U.S. patent application number 15/865602 was filed with the patent office on 2018-07-05 for combination of a pd-1 antagonist and a listeria-based vaccine for treating prostate cancer.
The applicant listed for this patent is Advaxis, Inc., Merck Sharp and Dohme Corp.. Invention is credited to David J. MAURO, Rodolfo F. PERINI, Robert PETIT.
Application Number | 20180185483 15/865602 |
Document ID | / |
Family ID | 55079087 |
Filed Date | 2018-07-05 |
United States Patent
Application |
20180185483 |
Kind Code |
A1 |
PETIT; Robert ; et
al. |
July 5, 2018 |
COMBINATION OF A PD-1 ANTAGONIST AND A LISTERIA-BASED VACCINE FOR
TREATING PROSTATE CANCER
Abstract
The present disclosure describes combination therapies
comprising an antagonist of Programmed Death 1 receptor (PD-1) and
a Listeria based strain that expresses prostate-tissue specific
antigen (PSA), and the use of the combination therapies for the
treatment of prostate cancer.
Inventors: |
PETIT; Robert; (Newtown
(Wrightstown), PA) ; MAURO; David J.; (Washington
Crossing, PA) ; PERINI; Rodolfo F.; (North Wales,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Advaxis, Inc.
Merck Sharp and Dohme Corp. |
Princeton
Rahway |
NJ
NJ |
US
US |
|
|
Family ID: |
55079087 |
Appl. No.: |
15/865602 |
Filed: |
January 9, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14802607 |
Jul 17, 2015 |
9907849 |
|
|
15865602 |
|
|
|
|
62039011 |
Aug 19, 2014 |
|
|
|
62026221 |
Jul 18, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/572 20130101;
A61K 2039/884 20180801; A61K 2039/523 20130101; A61K 39/001194
20180801; A61P 35/00 20180101; A61K 39/0011 20130101; A61K 39/39558
20130101; A61K 2039/522 20130101; A61K 39/39558 20130101; A61K
2300/00 20130101; A61K 39/0011 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00 |
Claims
1.-164. (canceled)
165. A method for treating prostate cancer in a patient comprising
administering to the patient a combination therapy which comprises
an antagonist of a Programmed Death 1 protein (PD-1) and a
bioengineered live-attenuated Listeria monocytogenes strain
deficient in at least one gene selected from the group consisting
of a virulence gene, a metabolic gene and combinations thereof,
wherein the live-attenuated Listeria monocytogenes stimulates
Antigen Presenting Cells (APCs) capable of driving a cellular
immune response to PSA expressing cells.
166. The method of claim 165, wherein the at least one gene is
deleted resulting in the deficiency.
167. The method of claim 165, wherein the at least one gene is
mutated resulting in the deficiency.
168. The method of claim 165, wherein the live-attenuated Listeria
monocytogenes strain is deficient in at least two genes selected
from the group consisting of virulence genes, metabolic genes and
combinations thereof.
169. The method of claim 165, wherein the virulence gene is
selected from the group consisting of actA gene, inlA gene, inlB
gene, inlC gene, inlJ gene, bsh gene, prfA gene, plbC gene, plcA
gene, plcB gene, and combinations thereof, and wherein the
metabolic gene is selected from the group consisting of dal gene,
dat gene and combinations thereof.
170. The method of claim 165, wherein the virulence gene is
actA.
171. The method of claim 165, wherein the metabolic gene is
selected from the group consisting of dal gene, dat gene and
combinations thereof.
172. The method of claim 165, wherein the live-attenuated Listeria
monocytogenes strain is deficient in at least two virulence
genes.
173. The method of claim 165, wherein the PD-1 antagonist is an
anti-PD-1 monoclonal antibody.
174. The method of claim 173, wherein an anti-PD-1 monoclonal
antibody comprises a heavy chain and a light chain, wherein the
heavy and light chains comprise SEQ ID NO:21 and SEQ ID NO:22,
respectively.
175. The method of claim 165, wherein the PD-1 antagonist is
pembrolizumab.
176. The method of claim 165, wherein the PD-1 antagonist and the
live-attenuated Listeria monocytogenes strain are administered
simultaneously.
177. The method of claim 165, wherein the PD-1 antagonist and the
live-attenuated Listeria monocytogenes strain are administered
sequentially.
178. The method of claim 165, wherein said strain is administered
with an adjuvant.
179. The method of claim 178, wherein said adjuvant comprises
Montanide ISA 51, GM-CSF, KLH, a cytokine, a growth factor, a cell
population, QS21, Freund's incomplete adjuvant, aluminum phosphate,
aluminum hydroxide, BCG, alum, an interleukin, an unmethylated CpG
oligonucleotide, quill glycosides, monophosphoryl lipid A, a
liposomes, a bacterial mitogen, a bacterial toxin, or a chemokine,
or any combination thereof.
180. The method of claim 165, wherein the prostate cancer is
metastatic Castration-Resistant Prostate Cancer (mCRPC).
181. A kit comprising: a first container comprising at least one
dose of a medicament comprising an antagonist of a Programmed Death
1 protein (PD-1); a second container comprising at least one dose
of a medicament comprising a bioengineered live-attenuated Listeria
monocytogenes strain deficient in at least one gene selected from
the group consisting of a virulence gene, a metabolic gene and
combinations thereof; and a package insert comprising instructions
for treating a patient for prostate cancer using the
medicaments.
182. The kit of claim 181, wherein the second container comprises
at least one dose of a medicament comprising a bioengineered
live-attenuated Listeria monocytogenes comprising an LmddA-143
(10403 S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3 fused to
the hly gene in the chromosome) strain.
183. The kit of claim 181, wherein the second container comprises
at least one dose of a medicament comprising a bioengineered
live-attenuated Listeria monocytogenes comprising an LmddA-142
(10403 S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain.
184. A kit comprising: a first container comprising at least one
dose of a medicament comprising an antagonist of a Programmed Death
1 protein (PD-1); a second container comprising at least one dose
of a medicament comprising a bioengineered live-attenuated Listeria
monocytogenes strain transformed with an expression vector to
express a PSA antigen fused to a truncated Listeriolysin O (tLLO);
and a package insert comprising instructions for treating a patient
for prostate cancer using the medicaments.
185. The kit of claim 184, wherein the PD-1 antagonist is an
anti-PD-1 monoclonal antibody comprising a heavy chain and a light
chain, wherein the heavy and light chains comprise SEQ ID NO:21 and
SEQ ID NO:22, respectively.
186. The kit of claim 184, wherein said tLLO-PSA fusion polypeptide
consists of the sequence of SEQ ID NO: 54 or a sequence at least
99% homologous thereto wherein said N-terminal LLO peptide enhances
the immunogenicity of the fusion peptide.
187. The kit of claim 184, wherein said kit further comprises an
adjuvant, wherein said adjuvant comprises Montanide ISA 51, GM-CSF,
KLH, a cytokine, a growth factor, a cell population, QS21, Freund's
incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG,
alum, an interleukin, an unmethylated CpG oligonucleotide, quill
glycosides, monophosphoryl lipid A, a liposomes, a bacterial
mitogen, a bacterial toxin or a chemokine or any combination
thereof.
188. The kit of claim 184, wherein the prostate cancer is
metastatic Castration-Resistant Prostate Cancer (mCRPC).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 62/026,221, filed Jul. 18, 2014 and U.S.
Provisional Patent Application No. 62/039,011, filed Aug. 19, 2014,
both of which are incorporated in their entirety herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to combination therapies
useful for the treatment of cancer. In particular, the invention
relates to the treatment of prostate cancer using an antagonist of
a Programmed Death 1 protein (PD-1) in combination with a live
attenuated recombinant Listeria strain comprising a fusion protein
of a PEST sequence-containing polypeptide or PEST-sequence
containing peptide fused to a tumor-associated antigen.
BACKGROUND OF THE INVENTION
[0003] PD-1 is recognized as an important player in immune
regulation and the maintenance of peripheral tolerance. PD-1 is
moderately expressed on naive T, B and NKT cells and up-regulated
by T/B cell receptor signaling on lymphocytes, monocytes and
myeloid cells (1).
[0004] Two known ligands for PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC),
are expressed in human cancers arising in various tissues. In large
sample sets of e.g. ovarian, renal, colorectal, pancreatic, liver
cancers and melanoma, it was shown that PD-L1 expression correlated
with poor prognosis and reduced overall survival irrespective of
subsequent treatment (2-13). Similarly, PD-1 expression on tumor
infiltrating lymphocytes was found to mark dysfunctional T cells in
breast cancer and melanoma (14-15) and to correlate with poor
prognosis in renal cancer (16). Thus, it has been proposed that
PD-L1 expressing tumor cells interact with PD-1 expressing T cells
to attenuate T cell activation and evasion of immune surveillance,
thereby contributing to an impaired immune response against the
tumor.
[0005] Several monoclonal antibodies that inhibit the interaction
between PD-1 and one or both of its ligands PD-L1 and PD-L2 are in
clinical development for treating cancer. It has been proposed that
the efficacy of such antibodies might be enhanced if administered
in combination with other approved or experimental cancer
therapies, e.g., radiation, surgery, chemotherapeutic agents,
targeted therapies, agents that inhibit other signaling pathways
that are disregulated in tumors, and other immune enhancing
agents.
[0006] Listeria monocytogenes (Lm) is a Gram-positive facultative
intracellular pathogen that causes listeriolysis. Once invading a
host cell, Lm can escape from the phagolysosome through production
of a pore-forming protein listeriolysin O (LLO) to lyse the
vascular membrane, allowing it to enter the cytoplasm, where it
replicates and spreads to adjacent cells based on the mobility of
actin-polymerizing protein (ActA). In the cytoplasm, Lm-secreting
proteins are degraded by the proteasome and processed into peptides
that associate with MHC class I molecules in the endoplasmic
reticulum.
SUMMARY OF THE INVENTION
[0007] In one embodiment, the invention provides a method for
treating a prostate cancer in a human individual comprising
administering to the individual a combination therapy which
comprises a PD-1 antagonist and a live-attenuated bacterial strain
that is used to stimulate Antigen Presenting Cells (APCs) capable
of driving a cellular immune response to Prostate Specific Antigen
(PSA) expressing cells.
[0008] In another embodiment, the invention provides a method for
treating a prostate cancer in a human individual comprising
administering to the individual a combination therapy which
comprises a PD-1 antagonist and a live-attenuated Listeria
monocytogenes strain bioengineered, by transforming it with an
expression vector to express a PSA antigen fused to a truncated
Listeriolysin O (tLLO).
[0009] In yet another embodiment, the invention provides a method
for treating a prostate cancer in a human individual comprising
administering to the individual a combination therapy, which
comprises a PD-1 antagonist and an LmddA-142 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) pADV142) strain.
[0010] In a further embodiment, the invention provides a method for
treating a prostate cancer in a human individual comprising
administering to the individual a combination therapy, which
comprises a PD-1 antagonist and an LmddA-143 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome) strain.
[0011] In another embodiment, the invention provides a medicament
comprising a PD-1 antagonist for use in combination with a
live-attenuated bacterial strain that is used to stimulate Antigen
Presenting Cells (APCs) capable of driving a cellular immune
response to PSA expressing cells, for treating a prostate cancer in
a patient.
[0012] In yet another embodiment, the invention provides a
medicament comprising a PD-1 antagonist for use in combination with
a live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a truncated Listeriolysin O (tLLO), for treating a
prostate cancer in a patient.
[0013] In still another embodiment, the invention provides a
medicament comprising a PD-1 antagonist for use in combination with
an LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain, for treating a prostate cancer in a patient. In a further
embodiment, the invention provides a medicament comprising a PD-1
antagonist for use in combination with an LmddA-143 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3 fused to the hly
gene in the chromosome) strain, for treating a prostate cancer in a
patient.
[0014] In yet another embodiment, the invention provides a
medicament comprising a live-attenuated bacterial strain that is
used to stimulate Antigen Presenting Cells (APCs) capable of
driving a cellular immune response to PSA expressing cells for use
in combination with a PD-1 antagonist for treating a prostate
cancer in a patient.
[0015] In yet another embodiment, the invention provides a
medicament comprising a live-attenuated Listeria monocytogenes
strain bioengineered, by transforming it with an expression vector
to express a PSA antigen fused to a truncated Listeriolysin O
(tLLO) for use in combination with a PD-1 antagonist for treating a
prostate cancer in a patient.
[0016] In another embodiment, the invention provides a medicament
comprising an LmddA-142 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) pADV142) strain for use in combination with a PD-1
antagonist for treating a prostate cancer in a patient.
[0017] In yet embodiment, the invention provides a medicament
comprising an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain for use in combination with a PD-1 antagonist for treating a
prostate cancer in a patient.
[0018] Other embodiments provide for use of a PD-1 antagonist in
the manufacture of medicament for treating a prostate cancer in a
human when administered in combination with a live-attenuated
bacterial strain that is used to stimulate Antigen Presenting Cells
(APCs) capable of driving a cellular immune response to PSA
expressing cells and use of a live-attenuated bacterial strain that
is used to stimulate Antigen Presenting Cells (APCs) capable of
driving a cellular immune response to PSA expressing cells in the
manufacture of a medicament for treating a prostate cancer in a
patient when administered in combination with a PD-1
antagonist.
[0019] Other embodiments provide for use of a PD-1 antagonist in
the manufacture of a medicament for treating a prostate cancer in a
human when administered in combination with a live-attenuated
Listeria monocytogenes strain bioengineered, by transforming it
with an expression vector to express a PSA antigen fused to a
truncated Listeriolysin O (tLLO) and use of a live-attenuated
Listeria monocytogenes strain bioengineered, by transforming it
with an expression vector to express a PSA antigen fused to a
truncated Listeriolysin O (tLLO) in the manufacture of a medicament
for treating a prostate cancer in a patient when administered in
combination with a PD-1 antagonist.
[0020] Other embodiments provide for use of a PD-1 antagonist in
the manufacture of medicament for treating a prostate cancer in a
human when administered in combination with an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain and use of an
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain in the manufacture of a medicament for treating a prostate
cancer in a patient when administered in combination with a PD-1
antagonist.
[0021] Other embodiments provide for use of a PD-1 antagonist in
the manufacture of medicament for treating a prostate cancer in a
human when administered in combination with an LmddA-143 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3 fused to the hly
gene in the chromosome) strain and use of an LmddA-143 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3 fused to the hly
gene in the chromosome) strain in the manufacture of a medicament
for treating a prostate cancer in a patient when administered in
combination with a PD-1 antagonist.
[0022] In a still further embodiment, the invention provides for
use of a PD-1 antagonist and a live-attenuated bacterial strain
that is used to stimulate Antigen Presenting Cells (APCs) capable
of driving a cellular immune response to PSA-expressing cells in
the manufacture of medicaments for treating a prostate cancer in a
patient. In some embodiments, the medicaments comprise a kit, and
the kit also comprises a package insert comprising instructions for
using the PD-1 antagonist in combination with an a live-attenuated
bacterial strain that is used to stimulate Antigen Presenting Cells
(APCs) capable of driving a cellular immune response to PSA
expressing cells to treat a prostate cancer in a patient.
[0023] In another embodiment, the invention provides for use of a
PD-1 antagonist and a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a truncated Listeriolysin O (tLLO)
in the manufacture of medicaments for treating a prostate cancer in
a patient. In some embodiments, the medicaments comprise a kit, and
the kit also comprises a package insert comprising instructions for
using the PD-1 antagonist in combination with a live-attenuated
Listeria monocytogenes strain bioengineered, by transforming it
with an expression vector to express a PSA antigen fused to a
truncated Listeriolysin O (tLLO) to treat a prostate cancer in a
patient.
[0024] In yet another embodiment, the invention provides for use of
a PD-1 antagonist and an LmddA-142 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) pADV142) strain in the manufacture of medicaments for
treating a prostate cancer in a patient. In some embodiments, the
medicaments comprise a kit, and the kit also comprises a package
insert comprising instructions for using the PD-1 antagonist in
combination with an LmddA-142 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) pADV142) strain to treat a prostate cancer in a
patient.
[0025] In still another embodiment, the invention provides for use
of a PD-1 antagonist and an LmddA-143 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome) strain in the manufacture of medicaments for treating a
prostate cancer in a patient. In some embodiments, the medicaments
comprise a kit, and the kit also comprises a package insert
comprising instructions for using the PD-1 antagonist in
combination with an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain to treat a prostate cancer in a patient.
[0026] In all of the above treatment method, medicaments and uses,
the PD-1 antagonist inhibits the binding of PD-L1 to PD-1, and
preferably also inhibits the binding of PD-L2 to PD-1. In some
embodiments of the above treatment method, medicaments and uses,
the PD-1 antagonist is a monoclonal antibody, or an antigen binding
fragment thereof, which specifically binds to PD-1 or to PD-L1 and
blocks the binding of PD-L1 to PD-1. In one embodiment, the PD-1
antagonist is an anti-PD-1 antibody which comprises a heavy chain
and a light chain, and wherein the heavy and light chains comprise
the amino acid sequences shown in FIG. 6 (SEQ ID NO:21 and SEQ ID
NO:22).
[0027] In all of the above embodiments of the treatment method,
medicaments and uses, the live-attenuated bacterial strain
comprises a recombinant Listeria that is an attenuated auxotrophic
strain. In one embodiment, the attenuated strain is Lm dal(-)dat(-)
(Lmdd). In another embodiment, the attenuated strains is Lm
dal(-)dat(-).DELTA.actA (LmddA). LmddA is based on a Listeria
strain vector which is attenuated due to the deletion of virulence
gene actA and retains a plasmid for expression of a trunctated LLO
(tLLO) fused to a PSA antigen polypeptide in vivo and in vitro by
complementation of dal gene. In some embodiments, the klk3 gene is
fused to the hly gene in the chromosome for expression of a
tLLO-PSA fusion polypeptide. In some of the above embodiments of
the treatment method, medicaments and uses, the Listeria strain is
a Listeria monocytogenes. In some of the above embodiments of the
treatment method, medicaments and uses, the PSA antigen is fused to
a truncated Listeriolysin O (tLLO). In one embodiment, the tLLO is
an N-terminal LLO protein fragment. In one embodiment, the
N-terminal LLO protein fragment and the PSA antigen are fused
directly to one another. In another embodiment, the N-terminal LLO
protein fragment and the PSA antigen are operably attached via a
linker peptide. In another embodiment, the N-terminal LLO protein
fragment and the PSA antigen are attached via a heterologous
peptide. In another embodiment, the N-terminal LLO protein fragment
is N-terminal to the PSA antigen. In another embodiment, the
N-terminal LLO protein fragment is the N-terminal-most portion of
the fusion protein. In another embodiment, a truncated LLO is
truncated at the C-terminal to arrive at an N-terminal LLO. In all
of the above embodiments of the treatment method, medicaments and
uses, PSA is a kallikrein serine protease (KLK3) secreted by
prostatic epithelial cells, which in one embodiment, is widely used
as a marker for prostate cancer. In some embodiments, PSA is the
full-length polypeptide. In other embodiment, PSA is a fraction of
the full-length polypeptide. In one embodiment, a PSA antigen is
encoded by the klk3 gene.
[0028] In some embodiments of the above treatment method,
medicaments and uses, the live-attenuated bacterial strain is a
dal.sup.(-) dat.sup.(-) actA.sup.(-) Listeria monocytogenes strain
that episomally expresses the tLLO-PSA fusion protein. In one
embodiment, the tLLO consists of about the first 441 AA of the LLO
protein. In another embodiment, the LLO fragment is a non-hemolytic
form of the LLO protein. In one embodiment, the PSA consists of a
full-length protein. In another embodiment, the PSA consists of
less than the full-length protein.
[0029] In some embodiments of the above treatment method,
medicaments and uses of the invention, the prostate cancer is
metastatic.
[0030] In other embodiments of the above treatment method,
medicaments and uses of the invention, the prostate cancer is
Castration-Resistant Prostate Cancer (mCRPC).
[0031] In still other embodiments of the above treatment method,
medicaments and uses of the invention, the patient has been
diagnosed with metastatic Castration-Resistant Prostate Cancer
(mCRPC) following treatment with at least one previous therapeutic
agent.
[0032] Also, in some embodiments of any of the above treatment
method, medicaments and uses, the prostate cancer tests positive
for the expression of one or both of PD-L1 and PD-L2. In some
embodiments, the prostate cancer has elevated PD-L1 expression.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows amino acid sequences of the light chain and
heavy chain CDRs for an exemplary anti-PD-1 monoclonal antibody
useful in the present invention (SEQ ID NOs:1-6).
[0034] FIG. 2 shows amino acid sequences of the light chain and
heavy chain CDRs for another exemplary anti-PD-1 monoclonal
antibody useful in the present invention (SEQ ID NOs:7-12).
[0035] FIG. 3 shows amino acid sequences of the heavy chain
variable region and full length heavy chain for an exemplary
anti-PD-1 monoclonal antibody useful in the present invention (SEQ
ID NO:13 and SEQ ID NO:14).
[0036] FIG. 4 shows amino acid sequences of alternative light chain
variable regions for an exemplary anti-PD-1 monoclonal antibody
useful in the present invention (SEQ ID NOs:15-17).
[0037] FIGS. 5A and 5B show amino acid sequences of alternative
light chains for an exemplary anti-PD-1 monoclonal antibody useful
in the present invention (SEQ ID NOs:18-20). FIG. 5A shows the full
length sequence of K09A-L-11 light chain (SEQ ID NO: 18) and
K09A-L-16 light chain (SEQ ID NO: 19). FIG. 5B shows the full
length sequence of K09A-L-17 light chain (SEQ ID NO: 20).
[0038] FIG. 6 shows amino acid sequences of the heavy and light
chains for MK-3475 (SEQ ID NOs. 21 and 22, respectively).
[0039] FIG. 7 shows amino acid sequences of the heavy and light
chains for nivolumab (SEQ ID NOs. 23 and 24, respectively).
[0040] FIG. 8 shows a schematic representation of the chromosomal
region of the Lmdd-143 and LmddA-143 after klk3 integration and
actA deletion;
[0041] FIGS. 9A-9C. FIG. 9A shows a map of the pADV134 plasmid.
FIGS. 9B and 9C show a map of the antibiotic-independent episomal
expression vector for PSA delivery, pADV142 plasmid, wherein the
antigen expression cassette consists of of a hly promoter and
LLO-PSA fusion protein (FIG. 9B) and the nucleotide sequence of
pADV142 plasmid, wherein the underline section encodes a Homo
sapiens kallikrein 3, (prostate specific antigen) (FIG. 9C).
[0042] FIG. 10 shows a diagram of the two parts of the Phase 1-2
study designed to evaluate safety and tolerability of ADXS31-142
(Listeria monocytogenes (10403S dal dat.sup.(-) actA.sup.(-)
pADV142)) as monotherapy and in combination with pembrolizumab
(MK-3475) in subjects with mCRPC.
DETAILED DESCRIPTION
[0043] Abbreviations. Throughout the detailed description and
examples of the invention the following abbreviations will be
used:
[0044] APC antigen presenting cell
[0045] BID One dose twice daily
[0046] CBD Cholesterol Binding Domain
[0047] CDR Complementarity determining region
[0048] CFU Colony-forming units
[0049] CHO Chinese hamster ovary
[0050] DFS Disease free survival
[0051] DTR Dose limiting toxicity
[0052] FFPE formalin-fixed, paraffin-embedded
[0053] FR Framework region
[0054] IgG Immunoglobulin G
[0055] IHC Immunohistochemistry or immunohistochemical
[0056] KLK3 Kallikrein-related peptidase 3; also known as APS; PSA;
hK3; KLK2A1
[0057] LLO Listeriolysin O polypeptide
[0058] tLLO truncated Listeriolysin O polypeptide
[0059] Lm Listeria monocytogenes
[0060] LmddA-142 Listeria monocytogenes (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) pADV142); also known as ADXS31-142
[0061] LmddA-143 Listeria monocytogenes (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome)
[0062] MTD Maximum tolerated dose
[0063] NCBI National Center for Biotechnology Information
[0064] NCI National Cancer Institute
[0065] OR Overall response
[0066] ORF Open reading frame
[0067] OS Overall survival
[0068] PCR Polymerase chain reaction
[0069] PD Progressive disease
[0070] PSA Prostate specific antigen
[0071] PFS Progression free survival
[0072] PR Partial response
[0073] Q2W One dose every two weeks
[0074] Q3W One dose every three weeks
[0075] Q4W One dose every four weeks
[0076] QD One dose per day
[0077] RECIST Response Evaluation Criteria in Solid Tumors
[0078] SD Stable disease
[0079] SDS-PAGE Sodium dodecyl sulfate-Polyacrylamide gel
electrophoresis
[0080] TILs Tumor infiltrating lymphocytes
[0081] VH Immunoglobulin heavy chain variable region
[0082] VK Immunoglobulin kappa light chain variable region
I. Definitions
[0083] So that the invention may be more readily understood,
certain technical and scientific terms are specifically defined
below. Unless specifically defined elsewhere in this document, all
other technical and scientific terms used herein have the meaning
commonly understood by one of ordinary skill in the art to which
this invention belongs.
[0084] "About" when used to modify a numerically defined parameter
(e.g., the dose of a PD-1 antagonist or a live-attenuated bacterial
strain that is used to stimulate Antigen Presenting Cells (APCs)
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a truncated Listeriolysin O (tLLO)
or an LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-)
pADV142) strain or an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain, or the length of treatment time with a combination therapy
described herein) means that the parameter may vary by as much as
10% below or above the stated numerical value for that parameter.
For example, a dose of about 200 mg of the PD-1 antagonist, i.e.,
MK-3475, may vary between 180 mg and 220 mg.
[0085] As used herein, including the appended claims, the singular
forms of words such as "a," "an," and "the," include their
corresponding plural references unless the context clearly dictates
otherwise.
[0086] "Administration" and "treatment," as it applies to an
animal, human, experimental subject, cell, tissue, organ, or
biological fluid, refers to contact of an exogenous pharmaceutical,
therapeutic, diagnostic agent, or composition to the animal, human,
subject, cell, tissue, organ, or biological fluid. Treatment of a
cell encompasses contact of a reagent to the cell, as well as
contact of a reagent to a fluid, where the fluid is in contact with
the cell. "Administration" and "treatment" also means in vitro and
ex vivo treatments, e.g., of a cell, by a reagent, diagnostic,
binding compound, or by another cell. The term "subject" includes
any organism, preferably an animal, more preferably a mammal (e.g.,
rat, mouse, dog, cat, rabbit) and most preferably a human.
[0087] The term "pharmaceutically acceptable carrier" refers to any
inactive substance that is suitable for use in a formulation for
the administration to a human of a PD-1 antagonist or a
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain.
[0088] As used herein, the term "antibody" refers to any form of
immunoglobulin molecule that exhibits the desired biological or
binding activity. Thus, it is used in the broadest sense and
specifically covers, but is not limited to, monoclonal antibodies
(including full length monoclonal antibodies), polyclonal
antibodies, multispecific antibodies (e.g., bispecific antibodies),
humanized, human antibodies, chimeric antibodies and camelized
single domain antibodies. "Parental antibodies" are antibodies
obtained by exposure of an immune system to an antigen prior to
modification of the antibodies for an intended use, such as
humanization of an antibody for use as a human therapeutic. As used
herein, the term "antibody" encompasses not only intact polyclonal
or monoclonal antibodies, but also, unless otherwise specified, any
antigen binding portion thereof that competes with the intact
antibody for specific binding, fusion proteins comprising an
antigen binding portion, and any other modified configuration of
the immunoglobulin molecule that comprises an antigen recognition
site. Antigen binding portions include, for example, Fab, Fab',
F(ab').sub.2, Fd, Fv, domain antibodies (dAbs, e.g., shark and
camelid antibodies), fragments including complementarity
determining regions (CDRs), single chain variable fragment
antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies,
triabodies, tetrabodies, v-NAR and bis-scFv, and polypeptides that
contain at least a portion of an immunoglobulin that is sufficient
to confer specific antigen binding to the polypeptide. An antibody
includes an antibody of any class, such as IgG, IgA, or IgM (or
sub-class thereof), and the antibody need not be of any particular
class. Depending on the antibody amino acid sequence of the
constant region of its heavy chains, immunoglobulins can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1 and IgA.sub.2. The
heavy-chain constant regions that correspond to the different
classes of immunoglobulins are called alpha, delta, epsilon, gamma,
and mu, respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0089] "Variable regions" or "V region" as used herein means the
segment of IgG chains which is variable in sequence between
different antibodies. It extends to Kabat residue 109 in the light
chain and 113 in the heavy chain. A "variable region" of an
antibody refers to the variable region of the antibody light chain
or the variable region of the antibody heavy chain, either alone or
in combination. Typically, the variable regions of both the heavy
and light chains comprise three hypervariable regions, also called
complementarity determining regions (CDRs), which are located
within relatively conserved framework regions (FR). The CDRs are
usually aligned by the framework regions, enabling binding to a
specific epitope. In general, from N-terminal to C-terminal, both
light and heavy chains variable domains comprise FR1, CDR1, FR2,
CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each
domain is, generally, in accordance with the definitions of
Sequences of Proteins of Immunological Interest, Kabat, et al.;
National Institutes of Health, Bethesda, Md.; 5.sup.th ed.; NIH
Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32:1-75;
Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et
al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989)
Nature 342:878-883.
[0090] As used herein, the term "hypervariable region" refers to
the amino acid residues of an antibody that are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (i.e.
CDRL1, CDRL2 and CDRL3 in the light chain variable domain and
CDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). See
Kabat et al. (1991) Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (defining the CDR regions of an antibody by
sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196:
901-917 (defining the CDR regions of an antibody by structure). As
used herein, the term "framework" or "FR" residues refers to those
variable domain residues other than the hypervariable region
residues defined herein as CDR residues.
[0091] As used herein, unless otherwise indicated, "antibody
fragment" or "antigen binding fragment" refers to antigen binding
fragments of antibodies, i.e. antibody fragments that retain the
ability to bind specifically to the antigen bound by the
full-length antibody, e.g. fragments that retain one or more CDR
regions. Examples of antibody binding fragments include, but are
not limited to, Fab, Fab', F(ab').sub.2, and Fv fragments;
diabodies; linear antibodies; single-chain antibody molecules,
e.g., sc-Fv; nanobodies and multispecific antibodies formed from
antibody fragments.
[0092] An antibody that "specifically binds to" a specified target
protein is an antibody that exhibits preferential binding to that
target as compared to other proteins, but this specificity does not
require absolute binding specificity. An antibody is considered
"specific" for its intended target if its binding is determinative
of the presence of the target protein in a sample, e.g. without
producing undesired results such as false positives. Antibodies, or
binding fragments thereof, useful in the present invention will
bind to the target protein with an affinity that is at least two
fold greater, preferably at least ten times greater, more
preferably at least 20-times greater, and most preferably at least
100-times greater than the affinity with non-target proteins. As
used herein, an antibody is said to bind specifically to a
polypeptide comprising a given amino acid sequence, e.g. the amino
acid sequence of a mature human PD-1 or human PD-L1 molecule, if it
binds to polypeptides comprising that sequence but does not bind to
proteins lacking that sequence.
[0093] "Chimeric antibody" refers to an antibody in which a portion
of the heavy and/or light chain is identical with or homologous to
corresponding sequences in an antibody derived from a particular
species (e.g., human) or belonging to a particular antibody class
or subclass, while the remainder of the chain(s) is identical with
or homologous to corresponding sequences in an antibody derived
from another species (e.g., mouse) or belonging to another antibody
class or subclass, as well as fragments of such antibodies, so long
as they exhibit the desired biological activity.
[0094] "Human antibody" refers to an antibody that comprises human
immunoglobulin protein sequences only. A human antibody may contain
murine carbohydrate chains if produced in a mouse, in a mouse cell,
or in a hybridoma derived from a mouse cell. Similarly, "mouse
antibody" or "rat antibody" refer to an antibody that comprises
only mouse or rat immunoglobulin sequences, respectively.
[0095] "Humanized antibody" refers to forms of antibodies that
contain sequences from non-human (e.g., murine) antibodies as well
as human antibodies. Such antibodies contain minimal sequence
derived from non-human immunoglobulin. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the hypervariable loops correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin sequence. The humanized antibody
optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. The prefix "hum", "hu" or "h" is added to antibody
clone designations when necessary to distinguish humanized
antibodies from parental rodent antibodies. The humanized forms of
rodent antibodies will generally comprise the same CDR sequences of
the parental rodent antibodies, although certain amino acid
substitutions may be included to increase affinity, increase
stability of the humanized antibody, or for other reasons.
[0096] The terms "cancer", "cancerous", or "malignant" refer to or
describe the physiological condition in mammals that is typically
characterized by unregulated cell growth.
[0097] "Biotherapeutic agent" means a biological molecule, such as
an antibody or fusion protein, that blocks ligand/receptor
signaling in any biological pathway that supports tumor maintenance
and/or growth or suppresses the anti-tumor immune response.
[0098] "CDR" or "CDRs" as used herein means complementarity
determining region(s) in a immunoglobulin variable region, defined
using the Kabat numbering system, unless otherwise indicated.
[0099] "Chemotherapeutic agent" refers to a chemical or biological
substance that can cause death of cancer cells, or interfere with
growth, division, repair, and/or function of cancer cells. Classes
of chemotherapeutic agents include, but are not limited to:
alkylating agents, antimetabolites, kinase inhibitors, spindle
poison plant alkaloids, cytoxic/antitumor antibiotics, topisomerase
inhibitors, photosensitizers, anti-estrogens and selective estrogen
receptor modulators (SERMs), anti-progesterones, estrogen receptor
down-regulators (ERDs), estrogen receptor antagonists, leutinizing
hormone-releasing hormone agonists, anti-androgens, aromatase
inhibitors, EGFR inhibitors, VEGF inhibitors, anti-sense
oligonucleotides that inhibit expression of genes implicated in
abnormal cell proliferation or tumor growth. Chemotherapeutic
agents useful in the treatment methods of the present invention
include cytostatic and/or cytotoxic agents.
[0100] The antibodies and compositions provided by the present
disclosure can be administered via any suitable enteral route or
parenteral route of administration. The term "enteral route" of
administration refers to the administration via any part of the
gastrointestinal tract. Examples of enteral routes include oral,
mucosal, buccal, and rectal route, or intragastric route.
"Parenteral route" of administration refers to a route of
administration other than enteral route. Examples of parenteral
routes of administration include intravenous, intramuscular,
intradermal, intraperitoneal, intratumor, intravesical,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, transtracheal, intraarticular, subcapsular,
subarachnoid, intraspinal, epidural and intrastemal, subcutaneous,
or topical administration. The antibodies and compositions of the
disclosure can be administered using any suitable method, such as
by oral ingestion, nasogastric tube, gastrostomy tube, injection,
infusion, implantable infusion pump, and osmotic pump. The suitable
route and method of administration may vary depending on a number
of factors such as the specific antibody being used, the rate of
absorption desired, specific formulation or dosage form used, type
or severity of the disorder being treated, the specific site of
action, and conditions of the patient, and can be readily selected
by a person skilled in the art.
[0101] The term "simultaneous administration" as used herein in
relation to the administration of medicaments refers to the
administration of medicaments such that the individual medicaments
are present within a subject at the same time. In addition to the
concomitant administration of medicaments (via the same or
alternative routes), simultaneous administration may include the
administration of the medicaments (via the same or an alternative
route) at different times.
[0102] The Bliss independence combined response C for two single
compounds with effects A and B is C=A+B-A*B, where each effect is
expressed as a fractional inhibition between 0 and 1. (Reference:
Bliss (1939) Annals of Applied Biology) The Bliss value, defined to
be the difference between the experimental response and the
calculated Bliss Independence value, indicates whether the two
compounds in combination are additive or synergistic.
[0103] A Bliss value of zero (0) is considered additive. The term
"additive" means that the result of the combination of the two
targeted agents is the sum of each agent individually.
[0104] "Chothia" as used herein means an antibody numbering system
described in Al-Lazikani et al., JMB 273:927-948 (1997).
[0105] "Conservatively modified variants" or "conservative
substitution" refers to substitutions of amino acids in a protein
with other amino acids having similar characteristics (e.g. charge,
side-chain size, hydrophobicity/hydrophilicity, backbone
conformation and rigidity, etc.), such that the changes can
frequently be made without altering the biological activity or
other desired property of the protein, such as antigen affinity
and/or specificity. Those of skill in this art recognize that, in
general, single amino acid substitutions in non-essential regions
of a polypeptide do not substantially alter biological activity
(see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The
Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition,
substitutions of structurally or functionally similar amino acids
are less likely to disrupt biological activity. Exemplary
conservative substitutions are set forth in Table 1 below.
TABLE-US-00001 TABLE 1 Exemplary Conservative Amino Acid
Substitutions Original residue Conservative substitution Ala (A)
Gly; Ser Arg (R) Lys; His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C)
Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln
Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu;
Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser
Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu
[0106] "Consists essentially of," and variations such as "consist
essentially of" or "consisting essentially of," as used throughout
the specification and claims, indicate the inclusion of any recited
elements or group of elements, and the optional inclusion of other
elements, of similar or different nature than the recited elements,
that do not materially change the basic or novel properties of the
specified dosage regimen, method, or composition. As a non-limiting
example, an antibody that consists essentially of a recited amino
acid sequence may also include the addition and/or substitution of
one or more amino acids that do not materially affect the
properties of the antibody.
[0107] "Diagnostic anti-PD-L monoclonal antibody" means a mAb which
specifically binds to the mature form of the designated PD-L (PD-L1
or PDL2) that is expressed on the surface of certain mammalian
cells. A mature PD-L lacks the presecretory leader sequence, also
referred to as leader peptide The terms "PD-L" and "mature PD-L"
are used interchangeably herein, and shall be understood to mean
the same molecule unless otherwise indicated or readily apparent
from the context.
[0108] As used herein, a diagnostic anti-human PD-L1 mAb or an
anti-hPD-L1 mAb refers to a monoclonal antibody that specifically
binds to mature human PD-L1. Specific examples of diagnostic
anti-human PD-L1 mAbs useful as diagnostic mAbs for
immunohistochemistry (IHC) detection of PD-L1 expression in
formalin-fixed, paraffin-embedded (FFPE) tumor tissue sections are
antibody 20C3 and antibody 22C3, which are described in
WO2014/100079. Another anti-human PD-L1 mAb that has been reported
to be useful for IHC detection of PD-L1 expression in FFPE tissue
sections (Chen, B. J. et al., Clin Cancer Res 19: 3462-3473 (2013))
is a rabbit anti-human PD-L1 mAb publicly available from Sino
Biological, Inc. (Beijing, P.R. China; Catalog number
10084-R015).
[0109] "Framework region" or "FR" as used herein means the
immunoglobulin variable regions excluding the CDR regions.
[0110] "Homology" refers to sequence similarity between two
polypeptide sequences when they are optimally aligned. When a
position in both of the two compared sequences is occupied by the
same amino acid monomer subunit, e.g., if a position in a light
chain CDR of two different Abs is occupied by alanine, then the two
Abs are homologous at that position. The percent of homology is the
number of homologous positions shared by the two sequences divided
by the total number of positions compared .times.100. For example,
if 8 of 10 of the positions in two sequences are matched or
homologous when the sequences are optimally aligned then the two
sequences are 80% homologous. Generally, the comparison is made
when two sequences are aligned to give maximum percent homology.
For example, the comparison can be performed by a BLAST algorithm
wherein the parameters of the algorithm are selected to give the
largest match between the respective sequences over the entire
length of the respective reference sequences.
[0111] The following references relate to BLAST algorithms often
used for sequence analysis: BLAST ALGORITHMS: Altschul, S. F., et
al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993)
Nature Genet. 3:266-272; Madden, T. L., et al., (1996) Meth.
Enzymol. 266:131-141; Altschul, S. F., et al., (1997) Nucleic Acids
Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656;
Wootton, J. C., et al., (1993) Comput. Chem. 17:149-163; Hancock,
J. M. et al., (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT
SCORING SYSTEMS: Dayhoff, M. O., et al., "A model of evolutionary
change in proteins." in Atlas of Protein Sequence and Structure,
(1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, Natl.
Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al.,
"Matrices for detecting distant relationships." in Atlas of Protein
Sequence and Structure, (1978) vol. 5, suppl. 3.'' M. O. Dayhoff
(ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.;
Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J.,
et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc.
Natl. Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al.,
(1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S.,
et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S.,
et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A.,
et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S. F.
"Evaluating the statistical significance of multiple distinct local
alignments." in Theoretical and Computational Methods in Genome
Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, N.Y.
[0112] "Isolated antibody" and "isolated antibody fragment" refers
to the purification status and in such context means the named
molecule is substantially free of other biological molecules such
as nucleic acids, proteins, lipids, carbohydrates, or other
material such as cellular debris and growth media. Generally, the
term "isolated" is not intended to refer to a complete absence of
such material or to an absence of water, buffers, or salts, unless
they are present in amounts that substantially interfere with
experimental or therapeutic use of the binding compound as
described herein.
[0113] "Kabat" as used herein means an immunoglobulin alignment and
numbering system pioneered by Elvin A. Kabat ((1991) Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md.).
[0114] "Monoclonal antibody" or "mAb" or "Mab", as used herein,
refers to a population of substantially homogeneous antibodies,
i.e., the antibody molecules comprising the population are
identical in amino acid sequence except for possible naturally
occurring mutations that may be present in minor amounts. In
contrast, conventional (polyclonal) antibody preparations typically
include a multitude of different antibodies having different amino
acid sequences in their variable domains, particularly their CDRs,
which are often specific for different epitopes. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, a monoclonal antibody to be
used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al. (1975) Nature
256: 495, or may be made by recombinant DNA methods (see, e.g.,
U.S. Pat. No. 4,816,567). The monoclonal antibody may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al. (1991) Nature 352: 624-628 and Marks
et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also
Presta (2005) J. Allergy Clin. Immunol. 116:731.
[0115] "Patient" or "subject" refers to any single human subject
for which therapy is desired or that is participating in a clinical
trial, epidemiological study or used as a control.
[0116] "PD-1 antagonist" means any chemical compound or biological
molecule that blocks binding of human PD-L1 expressed on a cancer
cell to human PD-1 expressed on an immune cell (T cell, B cell or
NKT cell) and preferably also blocks binding of human PD-L2
expressed on a cancer cell to the immune-cell expressed PD-1.
Alternative names or synonyms for PD-1 and its ligands include:
PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4,
CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273
for PD-L2. Exemplary human PD-1 amino acid sequences can be found
in NCBI Locus No.: NP_005009. Exemplary human PD-L1 and PD-L2 amino
acid sequences can be found in NCBI Locus No.: NP_054862 and
NP_079515, respectively.
[0117] PD-1 antagonists useful in the any of the treatment method,
medicaments and uses of the present invention include a monoclonal
antibody (mAb), or antigen binding fragment thereof, which
specifically binds to human PD-1 or human PD-L1. The mAb may be a
human antibody, a humanized antibody or a chimeric antibody, and
may include a human constant region. In some embodiments, the human
constant region is selected from the group consisting of IgG1,
IgG2, IgG3 and IgG4 constant regions, and in some embodiments, the
human constant region is an IgG1 or IgG4 constant region. In some
embodiments, the antigen binding fragment is selected from the
group consisting of Fab, Fab'-SH, F(ab').sub.2, scFv and Fv
fragments.
