U.S. patent application number 12/688754 was filed with the patent office on 2010-09-09 for recombinant human albumin-human granulocyte colony stimulating factor for the prevention of neutropenia.
This patent application is currently assigned to TEVA Biopharmaceuticals USA, Inc.. Invention is credited to Adam Carr Bell, Jason Benjamin Bock, Jeffrey Herpst.
Application Number | 20100227818 12/688754 |
Document ID | / |
Family ID | 42260335 |
Filed Date | 2010-09-09 |
United States Patent
Application |
20100227818 |
Kind Code |
A1 |
Bock; Jason Benjamin ; et
al. |
September 9, 2010 |
Recombinant Human Albumin-Human Granulocyte Colony Stimulating
Factor For The Prevention Of Neutropenia
Abstract
Disclosed are compositions and methods for treating, preventing
and ameliorating conditions and diseases characterized by a lowered
white blood cell count. The methods and compositions described
herein include a fusion polypeptide formed from human serum albumin
protein ("HSA") and human granulocyte-colony stimulating factor
("G-CSF").
Inventors: |
Bock; Jason Benjamin; (North
Potomac, MD) ; Bell; Adam Carr; (Germantown, MD)
; Herpst; Jeffrey; (Ellicott City, MD) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
TEVA Biopharmaceuticals USA,
Inc.
|
Family ID: |
42260335 |
Appl. No.: |
12/688754 |
Filed: |
January 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61145440 |
Jan 16, 2009 |
|
|
|
61145436 |
Jan 16, 2009 |
|
|
|
Current U.S.
Class: |
424/85.1 ;
514/15.2 |
Current CPC
Class: |
A61K 38/385 20130101;
A61P 35/00 20180101; A61K 38/193 20130101; A61P 37/04 20180101;
A61P 43/00 20180101; A61P 7/00 20180101; A61K 38/38 20130101; A61P
31/00 20180101; A61K 47/643 20170801 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61P 37/04 20060101 A61P037/04 |
Claims
1. A method of treating or preventing neutropenia in a human
subject comprising administering to a human subject exhibiting
neutropenia or at risk of developing neutropenia, recombinant human
albumin-human granulocyte colony stimulating factor in an amount
effective to treat the subject.
2. A method of treating or preventing leukopenia in a human subject
comprising administering to a human subject exhibiting leukopenia
or at risk of developing leukopenia, recombinant human
albumin-human granulocyte colony stimulating factor in an amount
effective to treat the subject.
3. The method according to claim 1 wherein the human subject is
suffering from a non-myeloid malignancy and is receiving at least
one myelosuppressive anti-cancer drug associated with a clinically
significant incidence of febrile neutropenia.
4. A method of decreasing the incidence of infection, as manifested
by febrile neutropenia, in a human subject with non-myeloid
malignancies and receiving at least one myelosuppressive
anti-cancer drug associated with a clinically significant incidence
of febrile neutropenia, comprising administering to the subject
recombinant human albumin-human granulocyte colony stimulating
factor in an amount effective to treat the subject.
5. The method of claim 1, wherein: (a) grade 4 neutropenia in the
subject is eliminated; (b) grade 4 neutropenia in the subject is
reduced; (c) the duration of severe neutropenia is reduced in the
subject; (d) the duration of grade 4 neutropenia in the subject is
less than 5 days; (e) the duration of grade 3 neutropenia in the
subject is eliminated; (f) the duration of grade 3 neutropenia in
the subject is decreased; or (g) any combination thereof.
6. The method of claim 1, wherein administering recombinant human
albumin-human granulocyte colony stimulating factor induces a rise
in white blood cells (WBC).
7. The method according to claim 1, wherein: (a) the number of
neutrophils is increased in the subject; (b) a decrease in the
number of neutrophils is inhibited in the subject; (c) the nadir
absolute neutrophil count (ANC) is increased in the subject; (d)
the recovery ANC is increased in the subject; (e) the time to ANC
recovery is reduced in the subject; or (f) any combination
thereof.
8. The method according to claim 1, wherein the amount of
recombinant human albumin-human granulocyte colony stimulating
factor administered to the subject is selected from the group
consisting of: (a) from about 50 .mu.g/kg to about 450 .mu.g/kg;
(b) about 50 .mu.g/kg; (c) about 150 .mu.g/kg; (d) about 300
.mu.g/kg; (e) about 450 .mu.g/kg; (f) from about 30 mg to about 60
mg; (g) about 30 mg; (h) about 40 mg; (i) about 50 mg; (j) about 60
mg; or (k) any combination thereof.
9. The method of claim 1, wherein neutropenia is selected from the
group consisting of primary neutropenia, acute neutropenia, severe
chronic neutropenia (SCN), severe congenital neutropenia
(Kostmann's syndrome), severe infantile genetic agranulocytosis,
benign neutropenia, cyclic neutropenia, chronic idiopathic
neutropenia, secondary neutropenia, syndrome associated
neutropenia, and immune-mediated neutropenia.
10. The method of claim 1 wherein neutropenia is caused or
associated with radiation, alcoholism, drugs, allergic disorders,
aplastic anemia, autoimmune disease, T-.gamma. lymphoproliferative
disease (T-.gamma. LPD), myelodysplasia, myelofibrosis,
dysgammaglobulinemia, paroxysmal nocturnal hemoglobinuria, cancer,
vitamin B.sub.12 deficiency, folate deficiency, viral infection,
bacterial infection, spleen disorder, hemodialysis, or
transplantation, leukemia, myeloma, lymphoma, metastatic solid
tumors which infiltrate and replace the bone marrow, toxins, bone
marrow failure, Schwachman-Diamond syndrome, cartilage-hair
hypoplasia, dyskeratosis congenita, glycogen storage disease type
IB, splenomegaly of any cause, intrinsic defects in myeloid cells
or their precursors.
11. The method of claim 3, wherein recombinant human albumin-human
granulocyte colony stimulating factor is administered at a time
selected from the group consisting of: (a) at least 12 hours after
administration of the myelosuppressive anti-cancer drug; (b) at
least 18 hours after administration of the myelosuppressive
anti-cancer drug; (c) at least 24 hours after administration of the
myelosuppressive anti-cancer drug.
12. The method of claim 11 wherein administering recombinant human
albumin-human granulocyte colony stimulating factor prior to the
myelosuppressive anti-cancer drug induces a rise in WBC.
13. The method of claim 11 wherein administering recombinant human
albumin-human granulocyte colony stimulating factor prior to
chemotherapy induces a rise in ANC.
14. The method of claim 3 wherein the non-myeloid malignancy
comprises breast cancer.
15. The method of claim 3 wherein the myelosuppressive anticancer
drugs comprise doxorubicin and docetaxel.
16. The method of claim 15 wherein about 50 mg/m.sup.2 doxorubicin
and about 75 mg/m.sup.2 docetaxel are administered sequentially by
intravenous infusion on the same day for at least one treatment
cycle.
17. The method of claim 15 wherein about 60 mg/m.sup.2 doxorubicin
and about 75 mg/m.sup.2 docetaxel are administered sequentially by
intravenous infusion on the same day for at least one treatment
cycle.
18. The method of claim 3, wherein ANC and WBC return to normal at
a time period selected from the group consisting of: (a) by day 10
after chemotherapy; (b) by day 11 after chemotherapy; (c) by day 12
after chemotherapy; (d) by day 13 after chemotherapy: (e) by day 14
after chemotherapy; or (f) by day 15 after chemotherapy.
19. The method of claim 3, wherein on day 14 after chemotherapy
administration the rise in ANC in patients treated with recombinant
human albumin-human granulocyte colony stimulating factor is lower
than the rise in ANC in patients treated with an equivalent dose of
pegfilgrastim.
20. The method according to claim 1 wherein administering
recombinant human albumin-human granulocyte colony stimulating
factor induces a rise in lymphocytes, monocytes, eosinophils,
basophils, or any combination thereof.
21. The method according to claim 1 wherein the number of
lymphocytes, monocytes, eosinophils, basophils or any combination
thereof is increased in the subject.
22. The method according to claim 1, wherein a decrease in the
number of lymphocytes, monocytes, eosinophils, or basophils is
inhibited in the subject.
Description
[0001] The present application claims the benefit of U.S.
Provisional Application No. 61/145,440 filed on Jan. 16, 2009, and
U.S. Provisional Application No. 61/145,436 filed on Jan. 16, 2009.
The contents of U.S. Provisional Application No. 61/145,440 and
U.S. Provisional Application No. 61/145,436 are hereby incorporated
by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Mar. 4,
2010, is named 75977234.txt, and is 25,481 bytes in size.
BACKGROUND
[0003] Leukopenia is a reduction in the circulating White Blood
Cells (WBC) and is often defined as a WBC count<4000/mL. The
main cells involved in leukopenia are neutrophils. However a
reduced number of lymphocytes, monocytes, eosinophils, or basophils
may also contribute to the decreased total cell count (Merck
Manual, 17th edition).
[0004] Neutropenia is characterized by a reduction in the blood
neutrophil count, often leading to increased susceptibility to
bacterial and fungal infections. Neutropenia is classified by the
neutrophil count and the relative risk of infection: mild (1000 to
1500/mL), moderate (grade 3, 500 to 1000/mL), or severe (grade 4,
<500/mL). Acute and severe neutropenia is a life-threatening
condition as it predisposes the patient to rapidly fatal infections
(Merck Manual, 17th edition).
[0005] Neutropenia can be caused by impaired production of
neutrophils in the bone marrow, or by accelerated destruction of
neutrophils. Acute neutropenia may occur over a few days when
neutrophil use is rapid and production is severely impaired.
Chronic neutropenia may last for many months and is often caused by
reduced production or sequestration of neutrophils in the spleen.
Neutropenia may be classified by whether it arises secondary to
factors extrinsic to marrow myeloid cells or whether an intrinsic
defect appears to be present in the myeloid progenitors (Merck
Manual, 17th edition).
[0006] Neutropenia and its infectious complications are among the
most common and serious adverse effects of cytotoxic chemotherapy
and other cancer therapies such as radiation therapy, biotherapy,
targeted therapy and bone marrow transplantation. Cytotoxic
chemotherapy, which works by destroying fast-growing cells, induces
neutropenia because of the high proliferative rate of neutrophil
precursors and the rapid turnover of blood neutrophils (Merck
Manual, 17th edition). The most common symptoms of neutropenia in
patients with undergoing chemotherapy include fever, mouth sores,
and ear infections. Patients with profound neutropenia often suffer
from pyogenic infections such as septicemia, cutaneous cellulitis,
liver abscesses, furunculosis, pneumonia, stomatitis, gingivitis,
perirectal inflammation, colitis, sinusitis, and otitis media.
Chemotherapy may have to be delayed until the body can produce more
neutrophils and a lower dosage may have to be given, resulting in
the treatment being less effective.
SUMMARY
[0007] Described herein are methods and compositions useful for the
treatment, amelioration and prevention of conditions characterized
by a lower than normal white blood cell count. Such conditions
include but are not limited to leukopenia and neutropenia.
[0008] In a first embodiment, described is a method of treating or
preventing neutropenia in a human subject comprising administering
to a human subject exhibiting neutropenia or at risk of developing
neutropenia, recombinant human albumin-human granulocyte colony
stimulating factor in an amount effective to treat the subject. In
an exemplary embodiment, the human subject can be suffering from a
non-myeloid malignancy and receiving at least one myelosuppressive
anti-cancer drug associated with a clinically significant incidence
of febrile neutropenia.
[0009] In a second embodiment, described is a method of treating or
preventing leukopenia in a human subject comprising administering
to a human subject exhibiting leukopenia or at risk of developing
leukopenia; recombinant human albumin-human granulocyte colony
stimulating factor in an amount effective to treat the subject.
[0010] In a third embodiment, described is a method of decreasing
the incidence of infection, as manifested by febrile neutropenia,
in a human subject with non-myeloid malignancies and receiving at
least one myelosuppressive anti-cancer drug associated with a
clinically significant incidence of febrile neutropenia, comprising
administering to the subject recombinant human albumin-human
granulocyte colony stimulating factor in an amount effective to
treat the subject.
[0011] In some methods, compounds described herein are useful for
decreasing the incidence of infection, such as infection manifested
by febrile neutropenia. In some embodiments, the compositions and
methods include a fusion polypeptide formed from human serum
albumin protein ("HSA") and human granulocyte-colony stimulating
factor ("G-CSF"). The fusion polypeptide is 759 amino acids in
length; amino acids 1-585 of the fusion correspond to amino acids
from the mature form of HSA, and amino acids 586-759 of the fusion
correspond to amino acids of the mature form of human G-CSF. The
amino acid sequences of the fusion protein is presented in FIG. 1.
The fusion polypeptide, termed Neugranin.TM. ("NEUG") is
administered to patients exhibiting or at risk of exhibiting
leukopenia or neutropenia. For example, in some embodiments,
methods include treating leukopenia or neutropenia in a human
subject by administering recombinant human albumin-human
granulocyte colony stimulating factor in an amount effective to
treat the subject.
[0012] In some embodiments, the neutropenia is primary neutropenia,
acute neutropenia, severe chronic neutropenia (SCN), severe
congenital neutropenia (Kostmann's syndrome), severe infantile
genetic agranulocytosis, benign neutropenia, cyclic neutropenia,
chronic idiopathic neutropenia, secondary neutropenia, syndrome
associated neutropenia, or immune-mediated neutropenia.
[0013] In other embodiments, the neutropenia is caused or
associated with radiation, alcoholism, drugs, allergic disorders,
aplastic anemia, autoimmune disease, T-.gamma. lymphoproliferative
disease (T-.gamma. LPD), myelodysplasia, myelofibrosis,
dysgammaglobulinemia, paroxysmal nocturnal hemoglobinuria, cancer,
vitamin B.sub.12 deficiency, folate deficiency, viral infection,
bacterial infection, spleen disorder, hemodialysis,
transplantation, leukemia, myeloma, lymphoma, metastatic solid
tumors which infiltrate and replace the bone marrow, toxins, bone
marrow failure, Schwachman-Diamond syndrome, cartilage-hair
hypoplasia, dyskeratosis congenita, glycogen storage disease type
IB, splenomegaly of any cause, and intrinsic defects in myeloid
cells or their precursors. In some embodiments, the neutropenia is
caused or associated with cytotoxic chemotherapy.
[0014] In some embodiments, the human subject is suffering from a
non-myeloid malignancy, for example, breast cancer, and is
receiving cytotoxic chemotherapy. For example, in some embodiments
the patient is receiving at least one myelosuppressive anti-cancer
drug associated with a clinically significant incidence of febrile
neutropenia. In some embodiments, the myelosuppressive anticancer
drugs are doxorubicin and docetaxel. In further embodiments, about
50 mg/m.sup.2 doxorubicin and about 75 mg/m.sup.2 docetaxel are
administered sequentially by intravenous infusion on the same day
for at least one treatment cycle. In still other embodiments, about
60 mg/m.sup.2 doxorubicin and about 75 mg/m.sup.2 docetaxel are
administered sequentially by intravenous infusion on the same day
for at least one treatment cycle.
[0015] In other embodiments, methods include decreasing the
incidence of infection, as manifested by febrile neutropenia, in
human subjects. In some embodiments, the human subject is suffering
from non-myeloid malignancies and is receiving at least one
myelosuppressive anti-cancer drug associated with a clinically
significant incidence of febrile neutropenia. In some embodiments,
recombinant human albumin-human granulocyte colony stimulating
factor is administered to the subject in an amount effective to
treat the neutropenia in the subject.
[0016] In some embodiments, the duration or severity of neutropenia
is reduced or neutropenia is eliminated in a subject. For example,
in some embodiments, grade 4 or grade 3 neutropenia in the subject
is eliminated. In other embodiments, the duration of grade 4 or
grade 3 neutropenia is reduced. For example, in some embodiments
the duration of grade 4 neutropenia in the subject is less than 5
days; in some embodiments, the duration of grade 4 neutropenia in
the subject is less than 4 days, less then 3 days or less than 2
days. In other embodiments, the duration of grade 3 neutropenia in
the subject is eliminated, and/or the duration of grade 3
neutropenia in the subject is decreased as compared to subjects who
do not receive treatment with human albumin-human granulocyte
colony stimulating factor.
[0017] In some embodiments, administering recombinant human
albumin-human granulocyte colony stimulating factor induces a rise
in white blood cells ("WBC") or decreases a loss of WBC in a
subject. For example, in some embodiments, the number of
neutrophils is increased in the subject; the decrease in the number
of neutrophils is inhibited in the subject, the nadir absolute
neutrophil count ("ANC") is increased in the subject, the recovery
ANC is increased in the subject, and/or the time to ANC recovery is
reduced in the subject.
[0018] In some embodiments, the amount of recombinant human
albumin-human granulocyte colony stimulating factor administered to
the subject is from about 40 .mu.g/kg to about 500 .mu.g/kg; in
other embodiments, the amount of recombinant human albumin-human
granulocyte colony stimulating factor administered to the subject
is about 50 .mu.g/kg to about 450 .mu.g/kg. In still other
embodiments, the amount of recombinant human albumin-human
granulocyte colony stimulating factor administered to the subject
is about 50 .mu.g/kg, about 100 .mu.g/kg, about 150 .mu.g/kg, about
200 .mu.g/kg or about 250 .mu.g/kg. In further embodiments, the
amount of recombinant human albumin-human granulocyte colony
stimulating factor administered to the subject is about 300
.mu.g/kg, about 350 .mu.g/kg or about 400 .mu.g/kg. In alternative
embodiments, the amount of recombinant human albumin-human
granulocyte colony stimulating factor administered to the subject
is about 450 .mu.g/kg. In yet other embodiments, the amount of
recombinant human albumin-human granulocyte colony stimulating
factor administered to the subject is from about 20 to about 100
mg. In further embodiments, the amount of recombinant human
albumin-human granulocyte colony stimulating factor administered to
the subject is from about 30 mg to about 60 mg. In further
embodiments, the amount of recombinant human albumin-human
granulocyte colony stimulating factor administered to the subject
is about 30 mg, about 40 mg, about 50 mg, or about 60 mg.
[0019] In some embodiments, recombinant human albumin-human
granulocyte colony stimulating factor is administered after
chemotherapy (e.g., administration of a myelosuppressive
anti-cancer drug). For example, in some embodiments, recombinant
human albumin-human granulocyte colony stimulating factor is
administered during chemotherapy, or is administered within 2
hours, within 4 hours, within 6 hours, within 12 hours, within 18
hours, within 24 hours or within 48 hours of chemotherapy
administration.
[0020] In another embodiment of the invention, recombinant human
albumin-human granulocyte colony stimulating factor can be
administered after myelosuppressive anti-cancer drug
administration. For example, the recombinant human albumin-human
granulocyte colony stimulating factor can be administered at a time
selected from the group consisting of: (a) at least 2 hours after
administration of the myelosuppressive anti-cancer drug; (b) at
least 4 hours of administration of the myelosuppressive anti-cancer
drug; (c) at least 6 hours after administration of the
myelosuppressive anti-cancer drug; (d) at least 12 hours after
administration of the myelosuppressive anti-cancer drug; (e) at
least 18 hours after administration of the myelosuppressive
anti-cancer drug; (f) at least 24 hours after administration of the
myelosuppressive anti-cancer drug; (g) at least 48 hours after
administration of the myelosuppressive anti-cancer drug; or (h)
during, or substantially concurrently with, the administration of
the myelosuppressive anti-cancer drug.
[0021] In some embodiments, administering recombinant human
albumin-human granulocyte colony stimulating factor during or after
chemotherapy treatment induces a rise in WBC and/or induces a rise
in ANC. For example, in some embodiments, ANC and WBC return to
normal by day 10 after chemotherapy. In other embodiments ANC and
WBC return to normal by day 11 after chemotherapy, by day 12 after
chemotherapy, by day 13 after chemotherapy, by day 14 after
chemotherapy or by day 15 after chemotherapy. In some embodiments,
on day 14 after chemotherapy administration the rise in ANC in
patients treated with recombinant human albumin-human granulocyte
colony stimulating factor is lower than the rise in ANC in patients
treated with an equivalent dose of pegfilgrastim.
[0022] In some embodiments, administering recombinant human
albumin-human granulocyte colony stimulating factor induces a rise
in lymphocytes, monocytes, eosinophils, or basophils. For example,
in some embodiments, the number lymphocytes, monocytes,
eosinophils, or basophils is increased in the subject. In other
embodiments, the decrease in the number of lymphocytes, monocytes,
eosinophils, or basophils is inhibited in the subject.
[0023] In some embodiments, particularly for methods of treating or
preventing neutropenia, a result achieved can be selected from the
group consisting of: (a) grade 4 neutropenia in the subject is
eliminated; (b) grade 4 neutropenia in the subject is reduced; (c)
the duration of severe neutropenia is reduced in the subject; (d)
the duration of grade 3 neutropenia in the subject is eliminated;
(e) the duration of grade 3 neutropenia in the subject is
decreased; or (f) any combination thereof.
[0024] In some embodiments, particularly for methods of treating or
preventing neutropenia, administering recombinant human
albumin-human granulocyte colony stimulating factor induces a rise
in white blood cells (WBC). In yet other embodiments, particularly
for methods of treating or preventing neutropenia, the result
achieved is selected from the group consisting of (a) the number of
neutrophils is increased in the subject; (b) a decrease in the
number of neutrophils is inhibited in the subject; (c) the nadir
absolute neutrophil count (ANC) is increased in the subject; (d)
the recovery ANC is increased in the subject; (e) the time to ANC
recovery is reduced in the subject; or (f) any combination
thereof.
[0025] In some embodiments the amount of recombinant human
albumin-human granulocyte colony stimulating factor administered to
the subject is selected from the group consisting of: (a) from
about 50 .mu.g/kg to about 450 .mu.g/kg; (b) about 50 .mu.g/kg; (c)
about 150 .mu.g/kg; (d) about 300 .mu.g/kg; (e) about 450 .mu.g/kg;
(f) from about 30 mg to about 60 mg; (g) about 30 mg; (h) about 40
mg; (i) about 50 mg; (j) about 60 mg; or (k) any combination
thereof.
[0026] In some embodiments, the neutropenia to be treated or
prevented is selected from the group consisting of primary
neutropenia, acute neutropenia, severe chronic neutropenia (SCN),
severe congenital neutropenia (Kostmann's syndrome), severe
infantile genetic agranulocytosis, benign neutropenia, cyclic
neutropenia, chronic idiopathic neutropenia, secondary neutropenia,
syndrome associated neutropenia, and immune-mediated neutropenia.
In addition, the eutropenia can be caused or associated with, for
example, radiation, alcoholism, drugs, allergic disorders, aplastic
anemia, autoimmune disease, T-.gamma. lymphoproliferative disease
(T-.gamma. LPD), myelodysplasia, myelofibrosis,
dysgammaglobulinemia, paroxysmal nocturnal hemoglobinuria, cancer,
vitamin B.sub.12 deficiency, folate deficiency, viral infection,
bacterial infection, spleen disorder, hemodialysis, or
transplantation, leukemia, myeloma, lymphoma, metastatic solid
tumors which infiltrate and replace the bone marrow, toxins, bone
marrow failure, Schwachman-Diamond syndrome, cartilage-hair
hypoplasia, dyskeratosis congenita, glycogen storage disease type
IB, splenomegaly of any cause, intrinsic defects in myeloid cells
or their precursors.
[0027] In embodiments of the invention where a the human subject is
suffering from a non-myeloid malignancy, the non-myeloid malignancy
can comprise breast cancer.
[0028] In embodiments of the invention where a myelosuppressive
anticancer drugs is administered, the myelosuppressive anticancer
drugs can comprise doxorubicin and docetaxel. For example, about 50
mg/m.sup.2 doxorubicin and about 75 mg/m.sup.2 docetaxel can be
administered sequentially by intravenous infusion on the same day
for at least one treatment cycle. Alternatively, about 60
mg/m.sup.2 doxorubicin and about 75 mg/m.sup.2 docetaxel can be
administered sequentially by intravenous infusion on the same day
for at least one treatment cycle.
[0029] In some embodiments of the invention following treatment ANC
and WBC return to normal at a time period selected from the group
consisting of: (a) by day 10 after chemotherapy; (b) by day 11
after chemotherapy; (c) by day 12 after chemotherapy; (d) by day 13
after chemotherapy: (e) by day 14 after chemotherapy; or (f) by day
15 after chemotherapy. In yet another embodiment, on day 14 after
chemotherapy administration the rise in ANC in patients treated
with recombinant human albumin-human granulocyte colony stimulating
factor is lower than the rise in ANC in patients treated with an
equivalent dose of pegfilgrastim.
[0030] In some embodiments of the invention administering
recombinant human albumin-human granulocyte colony stimulating
factor induces a rise in lymphocytes, monocytes, eosinophils,
basophils, or any combination thereof. In other embodiments, the
number of lymphocytes, monocytes, eosinophils, basophils or any
combination thereof is increased in the subject. In yet further
embodiments of the invention, a decrease in the number of
lymphocytes, monocytes, eosinophils, or basophils is inhibited in
the subject.
