U.S. patent application number 14/634606 was filed with the patent office on 2015-08-27 for antibody drug conjugates (adc) that bind to 158p1d7 proteins.
The applicant listed for this patent is AGENSYS, INC., SEATTLE GENETICS, INC.. Invention is credited to Zili AN, Xiao-Chi JIA, Karen Jane Meyrick MORRISON, Robert Kendall MORRISON, Josh SNYDER.
Application Number | 20150238633 14/634606 |
Document ID | / |
Family ID | 50150421 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150238633 |
Kind Code |
A1 |
MORRISON; Robert Kendall ;
et al. |
August 27, 2015 |
ANTIBODY DRUG CONJUGATES (ADC) THAT BIND TO 158P1D7 PROTEINS
Abstract
Antibody drug conjugates (ADC's) that bind to 158P1D7 protein
and variants thereof are described herein. 158P1D7 exhibits tissue
specific expression in normal adult tissue, and is aberrantly
expressed in glioblastoma, lung cancer, bladder cancer, and breast
cancer. Consequently, the ADC's of the invention provide a
therapeutic composition for the treatment of cancer.
Inventors: |
MORRISON; Robert Kendall;
(Santa Monica, CA) ; AN; Zili; (Santa Monica,
CA) ; MORRISON; Karen Jane Meyrick; (Santa Monica,
CA) ; SNYDER; Josh; (Santa Monica, CA) ; JIA;
Xiao-Chi; (Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AGENSYS, INC.
SEATTLE GENETICS, INC. |
Santa Monica
Bothell |
CA
WA |
US
US |
|
|
Family ID: |
50150421 |
Appl. No.: |
14/634606 |
Filed: |
February 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13975214 |
Aug 23, 2013 |
8968742 |
|
|
14634606 |
|
|
|
|
61692448 |
Aug 23, 2012 |
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Current U.S.
Class: |
530/391.7 |
Current CPC
Class: |
C07K 16/3015 20130101;
C07K 16/18 20130101; A61K 47/6849 20170801; C07K 2317/76 20130101;
A61K 45/06 20130101; A61K 47/6855 20170801; C07K 2317/92 20130101;
A61K 47/6859 20170801; C07K 2317/21 20130101; A61K 47/6861
20170801; A61K 47/6865 20170801; C07K 2317/56 20130101; A61K
47/6803 20170801; A61K 47/6813 20170801; C07K 16/30 20130101; A61K
47/6817 20170801; A61K 47/6851 20170801; A61P 35/00 20180101; C07K
16/3053 20130101; C07K 2317/73 20130101; A61K 47/6857 20170801;
C07K 16/3038 20130101; C07K 2317/77 20130101; A61K 2039/505
20130101; C07K 16/3023 20130101; A61K 47/6811 20170801; A61K 39/395
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Claims
1. An antibody drug conjugate comprising an antibody or antigen
binding fragment to monomethyl auristatin E (MMAE), wherein the
antibody or fragment comprises the heavy chain variable region
consisting of the amino acid sequence ranging from 1.sup.st Q to
the 120.sup.th S of SEQ ID NO: 7 and the light chain variable
consisting of the amino acid sequence ranging from 1.sup.st D to
the 113.sup.th R SEQ ID NO: 8.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/975,214, filed 23 Aug. 2013, now U.S. Pat. No. 8,968,742,
which claims priority to U.S. Provisional Patent Application No.
61/692,448, filed 23 Aug. 2012. All applications are herein
incorporated by reference in their entirety for all purposes.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] 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:
511582505001SeqList.txt, date recorded: Jan. 28, 2015, size: 40,186
bytes.
FIELD OF THE INVENTION
[0003] The invention described herein relates to antibodies,
binding fragments, and antibody drug conjugates (ADCs) thereof,
that bind proteins, termed 158P1D7. The invention further relates
to prognostic, prophylactic and therapeutic methods and
compositions useful in the treatment of cancers that express
158P1D7.
BACKGROUND OF THE INVENTION
[0004] Cancer is the second leading cause of human death next to
coronary disease.
[0005] 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 modalities 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 158P1D7
proteins and polypeptide fragments of 158P1D7 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 158P1D7 such as
cancers of tissues listed in Table I, especially bladder
cancer.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1. The cDNA and amino acid sequence of 158P1D7 is shown
in FIG. 1. The start methionine is underlined. The open reading
frame extends from nucleic acid 23-2548 including the stop codon.
The nucleic acid and amino acid sequences begin on the first panel
of FIG. 1, and continue on FIG. 1-2 and FIG. 1-3. FIG. 1-2 shows
nucleic acids 961-2040 and amino acids 314-693. FIG. 1-3 shows
nucleic acids 2041-2555 and amino acids 694-841.
[0032] FIG. 2. Nucleic Acid and Amino Acid sequences of 158P1D7
antibodies. FIG. 2A shows the cDNA and amino acid sequence of
Ha15-10ac12 heavy chain. Double-underlined is the heavy chain
variable region, underlined is the heavy chain human IgG2 constant
region. FIG. 2B shows the cDNA and amino acid sequence of
Ha15-10ac12 light chain. Double-underlined is the light chain
variable region, underlined is the human kappa constant region.
[0033] FIG. 3. Amino Acid sequences of 158P1D7 antibodies. FIG. 3A
shows the amino acid sequence of Ha15-10ac12 heavy chain.
Double-underlined is the heavy chain variable region, and
underlined is the human IgG2 constant region. FIG. 3B the amino
acid sequence of Ha15-10ac12 light chain. Double-underlined is the
light chain variable region, and underlined is the human kappa
constant region.
[0034] FIG. 4. Alignment of Ha15-10ac12 antibodies to human Ig
germline. FIG. 4A and FIG. 4A-2 alignment of Ha15-10ac12 heavy
chain (SEQ ID NO:3, positions 1-360; SEQ ID NO:4, positions 1-120)
to human Ig germline. FIG. 4B alignment of Ha15-10ac12 light chain
(SEQ ID NO:5, positions 1-240; SEQ ID NO:6, positions 1-80) to
human Ig germline IGKV2D-28*01 (SEQ ID NO:10).
[0035] FIG. 5. Efficacy of Ha15-10ac12vcMMAE in a Subcutaneously
Established Xenograft Model of Human Bladder Cancer AG-B7 in SCID
Mice.
[0036] FIG. 6. Efficacy of Ha15-10ac12vcMMAE in Subcutaneously
Established Human Bladder Cancer RT-4-XCL Implanted in SCID
Mice.
[0037] FIG. 7. Efficacy of Ha15-10ac12vcMMAE in Subcutaneously
Established Human Lung Cancer NCI-H322M-XCL Implanted in SCID
Mice.
[0038] FIG. 8. Efficacy of Ha15-10ac12vcMMAE in a Subcutaneously
Established Xenograft Model of Human Bladder Cancer AG-B7 in SCID
Mice.
[0039] FIG. 9. Detection of 158P1D7 protein in cancer patient
specimens by IHC. FIG. 9A and FIG. 9B show bladder cancer
specimens. FIG. 9C and FIG. 9D show breast cancer specimens. FIG.
9E and FIG. 9F show lung cancer specimens. FIG. 9G and FIG. 9H show
glioblastoma cancer specimens.
[0040] FIG. 10. Efficacy of Ha15-10ac12vcMMAE in subcutaneous
established xenograft model of human bladder cancer SW780 in SCID
mice.
[0041] FIG. 11. In vitro efficacy of Ha15-10ac12vcMMAE in CHP-212
cytotoxicity assay compared with M15-68(2)18 (a.k.a. 68(18)1.1)
Mab.
[0042] FIG. 12. Efficacy of Ha15-10ac12vcMMAE in Subcutaneous
established Xenograft model of Patient Derived human bladder cancer
AG-B8 in SCID mice
[0043] FIG. 13. A saturation curve for Ha15-10ac12vcMMAE.
[0044] FIG. 14. A histogram showing Mean Florescence Intensity
(MFI) of Ha15-10ac12vcMMAE.
[0045] FIG. 15A and FIG. 15B. Evaluation of the In-vitro
cytotoxicity of Ha15-10ac12vcMMAE on CHP-212 cells (FIG. 15A) and
IGROV-1 cells (FIG. 15B).
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
I.) Definitions
II.) 158P1D7 Antibodies
III.) Antibody Drug Conjugates Generally
[0046] III(A). Maytansinoids
[0047] III(B). Auristatins and dolostatins
[0048] III(C). Calicheamicin
[0049] III(D). Other Cytotoxic Agents
IV.) Antibody Drug Conjugates which Bind 158P1D7
V.) Linker Units
VI.) The Stretcher Unit
VII.) The Amino Acid Unit
VIII.) The Spacer Unit
IX.) The Drug Unit
X.) Drug Loading
[0050] XI.) Methods of Determining Cytotoxic effect of ADCs
XII.) Treatment of Cancer(s) Expressing 158P1D7
XIII) 158P1D7 as a Target for Antibody-based Therapy
XIV.) 158P1D7 ADC Cocktails
XV.) Combination Therapy
XVI.) Kits/Articles of Manufacture
I.) DEFINITIONS
[0051] 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.
[0052] 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.
[0053] 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.
[0054] The abbreviation "AFP" refers to
dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylened-
iamine (see Formula XVI infra).
[0055] The abbreviation "MMAE" refers to monomethyl auristatin E
(see Formula XI infra).
[0056] The abbreviation "AEB" refers to an ester produced by
reacting auristatin E with paraacetyl benzoic acid (see Formula XX
infra).
[0057] The abbreviation "AEVB" refers to an ester produced by
reacting auristatin E with benzoylvaleric acid (see Formula XXI
infra).
[0058] The abbreviation "MMAF" refers to
dovaline-valine-dolaisoleuine-dolaproine-phenylalanine (see Formula
XVIV infra).
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.2C.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.
[0063] 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.
[0064] 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--).
[0065] 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--).
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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).
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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).
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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, triphneylmethoxyacetate 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.
[0083] "Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence 158P1D7 (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 158P1D7. 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.
[0084] The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g. a 158P1D7-related protein). For example, an analog
of a 158P1D7 protein can be specifically bound by an antibody or T
cell that specifically binds to 158P1D7.
[0085] 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. 158P1D7 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 158P1D7 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 158P1D7 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 an
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
158P1D7. 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 158P1D7 or its receptor.
[0086] 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 158P1D7 antibody that retain the
ability to specifically bind to an antigen (e.g., 158P1D7 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.
[0087] As used herein, any form of the "antigen" can be used to
generate an antibody that is specific for 158P1D7. 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 158P1D7 of interest.
[0088] 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.
[0089] "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).
[0090] 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)).
[0091] 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.
[0092] 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.
[0093] The terms "complementarity determining region," and "CDR,"
are known in the art to refer to non-contiguous sequences of amino
acids within antibody variable regions, which confer antigen
specificity and binding affinity. In general, there are three (3)
CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3)
and three (3) CDRs in each light chain variable region (CDR-L1,
CDR-L2, CDR-L3).
[0094] The precise amino acid sequence boundaries of a given CDR
can be readily determined using any of a number of well-known
schemes, including those described by Kabat et al. (1991),
"Sequences of Proteins of Immunological Interest," 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB
273,927-948 ("Chothia" numbering scheme), MacCallum et al., J. Mol.
Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact
analysis and binding site topography," J. Mol. Biol. 262, 732-745."
(Contact" numbering scheme), Lefranc M P et al., "IMGT unique
numbering for immunoglobulin and T cell receptor variable domains
and Ig superfamily V-like domains," Dev Comp Immunol, 2003 January;
27(1):55-77 ("IMGT" numbering scheme), and Honegger A and Pluckthun
A, "Yet another numbering scheme for immunoglobulin variable
domains: an automatic modeling and analysis tool," J Mol Biol, 2001
Jun. 8; 309(3):657-70, (AHo numbering scheme).