[0118] Examples of mAbs that bind to human PD-1, and useful in the
treatment method, medicaments and uses of the present invention,
are described in U.S. Pat. No. 7,488,802, U.S. Pat. No. 7,521,051,
U.S. Pat. No. 8,008,449, U.S. Pat. No. 8,354,509, U.S. Pat. No.
8,168,757, WO2004/004771, WO2004/072286, WO2004/056875, and
US2011/0271358. Specific anti-human PD-1 mAbs useful as the PD-1
antagonist in the treatment method, medicaments and uses of the
present invention include:MK-3475, a humanized IgG4 mAb with the
structure described in WHO Drug Information, Vol. 27, No. 2, pages
161-162 (2013) and which comprises the heavy and light chain amino
acid sequences shown in FIG. 6, nivolumab (BMS-936558), a human
IgG4 mAb with the structure described in WHO Drug Information, Vol.
27, No. 1, pages 68-69 (2013) and which comprises the heavy and
light chain amino acid sequences shown in FIG. 7; the humanized
antibodies h409A11, h409A16 and h409A17, which are described in
WO2008/156712, and AMP-514, which is being developed by
MedImmune.
[0119] Examples of mAbs that bind to human PD-L1, and useful in the
treatment method, medicaments and uses of the present invention,
are described in WO2013/019906, WO2010/077634 A1 and U.S. Pat. No.
8,383,796. Specific anti-human PD-L1 mAbs useful as the PD-1
antagonist in the treatment method, medicaments and uses of the
present invention include MPDL3280A, BMS-936559, MEDI4736,
MSB0010718C and an antibody which comprises the heavy chain and
light chain variable regions of SEQ ID NO:24 and SEQ ID NO:21,
respectively, of WO2013/019906.
[0120] Other PD-1 antagonists useful in the any of the treatment
method, medicaments and uses of the present invention include an
immunoadhesin that specifically binds to human PD-1 or human PD-L1,
e.g., a fusion protein containing the extracellular or PD-1 binding
portion of PD-L1 or PD-L2 fused to a constant region such as an Fc
region of an immunoglobulin molecule. Examples of immunoadhesion
molecules that specifically bind to PD-1 are described in
WO2010/027827 and WO2011/066342. Specific fusion proteins useful as
the PD-1 antagonist in the treatment method, medicaments and uses
of the present invention include AMP-224 (also known as B7-DCIg),
which is a PD-L2-FC fusion protein and binds to human PD-1.
[0121] In some embodiments of the treatment method, medicaments and
uses of the present invention, the PD-1 antagonist is a monoclonal
antibody, or antigen binding fragment thereof, which comprises: (a)
light chain CDRs SEQ ID NOs: 1, 2 and 3 and heavy chain CDRs SEQ ID
NOs: 4, 5 and 6; or (b) light chain CDRs SEQ ID NOs: 7, 8 and 9 and
heavy chain CDRs SEQ ID NOs: 10, 11 and 12.
[0122] In other embodiments of the treatment method, medicaments
and uses of the present invention, the PD-1 antagonist is a
monoclonal antibody, or antigen binding fragment thereof, which
specifically binds to human PD-1 and comprises (a) a heavy chain
variable region comprising SEQ ID NO:13 or a variant thereof, and
(b) a light chain variable region comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 15 or a variant
thereof; SEQ ID NO:16 or a variant thereof; and SEQ ID NO: 17 or a
variant thereof. A variant of a heavy chain variable region
sequence is identical to the reference sequence except having up to
17 conservative amino acid substitutions in the framework region
(i.e., outside of the CDRs), and preferably has less than ten,
nine, eight, seven, six or five conservative amino acid
substitutions in the framework region. A variant of a light chain
variable region sequence is identical to the reference sequence
except having up to five conservative amino acid substitutions in
the framework region (i.e., outside of the CDRs), and preferably
has less than four, three or two conservative amino acid
substitution in the framework region.
[0123] In another embodiment of the treatment method, medicaments
and uses of the present invention, the PD-1 antagonist is a
monoclonal antibody which specifically binds to human PD-1 and
comprises (a) a heavy chain comprising SEQ ID NO: 14 and (b) a
light chain comprising SEQ ID NO:18, SEQ ID NO:19 or SEQ ID
NO:20.
[0124] In yet another embodiment of the treatment method,
medicaments and uses of the present invention, the PD-1 antagonist
is a monoclonal antibody which specifically binds to human PD-1 and
comprises (a) a heavy chain comprising SEQ ID NO: 14 and (b) a
light chain comprising SEQ ID NO: 18.
[0125] Table 2 below provides a list of the amino acid sequences of
exemplary anti-PD-1 mAbs for use in the treatment method,
medicaments and uses of the present invention, and the sequences
are shown in FIGS. 1-5.
TABLE-US-00002 TABLE 2 EXEMPLARY ANTI-HUMAN PD-1 MONOCLONAL
ANTIBODIES A. Comprises light and heavy chain CDRs of hPD-1.08A in
WO2008/156712 CDRL1 SEQ ID NO: 1 CDRL2 SEQ ID NO: 2 CDRL3 SEQ ID
NO: 3 CDRH1 SEQ ID NO: 4 CDRH2 SEQ ID NO: 5 CDRH3 SEQ ID NO: 6 B.
Comprises light and heavy chain CDRs of hPD-1.09A in WO2008/156712
CDRL1 SEQ ID NO: 7 CDRL2 SEQ ID NO: 8 CDRL3 SEQ ID NO: 9 CDRH1 SEQ
ID NO: 10 CDRH2 SEQ ID NO: 11 CDRH3 SEQ ID NO: 12 C. Comprises the
mature h109A heavy chain variable region and one of the mature K09A
light chain variable regions in WO2008/156712 Heavy chain VR SEQ ID
NO: 13 Light chain VR SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO:
17 D. Comprises the mature 409 heavy chain and one of the mature
K09A light chains in WO2008/156712 Heavy chain SEQ ID NO: 14 Light
chain SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20
[0126] "PD-L1" expression or "PD-L2" expression as used herein
means any detectable level of expression of the designated PD-L
protein on the cell surface or of the designated PD-L mRNA within a
cell or tissue. PD-L protein expression may be detected with a
diagnostic PD-L antibody in an IHC assay of a tumor tissue section
or by flow cytometry. Alternatively, PD-L protein expression by
tumor cells may be detected by PET imaging, using a binding agent
(e.g., antibody fragment, affibody and the like) that specifically
binds to the desired PD-L target, e.g., PD-L1 or PD-L2. Techniques
for detecting and measuring PD-L mRNA expression include RT-PCR and
realtime quantitative RT-PCR.
[0127] Several approaches have been described for quantifying PD-L1
protein expression in IHC assays of tumor tissue sections. See,
e.g., Thompson, R. H., et al., PNAS 101 (49); 17174-17179 (2004);
Thompson, R. H. et al., Cancer Res. 66:3381-3385 (2006); Gadiot,
J., et al., Cancer 117:2192-2201 (2011); Taube, J. M. et al., Sci
Transl Med 4, 127ra37 (2012); and Toplian, S. L. et al., New Eng. J
Med. 366 (26): 2443-2454 (2012).
[0128] One approach employs a simple binary end-point of positive
or negative for PD-L1 expression, with a positive result defined in
terms of the percentage of tumor cells that exhibit histologic
evidence of cell-surface membrane staining. A tumor tissue section
is counted as positive for PD-L1 expression if IHC staining is
observed in at least 1%, and preferably 5% of total tumor cells. In
an embodiment, a prostate tumor sample is designated as having weak
PD-L1 expression if 1% to 49% of the total tumor cells in the
sample exhibit membrane staining and is designated as having strong
PD-L1 expression if at least 50% of the tumor cells in the sample
exhibit membrane staining, in each case as determined by IHC assay
using the antibody 22C3 described in WO2014/100079.
[0129] In another approach, PD-L1 expression in the tumor tissue
section is quantified in the tumor cells as well as in infiltrating
immune cells, which predominantly comprise lymphocytes. The
percentage of tumor cells and infiltrating immune cells that
exhibit membrane staining are separately quantified as <5%, 5 to
9%, and then in 10% increments up to 100%. For tumor cells, PD-L1
expression is counted as negative if the score is <5% score and
positive if the score is .gtoreq.5%. PD-L1 expression in the immune
infiltrate is reported as a semi-quantitative measurement called
the adjusted inflammation score (AIS), which is determined by
multiplying the percent of membrane staining cells by the intensity
of the infiltrate, which is graded as none (0), mild (score of 1,
rare lymphocytes), moderate (score of 2, focal infiltration of
tumor by lymphohistiocytic aggregates), or severe (score of 3,
diffuse infiltration). A tumor tissue section is counted as
positive for PD-L1 expression by immune infiltrates if the AIS is
.gtoreq.5.
[0130] The level of PD-L mRNA expression may be compared to the
mRNA expression levels of one or more reference genes that are
frequently used in quantitative RT-PCR, such as ubiquitin C.
[0131] In some embodiments, a level of PD-L1 expression (protein
and/or mRNA) by malignant cells and/or by infiltrating immune cells
within a tumor is determined to be "overexpressed" or "elevated"
based on comparison with the level of PD-L1 expression (protein
and/or mRNA) by an appropriate control. For example, a control
PD-L1 protein or mRNA expression level may be the level quantified
in nonmalignant cells of the same type or in a section from a
matched normal tissue. In some embodiments, PD-L1 expression in a
tumor sample is determined to be elevated if PD-L1 protein (and/or
PD-L1 mRNA) in the sample is at least 10%, 20%, or 30% greater than
in the control.
[0132] "Prostate Cancer Working Group 2 (PCWG2) criteria" as used
herein has the meaning as presented in Scher et al., (2008) "Design
and End Points of Clinical Trials for Patients With Progressive
Prostate Cancer and Castrate Levels of Testosterone:
Recommendations of the Prostate Cancer Clinical Trials Working
Group" J. Clin. Oncol. 26(7): 1148-159, incorporated in its
entirety herein.
[0133] "RECIST 1.1 Response Criteria" as used herein means the
definitions set forth in Eisenhauer et al., E. A. et al., Eur. J
Cancer 45:228-247 (2009) for target lesions or nontarget lesions,
as appropriate based on the context in which response is being
measured.
[0134] "Sustained response" means a sustained therapeutic effect
after cessation of treatment with a therapeutic agent, or a
combination therapy described herein. In some embodiments, the
sustained response has a duration that is at least the same as the
treatment duration, or at least 1.5, 2.0, 2.5 or 3 times longer
than the treatment duration.
[0135] The terms "synergy" or "synergistic" are used to mean that
the response of the combination of the two agents is more than the
sum of each agent's individual response. More specifically, in the
in vitro setting one measure of synergy is known as "Bliss
synergy." Bliss synergy refers to "excess over Bliss independence",
as determined by the Bliss value defined above. When the Bliss
value is greater than zero (0), or more preferably greater than
0.2, it is considered indicative of synergy. Of course, the use of
"synergy" herein also encompasses in vitro synergy as measured by
additional and/or alternate methods. References herein to a
combination's in vitro biological effects, including but not
limited to anti-cancer effects, being greater than, or equal to,
the sum of the combination's components individually, may be
correlated to Bliss values. Again, the use of "synergy" herein,
including whether a combination of components demonstrates activity
equal to or greater than the sum of the components individually,
may be measured by additional and/or alternate methods and are
known, or will be apparent, to those skilled in this art. In one
embodiment, the combination of a Listeria based immunotherapy, as
described herein, with an anti-PD-1 antibody, as described herein,
provides synergistic antitumor activities.
[0136] "Tissue Section" refers to a single part or piece of a
tissue sample, e.g., a thin slice of tissue cut from a sample of a
normal tissue or of a tumor.
[0137] "Treat" or "treating" a cancer as used herein means to
administer a combination therapy of a PD-1 antagonist and a
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain to a subject having
a prostate cancer, or diagnosed with a prostate cancer, to achieve
at least one positive therapeutic effect, such as for example,
reduced number of cancer cells, reduced tumor size, reduced rate of
cancer cell infiltration into peripheral organs, or reduced rate of
tumor metastasis or tumor growth. Positive therapeutic effects in
cancer can be measured in a number of ways (See, W. A. Weber, J.
Nucl. Med. 50:1S-10S (2009)). For example, with respect to tumor
growth inhibition, according to NCI standards, a T/C.ltoreq.42% is
the minimum level of anti-tumor activity. A T/C<10% is
considered a high anti-tumor activity level, with T/C (%)=Median
tumor volume of the treated/Median tumor volume of the
control.times.100. In some embodiments, the treatment achieved by a
combination of the invention is any of PR, CR, OR, PFS, DFS and OS.
PFS, also referred to as "Time to Tumor Progression" indicates the
length of time during and after treatment that the cancer does not
grow, and includes the amount of time patients have experienced a
CR or PR, as well as the amount of time patients have experienced
SD. DFS refers to the length of time during and after treatment
that the patient remains free of disease. OS refers to a
prolongation in life expectancy as compared to naive or untreated
individuals or patients. In some embodiments, response to a
combination of the invention is any of PR, CR, PFS, DFS, OR or OS
that is assessed using RECIST 1.1 response criteria. The treatment
regimen for a combination of the invention that is effective to
treat a cancer patient may vary according to factors such as the
disease state, age, and weight of the patient, and the ability of
the therapy to elicit an anti-cancer response in the subject. While
an embodiment of any of the aspects of the invention may not be
effective in achieving a positive therapeutic effect in every
subject, it should do so in a statistically significant number of
subjects as determined by any statistical test known in the art
such as the Student's t-test, the chi.sup.2-test, the U-test
according to Mann and Whitney, the Kruskal-Wallis test (H-test),
Jonckheere-Terpstra-test and the Wilcoxon-test.
[0138] The terms "treatment regimen", "dosing protocol" and dosing
regimen are used interchangeably to refer to the dose and timing of
administration of each therapeutic agent in a combination of the
invention.
[0139] "Tumor" as it applies to a subject diagnosed with, or
suspected of having, a cancer refers to a malignant or potentially
malignant neoplasm or tissue mass of any size, and includes primary
tumors and secondary neoplasms. A solid tumor is an abnormal growth
or mass of tissue that usually does not contain cysts or liquid
areas. Different types of solid tumors are named for the type of
cells that form them. Examples of solid tumors are sarcomas,
carcinomas, and lymphomas. Leukemias (cancers of the blood)
generally do not form solid tumors (National Cancer Institute,
Dictionary of Cancer Terms).
[0140] "Tumor burden" also referred to as "tumor load", refers to
the total amount of tumor material distributed throughout the body.
Tumor burden refers to the total number of cancer cells or the
total size of tumor(s), throughout the body, including lymph nodes
and bone narrow. Tumor burden can be determined by a variety of
methods known in the art, such as, e.g. by measuring the dimensions
of tumor(s) upon removal from the subject, e.g., using calipers, or
while in the body using imaging techniques, e.g., ultrasound, bone
scan, computed tomography (CT) or magnetic resonance imaging (MRI)
scans.
[0141] The term "tumor size" refers to the total size of the tumor
which can be measured as the length and width of a tumor. Tumor
size may be determined by a variety of methods known in the art,
such as, e.g. by measuring the dimensions of tumor(s) upon removal
from the subject, e.g., using calipers, or while in the body using
imaging techniques, e.g., bone scan, ultrasound, CT or MRI
scans.
[0142] In some embodiment, this invention provides an immunogenic
composition comprising a live attenuated bacteria strain, for
example a Listeria vaccine strain comprises a nucleic acid
molecule, wherein the nucleic acid molecule comprises a first open
reading frame encoding fusion polypeptide, wherein said fusion
polypeptide comprises a PEST sequence-containing polypeptide or a
PEST sequence-containing peptide fused to a PSA antigen or fragment
thereof. In one embodiment, an immunogenic composition comprises a
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells. In another embodiment, an immunogenic composition comprises
a live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a tLLO. In yet another embodiment, an immunogenic
composition comprises an LmddA-142 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) pADV142) strain. In still another embodiment, an
immunogenic composition comprises an LmddA-143 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome) strain. In some embodiments, the present invention
provides methods of treating, protecting against, and inducing an
immune response against a tumor or a cancer, for example treating a
prostate cancer comprising the step of administering to a subject
an immunogenic composition provided herein.
[0143] In some embodiments, the immunogenic compositions comprising
a live attenuated bacteria strain, for example a Listeria vaccine
strain comprises a nucleic acid molecule, wherein the nucleic acid
molecule comprises a first open reading frame encoding fusion
polypeptide, wherein said fusion polypeptide comprises a PEST
sequence-containing polypeptide or a PEST sequence-containing
peptide fused to a PSA antigen or fragment thereof may be used in a
method of preventing or treating a tumor or cancer in a human
subject, for example a prostate cancer, comprising the step of
administering to the subject the immunogenic composition strain
provided herein, the recombinant Listeria strain comprising a
recombinant polypeptide comprising an N-terminal fragment of an LLO
protein and tumor-associated antigen, whereby the recombinant
Listeria strain induces an immune response against the
tumor-associated antigen, thereby treating a tumor or cancer in a
human subject. In another embodiment, the immune response is a
T-cell response. In another embodiment, the T-cell response is a
CD4+FoxP3- T cell response. In another embodiment, the T-cell
response is a CD8+ T cell response. In another embodiment, the
T-cell response is a CD4+FoxP3- and CD8+ T cell response.
[0144] In some embodiments, the live-attenuated Listeria strain
comprises a nucleic acid molecule, the nucleic acid molecule
comprising a first open reading frame encoding a fusion
polypeptide, wherein said fusion polypeptide comprises a PEST
sequence-containing polypeptide or a PEST sequence-containing
peptide fused to a PSA antigen or fragment thereof. In some
embodiments, the nucleic acid molecule is comprised in an episomal
expression vector. In other embodiments, the nucleic acid molecule
is integrated into the chromosomal DNA.
[0145] In one embodiment, the nucleic acid molecule comprising a
first open reading frame encoding a fusion polypeptide is
integrated into the Listeria genome. In another embodiment, the
nucleic acid is in a plasmid in said attenuated Listeria vaccine
strain. In another embodiment, the nucleic acid molecule is in a
bacterial artificial chromosome in said attenuated Listeria vaccine
strain.
[0146] It will be well appreciated an "immunogenic composition" may
comprise the recombinant Listeria provided herein, and an adjuvant.
In another embodiment, an immunogenic composition comprises a
recombinant Listeria provided herein. In another embodiment, an
immunogenic composition comprises an adjuvant known in the art or
as provided herein. It is also to be understood that such
compositions enhance an immune response, or increase a T effector
cell to regulatory T cell ratio or elicit an anti-tumor immune
response, as further provided herein. As used throughout, the term
"immunogeneic composition" and "composition" may be used
interchangeably having all the same meanings and qualities.
[0147] Following the administration of the immunogenic compositions
provided herein, the methods provided herein induce the expansion
of T effector cells in peripheral lymphoid organs, leading to an
enhanced presence of T effector cells at the tumor site. In another
embodiment, the methods provided herein induce the expansion of T
effector cells in peripheral lymphoid organs, leading to an
enhanced presence of T effector cells at the periphery. Such
expansion of T effector cells leads to an increased ratio of T
effector cells to regulatory T cells in the periphery and at the
tumor site without affecting the number of Tregs. It will be
appreciated by the skilled artisan that peripheral lymphoid organs
include, but are not limited to, the spleen, peyer's patches, the
lymph nodes, the adenoids, etc. In one embodiment, the increased
ratio of T effector cells to regulatory T cells occurs in the
periphery without affecting the number of Tregs. In another
embodiment, the increased ratio of T effector cells to regulatory T
cells occurs in the periphery, the lymphoid organs and at the tumor
site without affecting the number of Tregs at these sites. In
another embodiment, the increased ratio of T effector cells
decrease the frequency of Tregs, but not the total number of Tregs
at these sites. The term "attenuation" as used herein, is meant a
diminution in the ability of the bacterium to cause disease in an
animal. In other words, the pathogenic characteristics of the
attenuated Listeria strain have been lessened compared with
wild-type Listeria, although the attenuated Listeria is capable of
growth and maintenance in culture. Using as an example the
intravenous inoculation of Balb/c mice with an attenuated Listeria,
the lethal dose at which 50% of inoculated animals survive
(LD.sub.50) is preferably increased above the LD.sub.50 of
wild-type Listeria by at least about 10-fold, more preferably by at
least about 100-fold, more preferably at least about 1,000 fold,
even more preferably at least about 10,000 fold, and most
preferably at least about 100,000-fold. An attenuated strain of
Listeria is thus one which does not kill an animal to which it is
administered, or is one which kills the animal only when the number
of bacteria administered is vastly greater than the number of wild
type non-attenuated bacteria which would be required to kill the
same animal. An attenuated bacterium should also be construed to
mean one which is incapable of replication in the general
environment because the nutrient required for its growth is not
present therein. Thus, the bacterium is limited to replication in a
controlled environment wherein the required nutrient is provided.
The attenuated strains of the present invention are therefore
environmentally safe in that they are incapable of uncontrolled
replication.
[0148] In one embodiment, the attenuated Listeria strain provided
herein lacks antibiotic resistance genes. In another embodiment,
the attenuated Listeria strain provided herein comprises a plasmid
comprising a nucleic acid encoding an antibiotic resistance gene.
In another embodiment, an attenuated Listeria strain expressing a
PSA polypeptide in which the nucleic acid encoding the polypeptide
is operably integrated into the Listeria genome in an open reading
frame with an LLO gene.
[0149] In one embodiment, the attenuated Listeria provided herein
is capable of escaping the phagolysosome.
[0150] In one embodiment, the Listeria genome comprises a deletion
of the endogenous ActA gene, which in one embodiment is a virulence
factor. In one embodiment, such a deletion provides a more
attenuated and thus safer Listeria strain for human use. According
to this embodiment, the PSA antigen or fragment thereof is
integrated in frame with LLO in the Listeria chromosome. In another
embodiment, the integrated nucleic acid molecule is integrated into
the ActA locus.
[0151] In one embodiment, an expression vector comprises at nucleic
acid molecule comprising a recombinant polypeptide comprising a PSA
antigen or fragment thereof. In another embodiment, the recombinant
polypeptide further comprises a truncated LLO protein, a truncated
ActA protein or a PEST sequence peptide fused to the PSA antigen.
In another embodiment, the truncated LLO protein is a N-terminal
LLO or fragment thereof. In another embodiment, the truncated ActA
protein is a N-terminal ActA protein or fragment thereof.
[0152] In another embodiment the attenuated strain is LmddA. In
another embodiment, the attenuated strain is LmAactA. In another
embodiment, the attenuated strain is LmAPrfA. In another
embodiment, the attenuated strain is LmAPlcB. In another
embodiment, the attenuated strain is LmAPlcA. In another
embodiment, the strain is the double mutant or triple mutant of any
of the above-mentioned strains. In another embodiment, this strain
exerts a strong adjuvant effect which is an inherent property of
Listeria-based strains. In another embodiment, this strain is
constructed from the EGD Listeria backbone. In another embodiment,
the strain used in the invention is a Listeria strain that
expresses a non-hemolytic LLO.
[0153] In another embodiment, the Listeria strain is an auxotrophic
mutant. In another embodiment, the Listeria strain is deficient in
a gene encoding a vitamin synthesis gene. In another embodiment,
the Listeria strain is deficient in a gene encoding pantothenic
acid synthase.
[0154] In one embodiment, the generation of AA strains of Listeria
deficient in D-alanine, for example, may be accomplished in a
number of ways that are well known to those of skill in the art,
including deletion mutagenesis, insertion mutagenesis, and
mutagenesis which results in the generation of frameshift
mutations, mutations which cause premature termination of a
protein, or mutation of regulatory sequences which affect gene
expression. In another embodiment, mutagenesis can be accomplished
using recombinant DNA techniques or using traditional mutagenesis
technology using mutagenic chemicals or radiation and subsequent
selection of mutants. In another embodiment, deletion mutants are
preferred because of the accompanying low probability of reversion
of the auxotrophic phenotype. In another embodiment, mutants of
D-alanine which are generated according to the protocols presented
herein may be tested for the ability to grow in the absence of
D-alanine in a simple laboratory culture assay. In another
embodiment, those mutants which are unable to grow in the absence
of this compound are selected for further study.
[0155] In another embodiment, in addition to the aforementioned
D-alanine associated genes, other genes involved in synthesis of a
metabolic enzyme, as provided herein, may be used as targets for
mutagenesis of Listeria.
[0156] In another embodiment, the metabolic enzyme complements an
endogenous metabolic gene that is lacking in the remainder of the
chromosome of the recombinant bacterial strain. In one embodiment,
the endogenous metabolic gene is mutated in the chromosome. In
another embodiment, the endogenous metabolic gene is deleted from
the chromosome. In another embodiment, said metabolic enzyme is an
amino acid metabolism enzyme. In another embodiment, said metabolic
enzyme catalyzes a formation of an amino acid used for a cell wall
synthesis in said recombinant Listeria strain. In another
embodiment, said metabolic enzyme is an alanine racemase enzyme. In
another embodiment, said metabolic enzyme is a D-amino acid
transferase enzyme. Each possibility represents a separate
embodiment of the methods and compositions as provided herein.
[0157] In one embodiment, said auxotrophic Listeria strain
comprises an episomal expression vector comprising a metabolic
enzyme that complements the auxotrophy of said auxotrophic Listeria
strain. In another embodiment, the construct is contained in the
Listeria strain in an episomal fashion. In another embodiment, the
foreign antigen is expressed from a vector harbored by the
recombinant Listeria strain. In another embodiment, said episomal
expression vector lacks an antibiotic resistance marker. In one
embodiment, an antigen of the methods and compositions as provided
herein is fused to an polypeptide comprising a PEST sequence. In
another embodiment, said polypeptide comprising a PEST sequence is
a truncated LLO. In another embodiment, said polypeptide comprising
a PEST sequence is ActA.
[0158] In another embodiment, the Listeria strain is deficient in
an AA metabolism enzyme. In another embodiment, the Listeria strain
is deficient in a D-glutamic acid synthase gene. In another
embodiment, the Listeria strain is deficient in the dat gene. In
another embodiment, the Listeria strain is deficient in the dal
gene. In another embodiment, the Listeria strain is deficient in
the dga gene. In another embodiment, the Listeria strain is
deficient in a gene involved in the synthesis of diaminopimelic
acid. CysK. In another embodiment, the gene is vitamin-B12
independent methionine synthase. In another embodiment, the gene is
trpA. In another embodiment, the gene is trpB. In another
embodiment, the gene is trpE. In another embodiment, the gene is
asnB. In another embodiment, the gene is gltD. In another
embodiment, the gene is gltB. In another embodiment, the gene is
leuA. In another embodiment, the gene is argG. In another
embodiment, the gene is thrC. In another embodiment, the Listeria
strain is deficient in one or more of the genes described
hereinabove.
[0159] In another embodiment, the Listeria strain is deficient in a
synthase gene. In another embodiment, the gene is an AA synthesis
gene. In another embodiment, the gene is folP. In another
embodiment, the gene is dihydrouridine synthase family protein. In
another embodiment, the gene is ispD. In another embodiment, the
gene is ispF. In another embodiment, the gene is
phosphoenolpyruvate synthase. In another embodiment, the gene is
hisF. In another embodiment, the gene is hisH. In another
embodiment, the gene is fliI. In another embodiment, the gene is
ribosomal large subunit pseudouridine synthase. In another
embodiment, the gene is ispD. In another embodiment, the gene is
bifunctional GMP synthase/glutamine amidotransferase protein. In
another embodiment, the gene is cobS. In another embodiment, the
gene is cobB. In another embodiment, the gene is cbiD. In another
embodiment, the gene is uroporphyrin-III
C-methyltransferase/uroporphyrinogen-III synthase. In another
embodiment, the gene is cobQ. In another embodiment, the gene is
uppS. In another embodiment, the gene is truB. In another
embodiment, the gene is dxs. In another embodiment, the gene is
mvaS. In another embodiment, the gene is dapA. In another
embodiment, the gene is ispG. In another embodiment, the gene is
folC. In another embodiment, the gene is citrate synthase. In
another embodiment, the gene is argJ. In another embodiment, the
gene is 3-deoxy-7-phosphoheptulonate synthase. In another
embodiment, the gene is indole-3-glycerol-phosphate synthase. In
another embodiment, the gene is anthranilate synthase/glutamine
amidotransferase component. In another embodiment, the gene is
menB. In another embodiment, the gene is menaquinone-specific
isochorismate synthase. In another embodiment, the gene is
phosphoribosylformylglycinamidine synthase I or II. In another
embodiment, the gene is
phosphoribosylaminoimidazole-succinocarboxamide synthase. In
another embodiment, the gene is carB. In another embodiment, the
gene is carA. In another embodiment, the gene is thyA. In another
embodiment, the gene is mgsA. In another embodiment, the gene is
aroB. In another embodiment, the gene is hepB. In another
embodiment, the gene is rluB. In another embodiment, the gene is
ilvB. In another embodiment, the gene is ilvN. In another
embodiment, the gene is alsS. In another embodiment, the gene is
fabF. In another embodiment, the gene is fabH. In another
embodiment, the gene is pseudouridine synthase. In another
embodiment, the gene is pyrG. In another embodiment, the gene is
truA. In another embodiment, the gene is pabB. In another
embodiment, the gene is an atp synthase gene (e.g. atpC, atpD-2,
aptG, atpA-2, etc).
[0160] In another embodiment, the gene is phoP. In another
embodiment, the gene is aroA. In another embodiment, the gene is
aroC. In another embodiment, the gene is aroD. In another
embodiment, the gene is plcB.
[0161] In another embodiment, the Listeria strain is deficient in a
peptide transporter. In another embodiment, the gene is ABC
transporter/ATP-binding/permease protein. In another embodiment,
the gene is oligopeptide ABC transporter/oligopeptide-binding
protein. In another embodiment, the gene is oligopeptide ABC
transporter/permease protein. In another embodiment, the gene is
zinc ABC transporter/zinc-binding protein. In another embodiment,
the gene is sugar ABC transporter. In another embodiment, the gene
is phosphate transporter. In another embodiment, the gene is ZIP
zinc transporter. In another embodiment, the gene is drug
resistance transporter of the EmrB/QacA family. In another
embodiment, the gene is sulfate transporter. In another embodiment,
the gene is proton-dependent oligopeptide transporter. In another
embodiment, the gene is magnesium transporter. In another
embodiment, the gene is formate/nitrite transporter. In another
embodiment, the gene is spermidine/putrescine ABC transporter. In
another embodiment, the gene is Na/Pi-cotransporter. In another
embodiment, the gene is sugar phosphate transporter. In another
embodiment, the gene is glutamine ABC transporter. In another
embodiment, the gene is major facilitator family transporter. In
another embodiment, the gene is glycine betaine/L-proline ABC
transporter. In another embodiment, the gene is molybdenum ABC
transporter. In another embodiment, the gene is techoic acid ABC
transporter. In another embodiment, the gene is cobalt ABC
transporter. In another embodiment, the gene is ammonium
transporter. In another embodiment, the gene is amino acid ABC
transporter. In another embodiment, the gene is cell division ABC
transporter. In another embodiment, the gene is manganese ABC
transporter. In another embodiment, the gene is iron compound ABC
transporter. In another embodiment, the gene is
maltose/maltodextrin ABC transporter. In another embodiment, the
gene is drug resistance transporter of the Bcr/CflA family. In
another embodiment, the gene is a subunit of one of the above
proteins.
[0162] In one embodiment, provided herein is a nucleic acid
molecule that is used to transform the Listeria in order to arrive
at a recombinant Listeria. In another embodiment, the nucleic acid
provided herein used to transform Listeria lacks a virulence gene.
In another embodiment, the nucleic acid molecule is integrated into
the Listeria genome and carries a non-functional virulence gene. In
another embodiment, the virulence gene is mutated in the
recombinant Listeria. In yet another embodiment, the nucleic acid
molecule is used to inactivate the endogenous gene present in the
Listeria genome. In yet another embodiment, the virulence gene is
an actA gene, an inlA gene, and inlB gene, an inlC gene, inlJ gene,
a plbC gene, a bsh gene, or a prfA gene. It is to be understood by
a skilled artisan, that the virulence gene can be any gene known in
the art to be associated with virulence in the recombinant
Listeria.
[0163] In yet another embodiment the Listeria strain is an inlA
mutant, an inlB mutant, an inlC mutant, an inlJ mutant, prfA
mutant, actA mutant, a dal/dat mutant, a prfA mutant, a plcB
deletion mutant, or a double mutant lacking both plcA and plcB. In
another embodiment, the Listeria comprise a deletion or mutation of
these genes individually or in combination. In another embodiment,
the Listeria provided herein lack each one of genes. In another
embodiment, the Listeria provided herein lack at least one and up
to ten of any gene provided herein, including the actA, prfA, and
dal/dat genes. In another embodiment, the prfA mutant is a D133V
prfA mutant.
[0164] In one embodiment, the live attenuated Listeria is a
recombinant Listeria. In another embodiment, the recombinant
Listeria comprises a mutation or a deletion of a genomic internalin
C (inlC) gene. In another embodiment, the recombinant Listeria
comprises a mutation or a deletion of a genomic actA gene and a
genomic internalin C gene. In one embodiment, translocation of
Listeria to adjacent cells is inhibited by the deletion of the actA
gene and/or the inlC gene, which are involved in the process,
thereby resulting in unexpectedly high levels of attenuation with
increased immunogenicity and utility as a strain backbone. Each
possibility represents a separate embodiment of the present
invention.
[0165] In one embodiment, the metabolic gene, the virulence gene,
etc. is lacking in a chromosome of the Listeria strain. In another
embodiment, the metabolic gene, virulence gene, etc. is lacking in
the chromosome and in any episomal genetic element of the Listeria
strain. In another embodiment, the metabolic gene, virulence gene,
etc. is lacking in the genome of the virulence strain. In one
embodiment, the virulence gene is mutated in the chromosome. In
another embodiment, the virulence gene is deleted from the
chromosome. Each possibility represents a separate embodiment of
the present invention
[0166] In one embodiment, the recombinant Listeria strain provided
herein is attenuated. In another embodiment, the recombinant
Listeria lacks the actA virulence gene. In another embodiment, the
recombinant Listeria lacks the prfA virulence gene. In another
embodiment, the recombinant Listeria lacks the inlB gene. In
another embodiment, the recombinant Listeria lacks both, the actA
and inlB genes. In another embodiment, the recombinant Listeria
strain provided herein comprise an inactivating mutation of the
endogenous actA gene. In another embodiment, the recombinant
Listeria strain provided herein comprise an inactivating mutation
of the endogenous inlB gene. In another embodiment, the recombinant
Listeria strain provided herein comprise an inactivating mutation
of the endogenous inlC gene. In another embodiment, the recombinant
Listeria strain provided herein comprise an inactivating mutation
of the endogenous actA and inlB genes. In another embodiment, the
recombinant Listeria strain provided herein comprise an
inactivating mutation of the endogenous actA and inlC genes. In
another embodiment, the recombinant Listeria strain provided herein
comprise an inactivating mutation of the endogenous actA, inlB, and
inlC genes. In another embodiment, the recombinant Listeria strain
provided herein comprise an inactivating mutation of the endogenous
actA, inlB, and inlC genes. In another embodiment, the recombinant
Listeria strain provided herein comprise an inactivating mutation
of the endogenous actA, inlB, and inlC genes. In another
embodiment, the recombinant Listeria strain provided herein
comprise an inactivating mutation in any single gene or combination
of the following genes: actA, dal, dat, inlB, inlC, prfA, plcA,
plcB.
[0167] It will be appreciated by the skilled artisan that the term
"mutation" and grammatical equivalents thereof, include any type of
mutation or modification to the sequence (nucleic acid or amino
acid sequence), and includes a deletion mutation, a truncation, an
inactivation, a disruption, or a translocation. These types of
mutations are readily known in the art.
[0168] In one embodiment, in order to select for an auxotrophic
bacteria comprising a plasmid encoding a metabolic enzyme or a
complementing gene provided herein, transformed auxotrophic
bacteria are grown on a media that will select for expression of
the amino acid metabolism gene or the complementing gene. In
another embodiment, a bacteria auxotrophic for D-glutamic acid
synthesis is transformed with a plasmid comprising a gene for
D-glutamic acid synthesis, and the auxotrophic bacteria will grow
in the absence of D-glutamic acid, whereas auxotrophic bacteria
that have not been transformed with the plasmid, or are not
expressing the plasmid encoding a protein for D-glutamic acid
synthesis, will not grow. In another embodiment, a bacterium
auxotrophic for D-alanine synthesis will grow in the absence of
D-alanine when transformed and expressing the plasmid of the
present invention if the plasmid comprises an isolated nucleic acid
encoding an amino acid metabolism enzyme for D-alanine synthesis.
Such methods for making appropriate media comprising or lacking
necessary growth factors, supplements, amino acids, vitamins,
antibiotics, and the like are well known in the art, and are
available commercially (Becton-Dickinson, Franklin Lakes, N.J.).
Each method represents a separate embodiment of the present
invention.
[0169] In another embodiment, once the auxotrophic bacteria
comprising the plasmid of the present invention have been selected
on appropriate media, the bacteria are propagated in the presence
of a selective pressure. Such propagation comprises growing the
bacteria in media without the auxotrophic factor. The presence of
the plasmid expressing an amino acid metabolism enzyme in the
auxotrophic bacteria ensures that the plasmid will replicate along
with the bacteria, thus continually selecting for bacteria
harboring the plasmid. The skilled artisan, when equipped with the
present disclosure and methods herein will be readily able to
scale-up the production of the Listeria strain vector by adjusting
the volume of the media in which the auxotrophic bacteria
comprising the plasmid are growing.
[0170] The skilled artisan will appreciate that, in another
embodiment, other auxotroph strains and complementation systems are
adopted for the use with this invention.
[0171] In one embodiment, the N-terminal LLO protein fragment and
PSA antigen are fused directly to one another. In another
embodiment, the genes encoding the N-terminal LLO protein fragment
and PSA antigen are not fused directly to one another. In another
embodiment, the N-terminal LLO protein fragment and PSA antigen are
operably attached via a linker peptide. In another embodiment, the
N-terminal LLO protein fragment and PSA antigen are attached via a
heterologous peptide. In another embodiment, the N-terminal LLO
protein fragment is N-terminal to the PSA antigen. In another
embodiment, the N-terminal LLO protein fragment is the
N-terminal-most portion of the fusion protein. In another
embodiment, a truncated LLO is truncated at the C-terminal to
arrive at an N-terminal LLO. Each possibility represents a separate
embodiment of the present invention.
[0172] The term "linker", as used herein refers to an amino acid
sequence that joins two heterologous polypeptides, or fragments or
domains thereof. For example, linking a tLLO and a PSA polypeptide.
In general, as used herein, a linker is an amino acid sequence that
covalently links the polypeptides to form a fusion polypeptide. A
linker typically includes the amino acids translated from the
remaining recombination signal after removal of a reporter gene
from a display vector to create a fusion protein comprising an
amino acid sequence encoded by an open reading frame and the
display protein. As appreciated by one of skill in the art, the
linker can comprise additional amino acids, such as glycine and
other small neutral amino acids.
[0173] The term "operably linked" as used herein means that the
transcriptional and translational regulatory nucleic acid, is
positioned relative to any coding sequences in such a manner that
transcription is initiated. Generally, this will mean that the
promoter and transcriptional initiation or start sequences are
positioned 5' to the coding region.
[0174] The term "open reading frame" or "ORF" is a portion of an
organism's genome which contains a sequence of bases that could
potentially encode a protein. In another embodiment, the start and
stop ends of the ORF are not equivalent to the ends of the mRNA,
but they are usually contained within the mRNA. In one embodiment,
ORFs are located between the start-code sequence (initiation codon)
and the stop-codon sequence (termination codon) of a gene. Thus, in
one embodiment, a nucleic acid molecule operably integrated into a
genome as an open reading frame with an endogenous polypeptide is a
nucleic acid molecule that has integrated into a genome in the same
open reading frame as an endogenous polypeptide.
[0175] In one embodiment, the attenuated Listeria strain provided
herein expresses a recombinant polypeptide. In another embodiment,
the attenuated Listeria strain comprises a plasmid that encodes a
recombinant polypeptide. In another embodiment, a recombinant
nucleic acid provided herein is in a plasmid in the attenuated
Listeria strain provided herein. In another embodiment, the plasmid
is an episomal plasmid that does not integrate into said attenuated
Listeria strain's chromosome. In another embodiment, the plasmid is
an integrative plasmid that integrates into said Listeria strain's
chromosome. In another embodiment, the plasmid is a multicopy
plasmid.
[0176] In one embodiment, the attenuated Listeria strain of the
compositions and methods as provided herein comprise a first or
second nucleic acid molecule that encodes a Prostate Specific
Antigen (PSA), which in one embodiment, is a marker for prostate
cancer that is highly expressed by prostate tumors. In one
embodiment, PSA is a kallikrein serine protease (KLK3) secreted by
prostatic epithelial cells, which in one embodiment, is widely used
as a marker for prostate cancer.
[0177] In one embodiment, the recombinant Listeria strain as
provided herein comprises a nucleic acid molecule encoding KLK3
protein.
[0178] In another embodiment, the KLK3 protein has the
sequence:
[0179] MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC
GGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLK
NRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEE
FLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCN
GVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 25; GenBank
Accession No. CAA32915). In another embodiment, the KLK3 protein is
a homologue of SEQ ID No: 25. In another embodiment, the KLK3
protein is a variant of SEQ ID No: 25. In another embodiment, the
KLK3 protein is an isomer of SEQ ID No: 25. In another embodiment,
the KLK3 protein is a fragment of SEQ ID No: 25. Each possibility
represents a separate embodiment of the methods and compositions as
provided herein.
[0180] In another embodiment, the KLK3 protein has the
sequence:
[0181] IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVIL
LGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAEL
TDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHP
QKVTKFMLCAGRWTGGKSTCSGDSGGPLVCYGVLQGITSWGSEPCALPERPSLYTK
VVHYRKWIKDTIVANP (SEQ ID No: 26). In another embodiment, the KLK3
protein is a homologue of SEQ ID No: 26. In another embodiment, the
KLK3 protein is a variant of SEQ ID No: 26. In another embodiment,
the KLK3 protein is an isomer of SEQ ID No: 26. In another
embodiment, the KLK3 protein is a fragment of SEQ ID No: 26. Each
possibility represents a separate embodiment of the methods and
compositions as provided herein.