[0031] Both the foregoing general description and the following
brief description of the drawings and the detailed description are
exemplary and explanatory and are intended to provide further
explanation of the invention as claimed. Other objects, advantages,
and novel features will be readily apparent to those skilled in the
art from the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1A-1C: FIG. 1A shows the nucleic acid sequence (SEQ ID
NO: 3) and the amino acid sequence (SEQ ID NO: 4) of the
recombinant human albumin-granulocyte colony stimulating factor
("rHA-G-CSF") fusion protein termed "Neugranin.TM." ("NEUG"); FIG.
1B shows the amino acid sequence (SEQ ID NO: 5) of human G-CSF;
FIG. 1C shows the amino acid sequence (SEQ ID NO: 6) of human serum
albumin.
[0033] FIG. 2 is a graph showing the absolute neutrophil count
("ANC") for subjects in Phase I. Subjects received 300 .mu.g/kg
NEUG (n=19), 450 .mu.g/kg NEUG (n=20) or 6 mg pegfilgrastim
(Neulasta.RTM.) (n=9) in cycle 1 following study chemotherapy.
[0034] FIG. 3 is a graph showing the pharmacokinetics of NEUG in
the Phase I study in human subjects. The serum concentration of
NEUG administered subcutaneously at the indicated doses (450
.mu.g/kg, 300 .mu.g/kg or 150 .mu.g/kg) was measured in subjects
with breast cancer in the absence of chemotherapy. Squares: 450
.mu.g/kg Cycle 0; triangles: 300 .mu.g/kg Cycle 0; circles: 150
.mu.g/kg Cycle 0.
[0035] FIG. 4 is a graph showing the
pharmacokinetics/pharmacodynamics ("PK/PD") of NEUG in cycle 1 of
chemotherapy (Phase I study). Patients received 450 .mu.g/kg of
NEUG one day after doxorubicin/docetaxel administration in cycle 1.
ANC is shown by the open diamonds; NEUG concentration is shown by
closed squares. Cut-offs for neutropenia grades 3 and 4 are shown
by the dashed lines. The Lower Limit of Quantitation ("LLOQ") for
NEUG is shown as a dotted line at 6 ng/ml.
[0036] FIG. 5 is a graph showing the ANC profile for patients who
received either 30 mg of NEUG or 6 mg of pegfilgrastim
(Neulasta.RTM.) one day after starting cycle 1 of chemotherapy
(Phase II study). Grade 3 and 4 neutropenia cut-off values are
shown by dashed lines.
[0037] FIG. 6 illustrates the chemotherapy cycles for the Phase I
studies.
[0038] FIGS. 7A and 7B are graphs showing the ANC and white blood
cell ("WBC") count for subjects in the Phase I study.
[0039] FIG. 8 shows a graph of NSF-60 cell proliferation with
either NEUG (Albugranin) or Neupogen.RTM..
[0040] FIG. 9 show a graph of NSF-60 cell proliferation with either
NEUG or Neulasta.
[0041] FIG. 10 shows a graph of levels of peripheral blood
neutrophils (Gr.1+) in BDF-1 mice after single SC administration of
NEUG (Albugranin) and Neupogen (time course). The total number of
Gr.1+ cells is expressed as the group mean+/-SEM.
[0042] FIG. 11 shows a graph of levels of peripheral hematopoietic
progenitors (c-kit+) after single SC administration of NEUG
(Albugranin) and Neupogen (time course). The total number of c-kit+
cells is expressed as the group mean+/-SEM.
[0043] FIGS. 12 A and 12 B are graphs showing the levels of
peripheral blood granulocytes (Gr.1+) after single subcutaneous
("SC") administration of NEUG (Albugranin) or Neulasta.RTM. in
BDF-1 mice. 12A shows a time course of response following single
dose of Neulasta or NEUG and 12B show relative potency of NEUG or
Neulasta.
[0044] FIG. 13 is a table showing the composition of the NEUG drug
product used in Phase I.
[0045] FIG. 14 is a table showing the composition of the NEUG drug
product used in Phase II.
[0046] FIG. 15 is a graph showing levels of peripheral
hematopoietic progenitor cells (c-kit+) after single subcutaneous
administration of NEUG (Albugranin) or Neulasta.RTM. (time course).
The total number of c-kit+ is expressed as the mean and standard
error of the mean calculated for each group. Differences among
treatment groups were analyzed by using heteroscedastic t-test.
[0047] FIG. 16 is a graph showing levels of peripheral blood
neutrophils (Gr.1+) after single subcutaneous administration of
NEUG (Albugranin) or Neulasta 1 day after 5-FU (150 .mu.g/kg) IP
injection. The total number of GR.1+ cells enumerated daily is
expressed as the mean and standard error of the mean calculated for
each group. Difference among treatment groups were assessed with
the 2-sample t-test with unequal variance. Treatment with either
agent at all dose levels resulted in statistically significant
increases in neutrophil count compared with the vehicle
control.
[0048] FIG. 17 is a graph showing the effect of NEUG (Albugranin)
on the relative percent of peripheral blood neutrophils. The
relative percent of neutrophils on each study day is presented as
the group mean+/-SEM. Data from days 8 and 9 for NEUG 100 .mu.g/kg
Q7 are presented as days 9 and 10 respectively to facilitate
comparison with other groups. Controls were saline vehicle
administered SC every 4 days.times.4 or Neupogen.RTM. administered
SC daily.times.14. The treatment period is considered days 1-14,
and the recovery period is days 15-28.
[0049] FIG. 18 is a graph showing a comparison of repeated
dose-administration of NEUG (Albugranin) SC, NEUG IV, or
Neulasta.RTM. SC on neutrophil mobilization in monkeys. The number
of neutrophils (K/.mu.l) on each study day through day 22 is
presented as the group mean+/-SEM. The arrows indicate dose
administration. NEUG was administered SC (n=6) or IV (n=6) at 1.0
mg/kg/dose, and Neulasta was administered SC (n=6) at 0.22
mg/kg/dose (equimolar dose to 1.0 mg/kg NEUG). The NEUG vehicle was
administered SC as a control (n=2).
[0050] FIGS. 19A and 19B is a table showing a summary of in vivo
pharmacokinetic studies.
[0051] FIG. 20 is a table showing a summary of in vivo non-clinical
studies that provide safety data.
[0052] FIG. 21 a flow chart showing an exemplary overview of
fermentation and purification of NEUG.
[0053] FIG. 22 is a graph showing the median absolute neutrophil
count (ANC) for subjects in Phase I, part A (cycle 0, or
pre-chemotherapy) from treatment to 14 days. At day 4, the lines,
from highest to lowest are: 300 .mu.g/kg, 450 .mu.g/kg, 150
.mu.g/kg and 50 .mu.g/kg.
[0054] FIGS. 23A and 23 B show the area the curve (AUC) for each
subject treated in Phase I, Part B, based on the ANC values
obtained for days 0 to 15. FIG. 23A is a graph; the data from FIG.
23A is summarized in the table, 23B.
[0055] FIG. 24 is a graph showing the area under the curve (AUC)
for subjects treated in Phase II, based on the ANC values obtained
for days 0 to 15 in cycle 1 (fixed dose cohorts). For all subjects
in Phase II, AUC.sub.ANC (days 0-15) was calculated. Patients
treated with Neugranin received a range of doses from 0.3 to 1
mg/kg (calculated as dose divided by baseline weight). The weight
adjusted dose range was divided into quartiles and plotted vs.
AUC.sub.ANC (left panel). For all subjects treated with
pegfilgrastim (N=112), 30 mg (N=10), 40 mg (N=105), or 50 mg
(N=105) of Neugranin AUC.sub.ANC was also calculated and compared.
Data shown in means.+-.SEM.
[0056] FIG. 25 is a diagram illustrating the Phase II study
design.
DETAILED DESCRIPTION
[0057] Disclosed herein are compositions and methods for treating,
preventing and ameliorating conditions and diseases characterized
by a lowered white blood cell count. The methods and compositions
described herein include a fusion polypeptide formed from human
serum albumin protein ("HSA") and human granulocyte-colony
stimulating factor ("G-CSF"). In a preferred embodiment, the fusion
polypeptide is about 759 amino acids in length; amino acids 1-585
of the fusion correspond to amino acids from the mature form of
HSA, and amino acids 586-759 of the fusion correspond to amino
acids of the mature form of human G-CSF. The amino acid sequence of
the fusion protein is presented in FIG. 1.
[0058] The invention also encompasses fusion proteins comprising
variants or fragments of G-CSF, and fusion proteins comprising
albumin or fragments or variants of albumin. The invention also
encompasses polynucleotides encoding the therapeutic albumin fusion
proteins of the invention, therapeutic albumin fusion proteins,
compositions, pharmaceutical compositions, formulations and kits.
Host cells transformed with the polynucleotides encoding
therapeutic albumin fusion proteins are also encompassed by the
invention, as are methods of making the albumin fusion proteins of
the invention using these polynucleotides, and/or host cells.
[0059] In one embodiment, an albumin fusion protein according to
the present invention has extended shelf fife.
[0060] In a second embodiment, an albumin fusion protein according
to the present invention is more stable than the corresponding
unfused G-CSF molecule.
[0061] The present invention further includes transgenic organisms
modified to contain the nucleic acid molecules of the invention,
preferably modified to express an albumin fusion protein of the
invention.
[0062] The present invention relates generally to polynucleotides
encoding albumin fusion proteins; albumin fusion proteins; and
methods of treating, preventing, or ameliorating diseases or
disorders using albumin fusion proteins or polynucleotides encoding
albumin fusion proteins. As used herein, "albumin fusion protein"
refers to a protein formed by the fusion of at least one molecule
of albumin (or a fragment or variant thereof) to at least one
molecule of G-CSF (or fragment or variant thereof). An albumin
fusion protein of the invention comprises at least a fragment or
variant of a G-CSF and at least a fragment or variant of human
serum albumin, which are associated with one another by genetic
fusion (i.e., the albumin fusion protein is generated by
translation of a nucleic acid in which a polynucleotide encoding
all or a portion of G-CSF is joined in-frame with a polynucleotide
encoding all or a portion of albumin). The G-CSF and albumin
protein, once part of the albumin fusion protein, may each be
referred to as a "portion," "region" or "moiety" of the albumin
fusion protein (e.g., a "G-CSF protein portion" or an "albumin
protein portion"). In a highly preferred embodiment, an albumin
fusion protein of the invention comprises at least one molecule of
G-CSF or fragment or variant of thereof (including, but not limited
to a mature form of the G-CSF protein) and at least one molecule of
albumin or fragment or variant thereof (including but not limited
to a mature form of albumin).
[0063] In a further preferred embodiment, an albumin fusion protein
of the invention is processed by a host cell and secreted into the
surrounding culture medium. Processing of the nascent albumin
fusion protein that occurs in the secretory pathways of the host
used for expression may include, but is not limited to signal
peptide cleavage; formation of disulfide bonds; proper folding;
addition and processing of carbohydrates (such as for example, N-
and O-linked glycosylation); specific proteolytic cleavages; and
assembly into multimeric proteins. An albumin fusion protein of the
invention is preferably in the processed form. In a most preferred
embodiment, the "processed form of an albumin fusion protein"
refers to an albumin fusion protein product which has undergone
N-terminal signal peptide cleavage, herein also referred to as a
"mature albumin fusion protein."
[0064] In one embodiment, the invention provides a polynucleotide
encoding an albumin fusion protein comprising, or alternatively
consisting of, G-CSF and a serum albumin protein. In a further
embodiment, the invention provides an albumin fusion protein
comprising, or alternatively consisting of, G-CSF protein and a
serum albumin protein. In other embodiments, the invention provides
an albumin fusion protein comprising, or alternatively consisting
of, a biologically active and/or therapeutically active fragment of
G-CSF protein and a serum albumin protein. In other embodiments,
the invention provides an albumin fusion protein comprising, or
alternatively consisting of, a biologically active and/or
therapeutically active variant of G-CSF protein and a serum albumin
protein. In preferred embodiments, the serum albumin protein
component of the albumin fusion protein is the mature portion of
serum albumin. The invention further encompasses polynucleotides
encoding these albumin fusion proteins.
[0065] In further embodiments, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, G-CSF
protein, and a biologically active and/or therapeutically active
fragment of serum albumin. In further embodiments, the invention
provides an albumin fusion protein comprising, or alternatively
consisting of, G-CSF protein and a biologically active and/or
therapeutically active variant of serum albumin. In preferred
embodiments, the G-CSF protein portion of the albumin fusion
protein is the mature portion of the G-CSF protein. In a further
preferred embodiment, the G-CSF protein portion of the albumin
fusion protein is the extracellular soluble domain of the G-CSF
protein. In an alternative embodiment, the G-CSF protein portion of
the albumin fusion protein is the active form of the G-CSF protein.
The invention further encompasses polynucleotides encoding these
albumin fusion proteins.
[0066] In further embodiments, the invention provides an albumin
fusion protein comprising, or alternatively consisting of, a
biologically active and/or therapeutically active fragment or
variant of G-CSF protein and a biologically active and/or
therapeutically active fragment or variant of serum albumin. In
preferred embodiments, the invention provides an albumin fusion
protein comprising, or alternatively consisting of, the mature
portion of G-CSF protein and the mature portion of serum albumin.
The invention further encompasses polynucleotides encoding these
albumin fusion proteins.
I. DEFINITIONS
[0067] As used herein, "polynucleotide" refers to a nucleic acid
molecule having a nucleotide sequence encoding a fusion protein
comprising, or alternatively consisting of, at least one molecule
of albumin (or a fragment or variant thereof) joined in frame to at
least one molecule of Granulocyte-colony stimulating factor (G-CSF)
(or fragment or variant thereof).
[0068] As used herein, "albumin fusion construct" refers to a
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide encoding at least one molecule of albumin (or a
fragment or variant thereof) joined in frame to at least one
polynucleotide encoding at least one molecule of G-CSF (or fragment
or variant thereof); and, further comprising, for example, one or
more of the following elements: (1) a functional self-replicating
vector (including but not limited to, a shuttle vector, an
expression vector, an integration vector, and/or a replication
system), (2) a region for initiation of transcription (e.g., a
promoter region, such as for example, a regulatable or inducible
promoter, a constitutive promoter), (3) a region for termination of
transcription, (4) a leader sequence, and (5) a selectable marker.
The polynucleotide encoding the G-CSF and albumin protein, once
part of the albumin fusion construct, may each be referred to as a
"portion," "region" or "moiety" of the albumin fusion
construct.
[0069] By a G-CSF polypeptide displaying a "therapeutic activity"
or a G-CSF protein that is "therapeutically active" is meant a
G-CSF polypeptide that possesses one or more known biological
and/or therapeutic activities associated with G-CSF protein. As a
non-limiting example, a "G-CSF therapeutic protein" is a G-CSF
protein that is useful to treat, prevent or ameliorate a disease,
condition or disorder. As a non-limiting example, a "G-CSF
therapeutic protein" may be one that binds specifically to a
particular cell type (normal (e.g., lymphocytes) or abnormal e.g.,
(cancer cells)) and therefore may be used to target a compound
(drug, or cytotoxic agent) to that cell type specifically.
II. GRANULOCYTE-COLONY STIMULATING FACTOR
[0070] Granulocyte-colony stimulating factor (G-CSF) is a
hematopoietic growth factor that stimulates the production of
neutrophils. Administration of G-CSF results in rapid induction of
a neutrophilic leukocytosis when there are viable precursor cells
to stimulate. Another important in vivo activity of G-CSF is
mobilization of hematopoietic progenitor cells into the peripheral
blood (Duhrsen et al., 1988; Molineux et al., 1999; Roberts et al.,
1994). This effect includes not only the neutrophil lineage but
extends to other single lineage and multi-lineage progenitors and
pluripotent hematopoietic stem cells (Molineux et al., 1999). G-CSF
also increases the cellular events that are part of the defense
mechanism against infections by priming neutrophils, thereby
increasing both their phagocytic and anti-bacterial activities
against opsonized Staphylococcus aureus. G-CSF also induces
chemotaxis of neutrophils and monocytes and adhesion of neutrophils
(Yuo et al., 1989; Wang et al., 1988).
[0071] Recombinant G-CSF products are currently approved for a
number of clinical indications to stimulate the proliferation and
differentiation of neutrophils. In clinical trials, filgrastim
(recombinant methionyl human G-CSF; Neupogen.RTM., Amgen, Thousand
Oaks, Calif.) increased the number of peripheral neutrophils and
thereby reduced the duration of neutropenia after myelosuppressive
chemotherapy. Recombinant G-CSF (filgrastim) is given by daily
subcutaneous (SC) injection.
[0072] Another recombinant form of G-CSF is pegfilgrastim, a
polyethylene glycol-conjugated rG-CSF (Neulasta.RTM.), which has
proven safe and effective as a once-per-cycle alternative to daily
rG-CSF therapy to decrease the incidence of febrile neutropenia in
patients receiving myelosuppressive anti-cancer drugs (Holmes,
O'Shaughnessy et al., 2002; Green et al., 2003; Neulasta.RTM. SmPC
2007).
[0073] Primary prophylaxis with G-CSF is recommended for the
prevention of febrile neutropenia in patients who are at high risk
based on age, medical history, disease characteristics, and
myelotoxicity of the chemotherapy regimen. The American Society of
Clinical Oncology and the European Organization for Research and
Treatment of Cancer recommend the use of G-CSF when the risk of
febrile neutropenia is approximately 20%. The U.S. National
Comprehensive Cancer Center Network recommends an optional
indication of G-CSF prophylaxis when the risk of febrile
neutropenia is 10% to 20% and a definite indication of G-CSF
prophylaxis when the risk of febrile neutropenia is at least 20%.
(Smith et al., 2006, Vogel et al., 2005, Timmer-Bonte et al., 2006,
NCCN Guidelines).
[0074] Prophylaxis with colony-stimulating factors is recommended
to alleviate the toxicity of certain chemotherapy regimens.
However, the added cost of these treatments is a significant
consideration both in the U.S. and especially in parts of the E.U.
and may lead to under-use of prophylactic G-CSF treatment and may
also limit patient eligibility for dose-intensive chemotherapy
regimens (Timmer-Bonte et al., 2006; Adams et al., 2006, NCCN
Guidelines).
[0075] The G-CSF protein may be encoded by a wild type
polynucleotide sequence (e.g., either full length or mature), or in
some instances the sequence may be a variant of the wild type
polynucleotide sequence (e.g., a polynucleotide which encodes the
wild type G-CSF protein, wherein the DNA sequence of the
polynucleotide has been optimized, for example, for expression in a
particular species; or a polynucleotide encoding a variant of the
wild type G-CSF protein (i.e., a site directed mutant; an allelic
variant)).
III. HUMAN SERUM ALBUMIN
[0076] Human serum albumin (HSA or HA) is the most prevalent
naturally occurring blood protein in the human circulatory system,
measured at approximately 40 grams of albumin/liter and persisting
in the circulation for over 20 days. Albumin is a carrier protein
with minimal activity at physiological concentrations. Even though
HSA lacks enzymatic or immunological function it is widely
distributed in vivo, and is know to be a carrier for various
substances in the blood (e.g., hormones, fatty acids, unconjugated
bilirubin, etc (Yeh et al., 1992). Both HSA and recombinant HA
(rHA) have the same long circulating half-life in humans.
[0077] Research has shown that therapeutic proteins genetically
fused to human albumin are able to take on the circulating
half-life characteristics of albumin (Syed et al., 1997). For
example, in rabbits, the half-life of CD4 fused to albumin is 140
fold greater than non-fused CD4 (Yeh et al., 1992).
[0078] Human serum albumin, a protein of 585 amino acids in its
mature form (as shown in FIG. 1 of U.S. Pat. No. 7,592,010, is
responsible for a significant proportion of the osmotic pressure of
serum and also functions as a carrier of endogenous and exogenous
ligands. At present, HA for clinical use is produced by extraction
from human blood. The production of recombinant HA (rHA) in
microorganisms has been disclosed in EP 330 451 and EP 361 991.
IV. POLYPEPTIDE AND POLYNUCLEOTIDE FRAGMENTS AND VARIANTS
[0079] A. Fragments
[0080] The present invention is further directed to fragments of
G-CSF protein, albumin proteins, and/or albumin fusion proteins of
the invention. The present invention is also directed to
polynucleotides encoding fragments of the G-CSF protein, albumin
proteins, and/or albumin fusion proteins of the invention. Even if
deletion of one or more amino acids from the N-terminus of a
protein results in modification or loss of one or more biological
functions of the G-CSF protein, albumin protein, and/or albumin
fusion protein of the invention, other therapeutic activities
and/or functional activities (e.g., biological activities, ability
to multimerize, ability to bind a ligand) may still be retained.
For example, the ability of polypeptides with N-terminal deletions
to induce and/or bind to antibodies which recognize the complete or
mature forms of the polypeptides generally will be retained when
less than the majority of the residues of the complete polypeptide
are removed from the N-terminus. Whether a particular polypeptide
lacking N-terminal residues of a complete polypeptide retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art. It is not unlikely
that a mutein with a large number of deleted N-terminal amino acid
residues may retain some biological or immunogenic activities. In
fact, peptides composed of as few as six amino acid residues may
often evoke an immune response.
[0081] Accordingly, fragments of G-CSF protein corresponding to a
G-CSF protein portion of an albumin fusion protein of the invention
include the full length protein as well as polypeptides having one
or more residues deleted from the amino terminus of the amino acid
sequence of the reference polypeptide (i.e., a G-CSF protein, or a
G-CSF protein portion of an albumin fusion protein encoded by a
polynucleotide or albumin fusion construct). In particular,
N-terminal deletions may be described by the general formula m to
q, where q is a whole integer representing the total number of
amino acid residues in a reference polypeptide (e.g., a G-CSF
protein, or a G-CSF protein portion of an albumin fusion protein of
the invention), and m is defined as any integer ranging from 2 to q
minus 6. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0082] In addition, fragments of serum albumin polypeptides
corresponding to an albumin protein portion of an albumin fusion
protein of the invention, include the full length protein as well
as polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the reference polypeptide
(i.e., serum albumin, or a serum albumin portion of an albumin
fusion protein). In preferred embodiments, N-terminal deletions may
be described by the general formula m to 585, where 585 is a whole
integer representing the total number of amino acid residues in
mature human serum albumin, and m is defined as any integer ranging
from 2 to 579. Polynucleotides encoding these polypeptides are also
encompassed by the invention. In additional embodiments, N-terminal
deletions may be described by the general formula m to 609, where
609 is a whole integer representing the total number of amino acid
residues in full length human serum albumin, and m is defined as
any integer ranging from 2 to 603. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0083] Moreover, fragments of albumin fusion proteins of the
invention, include the full length albumin fusion protein as well
as polypeptides having one or more residues deleted from the amino
terminus of the albumin fusion protein. In particular, N-terminal
deletions may be described by the general formula m to q, where q
is a whole integer representing the total number of amino acid
residues in the albumin fusion protein, and m is defined as any
integer ranging from 2 to q minus 6. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0084] Also as mentioned above, even if deletion of one or more
amino acids from the N-terminus or C-terminus of a reference
polypeptide (e.g., a G-CSF protein; serum albumin protein; or
albumin fusion protein of the invention) results in modification or
loss of one or more biological functions of the protein, other
functional activities (e.g., biological activities, ability to
multimerize, ability to bind a ligand) and/or therapeutic
activities may still be retained. For example, the ability of
polypeptides with C-terminal deletions to induce and/or bind to
antibodies which recognize the complete or mature forms of the
polypeptide generally will be retained when less than the majority
of the residues of the complete or mature polypeptide are removed
from the C-terminus. Whether a particular polypeptide lacking the
N-terminal and/or C-terminal residues of a reference polypeptide
retains therapeutic activity can readily be determined by routine
methods described herein and/or otherwise known in the art.
[0085] The present invention further provides polypeptides having
one or more residues deleted from the carboxy terminus of the amino
acid sequence of a G-CSF protein corresponding to a G-CSF protein
portion of an albumin fusion protein of the invention. In
particular, C-terminal deletions may be described by the general
formula 1 to n, where n is any whole integer ranging from 6 to q
minus 1, and where q is a whole integer representing the total
number of amino acid residues in a reference polypeptide (e.g., a
G-CSF protein, or a G-CSF protein portion of an albumin fusion
protein encoded by a polynucleotide or albumin fusion construct).
