U.S. patent application number 16/216841 was filed with the patent office on 2019-10-31 for antibody drug conjugates (adc) that bind to 161p2f10b proteins.
The applicant listed for this patent is AGENSYS, INC.. Invention is credited to Zili AN, Jean GUDAS, Aya JAKOBOVITS, Robert Kendall MORRISON, Michael TORGOV.
Application Number | 20190328898 16/216841 |
Document ID | / |
Family ID | 44355840 |
Filed Date | 2019-10-31 |
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United States Patent
Application |
20190328898 |
Kind Code |
A1 |
TORGOV; Michael ; et
al. |
October 31, 2019 |
ANTIBODY DRUG CONJUGATES (ADC) THAT BIND TO 161P2F10B PROTEINS
Abstract
Antibody drug conjugates (ADC's) that bind to 161P2F10B protein
are described herein. 161P2F10B exhibits tissue specific expression
in normal adult tissue, and is aberrantly expressed in the cancers
listed in Table I. Consequently, the ADC's of the invention provide
a therapeutic composition for the treatment of cancer.
Inventors: |
TORGOV; Michael; (Santa
Monica, CA) ; MORRISON; Robert Kendall; (Santa
Monica, CA) ; JAKOBOVITS; Aya; (South San Francisco,
CA) ; GUDAS; Jean; (Los Angeles, CA) ; AN;
Zili; (Santa Monica, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AGENSYS, INC. |
Santa Monica |
CA |
US |
|
|
Family ID: |
44355840 |
Appl. No.: |
16/216841 |
Filed: |
December 11, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15071146 |
Mar 15, 2016 |
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16216841 |
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14106444 |
Dec 13, 2013 |
9308278 |
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15071146 |
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13022905 |
Feb 8, 2011 |
8609092 |
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14106444 |
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61302489 |
Feb 8, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/3038 20130101;
A61P 1/16 20180101; C07K 16/28 20130101; C07K 2317/21 20130101;
A61K 39/39558 20130101; A61K 2039/505 20130101; A61P 35/00
20180101; A61K 47/6811 20170801; A61K 47/6861 20170801; A61K
47/6849 20170801; A61P 13/12 20180101; C07K 2317/56 20130101; C07K
2317/92 20130101 |
International
Class: |
A61K 47/68 20060101
A61K047/68; C07K 16/28 20060101 C07K016/28; C07K 16/30 20060101
C07K016/30 |
Claims
1. (canceled)
2. An antibody drug conjugate comprising an antibody or antigen
binding fragment thereof which binds to a 161P2F10B protein variant
of SEQ ID NO:2, wherein the antibody or antigen binding fragment
thereof is conjugated to 1 to 12 units of monomethyl auristatin F
(MMAF), each unit of MMAF being conjugated via a linker, and
wherein the antibody or antigen binding fragment thereof comprises
a heavy chain variable region consisting of the amino acid sequence
ranging from residue 20 to residue 142 of SEQ ID NO: 7 and a light
chain variable region consisting of the amino acid sequence ranging
from residue 20 to residue 127 of SEQ ID NO: 8.
3. A method of delivering a cytotoxic agent or a diagnostic agent
to a cell, comprising: providing MMAF(s) conjugated to an antibody
or fragment thereof that binds specifically to a 161P2F10B protein
comprising the amino acid sequence of SEQ ID NO:2, wherein the
antibody or antigen binding fragment thereof comprises a heavy
chain variable region consisting of the amino acid sequence ranging
from residue 20 to residue 142 of SEQ ID NO: 7 and a light chain
variable region consisting of the amino acid sequence ranging from
residue 20 to residue 127 of SEQ ID NO: 8, to form an antibody drug
or fragment drug conjugate; and exposing the cell to the antibody
drug or fragment drug conjugate.
4. A method of inhibiting growth of cancer cells in a subject,
comprising: administering to said subject an antibody drug
conjugate, wherein the antibody drug conjugate comprises an
antibody or antigen binding fragment thereof which binds to a
161P2F10B protein variant of SEQ ID NO:2, wherein the antibody or
antigen binding fragment thereof is conjugated to 1 to 12 units of
monomethyl auristatin F (MMAF), each unit of MMAF being conjugated
via a linker, and wherein the antibody or antigen binding fragment
thereof comprises a heavy chain variable region consisting of the
amino acid sequence ranging from residue 20 to residue 142 of SEQ
ID NO: 7 and a light chain variable region consisting of the amino
acid sequence ranging from residue 20 to residue 127 of SEQ ID NO:
8.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 14/106,444, filed on Dec. 13, 2013, now U.S. Pat. No. ______,
which is a continuation of U.S. application Ser. No. 13/022,905,
filed on Feb. 8, 2011, now U.S. Pat. No. 8,609,902, which claims
the benefit of priority to U.S. Provisional Application No.
61/302,489, filed on Feb. 8, 2010. The contents of these
applications are hereby incorporated by reference in their
entirety.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] Not applicable.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0003] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
511582006233SeqList.txt, date recorded: Mar. 15, 2016; size: 43,691
bytes).
FIELD OF THE INVENTION
[0004] The invention described herein relates to antibody drug
conjugates (ADCs) thereof that bind proteins, termed 161P2F10B. The
invention further relates to prognostic, prophylactic and
therapeutic methods and compositions useful in the treatment of
cancers.
BACKGROUND OF THE INVENTION
[0005] Cancer is the second leading cause of human death next to
coronary disease. Worldwide, millions of people die from cancer
every year. In the United States alone, as reported by the American
Cancer Society, cancer causes the death of well over a half-million
people annually, with over 1.2 million new cases diagnosed per
year. While deaths from heart disease have been declining
significantly, those resulting from cancer generally are on the
rise. In the early part of the next century, cancer is predicted to
become the leading cause of death.
[0006] Worldwide, several cancers stand out as the leading killers.
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] While previously identified markers such as PSA, PSM, PCTA
and 161P2F10B 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.
[0008] Renal Cell Cancer (RCC) presently ranks 10th as the leading
cause of cancer death in the United States. An estimated 51,190
people will be diagnosed annually with renal cell carcinoma in the
US and approximately 12,890 died from the disease in 2007 (American
Cancer Society). Historically, treatment has focused primarily on
nephrectomy, followed by nonspecific immunotherapy, and sometimes
radiation therapy (Hauke, 2006). Nonspecific immunotherapy includes
treatment with the cytokines interleukin-2 or interferon-.alpha. as
either single agents or in combination. After surgical excision,
20-30% of patients will develop metastatic disease within 1-3
years, often in the lung (Motzer, et al., 2006). Median survival
for patients with metastatic disease is approximately 13 months
(Cohen and McGovern, 2005).
[0009] Since 2005, six (6) agents have been approved by FDA for the
treatment advanced renal cell cancer. These advances include
several agents that target the specific pathways implicated in
renal cell cancer. These agents include sorafenib (Nexavar.RTM.,
FDA approved in December 2005), sunitinib (Sutent.RTM., FDA
approved in January 2006), temsirolimus (Torisel.RTM., FDA approved
in May 2007), everolimus (Affinitor.RTM., FDA approved in March,
2009), bevacizumab (Avastin.RTM. in combination with interferon
alpha, FDA approved in August 2009) and pazopanib (Votrient.RTM.
FDA approved in October 2009). However, despite advances in the
treatment, metastatic Renal cell cancer remains incurable and only
temsirolimus was approved based on an advantage in overall
survival.
[0010] Additionally, hepatocellular carcinoma (i.e., cancer of the
liver) accounts for 80-90% of all liver cancers. This type of liver
cancer occurs more often in men than in women. It is usually seen
in people ages 50-60. Generally, treatment of liver cancer is
aggressive surgery or a liver transplant which may successfully
treat small or slow growing tumors if they are diagnosed early.
However, few patients are diagnosed early. Chemotherapy and
radiation treatments are not usually effective. However, these
therapies are used to shrink tumors so surgery has a greater chance
of success. Sorafenib tosylate (Nexavar.RTM.) is now available for
patients with liver cancer. The prognosis for patients with liver
cancer is usually poor, since only 10-20% of hepatocellular
carcinomas can be removed using surgery. Accordingly, there is a
need to develop an agent used to treat liver cancer.
[0011] 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)).
[0012] 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.
[0013] 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
[0014] The invention provides antibody drug conjugates (ADCs) that
bind to 161P2F10B proteins. In some embodiments, the invention
comprises fully human antibodies conjugated with a therapeutic
agent.
[0015] The invention further provides various immunogenic or
therapeutic compositions, such as antibody drug conjugates, and
strategies for treating cancers such as cancers of tissues listed
in Table I.
[0016] The present invention relates to:
[0017] [1] An antibody drug conjugate comprising an antibody or
antigen binding fragment thereof that binds specifically to a
161P2F10B protein comprising the amino acid sequence of SEQ ID
NO:2, and wherein the antibody comprises the amino acid sequence of
the V.sub.H region of SEQ ID NO:7, from 20 to 142 and the V.sub.L
region of SEQ ID NO:8, from 20 to 127 and wherein said antibody is
conjugated to monomethyl auristatin F (MMAF).
[0018] [2] The antibody drug conjugate of [1], wherein the antigen
binding fragment is an Fab, F(ab').sub.2 or Fv fragment.
[0019] [3] The antibody drug conjugate of [1], wherein the antibody
is a fully human antibody.
[0020] [4] The antibody drug conjugate of [1], which is
recombinantly produced.
[0021] [5] A pharmaceutical composition that comprises the antibody
drug conjugate of [1] in a human unit dose form.
[0022] [6] The pharmaceutical composition of [5], wherein the
composition is for cancer treatment.
[0023] [7] The pharmaceutical composition of [6], wherein the
cancer is renal cancer or liver cancer.
[0024] [8] A method of inhibiting growth of cancer cells in a
subject, comprising: administering to said subject an antibody drug
conjugate of [1].
[0025] [9] A method of delivering a cytotoxic agent or a diagnostic
agent to a cell, comprising:
[0026] providing MMAF(s) conjugated to an antibody or antigen
binding fragment thereof that binds specifically to a 161P2F10B
protein comprising the amino acid sequence of SEQ ID NO:2, and
wherein the antibody comprises the amino acid sequence of the
V.sub.H region of SEQ ID NO:7, from 20 to 142 and the V.sub.L
region of SEQ ID NO:8, from 20 to 127, to form an antibody drug
conjugate; and,
[0027] exposing the cell to the antibody drug or fragment drug
conjugate.
[0028] [10] A method for treating tumor in a mammal comprising
treating the mammal with an effective amount of an antibody drug
conjugate of [1].
[0029] [11] A method for reducing tumor growth in a mammal
comprising treating the mammal with an effective amount of a
combination of an antibody drug conjugate of [1] and radiation.
[0030] [12] A method for reducing tumor growth in a mammal
comprising treating the mammal with an effective amount of a
combination of an antibody drug conjugate of [1] and a
chemotherapeutic agent.
[0031] [13] A method for reducing tumor growth in a mammal
comprising treating the mammal with an effective amount of a
combination of an antibody drug conjugate of [1] and a drug or
biologically active therapy.
[0032] [14] A method for treating cancer in a mammal, comprising
treating the mammal with an effective amount of a combination of an
antibody drug conjugate of [1] and a chemotherapeutic agent.
[0033] [15] An antibody drug conjugate (ADC), wherein the ADC
having the formula L-(LU-D)p, wherein: (a) L is the antibody
comprising an antibody or antigen binding fragment thereof that
binds specifically to a 161P2F10B protein comprising the amino acid
sequence of SEQ ID NO:2, and wherein the antibody comprises the
amino acid sequence of the V.sub.H region of SEQ ID NO:7, from 20
to 142 and the V.sub.L region of SEQ ID NO:8, from 20 to 127; (b)
LU is a linker; (c) D is a drug moiety wherein drug is monomethyl
auristatin F (MMAF); (d) p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or
12.
[0034] [16] An antibody drug conjugate (ADC), wherein the ADC
having the following formula, wherein Ab-S is the antibody
comprising an antibody or antigen binding fragment thereof that
binds specifically to a 161P2F10B protein comprising the amino acid
sequence of SEQ ID NO:2, and wherein the antibody comprises the
amino acid sequence of the V.sub.H region of SEQ ID NO:7, from 20
to 142 and the V.sub.L region of SEQ ID NO:8, from 20 to 127; p is
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.
##STR00001##
[0035] [17] An antibody drug conjugate comprising an antibody that
binds specifically to a 161P2F10B protein comprising the amino acid
sequence of SEQ ID NO:2, and wherein the antibody comprises the
amino acid sequence of the heavy chain of SEQ ID NO:7, from 20 to
468 and the light chain of SEQ ID NO:8, from 20 to 233 and wherein
said antibody is conjugated to monomethyl auristatin F (MMAF).
[0036] [18] An antibody drug conjugate (ADC), wherein the ADC
having the formula L-(LU-D)p, wherein: (a) L is the antibody
comprising an antibody that binds specifically to a 161P2F10B
protein comprising the amino acid sequence of SEQ ID NO:2, and
wherein the antibody comprises the amino acid sequence of the heavy
chain of SEQ ID NO:7, from 20 to 468 and the light chain of SEQ ID
NO:8, from 20 to 233; (b) LU is a linker; (c) D is a drug moiety
wherein drug is monomethyl auristatin F (MMAF); (d) p is 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11 or 12.
[0037] [19] An antibody drug conjugate (ADC), wherein the ADC
having the following formula, wherein Ab-S is the antibody
comprising an antibody that binds specifically to a 161P2F10B
protein comprising the amino acid sequence of SEQ ID NO:2, and
wherein the antibody comprises the amino acid sequence of the heavy
chain of SEQ ID NO:7, from 20 to 468 and the light chain of SEQ ID
NO:8, from 20 to 233; p is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or
12.
##STR00002##
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1. The cDNA and amino acid sequence of 161P2F10B is
shown in FIG. 1. The open reading frame extends from nucleic acid
44-2671 including the stop codon.
[0039] FIG. 2. Nucleic Acid and Amino Acid sequences of 161P2F10B
antibodies.
[0040] FIG. 2A. The cDNA and amino acid sequence of H16-7.8 heavy
chain. Underlined is the leader sequence and double-underlined is
the heavy chain variable region. Dashed underline shows the human
IgG2 constant region.
[0041] FIG. 2B. The cDNA and amino acid sequence of H16-7.8 light
chain. Underlined is the leader sequence and double-underlined is
the light chain variable region. Dashed underline shows the human
Ig kappa constant region.
[0042] FIG. 3. Amino Acid sequences of 161P2F10B antibodies.
[0043] FIG. 3A. The amino acid sequence of H16-7.8 heavy chain.
Underlined is the leader sequence and double-underlined is the
heavy chain variable region. Dashed underline shows the human IgG2
constant region.
[0044] FIG. 3B. The amino acid sequence of H16-7.8 light chain.
Underlined is the leader sequence and double-underlined is the
light chain variable region. Dashed underline shows the human Ig
kappa constant region.
[0045] FIG. 4. Alignment of H16-7.8 antibodies to human
germline.
[0046] FIG. 4A. Alignment of H16-7.8 heavy chain to human germline
VH4-31/D5-12/JH6.
[0047] FIG. 4B. Alignment of H16-7.8 light chain to human germline
A26/JK1.
[0048] FIG. 5. H16-7.8 Recombinant Expression in CHO Cells. H16-7.8
heavy and light chain sequences were cloned into expression
vectors. Vectors were transfected into CHO cells. 3T3-control and
3T3-161P2F10B cells were stained with H16-7.8 from either hybridoma
or from CHO cells. Binding was detected by flow cytometry. Results
show H16-7.8 recombinantly expressed in CHO cells is secreted and
binds specifically to cell-surface 161P2F10B.
[0049] FIG. 6. Cell Binding and Affinity of H16-7.8 and
H16-7.8mcMMAF. H16-7.8 and H16-7.8mcMMAF were tested for the
binding affinity to 161P2F10B endogenously expressed on Ku812
cells. Briefly, eleven (11) dilutions of H16-7.8 or H16-7.8mcMMAF
were incubated with Ku812 cells (50,000 cells per well) overnight
at 4.degree. C. at a final concentration of 160 nM to 0.0001 nM. At
the end of the incubation, cells were washed and incubated with
anti-hIgG-PE detection antibody for 45 min at 4.degree. C. After
washing the unbound detection antibodies, the cells were analyzed
by FACS. Mean Florescence Intensity (MFI) values are obtained (See,
Table IV). 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 H16-7.8 or H16-7.8mcMMAF saturation
curves shown in FIG. 6. Bmax is the MFI value at maximal binding of
H16-7.8 or H16-7.8mcMMAF to 161P2F10B; Kd is H16-7.8 or
H16-7.8mcMMAF binding affinity which is the concentration of
H16-7.8 or H16-7.8mcMMAF required to reach half-maximal binding.