[0182] In another embodiment, the KLK3 protein has the sequence:
IVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSL
FHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVK
VMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTK
FMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYR
KWIKDTIVANP (SEQ ID No: 27; GenBank Accession No. AAA59995.1). In
another embodiment, the KLK3 protein is a homologue of SEQ ID No:
27. In another embodiment, the KLK3 protein is a variant of SEQ ID
No: 27. In another embodiment, the KLK3 protein is an isomer of SEQ
ID No: 27. In another embodiment, the KLK3 protein is a fragment of
SEQ ID No: 27. Each possibility represents a separate embodiment of
the methods and compositions as provided herein.
[0183] In another embodiment, the KLK3 protein is encoded by a
nucleotide molecule having the sequence:
ggtgtcttaggcacactggtcttggagtgcaaaggatctaggcacgtgaggctttgtatgaagaatcggggat-
cgtacccaccccctgtt
tctgtttcatcctgggcatgtctcctctgcctttgtcccctagatgaagtctccatgagctacaagggcctgg-
tgcatccagggtgatctagt
aattgcagaacagcaagtgctagctctccctccccttccacagctctgggtgtgggagggggttgtccagcct-
ccagcagcatgggga
gggccttggtcagcctctgggtgccagcagggcaggggcggagtcctggggaatgaaggttttatagggctcc-
tgggggaggctcc
ccagccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctg-
tccgtgacgtggattg
gtgagaggggccatggttggggggatgcaggagagggagccagccctgactgtcaagctgaggctctttcccc-
cccaacccagcac
cccagcccagacagggagctgggctcttttctgtctctcccagccccacttcaagcccatacccccagtcccc-
tccatattgcaacagtc
ctcactcccacaccaggtccccccacttaccctcagaacttcttcccatttgcccagccagctccctgctccc-
agctgctttac
taaaggggaagttcctgggcatctccgtgtttctctttgtggggctcaaaacctccaaggacctctctcaatg-
ccattggttccttggaccg
tatcactggtccatctcctgagcccctcaatcctatcacagtctactgacttttcccattcagctgtgagtgt-
ccaaccctatcccagagacc
ttgatgcttggcctcccatcttgctaggatacccagatgccaaccagacacctccttctttctagaggtatct-
ggcctgagaca
acaaatgggtccctcagtctggcaatgggactctgagaactcctcattccctgactcttagccccagactctt-
cattcagtggcccacattt
tccttaggaaaaacatgagcatccccagccacaactgccagctctctgagtccccaaatctgcatccttttca-
aaacctaaaaacaaaaa
gaaaaacaaataaaacaaaaccaactcagaccagaactgttttctcaacctgggacttcctaaactttccaaa-
accttcctcttccagcaa
ctgaacctcgccataaggcacttatccctggttcctagcaccccttatcccctcagaatccacaacttgtacc-
aagtttcccttctcccagtc
caagaccccaaatcaccacaaaggacccaatccccagactcaagatatggtctgggcgcgctgtgtctcctac-
cctgatccctggg
ttcaactctgctcccagagcatgaagcctctccaccagcaccagccaccaacctgcaaacctagggaagattg-
acagaattcccagcc
tttcccagctccccctgcccatgtcccaggactcccagccttggttctctgcccccgtgtcttttcaaaccca-
catcctaaatccatctccta
tccgagtcccccagttccccctgtcaaccctgattcccctgatctagcaccccctctgcaggcgctgcgcccc-
tcatcctgtctcggattg
tgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggcagtctg-
cggcggtgttctggt
gcacccccagtgggtcctcacagctgcccactgcatcaggaagtgagtaggggcctggggtctggggagcagg-
tgtctgtgtcccag
aggaataacagctgggcattttccccaggataacctctaaggccagccttgggactgggggagagagggaaag-
ttctggttcaggtca
catggggaggcagggttggggctggaccaccctccccatggctgcctgggtctccatctgtgtccctctatgt-
ctctttgtgtcgctttcat
tatgtctcttggtaactggcttcggttgtgtctctccgtgtgactattttgtttctctcctctgtcttcagtc-
tccatatctccccct
ctctctgtcctctggttctctgtcctttagccagtgtgtctcaccctgtatctctctgccaggctctgtctct-
cggtctctgtctcacctgtgcctt
ctccctactgaacacacgcacgggatgggcctgggggaccctgagaaaaggaagggctttggctgggcgcggt-
ggctcacacctgt
aatcccagcactttgggaggccaaggcaggtagatcacctgaggtcaggagttcgagaccagcctggccaact-
ggtgaaaccccatc
tctactaaaaatacaaaaaattagccaggcgtggtggcgcatgcctgtagtcccagctactcaggagctgagg-
gaggagaatgcatg
aacctggaggttgaggttgcagtgagccgagaccgtgccactgcactccagcctgggtgacagagtgagactc-
cgcctcaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaagaaaagaaaagaaaagaaaaggaagtgttttatccctgatgtgtgtgggta-
tgagggtatgagag
ggcccctctcactccattccltctccaggacatccctccactcltgggagacacagagaagggctggtccagc-
tggagctgggaggg
gcaattgagggaggaggaaggagaagggggaaggaaaacagggtatgggggaaaggaccctggggagcgaagt-
ggaggatac
aaccttgggcctgcaggcaggctacctacccacttggaaacccacgccaaagccgcatctacag-
ctgagccactctgaggcctcccc
tccccggcggtccccactcagctccaaagtctctctcccttttctctcccacacttatcatcccccggatcct-
ctctactggtctcatct
ttcccttggttctctcagttctgtatctgcccttcaccctctcacactgctgtttcccaactcgttgtctgta-
tttggcctgaactgtgtctccc
gtctgcttcctccccagcaaaagcgtgatcttgctgggtcggcacagcctgtttcatcctgaagacacaggcc-
aggtattcaggtcagc
cacagcttcccacacccgctctacgatatgagcctcctgaagaatcgatcctcaggccaggtgatgactccag-
ccacgacctcatgct
gctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagca-
ctggggaccacct
gctacgcctcaggctggggcagcattgaaccagaggagtgtacgcctgggccagatggtgcagccgggagccc-
agatgcctgggt
ctgagggaggaggggacaggactcctgggtctgagggaggagggccaaggaaccaggtggggtccagcccaca-
acagtgttttg
cctggcccgtagtcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgc-
gcaagttcaccctcagaa
ggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcggtgagtcatccctac-
tcccaagatctgag
ggaaaggtgagtgggaccltaaltctgggctggggtctagaagccaacaaggcgtctgcctcccctgctcccc-
agctgtagccatgcc
acctccccgtgtctcatctcattccctccttccctcttctttgactccctcaaggcaataggttatctacagc-
acaactcatctgtcctgcgt
tcagcacacggttactaggcacctgctatgcacccagcactgccctagagcctgggacatagcagtgaacaga-
cagagagcagccc
ctcccttctgtagcccccaagccagtgaggggcacaggcaggaacagggaccacaacacagaaaagctggagg-
gtgtcaggaggt
gatcaggctctcggggagggagaaggggtggggagtgtgactgggaggagacatcctgcagaaggtgggagtg-
agcaaacacct
gcgcaggggaggggagggcctgcggcacctgggggagcagagggaacagcatctggccaggcctgggaggagg-
ggcctagag
ggcgtcaggagcagagaggaggttgcctggctggagtgaaggatcggggcagggtgcgagaggg-
aacaaaggacccctcctgca
gggcctcacctgggccacaggaggacactgcttttcctctgaggagtcaggaactgtggatggtgctggacag-
aagcaggacaggg
cctggctcaggtgtccagaggctgcgctggcctcctatgggatcagactgcagggagggagggcagcagggat-
gtggagggagtg
atgatggggctgacctgggggtggctccaggcattgtccccacctgggccctacccagcctccctcacaggct-
cctggccctcagtct
ctcccctccactccaltctccacctacccacagtgggtcaltctgatcaccgaactgaccatgccagccctgc-
cgatggtcctccatggct
ccctagtgccctggagaggaggtgtctagtcagagagtagtcctggaaggtggcctctgtgaggagccacggg-
gacagcatcctgca
gatggtcctggcccttgtcccaccgacctgtctacaaggactgtcctcgtggaccctcccctctgcacaggag-
ctggaccctgaagtcc
cttcctaccggccaggactggagcccctacccctctgttggaatccctgcccaccttcttctggaagtcggct-
ctggagacattctctct
cttccaaagctgggaactgctatctgttatctgcctgtccaggtctgaaagataggatgcccaggcagaaact-
gggactgacctatctc
actctctccctgcttttacccttagggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatc-
acgtcatggggcagtgaac
catgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccat-
cgtggccaacccctg
agcacccctatcaagtccctattgtagtaaacttggaaccttggaaatgaccaggccaagactcaagcctccc-
cagttctactgacctttg
tccttaggtgtgaggtccagggttgctaggaaaagaaatcagcagacacaggtgtagaccagagtgtttctta-
aatggtgtaattttgtcc
tctctgtgtcctggggaatactggccatgcctggagacatatcactcaatttctctgaggacacagttaggat-
ggggtgtctgtgttatttgt gggatacagagatgaaagaggggtgggatcc (SEQ ID No:
28; GenBank Accession No. X14810). In another embodiment, the KLK3
protein is encoded by residues 401 . . . 446, 1688 . . . 1847, 3477
. . . 3763, 3907 . . . 4043, and 5413 . . . 5568 of SEQ ID No: 28.
In another embodiment, the KLK3 protein is encoded by a homologue
of SEQ ID No: 28. In another embodiment, the KLK3 protein is
encoded by a variant of SEQ ID No: 28. In another embodiment, the
KLK3 protein is encoded by an isomer of SEQ ID No: 28. In another
embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No:
28. Each possibility represents a separate embodiment of the
methods and compositions as provided herein.
[0184] In another embodiment, the KLK3 protein has the sequence:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH
PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG
DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL
QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSWVILITELTMPALPMVL
HGSLVPWRGGV (SEQ ID No: 29; GenBank Accession No. NP_001025218) In
another embodiment, the KLK3 protein is a homologue of SEQ ID No:
29. In another embodiment, the KLK3 protein is a variant of SEQ ID
No: 29. In another embodiment, the KLK3 protein is an isomer of SEQ
ID No: 29. In another embodiment, the KLK3 protein is a fragment of
SEQ ID No: 29. Each possibility represents a separate embodiment as
provided herein.
[0185] In another embodiment, the KLK3 protein is encoded by a
nucleotide molecule having the sequence:
agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtc-
cgtgacgtggattggtg
ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgct-
tgtggcctctcgtggc
agggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaa-
gcgtgatcttgctgg
gtcggcacagcctgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgct-
ctacgatatgagcctc
ctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctg-
ccgagctcacggatg
ctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcag-
cattgaaccagag
gagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttc-
accctcagaaggtgacca
agttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgtgggtcattctgatcaccgaact-
gaccatgccagccct
gccgatggtcctccatggctccctagtgccctggagaggaggtgtctagtcagagagtagtcctggaaggtgg-
cctctgtgaggagcc
acggggacagcatcctgcagatggtcctggcccttgtcccaccgacctgtctacaaggactgtcctcgtggac-
cctcccctctgcacag
gagctggaccctgaagtcccttccccaccggccaggactggagcccctacccctctgttggaatccctgccca-
ccttcttctggaagtc
ggctctggagacatttctctcttcttccaaagctgggaactgctatctgttatctgcctgtccaggtctgaaa-
gataggattgcccaggcag
aaactgggactgacctatctcactctctccctgcttttacccttagggtgattctgggggcccacttgtctgt-
aatggtgtgcttcaaggtat
cacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccgg-
aagtggatcaagga
caccatcgtggccaacccctgagcacccctatcaaccccctattgtagtaaacttggaaccttggaaatgacc-
aggccaagactcaagc
ctccccagttctactgaggtccagggttgctaggaaaagaaatcagcagacacaggtgtagaccagagtgt
ttcttaaatggtgtaattttgtcctctctgtgtcctggggaatactggccatgcctggagacatatcactcaa-
tttctctgaggacacagatag
gatggggtgtctgtgttatttgtggggtacagagatgaaagaggggtgggatccacactgagagagtggagag-
tgacatgtgctggac
actgtccatgaagcactgagcagaagctggaggcacaacgcaccagacactcacagcaaggatggagctgaaa-
acataacccactc
tgtcctggaggcactgggaagcctagagaaggctgtgagccaaggagggagggtcttcctttggcatgggatg-
gggatgaagtaag
gagagggactggaccccctggaagctgattcactatggggggaggtgtattgaagtcctccagacaaccctca-
gatttgatgatttccta gtagaactcacagaaataaagagctgttatactgtg (SEQ ID No:
30; GenBank Accession No. NM_001030047). In another embodiment, the
KLK3 protein is encoded by residues 42-758 of SEQ ID No: 30. In
another embodiment, the KLK3 protein is encoded by a homologue of
SEQ ID No: 30. In another embodiment, the KLK3 protein is encoded
by a variant of SEQ ID No: 30. In another embodiment, the KLK3
protein is encoded by an isomer of SEQ ID No: 30. In another
embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No:
30. Each possibility represents a separate embodiment of the
methods and compositions as provided herein.
[0186] In another embodiment, the KLK3 protein has the sequence:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH
PQWVLTAAHCIRK (SEQ ID No: 31; GenBank Accession No. NP_001025221).
In another embodiment, the KLK3 protein is a homologue of SEQ ID
No: 31. In another embodiment, the KLK3 protein is a variant of SEQ
ID No: 31. In another embodiment, the sequence of the KLK3 protein
comprises SEQ ID No: 31. In another embodiment, the KLK3 protein is
an isomer of SEQ ID No: 31. In another embodiment, the KLK3 protein
is a fragment of SEQ ID No: 31. Each possibility represents a
separate embodiment of the methods and compositions as provided
herein.
[0187] In another embodiment, the KLK3 protein is encoded by a
nucleotide molecule having the sequence:
agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcttcacccttcc-
gtgacgtggattggtgc
tgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgctt-
gtggcctctcgtggca
gggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaagtgagt-
aggggcctggggtc
tggggagcaggtgtctgtgtcccagaggaataacagctgggcatttccccaggataacctctaaggccagcct-
tgggactggggga
gagagggaaagttctggttcaggtcacatggggaggcagggttggggctggaccaccctccccatggctgcct-
gggtctccatctgtg
ttcctctatgtcttttgtgtcgctttcattatgtctcttggtaactggcttcggttgtgttctccgtgtgact-
attttgttctctctctccctctcttc tctgtcttcagt (SEQ ID No: 32). In another
embodiment, the KLK3 protein is encoded by residues 42-758 of SEQ
ID No: 32. In another embodiment, the KLK3 protein is encoded by a
homologue of SEQ ID No: 32. In another embodiment, the KLK3 protein
is encoded by a variant of SEQ ID No: 32. In another embodiment,
the KLK3 protein is encoded by an isomer of SEQ ID No: 32. In
another embodiment, the KLK3 protein is encoded by a fragment of
SEQ ID No: 32. Each possibility represents a separate embodiment of
the methods and compositions as provided herein.
[0188] In another embodiment, the KLK3 protein that is the source
of the KLK3 peptide has the sequence:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH
PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG
DDSSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGD
SGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 33).
In another embodiment, the KLK3 protein is a homologue of SEQ ID
No: 33. In another embodiment, the KLK3 protein is a variant of SEQ
ID No: 33. In another embodiment, the KLK3 protein is an isomer of
SEQ ID No: 33. In another embodiment, the KLK3 protein is a
fragment of SEQ ID No: 33. Each possibility represents a separate
embodiment of the methods and compositions as provided herein.
[0189] In another embodiment, the KLK3 protein is encoded by a
nucleotide molecule having the sequence:
agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtc-
cgtgacgggtggtg
ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgct-
tgtggcctctcgtggc
agggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaa-
gcgtgatcttgctgg
gtcggcacagcctgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgct-
ctacgatatgagcctc
ctgaagaatcgattcctcaggccaggtgatgactccagcattgaaccagaggagttcttgaccccaaagaaac-
ttcagtgtgtggacct
ccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctgga-
cgctggacagggggca
aaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatgggg-
cagtgaaccatgtgccc
tgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaa-
cccctgagcacccc
tatcaaccccctattgtagtaaacttggaaccttggaaatgaccaggccaagactcaagcctccccagttcta-
ctgacctttgtccttaggt
gtgaggtccagggttgctaggaaaagaaatcagcagacacaggtgtagaccagagtgtttcttaaatggtgta-
attttgtcctctctgtgt
cctggggaatactggccatgcctggagacatatcactcaatttctctgaggacacagataggatggggtgtct-
gtgttatttgtggggtac
agagatgaaagaggggtgggatccacactgagagagtggagagtgacatgtgctggacactgtccatgaagca-
ctgagcagaagct
ggaggcacaacgcaccagacactcacagcaaggatggagctgaaaacataacccactctgtcctggaggcact-
gggaagcctaga
gaaggctgtgagccaaggagggagggtcttcctttggcatgggatggggatgaagtaaggagagggactggac-
cccctggaagctg
attcactatggggggaggtgtattgaagtcctccagacaaccctcagatttgatgatttcctagtagaactca-
cagaaataaagagctgtt atactgtg (SEQ ID No: 34). In another embodiment,
the KLK3 protein is encoded by residues 42-758 of SEQ ID No: 34. In
another embodiment, the KLK3 protein is encoded by a homologue of
SEQ ID No: 34. In another embodiment, the KLK3 protein is encoded
by a variant of SEQ ID No: 34. In another embodiment, the KLK3
protein is encoded by an isomer of SEQ ID No: 34. In another
embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No:
34. Each possibility represents a separate embodiment of the
methods and compositions as provided herein.
[0190] In another embodiment, the KLK3 protein has the sequence:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH
PQWVLTAAHCIRKPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYAS
GWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSG
DSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 35;
GenBank Accession No. NP_001025219). In another embodiment, the
KLK3 protein is a homologue of SEQ ID No: 35. In another
embodiment, the KLK3 protein is a variant of SEQ ID No: 35. In
another embodiment, the KLK3 protein is an isomer of SEQ ID No: 35.
In another embodiment, the KLK3 protein is a fragment of SEQ ID No:
35. Each possibility represents a separate embodiment of the
methods and compositions as provided herein.
[0191] In another embodiment, the KLK3 protein is encoded by a
nucleotide molecule having the sequence:
agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtc-
cgtgacgtggattggtg
ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgct-
tgtggcctctcgtggc
agggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaagccag-
gtgatgactccagc
cacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgccca-
cccaggagccagca
ctggggaccacctgctacgcctcaggctggggcagcattgaaccagaggagttcttgaccccaaagaaacttc-
agtgtgtggacctcc
atgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacg-
ctggacagggggcaaa
agcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggca-
gtgaaccatgtgccctg
cccgaaaggccttccctgtacaccaaggtggtgcattacccaaggacaccatcgtggccaacccctgagcacc-
cctatcaacccccta
ttgtagtaaacttggaaccttggaaatgaccaggccaagactcaagcctccccagttctactgacctttgtcc-
ttaggtgtgaggtccagg
gttgctaggaaaagaaatcagcagacacaggtgtagaccagagtgtttcttaaatggtgtaattttgtcctct-
ctgtgtcctggggaatact
ggccatgcctggagacatatcactcaatttctctgaggacacagataggatggggtgtctgtgttatttgtgg-
ggtacagagatgaaaga
ggggtgggatccacactgagagagtggagagtgacatgtgctggacactgtccatgaagcactgagcagaagc-
tggaggcacaac
gcaccagacactcacagcaaggatggagctgaaaacataacccactctgtcctggaggcactgggaagcctag-
agaaggctgtgag
ccaaggagggagggtcttcctttggcatgggatggggatgaagtaaggagagggactggaccccctggaagct-
gattcactatgggg
ggaggtgtattgaagtcctccagacaaccctcagatttgatgatttcctagtagaactcacagaaataaagag-
ctgttatactgtg (SEQ ID No: 36; GenBank Accession No. NM_001030048).
In another embodiment, the KLK3 protein is encoded by residues
42-758 of SEQ ID No: 36. In another embodiment, the KLK3 protein is
encoded by a homologue of SEQ ID No: 36. In another embodiment, the
KLK3 protein is encoded by a variant of SEQ ID No: 36. In another
embodiment, the KLK3 protein is encoded by an isomer of SEQ ID No:
36. In another embodiment, the KLK3 protein is encoded by a
fragment of SEQ ID No: 36. Each possibility represents a separate
embodiment of the methods and compositions as provided herein
[0192] In another embodiment, the KLK3 protein has the sequence:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH
PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG
DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL
QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGIT
SWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 37; GenBank
Accession No. NP_001639). In another embodiment, the KLK3 protein
is a homologue of SEQ ID No: 37. In another embodiment, the KLK3
protein is a variant of SEQ ID No: 37. In another embodiment, the
KLK3 protein is an isomer of SEQ ID No: 37. In another embodiment,
the KLK3 protein is a fragment of SEQ ID No: 37. Each possibility
represents a separate embodiment of the methods and compositions as
provided herein.
[0193] In another embodiment, the KLK3 protein is encoded by a
nucleotide molecule having the sequence:
agccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcaccctgtc-
cgtgacgggtggtg
ctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgct-
tgtggcctctcgtggc
agggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaa-
gcgtgatcttgctgg
gtcggcacagcctgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgct-
ctacgatatgagcctc
ctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctg-
ccgagctcacggatg
ctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcag-
cattgaaccagag
gagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttc-
accctcagaaggtgacca
agttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgt-
ctgtaatggtgtgcttc
aaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgca-
ttaccggaagtggat
caaggacaccatcgtggccaacccctgagcacccctatcaaccccctattgtagtaaacttggaaccttggaa-
atgaccaggccaaga
ctcaagcctccccagttctactgacctttgtccttaggtgtgaggtcagggttgctaggaaaagaaatcagca-
gacacaggtgtagacc
agagtgtttcttaaatggtgtaattttgtcctctctgtgtctggggaatactggccatgcctggagacatatc-
actcaatttctctgaggaca
cagataggatggggtgtctgtgttatttgtggggtacagagatgaaagaggggtgggatccacactgagagag-
tggagagtgacatgt
gctggacactgtccatgaagcactgagcagaagctggaggcacaacgcaccagacactcacagcaaggatgga-
gctgaaaacata
acccactctgtcctggaggcactgggaagcctagagaaggctgtgagccaaggagggagggtcttcctttggc-
atgggatggggatg
aagtaaggagagggactggaccccctggaagctgattcactatggggggaggtgtattgaagtcctccagaca-
accctcagatttgat gatttcctagtagaactcacagaaataaagagctgttatactgtg (SEQ
ID No: 38; GenBank Accession No. NM_001648). In another embodiment,
the KLK3 protein is encoded by residues 42-827 of SEQ ID No: 38. In
another embodiment, the KLK3 protein is encoded by a homologue of
SEQ ID No: 38. In another embodiment, the KLK3 protein is encoded
by a variant of SEQ ID No: 38. In another embodiment, the KLK3
protein is encoded by an isomer of SEQ ID No: 38. In another
embodiment, the KLK3 protein is encoded by a fragment of SEQ ID No:
38. Each possibility represents a separate embodiment of the
methods and compositions as provided herein.
[0194] In another embodiment, the KLK3 protein has the sequence:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH
PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG
DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL
QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGIT
SWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID No: 39 GenBank Accession
No. AAX29407.1). In another embodiment, the KLK3 protein is a
homologue of SEQ ID No: 39. In another embodiment, the KLK3 protein
is a variant of SEQ ID No: 39. In another embodiment, the KLK3
protein is an isomer of SEQ ID No: 39. In another embodiment, the
sequence of the KLK3 protein comprises SEQ ID No: 39. In another
embodiment, the KLK3 protein is a fragment of SEQ ID No: 39. Each
possibility represents a separate embodiment of the methods and
compositions as provided herein.
[0195] In another embodiment, the KLK3 protein is encoded by a
nucleotide molecule having the sequence:
gggggagccccaagcttaccacctgcacccggagagctgtgtcaccatgtgggtcccggttgtcttcctcacc-
ctgtccgtgacgtgg
attggtgctgcacccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggc-
aggtgcttgtggcctct
cgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcagga-
acaaaagcgtgatc
ttgctgggtcggcacagcctgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccac-
acccgctctacgatatg
agcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcag-
agcctgccgagctca
cggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctg-
gggcagcattgaa
ccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgc-
aagttcaccctcagaag
gtgaccaagttcatgctgtgtgctggacgctggacagggggcaaagcacctgctcgggtgattctgggggccc-
acttgtctgtaatg
gtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaa-
ggtggtgcattaccgg
aagtggatcaaggacaccatcgtggccaacccctgagcacccctatcaactccctattgtagtaaacttggaa-
ccttggaaatgaccag
gccaagactcaggcctccccagttctactgacctttgtccttaggtgtgaggtccagggttgctaggaaaaga-
aatcagcagacacagg
tgtagaccagagtgtttcttaaatggtgtaattttgtcctctctgtgtcctggggaatactggccatgcctgg-
agacatatcactcaatttctct
gaggacacagataggatggggtgtctgtgttatttgtggggtacagagatgaaagaggggtgggatccacact-
gagagagtggagag
tgacatgtgctggacactgtccatgaagcactgagcagaagctggaggcacaacgcaccagacactcacagca-
aggatggagctga
aaacataacccactctgtcctggaggcactgggaagcctagagaaggctgtgagccaaggagggagggtcttc-
ctttggcatgggat
ggggatgaagtagggagagggactggaccccctggaagctgattcactatggggggaggtgtattgaagtcct-
ccagacaaccctca
gatttgatgatttcctagtagaactcacagaaataaagagctgttatactgcgaaaaaaaaaaaaaaaaaaaa-
aaaaaa (SEQ ID No: 40; GenBank Accession No. BC056665). In another
embodiment, the KLK3 protein is encoded by residues 47-832 of SEQ
ID No: 40. In another embodiment, the KLK3 protein is encoded by a
homologue of SEQ ID No: 40. In another embodiment, the KLK3 protein
is encoded by a variant of SEQ ID No: 40. In another embodiment,
the KLK3 protein is encoded by an isomer of SEQ ID No: 40. In
another embodiment, the KLK3 protein is encoded by a fragment of
SEQ ID No: 40. Each possibility represents a separate embodiment of
the methods and compositions as provided herein.
[0196] In another embodiment, the KLK3 protein has the sequence:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH
PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG
DDSSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGD
SGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVA (SEQ ID No: 41;
GenBank Accession No. AJ459782). In another embodiment, the KLK3
protein is a homologue of SEQ ID No: 41. In another embodiment, the
KLK3 protein is a variant of SEQ ID No: 41. In another embodiment,
the KLK3 protein is an isomer of SEQ ID No: 41. In another
embodiment, the KLK3 protein is a fragment of SEQ ID No: 41. Each
possibility represents a separate embodiment of the methods and
compositions as provided herein.
[0197] In another embodiment, the KLK3 protein has the sequence:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH
PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG
DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL
QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSVSHPYSQDLEGKGEWG P (SEQ ID
No: 42, GenBank Accession No. AJ512346). In another embodiment, the
KLK3 protein is a homologue of SEQ ID No: 42. In another
embodiment, the KLK3 protein is a variant of SEQ ID No: 42. In
another embodiment, the KLK3 protein is an isomer of SEQ ID No: 42.
In another embodiment, the sequence of the KLK3 protein comprises
SEQ ID No: 42. In another embodiment, the KLK3 protein is a
fragment of SEQ ID No: 42. Each possibility represents a separate
embodiment of the methods and compositions as provided herein.
[0198] In another embodiment, the KLK3 protein has the sequence:
MWVPVVFLTLSVTWIGERGHGWGDAGEGASPDCQAEALSPPTQHPSPDRELGSFLSL
PAPLQAHTPSPSILQQSSLPHQVPAPSHLPQNFLPIAQPAPCSQLLY (SEQ ID No: 43
GenBank Accession No. AJ459784). In another embodiment, the KLK3
protein is a homologue of SEQ ID No 43. In another embodiment, the
KLK3 protein is a variant of SEQ ID No: 43. In another embodiment,
the sequence of the KLK3 protein comprises SEQ ID No: 43. In
another embodiment, the KLK3 protein is an isomer of SEQ ID No: 43.
In another embodiment, the KLK3 protein is a fragment of SEQ ID No:
43. Each possibility represents a separate embodiment of the
methods and compositions as provided herein.
[0199] In another embodiment, the KLK3 protein has the sequence:
MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVH
PQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPG
DDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL
QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGIT
SWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID NO: 44 GenBank Accession
No. AJ459783). In another embodiment, the KLK3 protein is a
homologue of SEQ ID No: 44. In another embodiment, the KLK3 protein
is a variant of SEQ ID No: 44. In another embodiment, the KLK3
protein is an isomer of SEQ ID No: 44. In another embodiment, the
KLK3 protein is a fragment of SEQ ID No: 44. Each possibility
represents a separate embodiment of the methods and compositions as
provided herein.
[0200] In another embodiment, the KLK3 protein is encoded by a
nucleotide molecule having the sequence:
aagtttcccttctcccagtccaagaccccaaatcaccacaaaggacccaatccccagactcaagatatggtct-
gggcgctgtcttgtgtc
tcctaccctgatccctgggttcaactctgctcccagagcatgaagcctctccaccagcaccagccaccaacct-
gcaaacctagggaag
attgacagaattcccagcctttcccagctccccctgcccatgtcccaggactcccagccttggttctctgccc-
ccgtgtcttttcaaaccca
catcctaaatccatctcctatccgagtcccccagttcctcctgtcaaccctgattcccctgatctagcacccc-
ctctgcaggtgctgcaccc
ctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtagcct-
ctcgtggcagggcagt
ctgcggcggtgttctggtgcacccccagtgggtcctcacagctacccactgcatcaggaacaaaagcgtgatc-
ttgctgggtcggcac
agcctgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgata-
tgagcctcctgaagaat
cgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctca-
cggatgctatgaagg
tcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaacc-
agaggagttcttga
ccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaa-
ggtgaccaagttcatgct
gtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatggt-
gtgcttcaaggtatca
cgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaa-
gtggatcaaggaca
ccatcgtggccaacccctgagcacccctatcaactccctattgtagtaaacttggaaccttggaaatgaccag-
gccaagactcaggcct
ccccagttctactgacctttgtccttaggtgtgaggtccagggttgctaggaaaagaaatcagcagacacagg-
tgtagaccagagtgttt
cttaaatggtgtaattttgtcctctctgtgtcctggggaatactggccatgcctggagacatatcactcaatt-
tctctgaggacacagatagg
atggggtgtctgtgttatttgtggggtacagagatgaaagaggggtgggatccacactgagagagtggagagt-
gacatgtgctggaca
ctgtccatgaagcactgagcagaagctggaggcacaacgcaccagacactcacagcaaggatggagctgaaaa-
cataacccactct
gtcctggaggcactgggaagcctagagaaggctgtgaaccaaggagggagggtcttcctttggcatgggatgg-
ggatgaagtaagg
agagggactgaccccctggaagctgattcactatggggggaggtgtattgaagtcctccagacaaccctcaga-
tttgatgatttcctagt agaactcacagaaataaagagctgttatactgtgaa (SEQ ID No:
45; GenBank Accession No. X07730). In another embodiment, the KLK3
protein is encoded by residues 67-1088 of SEQ ID No: 45. In another
embodiment, the KLK3 protein is encoded by a homologue of SEQ ID
No: 45. In another embodiment, the KLK3 protein is encoded by a
variant of SEQ ID No: 45. In another embodiment, the KLK3 protein
is encoded by an isomer of SEQ ID No: 45. In another embodiment,
the KLK3 protein is encoded by a fragment of SEQ ID No: 45. Each
possibility represents a separate embodiment of the methods and
compositions as provided herein.
[0201] In another embodiment, the KLK3 protein has the
sequence:
[0202] MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC
GGVLVHPQWVLTAAHCIRK (SEQ ID No: 63; GenBank Accession No.
NM_001030050). In another embodiment, the KLK3 protein is a
homologue of SEQ ID No: 63. In another embodiment, the KLK3 protein
is a variant of SEQ ID No: 63. In another embodiment, the sequence
of the KLK3 protein comprises SEQ ID No: 63. In another embodiment,
the KLK3 protein is an isomer of SEQ ID No: 63. In another
embodiment, the KLK3 protein is a fragment of SEQ ID No: 63. Each
possibility represents a separate embodiment of the present
invention.
[0203] In another embodiment, the KLK3 protein that is the source
of the KLK3 peptide has the sequence:
[0204] MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC
GGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLK
NRFLRPGDDSSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGG
KSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID
No: 64; GenBank Accession No. NM_001064049). In another embodiment,
the KLK3 protein is a homologue of SEQ ID No: 64. In another
embodiment, the KLK3 protein is a variant of SEQ ID No: 64. In
another embodiment, the KLK3 protein is an isomer of SEQ ID No: 64.
In another embodiment, the KLK3 protein is a fragment of SEQ ID No:
64. Each possibility represents a separate embodiment of the
present invention.
[0205] In another embodiment, the KLK3 protein has the
sequence:
[0206] MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC
GGVLVHPQWVLTAAHCIRKPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALG
TTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGG
KSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP (SEQ ID
No: 65; GenBank Accession No. NM_001030048). In another embodiment,
the KLK3 protein is a homologue of SEQ ID No: 65. In another
embodiment, the KLK3 protein is a variant of SEQ ID No: 65. In
another embodiment, the KLK3 protein is an isomer of SEQ ID No: 65.
In another embodiment, the KLK3 protein is a fragment of SEQ ID No:
65. Each possibility represents a separate embodiment of the
present invention.
[0207] In another embodiment, the KLK3 protein has the
sequence:
[0208] MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC
GGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLK
NRFLRPGDDSSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGG
KSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVA (SEQ ID No:
66; GenBank Accession No. AJ459782). In another embodiment, the
KLK3 protein is a homologue of SEQ ID No: 66. In another
embodiment, the KLK3 protein is a variant of SEQ ID No: 66. In
another embodiment, the KLK3 protein is an isomer of SEQ ID No: 66.
In another embodiment, the KLK3 protein is a fragment of SEQ ID No:
66. Each possibility represents a separate embodiment of the
present invention.
[0209] In another embodiment, the KLK3 protein has the
sequence:
[0210] MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVC
GGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLK
NRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEE
FLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSVSHPYSQDLE GKGEWGP
(SEQ ID NO: 50 GenBank Accession No. AJ512346). In another
embodiment, the KLK3 protein is a homologue of SEQ ID NO: 50. In
another embodiment, the KLK3 protein is a variant of SEQ ID NO: 50.
In another embodiment, the KLK3 protein is an isomer of SEQ ID NO:
50. In another embodiment, the sequence of the KLK3 protein
comprises SEQ ID NO: 50. In another embodiment, the KLK3 protein is
a fragment of SEQ ID NO: 50. Each possibility represents a separate
embodiment of the present invention.
[0211] In another embodiment, the KLK3 protein has the
sequence:
[0212] MWVPVVFLTLSVTWIGERGHGWGDAGEGASPDCQAEALSPPTQHPSPDRE
LGSFLSLPAPLQAHTPSPSILQQSSLPHQVPAPSHLPQNFLPIAQPAPCSQLLY (SEQ ID NO:
51 GenBank Accession No. AJ459784). In another embodiment, the KLK3
protein is a homologue of SEQ ID NO: 51. In another embodiment, the
KLK3 protein is a variant of SEQ ID NO: 51. In another embodiment,
the sequence of the KLK3 protein comprises SEQ ID NO: 51. In
another embodiment, the KLK3 protein is an isomer of SEQ ID NO: 51.
In another embodiment, the KLK3 protein is a fragment of SEQ ID NO:
51.
[0213] In another embodiment, the KLK3 protein is encoded by a
sequence set forth in one of the following GenBank Accession
Numbers: BC005307, AJ310938, AJ310937, AF335478, AF335477, M27274,
and M26663. In another embodiment, the KLK3 protein is encoded by a
sequence set forth in one of the above GenBank Accession Numbers.
Each possibility represents a separate embodiment of the methods
and compositions as provided herein.
[0214] In another embodiment, the KLK3 protein is encoded by a
sequence set forth in one of the following GenBank Accession
Numbers: NM_001030050, NM_001030049, NM_001030048, NM_001030047,
NM_001648, AJ459782, AJ512346, or AJ459784. Each possibility
represents a separate embodiment of the methods and compositions as
provided herein.
[0215] In one embodiment, the KLK3 protein is encoded by a
variation of any of the sequences described herein wherein the
sequence lacks MWVPVVFLTLSVTWIGAAPLILSR (SEQ ID NO: 52).
[0216] In another embodiment, the KLK3 protein has the sequence
that comprises a sequence set forth in one of the following GenBank
Accession Numbers: X13943, X13942, X13940, X13941, and X13944. Each
possibility represents a separate embodiment of the methods and
compositions as provided herein.
[0217] In another embodiment, the KLK3 protein is any other KLK3
protein known in the art. Each KLK3 protein represents a separate
embodiment of the methods and compositions as provided herein.
[0218] In another embodiment, the KLK3 peptide is any other KLK3
peptide known in the art. In another embodiment, the KLK3 peptide
is a fragment of any other KLK3 peptide known in the art. Each type
of KLK3 peptide represents a separate embodiment of the methods and
compositions as provided herein.
[0219] "KLK3 peptide" refers, in another embodiment, to a
full-length KLK3 protein. In another embodiment, the term refers to
a fragment of a KLK3 protein. In another embodiment, the term
refers to a fragment of a KLK3 protein that is lacking the KLK3
signal peptide. In another embodiment, the term refers to a KLK3
protein that contains the entire KLK3 sequence except the KLK3
signal peptide. "KLK3 signal sequence" refers, in another
embodiment, to any signal sequence found in nature on a KLK3
protein. In another embodiment, a KLK3 protein of methods and
compositions as provided herein does not contain any signal
sequence. Each possibility represents a separate embodiment of the
methods and compositions as provided herein.
[0220] In another embodiment, the kallikrein-related peptidase 3
(KLK3 protein) that is the source of a KLK3 peptide for use in the
methods and compositions as provided herein is a PSA protein. In
another embodiment, the KLK3 protein is a P-30 antigen protein. In
another embodiment, the KLK3 protein is a gamma-seminoprotein
protein. In another embodiment, the KLK3 protein is a kallikrein 3
protein. In another embodiment, the KLK3 protein is a semenogelase
protein. In another embodiment, the KLK3 protein is a seminin
protein. In another embodiment, the KLK3 protein is any other type
of KLK3 protein that is known in the art. Each possibility
represents a separate embodiment of the methods and compositions as
provided herein.
[0221] In another embodiment, the KLK3 protein is a splice variant
1 KLK3 protein. In another embodiment, the KLK3 protein is a splice
variant 2 KLK3 protein. In another embodiment, the KLK3 protein is
a splice variant 3 KLK3 protein. In another embodiment, the KLK3
protein is a transcript variant 1 KLK3 protein. In another
embodiment, the KLK3 protein is a transcript variant 2 KLK3
protein. In another embodiment, the KLK3 protein is a transcript
variant 3 KLK3 protein. In another embodiment, the KLK3 protein is
a transcript variant 4 KLK3 protein. In another embodiment, the
KLK3 protein is a transcript variant 5 KLK3 protein. In another
embodiment, the KLK3 protein is a transcript variant 6 KLK3
protein. In another embodiment, the KLK3 protein is a splice
variant RP5 KLK3 protein. In another embodiment, the KLK3 protein
is any other splice variant KLK3 protein known in the art. In
another embodiment, the KLK3 protein is any other transcript
variant KLK3 protein known in the art. Each possibility represents
a separate embodiment of the methods and compositions as provided
herein.
[0222] In another embodiment, the KLK3 protein is a mature KLK3
protein. In another embodiment, the KLK3 protein is a pro-KLK3
protein. In another embodiment, the leader sequence has been
removed from a mature KLK3 protein of methods and compositions as
provided herein. Each possibility represents a separate embodiment
of the methods and compositions as provided herein.
[0223] In another embodiment, the KLK3 protein that is the source
of a KLK3 peptide of methods and compositions as provided herein is
a human KLK3 protein. In another embodiment, the KLK3 protein is a
primate KLK3 protein. In another embodiment, the KLK3 protein is a
KLK3 protein of any other species known in the art. In another
embodiment, one of the above KLK3 proteins is referred to in the
art as a "KLK3 protein." Each possibility represents a separate
embodiment of the methods and compositions as provided herein.
[0224] The term "isoform" refers to a version of a molecule, for
example, a protein, with only slight differences compared to
another isoform, or version, of the same protein. In one
embodiment, isoforms may be produced from different but related
genes, or in another embodiment, may arise from the same gene by
alternative splicing. In another embodiment, isoforms are caused by
single nucleotide polymorphisms.
[0225] The term, "fragment" refers to a protein or polypeptide that
is shorter or comprises fewer amino acids than the full length
protein or polypeptide. In another embodiment, fragment refers to a
nucleic acid that is shorter or comprises fewer nucleotides than
the full length nucleic acid. In another embodiment, the fragment
is an N-terminal fragment. In another embodiment, the fragment is a
C-terminal fragment. In one embodiment, the fragment is an
intrasequential section of the protein, peptide, or nucleic acid.
In one embodiment, the fragment is a functional fragment. In
another embodiment, the fragment is an immunogenic fragment. In one
embodiment, a fragment has 10-20 nucleic or amino acids, while in
another embodiment, a fragment has more than 5 nucleic or amino
acids, while in another embodiment, a fragment has 100-200 nucleic
or amino acids, while in another embodiment, a fragment has 100-500
nucleic or amino acids, while in another embodiment, a fragment has
50-200 nucleic or amino acids, while in another embodiment, a
fragment has 10-250 nucleic or amino acids.
[0226] The term, "immunogenicity" or "immunogenic" is used herein
to refer to the innate ability of a protein, peptide, nucleic acid,
antigen or organism to elicit an immune response in an animal when
the protein, peptide, nucleic acid, antigen or organism is
administered to the animal. Thus, "enhancing the immunogenicity" in
one embodiment, refers to increasing the ability of a protein,
peptide, nucleic acid, antigen or organism to elicit an immune
response in an animal when the protein, peptide, nucleic acid,
antigen or organism is administered to an animal. The increased
ability of a protein, peptide, nucleic acid, antigen or organism to
elicit an immune response can be measured by, in one embodiment, a
greater number of antibodies to a protein, peptide, nucleic acid,
antigen or organism, a greater diversity of antibodies to an
antigen or organism, a greater number of T-cells specific for a
protein, peptide, nucleic acid, antigen or organism, a greater
cytotoxic or helper T-cell response to a protein, peptide, nucleic
acid, antigen or organism, and the like.
[0227] In one embodiment, a PSA antigen comprises a truncated PSA
open reading frame (GenBank Accession Number NM_001648), lacking
its secretory signal sequence the first 24 AA. The truncated PSA
may be amplified using the primers: Adv60-PSA(XhoI-no ATG)F:
gtgCTCGAGattgtgggaggctgggagtg (SEQ ID No: 46) and
Adv61-PSA(SpeI-Stop)R: gatACTAGTttaggggttggccacgatgg (SEQ ID No:
47) and may be subcloned in-frame with the first 441 amino acids of
LLO to create a tLLO-PSA fusion polypeptide of this invention. The
AA sequence of LLO-PSA is as follows:
TABLE-US-00003 (SEQ ID No: 48; PSA sequence is underlined)
MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASP
KTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEY
IVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPV
KRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQA
YPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQ
EEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYIS
SVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSF
KAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFL
KDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYD
LEIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKS
VILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHD
LMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPK
KLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLV
CYGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP.