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0086] In addition, the present invention provides polypeptides
having one or more residues deleted from the carboxy terminus of
the amino acid sequence of an albumin protein corresponding to an
albumin protein portion of an albumin fusion protein of the
invention. In particular, C-terminal deletions may be described by
the general formula 1 to n, where n is any whole integer ranging
from 6 to 584, where 584 is the whole integer representing the
total number of amino acid residues in mature human serum albumin
minus 1. Polynucleotides encoding these polypeptides are also
encompassed by the invention. In particular, C-terminal deletions
may be described by the general formula 1 to n, where n is any
whole integer ranging from 6 to 608, where 608 is the whole integer
representing the total number of amino acid residues in serum
albumin minus 1. Polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0087] Moreover, the present invention provides polypeptides having
one or more residues deleted from the carboxy terminus of an
albumin fusion protein of the invention. In particular, C-terminal
deletions may be described by the general formula 1 to n, where n
is any whole integer ranging from 6 to q minus 1, and where q is a
whole integer representing the total number of amino acid residues
in an albumin fusion protein of the invention. Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0088] In addition, any of the above described N- or C-terminal
deletions can be combined to produce a N- and C-terminal deleted
reference polypeptide. The invention also provides polypeptides
having one or more amino acids deleted from both the amino and the
carboxyl termini, which may be described generally as having
residues m to n of a reference polypeptide (e.g., a G-CSF protein,
or a G-CSF protein portion of an albumin fusion protein of the
invention, or serum albumin, or an albumin protein portion of an
albumin fusion protein of the invention, or an albumin fusion
protein, or an albumin fusion protein encoded by a polynucleotide
or albumin fusion construct of the invention) where n and m are
integers as described above. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0089] The present application is also directed to proteins
containing polypeptides at least about 80%, about 85%, about 90%,
about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
about 97%, about 98% or about 99% identical to a reference G-CSF
polypeptide or a reference albumin polypeptide set forth herein, or
fragments thereof. In preferred embodiments, the application is
directed to proteins comprising polypeptides at least about 80%,
about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,
about 95%, about 96%, about 97%, about 98% or about 99% identical
to reference polypeptides having the amino acid sequence of N- and
C-terminal deletions as described above. Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0090] Preferred polypeptide fragments of the invention are
fragments comprising, or alternatively, consisting of, an amino
acid sequence that displays a therapeutic activity and/or
functional activity (e.g. biological activity) of the polypeptide
sequence of the G-CSF protein or serum albumin protein of which the
amino acid sequence is a fragment.
[0091] Other preferred polypeptide fragments are biologically
active fragments. Biologically active fragments are those
exhibiting activity similar, but not necessarily identical, to an
activity of the polypeptide of the present invention. The
biological activity of the fragments may include an improved
desired activity, or a decreased undesirable activity.
[0092] B. Variants
[0093] "Variant" refers to a polynucleotide or nucleic acid
differing from a reference nucleic acid or polypeptide, but
retaining essential properties thereof. Generally, variants are
overall closely similar, and, in many regions, identical to the
reference nucleic acid or polypeptide.
[0094] As used herein, "variant," refers to a G-CSF protein portion
of an albumin fusion protein of the invention, albumin portion of
an albumin fusion protein of the invention, or albumin fusion
protein of the invention differing in sequence from a G-CSF
protein, albumin protein, and/or albumin fusion protein,
respectively, but retaining at least one functional and/or
therapeutic property thereof as described elsewhere herein or
otherwise known in the art. Generally, variants are overall very
similar, and, in many regions, identical to the amino acid sequence
of the G-CSF protein corresponding to a G-CSF protein portion of an
albumin fusion protein, albumin protein corresponding to an albumin
protein portion of an albumin fusion protein, and/or albumin fusion
protein. Nucleic acids encoding these variants are also encompassed
by the invention.
[0095] The present invention is also directed to proteins which
comprise, or alternatively consist of, an amino acid sequence which
is at least about 80%, about 85%, about 90%, about 91%, about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 99% or about 100%, identical to, for example, the amino acid
sequence of a G-CSF protein corresponding to a G-CSF protein
portion of an albumin fusion protein of the invention, albumin
proteins corresponding to an albumin protein portion of an albumin
fusion protein of the invention, and/or albumin fusion proteins.
Fragments of these polypeptides are also provided. Further
polypeptides encompassed by the invention are polypeptides encoded
by polynucleotides which hybridize to the complement of a nucleic
acid molecule encoding an albumin fusion protein of the invention
under stringent hybridization conditions (e.g., hybridization to
filter bound DNA in 6.times.. Sodium chloride/Sodium citrate (SSC)
at about 45 degrees Celsius, followed by one or more washes in
0.2.times.SSC, 0.1% SDS at about 50-65 degrees Celsius), under
highly stringent conditions (e.g., hybridization to filter bound
DNA in 6.times. sodium chloride/Sodium citrate (SSC) at about 45
degrees Celsius, followed by one or more washes in 0.1.times.SSC,
0.2% SDS at about 68 degrees Celsius), or under other stringent
hybridization conditions which are known to those of skill in the
art (see, for example, Ausubel, F. M. et al., eds., 1989 Current
protocol in Molecular Biology, Green publishing associates, Inc.,
and John Wiley & Sons Inc., New York, at pages 6.3.1-6.3.6 and
2.10.3). Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0096] By a polypeptide having an amino acid sequence at least, for
example, 95%-"identical" to a query amino acid sequence, it is
intended that the amino acid sequence of the subject polypeptide is
identical to the query sequence except that the subject polypeptide
sequence may include up to five amino acid alterations per each 100
amino acids of the query amino acid sequence. In other words, to
obtain a polypeptide having an amino acid sequence at least 95%
identical to a query amino acid sequence, up to 5% of the amino
acid residues in the subject sequence may be inserted, deleted, or
substituted with another amino acid. These alterations of the
reference sequence may occur at the amino- or carboxy-terminal
positions of the reference amino acid sequence or anywhere between
those terminal positions, interspersed either individually among
residues in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0097] As a practical matter, whether any particular polypeptide is
at least about 80%, about 85%, about 90%, about 91%, about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or
about 99% identical to, for instance, the amino acid sequence of an
albumin fusion protein of the invention or a fragment thereof (such
as a G-CSF protein portion of the albumin fusion protein or an
albumin portion of the albumin fusion protein), can be determined
conventionally using known computer programs. A preferred method
for determining the best overall match between a query sequence (a
sequence of the present invention) and a subject sequence, also
referred to as a global sequence alignment, can be determined using
the FASTDB computer program based on the algorithm of Brutlag et
al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment
the query and subject sequences are either both nucleotide
sequences or both amino acid sequences. The result of the global
sequence alignment is expressed as percent identity. Preferred
parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0,
k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization
Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap
Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of
the subject amino acid sequence, whichever is shorter.
[0098] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N- and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the query sequence, the percent identity is corrected
by calculating the number of residues of the query sequence that
are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence.
[0099] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C-termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are to made for the purposes of the
present invention.
[0100] The variant will usually have at least about 75% (in other
embodiments at least about 80%, about 85%, about 90%, about 91%,
about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,
about 98% or about 99%) sequence identity with a length of normal
HA or G-CSF protein which is the same length as the variant.
Homology or identity at the nucleotide or amino acid sequence level
is determined by BLAST (Basic Local Alignment Search Tool) analysis
using the algorithm employed by the programs blastp, blastn,
blastx, tblastn and tblastx (Karlin et al., Proc. Natl. Acad. Sci.
USA 87: 2264-2268 (1990) and Altschul, J. Mol. Evol. 36: 290-300
(1993), fully incorporated by reference) which are tailored for
sequence similarity searching.
[0101] The approach used by the BLAST program is to first consider
similar segments between a query sequence and a database sequence,
then to evaluate the statistical significance of all matches that
are identified and finally to summarize only those matches which
satisfy a preselected threshold of significance. For a discussion
of basic issues in similarity searching of sequence databases, see
Altschul et al., (Nature Genetics 6: 119-129 (1994)) which is fully
incorporated by reference. The search parameters for histogram,
descriptions, alignments, expect (i.e., the statistical
significance threshold for reporting matches against database
sequences), cutoff, matrix and filter are at the default settings.
The default scoring matrix used by blastp, blastx, tblastn, and
tblastx is the BLOSUM62 matrix (Henikoff et al., Proc. Natl. Acad.
Sci. USA 89: 10915-10919 (1992), fully incorporated by reference).
For blastn, the scoring matrix is set by the ratios of M (i.e., the
reward score for a pair of matching residues) to N (i.e., the
penalty score for mismatching residues), wherein the default values
for M and N are 5 and -4, respectively. Four blastn parameters may
be adjusted as follows: Q-10 (gap creation penalty); R=10 (gap
extension penalty); wink=1 (generates word hits at every
wink.sup.th position along the query); and gapw=16 (sets the window
width within which gapped alignments are generated). The equivalent
Blastp parameter settings were Q=9; R=2; wink=1; and gapw=32. A
Bestfit comparison between sequences, available in the GCG package
version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and
LEN=3 (gap extension penalty) and the equivalent settings in
protein comparisons are GAP=8 and LEN=2.
[0102] The polynucleotide variants of the invention may contain
alterations in the coding regions, non-coding regions, or both.
Especially preferred are polynucleotide variants containing
alterations which produce silent substitutions, additions, or
deletions, but do not alter the properties or activities of the
encoded polypeptide. Nucleotide variants produced by silent
substitutions due to the degeneracy of the genetic code are
preferred. Moreover, polypeptide variants in which less than 50,
less than 40, less than 30, less than 20, less than 10, or 5-50,
5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or
added in any combination are also preferred. Polynucleotide
variants can be produced for a variety of reasons, e.g., to
optimize codon expression for a particular host (change codons in
the human mRNA to those preferred by a bacterial host, such as,
yeast or E. coli).
[0103] In a preferred embodiment, a polynucleotide of the invention
which encodes the albumin portion of an albumin fusion protein is
optimized for expression in yeast or mammalian cells. In a further
preferred embodiment, a polynucleotide of the invention which
encodes the G-CSF protein portion of an albumin fusion protein is
optimized for expression in yeast or mammalian cells. In a still
further preferred embodiment, a polynucleotide encoding an albumin
fusion protein of the invention is optimized for expression in
yeast or mammalian cells.
[0104] In an alternative embodiment, a codon optimized
polynucleotide which encodes a G-CSF protein portion of an albumin
fusion protein does not hybridize to the wild type polynucleotide
encoding the G-CSF protein under stringent hybridization conditions
as described herein. In a further embodiment, a codon optimized
polynucleotide which encodes an albumin portion of an albumin
fusion protein does not hybridize to the wild type polynucleotide
encoding the albumin protein under stringent hybridization
conditions as described herein. In another embodiment, a codon
optimized polynucleotide which encodes an albumin fusion protein
does not hybridize to the wild type polynucleotide encoding the
G-CSF protein portion or the albumin protein portion under
stringent hybridization conditions as described herein.
[0105] In an additional embodiment, a polynucleotide which encodes
a G-CSF protein portion of an albumin fusion protein does not
comprise, or alternatively consist of, the naturally occurring
sequence of that G-CSF protein. In a further embodiment, a
polynucleotide which encodes an albumin protein portion of an
albumin fusion protein does not comprise, or alternatively consist
of, the naturally occurring sequence of albumin protein. In an
alternative embodiment, a polynucleotide which encodes an albumin
fusion protein does not comprise, or alternatively consist of, the
naturally occurring sequence of a G-CSF protein portion or the
albumin protein portion.
[0106] Using known methods of protein engineering and recombinant
DNA technology, variants may be generated to improve or alter the
characteristics of the polypeptides of the present invention. For
instance, one or more amino acids can be deleted from the
N-terminus or C-terminus of the polypeptide of the present
invention without substantial loss of biological function.
[0107] In preferred embodiments, the variants of the invention have
conservative substitutions. By "conservative substitutions" is
intended swaps within groups such as replacement of the aliphatic
or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of
the hydroxyl residues Ser and Tar; replacement of the acidic
residues Asp and Glu; replacement of the amide residues Asn and
Gln, replacement of the basic residues Lys, Arg, and His;
replacement of the aromatic residues Phe, Tyr, and Trp, and
replacement of the small-sized amino acids Ala, Ser, Thr, Met, and
Gly.
[0108] Guidance concerning how to make phenotypically silent amino
acid substitutions is provided, for example, in Bowie et al.,
"Deciphering the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990), wherein the
authors indicate that there are two main strategies for studying
the tolerance of an amino acid sequence to change.
[0109] The first strategy exploits the tolerance of amino acid
substitutions by natural selection during the process of evolution.
By comparing amino acid sequences in different species, conserved
amino acids can be identified. These conserved amino acids are
likely important for protein function. In contrast, the amino acid
positions where substitutions have been tolerated by natural
selection indicates that these positions are not critical for
protein function. Thus, positions tolerating amino acid
substitution could be modified while still maintaining biological
activity of the protein.
[0110] The second strategy uses genetic engineering to introduce
amino acid changes at specific positions of a cloned gene to
identify regions critical for protein function. For example, site
directed mutagenesis or alanine-scanning mutagenesis (introduction
of single alanine mutations at every residue in the molecule) can
be used. See Cunningham and Wells, Science 244:1081-1085 (1989).
The resulting mutant molecules can then be tested for biological
activity.
[0111] As the authors state, these two strategies have revealed
that proteins are surprisingly tolerant of amino acid
substitutions. The authors further indicate which amino acid
changes are likely to be permissive at certain amino acid positions
in the protein. For example, most buried (within the tertiary
structure of the protein) amino acid residues require nonpolar side
chains, whereas few features of surface side chains are generally
conserved. Moreover, tolerated conservative amino acid
substitutions involve replacement of the aliphatic or hydrophobic
amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl
residues Ser and Thr; replacement of the acidic residues Asp and
Glu; replacement of the amide residues Asn and Gln, replacement of
the basic residues Lys. Arg, and His; replacement of the aromatic
residues Phe, Tyr, and Trp, and replacement of the small-sized
amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino
acid substitution, variants of the present invention include (i)
polypeptides containing substitutions of one or more of the
non-conserved amino acid residues, where the substituted amino acid
residues may or may not be one encoded by the genetic code, or (ii)
polypeptides containing substitutions of one or more of the amino
acid residues having a substituent group, or (iii) polypeptides
which have been fused with or chemically conjugated to another
compound, such as a compound to increase the stability and/or
solubility of the polypeptide (for example, polyethylene glycol),
(iv) polypeptide containing additional amino acids, such as, for
example, an IgG Fc fusion region peptide. Such variant polypeptides
are deemed to be within the scope of those skilled in the art from
the teachings herein.
[0112] For example, polypeptide variants containing amino acid
substitutions of charged amino acids with other charged or neutral
amino acids may produce proteins with improved characteristics,
such as less aggregation. Aggregation of pharmaceutical
formulations both reduces activity and increases clearance due to
the aggregate's immunogenic activity. See Pinckard et al., Clin.
Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:
838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier
Systems 10:307-377 (1993).
[0113] In specific embodiments, the polypeptides of the invention
comprise, or alternatively, consist of, fragments or variants of
the amino acid sequence of an albumin fusion protein, the amino
acid sequence of a G-CSF protein and/or human serum albumin,
wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50
or 50-150, amino acid residue additions, substitutions, and/or
deletions when compared to the reference amino acid sequence. In
preferred embodiments, the amino acid substitutions are
conservative. Nucleic acids encoding these polypeptides are also
encompassed by the invention.
[0114] The polypeptide of the present invention can be composed of
amino acids joined to each other by peptide bonds or modified
peptide bonds, i.e., peptide isosteres, and may contain amino acids
other than the 20 gene-encoded amino acids. The polypeptides may be
modified by either natural processes, such as post-translational
processing, or by chemical modification techniques which are well
known in the art. Such modifications are well described in basic
texts and in more detailed monographs, as well as in a voluminous
research literature. Modifications can occur anywhere in a
polypeptide, including the peptide backbone, the amino acid
side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in
the same or varying degrees at several sites in a given
polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched, for example, as a
result of ubiquitination, and they may be cyclic, with or without
branching. Cyclic, branched, and branched cyclic polypeptides may
result from posttranslation natural processes or may be made by
synthetic methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. (See, for instance, PROTEINS--STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION
OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990);
Rattan et al., Ann. N.Y. Acad. Sci. 663:4862 (1992)).
[0115] C. Functional Activity
[0116] "A polypeptide having functional activity" refers to a
polypeptide capable of displaying one or more known functional
activities associated with the full-length, pro-protein, and/or
mature form of a G-CSF protein. Such functional activities include,
but are not limited to, biological activity, antigenicity [ability
to bind (or compete with a polypeptide for binding) to an
anti-polypeptide antibody], immunogenicity (ability to generate
antibody which binds to a specific polypeptide of the invention),
ability to form multimers with polypeptides of the invention, and
ability to bind to a receptor or ligand for a polypeptide.
[0117] "A polypeptide having biological activity" refers to a
polypeptide exhibiting activity similar to, but not necessarily
identical to, an activity of a G-CSF protein of the present
invention, including mature forms, as measured in a particular
biological assay, with or without dose dependency.
[0118] In preferred embodiments, an albumin fusion protein of the
invention has at least one biological and/or therapeutic activity
associated with the G-CSF protein portion (or fragment or variant
thereof) when it is not fused to albumin.
[0119] The albumin fusion proteins of the invention can be assayed
for functional activity (e.g., biological activity) using or
routinely modifying assays known in the art, as well as assays
described herein. Additionally, one of skill in the art may
routinely assay fragments of a G-CSF protein corresponding to a
G-CSF protein portion of an albumin fusion protein. Further, one of
skill in the art may routinely assay fragments of an albumin
protein corresponding to an albumin protein portion of an albumin
fusion protein, for activity using assays known in the art and/or
as described in the Examples section below.
[0120] For example, in one embodiment where one is assaying for the
ability of an albumin fusion protein to bind or compete with a
G-CSF protein for binding to an anti-G-CSF polypeptide antibody
and/or anti-albumin antibody, various immunoassays known in the art
can be used, including but not limited to, competitive and
non-competitive assay systems using techniques such as
radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitation reactions, immunodiffusion assays, in situ
immunoassays (using colloidal gold, enzyme or radioisotope labels,
for example), western blots, precipitation reactions, agglutination
assays (e.g., gel agglutination assays, hemagglutination assays),
complement fixation assays, immunofluorescence assays, protein A
assays, and immunoelectrophoresis assays, etc. In one embodiment,
antibody binding is detected by detecting a label on the primary
antibody. In another embodiment, the primary antibody is detected
by detecting binding of a secondary antibody or reagent to the
primary antibody. In a further embodiment, the secondary antibody
is labeled. Many means are known in the art for detecting binding
in an immunoassay and are within the scope of the present
invention.
[0121] In a preferred embodiment, where a binding partner (e.g., a
receptor or a ligand) of a G-CSF protein is identified, binding to
that binding partner by an albumin fusion protein which comprises
that G-CSF protein as the G-CSF protein portion of the fusion can
be assayed, e.g., by means well-known in the art, such as, for
example, reducing and non-reducing gel chromatography, protein
affinity chromatography, and affinity blotting. See generally,
Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another
embodiment, the ability of physiological correlates of an albumin
fusion protein to bind to a receptor(s) of the G-CSF polypeptide
corresponding to the G-CSF protein portion of the fusion can be
routinely assayed using techniques known in the art.
[0122] In an alternative embodiment, where the ability of an
albumin fusion protein to multimerize is being evaluated,
association with other components of the multimer can be assayed,
e.g., by means well-known in the art, such as, for example,
reducing and non-reducing gel chromatography, protein affinity
chromatography, and affinity blotting. See generally, Phizicky et
al., supra.
[0123] Immunoassays which can be used to analyze protein binding,
cross-reactivity or identity include, but are not limited to,
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, and
protein A immunoassays, to name but a few. Such assays are routine
and well known in the art (see, e.g., Ausubel et al, eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York, which is incorporated by reference herein in
its entirety).
[0124] Antibodies that bind a G-CSF protein corresponding to the
G-CSF protein portion of an albumin fusion protein may also be
described or specified in terms of their binding affinity for a
given protein or antigen, preferably the antigen which they
specifically bind. Preferred binding affinities include those with
a dissociation constant or Kd less than 5.times.10.sup.-2 M,
10.sup.-2 M, 5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M,
10.sup.-4 M. More preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-5 M,
10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7 M,
10.sup.-7 M, 5.times.10.sup.-8 M or 10.sup.-8 M. Even more
preferred binding affinities include those with a dissociation
constant or Kd less than 5.times.10.sup.-9 M, 10.sup.-9 M,
5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11 M,
10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M,
5.times.10.sup.-13 M, 10.sup.-13 M, 5.times.10.sup.-14 M,
10.sup.-14 M, 5.times.10.sup.-15 M, or 10.sup.-15 M. In addition,
assays described herein and otherwise known in the art may
routinely be applied to measure the ability of albumin fusion
proteins and fragments, variants and derivatives thereof to elicit
biological activity and/or G-CSF activity (either in vitro or in
vivo) related to either the G-CSF protein portion and/or albumin
portion of the albumin fusion protein. Other methods will be known
to the skilled artisan and are within the scope of the
invention.
V. FUSION PROTEINS OF G-CSF AND HAS
[0125] Recombinant human albumin-human granulocyte colony
stimulating factor (rHA-G-CSF) is a G-CSF analogue. Examples of
rHA-G-CSFs are described in U.S. Pat. No. 5,665,863 and in U.S.
Pat. No. 7,041,478, both of which are hereby incorporated by
reference.
[0126] Another example of rHA-G-CSF is Neugranin.TM. ("NEUG")
developed by Teva Biopharmaceuticals USA LTD. NEUG is a fusion
polypeptide with a molecular mass of approximately 85 kDa. NEUG is
a 759 amino acid single chain polypeptide, with residues 1-585
corresponding to the mature form of HSA, and residues 586-759
corresponding to the mature form of human G-CSF. The amino acid
sequence of the NEUG fusion protein is shown in FIG. 1.
VI. PRODUCING THE FUSION PROTEIN
[0127] Exemplary methods of synthetic processes of manufacture of
rHA-G-CSF are described in U.S. patent application Ser. No.
11/929,828 hereby incorporated by reference in its entirety. In
some embodiments, NEUG is produced using a yeast host system (e.g.,
Saccharomyces cerevisiae) genetically engineered to express the
NEUG fusion protein. NEUG is harvested from the fermentation medium
of the yeast culture and purified using methods well known in the
art (e.g., by a series of chromatography and filtration steps, such
as affinity chromatography and ion exchange chromatography).
[0128] In one non-limiting example, a NEUG fusion construct was
developed as follows. The full-length albumin cDNA was isolated
from a human cDNA library in the laboratory of Dr. F. E. Baralle at
the University of Oxford, UK. This clone was sent to Delta
Biotechnology Limited, Nottingham, UK, as the plasmid pAT153ALB. In
addition, the 6-amino acid HSA pro-peptide (RGVFRR (SEQ ID NO: 1))
was modified to facilitate more efficient processing in yeast
(RSLDKR (SEQ ID NO: 2)).
[0129] The NEUG production plasmid, a modified pSAC35-based
expression vector, is based on the 2-.mu. plasmid found in wild
type Saccharomyces cerevisiae. The pSAC35-based expression vector
(see e.g., patents EP 286 424 B, U.S. Pat. No. 5,637,504) contains
the LEU2 gene from Saccharomyces cerevisiae as a selectable marker
that complements the leucine-deficiency of the S. cerevisiae
production host. This production plasmid also contains a strong
yeast promoter, PRB1, and sequences from plasmid pUC9 that permit
cloning and propagation in E. coli. In addition, the plasmid
eliminates the pUC9-derived sequences required for propagation in
E. coli once transformed into yeast. This is accomplished by
flanking FLP recognition targets (FRT) and the expression of the
yeast FLP recombinase from the plasmid once in yeast. Thus, no
bacterial DNA is present in the organism used for production of
NEUG. This is confirmed by rescue and sequence of the 2 .mu.m
plasmid from the yeast after the master cell bank is generated.
[0130] As described above, the NEUG production plasmid, termed
CID1643 (pSAC35:HSA.GCSF(T31-P204)), was derived from the
pSAC35-based expression vector. The region corresponding to
T31-P204 of human G-CSF was amplified by PCR, while adding the
appropriate 5' and 3' restriction sites to permit a seamless fusion
to the 3'-end of the HSA open reading frame.
[0131] NEUG seed vials were used to prepare a cGMP master cell bank
at Human Genome Sciences, Inc., in Rockville, Md. The testing and
characterization of the NEUG master cell bank was undertaken at
Charles River Laboratories (Malvern, Pa., USA) and Lark
Technologies (Houston, Tex., USA) in compliance with the ICH
guideline Q5D (Derivation and Characterization of Cell Substrates
Used for Production of Biotechnological/Biologicals Products).
[0132] A cGMP working cell bank derived from this master cell bank
was subsequently generated and tested at Charles River Laboratories
(Malvern, Pa., USA).
[0133] All media components used in the manufacture of the NEUG
cell line banks were synthetic, biosynthetic or plant derived. No
components of animal or human origin were used during cell line or
cell bank preparation.
[0134] The cell banks is stored at <-135.degree. C. in a
cryopreservation media in pre-sterilized 1.8 mL Nunc polypropylene
tubes with internally threaded caps.
[0135] A non-limiting, exemplary method of isolating, purifying and
preparing the rHSA-G-CSF fusion protein for pharmaceutical use is
shown in FIG. 21. The formulated drug substance is aseptically
filtered using a 0.2 .mu.m filter into autoclaved Teflon bottles.
The liquid filled drug substance is stored frozen at about
-80.degree. C. (nominal value, acceptable range of storage
temperature is about -65.degree. C.).