The calculated affinity (Kd) of H16-7.8 and H16-7.8mcMMAF is 0.06
nM and 0.19 nM, respectively on 161P2F10B endogenously expressed on
the surface of Ku812 cells.
[0050] FIG. 7. Binding of H16-7.8 and H16-7.8mcMMAF to Renal Cancer
Cells. Human UGK-3 cells (patient derived clear cell renal cancer)
and RXF-393 cells (clear cell renal cancer) were stained with 10
.mu.g/ml of native H16-7.8, H16-7.8mcMMAF, or an isotype control
human IgG2 and evaluated by FACS. The results in FIG. 7 (left
panels) demonstrate strong staining of the two different renal
tumor cells with H16-7.8 (gray lines), but not with the control MAb
(filled histograms). The panels on the right demonstrate a similar
strong staining of the same renal tumor cells with H16-7.8mcMMAF
(gray lines). (FIG. 7; right panels). These results show that both
H16-7.8 and H16-7.8mcMMAF bind native 161P2F10B antigen expressed
on the surface of human cancer cells. Conjugation of native H16-7.8
to generate the H16-7.8mcMMAF did not alter its cell surface
binding to native 161P2F10B antigen expressed on human cancer
cells.
[0051] FIG. 8. Cell Cytotoxicity by H16-7.8mcMMAF. 2000 viable
KU812 cells were plated in triplicate on Day 0 and allowed to
recover overnight. The next day, serial 1:4 dilutions of different
lots of H16-7.8mcMMAF or a control MAb conjugated with mcMMAF were
added to yield the final concentrations. The cells were allowed to
incubate for six (6) days at which time 20 .mu.l of Alamar blue was
added to each well. The plates were incubated for an additional
four (4) hours and the fluorescence intensity read on a fluorescent
plate reader using an excitation wavelength of 540 nM and an
emission wavelength of 620 nM. The results show that both lots of
H16-7.8mcMMAF potently inhibited the proliferation of KU812 cells.
The IC 50 was determined to be 0.2 nM and 0.1 nM for Lots (1) and
Lot (2) respectively. A fully human Control MAb that does not bind
KU812 cells was conjugated with mcMMAF to yield a DAR of 3.9
(+/-0.2). The Control ADC (Control mcF) did not inhibit KU812 cell
proliferation further demonstrating the specificity of
cytotoxicity. Thus, these results indicate that H16-7.8mcMMAF can
selectively deliver a cytotoxic drug to 161P2F10B expressing cells,
leading to their killing.
[0052] FIG. 9. Efficacy of H16-7.8mcMMAF in subcutaneously
established human renal cancer xenograft UG-K3 in SCID mice. In
this experiment, patient-derived human renal cancer xenograft UG-K3
was maintained by serial passages in SCID mice. Stock tumors were
harvested sterilely and minced to small pieces. The tumor pieces
were enzymatically digested to single cell suspensions using
Liberase Blendzyme (Roche Applied Science, Indianapolis, Ind.).
1.5.times.10.sup.6 cells were injected into the flanks of
individual SCID mice and tumors were allowed to grow untreated
until they reached an approximate volume of 100 mm.sup.3. Animals
were randomly assigned to the following cohorts: an H16-7.8mcMMAF
treated group, an H16-7.8 control and a 5% Dextrose control.
H16-7.8mcMMAF and H16-7.8 were dosed at 10 mg/kg once on day 0 by
intravenous bolus injection. The amount of H16-7.8mcMMAF and
H16-7.8 administered was based on the individual body weight of
each animal obtained immediately prior to dosing. The 5% Dextrose
control was dosed at 150 .mu.L per animal. Tumor growth was
monitored using caliper measurements every 3 to 4 days until the
end of the study. Tumor volume is calculated as
Width.sup.2.times.Length/2, where width is the smallest dimension
and length is the largest. Animals in control groups were humanely
euthanized when tumors reached approximately 1000 mm.sup.3. Animals
in H16-7.8mcMMAF treated group were monitored for an additional two
weeks before sacrifice. Statistical analysis was performed at the
last time point when data for both control groups were available,
using Kruskal-Wallis test with =0.05.
[0053] The results demonstrated that treatment of UG-K3 renal clear
cell xenograft tumors with H16-7.8mcMMAF at all doses and schedules
examined resulted in significant inhibition of tumor growth in SCID
mice.
[0054] FIG. 10. Growth Inhibition of Established Orthotopic UG-K3
Xenografts by H16-7.8mcMMAF. The ability of H16-7.8mcMMAF to
inhibit the growth of established renal tumors grown orthotopically
was evaluated using patient-derived, UG-K3 tumor xenografts.
Briefly, stocks of UG-K3 tumors were digested enzymatically and 1.5
million viable cells were surgically implanted into the kidneys of
male SCID mice on Day 0. The tumors were allowed to grow for 7 days
at which time animals were randomized to 4 different treatment
groups (n=10 per group). Animals randomized to Group A received
Control ADC at 5 mpk, Group B received H16-7.8mcMMAF at 3 mg/kg and
Group C received H16-7.8mcMMAF at 5 mg/kg administered every 4 days
for a total of 4 doses. Group D received H16-7.8mcMMAF at 10 mg/kg
one time. At the end of the study (Day 41) the animals were
sacrificed and the right and left kidneys weighed on an electronic
balance. The tumor weights plotted on the graph were determined by
subtracting the weight of the tumor-free contralateral kidney from
the weight of the tumor-bearing right kidney.
[0055] The results demonstrated that treatment of UG-K3 renal clear
cell xenograft tumors with H16-7.8mcMMAF at all doses and schedules
examined resulted in dramatic inhibition of tumor growth. Tumor
weights in all H16-7.8mcMMAF treatment groups (B, C, and D) were
less than 1% of the tumor weights in the Control treated group.
These differences were highly statistically significant
(p<0.0001, ANOVA).
[0056] FIG. 11. Efficacy of H16-7.8mcMMAF in subcutaneously
established human renal cancer xenograft RXF-393 in SCID mice. In
this experiment, human renal cancer cells RXF-393
(0.5.times.10.sup.6 cells per mouse) were injected into the flanks
of individual mice and tumors were allowed to grow untreated until
they reached an approximate volume of 100 mm.sup.3. Animals were
then randomly assigned to the following cohorts: an H16-7.8mcMMAF
treated group, an H16-7.8 treated group and a 5% Dextrose control.
H16-7.8mcMMAF and H16-7.8 were dosed at 10 mg/kg once a week for a
total of two doses by intravenous bolus injection. The amount of
H16-7.8mcMMAF and H16-7.8 administered was based on the individual
body weight of each animal obtained immediately prior to dosing.
The 5% Dextrose control was dosed at 150 .mu.L per animal. Tumor
growth was monitored using caliper measurements every 3 to 4 days
until the end of the study. Tumor volume is calculated as
Width.sup.2.times.Length/2, where width is the smallest dimension
and length is the largest. Animals in control groups were humanely
euthanized when tumors reached approximately 1000 mm.sup.3. Animals
in H16-7.8mcMMAF treated group were monitored for an additional two
weeks before sacrifice.
[0057] The results demonstrated that treatment of RFX-393 human
renal cancer xenograft tumors with H16-7.8mcMMAF at all doses and
schedules examined resulted in significant inhibition of tumor
growth in SCID mice. Statistical analysis was performed at the last
time point when data in both control groups were available, using
Kruskal-Wallis test with .alpha.=0.05.
[0058] FIG. 12. Efficacy Study of H16-7.8 compared to H16-7.8mcMMAF
in subcutaneously established human renal cancer SKRC-01 in SCID
Mice. Human renal cancer cells SKRC-01 (0.8.times.10.sup.6 cells
per mouse) were injected into the flanks of individual mice. Tumors
were allowed to grow untreated until they reached an approximate
volume of 100 mm.sup.3. On day 0 when tumors reach 100 mm.sup.3,
animals were randomly assigned to the following cohorts: an
H16-7.8mcMMAF treated group, an H16-7.8 treated group and a 5%
Dextrose control. H16-7.8mcMMAF and H16-7.8 were dosed at 4 mg/kg
every four days for a total of four doses by intravenous bolus
injection. The amount of H16-7.8mcMMAF and H16-7.8 administered was
based on the individual body weight of each animal obtained
immediately prior to dosing. The 5% Dextrose control was dosed at
150 .mu.L per animal. Tumor growth was monitored using caliper
measurements every 3 to 4 days. Tumor volume was calculated as
Width.sup.2.times.Length/2, where width is the smallest dimension
and length is the largest.
[0059] The results show that the ADC H16-7.8mcMMAF significantly
inhibited the growth of SKRC-01 tumor formation while the naked MAb
H16-7.8 had no effect. Thus, the ADC H16-7.8mcMMAF had a
significantly more prominent effect that the naked antibody
H16-7.8.
[0060] FIG. 13. Efficacy Study of H16-7.8mcMMAF compared to other
161P2F10B Antibody Drug Conjugates (ADCs) in subcutaneous
established UG-K3 in SCID mice. In another experiment, human renal
cancer cells UG-K3 (1.5.times.10.sup.6 cells per mouse) were
injected into the flanks of individual mice. Tumors were allowed to
grow untreated until they reached an approximate volume of 100
mm.sup.3. On day 0 when tumors reach 100 mm.sup.3, animals were
randomly assigned to the following cohorts: an H16-7.8mcMMAF, an
H16-7.8vcMMAE, and H16-1.11 mcMMAF, and H16-1.11vcMMAE, a PBS
control, and a control MAb-vcMMAE treated group. All antibody drug
conjugates (ADCs) were dosed at 10 mg/kg once on day 0. The amount
of each ADC administered was based on the individual body weight of
each animal obtained immediately prior to dosing. The PBS control
was dosed at 150.mu./L per animal. Tumor growth was monitored using
caliper measurements every 3 to 4 days. Tumor volume was calculated
as Width.sup.2.times.Length/2, where width is the smallest
dimension and length is the largest.
[0061] The results show that the ADCs H16-7.8vcMMAE and
H16-1.11vcMMAE did not inhibit tumor formation growth.
Additionally, both the H16-7.8mcMMAF and H16-1.11mcMMAF
significantly inhibited the growth of UG-K3 tumor formation during
the first thirty (30) days. After day thirty (30) the H16-7.8mcMMAF
had a significantly more prominent effect when compared to
H16-1.11mcMMAF.
[0062] FIG. 14. Peptide maps of H16-7.8mcMMAF and H16-7.8. The
obtained H16-7.8mcMMAF and H16-7.8 were treated with dithiothreitol
(DTT) to reduce disulfide bonds, followed by alkylation of the
resulting free cysteines. Guanidine was used in this step to ensure
complete denaturation of the protein. After dialysis to remove the
guanidine, the samples were digested with a specific
endoproteinase, Lys-C. Lys-C cleaves peptide bonds on the
C-terminal side of lysine residues. The resulting peptides were
analyzed by reversed phase chromatography coupled to mass
spectrometry. The reversed phase retention times and the observed
mass to charge ratios of the peaks were compared between
H16-7.8mcMMAF and H16-7.8. LC-MS (liquid chromatography-mass
spectrometry) analysis was carried out using a WATERS Acquity UPLC
coupled to a WATERS Q-TOFp mass spectrometer. The digested sample
was applied to YMC C18 column and eluted with an acetonitrile
gradient containing trifluoroacetic acid. The results show, peak
intensities indicated by asterisk were reduced in the conjugated
antibody compared to the native antibody. The peaks marked with an
arrow represent new peaks that appeared on the conjugated antibody
peptide map. Specifically, the peaks marked with either an asterisk
or with an arrow are believed to be a peptide destined for
conjugation and the resulting conjugated peptide, respectively.
[0063] FIG. 15. Mass spectra of the (*) peak. The results show a
portion of the mass spectra of the peak marked with an asterisk in
FIG. 14. The mass value of the signal that changed during
conjugation is indicated by the "plus" sign. This peptide with an
approximate m/z of 970.4 (+3 charge state) was identified as
C225-K250 that originated from the hinge region of the heavy chain
and contains the expected conjugation sites.
[0064] FIG. 16. Extracted ion chromatograms (XIC) of MSE on peptide
maps for H16-7.8mcMMAF and H16-7.8 at 619.4 m/z. In order to
identify the newly appeared peaks which are believed to be
conjugated peptide in FIG. 14 above, LC-MS analysis was conducted
using the elevated-energy (MSE) data acquisition technique. This
Figure shows the extracted ion chromatograms (XIC) for peptide maps
of H16-7.8mcMMAF and H16-7.8 using the m/z of 619.4. This ion
corresponds to a fragment ion of the drug moiety. Peaks observed in
XIC at 619.4 are almost identical to the peaks marked with an arrow
in FIG. 14. Furthermore, no such peaks were detected in the
chromatogram of the native antibody. These observations suggest
that the detected peaks in the XIC at m/z of 619.4 were apparently
drug conjugated peptides and are identified by its intact mass
values. The result was summarized in Table V. These results suggest
that in case of the conjugate, predominant peptides are those
conjugated to 2 drugs on the hinge region of heavy chain. These
data are consistent with the data obtained by the other orthogonal
such as a DAR analysis.
[0065] FIG. 17(A). Example Spectra showing mass profiles of
deglycosylated H16-7.8mcMMAF ADC (Lot 059K204).
[0066] FIG. 17(B). Example Spectra showing mass profiles of
deglycosylated H16-7.8mcMMAF ADC (Lot 089K7251). The full mass of
the deglycosylated H16-7.8mcMMAF was determined by electrospray
ionization time-of-flight (ESI-TOF) mass spectrometry. Test samples
were diluted by 250 mM sodium phosphate buffer, pH 7.5 and then
incubated overnight at 37.degree. C. with glycopeptidase F. The
samples were injected onto a PLRPTM column (Varian Technology),
equilibrated at 90.degree. C., and eluted with an
acetonitrile/water gradient. The sample peaks were analyzed by an
Acquity UPLC system coupled to a WATERS Synapt mass spectrometer
(Waters) and masses were reconstructed from the raw data by a
MaxEnt1 software. An example mass spectral profile for the
deglycosylated H16-7.8mcMMAF is shown. The predominant drug
conjugated antibody was a 4-drug loading species. This observation
including an abundance of the unconjugated antibody in
H16-7.8mcMMAF was consistent with the results obtained by the other
orthogonal methods, such as DAR by RP-HPLC, peptide mapping, and
HIC assay.
[0067] FIG. 18. Drug Antibody Ratio (DAR) profile of H16-7.8mcMMAF.
DAR analysis was conducted for quantitative HPLC determination of
the relative amount of drug loading in each Light chain and Heavy
chain. DAR analyses were carried out using a PLRP-S analytical
column, 2.1 mm.times.50 mm, with mobile phase A consisting of 2.0%
formic acid and mobile phase B consisting of 2.0% formic acid plus
90% acetonitrile. A representative DAR profile for H16-7.8mcMMAF is
shown. DAR value is 4.0. The sample was subjected to LC-MS analysis
using same HPLC conditions of this method to identify the observed
peak. Results are summarized in Table VI. The peak identification
of the DAR results obtained during the qualification of this method
has been confirmed orthogonally by LC-MS.
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
[0068] I.) Definitions
[0069] II.) 161P2F10B Antibodies
[0070] III.) Antibody Drug Conjugates Generally [0071] III(A).
Auristatins and Dolostatins
[0072] IV.) Antibody Drug Conjugates which Bind 161P2F10B
[0073] V.) Linker Units
[0074] VI.) The Stretcher Unit
[0075] VII.) The Amino Acid Unit
[0076] VIII.) The Spacer Unit
[0077] IX.) The Drug Unit
[0078] X.) Drug Loading
[0079] XI.) Methods of Determining Cytotoxic effect of ADCs
[0080] XII.) Treatment of Cancer(s)
[0081] XIII.) 161P2F10B as a Target for Antibody-based Therapy
[0082] XIV.) 161P2F10B ADC Cocktails
[0083] XV.) Combination Therapy
[0084] XVI.) KITS/Articles of Manufacture
I.) Definitions
[0085] 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.
[0086] 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.
[0087] 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.
[0088] The abbreviation "AFP" refers to
dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylened-
iamine (see Formula XVI infra).
[0089] The abbreviation "MMAE" refers to monomethyl auristatin E
(see Formula XI infra).
[0090] The abbreviation "AEB" refers to an ester produced by
reacting auristatin E with paraacetyl benzoic acid (see Formula XX
infra).
[0091] The abbreviation "AEVB" refers to an ester produced by
reacting auristatin E with benzoylvaleric acid (see Formula XXI
infra).
[0092] The abbreviation "MMAF" refers to
dovaline-valine-dolaisoleuine-dolaproine-phenylalanine (see Formula
XVIV infra).
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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 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 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.
[0097] 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 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 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 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.
[0098] 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--).
[0099] 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--).
[0100] 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.
[0101] 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.
[0102] 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.
##STR00003##
[0103] 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.
[0104] 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 monocyclic 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.
[0105] 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).
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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 3-carboline. Still more typically, nitrogen bonded
heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,
1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
[0110] 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.
[0111] 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 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.
[0112] 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.
[0113] 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).
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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-fluorophneyl)-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.
[0118] "Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence 161P2F10B (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
161P2F10B. 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.
[0119] The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g., a 161P2F10B-related protein). For example, an
analog of a 161P2F10B protein can be specifically bound by an
antibody or T cell that specifically binds to 161P2F10B.
[0120] 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. 161P2F10B 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 161P2F10B 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 161P2F10B and/or exhibit the desired biological activity. Any
specific antibody can be used in the methods and compositions
provided herein. Thus, in one embodiment the term "antibody"
encompasses a molecule comprising at least one variable region from
a light chain immunoglobulin molecule and at least one variable
region from a heavy chain molecule that in combination form a
specific binding site for the target antigen. In one embodiment,
the antibody is an IgG antibody. For example, the antibody is a
IgG, 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, 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); 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. Nos. 5,624,821, 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
161P2F10B. See e.g., Antibody Engineering: A Practical Approach
(Oxford University Press, 1996). Suitable antibodies with the
desired biologic activities can be identified the following in
vitro assays including but not limited to: proliferation,
migration, adhesion, soft agar growth, angiogenesis, cell-cell
communication, apoptosis, transport, signal transduction,
liternalization, antibody mediated secondary killing, and the
following in vivo assays such as the inhibition of tumor growth.
The antibody provided herein can be useful as an intermediate of
ADC. 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 161P2F10B or its receptor.
[0121] 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 161P2F10B antibody that retain the
ability to specifically bind to an antigen (e.g., 161P2F10B; 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) an Fab
fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H,
C.sub.L and C.sub.H1 domains; (ii) an F(ab').sub.2 fragment, a
bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region; (iii) an Fd fragment
consisting of the V.sub.H and C.sub.H1 domains; (iv) an 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.
[0122] As used herein, any form of the "antigen" can be used to
generate an antibody that is specific for 161P2F10B. 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 161P2F10B of interest.
[0123] 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.
[0124] The monoclonal antibodies 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)).
[0125] 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.
[0126] 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.
[0127] 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,
combretastatin, 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, restrictocin,
phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria
officinalis inhibitor, and glucocorticoid and other
chemotherapeutic agents, as well as radioisotopes such as
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212 or .sup.213, P.sup.32 and radioactive
isotopes of Lu including Lu.sup.177. Antibodies may also be
conjugated to an anti-cancer pro-drug activating enzyme capable of
converting the pro-drug to its active form.
[0128] The term "deplete," in the context of the effect of a
161P2F10B binding agent on 161P2F10B-expressing cells, refers to a
reduction in the number of or elimination of the
161P2F10B-expressing cells.
[0129] The "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.
[0130] "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.
[0131] 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.
[0132] 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.
WO02/43478 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).
[0133] The terms "inhibit" or "inhibition of" as used herein means
to reduce by a measurable amount, or to prevent entirely.
[0134] 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).
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] 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.
[0140] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or composition that is physiologically compatible with humans
or other mammals.
[0141] 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).
[0142] The term "polypeptide" means a polymer of at least about 4,
5, 6, 7, or 8 amino acids. Throughout the specification, standard
three letter or single letter designations for amino acids are
used. In the art, this term is often used interchangeably with
"peptide" or "protein".
[0143] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
[0144] 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 binds
the 161P2F10B-related antigen (particularly 161P2F10B), but does
not bind to the irrelevant antigen.
[0145] 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 out albeit not a
requirement for a treatment act.
[0146] The "161P2F10B-related proteins" 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 161P2F10B proteins or fragments thereof, as well as
fusion proteins of a 161P2F10B protein and a heterologous
polypeptide are also included. Such 161P2F10B proteins are
collectively referred to as the 161P2F10B-related proteins, the
proteins of the invention, or 161P2F10B. The term
"161P2F10B-related protein" refers to a polypeptide fragment or a
161P2F10B protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino
acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90,
95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,
160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300,
325, 330, 335, 339 or more amino acids.
II.) 161P2F10B Antibodies
[0147] Antibodies for ADCs of the invention specifically bind to a
161P2F10B protein and do not bind (or bind weakly) to peptides or
proteins that are not 161P2F10B-related proteins under
physiological conditions.
[0148] 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 161P2F10B-related protein, peptide, or
fragment thereof, 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 161P2F10B can also be used, such as a
161P2F10B 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 161P2F10B-related
protein is synthesized and used as an immunogen.
[0149] In addition, naked DNA immunization techniques known in the
art are used (with or without purified 161P2F10B-related protein or
161P2F10B expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly, et al., 1997, Ann.
Rev. Immunol. 15: 617-648).
[0150] The amino acid sequence of a 161P2F10B protein as shown in
FIG. 1 can be analyzed to select specific regions of the 161P2F10B
protein for generating antibodies. For example, hydrophobicity and
hydrophilicity analyses of a 161P2F10B amino acid sequence are used
to identify hydrophilic regions in the 161P2F10B structure. Regions
of a 161P2F10B 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 ofKyte, 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 161P2F10B 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
161P2F10B 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.
[0151] In a preferred embodiment, a 161P2F10B MAb for ADCs of the
invention comprises heavy and light chain variable regions of an
antibody designated H16-7.8 (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 H16-7.8, and wherein the antibodies retain the
desired functional properties (particularly binding activity to
161P2F10B) of the 161P2F10B MAbs of the invention. The heavy chain
variable region of H16-7.8 consists of the amino acid sequence
ranging from 20.sup.th Q residue to the 142.sup.nd S residue of SEQ
ID NO:7, and the light chain variable region of H16-7.8 consists of
the amino acid sequence ranging from 20.sup.th E residue to the
127.sup.th R residue of SEQ ID NO:8. As the constant region of the
antibody of the invention, any subclass of constant region can be
chosen. Preferably, 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. Reactivity (binding activity) of
161P2F10B MAbs with a 161P2F10B protein can be established by a
number of well known means, including Western blot,
immunoprecipitation, ELISA, and FACS analyses using 161P2F10B
proteins, 161P2F10B-expressing cells or extracts thereof.
[0152] In one embodiment, the hinge region of CH 1 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 161P2F10B MAb.
[0153] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half life of the 161P2F10B 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 Staphylococcal 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.
[0154] In another embodiment, the 161P2F10B 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.
[0155] 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 161P2F10B
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.
[0156] In one embodiment, the antibody for ADC of the present
invention 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); Current
Protocols In Immunology (John Wiley & Sons, most recent
edition).
[0157] The antibody for ADC of the present invention can easily be
prepared by those skilled in the art on the basis of the sequence
information on the heavy-chain variable region and light-chain
variable region thereof disclosed herein, using a method commonly
known in the art. Specifically, a heavy-chain variable region gene
fragment having a base sequence that encodes the heavy-chain
variable region amino acid of the antibody for ADC of the present
invention (SEQ ID NO:7, from 20 to 142), and a light-chain variable
region gene fragment having a base sequence that encodes the
light-chain variable region amino acid of the antibody for ADC of
the present invention (SEQ ID NO:8, from 20 to 127) are prepared.
Then, the variable region genes are joined to a constant region
gene in an appropriate class of human antibody to prepare an
antibody gene. Next, this antibody gene is joined to an appropriate
expression vector, and introduced to a cultured cell. Finally, this
cultured cell is cultured, whereby the antibody can be obtained
from the culture supernatant.
[0158] Each of the above-described variable region gene fragments
that encode the heavy-chain and light-chain variable region amino
acids of the antibody of the present invention (SEQ ID NO:7, from
20 to 142 and SEQ ID NO:8, from 20 to 127) can be prepared by on
the basis of base sequences designed on the basis of the amino acid
sequences of the heavy-chain and light-chain variable regions (SEQ
ID NO:7, from 20 to 142 and SEQ ID NO:8, from 20 to 127), or on the
basis of the base sequences of the heavy-chain and light-chain
variable regions of the antibody of the present invention, shown by
SEQ ID NO:4, from 91 to 459 and SEQ ID NO:6, from 100 to 423, using
a method of gene synthesis commonly known in the art. As such a
method of gene synthesis, various methods obvious to those skilled
in the art, such as the antibody gene synthesis method described in
WO90/07861, can be used. Next, the above-described variable region
gene fragments and the constant region gene of the human antibody
are joined to prepare an antibody gene. Although any subclass of
constant region can be chosen as the constant region of the human
antibody used, human Ig[gamma]2 as the heavy-chain constant region,
and human Ig[kappa] as the light-chain constant region, can be
preferably used.
[0159] As the preferable antibody heavy-chain gene of the antibody
for ADC of present invention, obtained by joining the heavy-chain
variable region gene shown by SEQ ID NO:7, from 20 to 142 and the
human Ig[gamma]2 heavy-chain constant region gene, a gene
comprising a base sequence that encodes the amino acid sequence
shown by SEQ ID NO:7, from 20 to 468, more preferably a gene
comprising the base sequence shown by SEQ ID NO:4, from 91 to 1437,
can be mentioned. As the preferable antibody light-chain gene of
the antibody for ADC of present invention, obtained by joining the
light-chain variable region gene shown by SEQ ID NO:8, from 20 to
127 and the human Ig[kappa] light-chain constant region gene, a
gene comprising a base sequence that encodes the amino acid
sequence shown by SEQ ID NO:8, from 20 to 233, more preferably a
gene comprising the base sequence shown by SEQ ID NO:6, from 100 to
741, can be mentioned. As the antibody for ADC of the present
invention, encoded by a heavy-chain gene comprising the base
sequence shown by SEQ ID NO:4, from 91 to 1437 and a light-chain
gene comprising the base sequence shown by SEQ ID NO:6, from 100 to
741, H16-7.8, described in an Example below, can be mentioned.
[0160] Subsequent to the preparation of this antibody gene,
introduction of the antibody gene to an expression vector,
introduction of the expression vector to cultured cells,
cultivation of the cultured cells, purification of the antibody and
the like can be performed by using various methods commonly known
in the art. The expression vector is not subject to limitation, as
long as it is capable of expressing the antibody gene. It is
preferable to utilize an expression vector already having a human
Ig constant region gene such as AG-[gamma]2 or AG-[kappa], because
it would become an expression vector having the antibody gene
simply when the antibody variable region gene is inserted
thereto.
[0161] The above-described expression vector is introduced to
cultured cells by, for example, the calcium phosphate method and
the like. As examples of the cultured cells to which the expression
vector is introduced, cultured cells such as CHO cells can be used,
and they may be cultured by a conventional method. After the
above-described cultivation, the antibody accumulated in the
culture supernatant can be purified by, for example, various
chromatographies using a Protein A column.
[0162] Reactivity (binding activity) of 161P2F10B antibodies thus
obtained with a 161P2F10B protein can be established by a number of
well known means, including Western blot, immunoprecipitation,
ELISA, and FACS analyses using, as appropriate, 161P2F10B protein,
161P2F10B-expressing cells or extracts thereof. The antibody thus
obtained or an antibody fragment retaining an activity due to the
antibody after being further purified as required, can be prepared
as an antibody for ADC. A 161P2F10B 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 161P2F10B
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).
[0163] In yet another preferred embodiment, the 161P2F10B MAb for
ADCs of the invention is an antibody comprising heavy and light
chain of an antibody designated H16-7.8. The heavy chain of H16-7.8
consists of the amino acid sequence ranging from 20.sup.th Q
residue to the 468.sup.th K residue of SEQ ID NO:7 and the light
chain of H16-7.8 consists of amino acid sequence ranging from
20.sup.th E residue to the 233.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, H16-7.8 is conjugated to a cytotoxic
agent.
III.) Antibody-Drug Conjugates Generally
[0164] In another aspect, the invention provides antibody-drug
conjugates (ADCs), comprising an antibody conjugated to a cytotoxic
agent such as MMAF. In another aspect, the invention further
provides methods of using the ADCs. In one aspect, an ADC comprises
any of the above 161P2F10B MAbs covalently attached to a cytotoxic
agent.
[0165] The use of antibody-drug conjugates for the local delivery
of cytotoxic or cytostatic agents, i.e., drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos (1999)
Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997)
Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows
targeted delivery of the drug moiety to tumors, and intracellular
accumulation therein, where systemic administration of these
unconjugated drug agents may result in unacceptable levels of
toxicity to normal cells as well as the tumor cells sought to be
eliminated (Baldwin, et al., (1986) Lancet pp. (Mar. 15,
1986):603-05; Thorpe, (1985) "Antibody Carriers Of Cytotoxic Agents
In Cancer Therapy: A Review," in Monoclonal Antibodies '84:
Biological And Clinical Applications, A. Pinchera, et al. (ed.s),
pp. 475-506). Maximal efficacy with minimal toxicity is sought
thereby. Both polyclonal antibodies and monoclonal antibodies have
been reported as useful in these strategies (Rowland, et al. (1986)
Cancer Immunol. Immunother., 21:183-87). Drugs used in these
methods include daunomycin, doxorubicin, methotrexate, and
vindesine (Rowland, et al., (1986) supra). Toxins used in
antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Mandler, et al. (2000) Jour. of the Nat.
Cancer Inst. 92(19):1573-1581; Mandler, et al. (2000) Bioorganic
& Med. Chem. Letters 10:1025-1028; Mandler, et al. (2002)
Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu, et
al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and
calicheamicin (Lode, et al. (1998) Cancer Res. 58:2928; Hinman, et
al. (1993) Cancer Res. 53:3336-3342). The toxins may effect their
cytotoxic and cytostatic effects by mechanisms including tubulin
binding, DNA binding, or topoisomerase inhibition. Some cytotoxic
drugs tend to be inactive or less active when conjugated to large
antibodies or protein receptor ligands.
[0166] 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).
[0167] Additionally, MYLOTARG.TM. (gemtuzumab ozogamicin, Wyeth
Pharmaceuticals), an antibody drug conjugate composed of a hu CD33
antibody linked to calicheamicin, was approved in 2000 for the
treatment of acute myeloid leukemia by injection (Drugs of the
Future (2000) 25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233;
5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285; and
5,773,001).
[0168] 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, DM 1, is
advancing into Phase 11 trials for the treatment of cancers that
express CanAg, such as colon, pancreatic, gastric, and others.
[0169] 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.
[0170] Finally, the auristatin peptides, auristatin E (AE) and
monomethylauristatin (MMAE), synthetic analogs of dolastatin, were
conjugated to chimeric monoclonal antibodies cBR96 (specific to
Lewis Y on carcinomas) and cAC 10 (specific to CD30 on
hematological malignancies) (Doronin, et al. (2003) Nature
Biotechnology 21(7):778-784) and are under therapeutic
development.
[0171] III(A). Auristatins and Dolostatins
[0172] 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).
[0173] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties D.sub.E and D.sub.F,
disclosed in Senter, et al, "Proceedings of the American
Association for Cancer Research," Volume 45, Abstract Number 623,
and presented Mar. 28, 2004, the disclosure of which is expressly
incorporated by reference in its entirety.
[0174] An exemplary auristatin embodiment is MMAE (wherein the wavy
line indicates the covalent attachment to a linker (L) of an
antibody drug conjugate).
##STR00004##
[0175] 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):
##STR00005##
[0176] 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 12):
##STR00006##
[0177] ADC of the present invention include MMAF. The preferred
embodiment, the ADC of the present invention include
maleimidocaproyl (mc) as a linker and MMAF as a drug.
IV.) Antibody-Drug Conjugate Compounds which Bind 161P2F10B
[0178] 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 161P2F10B. 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-).
[0179] In some embodiments, the antibody drug conjugate compound
has the following formula:
L-(LU-D).sub.p (I)
[0180] or a pharmaceutically acceptable salt or solvate thereof;
wherein:
[0181] L is the Antibody unit, e.g., 161P2F10B MAb of the present
invention, and
[0182] (LU-D) is a Linker unit-Drug unit moiety, wherein:
[0183] LU- is a Linker unit, and
[0184] -D is a drug unit having cytostatic or cytotoxic activity
against a target cell; and
[0185] p is an integer from 1 to 20.
[0186] In some embodiments, p ranges from 1 to 12, 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.
[0187] 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)
[0188] or a pharmaceutically acceptable salt or solvate thereof,
wherein:
[0189] L is the Antibody unit, e.g., 161P2F10B MAb; and
[0190] -A.sub.a-W.sub.w--Y.sub.y-- is a Linker unit (LU),
wherein:
[0191] -A- is a Stretcher unit,
[0192] a is 0 or 1,
[0193] each --W-- is independently an Amino Acid unit,
[0194] w is an integer ranging from 0 to 12,
[0195] --Y-- is a self-immolative spacer unit,
[0196] y is 0, 1 or 2;
[0197] -D is a drug units having cytostatic or cytotoxic activity
against the target cell; and
[0198] p is an integer from 1 to 20.
[0199] 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 12, 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.
[0200] For compositions comprising a plurality of antibodies, the
drug loading is represented by p, the average number of drug
molecules per Antibody. Drug loading may range from 1 to 12 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.
[0201] 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 161P2F10B
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 161P2F10B MAb comprising heavy and light chain
variable regions of an antibody designated H16-7.8 described above.
In more preferred embodiment, the Antibody is 161P2F10B MAb
comprising heavy and light chain of an antibody designated H16-7.8
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.
[0202] 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 161P2F10B MAb under appropriate
conditions.
[0203] 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
[0204] Typically, the antibody-drug conjugate compounds comprise a
linker region 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.
[0205] 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 161P2F10B-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.
[0206] 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).
[0207] 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.)
[0208] 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).
[0209] 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). In preferred embodiment, linker
unit is maleimidocaproyl (mc).
[0210] 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.
[0211] 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 161P2F10B MAb.
[0212] A variety of exemplary linkers that can be used with the
present compositions and methods are described in WO2004-010957,
U.S. Publication No. 20060074008, U.S. Publication No. 20050238649,
and U.S. Publication No. 20060024317 (each of which is incorporated
by reference herein in its entirety and for all purposes).
[0213] 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--
[0214] wherein:-A- is a Stretcher unit,
[0215] a is 0 or 1,
[0216] each --W-- is independently an Amino Acid unit,
[0217] w is an integer ranging from 0 to 12,
[0218] --Y-- is a self-immolative Spacer unit, and
[0219] y is 0, 1 or 2.
[0220] 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
[0221] 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 161P2F10B MAb (e.g.,
H16-7.8), 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 161P2F10B MAb. In another embodiment, sulfhydryl groups can be
generated by reaction of an amino group of a lysine moiety of a
161P2F10B MAb with 2-iminothiolane (Traut's reagent) or other
sulfhydryl generating reagents. In certain embodiments, the
161P2F10B MAb is a recombinant antibody and is engineered to carry
one or more lysines. In certain other embodiments, the recombinant
161P2F10B MAb is engineered to carry additional sulfhydryl groups,
e.g., additional cysteines.
[0222] 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.rCH.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.rCH.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.
[0223] It is to be understood from all the exemplary embodiments
that even where not denoted expressly, from 1 to 12 drug moieties
can be linked to an Antibody (p=1-12).
##STR00007##
[0224] An illustrative Stretcher unit is that of Formula IIIa
wherein R.sup.17 is --(CH.sub.2).sub.5--:
##STR00008##
[0225] Another illustrative Stretcher unit is that of Formula IIIa
wherein R.sup.17 is --(CH.sub.2CH.sub.2O).sub.rCH.sub.2--; and r is
2:
##STR00009##
[0226] An illustrative Stretcher unit is that of Formula IIIa
wherein R.sup.17 is -arylene- or arylene-C.sub.1-C.sub.10
alkylene-. In some embodiments, the aryl group is an unsubstituted
phenyl group.
[0227] Still another illustrative Stretcher unit is that of Formula
IIIb wherein R.sup.17 is --(CH.sub.2).sub.5--:
##STR00010##
[0228] 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.
L-S S--R.sup.17--C(O) W.sub.w--Y.sub.y-D IV
[0229] 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.
##STR00011##
[0230] 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;
##STR00012##
[0231] 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.
##STR00013##
VII.) the Amino Acid Unit
[0232] 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.
[0233] 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:
##STR00014##
[0234] 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,
##STR00015##
[0235] 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).
[0236] 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):
##STR00016##
[0237] 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; ##STR00017##
(CH.sub.2).sub.3NHCONH.sub.2; benzyl methyl; benzyl
(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2;
##STR00018##
[0238] 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;
##STR00019##
[0239] 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.
[0240] 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.2 is benzyl, R.sup.21
is benzyl, and R.sup.22 is --(CH.sub.2).sub.4NH.sub.2.
[0241] 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.
[0242] 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.
[0243] Each carbon atom to which R.sup.19, R.sup.2, R.sup.21,
R.sup.22 or R.sup.23 is attached is independently in the (S) or (R)
configuration.
[0244] 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
[0245] 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.
[0246] 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.
##STR00020##
[0247] 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-.
[0248] 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.
[0249] 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.
[0250] In some embodiments, --Y.sub.y-- is a p-aminobenzyl alcohol
(PAB) unit (see Schemes 2 and 3) whose phenylene portion is
substituted with Q.sub.m wherein Q is --C.sub.1-C.sub.8 alkyl,
--C.sub.1-C.sub.8 alkenyl, --C.sub.1-C.sub.8 alkynyl,
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.1-C.sub.8 alkenyl),
--O--(C.sub.1-C.sub.8 alkynyl), -halogen, -nitro or -cyano; and m
is an integer ranging from 0-4. The alkyl, alkenyl and alkynyl
groups, whether alone or as part of another group, can be
optionally substituted.
[0251] 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.
##STR00021##
[0252] 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 12. The alkyl, alkenyl and alkynyl groups,
whether alone or as part of another group, can be optionally
substituted.
[0253] 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.
##STR00022##
[0254] 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 12. The alkyl, alkenyl and alkynyl groups,
whether alone or as part of another group, can be optionally
substituted.
[0255] 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 o-position of
glycine (Kingsbury et al., 1984, J. Med Chem. 27:1447) are also
examples of self-immolative spacers.
[0256] 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.
##STR00023##
[0257] 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 12. The alkyl, alkenyl
and alkynyl groups, whether alone or as part of another group, can
be optionally substituted.
[0258] In some embodiments, the -D moieties are the same. In yet
another embodiment, the -D moieties are different.
[0259] In one aspect, Spacer units (--Y.sub.y--) are represented by
Formulas (X)-(XII):
##STR00024##
[0260] 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.
##STR00025##
[0261] Embodiments of the Formula I and II comprising antibody-drug
conjugate compounds can include:
##STR00026##
[0262] wherein w and y are each 0, 1 or 2, and,
##STR00027##
[0263] wherein w and y are each 0,
##STR00028##
IX.) the Drug Unit
[0264] 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.
[0265] Useful classes of cytotoxic or immunomodulatory agents
include, for example, antitubulin agents, DNA minor groove binders,
DNA replication inhibitors, and alkylating agents.
[0266] 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. WO04/010957,
International Patent Publication No. WO02/088172, 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.
[0267] Auristatins have been shown to interfere with microtubule
dynamics and nuclear and cellular division and have anticancer
activity. MMAF bind tubulin and can exert a cytotoxic or cytostatic
effect on a 161P2F10B-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.
[0268] Methods for determining whether a compound binds tubulin are
known in the art. See, for example, Muller, et al., Anal. Chem
2006, 78, 4390-4397; Hamel, et al., Molecular Pharmacology, 1995
47: 965-976; and Hamel, et al., The Journal of Biological
Chemistry, 1990 265:28, 17141-17149. For purposes of the present
invention, the relative affinity of a compound to tubulin can be
determined. Some preferred auristatins of the present invention
bind tubulin with an affinity ranging from 10 fold lower (weaker
affinity) than the binding affinity of MMAE to tubulin to 10 fold,
20 fold or even 100 fold higher (higher affinity) than the binding
affinity of MMAE to tublin.
[0269] In some embodiments, -D is an auristatin of the formula
D.sub.E or D.sub.F:
##STR00029##
[0270] or a pharmaceutically acceptable salt or solvate form
thereof;
[0271] wherein, independently at each location:
[0272] the wavy line indicates a bond;
[0273] 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;
[0274] 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);
[0275] 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);
[0276] R.sup.5 is --H or --C.sub.1-C.sub.8 alkyl;
[0277] 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,
[0278] 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;
[0279] 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);
[0280] 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;
[0281] 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;
[0282] R.sup.24 is -aryl, -heterocycle, or -carbocycle;
[0283] 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;
[0284] 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;
[0285] R.sup.10 is -aryl or -heterocycle;
[0286] 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;
[0287] 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;
[0288] m is an integer ranging from 1-1000;
[0289] 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;
[0290] R.sup.14 is --H, --C.sub.1-C.sub.20 alkyl,
--C.sub.2-C.sub.20 alkenyl, or --C.sub.2-C.sub.20 alkynyl; each
occurrence of R.sup.is 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;
[0291] 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
[0292] n is an integer ranging from 0 to 6;
[0293] wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene,
alkynyklene, aryl, carbocyle, and heterocycle radicals, whether
alone or as part of another group, are optionally substituted.
[0294] Auristatins of the formula D.sub.E include those wherein
said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene,
aryl, carbocyle, and heterocycle radicals are unsubstituted.
[0295] 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.
[0296] Auristatins of the formula D.sub.E include those wherein
[0297] R.sup.2 is C.sub.1-C.sub.8 alkyl;
[0298] 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;
[0299] R.sup.5 is --H;
[0300] R.sup.6 is --C.sub.1-C.sub.8 alkyl;
[0301] 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;
[0302] R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl;
[0303] R.sup.24 is optionally substituted -phenyl;
[0304] 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;
[0305] 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.
[0306] Auristatins of the formula D.sub.E include those wherein
[0307] R.sup.2 is methyl;
[0308] 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;
[0309] 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;
[0310] R.sup.5 is --H;
[0311] R.sup.6 is methyl;
[0312] 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;
[0313] each R.sup.8 is methoxy;
[0314] R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl;
[0315] R.sup.24 is -phenyl;
[0316] 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;
[0317] R.sup.2 is methyl;
[0318] or a pharmaceutically acceptable salt form thereof.
[0319] Auristatins of the formula D.sub.E include those
wherein:
[0320] 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.8 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.is represents
.dbd.OO; and R.sup.26 is methyl; or a pharmaceutically acceptable
salt or solvate form thereof.
[0321] Auristatins of the formula D.sub.E include those
wherein:
[0322] R.sup.2 is methyl or C.sub.1-C.sub.3 alkyl,
[0323] R.sup.3 is --H or --C.sub.1-C.sub.3 alkyl;
[0324] R.sup.4 is --C.sub.1-C.sub.5 alkyl;
[0325] R.sup.5 is H;
[0326] R.sup.7 is C1-C3 alkyl;
[0327] R.sup.7 is --C.sub.1-C.sub.5 alkyl;
[0328] R.sup.8 is --C.sub.1-C.sub.3 alkoxy;
[0329] R.sup.9 is --H or --C.sub.1-C.sub.8 alkyl;
[0330] R.sup.24 is phenyl;
[0331] 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
[0332] R.sup.26 is --C.sub.1-C.sub.3 alkyl;
[0333] or a pharmaceutically acceptable salt form thereof.
[0334] Auristatins of the formula D.sub.1 include those wherein
[0335] R.sup.2 is methyl;
[0336] 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;
[0337] R.sup.5 is --H;
[0338] R.sup.6 is methyl;
[0339] each R.sup.8 is methoxy;
[0340] 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;
[0341] R.sup.10 is optionally substituted aryl or optionally
substituted heterocycle;
[0342] 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;
[0343] 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;
[0344] m is an integer ranging from 1-1000 or m=0;
[0345] 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;
[0346] 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;
[0347] 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;
[0348] 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;
[0349] n is an integer ranging from 0 to 6;
[0350] or a pharmaceutically acceptable salt thereof.
[0351] In certain of these embodiments, R.sup.1 is optionally
substituted phenyl.
[0352] 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.
[0353] Auristatins of the formula D.sub.F include those wherein
said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene,
aryl, carbocyle, and heterocycle radicals are unsubstituted.
[0354] Auristatins of the formula D.sub.F include those wherein
[0355] 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.8 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.8 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'' is as defined
herein; or a pharmaceutically acceptable salt thereof.
[0356] Auristatins of the formula D.sub.F include those wherein
[0357] 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.8 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'' is as
defined herein; or a pharmaceutically acceptable salt thereof.
[0358] Auristatins of the formula D.sub.F include those wherein
[0359] 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.8 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.n is as defined herein, preferably
hydrogen; or a pharmaceutically acceptable salt form thereof.
[0360] Auristatins of the formula D.sub.F include those wherein
[0361] R.sup.2 is --C.sub.1-C.sub.3 alkyl; R.sup.3 is --H or
--C.sub.1-C.sub.3 alkyl; R.sup.4 is --C.sub.1-C.sub.8 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.8 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.
[0362] 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.
[0363] 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.
[0364] 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.
[0365] 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.
[0366] Auristatins of the formula D.sub.F include those wherein Z
is --O-- or --NH--. The remainder of the substituents are as
defined herein.
[0367] Auristatins of the formula D.sub.F include those wherein
R.sup.10 is aryl. The remainder of the substituents are as defined
herein.
[0368] Auristatins of the formula D.sub.F include those wherein
R.sup.10 is -phenyl. The remainder of the substituents are as
defined herein.
[0369] 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.
[0370] 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.
[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--SO.sub.3H. The remainder of the substituents
are as defined herein.
[0372] 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.
[0373] In some embodiments, wherein D is an auristatin of formula
D.sub.F, a is 1 and w and y are 0.
[0374] Illustrative Drug units (-D) include the drug units having
the following structures:
##STR00030## ##STR00031## ##STR00032##
[0375] 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''. 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] In preferred embodiment, antibody-drug conjugate compounds
have the following structures wherein "L" or "mAb-s-" represents a
161P2F10B MAb designated H16-7.8 set forth herein:
##STR00033##
[0379] or pharmaceutically acceptable salt thereof.
[0380] 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.
[0381] 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.
[0382] 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.
[0383] 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).
[0384] In certain embodiments, the cytotoxic or cytostatic agent is
a dolastatin. In certain embodiments, the cytotoxic or cytostatic
agent is of the auristatin class. Thus, in a specific embodiment,
the cytotoxic or cytostatic agent is MMAE (Formula XI). In another
specific embodiment, the cytotoxic or cytostatic agent is AFP
(Formula XVI).
##STR00034##
[0385] In certain embodiments, the cytotoxic or cytostatic agent is
a compound of formulas XII-XXI or pharmaceutically acceptable salt
thereof:
##STR00035## ##STR00036##
[0386] p is 1 to 12. In more preferred embodiment, antibody-drug
conjugate compounds have the following structures wherein "mAb-s-"
represents an 161P2F10B MAb designated H16-7.8 set forth herein,
and p is 4:
##STR00037##
[0387] In a specific embodiment, the cytotoxic or cytostatic agent
is MMAF (Formula XVIV).
X.) Drug Loading
[0388] 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.
[0389] 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 12; 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).
[0390] 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.
[0391] 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)).
[0392] 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
[0393] 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.
[0394] For determining whether an 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.
[0395] 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.
[0396] Cell viability can be measured by determining in a cell the
uptake of a dye such as neutral red, trypan blue, or ALAMAR.TM.
blue (see, e.g., Page, et al., 1993, Intl. J. Oncology 3:473-476).
In such an assay, the cells are incubated in media containing the
dye, the cells are washed, and the remaining dye, reflecting
cellular uptake of the dye, is measured spectrophotometrically. The
protein-binding dye sulforhodamine B (SRB) can also be used to
measure cytotoxicity (Skehan, et al., 1990, J. Natl. Cancer Inst.
82:1107-12).
[0397] 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).
[0398] 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).
[0399] 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.
[0400] 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).
[0401] In vivo, the effect of a 161P2F10B 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.
[0402] 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.
[0403] 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).
[0404] 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.
[0405] 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)
[0406] The identification of 161P2F10B 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.
[0407] 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.
[0408] 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.
[0409] Accordingly, therapeutic approaches that inhibit the
activity of a 161P2F10B protein are useful for patients suffering
from a cancer that expresses 161P2F10B. These therapeutic
approaches generally fall into three classes. The first class
modulates 161P2F10B 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 161P2F10B protein
with its binding partner or with other proteins. The third class
comprises a variety of methods for inhibiting the transcription of
a 161P2F10B gene or translation of 161P2F10B mRNA.
[0410] Accordingly, Cancer patients can be evaluated for the
presence and level of 161P2F10B expression, preferably using
immunohistochemical assessments of tumor tissue, quantitative
161P2F10B imaging, or other techniques that reliably indicate the
presence and degree of 161P2F10B 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.) 161P2F10B as a Target for Antibody-Based Therapy
[0411] 161P2F10B 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 161P2F10B is expressed by
cancer cells of various lineages relative to corresponding normal
cells, systemic administration of 161P2F10B-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 161P2F10B
are useful to treat cancers (preferably cancer of Table I, more
preferably kidney and/or liver cancer) systemically, preferably as
antibody drug conjugates (i.e., ADCs) wherein the conjugate is with
a toxin or therapeutic agent.
[0412] 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 161P2F10B sequence shown in FIG. 1. In
addition, skilled artisans understand that it is routine to
conjugate antibodies to cytotoxic agents (see, e.g., Slevers, et
al., Blood 93:11 3678-3684 (Jun. 1, 1999)). When cytotoxic and/or
therapeutic agents are delivered directly to cells, such as by
conjugating them to antibodies specific for a molecule expressed by
that cell (e.g., 161P2F10B), the cytotoxic agent will exert its
known biological effect (i.e., cytotoxicity) on those cells.
[0413] 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 161P2F10B
MAb, preferably H16-7.8) that binds to an antigen (e.g., 161P2F10B)
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 161P2F10B, comprising
conjugating the cytotoxic agent to an antibody that
immunospecifically binds to a 161P2F10B 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.
[0414] Cancer immunotherapy using 161P2F10B 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. (trastuzumab) with paclitaxel (Genentech,
Inc.).
[0415] In a preferred embodiment, the antibodies will be conjugated
a cytotoxic agent, supra, preferably an auristatin derivative
designated MMAF (Seattle Genetics).
[0416] 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 161P2F10B antigen with high
affinity.
XIV.) 161P2F10B ADC Cocktails
[0417] Therapeutic methods of the invention contemplate the
administration of single 161P2F10B ADCs as well as combinations, or
cocktails, of different MAbs (i.e., 161P2F10B 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, 161P2F10B 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 161P2F10B MAbs
are administered in conjugated form.
[0418] 161P2F10B 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
161P2F10B 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.
[0419] 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 161P2F10B expression in the
patient, the extent of circulating shed 161P2F10B 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.
[0420] Optionally, patients should be evaluated for the levels of
161P2F10B in a given sample (e.g., the levels of circulating
161P2F10B antigen and/or 161P2F10B 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).
[0421] An object of the present invention is to provide 161P2F10B
ADCs, which inhibit or retard the growth of tumor cells, preferably
tumor cells of Table 1. 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 161P2F10B ADCs, and in particular using such
161P2F10B ADCs combined with other drugs or immunologically active
treatments.
XV.) Combination Therapy
[0422] In one embodiment, there is synergy when tumors, including
human tumors, are treated with 161P2F10B ADCs in conjunction with
chemotherapeutic agents or radiation or combinations thereof. In
other words, the inhibition of tumor growth by a 161P2F10B 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 161P2F10B
ADC or the additive effect of treatment with a 161P2F10B 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 161P2F10B ADC or with
treatment using an additive combination of a 161P2F10B ADC and a
chemotherapeutic agent or radiation.
[0423] The method for inhibiting growth of tumor cells using a
161P2F10B ADC and a combination of chemotherapy or radiation or
both comprises administering the 161P2F10B 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 161P2F10B
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.
[0424] 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 161P2F10B
ADCs and the chemotherapeutic agent are administered as separate
molecules. Particular examples of chemotherapeutic agents or
chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin,
mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide,
carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin),
daunorubicin, procarbazine, mitomycin, cytarabine, etoposide,
methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin,
paclitaxel (taxol), docetaxel (taxotere), aldesleukin,
asparaginase, busulfan, carboplatin, cladribine, dacarbazine,
floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon
alpha, leuprolide, megestrol, melphalan, mercaptopurine,
plicamycin, mitotane, pegaspargase, pentostatin, pipobroman,
plicamycin, streptozocin, tamoxifen, teniposide, testolactone,
thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil,
taxol and combinations thereof.
[0425] The source of radiation, used in combination with a
161P2F10B 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).
[0426] 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 WO2005/092380 (Pfizer)) or other ligands that inhibit
tumor growth by binding to IGF-1R, and cytokines.
[0427] 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.
[0428] 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
[0429] 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.
[0430] 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.
[0431] 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.
[0432] The terms "kit" and "article of manufacture" can be used as
synonyms.
[0433] In another embodiment of the invention, an article(s) of
manufacture containing compositions, such as 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 also comprises an antibody, binding
fragment thereof or specific binding protein for use in evaluating
protein expression of 161P2F10B 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.
[0434] 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 drug conjugate
specifically binding to 161P2F10B.
[0435] 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
[0436] 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 161P2F10B Antigen
[0437] The 161P2F10B gene sequence was discovered using Suppression
Subtractive Hybridization (SSH) methods known in the art. The
161P2F10B SSH sequence of 182 bp was identified from cDNA derived
from kidney cancer patients using standard methods. A full length
cDNA clone for 161P2F10B was isolated from kidney cancer patient
specimens. The cDNA is 3858 bp in length and encodes a 875 amino
acid ORF (See, FIG. 1). 161P2F10B showed homology to ENPP3 (See,
Buhring, et. al., Blood 97:3303-3305 (2001). 161P2F10B maps to
chromosome 6q22 using standard methods known in the art. For
further reference see, U.S. Pat. No. 7,279,556 (Agensys, Inc.,
Santa Monica, Calif.), U.S. Pat. No. 7,405,290 (Agensys, Inc.,
Santa Monica, Calif.), U.S. Pat. No. 7,067,130 (Agensys, Inc.,
Santa Monica, Calif.), and U.S. Pat. No. 7,226,594 (Agensys, Inc.,
Santa Monica, Calif.).
Example 2
Generation of 161P2F10B Monoclonal Antibodies (MAbs)
[0438] In one embodiment, therapeutic Monoclonal Antibodies
("MAbs") to 161P2F10B comprise those that react with epitopes
specific for protein that would bind, internalize, disrupt or
modulate the biological function of 161P2F10B, 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 161P2F10B protein sequence, and regions predicted to
contain functional motifs predicted to be antigenic from computer
analysis of the amino acid sequence. Immunogens include peptides
and recombinant proteins such as tag5-161P2F10B, a purified
mammalian cell derived His tagged protein. In addition, cells
engineered through retroviral transduction to express high levels
of 161P2F10B, such as RAT1-161P2F10B, are used to immunize
mice.
[0439] MAbs to 161P2F10B were generated using XenoMouse.RTM.
technology (Amgen 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 H16-7.8 was generated from immunization with human
72 producing XenoMice with Tag5-161P2F10B cells.
[0440] The 161P2F10B MAb H16-7.8 specifically binds to recombinant
161P2F10B (SEQ ID NO:2) expressing cells and endogenous cell
surface 161P2F10B expressed in cancer xenograft cells.
[0441] DNA coding sequences for 161P2F10B MAb H16-7.8 were
determined after isolating mRNA from the respective hybridoma cells
with TRIzol.RTM. reagent (Life Technologies, Gibco BRL).
[0442] Anti-161P2F10B H16-7.8 heavy and light chain variable
nucleic acid sequences were sequenced from the hybridoma cells
using the following protocol. H16-7.8 secreting hybridoma cells
were lysed with TRIzol.RTM. 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.RTM.-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.
[0443] The nucleic acid and amino acid sequences of the variable
heavy and light chain regions are listed in FIG. 2 and FIG. 3. The
heavy chain variable region of H16-7.8 consists of the amino acid
sequence ranging from 20.sup.th Q residue to the 142.sup.nd S
residue of SEQ ID NO:7, and the light chain variable region of
H16-7.8 consists of the amino acid sequence ranging from 20.sup.t E
residue to the 127.sup.th R residue of SEQ ID NO:8. The heavy chain
of H16-7.8 consists of the amino acid sequence ranging from
20.sup.th Q residue to the 468.sup.th K residue of SEQ ID NO:7 and
the light chain of H16-7.8 consists of amino acid sequence ranging
from 20.sup.th E residue to the 233.sup.th C residue of SEQ ID
NO:8. Alignment of H16-7.8 to human VH4-31/D5-12/JH6 germline and
human A26/JK1 germline is set forth in FIG. 4A-4B.
Example 3
Expression of H16-7.8 Using Recombinant DNA Methods
[0444] To express H16-7.8 recombinantly in transfected cells,
H16-7.8 heavy and light chain sequences (SEQ ID NO:7, from 20 to
468 and the light chain of SEQ ID NO:8, from 20 to 233) were cloned
into expression vectors. The complete H16-7.8 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
H16-7.8 expressing constructs were transfected into CHO cells and
recombinant H16-7.8 were secreted from CHO cells. IgG titers were
measured by ELISA. Results confirmed IgG and expression and good
co-expression of the heavy and light chains. Recombinant H16-7.8
were evaluated for binding to cell surface 161P2F10B by flow
cytometry (FIG. 5). 3T3-control and 3T3-161P2F10B cells were
stained with recombinant H16-7.8 from either hybridoma or from CHO
cells transfected with H16-7.8 heavy and light chain vector
constructs.
[0445] Binding was detected by flow cytometry. Results show that
the recombinantly expressed H16-7.8 in CHO cells is secreted and
binds specifically to cell surface 161P2F10B. (FIG. 5).
[0446] Recombinant H16-7.8 was characterized with respect to its
peptide sequence. The peptide mapping analysis of H16-7.8 confirmed
that the deduced amino acid sequence of H16-7.8 is correct versus
the sequence determined using Lys-C digestion with LC-MS/MS, Asp-N
digestion with LC-MS/MS, and N-terminal sequence by Edman
degradation.
Example 4
Antibody Drug Conjugation of H16-7.8
[0447] The H16-7.8 (FIG. 2) was conjugated to a
dolavaline-valine-dolaisoleucine-dolaproine-phenylalanine
(aurastatin derivative) designated MMAF (Formula XVIV; monomethyl
auristatin F) using a maleimidocaproyl (mc) non-cleavble linker set
forth below:
##STR00038##
[0448] The synthesis of the maleimidocaproyl (mc) non-cleavable
linker to the MMAF (Seattle Genetics, Seattle, Wash.) was completed
(SAFC, Madison, Wis.) using the synthesis method set forth in Table
VI to create the cytotoxic mcMMAF.
[0449] Then, the antibody drug conjugate (ADC) of the invention
designated H16-7.8mcMMAF was made using the following
protocols.
[0450] Briefly, a 10 mg/mL solution of the H16-7.8 in 10 mM
succinate, at pH 4.5 was buffer exchanged by diafiltration. The
purpose of the buffer exchange is to remove H16-7.8 formulation
buffer components and replace it with a more compatible buffer that
is optimized for the subsequent "reduction" step. The antibody was
diafiltered against 6 diavolumes (DV) of sodium borate buffer and
concentrated to 10.+-.1 mg/ml, flushed from the system, diluted to
7.5 mg/ml and 0.2 .mu.m filtered.
[0451] Subsequently, EDTA was added to 5 mM final concentration in
the reaction mixture. Next, the disulfide bonds of the H16-7.8 were
partially reduced with tris-(2-carboxyethyl)-phosphine
hydrochloride (TCEP) to form free thiols (SH). This process is
performed at 37.degree. C. EDTA was present at 5 mM concentration
during this reaction to chelate any divalent metal cations that
could cause unwanted SH re-oxidation. At the end of the reduction
step the temperature of the reaction solution was lowered to
20.degree. C. and analyzed to determine the molar ratio of free SH
and to ensure that 23.9 SH per MAb had been generated. For
conjugation, the drug-linker mcMMAF was weighed out in the isolator
and dissolved in DMSO to a concentration of 5.5 mg/ml.
[0452] The SH groups on the partially reduced H16-7.8 were reacted
with drug-linker mcMMAF to form the conjugate, H16-7.8mcMMAF. The
mcMMAF in DMSO was added at a set molar equivalent of drug to
antibody. This step was performed at 20.degree. C. After 1 h
incubation period any excess of the drug-linker in the reaction was
quenched with N-Acetyl-cystein, thus eliminating any reactive
drug-linker and turning it into and adduct that is easier to remove
and to detect by analytical methods. The mixture is then stirred
for fifteen (15) additional minutes following the addition of one
(1) molar equivalents of N-Acetyl-Cysteine relative to mcMMAF.
[0453] Next, ultrafiltration/dialfiltration is performed in order
to remove DMSO, process impurities, and buffer exchange the ADC
into formulation buffer.
[0454] Excess quenched mcMMAF are thus removed by
ultrafiltration/diafiltration of the antibody drug conjugate (ADC)
with 8 diavolumes of 20 mM histidine, pH 5.2 formulation buffer.
After the completion of the 8th diavolume, the solution is
recirculated and assayed for protein concentration.
[0455] The conjugate is adjusted to six (6).+-.0.5 mg/ml with 20 mM
Histidine, 10% trehalose, pH 5.2 buffer. Polysorbate 20 is then
added, mixed to homogeneity, and aseptically filtered through a 0.2
.mu.m filter.
[0456] The resulting antibody drug conjugate (ADC) is designated
H16-7.8mcMMAF and has the following formula:
##STR00039##
[0457] wherein MAb is H16-7.8 (FIG. 2 and FIG. 3) and p is from 1
to 12.
Example 5
Characterization of H16-7.8 and H16-7.8mcMMAF
[0458] The H16-7.8 was generated using the procedures set forth in
the example entitled Example 2 "Generation of 161P2F10B Monoclonal
Antibodies (MAbs)". Additionally, the Antibody Drug Conjugates that
bind 161P2F10B were generated using the procedures set forth in the
example entitled "Antibody Drug Conjugation of H16-7.8". The
H16-7.8 and H16-7.8mcMMAF ADC were screened, identified, and
characterized using a combination of assays known in the art.
[0459] A. Cell Binding and Affinity Determination by FACS
[0460] H16-7.8 and H16-7.8mcMMAF were tested for the binding
affinity to 161P2F10B endogenously expressed on Ku812 cells.
Briefly, eleven (11) dilutions of H16-7.8 or H16-7.8mcMMAF are
incubated with Ku812 cells (50,000 cells per well) overnight at
4.degree. C. at a final concentration of 160 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 are obtained (See,
Table IV). 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 H16-7.8 or H16-7.8mcMMAF saturation
curves shown in FIG. 6. Bmax is the MFI value at maximal binding of
H16-7.8 or H16-7.8mcMMAF to 161P2F10B; Kd is H16-7.8 or
H16-7.8mcMMAF binding affinity which is the concentration of
H16-7.8 or H16-7.8mcMMAF required to reach half-maximal
binding.
[0461] The calculated affinity (Kd) of H16-7.8 and H16-7.8mcMMAF is
0.06 nM and 0.19 nM, respectively on 161P2F10B-related protein
endogenously expressed on the surface of Ku812 cells.
[0462] To determine binding of H16-7.8 and H16-7.8mcMMAF to
endogenous 161P2F10B-related protein expressed on the surface of
renal cancer cells human UGK-3 cells (patient derived clear cell
renal cancer) and RXF-393 cells (clear cell renal cancer) were
stained with 10 .mu.g/ml of native H16-7.8, H16-7.8mcMMAF, or an
isotype control human IgG2 and evaluated by FACS.
[0463] The results in FIG. 7 (left panels) demonstrate strong
staining of the two different renal tumor cells with H16-7.8 (gray
lines), but not with the control MAb (filled histograms). The
panels on the right demonstrate a similar strong staining of the
same renal tumor cells with H16-7.8mcMMAF (gray lines). (FIG. 7;
right panels). These results show that both H16-7.8 and
H16-7.8mcMMAF bind native 161P2F10B antigen expressed on the
surface of human cancer cells. Conjugation of native H16-7.8 to
generate the H16-7.8mcMMAF did not alter its cell surface binding
to native 161P2F10B antigen expressed on human cancer cells.
Example 6
Cell Cytotoxicity Mediated by H16-7.8mcMMAF
[0464] The ability of H16-7.8mcMMAF to mediate 161P2F10B-dependent
cytotoxicity was evaluated in KU812 cells engineered to express
161P2F10B. For this assay 2000 viable KU812 cells were plated in
triplicate on Day 0 and allowed to recover overnight. The next day,
serial 1:4 dilutions of different lots of H16-7.8mcMMAF or a
control MAb conjugated with mcMMAF was added to yield the final
concentrations indicated in FIG. 8. The cells were allowed to
incubate for six (6) days at which time 20 .mu.l of Alamar blue was
added to each well. The plates were incubated for an additional
four (4) hours and the fluorescence intensity read on a fluorescent
plate reader using an excitation wavelength of 540 nM and an
emission wavelength of 620 nM.
[0465] The results in FIG. 8 show that both lots of H16-7.8mcMMAF
potently inhibited the proliferation of KU812 cells. The IC 50 was
determined to be 0.2 nM and 0.1 nM for Lot (1) and Lot (2)
respectively. A fully human Control MAb that does not bind KU812
cells was conjugated with mcMMAF to yield a DAR of 3.9 (+/-0.2).
The Control ADC did not inhibit KU812 cell proliferation further
demonstrating the specificity of cytotoxicity. Thus, these results
indicate that H16-7.8mcMMAF can selectively deliver a cytotoxic
drug to 161P2F10B expressing cells leading to their killing.
Example 7
H16-7.8mcMMAF Inhibit Growth of Tumors In Vivo
[0466] The significant expression of 161P2F10B on the cell surface
of tumor tissues, together with its restrictive expression in
normal tissues makes 161P2F10B a good target for antibody therapy
and similarly, therapy via ADC. Thus, the therapeutic efficacy of
H16-7.8mcMMAF in human kidney cancer xenograft mouse models is
evaluated.
[0467] Antibody drug conjugate efficacy on tumor growth and
metastasis formation is studied in mouse cancer xenograft models
(e.g., subcutaneous and orthotopically).
[0468] Subcutaneous (s.c.) tumors are generated by injection of
5.times.10.sup.4-10.sup.6 cancer cells mixed at a 1:1 dilution with
Matrigel (Collaborative Research) in the right flank of male SCID
mice. To test ADC efficacy on tumor formation, i.e., ADC injections
are started on the same day as tumor-cell injections. As a control,
mice are injected with either purified human IgG or PBS; or a
purified MAb that recognizes an irrelevant antigen not expressed in
human cells. In preliminary studies, no difference is found between
control IgG or PBS on tumor growth. Tumor sizes are determined by
caliper measurements, and the tumor volume is calculated as
length.times.width.times.height. Mice with subcutaneous tumors
greater than 1.5 cm in diameter are sacrificed.
[0469] Growth of kidney tumors in mice are performed by injection
of 1.5 million to 2 million cells implanted subcutaneously into
male SCID mice. Mice are monitored for general health, physical
activity, and appearance until they become moribund. At the time of
sacrifice, the mice can be examined to determine tumor burden and
other organs harvested to evaluate metastasis to distant sites.
Alternatively, death can be used as an endpoint. The mice are then
segregated into groups for the appropriate treatments, with
161P2F10B or control MAbs being administered via i.v.
injection.
[0470] 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.
[0471] H16-7.8mcMMAF inhibits formation of kidney cancer
xenografts. These results indicate the utility of H16-7.8mcMMAF in
the treatment of local and advanced stages of cancer and preferably
those cancers set forth in Table I.
[0472] 161P2F10B ADCs:
[0473] Monoclonal antibodies were raised against 161P2F10B as
described in the Example entitled "Generation of 161P2F10B
Monoclonal Antibodies (MAbs)." Further the MAbs are conjugated to a
toxin as described in the Example entitled "Antibody Drug
Conjugation of H16-7.8" to form H16-7.8mcMMAF. The H16-7.8mcMMAF is
characterized by FACS, and other methods known in the art to
determine its capacity to bind 161P2F10B.
[0474] Cell Lines and Xenografts:
[0475] The RFX-393 cells are maintained in RPMI, supplemented with
L-glutamine and 10% FBS. UG-K3 and SKRC-01 xenografts are
maintained by serial propagation in SCID mice.
[0476] Efficacy of H16-7.8mcMMAF in Subcutaneously Established
Human Renal Cancer Xenograft UG-K3 in SCID Mice.
[0477] In this experiment, patient-derived human renal cancer
xenograft UG-K3 was maintained by serial passages in SCID mice.
Stock tumors were harvested sterilely and minced to small pieces.
The tumor pieces were enzymatically digested to single cell
suspensions using Liberase Blendzyme (Roche Applied Science,
Indianapolis, Ind.). 1.5.times.10.sup.6 cells were injected into
the flanks of individual SCID mice and tumors were allowed to grow
untreated until they reached an approximate volume of 100 mm.sup.3.
Animals were randomly assigned to the following cohorts: an
H16-7.8mcMMAF treated group, an H16-7.8 control and a 5% Dextrose
control. H16-7.8mcMMAF and H16-7.8 were dosed at 10 mg/kg once on
day 0 by intravenous bolus injection. The amount of H16-7.8mcMMAF
and H16-7.8 administered was based on the individual body weight of
each animal obtained immediately prior to dosing. The 5% Dextrose
control was dosed at 150 .mu.L per animal. Tumor growth was
monitored using caliper measurements every 3 to 4 days until the
end of the study. Tumor volume is calculated as
Width.sup.2.times.Length/2, where width is the smallest dimension
and length is the largest. Animals in control groups were humanely
euthanized when tumors reached approximately 1000 mm.sup.3. Animals
in H16-7.8mcMMAF treated group were monitored for an additional two
weeks before sacrifice. Statistical analysis was performed at the
last time point when data for both control groups were available,
using Kruskal-Wallis test with .alpha.=-0.05.
[0478] The results demonstrated that treatment of UG-K3 renal clear
cell xenograft tumors with H16-7.8mcMMAF at all doses and schedules
examined resulted in significant inhibition of tumor growth in SCID
mice. (FIG. 9).
[0479] Growth Inhibition of Established Orthotopic UG-K3 Xenografts
by H16-7.8mcMMAF
[0480] In this experiment, the ability of H16-7.8mcMMAF to inhibit
the growth of established renal tumors grown orthotopically was
evaluated using patient-derived, UG-K3 tumor xenografts. Briefly,
stocks of UG-K3 tumors were digested enzymatically and 1.5 million
viable cells were surgically implanted into the kidneys of male
SCID mice on Day 0. The tumors were allowed to grow for 7 days at
which time animals were randomized to 4 different treatment groups
(n=10 per group). Animals randomized to Group A received Control
ADC at 5 mpk, Group B received H16-7.8mcMMAF at 3 mg/kg and Group C
received H16-7.8mcMMAF at 5 mg/kg administered every 4 days for a
total of 4 doses. Group D received H16-7.8mcMMAF at 10 mg/kg one
time. At the end of the study (Day 41) the animals were sacrificed
and the right and left kidneys weighed on an electronic balance.
The tumor weights plotted on the graph were determined by
subtracting the weight of the tumor-free contralateral kidney from
the weight of the tumor-bearing right kidney.
[0481] The results demonstrated that treatment of UG-K3 renal clear
cell xenograft tumors with H16-7.8mcMMAF at all doses and schedules
examined resulted in dramatic inhibition of tumor growth (FIG. 10).
Tumor weights in all H16-7.8mcMMAF treatment groups (B, C, and D)
were less than 1% of the tumor weights in the Control treated
group. These differences were highly statistically significant
(p<0.0001, ANOVA).
[0482] Efficacy of H16-7.8mcMMAF in Subcutaneously Established
Human Renal Cancer Xenograft RXF-393 in SCID Mice.
[0483] In this experiment, human renal cancer cells RXF-393
(0.5.times.10.sup.6 cells per mouse) were injected into the flanks
of individual mice and tumors were allowed to grow untreated until
they reached an approximate volume of 100 mm.sup.3. Animals were
then randomly assigned to the following cohorts: an H16-7.8mcMMAF
treated group, an H16-7.8 treated group and a 5% Dextrose control.
H16-7.8mcMMAF and H16-7.8 were dosed at 10 mg/kg once a week for a
total of two doses by intravenous bolus injection. The amount of
H16-7.8mcMMAF and H16-7.8 administered was based on the individual
body weight of each animal obtained immediately prior to dosing.
The 5% Dextrose control was dosed at 150 .mu.L per animal. Tumor
growth was monitored using caliper measurements every 3 to 4 days
until the end of the study. Tumor volume is calculated as
Width.sup.2.times.Length/2, where width is the smallest dimension
and length is the largest. Animals in control groups were humanely
euthanized when tumors reached approximately 1000 mm.sup.3. Animals
in H16-7.8mcMMAF treated group were monitored for an additional two
weeks before sacrifice.
[0484] The results demonstrated that treatment of RFX-393 human
renal cancer xenograft tumors with H16-7.8mcMMAF at all doses and
schedules examined (including single dose) resulted in significant
inhibition of tumor growth in SCID mice. Statistical analysis was
performed at the last time point when data in both control groups
were available, using Kruskal-Wallis test with .alpha.=0.05. (FIG.
11).
[0485] Efficacy Study of H16-7.8 Compared to H16-7.8mcMMAF in
Subcutaneously Established Human Renal Cancer SKRC-01 in SCID
Mice
[0486] In another experiment, human renal cancer cells SKRC-01
(0.8.times.10.sup.6 cells per mouse) were injected into the flanks
of individual mice. Tumors were allowed to grow untreated until
they reached an approximate volume of 100 mm.sup.3. On day 0 when
tumors reach 100 mm.sup.3, animals were randomly assigned to the
following cohorts: an H16-7.8mcMMAF treated group, an H16-7.8
treated group and a 5% Dextrose control. H16-7.8mcMMAF and H16-7.8
were dosed at 4 mg/kg every four days for a total of four doses by
intravenous bolus injection. The amount of H16-7.8mcMMAF and
H16-7.8 administered was based on the individual body weight of
each animal obtained immediately prior to dosing. The 5% Dextrose
control was dosed at 150 .mu.L per animal. Tumor growth was
monitored using caliper measurements every 3 to 4 days. Tumor
volume was calculated as Width.sup.2.times.Length/2, where width is
the smallest dimension and length is the largest.
[0487] The results show that the ADC H16-7.8mcMMAF significantly
inhibited the growth of SKRC-01 tumor formation at all doses
(including single dose), while the naked MAb H16-7.8 had no effect.
Thus, the ADC H16-7.8mcMMAF had a significantly more prominent
effect that the naked antibody H16-7.8. (FIG. 12).
[0488] Efficacy Study of H16-7.8mcMMAF Compared to Other 161P2F10B
Antibody Drug Conjugates (ADCs) in Subcutaneous Established UG-K3
in SCID Mice
[0489] In another experiment, human renal cancer cells UG-K3
(1.5.times.10.sup.6 cells per mouse) were injected into the flanks
of individual mice. Tumors were allowed to grow untreated until
they reached an approximate volume of 100 mm.sup.3. On day 0 when
tumors reach 100 mm.sup.3, animals were randomly assigned to the
following cohorts: an H16-7.8mcMMAF, an H16-7.8vcMMAE, and H16-1.11
mcMMAF, and H16-1.11vcMMAE, a PBS control, and a control MAb-vcMMAE
treated group. All antibody drug conjugates (ADCs) were dosed at 10
mg/kg once on day 0. The amount of each ADC administered was based
on the individual body weight of each animal obtained immediately
prior to dosing. The PBS control was dosed at 150.mu./L per animal.
Tumor growth was monitored using caliper measurements every 3 to 4
days. Tumor volume was calculated as Width.sup.2.times.Length/2,
where width is the smallest dimension and length is the
largest.
[0490] The results show that the ADCs H16-7.8vcMMAE and
H16-1.1vcMMAE did not inhibit tumor formation growth. Additionally,
both the H16-7.8mcMMAF and H16-1.11mcMMAF significantly inhibited
the growth of UG-K3 tumor formation during the first thirty (30)
days. After day thirty (30) the H16-7.8mcMMAF had a significantly
more prominent effect when compared to H16-1.11mcMMAF. (FIG.
13).
Example 8
Human Clinical Trials for the Treatment and Diagnosis of Human
Carcinomas Through Use of 161P2F10B ADCs
[0491] 161P2F10B ADCs are used in accordance with the present
invention which specifically bind to 161P2F10B, 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.
[0492] I.) Adjunctive therapy: In adjunctive therapy, patients are
treated with 161P2F10B 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 161P2F10B
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. 161P2F10B ADCs are utilized in
several adjunctive clinical trials in combination with the
chemotherapeutic or anti-neoplastic agents.
[0493] II.) Monotherapy: In connection with the use of the
161P2F10B ADCs in monotherapy of tumors, the 161P2F10B 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.
[0494] Dosage
[0495] Dosage regimens may be adjusted to provide the optimum
desired response. For example, a single bolus may be administered,
several divided doses may be administered over time or the dose may
be proportionally reduced or increased as indicated by the
exigencies of the therapeutic situation. It is especially
advantageous to formulate parenteral compositions in dosage unit
form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited
as unitary dosages for the mammalian subjects to be treated; each
unit containing a predetermined quantity of active compound
calculated to produce the desired therapeutic effect in association
with the required pharmaceutical carrier. The specification for the
dosage unit forms of the invention are dictated by and directly
dependent on (a) the unique characteristics of the antibody and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0496] An exemplary, non limiting range for a therapeutically
effective amount of an 161P2F10B 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.
[0497] Clinical Development Plan (CDP)
[0498] The CDP follows and develops treatments of 161P2F10B ADCs in
connection with adjunctive therapy or monotherapy. Initially,
Pre-clinical toxicology studies are performed in non-human subjects
(e.g., mice, monkeys, etc.) using standard protocols known in the
art. The H16-7.8mcMMAF was demonstrated to be well-tolerated in the
non-human toxicology studies. The human Clinical Trials initially
demonstrate safety and thereafter confirm efficacy in repeat doses.
Trials are open label comparing standard chemotherapy with standard
therapy plus 161P2F10B ADCs. As will be appreciated, one
non-limiting criteria that can be utilized in connection with
enrollment of patients is 161P2F10B expression levels in their
tumors as determined by biopsy.
[0499] As with any protein or antibody infusion-based therapeutic,
safety concerns are related primarily to (i) cytokine release
syndrome, i.e., hypotension, fever, shaking, chills; (ii) the
development of an immunogenic response to the material (i.e.,
development of human antibodies by the patient to the antibody
therapeutic, or HAHA response); and, (iii) toxicity to normal cells
that express 161P2F10B. Standard tests and follow-up are utilized
to monitor each of these safety concerns. 161P2F10B MAbs are found
to be safe upon human administration.
Example 9
Antibody Drug Conjugation of H16-7.8 MAb Characterization
[0500] I) Peptide Mapping by Mass Spectrometry
[0501] Peptide mapping analysis was conducted. This method is used
to confirm the identity of H16-7.8mcMMAF and distinguishes native
antibody (H16-7.8). The obtained H16-7.8mcMMAF and H16-7.8 were
treated with dithiothreitol (DTT) to reduce disulfide bonds,
followed by alkylation of the resulting free cysteines. Guanidine
was used in this step to ensure complete denaturation of the
protein. After dialysis to remove the guanidine, the samples were
digested with a specific endoproteinase, Lys-C. Lys-C cleaves
peptide bonds on the C-terminal side of lysine residues. The
resulting peptides were analyzed by reversed phase chromatography
coupled to mass spectrometry. The reversed phase retention times
and the observed mass to charge ratios of the peaks were compared
between H16-7.8mcMMAF and H16-7.8. LC-MS (liquid
chromatography-mass spectrometry) analysis was carried out using a
WATERS Acquity UPLC coupled to a WATERS Q-TOFp mass spectrometer.
The digested sample was applied to YMC C18 column and eluted with
an acetonitrile gradient containing trifluoroacetic acid.
Representative peptide maps for H16-7.8mcMMAF and H16-7.8 are shown
in FIG. 14.
[0502] All three chromatograms in FIG. 14 seems to be identical
except for the peaks indicated by asterisk and arrow. As can be
seen in the Figure, peak intensities indicated by asterisk were
reduced in the conjugated antibody compared to the native antibody.
The peaks marked with an arrow represent new peaks that appeared on
the conjugated antibody peptide map. Specifically, the peaks marked
with either an asterisk or with an arrow are believed to be a
peptide destined for conjugation and the resulting conjugated
peptide, respectively. FIG. 15 shows a portion of the mass spectra
of the peak marked with an asterisk. The mass value of the signal
that changed during conjugation is indicated by the "plus" sign.
This peptide with an approximate m/z of 970.4 (+3 charge state) was
identified as C225-K250 that originated from the hinge region of
the heavy chain and contains the expected conjugation sites.
[0503] In order to identify the newly appeared peaks which are
believed to be conjugated peptide in FIG. 14, LC-MS analysis was
conducted using the elevated-energy (MSE) data acquisition
technique. FIG. 16 shows the extracted ion chromatograms (XIC) for
peptide maps of H16-7.8mcMMAF and H16-7.8 using the m/z of 619.4.
This ion corresponds to a fragment ion of the drug moiety. Peaks
observed in XIC at 619.4 are almost identical to the peaks marked
with an arrow in FIG. 14. Furthermore, no such peaks were detected
in the chromatogram of the native antibody. These observations
suggest that the detected peaks in the XIC at m/z of 619.4 were
apparently drug conjugated peptides and are identified by its
intact mass values. The result was summarized in Table V. These
results suggest that in case of the conjugate, predominant peptides
are those conjugated to 2 drugs on the hinge region of heavy chain.
These data are consistent with the data obtained by the other
orthogonal such as a DAR analysis.
[0504] II) Intact Mass Analysis by LC-MS
[0505] The full mass of the deglycosylated H16-7.8mcMMAF was
determined by electrospray ionization time-of-flight (ESI-TOF) mass
spectrometry. This technique provides direct information about the
drug-to-antibody ratio (DAR) value. Test samples were diluted by
250 mM sodium phosphate buffer, pH 7.5 and then incubated overnight
at 37.degree. C. with Glycopeptidase F. The samples were injected
onto a PLRP.TM. column (Varian Technology), equilibrated at
90.degree. C., and eluted with an acetonitrile/water gradient. The
sample peaks were analyzed by an Acquity UPLC system coupled to an
WATERS Synapt mass spectrometer (Waters) and masses were
reconstructed from the raw data by an MaxEnt1 software. An example
mass spectral profile for the deglycosylated H16-7.8mcMMAF is shown
in FIG. 17. The predominant drug conjugated antibody was a 4-drug
loading species. This observation including an abundance of the
unconjugated antibody in H16-7.8mcMMAF was consistent with the
results obtained by the other orthogonal methods, such as DAR by
RP-HPLC, peptide mapping and HIC assay.
[0506] III) Drug to Antibody Ratio (DAR) Analysis by RP-HPLC
[0507] Drug to Antibody Ratio (DAR) analysis was conducted for
quantitative HPLC determination of the relative amount of drug
loading in each Light chain and Heavy chain. DAR analyses were
carried out using a PLRP-S analytical column, 2.1 mm.times.50 mm,
with mobile phase A consisting of 2.0% formic acid and mobile phase
B consisting of 2.0% formic acid plus 90% acetonitrile. For sample
preparation, the drug conjugated antibody was completely reduced by
DTT and then separated to the L chain, the drug conjugated L chain,
the H chain and the drug conjugated H chains based on the drug
loading amount. 50 .mu.g of sample was eluted using a flow rate of
0.5 ml/min, with detection at 280 nm. The molar ratio of drug to
antibody ratio (DAR) is defined by the following equation.
D A R = ( n = 0 1 ( AUC Light , n AUC Total , Light .times. n ) + n
= 0 5 ( AUC Heavy , n AUC Total , Heavy .times. n ) ) .times. 2
##EQU00001##
[0508] Where
[0509] DAR--drug to antibody molar ratio
[0510] n--number of mcMMAF drugs per Ab chain
[0511] AUCLight,n, AUCHeavy,n--area under curve for the light or
heavy antibody chain with n drugs, respectively;
[0512] AUCTotal, Light(Heavy)--peak area under curve of the light
or heavy chain.
[0513] This method has been qualified using material from
H16-7.8mcMMAF. Parameters evaluated included specificity, accuracy,
repeatability, intermediate precision. A representative DAR profile
for H16-7.8mcMMAF is shown in FIG. 18. DAR value is 4.0. Sample was
subjected to LC-MS analysis using same HPLC conditions of this
method to identify the observed peak. Results are summarized in
Table VI. The peak identification of the DAR results obtained
during the qualification of this method has been confirmed
orthogonally by LC-MS.
[0514] IV) Binding Affinity
[0515] H16-7.8 and H16-7.8mcMMAF were tested for their binding
affinity to 161P2F10B expressed on KU812 cells (human chronic
myelogenous leukemia cells, ATCC). Briefly, twelve (12) dilutions
of H16-7.8 or H16-7.8mcMMAF were incubated with KU812 cells (50,000
cells per well) overnight at 4.degree. C. at a final concentration
of 160 nM to 0.004 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.
[0516] 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 H16-7.8 and H16-7.8mcMMAF saturation
curves. Bmax is the MFI value at maximal binding of H16-7.8 or
H16-7.8mcMMAF to KU812; Kd is H16-7.8 or H16-7.8mcMMAF binding
affinity which are the concentration of H16-7.8 or H16-7.8mcMMAF
required to reach halfmaximal binding. The calculated affinity (Kd)
of H16-7.8 and H16-7.8mcMMAF is 0.08 nM and 0.25 nM on 161P2F10B
expressed on the surface of KU812 cells, respectively. (n=4).
[0517] V) Cytotoxicity
[0518] The H16-7.8, H16-7.8mcMMAF and a negative control ADC were
separately serially diluted and added to a 96-well plate containing
KU812 cells, which endogenously express 161P2F10B on the cell
surface. After six days of incubation, Alamar Blue.RTM. reagent is
added to the antibody-cell mixture. AlamarBlue.RTM. is a cell
viability indicator that uses the natural reducing power of living
cells to convert resazurin to the fluorescent molecule, resorufin.
Resazurin is reduced to resorufin, which produces very bright red
fluorescence. Viable cells continuously convert resazurin to
resorufin, thereby generating a quantitative measure of viability-
and cytotoxicity. The percent cytotoxicity of H16-7.8mcMMAF is
evaluated using fluorescence units obtained spectrophotometrically
using the Synergy 4 Hybrid Multi-Mode Microplate reader (540/35,
620/40 nm). The linear range of the assay is approximately 3.9 to
1000 ng/ml. There are 9 points in the standard curve: the highest
concentration of H16-7.8mcMMAF is 1000 ng/ml followed by eight
serial 1 to 2 dilutions and a blank (0).
[0519] Calculate % Survival using formula below:
% Survival=(X-Blank)/(No treated-Blank).times.100
[0520] Specific cytotoxicity activity of H16-7.8mcMMAF on KU812
cells: IC50 0.15 nM.
[0521] H16-7.8 and ADC control (antibody(non-anti-161P2F10B
antibody)-mcMMAF) did not have cytotoxicity activity on KU812
cells.
[0522] 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.
[0523] 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 [0524] TABLE I Tissues that express 161P2F10B when
malignant. Kidney Colon Lung Ovary Breast Lymphoma Bone Uterus
Pancreas Liver Prostate
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 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 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.
TABLE-US-00007 TABLE IV FACS MFI Values on Ku812 cells nM H16-7.8
H16-7.8mcMMAF 160 99 108 80 92 102 40 98 108 20 89 97 10 75 89 5 71
80 2.5 65 68 1.3 59 60 0.63 57 57 0.31 58 54 0.16 53 47 0.078 47 37
0.039 36 30 0.020 27 20 0.010 18 14 0.0049 13 11 0.0024 9 8 0.0012
7 7 0.0006 6 6 0.0003 6 6 0.0002 6 5 0.0001 5 5
TABLE-US-00008 TABLE V Summary of the peak identification results
on the drug conjugated peptides, potential conjugation sites
(cystein residues) are set forth in bold type and underlined. Peak
Predominant Calculate Sequence No. observed mass mass Tentative
Identification Identification 1 1754.81 1736.51
S.sub.208FNRGEC.sub.214 + 1mcMMAF + H.sub.2O, Light chain SEQ ID
NO: 9 2 1736.82 1736.51 S.sub.208FNRGEC.sub.214 + 1mcMMAF, Light
chain SEQ ID NO: 9 3 4679.29 4643.72
VECPPCPAPPVGPSVFLFPPKPK.sub.249 + 2mc SEQ ID NO: 10 MMAF +
2H.sub.20, Heavy chain 4 4661.29 4643.72
VECPPCPAPPVGPSVFLFPPKPK.sub.249 + 2mc SEQ ID NO: 10 MMAF +
H.sub.20, Heavy chain 5 4643.20 4643.72
VECPPCPAPPVGPSVFLFPPKPK.sub.249 + 2mc SEQ ID NO: 10 MMAF, Heavy
chain 6 4704.39 4643.72 VECPPCPAPPVGPSVFLFPPKPK.sub.249 + 2mc SEQ
ID NO: 10 MMAF, Heavy chain 7 4644.38 4643.72
VECPPCPAPPVGPSVFLFPPKPK.sub.249 + 2mc SEQ ID NO: 10 MMAF, Heavy
chain 8 4626.27 4643.72 VECPPCPAPPVGPSVFLFPPKPK.sub.249 + 2mc SEQ
ID NO: 10 MMAF-H.sub.2O, Heavy chain
TABLE-US-00009 TABLE VI Peak identification results of DAR analysis
by LC-MS Peak Observed Mass difference from No mass un-conjugated
peak Assignments L0 23596.1055 N/A Unconjucated L chain L1
24521.7031 925.6 1 drug conjugated L chain H0 50304.9102 N/A
Unconjugated H chain H1 51230.3984 925.5 1 drug conjugated H chain
H2 52155.9023 1851.0 2 drug conjugated H chain H3 53085.2813 2780.4
3 drug conjugated H chain H4 54006.7422 3701.8 4 drug conjugated H
chain H5 54929.6002 4624.7 5 drug conjugated H chain
TABLE-US-00010 TABLE VII Synthetic Scheme of mcMMAF ##STR00040##
##STR00041##
Sequence CWU 1
1
1213858DNAHomo sapiensmisc_feature(1)...(3858)161P2F10B variant
2CDS(44)...(2671) 1ctactttatt ctgataaaac aggtctatgc agctaccagg aca
atg gaa tct acg 55 Met Glu Ser Thr 1ttg act tta gca acg gaa caa cct
gtt aag aag aac act ctt aag aaa 103Leu Thr Leu Ala Thr Glu Gln Pro
Val Lys Lys Asn Thr Leu Lys Lys 5 10 15 20tat aaa ata gct tgc att
gtt ctt ctt gct ttg ctg gtg atc atg tca 151Tyr Lys Ile Ala Cys Ile
Val Leu Leu Ala Leu Leu Val Ile Met Ser 25 30 35ctt gga tta ggc ctg
ggg ctt gga ctc agg aaa ctg gaa aag caa ggc 199Leu Gly Leu Gly Leu
Gly Leu Gly Leu Arg Lys Leu Glu Lys Gln Gly 40 45 50agc tgc agg aag
aag tgc ttt gat gca tca ttt aga gga ctg gag aac 247Ser Cys Arg Lys
Lys Cys Phe Asp Ala Ser Phe Arg Gly Leu Glu Asn 55 60 65tgc cgg tgt
gat gtg gca tgt aaa gac cga ggt gat tgc tgc tgg gat 295Cys Arg Cys
Asp Val Ala Cys Lys Asp Arg Gly Asp Cys Cys Trp Asp 70 75 80ttt gaa
gac acc tgt gtg gaa tca act cga ata tgg atg tgc aat aaa 343Phe Glu
Asp Thr Cys Val Glu Ser Thr Arg Ile Trp Met Cys Asn Lys 85 90 95
100ttt cgt tgt gga gag acc aga tta gag gcc agc ctt tgc tct tgt tca
391Phe Arg Cys Gly Glu Thr Arg Leu Glu Ala Ser Leu Cys Ser Cys Ser
105 110 115gat gac tgt ttg cag agg aaa gat tgc tgt gct gac tat aag
agt gtt 439Asp Asp Cys Leu Gln Arg Lys Asp Cys Cys Ala Asp Tyr Lys
Ser Val 120 125 130tgc caa gga gaa acc tca tgg ctg gaa gaa aac tgt
gac aca gcc cag 487Cys Gln Gly Glu Thr Ser Trp Leu Glu Glu Asn Cys
Asp Thr Ala Gln 135 140 145cag tct cag tgc cca gaa ggg ttt gac ctg
cca cca gtt atc ttg ttt 535Gln Ser Gln Cys Pro Glu Gly Phe Asp Leu
Pro Pro Val Ile Leu Phe 150 155 160tct atg gat gga ttt aga gct gaa
tat tta tac aca tgg gat act tta 583Ser Met Asp Gly Phe Arg Ala Glu
Tyr Leu Tyr Thr Trp Asp Thr Leu165 170 175 180atg cca aat atc aat
aaa ctg aaa aca tgt gga att cat tca aaa tac 631Met Pro Asn Ile Asn
Lys Leu Lys Thr Cys Gly Ile His Ser Lys Tyr 185 190 195atg aga gct
atg tat cct acc aaa acc ttc cca aat cat tac acc att 679Met Arg Ala
Met Tyr Pro Thr Lys Thr Phe Pro Asn His Tyr Thr Ile 200 205 210gtc
acg ggc ttg tat cca gag tca cat ggc atc att gac aat aat atg 727Val
Thr Gly Leu Tyr Pro Glu Ser His Gly Ile Ile Asp Asn Asn Met 215 220
225tat gat gta aat ctc aac aag aat ttt tca ctt tct tca aag gaa caa
775Tyr Asp Val Asn Leu Asn Lys Asn Phe Ser Leu Ser Ser Lys Glu Gln
230 235 240aat aat cca gcc tgg tgg cat ggg caa cca atg tgg ctg aca
gca atg 823Asn Asn Pro Ala Trp Trp His Gly Gln Pro Met Trp Leu Thr
Ala Met245 250 255 260tat caa ggt tta aaa gcc gct acc tac ttt tgg
ccc gga tca gaa gtg 871Tyr Gln Gly Leu Lys Ala Ala Thr Tyr Phe Trp
Pro Gly Ser Glu Val 265 270 275gct ata aat ggc tcc ttt cct tcc ata
tac atg cct tac aac gga agt 919Ala Ile Asn Gly Ser Phe Pro Ser Ile
Tyr Met Pro Tyr Asn Gly Ser 280 285 290gtc cca ttt gaa gag agg att
tct aca ctg tta aaa tgg ctg gac ctg 967Val Pro Phe Glu Glu Arg Ile
Ser Thr Leu Leu Lys Trp Leu Asp Leu 295 300 305ccc aaa gct gaa aga
ccc agg ttt tat acc atg tat ttt gaa gaa cct 1015Pro Lys Ala Glu Arg
Pro Arg Phe Tyr Thr Met Tyr Phe Glu Glu Pro 310 315 320gat tcc tct
gga cat gca ggt gga cca gtc agt gcc aga gta att aaa 1063Asp Ser Ser
Gly His Ala Gly Gly Pro Val Ser Ala Arg Val Ile Lys325 330 335
340gcc tta cag gta gta gat cat gct ttt ggg atg ttg atg gaa ggc ctg
1111Ala Leu Gln Val Val Asp His Ala Phe Gly Met Leu Met Glu Gly Leu
345 350 355aag cag cgg aat ttg cac aac tgt gtc aat atc atc ctt ctg
gct gac 1159Lys Gln Arg Asn Leu His Asn Cys Val Asn Ile Ile Leu Leu
Ala Asp 360 365 370cat gga atg gac cag act tat tgt aac aag atg gaa
tac atg act gat 1207His Gly Met Asp Gln Thr Tyr Cys Asn Lys Met Glu
Tyr Met Thr Asp 375 380 385tat ttt ccc aga ata aac ttc ttc tac atg
tac gaa ggg cct gcc ccc 1255Tyr Phe Pro Arg Ile Asn Phe Phe Tyr Met
Tyr Glu Gly Pro Ala Pro 390 395 400cgc atc cga gct cat aat ata cct
cat gac ttt ttt agt ttt aat tct 1303Arg Ile Arg Ala His Asn Ile Pro
His Asp Phe Phe Ser Phe Asn Ser405 410 415 420gag gaa att gtt aga
aac ctc agt tgc cga aaa cct gat cag cat ttc 1351Glu Glu Ile Val Arg
Asn Leu Ser Cys Arg Lys Pro Asp Gln His Phe 425 430 435aag ccc tat
ttg act cct gat ttg cca aag cga ctg cac tat gcc aag 1399Lys Pro Tyr
Leu Thr Pro Asp Leu Pro Lys Arg Leu His Tyr Ala Lys 440 445 450aac
gtc aga atc gac aaa gtt cat ctc ttt gtg gat caa cag tgg ctg 1447Asn
Val Arg Ile Asp Lys Val His Leu Phe Val Asp Gln Gln Trp Leu 455 460
465gct gtt agg agt aaa tca aat aca aat tgt gga gga ggc aac cat ggt
1495Ala Val Arg Ser Lys Ser Asn Thr Asn Cys Gly Gly Gly Asn His Gly
470 475 480tat aac aat gag ttt agg agc atg gag gct atc ttt ctg gca
cat gga 1543Tyr Asn Asn Glu Phe Arg Ser Met Glu Ala Ile Phe Leu Ala
His Gly485 490 495 500ccc agt ttt aaa gag aag act gaa gtt gaa cca
ttt gaa aat att gaa 1591Pro Ser Phe Lys Glu Lys Thr Glu Val Glu Pro
Phe Glu Asn Ile Glu 505 510 515gtc tat aac cta atg tgt gat ctt cta
cgc att caa cca gca cca aac 1639Val Tyr Asn Leu Met Cys Asp Leu Leu
Arg Ile Gln Pro Ala Pro Asn 520 525 530aat gga acc cat ggt agt tta
aac cat ctt ctg aag gtg cct ttt tat 1687Asn Gly Thr His Gly Ser Leu
Asn His Leu Leu Lys Val Pro Phe Tyr 535 540 545gag cca tcc cat gca
gag gag gtg tca aag ttt tct gtt tgt ggc ttt 1735Glu Pro Ser His Ala
Glu Glu Val Ser Lys Phe Ser Val Cys Gly Phe 550 555 560gct aat cca
ttg ccc aca gag tct ctt gac tgt ttc tgc cct cac cta 1783Ala Asn Pro
Leu Pro Thr Glu Ser Leu Asp Cys Phe Cys Pro His Leu565 570 575
580caa aat agt act cag ctg gaa caa gtg aat cag atg cta aat ctc acc
1831Gln Asn Ser Thr Gln Leu Glu Gln Val Asn Gln Met Leu Asn Leu Thr
585 590 595caa gaa gaa ata aca gca aca gtg aaa gta aat ttg cca ttt
ggg agg 1879Gln Glu Glu Ile Thr Ala Thr Val Lys Val Asn Leu Pro Phe
Gly Arg 600 605 610cct agg gta ctg cag aag aac gtg gac cac tgt ctc
ctt tac cac agg 1927Pro Arg Val Leu Gln Lys Asn Val Asp His Cys Leu
Leu Tyr His Arg 615 620 625gaa tat gtc agt gga ttt gga aaa gct atg
agg atg ccc atg tgg agt 1975Glu Tyr Val Ser Gly Phe Gly Lys Ala Met
Arg Met Pro Met Trp Ser 630 635 640tca tac aca gtc ccc cag ttg gga
gac aca tcg cct ctg cct ccc act 2023Ser Tyr Thr Val Pro Gln Leu Gly
Asp Thr Ser Pro Leu Pro Pro Thr645 650 655 660gtc cca gac tgt ctg
cgg gct gat gtc agg gtt cct cct tct gag agc 2071Val Pro Asp Cys Leu
Arg Ala Asp Val Arg Val Pro Pro Ser Glu Ser 665 670 675caa aaa tgt
tcc ttc tat tta gca gac aag aat atc acc cac ggc ttc 2119Gln Lys Cys
Ser Phe Tyr Leu Ala Asp Lys Asn Ile Thr His Gly Phe 680 685 690ctc
tat cct cct gcc agc aat aga aca tca gat agc caa tat gat gct 2167Leu
Tyr Pro Pro Ala Ser Asn Arg Thr Ser Asp Ser Gln Tyr Asp Ala 695 700
705tta att act agc aat ttg gta cct atg tat gaa gaa ttc aga aaa atg
2215Leu Ile Thr Ser Asn Leu Val Pro Met Tyr Glu Glu Phe Arg Lys Met
710 715 720tgg gac tac ttc cac agt gtt ctt ctt ata aaa cat gcc aca
gaa aga 2263Trp Asp Tyr Phe His Ser Val Leu Leu Ile Lys His Ala Thr
Glu Arg725 730 735 740aat gga gta aat gtg gtt agt gga cca ata ttt
gat tat aat tat gat 2311Asn Gly Val Asn Val Val Ser Gly Pro Ile Phe
Asp Tyr Asn Tyr Asp 745 750 755ggc cat ttt gat gct cca gat gaa att
acc aaa cat tta gcc aac act 2359Gly His Phe Asp Ala Pro Asp Glu Ile
Thr Lys His Leu Ala Asn Thr 760 765 770gat gtt ccc atc cca aca cac
tac ttt gtg gtg ctg acc agt tgt aaa 2407Asp Val Pro Ile Pro Thr His
Tyr Phe Val Val Leu Thr Ser Cys Lys 775 780 785aac aag agc cac aca
ccg gaa aac tgc cct ggg tgg ctg gat gtc cta 2455Asn Lys Ser His Thr
Pro Glu Asn Cys Pro Gly Trp Leu Asp Val Leu 790 795 800ccc ttt atc
atc cct cac cga cct acc aac gtg gag agc tgt cct gaa 2503Pro Phe Ile
Ile Pro His Arg Pro Thr Asn Val Glu Ser Cys Pro Glu805 810 815
820ggt aaa cca gaa gct ctt tgg gtt gaa gaa aga ttt aca gct cac att
2551Gly Lys Pro Glu Ala Leu Trp Val Glu Glu Arg Phe Thr Ala His Ile
825 830 835gcc cgg gtc cgt gat gta gaa ctt ctc act ggg ctt gac ttc
tat cag 2599Ala Arg Val Arg Asp Val Glu Leu Leu Thr Gly Leu Asp Phe
Tyr Gln 840 845 850gat aaa gtg cag cct gtc tct gaa att ttg caa cta
aag aca tat tta 2647Asp Lys Val Gln Pro Val Ser Glu Ile Leu Gln Leu
Lys Thr Tyr Leu 855 860 865cca aca ttt gaa acc act att taa
cttaataatg tctacttaat atataattta 2701Pro Thr Phe Glu Thr Thr Ile
870 875ctgtataaag taattttggc aaaatataag tgattttttc tggagaattg
taaaataaag 2761ttttctattt ttccttaaaa aaaaaaccgg aattccgggc
ttgggaggct gaggcaggag 2821actcgcttga acccgggagg cagaggttgc
agtgagccaa gattgcgcca ttgcactcca 2881gagcctgggt gacagagcaa
gactacatct caaaaaataa ataaataaaa taaaagtaac 2941aataaaaata
aaaagaacag cagagagaat gagcaaggag aaatgtcaca aactattgca
3001aaatactgtt acactgggtt ggctctccaa gaagatactg gaatctcttc
agccatttgc 3061ttttcagaag tagaaaccag caaaccacct ctaagcggag
aacatacgat tctttattaa 3121gtagctctgg ggaaggaaag aataaaagtt
gatagctccc tgattgggaa aaaatgcaca 3181attaataaag aatgaagatg
aaagaaagca tgcttatgtt gtaacacaaa aaaaattcac 3241aaacgttggt
ggaaggaaaa cagtatagaa aacattactt taactaaaag ctggaaaaat
3301tttcagttgg gatgcgactg acaaaaagaa cgggatttcc aggcataaag
ttggcgtgag 3361ctacagaggg caccatgtgg ctcagtggaa gacccttcaa
gattcaaagt tccatttgac 3421agagcaaagg cacttcgcaa ggagaagggt
ttaaattatg ggtccaaaag ccaagtggta 3481aagcgagcaa tttgcagcat
aactgcttct cctagacagg gctgagtggg caaaatacga 3541cagtacacac
agtgactatt agccactgcc agaaacaggc tgaacagccc tgggagacaa
3601gggaaggcag gtggtgggag ttgttcatgg agagaaagga gagttttaga
accagcacat 3661ccactggaga tgctgggcca ccagacccct cccagtcaat
aaagtctggt gcctcatttg 3721atctcagcct catcatgacc ctggagagac
cctgatacca tctgccagtc cccgacagct 3781taggcactcc ttgccatcaa
cctgaccccc cgagtggttc tccaggctcc ctgccccacc 3841cattcaggcc ggaattc
38582875PRTHomo sapiensmisc_feature(1)...(875)161P2F10B variant 2
2Met Glu Ser Thr Leu Thr Leu Ala Thr Glu Gln Pro Val Lys Lys Asn1 5
10 15Thr Leu Lys Lys Tyr Lys Ile Ala Cys Ile Val Leu Leu Ala Leu
Leu 20 25 30Val Ile Met Ser Leu Gly Leu Gly Leu Gly Leu Gly Leu Arg
Lys Leu 35 40 45Glu Lys Gln Gly Ser Cys Arg Lys Lys Cys Phe Asp Ala
Ser Phe Arg 50 55 60Gly Leu Glu Asn Cys Arg Cys Asp Val Ala Cys Lys
Asp Arg Gly Asp65 70 75 80Cys Cys Trp Asp Phe Glu Asp Thr Cys Val
Glu Ser Thr Arg Ile Trp 85 90 95Met Cys Asn Lys Phe Arg Cys Gly Glu
Thr Arg Leu Glu Ala Ser Leu 100 105 110Cys Ser Cys Ser Asp Asp Cys
Leu Gln Arg Lys Asp Cys Cys Ala Asp 115 120 125Tyr Lys Ser Val Cys
Gln Gly Glu Thr Ser Trp Leu Glu Glu Asn Cys 130 135 140Asp Thr Ala
Gln Gln Ser Gln Cys Pro Glu Gly Phe Asp Leu Pro Pro145 150 155
160Val Ile Leu Phe Ser Met Asp Gly Phe Arg Ala Glu Tyr Leu Tyr Thr
165 170 175Trp Asp Thr Leu Met Pro Asn Ile Asn Lys Leu Lys Thr Cys
Gly Ile 180 185 190His Ser Lys Tyr Met Arg Ala Met Tyr Pro Thr Lys
Thr Phe Pro Asn 195 200 205His Tyr Thr Ile Val Thr Gly Leu Tyr Pro
Glu Ser His Gly Ile Ile 210 215 220Asp Asn Asn Met Tyr Asp Val Asn
Leu Asn Lys Asn Phe Ser Leu Ser225 230 235 240Ser Lys Glu Gln Asn
Asn Pro Ala Trp Trp His Gly Gln Pro Met Trp 245 250 255Leu Thr Ala
Met Tyr Gln Gly Leu Lys Ala Ala Thr Tyr Phe Trp Pro 260 265 270Gly
Ser Glu Val Ala Ile Asn Gly Ser Phe Pro Ser Ile Tyr Met Pro 275 280
285Tyr Asn Gly Ser Val Pro Phe Glu Glu Arg Ile Ser Thr Leu Leu Lys
290 295 300Trp Leu Asp Leu Pro Lys Ala Glu Arg Pro Arg Phe Tyr Thr
Met Tyr305 310 315 320Phe Glu Glu Pro Asp Ser Ser Gly His Ala Gly
Gly Pro Val Ser Ala 325 330 335Arg Val Ile Lys Ala Leu Gln Val Val
Asp His Ala Phe Gly Met Leu 340 345 350Met Glu Gly Leu Lys Gln Arg
Asn Leu His Asn Cys Val Asn Ile Ile 355 360 365Leu Leu Ala Asp His
Gly Met Asp Gln Thr Tyr Cys Asn Lys Met Glu 370 375 380Tyr Met Thr
Asp Tyr Phe Pro Arg Ile Asn Phe Phe Tyr Met Tyr Glu385 390 395
400Gly Pro Ala Pro Arg Ile Arg Ala His Asn Ile Pro His Asp Phe Phe
405 410 415Ser Phe Asn Ser Glu Glu Ile Val Arg Asn Leu Ser Cys Arg
Lys Pro 420 425 430Asp Gln His Phe Lys Pro Tyr Leu Thr Pro Asp Leu
Pro Lys Arg Leu 435 440 445His Tyr Ala Lys Asn Val Arg Ile Asp Lys
Val His Leu Phe Val Asp 450 455 460Gln Gln Trp Leu Ala Val Arg Ser
Lys Ser Asn Thr Asn Cys Gly Gly465 470 475 480Gly Asn His Gly Tyr
Asn Asn Glu Phe Arg Ser Met Glu Ala Ile Phe 485 490 495Leu Ala His
Gly Pro Ser Phe Lys Glu Lys Thr Glu Val Glu Pro Phe 500 505 510Glu
Asn Ile Glu Val Tyr Asn Leu Met Cys Asp Leu Leu Arg Ile Gln 515 520
525Pro Ala Pro Asn Asn Gly Thr His Gly Ser Leu Asn His Leu Leu Lys
530 535 540Val Pro Phe Tyr Glu Pro Ser His Ala Glu Glu Val Ser Lys
Phe Ser545 550 555 560Val Cys Gly Phe Ala Asn Pro Leu Pro Thr Glu
Ser Leu Asp Cys Phe 565 570 575Cys Pro His Leu Gln Asn Ser Thr Gln
Leu Glu Gln Val Asn Gln Met 580 585 590Leu Asn Leu Thr Gln Glu Glu
Ile Thr Ala Thr Val Lys Val Asn Leu 595 600 605Pro Phe Gly Arg Pro
Arg Val Leu Gln Lys Asn Val Asp His Cys Leu 610 615 620Leu Tyr His
Arg Glu Tyr Val Ser Gly Phe Gly Lys Ala Met Arg Met625 630 635
640Pro Met Trp Ser Ser Tyr Thr Val Pro Gln Leu Gly Asp Thr Ser Pro
645 650 655Leu Pro Pro Thr Val Pro Asp Cys Leu Arg Ala Asp Val Arg
Val Pro 660 665 670Pro Ser Glu Ser Gln Lys Cys Ser Phe Tyr Leu Ala
Asp Lys Asn Ile 675 680 685Thr His Gly Phe Leu Tyr Pro Pro Ala Ser
Asn Arg Thr Ser Asp Ser 690 695 700Gln Tyr Asp Ala Leu Ile Thr Ser
Asn Leu Val Pro Met Tyr Glu Glu705 710 715 720Phe Arg Lys Met Trp
Asp Tyr Phe His Ser Val Leu Leu Ile Lys His 725 730 735Ala Thr Glu
Arg Asn Gly Val Asn Val Val Ser Gly Pro Ile Phe Asp 740 745 750Tyr
Asn Tyr Asp Gly His Phe Asp Ala Pro Asp Glu Ile Thr Lys His 755 760
765Leu Ala Asn Thr Asp Val Pro Ile Pro Thr His Tyr Phe Val Val Leu
770 775 780Thr Ser Cys Lys Asn Lys Ser His Thr Pro Glu Asn Cys Pro
Gly Trp785 790 795 800Leu Asp Val Leu Pro Phe Ile Ile Pro His Arg
Pro Thr Asn Val Glu 805 810 815Ser Cys Pro Glu Gly Lys Pro
Glu Ala Leu Trp Val Glu Glu Arg Phe 820 825 830Thr Ala His Ile Ala
Arg Val Arg Asp Val Glu Leu Leu Thr Gly Leu 835 840 845Asp Phe Tyr
Gln Asp Lys Val Gln Pro Val Ser Glu Ile Leu Gln Leu 850 855 860Lys
Thr Tyr Leu Pro Thr Phe Glu Thr Thr Ile865 870 87531440DNAHomo
sapiensmisc_feature(1)...(1440)H16-7.8 heavy chainCDS(34)...(1440)
3tttctgagag tcctggacct cctgtgcaag aac atg aaa cac ctg tgg ttc ttc
54 Met Lys His Leu Trp Phe Phe 1 5ctc ctg ctg gtg gca gct ccc aga
tgg gtc ctg tcc cag gtg cag ctg 102Leu Leu Leu Val Ala Ala Pro Arg
Trp Val Leu Ser Gln Val Gln Leu 10 15