In some embodiments, a live-attenuated Listeria monocytogenes
comprises a tLLO-PSA fusion polypeptide comprising the sequence SEQ
ID NO: 48. In some embodiments, a live-attenuated Listeria
monocytogenes comprises a tLLO-PSA fusion polypeptide consisting
essentially of the sequence SEQ ID NO: 48. In some embodiments, a
live-attenuated Listeria monocytogenes comprises a tLLO-PSA fusion
polypeptide consisting of the sequence SEQ ID NO: 48.
[0228] There is one AA difference between this PSA and the sequence
in NM_001648, at position N 221 Y).
[0229] In one embodiment, a recombinant fusion polypeptide of
methods and compositions of the present invention is an LLO-KLK3
fusion polypeptide. In another embodiment, the fusion polypeptide
has the sequence set forth in SEQ ID No: 48. In another embodiment,
the fusion polypeptide is homologous to the sequence set forth in
SEQ ID No: 48. In another embodiment, the fusion polypeptide is a
variant of the sequence set forth in SEQ ID No: 48. In another
embodiment, "homology" refers to identity to one of SEQ ID No: 48
of greater than 72%. In another embodiment, the homology is greater
than 75%. In another embodiment, "homology" refers to identity to a
sequence of greater than 78%. In another embodiment, the homology
is greater than 80%. In another embodiment, the homology is greater
than 82%. In another embodiment, "homology" refers to identity to a
sequence of greater than 83%. In another embodiment, the homology
is greater than 85%. In another embodiment, the homology is greater
than 87%. In another embodiment, "homology" refers to identity to a
sequence of greater than 88%. In another embodiment, the homology
is greater than 90%. In another embodiment, the homology is greater
than 92%. In another embodiment, "homology" refers to identity to a
sequence of greater than 93%. In another embodiment, the homology
is greater than 95%. In another embodiment, "homology" refers to
identity to a sequence of greater than 96%. In another embodiment,
the homology is greater than 97%. In another embodiment, the
homology is greater than 98%. In another embodiment, the homology
is greater than 99%. Each possibility represents a separate
embodiment of the present invention.
[0230] In one embodiment, the truncated LLO comprises a PEST amino
acid (AA) sequence. In another embodiment, the PEST amino acid
sequence is KENSISSMAPPASPPASPKTPIEKKHADEIDK (SEQ ID NO: 49). In
another embodiment, fusion of an antigen to other LM PEST AA
sequences from Listeria will also enhance immunogenicity of the
antigen.
[0231] The N-terminal LLO protein fragment of methods and
compositions of the present invention comprises, in another
embodiment, SEQ ID No: 54. In another embodiment, the fragment
comprises an LLO signal peptide. In another embodiment, the
fragment comprises SEQ ID No: 55. In another embodiment, the
fragment consists approximately of SEQ ID No: 55. In another
embodiment, the fragment consists essentially of SEQ ID No: 54. In
another embodiment, the fragment corresponds to SEQ ID No: 55. In
another embodiment, the fragment is homologous to SEQ ID No: 55. In
another embodiment, the fragment is homologous to a fragment of SEQ
ID No: 55. In some embodiments, the ALLO fused to a PSA antigen is
416 AA long (exclusive of the signal sequence), as 88 residues from
the amino terminus which is inclusive of the activation domain
containing cysteine 484 were truncated. It will be clear to those
skilled in the art that any ALLO without the activation domain, and
in particular without cysteine 484, are suitable for methods and
compositions of the present invention. In another embodiment,
fusion of a heterologous antigen to any ALLO, including the PEST AA
sequence, SEQ ID NO: 49, enhances cell mediated and anti-tumor
immunity of the antigen. Each possibility represents a separate
embodiment of the present invention.
[0232] The LLO protein utilized to construct strains of the present
invention has, in another embodiment, the sequence:
[0233] MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIE
KKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQ
NNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKI
VVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGT
AFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQ
ALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNI
IKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNE
LAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNW
SENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNRNISI
WGTTLYPKYSNKVDNPIE (GenBank Accession No. P13128; SEQ ID NO: 53;
nucleic acid sequence is set forth in GenBank Accession No.
X15127). The first 25 AA of the proprotein corresponding to this
sequence are the signal sequence and are cleaved from LLO when it
is secreted by the bacterium. Thus, in this embodiment, the full
length active LLO protein is 504 residues long. In another
embodiment, the above LLO fragment is used as the source of the LLO
fragment incorporated in a strain of the present invention. Each
possibility represents a separate embodiment of the present
invention.
[0234] In another embodiment, the N-terminal fragment of an LLO
protein utilized in compositions and methods of the present
invention has the sequence:
TABLE-US-00004 (SEQ ID NO: 54)
MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASP
KTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEY
IVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPV
KRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQA
YSNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQ
EEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYIS
SVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSF
KAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFL
KDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYD.
[0235] In another embodiment, the LLO fragment corresponds to about
AA 20-442 of an LLO protein utilized herein.
[0236] In another embodiment, the LLO fragment has the
sequence:
TABLE-US-00005 (SEQ ID NO: 55)
MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSVAPPASPPASP
KTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEY
IVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPV
KRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQA
YSNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQ
EEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYIS
SVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSF
KAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFL
KDNELAVIKNNSEYIETTSKAYTD.
[0237] In another embodiment, "truncated LLO" or "ALLO" refers to a
fragment of LLO that comprises the PEST-like domain. In another
embodiment, the terms refer to an LLO fragment that comprises a
PEST amino acid sequence.
[0238] In another embodiment, the terms refer to an LLO fragment
that does not contain the activation domain at the amino terminus
and does not include cysteine 484. In another embodiment, the terms
refer to an LLO fragment that is not hemolytic. In another
embodiment, the LLO fragment is rendered non-hemolytic by deletion
or mutation of the activation domain. In another embodiment, the
LLO fragment is rendered non-hemolytic by deletion or mutation of
cysteine 484. In another embodiment, the LLO fragment is rendered
non-hemolytic by deletion or mutation at another location. In
another embodiment, the LLO is rendered non-hemolytic by a deletion
or mutation of the cholesterol binding domain (CBD) as detailed in
U.S. Pat. No. 8,771,702, which is incorporated by reference herein.
In another embodiment, there is a mutation in a cholesterol-binding
domain (CBD) of the LLO or a fragment thereof, wherein said
mutation comprises a substitution of a 1-50 amino acid peptide
comprising a CBD. In one embodiment, there is a mutation in a CBD
of the LLO or a fragment thereof, wherein said mutation comprises a
substitution of residue C484, W491, W492 of SEQ ID NO: 53, alone or
in combination, wherein said recombinant protein exhibits a greater
than 100-fold reduction in hemolytic activity relative to wild-type
LLO. Each possibility represents a separate embodiment of the
present invention.
[0239] In another embodiment, the LLO fragment consists of about
the first 441 AA of the LLO protein. In another embodiment, the LLO
fragment consists of about the first 420 AA of LLO. In another
embodiment, the LLO fragment is a non-hemolytic form of the LLO
protein.
[0240] In another embodiment, the LLO fragment consists of about
residues 1-25. In another embodiment, the LLO fragment consists of
about residues 1-50. In another embodiment, the LLO fragment
consists of about residues 1-75. In another embodiment, the LLO
fragment consists of about residues 1-100. In another embodiment,
the LLO fragment consists of about residues 1-125. In another
embodiment, the LLO fragment consists of about residues 1-150. In
another embodiment, the LLO fragment consists of about residues
1175. In another embodiment, the LLO fragment consists of about
residues 1-200. In another embodiment, the LLO fragment consists of
about residues 1-225. In another embodiment, the LLO fragment
consists of about residues 1-250. In another embodiment, the LLO
fragment consists of about residues 1-275. In another embodiment,
the LLO fragment consists of about residues 1-300. In another
embodiment, the LLO fragment consists of about residues 1-325. In
another embodiment, the LLO fragment consists of about residues
1-350. In another embodiment, the LLO fragment consists of about
residues 1-375. In another embodiment, the LLO fragment consists of
about residues 1-400. In another embodiment, the LLO fragment
consists of about residues 1-425. Each possibility represents a
separate embodiment of the present invention.
[0241] In another embodiment, the LLO fragment contains residues of
a homologous LLO protein that correspond to one of the above AA
ranges. The residue numbers need not, in another embodiment,
correspond exactly with the residue numbers enumerated above; e.g.
if the homologous LLO protein has an insertion or deletion,
relative to an LLO protein utilized herein, then the residue
numbers can be adjusted accordingly. In another embodiment, the LLO
fragment is any other LLO fragment known in the art.
[0242] In another embodiment, a homologous LLO refers to identity
to an LLO sequence (e.g. to one of SEQ ID No: 53-55) of greater
than 70%. In another embodiment, a homologous LLO refers to
identity to one of SEQ ID No: 53-55 of greater than 72%. In another
embodiment, a homologous refers to identity to one of SEQ ID No:
53-55 of greater than 75%. In another embodiment, a homologous
refers to identity to one of SEQ ID No: 53-55 of greater than 78%.
In another embodiment, a homologous refers to identity to one of
SEQ ID No: 53-55 of greater than 80%. In another embodiment, a
homologous refers to identity to one of SEQ ID No: 53-55 of greater
than 82%. In another embodiment, a homologous refers to identity to
one of SEQ ID No: 53-55 of greater than 83%. In another embodiment,
a homologous refers to identity to one of SEQ ID No: 53-55 of
greater than 85%. In another embodiment, a homologous refers to
identity to one of SEQ ID No: 53-55 of greater than 87%. In another
embodiment, a homologous refers to identity to one of SEQ ID No:
53-55 of greater than 88%. In another embodiment, a homologous
refers to identity to one of SEQ ID No: 53-55 of greater than 90%.
In another embodiment, a homologous refers to identity to one of
SEQ ID No: 53-55 of greater than 92%. In another embodiment, a
homologous refers to identity to one of SEQ ID No: 53-55 of greater
than 93%. In another embodiment, a homologous refers to identity to
one of SEQ ID No: 53-55 of greater than 95%. In another embodiment,
a homologous refers to identity to one of SEQ ID No: 53-55 of
greater than 96%. In another embodiment, a homologous refers to
identity to one of SEQ ID No: 53-55 of greater than 97%. In another
embodiment, a homologous refers to identity to one of SEQ ID No:
53-55 of greater than 98%. In another embodiment, a homologous
refers to identity to one of SEQ ID No: 53-55 of greater than 99%.
In another embodiment, a homologous refers to identity to one of
SEQ ID No: 53-55 of 100%. Each possibility represents a separate
embodiment of the present invention.
[0243] The amino acid and nucleotide sequences from United States
Publication Nos. US-2007-0253976-A1 and US-2011-0129499-A1 are
incorporated herein in their entirety.
[0244] The term "homologue" refers to a nucleic acid or amino acid
sequence which shares a certain percentage of sequence identity
with a particular nucleic acid or amino acid sequence. In one
embodiment, a sequence useful in the composition and methods as
provided herein may be a homologue of a particular LLO sequence or
N-terminal fragment thereof. In another embodiment, a sequence
useful in the composition and methods as provided herein may be a
homologue of an antigenic polypeptide, which in one embodiment, is
KLK3 or a functional fragment thereof. In one embodiment, a homolog
of a polypeptide and, in one embodiment, the nucleic acid encoding
such a homolog, of the present invention maintains the functional
characteristics of the parent polypeptide. For example, in one
embodiment, a homolog of an antigenic polypeptide of the present
invention maintains the antigenic characteristic of the parent
polypeptide. In another embodiment, a sequence useful in the
composition and methods as provided herein may be a homologue of
any sequence described herein. In one embodiment, a homologue
shares at least 70% identity with a particular sequence. In another
embodiment, a homologue shares at least 72% identity with a
particular sequence. In another embodiment, a homologue shares at
least 75% identity with a particular sequence. In another
embodiment, a homologue shares at least 78% identity with a
particular sequence. In another embodiment, a homologue shares at
least 80% identity with a particular sequence. In another
embodiment, a homologue shares at least 82% identity with a
particular sequence. In another embodiment, a homologue shares at
least 83% identity with a particular sequence. In another
embodiment, a homologue shares at least 85% identity with a
particular sequence. In another embodiment, a homologue shares at
least 87% identity with a particular sequence. In another
embodiment, a homologue shares at least 88% identity with a
particular sequence. In another embodiment, a homologue shares at
least 90% identity with a particular sequence. In another
embodiment, a homologue shares at least 92% identity with a
particular sequence. In another embodiment, a homologue shares at
least 93% identity with a particular sequence. In another
embodiment, a homologue shares at least 95% identity with a
particular sequence. In another embodiment, a homologue shares at
least 96% identity with a particular sequence. In another
embodiment, a homologue shares at least 97% identity with a
particular sequence. In another embodiment, a homologue shares at
least 98% identity with a particular sequence. In another
embodiment, a homologue shares at least 99% identity with a
particular sequence. In another embodiment, a homologue shares 100%
identity with a particular sequence. Each possibility represents a
separate embodiment as provided herein.
[0245] Homology is, in one embodiment, determined by computer
algorithm for sequence alignment, by methods well described in the
art. For example, computer algorithm analysis of nucleic acid
sequence homology may include the utilization of any number of
software packages available, such as, for example, the BLAST,
DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and
TREMBL packages.
[0246] In another embodiment, homology is determined via
determination of candidate sequence hybridization, methods of which
are well described in the art (See, for example, "Nucleic Acid
Hybridization" Hames, B. D., and Higgins S. J., Eds. (1985);
Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Press, N.Y.; and Ausubel et al., 1989, Current
Protocols in Molecular Biology, Green Publishing Associates and
Wiley Interscience, N.Y). For example methods of hybridization may
be carried out under moderate to stringent conditions, to the
complement of a DNA encoding a native caspase peptide.
Hybridization conditions being, for example, overnight incubation
at 42.degree. C. in a solution comprising: 10-20% formamide,
5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 5.times.Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA.
[0247] In one embodiment, it is to be understood that a homolog of
any of the sequences as provided herein and/or as described herein
is considered to be a part of the invention.
[0248] In another embodiment, a recombinant Listeria strain of the
methods and compositions as provided herein comprise a nucleic acid
molecule encoding a PSA fusion polypeptide operably integrated into
the Listeria genome as an open reading frame with an endogenous
ActA sequence. In another embodiment, a recombinant Listeria strain
of the methods and compositions as provided herein comprise an
episomal expression vector comprising a nucleic acid molecule
encoding PSA fusion protein comprising an antigen fused to an ActA
or a truncated ActA. In one embodiment, the expression and
secretion of the antigen is under the control of an actA promoter
and ActA signal sequence and it is expressed as fusion to 1-233
amino acids of ActA (truncated ActA or tActA). In another
embodiment, the truncated ActA consists of the first 390 amino
acids of the wild type ActA protein as described in U.S. Pat. No.
7,655,238, which is incorporated by reference herein in its
entirety. In another embodiment, the truncated ActA is an ActA-N100
or a modified version thereof (referred to as ActA-N100*) in which
a PEST motif has been deleted and containing the nonconservative
QDNKR substitution as described in US Patent Publication Serial No.
2014/0186387.
[0249] The term "functional" within the meaning of the invention,
is used herein to refer to the innate ability of a protein,
peptide, nucleic acid, fragment or a variant thereof to exhibit a
biological activity or function. In one embodiment, such a
biological function is its binding property to an interaction
partner, e.g., a membrane-associated receptor, and in another
embodiment, its trimerization property. In the case of functional
fragments and the functional variants of the invention, these
biological functions may in fact be changed, e.g., with respect to
their specificity or selectivity, but with retention of the basic
biological function.
[0250] In another embodiment, a "functional fragment" is an
immunogenic fragment and elicits an immune response when
administered to a subject alone or in a strain composition provided
herein. In another embodiment, a functional fragment has biological
activity as will be understood by a skilled artisan and as further
provided herein.
[0251] The term "nucleic acids" or "nucleotide" refers to a string
of at least two base-sugar-phosphate combinations. The term
includes, in one embodiment, DNA and RNA. "Nucleotides" refers, in
one embodiment, to the monomeric units of nucleic acid polymers.
RNA may be, in one embodiment, in the form of a tRNA (transfer
RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA
(messenger RNA), anti-sense RNA, small inhibitory RNA (siRNA),
micro RNA (miRNA) and ribozymes. The use of siRNA and miRNA has
been described (Caudy A A et al, Genes & Devel 16: 2491-96 and
references cited therein). DNA may be in form of plasmid DNA, viral
DNA, linear DNA, or chromosomal DNA or derivatives of these groups.
In addition, these forms of DNA and RNA may be single, double,
triple, or quadruple stranded. The term also includes, in another
embodiment, artificial nucleic acids that may contain other types
of backbones but the same bases. In one embodiment, the artificial
nucleic acid is a PNA (peptide nucleic acid). PNA contain peptide
backbones and nucleotide bases and are able to bind, in one
embodiment, to both DNA and RNA molecules. In another embodiment,
the nucleotide is oxetane modified. In another embodiment, the
nucleotide is modified by replacement of one or more phosphodiester
bonds with a phosphorothioate bond. In another embodiment, the
artificial nucleic acid contains any other variant of the phosphate
backbone of native nucleic acids known in the art. The use of
phosphothiorate nucleic acids and PNA are known to those skilled in
the art, and are described in, for example, Neilsen P E, Curr Opin
Struct Biol 9:353-57; and Raz N K et al Biochem Biophys Res Commun.
297:1075-84. The production and use of nucleic acids is known to
those skilled in art and is described, for example, in Molecular
Cloning, (2001), Sambrook and Russell, eds. and Methods in
Enzymology: Methods for molecular cloning in eukaryotic cells
(2003) Purchio and G. C. Fareed. Each nucleic acid derivative
represents a separate embodiment as provided herein.
[0252] The terms "polypeptide," "peptide" and "recombinant peptide"
refer, in another embodiment, to a peptide or polypeptide of any
length. In another embodiment, a peptide or recombinant peptide as
provided herein has one of the lengths enumerated above for an
HMW-MAA fragment. Each possibility represents a separate embodiment
of the methods and compositions as provided herein. In one
embodiment, the term "peptide" refers to native peptides (either
degradation products, synthetically synthesized peptides or
recombinant peptides) and/or peptidomimetics (typically,
synthetically synthesized peptides), such as peptoids and
semipeptoids which are peptide analogs, which may have, for
example, modifications rendering the peptides more stable while in
a body or more capable of penetrating into cells. Such
modifications include, but are not limited to N terminus
modification, C terminus modification, peptide bond modification,
including, but not limited to, CH2-NH, CH2-S, CH2-S.dbd.O,
O.dbd.C--NH, CH2-O, CH2-CH2, S.dbd.C--NH, CH.dbd.CH or CF.dbd.CH,
backbone modifications, and residue modification. Methods for
preparing peptidomimetic compounds are well known in the art and
are specified, for example, in Quantitative Drug Design, C.A.
Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which
is incorporated by reference as if fully set forth herein. Further
details in this respect are provided hereinunder.
[0253] The term "antigenic polypeptide" is used herein to refer to
a polypeptide, peptide or recombinant peptide as described
hereinabove that is foreign to a host and leads to the mounting of
an immune response when present in, or, in another embodiment,
detected by, the host.
[0254] In one embodiment, "antigenic polypeptide" is used herein to
refer to a polypeptide, peptide recombinant polypeptide or
recombinant peptide as described herein that is processed and
presented on MHC class I and/or class II molecules present in a
subject's cells leading to the mounting of an immune response when
present in, or, in another embodiment, detected by, the host. In
one embodiment, the antigen may be foreign to the host. In another
embodiment, the antigen might be present in the host but the host
does not elicit an immune response against it because of
immunologic tolerance.
[0255] In one embodiment, the antigen is a tumor-associated
antigen. In one embodiment, the tumor-associated antigen is PSA. In
one embodiment, the recombinant attenuated Listeria strain of the
compositions and methods as provided herein express a PSA
polypeptide that is expressed by a tumor cell. In one embodiment,
the recombinant Listeria strain of the compositions and methods as
provided herein comprise a first or second nucleic acid molecule
that encodes a PSA, which in one embodiment, is a marker for
prostate cancer that is highly expressed by prostate tumors. In one
embodiment, PSA is a kallikrein serine protease (KLK3) secreted by
prostatic epithelial cells, which in one embodiment, is widely used
as a marker for prostate cancer.
[0256] Peptide bonds (--CO--NH--) within the peptide may be
substituted, for example, by N-methylated bonds (--N(CH3)-CO--),
ester bonds (--C(R)H--C--O--O--C(R)--N--), ketomethylen bonds
(--CO--CH2-), *-aza bonds (--NH--N(R)--CO--), wherein R is any
alkyl, e.g., methyl, carba bonds (--CH2-NH--), hydroxyethylene
bonds (--CH(OH)--CH2-), thioamide bonds (--CS--NH--), olefinic
double bonds (--CH.dbd.CH--), retro amide bonds (--NH--CO--),
peptide derivatives (--N(R)--CH2-CO--), wherein R is the "normal"
side chain, naturally presented on the carbon atom.
[0257] These modifications can occur at any of the bonds along the
peptide chain and even at several (2-3) at the same time. Natural
aromatic amino acids, Trp, Tyr and Phe, may be substituted for
synthetic non-natural acid such as TIC, naphthylelanine (Nol),
ring-methylated derivatives of Phe, halogenated derivatives of Phe
or o-methyl-Tyr.
[0258] In addition to the above, the peptides as provided herein
may also include one or more modified amino acids or one or more
non-amino acid monomers (e.g. fatty acids, complex carbohydrates
etc).
[0259] The term "oligonucleotide" is interchangeable with the term
"nucleic acid", and may refer to a molecule, which may include, but
is not limited to, prokaryotic sequences, eukaryotic mRNA, cDNA
from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g.,
mammalian) DNA, and even synthetic DNA sequences. The term also
refers to sequences that include any of the known base analogs of
DNA and RNA.
[0260] It will be appreciated by the skilled artisan that the term
"PEST sequence-containing polypeptide" or "PEST sequence-containing
protein" may encompass a truncated LLO protein, which in one
embodiment is a N-terminal LLO, and a truncated ActA protein which
in one embodiment is an N-terminal LLO, or fragments thereof. It
will also be appreciated by the skilled artisan that the term
"PEST-sequence containing peptide" may encompass a PEST sequence
peptide or peptide fragments of an LLO protein or an ActA protein
thereof. PEST sequence peptides are known in the art and are
described in U.S. Pat. No. 7,635,479, and in US Patent Publication
Serial No. 2014/0186387, both of which are hereby incorporated in
their entirety herein.
[0261] In another embodiment, a PEST sequence of prokaryotic
organisms can be identified routinely in accordance with methods
such as described by Rechsteiner and Roberts (TBS 21:267-271, 1996)
for L. monocytogenes. Alternatively, PEST amino acid sequences from
other prokaryotic organisms can also be identified based by this
method. Other prokaryotic organisms wherein PEST amino acid
sequences would be expected to include, but are not limited to,
other Listeria species. For example, the L. monocytogenes protein
ActA contains four such sequences. These are KTEEQPSEVNTGPR (SEQ ID
NO: 56), KASVTDTSEGDLDSSMQSADESTPQPLK (SEQ ID NO: 57),
KNEEVNASDFPPPPTDEELR (SEQ ID NO: 58), and
RGGIPTSEEFSSLNSGDFTDDENSETTEEEIDR (SEQ ID NO: 59). Also
Streptolysin O from Streptococcus sp. contain a PEST sequence. For
example, Streptococcus pyogenes Streptolysin O comprises the PEST
sequence KQNTASTETTTTNEQPK (SEQ ID NO: 60) at amino acids 35-51 and
Streptococcus equisimilis Streptolysin O comprises the PEST-like
sequence KQNTANTETTTTNEQPK (SEQ ID NO: 61) at amino acids 38-54.
Further, it is believed that the PEST sequence can be embedded
within the antigenic protein. Thus, for purposes of the present
invention, by "fusion" when in relation to PEST sequence fusions,
it is meant that the antigenic protein comprises both the antigen,
for example PSA, and the PEST amino acid sequence either linked at
one end of the antigen or embedded within the antigen.
[0262] In another embodiment, the construct or nucleic acid
molecule is expressed from an episomal or plasmid vector, with a
nucleic acid sequence encoding a PEST sequence-containing
polypeptide or a PEST-sequence peptide. In another embodiment, the
plasmid is stably maintained in the recombinant Listeria strain
strain in the absence of antibiotic selection. In another
embodiment, the plasmid does not confer antibiotic resistance upon
the recombinant Listeria. In another embodiment, the fragment is a
functional fragment. In another embodiment, the fragment is an
immunogenic fragment.
[0263] The term "Stably maintained" refers, in another embodiment,
to maintenance of a nucleic acid molecule or plasmid in the absence
of selection (e.g. antibiotic selection) for 10 generations,
without detectable loss. In another embodiment, the period is 15
generations. In another embodiment, the period is 20 generations.
In another embodiment, the period is 25 generations. In another
embodiment, the period is 30 generations. In another embodiment,
the period is 40 generations. In another embodiment, the period is
50 generations. In another embodiment, the period is 60
generations. In another embodiment, the period is 80 generations.
In another embodiment, the period is 100 generations. In another
embodiment, the period is 150 generations. In another embodiment,
the period is 200 generations. In another embodiment, the period is
300 generations. In another embodiment, the period is 500
generations. In another embodiment, the period is more than 500
generations. In another embodiment, the nucleic acid molecule or
plasmid is maintained stably in vitro (e.g. in culture). In another
embodiment, the nucleic acid molecule or plasmid is maintained
stably in vivo. In another embodiment, the nucleic acid molecule or
plasmid is maintained stably both in vitro and in vitro. Each
possibility represents a separate embodiment of the methods and
compositions as provided herein.
[0264] The term "amino acid" or "amino acids" is understood to
include the 20 naturally occurring amino acids; those amino acids
often modified post-translationally in vivo, including, for
example, hydroxyproline, phosphoserine and phosphothreonine; and
other unusual amino acids including, but not limited to,
2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,
nor-leucine and omithine. Furthermore, the term "amino acid" may
include both D- and L-amino acids.
[0265] The term "nucleic acid" or "nucleic acid sequence" refers to
a deoxyribonucleotide or ribonucleotide oligonucleotide in either
single- or double-stranded form. The term encompasses nucleic
acids, i.e., oligonucleotides, containing known analogues of
natural nucleotides which have similar or improved binding
properties, for the purposes desired, as the reference nucleic
acid. The term also includes nucleic acids which are metabolized in
a manner similar to naturally occurring nucleotides or at rates
that are improved thereover for the purposes desired. The term also
encompasses nucleic-acid-like structures with synthetic backbones.
DNA backbone analogues provided by the invention include
phosphodiester, phosphorothioate, phosphorodithioate,
methylphosphonate, phosphoramidate, alkyl phosphotriester,
sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate,
morpholino carbamate, and peptide nucleic acids (PNAs); see, e.g.,
Oligonucleotides and Analogues, a Practical Approach, edited by F.
Eckstein, IRL Press at Oxford University Press (1991); Antisense
Strategies, Annals of the New York Academy of Sciences, Volume 600,
Eds. Baserga and Denhardt (NYAS 1992); Mulligan (1993) J. Med.
Chem. 36:1923-1937; Antisense Research and Applications (1993, CRC
Press). PNAs contain non-ionic backbones, such as N-(2-aminoethyl)
glycine units. Phosphorothioate linkages are described, e.g., in WO
97/03211; WO 96/39154; Mata (1997) Toxicol. Appi. Pharmacol.
144:189-197. Other synthetic backbones encompasses by the term
include methyl-phosphonate linkages or alternating
methyiphosphonate and phosphodiester linkages (Strauss-Soukup
(1997) Biochemistry 36:8692-8698), and benzylphosphonate linkages
(Samstag (1996) Antisense Nucleic Acid Drug Dev. 6:153-156). The
term nucleic acid is used interchangeably with gene, cDNA, mRNA,
oligonucleotide primer, probe and amplification product.
[0266] In another embodiment, the construct or nucleic acid
molecule provided herein is integrated into the Listerial
chromosome using homologous recombination. Techniques for
homologous recombination are well known in the art, and are
described, for example, in Baloglu S, Boyle S M, et al. (Immune
responses of mice to vaccinia virus recombinants expressing either
Listeria monocytogenes partial listeriolysin or Brucella abortus
ribosomal L7/L12 protein. Vet Microbiol 2005, 109(1-2): 11-7); and
Jiang L L, Song H H, et al., (Characterization of a mutant Listeria
monocytogenes strain expressing green fluorescent protein. Acta
Biochim Biophys Sin (Shanghai) 2005, 37(1): 19-24). In another
embodiment, homologous recombination is performed as described in
U.S. Pat. No. 6,855,320. In another embodiment, a temperature
sensitive plasmid is used to select the recombinants. Each
technique represents a separate embodiment of the present
invention.
[0267] In another embodiment, the construct or nucleic acid
molecule is integrated into the Listerial chromosome using
transposon insertion. Techniques for transposon insertion are well
known in the art, and are described, inter alia, by Sun et al.
(Infection and Immunity 1990, 58: 3770-3778) in the construction of
DP-L967. Transposon mutagenesis has the advantage, in another
embodiment, that a stable genomic insertion mutant can be formed
but the disadvantage that the position in the genome where the
foreign gene has been inserted is unknown.
[0268] In another embodiment, the construct or nucleic acid
molecule is integrated into the Listerial chromosome using phage
integration sites (Lauer P, Chow M Y et al, Construction,
characterization, and use of two Listeria monocytogenes
site-specific phage integration vectors. J Bacteriol 2002; 184(15):
4177-86). In certain embodiments of this method, an integrase gene
and attachment site of a bacteriophage (e.g. U153 or PSA
listeriophage) is used to insert the heterologous gene into the
corresponding attachment site, which may be any appropriate site in
the genome (e.g. comK or the 3' end of the arg tRNA gene). In
another embodiment, endogenous prophages are cured from the
attachment site utilized prior to integration of the construct or
heterologous gene. In another embodiment, this method results in
single-copy integrants. In another embodiment, the present
invention further comprises a phage based chromosomal integration
system for clinical applications, where a host strain that is
auxotrophic for essential enzymes, including, but not limited to,
d-alanine racemase can be used, for example Lmdal(-)dat(-). In
another embodiment, in order to avoid a "phage curing step," a
phage integration system based on PSA is used. This requires, in
another embodiment, continuous selection by antibiotics to maintain
the integrated gene. Thus, in another embodiment, the current
invention enables the establishment of a phage based chromosomal
integration system that does not require selection with
antibiotics. Instead, an auxotrophic host strain can be
complemented. Each possibility represents a separate embodiment of
the present invention.
[0269] The term "recombination site" or "site-specific
recombination site" refers to a sequence of bases in a nucleic acid
molecule that is recognized by a recombinase (along with associated
proteins, in some cases) that mediates exchange or excision of the
nucleic acid segments flanking the recombination sites. The
recombinases and associated proteins are collectively referred to
as "recombination proteins" see, e.g., Landy, A., (Current Opinion
in Genetics & Development) 3:699-707; 1993).
[0270] The term "phage expression vector" or "phagemid" refers to
any phage-based recombinant expression system for the purpose of
expressing a nucleic acid sequence of the methods and compositions
as provided herein in vitro or in vivo, constitutively or
inducibly, in any cell, including prokaryotic, yeast, fungal,
plant, insect or mammalian cell. A phage expression vector
typically can both reproduce in a bacterial cell and, under proper
conditions, produce phage particles. The term includes linear or
circular expression systems and encompasses both phage-based
expression vectors that remain episomal or integrate into the host
cell genome.
[0271] The term "episomal expression vector" as described herein
refers to a nucleic acid vector which may be linear or circular,
and which is usually double-stranded in form. In one embodiemnt, an
episomal expression vector comprises a gene of interest. In another
embodiment, the inserted gene of interest is not interrupted or
subjected to regulatory constraints which often occur from
integration into cellular DNA. In another embodiment, the presence
of the inserted heterologous gene does not lead to rearrangement or
interruption of the cell's own important regions. In another
embodiment, episomal vectors persist in multiple copies in the
bacterial cytoplasm, resulting in amplification of the gene of
interest, and, in another embodiment, viral trans-acting factors
are supplied when necessary. In another embodiment, in stable
transfection procedures, the use of episomal vectors often results
in higher transfection efficiency than the use of
chromosome-integrating plasmids (Belt, P.B.G.M., et al (1991)
Efficient cDNA cloning by direct phenotypic correction of a mutant
human cell line (HPRT2) using an Epstein-Barr virus-derived cDNA
expression vector. Nucleic Acids Res. 19, 4861-4866; Mazda, O., et
al. (1997) Extremely efficient gene transfection into
lympho-hematopoietic cell lines by Epstein-Barr virus-based
vectors. J. Immunol. Methods 204, 143-151). In one embodiment, the
episomal expression vectors of the methods and compositions as
provided herein may be delivered to cells in vivo, ex vivo, or in
vitro by any of a variety of the methods employed to deliver DNA
molecules to cells. The vectors may also be delivered alone or in
the form of a pharmaceutical composition that enhances delivery to
cells of a subject.
[0272] The term "fused" refers to linkage by covalent bonding.
[0273] The term "Transforming," refers to engineering a bacterial
cell to take up a plasmid or other heterologous DNA molecule. In
another embodiment, "transforming" refers to engineering a
bacterial cell to express a gene of a plasmid or other heterologous
DNA molecule. Each possibility represents a separate embodiment of
the methods and compositions as provided herein.
[0274] In another embodiment, conjugation is used to introduce
genetic material and/or plasmids into bacteria. Methods for
conjugation are well known in the art, and are described, for
example, in Nikodinovic J et al (A second generation snp-derived
Escherichia coli-Streptomyces shuttle expression vector that is
generally transferable by conjugation. Plasmid. 2006 November;
56(3):223-7) and Auchtung J M et al (Regulation of a Bacillus
subtilis mobile genetic element by intercellular signaling and the
global DNA damage response. Proc Natl Acad Sci USA. 2005 Aug. 30;
102(35):12554-9). Each method represents a separate embodiment of
the methods and compositions as provided herein.
[0275] In one embodiment, the expression vector comprising a
nucleic acid molecule provided herein further comprises a second
open reading frame encoding a metabolic enzyme. In another
embodiment, the metabolic enzyme complements an endogenous gene
that is lacking in the chromosome of the recombinant Listeria
strain. In another embodiment, the metabolic enzyme encoded by the
open reading frame is an alanine racemase enzyme (dal). In another
embodiment, the metabolic enzyme encoded by the open reading frame
is a D-amino acid transferase enzyme (dat). In another embodiment,
the Listeria strains provided herein comprise a mutation in the
endogenous dal/dat genes. In another embodiment, the Listeria lacks
the dal/dat genes. In another embodiment, the Listeria lacks the
dal/dat/and actA genes.
[0276] In another embodiment, a nucleic acid molecule of the
methods and compositions of the present invention is operably
linked to a promoter/regulatory sequence. In another embodiment,
the nucleic acid sequence encoding PSA of methods and compositions
of the present invention is operably linked to a
promoter/regulatory sequence. In another embodiment, the second
open reading frame of methods and compositions of the present
invention is operably linked to a promoter/regulatory sequence. In
another embodiment, each of the open reading frames are operably
linked to a promoter/regulatory sequence. Each possibility
represents a separate embodiment of the present invention.
[0277] The term "Metabolic enzyme" refers, in one embodiment, to an
enzyme involved in synthesis of a nutrient required by the host
bacteria. In another embodiment, the term refers to an enzyme
required for synthesis of a nutrient required by the host bacteria.
In another embodiment, the term refers to an enzyme involved in
synthesis of a nutrient utilized by the host bacteria. In another
embodiment, the term refers to an enzyme involved in synthesis of a
nutrient required for sustained growth of the host bacteria. In
another embodiment, the enzyme is required for synthesis of the
nutrient. Each possibility represents a separate embodiment of the
methods and compositions as provided herein.
[0278] In one embodiment, the PSA antigen used in the invention is
associated with prostate cancer.
[0279] In one embodiment, strains as provided herein generate
effector T cells that are able to infiltrate the tumor, destroy
tumor cells and eradicate the disease. In one embodiment, naturally
occurring tumor infiltrating lymphocytes (TILs) are associated with
better prognosis in several tumors. Moreover, the infiltration of
the tumor by T cells has been associated with success of
immunotherapeutic approaches in both pre-clinical and human trials.
In one embodiment, the infiltration of lymphocytes into the tumor
site is dependent on the up-regulation of adhesion molecules in the
endothelial cells of the tumor vasculature, generally by
proinflammatory cytokines, such as IFN-.gamma., TNF-.alpha. and
IL-1. Several adhesion molecules have been implicated in the
process of lymphocyte infiltration into tumors, including
intercellular adhesion molecule 1 (ICAM-1), vascular endothelial
cell adhesion molecule 1 (V-CAM-1), vascular adhesion protein 1
(VAP-1) and E-selectin. However, these cell-adhesion molecules are
commonly down-regulated in the tumor vasculature. Thus, in one
embodiment, strains as provided herein increase TILs, up-regulate
adhesion molecules (in one embodiment, ICAM-1, V-CAM-1, VAP-1,
E-selectin, or a combination thereof), up-regulate pro-inflammatory
cytokines (in one embodiment, IFN-.gamma., TNF-.alpha., IL-1, or a
combination thereof), or a combination thereof.
[0280] The attenuated Listeria strain of methods and compositions
of the present invention is, in another embodiment, a attenuated
Listeria monocytogenes strain. In another embodiment, the Listeria
strain is a attenuated Listeria seeligeri strain. In another
embodiment, the Listeria strain is a attenuated Listeria grayi
strain. In another embodiment, the Listeria strain is a attenuated
Listeria ivanovii strain. In another embodiment, the Listeria
strain is a attenuated Listeria murrayi strain. In another
embodiment, the Listeria strain is a attenuated Listeria welshimeri
strain. In another embodiment, the Listeria strain is a attenuated
strain of any other Listeria species known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0281] In another embodiment, a recombinant Listeria strain of the
present invention has been passaged through an animal host. In
another embodiment, the passaging maximizes efficacy of the strain
as a strain vector. In another embodiment, the passaging stabilizes
the immunogenicity of the Listeria strain. In another embodiment,
the passaging stabilizes the virulence of the Listeria strain. In
another embodiment, the passaging increases the immunogenicity of
the Listeria strain. In another embodiment, the passaging increases
the virulence of the Listeria strain. In another embodiment, the
passaging removes unstable sub-strains of the Listeria strain. In
another embodiment, the passaging reduces the prevalence of
unstable sub-strains of the Listeria strain. In another embodiment,
the Listeria strain contains a genomic insertion of the gene
encoding the antigen-containing recombinant peptide. In another
embodiment, the Listeria strain carries a plasmid comprising the
gene encoding the antigen-containing recombinant peptide. In
another embodiment, the passaging is performed as described herein.
In another embodiment, the passaging is performed by any other
method known in the art. Each possibility represents a separate
embodiment of the present invention.
[0282] It is understood that wherever embodiments are described
herein with the language "comprising", otherwise analogous
embodiments described in terms of "consisting of" and/or
"consisting essentially of" are also provided.
[0283] Where aspects or embodiments of the invention are described
in terms of a Markush group or other grouping of alternatives, the
present invention encompasses not only the entire group listed as a
whole, but each member of the group individually and all possible
subgroups of the main group, but also the main group absent one or
more of the group members. The present invention also envisages the
explicit exclusion of one or more of any of the group members in
the claimed invention.
[0284] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict, the present specification, including definitions, will
control. Throughout this specification and claims, the word
"comprise," or variations such as "comprises" or "comprising" will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers. Unless otherwise required by context, singular terms
shall include pluralities and plural terms shall include the
singular. Any example(s) following the term "e.g." or "for example"
is not meant to be exhaustive or limiting.
[0285] Exemplary methods and materials are described herein,
although methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present invention. The materials, methods, and examples are
illustrative only and not intended to be limiting.
II. Methods, Uses and Medicaments
[0286] In one aspect of the invention, the invention provides a
method for treating a cancer in an individual comprising
administering to the individual a combination therapy which
comprises a PD-1 antagonist and a live-attenuated bacterial strain
that is used to stimulate APCs capable of driving a cellular immune
response to PSA expressing cells. In another aspect of the
invention, the invention provides a method for treating a cancer in
an individual comprising administering to the individual a
combination therapy which comprises a PD-1 antagonist and a
live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a tLLO. In yet another aspect of the invention, the
invention provides a method for treating a cancer in an individual
comprising administering to the individual a combination therapy
which comprises a PD-1 antagonist and an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain. In still
another aspect of the invention, the invention provides a method
for treating a cancer in an individual comprising administering to
the individual a combination therapy which comprises a PD-1
antagonist and an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain.
[0287] The combination therapy may also comprise one or more
additional therapeutic agents. The additional therapeutic agent may
be, e.g., a chemotherapeutic, a biotherapeutic agent (including but
not limited to antibodies to VEGF, VEGFR, EGFR, Her2/neu, other
growth factor receptors, CD20, CD40, CD-40L, CTLA-4, OX-40, 4-1BB,
and ICOS), an immunogenic agent (for example, attenuated cancerous
cells, tumor antigens, antigen presenting cells such as dendritic
cells pulsed with tumor derived antigen or nucleic acids, immune
stimulating cytokines (for example, IL-2, IFN.alpha.2, GM-CSF), and
cells transfected with genes encoding immune stimulating cytokines
such as but not limited to GM-CSF).
[0288] Examples of chemotherapeutic agents include alkylating
agents such as thiotepa and cyclosphosphamide; alkyl sulfonates
such as busulfan, improsulfan and piposulfan; aziridines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlomaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, ranimustine; antibiotics such as
the enediyne antibiotics (e.g. calicheamicin, especially
calicheamicin gamma1I and calicheamicin phiIl, see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antibiotic chromomophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidamine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g. paclitaxel and doxetaxel; chlorambucil; gemcitabine;
6-thioguanine; mercaptopurine; methotrexate; platinum analogs such
as cisplatin and carboplatin; vinblastine; platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine;
novantrone; teniposide; edatrexate; daunomycin; aminopterin;
xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;
difluoromethylomithine (DMFO); retinoids such as retinoic acid;
capecitabine; and pharmaceutically acceptable salts, acids or
derivatives of any of the above. Also included are anti-hormonal
agents that act to regulate or inhibit hormone action on tumors
such as anti-estrogens and selective estrogen receptor modulators
(SERMs), including, for example, tamoxifen, raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and toremifene (Fareston); aromatase inhibitors that
inhibit the enzyme aromatase, which regulates estrogen production
in the adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, megestrol acetate, exemestane, formestane,
fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens
such as flutamide, nilutamide, bicalutamide, leuprolide, and
goserelin; and pharmaceutically acceptable salts, acids or
derivatives of any of the above.