[0136] To improve the robustness of the formulation for shipping
and storage at clinical sites as well as to provide a stable
product with an expected long shelf life, NEUG may also be
lyophilized by methods well known in the art.
VII. EXEMPLARY CAUSES OF LEUKOPENIA AND NEUTROPENIA
[0137] As described above, leukopenia is a reduction in the
circulating white blood cells (WBC) count and neutropenia is
characterized by a reduction in the blood neutrophil count, often
leading to increased susceptibility to bacterial and fungal
infections. The following is a non-comprehensive list of factors
that can place a human subject at risk of developing leukopenia or
neutropenia: drugs (e.g. phenyloin, chloramphenicol, sulfa drugs,
and chemotherapy); vitamin B12 or folate deficiency; excessive
alcohol consumption; cancer or other diseases which involve the
bone marrow (e.g. aplastic anemia, dysgammaglobulinemia, paroxysmal
nocturnal hemoglobinemia, myelodysplasia, myelodysplastic
syndromes, myelofibrosis, leukemia, myeloma, lymphoma, or
metastatic solid tumors which infiltrate and replace the bone
marrow); viral infections (e.g. influenza, HIV, early-stage
infectious mononucleosis, childhood viral diseases); bacterial
infections (e.g. tuberculosis); radiation; toxins (e.g., benzene
and insecticides); bone marrow failure (e.g. Schwachman-Diamond
syndrome, cartilage-hair hypoplasia, dyskeratosis congenita,
glycogen storage disease type IB); spleen disorder, splenomegaly of
any cause; intrinsic defects in myeloid cells or their precursors;
allergic disorders; autoimmune disease; T-.gamma.
lymphoproliferative disease (T-.gamma. LPD); hemodialysis or
transplantation; toxins.
[0138] Numerous drugs, such as many chemotherapy regimens (e.g.,
cytotoxic chemotherapy regimens), are associated with a high risk
of febrile neutropenia (e.g., >than 20% risk). In some
chemotherapy regimens, the incidence of febrile neutropenia in the
absence of G-CSF treatment is about 40% (e.g., a chemotherapy
regimen of intravenous doxorubicin and docetaxel). Non-limiting
examples of various cancers and treatment regimens associated with
febrile neutropenia risk are provided below in Table 1. In some
embodiments, the HSA-G-CSF fusion protein of FIG. 1 is administered
to a patient to prevent, treat or ameliorate neutropenia associated
with the administration of such drug therapies.
TABLE-US-00001 TABLE 1 Exemplary cancers and treatment regimens
associated with febrile neutropenia Cancer Treatment Bladder Cancer
MVAC (methotrexate, vinblastine, doxorubicin, cisplatin)
(neoadjuvant, adjuvant, metastatic) Breast Cancer Docetaxel +
trastuzumab (metastatic or relapsed) Dose dense AC-T (doxorubicin,
cyclophosphamide, paclitaxel) (adjuvant) AT (doxorubicin,
paclitaxel) (metastatic or relapsed) AT (doxorubicin, docetaxel)
(metastatic or relapsed) TAC (docetaxel, doxorubicin,
cyclophosphamide) (adjuvant) Esophageal and
Docetaxel/cisplatin/fluorouracil Gastric Cancer Non-Hodgkin's ICE
(ifosfamide, carboplatin, etoposide) (Diffuse Large B-Cell Lymphoma
Lymphoma, Peripheral T cell Lymphomas, 2nd line, salvage) RICE
(rituximab, ifosfamide, carboplatin, etoposide) CHOP-14
(cyclophosphamide, doxorubicin, vincristine, prednisone) MINE
(mesna, ifosfamide, novantrone and etoposide) (Diffuse Large B-
Cell Lymphoma, Peripheral T cell Lymphomas, 2nd line, refractory)
DHAP (dexamethasone, cisplatin, cytarabine) (Peripheral T cell
Lymphomas, Diffuse Large B-Cell Lymphoma, 2nd line) ESHAP
(etoposide, methylprednisolone, cisplatin, cytarabine) (Diffuse
Large B-Cell Lymphoma, Peripheral T cell Lymphoma, 2nd line,
recurrent) BEACOPP (bleomycin, etoposide, doxorubicin,
cyclophosphamide, vincristine, procarbazine, prednisone) HyperCVAD
+ Rituximab (cyclophosphamide, vincristine, doxorubicin,
dexamethasone + rituximab) (Burkitt's Lymphoma) Melanoma
Dacarbazine-based combination (dacarbazine, cisplatin, vinblastine)
(advanced, metastatic, or recurrent) Dacarbazine-based combination
with IL-2, interferon alfa (dacarbazine, cisplatin, vinblastine,
IL-2, interferon alfa) (advanced, metastatic, or recurrent)
Myelodysplastic Decitabine syndrome Ovarian Cancer Topotecan
Paclitaxel Docetaxe1 Pancreatic Cancer Gemcitabine/docetaxel
Sarcoma MAID (mesna, doxorubicin, ifosfamide, dacarbazine)
Doxorubicin Small Cell Lung Topotecan Cancer Testicular Cancer VeIP
(vinblastine, ifosfamide, cisplatin) VIP (etoposide, ifosfamide,
cisplatin) BEP (bleomycin, etoposide, cisplatin) TIP (paclitaxel,
ifosfamide, cisplatin)
[0139] Cytotoxic treatment regimens for small cell lung carcinoma
e.g., cisplatin plus etoposide, as well as CAE, are also associated
with febrile neutropenia.
[0140] Various netropenias are know, and in some embodiments, the
HSA-G-CSF fusion protein of FIG. 1 is used to prevent, treat or
ameliorate one or more neutropenias, including, but not limited to
chemotherapy induced neutropenia, primary neutropenia, acute
neutropenia, severe chronic neutropenia (SCN), severe congenital
neutropenia (Kostmann's syndrome), severe infantile genetic
agranulocytosis, benign neutropenia, cyclic neutropenia, chronic
idiopathic neutropenia, secondary neutropenia, syndrome associated
neutropenia, or immune-mediated neutropenia.
VIII. EXPERIMENTAL EXAMPLES
[0141] The following examples are given to illustrate the present
invention. It should be understood, however, that the invention is
not to be limited to the specific conditions or details described
in these examples. All publicly available documents referenced
herein, including but not limited to US patents, are hereby
incorporated by reference.
[0142] In the following non-limiting examples, Neugranin.TM.
("NEUG") was tested on cells and in mice and monkeys, and was also
used to prevent, treat or ameliorate neutropenia and/or leukopenia
in human subjects caused by drug therapy (e.g., chemotherapy) for
the treatment of breast cancer.
[0143] NEUG is a G-CSF analog with a reduced plasma clearance rate
attributed to fusion of the active G-CSF moiety to human serum
albumin. The resulting fully recombinant protein retains the
pharmacologic activity of G-CSF in vivo, i.e. it stimulates
neutrophil and hematopoietic stem cell mobilization from the bone
marrow to the peripheral blood stream. The activity of this protein
was assessed in mice and in monkeys (see Experimental Examples,
below). The onset of the observed increases in neutrophil and
hematopoietic progenitor counts was rapid and persisted for several
days after a single administration. The effects were
dose-dependent, with higher doses including a greater magnitude and
duration of the response to NEUG compared with lower does.
Clearance of NEUG was slower than Neupogen.RTM. (filgrastim). The
kinetics of blood neutrophilia following single and repeat
administration of NEUG was nearly identical to that observed in
animals treated with Neulasta.RTM. (pegfilgrastim). Because NEUG is
4.5-fold larger than Neulasta.RTM. (pegfilgrastim) (by mass), a
larger dose (by weight) but an equimolar dose of NEUG can be used
to achieve similar effect in vivo. In monkeys, equimolar doses of
NEUG had equivalent effect to pegfilgrastim, and in mice a 1.5-fold
higher does of NEUG was shown to achieve equivalent AUC.sub.ANC. In
these studies, 1 mg of Neulasta.RTM. (pegfilgrastim) was equivalent
in effect to 4.5-7.7 mg of NEUG. For context, the No Adverse Effect
Level ("NOAEL") of NEUG demonstrated in monkey was greater than 1
mg/kg/week. A 1 mg/kg dose in monkeys resulted in an exposure (Cmax
and AUC) .about.12-fold higher than was observed in human patients
receiving 0.45 mg/kg NEUG. Thus, the NOAEL demonstrated in monkey
is higher than the dose range used for clinical evaluation of NEUG
(see below).
[0144] In aggregate, the studies show that equimolar doses of NEUG
provide similar pharmacological effect to Neulasta.RTM.
(pegfilgrastim) and have a similar effects on granulocyte
populations in human.
[0145] A. In Vitro and In Vivo Studies of NEUG
[0146] Results of in vitro and in vivo studies of NEUG are
summarized as follows and are detailed in the Experimental
Examples, below.
In vitro pharmacology studies have shown the following: [0147] 1.
NEUG induces proliferation of NFS-60 cells in a dose-dependent
fashion. [0148] 2. NEUG is .about.3-fold less potent than
Neupogen.RTM. (filgrastim) in vitro. [0149] 3. NEUG is equipotent
with Neulasta.RTM. (pegfilgrastim) when evaluated on molar basis (1
mg of Neulasta.RTM. (pegfilgrastim) is equivalent in effect to 4.5
of NEUG). In vivo pharmacology studies have demonstrated the
following properties of NEUG: [0150] 1. NEUG was well-tolerated in
mice and cynomolgus monkeys. [0151] 2. In mice, a single
administration of NEUG induces a dose-dependent, rapid and
prolonged increase of neutrophils and hematopoietic progenitor
cells in the peripheral blood. When compared with current marketed
G-CSF products, the rise in neutrophil and progenitor cell count
was longer in duration than that achieved by an equimolar dose of
Neupogen.RTM. (filgrastim) and similar in duration and magnitude to
an equimolar dose of Neulasta.RTM. (pegfilgrastim). [0152] 3. In
mice, equivalent and AUC.sub.ANC were achieved with a 7.7-fold
higher milligram dose of NEUG than Neulasta.RTM. (pegfilgrastim).
[0153] 4. In 5-FU-induced neutropenic mice, single injections of
equimolar doses of NEUG and Neulasta.RTM. (pegfilgrastim)
effectively accelerated neutrophil recovery with similar kinetics
and magnitude of effect. [0154] 5. Single and repeat (once weekly)
doses of NEUG and Neulasta.RTM. (pegfilgrastim) induce similar
increases in peripheral neutrophil count in monkeys. At equimolar
doses, both the magnitude and the duration of neutrophil elevation
in monkeys were similar in animals treated with Neulasta.RTM.
(pegfilgrastim) and animals treated with NEUG. [0155] 6. In both
mice and monkeys, NEUG has a slower clearance, a longer terminal
half-life, and a greater mean residence time than Neupogen.RTM.
(filgrastim) when administered IV or SC. [0156] 7. In cynomolgus
monkeys, the terminal half-life of NEUG (12.6 hours) is
approximately 33% longer than that of Neulasta.RTM. (pegfilgrastim)
(9.49 hours) following SC injection and the clearance over
bioavailability ("CL/F") of NEUG is about half that of
Neulasta.RTM. (pegfilgrastim). [0157] 8. The clearance of both NEUG
and Neulasta.RTM. (pegfilgrastim) are slower in mice that have
undergone 5-FU-induced cytopenia than in normal mice suggesting
that receptor-mediated clearance of both proteins contributes to
their clearance. [0158] 9. Renal excretion (assessed in rats)
contributes significantly to the clearance of Neupogen.RTM.
(filgrastim), has a small effect on Neulasta.RTM. (pegfilgrastim),
and is without substantial contribution to the clearance of NEUG.
[0159] 10. NEUG, a human protein composed of human serum albumin
and human colony stimulation factor, is immunogenic in mice and
cynomolgus monkeys. Neulasta.RTM. (pegfilgrastim) induced a similar
incidence and titer of antibodies in monkeys. Antibodies from some
monkeys treated with NEUG and Neulasta.RTM. (pegfilgrastim) were
neutralizing in vitro and for both NEUG and Neulasta.RTM.
(pegfilgrastim), the neutrophil response diminished with repeat
exposure, through antibody positive animals had basal levels of
neutrophils similar to antibody negative animals during
recovery.
[0160] Taken together, the in vitro and in vivo pharmacological
properties of NEUG suggest that it acts in a manner similar to
Neupogen.RTM. (filgrastim) and Neulasta.RTM. (pegfilgrastim) in
that it similarly promotes mobilization of neutrophils and
hematopoietic cells into the bloodstream. Non-limiting in vitro and
in vivo Experimental Examples are provided below.
Example 1
NFS-60 Cell Proliferation
[0161] NSF-60 is a cell line routinely used in bioassay for the
measurement of G-CSF activity. This cell line increases
proliferation rate in response to G-CSF. The relative potency of
recombinant C-CSF (Neupogen.RTM., filgrastim) and NEUG were
compared.
[0162] To measure the effectiveness of NEUG and Neupogen.RTM.
(filgrastim) in stimulation of NFS-60 cell proliferation,
.sup.3H-thymidine incorporation was measured following 24-hours of
exposure to a range of concentrations of these analogs. EC.sub.50
values were obtained and expressed in units of mass (ng/ml).
[0163] Briefly, 1.times.10.sup.5 NFS-60 cells/well were seeded in
96-well plate in a final volume of 200 .mu.l of complete medium
containing the indicated amount of NEUG (also termed Albugranin) or
Neupogen.RTM. (filgrastim). All samples were run in triplicate. The
cells were incubated at 30.degree. C. for 24 hours and pulsed
during the last 4 hours with 0.5 .mu.Ci .sup.3H-thymidine/well.
Incorporation of thymidine was used as a measure of proliferation.
(FIG. 8).
[0164] NEUG and Neupogen.RTM. (filgrastim) stimulated proliferation
in a dose-dependent fashion. In this assay, Neupogen.RTM.
(filgrastim) was 15-fold more potent that NEUG when compared on a
mass basis. NEUG is .about.4.5 fold larger (in mass) than
Neupogen.RTM. (filgrastim), thus expressed on a molar basis, in
this assay, NEUG was .about.3-fold less potent than Neupogen.RTM.
(filgrastim) in vitro.
[0165] Neulasta.RTM. (pegfilgrastim) is vialed based on the weight
of recombinant G-CSF, the mass of the polyethylene glycol
modification is not included in dosage calculations. NEUG is
4.5.times. larger than recombinant G-CSF, because of the mass
contribution from HSA, and thus to compare the ability of NEUG and
Neulasta.RTM. (pegfilgrastim) to induce NFS-60 cell proliferation,
a titration of equimolar doses of NEUG and Neulasta.RTM.
(pegfilgrastim) were compared and EC.sub.50s were expressed as a
molar concentration. Briefly, 1.times.10.sup.5NFS-60 cells/well
were seeded in 96-well plate in a final volume of 200 .mu.l of
complete medium containing the indicated amount of NEUG
(Albugranin) or Neulasta.RTM. (pegfilgrastim). All samples were run
in triplicate. The cells were incubated at 37.degree. C. for 20
hours and pulsed during the last 4 hours with 0.5 .mu.Ci
.sup.3H-thymidine/well. Incorporation of thymidine was used as a
measure of proliferation. Results are shown in FIG. 9.
Example 2
Comparison of NEUG and Neupogen.RTM. (Filgrastim) in BDF-1 Mice
[0166] The objective of this study was to assess the effect of
single subcutaneous doses of NEUG on peripheral blood neutrophil
and hematopoietic stem cells in BDF-1 mice. BDF-1 mice were
injected subcutaneously ("SC") with a single administration of NEUG
at 3 dose levels (0.25 mg/kg, 1.25 mg/kg or 5.0 mg/kg) or
Neupogen.RTM. (filgrastim) at 2 dose levels (0.25 mg/kg and 1.25
mg/kg). Peripheral granulocytes (Gr.1+) and hematopoietic
progenitor cells (c-kit+) were quantified by flow cytometry daily
from day 1 until day 5 and were compared to the levels obtained in
vehicle treated animals.
[0167] Both NEUG and Neupogen.RTM. (filgrastim) caused an elevation
in peripheral neutrophil counts when compared to vehicle treated
animals, but the kinetics and the magnitude of the responses were
different (FIG. 10). In the Neupogen.RTM. (pegfilgrastim) groups, a
maximum 3-fold increase in neutrophil count occurred on day 1 and
neutrophils returned to normal levels by day 2. In contrast, while
a single administration of NEUG elevated neutrophil counts to a
similar extent as comparable doses of Neupogen.RTM. (filgrastim),
neutrophil counts continued to rise in the NEUG groups peaking with
a kinetic and magnitude that was dose dependent. Doses of 0.25,
1.25 and 5.0 mg/kg NEUG resulted in peak neutrophil counts 5.4, 10
and 24 fold over those obtained in vehicle treated animals. The
lower two doses caused a peak elevation in neutrophil count on day
2, while the highest dose tested resulted in a peak on day 4.
Neutrophils returned to normal levels on days 3, 4 and 5 for NEUG
at 0.25, 1.25 and 5.0 mg/kg respectively (FIG. 10). As shown in
FIG. 10, the NEUG induced increases in peripheral blood neutrophils
were of greater magnitude and longer duration than those induced by
Neupogen.RTM. (filgrastim).
[0168] The results of NEUG and Neupogen' (filgrastim) treatment on
peripheral hematopoietic (c-kit+) stem cell counts in this study
were very similar to those obtained for peripheral neutrophils
(FIG. 11). The effect of NEUG was dose-dependent, similar to
comparable doses of Neupogen.RTM. (filgrastim) on day 1, but
continued to rise on days 2-4 with all treatment groups returning
to vehicle-defined baseline by day 5. As shown in FIG. 11, the
NEUG-induced increases in c-kit+ cells were of greater magnitude
and longer duration than those induced by Neupogen.RTM.
(filgrastim).
Example 3
Comparison of NEUG and Neulasta.RTM. (Pegfilgrastim) in BDF-1
Mice
[0169] The objective of this study was to compare the effect of
single subcutaneous (SC) injections of NEUG and Neulasta.RTM.
(pegfilgrastim) on peripheral blood neutrophils and hematopoietic
progenitor cells in peripheral blood of BDF-1 mice. This was
evaluated by injecting BDF-1 mice (n=5) with a single dose of NEUG
at 5 or 10 mg/kg. The effect of NEUG was compared with the effect
of equimolar doses of pegfilgrastim (Neulasta.RTM.) (1.12 mg/kg and
2.24 mg/kg) given as a single administration. These two doses of
Neulasta.RTM. (pegfilgrastim) and NEUG are approximately
equimolar.
[0170] Results are shown in FIG. 12. A single administration of
NEUG (5 and 10 mg/kg) or Neulasta.RTM. (1.12 mg/kg and 2.24 mg/kg)
effectively increased the number of peripheral granulocytes and
hematopoietic progenitor cells in BDF-1 mice. In this experiment
the maximum mobilization of peripheral granulocytes occurred on day
3 for NEUG at 5 mg/kg and on day 4 for NEUG at 10 mg/kg.
Granulocytes returned to normal levels on day 6 post-NEUG
treatment. In mice administered with a single dose of Neulasta.RTM.
(pegfilgrastim), the maximum mobilization of granulocytes occurred
on day 4. ANC in mice receiving Neulasta.RTM. (pegfilgrastim) at
2.25 mg/kg was still significantly (p=0.036) higher than the
baseline on day 6 post drug administration, while the absolute
neutrophil count (ANC) in mice receiving 1.12 mg/kg returned to
normal on day 6 (FIG. 12A).
[0171] To evaluate relative potency in mice, areas under the PD
curves (AUC.sub.ANC) versus molar equivalent dose (nmol/kg) were
calculated (FIG. 12B). The lines for the dose response are
parallel, suggesting that AUC.sub.ANC provides an appropriate means
of comparison in this model. AUC.sub.ANC was clearly dose-dependent
for both G-CSF analogs. In this experiment, the relative potency of
Neulasta to NEUG on weight bases was 7.7. That is, 1 mg or
Neulasta.RTM. (pegfilgrastim) (as dosed in clinic on the bases of
rhG-CSF weight) is equivalent in effect to 7.7 mg of NEUG. This is
.about.1.5 fold higher than the 4.5 fold molecular weight
difference between NEUG and Neulasta.RTM. (pegfilgrastim).
[0172] A single administration of NEUG or Neulasta.RTM.
(pegfilgrastim) significantly (p<0.05) increased the total
number of hematopoietic progenitors in peripheral blood (FIG. 15).
The maximum mobilization of hematopoietic progenitor cells occurred
on day 4 in both NEUG and Neulasta.RTM. (pegfilgrastim)-treated
groups. NEUG at 10 mg/kg and Neulasta at 1.12 and 2.24 mg/kg
induced a similar increase in c-kit+ cells (p<0.0001). A 5 mg/kg
dose of NEUG resulted in statistically significant (p<0.0001)
increased in c-kit+ cells compared to HSA control, however, this
increase was about 50% less than that observed in both
Neulasta.RTM. (pegfilgrastim) groups and appeared sub-maximal since
a 2-fold higher dose resulted in an increase in maximal c-kit+
cells count (FIG. 15).
Example 4
Comparison of NEUG and Neulasta.RTM. (Pegfilgrastim) in 5-FU
Induced Neutropenic BDF-1 Mice
[0173] G-CSF products are used clinically to accelerate the
recovery of neutrophils after myelosuppressive chemotherapy. The
objective of this study was to compare the effect of single
subcutaneous (SC) injection of NEUG and Neulasta.RTM.
(pegfilgrastim) on neutrophil recovery in a model where neutropenia
was induced by a sub-lethal dose of 5-FU (150 mg/kg). BDF1 mice
were given a single administration of NEUG at 5 or 10 mg/kg. The
effect of NEUG was compared with the effect of a single
administration of Neulasta (1.12 mg/kg--equimolar dose to NEUG at 5
mg/kg). Both agents were given 1 day after a single dose of 5-FU.
The number of peripheral blood neutrophils was determined daily
from day 6 until day 10. In this period of time, mice receiving
5-FU were characterized by a neutrophil nadir followed by a slow
recovery phase. The experiment was designed to determine the
effects of NEUG on the time and magnitude of neutrophil
recovery.
[0174] Mice injected with vehicle control or HSA 1 day post 5-FU
administration reached a neutropenic nadir by day 6 (FIG. 16).
Neutrophil levels began to recover by day 10. In contrast, recovery
from neutropenia was accelerated when mice were treated with either
NEUG or Neulasta on day 1 post 5-FU administration. Treatment with
either agent at all dose levels resulted in statistically
significant increases in neutrophil count compared with the vehicle
control. On day 9 the effect of NEUG given at 5 mg/kg was lower
(p=0.0048) compared with the effect achieved by an equimolar dose
of Neulasta (1.12 mg/kg). However, by day 10 both agents caused
similar increases in the total peripheral neutrophils.
[0175] To summarize the mouse study data, a single administration
of NEUG to normal mice effectively induced a dose-dependent, rapid
and prolonged increase of neutrophils (Gr.1+ cells) and
hematopoietic progenitor cells (c-kit+) in the peripheral blood.
The response to NEUG was similar to that induced by Neulasta.RTM.
(pegfilgrastim), however in this study the maximal response to NEUG
was slightly delayed relative to Neulasta.RTM. (pegfilgrastim). A
single administration of Neupogen.RTM. (filgrastim) elicited only a
transient increase in neutrophil and hematopoietic progenitor cell
count in peripheral blood. Using a clinically relevant mouse model
of cytopenia in which a sub-lethal dose of 5-FU injected IP induced
myelosuppression and peripheral neutropenia, a single
administration of NEUG or Neulasta.RTM. (pegfilgrastim) effectively
enhanced neutrophil recovery.
Example 5
NEUG Test in Cynomolgus Monkeys
[0176] Cynomolgus monkeys were chosen to determine the effects of
repeated administration of NEUG. Two monkey studies were performed
with serial evaluation of hematology after repeated administrations
of NEUG: a 2-week pharmacology study comparing subcutaneous doses
of NEUG and Neupogen.RTM. (filgrastim), and a longer (5 month)
immunogenicity study comparing the effect of subcutaneous and
intravenous NEUG with subcutaneous Neulasta.RTM. (filgrastim). Both
studies show that NEUG causes a prolonged elevation in peripheral
blood neutrophils in monkeys with a potency and pharmacodynamic
profile similar to Neulasta.RTM. (pegfilgrastim).
Example 6
2-Week Pharmacology Study of NEUG in Monkeys
[0177] To evaluate the pharmacodynamics of NEUG in monkeys, a
2-week repeat dose study was performed with hematology parameters
as an efficacy endpoint. Twenty experimentally naive male and
female cynomolgus monkeys were randomized into 5 treatment groups
of 2 male and 2 female monkeys each. Monkeys were injected
subcutaneously ("SC") in the mid-scapular region with vehicle,
NEUG, or Neupogen.RTM. (filgrastim). During the 14 day treatment
phase of the study, vehicle was administered every 4 days (Q4D),
NEUG was administered at 25 .mu.g/kg every 4 days (Q4D), or 100
.mu.g/kg every 4 or 7 days (Q4D or Q7D, respectively), and
Neupogen.RTM. (filgrastim) was administered at 5 .mu.g/kg
daily.