[0095] The boundaries of a given CDR may vary depending on the
scheme used for identification. For example, the Kabat scheme is
based structural alignments, while the Chothia scheme is based on
structural information. Numbering for both the Kabat and Chothia
schemes is based upon the most common antibody region sequence
lengths, with insertions accommodated by insertion letters, for
example, "30a," and deletions appearing in some antibodies. The two
schemes place certain insertions and deletions ("indels") at
different positions, resulting in differential numbering. The
Contact scheme is based on analysis of complex crystal structures
and is similar in many respects to the Chothia numbering scheme.
Table V, infra, lists the positions of CDR-L1, CDR-L2, CDR-L3 and
CDR-H1, CDR-H2, CDR-H3 as identified by the Kabat, Chothia, and
Contact schemes, respectively. For CDR-H1, residue numbering is
given listed using both the Kabat and Chothia numbering
schemes.
[0096] Thus, unless otherwise specified, the terms "CDR" and
"complementary determining region" of a given antibody or region
thereof, such as a variable region, as well as individual CDRs
(e.g., "CDR-H1, CDR-H2) of the antibody or region thereof, should
be understood to encompass the complementary determining region as
defined by any of the known schemes described herein above. In some
instances, the scheme for identification of a particular CDR or
CDRs is specified, such as the CDR as defined by the Kabat,
Chothia, or Contact method. In other cases, the particular amino
acid sequence of a CDR is given.
[0097] 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.
[0098] 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.
[0099] Antibodies may also be conjugated to an anti-cancer pro-drug
activating enzyme capable of converting the pro-drug to its active
form.
[0100] 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).
[0101] The term "deplete," in the context of the effect of a
158P1D7 binding agent on 158P1D7-expressing cells, refers to a
reduction in the number of or elimination of the 158P1D7-expressing
cells.
[0102] 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.
[0103] "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.
[0104] 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.
[0105] 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).
[0106] 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).
[0107] The terms "inhibit" or "inhibition of" as used herein means
to reduce by a measurable amount, or to prevent entirely.
[0108] 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 158P1D7 genes or that encode
polypeptides other than 158P1D7 gene product or fragments thereof.
A skilled artisan can readily employ nucleic acid isolation
procedures to obtain an isolated 158P1D7 polynucleotide. A protein
is said to be "isolated," for example, when physical, mechanical or
chemical methods are employed to remove the 158P1D7 proteins from
cellular constituents that are normally associated with the
protein. A skilled artisan can readily employ standard purification
methods to obtain an isolated 158P1D7 protein. Alternatively, an
isolated protein can be prepared by chemical means.
[0109] 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).
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or composition that is physiologically compatible with humans
or other mammals.
[0117] 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).
[0118] The term "polypeptide" means a polymer of at least about 4,
5, 6, 7, or 8 amino acids. Throughout the specification, standard
three letter (See, Table III) or single letter designations for
amino acids are used. In the art, this term is often used
interchangeably with "peptide" or "protein".
[0119] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
[0120] 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).
[0121] 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 158P1D7 antigen, but does not bind to the irrelevant
antigen. In another embodiment, a specific antibody is one that
binds human 158P1D7 antigen but does not bind a non-human 158P1D7
antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or greater amino acid homology with the 158P1D7
antigen. In another embodiment, a specific antibody is one that
binds human 158P1D7 antigen and binds murine 158P1D7 antigen, but
with a higher degree of binding the human antigen. In another
embodiment, a specific antibody is one that binds human 158P1D7
antigen and binds primate 158P1D7 antigen, but with a higher degree
of binding the human antigen. In another embodiment, the specific
antibody binds to human 158P1D7 antigen and any non-human 158P1D7
antigen, but with a higher degree of binding the human antigen or
any combination thereof.
[0122] 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.
[0123] 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 158P1D7
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.
[0124] The "158P1D7 proteins" and/or "158P1D7 related proteins" of
the invention include those specifically identified herein (see,
FIG. 1), 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 158P1D7 proteins or fragments
thereof, as well as fusion proteins of a 158P1D7 protein and a
heterologous polypeptide are also included. Such 158P1D7 proteins
are collectively referred to as the 158P1D7-related proteins, the
proteins of the invention, or 158P1D7. The term "158P1D7-related
protein" refers to a polypeptide fragment or a 158P1D7 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,
345, 355, 365, 375, 385, 395, 405, 425, 450, 475, 500, 525, 550,
575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 830, 835,
840, 841 or more amino acids.
II.) 158P1D7 ANTIBODIES
[0125] Another aspect of the invention provides antibodies that
bind to 158P1D7-related proteins (See FIG. 1). In one embodiment,
the antibody that binds to 158P1D7-related proteins is an antibody
that specifically binds to 158P1D7 protein comprising amino acid
sequence of SEQ ID NO.: 2. The antibody that specifically binds to
158P1D7 protein comprising amino acid sequence of SEQ ID NO.: 2
includes antibodies that can bind to other 158P1D7-related
proteins. For example, antibodies that bind 158P1D7 protein
comprising amino acid sequence of SEQ ID NO.: 2 can bind
158P1D7-related proteins such as 158P1D7 variants and the homologs
or analogs thereof.
[0126] 158P1D7 antibodies of the invention are particularly useful
in cancer (see, e.g., Table I) prognostic assays, imaging,
diagnostic, and therapeutic methodologies. Similarly, such
antibodies are useful in the treatment, and/or prognosis of bladder
and other cancers, to the extent 158P1D7 is also expressed or
overexpressed in these other cancers. Moreover, 158P1D7 antibodies
of the invention are therapeutically useful in treating cancers in
which the expression of 158P1D7 is involved especially bladder
cancer, such as advanced or metastatic bladder cancers when the
antibodies are conjugated to monomethyl auristatin E (MMAE)
described herein.
[0127] 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 158P1D7-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 158P1D7 can also be used, such as a
158P1D7 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 158P1D7-related
protein is synthesized and used as an immunogen.
[0128] In addition, naked DNA immunization techniques known in the
art are used (with or without purified 158P1D7-related protein or
158P1D7 expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev.
Immunol. 15: 617-648).
[0129] The amino acid sequence of a 158P1D7 protein as shown in
FIG. 1 can be analyzed to select specific regions of the 158P1D7
protein for generating antibodies. For example, hydrophobicity and
hydrophilicity analyses of a 158P1D7 amino acid sequence are used
to identify hydrophilic regions in the 158P1D7 structure. Regions
of a 158P1D7 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 158P1D7 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
158P1D7 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.
[0130] 158P1D7 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
158P1D7-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.
[0131] The antibodies or fragments of the invention can also be
produced by recombinant means. Regions that bind specifically to
the desired regions of a 158P1D7 protein can also be produced in
the context of chimeric or complementarity-determining region (CDR)
grafted antibodies of multiple species origin. Humanized or human
158P1D7 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.
[0132] In a preferred embodiment, the antibodies of the present
invention comprise fully human 158P1D7 antibodies (158P1D7 MAbs).
Various methods in the art provide means for producing fully human
158P1D7 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).
[0133] 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).
[0134] In another embodiment, fully human antibodies of the
invention can be raised using a mouse that carries human
immunoglobulin sequences on transgenes and transchromosomes, 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.
[0135] 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.
[0136] 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.
[0137] Human monoclonal antibodies of the invention can also be
prepared using mice into which genomic sequences bearing endogenous
mouse variable segments at the immunoglobulin heavy chain (VH, DH,
and JH segments) and/or kappa light chain (VK and JK) loci have
been replaced, in whole or in part, with human genomic sequences
bearing unrearranged germline variable segments of the human
immunoglobulin heavy chain (VH, DH, and JH) and/or kappa light
chain (VK and JK) loci (Regeneron, Tarrytown, N.Y.). See, for
example, U.S. Pat. Nos. 6,586,251, 6,596,541, 7,105,348, 6,528,313,
6,638,768, and 6,528,314.
[0138] In one embodiment, an 158P1D7 MAbs of the invention
comprises heavy and light chain variable regions of an antibody
designated Ha15-10ac12 produced by a Chinese Hamster Ovary (CHO)
cell deposited under the American Type Culture Collection (ATCC)
Accession No.: PTA-13102 (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 Ha15-10ac12, such as functional fragments thereof, and wherein
the antibodies retain the desired functional properties of the
158P1D7 MAbs of the invention. The heavy chain variable region of
Ha15-10ac12 has the amino acid sequence ranging from the 1.sup.st Q
residue to the 120.sup.th S residue of SEQ ID NO: 7, and the light
chain variable region of Ha15-10ac12 has the amino acid sequence
ranging from the 1.sup.st D residue to the 113.sup.th R residue of
SEQ ID NO: 8.
[0139] In one embodiment, the 158P1D7 antibody contains a heavy
chain CDR of the heavy chain variable region of HA15-10ac12, such
as heavy chain CDR 1, 2, and/or 3 of the heavy chain variable
region of HA15-10ac12, e.g., CDR1, CDR2, and/or CDR3 of the amino
acid sequence set forth as SEQ ID NO: 7, determined by any known
numbering scheme for identifying CDRs, such as any described
herein. In one embodiment, the 158P1D7 antibody contains a light
chain CDR of the light chain variable region of HA15-10ac12, such
as light chain CDR 1, 2, and/or 3 of the light chain variable
region of HA15-10ac12, e.g., CDR1, CDR2, and/or CDR3 of the amino
acid sequence set forth as SEQ ID NO: 8, as determined by any known
numbering scheme for identifying CDRs, such as any described
herein. In one aspect, CDRs 1-3 of the heavy chain variable region
of Ha15-10ac12 contain the amino acid sequences ranging from
residues 31-35, from residues 50-66, and from residues 99-108,
respectively, of SEQ ID NO: 7. In one aspect, CDRs 1-3 of the light
chain variable region of Ha15-10ac12 contain the amino acid
sequences ranging from residues 24-39, from residues 55-61, and
from residues 94-102, respectively, of SEQ ID NO: 8. Thus, in some
aspects, the 158P1D7 antibody contains a heavy chain CDR1 having
residues 31-35 of SEQ ID NO: 7, a heavy chain CDR2 having residues
50-66 of SEQ ID NO: 7, and/or a heavy chain CDR3 having residues
99-108 of SEQ ID NO: 7 and/or a light chain CDR1 having residues
24-39 of SEQ ID NO: 8, a CDR2 having residues 55-61 of SEQ ID NO:
8, and/or a CDR3 having residues 94-102 of SEQ ID NO: 8. Also among
the provided embodiments are antibodies that compete for binding to
antigen with antibodies having such variable region and/or CDR
sequences. In some embodiments, the provided antibody includes a
constant region. The constant region can be any subclass of
constant region. 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.
[0140] 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:
[0141] (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
[0142] (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.
[0143] 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.
[0144] 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:
[0145] (a) the heavy chain variable region comprises one, two, or
three complementarity determining regions (CDRs) having the amino
acid sequence(s) of the heavy chain variable region CDRs 1, 2,
and/or 3, of the heavy chain variable region set forth in FIG. 3,
as determined using any known numbering scheme, such as any
described herein;
[0146] (b) the light chain variable region comprises one, two, or
three CDRs having the amino acid sequence(s) of the light chain
variable region CDRs 1, 2, and/or 3, of the light chain variable
region set forth in FIG. 3, as determined using any known numbering
scheme, such as any described herein.
[0147] In another embodiment, the antibody or antigen binding
portion thereof competes for binding with an antibody having such
heavy and/or light chain CDR(s).
[0148] 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.
[0149] 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 Can et al.
[0150] 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 158P1D7 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.
[0151] 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 158P1D7 MAb.
[0152] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half life of the 158P1D7 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.
[0153] In another embodiment, the 158P1D7 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.
[0154] 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 158P1D7 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.
[0155] Reactivity of 158P1D7 antibodies with a 158P1D7-related
protein can be established by a number of well known means,
including Western blot, immunoprecipitation, ELISA, and FACS
analyses using, as appropriate, 158P1D7-related proteins,
158P1D7-expressing cells or extracts thereof. A 158P1D7 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 158P1D7 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).
