatory agents can also be
administered in combination therapy with, e.g., chemotherapeutic
agents, hormones, antiangiogens, radiolabelled, compounds, or with
surgery, cryotherapy, and/or radiotherapy. The preceding treatment
methods can be administered in conjunction with other forms of
conventional therapy (e.g. standard-of-care treatments for cancer
well known to the skilled artisan), either consecutively with, pre-
or post-conventional therapy. For example, these modulatory agents
can be administered with a therapeutically effective dose of
chemotherapeutic agent. In another embodiment, these modulatory
agents are administered in conjunction with chemotherapy to enhance
the activity and efficacy of the chemotherapeutic agent. The
Physicians' Desk Reference (PDR) discloses dosages of
chemotherapeutic agents that have been used in the treatment of
various cancers. The dosing regimen and dosages of these
aforementioned chemotherapeutic drugs that are therapeutically
effective will depend on the particular cancer (e.g., head, neck,
and/or lung cancers), being treated, the extent of the disease and
other factors familiar to the physician of skill in the art and can
be determined by the physician.
[0285] In some embodiments, it is useful to downregulate immune
responses. In other embodiments, it is useful to upregulate immune
responses.
[0286] a. Downregulation of Immune Responses by Modulation
[0287] There are numerous embodiments of the invention for
upregulating the inhibitory function or downregulating the
costimulatory function of a PD-1 ligand (e.g., soluble PD-L1) to
thereby downregulate immune responses. Downregulation can be in the
form of inhibiting or blocking an immune response already in
progress, or may involve preventing the induction of an immune
response. The functions of activated immune cells can be inhibited
by down-regulating immune cell responses, or by inducing specific
anergy in immune cells, or both.
[0288] For example, the immune response can be downmodulated using
PD-1 ligand (e.g., soluble PD-L1) polypeptides (e.g., soluble,
monomeric forms of a PD-1 ligand (e.g., soluble PD-L1) polypeptide
such as PD-1 ligand (e.g., soluble PD-L1)-Ig) and/or anti-PD-1
ligand (e.g., soluble PD-L1) antibodies that block the interaction
of PD-1 ligand (e.g., soluble PD-L1) with a B7 polypeptide (e.g.,
while not affecting or increasing the interaction between PD-L1 and
PD-1) or which promote the binding of a PD-1 ligand (e.g., soluble
PD-L1) with PD-1, (e.g., while not affecting or while inhibiting
the interaction between a B7 polypeptide and the PD-1 ligand (e.g.,
soluble PD-L1)). Other exemplary agents which can block these
interactions include anti-B7 polypeptide, a B7 polypeptide, or a
blocking small molecule.
[0289] In addition, in embodiments where PD-L1 binds to an
inhibitory receptor, forms of PD-L1 that bind to the inhibitory
receptor, e.g., multivalent PD-L1 on a cell surface, can be used to
downmodulate the immune response.
[0290] Likewise, the PD-1 pathway can also be stimulated by the use
of an agent to thereby downmodulate the immune response. Inhibition
of the interaction of B7-4 with a stimulatory receptor on an immune
cell (e.g., by using a soluble form of PD-1 and/or CTLA4) or
activation of PD-1 (e.g., using an activating antibody which
cross-links PD-1) may provide negative signals to immune cells.
[0291] Agents that promote binding of a PD-1 ligand (e.g., soluble
PD-L1) to PD-1 or a B7 polypeptide to CTLA4, while not affecting or
reducing the binding of the PD-1 ligand (e.g., soluble PD-L1) to
the B7 polypeptide, can also be used to down modulate the immune
response. Exemplary agents include PD-1 peptide mimetics,
identified by the methods described herein.
[0292] In one embodiment of the invention, an activating antibody
used to stimulate PD-1 activity is a bispecific antibody. For
example, such an antibody can comprise a PD-1 binding site and
another binding site which targets a cell surface receptor on an
immune cell, e.g., on a T cell, a B cell, or a myeloid cell. In one
embodiment, such an antibody, in addition to comprising a PD-1
binding site can further comprise a binding site which binds to a
molecule which is in proximity to an activating or inhibitory
receptor, e.g., B-cell antigen receptor, a T-cell antigen receptor,
or an Fe receptor in order to target the molecule to a specific
cell population. For example, a CD3 antigen, a T-cell receptor
chain, LFA-1, CD2, CTLA-4, immunoglobulin, B cell receptor, Ig
alpha, Ig beta, CD22, or Fc receptor could be used. Such antibodies
(or other bispecific agents) are art recognized and can be
produced, e.g., as described herein. Selection of this second
antigen for the bispecific antibody provides flexibility in
selection of cell population to be targeted for inhibition.
[0293] In another embodiment, the co-ligation of PD-1 and an
activating or inhibitory receptor on a cell can enhance the
generation of a negative signal via PD-1. Such co-ligation can be
accomplished e.g., by use of a bispecific agent, e.g., a bispecific
antibody as described herein having specificity for both PD-1 and a
molecule associated with a receptor. In another embodiment, the use
of a multivalent form of an agent that transmits a negative signal
via PD-1 can be used to enhance the transmission of a negative
signal via PD-1, e.g., an agent presented on a bead or on a
surface. In another embodiment, a such a multivalent agent can
comprise two specificities to achieve co-ligation of PD-1 and a
receptor or a receptor associated molecule (e.g., a bead comprising
anti CD3 and PD-L1).
[0294] Agents that block or inhibit interaction of PD-L1 with a
costimulatory receptor (e.g., soluble forms of PD-L1 or blocking
antibodies to PD-L1) as well as agents that promote a
PD-L1-mediated inhibitory signal or agonists of PD-1 which activate
PD-1 (e.g., PD-1 activating antibodies or PD-1 activating small
molecules) can be identified by their ability to inhibit immune
cell proliferation and/or effector function or to induce anergy
when added to an in vitro assay. For example, cells can cultured in
the presence of an agent that stimulates signal transduction via an
activating receptor. A number of art recognized readouts of cell
activation can be employed to measure, e.g., cell proliferation or
effector function (e.g., antibody production, cytokine production,
phagocytosis) in the presence of the activating agent. The ability
of a test agent to block this activation can be readily determined
by measuring the ability of the agent to effect a decrease in
proliferation or effector function being measured.
[0295] In one embodiment of the invention, tolerance is induced
against specific antigens by co-administering an antigen with an
agent (e.g., antibody, peptide, fusion protein, or small molecule)
which blocks the interaction between a PD-1 ligand (e.g., soluble
PD-L1) and a B7 polypeptide. For example, tolerance can be induced
to specific proteins. In one embodiment, immune responses to
allergens, or to foreign proteins to which an immune response is
undesirable, can be inhibited. For example, patients that receive
Factor VIII frequently generate antibodies against this clotting
factor. Co-administration of an agent that blocks a PD-1 ligand
(e.g., soluble PD-L1)-mediated costimulatory signal or an agent
that stimulates a PD-1 mediated inhibitory signal in combination
with recombinant factor VIII (or by physically linked to Factor
VIII, e.g., by cross-linking) can result in downmodulation.