[0178] NEUG was well tolerated by cynomolgus monkeys at 25 .mu.g/kg
or 100 .mu.g/kg administered as frequently as every 4 days, and
resulted in no adverse effects. The hematologic changes primarily
consisted of NEUG-induced increases in peripheral blood
neutrophils, with a less prominent increase in peripheral blood
monocytes. The increase in neutrophils peaked 24 hours following SC
administration of 100 .mu.g/kg (FIG. 17). Administration of NEUG at
25 .mu.g/kg every 4 days, or of 5 .mu.g/kg Neupogen.RTM.
(filgrastim) daily, resulted in moderate increases in neutrophils
that reached significance when compared with vehicle during the
second week of administration. All hematology changes attributed to
NEUG completely reversed during the 2-week treatment-free recovery
period.
Other Observations
[0179] Monocyte numbers are reported to increase in the periphery
in response to G-CSF, but to a lesser degree than is observed with
neutrophils. In this study, only NEUG at 100 .mu.g/kg administered
every 4 days induced increases in absolute numbers of monocytes.
The absolute number of peripheral blood lymphocytes were not
affected by treatment with either NEUG or Neupogen.RTM.
(filgrastim).
Example 7
Comparison of IV and SC NEUG with SC Neulasta.RTM. (Pegfilgrastim)
in Monkeys
[0180] A non-GLP repeat dose administration study of NEUG in
cynomolgus monkeys was conducted with the primary objective of
assessing immunogenicity (Covance Study No. 6962-129). Hematology
parameters were evaluated as a study endpoint and this study also
provides useful pharmacology information in comparing equimolar
doses of NEUG and Neulasta.RTM. (pegfilgrastim). Both NEUG and
Neulasta were administered once weekly for 3 weeks. FIG. 18
illustrates the ANC following each of the first 3 dose
administrations of NEUG or Neulasta.RTM. (pegfilgrastim).
[0181] In this study, Neugranin administered SC and IV and
Neulasta.RTM. (pegfilgrastim), administered SC at an equimolar
dose, resulted in significant (<0.0001 compared to vehicle)
elevation of peripheral blood neutrophils. Both the magnitude and
the kinetics of neutrophil response were nearly identical among
these three groups.
Example 8
Pharmacokinetics
[0182] The pharmacokinetics of NEUG were evaluated in normal BDF-1
mice following a single IV or SC injection, in 5-FU-treated,
neutropenic BDF-1 mice following SC injection, in nephrectomized
rats following IV injection, and in cynomolgus monkeys following
single and multiple IV and SC injections. In addition, comparisons
were made to the PK of rhG-CSF (Neupogen.RTM.) and pegylated
rhG-CSF (Neulasta.RTM.). These studies are summarized below.
[0183] In all studies, plasma NEUG concentration was measured by
"sandwich" ELISA with a G-CSF capture and HSA detection. This assay
format allows intact NEUG to be quantified without interference or
cross reactivity with endogenous G-CSF and albumin. The
pharmacokinetics studies are summarized in tabular form in FIG.
19.
[0184] In these studies, NEUG has a slower clearance and longer
terminal half-life and mean residence time (MRT) than Neupogen.RTM.
(filgrastim) when administered IV or SC to BDF-1 mice. The
clearance of NEUG is approximately 8 times slower than the
clearance of Neupogen.RTM. (filgrastim) and the MRT (11.2-20.7
hours) is approximately 4 times longer. The clearance of both NEUG
and Neulasta.RTM. (pegfilgrastim) are slower in mice that have
undergone 5-FU induced cytopenia than in normal mice. This is most
likely due to the smaller number of neutrophils (following 5-FU
treatment), which play a role in the clearance of G-CSF. In
cynomolgus monkeys, the MRT for NEUG is 17.9-27.2 hours. In
addition, Cmax following the last of 5 weekly SC doses appears to
decrease compared with the Cmax following the first dose. The SC
bioavailability of NEUG in cynomolgus monkeys is approximately 22%.
In cynomolgus monkeys, the terminal half-life of NEUG (12.6 hours)
is approximately 33% longer than that of Neulasta.RTM.
(pegfilgrastim) (9.49 hours) following SC injection. Renal
clearance does not appear to play a significant role in the
elimination of NEUG (determined in rats).
Example 9
Non-Clinical Toxicology Summary
[0185] NEUG was well tolerated in mice and monkeys. There were no
adverse finding in monkeys administered NEUG subcutaneously at 100,
500, or 1000 .mu.g/kg/dose once weekly for 4 weeks. Pharmacodynamic
responses to NEUG treatment were observed after multiple SC or IV
dose administrations in cynomolgus monkeys, and were consistent
with previously reported effects of G-CSF. NEUG consistently
induced a marked and dose-dependent leukocytosis and neutrophilia,
with less pronounced increases in monocytes, eosinophils and
basophils, and inconsistent increases in lymphocytes. A no
observable effect level (NOEL) for NEUG in monkeys was not
identified in the GLP or non-GLP studies, and is therefore
considered to be less than 25 .mu.g/kg/dose for subcutaneous
administration. No adverse effects were observed in NEUG-treated
monkeys, therefore the no observable adverse effect level (NOAEL)
for subcutaneous administration of NEUG in monkeys is greater than
1000 .mu.g/kg/dose. Additional findings consistent with the
pharmacology of NEUG included: increased splenic weight,
microscopic evidence of myeloid hyperplasia and leukocytosis.
[0186] Non-clinical safety studies are summarized in the tables of
FIG. 20.
Example 10
Immunogenicity
[0187] NEUG could (in theory) induce an immune response in patients
that was neutralizing to G-CSF. Antibodies to HSA are also
possible, though their clinical significance is uncertain given the
extremely high concentration of HSA in the blood (40 mg/mL). A
series of highly sensitive assays able to detect antibodies to all
components of NEUG was used to assess immunogenicity in man.
[0188] To determine the safety and toxicology for NEUG,
immunogenicity was assessed in several studies. These studies
demonstrate that human G-CSF (Neulasta.RTM., pegfilgrastim), human
albumin, and NEUG are all immunogenic in monkeys. In an
immunogenicity study that included a Neulasta.RTM. (pegfilgrastim)
treatment arm, the majority of animals treated with weekly IV or SC
doses of NEUG developed antibodies to Neulasta.RTM.
(pegfilgrastim). Antibodies to NEUG (or Neulasta.RTM.
pegfilgrastim) were first detected on or after day 22 (following
the 3rd weekly dose). In many cases, these antibodies had a
neutralizing effect in an in vitro assay, though the presence of
neutralizing antibodies did not cause neutropenia and did not
prevent the pharmacological effects of NEUG or Neulasta.RTM.
(pegfilgrastim) in monkeys. Furthermore, following a non-dosing
periods of 2 weeks and 2 months, ANCs in all groups were within a
normal range and there was no significant difference in ANC
profiles regardless of antibody status.
[0189] Human albumin, like most human proteins, is immunogenic in
animals. Experience with other albumin fusion proteins has
demonstrated that immunogenicity in monkeys is not predictive of
the incidence (or consequence) of immunogenicity in man. For
example, Albuferon.RTM. (a fusion protein composed of human serum
albumin and interferon-alpha) was highly immunogenic in monkeys
(10/12 monkeys positive for antibody following a single injection)
and the immune response was both neutralizing and substantially
impacted exposure. In contrast, in a recent 458 patient Phase 2
study of Albuferon.RTM., the rate of emergent antibodies was very
low and was significantly lower in the Albuferon.RTM. treatment
groups (3%) compared with a pegylated interferon treatment group
(18%) through the first 12 weeks of treatment. Furthermore,
antibodies were without apparent consequence.
[0190] Antibodies to NEUG have not been observed in human patients
receiving up to 3 doses of NEUG (see e.g., section Phase I.5.c and
Phase II.5.e, below). With regard to NEUG risk assessment,
available human and animal data suggest that neutralization of
G-CSF would not preclude response to pharmacologic doses of G-CSF,
nor would it preclude normal response to challenge by an infectious
agent. In mouse models in which G-CSF is eliminated it has been
shown that neutrophilia can still develop in response to challenge
by an infectious agent, suggesting redundancies in the
granulopoietic system. In addition, there are reports of humans
with auto-antibodies against G-CSF in cases of Felty's syndrome and
systemic lupus erythematosus. These patients develop neutropenia;
however, treatment with G-CSF or GM-CSF remains effective in the
majority of patients.
[0191] In summary, the in vitro and in vivo data and the
pharmacokinetic characteristics of NEUG support NEUG use as a
single dose prophylactic against febrile neutropenia in patients
undergoing myelosuppressive anti-tumor therapy. Its ability to
induce high levels of hematopoietic progenitors in peripheral blood
following a single dose may also be beneficial in patients or
donors for both autologous and allogenic hematopoietic stem cell
transplantation.
[0192] B. Human Studies of NEUG
[0193] The following examples are provided in two main sections
entitled "Phase I" and "Phase II." Each phase includes two parts,
Part A and Part B. The Phase I and Phase II examples are summarized
in Table 2 below.
TABLE-US-00002 TABLE 2 Summary of human clinical NEUG studies
(Part)/Tumor No. of Trial Type Objective Chemo. Subjects Treatment
Arms Phase I (A)/breast Initial dose-finding none 13 50, 150, 300,
450 .mu.g/kg in absence of NEUG chemotherapy (B)/breast Initial
dose-finding Doxorubicin 51 300 or 450 .mu.g/kg in presence of
Docetaxel NEUG vs. 6 mg chemotherapy 2 Cycles pegfilgrastim Phase
II (A)/breast Dose-finding for Doxorubicin 78 30, 40, 50 mg fixed
doses of Docetaxel NEUG vs. 6 mg Neugranin 4 Cycles pegfilgrastim
(B)/breast Demonstration of Doxorubicin 256 40 and 50 mg
non-inferiority of Docetaxel NEUG vs. 6 mg NEUG vs 4 Cycles
pegfilgrastim pegfilgrastim
[0194] Each Phase is divided into five sections: 1) objectives, 2)
patient characteristics, 3) study agent, 4) study characteristics,
and 5) results of Parts A and B.
Example 11
Phase I
[0195] 1. Objective
[0196] The Phase IA/B, IIA/B study was performed to evaluate the
safety, tolerability, immunogenicity, pharmacokinetics and
pharmacodynamics of subcutaneously administered Neugranin.TM.
("NEUG") (recombinant human albumin-human granulocyte colony
stimulating factor) in subjects receiving myelosuppressive
chemotherapy (doxorubicin/docetaxel).
[0197] For Phase I, the primary study objectives were to assess the
safety profile of NEUG given subcutaneously over a range of
potential therapeutic doses compared to pegfilgrastim by measuring
the frequency, severity, and duration of treatment-emergent adverse
events and correlating them with the time and dose of NEUG
administration.
[0198] Secondary study objectives were to assess the
pharmacokinetics and immunogenicity of NEUG, and to compare the
effect of NEUG administration on the incidence, severity and
duration of neutropenia to pegfilgrastim in patients receiving
doxorubicin/docetaxel.
[0199] Phase I was performed as two parts, Part A and Part B as
noted in Table 2 above.
[0200] 2. Patient Characteristics
[0201] For Phase I, patients were screened based on the following
characteristics or parameters:
Inclusion:
[0202] 1. Patients with histologically-confirmed breast cancer
scheduled to receive doxorubicin and docetaxel. [0203] 2. 18 years
of age or older. [0204] 3. Adequate hematologic function. [0205] 4.
ANC>1500/mm.sup.3 [0206] 5. Platelets>100,000/mm.sup.3 [0207]
6. Adequate hepatic and renal function: [0208] 7. Serum
creatinine<2.0.times.upper limit normal [0209] 8. Total
bilirubin within normal limits (WNL) for local laboratory [0210] 9.
Serum transaminases (SGOT/SGPT)<1.5.times.upper limit normal
[0211] 10. Alkaline phosphatase<2.5.times.upper limit normal
[0212] 11. ECOG performance status 0 or 1. [0213] 12. Eligible to
receive doxorubicin based on a left ventricular ejection fraction
(LVEF) within normal limits. [0214] 13. Have the ability to
understand the requirements of the study, provide written informed
consent (including consent for use and disclosure of
research-related health information) and comply with the study
protocol procedures.
Exclusion:
[0214] [0215] 1. More than 1 prior chemotherapy regimen (including
adjuvant therapy if given within the last 12 months); any
chemotherapy/immunotherapy within 4 weeks prior to study entry;
cumulative anthracycline dose that would preclude 2 full-dose
cycles of doxorubicin in this study. [0216] 2. Prior use of any
nitrosoureas (BCNU, CCNU) or mitomycin-C within 6 weeks of study
chemotherapy. [0217] 3. Cardiac history, signs or symptoms that, in
the Investigator's opinion, preclude the use of an
anthracycline-based chemotherapy regimen. [0218] 4. Prior surgery
or radiation therapy within 2 weeks of study chemotherapy. [0219]
5. Prior wide field irradiation to the pelvis or to greater than
20% of the marrow-bearing areas, or bone marrow involvement. [0220]
6. Prior high-dose chemotherapy with hematopoietic stem cell
transplant. [0221] 7. Prior use of myeloid (G-CSF or GM-CSF) growth
factors within 4 weeks of study chemotherapy. [0222] 8. Prior use
of erythropoietin within 4 weeks of study chemotherapy. [0223] 9.
History of myeloid malignancy or myelodysplasia. [0224] 10. Known
brain metastases unless adequately treated (surgery or
radiotherapy), no evidence of progression with a minimum of 3 weeks
observation and neurologically stable off anticonvulsants and
steroids. [0225] 11. Known sickle cell disease. [0226] 12.
Diagnosis of adult respiratory distress syndrome (ARDS). [0227] 13.
Current infection requiring intravenous or oral antibiotics. [0228]
14. Known history of allergies to yeast-derived products. [0229]
15. Known hypersensitivity to E coli-derived proteins,
pegfilgrastim, filgrastim, or any other component of pegfilgrastim
(phase 2 only). [0230] 16. Pregnant female or nursing mother (over
the course of the study, all females must practice a method of
contraception with greater than 90% reliability, or be sterile or
postmenopausal). [0231] 17. Known HIV positive or active hepatitis
(patients with unknown status will not be tested). [0232] 18. Males
who do not agree to use effective contraception throughout the
study and for a period of 30 days after the last dose of study
agent. Subjects were removed from further treatment for the
following reasons: [0233] 1. Disease progression [0234] 2.
Unacceptable toxicities despite optimal treatment [0235] 3.
Intercurrent illness at the investigator's discretion [0236] 4.
Doxorubicin regimen--Maximum lifetime permissible cumulative dose
reached (see eligibility criteria) [0237] 5. Withdrawal of consent
[0238] 6. Non-compliance/Loss to follow-up [0239] 7. Pregnancy
[0240] If treatment with NEUG was stopped, subjects remained on
study and were followed at least 30 days following the final dose
of any study drug for scheduled safety and PK assessments.
[0241] 3. Study Agent
[0242] NEUG (recombinant human albumin-human granulocyte colony
stimulating factor, rHSA-G-CSF), is a fusion protein with a
molecular mass of approximately 85 kDa connected in a single chain
comprising residues 1-585 corresponding to the mature form of HSA
and residues 586-759 corresponding to the mature form of human
G-CSF. The therapeutic moiety of NEUG is recombinant human
DNA-derived G-CSF.
[0243] NEUG was supplied as a sterile, lyophilized formulation in
single-use Type 1 glass vials and stored at 2-8.degree. C. Upon
reconstitution with 1.1 ml of sterile water for injection, each
vial contained 15 mg/ml (15 mg/vial deliverable) NEUG in 10 mM
sodium phosphate, 200 mM mannitol, 60 mM trehalose dehydrate, 0.01%
(w/v) polysorbate 80, pH 7.2.
[0244] The composition of the NEUG drug product used in Phase I is
presented in FIG. 13.
[0245] Commercially available Neulasta.RTM. (pegfilgrastim) was
supplied in 0.6 ml prefilled syringes for subcutaneous injection.
Each syringe contains 6 mg pegfilgrastim (based on protein weight),
in a sterile, clear, colorless, preservative-free solution (pH 4.0)
containing acetate (0.35 mg), sorbitol (30.0 mg), polysorbate 20
(0.02 mg), sodium (0.102 mg) in water for injection. USP.
[0246] NEUG (50, 150, 300 or 450 .mu.g) or Neulasta.RTM.
(pegfilgrastim) (6 mg) was administered by subcutaneous
administration.
[0247] 4. Study Characteristics
[0248] a. Study Schedule and Duration
[0249] This study was a first-in-man, multi-center, open-label
non-controlled sequential dose escalation of a followed by a
controlled, randomized trial conducted in 62 subjects with breast
cancer scheduled to receive doxorubicin/docetaxel. The study
consisted of 2 parts. Part A was a sequential dose escalation in 13
subjects, 4 dose cohorts (50, 150, 300, or 450 .mu.g/kg) with 3
subjects in each of the 50, 150 and 450 .mu.g/kg cohorts and 4
subjects in the 300 .mu.g/kg cohort, to evaluate safety prior to
the randomized, Part B of the trial.
[0250] In Part A, subjects received the first dose of NEUG at least
2 weeks prior to the start of chemotherapy (cycle 0) for an initial
assessment of safety and effects on absolute neutrophil count
("ANC") in the absence of cytotoxic chemotherapy. After a minimum
of 2 weeks follow-up, subjects received NEUG at the same dose
following chemotherapy in cycles 1 and 2 if there were no
dose-limiting adverse events considered related to NEUG in cycle 0
and the subject continued to meet all eligibility criteria.
[0251] In Part A, dose limiting toxicity (DLT) was defined as grade
2 or greater clinically significant adverse event(s) considered
possibly, probably or definitely related to the study agent with
the exception of grade 2 bone pain. Within each Part A cohort, the
initial study drug administration to each subject entering the
trial was separated by a minimum of 24 hours to monitor for acute
adverse events.
[0252] The decision to escalate to the next dose level was based
upon the review of the safety data for at least 7 days after the
first dose administration of NEUG for all subjects in a given
cohort. If none of the 3 subjects experienced a DLT, dose
escalation continued with the enrollment of 3 subjects at the next
dose level. If 1 of 3 subjects in a given cohort exhibited evidence
of a DLT, another 3 subjects were recruited at that dose level for
a total of 6 subjects per cohort. Dose escalation continued to
occur if only 1 of 6 subjects experienced a DLT. If 2 of 6 subjects
develop a DLT, dose escalation stopped and no further NEUG
treatments were administered.
[0253] The remaining subjects completed their scheduled safety,
pharmacokinetic and pharmacodynamic evaluations.
[0254] Following demonstration of safety in the initial Part A
cohorts, Part B was performed. In Part B, subjects were randomized
in a parallel fashion to 1 of 3 treatment groups: NEUG 300 .mu.g/kg
(n=20), NEUG 450 .mu.g/kg (n=21), or pegfilgrastim (n=10) at the
approved dose of 6 mg administered approximately 24 hours after
study chemotherapy.
[0255] Tables 3 and 4 below summarize the disposition of the
subjects in Phase I, Parts A and B. FIG. 6 shows the chemotherapy
cycles for Phase I study, Parts A and B.
TABLE-US-00003 TABLE 3 Disposition of Subjects in Phase I Dose N
(NEUG/pegfilgrastim) Part A Sequential Dose Escalation NEUG 50
.mu.g/kg 3/0 NEUG 150 .mu.g/kg 3/0 NEUG 300 .mu.g/kg 4/0 NEUG 450
.mu.g/kg 3/0 Part B Parallel Randomization NEUG 300 .mu.g/kg 20/5
NEUG 450 .mu.g/kg 21/5
TABLE-US-00004 TABLE 4 Disposition of Subjects in Phase I Treatment
Part A (N) Part B (N) Total Neulasta .RTM. (pegfilgrastim) 0 10 10
NEUG 50 3 0 3 NEUG 150 3 0 3 NEUG 300 4 20 23 NEUG 450 3 21 24
Total 13 51 64
[0256] b. Concomitant Therapy During Phase I, Parts A and B
Chemotherapy
[0257] The chemotherapy regimen for this trial consisted of
doxorubicin 50 mg/m.sup.2 and docetaxel 75 mg/m.sup.2 administered
sequentially by intravenous infusion on day 1 of treatment for up
to two 21-day cycles.
[0258] Prior to receiving each cycle of therapy, subjects had to
have an absolute neutrophil count (ANC)>1.5.times.10.sup.9/L and
platelets>100.times.10.sup.9/L. Treatment could be delayed up to
two weeks for hematologic recovery.
[0259] The combination of doxorubicin and docetaxel has been
reported to have significant clinical activity in patients with
breast cancer. However, the combination is highly myelosuppressive
with higher rates of grade 3 or 4 neutropenia than other standard
regimens.
[0260] Even with the addition of CSFs, the combination of
doxorubicin and docetaxel has induced grade 4 neutropenia in 79% of
patients and febrile neutropenia rates of 9-18%. This
doxorubicin/docetaxel regimen has been used in studies of new
agents to prevent neutropenia and its complications. Therefore, the
combination of doxorubicin and docetaxel is an appropriate
chemotherapy regimen to study the potential of a new agent like
NEUG.
Doxorubicin
[0261] Pharmacologic Data
[0262] Doxorubicin hydrochloride is an anthracycline antibiotic
obtained from streptomyces peucetius var caesius which inhibits DNA
and DNA-dependent RNA synthesis, as well as protein synthesis.
Doxorubicin is active in all phases of the cell cycle but maximally
cytotoxic in S phase. Excretion of the drug is predominately by the
liver; renal clearance is minor.
Pharmaceutical Data
[0263] The drug is marketed commercially in 10, 20 50, 100 or 200
mg vials. Lyophilized preparations may be reconstituted with
sterile water for injection, dextrose 5% solution, or 0.9% saline
for injection.
[0264] Side Effects and Toxicity
[0265] Myelosuppression, primarily leukopenia, with a nadir of
approximately 10-14 days, and cardiotoxicity, including a rare,
acute pericarditis-myocarditis syndrome and a delayed, cumulative
dose related cardiomyopathy are the dose-limiting toxicities of
doxorubicin. Marked alopecia and moderate nausea/vomiting are
expected toxicities. Extravasation reactions producing local skin
and tissue damage at the site of inadvertent extravasation,
stomatitis, hyperpigmentation of the skin (particularly the
nailbeds), and a "recall" phenomenon at sites of previous
irradiation have been reported.
Docetaxel
[0266] Pharmacologic Data
[0267] Docetaxel is a semisynthetic taxoid that binds to free
tubulin and promotes assembly of stable microtubules, interfering
with mitosis and cell replication (cell cycle specific for M
phase). Docetaxel is extensively protein-bound, extensively
metabolized in the liver, with fecal excretion of approximately 75%
of the dose within 7 days.
Pharmaceutical Data
[0268] Docetaxel (Taxotere.TM., Sanofi Aventis) is provided in 80
mg/2 mL or 20 mg/0.5 ml single-dose vials with an accompanying
diluent (13% ethanol in Water for Injection) vial. Each ml of
Taxotere contains 40 mg of docetaxel (anhydrous) and 1080 mg
polysorbate 80.
[0269] Side Effects and Toxicity
[0270] Docetaxel should not be given to patients who have a history
of severe hypersensitivity reactions to docetaxel or other drugs
formulated with polysorbate 80 such as etoposide and vitamin E.
[0271] Patients who experience severe hypersensitivity reactions
should not be rechallenged. Patients receiving docetaxel should be
premedicated with corticosteroids as outlined below.
[0272] Mild to moderate liver impairment results in delayed
metabolism by 27% and a 38% increase in systemic exposure (AUC).
Docetaxel should not be given to patients with SGOT and/or
SGPT>1.5 times normal limits and alkaline phosphatase>2.5
times normal limits. Fluid retention occurred in 17% (moderate) and
6% (severe retention) of patients in Phase III studies despite
corticosteroid premedication. Severe neurosensory symptoms
(paresthesia, dyesthesia, pain) have been observed.
[0273] Expected side effects include myelosuppression, primarily
leukopenia, with a nadir of approximately 9 days with recovery by
day 15-21. Alopecia, nail and cutaneous changes, stomatitis,
myalgia/arthralgia, nausea/vomiting, and hypotension have been
reported.
[0274] Chemotherapy Dosage, Administration and Dose
Modifications
[0275] On day 1 of each treatment cycle, chemotherapy (doxorubicin
followed by docetaxel) was administered.
[0276] Doxorubicin was administered at a dose of 50 mg/m.sup.2 by
IV bolus through the side arm of an infusing intravenous line or
central venous catheter to avoid extravasation injury.
[0277] Docetaxel 75 mg/m.sup.2 was diluted in 250 mL 0.9% saline or
5% dextrose solution and administered intravenously over
approximately 1 hour via a polyethylene-lined infusion set. Vital
signs were obtained immediately prior to and after the end of the
docetaxel infusion.