[0156] In yet another preferred embodiment, the 158P1D7 MAb of the
invention is an antibody comprising heavy and light chain of an
antibody designated Ha15-10ac12. The heavy chain of Ha15-10ac12
consists of the amino acid sequence ranging from 1.sup.st Q residue
to the 446.sup.th K residue of SEQ ID NO: 7 and the light chain of
Ha15-10ac12 consists of amino acid sequence ranging from 1.sup.st D
residue to the 219.sup.th C residue of SEQ ID NO: 8 sequence. The
sequence of which is set forth in FIG. 2 and FIG. 3. In a preferred
embodiment, Ha15-10ac12 is conjugated to a cytotoxic agent.
[0157] The Chinese Hamster Ovary (CHO) cell producing the antibody
designated Ha15-10ac12 was sent (via Federal Express) to the
American Type Culture Collection (ATCC), P.O. Box 1549, Manassas,
Va. 20108 on 25 Jul. 2012 and assigned Accession number
PTA-13102.
III.) ANTIBODY-DRUG CONJUGATES GENERALLY
[0158] 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 158P1D7 MAbs covalently
attached to a cytotoxic agent or a detectable agent.
[0159] 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 affect 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.
[0160] 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.111In 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).
[0161] Additionally, MYLOTARG.TM. (gemtuzumab ozogamicin, Wyeth
Pharmaceuticals), an antibody drug conjugate composed of a human
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;
5585089; 5606040; 5693762; 5739116; 5767285; 5773001).
[0162] 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.
[0163] 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.
[0164] Finally, the auristatin peptides, such as monomethyl
auristatin E (MMAE), synthetic analogs of dolastatin, were
conjugated to chimeric monoclonal antibodies cBR96 (specific to
Lewis Y on carcinomas) and cAzC10 (specific to CD30 on
hematological malignancies) (Doronina et al (2003) Nature
Biotechnology 21(7):778-784). The cAC10 is under therapeutic
development.
[0165] 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.122Bi, .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).
[0166] 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.
[0167] III(A). Maytansinoids
[0168] 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.
[0169] 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
[0170] 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.2 OR)(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).
[0171] 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.
[0172] III(B). Auristatins and Dolastatins
[0173] 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).
[0174] 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. 2005/0238649,
the disclosure of which is expressly incorporated by reference in
its entirety.
[0175] An exemplary auristatin embodiment is MMAE (wherein the wavy
line indicates the covalent attachment to a linker (L) of an
antibody drug conjugate).
##STR00002##
[0176] 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##
[0177] 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##
[0178] 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.
[0179] III(C). Calicheamicin
[0180] 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, and 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.
[0181] III(D). Other Cytotoxic Agents
[0182] 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).
[0183] 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.
[0184] 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).
[0185] 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.
[0186] 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 158P1D7
[0187] 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 158P1D7. 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-).
[0188] In some embodiments, the antibody drug conjugate compound
has the following formula:
L-(LU-D).sub.p (I)
[0189] or a pharmaceutically acceptable salt or solvate thereof;
wherein: [0190] L is the Antibody unit, e.g., 158P1D7 MAb of the
present invention, and [0191] (LU-D) is a Linker unit-Drug unit
moiety, wherein: [0192] LU- is a Linker unit, and [0193] -D is a
drug unit having cytostatic or cytotoxic activity against a target
cell; and [0194] p is an integer from 1 to 20.
[0195] 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.
[0196] 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)
[0197] or a pharmaceutically acceptable salt or solvate thereof,
wherein: [0198] L is the Antibody unit, e.g., 158P1D7 MAb; and
[0199] -A.sub.a-W.sub.w--Y.sub.y-- is a Linker unit (LU), wherein:
[0200] -A- is a Stretcher unit, [0201] a is 0 or 1,
[0202] each --W-- is independently an Amino Acid unit, [0203] w is
an integer ranging from 0 to 12, [0204] --Y-- is a self-immolative
spacer unit, [0205] y is 0, 1 or 2; [0206] -D is a drug units
having cytostatic or cytotoxic activity against the target cell;
and [0207] p is an integer from 1 to 20.
[0208] 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.
[0209] 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.
[0210] 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 158P1D7 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 158P1D7 MAb comprising heavy and light chain variable
regions of an antibody designated Ha15-10ac12 described above. In
more preferred embodiment, the Antibody is 158P1D7 MAb comprising
heavy and light chain of an antibody designated Ha15-10ac12
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.
[0211] 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 158P1D7 MAb under appropriate
conditions.
[0212] 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
[0213] 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.
[0214] 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 158P1D7-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: 9)).
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.
[0215] 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).
[0216] 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-S-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.)
[0217] 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).
[0218] 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).
[0219] 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.
[0220] 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 158P1D7 MAb.
[0221] 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).
[0222] 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--
[0223] wherein:
[0224] -A- is a Stretcher unit,
[0225] a is 0 or 1,
[0226] each --W-- is independently an Amino Acid unit,
[0227] w is an integer ranging from 0 to 12,
[0228] --Y-- is a self-immolative Spacer unit, and
[0229] y is 0, 1 or 2.
[0230] 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
[0231] 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 158P1D7 MAb (e.g.
Ha15-10ac12), 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 158P1D7 MAb. In another
embodiment, sulfhydryl groups can be generated by reaction of an
amino group of a lysine moiety of a 158P1D7 MAb with
2-iminothiolane (Traut's reagent) or other sulfhydryl generating
reagents. In certain embodiments, the 158P1D7 MAb is a recombinant
antibody and is engineered to carry one or more lysines. In certain
other embodiments, the recombinant 158P1D7 MAb is engineered to
carry additional sulfhydryl groups, e.g., additional cysteines.
[0232] 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.
[0233] 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##
[0234] An illustrative Stretcher unit is that of Formula Ma wherein
R.sup.17 is --(CH.sub.2).sub.5--:
##STR00006##
[0235] 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##
[0236] An illustrative Stretcher unit is that of Formula Ma 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.
[0237] Still another illustrative Stretcher unit is that of Formula
IIIb wherein R.sup.17 is --(CH.sub.2).sub.5--:
##STR00008##
[0238] 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##
[0239] 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##
[0240] 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##
[0241] 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
[0242] 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.
[0243] 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##
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##
[0244] 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).
[0245] 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):
##STR00015##
[0246] wherein R.sup.20 and R.sup.21 are as follows:
TABLE-US-00001 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;
##STR00017##
wherein R.sup.20, R.sup.21 and R.sup.22 are as follows:
TABLE-US-00002 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;
##STR00018##
wherein R.sup.20, R.sup.21, R.sup.22 and R.sup.23 are as
follows:
TABLE-US-00003 R.sup.20 R.sup.21 R.sup.22 R.sup.23 H benzyl
isobutyl H; and methyl isobutyl methyl isobutyl.
[0247] 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.
[0248] 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.
[0249] 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.
[0250] 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.
[0251] 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
[0252] 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.
[0253] 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##
[0254] 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-.
[0255] 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.
[0256] 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.
[0257] In some embodiments, --Y.sub.r-- 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.
[0258] 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##
[0259] 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.
[0260] 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##
[0261] 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.
[0262] 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.
[0263] 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##
[0264] 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.
[0265] In some embodiments, the -D moieties are the same. In yet
another embodiment, the -D moieties are different.
[0266] In one aspect, Spacer units (--Y.sub.r--) are represented by
Formulas (X)-(XII):
##STR00023##
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.
##STR00024##
[0267] Embodiments of the Formula I and II comprising antibody-drug
conjugate compounds can include:
##STR00025##
wherein w and y are each 0, 1 or 2, and,
##STR00026##
wherein w and y are each 0,
##STR00027##
IX.) THE DRUG UNIT
[0268] 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.
[0269] Useful classes of cytotoxic or immunomodulatory agents
include, for example, antitubulin agents, DNA minor groove binders,
DNA replication inhibitors, and alkylating agents.
[0270] 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.
[0271] 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 158P1D7-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.
[0272] 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 tubulin.
[0273] 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:
[0274] the wavy line indicates a bond;
[0275] 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;
[0276] 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);
[0277] 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);
[0278] R.sup.5 is --H or --C.sub.1-C.sub.8 alkyl;
[0279] 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,
[0280] 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; 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);
[0281] 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;
[0282] 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;
[0283] R.sup.24 is aryl, heterocycle, or -carbocycle;
[0284] 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;
[0285] 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;
[0286] R.sup.10 is -aryl or -heterocycle;
[0287] 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;
[0288] 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;
[0289] m is an integer ranging from 1-1000;
[0290] 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;
[0291] 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;
[0292] 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;
[0293] 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
[0294] n is an integer ranging from 0 to 6;
[0295] wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene,
alkynyklene, aryl, carbocyle, and heterocycle radicals, whether
alone or as part of another group, are optionally substituted.
[0296] Auristatins of the formula D.sub.E include those wherein
said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene,
aryl, carbocyle, and heterocycle radicals are unsubstituted.
[0297] 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.
[0298] Auristatins of the formula D.sub.E include those
wherein:
[0299] R.sup.2 is C.sub.1-C.sub.8 alkyl;
[0300] 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;
[0301] R.sup.5 is --H;
[0302] R.sup.6 is --C.sub.1-C.sub.8 alkyl;
[0303] 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;
[0304] R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl;
[0305] R.sup.24 is optionally substituted -phenyl;
[0306] 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;
[0307] 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.
[0308] Auristatins of the formula D.sub.E include those
wherein:
[0309] R.sup.2 is methyl;
[0310] 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;
[0311] 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;
[0312] R.sup.5 is --H;
[0313] R.sup.6 is methyl;
[0314] 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;
[0315] each R.sup.8 is methoxy;
[0316] R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl;
[0317] R.sup.24 is -phenyl;
[0318] 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;
[0319] R.sup.26 is methyl;
[0320] or a pharmaceutically acceptable salt form thereof.
[0321] 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.
[0322] Auristatins of the formula D.sub.E include those
wherein:
[0323] R.sup.2 is methyl or C.sub.1-C.sub.3 alkyl,
[0324] R.sup.3 is --H or --C.sub.1-C.sub.3 alkyl;
[0325] R.sup.4 is --C.sub.1-C.sub.5 alkyl;
[0326] R.sup.5 is H;
[0327] R.sup.6 is C.sub.1-C.sub.3 alkyl;
[0328] R.sup.7 is --C.sub.1-C.sub.5 alkyl;
[0329] R.sup.8 is --C.sub.1-C.sub.3 alkoxy;
[0330] R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl;
[0331] R.sup.24 is phenyl;
[0332] 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
[0333] R.sup.26 is --C.sub.1-C.sub.3 alkyl;
[0334] or a pharmaceutically acceptable salt form thereof.
[0335] Auristatins of the formula D.sub.F include those
wherein:
[0336] R.sup.2 is methyl;
[0337] 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;
[0338] R.sup.5 is --H;
[0339] R.sup.6 is methyl;
[0340] each R.sup.8 is methoxy;
[0341] 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;
[0342] R.sup.10 is optionally substituted aryl or optionally
substituted heterocycle;
[0343] 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;
[0344] 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;
[0345] m is an integer ranging from 1-1000 or m=0;
[0346] 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;
[0347] 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;
[0348] 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;
[0349] 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;
[0350] n is an integer ranging from 0 to 6;
[0351] or a pharmaceutically acceptable salt thereof.
[0352] In certain of these embodiments, R.sup.10 is optionally
substituted phenyl.
[0353] 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.
[0354] Auristatins of the formula D.sub.F include those wherein
said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene,
aryl, carbocyle, and heterocycle radicals are unsubstituted.
[0355] Auristatins of the formula D.sub.F include those
wherein:
[0356] 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.
[0357] Auristatins of the formula D.sub.F include those
wherein:
[0358] 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.
[0359] Auristatins of the formula D.sub.F include those
wherein:
[0360] 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.
[0361] Auristatins of the formula D.sub.F include those
wherein:
[0362] 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.
[0363] 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.
[0364] 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.
[0365] Auristatins of the formula D.sub.E or D.sub.F include those
wherein each occurrence of R.sup.8 is --OCH.sub.3. The remainder of
the substituents are as defined herein.
[0366] Auristatins of the formula D.sub.E or D.sub.F 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.
[0367] Auristatins of the formula D.sub.F include those wherein Z
is --O-- or --NH--. The remainder of the substituents are as
defined herein.
[0368] Auristatins of the formula D.sub.F include those wherein
R.sup.10 is aryl. The remainder of the substituents are as defined
herein.
[0369] Auristatins of the formula D.sub.F include those wherein
R.sup.10 is -phenyl. The remainder of the substituents are as
defined herein.
[0370] Auristatins of the formula D.sub.F 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.
[0371] Auristatins of the formula D.sub.F 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.
[0372] Auristatins of the formula D.sub.F 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.
[0373] 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.
[0374] In some embodiments, wherein D is an auristatin of formula
D.sub.F, a is 1 and w and y are 0.
[0375] Illustrative Drug units (-D) include the drug units having
the following structures:
##STR00029## ##STR00030## ##STR00031##
or pharmaceutically acceptable salts or solvates thereof.
[0376] 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.
[0377] 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.
[0378] Exemplary antibody-drug conjugate compounds have the
following structures wherein "L" or "mAb-s-" represents an 158P1D7
MAb designated Ha15-10ac12 set forth herein:
##STR00032##
or pharmaceutically acceptable salt thereof.
[0379] 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.
[0380] 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.
[0381] 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.
[0382] 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).
[0383] 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).
##STR00033##
[0384] In certain embodiments, the cytotoxic or cytostatic agent is
a compound of formulas XII-XXI or pharmaceutically acceptable salt
thereof:
##STR00034## ##STR00035##
X.) DRUG LOADING
[0385] 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.
[0386] 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).
[0387] 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.
[0388] 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)).
[0389] 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
[0390] 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.
[0391] 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 .sup.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.
[0392] 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.
[0393] 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.
[0394] The protein-binding dye sulforhodamine B (SRB) can also be
used to measure cytotoxicity (Skehan et al., 1990, J. Natl. Cancer
Inst. 82:1107-12).
[0395] 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).
[0396] 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).
[0397] 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.
[0398] 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).
[0399] In vivo, the effect of a 158P1D7 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.
[0400] 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.
[0401] 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).
[0402] 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.
[0403] 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 158P1D7
[0404] The identification of 158P1D7 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.
[0405] 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.
[0406] 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 dispensable, 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.
[0407] Accordingly, therapeutic approaches that inhibit the
activity of a 158P1D7 protein are useful for patients suffering
from a cancer that expresses 158P1D7. These therapeutic approaches
generally fall into three classes. The first class modulates
158P1D7 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 158P1D7 protein with its binding
partner or with other proteins. The third class comprises a variety
of methods for inhibiting the transcription of a 158P1D7 gene or
translation of 158P1D7 mRNA.
[0408] Accordingly, Cancer patients can be evaluated for the
presence and level of 158P1D7 expression, preferably using
immunohistochemical assessments of tumor tissue, quantitative
158P1D7 imaging, or other techniques that reliably indicate the
presence and degree of 158P1D7 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.) 158P1D7 AS A TARGET FOR ANTIBODY-BASED THERAPY
[0409] 158P1D7 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 158P1D7 is expressed by cancer
cells of various lineages relative to corresponding normal cells,
systemic administration of 158P1D7-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 158P1D7 are useful
to treat 158P1D7-expressing cancers systemically, preferably as
antibody drug conjugates (i.e. ADCs) wherein the conjugate is with
a toxin or therapeutic agent.
[0410] 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 158P1D7 sequence shown in FIG. 1. In
addition, skilled artisans understand that it is routine to
conjugate antibodies to cytotoxic agents (see, e.g., Sievers 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. 158P1D7), the cytotoxic agent will exert its known
biological effect (i.e. cytotoxicity) on those cells.
[0411] 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 158P1D7 MAb,
preferably Ha15-10ac12) that binds to an antigen (e.g. 158P1D7)
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 158P1D7, comprising
conjugating the cytotoxic agent to an antibody that
immunospecifically binds to a 158P1D7 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.
[0412] Cancer immunotherapy using 158P1D7 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).
[0413] Although 158P1D7 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.
[0414] 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 158P1D7
antigen with high affinity. Although the antibody drug conjugate of
the invention is useful for treating cancers in which the 158P1D7
is expressed, the antibody drug conjugate of the invention can be
particulary therapeutically useful in treating bladder cancers.
XIV.) 158P1D7 ADC COCKTAILS
[0415] Therapeutic methods of the invention contemplate the
administration of single 158P1D7 ADCs as well as combinations, or
cocktails, of different MAbs (i.e. 158P1D7 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, 158P1D7 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 158P1D7 MAbs are administered in
conjugated form.
[0416] 158P1D7 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
158P1D7 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.
[0417] 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 158P1D7 expression in the
patient, the extent of circulating shed 158P1D7 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.
[0418] Optionally, patients should be evaluated for the levels of
158P1D7 in a given sample (e.g. the levels of circulating 158P1D7
antigen and/or 158P1D7 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).
[0419] An object of the present invention is to provide 158P1D7
ADCs, which inhibit or retard the growth of tumor cells expressing
158P1D7. 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
158P1D7 ADCs, and in particular using such 158P1D7 ADCs combined
with other drugs or immunologically active treatments.
XV.) COMBINATION THERAPY
[0420] In one embodiment, there is synergy when tumors, including
human tumors, are treated with 158P1D7 ADCs in conjunction with
chemotherapeutic agents or radiation or combinations thereof. In
other words, the inhibition of tumor growth by a 158P1D7 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 158P1D7
ADC or the additive effect of treatment with a 158P1D7 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 158P1D7 ADC or with treatment
using an additive combination of a 158P1D7 ADC and a
chemotherapeutic agent or radiation.
[0421] The method for inhibiting growth of tumor cells using a
158P1D7 ADC and a combination of chemotherapy or radiation or both
comprises administering the 158P1D7 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 158P1D7
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.
[0422] 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 158P1D7 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, gemcitabine,
chlorambucil, taxol and combinations thereof.
[0423] The source of radiation, used in combination with a 158P1D7
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).
[0424] 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.
[0425] 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.
[0426] 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
[0427] 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.
[0428] 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.
[0429] 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.
[0430] The terms "kit" and "article of manufacture" can be used as
synonyms.
[0431] 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 158P1D7 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.
[0432] 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 158P1D7 or an antibody drug conjugate
specifically binding to 158P1D7.
[0433] 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
[0434] 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 158P1D7 Antigen
[0435] The 158P1D7 gene sequence was discovered using Suppression
Subtractive Hybridization (SSH) methods known in the art. The
158P1D7 SSH sequence of 223 bp was identified from a bladder cancer
pool minus normal bladder cDNA substraction. A full length cDNA
clone for 158P1D7 was isolated from a bladder cancer tissue pool.
The cDNA is 2,555 bp in length and encodes an 841 amino acid ORF
(See, FIG. 1). The 158P1D7 gene shows homology to SLITRK6 gene. For
further reference see, U.S. Pat. No. 6,863,892 (Agensys, Inc.,
Santa Monica, Calif.), U.S. Pat. No. 7,358,353 (Agensys, Inc.,
Santa Monica, Calif.) and EP 1,311,675 (Agensys, Inc., Santa
Monica, Calif.). For exemplary embodiments of the 158P1D7 antigen,
see FIG. 1.
Example 2
Generation of 158P1D7 Monoclonal Antibodies (MAbs)
[0436] In one embodiment, therapeutic Monoclonal Antibodies
("MAbs") to 158P1D7 and 158P1D7 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 158P1D7 or 158P1D7 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 158P1D7 protein sequence, regions predicted
to contain functional motifs, and regions of the 158P1D7 protein
variants predicted to be antigenic from computer analysis of the
amino acid sequence. Immunogens include peptides and recombinant
proteins such as tag5-158P1D7, a purified mammalian cell derived
His tagged protein. In addition, cells engineered to express high
levels of 158P1D7, such as UMUC-158P1D7 or 3T3-158P1D7, are used to
immunize mice.
[0437] MAbs to 158P1D7 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 Ha15-10ac12 was generated from immunization of
human .gamma.2 producing XenoMice with recombinant 3T3 cells
expressing 158P1D7.
[0438] The 158P1D7 MAb Ha15-10ac12 specifically binds to 158P1D7
expressing cells (recombinant and endogenous) as well as
recombinant 158P1D7 protein by ELISA.
[0439] DNA coding sequences for 158P1D7 MAb Ha15-10ac12 was
determined after isolating mRNA from the respective hybridoma cells
with Trizol reagent (Life Technologies, Gibco BRL).
[0440] Anti-158P1D7 Ha15-10ac12 heavy and light chain variable
nucleic acid sequences were sequenced from the hybridoma cells
using the following protocol. Ha15-10ac12 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.
[0441] 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 Ha15-10ac12 MAb to human Ig germline is set forth in
FIG. 4A-4B.
Example 3
Expression of Ha15-10Ac12 Using Recombinant DNA Methods
[0442] To express Ha15-10ac12 MAb recombinantly in transfected
cells, Ha15-10ac12 MAb variable heavy and light chain sequences
were cloned upstream of the human heavy chain IgG2 and human light
chain Ig.kappa. constant regions respectively. The complete
Ha15-10ac12 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 Ha15-10ac12 MAb expressing constructs
were transfected into CHO-K1SV cells. The Ha15-10ac12 related MAb
secreted from recombinant CHO cells was evaluated for binding to
cell surface 158P1D7 by flow cytometry. UMUC-control and
UMUC-158P1D7 cells were stained with Ha15-10ac12 MAb from either
hybridoma or from CHO cells transfected with Ha15-10ac12 heavy and
light chain vector constructs. Binding was detected by flow
cytometry.
[0443] Results show that the recombinantly expressed Ha15-10ac12
expressed in CHO cells binds 158P1D7 similarly to the Ha15-10ac12
purified from hybridoma. The Ha15-10ac12 MAb secreted from
recombinant cells was also evaluated for binding to 158P1D7
recombinant protein by ELISA. Binding of Ha15-10ac12 to 158P1D7
protein was identical between MAb material derived from CHO and
from hybridoma cells.
[0444] The Chinese Hamster Ovary (CHO) cell producing an antibody
designated Ha15-10ac12 was sent (via Federal Express) to the
American Type Culture Collection (ATCC), P.O. Box 1549, Manassas,
Va. 20108 on 25 Jul. 2012 and assigned Accession number
PTA-13102.
Example 4
Antibody Drug Conjugation of Ha15-10Ac12 MAb
[0445] The Ha15-10ac12 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 Ha15-10ac12vcMMAE 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 IV to create the cytotoxic vcMMAE (see,
US/2006/0074008).
[0446] Next, the antibody drug conjugate (ADC) of the invention
designated Ha15-10ac12vcMMAE was made using the following
protocols.
[0447] Briefly, the Ha15-10ac12 MAb in formulation buffer (10 mM
acetate pH 5.0 with 5% sorbitol) is buffer-exchanged to reduction
buffer (25 mM sodium borate, 300 mM sodium chloride, pH of
9.0.+-.0.1). The Ha15-10ac12 MAb is then partially reduced by
adding 5 mM EDTA and 2.65 molar equivalents of TCEP (relative to
moles of Ha15-10ac12 MAb). This mixture is then stirred at
37.degree. C. for three (3) hours. Following disulfide reduction,
the mixture is cooled to a target temperature of 15-17.degree. C.
and five (5) drug equivalents of vcMMAE per mole are added as a 4%
(v/v) solution of DMSO. After 60 to 75 minutes, the excess
unreacted vc is quenched by adding N-Acetyl-L-Cysteine in the
amount of 1 mole per mole of vcMMAE added at the beginning of
conjugation. After 15 minutes, the Ha15-10ac12vcMMAE is adjusted to
a target pH of 6.0-6.4 using a concentrated histidine pH 5.2 buffer
stock and filtered through a 0.5/0.2 .mu.m PES membrane to remove
aggregated antibody-drug-conjugate Immediately following
filtration, tangential flow filtration is performed to remove DMSO
and quenched drug linker and to exchange the
antibody-drug-conjugate to the buffer, 20 mM histidine (pH
6.0.+-.0.1) containing 5.5% trehalose dehydrate. After tangential
flow filtration, the Ha15-10ac12vcMMAE is diluted to a final
concentration of 6.+-.1 mg/mL and polysorbate 20 is added to a
final concentration of 0.01%.
[0448] The resulting antibody drug conjugate (ADC) is designated
Ha15-10ac12vcMMAE and has the following formula:
##STR00036##
wherein MAb is Ha15-10ac12 (FIG. 2 and FIG. 3) and p is from 1 to
12. The preferred p value of the antibody drug conjugate set forth
in this Example is between 3.5 and 3.7.
Example 5
Characterization of Ha15-10ac12vcMMAE
[0449] Antibody Drug Conjugates that bind 158P1D7 were generated
using the procedures set forth in the example entitled "Antibody
Drug Conjugation of Ha15-10ac12 MAb" and were screened, identified,
and characterized using a combination of assays known in the
art.
[0450] A. Affinity Determination by FACS
[0451] Ha15-10ac12vcMMAE was tested for its binding affinity to
158P1D7 expressed on the surface of SW780 cells. Briefly, eleven
(11) dilutions of Ha15-10ac12vcMMAE are incubated with SW780 cells
(50,000 cells per well) overnight at 4.degree. C. at a final
concentration of 6.67 nM to 0.0001 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 VI). 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 Ha15-10ac12vcMMAE saturation curve
shown in (FIG. 13). Bmax is the MFI value at maximal binding of
Ha15-10ac12vcMMAE to 158P1D7; Kd is the Ha15-10ac12vcMMAE binding
affinity which is the concentration of Ha15-10ac12vcMMAE required
to reach half-maximal binding.
[0452] The calculated affinity (Kd) of Ha15-10ac12vcMMAE to 158P1D7
expressed on the surface of SW780 cell is 0.005 nM.
[0453] B. FACS Binding
[0454] Ha15-10ac12vcMMAE was tested by FACS for its binding to
158P1D7 expressed on the surface of UMUC, SW780 and CHP-212 cells.
Briefly, the cells were harvested and plated at a concentration of
50,000 cells per well. Ha15-10ac12vcMMAE was diluted to either 3
.mu.g/mL (for UMUC and SW780 cells) or 10 .mu.g/mL (for CHP-212
cells) and incubated with the cells (1 hour at 4.degree. C.). At
the end of the incubation, cells are washed and incubated with
anti-hIgG-PE detection antibody for 1 hour at 4.degree. C. After
washing the unbound detection antibodies, the cells are analyzed by
FACS. Mean Florescence Intensity (MFI) values were obtained (Table
VII) and histograms are shown (FIG. 14).
Example 6
In Vitro Cell Cytotoxicity Mediated by Ha15-10ac12vcMMAE
[0455] The ability of Ha15-10ac12vcMMAE to mediate SLITRK-6
dependent cytotoxicity was evaluated using the human neuroblastoma
CHP-212 cell line, which endogenously express SLITRK-6 and the
human ovarian cancer cell line, IGROV-1, which does not express
SLITRK-6.
[0456] Briefly, the CHP-212 and IGROV-1 cells were seeded in 50
.mu.l of complete media, at a density of 1000 cell/well, onto 96
well plates and placed in a tissue culture incubator at 37 degrees
C.; 5% CO.sub.2. The next day a 2.times. stock solution of
Ha15-10ac12vcMMAE and isotype control antibody conjugated to vcMMAE
were prepared in complete media and 50 .mu.l of the serial
dilutions of the ADCs were added to the appropriate wells. The
cells were treated with Ha15-10ac12vcMMAE and the isotype control
antibody conjugated to vcMMAE for six (6) days in a tissue culture
incubator at 37 degrees C.; 5% CO.sub.2. At the end of the
incubation period, 12 .mu.l of Alamar Blue or Presto Blue was added
to each well and incubated for four (4) hours. The plates were read
using a BioTek Synergy H4 plate reader using 540 Excitation and 590
Emission wavelengths.
[0457] These data show that the ADC entitled Ha15-10ac12vcMMAE can
selectively induce the cytotoxicity of the SLITRK-6 expressing
CHP-212 cell line while it is unable to induce the cytotoxicity of
the SLITRK-6 non-expressing IGROV-1 cell line. Thus, these results
indicate that Ha15-10ac12vcMMAE can selectively deliver a cytotoxic
drug to 158P1D7 expressing cells leading to their killing (FIG.
15).
[0458] In another experiment, human neuroblastoma cells, CHP-212,
which express SLITRK6 (target for Ha15-10ac12 MAb and M15-68(2)18)
(a.k.a. 68(18)1.1, See, WO 2004/072263) MAb), were incubated with
the test articles, Ha15-10ac12vcMMAE and M15-68(2)18, to
demonstrate any in vitro cell killing (cytotoxicity) activity of
these articles. Briefly, ninety-six (96) well assay plates were
seeded with 4000 CHP-212 cells/ml and test antibodies were added in
a dilution series from 10,000 ng/ml down to 0.006 ng/ml using a six
(6) fold dilution over nine (9) points plus a 0.0 ng/ml final test
well. Wells were set up in triplicate. In addition, control
antibodies and control cells (IGROV-1) were set up in a similar way
for comparison with the test articles. The assay was allowed to run
for four (4) days before PrestoBlue (pigment that stains live
cells) was added to the wells to give a colorimetric readout of
cell viability. Percentage cell survival was then calculated in
each well and data was analyzed using non-linear fitting of
sigmoidal dose response. IC50 values were calculated using Prism
graphing software and cytotoxic effects of Ha15-10ac12vcMMAE were
compared with M15-68(2)18 and other controls used in the
experiment.
[0459] The results show that Ha15-10ac12vcMMAE possesses superior
in vitro cytotoxicity when compared with the M15-68(2)18 antibody
and other controls. (FIG. 11). The IC50 for Ha15-10ac12vcMMAE was
shown to be 0.9076 and the IC50 for M15-68(2)18 could not be
calculated.
Example 7
Ha15-10ac12vcMMAE Inhibit Growth of Tumors In Vivo
[0460] The significant expression of 158P1D7 on the cell surface of
tumor tissues, together with its restrictive expression in normal
tissues makes 158P1D7 a good target for antibody therapy and
similarly, therapy via ADC. Thus, the therapeutic efficacy of
Ha15-10ac12vcMMAE in human bladder, lung, breast, and glioblastoma
cancer xenograft mouse models is evaluated.
[0461] Antibody drug conjugate efficacy on tumor growth and
metastasis formation is studied in mouse cancer xenograft models
(e.g. subcutaneous and orthotopically).
[0462] 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
width.sup.2.times.Length/2, wherein width is the smallest dimension
and length is the largest dimension. Mice with subcutaneous tumors
greater than 1.5 cm in diameter are sacrificed.
[0463] 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.
[0464] 158P1D7 ADCs:
[0465] Monoclonal antibodies were raised against 158P1D7 as
described in the Example entitled "Generation of 158P1D7 Monoclonal
Antibodies (MAbs)." Further the MAbs are conjugated to a toxin as
described in the Example entitled "Antibody Drug Conjugation of
Ha15-10ac12 MAb" to form Ha15-10ac12vcMMAE. The Ha15-10ac12vcMMAE
is characterized by FACS, and other methods known in the art to
determine its capacity to bind 158P1D7.
[0466] Cell Lines and Xenografts:
[0467] The cells are maintained in DMEM, supplemented with
L-glutamine and 10% FBS, as known in the art. The AG-B7, RT-4-XCL,
and NCI-H322M-XCL xenografts are maintained by serial propagation
in SCID mice. The SW780 and RT-4-XCL are cell derived bladder
cancer line(s) that were obtained via the A.T.C.C. (Manassas, Va.).
The AG-B7 and AG-B8 are patient derived xenograft(s) derived from
human bladder cancer specimens. One of skill in the art will
appreciate that a plurality of experiments are performed in vivo on
the ADCs of the invention. This is due, in part, that every in vivo
model, even if in the same disease indication, exhibits a level of
unpredictability to those of skill in the art. Thus, one of skill
in the art will appreciate that utilization of several types of in
vivo models will allow one of skill in the art to better assess the
ADCs of the invention.
[0468] Evaluation of Ha15-10ac12vcMMAE in the Subcutaneous
Established Xenograft Model of Human Bladder Cancer AG-B7 in SCID
Mice
[0469] In this experiment, human bladder cancer AG-B7 cells
(5.times.10.sup.6 cells per mouse) were injected into the flanks of
individual SCID mice and tumors were allowed to grow untreated
until they reached an approximate volume of 250 mm.sup.3. At that
point, animals were allocated to each group based on tumor volume
at the time of treatment initiation to ensure similar mean tumor
size and variation in each group using Study Director Software
(v.1.7; Studylog Systems, Inc., South San Francisco, Calif.). All
ADC treated groups received a single dose at 10 mg/kg by
intravenous bolus injection. Tumor growth in each group was
monitored twice weekly using caliper measurements until study
termination. Statistical analysis of tumor volumes was performed at
the last time point when data from all groups were available using
a nonparametric analysis of variance (ANOVA) on the ranked
data.
[0470] The results show that Ha15-10ac12vcMMAE demonstrated a
potent inhibitory effect when compared to the non-treated control
(p<0.0001) (FIG. 5).
[0471] Evaluation of Ha15-10ac12vcMMAE in the Subcutaneous
Established Human Bladder Cancer RT-4-XCL Implanted in SCID
Mice
[0472] In another experiment, human bladder cancer xenograft AG-B7
stock tumors were harvested sterilely and minced in to small pieces
(approximately 1 mm.sup.3) Six pieces were implanted subcutaneously
into the flanks of individual SCID mice. When the average tumor
volumes reached a predetermined size of 100 mm.sup.3 in volume,
animals were randomized into ADC treated groups and a non-treated
control group (see tumor volume graph) with similar mean tumor size
and variation in each group using Study Director Software (v.1.7;
Studylog Systems, Inc., South San Francisco, Calif.). All ADC
treated groups, including two control ADCs, received a single dose
at 5 mg/kg by intravenous bolus injection. Tumor growth in each
group was monitored twice weekly using caliper measurements until
study termination. Statistical analysis of tumor volumes was
performed at the last time point when data from all groups were
available using a nonparametric analysis of variance (ANOVA) on the
ranked data.
[0473] The results show that Ha15-10ac12vcMMAE demonstrated a
potent tumor inhibitory effect when compared to the non-treated
control or to the corresponding ADC control H3-1.4.1.2vcMMAE (both
p<0.0001) (FIG. 6).
[0474] Evaluation of Ha15-10ac12vcMMAE in the Subcutaneous
Established Human Lung Cancer NCI-H322M-XCL Implanted in SCID
Mice
[0475] In another experiment, Human lung cancer NCI-H322M-XCL cells
(2.5.times.10.sup.6 cells per mouse) were injected into the flanks
of individual SCID mice and tumors were allowed to grow untreated
until they reached an approximate volume of 200 mm.sup.3. At that
point, animals were allocated to each group based on tumor volume
at the time of treatment initiation to ensure similar mean tumor
size and variation in each group using Study Director Software
(v.1.7; Studylog Systems, Inc., South San Francisco, Calif.). All
ADC groups including the two control ADCs were dosed at 3 mg/kg two
times per week for a total of five doses by intravenous bolus
injection. Tumor growth in each group was monitored twice weekly
using caliper measurements until study termination. Statistical
analysis of tumor volumes was performed at the last time point when
data from all groups were available using a nonparametric analysis
of variance (ANOVA) on the ranked data.
[0476] The results show that Ha15-10ac12vcMMAE demonstrated a
potent inhibitory effect when compared to the vehicle control
(p<0.0001) or to the corresponding ADC control Ha3-12bc1.1vcMMAE
(p=0.0041) (FIG. 7).
[0477] Efficacy of Ha15-10ac12vcMMAE in Subcutaneous Established
Xenograft Model of Human Bladder Cancer AG-B7 in SCID Mice
[0478] In another experiment, Human bladder cancer xenograft AG-B7
stock tumors were harvested sterilely and minced into small pieces
(approximately 1 mm.sup.3) Five pieces were implanted
subcutaneously into the flanks of individual SCID mice. Tumors were
allowed to grow untreated until they reached an approximate volume
of 200 mm.sup.3. At that point, animals were allocated to each
group based on tumor volume at the time of treatment initiation to
ensure similar mean tumor size and variation in each group using
Study Director Software (v.1.7; Studylog Systems, Inc., South San
Francisco Calif.). All ADC groups including the two control ADCs
were dosed at 0.5 mg/kg twice a week for a total of seven doses by
intravenous bolus injection. Tumor growth in each group was
monitored twice weekly using caliper measurements until study
termination. Statistical analysis of tumor volumes was performed at
the last time point when data from all groups were available using
a nonparametric analysis of variance (ANOVA) on the ranked
data.
[0479] The results show that Ha15-10ac12vcMMAE produced from
hybridoma (Ha15-10ac12.1vcMMAE) and CHO cells (Ha15-10ac12vcMMAE)
both demonstrated a potent tumor inhibitory effect when compared to
either the vehicle control (p<0.0001) or to the corresponding
ADC control Ha3-12abc1vcMMAE (p<0.0001) (FIG. 8).
[0480] Efficacy of Ha15-10ac12vcMMAE in Subcutaneous Established
Xenograft Model of Human Bladder Cancer SW780 in SCID Mice
[0481] In another experiment, human bladder cancer xenograft SW780
cells were implanted into the flanks of SCID mice and tumors were
allowed to grow until they reached approximate volume of 200
mm.sup.3. At that point, animals were allocated to the treatment
groups according to tumor volume at the time of treatment
initiation to ensure similar mean tumor size and variation in each
group. Allocation of mice into treatment groups was aided by Study
Director Software (v.1.7; Studylog Systems, Inc., South San
Francisco Calif.) to help sixe match the mice. All ADC groups
including the two control ADCs were dosed, by intravenous bolus
injection, at 1 mg/kg at the beginning of the study. Tumor growth
in each group was monitored twice weekly using caliper measurements
until study termination. Statistical analysis of tumor volumes was
performed at the last time point when data from all groups were
available using a nonparametric analysis of variance (ANOVA) on the
ranked data.
[0482] The results show that Ha15-10ac12vcMMAE has superior tumor
inhibitory activity than the Ha15-10ac12 MAb. Further, it can be
concluded that Ha15-10ac12 MAb has no tumor inhibitory effect as
the growth dynamics of the tumor in this treatment group follows
that of the isotype control antibodies. Further, after a single
dose of 1 mg/kg, Ha15-10ac12vcMMAE showed statistically significant
growth inhibition when compared to the isotype control ADC
(p<0.001) (FIG. 10).
[0483] Efficacy of Ha15-10ac12vcMMAE in Subcutaneous Established
Xenograft Model of Patient Derived Human Bladder Cancer AG-B8 in
SCID Mice
[0484] In another experiment, patient derived human bladder cancer
AG-B8 tumor pieces were implanted into the flanks of SCID mice and
tumors were allowed to grow until they reached approximate volume
of 200 mm.sup.3. At that point, animals were allocated to the
treatment groups according to tumor volume at the time of treatment
initiation to ensure similar mean tumor size and variation in each
group. Allocation of mice into treatment groups was aided by Study
Director Software (v.1.7; Studylog Systems, Inc., South San
Francisco Calif.) to help sixe match the mice. All ADC groups
including the two control ADCs were dosed, by intravenous bolus
injection, at 5 mg/kg at the beginning of the study (day zero (0)).
Tumor growth in each group was monitored twice weekly using caliper
measurements until study termination. Statistical analysis of tumor
volumes was performed at the last time point when data from all
groups were available using a nonparametric analysis of variance
(ANOVA) on the ranked data.
[0485] The results show that Ha15-10ac12vcMMAE has superior tumor
inhibitory activity than either non-treated control(s)
(p<0.0001) or to other corresponding ADC gamma-2 control(s)
(p<0.0001). Additionally, Ha15ac12vcMMAE was also showed
superior statistically significant effect when compared to
Ha15-10ac12mcMMAF (p<0.0458) (FIG. 12).
CONCLUSION
[0486] In summary, FIGS. 5-8, 10, and 12, show that the 158P1D7 ADC
entitled Ha15-10ac12vcMMAE significantly inhibited the growth of
tumors cells that express 158P1D7 when compared to control ADCs.
Thus, the Ha15-10ac12vcMMAE can be used for therapeutic purposes to
treat and manage cancers set forth in Table I Specifically, these
results indicate the Ha15-10ac12vcMMAE had inhibitory effect on
various types of bladder cancer models, showing it can be
particularly therapeutically useful in the treatment of bladder
cancer.
Example 8
Human Clinical Trials for the Treatment and Diagnosis of Human
Carcinomas Through Use of 158P1D7 ADCs
[0487] 158P1D7 ADCs are used in accordance with the present
invention which specifically bind to 158P1D7, 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.
[0488] I.) Adjunctive therapy: In adjunctive therapy, patients are
treated with 158P1D7 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 158P1D7
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. 158P1D7 ADCs are utilized in
several adjunctive clinical trials in combination with the
chemotherapeutic or anti-neoplastic agents.
[0489] II.) Monotherapy: In connection with the use of the 158P1D7
ADCs in monotherapy of tumors, the 158P1D7 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.
[0490] Dosage
[0491] 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/or
ADC 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.
[0492] An exemplary, non limiting range for a therapeutically
effective amount of an 158P1D7 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.
[0493] Clinical Development Plan (CDP)
[0494] The CDP follows and develops treatments of 158P1D7 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 158P1D7 ADCs. As will be appreciated, one non-limiting
criteria that can be utilized in connection with enrollment of
patients is 158P1D7 expression levels in their tumors as determined
by biopsy.
[0495] 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 HAMA response); and, (iii) toxicity to normal cells
that express 158P1D7. Standard tests and follow-up are utilized to
monitor each of these safety concerns. 158P1D7 ADCs are found to be
safe upon human administration.
Example 9
Detection of 158P1D7 Protein in Cancer Patient Specimens by IHC
[0496] Expression of 158P1D7 protein by immunohistochemistry was
tested in patient tumor specimens from (i) bladder, (ii) breast,
(iii) lung, and (iv) glioblastoma cancer patients. Briefly,
formalin fixed, paraffin wax-embedded tissues were cut into four
(4) micron sections and mounted on glass slides. The sections were
de-waxed, rehydrated and treated with citra antigen retrieval
solution (Biogenex, San Ramon, Calif.) in the EZ-Retriever
microwave (Biogenex, San Ramon, Calif.) for 15 minutes at
95.degree. C. 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-158P1D7 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
158P1D7 immunoreactive antibody, as indicated by the brown
staining. (See, FIGS. 9(A), 9(C), 9(E), and 9(G). In contrast, the
control antibody did not stain either patient specimen. (See, FIGS.
9(B), 9(D), 9(F), and 9(H).
[0497] The results show expression of 158P1D7 in the tumor cells of
patient bladder, breast, lung, and glioblastoma cancer tissues.
These results indicate that 158P1D7 is expressed in human cancers
and that antibodies directed to this antigen and the antibody drug
conjugate designated Ha15-10ac12vcMMAE are useful for diagnostic
and therapeutic purposes. (FIG. 9).
[0498] 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.
[0499] 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-00004 [0500] TABLE I Tissues that express 158P1D7 when
malignant. Glioblastoma Lung Bladder Breast
TABLE-US-00005 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-00006 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-00007 TABLE IV General Method for Synthesis of vcMMAE
Where: AA1 = Amino Acid 1 AA2 = Amino Acid 2 AA5 = Amino Acid 5 DIL
= Dolaisoleucine DAP = Dolaproine Linker = Val-Cit (vc)
##STR00037##
TABLE-US-00008 TABLE V Positions CDR-L1, CDR-L2, CDR-L3 and CDR-H1,
CDR-H2, CDR-H3 as identified by the Kabat, Chothia, and Contact
schemes, respectively. For CDR-H1, residue numbering is given
listed using both the Kabat and Chothia numbering schemes. CDR
Kabat Chothia Contact CDR-L1 L24--L34 L24--L34 L30--L36 CDR-L2
L50--L56 L50--L56 L46--L55 CDR-L3 L89--L97 L89--L97 L89--L96 CDR-H1
H31--H35B H26--H32 . . . 34 H30--H35B (Kabat Numbering.sup.1)
CDR-H1 H31--H35 H26--H32 H30--H35 (Chothia Numbering.sup.2) CDR-H2
H50--H65 H52--H56 H47--H58 CDR-H3 H95--H102 H95--H102 H93--H101
TABLE-US-00009 TABLE VI Mean Florescence Intensity (MFI) values in
FACS assay. nM Ha15-10ac12vcMMAE 6.667 155.0 2.222 149.6 0.741
140.9 0.247 133.8 0.082 124.0 0.027 107.6 0.0091 87.0 0.0030 60.8
0.0010 25.7 0.0003 14.9 0.0001 4.3
TABLE-US-00010 TABLE VII Ha15-10ac12vcMMAE FACS MFA Values for
Binding Assay UMUC- AGS15 SW780 CHP-212 MFI 115 159 142
Sequence CWU 1
1
1012555DNAHomo
sapiensCDS(23)...(2548)misc_feature(1)...(2555)158P1D7 1tcggatttca
tcacatgaca ac atg aag ctg tgg att cat ctc ttt tat tca 52 Met Lys
Leu Trp Ile His Leu Phe Tyr Ser 1 5 10tct ctc ctt gcc tgt ata tct
tta cac tcc caa act cca gtg ctc tca 100Ser Leu Leu Ala Cys Ile Ser
Leu His Ser Gln Thr Pro Val Leu Ser 15 20 25tcc aga ggc tct tgt gat
tct ctt tgc aat tgt gag gaa aaa gat ggc 148Ser Arg Gly Ser Cys Asp
Ser Leu Cys Asn Cys Glu Glu Lys Asp Gly 30 35 40aca atg cta ata aat
tgt gaa gca aaa ggt atc aag atg gta tct gaa 196Thr Met Leu Ile Asn
Cys Glu Ala Lys Gly Ile Lys Met Val Ser Glu 45 50 55ata agt gtg cca
cca tca cga cct ttc caa cta agc tta tta aat aac 244Ile Ser Val Pro
Pro Ser Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn 60 65 70ggc ttg acg
atg ctt cac aca aat gac ttt tct ggg ctt acc aat gct 292Gly Leu Thr
Met Leu His Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala75 80 85 90att
tca ata cac ctt gga ttt aac aat att gca gat att gag ata ggt 340Ile
Ser Ile His Leu Gly Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly 95 100
105gca ttt aat ggc ctt ggc ctc ctg aaa caa ctt cat atc aat cac aat
388Ala Phe Asn Gly Leu Gly Leu Leu Lys Gln Leu His Ile Asn His Asn
110 115 120tct tta gaa att ctt aaa gag gat act ttc cat gga ctg gaa
aac ctg 436Ser Leu Glu Ile Leu Lys Glu Asp Thr Phe His Gly Leu Glu
Asn Leu 125 130 135gaa ttc ctg caa gca gat aac aat ttt atc aca gtg
att gaa cca agt 484Glu Phe Leu Gln Ala Asp Asn Asn Phe Ile Thr Val
Ile Glu Pro Ser 140 145 150gcc ttt agc aag ctc aac aga ctc aaa gtg
tta att tta aat gac aat 532Ala Phe Ser Lys Leu Asn Arg Leu Lys Val
Leu Ile Leu Asn Asp Asn155 160 165 170gct att gag agt ctt cct cca
aac atc ttc cga ttt gtt cct tta acc 580Ala Ile Glu Ser Leu Pro Pro
Asn Ile Phe Arg Phe Val Pro Leu Thr 175 180 185cat cta gat ctt cgt
gga aat caa tta caa aca ttg cct tat gtt ggt 628His Leu Asp Leu Arg
Gly Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly 190 195 200ttt ctc gaa
cac att ggc cga ata ttg gat ctt cag ttg gag gac aac 676Phe Leu Glu
His Ile Gly Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn 205 210 215aaa
tgg gcc tgc aat tgt gac tta ttg cag tta aaa act tgg ttg gag 724Lys
Trp Ala Cys Asn Cys Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu 220 225
230aac atg cct cca cag tct ata att ggt gat gtt gtc tgc aac agc cct
772Asn Met Pro Pro Gln Ser Ile Ile Gly Asp Val Val Cys Asn Ser
Pro235 240 245 250cca ttt ttt aaa gga agt ata ctc agt aga cta aag
aag gaa tct att 820Pro Phe Phe Lys Gly Ser Ile Leu Ser Arg Leu Lys
Lys Glu Ser Ile 255 260 265tgc cct act cca cca gtg tat gaa gaa cat
gag gat cct tca gga tca 868Cys Pro Thr Pro Pro Val Tyr Glu Glu His
Glu Asp Pro Ser Gly Ser 270 275 280tta cat ctg gca gca aca tct tca
ata aat gat agt cgc atg tca act 916Leu His Leu Ala Ala Thr Ser Ser
Ile Asn Asp Ser Arg Met Ser Thr 285 290 295aag acc acg tcc att cta
aaa cta ccc acc aaa gca cca ggt ttg ata 964Lys Thr Thr Ser Ile Leu
Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile 300 305 310cct tat att aca
aag cca tcc act caa ctt cca gga cct tac tgc cct 1012Pro Tyr Ile Thr
Lys Pro Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro315 320 325 330att
cct tgt aac tgc aaa gtc cta tcc cca tca gga ctt cta ata cat 1060Ile
Pro Cys Asn Cys Lys Val Leu Ser Pro Ser Gly Leu Leu Ile His 335 340
345tgt cag gag cgc aac att gaa agc tta tca gat ctg aga cct cct ccg
1108Cys Gln Glu Arg Asn Ile Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro
350 355 360caa aat cct aga aag ctc att cta gcg gga aat att att cac
agt tta 1156Gln Asn Pro Arg Lys Leu Ile Leu Ala Gly Asn Ile Ile His
Ser Leu 365 370 375atg aag tct gat cta gtg gaa tat ttc act ttg gaa
atg ctt cac ttg 1204Met Lys Ser Asp Leu Val Glu Tyr Phe Thr Leu Glu
Met Leu His Leu 380 385 390gga aac aat cgt att gaa gtt ctt gaa gaa
gga tcg ttt atg aac cta 1252Gly Asn Asn Arg Ile Glu Val Leu Glu Glu
Gly Ser Phe Met Asn Leu395 400 405 410acg aga tta caa aaa ctc tat
cta aat ggt aac cac ctg acc aaa tta 1300Thr Arg Leu Gln Lys Leu Tyr
Leu Asn Gly Asn His Leu Thr Lys Leu 415 420 425agt aaa ggc atg ttc
ctt ggt ctc cat aat ctt gaa tac tta tat ctt 1348Ser Lys Gly Met Phe
Leu Gly Leu His Asn Leu Glu Tyr Leu Tyr Leu 430 435 440gaa tac aat
gcc att aag gaa ata ctg cca gga acc ttt aat cca atg 1396Glu Tyr Asn
Ala Ile Lys Glu Ile Leu Pro Gly Thr Phe Asn Pro Met 445 450 455cct
aaa ctt aaa gtc ctg tat tta aat aac aac ctc ctc caa gtt tta 1444Pro
Lys Leu Lys Val Leu Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu 460 465
470cca cca cat att ttt tca ggg gtt cct cta act aag gta aat ctt aaa
1492Pro Pro His Ile Phe Ser Gly Val Pro Leu Thr Lys Val Asn Leu
Lys475 480 485 490aca aac cag ttt acc cat cta cct gta agt aat att
ttg gat gat ctt 1540Thr Asn Gln Phe Thr His Leu Pro Val Ser Asn Ile
Leu Asp Asp Leu 495 500 505gat tta cta acc cag att gac ctt gag gat
aac ccc tgg gac tgc tcc 1588Asp Leu Leu Thr Gln Ile Asp Leu Glu Asp
Asn Pro Trp Asp Cys Ser 510 515 520tgt gac ctg gtt gga ctg cag caa
tgg ata caa aag tta agc aag aac 1636Cys Asp Leu Val Gly Leu Gln Gln
Trp Ile Gln Lys Leu Ser Lys Asn 525 530 535aca gtg aca gat gac atc
ctc tgc act tcc ccc ggg cat ctc gac aaa 1684Thr Val Thr Asp Asp Ile
Leu Cys Thr Ser Pro Gly His Leu Asp Lys 540 545 550aag gaa ttg aaa
gcc cta aat agt gaa att ctc tgt cca ggt tta gta 1732Lys Glu Leu Lys
Ala Leu Asn Ser Glu Ile Leu Cys Pro Gly Leu Val555 560 565 570aat
aac cca tcc atg cca aca cag act agt tac ctt atg gtc acc act 1780Asn
Asn Pro Ser Met Pro Thr Gln Thr Ser Tyr Leu Met Val Thr Thr 575 580
585cct gca aca aca aca aat acg gct gat act att tta cga tct ctt acg
1828Pro Ala Thr Thr Thr Asn Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr
590 595 600gac gct gtg cca ctg tct gtt cta ata ttg gga ctt ctg att
atg ttc 1876Asp Ala Val Pro Leu Ser Val Leu Ile Leu Gly Leu Leu Ile
Met Phe 605 610 615atc act att gtt ttc tgt gct gca ggg ata gtg gtt
ctt gtt ctt cac 1924Ile Thr Ile Val Phe Cys Ala Ala Gly Ile Val Val
Leu Val Leu His 620 625 630cgc agg aga aga tac aaa aag aaa caa gta
gat gag caa atg aga gac 1972Arg Arg Arg Arg Tyr Lys Lys Lys Gln Val
Asp Glu Gln Met Arg Asp635 640 645 650aac agt cct gtg cat ctt cag
tac agc atg tat ggc cat aaa acc act 2020Asn Ser Pro Val His Leu Gln
Tyr Ser Met Tyr Gly His Lys Thr Thr 655 660 665cat cac act act gaa
aga ccc tct gcc tca ctc tat gaa cag cac atg 2068His His Thr Thr Glu
Arg Pro Ser Ala Ser Leu Tyr Glu Gln His Met 670 675 680gtg agc ccc
atg gtt cat gtc tat aga agt cca tcc ttt ggt cca aag 2116Val Ser Pro
Met Val His Val Tyr Arg Ser Pro Ser Phe Gly Pro Lys 685 690 695cat
ctg gaa gag gaa gaa gag agg aat gag aaa gaa gga agt gat gca 2164His
Leu Glu Glu Glu Glu Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala 700 705
710aaa cat ctc caa aga agt ctt ttg gaa cag gaa aat cat tca cca ctc
2212Lys His Leu Gln Arg Ser Leu Leu Glu Gln Glu Asn His Ser Pro
Leu715 720 725 730aca ggg tca aat atg aaa tac aaa acc acg aac caa
tca aca gaa ttt 2260Thr Gly Ser Asn Met Lys Tyr Lys Thr Thr Asn Gln
Ser Thr Glu Phe 735 740 745tta tcc ttc caa gat gcc agc tca ttg tac
aga aac att tta gaa aaa 2308Leu Ser Phe Gln Asp Ala Ser Ser Leu Tyr
Arg Asn Ile Leu Glu Lys 750 755 760gaa agg gaa ctt cag caa ctg gga
atc aca gaa tac cta agg aaa aac 2356Glu Arg Glu Leu Gln Gln Leu Gly
Ile Thr Glu Tyr Leu Arg Lys Asn 765 770 775att gct cag ctc cag cct
gat atg gag gca cat tat cct gga gcc cac 2404Ile Ala Gln Leu Gln Pro
Asp Met Glu Ala His Tyr Pro Gly Ala His 780 785 790gaa gag ctg aag
tta atg gaa aca tta atg tac tca cgt cca agg aag 2452Glu Glu Leu Lys
Leu Met Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys795 800 805 810gta
tta gtg gaa cag aca aaa aat gag tat ttt gaa ctt aaa gct aat 2500Val
Leu Val Glu Gln Thr Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn 815 820
825tta cat gct gaa cct gac tat tta gaa gtc ctg gag cag caa aca tag
2548Leu His Ala Glu Pro Asp Tyr Leu Glu Val Leu Glu Gln Gln Thr 830
835 840atggaga 25552841PRTHomo
sapiensmisc_feature(1)...(842)158P1D7 2Met Lys Leu Trp Ile His Leu
Phe Tyr Ser Ser Leu Leu Ala Cys Ile1 5 10 15 Ser Leu His Ser Gln
Thr Pro Val Leu Ser Ser Arg Gly Ser Cys Asp 20 25 30 Ser Leu Cys
Asn Cys Glu Glu Lys Asp Gly Thr Met Leu Ile Asn Cys 35 40 45 Glu
Ala Lys Gly Ile Lys Met Val Ser Glu Ile Ser Val Pro Pro Ser 50 55
60 Arg Pro Phe Gln Leu Ser Leu Leu Asn Asn Gly Leu Thr Met Leu
His65 70 75 80 Thr Asn Asp Phe Ser Gly Leu Thr Asn Ala Ile Ser Ile
His Leu Gly 85 90 95 Phe Asn Asn Ile Ala Asp Ile Glu Ile Gly Ala
Phe Asn Gly Leu Gly 100 105 110 Leu Leu Lys Gln Leu His Ile Asn His
Asn Ser Leu Glu Ile Leu Lys 115 120 125 Glu Asp Thr Phe His Gly Leu
Glu Asn Leu Glu Phe Leu Gln Ala Asp 130 135 140 Asn Asn Phe Ile Thr
Val Ile Glu Pro Ser Ala Phe Ser Lys Leu Asn145 150 155 160 Arg Leu
Lys Val Leu Ile Leu Asn Asp Asn Ala Ile Glu Ser Leu Pro 165 170 175
Pro Asn Ile Phe Arg Phe Val Pro Leu Thr His Leu Asp Leu Arg Gly 180
185 190 Asn Gln Leu Gln Thr Leu Pro Tyr Val Gly Phe Leu Glu His Ile
Gly 195 200 205 Arg Ile Leu Asp Leu Gln Leu Glu Asp Asn Lys Trp Ala
Cys Asn Cys 210 215 220 Asp Leu Leu Gln Leu Lys Thr Trp Leu Glu Asn
Met Pro Pro Gln Ser225 230 235 240 Ile Ile Gly Asp Val Val Cys Asn
Ser Pro Pro Phe Phe Lys Gly Ser 245 250 255 Ile Leu Ser Arg Leu Lys
Lys Glu Ser Ile Cys Pro Thr Pro Pro Val 260 265 270 Tyr Glu Glu His
Glu Asp Pro Ser Gly Ser Leu His Leu Ala Ala Thr 275 280 285 Ser Ser
Ile Asn Asp Ser Arg Met Ser Thr Lys Thr Thr Ser Ile Leu 290 295 300
Lys Leu Pro Thr Lys Ala Pro Gly Leu Ile Pro Tyr Ile Thr Lys Pro305
310 315 320 Ser Thr Gln Leu Pro Gly Pro Tyr Cys Pro Ile Pro Cys Asn
Cys Lys 325 330 335 Val Leu Ser Pro Ser Gly Leu Leu Ile His Cys Gln
Glu Arg Asn Ile 340 345 350 Glu Ser Leu Ser Asp Leu Arg Pro Pro Pro
Gln Asn Pro Arg Lys Leu 355 360 365 Ile Leu Ala Gly Asn Ile Ile His
Ser Leu Met Lys Ser Asp Leu Val 370 375 380 Glu Tyr Phe Thr Leu Glu
Met Leu His Leu Gly Asn Asn Arg Ile Glu385 390 395 400 Val Leu Glu
Glu Gly Ser Phe Met Asn Leu Thr Arg Leu Gln Lys Leu 405 410 415 Tyr
Leu Asn Gly Asn His Leu Thr Lys Leu Ser Lys Gly Met Phe Leu 420 425
430 Gly Leu His Asn Leu Glu Tyr Leu Tyr Leu Glu Tyr Asn Ala Ile Lys
435 440 445 Glu Ile Leu Pro Gly Thr Phe Asn Pro Met Pro Lys Leu Lys
Val Leu 450 455 460 Tyr Leu Asn Asn Asn Leu Leu Gln Val Leu Pro Pro
His Ile Phe Ser465 470 475 480 Gly Val Pro Leu Thr Lys Val Asn Leu
Lys Thr Asn Gln Phe Thr His 485 490 495 Leu Pro Val Ser Asn Ile Leu
Asp Asp Leu Asp Leu Leu Thr Gln Ile 500 505 510 Asp Leu Glu Asp Asn
Pro Trp Asp Cys Ser Cys Asp Leu Val Gly Leu 515 520 525 Gln Gln Trp
Ile Gln Lys Leu Ser Lys Asn Thr Val Thr Asp Asp Ile 530 535 540 Leu
Cys Thr Ser Pro Gly His Leu Asp Lys Lys Glu Leu Lys Ala Leu545 550
555 560 Asn Ser Glu Ile Leu Cys Pro Gly Leu Val Asn Asn Pro Ser Met
Pro 565 570 575 Thr Gln Thr Ser Tyr Leu Met Val Thr Thr Pro Ala Thr
Thr Thr Asn 580 585 590 Thr Ala Asp Thr Ile Leu Arg Ser Leu Thr Asp
Ala Val Pro Leu Ser 595 600 605 Val Leu Ile Leu Gly Leu Leu Ile Met
Phe Ile Thr Ile Val Phe Cys 610 615 620 Ala Ala Gly Ile Val Val Leu
Val Leu His Arg Arg Arg Arg Tyr Lys625 630 635 640 Lys Lys Gln Val
Asp Glu Gln Met Arg Asp Asn Ser Pro Val His Leu 645 650 655 Gln Tyr
Ser Met Tyr Gly His Lys Thr Thr His His Thr Thr Glu Arg 660 665 670
Pro Ser Ala Ser Leu Tyr Glu Gln His Met Val Ser Pro Met Val His 675
680 685 Val Tyr Arg Ser Pro Ser Phe Gly Pro Lys His Leu Glu Glu Glu
Glu 690 695 700 Glu Arg Asn Glu Lys Glu Gly Ser Asp Ala Lys His Leu
Gln Arg Ser705 710 715 720 Leu Leu Glu Gln Glu Asn His Ser Pro Leu
Thr Gly Ser Asn Met Lys 725 730 735 Tyr Lys Thr Thr Asn Gln Ser Thr
Glu Phe Leu Ser Phe Gln Asp Ala 740 745 750 Ser Ser Leu Tyr Arg Asn
Ile Leu Glu Lys Glu Arg Glu Leu Gln Gln 755 760 765 Leu Gly Ile Thr
Glu Tyr Leu Arg Lys Asn Ile Ala Gln Leu Gln Pro 770 775 780 Asp Met
Glu Ala His Tyr Pro Gly Ala His Glu Glu Leu Lys Leu Met785 790 795
800 Glu Thr Leu Met Tyr Ser Arg Pro Arg Lys Val Leu Val Glu Gln Thr
805 810 815 Lys Asn Glu Tyr Phe Glu Leu Lys Ala Asn Leu His Ala Glu
Pro Asp 820 825 830 Tyr Leu Glu Val Leu Glu Gln Gln Thr 835 840
31341DNAHomo
sapiensCDS(1)...(1341)misc_feature(1)...(1341)Ha15-10ac12 heavy
chain 3cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg
agg 48Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly
Arg1 5 10 15tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc agt
agc tat 96Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg
gaa tgg gtg 144Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45gca gtt ata tgg tat gat gga agt aat caa tat tat
gca gac tcc gtg 192Ala Val Ile Trp Tyr Asp Gly Ser Asn Gln Tyr Tyr
Ala Asp Ser Val 50 55 60aag ggc cga ttc acc atc tcc aga gac aat tcc
aag aac acg ctg ttt 240Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Phe65 70 75 80ctg caa atg cac agc ctg aga gcc gag
gac acg gct gtg tat tac tgt 288Leu Gln Met His Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95gcg aga ggt ctg act tct gga cgg
tac ggt atg gac gtc tgg ggc caa 336Ala Arg Gly Leu Thr Ser Gly Arg
Tyr Gly Met Asp Val Trp Gly Gln 100
105 110ggg acc acg gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg
gtc 384Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val 115 120 125ttc ccc ctg gcg ccc tgc tcc agg agc acc tcc gag agc
aca gcg gcc 432Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
Thr Ala Ala 130 135 140ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa
ccg gtg acg gtg tcg 480Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser145 150 155 160tgg aac tca ggc gct ctg acc agc
ggc gtg cac acc ttc cca gct gtc 528Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val 165 170 175cta cag tcc tca gga ctc
tac tcc ctc agc agc gtg gtg acc gtg ccc 576Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190tcc agc aac ttc
ggc acc cag acc tac acc tgc aac gta gat cac aag 624Ser Ser Asn Phe
Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205ccc agc
aac acc aag gtg gac aag aca gtt gag cgc aaa tgt tgt gtc 672Pro Ser
Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val 210 215
220gag tgc cca ccg tgc cca gca cca cct gtg gca gga ccg tca gtc ttc
720Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
Phe225 230 235 240ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc
tcc cgg acc cct 768Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro 245 250 255gag gtc acg tgc gtg gtg gtg gac gtg agc
cac gaa gac ccc gag gtc 816Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val 260 265 270cag ttc aac tgg tac gtg gac ggc
gtg gag gtg cat aat gcc aag aca 864Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr 275 280 285aag cca cgg gag gag cag
ttc aac agc acg ttc cgt gtg gtc agc gtc 912Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Phe Arg Val Val Ser Val 290 295 300ctc acc gtt gtg
cac cag gac tgg ctg aac ggc aag gag tac aag tgc 960Leu Thr Val Val
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320aag
gtc tcc aac aaa ggc ctc cca gcc ccc atc gag aaa acc atc tcc 1008Lys
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330
335aaa acc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca
1056Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350tcc cgg gag gag atg acc aag aac cag gtc agc ctg acc tgc
ctg gtc 1104Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val 355 360 365aaa ggc ttc tac ccc agc gac atc gcc gtg gag tgg
gag agc aat ggg 1152Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly 370 375 380cag ccg gag aac aac tac aag acc aca cct
ccc atg ctg gac tcc gac 1200Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp385 390 395 400ggc tcc ttc ttc ctt tac agc
aag ctc acc gtg gac aag agc agg tgg 1248Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415cag cag ggg aac gtc
ttc tca tgc tcc gtg atg cat gag gct ctg cac 1296Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430aac cac tac
acg cag aag agc ctc tcc ctg tct ccg ggt aaa taa 1341Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 4454446PRTHomo
sapiensCHAIN(1)...(446)Ha15-10ac12 heavy chain 4Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Gln Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Phe65 70 75 80 Leu Gln Met His Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Thr Ser Gly Arg Tyr Gly
Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160 Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190 Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp
His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg
Lys Cys Cys Val 210 215 220 Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly Pro Ser Val Phe225 230 235 240 Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val 290 295
300 Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser 325 330 335 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro 340 345 350 Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp385 390 395 400 Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410
415 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445 5660DNAHomo
sapiensCDS(1)...(660)misc_feature(1)...(660)Ha15-10ac12 light chain
5gat att gtg atg act cag tct cca ctc tcc ctg ccc gtc acc cct gga
48Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15gag ccg gcc tcc atc tcc tgc agg tct agt cag agc ctc ctg ctt
agt 96Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Leu
Ser 20 25 30cat gga ttc aac tat ttg gat tgg tac ctg cag aag cca ggg
cag tct 144His Gly Phe Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45cca caa ctc ctg atc tat ttg ggt tct agt cgg gcc tcc
ggg gtc cct 192Pro Gln Leu Leu Ile Tyr Leu Gly Ser Ser Arg Ala Ser
Gly Val Pro 50 55 60gac agg ttc agt ggc agt gga tca ggc aca gat ttt
aca ctg aaa atc 240Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80agc aga gtg gag gct gag gat gtt ggg ctt
tat tac tgc atg caa ccc 288Ser Arg Val Glu Ala Glu Asp Val Gly Leu
Tyr Tyr Cys Met Gln Pro 85 90 95cta caa att ccg tgg acg ttc ggc caa
ggg acc aag gtg gaa atc aaa 336Leu Gln Ile Pro Trp Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 110cga act gtg gct gca cca tct
gtc ttc atc ttc ccg cca tct gat gag 384Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125cag ttg aaa tct gga
act gcc tct gtt gtg tgc ctg ctg aat aac ttc 432Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140tat ccc aga
gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa 480Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155
160tcg ggt aac tcc cag gag agt gtc aca gag cag gac agc aag gac agc
528Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
165 170 175acc tac agc ctc agc agc acc ctg acg ctg agc aaa gca gac
tac gag 576Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu 180 185 190aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag
ggc ctg agc tcg 624Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 195 200 205ccc gtc aca aag agc ttc aac agg gga gag
tgt tag 660Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
2156219PRTHomo sapiensCHAIN(1)...(219)Ha15-10ac12 light chain 6Asp
Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10
15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Leu Ser
20 25 30 His Gly Phe Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Ser Arg Ala
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val
Gly Leu Tyr Tyr Cys Met Gln Pro 85 90 95 Leu Gln Ile Pro Trp Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145
150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 210 215 7446PRTHomo sapiensCHAIN(1)...(446)Ha15-10ac12
lheavy chain 7Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly
Ser Asn Gln Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe65 70 75 80 Leu Gln Met
His Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Gly Leu Thr Ser Gly Arg Tyr Gly Met Asp Val Trp Gly Gln 100 105
110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Asn Phe Gly
Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn
Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val 210 215 220 Glu
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe225 230
235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Phe Arg Val Val Ser Val 290 295 300 Leu Thr Val Val His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320 Lys Val Ser Asn
Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335 Lys Thr
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355
360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu
Asp Ser Asp385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp 405 410 415 Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 8219PRTHomo
sapiensCHAIN(1)...(219)Ha15-10ac12 light chain 8Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15 Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Leu Ser 20 25 30 His
Gly Phe Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Ser Arg Ala Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr
Cys Met Gln Pro 85 90 95 Leu Gln Ile Pro Trp Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160 Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170
175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215 94PRTArtificial Sequencesynthetic construct 9Gly Phe Leu Gly1
10100PRTHomo sapiensDOMAIN(1)...(100)IGKV2D-28*01 10Asp Ile Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met
Gln Ala 85 90 95 Leu Gln Thr Pro 100
* * * * *