[0296] In one embodiment, fusion proteins comprising a first PD-1
ligand (e.g., soluble PD-L1) peptide fused to a second peptide can
be used to block the interaction of the PD-1 ligand (e.g., soluble
PD-L1) with a B7 polypeptide on an immune cell, to thereby
downmodulate immune responses. In one embodiment, the second
peptide blocks an activity of another B lymphocyte antigen (e.g.,
B7-1, B7-2, or B7-3) to further downmodulate immune responses.
Alternatively, two separate agents that downmodulate immune
responses can be combined as a single composition or administered
separately (simultaneously or sequentially) to more effectively
downregulate immune cell mediated immune responses in a subject.
For instance, a PD-1 ligand (e.g., soluble PD-L1) can be combined
with a B7 polypeptide, or with a combination of blocking antibodies
(e.g., antibodies against a PD-1 ligand (e.g., soluble PD-L1)
polypeptide with anti-B7-1 and/or anti-B7-2 monoclonal antibodies).
Furthermore, a therapeutically active amount of one or more of the
subject agents, can be used in conjunction with other
downmodulating reagents to influence immune responses. Examples of
other immunomodulating reagents include, without limitation,
antibodies that block a costimulatory signal, (e.g., against CD28
or ICOS), antibodies that act as agonists of CTLA4, and/or
antibodies against other immune cell markers (e.g., against CD40,
against CD40 ligand, or against cytokines), fusion proteins (e.g.,
CTLA4-Fc), and immunosuppressive drugs, (e.g., rapamycin,
cyclosporine A or FK506).
[0297] The agents described herein can also be useful in the
construction of therapeutic agents which block immune cell function
by destruction of cells. For example, portions of a PD-L1 or PD-1
polypeptide can be linked to a toxin to make a cytotoxic agent
capable of triggering the destruction of cells to which it
binds.
[0298] For making cytotoxic agents, polypeptides of the invention
may be linked, or operatively attached, to toxins using techniques
that are known in the art, e.g., crosslinking or via recombinant
DNA techniques. The preparation of immunotoxins is, in general,
well known in the art (see, e.g., U.S. Pat. No. 4,340,535, and EP
44167, both incorporated herein by reference). Numerous types of
disulfide-bond containing linkers are known which can successfully
be employed to conjugate the toxin moiety with a polypeptide. In
one embodiment, linkers that contain a disulfide bond that is
sterically "hindered" are to be preferred, due to their greater
stability in vivo, thus preventing release of the toxin moiety
prior to binding at the site of action.
[0299] A wide variety of toxins are known that may be conjugated to
polypeptides or antibodies of the invention. Examples include:
numerous useful plant-, fungus- or even bacteria-derived toxins,
which, by way of example, include various A chain toxins,
particularly ricin A chain, ribosome inactivating proteins such as
saporin or gelonin, .alpha.-sarcin, aspergillin, restrictocin,
ribonucleases such as placental ribonuclease, angiogenic,
diphtheria toxin, and pseudomonas exotoxin, etc. A preferred toxin
moiety for use in connection with the invention is toxin A chain
which has been treated to modify or remove carbohydrate residues,
deglycosylated A chain. (U.S. Pat. No. 5,776,427).
[0300] Infusion of one or a combination of such cytotoxic agents,
(e.g., PD-L1 ricin (alone or in combination with B7-2-ricin or
B7-1-ricin), into a patient may result in the death of immune
cells, particularly in light of the fact that activated immune
cells that express higher amounts of PD-L1 ligands. For example,
because PD-1 is induced on the surface of activated lymphocytes, an
antibody against PD-1 can be used to target the depletion of these
specific cells by Fc-R dependent mechanisms or by ablation by
conjugating a cytotoxic drug (e.g., ricin, saporin, or
calicheamicin) to the antibody. In one embodiment, the antibody
toxin can be a bispecific antibody. Such bispecific antibodies are
useful for targeting a specific cell population, e.g., using a
marker found only on a certain type of cell, e.g., a TCR, BCR, or
FcR molecule.
[0301] Downregulating or preventing a PD-1 ligand (e.g., soluble
PD-L1) interaction with a B7 polypeptide, or promoting an
interaction between a PD-1 ligand (e.g., soluble PD-L1) and PD-1
(for example, without modulating, or by additionally enhancing) the
interaction between the PD-1 ligand (e.g., soluble PD-L1) and the
B7 polypeptide (e.g., by stimulation of the negative signaling
function of PD-1) is useful to downmodulate the immune response,
e.g., in situations of excess inflammation; in tissue, skin and
organ transplantation; in graft-versus-host disease (GVHD); or in
autoimmune diseases such as systemic lupus erythematosus, and
multiple sclerosis. For example, blockage of immune cell function
results in reduced tissue destruction in tissue transplantation.
Typically, in tissue transplants, rejection of the transplant is
initiated through its recognition as foreign by immune cells,
followed by an immune reaction that destroys the transplant. The
administration of a polypeptide which inhibits or blocks
interaction of a PD-1 ligand (e.g., soluble PD-L1) with a B7
polypeptide (such as a soluble, monomeric form of the PD-1 ligand
(e.g., soluble PD-L1) or PD-1), alone or in conjunction with
another downmodulatory agent, prior to or at the time of
transplantation can promote the generation of an inhibitory signal.
Moreover, inhibition of PD-1 ligand (e.g., soluble PD-L1)
costimulatory signals, or promotion of a PD-1 ligand (e.g., soluble
PD-L, 1) or PD-1 inhibitory signals, may also be sufficient to
anergize the immune cells, thereby inducing tolerance in a subject.
Induction of long-term tolerance by blocking a PD-1 ligand (e.g.,
soluble PD-L1) mediated costimulatory signal may avoid the
necessity of repeated administration of these blocking
reagents.
[0302] To achieve sufficient immunosuppression or tolerance in a
subject, it may also be desirable to block the costimulatory
function of other polypeptides. For example, it may be desirable to
block the function of B7-1 and PD-L1; B7-2 and PD-L1; B7-1 and B7-2
and PD-L1; B7-1; B7-2; or B7-1 and B7-2 by administering a soluble
form of a combination of peptides having an activity of each of
these antigens, blocking antibodies against these antigens or
blocking small molecules (separately or together in a single
composition) prior to or at the time of transplantation.
