U.S. patent application number 13/672419 was filed with the patent office on 2013-05-30 for antibody drug conjugates (adc) that bind to 24p4c12 proteins.
This patent application is currently assigned to SEATTLE GENETICS, INC.. The applicant listed for this patent is Agensys, Inc., Seattle Genetics, Inc.. Invention is credited to Zili AN, Dennis BENJAMIN, Jean GUDAS, Aya JAKOBOVITS, Xiao-chi JIA, Karen Jane Meyrick MORRISON, Robert Kendall MORRISON, Ruth MOSER, Peter SENTER.
Application Number | 20130136756 13/672419 |
Document ID | / |
Family ID | 42781369 |
Filed Date | 2013-05-30 |
United States Patent
Application |
20130136756 |
Kind Code |
A1 |
GUDAS; Jean ; et
al. |
May 30, 2013 |
ANTIBODY DRUG CONJUGATES (ADC) THAT BIND TO 24P4C12 PROTEINS
Abstract
Antibody drug conjugates (ADC's) that bind to 24P4C12 protein
and variants thereof are described herein. 24P4C12 exhibits tissue
specific expression in normal adult tissue, and is aberrantly
expressed in the cancers listed in Table I. Consequently, the ADC's
of the invention provide a therapeutic composition for the
treatment of cancer.
Inventors: |
GUDAS; Jean; (Los Angeles,
CA) ; JAKOBOVITS; Aya; (Beverly Hills, CA) ;
AN; Zili; (Santa Monica, CA) ; MORRISON; Robert
Kendall; (Santa Monica, CA) ; MORRISON; Karen Jane
Meyrick; (Santa Monica, CA) ; JIA; Xiao-chi;
(Los Angeles, CA) ; BENJAMIN; Dennis; (Redmond,
WA) ; MOSER; Ruth; (Bellevue, WA) ; SENTER;
Peter; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Agensys, Inc.;
Seattle Genetics, Inc.; |
Santa Monica
Bothell |
CA
WA |
US
US |
|
|
Assignee: |
SEATTLE GENETICS, INC.
Bothell
WA
AGENSYS, INC.
Santa Monica
CA
|
Family ID: |
42781369 |
Appl. No.: |
13/672419 |
Filed: |
November 8, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12718902 |
Mar 5, 2010 |
8309093 |
|
|
13672419 |
|
|
|
|
61158143 |
Mar 6, 2009 |
|
|
|
Current U.S.
Class: |
424/178.1 |
Current CPC
Class: |
C07K 16/30 20130101;
A61K 2039/505 20130101; C07K 16/3046 20130101; A61K 47/6803
20170801; A61K 45/06 20130101; A61P 35/00 20180101; A61K 47/6851
20170801; A61K 47/6863 20170801 |
Class at
Publication: |
424/178.1 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 45/06 20060101 A61K045/06; A61K 47/48 20060101
A61K047/48 |
Claims
1. A method for inhibiting growth of a cancer cell in a human
subject, comprising: administering to the subject an effective
amount of an antibody drug conjugate, wherein the conjugate
comprises an antibody or antigen binding fragment thereof
conjugated to monomethyl auristatin E (MMAE), and wherein the
antibody or fragment thereof comprises a heavy chain variable
region amino acid sequence of SEQ ID NO: 20, from residue 20 to
143, and a light chain variable region amino acid sequence of SEQ
ID NO: 22, from residue 23 to 130.
2. The method of claim 1, wherein the antibody or antigen binding
fragment thereof comprises the heavy chain amino acid sequence of
SEQ ID NO: 20, from residue 20 to 469 and the light chain amino
acid sequence of SEQ ID NO: 22, from residue 23 to 236
3. The method of claim 1, wherein the cancer cell is a prostate
cancer cell.
4. The method of claim 1, wherein the cancer cell is a gastric
cancer cell.
5. The method of claim 1, wherein the cancer cell is a pancreatic
cancer cell.
6. The method of claim 1, wherein the cancer is ovarian cancer.
7. The method of claim 1, further comprising administering
radiotherapy to the subject.
8. The method of claim 1, further comprising administering a
chemotherapeutic agent other than the conjugate to the subject.
9. A method for inhibiting growth of a cancer cell in a human
subject, comprising: administering to the subject an effective
amount of an antibody drug conjugate, wherein the conjugate
comprises an antibody or antigen binding fragment thereof
conjugated to monomethyl auristatin E (MMAE), and wherein the
antibody or fragment thereof comprises the variable heavy chain and
variable light chain amino acid sequences of an antibody produced
by the hybridoma deposited under American Type Culture Collection
(ATCC) Accession No. PTA-8602.
10. The method of claim 9, wherein the antibody or antigen binding
fragment thereof comprises the amino acid sequences of the heavy
chain and light chain of an antibody produced by a hybridoma
deposited under A.T.C.C. Accession No.: PTA-8602.
11. The method of claim 9, wherein the cancer cell is a prostate
cancer cell.
12. The method of claim 9, wherein the cancer cell is a gastric
cancer cell.
13. The method of claim 9, wherein the cancer cell is a pancreatic
cancer cell.
14. The method of claim 9, wherein the cancer cell is ovarian
cancer cell.
15. The method of claim 9, further comprising administering
radiotherapy to the subject.
16. The method of claim 9, further comprising administering a
chemotherapeutic agent other than the conjugate to the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a continuation of U.S.
application Ser. No. 12/718,902, filed on Mar. 5, 2010, now U.S.
Pat. No. 8,309,093, which claims the benefit of U.S. Provisional
Application No. 61/158,143, filed Mar. 6, 2009. The contents of
each application are fully incorporated by reference herein.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] Not applicable.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0003] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
511582001108Seqlist.txt, date recorded: 8 Oct. 2012, size: 160,274
bytes).
FIELD OF THE INVENTION
[0004] The invention described herein relates to antibodies,
binding fragments, and antibody drug conjugates (ADCs) thereof,
that bind proteins, termed 24P4C12. The invention further relates
to prognostic, prophylactic and therapeutic methods and
compositions useful in the treatment of cancers that express
24P4C12.
BACKGROUND OF THE INVENTION
[0005] Cancer is the second leading cause of human death next to
coronary disease. Worldwide, millions of people die from cancer
every year. In the United States alone, as reported by the American
Cancer Society, cancer causes the death of well over a half-million
people annually, with over 1.2 million new cases diagnosed per
year. While deaths from heart disease have been declining
significantly, those resulting from cancer generally are on the
rise. In the early part of the next century, cancer is predicted to
become the leading cause of death.
[0006] Worldwide, several cancers stand out as the leading killers.
In particular, carcinomas of the lung, prostate, breast, colon,
pancreas, ovary, and bladder represent the primary causes of cancer
death. These and virtually all other carcinomas share a common
lethal feature. With very few exceptions, metastatic disease from a
carcinoma is fatal. Moreover, even for those cancer patients who
initially survive their primary cancers, common experience has
shown that their lives are dramatically altered. Many cancer
patients experience strong anxieties driven by the awareness of the
potential for recurrence or treatment failure. Many cancer patients
experience physical debilitations following treatment. Furthermore,
many cancer patients experience a recurrence.
[0007] Worldwide, prostate cancer is the fourth most prevalent
cancer in men. In North America and Northern Europe, it is by far
the most common cancer in males and is the second leading cause of
cancer death in men. In the United States alone, well over 30,000
men die annually of this disease--second only to lung cancer.
Despite the magnitude of these figures, there is still no effective
treatment for metastatic prostate cancer. Surgical prostatectomy,
radiation therapy, hormone ablation therapy, surgical castration
and chemotherapy continue to be the main treatment modalities.
Unfortunately, these treatments are ineffective for many and are
often associated with undesirable consequences.
[0008] On the diagnostic front, the lack of a prostate tumor marker
that can accurately detect early-stage, localized tumors remains a
significant limitation in the diagnosis and management of this
disease. Although the serum prostate specific antigen (PSA) assay
has been a very useful tool, its specificity and general utility is
widely regarded as lacking in several important respects.
[0009] Progress in identifying additional specific markers for
prostate cancer has been improved by the generation of prostate
cancer xenografts that can recapitulate different stages of the
disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts
are prostate cancer xenografts that have survived passage in severe
combined immune deficient (SCID) mice and have exhibited the
capacity to mimic the transition from androgen dependence to
androgen independence (Klein et al., 1997, Nat. Med. 3:402). More
recently identified prostate cancer markers include PCTA-1 (Su et
al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific
membrane antigen (PSMA) (Pinto et al., Clin Cancer Res 1996 Sep. 2
(9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. 1999
Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA)
(Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
[0010] While previously identified markers such as PSA have
facilitated efforts to diagnose and treat prostate cancer, there is
need for the identification of additional markers and therapeutic
targets for prostate and related cancers in order to further
improve diagnosis and therapy. An estimated 130,200 cases of
colorectal cancer occurred in 2000 in the United States, including
93,800 cases of colon cancer and 36,400 of rectal cancer.
[0011] Colorectal cancers are the third most common cancers in men
and women. Incidence rates declined significantly during 1992-1996
(-2.1% per year). Research suggests that these declines have been
due to increased screening and polyp removal, preventing
progression of polyps to invasive cancers. There were an estimated
56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer)
in 2000, accounting for about 11% of all U.S. cancer deaths.
[0012] At present, surgery is the most common form of therapy for
colorectal cancer, and for cancers that have not spread, it is
frequently curative. Chemotherapy, or chemotherapy plus radiation,
is given before or after surgery to most patients whose cancer has
deeply perforated the bowel wall or has spread to the lymph nodes.
A permanent colostomy (creation of an abdominal opening for
elimination of body wastes) is occasionally needed for colon cancer
and is infrequently required for rectal cancer. There continues to
be a need for effective diagnostic and treatment modalities for
colorectal cancer.
[0013] Of all new cases of cancer in the United States, bladder
cancer represents approximately 5 percent in men (fifth most common
neoplasm) and 3 percent in women (eighth most common neoplasm). The
incidence is increasing slowly, concurrent with an increasing older
population. In 1998, there were an estimated 54,500 cases,
including 39,500 in men and 15,000 in women. The age-adjusted
incidence in the United States is 32 per 100,000 for men and eight
per 100,000 in women. The historic male/female ratio of 3:1 may be
decreasing related to smoking patterns in women. There were an
estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men
and 3,900 in women). Bladder cancer incidence and mortality
strongly increase with age and will be an increasing problem as the
population becomes more elderly.
[0014] Most bladder cancers recur in the bladder. Bladder cancer is
managed with a combination of transurethral resection of the
bladder (TUR) and intravesical chemotherapy or immunotherapy. The
multifocal and recurrent nature of bladder cancer points out the
limitations of TUR. Most muscle-invasive cancers are not cured by
TUR alone. Radical cystectomy and urinary diversion is the most
effective means to eliminate the cancer but carry an undeniable
impact on urinary and sexual function. There continues to be a
significant need for treatment modalities that are beneficial for
bladder cancer patients.
[0015] There were an estimated 164,100 new cases of lung and
bronchial cancer in 2000, accounting for 14% of all U.S. cancer
diagnoses. The incidence rate of lung and bronchial cancer is
declining significantly in men, from a high of 86.5 per 100,000 in
1984 to 70.0 in 1996. In the 1990s, the rate of increase among
women began to slow. In 1996, the incidence rate in women was 42.3
per 100,000.
[0016] Lung and bronchial cancer caused an estimated 156,900 deaths
in 2000, accounting for 28% of all cancer deaths. During 1992-1996,
mortality from lung cancer declined significantly among men (-1.7%
per year) while rates for women were still significantly increasing
(0.9% per year). Since 1987, more women have died each year of lung
cancer than breast cancer, which, for over 40 years, was the major
cause of cancer death in women. Decreasing lung cancer incidence
and mortality rates most likely resulted from decreased smoking
rates over the previous 30 years; however, decreasing smoking
patterns among women lag behind those of men. Of concern, although
the declines in adult tobacco use have slowed, tobacco use in youth
is increasing again.
[0017] Treatment options for lung and bronchial cancer are
determined by the type and stage of the cancer and include surgery,
radiation therapy, and chemotherapy. For many localized cancers,
surgery is usually the treatment of choice. Because the disease has
usually spread by the time it is discovered, radiation therapy and
chemotherapy are often needed in combination with surgery.
Chemotherapy alone or combined with radiation is the treatment of
choice for small cell lung cancer; on this regimen, a large
percentage of patients experience remission, which in some cases is
long lasting. There is however, an ongoing need for effective
treatment and diagnostic approaches for lung and bronchial
cancers.
[0018] An estimated 182,800 new invasive cases of breast cancer
were expected to occur among women in the United States during
2000. Additionally, about 1,400 new cases of breast cancer were
expected to be diagnosed in men in 2000. After increasing about 4%
per year in the 1980s, breast cancer incidence rates in women have
leveled off in the 1990s to about 110.6 cases per 100,000.
[0019] In the U.S. alone, there were an estimated 41,200 deaths
(40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer
ranks second among cancer deaths in women. According to the most
recent data, mortality rates declined significantly during
1992-1996 with the largest decreases in younger women, both white
and black. These decreases were probably the result of earlier
detection and improved treatment.
[0020] Taking into account the medical circumstances and the
patient's preferences, treatment of breast cancer may involve
lumpectomy (local removal of the tumor) and removal of the lymph
nodes under the arm; mastectomy (surgical removal of the breast)
and removal of the lymph nodes under the arm; radiation therapy;
chemotherapy; or hormone therapy. Often, two or more methods are
used in combination. Numerous studies have shown that, for early
stage disease, long-term survival rates after lumpectomy plus
radiotherapy are similar to survival rates after modified radical
mastectomy. Significant advances in reconstruction techniques
provide several options for breast reconstruction after mastectomy.
Recently, such reconstruction has been done at the same time as the
mastectomy.
[0021] Local excision of ductal carcinoma in situ (DCIS) with
adequate amounts of surrounding normal breast tissue may prevent
the local recurrence of the DCIS. Radiation to the breast and/or
tamoxifen may reduce the chance of DCIS occurring in the remaining
breast tissue. This is important because DCIS, if left untreated,
may develop into invasive breast cancer. Nevertheless, there are
serious side effects or sequelae to these treatments. There is,
therefore, a need for efficacious breast cancer treatments.
[0022] There were an estimated 23,100 new cases of ovarian cancer
in the United States in 2000. It accounts for 4% of all cancers
among women and ranks second among gynecologic cancers. During
1992-1996, ovarian cancer incidence rates were significantly
declining. Consequent to ovarian cancer, there were an estimated
14,000 deaths in 2000. Ovarian cancer causes more deaths than any
other cancer of the female reproductive system.
[0023] Surgery, radiation therapy, and chemotherapy are treatment
options for ovarian cancer. Surgery usually includes the removal of
one or both ovaries, the fallopian tubes (salpingo-oophorectomy),
and the uterus (hysterectomy). In some very early tumors, only the
involved ovary will be removed, especially in young women who wish
to have children. In advanced disease, an attempt is made to remove
all intra-abdominal disease to enhance the effect of chemotherapy.
There continues to be an important need for effective treatment
options for ovarian cancer.
[0024] There were an estimated 28,300 new cases of pancreatic
cancer in the United States in 2000. Over the past 20 years, rates
of pancreatic cancer have declined in men. Rates among women have
remained approximately constant but may be beginning to decline.
Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the
United States. Over the past 20 years, there has been a slight but
significant decrease in mortality rates among men (about -0.9% per
year) while rates have increased slightly among women.
[0025] Surgery, radiation therapy, and chemotherapy are treatment
options for pancreatic cancer. These treatment options can extend
survival and/or relieve symptoms in many patients but are not
likely to produce a cure for most. There is a significant need for
additional therapeutic and diagnostic options for cancers. These
include the use of antibodies, vaccines, and small molecules as
treatment modalities. Additionally, there is also a need to use
these modilities as research tools to diagnose, detect, monitor,
and further the state of the art in all areas of cancer treatment
and studies.
[0026] The therapeutic utility of monoclonal antibodies (mAbs) (G.
Kohler and C. Milstein, Nature 256:495-497 (1975)) is being
realized. Monoclonal antibodies have now been approved as therapies
in transplantation, cancer, infectious disease, cardiovascular
disease and inflammation. Different isotypes have different
effector functions. Such differences in function are reflected in
distinct 3-dimensional structures for the various immunoglobulin
isotypes (P. M. Alzari et al., Annual Rev. Immunol., 6:555-580
(1988)).
[0027] Because mice are convenient for immunization and recognize
most human antigens as foreign, mAbs against human targets with
therapeutic potential have typically been of murine origin.
However, murine mAbs have inherent disadvantages as human
therapeutics. They require more frequent dosing as mAbs have a
shorter circulating half-life in humans than human antibodies. More
critically, the repeated administration of murine antibodies to the
human immune system causes the human immune system to respond by
recognizing the mouse protein as a foreign and generating a human
anti-mouse antibody (HAMA) response. Such a HAMA response may
result in allergic reaction and the rapid clearing of the murine
antibody from the system thereby rendering the treatment by murine
antibody useless. To avoid such affects, attempts to create human
immune systems within mice have been attempted.
[0028] Initial attempts hoped to create transgenic mice capable of
responding to antigens with antibodies having human sequences (See
Bruggemann et al., Proc. Nat'l. Acad. Sci. USA 86:6709-6713
(1989)), but were limited by the amount of DNA that could be stably
maintained by available cloning vehicles. The use of yeast
artificial chromosome (YAC) cloning vectors led the way to
introducing large germline fragments of human Ig locus into
transgenic mammals. Essentially a majority of the human V, D, and J
region genes arranged with the same spacing found in the human
genome and the human constant regions were introduced into mice
using YACs. One such transgenic mouse strain is known as
XenoMouse.RTM. mice and is commercially available from Amgen
Fremont, Inc. (Fremont Calif.).
SUMMARY OF THE INVENTION
[0029] The invention provides antibodies, binding fragments, and
antibody drug conjugates (ADCs) thereof that bind to 24P4C12
proteins and polypeptide fragments of 24P4C12 proteins. In some
embodiments, the invention comprises fully human antibodies
conjugated with a therapeutic agent. In certain embodiments, there
is a proviso that the entire nucleic acid sequence of FIG. 3 is not
encoded and/or the entire amino acid sequence of FIG. 2 is not
prepared. In certain embodiments, the entire nucleic acid sequence
of FIG. 3 is encoded and/or the entire amino acid sequence of FIG.
2 is prepared, either of which are in respective human unit dose
forms.
[0030] The invention further provides various immunogenic or
therapeutic compositions, such as antibody drug conjugates, and
strategies for treating cancers that express 24P4C12 such as
cancers of tissues listed in Table I.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1. Nucleic Acid and Amino Acid Sequences of 24P4C12.
FIG. 1A. The cDNA and amino acid sequence of 24P4C12 variant 1
(also called "24P4C12 v.1" or "24P4C12 variant 1") is shown in FIG.
1A. The start methionine is underlined. The open reading frame
extends from nucleic acid 6-2138 including the stop codon.
[0032] FIG. 1B. The cDNA and amino acid sequence of 24P4C12 variant
2 (also called "24P4C12 v.2") is shown in FIG. 1B. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 6-2138 including the stop codon.
[0033] FIG. 1C. The cDNA and amino acid sequence of 24P4C12 variant
3 (also called "24P4C12 v.3") is shown in FIG. 1C. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 6-2138 including the stop codon.
[0034] FIG. 1D. The cDNA and amino acid sequence of 24P4C12 variant
4 (also called "24P4C12 v.4") is shown in FIG. 1D. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 6-2138 including the stop codon.
[0035] FIG. 1E. The cDNA and amino acid sequence of 24P4C12 variant
5 (also called "24P4C12 v.5") is shown in FIG. 1E. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 6-2138 including the stop codon.
[0036] FIG. 1F. The cDNA and amino acid sequence of 24P4C12 variant
6 (also called "24P4C12 v.6") is shown in FIG. 1F. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 6-2138 including the stop codon.
[0037] FIG. 1G. The cDNA and amino acid sequence of 24P4C12 variant
7 (also called "24P4C12 v.7") is shown in FIG. 1G. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 6-1802 including the stop codon.
[0038] FIG. 1H. The cDNA and amino acid sequence of 24P4C12 variant
8 (also called "24P4C12 v.8") is shown in FIG. 1H. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 6-2174 including the stop codon.
[0039] FIG. 1I. The cDNA and amino acid sequence of 24P4C12 variant
9 (also called "24P4C12 v.9") is shown in FIG. 1I. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 6-2144 including the stop codon.
[0040] FIG. 2. Nucleic Acid and Amino Acid sequences of 24P4C12
antibodies.
[0041] FIG. 2A. The cDNA and amino acid sequence of Ha5-1(5)2.1
heavy chain. Double-underlined is the leader sequence, underlined
is the heavy chain variable region, and underlined with a dashed
line is the human IgG2 constant region.
[0042] FIG. 2B. The cDNA and amino acid sequence of Ha5-1(5)2.1
light chain. Double-underlined is the leader sequence, underlined
is the light chain variable region, and underlined with a dashed
line is the human kappa constant region.
[0043] FIG. 3. Amino Acid sequences of 24P4C12 antibodies.
[0044] FIG. 3A. The amino acid sequence of Ha5-1(5)2.1 heavy chain.
Double-underlined is the leader sequence, underlined is the heavy
chain variable region, and underlined with a dashed line is the
human IgG2 constant region.
[0045] FIG. 3B. The amino acid sequence of Ha5-1(5)2.1 light chain.
Double-underlined is the leader sequence, underlined is the light
chain variable region, and underlined with a dashed line is the
human kappa constant region.
[0046] FIG. 4A. Alignment of Ha5-1(5)2.1 heavy chain to human Ig
germline.
[0047] FIG. 4B. Alignment of Ha5-1(5)2.1 light chain to human Ig
germline.
[0048] FIG. 5A-B Ha5-1(5)2.1 MAb binds to cell surface of 24P4C12.
PC3-control and PC3-24P4C12 cells were stained with Ha5-1(5)2.1 MAb
purified from either hybridoma or from CHO cells transfected with
Ha5-1(5)2.1 heavy and light chain vector constructs. Binding was
detected by flow cytometry. Results show Ha5-1(5)2.1 produced by
CHO cells bind 24P4C12 similarly to the Ha5-1(5)2.1 hybridoma
product.
[0049] FIG. 6. Cell Cytotoxicity by Ha5-1(5)2.1-vcMMAE.
Cytotoxicity by Ha5-1(5)2.1-vcMMAE was evaluated in PC3 cells
engineered to express 24P4C12. PC3- Neo or PC3-24P4C12 cells (1000
cells/well) were seeded into a 96 well plate on day 1. The
following day an equal volume of medium containing the indicated
concentration of Ha5-1(5)2.1-vcMMAE or a Control MAb conjugated
with vc-MMAE was added to each well. The cells were allowed to
incubate for 4 days at 37 degrees C. At the end of the incubation
period, Alamar Blue was added to each well and incubation continued
for an additional 4 hours. The resulting fluorescence was detected
using a Biotek plate reader with an excitation wavelength of 620 nm
and an emission wavelength of 540 nm. The results in show that
Ha5-1(5)2.1-vcMMAE mediated cytotoxicity in PC3-24P4C12 cells while
a control human IgG conjugated with vcMMAE had no effect. These
results indicate that Ha5-1(5)2.1-vcMMAE can selectively deliver a
cytotoxic drug to 24P4C12 expressing cells leading to their
killing.
[0050] FIG. 7. Ha5-1(5)2.1vcMMAE inhibits the growth of
subcutaneous established human androgen-independent prostate cancer
xenograft in SCID mice. In this experiment, androgen-independent
human prostate cancer PC-3-Hu24P4C12 tumor cells
(3.0.times.10.sup.6 cells/mouse) were injected subcutaneously into
male SCID mice. Mice were randomized into Ha5-1(5)2.1-vcMMAE and
PBS control groups (n=5 in each group) when tumors reached 100
mm.sup.3. Mice were treated with a single dose of
Ha5-1(5)2.1-vcMMAE (10 mg/kg) or PBS administered intravenously
(i.v.) on Day 0. Tumor growth was monitored using caliper
measurements every 3 to 4 days as indicated. Tumor volume was
calculated as Width2.times.Length/2, where width is the smallest
dimension and length is the largest. The results show that
treatment with Ha5-1(5)2.1-vcMMAE significantly inhibited the
growth of PC-3-Hu24P4C12 prostate tumors in SCID mice (p<0.01)
and resulted in complete tumor regression in most animals.
[0051] FIG. 8. Ha5-1(5)2.1vcMMAE inhibits the growth of
orthotopically established human androgen-independent prostate
cancer xenograft in SCID mice. LAPC-9AI androgen-independent human
prostate cancer cells (2.0.times.10.sup.6 cells/mouse) were
implanted into the prostates of male SCID mice. Fifteen (15) days
after implantation when tumors were well established and palpable,
the mice were randomized into two groups (n=8 in each group). Mice
were treated with either Ha5-1(5)2.1-vcMMAE or isotype control MAb
conjugated with vcMMAE administered i.v. at 3 mg/kg every 4 days
for a total of 4 doses. At the end of study tumors in the mouse
prostate were excised and weighed using an electronic balance. The
results show that treatment with Ha5-1(5)2.1-vcMMAE significantly
inhibited the growth of LAPC9-AI human prostate tumors implanted
orthotopically in SCID mice (p<0.01).
[0052] FIG. 9. Ha5-1(5)2.1vcMMAE inhibits the growth of
subcutaneous established human androgen-independent human colon
cancer xenograft in SCID mice. HT-29 human colon cancer cells
(1.0.times.10.sup.6 cells/mouse) were injected subcutaneously into
SCID mice. Mice were randomized into two groups (n=6 in each group)
when tumors reached 100 mm.sup.3. Ha5-1(5)2.1-vcMMAE (3 mg/kg) or
PBS was administered intravenously every 4 days for a total of 4
doses beginning on Day 0. Tumor growth was monitored using caliper
measurements every 3 to 4 days as indicated. Tumor volume was
calculated as Width2.times.Length/2, where width is the smallest
dimension and length is the largest. The results show that
treatment with Ha5-1(5)2.1-vcMMAE significantly inhibited the
growth of HT-29 human colon tumor xenografts implanted
subcutaneously in SCID mice (p<0.01).
[0053] FIG. 10. Ha5-1(5)2.1vcMMAE inhibits the growth of
subcutaneous established patient-derived colon cancer xenograft in
SCID mice. AG-C4, patient-derived colon cancer xenograft tumor
pieces, were implanted subcutaneously into SCID mice. Mice were
randomized into two groups (n=6 in each group) when tumors reached
100 mm.sup.3. Ha5-1(5)2.1-vcMMAE (3 mg/kg) or PBS was administered
intravenously every 3-4 days for a total of 4 doses starting on Day
0. Tumor growth was monitored using caliper measurements every 3 to
4 days as indicated. Tumor volume was calculated as
Width2.times.Length/2, where width is the smallest dimension and
length is the largest. The results show that treatment with
Ha5-1(5)2.1-vcMMAE significantly inhibited the growth of AG-C4
human colon tumor xenografts implanted subcutaneously in SCID mice
(p<0.05).
[0054] FIG. 11. Ha5-1(5)2.1vcMMAE inhibits the growth of
subcutaneous established human ovarian cancer xenograft in nude
mice. OVCAR-5 human ovarian cancer tumor cells (2.0.times.10.sup.6
cells/mouse) were injected subcutaneously into the nude mice. Mice
were randomized into two groups (n=6 in each group) when tumors
reached 100 mm3. Ha5-1(5)2.1-vcMMAE (5 mg/kg) or PBS was
administered intravenously once every 3-4 days for a total of 4
doses starting on Day 0. Tumor growth was monitored using caliper
measurements every 3 to 4 days as indicated. Tumor volume was
calculated as Width2.times.Length/2, where width is the smallest
dimension and length is the largest. The results show that
treatment with Ha5-1(5)2.1-vcMMAE significantly inhibited the
growth of OVCAR-5 ovarian cancer xenografts implanted
subcutaneously in nude mice (p<0.01).
[0055] FIG. 12. Ha5-1(5)2.1vcMMAE inhibits the growth of
subcutaneous established patient-derived pancreatic cancer
xenograft in SCID mice. AG-Panc3 patient-derived pancreatic tumor
pieces were implanted subcutaneously into SCID mice. Mice were
randomized into two groups (n=6 in each group) when tumors reached
85 mm.sup.3. Ha5-1(5)2.1-vcMMAE (5 mg/kg) or PBS was administered
intravenously once every 3-4 days for a total of 4 doses beginning
on Day 0. Tumor growth was monitored using caliper measurements
every 3 to 4 days as indicated. Tumor volume was calculated as
Width2.times.Length/2, where width is the smallest dimension and
length is the largest. The results show that treatment with
Ha5-1(5)2.1-vcMMAE significantly inhibited the growth of AG-Panc3
tumor xenografts implanted subcutaneously in SCID mice
(p<0.01).
[0056] FIG. 13. Efficacy of Ha5-1(5)2.1vcMMAE compared to other
24P4C12 Antibody Drug Conjugates (ADCs) in Prostate Cancer LAPC9-AD
Xenografts. LAPC-9AD androgen-dependent human prostate cancer cells
(1.5.times.10.sup.6 cells/mouse) were injected subcutaneously into
male SCID mice. Mice were randomized into Ha5-1(5)2.1-vcMMAE,
Ha5-1(5)2.1-mcMMAF and other Antibody Drug Conjugate (ADC) groups
including a PBS control group (n=6 in each group), as shown in
graph (FIG. 13). When tumors reached 100 mm.sup.3,
Ha5-1(5)2.1-vcMMAE, Ha5-1(5)2.1-mcMMAF and all other ADCs were
administered intravenously at 10 mg/kg once on day 0. Tumor growth
was monitored using caliper measurements every 3 to 4 days as
indicated. Tumor volume was calculated as Width2.times.Length/2,
where width is the smallest dimension and length is the largest.
The results show that treatment with Ha5-1(5)2.1-vcMMAE
significantly inhibited the growth of LAPC9-AD prostate cancer
xenografts as compared to Ha5-1(5)2.1-mcMMAF (p=0.0048). (FIG. 13).
Other antibodies conjugated to -vcMMAE and -mcMMAF did not have any
tumor inhibitory activity which shows that Ha5-1(5)2.1 possesses a
significant prominent effect of inhibiting tumor growth and can be
used for therapeutic purposes to treat and manage cancers set forth
in Table I.
[0057] FIG. 14. Detection of 24P4C12 protein in gastric cancer
patient specimens by IHC. Expression of 24P4C12 protein by
immunohistochemistry was tested in two (2) different tumor
specimens from gastric cancer patients. Briefly, formalin fixed,
paraffin wax-embedded tissues were cut into 4 micron sections and
mounted on glass slides. The sections were de-waxed, rehydrated and
treated with trypsin solution (0.05% trypsin (ICN, Aurora, Ohio) in
0.05% calcium chloride, with pH adjusted to 7.8) at 37.degree. C.
for 10 minutes. Sections were then treated with 3% hydrogen
peroxide solution to inactivate endogenous peroxidase activity.
Serum-free protein block (Dako, Carpenteria, Calif.) was used to
inhibit non-specific binding prior to incubation with monoclonal
mouse anti-24P4C12 antibody or an isotype control. Subsequently,
the sections were treated with the Super Sensitive.TM.
Polymer-horseradish peroxidase (HRP) Detection System which
consists of an incubation in Super Enhancer.TM. reagent followed by
an incubation with polymer-HRP secondary antibody conjugate
(BioGenex, San Ramon, Calif.). The sections were then developed
using the DAB kit (BioGenex, San Ramon, Calif.), nuclei were
stained using hematoxylin, and analyzed by bright field microscopy.
Specific staining was detected in patient specimens using the
24P4C12 immunoreactive antibody, as indicated by the brown
staining. (See, FIGS. 14(A) and 14(C). In contrast, the control
antibody did not stain either patient specimen. (See, FIGS. 14(B)
and 14(D). The results show expression of 24P4C12 in the tumor
cells of patient gastric cancer tissues. These results indicate
that 24P4C12 is expressed in human cancers and that antibodies
directed to this antigen (e.g. Ha5-1(5)2.1) are useful for
diagnostic and therapeutic purposes. (FIG. 14(A)-14(D)).
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
I.) Definitions
II.) 24P4C12 Antibodies
III.) Antibody Drug Conjugates Generally
[0058] III(A). Maytansinoids
[0059] III(B). Auristatins and dolostatins
[0060] III(C). Calicheamicin
[0061] III(D). Other Cytotoxic Agents
IV.) Antibody Drug Conjugates which Bind 24P4C12
V.) Linker Units
VI.) The Stretcher Unit
VII.) The Amino Acid Unit
VIII.) The Spacer Unit
IX.) The Drug Unit
X.) Drug Loading
[0062] XI.) Methods of Determining Cytotoxic effect of ADCs
XII.) Treatment of Cancer(s) Expressing 24P4C12
XIII.) 24P4C12 as a Target for Antibody-based Therapy
XIV.) 24P4C12 ADC Cocktails
XV.) Combination Therapy
XVI.) KITS/Articles of Manufacture
I.) DEFINITIONS
[0063] Unless otherwise defined, all terms of art, notations and
other scientific terms or terminology used herein are intended to
have the meanings commonly understood by those of skill in the art
to which this invention pertains. In some cases, terms with
commonly understood meanings are defined herein for clarity and/or
for ready reference, and the inclusion of such definitions herein
should not necessarily be construed to represent a substantial
difference over what is generally understood in the art. Many of
the techniques and procedures described or referenced herein are
well understood and commonly employed using conventional
methodology by those skilled in the art, such as, for example, the
widely utilized molecular cloning methodologies described in
Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd.
edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. As appropriate, procedures involving the use of
commercially available kits and reagents are generally carried out
in accordance with manufacturer defined protocols and/or parameters
unless otherwise noted.
[0064] When a trade name is used herein, reference to the trade
name also refers to the product formulation, the generic drug, and
the active pharmaceutical ingredient(s) of the trade name product,
unless otherwise indicated by context.
[0065] The terms "advanced cancer", "locally advanced cancer",
"advanced disease" and "locally advanced disease" mean cancers that
have extended through the relevant tissue capsule, and are meant to
include stage C disease under the American Urological Association
(AUA) system, stage C1-C2 disease under the Whitmore-Jewett system,
and stage T3-T4 and N+ disease under the TNM (tumor, node,
metastasis) system. In general, surgery is not recommended for
patients with locally advanced disease, and these patients have
substantially less favorable outcomes compared to patients having
clinically localized (organ-confined) cancer.
[0066] The abbreviation "AFP" refers to
dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylened-
iamine (see Formula XVI infra).
[0067] The abbreviation "MMAE" refers to monomethyl auristatin E
(see Formula XI infra).
[0068] The abbreviation "AEB" refers to an ester produced by
reacting auristatin E with paraacetyl benzoic acid (see Formula XX
infra).
[0069] The abbreviation "AEVB" refers to an ester produced by
reacting auristatin E with benzoylvaleric acid (see Formula XXI
infra).
[0070] The abbreviation "MMAF" refers to
dovaline-valine-dolaisoleuine-dolaproine-phenylalanine (see Formula
XVIV infra).
[0071] Unless otherwise noted, the term "alkyl" refers to a
saturated straight or branched hydrocarbon having from about 1 to
about 20 carbon atoms (and all combinations and subcombinations of
ranges and specific numbers of carbon atoms therein), with from
about 1 to about 8 carbon atoms being preferred. Examples of alkyl
groups are methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,
sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl,
2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl,
3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl,
2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,
4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,
2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.
[0072] Alkyl groups, whether alone or as part of another group, can
be optionally substituted with one or more groups, preferably 1 to
3 groups (and any additional substituents selected from halogen),
including, but not limited to, -halogen, --O--(C.sub.1-C.sub.8
alkyl), --O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8
alkynyl), --aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R',
--S(O).sub.2R', --S(O)R', --OH, .dbd.O, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN, where each R' is independently
selected from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8
alkenyl, --C.sub.2-C.sub.8 alkynyl, or -aryl, and wherein said
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8 alkenyl),
--O--(C.sub.2-C.sub.8 alkynyl), -aryl, --C.sub.1-C.sub.8 alkyl,
--C.sub.2-C.sub.8 alkenyl, and --C.sub.2-C.sub.8 alkynyl groups can
be optionally further substituted with one or more groups
including, but not limited to, --C.sub.1-C.sub.8 alkyl,
--C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl, -halogen,
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8 alkenyl),
--O--(C.sub.2-C.sub.8 alkynyl), -aryl, --C(O)R'', --OC(O)R'',
--C(O)OR'', --C(O)NH.sub.2, --C(O)NHR'', --C(O)N(R'').sub.2,
--NHC(O)R'', --SR'', --SO.sub.3R'', --S(O).sub.2R'', --S(O)R'',
--OH, --N.sub.3, --NH.sub.2, --NH(R''), --N(R'').sub.2 and --CN,
where each R'' is independently selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or -aryl.
[0073] Unless otherwise noted, the terms "alkenyl" and "alkynyl"
refer to straight and branched carbon chains having from about 2 to
about 20 carbon atoms (and all combinations and subcombinations of
ranges and specific numbers of carbon atoms therein), with from
about 2 to about 8 carbon atoms being preferred. An alkenyl chain
has at least one double bond in the chain and an alkynyl chain has
at least one triple bond in the chain. Examples of alkenyl groups
include, but are not limited to, ethylene or vinyl, allyl,
-1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,
-3-methyl-1-butenyl, -2-methyl-2-butenyl, and
-2,3-dimethyl-2-butenyl. Examples of alkynyl groups include, but
are not limited to, acetylenic, propargyl, acetylenyl, propynyl,
-1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1
butynyl.
[0074] Alkenyl and alkynyl groups, whether alone or as part of
another group, can be optionally substituted with one or more
groups, preferably 1 to 3 groups (and any additional substituents
selected from halogen), including but not limited to, -halogen,
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8 alkenyl),
--O--(C.sub.2-C.sub.8 alkynyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2,
--NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R', --S(O)R', --OH,
.dbd.O, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and --CN,
where each R' is independently selected from --H, --C.sub.1-C.sub.8
alkyl, --C.sub.2-C.sub.8 alkyenl, --C.sub.2-C.sub.8 alkynyl, or
-aryl and wherein said --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl, and
--C.sub.2-C.sub.8 alkynyl groups can be optionally further
substituted with one or more substituents including, but not
limited to, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, -halogen, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C(O)R'', --OC(O)R'', --C(O)OR'', --C(O)NH.sub.2,
--C(O)NHR'', --C(O)N(R'').sub.2, --NHC(O)R'', --SR'',
--SO.sub.3R'', --S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3,
--NH.sub.2, --NH(R''), --N(R'').sub.2 and --CN, where each R'' is
independently selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl, or -aryl.
[0075] Unless otherwise noted, the term "alkylene" refers to a
saturated branched or straight chain hydrocarbon radical having
from about 1 to about 20 carbon atoms (and all combinations and
subcombinations of ranges and specific numbers of carbon atoms
therein), with from about 1 to about 8 carbon atoms being preferred
and having two monovalent radical centers derived by the removal of
two hydrogen atoms from the same or two different carbon atoms of a
parent alkane. Typical alkylenes include, but are not limited to,
methylene, ethylene, propylene, butylene, pentylene, hexylene,
heptylene, ocytylene, nonylene, decalene, 1,4-cyclohexylene, and
the like. Alkylene groups, whether alone or as part of another
group, can be optionally substituted with one or more groups,
preferably 1 to 3 groups (and any additional substituents selected
from halogen), including, but not limited to, -halogen,
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8 alkenyl),
--O--(C.sub.2-C.sub.8 alkynyl), -aryl, --C(O)R', --OC(O)R',
--C(O)OR', --C(O)NH.sub.2, --C(O)NHR', --C(O)N(R').sub.2,
--NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R', --S(O)R', --OH,
.dbd.O, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and --CN,
where each R' is independently selected from --H, --C.sub.1-C.sub.8
alkyl, --C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl, or
-aryl and wherein said --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl, and
--C.sub.2-C.sub.8 alkynyl groups can be further optionally
substituted with one or more substituents including, but not
limited to, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, -halogen, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C(O)R'', --OC(O)R'', --C(O)OR'', --C(O)NH.sub.2,
--C(O)NHR'', --C(O)N(R'').sub.2, --NHC(O)R'', --SR'',
--SO.sub.3R'', --S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3,
--NH.sub.2, --NH(R''), --N(R'').sub.2 and --CN, where each R'' is
independently selected from --H, --C.sub.1-C.sub.8 alkyl,
--C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl, or -aryl.
[0076] Unless otherwise noted, the term "alkenylene" refers to an
optionally substituted alkylene group containing at least one
carbon-carbon double bond. Exemplary alkenylene groups include, for
example, ethenylene (--CH.dbd.CH--) and propenylene
(--CH.dbd.CHCH.sub.2--).
[0077] Unless otherwise noted, the term "alkynylene" refers to an
optionally substituted alkylene group containing at least one
carbon-carbon triple bond. Exemplary alkynylene groups include, for
example, acetylene (--C.ident.C--), propargyl
(--CH.sub.2C.ident.C--), and 4-pentynyl
(--CH.sub.2CH.sub.2CH.sub.2C.ident.CH--).
[0078] Unless otherwise noted, the term "aryl" refers to a
monovalent aromatic hydrocarbon radical of 6-20 carbon atoms (and
all combinations and subcombinations of ranges and specific numbers
of carbon atoms therein) derived by the removal of one hydrogen
atom from a single carbon atom of a parent aromatic ring system.
Some aryl groups are represented in the exemplary structures as
"Ar". Typical aryl groups include, but are not limited to, radicals
derived from benzene, substituted benzene, phenyl, naphthalene,
anthracene, biphenyl, and the like.
[0079] An aryl group, whether alone or as part of another group,
can be optionally substituted with one or more, preferably 1 to 5,
or even 1 to 2 groups including, but not limited to, -halogen,
--C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, --NO.sub.2, --N.sub.3, --NH.sub.2, --NH(R'),
--N(R').sub.2 and --CN, where each R' is independently selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or -aryl and wherein said
--C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl), and
-aryl groups can be further optionally substituted with one or more
substituents including, but not limited to, --C.sub.1-C.sub.8
alkyl, --C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl,
-halogen, --O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8
alkenyl), --O--(C.sub.2-C.sub.8 alkynyl), -aryl, --C(O)R'',
--OC(O)R'', --C(O)OR'', --C(O)NH.sub.2, --C(O)NHR'',
--C(O)N(R'').sub.2, --NHC(O)R'', --SR'', --SO.sub.3R'',
--S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3, --NH.sub.2, --NH(R''),
--N(R'').sub.2 and --CN, where each R'' is independently selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or -aryl.
[0080] Unless otherwise noted, the term "arylene" refers to an
optionally substituted aryl group which is divalent (i.e., derived
by the removal of two hydrogen atoms from the same or two different
carbon atoms of a parent aromatic ring system) and can be in the
ortho, meta, or para configurations as shown in the following
structures with phenyl as the exemplary aryl group.
##STR00001##
Typical "--(C.sub.1-C.sub.8 alkylene)aryl," "--(C.sub.2-C.sub.8
alkenylene)aryl", "and --(C.sub.2-C.sub.8 alkynylene)aryl" groups
include, but are not limited to, benzyl, 2-phenylethan-1-yl,
2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,
2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and
the like.
[0081] Unless otherwise noted, the term "heterocycle," refers to a
monocyclic, bicyclic, or polycyclic ring system having from 3 to 14
ring atoms (also referred to as ring members) wherein at least one
ring atom in at least one ring is a heteroatom selected from N, O,
P, or S (and all combinations and subcombinations of ranges and
specific numbers of carbon atoms and heteroatoms therein). The
heterocycle can have from 1 to 4 ring heteroatoms independently
selected from N, O, P, or S. One or more N, C, or S atoms in a
heterocycle can be oxidized. A monocylic heterocycle preferably has
3 to 7 ring members (e.g., 2 to 6 carbon atoms and 1 to 3
heteroatoms independently selected from N, O, P, or S), and a
bicyclic heterocycle preferably has 5 to 10 ring members (e.g., 4
to 9 carbon atoms and 1 to 3 heteroatoms independently selected
from N, O, P, or S). The ring that includes the heteroatom can be
aromatic or non-aromatic. Unless otherwise noted, the heterocycle
is attached to its pendant group at any heteroatom or carbon atom
that results in a stable structure.