[0289] Each therapeutic agent in a combination therapy of the
invention may be administered either alone or in a medicament (also
referred to herein as a pharmaceutical composition) which comprises
the therapeutic agent and one or more pharmaceutically acceptable
carriers, excipients and diluents, according to standard
pharmaceutical practice.
[0290] Each therapeutic agent in a combination therapy of the
invention may be administered simultaneously (i.e., in the same
medicament), concurrently (i.e., in separate medicaments
administered one right after the other in any order) or
sequentially in any order. Sequential administration is
particularly useful when the therapeutic agents in the combination
therapy are in different dosage forms (one agent is a tablet or
capsule and another agent is a sterile liquid) and/or are
administered on different dosing schedules, e.g., a
chemotherapeutic that is administered at least daily and a
biotherapeutic that is administered less frequently, such as once
weekly, once every two weeks, or once every three weeks.
[0291] In another embodiment, administration of a combination
therapy comprising Pembrolizumab (MK-3475) and a live-attenuated
bacterial strain that is used to stimulate APCs capable of driving
a cellular immune response to PSA expressing cells provides
synergistic antitumor activity. In another embodiment,
administration of a combination therapy comprising Pembrolizumab
(MK-3475) and a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO provides synergistic
antitumor activity. In another embodiment, administration of a
combination therapy comprising Pembrolizumab (MK-3475) and an
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain provides synergistic antitumor activity. In another
embodiment, administration of a combination therapy comprising
Pembrolizumab (MK-3475) and an LmddA-143 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome) strain provides synergistic antitumor activity.
[0292] Dosage units for a PD-1 antagonist (e.g., MK-3475) may be
expressed as a flat dose, i.e., 100 mg, 200 mg, 300 mg, or as a
patient-specific dose, i.e., mg/kg (mg therapeutic agent/kg of body
weight) or mg/m.sup.2 (quantity in milligrams per square meter of
body surface area).
[0293] In one embodiment, the dose of the attenuated Listeria
strain comprised by the immunogenic composition provided herein is
administered to a subject at a dose of
1.times.10.sup.7-3.31.times.10.sup.10 CFU. In another embodiment,
the dose is 1.times.10.sup.8-3.31.times.10.sup.10 CFU. In another
embodiment, the dose is 1.times.10.sup.9-3.31.times.10.sup.10 CFU.
In another embodiment, the dose is 5-500.times.10.sup.8 CFU. In
another embodiment, the dose is 7-500.times.10.sup.8 CFU. In
another embodiment, the dose is 10-500.times.10.sup.8 CFU. In
another embodiment, the dose is 20-500.times.10.sup.8 CFU. In
another embodiment, the dose is 30-500.times.10.sup.8 CFU. In
another embodiment, the dose is 50-500.times.10.sup.8 CFU. In
another embodiment, the dose is 70-500.times.10.sup.8 CFU. In
another embodiment, the dose is 100-500.times.10.sup.8 CFU. In
another embodiment, the dose is 150-500.times.10.sup.8 CFU. In
another embodiment, the dose is 5-300.times.10.sup.8 CFU. In
another embodiment, the dose is 5-200.times.10.sup.8 CFU. In
another embodiment, the dose is 5-150.times.10.sup.8 CFU. In
another embodiment, the dose is 5-100.times.10.sup.8 CFU. In
another embodiment, the dose is 5-70.times.10.sup.8 CFU. In another
embodiment, the dose is 5-50.times.10.sup.8 CFU. In another
embodiment, the dose is 5-30.times.10.sup.8 CFU. In another
embodiment, the dose is 5-20.times.10.sup.8 CFU. In another
embodiment, the dose is 1-30.times.10.sup.9 CFU. In another
embodiment, the dose is 1-20.times.10.sup.9 CFU. In another
embodiment, the dose is 2-30.times.10.sup.9 CFU. In another
embodiment, the dose is 1-10.times.10.sup.9 CFU. In another
embodiment, the dose is 2-10.times.10.sup.9 CFU. In another
embodiment, the dose is 3-10.times.10.sup.9 CFU. In another
embodiment, the dose is 2-7.times.10.sup.9 CFU. In another
embodiment, the dose is 2-5.times.10.sup.9 CFU. In another
embodiment, the dose is 3-5.times.10.sup.9 CFU. In another
embodiment, the dose is 0.5.times.10.sup.9 CFU. In another
embodiment, the dose is 1.times.10.sup.9 CFU. In another
embodiment, the dose is 5.times.10.sup.9 CFU. In another
embodiment, the dose is 1.times.10.sup.10 CFU.
[0294] In another embodiment, the dose is 1.times.10.sup.7
organisms. In another embodiment, the dose is 1.times.10.sup.8
organisms. In another embodiment, the dose is 1.times.10.sup.9
organisms. In another embodiment, the dose is 1.5.times.10.sup.9
organisms. In another embodiment, the dose is 2.times.10.sup.9
organisms. In another embodiment, the dose is 3.times.10.sup.9
organisms. In another embodiment, the dose is 4.times.10.sup.9
organisms. In another embodiment, the dose is 5.times.10.sup.9
organisms. In another embodiment, the dose is 6.times.10.sup.9
organisms. In another embodiment, the dose is 7.times.10.sup.9
organisms. In another embodiment, the dose is 8.times.10.sup.9
organisms. In another embodiment, the dose is 10.times.10.sup.9
organisms. In another embodiment, the dose is 1.5.times.10.sup.10
organisms. In another embodiment, the dose is 2.times.10.sup.10
organisms. In another embodiment, the dose is 2.5.times.10.sup.10
organisms. In another embodiment, the dose is 3.times.10.sup.10
organisms. In another embodiment, the dose is 3.3.times.10.sup.10
organisms. In another embodiment, the dose is 4.times.10.sup.10
organisms. In another embodiment, the dose is 5.times.10.sup.10
organisms. Each dose and range of doses represents a separate
embodiment of the present invention.
[0295] It will be appreciated by the skilled artisan that the term
"Boosting" may encompass administering an additional strain or
immunogenic composition or recombinant Listeria strain dose or
immune checkpoint inhibitor alone or in combination to a subject.
In another embodiment of methods of the present invention, 2 boosts
(or a total of 3 inoculations) are administered. In another
embodiment, 3 boosts are administered. In another embodiment, 4
boosts are administered. In another embodiment, 5 boosts are
administered. In another embodiment, 6 boosts are administered. In
another embodiment, more than 6 boosts are administered. Each
possibility represents a separate embodiment of the present
invention.
[0296] In another embodiment, a method of present invention further
comprises the step of boosting the subject with a recombinant
Listeria strain or immune checkpoint inhibitor as provided herein.
In another embodiment, the recombinant Listeria strain used in the
booster inoculation is the same as the strain used in the initial
"priming" inoculation. In another embodiment, the booster strain is
different from the priming strain. In another embodiment, the
recombinant immune checkpoint inhibitor used in the booster
inoculation is the same as the inhibitor used in the initial
"priming" inoculation. In another embodiment, the booster inhibitor
is different from the priming inhibitor. In another embodiment, the
same doses are used in the priming and boosting inoculations. In
another embodiment, a larger dose is used in the booster. In
another embodiment, a smaller dose is used in the booster. In
another embodiment, the methods of the present invention further
comprise the step of administering to the subject a booster
vaccination. In one embodiment, the booster vaccination follows a
single priming vaccination. In another embodiment, a single booster
vaccination is administered after the priming vaccinations. In
another embodiment, two booster vaccinations are administered after
the priming vaccinations. In another embodiment, three booster
vaccinations are administered after the priming vaccinations. In
one embodiment, the period between a prime and a boost strain is
experimentally determined by the skilled artisan. In another
embodiment, the period between a prime and a boost strain is 1
week, in another embodiment it is 2 weeks, in another embodiment,
it is 3 weeks, in another embodiment, it is 4 weeks, in another
embodiment, it is 5 weeks, in another embodiment it is 6-8 weeks,
in yet another embodiment, the boost strain is administered 8-10
weeks after the prime strain.
[0297] In another embodiment, a method of the present invention
further comprises boosting the subject with a immunogenic
composition comprising an attenuated Listeria strain provided
herein. In another embodiment, a method of the present invention
comprises the step of administering a booster dose of the
immunogenic composition comprising the attenuated Listeria strain
provided herein. In another embodiment, the booster dose is an
alternate form of said immunogenic composition. In another
embodiment, the methods of the present invention further comprise
the step of administering to the subject a booster immunogenic
composition. In one embodiment, the booster dose follows a single
priming dose of said immunogenic composition. In another
embodiment, a single booster dose is administered after the priming
dose. In another embodiment, two booster doses are administered
after the priming dose. In another embodiment, three booster doses
are administered after the priming dose. In one embodiment, the
period between a prime and a boost dose of an immunogenic
composition comprising the attenuated Listeria provided herein is
experimentally determined by the skilled artisan. In another
embodiment, the dose is experimentally determined by a skilled
artisan. In another embodiment, the period between a prime and a
boost dose is 1 week, in another embodiment it is 2 weeks, in
another embodiment, it is 3 weeks, in another embodiment, it is 4
weeks, in another embodiment, it is 5 weeks, in another embodiment
it is 6-8 weeks, in yet another embodiment, the boost dose is
administered 8-10 weeks after the prime dose of the immunogenic
composition.
[0298] Heterologous "prime boost" strategies have been effective
for enhancing immune responses and protection against numerous
pathogens. Schneider et al., Immunol. Rev. 170:29-38 (1999);
Robinson, H. L., Nat. Rev. Immunol. 2:239-50 (2002); Gonzalo, R. M.
et al., Strain 20:1226-31 (2002); Tanghe, A., Infect. Immun.
69:3041-7 (2001). Providing antigen in different forms in the prime
and the boost injections appears to maximize the immune response to
the antigen. DNA strain priming followed by boosting with protein
in adjuvant or by viral vector delivery of DNA encoding antigen
appears to be the most effective way of improving antigen specific
antibody and CD4+ T-cell responses or CD8+ T-cell responses
respectively. Shiver J. W. et al., Nature 415: 331-5 (2002);
Gilbert, S. C. et al., Strain 20:1039-45 (2002); Billaut-Mulot, O.
et al., Strain 19:95-102 (2000); Sin, J. I. et al., DNA Cell Biol.
18:771-9 (1999). Recent data from monkey vaccination studies
suggests that adding CRL1005 poloxamer (12 kDa, 5% POE), to DNA
encoding the HIV gag antigen enhances T-cell responses when monkeys
are vaccinated with an HIV gag DNA prime followed by a boost with
an adenoviral vector expressing HIV gag (Ad5-gag). The cellular
immune responses for a DNA/poloxamer prime followed by an Ad5-gag
boost were greater than the responses induced with a DNA (without
poloxamer) prime followed by Ad5-gag boost or for Ad5-gag only.
Shiver, J. W. et al. Nature 415:331-5 (2002). U.S. Patent Appl.
Publication No. US 2002/0165172 A1 describes simultaneous
administration of a vector construct encoding an immunogenic
portion of an antigen and a protein comprising the immunogenic
portion of an antigen such that an immune response is generated.
The document is limited to hepatitis B antigens and HIV antigens.
Moreover, U.S. Pat. No. 6,500,432 is directed to methods of
enhancing an immune response of nucleic acid vaccination by
simultaneous administration of a polynucleotide and polypeptide of
interest. According to the patent, simultaneous administration
means administration of the polynucleotide and the polypeptide
during the same immune response, preferably within 0-10 or 3-7 days
of each other. The antigens contemplated by the patent include,
among others, those of Hepatitis (all forms), HSV, HIV, CMV, EBV,
RSV, VZV, HPV, polio, influenza, parasites (e.g., from the genus
Plasmodium), and pathogenic bacteria (including but not limited to
M. tuberculosis, M. leprae, Chlamydia, Shigella, B. burgdorferi,
enterotoxigenic E. coli, S. typhosa, H. pylori, V. cholerae, B.
pertussis, etc.). All of the above references are herein
incorporated by reference in their entireties.
[0299] In another embodiment, the recombinant polypeptide of
methods of the present invention is expressed by the recombinant
Listeria strain. In another embodiment, the expression is mediated
by a nucleotide molecule carried by the recombinant Listeria
strain. Each possibility represents a separate embodiment of the
present invention.
[0300] As used herein, the term "recombinant Listeria" in some
embodiments refers to an attenuated Listeria having all the same
meanings and qualities described throughout.
[0301] In another embodiment, a composition comprising a strain of
the present invention further comprises an adjuvant. In yet another
embodiment, a strain of the present invention may be administered
with an adjuvant. The adjuvant utilized in methods and compositions
of the present invention is, in another embodiment, a
granulocyte/macrophage colony-stimulating factor (GM-CSF) protein.
In another embodiment, the adjuvant comprises a GM-CSF protein. In
another embodiment, the adjuvant is a nucleotide molecule encoding
GM-CSF. In another embodiment, the adjuvant comprises a nucleotide
molecule encoding GM-CSF. In another embodiment, the adjuvant is
saponin QS21. In another embodiment, the adjuvant comprises saponin
QS21. In another embodiment, the adjuvant is monophosphoryl lipid
A. In another embodiment, the adjuvant comprises monophosphoryl
lipid A. In another embodiment, the adjuvant is SBAS2. In another
embodiment, the adjuvant comprises SBAS2. In another embodiment,
the adjuvant is an unmethylated CpG-containing oligonucleotide. In
another embodiment, the adjuvant comprises an unmethylated
CpG-containing oligonucleotide. In another embodiment, the adjuvant
is an immune-stimulating cytokine. In another embodiment, the
adjuvant comprises an immune-stimulating cytokine. In another
embodiment, the adjuvant is a nucleotide molecule encoding an
immune-stimulating cytokine. In another embodiment, the adjuvant
comprises a nucleotide molecule encoding an immune-stimulating
cytokine. In another embodiment, the adjuvant is or comprises a
quill glycoside. In another embodiment, the adjuvant is or
comprises a bacterial mitogen. In another embodiment, the adjuvant
is or comprises a bacterial toxin. In another embodiment, the
adjuvant is or comprises any other adjuvant known in the art. Each
possibility represents a separate embodiment of the present
invention.
[0302] In one embodiment, the method provided herein further
comprises the step of co-administering with, prior to or following
the administration of said recombinant Listeria strain an an immune
checkpoint protein inhibitor. In one embodiment, an adjuvant is
selected from the group comprising Montanide ISA 51, GM-CSF, KLH, a
cytokine, a growth factor, a cell population, QS21, Freund's
incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG,
alum, an interleukin, an unmethylated CpG oligonucleotide, quill
glycosides, monophosphoryl lipid A, a liposome, a bacterial
mitogen, a bacterial toxin, or a chemokine, or any combination
thereof.
[0303] In some instances, the PD-1 antagonist and the
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells are combined in a single dosage form. In some instances, the
PD-1 antagonist and the live-attenuated Listeria monocytogenes
strain bioengineered, by transforming it with an expression vector
to express a PSA antigen fused to a tLLO are combined in a single
dosage form. In some instances, the PD-1 antagonist and the an
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain are combined in a single dosage form. In some instances, the
PD-1 antagonist and the LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain are combined in a single dosage form.
[0304] Although the simultaneous administration of the PD-1
antagonist and the live-attenuated bacterial strain that is used to
stimulate APCs capable of driving a cellular immune response to PSA
expressing cells may be maintained throughout a period of treatment
or prevention, anti-cancer activity may also be achieved by
subsequent administration of one compound in isolation (for
example, PD-1 antagonist without the live-attenuated bacterial
strain that is used to stimulate APCs capable of driving a cellular
immune response to PSA expressing cells, following combination
treatment, or alternatively the live-attenuated bacterial strain
that is used to stimulate APCs capable of driving a cellular immune
response to PSA expressing cells, without PD-1 antagonist,
following combination treatment). In addition, although the
simultaneous administration of the PD-1 antagonist and the
live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a tLLO may be maintained throughout a period of treatment
or prevention, anti-cancer activity may also be achieved by
subsequent administration of one compound in isolation (for
example, PD-1 antagonist without the live-attenuated Listeria
monocytogenes strain bioengineered, by transforming it with an
expression vector to express a PSA antigen fused to a tLLO,
following combination treatment, or alternatively the
live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a tLLO, without PD-1 antagonist, following combination
treatment). Further, although the simultaneous administration of
the PD-1 antagonist and the LmddA-142 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) pADV142) strain may be maintained
throughout a period of treatment or prevention, anti-cancer
activity may also be achieved by subsequent administration of one
compound in isolation (for example, PD-1 antagonist without the
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain, following combination treatment, or alternatively the
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain, without PD-1 antagonist, following combination treatment).
In addition, although the simultaneous administration of the PD-1
antagonist and the LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain may be maintained throughout a period of treatment or
prevention, anti-cancer activity may also be achieved by subsequent
administration of one compound in isolation (for example, PD-1
antagonist without the LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain, following combination treatment, or alternatively the
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain, without PD-1
antagonist, following combination treatment).
[0305] In some embodiments, the live-attenuated bacterial strain
that is used to stimulate APCs capable of driving a cellular immune
response to PSA expressing cells or the live-attenuated Listeria
monocytogenes strain bioengineered, by transforming it with an
expression vector to express a PSA antigen fused to a tLLO or the
LmddA-142 (10403S dal.sup.(-) dat actA.sup.(-) pADV142) strain or
the LmddA-143 (10403S dal.sup.(-) dat actA.sup.(-) with klk3 fused
to the hly gene in the chromosome) strain is administered before
administration of the PD-1 antagonist, while in other embodiments,
the live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or the live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or the LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or the
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain is administered
after administration of the PD-1 antagonist. Each possibility
represents a separate embodiment of the methods and compositions as
provided herein.
[0306] In some embodiments, at least one of the therapeutic agents
in the combination therapy is administered using the same dosage
regimen (dose, frequency and duration of treatment) that is
typically employed when the agent is used as monotherapy for
treating the same cancer. In other embodiments, the patient
receives a lower total amount of at least one of the therapeutic
agents in the combination therapy than when the agent is used as
monotherapy, e.g., smaller doses, less frequent doses, and/or
shorter treatment duration.
[0307] A combination therapy of the invention may be used prior to
or following surgery to remove a tumor and may be used prior to,
during or after radiation therapy.
[0308] In some embodiments, a combination therapy of the invention
is administered to a patient who has not been previously treated
with a biotherapeutic or chemotherapeutic agent, i.e., is
treatment-naive. In other embodiments, the combination therapy is
administered to a patient who failed to achieve a sustained
response after prior therapy with a biotherapeutic or
chemotherapeutic agent, i.e., is treatment-experienced. In certain
embodiments, a combination therapy of the invention is administered
to a patient with previously treated metastatic
Castration-Resistant Prostate Cancer (mCRPC).
[0309] A combination therapy of the invention is typically used to
treat a tumor that is large enough to be found by palpation or by
imaging techniques well known in the art, such as MRI, ultrasound,
or CAT scan. In some embodiments, a combination therapy of the
invention is used to treat an advanced stage tumor having
dimensions of at least about 200 mm.sup.3, 300 mm.sup.3, 400
mm.sup.3, 500 mm.sup.3, 750 mm.sup.3, or up to 1000 mm.sup.3.
[0310] A combination therapy of the invention is preferably
administered to a patient diagnosted with a prostate cancer that
tests positive for PD-L1 expression. In some embodiments, PD-L1
expression is detected using a diagnostic anti-human PD-L1
antibody, or antigen binding fragment thereof, in an IHC assay on
an FFPE or frozen tissue section of a tumor sample removed from the
patient. Typically, the patient's physician would order a
diagnostic test to determine PD-L1 expression in a tumor tissue
sample removed from the patient prior to initiation of treatment
with the PD-1 antagonist and the live-attenuated bacterial strain
that is used to stimulate APCs capable of driving a cellular immune
response to PSA expressing cells or the live-attenuated Listeria
monocytogenes strain bioengineered, by transforming it with an
expression vector to express a PSA antigen fused to a tLLO or the
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain or the LmddmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain, but it is envisioned that the physician could order the
first or subsequent diagnostic tests at any time after initiation
of treatment, such as for example after completion of a treatment
cycle. Each possibility represents a separate embodiment of the
methods and compositions as provided herein.
[0311] In one embodiment, the dosage regimen is tailored to the
particular patient's conditions, response and associate treatments,
in a manner which is conventional for any therapy, and may need to
be adjusted in response to changes in conditions and/or in light of
other clinical conditions.
[0312] In some embodiments, selecting a dosage regimen (also
referred to herein as an administration regimen) for a combination
therapy of the invention depends on several factors, including the
serum or tissue turnover rate of the entity, the level of symptoms,
the immunogenicity of the entity, and the accessibility of the
target cells, tissue or organ in the individual being treated.
Preferably, a dosage regimen maximizes the amount of each
therapeutic agent delivered to the patient consistent with an
acceptable level of side effects. Accordingly, the dose amount and
dosing frequency of each biotherapeutic and chemotherapeutic agent
in the combination depends in part on the particular therapeutic
agent, the severity of the cancer being treated, and patient
characteristics. Guidance in selecting appropriate doses of
antibodies, cytokines, and small molecules are available. See,
e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub.
Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.)
(1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y.; Baert et al. (2003) New
Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med.
341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792;
Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et
al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New
Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003
(Physicians' Desk Reference, 57th Ed); Medical Economics Company;
ISBN: 1563634457; 57th edition (November 2002). Determination of
the appropriate dosage regimen may be made by the clinician, e.g.,
using parameters or factors known or suspected in the art to affect
treatment or predicted to affect treatment, and will depend, for
example, the patient's clinical history (e.g., previous therapy),
the type and stage of the cancer to be treated and biomarkers of
response to one or more of the therapeutic agents in the
combination therapy.
[0313] Biotherapeutic agents in a combination therapy of the
invention may be administered by continuous infusion, or by doses
at intervals of, e.g., daily, every other day, three times per
week, or one time each week, two weeks, three weeks, monthly,
bimonthly, etc. A total weekly dose is generally at least 0.05
.mu.g/kg, 0.2 .mu.g/kg, 0.5 .mu.g/kg, 1 .mu.g/kg, 10 .mu.g/kg, 100
.mu.g/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10 mg/kg, 25 mg/kg, 50
mg/kg body weight or more. See, e.g., Yang et al. (2003) New Engl.
J. Med. 349:427-434; Herold et al. (2002) New Engl. J. Med.
346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych.
67:451-456; Portielji et al. (20003) Cancer Immunol. Immunother.
52:133-144.
[0314] In some embodiments that employ an anti-human PD-1 mAb as
the PD-1 antagonist in the combination therapy, the dosing regimen
will comprise administering the anti-human PD-1 mAb at a flat dose
of 100 to 500 mg or a weight-based dose of 1 to 10 mg/kg at
intervals of about 14 days (+2 days) or about 21 days (+2 days) or
about 30 days (+2 days) throughout the course of treatment.
[0315] In other embodiments that employ an anti-human PD-1 mAb as
the PD-1 antagonist in the combination therapy, the dosing regimen
will comprise administering the anti-human PD-1 mAb at a dose of
from about 0.005 mg/kg to about 10 mg/kg, with intra-patient dose
escalation. In other escalating dose embodiments, the interval
between doses will be progressively shortened, e.g., about 30 days
(.+-.2 days) between the first and second dose, about 14 days
(.+-.2 days) between the second and third doses. In certain
embodiments, the dosing interval will be about 14 days (.+-.2
days), for doses subsequent to the second dose.
[0316] In certain embodiments, a subject will be administered an
intravenous (IV) infusion of a medicament comprising any of the
PD-1 antagonists described herein.
[0317] In one embodiment of the invention, the PD-1 antagonist in
the combination therapy is nivolumab, which is administered
intravenously at a dose selected from the group consisting of: 1
mg/kg Q2W, 2 mg/kg Q2W, 3 mg/kg Q2W, 5 mg/kg Q2W, 10 mg Q2W, 1
mg/kg Q3W, 2 mg/kg Q3W, 3 mg/kg Q3W, 5 mg/kg Q3W, and 10 mg
Q3W.
[0318] In another embodiment of the invention, the PD-1 antagonist
in the combination therapy is MK-3475, which is administered in a
liquid medicament at a dose selected from the group consisting of
200 mg Q3W, 1 mg/kg Q2W, 2 mg/kg Q2W, 3 mg/kg Q2W, 5 mg/kg Q2W, 10
mg Q2W, 1 mg/kg Q3W, 2 mg/kg Q3W, 3 mg/kg Q3W, 5 mg/kg Q3W, and 10
mg Q3W or equivalents of any of these doses (e.g., a PK model of
MK-3475 estimates that the fixed dose of 200 mg Q3W provides
exposures that are consistent with those obtained with 2 mg/kg
Q3W). In some embodiments, MK-3475 is administered as a liquid
medicament which comprises 25 mg/ml MK-3475, 7% (w/v) sucrose,
0.02% (w/v) polysorbate 80 in 10 mM histidine buffer pH 5.5, and
the selected dose of the medicament is administered by IV infusion
over a time period of 30 minutes+/-10 min.
[0319] In another embodiment of the invention, the attenuated
bacterial or attenuated Listeria in the combination therapy is a
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain, which is
administered in a liquid medicament at a dose selected from the
group consisting of 0.5.times.10.sup.9, 1.times.10.sup.9,
5.times.10.sup.9 and 1.times.10.sup.10 cfu. In some embodiments, a
dose is selected from the group consisting of of
1.times.10.sup.9-3.31.times.10.sup.10 CFU, 5-500.times.10.sup.8
CFU, 7-500.times.10.sup.8 CFU, 10-500.times.10.sup.8 CFU,
20-500.times.10.sup.8 CFU, 30-500.times.10.sup.8 CFU,
50-500.times.10.sup.8 CFU, 70-500.times.10.sup.8 CFU,
100-500.times.10.sup.8 CFU, 150-500.times.10.sup.8 CFU,
5-300.times.10.sup.8 CFU, 5-200.times.10.sup.8 CFU,
5-150.times.10.sup.8 CFU, 5-100.times.10.sup.8 CFU,
5-70.times.10.sup.8 CFU, 5-50.times.10.sup.8 CFU,
5-30.times.10.sup.8 CFU, 5-20.times.10.sup.8 CFU,
1-30.times.10.sup.9 CFU, 1-20.times.10.sup.9 CFU,
2-30.times.10.sup.9 CFU, 1-10.times.10.sup.9 CFU,
2-10.times.10.sup.9 CFU, 3-10.times.10.sup.9 CFU,
2-7.times.10.sup.9 CFU, 2-5.times.10.sup.9 CFU, or
3-5.times.10.sup.9 CFU. In another embodiment, the dose is
0.5.times.10.sup.9 CFU. In another embodiment, the dose is
1.times.10.sup.9 CFU. In another embodiment, the dose is
5.times.10.sup.9 CFU. In another embodiment, the dose is
1.times.10.sup.10 CFU.
[0320] In other embodiments, a dose is selected from the group
consisting of 1.times.10.sup.9 organisms, 1.5.times.10.sup.9
organisms, 2.times.10.sup.9 organisms, 3.times.10.sup.9 organisms,
4.times.10.sup.9 organisms, 5.times.10.sup.9 organisms,
6.times.10.sup.9 organisms, 7.times.10.sup.9 organisms,
8.times.10.sup.9 organisms, 10.times.10.sup.9 organisms,
1.5.times.10.sup.10 organisms, .times.10.sup.10 organisms,
2.5.times.10.sup.10 organisms, 3.times.10.sup.10 organisms,
0.3.times.10.sup.10 organisms, 4.times.10.sup.10 organisms or
5.times.10.sup.10 organisms.
[0321] Each dose and range of doses represents a separate
embodiment of the present invention.
[0322] In some embodiments, pharmaceutical compositions containing
strains and compositions of the present invention areadministered
to a subject by any method known to a person skilled in the art,
such as parenterally, paracancerally, transmucosally,
transdermally, intramuscularly, intravenously, intra-dermally,
subcutaneously, intra-peritonealy, intra-ventricularly,
intra-cranially, intra-vaginally or intra-tumorally.
[0323] In another embodiment of the methods and compositions
provided herein, the strains or compositions are administered
orally, and are thus formulated in a form suitable for oral
administration, i.e. as a solid or a liquid preparation. Suitable
solid oral formulations include tablets, capsules, pills, granules,
pellets and the like. Suitable liquid oral formulations include
solutions, suspensions, dispersions, emulsions, oils and the like.
In another embodiment of the present invention, the active
ingredient is formulated in a capsule. In accordance with this
embodiment, the compositions of the present invention comprise, in
addition to the active compound and the inert carrier or diluent, a
hard gelating capsule.
[0324] In another embodiment, the strains or compositions are
administered by intravenous, intra-arterial, or intra-muscular
injection of a liquid preparation. Suitable liquid formulations
include solutions, suspensions, dispersions, emulsions, oils and
the like. In one embodiment, the pharmaceutical compositions are
administered intravenously and are thus formulated in a form
suitable for intravenous administration. In another embodiment, the
pharmaceutical compositions are administered intra-arterially and
are thus formulated in a form suitable for intra-arterial
administration. In another embodiment, the pharmaceutical
compositions are administered intra-muscularly and are thus
formulated in a form suitable for intra-muscular
administration.
[0325] In another embodiment, the vaccines or compositions are
administered by intravenous, intra-arterial, or intra-muscular
injection of a liquid preparation. Suitable liquid formulations
include solutions, suspensions, dispersions, emulsions, oils and
the like. In one embodiment, the pharmaceutical compositions are
administered intravenously and are thus formulated in a form
suitable for intravenous administration. In another embodiment, the
pharmaceutical compositions are administered intra-arterially and
are thus formulated in a form suitable for intra-arterial
administration. In another embodiment, the pharmaceutical
compositions are administered intra-muscularly and are thus
formulated in a form suitable for intra-muscular
administration.
[0326] In one embodiment, the vaccines of the methods and
compositions as provided herein may be administered to a host
vertebrate animal, preferably a mammal, and more preferably a
human, either alone or in combination with a pharmaceutically
acceptable carrier. In another embodiment, the vaccine is
administered in an amount effective to induce an immune response to
the Listeria strain itself or to a heterologous antigen which the
Listeria species has been modified to express. In another
embodiment, the amount of vaccine or immunogenic composition to be
administered may be routinely determined by one of skill in the art
when in possession of the present disclosure. In another
embodiment, a pharmaceutically acceptable carrier may include, but
is not limited to, sterile distilled water, saline, phosphate
buffered solutions or bicarbonate buffered solutions. In another
embodiment, the pharmaceutically acceptable carrier selected and
the amount of carrier to be used will depend upon several factors
including the mode of administration, the strain of Listeria and
the age and disease state of the vaccinee. In another embodiment,
administration of the vaccine may be by an oral route, or it may be
parenteral, intranasal, intramuscular, intravascular, intrarectal,
intraperitoneal, or any one of a variety of well-known routes of
administration. In another embodiment, the route of administration
may be selected in accordance with the type of infectious agent or
tumor to be treated.
[0327] In another embodiment, the present invention provides a
method of treating, suppressing, or inhibiting at least one tumor
in a subject comprising administering the immunogenic composition
provided herein.
[0328] In some embodiments an attenuated bacteria, or attenuated
Listeria, or LmddA-142 or LmddA-143 is administered as a liquid
medicament, and the selected dose of the medicament is administered
by IV infusion over a time period of 30 minutes+/-10 min.
[0329] The optimal dose for MK-3475 in combination with a
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an
LmddA-143 (10403S dal.sup.(-) dat.sup.(actA.sup.(-) with klk3 fused
to the hly gene in the chromosome) strain may be identified by dose
escalation of one or both of these agents.
[0330] In one embodiment, the patient is treated with the
combination therapy on day 1 of weeks 1, 4 and 7 in a 12 week
cycle, with MK-3475 administered at a starting dose of 200 mg and a
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain administered at a
starting dose of 1.times.10.sup.9 cfu. In another embodiment, the
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain is administered at
a starting dose of 0.5.times.10.sup.9 CFU. In another embodiment,
the live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain administered at a
starting dose of 5.times.10.sup.9 CFU. In another embodiment, the
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain is administered at
a starting dose of 1.times.10.sup.10 CFU.
[0331] In one embodiment, the patient is treated with the
combination therapy, wherein MK-3475 is administered on Day 1 Q3W
of a 12-week cycle at a starting dose of 200 mg, and a
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain is administered on
Day 1 of Weeks 1, 4, and 7 of the 12-week cycle at a starting dose
of 1.times.10.sup.9 cfu. In another embodiment, the live-attenuated
bacterial strain that is used to stimulate APCs capable of driving
a cellular immune response to PSA expressing cells or a
live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a tLLO or an LmddA-142 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) pADV142) strain or an an LmddA-143 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome) strain is administered at a starting dose of
0.5.times.10.sup.9 CFU. In another embodiment, the live-attenuated
bacterial strain that is used to stimulate APCs capable of driving
a cellular immune response to PSA expressing cells or a
live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a tLLO or an LmddA-142 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) pADV142) strain or an an LmddA-143 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome) strain administered at a starting dose of
5.times.10.sup.9 CFU. In another embodiment, the live-attenuated
bacterial strain that is used to stimulate APCs capable of driving
a cellular immune response to PSA expressing cells or a
live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a tLLO or an LmddA-142 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) pADV142) strain or an an LmddA-143 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome) strain is administered at a starting dose of
1.times.10.sup.10 CFU. In another embodiment, administration is
administered up to 3 days before or 3 days after the scheduled Day
1 of each cycle.
[0332] In one embodiment, the patient is treated with the
combination therapy, wherein MK-3475 is administered on Day 1 of
week 1, 4, 7 and 10 of a 12-week cycle at a starting dose of 200
mg, and a live-attenuated bacterial strain that is used to
stimulate APCs capable of driving a cellular immune response to PSA
expressing cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain is administered on
Day 1 of Weeks 1, 4, and 7 of the 12-week cycle at a starting dose
of 1.times.10.sup.9 cfu. In another embodiment, the live-attenuated
bacterial strain that is used to stimulate APCs capable of driving
a cellular immune response to PSA expressing cells or a
live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a tLLO or an LmddA-142 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) pADV142) strain or an an LmddA-143 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome) strain is administered at a starting dose of
0.5.times.10.sup.9 CFU. In another embodiment, the live-attenuated
bacterial strain that is used to stimulate APCs capable of driving
a cellular immune response to PSA expressing cells or a
live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a tLLO or an LmddA-142 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) pADV142) strain or an an LmddA-143 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome) strain administered at a starting dose of
5.times.10.sup.9 CFU. In another embodiment, the live-attenuated
bacterial strain that is used to stimulate APCs capable of driving
a cellular immune response to PSA expressing cells or a
live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a tLLO or an LmddA-142 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) pADV142) strain or an an LmddA-143 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome) strain is administered at a starting dose of
1.times.10.sup.10 CFU. In another embodiment, administration is
administered up to 3 days before or 3 days after the scheduled Day
1 of each cycle.
[0333] In an embodiment, the MK-3475 infusion is administered
first, followed by a NSAIDS, e.g., naproxen or ibuprofen, and oral
antiemetic medication within 30 minutes prior to the
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or the live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or the LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or the
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain infusion.
[0334] In another embodiment, MK-3475 is administered at a starting
dose of 200 mg Q3W and a live-attenuated bacterial strain that is
used to stimulate APCs capable of driving a cellular immune
response to PSA expressing cells or a live-attenuated Listeria
monocytogenes strain bioengineered, by transforming it with an
expression vector to express a PSA antigen fused to a tLLO or an
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain or an an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain is administered Q3W at a starting dose of between
1.times.10.sup.9 and 1.times.10.sup.10 cfu. In another embodiment,
MK-3475 is administered at a starting dose of 200 mg Q3W and a
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain is administered Q3W
at a starting dose of between 0.5.times.10.sup.9 and
1.times.10.sup.10 cfu.
[0335] In yet another embodiment, a live-attenuated bacterial
strain that is used to stimulate APCs capable of driving a cellular
immune response to PSA expressing cells or a live-attenuated
Listeria monocytogenes strain bioengineered, by transforming it
with an expression vector to express a PSA antigen fused to a tLLO
or an LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-)
pADV142) strain or an an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain is administered at a starting dose of 5.times.10.sup.9 Q3W
and MK-3475 is administered at a starting dose of 200 mg Q3W, and
if the starting dose of the combination is not tolerated by the
patient, then the dose of a live-attenuated bacterial strain that
is used to stimulate APCs capable of driving a cellular immune
response to PSA expressing cells or a live-attenuated Listeria
monocytogenes strain bioengineered, by transforming it with an
expression vector to express a PSA antigen fused to a tLLO or an
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain or an an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain is reduced to 1.times.10.sup.9 cfu Q3W.
[0336] In some embodiments, dosage levels below the lower limit of
the aforesaid range may be more than adequate, while in other cases
still larger doses may be employed, as determined by those skilled
in the art.
[0337] In some embodiments, a treatment cycle begins with the first
day of combination treatment and lasts for at least 12 weeks, 24
weeks or 48 weeks. On any day of a treatment cycle that the drugs
are co-administered, the timing between the separate IV infusions
of MK-3475 and a live-attenuated bacterial strain that is used to
stimulate APCs capable of driving a cellular immune response to PSA
expressing cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain is between about 15
minutes to about 45 minutes. The invention contemplates that
MK-3475 and a live-attenuated bacterial strain that is used to
stimulate APCs capable of driving a cellular immune response to PSA
expressing cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain may be administered
in either order or by simultaneous IV infusion.
[0338] In some embodiments, the combination therapy is administered
for at least 2 to 4 weeks after the patient achieves a CR.
[0339] In some embodiments, the patient selected for treatment with
the combination therapy of the invention has been diagnosed with a
metastatic prostate cancer and the patient has progressed or become
resistant to no more than 3 prior systemic treatment regimens.
[0340] In an embodiment, the patient selected for treatment with
the combination therapy of the invention had a serum PSA
level.gtoreq.5 ng/mL within 1 week prior to starting the
combination therapy.
[0341] In another embodiment, the patient selected for treatment
with the combination therapy of the invention had a rising PSA
level within the 4 weeks prior to starting the combination
therapy.
[0342] The present invention also provides a medicament which
comprises a PD-1 antagonist as described above and a
pharmaceutically acceptable excipient. When the PD-1 antagonist is
a biotherapeutic agent, e.g., a mAb, the antagonist may be produced
in CHO cells using conventional cell culture and
recovery/purification technologies.
[0343] In some embodiments, a medicament comprising an anti-PD-1
antibody as the PD-1 antagonist may be provided as a liquid
formulation or prepared by reconstituting a lyophilized powder with
sterile water for injection prior to use. WO 2012/135408 describes
the preparation of liquid and lyophilized medicaments comprising
MK-3475 that are suitable for use in the present invention. In some
embodiments, a medicament comprising MK-3475 is provided in a glass
vial which contains about 50 mg of MK-3475.
[0344] The present invention also provides a medicament which
comprises a live-attenuated bacterial strain that is used to
stimulate APCs capable of driving a cellular immune response to PSA
expressing cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain and a
pharmaceutically acceptable excipient. A live-attenuated bacterial
strain that is used to stimulate APCs capable of driving a cellular
immune response to PSA expressing cells or a live-attenuated
Listeria monocytogenes strain bioengineered, by transforming it
with an expression vector to express a PSA antigen fused to a tLLO
or an LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-)
pADV142) strain or an an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain may be prepared as described in Wallecha et al. CLINICAL
ANDSTRAINIMMUNOLOGY, January 2009, p. 96-103.
[0345] The PD-1 antagonist medicament and the a live-attenuated
bacterial strain that is used to stimulate APCs capable of driving
a cellular immune response to PSA expressing cells or a
live-attenuated Listeria monocytogenes strain bioengineered, by
transforming it with an expression vector to express a PSA antigen
fused to a tLLO or an LmddA-142 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) pADV142) strain or an an LmddA-143 (10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome) strain medicament may be provided as a kit which
comprises a first container and a second containiner and a package
insert. The first container contains at least one dose of a
medicament comprising an anti-PD-1 antibody, the second container
contains at least one dose of a medicament comprising a
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain, and the package
insert, or label, which comprises instructions for treating a
patient for a prostate cancer using the medicaments. The first and
second containers may be comprised of the same or different shape
(e.g., vials, syringes and bottles) and/or material (e.g., plastic
or glass). The kit may further comprise other materials that may be
useful in administering the medicaments, such as diluents, filters,
IV bags and lines, needles and syringes. In some embodiments of the
kit, the anti-PD-1 antagonist is an anti-PD-1 antibody and the
instructions state that the medicaments are intended for use in
treating a patient having a prostate cancer that tests positive for
PD-L1 expression by an IHC assay.
[0346] These and other aspects of the invention, including the
exemplary specific embodiments listed below, will be apparent from
the teachings contained herein.
Exemplary Specific Embodiments of the Invention
[0347] 1. A method for treating a prostate cancer in a patient
comprising administering to the individual a combination therapy
which comprises a PD-1 antagonist and a live-attenuated bacterial
strain that is used to stimulate APCs capable of driving a cellular
immune response to PSA expressing cells or a live-attenuated
Listeria monocytogenes strain bioengineered, by transforming it
with an expression vector to express a PSA antigen fused to a tLLO
or an LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-)
pADV142) strain or an an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain. Each possibility represents a separate embodiment as
provided herein. 2. A medicament comprising a PD-1 antagonist for
use in combination with a live-attenuated bacterial strain that is
used to stimulate APCs capable of driving a cellular immune
response to PSA expressing cells or a live-attenuated Listeria
monocytogenes strain bioengineered, by transforming it with an
expression vector to express a PSA antigen fused to a tLLO or an
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain or an an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain for treating a prostate cancer in a patient. Each
possibility represents a separate embodiment as provided herein. 3.
A medicament comprising a live-attenuated bacterial strain that is
used to stimulate APCs capable of driving a cellular immune
response to PSA expressing cells or a live-attenuated Listeria
monocytogenes strain bioengineered, by transforming it with an
expression vector to express a PSA antigen fused to a tLLO or an
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain or an an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain for use in combination with a PD-1 antagonist for treating a
prostate cancer in a patient. Each possibility represents a
separate embodiment as provided herein. 4. The medicament of
embodiment 3 or 4, which further comprises a pharmaceutically
acceptable excipient or adjuvant, or a combination thereof. 5. Use
of a PD-1 antagonist in the manufacture of medicament for treating
a prostate cancer in a patient when administered in combination
with a live-attenuated bacterial strain that is used to stimulate
APCs capable of driving a cellular immune response to PSA
expressing cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain. Each possibility
represents a separate embodiment as provided herein. 6. Use of a
live-attenuated bacterial strain that is used to stimulate APCs
capable of driving a cellular immune response to PSA expressing
cells or a live-attenuated Listeria monocytogenes strain
bioengineered, by transforming it with an expression vector to
express a PSA antigen fused to a tLLO or an LmddA-142 (10403S
dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142) strain or an an
LmddA-143 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) with klk3
fused to the hly gene in the chromosome) strain in the manufacture
of a medicament for treating a prostate cancer in a patient when
administered in combination with a PD-1 antagonist. Each
possibility represents a separate embodiment as provided herein. 7.