Alternatively, it may be desirable to promote inhibitory activity
of a PD-1 ligand (e.g., soluble PD-L1) or PD-1 and inhibit a
costimulatory activity of B7-1 and/or B7-2. Other downmodulatory
agents that can be used in connection with the downmodulatory
methods of the invention include, for example, agents that transmit
an inhibitory signal via CTLA4, soluble forms of CTLA4, antibodies
that activate an inhibitory signal via CTLA4, blocking antibodies
against other immune cell markers or soluble forms of other
receptor ligand pairs (e.g., agents that disrupt the interaction
between CD40 and CD40 ligand (e.g., anti CD40 ligand antibodies)),
antibodies against cytokines, or immunosuppressive drugs. In
another embodiment, a combination of at least two different PD-L1
antibodies can be administered to achieve optimal blocking
activity.
[0303] Downmodulation of immune responses are also useful in
treating autoimmune disease. Many autoimmune disorders are the
result of inappropriate activation of immune cells that are
reactive against self tissue and which promote the production of
cytokines and autoantibodies involved in the pathology of the
diseases. Preventing the activation of autoreactive immune cells
may reduce or eliminate disease symptoms. Administration of
reagents which block costimulation of immune cells by disrupting
interactions between PD-1 ligand (e.g., soluble PD-L1) and B7
polypeptides, or by promoting the interaction between PD-1 ligand
(e.g., soluble PD-L1) and PD-1, without modulating or while
downmodulating the interaction between PD-1 ligand (e.g., soluble
PD-L1) and a B7 polypeptide, are useful for inhibiting immune cell
activation and preventing production of autoantibodies or cytokines
which may be involved in the disease process. Additionally, agents
that promote an inhibitory function of a PD-1 ligand (e.g., soluble
PD-L1) or PD-1 may induce antigen-specific tolerance of
autoreactive immune cells, which could lead to long-term relief
from the disease. The efficacy of reagents in preventing or
alleviating autoimmune disorders can be determined using a number
of well-characterized animal models of human autoimmune diseases.
Examples include murine experimental autoimmune encephalitis,
systemic lupus erythematosus in MRL/lpr/lpr mice or NZB hybrid
mice, murine autoimmune collagen arthritis, diabetes mellitus in
NOD mice and BB rats, and murine experimental myasthenia gravis
(see. e.g., Paul ed., Fundamental Immunology, Raven Press, New
York, Third Edition 1993, chapter 30).
[0304] Inhibition of immune cell activation is useful
therapeutically in the treatment of allergy and allergic reactions,
e.g., by inhibiting IgE production. An agent that promotes a PD-1
ligand (e.g., soluble PD-L1) or PD-1 inhibitory function can be
administered to an allergic subject to inhibit immune cell mediated
allergic responses in the subject. Inhibition of PD-1 ligand (e.g.,
soluble PD-L1) costimulation of immune cells or stimulation of a
PD-1 ligand (e.g., soluble PD-L1) or PD-1 inhibitory pathway can be
accompanied by exposure to allergen in conjunction with appropriate
MHC polypeptides. Allergic reactions can be systemic or local in
nature, depending on the route of entry of the allergen and the
pattern of deposition of IgE on mast cells or basophils. Thus,
inhibition of immune cell mediated allergic responses locally or
systemically by administration of an inhibitory form of an agent
that inhibits the interaction of a PD-1 ligand (e.g., soluble
PD-L1) with a costimulatory receptor, or an agent that promotes an
inhibitory function of a PD-1 ligand (e.g., soluble PD-L1) or
PD-1.
[0305] Inhibition of immune cell activation through blockage of the
interaction of a PD-1 ligand (e.g., soluble PD-L1) and a B7
polypeptide, or through promotion of the interaction between a PD-1
ligand (e.g., soluble PD-L1) and PD-1, without modulating or while
downmodulating the interaction between the PD-1 ligand (e.g.,
soluble PD-L1) and a B7 polypeptide, may also be important
therapeutically in viral infections of immune cells. For example,
in the acquired immune deficiency syndrome (AIDS), viral
replication is stimulated by immune cell activation. Modulation of
these interactions may result in inhibition of viral replication
and thereby ameliorate the course of AIDS. Modulation of these
interactions may also be useful in promoting the maintenance of
pregnancy. PD-1 ligand (e.g., soluble PD-L1) is normally highly
expressed in placental trophoblasts, the layer of cells that forms
the interface between mother and fetus and may play a role in
preventing maternal rejection of the fetus. Females at risk for
spontaneous abortion (e.g., those who have previously had a
spontaneous abortion or those who have had difficulty conceiving)
because of immunologic rejection of the embryo or fetus can be
treated with agents that modulate these interactions.
[0306] Downregulation of an immune response by modulation of PD-1
ligand (e.g., soluble PD-L1)/B7 polypeptide, binding or by
modulation of PD-1 ligand (e.g., soluble PD-L1)/PD-1 binding may
also be useful in treating an autoimmune attack of autologous
tissues. For example, PD-1 ligand (e.g., soluble PD-L1) is normally
highly expressed in the heart and may protect the heart from
autoimmune attack. This is evidenced by the fact that the Balb/c
PD-1 knockout mouse exhibits massive autoimmune attack on the heart
with thrombosis. Thus, conditions that are caused or exacerbated by
autoimmune attack (e.g., in this example, heart disease, myocardial
infarction or atherosclerosis) may be ameliorated or improved by
modulation of these interactions. It is therefore within the scope
of the invention to modulate conditions exacerbated by autoimmune
attack, such as autoimmune disorders (as well as conditions such as
heart disease, myocardial infarction, and atherosclerosis).
[0307] b. Upregulation of Immune Responses
[0308] Also useful therapeutically is the blockage of the
interaction of a PD-1 ligand (e.g., soluble PD-L1) with PD-1,
and/or a B7 polypeptide with CTLA4, without modulating or while
upregulating the interaction between the B7 polypeptide and the
PD-1 ligand (e.g., soluble PD-L1), or by promoting the interaction
of the PD-1 ligand (e.g., soluble PD-L1) with the B7 polypeptide
(e.g., while not affecting or while inhibiting the interaction
between the PD-1 ligand (e.g., soluble PD-L1) and PD-1) as a means
of upregulating an immune response. Blocking the interaction
between a B7 polypeptide and a PD-1 ligand (e.g., soluble PD-L1) to
thereby increase the interaction between the B7 polypeptide and
CD28, is also useful to upregulate immune responses. Upregulation
of immune responses can be in the form of enhancing an existing
immune response or eliciting an initial immune response. For
instance, enhancing an immune response using the subject
compositions and methods is useful in cases of infections with
microbes (e.g., bacteria, viruses, or parasites). In one
embodiment, an agent that blocks the interaction of a PD-1 ligand
(e.g., soluble PD-L1) with PD-1, without modulating or while
upregulating the interaction between a B7 polypeptide and the PD-1
ligand (e.g., soluble PD-L1), or by promoting the interaction of
the PD-1 ligand (e.g., soluble PD-L1) with the B7 polypeptide, is
used to enhance the immune response. Such an agent (e.g., a
non-activating antibody that blocks PD-L1 binding to PD-1) is
therapeutically useful in situations where upregulation of antibody
and cell-mediated responses would be beneficial. In a preferred
embodiment, the agent inhibits the interaction between PD-1 and a
PD-1 ligand (e.g., soluble PD-L1), without inhibiting the
interaction between the PD-1 ligand (e.g., soluble PD-L1) and a B7
polypeptide (e.g., an interaction which prevents PD-L1 from binding
to PD-1). Exemplary disorders include viral skin diseases, such as
Herpes or shingles, in which case such an agent can be delivered
topically to the skin. In addition, systemic viral diseases such as
influenza, the common cold, and encephalitis might be alleviated by
systemic administration of such agents.