[0082] Heterocycles are described in Paquette, "Principles of
Modern Heterocyclic Chemistry" (W. A. Benjamin, New York, 1968),
particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of
Heterocyclic Compounds, A series of Monographs" (John Wiley &
Sons, New York, 1950 to present), in particular Volumes 13, 14, 16,
19, and 28; and J. Am. Chem. Soc. 82:5566 (1960).
[0083] Examples of "heterocycle" groups include by way of example
and not limitation pyridyl, dihydropyridyl,
tetrahydropyridyl(piperidyl), thiazolyl, pyrimidinyl, furanyl,
thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl,
thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl,
benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,
2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl,
bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,
octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,
2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,
isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,
isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl,
phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,
cinnolinyl, pteridinyl, 4H-carbazolyl, carbazolyl,
.beta.-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,
phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl,
imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl,
isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl,
benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and
isatinoyl. Preferred "heterocycle" groups include, but are not
limited to, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl,
coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl,
thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl,
pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl,
isothiazolyl, isoxazolyl and tetrazolyl.
[0084] A heterocycle group, whether alone or as part of another
group, can be optionally substituted with one or more groups,
preferably 1 to 2 groups, including but not limited to,
--C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, -halogen, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and
--CN, where each R' is independently selected from --H,
--C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or -aryl and wherein said
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8 alkenyl),
--O--(C.sub.2-C.sub.8 alkynyl), --C.sub.1-C.sub.8 alkyl,
--C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl, and -aryl
groups can be further optionally substituted with one or more
substituents including, but not limited to, --C.sub.1-C.sub.8
alkyl, --C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl,
-halogen, --O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8
alkenyl), --O--(C.sub.2-C.sub.8 alkynyl), -aryl, --C(O)R'',
--OC(O)R'', --C(O)OR'', --C(O)NH.sub.2, --C(O)NHR'',
--C(O)N(R'').sub.2, --NHC(O)R'', --SR'', --SO.sub.3R'',
--S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3, --NH.sub.2, --NH(R''),
--N(R'').sub.2 and --CN, where each R'' is independently selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or aryl.
[0085] By way of example and not limitation, carbon-bonded
heterocycles can be bonded at the following positions: position 2,
3, 4, 5, or 6 of a pyridine; position 3, 4, 5, or 6 of a
pyridazine; position 2, 4, 5, or 6 of a pyrimidine; position 2, 3,
5, or 6 of a pyrazine; position 2, 3, 4, or 5 of a furan,
tetrahydrofuran, thiofuran, thiophene, pyrrole or
tetrahydropyrrole; position 2, 4, or 5 of an oxazole, imidazole or
thiazole; position 3, 4, or 5 of an isoxazole, pyrazole, or
isothiazole; position 2 or 3 of an aziridine; position 2, 3, or 4
of an azetidine; position 2, 3, 4, 5, 6, 7, or 8 of a quinoline; or
position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more
typically, carbon bonded heterocycles include 2-pyridyl, 3-pyridyl,
4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,
5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,
5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,
5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or
5-thiazolyl.
[0086] By way of example and not limitation, nitrogen bonded
heterocycles can be bonded at position 1 of an aziridine,
azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline,
imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole,
pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine,
indole, indoline, or 1H-indazole; position 2 of a isoindole, or
isoindoline; position 4 of a morpholine; and position 9 of a
carbazole, or .beta.-carboline. Still more typically, nitrogen
bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,
1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
[0087] Unless otherwise noted, the term "carbocycle," refers to a
saturated or unsaturated non-aromatic monocyclic, bicyclic, or
polycyclic ring system having from 3 to 14 ring atoms (and all
combinations and subcombinations of ranges and specific numbers of
carbon atoms therein) wherein all of the ring atoms are carbon
atoms. Monocyclic carbocycles preferably have 3 to 6 ring atoms,
still more preferably 5 or 6 ring atoms. Bicyclic carbocycles
preferably have 7 to 12 ring atoms, e.g., arranged as a bicyclo
[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged
as a bicyclo[5,6] or [6,6] system. The term "carbocycle" includes,
for example, a monocyclic carbocycle ring fused to an aryl ring
(e.g., a monocyclic carbocycle ring fused to a benzene ring).
Carbocyles preferably have 3 to 8 carbon ring atoms.
[0088] Carbocycle groups, whether alone or as part of another
group, can be optionally substituted with, for example, one or more
groups, preferably 1 or 2 groups (and any additional substituents
selected from halogen), including, but not limited to, -halogen,
--C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, .dbd.O, --N.sub.3, --NH.sub.2, --NH(R'),
--N(R').sub.2 and --CN, where each R' is independently selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or -aryl and wherein said
--C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl), and
-aryl groups can be further optionally substituted with one or more
substituents including, but not limited to, --C.sub.1-C.sub.8
alkyl, --C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl,
-halogen, --O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8
alkenyl), --O--(C.sub.2-C.sub.8 alkynyl), -aryl, --C(O)R'',
--OC(O)R'', --C(O)OR'', --C(O)NH.sub.2, --C(O)NHR'',
--C(O)N(R'').sub.2, --NHC(O)R'', --SR'', --SO.sub.3R'',
--S(O).sub.2R'', S(O)R'', --OH, --N.sub.3, --NH.sub.2, --NH(R''),
--N(R'').sub.2 and --CN, where each R'' is independently selected
from --H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or -aryl.
[0089] Examples of monocyclic carbocylic substituents include
-cyclopropyl, -cyclobutyl, -cyclopentyl, -1-cyclopent-1-enyl,
-1-cyclopent-2-enyl, -1-cyclopent-3-enyl, cyclohexyl,
-1-cyclohex-1-enyl, -1-cyclohex-2-enyl, -1-cyclohex-3-enyl,
-cycloheptyl, -cyclooctyl, -1,3-cyclohexadienyl,
-1,4-cyclohexadienyl, -1,3-cycloheptadienyl,
-1,3,5-cycloheptatrienyl, and -cyclooctadienyl.
[0090] A "carbocyclo," whether used alone or as part of another
group, refers to an optionally substituted carbocycle group as
defined above that is divalent (i.e., derived by the removal of two
hydrogen atoms from the same or two different carbon atoms of a
parent carbocyclic ring system).
[0091] Unless otherwise indicated by context, a hyphen (-)
designates the point of attachment to the pendant molecule.
Accordingly, the term "-(C.sub.1-C.sub.8 alkylene)aryl" or
"--C.sub.1-C.sub.8 alkylene(aryl)" refers to a C.sub.1-C.sub.8
alkylene radical as defined herein wherein the alkylene radical is
attached to the pendant molecule at any of the carbon atoms of the
alkylene radical and one of the hydrogen atoms bonded to a carbon
atom of the alkylene radical is replaced with an aryl radical as
defined herein.
[0092] When a particular group is "substituted", that group may
have one or more substituents, preferably from one to five
substituents, more preferably from one to three substituents, most
preferably from one to two substituents, independently selected
from the list of substituents. The group can, however, generally
have any number of substituents selected from halogen. Groups that
are substituted are so indicated.
[0093] It is intended that the definition of any substituent or
variable at a particular location in a molecule be independent of
its definitions elsewhere in that molecule. It is understood that
substituents and substitution patterns on the compounds of this
invention can be selected by one of ordinary skill in the art to
provide compounds that are chemically stable and that can be
readily synthesized by techniques known in the art as well as those
methods set forth herein.
[0094] Protective groups as used herein refer to groups which
selectively block, either temporarily or permanently, one reactive
site in a multifunctional compound. Suitable hydroxy-protecting
groups for use in the present invention are pharmaceutically
acceptable and may or may not need to be cleaved from the parent
compound after administration to a subject in order for the
compound to be active. Cleavage is through normal metabolic
processes within the body. Hydroxy protecting groups are well known
in the art, see, Protective Groups in Organic Synthesis by T. W.
Greene and P. G. M. Wuts (John Wiley & sons, 3.sup.rd Edition)
incorporated herein by reference in its entirety and for all
purposes and include, for example, ether (e.g., alkyl ethers and
silyl ethers including, for example, dialkylsilylether,
trialkylsilylether, dialkylalkoxysilylether), ester, carbonate,
carbamates, sulfonate, and phosphate protecting groups. Examples of
hydroxy protecting groups include, but are not limited to, methyl
ether; methoxymethyl ether, methylthiomethyl ether,
(phenyldimethylsilyl)methoxymethyl ether, benzyloxymethyl ether,
p-methoxybenzyloxymethyl ether, p-nitrobenzyloxymethyl ether,
o-nitrobenzyloxymethyl ether, (4-methoxyphenoxy)methyl ether,
guaiacolmethyl ether, t-butoxymethyl ether, 4-pentenyloxymethyl
ether, siloxymethyl ether, 2-methoxyethoxymethyl ether,
2,2,2-trichloroethoxymethyl ether, bis(2-chloroethoxy)methyl ether,
2-(trimethylsilyl)ethoxymethyl ether, menthoxymethyl ether,
tetrahydropyranyl ether, 1-methoxycylcohexyl ether,
4-methoxytetrahydrothiopyranyl ether,
4-methoxytetrahydrothiopyranyl ether S,S-Dioxide,
1-[(2-choro-4-methyl)phenyl]-4-methoxypiperidin-4-yl ether,
1-(2-fluorophenyl)-4-methoxypiperidin-4-yl ether, 1,4-dioxan-2-yl
ether, tetrahydrofuranyl ether, tetrahydrothiofuranyl ether;
substituted ethyl ethers such as 1-ethoxyethyl ether,
1-(2-chloroethoxy)ethyl ether, 1-[2-(trimethylsilyl)ethoxy]ethyl
ether, 1-methyl-1-methoxyethyl ether, 1-methyl-1-benzyloxyethyl
ether, 1-methyl-1-benzyloxy-2-fluoroethyl ether,
1-methyl-1phenoxyethyl ether, 2-trimethylsilyl ether, t-butyl
ether, allyl ether, propargyl ethers, p-chlorophenyl ether,
p-methoxyphenyl ether, benzyl ether, p-methoxybenzyl ether
3,4-dimethoxybenzyl ether, trimethylsilyl ether, triethylsilyl
ether, tripropylsilylether, dimethylisopropylsilyl ether,
diethylisopropylsilyl ether, dimethylhexylsilyl ether,
t-butyldimethylsilyl ether, diphenylmethylsilyl ether,
benzoylformate ester, acetate ester, chloroacetate ester,
dichloroacetate ester, trichloroacetate ester, trifluoroacetate
ester, methoxyacetate ester, triphenylmethoxyacetate ester,
phenylacetate ester, benzoate ester, alkyl methyl carbonate, alkyl
9-fluorenylmethyl carbonate, alkyl ethyl carbonate, alkyl
2,2,2,-trichloroethyl carbonate, 1,1,-dimethyl-2,2,2-trichloroethyl
carbonate, alkylsulfonate, methanesulfonate, benzylsulfonate,
tosylate, methylene acetal, ethylidene acetal, and
t-butylmethylidene ketal. Preferred protecting groups are
represented by the formulas --R.sup.a,
--Si(R.sup.a)(R.sup.a)(R.sup.a), --C(O)R.sup.a, --C(O)OR.sup.a,
--C(O)NH(R.sup.a), --S(O).sub.2R.sup.a, --S(O).sub.2OH,
P(O)(OH).sub.2, and --P(O)(OH)OR.sup.a, wherein R.sup.a is
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl, --C.sub.1-C.sub.20 alkylene(carbocycle),
--C.sub.2-C.sub.20 alkenylene(carbocycle), --C.sub.2-C.sub.20
alkynylene(carbocycle), --C.sub.6-C.sub.10 aryl, --C.sub.1-C.sub.20
alkylene(aryl), --C.sub.2-C.sub.20 alkenylene(aryl),
--C.sub.2-C.sub.20 alkynylene(aryl), --C.sub.1-C.sub.20
alkylene(heterocycle), --C.sub.2-C.sub.20 alkenylene(heterocycle),
or --C.sub.2-C.sub.20 alkynylene(heterocycle) wherein said alkyl,
alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl,
carbocycle, and heterocycle radicals whether alone or as part of
another group are optionally substituted.
[0095] "Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence 24P4C12 (either by removing the underlying
glycosylation site or by deleting the glycosylation by chemical
and/or enzymatic means), and/or adding one or more glycosylation
sites that are not present in the native sequence 24P4C12. In
addition, the phrase includes qualitative changes in the
glycosylation of the native proteins, involving a change in the
nature and proportions of the various carbohydrate moieties
present.
[0096] The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g. a 24P4C12-related protein). For example, an analog
of a 24P4C12 protein can be specifically bound by an antibody or T
cell that specifically binds to 24P4C12.
[0097] The term "antibody" is used in the broadest sense unless
clearly indicated otherwise. Therefore, an "antibody" can be
naturally occurring or man-made such as monoclonal antibodies
produced by conventional hybridoma technology. 24P4C12 antibodies
comprise monoclonal and polyclonal antibodies as well as fragments
containing the antigen-binding domain and/or one or more
complementarity determining regions of these antibodies. As used
herein, the term "antibody" refers to any form of antibody or
fragment thereof that specifically binds 24P4C12 and/or exhibits
the desired biological activity and specifically covers monoclonal
antibodies (including full length monoclonal antibodies),
polyclonal antibodies, multispecific antibodies (e.g., bispecific
antibodies), and antibody fragments so long as they specifically
bind 24P4C12 and/or exhibit the desired biological activity. Any
specific antibody can be used in the methods and compositions
provided herein. Thus, in one embodiment the term "antibody"
encompasses a molecule comprising at least one variable region from
a light chain immunoglobulin molecule and at least one variable
region from a heavy chain molecule that in combination form a
specific binding site for the target antigen. In one embodiment,
the antibody is an IgG antibody. For example, the antibody is a
IgG1, IgG2, IgG3, or IgG4 antibody. The antibodies useful in the
present methods and compositions can be generated in cell culture,
in phage, or in various animals, including but not limited to cows,
rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs,
cats, monkeys, chimpanzees, and apes. Therefore, in one embodiment,
an antibody of the present invention is a mammalian antibody. Phage
techniques can be used to isolate an initial antibody or to
generate variants with altered specificity or avidity
characteristics. Such techniques are routine and well known in the
art. In one embodiment, the antibody is produced by recombinant
means known in the art. For example, a recombinant antibody can be
produced by transfecting a host cell with a vector comprising a DNA
sequence encoding the antibody. One or more vectors can be used to
transfect the DNA sequence expressing at least one VL and one VH
region in the host cell. Exemplary descriptions of recombinant
means of antibody generation and production include Delves,
ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard,
et al., MONOCLONAL ANTIBODIES (Oxford University Press, 2000);
Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic
Press, 1993); and CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley &
Sons, most recent edition). An antibody of the present invention
can be modified by recombinant means to increase efficacy of the
antibody in mediating the desired function. Thus, it is within the
scope of the invention that antibodies can be modified by
substitutions using recombinant means. Typically, the substitutions
will be conservative substitutions. For example, at least one amino
acid in the constant region of the antibody can be replaced with a
different residue. See, e.g., U.S. Pat. No. 5,624,821, U.S. Pat.
No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol.
Immunol. 30: 105-08 (1993). The modification in amino acids
includes deletions, additions, and substitutions of amino acids. In
some cases, such changes are made to reduce undesired activities,
e.g., complement-dependent cytotoxicity. Frequently, the antibodies
are labeled by joining, either covalently or non-covalently, a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and are reported
extensively in both the scientific and patent literature. These
antibodies can be screened for binding to normal or defective
24P4C12. See e.g., ANTIBODY ENGINEERING: A PRACTICAL APPROACH
(Oxford University Press, 1996). Suitable antibodies with the
desired biologic activities can be identified using the following
in vitro assays including but not limited to: proliferation,
migration, adhesion, soft agar growth, angiogenesis, cell-cell
communication, apoptosis, transport, signal transduction, and the
following in vivo assays such as the inhibition of tumor growth.
The antibodies provided herein can also be useful in diagnostic
applications. As capture or non-neutralizing antibodies, they can
be screened for the ability to bind to the specific antigen without
inhibiting the receptor-binding or biological activity of the
antigen. As neutralizing antibodies, the antibodies can be useful
in competitive binding assays. They can also be used to quantify
the 24P4C12 or its receptor.
[0098] The term "antigen-binding portion" or "antibody fragment" of
an antibody (or simply "antibody portion"), as used herein, refers
to one or more fragments of a 24P4C12 antibody that retain the
ability to specifically bind to an antigen (e.g., 24P4C12 and
variants; FIG. 1). It has been shown that the antigen-binding
function of an antibody can be performed by fragments of a
full-length antibody. Examples of binding fragments encompassed
within the term "antigen-binding portion" of an antibody include
(i) a Fab fragment, a monovalent fragment consisting of the
V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the V.sub.H and C.sub.H1 domains; (iv) a Fv fragment
consisting of the V.sub.L and V.sub.H domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a V.sub.H domain; and (vi) an
isolated complementarily determining region (CDR). Furthermore,
although the two domains of the Fv fragment, V.sub.L and V.sub.H,
are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be
made as a single protein chain in which the V.sub.L and V.sub.H
regions pair to form monovalent molecules (known as single chain Fv
(scFv); see e.g., Bird et al. (1988) Science 242:423-426; and
Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such
single chain antibodies are also intended to be encompassed within
the term "antigen-binding portion" of an antibody. These antibody
fragments are obtained using conventional techniques known to those
with skill in the art, and the fragments are screened for utility
in the same manner as are intact antibodies.
[0099] As used herein, any form of the "antigen" can be used to
generate an antibody that is specific for 24P4C12. Thus, the
eliciting antigen may be a single epitope, multiple epitopes, or
the entire protein alone or in combination with one or more
immunogenicity enhancing agents known in the art. The eliciting
antigen may be an isolated full-length protein, a cell surface
protein (e.g., immunizing with cells transfected with at least a
portion of the antigen), or a soluble protein (e.g., immunizing
with only the extracellular domain portion of the protein). The
antigen may be produced in a genetically modified cell. The DNA
encoding the antigen may be genomic or non-genomic (e.g., cDNA) and
encodes at least a portion of the extracellular domain. As used
herein, the term "portion" refers to the minimal number of amino
acids or nucleic acids, as appropriate, to constitute an
immunogenic epitope of the antigen of interest. Any genetic vectors
suitable for transformation of the cells of interest may be
employed, including but not limited to adenoviral vectors,
plasmids, and non-viral vectors, such as cationic lipids. In one
embodiment, the antibody of the methods and compositions herein
specifically bind at least a portion of the extracellular domain of
the 24P4C12 of interest.
[0100] The antibodies or antigen binding fragments thereof provided
herein may be conjugated to a "bioactive agent." As used herein,
the term "bioactive agent" refers to any synthetic or naturally
occurring compound that binds the antigen and/or enhances or
mediates a desired biological effect to enhance cell-killing
toxins. In one embodiment, the binding fragments useful in the
present invention are biologically active fragments. As used
herein, the term "biologically active" refers to an antibody or
antibody fragment that is capable of binding the desired antigenic
epitope and directly or indirectly exerting a biologic effect.
Direct effects include, but are not limited to the modulation,
stimulation, and/or inhibition of a growth signal, the modulation,
stimulation, and/or inhibition of an anti-apoptotic signal, the
modulation, stimulation, and/or inhibition of an apoptotic or
necrotic signal, modulation, stimulation, and/or inhibition the
ADCC cascade, and modulation, stimulation, and/or inhibition the
CDC cascade.
[0101] "Bispecific" antibodies are also useful in the present
methods and compositions. As used herein, the term "bispecific
antibody" refers to an antibody, typically a monoclonal antibody,
having binding specificities for at least two different antigenic
epitopes. In one embodiment, the epitopes are from the same
antigen. In another embodiment, the epitopes are from two different
antigens. Methods for making bispecific antibodies are known in the
art. For example, bispecific antibodies can be produced
recombinantly using the co-expression of two immunoglobulin heavy
chain/light chain pairs. See, e.g., Milstein et al., Nature
305:537-39 (1983). Alternatively, bispecific antibodies can be
prepared using chemical linkage. See, e.g., Brennan, et al.,
Science 229:81 (1985). Bispecific antibodies include bispecific
antibody fragments. See, e.g., Hollinger, et al., Proc. Natl. Acad.
Sci. U.S.A. 90:6444-48 (1993), Gruber, et al., J. Immunol. 152:5368
(1994).
[0102] The monoclonal antibodies described herein specifically
include "chimeric" antibodies in which a portion of the heavy
and/or light chain is identical with or homologous to corresponding
sequences in antibodies derived from a particular species or
belonging to a particular antibody class or subclass, while the
remainder of the chain(s) is identical with or homologous to
corresponding sequences in antibodies derived from another species
or belonging to another antibody class or subclass, as well as
fragments of such antibodies, so long as they specifically bind the
target antigen and/or exhibit the desired biological activity (U.S.
Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA
81: 6851-6855 (1984)).
[0103] The term "Chemotherapeutic Agent" refers to all chemical
compounds that are effective in inhibiting tumor growth.
Non-limiting examples of chemotherapeutic agents include alkylating
agents; for example, nitrogen mustards, ethyleneimine compounds and
alkyl sulphonates; antimetabolites, for example, folic acid, purine
or pyrimidine antagonists; mitotic inhibitors, for example,
anti-tubulin agents such as vinca alkaloids, auristatins and
derivatives of podophyllotoxin; cytotoxic antibiotics; compounds
that damage or interfere with DNA expression or replication, for
example, DNA minor groove binders; and growth factor receptor
antagonists. In addition, chemotherapeutic agents include cytotoxic
agents (as defined herein), antibodies, biological molecules and
small molecules.
[0104] The term "compound" refers to and encompasses the chemical
compound itself as well as, whether explicitly stated or not, and
unless the context makes clear that the following are to be
excluded: amorphous and crystalline forms of the compound,
including polymorphic forms, where these forms may be part of a
mixture or in isolation; free acid and free base forms of the
compound, which are typically the forms shown in the structures
provided herein; isomers of the compound, which refers to optical
isomers, and tautomeric isomers, where optical isomers include
enantiomers and diastereomers, chiral isomers and non-chiral
isomers, and the optical isomers include isolated optical isomers
as well as mixtures of optical isomers including racemic and
non-racemic mixtures; where an isomer may be in isolated form or in
a mixture with one or more other isomers; isotopes of the compound,
including deuterium- and tritium-containing compounds, and
including compounds containing radioisotopes, including
therapeutically- and diagnostically-effective radioisotopes;
multimeric forms of the compound, including dimeric, trimeric, etc.
forms; salts of the compound, preferably pharmaceutically
acceptable salts, including acid addition salts and base addition
salts, including salts having organic counterions and inorganic
counterions, and including zwitterionic forms, where if a compound
is associated with two or more counterions, the two or more
counterions may be the same or different; and solvates of the
compound, including hemisolvates, monosolvates, disolvates, etc.,
including organic solvates and inorganic solvates, said inorganic
solvates including hydrates; where if a compound is associated with
two or more solvent molecules, the two or more solvent molecules
may be the same or different. In some instances, reference made
herein to a compound of the invention will include an explicit
reference to one or of the above forms, e.g., salts and/or
solvates; however, this reference is for emphasis only, and is not
to be construed as excluding other of the above forms as identified
above.
[0105] As used herein, the term "conservative substitution" refers
to substitutions of amino acids are known to those of skill in this
art and may be made generally without altering the biological
activity of the resulting molecule. Those of skill in this art
recognize that, in general, single amino acid substitutions in
non-essential regions of a polypeptide do not substantially alter
biological activity (see, e.g., Watson, et al., MOLECULAR BIOLOGY
OF THE GENE, The Benjamin/Cummings Pub. Co., p. 224 (4th Edition
1987)). Such exemplary substitutions are preferably made in
accordance with those set forth in Table II and Table(s) III(a-b).
For example, such changes include substituting any of isoleucine
(I), valine (V), and leucine (L) for any other of these hydrophobic
amino acids; aspartic acid (D) for glutamic acid (E) and vice
versa; glutamine (Q) for asparagine (N) and vice versa; and serine
(S) for threonine (T) and vice versa. Other substitutions can also
be considered conservative, depending on the environment of the
particular amino acid and its role in the three-dimensional
structure of the protein. For example, glycine (G) and alanine (A)
can frequently be interchangeable, as can alanine (A) and valine
(V). Methionine (M), which is relatively hydrophobic, can
frequently be interchanged with leucine and isoleucine, and
sometimes with valine. Lysine (K) and arginine (R) are frequently
interchangeable in locations in which the significant feature of
the amino acid residue is its charge and the differing pK's of
these two amino acid residues are not significant. Still other
changes can be considered "conservative" in particular environments
(see, e.g. Table III(a) herein; pages 13-15 "Biochemistry" 2nd ED.
Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992
Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;
270(20):11882-6). Other substitutions are also permissible and may
be determined empirically or in accord with known conservative
substitutions.
[0106] The term "cytotoxic agent" refers to a substance that
inhibits or prevents the expression activity of cells, function of
cells and/or causes destruction of cells. The term is intended to
include radioactive isotopes, chemotherapeutic agents, and toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof. Examples of cytotoxic agents include, but
are not limited to auristatins (e.g., auristatin E, auristatin F,
MMAE and MMAF), auromycins, maytansinoids, ricin, ricin A-chain,
combrestatin, duocarmycins, dolastatins, doxorubicin, daunorubicin,
taxols, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide,
tenoposide, vincristine, vinblastine, colchicine, dihydroxy
anthracin dione, actinomycin, diphtheria toxin, Pseudomonas
exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain,
alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin,
enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis
inhibitor, and glucocorticoid and other chemotherapeutic agents, as
well as radioisotopes such as At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212 or
.sup.213, P.sup.32 and radioactive isotopes of Lu including
Lu.sup.177. Antibodies may also be conjugated to an anti-cancer
pro-drug activating enzyme capable of converting the pro-drug to
its active form.
[0107] As used herein, the term "diabodies" refers to small
antibody fragments with two antigen-binding sites, which fragments
comprise a heavy chain variable domain (V.sub.H) connected to a
light chain variable domain (V.sub.L) in the same polypeptide chain
(V.sub.H--V.sub.L). By using a linker that is too short to allow
pairing between the two domains on the same chain, the domains are
forced to pair with the complementary domains of another chain and
create two antigen-binding sites. Diabodies are described more
fully in, e.g., EP 404,097; WO 93/11161; and Hollinger et al.,
Proc. Natl. Acad. Sci. USA 90:6444-48 (1993).
[0108] The term "deplete," in the context of the effect of a
24P4C12 binding agent on 24P4C12-expressing cells, refers to a
reduction in the number of or elimination of the 24P4C12-expressing
cells.
[0109] The term "gene product" is used herein to indicate a
peptide/protein or mRNA. For example, a "gene product of the
invention" is sometimes referred to herein as a "cancer amino acid
sequence", "cancer protein", "protein of a cancer listed in Table
I", a "cancer mRNA", "mRNA of a cancer listed in Table I", etc. In
one embodiment, the cancer protein is encoded by a nucleic acid of
FIG. 1. The cancer protein can be a fragment, or alternatively, be
the full-length protein encoded by nucleic acids of FIG. 1. In one
embodiment, a cancer amino acid sequence is used to determine
sequence identity or similarity. In another embodiment, the
sequences are naturally occurring allelic variants of a protein
encoded by a nucleic acid of FIG. 1. In another embodiment, the
sequences are sequence variants as further described herein.
[0110] "Heteroconjugate" antibodies are useful in the present
methods and compositions. As used herein, the term "heteroconjugate
antibody" refers to two covalently joined antibodies. Such
antibodies can be prepared using known methods in synthetic protein
chemistry, including using crosslinking agents. See, e.g., U.S.
Pat. No. 4,676,980.
[0111] The term "homolog" refers to a molecule which exhibits
homology to another molecule, by for example, having sequences of
chemical residues that are the same or similar at corresponding
positions.
[0112] In one embodiment, the antibody provided herein is a "human
antibody." As used herein, the term "human antibody" refers to an
antibody in which essentially the entire sequences of the light
chain and heavy chain sequences, including the complementary
determining regions (CDRs), are from human genes. In one
embodiment, human monoclonal antibodies are prepared by the trioma
technique, the human B-cell technique (see, e.g., Kozbor, et al.,
Immunol. Today 4: 72 (1983), EBV transformation technique (see,
e.g., Cole et al. MONOCLONAL ANTIBODIES AND CANCER THERAPY 77-96
(1985)), or using phage display (see, e.g., Marks et al., J. Mol.
Biol. 222:581 (1991)). In a specific embodiment, the human antibody
is generated in a transgenic mouse. Techniques for making such
partially to fully human antibodies are known in the art and any
such techniques can be used. According to one particularly
preferred embodiment, fully human antibody sequences are made in a
transgenic mouse engineered to express human heavy and light chain
antibody genes. An exemplary description of preparing transgenic
mice that produce human antibodies found in Application No. WO
02/43478 and U.S. Pat. No. 6,657,103 (Abgenix) and its progeny. B
cells from transgenic mice that produce the desired antibody can
then be fused to make hybridoma cell lines for continuous
production of the antibody. See, e.g., U.S. Pat. Nos. 5,569,825;
5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Jakobovits,
Adv. Drug Del. Rev. 31:33-42 (1998); Green, et al., J. Exp. Med.
188:483-95 (1998).
[0113] As used herein, the term "humanized antibody" refers to
forms of antibodies that contain sequences from non-human (e.g.,
murine) antibodies as well as human antibodies. Such antibodies are
chimeric antibodies which contain minimal sequence derived from
non-human immunoglobulin. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the
hypervariable loops correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin sequence. The humanized antibody
optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. See e.g., Cabilly U.S. Pat. No. 4,816,567; Queen et
al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; and ANTIBODY
ENGINEERING: A PRACTICAL APPROACH (Oxford University Press
1996).
[0114] The terms "inhibit" or "inhibition of" as used herein means
to reduce by a measurable amount, or to prevent entirely.
[0115] The phrases "isolated" or "biologically pure" refer to
material which is substantially or essentially free from components
which normally accompany the material as it is found in its native
state. Thus, isolated peptides in accordance with the invention
preferably do not contain materials normally associated with the
peptides in their in situ environment. For example, a
polynucleotide is said to be "isolated" when it is substantially
separated from contaminant polynucleotides that correspond or are
complementary to genes other than the 24P4C12 genes or that encode
polypeptides other than 24P4C12 gene product or fragments thereof.
A skilled artisan can readily employ nucleic acid isolation
procedures to obtain an isolated 24P4C12 polynucleotide. A protein
is said to be "isolated," for example, when physical, mechanical or
chemical methods are employed to remove the 24P4C12 proteins from
cellular constituents that are normally associated with the
protein. A skilled artisan can readily employ standard purification
methods to obtain an isolated 24P4C12 protein. Alternatively, an
isolated protein can be prepared by chemical means.
[0116] Suitable "labels" include radionuclides, enzymes,
substrates, cofactors, inhibitors, fluorescent moieties,
chemiluminescent moieties, magnetic particles, and the like.
Patents teaching the use of such labels include U.S. Pat. Nos.
3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149;
and 4,366,241. In addition, the antibodies provided herein can be
useful as the antigen-binding component of fluorobodies. See e.g.,
Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003).
[0117] The term "mammal" refers to any organism classified as a
mammal, including mice, rats, rabbits, dogs, cats, cows, horses and
humans. In one embodiment of the invention, the mammal is a mouse.
In another embodiment of the invention, the mammal is a human.
[0118] The terms "metastatic cancer" and "metastatic disease" mean
cancers that have spread to regional lymph nodes or to distant
sites, and are meant to include stage D disease under the AUA
system and stage T.times.N.times.M+ under the TNM system.
[0119] The term "modulator" or "test compound" or "drug candidate"
or grammatical equivalents as used herein describe any molecule,
e.g., protein, oligopeptide, small organic molecule,
polysaccharide, polynucleotide, etc., to be tested for the capacity
to directly or indirectly alter the cancer phenotype or the
expression of a cancer sequence, e.g., a nucleic acid or protein
sequences, or effects of cancer sequences (e.g., signaling, gene
expression, protein interaction, etc.) In one aspect, a modulator
will neutralize the effect of a cancer protein of the invention. By
"neutralize" is meant that an activity of a protein is inhibited or
blocked, along with the consequent effect on the cell. In another
aspect, a modulator will neutralize the effect of a gene, and its
corresponding protein, of the invention by normalizing levels of
said protein. In preferred embodiments, modulators alter expression
profiles, or expression profile nucleic acids or proteins provided
herein, or downstream effector pathways. In one embodiment, the
modulator suppresses a cancer phenotype, e.g. to a normal tissue
fingerprint. In another embodiment, a modulator induced a cancer
phenotype. Generally, a plurality of assay mixtures is run in
parallel with different agent concentrations to obtain a
differential response to the various concentrations. Typically, one
of these concentrations serves as a negative control, i.e., at zero
concentration or below the level of detection.
[0120] Modulators, drug candidates, or test compounds encompass
numerous chemical classes, though typically they are organic
molecules, preferably small organic compounds having a molecular
weight of more than 100 and less than about 2,500 Daltons.
Preferred small molecules are less than 2000, or less than 1500 or
less than 1000 or less than 500 D. Candidate agents comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and typically include at
least an amine, carbonyl, hydroxyl or carboxyl group, preferably at
least two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Modulators also comprise biomolecules
such as peptides, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs or combinations
thereof. Particularly preferred are peptides. One class of
modulators are peptides, for example of from about five to about 35
amino acids, with from about five to about 20 amino acids being
preferred, and from about 7 to about 15 being particularly
preferred. Preferably, the cancer modulatory protein is soluble,
includes a non-transmembrane region, and/or, has an N-terminal Cys
to aid in solubility. In one embodiment, the C-terminus of the
fragment is kept as a free acid and the N-terminus is a free amine
to aid in coupling, i.e., to cysteine. In one embodiment, a cancer
protein of the invention is conjugated to an immunogenic agent as
discussed herein. In one embodiment, the cancer protein is
conjugated to BSA. The peptides of the invention, e.g., of
preferred lengths, can be linked to each other or to other amino
acids to create a longer peptide/protein. The modulatory peptides
can be digests of naturally occurring proteins as is outlined
above, random peptides, or "biased" random peptides. In a preferred
embodiment, peptide/protein-based modulators are antibodies, and
fragments thereof, as defined herein.
[0121] The term "monoclonal antibody", as used herein, refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic epitope. In contrast, conventional (polyclonal) antibody
preparations typically include a multitude of antibodies directed
against (or specific for) different epitopes. In one embodiment,
the polyclonal antibody contains a plurality of monoclonal
antibodies with different epitope specificities, affinities, or
avidities within a single antigen that contains multiple antigenic
epitopes. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler et al., Nature 256: 495 (1975), or may be made by
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The
"monoclonal antibodies" may also be isolated from phage antibody
libraries using the techniques described in Clackson et al., Nature
352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597
(1991), for example. These monoclonal antibodies will usually bind
with at least a Kd of about 1 .mu.M, more usually at least about
300 nM, typically at least about 30 nM, preferably at least about
10 nM, more preferably at least about 3 nM or better, usually
determined by ELISA.
[0122] A "pharmaceutical excipient" comprises a material such as an
adjuvant, a carrier, pH-adjusting and buffering agents, tonicity
adjusting agents, wetting agents, preservative, and the like.
[0123] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or composition that is physiologically compatible with humans
or other mammals.
[0124] The term "polynucleotide" means a polymeric form of
nucleotides of at least 10 bases or base pairs in length, either
ribonucleotides or deoxynucleotides or a modified form of either
type of nucleotide, and is meant to include single and double
stranded forms of DNA and/or RNA. In the art, this term if often
used interchangeably with "oligonucleotide". A polynucleotide can
comprise a nucleotide sequence disclosed herein wherein thymidine
(T), as shown for example in FIG. 1, can also be uracil (U); this
definition pertains to the differences between the chemical
structures of DNA and RNA, in particular the observation that one
of the four major bases in RNA is uracil (U) instead of thymidine
(T).
[0125] The term "polypeptide" means a polymer of at least about 4,
5, 6, 7, or 8 amino acids. Throughout the specification, standard
three letter or single letter designations for amino acids are
used. In the art, this term is often used interchangeably with
"peptide" or "protein".
[0126] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
[0127] As used herein, the term "single-chain Fv" or "scFv" or
"single chain" antibody refers to antibody fragments comprising the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113,
Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315
(1994).
[0128] As used herein, the terms "specific", "specifically binds"
and "binds specifically" refer to the selective binding of the
antibody to the target antigen epitope. Antibodies can be tested
for specificity of binding by comparing binding to appropriate
antigen to binding to irrelevant antigen or antigen mixture under a
given set of conditions. If the antibody binds to the appropriate
antigen at least 2, 5, 7, and preferably 10 times more than to
irrelevant antigen or antigen mixture then it is considered to be
specific. In one embodiment, a specific antibody is one that only
binds the 24P4C12 antigen, but does not bind to the irrelevant
antigen. In another embodiment, a specific antibody is one that
binds human 24P4C12 antigen but does not bind a non-human 24P4C12
antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or greater amino acid homology with the 24P4C12
antigen. In another embodiment, a specific antibody is one that
binds human 24P4C12 antigen and binds murine 24P4C12 antigen, but
with a higher degree of binding the human antigen. In another
embodiment, a specific antibody is one that binds human 24P4C12
antigen and binds primate 24P4C12 antigen, but with a higher degree
of binding the human antigen. In another embodiment, the specific
antibody binds to human 24P4C12 antigen and any non-human 24P4C12
antigen, but with a higher degree of binding the human antigen or
any combination thereof.
[0129] As used herein "to treat" or "therapeutic" and grammatically
related terms, refer to any improvement of any consequence of
disease, such as prolonged survival, less morbidity, and/or a
lessening of side effects which are the byproducts of an
alternative therapeutic modality; as is readily appreciated in the
art, full eradication of disease is a preferred but albeit not a
requirement for a treatment act.
[0130] The term "variant" refers to a molecule that exhibits a
variation from a described type or norm, such as a protein that has
one or more different amino acid residues in the corresponding
position(s) of a specifically described protein (e.g. the 24P4C12
protein shown in FIG. 1.) An analog is an example of a variant
protein. Splice isoforms and single nucleotides polymorphisms
(SNPs) are further examples of variants.
[0131] The "24P4C12-related proteins" of the invention include
those specifically identified herein (see, FIG. 1A-1I), as well as
allelic variants, conservative substitution variants, analogs and
homologs that can be isolated/generated and characterized without
undue experimentation following the methods outlined herein or
readily available in the art. Fusion proteins that combine parts of
different 24P4C12 proteins or fragments thereof, as well as fusion
proteins of a 24P4C12 protein and a heterologous polypeptide are
also included. Such 24P4C12 proteins are collectively referred to
as the 24P4C12-related proteins, the proteins of the invention, or
24P4C12. The term "24P4C12-related protein" refers to a polypeptide
fragment or a 24P4C12 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more
than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65,
70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,
145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225,
250, 275, 300, 325, 330, 335, 339 or more amino acids.
II.) 24P4C12 ANTIBODIES
[0132] Another aspect of the invention provides antibodies that
bind to 24P4C12-related proteins (See FIG. 1). Preferred antibodies
specifically bind to a 24P4C12-related protein and do not bind (or
bind weakly) to peptides or proteins that are not 24P4C12-related
proteins under physiological conditions. For example, antibodies
that bind 24P4C12 can bind 24P4C12-related proteins such as 24P4C12
variants and the homologs or analogs thereof.
[0133] 24P4C12 antibodies of the invention are particularly useful
in cancer (see, e.g., Table I) prognostic assays, imaging, and
therapeutic methodologies. Similarly, such antibodies are useful in
the treatment, and/or prognosis of colon and other cancers, to the
extent 24P4C12 is also expressed or overexpressed in these other
cancers. Moreover, intracellularly expressed antibodies (e.g.,
single chain antibodies) are therapeutically useful in treating
cancers in which the expression of 24P4C12 is involved, such as
advanced or metastatic colon cancers or other advanced or
metastatic cancers.
[0134] Various methods for the preparation of antibodies,
specifically monoclonal antibodies, are well known in the art. For
example, antibodies can be prepared by immunizing a suitable
mammalian host using a 24P4C12-related protein, peptide, or
fragment, in isolated or immunoconjugated form (Antibodies: A
Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988);
Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In
addition, fusion proteins of 24P4C12 can also be used, such as a
24P4C12 GST-fusion protein. In a particular embodiment, a GST
fusion protein comprising all or most of the amino acid sequence of
FIG. 1 is produced, and then used as an immunogen to generate
appropriate antibodies. In another embodiment, a 24P4C12-related
protein is synthesized and used as an immunogen.
[0135] In addition, naked DNA immunization techniques known in the
art are used (with or without purified 24P4C12-related protein or
24P4C12 expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev.
Immunol. 15: 617-648).
[0136] The amino acid sequence of a 24P4C12 protein as shown in
FIG. 1 can be analyzed to select specific regions of the 24P4C12
protein for generating antibodies. For example, hydrophobicity and
hydrophilicity analyses of a 24P4C12 amino acid sequence are used
to identify hydrophilic regions in the 24P4C.sub.1-2 structure.
Regions of a 24P4C12 protein that show immunogenic structure, as
well as other regions and domains, can readily be identified using
various other methods known in the art, such as Chou-Fasman,
Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or
Jameson-Wolf analysis. Hydrophilicity profiles can be generated
using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl.
Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be
generated using the method of Kyte, J. and Doolittle, R. F., 1982,
J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles
can be generated using the method of Janin J., 1979, Nature
277:491-492. Average Flexibility profiles can be generated using
the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept.
Protein Res. 32:242-255. Beta-turn profiles can be generated using
the method of Deleage, G., Roux B., 1987, Protein Engineering
1:289-294. Thus, each region identified by any of these programs or
methods is within the scope of the present invention. Preferred
methods for the generation of 24P4C12 antibodies are further
illustrated by way of the examples provided herein. Methods for
preparing a protein or polypeptide for use as an immunogen are well
known in the art. Also well known in the art are methods for
preparing immunogenic conjugates of a protein with a carrier, such
as BSA, KLH or other carrier protein. In some circumstances, direct
conjugation using, for example, carbodiimide reagents are used; in
other instances linking reagents such as those supplied by Pierce
Chemical Co., Rockford, Ill., are effective. Administration of a
24P4C12 immunogen is often conducted by injection over a suitable
time period and with use of a suitable adjuvant, as is understood
in the art. During the immunization schedule, titers of antibodies
can be taken to determine adequacy of antibody formation.
[0137] 24P4C12 monoclonal antibodies can be produced by various
means well known in the art. For example, immortalized cell lines
that secrete a desired monoclonal antibody are prepared using the
standard hybridoma technology of Kohler and Milstein or
modifications that immortalize antibody-producing B cells, as is
generally known. Immortalized cell lines that secrete the desired
antibodies are screened by immunoassay in which the antigen is a
24P4C12-related protein. When the appropriate immortalized cell
culture is identified, the cells can be expanded and antibodies
produced either from in vitro cultures or from ascites fluid.
[0138] The antibodies or fragments of the invention can also be
produced by recombinant means. Regions that bind specifically to
the desired regions of a 24P4C12 protein can also be produced in
the context of chimeric or complementarity-determining region (CDR)
grafted antibodies of multiple species origin. Humanized or human
24P4C12 antibodies can also be produced, and are preferred for use
in therapeutic contexts. Methods for humanizing murine and other
non-human antibodies, by substituting one or more of the non-human
antibody CDRs for corresponding human antibody sequences, are well
known (see for example, Jones et al., 1986, Nature 321: 522-525;
Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al.,
1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.
Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol.
151: 2296.