Use of a PD-1 antagonist and a live-attenuated bacterial strain
that is used to stimulate APCs capable of driving a cellular immune
response to PSA expressing cells or a live-attenuated Listeria
monocytogenes strain bioengineered, by transforming it with an
expression vector to express a PSA antigen fused to a tLLO or an
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain or an an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain in the manufacture of medicaments for treating a cancer in a
patient. Each possibility represents a separate embodiment as
provided herein. 8. A kit which comprises a first container, a
second container and a package insert, wherein the first container
comprises at least one dose of a medicament comprising an anti-PD-1
antagonist, the second container comprises at least one dose of a
medicament comprising a live-attenuated bacterial strain that is
used to stimulate APCs capable of driving a cellular immune
response to PSA expressing cells or a live-attenuated Listeria
monocytogenes strain bioengineered, by transforming it with an
expression vector to express a PSA antigen fused to a tLLO or an
LmddA-142 (10403S dal.sup.(-) dat.sup.(-) actA.sup.(-) pADV142)
strain or an an LmddA-143 (10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) with klk3 fused to the hly gene in the chromosome)
strain, and the package insert comprises instructions for treating
a patient for prostate cancer using the medicaments. Each
possibility represents a separate embodiment as provided herein. 9.
The kit of embodiment 8, wherein the instructions state that the
medicaments are intended for use in treating a patient having a
prostate cancer that tests positive for PD-L1 expression by an
immunohistochemical (IHC) assay. 10. The method, medicament, use or
kit of any of embodiments 1 to 9, wherein the PD-1 antagonist is a
monoclonal antibody, or an antigen binding fragment thereof. 11.
The method, medicament, use or kit of embodiment 9, wherein the
PD-1 antagonist is MPDL3280A, BMS-936559, MEDI4736, MSB0010718C or
a monoclonal antibody which comprises the heavy chain and light
chain variable regions of SEQ ID NO:24 and SEQ ID NO:21,
respectively, of WO2013/019906. 12. The method, medicament, use or
kit of embodiment 10, wherein the PD-1 antagonist is a monoclonal
antibody, or an antigen binding fragment thereof, and blocks
binding of PD-L1 and PD-L2 to PD-1. 13. The method, medicament, use
or kit of embodiment 12, wherein the monoclonal antibody, or
antigen binding fragment thereof, comprises: (a) light chain CDRs
of SEQ ID NOs: 1, 2 and 3 and heavy chain CDRs of SEQ ID NOs: 4, 5
and 6; or (b) light chain CDRs of SEQ ID NOs: 7, 8 and 9 and heavy
chain CDRs of SEQ ID NOs: 10, 11 and 12. 14. The method,
medicament, use or kit of embodiment 12, wherein the monoclonal
antibody, or antigen binding fragment thereof, comprises light
chain CDRs of SEQ ID NOs: 7, 8 and 9 and heavy chain CDRs of SEQ ID
NOs: 10, 11 and 12. 15. The method, medicament, use or kit of
embodiment 12, wherein the PD-1 antagonist is an anti-PD-1
monoclonal antibody which comprises a heavy chain and a light
chain, and wherein the heavy chain comprises SEQ ID NO:21 and the
light chain comprises SEQ ID NO:22. 16. The method, medicament, use
or kit of embodiment 12, wherein the PD-1 antagonist is an
anti-PD-1 monoclonal antibody which comprises a heavy chain and a
light chain, and wherein the heavy chain comprises SEQ ID NO:23 and
the light chain comprises SEQ ID NO:24. 17. The method, medicament,
use or kit of any of embodiments 10-16, wherein the prostate cancer
is metastatic. 18. The method, medicament, use or kit of embodiment
17, wherein the cancer is metastatic Castration-resistant Prostate
Cancer (mCRPC). 19. The method, medicament, use or kit of any of
embodiments 10-18, wherein the patient has not been previously
treated for prostate cancer. 20. The method, medicament, use or kit
of any of embodiments 10-18, wherein the patient has previously
been treated for prostate cancer. 21. The method, medicament, use
or kit of any of embodiments 10-20, wherein the prostate cancer
tests positive for PD-L1. 22. The method, medicament, use or kit of
embodiment 21, wherein the PD-L1 expression is elevated. 23. The
method, medicament, use or kit of embodiment 12, wherein the PD-1
antagonist is MK-3475 or nivolumab. 24. The method, medicament, use
or kit of embodiment 25, wherein MK-3475 is formulated as a liquid
medicament which comprises 25 mg/ml MK-3475, 7% (w/v) sucrose,
0.02% (w/v) polysorbate 80 in 10 mM histidine buffer pH 5.5. 25.
The method, medicament, use or kit of any of embodiments 1 to 24,
wherein the attenuated Listeria is LmddA-142 or LmddA-143 26. The
method, medicament, use of kit of any of embodiments 1 to 25,
wherein the PD-1 antagonist is MK-3475, the attenuated Listeria is
LmddA-142 or LmddA-143, the patient is diagnosed with a metastatic
prostate cancer, and doses of the PD-1 antagonist and the
attenuated Listeria are selected from the group consisting of one
of the combinations in the table below:
TABLE-US-00006 MK-3475 LmddA-142 LmddA-143 200 mg Q3W 1 .times.
10.sup.9 cfu 1 .times. 10.sup.9 cfu 200 mg Q3W 2 .times. 10.sup.9
cfu 2 .times. 10.sup.9 cfu 200 mg Q3W 5 .times. 10.sup.9 cfu 5
.times. 10.sup.9 cfu 200 mg Q3W 1 .times. 10.sup.10 cfu.sup. 1
.times. 10.sup.10 cfu.sup.
General Methods
[0348] Standard methods in molecular biology are described
Sambrook, Fritsch and Maniatis (1982 & 1989 2.sup.nd Edition,
2001 3.sup.rd Edition) Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook
and Russell (2001) Molecular Cloning, 3.sup.rd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993)
Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.).
Standard methods also appear in Ausbel, et al. (2001) Current
Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons,
Inc. New York, N.Y., which describes cloning in bacterial cells and
DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast
(Vol. 2), glycoconjugates and protein expression (Vol. 3), and
bioinformatics (Vol. 4).
[0349] Methods for protein purification including
immunoprecipitation, chromatography, electrophoresis,
centrifugation, and crystallization are described (Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 1, John Wiley and
Sons, Inc., New York). Chemical analysis, chemical modification,
post-translational modification, production of fusion proteins,
glycosylation of proteins are described (see, e.g., Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 2, John Wiley and
Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in
Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp.
16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life
Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia
Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391).
Production, purification, and fragmentation of polyclonal and
monoclonal antibodies are described (Coligan, et al. (2001) Current
Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New
York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane,
supra). Standard techniques for characterizing ligand/receptor
interactions are available (see, e.g., Coligan, et al. (2001)
Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New
York).
[0350] Monoclonal, polyclonal, and humanized antibodies can be
prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal
Antibodies, Oxford Univ. Press, New York, N.Y.; Kontermann and
Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New
York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp.
139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al.
(1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem.
274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684;
Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992)
J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).
[0351] An alternative to humanization is to use human antibody
libraries displayed on phage or human antibody libraries in
transgenic mice (Vaughan et al. (1996) Nature Biotechnol.
14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al.
(1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000)
Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; Kay et al. (1996) Phage Display of Peptides and
Proteins: A Laboratory Manual, Academic Press, San Diego, Calif.;
de Bruin et al. (1999) Nature Biotechnol. 17:397-399).
[0352] Purification of antigen is not necessary for the generation
of antibodies. Animals can be immunized with cells bearing the
antigen of interest. Splenocytes can then be isolated from the
immunized animals, and the splenocytes can fused with a myeloma
cell line to produce a hybridoma (see, e.g., Meyaard et al. (1997)
Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242;
Preston et al., supra; Kaithamana et al. (1999) J. Immunol.
163:5157-5164).
[0353] Antibodies can be conjugated, e.g., to small drug molecules,
enzymes, liposomes, polyethylene glycol (PEG). Antibodies are
useful for therapeutic, diagnostic, kit or other purposes, and
include antibodies coupled, e.g., to dyes, radioisotopes, enzymes,
or metals, e.g., colloidal gold (see, e.g., Le Doussal et al.
(1991) J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol.
160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811;
Everts et al. (2002) J. Immunol. 168:883-889).
[0354] Methods for flow cytometry, including fluorescence activated
cell sorting (FACS), are available (see, e.g., Owens, et al. (1994)
Flow Cytometry Principles for Clinical Laboratory Practice, John
Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry,
2.sup.nd ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical
Flow Cytometry, John Wiley and Sons, Hoboken, N.J.). Fluorescent
reagents suitable for modifying nucleic acids, including nucleic
acid primers and probes, polypeptides, and antibodies, for use,
e.g., as diagnostic reagents, are available (Molecular Probesy
(2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.;
Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).
[0355] Standard methods of histology of the immune system are
described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus:
Histopathology and Pathology, Springer Verlag, New York, N.Y.;
Hiatt, et al. (2000) Color Atlas of Histology, Lippincott,
Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic
Histology: Text and Atlas, McGraw-Hill, New York, N.Y.).
[0356] Software packages and databases for determining, e.g.,
antigenic fragments, leader sequences, protein folding, functional
domains, glycosylation sites, and sequence alignments, are
available (see, e.g., GenBank, Vector NTI.RTM. Suite (Informax,
Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San
Diego, Calif.); DeCypher.RTM. (TimeLogic Corp., Crystal Bay, Nev.);
Menne, et al. (2000) Bioinformatics 16: 741-742; Menne, et al.
(2000) Bioinformatics Applications Note 16:741-742; Wren, et al.
(2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne
(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids
Res. 14:4683-4690).
TABLE-US-00007 SEQ ID NO: Description 1 hPD-1.08A light chain CDR1
2 hPD-1.08A light chain CDR2 3 hPD-1-08A light chain CDR3 4
hPD-1.08A heavy chain CDR1 5 hPD-1.08A heavy chain CDR2 6 hPD-1.08A
heavy chain CDR3 7 hPD-1.09A light chain CDR1 8 hPD-1.09A light
chain CDR2 9 hPD-1.09A light chain CDR3 10 hPD-1.09A heavy chain
CDR1 11 hPD-1.09A heavy chain CDR2 12 hPD-1.09A heavy chain CDR3 13
109A-H heavy chain variable region 14 409A-H heavy chain full
length 15 K09A-L-11 light chain variable region 16 K09A-L-16 light
chain variable region 17 K09A-L-17 light chain variable region 18
K09A-L-11 light chain full length 19 K09A-L-16 light chain full
length 20 K09A-L-17 light chain full length 21 MK-3475 Heavy chain
22 MK-3475 Light chain 23 Nivolumab Heavy chain 24 Nivolumab light
chain 25 KLK3 protein 26 KLK3 protein 27 KLK3 protein 28 Nucleotide
encoding KLK3 protein 29 KLK3 protein 30 Nucleotide encoding KLK3
protein 31 KLK3 protein 32 Nucleotide encoding KLK3 protein 33 KLK3
protein 34 Nucleotide encoding KLK3 protein 35 KLK3 protein 36
Nucleotide encoding KLK3 protein 37 KLK3 protein 38 Nucleotide
encoding KLK3 protein 39 KLK3 protein 40 Nucleotide encoding KLK3
protein 41 KLK3 protein 42 KLK3 protein 43 KLK3 protein 44 KLK3
protein 45 Nucleotide encoding KLK3 protein 46 Primer Adv60-PSA 47
Primer Adv61-PSA 48 tLLO-PSA fusion polypeptide 49 LLO PEST
seqeunce 50 KLK3 protein 51 KLK3 protein 52 KLK3 protein 53 LLO
polypeptide 54 LLO polypeptide 55 LLO polypeptide 56 ActA PEST
sequence 57 ActA PEST sequence 58 ActA PEST sequence 59 ActA PEST
sequence 60 Streptococcus pyogenes Streptolysin O PEST sequence 61
Streptococcus equisimilis Streptolysin O PEST-like sequence 62
pADV142 nucleic acid sequence 63 KLK3 protein 64 KLK3 protein 65
KLK3 protein 66 KLK3 protein
[0357] A recombinant Lm of this invention secretes PSA fused to
tLLO (Lm-LLO-PSA), which elicits a potent PSA-specific immune
response associated with regression of tumors in a mouse model for
prostate cancer. Details for the vectors used to create the
LmddA-142 strain and the LmddA-143 strain are provided in Table 4
below. The pADV142 plasmid, which has no antibiotic resistance
markers, was used to create the LmddA-142 strain. This new strain
is 10 times more attenuated than Lm-LLO-PSA. In addition, LmddA-142
is slightly more immunogenic and significantly more efficacious in
regressing PSA expressing tumors than the Lm-LLO-PSA.
TABLE-US-00008 TABLE 4 Plasmids and strains Plasmids Features
pADV119 Derived from pTV3 by deleting the prfA gene pADV134 Derived
from pADV119 by replacing the Lm dal gene by the Bacillus dal gene
pADV142 Derived from pADV134 by replacing HPV16 e7 with klk3 (Map
at FIG. 9B; sequence FIG. 9C) Strains Genotype 10403S Wild-type
Listeria monocytogenes:: str XFL-7 10403S prfA.sup.(-) Lmdd 10403S
dal.sup.(-) dat.sup.(-) LmddA 10403S dal.sup.(-) dat.sup.(-)
actA.sup.(-) LmddA-142 10403S dal.sup.(-) dat.sup.(-) actA.sup.(-)
pADV142 Lmdd-143 10403S dal.sup.(-) dat.sup.(-) with klk3 fused to
the hly gene in the chromosome LmddA-143 10403S dal.sup.(-)
dat.sup.(-) actA.sup.(-) with klk3 fused to the hly gene in the
chromosome (FIG. 8 shows the chromosomal structure and FIG. 9A
shows a map of the pADV143 plasmid)
[0358] The sequence of the plasmid pAdv142 (6523 bp) was as
follows:
TABLE-US-00009 (SEQ ID NO: 62)
cggagtgtatactggcttactatgttggcactgatgagggtgtcagtga
agtgcttcatgtggcaggagaaaaaaggctgcaccggtgcgtcagcaga
atatgtgatacaggatatattccgcttcctcgctcactgactcgctacg
ctcggtcgttcgactgcggcgagcggaaatggcttacgaacggggcgga
gatttcctggaagatgccaggaagatacttaacagggaagtgagagggc
cgcggcaaagccgtttttccataggctccgcccccctgacaagcatcac
gaaatctgacgctcaaatcagtggtggcgaaacccgacaggactataaa
gataccaggcgtttccccctggcggctccctcgtgcgctctcctgttcc
tgcctttcggtttaccggtgtcattccgctgttatggccgcgtttgtct
cattccacgcctgacactcagttccgggtaggcagttcgctccaagctg
gactgtatgcacgaaccccccgttcagtccgaccgctgcgccttatccg
gtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccact
ggcagcagccactggtaattgatttagaggagttagtcttgaagtcatg
cgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcct
ccaagccagttacctcggttcaaagagttggtagctcagagaaccttcg
aaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattac
gcgcagaccaaaacgatctcaagaagatcatcttattaatcagataaaa
tatttctagccctcctttgattagtatattcctatcttaaagttacttt
tatgtggaggcattaacatttgttaatgacgtcaaaaggatagcaagac
tagaataaagctataaagcaagcatataatattgcgtttcatctttaga
agcgaatttcgccaatattataattatcaaaagagaggggtggcaaacg
gtatttggcattattaggttaaaaaatgtagaaggagagtgaaacccat
gaaaaaaataatgctagtttttattacacttatattagttagtctacca
attgcgcaacaaactgaagcaaaggatgcatctgcattcaataaagaaa
attcaatttcatccatggcaccaccagcatctccgcctgcaagtcctaa
gacgccaatcgaaaagaaacacgcggatgaaatcgataagtatatacaa
ggattggattacaataaaaacaatgtattagtataccacggagatgcag
tgacaaatgtgccgccaagaaaaggttacaaagatggaaatgaatatat
tgttgtggagaaaaagaagaaatccatcaatcaaaataatgcagacatt
caagttgtgaatgcaatttcgagcctaacctatccaggtgctctcgtaa
aagcgaattcggaattagtagaaaatcaaccagatgttctccctgtaaa
acgtgattcattaacactcagcattgatttgccaggtatgactaatcaa
gacaataaaatagttgtaaaaaatgccactaaatcaaacgttaacaacg
cagtaaatacattagtggaaagatggaatgaaaaatatgctcaagctta
tccaaatgtaagtgcaaaaattgattatgatgacgaaatggcttacagt
gaatcacaattaattgcgaaataggtacagcatttaaagctgtaaataa
tagcttgaatgtaaacttcggcgcaatcagtgaagggaaaatgcaagaa
gaagtcattagttttaaacaaatttactataacgtgaatgttaatgaac
ctacaagaccttccagatttttcggcaaagctgttactaaagagcagtt
gcaagcgcttggagtgaatgcagaaaatcctcctgcatatatctcaagt
gtggcgtatggccgtcaagtttatttgaaattatcaactaattcccata
gtactaaagtaaaagctgcttttgatgctgccgtaagcggaaaatctgt
ctcaggtgatgtagaactaacaaatatcatcaaaaattcttccttcaaa
gccgtaatttacggaggttccgcaaaagatgaagttcaaatcatcgacg
gcaacctcggagacttacgcgatattttgaaaaaaggcgctacttttaa
tcgagaaacaccaggagttcccattgcttatacaacaaacttcctaaaa
gacaatgaattagctgttattaaaaacaactcagaatatattgaaacaa
cttcaaaagcttatacagatggaaaaattaacatcgatcactctggagg
atacgttgctcaattcaacatttcttgggatgaagtaaattatgatctc
gagattgtgggaggctgggagtgcgagaagcattcccaaccctggcagg
tgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggtgca
cccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtg
atcttgctgggtcggcacagcctgtttcatcctgaagacacaggccagg
tatttcaggtcagccacagcttcccacacccgctctacgatatgagcct
cctgaagaatcgattcctcaggccaggtgatgactccagccacgacctc
atgctgctccgcctgtcagagcctgccgagctcacggatgctgtgaagg
tcatggacctgcccacccaggagccagcactggggaccacctgctacgc
ctcaggctggggcagcattgaaccagaggagttcttgaccccaaagaaa
cttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaag
ttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggac
agggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgt
tatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccc
tgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtg
gatcaaggacaccatcgtggccaaccccTAAcccgggccactaactcaa
cgctagtagtggatttaatcccaaatgagccaacagaaccagaaccaga
aacagaacaagtaacattggagttagaaatggaagaagaaaaaagcaat
gatttcgtgtgaataatgcacgaaatcattgcttatttttttaaaaagc
gatatactagatataacgaaacaacgaactgaataaagaatacaaaaaa
agagccacgaccagttaaagcctgagaaactttaactgcgagccttaat
tgattaccaccaatcaattaaagaagtcgagacccaaaatttggtaaag
tatttaattactttattaatcagatacttaaatatctgtaaacccatta
tatcgggattgaggggatttcaagtctttaagaagataccaggcaatca
attaagaaaaacttagttgattgccttttagttgtgattcaactttgat
cgtagcttctaactaattaattttcgtaagaaaggagaacagctgaatg
aatatcccttttgttgtagaaactgtgcttcatgacggcttgttaaagt
acaaatttaaaaatagtaaaattcgctcaatcactaccaagccaggtaa
aagtaaaggggctatttttgcgtatcgctcaaaaaaaagcatgattggc
ggacgtggcgttgttctgacttccgaagaagcgattcacgaaaatcaag
atacatttacgcattggacaccaaacgtttatcgttatggtacgtatgc
agacgaaaaccgttcatacactaaaggacattctgaaaacaatttaaga
caaatcaataccttctttattgagatattcacacggaaaaagaaactat
ttcagcaagcgatattttaacaacagctattgatttaggttttatgcct
acgttaattatcaaatctgataaaggttatcaagcatattttgttttag
aaacgccagtctatgtgacttcaaaatcagaatttaaatctgtcaaagc
agccaaaataatctcgcaaaatatccgagaatattttggaaagtctttg
ccagttgatctaacgtgcaatcattagggattgctcgtataccaagaac
ggacaatgtagaattattgatcccaattaccgttattctttcaaagaat
ggcaagattggtctttcaaacaaacagataataagggctttactcgttc
aagtctaacggttttaagcggtacagaaggcaaaaaacaagtagatgaa
ccctggtttaatctcttattgcacgaaacgaaattttcaggagaaaagg
gtttagtagggcgcaatagcgttatgtttaccctctctttagcctactt
tagttcaggctattcaatcgaaacgtgcgaatataatatgtttgagttt
aataatcgattagatcaacccttagaagaaaaagaagtaatcaaaattg
ttagaagtgcctattcagaaaactatcaaggggctaatagggaatacat
taccattctttgcaaagcttgggtatcaagtgatttaaccagtaaagat
ttatttgtccgtcaagggtggtttaaattcaagaaaaaaagaagcgaac
gtcaacgtgttcatttgtcagaatggaaagaagatttaatggcttatat
tagcgaaaaaagcgatgtatacaagccttatttagcgacgaccaaaaaa
gagattagagaagtgctaggcattcctgaacggacattagataaattgc
tgaaggtactgaaggcgaatcaggaaattttctttaagattaaaccagg
aagaaatggtggcattcaacttgctagtgttaaatcattgttgctatcg
atcattaaattaaaaaaagaagaacgagaaagctatataaaggcgctga
cagcttcgtttaatttagaacgtacatttattcaagaaactctaaacaa
attggcagaacgccccaaaacggacccacaactcgatttgtttagctac
gatacaggctgaaaataaaacccgcactatgccattacatttatatcta
tgatacgtgtttgtttttctttgctggctagcttaattgcttatattta
cctgcaataaaggatacttacttccattatactcccattttccaaaaac
atacggggaacacgggaacttattgtacaggccacctcatagttaatgg
tttcgagccttcctgcaatctcatccatggaaatatattcatccccctg
ccggcctattaatgtgacttttgtgcccggcggatattcctgatccagc
tccaccataaattggtccatgcaaattcggccggcaattttcaggcgtt
ttcccttcacaaggatgtcggtccctttcaattttcggagccagccgtc
cgcatagcctacaggcaccgtcccgatccatgtgtctttttccgctgtg
tactcggctccgtagctgacgctctcgccttttctgatcagtttgacat
gtgacagtgtcgaatgcagggtaaatgccggacgcagctgaaacggtat
ctcgtccgacatgtcagcagacgggcgaaggccatacatgccgatgccg
aatctgactgcattaaaaaagccattacagccggagtccagcggcgctg
ttcgcgcagtggaccattagattctttaacggcagcggagcaatcagct
ctttaaagcgctcaaactgcattaagaaatagcctattctattcatccg
ctgtcgcaaaatgggtaaatacccctttgcactttaaacgagggttgcg
gtcaagaattgccatcacgttctgaacttcttcctctgtttttacacca
agtctgttcatccccgtatcgaccttcagatgaaaatgaagagaacctt
ttttcgtgtggcgggctgcctcctgaagccattcaacagaataacctgt
taaggtcacgtcatactcagcagcgattgccacatactccgggggaacc
gcgccaagcaccaatataggcgccttcaatcccatagcgcagtgaaatc
gcttcatccaaaatggccacggccaagcatgaagcacctgcgtcaagag
cagcattgctgtttctgcatcaccatgcccgtaggcgtttgctttcaca
actgccatcaagtggacatgttcaccgatatgttttttcatattgctga
cattacctttatcgcggacaagtcaatttccgcccacgtatctctgtaa
aaaggttttgtgctcatggaaaactcctctcattttcagaaaatcccag
tacgtaattaagtatttgagaattaattttatattgattaatactaagt
ttacccagttacacctaaaaaacaaatgatgagataatagctccaaagg
ctaaagaggactataccaactatttgttaattaa.
(FIG. 9C) This plasmid was sequenced at Genewiz facility from the
E. coli strain on 2-20-08.A map of the plasmid is presented as FIG.
9B.
REFERENCES
[0359] 1. Sharpe, A. H, Wherry, E. J., Ahmed R., and Freeman G. J.
The function of programmed cell death 1 and its ligands in
regulating autoimmunity and infection. Nature Immunology (2007);
8:239-245. [0360] 2. Dong H et al. Tumor-associated B7-H1 promotes
T-cell apoptosis: a potential mechanism of immune evasion. Nat Med.
2002 August; 8(8):793-800. [0361] 3. Yang et al. PD-1 interaction
contributes to the functional suppression of T-cell responses to
human uveal melanoma cells in vitro. Invest Ophthalmol Vis Sci.
2008 June; 49(6 (2008): 49: 2518-2525. [0362] 4. Ghebeh et al. The
B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in
breast cancer patients with infiltrating ductal carcinoma:
correlation with important high-risk propgnostic factors. Neoplasia
(2006) 8: 190-198. [0363] 5. Hamanishi J et al. Programmed cell
death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are
prognostic factors of human ovarian cancer. Proceeding of the
National Academy of Sciences (2007): 104: 3360-3365. [0364] 6.
Thompson R H et al. Significance of B7-H1 overexpression in kidney
cancer. Clinical genitourin Cancer (2006): 5: 206-211. [0365] 7.
Nomi, T. Sho, M., Akahori, T., et al. Clinical significance and
therapeutic potential of the programmed death-1 ligand/programmed
death-1 pathway in human pancreatic cancer. Clinical Cancer
Research (2007); 13:2151-2157. [0366] 8. Ohigashi Y et al. Clinical
significance of programmed death-1 ligand-1 and programmed death-1
ligand 2 expression in human esophageal cancer. Clin. Cancer
Research (2005): 11: 2947-2953. [0367] 9. Inman et al. PD-L1
(B7-H1) expression by urothelial carcinoma of the bladder and
BCG-induced granulomata: associations with localized stage
progression. Cancer (2007): 109: 1499-1505. [0368] 10. Shimauchi T
et al. Augmented expression of programmed death-1 in both
neoplasmatic and nonneoplastic CD4+ T-cells in adult T-cell
Leukemia/Lymphoma. Int. J. Cancer (2007): 121:2585-2590. [0369] 11.
Gao et al. Overexpression of PD-L1 significantly associates with
tumor aggressiveness and postoperative recurrence in human
hepatocellular carcinoma. Clinical Cancer Research (2009) 15:
971-979. [0370] 12. Nakanishi J. Overexpression of B7-H1 (PD-L1)
significantly associates with tumor grade and postoperative
prognosis in human urothelial cancers. Cancer Immunol Immunother.
(2007) 56: 1173-1182. [0371] 13. Hino et al. Tumor cell expression
of programmed cell death-1 is a prognostic factor for malignant
melanoma. Cancer (2010): 00: 1-9. [0372] 14. Ghebeh H. Foxp3+ tregs
and B7-H1+/PD-1+ T lymphocytes co-infiltrate the tumor tissues of
high-risk breast cancer patients: implication for immunotherapy.
BMC Cancer. 2008 Feb. 23; 8:57. [0373] 15. Ahmadzadeh M et al.
Tumor antigen-specific CD8 T cells infiltrating the tumor express
high levels of PD-1 and are functionally impaired. Blood (2009)
114: 1537-1544. [0374] 16. Thompson R H et al. PD-1 is expressed by
tumor infiltrating cells and is associated with poor outcome for
patients with renal carcinoma. Clinical Cancer Research (2007) 15:
1757-1761.
[0375] All references cited herein are incorporated by reference to
the same extent as if each individual publication, database entry
(e.g. Genbank sequences or GeneID entries), patent application, or
patent, was specifically and individually indicated to be
incorporated by reference. This statement of incorporation by
reference is intended by Applicants, pursuant to 37 C.F.R. .sctn.
1.57(b)(1), to relate to each and every individual publication,
database entry (e.g. Genbank sequences or GeneID entries), patent
application, or patent, each of which is clearly identified in
compliance with 37 C.F.R. .sctn. 1.57(b)(2), even if such citation
is not immediately adjacent to a dedicated statement of
incorporation by reference. The inclusion of dedicated statements
of incorporation by reference, if any, within the specification
does not in any way weaken this general statement of incorporation
by reference. Citation of the references herein is not intended as
an admission that the reference is pertinent prior art, nor does it
constitute any admission as to the contents or date of these
publications or documents.
[0376] In the following example, numerous specific details are set
forth in order to provide a thorough understanding of the
invention. However, it will be understood by those skilled in the
art that the present invention may be practiced without these
specific details. In other instances, well-known methods,
procedures, and components have not been described in detail so as
not to obscure the present invention.
Example
A Phase 1-2 Dose-Escalation and Safety Study of ADXS31-142 Alone
and in Combination with Pembrolizumab (MK-3475) in Patients with
Previously Treated Metastatic Castration-Resistant Prostate Cancer
(mCRPC)
[0377] This is a phase 1-2, open-label, multicenter, nonrandomized,
2-part study in patients with metastatic Castration-Resistant
Prostate Cancer (mCRPC). Part A of the study will be an open-label,
Phase 1, multicenter, non-randomized, dose-determining trial of
ADX31-142 monotherapy in subjects with metastatic
castration-resistant prostate cancer (mCRPC). Part B of the study
will be an open-label, Phase 1-2, multicenter, non-randomized
dose-determining trial of ADXS31-142 in combination with
pembrolizumab (MK-3475) in subjects with mCRPC. FIG. 10 presents a
diagram of the monotherapy and combination portions of this
study.
Objectives:
[0378] Part A: to evaluate safety and tolerability of ADXS31-142
monotherapy and select the recommended phase 2 dose (RP2D) in
subjects with mCRPC
[0379] Part B: to evaluate safety and tolerability of ADXS31-142 in
combination with pembrolizumab (MK-3475) and to establish the RP2D
for this combination in subjects with mCRPC
[0380] In addition, objectives of the Study include evaluating
anti-tumor activity and progression free survival (PFS) signal of
ADXS31-142 monotherapy and ADXS31-142+pembrolizumab (MK-3475)
combination therapy using RECIST 1.1, immune-related Response
Evaluation Criteria in Solid Tumors (irRECIST) and Prostate Cancer
Working Group 2 (PCWG2) criteria to inform design of a subsequent
randomized Phase 2 trial. The effects on serum prostate specific
antigen (PSA) and periphaeral immunologic measures of ADXS31-142
montherapy and ADXS31-142+pembrolizumab (MK-3475) combination
therapy will be determined.
Product Description
[0381] Each of these products is described in detail above.
[0382] ADXS31-142 will be provided as a concentrated suspension for
injection, at a concentration of 2.7.times.10.sup.9 cfu/mL; 1.2
mL/vial.
[0383] Pembrolizumab (MK-3475) may be provided in two different
forms: (1) as a lyophilized powder for injection (50 mg); and (2)
as a solution for infusion at a concentration of 25 mg/mL.
Part A--ADXS31-124 Monotherapy
[0384] Materials and Methods
[0385] Subjects:
[0386] The study will be conducted in male subjects (.gtoreq.18
years) with histologically confirmed mCRPC who have progressed or
become resistant to no more than 3 prior systemic treatment
regimens with chemotherapy, hormonal, or immunotherapy in the
metastatic setting; and with an Eastern Cooperative Oncology Group
(ECOG) performance status of 0-1 are eligble. However, subjects can
not have had more than 1 prior chemotherapeutic regimen in the
metastatic setting. Subjects with evidence of progressive bone or
other metastases are acceptable. Subjects may remain on castration
therapy (luteinizing-hormone-releasing hormone [LHRH] agonist or
antagonist) during the trial.
[0387] Dose:
[0388] The dose determining phase is intended to select a
recommended Phase 2 dose (RP2D) for Part B. The starting dose level
(DL) of ADSX31-142 monotherapy will be 1.times.10.sup.9 colony
forming units (cfu) (DL 1). The dose will be escalated
(5.times.10.sup.9 cfu, 1.times.10.sup.10 cfu), remain the same or
be de-escalated according to pre-defined dose-limiting toxicity
(DLT) criteria associated with a DLT rate.ltoreq.0.25 by applying
the modified toxicity probability interval (mTPI) design. Table 5
summarizes ADXS31-142 Monotherapy Doses to be used.
TABLE-US-00010 TABLE 5 Route of Dose Level Dose Administration
Regimen 1 1 .times. 10.sup.9 cfu IV infusion Day 1 of Weeks 1, 4
and 7 of 12-week cycle 2 5 .times. 10.sup.9 cfu IV infusion Day 1
of Weeks 1, 4 and 7 of 12-week cycle 3 1 .times. 10.sup.10 cfu.sup.
IV infusion Day 1 of Weeks 1, 4 and 7 of 12-week cycle -1 .5
.times. 10.sup.9 cfu IV infusion Day 1 of Weeks 1, 4 and 7 of
12-week cycle
[0389] Up to 21 subjects will be entered (with a minimum of 6
subjects treated at the recommended dose before proceeding to the
next phase). A minimum of 3 subjects will be evaluated in each
cohort before dose-escalation decisions are made.
[0390] Table 6 below presents Dose Decisions for
ADSX31-142--Monotherapy.
[0391] Table 6 is based on a sample size of 21 subjects. Two
parameters epsilon1 and epsilon2 are set at default values of 0.05.
x-axis is number of subjects treated at current dose; y-axis is
number of toxicities.
[0392] The Targeted dose limiting toxicity (DLT) Rate will increase
to 30% for the combination regimen (Part B) in the event that a 25%
DLT rate is observed at the recommended ADXS31-142 monotherapy
dose.
[0393] The same DLT criteria will be utilized for study Part A and
Part B. All toxicities will be graded using CTCAE Version 4.0. The
DLT window of observation will be 4 weeks (after 2 doses of each
drug). The occurrence of any of the following toxicities will be
considered a DLT, if judged by the investigator to be possibly,
probably or definitely related to study treatment
administration.
[0394] Endpoints
[0395] Efficacy Endpoints--The efficacy endpoints to be used in
this study (PSA/PAP, other serum markers for prostate cancer,
scans, and measureable and evaluable disease assessments) are those
typically used to assess anti-tumor activity of mCRPC.
[0396] Safety Endpoint--The primary safety objective of this trial
is to characterize the safety and tolerability of ADXS31-142 alone.
The primary safety analysis will be based on subjects who
experienced toxicities as defined by CTCAE criteria. Safety will be
assessed by quantifying the toxicities and grades experienced by
subjects who have received ADXS31-142 alone, including serious
adverse events (SAEs) and events of clinical interest (ECIs).
[0397] Biomarkers
[0398] T-cells will be assessed for their specific response to PSA
and other prostate cancer antigens which may include PSMA, PAP, and
prostate stem cell antigen (PSCA). T-cell responses will be
determined by enzyme-linked immunosorbent assay (ELISA) and/or
ELISpot. PBMC immunologic gene expression analysis may also be
conducted.
[0399] Serum cytokine and chemokine changes will be determined to
assess immune stimulation as a result of treatment. Serum cytokine
levels will include IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15,
IL-18, transforming growth factor beta (TGF.beta.), and tumor
necrosis factor alpha (TNF.alpha.). Serum chemokines will include
CXCL 9, 10, and 11.
[0400] Route of Administration
[0401] ADSX31-142 monotherapy will be administered by IV infusion
by medically trained personnel.
[0402] Regimen
[0403] ADSX31-142 monotherapy will be administered on Day 1 of
weeks 1, 4, and 7, of a once every 3 weeks in a 12-week treatment
cycle. Trial treatment may be administered up to 3 days before or
after the scheduled Day 1 of each cycle due to administrative
reasons.
[0404] All trial treatments will be administered as a 30 minute IV
infusion (treatment cycle intervals may be increased due to
toxicity). In Part A, NSAIDs (naproxen or ibuprofen) and antiemetic
medication should be administered within 30 minutes prior to the
ADXS31-142 infusion.
[0405] Tumor Imaging and Assessment of Disease
[0406] Computed tomography (CT), magnetic resonance imaging (MRI)
or bone scan will be considered the best currently available and
reproducible methods to measure target lesions selected for
response assessment. Conventional CT and MRI of the abdomen/pelvis
should be performed with contiguous cuts of 10 mm or less. Spiral
CT scan should be performed using a 5 mm contiguous reconstruction
algorithm (as a general rule, lesion diameter should be no less
than double the slice thickness). Lesions on chest x-rays will be
acceptable as measurable lesions when they are clearly defined and
surrounded by aerated lung; however, CT is preferable. Ultrasound
will not be an acceptable method to measure disease.
[0407] Measurable and Non-Measurable Lesions and Disease
[0408] Measurable lesions will be those that can be accurately
measured in at least one dimension with the longest
diameter.gtoreq.2.0 cm (for spiral CT scan or MRI scan, .gtoreq.1.0
cm). Measurable disease will be present if the subject has 1 or
more measurable lesions.
[0409] Non-measurable lesions/disease will be all other lesions (or
sites of disease), including small lesions (those with all
measurements<2.0 cm with spiral CT or <1.0 cm with MRI), or
any of the following: bone lesions, leptomeningeal disease,
ascites, pleural or pericardial effusion, lymphangitis,
cutis/pulmonis, abdominal masses that are not confirmed and
followed by imaging techniques, cystic lesions, and lesions
occurring within a previously irradiated area unless they are
documented as new lesions since the completion of radiation
therapy.
[0410] Target/Non-Target Lesions
[0411] All measurable lesions, up to a maximum of 2 per organ and 5
in total, should be identified as target lesions to be measured and
recorded at baseline. The target lesions should be representative
of all involved organs. Target lesions will be selected based on
their size (the lesion with the longest diameter) and suitability
for accurate repeated measurements. At baseline, a sum of the
longest diameters for all target lesions will be calculated and
recorded as the baseline tumor burden. The baseline sum will be
used as the reference point to determine the objective tumor
response of the target lesions.
[0412] Measurable lesions other than the target lesions and all
sites of non-measurable disease will be identified as non-target
lesions and will be recorded at baseline. Non-target lesions will
be evaluated at the same timepoints as target lesions.
[0413] Response in Measureable Lesions (RECIST 1.1)
[0414] At baseline, the sum of the longest diameters (SumD) of all
target lesions (up to 2 lesions per organ, up to total 5 lesions)
is measured. At each subsequent tumor assessment (TA), the SumD of
the target lesions and of new, measurable lesions are added
together to provide the total measurable tumor burden (TMTB):
TMTB=SumD target lesions+SumD new,measurable lesions
[0415] Percentage changes in TMTB per assessment time point
describe the size and growth kinetics of both old and new
measurable lesions as they appear. At each TA, the response in
target and new measurable lesions is defined based on the change in
TMTB (after ruling out irPD) as follows:
[0416] Complete Response (CR): disappearance of all target lesions.
Any pathological lymph nodes (whether target or non-target) must
have reduction in short axis to .ltoreq.10 mm.
[0417] Partial Response (PR): At least a 30% decrease in sum of
diameter of target lesions, taking as reference the baseline sum
diameter.
[0418] Stable Disease (SD): Neither sufficient shrinkage to qualify
for PR nor sufficient increa
[0419] Progressive Disease (PD): At least a 20% increase in the sum
of diameters of target lesions, taking as reference the smallest
sum on study (this includes the baseline sum if that is the
smallest on study). In addition to the relative increase of 20%,
the sum must also demonstrate an absolute increase of at least 5
mm. (Note: the appearance of one or more new lesions is also
considered progression).
[0420] Response in non-measurable lesions will also be
assessed.
[0421] Part B--ADSX31-142+Pembrolizumab (MK-3475) Combination
Therapy
[0422] Materials and Methods
[0423] Subjects
[0424] Subjects have been described above in Part A of the study.
The subjects for Part B will be included in the study based on the
same population criteria. The plan is to treat a total of 30
subject at RP2D.
[0425] Dose
[0426] Part B will consist of a dose-determination phase followed
by an expansion cohort phase. The dose-determining phase is
intended to select a RP2D for the combination. Dose
escalation/de-escalation will be explored by applying the mTPI
design.
[0427] During the dose-determining stage, up to 21 subjects will be
entered at escalating doses of ADXS31-142 (see Table 7) in
combination with pembrolizumab (MK-3475) at 200 mg (with a minimum
of 6 subjects treated at the RP2D before proceeding to expansion).
Dose-determination will continue until identification of a
preliminary maximum tolerated dose/maximum allowable dose
(MTD/MAD), up to a maximum dose of 1.times.10.sup.10 cfu of
ADXS31-142. The MTD/MAD will be the RP2D for the dose expansion
portion. If a MTD is not identified, then the highest planned dose
level of ADXS31-142 in combination with pembrolizumab (MK-3475)
will be considered the RP2D. The pembrolizumab (MK-3475) will
remain constant at 200 mg.
TABLE-US-00011 TABLE 7 ADXS31-142 and Pembrolizumab (MK-3475)
Combination Therapy Doses to be used in Trial Part B Dose Route of
Drug Level Dose Administration Regimen ADXS31-142 1 One level below
RP2D in Part A IV infusion Day 1 of Weeks 1, 4 and 7 of 12-week
cycle 2 RP2D in Part A IV infusion Day 1 of Weeks 1, 4 and 7 of
12-week cycle -1 Two dose levels below RP2D IV infusion Day 1 of
Weeks 1, 4 and 7 of in Part A 12-week cycle Pembrolizumab 200 mg IV
infusion Day 1 Q3W of 12-week cycle (MK-3475)
[0428] As for Part A of this study, the expected doses for
ADXS31-142 will be 0.5.times.10.sup.9, 1.times.10.sup.9,
5.times.10.sup.9, and 1.times.10.sup.10
[0429] The expansion cohort will be open for enrollment once the
RP2D of ADXS31-142 in combination with pembrolizumab (MK-3475) is
selected in the Part B dose determination phase. Further assessment
of the RP2D will be explored in up to 30 patients with mCRPC to
evaluate the safety and clinical activity of ADXS31-142 in
combination with pembrolizumab (MK-3475).
[0430] Table 8 below presents Dose Decisions for Combination
Therapy
[0431] Adverse events will be monitored from the time informed
consent is obtained and graded in severity according to the
guidelines outlined in the National Cancer Institute (NCI) Common
Terminology Criteria for Adverse Events (CTCAE) Version 4.0.