[0309] Alternatively, immune responses can be enhanced in an
infected patient through an er vivo approach, for instance, by
removing immune cells from the patient, contacting immune cells in
vitro with an agent that blocks the interaction of a PD-1 ligand
(e.g., soluble PD-L1) with PD-1, without modulating or while
upmodulating the interaction between a B7 polypeptide and the PD-1
ligand (e.g., soluble PD-L1), or by promoting the interaction of
the PD-1 ligand (e.g., soluble PD-L1) with the B7 polypeptide, and
reintroducing the in vitro stimulated immune cells into the
patient.
[0310] In certain instances, it may be desirable to further
administer other agents that upregulate immune responses, for
example, forms of other B7 family members that transduce signals
via costimulatory receptors, in order to further augment the immune
response.
[0311] An agent that blocks the interaction of a PD-1 ligand (e.g.,
soluble PD-L1) with PD-1 (e.g., without modulating or while
upmodulating the interaction between a B7 polypeptide and the PD-1
ligand (e.g., soluble PD-L1) or by enhancing the interaction of the
PD-1 ligand (e.g., soluble PD-L1) with the B7 polypeptide) can be
used prophylactically in vaccines against various polypeptides
(e.g., polypeptides derived from pathogens). Immunity against a
pathogen (e.g., a virus) can be induced by vaccinating with a viral
protein along with an agent that blocks the interaction of a PD-1
ligand (e.g., soluble PD-L1) with PD-1, without modulating or while
upmodulating the interaction between a B7 polypeptide and the PD-1
ligand (e.g., soluble PD-L1), or by promoting the interaction of
the PD-1 ligand (e.g., soluble PD-L1) with the B7 polypeptide, in
an appropriate adjuvant.
[0312] In another embodiment, upregulation or enhancement of an
immune response function, as described herein, is useful in the
induction of tumor immunity
[0313] In another embodiment, the immune response can be stimulated
by the methods described herein, such that preexisting tolerance is
overcome. For example, immune responses against antigens to which a
subject cannot mount a significant immune response, e.g., to an
autologous antigen, such as a tumor specific antigens can be
induced by administering an agent that blocks the interaction of a
PD-1 ligand (e.g., soluble PD-L1) with PD-1 (e.g., without
modulating or while upmodulating the interaction between a B7
polypeptide and the PD-1 ligand (e.g., soluble PD-L1) or by
promoting the interaction of the PD-1 ligand (e.g., soluble PD-L1)
with the B7 polypeptide). In one embodiment, a soluble PD-1 or a
soluble PD-1 ligand (e.g., soluble PD-L1) that inhibits the
interaction of a PD-1 ligand (e.g., soluble PD-L1) with PD-1,
without modulating or while upmodulating the interaction between a
B7 polypeptide and the PD-1 ligand (e.g., soluble PD-L1), or by
promoting the interaction of the PD-1 ligand (e.g., soluble PD-L1)
with the B7 polypeptide, can be used to enhance an immune response
(e.g., to a tumor cell). In one embodiment, an autologous antigen,
such as a tumor-specific antigen can be coadministered. In another
embodiment, the subject agents can be used as adjuvants to boost
responses to foreign antigens in the process of active
immunization. In yet another embodiment, the production of a form
of PD-L1 that binds to an inhibitory receptor or that competes with
the binding of PD-L1 to a costimulatory receptor (e.g., a form of
PD-L1 that binds to PD-1 or a naturally occurring soluble molecule)
can be inhibited, e.g., using antisense RNA, in order to upregulate
the immune response. For example, in one embodiment, the production
of inhibitory PD-L1 molecules by a rumor cell can be inhibited in
order to increase anti-tumor immunity.
[0314] In one embodiment, immune cells are obtained from a subject
and cultured ex vivo in the presence of an agent as described
herein, to expand the population of immune cells and/or to enhance
immune cell activation. In a further embodiment the immune cells
are then administered to a subject. Immune cells can be stimulated
in vitro by, for example, providing to the immune cells a primary
activation signal and a costimulatory signal, as is known in the
art. Various agents can also be used to costimulate proliferation
of immune cells. In one embodiment immune cells are cultured ex
vivo according to the method described in PCT Application No. WO
94/29436. The costimulatory polypeptide can be soluble, attached to
a cell membrane, or attached to a solid surface, such as a
bead.
IV. PHARMACEUTICAL COMPOSITIONS
[0315] In another aspect, the present invention provides
pharmaceutically acceptable compositions which comprise a
therapeutically-effective amount of an agent that modulates (e.g.,
increases or decreases) PD-1, membrane-bound PD-L1, and/or soluble
PD-L1 levels, formulated together with one or more pharmaceutically
acceptable carriers (additives) and/or diluents. As described in
detail below, the pharmaceutical compositions of the present
invention may be specially formulated for administration in solid
or liquid form, including those adapted for the following: (1) oral
administration, for example, drenches (aqueous or non-aqueous
solutions or suspensions), tablets, boluses, powders, granules,
pastes; (2) parenteral administration, for example, by
subcutaneous, intramuscular or intravenous injection as, for
example, a sterile solution or suspension: (3) topical application,
for example, as a cream, ointment or spray applied to the skin; (4)
intravaginally or intrarectally, for example, as a pessary, cream
or foam; or (5) aerosol, for example, as an aqueous aerosol,
liposomal preparation or solid particles containing the
compound.
[0316] The phrase "therapeutically-effective amount" as used herein
means that amount of an agent that modulates (e.g., inhibits) PD-1,
membrane-bound PD-L1, and/or soluble PD-L1 levels, or expression
and/or activity of the receptor/ligand complex, or composition
comprising an agent that modulates (e.g., inhibits) PD-1,
membrane-bound PD-L1, and/or soluble PD-L1 levels, or expression
and/or activity of the receptor/ligand complex, which is effective
for producing some desired therapeutic effect, e.g., cancer
treatment, at a reasonable benefit/risk ratio.
