U.S. patent application number 12/567040 was filed with the patent office on 2011-01-20 for combination treatment for non-hematologic malignancies.
This patent application is currently assigned to PFIZER INC.. Invention is credited to Bruce D. Cohen, Antonio Gualberto, Carrie L. Melvin, M. Luisa Roberts.
Application Number | 20110014207 12/567040 |
Document ID | / |
Family ID | 35058695 |
Filed Date | 2011-01-20 |
United States Patent
Application |
20110014207 |
Kind Code |
A1 |
Gualberto; Antonio ; et
al. |
January 20, 2011 |
COMBINATION TREATMENT FOR NON-HEMATOLOGIC MALIGNANCIES
Abstract
The present invention relates to a therapeutic method for the
treatment of non-hematologic malignancies comprising administering
anti-IGF-1R antibodies, particularly human anti-IGF-1R antibodies,
to a patient, in conjunction with the administration of at least
one other therapeutic agent. The invention further relates to
pharmaceutical compositions comprising these antibodies and methods
of using such compositions thereof for treatment.
Inventors: |
Gualberto; Antonio; (East
Greenwich, RI) ; Cohen; Bruce D.; (East Lyme, CT)
; Melvin; Carrie L.; (Wayland, MA) ; Roberts; M.
Luisa; (Noank, CT) |
Correspondence
Address: |
PFIZER INC;Mary J Hosley
150 EAST 42ND STREET, MS: 150/02/E112
NEW YORK
NY
10017-5612
US
|
Assignee: |
PFIZER INC.
|
Family ID: |
35058695 |
Appl. No.: |
12/567040 |
Filed: |
September 25, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11182343 |
Jul 15, 2005 |
7618626 |
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12567040 |
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60588721 |
Jul 16, 2004 |
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Current U.S.
Class: |
424/158.1 |
Current CPC
Class: |
A61K 31/28 20130101;
A61K 31/28 20130101; A61K 31/337 20130101; A61K 45/06 20130101;
A61K 39/39541 20130101; A61K 31/573 20130101; A61K 31/337 20130101;
A61K 39/39541 20130101; A61P 35/00 20180101; A61K 2300/00 20130101;
A61K 31/573 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/158.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method for the treatment of lung cancer in a patient in need
of such treatment comprising the step of administering to the
patient a therapeutically effective amount of an antibody that
specifically binds to IGF-1R in combination with a therapeutically
effective amount of a taxane and a therapeutically effective amount
of carboplatin, wherein the antibody is selected from the group
consisting of 2.12.1, 2.13.2, 2.14.3, 4.9.2, 4.17.3, and 6.1.1.
2. (canceled)
3. The method of claim 1, wherein the taxane is docetaxel.
4. The method of claim 1, wherein the taxane is paclitaxel.
5-16. (canceled)
17. The method according to claim 1, where in the amount of the
antibody is in the range of 0.05-20 mg/kg.
18. The method according to claim 1, where in the amount of the
antibody is in the range of 0.5-10 mg/kg.
19. The method according to claim 4, wherein the antibody is
2.13.2.
20. The method according to claim 4, wherein the cancer is
non-small cell lung cancer.
21. A method of treatment for non-small cell lung cancer in a
patient in need thereof, comprising administering to the patient a
therapeutically effective amount of antibody 2.13.2 in combination
of carboplatin and a therapeutically effective amount of
paclitaxel.
22. The method according to claim 21, wherein the amount of the
antibody is in the range of 0.05-20 mg/kg.
23. The method according to claim 21, wherein the antibody is
administered once every two weeks, once every three weeks, or once
every month.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a method of treatment for
non-hematologic malignancies comprising the administration of
anti-insulin-like growth factor I receptor (IGF-1R) antibodies, in
conjunction with other therapeutic agents such as chemotherapeutic
agents and hormonal therapy.
[0002] The insulin-like growth factor (IGF) signaling system plays
an important role in the growth and development of many tissues and
regulates overall growth. Insulin-like growth factor (IGF-1) is a
7.5-kD polypeptide that circulates in plasma in high concentrations
and is detectable in most tissues. IGF-1 stimulates cell
differentiation and cell proliferation, and is required by most
mammalian cell types for sustained proliferation. These cell types
include, among others, human diploid fibroblasts, epithelial cells,
smooth muscle cells, T lymphocytes, neural cells, myeloid cells,
chondrocytes, osteoblasts and bone marrow stem cells.
[0003] The first step in the transduction pathway leading to
IGF-1-stimulated cellular proliferation or differentiation is
binding of IGF-1 or IGF-2 (or insulin at supraphysiological
concentrations) to the IGF-1 receptor. The IGF-1 receptor (IGF-1R)
is composed of two types of subunits: an alpha subunit (a 130-135
kD protein that is entirely extracellular and functions in ligand
binding) and a beta subunit (a 95-kD transmembrane protein, with
transmembrane and cytoplasmic domains). IGF binding proteins
(IGFBPs) have growth inhibiting effects by, at least in part,
competitively binding IGFs and preventing their association with
IGF-1F. The interactions between IGF-1, IGF-2, IGF1R, and IGFBPs
affect many physiological and pathological processes such as
development, growth and metabolic regulation.
[0004] The IGF-1R is initially synthesized as a single chain
proreceptor polypeptide that is processed by glycosylation,
proteolytic cleavage, and covalent bonding to assemble into a
mature 460-kD heterotetramer comprising two alpha-subunits and two
beta-subunits. The beta subunit(s) possesses ligand-activated
tyrosine kinase activity. This activity is implicated in the
signaling pathways mediating ligand action which involve
autophosphorylation of the beta-subunit and phosphorylation of
IGF-1R substrates.
[0005] There is considerable evidence for a role for IGF-1 and/or
IGF-1R in the maintenance of tumor cells in vitro and in vivo.
IGF-1R levels are elevated in tumors of lung (Kaiser et al., J.
Cancer Res. Clin. Oncol. 119: 665-668, 1993; Moody et al., Life
Sciences 52: 1161-1173, 1993; Macauley et al., Cancer Res., 50:
2511-2517, 1990), breast (Pollack et al., Cancer Lett. 38: 223-230,
1987; Foekens et al., Cancer Res. 49: 7002-7009, 1989; Cullen et
al., Cancer Res. 49: 7002-7009, 1990; Arteaga et al., J. Clin.
Invest. 84: 1418-1423, 1989), prostate and colon (Remaole-Bennet et
al., J. Clin. Endocrinol. Metab. 75: 609-616, 1992; Guo et al.,
Gastroenterol. 102: 1101-1108, 1992). In addition, IGF-1 appears to
be an autocrine stimulator of human gliomas (Sandberg-Nordqvist et
al., Cancer Res. 53: 2475-2478, 1993), while IGF-1 stimulated the
growth of fibrosarcomas that overexpressed IGF-1R (Butler et al.,
Cancer Res. 58: 3021-27, 1998). In addition, individuals with "high
normal" levels of IGF-1 have an increased risk of common cancers
compared to individuals with IGF-1 levels in the "low normal" range
(Rosen et al., Trends Endocrinol. Metab. 10: 136-41, 1999). For a
review of the role IGF-1/IGF-1 receptor interaction plays in the
growth of a variety of human tumors, see Macaulay, Br. J. Cancer,
65: 311-320, 1992.
[0006] Numerous classes of antineoplastic agents are currently in
use. Docetaxel, one of a group of drugs called "taxanes," which are
derived from the bark and needles of yew trees, is the first
anticancer agent to show a significantly higher response rate than
doxorubicin, a very active agent and widely used chemotherapy in
the first-line treatment of metastatic breast cancer. Docetaxel
also is the first chemotherapy drug as a single agent to
demonstrate increased survival among patients with advanced breast
cancer compared to the combination of mitomycin C and vinblastine,
a commonly used regimen in this patient population. Median time to
progression and time to treatment failure were significantly longer
for docetaxel than for mitomycin C in combination with vinblastine,
and the one-year survival rate significantly greater. Promising
results have also been recorded for docetaxel in other human
malignancies, such as ovarian, lung, head and neck, gastric and
pancreatic cancers.
[0007] Paclitaxel, also a taxane, binds to microtubules and
prevents their molecular disassembly, thereby inhibiting mitosis
(cell division). With the spindle still in place the cell cannot
divide into daughter cells. Paclitaxel is most effective against
ovarian carcinomas and advanced breast carcinomas.
[0008] Hormonal therapy can be very effective in lowering the risk
of recurrence for women with hormone-receptor-positive breast
cancer. Tamoxifen is the hormonal therapy that has been around the
longest--nearly 30 years. It blocks the effect of estrogen on
breast cancer cells, keeping the cells from growing. Tamoxifen can
reduce recurrence by 40-50% in post-menopausal women, and by 30-50%
in pre-menopausal women. It also lowers the risk of a new breast
cancer developing in the unaffected breast, and can slow down the
progression of advanced disease.
[0009] In recent years, aromatase inhibitors have been used as
hormonal therapy. This type of therapy is recommended only for
postmenopausal women with hormone-receptor-positive breast cancer.
It works by blocking the production of estrogen in muscle and fat
tissue, which is the main source of estrogen in women beyond
menopause, after which the ovaries stop making significant levels
of estrogen.
[0010] Prostate cancer is the most common cancer and the second
cause of cancer death in men in the United States. About 10% of the
initial cases of prostate cancer present with metastatic disease.
However, in the rest, metastases will develop despite treatment
with surgery, radiation or medical therapy, and those metastases
will eventually become refractory to hormonal treatment. The use of
chemotherapy in hormone refractory (androgen independent)
progressive prostate cancer (HRPC) has been characterized
historically by poor efficacy and high toxicity. Newer regimens
containing docetaxel have shown a survival benefit over previous
palliative regimens. Despite this positive trend, the median
survival of HRPC patients treated with docetaxel and prednisone is
only 18.9 months; clearly, more effective regimens are required for
the treatment of HRPC patients.
[0011] Although some currently available anti-cancer treatments
have been successful, complete responses to these treatments are
infrequently observed, and the patient population refractory to
these treatments is still large. Thus, development of new
therapeutic regimens, particularly those capable of augmenting or
potentiating the anti-tumor activity of other anti-neoplastic
agents, is necessary.
[0012] In view of the roles that IGF-1 and IGF-1R have in such
disorders as cancer and other proliferative disorders when IGF-1
and/or IGF-1R are overexpressed, antibodies to IGF-1R have been
produced that block binding of IGF-1 or IGF-2 to IGF-1R. Such
antibodies are described, for example, in International Patent
Application No. WO 02/053596, published Jul. 11, 2002;
International Patent Application Nos. WO 05/016967 and WO
05/016970, both published Feb. 24, 2005; International Patent
Application No. WO 03/106621, published Dec. 24, 2003;
International Patent Application No. WO 04/083248, published Sep.
30, 2004; International Patent Application No. WO 03/100008,
published Dec. 4, 2003; International Patent Publication WO
04/087756, published Oct. 14, 2004; and International Patent
Application No WO 05/005635, published Jan. 26, 2005. Because of
their ability to block a tumor cell survival pathway, it is
desirable to use such anti-IGF-1R antibodies to treat cancer,
particularly non-hematological malignancies, in patients to obtain
an improved clinical benefit relative to standard cancer treatment
regimes alone.
SUMMARY OF THE INVENTION
[0013] The present invention is directed to a method for the
treatment of an advanced non-hematologic malignancy in a patient in
need of such treatment comprising the step of administering to the
patient a therapeutically effective amount of an anti-IGF-1R
antibody.
[0014] More particularly, the present invention is directed to a
method comprising the step of administering to the patient an
antibody that specifically binds to IGF-1R in combination with a
therapeutically effective amount of at least one agent selected
from the group consisting of an alkylating agent, a folate
antagonist, a pyrimidine antagonist, a cytotoxic antibiotic, a
platinum compound, a taxane, a vinca alkaloid, a topoisomerase
inhibitor, an EGFR inhibitor, and a hormonal therapy agent.
Preferably the antibody is one that specifically binds to human
IGF-1R.
[0015] In a preferred embodiment of the present invention, the
anti-IGF-1R antibody has the following properties: (a) a binding
affinity for human IGF-1R of K.sub.d of 8.times.10.sup.-9 or less,
and (b) inhibition of binding between human IGF-1R and IGF-1 with
an IC.sub.50 of less than 100 nM.
[0016] In another preferred embodiment of the present invention,
the anti-IGF-1R antibody comprises (a) a heavy chain comprising the
amino acid sequences of CDR-1, CDR-2, and CDR-3 of an antibody
selected from the group consisting of 2.12.1, 2.13.2, 2.14.3,
4.9.2, 4.17.3, and 6.1.1, and (b) a light chain comprising the
amino acid sequences of CDR-1, CDR-2, and CDR-3 of an antibody
selected from the group consisting of 2.12.1, 2.13.2, 2.14.3,
4.9.2, 4.17.3, and 6.1.1, or (c) sequences having changes from the
CDR sequences of an antibody selected from the group consisting of
2.12.1, 2.13.2, 2.14.3, 4.9.2, 4.17.3, and 6.1.1, said sequences
being selected from the group consisting of conservative changes,
wherein the conservative changes are selected from the group
consisting of replacement of nonpolar residues by other nonpolar
residues, replacement of polar charged residues by other polar
uncharged residues, replacement of polar charged residues by other
polar charged residues, and substitution of structurally similar
residues; and non-conservative substitutions, wherein the
non-conservative substitutions are selected from the group
consisting of substitution of polar charged residue for polar
uncharged residues and substitution of nonpolar residues for polar
residues, additions and deletions.
[0017] The present invention is also directed to a pharmaceutical
composition for the treatment of a non-hematologic malignancy
comprising (a) a therapeutically effective amount of an antibody
that specifically binds IGF-1R, (b) a therapeutically effective
amount of at least one agent selected from the group consisting of
an alkylating agent, a folate antagonist, a pyrimidine antagonist,
a cytotoxic antibiotic, a platinum compound, a taxane, a vinca
alkaloid, a topoisomerase inhibitor, an EGFR inhibitor, and a
hormonal therapy agent; and (c) a pharmaceutically acceptable
carrier.
DETAILED DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-10 show alignments of the nucleotide sequences of
the light chain variable regions from six human anti-IGF-1R
antibodies to each other and to germline sequences. FIG. 1A shows
the alignment of the nucleotide sequences of the variable region of
the light chain (VL) of antibodies 2.12.1 (SEQ ID NO: 1) 2.13.2
(SEQ ID NO: 5), 2.1-4.3 (SEQ ID NO: 9) and 4.9.2 (SEQ ID NO: 13) to
each other and to the germline V.kappa. A30 sequence (SEQ ID NO:
39). FIG. 1B shows the alignment of the nucleotide sequence of VL
of antibody 4.17.3 (SEQ ID NO: 17) to the germline V.kappa. O12
sequence (SEQ ID NO: 41). FIG. 10 shows the alignment of the
nucleotide sequence of VL of antibody 6.1.1 (SEQ ID NO: 21) to the
germline V.kappa. A27 sequence (SEQ ID NO: 37). The alignments also
show the CDR regions of the VL from each antibody. The consensus
sequences for FIGS. 1A-10 are shown in SEQ ID NOS: 53-55,
respectively.
[0019] FIGS. 2A-2D show alignments of the nucleotide sequences of
the heavy chain variable regions from six human anti-IGF-1R
antibodies to each other and to germline sequences. FIG. 2A shows
the alignment of the nucleotide sequence of the VH of antibody
2.12.1 (SEQ ID NO: 3) to the germline VH DP-35 sequence (SEQ ID NO:
29). FIG. 2B shows the alignment of the nucleotide sequence of the
VH of antibody 2.14.3 (SEQ ID NO: 11) to the germline VIV-4/4.35
sequence (SEQ ID NO: 43). FIGS. 2C-1 and 2C-2 show the alignments
of the nucleotide sequences of the VH of antibodies 2.13.2 (SEQ ID
NO: 7), 4.9.2 (SEQ ID NO: 15) and 6.1.1 (SEQ ID NO: 23) to each
other and to the germline VH DP-47 sequence (SEQ ID NO: 31). FIG.
2D shows the alignment of the nucleotide sequence of the VH of
antibody 4.17.3 (SEQ ID NO: 19) to the germline VH DP-71 sequence
(SEQ ID NO: 35). The alignment also shows the CDR regions of the
antibodies. The consensus sequences for FIGS. 2A-2D are shown in
SEQ ID NOS: 56-59, respectively.
[0020] FIG. 3A shows the number of mutations in different regions
of the heavy and light chains of 2.13.2 and 2.12.1 compared to the
germline sequences. FIGS. 3A-D show alignments of the amino acid
sequences from the heavy and light chains of antibodies 2.13.2 and
2.12.1 with the germline sequences from which they are derived.
FIG. 3B shows an alignment of the amino acid sequence of the heavy
chain of antibody 2.13.2 (SEQ ID NO: 45) with that of germline
sequence DP-47(3-23)/D6-19/JH6 (SEQ ID NO: 46). FIG. 3C shows an
alignment of the amino acid sequence of the light chain of antibody
2.13.2 (SEQ ID NO: 47) with that of germline sequence A30/Jk2 (SEQ
ID NO: 48). FIG. 3D shows an alignment of the amino acid sequence
of the heavy chain of antibody 2.12.1 (SEQ ID NO: 49) with that of
germline sequence DP-35(3-11)/D3-3/JH6 (SEQ ID NO: 50). FIG. 3E
shows an alignment of the amino acid sequence of the light chain of
antibody 2.12.1 (SEQ ID NO: 51) with that of germline sequence
A30/Jk1 (SEQ ID NO: 52). For FIGS. 3B-E, the signal sequences are
in italic, the CDRs are underlined, the constant domains are bold,
the framework (FR) mutations are highlighted with a plus sign ("+")
above the amino acid residue and CDR mutations are highlighted with
an asterisk above the amino acid residue.
[0021] FIG. 4 shows that anti-IGF-1R antibodies 2.13.2 and 4.9.2
reduce IGF-1R phosphotyrosine signal in 3T3-IGF-1R tumors.
[0022] FIG. 5 shows that anti-IGF-1R antibody 2.13.2 inhibits
3T3-IGF-1R tumor growth in vivo.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention are directed to the treatment of
non-hematologic malignancies, including breast, lung, brain, skin,
ovarian, prostate, head and neck, colorectal, gastric, bladder,
renal, esophageal, and pancreatic cancers, as well as solid tumors
of childhood. Treatment of both early stage and advanced
(metastatic) cancers are within the scope of the present invention.
In preferred embodiments, the method of the present invention is
used in the treatment of breast cancer, prostate cancer, and
non-small cell lung cancer (NSCLC).
[0024] There are many classes of chemotherapeutic drugs currently
in use for the treatment of non-hematological malignancies that are
suitable for use in the combination therapy of the present
invention. For example, alkylating agents are a class of drugs that
alkylate DNA, restricting uncoiling and replication of strands.
Alkylating agents include cyclophosphamide (CYTOXAN), ifosfamide
(IFEX), mechlorethamine hydrochloride (MUSTARGEN), thiotepa
(THIOPLEX), streptozotocin (ZANOSAR), carmustine (BICNU, GLIADEL
WAFER), lomustine (CEENU), and dacarbazine (DTIC-DOME). A preferred
alkylating agent for use in the methods of the present invention is
cyclophosphamide.
[0025] Folate antagonists bind to dihydrofolate reductase (DHFR)
and interfere with pyrimidine (thymidine) synthesis. Methotrexate
(MATREX, FOLEX, TREXALL), trimetrexate (NEUTREXIN) and pemetrexed
(ARIMTA) are folate antagonists suitable for use in the methods of
the present invention. In addition to DHFR, pemetrexed also
inhibits thymidylate synthase and glycinamide ribonucleotide formyl
transferase, two other folate-dependent enzymes involved in
thymidine synthesis.
[0026] Pyrimidine antagonists inhibit enzymes involved in
pyrimidine synthesis. As pyrimidine analogs, they also interfere
with DNA production by competing with normal nucleotides for
incorporation into the DNA molecule. Pyrimidine antagonists
suitable for use in the methods of the present invention include
5-fluorouracil (5-FU); capecitabine (XELODA), a prodrug of
5'-deoxy-5-fluorouridine (5'-FDUR), which is enzymatically
converted to 5-FU in vivo; raltitrexed (TOMUDEX); tegafur-uracil
(UFTORAL); and gemcitabine (GEMZAR).
[0027] Anthracycline antibiotics exert a cytotoxic effect by
inhibiting the uncoiling of DNA by intercalation between DNA
strands. Anthracyclines and anthracyclines derivatives include
doxorubicin hydrochloride (ADRIAMYCIN, RUBEX, DOXIL), epirubicin
hydrochloride (ELLENCE, PHARMORUBICIN), daunorubicin (CERUBIDINE,
DAUNOXOME), nemorubicin, idarubicin hydrochloride (IDAMYCIN PFS,
ZAVEDOS) and mitoxantrone (DHAD, NOVANTRONE). Preferred
anthracyclines for use with the present invention include
doxorubicin and epirubicin.
[0028] Other cytotoxic antibiotics are useful as cancer
chemotherapeutic agents and suitable for use in the present
invention. These include dactinomycin (actinomycin D, COSMEGEN),
plicamycin (MITHRACIN), mitomycin (MUTAMYCIN), and bleomycin
(BLENOXANE). Dactinomycin is particularly preferred.
[0029] Platinum compounds exert their anti-neoplastic effect by
intercalation and intracalation between DNA strands, which inhibits
uncoiling of the DNA. Platinum compounds useful in the methods of
the present invention include cisplatin (PLATINOL) and carboplatin
(PARAPLATIN).
[0030] Taxanes promote assembly of microtubules while inhibiting
their disassembly into tubulin, thereby blocking a cell's ability
to break down the mitotic spindle during mitosis. They have
demonstrated significant activity against many solid tumors as
single agent therapy and in combination with other chemotherapy
agents. One embodiment of the combination therapy of the present
invention includes the use of one or more taxanes in combination
with the IGF-1R antibody. Suitable taxanes for use in combination
with the IGF-1R antibody include docetaxel (TAXOTERE) and
paclitaxel (TAXOL).
[0031] Vinca alkaloids, like taxanes, are "spindle poisons," acting
on the microtubules that form the mitotic spindle. They inhibit
mitosis by interfering with microtubule assembly, keeping the
spindle from being formed. Vinca alkaloids include vindesine
(ELDISINE), vinblastine sulfate (VELBAN), vincristine sulfate
(ONCOVIN) and vinorelbine tartrate (NAVELBINE). A preferred vinca
alkaloid for use in the methods of the present invention is
vinorelbine.
[0032] The camptothecin analogs act through inhibition of
topoisomerase I, an enzyme critical for DNA replication and
packaging. Levels of topoisomerase I are higher in tumor cells than
in normal tissue. Camptothecin analogs useful in the methods of the
present invention include irinotecan (CAMPTOSAR) and topotecan
(HYCAMTIN). Irinotecan is particularly preferred.
[0033] Inhibitors of topoisomerase II interfere with the normal DNA
breakage resealing process (as do inhibitors of topoisomerase I),
and they also interfere with the separation of newly replicated
chromosomes, resulting in clastogenic mutation and potential cell
death. The anthracyline antibiotics discussed above exhibit
topoisomerase II inhibitory activity. Derivatives of
podophyllotoxin, an extract of the mayapple that is an antimitotic
glucoside) are also topoisomerase II inhibitors. Podophyllotoxin
derivatives suitable for use in the present invention include
etoposide (VEPESID), etoposide phosphate (ETOPOPHOS), and
teniposide (VUMON). Etoposide is particularly preferred.
[0034] Compounds directed at inhibition of epidermal growth factor
receptor (EGFR) tyrosine kinase (TK) represent a relatively new
class of antineoplastic drugs that are useful in the method of the
present invention. Many human cancers express members of the EGFR
family on the cell surface. When a ligand binds to EGFR, it sets
off a cascade of cellular reactions that result in increased cell
division and influence other aspects of cancer development and
progression, including angiogenesis, metastatic spread, and
inhibition of apoptosis. EGFR-TK inhibitors may selectively target
one of the members of the EGFR family (EGFR (also known as HER1 or
ErbB-1), HER2/neu (also known as ErbB-2), HER3 (also known as
ErbB-3), or HER4 (also known as ErbB-4)), or may target two or more
of them. EGFR-TK inhibitors suitable for use in the present
invention include gefitinib (IRESSA), erlotinib (TARCEVA),
trastuzumab (HERCEPTIN), panitumumab (ABX-EGF; Abgenix/Amgen),
lapatinib (GlaxoSmithKline), CI-1033 (Pfizer), GW2016
(GlaxoSmithKline), EKB-569 (Wyeth), PKI-166 (Novartis), CP-724,714
(Pfizer), and BIBX-1382 (Boeringer-Ingelheim). Additional EGFR-TK
inhibitors are described in United States Patent Publication No. US
2002-0169165A1, published Nov. 14, 2002.
[0035] Another embodiment of the combination therapy of the present
invention includes the use of hormonal therapy in combination with
the IGF-1R antibody, particularly anti-estrogens in the treatment
of breast cancer. Some hormonal treatments compete with estrogen
for binding sites in breast tissue. These include tamoxifen citrate
(NOLVADEX) and fulvestrant (FASLODEX). Similarly, anti-androgens
block testosterone receptors and therefore are useful in the
treatment of androgen-dependent prostate cancer.
[0036] Other hormone treatments include aromatase inhibitors. This
class of hormonal agents inactivate aromatase, the enzyme which
converts androgens to estrogens. Examples of aromatase inhibitors
suitable for use in combination with the IGF-1R antibody include
anastrozole (ARIMIDEX), letrozole (FEMARA), exemestane (AROMASIN),
and fadrozole hydrochloride. Exemestane is a particularly preferred
aromatase inhibitor for use in the methods of the present
invention.
[0037] Co-administration of the antibody with an additional
therapeutic agent (combination therapy) encompasses administering a
pharmaceutical composition comprising both the anti-IGF-1R antibody
and one or more additional therapeutic agents, and administering
two or more separate pharmaceutical compositions, one comprising
the anti-IGF-1R antibody and the other(s) comprising the additional
therapeutic agent(s). Further, although co-administration or
combination (conjoint) therapy generally mean that the antibody and
additional therapeutic agents are administered at the same time as
one another, it also encompasses simultaneous, sequential or
separate dosing of the individual components of the treatment.
[0038] The present invention also encompasses the administration of
other therapeutic agents in addition to the first and second
components, either concurrently with one or more of those
components, or sequentially. Such therapeutic agents include
analgesics, cancer vaccines, anti-vascular agents,
anti-proliferative agents, and anti-emetic agents. Preferred
anti-emetic agents include aprepitant, ondansetron hydrochloride,
granisetron hydrochloride, and metoclopramide.
[0039] Each administration may vary in its duration from a rapid
administration to a continuous perfusion. As a result, for the
purposes of the present invention, the combinations are not
exclusively limited to those that are obtained by physical
association of the constituents, but also to those that permit a
separate administration, which can be simultaneous or spaced out
over a period of time. The compositions according to the invention
are preferably compositions which can be administered parentally.
However, these compositions may be administered orally or
intraperitoneally in the case of localized regional therapies.
[0040] As will be appreciated by one of skill in the art, the
choice of therapeutic agents to be used in combination with IGF-1R
antibodies, and the timing of their use, will be determined in part
by the type and stage of the cancer that is being treated. For
example, in early breast cancer (where the cancer has not spread
outside the breast), surgery and radiation are generally followed
by adjuvant chemotherapy or adjuvant hormonal therapy, either of
which may be combined with IGF-1R antibodies in the methods of the
present invention. Typical adjuvant chemotherapy for early breast
cancer includes cyclophosphamide, methotrexate and 5-FU ("CMF");
5-FU, doxorubicin, and cyclophosphamide ("FAC"); docetaxel,
doxorubicin, and cyclophosphamide ("TAC"); doxorubicin and
cyclophosphamide ("AC"); doxorubicin and cyclophosphamide followed
by paclitaxel ("AC and T"); and 5-FU, epirubicin, and
cyclophosphamide ("FEC"). Tamoxifen is a preferred hormonal
treatment at this stage.
[0041] In locally advanced breast cancer, wherein the cancer has
spread only to nearby tissues or lymph nodes, the patient is often
given chemotherapy prior to surgery and radiation, which are then
followed by adjuvant hormonal therapy. Alternatively,
surgery/radiation is followed by adjuvant chemotherapy, then
adjuvant hormonal therapy. IGF-1R antibodies may be administered in
conjunction with the chemotherapeutic or hormonal therapy agents
whether they are used either before or after surgery/radiation.
Typical chemotherapy regimes for locally advanced breast cancer
include FAC, AC, FEC, and doxorubicin plus docetaxel ("AT").
[0042] Metastatic breast cancer has spread to other parts of the
body from the breast in which it started. Chemotherapy optionally
may be preceded by hormonal therapy. First line hormonal therapy
currently includes tamoxifen and anastrozole. First line
chemotherapy regimens currently include FAC, TAC, docetaxel plus
epirubicin, docetaxel, paclitaxel, capecitabine, vinorelbine, and
trastuzumab. Second line chemotherapy treatments include docetaxel,
alone or in combination with capecitabine. The methods of the
present invention are suitable for use both as first line therapy
and second line therapy.
[0043] In the United States, the combination of paclitaxel and
carboplatin has become accepted as the standard of care for first
line treatment of inoperable Stage IIIB (i.e. cancer has spread to
structures near the lung, to lymph nodes in the mediastinum, or to
lymph nodes on the other side of the chest or in the lower neck)
and Stage IV (i.e. cancer has spread to other parts of the body or
to another lobe of the lungs) non-small cell lung cancer (NSCLC).
But the overall response rate is only approximately 28% for
patients with performance status 0-1 in efficacy studies with a
predominantly Stage IV population. In Europe, first line treatment
for NSCLC is gemcitabine and cisplatin. Other treatment regimens
for NSCLC include paclitaxel alone or with cisplatin or
gemcitabine; docetaxel alone or with cisplatin or gemcitabine;
vinorelbine alone or with gemcitabine; irinotecan alone or with
gemcitabine; pemetrexed; and gefitinib.
[0044] It is known that signaling through IGF-1R is required for
the tumorgenicity of cell lines and has been shown to decrease the
cytotoxicity of chemotherapy, and that blocking IGF-1R activity
enhances the effectiveness of current therapies and prevents tumor
progression in animal models. It was therefore expected that an
inhibitor of IGF-1R such as the antibodies of the present invention
would reduce tumor cell survival and enhance the efficacy of
chemotherapy when given in combination.
[0045] When incubated with cells, fully human monoclonal antibodies
that are highly specific and potent inhibitors of IGF-1-induced
receptor autophosphorylation induced down-regulation of IGF-1R by
receptor internalization. The doses that down-regulated IGF-1R in
solid tumor ex vivo models (31.25-125 .mu.g) corresponded to
antibody concentrations of 8-40 .mu.g/ml at Day 1 and 2-20 .mu.g/ml
at Day 9. Intraperitoneal administration of the anti-IGF-1R
antibodies to athymic mice bearing tumors of the transfectant
IGF-1R over-expressing NIH-3T3 cell line resulted in a dose
dependent inhibition of tumor growth. The serum concentration of
anti-IGF-1R antibodies that led to 50% growth inhibition was 20
.mu.g/ml at Day 1, and 13 .mu.g/ml at Day 9. Similar anti-tumor
studies were extended to human tumor xenograft models. As a single
agent, anti-IGF-1R antibodies inhibited the growth of several
xenograft models including breast, lung and colorectal
carcinomas.
[0046] The combination of anti-IGF-1R antibodies with paclitaxel or
carboplatin was tested in the H460 and EBC-1 human NSCLC tumor
xenograft models. Combination of anti-IGF-1R antibodies with those
agents increased their tumor growth inhibition compared to each
agent alone.
[0047] Unless otherwise defined herein, scientific, technical, and
medical terms used in connection with the present invention shall
have the meanings that are commonly understood by those of ordinary
skill in the art. Generally, nomenclatures used in connection with,
and techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics and protein and nucleic acid
chemistry described herein are those well known and commonly used
in the art.
[0048] The following terms, unless otherwise indicated, shall be
understood to have the following meanings:
[0049] An "antibody" refers to an intact immunoglobulin or to an
antigen-binding portion thereof that competes with the intact
antibody for specific binding. Antigen-binding portions may be
produced by recombinant DNA techniques or by enzymatic or chemical
cleavage of intact antibodies. Antigen-binding portions include,
inter alia, Fab, Fab', F(ab').sub.2, Fv, dAb, and complementarity
determining region (CDR) fragments, single-chain antibodies (scFv),
chimeric antibodies, diabodies and polypeptides that contain at
least a portion of an immunoglobulin that is sufficient to confer
specific antigen binding to the polypeptide.
[0050] Immunoglobulin chains exhibit the same general structure of
relatively conserved framework regions (FR) joined by three
hypervariable regions, also called complementarity determining
regions or CDRs. The CDRs from the two chains of each pair are
aligned by the framework regions, enabling binding to a specific
epitope. From N-terminus to C-terminus, both light and heavy chains
comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The
assignment of amino acids to each domain is in accordance with the
definitions of Kabat, Sequences of Proteins of Immunological
Interest (National Institutes of Health, Bethesda, Md. (1987 and
1991)), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987);
Chothia et al., Nature 342:878-883 (1989).
[0051] An "isolated antibody" is an antibody that (1) is not
associated with naturally-associated components, including other
naturally-associated antibodies, that accompany it in its native
state, (2) is free of other proteins from the same species, (3) is
expressed by a cell from a different species, or (4) does not occur
in nature. Examples of isolated antibodies include an anti-IGF-1R
antibody that has been affinity purified using IGF-1R is an
isolated antibody, an anti-IGF-1R antibody that has been
synthesized by a hybridoma or other cell line in vitro, and a human
anti-IGF-1R antibody derived from a transgenic mouse.
[0052] The term "chimeric antibody" refers to an antibody that
contains one or more regions from one antibody and one or more
regions from one or more other antibodies. In a preferred
embodiment, one or more of the CDRs are derived from a human
anti-IGF-1R antibody. In a more preferred embodiment, all of the
CDRs are derived from a human anti-IGF-1R antibody. In another
preferred embodiment, the CDRs from more than one human anti-IGF-1R
antibodies are mixed and matched in a chimeric antibody. Further,
the framework regions may be derived from one of the same
anti-IGF-1R antibodies, from one or more different antibodies, such
as a human antibody, or from a humanized antibody.
[0053] The term "epitope" includes any protein determinant capable
of specific binding to an immunoglobulin or T-cell receptor.
Epitopic determinants usually consist of chemically active surface
groupings of molecules such as amino acids or sugar sides chains
and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics. An
antibody is said to specifically bind an antigen when the
dissociation constant is preferably .ltoreq.100 nM and most
preferably .ltoreq.10 nM.
[0054] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 75% or 80% sequence identity, preferably at least 90% or 95%
sequence identity, even more preferably at least 98% or 99%
sequence identity. Preferably, residue positions that are not
identical differ by conservative amino acid substitutions. A
"conservative amino acid substitution" is one in which an amino
acid residue is substituted by another amino acid residue having a
side chain (R group) with similar chemical properties (e.g., charge
or hydrophobicity). In general, a conservative amino acid
substitution will not substantially change the functional
properties of a protein. In cases where two or more amino acid
sequences differ from each other by conservative substitutions, the
percent sequence identity or degree of similarity may be adjusted
upwards to correct for the conservative nature of the substitution.
Means for making this adjustment are well-known to those of skill
in the art. See, e.g., Pearson, Methods Mol. Biol. 24: 307-31
(1994). Examples of groups of amino acids that have side chains
with similar chemical properties include 1) aliphatic side chains:
glycine, alanine, valine, leucine and isoleucine; 2)
aliphatic-hydroxyl side chains: serine and threonine; 3)
amide-containing side chains: asparagine and glutamine; 4) aromatic
side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side
chains: lysine, arginine, and histidine; and 6) sulfur-containing
side chains are cysteine and methionine. Conservative amino acids
substitution groups include: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine,
glutamate-aspartate, and asparagine-glutamine.
[0055] Preferred amino acid substitutions are those which: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to
oxidation, (3) alter binding affinity for forming protein
complexes, (4) alter binding affinities, and (4) confer or modify
other physicochemical or functional properties of such analogs.
Analogs can include various mutations of a sequence other than the
naturally-occurring peptide sequence. For example, single or
multiple amino acid substitutions (preferably conservative amino
acid substitutions) may be made in the naturally-occurring sequence
(preferably in the portion of the polypeptide outside the domain(s)
forming intermolecular contacts. A conservative amino acid
substitution should not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that
characterizes the parent sequence).
[0056] The phrase "in combination with" encompasses simultaneous,
sequential or separate dosing of the individual components of the
treatment. For example, the antibody may be administered once every
three days, while the additional therapeutic agent is administered
once daily. The antibody may be administered prior to or subsequent
to treatment of the disorder with the additional therapeutic agent.
Similarly, the anti-IGF-1R antibody may be administered prior to or
subsequent to other therapy, such as radiotherapy, chemotherapy,
photodynamic therapy, surgery or other immunotherapy.
[0057] The terms "concurrently" and "simultaneously" are used
interchangeably and mean the compounds of the combination therapy
of the present invention are administered (1) simultaneously in
time, or (2) at different times during the course of a common
treatment schedule. The term "sequentially" as used herein means
administration of the a first component, followed by administration
of a second component. Anti-IGF-1R antibodies may be the first
component or the second component. After administration of one
component, the second component can be administered substantially
immediately after the first component, or the second component can
be administered an effective time period after the first component;
the effective time period is the amount of time given for
realization of maximum benefit from the administration of the first
component.
[0058] The term "patient" includes mammals. In a preferred
embodiment, the mammal is a human.
[0059] The term "treating," as used herein, unless otherwise
indicated, means reversing, alleviating, inhibiting the progress
of, or preventing the disorder or condition to which such term
applies, or one or more symptoms of such disorder or condition. The
term "treatment," as used herein, unless otherwise indicated,
refers to the act of treating as "treating" is defined immediately
above.
[0060] Human antibodies avoid certain of the problems associated
with antibodies that possess mouse or rat variable and/or constant
regions. More preferred are fully human anti-human IGF-1R
antibodies. Fully human anti-IGF-1R antibodies are expected to
minimize the immunogenic and allergic responses intrinsic to mouse
or mouse-derivatized monoclonal antibodies (Mabs) and thus to
increase the efficacy and safety of the administered antibodies.
The use of fully human antibodies can be expected to provide a
substantial advantage in the treatment of chronic and recurring
human diseases, such as inflammation and cancer, which may require
repeated antibody administrations. In another embodiment, the
invention provides an anti-IGF-1R antibody that does not bind
complement.
[0061] In another aspect of the invention, the anti-IGF-1R
antibodies bind to IGF-1R with high affinity. In one embodiment,
the anti-IGF-1R antibody binds to IGF-1R with a K.sub.d of
1.times.10.sup.-8 M or less. In a more preferred embodiment, the
antibody binds to IGF-1R with a K.sub.d or 1.times.10.sup.-9 M or
less. In an even more preferred embodiment, the antibody binds to
IGF-1R with a K.sub.d or 5.times.10.sup.-10 M or less. In another
preferred embodiment, the antibody binds to IGF-1R with a K.sub.d
or 1.times.10.sup.-10 M or less. In another preferred embodiment,
the antibody binds to IGF-1R with substantially the same K.sub.d as
an antibody selected from 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2,
4.17.3 or 6.1.1. In another preferred embodiment, the antibody
binds to IGF-1R with substantially the same K.sub.d as an antibody
that comprises one or more CDRs from an antibody selected from
2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1.
[0062] The invention also employs an anti-IGF-1R antibody that
binds the same antigen or epitope as a human anti-IGF-1R antibody.
The invention may also employ an anti-IGF-1R antibody that
cross-competes with a human anti-IGF-1R antibody. In a preferred
embodiment, the human anti-IGF-1R antibody is 2.12.1, 2.13.2,
2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another preferred
embodiment, the human anti-IGF-1R comprises one or more CDRs from
an antibody selected from 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2,
4.17.3 or 6.1.1
[0063] The invention can also be practiced using an anti-IGF-1R
antibody that comprises variable sequences encoded by a human
.kappa. gene. In a preferred embodiment, the variable sequences are
encoded by either the V.kappa. A27, A30 or O12 gene family. In a
preferred embodiment, the variable sequences are encoded by a human
V.kappa. A30 gene family. In a more preferred embodiment, the light
chain comprises no more than ten amino acid substitutions from the
germline V.kappa. A27, A30 or O12, preferably no more than six
amino acid substitutions, and more preferably no more than three
amino acid substitutions. In a preferred embodiment, the amino acid
substitutions are conservative substitutions.
[0064] In a preferred embodiment, the VL of the anti-IGF-1R
antibody contains the same amino acid substitutions, relative to
the germline amino acid sequence, as any one or more of the VL of
antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or
6.1.1.
[0065] In another preferred embodiment, the light chain comprises
an amino acid sequence that is the same as the amino acid sequence
of the VL of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1.
In another highly preferred embodiment, the light chain comprises
amino acid sequences that are the same as the CDR regions of the
light chain of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or
6.1.1. In another preferred embodiment, the light chain comprises
an amino acid sequence from at least one CDR region of the light
chain of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1.
[0066] The present invention can also be carried out using an
anti-IGF-1R antibody or portion thereof comprising a human heavy
chain or a sequence derived from a human heavy chain. In one
embodiment, the heavy chain amino acid sequence is derived from a
human V.sub.H DP-35, DP-47, DP-70, DP-71 or VIV-4/4.35 gene family.
In a preferred embodiment, the heavy chain amino acid sequence is
derived from a human V.sub.H DP-47 gene family. In a more preferred
embodiment, the heavy chain comprises no more than eight amino acid
changes from germline V.sub.H DP-35, DP-47, DP-70, DP-71 or
VIV-4/4.35, more preferably no more than six amino acid changes,
and even more preferably no more than three amino acid changes.
[0067] In a preferred embodiment, the VH of the anti-IGF-1R
antibody contains the same amino acid substitutions, relative to
the germline amino acid sequence, as any one or more of the VH of
antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1.
In another embodiment, the amino acid substitutions are made in the
same position as those found in any one or more of the VH of
antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.17.3., 4.9.2 or 6.1.1,
but conservative amino acid substitutions are made rather than
using the same amino acid.
[0068] In another preferred embodiment, the heavy chain comprises
an amino acid sequence that is the same as the amino acid sequence
of the VH of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1.
In another highly preferred embodiment, the heavy chain comprises
amino acid sequences that are the same as the CDR regions of the
heavy chain of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or
6.1.1. In another preferred embodiment, the heavy chain comprises
an amino acid sequence from at least one CDR region of the heavy
chain of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In
another preferred embodiment, the heavy chain comprises amino acid
sequences from CDRs from different heavy chains. In a more
preferred embodiment, the CDRs from different heavy chains are
obtained from 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or
6.1.1.
[0069] In another embodiment, the invention employs an anti-IGF-1R
antibody that inhibits the binding of IGF-1 to IGF-1R or the
binding of IGF-2 to IGF-1R. In a preferred embodiment, the IGF-1R
is human. In another preferred embodiment, the anti-IGF-1R antibody
is a human antibody. In another embodiment, the antibody or portion
thereof inhibits binding between IGF-1R and IGF-1 with an IC.sub.50
of no more than 100 nM. In a preferred embodiment, the IC.sub.50 is
no more than 10 nM. In a more preferred embodiment, the IC.sub.50
is no more than 5 nM. The IC.sub.50 can be measured by any method
known in the art. Typically, an IC.sub.50 can be measured by ELISA
or RIA. In a preferred embodiment, the IC.sub.50 is measured by
RIA.
[0070] In another embodiment, the invention employs an anti-IGF-1R
antibody that prevents activation of the IGF-1R in the presence of
IGF-i. In another aspect of the invention, the antibody causes the
downregulation of IGF-1R from a cell treated with the antibody. In
a preferred embodiment, the antibody is selected 2.12.1, 2.13.2,
2.14.3, 3.1.1, 4.9.2, or 6.1.1, or comprises a heavy chain, light
chain or antigen-binding region thereof.
[0071] Human antibodies can be produced by immunizing a non-human
animal comprising of some or all of the human immunoglobulin locus
with an IGF-1R antigen. In a preferred embodiment, the non-human
animal is a XENOMOUSE.TM., which is an engineered mouse strain that
comprises large fragments of the human immunoglobulin loci and is
deficient in mouse antibody production. See, e.g., Green et al.
Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771,
5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598,
and 6,130,364. See also International Patent Application Nos. WO
91/10741, published Jul. 25, 1991; WO 94/02602, published Feb. 3,
1994; WO 96/34096 and WO 96/33735, both published Oct. 31, 1996; WO
98/16654, published Apr. 23, 1998; WO 98/24893, published Jun. 11,
1998; WO 98/50433, published Nov. 12, 1998; WO 99/45031, published
Sep. 10, 1999; WO 99/53049, published Oct. 21, 1999; WO 00/09560,
published Feb. 24, 2000; and WO 00/037504, published Jun. 29, 2000.
The XENOMOUSE.TM. produces an adult-like human repertoire of fully
human antibodies, and generates antigen-specific human monoclonal
antibodies. A second generation XENOMOUSE.TM. contains
approximately 80% of the human antibody repertoire through
introduction of megabase sized, germline configuration YAC
fragments of the human heavy chain loci and .kappa. light chain
loci. See Mendez et al. Nature Genetics 15:146-156 (1997), Green
and Jakobovits J. Exp. Med. 188:483-495 (1998).
[0072] The IGF-1R antigen can be administered with an adjuvant to
stimulate the immune response. Such adjuvants include complete or
incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM
(immunostimulating complexes). Such adjuvants may protect the
polypeptide from rapid dispersal by sequestering it in a local
deposit, or they may contain substances that stimulate the host to
secrete factors that are chemotactic for macrophages and other
components of the immune system.
[0073] The nucleic acid molecule encoding the variable region of
the light chain may be derived from the A30, A27 or O12 V.kappa.
gene. In a preferred embodiment, the light chain is derived from
the A30 V.kappa. gene. In an even more preferred embodiment, the
nucleic acid molecule encoding the light chain contains no more
than ten amino acid changes from the germline A30 V.kappa. gene,
preferably no more than six amino acid changes, and even more
preferably no more than three amino acid changes.
[0074] In one embodiment, the antibody contains no greater than ten
amino acid changes in either the VH or VL regions of the mutated
anti-IGF-1R antibody compared to the anti-IGF-1R antibody prior to
mutation. In a more preferred embodiment, there are no more than
five amino acid changes in either the VH or VL regions of the
mutated anti-IGF-1R antibody, more preferably no more than three
amino acid changes. In another embodiment, there are no more than
fifteen amino acid changes in the constant domains, more
preferably, no more than ten amino acid changes, even more
preferably, no more than five amino acid changes.
[0075] SEQ ID NOS: 2, 6, 10, 14, 18 and 22 provide the amino acid
sequences of the variable regions of six anti-IGF-1R K light
chains. SEQ ID NOS: 4, 8, 12, 16, 20 and 24 provide the amino acid
sequences of the variable regions of six anti-IGF-1R heavy chains.
SEQ ID NO: 26 depicts the amino acid sequence and SEQ ID NO: 25
depicts the nucleic acid sequence encoding the constant region of
the light chain of the anti-IGF-1R antibodies 2.12.1, 2.13.2,
2.14.3, 3.1.1, 4.9.2, 4.17.3 and 6.1.1. SEQ ID NO: 28 depicts the
amino acid sequence and SEQ ID NO: 27 depicts the nucleic acid
sequence encoding the constant region of the heavy chain of the
anti-IGF-1R antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3
and 6.1.1. SEQ ID NOS: 30, 32, 34, 36 and 44 provide the amino acid
sequences of the germline heavy chains DP-35, DP-47, DP-70, DP-71
and VIV-4, respectively. SEQ ID NO: 33 provides the nucleotide
sequence of the germline heavy chain DP-70. SEQ ID NOS: 38, 40 and
42 provide the amino acid sequences of the three germline K light
chains from which the six anti-IGF-1R K light chains are
derived.
[0076] The anti-IGF-1R antibodies can be incorporated into
pharmaceutical compositions suitable for administration to a
subject. Typically, the pharmaceutical composition comprises an
antibody and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Examples of pharmaceutically
acceptable carriers include water, saline, phosphate buffered
saline, dextrose, glycerol, ethanol and the like, as well as
combinations thereof. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition. Minor
amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the antibody or antibody portion, may also be
included.
[0077] The pharmaceutical compositions may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans with other antibodies. A preferred
mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular, or infusion). In a
preferred embodiment, the antibody is administered by intravenous
infusion or injection. In another preferred embodiment, the
antibody is administered by intramuscular or subcutaneous
injection. As will be appreciated by the skilled artisan, the route
and/or mode of administration will vary depending upon the desired
results.
[0078] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the anti-IGF-1R antibody in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying that yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. The proper fluidity
of a solution can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Prolonged absorption of injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin.
[0079] In certain embodiments, the active compound may be prepared
with a carrier that will protect the compound against rapid
release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
[0080] The pharmaceutical composition may include a
"therapeutically effective amount" or a "prophylactically effective
amount" of an antibody or antibody portion of the invention. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired therapeutic result. A therapeutically effective amount of
the antibody or antibody portion may vary according to factors such
as the disease state, age, sex, and weight of the individual, and
the ability of the antibody or antibody portion to elicit a desired
response in the individual. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the antibody
or antibody portion are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0081] Dosage regimens may be adjusted to provide the optimum
desired response. For example, a single bolus may be administered,
several divided doses may be administered over time or the dose may
be proportionally reduced or increased as indicated by the
exigencies of the therapeutic situation. Pharmaceutical composition
comprising the antibody or comprising a combination therapy
comprising the antibody and one or more additional therapeutic
agents may be formulated for single or multiple doses. It is
especially advantageous to formulate parenteral compositions in
dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the mammalian subjects
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic or prophylactic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
compound for the treatment of sensitivity in individuals. A
particularly useful formulation is 5 mg/ml anti-IGF-1R antibody in
a buffer of 20 mM sodium citrate, pH 5.5, 140 mM NaCl, and 0.2
mg/ml polysorbate 80.
[0082] The antibody, with or without an additional agent, may be
administered once, or more than once for at least the period of
time until the condition is treated, palliated or cured. The
antibody generally will be administered for as long as the tumor is
present provided that the antibody causes the tumor or cancer to
stop growing or to decrease in weight or volume. The antibody will
generally be administered as part of a pharmaceutical composition
as described supra. The dosage of antibody will generally be in the
range of 0.025-100 mg/kg, more preferably 0.05-50 mg/kg, more
preferably 0.05-20 mg/kg, and even more preferably 0.1-10 mg/kg. It
is to be noted that dosage values may vary with the type and
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition.
[0083] The antibody may be administered from three times daily to
once every six months. The administration may be on a schedule such
as three times daily, twice daily, once daily, once every two days,
once every three days, once weekly, once every two weeks, once
every month, once every two months, once every three months and
once every six months. The antibody may be administered via an
oral, mucosal, buccal, intranasal, inhalable, intravenous,
subcutaneous, intramuscular, parenteral, intratumor or topical
route.
[0084] The antibody may be administered at a site distant from the
site of the tumor. The antibody may also be administered
continuously via a minipump.
[0085] In certain embodiments, the antibody may be administered in
an aerosol or inhalable form. Dry aerosol in the form of finely
divided solid particles that are not dissolved or suspended in a
liquid are also useful in the practice of the present invention.
The pharmaceutical formulations of the present invention may be
administered in the form of an aerosol spray using for example, a
nebulizer such as those described in U.S. Pat. Nos. 4,624,251;
3,703,173; 3,561,444; and 4,635,627.
[0086] The serum concentration of the antibody may be measured by
any method known in the art. The antibody may also be administered
prophylactically in order to prevent a cancer or tumor from
occurring. This may be especially useful in patients that have a
"high normal" level of IGF-1 because these patients have been shown
to have a higher risk of developing common cancers. See Rosen et
al., supra.
[0087] The antibody employed in the method of the invention can be
labeled. This can be done by incorporation of a detectable marker,
e.g., incorporation of a radiolabeled amino acid or attachment to a
polypeptide of biotinyl moieties that can be detected by marked
avidin (e.g., streptavidin containing a fluorescent marker or
enzymatic activity that can be detected by optical or colorimetric
methods). In certain situations, the label or marker can also be
therapeutic. Various methods of labeling polypeptides and
glycoproteins are known in the art and may be used. Examples of
labels for polypeptides include, but are not limited to, the
following: radioisotopes or radionuclides (e.g., .sup.3H, .sup.14C,
.sup.15N, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In, .sup.125I,
.sup.131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide
phosphors), enzymatic labels (e.g., horseradish peroxidase,
.beta.-galactosidase, luciferase, alkaline phosphatase),
chemiluminescent, biotinyl groups, predetermined polypeptide
epitopes recognized by a secondary reporter (e.g., leucine zipper
pair sequences, binding sites for secondary antibodies, metal
binding domains, epitope tags). In some embodiments, labels are
attached by spacer arms of various lengths to reduce potential
steric hindrance.
[0088] The antibodies employed in the present invention are
preferably derived from cells that express human immunoglobulin
genes. Use of transgenic mice is known in the art to produce such
"human" antibodies. One such method is described in U.S. Patent
Application Ser. No. 08/759,620, filed Dec. 3, 1996. See also
Mendez et al. Nature Genetics 15:146-156 (1997); Green and
Jakobovits J. Exp. Med. 188:483-495 (1998); European Patent No. EP
0 463 151 (grant published Jun. 12, 1996); and International Patent
Application Nos. WO 94/02602, published Feb. 3, 1994; WO 96/34096,
published Oct. 31, 1996; and WO 98/24893, published Jun. 11,
1998.
[0089] As noted above, the invention encompasses use of antibody
fragments. Antibody fragments, such as Fv, F(ab').sub.2 and Fab may
be prepared by cleavage of the intact protein, e.g. by protease or
chemical cleavage. Alternatively, a truncated gene is designed. For
example, a chimeric gene encoding a portion of the F(ab').sub.2
fragment would include DNA sequences encoding the CH1 domain and
hinge region of the H chain, followed by a translational stop codon
to yield the truncated molecule.
[0090] In one approach, consensus sequences encoding the heavy and
light chain J regions may be used to design oligonucleotides for
use as primers to introduce useful restriction sites into the J
region for subsequent linkage of V region segments to human C
region segments. C region cDNA can be modified by site directed
mutagenesis to place a restriction site at the analogous position
in the human sequence.
[0091] Expression vectors for use in obtaining the antibodies
employed in the invention include plasmids, retroviruses, cosmids,
YACs, EBV derived episomes, and the like. A convenient vector is
normally one that encodes a functionally complete human CH or CL
immunoglobulin sequence, with appropriate restriction sites
engineered so that any VH or VL sequence can be easily inserted and
expressed. In such vectors, splicing usually occurs between the
splice donor site in the inserted J region and the splice acceptor
site preceding the human C region, and also at the splice regions
that occur within the human CH exons. Polyadenylation and
transcription termination occur at native chromosomal sites
downstream of the coding regions. The resulting chimeric antibody
may be joined to any strong promoter, including retroviral LTRs,
e.g. SV-40 early promoter (Okayama et al. Mol. Cell. Bio. 3:280
(1983)), Rous sarcoma virus LTR (Gorman et al. Proc. Natl. Acad.
Sci. 79:6777 (1982)), and moloney murine leukemia virus LTR
(Grosschedl et al. Cell 41:885 (1985)); native Ig promoters,
etc.
[0092] Antibodies that are generated for use in the invention need
not initially possess a particular desired isotype. Rather, the
antibody as generated can possess any isotype and can be isotype
switched thereafter using conventional techniques. These include
direct recombinant techniques (see e.g., U.S. Pat. No. 4,816,397),
and cell-cell fusion techniques (see e.g., U.S. Pat. No.
5,916,771).
[0093] As noted above, the effector function of the antibodies of
the invention may be changed by isotype switching to an IgG1, IgG2,
IgG3, IgG4, IgD, IgA, IgE, or IgM for various therapeutic uses.
Furthermore, dependence on complement for cell killing can be
avoided through the use of bispecifics, immunotoxins, or
radiolabels, for example.
[0094] Bispecific antibodies can be generated that comprise (i) two
antibodies: one with a specificity for IGF-1R and the other for a
second molecule (ii) a single antibody that has one chain specific
for IGF-1R and a second chain specific for a second molecule, or
(iii) a single chain antibody that has specificity for IGF-1R and
the other molecule. Such bispecific antibodies can be generated
using well known techniques, e.g., Fanger et al. Immunol. Methods
4:72-81 (1994); Wright and Harris, supra; and Traunecker et al.
Int. J. Cancer (Suppl.) 7:51-52 (1992).
[0095] Antibodies for use in the invention also include
"kappabodies" (Ill et al. Protein Eng. 10:949-57 (1997)),
"minibodies" (Martin et al. EMBO J. 13:5303-9 (1994)), "diabodies"
(Holliger et al. Proc. Natl. Acad. Sci. (USA) 90:6444-6448 (1993)),
and "janusins" (Traunecker et al. EMBO J. 10:3655-3659 (1991) and
Traunecker et al. Int. J. Cancer Suppl. 7:51-52 (1992)) may also be
prepared.
[0096] The antibodies employed can be modified to act as
immunotoxins by conventional techniques. See e.g., Vitetta Immunol.
Today 14:252 (1993). See also U.S. Pat. No. 5,194,594. Radiolabeled
antibodies can also be prepared using well-known techniques. See
e.g., Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686
(2d edition, Chafner and Longo, eds., Lippincott Raven (1996)). See
also U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (Re.
35,500), 5,648,471, and 5,697,902.
[0097] Particular antibodies useful in practice of the invention
include those described in International Patent Application No. WO
02/053596, which further describes antibodies 2.12.1, 2.13.2.,
2.14.3, 3.1.1, 4.9.2, and 4.17.3. As disclosed in that published
application, hybridomas producing these antibodies were deposited
in the American Type Culture Collection (ATCC), 10801 University
Boulevard, Manassas, Va. 20110-2209, on Dec. 12, 2000 with the
following deposit numbers:
TABLE-US-00001 Hybridoma Deposit No. 2.12.1 PTA-2792 2.13.2
PTA-2788 2.14.3 PTA-2790 3.1.1 PTA-2791 4.9.2 PTA-2789 4.17.3
PTA-2793
[0098] These antibodies are either fully human IgG2 or IgG4 heavy
chains with human kappa light chains. In particular the invention
concerns use of antibodies having amino acid sequences of these
antibodies.
[0099] Antibodies employed in the invention preferably possess very
high affinities, typically possessing Kds of from about 10.sup.-9
through about 10.sup.-11 M, when measured by either solid phase or
solution phase.
[0100] Antibodies used in the present invention can be expressed in
cell lines other than hybridoma cell lines. Sequences encoding the
cDNAs or genomic clones for the particular antibodies can be used
for transformation of suitable mammalian or nonmammalian host
cells. Transformation can be by any known method for introducing
polynucleotides into a host cell, including, for example packaging
the polynucleotide in a virus (or into a viral vector) and
transducing a host cell with the virus (or vector) or by
transfection procedures known in the art, as exemplified by U.S.
Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods
for introduction of heterologous polynucleotides into mammalian
cells are well known in the art and include, but are not limited
to, dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion,
electroporation, particle bombardment, encapsulation of the
polynucleotide(s) in liposomes, peptide conjugates, dendrimers, and
direct microinjection of the DNA into nuclei.
[0101] Mammalian cell lines available as hosts for expression are
well known in the art and include many immortalized cell lines
available from the American Type Culture Collection (ATCC),
including but not limited to Chinese hamster ovary (CHO) cells,
NSO.sub.0, HeLa cells, baby hamster kidney (BHK) cells, monkey
kidney cells (COS), and human hepatocellular carcinoma cells (e.g.,
Hep G2). Non-mammalian cells can also be employed, including
bacterial, yeast, insect, and plant cells. Site directed
mutagenesis of the antibody CH2 domain to eliminate glycosylation
may be preferred in order to prevent changes in either the
immunogenicity, pharmacokinetic, and/or effector functions
resulting from non-human glycosylation. The glutamine synthase
system of expression is discussed in whole or part in connection
with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997, and
European Patent Application No. 89303964.4.
[0102] Antibodies for use in the invention can also be produced
transgenically through the generation of a mammal or plant that is
transgenic for the immunoglobulin heavy and light chain sequences
of interest and production of the antibody in a recoverable form
therefrom. Transgenic antibodies can be produced in, and recovered
from, the milk of goats, cows, or other mammals. See, e.g., U.S.
Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957.
[0103] The advantages of the present invention can be further
appreciated by reference to the following examples. These examples
serve intended to illustrate preferred embodiments of the invention
and are by no means intended to limit the effective scope of the
claims.
Example I
Anti-IGF-1R Antibodies in Combination with Docetaxel in the
Treatment of Advanced Non-Hematologic Malignancies
[0104] Patients with advanced-stage non-hematologic malignancies
(measurable disease defined by at least one lesion that can be
accurately measured and whose size is .gtoreq.2 cm.times.1 cm by
conventional computed tomography (CT) scan or .gtoreq.1 cm.times.1
cm by spiral CT scan) received a standard dose of docetaxel
(TAXOTERE) (up to 75 mg/m.sup.2, using actual body weight to
calculate body surface area (BSA)) by intravenous (IV) infusion
over 1 hour on Day 1 only of each cycle. After the docetaxel
infusion was completed, anti-IGF-1R antibodies as described herein
were administered intravenously in a 5 mg/ml liquid formulation at
a dose between 0.1 mg/kg and 10 mg/kg. The treatment regimen was
repeated after 21 days, with escalation of the anti-IGF-1R antibody
dose, and every 21 days thereafter until disease progression or
unacceptable toxicity develops for a minimum of 2 cycles and a
maximum of 17 cycles. The pre-medication regimen for docetaxel
included oral dexamethasone 8 mg twice daily for three days
starting one day prior to docetaxel administration. Prophylactic
anti-emetics were provided as appropriate.
[0105] Dose escalation used an accelerated titration design
utilizing a dose-doubling schema with 3-6 subjects per dose level
(cohort). Within each new cohort there was no required waiting
period between subjects. Subsequent cohorts were not opened until
the first subject at the current dose level had been observed for
21 days and subsequent subjects had been observed for 14 days.
[0106] The following endpoints were measured: safety, tolerability,
pharmacokinetic (PK) parameters of the anti-IGF-1R antibody; human
anti-human antibody response (HAHA); response rate and time to
progression; and number of circulating tumor cells (CTC) and
circulating soluble IGF-1R.
Example II
Anti-IGF-1R Antibodies in Combination with Paclitaxel and
Carboplatin in the Treatment of Advanced Non-Small Cell Lung
Cancer
[0107] In Part 1 of the study, patients with Stage IIIB or Stage IV
or recurrent (after surgery/radiation), measurable, non-small cell
lung cancer (NSCLC) who have received no prior chemotherapy
received paclitaxel (TAXOL) at a standard dose of 200 mg/m.sup.2 by
IV infusion over 3 hours. Prior to receiving paclitaxel, all
patients received prophylactic anti-allergic/emetic medicines.
Carboplatin (PARAPLATIN) was administered by IV infusion over 15-30
minutes; the dose was calculated based on the Calvert formula with
a target area under the curve (AUC) of 6 mg/ml.times.min. After the
carboplatin infusion was completed, anti-IGF-1R antibodies as
described herein were administered intravenously in a 5 mg/ml
formulation at a dose between 0.05 mg/kg and 10 mg/kg. The
treatment regimen was repeated after 21 days, with escalation of
the anti-IGF-1R antibody dose, and every 21 days thereafter until
disease progression or unacceptable toxicity develops, for a
minimum of 1 cycle and a maximum of 6 cycles.
[0108] Doses were escalated using an accelerated titration design
utilizing a dose-doubling schema with 3-6 subjects per cohort.
Within each new cohort there was no required waiting period between
subjects. Subsequent cohorts were not opened until the first
subject at the current dose level has been observed for 21 days and
subsequent subjects have been observed for 14 days.
[0109] Once at least six patients have been observed for 21 days
(i.e., completed one cycle), the randomized second portion of the
study will begin.
[0110] Part 2 of the study is a two-arm randomized, non-comparative
study of anti-IGF-1R antibody in combination with paclitaxel and
carboplatin (Arm A) and of paclitaxel and carboplatin alone (Arm
B). On Day 1 of Part 2, the patients in both arms are given the
same dosages of paclitaxel and carboplatin, over the same time
periods, as in the first part. After administration of carboplatin,
patients in Arm A are also given the same anti-IGF-1R antibody dose
they were given in Part 1. The dose is determined in view of the
safety and tolerability demonstrated in Part 1. The treatment is
repeated after 21 days, and every 21 days thereafter, until
progression or unacceptable toxicity occurs for a minimum of 2
cycles and a maximum of 6.
[0111] The following endpoints are measured: PK parameters of the
anti-IGF-1R antibody, HAHA, response rate and time to progression,
CTC, circulating IGF-1, IGFBPs, and soluble circulating IGF-1R.
Example III
Anti-IGF-1R in Combination with Docetaxel and Epirubicin in
Metastatic Breast Cancer
[0112] Patients having metastatic breast cancer with at least one
lesion that can be accurately measured in two dimensions and whose
size is 2 cm.times.1 cm by conventional CT scan or 1 cm.times.1 cm
by spiral CT scan are given epirubicin 75 mg/m.sup.2 as a single 15
minute infusion. After a one hour pause, docetaxel (TAXOTERE) 75
mg/m.sup.2 is administered as a single IV infusion, followed by IV
infusion of anti-IGF-1R antibodies as described herein at a dose
between 0.05 mg/kg and 10 mg/kg. Prophylactic anti-emetics are
given as appropriate. The treatment is repeated after 21 days with
escalation of the anti-IGF-1R antibody dose, and every 21 days
thereafter until disease progression or unacceptable toxicity
develops for a minimum of 2 cycles and a maximum of 6.
[0113] Doses are escalated using an accelerated titration design
utilizing a dose-doubling schema with 3-6 subjects per cohort.
Within each new cohort there is no required waiting period between
subjects. Subsequent cohorts may not be opened until the first
subject at the current dose level has been observed for 21 days and
subsequent subjects have been observed for 14 days.
[0114] The following endpoints are measured: PK parameters, HAHA,
response rate and time to progression. Time to progression and
overall survival are calculated using the Kaplan-Meier product
limit method.
Example IV
Anti-IGF-1R in Combination with Docetaxel and Prednisone in
Hormone-Refractory Prostate Cancer
[0115] Subjects are patients with metastatic adenocarcinoma of the
prostate who, after at least one hormonal treatment (orchiectomy,
estrogens, LHRH therapy, etc.), have testosterone levels less than
50 ng/dL, prostate-specific antigen (PSA) above 20 ng/mL, and an
increase in PSA >50% over nadir value on hormonal therapy
measured on 3 successive occasions at least 1 week apart. A
pre-medication regimen for docetaxel includes oral dexamethasone 8
mg twice a day given for 3 days starting one day prior to docetaxel
administration. A 75 mg/m.sup.2 dose of docetaxel (TAXOTERE) (using
actual body weight to calculate BSA) is administered by IV infusion
over 1 hour on Day 1 only of each cycle. After the docetaxel
infusion is completed, anti-IGF-1R antibodies as described herein
are administered intravenously in a 5 mg/ml liquid formulation.
Prednisone is given daily in two oral 5 mg doses per day, starting
on Day 1. Prophylactic anti-emetics may be given as appropriate.
The treatment regimen is repeated every 21 days (.+-.3 days) until
disease progression or unacceptable toxicity develops, for a
maximum of 10 cycles.
[0116] The following endpoints are measured: PSA response,
population PK parameters of the anti-IGF-1R antibody, HAHA, total
number of CTCs and CTCs expressing IGF-1R.
[0117] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Sequence CWU 1
1
601291DNAHomo sapiens 1tgcatctgta ggagacagag tcaccttcac ttgccgggca
agtcaggaca ttagacgtga 60tttaggctgg tatcagcaga aaccagggaa agctcctaag
cgcctgatct atgctgcatc 120ccgtttacaa agtggggtcc catcaaggtt
cagcggcagt ggatctggga cagaattcac 180tctcacaatc agcagcctgc
agcctgaaga ttttgcaact tattactgtc tacagcataa 240taattatcct
cggacgttcg gccaagggac cgaggtggaa atcatacgaa c 2912136PRTHomo
sapiens 2Ala Ser Val Gly Asp Arg Val Thr Phe Thr Cys Arg Ala Ser
Gln Asp1 5 10 15Ile Arg Arg Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro 20 25 30Lys Arg Leu Ile Tyr Ala Ala Ser Arg Leu Gln Ser
Gly Val Pro Ser 35 40 45Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser 50 55 60Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln His Asn65 70 75 80Asn Tyr Pro Arg Thr Phe Gly Gln
Gly Thr Glu Val Glu Ile Ile Arg 85 90 95Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln 100 105 110Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 115 120 125Pro Arg Glu
Ala Lys Val Gln Trp 130 1353352DNAHomo sapiens 3gggaggcttg
gtcaagcctg gaggtccctg agactctcct gtgcagcctc tggattcact 60ttcagtgact
actatatgag ctggatccgc caggctccag ggaaggggct ggaatgggtt
120tcatacatta gtagtagtgg tagtaccaga gactacgcag actctgtgaa
gggccgattc 180accatctcca gggacaacgc caagaactca ctgtatctgc
aaatgaacag cctgagagcc 240gaggacacgg ccgtgtatta ctgtgtgaga
gatggagtgg aaactacttt ttactactac 300tactacggta tggacgtctg
gggccaaggg accacggtca ccgtctcctc ag 3524174PRTHomo sapiens 4Gly Arg
Leu Gly Gln Ala Trp Arg Ser Leu Arg Leu Ser Cys Ala Ala1 5 10 15Ser
Gly Phe Thr Phe Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala 20 25
30Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser
35 40 45Thr Arg Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg 50 55 60Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala65 70 75 80Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg Asp Gly
Val Glu Thr Thr 85 90 95Phe Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr Thr 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ser Cys Ala 165 1705322DNAHomo
sapiens 5gacatccaga tgacccagtt tccatcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca
gcagaaacca 120gggaaagccc ctaagcgcct gatctatgct gcatcccgtt
tgcacagagg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa
ttcactctca caatcagcag cctgcagcct 240gaagattttg caacttatta
ctgtttacaa cataatagtt acccgtgcag ttttggccag 300gggaccaagc
tggagatcaa ac 3226107PRTHomo sapiens 6Asp Ile Gln Met Thr Gln Phe
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Ala Ala Ser
Arg Leu His Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Cys 85 90
95Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 1057375DNAHomo
sapiens 7aggtgcagct gttggagtct gggggaggct tggtacagcc tggggggtcc
ctgagactct 60cctgtacagc ctctggattc acctttagca gctatgccat gaactgggtc
cgccaggctc 120cagggaaggg gctggagtgg gtctcagcta ttagtggtag
tggtggtacc acattctacg 180cagactccgt gaagggccgg ttcaccatct
ccagagacaa ttccaggacc acgctgtatc 240tgcaaatgaa cagcctgaga
gccgaggaca cggccgtata ttactgtgcg aaagatcttg 300gctggtccga
ctcttactac tactactacg gtatggacgt ctggggccaa gggaccacgg
360tcaccgtctc ctcag 3758124PRTHomo sapiens 8Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser1 5 10 15Leu Arg Leu Ser Cys
Thr Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala 20 25 30Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45Ala Ile Ser
Gly Ser Gly Gly Thr Thr Phe Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Arg Thr Thr Leu Tyr Leu65 70 75
80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Lys Asp Leu Gly Trp Ser Asp Ser Tyr Tyr Tyr Tyr Tyr Gly Met
Asp 100 105 110Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
1209302DNAHomo sapiens 9tcctccctgt ctgcatctgt aggagacaga gtcaccttca
cttgccgggc aagtcaggac 60attagacgtg atttaggctg gtatcagcag aaaccaggga
aagctcctaa gcgcctgatc 120tatgctgcat cccgtttaca aagtggggtc
ccatcaaggt tcagcggcag tggatctggg 180acagaattca ctctcacaat
cagcagcctg cagcctgaag attttgcaac ttattactgt 240ctacagcata
ataattatcc tcggacgttc ggccaaggga ccgaggtgga aatcatacga 300ac
30210100PRTHomo sapiens 10Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
Val Thr Phe Thr Cys Arg1 5 10 15Ala Ser Gln Asp Ile Arg Arg Asp Leu
Gly Trp Tyr Gln Gln Lys Pro 20 25 30Gly Lys Ala Pro Lys Arg Leu Ile
Tyr Ala Ala Ser Arg Leu Gln Ser 35 40 45Gly Val Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr Glu Phe Thr 50 55 60Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys65 70 75 80Leu Gln His Asn
Asn Tyr Pro Arg Thr Phe Gly Gln Gly Thr Glu Val 85 90 95Glu Ile Ile
Arg 10011338DNAHomo sapiens 11gggcccagga ctggtgaagc cttcggagac
cctgtccctc acctgcactg tctctggtgg 60ctccatcagt aattactact ggagctggat
ccggcagccc gccgggaagg gactggagtg 120gattgggcgt atctatacca
gtgggagccc caactacaac ccctccctca agagtcgagt 180caccatgtca
gtagacacgt ccaagaacca gttctccctg aagctgaact ctgtgaccgc
240cgcggacacg gccgtgtatt actgtgcggt aacgattttt ggagtggtta
ttatctttga 300ctactggggc cagggaaccc tggtcaccgt ctcctcag
33812112PRTHomo sapiens 12Gly Pro Gly Leu Val Lys Pro Ser Glu Thr
Leu Ser Leu Thr Cys Thr1 5 10 15Val Ser Gly Gly Ser Ile Ser Asn Tyr
Tyr Trp Ser Trp Ile Arg Gln 20 25 30Pro Ala Gly Lys Gly Leu Glu Trp
Ile Gly Arg Ile Tyr Thr Ser Gly 35 40 45Ser Pro Asn Tyr Asn Pro Ser
Leu Lys Ser Arg Val Thr Met Ser Val 50 55 60Asp Thr Ser Lys Asn Gln
Phe Ser Leu Lys Leu Asn Ser Val Thr Ala65 70 75 80Ala Asp Thr Ala
Val Tyr Tyr Cys Ala Val Thr Ile Phe Gly Val Val 85 90 95Ile Ile Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105
11013322DNAHomo sapiens 13gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gggcattaga
agtgatttag gctggtttca gcagaaacca 120gggaaagccc ctaagcgcct
gatctatgct gcatccaaat tacaccgtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca caatcagccg cctgcagcct
240gaagattttg caacttatta ctgtctacag cataatagtt accctctcac
tttcggcgga 300gggaccaagg tggagatcaa ac 32214107PRTHomo sapiens
14Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Ser
Asp 20 25 30Leu Gly Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg
Leu Ile 35 40 45Tyr Ala Ala Ser Lys Leu His Arg Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Arg Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
His Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 10515376DNAHomo sapiens 15gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc agctatgcca tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attagtggta gtggtggtat cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat
attactgtgc gaaagatctg 300ggctacggtg acttttacta ctactactac
ggtatggacg tctggggcca agggaccacg 360gtcaccgtct cctcag
37616125PRTHomo sapiens 16Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly
Gly Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp
Leu Gly Tyr Gly Asp Phe Tyr Tyr Tyr Tyr Tyr Gly Met 100 105 110Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
12517279DNAHomo sapiens 17caggagacag agtcaccatc acttgccggg
caagtcagag cattagtacc tttttaaatt 60ggtatcagca gaaaccaggg aaagccccta
aactcctgat ccatgttgca tccagtttac 120aaggtggggt cccatcaagg
ttcagtggca gtggatctgg gacagatttc actctcacca 180tcagcagtct
gcaacctgaa gattttgcaa cttactactg tcaacagagt tacaatgccc
240cactcacttt cggcggaggg accaaggtgg agatcaaac 2791892PRTHomo
sapiens 18Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Thr1 5 10 15Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu 20 25 30Ile His Val Ala Ser Ser Leu Gln Gly Gly Val Pro
Ser Arg Phe Ser 35 40 45Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln 50 55 60Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Ser Tyr Asn Ala Pro65 70 75 80Leu Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 85 9019341DNAHomo sapiens 19cccaggactg gtgaagcctt
cggagaccct gtccctcacc tgcactgtct ctggtggctc 60catcagtagt tactactgga
gttggatccg gcagccccca gggaagggac tggagtggat 120tgggtatatc
tattacagtg ggagcaccaa ctacaacccc tccctcaaga gtcgagtcac
180catatcagta gacacgtcca agaaccagtt ctccctgaag ctgagttctg
tgaccgctgc 240ggacacggcc gtgtattact gtgccaggac gtatagcagt
tcgttctact actacggtat 300ggacgtctgg ggccaaggga ccacggtcac
cgtctcctca g 34120113PRTHomo sapiens 20Pro Gly Leu Val Lys Pro Ser
Glu Thr Leu Ser Leu Thr Cys Thr Val1 5 10 15Ser Gly Gly Ser Ile Ser
Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro 20 25 30Pro Gly Lys Gly Leu
Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser 35 40 45Thr Asn Tyr Asn
Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp 50 55 60Thr Ser Lys
Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala65 70 75 80Asp
Thr Ala Val Tyr Tyr Cys Ala Arg Thr Tyr Ser Ser Ser Phe Tyr 85 90
95Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser
100 105 110Ser21274PRTHomo sapiens 21Ala Gly Ala Gly Cys Cys Ala
Cys Cys Cys Thr Cys Thr Cys Cys Thr1 5 10 15Gly Thr Ala Gly Gly Gly
Cys Cys Ala Gly Thr Cys Ala Gly Ala Gly 20 25 30Thr Gly Thr Thr Cys
Gly Cys Gly Gly Cys Ala Gly Gly Thr Ala Cys 35 40 45Thr Thr Ala Gly
Cys Cys Thr Gly Gly Thr Ala Cys Cys Ala Gly Cys 50 55 60Ala Gly Ala
Ala Ala Cys Cys Thr Gly Gly Cys Cys Ala Gly Gly Cys65 70 75 80Thr
Cys Cys Cys Ala Gly Gly Cys Thr Cys Cys Thr Cys Ala Thr Cys 85 90
95Thr Ala Thr Gly Gly Thr Gly Cys Ala Thr Cys Cys Ala Gly Cys Ala
100 105 110Gly Gly Gly Cys Cys Ala Cys Thr Gly Gly Cys Ala Thr Cys
Cys Cys 115 120 125Ala Gly Ala Cys Ala Gly Gly Thr Thr Cys Ala Gly
Thr Gly Gly Cys 130 135 140Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly
Gly Gly Ala Cys Ala Gly145 150 155 160Ala Cys Thr Thr Cys Ala Cys
Thr Cys Thr Cys Ala Cys Cys Ala Thr 165 170 175Cys Ala Gly Cys Ala
Gly Ala Cys Thr Gly Gly Ala Gly Cys Cys Thr 180 185 190Gly Ala Ala
Gly Ala Thr Thr Thr Thr Gly Cys Ala Gly Thr Gly Thr 195 200 205Thr
Thr Thr Ala Cys Thr Gly Thr Cys Ala Gly Cys Ala Gly Thr Ala 210 215
220Thr Gly Gly Thr Ala Gly Thr Thr Cys Ala Cys Cys Thr Cys Gly
Asn225 230 235 240Ala Cys Gly Thr Thr Cys Gly Gly Cys Cys Ala Ala
Gly Gly Gly Ala 245 250 255Cys Cys Ala Ala Gly Gly Thr Gly Gly Ala
Ala Ala Thr Cys Ala Ala 260 265 270Ala Cys2291PRTHomo sapiens 22Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Gly Arg Tyr1 5 10
15Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
20 25 30Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
Gly 35 40 45Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu Pro 50 55 60Glu Asp Phe Ala Val Phe Tyr Cys Gln Gln Tyr Gly Ser
Ser Pro Arg65 70 75 80Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
85 9023367DNAHomo sapiens 23gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagc
agctatgcca tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtctcaggt attactggga gtggtggtag tacatactac 180gcagactccg
tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag agccgaggac acggccgtat attactgtgc
gaaagatcca 300gggactacgg tgattatgag ttggttcgac ccctggggcc
agggaaccct ggtcaccgtc 360tcctcag 36724122PRTHomo sapiens 24Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Gly Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Lys Asp Pro Gly Thr Thr Val Ile Met Ser
Trp Phe Asp Pro Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 12025320DNAHomo sapiens 25gaactgtggc tgcaccatct gtcttcatct
tcccgccatc tgatgagcag ttgaaatctg 60gaactgcctc tgttgtgtgc ctgctgaata
acttctatcc cagagaggcc aaagtacagt 120ggaaggtgga taacgccctc
caatcgggta actcccagga gagtgtcaca gagcaggaca 180gcaaggacag
cacctacagc ctcagcagca ccctgacgct gagcaaagca gactacgaga
240aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc
gtcacaaaga 300gcttcaacag gggagagtgt 32026106PRTHomo sapiens 26Thr
Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln1 5 10 15Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys65 70 75 80His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 100 10527978DNAHomo sapiens
27gcctccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag
60agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
120tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct
acagtcctca 180ggactctact ccctcagcag cgtggtgacc gtgccctcca
gcaacttcgg cacccagacc 240tacacctgca acgtagatca caagcccagc
aacaccaagg tggacaagac agttgagcgc 300aaatgttgtg tcgagtgccc
accgtgccca gcaccacctg tggcaggacc gtcagtcttc 360ctcttccccc
caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc
420gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta
cgtggacggc 480gtggaggtgc ataatgccaa gacaaagcca cgggaggagc
agttcaacag cacgttccgt 540gtggtcagcg tcctcaccgt tgtgcaccag
gactggctga acggcaagga gtacaagtgc 600aaggtctcca acaaaggcct
cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 660cagccccgag
aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac
720caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc
cgtggagtgg 780gagagcaatg ggcagccgga gaacaactac aagaccacac
ctcccatgct ggactccgac 840ggctccttct tcctctacag caagctcacc
gtggacaaga gcaggtggca gcaggggaac 900gtcttctcat gctccgtgat
gcatgaggct ctgcacaacc actacacgca gaagagcctc 960tccctgtctc cgggtaaa
97828326PRTHomo sapiens 28Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Thr Val Glu
Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110Pro
Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120
125Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
130 135 140Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly145 150 155 160Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn 165 170 175Ser Thr Phe Arg Val Val Ser Val Leu
Thr Val Val His Gln Asp Trp 180 185 190Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205Ala Pro Ile Glu Lys
Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn225 230 235
240Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
245 250 255Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 260 265 270Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys 275 280 285Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys 290 295 300Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu305 310 315 320Ser Leu Ser Pro Gly
Lys 32529296DNAHomo sapiens 29caggtgcagc tggtggagtc tgggggaggc
ttggtcaagc ctggagggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt
gactactaca tgagctggat ccgccaggct 120ccagggaagg ggctggagtg
ggtttcatac attagtagta gtggtagtac catatactac 180gcagactctg
tgaagggccg attcaccatc tccagggaca acgccaagaa ctcactgtat
240ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaga
2963098PRTHomo sapiens 30Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25 30Tyr Met Ser Trp Ile Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Tyr Ile Ser Ser Ser Gly
Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg31296PRTHomo sapiens 31Gly Ala Gly Gly Thr Gly Cys Ala Gly Cys
Thr Gly Thr Thr Gly Gly1 5 10 15Ala Gly Thr Cys Thr Gly Gly Gly Gly
Gly Ala Gly Gly Cys Thr Thr 20 25 30Gly Gly Thr Ala Cys Ala Gly Cys
Cys Thr Gly Gly Gly Gly Gly Gly 35 40 45Thr Cys Cys Cys Thr Gly Ala
Gly Ala Cys Thr Cys Thr Cys Cys Thr 50 55 60Gly Thr Gly Cys Ala Gly
Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr65 70 75 80Cys Ala Cys Cys
Thr Thr Thr Ala Gly Cys Ala Gly Cys Thr Ala Thr 85 90 95Gly Cys Cys
Ala Thr Gly Ala Gly Cys Thr Gly Gly Gly Thr Cys Cys 100 105 110Gly
Cys Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Gly Gly Ala Ala 115 120
125Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Gly Thr Cys
130 135 140Thr Cys Ala Gly Cys Thr Ala Thr Thr Ala Gly Thr Gly Gly
Thr Ala145 150 155 160Gly Thr Gly Gly Thr Gly Gly Thr Ala Gly Cys
Ala Cys Ala Thr Ala 165 170 175Cys Thr Ala Cys Gly Cys Ala Gly Ala
Cys Thr Cys Cys Gly Thr Gly 180 185 190Ala Ala Gly Gly Gly Cys Cys
Gly Gly Thr Thr Cys Ala Cys Cys Ala 195 200 205Thr Cys Thr Cys Cys
Ala Gly Ala Gly Ala Cys Ala Ala Thr Thr Cys 210 215 220Cys Ala Ala
Gly Ala Ala Cys Ala Cys Gly Cys Thr Gly Thr Ala Thr225 230 235
240Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Ala Cys Ala Gly Cys Cys
245 250 255Thr Gly Ala Gly Ala Gly Cys Cys Gly Ala Gly Gly Ala Cys
Ala Cys 260 265 270Gly Gly Cys Cys Gly Thr Ala Thr Ala Thr Thr Ala
Cys Thr Gly Thr 275 280 285Gly Cys Gly Ala Ala Ala Gly Ala 290
2953298PRTHomo sapiens 32Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Lys33296DNAHomo sapiens 33caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggggac cctgtccctc 60acctgcgctg tctctggtgg ctccatcagc
agtagtaact ggtggagttg ggtccgccag 120cccccaggga aggggctgga
gtggattggg gaaatctatc atagtgggag caccaactac 180aacccgtccc
tcaagagtcg agtcaccata tcagtagaca agtccaagaa ccagttctcc
240ctgaagctga gctctgtgac cgccgcggac acggccgtgt attactgtgc gagaga
2963498PRTHomo sapiens 34Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gly1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Ser
Gly Gly Ser Ile Ser Ser Ser 20 25 30Asn Trp Trp Ser Trp Val Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Glu Ile Tyr His Ser
Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Ile
Ser Val Asp Lys Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg35293DNAHomo sapiens 35caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
agttactact ggagctggat ccggcagccc 120ccagggaagg gactggagtg
gattgggtat atctattaca gtgggagcac caactacaac 180ccctccctca
agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg
240aagctgagct ctgtgaccgc tgcggacacg gccgtgtatt actgtgcgag aga
2933697PRTHomo sapiens 36Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Gly Ser Ile Ser Ser Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Tyr Tyr Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95Arg37290PRTHomo sapiens 37Gly Ala Ala Ala Thr Thr Gly Thr Gly Thr
Thr Gly Ala Cys Gly Cys1 5 10 15Ala Gly Thr Cys Thr Cys Cys Ala Gly
Gly Cys Ala Cys Cys Cys Thr 20 25 30Gly Thr Cys Thr Thr Thr Gly Thr
Cys Thr Cys Cys Ala Gly Gly Gly 35 40 45Gly Ala Ala Ala Gly Ala Gly
Cys Cys Ala Cys Cys Cys Thr Cys Thr 50 55 60Cys Cys Thr Gly Cys Ala
Gly Gly Gly Cys Cys Ala Gly Thr Cys Ala65 70 75 80Gly Ala Gly Thr
Gly Thr Thr Ala Gly Cys Ala Gly Cys Ala Gly Cys 85 90 95Thr Ala Cys
Thr Thr Ala Gly Cys Cys Thr Gly Gly Thr Ala Cys Cys 100 105 110Ala
Gly Cys Ala Gly Ala Ala Ala Cys Cys Thr Gly Gly Cys Cys Ala 115 120
125Gly Gly Cys Thr Cys Cys Cys Ala Gly Gly Cys Thr Cys Cys Thr Cys
130 135 140Ala Thr Cys Thr Ala Thr Gly Gly Thr Gly Cys Ala Thr Cys
Cys Ala145 150 155 160Gly Cys Ala Gly Gly Gly Cys Cys Ala Cys Thr
Gly Gly Cys Ala Thr 165 170 175Cys Cys Cys Ala Gly Ala Cys Ala Gly
Gly Thr Thr Cys Ala Gly Thr 180 185 190Gly Gly Cys Ala Gly Thr Gly
Gly Gly Thr Cys Thr Gly Gly Gly Ala 195 200 205Cys Ala Gly Ala Cys
Thr Thr Cys Ala Cys Thr Cys Thr Cys Ala Cys 210 215 220Cys Ala Thr
Cys Ala Gly Cys Ala Gly Ala Cys Thr Gly Gly Ala Gly225 230 235
240Cys Cys Thr Gly Ala Ala Gly Ala Thr Thr Thr Thr Gly Cys Ala Gly
245 250 255Thr Gly Thr Ala Thr Thr Ala Cys Thr Gly Thr Cys Ala Gly
Cys Ala 260 265 270Gly Thr Ala Thr Gly Gly Thr Ala Gly Cys Thr Cys
Ala Cys Cys Thr 275 280 285Cys Cys 2903896PRTHomo sapiens 38Glu Ile
Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg
Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
Gly Ser Ser Pro 85 90 9539288DNAHomo
sapiensmisc_feature(288)..(288)n is a, c, g, or t 39gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca
120gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca
caatcagcag cctgcagcct 240gaagattttg caacttatta ctgtctacag
cataatagtt accctccn 2884096PRTHomo sapiens 40Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu Gly Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Pro
85 90 9541288DNAHomo sapiens 41gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc
agctatttaa attggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg caacttacta ctgtcaacag agttacagta cccctcch
2884296PRTHomo sapiens 42Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90
9543293DNAHomo sapiens 43caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt
agttactact ggagctggat ccggcagccc 120gccgggaagg gactggagtg
gattgggcgt atctatacca gtgggagcac caactacaac 180ccctccctca
agagtcgagt caccatgtca gtagacacgt ccaagaacca gttctccctg
240aagctgagct ctgtgaccgc cgcggacacg gccgtgtatt actgtgcgag aga
2934497PRTHomo sapiens 44Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Gly Ser Ile Ser Ser Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro
Ala Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly Arg Ile Tyr Thr Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Met Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95Arg45470PRTHomo sapiens 45Met Glu Phe Gly Leu Ser Trp Leu Phe Leu
Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser
Cys Thr Ala Ser Gly Phe Thr Phe 35 40 45Ser Ser Tyr Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ser Ala Ile
Ser Gly Ser Gly Gly Thr Thr Phe Tyr Ala65 70 75 80Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Thr 85 90 95Thr Leu Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110Tyr
Tyr Cys Ala Lys Asp Leu Gly Trp Ser Asp Ser Tyr Tyr Tyr Tyr 115 120
125Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
130 135 140Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg145 150 155 160Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr
165 170 175Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 180 185 190Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 195 200 205Leu Ser Ser Val Val Thr Val Pro Ser Ser
Asn Phe Gly Thr Gln Thr 210 215 220Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys225 230 235 240Thr Val Glu Arg Lys
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 245 250 255Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280
285Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
290 295 300Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn305 310 315 320Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp 325 330 335Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro 340 345 350Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu 355 360 365Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile385 390 395
400Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
405 410 415Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys 420 425 430Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys 435 440 445Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu 450 455 460Ser Leu Ser Pro Gly Lys465
47046470PRTHomo sapiens 46Met Glu Phe Gly Leu Ser Trp Leu Phe Leu
Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45Ser Ser Tyr Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ser Ala Ile
Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala65 70 75 80Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95Thr Leu Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110Tyr
Tyr Cys Ala Lys Gly Tyr Ser Ser Gly Trp Tyr Tyr Tyr Tyr Tyr 115 120
125Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
130 135 140Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg145 150 155 160Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 165 170 175Phe Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser 180 185 190Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 195 200 205Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 210 215 220Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys225 230 235
240Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
245 250 255Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp 260 265 270Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp 275 280 285Val Ser His Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly 290 295 300Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe Asn305 310 315 320Ser Thr Phe Arg Val
Val Ser Val Leu Thr Val Val His Gln Asp Trp 325 330 335Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 340 345 350Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 355 360
365Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
370 375 380Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile385 390 395 400Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr 405 410 415Thr Pro Pro Met Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys 420 425 430Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460Ser Leu Ser
Pro Gly Lys465 47047236PRTHomo sapiens 47Met Asp Met Arg Val Pro
Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro Gly Ala Arg
Cys Asp Ile Gln Met Thr Gln Phe Pro Ser Ser 20 25 30Leu Ser Ala Ser
Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45Gln Gly Ile
Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro
Lys Arg Leu Ile Tyr Ala Ala Ser Arg Leu His Arg Gly Val65 70 75
80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu
Gln 100 105 110His Asn Ser Tyr Pro Cys Ser Phe Gly Gln Gly Thr Lys
Leu Glu Ile 115 120 125Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp 130 135 140Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn145 150 155 160Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200
205Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
210 215 220Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230
23548236PRTHomo sapiens 48Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro Gly Ala Arg Cys Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser 20 25 30Leu Ser Ala Ser Val Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser 35 40 45Gln Gly Ile Arg Asn Asp Leu
Gly Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys Arg Leu Ile
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val65 70 75 80Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110His
Asn Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120
125Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
130 135 140Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn145 150 155 160Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu 165 170 175Gln Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp 180 185 190Ser Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205Glu Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230 23549470PRTHomo sapiens
49Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Ile Ile Lys Gly1
5 10 15Val Gln Cys Gln Ala Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Lys 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe 35 40 45Ser Asp Tyr Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly
Lys Gly Leu 50 55 60Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr
Arg Asp Tyr Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn 85 90 95Ser Leu Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val 100 105 110Tyr Tyr Cys Val Arg Asp Gly
Val Glu Thr Thr Phe Tyr Tyr Tyr Tyr 115 120 125Tyr Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 130 135 140Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg145 150 155
160Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
165 170 175Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 180 185 190Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 195 200 205Leu Ser Ser Val Val Thr Val Pro Ser Ser
Asn Phe Gly Thr Gln Thr 210 215 220Tyr Thr Cys Asn Val Asp His Lys
Pro Ser Asn Thr Lys Val Asp Lys225 230 235 240Thr Val Glu Arg Lys
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 245 250 255Pro Val Ala
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280
285Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
290 295 300Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn305 310 315 320Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp 325 330 335Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro 340 345 350Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu 355 360 365Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 370 375 380Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile385 390 395
400Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
405 410 415Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys 420 425 430Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys 435 440 445Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu 450 455 460Ser Leu Ser Pro Gly Lys465
47050473PRTHomo sapiens 50Met Glu Phe Gly Leu Ser Trp Val Phe Leu
Val Ala Ile Ile Lys Gly1 5 10 15Val Gln Cys Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45Ser Asp Tyr Tyr Met Ser Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ser Tyr Ile
Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala65 70 75 80Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Ser Leu Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110Tyr
Tyr Cys Ala Arg Val Leu Arg Phe Leu Glu Trp Leu Leu Tyr Tyr 115 120
125Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
130 135 140Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro145 150 155 160Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val 165 170 175Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala 180 185 190Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly 195 200 205Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly 210 215 220Thr Gln Thr
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys225 230 235
240Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys
245 250 255Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys 260 265 270Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 275 280 285Val Val Asp Val Ser His Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr 290 295 300Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu305 310 315 320Gln Phe Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His 325 330 335Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 340 345 350Gly
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 355 360
365Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
370 375 380Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro385 390 395 400Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn 405 410 415Tyr Lys Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu 420 425 430Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 435 440 445Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 450 455 460Lys Ser Leu
Ser Leu Ser Pro Gly Lys465 47051236PRTHomo sapiens 51Met Asp Met
Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro
Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30Leu
Ser Ala Ser Val Gly Asp Arg Val Thr Phe Thr Cys Arg Ala Ser 35 40
45Gln Asp Ile Arg Arg Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Arg Leu Gln Ser Gly
Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr 85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln 100 105 110His Asn Asn Tyr Pro Arg Thr Phe Gly Gln
Gly Thr Glu Val Glu Ile 115 120 125Ile Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp 130 135 140Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150 155 160Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185
190Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser 210 215 220Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys225 230 23552236PRTHomo sapiens 52Met Asp Met Arg Val Pro Ala
Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro Gly Ala Arg Cys
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30Leu Ser Ala Ser Val
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40
45Gln Gly Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60Ala Pro Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe
Thr Leu Thr 85 90 95Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Leu Gln 100 105 110His Asn Ser Tyr Pro Trp Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile 115 120 125Lys Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp 130 135 140Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150 155 160Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185
190Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
195 200 205Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser 210 215 220Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys225 230 23553326DNAArtificial SequenceDescription of Artificial
Sequence Consensus Sequence 53gacatccaga tgacccagty tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60wtcacttgcc gggcaagtca ggrcattaga
mrtgatttag gctggtwtca gcagaaacca 120gggaaagcyc ctaagcgcct
gatctatgct gcatccmrwt trcammgwgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca caatcagcmg cctgcagcct
240gaagattttg caacttatta ctgtytacar cataatartt aycckybsns
kttyggcsrr 300gggaccrags tggaratcaw acgaac 32654322DNAArtificial
SequenceDescription of Artificial Sequence Consensus Sequence
54gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgyaggaga cagagtcacc
60atcacttgcc gggcaagtca gagcattagy asctwtttaa attggtatca gcagaaacca
120gggaaagccc ctaarctcct gatcyatgyt gcatccagtt trcaargtgg
ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caacttacta ctgtcaacag
agttacartr ccccayychc tttcggcgga 300gggaccaagg tggagatcaa ac
32255325DNAArtificial SequenceDescription of Artificial Sequence
Consensus Sequence 55gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgya gggccagtca gagtgttmgc rgcagstact
tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat
ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg
gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgtw ttactgtcag cagtatggta gytcacctcs nacgttcggc
300caagggacca aggtggaaat caaac 32556376DNAArtificial
SequenceDescription of Artificial Sequence Consensus Sequence
56caggtgcagc tggtggagtc tgggggaggc ttggtcaagc ctggagggtc cctgagactc
60tcctgtgcag cctctggatt cacyttcagt gactactaya tgagctggat ccgccaggct
120ccagggaagg ggctggartg ggtttcatac attagtagta gtggtagtac
cakakactac 180gcagactctg tgaagggccc attcaccatc tccagggaca
acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtgt attactgtgy gagagatgga 300gtggaaacta ctttttacta
ctactactac ggtatggacg tctggggcca agggaccacg 360gtcaccgtct cctcag
37657358DNAArtificial SequenceDescription of Artificial Seqence
Consensus Sequence 57caggtgcagc tgcaggagtc gggcccagga ctggtgaagc
cttcggagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagt arttactact
ggagctggat ccggcagccc 120gccgggaagg gactggagtg gattgggcgt
atctatacca gtgggagcmc caactacaac 180ccctccctca agagtcgagt
caccatgtca gtagacacgt ccaagaacca gttctccctg 240aagctgarct
ctgtgaccgc cgcggacacg gccgtgtatt actgtgcggt aacgattttt
300ggagtggtta ttatctttga ctactggggc cagrganccc tggtcaccgt ctcctcag
35858418DNAArtificial SequenceDescription of Artificial Sequence
Consensus Sequence 58caggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgagactc 60tcctgtrcag cctctggatt cacctttagc agctatgcca
tgarctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagst
attastggka gtggtggtab yacatwctac 180gcagactccg tgaagggccc
gttcaccatc tccagagaca attccargam cacgctgtat 240ctgcaaatga
acagcctgag agccgaggac acggccgtat attactgtgc gaaagatctk
300ggctrsksyg actyttacta ctactactac ggtatggacg tctggggcca
agggacyacg 360gtgattatga gttggttcga cccctggggc cagggaaccc
tggtcaccgt ctcctcag 41859364DNAArtificial SequenceDescription of
Artificial Sequence Consensus Sequence 59caggtgcagc tgcaggagtc
gggcccagga ctggtgaagc cttcggagac cctgtccctc 60acctgcactg tctctggtgg
ctccatcagt agttactact ggagytggat ccggcagccc 120ccagggaagg
gactggagtg gattgggtat atctattaca gtgggagcac caactacaac
180ccctccctca agagtcgact caccatatca gtagacacgt ccaagaacca
gttctccctg 240aagctgagyt ctgtgaccgc tgcggacacg gccgtgtatt
actgtgccag gacgtatagc 300agttcgttct actactacgg tatggacgtc
tggggccaag ggaccacggt caccgtctcc 360tcag 3646015PRTArtificial
SequenceDescription of Artificial Sequence Consensus Sequence 60Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10
15
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