[0432] Treatment with ADXS31-142 in combination with pembrolizumab
(MK3475) will continue until documented disease progression,
unacceptable adverse event(s), intercurrent illness that prevents
further administration of treatment, investigator's decision to
withdraw the subject, subject withdraws consent, subject
experiences a complete response (irCR) and receives one additional
cycle of treatment, noncompliance with trial treatment or procedure
requirements, completion of 24 months of treatment with ADXS31142
and pembrolizumab (MK-3475), or administrative reasons. Subjects
who attain an investigator confirmed irCR, after receiving at least
2 cycles of therapy, may consider stopping pembrolizumab (MK-3475)
and continue treatment with ADXS31-142 only. After the end of
treatment, each subject will be followed for 30 days after the last
study drug administration for adverse event and 90 days for serious
adverse events or events of clinical interest monitoring. Subjects
who discontinue treatment for reasons other than disease
progression will have post-treatment follow-up for disease status
until disease progression, initiating a non-study cancer treatment,
withdrawing consent, becoming lost to follow-up, or until the
sponsor ends the study. The primary objectives of the trial are to
establish a MTD or MAD and to determine safety and tolerability of
ADXS31-142 in combination with pembrolizumab (MK-3475) in subjects
with mCRPC.
[0433] Efficcacy and Safety Endpoints will be as described for Part
A of this Study.
[0434] Biomarker Research will be as described for Part A of this
Study.
[0435] Route of Administration
[0436] ADSX31-142 and Pembrolizumab (MK-3475 will each be
administered by IV infusion by medically trained personnel.
[0437] Regimen
[0438] Trial treatment should be administered on Day 1 of Week 1, 4
and 7 (for ADXS31-142) or Q3W (for pembrolizumab [MK-3475]) in each
12-week cycle after all procedures/assessments have been completed.
Trial treatment may be administered up to 3 days before or after
the scheduled Day 1 of each cycle due to administrative
reasons.
[0439] All trial treatments will be administered as a 30 minute IV
infusion (treatment cycle intervals may be increased due to
toxicity. there should be approximately 60 minutes between the end
of the first infusion and the start of the second infusion.
Pembrolizumab (MK-3475) infusion will be administered first.
[0440] Assessment in target and non-target lesions is as described
above for Part A.
Sequence CWU 1
1
66115PRTArtificial SequenceAntibody Light Chain CDR 1Arg Ala Ser
Lys Ser Val Ser Thr Ser Gly Phe Ser Tyr Leu His 1 5 10 15
27PRTArtificial SequenceAntibody Light Chain CDR 2Leu Ala Ser Asn
Leu Glu Ser 1 5 39PRTArtificial SequenceAntibody Light Chain CDR
3Gln His Ser Trp Glu Leu Pro Leu Thr 1 5 45PRTArtificial
SequenceAntibody Heavy Chain CDR 4Ser Tyr Tyr Leu Tyr 1 5
517PRTArtificial SequenceAntibody Heavy Chain CDR 5Gly Val Asn Pro
Ser Asn Gly Gly Thr Asn Phe Ser Glu Lys Phe Lys 1 5 10 15 Ser
611PRTArtificial SequenceAntibody Heavy Chain CDR 6Arg Asp Ser Asn
Tyr Asp Gly Gly Phe Asp Tyr 1 5 10 715PRTArtificial
SequenceAntibody Light Chain CDR 7Arg Ala Ser Lys Gly Val Ser Thr
Ser Gly Tyr Ser Tyr Leu His 1 5 10 15 87PRTArtificial
SequenceAntibody Light Chain CDR 8Leu Ala Ser Tyr Leu Glu Ser 1 5
99PRTArtificial SequenceAntibody Light Chain CDR 9Gln His Ser Arg
Asp Leu Pro Leu Thr 1 5 105PRTArtificial SequenceAntibody Heavy
Chain CDR 10Asn Tyr Tyr Met Tyr 1 5 1117PRTArtificial
SequenceAntibody Heavy Chain CDR 11Gly Ile Asn Pro Ser Asn Gly Gly
Thr Asn Phe Asn Glu Lys Phe Lys 1 5 10 15 Asn 1211PRTArtificial
SequenceAntibody Heavy Chain CDR 12Arg Asp Tyr Arg Phe Asp Met Gly
Phe Asp Tyr 1 5 10 13120PRTArtificial SequenceHumanized Antibody
Heavy Chain Variable Region 13Gln Val Gln Leu Val Gln Ser Gly Val
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile
Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys
Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70
75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr
Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120
14447PRTArtificial SequenceHumanized Antibody Heavy Chain 14Gln Val
Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20
25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn
Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser
Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe
Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150
155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala
Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275
280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395
400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys 435 440 445 15111PRTArtificial SequenceHumanized
Antibody Light Chain Variable Region 15Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser
Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg
Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55
60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His
Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 110 16111PRTArtificial SequenceHumanized
Antibody Light Chain Variable Region 16Glu Ile Val Leu Thr Gln Ser
Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile
Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser
Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 35 40 45 Gln
Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp 50 55
60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser
65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His
Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 100 105 110 17111PRTArtificial SequenceHumanized
Antibody Light Chain Variable Region 17Asp Ile Val Met Thr Gln Thr
Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile
Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser
Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 35 40 45 Gln
Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp 50 55
60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile Ser
65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Gln His
Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys 100 105 110 18218PRTArtificial SequenceHumanized
Antibody Light Chain 18Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr
Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser
Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90
95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 19218PRTArtificial
SequenceHumanized Antibody Light Chain 19Glu Ile Val Leu Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser
Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr
Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro 35 40 45
Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp 50
55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln
His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180
185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
20218PRTArtificial SequenceHumanized Antibody Light Chain 20Asp Ile
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20
25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
Pro 35 40 45 Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly
Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe
Thr Leu Lys Ile Ser 65 70 75 80 Arg Val Glu Ala Glu Asp Val Gly Leu
Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150
155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 210 215 21447PRTArtificial SequenceHumanized Antibody Heavy
Chain 21Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly
Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly
Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr
Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser
Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg
Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120
125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys
Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245
250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370
375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys 435 440 445 22218PRTArtificial
SequenceHumanized Antibody Light Chain 22Glu Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25
30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val
Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155
160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 23440PRTHomo sapiens 23Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Asp Cys Lys
Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30 Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile
Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys 180 185 190
Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp 195
200 205 Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
Ala 210 215 220 Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro 225 230 235 240 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val 245 250 255 Val Asp Val Ser Gln Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val 260 265 270 Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 275 280 285 Phe Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 290 295 300 Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 305 310 315
320 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
325 330 335 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr 340 345 350 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser 355 360 365 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr 370 375 380 Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr 385 390 395 400 Ser Arg Leu Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 405 410 415 Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 420 425 430 Ser
Leu Ser Leu Ser Leu Gly Lys 435 440 24214PRTHomo sapiens 24Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150
155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
25261PRTHomo sapiens 25Met Trp Val Pro Val Val Phe Leu Thr Leu Ser
Val Thr Trp Ile Gly 1 5 10 15 Ala Ala Pro Leu Ile Leu Ser Arg Ile
Val Gly Gly Trp Glu Cys Glu 20 25 30 Lys His Ser Gln Pro Trp Gln
Val Leu Val Ala Ser Arg Gly Arg Ala 35 40 45 Val Cys Gly Gly Val
Leu Val His Pro Gln Trp Val Leu Thr Ala Ala 50 55 60 His Cys Ile
Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu 65 70 75 80 Phe
His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser His Ser Phe 85 90
95 Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg
100 105 110 Pro Gly Asp Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu
Ser Glu 115 120 125 Pro Ala Glu Leu Thr Asp Ala Val Lys Val Met Asp
Leu Pro Thr Gln 130 135 140 Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala
Ser Gly Trp Gly Ser Ile 145 150 155 160 Glu Pro Glu Glu Phe Leu Thr
Pro Lys Lys Leu Gln Cys Val Asp Leu 165 170 175 His Val Ile Ser Asn
Asp Val Cys Ala Gln Val His Pro Gln Lys Val 180 185 190 Thr Lys Phe
Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr 195 200 205 Cys
Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln 210 215
220 Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro
225 230 235 240 Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile
Lys Asp Thr 245 250 255 Ile Val Ala Asn Pro 260 26237PRTHomo
sapiens 26Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln Pro Trp
Gln Val 1 5 10 15 Leu Val Ala Ser Arg Gly Arg Ala Val Cys Gly Gly
Val Leu Val His 20 25 30 Pro Gln Trp Val Leu Thr Ala Ala His Cys
Ile Arg Asn Lys Ser Val 35 40 45 Ile Leu Leu Gly Arg His Ser Leu
Phe His Pro Glu Asp Thr Gly Gln 50 55 60 Val Phe Gln Val Ser His
Ser Phe Pro His Pro Leu Tyr Asp Met Ser 65 70 75 80 Leu Leu Lys Asn
Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His Asp 85 90 95 Leu Met
Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu Thr Asp Ala Val 100 105 110
Lys Val Met Asp Leu Pro Thr Gln Glu Pro Ala Leu Gly Thr Thr Cys 115
120 125 Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Glu Phe Leu Thr
Pro 130 135 140 Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser Asn
Asp Val Cys 145 150 155 160 Ala Gln Val His Pro Gln Lys Val Thr Lys
Phe Met Leu Cys Ala Gly 165 170 175 Arg Trp Thr Gly Gly Lys Ser Thr
Cys Ser Gly Asp Ser Gly Gly Pro 180 185 190 Leu Val Cys Tyr Gly Val
Leu Gln Gly Ile Thr Ser Trp Gly Ser Glu 195 200 205 Pro Cys Ala Leu
Pro Glu Arg Pro Ser Leu Tyr Thr Lys Val Val His 210 215 220 Tyr Arg
Lys Trp Ile Lys Asp Thr Ile Val Ala Asn Pro 225 230 235
27237PRTHomo sapiens 27Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser
Gln Pro Trp Gln Val 1 5 10 15 Leu Val Ala Ser Arg Gly Arg Ala Val
Cys Gly Gly Val Leu Val His 20 25 30 Pro Gln Trp Val Leu Thr Ala
Ala His Cys Ile Arg Asn Lys Ser Val 35 40 45 Ile Leu Leu Gly Arg
His Ser Leu Phe His Pro Glu Asp Thr Gly Gln 50 55 60 Val Phe Gln
Val Ser His Ser Phe Pro His Pro Leu Tyr Asp Met Ser 65 70 75 80 Leu
Leu Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His Asp 85 90
95 Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu Thr Asp Ala Val
100 105 110 Lys Val Met Asp Leu Pro Thr Gln Glu Pro Ala Leu Gly Thr
Thr Cys 115 120 125 Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Glu
Phe Leu Thr Pro 130 135 140 Lys Lys Leu Gln Cys Val Asp Leu His Val
Ile Ser Asn Asp Val Cys 145 150 155 160 Ala Gln Val His Pro Gln Lys
Val Thr Lys Phe Met Leu Cys Ala Gly 165 170 175 Arg Trp Thr Gly Gly
Lys Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro 180 185 190 Leu Val Cys
Asn Gly Val Leu Gln Gly Ile Thr Ser Trp Gly Ser Glu 195 200 205 Pro
Cys Ala Leu Pro Glu Arg Pro Ser Leu Tyr Thr Lys Val Val His 210 215
220 Tyr Arg Lys Trp Ile Lys Asp Thr Ile Val Ala Asn Pro 225 230 235
285873DNAHomo sapiens 28ggtgtcttag gcacactggt cttggagtgc aaaggatcta
ggcacgtgag gctttgtatg 60aagaatcggg gatcgtaccc accccctgtt tctgtttcat
cctgggcatg tctcctctgc 120ctttgtcccc tagatgaagt ctccatgagc
tacaagggcc tggtgcatcc agggtgatct 180agtaattgca gaacagcaag
tgctagctct ccctcccctt ccacagctct gggtgtggga 240gggggttgtc
cagcctccag cagcatgggg agggccttgg tcagcctctg ggtgccagca
300gggcaggggc ggagtcctgg ggaatgaagg ttttataggg ctcctggggg
aggctcccca 360gccccaagct taccacctgc acccggagag ctgtgtcacc
atgtgggtcc cggttgtctt 420cctcaccctg tccgtgacgt ggattggtga
gaggggccat ggttgggggg atgcaggaga 480gggagccagc cctgactgtc
aagctgaggc tctttccccc ccaacccagc accccagccc 540agacagggag
ctgggctctt ttctgtctct cccagcccca cttcaagccc atacccccag
600tcccctccat attgcaacag tcctcactcc cacaccaggt ccccgctccc
tcccacttac 660cccagaactt tcttcccatt tgcccagcca gctccctgct
cccagctgct ttactaaagg 720ggaagttcct gggcatctcc gtgtttctct
ttgtggggct caaaacctcc aaggacctct 780ctcaatgcca ttggttcctt
ggaccgtatc actggtccat ctcctgagcc cctcaatcct 840atcacagtct
actgactttt cccattcagc tgtgagtgtc caaccctatc ccagagacct
900tgatgcttgg cctcccaatc ttgccctagg atacccagat gccaaccaga
cacctccttc 960tttcctagcc aggctatctg gcctgagaca acaaatgggt
ccctcagtct ggcaatggga 1020ctctgagaac tcctcattcc ctgactctta
gccccagact cttcattcag tggcccacat 1080tttccttagg aaaaacatga
gcatccccag ccacaactgc cagctctctg agtccccaaa 1140tctgcatcct
tttcaaaacc taaaaacaaa aagaaaaaca aataaaacaa aaccaactca
1200gaccagaact gttttctcaa cctgggactt cctaaacttt ccaaaacctt
cctcttccag 1260caactgaacc tcgccataag gcacttatcc ctggttccta
gcacccctta tcccctcaga 1320atccacaact tgtaccaagt ttcccttctc
ccagtccaag accccaaatc accacaaagg 1380acccaatccc cagactcaag
atatggtctg ggcgctgtct tgtgtctcct accctgatcc 1440ctgggttcaa
ctctgctccc agagcatgaa gcctctccac cagcaccagc caccaacctg
1500caaacctagg gaagattgac agaattccca gcctttccca gctccccctg
cccatgtccc 1560aggactccca gccttggttc tctgcccccg tgtcttttca
aacccacatc ctaaatccat 1620ctcctatccg agtcccccag ttccccctgt
caaccctgat tcccctgatc tagcaccccc 1680tctgcaggcg ctgcgcccct
catcctgtct cggattgtgg gaggctggga gtgcgagaag 1740cattcccaac
cctggcaggt gcttgtggcc tctcgtggca gggcagtctg cggcggtgtt
1800ctggtgcacc cccagtgggt cctcacagct gcccactgca tcaggaagtg
agtaggggcc 1860tggggtctgg ggagcaggtg tctgtgtccc agaggaataa
cagctgggca ttttccccag 1920gataacctct aaggccagcc ttgggactgg
gggagagagg gaaagttctg gttcaggtca 1980catggggagg cagggttggg
gctggaccac cctccccatg gctgcctggg tctccatctg 2040tgtccctcta
tgtctctttg tgtcgctttc attatgtctc ttggtaactg gcttcggttg
2100tgtctctccg tgtgactatt ttgttctctc tctccctctc ttctctgtct
tcagtctcca 2160tatctccccc tctctctgtc cttctctggt ccctctctag
ccagtgtgtc tcaccctgta 2220tctctctgcc aggctctgtc tctcggtctc
tgtctcacct gtgccttctc cctactgaac 2280acacgcacgg gatgggcctg
ggggaccctg agaaaaggaa gggctttggc tgggcgcggt 2340ggctcacacc
tgtaatccca gcactttggg aggccaaggc aggtagatca cctgaggtca
2400ggagttcgag accagcctgg ccaactggtg aaaccccatc tctactaaaa
atacaaaaaa 2460ttagccaggc gtggtggcgc atgcctgtag tcccagctac
tcaggagctg agggaggaga 2520attgcattga acctggaggt tgaggttgca
gtgagccgag accgtgccac tgcactccag 2580cctgggtgac agagtgagac
tccgcctcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaga 2640aaagaaaaga
aaagaaaagg aagtgtttta tccctgatgt gtgtgggtat gagggtatga
2700gagggcccct ctcactccat tccttctcca ggacatccct ccactcttgg
gagacacaga 2760gaagggctgg ttccagctgg agctgggagg ggcaattgag
ggaggaggaa ggagaagggg 2820gaaggaaaac agggtatggg ggaaaggacc
ctggggagcg aagtggagga tacaaccttg 2880ggcctgcagg caggctacct
acccacttgg aaacccacgc caaagccgca tctacagctg 2940agccactctg
aggcctcccc tccccggcgg tccccactca gctccaaagt ctctctccct
3000tttctctccc acactttatc atcccccgga ttcctctcta cttggttctc
attcttcctt 3060tgacttcctg cttccctttc tcattcatct gtttctcact
ttctgcctgg ttttgttctt 3120ctctctctct ttctctggcc catgtctgtt
tctctatgtt tctgtctttt ctttctcatc 3180ctgtgtattt tcggctcacc
ttgtttgtca ctgttctccc ctctgccctt tcattctctc 3240tgccctttta
ccctcttcct tttcccttgg ttctctcagt tctgtatctg cccttcaccc
3300tctcacactg ctgtttccca actcgttgtc tgtattttgg cctgaactgt
gtcttcccaa 3360ccctgtgttt tctcactgtt tctttttctc ttttggagcc
tcctccttgc tcctctgtcc 3420cttctctctt tccttatcat cctcgctcct
cattcctgcg tctgcttcct ccccagcaaa 3480agcgtgatct tgctgggtcg
gcacagcctg tttcatcctg aagacacagg ccaggtattt 3540caggtcagcc
acagcttccc acacccgctc tacgatatga gcctcctgaa gaatcgattc
3600ctcaggccag gtgatgactc cagccacgac ctcatgctgc tccgcctgtc
agagcctgcc 3660gagctcacgg atgctgtgaa ggtcatggac ctgcccaccc
aggagccagc actggggacc 3720acctgctacg cctcaggctg gggcagcatt
gaaccagagg agtgtacgcc tgggccagat 3780ggtgcagccg ggagcccaga
tgcctgggtc tgagggagga ggggacagga ctcctgggtc 3840tgagggagga
gggccaagga accaggtggg gtccagccca caacagtgtt tttgcctggc
3900ccgtagtctt gaccccaaag aaacttcagt gtgtggacct ccatgttatt
tccaatgacg 3960tgtgtgcgca agttcaccct cagaaggtga ccaagttcat
gctgtgtgct ggacgctgga 4020cagggggcaa aagcacctgc tcggtgagtc
atccctactc ccaagatctt gagggaaagg 4080tgagtgggac cttaattctg
ggctggggtc tagaagccaa caaggcgtct gcctcccctg 4140ctccccagct
gtagccatgc cacctccccg tgtctcatct cattccctcc ttccctcttc
4200tttgactccc tcaaggcaat aggttattct tacagcacaa ctcatctgtt
cctgcgttca 4260gcacacggtt actaggcacc tgctatgcac ccagcactgc
cctagagcct gggacatagc 4320agtgaacaga cagagagcag cccctccctt
ctgtagcccc caagccagtg aggggcacag 4380gcaggaacag ggaccacaac
acagaaaagc tggagggtgt caggaggtga tcaggctctc 4440ggggagggag
aaggggtggg gagtgtgact gggaggagac atcctgcaga aggtgggagt
4500gagcaaacac ctgcgcaggg gaggggaggg cctgcggcac ctgggggagc
agagggaaca 4560gcatctggcc aggcctggga ggaggggcct agagggcgtc
aggagcagag aggaggttgc 4620ctggctggag tgaaggatcg gggcagggtg
cgagagggaa caaaggaccc ctcctgcagg 4680gcctcacctg ggccacagga
ggacactgct tttcctctga ggagtcagga actgtggatg 4740gtgctggaca
gaagcaggac agggcctggc tcaggtgtcc agaggctgcg ctggcctcct
4800atgggatcag actgcaggga gggagggcag cagggatgtg gagggagtga
tgatggggct 4860gacctggggg tggctccagg cattgtcccc acctgggccc
ttacccagcc tccctcacag 4920gctcctggcc ctcagtctct cccctccact
ccattctcca cctacccaca gtgggtcatt 4980ctgatcaccg aactgaccat
gccagccctg ccgatggtcc tccatggctc cctagtgccc 5040tggagaggag
gtgtctagtc agagagtagt cctggaaggt ggcctctgtg aggagccacg
5100gggacagcat cctgcagatg gtcctggccc ttgtcccacc gacctgtcta
caaggactgt 5160cctcgtggac cctcccctct gcacaggagc tggaccctga
agtcccttcc taccggccag 5220gactggagcc cctacccctc tgttggaatc
cctgcccacc ttcttctgga agtcggctct 5280ggagacattt ctctcttctt
ccaaagctgg gaactgctat ctgttatctg cctgtccagg 5340tctgaaagat
aggattgccc aggcagaaac tgggactgac ctatctcact ctctccctgc
5400ttttaccctt agggtgattc tgggggccca cttgtctgta atggtgtgct
tcaaggtatc 5460acgtcatggg gcagtgaacc atgtgccctg cccgaaaggc
cttccctgta caccaaggtg 5520gtgcattacc ggaagtggat caaggacacc
atcgtggcca acccctgagc acccctatca 5580agtccctatt gtagtaaact
tggaaccttg gaaatgacca ggccaagact caagcctccc 5640cagttctact
gacctttgtc cttaggtgtg aggtccaggg ttgctaggaa aagaaatcag
5700cagacacagg tgtagaccag agtgtttctt aaatggtgta attttgtcct
ctctgtgtcc 5760tggggaatac tggccatgcc tggagacata tcactcaatt
tctctgagga cacagttagg 5820atggggtgtc tgtgttattt gtgggataca
gagatgaaag aggggtggga tcc 587329238PRTHomo sapiens 29Met Trp Val
Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala
Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 20 25
30 Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala
35 40 45 Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr
Ala Ala 50 55 60 His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly
Arg His Ser Leu 65 70 75 80 Phe His Pro Glu Asp Thr Gly Gln Val Phe
Gln Val Ser His Ser Phe 85 90 95 Pro His Pro Leu Tyr Asp Met Ser
Leu Leu Lys Asn Arg Phe Leu Arg 100 105 110 Pro Gly Asp Asp Ser Ser
His Asp Leu Met Leu Leu Arg Leu Ser Glu 115 120 125 Pro Ala Glu Leu
Thr Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln 130 135 140 Glu Pro
Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile 145 150 155
160 Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu
165 170 175 His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln
Lys Val 180 185 190 Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly
Gly Lys Ser Thr 195 200 205 Cys Ser Trp Val Ile Leu Ile Thr Glu Leu
Thr Met Pro Ala Leu Pro 210 215 220 Met Val Leu His Gly Ser Leu Val
Pro Trp Arg Gly Gly Val 225 230 235 301906DNAHomo sapiens
30agccccaagc ttaccacctg cacccggaga gctgtgtcac catgtgggtc ccggttgtct
60tcctcaccct gtccgtgacg tggattggtg ctgcacccct catcctgtct cggattgtgg
120gaggctggga gtgcgagaag cattcccaac cctggcaggt gcttgtggcc
tctcgtggca 180gggcagtctg cggcggtgtt ctggtgcacc cccagtgggt
cctcacagct gcccactgca 240tcaggaacaa aagcgtgatc ttgctgggtc
ggcacagcct gtttcatcct gaagacacag 300gccaggtatt tcaggtcagc
cacagcttcc cacacccgct ctacgatatg agcctcctga 360agaatcgatt
cctcaggcca ggtgatgact ccagccacga cctcatgctg ctccgcctgt
420cagagcctgc cgagctcacg gatgctgtga aggtcatgga cctgcccacc
caggagccag 480cactggggac cacctgctac gcctcaggct ggggcagcat
tgaaccagag gagttcttga 540ccccaaagaa acttcagtgt gtggacctcc
atgttatttc caatgacgtg tgtgcgcaag 600ttcaccctca gaaggtgacc
aagttcatgc tgtgtgctgg acgctggaca gggggcaaaa 660gcacctgctc
gtgggtcatt ctgatcaccg aactgaccat gccagccctg ccgatggtcc
720tccatggctc cctagtgccc tggagaggag gtgtctagtc agagagtagt
cctggaaggt 780ggcctctgtg aggagccacg gggacagcat cctgcagatg
gtcctggccc ttgtcccacc 840gacctgtcta caaggactgt cctcgtggac
cctcccctct gcacaggagc tggaccctga 900agtcccttcc ccaccggcca
ggactggagc ccctacccct ctgttggaat ccctgcccac 960cttcttctgg
aagtcggctc tggagacatt tctctcttct tccaaagctg ggaactgcta
1020tctgttatct gcctgtccag gtctgaaaga taggattgcc caggcagaaa
ctgggactga 1080cctatctcac tctctccctg cttttaccct tagggtgatt
ctgggggccc acttgtctgt 1140aatggtgtgc ttcaaggtat cacgtcatgg
ggcagtgaac catgtgccct gcccgaaagg 1200ccttccctgt acaccaaggt
ggtgcattac cggaagtgga tcaaggacac catcgtggcc 1260aacccctgag
cacccctatc aaccccctat tgtagtaaac ttggaacctt ggaaatgacc
1320aggccaagac tcaagcctcc ccagttctac tgacctttgt ccttaggtgt
gaggtccagg 1380gttgctagga aaagaaatca gcagacacag gtgtagacca
gagtgtttct taaatggtgt 1440aattttgtcc tctctgtgtc ctggggaata
ctggccatgc ctggagacat atcactcaat 1500ttctctgagg acacagatag
gatggggtgt ctgtgttatt tgtggggtac agagatgaaa 1560gaggggtggg
atccacactg agagagtgga gagtgacatg tgctggacac tgtccatgaa
1620gcactgagca gaagctggag gcacaacgca ccagacactc acagcaagga
tggagctgaa 1680aacataaccc actctgtcct ggaggcactg ggaagcctag
agaaggctgt gagccaagga 1740gggagggtct tcctttggca tgggatgggg
atgaagtaag gagagggact ggaccccctg 1800gaagctgatt cactatgggg
ggaggtgtat tgaagtcctc cagacaaccc tcagatttga 1860tgatttccta
gtagaactca cagaaataaa gagctgttat actgtg 19063169PRTHomo sapiens
31Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1
5 10 15 Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys
Glu 20 25 30 Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg
Gly Arg Ala 35 40 45 Val Cys Gly Gly Val Leu Val His Pro Gln Trp
Val Leu Thr Ala Ala 50 55 60 His Cys Ile Arg Lys 65 32554DNAHomo
sapiens 32agccccaagc ttaccacctg cacccggaga gctgtgtcac catgtgggtc
ccggttgtct 60tcctcaccct tccgtgacgt ggattggtgc tgcacccctc atcctgtctc
ggattgtggg 120aggctgggag tgcgagaagc attcccaacc ctggcaggtg
cttgtggcct ctcgtggcag 180ggcagtctgc ggcggtgttc tggtgcaccc
ccagtgggtc ctcacagctg cccactgcat 240caggaagtga gtaggggcct
ggggtctggg gagcaggtgt ctgtgtccca gaggaataac 300agctgggcat
tttccccagg ataacctcta aggccagcct tgggactggg ggagagaggg
360aaagttctgg ttcaggtcac atggggaggc agggttgggg ctggaccacc
ctccccatgg 420ctgcctgggt ctccatctgt gttcctctat gtctctttgt
gtcgctttca ttatgtctct 480tggtaactgg cttcggttgt gtctctccgt
gtgactattt tgttctctct ctccctctct 540tctctgtctt cagt 55433220PRTHomo
sapiens 33Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp
Ile Gly 1 5 10 15 Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly
Trp Glu Cys Glu 20 25 30 Lys His Ser Gln Pro Trp Gln Val Leu Val
Ala Ser Arg Gly Arg Ala 35 40 45 Val Cys Gly Gly Val Leu Val His
Pro Gln Trp Val Leu Thr Ala Ala 50 55 60 His Cys Ile Arg Asn Lys
Ser Val Ile Leu Leu Gly Arg His Ser Leu 65 70 75 80 Phe His Pro Glu
Asp Thr Gly Gln Val Phe Gln Val Ser His Ser Phe 85 90 95 Pro His
Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg 100 105 110
Pro Gly Asp Asp Ser Ser Ile Glu Pro Glu Glu Phe Leu Thr Pro Lys 115
120 125 Lys Leu Gln Cys Val Asp Leu His Val Ile Ser Asn Asp Val Cys
Ala 130 135 140 Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys
Ala Gly Arg 145 150 155 160 Trp Thr Gly Gly Lys Ser Thr Cys Ser Gly
Asp Ser Gly Gly Pro Leu 165 170 175 Val Cys Asn Gly Val Leu Gln Gly
Ile Thr Ser Trp Gly Ser Glu Pro 180 185 190 Cys Ala Leu Pro Glu Arg
Pro Ser Leu Tyr Thr Lys Val Val His Tyr 195 200 205 Arg Lys Trp Ile
Lys Asp Thr Ile Val Ala Asn Pro 210 215 220 341341DNAHomo sapiens
34agccccaagc ttaccacctg cacccggaga gctgtgtcac catgtgggtc ccggttgtct
60tcctcaccct gtccgtgacg tggattggtg ctgcacccct catcctgtct cggattgtgg
120gaggctggga gtgcgagaag cattcccaac cctggcaggt gcttgtggcc
tctcgtggca 180gggcagtctg cggcggtgtt ctggtgcacc cccagtgggt
cctcacagct gcccactgca 240tcaggaacaa aagcgtgatc ttgctgggtc
ggcacagcct gtttcatcct gaagacacag 300gccaggtatt tcaggtcagc
cacagcttcc cacacccgct ctacgatatg agcctcctga 360agaatcgatt
cctcaggcca ggtgatgact ccagcattga accagaggag ttcttgaccc
420caaagaaact tcagtgtgtg gacctccatg ttatttccaa tgacgtgtgt
gcgcaagttc 480accctcagaa ggtgaccaag ttcatgctgt gtgctggacg
ctggacaggg ggcaaaagca 540cctgctcggg tgattctggg ggcccacttg
tctgtaatgg tgtgcttcaa ggtatcacgt 600catggggcag tgaaccatgt
gccctgcccg aaaggccttc cctgtacacc aaggtggtgc 660attaccggaa
gtggatcaag gacaccatcg tggccaaccc ctgagcaccc ctatcaaccc
720cctattgtag taaacttgga accttggaaa tgaccaggcc aagactcaag
cctccccagt 780tctactgacc tttgtcctta ggtgtgaggt ccagggttgc
taggaaaaga aatcagcaga 840cacaggtgta gaccagagtg tttcttaaat
ggtgtaattt tgtcctctct gtgtcctggg 900gaatactggc catgcctgga
gacatatcac tcaatttctc tgaggacaca gataggatgg 960ggtgtctgtg
ttatttgtgg ggtacagaga tgaaagaggg gtgggatcca cactgagaga
1020gtggagagtg acatgtgctg gacactgtcc atgaagcact gagcagaagc
tggaggcaca 1080acgcaccaga cactcacagc aaggatggag ctgaaaacat
aacccactct gtcctggagg 1140cactgggaag cctagagaag gctgtgagcc
aaggagggag ggtcttcctt tggcatggga 1200tggggatgaa gtaaggagag
ggactggacc ccctggaagc tgattcacta tggggggagg 1260tgtattgaag
tcctccagac aaccctcaga tttgatgatt tcctagtaga actcacagaa
1320ataaagagct gttatactgt g 134135218PRTHomo sapiens 35Met Trp Val
Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala
Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 20 25
30 Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala
35 40 45 Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr
Ala Ala 50 55 60 His Cys Ile Arg Lys Pro Gly Asp Asp Ser Ser His
Asp Leu Met Leu 65 70 75 80 Leu Arg Leu Ser Glu Pro Ala Glu Leu Thr
Asp Ala Val Lys Val Met 85 90 95 Asp Leu Pro Thr Gln Glu Pro Ala
Leu Gly Thr Thr Cys Tyr Ala Ser 100 105 110 Gly Trp Gly Ser Ile Glu
Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu 115 120 125 Gln Cys Val Asp
Leu His Val Ile Ser Asn Asp Val Cys Ala Gln Val 130 135 140 His Pro
Gln Lys Val Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr 145 150 155
160 Gly Gly Lys Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys
165 170 175 Asn Gly Val Leu Gln Gly Ile Thr Ser Trp Gly Ser Glu Pro
Cys Ala 180 185 190 Leu Pro Glu Arg Pro Ser Leu Tyr Thr Lys Val Val
His Tyr Arg Lys 195 200 205 Trp Ile Lys Asp Thr Ile Val Ala Asn Pro
210 215 361325DNAHomo sapiens 36agccccaagc ttaccacctg cacccggaga
gctgtgtcac catgtgggtc ccggttgtct 60tcctcaccct gtccgtgacg tggattggtg
ctgcacccct catcctgtct cggattgtgg 120gaggctggga gtgcgagaag
cattcccaac cctggcaggt gcttgtggcc tctcgtggca 180gggcagtctg
cggcggtgtt ctggtgcacc cccagtgggt cctcacagct gcccactgca
240tcaggaagcc aggtgatgac tccagccacg acctcatgct gctccgcctg
tcagagcctg 300ccgagctcac ggatgctgtg aaggtcatgg acctgcccac
ccaggagcca gcactgggga 360ccacctgcta cgcctcaggc tggggcagca
ttgaaccaga ggagttcttg accccaaaga 420aacttcagtg tgtggacctc
catgttattt ccaatgacgt gtgtgcgcaa gttcaccctc 480agaaggtgac
caagttcatg ctgtgtgctg gacgctggac agggggcaaa agcacctgct
540cgggtgattc tgggggccca cttgtctgta atggtgtgct tcaaggtatc
acgtcatggg 600gcagtgaacc atgtgccctg cccgaaaggc cttccctgta
caccaaggtg gtgcattacc 660caaggacacc atcgtggcca acccctgagc
acccctatca accccctatt gtagtaaact 720tggaaccttg gaaatgacca
ggccaagact caagcctccc cagttctact gacctttgtc 780cttaggtgtg
aggtccaggg ttgctaggaa aagaaatcag cagacacagg tgtagaccag
840agtgtttctt aaatggtgta attttgtcct ctctgtgtcc tggggaatac
tggccatgcc 900tggagacata tcactcaatt tctctgagga cacagatagg
atggggtgtc tgtgttattt 960gtggggtaca gagatgaaag aggggtggga
tccacactga gagagtggag agtgacatgt 1020gctggacact gtccatgaag
cactgagcag aagctggagg cacaacgcac cagacactca 1080cagcaaggat
ggagctgaaa acataaccca ctctgtcctg gaggcactgg gaagcctaga
1140gaaggctgtg agccaaggag ggagggtctt cctttggcat gggatgggga
tgaagtaagg 1200agagggactg gaccccctgg aagctgattc actatggggg
gaggtgtatt gaagtcctcc 1260agacaaccct cagatttgat gatttcctag
tagaactcac agaaataaag agctgttata 1320ctgtg 132537261PRTHomo sapiens
37Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1
5 10 15 Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys
Glu 20 25 30 Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg
Gly Arg Ala 35 40 45 Val Cys Gly Gly Val Leu Val His Pro Gln Trp
Val Leu Thr Ala Ala 50 55 60 His Cys Ile Arg Asn Lys Ser Val Ile
Leu Leu Gly Arg His Ser Leu 65 70 75 80 Phe His Pro Glu Asp Thr Gly
Gln Val Phe Gln Val Ser His Ser Phe 85 90 95 Pro His Pro Leu Tyr
Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg 100 105 110 Pro Gly Asp
Asp Ser Ser His Asp Leu Met Leu Leu Arg Leu Ser Glu 115 120 125 Pro
Ala Glu Leu Thr Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln 130 135
140 Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile
145 150 155 160 Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys
Val Asp Leu 165 170 175 His Val Ile Ser Asn Asp Val Cys Ala Gln Val
His Pro Gln Lys Val 180 185 190 Thr Lys Phe Met Leu Cys Ala Gly Arg
Trp Thr Gly Gly Lys Ser Thr 195 200 205 Cys Ser Gly Asp Ser Gly Gly
Pro Leu Val Cys Asn Gly Val Leu Gln 210 215 220 Gly Ile Thr Ser Trp
Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro 225 230 235 240 Ser Leu
Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp Thr 245 250 255
Ile Val Ala Asn Pro 260 381464DNAHomo sapiens 38agccccaagc
ttaccacctg cacccggaga gctgtgtcac catgtgggtc ccggttgtct 60tcctcaccct
gtccgtgacg tggattggtg ctgcacccct catcctgtct cggattgtgg
120gaggctggga gtgcgagaag cattcccaac cctggcaggt gcttgtggcc
tctcgtggca 180gggcagtctg cggcggtgtt ctggtgcacc cccagtgggt
cctcacagct gcccactgca 240tcaggaacaa aagcgtgatc ttgctgggtc
ggcacagcct gtttcatcct gaagacacag 300gccaggtatt tcaggtcagc
cacagcttcc cacacccgct ctacgatatg agcctcctga 360agaatcgatt
cctcaggcca ggtgatgact ccagccacga cctcatgctg ctccgcctgt
420cagagcctgc cgagctcacg gatgctgtga aggtcatgga cctgcccacc
caggagccag 480cactggggac cacctgctac gcctcaggct ggggcagcat
tgaaccagag gagttcttga 540ccccaaagaa acttcagtgt gtggacctcc
atgttatttc caatgacgtg tgtgcgcaag 600ttcaccctca gaaggtgacc
aagttcatgc tgtgtgctgg acgctggaca gggggcaaaa 660gcacctgctc
gggtgattct gggggcccac ttgtctgtaa tggtgtgctt caaggtatca
720cgtcatgggg cagtgaacca tgtgccctgc ccgaaaggcc ttccctgtac
accaaggtgg 780tgcattaccg gaagtggatc aaggacacca tcgtggccaa
cccctgagca cccctatcaa 840ccccctattg tagtaaactt ggaaccttgg
aaatgaccag gccaagactc aagcctcccc 900agttctactg acctttgtcc
ttaggtgtga ggtccagggt tgctaggaaa agaaatcagc 960agacacaggt
gtagaccaga gtgtttctta aatggtgtaa ttttgtcctc tctgtgtcct
1020ggggaatact ggccatgcct ggagacatat cactcaattt ctctgaggac
acagatagga 1080tggggtgtct gtgttatttg tggggtacag agatgaaaga
ggggtgggat ccacactgag 1140agagtggaga gtgacatgtg ctggacactg
tccatgaagc actgagcaga agctggaggc 1200acaacgcacc agacactcac
agcaaggatg gagctgaaaa cataacccac tctgtcctgg 1260aggcactggg
aagcctagag aaggctgtga gccaaggagg gagggtcttc ctttggcatg
1320ggatggggat gaagtaagga gagggactgg accccctgga agctgattca
ctatgggggg 1380aggtgtattg aagtcctcca gacaaccctc agatttgatg
atttcctagt agaactcaca 1440gaaataaaga gctgttatac tgtg
146439261PRTArtificial Sequencekallikrein 3, partial 39Met Trp Val
Pro Val Val Phe Leu Thr Leu
Ser Val Thr Trp Ile Gly 1 5 10 15 Ala Ala Pro Leu Ile Leu Ser Arg
Ile Val Gly Gly Trp Glu Cys Glu 20 25 30 Lys His Ser Gln Pro Trp
Gln Val Leu Val Ala Ser Arg Gly Arg Ala 35 40 45 Val Cys Gly Gly
Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala 50 55 60 His Cys
Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu 65 70 75 80
Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser His Ser Phe 85
90 95 Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu
Arg 100 105 110 Pro Gly Asp Asp Ser Ser His Asp Leu Met Leu Leu Arg
Leu Ser Glu 115 120 125 Pro Ala Glu Leu Thr Asp Ala Val Lys Val Met
Asp Leu Pro Thr Gln 130 135 140 Glu Pro Ala Leu Gly Thr Thr Cys Tyr
Ala Ser Gly Trp Gly Ser Ile 145 150 155 160 Glu Pro Glu Glu Phe Leu
Thr Pro Lys Lys Leu Gln Cys Val Asp Leu 165 170 175 His Val Ile Ser
Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val 180 185 190 Thr Lys
Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr 195 200 205
Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln 210
215 220 Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg
Pro 225 230 235 240 Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp
Ile Lys Asp Thr 245 250 255 Ile Val Ala Asn Pro 260 401495DNAHomo
sapiens 40gggggagccc caagcttacc acctgcaccc ggagagctgt gtcaccatgt
gggtcccggt 60tgtcttcctc accctgtccg tgacgtggat tggtgctgca cccctcatcc
tgtctcggat 120tgtgggaggc tgggagtgcg agaagcattc ccaaccctgg
caggtgcttg tggcctctcg 180tggcagggca gtctgcggcg gtgttctggt
gcacccccag tgggtcctca cagctgccca 240ctgcatcagg aacaaaagcg
tgatcttgct gggtcggcac agcctgtttc atcctgaaga 300cacaggccag
gtatttcagg tcagccacag cttcccacac ccgctctacg atatgagcct
360cctgaagaat cgattcctca ggccaggtga tgactccagc cacgacctca
tgctgctccg 420cctgtcagag cctgccgagc tcacggatgc tgtgaaggtc
atggacctgc ccacccagga 480gccagcactg gggaccacct gctacgcctc
aggctggggc agcattgaac cagaggagtt 540cttgacccca aagaaacttc
agtgtgtgga cctccatgtt atttccaatg acgtgtgtgc 600gcaagttcac
cctcagaagg tgaccaagtt catgctgtgt gctggacgct ggacaggggg
660caaaagcacc tgctcgggtg attctggggg cccacttgtc tgtaatggtg
tgcttcaagg 720tatcacgtca tggggcagtg aaccatgtgc cctgcccgaa
aggccttccc tgtacaccaa 780ggtggtgcat taccggaagt ggatcaagga
caccatcgtg gccaacccct gagcacccct 840atcaactccc tattgtagta
aacttggaac cttggaaatg accaggccaa gactcaggcc 900tccccagttc
tactgacctt tgtccttagg tgtgaggtcc agggttgcta ggaaaagaaa
960tcagcagaca caggtgtaga ccagagtgtt tcttaaatgg tgtaattttg
tcctctctgt 1020gtcctgggga atactggcca tgcctggaga catatcactc
aatttctctg aggacacaga 1080taggatgggg tgtctgtgtt atttgtgggg
tacagagatg aaagaggggt gggatccaca 1140ctgagagagt ggagagtgac
atgtgctgga cactgtccat gaagcactga gcagaagctg 1200gaggcacaac
gcaccagaca ctcacagcaa ggatggagct gaaaacataa cccactctgt
1260cctggaggca ctgggaagcc tagagaaggc tgtgagccaa ggagggaggg
tcttcctttg 1320gcatgggatg gggatgaagt agggagaggg actggacccc
ctggaagctg attcactatg 1380gggggaggtg tattgaagtc ctccagacaa
ccctcagatt tgatgatttc ctagtagaac 1440tcacagaaat aaagagctgt
tatactgcga aaaaaaaaaa aaaaaaaaaa aaaaa 149541218PRTHomo sapiens
41Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1
5 10 15 Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys
Glu 20 25 30 Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg
Gly Arg Ala 35 40 45 Val Cys Gly Gly Val Leu Val His Pro Gln Trp
Val Leu Thr Ala Ala 50 55 60 His Cys Ile Arg Asn Lys Ser Val Ile
Leu Leu Gly Arg His Ser Leu 65 70 75 80 Phe His Pro Glu Asp Thr Gly
Gln Val Phe Gln Val Ser His Ser Phe 85 90 95 Pro His Pro Leu Tyr
Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg 100 105 110 Pro Gly Asp
Asp Ser Ser Ile Glu Pro Glu Glu Phe Leu Thr Pro Lys 115 120 125 Lys
Leu Gln Cys Val Asp Leu His Val Ile Ser Asn Asp Val Cys Ala 130 135
140 Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala Gly Arg
145 150 155 160 Trp Thr Gly Gly Lys Ser Thr Cys Ser Gly Asp Ser Gly
Gly Pro Leu 165 170 175 Val Cys Asn Gly Val Leu Gln Gly Ile Thr Ser
Trp Gly Ser Glu Pro 180 185 190 Cys Ala Leu Pro Glu Arg Pro Ser Leu
Tyr Thr Lys Val Val His Tyr 195 200 205 Arg Lys Trp Ile Lys Asp Thr
Ile Val Ala 210 215 42227PRTHomo sapiens 42Met Trp Val Pro Val Val
Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala Ala Pro Leu
Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 20 25 30 Lys His
Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala 35 40 45
Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala 50
55 60 His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser
Leu 65 70 75 80 Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser
His Ser Phe 85 90 95 Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys
Asn Arg Phe Leu Arg 100 105 110 Pro Gly Asp Asp Ser Ser His Asp Leu
Met Leu Leu Arg Leu Ser Glu 115 120 125 Pro Ala Glu Leu Thr Asp Ala
Val Lys Val Met Asp Leu Pro Thr Gln 130 135 140 Glu Pro Ala Leu Gly
Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile 145 150 155 160 Glu Pro
Glu Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu 165 170 175
His Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val 180
185 190 Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser
Thr 195 200 205 Cys Ser Val Ser His Pro Tyr Ser Gln Asp Leu Glu Gly
Lys Gly Glu 210 215 220 Trp Gly Pro 225 43104PRTHomo sapiens 43Met
Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10
15 Glu Arg Gly His Gly Trp Gly Asp Ala Gly Glu Gly Ala Ser Pro Asp
20 25 30 Cys Gln Ala Glu Ala Leu Ser Pro Pro Thr Gln His Pro Ser
Pro Asp 35 40 45 Arg Glu Leu Gly Ser Phe Leu Ser Leu Pro Ala Pro
Leu Gln Ala His 50 55 60 Thr Pro Ser Pro Ser Ile Leu Gln Gln Ser
Ser Leu Pro His Gln Val 65 70 75 80 Pro Ala Pro Ser His Leu Pro Gln
Asn Phe Leu Pro Ile Ala Gln Pro 85 90 95 Ala Pro Cys Ser Gln Leu
Leu Tyr 100 44261PRTHomo sapiens 44Met Trp Val Pro Val Val Phe Leu
Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala Ala Pro Leu Ile Leu
Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 20 25 30 Lys His Ser Gln
Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala 35 40 45 Val Cys
Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala 50 55 60
His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu 65
70 75 80 Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser His
Ser Phe 85 90 95 Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn
Arg Phe Leu Arg 100 105 110 Pro Gly Asp Asp Ser Ser His Asp Leu Met
Leu Leu Arg Leu Ser Glu 115 120 125 Pro Ala Glu Leu Thr Asp Ala Val
Lys Val Met Asp Leu Pro Thr Gln 130 135 140 Glu Pro Ala Leu Gly Thr
Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile 145 150 155 160 Glu Pro Glu
Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu 165 170 175 His
Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val 180 185
190 Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr
195 200 205 Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val
Leu Gln 210 215 220 Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu
Pro Glu Arg Pro 225 230 235 240 Ser Leu Tyr Thr Lys Val Val His Tyr
Arg Lys Trp Ile Lys Asp Thr 245 250 255 Ile Val Ala Asn Pro 260
451729DNAHomo sapiens 45aagtttccct tctcccagtc caagacccca aatcaccaca
aaggacccaa tccccagact 60caagatatgg tctgggcgct gtcttgtgtc tcctaccctg
atccctgggt tcaactctgc 120tcccagagca tgaagcctct ccaccagcac
cagccaccaa cctgcaaacc tagggaagat 180tgacagaatt cccagccttt
cccagctccc cctgcccatg tcccaggact cccagccttg 240gttctctgcc
cccgtgtctt ttcaaaccca catcctaaat ccatctccta tccgagtccc
300ccagttcctc ctgtcaaccc tgattcccct gatctagcac cccctctgca
ggtgctgcac 360ccctcatcct gtctcggatt gtgggaggct gggagtgcga
gaagcattcc caaccctggc 420aggtgcttgt agcctctcgt ggcagggcag
tctgcggcgg tgttctggtg cacccccagt 480gggtcctcac agctacccac
tgcatcagga acaaaagcgt gatcttgctg ggtcggcaca 540gcctgtttca
tcctgaagac acaggccagg tatttcaggt cagccacagc ttcccacacc
600cgctctacga tatgagcctc ctgaagaatc gattcctcag gccaggtgat
gactccagcc 660acgacctcat gctgctccgc ctgtcagagc ctgccgagct
cacggatgct atgaaggtca 720tggacctgcc cacccaggag ccagcactgg
ggaccacctg ctacgcctca ggctggggca 780gcattgaacc agaggagttc
ttgaccccaa agaaacttca gtgtgtggac ctccatgtta 840tttccaatga
cgtgtgtgcg caagttcacc ctcagaaggt gaccaagttc atgctgtgtg
900ctggacgctg gacagggggc aaaagcacct gctcgggtga ttctgggggc
ccacttgtct 960gtaatggtgt gcttcaaggt atcacgtcat ggggcagtga
accatgtgcc ctgcccgaaa 1020ggccttccct gtacaccaag gtggtgcatt
accggaagtg gatcaaggac accatcgtgg 1080ccaacccctg agcaccccta
tcaactccct attgtagtaa acttggaacc ttggaaatga 1140ccaggccaag
actcaggcct ccccagttct actgaccttt gtccttaggt gtgaggtcca
1200gggttgctag gaaaagaaat cagcagacac aggtgtagac cagagtgttt
cttaaatggt 1260gtaattttgt cctctctgtg tcctggggaa tactggccat
gcctggagac atatcactca 1320atttctctga ggacacagat aggatggggt
gtctgtgtta tttgtggggt acagagatga 1380aagaggggtg ggatccacac
tgagagagtg gagagtgaca tgtgctggac actgtccatg 1440aagcactgag
cagaagctgg aggcacaacg caccagacac tcacagcaag gatggagctg
1500aaaacataac ccactctgtc ctggaggcac tgggaagcct agagaaggct
gtgaaccaag 1560gagggagggt cttcctttgg catgggatgg ggatgaagta
aggagaggga ctgaccccct 1620ggaagctgat tcactatggg gggaggtgta
ttgaagtcct ccagacaacc ctcagatttg 1680atgatttcct agtagaactc
acagaaataa agagctgtta tactgtgaa 17294629DNAHomo sapiens
46gtgctcgaga ttgtgggagg ctgggagtg 294729DNAHomo sapiens
47gatactagtt taggggttgg ccacgatgg 2948680PRTListeria monocytogenes
48Met Lys Lys Ile Met Leu Val Phe Ile Thr Leu Ile Leu Val Ser Leu 1
5 10 15 Pro Ile Ala Gln Gln Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn
Lys 20 25 30 Glu Asn Ser Ile Ser Ser Met Ala Pro Pro Ala Ser Pro
Pro Ala Ser 35 40 45 Pro Lys Thr Pro Ile Glu Lys Lys His Ala Asp
Glu Ile Asp Lys Tyr 50 55 60 Ile Gln Gly Leu Asp Tyr Asn Lys Asn
Asn Val Leu Val Tyr His Gly 65 70 75 80 Asp Ala Val Thr Asn Val Pro
Pro Arg Lys Gly Tyr Lys Asp Gly Asn 85 90 95 Glu Tyr Ile Val Val
Glu Lys Lys Lys Lys Ser Ile Asn Gln Asn Asn 100 105 110 Ala Asp Ile
Gln Val Val Asn Ala Ile Ser Ser Leu Thr Tyr Pro Gly 115 120 125 Ala
Leu Val Lys Ala Asn Ser Glu Leu Val Glu Asn Gln Pro Asp Val 130 135
140 Leu Pro Val Lys Arg Asp Ser Leu Thr Leu Ser Ile Asp Leu Pro Gly
145 150 155 160 Met Thr Asn Gln Asp Asn Lys Ile Val Val Lys Asn Ala
Thr Lys Ser 165 170 175 Asn Val Asn Asn Ala Val Asn Thr Leu Val Glu
Arg Trp Asn Glu Lys 180 185 190 Tyr Ala Gln Ala Tyr Pro Asn Val Ser
Ala Lys Ile Asp Tyr Asp Asp 195 200 205 Glu Met Ala Tyr Ser Glu Ser
Gln Leu Ile Ala Lys Phe Gly Thr Ala 210 215 220 Phe Lys Ala Val Asn
Asn Ser Leu Asn Val Asn Phe Gly Ala Ile Ser 225 230 235 240 Glu Gly
Lys Met Gln Glu Glu Val Ile Ser Phe Lys Gln Ile Tyr Tyr 245 250 255
Asn Val Asn Val Asn Glu Pro Thr Arg Pro Ser Arg Phe Phe Gly Lys 260
265 270 Ala Val Thr Lys Glu Gln Leu Gln Ala Leu Gly Val Asn Ala Glu
Asn 275 280 285 Pro Pro Ala Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln
Val Tyr Leu 290 295 300 Lys Leu Ser Thr Asn Ser His Ser Thr Lys Val
Lys Ala Ala Phe Asp 305 310 315 320 Ala Ala Val Ser Gly Lys Ser Val
Ser Gly Asp Val Glu Leu Thr Asn 325 330 335 Ile Ile Lys Asn Ser Ser
Phe Lys Ala Val Ile Tyr Gly Gly Ser Ala 340 345 350 Lys Asp Glu Val
Gln Ile Ile Asp Gly Asn Leu Gly Asp Leu Arg Asp 355 360 365 Ile Leu
Lys Lys Gly Ala Thr Phe Asn Arg Glu Thr Pro Gly Val Pro 370 375 380
Ile Ala Tyr Thr Thr Asn Phe Leu Lys Asp Asn Glu Leu Ala Val Ile 385
390 395 400 Lys Asn Asn Ser Glu Tyr Ile Glu Thr Thr Ser Lys Ala Tyr
Thr Asp 405 410 415 Gly Lys Ile Asn Ile Asp His Ser Gly Gly Tyr Val
Ala Gln Phe Asn 420 425 430 Ile Ser Trp Asp Glu Val Asn Tyr Asp Leu
Glu Ile Val Gly Gly Trp 435 440 445 Glu Cys Glu Lys His Ser Gln Pro
Trp Gln Val Leu Val Ala Ser Arg 450 455 460 Gly Arg Ala Val Cys Gly
Gly Val Leu Val His Pro Gln Trp Val Leu 465 470 475 480 Thr Ala Ala
His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg 485 490 495 His
Ser Leu Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser 500 505
510 His Ser Phe Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg
515 520 525 Phe Leu Arg Pro Gly Asp Asp Ser Ser His Asp Leu Met Leu
Leu Arg 530 535 540 Leu Ser Glu Pro Ala Glu Leu Thr Asp Ala Val Lys
Val Met Asp Leu 545 550 555 560 Pro Thr Gln Glu Pro Ala Leu Gly Thr
Thr Cys Tyr Ala Ser Gly Trp 565 570 575 Gly Ser Ile Glu Pro Glu Glu
Phe Leu Thr Pro Lys Lys Leu Gln Cys 580 585 590 Val Asp Leu His Val
Ile Ser Asn Asp Val Cys Ala Gln Val His Pro 595 600 605 Gln Lys Val
Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly 610 615 620 Lys
Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro Leu Val Cys Tyr Gly 625 630
635 640 Val Leu Gln Gly Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu
Pro 645 650 655 Glu Arg Pro Ser Leu Tyr Thr Lys Val Val His Tyr Arg
Lys Trp Ile 660 665 670 Lys Asp Thr Ile Val Ala Asn Pro 675 680
4932PRTListeria monocytogenes 49Lys Glu Asn Ser Ile Ser Ser Met Ala
Pro Pro Ala Ser Pro Pro Ala 1 5
10 15 Ser Pro Lys Thr Pro Ile Glu Lys Lys His Ala Asp Glu Ile Asp
Lys 20 25 30 50227PRTHomo sapiens 50Met Trp Val Pro Val Val Phe Leu
Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala Ala Pro Leu Ile Leu
Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 20 25 30 Lys His Ser Gln
Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala 35 40 45 Val Cys
Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala 50 55 60
His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser Leu 65
70 75 80 Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser His
Ser Phe 85 90 95 Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn
Arg Phe Leu Arg 100 105 110 Pro Gly Asp Asp Ser Ser His Asp Leu Met
Leu Leu Arg Leu Ser Glu 115 120 125 Pro Ala Glu Leu Thr Asp Ala Val
Lys Val Met Asp Leu Pro Thr Gln 130 135 140 Glu Pro Ala Leu Gly Thr
Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile 145 150 155 160 Glu Pro Glu
Glu Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu 165 170 175 His
Val Ile Ser Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val 180 185
190 Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr
195 200 205 Cys Ser Val Ser His Pro Tyr Ser Gln Asp Leu Glu Gly Lys
Gly Glu 210 215 220 Trp Gly Pro 225 51104PRTHomo sapiens 51Met Trp
Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15
Glu Arg Gly His Gly Trp Gly Asp Ala Gly Glu Gly Ala Ser Pro Asp 20
25 30 Cys Gln Ala Glu Ala Leu Ser Pro Pro Thr Gln His Pro Ser Pro
Asp 35 40 45 Arg Glu Leu Gly Ser Phe Leu Ser Leu Pro Ala Pro Leu
Gln Ala His 50 55 60 Thr Pro Ser Pro Ser Ile Leu Gln Gln Ser Ser
Leu Pro His Gln Val 65 70 75 80 Pro Ala Pro Ser His Leu Pro Gln Asn
Phe Leu Pro Ile Ala Gln Pro 85 90 95 Ala Pro Cys Ser Gln Leu Leu
Tyr 100 5224PRTHomo sapiens 52Met Trp Val Pro Val Val Phe Leu Thr
Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala Ala Pro Leu Ile Leu Ser
Arg 20 53529PRTListeria monocytogenes 53Met Lys Lys Ile Met Leu Val
Phe Ile Thr Leu Ile Leu Val Ser Leu 1 5 10 15 Pro Ile Ala Gln Gln
Thr Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30 Glu Asn Ser
Ile Ser Ser Met Ala Pro Pro Ala Ser Pro Pro Ala Ser 35 40 45 Pro
Lys Thr Pro Ile Glu Lys Lys His Ala Asp Glu Ile Asp Lys Tyr 50 55
60 Ile Gln Gly Leu Asp Tyr Asn Lys Asn Asn Val Leu Val Tyr His Gly
65 70 75 80 Asp Ala Val Thr Asn Val Pro Pro Arg Lys Gly Tyr Lys Asp
Gly Asn 85 90 95 Glu Tyr Ile Val Val Glu Lys Lys Lys Lys Ser Ile
Asn Gln Asn Asn 100 105 110 Ala Asp Ile Gln Val Val Asn Ala Ile Ser
Ser Leu Thr Tyr Pro Gly 115 120 125 Ala Leu Val Lys Ala Asn Ser Glu
Leu Val Glu Asn Gln Pro Asp Val 130 135 140 Leu Pro Val Lys Arg Asp
Ser Leu Thr Leu Ser Ile Asp Leu Pro Gly 145 150 155 160 Met Thr Asn
Gln Asp Asn Lys Ile Val Val Lys Asn Ala Thr Lys Ser 165 170 175 Asn
Val Asn Asn Ala Val Asn Thr Leu Val Glu Arg Trp Asn Glu Lys 180 185
190 Tyr Ala Gln Ala Tyr Pro Asn Val Ser Ala Lys Ile Asp Tyr Asp Asp
195 200 205 Glu Met Ala Tyr Ser Glu Ser Gln Leu Ile Ala Lys Phe Gly
Thr Ala 210 215 220 Phe Lys Ala Val Asn Asn Ser Leu Asn Val Asn Phe
Gly Ala Ile Ser 225 230 235 240 Glu Gly Lys Met Gln Glu Glu Val Ile
Ser Phe Lys Gln Ile Tyr Tyr 245 250 255 Asn Val Asn Val Asn Glu Pro
Thr Arg Pro Ser Arg Phe Phe Gly Lys 260 265 270 Ala Val Thr Lys Glu
Gln Leu Gln Ala Leu Gly Val Asn Ala Glu Asn 275 280 285 Pro Pro Ala
Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu 290 295 300 Lys
Leu Ser Thr Asn Ser His Ser Thr Lys Val Lys Ala Ala Phe Asp 305 310
315 320 Ala Ala Val Ser Gly Lys Ser Val Ser Gly Asp Val Glu Leu Thr
Asn 325 330 335 Ile Ile Lys Asn Ser Ser Phe Lys Ala Val Ile Tyr Gly
Gly Ser Ala 340 345 350 Lys Asp Glu Val Gln Ile Ile Asp Gly Asn Leu
Gly Asp Leu Arg Asp 355 360 365 Ile Leu Lys Lys Gly Ala Thr Phe Asn
Arg Glu Thr Pro Gly Val Pro 370 375 380 Ile Ala Tyr Thr Thr Asn Phe
Leu Lys Asp Asn Glu Leu Ala Val Ile 385 390 395 400 Lys Asn Asn Ser
Glu Tyr Ile Glu Thr Thr Ser Lys Ala Tyr Thr Asp 405 410 415 Gly Lys
Ile Asn Ile Asp His Ser Gly Gly Tyr Val Ala Gln Phe Asn 420 425 430
Ile Ser Trp Asp Glu Val Asn Tyr Asp Pro Glu Gly Asn Glu Ile Val 435
440 445 Gln His Lys Asn Trp Ser Glu Asn Asn Lys Ser Lys Leu Ala His
Phe 450 455 460 Thr Ser Ser Ile Tyr Leu Pro Gly Asn Ala Arg Asn Ile
Asn Val Tyr 465 470 475 480 Ala Lys Glu Cys Thr Gly Leu Ala Trp Glu
Trp Trp Arg Thr Val Ile 485 490 495 Asp Asp Arg Asn Leu Pro Leu Val
Lys Asn Arg Asn Ile Ser Ile Trp 500 505 510 Gly Thr Thr Leu Tyr Pro
Lys Tyr Ser Asn Lys Val Asp Asn Pro Ile 515 520 525 Glu
54441PRTListeria monocytogenes 54Met Lys Lys Ile Met Leu Val Phe
Ile Thr Leu Ile Leu Val Ser Leu 1 5 10 15 Pro Ile Ala Gln Gln Thr
Glu Ala Lys Asp Ala Ser Ala Phe Asn Lys 20 25 30 Glu Asn Ser Ile
Ser Ser Val Ala Pro Pro Ala Ser Pro Pro Ala Ser 35 40 45 Pro Lys
Thr Pro Ile Glu Lys Lys His Ala Asp Glu Ile Asp Lys Tyr 50 55 60
Ile Gln Gly Leu Asp Tyr Asn Lys Asn Asn Val Leu Val Tyr His Gly 65
70 75 80 Asp Ala Val Thr Asn Val Pro Pro Arg Lys Gly Tyr Lys Asp
Gly Asn 85 90 95 Glu Tyr Ile Val Val Glu Lys Lys Lys Lys Ser Ile
Asn Gln Asn Asn 100 105 110 Ala Asp Ile Gln Val Val Asn Ala Ile Ser
Ser Leu Thr Tyr Pro Gly 115 120 125 Ala Leu Val Lys Ala Asn Ser Glu
Leu Val Glu Asn Gln Pro Asp Val 130 135 140 Leu Pro Val Lys Arg Asp
Ser Leu Thr Leu Ser Ile Asp Leu Pro Gly 145 150 155 160 Met Thr Asn
Gln Asp Asn Lys Ile Val Val Lys Asn Ala Thr Lys Ser 165 170 175 Asn
Val Asn Asn Ala Val Asn Thr Leu Val Glu Arg Trp Asn Glu Lys 180 185
190 Tyr Ala Gln Ala Tyr Ser Asn Val Ser Ala Lys Ile Asp Tyr Asp Asp
195 200 205 Glu Met Ala Tyr Ser Glu Ser Gln Leu Ile Ala Lys Phe Gly
Thr Ala 210 215 220 Phe Lys Ala Val Asn Asn Ser Leu Asn Val Asn Phe
Gly Ala Ile Ser 225 230 235 240 Glu Gly Lys Met Gln Glu Glu Val Ile
Ser Phe Lys Gln Ile Tyr Tyr 245 250 255 Asn Val Asn Val Asn Glu Pro
Thr Arg Pro Ser Arg Phe Phe Gly Lys 260 265 270 Ala Val Thr Lys Glu
Gln Leu Gln Ala Leu Gly Val Asn Ala Glu Asn 275 280 285 Pro Pro Ala
Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu 290 295 300 Lys
Leu Ser Thr Asn Ser His Ser Thr Lys Val Lys Ala Ala Phe Asp 305 310
315 320 Ala Ala Val Ser Gly Lys Ser Val Ser Gly Asp Val Glu Leu Thr
Asn 325 330 335 Ile Ile Lys Asn Ser Ser Phe Lys Ala Val Ile Tyr Gly
Gly Ser Ala 340 345 350 Lys Asp Glu Val Gln Ile Ile Asp Gly Asn Leu
Gly Asp Leu Arg Asp 355 360 365 Ile Leu Lys Lys Gly Ala Thr Phe Asn
Arg Glu Thr Pro Gly Val Pro 370 375 380 Ile Ala Tyr Thr Thr Asn Phe
Leu Lys Asp Asn Glu Leu Ala Val Ile 385 390 395 400 Lys Asn Asn Ser
Glu Tyr Ile Glu Thr Thr Ser Lys Ala Tyr Thr Asp 405 410 415 Gly Lys
Ile Asn Ile Asp His Ser Gly Gly Tyr Val Ala Gln Phe Asn 420 425 430
Ile Ser Trp Asp Glu Val Asn Tyr Asp 435 440 55416PRTListeria
monocytogenes 55Met Lys Lys Ile Met Leu Val Phe Ile Thr Leu Ile Leu
Val Ser Leu 1 5 10 15 Pro Ile Ala Gln Gln Thr Glu Ala Lys Asp Ala
Ser Ala Phe Asn Lys 20 25 30 Glu Asn Ser Ile Ser Ser Val Ala Pro
Pro Ala Ser Pro Pro Ala Ser 35 40 45 Pro Lys Thr Pro Ile Glu Lys
Lys His Ala Asp Glu Ile Asp Lys Tyr 50 55 60 Ile Gln Gly Leu Asp
Tyr Asn Lys Asn Asn Val Leu Val Tyr His Gly 65 70 75 80 Asp Ala Val
Thr Asn Val Pro Pro Arg Lys Gly Tyr Lys Asp Gly Asn 85 90 95 Glu
Tyr Ile Val Val Glu Lys Lys Lys Lys Ser Ile Asn Gln Asn Asn 100 105
110 Ala Asp Ile Gln Val Val Asn Ala Ile Ser Ser Leu Thr Tyr Pro Gly
115 120 125 Ala Leu Val Lys Ala Asn Ser Glu Leu Val Glu Asn Gln Pro
Asp Val 130 135 140 Leu Pro Val Lys Arg Asp Ser Leu Thr Leu Ser Ile
Asp Leu Pro Gly 145 150 155 160 Met Thr Asn Gln Asp Asn Lys Ile Val
Val Lys Asn Ala Thr Lys Ser 165 170 175 Asn Val Asn Asn Ala Val Asn
Thr Leu Val Glu Arg Trp Asn Glu Lys 180 185 190 Tyr Ala Gln Ala Tyr
Ser Asn Val Ser Ala Lys Ile Asp Tyr Asp Asp 195 200 205 Glu Met Ala
Tyr Ser Glu Ser Gln Leu Ile Ala Lys Phe Gly Thr Ala 210 215 220 Phe
Lys Ala Val Asn Asn Ser Leu Asn Val Asn Phe Gly Ala Ile Ser 225 230
235 240 Glu Gly Lys Met Gln Glu Glu Val Ile Ser Phe Lys Gln Ile Tyr
Tyr 245 250 255 Asn Val Asn Val Asn Glu Pro Thr Arg Pro Ser Arg Phe
Phe Gly Lys 260 265 270 Ala Val Thr Lys Glu Gln Leu Gln Ala Leu Gly
Val Asn Ala Glu Asn 275 280 285 Pro Pro Ala Tyr Ile Ser Ser Val Ala
Tyr Gly Arg Gln Val Tyr Leu 290 295 300 Lys Leu Ser Thr Asn Ser His
Ser Thr Lys Val Lys Ala Ala Phe Asp 305 310 315 320 Ala Ala Val Ser
Gly Lys Ser Val Ser Gly Asp Val Glu Leu Thr Asn 325 330 335 Ile Ile
Lys Asn Ser Ser Phe Lys Ala Val Ile Tyr Gly Gly Ser Ala 340 345 350
Lys Asp Glu Val Gln Ile Ile Asp Gly Asn Leu Gly Asp Leu Arg Asp 355
360 365 Ile Leu Lys Lys Gly Ala Thr Phe Asn Arg Glu Thr Pro Gly Val
Pro 370 375 380 Ile Ala Tyr Thr Thr Asn Phe Leu Lys Asp Asn Glu Leu
Ala Val Ile 385 390 395 400 Lys Asn Asn Ser Glu Tyr Ile Glu Thr Thr
Ser Lys Ala Tyr Thr Asp 405 410 415 5614PRTListeria monocytogenes
56Lys Thr Glu Glu Gln Pro Ser Glu Val Asn Thr Gly Pro Arg 1 5 10
5728PRTListeria monocytogenes 57Lys Ala Ser Val Thr Asp Thr Ser Glu
Gly Asp Leu Asp Ser Ser Met 1 5 10 15 Gln Ser Ala Asp Glu Ser Thr
Pro Gln Pro Leu Lys 20 25 5820PRTListeria monocytogenes 58Lys Asn
Glu Glu Val Asn Ala Ser Asp Phe Pro Pro Pro Pro Thr Asp 1 5 10 15
Glu Glu Leu Arg 20 5933PRTListeria monocytogenes 59Arg Gly Gly Ile
Pro Thr Ser Glu Glu Phe Ser Ser Leu Asn Ser Gly 1 5 10 15 Asp Phe
Thr Asp Asp Glu Asn Ser Glu Thr Thr Glu Glu Glu Ile Asp 20 25 30
Arg 6017PRTStreptococcus pyogenes 60Lys Gln Asn Thr Ala Ser Thr Glu
Thr Thr Thr Thr Asn Glu Gln Pro 1 5 10 15 Lys 6117PRTStreptococcus
equisimilis 61Lys Gln Asn Thr Ala Asn Thr Glu Thr Thr Thr Thr Asn
Glu Gln Pro 1 5 10 15 Lys 626523DNAHomo sapiens 62cggagtgtat
actggcttac tatgttggca ctgatgaggg tgtcagtgaa gtgcttcatg 60tggcaggaga
aaaaaggctg caccggtgcg tcagcagaat atgtgataca ggatatattc
120cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag
cggaaatggc 180ttacgaacgg ggcggagatt tcctggaaga tgccaggaag
atacttaaca gggaagtgag 240agggccgcgg caaagccgtt tttccatagg
ctccgccccc ctgacaagca tcacgaaatc 300tgacgctcaa atcagtggtg
gcgaaacccg acaggactat aaagatacca ggcgtttccc 360cctggcggct
ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg
420ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt
aggcagttcg 480ctccaagctg gactgtatgc acgaaccccc cgttcagtcc
gaccgctgcg ccttatccgg 540taactatcgt cttgagtcca acccggaaag
acatgcaaaa gcaccactgg cagcagccac 600tggtaattga tttagaggag
ttagtcttga agtcatgcgc cggttaaggc taaactgaaa 660ggacaagttt
tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag
720ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca
gagcaagaga 780ttacgcgcag accaaaacga tctcaagaag atcatcttat
taatcagata aaatatttct 840agccctcctt tgattagtat attcctatct
taaagttact tttatgtgga ggcattaaca 900tttgttaatg acgtcaaaag
gatagcaaga ctagaataaa gctataaagc aagcatataa 960tattgcgttt
catctttaga agcgaatttc gccaatatta taattatcaa aagagagggg
1020tggcaaacgg tatttggcat tattaggtta aaaaatgtag aaggagagtg
aaacccatga 1080aaaaaataat gctagttttt attacactta tattagttag
tctaccaatt gcgcaacaaa 1140ctgaagcaaa ggatgcatct gcattcaata
aagaaaattc aatttcatcc atggcaccac 1200cagcatctcc gcctgcaagt
cctaagacgc caatcgaaaa gaaacacgcg gatgaaatcg 1260ataagtatat
acaaggattg gattacaata aaaacaatgt attagtatac cacggagatg
1320cagtgacaaa tgtgccgcca agaaaaggtt acaaagatgg aaatgaatat
attgttgtgg 1380agaaaaagaa gaaatccatc aatcaaaata atgcagacat
tcaagttgtg aatgcaattt 1440cgagcctaac ctatccaggt gctctcgtaa
aagcgaattc ggaattagta gaaaatcaac 1500cagatgttct ccctgtaaaa
cgtgattcat taacactcag cattgatttg ccaggtatga 1560ctaatcaaga
caataaaata gttgtaaaaa atgccactaa atcaaacgtt aacaacgcag
1620taaatacatt agtggaaaga tggaatgaaa aatatgctca agcttatcca
aatgtaagtg 1680caaaaattga ttatgatgac gaaatggctt acagtgaatc
acaattaatt gcgaaatttg 1740gtacagcatt taaagctgta aataatagct
tgaatgtaaa cttcggcgca atcagtgaag 1800ggaaaatgca agaagaagtc
attagtttta aacaaattta ctataacgtg aatgttaatg 1860aacctacaag
accttccaga tttttcggca aagctgttac taaagagcag ttgcaagcgc
1920ttggagtgaa tgcagaaaat cctcctgcat atatctcaag tgtggcgtat
ggccgtcaag 1980tttatttgaa attatcaact aattcccata gtactaaagt
aaaagctgct tttgatgctg 2040ccgtaagcgg aaaatctgtc tcaggtgatg
tagaactaac aaatatcatc aaaaattctt 2100ccttcaaagc cgtaatttac
ggaggttccg caaaagatga agttcaaatc atcgacggca 2160acctcggaga
cttacgcgat attttgaaaa aaggcgctac ttttaatcga gaaacaccag
2220gagttcccat tgcttataca acaaacttcc taaaagacaa tgaattagct
gttattaaaa 2280acaactcaga atatattgaa acaacttcaa aagcttatac
agatggaaaa attaacatcg 2340atcactctgg aggatacgtt gctcaattca
acatttcttg ggatgaagta
aattatgatc 2400tcgagattgt gggaggctgg gagtgcgaga agcattccca
accctggcag gtgcttgtgg 2460cctctcgtgg cagggcagtc tgcggcggtg
ttctggtgca cccccagtgg gtcctcacag 2520ctgcccactg catcaggaac
aaaagcgtga tcttgctggg tcggcacagc ctgtttcatc 2580ctgaagacac
aggccaggta tttcaggtca gccacagctt cccacacccg ctctacgata
2640tgagcctcct gaagaatcga ttcctcaggc caggtgatga ctccagccac
gacctcatgc 2700tgctccgcct gtcagagcct gccgagctca cggatgctgt
gaaggtcatg gacctgccca 2760cccaggagcc agcactgggg accacctgct
acgcctcagg ctggggcagc attgaaccag 2820aggagttctt gaccccaaag
aaacttcagt gtgtggacct ccatgttatt tccaatgacg 2880tgtgtgcgca
agttcaccct cagaaggtga ccaagttcat gctgtgtgct ggacgctgga
2940cagggggcaa aagcacctgc tcgggtgatt ctgggggccc acttgtctgt
tatggtgtgc 3000ttcaaggtat cacgtcatgg ggcagtgaac catgtgccct
gcccgaaagg ccttccctgt 3060acaccaaggt ggtgcattac cggaagtgga
tcaaggacac catcgtggcc aacccctaac 3120ccgggccact aactcaacgc
tagtagtgga tttaatccca aatgagccaa cagaaccaga 3180accagaaaca
gaacaagtaa cattggagtt agaaatggaa gaagaaaaaa gcaatgattt
3240cgtgtgaata atgcacgaaa tcattgctta tttttttaaa aagcgatata
ctagatataa 3300cgaaacaacg aactgaataa agaatacaaa aaaagagcca
cgaccagtta aagcctgaga 3360aactttaact gcgagcctta attgattacc
accaatcaat taaagaagtc gagacccaaa 3420atttggtaaa gtatttaatt
actttattaa tcagatactt aaatatctgt aaacccatta 3480tatcgggttt
ttgaggggat ttcaagtctt taagaagata ccaggcaatc aattaagaaa
3540aacttagttg attgcctttt ttgttgtgat tcaactttga tcgtagcttc
taactaatta 3600attttcgtaa gaaaggagaa cagctgaatg aatatccctt
ttgttgtaga aactgtgctt 3660catgacggct tgttaaagta caaatttaaa
aatagtaaaa ttcgctcaat cactaccaag 3720ccaggtaaaa gtaaaggggc
tatttttgcg tatcgctcaa aaaaaagcat gattggcgga 3780cgtggcgttg
ttctgacttc cgaagaagcg attcacgaaa atcaagatac atttacgcat
3840tggacaccaa acgtttatcg ttatggtacg tatgcagacg aaaaccgttc
atacactaaa 3900ggacattctg aaaacaattt aagacaaatc aataccttct
ttattgattt tgatattcac 3960acggaaaaag aaactatttc agcaagcgat
attttaacaa cagctattga tttaggtttt 4020atgcctacgt taattatcaa
atctgataaa ggttatcaag catattttgt tttagaaacg 4080ccagtctatg
tgacttcaaa atcagaattt aaatctgtca aagcagccaa aataatctcg
4140caaaatatcc gagaatattt tggaaagtct ttgccagttg atctaacgtg
caatcatttt 4200gggattgctc gtataccaag aacggacaat gtagaatttt
ttgatcccaa ttaccgttat 4260tctttcaaag aatggcaaga ttggtctttc
aaacaaacag ataataaggg ctttactcgt 4320tcaagtctaa cggttttaag
cggtacagaa ggcaaaaaac aagtagatga accctggttt 4380aatctcttat
tgcacgaaac gaaattttca ggagaaaagg gtttagtagg gcgcaatagc
4440gttatgttta ccctctcttt agcctacttt agttcaggct attcaatcga
aacgtgcgaa 4500tataatatgt ttgagtttaa taatcgatta gatcaaccct
tagaagaaaa agaagtaatc 4560aaaattgtta gaagtgccta ttcagaaaac
tatcaagggg ctaataggga atacattacc 4620attctttgca aagcttgggt
atcaagtgat ttaaccagta aagatttatt tgtccgtcaa 4680gggtggttta
aattcaagaa aaaaagaagc gaacgtcaac gtgttcattt gtcagaatgg
4740aaagaagatt taatggctta tattagcgaa aaaagcgatg tatacaagcc
ttatttagcg 4800acgaccaaaa aagagattag agaagtgcta ggcattcctg
aacggacatt agataaattg 4860ctgaaggtac tgaaggcgaa tcaggaaatt
ttctttaaga ttaaaccagg aagaaatggt 4920ggcattcaac ttgctagtgt
taaatcattg ttgctatcga tcattaaatt aaaaaaagaa 4980gaacgagaaa
gctatataaa ggcgctgaca gcttcgttta atttagaacg tacatttatt
5040caagaaactc taaacaaatt ggcagaacgc cccaaaacgg acccacaact
cgatttgttt 5100agctacgata caggctgaaa ataaaacccg cactatgcca
ttacatttat atctatgata 5160cgtgtttgtt tttctttgct ggctagctta
attgcttata tttacctgca ataaaggatt 5220tcttacttcc attatactcc
cattttccaa aaacatacgg ggaacacggg aacttattgt 5280acaggccacc
tcatagttaa tggtttcgag ccttcctgca atctcatcca tggaaatata
5340ttcatccccc tgccggccta ttaatgtgac ttttgtgccc ggcggatatt
cctgatccag 5400ctccaccata aattggtcca tgcaaattcg gccggcaatt
ttcaggcgtt ttcccttcac 5460aaggatgtcg gtccctttca attttcggag
ccagccgtcc gcatagccta caggcaccgt 5520cccgatccat gtgtcttttt
ccgctgtgta ctcggctccg tagctgacgc tctcgccttt 5580tctgatcagt
ttgacatgtg acagtgtcga atgcagggta aatgccggac gcagctgaaa
5640cggtatctcg tccgacatgt cagcagacgg gcgaaggcca tacatgccga
tgccgaatct 5700gactgcatta aaaaagcctt ttttcagccg gagtccagcg
gcgctgttcg cgcagtggac 5760cattagattc tttaacggca gcggagcaat
cagctcttta aagcgctcaa actgcattaa 5820gaaatagcct ctttcttttt
catccgctgt cgcaaaatgg gtaaataccc ctttgcactt 5880taaacgaggg
ttgcggtcaa gaattgccat cacgttctga acttcttcct ctgtttttac
5940accaagtctg ttcatccccg tatcgacctt cagatgaaaa tgaagagaac
cttttttcgt 6000gtggcgggct gcctcctgaa gccattcaac agaataacct
gttaaggtca cgtcatactc 6060agcagcgatt gccacatact ccgggggaac
cgcgccaagc accaatatag gcgccttcaa 6120tccctttttg cgcagtgaaa
tcgcttcatc caaaatggcc acggccaagc atgaagcacc 6180tgcgtcaaga
gcagcctttg ctgtttctgc atcaccatgc ccgtaggcgt ttgctttcac
6240aactgccatc aagtggacat gttcaccgat atgttttttc atattgctga
cattttcctt 6300tatcgcggac aagtcaattt ccgcccacgt atctctgtaa
aaaggttttg tgctcatgga 6360aaactcctct cttttttcag aaaatcccag
tacgtaatta agtatttgag aattaatttt 6420atattgatta atactaagtt
tacccagttt tcacctaaaa aacaaatgat gagataatag 6480ctccaaaggc
taaagaggac tataccaact atttgttaat taa 65236369PRTHomo sapiens 63Met
Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10
15 Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu
20 25 30 Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly
Arg Ala 35 40 45 Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val
Leu Thr Ala Ala 50 55 60 His Cys Ile Arg Lys 65 64220PRTOryza
sativa 64Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp
Ile Gly 1 5 10 15 Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly
Trp Glu Cys Glu 20 25 30 Lys His Ser Gln Pro Trp Gln Val Leu Val
Ala Ser Arg Gly Arg Ala 35 40 45 Val Cys Gly Gly Val Leu Val His
Pro Gln Trp Val Leu Thr Ala Ala 50 55 60 His Cys Ile Arg Asn Lys
Ser Val Ile Leu Leu Gly Arg His Ser Leu 65 70 75 80 Phe His Pro Glu
Asp Thr Gly Gln Val Phe Gln Val Ser His Ser Phe 85 90 95 Pro His
Pro Leu Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg 100 105 110
Pro Gly Asp Asp Ser Ser Ile Glu Pro Glu Glu Phe Leu Thr Pro Lys 115
120 125 Lys Leu Gln Cys Val Asp Leu His Val Ile Ser Asn Asp Val Cys
Ala 130 135 140 Gln Val His Pro Gln Lys Val Thr Lys Phe Met Leu Cys
Ala Gly Arg 145 150 155 160 Trp Thr Gly Gly Lys Ser Thr Cys Ser Gly
Asp Ser Gly Gly Pro Leu 165 170 175 Val Cys Asn Gly Val Leu Gln Gly
Ile Thr Ser Trp Gly Ser Glu Pro 180 185 190 Cys Ala Leu Pro Glu Arg
Pro Ser Leu Tyr Thr Lys Val Val His Tyr 195 200 205 Arg Lys Trp Ile
Lys Asp Thr Ile Val Ala Asn Pro 210 215 220 65218PRTHomo sapiens
65Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1
5 10 15 Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys
Glu 20 25 30 Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg
Gly Arg Ala 35 40 45 Val Cys Gly Gly Val Leu Val His Pro Gln Trp
Val Leu Thr Ala Ala 50 55 60 His Cys Ile Arg Lys Pro Gly Asp Asp
Ser Ser His Asp Leu Met Leu 65 70 75 80 Leu Arg Leu Ser Glu Pro Ala
Glu Leu Thr Asp Ala Val Lys Val Met 85 90 95 Asp Leu Pro Thr Gln
Glu Pro Ala Leu Gly Thr Thr Cys Tyr Ala Ser 100 105 110 Gly Trp Gly
Ser Ile Glu Pro Glu Glu Phe Leu Thr Pro Lys Lys Leu 115 120 125 Gln
Cys Val Asp Leu His Val Ile Ser Asn Asp Val Cys Ala Gln Val 130 135
140 His Pro Gln Lys Val Thr Lys Phe Met Leu Cys Ala Gly Arg Trp Thr
145 150 155 160 Gly Gly Lys Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro
Leu Val Cys 165 170 175 Asn Gly Val Leu Gln Gly Ile Thr Ser Trp Gly
Ser Glu Pro Cys Ala 180 185 190 Leu Pro Glu Arg Pro Ser Leu Tyr Thr
Lys Val Val His Tyr Arg Lys 195 200 205 Trp Ile Lys Asp Thr Ile Val
Ala Asn Pro 210 215 66218PRTHomo sapiens 66Met Trp Val Pro Val Val
Phe Leu Thr Leu Ser Val Thr Trp Ile Gly 1 5 10 15 Ala Ala Pro Leu
Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu 20 25 30 Lys His
Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala 35 40 45
Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala 50
55 60 His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu Gly Arg His Ser
Leu 65 70 75 80 Phe His Pro Glu Asp Thr Gly Gln Val Phe Gln Val Ser
His Ser Phe 85 90 95 Pro His Pro Leu Tyr Asp Met Ser Leu Leu Lys
Asn Arg Phe Leu Arg 100 105 110 Pro Gly Asp Asp Ser Ser Ile Glu Pro
Glu Glu Phe Leu Thr Pro Lys 115 120 125 Lys Leu Gln Cys Val Asp Leu
His Val Ile Ser Asn Asp Val Cys Ala 130 135 140 Gln Val His Pro Gln
Lys Val Thr Lys Phe Met Leu Cys Ala Gly Arg 145 150 155 160 Trp Thr
Gly Gly Lys Ser Thr Cys Ser Gly Asp Ser Gly Gly Pro Leu 165 170 175
Val Cys Asn Gly Val Leu Gln Gly Ile Thr Ser Trp Gly Ser Glu Pro 180
185 190 Cys Ala Leu Pro Glu Arg Pro Ser Leu Tyr Thr Lys Val Val His
Tyr 195 200 205 Arg Lys Trp Ile Lys Asp Thr Ile Val Ala 210 215
* * * * *