[0317] The phrase "pharmaceutically acceptable" is employed herein
to refer to those agents, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0318] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject chemical from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the subject.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth: (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (1) polyols, such as glycerin, sorbitol, mannitol
and polyethylene glycol; (12) esters, such as ethyl oleate and
ethyl laurate; (13) agar; (14) buffering agents, such as magnesium
hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0319] The term "pharmaceutically-acceptable salts" refers to the
relatively non-toxic, inorganic and organic acid addition salts of
the agents that modulates (e.g., inhibits) PD-1, membrane-bound
PD-L1, and/or soluble PD-L1 levels, or expression and/or activity
of the receptor/ligand complex encompassed by the invention. These
salts can be prepared in situ during the final isolation and
purification of the respiration uncoupling agents, or by separately
reacting a purified respiration uncoupling agent in its free base
form with a suitable organic or inorganic acid, and isolating the
salt thus formed. Representative salts include the hydrobromide,
hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,
valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,
phosphate, tosylate, citrate, maleate, fumarate, succinate,
tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and
laurylsulphonate salts and the like (See, for example, Berge et al.
(1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).
[0320] In other cases, the agents useful in the methods of the
present invention may contain one or more acidic functional groups
and, thus, are capable of forming pharmaceutically-acceptable salts
with pharmaceutically-acceptable bases. The term
"pharmaceutically-acceptable salts" in these instances refers to
the relatively non-toxic, inorganic and organic base addition salts
of agents that modulates (e.g., inhibits) PD-1, membrane-bound
PD-L1, and/or soluble PD-L1 levels, or expression and/or activity
of the receptor/ligand complex. These salts can likewise be
prepared in situ during the final isolation and purification of the
respiration uncoupling agents, or by separately reacting the
purified respiration uncoupling agent in its free acid form with a
suitable base, such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically-acceptable metal cation, with ammonia, or with a
pharmaceutically-acceptable organic primary, secondary or tertiary
amine. Representative alkali or alkaline earth salts include the
lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like. Representative organic amines useful for the
formation of base addition salts include ethylamine, diethylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine and the
like (see, for example, Berge et al., supra).
[0321] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0322] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BIA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
[0323] Formulations useful in the methods of the present invention
include those suitable for oral, nasal, topical (including buccal
and sublingual), rectal, vaginal, aerosol and/or parenteral
administration. The formulations may conveniently be presented in
unit dosage form and may be prepared by any methods well known in
the art of pharmacy. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will vary depending upon the host being treated, the particular
mode of administration. The amount of active ingredient, which can
be combined with a carrier material to produce a single dosage form
will generally be that amount of the compound which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 1% to about 99% of active ingredient,
preferably from about 5% to about 70%, most preferably from about
10% to about 30%.
[0324] Methods of preparing these formulations or compositions
include the step of bringing into association an agent that
modulates (e.g., increases or decreases) PD-1, membrane-bound
PD-L1, and/or soluble PD-L1 levels, with the carrier and,
optionally, one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association a respiration uncoupling agent with liquid carriers, or
finely divided solid carriers, or both, and then, if necessary,
shaping the product.
[0325] Formulations suitable for oral administration may be in the
form of capsules, cachets, pills, tablets, lozenges (using a
flavored basis, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or
non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin, or sucrose and acacia) and/or
as mouth washes and the like, each containing a predetermined
amount of a respiration uncoupling agent as an active ingredient. A
compound may also be administered as a bolus, electuary or
paste.
[0326] In solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules and the like), the
active ingredient is mixed with one or more
pharmaceutically-acceptable carriers, such as sodium citrate or
dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, acetyl alcohol and glycerol
monostearate; (8) absorbents, such as kaolin and bentonite clay;
(9) lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may also be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0327] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered peptide or peptidomimetic moistened with an
inert liquid diluent.
[0328] Tablets, and other solid dosage forms, such as dragees,
capsules, pills and granules, may optionally be scored or prepared
with coatings and shells, such as enteric coatings and other
coatings well known in the pharmaceutical-formulating art. They may
also be formulated so as to provide slow or controlled release of
the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions, which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions, which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0329] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
ingredient, the liquid dosage forms may contain inert diluents
commonly used in the art, such as, for example, water or other
solvents, solubilizing agents and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof.
[0330] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0331] Suspensions, in addition to the active agent may contain
suspending agents as, for example, ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol and sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar-agar and
tragacanth, and mixtures thereof.
[0332] Formulations for rectal or vaginal administration may be
presented as a suppository, which may be prepared by mixing one or
more respiration uncoupling agents with one or more suitable
nonirritating excipients or carriers comprising, for example, cocoa
butter, polyethylene glycol, a suppository wax or a salicylate, and
which is solid at room temperature, but liquid at body temperature
and, therefore, will melt in the rectum or vaginal cavity and
release the active agent.
[0333] Formulations which are suitable for vaginal administration
also include pessaries, tampons, creams, gels, pastes, foams or
spray formulations containing such carriers as are known in the art
to be appropriate.
[0334] Dosage forms for the topical or transdermal administration
of an agent that modulates (e.g., increases or decreases) PD-1,
membrane-bound PD-L1, and/or soluble PD-L1 levels include powders,
sprays, ointments, pastes, creams, lotions, gels, solutions,
patches and inhalants. The active component may be mixed under
sterile conditions with a pharmaceutically-acceptable carrier, and
with any preservatives, buffers, or propellants which may be
required.
[0335] The ointments, pastes, creams and gels may contain, in
addition to a respiration uncoupling agent, excipients, such as
animal and vegetable fats, oils, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and zinc oxide, or mixtures
thereof.
[0336] Powders and sprays can contain, in addition to an agent that
modulates (e.g., increases or decreases) PD-1, membrane-bound
PD-L1, and/or soluble PD-L1 levels, excipients such as lactose,
talc, silicic acid, aluminum hydroxide, calcium silicates and
polyamide powder, or mixtures of these substances. Sprays can
additionally contain customary propellants, such as
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,
such as butane and propane.
[0337] The agent that modulates (e.g., increases or decreases)
PD-1, membrane-bound PD-L1, and/or soluble PD-L1 levels, can be
alternatively administered by aerosol. This is accomplished by
preparing an aqueous aerosol, liposomal preparation or solid
particles containing the compound. A nonaqueous (e.g., fluorocarbon
propellant) suspension could be used. Sonic nebulizers are
preferred because they minimize exposing the agent to shear, which
can result in degradation of the compound.