[0278] Prior to receiving each cycle of therapy, subjects had to
have an absolute neutrophil count (ANC)>1500/mm.sup.3 and
platelets>100,000/mm.sup.3. Treatment could be delayed up to two
weeks for hematologic recovery. A 25% dose reduction of
chemotherapy doses was allowed for grade 3-4 non-hematologic
toxicities, two grade 3-4 infectious episodes, or grade 4
thrombocytopenia.
[0279] Subjects experiencing severe hypersensitivity reactions or
non-hematologic toxicities that preclude further cycles of
chemotherapy were removed from study treatment but completed
follow-up.
[0280] Chemotherapy Pre-Medication
[0281] Oral (IV as needed) corticosteroids (such as dexamethasone 8
mg BID) were administered for three days starting 1 day prior to
docetaxel administration in order to reduce the incidence and
severity of fluid retention and hypersensitivity reactions.
[0282] The use and selection of anti-emetic agents or other
pre-medications (e.g. H.sub.2 antagonists) was left to the
discretion of the treating physician.
Prohibited Medications
[0283] Subjects should not have received any of the following
medications and or procedures during this study and for the
additional times specified below: [0284] 1. Other investigational
agents within 30 days of initiating study agent and for the
duration of the trial. [0285] 2. Subsequent cycles of chemotherapy
should not be initiated until 14 days following dosing with NEUG.
[0286] 3. Cytokines, other hematopoietic growth factors and
prophylactic antibiotics for the duration of the trial unless
prolonged or febrile neutropenia occurs. If the subject was treated
with G-CSF at any time between the screening period and Day 0 they
were not eligible to receive NEUG and were discontinued from the
study.
Allowed Medications
[0287] Subjects were allowed to continue their baseline
medications(s). The daily dose of each medication was maintained
throughout the study if possible. If for any reason deemed
necessary by the investigator, a subject required additional
medication(s) or change of dose, the medication(s), route of
administration, and the indication for which it was given was
recorded on the appropriate pages of the CRF.
Antibiotics
[0288] All subjects received prophylactic oral antibiotics (e.g.
ciprofloxacin) following each course of chemotherapy to reduce the
likelihood of infection. If febrile neutropenia or persistent
severe neutropenia (ANC<0.5.times.10.sup.9/L for .gtoreq.5 days)
occurred, the subject was considered a treatment failure, removed
from the study, completed study follow-up and received all standard
supportive care, including growth factor support at the
Investigator's discretion.
[0289] Subjects who experienced severe hypersensitivity reactions
or non-hematologic toxicities that precluded further cycles of
chemotherapy were also removed from study treatment and completed
follow-up.
[0290] c. Safety Assessments
[0291] The safety of NEUG was assessed by evaluation of the type,
frequency, and severity of adverse events ("AEs"), changes in
clinical laboratory tests (hematology and clinical chemistry),
immunogenicity, physical examinations, and the monitoring of vital
signs over time. All AEs and laboratory toxicities were graded
based on the National Cancer Institute Common Terminology Criteria
for Adverse Events (NCI-CTCAE Version 3.0, 12 Dec. 2003). Adverse
events (to include serious adverse events, "SAEs") were captured
from the start of study drug administration through 30 days
following the final dose of any study drug. Laboratory assessments
were obtained as outlined in the Schedule of Assessments. In the
event of any Grade 4 neutropenia toxicity, labs were obtained every
day until ANC>500. If the subject's next cycle of therapy was
delayed (and after the last cycle of treatment), complete blood
count (CBC) with differential was obtained at least twice weekly
until ANC>1500.
[0292] 5. Results of Phase I, Parts A and B
[0293] a. General
Statistical Methods:
[0294] The data related to safety, pharmacokinetics (PK),
pharmacodynamics (PD) and immunogenicity parameters were analyzed
using descriptive statistical methods.
[0295] For frequency and severity of adverse events, and for
laboratory toxicity grading, counts and rates are presented.
[0296] Efficacy analyses included the incidence and duration of
grade 4 and grade 3-4 neutropenia, nadir ANC, time to nadir ANC,
time to recovery (to ANC>0.5.times.10.sup.9/L and
ANC>1.0.times.10.sup.9/L), and the incidence of febrile
neutropenia. No strict statistical power requirement was used to
select the sample size for this study. A study with a power of 80%
to demonstrate non-inferiority of NEUG to pegfilgrastim at a
significance level of 5% was calculated to require approximately 37
subjects per treatment arm. As this was a phase 1/2a study
conducted primarily for safety, it was determined that the required
sample size to be powered for effect was larger than appropriate.
As such, efficacy trends were evaluated.
Disposition/Demographics:
[0297] A total of 13 subjects were enrolled in the Part A,
sequential dose escalation portion of the trial. A total of 51
subjects were enrolled in the Part B portion, and randomized to
NEUG 300 .mu.g/kg (n=20), NEUG 450 .mu.g/kg (N=21), or
pegfilgrastim 6 mg (n=10).
[0298] b. Study Results
[0299] In initial dose-finding, in the absence of chemotherapy,
NEUG was well tolerated and resulted in the expected rise in ANC,
which peaked between days 2 and 4 and returned to normal by day 14
(FIG. 22).
[0300] In Part A, all three subjects in the 50 .mu.g/kg NEUG dose
group and 1 subject in the 450 .mu.g/kg Neugranin dose group
experienced febrile neutropenia or severe neutropenia lasting
greater than 5 days. In Part B, one subject in the 300 .mu.g/kg
NEUG dose group and 2 subject in the 450 .mu.g/kg NEUG dose group
experienced febrile neutropenia or severe neutropenia lasting
greater than 5 days. One subject in the pegfilgrastim group
experienced febrile neutropenia or severe neutropenia lasting
greater than 5 days.
[0301] c. Immunogenicity
[0302] Serum samples for antibodies to NEUG were obtained prior to
dosing on Day 1 of every NEUG cycle and at the end of treatment
visit (at least 15 days after the last dose) in subjects receiving
NEUG. If at any time during the study a subject developed a
positive anti-NEUG antibody response, a repeat sample was obtained
approximately 6 months after the final NEUG dose.
[0303] Testing was completed on all subjects through the end of
treatment for both Part A and B. All samples were negative for
antibodies to NEUG.
[0304] d. Adverse Events
[0305] During Part A, dose-limiting toxicity (DLT) was defined as
grade 2 or greater clinically significant adverse event(s),
considered possibly, probably or definitely related to the study
agent with the exception of grade 2 medullary bone pain.
[0306] No DLT was encountered in cycle 0 in any of the Part A
cohorts. Only 2 adverse event were reported as related to NEUG
administration: bone pain and exacerbation of pre-existing
hypertension, the latter occurring 7 days after NEUG
administration. Both events resolved without sequeale.
[0307] Thirty one of the 41 NEUG-treated subjects experienced at
least 1 adverse event. The incidence of AEs among NEUG- and
pegfilgrastim-treated subjects was comparable (75.6% and 70%
respectively).
[0308] A summary of commonly reported adverse events (AEs greater
than or equal to 5% of all subjects) for Part B is provided in
Table 5.
TABLE-US-00005 TABLE 5 Summary of Treatment-Emergent Adverse Events
in the Phase 1, Part B Population Med DRA NEUG 300 NEUG 450
Pegfilgrastim Preferred Term (N = 20) (N = 21) (N = 10) Related
AE.sup.1: Bone Pain 1 (4.5%) 3 (14.3%) 0 (0%) Unrelated AE.sup.2:
Nausea 3 (15%) 3 (14.3%) 3 (30%) Vomiting 1 (5%) 3 (14.3%) 3 (30%)
Diarrhea 1 (5%) 1 (4.8%) 1 (10%) Stomatitis 0 (0%) 3 (14.3%) 0 (0%)
Fatigue 0 (0%) 0 (0%) 1 (10%) Pharyngitis 2 (10%) 0 (0%) 1 (10%)
Alopecia 4 (20%) 7 (33%) 2 (20%) Thrombocytopenia 0 (0%) 0 (0%) 1
(10%) Headache 0 (0%) 1 (4.8%) 1 (10%) Hypokalaemia 0 (0%) 2 (10%)
1 (10%) Vitamin D Deficiency 3 (15%) 0 (0%) 1 (10%) Hypertension 0
(0%) 1 (4.8%) 1 (10%) .sup.1Related = considered possibly, probably
or definitely related .sup.2Unrelated = considered probably not
related or not related
[0309] The most commonly reported adverse event considered related
to NEUG was bone pain, a typical adverse reaction associated with
all G-CSF products, which was reported in 5 patients 4 listed in
the table above, plus one Part A subject receiving 450 .mu.g/kg).
In all cases, the bone pain was NCI-CTCAE grade 1-2 in intensity,
transient in duration and resolved without sequelae. Grade 1
elevations in alkaline phosphatase and uric acid occurred following
administration of NEUG in Cycle 0; these events were deemed to be
not clinically significant by the Investigators and resolved
without intervention. These are expected effects in patients
receiving a G-CSF (e.g., Neulasta.RTM.).
[0310] Other commonly reported adverse events during chemotherapy
cycles (nausea, vomiting, alopecia, stomatitis) were consistent
with anticipated adverse events in patients receiving the
doxorubicin/docetaxel regimen.
[0311] The majority of reported AEs were of NCl CTC Grade 1 or 2
severity. Four AEs were reported as serious adverse events. Two
subjects, one receiving 150 .mu.g/kg and one 450 .mu.g/kg,
experienced vomiting that caused hospitalization and one of these
subjects experienced a second SAE in the following chemotherapy
cycle; vomiting that was mild in intensity but caused or prolonged
hospitalization. A third subject received 450 .mu.g/kg was
hospitalized for febrile neutropenia. The events were considered
unrelated to NEUG.
[0312] e. Pharmacokinetics
[0313] All subjects receiving NEUG were sampled for serum NEUG
concentrations over the course of the study. The drug was detected
using a sandwich enzyme-linked immunosorbent assay (ELISA) specific
for NEUG. The serum drug concentration-time data was subjected to
PK analysis using WinNonlin Enterprise Edition, Version 4.1 or
higher, using noncompartmental or model-based analysis.
[0314] The following PK parameters were obtained: area under the
curve (AUC.sub.0-.infin.), clearance (CL/F), volume of distribution
(Vz/F), maximum concentration (Cmax), absorption half-life (t1/2,
abs), elimination half-life (t1/2, elim), and mean residence time
(MRT). Pharmacokinetic data were assessed for linearity across the
dose range employed in the protocol.
[0315] Pharmacokinetic parameters from cycle 0 (pre-chemotherapy)
are summarized in Table 6 and the cycle 0 PK profile is illustrated
in FIG. 3.
TABLE-US-00006 TABLE 6 Neugranin Pharmacokinetics in Human Subjects
(Phase 1 Cycle 0) Parameter NEUG NEUG NEUG Dose (mcg/kg) 150
.mu.g/kg 300 .mu.g/kg 450 .mu.g/kg Number of Subjects 3 4 3 AUC (hr
* ng/mL) 1758 .+-. 1675 3390 .+-. 2003 10131 .+-. 9563 (mean .+-.
SD) t.sub.1/2,term(hr) (mean .+-. SD) 14.4 .+-. 4.0 23.5 .+-. 10 29
.+-. 9.3 C.sub.max (ng/mL) 72.7 .+-. 59.7 108.9 .+-. 50.5 294 .+-.
351 (mean .+-. SD) t.sub.max (hr) (mean) 12 15 18
[0316] Drug exposure as measured by maximum serum NEUG
concentration and area under the time-concentration curve increased
in a dose-dependent manner. Serum concentrations for subjects in
the initial 50 .mu.g/kg dose cohort were consistently below the
lower limit of quantization (6.3 ng/mL). Tmax was in the range of
6-24 hours for all doses from 150 through 450 .mu.g/kg. Cmax ranged
from 72.7.+-.59.7 (mean.+-.SD) ng/mL at a dose of 150 .mu.g/kg to
294.+-.351 ng/mL, at a dose of 450 .mu.g/kg. Correspondingly,
AUC.sub.0-.infin. ranged from 1758.+-.1675 ng/mL*hr at a dose of
150 mcg/kg to 10131.+-.9563 ng/mL*hr at a dose of 450 .mu.g/kg.
Cycle 1 ranges were similar. The mean elimination half-life of NEUG
ranged from 14-30 hours.
[0317] As noted in "Study Characteristics" (section 4, above),
subjects in Part A received the first dose of NEUG at least 2 weeks
prior to the start of chemotherapy (cycle 0) for an initial
assessment of safety and effects on absolute neutrophil count
("ANC") in the absence of cytotoxic chemotherapy. After a minimum
of 2 weeks follow-up, subjects received NEUG at the same dose
following chemotherapy in cycles 1 and 2 if there were no
dose-limiting adverse events considered related to NEUG in cycle 0
and the subject continued to meet all eligibility criteria. NEUG
was administered 24 hours following chemotherapy administration.
FIG. 7 shows the ANC and WBC for subjects that received NEUG during
cycles 1 and 2.
[0318] f. Pharmacodynamics and Establishment of Part B Dosages
[0319] Analysis of the data from Part A of Phase I of the study
yielded the following observations: [0320] 1. NEUG induces a
dose-dependent rise in WBC and ANC rise in Cycle 0 (prior to
chemotherapy) (see cycle 0 data at FIG. 7A). [0321] 2. ANC
increases in Cycle 0 were comparable to historical data for
pegfilgrastim at equimolar doses [0322] 3. As anticipated, WBC and
ANC drop following chemotherapy [0323] 4. Recovery from Nadir ANC
appears dose related [0324] 5. ANC and WBC returned to normal by
day 15
[0325] Based on these observations and demonstration of safety at
all dose levels in Part A, the doses chosen for the Part B
evaluation were 300 and 450 .mu.g/kg. As described above, subjects
were randomized to NEUG 300 .mu.g/kg, NEUG 450 .mu.g/kg, or
pegfilgrastim at the approved fixed dose of 6 mg. Subjects received
the NEUG or pegfilgrastim one day following doxorubicin/docetaxel
(administered for 2 cycles, 21 days apart). Data for Part B
includes the cycle 1 ANC profiles of the population. Results are
summarized in FIG. 2 and Table 5, below.
[0326] The incidence of grade 3 and 4 neutropenia, and the ANC
profiles during Cycle 1 were determined in 48 of 51 treated
subjects as show in Table 7, below. Note that 70-80% of patients
treated with doxorubicin/docetaxel get Grade 4 neutropenia with
durations average of 5 days in the absence of prophylactic G-CSF
treatment.
TABLE-US-00007 TABLE 7 Incidence and duration of Grade 4
neutropenia in Phase I, Part B after cycle 1 of chemotherapy
Treatment NEUG Pegfilgrastim Dose 300 .mu.g/kg 450 .mu.g/kg 6 mg
Number of subjects 20 21 10 Grade 4 9 6 3 Neutropenia % grade 4
45.0% 28.6% 30.0% Neutropenia Mean (days) 1.1 1.0 0.7 SD (days)
1.33 1.67 1.16 Range (days) 0-4 0-5 0-3
[0327] Mean ANC curves for the treatment groups are presented in
FIG. 2.
[0328] NEUG is effective for treating grade 3, grade 4 and febrile
neutropenia. In the absence of G-CSF treatment for this
chemotherapy regimen, the incidence of febrile neutropenia is about
40%. A dose-related elevation in ANC and a lower rate of
neutropenia than is expected with doxorubicin/docetaxel were
observed following administration of NEUG. There were no unexpected
or serious adverse events attributed to NEUG.
[0329] The incidence of grade 3 and grade 4 neutropenia was higher
in patients receiving 300 .mu.g/kg NEUG than those receiving
pegfilgrastim (Neulasta.RTM.) and the rate of return to normal ANC
also appeared slower in patients who received 300 .mu.g/kg NEUG
than in those subjects who received pegfilgrastim. The ANC profiles
in patients who received NEUG at 450 .mu.g/kg and those who
received pegfilgrastim were similar, though the ANCs during
recovery from neutropenia were generally lower in patients who
received NEUG than in patients receiving pegfilgrastim. In summary,
NEUG at these doses appears to provide similar effect as
pegfilgrastim.
[0330] g. PK/PD Profile, Phase I, Part B
[0331] The PK/PD profile from patients receiving 450 .mu.g/kg NEUG
one day after doxorubicin/docetaxel administration in cycle 1 of
treatment for breast cancer is shown in FIG. 4. Cmax for NEUG is
achieved within one day of administration and gradually falls to
undetectable levels by day 10. Following administration of NEUG,
the ANC rises to a peak by day 4 and then, as expected in patients
receiving doxorubicin/docetaxel and G-CSF treatment, the ANCs fall
to a nadir on day 8 and return to normal on day 10. By day 12, ANC
values are in the normal range and NEUG is undetectable. Note that
in patients who do not receive prophylactic G-CSF treatment, the
duration of nadir ANC and time to reach recovery ANC are much
longer (e.g., 5-7 days). After a dose of 450 .mu.g/kg, the NEUG
median elimination half-life was approximately 30 hours, as
compared to the 15-80 hours reported for a standard dose of
pegfilgrastim.
[0332] h. Additional Differences Between NEUG and Pegfilgrastim
[0333] More detail of the differences between NEUG and
pegfilgrastim at the tested doses in effectiveness in hastening the
recovery form neutropenia is evident in comparison of the
individuals ANC profiles in cycle 1 of treatment. The peak ANCs in
all groups were very similar, nadir ANCs in subjects receiving NEUG
at 300 .mu.g/kg were lower than in subjects receiving NEUG at 450
.mu.g/kg, and the ANC nadirs in subjects receiving pegfilgrastim
were on average the highest. Recovery from nadir ANC to baseline
occurred by day 14 in all treatment groups, but was slower for
those receiving 300 .mu.g/kg NEUG, than 450 .mu.g/kg NEUG, and most
rapid for subjects receiving pegfilgrastim.
[0334] Available published data for a pegfilgrastim trial with a
similar prechemotherapy administration were compared to NEUG PK/PD
data from patients who completed the Phase I through the scheduled
cycle 0 (pre-chemotherapy). Results of this comparison were as
follows: [0335] 1. Emax (maximum observed ANCs) at NEUG dose of 150
.mu.g/kg matches the 30 .mu.g/kg dose of pegfilgrastim in Cycle 0,
a dose later demonstrated to be inferior for efficacy to the
confirmed efficacious pegfilgrastim dose of 100 .mu.g/kg. [0336] 2.
Emax for 300 and 450 .mu.g/kg Neugranin doses are more consistent
with Cycle 0 levels for 100 .mu.g/kg dose of pegfilgrastim. [0337]
3. At 300 and 450 .mu.g/kg NEUG median Cmax and median Emax are
nearly the same, thus Cmax continued to predict Emax. [0338] 4. ANC
increases were comparable to published data for pegfilgrastim at
equimolar doses.
[0339] As discussed above, PK/PD assessment in animals and in man
was consistent with an estimate of NEUG and pegfilgrastim dose
equivalence when dosed on an equimolar basis. In mice, equivalent
AUC.sub.ANC were achieved with a 7.7 fold higher dose thane
pegfilgrastim. Because albumin contributes significantly to the
molecular weight of NEUG, and Neulasta.RTM. (pegfilgrastim) is
dosed base on the weight of the rhG-CSF (not including the
contribution of the polyethylene glycol in pegfilgrastim), a 4.5
fold greater dose of NEUG (based on weight) is predicted to be as
effective as an equal dose of Neulasta.RTM. (pegfilgrastim).
Efficacy data in animals were consistent with a 4.5-7.7 fold
equivalence to pegfilgrastim (1 mg pegfilgrastim=4.5-7.7 NEUG).
Non-clinical safety and effect data are consistent with this dose
estimate and when considered with available clinical data, form the
basis for the doses elected for clinical evaluation.
[0340] i. Results of Phase I
[0341] The Results from the Phase I pharmacokinetic evaluation are
as follows. [0342] NEUG was detected in serum samples from all
subjects treated with NEUG at doses of 150 .mu.g/kg, 300 .mu.g/kg
and 450 .mu.g/kg on Cycle 0 and Cycle 1. [0343] In Cycle 1, NEUG
was detected uptown 144 hours in most subjects (45/50 sampled) in
the 150 mg/kg, 300 .mu.g/kg and 450 .mu.g/kg dose groups. Virtually
no cycle to cycle drug accumulation was observed. [0344] Drug
exposure was higher in Cycle 1 and in Cycle 0 (pre-chemotherapy)
with each dose group. The increased exposure to NEUG in Cycle 1 is
likely due to the decreased number of neutrophils, which plan a
role in the receptor-mediated clearance of G-CSF. The median
elimination half-life of NEUG in Cycle 1 was about 36 hours for
dose group 300 .mu.g/kg and 30 hour for dose group 450 .mu.g/kg.
The elimination half-life is reported to be 3-4 hours for
filgrastim and 42-67.5 hours, depending on dose, for pegfilgrastim.
Statistically significant differences across doses were observed in
the time to maximal serum concentration (t.sub.max) and the
absorption half-life (t.sub.1/2,abs). Both of these parameters
increased with increasing NEUG dose. No other dose normalized PK
parameters showed statistically significant differences across
doses.
Example 12
Phase II
[0345] Phase II of the study was a controlled, randomized trial,
conducted in 334 subjects with breast cancer who received up to 4
doses of doxorubicin/docetaxel. The study, was conducted at 45
clinical sites, and consisted of a two-way randomized pilot phase
to assess the safety and effect of subcutaneously administered NEUG
versus pegfilgrastim, followed by a main phase in which subjects
were randomized to pegfilgrastim and two, well-tolerated doses of
NEUG (1:1:1) selected based on the pilot phase. The sample size for
the main phase was powered to establish non-inferiority of NEUG to
pegfilgrastim with regard to the primary endpoint, duration of
sever (grade 4) neutropenia (DSN) during chemotherapy cycle 1. The
study design is shown schematically in FIG. 25.
[0346] 1. Objectives
[0347] The primary objectives of Phase II were to select doses of
NEUG demonstrating a comparable effect to pegfilgrastim and to
assess the duration of severe neutropenia (DSN) in cycle 1 of
chemotherapy after treatment with NEUG. Secondary objectives were
to assess the DSN in cycles 2-4, to assess the time to absolute
neutrophil count recovery and rates of febrile entroopenia in
cycles 1-4; and to assess the safety, tolerability,
pharmacokinetics (in cycle 1), and immunogenicity of NEUG.
[0348] 2. Patient Characteristics
[0349] For Phase II, patients were screened based on the following
characteristics or parameters:
Inclusion:
[0350] 1. Patients with histologically-confirmed breast cancer
scheduled to receive doxorubicin 60 mg/m.sup.2 and docetaxel 75
mg/m.sup.2 [0351] 2. 18 years of age or older [0352] 3. Adequate
hematologic function: [0353] 4. ANC>1500/mm.sup.3 [0354] 5.
Platelets>100,000/mm.sup.3 [0355] 6. Adequate hepatic and renal
function: [0356] 7. Serum creatinine<1.5.times.upper limit
normal [0357] 8. Total bilirubin within normal limits (WNL) for
local laboratory [0358] 9. Serum transaminases
(SGOT/SGPT)<1.5.times.upper limit normal [0359] 10. Alkaline
phosphatase<2.5.times.upper limit normal [0360] 11. Eastern
Cooperative Oncology Group ("ECOG") performance status 0-2 [0361]
12. Eligible to receive doxorubicin based on a left ventricular
ejection fraction (LVEF) within normal limits [0362] 13. Have the
ability to understand the requirements of the study, provide
written informed consent (including consent for use and disclosure
of research-related health information) and comply with the study
protocol procedures.
Exclusion:
[0362] [0363] 1. More than 1 prior chemotherapy regimen (including
adjuvant therapy if given within the last 12 months) [0364] 2. A
cumulative anthracycline dose that would preclude 4 full-dose
cycles of doxorubicin in this study [0365] 3. Prior
chemotherapy/immunotherapy within 30 days prior of study
chemotherapy (within 6 weeks of study chemotherapy for nitrosoureas
(BCNU, CCNU) or mitomycin-C) [0366] 4. Concomitant trastuzumab
(Herceptin) [0367] 5. Received any investigational agent in the
past 30 days [0368] 6. Cardiac history, signs or symptoms that, in
the Investigator's opinion, preclude the use of an
anthracycline-based chemotherapy regimen [0369] 7. Prior surgery
within 2 weeks of study chemotherapy [0370] 8. Prior radiation
therapy within 4 weeks of study chemotherapy (except spot
irradiation for bone metastases) [0371] 9. Prior high-dose
chemotherapy with hematopoietic stem cell transplant [0372] 10.
Prior use of G-CSF, GM-CSF or erythropoietin within 4 weeks of
study chemotherapy [0373] 11. Received systemic antibiotics within
72 hours of study chemotherapy [0374] 12. History of myeloid
malignancy or myelodysplasia [0375] 13. Known brain metastases
unless adequately treated (surgery or radiotherapy), no evidence of
progression with a minimum of 3 weeks observation and
neurologically stable off anticonvulsants and steroids. [0376] 14.