[0139] In a preferred embodiment, the antibodies of the present
invention comprise fully human 24P4C12 antibodies (24P4C12 MAbs).
Various methods in the art provide means for producing fully human
24P4C12 MAbs. For example, a preferred embodiment provides for
techniques using transgenic mice, inactivated for antibody
production, engineered with human heavy and light chains loci
referred to as Xenomouse (Amgen Fremont, Inc.). An exemplary
description of preparing transgenic mice that produce human
antibodies can be found in U.S. Pat. No. 6,657,103. See, also, U.S.
Pat. Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and
5,545,806; and Mendez, et. al. Nature Genetics, 15: 146-156 (1998);
Kellerman, S. A. & Green, L. L., Curr. Opin. Biotechnol 13,
593-597 (2002).
[0140] In addition, human antibodies of the invention can be
generated using the HuMAb mouse (Medarex, Inc.) which contains
human immunoglobulin gene miniloci that encode unrearranged human
heavy (mu and gamma) and kappa light chain immunoglobulin
sequences, together with targeted mutations that inactivate the
endogenous mu and kappa chain loci (see e.g., Lonberg, et al.
(1994) Nature 368(6474): 856-859).
[0141] In another embodiment, fully human antibodies of the
invention can be raised using a mouse that carries human
immunoglobulin sequences on transgenes and transchomosomes, such as
a mouse that carries a human heavy chain transgene and a human
light chain transchromosome. Such mice, referred to herein as "KM
mice", such mice are described in Tomizuka et al. (2000) Proc.
Natl. Acad. Sci. USA 97:722-727 and PCT Publication WO 02/43478 to
Tomizuka, et al.
[0142] Human monoclonal antibodies of the invention can also be
prepared using phage display methods for screening libraries of
human immunoglobulin genes. Such phage display methods for
isolating human antibodies are established in the art. See for
example: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to
Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et
al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.;
and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;
6,582,915 and 6,593,081 to Griffiths et al.
[0143] Human monoclonal antibodies of the invention can also be
prepared using SCID mice into which human immune cells have been
reconstituted such that a human antibody response can be generated
upon immunization. Such mice are described in, for example, U.S.
Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.
[0144] In a preferred embodiment, an 24P4C12 MAbs of the invention
comprises heavy and light chain variable regions of an antibody
designated Ha5-1(5)2.1 produced by a hybridoma deposited under the
American Type Culture Collection (ATCC) Accession No.: PTA-8602
(See, FIG. 3), or heavy and light variable regions comprising amino
acid sequences that are homologous to the amino acid sequences of
the heavy and light chain variable regions of Ha5-1(5)2.1, and
wherein the antibodies retain the desired functional properties of
the 24P4C12 MAbs of the invention. The heavy chain variable region
of Ha5-1(5)2.1 consists of the amino acid sequence ranging from
20.sup.th Q residue to the 143.sup.th S residue of SEQ ID NO: 20,
and the light chain variable region of Ha5-1(5)2.1 consists of the
amino acid sequence ranging from 23.sup.th D residue to the
130.sup.th R residue of SEQ ID NO: 22. As the constant region of
the antibody of the invention, any subclass of constant region can
be chosen. In one embodiment, human IgG2 constant region as the
heavy chain constant region and human Ig kappa constant region as
the light chain constant region can be used.
[0145] For example, the invention provides an isolated monoclonal
antibody, or antigen binding portion thereof, comprising a heavy
chain variable region and a light chain variable region,
wherein:
[0146] (a) the heavy chain variable region comprises an amino acid
sequence that is at least 80% homologous to heavy chain variable
region amino acid sequence set forth in FIG. 3; and
[0147] (b) the light chain variable region comprises an amino acid
sequence that is at least 80% homologous to the light chain
variable region amino acid sequence set forth in FIG. 3.
[0148] In other embodiments, the V.sub.H and/or V.sub.L amino acid
sequences may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98% or 99% homologous to the V.sub.H and V.sub.L
sequences set forth in FIG. 3.
[0149] In another embodiment, the invention provides an isolated
monoclonal antibody, or antigen binding portion thereof, comprising
a humanized heavy chain variable region and a humanized light chain
variable region, wherein:
[0150] (a) the heavy chain variable region comprises
complementarity determining regions (CDRs) having the amino acid
sequences of the heavy chain variable region CDRs set forth in FIG.
3;
[0151] (b) the light chain variable region comprises CDRs having
the amino acid sequences of the light chain variable region CDRs
set forth in FIG. 3.
[0152] Engineered antibodies of the invention include those in
which modifications have been made to framework residues within
V.sub.H and/or V.sub.L (e.g. to improve the properties of the
antibody). Typically such framework modifications are made to
decrease the immunogenicity of the antibody. For example, one
approach is to "backmutate" one or more framework residues to the
corresponding germline sequence. More specifically, an antibody
that has undergone somatic mutation may contain framework residues
that differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived. To return the framework region sequences to
their germline configuration, the somatic mutations can be
"backmutated" to the germline sequence by, for example,
site-directed mutagenesis or PCR-mediated mutagenesis (e.g.,
"backmutated" from leucine to methionine). Such "backmutated"
antibodies are also intended to be encompassed by the
invention.
[0153] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T-cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Patent Publication No. 2003/0153043 by Carr et al.
[0154] In addition or alternative to modifications made within the
framework or CDR regions, antibodies of the invention may be
engineered to include modifications within the Fc region, typically
to alter one or more functional properties of the antibody, such as
serum half-life, complement fixation, Fc receptor binding, and/or
antigen-dependent cellular cytotoxicity. Furthermore, a 24P4C12 MAb
of the invention may be chemically modified (e.g., one or more
chemical moieties can be attached to the antibody) or be modified
to alter its glycosylation, again to alter one or more functional
properties of the MAb. Each of these embodiments is described in
further detail below.
[0155] In one embodiment, the hinge region of CH1 is modified such
that the number of cysteine residues in the hinge region is
altered, e.g., increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of
cysteine residues in the hinge region of CH1 is altered to, for
example, facilitate assembly of the light and heavy chains or to
increase or decrease the stability of the 24P4C12 MAb.
[0156] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half life of the 24P4C12 MAb.
More specifically, one or more amino acid mutations are introduced
into the CH2-CH3 domain interface region of the Fc-hinge fragment
such that the antibody has impaired Staphylococcyl protein A (SpA)
binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Pat. No. 6,165,745
by Ward et al.
[0157] In another embodiment, the 24P4C12 MAb is modified to
increase its biological half life. Various approaches are possible.
For example, mutations can be introduced as described in U.S. Pat.
No. 6,277,375 to Ward. Alternatively, to increase the biological
half life, the antibody can be altered within the CH1 or CL region
to contain a salvage receptor binding epitope taken from two loops
of a CH2 domain of an Fc region of an IgG, as described in U.S.
Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.
[0158] In yet other embodiments, the Fc region is altered by
replacing at least one amino acid residue with a different amino
acid residue to alter the effector function(s) of the 24P4C12 MAb.
For example, one or more amino acids selected from amino acid
specific residues can be replaced with a different amino acid
residue such that the antibody has an altered affinity for an
effector ligand but retains the antigen-binding ability of the
parent antibody. The effector ligand to which affinity is altered
can be, for example, an Fc receptor or the C1 component of
complement. This approach is described in further detail in U.S.
Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
[0159] Reactivity of 24P4C12 antibodies with a 24P4C12-related
protein can be established by a number of well known means,
including Western blot, immunoprecipitation, ELISA, and FACS
analyses using, as appropriate, 24P4C12-related proteins,
24P4C12-expressing cells or extracts thereof. A 24P4C12 antibody or
fragment thereof can be labeled with a detectable marker or
conjugated to a second molecule. Suitable detectable markers
include, but are not limited to, a radioisotope, a fluorescent
compound, a bioluminescent compound, chemiluminescent compound, a
metal chelator or an enzyme. Further, bi-specific antibodies
specific for two or more 24P4C12 epitopes are generated using
methods generally known in the art. Homodimeric antibodies can also
be generated by cross-linking techniques known in the art (e.g.,
Wolff et al., Cancer Res. 53: 2560-2565).
[0160] In yet another preferred embodiment, the 24P4C12 MAb of the
invention is an antibody comprising heavy and light chain of an
antibody designated Ha5-1(5)2.1. The heavy chain of Ha5-1(5)2.1
consists of the amino acid sequence ranging from 20.sup.th Q
residue to the 469.sup.th K residue of SEQ ID NO: 20 and the light
chain of Ha5-1(5)2.1 consists of amino acid sequence ranging from
23.sup.th D residue to the 236.sup.th C residue of SEQ ID NO: 22
sequence. The sequence of which is set forth in FIG. 2 and FIG. 3.
In a preferred embodiment, Ha5-1(5)2.1 is conjugated to a cytotoxic
agent.
[0161] The hybridoma producing the antibody designated Ha5-1(5)2.1
was sent (via Federal Express) to the American Type Culture
Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108 on 8 Aug.
2007 and assigned Accession number PTA-8602.
III.) ANTIBODY-DRUG CONJUGATES GENERALLY
[0162] In another aspect, the invention provides antibody-drug
conjugates (ADCs), comprising an antibody conjugated to a cytotoxic
agent such as a chemotherapeutic agent, a drug, a growth inhibitory
agent, a toxin (e.g., an enzymatically active toxin of bacterial,
fungal, plant, or animal origin, or fragments thereof), or a
radioactive isotope (i.e., a radioconjugate). In another aspect,
the invention further provides methods of using the ADCs. In one
aspect, an ADC comprises any of the above 24P4C12 MAbs covalently
attached to a cytotoxic agent or a detectable agent.
[0163] The use of antibody-drug conjugates for the local delivery
of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos (1999)
Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997)
Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows
targeted delivery of the drug moiety to tumors, and intracellular
accumulation therein, where systemic administration of these
unconjugated drug agents may result in unacceptable levels of
toxicity to normal cells as well as the tumor cells sought to be
eliminated (Baldwin et al., (1986) Lancet pp. (Mar. 15,
1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents
In Cancer Therapy: A Review," in Monoclonal Antibodies '84:
Biological And Clinical Applications, A. Pinchera et al. (ed.s),
pp. 475-506). Maximal efficacy with minimal toxicity is sought
thereby. Both polyclonal antibodies and monoclonal antibodies have
been reported as useful in these strategies (Rowland et al., (1986)
Cancer Immunol. Immunother., 21:183-87). Drugs used in these
methods include daunomycin, doxorubicin, methotrexate, and
vindesine (Rowland et al., (1986) supra). Toxins used in
antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer
Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.
Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.
13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc.
Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al
(1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.
53:3336-3342). The toxins may effect their cytotoxic and cytostatic
effects by mechanisms including tubulin binding, DNA binding, or
topoisomerase inhibition. Some cytotoxic drugs tend to be inactive
or less active when conjugated to large antibodies or protein
receptor ligands.
[0164] Examples of antibody drug conjugates are, ZEVALIN.RTM.
(ibritumomab tiuxetan, Biogen/Idec) which is an
antibody-radioisotope conjugate composed of a murine IgG1 kappa
monoclonal antibody directed against the CD20 antigen found on the
surface of normal and malignant B lymphocytes and .sup.1111n or
.sup.90Y radioisotope bound by a thiourea linker-chelator (Wiseman
et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al
(2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol.
20(10):2453-63; Witzig et al (2002) J. Clin. Oncol.
20(15):3262-69).
[0165] Additionally, MYLOTARG.TM. (gemtuzumab ozogamicin, Wyeth
Pharmaceuticals), an antibody drug conjugate composed of a hu CD33
antibody linked to calicheamicin, was approved in 2000 for the
treatment of acute myeloid leukemia by injection (Drugs of the
Future (2000) 25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233;
5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285;
5,773,001).
[0166] In addition, Cantuzumab mertansine (Immunogen, Inc.), an
antibody drug conjugate composed of the huC242 antibody linked via
the disulfide linker SPP to the maytansinoid drug moiety, DM1, is
advancing into Phase II trials for the treatment of cancers that
express CanAg, such as colon, pancreatic, gastric, and others.
[0167] Additionally, MLN-2704 (Millennium Pharm., BZL Biologics,
Immunogen Inc.), an antibody drug conjugate composed of the
anti-prostate specific membrane antigen (PSMA) monoclonal antibody
linked to the maytansinoid drug moiety, DM1, is under development
for the potential treatment of prostate tumors.
[0168] Finally, the auristatin peptides, auristatin E (AE) and
monomethylauristatin (MMAE), synthetic analogs of dolastatin, were
conjugated to chimeric monoclonal antibodies cBR96 (specific to
Lewis Y on carcinomas) and cAC10 (specific to CD30 on hematological
malignancies) (Doronina et al (2003) Nature Biotechnology
21(7):778-784) and are under therapeutic development.
[0169] Further, chemotherapeutic agents useful in the generation of
ADCs are described herein. Enzymatically active toxins and
fragments thereof that can be used include diphtheria A chain,
nonbinding active fragments of diphtheria toxin, exotoxin A chain
(from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. See, e.g., WO 93/21232
published Oct. 28, 1993. A variety of radionuclides are available
for the production of radioconjugated antibodies. Examples include
.sup.212Bi, .sup.131I, .sup.131In, .sup.90Y, and .sup.186Re.
Conjugates of the antibody and cytotoxic agent are made using a
variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al (1987) Science, 238:1098. Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody (WO94/11026).
[0170] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, dolastatins,
auristatins, a trichothecene, and CC1065, and the derivatives of
these toxins that have toxin activity, are also contemplated
herein.
[0171] III(A). Maytansinoids
[0172] Maytansine compounds suitable for use as maytansinoid drug
moieties are well known in the art, and can be isolated from
natural sources according to known methods, produced using genetic
engineering techniques (see Yu et al (2002) PNAS 99:7968-7973), or
maytansinol and maytansinol analogues prepared synthetically
according to known methods.
[0173] Exemplary maytansinoid drug moieties include those having a
modified aromatic ring, such as: C-19-dechloro (U.S. Pat. No.
4,256,746) (prepared by lithium aluminum hydride reduction of
ansamytocin P2); C-20-hydroxy (or C-20-demethyl) +/-C-19-dechloro
(U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation
using Streptomyces or Actinomyces or dechlorination using LAH); and
C-20-demethoxy, C-20-acyloxy (--OCOR), +/-dechloro (U.S. Pat. No.
4,294,757) (prepared by acylation using acyl chlorides). and those
having modifications at other positions
[0174] Exemplary maytansinoid drug moieties also include those
having modifications such as: C-9-SH (U.S. Pat. No. 4,424,219)
(prepared by the reaction of maytansinol with H.sub.2S or
P.sub.2S.sub.5); C-14-alkoxymethyl(demethoxy/CH.sub.2OR)(U.S. Pat.
No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH.sub.2OH or
CH.sub.2OAc) (U.S. Pat. No. 4,450,254) (prepared from Nocardia);
C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by the
conversion of maytansinol by Streptomyces); C-15-methoxy (U.S. Pat.
Nos. 4,313,946 and 4,315,929) (isolated from Trewia nudlflora);
C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared
by the demethylation of maytansinol by Streptomyces); and 4,5-deoxy
(U.S. Pat. No. 4,371,533) (prepared by the titanium trichloride/LAH
reduction of maytansinol).
[0175] ADCs containing maytansinoids, methods of making same, and
their therapeutic use are disclosed, for example, in U.S. Pat. Nos.
5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235
B1, the disclosures of which are hereby expressly incorporated by
reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described ADCs comprising a maytansinoid designated DM1
linked to the monoclonal antibody C242 directed against human
colorectal cancer. The conjugate was found to be highly cytotoxic
towards cultured colon cancer cells, and showed antitumor activity
in an in vivo tumor growth assay. Chari et al., Cancer Research
52:127-131 (1992) describe ADCs in which a maytansinoid was
conjugated via a disulfide linker to the murine antibody A7 binding
to an antigen on human colon cancer cell lines, or to another
murine monoclonal antibody TA.1 that binds the HER-2/neu oncogene.
The cytotoxicity of the TA.1-maytansonoid conjugate was tested in
vitro on the human breast cancer cell line SK-BR-3, which expresses
3.times.10.sup.5 HER-2 surface antigens per cell. The drug
conjugate achieved a degree of cytotoxicity similar to the free
maytansinoid drug, which could be increased by increasing the
number of maytansinoid molecules per antibody molecule. The
A7-maytansinoid conjugate showed low systemic cytotoxicity in
mice.
[0176] III(B). Auristatins and Dolastatins
[0177] In some embodiments, the ADC comprises an antibody of the
invention conjugated to dolastatins or dolostatin peptidic analogs
and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483;
5,780,588). Dolastatins and auristatins have been shown to
interfere with microtubule dynamics, GTP hydrolysis, and nuclear
and cellular division (Woyke et al (2001) Antimicrob. Agents and
Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No.
5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob.
Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug
moiety may be attached to the antibody through the N (amino)
terminus or the C (carboxyl) terminus of the peptidic drug moiety
(WO 02/088172).
[0178] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, disclosed in
"Senter et al, Proceedings of the American Association for Cancer
Research, Volume 45, Abstract Number 623, presented Mar. 28, 2004
and described in United States Patent Publication No. 2005/0238648,
the disclosure of which is expressly incorporated by reference in
its entirety.
[0179] An exemplary auristatin embodiment is MMAE (wherein the wavy
line indicates the covalent attachment to a linker (L) of an
antibody drug conjugate).
##STR00002##
[0180] Another exemplary auristatin embodiment is MMAF, wherein the
wavy line indicates the covalent attachment to a linker (L) of an
antibody drug conjugate (US 2005/0238649):
##STR00003##
[0181] Additional exemplary embodiments comprising MMAE or MMAF and
various linker components (described further herein) have the
following structures and abbreviations (wherein Ab means antibody
and p is 1 to about 8):
##STR00004##
[0182] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug moieties may be prepared according to
the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;
Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al
(1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al.
Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin
Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol
21(7):778-784.
[0183] III(C). Calicheamicin
[0184] In other embodiments, the ADC comprises an antibody of the
invention conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma..sub.1.sup.I, .alpha..sub.2.sup.I,
.alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I, PSAG and
.theta..sup.I.sub.1 (Hinman et al., Cancer Research 53:3336-3342
(1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the
aforementioned U.S. patents to American Cyanamid). Another
anti-tumor drug that the antibody can be conjugated is QFA which is
an antifolate. Both calicheamicin and QFA have intracellular sites
of action and do not readily cross the plasma membrane. Therefore,
cellular uptake of these agents through antibody mediated
internalization greatly enhances their cytotoxic effects.
[0185] III(D). Other Cytotoxic Agents
[0186] Other antitumor agents that can be conjugated to the
antibodies of the invention include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known
collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0187] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0188] The present invention further contemplates an ADC formed
between an antibody and a compound with nucleolytic activity (e.g.,
a ribonuclease or a DNA endonuclease such as a deoxyribonuclease;
DNase).
[0189] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
antibodies. Examples include At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu. When the conjugate is
used for detection, it may comprise a radioactive atom for
scintigraphic studies, for example tc.sup.99m or I.sup.123, or a
spin label for nuclear magnetic resonance (NMR) imaging (also known
as magnetic resonance imaging, mri), such as iodine-123 again,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
[0190] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods in detail.
IV.) ANTIBODY-DRUG CONJUGATE COMPOUNDS WHICH BIND 24P4C12
[0191] The present invention provides, inter alia, antibody-drug
conjugate compounds for targeted delivery of drugs. The inventors
have made the discovery that the antibody-drug conjugate compounds
have potent cytotoxic and/or cytostatic activity against cells
expressing 24P4C12. The antibody-drug conjugate compounds comprise
an Antibody unit covalently linked to at least one Drug unit. The
Drug units can be covalently linked directly or via a Linker unit
(-LU-).
[0192] In some embodiments, the antibody drug conjugate compound
has the following formula:
L-(LU-D).sub.p (I)
[0193] or a pharmaceutically acceptable salt or solvate thereof;
wherein: [0194] L is the Antibody unit, e.g., 24P4C12 MAb of the
present invention, and (LU-D) is a Linker unit-Drug unit moiety,
wherein: [0195] LU- is a Linker unit, and [0196] -D is a drug unit
having cytostatic or cytotoxic activity against a target cell; and
[0197] p is an integer from 1 to 20.
[0198] In some embodiments, p ranges from 1 to 10, 1 to 9, 1 to 8,
1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some
embodiments, p ranges from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6,
2 to 5, 2 to 4 or 2 to 3. In other embodiments, p is 1, 2, 3, 4, 5
or 6. In some embodiments, p is 2 or 4.
[0199] In some embodiments, the antibody drug conjugate compound
has the following formula:
L-(A.sub.a-W.sub.w--Y.sub.y-D).sub.p (II)
[0200] or a pharmaceutically acceptable salt or solvate thereof,
wherein: [0201] L is the Antibody unit, e.g., 24P4C12 MAb; and
[0202] -A.sub.a-W.sub.w--Y.sub.y-- is a Linker unit (LU), wherein:
[0203] -A- is a Stretcher unit, [0204] a is 0 or 1, [0205] each
--W-- is independently an Amino Acid unit, [0206] w is an integer
ranging from 0 to 12, [0207] --Y-- is a self-immolative spacer
unit, [0208] y is 0, 1 or 2; [0209] -D is a drug units having
cytostatic or cytotoxic activity against the target cell; and
[0210] p is an integer from 1 to 20.
[0211] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1
or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or
1. In some embodiments, p ranges from 1 to 10, 1 to 9, 1 to 8, 1 to
7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments,
p ranges from 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In
other embodiments, p is 1, 2, 3, 4, 5 or 6. In some embodiments, p
is 2 or 4. In some embodiments, when w is not zero, y is 1 or 2. In
some embodiments, when w is 1 to 12, y is 1 or 2. In some
embodiments, w is 2 to 12 and y is 1 or 2. In some embodiments, a
is 1 and w and y are 0.
[0212] For compositions comprising a plurality antibodies, the drug
loading is represented by p, the average number of drug molecules
per Antibody. Drug loading may range from 1 to 20 drugs (D) per
Antibody. The average number of drugs per antibody in preparation
of conjugation reactions may be characterized by conventional means
such as mass spectroscopy, ELISA assay, and HPLC. The quantitative
distribution of Antibody-Drug-Conjugates in terms of p may also be
determined. In some instances, separation, purification, and
characterization of homogeneous Antibody-Drug-conjugates where p is
a certain value from Antibody-Drug-Conjugates with other drug
loadings may be achieved by means such as reverse phase HPLC or
electrophoresis. In exemplary embodiments, p is from 2 to 8.
[0213] The generation of Antibody-drug conjugate compounds can be
accomplished by any technique known to the skilled artisan.
Briefly, the Antibody-drug conjugate compounds comprise 24P4C12 MAb
as the Antibody unit, a drug, and optionally a linker that joins
the drug and the binding agent. In a preferred embodiment, the
Antibody is 24P4C12 MAb comprising heavy and light chain variable
regions of an antibody designated Ha5-1(5)2.1 described above. In
more preferred embodiment, the Antibody is 24P4C12 MAb comprising
heavy and light chain of an antibody designated Ha5-1(5)2.1
described above. A number of different reactions are available for
covalent attachment of drugs and/or linkers to binding agents. This
is often accomplished by reaction of the amino acid residues of the
binding agent, e.g., antibody molecule, including the amine groups
of lysine, the free carboxylic acid groups of glutamic and aspartic
acid, the sulfhydryl groups of cysteine and the various moieties of
the aromatic amino acids. One of the most commonly used
non-specific methods of covalent attachment is the carbodiimide
reaction to link a carboxy (or amino) group of a compound to amino
(or carboxy) groups of the antibody. Additionally, bifunctional
agents such as dialdehydes or imidoesters have been used to link
the amino group of a compound to amino groups of an antibody
molecule. Also available for attachment of drugs to binding agents
is the Schiff base reaction. This method involves the periodate
oxidation of a drug that contains glycol or hydroxy groups, thus
forming an aldehyde which is then reacted with the binding agent.
Attachment occurs via formation of a Schiff base with amino groups
of the binding agent. Isothiocyanates can also be used as coupling
agents for covalently attaching drugs to binding agents. Other
techniques are known to the skilled artisan and within the scope of
the present invention.
[0214] In certain embodiments, an intermediate, which is the
precursor of the linker, is reacted with the drug under appropriate
conditions. In certain embodiments, reactive groups are used on the
drug and/or the intermediate. The product of the reaction between
the drug and the intermediate, or the derivatized drug, is
subsequently reacted with the 24P4C12 MAb under appropriate
conditions.
[0215] Each of the particular units of the Antibody-drug conjugate
compounds is described in more detail herein. The synthesis and
structure of exemplary Linker units, Stretcher units, Amino Acid
units, self-immolative Spacer unit, and Drug units are also
described in U.S. Patent Application Publication Nos. 2003-0083263,
2005-0238649 and 2005-0009751, each if which is incorporated herein
by reference in its entirety and for all purposes.
V.) LINKER UNITS
[0216] Typically, the antibody-drug conjugate compounds comprise a
Linker unit between the drug unit and the antibody unit. In some
embodiments, the linker is cleavable under intracellular
conditions, such that cleavage of the linker releases the drug unit
from the antibody in the intracellular environment. In yet other
embodiments, the linker unit is not cleavable and the drug is
released, for example, by antibody degradation.
[0217] In some embodiments, the linker is cleavable by a cleaving
agent that is present in the intracellular environment (e.g.,
within a lysosome or endosome or caveolea). The linker can be,
e.g., a peptidyl linker that is cleaved by an intracellular
peptidase or protease enzyme, including, but not limited to, a
lysosomal or endosomal protease. In some embodiments, the peptidyl
linker is at least two amino acids long or at least three amino
acids long. Cleaving agents can include cathepsins B and D and
plasmin, all of which are known to hydrolyze dipeptide drug
derivatives resulting in the release of active drug inside target
cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics
83:67-123). Most typical are peptidyl linkers that are cleavable by
enzymes that are present in 24P4C12-expressing cells. For example,
a peptidyl linker that is cleavable by the thiol-dependent protease
cathepsin-B, which is highly expressed in cancerous tissue, can be
used (e.g., a Phe-Leu or a Gly-Phe-Leu-Gly linker (SEQ ID NO: 25)).
Other examples of such linkers are described, e.g., in U.S. Pat.
No. 6,214,345, incorporated herein by reference in its entirety and
for all purposes. In a specific embodiment, the peptidyl linker
cleavable by an intracellular protease is a Val-Cit linker or a
Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes
the synthesis of doxorubicin with the val-cit linker). One
advantage of using intracellular proteolytic release of the
therapeutic agent is that the agent is typically attenuated when
conjugated and the serum stabilities of the conjugates are
typically high.
[0218] In other embodiments, the cleavable linker is pH-sensitive,
i.e., sensitive to hydrolysis at certain pH values. Typically, the
pH-sensitive linker hydrolyzable under acidic conditions. For
example, an acid-labile linker that is hydrolyzable in the lysosome
(e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic
amide, orthoester, acetal, ketal, or the like) can be used. (See,
e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and
Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989,
Biol. Chem. 264:14653-14661). Such linkers are relatively stable
under neutral pH conditions, such as those in the blood, but are
unstable at below pH 5.5 or 5.0, the approximate pH of the
lysosome. In certain embodiments, the hydrolyzable linker is a
thioether linker (such as, e.g., a thioether attached to the
therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat.
No. 5,622,929).
[0219] In yet other embodiments, the linker is cleavable under
reducing conditions (e.g., a disulfide linker). A variety of
disulfide linkers are known in the art, including, for example,
those that can be formed using SATA
(N-succinimidyl-5-acetylthioacetate), SPDP
(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB
(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT
(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-
, SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.
47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody
Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,
Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935).
[0220] In yet other specific embodiments, the linker is a malonate
linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a
maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem.
3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995,
Bioorg-Med-Chem. 3(10):1305-12).
[0221] In yet other embodiments, the linker unit is not cleavable
and the drug is released by antibody degradation. (See U.S.
Publication No. 2005/0238649 incorporated by reference herein in
its entirety and for all purposes).
[0222] Typically, the linker is not substantially sensitive to the
extracellular environment. As used herein, "not substantially
sensitive to the extracellular environment," in the context of a
linker, means that no more than about 20%, typically no more than
about 15%, more typically no more than about 10%, and even more
typically no more than about 5%, no more than about 3%, or no more
than about 1% of the linkers, in a sample of antibody-drug
conjugate compound, are cleaved when the antibody-drug conjugate
compound presents in an extracellular environment (e.g., in
plasma). Whether a linker is not substantially sensitive to the
extracellular environment can be determined, for example, by
incubating with plasma the antibody-drug conjugate compound for a
predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and then
quantitating the amount of free drug present in the plasma.
[0223] In other, non-mutually exclusive embodiments, the linker
promotes cellular internalization. In certain embodiments, the
linker promotes cellular internalization when conjugated to the
therapeutic agent (i.e., in the milieu of the linker-therapeutic
agent moiety of the antibody-drug conjugate compound as described
herein). In yet other embodiments, the linker promotes cellular
internalization when conjugated to both the auristatin compound and
the 24P4C12 MAb.
[0224] A variety of exemplary linkers that can be used with the
present compositions and methods are described in WO 2004-010957,
U.S. Publication No. 2006/0074008, U.S. Publication No.
20050238649, and U.S. Publication No. 2006/0024317 (each of which
is incorporated by reference herein in its entirety and for all
purposes).
[0225] A "Linker unit" (LU) is a bifunctional compound that can be
used to link a Drug unit and a Antibody unit to form an
antibody-drug conjugate compound. In some embodiments, the Linker
unit has the formula:
-A.sub.a-W.sub.w--Y.sub.y-- [0226] wherein: -A- is a Stretcher
unit, [0227] a is 0 or 1, [0228] each --W-- is independently an
Amino Acid unit, [0229] w is an integer ranging from 0 to 12,
[0230] --Y-- is a self-immolative Spacer unit, and [0231] y is 0, 1
or 2.
[0232] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1
or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or
1. In some embodiments, when w is 1 to 12, y is 1 or 2. In some
embodiments, w is 2 to 12 and y is 1 or 2. In some embodiments, a
is 1 and w and y are 0.
VI.) THE STRETCHER UNIT
[0233] The Stretcher unit (A), when present, is capable of linking
an Antibody unit to an Amino Acid unit (--W--), if present, to a
Spacer unit (--Y--), if present; or to a Drug unit (-D). Useful
functional groups that can be present on a 24P4C12 MAb (e.g.
Ha5-1(5)2.1), either naturally or via chemical manipulation
include, but are not limited to, sulfhydryl, amino, hydroxyl, the
anomeric hydroxyl group of a carbohydrate, and carboxyl. Suitable
functional groups are sulfhydryl and amino. In one example,
sulfhydryl groups can be generated by reduction of the
intramolecular disulfide bonds of a 24P4C12 MAb. In another
embodiment, sulfhydryl groups can be generated by reaction of an
amino group of a lysine moiety of a 24P4C12 MAb with
2-iminothiolane (Traut's reagent) or other sulfhydryl generating
reagents. In certain embodiments, the 24P4C12 MAb is a recombinant
antibody and is engineered to carry one or more lysines. In certain
other embodiments, the recombinant 24P4C12 MAb is engineered to
carry additional sulfhydryl groups, e.g., additional cysteines.
[0234] In one embodiment, the Stretcher unit forms a bond with a
sulfur atom of the Antibody unit. The sulfur atom can be derived
from a sulfhydryl group of an antibody. Representative Stretcher
units of this embodiment are depicted within the square brackets of
Formulas IIIa and IIIb, wherein L--, --W--, --Y--, -D, w and y are
as defined above, and R.sup.17 is selected from --C.sub.1-C.sub.10
alkylene-, --C.sub.1-C.sub.10 alkenylene-, --C.sub.1-C.sub.10
alkynylene-, carbocyclo-, --O--(C.sub.1-C.sub.8 alkylene)-,
O--(C.sub.1-C.sub.8 alkenylene)-, --O--(C.sub.1-C.sub.8
alkynylene)-, -arylene-, --C.sub.1-C.sub.10 alkylene-arylene-,
--C.sub.2-C.sub.10 alkenylene-arylene, --C.sub.2-C.sub.10
alkynylene-arylene, -arylene-C.sub.1-C.sub.10 alkylene-,
-arylene-C.sub.2-C.sub.10 alkenylene-, -arylene-C.sub.2-C.sub.10
alkynylene-, --C.sub.1-C.sub.10 alkylene-(carbocyclo)-,
--C.sub.2-C.sub.10 alkenylene-(carbocyclo)-, --C.sub.2-C.sub.10
alkynylene-(carbocyclo)-, -(carbocyclo)-C.sub.1-C.sub.10 alkylene-,
-(carbocyclo)-C.sub.2-C.sub.10 alkenylene-,
-(carbocyclo)-C.sub.2-C.sub.10 alkynylene, -heterocyclo-,
--C.sub.1-C.sub.10 alkylene-(heterocyclo)-, --C.sub.2-C.sub.10
alkenylene-(heterocyclo)-, --C.sub.2-C.sub.10
alkynylene-(heterocyclo)-, -(heterocyclo)-C.sub.1-C.sub.10
alkylene-, -(heterocyclo)-C.sub.2-C.sub.10 alkenylene-,
-(heterocyclo)-C.sub.1-C.sub.10 alkynylene-,
--(CH.sub.2CH.sub.2O).sub.r--, or
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--, and r is an integer
ranging from 1-10, wherein said alkyl, alkenyl, alkynyl, alkylene,
alkenylene, alkynyklene, aryl, carbocycle, carbocyclo, heterocyclo,
and arylene radicals, whether alone or as part of another group,
are optionally substituted. In some embodiments, said alkyl,
alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl,
carbocyle, carbocyclo, heterocyclo, and arylene radicals, whether
alone or as part of another group, are unsubstituted. In some
embodiments, R.sup.17 is selected from --C.sub.1-C.sub.10
alkylene-, -carbocyclo-, --O--(C.sub.1-C.sub.8 alkylene)-,
-arylene-, --C.sub.1-C.sub.10 alkylene-arylene-,
-arylene-C.sub.1-C.sub.10 alkylene-, --C.sub.1-C.sub.10
alkylene-(carbocyclo)-, -(carbocyclo)-C.sub.1-C.sub.10 alkylene-,
--C.sub.3-C.sub.8 heterocyclo-, --C.sub.1-C.sub.10
alkylene-(heterocyclo)-, -(heterocyclo)-C.sub.1-C.sub.10 alkylene-,
--(CH.sub.2CH.sub.2O).sub.r--, and
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r is an integer
ranging from 1-10, wherein said alkylene groups are unsubstituted
and the remainder of the groups are optionally substituted.
[0235] It is to be understood from all the exemplary embodiments
that even where not denoted expressly, from 1 to 20 drug moieties
can be linked to an Antibody (p=1-20).
##STR00005##
[0236] An illustrative Stretcher unit is that of Formula IIIa
wherein R.sup.17 is --(CH.sub.2).sub.5--:
##STR00006##
[0237] Another illustrative Stretcher unit is that of Formula IIIa
wherein R.sup.17 is --(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r
is 2:
##STR00007##
[0238] An illustrative Stretcher unit is that of Formula IIIa
wherein R.sup.17 is arylene- or arylene-C.sub.1-C.sub.10 alkylene-.
In some embodiments, the aryl group is an unsubstituted phenyl
group.
[0239] Still another illustrative Stretcher unit is that of Formula
IIIb wherein R.sup.17 is --(CH.sub.2).sub.5--:
##STR00008##
[0240] In certain embodiments, the Stretcher unit is linked to the
Antibody unit via a disulfide bond between a sulfur atom of the
Antibody unit and a sulfur atom of the Stretcher unit. A
representative Stretcher unit of this embodiment is depicted within
the square brackets of Formula IV, wherein R.sup.17, L-, --W--,
--Y--, -D, w and y are as defined above.
##STR00009##
[0241] It should be noted that throughout this application, the S
moiety in the formula below refers to a sulfur atom of the Antibody
unit, unless otherwise indicated by context.
##STR00010##
[0242] In yet other embodiments, the Stretcher contains a reactive
site that can form a bond with a primary or secondary amino group
of an Antibody. Examples of these reactive sites include, but are
not limited to, activated esters such as succinimide esters, 4
nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl
esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates
and isothiocyanates. Representative Stretcher units of this
embodiment are depicted within the square brackets of Formulas Va
and Vb, wherein --R.sup.17--, L-, --W--, --Y--, -D, w and y are as
defined above;
##STR00011##
[0243] In some embodiments, the Stretcher contains a reactive site
that is reactive to a modified carbohydrate's (--CHO) group that
can be present on an Antibody. For example, a carbohydrate can be
mildly oxidized using a reagent such as sodium periodate and the
resulting (--CHO) unit of the oxidized carbohydrate can be
condensed with a Stretcher that contains a functionality such as a
hydrazide, an oxime, a primary or secondary amine, a hydrazine, a
thiosemicarbazone, a hydrazine carboxylate, and an arylhydrazide
such as those described by Kaneko et al., 1991, Bioconjugate Chem.
2:133-41. Representative Stretcher units of this embodiment are
depicted within the square brackets of Formulas VIa, VIb, and VIc,
wherein --R.sup.17--, L-, --W--, --Y--, -D, w and y are as defined
as above.
##STR00012##
VII.) THE AMINO ACID UNIT
[0244] The Amino Acid unit (--W--), when present, links the
Stretcher unit to the Spacer unit if the Spacer unit is present,
links the Stretcher unit to the Drug moiety if the Spacer unit is
absent, and links the Antibody unit to the Drug unit if the
Stretcher unit and Spacer unit are absent.
[0245] W.sub.w-- can be, for example, a monopeptide, dipeptide,
tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide,
octapeptide, nonapeptide, decapeptide, undecapeptide or
dodecapeptide unit. Each --W-- unit independently has the formula
denoted below in the square brackets, and w is an integer ranging
from 0 to 12:
##STR00013##
[0246] wherein R.sup.19 is hydrogen, methyl, isopropyl, isobutyl,
sec-butyl, benzyl, p-hydroxybenzyl, --CH.sub.2OH, --CH(OH)CH.sub.3,
--CH.sub.2CH.sub.2SCH.sub.3, --CH.sub.2CONH.sub.2, --CH.sub.2COOH,
--CH.sub.2CH.sub.2CONH.sub.2, --CH.sub.2CH.sub.2COOH,
--(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.3NH.sub.2,
--(CH.sub.2).sub.3NHCOCH.sub.3, --(CH.sub.2).sub.3NHCHO,
--(CH.sub.2).sub.4NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.4NH.sub.2,
(CH.sub.2).sub.4NHCOCH.sub.3, --(CH.sub.2).sub.4NHCHO,
--(CH.sub.2).sub.3NHCONH.sub.2, --(CH.sub.2).sub.4NHCONH.sub.2,
--CH.sub.2CH.sub.2CH(OH)CH.sub.2NH.sub.2, 2-pyridylmethyl-,
3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,
##STR00014##
[0247] In some embodiments, the Amino Acid unit can be
enzymatically cleaved by one or more enzymes, including a cancer or
tumor-associated protease, to liberate the Drug unit (-D), which in
one embodiment is protonated in vivo upon release to provide a Drug
(D).
[0248] In certain embodiments, the Amino Acid unit can comprise
natural amino acids. In other embodiments, the Amino Acid unit can
comprise non-natural amino acids. Illustrative Ww units are
represented by formulas (VII)-(IX):
TABLE-US-00001 (VII) ##STR00015## wherein R.sup.20 and R.sup.21 are
as follows: R.sup.20 R.sup.21 Benzyl (CH.sub.2).sub.4NH.sub.2;
methyl (CH.sub.2).sub.4NH.sub.2; isopropyl
(CH.sub.2).sub.4NH.sub.2; isopropyl (CH.sub.2).sub.3NHCONH.sub.2;
benzyl (CH.sub.2).sub.3NHCONH.sub.2; isobutyl
(CH.sub.2).sub.3NHCONH.sub.2; sec-butyl
(CH.sub.2).sub.3NHCONH.sub.2; ##STR00016##
(CH.sub.2).sub.3NHCONH.sub.2; benzyl methyl; benzyl
(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2; (VIII) ##STR00017## wherein
R.sup.20, R.sup.21 and R.sup.22 are as follows: R.sup.20 R.sup.21
R.sup.22 benzyl benzyl (CH.sub.2).sub.4NH.sub.2; isopropyl benzyl
(CH.sub.2).sub.4NH.sub.2; and H benzyl (CH.sub.2).sub.4NH.sub.2;
(IX) ##STR00018## wherein R.sup.20, R.sup.21, R.sup.22 and R.sup.23
are as follows: R.sup.20 R.sup.21 R.sup.22 R.sup.23 H benzyl
isobutyl H; and methyl isobutyl methyl isobutyl.
[0249] Exemplary Amino Acid units include, but are not limited to,
units of formula VII where: R.sup.20 is benzyl and R.sup.21 is
--(CH.sub.2).sub.4NH.sub.2; R.sup.20 is isopropyl and R.sup.21 is
--(CH.sub.2).sub.4NH.sub.2; or R.sup.20 is isopropyl and R.sup.21
is --(CH.sub.2).sub.3NHCONH.sub.2. Another exemplary Amino Acid
unit is a unit of formula VIII wherein R.sup.20 is benzyl, R.sup.21
is benzyl, and R.sup.22 is --(CH.sub.2).sub.4NH.sub.2.
[0250] Useful --W.sub.w-- units can be designed and optimized in
their selectivity for enzymatic cleavage by a particular enzyme,
for example, a tumor-associated protease. In one embodiment, a
--W.sub.w-- unit is that whose cleavage is catalyzed by cathepsin
B, C and D, or a plasmin protease.
[0251] In one embodiment, --W.sub.w-- is a dipeptide, tripeptide,
tetrapeptide or pentapeptide. When R.sup.19, R.sup.20, R.sup.21,
R.sup.22 or R.sup.23 is other than hydrogen, the carbon atom to
which R.sup.19, R.sup.20, R.sup.21, R.sup.22 or R.sup.23 is
attached is chiral.
[0252] Each carbon atom to which R.sup.19, R.sup.20, R.sup.21,
R.sup.22 or R.sup.23 is attached is independently in the (S) or (R)
configuration.
[0253] In one aspect of the Amino Acid unit, the Amino Acid unit is
valine-citrulline (vc or val-cit). In another aspect, the Amino
Acid unit is phenylalanine-lysine (i.e., fk). In yet another aspect
of the Amino Acid unit, the Amino Acid unit is
N-methylvaline-citrulline. In yet another aspect, the Amino Acid
unit is 5-aminovaleric acid, homo phenylalanine lysine,
tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine,
isonepecotic acid lysine, beta-alanine lysine, glycine serine
valine glutamine and isonepecotic acid.
VIII.) THE SPACER UNIT
[0254] The Spacer unit (--Y--), when present, links an Amino Acid
unit to the Drug unit when an Amino Acid unit is present.
Alternately, the Spacer unit links the Stretcher unit to the Drug
unit when the Amino Acid unit is absent. The Spacer unit also links
the Drug unit to the Antibody unit when both the Amino Acid unit
and Stretcher unit are absent.
[0255] Spacer units are of two general types: non self-immolative
or self-immolative. A non self-immolative Spacer unit is one in
which part or all of the Spacer unit remains bound to the Drug
moiety after cleavage, particularly enzymatic, of an Amino Acid
unit from the antibody-drug conjugate. Examples of a non
self-immolative Spacer unit include, but are not limited to a
(glycine-glycine) Spacer unit and a glycine Spacer unit (both
depicted in Scheme 1) (infra). When a conjugate containing a
glycine-glycine Spacer unit or a glycine Spacer unit undergoes
enzymatic cleavage via an enzyme (e.g., a tumor-cell
associated-protease, a cancer-cell-associated protease or a
lymphocyte-associated protease), a glycine-glycine-Drug moiety or a
glycine-Drug moiety is cleaved from L-Aa-Ww-. In one embodiment, an
independent hydrolysis reaction takes place within the target cell,
cleaving the glycine-Drug moiety bond and liberating the Drug.
##STR00019##
[0256] In some embodiments, a non self-immolative Spacer unit
(--Y--) is -Gly-. In some embodiments, a non self-immolative Spacer
unit (--Y--) is -Gly-Gly-.
[0257] In one embodiment, a Drug-Linker conjugate is provided in
which the Spacer unit is absent (y=0), or a pharmaceutically
acceptable salt or solvate thereof.
[0258] Alternatively, a conjugate containing a self-immolative
Spacer unit can release -D. As used herein, the term
"self-immolative Spacer" refers to a bifunctional chemical moiety
that is capable of covalently linking together two spaced chemical
moieties into a stable tripartite molecule. It will spontaneously
separate from the second chemical moiety if its bond to the first
moiety is cleaved.
[0259] In some embodiments, --Y.sub.y-- is a p-aminobenzyl alcohol
(PAB) unit (see Schemes 2 and 3) whose phenylene portion is
substituted with Q.sub.m wherein Q is --C.sub.1-C.sub.8 alkyl,
--C.sub.1-C.sub.8 alkenyl, --C.sub.1-C.sub.8 alkynyl,
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.1-C.sub.8 alkenyl),
--O--(C.sub.1-C.sub.8 alkynyl), -halogen, -nitro or -cyano; and m
is an integer ranging from 0-4. The alkyl, alkenyl and alkynyl
groups, whether alone or as part of another group, can be
optionally substituted.
[0260] In some embodiments, --Y-- is a PAB group that is linked to
--W.sub.w-- via the amino nitrogen atom of the PAB group, and
connected directly to -D via a carbonate, carbamate or ether group.
Without being bound by any particular theory or mechanism, Scheme 2
depicts a possible mechanism of Drug release of a PAB group which
is attached directly to -D via a carbamate or carbonate group as
described by Toki et al., 2002, J. Org. Chem. 67:1866-1872.
##STR00020##
[0261] In Scheme 2, Q is --C.sub.1-C.sub.8 alkyl, --C.sub.1-C.sub.8
alkenyl, --C.sub.1-C.sub.8 alkynyl, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.1-C.sub.8 alkenyl), --O--(C.sub.1-C.sub.8 alkynyl),
-halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p
ranges from 1 to about 20. The alkyl, alkenyl and alkynyl groups,
whether alone or as part of another group, can be optionally
substituted.
[0262] Without being bound by any particular theory or mechanism,
Scheme 3 depicts a possible mechanism of Drug release of a PAB
group which is attached directly to -D via an ether or amine
linkage, wherein D includes the oxygen or nitrogen group that is
part of the Drug unit.
##STR00021##
[0263] In Scheme 3, Q is --C.sub.1-C.sub.8 alkyl, --C.sub.1-C.sub.8
alkenyl, --C.sub.1-C.sub.8 alkynyl, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.1-C.sub.8 alkenyl), --O--(C.sub.1-C.sub.8 alkynyl),
-halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p
ranges from 1 to about 20. The alkyl, alkenyl and alkynyl groups,
whether alone or as part of another group, can be optionally
substituted.
[0264] Other examples of self-immolative spacers include, but are
not limited to, aromatic compounds that are electronically similar
to the PAB group such as 2-aminoimidazol-5-methanol derivatives
(Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho or
para-aminobenzylacetals. Spacers can be used that undergo
cyclization upon amide bond hydrolysis, such as substituted and
unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995,
Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1]
and bicyclo[2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem.
Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry et
al., 1990, J. Org. Chem. 55:5867). Elimination of amine-containing
drugs that are substituted at the .alpha.-position of glycine
(Kingsbury et al., 1984, J. Med. Chem. 27:1447) are also examples
of self-immolative spacers.
[0265] In one embodiment, the Spacer unit is a branched
bis(hydroxymethyl)-styrene (BHMS) unit as depicted in Scheme 4,
which can be used to incorporate and release multiple drugs.
##STR00022##
[0266] In Scheme 4, Q is --C.sub.1-C.sub.8 alkyl, --C.sub.1-C.sub.8
alkenyl, --C.sub.1-C.sub.8 alkynyl, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.1-C.sub.8 alkenyl), --O--(C.sub.1-C.sub.8 alkynyl),
-halogen, -nitro or -cyano; m is an integer ranging from 0-4; n is
0 or 1; and p ranges raging from 1 to about 20. The alkyl, alkenyl
and alkynyl groups, whether alone or as part of another group, can
be optionally substituted.
[0267] In some embodiments, the -D moieties are the same. In yet
another embodiment, the -D moieties are different.
[0268] In one aspect, Spacer units (--Y.sub.y-) are represented by
Formulas (X)-(XII):
##STR00023##
[0269] wherein Q is --C.sub.1-C.sub.8 alkyl, --C.sub.1-C.sub.8
alkenyl, --C.sub.1-C.sub.8 alkynyl, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.1-C.sub.8 alkenyl), --O--(C.sub.1-C.sub.8 alkynyl),
-halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
The alkyl, alkenyl and alkynyl groups, whether alone or as part of
another group, can be optionally substituted.
and
##STR00024##
[0270] Embodiments of the Formula I and II comprising antibody-drug
conjugate compounds can include:
##STR00025##
[0271] wherein w and y are each 0, 1 or 2, and,
##STR00026##
[0272] wherein w and y are each 0,
##STR00027##
IX.) THE DRUG UNIT
[0273] The Drug moiety (D) can be any cytotoxic, cytostatic or
immunomodulatory (e.g., immunosuppressive) or drug. D is a Drug
unit (moiety) having an atom that can form a bond with the Spacer
unit, with the Amino Acid unit, with the Stretcher unit or with the
Antibody unit. In some embodiments, the Drug unit D has a nitrogen
atom that can form a bond with the Spacer unit. As used herein, the
terms "Drug unit" and "Drug moiety" are synonymous and used
interchangeably.
[0274] Useful classes of cytotoxic or immunomodulatory agents
include, for example, antitubulin agents, DNA minor groove binders,
DNA replication inhibitors, and alkylating agents.
[0275] In some embodiments, the Drug is an auristatin, such as
auristatin E (also known in the art as a derivative of
dolastatin-10) or a derivative thereof. The auristatin can be, for
example, an ester formed between auristatin E and a keto acid. For
example, auristatin E can be reacted with paraacetyl benzoic acid
or benzoylvaleric acid to produce AEB and AEVB, respectively. Other
typical auristatins include AFP, MMAF, and MMAE. The synthesis and
structure of exemplary auristatins are described in U.S. Patent
Application Publication Nos. 2003-0083263, 2005-0238649 and
2005-0009751; International Patent Publication No. WO 04/010957,
International Patent Publication No. WO 02/088172, and U.S. Pat.
Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860;
5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284;
5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744;
4,879,278; 4,816,444; and 4,486,414, each of which is incorporated
by reference herein in its entirety and for all purposes.
[0276] Auristatins have been shown to interfere with microtubule
dynamics and nuclear and cellular division and have anticancer
activity. Auristatins bind tubulin and can exert a cytotoxic or
cytostatic effect on a 24P4C12-expressing cell. There are a number
of different assays, known in the art, which can be used for
determining whether an auristatin or resultant antibody-drug
conjugate exerts a cytostatic or cytotoxic effect on a desired cell
line.
[0277] Methods for determining whether a compound binds tubulin are
known in the art. See, for example, Muller et al., Anal. Chem.
2006, 78, 4390-4397; Hamel et al., Molecular Pharmacology, 1995 47:
965-976; and Hamel et al., The Journal of Biological Chemistry,
1990 265:28, 17141-17149. For purposes of the present invention,
the relative affinity of a compound to tubulin can be determined.
Some preferred auristatins of the present invention bind tubulin
with an affinity ranging from 10 fold lower (weaker affinity) than
the binding affinity of MMAE to tubulin to 10 fold, 20 fold or even
100 fold higher (higher affinity) than the binding affinity of MMAE
to tublin.
[0278] In some embodiments, -D is an auristatin of the formula
D.sub.E or D.sub.F:
##STR00028##
or a pharmaceutically acceptable salt or solvate form thereof;
wherein, independently at each location:
[0279] the wavy line indicates a bond;
[0280] R.sup.2 is --C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20
alkenyl, or --C.sub.2-C.sub.20 alkynyl;
[0281] R.sup.3 is --H, --C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20
alkenyl, --C.sub.2-C.sub.20 alkynyl, -carbocycle,
--C.sub.1-C.sub.20 alkylene (carbocycle), --C.sub.2-C.sub.20
alkenylene(carbocycle), --C.sub.2-C.sub.20 alkynylene(carbocycle),
-aryl, --C.sub.1-C.sub.20 alkylene(aryl), --C.sub.2-C.sub.20
alkenylene(aryl), --C.sub.2-C.sub.20 alkynylene(aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle);
[0282] R.sup.4 is --H, --C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20
alkenyl, --C.sub.2-C.sub.20 alkynyl, carbocycle, --C.sub.1-C.sub.20
alkylene (carbocycle), --C.sub.2-C.sub.20 alkenylene(carbocycle),
--C.sub.2-C.sub.20 alkynylene(carbocycle), aryl, --C.sub.1-C.sub.20
alkylene(aryl), --C.sub.2-C.sub.20 alkenylene(aryl),
--C.sub.2-C.sub.20 alkynylene(aryl), -heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle);
[0283] R.sup.5 is --H or --C.sub.1-C.sub.8 alkyl;
[0284] or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.s-- wherein R.sup.a and
R.sup.b are independently --H, --C.sub.1-C.sub.20 alkyl,
--C.sub.2-C.sub.20 alkenyl, --C.sub.2-C.sub.20 alkynyl, or
-carbocycle and s is 2, 3, 4, 5 or 6,
[0285] R.sup.6 is --H, --C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20
alkenyl, or --C.sub.2-C.sub.20 alkynyl;
[0286] R.sup.7 is --H, --C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20
alkenyl, --C.sub.2-C.sub.20 alkynyl, carbocycle, --C.sub.1-C.sub.20
alkylene (carbocycle), --C.sub.2-C.sub.20 alkenylene(carbocycle),
--C.sub.2-C.sub.20 alkynylene(carbocycle), -aryl,
--C.sub.1-C.sub.20 alkylene(aryl), --C.sub.2-C.sub.20
alkenylene(aryl), --C.sub.2-C.sub.20 alkynylene(aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle);
[0287] each R.sup.8 is independently --H, --OH, --C.sub.1-C.sub.20
alkyl, --C.sub.2-C.sub.20 alkenyl, --C.sub.2-C.sub.20 alkynyl,
--O--(C.sub.1-C.sub.20 alkyl), --O--(C.sub.2-C.sub.20 alkenyl),
--O--(C.sub.1-C.sub.20 alkynyl), or -carbocycle;
[0288] R.sup.9 is --H, --C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20
alkenyl, or --C.sub.2-C.sub.20 alkynyl;
[0289] R.sup.24 is -aryl, -heterocycle, or -carbocycle;
[0290] R.sup.25 is --H, C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20
alkenyl, --C.sub.2-C.sub.20 alkynyl, -carbocycle,
--O--(C.sub.1-C.sub.20 alkyl), --O--(C.sub.2-C.sub.20 alkenyl),
--O--(C.sub.2-C.sub.20 alkynyl), or OR.sup.18 wherein R.sup.18 is
--H, a hydroxyl protecting group, or a direct bond where OR.sup.18
represents .dbd.O;
[0291] R.sup.26 is --H, --C.sub.1-C.sub.20 alkyl,
--C.sub.2-C.sub.20 alkenyl, or --C.sub.2-C.sub.20 alkynyl, -aryl,
-heterocycle, or -carbocycle;
[0292] R.sup.10 is -aryl or -heterocycle;
[0293] Z is --O, --S, --NH, or --NR.sup.12, wherein R.sup.12 is
--C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20 alkenyl, or
--C.sub.2-C.sub.20 alkynyl;
[0294] R.sup.11 is --H, --C.sub.1-C.sub.20 alkyl,
--C.sub.2-C.sub.20 alkenyl, --C.sub.2-C.sub.20 alkynyl, -aryl,
-heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2;
[0295] m is an integer ranging from 1-1000;
[0296] R.sup.13 is --C.sub.2-C.sub.20 alkylene, --C.sub.2-C.sub.20
alkenylene, or --C.sub.2-C.sub.20 alkynylene;
[0297] R.sup.14 is --H, --C.sub.1-C.sub.20 alkyl,
--C.sub.2-C.sub.20 alkenyl, or --C.sub.2-C.sub.20 alkynyl; each
occurrence of R.sup.15 is independently --H, --COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.N--SO.sub.3H,
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.20 alkyl,
--(CH.sub.2).sub.N--SO.sub.3--C.sub.2-C.sub.20 alkenyl, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.2-C.sub.20 alkynyl;
[0298] each occurrence of R.sup.16 is independently --H,
--C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20 alkenyl,
--C.sub.2-C.sub.20 alkynyl or --(CH.sub.2).sub.n--COOH; and
[0299] n is an integer ranging from 0 to 6;
[0300] wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene,
alkynyklene, aryl, carbocyle, and heterocycle radicals, whether
alone or as part of another group, are optionally substituted.
[0301] Auristatins of the formula D.sub.E include those wherein
said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene,
aryl, carbocyle, and heterocycle radicals are unsubstituted.
[0302] Auristatins of the formula D.sub.E include those wherein the
groups of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, and R.sup.9 are unsubstituted and the groups of R.sup.19,
R.sup.20 and R.sup.21 are optionally substituted as described
herein.
[0303] Auristatins of the formula D.sub.E include those wherein
[0304] R.sup.2 is C.sub.1-C.sub.8 alkyl;
[0305] R.sup.3, R.sup.4 and R.sup.7 are independently selected from
--H, --C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20 alkenyl,
--C.sub.2-C.sub.20 alkynyl, monocyclic C.sub.3-C.sub.6 carbocycle,
--C.sub.1-C.sub.20 alkylene(monocyclic C.sub.3-C.sub.6 carbocycle),
--C.sub.2-C.sub.20 alkenylene(monocyclic C.sub.3-C.sub.6
carbocycle), --C.sub.2-C.sub.20 alkynylene(monocyclic
C.sub.3-C.sub.6 carbocycle), C.sub.6-C.sub.10 aryl,
--C.sub.1-C.sub.20 alkylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkenylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkynylene(C.sub.6-C.sub.10 aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,
alkylene, alkenylene, alkynylene, carbocycle, aryl and heterocycle
radicals are optionally substituted;
[0306] R.sup.5 is --H;
[0307] R.sup.6 is --C.sub.1-C.sub.8 alkyl;
[0308] each R.sup.8 is independently selected from --OH,
--O--(C.sub.1-C.sub.20 alkyl), --O--(C.sub.2-C.sub.20 alkenyl), or
--O--(C.sub.2-C.sub.20 alkynyl) wherein said alkyl, alkenyl, and
alkynyl radicals are optionally substituted;
[0309] R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl;
[0310] R.sup.24 is optionally substituted -phenyl;
[0311] R.sup.25 is --OR.sup.18; wherein R.sup.18 is H, a hydroxyl
protecting group, or a direct bond where OR.sup.18 represents
.dbd.O;
[0312] R.sup.26 is selected from --H, --C.sub.1-C.sub.20 alkyl,
--C.sub.2-C.sub.20 alkenyl, --C.sub.2-C.sub.20 alkynyl, or
-carbocycle; wherein said alkyl, alkenyl, alkynyl and carbocycle
radicals are optionally substituted; or a pharmaceutically
acceptable salt or solvate form thereof.
[0313] Auristatins of the formula D.sub.E include those wherein
[0314] R.sup.2 is methyl;
[0315] R.sup.3 is --H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8
alkenyl, or C.sub.2-C.sub.8 alkynyl, wherein said alkyl, alkenyl
and alkynyl radicals are optionally substituted;
[0316] R.sup.4 is --H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8
alkenyl, --C.sub.2-C.sub.8 alkynyl, monocyclic C.sub.3-C.sub.6
carbocycle, --C.sub.6-C.sub.10 aryl, --C.sub.1-C.sub.8
alkylene(C.sub.6-C.sub.10 aryl), --C.sub.2-C.sub.8
alkenylene(C.sub.6-C.sub.10 aryl), --C.sub.2-C.sub.8
alkynylene(C.sub.6-C.sub.10 aryl), --C.sub.1-C.sub.8 alkylene
(monocyclic C.sub.3-C.sub.6 carbocycle), --C.sub.2-C.sub.8
alkenylene (monocyclic C.sub.3-C.sub.6 carbocycle),
--C.sub.2-C.sub.8 alkynylene(monocyclic C.sub.3-C.sub.6
carbocycle); wherein said alkyl, alkenyl, alkynyl, alkylene,
alkenylene, alkynylene, aryl and carbocycle radicals whether alone
or as part of another group are optionally substituted;
[0317] R.sup.5 is --H;
[0318] R.sup.6 is methyl;
[0319] R.sup.7 is --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8
alkenyl or --C.sub.2-C.sub.8 alkynyl;
[0320] each R.sup.8 is methoxy;
[0321] R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl;
[0322] R.sup.24 is -phenyl;
[0323] R.sup.25 is --OR.sup.18; wherein R.sup.18 is H, a hydroxyl
protecting group, or a direct bond where OR.sup.18 represents
.dbd.O;
[0324] R.sup.26 is methyl;
[0325] or a pharmaceutically acceptable salt form thereof.
[0326] Auristatins of the formula D.sub.E include those
wherein:
R.sup.2 is methyl; R.sup.3 is --H or --C.sub.1-C.sub.3 alkyl;
R.sup.4 is --C.sub.1-C.sub.5 alkyl; R.sup.5 is --H; R.sup.6 is
methyl; R.sup.7 is isopropyl or sec-butyl; R.sup.8 is methoxy;
R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl; R.sup.24 is phenyl;
R.sup.25 is --OR.sup.18; wherein R.sup.18 is --H, a hydroxyl
protecting group, or a direct bond where OR.sup.18 represents
.dbd.O; and R.sup.26 is methyl; or a pharmaceutically acceptable
salt or solvate form thereof.
[0327] Auristatins of the formula D.sub.E include those
wherein:
[0328] R.sup.2 is methyl or C.sub.1-C.sub.3 alkyl,
[0329] R.sup.3 is --H or --C.sub.1-C.sub.3 alkyl;
[0330] R.sup.4 is --C.sub.1-C.sub.5 alkyl;
[0331] R.sup.5 is H;
[0332] R.sup.6 is C1-C3 alkyl;
[0333] R.sup.7 is --C.sub.1-C.sub.5 alkyl;
[0334] R.sup.8 is --C.sub.1-C.sub.3 alkoxy;
[0335] R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl;
[0336] R.sup.24 is phenyl;
[0337] R.sup.25 is --OR.sup.18; wherein R.sup.18 is --H, a hydroxyl
protecting group, or a direct bond where OR.sup.18 represents
.dbd.O; and
[0338] R.sup.26 is --C.sub.1-C.sub.3 alkyl;
[0339] or a pharmaceutically acceptable salt form thereof.
[0340] Auristatins of the formula D.sub.E include those wherein
[0341] R.sup.2 is methyl;
[0342] R.sup.3, R.sup.4, and R.sup.7 are independently selected
from --H, --C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20 alkenyl,
--C.sub.2-C.sub.20 alkynyl, monocyclic C.sub.3-C.sub.6 carbocycle,
--C.sub.1-C.sub.20 alkylene(monocyclic C.sub.3-C.sub.6 carbocycle),
--C.sub.2-C.sub.20 alkenylene(monocyclic C.sub.3-C.sub.6
carbocycle), --C.sub.2-C.sub.20 alkynylene(monocyclic
C.sub.3-C.sub.6 carbocycle), --C.sub.6-C.sub.10 aryl,
--C.sub.1-C.sub.20 alkylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkenylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkynylene(C.sub.6-C.sub.10 aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,
alkylene, alkenylene, alkynylene, carbocycle, aryl and heterocycle
radicals whether alone or as part of another group are optionally
substituted;
[0343] R.sup.5 is --H;
[0344] R.sup.6 is methyl;
[0345] each R.sup.8 is methoxy;
[0346] R.sup.9 is --H, --C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20
alkenyl, or --C.sub.2-C.sub.20 alkynyl; wherein said alkyl, alkenyl
and alkynyl radical are optionally substituted;
[0347] R.sup.10 is optionally substituted aryl or optionally
substituted heterocycle;
[0348] Z is --O--, --S--, --NH--, or --NR.sup.12, wherein R.sup.12
is --C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20 alkenyl, or
--C.sub.2-C.sub.20 alkynyl, each of which is optionally
substituted;
[0349] R.sup.11 is --H, --C.sub.1-C.sub.20 alkyl,
--C.sub.2-C.sub.20 alkenyl, --C.sub.2-C.sub.20 alkynyl, -aryl,
-heterocycle, --(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2, wherein said alkyl,
alkenyl, alkynyl, aryl and heterocycle radicals are optionally
substituted;
[0350] m is an integer ranging from 1-1000 or m=O;
[0351] R.sup.13 is --C.sub.2-C.sub.20 alkylene, --C.sub.2-C.sub.20
alkenylene, or --C.sub.2-C.sub.20 alkynylene, each of which is
optionally substituted;
[0352] R.sup.14 is --H, --C.sub.1-C.sub.20 alkyl,
--C.sub.2-C.sub.20 alkenyl, or --C.sub.2-C.sub.20 alkynyl wherein
said alkyl, alkenyl and alkynyl radicals are optionally
substituted;
[0353] each occurrence of R.sup.15 is independently --H, --COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H,
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.20 alkyl,
--(CH.sub.2).sub.n--SO.sub.3--C.sub.2-C.sub.20 alkenyl, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.2-C.sub.20 alkynyl wherein said
alkyl, alkenyl and alkynyl radicals are optionally substituted;
[0354] each occurrence of R.sup.16 is independently --H,
--C.sub.1-C.sub.20 alkyl, --C.sub.2-C.sub.20 alkenyl,
--C.sub.2-C.sub.20 alkynyl or --(CH.sub.2).sub.n--COOH wherein said
alkyl, alkenyl and alkynyl radicals are optionally substituted;
[0355] n is an integer ranging from 0 to 6;
[0356] or a pharmaceutically acceptable salt thereof.
[0357] In certain of these embodiments, R.sup.10 is optionally
substituted phenyl.
[0358] Auristatins of the formula D.sub.F include those wherein the
groups of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, and R.sup.9 are unsubstituted and the groups of R.sup.10
and R.sup.11 are as described herein.
[0359] Auristatins of the formula D.sub.F include those wherein
said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene,
aryl, carbocyle, and heterocycle radicals are unsubstituted
[0360] Auristatins of the formula D.sub.F include those wherein
[0361] R.sup.2 is --C.sub.1-C.sub.3 alkyl; R.sup.3 is --H or
--C.sub.1-C.sub.3 alkyl; R.sup.4 is --C.sub.1-C.sub.5 alkyl;
R.sup.5 is --H; R.sup.6 is --C.sub.1-C.sub.3 alkyl; R.sup.7 is
--C.sub.1-C.sub.5 alkyl; R.sup.8 is --C.sub.1-C.sub.3 alkoxy;
R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl; R.sup.10 is optionally
substituted phenyl; Z is --O--, --S--, or --NH--; R.sup.11 is as
defined herein; or a pharmaceutically acceptable salt thereof.
[0362] Auristatins of the formula D.sub.F include those wherein
[0363] R.sup.2 is methyl; R.sup.3 is --H or --C.sub.1-C.sub.3
alkyl; R.sup.4 is --C.sub.1-C.sub.5 alkyl; R.sup.5 is --H; R.sup.6
is methyl; R.sup.7 is isopropyl or sec-butyl; R.sup.8 is methoxy;
R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl; R.sup.10 is optionally
substituted phenyl; Z is --O--, --S--, or --NH--; and R.sup.11 is
as defined herein; or a pharmaceutically acceptable salt
thereof.
[0364] Auristatins of the formula D.sub.F include those wherein
[0365] R.sup.2 is methyl; R.sup.3 is --H or --C.sub.1-C.sub.3
alkyl; R.sup.4 is --C.sub.1-C.sub.5 alkyl; R.sup.5 is --H; R.sup.6
is methyl; R.sup.7 is isopropyl or sec-butyl; R.sup.8 is methoxy;
R.sup.9 is --H or C.sub.1-C.sub.8 alkyl; R.sup.10 is phenyl; and Z
is --O-- or --NH-- and R.sup.11 is as defined herein, preferably
hydrogen; or a pharmaceutically acceptable salt form thereof.
[0366] Auristatins of the formula DF include those wherein
[0367] R.sup.2 is --C.sub.1-C.sub.3 alkyl; R.sup.3 is --H or
--C.sub.1-C.sub.3 alkyl; R.sup.4 is --C.sub.1-C.sub.5 alkyl;
R.sup.5 is --H; R.sup.6 is --C.sub.1-C.sub.3 alkyl; R.sup.7 is
--C.sub.1-C.sub.5 alkyl; R.sup.8 is --C.sub.1-C.sub.3 alkoxy;
R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl; R.sup.10 is phenyl; and
Z is --O-- or --NH-- and R.sup.11 is as defined herein, preferably
hydrogen; or a pharmaceutically acceptable salt form thereof.
[0368] Auristatins of the formula D.sub.E or D.sub.F include those
wherein R.sup.3, R.sup.4 and R.sup.7 are independently isopropyl or
sec-butyl and R.sup.5 is --H. In an exemplary embodiment, R.sup.3
and R.sup.4 are each isopropyl, R.sup.5 is H, and R.sup.7 is
sec-butyl. The remainder of the substituents are as defined
herein.
[0369] Auristatins of the formula D.sub.E or D.sub.F include those
wherein R.sup.2 and R.sup.6 are each methyl, and R.sup.9 is H. The
remainder of the substituents are as defined herein.
[0370] Auristatins of the formula D.sub.E or D.sub.E include those
wherein each occurrence of R.sup.8 is --OCH.sub.3. The remainder of
the substituents are as defined herein.
[0371] Auristatins of the formula D.sub.E or D.sub.E include those
wherein R.sup.3 and R.sup.4 are each isopropyl, R.sup.2 and R.sup.6
are each methyl, R.sup.5 is H, R.sup.7 is sec-butyl, each
occurrence of R.sup.8 is --OCH.sub.3, and R.sup.9 is H. The
remainder of the substituents are as defined herein.
[0372] Auristatins of the formula D.sub.F include those wherein Z
is --O-- or --NH--. The remainder of the substituents are as
defined herein.
[0373] Auristatins of the formula D.sub.E include those wherein
R.sup.10 is aryl. The remainder of the substituents are as defined
herein.
[0374] Auristatins of the formula D.sub.E include those wherein
R.sup.10 is -phenyl. The remainder of the substituents are as
defined herein.
[0375] Auristatins of the formula D.sub.E include those wherein Z
is --O--, and R.sup.11 is H, methyl or t-butyl. The remainder of
the substituents are as defined herein.
[0376] Auristatins of the formula D.sub.E include those wherein,
when Z is --NH--, R.sup.11 is
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--N(R.sup.16).sub.2, and R.sup.16 is
--C.sub.1-C.sub.8 alkyl or --(CH.sub.2).sub.n--COOH. The remainder
of the substituents are as defined herein.
[0377] Auristatins of the formula D.sub.E include those wherein
when Z is --NH--, R.sup.11 is
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2, wherein R.sup.15 is
--(CH.sub.2).sub.n--SO.sub.3H. The remainder of the substituents
are as defined herein.
[0378] In preferred embodiments, when D is an auristatin of formula
D.sub.E, w is an integer ranging from 1 to 12, preferably 2 to 12,
y is 1 or 2, and a is preferably 1.
[0379] In some embodiments, wherein D is an auristatin of formula
D.sub.F, a is 1 and w and y are 0.
[0380] Illustrative Drug units (-D) include the drug units having
the following structures:
##STR00029## ##STR00030## ##STR00031##
or pharmaceutically acceptable salts or solvates thereof.
[0381] In one aspect, hydrophilic groups, such as but not limited
to triethylene glycol esters (TEG) can be attached to the Drug Unit
at R.sup.11. Without being bound by theory, the hydrophilic groups
assist in the internalization and non-agglomeration of the Drug
Unit.
[0382] In some embodiments, the Drug unit is not TZT-1027. In some
embodiments, the Drug unit is not auristatin E, dolastatin 10, or
auristatin PE.
[0383] Exemplary antibody-drug conjugate compounds have the
following structures wherein "L" or "mAb-s-" represents an 24P4C12
MAb designated Ha5-1(5)2.1 set forth herein:
##STR00032## ##STR00033##
[0384] or pharmaceutically acceptable salt thereof.
[0385] In some embodiments, the Drug Unit is a calicheamicin,
camptothecin, a maytansinoid, or an anthracycline. In some
embodiments the drug is a taxane, a topoisomerase inhibitor, a
vinca alkaloid, or the like.
[0386] In some typical embodiments, suitable cytotoxic agents
include, for example, DNA minor groove binders (e.g., enediynes and
lexitropsins, a CBI compound; see also U.S. Pat. No. 6,130,237),
duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins,
and vinca alkaloids. Other cytotoxic agents include, for example,
CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin,
cyanomorpholino-doxorubicin, echinomycin, combretastatin,
netropsin, epothilone A and B, estramustine, cryptophysins,
cemadotin, maytansinoids, discodermolide, eleutherobin, and
mitoxantrone.
[0387] In some embodiments, the Drug is an anti-tubulin agent.
Examples of anti-tubulin agents include, auristatins, taxanes
(e.g., Taxol.RTM. (paclitaxel), Taxotere.RTM. (docetaxel)), T67
(Tularik) and vinca alkyloids (e.g., vincristine, vinblastine,
vindesine, and vinorelbine). Other antitubulin agents include, for
example, baccatin derivatives, taxane analogs (e.g., epothilone A
and B), nocodazole, colchicine and colcimid, estramustine,
cryptophycins, cemadotin, maytansinoids, combretastatins,
discodermolide, and eleutherobin.
[0388] In certain embodiments, the cytotoxic agent is a
maytansinoid, another group of anti-tubulin agents. For example, in
specific embodiments, the maytansinoid is maytansine or DM-1
(ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.
52:127-131).
[0389] In certain embodiments, the cytotoxic or cytostatic agent is
a dolastatin. In certain embodiments, the cytotoxic or cytostatic
agent is of the auristatin class. Thus, in a specific embodiment,
the cytotoxic or cytostatic agent is MMAE (Formula XI). In another
specific embodiment, the cytotoxic or cytostatic agent is AFP
(Formula XVI).
##STR00034##
[0390] In certain embodiments, the cytotoxic or cytostatic agent is
a compound of formulas XII-XXI or pharmaceutically acceptable salt
thereof:
##STR00035## ##STR00036##
X.) DRUG LOADING
[0391] Drug loading is represented by p and is the average number
of Drug moieties per antibody in a molecule. Drug loading may range
from 1 to 20 drug moieties (D) per antibody. ADCs of the invention
include collections of antibodies conjugated with a range of drug
moieties, from 1 to 20. The average number of drug moieties per
antibody in preparations of ADC from conjugation reactions may be
characterized by conventional means such as mass spectroscopy and,
ELISA assay. The quantitative distribution of ADC in terms of p may
also be determined. In some instances, separation, purification,
and characterization of homogeneous ADC where p is a certain value
from ADC with other drug loadings may be achieved by means such as
electrophoresis.
[0392] For some antibody-drug conjugates, p may be limited by the
number of attachment sites on the antibody. For example, where the
attachment is a cysteine thiol, as in the exemplary embodiments
above, an antibody may have only one or several cysteine thiol
groups, or may have only one or several sufficiently reactive thiol
groups through which a linker may be attached. In certain
embodiments, higher drug loading, e.g. p>5, may cause
aggregation, insolubility, toxicity, or loss of cellular
permeability of certain antibody-drug conjugates. In certain
embodiments, the drug loading for an ADC of the invention ranges
from 1 to about 8; from about 2 to about 6; from about 3 to about
5; from about 3 to about 4; from about 3.1 to about 3.9; from about
3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to
about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about
3.7. Indeed, it has been shown that for certain ADCs, the optimal
ratio of drug moieties per antibody may be less than 8, and may be
about 2 to about 5. See US 2005-0238649 A1 (herein incorporated by
reference in its entirety).
[0393] In certain embodiments, fewer than the theoretical maximum
of drug moieties are conjugated to an antibody during a conjugation
reaction. An antibody may contain, for example, lysine residues
that do not react with the drug-linker intermediate or linker
reagent, as discussed below. Generally, antibodies do not contain
many free and reactive cysteine thiol groups which may be linked to
a drug moiety; indeed most cysteine thiol residues in antibodies
exist as disulfide bridges. In certain embodiments, an antibody may
be reduced with a reducing agent such as dithiothreitol (DTT) or
tricarbonylethylphosphine (TCEP), under partial or total reducing
conditions, to generate reactive cysteine thiol groups. In certain
embodiments, an antibody is subjected to denaturing conditions to
reveal reactive nucleophilic groups such as lysine or cysteine.
[0394] The loading (drug/antibody ratio) of an ADC may be
controlled in different ways, e.g., by: (i) limiting the molar
excess of drug-linker intermediate or linker reagent relative to
antibody, (ii) limiting the conjugation reaction time or
temperature, (iii) partial or limiting reductive conditions for
cysteine thiol modification, (iv) engineering by recombinant
techniques the amino acid sequence of the antibody such that the
number and position of cysteine residues is modified for control of
the number and/or position of linker-drug attachments (such as
thioMab or thioFab prepared as disclosed herein and in
WO2006/034488 (herein incorporated by reference in its
entirety)).
[0395] It is to be understood that where more than one nucleophilic
group reacts with a drug-linker intermediate or linker reagent
followed by drug moiety reagent, then the resulting product is a
mixture of ADC compounds with a distribution of one or more drug
moieties attached to an antibody. The average number of drugs per
antibody may be calculated from the mixture by a dual ELISA
antibody assay, which is specific for antibody and specific for the
drug. Individual ADC molecules may be identified in the mixture by
mass spectroscopy and separated by HPLC, e.g. hydrophobic
interaction chromatography (see, e.g., Hamblett, K. J., et al.
"Effect of drug loading on the pharmacology, pharmacokinetics, and
toxicity of an anti-CD30 antibody-drug conjugate," Abstract No.
624, American Association for Cancer Research, 2004 Annual Meeting,
Mar. 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004;
Alley, S. C., et al. "Controlling the location of drug attachment
in antibody-drug conjugates," Abstract No. 627, American
Association for Cancer Research, 2004 Annual Meeting, Mar. 27-31,
2004, Proceedings of the AACR, Volume 45, March 2004). In certain
embodiments, a homogeneous ADC with a single loading value may be
isolated from the conjugation mixture by electrophoresis or
chromatography.
XI.) METHODS OF DETERMINING CYTOTOXIC EFFECT OF ADCS
[0396] Methods of determining whether a Drug or Antibody-Drug
conjugate exerts a cytostatic and/or cytotoxic effect on a cell are
known. Generally, the cytotoxic or cytostatic activity of a
Antibody Drug conjugate can be measured by: exposing mammalian
cells expressing a target protein of the Antibody Drug conjugate in
a cell culture medium; culturing the cells for a period from about
6 hours to about 5 days; and measuring cell viability. Cell-based
in vitro assays can be used to measure viability (proliferation),
cytotoxicity, and induction of apoptosis (caspase activation) of
the Antibody Drug conjugate.
[0397] For determining whether a Antibody Drug conjugate exerts a
cytostatic effect, a thymidine incorporation assay may be used. For
example, cancer cells expressing a target antigen at a density of
5,000 cells/well of a 96-well plated can be cultured for a 72-hour
period and exposed to 0.5 .mu.Ci of 3H-thymidine during the final 8
hours of the 72-hour period. The incorporation of .sup.3H-thymidine
into cells of the culture is measured in the presence and absence
of the Antibody Drug conjugate.
[0398] For determining cytotoxicity, necrosis or apoptosis
(programmed cell death) can be measured. Necrosis is typically
accompanied by increased permeability of the plasma membrane;
swelling of the cell, and rupture of the plasma membrane. Apoptosis
is typically characterized by membrane blebbing, condensation of
cytoplasm, and the activation of endogenous endonucleases.
Determination of any of these effects on cancer cells indicates
that a Antibody Drug conjugate is useful in the treatment of
cancers.
[0399] Cell viability can be measured by determining in a cell the
uptake of a dye such as neutral red, trypan blue, or ALAMAR.TM.
blue (see, e.g., Page et al., 1993, Intl. J. Oncology 3:473-476).
In such an assay, the cells are incubated in media containing the
dye, the cells are washed, and the remaining dye, reflecting
cellular uptake of the dye, is measured spectrophotometrically. The
protein-binding dye sulforhodamine B (SRB) can also be used to
measure cytoxicity (Skehan et al., 1990, J. Natl. Cancer Inst.
82:1107-12).
[0400] Alternatively, a tetrazolium salt, such as MTT, is used in a
quantitative colorimetric assay for mammalian cell survival and
proliferation by detecting living, but not dead, cells (see, e.g.,
Mosmann, 1983, J. Immunol. Methods 65:55-63).
[0401] Apoptosis can be quantitated by measuring, for example, DNA
fragmentation. Commercial photometric methods for the quantitative
in vitro determination of DNA fragmentation are available. Examples
of such assays, including TUNEL (which detects incorporation of
labeled nucleotides in fragmented DNA) and ELISA-based assays, are
described in Biochemica, 1999, no. 2, pp. 34-37 (Roche Molecular
Biochemicals).
[0402] Apoptosis can also be determined by measuring morphological
changes in a cell. For example, as with necrosis, loss of plasma
membrane integrity can be determined by measuring uptake of certain
dyes (e.g., a fluorescent dye such as, for example, acridine orange
or ethidium bromide). A method for measuring apoptotic cell number
has been described by Duke and Cohen, Current Protocols in
Immunology (Coligan et al. eds., 1992, pp. 3.17.1-3.17.16). Cells
also can be labeled with a DNA dye (e.g., acridine orange, ethidium
bromide, or propidium iodide) and the cells observed for chromatin
condensation and margination along the inner nuclear membrane.
Other morphological changes that can be measured to determine
apoptosis include, e.g., cytoplasmic condensation, increased
membrane blebbing, and cellular shrinkage.
[0403] The presence of apoptotic cells can be measured in both the
attached and "floating" compartments of the cultures. For example,
both compartments can be collected by removing the supernatant,
trypsinizing the attached cells, combining the preparations
following a centrifugation wash step (e.g., 10 minutes at 2000
rpm), and detecting apoptosis (e.g., by measuring DNA
fragmentation). (See, e.g., Piazza et al., 1995, Cancer Research
55:3110-16).
[0404] In vivo, the effect of a 24P4C12 therapeutic composition can
be evaluated in a suitable animal model. For example, xenogenic
cancer models can be used, wherein cancer explants or passaged
xenograft tissues are introduced into immune compromised animals,
such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3:
402-408). For example, PCT Patent Application WO98/16628 and U.S.
Pat. No. 6,107,540 describe various xenograft models of human
prostate cancer capable of recapitulating the development of
primary tumors, micrometastasis, and the formation of osteoblastic
metastases characteristic of late stage disease. Efficacy can be
predicted using assays that measure inhibition of tumor formation,
tumor regression or metastasis, and the like.
[0405] In vivo assays that evaluate the promotion of apoptosis are
useful in evaluating therapeutic compositions. In one embodiment,
xenografts from tumor bearing mice treated with the therapeutic
composition can be examined for the presence of apoptotic foci and
compared to untreated control xenograft-bearing mice. The extent to
which apoptotic foci are found in the tumors of the treated mice
provides an indication of the therapeutic efficacy of the
composition.
[0406] The therapeutic compositions used in the practice of the
foregoing methods can be formulated into pharmaceutical
compositions comprising a carrier suitable for the desired delivery
method. Suitable carriers include any material that when combined
with the therapeutic composition retains the anti-tumor function of
the therapeutic composition and is generally non-reactive with the
patient's immune system. Examples include, but are not limited to,
any of a number of standard pharmaceutical carriers such as sterile
phosphate buffered saline solutions, bacteriostatic water, and the
like (see, generally, Remington's Pharmaceutical Sciences 16th
Edition, A. Osal., Ed., 1980).
[0407] Therapeutic formulations can be solubilized and administered
via any route capable of delivering the therapeutic composition to
the tumor site. Potentially effective routes of administration
include, but are not limited to, intravenous, parenteral,
intraperitoneal, intramuscular, intratumor, intradermal,
intraorgan, orthotopic, and the like. A preferred formulation for
intravenous injection comprises the therapeutic composition in a
solution of preserved bacteriostatic water, sterile unpreserved
water, and/or diluted in polyvinylchloride or polyethylene bags
containing 0.9% sterile Sodium Chloride for Injection, USP.
Therapeutic protein preparations can be lyophilized and stored as
sterile powders, preferably under vacuum, and then reconstituted in
bacteriostatic water (containing for example, benzyl alcohol
preservative) or in sterile water prior to injection.
[0408] Dosages and administration protocols for the treatment of
cancers using the foregoing methods will vary with the method and
the target cancer, and will generally depend on a number of other
factors appreciated in the art.
XII.) TREATMENT OF CANCER(S) EXPRESSING 24P4C12
[0409] The identification of 24P4C12 as a protein that is normally
expressed in a restricted set of tissues, but which is also
expressed in cancers such as those listed in Table I, opens a
number of therapeutic approaches to the treatment of such
cancers.
[0410] Of note, targeted antitumor therapies have been useful even
when the targeted protein is expressed on normal tissues, even
vital normal organ tissues. A vital organ is one that is necessary
to sustain life, such as the heart or colon. A non-vital organ is
one that can be removed whereupon the individual is still able to
survive. Examples of non-vital organs are ovary, breast, and
prostate.
[0411] Expression of a target protein in normal tissue, even vital
normal tissue, does not defeat the utility of a targeting agent for
the protein as a therapeutic for certain tumors in which the
protein is also overexpressed. For example, expression in vital
organs is not in and of itself detrimental. In addition, organs
regarded as dispensible, such as the prostate and ovary, can be
removed without affecting mortality. Finally, some vital organs are
not affected by normal organ expression because of an
immunoprivilege. Immunoprivileged organs are organs that are
protected from blood by a blood-organ barrier and thus are not
accessible to immunotherapy. Examples of immunoprivileged organs
are the brain and testis.
[0412] Accordingly, therapeutic approaches that inhibit the
activity of a 24P4C12 protein are useful for patients suffering
from a cancer that expresses 24P4C12. These therapeutic approaches
generally fall into three classes. The first class modulates
24P4C12 function as it relates to tumor cell growth leading to
inhibition or retardation of tumor cell growth or inducing its
killing. The second class comprises various methods for inhibiting
the binding or association of a 24P4C12 protein with its binding
partner or with other proteins. The third class comprises a variety
of methods for inhibiting the transcription of a 24P4C12 gene or
translation of 24P4C12 mRNA.
[0413] Accordingly, Cancer patients can be evaluated for the
presence and level of 24P4C12 expression, preferably using
immunohistochemical assessments of tumor tissue, quantitative
24P4C12 imaging, or other techniques that reliably indicate the
presence and degree of 24P4C12 expression. Immunohistochemical
analysis of tumor biopsies or surgical specimens is preferred for
this purpose. Methods for immunohistochemical analysis of tumor
tissues are well known in the art.
XIII) 24P4C12 AS A TARGET FOR ANTIBODY-BASED THERAPY
[0414] 24P4C12 is an attractive target for antibody-based
therapeutic strategies. A number of antibody strategies are known
in the art for targeting both extracellular and intracellular
molecules (see, e.g., complement and ADCC mediated killing as well
as the use of intrabodies). Because 24P4C12 is expressed by cancer
cells of various lineages relative to corresponding normal cells,
systemic administration of 24P4C12-immunoreactive compositions are
prepared that exhibit excellent sensitivity without toxic,
non-specific and/or non-target effects caused by binding of the
immunoreactive composition to non-target organs and tissues.
Antibodies specifically reactive with domains of 24P4C12 are useful
to treat 24P4C12-expressing cancers systemically, preferably as
antibody drug conjugates (i.e. ADCs) wherein the conjugate is with
a toxin or therapeutic agent.
[0415] Those skilled in the art understand that antibodies can be
used to specifically target and bind immunogenic molecules such as
an immunogenic region of a 24P4C12 sequence shown in FIG. 1. In
addition, skilled artisans understand that it is routine to
conjugate antibodies to cytotoxic agents (see, e.g., Slevers et al.
Blood 93:11 3678-3684 (Jun. 1, 1999)). When cytotoxic and/or
therapeutic agents are delivered directly to cells, such as by
conjugating them to antibodies specific for a molecule expressed by
that cell (e.g. 24P4C12), the cytotoxic agent will exert its known
biological effect (i.e. cytotoxicity) on those cells.
[0416] A wide variety of compositions and methods for using
antibody-cytotoxic agent conjugates to kill cells are known in the
art. In the context of cancers, typical methods entail
administering to an mammal having a tumor a biologically effective
amount of a conjugate comprising a selected cytotoxic and/or
therapeutic agent linked to a targeting agent (e.g. a 24P4C12 MAb,
preferably Ha5-1(5)2.1) that binds to an antigen (e.g. 24P4C12)
expressed, accessible to binding or localized on the cell surfaces.
A typical embodiment is a method of delivering a cytotoxic and/or
therapeutic agent to a cell expressing 24P4C12, comprising
conjugating the cytotoxic agent to an antibody that
immunospecifically binds to a 24P4C12 epitope, and, exposing the
cell to the antibody drug conjugate (ADC). Another illustrative
embodiment is a method of treating an individual suspected of
suffering from metastasized cancer, comprising a step of
administering parenterally to said individual a pharmaceutical
composition comprising a therapeutically effective amount of an
antibody conjugated to a cytotoxic and/or therapeutic agent.
[0417] Cancer immunotherapy using 24P4C12 antibodies can be done in
accordance with various approaches that have been successfully
employed in the treatment of other types of cancer, including but
not limited to colon cancer (Arlen et al., 1998, Crit. Rev.
Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood
90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric
cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell
lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor
Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res.
20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res.
54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), and
breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127).
Some therapeutic approaches involve conjugation of naked antibody
to a toxin or radioisotope, such as the conjugation of Y.sup.91 or
I.sup.131 to anti-CD20 antibodies (e.g., Zevalin.TM., IDEC
Pharmaceuticals Corp. or Bexxar.TM., Coulter Pharmaceuticals)
respectively, while others involve co-administration of antibodies
and other therapeutic agents, such as Herceptin.TM. (trastuzu MAb)
with paclitaxel (Genentech, Inc.). In a preferred embodiment, the
antibodies will be conjugated a cytotoxic agent, supra, preferably
an aurastatin derivative designated MMAE (Seattle Genetics).
[0418] Although 24P4C12 antibody therapy is useful for all stages
of cancer, antibody therapy can be particularly appropriate in
advanced or metastatic cancers. Treatment with the antibody therapy
of the invention is indicated for patients who have received one or
more rounds of chemotherapy. Alternatively, antibody therapy of the
invention is combined with a chemotherapeutic or radiation regimen
for patients who have not received chemotherapeutic treatment.
Additionally, antibody therapy can enable the use of reduced
dosages of concomitant chemotherapy, particularly for patients who
do not tolerate the toxicity of the chemotherapeutic agent very
well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al.
(International J. of Onco. 9:217-224, 1996), and Hancock et al.
(Cancer Res. 51:4575-4580, 1991) describe the use of various
antibodies together with chemotherapeutic agents.
[0419] 24P4C12 monoclonal antibodies that treat colon and other
cancers (Table I) include those that initiate a potent immune
response against the tumor or those that are directly cytotoxic. In
this regard, 24P4C12 monoclonal antibodies (MAbs) can elicit tumor
cell lysis by either complement-mediated or antibody-dependent cell
cytotoxicity (ADCC) mechanisms, both of which require an intact Fc
portion of the immunoglobulin molecule for interaction with
effector cell Fc receptor sites on complement proteins. In
addition, 24P4C12 MAbs that exert a direct biological effect on
tumor growth are useful to treat cancers that express 24P4C12.
Mechanisms by which directly cytotoxic MAbs act include: inhibition
of cell growth, modulation of cellular differentiation, modulation
of tumor angiogenesis factor profiles, and the induction of
apoptosis. The mechanism(s) by which a particular 24P4C12 MAb
exerts an anti-tumor effect is evaluated using any number of in
vitro assays that evaluate cell death such as ADCC,
complement-mediated cell lysis, and so forth, as is generally known
in the art.
[0420] Accordingly, preferred monoclonal antibodies used in the
therapeutic methods of the invention are those that are either
fully human and that bind specifically to the target 24P4C12
antigen with high affinity.
XIV.) 24P4C12 ADC COCKTAILS
[0421] Therapeutic methods of the invention contemplate the
administration of single 24P4C12 ADCs as well as combinations, or
cocktails, of different MAbs (i.e. 24P4C12 MAbs or Mabs that bind
another protein). Such MAb cocktails can have certain advantages
inasmuch as they contain MAbs that target different epitopes,
exploit different effector mechanisms or combine directly cytotoxic
MAbs with MAbs that rely on immune effector functionality. Such
MAbs in combination can exhibit synergistic therapeutic effects. In
addition, 24P4C12 MAbs can be administered concomitantly with other
therapeutic modalities, including but not limited to various
chemotherapeutic and biologic agents, androgen-blockers, immune
modulators (e.g., IL-2, GM-CSF), surgery or radiation. In a
preferred embodiment, the 24P4C12 MAbs are administered in
conjugated form.
[0422] 24P4C12 ADC formulations are administered via any route
capable of delivering the antibodies to a tumor cell. Routes of
administration include, but are not limited to, intravenous,
intraperitoneal, intramuscular, intratumor, intradermal, and the
like. Treatment generally involves repeated administration of the
24P4C12 ADC preparation, via an acceptable route of administration
such as intravenous injection (IV), typically at a dose in the
range, including but not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg
body weight. In general, doses in the range of 10-1000 mg MAb per
week are effective and well tolerated.
[0423] Based on clinical experience with the Herceptin.RTM.
(Trastuzumab) in the treatment of metastatic breast cancer, an
initial loading dose of approximately 4 mg/kg patient body weight
IV, followed by weekly doses of about 2 mg/kg IV of the MAb
preparation represents an acceptable dosing regimen. Preferably,
the initial loading dose is administered as a 90-minute or longer
infusion. The periodic maintenance dose is administered as a 30
minute or longer infusion, provided the initial dose was well
tolerated. As appreciated by those of skill in the art, various
factors can influence the ideal dose regimen in a particular case.
Such factors include, for example, the binding affinity and half
life of the MAbs used, the degree of 24P4C12 expression in the
patient, the extent of circulating shed 24P4C12 antigen, the
desired steady-state antibody concentration level, frequency of
treatment, and the influence of chemotherapeutic or other agents
used in combination with the treatment method of the invention, as
well as the health status of a particular patient.
[0424] Optionally, patients should be evaluated for the levels of
24P4C12 in a given sample (e.g. the levels of circulating 24P4C12
antigen and/or 24P4C12 expressing cells) in order to assist in the
determination of the most effective dosing regimen, etc. Such
evaluations are also used for monitoring purposes throughout
therapy, and are useful to gauge therapeutic success in combination
with the evaluation of other parameters (for example, urine
cytology and/or ImmunoCyt levels in bladder cancer therapy, or by
analogy, serum PSA levels in prostate cancer therapy).
[0425] An object of the present invention is to provide 24P4C12
ADCs, which inhibit or retard the growth of tumor cells expressing
24P4C12. A further object of this invention is to provide methods
to inhibit angiogenesis and other biological functions and thereby
reduce tumor growth in mammals, preferably humans, using such
24P4C12 ADCs, and in particular using such 24P4C12 ADCs combined
with other drugs or immunologically active treatments.
XV.) COMBINATION THERAPY
[0426] In one embodiment, there is synergy when tumors, including
human tumors, are treated with 24P4C12 ADCs in conjunction with
chemotherapeutic agents or radiation or combinations thereof. In
other words, the inhibition of tumor growth by a 24P4C12 ADC is
enhanced more than expected when combined with chemotherapeutic
agents or radiation or combinations thereof. Synergy may be shown,
for example, by greater inhibition of tumor growth with combined
treatment than would be expected from a treatment of only 24P4C12
ADC or the additive effect of treatment with a 24P4C12 ADC and a
chemotherapeutic agent or radiation. Preferably, synergy is
demonstrated by remission of the cancer where remission is not
expected from treatment either from a 24P4C12 ADC or with treatment
using an additive combination of a 24P4C12 ADC and a
chemotherapeutic agent or radiation.
[0427] The method for inhibiting growth of tumor cells using a
24P4C12 ADC and a combination of chemotherapy or radiation or both
comprises administering the 24P4C12 ADC before, during, or after
commencing chemotherapy or radiation therapy, as well as any
combination thereof (i.e. before and during, before and after,
during and after, or before, during, and after commencing the
chemotherapy and/or radiation therapy). For example, the 24P4C12
ADC is typically administered between 1 and 60 days, preferably
between 3 and 40 days, more preferably between 5 and 12 days before
commencing radiation therapy and/or chemotherapy. However,
depending on the treatment protocol and the specific patient needs,
the method is performed in a manner that will provide the most
efficacious treatment and ultimately prolong the life of the
patient.
[0428] The administration of chemotherapeutic agents can be
accomplished in a variety of ways including systemically by the
parenteral and enteral routes. In one embodiment, the 24P4C12 ADCs
and the chemotherapeutic agent are administered as separate
molecules. Particular examples of chemotherapeutic agents or
chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin,
mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide,
carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin),
daunorubicin, procarbazine, mitomycin, cytarabine, etoposide,
methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin,
paclitaxel (taxol), docetaxel (taxotere), aldesleukin,
asparaginase, busulfan, carboplatin, cladribine, dacarbazine,
floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon
alpha, leuprolide, megestrol, melphalan, mercaptopurine,
plicamycin, mitotane, pegaspargase, pentostatin, pipobroman,
plicamycin, streptozocin, tamoxifen, teniposide, testolactone,
thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil,
taxol and combinations thereof.
[0429] The source of radiation, used in combination with a 24P4C12
ADC, can be either external or internal to the patient being
treated. When the source is external to the patient, the therapy is
known as external beam radiation therapy (EBRT). When the source of
radiation is internal to the patient, the treatment is called
brachytherapy (BT).
[0430] The above described therapeutic regimens may be further
combined with additional cancer treating agents and/or regimes, for
example additional chemotherapy, cancer vaccines, signal
transduction inhibitors, agents useful in treating abnormal cell
growth or cancer, antibodies (e.g. Anti-CTLA-4 antibodies as
described in WO/2005/092380 (Pfizer)) or other ligands that inhibit
tumor growth by binding to IGF-1R, and cytokines.
[0431] When the mammal is subjected to additional chemotherapy,
chemotherapeutic agents described above may be used. Additionally,
growth factor inhibitors, biological response modifiers,
anti-hormonal therapy, selective estrogen receptor modulators
(SERMs), angiogenesis inhibitors, and anti-androgens may be used.
For example, anti-hormones, for example anti-estrogens such as
Nolvadex (tamoxifen) or, anti-androgens such as Casodex
(4'-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3-'-(tri-
fluoromethyl)propionanilide) may be used.
[0432] The above therapeutic approaches can be combined with any
one of a wide variety of surgical, chemotherapy or radiation
therapy regimens. The therapeutic approaches of the invention can
enable the use of reduced dosages of chemotherapy (or other
therapies) and/or less frequent administration, an advantage for
all patients and particularly for those that do not tolerate the
toxicity of the chemotherapeutic agent well.
XVI.) KITS/ARTICLES OF MANUFACTURE
[0433] For use in the laboratory, prognostic, prophylactic,
diagnostic and therapeutic applications described herein, kits are
within the scope of the invention. Such kits can comprise a
carrier, package, or container that is compartmentalized to receive
one or more containers such as vials, tubes, and the like, each of
the container(s) comprising one of the separate elements to be used
in the method, along with a label or insert comprising instructions
for use, such as a use described herein. For example, the
container(s) can comprise an antibody that is or can be detectably
labeled. Kits can comprise a container comprising a Drug Unit. The
kit can include all or part of the amino acid sequences in FIG. 2,
or FIG. 3 or analogs thereof, or a nucleic acid molecule that
encodes such amino acid sequences.
[0434] The kit of the invention will typically comprise the
container described above and one or more other containers
associated therewith that comprise materials desirable from a
commercial and user standpoint, including buffers, diluents,
filters, needles, syringes; carrier, package, container, vial
and/or tube labels listing contents and/or instructions for use,
and package inserts with instructions for use.
[0435] A label can be present on or with the container to indicate
that the composition is used for a specific therapy or
non-therapeutic application, such as a prognostic, prophylactic,
diagnostic or laboratory application, and can also indicate
directions for either in vivo or in vitro use, such as those
described herein. Directions and or other information can also be
included on an insert(s) or label(s) which is included with or on
the kit. The label can be on or associated with the container. A
label a can be on a container when letters, numbers or other
characters forming the label are molded or etched into the
container itself; a label can be associated with a container when
it is present within a receptacle or carrier that also holds the
container, e.g., as a package insert. The label can indicate that
the composition is used for diagnosing, treating, prophylaxing or
prognosing a condition, such as a cancer of a tissue set forth in
Table I.
[0436] The terms "kit" and "article of manufacture" can be used as
synonyms.
[0437] In another embodiment of the invention, an article(s) of
manufacture containing compositions, such as antibody(s), or
antibody drug conjugates (ADCs) e.g., materials useful for the
diagnosis, prognosis, prophylaxis and/or treatment of cancers of
tissues such as those set forth in Table I is provided. The article
of manufacture typically comprises at least one container and at
least one label. Suitable containers include, for example, bottles,
vials, syringes, and test tubes. The containers can be formed from
a variety of materials such as glass, metal or plastic. The
container can hold amino acid sequence(s), small molecule(s),
nucleic acid sequence(s), cell population(s) and/or antibody(s). In
another embodiment a container comprises an antibody, binding
fragment thereof or specific binding protein for use in evaluating
protein expression of 24P4C12 in cells and tissues, or for relevant
laboratory, prognostic, diagnostic, prophylactic and therapeutic
purposes; indications and/or directions for such uses can be
included on or with such container, as can reagents and other
compositions or tools used for these purposes.
[0438] The container can alternatively hold a composition that is
effective for treating, diagnosis, prognosing or prophylaxing a
condition and can have a sterile access port (for example the
container can be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The active
agents in the composition can be an antibody capable of
specifically binding 24P4C12 or an antibody drug conjugate
specifically binding to 24P4C12.
[0439] The article of manufacture can further comprise a second
container comprising a pharmaceutically-acceptable buffer, such as
phosphate-buffered saline, Ringer's solution and/or dextrose
solution. It can further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, stirrers, needles, syringes, and/or package inserts with
indications and/or instructions for use.
EXAMPLES
[0440] Various aspects of the invention are further described and
illustrated by way of the several examples that follow, none of
which is intended to limit the scope of the invention.
Example 1
The 24P4C12 Antigen
[0441] The novel 24P4C12 gene sequence was discovered using
Suppression Subtractive Hybridization (SSH) methods known in the
art. The 24P4C12 SSH sequence of 160 bp was identified from a LAPC
xenograft SSH experiment using standard methods. A full length cDNA
clone for 24P4C12 was isolated from a LAPC-9 AD minus benign
prostatic hyperplasia experiment. The cDNA is 2587 bp in length and
encodes a 710 amino acid ORF (See, FIG. 1A). For further reference
see, U.S. Pat. No. 6,943,235 (Agensys, Inc., Santa Monica, Calif.),
U.S. Pat. No. 7,220,823 (Agensys, Inc., Santa Monica, Calif.), U.S.
Pat. No. 7,227,008 (Agensys, Inc., Santa Monica, Calif.), and U.S.
Pat. No. 7,244,827 (Agensys, Inc., Santa Monica, Calif.). For
exemplary embodiments of the 24P4C12 antigen and variants thereof,
see FIG. 1.
Example 2
Generation of 24P4C12 Monoclonal Antibodies (MAbs)
[0442] In one embodiment, therapeutic Monoclonal Antibodies
("MAbs") to 24P4C12 and 24P4C12 variants comprise those that react
with epitopes specific for each protein or specific to sequences in
common between the variants that would bind, internalize, disrupt
or modulate the biological function of 24P4C12 or 24P4C12 variants,
for example, those that would disrupt the interaction with ligands,
substrates, and binding partners. Immunogens for generation of such
MAbs include those designed to encode or contain the extracellular
domains or the entire 24P4C12 protein sequence, regions predicted
to contain functional motifs, and regions of the 24P4C12 protein
variants predicted to be antigenic from computer analysis of the
amino acid sequence. Immunogens include peptides and recombinant
proteins such as tag5-24P4C12, a purified mammalian cell derived
His tagged protein. In addition, cells engineered to express high
levels of 24P4C12, such as RAT1-24P4C12 or 300.19-24P4C12, are used
to immunize mice.
[0443] MAbs to 24P4C12 were generated using XenoMouse
Technology.RTM. (Amgem Fremont) wherein the murine heavy and kappa
light chain loci have been inactivated and a majority of the human
heavy and kappa light chain immunoglobulin loci have been inserted.
The MAb designated Ha5-1(5)2.1 was generated from immunization of
human .gamma.2 producing XenoMice with RAT(E)-24P4C12 cells.
[0444] The 24P4C12 MAb Ha5-1(5)2.1 specifically binds to
recombinant 24P4C12 expressing cells (PC3-24P4C12) and multiple
cancer cell lines expressing 24P4C12.
[0445] The hybridoma producing an antibody designated Ha5-1(5)2.1
was sent (via Federal Express) to the American Type Culture
Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108 on 8 Aug.
2007 and assigned Accession numbers PTA-8602.
[0446] DNA coding sequences for 24P4C12 MAb Ha5-1(5)2.1 was
determined after isolating mRNA from the respective hybridoma cells
with Trizol reagent (Life Technologies, Gibco BRL).
[0447] Anti-24P4C12 Ha5-1(5)2.1 heavy and light chain variable
nucleic acid sequences were sequenced from the hybridoma cells
using the following protocol. Ha5-1(5)2.1 secreting hybridoma cells
were lysed with Trizol reagent (Life Technologies, Gibco BRL).
Total RNA was purified and quantified. First strand cDNAs was
generated from total RNA with oligo (dT)12-18 priming using the
Gibco-BRL Superscript Preamplification system. First strand cDNA
was amplified using human immunoglobulin variable heavy chain
primers, and human immunoglobulin variable light chain primers. PCR
products were sequenced and the variable heavy and light chain
regions determined.
[0448] The nucleic acid and amino acid sequences of the variable
heavy and light chain regions are listed in FIG. 2 and FIG. 3.
Alignment of Ha5-1(5)2.1 MAb to human Ig germline is set forth in
FIG. 4A-4B.
Example 3
Expression of Ha5-1(5)2.1 using Recombinant DNA Methods
[0449] To express Ha5-1(5)2.1 MAb recombinantly in transfected
cells, Ha5-1(5)2.1 MAb variable heavy and light chain sequences
were cloned upstream of the human heavy chain IgG2 and light chain
Ig.kappa. constant regions, respectively. The complete Ha5-1(5)2.1
MAb human heavy chain and light chain cassettes were cloned
downstream of the CMV promoter/enhancer in a cloning vector. A
polyadenylation site was included downstream of the MAb coding
sequence. The recombinant Ha5-1(5)2.1 MAb expressing constructs
were transfected into 293T, Cos and CHO cells. The Ha5-1(5)2.1 MAb
secreted from recombinant cells was evaluated for binding to cell
surface 24P4C12 by flow cytometry (FIG. 5). PC3-control and
PC3-24P4C12 cells were stained with Ha5-1(5)2.1 MAb from either
hybridoma or from CHO cells transfected with Ha5-1(5)2.1 heavy and
light chain vector constructs.
[0450] Binding was detected by flow cytometry. Results show that
the recombinantly expressed Ha5-1(5)2.1 expressed in CHO cells
binds 24P4C12 similarly to the Ha5-1(5)2.1 purified from hybridoma
(FIG. 5).
Example 4
Antibody Drug Conjugation of Ha5-1(5)2.1 MAb
[0451] The Ha5-1(5)2.1 Mab (FIG. 2) was conjugated to an auristatin
derivative designated MMAE (Formula XI) using a vc (Val-Cit) linker
described herein to create the antibody drug conjugate (ADC) of the
invention designated Ha5-1(5)2.1vcMMAE using the following
protocols. The conjugation of the vc (Val-Cit) linker to the MMAE
(Seattle Genetics, Seattle, Wash.) was completed using the general
method set forth in Table V to create the cytotoxic vcMMAE (see,
US/2006/0074008).
[0452] Next, the antibody drug conjugate (ADC) of the invention
designated H5-1(5)2.1vcMMAE was made using the following
protocols.
[0453] Briefly, a 10 mg/mL solution of the Ha5-1(5)2.1 MAb in 20 mM
histidine at pH 5.2 is added with a 15% volume of 0.5 M Tris at pH
8.8 to adjust the pH of the solution to 8.0-8.2. Then, EDTA and
sodium chloride are added to 5 mM and 250 mM final concentration,
respectively, in the reaction mixture. The MAb is then partially
reduced by adding 2.3 molar equivalents of TCEP (relative to moles
of MAb) and then stirred at 37.degree. C. for 3 hours. The
partially reduced MAb solution is then cooled to 22.degree. C. and
5.1 molar equivalents of vcMMAE (relative to moles of antibody) are
added as a 7 mg/mL solution in DMSO. The mixture is stirred for 30
minutes at 22.degree. C., then for 15 additional minutes following
the addition of 2 molar equivalents of N-acetylcysteine relative to
vcMMAE. Excess quenched vcMMAE and other reaction components are
removed by ultrafiltration/diafiltration of the antibody drug
conjugate (ADC) with 10 diavolumes of 20 mM histidine, pH 5.2.
[0454] The resulting antibody drug conjugate (ADC) is designated
Ha5-1(5)2.1vcMMAE and has the following formula:
##STR00037##
Wherein MAb is Ha5-1(5)2.1 (FIG. 2 and FIG. 3) and p is from 1 to
8. The p value of the antibody drug conjugate set forth in this
Example was about 3.6.
Example 5
Characterization of HA5-1(5)2.1vcMMAE
[0455] Antibody Drug Conjugates that bind 24P4C12 were generated
using the procedures set forth in the example entitled "Antibody
Drug Conjugation of Ha5-1(5)2.1 MAb" and were screened, identified,
and characterized using a combination of assays known in the
art.
[0456] A. Affinity Determination by FACS
[0457] Ha5-1(5)2.1vcMMAE was tested for its binding affinity to
24P4C12 endogenously expressed on LNCaP cells. Briefly, eleven (11)
dilutions of Ha5-1(5)2.1vcMMAE are incubated with LNCaP cells
(50,000 cells per well) overnight at 4.degree. C. at a final
concentration of 160 nM to 0.011 nM. At the end of the incubation,
cells are washed and incubated with anti-hIgG-PE detection antibody
for 45 min at 4.degree. C. After washing the unbound detection
antibodies, the cells are analyzed by FACS. Mean Florescence
Intensity (MFI) values were obtained as listed in (Table IV(A)).
MFI values were entered into Graphpad Prisim software and analyzed
using the one site binding (hyperbola) equation of Y=Bmax*X/(Kd+X)
to generate Ha5-1(5)2.1vcMMAE saturation curves shown in (Table
IV(B)). Bmax is the MFI value at maximal binding of
Ha5-1(5)2.1vcMMAE to 24P4C12; Kd is Ha5-1(5)2.1vcMMAE binding
affinity which is the concentration of Ha5-1(5)2.1vcMMAE required
to reach half-maximal binding.
[0458] The calculated affinity (Kd) of Ha5-1(5)2.1vcMMAE is 1.05 nM
on 24P4C12 endogenously expressed on the surface of LNCaP
cells.
Example 6
Cell Cytotoxicity Mediated by Ha5-1(5)2.1vcMMAE
[0459] The ability of Ha5-1(5)2.1-vcMMAE to mediate
24P4C12-dependent cytoxicity was evaluated in PC3 cells engineered
to express 24P4C12. PC3-Neo or PC3-24P4C12 cells (1000 cells/well)
were seeded into a 96 well plate on day 1. The following day an
equal volume of medium containing the indicated concentration of
Ha5-1(5)2.1-vcMMAE or a Control MAb conjugated with vc-MMAE (i.e.
Control-vcMMAE) was added to each well. The cells were allowed to
incubate for 4 days at 37 degrees C. At the end of the incubation
period, Alamar Blue was added to each well and incubation continued
for an additional 4 hours. The resulting fluorescence was detected
using a Biotek plate reader with an excitation wavelength of 620 nm
and an emission wavelength of 540 nm.
[0460] The results in FIG. 6 show that Ha5-1(5)2.1-vcMMAE mediated
cytotoxicity in PC3-24P4C12 cells while a control human IgG
conjugated with vcMMAE had no effect. The specificity of
Ha5-1(5)2.1-vcMMAE was further demonstrated by the lack of toxicity
for PC3-Neo cells that do not express 24P4C12. Thus, these results
indicate that Ha5-1(5)2.1-vcMMAE can selectively deliver a
cytotoxic drug to 24P4C12 expressing cells leading to their
killing.
Example 7
Ha5-1(5)2.1vcMMAE Inhibit Growth of Tumors In Vivo
[0461] The significant expression of 24P4C12 on the cell surface of
tumor tissues, together with its restrictive expression in normal
tissues makes 24P4C12 a good target for antibody therapy and
similarly, therapy via ADC. Thus, the therapeutic efficacy of
Ha5-1(5)2.1vcMMAE in human ovarian, prostate, colon, and pancreatic
cancer xenograft mouse models is evaluated.
[0462] Antibody drug conjugate efficacy on tumor growth and
metastasis formation is studied in mouse cancer xenograft models
(e.g. subcutaneous and orthotopically).
[0463] Subcutaneous (s.c.) tumors are generated by injection of
5.times.10.sup.4-10.sup.6 cancer cells mixed at a 1:1 dilution with
Matrigel (Collaborative Research) in the right flank of male SCID
mice. To test ADC efficacy on tumor formation, i.e. ADC injections
are started on the same day as tumor-cell injections. As a control,
mice are injected with either purified human IgG or PBS; or a
purified MAb that recognizes an irrelevant antigen not expressed in
human cells. In preliminary studies, no difference is found between
control IgG or PBS on tumor growth. Tumor sizes are determined by
caliper measurements, and the tumor volume is calculated as
length.times.width.times.height. Mice with subcutaneous tumors
greater than 1.5 cm in diameter are sacrificed.
[0464] Ovarian tumors often metastasize and grow within the
peritoneal cavity. Accordingly, intraperitoneal growth of ovarian
tumors in mice are performed by injection of 2 million cells
directly into the peritoneum of female mice. Mice are monitored for
general health, physical activity, and appearance until they become
moribund. At the time of sacrifice, the peritoneal cavity can be
examined to determine tumor burden and lungs harvested to evaluate
metastasis to distant sites. Alternatively, death can be used as an
endpoint. The mice are then segregated into groups for the
appropriate treatments, with 24P4C12 or control MAbs being injected
i.p.
[0465] An advantage of xenograft cancer models is the ability to
study neovascularization and angiogenesis. Tumor growth is partly
dependent on new blood vessel development. Although the capillary
system and developing blood network is of host origin, the
initiation and architecture of the neovasculature is regulated by
the xenograft tumor (Davidoff et al., Clin Cancer Res. (2001)
7:2870; Solesvik et al., Eur J Cancer Clin Oncol. (1984) 20:1295).
The effect of antibody and small molecule on neovascularization is
studied in accordance with procedures known in the art, such as by
IHC analysis of tumor tissues and their surrounding
microenvironment.
[0466] Ha5-1(5)2.1ADC inhibits formation colon, pancreatic,
ovarian, and prostate cancer xenografts. These results indicate the
utility of Ha5-1(5)2.1ADC in the treatment of local and advanced
stages of cancer and preferably those cancers set forth in Table
I.
[0467] 24P4C12 ADCs:
[0468] Monoclonal antibodies were raised against 24P4C12 as
described in the Example entitled "Generation of 24P4C12 Monoclonal
Antibodies (MAbs)." Further the MAbs are conjugated to a toxin as
described in the Example entitled "Antibody Drug Conjugation of
Ha5-1(5)2.1 MAb" to form AGS-5M2.1vcMMAE. The Ha5-1(5)2.1vcMMAE is
characterized by FACS, and other methods known in the art to
determine its capacity to bind 24P4C12.
[0469] Cell Lines and Xenografts:
[0470] The PC3-24P4C12, LAPC9, HT-29, AG-C4, OVCARS-24P4C12, and
AG-Panc3 cells are maintained in DMEM and RPMI respectively,
supplemented with L-glutamine and 10% FBS. LAPC9, AG-C4, and
AG-PAnc3 xenografts are maintained by serial propogation in SCID
mice.
[0471] Ha5-1(5)2.1vcMMAE Inhibits the Growth of Subcutaneous
Established Human Androgen-Independent Prostate Cancer Xenograft in
SCID Mice
[0472] In this experiment, androgen-independent human prostate
cancer PC3-24P4C12 tumor cells (3.0.times.10.sup.6 cells/mouse)
were injected subcutaneously into male SCID mice. Mice were
randomized into Ha5-1(5)2.1-vcMMAE and PBS control groups (n=5 in
each group) when tumors reached 100 mm.sup.3. Mice were treated
with a single dose of Ha5-1(5)2.1-vcMMAE (10 mg/kg) or PBS
administered intravenously (i.v.) on Day 0. Tumor growth was
monitored using caliper measurements every 3 to 4 days as
indicated. Tumor volume was calculated as Width2.times.Length/2,
where width is the smallest dimension and length is the
largest.
[0473] The results show that treatment with Ha5-1(5)2.1-vcMMAE
significantly inhibited the growth of PC-3-Hu24P4C12 prostate
tumors in SCID mice (p<0.01) and resulted in complete tumor
regression in most animals. (FIG. 7)
[0474] Ha5-1(5)2.1vcMMAE Inhibits the Growth of Orthotopically
Established Human Androgen-Independent Prostate Cancer Xenograft in
SCID Mice
[0475] In another experiment, LAPC-9AI androgen-independent human
prostate cancer cells (2.0.times.106 cells/mouse) were implanted
into the prostates of male SCID mice. Fifteen (15) days after
implantation when tumors were well established and palpable, the
mice were randomized into two groups (n=8 in each group). Mice were
treated with either Ha5-1(5)2.1-vcMMAE or isotype control MAb
conjugated with vcMMAE administered i.v. at 3 mg/kg every 4 days
for a total of 4 doses. At the end of study tumors in the mouse
prostate were excised and weighed using an electronic balance.
[0476] The results show that treatment with Ha5-1(5)2.1-vcMMAE
significantly inhibited the growth of LAPC9-AI human prostate
tumors implanted orthotopically in SCID mice (p<0.01). (FIG.
8).
[0477] Ha5-1(5)2.1vcMMAE Inhibits the Growth of Subcutaneous
Established Human Androgen-Independent Human Colon Cancer Xenograft
in SCID Mice
[0478] In another experiment, HT-29 human colon cancer cells
(1.0.times.10.sup.6 cells/mouse) were injected subcutaneously into
SCID mice. Mice were randomized into two groups (n=6 in each group)
when tumors reached 100 mm.sup.3. Ha5-1(5)2.1-vcMMAE (3 mg/kg) or
PBS was administered intravenously every 4 days for a total of 4
doses beginning on Day 0. Tumor growth was monitored using caliper
measurements every 3 to 4 days as indicated. Tumor volume was
calculated as Width2.times.Length/2, where width is the smallest
dimension and length is the largest.
[0479] The results show that treatment with Ha5-1(5)2.1-vcMMAE
significantly inhibited the growth of HT-29 human colon tumor
xenografts implanted subcutaneously in SCID mice (p<0.01). (FIG.
9).
[0480] Ha5-1(5)2.1vcMMAE Inhibits the Growth of Subcutaneous
Established Human Androgen-Independent Patient-Derived Colon Cancer
Xenograft in SCID Mice
[0481] In another experiment, AG-C4, patient-derived colon cancer
xenograft tumor pieces, were implanted subcutaneously into SCID
mice. Mice were randomized into two groups (n=6 in each group) when
tumors reached 100 mm.sup.3. Ha5-1(5)2.1-vcMMAE (3 mg/kg) or PBS
was administered intravenously every 3-4 days for a total of 4
doses starting on Day 0. Tumor growth was monitored using caliper
measurements every 3 to 4 days as indicated. Tumor volume was
calculated as Width2.times.Length/2, where width is the smallest
dimension and length is the largest.
[0482] The results show that treatment with Ha5-1(5)2.1-vcMMAE
significantly inhibited the growth of AG-C4 human colon tumor
xenografts implanted subcutaneously in SCID mice (p<0.05). (FIG.
10).
[0483] Ha5-1(5)2.1vcMMAE Inhibits the Growth of Subcutaneous
Established Human Ovarian Cancer Xenograft in Nude Mice
[0484] In another experiment, OVCAR-5 human ovarian cancer tumor
cells (2.0.times.10.sup.6 cells/mouse) were injected subcutaneously
into the nude mice. Mice were randomized into two groups (n=6 in
each group) when tumors reached 100 mm3. Ha5-1(5)2.1-vcMMAE (5
mg/kg) or PBS was administered intravenously once every 3-4 days
for a total of 4 doses starting on Day 0. Tumor growth was
monitored using caliper measurements every 3 to 4 days as
indicated. Tumor volume was calculated as Width2.times.Length/2,
where width is the smallest dimension and length is the
largest.
[0485] The results show that treatment with Ha5-1(5)2.1-vcMMAE
significantly inhibited the growth of OVCAR-5 ovarian cancer
xenografts implanted subcutaneously in nude mice (p<0.01). (FIG.
11).
[0486] Ha5-1(5)2.1vcMMAE Inhibits the Growth of Subcutaneous
Established Patient-Derived Pancreatic Cancer Xenograft in SCID
Mice
[0487] In this experiment, AG-Panc3 patient-derived pancreatic
tumor pieces were implanted subcutaneously into SCID mice. Mice
were randomized into two groups (n=6 in each group) when tumors
reached 85 mm.sup.3. Ha5-1(5)2.1-vcMMAE (5 mg/kg) or PBS was
administered intravenously once every 3-4 days for a total of 4
doses beginning on Day 0. Tumor growth was monitored using caliper
measurements every 3 to 4 days as indicated. Tumor volume was
calculated as Width2.times.Length/2, where width is the smallest
dimension and length is the largest.
[0488] The results show that treatment with Ha5-1(5)2.1-vcMMAE
significantly inhibited the growth of AG-Panc3 tumor xenografts
implanted subcutaneously in SCID mice (p<0.01). (FIG. 12).
[0489] The results of these experiments show that 24P4C12 ADC
designated Ha5-1(5)2.1vcMMAE can be used for therapeutic purposes
to treat and manage cancers set forth in Table I.
[0490] Efficacy of Ha5-1(5)2.1vcMMAE Compared to Other 24P4C12
Antibody Drug Conjugates (ADCs) in Prostate Cancer LAPC9-AD
Xenografts
[0491] In another experiment, LAPC-9AD androgen-dependent human
prostate cancer cells (1.5.times.10.sup.6 cells/mouse) were
injected subcutaneously into male SCID mice. Mice were randomized
into Ha5-1(5)2.1-vcMMAE, Ha5-1(5)2.1-mcMMAF and other Antibody Drug
Conjugate (ADC) groups including a PBS control group (n=6 in each
group), as shown in graph (FIG. 13). When tumors reached 100
mm.sup.3, Ha5-1(5)2.1-vcMMAE, Ha5-1(5)2.1-mcMMAF and all other ADCs
were administered intravenously at 10 mg/kg once on day 0. Tumor
growth was monitored using caliper measurements every 3 to 4 days
as indicated. Tumor volume was calculated as Width2.times.Length/2,
where width is the smallest dimension and length is the
largest.
[0492] The results show that treatment with Ha5-1(5)2.1-vcMMAE
significantly inhibited the growth of LAPC9-AD prostate cancer
xenografts as compared to Ha5-1(5)2.1-mcMMAF (p=0.0048). (FIG. 13).
Other antibodies conjugated to -vcMMAE and -mcMMAF did not have any
tumor inhibitory activity which shows that Ha5-1(5)2.1 possesses a
significant prominent effect of inhibiting tumor growth and can be
used for therapeutic purposes to treat and manage cancers set forth
in Table I.
Example 8
Human Clinical Trials for the Treatment and Diagnosis of Human
Carcinomas Through Use of 24P4C12 ADCs
[0493] 24P4C12 ADCs are used in accordance with the present
invention which specifically bind to 24P4C12, and are used in the
treatment of certain tumors, preferably those listed in Table I. In
connection with each of these indications, two clinical approaches
are successfully pursued.
[0494] I.) Adjunctive therapy: In adjunctive therapy, patients are
treated with 24P4C12 ADCs in combination with a chemotherapeutic or
anti-neoplastic agent and/or radiation therapy or a combination
thereof. Primary cancer targets, such as those listed in Table I,
are treated under standard protocols by the addition of 24P4C12
ADCs to standard first and second line therapy. Protocol designs
address effectiveness as assessed by the following examples,
including but not limited to, reduction in tumor mass of primary or
metastatic lesions, increased progression free survival, overall
survival, improvement of patients health, disease stabilization, as
well as the ability to reduce usual doses of standard chemotherapy
and other biologic agents. These dosage reductions allow additional
and/or prolonged therapy by reducing dose-related toxicity of the
chemotherapeutic or biologic agent. 24P4C12 ADCs are utilized in
several adjunctive clinical trials in combination with the
chemotherapeutic or anti-neoplastic agents.
[0495] II.) Monotherapy: In connection with the use of the 24P4C12
ADCs in monotherapy of tumors, the 24P4C12 ADCs are administered to
patients without a chemotherapeutic or anti-neoplastic agent. In
one embodiment, monotherapy is conducted clinically in end-stage
cancer patients with extensive metastatic disease. Protocol designs
address effectiveness as assessed by the following examples,
including but not limited to, reduction in tumor mass of primary or
metastatic lesions, increased progression free survival, overall
survival, improvement of patients health, disease stabilization, as
well as the ability to reduce usual doses of standard chemotherapy
and other biologic agents.
[0496] Dosage
[0497] 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. 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 antibody 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.
[0498] An exemplary, non limiting range for a therapeutically
effective amount of an 24P4C12 ADC administered in combination
according to the invention is about 0.5 to about 10 mg/kg, about 1
to about 5 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3
mg/kg, or at least 4 mg/kg. Other exemplary non-limiting ranges are
for example about 0.5 to about 5 mg/kg, or for example about 0.8 to
about 5 mg/kg, or for example about 1 to about 7.5 mg/kg. The high
dose embodiment of the invention relates to a dosage of more than
10 mg/kg. It is to be noted that dosage values may vary with the
type and severity of the condition to be alleviated, and may
include single or multiple doses. 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.
[0499] Clinical Development Plan (CDP)
[0500] The CDP follows and develops treatments of 24P4C12 ADcs in
connection with adjunctive therapy or monotherapy. Trials initially
demonstrate safety and thereafter confirm efficacy in repeat doses.
Trials are open label comparing standard chemotherapy with standard
therapy plus 24P4C12 ADCs. As will be appreciated, one non-limiting
criteria that can be utilized in connection with enrollment of
patients is 24P4C12 expression levels in their tumors as determined
by biopsy.
[0501] As with any protein or antibody infusion-based therapeutic,
safety concerns are related primarily to (i) cytokine release
syndrome, i.e., hypotension, fever, shaking, chills; (ii) the
development of an immunogenic response to the material (i.e.,
development of human antibodies by the patient to the antibody
therapeutic, or HAHA response); and, (iii) toxicity to normal cells
that express 24P4C12. Standard tests and follow-up are utilized to
monitor each of these safety concerns. 24P4C12 MAbs are found to be
safe upon human administration.
Example 9
Detection of 24P4C12 Protein in Gastric Cancer Patient Specimens by
IHC
[0502] Expression of 24P4C12 protein by immunohistochemistry was
tested in two (2) different tumor specimens from gastric cancer
patients. Briefly, formalin fixed, paraffin wax-embedded tissues
were cut into 4 micron sections and mounted on glass slides. The
sections were de-waxed, rehydrated and treated with trypsin
solution (0.05% trypsin (ICN, Aurora, Ohio) in 0.05% calcium
chloride, with pH adjusted to 7.8) at 37.degree. C. for 10 minutes.
Sections were then treated with 3% hydrogen peroxide solution to
inactivate endogenous peroxidase activity. Serum-free protein block
(Dako, Carpenteria, Calif.) was used to inhibit non-specific
binding prior to incubation with monoclonal mouse anti-24P4C12
antibody or an isotype control. Subsequently, the sections were
treated with the Super Sensitive.TM. Polymer-horseradish peroxidase
(HRP) Detection System which consists of an incubation in Super
Enhancer.TM. reagent followed by an incubation with polymer-HRP
secondary antibody conjugate (BioGenex, San Ramon, Calif.). The
sections were then developed using the DAB kit (BioGenex, San
Ramon, Calif.), nuclei were stained using hematoxylin, and analyzed
by bright field microscopy. Specific staining was detected in
patient specimens using the 24P4C12 immunoreactive antibody, as
indicated by the brown staining. (See, FIGS. 14(A) and 14(C). In
contrast, the control antibody did not stain either patient
specimen. (See, FIGS. 14(B) and 14(D). The results show expression
of 24P4C12 in the tumor cells of patient gastric cancer tissues.
These results indicate that 24P4C12 is expressed in human cancers
and that antibodies directed to this antigen (e.g. Ha5-1(5)2.1) are
useful for diagnostic and therapeutic purposes. (FIG.
14(A)-14(D)).
[0503] Throughout this application, various website data content,
publications, patent applications and patents are referenced.
(Websites are referenced by their Uniform Resource Locator, or URL,
addresses on the World Wide Web). The disclosures of each of these
references are hereby incorporated by reference herein in their
entireties.
[0504] The present invention is not to be limited in scope by the
embodiments disclosed herein, which are intended as single
illustrations of individual aspects of the invention, and any that
are functionally equivalent are within the scope of the invention.
Various modifications to the models and methods of the invention,
in addition to those described herein, will become apparent to
those skilled in the art from the foregoing description and
teachings, and are similarly intended to fall within the scope of
the invention. Such modifications or other embodiments can be
practiced without departing from the true scope and spirit of the
invention.
Tables
TABLE-US-00002 [0505] TABLE I Tissues that express 24P4C12 when
malignant. Colon Pancreas Ovarian Breast Lung Prostate Gastric
TABLE-US-00003 TABLE II Amino Acid Abbreviations SINGLE LETTER
THREE LETTER FULL NAME F Phe phenylalanine L Leu leucine S Ser
serine Y Tyr tyrosine C Cys cysteine W Trp tryptophan P Pro proline
H His histidine Q Gln glutamine R Arg arginine I Ile isoleucine M
Met methionine T Thr threonine N Asn asparagine K Lys lysine V Val
valine A Ala alanine D Asp aspartic acid E Glu glutamic acid G Gly
glycine
TABLE-US-00004 TABLE III Amino Acid Substitution Matrix Adapted
from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix
(block substitution matrix). The higher the value, the more likely
a substitution is found in related, natural proteins. A C D E F G H
I K L M N P Q R S T V W Y . 4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1
-1 1 0 0 -3 -2 A 9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1
-2 -2 C 6 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3 D 5 -3 -2
0 -3 1 -3 -2 0 -1 2 0 0 -1 -2 -3 -2 E 6 -3 -1 0 -3 0 0 -3 -4 -3 -3
-2 -2 -1 1 3 F 6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3 G 8 -3 -1
-3 -2 1 -2 0 0 -1 -2 -3 -2 2 H 4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3 -1 I
5 -2 -1 0 -1 1 2 0 -1 -2 -3 -2 K 4 2 -3 -3 -2 -2 -2 -1 1 -2 -1 L 5
-2 -2 0 -1 -1 -1 1 -1 -1 M 6 -2 0 0 1 0 -3 -4 -2 N 7 -1 -2 -1 -1 -2
-4 -3 P 5 1 0 -1 -2 -2 -1 Q 5 -1 -1 -3 -3 -2 R 4 1 -2 -3 -2 S 5 0
-2 -2 T 4 -3 -1 V 11 2 W 7 Y
TABLE-US-00005 TABLE IV(A) FACS MFI of AGS-5M2.1vcMMAE on LnCAP
cells Ha5- MFI on 1(5)2.1vcMMAE LNCaP Conc. (nM) cells 160.000 116
106.667 114 71.111 108 23.704 97 7.901 86 2.634 70 0.878 54 0.293
28 0.098 15 0.033 9 0.011 7
TABLE-US-00006 TABLE V General Method for Synthesis of vcMMAE
##STR00038## Where: AA1 = Amino Acid 1 AA2 = Amino Acid 2 AA5 =
Amino Acid 5 DIL = Dolaisoleuine DAP = Dolaproine Linker = Val-Cit
(vc)
Sequence CWU 1
1
2512587DNAHomo sapiensmisc_feature(1)...(2587)24P4C12 variant 1
1gagcc atg ggg gga aag cag cgg gac gag gat gac gag gcc tac ggg aag
50 Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly Lys 1 5
10 15cca gtc aaa tac gac ccc tcc ttt cga ggc ccc atc aag aac aga
agc 98Pro Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg
Ser 20 25 30tgc aca gat gtc atc tgc tgc gtc ctc ttc ctg ctc ttc att
cta ggt 146Cys Thr Asp Val Ile Cys Cys Val Leu Phe Leu Leu Phe Ile
Leu Gly 35 40 45tac atc gtg gtg ggg att gtg gcc tgg ttg tat gga gac
ccc cgg caa 194Tyr Ile Val Val Gly Ile Val Ala Trp Leu Tyr Gly Asp
Pro Arg Gln 50 55 60gtc ctc tac ccc agg aac tct act ggg gcc tac tgt
ggc atg ggg gag 242Val Leu Tyr Pro Arg Asn Ser Thr Gly Ala Tyr Cys
Gly Met Gly Glu 65 70 75aac aaa gat aag ccg tat ctc ctg tac ttc aac
atc ttc agc tgc atc 290Asn Lys Asp Lys Pro Tyr Leu Leu Tyr Phe Asn
Ile Phe Ser Cys Ile80 85 90 95ctg tcc agc aac atc atc tca gtt gct
gag aac ggc cta cag tgc ccc 338Leu Ser Ser Asn Ile Ile Ser Val Ala
Glu Asn Gly Leu Gln Cys Pro 100 105 110aca ccc cag gtg tgt gtg tcc
tcc tgc ccg gag gac cca tgg act gtg 386Thr Pro Gln Val Cys Val Ser
Ser Cys Pro Glu Asp Pro Trp Thr Val 115 120 125gga aaa aac gag ttc
tca cag act gtt ggg gaa gtc ttc tat aca aaa 434Gly Lys Asn Glu Phe
Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys 130 135 140aac agg aac
ttt tgt ctg cca ggg gta ccc tgg aat atg acg gtg atc 482Asn Arg Asn
Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile 145 150 155aca
agc ctg caa cag gaa ctc tgc ccc agt ttc ctc ctc ccc tct gct 530Thr
Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu Pro Ser Ala160 165
170 175cca gct ctg ggg cgc tgc ttt cca tgg acc aac gtt act cca ccg
gcg 578Pro Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn Val Thr Pro Pro
Ala 180 185 190ctc cca ggg atc acc aat gac acc acc ata cag cag ggg
atc agc ggt 626Leu Pro Gly Ile Thr Asn Asp Thr Thr Ile Gln Gln Gly
Ile Ser Gly 195 200 205ctt att gac agc ctc aat gcc cga gac atc agt
gtt aag atc ttt gaa 674Leu Ile Asp Ser Leu Asn Ala Arg Asp Ile Ser
Val Lys Ile Phe Glu 210 215 220gat ttt gcc cag tcc tgg tat tgg att
ctt gtt gcc ctg ggg gtg gct 722Asp Phe Ala Gln Ser Trp Tyr Trp Ile
Leu Val Ala Leu Gly Val Ala 225 230 235ctg gtc ttg agc cta ctg ttt
atc ttg ctt ctg cgc ctg gtg gct ggg 770Leu Val Leu Ser Leu Leu Phe
Ile Leu Leu Leu Arg Leu Val Ala Gly240 245 250 255ccc ctg gtg ctg
gtg ctg atc ctg gga gtg ctg ggc gtg ctg gca tac 818Pro Leu Val Leu
Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr 260 265 270ggc atc
tac tac tgc tgg gag gag tac cga gtg ctg cgg gac aag ggc 866Gly Ile
Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp Lys Gly 275 280
285gcc tcc atc tcc cag ctg ggt ttc acc acc aac ctc agt gcc tac cag
914Ala Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser Ala Tyr Gln
290 295 300agc gtg cag gag acc tgg ctg gcc gcc ctg atc gtg ttg gcg
gtg ctt 962Ser Val Gln Glu Thr Trp Leu Ala Ala Leu Ile Val Leu Ala
Val Leu 305 310 315gaa gcc atc ctg ctg ctg atg ctc atc ttc ctg cgg
cag cgg att cgt 1010Glu Ala Ile Leu Leu Leu Met Leu Ile Phe Leu Arg
Gln Arg Ile Arg320 325 330 335att gcc atc gcc ctc ctg aag gag gcc
agc aag gct gtg gga cag atg 1058Ile Ala Ile Ala Leu Leu Lys Glu Ala
Ser Lys Ala Val Gly Gln Met 340 345 350atg tct acc atg ttc tac cca
ctg gtc acc ttt gtc ctc ctc ctc atc 1106Met Ser Thr Met Phe Tyr Pro
Leu Val Thr Phe Val Leu Leu Leu Ile 355 360 365tgc att gcc tac tgg
gcc atg act gct ctg tac ctg gct aca tcg ggg 1154Cys Ile Ala Tyr Trp
Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly 370 375 380caa ccc cag
tat gtg ctc tgg gca tcc aac atc agc tcc ccc ggc tgt 1202Gln Pro Gln
Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys 385 390 395gag
aaa gtg cca ata aat aca tca tgc aac ccc acg gcc cac ctt gtg 1250Glu
Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala His Leu Val400 405
410 415aac tcc tcg tgc cca ggg ctg atg tgc gtc ttc cag ggc tac tca
tcc 1298Asn Ser Ser Cys Pro Gly Leu Met Cys Val Phe Gln Gly Tyr Ser
Ser 420 425 430aaa ggc cta atc caa cgt tct gtc ttc aat ctg caa atc
tat ggg gtc 1346Lys Gly Leu Ile Gln Arg Ser Val Phe Asn Leu Gln Ile
Tyr Gly Val 435 440 445ctg ggg ctc ttc tgg acc ctt aac tgg gta ctg
gcc ctg ggc caa tgc 1394Leu Gly Leu Phe Trp Thr Leu Asn Trp Val Leu
Ala Leu Gly Gln Cys 450 455 460gtc ctc gct gga gcc ttt gcc tcc ttc
tac tgg gcc ttc cac aag ccc 1442Val Leu Ala Gly Ala Phe Ala Ser Phe
Tyr Trp Ala Phe His Lys Pro 465 470 475cag gac atc cct acc ttc ccc
tta atc tct gcc ttc atc cgc aca ctc 1490Gln Asp Ile Pro Thr Phe Pro
Leu Ile Ser Ala Phe Ile Arg Thr Leu480 485 490 495cgt tac cac act
ggg tca ttg gca ttt gga gcc ctc atc ctg acc ctt 1538Arg Tyr His Thr
Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu 500 505 510gtg cag
ata gcc cgg gtc atc ttg gag tat att gac cac aag ctc aga 1586Val Gln
Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys Leu Arg 515 520
525gga gtg cag aac cct gta gcc cgc tgc atc atg tgc tgt ttc aag tgc
1634Gly Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys Cys Phe Lys Cys
530 535 540tgc ctc tgg tgt ctg gaa aaa ttt atc aag ttc cta aac cgc
aat gca 1682Cys Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe Leu Asn Arg
Asn Ala 545 550 555tac atc atg atc gcc atc tac ggg aag aat ttc tgt
gtc tca gcc aaa 1730Tyr Ile Met Ile Ala Ile Tyr Gly Lys Asn Phe Cys
Val Ser Ala Lys560 565 570 575aat gcg ttc atg cta ctc atg cga aac
att gtc agg gtg gtc gtc ctg 1778Asn Ala Phe Met Leu Leu Met Arg Asn
Ile Val Arg Val Val Val Leu 580 585 590gac aaa gtc aca gac ctg ctg
ctg ttc ttt ggg aag ctg ctg gtg gtc 1826Asp Lys Val Thr Asp Leu Leu
Leu Phe Phe Gly Lys Leu Leu Val Val 595 600 605gga ggc gtg ggg gtc
ctg tcc ttc ttt ttt ttc tcc ggt cgc atc ccg 1874Gly Gly Val Gly Val
Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro 610 615 620ggg ctg ggt
aaa gac ttt aag agc ccc cac ctc aac tat tac tgg ctg 1922Gly Leu Gly
Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu 625 630 635ccc
atc atg acc tcc atc ctg ggg gcc tat gtc atc gcc agc ggc ttc 1970Pro
Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala Ser Gly Phe640 645
650 655ttc agc gtt ttc ggc atg tgt gtg gac acg ctc ttc ctc tgc ttc
ctg 2018Phe Ser Val Phe Gly Met Cys Val Asp Thr Leu Phe Leu Cys Phe
Leu 660 665 670gaa gac ctg gag cgg aac aac ggc tcc ctg gac cgg ccc
tac tac atg 2066Glu Asp Leu Glu Arg Asn Asn Gly Ser Leu Asp Arg Pro
Tyr Tyr Met 675 680 685tcc aag agc ctt cta aag att ctg ggc aag aag
aac gag gcg ccc ccg 2114Ser Lys Ser Leu Leu Lys Ile Leu Gly Lys Lys
Asn Glu Ala Pro Pro 690 695 700gac aac aag aag agg aag aag tga
cagctccggc cctgatccag gactgcaccc 2168Asp Asn Lys Lys Arg Lys Lys
705 710cacccccacc gtccagccat ccaacctcac ttcgccttac aggtctccat
tttgtggtaa 2228aaaaaggttt taggccaggc gccgtggctc acgcctgtaa
tccaacactt tgagaggctg 2288aggcgggcgg atcacctgag tcaggagttc
gagaccagcc tggccaacat ggtgaaacct 2348ccgtctctat taaaaataca
aaaattagcc gagagtggtg gcatgcacct gtcatcccag 2408ctactcggga
ggctgaggca ggagaatcgc ttgaacccgg gaggcagagg ttgcagtgag
2468ccgagatcgc gccactgcac tccaacctgg gtgacagact ctgtctccaa
aacaaaacaa 2528acaaacaaaa agattttatt aaagatattt tgttaactca
gtaaaaaaaa aaaaaaaaa 25872710PRTHomo
sapiensmisc_feature(1)...(710)24P4C12 variant 1 coding sequence
2Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly Lys Pro1 5
10 15 Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg Ser
Cys 20 25 30 Thr Asp Val Ile Cys Cys Val Leu Phe Leu Leu Phe Ile
Leu Gly Tyr 35 40 45 Ile Val Val Gly Ile Val Ala Trp Leu Tyr Gly
Asp Pro Arg Gln Val 50 55 60 Leu Tyr Pro Arg Asn Ser Thr Gly Ala
Tyr Cys Gly Met Gly Glu Asn65 70 75 80 Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile Leu 85 90 95 Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro Thr 100 105 110 Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val Gly 115 120 125 Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys Asn 130 135
140 Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
Thr145 150 155 160 Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu
Pro Ser Ala Pro 165 170 175 Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Val Thr Pro Pro Ala Leu 180 185 190 Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly Leu 195 200 205 Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu Asp 210 215 220 Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala Leu225 230 235 240 Val
Leu Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly Pro 245 250
255 Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr Gly
260 265 270 Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp Lys
Gly Ala 275 280 285 Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser
Ala Tyr Gln Ser 290 295 300 Val Gln Glu Thr Trp Leu Ala Ala Leu Ile
Val Leu Ala Val Leu Glu305 310 315 320 Ala Ile Leu Leu Leu Met Leu
Ile Phe Leu Arg Gln Arg Ile Arg Ile 325 330 335 Ala Ile Ala Leu Leu
Lys Glu Ala Ser Lys Ala Val Gly Gln Met Met 340 345 350 Ser Thr Met
Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile Cys 355 360 365 Ile
Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly Gln 370 375
380 Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys
Glu385 390 395 400 Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala
His Leu Val Asn 405 410 415 Ser Ser Cys Pro Gly Leu Met Cys Val Phe
Gln Gly Tyr Ser Ser Lys 420 425 430 Gly Leu Ile Gln Arg Ser Val Phe
Asn Leu Gln Ile Tyr Gly Val Leu 435 440 445 Gly Leu Phe Trp Thr Leu
Asn Trp Val Leu Ala Leu Gly Gln Cys Val 450 455 460 Leu Ala Gly Ala
Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro Gln465 470 475 480 Asp
Ile Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr Leu Arg 485 490
495 Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu Val
500 505 510 Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys Leu
Arg Gly 515 520 525 Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys Cys
Phe Lys Cys Cys 530 535 540 Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe
Leu Asn Arg Asn Ala Tyr545 550 555 560 Ile Met Ile Ala Ile Tyr Gly
Lys Asn Phe Cys Val Ser Ala Lys Asn 565 570 575 Ala Phe Met Leu Leu
Met Arg Asn Ile Val Arg Val Val Val Leu Asp 580 585 590 Lys Val Thr
Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val Gly 595 600 605 Gly
Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro Gly 610 615
620 Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu
Pro625 630 635 640 Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala
Ser Gly Phe Phe 645 650 655 Ser Val Phe Gly Met Cys Val Asp Thr Leu
Phe Leu Cys Phe Leu Glu 660 665 670 Asp Leu Glu Arg Asn Asn Gly Ser
Leu Asp Arg Pro Tyr Tyr Met Ser 675 680 685 Lys Ser Leu Leu Lys Ile
Leu Gly Lys Lys Asn Glu Ala Pro Pro Asp 690 695 700 Asn Lys Lys Arg
Lys Lys705 710 32587DNAHomo sapiensmisc_feature(1)...(2587)24P4C12
variant 2 3gagcc atg ggg gga aag cag cgg gac gag gat gac gag gcc
tac ggg aag 50 Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr
Gly Lys 1 5 10 15cca gtc aaa tac gac ccc tcc ttt cga ggc ccc atc
aag aac aga agc 98Pro Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile
Lys Asn Arg Ser 20 25 30tgc aca gat gtc atc tgc tgc gtc ctc ttc ctg
ctc ttc att cta ggt 146Cys Thr Asp Val Ile Cys Cys Val Leu Phe Leu
Leu Phe Ile Leu Gly 35 40 45tac atc gtg gtg ggg att gtg gcc tgg ttg
tat gga gac ccc cgg caa 194Tyr Ile Val Val Gly Ile Val Ala Trp Leu
Tyr Gly Asp Pro Arg Gln 50 55 60gtc ctc tac ccc agg aac tct act ggg
gcc tac tgt ggc atg ggg gag 242Val Leu Tyr Pro Arg Asn Ser Thr Gly
Ala Tyr Cys Gly Met Gly Glu 65 70 75aac aaa gat aag ccg tat ctc ctg
tac ttc aac atc ttc agc tgc atc 290Asn Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile80 85 90 95ctg tcc agc aac atc atc
tca gtt gct gag aac ggc cta cag tgc ccc 338Leu Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro 100 105 110aca ccc cag gtg
tgt gtg tcc tcc tgc ccg gag gac cca tgg act gtg 386Thr Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val 115 120 125gga aaa
aac gag ttc tca cag act gtt ggg gaa gtc ttc tat aca aaa 434Gly Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys 130 135
140aac agg aac ttt tgt ctg cca ggg gta ccc tgg aat atg acg gtg atc
482Asn Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
145 150 155aca agc ctg caa cag gaa ctc tgc ccc agt ttc ctc ctc ccc
tct gct 530Thr Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu Pro
Ser Ala160 165 170 175cca gct ctg gga cgc tgc ttt cca tgg acc aac
gtt act cca ccg gcg 578Pro Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Val Thr Pro Pro Ala 180 185 190ctc cca ggg atc acc aat gac acc acc
ata cag cag ggg atc agc ggt 626Leu Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly 195 200 205ctt att gac agc ctc aat gcc
cga gac atc agt gtt aag atc ttt gaa 674Leu Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu 210 215 220gat ttt gcc cag tcc
tgg tat tgg att ctt gtt gcc ctg ggg gtg gct 722Asp Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala 225 230 235ctg gtc ttg
agc cta ctg ttt atc ttg ctt ctg cgc ctg gtg gct ggg 770Leu Val Leu
Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly240 245 250
255ccc ctg gtg ctg gtg ctg atc ctg gga gtg ctg ggc gtg ctg gca tac
818Pro Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr
260 265 270ggc atc tac tac tgc tgg gag gag tac cga gtg ctg cgg gac
aag ggc 866Gly Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp
Lys Gly 275 280
285gcc tcc atc tcc cag ctg ggt ttc acc acc aac ctc agt gcc tac cag
914Ala Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser Ala Tyr Gln
290 295 300agc gtg cag gag acc tgg ctg gcc gcc ctg atc gtg ttg gcg
gtg ctt 962Ser Val Gln Glu Thr Trp Leu Ala Ala Leu Ile Val Leu Ala
Val Leu 305 310 315gaa gcc atc ctg ctg ctg atg ctc atc ttc ctg cgg
cag cgg att cgt 1010Glu Ala Ile Leu Leu Leu Met Leu Ile Phe Leu Arg
Gln Arg Ile Arg320 325 330 335att gcc atc gcc ctc ctg aag gag gcc
agc aag gct gtg gga cag atg 1058Ile Ala Ile Ala Leu Leu Lys Glu Ala
Ser Lys Ala Val Gly Gln Met 340 345 350atg tct acc atg ttc tac cca
ctg gtc acc ttt gtc ctc ctc ctc atc 1106Met Ser Thr Met Phe Tyr Pro
Leu Val Thr Phe Val Leu Leu Leu Ile 355 360 365tgc att gcc tac tgg
gcc atg act gct ctg tac ctg gct aca tcg ggg 1154Cys Ile Ala Tyr Trp
Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly 370 375 380caa ccc cag
tat gtg ctc tgg gca tcc aac atc agc tcc ccc ggc tgt 1202Gln Pro Gln
Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys 385 390 395gag
aaa gtg cca ata aat aca tca tgc aac ccc acg gcc cac ctt gtg 1250Glu
Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala His Leu Val400 405
410 415aac tcc tcg tgc cca ggg ctg atg tgc gtc ttc cag ggc tac tca
tcc 1298Asn Ser Ser Cys Pro Gly Leu Met Cys Val Phe Gln Gly Tyr Ser
Ser 420 425 430aaa ggc cta atc caa cgt tct gtc ttc aat ctg caa atc
tat ggg gtc 1346Lys Gly Leu Ile Gln Arg Ser Val Phe Asn Leu Gln Ile
Tyr Gly Val 435 440 445ctg ggg ctc ttc tgg acc ctt aac tgg gta ctg
gcc ctg ggc caa tgc 1394Leu Gly Leu Phe Trp Thr Leu Asn Trp Val Leu
Ala Leu Gly Gln Cys 450 455 460gtc ctc gct gga gcc ttt gcc tcc ttc
tac tgg gcc ttc cac aag ccc 1442Val Leu Ala Gly Ala Phe Ala Ser Phe
Tyr Trp Ala Phe His Lys Pro 465 470 475cag gac atc cct acc ttc ccc
tta atc tct gcc ttc atc cgc aca ctc 1490Gln Asp Ile Pro Thr Phe Pro
Leu Ile Ser Ala Phe Ile Arg Thr Leu480 485 490 495cgt tac cac act
ggg tca ttg gca ttt gga gcc ctc atc ctg acc ctt 1538Arg Tyr His Thr
Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu 500 505 510gtg cag
ata gcc cgg gtc atc ttg gag tat att gac cac aag ctc aga 1586Val Gln
Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys Leu Arg 515 520
525gga gtg cag aac cct gta gcc cgc tgc atc atg tgc tgt ttc aag tgc
1634Gly Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys Cys Phe Lys Cys
530 535 540tgc ctc tgg tgt ctg gaa aaa ttt atc aag ttc cta aac cgc
aat gca 1682Cys Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe Leu Asn Arg
Asn Ala 545 550 555tac atc atg atc gcc atc tac ggg aag aat ttc tgt
gtc tca gcc aaa 1730Tyr Ile Met Ile Ala Ile Tyr Gly Lys Asn Phe Cys
Val Ser Ala Lys560 565 570 575aat gcg ttc atg cta ctc atg cga aac
att gtc agg gtg gtc gtc ctg 1778Asn Ala Phe Met Leu Leu Met Arg Asn
Ile Val Arg Val Val Val Leu 580 585 590gac aaa gtc aca gac ctg ctg
ctg ttc ttt ggg aag ctg ctg gtg gtc 1826Asp Lys Val Thr Asp Leu Leu
Leu Phe Phe Gly Lys Leu Leu Val Val 595 600 605gga ggc gtg ggg gtc
ctg tcc ttc ttt ttt ttc tcc ggt cgc atc ccg 1874Gly Gly Val Gly Val
Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro 610 615 620ggg ctg ggt
aaa gac ttt aag agc ccc cac ctc aac tat tac tgg ctg 1922Gly Leu Gly
Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu 625 630 635ccc
atc atg acc tcc atc ctg ggg gcc tat gtc atc gcc agc ggc ttc 1970Pro
Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala Ser Gly Phe640 645
650 655ttc agc gtt ttc ggc atg tgt gtg gac acg ctc ttc ctc tgc ttc
ctg 2018Phe Ser Val Phe Gly Met Cys Val Asp Thr Leu Phe Leu Cys Phe
Leu 660 665 670gaa gac ctg gag cgg aac aac ggc tcc ctg gac cgg ccc
tac tac atg 2066Glu Asp Leu Glu Arg Asn Asn Gly Ser Leu Asp Arg Pro
Tyr Tyr Met 675 680 685tcc aag agc ctt cta aag att ctg ggc aag aag
aac gag gcg ccc ccg 2114Ser Lys Ser Leu Leu Lys Ile Leu Gly Lys Lys
Asn Glu Ala Pro Pro 690 695 700gac aac aag aag agg aag aag tga
cagctccggc cctgatccag gactgcaccc 2168Asp Asn Lys Lys Arg Lys Lys
705 710cacccccacc gtccagccat ccaacctcac ttcgccttac aggtctccat
tttgtggtaa 2228aaaaaggttt taggccaggc gccgtggctc acgcctgtaa
tccaacactt tgagaggctg 2288aggcgggcgg atcacctgag tcaggagttc
gagaccagcc tggccaacat ggtgaaacct 2348ccgtctctat taaaaataca
aaaattagcc gagagtggtg gcatgcacct gtcatcccag 2408ctactcggga
ggctgaggca ggagaatcgc ttgaacccgg gaggcagagg ttgcagtgag
2468ccgagatcgc gccactgcac tccaacctgg gtgacagact ctgtctccaa
aacaaaacaa 2528acaaacaaaa agattttatt aaagatattt tgttaactca
gtaaaaaaaa aaaaaaaaa 25874710PRTHomo
sapiensmisc_feature(1)...(710)24P4C12 variant 2 coding sequence
4Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly Lys Pro1 5
10 15 Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg Ser
Cys 20 25 30 Thr Asp Val Ile Cys Cys Val Leu Phe Leu Leu Phe Ile
Leu Gly Tyr 35 40 45 Ile Val Val Gly Ile Val Ala Trp Leu Tyr Gly
Asp Pro Arg Gln Val 50 55 60 Leu Tyr Pro Arg Asn Ser Thr Gly Ala
Tyr Cys Gly Met Gly Glu Asn65 70 75 80 Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile Leu 85 90 95 Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro Thr 100 105 110 Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val Gly 115 120 125 Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys Asn 130 135
140 Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
Thr145 150 155 160 Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu
Pro Ser Ala Pro 165 170 175 Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Val Thr Pro Pro Ala Leu 180 185 190 Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly Leu 195 200 205 Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu Asp 210 215 220 Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala Leu225 230 235 240 Val
Leu Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly Pro 245 250
255 Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr Gly
260 265 270 Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp Lys
Gly Ala 275 280 285 Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser
Ala Tyr Gln Ser 290 295 300 Val Gln Glu Thr Trp Leu Ala Ala Leu Ile
Val Leu Ala Val Leu Glu305 310 315 320 Ala Ile Leu Leu Leu Met Leu
Ile Phe Leu Arg Gln Arg Ile Arg Ile 325 330 335 Ala Ile Ala Leu Leu
Lys Glu Ala Ser Lys Ala Val Gly Gln Met Met 340 345 350 Ser Thr Met
Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile Cys 355 360 365 Ile
Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly Gln 370 375
380 Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys
Glu385 390 395 400 Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala
His Leu Val Asn 405 410 415 Ser Ser Cys Pro Gly Leu Met Cys Val Phe
Gln Gly Tyr Ser Ser Lys 420 425 430 Gly Leu Ile Gln Arg Ser Val Phe
Asn Leu Gln Ile Tyr Gly Val Leu 435 440 445 Gly Leu Phe Trp Thr Leu
Asn Trp Val Leu Ala Leu Gly Gln Cys Val 450 455 460 Leu Ala Gly Ala
Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro Gln465 470 475 480 Asp
Ile Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr Leu Arg 485 490
495 Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu Val
500 505 510 Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys Leu
Arg Gly 515 520 525 Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys Cys
Phe Lys Cys Cys 530 535 540 Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe
Leu Asn Arg Asn Ala Tyr545 550 555 560 Ile Met Ile Ala Ile Tyr Gly
Lys Asn Phe Cys Val Ser Ala Lys Asn 565 570 575 Ala Phe Met Leu Leu
Met Arg Asn Ile Val Arg Val Val Val Leu Asp 580 585 590 Lys Val Thr
Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val Gly 595 600 605 Gly
Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro Gly 610 615
620 Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu
Pro625 630 635 640 Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala
Ser Gly Phe Phe 645 650 655 Ser Val Phe Gly Met Cys Val Asp Thr Leu
Phe Leu Cys Phe Leu Glu 660 665 670 Asp Leu Glu Arg Asn Asn Gly Ser
Leu Asp Arg Pro Tyr Tyr Met Ser 675 680 685 Lys Ser Leu Leu Lys Ile
Leu Gly Lys Lys Asn Glu Ala Pro Pro Asp 690 695 700 Asn Lys Lys Arg
Lys Lys705 710 52587DNAHomo sapiensmisc_feature(1)...(2587)24P4C12
variant 3 5gagcc atg ggg gga aag cag cgg gac gag gat gac gag gcc
tac ggg aag 50 Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr
Gly Lys 1 5 10 15cca gtc aaa tac gac ccc tcc ttt cga ggc ccc atc
aag aac aga agc 98Pro Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile
Lys Asn Arg Ser 20 25 30tgc aca gat gtc atc tgc tgc gtc ctc ttc ctg
ctc ttc att cta ggt 146Cys Thr Asp Val Ile Cys Cys Val Leu Phe Leu
Leu Phe Ile Leu Gly 35 40 45tac atc gtg gtg ggg att gtg gcc tgg ttg
tat gga gac ccc cgg caa 194Tyr Ile Val Val Gly Ile Val Ala Trp Leu
Tyr Gly Asp Pro Arg Gln 50 55 60gtc ctc tac ccc agg aac tct act ggg
gcc tac tgt ggc atg ggg gag 242Val Leu Tyr Pro Arg Asn Ser Thr Gly
Ala Tyr Cys Gly Met Gly Glu 65 70 75aac aaa gat aag ccg tat ctc ctg
tac ttc aac atc ttc agc tgc atc 290Asn Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile80 85 90 95ctg tcc agc aac atc atc
tca gtt gct gag aac ggc cta cag tgc ccc 338Leu Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro 100 105 110aca ccc cag gtg
tgt gtg tcc tcc tgc ccg gag gac cca tgg act gtg 386Thr Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val 115 120 125gga aaa
aac gag ttc tca cag act gtt ggg gaa gtc ttc tat aca aaa 434Gly Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys 130 135
140aac agg aac ttt tgt ctg cca ggg gta ccc tgg aat atg acg gtg atc
482Asn Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
145 150 155aca agc ctg caa cag gaa ctc tgc ccc agt ttc ctc ctc ccc
tct gct 530Thr Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu Pro
Ser Ala160 165 170 175cca gct ctg ggg cgc tgc ttt cca tgg acc aac
att act cca ccg gcg 578Pro Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Ile Thr Pro Pro Ala 180 185 190ctc cca ggg atc acc aat gac acc acc
ata cag cag ggg atc agc ggt 626Leu Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly 195 200 205ctt att gac agc ctc aat gcc
cga gac atc agt gtt aag atc ttt gaa 674Leu Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu 210 215 220gat ttt gcc cag tcc
tgg tat tgg att ctt gtt gcc ctg ggg gtg gct 722Asp Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala 225 230 235ctg gtc ttg
agc cta ctg ttt atc ttg ctt ctg cgc ctg gtg gct ggg 770Leu Val Leu
Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly240 245 250
255ccc ctg gtg ctg gtg ctg atc ctg gga gtg ctg ggc gtg ctg gca tac
818Pro Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr
260 265 270ggc atc tac tac tgc tgg gag gag tac cga gtg ctg cgg gac
aag ggc 866Gly Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp
Lys Gly 275 280 285gcc tcc atc tcc cag ctg ggt ttc acc acc aac ctc
agt gcc tac cag 914Ala Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu
Ser Ala Tyr Gln 290 295 300agc gtg cag gag acc tgg ctg gcc gcc ctg
atc gtg ttg gcg gtg ctt 962Ser Val Gln Glu Thr Trp Leu Ala Ala Leu
Ile Val Leu Ala Val Leu 305 310 315gaa gcc atc ctg ctg ctg atg ctc
atc ttc ctg cgg cag cgg att cgt 1010Glu Ala Ile Leu Leu Leu Met Leu
Ile Phe Leu Arg Gln Arg Ile Arg320 325 330 335att gcc atc gcc ctc
ctg aag gag gcc agc aag gct gtg gga cag atg 1058Ile Ala Ile Ala Leu
Leu Lys Glu Ala Ser Lys Ala Val Gly Gln Met 340 345 350atg tct acc
atg ttc tac cca ctg gtc acc ttt gtc ctc ctc ctc atc 1106Met Ser Thr
Met Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile 355 360 365tgc
att gcc tac tgg gcc atg act gct ctg tac ctg gct aca tcg ggg 1154Cys
Ile Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly 370 375
380caa ccc cag tat gtg ctc tgg gca tcc aac atc agc tcc ccc ggc tgt
1202Gln Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys
385 390 395gag aaa gtg cca ata aat aca tca tgc aac ccc acg gcc cac
ctt gtg 1250Glu Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala His
Leu Val400 405 410 415aac tcc tcg tgc cca ggg ctg atg tgc gtc ttc
cag ggc tac tca tcc 1298Asn Ser Ser Cys Pro Gly Leu Met Cys Val Phe
Gln Gly Tyr Ser Ser 420 425 430aaa ggc cta atc caa cgt tct gtc ttc
aat ctg caa atc tat ggg gtc 1346Lys Gly Leu Ile Gln Arg Ser Val Phe
Asn Leu Gln Ile Tyr Gly Val 435 440 445ctg ggg ctc ttc tgg acc ctt
aac tgg gta ctg gcc ctg ggc caa tgc 1394Leu Gly Leu Phe Trp Thr Leu
Asn Trp Val Leu Ala Leu Gly Gln Cys 450 455 460gtc ctc gct gga gcc
ttt gcc tcc ttc tac tgg gcc ttc cac aag ccc 1442Val Leu Ala Gly Ala
Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro 465 470 475cag gac atc
cct acc ttc ccc tta atc tct gcc ttc atc cgc aca ctc 1490Gln Asp Ile
Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr Leu480 485 490
495cgt tac cac act ggg tca ttg gca ttt gga gcc ctc atc ctg acc ctt
1538Arg Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu
500 505 510gtg cag ata gcc cgg gtc atc ttg gag tat att gac cac aag
ctc aga 1586Val Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys
Leu Arg 515 520 525gga gtg cag aac cct gta gcc cgc tgc atc atg tgc
tgt ttc aag tgc 1634Gly Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys
Cys Phe Lys Cys 530 535 540tgc ctc tgg tgt ctg gaa aaa ttt atc aag
ttc cta aac cgc aat gca 1682Cys Leu Trp Cys Leu Glu Lys Phe Ile Lys
Phe Leu Asn Arg Asn Ala 545 550 555tac atc atg atc gcc atc tac ggg
aag aat ttc tgt gtc tca gcc aaa 1730Tyr Ile Met Ile Ala Ile Tyr Gly
Lys Asn Phe Cys Val Ser Ala Lys560 565 570 575aat gcg ttc atg cta
ctc atg cga aac att gtc agg gtg gtc gtc ctg 1778Asn Ala Phe Met
Leu Leu Met Arg Asn Ile Val Arg Val Val Val Leu 580 585 590gac aaa
gtc aca gac ctg ctg ctg ttc ttt ggg aag ctg ctg gtg gtc 1826Asp Lys
Val Thr Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val 595 600
605gga ggc gtg ggg gtc ctg tcc ttc ttt ttt ttc tcc ggt cgc atc ccg
1874Gly Gly Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro
610 615 620ggg ctg ggt aaa gac ttt aag agc ccc cac ctc aac tat tac
tgg ctg 1922Gly Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr
Trp Leu 625 630 635ccc atc atg acc tcc atc ctg ggg gcc tat gtc atc
gcc agc ggc ttc 1970Pro Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile
Ala Ser Gly Phe640 645 650 655ttc agc gtt ttc ggc atg tgt gtg gac
acg ctc ttc ctc tgc ttc ctg 2018Phe Ser Val Phe Gly Met Cys Val Asp
Thr Leu Phe Leu Cys Phe Leu 660 665 670gaa gac ctg gag cgg aac aac
ggc tcc ctg gac cgg ccc tac tac atg 2066Glu Asp Leu Glu Arg Asn Asn
Gly Ser Leu Asp Arg Pro Tyr Tyr Met 675 680 685tcc aag agc ctt cta
aag att ctg ggc aag aag aac gag gcg ccc ccg 2114Ser Lys Ser Leu Leu
Lys Ile Leu Gly Lys Lys Asn Glu Ala Pro Pro 690 695 700gac aac aag
aag agg aag aag tga cagctccggc cctgatccag gactgcaccc 2168Asp Asn
Lys Lys Arg Lys Lys 705 710cacccccacc gtccagccat ccaacctcac
ttcgccttac aggtctccat tttgtggtaa 2228aaaaaggttt taggccaggc
gccgtggctc acgcctgtaa tccaacactt tgagaggctg 2288aggcgggcgg
atcacctgag tcaggagttc gagaccagcc tggccaacat ggtgaaacct
2348ccgtctctat taaaaataca aaaattagcc gagagtggtg gcatgcacct
gtcatcccag 2408ctactcggga ggctgaggca ggagaatcgc ttgaacccgg
gaggcagagg ttgcagtgag 2468ccgagatcgc gccactgcac tccaacctgg
gtgacagact ctgtctccaa aacaaaacaa 2528acaaacaaaa agattttatt
aaagatattt tgttaactca gtaaaaaaaa aaaaaaaaa 25876710PRTHomo
sapiensmisc_feature(1)...(710)24P4C12 variant 3 coding sequence
6Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly Lys Pro1 5
10 15 Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg Ser
Cys 20 25 30 Thr Asp Val Ile Cys Cys Val Leu Phe Leu Leu Phe Ile
Leu Gly Tyr 35 40 45 Ile Val Val Gly Ile Val Ala Trp Leu Tyr Gly
Asp Pro Arg Gln Val 50 55 60 Leu Tyr Pro Arg Asn Ser Thr Gly Ala
Tyr Cys Gly Met Gly Glu Asn65 70 75 80 Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile Leu 85 90 95 Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro Thr 100 105 110 Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val Gly 115 120 125 Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys Asn 130 135
140 Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
Thr145 150 155 160 Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu
Pro Ser Ala Pro 165 170 175 Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Ile Thr Pro Pro Ala Leu 180 185 190 Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly Leu 195 200 205 Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu Asp 210 215 220 Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala Leu225 230 235 240 Val
Leu Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly Pro 245 250
255 Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr Gly
260 265 270 Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp Lys
Gly Ala 275 280 285 Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser
Ala Tyr Gln Ser 290 295 300 Val Gln Glu Thr Trp Leu Ala Ala Leu Ile
Val Leu Ala Val Leu Glu305 310 315 320 Ala Ile Leu Leu Leu Met Leu
Ile Phe Leu Arg Gln Arg Ile Arg Ile 325 330 335 Ala Ile Ala Leu Leu
Lys Glu Ala Ser Lys Ala Val Gly Gln Met Met 340 345 350 Ser Thr Met
Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile Cys 355 360 365 Ile
Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly Gln 370 375
380 Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys
Glu385 390 395 400 Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala
His Leu Val Asn 405 410 415 Ser Ser Cys Pro Gly Leu Met Cys Val Phe
Gln Gly Tyr Ser Ser Lys 420 425 430 Gly Leu Ile Gln Arg Ser Val Phe
Asn Leu Gln Ile Tyr Gly Val Leu 435 440 445 Gly Leu Phe Trp Thr Leu
Asn Trp Val Leu Ala Leu Gly Gln Cys Val 450 455 460 Leu Ala Gly Ala
Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro Gln465 470 475 480 Asp
Ile Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr Leu Arg 485 490
495 Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu Val
500 505 510 Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys Leu
Arg Gly 515 520 525 Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys Cys
Phe Lys Cys Cys 530 535 540 Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe
Leu Asn Arg Asn Ala Tyr545 550 555 560 Ile Met Ile Ala Ile Tyr Gly
Lys Asn Phe Cys Val Ser Ala Lys Asn 565 570 575 Ala Phe Met Leu Leu
Met Arg Asn Ile Val Arg Val Val Val Leu Asp 580 585 590 Lys Val Thr
Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val Gly 595 600 605 Gly
Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro Gly 610 615
620 Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu
Pro625 630 635 640 Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala
Ser Gly Phe Phe 645 650 655 Ser Val Phe Gly Met Cys Val Asp Thr Leu
Phe Leu Cys Phe Leu Glu 660 665 670 Asp Leu Glu Arg Asn Asn Gly Ser
Leu Asp Arg Pro Tyr Tyr Met Ser 675 680 685 Lys Ser Leu Leu Lys Ile
Leu Gly Lys Lys Asn Glu Ala Pro Pro Asp 690 695 700 Asn Lys Lys Arg
Lys Lys705 710 72587DNAHomo sapiensmisc_feature(1)...(2587)24P4C12
variant 4 7gagcc atg ggg gga aag cag cgg gac gag gat gac gag gcc
tac ggg aag 50 Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr
Gly Lys 1 5 10 15cca gtc aaa tac gac ccc tcc ttt cga ggc ccc atc
aag aac aga agc 98Pro Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile
Lys Asn Arg Ser 20 25 30tgc aca gat gtc atc tgc tgc gtc ctc ttc ctg
ctc ttc att cta ggt 146Cys Thr Asp Val Ile Cys Cys Val Leu Phe Leu
Leu Phe Ile Leu Gly 35 40 45tac atc gtg gtg ggg att gtg gcc tgg ttg
tat gga gac ccc cgg caa 194Tyr Ile Val Val Gly Ile Val Ala Trp Leu
Tyr Gly Asp Pro Arg Gln 50 55 60gtc ctc tac ccc agg aac tct act ggg
gcc tac tgt ggc atg ggg gag 242Val Leu Tyr Pro Arg Asn Ser Thr Gly
Ala Tyr Cys Gly Met Gly Glu 65 70 75aac aaa gat aag ccg tat ctc ctg
tac ttc aac atc ttc agc tgc atc 290Asn Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile80 85 90 95ctg tcc agc aac atc atc
tca gtt gct gag aac ggc cta cag tgc ccc 338Leu Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro 100 105 110aca ccc cag gtg
tgt gtg tcc tcc tgc ccg gag gac cca tgg act gtg 386Thr Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val 115 120 125gga aaa
aac gag ttc tca cag act gtt ggg gaa gtc ttc tat aca aaa 434Gly Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys 130 135
140aac agg aac ttt tgt ctg cca ggg gta ccc tgg aat atg acg gtg atc
482Asn Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
145 150 155aca agc ctg caa cag gaa ctc tgc ccc agt ttc ctc ctc ccc
tct gct 530Thr Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu Pro
Ser Ala160 165 170 175cca gct ctg ggg cgc tgc ttt cca tgg acc aac
gtt act cca ccg gcg 578Pro Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Val Thr Pro Pro Ala 180 185 190ctc cca ggg atc acc aat gac acc acc
ata cag cag ggg atc agc ggt 626Leu Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly 195 200 205ctt att gac agc ctc aat gcc
cga gac atc agt gtt aag atc ttt gaa 674Leu Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu 210 215 220gat ttt gcc cag tcc
tgg tat tgg att ctt gtt gcc ctg ggg gtg gct 722Asp Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala 225 230 235ctg gtc ttg
agc cta ctg ttt atc ttg ctt ctg cgc ctg gtg gct ggg 770Leu Val Leu
Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly240 245 250
255ccc ctg gtg ctg gtg ctg atc ctg gga gtg ctg ggc gtg ctg gca tat
818Pro Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr
260 265 270ggc atc tac tac tgc tgg gag gag tac cga gtg ctg cgg gac
aag ggc 866Gly Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp
Lys Gly 275 280 285gcc tcc atc tcc cag ctg ggt ttc acc acc aac ctc
agt gcc tac cag 914Ala Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu
Ser Ala Tyr Gln 290 295 300agc gtg cag gag acc tgg ctg gcc gcc ctg
atc gtg ttg gcg gtg ctt 962Ser Val Gln Glu Thr Trp Leu Ala Ala Leu
Ile Val Leu Ala Val Leu 305 310 315gaa gcc atc ctg ctg ctg atg ctc
atc ttc ctg cgg cag cgg att cgt 1010Glu Ala Ile Leu Leu Leu Met Leu
Ile Phe Leu Arg Gln Arg Ile Arg320 325 330 335att gcc atc gcc ctc
ctg aag gag gcc agc aag gct gtg gga cag atg 1058Ile Ala Ile Ala Leu
Leu Lys Glu Ala Ser Lys Ala Val Gly Gln Met 340 345 350atg tct acc
atg ttc tac cca ctg gtc acc ttt gtc ctc ctc ctc atc 1106Met Ser Thr
Met Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile 355 360 365tgc
att gcc tac tgg gcc atg act gct ctg tac ctg gct aca tcg ggg 1154Cys
Ile Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly 370 375
380caa ccc cag tat gtg ctc tgg gca tcc aac atc agc tcc ccc ggc tgt
1202Gln Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys
385 390 395gag aaa gtg cca ata aat aca tca tgc aac ccc acg gcc cac
ctt gtg 1250Glu Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala His
Leu Val400 405 410 415aac tcc tcg tgc cca ggg ctg atg tgc gtc ttc
cag ggc tac tca tcc 1298Asn Ser Ser Cys Pro Gly Leu Met Cys Val Phe
Gln Gly Tyr Ser Ser 420 425 430aaa ggc cta atc caa cgt tct gtc ttc
aat ctg caa atc tat ggg gtc 1346Lys Gly Leu Ile Gln Arg Ser Val Phe
Asn Leu Gln Ile Tyr Gly Val 435 440 445ctg ggg ctc ttc tgg acc ctt
aac tgg gta ctg gcc ctg ggc caa tgc 1394Leu Gly Leu Phe Trp Thr Leu
Asn Trp Val Leu Ala Leu Gly Gln Cys 450 455 460gtc ctc gct gga gcc
ttt gcc tcc ttc tac tgg gcc ttc cac aag ccc 1442Val Leu Ala Gly Ala
Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro 465 470 475cag gac atc
cct acc ttc ccc tta atc tct gcc ttc atc cgc aca ctc 1490Gln Asp Ile
Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr Leu480 485 490
495cgt tac cac act ggg tca ttg gca ttt gga gcc ctc atc ctg acc ctt
1538Arg Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu
500 505 510gtg cag ata gcc cgg gtc atc ttg gag tat att gac cac aag
ctc aga 1586Val Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys
Leu Arg 515 520 525gga gtg cag aac cct gta gcc cgc tgc atc atg tgc
tgt ttc aag tgc 1634Gly Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys
Cys Phe Lys Cys 530 535 540tgc ctc tgg tgt ctg gaa aaa ttt atc aag
ttc cta aac cgc aat gca 1682Cys Leu Trp Cys Leu Glu Lys Phe Ile Lys
Phe Leu Asn Arg Asn Ala 545 550 555tac atc atg atc gcc atc tac ggg
aag aat ttc tgt gtc tca gcc aaa 1730Tyr Ile Met Ile Ala Ile Tyr Gly
Lys Asn Phe Cys Val Ser Ala Lys560 565 570 575aat gcg ttc atg cta
ctc atg cga aac att gtc agg gtg gtc gtc ctg 1778Asn Ala Phe Met Leu
Leu Met Arg Asn Ile Val Arg Val Val Val Leu 580 585 590gac aaa gtc
aca gac ctg ctg ctg ttc ttt ggg aag ctg ctg gtg gtc 1826Asp Lys Val
Thr Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val 595 600 605gga
ggc gtg ggg gtc ctg tcc ttc ttt ttt ttc tcc ggt cgc atc ccg 1874Gly
Gly Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro 610 615
620ggg ctg ggt aaa gac ttt aag agc ccc cac ctc aac tat tac tgg ctg
1922Gly Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu
625 630 635ccc atc atg acc tcc atc ctg ggg gcc tat gtc atc gcc agc
ggc ttc 1970Pro Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala Ser
Gly Phe640 645 650 655ttc agc gtt ttc ggc atg tgt gtg gac acg ctc
ttc ctc tgc ttc ctg 2018Phe Ser Val Phe Gly Met Cys Val Asp Thr Leu
Phe Leu Cys Phe Leu 660 665 670gaa gac ctg gag cgg aac aac ggc tcc
ctg gac cgg ccc tac tac atg 2066Glu Asp Leu Glu Arg Asn Asn Gly Ser
Leu Asp Arg Pro Tyr Tyr Met 675 680 685tcc aag agc ctt cta aag att
ctg ggc aag aag aac gag gcg ccc ccg 2114Ser Lys Ser Leu Leu Lys Ile
Leu Gly Lys Lys Asn Glu Ala Pro Pro 690 695 700gac aac aag aag agg
aag aag tga cagctccggc cctgatccag gactgcaccc 2168Asp Asn Lys Lys
Arg Lys Lys 705 710cacccccacc gtccagccat ccaacctcac ttcgccttac
aggtctccat tttgtggtaa 2228aaaaaggttt taggccaggc gccgtggctc
acgcctgtaa tccaacactt tgagaggctg 2288aggcgggcgg atcacctgag
tcaggagttc gagaccagcc tggccaacat ggtgaaacct 2348ccgtctctat
taaaaataca aaaattagcc gagagtggtg gcatgcacct gtcatcccag
2408ctactcggga ggctgaggca ggagaatcgc ttgaacccgg gaggcagagg
ttgcagtgag 2468ccgagatcgc gccactgcac tccaacctgg gtgacagact
ctgtctccaa aacaaaacaa 2528acaaacaaaa agattttatt aaagatattt
tgttaactca gtaaaaaaaa aaaaaaaaa 25878710PRTHomo
sapiensmisc_feature(1)...(710)24P4C12 variant 4 coding sequence
8Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly Lys Pro1 5
10 15 Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg Ser
Cys 20 25 30 Thr Asp Val Ile Cys Cys Val Leu Phe Leu Leu Phe Ile
Leu Gly Tyr 35 40 45 Ile Val Val Gly Ile Val Ala Trp Leu Tyr Gly
Asp Pro Arg Gln Val 50 55 60 Leu Tyr Pro Arg Asn Ser Thr Gly Ala
Tyr Cys Gly Met Gly Glu Asn65 70 75 80 Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile Leu 85 90 95 Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro Thr 100 105 110 Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val Gly 115 120 125 Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys Asn 130 135
140 Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
Thr145 150 155 160 Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu
Leu
Pro Ser Ala Pro 165 170 175 Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Val Thr Pro Pro Ala Leu 180 185 190 Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly Leu 195 200 205 Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu Asp 210 215 220 Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala Leu225 230 235 240 Val
Leu Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly Pro 245 250
255 Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr Gly
260 265 270 Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp Lys
Gly Ala 275 280 285 Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser
Ala Tyr Gln Ser 290 295 300 Val Gln Glu Thr Trp Leu Ala Ala Leu Ile
Val Leu Ala Val Leu Glu305 310 315 320 Ala Ile Leu Leu Leu Met Leu
Ile Phe Leu Arg Gln Arg Ile Arg Ile 325 330 335 Ala Ile Ala Leu Leu
Lys Glu Ala Ser Lys Ala Val Gly Gln Met Met 340 345 350 Ser Thr Met
Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile Cys 355 360 365 Ile
Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly Gln 370 375
380 Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys
Glu385 390 395 400 Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala
His Leu Val Asn 405 410 415 Ser Ser Cys Pro Gly Leu Met Cys Val Phe
Gln Gly Tyr Ser Ser Lys 420 425 430 Gly Leu Ile Gln Arg Ser Val Phe
Asn Leu Gln Ile Tyr Gly Val Leu 435 440 445 Gly Leu Phe Trp Thr Leu
Asn Trp Val Leu Ala Leu Gly Gln Cys Val 450 455 460 Leu Ala Gly Ala
Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro Gln465 470 475 480 Asp
Ile Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr Leu Arg 485 490
495 Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu Val
500 505 510 Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys Leu
Arg Gly 515 520 525 Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys Cys
Phe Lys Cys Cys 530 535 540 Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe
Leu Asn Arg Asn Ala Tyr545 550 555 560 Ile Met Ile Ala Ile Tyr Gly
Lys Asn Phe Cys Val Ser Ala Lys Asn 565 570 575 Ala Phe Met Leu Leu
Met Arg Asn Ile Val Arg Val Val Val Leu Asp 580 585 590 Lys Val Thr
Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val Gly 595 600 605 Gly
Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro Gly 610 615
620 Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu
Pro625 630 635 640 Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala
Ser Gly Phe Phe 645 650 655 Ser Val Phe Gly Met Cys Val Asp Thr Leu
Phe Leu Cys Phe Leu Glu 660 665 670 Asp Leu Glu Arg Asn Asn Gly Ser
Leu Asp Arg Pro Tyr Tyr Met Ser 675 680 685 Lys Ser Leu Leu Lys Ile
Leu Gly Lys Lys Asn Glu Ala Pro Pro Asp 690 695 700 Asn Lys Lys Arg
Lys Lys705 710 92587DNAHomo sapiensmisc_feature(1)...(2587)24P4C12
variant 5 9gagcc atg ggg gga aag cag cgg gac gag gat gac gag gcc
tac ggg aag 50 Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr
Gly Lys 1 5 10 15cca gtc aaa tac gac ccc tcc ttt cga ggc ccc atc
aag aac aga agc 98Pro Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile
Lys Asn Arg Ser 20 25 30tgc aca gat gtc atc tgc tgc gtc ctc ttc ctg
ctc ttc att cta ggt 146Cys Thr Asp Val Ile Cys Cys Val Leu Phe Leu
Leu Phe Ile Leu Gly 35 40 45tac atc gtg gtg ggg att gtg gcc tgg ttg
tat gga gac ccc cgg caa 194Tyr Ile Val Val Gly Ile Val Ala Trp Leu
Tyr Gly Asp Pro Arg Gln 50 55 60gtc ctc tac ccc agg aac tct act ggg
gcc tac tgt ggc atg ggg gag 242Val Leu Tyr Pro Arg Asn Ser Thr Gly
Ala Tyr Cys Gly Met Gly Glu 65 70 75aac aaa gat aag ccg tat ctc ctg
tac ttc aac atc ttc agc tgc atc 290Asn Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile80 85 90 95ctg tcc agc aac atc atc
tca gtt gct gag aac ggc cta cag tgc ccc 338Leu Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro 100 105 110aca ccc cag gtg
tgt gtg tcc tcc tgc ccg gag gac cca tgg act gtg 386Thr Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val 115 120 125gga aaa
aac gag ttc tca cag act gtt ggg gaa gtc ttc tat aca aaa 434Gly Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys 130 135
140aac agg aac ttt tgt ctg cca ggg gta ccc tgg aat atg acg gtg atc
482Asn Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
145 150 155aca agc ctg caa cag gaa ctc tgc ccc agt ttc ctc ctc ccc
tct gct 530Thr Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu Pro
Ser Ala160 165 170 175cca gct ctg ggg cgc tgc ttt cca tgg acc aac
gtt act cca ccg gcg 578Pro Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Val Thr Pro Pro Ala 180 185 190ctc cca ggg atc acc aat gac acc acc
ata cag cag ggg atc agc ggt 626Leu Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly 195 200 205ctt att gac agc ctc aat gcc
cga gac atc agt gtt aag atc ttt gaa 674Leu Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu 210 215 220gat ttt gcc cag tcc
tgg tat tgg att ctt gtt gcc ctg ggg gtg gct 722Asp Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala 225 230 235ctg gtc ttg
agc cta ctg ttt atc ttg ctt ctg cgc ctg gtg gct ggg 770Leu Val Leu
Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly240 245 250
255ccc ctg gtg ctg gtg ctg atc ctg gga gtg ctg ggc gtg ctg gca tac
818Pro Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr
260 265 270ggc atc tac tac tgc tgg gag gag tac cga gtg ctg cgg gac
aag ggc 866Gly Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp
Lys Gly 275 280 285gcc tcc atc tcc cag ctg ggt ttc acc acc aac ctc
agt gcc tac cag 914Ala Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu
Ser Ala Tyr Gln 290 295 300agc gtg cag gag acc tgg ctg gcc gcc ctg
atc gtg ttg gcg gtg ctt 962Ser Val Gln Glu Thr Trp Leu Ala Ala Leu
Ile Val Leu Ala Val Leu 305 310 315gaa gcc atc ctg ctg ctg gtg ctc
atc ttc ctg cgg cag cgg att cgt 1010Glu Ala Ile Leu Leu Leu Val Leu
Ile Phe Leu Arg Gln Arg Ile Arg320 325 330 335att gcc atc gcc ctc
ctg aag gag gcc agc aag gct gtg gga cag atg 1058Ile Ala Ile Ala Leu
Leu Lys Glu Ala Ser Lys Ala Val Gly Gln Met 340 345 350atg tct acc
atg ttc tac cca ctg gtc acc ttt gtc ctc ctc ctc atc 1106Met Ser Thr
Met Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile 355 360 365tgc
att gcc tac tgg gcc atg act gct ctg tac ctg gct aca tcg ggg 1154Cys
Ile Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly 370 375
380caa ccc cag tat gtg ctc tgg gca tcc aac atc agc tcc ccc ggc tgt
1202Gln Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys
385 390 395gag aaa gtg cca ata aat aca tca tgc aac ccc acg gcc cac
ctt gtg 1250Glu Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala His
Leu Val400 405 410 415aac tcc tcg tgc cca ggg ctg atg tgc gtc ttc
cag ggc tac tca tcc 1298Asn Ser Ser Cys Pro Gly Leu Met Cys Val Phe
Gln Gly Tyr Ser Ser 420 425 430aaa ggc cta atc caa cgt tct gtc ttc
aat ctg caa atc tat ggg gtc 1346Lys Gly Leu Ile Gln Arg Ser Val Phe
Asn Leu Gln Ile Tyr Gly Val 435 440 445ctg ggg ctc ttc tgg acc ctt
aac tgg gta ctg gcc ctg ggc caa tgc 1394Leu Gly Leu Phe Trp Thr Leu
Asn Trp Val Leu Ala Leu Gly Gln Cys 450 455 460gtc ctc gct gga gcc
ttt gcc tcc ttc tac tgg gcc ttc cac aag ccc 1442Val Leu Ala Gly Ala
Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro 465 470 475cag gac atc
cct acc ttc ccc tta atc tct gcc ttc atc cgc aca ctc 1490Gln Asp Ile
Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr Leu480 485 490
495cgt tac cac act ggg tca ttg gca ttt gga gcc ctc atc ctg acc ctt
1538Arg Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu
500 505 510gtg cag ata gcc cgg gtc atc ttg gag tat att gac cac aag
ctc aga 1586Val Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys
Leu Arg 515 520 525gga gtg cag aac cct gta gcc cgc tgc atc atg tgc
tgt ttc aag tgc 1634Gly Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys
Cys Phe Lys Cys 530 535 540tgc ctc tgg tgt ctg gaa aaa ttt atc aag
ttc cta aac cgc aat gca 1682Cys Leu Trp Cys Leu Glu Lys Phe Ile Lys
Phe Leu Asn Arg Asn Ala 545 550 555tac atc atg atc gcc atc tac ggg
aag aat ttc tgt gtc tca gcc aaa 1730Tyr Ile Met Ile Ala Ile Tyr Gly
Lys Asn Phe Cys Val Ser Ala Lys560 565 570 575aat gcg ttc atg cta
ctc atg cga aac att gtc agg gtg gtc gtc ctg 1778Asn Ala Phe Met Leu
Leu Met Arg Asn Ile Val Arg Val Val Val Leu 580 585 590gac aaa gtc
aca gac ctg ctg ctg ttc ttt ggg aag ctg ctg gtg gtc 1826Asp Lys Val
Thr Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val 595 600 605gga
ggc gtg ggg gtc ctg tcc ttc ttt ttt ttc tcc ggt cgc atc ccg 1874Gly
Gly Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro 610 615
620ggg ctg ggt aaa gac ttt aag agc ccc cac ctc aac tat tac tgg ctg
1922Gly Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu
625 630 635ccc atc atg acc tcc atc ctg ggg gcc tat gtc atc gcc agc
ggc ttc 1970Pro Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala Ser
Gly Phe640 645 650 655ttc agc gtt ttc ggc atg tgt gtg gac acg ctc
ttc ctc tgc ttc ctg 2018Phe Ser Val Phe Gly Met Cys Val Asp Thr Leu
Phe Leu Cys Phe Leu 660 665 670gaa gac ctg gag cgg aac aac ggc tcc
ctg gac cgg ccc tac tac atg 2066Glu Asp Leu Glu Arg Asn Asn Gly Ser
Leu Asp Arg Pro Tyr Tyr Met 675 680 685tcc aag agc ctt cta aag att
ctg ggc aag aag aac gag gcg ccc ccg 2114Ser Lys Ser Leu Leu Lys Ile
Leu Gly Lys Lys Asn Glu Ala Pro Pro 690 695 700gac aac aag aag agg
aag aag tga cagctccggc cctgatccag gactgcaccc 2168Asp Asn Lys Lys
Arg Lys Lys 705 710cacccccacc gtccagccat ccaacctcac ttcgccttac
aggtctccat tttgtggtaa 2228aaaaaggttt taggccaggc gccgtggctc
acgcctgtaa tccaacactt tgagaggctg 2288aggcgggcgg atcacctgag
tcaggagttc gagaccagcc tggccaacat ggtgaaacct 2348ccgtctctat
taaaaataca aaaattagcc gagagtggtg gcatgcacct gtcatcccag
2408ctactcggga ggctgaggca ggagaatcgc ttgaacccgg gaggcagagg
ttgcagtgag 2468ccgagatcgc gccactgcac tccaacctgg gtgacagact
ctgtctccaa aacaaaacaa 2528acaaacaaaa agattttatt aaagatattt
tgttaactca gtaaaaaaaa aaaaaaaaa 258710710PRTHomo
sapiensmisc_feature(1)...(710)24P4C12 variant 5 coding sequence
10Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly Lys Pro1
5 10 15 Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg Ser
Cys 20 25 30 Thr Asp Val Ile Cys Cys Val Leu Phe Leu Leu Phe Ile
Leu Gly Tyr 35 40 45 Ile Val Val Gly Ile Val Ala Trp Leu Tyr Gly
Asp Pro Arg Gln Val 50 55 60 Leu Tyr Pro Arg Asn Ser Thr Gly Ala
Tyr Cys Gly Met Gly Glu Asn65 70 75 80 Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile Leu 85 90 95 Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro Thr 100 105 110 Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val Gly 115 120 125 Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys Asn 130 135
140 Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
Thr145 150 155 160 Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu
Pro Ser Ala Pro 165 170 175 Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Val Thr Pro Pro Ala Leu 180 185 190 Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly Leu 195 200 205 Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu Asp 210 215 220 Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala Leu225 230 235 240 Val
Leu Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly Pro 245 250
255 Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr Gly
260 265 270 Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp Lys
Gly Ala 275 280 285 Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser
Ala Tyr Gln Ser 290 295 300 Val Gln Glu Thr Trp Leu Ala Ala Leu Ile
Val Leu Ala Val Leu Glu305 310 315 320 Ala Ile Leu Leu Leu Val Leu
Ile Phe Leu Arg Gln Arg Ile Arg Ile 325 330 335 Ala Ile Ala Leu Leu
Lys Glu Ala Ser Lys Ala Val Gly Gln Met Met 340 345 350 Ser Thr Met
Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile Cys 355 360 365 Ile
Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly Gln 370 375
380 Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys
Glu385 390 395 400 Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala
His Leu Val Asn 405 410 415 Ser Ser Cys Pro Gly Leu Met Cys Val Phe
Gln Gly Tyr Ser Ser Lys 420 425 430 Gly Leu Ile Gln Arg Ser Val Phe
Asn Leu Gln Ile Tyr Gly Val Leu 435 440 445 Gly Leu Phe Trp Thr Leu
Asn Trp Val Leu Ala Leu Gly Gln Cys Val 450 455 460 Leu Ala Gly Ala
Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro Gln465 470 475 480 Asp
Ile Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr Leu Arg 485 490
495 Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu Val
500 505 510 Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys Leu
Arg Gly 515 520 525 Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys Cys
Phe Lys Cys Cys 530 535 540 Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe
Leu Asn Arg Asn Ala Tyr545 550 555 560 Ile Met Ile Ala Ile Tyr Gly
Lys Asn Phe Cys Val Ser Ala Lys Asn 565 570 575 Ala Phe Met Leu Leu
Met Arg Asn Ile Val Arg Val Val Val Leu Asp 580 585 590 Lys Val Thr
Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val Gly 595 600 605
Gly
Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro Gly 610 615
620 Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu
Pro625 630 635 640 Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala
Ser Gly Phe Phe 645 650 655 Ser Val Phe Gly Met Cys Val Asp Thr Leu
Phe Leu Cys Phe Leu Glu 660 665 670 Asp Leu Glu Arg Asn Asn Gly Ser
Leu Asp Arg Pro Tyr Tyr Met Ser 675 680 685 Lys Ser Leu Leu Lys Ile
Leu Gly Lys Lys Asn Glu Ala Pro Pro Asp 690 695 700 Asn Lys Lys Arg
Lys Lys705 710 112587DNAHomo sapiensmisc_feature(1)...(2587)24P4C12
variant 6 11gagcc atg ggg gga aag cag cgg gac gag gat gac gag gcc
tac ggg aag 50 Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr
Gly Lys 1 5 10 15cca gtc aaa tac gac ccc tcc ttt cga ggc ccc atc
aag aac aga agc 98Pro Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile
Lys Asn Arg Ser 20 25 30tgc aca gat gtc atc tgc tgc gtc ctc ttc ctg
ctc ttc att cta ggt 146Cys Thr Asp Val Ile Cys Cys Val Leu Phe Leu
Leu Phe Ile Leu Gly 35 40 45tac atc gtg gtg ggg att gtg gcc tgg ttg
tat gga gac ccc cgg caa 194Tyr Ile Val Val Gly Ile Val Ala Trp Leu
Tyr Gly Asp Pro Arg Gln 50 55 60gtc ctc tac ccc agg aac tct act ggg
gcc tac tgt ggc atg ggg gag 242Val Leu Tyr Pro Arg Asn Ser Thr Gly
Ala Tyr Cys Gly Met Gly Glu 65 70 75aac aaa gat aag ccg tat ctc ctg
tac ttc aac atc ttc agc tgc atc 290Asn Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile80 85 90 95ctg tcc agc aac atc atc
tca gtt gct gag aac ggc cta cag tgc ccc 338Leu Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro 100 105 110aca ccc cag gtg
tgt gtg tcc tcc tgc ccg gag gac cca tgg act gtg 386Thr Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val 115 120 125gga aaa
aac gag ttc tca cag act gtt ggg gaa gtc ttc tat aca aaa 434Gly Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys 130 135
140aac agg aac ttt tgt ctg cca ggg gta ccc tgg aat atg acg gtg atc
482Asn Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
145 150 155aca agc ctg caa cag gaa ctc tgc ccc agt ttc ctc ctc ccc
tct gct 530Thr Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu Pro
Ser Ala160 165 170 175cca gct ctg ggg cgc tgc ttt cca tgg acc aac
gtt act cca ccg gcg 578Pro Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Val Thr Pro Pro Ala 180 185 190ctc cca ggg atc acc aat gac acc acc
ata cag cag ggg atc agc ggt 626Leu Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly 195 200 205ctt att gac agc ctc aat gcc
cga gac atc agt gtt aag atc ttt gaa 674Leu Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu 210 215 220gat ttt gcc cag tcc
tgg tat tgg att ctt gtt gcc ctg ggg gtg gct 722Asp Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala 225 230 235ctg gtc ttg
agc cta ctg ttt atc ttg ctt ctg cgc ctg gtg gct ggg 770Leu Val Leu
Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly240 245 250
255ccc ctg gtg ctg gtg ctg atc ctg gga gtg ctg ggc gtg ctg gca tac
818Pro Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr
260 265 270ggc atc tac tac tgc tgg gag gag tac cga gtg ctg cgg gac
aag ggc 866Gly Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp
Lys Gly 275 280 285gcc tcc atc tcc cag ctg ggt ttc acc acc aac ctc
agt gcc tac cag 914Ala Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu
Ser Ala Tyr Gln 290 295 300agc gtg cag gag acc tgg ctg gcc gcc ctg
atc gtg ttg gcg gtg ctt 962Ser Val Gln Glu Thr Trp Leu Ala Ala Leu
Ile Val Leu Ala Val Leu 305 310 315gaa gcc atc ctg ctg ctg atg ctc
atc ttc ctg cgg cag cgg att cgt 1010Glu Ala Ile Leu Leu Leu Met Leu
Ile Phe Leu Arg Gln Arg Ile Arg320 325 330 335att gcc atc gcc ctc
ctg aag gag gcc agc aag gct gtg gga cag atg 1058Ile Ala Ile Ala Leu
Leu Lys Glu Ala Ser Lys Ala Val Gly Gln Met 340 345 350atg tct acc
atg ttc tac cca ctg gtc acc ttt gtc ctc ctc ctc atc 1106Met Ser Thr
Met Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile 355 360 365tgc
att gcc tac tgg gcc atg act gct ctg tac ctg gct aca tcg ggg 1154Cys
Ile Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly 370 375
380caa ccc cag tat gtg ctc tgg gca tcc aac atc agc tcc ccc ggc tgt
1202Gln Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys
385 390 395gag aaa gtg cca ata aat aca tca tgc aac ccc acg gcc cac
ctt gtg 1250Glu Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala His
Leu Val400 405 410 415aac tcc tcg tgc cca ggg ctg atg tgc gtc ttc
cag ggc tac tca tcc 1298Asn Ser Ser Cys Pro Gly Leu Met Cys Val Phe
Gln Gly Tyr Ser Ser 420 425 430aaa ggc cta atc cca cgt tct gtc ttc
aat ctg caa atc tat ggg gtc 1346Lys Gly Leu Ile Pro Arg Ser Val Phe
Asn Leu Gln Ile Tyr Gly Val 435 440 445ctg ggg ctc ttc tgg acc ctt
aac tgg gta ctg gcc ctg ggc caa tgc 1394Leu Gly Leu Phe Trp Thr Leu
Asn Trp Val Leu Ala Leu Gly Gln Cys 450 455 460gtc ctc gct gga gcc
ttt gcc tcc ttc tac tgg gcc ttc cac aag ccc 1442Val Leu Ala Gly Ala
Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro 465 470 475cag gac atc
cct acc ttc ccc tta atc tct gcc ttc atc cgc aca ctc 1490Gln Asp Ile
Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr Leu480 485 490
495cgt tac cac act ggg tca ttg gca ttt gga gcc ctc atc ctg acc ctt
1538Arg Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu
500 505 510gtg cag ata gcc cgg gtc atc ttg gag tat att gac cac aag
ctc aga 1586Val Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys
Leu Arg 515 520 525gga gtg cag aac cct gta gcc cgc tgc atc atg tgc
tgt ttc aag tgc 1634Gly Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys
Cys Phe Lys Cys 530 535 540tgc ctc tgg tgt ctg gaa aaa ttt atc aag
ttc cta aac cgc aat gca 1682Cys Leu Trp Cys Leu Glu Lys Phe Ile Lys
Phe Leu Asn Arg Asn Ala 545 550 555tac atc atg atc gcc atc tac ggg
aag aat ttc tgt gtc tca gcc aaa 1730Tyr Ile Met Ile Ala Ile Tyr Gly
Lys Asn Phe Cys Val Ser Ala Lys560 565 570 575aat gcg ttc atg cta
ctc atg cga aac att gtc agg gtg gtc gtc ctg 1778Asn Ala Phe Met Leu
Leu Met Arg Asn Ile Val Arg Val Val Val Leu 580 585 590gac aaa gtc
aca gac ctg ctg ctg ttc ttt ggg aag ctg ctg gtg gtc 1826Asp Lys Val
Thr Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val 595 600 605gga
ggc gtg ggg gtc ctg tcc ttc ttt ttt ttc tcc ggt cgc atc ccg 1874Gly
Gly Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro 610 615
620ggg ctg ggt aaa gac ttt aag agc ccc cac ctc aac tat tac tgg ctg
1922Gly Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu
625 630 635ccc atc atg acc tcc atc ctg ggg gcc tat gtc atc gcc agc
ggc ttc 1970Pro Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala Ser
Gly Phe640 645 650 655ttc agc gtt ttc ggc atg tgt gtg gac acg ctc
ttc ctc tgc ttc ctg 2018Phe Ser Val Phe Gly Met Cys Val Asp Thr Leu
Phe Leu Cys Phe Leu 660 665 670gaa gac ctg gag cgg aac aac ggc tcc
ctg gac cgg ccc tac tac atg 2066Glu Asp Leu Glu Arg Asn Asn Gly Ser
Leu Asp Arg Pro Tyr Tyr Met 675 680 685tcc aag agc ctt cta aag att
ctg ggc aag aag aac gag gcg ccc ccg 2114Ser Lys Ser Leu Leu Lys Ile
Leu Gly Lys Lys Asn Glu Ala Pro Pro 690 695 700gac aac aag aag agg
aag aag tga cagctccggc cctgatccag gactgcaccc 2168Asp Asn Lys Lys
Arg Lys Lys 705 710cacccccacc gtccagccat ccaacctcac ttcgccttac
aggtctccat tttgtggtaa 2228aaaaaggttt taggccaggc gccgtggctc
acgcctgtaa tccaacactt tgagaggctg 2288aggcgggcgg atcacctgag
tcaggagttc gagaccagcc tggccaacat ggtgaaacct 2348ccgtctctat
taaaaataca aaaattagcc gagagtggtg gcatgcacct gtcatcccag
2408ctactcggga ggctgaggca ggagaatcgc ttgaacccgg gaggcagagg
ttgcagtgag 2468ccgagatcgc gccactgcac tccaacctgg gtgacagact
ctgtctccaa aacaaaacaa 2528acaaacaaaa agattttatt aaagatattt
tgttaactca gtaaaaaaaa aaaaaaaaa 258712710PRTHomo
sapiensmisc_feature(1)...(710)24P4C12 variant 6 coding sequence
12Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly Lys Pro1
5 10 15 Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg Ser
Cys 20 25 30 Thr Asp Val Ile Cys Cys Val Leu Phe Leu Leu Phe Ile
Leu Gly Tyr 35 40 45 Ile Val Val Gly Ile Val Ala Trp Leu Tyr Gly
Asp Pro Arg Gln Val 50 55 60 Leu Tyr Pro Arg Asn Ser Thr Gly Ala
Tyr Cys Gly Met Gly Glu Asn65 70 75 80 Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile Leu 85 90 95 Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro Thr 100 105 110 Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val Gly 115 120 125 Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys Asn 130 135
140 Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
Thr145 150 155 160 Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu
Pro Ser Ala Pro 165 170 175 Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Val Thr Pro Pro Ala Leu 180 185 190 Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly Leu 195 200 205 Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu Asp 210 215 220 Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala Leu225 230 235 240 Val
Leu Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly Pro 245 250
255 Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr Gly
260 265 270 Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp Lys
Gly Ala 275 280 285 Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser
Ala Tyr Gln Ser 290 295 300 Val Gln Glu Thr Trp Leu Ala Ala Leu Ile
Val Leu Ala Val Leu Glu305 310 315 320 Ala Ile Leu Leu Leu Met Leu
Ile Phe Leu Arg Gln Arg Ile Arg Ile 325 330 335 Ala Ile Ala Leu Leu
Lys Glu Ala Ser Lys Ala Val Gly Gln Met Met 340 345 350 Ser Thr Met
Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu Ile Cys 355 360 365 Ile
Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala Thr Ser Gly Gln 370 375
380 Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys
Glu385 390 395 400 Lys Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala
His Leu Val Asn 405 410 415 Ser Ser Cys Pro Gly Leu Met Cys Val Phe
Gln Gly Tyr Ser Ser Lys 420 425 430 Gly Leu Ile Pro Arg Ser Val Phe
Asn Leu Gln Ile Tyr Gly Val Leu 435 440 445 Gly Leu Phe Trp Thr Leu
Asn Trp Val Leu Ala Leu Gly Gln Cys Val 450 455 460 Leu Ala Gly Ala
Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro Gln465 470 475 480 Asp
Ile Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr Leu Arg 485 490
495 Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile Leu Thr Leu Val
500 505 510 Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys Leu
Arg Gly 515 520 525 Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys Cys
Phe Lys Cys Cys 530 535 540 Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe
Leu Asn Arg Asn Ala Tyr545 550 555 560 Ile Met Ile Ala Ile Tyr Gly
Lys Asn Phe Cys Val Ser Ala Lys Asn 565 570 575 Ala Phe Met Leu Leu
Met Arg Asn Ile Val Arg Val Val Val Leu Asp 580 585 590 Lys Val Thr
Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val Gly 595 600 605 Gly
Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg Ile Pro Gly 610 615
620 Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu
Pro625 630 635 640 Ile Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala
Ser Gly Phe Phe 645 650 655 Ser Val Phe Gly Met Cys Val Asp Thr Leu
Phe Leu Cys Phe Leu Glu 660 665 670 Asp Leu Glu Arg Asn Asn Gly Ser
Leu Asp Arg Pro Tyr Tyr Met Ser 675 680 685 Lys Ser Leu Leu Lys Ile
Leu Gly Lys Lys Asn Glu Ala Pro Pro Asp 690 695 700 Asn Lys Lys Arg
Lys Lys705 710 132251DNAHomo sapiensmisc_feature(1)...(2251)24P4C12
variant 7 13gagcc atg ggg gga aag cag cgg gac gag gat gac gag gcc
tac ggg aag 50 Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr
Gly Lys 1 5 10 15cca gtc aaa tac gac ccc tcc ttt cga ggc ccc atc
aag aac aga agc 98Pro Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile
Lys Asn Arg Ser 20 25 30tgc aca gat gtc atc tgc tgc gtc ctc ttc ctg
ctc ttc att cta ggt 146Cys Thr Asp Val Ile Cys Cys Val Leu Phe Leu
Leu Phe Ile Leu Gly 35 40 45tac atc gtg gtg ggg att gtg gcc tgg ttg
tat gga gac ccc cgg caa 194Tyr Ile Val Val Gly Ile Val Ala Trp Leu
Tyr Gly Asp Pro Arg Gln 50 55 60gtc ctc tac ccc agg aac tct act ggg
gcc tac tgt ggc atg ggg gag 242Val Leu Tyr Pro Arg Asn Ser Thr Gly
Ala Tyr Cys Gly Met Gly Glu 65 70 75aac aaa gat aag ccg tat ctc ctg
tac ttc aac atc ttc agc tgc atc 290Asn Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile80 85 90 95ctg tcc agc aac atc atc
tca gtt gct gag aac ggc cta cag tgc ccc 338Leu Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro 100 105 110aca ccc cag gtg
tgt gtg tcc tcc tgc ccg gag gac cca tgg act gtg 386Thr Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val 115 120 125gga aaa
aac gag ttc tca cag act gtt ggg gaa gtc ttc tat aca aaa 434Gly Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys 130 135
140aac agg aac ttt tgt ctg cca ggg gta ccc tgg aat atg acg gtg atc
482Asn Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
145 150 155aca agc ctg caa cag gaa ctc tgc ccc agt ttc ctc ctc ccc
tct gct 530Thr Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu Pro
Ser Ala160 165 170 175cca gct ctg ggg cgc tgc ttt cca tgg acc aac
gtt act cca ccg gcg 578Pro Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Val Thr Pro Pro Ala 180 185 190ctc cca ggg atc acc aat gac acc acc
ata cag cag ggg atc agc ggt 626Leu Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly 195 200 205ctt att gac agc ctc aat gcc
cga gac atc agt gtt aag atc ttt gaa 674Leu Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu
210 215 220gat ttt gcc cag tcc tgg tat tgg att ctt gtg gct gtg gga
cag atg 722Asp Phe Ala Gln Ser Trp Tyr Trp Ile Leu Val Ala Val Gly
Gln Met 225 230 235atg tct acc atg ttc tac cca ctg gtc acc ttt gtc
ctc ctc ctc atc 770Met Ser Thr Met Phe Tyr Pro Leu Val Thr Phe Val
Leu Leu Leu Ile240 245 250 255tgc att gcc tac tgg gcc atg act gct
ctg tac ctg gct aca tcg ggg 818Cys Ile Ala Tyr Trp Ala Met Thr Ala
Leu Tyr Leu Ala Thr Ser Gly 260 265 270caa ccc cag tat gtg ctc tgg
gca tcc aac atc agc tcc ccc ggc tgt 866Gln Pro Gln Tyr Val Leu Trp
Ala Ser Asn Ile Ser Ser Pro Gly Cys 275 280 285gag aaa gtg cca ata
aat aca tca tgc aac ccc acg gcc cac ctt gtg 914Glu Lys Val Pro Ile
Asn Thr Ser Cys Asn Pro Thr Ala His Leu Val 290 295 300aac tcc tcg
tgc cca ggg ctg atg tgc gtc ttc cag ggc tac tca tcc 962Asn Ser Ser
Cys Pro Gly Leu Met Cys Val Phe Gln Gly Tyr Ser Ser 305 310 315aaa
ggc cta atc caa cgt tct gtc ttc aat ctg caa atc tat ggg gtc 1010Lys
Gly Leu Ile Gln Arg Ser Val Phe Asn Leu Gln Ile Tyr Gly Val320 325
330 335ctg ggg ctc ttc tgg acc ctt aac tgg gta ctg gcc ctg ggc caa
tgc 1058Leu Gly Leu Phe Trp Thr Leu Asn Trp Val Leu Ala Leu Gly Gln
Cys 340 345 350gtc ctc gct gga gcc ttt gcc tcc ttc tac tgg gcc ttc
cac aag ccc 1106Val Leu Ala Gly Ala Phe Ala Ser Phe Tyr Trp Ala Phe
His Lys Pro 355 360 365cag gac atc cct acc ttc ccc tta atc tct gcc
ttc atc cgc aca ctc 1154Gln Asp Ile Pro Thr Phe Pro Leu Ile Ser Ala
Phe Ile Arg Thr Leu 370 375 380cgt tac cac act ggg tca ttg gca ttt
gga gcc ctc atc ctg acc ctt 1202Arg Tyr His Thr Gly Ser Leu Ala Phe
Gly Ala Leu Ile Leu Thr Leu 385 390 395gtg cag ata gcc cgg gtc atc
ttg gag tat att gac cac aag ctc aga 1250Val Gln Ile Ala Arg Val Ile
Leu Glu Tyr Ile Asp His Lys Leu Arg400 405 410 415gga gtg cag aac
cct gta gcc cgc tgc atc atg tgc tgt ttc aag tgc 1298Gly Val Gln Asn
Pro Val Ala Arg Cys Ile Met Cys Cys Phe Lys Cys 420 425 430tgc ctc
tgg tgt ctg gaa aaa ttt atc aag ttc cta aac cgc aat gca 1346Cys Leu
Trp Cys Leu Glu Lys Phe Ile Lys Phe Leu Asn Arg Asn Ala 435 440
445tac atc atg atc gcc atc tac ggg aag aat ttc tgt gtc tca gcc aaa
1394Tyr Ile Met Ile Ala Ile Tyr Gly Lys Asn Phe Cys Val Ser Ala Lys
450 455 460aat gcg ttc atg cta ctc atg cga aac att gtc agg gtg gtc
gtc ctg 1442Asn Ala Phe Met Leu Leu Met Arg Asn Ile Val Arg Val Val
Val Leu 465 470 475gac aaa gtc aca gac ctg ctg ctg ttc ttt ggg aag
ctg ctg gtg gtc 1490Asp Lys Val Thr Asp Leu Leu Leu Phe Phe Gly Lys
Leu Leu Val Val480 485 490 495gga ggc gtg ggg gtc ctg tcc ttc ttt
ttt ttc tcc ggt cgc atc ccg 1538Gly Gly Val Gly Val Leu Ser Phe Phe
Phe Phe Ser Gly Arg Ile Pro 500 505 510ggg ctg ggt aaa gac ttt aag
agc ccc cac ctc aac tat tac tgg ctg 1586Gly Leu Gly Lys Asp Phe Lys
Ser Pro His Leu Asn Tyr Tyr Trp Leu 515 520 525ccc atc atg acc tcc
atc ctg ggg gcc tat gtc atc gcc agc ggc ttc 1634Pro Ile Met Thr Ser
Ile Leu Gly Ala Tyr Val Ile Ala Ser Gly Phe 530 535 540ttc agc gtt
ttc ggc atg tgt gtg gac acg ctc ttc ctc tgc ttc ctg 1682Phe Ser Val
Phe Gly Met Cys Val Asp Thr Leu Phe Leu Cys Phe Leu 545 550 555gaa
gac ctg gag cgg aac aac ggc tcc ctg gac cgg ccc tac tac atg 1730Glu
Asp Leu Glu Arg Asn Asn Gly Ser Leu Asp Arg Pro Tyr Tyr Met560 565
570 575tcc aag agc ctt cta aag att ctg ggc aag aag aac gag gcg ccc
ccg 1778Ser Lys Ser Leu Leu Lys Ile Leu Gly Lys Lys Asn Glu Ala Pro
Pro 580 585 590gac aac aag aag agg aag aag tga cagctccggc
cctgatccag gactgcaccc 1832Asp Asn Lys Lys Arg Lys Lys 595cacccccacc
gtccagccat ccaacctcac ttcgccttac aggtctccat tttgtggtaa
1892aaaaaggttt taggccaggc gccgtggctc acgcctgtaa tccaacactt
tgagaggctg 1952aggcgggcgg atcacctgag tcaggagttc gagaccagcc
tggccaacat ggtgaaacct 2012ccgtctctat taaaaataca aaaattagcc
gagagtggtg gcatgcacct gtcatcccag 2072ctactcggga ggctgaggca
ggagaatcgc ttgaacccgg gaggcagagg ttgcagtgag 2132ccgagatcgc
gccactgcac tccaacctgg gtgacagact ctgtctccaa aacaaaacaa
2192acaaacaaaa agattttatt aaagatattt tgttaactca gtaaaaaaaa
aaaaaaaaa 225114598PRTHomo sapiensVARIANT(1)...(598)24P4C12 variant
7 coding sequence 14Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala
Tyr Gly Lys Pro1 5 10 15 Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro
Ile Lys Asn Arg Ser Cys 20 25 30 Thr Asp Val Ile Cys Cys Val Leu
Phe Leu Leu Phe Ile Leu Gly Tyr 35 40 45 Ile Val Val Gly Ile Val
Ala Trp Leu Tyr Gly Asp Pro Arg Gln Val 50 55 60 Leu Tyr Pro Arg
Asn Ser Thr Gly Ala Tyr Cys Gly Met Gly Glu Asn65 70 75 80 Lys Asp
Lys Pro Tyr Leu Leu Tyr Phe Asn Ile Phe Ser Cys Ile Leu 85 90 95
Ser Ser Asn Ile Ile Ser Val Ala Glu Asn Gly Leu Gln Cys Pro Thr 100
105 110 Pro Gln Val Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val
Gly 115 120 125 Lys Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr
Thr Lys Asn 130 135 140 Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn
Met Thr Val Ile Thr145 150 155 160 Ser Leu Gln Gln Glu Leu Cys Pro
Ser Phe Leu Leu Pro Ser Ala Pro 165 170 175 Ala Leu Gly Arg Cys Phe
Pro Trp Thr Asn Val Thr Pro Pro Ala Leu 180 185 190 Pro Gly Ile Thr
Asn Asp Thr Thr Ile Gln Gln Gly Ile Ser Gly Leu 195 200 205 Ile Asp
Ser Leu Asn Ala Arg Asp Ile Ser Val Lys Ile Phe Glu Asp 210 215 220
Phe Ala Gln Ser Trp Tyr Trp Ile Leu Val Ala Val Gly Gln Met Met225
230 235 240 Ser Thr Met Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu
Ile Cys 245 250 255 Ile Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala
Thr Ser Gly Gln 260 265 270 Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile
Ser Ser Pro Gly Cys Glu 275 280 285 Lys Val Pro Ile Asn Thr Ser Cys
Asn Pro Thr Ala His Leu Val Asn 290 295 300 Ser Ser Cys Pro Gly Leu
Met Cys Val Phe Gln Gly Tyr Ser Ser Lys305 310 315 320 Gly Leu Ile
Gln Arg Ser Val Phe Asn Leu Gln Ile Tyr Gly Val Leu 325 330 335 Gly
Leu Phe Trp Thr Leu Asn Trp Val Leu Ala Leu Gly Gln Cys Val 340 345
350 Leu Ala Gly Ala Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro Gln
355 360 365 Asp Ile Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile Arg Thr
Leu Arg 370 375 380 Tyr His Thr Gly Ser Leu Ala Phe Gly Ala Leu Ile
Leu Thr Leu Val385 390 395 400 Gln Ile Ala Arg Val Ile Leu Glu Tyr
Ile Asp His Lys Leu Arg Gly 405 410 415 Val Gln Asn Pro Val Ala Arg
Cys Ile Met Cys Cys Phe Lys Cys Cys 420 425 430 Leu Trp Cys Leu Glu
Lys Phe Ile Lys Phe Leu Asn Arg Asn Ala Tyr 435 440 445 Ile Met Ile
Ala Ile Tyr Gly Lys Asn Phe Cys Val Ser Ala Lys Asn 450 455 460 Ala
Phe Met Leu Leu Met Arg Asn Ile Val Arg Val Val Val Leu Asp465 470
475 480 Lys Val Thr Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val Val
Gly 485 490 495 Gly Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly Arg
Ile Pro Gly 500 505 510 Leu Gly Lys Asp Phe Lys Ser Pro His Leu Asn
Tyr Tyr Trp Leu Pro 515 520 525 Ile Met Thr Ser Ile Leu Gly Ala Tyr
Val Ile Ala Ser Gly Phe Phe 530 535 540 Ser Val Phe Gly Met Cys Val
Asp Thr Leu Phe Leu Cys Phe Leu Glu545 550 555 560 Asp Leu Glu Arg
Asn Asn Gly Ser Leu Asp Arg Pro Tyr Tyr Met Ser 565 570 575 Lys Ser
Leu Leu Lys Ile Leu Gly Lys Lys Asn Glu Ala Pro Pro Asp 580 585 590
Asn Lys Lys Arg Lys Lys 595 152623DNAHomo
sapiensmisc_feature(1)...(2623)24P4C12 variant 8 15gagcc atg ggg
gga aag cag cgg gac gag gat gac gag gcc tac ggg aag 50 Met Gly Gly
Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly Lys 1 5 10 15cca gtc
aaa tac gac ccc tcc ttt cga ggc ccc atc aag aac aga agc 98Pro Val
Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg Ser 20 25 30tgc
aca gat gtc atc tgc tgc gtc ctc ttc ctg ctc ttc att cta ggt 146Cys
Thr Asp Val Ile Cys Cys Val Leu Phe Leu Leu Phe Ile Leu Gly 35 40
45tac atc gtg gtg ggg att gtg gcc tgg ttg tat gga gac ccc cgg caa
194Tyr Ile Val Val Gly Ile Val Ala Trp Leu Tyr Gly Asp Pro Arg Gln
50 55 60gtc ctc tac ccc agg aac tct act ggg gcc tac tgt ggc atg ggg
gag 242Val Leu Tyr Pro Arg Asn Ser Thr Gly Ala Tyr Cys Gly Met Gly
Glu 65 70 75aac aaa gat aag ccg tat ctc ctg tac ttc aac atc ttc agc
tgc atc 290Asn Lys Asp Lys Pro Tyr Leu Leu Tyr Phe Asn Ile Phe Ser
Cys Ile80 85 90 95ctg tcc agc aac atc atc tca gtt gct gag aac ggc
cta cag tgc ccc 338Leu Ser Ser Asn Ile Ile Ser Val Ala Glu Asn Gly
Leu Gln Cys Pro 100 105 110aca ccc cag gtg tgt gtg tcc tcc tgc ccg
gag gac cca tgg act gtg 386Thr Pro Gln Val Cys Val Ser Ser Cys Pro
Glu Asp Pro Trp Thr Val 115 120 125gga aaa aac gag ttc tca cag act
gtt ggg gaa gtc ttc tat aca aaa 434Gly Lys Asn Glu Phe Ser Gln Thr
Val Gly Glu Val Phe Tyr Thr Lys 130 135 140aac agg aac ttt tgt ctg
cca ggg gta ccc tgg aat atg acg gtg atc 482Asn Arg Asn Phe Cys Leu
Pro Gly Val Pro Trp Asn Met Thr Val Ile 145 150 155aca agc ctg caa
cag gaa ctc tgc ccc agt ttc ctc ctc ccc tct gct 530Thr Ser Leu Gln
Gln Glu Leu Cys Pro Ser Phe Leu Leu Pro Ser Ala160 165 170 175cca
gct ctg ggg cgc tgc ttt cca tgg acc aac gtt act cca ccg gcg 578Pro
Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn Val Thr Pro Pro Ala 180 185
190ctc cca ggg atc acc aat gac acc acc ata cag cag ggg atc agc ggt
626Leu Pro Gly Ile Thr Asn Asp Thr Thr Ile Gln Gln Gly Ile Ser Gly
195 200 205ctt att gac agc ctc aat gcc cga gac atc agt gtt aag atc
ttt gaa 674Leu Ile Asp Ser Leu Asn Ala Arg Asp Ile Ser Val Lys Ile
Phe Glu 210 215 220gat ttt gcc cag tcc tgg tat tgg att ctt gtt gcc
ctg ggg gtg gct 722Asp Phe Ala Gln Ser Trp Tyr Trp Ile Leu Val Ala
Leu Gly Val Ala 225 230 235ctg gtc ttg agc cta ctg ttt atc ttg ctt
ctg cgc ctg gtg gct ggg 770Leu Val Leu Ser Leu Leu Phe Ile Leu Leu
Leu Arg Leu Val Ala Gly240 245 250 255ccc ctg gtg ctg gtg ctg atc
ctg gga gtg ctg ggc gtg ctg gca tac 818Pro Leu Val Leu Val Leu Ile
Leu Gly Val Leu Gly Val Leu Ala Tyr 260 265 270ggc atc tac tac tgc
tgg gag gag tac cga gtg ctg cgg gac aag ggc 866Gly Ile Tyr Tyr Cys
Trp Glu Glu Tyr Arg Val Leu Arg Asp Lys Gly 275 280 285gcc tcc atc
tcc cag ctg ggt ttc acc acc aac ctc agt gcc tac cag 914Ala Ser Ile
Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser Ala Tyr Gln 290 295 300agc
gtg cag gag acc tgg ctg gcc gcc ctg atc gtg ttg gcg gtg ctt 962Ser
Val Gln Glu Thr Trp Leu Ala Ala Leu Ile Val Leu Ala Val Leu 305 310
315gaa gcc atc ctg ctg ctg atg ctc atc ttc ctg cgg cag cgg att cgt
1010Glu Ala Ile Leu Leu Leu Met Leu Ile Phe Leu Arg Gln Arg Ile
Arg320 325 330 335att gcc atc gcc ctc ctg aag gag gcc agc aag gct
gtg gga cag atg 1058Ile Ala Ile Ala Leu Leu Lys Glu Ala Ser Lys Ala
Val Gly Gln Met 340 345 350atg tct acc atg ttc tac cca ctg gtc acc
ttt gtc ctc ctc ctc atc 1106Met Ser Thr Met Phe Tyr Pro Leu Val Thr
Phe Val Leu Leu Leu Ile 355 360 365tgc att gcc tac tgg gcc atg act
gct ctg tac ctg gct aca tcg ggg 1154Cys Ile Ala Tyr Trp Ala Met Thr
Ala Leu Tyr Leu Ala Thr Ser Gly 370 375 380caa ccc cag tat gtg ctc
tgg gca tcc aac atc agc tcc ccc ggc tgt 1202Gln Pro Gln Tyr Val Leu
Trp Ala Ser Asn Ile Ser Ser Pro Gly Cys 385 390 395gag aaa gtg cca
ata aat aca tca tgc aac ccc acg gcc cac ctt gtg 1250Glu Lys Val Pro
Ile Asn Thr Ser Cys Asn Pro Thr Ala His Leu Val400 405 410 415aac
tcc tcg tgc cca ggg ctg atg tgc gtc ttc cag ggc tac tca tcc 1298Asn
Ser Ser Cys Pro Gly Leu Met Cys Val Phe Gln Gly Tyr Ser Ser 420 425
430aaa ggc cta atc caa cgt tct gtc ttc aat ctg caa atc tat ggg gtc
1346Lys Gly Leu Ile Gln Arg Ser Val Phe Asn Leu Gln Ile Tyr Gly Val
435 440 445ctg ggg ctc ttc tgg acc ctt aac tgg gta ctg gcc ctg ggc
caa tgc 1394Leu Gly Leu Phe Trp Thr Leu Asn Trp Val Leu Ala Leu Gly
Gln Cys 450 455 460gtc ctc gct gga gcc ttt gcc tcc ttc tac tgg gcc
ttc cac aag ccc 1442Val Leu Ala Gly Ala Phe Ala Ser Phe Tyr Trp Ala
Phe His Lys Pro 465 470 475cag gac atc cct acc ttc ccc tta atc tct
gcc ttc atc cgc aca ctc 1490Gln Asp Ile Pro Thr Phe Pro Leu Ile Ser
Ala Phe Ile Arg Thr Leu480 485 490 495cgt tac cac act ggg tca ttg
gca ttt gga gcc ctc atc ctg acc ctt 1538Arg Tyr His Thr Gly Ser Leu
Ala Phe Gly Ala Leu Ile Leu Thr Leu 500 505 510gtg cag ata gcc cgg
gtc atc ttg gag tat att gac cac aag ctc aga 1586Val Gln Ile Ala Arg
Val Ile Leu Glu Tyr Ile Asp His Lys Leu Arg 515 520 525gga gtg cag
aac cct gta gcc cgc tgc atc atg tgc tgt ttc aag tgc 1634Gly Val Gln
Asn Pro Val Ala Arg Cys Ile Met Cys Cys Phe Lys Cys 530 535 540tgc
ctc tgg tgt ctg gaa aaa ttt atc aag ttc cta aac cgc aat gca 1682Cys
Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe Leu Asn Arg Asn Ala 545 550
555tac atc atg atc gcc atc tac ggg aag aat ttc tgt gtc tca gcc aaa
1730Tyr Ile Met Ile Ala Ile Tyr Gly Lys Asn Phe Cys Val Ser Ala
Lys560 565 570 575aat gcg ttc atg cta ctc atg cga aac att gtc agg
gtg gtc gtc ctg 1778Asn Ala Phe Met Leu Leu Met Arg Asn Ile Val Arg
Val Val Val Leu 580 585 590gac aaa gtc aca gac ctg ctg ctg ttc ttt
ggg aag ctg ctg gtg gtc 1826Asp Lys Val Thr Asp Leu Leu Leu Phe Phe
Gly Lys Leu Leu Val Val 595 600 605gga ggc gtg ggg gtc ctg tcc ttc
ttt ttt ttc tcc ggt cgc atc ccg 1874Gly Gly Val Gly Val Leu Ser Phe
Phe Phe Phe Ser Gly Arg Ile Pro 610 615 620ggg ctg ggt aaa gac ttt
aag agc ccc cac ctc aac tat tac tgg ctg 1922Gly Leu Gly Lys Asp Phe
Lys Ser Pro His Leu Asn Tyr Tyr Trp Leu 625 630 635ccc atc atg agg
aac cca ata acc cca acg ggt cat gtc ttc cag acc 1970Pro Ile Met Arg
Asn Pro Ile Thr Pro Thr Gly His Val Phe Gln Thr640 645 650 655tcc
atc ctg ggg gcc tat gtc atc gcc agc ggc ttc ttc agc gtt ttc 2018Ser
Ile Leu Gly Ala Tyr Val Ile Ala Ser Gly Phe Phe Ser Val Phe 660 665
670ggc atg tgt gtg gac acg ctc ttc ctc tgc ttc ctg gaa gac ctg gag
2066Gly Met Cys Val Asp Thr Leu Phe Leu Cys Phe Leu Glu Asp Leu Glu
675 680 685cgg aac aac ggc tcc ctg gac cgg ccc tac tac atg tcc aag
agc ctt 2114Arg Asn Asn Gly Ser Leu Asp Arg Pro Tyr Tyr Met Ser Lys
Ser Leu 690 695 700cta aag att ctg ggc aag aag
aac gag gcg ccc ccg gac aac aag aag 2162Leu Lys Ile Leu Gly Lys Lys
Asn Glu Ala Pro Pro Asp Asn Lys Lys 705 710 715agg aag aag tga
cagctccggc cctgatccag gactgcaccc cacccccacc 2214Arg Lys Lys
720gtccagccat ccaacctcac ttcgccttac aggtctccat tttgtggtaa
aaaaaggttt 2274taggccaggc gccgtggctc acgcctgtaa tccaacactt
tgagaggctg aggcgggcgg 2334atcacctgag tcaggagttc gagaccagcc
tggccaacat ggtgaaacct ccgtctctat 2394taaaaataca aaaattagcc
gagagtggtg gcatgcacct gtcatcccag ctactcggga 2454ggctgaggca
ggagaatcgc ttgaacccgg gaggcagagg ttgcagtgag ccgagatcgc
2514gccactgcac tccaacctgg gtgacagact ctgtctccaa aacaaaacaa
acaaacaaaa 2574agattttatt aaagatattt tgttaactca gtaaaaaaaa
aaaaaaaaa 262316722PRTHomo sapiensmisc_feature(1)...(722)24P4C12
variant 8 coding sequence 16Met Gly Gly Lys Gln Arg Asp Glu Asp Asp
Glu Ala Tyr Gly Lys Pro1 5 10 15 Val Lys Tyr Asp Pro Ser Phe Arg
Gly Pro Ile Lys Asn Arg Ser Cys 20 25 30 Thr Asp Val Ile Cys Cys
Val Leu Phe Leu Leu Phe Ile Leu Gly Tyr 35 40 45 Ile Val Val Gly
Ile Val Ala Trp Leu Tyr Gly Asp Pro Arg Gln Val 50 55 60 Leu Tyr
Pro Arg Asn Ser Thr Gly Ala Tyr Cys Gly Met Gly Glu Asn65 70 75 80
Lys Asp Lys Pro Tyr Leu Leu Tyr Phe Asn Ile Phe Ser Cys Ile Leu 85
90 95 Ser Ser Asn Ile Ile Ser Val Ala Glu Asn Gly Leu Gln Cys Pro
Thr 100 105 110 Pro Gln Val Cys Val Ser Ser Cys Pro Glu Asp Pro Trp
Thr Val Gly 115 120 125 Lys Asn Glu Phe Ser Gln Thr Val Gly Glu Val
Phe Tyr Thr Lys Asn 130 135 140 Arg Asn Phe Cys Leu Pro Gly Val Pro
Trp Asn Met Thr Val Ile Thr145 150 155 160 Ser Leu Gln Gln Glu Leu
Cys Pro Ser Phe Leu Leu Pro Ser Ala Pro 165 170 175 Ala Leu Gly Arg
Cys Phe Pro Trp Thr Asn Val Thr Pro Pro Ala Leu 180 185 190 Pro Gly
Ile Thr Asn Asp Thr Thr Ile Gln Gln Gly Ile Ser Gly Leu 195 200 205
Ile Asp Ser Leu Asn Ala Arg Asp Ile Ser Val Lys Ile Phe Glu Asp 210
215 220 Phe Ala Gln Ser Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala
Leu225 230 235 240 Val Leu Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu
Val Ala Gly Pro 245 250 255 Leu Val Leu Val Leu Ile Leu Gly Val Leu
Gly Val Leu Ala Tyr Gly 260 265 270 Ile Tyr Tyr Cys Trp Glu Glu Tyr
Arg Val Leu Arg Asp Lys Gly Ala 275 280 285 Ser Ile Ser Gln Leu Gly
Phe Thr Thr Asn Leu Ser Ala Tyr Gln Ser 290 295 300 Val Gln Glu Thr
Trp Leu Ala Ala Leu Ile Val Leu Ala Val Leu Glu305 310 315 320 Ala
Ile Leu Leu Leu Met Leu Ile Phe Leu Arg Gln Arg Ile Arg Ile 325 330
335 Ala Ile Ala Leu Leu Lys Glu Ala Ser Lys Ala Val Gly Gln Met Met
340 345 350 Ser Thr Met Phe Tyr Pro Leu Val Thr Phe Val Leu Leu Leu
Ile Cys 355 360 365 Ile Ala Tyr Trp Ala Met Thr Ala Leu Tyr Leu Ala
Thr Ser Gly Gln 370 375 380 Pro Gln Tyr Val Leu Trp Ala Ser Asn Ile
Ser Ser Pro Gly Cys Glu385 390 395 400 Lys Val Pro Ile Asn Thr Ser
Cys Asn Pro Thr Ala His Leu Val Asn 405 410 415 Ser Ser Cys Pro Gly
Leu Met Cys Val Phe Gln Gly Tyr Ser Ser Lys 420 425 430 Gly Leu Ile
Gln Arg Ser Val Phe Asn Leu Gln Ile Tyr Gly Val Leu 435 440 445 Gly
Leu Phe Trp Thr Leu Asn Trp Val Leu Ala Leu Gly Gln Cys Val 450 455
460 Leu Ala Gly Ala Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro
Gln465 470 475 480 Asp Ile Pro Thr Phe Pro Leu Ile Ser Ala Phe Ile
Arg Thr Leu Arg 485 490 495 Tyr His Thr Gly Ser Leu Ala Phe Gly Ala
Leu Ile Leu Thr Leu Val 500 505 510 Gln Ile Ala Arg Val Ile Leu Glu
Tyr Ile Asp His Lys Leu Arg Gly 515 520 525 Val Gln Asn Pro Val Ala
Arg Cys Ile Met Cys Cys Phe Lys Cys Cys 530 535 540 Leu Trp Cys Leu
Glu Lys Phe Ile Lys Phe Leu Asn Arg Asn Ala Tyr545 550 555 560 Ile
Met Ile Ala Ile Tyr Gly Lys Asn Phe Cys Val Ser Ala Lys Asn 565 570
575 Ala Phe Met Leu Leu Met Arg Asn Ile Val Arg Val Val Val Leu Asp
580 585 590 Lys Val Thr Asp Leu Leu Leu Phe Phe Gly Lys Leu Leu Val
Val Gly 595 600 605 Gly Val Gly Val Leu Ser Phe Phe Phe Phe Ser Gly
Arg Ile Pro Gly 610 615 620 Leu Gly Lys Asp Phe Lys Ser Pro His Leu
Asn Tyr Tyr Trp Leu Pro625 630 635 640 Ile Met Arg Asn Pro Ile Thr
Pro Thr Gly His Val Phe Gln Thr Ser 645 650 655 Ile Leu Gly Ala Tyr
Val Ile Ala Ser Gly Phe Phe Ser Val Phe Gly 660 665 670 Met Cys Val
Asp Thr Leu Phe Leu Cys Phe Leu Glu Asp Leu Glu Arg 675 680 685 Asn
Asn Gly Ser Leu Asp Arg Pro Tyr Tyr Met Ser Lys Ser Leu Leu 690 695
700 Lys Ile Leu Gly Lys Lys Asn Glu Ala Pro Pro Asp Asn Lys Lys
Arg705 710 715 720 Lys Lys172593DNAHomo
sapiensmisc_feature(1)...(2593)24P4C12 variant 9 17gagcc atg ggg
gga aag cag cgg gac gag gat gac gag gcc tac ggg aag 50 Met Gly Gly
Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly Lys 1 5 10 15cca gtc
aaa tac gac ccc tcc ttt cga ggc ccc atc aag aac aga agc 98Pro Val
Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg Ser 20 25 30tgc
aca gat gtc atc tgc tgc gtc ctc ttc ctg ctc ttc att cta ggt 146Cys
Thr Asp Val Ile Cys Cys Val Leu Phe Leu Leu Phe Ile Leu Gly 35 40
45tac atc gtg gtg ggg att gtg gcc tgg ttg tat gga gac ccc cgg caa
194Tyr Ile Val Val Gly Ile Val Ala Trp Leu Tyr Gly Asp Pro Arg Gln
50 55 60gtc ctc tac ccc agg aac tct act ggg gcc tac tgt ggc atg ggg
gag 242Val Leu Tyr Pro Arg Asn Ser Thr Gly Ala Tyr Cys Gly Met Gly
Glu 65 70 75aac aaa gat aag ccg tat ctc ctg tac ttc aac atc ttc agc
tgc atc 290Asn Lys Asp Lys Pro Tyr Leu Leu Tyr Phe Asn Ile Phe Ser
Cys Ile80 85 90 95ctg tcc agc aac atc atc tca gtt gct gag aac ggc
cta cag tgc ccc 338Leu Ser Ser Asn Ile Ile Ser Val Ala Glu Asn Gly
Leu Gln Cys Pro 100 105 110aca ccc cag gtg tgt gtg tcc tcc tgc ccg
gag gac cca tgg act gtg 386Thr Pro Gln Val Cys Val Ser Ser Cys Pro
Glu Asp Pro Trp Thr Val 115 120 125gga aaa aac gag ttc tca cag act
gtt ggg gaa gtc ttc tat aca aaa 434Gly Lys Asn Glu Phe Ser Gln Thr
Val Gly Glu Val Phe Tyr Thr Lys 130 135 140aac agg aac ttt tgt ctg
cca ggg gta ccc tgg aat atg acg gtg atc 482Asn Arg Asn Phe Cys Leu
Pro Gly Val Pro Trp Asn Met Thr Val Ile 145 150 155aca agc ctg caa
cag gaa ctc tgc ccc agt ttc ctc ctc ccc tct gct 530Thr Ser Leu Gln
Gln Glu Leu Cys Pro Ser Phe Leu Leu Pro Ser Ala160 165 170 175cca
gct ctg ggg cgc tgc ttt cca tgg acc aac gtt act cca ccg gcg 578Pro
Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn Val Thr Pro Pro Ala 180 185
190ctc cca ggg atc acc aat gac acc acc ata cag cag ggg atc agc ggt
626Leu Pro Gly Ile Thr Asn Asp Thr Thr Ile Gln Gln Gly Ile Ser Gly
195 200 205ctt att gac agc ctc aat gcc cga gac atc agt gtt aag atc
ttt gaa 674Leu Ile Asp Ser Leu Asn Ala Arg Asp Ile Ser Val Lys Ile
Phe Glu 210 215 220gat ttt gcc cag tcc tgg tat tgg att ctt gtt gcc
ctg ggg gtg gct 722Asp Phe Ala Gln Ser Trp Tyr Trp Ile Leu Val Ala
Leu Gly Val Ala 225 230 235ctg gtc ttg agc cta ctg ttt atc ttg ctt
ctg cgc ctg gtg gct ggg 770Leu Val Leu Ser Leu Leu Phe Ile Leu Leu
Leu Arg Leu Val Ala Gly240 245 250 255ccc ctg gtg ctg gtg ctg atc
ctg gga gtg ctg ggc gtg ctg gca tac 818Pro Leu Val Leu Val Leu Ile
Leu Gly Val Leu Gly Val Leu Ala Tyr 260 265 270ggc atc tac tac tgc
tgg gag gag tac cga gtg ctg cgg gac aag ggc 866Gly Ile Tyr Tyr Cys
Trp Glu Glu Tyr Arg Val Leu Arg Asp Lys Gly 275 280 285gcc tcc atc
tcc cag ctg ggt ttc acc acc aac ctc agt gcc tac cag 914Ala Ser Ile
Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser Ala Tyr Gln 290 295 300agc
gtg cag gag acc tgg ctg gcc gcc ctg atc gtg ttg gcg gtg ctt 962Ser
Val Gln Glu Thr Trp Leu Ala Ala Leu Ile Val Leu Ala Val Leu 305 310
315gaa gcc atc ctg ctg ctg atg ctc atc ttc ctg cgg cag cgg att cgt
1010Glu Ala Ile Leu Leu Leu Met Leu Ile Phe Leu Arg Gln Arg Ile
Arg320 325 330 335att gcc atc gcc ctc ctg aag gag gcc agc aag gct
gtg gga cag atg 1058Ile Ala Ile Ala Leu Leu Lys Glu Ala Ser Lys Ala
Val Gly Gln Met 340 345 350atg tct acc atg ttc tac cca ctg gtc acc
ttt gtc ctc ctc ctc atc 1106Met Ser Thr Met Phe Tyr Pro Leu Val Thr
Phe Val Leu Leu Leu Ile 355 360 365tgc att gcc tac tgg gcc atg act
gct ctg tat cct ctg ccc acg cag 1154Cys Ile Ala Tyr Trp Ala Met Thr
Ala Leu Tyr Pro Leu Pro Thr Gln 370 375 380cca gcc act ctt gga tat
gtg ctc tgg gca tcc aac atc agc tcc ccc 1202Pro Ala Thr Leu Gly Tyr
Val Leu Trp Ala Ser Asn Ile Ser Ser Pro 385 390 395ggc tgt gag aaa
gtg cca ata aat aca tca tgc aac ccc acg gcc cac 1250Gly Cys Glu Lys
Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala His400 405 410 415ctt
gtg aac tcc tcg tgc cca ggg ctg atg tgc gtc ttc cag ggc tac 1298Leu
Val Asn Ser Ser Cys Pro Gly Leu Met Cys Val Phe Gln Gly Tyr 420 425
430tca tcc aaa ggc cta atc caa cgt tct gtc ttc aat ctg caa atc tat
1346Ser Ser Lys Gly Leu Ile Gln Arg Ser Val Phe Asn Leu Gln Ile Tyr
435 440 445ggg gtc ctg ggg ctc ttc tgg acc ctt aac tgg gta ctg gcc
ctg ggc 1394Gly Val Leu Gly Leu Phe Trp Thr Leu Asn Trp Val Leu Ala
Leu Gly 450 455 460caa tgc gtc ctc gct gga gcc ttt gcc tcc ttc tac
tgg gcc ttc cac 1442Gln Cys Val Leu Ala Gly Ala Phe Ala Ser Phe Tyr
Trp Ala Phe His 465 470 475aag ccc cag gac atc cct acc ttc ccc tta
atc tct gcc ttc atc cgc 1490Lys Pro Gln Asp Ile Pro Thr Phe Pro Leu
Ile Ser Ala Phe Ile Arg480 485 490 495aca ctc cgt tac cac act ggg
tca ttg gca ttt gga gcc ctc atc ctg 1538Thr Leu Arg Tyr His Thr Gly
Ser Leu Ala Phe Gly Ala Leu Ile Leu 500 505 510acc ctt gtg cag ata
gcc cgg gtc atc ttg gag tat att gac cac aag 1586Thr Leu Val Gln Ile
Ala Arg Val Ile Leu Glu Tyr Ile Asp His Lys 515 520 525ctc aga gga
gtg cag aac cct gta gcc cgc tgc atc atg tgc tgt ttc 1634Leu Arg Gly
Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys Cys Phe 530 535 540aag
tgc tgc ctc tgg tgt ctg gaa aaa ttt atc aag ttc cta aac cgc 1682Lys
Cys Cys Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe Leu Asn Arg 545 550
555aat gca tac atc atg atc gcc atc tac ggg aag aat ttc tgt gtc tca
1730Asn Ala Tyr Ile Met Ile Ala Ile Tyr Gly Lys Asn Phe Cys Val
Ser560 565 570 575gcc aaa aat gcg ttc atg cta ctc atg cga aac att
gtc agg gtg gtc 1778Ala Lys Asn Ala Phe Met Leu Leu Met Arg Asn Ile
Val Arg Val Val 580 585 590gtc ctg gac aaa gtc aca gac ctg ctg ctg
ttc ttt ggg aag ctg ctg 1826Val Leu Asp Lys Val Thr Asp Leu Leu Leu
Phe Phe Gly Lys Leu Leu 595 600 605gtg gtc gga ggc gtg ggg gtc ctg
tcc ttc ttt ttt ttc tcc ggt cgc 1874Val Val Gly Gly Val Gly Val Leu
Ser Phe Phe Phe Phe Ser Gly Arg 610 615 620atc ccg ggg ctg ggt aaa
gac ttt aag agc ccc cac ctc aac tat tac 1922Ile Pro Gly Leu Gly Lys
Asp Phe Lys Ser Pro His Leu Asn Tyr Tyr 625 630 635tgg ctg ccc atc
atg acc tcc atc ctg ggg gcc tat gtc atc gcc agc 1970Trp Leu Pro Ile
Met Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala Ser640 645 650 655ggc
ttc ttc agc gtt ttc ggc atg tgt gtg gac acg ctc ttc ctc tgc 2018Gly
Phe Phe Ser Val Phe Gly Met Cys Val Asp Thr Leu Phe Leu Cys 660 665
670ttc ctg gaa gac ctg gag cgg aac aac ggc tcc ctg gac cgg ccc tac
2066Phe Leu Glu Asp Leu Glu Arg Asn Asn Gly Ser Leu Asp Arg Pro Tyr
675 680 685tac atg tcc aag agc ctt cta aag att ctg ggc aag aag aac
gag gcg 2114Tyr Met Ser Lys Ser Leu Leu Lys Ile Leu Gly Lys Lys Asn
Glu Ala 690 695 700ccc ccg gac aac aag aag agg aag aag tga
cagctccggc cctgatccag 2164Pro Pro Asp Asn Lys Lys Arg Lys Lys 705
710gactgcaccc cacccccacc gtccagccat ccaacctcac ttcgccttac
aggtctccat 2224tttgtggtaa aaaaaggttt taggccaggc gccgtggctc
acgcctgtaa tccaacactt 2284tgagaggctg aggcgggcgg atcacctgag
tcaggagttc gagaccagcc tggccaacat 2344ggtgaaacct ccgtctctat
taaaaataca aaaattagcc gagagtggtg gcatgcacct 2404gtcatcccag
ctactcggga ggctgaggca ggagaatcgc ttgaacccgg gaggcagagg
2464ttgcagtgag ccgagatcgc gccactgcac tccaacctgg gtgacagact
ctgtctccaa 2524aacaaaacaa acaaacaaaa agattttatt aaagatattt
tgttaactca gtaaaaaaaa 2584aaaaaaaaa 259318712PRTHomo
sapiensmisc_feature(1)...(712)24P4C12 variant 9 coding sequence
18Met Gly Gly Lys Gln Arg Asp Glu Asp Asp Glu Ala Tyr Gly Lys Pro1
5 10 15 Val Lys Tyr Asp Pro Ser Phe Arg Gly Pro Ile Lys Asn Arg Ser
Cys 20 25 30 Thr Asp Val Ile Cys Cys Val Leu Phe Leu Leu Phe Ile
Leu Gly Tyr 35 40 45 Ile Val Val Gly Ile Val Ala Trp Leu Tyr Gly
Asp Pro Arg Gln Val 50 55 60 Leu Tyr Pro Arg Asn Ser Thr Gly Ala
Tyr Cys Gly Met Gly Glu Asn65 70 75 80 Lys Asp Lys Pro Tyr Leu Leu
Tyr Phe Asn Ile Phe Ser Cys Ile Leu 85 90 95 Ser Ser Asn Ile Ile
Ser Val Ala Glu Asn Gly Leu Gln Cys Pro Thr 100 105 110 Pro Gln Val
Cys Val Ser Ser Cys Pro Glu Asp Pro Trp Thr Val Gly 115 120 125 Lys
Asn Glu Phe Ser Gln Thr Val Gly Glu Val Phe Tyr Thr Lys Asn 130 135
140 Arg Asn Phe Cys Leu Pro Gly Val Pro Trp Asn Met Thr Val Ile
Thr145 150 155 160 Ser Leu Gln Gln Glu Leu Cys Pro Ser Phe Leu Leu
Pro Ser Ala Pro 165 170 175 Ala Leu Gly Arg Cys Phe Pro Trp Thr Asn
Val Thr Pro Pro Ala Leu 180 185 190 Pro Gly Ile Thr Asn Asp Thr Thr
Ile Gln Gln Gly Ile Ser Gly Leu 195 200 205 Ile Asp Ser Leu Asn Ala
Arg Asp Ile Ser Val Lys Ile Phe Glu Asp 210 215 220 Phe Ala Gln Ser
Trp Tyr Trp Ile Leu Val Ala Leu Gly Val Ala Leu225 230 235 240 Val
Leu Ser Leu Leu Phe Ile Leu Leu Leu Arg Leu Val Ala Gly Pro 245 250
255 Leu Val Leu Val Leu Ile Leu Gly Val Leu Gly Val Leu Ala Tyr Gly
260 265 270 Ile Tyr Tyr Cys Trp Glu Glu Tyr Arg Val Leu Arg Asp Lys
Gly Ala 275 280 285 Ser Ile Ser Gln Leu Gly Phe Thr Thr Asn Leu Ser
Ala Tyr Gln Ser 290 295 300 Val Gln Glu Thr Trp Leu Ala Ala Leu Ile
Val Leu Ala Val Leu Glu305
310 315 320 Ala Ile Leu Leu Leu Met Leu Ile Phe Leu Arg Gln Arg Ile
Arg Ile 325 330 335 Ala Ile Ala Leu Leu Lys Glu Ala Ser Lys Ala Val
Gly Gln Met Met 340 345 350 Ser Thr Met Phe Tyr Pro Leu Val Thr Phe
Val Leu Leu Leu Ile Cys 355 360 365 Ile Ala Tyr Trp Ala Met Thr Ala
Leu Tyr Pro Leu Pro Thr Gln Pro 370 375 380 Ala Thr Leu Gly Tyr Val
Leu Trp Ala Ser Asn Ile Ser Ser Pro Gly385 390 395 400 Cys Glu Lys
Val Pro Ile Asn Thr Ser Cys Asn Pro Thr Ala His Leu 405 410 415 Val
Asn Ser Ser Cys Pro Gly Leu Met Cys Val Phe Gln Gly Tyr Ser 420 425
430 Ser Lys Gly Leu Ile Gln Arg Ser Val Phe Asn Leu Gln Ile Tyr Gly
435 440 445 Val Leu Gly Leu Phe Trp Thr Leu Asn Trp Val Leu Ala Leu
Gly Gln 450 455 460 Cys Val Leu Ala Gly Ala Phe Ala Ser Phe Tyr Trp
Ala Phe His Lys465 470 475 480 Pro Gln Asp Ile Pro Thr Phe Pro Leu
Ile Ser Ala Phe Ile Arg Thr 485 490 495 Leu Arg Tyr His Thr Gly Ser
Leu Ala Phe Gly Ala Leu Ile Leu Thr 500 505 510 Leu Val Gln Ile Ala
Arg Val Ile Leu Glu Tyr Ile Asp His Lys Leu 515 520 525 Arg Gly Val
Gln Asn Pro Val Ala Arg Cys Ile Met Cys Cys Phe Lys 530 535 540 Cys
Cys Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe Leu Asn Arg Asn545 550
555 560 Ala Tyr Ile Met Ile Ala Ile Tyr Gly Lys Asn Phe Cys Val Ser
Ala 565 570 575 Lys Asn Ala Phe Met Leu Leu Met Arg Asn Ile Val Arg
Val Val Val 580 585 590 Leu Asp Lys Val Thr Asp Leu Leu Leu Phe Phe
Gly Lys Leu Leu Val 595 600 605 Val Gly Gly Val Gly Val Leu Ser Phe
Phe Phe Phe Ser Gly Arg Ile 610 615 620 Pro Gly Leu Gly Lys Asp Phe
Lys Ser Pro His Leu Asn Tyr Tyr Trp625 630 635 640 Leu Pro Ile Met
Thr Ser Ile Leu Gly Ala Tyr Val Ile Ala Ser Gly 645 650 655 Phe Phe
Ser Val Phe Gly Met Cys Val Asp Thr Leu Phe Leu Cys Phe 660 665 670
Leu Glu Asp Leu Glu Arg Asn Asn Gly Ser Leu Asp Arg Pro Tyr Tyr 675
680 685 Met Ser Lys Ser Leu Leu Lys Ile Leu Gly Lys Lys Asn Glu Ala
Pro 690 695 700 Pro Asp Asn Lys Lys Arg Lys Lys705 710
191410DNAHomo sapiensmisc_feature(1)...(1410)Ha5-1(5)2.1 heavy
chain 19atg gag ttt ggg ctg acc tgg gtt ttc ctc gtt gct ctt tta aga
ggt 48Met Glu Phe Gly Leu Thr Trp Val Phe Leu Val Ala Leu Leu Arg
Gly1 5 10 15gtc cag tgt cag gtg cag ctg gtg gag tct ggg gga ggc gtg
gtc cag 96Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln 20 25 30cct ggg agg tcc ctg aga ctc tcc tgt gca gcc tct gga
ttc acc ttc 144Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe 35 40 45agt agt tat ggc atg cac tgg gtc cgc cag gct cca
ggc aag ggg ctg 192Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu 50 55 60gag tgg gtg gca gtt atg tca tat gat gga agt
aaa aaa ttc tat aca 240Glu Trp Val Ala Val Met Ser Tyr Asp Gly Ser
Lys Lys Phe Tyr Thr65 70 75 80gac tcc gtg aag ggc cga ttc acc atc
tcc aga gac aat tcc aag aac 288Asp Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn 85 90 95acg ctg tat ctg caa atg aac agc
ctg aga gct gag gac acg gct gtg 336Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val 100 105 110tat tac tgt gcg aga gat
ggg ggt gac tat gtc cgc tac cac tac tac 384Tyr Tyr Cys Ala Arg Asp
Gly Gly Asp Tyr Val Arg Tyr His Tyr Tyr 115 120 125ggt atg gac gtc
tgg ggc caa ggg acc acg gtc acc gtc tcc tca gcc 432Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 130 135 140tcc acc
aag ggc cca tcg gtc ttc ccc ctg gcg ccc tgc tcc agg agc 480Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser145 150 155
160acc tcc gag agc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc
528Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
165 170 175ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gct ctg acc
agc ggc 576Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly 180 185 190gtg cac acc ttc cca gct gtc cta cag tcc tca gga
ctc tac tcc ctc 624Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu 195 200 205agc agc gtg gtg acc gtg ccc tcc agc aac
ttc ggc acc cag acc tac 672Ser Ser Val Val Thr Val Pro Ser Ser Asn
Phe Gly Thr Gln Thr Tyr 210 215 220acc tgc aac gta gat cac aag ccc
agc aac acc aag gtg gac aag aca 720Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys Thr225 230 235 240gtt gag cgc aaa tgt
tgt gtc gag tgc cca ccg tgc cca gca cca cct 768Val Glu Arg Lys Cys
Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro 245 250 255gtg gca gga
ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc 816Val Ala Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270ctc
atg atc tcc cgg acc cct gag gtc acg tgc gtg gtg gtg gac gtg 864Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280
285agc cac gaa gac ccc gag gtc cag ttc aac tgg tac gtg gac ggc gtg
912Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
290 295 300gag gtg cat aat gcc aag aca aag cca cgg gag gag cag ttc
aac agc 960Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser305 310 315 320acg ttc cgt gtg gtc agc gtc ctc acc gtt gtg
cac cag gac tgg ctg 1008Thr Phe Arg Val Val Ser Val Leu Thr Val Val
His Gln Asp Trp Leu 325 330 335aac ggc aag gag tac aag tgc aag gtc
tcc aac aaa ggc ctc cca gcc 1056Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ala 340 345 350ccc atc gag aaa acc atc tcc
aaa acc aaa ggg cag ccc cga gaa cca 1104Pro Ile Glu Lys Thr Ile Ser
Lys Thr Lys Gly Gln Pro Arg Glu Pro 355 360 365cag gtg tac acc ctg
ccc cca tcc cgg gag gag atg acc aag aac cag 1152Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 370 375 380gtc agc ctg
acc tgc ctg gtc aaa ggc ttc tac ccc agc gac atc gcc 1200Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395
400gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc aca
1248Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
405 410 415cct ccc atg ctg gac tcc gac ggc tcc ttc ttc ctt tac agc
aag ctc 1296Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu 420 425 430acc gtg gac aag agc agg tgg cag cag ggg aac gtc
ttc tca tgc tcc 1344Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser 435 440 445gtg atg cat gag gct ctg cac aac cac tac
acg cag aag agc ctc tcc 1392Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 450 455 460ctg tct ccg ggt aaa tga 1410Leu
Ser Pro Gly Lys 46520469PRTHomo
sapiensmisc_feature(1)...(469)Ha5-1(5)2.1 heavy chain coding
sequence 20Met Glu Phe Gly Leu Thr Trp Val Phe Leu Val Ala Leu Leu
Arg Gly1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Met
Ser Tyr Asp Gly Ser Lys Lys Phe Tyr Thr65 70 75 80 Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110
Tyr Tyr Cys Ala Arg Asp Gly Gly Asp Tyr Val Arg Tyr His Tyr Tyr 115
120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
Ala 130 135 140 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser145 150 155 160 Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe 165 170 175 Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly 180 185 190 Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 195 200 205 Ser Ser Val Val
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr 210 215 220 Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr225 230 235
240 Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro
245 250 255 Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val 275 280 285 Ser His Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val 290 295 300 Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser305 310 315 320 Thr Phe Arg Val Val
Ser Val Leu Thr Val Val His Gln Asp Trp Leu 325 330 335 Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala 340 345 350 Pro
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro 355 360
365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Met Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 Leu Ser Pro
Gly Lys465 21711DNAHomo sapiensmisc_feature(1)...(711)Ha5-1(5)2.1
light chain 21atg gac atg agg gtc cct gct cag ctc ctg gga ctc ctg
ctg ctc tgg 48Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu
Leu Leu Trp1 5 10 15ctc cca gat acc aga tgt gac atc cag atg acc cag
tct cca tcc acc 96Leu Pro Asp Thr Arg Cys Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr 20 25 30ctg tct gca tct ata gga gac aga gtc acc atc
act tgc cgg gcg agt 144Leu Ser Ala Ser Ile Gly Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser 35 40 45cag ggc att agc tat tat tta gcc tgg tat
cag cag aaa ccg ggg aaa 192Gln Gly Ile Ser Tyr Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys 50 55 60att cct aag ctc ctg atc tat gat aca
tcc tct ttg caa tca ggg gtc 240Ile Pro Lys Leu Leu Ile Tyr Asp Thr
Ser Ser Leu Gln Ser Gly Val65 70 75 80cca tct cga ttc agt ggc agt
aga tct ggg aca gat ctc tct ctc acc 288Pro Ser Arg Phe Ser Gly Ser
Arg Ser Gly Thr Asp Leu Ser Leu Thr 85 90 95atc agc agc ctg cag cct
gaa gat gtt gca act tat tac tgt caa agg 336Ile Ser Ser Leu Gln Pro
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg 100 105 110tat gac agt gcc
ccg ctc act ttc ggc gga ggg acc aag gtg gag atc 384Tyr Asp Ser Ala
Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125aaa cga
act gtg gct gca cca tct gtc ttc atc ttc ccg cca tct gat 432Lys Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135
140gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac
480Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn145 150 155 160ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg
gat aac gcc ctc 528Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu 165 170 175caa tcg ggt aac tcc cag gag agt gtc aca
gag cag gac agc aag gac 576Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp 180 185 190agc acc tac agc ctc agc agc acc
ctg acg ctg agc aaa gca gac tac 624Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205gag aaa cac aaa gtc tac
gcc tgc gaa gtc acc cat cag ggc ctg agc 672Glu Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220tcg ccc gtc aca
aag agc ttc aac agg gga gag tgt tag 711Ser Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 225 230 23522236PRTHomo
sapiensmisc_feature(1)...(236)Ha5-1(5)2.1 light chain coding
sequence 22Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu
Leu Trp1 5 10 15 Leu Pro Asp Thr Arg Cys Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr 20 25 30 Leu Ser Ala Ser Ile Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser 35 40 45 Gln Gly Ile Ser Tyr Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ile Pro Lys Leu Leu Ile
Tyr Asp Thr Ser Ser Leu Gln Ser Gly Val65 70 75 80 Pro Ser Arg Phe
Ser Gly Ser Arg Ser Gly Thr Asp Leu Ser Leu Thr 85 90 95 Ile Ser
Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg 100 105 110
Tyr Asp Ser Ala Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115
120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230 235 23469PRTHomo
sapiensSITE(1)...(469)Ha5-1(5)2.1 heavy chain 23Met Glu Phe Gly Leu
Thr Trp Val Phe Leu Val Ala Leu Leu Arg Gly1 5 10 15 Val Gln Cys
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro
Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40
45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60 Glu Trp Val Ala Val Met Ser Tyr Asp Gly Ser Lys Lys Phe
Tyr Thr65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn 85 90
95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
100 105 110 Tyr Tyr Cys Ala Arg Asp Gly Gly Asp Tyr Val Arg Tyr His
Tyr Tyr 115 120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser Ala 130 135 140 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg Ser145 150 155 160 Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe 165 170 175 Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 180 185 190 Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 195 200 205 Ser
Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr 210 215
220 Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys
Thr225 230 235 240 Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys
Pro Ala Pro Pro 245 250 255 Val Ala Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val 275 280 285 Ser His Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser305 310 315 320 Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu 325 330
335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala385 390 395 400 Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455
460 Leu Ser Pro Gly Lys465 24236PRTHomo
sapiensmisc_feature(1)...(236)Ha5-1(5)2.1 light chain 24Met Asp Met
Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15 Leu
Pro Asp Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Thr 20 25
30 Leu Ser Ala Ser Ile Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45 Gln Gly Ile Ser Tyr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys 50 55 60 Ile Pro Lys Leu Leu Ile Tyr Asp Thr Ser Ser Leu
Gln Ser Gly Val65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly
Thr Asp Leu Ser Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp
Val Ala Thr Tyr Tyr Cys Gln Arg 100 105 110 Tyr Asp Ser Ala Pro Leu
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn145 150 155
160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys225 230 235 254PRTArtificial Sequencesynthetically
constructed linker 25Gly Phe Leu Gly1
* * * * *