[0338] Ordinarily, an aqueous aerosol is made by formulating an
aqueous solution or suspension of the agent together with
conventional pharmaceutically acceptable carriers and stabilizers.
The carriers and stabilizers vary with the requirements of the
particular compound, but typically include nonionic surfactants
(Tweens, Pluronics, or polyethylene glycol), innocuous proteins
like serum albumin, sorbitan esters, oleic acid, lecithin, amino
acids such as glycine, buffers, salts, sugars or sugar alcohols.
Aerosols generally are prepared from isotonic solutions.
[0339] Transdermal patches have the added advantage of providing
controlled delivery of a respiration uncoupling agent to the body.
Such dosage forms can be made by dissolving or dispersing the agent
in the proper medium. Absorption enhancers can also be used to
increase the flux of the peptidomimetic across the skin. The rate
of such flux can be controlled by either providing a rate
controlling membrane or dispersing the peptidomimetic in a polymer
matrix or gel.
[0340] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0341] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more respiration
uncoupling agents in combination with one or more
pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents.
[0342] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0343] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0344] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution, which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0345] Injectable depot forms are made by forming microencapsule
matrices of an agent that modulates (e.g., increases or decreases)
PD-1, membrane-bound PD-L1, and/or soluble PD-L1 levels, in
biodegradable polymers such as polylactide-polyglycolide. Depending
on the ratio of drug to polymer, and the nature of the particular
polymer employed, the rate of drug release can be controlled.
Examples of other biodegradable polymers include poly(orthoesters)
and poly(anhydrides). Depot injectable formulations are also
prepared by entrapping the drug in liposomes or microemulsions,
which are compatible with body tissue.
[0346] When the respiration uncoupling agents of the present
invention are administered as pharmaceuticals, to humans and
animals, they can be given per se or as a pharmaceutical
composition containing, for example, 0.1 to 99.5% (more preferably,
0.5 to 90%) of active ingredient in combination with a
pharmaceutically acceptable carrier.
[0347] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of this invention may be determined by
the methods of the present invention so as to obtain an amount of
the active ingredient, which is effective to achieve the desired
therapeutic response for a particular subject, composition, and
mode of administration, without being toxic to the subject.
[0348] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3054 3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g., retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
V. ADMINISTRATION OF AGENTS
[0349] The cancer diagnostic, prognostic, prevention, and/or
treatment modulating agents of the invention are administered to
subjects in a biologically compatible form suitable for
pharmaceutical administration in vivo, to either enhance or
suppress immune cell mediated immune responses. By "biologically
compatible form suitable for administration in vivo" is meant a
form of the protein to be administered in which any toxic effects
are outweighed by the therapeutic effects of the protein. The term
"subject" is intended to include living organisms in which an
immune response can be elicited, e.g., mammals. Examples of
subjects include humans, dogs, cats, mice, rats, and transgenic
species thereof. Administration of an agent as described herein can
be in any pharmacological form including a therapeutically active
amount of an agent alone or in combination with a pharmaceutically
acceptable carrier.
[0350] Administration of a therapeutically active amount of the
therapeutic composition of the present invention is defined as an
amount effective, at dosages and for periods of time necessary, to
achieve the desired result. For example, a therapeutically active
amount of a blocking antibody may vary according to factors such as
the disease state, age, sex, and weight of the individual, and the
ability of peptide to elicit a desired response in the individual.
Dosage regimens can be adjusted to provide the optimum therapeutic
response. For example, several divided doses can be administered
daily or the dose can be proportionally reduced as indicated by the
exigencies of the therapeutic situation.
[0351] The agents of the invention described herein can be
administered in a convenient manner such as by injection
(subcutaneous, intravenous, etc.), oral administration, inhalation,
transdermal application, or rectal administration. Depending on the
route of administration, the active compound can be coated in a
material to protect the compound from the action of enzymes, acids
and other natural conditions which may inactivate the compound. For
example, for administration of agents, by other than parenteral
administration, it may be desirable to coat the agent with, or
co-administer the agent with, a material to prevent its
inactivation.
[0352] An agent can be administered to an individual in an
appropriate carrier, diluent or adjuvant, co-administered with
enzyme inhibitors or in an appropriate carrier such as liposomes.
Pharmaceutically acceptable diluents include saline and aqueous
buffer solutions. Adjuvant is used in its broadest sense and
includes any immune stimulating compound such as interferon.
Adjuvants contemplated herein include resorcinols, non-ionic
surfactants such as polyoxyethylene oleyl ether and n-hexadecyl
polyethylene ether. Enzyme inhibitors include pancreatic trypsin
inhibitor, diisopropylfluorophosphate (DEEP) and trasylol.
Liposomes include water-in-oil-in-water emulsions as well as
conventional liposomes (Sterna et al. (1984) J. Neuroimmunol.
7:27).
[0353] The agent may also be administered parenterally or
intraperitoneally. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, and mixtures thereof, and in oils.
Under ordinary conditions of storage and use, these preparations
may contain a preservative to prevent the growth of
microorganisms.
[0354] Pharmaceutical compositions of agents suitable for
injectable use include sterile aqueous solutions (where water
soluble) or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. In all
cases the composition will preferably be sterile and must be fluid
to the extent that easy syringeability exists. It will preferably
be stable under the conditions of manufacture and storage and
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prevention
of the action of microorganisms can be achieved by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In
many cases, it is preferable to include isotonic agents, for
example, sugars, polyalcohols such as manitol, sorbitol, sodium
chloride in the composition. Prolonged absorption of the injectable
compositions can be brought about by including in the composition
an agent which delays absorption, for example, aluminum
monostearate and gelatin.
[0355] Sterile injectable solutions can be prepared by
incorporating an agent of the invention (e.g., an antibody,
peptide, fusion protein or small molecule) in the required amount
in an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
agent plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0356] When the agent is suitably protected, as described above,
the protein can be orally administered, for example, with an inert
diluent or an assimilable edible carrier. As used herein
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active compound, use thereof in
the therapeutic compositions is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
[0357] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. "Dosage unit form", as used herein, refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by, and directly dependent on, (a)
the unique characteristics of the active compound and the
particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
compound for the treatment of sensitivity in individuals.
[0358] In one embodiment, an agent of the invention is an antibody.