Known sickle cell disease [0377] 15. Diagnosis of adult respiratory
distress syndrome (ARDS) [0378] 16. Known history of allergies to
yeast-derived products [0379] 17. Known hypersensitivity to E
coli-derived proteins, pegfilgrastim, filgrastim, or any other
component of pegfilgrastim [0380] 18. Pregnant female or nursing
mother. (All females with an intact uterus must have a negative
serum pregnancy test at screening. All non-sterile or
non-postmenopausal females must practice a medically accepted
method of contraception over the course of the study and for 30
days after the last dose of study agent.) [0381] 19. Males who do
not agree to use effective contraception throughout the study and
for a period of 30 days after the last dose of study agent [0382]
20. Known HIV positive or active hepatitis (Patients with unknown
status will not be tested) Subjects were removed from further
treatment for the following reasons: [0383] 1. Disease progression
[0384] 2. Unacceptable toxicities despite optimal treatment [0385]
3. Intercurrent illness at the investigator's discretion [0386] 4.
Doxorubicin regimen--Maximum lifetime permissible cumulative dose
reached (see eligibility criteria) [0387] 5. Withdrawal of consent
[0388] 6. Non-compliance/Loss to follow-up [0389] 7. Pregnancy
[0390] If treatment with study drug was stopped, subjects remained
on study were followed at least 30 days following the final dose of
any study drug for scheduled safety and PK assessments.
[0391] 3. Study Agent
[0392] NEUG (recombinant human albumin-human granulocyte colony
stimulating factor, rHSA-GCSF), is a fusion protein with a
molecular mass of approximately 85 kDa connected in a single chain
comprising residues 1-585 corresponding to the mature form of HSA
and residues 586-759 corresponding to the mature form of human
G-CSF. The therapeutic moiety of NEUG is recombinant human
DNA-derived G-CSF.
[0393] NEUG was supplied as a sterile, lyophilized formulation in
single-use Type 1 glass vials and stored at 2-8.degree. C. Upon
reconstitution with 1.0 ml of sterile water for injection, each
vial contains 50 mg/ml (50 mg/vial deliverable) NEUG in 20 mM
sodium phosphate, 180 mM, mannitol, 60 mM trehalose dehydrate,
0.01% (w/v) polysorbate 80, pH 6.0. Note that NEUG is also be
provided as a liquid, either in vials or in pre-filled
syringes.
[0394] The composition of the drug product used in Phase II is
shown in FIG. 14. Difference between the NEUG formulations used in
Phase I and Phase II are shown below in Table 8.
TABLE-US-00008 TABLE 8 cGMP formulation comparison Excipient
Formulation Phase I Phase II Attribute formulation formulation
Rationale for change API 15.0 mg/mL 50 mg/mL Increased API
concentration to reduce volume of injection Sodium 10 mM 20 mM
Higher ionic strength reduces Phosphate concentration dependent
aggregation Mannitol 200 mM 180 mM Reduced to provide iso- osmotic
solution Trehalose 60 mM 60 mM Unchanged-acts as robust dihydrate
cryo/lyo protectant. Polysorbate 0.01% 0.01% Unchanged-inhibits 80
nonspecific aggregation and adsorption pH 7.2 6.0 Lower pH reduces
concentration dependent aggregation
[0395] The formulation used in Phase I was quite stable, with a
shelf-life of at least 2 years. Studies demonstrated that higher
ionic strength and lower pH further stabilized the API at higher
concentration (>25 mg/mL) (data not shown). To this end, the
Phase II formulation has a lower pH (6.0 vs 7.2) and higher
phosphate concentration (20 vs. 10 mM). Forced degradation studies
demonstrate that this formulation protects the drug substance in
the liquid state from vigorous shaking, repeated freeze-thawing,
and concentration induced aggregation. Freeze drying of the Phase
II formulation also produces well-formed cakes.
[0396] Commercially available Neulasta.RTM. (pegfilgrastim) is
supplied in 0.6 ml prefilled syringes for subcutaneous injection.
Each syringe contains 6 mg pegfilgrastim (based on protein weight),
in a sterile, clear, colorless, preservative-free solution (pH 4.0)
containing acetate (0.35 mg), sorbitol (30.0 mg), polysorbate 20
(0.02 mg), sodium (0.102 mg) in water for injection USP.
[0397] NEUG (30, 40, 50, or 60 mg) or Neulasta.RTM. (pegfilgrastim)
(6 mg) was administered by subcutaneous administration.
Dose Rationale:
[0398] The data from Phase I demonstrated that doses of NEUG of 300
and 450 .mu.g/kg were safe and well tolerated. Moreover, compared
to the approved fixed doses of pegfilgrastim, both doses of NEUG
resulted in similar effects on ANC profiles in breast cancer
patients receiving cytotoxic chemotherapy. The AUC for the ANC
profiles serves as a single-point measure of effect. There was no
statistically significant difference among these treatment groups
in terms of AUC.sub.ANC, however, the AUC for the 450 .mu.g/kg
group is slightly higher than that observed for the 300 .mu.g/kg
group and nearly identical to that observed for the pegfilgrastim
group (FIG. 23). Based on available data, it was estimated that 300
.mu.g/kg NEUG was less effective than pegfilgrastim and 450
.mu.g/kg approximates a minimum necessary dose to provide
equivalent effect to pegfilgrastim.
[0399] The intent of a fixed dose is to identify doses that will
provide patients with a dose sufficient to provide efficacy and
safety regardless of patient weight. Based on the results of Phase
I, it as estimated that 450 .mu.g/kg NEUG may be a minimum dose
necessary to provide similar effect as pegfilgrastim, and >300
.mu.g/kg was set as the minimum dose for further evaluation in
Phase II. To select fixed doses of NEUG, the patient population
(breast cancer) for Phase II was modeled. Using 40-100 kg weight
range, a 30 mg fixed dose provides the heaviest patient with a
minimum dose (300 .mu.g/kg or 0.3 mg/kg), while approximately 75%
of patients receive at least the target dose, 450 mg/kg, at a fixed
dose of 40 mg. Thus, the doses selected for evaluation in Phase II
were 30 mg, 40 mg and 50 mg. These provide an average 70 kg patient
with 0.42, 0.57 and 0.71 mg/kg doses, respectively.
[0400] The equivalent dose per kilogram based on the fixed doses
evaluated in this trial is provided in Table 9.
TABLE-US-00009 TABLE 9 Equivalent dose per kilogram for the
anticipated subject weight range 50 kg 60 kg 70 kg 80 kg 90 kg 100
kg 30 mg 0.600 0.500 0.429 0.375 0.333 0.300 40 mg 0.800 0.667
0.571 0.500 0.444 0.400 50 mg 1.000 0.833 0.714 0.625 0.556 0.500
60 mg 1.200 1.000 0.857 0.750 0.666 0.600
[0401] The nonclinical safety for NEUG provides additional support
for the expectation of safety at these doses. Exposure in patients
at these fixed doses (AUC and Cmax) is expected to be lower than
exposure at well tolerated doses in monkeys. For example, Cmax and
AUC in the monkey at the well-tolerated dose of 1 mg/kg was 12-fold
higher than exposure in patients at 0.45 mg/kg suggesting a further
margin of safety exists for higher dose evaluation in patients and
in a repeat-dose toxicology study in monkey, doses up to and
including 10 mg/kg were well tolerated. Doses of pegfilgrastim as
high as 0.3 mg/kg have been demonstrated to be safe in
patients.
[0402] 6. Study Characteristics
[0403] a. Study Schedule and Duration
[0404] This study was a controlled, randomized trial conducted in
approximately 330 subjects with breast cancer scheduled to receive
up to 4 doses of doxorubicin/docetaxel. The study, which was
conducted at 45 clinical sites, consisted of two phases, a pilot
phase and a main phase.
[0405] The pilot phase, Part A, consisted of a two-way randomized
study to assess the safety and effect of NEUG versus pegfilgrastim,
with sequential enrollment to the following doses: NEUG 30 mg
(N=10) vs. pegfilgrastim (N=5); NEUG 40 mg (N=20) vs. pegfilgrastim
(N=10), and NEUG 50 mg (N=20) vs. pegfilgrastim (N=10). In a
further study, NEUG 60 mg (N=20) vs. pegfilgrastim (N=10) could
also be tested. In the Part A pilot phase, subjects were randomized
in a 2:1 ratio of NEUG to pegfilgrastim with a total of 10 subjects
in the 30 mg cohort and 20 subjects for each of the other cohorts.
NEUG or pegfilgrastim was administered to subjects 24 hours after
the chemotherapy treatment in each cycle. Subjects were assigned to
treatment groups using a stratified randomization for balance among
treatment groups based on weigh (<50 kg, .gtoreq.50 kg and
<80 kg, or .gtoreq.80 kg), prior chemotherapy exposure and
global location.
[0406] Following the pilot phase, 255 subjects were randomized
(1:1:1) to pegfilgrastim and the two well tolerated doses of NEUG
with the more comparable effect to pegfilgrastim in the pilot phase
(a 3-arm, balanced parallel-randomized phase). NEUG or
pegfilgrastim was administered 24 hours after the chemotherapy
treatments in each cycle. Subjects were assigned to treatment
groups using a stratified randomization for balance among treatment
groups based on weight (<50 kg, .gtoreq.50 kg and <80 kg, or
.gtoreq.80 kg).
[0407] During the pilot phase, adverse events were reviewed on an
ongoing basis. Escalation of the dose from 30 through 50 mg occurs
unless the ongoing review of data suggested a safety concern. If
the Cycle 1 ANC profile for Neugranin at 40 mg appeared inferior to
the profile observed from pegfilgrastim patients and 50 mg of
Neugranin is safe, then an additional arm may be randomized in a
2:1 ratio of Neugranin at 60 mg to pegfilgrastim with a total of 30
patients in the cohort.
[0408] Each dose level of NEUG is compared to pegfilgrastim for
safety and efficacy. Table 10 summarize the patient allocation for
Phase II, Part A and Part B.
TABLE-US-00010 TABLE 10 Allocation of Subjects in Phase II, Parts A
and B NEUG NEUG NEUG Pegfilgrastim Phase 30 mg 40 mg 50 mg 6 mg
Pilot 30 10 -- -- 5 Pilot 40 -- 20 -- 10 Pilot 50 -- -- 20 10 3-Arm
-- 85 85 85 Randomized Total 10 105 105 110
Safety Evaluation:
[0409] The safety of NEUG was assessed by evaluation of the type,
frequency, and severity of AEs, changes in clinical laboratory
tests (hematology and clinical chemistry), immunogenicity, physical
examinations, and the monitoring of vital signs over time. All AEs
and laboratory toxicities were graded based on the National Cancer
Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE
Version 3.0, 12 Dec. 2003).
[0410] Adverse events were captured from the start of study drug
administration through 30 days following the final dose of any
study drug. Serious adverse events (SAE) were captured from the
time of consent through 30 days following the final dose of any
study drug. Laboratory assessments were obtained as outlined in the
Schedule of Assessments.
[0411] b. Concomitant Therapy
Chemotherapy
[0412] The chemotherapy regimen for this trial consisted of
doxorubicin 60 mg/m.sup.2 and docetaxel 75 mg/m.sup.2 administered
sequentially by intravenous infusion on day 1 of treatment for up
to four 21-day cycles.
[0413] Prior to receiving each cycle of therapy, subjects were
required to have an absolute neutrophil count
(ANC)>1000/mm.sup.3 and platelets>100,000/mm.sup.3. Treatment
could be delayed up to two weeks for hematologic recovery. A 25%
dose reduction of chemotherapy doses was allowed for grade 3-4
non-hematologic toxicities, two grade 3-4 infectious episodes, or
grade 4 thrombocytopenia. The use of prophylactic antibiotics or
other hematopoietic growth factors was prohibited during trial
participation.
[0414] The combination of doxorubicin and docetaxel has been
reported to have significant clinical activity in patients with
breast cancer. However, the combination is highly myelosuppressive
with higher rates of grade 3 or 4 neutropenia than other standard
regimens.
[0415] Even with the addition of CSFs, the combination of
doxorubicin and docetaxel has induced Grade 4 neutropenia in 79% of
patients and febrile neutropenia rates of 9-18%. This
doxorubicin/docetaxel regimen has been used in studies of new
agents to prevent neutropenia and its complications. Therefore, the
combination of doxorubicin and docetaxel is an appropriate
chemotherapy regimen to study the potential of a new agent like
NEUG.
Doxorubicin
[0416] Pharmacologic Data
[0417] Doxorubicin hydrochloride is an anthracycline antibiotic
obtained from streptomyces peucetius var caesius which binds
directly to DNA base pairs (intercalates) and inhibits DNA and
DNA-dependent RNA synthesis, as well as protein synthesis.
Doxorubicin is active in all phases of the cell cycle but maximally
cytotoxic in S phase. Excretion of the drug is predominately by the
liver; renal clearance is minor.
[0418] Pharmaceutical Data
[0419] The drug is marketed commercially in 10, 20 50, 100 or 200
mg vials. Lyophilized preparations may be reconstituted with
sterile water for injection, dextrose 5% solution, or 0.9% saline
for injection.
[0420] Side Effects and Toxicity
[0421] Myelosuppression, primarily leukopenia, with a nadir of
approximately 10-14 days, and cardiotoxicity, including a rare,
acute pericarditis-myocarditis syndrome and a delayed, cumulative
dose related cardiomyopathy are the dose-limiting toxicities of
doxorubicin.
[0422] Marked alopecia and moderate nausea/vomiting are expected
toxicities. Extravasation reactions producing local skin and tissue
damage at the site of inadvertent extravasation, stomatitis,
hyperpigmentation of the skin (particularly the nailbeds), and a
"recall" phenomenon at sites of previous irradiation have been
reported.
Docetaxel
[0423] Pharmacologic Data
[0424] Docetaxel is a semisynthetic taxoid that binds to free
tubulin and promotes assembly of stable microtubules, interfering
with mitosis and cell replication (cell cycle specific for M
phase). Docetaxel is extensively protein-bound, extensively
metabolized in the liver, with fecal excretion of approximately 75%
of the dose within 7 days.
[0425] Pharmaceutical Data
[0426] Docetaxel (Taxotere.TM., Sanofi Aventis) is provided in 80
mg/2 mL or 20 mg/0.5 ml single-dose vials with an accompanying
diluent (13% ethanol in Water for Injection) vial. Each ml of
Taxotere contains 40 mg of docetaxel (anhydrous) and 1080 mg
polysorbate 80.
[0427] Side Effects and Toxicity
[0428] Docetaxel should not be given to patients who have a history
of severe hypersensitivity reactions to docetaxel or other drugs
formulated with polysorbate 80 such as etoposide and vitamin E.
[0429] Patients who experience severe hypersensitivity reactions
should not be rechallenged. All patients receiving docetaxel should
be premedicated with corticosteroids as outlined below.
[0430] Mild to moderate liver impairment results in delayed
metabolism by 27% and a 38% increase in systemic exposure (AUC).
Docetaxel should not be given to patients with SGOT and/or
SGPT>1.5 times normal limits and alkaline phosphatase>2.5
times normal limits. Fluid retention occurred in 17% (moderate) and
6% (severe retention) of patients in phase III studies despite
corticosteroid premedication. Severe neurosensory symptoms
(paresthesia, dyesthesia, pain) have been observed.
[0431] Expected side effects include myelosuppression, primarily
leukopenia, with a nadir of approximately 9 days with recovery by
day 15-21. Alopecia, nail and cutaneous changes, stomatitis,
myalgia/arthralgia, nausea/vomiting, and hypotension have been
reported.
[0432] Chemotherapy Dosage, Administration and Dose
Modifications
[0433] On day 1 of each treatment cycle, chemotherapy (doxorubicin
followed by docetaxel) was be administered.
[0434] Doxorubicin was administered at a dose of 60 mg/m.sup.2 by
IV bolus through the side arm of an infusing intravenous line or
central venous catheter to avoid extravasation injury.
[0435] Docetaxel 75 mg/m.sup.2 was diluted in 250 ml 0.9% saline or
5% dextrose solution and administered intravenously over
approximately 1 hour via a polyethylene-lined infusion set. Vital
signs were obtained immediately prior to and after the end of the
docetaxel infusion.
[0436] Subjects experiencing severe hypersensitivity reactions or
non-hematologic toxicities that preclude further cycles of
chemotherapy were be removed from study treatment and complete
follow-up.
[0437] Chemotherapy Pre-Medication
[0438] Oral (IV as needed) corticosteroids (such as dexamethasone 8
mg BID) was administered for three days starting 1 day prior to
docetaxel administration in order to reduce the incidence and
severity of fluid retention and hypersensitivity reactions.
[0439] The use and selection of anti-emetic agents or other
pre-medications (e.g. H.sub.2 antagonists) was left to the
discretion of the treating physician.
Prohibited Medications
[0440] Subjects were not to receive any of the following
medications and or procedures during this study and for the
additional times specified below: [0441] 1. Systemic antibiotics
within 72 hours of cycle 1 chemotherapy. [0442] 2. Other
investigational agents within 30 days of initiating study agent and
for the duration of the trial [0443] 3. Subsequent cycles of
chemotherapy should not be initiated until 14 days following dosing
with NEUG. [0444] 4. Cytokines, other hematopoietic growth factors
and prophylactic antibiotics for the duration of the trial unless
prolonged or febrile neutropenia occurs. If the subject is treated
with G-CSF at any time between the screening period and Day 0 they
will not be eligible to receive NEUG and will be discontinued from
the study.
Allowed Medications
[0445] Subjects were allowed to continue their baseline
medications(s). The daily dose of each medication was maintained
throughout the study if possible. If for any reason deemed
necessary by the investigator, a subject required additional
medication(s) or change of dose, the medication(s), route of
administration, and the indication for which it was given was be
recorded.
[0446] Subjects experiencing severe hypersensitivity reactions or
non-hematologic toxicities that preclude further cycles of
chemotherapy were removed from study treatment and completed
follow-up.
[0447] d. Pharmacokinetics
[0448] All subjects receiving NEUG were sampled for serum NEUG
concentrations during cycle 1. The drug was detected using a
sandwich enzyme-linked immunosorbent assay (ELISA) specific for
NEUG. The serum drug concentration-time data was subjected to PK
analysis using WinNonlin Enterprise Edition, Version 5.0 or higher,
using noncompartmental or model-based analysis. The following PK
parameters were determined: area under the curve
(AUC.sub.0-.infin.), clearance (CL/F), volume of distribution
(Vz/F), maximum concentration (Cmax), absorption half-life (t1/2,
abs), elimination half-life (t1/2, elim), and mean residence time
(MRT).
[0449] e. Immunogenicity
[0450] Serum samples for antibodies to NEUG were obtained prior to
dosing on Day 1 of every NEUG cycle and at the end of treatment
visit (approximately 30 days after the last dose) in subjects
receiving NEUG. If at any time during the study a subject developed
a positive anti-NEUG antibody response, a repeat sample was
obtained approximately 6 months after the final NEUG dose; if this
sample was positive, a sample was obtained at 12 months. The
protocol was later amended to require 6 and 12 month immunogenicity
samples from all subjects.
[0451] 7. Results
[0452] a. General
Statistical Methods
[0453] The sample size of about 85 subjects per arm in the main
phase of this trial (Part B) was chosen to provide 91% power to
establish non-inferiority of NEUG to pegfilgrastim with regard to
the primary endpoint of mean duration of severe neutropenia (DSN)
in cycle 1, with a non-inferiority margin of 1 day and an overall
1-sided significance level adjusted for multiple testing (by the
Hochberg method) of 0.025. Sample sizes were calculated based on
the normal approximation for two independent groups, an estimate of
1.6 days as the within-treatment standard deviation of cycle 1 DSN,
and a maximum rate of 20% not evaluable for the primary endpoint of
cycle 1 DSN.
[0454] Efficacy comparison was made between the two selected NEUG
doses (either 40 mg and 50 mg) and pegfilgrastim, based on subjects
in the 3-arm randomized phase (Part A).
[0455] Secondary efficacy analyses include the DSN in each of
chemotherapy cycles 2 through 4, depth of ANC nadir in each of the
cycles 1 through 4, rates of FN (defined as
ANC<0.5.times.10.sup.9/L with coincidental oral equivalent
temperature>38.2.degree. C.) by cycle and across all cycles, and
times to ANC recovery to >1.5.times.10.sup.9/L in all
cycles.
[0456] The data related to secondary efficacy analysis was analyzed
using appropriate statistical methods. Safety, PK, and
immunogenicity parameters were analyzed by descriptive statistical
methods.
[0457] For frequency and severity of adverse events, and for
laboratory toxicity grading, counts and rates are presented.
Efficacy Measures
[0458] Complete blood counts ("CBC") were obtained on day 1, 3 and
daily from day 5 until ANC>2.0.times.10.sup.9/L after the nadir,
then twice weekly, and at the end of treatment.
[0459] b. Efficacy of Phase II, Part A
[0460] Of the 78 subjects enrolled in the pilot phase of the study,
13 subjects did not complete the study, 3 (27.3%) treated with NEUG
30 mg, 3 (14.3%) treated with NEUG 40 mg, 3 (15.0%) treated with
NEUG 50 mg, and 4 (15.4%) treated with pegfilgrastim. The most
frequent reasons for early discontinuation were withdrawal of
consent (7 subjects) and decision of the investigator (3 subjects).
One NEUG 30 mg subject was withdrawn due to an adverse event
(diabetic foot).
[0461] The incidence of severe neutropenia and the mean duration of
severe neutropenia (DSN) were similar across treatment groups in
each chemotherapy cycle; however, the time to ANC recovery and the
incidence of febrile neutropenia suggested that NEUG 30 mg was not
quite as effective as NEUG 40 mg, NEUG 50 mg, or pegfilgrastim.
[0462] During Cycle 1, the proportion of subjects experiencing
febrile neutropenia was 20.0%, 9.5%, 10.0% and 8.0% for the NEUG 30
mg, 40 mg, 50 mg and pegfilgrastim group, respectively. Febrile
neutropenia was observed for only three additional subjects during
Cycles 2-4, one each in the NEUG 30 mg, NEUG 40 mg and
pegfilgrastim groups. FIG. 5 shows the ANC profile of a subset of
patients receiving either NEUG 30 or pegfilgrastim and who later
presented with grade 4 neutropenia.
[0463] In Cycle 1, the mean DSN was similar for NEUG 30 mg (0.9
days), NEUG 50 mg (1.1 days), and pegfilgrastim (0.9 days).
Although the mean DSN was slightly longer for NEUG 40 mg (1.6 days)
than the other three treatments, the differences among treatments
were all less than 1 day, the criterion to consider the treatments
equivalent in the main phase. The median DSN was 0 or 1 day in all
four treatment groups.
[0464] Summary statistics for the incidence and duration of Grade 3
or 4 neutropenia followed a similar pattern, i.e., the NEUG 30 mg,
NEUG 50 mg, and pegfilgrastim groups had similar outcomes, while
the incidence and duration of Grade 3 or 4 neutropenia were
slightly higher for the NEUG 40 mg group than for the other
treatment groups. The number of subjects in the pilot phase (Part
A) was fairly small, and the observed differences were not
statistically significant. NEUG 40 mg and NEUG 50 mg were selected
for further evaluation in Part B, the 3-arm randomized phase of the
study.
[0465] c. Efficacy of Phase II, Part B
[0466] Of the 256 subjects enrolled in the main phase of the study,
18 subjects did not complete the study; 10 (11.6%) treated with
NEUG 40 mg, 5 (6.0%) treated with NEUG 50 mg, and 3 (3.5%) treated
with pegfilgrastim. The most frequent reasons for early
discontinuation were withdrawal of consent (7 subjects) and AEs (4
subjects), including 2 deaths. The investigator considered all of
these AEs to be not related to study medication or chemotherapy. In
the main phase, 1 (1.2%) NEUG 40 mg subject was withdrawn before
being treated with study drug.
[0467] The incidence and duration of severe neutropenia in Cycle 1
are summarized in Table 11.
TABLE-US-00011 TABLE 11 Phase II, Part B: Incidence and duration of
severe neutropenia in Cycle 1 Neugranin Pegfil- 40 mg 50 mg All
Neug. grastim 95% CI (N = 85) (N = 84) (N = 169) (N = 86) 97.5% CI
Incidence of severe neutropenia n (%) 50 55 105 50 (58.8%) (65.5%)
(62.1%) (58.1%) NEUG 50 mg - NEUG 40 mg (-7.94; 21.24) NEUG 40 mg -
Pegfilgrastim (-14.09; 15.45) NEUG 50 mg - Pegfilgrastim (-7.23;
21.90) Duration (days) of severe neutropenia n 84 84 168 86 Mean
(SD) 1.0 (1.09) 1.3 (1.22) 1.2 (1.16) 1.2 (1.34) Median 1 1 1 1
Min/Max 0/4 0/5 0/5 0/5 95% CI NEUG 50 mg - NEUG 40 mg (-0.07;
0.58) NEUG 40 mg - Pegfilgrastim (-0.57; 0.15) NEUG 50 mg -
Pegfilgrastim (-0.31; 0.41) 97.5% CI NEUG 50 mg - NEUG 40 mg
(-0.12; 0.63) NEUG 40 mg - Pegfilgrastim (-0.62; 0.21) NEUG 50 mg -
Pegfilgrastim (-0.37; 0.46)
[0468] The incidence of severe neutropenia ranged from 58.1% in the
pegfilgrastim group to 65.5% in the NEUG 50 mg group. The treatment
effect was not statistically significant (p=0.559). The treatment
groups were comparable for Cycle 1 DSN, with mean values of 1.0,
1.3, and 1.2 days for the NEUG 40 mg, NEUG 50 mg, and pegfilgrastim
groups, respectively. The 95% and 97.5% two-sided confidence
intervals for differences between NEUG and pegfilgrastim were
strictly less than 1 day for both NEUG doses. This analysis
established non-inferiority of NEUG to pegfilgrastim. Across
treatment cycles, the incidences of severe neutropenia and Grade 3
or 4 neutropenia were lower in Cycles 2-4 than in Cycle 1. The mean
DSN and mean duration of Grade 3 or 4 neutropenia were smaller in
Cycles 2-4 than in Cycle 1. Within treatment cycles, the treatments
were similar, and treatment effect was not significantly different
for any of these parameters in any chemotherapy cycle.
[0469] The DSN were compared in patients grouped into weight
quartiles to determine if the fixed doses of NEUG provided adequate
support for patients of all weights. These results show that all
weight groups were adequately supported, as there is no significant
difference in the mean DSN among weight subgroups (Table 12).
TABLE-US-00012 TABLE 12 Cycle 1 duration of sever neutropenia (in
days), by weight Baseline weight (kg) 40-62 63-71 72-80 81-127
Pegfil- Mean (SD) 1.1 (1.3) 1.3 (1.4) 1.5 (1.6) 1.0 (1.0) grastim 6
mg N 16 21 26 23 Neugranin Mean (SD) 1.0 (1.0) 1.0 (1.2) 0.9 (1.0)
1.4 (1.4) 40 mg N 22 21 21 21 Neugranin Mean (SD) 1.3 (1.1) 1.0
(1.4) 1.4 (1.3) 1.5 (1.1) 50 mg N 15 26 20 23
[0470] Febrile neutropenia is summarized for all cycles in Table
13. During Cycle 1, the proportion of subjects experiencing febrile
neutropenia was 2 subjects (3.5%), 5 subjects (6.0%), and 2
subjects (2.3%) in the NEUG 40 mg, NEUG 50 mg, and pegfilgrastim
groups, respectively. Febrile neutropenia was observed for only
three additional subjects during Cycles 2-4, 2 subjects in the NEUG
40 mg group and 1 subject in the pegfilgrastim group. The treatment
effect was not statistically significant in any chemotherapy
cycle.
TABLE-US-00013 TABLE 13 Incidence of febrile neutropenia in cycles
1-4 Treatment Overall Cycle 1 Cycle 2 Cycle 3 Cycle 4 Neugranin 40
mg 4.7% (4/85) 3.5% 0.0% 2.4% 0.0% Neugranin 50 mg 6.0% (5/85) 6.0%
0.0% 0.0% 0.0% Pegfilgrastim 3.5% (3/86) 2.3% 0.0% 0.0% 1.2%
[0471] There were no significant differences between treatments for
duration of severe neutropenia in cycles 2-4 (Table 14).
TABLE-US-00014 TABLE 14 Mean duration of severe neutropenia in
cycles 2-4 Treatment Cycle 2 Cycle 3 Cycle 4 Neugranin 40 mg 0.5
0.4 0.4 Neugranin 50 mg 0.4 0.5 0.6 Pegfilgrastim 0.5 0.4 0.6
[0472] The mean time to ANC recovery (>1.5.times.109/L) was 2.0,
2.1, and 2.6 days for the Neugranin 40 mg, NEUG 50 mg, and
pegfilgrastim groups, respectively (Table 15). There were no
significant differences between treatment groups for the depth of
ANC nadir or time to nadir.
TABLE-US-00015 TABLE 15 ANC nadir, time to ANC nadir and time to
recovery Neugranin Pegfil- 40 mg 50 mg All Neug. grastim Parameter
(N = 85) (N = 84) N = 169) (N = 86) 95% CI p-value Nadir ANC
(10.sup.9/L) n 85 84 169 86 0.423 Mean (SD) 0.7 (0.88) 0.6 (0.68)
0.6 (0.79) 0.7 (1.04) Median 0 0 0 0 Min/Max 0/5 0/3 0/5 0/7 Time
(days) to Nadir ANC n 85 84 169 86 0.610 Mean (SD) 604 (1.38) 6.7
(2.62) 6.5 (2.09) 6.5 (2.05) Median 6 6 6 6 Min/Max 5/18 5/20 5/20
4/17 Time (days to ANC recover >1500 N 71 73 144 72 0.005 Mean
(SD) 2.0 (0.94) 2.1 (1.03.sup. 2.0 (0.98) 2.6 (1.23) Median 2 2 2 2
Min/Max 1/6 1/6 1/6 1/6 Treatment comparisons NEUG 50 mg - (-0.31;
0.39) NEUG 40 mg NEUG 40 mg - (-0.88; -0.17) Pegfilgrastim NEUG 50
mg - (-0.84; -0.13) Pegfilgrastim
[0473] d. Pharmacokinetics of Phase II, Part B
[0474] Serum Neugranin concentrations were determined using a
validated sandwich ELISA with a lower limit of quantification (LLQ)
of 6.312 ng/mL. Pharmacokinetic parameters were calculated using
noncompartmental modeling techniques, with the exception of the
absorption half-life, which was determined using a first-order
absorption, first-order elimination one-compartment model. Modeling
was performed with WinNonlin Professional (version 5.0.1). Serum
NEUG concentrations were determined in chemotherapy Cycle 1 in all
subjects treated with NEUG in Phase II. In the Part A of Phase II,
the median elimination half-life of NEUG was 33 hours in the 30 mg
dose group, 46 hours in the 40 mg dose group, and 18 hours in the
50 mg dose group (Table 16). In Part B, the median elimination
half-life of NEUG was 40 hours for 40 mg dose group, and 39 hours
for the 50 mg dose group (Table 17). During the Part A, PK sampling
was more frequent (pre-dose, 3 h, 6 h, 12 h, 24 h Day 3, Day 5-9,
Day 11) than for Part B (pre-dose, Day 3, Day 5-8).
TABLE-US-00016 TABLE 16 Median elimination half-life by treatment,
Phase II, Part A NEUG NEUG NEUG Pegfilgrastim 30 mg 40 mg 50 mg 6
mg Number of subjects 10 20 20 26 Number of subjects 3 12 16 19
evaluated for elimination half-life Median half-life (hr) 33 46 18
40
TABLE-US-00017 TABLE 17 Median elimination half-life by treatment,
Phase II, Part B NEUG NEUG Pegfilgrastim 40 mg 50 mg 6 mg Number of
subjects 85 84 84 Number of subjects 48 54 52 evaluated for
elimination half-life Median half-life (hr) 40 39 50
[0475] Serum pegfilgrastim concentrations were determined using a
validated sandwich ELISA in chemotherapy Cycle 1 in all subjects
treated with pegfilgrastim in Phase II. In Part A, the median
elimination half-life of pegfilgrastim was about 40 hours. In Part
B, the median elimination half-life of pegfilgrastim was about 50
hours. The elimination half-life is reported to be 3-4 hours for
filgrastim and 42-67.5 hours (depending on dose) for
pegfilgrastim.
[0476] e. Immunogenicity
[0477] Among the study participants, there was one confirmed
anti-G-CSF/neoepitope antibody response in the Neugranin-treated
subjects and one anti-G-CSF response in the pegfilgrastim-treated
group, or 0.5% and 0.9%, respectively (Table 18). In both cases,
the subjects had elevated non-specific binding in pre-dose
samples.
TABLE-US-00018 TABLE 18 Summary of G-CSF specific treatment
emergent immune responses to NEUG and Pegfilgrastim NEUG
Pegfilgrastim Positive response/number Positive response/number of
subjects of subjects Phase II, Part A 0/50 0/26 (4 cycles maximum)
Phase II, Part B 1/169 1/86 (4 cycles maximum) Total number of
1/219 1/112 subjects
[0478] After NEUG treatment, very low levels of confirmed positive
antibodies were seen in the patient, with no apparent increase in
the magnitude of the response after repeated doses (data not
shown). In the pegfilgrastim-treated patient, an unusually high
non-specific background binding was observed; however, only a
transient confirmed antibody response was seen after Cycle 2
treatment (data not shown). No antibody response was
neutralizing.
[0479] Anti-HSA antibodies were naturally occurring at a low level
in this population, with 6.9% of the subjects testing positive for
HSA antibodies in the pre-dose evaluation. Treatment emergent
anti-HSA antibodies were observed in four NEUG-treated subjects,
1.8% (Table 19). All responses were transient and weak. Three
responses emerged after the first treatment cycle and were
undetectable after Cycles 2, 3 and 4. One response occurred after
the third treatment but was undetectable at the 30 day follow-up
after the 4th treatment (data not shown).
TABLE-US-00019 TABLE 19 Summary of HSA-specific treatment emergent
immune responses to NEUG NEUG Positive response/number of subjects
Phase II, Part A 0/50 (4 cycles maximum) Phase II, Part B 4/169 (4
cycles maximum) Total number of subjects 4/219
[0480] f. Treatment-Emergent Adverse Events in Phase II, Part B
[0481] In Phase II, Part B, .gtoreq.90% of subjects in each
treatment group experienced at least one treatment-emergent adverse
event (TEAE), and the percent of subjects with at least one TEAE
related to study medication ranged from 23.1% in the pegfilgrastim
group to 35.0% in the Neugranin 50 mg group. The percent of
subjects with at least one SAE was highest in the NEUG 30 mg group
(30%), but was approximately 15% in the other three treatment
groups. None of the SAEs were related to study medication. One
patient (NEUG 30 mg) was withdrawn from the study due to diabetic
foot, which was considered to be not related to study medication.
In the Part B, all except 8 subjects (2 NEUG 40 mg, 3 NEUG 50 mg, 3
pegfilgrastim) had at least one TEAE. The percent of subjects with
at least one TEAE related to study medication was 20.2% in the NEUG
50 mg group, 22.4% in the NEUG 40 mg group and 22.1% in subjects
receiving pegfilgrastim. Two subjects (1 NEUG 40 mg, 1
pegfilgrastim) died during the study, and 6-8 subjects in each
treatment group experienced at least one SAE. No deaths or SAEs
were considered to be related to study medication.
[0482] The total number of TEAEs was similar across treatment
groups in Part A, when sample size is taken into consideration for
the NEUG 30 mg dose, and in Part B. In both Parts A and B, the
percent of TEAEs with CTC Grade 3 or higher was similar for NEUG
and pegfilgrastim as was the percent of TEAEs related to study
medication.
[0483] g. Dose Response
[0484] The results of Phase II demonstrated that both 40 and 50 mg
fixed doses of NEUG provided equivalent safety and efficacy to 6 mg
of pegfilgrastim in breast cancer subjects treated with myelotoxic
chemotherapy. While the mean DSN for the 40 mg treatment group was
slightly lower than the mean DSN of the 50 mg group, these
differences were not statistically significant. A dose response was
observed for AUC.sub.ANC (Days 0-15 in cycle 1) both when
weight-adjusted dose was considered and for fixed dose cohorts
(FIG. 24). The AUC.sub.ANC for the 30 mg cohort was slightly lower
than that of pegfilgrastim, indicating that the 30 mg fixed dose
was less effective in this study, whereas AUC.sub.ANC for the 40 mg
and the 50 mg cohorts were dose-related and higher (although not
significantly) than the AUC.sub.ANC for pegfilgrastim treated
subjects. From the above analysis, a dose response is apparent when
NEUG is administered on a weight adjusted basis (mg/kg). However,
comparison of DSN in cycle 1 for Phase II, Part B suggested that
patients of all weight quartiles were adequately supported as DSN
did not vary significantly among the treatment arms (40 and 50 mg
NEUG and pegfilgrastim) nor with weight-adjusted dose (mg/kg).
Further, there was no evidence that a fixed dose might result in an
altered safety profile in lighter patients as the incidence and
severity of related adverse events (bone pain in particular; data
not shown) did not correlate with dose received per kilogram body
weight, nor were they different from those with pegfilgrastim.
[0485] It will be apparent to those skilled in the art that various
modifications and variations can be made in the methods and
compositions of the present invention without departing from the
spirit or scope of the invention. Thus, it is intended that the
present invention cover the modifications and variations of this
invention provided they come within the scope of the appended
claims and their equivalents.
Sequence CWU 1
1
616PRTUnknownDescription of Unknown HSA pro-peptide sequence 1Arg
Gly Val Phe Arg Arg1 526PRTUnknownDescription of Unknown Yeast
sequence 2Arg Ser Leu Asp Lys Arg1 532277DNAHomo
sapiensCDS(1)..(2277) 3gat gca cac aag agt gag gtt gct cat cgg ttt
aaa gat ttg gga gaa 48Asp Ala His Lys Ser Glu Val Ala His Arg Phe
Lys Asp Leu Gly Glu1 5 10 15gaa aat ttc aaa gcc ttg gtg ttg att gcc
ttt gct cag tat ctt cag 96Glu Asn Phe Lys Ala Leu Val Leu Ile Ala
Phe Ala Gln Tyr Leu Gln 20 25 30cag tgt cca ttt gaa gat cat gta aaa
tta gtg aat gaa gta act gaa 144Gln Cys Pro Phe Glu Asp His Val Lys
Leu Val Asn Glu Val Thr Glu 35 40 45ttt gca aaa aca tgt gtt gct gat
gag tca gct gaa aat tgt gac aaa 192Phe Ala Lys Thr Cys Val Ala Asp
Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60tca ctt cat acc ctt ttt gga
gac aaa tta tgc aca gtt gca act ctt 240Ser Leu His Thr Leu Phe Gly
Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80cgt gaa acc tat ggt
gaa atg gct gac tgc tgt gca aaa caa gaa cct 288Arg Glu Thr Tyr Gly
Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95gag aga aat gaa
tgc ttc ttg caa cac aaa gat gac aac cca aac ctc 336Glu Arg Asn Glu
Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110ccc cga
ttg gtg aga cca gag gtt gat gtg atg tgc act gct ttt cat 384Pro Arg
Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120
125gac aat gaa gag aca ttt ttg aaa aaa tac tta tat gaa att gcc aga
432Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
130 135 140aga cat cct tac ttt tat gcc ccg gaa ctc ctt ttc ttt gct
aaa agg 480Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala
Lys Arg145 150 155 160tat aaa gct gct ttt aca gaa tgt tgc caa gct
gct gat aaa gct gcc 528Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala
Ala Asp Lys Ala Ala 165 170 175tgc ctg ttg cca aag ctc gat gaa ctt
cgg gat gaa ggg aag gct tcg 576Cys Leu Leu Pro Lys Leu Asp Glu Leu
Arg Asp Glu Gly Lys Ala Ser 180 185 190tct gcc aaa cag aga ctc aag
tgt gcc agt ctc caa aaa ttt gga gaa 624Ser Ala Lys Gln Arg Leu Lys
Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205aga gct ttc aaa gca
tgg gca gta gct cgc ctg agc cag aga ttt ccc 672Arg Ala Phe Lys Ala
Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220aaa gct gag
ttt gca gaa gtt tcc aag tta gtg aca gat ctt acc aaa 720Lys Ala Glu
Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235
240gtc cac acg gaa tgc tgc cat gga gat ctg ctt gaa tgt gct gat gac
768Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
245 250 255agg gcg gac ctt gcc aag tat atc tgt gaa aat caa gat tcg
atc tcc 816Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser
Ile Ser 260 265 270agt aaa ctg aag gaa tgc tgt gaa aaa cct ctg ttg
gaa aaa tcc cac 864Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu
Glu Lys Ser His 275 280 285tgc att gcc gaa gtg gaa aat gat gag atg
cct gct gac ttg cct tca 912Cys Ile Ala Glu Val Glu Asn Asp Glu Met
Pro Ala Asp Leu Pro Ser 290 295 300tta gct gct gat ttt gtt gaa agt
aag gat gtt tgc aaa aac tat gct 960Leu Ala Ala Asp Phe Val Glu Ser
Lys Asp Val Cys Lys Asn Tyr Ala305 310 315 320gag gca aag gat gtc
ttc ctg ggc atg ttt ttg tat gaa tat gca aga 1008Glu Ala Lys Asp Val
Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335agg cat cct
gat tac tct gtc gtg ctg ctg ctg aga ctt gcc aag aca 1056Arg His Pro
Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350tat
gaa acc act cta gag aag tgc tgt gcc gct gca gat cct cat gaa 1104Tyr
Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360
365tgc tat gcc aaa gtg ttc gat gaa ttt aaa cct ctt gtg gaa gag cct
1152Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
370 375 380cag aat tta atc aaa caa aat tgt gag ctt ttt gag cag ctt
gga gag 1200Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu
Gly Glu385 390 395 400tac aaa ttc cag aat gcg cta tta gtt cgt tac
acc aag aaa gta ccc 1248Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr
Thr Lys Lys Val Pro 405 410 415caa gtg tca act cca act ctt gta gag
gtc tca aga aac cta gga aaa 1296Gln Val Ser Thr Pro Thr Leu Val Glu
Val Ser Arg Asn Leu Gly Lys 420 425 430gtg ggc agc aaa tgt tgt aaa
cat cct gaa gca aaa aga atg ccc tgt 1344Val Gly Ser Lys Cys Cys Lys
His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445gca gaa gac tat cta
tcc gtg gtc ctg aac cag tta tgt gtg ttg cat 1392Ala Glu Asp Tyr Leu
Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460gag aaa acg
cca gta agt gac aga gtc acc aaa tgc tgc aca gaa tcc 1440Glu Lys Thr
Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475
480ttg gtg aac agg cga cca tgc ttt tca gct ctg gaa gtc gat gaa aca
1488Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495tac gtt ccc aaa gag ttt aat gct gaa aca ttc acc ttc cat
gca gat 1536Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His
Ala Asp 500 505 510ata tgc aca ctt tct gag aag gag aga caa atc aag
aaa caa act gca 1584Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys
Lys Gln Thr Ala 515 520 525ctt gtt gag ctc gtg aaa cac aag ccc aag
gca aca aaa gag caa ctg 1632Leu Val Glu Leu Val Lys His Lys Pro Lys
Ala Thr Lys Glu Gln Leu 530 535 540aaa gct gtt atg gat gat ttc gca
gct ttt gta gag aag tgc tgc aag 1680Lys Ala Val Met Asp Asp Phe Ala
Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560gct gac gat aag gag
acc tgc ttt gcc gag gag ggt aaa aaa ctt gtt 1728Ala Asp Asp Lys Glu
Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575gct gca agt
caa gct gcc tta ggc tta acc ccc ctg ggc cct gcc agc 1776Ala Ala Ser
Gln Ala Ala Leu Gly Leu Thr Pro Leu Gly Pro Ala Ser 580 585 590tcc
ctg ccc cag agc ttc ctg ctc aag tgc tta gag caa gtg agg aag 1824Ser
Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys 595 600
605atc cag ggc gat ggc gca gcg ctc cag gag aag ctg tgt gcc acc tac
1872Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr
610 615 620aag ctg tgc cac ccc gag gag ctg gtg ctg ctc gga cac tct
ctg ggc 1920Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser
Leu Gly625 630 635 640atc ccc tgg gct ccc ctg agc agc tgc ccc agc
cag gcc ctg cag ctg 1968Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser
Gln Ala Leu Gln Leu 645 650 655gca ggc tgc ttg agc caa ctc cat agc
ggc ctt ttc ctc tac cag ggg 2016Ala Gly Cys Leu Ser Gln Leu His Ser
Gly Leu Phe Leu Tyr Gln Gly 660 665 670ctc ctg cag gcc ctg gaa ggg
atc tcc ccc gag ttg ggt ccc acc ttg 2064Leu Leu Gln Ala Leu Glu Gly
Ile Ser Pro Glu Leu Gly Pro Thr Leu 675 680 685gac aca ctg cag ctg
gac gtc gcc gac ttt gcc acc acc atc tgg cag 2112Asp Thr Leu Gln Leu
Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln 690 695 700cag atg gaa
gaa ctg gga atg gcc cct gcc ctg cag ccc acc cag ggt 2160Gln Met Glu
Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly705 710 715
720gcc atg ccg gcc ttc gcc tct gct ttc cag cgc cgg gca gga ggg gtc
2208Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val
725 730 735ctg gtt gcc tcc cat ctg cag agc ttc ctg gag gtg tcg tac
cgc gtt 2256Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr
Arg Val 740 745 750cta cgc cac ctt gcc cag ccc 2277Leu Arg His Leu
Ala Gln Pro 7554759PRTHomo sapiens 4Asp Ala His Lys Ser Glu Val Ala
His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu Val
Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe Glu Asp
His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys Thr Cys
Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu His Thr
Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80Arg Glu
Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95Glu
Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105
110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile
Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe
Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys
Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp
Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys Gln Arg
Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe
Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys
Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230
235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp
Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp
Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu
Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu
Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu
Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp
Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His
Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345
350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu
355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu
Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu
Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val
Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro Thr Leu
Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser Lys Cys
Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala Glu Asp
Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460Glu
Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470
475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu
Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe
His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile
Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro
Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe
Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys
Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala
Ser Gln Ala Ala Leu Gly Leu Thr Pro Leu Gly Pro Ala Ser 580 585
590Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys
595 600 605Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala
Thr Tyr 610 615 620Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly
His Ser Leu Gly625 630 635 640Ile Pro Trp Ala Pro Leu Ser Ser Cys
Pro Ser Gln Ala Leu Gln Leu 645 650 655Ala Gly Cys Leu Ser Gln Leu
His Ser Gly Leu Phe Leu Tyr Gln Gly 660 665 670Leu Leu Gln Ala Leu
Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu 675 680 685Asp Thr Leu
Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln 690 695 700Gln
Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly705 710
715 720Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly
Val 725 730 735Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser
Tyr Arg Val 740 745 750Leu Arg His Leu Ala Gln Pro 7555204PRTHomo
sapiens 5Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala
Leu Gln1 5 10 15Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu
Ala Thr Pro 20 25 30Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu
Leu Lys Cys Leu 35 40 45Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala
Ala Leu Gln Glu Lys 50 55 60Leu Cys Ala Thr Tyr Lys Leu Cys His Pro
Glu Glu Leu Val Leu Leu65 70 75 80Gly His Ser Leu Gly Ile Pro Trp
Ala Pro Leu Ser Ser Cys Pro Ser 85 90 95Gln Ala Leu Gln Leu Ala Gly
Cys Leu Ser Gln Leu His Ser Gly Leu 100 105 110Phe Leu Tyr Gln Gly
Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu 115 120 125Leu Gly Pro
Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala 130 135 140Thr
Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu145 150
155 160Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln
Arg 165 170 175Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
Phe Leu Glu 180 185 190Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln
Pro 195 2006609PRTHomo sapiens 6Met Lys Trp Val Thr Phe Ile Ser Leu
Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Arg Gly Val Phe Arg Arg
Asp Ala His Lys Ser Glu Val Ala 20 25 30His Arg Phe Lys Asp Leu Gly
Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45Ile Ala Phe Ala Gln Tyr
Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60Lys Leu Val Asn Glu
Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp65 70 75 80Glu Ser Ala
Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95Lys Leu
Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105
110Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln
115 120 125His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro
Glu Val 130 135 140Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu
Thr Phe Leu Lys145 150 155 160Lys Tyr Leu Tyr Glu Ile Ala Arg Arg
His Pro Tyr Phe Tyr Ala Pro 165 170 175Glu Leu Leu Phe Phe Ala Lys
Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190Cys Gln Ala Ala Asp
Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205Leu Arg Asp
Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220Ala
Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val225 230
235 240Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val
Ser 245
250 255Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His
Gly 260 265 270Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala
Lys Tyr Ile 275 280 285Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu
Lys Glu Cys Cys Glu 290 295 300Lys Pro Leu Leu Glu Lys Ser His Cys
Ile Ala Glu Val Glu Asn Asp305 310 315 320Glu Met Pro Ala Asp Leu
Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335Lys Asp Val Cys
Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350Met Phe
Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360
365Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys
370 375 380Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe
Asp Glu385 390 395 400Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu
Ile Lys Gln Asn Cys 405 410 415Glu Leu Phe Glu Gln Leu Gly Glu Tyr
Lys Phe Gln Asn Ala Leu Leu 420 425 430Val Arg Tyr Thr Lys Lys Val
Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445Glu Val Ser Arg Asn
Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460Pro Glu Ala
Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val465 470 475
480Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg
485 490 495Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro
Cys Phe 500 505 510Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys
Glu Phe Asn Ala 515 520 525Glu Thr Phe Thr Phe His Ala Asp Ile Cys
Thr Leu Ser Glu Lys Glu 530 535 540Arg Gln Ile Lys Lys Gln Thr Ala
Leu Val Glu Leu Val Lys His Lys545 550 555 560Pro Lys Ala Thr Lys
Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575Ala Phe Val
Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590Ala
Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600
605Leu
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