As defined herein, a therapeutically effective amount of antibody
(i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg
body weight, preferably about 0.01 to 25 mg/kg body weight, more
preferably about 0.1 to 20 mg/kg body weight, and even more
preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7
mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will
appreciate that certain factors may influence the dosage required
to effectively treat a subject, including but not limited to the
severity of the disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Moreover, treatment of a subject with a therapeutically
effective amount of an antibody can include a single treatment or,
preferably, can include a series of treatments. In a preferred
example, a subject is treated with antibody in the range of between
about 0.1 to 20 mg/kg body weight, one time per week for between
about 1 to 10 weeks, preferably between 2 to 8 weeks, more
preferably between about 3 to 7 weeks, and even more preferably for
about 4, 5, or 6 weeks. It will also be appreciated that the
effective dosage of antibody used for treatment may increase or
decrease over the course of a particular treatment. Changes in
dosage may result from the results of diagnostic assays. In
addition, an antibody of the invention can also be administered in
combination therapy with, e.g., chemotherapeutic agents, hormones,
antiangiogens, radiolabelled, compounds, or with surgery,
cryotherapy, and/or radiotherapy. An antibody of the invention can
also be administered in conjunction with other forms of
conventional therapy, either consecutively with, pre- or
post-conventional therapy. For example, the antibody can be
administered with a therapeutically effective dose of
chemotherapeutic agent. In another embodiment, the antibody can be
administered in conjunction with chemotherapy to enhance the
activity and efficacy of the chemotherapeutic agent. The
Physicians' Desk Reference (PDR) discloses dosages of
chemotherapeutic agents that have been used in the treatment of
various cancers. The dosing regimen and dosages of these
aforementioned chemotherapeutic drugs that are therapeutically
effective will depend on the particular immune disorder, e.g.,
Hodgkin lymphoma, being treated, the extent of the disease and
other factors familiar to the physician of skill in the art and can
be determined by the physician.
[0359] In addition, the agents of the invention described herein
can be administered using nanoparticle-based composition and
delivery methods well known to the skilled artisan. For example,
nanoparticle-based delivery for improved nucleic acid (e.g., small
RNAs) therapeutics are well known in the art (Expert Opinion on
Biological Therapy 7:1811-1822).
EXEMPLIFICATION
[0360] This invention is further illustrated by the following
examples, which should not be construed as limiting.
Example 1
HPV-Mediated PD-L1 Splice Variants are Associated with Head, Neck,
and Lung Cancers and Other Cancers
[0361] FIG. 1 shows a schematic of a mutational landscape of head
and neck cancers analyzed by The Cancer Genome Atlas (TCGA)
project. Sequencing results of 279 tumors using whole-exome hybrid
capture identified genes displaying significant enrichment for
mutation in this dataset. The start indicates samples with evidence
of HPV infection. Each column represents one tumor and the
different types of tumors are shown by the different marks. Also
listed are the number of mutations per sample, the percent of
samples with mutations in each of the listed genes and whether
these genes have been reported in the COSMIC (Sanger Cancer Gene
Census) database.
[0362] FIG. 2 shows data in the same format as for FIG. 1, except
that the data is drawn from an independent cohort of HPV- (left
half) and HPV+ (right half) head and neck cancers in which 700
cancer-related genes were sequenced. FIG. 2 further shows amplified
(middle box) or deleted genes (bottom box).
[0363] FIG. 3 shows a representation of sites of HPV integration in
the host genome in head and neck cancers analyzed as part of the
TCGA project described in FIG. 1. The circle shows the human
chromosomes around the perimeter and the green lines show sites of
integration.
[0364] FIGS. 4 and 5 show sequencing reads for the CD274 (PD-L1)
gene in a squamous head and neck cancer tumor from the larynx
(CV-5433) with a detected HPV integration in the PD-L1 gene. On the
right panel is seen a white space in the center of the reads which
corresponds to where HPV is detected (see left panel). The site of
integrations spans positions 5,464,244 to 5,464,509 on chromosome 9
of the Human Genome Reference Consortium Human Build 37
(GRCh37/hg19) assembly publicly available as GenBank Assembly ID:
GCA_000001405.1 and RefSeq Assembly ID: GCF_00001405.13 dated Feb.
27, 2009. FIG. 5 shows a zoomed in view of FIG. 4 according to a
log scale.
[0365] FIG. 6 shows a consolidation of the sequencing read data of
FIGS. 4 and 5 into a schematic showing the HPV integration within
the PD-L1 gene of tumor CV-5433.
[0366] FIG. 7 shows predicted protein products following HPV
integration in tumor CV-5433. One protein is a short form of PD-L1
(soluble 1) made of 227 amino acids (plus a stop codon) instead of
the full-length, membrane-bound form having 290 amino acids (i.e.,
the nucleic acid encodes from 5' to 3' the following domains: Exon
1 (L-region), Exon 2 (IgV-like-domain), Exon 3 (IgC-like-domain),
Exon 4 (the connecting-region, transmembrane-region, and the start
of the intracytoplasmic region), and Exons 5 and 6
(intracytoplasmic-region). The identified short form of PD-L1
results in loss of the transmembrane and intracellular domains to
generate a soluble/secreted polypeptide. In addition, another short
form of PD-L1 (soluble 2) was identified and confirmed via RNA
sequencing as being identical to the soluble 1 form, but further
containing an additional C-terminal sequence (FIG. 7) that is not
contained in the wild-type, full-length, membrane-bound PD-L1
form.
[0367] FIG. 8 shows expression of each exon of full-length,
membrane-bound PD-L1 on a log scale from the CV-5433 sample and
demonstrates a dramatic drop in exons following exon 4 which is the
site of HPV integration.
[0368] FIGS. 9-10 show transcript variants of PD-L1 expressed by
head, neck, and lung cancers. FIG. 9 shows that samples from the
TCGA head and neck (left panel) and lung cancer (right panel)
projects display a diversity of splicing in PD-L1 indicating that
many tumors produce soluble PD-L1. FIG. 10 shows the data from FIG.
9 for head and neck cancers in a different manner. Each head and
neck tumor is represented as a circle and the tumors are ordered
left to right according to total PD-L1 expression. Tumors which
favor the short form are lower on the y-axis and normal samples are
shown as square boxes. The sample with the HPV integration is
labeled as the index case and is a clear outlier in terms of high
PD-L1 expression favoring the soluble form.
[0369] FIG. 11 shows that, in addition to head and neck cancer
(HNSC), RNA sequencing analyses generated by TCGA (The Cancer
Genome Atlas) using computational methods to detect PD-L1 short
forms identified expression of such forms in bladder cancer (BLCA),
breast cancer (BRCA), glioblastoma (GBM), kidney cancer (KIRC),
acute myelogenous leukemia (LAML), lung adenocarcinoma (LUAD), lung
squamous cell carcinoma (LUSC), ovarian cancer (OV), and also
summarized in a pan-cancer graph. Tumors are shown in dark gray and
normal samples, when available, are shown in light gray. The y-axis
indicates total PD-L1 expression and the x-axis indicates the
fraction of short PD-L1. Amplification of PD-L1 is shown by the
intensity of the circle. The results indicate that there is a
spectrum of PD-L1 expression across many cancer types with many
tumors displaying evidence of at least some production of the short
form, typically consistent with the soluble 2 form having the
C-terminal sequence, VIPGNILNVSIKICLTLSPST*. The soluble 2 form
sequences were identified by analysis of an index case of head and
neck cancer from The Cancer Genome Atlas project (TCGA-CV-5443) in
which we identified integration of HPV in the PDL1 (CD274) gene
using the Pathseq algorithm as described above. Assembly of PDL1
transcripts from RNA sequencing data from this tumor using
Cufflinks demonstrated a proportion of transcripts with evidence of
sequence beyond the normal exon boundary. Manual assembly and
inspection of these transcripts revealed the presence of sequence
corresponding to the downstream intron as well as alternative
polyadenylation of truncated PDL1 mRNA leading to the translation
of the amino acids "G N I L N V S I K I C L T L S P S T" which are
not part of full-length, wild-type PDL1. Evidence of similar
transcripts was identified in other primary tumors and cancer
derived cell lines.
[0370] FIG. 12 shows similar results using similar analyses as
those conducted in FIG. 11 using the Cancer Cell Line Encyclopedia
dataset. Since the TCGA samples shown in FIG. 11 are bulk tumors
which contain both tumor and normal stroma, the source of
soluble/short PD-L1 could be either the tumor or normal immune
cells. However, FIG. 12 demonstrates that cancer cells grown
without any stroma produce short PD-L1. The circle at approximately
-12 on the y-axis and 0.38 on the x-axis and within the circles at
the very top row of circles on the y-axis is the index head and
neck case for comparison.
Example 2
Soluble PD-L1 Isoforms Associated with Head, Neck, and Lung Cancers
and Other Cancers are Immunologically Active
[0371] FIGS. 13-14 show Western blot results 293T cells transfected
with expression vectors encoding the full-length or short PD-L1
forms. FIG. 13 demonstrates that full-length PD-L1 is only found in
the cells (left) and the short form is only found in the media
(right), thereby confirming that the short form is soluble. FIG. 14
demonstrates that the short form of PD-L1 was produced and affinity
purified. Specifically, nucleic acid molecules encoding full-length
PD-L1 were purchased from Origene and PCR was used to introduce an
early termination codon. PCR was also used to add 3' HA epitope
tags. The short and long forms were cloned into the expression
vector pCDNA3 and also pBabe Puro and pMSCV puro. Cell lines
expressing short and long PD-L1 forms were created by introducing
the PD-L1 transgene by transient transfection (293T cells) or
retroviral infection (K562 and Ba/F3 cells). HA. 11 Clone 16B12
monoclonal antibody was used to detect the HA epitope tag and the
ab58810 polyclonal antibody to detect PD-L1 (Abcam).
[0372] FIG. 15 shows the detection of short PDL1 in culture media
obtained from cell lines predicted to make higher amounts of using
immunoblot analyses (e.g., the RKO colon cancer cell lines, the
BDCM acute myclogenous leukemia cell line, and the CAL62 thyroid
anaplastic carcinoma cell line). This was confirmed by ELISA with
cells engineered to make the short form (293T, 293T PDL1 long
(i.e., wild-type, full-length, membrane-bound PD-L1), PDL1 short),
as well as cancer cell lines predicted to produce the soluble form
(e.g., the RKO and CAL62 cell lines). For recombinant production of
full-length and soluble PDL1, a c-terminal HA tag was attached and
an anti-HA antibody (Thermo 26183) was used for detection. For
endogenous PDL1, the 368A.5A4 antibody was used for Western
blotting and the EISA 29E.12B1 antibody was used for ELISA assays
(courtesy of Gordon Freeman's laboratory; see also Brown et al.
(2003) J. Immunol. 170:1257-1266. FIG. 15 further shows the
distribution of PD-L1 in cells engineered to make the wild-type or
short forms, which indicates that the short form is present in the
media.
[0373] FIG. 16 shows the results of T cell viability assays using T
cells obtained from two independent donors and incubated in the
presence of vehicle or either of the two short PDL1 forms (i.e.,
PD-L1S-Soluble 1 or PD-L1S2-Soluble 2) or media from cells
engineered to make wild-type PD-L1 in which most should have been
retained in the cell. Briefly, T cells were isolated from
peripheral blood from healthy blood donors at the Dana-Farber
Cancer Institute using the auto-MACS system. T cells were cultured
in RPMI media and stimulated with CD3 and CD28 conjugated antibody
beads for 48 hours. Recombinant soluble PDL1 (both forms) produced
via HA-tag affinity purification from transiently transfected
HEK293T cells was then as added at 10 micrograms/ml and cells were
cultured for an additional 48 hours. Cell viability was measured by
propidium iodide exclusion using a flow cytometer. As a control,
HA-affinity purification was performed from the supernatant of 293T
cells expressing full-length PDL1 using exactly the same conditions
as for the soluble form. Etoposide at 10 micromolar was used as a
positive control for cell death. For conditions in which blocking
antibodies were used, the blocking antibodies were added at 10
micrograms per ml and the following antibodies from the Freeman
laboratory were utilized: anti-human PD-L1 29E.2A3 and anti-human
PD-1 EH12 (see, Brown et al. (2003) J. Immunol. 170:1257-1266;
Rodig et al. (2003) Eur. J. Immunol. 33:3117-3126; Cai et al.
(2004) Cell. Immunol. 230:89-98; Dorfman et al. (2006) Amer. J.
Surg. Pathol. 30:802-810; Day et al. (2006) Nature 443:350-354).
FIG. 16 shows a reduction in T cell viability by approximately
10-20% with soluble PD-L1 treatment, but further shows that this
effect can be blunted by addition of anti-PD1 or anti-PDL1
antibodies.
[0374] FIG. 17-18 shows that the short form of PD-L1 can
differentially kill proliferating T cells. FIG. 17 shows the
results of T cells obtained from a healthy donor and stimulated to
proliferate and then treated with either a) the short form of PD-L1
(undiluted, "high input"), b) the short form of PD-L1 (diluted,
"low input"), c) the long form of PD-L1 or d) the chemotherapeutic
topoisomerase inhibitor, etoposide. The treated T cells then
underwent flow cytometry analysis to determine the proportion of
live cells. CD8 staining is shown on the x-axis and cell viability
is shown on the y-axis. The results show that the short form of
PD-L1 kills T cells similar to etoposide and likely CD4 and not CD8
cells. FIG. 18 shows the results of the experiment described in
FIG. 17, except that the T cells were not stimulated to
proliferate.
INCORPORATION BY REFERENCE
[0375] The contents of all references, patent applications,
patents, and published patent applications, as well as the Figures
and the Sequence Listing, cited throughout this application are
hereby incorporated by reference.
EQUIVALENTS
[0376] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *