full text.txt

Atty. Dkt. No. 111119-0105
Description
Title of Invention: ANTI-HER2 ANTIBODY-DRUG CONJUGATE
Cross-Reference to Related Applications
[0000]
This application is a continuation application
filed under 35 U.S.C. § 111 (a) claiming the benefit
under 35 U.S.C. §§ 120 and 365(c) of International
Application No. PCT/JP2015/000355 filed on January 28,
2015, which is based upon and claims the benefit of
priority of Japanese Patent Application No. 2014-017777,
filed on January 31, 2014, Japanese Patent Application
No. 2014-168944, filed on August 22, 2014, and Japanese
Patent Application No. 2014-227886, filed on November
10, 2014, the contents of which are hereby incorporated
by reference in their entireties.
Technical Field
[0001]
The present invention relates to an antibody-drug
conjugate having an antitumor drug conjugated to an
anti-HER2 antibody via a linker structure moiety, the
conjugate being useful as an antitumor drug.
Background Art
[0002]
4833-0752-7733.1
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Atty. Dkt. No. 111119-0105
An antibody-drug conjugate (ADC)
with cytotoxicity conjugated to an
having a drug
antibody, whose
antigen is expressed on the surface of cancer cells and
which also binds to an antigen capable of cellular
internalization, and therefore can deliver the drug
selectively to cancer cells, is thus expected to cause
accumulation of the drug within cancer cells and to
kill the cancer cells (see, Non-patent Literatures 1 to
3) . As Mylotarg (registered trademark;
Gemtuzumab
conjugated
an ADC,
ozogamicin) in
to an anti-CD33
which
antibody
cal icheamicin is
is approved as a
therapeutic agent for acute myeloid leukemia. Further,
Adcetris (registered trademark; Brentuximab vedotin),
in which auristatin E is conjugated to an anti-CD30
antibody, has recently been approved as a therapeutic
agent for Hodgkin's lymphoma and anaplastic large cell
lymphoma (see, Non-patent Literature 4). The drugs
contained in ADCs which have been approved until now
target DNA or tubulin.
[0003]
With regard to an antitumor agent, camptothecin
derivatives, low-molecular-weight compounds that
inhibit topoisomerase I to exhibit an antitumor effect,
are known. Among these, an anti tumor compound
represented by the formula below
[0004]
[Formula 1]
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Me
F
[0005]
(exatecan, chemical name: (1S,9S)-1-amino-9-ethyl-5-
fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-
10, 13 (9H, 15H) -dione) is a water soluble derivative of
camptothecin (Patent Literatures 1 and 2). Unlike
irinotecan currently used in clinical settings, this
compound does not require activation by an enzyme for
exhibiting its anti tumor effect. Further, its
inhibitory activity on topoisomerase I was observed to
be higher than SN-38 which is the main pharmaceutically
active substance of irinotecan and topotecan also used
in clinical settings, and higher in vitro cytocidal
activity was confirmed against various cancer eel ls.
In particular, it was confirmed to have the effect
against cancer cells that have resistance to SN-38 or
the like due to expression of P-glycoprotein. Further,
in a human tumor subcutaneously transplanted mouse
model, it was confirmed to have a potent antitumor
effect, and thus has undergone clinical studies, but
has not been placed on the market yet (see, Non-patent
Literatures 5 to 10) . It remains unclear whether or
not exatecan acts effectively as an ADC.
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Atty. Dkt. No. 111119-0105
[0006]
DE-310 is a complex in which exatecan is
conjugated to a biodegradable carboxymethyldextran
polyalcohol polymer via a GGFG peptide spacer (Patent
Literature 3). By converting exatecan into the form of
a polymer prodrug, a high blood retention property can
be maintained and also a high targeting property to
tumor areas is passively increased by utilizing the
increased permeability of newly formed blood vessels
within tumors and retention property in tumor tissues.
With DE-310, through cleavage of the peptide spacer by
enzyme, exatecan and exatecan with glycine connected to
an amino group are continuously released as main active
substance, and as a result, the pharmacokinet ics are
improved. DE-310 was found to have higher
effectiveness than exatecan administered alone even
though the total dosage of exatecan contained in D310
is lower than in the case of administration of exatecan
alone according to various tumor evaluation models in
non-clinical studies. A clinical study was conducted
for DE-310, and effective cases were also confirmed,
including a report suggesting that the main active
substance accumulates in tumors more than in normal
tissues. However, there is also a
that accumulation of DE-310
substance in tumors is not
and
much
report indicating
the main active
different from
accumulation in normal tissues in humans, and thus no
passive targeting is observed in humans (see, Non-
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Atty. Dkt. No. 111119-0105
patent Literatures 11 to 14) As a result, DE-310 was
not also commercialized, and it remains unclear whether
or not exatecan effectively acts as a drug directed to
such targeting.
[0007]
As a compound relating to DE-310, a complex in
which a structure moiety represented by -NH-(CH2) 4-
C(=O)- is inserted between the -GGFG- spacer and
exatecan to form -GGFG-NH-(CH2)4-C{=O)- used as a
spacer structure is also known (Patent Literature 4).
However, the anti tumor effect of said complex is not
known at all.
[0008]
HER2 is one
factor receptor
epidermal cell
of the products
type oncogene
growth factor
of a typical growth
identified as human
receptor 2-related
oncogene, and is a transmembrane receptor protein
having a molecular weight of 185 kDa and having a
tyrosine kinase domain (Non-patent Literature 15). The
DNA sequence and amino acid sequence of HER2 are
disclosed on a public database, and can be referred to,
for example, under Accession No. M11730 (GenBank),
NP 004439.2 (NCBI), or the like.
HER2 (neu, ErbB-2) is one of the members of the
EGFR (epidermal growth factor receptor) family and is
activated by autophosphorylation at intracellular
tyrosine residues by its homodimer formation or
heterodimer formation with another EGFR receptor HERl
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(EGFR, ErbB-1), HER3 (ErbB-3), or HER4 (ErbB-4) (Nonpatent
Literatures 16 to 18), thereby playing an
important role in cell growth, differentiation, and
survival in normal cells and cancer cells (Non-patent
Literatures 19 and 20). HER2 is overexpressed in
various cancer types such as breast cancer, gastric
cancer, and ovarian cancer (Non-patent Literatures 21
to 26) and has been reported to be a negative prognosis
factor for breast cancer (Non-patent Literatures 27 and
28) .
[0009]
Trastuzumab is a humanized antibody of a mouse
anti-HER2 antibody 4D5 (Non-patent Literature 29 and
Patent Literature 5), named as recombinant humanized
anti-HER2 monoclonal antibody (huMAb4D5-8, rhuMAb HER2,
Herceptin (R)) (Patent Literature 6) . Trastuzumab
specifically binds to
HER2 and induces
the extracellular domain IV of
antibody-dependent cellular
cytotoxicity (ADCC) or exerts an anticancer effect via
the inhibition of signal transduction from HER2 (Nonpatent
Literatures 30 and 31). Trastuzumab is highly
effective for tumors overexpressing HER2 (Non-patent
Literature 32) and as such, was launched in 1999 in the
USA and in 2001 in Japan as a therapeutic agent for
patients with metastatic breast cancer overexpressing
HER2.
Although the therapeutic effect of trastuzumab on
breast cancer has been adequately proven (Non-patent
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Literature 3 3) , allegedly about 15% of patients with
breast cancer overexpressing HER2 who have received a
wide range of conventional anticancer therapies are
responders to trastuzumab. About 85% of patients of
this population have no or merely weak response to
trastuzumab treatment.
[0010]
Thus, the need for a therapeutic agent targeting
HER2 expression-related diseases has been recognized
for patients affected by tumors overexpressing HER2
with no or weak response to trastuzumab or HER2-related
disorders. T-DMl (trastuzumab emtansine, Kadcyla (R);
Non-patent Literature 34) having an antitumor drug
conjugated to trastuzumab via a linker structure, and
pertuzumab (Perjeta(R); Non-patent Literature 35 and
Patent Literature 7) designed to target the
extracellular domain II of HER2 and inhibit heterodimer
formation have been developed. However, their
responsiveness, activity strength,
indications are still insufficient,
unsatisfied needs for targeting HER2.
[0011]
[Ci tat ion List]
[Patent Literatures]
and accepted
and there are
[Patent Literature 1] Japanese Patent Laid-Open No. 5-
59061
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Atty. Dkt. No. 111119-0105
[Patent Literature 2] Japanese Patent Laid-Open No. 8-
337584
[Patent Literature 3] International Publication No. WO
1997/46260
[Patent Literature 4] International Publication No. WO
2000/25825
[Patent Literature 5] U.S. Patent No. 5677171
[Patent Literature 6] U.S. Patent No. 5821337
[Patent Literature 7] International Publication No. WO
01/00244
[Non-patent Literatures]
[0012]
[Non-patent Literature 1] Ducry,
Bioconjugate Chem. (2010) 21, 5-13.
L., et al.,
[Non-patent Literature 2] Alley, S. C., et al., Current
Opinion in Chemical Biology (2010) 14, 529-537.
[Non-patent Literature 3] Damle N.K. Expert Opin. Biol.
The r. ( 2 0 0 4) 4 , 14 4 5 -14 5 2 .
[Non-patent Literature 4] Senter P. D., et al., Nature
Biotechnology (2012) 30, 631-637.
[Non-patent Literature 5] Kumazawa, E., Tohgo, A., Exp.
Opin. Invest. Drugs (1998) 7, 625-632.
[Non-patent Literature 6] Mitsui, I., et al., Jpn J.
Cancer Res. (1995) 86, 776-782.
[Non-patent Literature 7] Takiguchi, S., et al., Jpn J.
Cancer Res. (1997) 88, 760-769.
[Non-patent Literature 8] Joto, N. et al. Int J Cancer
(1997) 72, 680-686.
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[Non-patent Literature 9] Kumazawa, E. et al., Cancer
Chemother. Pharmacol. (1998) 42, 210-220.
[Non-patent Literature 10] De Jager, R., et al., Ann N
Y Acad Sci (2000) 922, 260-273.
[Non-patent Literature 11] Inoue, K. et al., Polymer
Drugs in the Clinical Stage, Edited by Maeda et al.
(2003) 145-153.
[Non-patent Literature 12] Kumazawa, E. et al., Cancer
Sci (2004) 95, 168-175.
[Non-patent Literature 13] Soepenberg, 0. et al.,
Clinical Cancer Research, (2005) 11, 703-711.
[Non-patent Literature 14] Wente M. N.
Investigational New Drugs (2005) 23, 339-347.
[Non-patent Literature 15] Coussens L, et al.,
1985;230 (4730) :1132-1139.
et al.,
Science.
[Non-patent Literature 16] Graus-Porta G, et al., EMBO
J. 1997;16;1647-1655.
[Non-patent Literature 17] Karunagaran D, et al., EMBO
J. 1996;15:254-264.
[Non-patent Literature 18] Sliwkowski MX, et al., J.
Biol. Chem. 1994;269:14661-14665.
[Non-patent Literature 19] Di Fore PP, et al., Science.
1987;237:178-182.
[Non-patent Literature 20] Hudziak RM, et al., Proc
Natl Acad Sci US A. 1987;84:7159-7163.
[Non-patent Literature 21] Hardwick R, et al., Eur. J
Surg Oncol. 1997 (23) :30-35.
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[Non-patent Literature 22] Korkaya H, et al., Oncogene.
2008;27(47) :6120-6130.
[Non-patent Literature 23] Yano T, et al., Oncol Rep.
2006;15(1) :65-71.
[Non-patent Literature 24] Slamon DJ, et al., Science.
1987;235:177-182.
[Non-patent Literature 25] Gravalos C, et al., Ann
Oncol 19: 1523-1529, 2008.
[Non-patent Literature 26] Fukushige Set al., Mol Cell
Biol 6: 955-958, 1986.
[Non-patent Literature 27] Slamon DJ, et al. Science.
1989;244:707-712.
[Non-patent Literature 28] Kaptain Set al., Diagn Mol
Pathol 10:139-152, 2001.
[Non-patent Literature 29]
Research 1990 (50): 1550-1558.
Fendly. et al., Cancer
[Non-patent Literature 30] Sliwkowski MX, et al., Semin
Oncol. 1999;26(4,Suppl 12) :60-70.
[Non-patent Literature 31] Hudis CA, et al., N Engl J
Med. 357: 39-51, 2007.
[Non-patent Literature 32] Vogel CL, et al., J Clin
Oncol. 2002;20(3) :719-726.
[Non-patent Literature 33] Baselga et al., J. Clin.
Oncol. 14:737-744 (1996).
[Non-patent Literature 34] Burris III et al., J Clin
Oncol 2011;29:398-405.
[Non-patent Literature 35] Adams CW, et al., Cancer
Immunol Immunother. 2006;6:717-727.
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[Summary of Invention]
[Technical Problem]
[0013]
With regard to the treatment of tumors by
antibodies, an insufficient antitumor effect may be
observed even when the
to bind to tumor cells,
antibody recognizes
and there are cases
an antigen
in which a
more effective antitumor antibody is needed. Further,
many antitumor low-molecular-weight compounds have
problems in safety like side effects and toxicity even
if the compounds have an excellent antitumor effect. It
has remained an objective to achieve a superior
therapeutic effect by further enhancing safety. Thus,
an object of the present invention is to provide an
antitumor drug having an excellent therapeutic effect,
which is excellent in terms of antitumor effect and
safety.
[Solution to Problem]
[ 0014]
The inventors considered that an anti-HER2
antibody is an antibody which is capable of targeting
tumor cells, that is, having a property of recognizing
tumor cells, a property of binding to tumor cells, a
property of internalizing within tumor
cytotoxic activity against tumor cells, a
activity against tumor cells, or the like;
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cells, a
cytocidal
thus, when
Atty. Dkt. No. 111119-0105
the antitumor compound exatecan is converted into an
antibody-drug conjugate, via a linker structure moiety,
by conjugation to this antibody, the antitumor compound
can be more surely delivered to tumor cells to
specifically exhibit the antitumor effect of the
compound in tumor cells, and thus the antitumor effect
can be surely exhibited and also an enhanced cytocidal
effect of the anti-HER2 antibody can be expected, and
the dose of the antitumor compound can be reduced
compared to the case of administering the compound
alone, and thus influences of the antitumor compound on
normal cells can be alleviated so that a higher safety
can be achieved.
In this connection, the inventors created a linker
with a specific structure and succeeded in obtaining an
antibody-drug conjugate in which the anti-HER2 antibody
and exatecan are conjugated to each other via the
linker, and confirmed an excellent antitumor effect
exhibited by the conjugate to thereby complete the
present invention.
[0015]
Specifically, the present invention relates to the
following.
[1] An antibody-drug conjugate wherein an antitumor
compound represented by the following formula:
[Formula 2]
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4833-0752-7733.1
Me
F
is conjugated to an
having a structure
formula:
Atty. Dkt. No. 111119-0105
anti-HER2 antibody via a linker
represented by the following
-L1 -L2 -LP-NH- (CH2 )n1 -La-(CH2 )n2 -C(=O) -
via a thioether bond which is formed at a disulfide
bond moiety present in the hinge part of the anti-HER2
antibody.
[0016]
Here, the ant i-HER2 antibody is connected to the
terminal L1
,
the antitumor compound is connected to the carbonyl
group of the - (CH2 ) n2 -C (=0) - moiety with the nitrogen
atom of the amino group at position 1 as the connecting
position,
wherein
n 1 represents an integer of Oto 6,
n 2 represents an integer of Oto 5,
L1 represents -(Succinimid-3-yl-N)-(CH2 )n3 -C(=O)-,
wherein n 3 represents an integer of 2 to 8,
L2 represents -NH- (CH2CHrO) n 4 -CH2CHrC (=0) - or a single
bond,
wherein n 4 represents an integer of 1 to 6,
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LP represents a peptide residue consisting of 2 to 7
amino acids,
La represents -0- or a single bond, and
- (Succinimid-3 -yl-N) - has a structure represented by
the following formula:
[Formula 3]
0
which is connected to the anti-HER2 antibody at
position 3 thereof and is connected to the methylene
group in the linker structure containing this structure
on the nitrogen atom at position 1.
[0017]
The present invention further relates to each of
the following.
[2] The antibody-drug conjugate according to [1],
wherein the peptide residue LP is a peptide residue
comprising an amino acid selected from phenylalanine,
glycine, valine, lysine, ci trulline, serine, glutamic
acid, and aspartic acid.
[3] The antibody-drug conjugate according to [1] or
[2], wherein LP is a peptide residue selected from the
following group:
-GGF-,
-DGGF-,
-(D-)D-GGF-,
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-EGGF-,
-GGFG-,
-SGGF-,
-KGGF-,
-DGGFG-,
-GGFGG-,
-DDGGFG-,
-KDGGFG-, and
-GGFGGGF-;
wherein "(D-)D" represents D-aspartic acid.
[4] The antibody-drug conjugate according to [1] or
[2], wherein LP is a peptide residue consisting of 4
amino acids.
[5] The antibody-drug conjugate according to any one
of [1] to [4], wherein LP is the tetrapeptide residue -
GGFG-.
[0018]
[6] The antibody-drug conjugate according to any one
of [1] to [5], wherein n 3 is an integer of 2 to 5, and
L2 is a single bond.
[7] The antibody-drug conjugate according to any one
of [1] to [5] , wherein n 3 is an integer of 2 to 5, L2
is -NH-(CH2 CH2 -0)n4 -CH2 CH2 -C(=0)-, and n 4 is 2 or 4.
[8] The antibody-drug conjugate according to any one
of [1] to [7], wherein -NH- (CH2 ) n 1 -La- (CH2 ) n 2 -C (=0) - is
a partial structure having a chain length of 4 to 7
atoms.
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[9] The antibody-drug conjugate according to any one
of [1] to [7], wherein -NH- (CH2) n 1 -La- (CH2) n2-C (=0) - is
a partial structure having a chain length of 5 or 6
atoms.
[10] The antibody-drug conjugate according to any one
of [1] to [9], wherein -NH- (CH2) n 1 -La- (CH2) n2-C (=0) - is
-NH-CH2CH2-C(=0)-,
-NH-CH2CH2CH2-C(=0) -,
-NH-CH2CH2CH2CH2-C(=0) -,
-NH-CH2CH2CH2CH2CH2-C(=0) -,
-NH-CH2-0-CH2-C(=0)-,
-NH-CH2CH2-0-CH2-C(=0)-, or
-NH-CH2CH2-0-C(=0)-.
[11] The antibody-drug conjugate according to any one
of [1] to [9], wherein -NH- (CH2) n 1 -La- (CH2) n2-C (=0) - is
-NH-CH2CH2CH2-C(=0) -,
-NH-CH2-0-CH2-C(=0)-, or
-NH-CH2CH2-0-CH2-C(=0) -.
[0019]
[12] The antibody-drug conjugate according to any one
of [1] to [9], wherein the drug-linker structure moiety
having the drug connected to -L1 -L2-LP-NH-(CH2)n1 -La(
CH2)n2-C(=0) - is one drug-linker structure selected
from the following group:
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-GGFG-NH-CH2CH2-C(=0)(
NH-DX) ,
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-GGFG-NH-CH2CH2CH2-
C (=0) - (NH-DX),
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-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
C(=O) -(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2CHrC
( =0) - (NH-DX) ,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2CH2CH2CH2-
C(=O) -(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2-OCH2-
C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
O-CH2-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
O-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
C(=O) -GGFG-NH-CH2CH2-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
C(=O) -GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2-C(=O) -(NHDX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-C(=O) -
(NH-DX),
[0020]
wherein -(Succinimid-3-yl-N)- has
represented by the following formula:
[Formula 4]
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a structure
Atty. Dkt. No. 111119-0105
0
"(NO
which is connected to the anti-HER2 antibody at
position 3 thereof and is connected to the methylene
group in the linker structure containing this structure
on the nitrogen atom at position 1,
- (NH-DX) represents
following formula:
[Formula 5]
H
Me 0
F
HO
1
Me
0
\
0
a group represented by the
wherein the nitrogen atom of the amino group at
position 1 is the connecting position, and
-GGFG- represents the tetrapeptide residue -Gly-GlyPhe-
Gly-.
[0021]
[13] The antibody-drug conjugate according to any one
of [1] to [9], wherein the drug-linker structure moiety
having the drug connected to -L1 -L2 -LP-NH-(CH2 )n1 -La(
CH2 )n2 -C(=O) - is one drug-linker structure selected
from the following group:
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Atty. Dkt. No. 111119-0105
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2-OCH2-
C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
O-CH2-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
C(=O) -GGFG-NH-CH2CH2CH2-C(=O) -(NH-DX)
[0022]
Here, - (Succinimid-3-yl-N) - , - (NH-DX), and -GGFGare
as defined above.
[0023]
[14] An antibody-drug conjugate wherein an antitumor
compound represented by the following formula:
[Formula 6]
Me
F
! 0
Me
is conjugated to an
having a
formula:
structure
anti-HER2 antibody via a linker
represented by the following
-L1 -L2-LP-NH- (CH2)n1 -La-(CH2)n2-C(=O) -
via a thioether bond which is formed at a disulfide
bond moiety present in the hinge part of the anti-HER2
antibody,
wherein
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the anti-HER2 antibody is connected to the terminal L1
,
the antitumor compound is connected to the carbonyl
group of the -(CH2 )n2 -C(=O) - moiety,
wherein
n 1 represents an integer of Oto 6,
n 2 represents an integer of Oto 5,
L1 represents -(Succinimid-3-yl-N)-(CH2 )n3 -C(=O)-,
wherein n 3 represents an integer of 2 to 8,
bond,
wherein n 4 represents an integer of 1 to 6,
LP represents the tetrapeptide residue -GGFG-,
La represents -0- or a single bond, and
- (Succinimid-3 -yl-N) - has a structure represented by
the following formula:
[Formula 7]
0
which is connected to the anti-HER2 antibody at
position 3 thereof and is connected to the methylene
group in the linker structure containing this structure
on the nitrogen atom at position 1.
[0024]
[15] The antibody-drug conjugate according to
wherein
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[ 14] ,
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n 1 is 3, n2 is O, n 3 is 2, L2 is -NH- (CH2CHr0) n4 -CH2CHr
C(=0)-, n4 is 2, and La is a single bond,
n 1 is 1, n2 is 1, n 3 is 5, L2 is a single bond, and La
is -0-, or
n 1 is 2, n2 is 1, n 3 is 5, L2 is a single bond, and La
is -0-.
[16] The antibody-drug conjugate according to [14] or
[15], wherein n 3 is 2 or 5, and L2 is a single bond.
[17] The antibody-drug conjugate according to [14] or
[15], wherein n 3 is 2 or 5, L2 is -NH- (CH2CHr0) n4
-
CH2CH2-C (=0) -, and n4 is 2 or 4.
[18] The antibody-drug conjugate according to any one
of [14] to [17], wherein -NH- (CH2) n 1 -La- (CH2) n2-C (=0) -
is
-NH-CH2CH2CH2-C(=0) -,
-NH-CH2-0-CH2-C(=0)-, or
-NH-CH2CH2-0-CH2-C(=0) -.
[0025]
[19] The antibody-drug conjugate according to any one
of [14] to [18], wherein the drug-linker structure
moiety having the drug connected to -L1 -L2-LP-NH(
CH2)n1 -La-(CH2)n2-C(=0) - is one drug-linker structure
selected from the group consisting of the following:
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-GGFG-NH-CH2CH2-C(=0)(
NH-DX) ,
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-GGFG-NH-CH2CH2CH2-
C (=0) - (NH-DX),
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-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
C(=O) -(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2CHrC
( =0) - (NH-DX) ,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2CH2CH2CH2-
C(=O) -(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2-OCH2-
C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
O-CH2-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
O-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
C(=O) -GGFG-NH-CH2CH2-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
C(=O) -GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2-C(=O) -(NHDX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-C(=O) -
(NH-DX),
[0026]
wherein, -(Succinimid-3-yl-N)- has a structure
represented by the following formula:
[Formula 8]
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Atty. Dkt. No. 111119-0105
~NO
which is connected to the anti-HER2 antibody at
position 3 thereof and is connected to the methylene
group in the linker structure containing this structure
on the nitrogen atom at position 1,
- (NH-DX) represents
following formula:
[Formula 9]
H
Me
F
a group represented by the
wherein the nitrogen atom of the amino group at
position 1 is the connecting position, and
-GGFG- represents the tetrapeptide residue -Gly-GlyPhe-
Gly-.
[0027]
[20] The antibody-drug conjugate according to any one
of [14] to [18], wherein the drug-linker structure
moiety having the drug connected to -L1 -L2 -LP-NH(
CH 2 )n1 -La-(CH2 )n2 -C(=O) - is one drug-linker structure
selected from the following group:
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-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
O-CH2-C(=O)-(NH-DX), and
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CHrC
(=0) -GGFG-NH-CH2CH2CHrC (=0) - (NH-DX) .
Here, - (Succinimid-3-yl-N) - , - (NH-DX), and -GGFGare
as defined above.
[0028]
[21] The antibody-drug conjugate according to any one
of [1] to [20], wherein the average number of units of
the selected one drug-linker structure conjugated per
antibody molecule is in the range of from 1 to 10.
[22] The antibody-drug conjugate according to any one
of [1] to [20], wherein the average number of units of
the selected one drug-linker structure conjugated per
antibody molecule is in the range of from 2 to 8.
[23] The antibody-drug conjugate according to any one
of [1] to [20], wherein the average number of units of
the selected one drug-linker structure conjugated per
antibody molecule is in the range of from 3 to 8.
[0029]
[24] A drug containing
according to any one of
a hydrate thereof.
the
[1] to
antibody-drug conjugate
[23], a salt thereof or
[25] An antitumor drug and/or anticancer drug
containing the antibody-drug conjugate according to any
one of [1] to [23], a salt thereof or a hydrate thereof.
[26] The antitumor drug and/or anticancer drug
according to [25], which is for use against lung cancer,
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urothelial cancer, colorectal cancer, prostate cancer,
ovarian
bladder
cancer,
cancer,
pancreatic
gastric
cancer, breast cancer,
cancer,
stromal tumor, uterine cervix cancer,
gastrointestinal
esophageal cancer,
squamous
cancer,
cell carcinoma, peritoneal cancer,
hepatocellular cancer, colon cancer,
liver
rectal
cancer, colorectal cancer, endometrial cancer, uterine
cancer, salivary gland cancer, kidney cancer, vul val
cancer, thyroid cancer, penis cancer, leukemia,
malignant lymphoma, plasmacytoma, myeloma, or sarcoma.
[27] A pharmaceutical composition containing the
antibody-drug conjugate according to any one of [1] to
[23], a salt thereof or a hydrate thereof as an active
component, and a pharmaceutically acceptable
formulation component.
[28] The pharmaceutical composition according to [27],
which is for use against lung cancer, urothelial cancer,
colorectal cancer, prostate cancer, ovarian cancer,
pancreatic cancer, breast cancer, bladder cancer,
gastric cancer, gastrointestinal stromal tumor, uterine
cervix cancer, esophageal cancer, squamous cell
carcinoma, peritoneal cancer, liver cancer,
hepatocellular cancer, colon cancer, rectal cancer,
colorectal cancer, endometrial cancer, uterine cancer,
salivary
thyroid
gland cancer,
cancer, penis
kidney cancer, vul val cancer,
cancer, leukemia, malignant
lymphoma, plasmacytoma, myeloma, or sarcoma.
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[29] A method for treating tumor and/or cancer
comprising administering the antibody-drug conjugate
according to any one of [1] to [23], a salt thereof or
a hydrate thereof.
[0030]
[30] A method for producing an antibody-drug conjugate
comprising reacting a compound represented by the
following formula:
(maleimid-N-yl) -(CH2)n3 -C(=O) -L2-LP-NH-(CH2)n1 -La-(CH2)n2-
C(=O)-(NH-DX)
with an anti-HER2 antibody or a reactive derivative
thereof and conjugating a drug-linker moiety to the
antibody by a method for forming a thioether bond at a
disulfide bond site present in the hinge part of the
antibody.
[0031]
In the formula, n 3 represents an integer of 2 to 8,
L2 represents -NH- (CH2CHrO) n 4 -CH2CHrC (=0) - or a single
bond,
wherein n 4 represents an integer of 1 to 6,
LP represents a peptide residue consisting of 2 to 7
amino acids selected from phenylalanine, glycine,
valine, lysine, citrulline, serine, glutamic acid, and
aspartic acid,
nl represents an integer of 0 to 6,
n2 represents an integer of 0 to 5,
La represents -0- or a single bond,
- 26 -
4833-0752-7733.1
(maleimid-N-yl)following
formula:
[Formula 10]
0
is a
Atty. Dkt. No. 111119-0105
group represented by the
wherein the nitrogen atom is the connecting position,
and
- (NH-DX) is a group represented by the following
formula:
[Formula 11]
_,,
Me
F
H
N-
0
I 0
Me
wherein the nitrogen atom of the amino group at
position 1 is the connecting position.
[0032]
[31] The production method according to [30], wherein
the method for conjugating a drug-linker moiety to an
anti-HER2 antibody is a method of reducing the antibody
to convert the antibody to a reactive derivative.
[0033]
[32] The production method according to [30] or [31],
wherein the average number of units of the selected one
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drug-linker structure conjugated per antibody molecule
is in the range of from 1 to 10.
[33] The production method according to [30] or [31],
wherein the average number of units of the selected one
drug-linker structure conjugated per antibody molecule
is in the range of from 2 to 8.
[34] The production method according to [30] or [31],
wherein the average number of units of the selected one
drug-linker structure conjugated per antibody molecule
is in the range of from 3 to 8.
[35] An antibody-drug conjugate obtained by the
production method according to any of [30] to [34]
[0034]
[36] An antibody-drug conjugate obtained by forming a
thioether bond at a sulfide bond site in the hinge part
of the antibody, wherein the anti-HER2 antibody is
treated in a reducing condition and thereafter reacted
with a compound selected from the group shown below:
(maleimid-N-yl)-CH2CH2-C(=0)-GGFG-NH-CH2CH2-C(=0)-(NHDX),
(maleimid-N-yl)-CH2CH2CH2-C(=0)-GGFG-NH-CH2CH2-C(=0)(
NH-DX),
(maleimid-N-yl)-CH2CH2CH2CH2-C(=0)-GGFG-NH-CH2CH2-C(=0)(
NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl)-CH2CH2-C(=0)-GGFG-NH-CH2CH2CH2-C(=0)(
NH-DX),
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(maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)(
NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2CH2CH2-
C (=0) - (NH-DX),
(maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NHCH2CH2CH2CH2CH2-
C(=O) -(NH-DX),
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2CH2CH2CH2-
C(=O) -(NH-DX),
(maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O) -
(NH-DX) ,
(maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)(
NH-DX),
(maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-
C (=0) - (NH-DX),
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2-O-CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O) -
(NH-DX),
(maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-
C (=0) - (NH-DX),
(maleimid-N-yl) -CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2-O-CH2-
C (=0) - (NH-DX) ,
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Atty. Dkt. No. 111119-0105
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2-OCH2-
C(=O)-(NH-DX),
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2-OC
(=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O) -(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O) -(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
O-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
O-CH2CH2-O-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
O-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX), and
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Atty. Dkt. No. 111119-0105
DX).
[0035]
In the above, (maleimid-N-yl)-
represented by the following formula:
[ Formula 12]
is a group
wherein the nitrogen atom is the connecting position,
and
- (NH-DX) is a group represented by the following
formula:
[ Formula 13]
Me
F
H
i 0
Me
wherein the nitrogen atom of the amino group at
position 1 is the connecting position, and
-GGFG- represents the tetrapeptide residue -Gly-GlyPhe-
Gly-.
[0036]
[37] An antibody-drug conjugate obtained by forming a
thioether bond at a sulfide bond site present in the
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Atty. Dkt. No. 111119-0105
hinge part
antibody is
of the antibody, wherein the anti-HER2
treated in
thereafter reacted with a
group shown below:
a reducing condition
compound selected from
and
the
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-0-CH2CH2-0-CH2CH2-
C(=0)-GGFG-NH-CH2CH2CH2-C(=0)-(NH-DX),
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2-0-CH2-
C(=0)-(NH-DX), and
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2-0-
CHrC (=0) - (NH-DX) .
Here, (maleimid-N-yl) - , - (NH-DX) , and -GGFG- are
as defined above.
[0037]
[38] The antibody-drug conjugate according to [36] or
[37], wherein the average number of units of the
selected one drug-linker structure conjugated per
antibody molecule is in the range of from 1 to 10.
[39] The antibody-drug conjugate according to [36] or
[37], wherein the average number of units of the
selected one drug-linker structure conjugated per
antibody molecule is in the range of from 2 to 8.
[40] The antibody-drug conjugate according to [36] or
[37], wherein the average number of units of the
selected one drug-linker structure conjugated per
antibody molecule is in the range of from 3 to 8.
Advantageous Effects of Invention
[0038]
- 32 -
4833-0752-7733.1
Atty. Dkt. No. 111119-0105
With an anti-HER2 antibody-drug conjugate having
the antitumor compound exatecan conjugated via a linker
with a specific structure, an excellent antitumor
effect and safety can be achieved.
Brief Description of Drawings
[0039]
[Fig. 1] Fig. 1 shows an amino acid sequence of a heavy
chain of a humanized anti-HER2 monoclonal antibody (SEQ
ID NO: 1) .
[Fig. 2] Fig. 2 shows an amino acid sequence of a light
chain of a humanized anti-HER2 monoclonal antibody (SEQ
ID NO: 2)
[Fig. 3] Fig. 3
effect of an
is a diagram
antibody-drug
showing the
conjugate
anti tumor
( 2 7) or
trastuzumab on a nude mouse with subcutaneously
transplanted human breast cancer line KPL-4 cells. In
the drawing, the abscissa depicts days after tumor
inoculation, and the ordinate depicts tumor volume.
[Fig. 4] Fig. 4 is a diagram showing the antitumor
effect of an antibody-drug conjugate (8), (28) or
trastuzumab emtansine on a nude mouse with
subcutaneously transplanted human gastric cancer line
NCI-N87 cells. In the drawing, the abscissa depicts
days after tumor inoculation, and the ordinate depicts
tumor volume.
[Fig. 5] Fig. 5 is a diagram showing the antitumor
effect of an antibody-drug conjugate (8), (29), (30),
- 33 -
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Atty. Dkt. No. 111119-0105
trastuzumab, or trastuzumab emtansine on a nude mouse
with subcutaneously transplanted human breast cancer
line JIMT-1 cells. In the drawing, the abscissa
depicts days after tumor inoculation, and the ordinate
depicts tumor volume.
[Fig. 6] Fig. 6 is a diagram showing the antitumor
effect of an antibody-drug conjugate (31), trastuzumab,
or trastuzumab emtansine on a nude mouse with
subcutaneously transplanted human pancreatic cancer
line Capan-1 cells. In the drawing, the abscissa
depicts days after tumor inoculation, and the ordinate
depicts tumor volume.
[Fig. 7] Fig. 7 is a diagram showing the antitumor
effect of an antibody-drug conjugate (50) on a nude
mouse with subcutaneously transplanted human gastric
cancer line NCI-N87 cells. In the drawing, the
abscissa depicts days after tumor inoculation, and the
ordinate depicts tumor volume.
[Fig. 8] Fig. 8 is a diagram showing the antitumor
effect of an antibody-drug conjugate (50), trastuzumab,
or trastuzumab emtansine on a nude mouse with
subcutaneously transplanted human breast cancer ST225
cells. In the drawing, the abscissa depicts days after
tumor inoculation, and the ordinate depicts tumor
volume.
[Fig. 9] Fig. 9 is a diagram showing the antitumor
effect of an antibody-drug conjugate (50), trastuzumab,
or trastuzumab emtansine on a nude mouse with
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Atty. Dkt. No. 111119-0105
subcutaneously transplanted human breast cancer ST910
cells. In the drawing, the abscissa depicts days after
tumor inoculation, and the ordinate depicts tumor
volume.
[Fig. 10] Fig. 10 is a diagram showing the antitumor
effect of an antibody-drug conjugate (50), trastuzumab,
or trastuzumab emtansine on a nude mouse with
subcutaneously transplanted human colorectal cancer
line CTG-0401 cells. In the drawing, the abscissa
depicts days after tumor inoculation, and the ordinate
depicts tumor volume.
[Fig. 11] Fig. 11 is a diagram showing the anti tumor
effect of an antibody-drug conjugate (50), trastuzumab,
or trastuzumab emtansine on a nude mouse
cell
with
subcutaneously transplanted human non-small lung
cancer CTG- 0 8 6 0 cells. In the drawing, the abscissa
depicts days after tumor inoculation, and the ordinate
depicts tumor volume.
[Fig. 12] Fig. 12 is a diagram showing the anti tumor
effect of an antibody-drug conjugate (50), trastuzumab,
or trastuzumab emtansine on a nude mouse with
subcutaneously transplanted human bile duct cancer line
CTG- 0 92 7 eel ls. In the drawing, the abscissa depicts
days after tumor inoculation, and the ordinate depicts
tumor volume.
[Fig. 13] Fig. 13 is a diagram showing the anti tumor
effect of an antibody-drug conjugate (50), trastuzumab,
or trastuzumab emtansine on a nude mouse with
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Atty. Dkt. No. 111119-0105
subcutaneously transplanted human esophageal cancer
line CTG-0137 cells. In the drawing, the abscissa
depicts days after tumor inoculation, and the ordinate
depicts tumor volume.
[Fig. 14] Fig. 14 is a diagram showing the anti tumor
effect of an antibody-drug conjugate (50), trastuzumab,
or trastuzumab emtansine on a nude mouse with
subcutaneously transplanted human ovarian cancer line
SK-OV-3 cells. In the drawing, the abscissa depicts
days after tumor inoculation, and the ordinate depicts
tumor volume.
Description of Embodiments
[0040]
Hereinafter, preferred modes for carrying out the
present invention are described with reference to the
drawings.
merely for
embodiment
The embodiments described below are given
illustrating one example of a typical
of the present invention and are not
intended to limit the scope of the present invention.
[0041]
The anti-HER2 antibody-drug
present invention is an antitumor
anti-HER2 antibody is conjugated
conjugate of the
drug in which an
to an antitumor
compound via a linker structure moiety and is explained
in detail hereinbelow.
[0042]
[Antibody]
4833-0752-7733.1
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Atty. Dkt. No. 111119-0105
The anti-HER2 antibody used in the anti-HER2
antibody-drug conjugate of the present invention may be
derived from any species, and preferred examples of the
species can include humans, rats, mice, and rabbits.
In case when the antibody is derived from other than
human species, it is preferably chimerized or humanized
using a well known technique. The antibody of the
present invention may be a polyclonal antibody or a
monoclonal antibody and is preferably a monoclonal
antibody.
The anti-HER2 antibody is the antibody, which is
capable of targeting tumor cells, that is, possesses a
property of recognizing a tumor cell, a property of
binding to a tumor cell, a property of internalizing in
a tumor cell, cytocidal activity against tumor cells,
or the like, and can be conjugated with a drug having
antitumor activity via a linker to form an antibodydrug
conjugate.
The binding activity of the antibody against tumor
cells can be confirmed using flow cytometry. The
internalization of the antibody into tumor cells can be
confirmed using (1) an assay of visualizing an antibody
incorporated in cells under a
using a secondary antibody
binding to the therapeutic
fluorescence microscope
Differentiation (2008) 15,
measuring a fluorescence
cells using a secondary
(fluorescently
antibody (Cell
7 51 - 7 61 ) , ( 2 ) an
labeled)
Death
assay
and
of
intensity incorporated in
antibody (fluorescently
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Atty. Dkt. No. 111119-0105
labeled) binding to the therapeutic antibody (Molecular
Biology of the Cell, Vol. 15, 5268-5282, December 2004),
or (3) a Mab-ZAP assay using an immunotoxin binding to
the therapeutic antibody wherein the toxin is released
upon incorporation into cells to inhibit cell growth
(Bio Techniques 28: 162-165, January 2000). As the
immunotoxin, a recombinant complex protein of a
diphtheria toxin catalytic domain and protein G may be
used.
The antitumor activity of the antibody can be
confirmed in vitro by determining inhibitory activity
against cell growth. For example, a cancer cell line
overexpressing a target protein for the antibody is
cultured, and the antibody is added at varying
concentrations into the culture system to determine an
inhibitory activity against focus formation, colony
formation, and spheroid growth. The antitumor activity
can be confirmed in vivo, for example, by administering
the antibody to a nude mouse with a transplanted tumor
cell line highly expressing the target protein, and
determining change in the cancer cell.
Since the compound conjugated in the antibody-drug
conjugate exerts an antitumor effect, it is preferred
but not essential that the antibody itself should have
an antitumor effect. For the purpose of specifically
and selectively exerting the cytotoxic activity of the
antitumor compound against tumor cells, it is important
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and also preferred that the antibody should have the
property of internalizing to migrate into tumor cells.
[0043]
The anti-HER2 antibody can be obtained by a
procedure known in the art. For example, the antibody
of the present invention can be obtained using a method
usually carried out in the art, which involves
immunizing animals with an antigenic polypeptide and
collecting and purifying antibodies produced in vivo.
The origin of the antigen is not limited to humans, and
the animals may be immunized with an antigen derived
from a non-human animal such as a mouse, a rat and the
like. In this case, the cross-reactivity of antibodies
binding to the obtained heterologous antigen with human
antigens can be tested to screen for an antibody
applicable to a human disease.
Alternatively,
produce antibodies
antibody-producing
against the antigen
cells which
are
myeloma cells according to a method known
fused with
in the art
(e.g., Kohler and Milstein, Nature (1975) 256, p. 495-
497; and Kennet, R. ed., Monoclonal Antibodies, p. 365-
367, Plenum Press, N.Y. (1980)) to establish hybridomas,
from which monoclonal antibodies can in turn be
obtained.
The antigen can be obtained by genetically
engineering host cells to produce a gene encoding the
antigenic protein. Specifically, vectors that permit
expression of the antigen gene are prepared and
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transferred to host cells so that the gene is expressed.
The antigen thus expressed can be purified. The
antibody can also be obtained by a method of immunizing
animals with the above-described genetically engineered
antigen-expressing cells or a cell line expressing the
antigen.
[0044]
The anti-HER2 antibodies that can be used in the
present invention are not particularly limited and are
preferably, for example, those having properties as
described below.
(1) An anti-HER2 antibody having the following
properties:
(a) specifically binding to HER2, and
(b) having an activity of internalizing in HER2-
expressing cells by binding to HER2.
(2) The antibody according to (1) above, wherein the
antibody binds to the extracellular domain of HER2.
(3) The antibody according to (1) or (2) above, wherein
the antibody is a monoclonal antibody.
(4) The antibody according to any of (1) to (3) above,
wherein the antibody has an antibody-dependent cellular
cytotoxici ty (ADCC) activity and/ or a complement -
dependent cytotoxicity (CDC) activity
(5) The antibody according to any of (1) to (4) above,
wherein the antibody is a mouse monoclonal antibody, a
chimeric monoclonal antibody, or a humanized monoclonal
antibody.
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(6) The antibody according to any of (1) to (5) above,
wherein the antibody is a humanized monoclonal antibody
comprising a heavy chain consisting of the amino acid
sequence represented by SEQ ID NO: 1 and a light chain
consisting of the amino acid sequence represented by
SEQ ID NO: 2.
(7) The antibody according to any of (1) to (6) above,
wherein the antibody lacks a lysine residue at the
carboxyl terminus of the heavy chain.
(8) The antibody according to (7) above, wherein the
antibody comprises a heavy chain consisting of an amino
acid sequence consisting of amino acid residues 1 to
449 of SEQ ID NO: 1 and a light chain consisting of an
amino acid sequence consisting of amino acid residues 1
to 214 of SEQ ID NO: 2.
(9) An antibody obtained by a method for producing the
antibody according to any of (1) to (8) above, the
method comprising the steps of: culturing a host cell
transformed with an expression vector containing a
polynucleotide encoding the antibody; and collecting
the antibody of interest from the cultures obtained in
the preceding step.
[0045]
Hereinafter, the anti-HER2 antibody used in the
invention is described.
The terms "cancer" and "tumor" as used herein are
used with the same meaning.
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The term "gene" as used herein includes not only
DNA, but also mRNA thereof, cDNA thereof and cRNA
thereof.
The term "polynucleotide" as used herein is used
with the same meaning as a nucleic acid and also
includes DNA, RNA, probes, oligonucleotides, and
primers.
The terms "polypeptide", "protein" and "protein"
as used herein are used without distinction.
The term "cell" as used herein also includes cells
in an animal individual and cultured cells.
The term "HER2" as used herein is used with the
same meaning as HER2 protein.
Examples of the anti-HER2 antibody as used herein
can include, but not particularly limited to,
pertuzumab (International Patent Publication No. WO
01/00245) and trastuzumab (U.S. Patent No. 5821337).
Trastuzumab is preferred. However, the anti-HER2
antibody of the present invention is not limited
thereto as long as it is an anti-HER2 antibody
specifically binding to HER2, and more preferably
having an activity of internalizing in HER2-expressing
cells by binding to HER2.
The term "trastuzumab" as used herein is also
called HERCEPTIN(R), huMAb4D5-8, or rhuMAb4D5-8 and is
a humanized antibody comprising a heavy chain
consisting of an amino acid sequence consisting of
amino acid residues 1 to 449 of SEQ ID NO: 1 (Fig. 1)
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and a light chain consisting of an amino acid sequence
consisting of amino acid residues 1 to 214 of SEQ ID
NO: 2 (Fig. 2)
The term "specifically binding" as used herein
means binding that is not nonspecific adsorption.
Examples of the criterion for determining whether the
binding is specific or not can include dissociation
constant (hereinafter referred to as "KD"). The KD
value of the antibody for the HER2 protein is
preferably 1 X 10-5 M or smaller, 5 X 10-6 M or smaller,
2 X 10-6 M or smaller, or 1 X 10-6 M or smaller, more
preferably 5 X 10-7 M or smaller, 2 X 10-7 M or smaller,
or 1 X 10-7 M or smaller, further preferably 5 X 10-8 M
or smaller, 2 X 10-8 M or smaller, or 1 x 10-8 M or
smaller, and most preferably 5 X 10-9 Mor smaller, 2 x
10-9 M or smaller, or 1 X 10-9 M or smaller. The
binding between the HER2 protein and the antibody can
be measured using a method known in the art, such as
surface plasmon resonance, ELISA, or RIA.
The term "CDR" as used herein refers to a
complementarity determining region (CDR) It is known
that each heavy and light chain of an antibody molecule
has three complementarity determining regions ( CDRs) .
The CDR is also called the hypervariable domain, and is
present in a variable region of each heavy and light
chain of an antibody. It is a site which has unusually
high variability in its primary structure, and there
are three separate CDRs in the primary structure of
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each heavy and light polypeptide chain. In this
specification, as for the CDRs of an antibody, the CDRs
of the heavy chain are represented by CDRHl, CDRH2, and
CDRH3 from the amino-terminal side of the amino acid
sequence of the heavy chain, and the CDRs of the light
chain are represented by CDRLl, CDRL2, and CDRL3 from
the amino-terminal side of the amino acid sequence of
the light chain. These sites are proximate to one
another in the tertiary structure and determine the
specificity for an antigen to which the antibody binds.
The phrase "hybridization is performed under
stringent conditions" as used herein refers to a
process in which hybridization is performed under
conditions under which identification can be achieved
by performing hybridization at 68°C in a commercially
available hybridization solution ExpressHyb
Hybridization Solution (manufactured by Clontech, Inc.)
or by performing hybridization at 68°C in the presence
of 0.7 to 1.0 M NaCl using a filter having DNA
immobilized thereon, followed by performing washing at
68°C using 0.1 to 2 x SSC solution (1 x SSC solution is
composed of 150 mM NaCl and 15 mM sodium citrate) or
under conditions equivalent thereto.
[0046]
1. HER2
HER2 is one of the oncogene products of a typical
growth factor
epidermal cell
4833-0752-7733.1
receptor
growth
oncogene
factor
- 44 -
identified
receptor
as human
2-related
Atty. Dkt. No. 111119-0105
oncogene, and is a transmembrane receptor protein
having a molecular weight of 185 kDa and having a
tyrosine kinase domain. HER2 is a member of the EGFR
family consisting of HERl (EGFR, ErbB-1), HER2 (neu,
ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4) and is known
to be autophosphorylated at intracellular tyrosine
residues by its homodimer formation or heterodimer
formation with another EGFR receptor HERl, HER3, or
HER4 and is itself activated in that manner, thereby
playing an important role in cell growth,
differentiation, and survival in normal cells and tumor
cells.
As for the HER2 protein to be used in the present
invention, the HER2 protein can be directly purified
from HER2-expressing cells of a human or a non-human
mammal (such as a rat or a mouse) and used, or a cell
membrane fraction of the above-described eel ls can be
prepared and used. Further, HER2 can be obtained by in
vitro synthesis thereof or production thereof in a host
cell through genetic engineering. In the genetic
engineering, specifically, after HER2 cDNA is
integrated into a vector capable of expressing HER2
cDNA, the HER2 protein can be obtained by synthesizing
it in a solution containing an enzyme, a substrate and
an energy substance required for transcription and
translation, or by expressing HER2 in another
prokaryotic or eucaryotic transformed host cell.
Alternatively, the above-described genetically
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engineered HER2-expressing cells, or a cell line
expressing HER2 may be used as the HER2 protein.
The DNA sequence and amino acid sequence of HER2
are disclosed on a public database, and can be referred
to, for example, under Accession No. M11730 (GenBank),
NP 004439.2 (NCBI), or the like.
Further, a protein which consists of an amino acid
sequence wherein one or several
substituted, deleted and/or added in
amino acids are
any of the abovedescribed
amino acid sequences of HER2 and also has a
biological activity equivalent to that of the protein
is also included in HER2.
Human HER2 protein is composed of a signal
sequence consisting of N-terminal 22 amino acid
residues, an extracellular domain consisting of 630
amino acid residues, a transmembrane domain consisting
of 23 amino acid residues, and an intracellular domain
consisting of 580 amino acid residues.
[0047]
2. Production of anti-HER2 antibody
The antibody against HER2 of the present invention
can be obtained according to, for example, a method
usually carried out in the art, which involves
immunizing animals with HER2 or an arbitrary
polypeptide selected from the amino acid sequence of
HER2 and collecting and purifying antibodies produced
in vivo. The biological species of HER2 to be used as
an antigen is not limited to being human, and an animal
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can be immunized with HER2 derived from an animal other
than humans such as a mouse or a rat or with rat
pl85neu. In this case, by examining the crossreactivity
between an antibody binding to the obtained
heterologous HER2 and human HER2, an antibody
applicable to a human disease can be selected.
Further, a monoclonal antibody can be obtained
from a hybridoma established by fusing antibodyproducing
cells which produce an antibody against HER2
with myeloma cells according to a known method ( for
example, Kohler and Milstein, Nature, (1975) 256, pp.
495-497; Kennet, R. ed., Monoclonal Antibodies, pp.
365-367, Plenum Press, N.Y. (1980)).
HER2 to be used as an antigen can be obtained by
expressing HER2 gene in a host cell using genetic
engineering.
Specifically, a vector capable of expressing HER2
gene is produced, and the resulting vector is
transfected into a host cell to express the gene, and
then, the expressed HER2 is purified.
Alternatively, the above-described genetically
engineered HER2-expressing cells, or a cell line
expressing HER2 may be used as the HER2 protein. The
anti-HER2 antibody can be obtained by a preocedure
known in the art. Hereinafter, a method of obtaining
an antibody against HER2 is specifically described.
[0048]
(1) Preparation of antigen
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4833-0752-7733.1
the
Examples
anti-HER2
Atty. Dkt. No. 111119-0105
of the antigen to be used
antibody include HER2, or
for producing
a polypeptide
consisting of a partial amino acid sequence comprising
at least 6 consecutive amino acids of HER2, or a
derivative obtained by adding a given amino acid
sequence or carrier thereto.
HER2 can be purified directly from human tumor
tissues or tumor cells and used. Further, HER2 can be
obtained by synthesizing it in vitro or by producing it
in a host cell by genetic engineering.
With respect to the genetic engineering,
specifically, after HER2 cDNA is integrated into a
vector capable of expressing HER2 cDNA, HER2 can be
obtained by synthesizing it in a solution containing an
enzyme, a substrate and an energy substance required
for transcription and translation, or by expressing
HER2 in another prokaryotic or eucaryotic transformed
host cell.
Further, the antigen can also be obtained as a
secretory protein by expressing a fusion protein
obtained by ligating the extracellular domain of HER2,
which is a membrane protein, to the constant region of
an antibody in an appropriate host-vector system.
HER2 cDNA can be obtained by, for example, a socalled
PCR method in which a polymerase chain reaction
is performed using a cDNA library expressing HER2 cDNA
as a template and primers which specifically amplify
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HER2 cDNA (PCR; Saiki, R. K., et al., Science, (1988)
2 3 9 , pp . 4 8 7 - 4 8 9 ) .
As the in vitro synthesis of the polypeptide, for
example, Rapid Translation System (RTS) manufactured by
Roche Diagnostics, Inc. can be exemplified, but it is
not limited thereto.
Examples of the prokaryotic host cells include
Escherichia coli and Bacillus subtilis. In
transform the host cells with a target gene,
order to
the host
cells are transformed by a plasmid vector comprising a
replicon,
species
sequence.
i.e., a replication
compatible with the
origin derived from a
Further, the
host,
vector
and a regulatory
preferably has a
sequence capable of imposing phenotypic selectivity on
the transformed cell.
Examples of
vertebrate cells,
the eucaryotic
insect eel ls,
host cells include
and yeast cells.
the vertebrate cells, for example, simian cos
As
cells
(Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-
1650; ATCC: American Type Culture Collection), murine
fibroblasts NIH3T3 (ATCC No. CRL-1658), and
dihydrofolate reductase-deficient strains (Urlaub, G.
and Chasin, L.A., Proc. Natl. Acad. Sci. USA (1980) 77,
pp. 4126-4220) of Chinese hamster ovarian cells (CHO
cells; ATCC: CCL-61); and the like are often used,
however, the cells are not limited thereto.
The thus obtained transformant can be cultured
according to a method usually carried out in the art,
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Atty. Dkt. No. 111119-0105
and by the culturing of the transformant, a target
polypeptide is produced intracellularly or
extracellularly.
A suitable medium to be used for the culturing can
be selected
depending on
from various commonly used
the employed host cells.
culture media
If Escherichia
coli is employed,
supplemented with an
for example, an LB medium
antibiotic such as ampicillin or
IPMG as needed can be used.
A recombinant protein produced intracellularly or
extracellularly by the trans formant through such
culturing can be separated and purified by any of
various known separation methods utilizing the physical
or chemical property of the protein.
Specific examples of the methods include treatment
with a common protein precipitant, ultrafiltration,
various types of liquid chromatography such as
molecular sieve chromatography (gel filtration),
adsorption chromatography, ion exchange chromatography,
and affinity chromatography, dialysis, and a
combination thereof.
Further, by attaching a tag of six histidine
residues to a recombinant protein to be expressed, the
protein can be efficiently purified with a nickel
affinity column. Alternatively, by attaching the IgG
Fe region to a recombinant protein to be expressed, the
protein can be efficiently purified with a protein A
column.
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By combining the above-described methods, a large
amount of a target polypeptide can be easily produced
in high yield and high purity.
The above-described transformant itself may be
used as the antigen. A cell line expressing HER2 may
also be used as the antigen. Examples of such a cell
line can include human breast cancer lines SK-BR-3, BT-
474, KPL-4, and JIMT-1, a human gastric cancer line
NCI-N87, and a human ovarian cancer line SK-OV-3. The
cell line of the present invention is not limited to
these cell lines as long as it expresses HER2.
[0049]
(2) Production of anti-HER2 monoclonal antibody
Examples of the antibody specifically bind to HER2
include a monoclonal antibody specifically bind to HER2,
and a method of obtaining such antibody is as described
below.
The production of a monoclonal antibody generally
requires the following operational steps of:
(a) purifying a biopolymer to be used as an
antigen, or preparing antigen-expressing cells;
(b) preparing antibody-producing cells by
immunizing an animal by injection of the antigen,
collecting the blood, assaying its antibody titer to
determine when the spleen is excised;
(c) preparing myeloma cells (hereinafter referred
to as "myeloma") ;
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(d) fusing the antibody-producing cells with the
myeloma;
(e) screening a group of hybridomas producing a
desired antibody;
(f) dividing the hybridomas
clones (cloning);
into single cell
(g) optionally, culturing the hybridoma or rearing
an animal implanted with the hybridoma for producing a
large amount of monoclonal antibody;
(h) examining the thus produced monoclonal
antibody for biological activity and binding
specificity, or assaying the same for properties as a
labeled reagent; and the like.
Hereinafter, the method of producing a monoclonal
antibody will be described in detail following the
above steps, however, the method is not limited thereto,
and, for example, antibody-producing cells other than
spleen cells and myeloma can be used.
[0050]
(a) Purification of antigen
As the antigen, HER2 prepared by the method as
described above or a partial peptide thereof can be
used.
Further, a membrane fraction prepared from
recombinant cells expressing HER2 the recombinant
cells expressing HER2 themselves,
peptide of the protein of the
- 52 -
4833-0752-7733.1
or
and also
invention
a partial
chemically
Atty. Dkt. No. 111119-0105
synthesized by a method known to those skilled in the
art can also be used as the antigen.
Furthermore, a HER2-expressing cell line can also
be used as the antigen.
[0051]
(b) Preparation of antibody-producing cells
The antigen obtained in the step (a) is mixed with
an adj uvant such as Freund' s complete or incomplete
adjuvant or auxiliary agent such as aluminum potassium
sulfate and the resulting mixture is used as an
immunogen to immunize an experimental animal. Another
method involves immunizing an experimental animal with
antigen-expressing cells as an immunogen. As the
experimental animal, any animal used in a known
hybridoma production method can be used without
hindrance. Specifically, for example, a mouse, a rat,
a goat, sheep, cattle, a horse, or the like can be used.
However, from the viewpoint of ease of availability of
myeloma cells to be fused with the extracted antibodyproducing
cells, a mouse or a rat is preferably used as
the animal to be immunized.
Further, the strain of a mouse or a rat to be used
is not particularly limited, and in the case of a mouse,
for example, various strains such as A, AKR, BALB/c,
BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA, FL, HTH, HTl,
LP, NZB, NZW, RF, R III, SJL, SWR, WB, and 129 and the
like can be used, and in the case of a rat, for example,
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Wistar, Low, Lewis, Sprague, Dawley, ACI, BN, Fischer
and the like can be used.
These mice and rats are commercially available
from breeders/distributors of experimental animals, for
example, CLEA Japan, Inc. and Charles River
Laboratories Japan, Inc.
As the animal to be immunized, in consideration of
compatibility of fusing with myeloma cells described
below, in the case of a mouse, BALB/ c strain, and in
the case of a rat, Wistar and Low strains are
particularly preferred.
Further, in consideration of antigenic homology
between humans and mice, it is also preferred to use a
mouse having decreased biological function to remove
auto-antibodies, that is, a mouse with an autoimmune
disease.
The age of such mouse or rat at the time of
immunization is preferably 5 to 12 weeks of age, more
preferably 6 to 8 weeks of age.
In order to immunize an animal with HER2 or a
recombinant thereof,
described in detail
for
in,
example, a
for example,
known
Weir,
method
D. M.,
Handbook of Experimental Immunology Vol. I. I I. I I I. ,
Blackwell Scientific Publications, Oxford (1987); Kabat,
E. A. and Mayer, M. M., Experimental Immunochemistry,
Charles C Thomas Publisher Springfield, Illinois (1964)
or the like can be used.
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Among these
specific method
immunization methods, a preferred
in the present invention is, for
example, as follows.
That is, first, a membrane protein fraction
serving as the antigen or cells caused to express the
antigen is/are intradermally or intraperitoneally
administrated to an animal. However, the combination
of both routes of administration is preferred for
increasing
intradermal
the immunization
administration is
efficiency,
performed in
and when
the first
half and intraperitoneal administration is performed in
the latter half or only at the last dosing, the
immunization efficiency can be particularly increased.
The administration schedule of the antigen varies
depending on the type of animal to be immunized,
individual difference or the like. However, in general,
an administration schedule in which the frequency of
administration of the antigen is 3
dosing interval is 2 to 6 weeks is
administration schedule in which
administration of the antigen is 3
to 6 times and the
preferred, and an
the frequency of
to 4 times and the
dosing interval is 2 to 4 weeks is more preferred.
Further, the dose of the antigen varies depending
on the type of animal, individual differences or the
like, however, the dose is generally set to 0.05 to 5
mg, preferably about 0.1 to 0.5 mg.
A booster immunization is performed 1 to 6 weeks,
preferably 1 to 4 weeks, more preferably 1 to 3 weeks
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after the administration of the antigen as described
above. When the immunogen is cells, 1 X 10 6 to 1 X 10 7
cells are used.
The dose of the antigen at the time of performing
the booster immunization varies depending on the type
or size of animal or the like, however, in the case of,
for example, a mouse, the dose is generally set to 0.05
to 5 mg, preferably 0. 1 to 0. 5 mg, more preferably
about 0.1 to 0.2 mg. When the immunogen is cells, 1 X
10 6 to 1 X 10 7 cells are used.
Spleen cells or lymphocytes including antibodyproducing
cells are aseptically from the
immunized animal after 1 to 10 days,
days, more preferably 2 to 3 days
immunization. At this time, the
removed
preferably 2 to 5
from the booster
antibody titer is
measured, and if an animal having a sufficiently
increased antibody titer is used as a supply source of
the antibody-producing cells, the subsequent procedure
can be carried out more efficiently.
Examples of the method of measuring the antibody
titer to be used here include an RIA method and an
ELISA method, but the method is not limited thereto.
For example, if an ELISA method is employed, the
measurement of the antibody titer in the invention can
be carried out according to the procedures as described
below.
First, a purified or partially purified antigen is
adsorbed to the surface of a solid phase such as a 96-
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well plate for ELISA, and the surface of the solid
phase having no antigen adsorbed thereto is covered
with a protein unrelated to the antigen such as bovine
serum albumin (BSA) . After washing the surface, the
surface is brought into contact with a serially-diluted
sample (for example, mouse serum) as a primary antibody
to allow the antibody in the sample to bind to the
antigen.
Further,
labeled with
added and is
After washing,
a change in
as a secondary antibody, an antibody
an enzyme against a mouse antibody is
allowed to bind to the mouse antibody.
a substrate for the enzyme is added and
absorbance which occurs due to color
development induced by degradation of the substrate or
the like is measured and the antibody titer is
calculated based on the measurement.
The separation of the antibody-producing cells
from the spleen cells or lymphocytes of the immunized
animal can be carried out according to a known method
(for example, Kohler et al., Nature (1975), 256, p.
495; Kohler et al., Eur. J. Immunol. (1977), 6, p. 511;
Milstein et al., Nature (1977), 266, p. 550; Walsh,
Nature (1977), 266, p. 495). For example, in the case
of spleen cells, a general method in which the
antibody-producing cells are separated by homogenizing
the spleen to obtain the cells through filtration with
a stainless steel mesh and suspending the cells in
Eagle's Minimum Essential Medium (MEM) can be employed.
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[0052]
(c) Preparation of myeloma cells (hereinafter referred
to as "myeloma")
The myeloma cells to be used for cell fusion are
not particularly limited and suitable cells can be
selected from
consideration of
known cell lines. However,
convenience when a hybridoma
in
is
selected from fused cells, it is preferred to use an
HGPRT (hypoxanthine-guanine phosphoribosyl transferase)
deficient strain whose selection procedure has been
established.
More specifically, examples of the HGPRT-deficient
strain include X63-Ag8(X63), NSl-ANS/l(NSl), P3X63-
Ag8.Ul(P3Ul), X63-Ag8.653(X63.653), SP2/0-Ag14(SP2/0),
MPC11-45.6TG1.7(45.6TG), FO, S149/5XXO, and BU.1
derived from mice; 210.RSY3.Ag.1.2.3(Y3) derived from
rats; and U266AR(SKO-007), GM1500·GTG-A12(GM1500),
UC729-6, LICR-LOW-HMy2(HMy2) and 8226AR/NIP4-l(NP41)
derived from humans. These HGPRT-deficient strains are
available from, for example, ATCC or the like.
These cell strains are subcultured in an
appropriate medium such as an 8-azaguanine medium [a
medium obtained by adding 8-azaguanine to an RPMI 1640
medium supplemented with glutamine, 2-mercaptoethanol,
gentamicin, and fetal calf serum (hereinafter referred
to as "FCS")], Iscove's Modified Dulbecco's Medium
(hereinafter referred to as "IMDM"), or Dulbecco's
Modified Eagle Medium (hereinafter referred to as
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"DMEM"). In this case, 3 to 4 days before performing
cell fusion, the cells are subcultured in a normal
medium (for example, an ASF104 medium (manufactured by
Ajinomoto Co., Ltd.) containing 10% FCS) to ensure not
less than 2 x 10 7 cells on the day of cell fusion.
[0053]
(d) Cell fusion
Fusion between the antibody-producing cells and
the myeloma cells can be appropriately performed
according to a known method (Weir, D. M. Handbook of
Experimental Immunology Vol. I. II. III., Blackwell
Scientific Publications, Oxford (1987); Kabat, E. A.
and Mayer, M. M., Experimental Immunochemistry, Charles
C Thomas Publisher, Springfield, Illinois (1964), etc.),
under conditions such that the survival rate of cells
is not excessively reduced.
As such a method, for example, a chemical method
in which the antibody-producing cells and the myeloma
cells are mixed in a solution containing a polymer such
as polyethylene glycol at a high concentration, a
physical method using electric stimulation, or the like
can be used. Among these methods, a specific example
of the chemical method is as described below.
That is, in the case where polyethylene glycol is
used in the solution containing a polymer at a high
concentration, the antibody-producing cells and the
myeloma cells are mixed in a solution of polyethylene
glycol having a molecular weight of 1500 to 6000, more
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preferably 2000 to 4000 at a temperature of from 30 to
4 0°C, preferably from 3 5 to 3 8°C for 1 to 10 minutes,
preferably 5 to 8 minutes.
[0054]
(e) Selection of a group of hybridomas
The method of selecting hybridomas obtained by the
above-described cell fusion is not particularly limited.
Usually, an HAT (hypoxanthine, aminopterin, thymidine)
selection method (Kohler et al., Nature (1975), 256, p.
495; Milstein et al., Nature (1977), 266, p. 550) is
used.
This method is effective when hybridomas are
obtained using the myeloma cells of an HGPRT-deficient
strain which cannot survive in the presence of
aminopterin. That is, by culturing unfused cells and
hybridomas in an HAT medium, only hybridomas resistant
to aminopterin are selectively allowed to survive and
proliferate.
[0055]
(f) Division into single cell clone (cloning)
As a cloning method for hybridomas, a known method
such as a methylcellulose method, a soft agarose method,
or a limiting dilution method can be used (see, for
example, Barbara, B. M. and Stanley, M. S.: Selected
Methods in Cellular Immunology, W. H. Freeman and
Company, San Francisco (1980)). Among these methods,
particularly, a three-dimensional culture method such
as a methylcellulose method is preferred. For example,
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the group of hybridomas produced by cell fusion are
suspended in a methylcellulose medium such as
ClonaCell-HY Selection Medium D (manufactured by
StemCell Technologies, Inc., #03804) and cultured.
Then, the formed hybridoma colonies are collected,
whereby monoclonal hybridomas can be obtained. The
collected respective hybridoma colonies are cultured,
and a hybridoma which has been confirmed to have a
stable antibody titer in an obtained hybridoma culture
supernatant is selected as a HER2 monoclonal antibodyproducing
hybridoma strain.
[0056]
(g) Preparation of monoclonal antibody by culturing
hybridoma
By culturing the
monoclonal antibody
thus
can be
selected hybridoma, a
efficiently obtained.
However, prior to culturing, it is preferred to perform
screening of a hybridoma which produces a target
monoclonal antibody.
In such screening, a known method can be employed.
The measurement of the antibody titer in the
invention can be carried out by, for example, an ELISA
method explained in item (b)
The hybridoma obtained
above can be stored in a
described above.
by the method
frozen state
nitrogen or in a freezer at -80°C or below.
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described
in liquid
Atty. Dkt. No. 111119-0105
After completion of cloning, the medium is changed
from an HT medium to a normal medium, and the hybridoma
is cultured.
Large-scale
culture using a
culture is
large culture
performed
bottle
by
or by
rotation
spinner
culture. From the supernatant obtained by the largescale
culture, a monoclonal antibody which specifically
binds to the protein of the invention can be obtained
by purification using a method known to those skilled
in the art such as gel filtration.
Further, the hybridoma is injected into the
abdominal cavity of a mouse of the same strain as the
hybridoma (for example, the above-described BALB/c) or
a Nu/Nu mouse to proliferate the hybridoma, whereby the
ascites containing a large amount of the monoclonal
antibody of the invention can be obtained.
In the case where the hybridoma is administrated
in the abdominal cavity, if a mineral oil such as
2,6,10,14-tetramethyl pentadecane (pristane) is
administrated 3 to 7 days prior thereto, a larger
amount of the ascites can be obtained.
For example, an immunosuppressant is previously
injected into the abdominal cavity of a mouse of the
same strain as the hybridoma to inactivate T cells. 20
days thereafter,
suspended in a
10 6 to 10 7 hybridoma clone cells
serum-free medium (0.5 ml), and
are
the
suspension is administrated in the abdominal cavity of
the mouse. In general, when the abdomen is expanded
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and filled with the
from the mouse.
ascites,
By this
Atty. Dkt. No. 111119-0105
the ascites
method, the
is collected
monoclonal
antibody can be obtained at a concentration which is
about 100 times or much higher than that in the culture
solution.
The monoclonal antibody obtained by the abovedescribed
method can be purified by a method described
in, for example, Weir, D. M.: Handbook of Experimental
Immunology Vol. I, II, III, Blackwell Scientific
Publications, Oxford (1978)
The thus obtained monoclonal antibody has high
antigen specificity for HER2. Examples of the
monoclonal antibody of the present invention can
include, but are not particularly limited to, a mouse
monoclonal antibody 4D5 (ATCC CRL 10463).
[0057]
(h) Assay of monoclonal antibody
The isotype and subclass of the thus obtained
monoclonal antibody can be determined as follows.
First, examples of the identification method
include an Ouchterlony method, an ELISA method, and an
RIA method.
An Ouchterlony method is simple, but when the
concentration of the monoclonal antibody is low, a
condensation operation is required.
On the other hand, when an ELISA method or an RIA
method is used, by directly reacting the culture
supernatant with an antigen-adsorbed solid phase and
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using antibodies corresponding to various types of
immunoglobulin isotypes and subclasses as secondary
antibodies, the isotype and subclass of the monoclonal
antibody can be identified.
In addition, as a simpler method, a commercially
available identification kit (for example, Mouse Typer
Kit manufactured by Bio-Rad Laboratories, Inc.) or the
like can also be used.
Further, the quantitative determination of a
protein can be performed by the Folin Lowry method and
a method of calculation based on the absorbance at 280
nm (1.4 (OD 280) = Immunoglobulin 1 mg/ml)
Further, even when the monoclonal antibody is
separately and independently obtained by performing
again the steps of (a) to (h) in (2), it is possible to
obtain an antibody having a cytotoxic activity
equivalent to that of the HER2 antibody obtained in the
the step of (g). As one example of such an antibody,
an antibody which binds to the same epitope as the HER2
antibody obtained in the step of (g) can be exemplified.
If a newly produced monoclonal antibody binds to a
partial peptide or a partial tertiary structure to
which the anti-HER2 antibody binds, it can be
determined that the monoclonal antibody binds to the
same epi tope as the anti-HER2 antibody. Further, by
confirming that the monoclonal antibody competes with
the anti-HER2 antibody for the binding to HER2 (that is,
the monoclonal antibody inhibits the binding between
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the anti-HER2 antibody and HER2), it can be determined
that the monoclonal antibody binds to the same epitope
as the anti-HER2 antibody even if the specific epitope
sequence or structure has not been determined. When it
is confirmed that the monoclonal antibody binds to the
same epitope as the anti-HER2 antibody, the monoclonal
antibody is strongly expected to have an antigenbinding
affinity or biological activity equivalent to
that of the anti-HER2 antibody.
[0058]
(3) Other antibodies
The antibody of the invention includes not only
the above-described monoclonal antibody against HER2
but also a recombinant antibody obtained by artificial
modification for the purpose of decreasing heterologous
antigenicity to humans such
humanized antibody and a
as a chimeric antibody, a
human antibody. These
antibodies can be produced using a known method.
As the chimeric antibody, an antibody in which
antibody variable and constant regions are derived from
different species, for example, a chimeric antibody in
which a mouse- or rat-derived antibody variable region
is connected to a human-derived antibody constant
region can be exemplified (see Proc. Natl. Acad. Sci.
USA, 81, 6851-6855, (1984)). Examples of the chimeric
antibody of the present invention can include, but are
not particularly limited to, a chimeric antibody 4D5
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comprising a heavy chain constant region of human IgGl
or IgG2.
As the humanized antibody, an antibody obtained by
integrating only a complementarity determining region
(CDR) into a human-derived antibody (see Nature (1986)
321, pp. 522-525), and an antibody obtained by grafting
a part of the amino acid residues of the framework as
well as the CDR sequence to a human antibody by a CDRgrafting
method (WO 90/07861), and an antibody
humanized using gene conversion mutagenesis strategy
(U.S. Patent No. 5821337) can be exemplified.
[0059]
The term "several" as used herein refers to 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 or 2.
[0060]
As the amino acid substitution in this
specification, a conservative amino acid substitution
is preferred. The conservative amino acid substitution
refers
amino
to a
acids
substitution
related to
occurring within a
amino acid side
group of
chains.
Preferred amino acid groups are as follows: an acidic
group (aspartic acid and glutamic acid); a basic group
(lysine, arginine, and histidine); a non-polar group
(alanine, valine, leucine,
phenylalanine, methionine, and
uncharged polar family (glycine,
cysteine, serine, threonine,
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isoleucine,
tryptophan)
praline,
and an
asparagine, glutamine,
and tyrosine). More
Atty. Dkt. No. 111119-0105
preferred amino acid groups are as follows: an
aliphatic hydroxy group (serine and threonine); an
amide-containing group (asparagine and glutamine) an
aliphatic group (alanine, valine, leucine, and
isoleucine); and an aromatic group (phenylalanine,
tryptophan, and tyrosine). Such an amino acid
substitution is preferably performed within a range
which does not impair the properties of a substance
having the original amino acid sequence.
[0061]
with
By combining a sequence having a
the above-described heavy chain
high homology
amino acid
sequence with a sequence having a high homology with
the above-described light chain amino acid sequence, it
is possible to select an antibody having a biological
activity equivalent to that of each of the abovedescribed
antibodies. Such a homology is generally a
homology of 80% or more, preferably a homology of 90%
or more, more preferably a homology of 95% or more,
most preferably a homology of 99% or more. Further, by
combining an amino acid sequence wherein one to several
amino acid residues are substituted, deleted or added
in the heavy chain or light chain amino acid sequence,
it is also possible to select an antibody having a
biological activity equivalent to that of each of the
above-described antibodies. The term "homology" as
used herein is used with the same meaning as "identity".
[0062]
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The homology between two amino acid sequences can
be determined using default parameters of Blast
algorithm version 2.2.2 (Altschul, Stephen F., Thomas L.
Madden, Alejandro A. Schaeffer, Jinghui Zhang, Zheng
Zhang, Webb Miller, and David J. Lipman (1997), "Gapped
BLAST and PSI-BLAST: a new generation of protein
database search programs'', Nucleic Acids Res. 25: 3389-
3402) The Blast algorithm can be used also through
the Internet by accessing the site
www.ncbi.nlm.nih.gov/blast.
[0063]
Further, the antibody of the invention includes a
human antibody which binds to HER2. An anti-HER2 human
antibody refers to a human antibody having only a
sequence of an antibody derived from a human chromosome.
The anti-HER2 human antibody can be obtained by a
method using a human antibody-producing mouse having a
human chromosome fragment comprising heavy and light
chain genes of a human antibody (see Tomizuka, K. et
al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y.
et al., Nucl. Acids Res. (1998) 26, pp. 3447-3448;
Yoshida, H. et al., Animal Cell Technology: Basic and
Applied Aspects vol. 10, pp. 69-73 (Kitagawa, Y.,
Matuda, T. and Iijima, s. eds.), Kluwer Academic
Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad.
Sci. USA (2000) 97, pp. 722-727, etc.).
[0064]
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Such a human antibody-producing
created specifically as follows.
mouse can be
A genetically
modified animal in which endogenous immunoglobulin
heavy and light chain gene loci have been disrupted,
and instead, human immunoglobulin heavy and light chain
gene loci have been introduced via a yeast artificial
chromosome (YAC) vector or the like is created by
producing a knockout animal and a transgenic animal and
mating these animals.
Further, according to a recombinant DNA technique,
by using cDNAs encoding each of such a heavy chain and
a light chain of a human antibody, and preferably a
vector comprising such cDNAs,
transformed, and a transformant
recombinant human monoclonal
whereby the antibody can also
culture supernatant.
eukaryotic cells are
cell which produces a
antibody is cultured,
be obtained from the
Here, as the host, for example,
preferably mammalian cells such
eukaryotic cells,
as CHO cells,
lymphocytes, or myeloma cells can be used.
[0065]
Further, a method of obtaining a phage displayderived
human antibody selected from a human antibody
library (see Wormstone, I. M. et al., Investigative
Ophthalmology & Visual Science. (2002) 43 (7), pp.
2301-2308; Carmen, S. et al., Briefings in Functional
Genomics and Proteomics (2002), 1 (2), pp. 189-203;
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Siriwardena, D. et al., Ophthalmology (2002) 109 (3),
pp. 427-431, etc.) is also known.
For example, a phage display method in which a
variable region of a human antibody is expressed on the
surface of a phage as a single-chain antibody (scFv),
and a phage which binds to an antigen is selected
(Nature Biotechnology (2005), 23, (9), pp. 1105-1116)
can be used.
By analyzing the gene of the phage selected based
on the binding to an antigen, a DNA sequence encoding
the variable region of a human antibody which binds to
an antigen can be determined.
If the DNA sequence of scFv which binds to an
antigen is determined, a human antibody can be obtained
by preparing an expression vector comprising the
sequence and introducing the vector into an appropriate
host to express it (WO 92/01047, WO 92/20791, WO
93/06213, WO 93/11236, WO 93/19172,
95/15388; Annu. Rev. Immunol. (1994)
WO 95/01438, WO
12, pp. 4 3 3 - 4 5 5,
Nature Biotechnology (2005) 23 (9), pp. 1105-1116).
[0066]
As one example of another index for use in the
comparison of the properties of antibodies, the
stability of antibodies can be exemplified. The
differential scanning calorimetry (DSC) is a device
capable of quickly and accurately measuring a thermal
denaturation midpoint temperature (Tm) to be used as a
favorable index of the relative conformational
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stability of proteins. By measuring the Tm values
using DSC and comparing the values, a difference in
thermal stability can be compared. It is known that
the storage stability of antibodies shows some
correlation with the thermal stability of antibodies
(Lori Burton, et. al., Pharmaceutical Development and
Technology (2007) 12, pp. 265-273), and a preferred
antibody can be selected by using thermal stability as
an index. Examples of other indices for selecting
antibodies include the following features: the yield in
an appropriate host cell is high; and the aggregability
in an aqueous solution is low. For example, an
antibody which shows the highest yield does not always
show the highest thermal stability, and therefore, it
is necessary to select an antibody most suitable for
the administration to humans by making comprehensive
evaluation based on the above-described indices.
[0067]
In the present invention, a modified variant of
the antibody is also included. The modified variant
refers to a variant obtained by subjecting the antibody
of the present invention to chemical or biological
modification. Examples of the chemically modified
variant include variants chemically modified by linking
a chemical moiety to an amino acid skeleton, variants
chemically modified with an N-linked or 0-linked
carbohydrate chain, etc. Examples of the biologically
modified variant include variants obtained by post-
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translational modification (such as N-linked or 0-
linked glycosylation, N- or C-terminal processing,
deamidation, isomerization of aspartic acid, or
oxidation of methionine), and variants in which a
methionine residue has been added to the N terminus by
being expressed in a prokaryotic host cell. Further,
an antibody labeled so as to enable the detection or
isolation of the antibody or an antigen of the
invention, for example, an enzyme-labeled antibody, a
fluorescence-labeled antibody, and an affinity-labeled
antibody are also included in the meaning of the
modified variant. Such a modified variant of the
antibody of the invention is useful for improving the
stability and blood retention of the antibody, reducing
the antigenicity thereof, detecting or isolating an
antibody or an antigen, and so on.
[0068]
Further, by regulating the modification of a
glycan which is linked to the antibody of the invention
(glycosylation, defucosylation, etc.), it is possible
to enhance an antibody-dependent cellular cytotoxic
activity. As the technique for regulating the
modification of a glycan of antibodies, WO 99/54342, WO
00/61739, WO 02/31140, etc. are known. However, the
technique is not limited thereto. In the antibody of
the present invention, an antibody in which the
modification of a glycan is regulated is also included.
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In the case where an antibody is produced by first
isolating an antibody gene and then introducing the
gene into an appropriate host, a combination of an
appropriate host and an appropriate expression vector
can be used. Specific examples of the antibody gene
include a combination of a gene encoding a heavy chain
sequence of an antibody described in this specification
and a gene encoding a light chain sequence thereof.
When a host cell is transformed, it is possible to
insert the heavy chain sequence gene and the light
chain sequence gene into the same expression vector,
and also into different expression vectors separately.
In the case where eukaryotic cells are used as the
host, animal cells, plant cells, and eukaryotic
microorganisms can be used. As the animal cells,
mammalian cells, for example, simian COS cells (Gluzman,
Y., Cell, (1981)
murine fibroblasts
23, pp.
NIH3T3
175-182,
(ATCC No.
ATCC CRL-1650),
CRL -16 5 8) , and
dihydrofolate reductase-deficient strains (Urlaub, G.
and Chasin, L.A., Proc. Natl. Acad. Sci. USA (1980) 77,
pp. 4126-4220) of Chinese hamster ovarian cells (CHO
cells; ATCC: CCL-61) can be exemplified.
In the case where prokaryotic cells are used, for
example, Escherichia coli and Bacillus subtilis can be
exemplified.
By introducing a desired antibody gene into these
cells through transformation, and culturing the thus
transformed cells in vitro, the antibody can be
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obtained. In the above-described culture method, the
yield may sometimes vary depending on the sequence of
the antibody, and therefore, it is possible to select
an antibody which is easily produced as a
pharmaceutical by using the yield as an index among the
antibodies having an equivalent binding activity.
Therefore, in the antibody of the present invention, an
antibody obtained by a method of producing an antibody,
characterized by including a step of culturing the
transformed host cell and a step of collecting a
desired antibody from a cultured product obtained in
the culturing step is also included.
[0069]
It is known that a lysine residue at the carboxyl
terminus of the heavy chain of an antibody produced in
a cultured mammalian cell is deleted (Journal of
Chromatography A, 705: 129-134 (1995)), and it is also
known that two amino acid residues (glycine and lysine)
heavy chain of
mammalian cell
at the carboxyl terminus of the
antibody produced in a cultured
deleted and a praline residue newly located at
an
are
the
carboxyl terminus is amidated (Analytical Biochemistry,
360: 75-83 (2007)) However, such deletion and
modification of the heavy chain sequence do not affect
the antigen-binding affinity and the effector function
(the activation of a complement, the antibody-dependent
cellular cytotoxicity, etc.) of the antibody.
Therefore, in the antibody according to the present
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invention, an antibody subjected to such modification
and a functional fragment of the antibody is also
included, and a deletion variant in which one or two
amino acids have been deleted at the carboxyl terminus
of the heavy chain, a variant obtained by amidation of
the deletion variant ( for example, a heavy chain in
which the carboxyl terminal praline residue has been
amidated), and the like are also included. The type of
deletion variant having a deletion at the carboxyl
terminus of the heavy chain of the antibody according
to the invention is not limited to the above variants
as long as the antigen-binding affinity and the
effector function are conserved. The two heavy chains
constituting the antibody according to the invention
may be of one type selected from the group consisting
of a full-length heavy chain and the above-described
deletion variant, or may be of two types in combination
selected therefrom. The ratio of the amount of each
deletion variant can be affected by the type of
cultured mammalian cells which produce the antibody
according to the invention and the culture conditions,
however, a case where one amino acid residue at the
carboxyl terminus has been deleted in both of the two
heavy chains contained as main components in the
antibody according to the invention can be exemplified.
[0070]
As isotype of the antibody of the invention, for
example, IgG ( IgGl, IgG2, IgG3, IgG4) can be
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exemplified,
preferably.
[0071]
and IgGl or IgG2 can be exemplified
As the biological activity of the antibody,
generally an antigen-binding activity, an activity of
internalizing in cells expressing an antigen by binding
to the antigen, an activity of neutralizing the
activity of an antigen, an activity of enhancing the
activity of an antigen, an antibody-dependent cellular
cytotoxicity (ADCC) activity, a complement-dependent
cytotoxicity (CDC) activity, and an antibody-dependent
cell-mediated phagocytosis (ADCP) can be exemplified.
The biological activity of the antibody of the present
invention is a binding activity to HER2, and preferably
an activity of internalizing in HER2-expressing cells
by binding to HER2. Further, the antibody of the
present invention may have an ADCC activity, a CDC
activity, and/ or an ADCP activity in addition to an
activity of internalizing in cells.
[0072]
The obtained
homogeneity. The
antibody may be
antibody can be purified to
separation and purification of the
performed employing a conventional
protein separation and purification method. For
example, the antibody can be separated and purified by
appropriately
chromatography,
selecting
filter
salt precipitation,
and combining column
filtration,
dialysis,
ultrafiltration,
preparative
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polyacrylamide gel electrophoresis, isoelectric
focusing electrophoresis, and the like (Strategies for
Protein Purification and Characterization: A Laboratory
Course Manual, Daniel R. Marshak et al. eds., Cold
Spring Harbor Laboratory Press (1996) Antibodies: A
Laboratory Manual. Ed Harlow and David Lane, Cold
Spring Harbor Laboratory (1988)), but the method is not
limited thereto.
Examples of
chromatography,
hydrophobic
chromatography,
such chromatography include affinity
ion exchange chromatography,
chromatography, gel filtration
reverse phase chromatography, and
adsorption chromatography.
Such chromatography can be performed employing
liquid chromatography such as HPLC or FPLC.
As a column to be used in affinity chromatography,
a Protein A column and a Protein G column can be
exemplified. For example, as a column using a Protein
A column, Hyper D, POROS, Sepharose FF (Pharmacia
Corporation) and the like can be exemplified.
Further, by using a carrier having an antigen
immobilized thereon, the antibody can also be purified
utilizing the binding property of the antibody to the
antigen.
[0073]
[Antitumor compound]
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The antitumor compound to be conjugated to the
anti-HER2 antibody-drug conjugate of the present
invention is explained. The antitumor compound used in
the present invention is not particularly limited
provided it is a compound having an antitumor effect
and a substituent group or a partial structure allowing
connection to a linker structure. When a part or whole
linker is cleaved in tumor cells, the antitumor
compound moiety is released to exhibit the anti tumor
effect of the anti tumor compound. As the linker is
cleaved at a connecting position to the drug, the
anti tumor compound is released in an unmodified
structure to exhibit its intrinsic anti tumor effect.
As the anti tumor compound used in the present
invention, exatecan (((1S,9S)-1-amino-9-ethyl-5-fluoro-
2,3-dihydro-9-hydroxy-4-methyl-1H,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinoline-
10,13(9H,15H)-dione; shown in the following formula),
one of the camptothecin derivatives, can be preferably
used.
[0074]
[Formula 14]
Me
F
[0075]
4833-0752-7733.1
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Atty. Dkt. No. 111119-0105
Although having an excellent antitumor effect, exatecan
has not been commercialized as an antitumor drug. The
compound can be easily obtained by a known method and
the amino group at position 1 can be preferably used as
the connecting position to the linker structure.
Further, although exatecan can be also released in
tumor cells while part of the linker is still attached
thereto, it is an excellent compound exhibiting an
excellent antitumor effect even in such a structure.
Because exatecan has a camptothecin structure, it
is known that the equilibrium shifts to a structure
with a closed lactone ring (closed ring) in an aqueous
acidic medium (for example, pH 3 or so) but it shifts
to a structure with an open lactone ring (open ring) in
an aqueous basic medium (for example, pH 10 or so). A
drug conjugate being introduced with an exatecan
residue corresponding to the closed ring structure and
the open ring structure is also expected to have the
same antitumor effect and it is needless to say that
any of these structures is within the scope of the
present invention.
[0076]
Further examples of the antitumor compound can
include
bleomycin,
doxorubicin, daunorubicin,
cyclocytidine, vincristine,
mitomycin C,
vinblastine,
methotrexate, platinum-based antitumor agent (cisplatin
or derivatives thereof), taxol or derivatives thereof,
and other camptothecins or derivatives thereof
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(antitumor agent described in Japanese Patent Laid-Open
No. 6-87746)
[0077]
With regard to the antibody-drug conjugate, the
number of conjugated drug molecules per antibody
molecule is a key factor having an influence on
efficacy and safety. Production of the antibody-drug
conjugate is performed by defining the reaction
conditions including the amounts of raw materials and
reagents used for the reaction so as to have a constant
number of conjugated drug molecules. A mixture
containing
molecules
different
is generally
numbers
obtained
of conjugated drug
unlike the chemical
reaction of a low-molecular-weight compound. The
number of drugs conjugated in an antibody molecule is
expressed or specified by the average value, that is,
the average number of conjugated drug molecules.
Unless specifically described otherwise as a principle,
the number of conjugated drug molecules means the
average value except in the case in which it represents
an antibody-drug conjugate having a specific number of
conjugated drug molecules that is included in an
antibody-drug conjugate mixture having different
numbers of conjugated drug molecules.
The number of exatecan molecules conjugated to an
antibody molecule is controllable, and as the average
number of conjugated drug molecules per antibody
molecule, about 1 to 10 exatecans can be connected.
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Preferably, it is 2 to 8, and more preferably 3 to 8.
Meanwhile, a person skilled in the art can design a
reaction for conjugating a required number of drug
molecules to an antibody molecule based on the
description of the Examples of the present application
and can obtain an antibody-drug conjugate conjugated
with a controlled number of exatecan molecules.
[0078]
[Linker structure]
With regard
conjugate of the
to the
present
anti-HER2
invention,
antibody-drug
the linker
structure for conjugating an antitumor compound to the
anti-HER2 antibody is explained. The linker has a
structure of the following formula:
-L1 -L2-LP-NH- (CH2)n1 -La-(CH2)n2-C(=0) -.
The antibody is connected to the terminal L1 (terminal
opposite to the connection to L2
), and the anti tumor
compound is connected to the carbonyl group of the -La(
CH2)n2-C(=0)- moiety.
n1 represents an integer of O to 6 and is
preferably an integer of 1 to 5, and more preferably 1
to 3.
[0079]
1. Ll
L1 is represented by the following structure:
-(Succinimid-3-yl-N)-(CH2)n3 -C(=0)-.
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In the above, n 3 is an integer of 2 to 8, "
(Succinimid-3-yl-N) - " has a structure represented by
the following formula:
[0080]
[Formula 15]
[0081]
Position 3 of the above partial structure is the
connecting position to the anti-HER2 antibody. The
bond to the antibody at position 3 is characterized by
bonding with thioether formation. The nitrogen atom at
position 1 of the structure moiety is connected to the
carbon atom of the methylene which is present within
the linker including the structure.
(Succinimid-3-yl-N)-(CH2 )n3 -C(=O)-L2
-
represented by the following
Specifically,
is a
formula
structure
(herein,
"antibody-8-" originates from an antibody).
[0082]
[Formula 16]
0
Antibody -s~
~N-(CH2)n 3-C(=O)-L2-
0
[0083]
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Atty. Dkt. No. 111119-0105
In the formula, n 3 is an integer of 2 to 8, and
preferably 2 to 5.
[0084]
Specific examples of L1 can include
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-,
-(Succinimid-3-yl-N)-CH2CH2CH2-C(=O)-,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2-C(=O)-, and
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -.
[0085]
2. L2
L2 is represented by the following structure:
-NH-(CH2CH2-0)n4 -CH2CH2-C(=0) -,
L 2 may not be present, and in such a case, L 2 is a
single bond. n 4 is an integer of 1 to 6, and
preferably 2 to 4. L 2 is connected to L 1 at its
terminal amino group and is connected to LP at its
carbonyl group at the other terminal.
[0086]
Specific examples of L2 can include
-NH-CH2CH2-0-CH2CH2-C(=0) -,
-NH-CH2CH2-0-CH2CH2-0-CH2CH2-C(=0) -,
-NH-CH2CH2-0-CH2CH2-0-CH2CH2-0-CH2CH2-C(=0) -,
-NH-CH2CH2-0-CH2CH2-0-CH2CH2-0-CH2CH2-0-CH2CH2-C(=O) -,
-NH-CH2CH2-0-CH2CH2-0-CH2CH2-0-CH2CH2-0-CH2CH2-0-CH2CH2-
C(=O)-,
-NH-CH2CH2-0-CH2CH2-0-CH2CH2-0-CH2CH2-0-CH2CH2-0-CH2CH2-0-
CH2CHrC (=0) - .
[0087]
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3. LP
LP is a peptide residue consisting of 2 to 7 amino
acids. Specifically, it consists of an oligopeptide
residue in which 2 to 7 amino acids are linked by
peptide bonding. LP is connected to L2 at its N
terminus and is connected to the amino group of the -
NH-(CH2 )n1 -La-(CH2 )n2 -C(=0)- moiety of the linker at its
C terminus. Here, the term "peptide residue" or
"oligopeptide residue" is a group derived from a
peptide consisting of two or more amino acid residues
and refers to a divalent group whose N terminus and C
terminus are connecting positions.
[0088]
The amino acid cons ti tu ting LP in the linker is
not particularly limited, however, examples thereof
include an L- or a D-amino acid, preferably an L-amino
acid. And, it can be an amino acid having a structure
such as P-alanine,
aminobutyric acid in
£-aminocaproic
addition to an
acid,
a-amino
or yacid,
further, it can be a non-natural type amino acid such
as N-methylated amino acid.
The amino acid sequence of LP is not particularly
limited, but examples of the constituting amino acids
include phenylalanine (Phe; F), tyrosine (Tyr; Y),
leucine (Leu; L), glycine (Gly; G), alanine (Ala; A),
valine (Val; V), lysine (Lys; K), citrulline (Cit),
serine (Ser; S) glutamic acid (Glu; E), and aspartic
acid (Asp; D) Among them, preferred examples include
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phenylalanine, glycine, valine, lysine, citrulline,
serine, glutamic acid, and aspartic acid. From these
amino acids, LP having a sequence of amino acids
optionally selected with or without overlaps may be
constructed. Depending on the type of the amino acids,
the drug release pattern can be controlled. The number
of amino acids can be between 2 to 7.
[0089]
Specific examples of LP can include
-GGF-,
-DGGF-,
-(D-)D-GGF-,
-EGGF-,
-GGFG-,
-SGGF-,
-KGGF-,
-DGGFG-,
-GGFGG-,
-DDGGFG-,
-KDGGFG-, and
-GGFGGGF-.
In the above, "(D-)D" represents a D-aspartic acid.
Particularly preferred examples of LP for the antibodydrug
conjugate of the present invention can include the
tetrapeptide residue -GGFG-.
[0090]
4. La- (CH2 ) n 2 -C (=0) -
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La in La- (CH2) n 2-C (=0) - is a structure of -0- or a
single bond. n2 is an integer of Oto 5, preferably 0
to 3, and more preferably 0 or 1.
Examples of La- (CH2) n 2-C (=0) -
following structures:
-O-CHrC (=0) - ,
-O-CH2CH2-C(=O)-,
-O-CH2CH2CH2-C(=O) -,
-O-CH2CH2CH2CHrC (=0) - ,
-O-CH2CH2CH2CH2CH2-C(=O) -,
-CHrC (=0) - ,
-CH2CHrC (=0) - ,
-CH2CH2CH2-C(=O) -,
-CH2CH2CH2CH2-C(=O) -,
-CH2CH2CH2CH2CH2-C(=O) -,
-O-C (=0) - .
Among these,
-O-CHrC (=0) - ,
-O-CH2CH2-C(=O)-,
-O-C (=0) - ,
can include the
or a case in which La is a single bond, and n2 is 0 is
preferred.
[0091]
Specific examples of the structure represented by
-NH-(CH2)n1 -La-(CH2)n2-C(=O)- of the linker can include
-NH-CHrC (=0) - ,
-NH-CH2CH2-C(=O)-,
-NH-CH2-O-CH2-C(=O)-,
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4833-0752-7733.1
-NH-CH2 CH2 -O-CH2 -C(=O) -,
-NH-CH2 CH2CH2 -C(=O) -,
-NH-CH2 CH2CH2 CH2 -C(=O) -,
-NH-CH2 CH2CH2 CH2 CH2 -C(=O) -,
-NH-CH2 -O-C(=O)-,
-NH-CH2 CH2 -O-C(=O)-,
-NH-CH2 CH2CH2 -O-C(=O) -,
-NH-CH2 CH2CH2 CH2 -O-C(=O) -.
[0092]
Among these,
-NH-CH2 CH2CH2 -C(=O) -,
-NH-CH2 -O-CH2 -C(=O)-,
-NH-CH2 CH2 -O-CH2 -C(=O) -
are more preferred.
[0093]
Atty. Dkt. No. 111119-0105
In the linker, the chain length of -NH- ( CH2 ) n1 -La(
CH2 ) n2 -C (=0) - is preferably a chain length of 4 to 7
atoms, and more preferably a chain length of 5 or 6
atoms.
[ 0 0 94]
With regard to the
conjugate of the present
transferred to the inside of
anti-HER2
invention,
tumor cells,
antibody-drug
when it is
it has been
suggested that the linker moiety is cleaved and the
drug derivative having a structure represented by NH2 -
(CH2 ) n1 -La- (CH2 ) n2 -C (=0) - (NH-DX) is released to express
an anti tumor action. Examples of the antitumor
derivative exhibiting an antitumor effect by releasing
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Atty. Dkt. No. 111119-0105
from the antibody-drug conjugate of the present
invention include an antitumor derivative having a
structure moiety in which a terminal of the structure
represented by -NH- (CH2 ) n1 -La- (CH2 ) n2 -C (=0) - of the
linker has an amino group, and those particularly
preferred include the following.
NH2 -CH2 CH2 -C(=0) -(NH-DX),
NH2 -CH2 CH2 CH2 -C(=0) -(NH-DX),
NH2 -CH2 -0-CH2 -C(=0) -(NH-DX),
NH2 -CHCH2 -0-CH2 -C(=0)-(NH-DX)
Meanwhile, in case of NHrCHr0-CHrC (=0) - (NH-DX),
it was confirmed that, as the aminal structure
molecule is unstable, it again undergoes a
degradation to release the following
in the
self-
Those compounds can be also
preferably used as a production intermediate of the
antibody-drug conjugate of the present invention.
[0095]
For the antibody-drug conjugate of the present
invention in which exatecan is used as the drug, it is
preferable that the drug-linker structure moiety [-L1
-
L2 -LP -NH- ( CH2 ) n1 -La- ( CH2 ) n2 -C ( =0) - (NH-DX)] having the
following structure is connected to an antibody. The
average conjugated number of said drug-linker structure
moieties per antibody molecule can be 1 to 10.
Preferably, it is 2 to 8, and more preferably 3 to 8.
-(Succinimid-3-yl-N)-CH2 CH2 -C(=0)-GGFG-NH-CH2 CH2 -C(=0)(
NH-DX) ,
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Atty. Dkt. No. 111119-0105
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-
C (=0) - (NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
C(=O) -(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2CHrC
( =0) - (NH-DX) ,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2CH2CH2CH2-
C(=O) -(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2-OCH2-
C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
O-CH2-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
O-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
C(=O) -GGFG-NH-CH2CH2-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
C(=O) -GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2-C(=O) -(NHDX),
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2-O-CH2CH2-OCH2CH2-
O-CH2CH2-O-CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-C(=O) -
(NH-DX) .
Among these, more preferred are the following.
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-
C (=0) - (NH-DX),
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Atty. Dkt. No. 111119-0105
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2CHrC
( =0) - (NH-DX) ,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2-0-
CH2 -C(=0)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
0-CH2-C(=0)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
0-C(=O)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-NH-CH2CH2-0-CH2CH2-0-
CH2CH2-C(=0) -GGFG-NH-CH2CH2CH2-C(=0)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-NH-CH2CH2-0-CH2CH2-0-
CH2CH2-0-CH2CH2-0-CH2CH2-C(=0) -GGFG-NH-CH2CH2CH2-C(=0) -
(NH-DX) .
Particularly preferred are the following.
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2-0-
CH2 -C(=0)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2-
0-CH2-C(=0)-(NH-DX),
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-NH-CH2CH2-0-CH2CH2-0-
CH2CH2-C(=0) -GGFG-NH-CH2CH2CH2-C(=0) -(NH-DX)
[0096]
With regard to the linker structure for
conjugating the anti-HER2 antibody and the drug in the
antibody-drug conjugate of the present invention, the
preferred linker can be constructed by connecting
preferred structures shown for each part of the linker
explained above. As for the linker structure, those
with the following structure can be preferably used.
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Atty. Dkt. No. 111119-0105
Meanwhile, the left terminal of these structures is the
connecting position with the antibody and the right
terminal is the connecting position with the drug.
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-GGFG-NH-CH2CH2-C(=0)-,
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-GGFG-NH-CH2CH2CH2-
C(=0)-,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NHCH2CHrC
(=0) - ,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NHCH2CH2CH2-
C(=0) -,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NHCH2CH2CH2CH2CHrC
(=0) - ,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NH-CH2-0-
CHrC (=0) - ,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NHCH2CH2-
0-CH2-C(=0) -,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NHCH2CH2-
0-C(=0) -,
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-NH-CH2CH2-0-CH2CH2-0-
CH2CH2-C(=0) -GGFG-NH-CH2CH2-C(=0)-,
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-NH-CH2CH2-0-CH2CH2-0-
CH2CH2-C(=0) -GGFG-NH-CH2CH2CH2-C(=0) -,
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-NH-CH2CH2-0-CH2CH2-0-
CH2CH2-0-CH2CH2-0-CH2CH2-C(=0) -GGFG-NH-CH2CH2-C(=0) -,
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-NH-CH2CH2-0-CH2CH2-0-
CH2CH2-0-CH2CH2-0-CH2CH2-C(=0) -GGFG-NH-CH2CH2CH2-C(=0) -.
Among these, more preferred are the following.
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Atty. Dkt. No. 111119-0105
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-GGFG-NH-CH2CH2CH2-
C(=0)-,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NHCH2CH2CH2-
C(=0) -,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NH-CH2-0-
CHrC (=0) - ,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NHCH2CH2-
0-CH2-C(=0) -,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NHCH2CH2-
0-C(=0) -,
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-NH-CH2CH2-0-CH2CH2-0-
CH2CH2-C(=0) -GGFG-NH-CH2CH2CH2-C(=0) -,
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-NH-CH2CH2-0-CH2CH2-0-
CH2CH2-0-CH2CH2-0-CH2CH2-C(=0) -GGFG-NH-CH2CH2CH2-C(=0) -.
Particularly preferred include the following.
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NH-CH2-0-
CHrC (=0) - ,
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=0) -GGFG-NHCH2CH2-
0-CH2-C(=0) -,
-(Succinimid-3-yl-N)-CH2CH2-C(=0)-NH-CH2CH2-0-CH2CH2-0-
CH2CH2-C(=0) -GGFG-NH-CH2CH2CH2-C(=0) -.
[0097]
[Production method]
Next, explanations are given for the
representative method for producing the antibody-drug
conjugate of the present invention or a production
intermediate thereof. Meanwhile, the compounds are
hereinbelow described with the compound number shown in
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Atty. Dkt. No. 111119-0105
each reaction formula. Specifically, they are referred
to as a "compound of the formula (1) ", a "compound (1) ",
or the like. The compounds with numbers other than
those are also described similarly.
[0098]
1. Production method 1
The antibody-drug conjugate represented by the
formula (1) in which the antibody is connected to the
drug-linker structure via thioether can be produced by
the following method, for example.
[0099]
[Formula 1 7]
L 1'-L 2-LP-NH-(CH2)n 1-L a-(CH2)n2-C(=O)-(NH-DX)
2
[0100]
AB
3a
_____ AB-L 1-L2-LP-NH-(CH2)n1-L 8-(CH2)n2-C(=O)-(NH-DX)
1
[In the formula, AB represents an antibody having a
sulfhydryl group, and L 1
' represents L 1 linker structure
in which the linker terminal is a maleimidyl group
(formula shown below)
[0101]
[Formula 18]
-N~
0
[0102]
(in the formula, the nitrogen atom is the connecting
position), and specifically represents a group in which
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the -(Succinimid-3-yl-N)- moiety in -(Succinimid-3-ylN)-(
CH2 )n3 -C(=O)- of L1 is a maleimidyl group. Further,
the -(NH-DX) represents a structure represented by the
following formula:
[0103]
[Formula 19]
Me
F
[0104]
and it represents a group that is derived by removing
one hydrogen atom of the amino group at position 1 of
exatecan.]
[0105]
Further, the compound of the formula ( 1) in the
above reaction formula may be interpreted as a
structure in which one structure moiety corresponding
from the drug to the linker terminal connects to one
antibody. However, it is only a description given for
the sake of convenience, and there are actually many
cases in which a plurality of the structure moieties
are connected to one antibody
applies to the explanation of
described below.
[0106]
- 94 -
4833-0752-7733.1
molecule. The same
the product ion method
Atty. Dkt. No. 111119-0105
The antibody-drug conjugate (1) can be produced by
reacting the compound (2), which is obtainable by the
method described below, with the antibody (3a) having a
sulfhydryl group.
The antibody (3a) having a sulfhydryl group can be
obtained by a method well known in the art (Hermanson,
G.T, Bioconjugate Techniques, pp. 56-136, pp. 456-493,
Academic Press (1996)). Examples include: Traut's
reagent is reacted with the amino group of the
antibody; N-succinimidyl s-acetylthioalkanoates are
reacted with the amino group of the antibody followed
by reaction with hydroxylamine; after reacting with Nsuccinimidyl
3 - (pyridyldi thio) propionate, the antibody
is reacted with a reducing agent; the antibody is
reacted with a reducing agent such as di thiothrei tol,
2-mercaptoethanol, and tris(2-carboxyethyl)phosphine
hydrochloride (TCEP) to reduce the disulfide bond in
the hinge part in the antibody to form a sulfhydryl
group, but it is not limited thereto.
Specifically, using 0.3 to 3 molar equivalents of
TCEP as a reducing agent per disulfide at the hinge
part in the antibody and reacting with the antibody in
a buffer solution containing a chelating agent, the
antibody with partially or completely reduced disulfide
at the hinge part in the antibody can be obtained.
Examples of the chelating agent include ethylenediamine
tetraacetic acid (EDTA)
pentaacetic acid (DTPA).
and diethylenetriamine
It can be used at the
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concentration of 1 mM to 20 mM. Examples of the buffer
solution which may be used include a solution of sodium
phosphate, sodium borate, or sodium acetate.
Specifically, by reacting the antibody with TCEP at 4°C
to 37°C for 1 to 4 hours, the antibody (3a) having a
partially or completely reduced sulfhydryl group can be
obtained.
Meanwhile, by conducting the reaction for adding a
sulfhydryl group to a drug-linker moiety, the druglinker
moiety can be conjugated by a thioether bond.
Using 2 to 20 molar equivalents of the compound
(2) per the antibody (3a) having a sulfhydryl group,
the antibody-drug conjugate (1) in which 2 to 8 drug
molecules are conjugated per antibody molecule can be
produced. Specifically, it is sufficient that the
solution containing the compound (2) dissolved therein
is added to a buffer solution containing the antibody
(3a) having a sulfhydryl group for the reaction.
Herein, examples of the buffer solution which may be
used include sodium acetate solution, sodium phosphate,
and sodium borate. The pH for the reaction is 5 to 9,
and more preferably the reaction is performed near pH 7.
Examples of the solvent for dissolving the compound (2)
include an organic solvent such as dimethyl sulfoxide
(DMSO), dimethylformamide (DMF), dimethyl acetamide
(DMA), and N-methyl-2-pyridone (NMP).
It is sufficient that the organic solvent solution
containing the compound (2) dissolved therein is added
- 96 -
4833-0752-7733.1
at 1 to
antibody
20% v/v to a
(3a) having
Atty. Dkt. No. 111119-0105
buffer solution containing
a sulfhydryl group for
the
the
reaction. The reaction temperature is O to 37°C, more
preferably 10 to 25°C, and the reaction time is 0.5 to
2 hours. The reaction can be terminated by
deactivating the reactivity of unreacted compound (2)
with a thiol-containing reagent. Examples of the
thiol-containing reagent include cysteine and N-acetylL-
cysteine (NAC) More specifically, 1 to 2 molar
equivalents of NAC are added to the compound (2) used
and, by incubating at room temperature for 10 to 30
minutes, the reaction can be terminated.
The produced antibody-drug conjugate ( 1) can,
after concentration, buffer exchange, purification, and
measurement
number of
of antibody concentration and average
conjugated drug molecules per antibody
molecule according to common procedures described below,
be subjected to identification of the antibody-drug
conjugate (1).
[0107]
Common procedure A: Concentration of
of antibody or antibody-drug conjugate
To a Amicon Ultra (50,000 MWCO,
aqueous solution
container, a solution of antibody or
Millipore Co.)
antibody-drug
conjugate was added and the solution of the antibody or
antibody-drug conjugate was concentrated by
centrifugation (centrifuge for 5 to 20 minutes at 2000
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G to 3800 G) using a centrifuge (Allegra X-15R, Beckman
Coulter, Inc.)
[0108]
Common procedure B: Measurement of antibody
concentration
Using a UV detector (Nanodrop 1000, Thermo Fisher
Scientific Inc.), measurement of
concentration was performed according
the antibody
to the method
defined by the manufacturer. At that time, a 280 nm
absorption coefficient different for each antibody was
used ( 1. 3 mLmg- 1 cm- 1 to 1. 8 mLmg- 1 cm- 1
) .
[0109]
Common procedure C-1: Buffer Exchange for antibody
NAP-25 column (Cat. No. 17-0852-02, GE Healthcare
Japan Corporation) using Sephadex G-25 carrier was
equilibrated with phosphate buffer (10 mM, pH 6. 0; it
is referred to as PBS6. 0/EDTA in the specification)
containing sodium chloride ( 13 7 mM) and ethylene
diamine tetraacetic acid (EDTA, 5 mM) according to the
method defined by the manufacturer. Aqueous solution
of the antibody was applied in an amount of 2.5 mL to
single NAP-25 column, and then the fraction (3. 5 mL)
eluted with 3.5 mL of PBS6.0/EDTA was collected. The
resulting fraction was concentrated by the Common
procedure A. After measuring the concentration of the
antibody using the Common procedure B, the antibody
concentration was adjusted to 10 mg/mL using
PBS6.0/EDTA.
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Atty. Dkt. No. 111119-0105
Common procedure C-2: Buffer Exchange for antibody
NAP-25 column (Cat. No. 17-0852-02, GE Healthcare
Japan Corporation) using Sephadex G-25 carrier was
equilibrated with phosphate buffer (50 mM, pH 6.5; it
is referred to as PBS6. 5/EDTA in the specification)
containing sodium chloride (50 mM) and EDTA (2 mM)
according to the method defined by the manufacturer.
Aqueous solution of the antibody was applied in an
amount of 2.5 mL to single NAP-25 column, and then the
fraction (3.5 mL) eluted with 3.5 mL of PBS6.5/EDTA was
collected. The resulting fraction was concentrated by
the Common procedure A. After measuring the
concentration of the antibody using the Common
procedure B, the antibody concentration was adjusted to
20 mg/mL using PBS6.5/EDTA.
[0110]
Common procedure D: Purification of antibody-drug
conjugate
NAP-25 column was equilibrated with any buffer
selected from commercially available phosphate buffer
(PBS7.4, Cat. No. 10010-023, Invitrogen), sodium
phosphate buffer (10 mM, pH 6.0; it is referred to as
PBS6. 0) containing sodium chloride ( 13 7 mM) , and
acetate buffer containing sorbitol (5%) (10 mM, pH 5. 5;
it is referred to as ABS in the specification).
Aqueous solution of the antibody-drug conjugate
reaction was applied in an amount of about 1. 5 mL to
the NAP-25 column, and then eluted with the buffer in
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an amount defined by the manufacturer to collect the
antibody fraction. The collected fraction was again
applied to the NAP-25 column and, by repeating 2 to 3
times in total the gel filtration purification process
for eluting with buffer, the antibody-drug conjugate
excluding non-conjugated drug linker and a lowmolecular-
weight compound (tris(2-
carboxyethyl) phosphine hydrochloride (TCEP) , N-acetylL-
cysteine (NAC), and dimethyl sulfoxide) was obtained.
[0111]
Common procedure E: Measurement
concentration in antibody-drug conjugate
number of conjugated drug molecules
molecule (1)
of antibody
and average
per antibody
The conjugated drug concentration in the antibodydrug
conjugate can be calculated by measuring UV
absorbance of an aqueous solution of the antibody-drug
conjugate at two wavelengths of 280 nm and 370 nm,
followed by performing the calculation shown below.
Because the total absorbance at any wavelength is
equal to the sum of the absorbance of every lightabsorbing
chemical species that are present in the
system (additivity of absorbance), when the molar
absorption coefficients of the antibody and the drug
remain the same before and after conjugation between
the antibody and the drug, the antibody concentration
and the drug concentration in the antibody-drug
conjugate are expressed by the following equations.
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4833-0752-7733.1
Atty. Dkt. No. 111119-0105
AD,280 + AA,280 = ED,280CD + EA,280CA
AD, 370+ AA, 370 = ED, 370CD + EA, 370CA
Equation (I)
Equation (II)
In the above, A230 represents the absorbance of an
aqueous solution of the antibody-drug conjugate at 280
nm, A370 represents the absorbance of an aqueous
solution of the antibody-drug conjugate at 370 nm,
AA,280 represents the absorbance of an antibody at 280
nm, AA,37o represents the absorbance of an antibody at
370 nm, AD,280 represents the absorbance of a conjugate
precursor at 280 nm, AD,37o represents the absorbance of
a conjugate precursor at 370 nm, EA,280 represents the
molar absorption coefficient of an antibody at 280 nm,
EA,37o represents the molar absorption coefficient of an
antibody at 370 nm, ED,280 represents the molar
absorption coefficient of a conjugate precursor at 280
nm, ED,37o represents the molar absorption coefficient of
a conjugate precursor at 370 nm, CA represents the
antibody concentration in an antibody-drug conjugate,
and CD represent the drug concentration in an antibodydrug
conjugate.
As for EA, 280, EA, 370, ED, 280, and ED, 370 in the above,
previously prepared values (estimated values based on
calculation or measurement values obtained by UV
measurement of the compounds) are used. For example,
EA,280 can be estimated from the amino acid sequence of
an antibody using a known calculation method (Protein
- 101 -
4833-0752-7733.1
Atty. Dkt. No. 111119-0105
Science, 1995, vol. 4, 2411-2423). EA,37o is generally
zero. In Examples, as for the molar absorption
coefficient of trastuzumab, EA, 280 2154 0 0 ( estimated
value based on calculation) and EA,37o O were used.
ED,280 and ED,37o can be obtained based on Lambert-Beer's
law (Absorbance molar concentration X molar
absorption coefficient X cell path length) by measuring
the absorbance of a solution in which the conjugate
precursor to be used is dissolved at a certain molar
concentration. As for the molar absorption coefficient
of a drug linker in the Examples, ED, 280 = 50 0 0 (measured
average value) and ED,37o 19000 (measured average
value) were used, unless otherwise specified. By
measuring A230 and A370 of an aqueous solution of the
antibody-drug conjugate and solving the simultaneous
equations (I)
be obtained.
and (II) using the values, CA and CD can
Further, by dividing CD by CA, the average
number of conjugated drug molecules per antibody
molecule can be obtained.
[0112]
Common procedure F: Measurement ( 2) of average number
of conjugated drug molecules per antibody molecule in
antibody-drug conjugate.
The average number of conjugated drug molecules
per antibody molecule in the antibody-drug conjugate
can also be determined by high-performance liquid
chromatography (HPLC) analysis using the following
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4833-0752-7733.1
Atty. Dkt. No. 111119-0105
method in addition to the aforementioned Common
procedure E.
[F-1. Preparation of sample for HPLC
(reduction of antibody-drug conjugate)]
analysis
An antibody-drug conjugate solution (about 1 mg/mL,
60 μL) is mixed with an aqueous solution of
dithiothreitol (DTT) (100 mM, 15 μL) . A sample in
which the disulfide bond between the L chain and the H
chain of the antibody-drug conjugate has been cleaved
by incubating the mixture for 30 minutes at 37°C is
used in HPLC analysis.
[F-2. HPLC analysis]
The HPLC analysis is performed under the following
measurement conditions:
HPLC system: Agilent 1290 HPLC system (Agilent
Technologies, Inc.)
Detector: ultraviolet absorption spectrometer
(measurement wavelength: 280 nm)
Column: PLRP-S (2.1 X 50 mm, 8 μm, 1000 angstroms;
Agilent Technologies, Inc., P/N PL1912-1802)
Column temperature: 80°C
Mobile phase A: aqueous solution containing 0.04%
trifluoroacetic acid (TFA)
Mobile phase B: acetoni trile solution containing
0.04% TFA
Gradient program: 29%-36% (0-12.5 min), 36%-42%
(12.5-15 min), 42%-29%
(15.1-25 min)
4833-0752-7733.1
(15-15.1 min), and 29%-29%
- 103 -
Atty. Dkt. No. 111119-0105
Sample injection volume: 15 μL
[F-3. Data analysis]
[F-3-1] Compared with non-conjugated antibody L (L 0 )
and H (H 0 ) chains, drug-conjugated L (L chain connected
to one drug molecule: Li) and H (H chain connected to
one drug molecule: H1, H chain connected to two drug
molecule: H chain connected to three drug
molecules: H3) chains exhibit higher hydrophobicity in
proportion to the number of conjugated drug molecules
and thus have a larger retention time. These chains
are therefore eluted in the order of Lo and L1 or Ho, H1,
H2, and H3. Detection peaks can be assigned to any of
Lo, L1, Ho, H1, H2, and H3 by the comparison of retention
times with L0 and Ho.
[F-3-2] Since the drug linker has UV absorption, peak
area values are corrected in response to the number of
conjugated drug linker molecules according to the
following expression using the molar absorption
coefficients of the Lor H chain and the drug linker.
[0113]
[Express ion 1]
Corrected value of the peak area of the L chain (L1)
= Peak area
Molar absorption coefficient of the L chain
x---------------------------
Molar absorption coefficient of the L chain+ The number of conjugated drug molecules x Molar absorption coefficient of the drug linker
[0114]
[Expression 2]
4833-0752-7733.1
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Atty. Dkt. No. 111119-0105
Corrected value of the peak area of the H chain (H1)
Peak area
Molar absorption coefficient of the H chain
x---------------------------
Molar absorption coefficient of the H chain+ The number of conjugated drug molecules x Molar absorption coefficient of the drug linker
[0115]
Here, as for the molar absorption coefficient (280
nm) of the L or H chain of each antibody, a value
estimated from the amino acid sequence of the L or H
chain of each antibody by a known calculation method
(Protein Science, 1995, vol. 4, 2411-2423) can be used.
In the case of trastuzumab, a molar absorption
coefficient of 26150 and a molar absorption coefficient
of 81290 were used as estimated values for the Land H
chains, respectively, according to its amino acid
sequence. As for the molar absorption coefficient (280
nm) of the drug linker, the measured molar absorption
coefficient (280 nm) of a compound in which the
maleimide group was converted to succinimide thioether
by the reaction of each drug linker with
mercaptoethanol or N-acetylcysteine was used.
[F-3-3] The peak area ratio (%) of each chain is
calculated for the total of the corrected values of
peak areas according to the following expression.
[0116]
[Expression 3]
4833-0752-7733.1
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Atty. Dkt. No. 111119-0105
Peak area ratio of the L chain
Au
----XlOO
ALO+ AL1
AHi
Peak area ratio of the H chain
A
X 100
+ H3
Corrected values of respective peak areas of Au, AHi:L, Hi
[0117]
[F-3-4] The average number of conjugated drug molecules
per antibody molecule in the antibody-drug conjugate is
calculated according to the following expression.
Average number of conjugated drug molecules (L 0
peak area ratio X 0 + Lo peak area ratio X 1 + Ho peak
area ratio X 0 + H1 peak area ratio X 1 + H2 peak area
ratio X 2 + H3 peak area ratio X 3) I 100 X 2
[0118]
The production intermediate compound used in
Production method 1 is described below. The compound
represented by the formula (2) in Production method 1
is a compound represented by the following formula:
C (=0) - (NH-DX) .
In the formula,
n 3 represents an integer of 2 to 8,
bond,
wherein n 4 represents an integer of 1 to 6,
- 106 -
4833-0752-7733.1
Atty. Dkt. No. 111119-0105
LP represents a peptide residue consisting of 2 to 7
amino acids selected from phenylalanine, glycine,
valine, lysine, citrulline, serine, glutamic acid, and
aspartic acid,
nl represents an integer of 0 to 6,
n2 represents an integer of 0 to 5,
La represents -0- or a single bond,
(maleimid-N-yl) - is a maleimidyl group ( 2, 5 -dioxo-2, 5 -
dihydro-lH-pyrrol-1-yl
following formula:
[0119]
[ Formula 2 0]
0
[0120]
group) represented by the
wherein the nitrogen atom is the connecting position,
and
- (NH-DX) is a group represented by the following
formula:
[0121]
[Formula 21]
4833-0752-7733.1
- 107 -
Atty. Dkt. No. 111119-0105
H
Me
F
[0122]
0
HO
l
Me
0
I
0
wherein the nitrogen atom of the amino group at
position 1 is the connecting position.
[0123]
C (=0) - , it is preferred as a production intermediate
that n4 should be an integer of 2 to 4.
As for the peptide residue of a compound
having a peptide residue consisting of an amino acid
selected from phenylalanine, glycine, valine, lysine,
citrulline, serine, glutamic acid, and aspartic acid is
preferred as a production intermediate. Among those
peptide residues, a compound in which LP is a peptide
residue consisting of 4 amino acids is preferred as a
production intermediate. More specifically, a compound
in which LP is the tetrapeptide residue -GGFG- is
preferred as a production intermediate.
[0124]
Further, as for the a
compound having -NHis
preferred as a production
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4833-0752-7733.1
Atty. Dkt. No. 111119-0105
intermediate. A compound having -NH-CH2CH2CHr, -NHCH2-
O-CH2-, or -NH-CH2CH2-O-CH2 is more preferred.
[0125]
A compound represented by the formula (2) in which
n 3 is an integer of 2 to 5, L 2 is a single bond, and -
NH- (CH2) n 1 -La- (CH2) n 2- is -NH-CH2CHr, -NH-CH2CH2CHr,
NH-CH2CH2CH2CHr, -NH-CH2CH2CH2CH2CHr, -NH-CHrO-CHr, or
is preferred as
intermediate. A compound in which
( CH2) n2 - is -NH-CH2CHr, -NH-CH2CH2CHr,
or -NH-CH2CHrO-CHr is more preferred.
a production
-NH- ( CH2) n 1 -La-
-NH-CHrO-CHr,
A compound in
which n 3 is an integer of 2 or 5 is further preferred.
[0126]
A compound represented by the formula (2) in which
n 3 is an integer of 2 to 5, L 2 is -NH- (CH2CHrO) n 4
-
CH2CHrC (=0) - , n 4 is an integer of 2 to 4, and -NH(
CH2) n 1 -La- (CH2) n 2- is -NH-CH2CHr, -NH-CH2CH2CHr, -NHCH2
CH2 CH2 CH2 - , - NH - CH2 CH2 CH2 CH2 CH2 - , -NH-CHrO-CHr, or -
NH-CH2CHrO-CHr is preferred as a production
intermediate. A compound in which n 4 is an integer of
2 or 4 is more preferred. A compound in which -NH(
CH2) n 1 -La- (CH2) n 2- is -NH-CH2CH2CHr, -NH-CHrO-CHr, or
-NH-CH2CH2-O-CH2- is further preferred.
[0127]
Preferred examples of an intermediate useful in
producing such a compound of the present invention can
include the following.
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Atty. Dkt. No. 111119-0105
(maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NHDX),
(maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)(
NH-DX),
(maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)(
NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)(
NH-DX),
(maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)(
NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2CH2-C(=O)(
NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2CH2CHrC
(=0) - (NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2CH2CH2-
C (=0) - (NH-DX),
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4833-0752-7733.1
Atty. Dkt. No. 111119-0105
(maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NHCH2CH2CH2CH2CH2-
C(=O) -(NH-DX),
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2CH2CH2CH2-
C(=O) -(NH-DX),
(maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O) -
(NH-DX) ,
(maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)(
NH-DX),
(maleimid-N-yl)-CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-
C (=0) - (NH-DX),
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2-O-CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O) -
(NH-DX),
(maleimid-N-yl)-CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-
C (=0) - (NH-DX),
(maleimid-N-yl) -CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2-O-CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2-OCH2-
C(=O)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O) -(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-O-CH2CH2-O-CH2CH2-
O-CH2CHrO-CH2CHrC (=0) -GGFG-NH-CH2CH2CHrC (=0) - (NH-DX).
[0128]
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Atty. Dkt. No. 111119-0105
The anti-HER2 antibody-drug conjugate of the
present
linker
invention can be produced by reacting a drugcompound
selected from the above-mentioned
production intermediate compound group with an antiHER2
antibody or a reactive derivative thereof to
thereby form a thioether bond at a disulfide bond site
present in the hinge part of the anti-HER2 antibody.
In this case, the reactive derivative of the anti-HER2
antibody is preferably used, and a reactive derivative
obtained by reducing the anti-HER2 antibody is
particularly preferred.
[0129]
The following are compounds more preferred as a
production intermediate.
(maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2-C(=O)-(NHDX),
(maleimid-N-yl)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)(
NH-DX),
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NHCH2CH2CH2CH2CH2-
C(=O) -(NH-DX),
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2-O-CH2-
C (=0) - (NH-DX) ,
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2-OCH2-
C(=O)-(NH-DX),
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Atty. Dkt. No. 111119-0105
(maleimid-N-yl)-CH2CH2-C(=0)-NH-CH2-CH2-0-CH2CH2-0-
CH2CH2-C(=0) -GGFG-NH-CH2CH2-C(=0)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-0-CH2CH2-0-CH2CH2-
C(=0)-GGFG-NH-CH2CH2CH2-C(=0)-(NH-DX),
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-0-CH2CH2-0-CH2CH2-
0-CH2CH2-0-CH2CH2-C(=0)-GGFG-NH-CH2CH2CH2-C(=0) -(NH-DX)
[0130]
Among the above-mentioned intermediate compound
group, a compound represented by the following formula:
(maleimid-N-yl) -CH2CH2-C(=O) -NH-CH2CH2-0-CH2CH2-0-CH2CH2-
C(=0)-GGFG-NH-CH2CH2CH2-C(=0)-(NH-DX),
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2-0-CH2-
C(=0)-(NH-DX), or
(maleimid-N-yl) -CH2CH2CH2CH2CH2-C(=O) -GGFG-NH-CH2CH2-0-
CH2-C(=0)-(NH-DX),
is a further preferred compound.
[0131]
In order to secure the amount of the conjugate, a
plurality of conjugates obtained under similar
production conditions to have an equivalent number of
drugs (e.g. , about ± 1) can be mixed to prepare new
lots. In this case, the average number of drugs falls
between the average numbers of drugs in the conjugates
before the mixing.
[0132]
2. Production method 2
The compound represented by the formula (2) as an
intermediate used in the previous production method and
- 113 -
4833-0752-7733.1
Atty. Dkt. No. 111119-0105
a pharmacologically acceptable salt thereof can be
produced by the following method, for example.
[0133]
[Formula 22]
NH2-DX
4
l P'-Nt(CH2)n2-C(=O)-OH
P1-NH-(CH2)n 1-L '-(CH2Jn2-C(=O)-(NH-DX)
6
NH2-(CH2)n 1-L "-(CH2)n2-C(=O)-(NH-DX)
l P'-L;-oH
7
P2-LP-NH-(CH2)n1-L"-(CH2)n'-C(=O)-(NH-DX)
g
NH2-(CH,)n1-L "-(CH2)n2-C(=O)-OP3
12 l P'-L;-oH
P2-L'-NH-(CH2)n1-L'-(CH2)n2-C(~O)-OP 3
13
NH2-DX -~ j
.... _ 4 ___ p2-LP-NH-(CH2)n 1-L'-(CH2)n2-C(=O)-OH
14
H-LP-NH-(CH2)n 1-L '-(CH2)n2-C(=O)-OP3
l
L1 -L2-0H
15
H-L'-NH-(CH 11 2)n1-L'-(CH2)n2-C(=O)-(NH-DX)
i L1'_L,-OH 10 L r-L2-LP-NH-(CH2)n 1-L '-(CH2)n2-C(=O)-OP3
t 11 NH~DX
L1-L 2-LP-NH-(CH2)n1-L "-(CH2)n2-C(=O)-(NH-DX) ~
2
[0134]
In the formula,
maleimidyl group,
protecting group.
[0135]
and pl ,
~ 16
L 1'-L2-LP-NH-(CH2)n1-L '-(CH2)n2-C(=O)-OH
17
Ll I represents a terminal
p2 , and P3 each represent a
The compound (6) can be produced by derivatizing
the carboxylic acid (5) into an active ester, mixed
acid anhydride, acid halide, or the like and reacting
it with NH2 -DX(4) or a pharmacologically acceptable
salt thereof. NHrDX (4) indicates exatecan (chemical
name: (1S,9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-
hydroxy-4-methyl-1H,12H-
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benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-
10, 13 (9H, 15H) -dione).
Reaction reagents and conditions that are commonly
used for peptide synthesis can be employed for the
reaction. There are various kinds
For example, it can be produced by
such as p-nitrophenol, N-hydroxy
of active ester.
reacting phenols
benzotriazole, Nhydroxy
succinimide, or the like, with the carboxylic
acid (5) using a condensing agent such as N,N'-
dicyclohexylcarbodiimide or 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride.
Further, the active ester can be also produced by a
reaction of the carboxylic acid ( 5) with
pentafluorophenyl
reaction of the
benzotriazolyl
trifluoroacetate or the like; a
carboxylic acid (5) with 1-
oxytripyrrolidinophosphonium
hexafluorophosphite; a reaction of the carboxylic acid
(5) with diethyl cyanophosphonate (salting-in method);
a reaction of the carboxylic acid (5) with
triphenylphosphine and 2,2'-dipyridyl disulfide
(Mukaiyama's method) a reaction of the carboxylic acid
( 5) with a triazine derivative such as 4-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride (DMTMM); or the like. Further, the reaction
can be also performed by, e.g., an acid halide method
by which the carboxylic acid (5) is treated with an
acid halide such as thionyl chloride and oxalyl
chloride in the presence of a base.
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By reacting the active ester, mixed acid anhydride,
or acid halide of the carboxylic acid (5) obtained as
above with the compound (4) in the presence of a
suitable base in an inert sol vent at - 7 8°C to 15 0°C,
the compound ( 6) can be produced. (Meanwhile, 11 inert
sol vent II indicates a sol vent which does not inhibit a
reaction for which the solvent is used.)
[0136]
Specific examples of the base used for each step
described
hydroxide
above include
or hydride of an
a carbonate, alkoxide,
alkali metal or an alkali
earth metal such as sodium carbonate, potassium
carbonate, sodium ethoxide, potassium butoxide, sodium
hydroxide, potassium hydroxide, sodium hydride, and
potassium hydride; an
by an alkyl lithium
organometallic base
such as n-butyl
represented
lithium, or
dialkylamino lithium such as lithium diisopropylamide;
an organometallic base of bissilylamine such as lithium
bis(trimethylsilyl)amide; and an organic base including
a tertiary amine or a nitrogen-containing heterocyclic
compound such as pyridine, 2,6-lutidine, collidine, 4-
dimethylaminopyridine, triethylamine, N-methyl
morpholine, diisopropylethylamine, and
diazabicyclo[5.4.0]undec-7-ene (DBU).
[0137]
Examples of the inert solvent which is used for
the reaction of the present invention include a
halogenated hydrocarbon solvent such as dichloromethane,
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chloroform, and carbon tetrachloride; an ether solvent
such as
dioxane;
tetrahydrofuran, 1,2-dimethoxyethane, and
an aromatic hydrocarbon solvent such as
benzene and toluene; and an amide solvent such as N,Ndimethylformamide,
N,N-dimethylacetamide, and Nmethylpyrrolidin-
2 -one. In addition to these, a
sulfoxide solvent such as dimethyl sulfoxide and
sul folane; and a ketone sol vent such as acetone and
methyl ethyl solvent such as
methanol and
ketone and an
ethanol may
alcohol
be used in some cases.
Further, a mixed solvent thereof can also be used.
[0138]
As for the protecting group P1 for the terminal
amino group of the compound (6), a protecting group for
an amino group which is generally used for peptide
synthesis, for example, a tert-butyloxy carbonyl group,
a 9-fluorenylmethyloxy carbonyl group, or a benzyloxy
carbonyl group, can be used. Examples of other
protecting groups for an
alkanoyl group such as
alkoxycarbonyl group such
amino group include an
an acetyl group; an
as a methoxycarbonyl group
and an ethoxycarbonyl group; an arylmethoxy carbonyl
group such as a paramethoxybenzyloxy carbonyl group,
and a para (or ortho)nitrobenzyloxy carbonyl group; an
arylmethyl group such as a benzyl group and a triphenyl
methyl group; an aroyl group such as a benzoyl group;
and an aryl sulfonyl group such as a 2,4-dinitrobenzene
sulfonyl group and a orthonitrobenzene sulfonyl group.
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The protecting group P1 can be selected depending on,
e.g., the properties of the compound having the amino
group to be protected.
By deprotecting the protecting group P1 for the
terminal amino group of the compound (6) obtained, the
compound (7) can be produced. In the deprotection,
reagents and conditions can be selected depending on
the protecting group.
The compound (9) can be produced by derivatizing
the peptide carboxylic acid (8) having the N terminal
protected with P2 into an active ester, mixed acid
anhydride, or the like and reacting it with the
compound (7) obtained. The reaction conditions,
reagents, base, and inert sol vent used for forming a
peptide bond between the peptide carboxylic acid ( 8)
and the compound ( 7) can be suitably selected from
those described for the synthesis of the compound (6).
The protecting group P2 can
those described for the
be suitably selected from
protecting group of the
compound (6), and the selection can be made based on,
e.g., the properties of the compound having the amino
group to be protected. As it is generally used for
peptide synthesis, by repeating sequentially the
reaction and deprotection of the amino acid or peptide
constituting the peptide carboxylic acid (8) for
elongation, the compound (9) can be also produced.
By deprotecting the protecting group P2 for the
amino group of the compound (9) obtained, the compound
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Atty. Dkt. No. 111119-0105
(10)
and
can be produced. In the deprotection,
conditions can be selected depending
protecting group.
reagents
on the
It is possible to produce the compound (2) by
derivatizing the carboxylic acid (11) into an active
ester, mixed acid anhydride, acid halide, or the like
and reacting it with the compound (10) obtained. The
reaction conditions, reagents, base, and inert solvent
used for forming a peptide bond between the carboxylic
acid (11) and the compound (10) can be suitably
selected from those described for the synthesis of the
compound (6).
[0139]
The compound (9) can be also produced by the
following method, for example.
The compound (13) can be produced by derivatizing
the peptide carboxylic acid (8) having the N terminal
protected with P2 into an active ester, mixed acid
anhydride, or the like and reacting it in the presence
of a base with the amine compound ( 12) having the
car boxy group protected with P3
• The react ion
conditions, reagents, base, and inert solvent used for
forming a peptide bond between the peptide carboxylic
acid (8) and the compound (12) can be suitably selected
from those described for the synthesis of the compound
( 6) . The protecting group P2 for the amino group of
the compound ( 13) is not particularly 1 imi ted as long
as it is a protecting group generally used.
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Specifically, examples of the protecting group for
a hydroxyl group can include an alkoxymethyl group such
as a methoxymethyl group; an arylmethyl group such as a
benzyl group, a 4-methoxybenzyl group, and a
triphenylmethyl group; an alkanoyl group such as an
acetyl group; an aroyl group such as a benzoyl group;
and a silyl group such as a tert-butyl diphenylsilyl
group. Car boxy group can be protected, e.g. , as an
ester with an alkyl group such as a methyl group, an
ethyl group, and a tert-butyl group, an allyl group, or
an arylmethyl group such as a benzyl group. Examples
of the protecting group for an amino group can include:
an alkyloxy carbonyl group such as a tert-butyloxy
carbonyl group, a
ethoxycarbonyl group;
methoxycarbonyl group, and an
an allyloxycarbonyl group, or an
arylmethoxy carbonyl group such as a 9-
fluorenylmethyloxy carbonyl group, a benzyloxy carbonyl
group, a paramethoxybenzyloxy carbonyl group, and a
para (or ortho) ni trobenzyloxy carbonyl group; an
alkanoyl group such as an acetyl group; an arylmethyl
group such as a benzyl group and a triphenyl methyl
group; an aroyl group such as a benzoyl group; and an
aryl sulfonyl group such as a 2,4-dinitrobenzene
sulfonyl group or an orthonitrobenzene sulfonyl group.
As for the protecting group P3 for a carboxy group,
a protecting group commonly used as a protecting group
for a carboxy group in organic synthetic chemistry, in
particular, peptide synthesis can be used.
- 120 -
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Atty. Dkt. No. 111119-0105
Specifically, it can be suitably selected from the
protecting groups described above, for example, esters
with an alkyl group such as a methyl group, an ethyl
group, or a tert-butyl, allyl esters, and benzyl esters.
In such cases, the protecting group for an amino
group and the protecting group for a carboxy group can
be those preferably removed by a different method or
different conditions. For example, a representative
example includes a combination in which P2 is a tertbutyloxy
carbonyl group and P3 is a benzyl group. The
protecting groups can be selected from the
aforementioned ones depending on, e.g., the properties
of the compounds having the amino group and the carboxy
group to be protected. For removal of the protecting
groups, reagents and conditions can be selected
depending on the protecting group.
By deprotecting the protecting group P3 for the
carboxy group of the compound (13) obtained, the
compound ( 14) can be produced. In the deprotection,
reagents and conditions are selected depending on the
protecting group.
The compound (9) can be produced by derivatizing
the compound (14) obtained into an active ester, mixed
acid anhydride, acid halide, or the like and reacting
with the compound (4) in the presence of a base. For
the reaction, reaction reagents and conditions that are
generally used for peptide synthesis can be also used,
and the reaction conditions, reagents, base, and inert
- 121 -
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solvent used for the reaction can be suitably selected
from those described for the synthesis of the compound
( 6) .
[0140]
The compound (2) can be also produced by the
following method, for example.
By deprotecting the protecting group P2 for the
amino group of the compound (13), the compound (15) can
be produced. In the deprotection, reagents and
conditions can be selected depending on the protecting
group.
The compound (16) can be produced by derivatizing
the carboxylic acid derivative (11) into an active
ester, mixed acid anhydride, acid halide, or the like
and reacting it in the presence of a base with the
compound (15) obtained. The reaction conditions,
reagents, base, and inert solvent used for forming an
amide bond between the peptide carboxylic acid (11) and
the compound ( 15) can be suitably selected from those
described for the synthesis of the compound (6).
By deprotecting the protecting group for the
carboxy group of the compound (16) obtained, the
compound (17) can be produced. The deprotection can be
carried out similarly to the deprotection at the
carboxy group for producing the compound (14).
The compound (2) can be produced by derivatizing
the compound (17) into an active ester, mixed acid
anhydride, acid halide, or the like and reacting it
- 122 -
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with the compound (4) in the presence of a base. For
the reaction, reaction reagents and conditions that are
generally used for peptide synthesis can be also used,
and the reaction conditions, reagents, base, and inert
solvent used for the reaction can be suitably selected
from those described for the synthesis of the compound
( 6) .
[0141]
3. Production method 3
The compound represented by the formula (2) of an
intermediate can be also produced by the following
method.
[0142]
[ Formula 2 3]
L 1 '_L 2_LP-Qp4
19 l L 1'-L 2-LP-QH
2l0 NH2-(C H,)n 1-L '-(CH/n'-C(=O)-(NH-D X)
L 1 '_L 2-LP-NH-(C H2)n 1-L a-(C H2)n2-C(::::O)-(NH-DX)
2
- 123 -
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Atty. Dkt. No. 111119-0105
[0143]
In the formula, L 1
' corresponds to L 1 having a
structure in which the terminal is converted to a
maleimidyl group, and P4 represents a protecting group.
[0144]
The compound (19) can be produced by derivatizing
the compound (11) into an active ester, mixed acid
anhydride, or the like and reacting it in the presence
of a base with the peptide carboxylic acid (18) having
the C terminal protected with P4
• The reaction
conditions, reagents, base, and inert solvent used for
forming a peptide bond between the peptide carboxylic
acid (18) and the compound (11) can be suitably
selected from those described for the synthesis of the
compound (6). The protecting group P4 for the carboxy
group of the compound ( 18) can be suitably selected
from the protecting groups described above.
By deprotecting the protecting group for the
carboxy group of the compound (19) obtained, the
compound (20) can be produced. The deprotection can be
performed similar to the deprotection of the carboxy
group for producing the compound (14).
The compound (2) can be produced by derivatizing
the compound (20) obtained into an active ester, mixed
acid anhydride, or the
compound (7) For the
conditions that are
like and reacting it with the
reaction, reaction reagents and
generally used for peptide
synthesis can be also used, and the reaction conditions,
- 124 -
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reagents, base, and inert solvent used for the reaction
can be suitably selected from those described for the
synthesis of the compound (6).
[0145]
4. Production method 4
Hereinbelow, the method for producing the compound
( 10b) having n1 1, 0 in the production
intermediate ( 10) described in Production method 2 is
described in detail. The compound represented by the
formula (10b), a salt or a solvate thereof can be
produced according to the following method, for example.
[ 014 6]
[ Formula 24]
HO-CH2-C(=O)-OP6
po_X-NH-CH2-0-L
21
22 P5-X-NH-CH2-0-CH2-C(=O)-OP6
23
__ _______,_ P5-X-NH-CH2-0-CH2-C(=O)-OH
24
H2N-DX
4 P5-X-NH-CH2-0-CH2-C(=O)-(NH-DX) H-X-NH-CH2-0-CH2~C(=O)-(NH-DX)
25 --------- 26
P7-LP-NH-CH2-0-CH[C(=O)-(NH-DX)
9b
---->- H-LP-NH-CHz-O-CH2-C(=O)-(NH-DX)
10b
[0147]
In the formula, is as defined
P7-Y-OH
27
above, L
represents an acyl group which is an alkanoyl group
such as an acetyl group or an aroyl group such as
benzoyl group, or a hydrogen atom, X and Y each
represent an oligopeptide consisting of 1 to 3 amino
acids, P5 and P7 each represent a protecting group for
an amino group, and P6 represents a protecting group
for a carboxy group.
- 125 -
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Atty. Dkt. No. 111119-0105
[0148]
A compound represented by the formula (21) can be
produced by using or applying the method described in
Japanese Patent Laid-Open No. 2002-60351 or the
literature (J. Org. Chem., Vol. 51, page 3196, 1986),
and by conducting removal of the protecting groups or
modification of the functional groups, if necessary.
Furthermore, it can be also obtained by treating an
amino acid with a protected terminal amino group or an
acid amide of an oligopeptide with protected amino
group with an aldehyde or a ketone.
By reacting the compound ( 21) with the compound
(22) having a hydroxyl group at a temperature ranging
from under temperature conditions of cooling to room
temperature in an inert solvent in the presence of an
acid or a base, the compound (23) can be produced.
Here, examples of the acid which may be used
include an inorganic acid such as hydrofluoric acid,
hydrochloric acid, sulfuric acid, nitric acid,
phosphoric acid, and boric acid; an organic acid such
as acetic acid, citric acid, paratoluene sulfonic acid,
and methanesulfonic acid; and a Lewis acid such as
tetrafluoroborate, zinc chloride, tin chloride,
aluminum chloride, and iron chloride. Among these, a
sulfonic acid, in particular, paratoluene sulfonic acid
is preferable. As for the base, any one of the already
mentioned bases can be suitably selected and used.
Preferred examples thereof include an alkali metal
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alkoxide such as potassium tert-butoxide, an alkali
metal hydroxide such as sodium hydroxide and potassium
hydroxide; an alkali metal or alkaline earth metal
hydride such as sodium hydride and potassium hydride;
an organometallic
lithium such as
base represented by dialkylamino
lithium diisopropylamide; and an
organometallic base of bissilylamine such as lithium
bis(trimethylsilyl)amide.
Examples of the solvent to be used for the
reaction include an ether
tetrahydrofuran and 1,4-dioxane;
solvent
and an
such as
aromatic
hydrocarbon solvent such as benzene and toluene. Those
solvents can be prepared as a mixture with water.
Further, the protecting group for an amino group
as exemplified by P5 is not particularly limited
provided it is a group commonly used for protection of
an amino group. Representative examples include the
protecting groups for an amino group that are described
in Production method 2. However, in the present
reaction, there may be cases in which the protecting
group for an amino group as exemplified by P5 is
cleaved off. In such cases, a protecting group can be
introduced again by appropriately performing a reaction
with a suitable reagent for protecting an amino group
as may be required.
The compound (24) can be produced by removing the
protecting group P6 of the compound (23) . Herein,
representative examples of the protecting group for a
- 127 -
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carboxy group as
Production method
exemplified
2, and it
by p6
can
are
be
described in
appropriately
selected from these. In the compound (23), it is
desirable in this case that the protecting group P5 for
an amino group and protecting group P6 for a carboxy
group are the protecting groups that can be removed by
a different method or different conditions. For
example, a representative example includes a
combination in which P5 is a 9-fluorenylmethyloxy
carbonyl group and P6 is a benzyl group. The
protecting groups can be selected depending on, e.g.,
the properties of a compound having the amino group and
the carboxy group to be protected. For removal of the
protecting groups, reagents and conditions are selected
depending on the protecting group.
The compound (26) can be produced by derivatizing
the carboxylic acid (24) into an active ester, mixed
acid anhydride, acid halide, or the like and reacting
it with the compound (4) or a pharmacologically
acceptable salt thereof to produce the compound ( 2 5)
followed by removing the protecting group P5 of the
compound (25) obtained. For the reaction between the
compound (4) and the carboxylic acid (24) and the
reaction for removing the protecting group P6
, the same
reagents and reaction conditions as those described for
Production method 2 can be used.
The compound (10b)
compound (26) with an
4833-0752-7733.1
can be produced by reacting the
amino acid with a protected
- 128 -
Atty. Dkt. No. 111119-0105
terminal amino group or the oligopeptide (27) with a
protected amino group to produce the compound (9b) and
removing the protecting group P7 of the compound (9b)
obtained. The protecting group for an amino group as
represented by P7 is not particularly limited provided
it is generally used for protection of an amino group.
Representative examples thereof include the protecting
groups for an amino group that are described in
Production method 2. For removing the protecting group,
reagents and conditions are selected depending on the
protecting group. For the reaction between the
compound (26) and the compound (27), reaction reagents
and conditions that are commonly used for peptide
synthesis can be employed. The compound (10b) produced
by the aforementioned method can be derivatized into
the compound (1) of the present invention according to
the method described above.
[0149]
5. Production method 5
Hereinbelow, the method for producing the compound
(2) having n 1
intermediate (2)
1, n2 1, = 0 in the production
described in Production method 2 is
described in detail.
formula (2), a salt
The compound represented by the
or a solvate thereof can be
produced according to the following method, for example.
[0150]
[Formula 25]
- 129 -
4833-0752-7733.1
28
H-Z-OP9
29
H-X-NH-CH2-O-CH2-C( =0 )-(NH-DX)
26
[0151]
Atty. Dkt. No. 111119-0105
30
L 1'-L 2-LP-N H-CH2-O-CH2-C( =0)-( NH-DX)
2b
31
In the formula, L 1
', L 2
, LP are as defined above, Z
represents an oligopeptide consisting of 1 to 3 amino
acids, P8 represents a protecting group for an amino
group, and P9 represents a protecting group for a
carboxy group.
[0152]
The compound (30) can be produced by removing the
protecting group P8 of the amino acid or oligopeptide
(28) with the protected terminal amino group and
carboxy group to produce the compound (29) and reacting
the obtained amine form (29) with the compound (11).
The protecting group for an amino group as represented
by P8 is not particularly limited provided it is a
group commonly used for protection of an amino group.
Representative examples include the protecting groups
for an amino group that are described in Production
method 2. Further, for removing the protecting group
P8
, reagents and conditions can be selected depending
on the protecting group. For the reaction between the
compound (29) and the carboxylic acid (11), the same
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reagents and reaction conditions as those described for
Production method 2 can be used.
The production intermediate (2b) can be produced
by removing the protecting group P9 of the compound
(30) to produce the compound (31) and reacting the
obtained carboxylic acid (31) with the compound (26).
The representative examples of the protecting group for
a carboxy group as represented by P8 are described in
Production method 2. For the deprotection reaction
thereof, the same reagents and reaction conditions as
those described for Production method 2 can be used.
For the reaction between the compound (26) and the
carboxylic acid (31), reaction reagents and conditions
that are generally used for peptide synthesis can be
also used. The compound (2b) produced by the
aforementioned method can be derivatized into the
compound (1) of the present invention according to the
method described above.
[0153]
6. Production method 6
Hereinbelow, a method for producing the compound
( 1 7b) having n 1 = 1, n 2 = 1, La = 0 in the production
intermediate ( 1 7) described in Production method 2 is
described in detail. The compound represented by the
formula (17b), a salt or a solvate thereof can be also
produced according to the following method, for example.
[0154]
[ Formula 2 6]
- 131 -
4833-0752-7733.1
P5-X-N H-CH2-O-CH2-C( =O)-OP6
23
P 7 -LP-N H-CH2-O-CH 2-C( =0 )-OP6
33
L 1' -L 2-LP- NH-CH 2-0-CH2-C( =O )-OH
17b
[0155]
In the formula,
are as defined above.
[0156]
L 1' ,
Atty. Dkt. No. 111119-0105
H-X-N H-CH2-O-CH2-C( =0 )-OP6
32
H-LP-NH-CH2-0-CH2-C(=O)-OH
34
X, Y, p5 ,
11
----►
p6 , and P7
The compound (33) can be produced by deprotecting
the protecting group P5 for the amino group of the
compound (23) having the protected terminal amino group
and car boxy group to produce the compound ( 3 2) and
reacting the obtained amine derivative (32) with the
oligopeptide (27) having a protected terminal amino
group or a protected amino group. The protecting group
for an amino group as represented by P5 is not
particularly limited provided it is a group commonly
used for protection of an amino group. Representative
examples include the protecting groups for an amino
group that are described in Production method 2.
Further, for removing the protecting group P5
, reagents
and conditions can be selected depending on the
protecting group. Herein, although representative
examples of the protecting group for a carboxy group as
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represented by P6 and the protecting group for an amino
group as represented by P7 include the protecting
groups for a carboxy group and an amino group that are
described in Production method 2. It is desirable that
in the compound (33), the protecting group P6 for a
carboxy group and the protecting group P7 for an amino
group are protecting groups that can be removed by the
same method or the same conditions. For example, a
representative example includes a combination in which
P6 is a benzyl ester group and P7 is a benzyloxy
carbonyl group.
The compound (34) can be produced by removing the
protecting group P6 for the carboxy group of the
compound (33) and the protecting group P7 for the amino
group of the compound (33). The compound (37) can be
also produced by sequentially removing the protecting
group P6 for the carboxy group and the protecting group
P7 for the amino group, and furthermore, the compound
(34) can be produced simply by removing at once both of
the protecting groups P6 and P7 that can be removed by
the same method or the same conditions.
The compound (17b) can be produced by reacting the
obtained compound (34) with the compound (11). For the
reaction between the compound (34) and the compound
(11), the same reagents and reaction conditions as
those described for Production method 2 can be used.
[0157]
- 133 -
4833-0752-7733.1
The anti-HER2
present invention,
Atty. Dkt. No. 111119-0105
antibody-drug
when it is
conjugate
left in
of the
air or
recrystallized or purified, may absorb moisture or have
adsorption water or turn into a hydrate, and such
compounds or salts containing water are also included
in the present invention.
Compounds labeled with various radioactive or nonradioactive
isotopes are also included in the present
invention. One or more atoms constituting the
antibody-drug conjugate of the present invention may
contain an atomic isotope at a non-natural ratio.
Examples of atomic isotopes include deuterium (2H),
tritium (3H), iodine-125 (1 251), and carbon-14 (1 4 C)
Further, the compound of the present invention may be
radioactive-labeled with a radioactive isotope such as
tritium (3H), iodine-125 (1 251), carbon-14 (1 4C), copper-
64 (64Cu), zirconium-89 (89Zr), iodine-124 (1 241),
fluorine-18 (1 8 F), indium-111 (1 111), carbon-11 (1 1c) and
iodine-131 (1 311). The compound labeled with a
radioactive isotope is useful as a therapeutic or
prophylactic agent, a reagent for research such as an
assay reagent and an agent for diagnosis such as an in
vivo diagnostic imaging agent. Without being related
to radioactivity, any isotope variant type of the
antibody-drug conjugate of the present invention is
within the scope of the present invention.
[0158]
[Drugs]
4833-0752-7733.1
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The anti-HER2 antibody-drug conjugate of the
present invention exhibits cytotoxic activity against
cancer cells, and thus, it can be used as a drug,
particularly as a therapeutic agent and/or prophylactic
agent for cancer.
That is, the anti-HER2 antibody-drug conjugate of
the present invention can be selectively used as a drug
for chemotherapy, which is a main method for treating
cancer, and as a result, can delay development of
cancer cells, inhibit growth thereof, and further kill
cancer cells. This can allow cancer patients to be
free from symptoms caused by cancer or achieve
improvement
therapeutic
in QOL of
effect by
cancer patients. Even
cancer patients and attains a
sustaining the lives of the
if the anti-HER2 antibody-drug
conjugate of the present invention does not accomplish
killing cancer cells, it can achieve higher QOL of
cancer patients while achieving longer-term survival,
by inhibiting or controlling the growth of cancer cells.
In such drug therapy, it can be used as a drug
alone and in addition, it can be used as a drug in
combination with an additional therapy in adjuvant
therapy and can be combined with surgical operation,
radiotherapy, hormone therapy, or the like.
Furthermore, it can also be used as a drug for drug
therapy in neoadjuvant therapy.
In addition to the therapeutic use as described
above, an effect of suppressing the growth of small
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metastatic cancer cells and further killing them can
also be expected. Particularly, when the expression of
HER2 is confirmed in primary cancer cells, inhibition
of cancer metastasis or a prophylactic effect can be
expected by administering the anti-HER2 antibody-drug
conjugate of the present invention. For example, an
effect of inhibiting and killing cancer cells in a body
fluid in the course of metastasis or an effect of, for
example, inhibiting and killing small cancer cells
immediately after implantation in any tissue can be
expected. Accordingly, inhibition of cancer metastasis
or a prophylactic effect can be expected, particularly,
after surgical removal of cancer.
The anti-HER2 antibody-drug conjugate
present invention can be expected to
therapeutic effect by administration as
therapy to patients, and addtionally,
administration to cancer tissues.
[0159]
of the
exert a
systemic
by local
Examples of the cancer type to which the anti-HER2
antibody-drug conjugate of the present invention is
applied can include lung cancer, urothelial cancer,
colorectal cancer, prostate cancer, ovarian cancer,
pancreatic cancer, breast cancer, bladder cancer,
gastric cancer, gastrointestinal stromal tumor, uterine
cervix cancer, esophageal cancer, squamous cell
carcinoma, peritoneal cancer, liver cancer,
hepatocellular cancer, colon cancer, rectal cancer,
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colorectal cancer, endometrial cancer, uterine cancer,
salivary gland cancer, kidney cancer, vul val cancer,
thyroid cancer, or penis cancer. The treatment subject
of the anti-HER2 antibody-drug conjugate of the present
invention is a cancer cell expressing, in a cancer cell
as a treatment subject, HER2 protein which the antibody
within the antibody-drug conjugate can recognize. The
term "cancer expressing HER2 protein" as used in the
present specification
having HER2 protein on
is a cancer containing cells
their cell surface. The HER2
protein is overexpressed in various human tumors and
can be evaluated using a method generally carried out
in the art, such as immunohistochemical staining method
(IHC) for evaluating the overexpression of the HER2
protein, or fluorescence in situ hybridization method
(FISH) for evaluating amplification of the HER2 gene.
Further, the anti-HER2 antibody-drug conjugate of
the present invention exhibits an antitumor effect by
recognizing, through its anti-HER2 antibody, the HER2
protein expressed on the surface of cancer cells and
internalizing in the cancer cells. Thus, the treatment
subject of the anti-HER2 antibody-drug conjugate of the
present invention is not limited to the "cancer
expressing HER2 protein" and can also be, for example,
leukemia, malignant lymphoma, plasmacytoma, myeloma, or
sarcoma.
[0160]
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The anti-HER2 antibody-drug conjugate of the
present invention can be preferably administered to a
mammal, but it is more preferably administered to a
human.
[0161]
Substances used in a pharmaceutical composition
containing anti-HER2 antibody-drug conjugate of the
present invention can be suitably selected and applied
from formulation additives or the like that are
generally used in the art, in view of the dosage or
administration concentration.
[0162]
The
present
anti-HER2 antibody-drug conjugate of the
invention can be administered as a
pharmaceutical composition containing at least one
pharmaceutically suitable ingredient. For example, the
pharmaceutical composition above typically contains at
least one pharmaceutical carrier (for example,
sterilized liquid) . Herein, the liquid includes, for
example, water and oil (petroleum oil and oil of animal
origin, plant origin, or synthetic origin. The oil may
be, for example,
or sesame oil.
peanut oil, soybean oil,
Water is a more typical
mineral oil,
carrier when
the pharmaceutical composition above is intravenously
administered. Saline solution, an aqueous dextrose
solution, and an aqueous glycerol solution can be also
used as a liquid carrier, in particular, for an
injection solution. A suitable pharmaceutical vehicle
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can be selected from ones known in the art. If desired,
the composition above may also contain a trace amount
of a moisturizing agent, an emulsifying agent, or a pH
buffering agent. Examples of suitable pharmaceutical
carrier are disclosed in "Remington's Pharmaceutical
Sciences" by E. W. Martin. The formulations correspond
to an administration mode.
[0163]
Various delivery systems are known and they can be
used for administering the anti-HER2 antibody-drug
conjugate of the present invention. Examples of the
administration route can include intradermal,
intramuscular, intraperitoneal, intravenous, and
subcutaneous routes, but not limited thereto. The
administration can be made by injection or bolus
injection, for example. According to a specific
preferred embodiment, the administration of the
antibody-drug conjugate is performed by injection.
Parenteral administration is a preferred administration
route.
[0164]
According to a representative embodiment, the
pharmaceutical composition is prescribed, as a
pharmaceutical composition suitable for intravenous
administration to human, according to the conventional
procedures. The composition for intravenous
administration is typically a solution in a sterile and
isotonic aqueous buffer solution. If necessary, the
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drug may contain a solubilizing agent and local
anesthetics to alleviate pain at injection site ( for
example, lignocaine) . Generally, the ingredient above
is provided individually as any one of lyophilized
powder or an anhydrous concentrate contained in a
container which is obtained by sealing in an ampoule or
a sachet having an amount of the active agent or as a
mixture in a unit dosage form. When the drug is to be
administered by injection, it may be administered from
an injection bottle containing water or saline of
sterile pharmaceutical grade. When the drug is
administered by injection, an ampoule of sterile water
or saline for injection may be provided such that the
aforementioned ingredients are admixed with each other
before administration.
[0165]
The pharmaceutical composition of the present
invention may be a pharmaceutical composition
containing only the anti-HER2 antibody-drug conjugate
of the present invention or a pharmaceutical
composition containing the anti-HER2 antibody-drug
conjugate and at least one cancer treating agent other
than the conjugate. The anti-HER2 antibody-drug
conjugate of the present invention can be administered
with other cancer treating agents.
effect may be enhanced accordingly.
The anti-cancer
Other anti-cancer
agents used for such purpose may be administered to an
individual simultaneously with, separately from, or
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subsequently to the antibody-drug conjugate, and may be
administered while varying the administration interval
for each. Examples of cancer treating agents include
5-FU, pertuzumab, paclitaxel, carboplatin, cisplatin,
gemcitabine,
paclitaxel,
vinblastin,
capecitabine, irinotecan
docetaxel, pemetrexed,
vinorelbine, everolims,
(CPT-11),
sorafenib,
bevacizumab, oxaliplatin, lapatinib,
emtansine (T-DMl), or drugs described
Publication No. WO 2003/038043,
tanespimycin,
ado-trastuzumab
in International
LH-RH analogues
(leuprorelin, goserelin, or the like), estramustine
phosphate, estrogen antagonists (tamoxifen, raloxifene,
or the 1 ike) , and aroma tase inhibitors ( anastrozole,
letrozole, exemestane, or the like), but are not
limited as long as they are drugs having an antitumor
activity.
[0166]
The pharmaceutical composition can be
into a lyophilization formulation or
formulated
a liquid
formulation as a formulation
composition and required purity.
lyophilization formulation, it
having the desired
When formulated as a
may be a formulation
containing suitable formulation additives that are used
in the art. Also for a liquid formulation, it can be
formulated as a liquid formulation containing various
formulation additives that are used in the art.
[0167]
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The composition and concentration of the
pharmaceutical composition
administration method.
may vary
However,
depending on
the anti-HER2
antibody-drug conjugate contained in the pharmaceutical
composition of the present invention can exhibit a
pharmaceutical effect even at a small dosage when the
antibody-drug conjugate has a higher affinity for an
antigen, that is, a higher affinity (= lower Kd value)
in terms of the dissociation constant (that is, Kd
value) for the antigen. Thus, for determining the
dosage of the antibody-drug conjugate, the dosage can
be determined in view of the situation relating to the
affinity between the antibody-drug conjugate and
antigen. When the antibody-drug conjugate of the
present invention is administered to a human, for
example, about 0.001 to 100 mg/kg can be administered
once or administered several times with an interval of
for 1 to 180 days.
at one time with an interval of 1 to 180 days
Examples
[0168]
The present invention is specifically described in
view of the examples shown below. However, the present
invention is not limited to these. Further, it is by
no means interpreted in a limited way. Further, unless
specifically described otherwise,
and starting material described
the reagent, solvent,
in the specification
can be easily obtained from a commercial supplier.
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[0169]
Reference Example 1 Preparation of trastuzumab
Fourteen vials of 440 mg/vial Herceptin (Genentech,
Inc.) were dissolved in 2 L of cation-exchange
chromatography buffer A (25 mM citrate buffer, 30 mM
NaCl, pH 5.0) and filtered through a 0.2 μm filter
(Millipore Corp.: Stericup 0.22 μm, GVPVDF Membrane).
The samples were applied to a cation-exchange
chromatography column (SP Sepharose HP 240 ml, XK50
column), followed by elution under a NaCl concentration
linear gradient from 30 mM to 500 mM using cationexchange
chromatography buffer B (25 mM citrate buffer,
500 mM NaCl, pH 5.0) to separate IgG monomer fractions.
Monomer samples having a higher purity over 98% by size
exclusion chromatography analysis were combined and
concentrated with UF3 OK (Millipore Corp. : PELL ICON XL
Filter, BIOMAX 30K, PXB030A50), and the buffer was
replaced with CBS buffer (10 mM citrate/140 mM NaCl, pH
6. 0). The CBS buffer-replaced samples were filtered
through a 0.2 μm filter (Sartorius AG: Minisart-Plus
0. 2 μm, 17823K)
[0170]
Reference Example 2 Production of trastuzumab emtansine
T-DMl
SMCC derivatization of antibody: By using the Common
procedure C-2 (PBS6.5/EDTA was used as a buffer
solution), Common procedure A, and Common procedure B
(as absorption coefficient at 280 nm, 1.37 mLmg- 1 cm- 1
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was used) described in Production method 1, replacement
of buffer with PBS6.5/EDTA was conducted on the
trastuzumab produced in Reference Example 1 to prepare
a solution containing trastuzumab (160.0 mg) dissolved
in PBS6.5/EDTA (7.60 mL) in a 15 mL polypropylene tube.
Subsequently, SMCC (1.84 mg) DMSO solution (0.40 mL;
which corresponds to about 5.1 equivalents per antibody
molecule) was added at room temperature. The reaction
mixture was adjusted to have an antibody concentration
of 20mg/mL, and the reaction was carried out at room
temperature by using a tube rotator
manufactured by AS ONE Corporation)
reaction solution was subjected
according to the Common procedure D-2
(MTR-103,
for 2 hours. This
to purification
(PBS6.5/EDTA was
used as a buffer solution) to yield 12 mL of a solution
containing 154.9 mg of the SMCC-derivatized antibody.
Conjugation between antibody and drug linker: Adding
PBS6.5/EDTA (2.56 mL) and N2 -deacetyl-N2 -(3-mercapto-1-
oxopropyl) -maytansine (4.67 mg; DMl, Journal of
Medicinal Chemistry, 2006, Vol. 49, No. 14, p. 4392)
DMA (dimethylacetamide) solution ( 0. 93 mL; which
corresponds to about 5. 8 equivalents per SMCCderivatized
antibody molecule) to the solution obtained
above in the 50 mL polypropylene tube at room
temperature, the reaction
antibody concentration of
solution was adjusted to an
10 mg/mL, and the reaction
was carried out at room temperature by using a tube
rotator for 16.5 hours.
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Purification procedure: The above solution was
subjected to purification using the Common procedure D-
1 using a sodium phosphate buffer solution (10 mM, pH
6.5) containing sodium chloride (137 mM) to yield 35 mL
of a solution containing the target Reference Example
compound.
Physicochemical characterization: By using the Common
procedure E using UV absorbance at two wavelengths of
252 nm and 280 nm, the following characteristic values
were obtained.
Antibody concentration: 4.14 mg/mL, antibody yield:
144.9 mg (91%), and average number of conjugated drug
molecules (n) per antibody molecule: 3.0.
[0171]
Example 1 Intermediate (1)
[Formula 27]
[0172]
Process 2
HzN~o
NH TFA
0
N---f
F N ::C;o
01{)
Process 1: tert-Butyl (4-{ [(1S,9S)-9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl]amino}-4-oxobutyl)carbamate
4-(tert-Butoxycarbonylamino)butanoic acid (0.237 g,
1.13 mmol) was dissolved in dichloromethane (10 mL), N-
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hydroxysuccinimide (0.130 g, 1.13 mmol) and 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride
(0.216 g, 1.13 mmol) were added and stirred for 1 hour.
The reaction solution was added dropwise to an N, Ndimethylformamide
solution (10 mL) charged with
methanesul fonic acid salt of exatecan ( 0. 50 0 g, 0. 94
mmol) and triethylamine (0.157 mL, 1.13 mmol), and
stirred at room temperature for 1 day.
removed under reduced pressure and
by silica
The solvent was
the
gel
residues
obtained were purified column
chromatography [chloroform -
8 2 (v/v)] to yield the
chloroform
titled compound
methanol
(0.595 g,
quantitative).
1H-NMR (400 MHz, DMSO-dG) 8: 0.87 (3H, t, J=7.2 Hz),
1.31 (9H, s), 1.58 (lH, t, J=7.2 Hz), 1.66 (2H, t,
J=7.2 Hz), 1.82-1.89 (2H, m), 2.12-2.21 (3H, m), 2.39
(3H, s), 2.92 (2H, t, J=6.5 Hz), 3.17 (2H, s), 5.16 (lH,
d, J=18.8 Hz), 5.24 (lH, d, J=18.8 Hz), 5.42 (2H, s),
5.59-5.55 (lH, m), 6.53 (lH, s), 6.78 (lH, t, J=6.3 Hz),
7.30 (lH, s), 7.79 (lH, d, J=ll.0 Hz), 8.40 (lH, d,
J=8.6 Hz).
MS (APCI) m/z: 621 (M+H)+
[0173]
Process 2: 4-Amino-N- [(1S,9S) -9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl]butanamide
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The compound (0.388 g, 0.61 mmol) obtained in
Process 1 above was dissolved in dichloromethane (9 mL).
Trifluoroacetic acid (9 mL) was added and it was
stirred for 4 hours. The solvent was removed under
reduced
purified
pressure and
by silica
the
gel
residues
column
obtained were
chromatography
[chloroform - partitioned organic layer of chloroform :
methanol water 7 3 1 (v/v/v)] to yield
trifluoroacetate of the titled compound (0.343 g,
quantitative).
1H-NMR (400 MHz, DMSO-dd 8: 0.87 (3H, t, J=7.2 Hz),
1.79-1.92 (4H, m), 2.10-2.17 (2H, m), 2.27 (2H, t,
J=7.0 Hz), 2.40 (3H, s), 2.80-2.86 (2H, m), 3.15-3.20
(2H, m), 5 .15 (lH, d, J=18. 8 Hz), 5. 26 (lH, d, J=18. 8
Hz), 5.42 (2H, s), 5.54-5.61 (lH, m), 6.55 (lH, s),
7.32 (lH, s), 7.72 (3H, brs), 7.82 (lH, d, J=ll.0 Hz),
8.54 (lH, d, J=8.6 Hz).
MS (APCI) m/z: 521 (M+H)+
[0174]
Example 2 Antibody-drug conjugate (2)
- 147 -
4833-0752-7733.1
[Formula 28]
H2N~O
NH TFA
' ' 0
N
F N (;,c{o
"'•" )---,(
OHJ
Atty. Dkt. No. 111119-0105
0 Process 2
Process 1 0
's ..
OHJ
i
Process 4
I O {~
4
~ 0 H O H 0
Trastuzumab~N~N~NJN NJN~O
l O H O H O H ,NH
I
I 2 6
[0175]
Process 1: N-(tert-Butoxycarbonyl)glycylglycyl-Lphenylalanyl-
N-(4-{ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-
4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-4-oxobutyl)glycinamide
N-(tert-Butoxycarbonyl)glycylglycyl-Lphenylalanylglycine
(0.081 g, 0.19 mmol) was dissolved
in dichloromethane (3 mL) N-hydroxysuccinimide ( 0. 0 21
g, 0.19 mmol) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (0.036 g,
0.19 mmol) were added and then stirred for 3.5 hours.
The reaction solution was added dropwise to an N, N-
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dimethylformamide solution (1.5 mL) charged with the
compound (0.080 g, 0.15 mmol) obtained in Process 2 of
Example 1, and stirred at room temperature for 4 hours.
The solvent was removed under reduced pressure and the
residues obtained were purified by silica gel column
chromatography [chloroform - chloroform methanol
8 2 (v/v)] to yield the titled compound (0.106 g,
73%) .
1H-NMR ( 4 0 0 MHz, DMSO-d6) 8: 0.87 (3H, t, J=7.4 Hz),
1. 3 6 (9H, s), 1. 71 (2H, m), 1. 86 (2H, t, J=7.8 Hz),
2.15-2.19 (4H, m), 2.40 (3H, s), 2.77 (lH, dd, J=12.7,
8.8 Hz), 3.02 (lH, dd, J=14.1, 4.7 Hz), 3.08-3.11 (2H,
m), 3.16-3.19 (2H, m), 3.54 (2H, d, J=5.9 Hz), 3.57-
3.77 (4H, m), 4.46-4.48 (lH, m), 5.16 (lH, d, J=19.2
Hz), 5.25 (lH, d, J=18.8 Hz), 5.42 (2H, s), 5.55-5.60
( lH, m), 6.53 (lH, s), 7.00 (lH, t, J=6.3 Hz), 7.17-
7.26 (SH, m), 7.31 (lH, s), 7.71 (lH, t, J=5.7 Hz),
7.80 (lH, d, J=ll.0 Hz), 7.92 (lH, t, J=5.7 Hz), 8.15
(lH, d, J=8.2 Hz), 8.27 (lH, t, J=5.5 Hz), 8.46 (lH, d,
J=8.2 Hz).
MS (APCI) m/z: 939 (M+H)+
[0176]
Process 2: Glycylglycyl-L-phenylalanyl-N-(4-{ [(1S,9S) -
9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-
2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-4-oxobutyl)glycinamide
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The compound (1.97 g, 2.10 mmol) obtained in
Process 1 above was dissolved in dichloromethane (7 mL).
After adding trifluoroacetic acid (7 mL), it was
stirred for 1 hour. The sol vent was removed under
reduced pressure, and the residues were charged with
toluene for azeotropic distillation. The residues
obtained were purified by silica gel column
chromatography [chloroform - partitioned organic layer
of chloroform : methanol water = 7 : 3 : 1 (v/v/v)]
to yield trifluoroacetate of the titled compound (1.97
g, 99%).
1H-NMR (400 MHz, DMSO-dd 8: 0.87 (3H, t, J=7.4 Hz),
1.71-1.73 (2H, m), 1.82-1.90 (2H, m), 2.12-2.20 (4H, m),
2.40 (3H, s), 2.75 (lH, dd, J=13.7, 9.4 Hz), 3.03-3.09
(3H, m), 3.18-3.19 (2H, m), 3.58-3.60 (2H, m), 3.64 (lH,
d, J=5.9 Hz), 3.69 (lH, d, J=5.9 Hz), 3.72 (lH, d,
J=5.5 Hz), 3.87 (lH, dd, J=16.8, 5.9 Hz), 4.50-4.56 (lH,
m), 5.16 (lH, d, J=19.2 Hz), 5.25 (lH, d, J=18.8 Hz),
5.42 (2H, s), 5.55-5.60 (lH, m), 7.17-7.27 (SH, m),
7.32 (lH, s), 7.78-7.81 (2H, m), 7.95-7.97 (3H, m),
8.33-8.35 (2H, m), 8.48-8.51 (2H, m).
MS (APCI) m/z: 839 (M+H) +
[0177]
Process 3: N- [6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(4-{ [(1S,9S) -
9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-
2,3,9,10,13,15-hexahydro-lH,12H-
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benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-4-oxobutyl)glycinamide
To an N,N-dimethylformamide (1.2 mL) solution of
the compound (337 mg, 0.353 mmol) obtained in Process 2
above, triethylamine (44.3 mL, 0.318 mmol) and Nsuccinimidyl
6-maleimide hexanoate (119.7 mg, 0.388
mmol) were added and stirred at room temperature for 1
hour. The sol vent was removed under reduced pressure
and the residues obtained were purified by silica gel
column chromatography [chloroform chloroform
methanol= 5 : 1 (v/v)] to yield the titled compound as
a pale yellow solid (278.0 mg, 76%).
1H-NMR (400 MHz, DMSO-dG) 8: 0.87 (3H, t, J=7.3 Hz),
1.12-1.22 (2H, m), 1.40-1.51 (4H, m), 1.66-1.76 (2H, m),
1.80-1.91 (2H, m), 2.05-2.21 (6H, m), 2.39 (3H, s),
2.79 (lH, dd, J=14.0, 9.8 Hz), 2.98-3.21 (SH, m), 3.55-
3.77 (8H, m), 4.41-4.48 (lH, m), 5.15 (lH, d, J=18.9
Hz), 5.24 (lH, d, J=18.9 Hz), 5.40 (lH, d, J=17.1 Hz),
5.44 (lH, d, J=17.1 Hz), 5.54-5.60 (lH, m), 6.53 (lH,
s), 6.99 (2H, s), 7.20-7.27 (SH, m), 7.30 (lH, s), 7.70
(lH, t, J=5.5 Hz), 7.80 (lH, d, J=ll.0 Hz), 8.03 (lH, t,
J=5.8 Hz), 8.08 (lH, t, J=5.5 Hz), 8.14 (lH, d, J=7.9
Hz), 8.25 (lH, t, J=6.1 Hz), 8.46 (lH, d, J=8.5 Hz).
MS (APCI) m/z: 1032 (M+H)+
[0178]
Process 4: Antibody-drug conjugate (2)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
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concentration of 10 mg/mL with PBS6.0/EDTA by using the
Common procedure C-1 and Common procedure B (as
absorption coefficient at 280 nm, 1. 37 mLmg- 1 cm- 1 was
used) described in Production method 1. The solution
(3.0 mL) was placed in a 15 mL polypropylene tube and
charged with an aqueous solution of 10 mM tris(2-
carboxyethyl)phosphine hydrochloride (TCEP, Tokyo
Chemical Industry Co., Ltd.) (0.0934 mL; 4.6
equivalents per antibody molecule) and an aqueous
solution of 1 M dipotassium hydrogen phosphate (Nacalai
Tesque, Inc.; 0 .150 mL) . After confirming that the
solution had a pH of 7.4 ± 0.1, the disulfide bond at
the hinge part in the antibody was reduced by
incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
incubating the solution above at 22°C for 10 minutes, a
DMSO solution ( 0. 18 7 mL; 9. 2 equivalents per antibody
molecule) containing 10 mM of the compound obtained in
Process 3 was added thereto and incubated for
conjugating the drug linker to the antibody at 22°C for
40 minutes. Next, an aqueous solution (0.0374 mL; 18.4
equivalents per antibody molecule)
(NAC, Sigma-Aldrich Co. LLC) was
of N-acetylcysteine
added thereto and
incubated at 22°C to terminate the reaction of the drug
linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D-1 (PBS6.0 was
used as buffer solution) described in Production method
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1 to yield 6 mL of a solution containing the titled
antibody-drug conjugate. After that, the solution was
concentrated by the Common procedure A.
Physicochemical characterization: By using the Common
procedure E described in Production method 1, the
following characteristic values were obtained.
Antibody concentration: 3.21 mg/mL, antibody yield:
22. 5 mg (75%), and average number of conjugated drug
molecules (n) per antibody molecule: 2.6.
[0179]
Example 3 Antibody-drug conjugate (3)
[Formula 29]
Trastuzurnab -
[0180]
Process 1: Antibody-drug conjugate (3)
7
73
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
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Common procedure B (as absorption coefficient at 280 nm,
1. 487 mLmg- 1 cm- 1 was used). The solution (1. 25 mL) was
placed in a 1.5 mL polypropylene tube and charged with
an aqueous solution of 10 mM TCEP (0.039 mL; 4.6
equivalents per antibody molecule) and an aqueous
solution of 1 M dipotassium hydrogen phosphate (0.0625
mL) . After confirming that the solution had a pH of
7.4 ± 0.1, the disulfide bond at the hinge part in the
antibody was reduced by incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
adding DMSO ( 0. 072 mL) and a DMSO solution containing
10 mM of the compound of Process 3 of Example 2 (0.078
mL; 9.2 equivalents per antibody molecule) to the above
solution at room temperature, it was stirred by using a
tube rotator for conjugating the drug linker to the
antibody at room temperature for 40 minutes. Next, an
aqueous solution (0.0155 mL; 18.4 equivalents per
antibody molecule) of 100 mM NAC was added thereto and
stirred at room temperature to terminate the reaction
of the drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D (ABS was used
as buffer solution) described in Production method 1 to
yield 6 mL of a solution containing the compound of
interest. The solution was further concentrated by the
Common procedure A. After that, by using the Common
procedure E, the following characteristic values were
obtained.
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Antibody concentration: 9.85 mg/mL, antibody yield: 6.9
mg (55%) , and average number of conjugated drug
molecules (n) per antibody molecule: 7.3.
[0181]
Example 4 Antibody-drug conjugate (4)
[Formula 30]
Trastuzumab
Process 2
[0182]
Process 1: N- [3-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)propanoyl]glycylglycyl-L-phenylalanyl-N-(4-
{ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-4-oxobutyl)glycinamide
The compound (80 mg, 0.084 mmol) of Example 1 was
reacted in the same manner as Process 3 of Example 2 by
using N-succinimidyl 3-maleimide propioate (24.6 mg,
0 . 0 9 2 4 mmo 1 ) instead of N-succinimidyl 6-maleimide
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hexanoate to yield the titled compound as a pale yellow
solid (60.0 mg, 73%).
1H-NMR (400 MHz, DMSO-dd 8: 0.89 (3H, t, J=7.3 Hz),
1.70-1.78 (2H, m), 1.81-1.94 (2H, m), 2.12-2.23 (4H, m),
2.42 (3H, s), 2.81 (lH, dd, J=13.7, 9.8 Hz), 3.01-3.15
(3H, m), 3.16-3.23 (2H, m), 3.30-3.35 (lH, m), 3.58-
3.71 (6H, m), 3.71-3.79 (lH, m), 4.44-4.51 (lH, m),
5.19 (lH, d, J=19.0 Hz), 5.27 (lH, d, J=19.0 Hz), 5.43
(lH, d, J=17.6 Hz), 5.47 (lH, d, J=17.6 Hz), 5.57-5.63
(lH, m), 6.56 (lH, s), 7.02 (2H, s), 7.17-7.22 (lH, m),
7.22-7.30 (SH, m), 7.34 (lH, s), 7.73 (lH, t, J=5.6 Hz),
7.83 (lH, d, J=l0.7 Hz), 8.08 (lH, t, J=5.6 Hz), 8.15
(lH, d, J=7.8 Hz), 8.30 (2H, dt, J=18.7, 5.7 Hz), 8.49
(lH, d, J=8.8 Hz).
MS (APCI) m/z: 990 (M+H)+
[0183]
Process 2: Antibody-drug conjugate (4)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg-1 cm-1 was used) . The solution (1 mL) was
placed in a 1.5 mL polypropylene tube and charged with
an aqueous solution of 10 mM TCEP (0.0155 mL; 2.3
equivalents per antibody molecule) and an aqueous
solution of 1 M dipotassium hydrogen phosphate ( 0. 050
mL). After confirming that the solution had a pH of
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7.4 ± 0.1, the disulfide bond at the hinge part in the
antibody was reduced by incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
adding DMSO ( 0. 072 mL) and a DMSO solution containing
10 mM of the compound of Process 3 of Example 2 (0.031
mL; 4.6 equivalents per antibody molecule) to the above
solution at room temperature, it was stirred by using a
tube rotator for conjugating the drug linker to the
antibody at room temperature for 40 minutes. Next, an
aqueous solution
antibody molecule)
(0.0078 mL; 9.2 equivalents per
of 100 mM NAC was added thereto and
stirred at room temperature to terminate the reaction
of the drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D (ABS was used
as buffer solution) to yield 6 mL of a solution
containing the compound of interest. By using the
Common procedure E, the following characteristic values
were obtained.
Antibody concentration: 1.32 mg/mL, antibody yield: 7.9
mg (79%), and average number of conjugated drug
molecules (n) per antibody molecule: 3.1.
[0184]
Example 5 Antibody-drug conjugate (5)
[ Formula 31]
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Trastuwrnab
[0185]
0
N---<('
Process 1
\ ,; ·,
0
~-............ \
Of-0
Process 1: Antibody-drug conjugate (5)
The amount of the aqueous solution of 10 mM TCEP
was adjusted such that the molar ratio of TCEP to the
antibody at the antibody reduction was 4.6. And the
amount of the 10 mM drug linker solution added was
adjusted such that the molar ratio of the compound of
Process 1 of Example 4 to the antibody at the drug
1 inker conjugation was 9 . 2 . Then the amount of the
aqueous solution of 100 mM NAC added was adjusted such
that the molar ratio of NAC to the antibody at the
termination of the reaction was 18. 4. By the same
procedures as Process 2 of Example 4, 6 mL of a
solution containing the titled antibody-drug conjugate
was obtained, and the following characteristic values
were obtained.
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Antibody concentration: 1.23 mg/mL, antibody yield: 7.4
mg ( 74 % ) , and average number of conjugated drug
molecules (n) per antibody molecule: 6.1.
[0186]
Example 6: Antibody-drug conjugate (6)
[Formula 32]
Process 2 Trastuzumab
[0187]
3.6
Process 1: N-{3- [2- (2-{ [3- (2,5-Dioxo-2,5-dihydro-lHpyrrol-
1-
yl)propanoyl]amino}ethoxy)ethoxy]propanoyl}glycylglycyl
-L-phenylalanyl-N-(4-{ [(1S,9S)-9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-
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1H,12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl]amino}-4-oxobutyl)glycinamide
The compound (100 mg, 0.119 mmol) obtained in
Process 2 of Example 2 was reacted in the same manner
as Process 3 of Example 2 by using
diisopropylethylamine (20.8 μL, 0.119 mmol) instead of
triethylamine and N-succinimidyl 3-(2-(2-(3-
maleinimidepropanamide)ethoxy)ethoxy)propanoate (50.7
mg, 0.119 mmol) instead of N-succinimidyl 6-maleimide
hexanoate to yield the titled compound as a pale yellow
solid (66.5 mg, 48%).
1H-NMR (400 MHz, DMSO-dd 8: 0.85 (3H, t, J=7.4 Hz),
1.65-1.74 (2H, m), 1.77-1.90 (2H, m), 2.07-2.19 (4H, m),
2.30 (2H, t, J=7.2 Hz), 2.33-2.36 (2H, m), 2.38 (3H, s),
2.76 (lH, dd, J=13.7, 9.8 Hz), 2.96-3.18 (9H, m), 3.42-
3.44 (4H, m), 3.53-3.76 (l0H, m), 4.43 (lH, td, J=8.6,
4.7 Hz), 5.14 (lH, d, J=18.8 Hz), 5.23 (lH, d, J=18.8
Hz), 5.38 (lH, d, J=17.2 Hz), 5.42 (lH, d, J=17.2 Hz),
5.52-5.58 (lH, m), 6.52 (lH, s), 6.98 (2H, s), 7.12-
7.17 (lH, m), 7.18-7.25 (4H, m), 7.29 (lH, s), 7.69 (lH,
t, J=5.5 Hz), 7.78 (lH, d, J=ll.3 Hz), 7.98-8.03 (2H,
m), 8.11 (lH, d, J=7.8 Hz), 8.16 (lH, t, J=5.7 Hz),
8.23 (lH, t, J=5.9 Hz), 8.44 (lH, d, J=9.0 Hz).
MS (APCI) m/z: 1149 (M+H)+
[0188]
Process 2: Antibody-drug conjugate (6)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
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concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg- 1 cm- 1 was used) . The solution ( 1. 25 mL) was
placed in a 1.5 mL polypropylene tube and charged with
an aqueous solution of 10 mM TCEP (0.019 mL; 2.3
equivalents per antibody molecule) and an aqueous
solution of 1 M dipotassium hydrogen phosphate (0.0625
mL) . After confirming that the solution had a pH of
7.4 ± 0.1, the disulfide bond at the hinge part in the
antibody was reduced by incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
adding DMSO (Sigma-Aldrich Co. LLC; 0.109 mL) and a
DMSO solution containing 10 mM of the compound of
Process 1 (0.039 mL; 4.6 equivalents per antibody
molecule) to the above solution at room temperature, it
was stirred by using a tube rotator for conjugating the
drug linker to the antibody at room temperature for 40
minutes. Next, an aqueous solution (0.008 mL) of 100
mM NAC was added thereto and stirred at room
temperature to terminate the reaction of the drug
linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D (ABS was used
as buffer solution) described in Production method 1 to
yield 6 mL of a solution containing the compound of
interest.
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Physicochemical characterization: By using the Common
procedure E, the following characteristic values were
obtained.
Antibody concentration: 1.76 mg/mL, antibody yield:
10. 6 mg (85%), and average number of conjugated drug
molecules (n) per antibody molecule: 3.6.
[0189]
Example 7 Antibody-drug conjugate (7)
[Formula 33]
Trastuzumab «~' O O O H O 0
N~N~0~o~N~NJN JN~r0
, H H O H o H .NH
0
[0190]
Process 1: Antibody-drug conjugate (7)
69
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg- 1 cm- 1 was used) . The solution (1.25 mL) was
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placed in a 1.5 mL polypropylene tube and charged with
an aqueous solution of 10 mM TCEP (0.039 mL; 4.6
equivalents per antibody molecule) and an aqueous
solution of 1 M dipotassium hydrogen phosphate (0.0625
mL) . After confirming that the solution had a pH of
7.4 ± 0.1, the disulfide bond at the hinge part in the
antibody was reduced by incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
adding DMSO ( 0. 072 mL) and a DMSO solution containing
10 mM of the compound of Process 1 of Example 6 (0.078
mL; 9.2 equivalents per antibody molecule) to the above
solution at room temperature, it was stirred by using a
tube rotator for conjugating the drug linker to the
antibody at room temperature for 40 minutes. Next, an
aqueous solution (0.0155 mL) of 100 mM NAC was added
thereto and stirred at room temperature to terminate
the reaction of the drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D (ABS was used
as buffer solution) to yield 6 mL of a solution
containing the compound of interest.
Physicochemical characterization: By using the Common
procedure E, the following characteristic values were
obtained.
Antibody
11. 6 mg
molecules
[0191]
4833-0752-7733.1
concentration: 1.93 mg/mL, antibody yield:
(93%), and average number of conjugated drug
(n) per antibody molecule: 6.9.
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Example 8 Antibody-drug conjugate (8)
[Formula 34]
Trastu.::um~b
[0192]
Process 1: Antibody-drug conjugate (8)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg- 1 cm- 1 was used) . The solution (1.25 mL) was
placed in a 1.5 mL polypropylene tube and charged with
an aqueous solution of 10 mM TCEP (0.039 mL; 4.6
equivalents per antibody molecule) and an aqueous
solution of 1 M dipotassium hydrogen phosphate (0.0625
mL). After confirming that the solution had a pH of
7.4 ± 0.1, the disulfide bond at the hinge part in the
antibody was reduced by incubating at 37°C for 1 hour.
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Conjugation
adding DMSO
between antibody and drug linker: After
( 0. 072 mL) and a DMSO solution containing
10 mM of the compound of Process 1 of Example 6 (0.078
mL; 9.2 equivalents per antibody molecule) to the above
solution at room temperature, it was stirred by using a
tube rotator for conjugating the drug linker to the
antibody at room temperature for 40 minutes. Next, an
aqueous solution (0.0155 mL) of 100 mM NAC was added
thereto and stirred at room temperature to terminate
the reaction of the drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D-1 (ABS was
used as buffer solution) to yield 5.7 mL of a solution
containing the compound of interest.
Physicochemical characterization: By using the Common
procedure E, the following characteristic values were
obtained.
Antibody
8. 55 mg
molecules
[0193]
concentration: 1.50 mg/mL, antibody yield:
(86%), and average number of conjugated drug
(n) per antibody molecule: 6.2.
Example 9 Antibody-drug conjugate (9)
[Formula 35]
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0
~ H 0
\,,N,~N~O~"O.J~o,"',0~'70 ~)
o o o
0
,-
Process 1
Trastuzum-ab
3-4
[0194]
Process 1: N- [19-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl) -17-oxo-4,7,10,13-tetraoxo-16-azanonadecan-1-
oyl]glycylglycyl-L-phenylalanyl-N-(4-{ [(1S,9S)-9-ethyl-
5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-
hexahydro-1H,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-4-oxobutyl)glycinamide
The compound (90 mg, 0.107 mmol) obtained in
Process 2 of Example 2 was reacted in the same manner
as Process 3 of Example 2 by using
diisopropylethylamine (18. 7 μL, 0 .107 mmol) instead of
triethylamine and N-succinimidyl 1-maleinimide-3-oxo-
7,10,13,16-tetraoxa-4-azanonadecan-19-oate (55.1 mg,
0.107 mmol) instead of N-succinimidyl 6-maleimide
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hexanoate to yield the titled compound as a pale yellow
so 1 id ( 5 0 mg , 3 7 % ) .
1H-NMR (400 MHz, DMSO-dd 8: 0.85 (3H, t, J=7.2 Hz),
1.64-1.74 (2H, m), 1.77-1.90 (2H, m), 2.06-2.19 (4H, m),
2.27-2.32 (2H, m), 2.33-2.37 (2H, m), 2.38 (3H, s),
2.72-2.80 (3H, m), 2.96-3.19 (6H, m), 3.39-3.48 (l0H,
m), 3.52-3.75 (l0H, m), 4.39-4.48 (lH, m), 5.14 (lH, d,
J=18.8 Hz), 5.23 (lH, d, J=18.8 Hz), 5.38 (lH, d,
J=17.0 Hz), 5.42 (lH, d, J=17.0 Hz), 5.52-5.58 (lH, m),
6.52 (lH, s), 6.98 (lH, s), 7.13-7.24 (SH, m), 7.29 (lH,
s), 7.69 (lH, t, J=5.5 Hz), 7.78 (lH, d, J=l0.9 Hz),
7.98-8.03 (2H, m), 8.10 (lH, d, J=7.8 Hz), 8.16 (lH, t,
J=5.7 Hz), 8.23 (lH, t, J=5.7 Hz), 8.44 (lH, d, J=8.6
Hz)
MS (APCI) m/z: 1237 (M+H)+
[0195]
Process 2: Antibody-drug conjugate (9)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 1, the
titled antibody-drug conjugate was obtained in the same
manner as Process 2 of Example 6.
Antibody concentration: 1.75 mg/mL, antibody yield:
10. 5 mg (84%), and average number of conjugated drug
molecules (n) per antibody molecule: 3.4.
[0196]
Example 10 Antibody-drug conjugate (10)
[Formula 36]
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Trastuzumab-
6. 0
[0197]
Process 1: Antibody-drug conjugate (10)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 1 of
Example 9, the titled antibody-drug conjugate was
obtained in the same manner as Process 1 of Example 7.
Antibody concentration: 1.79 mg/mL, antibody yield:
10. 7 mg (86%), and average number of conjugated drug
molecules (n) per antibody molecule: 6.0.
[0198]
Example 11 Intermediate (11)
[ Formula 3 7]
[0199]
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Process 1: tert-Butyl [2- (2-{ [ (1S, 9S) -9-ethyl-5-fluoro-
9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-
hexahydro-1H,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethoxy)ethyl]carbamate
Methanesulfonic acid salt of exatecan (3.10 g,
5.47 mol) was reacted in the same manner as Process 1
of Example 1 by using {2-[(tertbutoxycarbonyl)
amino]ethoxy}acetic acid (J. Med. Chem.,
1992, vol. 35, pp. 292; 1.55 g, 6.01 mmol) instead of
4-(tert-butoxycarbonylamino)butanoic acid to yield the
titled compound as a pale yellow solid (2.56 g, 73%).
1H-NMR (400 MHz, DMSO-dG) 8: 0.87 (3H, t, J=7.3 Hz),
1. 26 (9H, s), 1. 81-1. 91 (2H, m), 2 .13-2. 22 (2H, m),
2.40 (3H, s), 3.08-3.26 (4H, m), 3.43-3.53 (2H, m),
4.00 (lH, d, J=15.1 Hz), 4.05 (lH, d, J=15.1 Hz), 5.14
(lH, d, J=18.7 Hz), 5.22 (lH, d, J=18.7 Hz), 5.40 (lH,
d, J=16.6 Hz), 5.44 (lH, d, J=16.6 Hz), 5.59-5.66 (lH,
m), 6.53 (lH, s), 6.86 (lH, t, J=5.4 Hz), 7.31 (lH, s),
7. 7 9 ( lH, d, J = 10. 9 Hz) , 8. 4 9 ( lH, d, J = 9. 1 Hz) .
MS (APCI) m/z: 637 (M+H)+
[0200]
Process 2: 2-(2-Aminoethoxy)-N- [(1S,9S) -9-ethyl-5-
fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-
hexahydro-1H,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]acetamide
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The compound (1.50 g, 2.36 mol) obtained in
Process 1 above was reacted in the same manner as
Process 2 of Example 1 to yield trifluoroacetic acid
salt of the titled compound as a pale yellow solid
(1.50 g, quantitative).
1H-NMR (400 MHz, DMSO-dd 8: 0.87 (3H, t, J=7.5 Hz),
1.81-1.92 (2H, m), 2.15-2.23 (2H, m), 2.41 (3H, s),
3.05 (2H, t, J=5.1 Hz), 3.15-3.23 (2H, m), 3.71 (2H, t,
J=5.1 Hz), 4.10 (2H, s), 5.19 (lH, d, J=18.7 Hz), 5.24
(lH, d, J=18.7 Hz), 5.43 (2H, s), 5.58-5.66 (lH, m),
6.55 (lH, s), 7.33 (lH, s), 7.73-7.84 (4H, m), 8.55 (lH,
d, J=9.1 Hz).
MS (APCI) m/z: 537 (M+H)+
[0201]
Example 12 Antibody-drug conjugate (12)
[Formula 38]
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Process 2
lrnsluzumab -
[0202]
Atty. Dkt. No. 111119-0105
Process 1
0
OH 0
Process 3
Process 4
O H [! H O K
N~N~N"'r(N~N"'r(N-.__/'-0~0
D O H O H O .. ,NH
,,.,..
OH 0
3.7
N-(tert-Butoxycarbonyl)glycylglycyl-Lphenylalanyl-
N- [2-(2-{ [(1S,9S)-9-ethyl-5-fluoro-9-
Process 1:
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl]amino}-2-oxoethoxy)ethyl]glycinamide
The compound (554 mg, 0.85 mmol) of Process 2 of
Example 11 was reacted in the same manner as Process 1
of Example 2 to yield the titled compound (775 mg, 95%)
1H-NMR (400 MHz, DMSO-dd 8: 0.85 (3H, t, J=7.3 Hz),
1.36 (9H, s), 1.78-1.89 (2H, m), 2.13-2.22 (2H, m),
2.39 (3H, s), 2.71 (lH, dd, J=13.4, 9.8 Hz), 2.95 (lH,
dd, J=13.4, 4.3 Hz), 3.09-3.23 (lH, m), 3.23-3.32 (2H,
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m), 3.40-3.62 ( 8H, m), 3.73 (lH, dd, J=16.5, 5.5 Hz),
4.03 (2H, s), 4.39-4.47 (lH, m), 5.17 (lH, d, J=18.9
Hz), 5.25 (lH, d, J=18.9 Hz), 5.41 (lH, d, J=16.8 Hz),
5.45 (lH, d, J=16.8 Hz), 5.57-5.64 (lH, m), 6.54 (lH,
s), 6.99 (lH, t, J=5.8 Hz), 7.13-7.26 (SH, m), 7.31 (lH,
s), 7.76-7.82 (2H, m), 7.90
d, J=7.9 Hz), 8.27 (lH,
J=8.5 Hz).
MS (APCI) m/z: 955 (M+H)+
[0203]
t,
(lH, t, J=5.2 Hz), 8.13 (lH,
J=5.8 Hz), 8.49 (lH, d,
Process 2: Glycylglycyl-L-phenylalanyl-N- [2-(2-
{ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethoxy)ethyl]glycinamide
The compound (630 mg, 0.659 mmol) obtained in
Process 1 above was reacted in the same manner as
Process 2 of Example 2 to yield trifluoroacetic acid
salt of the titled compound (588 mg, 92%).
1H-NMR (400 MHz, DMSO-dd 8: 0.86 (3H, t, J=7.3 Hz),
1.79-1.90 (2H, m), 2.13-2.22 (2H, m), 2.39 (3H, s),
2.71 (lH, dd, J=13.4, 10.1 Hz), 2.99 (lH, dd, J=13.4,
4.3 Hz), 3.09-3.23 ( lH, m), 3.24-3.32 (3H, m), 3.41-
3.71 ( 7H, m), 3.86 ( lH, dd, J=16.8, 5.8 Hz), 4.04 (2H,
s), 4.52 (lH, td, J=9.0, 4.1 Hz), 5.17 (lH, d, J=18.9
Hz), 5.25 (lH, d, J=18.9 Hz), 5.41 (lH, d, J=16.5 Hz),
5.45 (lH, d, J=16.5 Hz), 5.56-5.65 (lH, m), 6.55 (lH,
s), 7.13-7.26 (SH, m), 7.32 (lH, s), 7.80 (lH, d,
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J=ll.0 Hz), 7.87-8.01 (4H, m), 8.29-8.36 (2H, m), 8.46-
8.55 (2H, m).
MS (APCI) m/z: 855 (M+H)+
[0204]
Process 3: N- [6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-L-phenylalanyl-N- [2-(2-
{ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethoxy)ethyl]glycinamide
The compound (240 mg, 0.247 mmol) obtained in
Process 2 above was reacted in the same manner as
Process 3 of Example 2 to yield the titled compound
( 16 2 mg , 6 2 % ) .
1H-NMR (400 MHz, DMSO-dG) 8: 0.86 (3H, t, J=7.6 Hz),
1.13-1.22 (2H, m), 1.40-1.51 (4H, m), 1.78-1.90 (2H, m),
2.09 (2H, t, J=7.6 Hz), 2.14-2.21 (2H, m), 2.39 (3H, s),
2.74 (lH, dd, J=13.6, 9.7 Hz), 2.96 (lH, dd, J=13.6,
4.5 Hz), 3.08-3.24 (lH, m), 3.24-3.30 (lH, m), 3.33-
3.40 (4H, m), 3.47-3.68 (7H, m), 3.72 (lH, dd, J=16.6,
5.7 Hz), 4.03 (2H, s), 4.42 (lH, td, J=8.6, 4.2 Hz),
5.17 (lH, d, J=18.7 Hz), 5.25 (lH, d, J=18.7 Hz), 5.40
(lH, d, J=17.2 Hz), 5.44 (lH, d, J=17.2 Hz), 5.57-5.64
(lH, m), 6.52 (lH, s), 6.99 (2H, s), 7.13-7.25 (SH, m),
7.31 (lH, s), 7.74-7.81 (2H, m), 7.99 (lH, t, J=5.7 Hz),
8.03-8.11 (2H, m), 8.22 (lH, t, J=5.7 Hz), 8.47 (lH, d,
J=9.1 Hz).
MS (APCI) m/z: 1048 (M+H)+
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[0205]
Process 4: Antibody-drug conjugate (12)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 3, the
titled antibody-drug conjugate was obtained in the same
manner as Process 2 of Example 6. The solution was
further concentrated by the Common procedure A. After
that, by using the Common procedure E, the following
characteristic values were obtained.
Antibody concentration: 10.77 mg/mL, antibody yield:
7.5 mg (60%), and average number of conjugated drug
molecules (n) per antibody molecule: 3.7.
[0206]
Example 13 Antibody-drug conjugate (13)
[ Formula 3 9]
[0207]
Process 1: Antibody-drug conjugate (13)
69
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 3 of
Example 12, the titled antibody-drug conjugate was
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obtained in the same manner as Process 1 of Example 7.
The solution was further concentrated by the Common
procedure A. After that, by using the Common procedure
E, the following characteristic values were obtained.
Antibody concentration: 10.69 mg/mL, antibody yield:
7. 5 mg ( 6 0 % ) , and average number of conjugated drug
molecules (n) per antibody molecule: 6.9.
[0208]
Example 14 Intermediate (14)
[Formula 40]
[0209]
Process 2
Process 1: tert-Butyl (3-{ [(1S,9S)-9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl]amino}-3-oxopropyl)carbamate
Methanesulfonic acid salt of exatecan (500 mg,
0.941 mmol) was reacted in the same manner as Process 1
of Example 1 by using N-(tert-butoxycarbonyl)-P-alanine
instead of 4-(tert-butoxycarbonylamino)butanoic acid to
yield the titled compound as a yellow-brown solid (616
mg, quantitative)
1H-NMR (400 MHz, DMSO-dd 8: 0.87 (3H, t, J=7.2 Hz),
1.29 (9H, s), 1.86 (2H, dt, J=15.1, 7.3 Hz), 2.04-2.22
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(2H, m), 2.31 (2H, t, J=6.8 Hz), 2.40 (3H, s), 3.10-
3.26 (4H, m), 5.15 (lH, d, J=18.8 Hz), 5.26 (lH, d,
J=19.2 Hz), 5.42 (2H, dd, J=18.8, 16.4 Hz), 5.57 (lH,
dt, J=8.5, 4.2 Hz), 6.53 (lH, s), 6.78 (lH, t, J=5.5
Hz), 7.30 (lH, s), 7.80 (lH, d, J=ll.0 Hz), 8.46 (lH, d,
J=8.6 Hz).
MS (ESI) m/z: 607 (M+H)+
[0210]
Process 2 : N- [(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-
methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl] -P-alaninamide
The compound obtained in Process 1 above was
reacted in the same manner as Process 2 of Example 1 to
yield trifluoroacetic acid salt of the titled compound
as a yellow solid (499 mg, 86%).
1H-NMR (400 MHz, DMSO-dd 8: 0. 87 (3H,
1.86 (2H, dquin, J=14.6, 7.2, 7.2, 7.2,
t, J=7.2 Hz),
7. 2 Hz) , 2. 0 6 -
2.27 (lH, m), 2.41 (3H, s), 2.46-2.57 (2H, m), 3.08 (2H,
t, J=6.8 Hz), 3.14-3.24 (2H, m), 5.22 (lH, d, J=18.8
Hz), 5.29 (lH, d, J=18.8 Hz), 5.43 (2H, s), 5.58 (lH,
dt, J=8.5, 4.5 Hz), 6.55 (lH, s), 7.32 (lH, s), 7.74
(3H, brs), 7. 82 (lH, d, J=ll. 0 Hz), 8. 67 (lH, d, J=8. 6
Hz).
MS (ESI) m/z: 507 (M+H)+
[0211]
Example 15 Antibody-drug conjugate (15)
[Formula 41]
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4833-0752-7733.1
f1 2N~O
N" TFA
F,-.. N.,.,\"_11/0' ', ,6<
OH 0
Process 4
[0212]
Process
Atty. Dkt. No. 111119-0105
Process 1
Tmstu:-:umab
1: N-(tert-Butoxycarbonyl)glycylglycyl-Lphenylalanylglycyl-
N- [(1S,9S)-9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl] -P-alaninamide
The compound (484 mg, 0.780 mmol) obtained in
Process 2 of Example 14 was reacted in the same manner
as Process 1 of Example 2 to yield the titled compound
as a pale yellow solid (626 mg, 87%)
1H-NMR (400 MHz, DMSO-dd 8: 0.87 (3H, t, J=7.4 Hz),
1.27-1.42 (9H, m), 1.77-1.93 (2H, m), 2.06-2.22 (2H, m),
2.36 (2H, t, J=7.2 Hz), 2.40 (3H, d, J=l.6 Hz), 2.44-
2.54 (2H, m), 2.76 (lH, dd, J=14.5, 10.2 Hz), 3.02 (lH,
dd, J=13.9, 4.5 Hz), 3.12-3.22 (2H, m), 3.52 (6H, d,
J=6.3 Hz), 4.42-4.54 (lH, m), 5.19 (lH, d, J=19.2 Hz),
5.26 (lH, d, J=18.4 Hz), 5.42 (lH, dd, J=18.4, 16.4 Hz),
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5.57 (lH, dt, J=8.7, 4.4 Hz), 6.53 (lH, s), 6.98 (lH, t,
J=5.9 Hz), 7.14-7.28 (SH, m), 7.31 (lH, s), 7.77-7.84
(lH, m), 7.91 (lH, t, J=5.5 Hz), 8.16 (lH, d, J=7.8 Hz),
8.27 (lH, t, J=5.1 Hz), 8.52 (lH, d, J=9.0 Hz).
[0213]
Process 2: Glycylglycyl-L-phenylalanylglycyl-N-
[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl] -P-alaninamide trifluoroacetate
The compound (624 mg, 0.675 mmol) obtained in
Process 1 above was reacted in the same manner as
Process 2 of Example 2 to yield the titled compound as
a yellow solid (626 mg, 92%).
1H-NMR (400 MHz, DMSO-dG) 8: 0.87 (3H, t, J=7.4 Hz),
1.86 (2H, tt, J=14.5, 7.2 Hz), 2.07-2.22 (2H, m), 2.36
(2H, t, J=7.2 Hz), 2.40 (3H, s), 2.44-2.54 (2H, m),
2.75 (lH, dd, J=13.7, 9.8 Hz), 3.04 (lH, dd, J=13.7,
4.3 Hz), 3.12-3.22 (2H, m), 3.58 (2H, d, J=4.7 Hz),
3.69 (3H, td, J=ll.2, 5.7 Hz), 3.87 (lH, dd, J=17.0,
5.7 Hz), 4.54 (lH, m, J=17.8, 4.5 Hz), 5.19 (lH, d,
J=19.2 Hz), 5.26 (lH, d, J=18.8 Hz), 5.43 (2H, s),
5.51-5.60 (lH, m), 6.55 (lH, s), 7.14-7.29 (SH, m),
7.32 (lH, s), 7.81 (lH, d, J=l0.9 Hz), 7.88 (lH, t,
J=5.7 Hz), 7.97 (3H, brs), 8.29-8.38 (2H, m), 8.50 (lH,
t, J=5.7 Hz), 8.55 (lH, d, J=8.6 Hz).
MS (ESI) m/z: 825 (M+H)+
[0214]
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Process 3: N- [6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-L-phenylalanylglycyl-N-
[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl] -P-alaninamide
The compound (60.0 mg, 0.0646 mmol) obtained in
Process 2 above was reacted in the same manner as
Process 3 of Example 2 to yield the titled compound as
a so 1 id ( 14 . 0 mg, 21 % ) .
1H-NMR (400 MHz, DMSO-dd 8: 0.86 (3H, t, J=7.2 Hz),
1.12-1.22 (2H, m), 1.39-1.51 (4H, m), 1.79-1.91 (2H, m),
2.02-2.20 (2H, m), 2.07 (2H, t, J=7.4 Hz), 2.30-2.42
(4H, m), 2.40 (3H, s), 2.78 (lH, dd, J=14.1, 9.4 Hz),
3.02 (lH, dd, J=14.7, 4.9 Hz), 3.12-3.21 (2H, m), 3.26-
3.42 (2H, m), 3.50-3.80 (6H, m), 4.40-4.51 (lH, m),
5.19 (lH, d, J=19.6 Hz), 5.26 (lH, d, J=19.2 Hz), 5.42
(2H, brs), 5.51-5.62 (lH, m), 6.53 (lH, s), 6.99 (2H,
s), 7.13-7.28 (SH, m), 7.31 (lH, s), 7.74-7.84 (2H, m),
8.01 (lH, t, J=5.3 Hz), 8.06 (lH, t, J=5.7 Hz), 8.14
(lH, d, J=8.2 Hz), 8.25 (lH, t, J=5.7 Hz), 8.53 (lH, d,
J=8.6 Hz).
MS (ESI) m/z: 1018 (M+H)+
[0215]
Process 4: Antibody-drug conjugate (15)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL with PBS6.0/EDTA by using the
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Common procedure C-1 and Common procedure B (as
absorption coefficient at 280 nm, 1. 37 mLmg- 1 cm- 1 was
used). The solution (1.0 mL) was collected into a 2 mL
tube and charged with an aqueous solution of 10 mM TCEP
(0.0155 mL; 2.3 equivalents per antibody molecule) and
an aqueous solution of 1 M dipotassium hydrogen
phosphate (0.050 mL) After confirming that the
solution had a pH of 7.4 ± 0.1, the disulfide bond at
the hinge part in the antibody was reduced by
incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
incubating the solution at 22°C for 10 minutes, a DMSO
solution (0.0311 mL; 4.6 equivalents per antibody
molecule) containing 10 mM of the compound obtained in
Process 3 was added thereto and incubated for
conjugating the drug linker to the antibody at 22°C for
40 minutes. Next, an aqueous solution (0.00622 mL; 9.2
equivalents per antibody molecule) of 100 mM NAC was
added thereto and incubated at 22°C to terminate the
reaction of the drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D-1 (PBS6.0 was
used as buffer solution) to yield 6 mL of a solution
containing the titled antibody-drug conjugate.
Physicochemical characterization: By using the Common
procedure E, the following characteristic values were
obtained.
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Antibody concentration: 1.18 mg/mL, antibody yield:
7. 08 mg (71%), and average number of conjugated drug
molecules (n) per antibody molecule: 2.0.
[0216]
Example 16 Antibody-drug conjugate (16)
[Formula 42]
0 H O H O H
l"N~N ~N"yN~N"yN~ O
U O H 0 • H EO .NH
0 1/' \
1-• 1/ I ~N \0
F ~ N' ~
OH)
Trastuzumab
Process 1
0 H O H O H
N"'',,,.., .. v,.."yN J .. N"yN~N'YN~ 0
1 O H O " H O NH
0 0 .
[0217]
Process 1: Antibody-drug conjugate (16)
3.B
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL with PBS6.0/EDTA by using the
Common procedure C-1 and Common procedure B (as
absorption coefficient at 280 nm, 1. 37 mLmg- 1 cm- 1 was
used). The solution (1.0 mL) was collected into a 2 mL
tube and charged with an aqueous solution of 10 mM TCEP
(0.0311 mL; 4.6 equivalents per antibody molecule) and
an aqueous
phosphate
4833-0752-7733.1
solution of
( 0. 050 mL)
1 M dipotassium hydrogen
After confirming that the
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Atty. Dkt. No. 111119-0105
solution had a pH of 7.4 ± 0.1, the disulfide bond at
the hinge part in the antibody was reduced by
incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
incubating the solution at 22°C for 10 minutes, a DMSO
solution (0.0622 mL; 9.2 equivalents per antibody
molecule) containing 10 mM of the compound obtained in
Process 3 of Example 15 was added thereto and incubated
for conjugating the drug linker to the antibody at 22°C
for 40 minutes. Next, an aqueous solution (0.0124 mL;
18. 4 equivalents per antibody molecule) of 100 mM NAC
was added thereto and incubated at 22°C to terminate
the reaction of the drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D-1 (PBS6.0 was
used as buffer solution) to yield 6 mL of a solution
containing the titled antibody-drug conjugate.
Physicochemical characterization: By using the Common
procedure E, the following characteristic values were
obtained.
Antibody
6 .18 mg
molecules
[0218]
concentration: 1.03 mg/mL, antibody yield:
(62%), and average number of conjugated drug
(n) per antibody molecule: 3.8.
Example 17 Antibody-drug conjugate (17)
[ Formula 4 3]
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Trastuzuma b
Process 2
[0219]
Process 1: N- [3-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)propanoyl]glycylglycyl-L-phenylalanylglycyl-N-
[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl] -P-alaninamide
The compound (60.0 mg, 0.0646 mmol) obtained in
Process 2 of Example 15 was reacted in the same manner
as Process 3 of Example 2 by using N-succinimidyl 3 -
maleimide propionate instead of N-succinimidyl 6-
maleimide hexanoate to yield the titled compound as a
pale yellow solid (36.0 mg, 57%).
1H-NMR (400 MHz, DMSO-dd 8: 0.86 (3H, t, J=7.4 Hz),
1.85 (2H, dt, J=14.4, 7.5 Hz), 2.05-2.22 (2H, m), 2.40
(3H, s), 2.30-2.44 (SH, m), 2.73-2.84 (lH, m), 3.02 (lH,
dd, J=13.9, 4.5 Hz), 3.17 (3H, d, J=5.1 Hz), 3.26-3.40
(2H, m), 3.41-3.81 (6H, m), 4.40-4.51 (lH, m), 5.19 (lH,
d, J=19.2 Hz), 5.26 (lH, d, J=18.8 Hz), 5.42 (2H, brs),
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5.52-5.61 (lH, m), 6.53 (lH, s), 6.99 (2H, s), 7.13-
7.28 (SH, m), 7.31 (lH, s), 7.80 (2H, d, J=l0.2 Hz),
8.03 (lH, t, J=5.5 Hz), 8.12 (lH, d, J=8.2 Hz), 8.20-
8.31 (2H, m), 8.52 (lH, d, J=8.6 Hz).
MS (ESI) m/z: 976 (M+H)+
[0220]
Process 2: Antibody-drug conjugate (17)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 1 above,
the titled antibody-drug conjugate was obtained in the
same manner as Process 2 of Example 6.
Antibody concentration: 1.74 mg/mL, antibody yield:
10.4 mg (83%), and average number of conjugated drug
molecules (n) per antibody molecule: 3.7.
[0221]
Example 18 Antibody-drug conjugate (18)
[ Formula 44]
0 (if H O H O H
)rN~NJN¥NJN¥N~O
0 O H 00 · H O NH
{" \
~ 1/ " N
Process 2
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6. 6
Atty. Dkt. No. 111119-0105
[0222]
Process 1: Antibody-drug conjugate (18)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 1 of
Example 17, the titled antibody-drug conjugate was
obtained in the same manner as Process 1 of Example 7.
Antibody concentration: 1.98 mg/mL, antibody yield:
11. 9 mg (95%), and average number of conjugated drug
molecules (n) per antibody molecule: 6.6.
[0223]
Example 19 Antibody-drug conjugate (19)
[Formula 45]
[0224]
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4833-0752-7733.1
Process 2
1.9
Atty. Dkt. No. 111119-0105
Process 1: N-{3- [2- (2-{ [3- (2,5-Dioxo-2,5-dihydro-lHpyrrol-
1-
yl)propanoyl]amino})ethoxy]propanoyl}glycylglycyl-Lphenylalanylglycyl-
N- [(1S,9S)-9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl] -P-alaninamide
The compound (60.0 mg, 0.0646 mmol) obtained in
Process 2 of Example 15 was reacted in the same manner
as Process 3 of Example 2 by using N-succinimidyl 3-(2-
(2-(3-maleinimidepropanamide)ethoxy)ethoxy)propanoate
instead of N-succinimidyl 6-maleimide hexanoate to
yield the titled compound as a solid (23.0 mg, 31%).
1H-NMR (400 MHz, DMSO-dG) 8: 0.86 (3H, t, J=7.4 Hz),
1.77-1.92 (2H, m), 2.07-2.21 (2H, m), 2.27-2.42 (6H, m),
2 .40 (3H, s), 2. 74-2. 84 (lH, m), 2. 97-3. 06 (lH, m),
3 .09-3 .21 (4H, m), 3 .25-3 .39 (6H, m), 3 .45 (4H, s),
3.50-3.80 (8H, m), 4.41-4.51 (lH, m), 5.19 (lH, d,
J=18.4 Hz), 5.26 (lH, m, J=18.4 Hz), 5.42 (2H, brs),
5.51-5.61 (lH, m), 6.54 (lH, s), 7.00 (2H, s), 7.13-
7.28 (SH, m), 7.31 (lH, s), 7.74-7.87 (2H, m), 7.93-
8 . 0 7 ( 2H, m) , 8. 0 9 - 8. 21 ( 2H, m) , 8. 2 6 ( lH, brs) , 8. 54
(lH, d, J=8.6 Hz).
MS (ESI) m/z: 1135 (M+H)+
[0225]
Process 2: Antibody-drug conjugate (19)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 1 above,
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the titled antibody-drug conjugate was obtained in the
same manner as Process 2 of Example 6.
Antibody concentration: 1.60 mg/mL, antibody yield: 9.6
mg ( 77%) , and average number of conjugated drug
molecules (n) per antibody molecule: 1.9.
[0226]
Example 20 Antibody-drug conjugate (20)
[Formula 46]
Trastuzumab
[0227]
Process 1: Antibody-drug conjugate (20)
Process 1
3. 0
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 1 of
Example 19, the titled antibody-drug conjugate was
obtained in the same manner as Process 1 of Example 7.
Antibody concentration: 1.69 mg/mL, antibody yield:
10 .1 mg (81%), and average number of conjugated drug
molecules (n) per antibody molecule: 3.0.
[0228]
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Example 21 Antibody-drug conjugate (21)
[Formula 47]
Process 1
Trastuzumab
[0229]
Process 1: N- [19-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl) -17-oxo-4,7,10,13-tetraoxa-16-azanonadecan-1-
oyl]glycylglycyl-L-phenylalanylglycyl-N- [(1S,9S) -9-
ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-
2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl] -P-alaninamide
The compound (60.0 mg, 0.0646 mmol) obtained in
Process 2 of Example 15 was reacted in the same manner
as Process 3 of Example 2 by using N-succinimidyl 1-
maleinimide-3 -oxo- 7, 10, 13, 16 -tetraoxa-4 -
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azanonadecanoate instead of N-succinimidyl 6-maleimide
hexanoate to yield the titled compound as a solid (23.0
mg, 29%) .
1H-NMR (400 MHz, DMSO-dd 8: 0.86 (3H, t, J=7.0 Hz),
1.85 (2H, tt, J=14.6, 7.1 Hz), 2.06-2.22 (2H, m), 2.40
(3H, s), 2.28-2.43 (6H, m), 2.78 (lH, dd, J=13.7, 9.4
Hz), 3.02 (lH, dd, J=14.1, 3.9 Hz), 3.09-3.22 (4H, m),
3.27-3.41 (4H, m), 3.47 (12H, d, J=8.6 Hz), 3.53-3.81
(l0H, m), 4.41-4.51 (lH, m), 5.19 (lH, d, J=19.2 Hz),
5.26 (lH, d, J=18.8 Hz), 5.42 (2H, brs), 5.53-5.61 (lH,
m), 6.54 (lH, s), 7.00 (2H, s), 7.12-7.29 (SH, m), 7.31
(lH, s), 7.74-7.85 (2H, m), 8.03 (2H, d, J=6.6 Hz),
8.11-8.21 (2H, m), 8.27 (lH, t, J=5.9 Hz), 8.54 (lH, d,
J=8.6 Hz).
MS (ESI) m/z: 1224 (M+H)+
[0230]
Process 2: Antibody-drug conjugate (21)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 1, the
titled antibody-drug conjugate was obtained in the same
manner as Process 2 of Example 6.
Antibody concentration: 1.77 mg/mL, antibody yield:
10. 6 mg (85%), and average number of conjugated drug
molecules (n) per antibody molecule: 3.2.
[0231]
Example 22 Antibody-drug conjugate (22)
[ Formula 4 8]
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Trastuzumab
[0232]
Process 1: Antibody-drug conjugate (22)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 1 of
Example 21, the titled antibody-drug conjugate was
obtained in the same manner as Process 1 of Example 7.
Antibody concentration: 1.89 mg/mL, antibody yield:
11.3 mg (90%), and average number of conjugated drug
molecules (n) per antibody molecule: 6.2.
[0233]
Example 23 Intermediate (23)
[Formula 49]
[0234]
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Process 1: tert-Butyl (6-{ [(1S,9S)-9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl]amino}-6-oxohexyl)carbamate
Methanesulfonic acid salt of exatecan (0.500 g,
0.882 mmol) was reacted in the same manner as Process 1
of Example 1 by using 6-(tertbutoxycarbonylamino)
hexanoic acid instead of 4- (tertbutoxycarbonylamino)
butanoic acid to yield the titled
compound (0.620 g, quantitative).
1H-NMR (DMSO-dd 8: 0.83 (3H, t, J=7.8 Hz), 1.14-1.28
(2H, m), 1.31 (9H, s), 1.47-1.61 (2H, m), 1.75-1.89 (2H,
m), 2.04-2.17 (4H, m), 2.35 (3H, s), 2.81-2.88 (2H, m),
3.09-3.16 (2H, m), 5.10 (lH, d, J=19.4 Hz), 5.16 (lH, d,
J=19.4 Hz), 5.39 (2H, s), 5.48-5.55 (lH, m), 6.50 (lH,
s), 6.73-6.78 (lH, m), 7.26 (lH, s), 7.74 (lH, d,
J=l0.9 Hz), 8.39 (lH, d, J=9.0 Hz).
[0235]
Process 2: 6-Amino-N- [(1S,9S) -9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl]hexanamide
The compound (0.397 g, 0.611 mmol) obtained in
Process 1 above was reacted in the same manner as
Process 2 of Example 1 to yield trifluoroacetic
salt of the titled compound (0.342 g, 84%).
acid
1H-NMR (DMSO-dd 8: 0.88 (3H, t, J=7.2 Hz),
(2H, m), 1.52-1.70 (4H, m), 1.80-1.94 (2H,
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2.18 (2H, m), 2.21 (2H, t, J=7.4 Hz), 2.40 (3H, s),
2.81 (2H, t, J=7.4 Hz), 3.10-3.25 (2H, m), 3.33 (2H,
brs), 5.18 (lH, d, J=19.8 Hz), 5.22 (lH, d, J=19.8 Hz),
5.41 (2H, d, J=16.6 Hz), 5.45 (2H, d, J=16.6 Hz), 5.53-
5.60 (lH, m), 6.55 (lH, s), 7.32 (lH, s), 7.80 (lH, d,
J=l0.9 Hz), 8.49 (lH, d, J=9.2 Hz).
[0236]
Example 24 Antibody-drug conjugate (24)
[Formula 50]
Process 4
0 {;!~ 1 0 H O H 0
()~)lN~NJN NJN~O
0 H O H cj H .NH
Trastu,wm.ab
[0237]
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4833-0752-7733.1
/fi.O o OT,'\
YN~O N--,{
0 0
Process 5
Process 6
1.7
Atty. Dkt. No. 111119-0105
Process 1: N-(tert-Butoxycarbonyl)glycylglycyl-Lphenylalanyl-
N-(6-{ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-
4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-6-oxohexyl)glycinamide
The compound (0.170 g, 0.516 mmol) obtained in
Process 2 of Example 23 was reacted in the same manner
as Process 1 of Example 2 to yield the titled compound
(0.225 g, 91%).
1H-NMR (DMSO-dd 8: 0.88 (3H, t, J=7.4 Hz), 1.43-1.70
(6H, m), 1.87 (2H, td, J=15.0, 7.4 Hz), 2.10-2.22 (3H,
m), 2.28-2.37 (lH, m), 2.42 (3H, s), 2.78-2.85 (lH, m),
3.01-3.10 (3H, m), 3.15-3.22 (2H, m), 3.54-3.61 (SH, m),
3.62-3.69 (lH, m), 4.44-4.53 (lH, m), 5.17 (lH, d,
J=19.2 Hz), 5.25 (lH, d, J=19.2 Hz), 5.45 (2H, s),
5.54-5.61 (lH, m), 6.55 (lH, s), 7.02 (lH, t, J=6.1 Hz),
7.11-7.28 (SH, m), 7.33 (lH, s), 7.63-7.69 (lH, m),
7.82 (lH, d, J=ll.0 Hz), 7.90-7.96 (lH, m), 8.17 (lH, d,
J=7.8 Hz), 8.28 (lH, t, J=5.5 Hz), 8.46 (lH, d, J=9.0
Hz).
[0238]
Process 2: Glycylglycyl-L-phenylalanyl-N-(6-{ [(1S,9S) -
9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-
hexahydro-1H,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-6-oxohexyl)glycinamide
The compound (0.105 g, 0.108 mmol) obtained in
Process 1 above was reacted in the same manner as
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Process 2 of Example 2 to yield the titled compound
( 0 . 0 6 8 mg, 6 5 % ) .
1H-NMR (DMSO-dd 8: 0.89 (3H, t, J=7.4 Hz), 1.15-1.67
(6H, m), 1. 79-1. 97 (2H, m), 2. 08-2. 24 (4H, m), 2. 42 (3H,
s), 2.76-2.82 (lH, m), 3.00-3.10 (SH, m), 3.19 (lH, s),
3.50-3.63 (2H, m), 3.64-3.76 (3H, m), 3.84-3.92 (lH, m),
4.51-4.59 (lH, m), 5.17 (lH, d, J=19.4 Hz), 5.24 (lH, d,
J=19.4 Hz), 5.44 (2H, s), 5.53-5.61 (lH, m), 6.55 (lH,
brs), 7.15-7.29 (SH, m), 7.33 (lH, s), 7.72-7.78 (lH,
m), 7.82 (lH, d, J=ll.0 Hz), 7.96-8.08 (2H, m), 8.30-
8.38 (2H, m), 8.46-8.56 (2H, m).
[0239]
Process 3: N- [6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(6-{ [(1S,9S) -
9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-
hexahydro-1H,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-6-oxohexyl)glycinamide
The compound (58 mg, 0.060 mmol) obtained in
Process 2 above was reacted in the same manner as
Process 3 of Example 2 to yield the titled compound (39
mg, 62%) .
1H-NMR (CD3OD) 8: 0.99 (3H, t, J=7.4 Hz), 1.27 (2H, td,
J=ll.6, 6.1 Hz), 1.38-1.44 (2H, m), 1.50-1.63 (6H, m),
1.65-1.80 (2H, m), 1.89-1.98 (2H, m), 2.17-2.25 (3H, m),
2.26-2.36 (3H, m), 2.40 (3H, s), 2.95 (lH, dd, J=14.3,
9.2 Hz), 3.12 (lH, dd, J=13.7, 5.7 Hz), 3.15-3.25 (4H,
m), 3.44 (2H, t, J=7.2 Hz), 3.65 (lH, d, J=17.2 Hz),
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3.76 (lH, d, J=17.2 Hz), 3.79-3.86 (4H, m), 4.43 (lH,
dd, J=8.9, 6.0 Hz), 5.10 (lH, d, J=18.9 Hz), 5.25 (lH,
d, J=18.9 Hz), 5.35 (lH, d, J=16.6 Hz), 5.56 (lH, d,
J=16.0 Hz), 5.60-5.64 (lH, m), 6.76 (2H, s), 7.12-7.24
(6H, m), 7.58 (lH, s), 7.60 (lH, d, J=l0.9 Hz), 7.68
(lH, t, J=5.7 Hz).
MS (ESI) m/z: 1060 (M+H)+
[0240]
Process 4: Antibody-drug conjugate (24)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL with PBS6.0/EDTA by using the
Common procedure C-1 and Common procedure B (as
absorption coefficient at 280 nm,
used). The solution (9.0 mL) was
1. 3 7 mLmg- 1 cm- 1 was
collected into a 50
mL tube and charged with an aqueous solution of 10 mM
TCEP (0.140 mL; 2.3 equivalents per antibody molecule)
and an aqueous solution of 1 M dipotassium hydrogen
phosphate (0.450 mL). After confirming that the
solution had a pH of 7.4 ± 0.1, the disulfide bond at
the hinge part in the antibody was reduced by
incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
incubating the solution at 22°C for 10 minutes, a DMSO
solution (0.280 mL; 4.6 equivalents per antibody
molecule) containing 10 mM of the compound in Process 3
was added thereto and incubated for conjugating the
drug linker to the antibody at 22°C for 40 minutes.
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Next, an aqueous solution (0.0559 mL; 9.2 equivalents
per antibody molecule) of 100 mM NAC was added thereto
and incubated at 22°C to terminate the reaction of the
drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D-1 (PBS7.4 was
used as buffer solution) to yield a solution containing
the titled antibody-drug conjugate.
Physicochemical characterization: By using the Common
procedure E, the following characteristic values were
obtained.
Antibody concentration: 3.30 mg/mL, antibody yield:
53. 5 mg (59%), and average number of conjugated drug
molecules (n) per antibody molecule: 1.7.
[0241]
Example 25 Antibody-drug conjugate (25)
[Formula 51]
Process 1
Trastuzumab
[0242]
Process 1: Antibody-drug conjugate (25)
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25
Atty. Dkt. No. 111119-0105
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL with PBS6.0/EDTA by using the
Common procedure C-1 and Common procedure B (as
absorption coefficient at 280 nm, 1. 37 mLmg- 1 cm- 1 was
used). The solution (9.0 mL) was collected into a 50
mL tube and charged with an aqueous solution of 10 mM
TCEP (0.280 mL; 4.6 equivalents per antibody molecule)
and an aqueous solution of 1 M dipotassium hydrogen
phosphate (0.450 mL). After confirming that the
solution had a pH of 7.4 ± 0.1, the disulfide bond at
the hinge part in the antibody was reduced by
incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
incubating the solution at 22°C for 10 minutes, a DMSO
solution (0.559 mL; 9.2 equivalents per antibody
molecule) containing 10 mM of the compound in Process 3
of Example 24 was added thereto and incubated for
conjugating the drug linker to the antibody at 22°C for
40 minutes. Next, an aqueous solution (0.112 mL; 18.4
equivalents per antibody molecule) of 100 mM NAC was
added thereto and incubated at 22°C to terminate the
reaction of the drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D-1 (PBS6.0 was
used as buffer solution) to yield a solution containing
the titled antibody-drug conjugate.
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Physicochemical characterization: By using the Common
procedure E, the following characteristic values were
obtained.
Antibody concentration: 10.65 mg/mL, antibody yield:
55 .1 mg (61%), and average number of conjugated drug
molecules (n) per antibody molecule: 2.5.
[0243]
Example 26 Antibody-drug conjugate (26)
[Formula 52]
Process 3
4833-0752-7733.1
Process 4
0
HOJ0 ~
Process 2
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[0244]
Process 1: ({N- [(9H-Fluoren-9-
ylmethoxy)carbonyl]glycyl}amino)methyl acetate
To a mixture containing N-9-
f luorenylmethoxycarbonylglycylglycine ( 4. 33 g, 12. 2
mmol), tetrahydrofuran (THF; 120 ml), and toluene (40.0
ml), pyridine (1.16 ml, 14.7 mmol) and lead
tetraacetate (6.84 g, 14.7 mmol) were added and heated
under reflux for 5 hours. After the reaction solution
was cooled to room temperature, the insolubles were
removed by filtration through Celite, and concentrated
under reduced pressure. The residues obtained were
dissolved in ethyl acetate and washed with water and
saturated brine, and then the organic layer was dried
over anhydrous magnesium sulfate. After the solvent
was removed under reduced pressure, the residues
obtained were purified by silica gel column
chromatography [hexane : ethyl acetate 9 : 1 (v/v) -
ethyl acetate] to yield the titled compound as a
colorless solid (3. 00 g, 67%) .
1H-NMR (400 MHz, CDC1 3 ) 8: 2.07 (3H, s), 3.90 (2H, d,
J=5.1 Hz), 4.23 (lH, t, J=7.0 Hz), 4.46 (2H, d, J=6.6
Hz), 5.26 (2H, d, J=7.0 Hz), 5.32 (lH, brs), 6.96 (lH,
brs), 7.32 (2H, t, J=7.3 Hz), 7.41 (2H, t, J=7.3 Hz),
7.59 (2H, d, J=7.3 Hz), 7.77 (2H, d, J=7.3 Hz).
[0245]
Process 2: Benzyl [({N- [(9H-fluoren-9-
ylmethoxy)carbonyl]glycyl}amino)methoxy]acetate
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To a THF (40.0 mL) solution of the compound (3.68
g, 10. 0 mmol) obtained in Process 1 above and benzyl
glycolate (4.99 g, 30.0 mmol), potassium tert-butoxide
(2.24 g, 20.0 mmol) was added at 0°C and stirred at
room temperature for 15 minutes. The reaction solution
was charged with ethyl acetate and
extracted with ethyl acetate and
water at 0°C
chloroform.
and
The
obtained organic layer was dried over sodium sulfate
and filtered. The solvent was removed under reduced
pressure. The residues obtained were dissolved in
dioxane (40.0 mL) and water (10.0 mL), charged with
sodium hydrogen carbonate (1.01 g, 12.0 mmol) and 9-
fluorenylmethyl chloroformate (2.59 g, 10.0 mmol), and
stirred at room temperature for 2 hours. The reaction
solution was charged with water and extracted with
ethyl acetate. The obtained organic layer was dried
over sodium sulfate and filtered. The solvent was
removed under reduced pressure and the residues
obtained were purified by silica gel column
chromatography [hexane : ethyl acetate= 100 0 (v/v)
- 0 : 100] to yield the titled compound as a colorless
oily substance (1. 88 g, 40%) .
1H-NMR (400 MHz, CDC1 3 ) 8: 3. 84 (2H, d, J=5. 5 Hz), 4. 24
(3H, t, J=6.5 Hz), 4.49 (2H, d, J=6.7 Hz), 4.88 (2H, d,
J=6.7 Hz), 5.15-5.27 (lH, m), 5.19 (2H, s), 6.74 (lH,
brs), 7.31-7.39 (7H, m), 7.43 (2H, t, J=7.4 Hz), 7.61
(2H, d, J=7 .4 Hz), 7. 79 (2H, d, J=7 .4 Hz).
[0246]
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Process 3 : [({N- [(9H-Fluoren-9-
ylmethoxy)carbonyl]glycyl}amino)methoxy]acetic acid
The compound (1.88 g, 3.96 mmol) obtained in
Process 2 above was dissolved in ethanol (40.0 mL) and
ethyl acetate (20.0 ml). After adding palladium carbon
catalyst (376 mg), it was stirred under hydrogen
atmosphere at room temperature for 2 hours. The
insolubles were removed by filtration through Celite,
and the solvent was removed under reduced pressure to
yield the titled compound as a colorless solid (1.52 g,
quantitative).
1H-NMR (400 MHz, DMSO-dd 8: 3.62 (2H, d, J=6.3 Hz),
3.97 (2H, s), 4.18-4.32 (3H, m), 4.60 (2H, d, J=6.7 Hz),
7.29-7.46 (4H, m), 7.58 (lH, t, J=5.9 Hz), 7.72 (2H, d,
J=7.4 Hz), 7.90 (2H, d, J=7.4 Hz), 8.71 (lH, t, J=6.5
Hz).
[0247]
Process 4 : 9H-Fluoren-9-ylmethyl (2-{ [ (2-{ [ (1S, 9S) -9-
ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-
2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethoxy)methyl]amino}-2-
oxoethyl)carbamate
Under ice cooling, to an N,N-dimethylformamide
(10.0 mL) solution of methanesulfonic acid salt of
exatecan (0.283 g, 0.533 mmol), N-hydroxysuccinimide
(61.4 mg, 0.533 mmol), and the compound (0.205 g, 0.533
mmol) obtained in Process 3 above, N,N-
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diisopropylethylamine ( 92. 9 μL,
dicyclohexylcarbodiimide (0.143
0. 533 mmol)
g, 0.693
and
mmol)
N,N' -
were
added and stirred at room temperature for 3 days. The
solvent was removed under reduced pressure and the
residues obtained were purified by silica gel column
chromatography [chloroform - partitioned organic layer
of chloroform : methanol : water= 7 : 3 : 1 (v/v/v)]
to yield the titled compound as a pale brown solid
(0.352 g, 82%).
1H-NMR (400 MHz, DMSO-dd 8: 0.81 (3H, t, J=7.4 Hz),
1.73-1.87 (2H, m), 2.06-2.20 (2H, m), 2.34 (3H, s),
3.01-3.23 (2H, m), 3.58 (2H, d, J=6.7 Hz), 3.98 (2H, s),
4.13-4.25 (3H, m), 4.60 (2H, d, J=6.7 Hz), 5.09-5.22
(2H, m), 5.32-5.42 (2H, m), 5.50-5.59 (lH, m), 6.49 (lH,
s), 7.24-7.30 (3H, m), 7.36 (2H, t, J=7.4 Hz), 7.53 (lH,
t, J=6.3 Hz), 7.66 (2H, d, J=7.4 Hz), 7.75 (lH, d,
J=ll.0 Hz), 7.84 (2H, d, J=7.4 Hz), 8.47 (lH, d, J=8.6
Hz), 8.77 (lH, t, J=6.7 Hz).
MS (ESI) m/z: 802 (M+H)+
[0248]
Process 5: N- [(2-{ [(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-
4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethoxy)methyl]glycinamide
To an N,N-dimethylformamide (11.0 mL) solution of
the compound (0.881 g, 1.10 mmol) obtained in Process 4
above, piperidine ( 1 .1 mL) was added and stirred at
room temperature for 2 hours. The solvent was removed
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under reduced pressure to yield a mixture containing
the titled compound. The mixture was used for the next
reaction without further purification.
[0249]
Process 6: N-[(9H-Fluoren-9-
ylmethoxy)carbonyl]glycylglycyl-L-phenylalanyl-N- [(2-
{ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethoxy)methyl]glycinamide
Under ice cooling, to an N,N-dimethylformamide
(50.0 mL) solution of the mixture (0.439 mmol) obtained
in Process 5 above, N-hydroxysuccinimide (0.101 g,
0.878 mmol), and N- [(9H-fluoren-9-
ylmethoxy)carbonyl]glycylglycyl-L-phenylalanine (the
compound described in Japanese Patent Laid-Open No.
2002-60351; 0.440 g,
dicyclohexylcarbodiimide
added and stirred at room
0.878
(0.181 g,
mmol),
0. 878 mmol)
temperature for 4 days.
N,N' -
was
The
solvent was removed under reduced pressure and the
residues obtained were purified by silica gel column
chromatography [chloroform - chloroform methanol
9 1 (v/v)] to yield the titled compound as a pale
orange solid (0.269 g, 58%)
MS (ESI) m/z: 1063 (M+H)+
[0250]
Process 7: Glycylglycyl-L-phenylalanyl-N- [(2-{ [(1S,9S) -
9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-
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2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethoxy)methyl]glycinamide
To an N,N-dimethylformamide (4.00 mL) solution of
the compound (0.269 g, 0.253 mmol) obtained in Process
6 above, piperidine (0.251 mL, 2.53 mmol) was added and
stirred at room temperature for 2 hours. The solvent
was removed under reduced pressure to yield a mixture
containing the titled compound. The mixture was used
for the next reaction without further purification.
[0251]
Process 8: N-[6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-L-phenylalanyl-N- [(2-
{ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethoxy)methyl]glycinamide
To an N,N-dimethylformamide (10.0 mL) solution of
the compound (0.253 mmol) obtained in Process 7 above,
N-succinimidyl 6-maleimide hexanoate (0.156 g, 0.506
mmol) was added and stirred at room temperature for 3
days. The solvent was removed under reduced pressure
and the residues obtained were purified by silica gel
column chromatography [chloroform chloroform
methanol= 9 : 1 (v/v)] to yield the titled compound as
a pale yellow solid (0.100 g, 38%).
1H-NMR (400 MHz, DMSO-dd 8: 0.83 (3H, t, J=7.2 Hz),
1.09-1.21 (2H, m), 1.33-1.47 (4H, m), 1.75-1.90 (2H, m),
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2.00-2.23 (4H, m), 2.36 (3H, s), 2.69-2.81 (lH, m),
2.94-3.03 (lH, m), 3.06-3.22 (2H, m), 3.23-3.74 ( 6H, m),
3.98 (2H, s), 4.39-4.50 (lH, m), 4.60 (2H, d, J=6.7 Hz),
5.17 (2H, s), 5.39 (2H, s), 5.53-5.61 (lH, m), 6.50 (lH,
s), 6.96 (2H, s), 7.11-7.24 (SH, m), 7.28 (lH, s), 7.75
(lH, d, J=ll.0 Hz), 7.97 ( lH, t, J=5.7 Hz), 8.03 ( lH, t,
J=5.9 Hz), 8.09 (lH, d, J=7.8 Hz), 8.27 (lH, t, J=6.5
Hz) , 8. 4 8 ( lH, d, J = 9. 0 Hz) , 8. 6 0 ( lH, t, J = 6. 5 Hz) .
MS (ESI) m/z: 1034 (M+H)+
[0252]
Process 9: Antibody-drug conjugate (26)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 8 above,
the titled antibody-drug conjugate was obtained in the
same manner as Process 2 of Example 6.
Antibody concentration: 1.61 mg/mL, antibody yield: 9.7
mg ( 77%) , and average number of conjugated drug
molecules (n) per antibody molecule: 2.9.
[0253]
Example 27 Antibody-drug conjugate (27)
[ Formula 53]
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Process 1 Trastuzumab
56
[0254]
Process 1: Antibody-drug conjugate (27)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 8 of
Example 26, the titled antibody-drug conjugate was
obtained in the same manner as Process 1 of Example 7.
Antibody concentration: 1.58 mg/mL, antibody yield: 9.5
mg ( 76 % ) , and average number of conjugated drug
molecules (n) per antibody molecule: 5.6.
[0255]
Example 28 Antibody-drug conjugate (28)
[ Formula 54]
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Process 1
[0256]
Process 1: Antibody-drug conjugate (28)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg- 1 cm- 1 was used) . The solution (1.25 mL) was
placed in two 1. 5 mL polypropylene tubes and charged
with an aqueous solution of 10 mM TCEP (0.039 mL; 4.6
equivalents per antibody molecule) and an aqueous
solution of 1 M dipotassium hydrogen phosphate (0.0625
mL). After confirming that the solution had a pH of
7.4 ± 0.1, the disulfide bond at the hinge part in the
antibody was reduced by incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
adding DMSO ( 0. 072 mL) and a DMSO solution containing
10 mM of the compound of Process 8 of Example 26 (0.078
mL; 9.2 equivalents per antibody molecule) to the above
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solution at room temperature, it was stirred by using a
tube rotator for conjugating the drug linker to the
antibody at room temperature for 40 minutes. Next, an
aqueous solution of 100 mM NAC (0.0155 mL) was added
thereto and stirred at room temperature to terminate
the reaction of the drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D-1 (ABS was
used as a buffer solution) to yield 11.7 mL in total of
a solution containing the compound of interest.
Physicochemical characterization: By using the Common
procedure E, the following characteristic values were
obtained.
Antibody concentration: 1.60 mg/mL, antibody yield:
18. 7 mg (94%), and average number of conjugated drug
molecules (n) per antibody molecule: 5.2.
[0257]
Example 29 Antibody-drug conjugate (29)
[Formula 55]
Process 1 Trastuzumab
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[0258]
Process 1: Antibody-drug conjugate (29)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg- 1 cm- 1 was used) . The solution ( 6 mL) was
placed in a polypropylene tube and charged with an
aqueous solution of 10 mM TCEP (0.108 mL; 2.5
equivalents per antibody molecule) and an aqueous
solution of 1 M dipotassium hydrogen phosphate (0.091
mL) . After confirming that the solution had a pH of
7.0 ± 0.1, the disulfide bond at the hinge part in the
antibody was reduced by incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
adding DMSO ( 0 .146 mL) and a DMSO solution containing
10 mM of the compound of Process 8 of Example 26 (0.193
mL; 4.5 equivalents per antibody molecule) to the above
solution at room temperature, it was incubated for
conjugating the drug linker to the antibody at 15°C for
1 hour. Next, an aqueous solution (0.029 mL) of 100 mM
NAC was added thereto and stirred at room temperature
to terminate the reaction of the drug linker for
another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D (ABS was used
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as buffer solution) to yield 24 mL of a solution
containing the compound of interest.
Physicochemical characterization: By using the Common
procedures E and F (ED,2so = 5178 (measured value), and
ED,37o = 20217 (measured value) were used), the following
characteristic values were obtained.
Antibody concentration: 1.77 mg/mL, antibody yield: 42
mg (85%), average number of conjugated drug molecules
(n) per antibody molecule measured by the Common
procedure E: 3.0, and average number of conjugated drug
molecules (n) per antibody molecule measured by the
Common procedure F: 3.4.
[0259]
Example 30 Antibody-drug conjugate (30)
[Formula 56]
[0260]
Process 1: Antibody-drug conjugate (30)
62
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
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PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg- 1 cm- 1 was used) . The solution ( 6 mL) was
placed in a polypropylene tube and charged with an
aqueous solution of 10 mM TCEP (0.215 mL; 5 equivalents
per antibody molecule) and an aqueous solution of 1 M
dipotassium hydrogen phosphate (0.094 mL). After
confirming that the solution had a pH of 7.0 ± 0.1, the
disulfide bond at the hinge part in the antibody was
reduced by incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
adding a DMSO solution containing 10 mM of the compound
of Process 8 of Example 2 6 ( 0. 3 70 mL; 8. 6 equivalents
per antibody molecule) to the above solution at room
temperature, it was incubated for conjugating the drug
linker to the antibody at 15°C for 1 hour. Next, an
aqueous solution (0.056 mL) of 100 mM NAC was added
thereto and stirred at room temperature to terminate
the reaction of the drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D (ABS was used
as buffer solution) to yield 24 mL of a solution
containing the compound of interest.
Physicochemical characterization: By using the Common
procedures E and F (ED, 280 = 5178 (measured value), and
ED,37o = 20217 (measured value) were used), the following
characteristic values were obtained.
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Antibody concentration: 1.92 mg/mL, antibody yield: 46
mg (92%), average number of conjugated drug molecules
(n) per antibody molecule measured by the Common
procedure E: 6.2, and average number of conjugated drug
molecules (n) per antibody molecule measured by the
Common procedure F: 7.1.
[0261]
Example 31 Antibody-drug conjugate (31)
[Formula 57]
Process 1 Trastuz11mJC1b ---
[0262]
Process 1: Antibody-drug conjugate (31)
:6.6
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg- 1 cm- 1 was used) . The solution (50.00 mL) was
placed in a polypropylene container and charged with an
aqueous solution of 1 M dipotassium hydrogen phosphate
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( 0. 745 mL) at room temperature with stirring and then
with an aqueous solution of 10 mM TCEP (1.868 mL; 5.4
equivalents per antibody molecule) After confirming
that the solution had a pH of 7.0 ± 0.1, stirring was
terminated, and the disulfide bond at the hinge part in
the antibody was reduced by incubating at 37°C for 1
hour.
Conjugation between antibody and drug linker: After
cooling the above solution to 15°C, a DMSO solution
containing 10 mM of the compound of Process 8 of
Example 26 (2.958 mL; 8.6 equivalents per antibody
molecule) was gradually added dropwise thereto with
stirring. While the temperature was kept at 15°C, the
reaction solution was stirred for the first 30 minutes
and incubated without stirring for conjugating the drug
Next, an
was added
linker to the antibody for the next 1 hour.
aqueous solution (0.444 mL) of 100 mM NAC
thereto with stirring and stirred at room
to terminate the reaction of the drug
temperature
linker for
another 20 minutes.
Purification: By gradually adding 20% aqueous acetic
acid solution (about O. 25 mL) and ABS (50 mL) to the
above solution with stirring, the pH of the solution
was adjusted to 5.5 ± 0.1. This solution was subjected
to microfiltration (Millipore Corp., Millex-HV filter,
0.45 μm, PVDF membrane) to remove whitish matter. This
solution was subjected to ultrafiltration purification
using an ultrafiltration apparatus composed of an
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ultrafiltration membrane (Merck Japan, Pellicon XL
Cassette, Biomax 50 KDa), a tube pump (Cole-Parmer
International, MasterFlex Pump model 77521-40, Pump
Head model 7518-00), and a tube (Cole-Parmer
International, MasterFlex Tube L/S16) Specifically,
while ABS was added dropwise (a total of 800 mL) as a
buffer solution for purification to the reaction
solution, ultrafiltration purification was performed
for removing unconjugated drug linkers and other lowmolecular-
weight reagents, also replacing the buffer
solution with ABS, and further concentrating the
solution. The purified solution obtained was subjected
to microfiltration (0.22 μm (Millipore Corp., Millex-GV
filter, PVDF membrane) and 0.10 μm (Millipore Corp.,
Millex-VV filter, PVDF membrane)) to yield a solution
containing the titled antibody-drug conjugate.
Physicochemical characterization: By using the Common
procedures E and F (ED,2so = 5178 (measured value), and
ED,37o = 20217 (measured value) were used), the following
characteristic values were obtained.
Antibody
451 mg
molecules
concentration: 11.28 mg/mL, antibody
conjugated
yield:
( 90 % ) ,
(n)
average
per
number of
molecule measured by
Common procedure
conjugated drug
antibody
E: 6. 6, and average number
drug
the
of
molecules (n) per antibody
measured by the Common procedure F: 7.7.
[0263]
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molecule
Atty. Dkt. No. 111119-0105
Example 32 (Alternative method
compound of Process 8 of Example 26)
[Formula 58]
Process 3
[0264]
for synthesizing
Process 1: tert-Butyl N- [6-(2,5-Dioxo-2,5-dihydro-lHpyrrol-
1-yl)hexanoyl]glycylglycyl-L-phenyl alaninate
Under ice cooling, to a THF (12.0 ml) solution of
tert-butyl N- [(9H-fluoren-9-
ylmethoxy)carbonyl]glycylglycyl-L-phenyl alaninate (J.
Pept. Res., 1999, vol. 53, pp. 393; 0.400 g, 0.717
mmol), 1,8-diazabicyclo[5.4.0] -7-undecene (0.400 ml)
was added and stirred at room temperature for 4 days,
and then N-succinimidyl 6-maleimide hexanoate (0.221 g,
0.717 mmol) was further added and stirred for 3 hours.
The reaction solution was diluted with ethyl acetate
and washed with an aqueous solution of 10% citric acid,
a saturated aqueous solution of sodium hydrogen
carbonate, and saturated brine, and then the organic
layer was dried over anhydrous magnesium sulfate.
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After the sol vent was removed under reduced pressure,
the residues obtained were purified by silica gel
column chromatography [chloroform chloroform
methanol= 9 : 1 (v/v)] to yield the titled compound as
a pale yellow solid (0.295 g, 78%).
1H-NMR (400 MHz, CDC1 3 ) 8: 1.28-1.36 (2H, m), 1.41 (9H,
s), 1.57-1.71 (4H, m), 2.23 (2H, t, J=7.6 Hz), 3.09 (2H,
d, J=6.0 Hz), 3.51 (2H, t, J=7.6 Hz), 3.85-4.02 (4H, m),
4.69-4.78 (lH, m), 6.15 (lH, t, J=4.6 Hz), 6.33 (lH, d,
J=7.3 Hz), 6.60 (lH, t, J=5.0 Hz), 6.68 (2H, s), 7.10-
7.16 (2H, m), 7.22-7.31 (3H, m).
MS (ESI) m/z: 529 (M+H)+
[0265]
Process 2 : N- [6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-L-phenylalanine
To a dichloromethane (8.00 ml) solution of the
compound (0.295 g, 0.558 mmol) obtained in Process 1
above, trifluoroacetic acid (4.00 mL) was added and
stirred at room temperature for 18 hours. The solvent
was removed under reduced pressure to yield the titled
compound as a pale yellow solid (0.240 g, 91%).
1H-NMR (400 MHz, DMSO-dd 8: 1.15-1.23 (2H, m), 1.40-
1.53 (4H, m), 2.10 (2H, t, J=7.6 Hz), 2.88 (lH, dd,
J=13.7, 8.9 Hz), 3.04 (lH, dd, J=13.7, 5.0 Hz), 3.35-
3.43 (2H, m), 3.58-3.77 (4H, m), 4.41 (lH, td, J=7.8,
5.0 Hz), 7.00 (2H, s), 7.16-7.31 (SH, m), 8.00 (lH, t,
J=5.7 Hz), 8.06 (lH, t, J=5.7 Hz), 8.13 (lH, d, J=7.8
Hz).
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[0266]
Process 3: N- [6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-L-phenylalanyl-N- [(2-
{ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethoxy)methyl]glycinamide
The compound (0.572 g, 1.21 mmol) obtained in
Process 2 above was dissolved in dichloromethane (12.0
mL), charged with N-hydroxysuccinimide (0.152 g, 1.32
mmol) and 1-(3-dimethylaminopropyl) -3-ethylcarbodiimide
hydrochloride (0.253 g, 1.32 mmol), and stirred for 1
hour. The react ion so 1 u t ion was added to an N, N -
dimethylformamide (22.0 mL) solution of the mixture
(1.10 mmol) obtained in Process 5 of Example 26, and
stirred at room temperature for 3 hours. The reaction
solution was charged with an aqueous solution of 10%
citric acid and extracted with chloroform. The
obtained organic layer was dried over sodium sulfate
and filtered. The solvent was removed under reduced
pressure and
silica gel
chloroform
the residues obtained
column
methanol
chromatography
were purified
[chloroform
8 2 (v/v)] to yield
by
the
titled compound as a pale yellow solid (0.351 g, 31%).
The instrumental data of the compound was the same as
that of the compound of Process 8 of Example 26.
[0267]
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Example 33 (Alternative method for synthesizing
compound of Process 8 of Example 26)
[Formula 59]
~OH
HOJov O ~O 0~ 0
I % II V OJ-.N~NH
,,,, ,9 o--'ft"f0 o~ H o
----- ~ H HN..._,.O~,-, V --------
Process 1 ~ Ii O Process 2
Process 4
Process 5
[0268]
Process 1: Benzyl [ ( {N- [ (9H-fluoren-9-
ylmethoxy)carbonyl]glycyl}amino)methoxy]acetate
To a THF (200 ml) solution of the compound (7.37 g,
20.0 mmol) obtained in Process 1 of Example 26, benzyl
glycolate (6. 65 g, 40. 0 mmol) and p-toluene sulfonic
acid monohydrate (0.381 g, 2.00 mmol) were added at 0°C
and stirred at room temperature for 2 hours and 3 0
minutes. The reaction solution was charged with a
saturated aqueous solution of sodium hydrogen carbonate
and extracted with ethyl acetate. The obtained organic
layer was dried over sodium sulfate and filtered. The
solvent was removed under reduced pressure and the
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residues obtained were purified by silica gel column
chromatography [hexane ethyl acetate= 100 : 0 (v/v)
- 0 : 100] to yield the titled compound as a colorless
solid (6.75 g, 71%). The instrumental data of the
compound was the same as that of the compound of
Process 2 of Example 26.
[0269]
Process 2: N- [(Benzyloxy)carbonyl]glycylglycyl-Lphenylalanine-
N-{ [(2-(benzyloxy)-2-
oxoethoxy]methyl}glycinamide
To an N,N-dimethylformamide (140 mL) solution of
the compound (6.60 g, 13.9 mmol) obtained in Process 1
above, 1,8-diazabicyclo[5.4.0]undec-7-ene (2.22 g, 14.6
mmol) was added at 0°C and stirred at room temperature
for 15 minutes. The reaction solution was charged with
an N,N-dimethylformamide (140 mL) solution of N[
(benzyloxy) carbonyl] glycylglycyl-L-phenylalanine (6.33
g, 15.3 mmol), N-hydroxysuccinimide (1.92 g, 16.7 mmol),
and 1-(3-dimethylaminopropyl) -3-ethylcarbodiimide
hydrochloride (3.20 g, 16.7 mmol) stirred in advance at
room temperature for 1 hour, and stirred at room
temperature for 4 hours. The reaction solution was
charged with 0.1 N hydrochloric acid and extracted with
chloroform. The obtained organic layer was dried over
sodium sulfate and filtered. The sol vent was removed
under reduced pressure
purified by silica
and the residues
gel column
[chloroform - chloroform : methanol = 8
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obtained were
chromatography
2 (v/v)] to
Atty. Dkt. No. 111119-0105
yield the titled compound as a colorless solid (7.10 g,
79%) .
1H-NMR (DMSO-dd 8: 2.78 (lH, dd, J=13.9, 9.6 Hz), 3.05
(lH, dd, J=13.9, 4.5 Hz), 3.56-3.80 (6H, m), 4.15 (2H,
s), 4.47-4.55 (lH, m), 4.63 (2H, d, J=6.6 Hz), 5.03 (2H,
s), 5.15 (2H, s), 7.16-7.38 (15H, m), 7.52 (lH, t,
J=5.9 Hz), 8.03 (lH, t, J=5.5 Hz), 8.17 (lH, d, J=8.2
Hz), 8.36 (lH, t, J=5.7 Hz), 8.61 (lH, t, J=6.6 Hz).
[0270]
Process 3: Glycylglycyl-L-phenylalanyl-N-
[(carboxymethoxy)methyl]glycinamide
To an N,N-dimethylformamide (216 mL) solution of
the compound (7.00 g, 10.8 mmol) obtained in Process 2
above, palladium carbon catalyst (7.00 g) was added and
stirred under a hydrogen atmosphere at room temperature
for 24 hours. The insolubles were removed by
filtration through Celite, and the solvent was removed
under reduced pressure. The residues obtained were
dissolved in water, the insoluble material was removed
by filtration through Celite, and the solvent was
removed under reduced pressure. This procedure was
repeated twice to yield the titled compound as a
colorless solid (3. 77 g, 82%).
1H-NMR (DMSO-dd 8: 2.84 (lH, dd, J=13.7, 9.8 Hz), 3.08
(lH, dd, J=13.7, 4.7 Hz), 3.50-3.72 (4H, m), 3.77-3.86
(2H, m), 3.87 (2H, s), 4.52-4.43 (lH, m), 4.61 (2H, d,
J=6.6 Hz), 7.12-7.30 (SH, m), 8.43 (lH, t, J=5.9 Hz),
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8.54 (lH, d, J=7.8 Hz), 8.70 (lH, t, J=6.3 Hz), 8.79
(lH, t, J=5.5 Hz).
[0271]
Process 4: N- [6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-L-phenylalanyl-N[(
carboxymethoxy)methyl]glycinamide
To an N,N-dimethylformamide (85.0 mL) solution of
the compound (3.59 g, 8.48 mmol) obtained in Process 3
above, N-succinimidyl 6-maleimide hexanoate (2.88 g,
9.33 mmol) and triethylamine (0.858 g, 8.48 mmol) were
added and stirred at room temperature for 1 hour. The
reaction solution was charged with 0.1 N hydrochloric
acid and extracted with chloroform and a mixed solvent
of chloroform and methanol [chloroform : methanol= 4 :
1 (v/v)]. The obtained organic layer was dried over
sodium sulfate and filtered. The sol vent was removed
under reduced pressure and the residues obtained were
purified by silica gel column chromatography
[chloroform - partitioned organic layer of chloroform :
methanol water 7 3: 1 (v /v /v)] to yield the
titled compound as a colorless solid (3.70 g, 71%)
1H-NMR (DMSO-dd 8: 1.13-1.24 (2H, m), 1.42-1.53 (4H,
m), 2.11 (2H, t, J=7.4 Hz), 2.80 (lH, dd, J=13.7, 9.8
Hz), 3.06 (lH, dd, J=13.9, 4.5 Hz), 3.37 (2H, t, J=7.2
Hz), 3.56-3.78 ( 6H, m), 3.97 (2H, s), 4.46-4.53 (lH, m),
4.61 (2H, d, J=6.3 Hz), 7.00 (2H, s), 7.15-7.29 (SH, m),
8.03-8.20 (3H, m), 8.32 (lH, t, J=5.9 Hz), 8.60 (lH, t,
J=6.7 Hz).
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[0272]
Process 5: N- [6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-L-phenylalanyl-N- [(2-
{ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethoxy)methyl]glycinamide
To an N,N-dimethylformamide (40.0 mL) solution of
methanesulfonic acid salt of exatecan ( 1. 14 g, 2. 0 0
mmol), triethylamine (0.202 g, 2.00 mmol), the compound
(1.48 g, 2.40 mmol) obtained in Process 4 above, and 4-
(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride (0.993 g, 3.00 mmol) containing 16.4% water
were added at 0°C and stirred at room temperature for 1
hour. The sol vent was removed under reduced pressure
and the residues obtained were purified by silica gel
column chromatography [chloroform chloroform
methanol= 8 : 2 (v/v)] to yield the titled compound as
a pale yellow solid (1.69 g, 82%). The spectral data
of the compound was the same as that of the compound of
Process 8 of Example 26.
[0273]
Example 34 Intermediate (34)
[ Formula 6 0]
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MsOH
Process 1
F
[0274]
0
)lo~o
NH
I
Atty. Dkt. No. 111119-0105
Process 2
0
N~✓
F ;f-io
01-0
Process 1: 2-{ [(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-
methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethylacetate
Under ice cooling, to an N,N-dimethylformamide
(20. 0 mL) suspension of methanesulfonic acid salt of
exatecan (0.500 g,
diisopropylethylamine (0.492
0.941
mL,
mmol),
2.82 mmol)
N,Nand
acetoxyacetyl chloride (0.121 ml, 1.13 mmol) were added
and stirred at room temperature for 1 hour. The
solvent was removed under reduced pressure and the
residues obtained were purified by silica gel column
chromatography [chloroform - partitioned organic layer
of chloroform : methanol : water= 7 : 3: 1 (v/v/v)] to
yield the titled compound as a pale yellow solid (0.505
g, quantitative).
1H-NMR (400 MHz, DMSO-dd 8: 0.87 (3H, t, J=7.4 Hz),
1.81-1.92 (2H, m), 2.08 (3H, s), 2.08-2.22 (2H, m),
2.41 (3H, s), 3.14-3.21 (2H, m), 4.51 (2H, dd, J=19.4,
14.7 Hz), 5.22 (2H, dd, J=40.1, 19.0 Hz), 5.43 (2H, s),
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5.56-5.61 (lH, m), 6.53 (lH, s), 7.31 (lH, s), 7.81 (lH,
d, J=ll. 0 Hz), 8. 67 (lH, d, J=8. 6 Hz).
MS (ESI) m/z: 536 (M+H)+
[0275]
Process 2 : N- [(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-
methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl] -2-hydroxyacetamide
To a methanol (50.0 mL) suspension of the compound
( 0. 504 g, 0. 941 mmol) obtained in Process 1 above, a
THF (20. 0 ml) and an aqueous solution of 1 N sodium
hydroxide (4.00 ml, 4.00 mmol) were added and stirred
at room temperature for 1 hour. The reaction was
terminated by the addition of 1 N hydrochloric acid
(5.00 ml, 5.00 mmol), and the solvent was removed under
reduced pressure. The residues obtained were purified
by silica gel column chromatography [chloroform
partitioned organic layer of chloroform methanol
water= 7 3: 1 (v/v/v)] to yield the titled compound
as a pale yellow solid ( 0. 412 g, 8 9%) . This compound
was confirmed in the tumor of a mouse that received the
antibody-drug conjugate (45) or (46).
1H-NMR (400 MHz, DMSO-dd 8: 0.87 (3H, t, J=7.3 Hz),
1.78-1.95 (2H, m), 2.09-2.28 (2H, m), 2.39 (3H, s),
3.07-3.27 (2H, m), 3.96 (2H, d, J=6.0 Hz), 5.11-5.26
(2H, m), 5.42 (2H, s), 5.46-5.54 (lH, m), 5.55-5.63 (lH,
m), 6.52 (lH, s), 7.30 (lH, s), 7.78 (lH, d, J=l0.9 Hz),
8 .41 (lH, d, J=9 .1 Hz). MS (ESI) m/z: 494 (M+H) +
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[0276]
Example 35 (Alternative
compound of Example 34)
[Formula 61]
Process 1
[0277]
method for synthesizing
Process 1: N- [(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-
methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl] -2-hydroxyacetamide
Glycolic acid (0.0201 g, 0.27 mmol) was dissolved
in N,N-dimethylformamide (1.0 mL), charged with Nhydroxysuccinimide
(0.0302 g, 0.27 mmol) and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(0.0508 g, 0.27 mmol), and stirred for 1 hour. The
reaction solution was added to an N,N-dimethylformamide
suspension (1. 0 mL) charged with methanesulfonic acid
salt of exatecan (0.1 g, 0.176 mmol) and triethylamine
(0. 025 mL, 0 .18 mmol) and stirred at room temperature
for 24 hours. The sol vent was removed under reduced
pressure and
silica gel
chloroform
the residues obtained were purified
[chloroform
to yield
column
methanol
chromatography
10 1 (v/v)]
by
the
titled compound as a pale yellow solid (0.080 g, 92%).
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The spectral data of the compound was the same as that
of the compound obtained in Process 2 of Example 34.
[0278]
Example 36 Antibody-drug conjugate (36)
[Formula 62]
Process 1
[0279]
"'-"·
0 f-r)
4.7
Process 1: N- [4-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)butanoyl]glycylglycyl-L-phenylalanylglycyl-N-
[(1S,9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl] -P-alaninamide
The compound (60.0 mg, 0.0646 mmol) obtained in
Process 2 of Example 15 was reacted in the same manner
as Process 3 of Example 2 by using N-succinimidyl 4-
maleimide butyrate instead of N-succinimidyl 6-
maleimide hexanoate to yield the titled compound as a
pale white solid (24.0 mg, 38%).
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1H-NMR (400 MHz, DMSO-dd 8: 0.86 (3H, t, J = 7.2 Hz),
1 . 6 8 ( 2 H , qui n , J = 7 . 4 Hz ) , 1 . 7 8 - 1 . 9 2 ( 2 H , m) , 2 . 0 6 -
2.22 (2H, m), 2.10 (2H, t, J 7. 8 Hz) , 2. 31- 2. 4 3 ( 2H,
m), 2.40 (3H, s), 2.78 (lH, dd, J 13.7, 9.4 Hz), 3.01
( lH, dd, J = 13.7, 4.7 Hz), 3.17 (4H, d, J = 5.1 Hz),
3.29-3.40 (2H, m), 3.52-3.80 ( 6H, m), 4.40-4.51 (lH, m),
5.19 (lH, d, J = 18.4 Hz), 5.26 (lH, d, J = 18.8 Hz),
5.42 (2H, s), 5.52-5.61 (lH, m), 6.53 (lH, s), 6.99 (2H,
s), 7.12-7.28 (SH, m), 7.31 (lH, s), 7.74-7.84 (2H, m),
8.02 (lH, t, J = 5.9 Hz), 8.08-8.16 (2H, m), 8.25 (lH,
t, J = 5.9 Hz), 8.52 (lH, d, J = 8.2 Hz).
MS (ESI) m/z: 990 (M+H)+
[0280]
Process 2: Antibody-drug conjugate (33)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 1 above,
the titled antibody-drug conjugate was obtained in the
same manner as Process 2 of Example 6.
Antibody concentration: 1.75 mg/mL, antibody yield:
10. 5 mg (84%), and average number of conjugated drug
molecules (n) per antibody molecule: 4.7.
[0281]
Example 37 Antibody-drug conjugate (37)
[ Formula 6 3]
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Trastuzumab
Process 1
l 8.5
[0282]
Process 1: Antibody-drug conjugate (37)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 1 of
Example 36, the titled antibody-drug conjugate was
obtained in the same manner as Process 1 of Example 7.
Antibody concentration: 1.89 mg/mL, antibody yield:
11.3 mg (90%), and average number of conjugated drug
molecules (n) per antibody molecule: 8.5.
[0283]
Example 38 Intermediate (38)
[ Formula 64]
MsOH
NH
10
F ;! -,
4833-0752-7733.1
'-..._ .... ---t
0 l-0
H
x O ,(N ~ 0
, I O OH
Process 1
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Atty. Dkt. No. 111119-0105
[0284]
Process 1: tert-Butyl (5-{ [ (1S, 9S) -9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl]amino}-5-oxopentyl)carbamate
Methanesulfonic acid salt of exatecan (500 mg,
0.941 mmol) was reacted in the same manner as Process 1
of Example 1 by using 5-(tertbutoxycarbonylamino)
valeric acid instead of 4-(tertbutoxycarbonylamino)
butanoic acid to yield the titled
compound as a yellow-brown solid (571 mg, 96%) The
compound was used for the next reaction without
performing further purification.
MS(ESI)m/z:635(M+H)+
[0285]
Process 2: 5-Amino-N- [(1S,9S) -9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl]pentanamide
The compound (558 mg, 0.879 mmol) obtained in
Process 1 above was reacted in the same manner as
Process 2 of Example 1 to yield trifluoroacetate of the
titled compound as a yellow solid (363 mg, 64%).
1H-NMR (400 MHz, DMSO-dd 8: 0.88 (3H, t, J = 7.4 Hz),
1.52-1.71 (4H, m), 1.87 (2H, tt, J = 14.4, 6.9 Hz),
2.07-2.18 (2H, m), 2.22 (2H, t, J = 7.0 Hz), 2.40 (3H,
s), 2.76-2.88 (2H, m), 3.13-3.22 (2H, m), 5.18 (lH, d,
J = 18.8 Hz), 5.24 (lH, d, J = 18.8 Hz), 5.43 (2H, s),
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5.53-5.61 (lH, m), 6.55 (lH, s), 7.33 (lH, s), 7.65 (3H,
br.s.), 7.81 (lH, d, J = 11.3 Hz), 8.49 (lH, d, J = 8.6
Hz)
MS (ESI) m/z: 535 (M+H)+
[0286]
Example 39 Antibody-drug conjugate (39)
[Formula 65]
Process 1
Process 4
[0287]
Process 1: N-(tert-Butoxycarbonyl)glycylglycyl-Lphenylalanyl-
N-(5-{ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-
4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-5-oxopentyl)glycinamide
The compound (348 mg, 0.537 mmol) obtained in
Process 2 of Example 38 was reacted in the same manner
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as Process 1 of Example 2 to yield the titled compound
as a pale yellow solid (429 mg, 84%). The compound was
used for the next reaction without performing further
purification.
[0288]
Process 2: Glycylglycyl-L-phenylalanyl-N-(5-{ [(1S,9S) -
9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-
hexahydro-1H,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-5-oxopentyl)glycinamide
The compound (427 mg, 0.448 mmol) obtained in
Process 1 above was reacted in the same manner as
Process 2 of Example 2 to yield trifluoroacetate of the
titled compound as a yellow solid (430 mg, 99%).
1H-NMR (400 MHz, DMSO-dG) 8: 0.87 (3H, t, J = 7.2 Hz),
1.38-1.49 (2H, m), 1.54-1.66 (2H, m), 1.86 (2H, tt, J =
14.5, 7.0 Hz), 2.08-2.16 (2H, m), 2.19 (2H, t, J = 7.2
Hz), 2.40 (3H, s), 2.76 (lH, dd, J = 13.9, 10.0 Hz),
3.00-3.12 (3H, m), 3.14-3.21 (2H, m), 3.57 (2H, d, J =
4.7 Hz), 3.60-3.75 (3H, m), 3.87 (lH, dd, J = 16.8, 5.9
Hz), 4.55 (lH, td, J 9.0, 4.7 Hz), 5.16 (lH, d, J =
18.8 Hz), 5.23 (lH, d, J = 18.4 Hz), 5.44 (2H, s),
5.53-5.60 (lH, m), 6.55 (lH, s), 7.14-7.29 (SH, m),
7 . 3 2 ( lH, s) , 7 . 7 4 ( lH, t, J = 5 . 5 Hz) , 7 . 81 ( lH, d, J
= 10.9 Hz), 7.96 (3H, br.s.), 8.30-8.37 (lH, m), 8.44-
8.53 (2H, m).
MS (ESI) m/z: 853 (M+H)+
[0289]
4833-0752-7733.1
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Process 3: N- [6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-L-phenylalanyl-N-(5-{ [(1S,9S) -
9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-
hexahydro-1H,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-5-oxopentyl)glycinamide
The compound (60.0 mg, 0.0621 mmol) obtained in
Process 2 above was reacted in the same manner as
Process 3 of Example 2 to yield the titled compound as
a so 1 id ( 16 . 0 mg, 2 5 % ) .
1H-NMR (400 MHz, DMSO-dd 8: 0.87 (3H, t, J = 7.4 Hz),
1.13-1.21 (2H, m), 1.36-1.52 (6H, m), 1.53-1.65 (2H, m),
1. 79-1. 92 (2H, m), 2. 05-2 .15 (4H, m), 2 .19 (2H, s),
2.40 (3H, s), 2.79 (lH, dd, J = 13.7, 10.2 Hz), 2.98-
3.10 (3H, m), 3.12-3.21 (2H, m), 3.29-3.37 (2H, m),
3.53-3.79 (6H, m), 4.41-4.50 (lH, m), 5.16 (lH, d, J =
18.8 Hz), 5.23 (lH, d, J = 18.8 Hz), 5.43 (2H, s),
5.52-5.60 ( lH, m), 6.53 (lH, s), 6.99 (2H, s), 7.12-
7.28 (SH, m), 7.31 (lH, s), 7.63 (lH, t, J 5.7
7.80 (lH, d, J 10.6 Hz), 8.02 (lH, t, J 5.9
8.08 ( lH, t, J 5.7 Hz), 8.12 (lH, d, J 7.8
8.24 (lH, t, J = 5.7 Hz), 8.45 (lH, d, J = 8.6 Hz).
MS (ESI) m/z: 1046 (M+H)+
[0290]
Process 4: Antibody-drug conjugate (39)
Hz),
Hz),
Hz),
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL with PBS6.0/EDTA by using the
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Common procedure C-1 and Common procedure B (as
absorption coefficient at 280 nm, 1. 37 mLmg- 1 cm- 1 was
used). The solution (1.0 mL) was collected into a 2 mL
tube and charged with an aqueous solution of 10 mM TCEP
(0.0155 mL; 2.3 equivalents per antibody molecule) and
an aqueous solution of 1 M dipotassium hydrogen
phosphate (0.050 mL) After confirming that the
solution had a pH of 7.4 ± 0.1, the disulfide bond at
the hinge part in the antibody was reduced by
incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
incubating the solution for 10 minutes at 22°C, a DMSO
solution (0.0311 mL; 4.6 equivalents per antibody
molecule) containing 10 mM of the compound obtained in
Process 3 was added thereto and incubated for
conjugating the drug linker to the antibody at 22°C for
40 minutes. Next, an aqueous solution (0.00622 mL; 9.2
equivalents per antibody molecule) of 100 mM NAC was
added thereto and incubated at 22°C to terminate the
reaction of the drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D-1 (PBS6.0 was
used as buffer solution) to yield 6 mL of a solution
containing the titled antibody-drug conjugate.
Physicochemical characterization: By using the Common
procedure E, the following characteristic values were
obtained.
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Antibody concentration: 1.12 mg/mL, antibody yield:
6. 72 mg (67%), and average number of conjugated drug
molecules (n) per antibody molecule: 1.8.
[0291]
Example 40 Antibody-drug conjugate (40)
[Formula 66]
Trastuzumab
Process 1
[0292]
Process 1: Antibody-drug conjugate (40)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL with PBS6.0/EDTA by using the
Common procedure C-1 and Common procedure B (as
absorption coefficient at 280 nm, 1. 37 mLmg- 1 cm- 1 was
used). The solution (1.0 mL) was collected into a 2 mL
tube and charged with an aqueous solution of 10 mM TCEP
(0.0311 mL; 4.6 equivalents per antibody molecule) and
an aqueous solution of 1 M dipotassium hydrogen
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phosphate ( 0. 050 mL) . After confirming that the
solution had a pH of 7.4 ± 0.1, the disulfide bond at
the hinge part in the antibody was reduced by
incubating at 37°C for 1 hour.
Conjugation between antibody and drug linker: After
incubating the solution at 22°C for 10 minutes, a DMSO
solution (0.0622 mL; 9.2 equivalents per antibody
molecule) containing 10 mM of the compound obtained in
Process 3 of Example 39 was added thereto and incubated
for conjugating the drug linker to the antibody at 22°C
for 40 minutes. Next, an aqueous solution (0.0124 mL;
18. 4 equivalents per antibody molecule) of 100 mM NAC
was added thereto and incubated at 22°C to terminate
the reaction of the drug linker for another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D-1 (PBS6.0 was
used as buffer solution) to yield 6 mL of a solution
containing the titled antibody-drug conjugate.
Physicochemical characterization: By using the Common
procedure E, the following characteristic values were
obtained.
Antibody
5. 88 mg
molecules
[0293]
concentration: 0.98 mg/mL, antibody yield:
(59%), and average number of conjugated drug
(n) per antibody molecule: 3.4.
Example 41 Antibody-drug conjugate (41)
[Formula 67]
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H ~ Process 1
>'or(N,/'•oH
0
Process 5
Process 7
Tras1uz:umab -
Process 8
__- _ _ _____
[0294]
tert-Butyl
oxoethyl}carbamate
Process 1: {2- [(2-hydroxyethyl)amino] -2-
N-(tert-Butoxycarbonyl)glycine
was dissolved in dimethylformamide
adding aminoethanol g, 48
( 4. 2 g,
( 40 mL)
mmol)
24 mmol)
After
and 1-
hydroxybenzotriazole
( 2. 9
(3. 7 g, 24 mmol) and adding 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (6.9 g, 36 mmol), it was stirred at room
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temperature for 12 hours. The solvent was removed
under reduced pressure, and the residue was charged
with toluene for azeotropic distillation. The residue
obtained were purified by silica gel column
chromatography [ethyl acetate ethyl acetate
methanol = 10 1 (v/v)] to yield the titled compound
as a colorless oily substance (3.8 g, 72%).
1H-NMR (400 MHz, CDC1 3 ) 8: 1. 44 (9H, s), 1. 69 (lH, brs),
3.43 (2H, td, J = 5.9, 5.1 Hz), 3.71 (2H, t, J = 5.1
Hz), 3.79 (2H, d, J = 5.9 Hz), 5.22 (lH, brs), 6.62 (lH,
brs).
[0295]
Process 2 : 2-{[N-(tert-
Butoxycarbonyl)glycyl]amino}ethyl4-nitrophenylcarbonate
To a THF (23 mL) solution of the compound (1.0 g,
4.59 mmol) obtained in Process 1 above,
diisopropylethylamine (0. 80 mL,
nitrophenyl) carbonate (1.32 g,
4. 59 mmol)
6. 88 mmol)
and bis(4-
were added
and stirred at room temperature for 12 hours. The
sol vent was removed under reduced pressure, and the
residue obtained was purified by silica gel column
chromatography [hexane - hexane : ethyl acetate= 1 : 3
(v/v)] to yield the titled compound as a pale yellow
solid (1.13 g, 64%).
1H-NMR (400 MHz, CDC1 3 ) 8: 1.44 (lH, s), 3.66 (2H, td,
J 5. 1, 5. 9 Hz) , 3. 81 ( 2H, d, J = 5. 9 Hz) , 4. 3 6 ( 2H, t,
J 5.1 Hz), 5.07 (lH, s), 6.48-6.53 (lH, m), 7.38 (2H,
dt, J = 9.9, 2.7 Hz), 8.27 (2H, dt, J = 9.9, 2.7 Hz).
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[0296]
Process 3 : 2- ( { [ (tert-
Butoxycarbonyl)amino]acetyl}amino)ethyl[(lS,9S)-9-
ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-
2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]carbamate
To methanesulfonic acid salt of exatecan (0.70 g,
1.2 mmol), the compound (0.57 g, 1.5 mmol) obtained in
Process 2, and 1-hydroxybenzotriazole (3.7 g, 24 mmol),
dimethylformamide ( 23 mL) was added, and
diisopropylethylamine (0.43 mL, 2.5 mmol) was added and
stirred at room temperature for 12 hours. The solvent
was removed under reduced pressure, and the residue was
charged with toluene for azeotropic distillation. The
residue obtained were purified by silica gel column
chromatography [chloroform - chloroform methanol
10 1 (v/v)] to yield the titled compound (0.86 g,
quantitative) as a pale yellow solid.
1H-NMR (400 MHz, DMSO-dd 8: 0.87 (3H, t, J = 7.4 Hz),
1.35 (9H, s), 1.78-1.94 (lH, m), 2.07-2.17 (lH, m),
2.17-2.27 (lH, m), 2.37 (3H, s), 3.05-3.16 (lH, m),
3.19-3.26 (lH, m), 3.34-3.39 (2H, m), 3.50-3.56 (2H, m),
4.00-4.07 (lH, m), 4.13-4.21 (lH, m), 5.15-5.34 (3H, m),
5.44 (2H, s), 6.54 (lH, s), 6.90-6.96 (lH, m), 7.32 (lH,
s), 7.78 (lH, d, J = 11. 0 Hz), 7.93-8.07 (2H, m).
[0297]
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Process 4 : 2-(Glycylamino)ethyl[(lS,9S) -9-ethyl-5-
fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-
hexahydro-1H,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]carbamate
The compound (0.86 g, 2.1 mmol) obtained in
Process 3 above was dissolved in dichloromethane ( 15
mL). After adding trifluoroacetic acid (15 mL), it was
stirred for 1 hour. The sol vent was removed under
reduced pressure, and the residue was charged with
toluene for azeotropic distillation. The residue
obtained were purified by silica gel column
chromatography [chloroform - partitioned organic layer
of chloroform : methanol: water= 7 : 3 : 1 (v/v/v)] to
yield the titled compound as a pale yellow solid (0.86
g, 99%).
1H-NMR (400 MHz, DMSO-dd 8: 0.87 (3H, t, J = 7.2 Hz),
1.79-1.95 (2H, m), 2.06-2.18 (lH, m), 2.18-2.29 (lH, m),
2.38 (3H, s), 3.07-3.17 (lH, m), 3.20-3.29 (lH, m),
3.36-3.50 (2H, m), 3.51-3.62 (2H, m), 3.99-4.08 (lH, m),
4.22-4.31 ( lH, m), 5.16-5.35 ( 3H, m), 5.42 ( lH, d, J =
18.8 Hz), 5.46 ( lH, d, J = 18.8 Hz), 6.56 (lH, s), 7.34
( lH, s), 7.65 (2H, brs), 7.79 ( lH, d, J = 10.6 Hz),
7.99-8.06 (lH, m), 8.51 (lH, t, J = 5.5 Hz).
MS (APCI) m/z: 939 (M+H) +
[0298]
Process 5: N- [(9H-Fluoren-9-
ylmethoxy)carbonyl]glycylglycyl-L-phenylalanyl-N- [2-
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({ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]carbamoyl}oxy)ethyl]glycinamide
N- [(9H-fluoren-9-ylmethoxy)carbonyl]glycylglycylL-
phenylalanine (Japanese Patent Laid-Open No. 2002-
60351; 0.21 g, 0.41 mmol) was dissolved in N,Ndimethylformamide
(3 mL). After adding Nhydroxysuccinimide
(0.052 g, 0.45 mmol) and 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride
(0.086 g, 0.45 mmol), it was stirred for 1 hour. The
reaction solution was added dropwise to a N,Ndimethylformamide
solution (2 mL) charged with the
compound (0.24 g, 0.35 mmol) obtained in Process 4 and
triethylamine (0.078 mL, 0.45 mmol), and stirred at
room temperature for 1 hour. The solvent was removed
under reduced pressure, and the residue obtained was
purified by silica gel column chromatography
[chloroform - chloroform : methanol 8 2 (v/v)] to
yield the titled compound as a pale yellow solid (0.24
g, 65%).
1H-NMR (400 MHz, DMSO-dd 8: 0.86 (3H, t, J = 7.3 Hz),
1.79-1.90 (2H, m), 2.05-2.27 (2H, m), 2.36 (3H, s),
2.73-2.81 (lH, m), 2.98-3.12 (2H, m), 3.17-3.26 (lH, m),
3.35-3.42 (2H, m), 3.55-3.79 (6H, m), 4.00-4.10 (lH, m),
4.12-4.23 (2H, m), 4.23-4.29 (2H, m), 4.45-4.55 (lH, m),
5.13-5.33 (3H, m), 5.40 (lH, d, J = 17.2 Hz), 5.44 (lH,
d, J = 17.2 Hz), 6.53 (lH, s), 7.11-7.26 (SH, m), 7.26-
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7.33 (3H, m), 7.38 (2H, t, J = 7.6 Hz), 7.57 (lH, t, J
= 5. 9 Hz) , 7. 6 8 ( 2H, d, J 7. 4 Hz) , 7. 7 7 ( lH, d, J
11.0 Hz), 7.85 (2H, d, J = 9.0 Hz), 7.91-7.97 (lH, m),
7.98-8.05 (2H, m), 8.14 (lH, d, J = 7.8 Hz), 8.31-8.26
(lH, m).
MS (APCI) m/z: 1063 (M+H)+
[0299]
Process 6: Glycylglycyl-L-phenylalanyl-N- [2-({ [(1S,9S) -
9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-
2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]carbamoyl}oxy)ethyl]glycinamide
The compound (0.24 g, 0.35 mmol) obtained in
Process 5 above was reacted in the same manner as
Process 7 of Example 26 to yield the titled compound as
a pale yellow solid (0.12 g, 65%).
1H-NMR (400 MHz, DMSO-dd 8: 0.86 (3H, t, J = 7.4 Hz),
1.78-1.94 (2H, m), 2.06-2.27 (2H, m), 2.37 (3H, s),
2.72-2.81 (lH, m), 2.98-3.07 (lH, m), 3.12-3.17 (2H, m),
3.57-3.81 ( 6H, m), 4.00-4.21 (3H, m), 4.45-4.54 (lH, m),
5.15-5.35 (3H, m), 5.41 (lH, d, J = 17.2 Hz), 5.45 (lH,
d, J = 17.2 Hz), 6.54 ( lH, s), 7.11-7.26 ( 6H, m), 7.32
( lH, s), 7.78 ( lH, d, J = 11. 0 Hz), 7.93-8.00 (lH, m),
8.03 (lH, d, J = 9.4 Hz), 8.06-8.13 (lH, m), 8.21-8.27
(2H, m), 8.30-8.36 (lH, m).
MS (APCI) m/z: 841 (M+H)+
[0300]
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Process 7: N- [6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-L-phenylalanyl-N- [2-
({ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]carbamoyl}oxy)ethyl]glycinamide
The compound (42.0 mg, 0.0499 mmol) obtained in
Process 6 was reacted in the same manner as Process 3
of Example 2 to yield the titled compound as a pale
yellow solid (38.3 mg, 74%).
1H-NMR (400 MHz, DMSO-dd 8: 0.87 (3H, t, J = 7.4 Hz),
1.12-1.23 (2H, m), 1.40-1.51 (4H, m), 1.80-1.95 (2H, m),
2.05-2.27 (4H, m), 2.38 (3H, s), 3.43-2.40 (8H, m),
3.53-3.78 (6H, m), 4.00-4.21 (2H, m), 4.44-4.55 (lH, m),
5.17-5.36 (3H, m), 5.43 (2H, s), 6.54 (lH, s), 6.99 (2H,
s), 7.19 (SH, d, J = 23.9 Hz), 7.33 (lH, s), 7.78 (lH,
d, J = 10.6 Hz), 7.91-8.16 (SH, m), 8.24-8.31 (lH, m).
MS (ESI) m/z: 1034 (M+H)+
[0301]
Process 8: Antibody-drug conjugate (41)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 7 above,
the titled antibody-drug conjugate was obtained in the
same manner as Process 2 of Example 6.
Antibody concentration: 1.54 mg/mL, antibody yield: 9.2
mg (74%), and average number of conjugated drug
molecules (n) per antibody molecule: 3.7.
[0302]
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Example 42 Antibody-drug conjugate (42)
[Formula 68]
Trastuzumab
[0303]
Process 1: Antibody-drug conjugate (42)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 7 of
Example 41, the titled antibody-drug conjugate was
obtained in the same manner as Process 1 of Example 7.
Antibody concentration: 1.47 mg/mL, antibody yield: 8.8
mg ( 71%) , and average number of conjugated drug
molecules (n) per antibody molecule: 7.0.
[0304]
Example 43 Antibody-drug conjugate (43)
[Formula 69]
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Process 2
[0305]
Process 1: N-{3- [2- (2-{ [3- (2,5-Dioxo-2,5-dihydro-lHpyrrol-
1-
yl)propanoyl]amino}ethoxy)ethoxy]propanoyl}glycylglycyl
-L-phenylalanyl-N- [(2-{ [(1S,9S)-9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl]amino}-2-oxoethoxy)methyl]glycinamide
The compound (53.7 mg, 50.5 μmol) obtained in
Process 6 of Example
dimethylformamide (1.50
26
mL)
was dissolved in N, NAfter
adding 1,8-
diazabicyclo(5.4.0)-7-undecene (7.5 μL, 50.5 μmol), it
was stirred at room temperature for 30 minutes. The
reaction solution was charged with pyridinium ptoluenesulfonate
(14.0 mg, 5.56 μmol), then charged
with N-succinimidyl 3- (2- (2- (3-
maleinimidopropanamido)ethoxy)ethoxy)propanoate ( 3 2. 3
mg, 75.8 μmol), and stirred at room temperature for
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2.25 hours. The solvent was removed under reduced
pressure, and the residue obtained was purified by
silica gel column chromatography [ [chloroform
partitioned organic layer of chloroform methanol
water= 7 3 : 1 (v/v/v)] to yield the titled compound
as a pale yellow solid (27.1 mg, 47%) .
1H-NMR (DMSO-dd 8: 0.87 (3H, t, J = 7.0 Hz), 1. 79-1. 91
(2H, m), 2.18 (2H, t, J = 15.1 Hz), 2.29-2.33 (4H, m),
2.39 ( 3H, s), 2.76 (lH, dd, J = 13.9, 9.2 Hz), 3.02 (lH,
dd, J = 13.7, 3.9 Hz), 3.13-3.15 (2H, m), 3.44-3.46 ( 6H,
m), 3.57-3.59 ( 6H, m), 3.69-3.75 ( 6H, m), 4.01 (2H, s),
4.46-4.48 (lH, m), 4.63 (2H, d, J = 6.3 Hz), 5.21 (2H,
s), 5.42 (2H, s), 5.60 (lH, dd, J = 13.5, 5.7 Hz), 6.54
(lH, s), 7.00 (2H, s), 7.17-7.24 (6H, m), 7.31 (lH, s),
7.79 (lH, d, J = 11.0 Hz), 8.00-8.02 (2H, m), 8.13 (lH,
d, J = 7. 8 Hz) , 8. 1 7 ( lH, t, J = 6. 3 Hz) , 8. 5 2 ( lH, d,
J = 9.0 Hz), 8.65 (lH, t, J
MS (ESI) m/z = 1151 (M+H)+
[0306]
6. 5 Hz).
Process 2: Antibody-drug conjugate (43)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 1, the
titled antibody-drug conjugate was obtained in the same
manner as Process 1 of Example 7.
Antibody concentration: 1.96 mg/mL, antibody yield:
17. 6 mg (88%), and average number of conjugated drug
molecules (n) per antibody molecule: 5.6.
[0307]
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Example 44 Antibody-drug conjugate (44)
[Formula 70]
Process 5
F
Process 6
Trastuzumab
Process 7
[0308]
Process 2
Process tert-Butyl
[(benzyloxy)carbonyl]glycylglycyl-D-phenylalaninate
1:
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NAtty.
Dkt. No. 111119-0105
N- [(Benzyloxy)carbonyl]glycylglycine (3.00 g, 11.3
mmol) was dissolved in N,N-dimethylformamide (20.0 mL).
After adding N-hydroxysuccinimide (1.43 g, 12.4 mmol)
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (2.37 g, 12.4 mmol), it was stirred for 1
hour. A N,N-dimethylformamide solution (10 mL) charged
with D-phenylalanine tert-butyl (2. 74 g, 12. 38 mmol)
and triethylamine (1.73 mL, 12.4 mmol) was added
dropwise to the reaction solution and stirred at room
temperature for 2 hours. The reaction solution was
charged with dichloromethane and washed with water, 1 N
hydrochloric acid, and a saturated aqueous solution of
sodium bicarbonate, and then the organic layer was
dried over anhydrous sodium sulfate. The solvent was
removed under reduced pressure, and the residue
obtained was purified by silica gel column
chromatography [chloroform - chloroform methanol
9 1 (v/v)] to yield the titled compound as a
colorless solid (4.21 g, 80%).
1H-NMR (CDCl3) 8: 1. 41 (9H, s), 3. 03-3 .14 (2H, m),
3.86-3.97 (4H, m), 4.70-4.77
5 . 4 3 ( lH, brs) , 6 . 4 2 ( lH, d,
(lH, m), 7.11-7.15 (2H, m),
7.38 (4H, m).
MS (APCI) m/z: 470 (M+H)+
[0309]
(lH, m), 5.13
J = 10.0 Hz),
7. 2 0 - 7. 31 ( 4H,
(2H, s),
6.64-6.71
m), 7.31-
Process 2: N- [(Benzyloxy)carbonyl]glycylglycyl-Dphenylalanine
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The compound (4.21 g,
Process 1 was dissolved in
8.97 mmol) obtained in
ethyl acetate (20 mL).
After adding an ethyl acetate solution (20.0 mL) of 4 N
hydrogen chloride, it was left overnight at room
temperature. The solvent was removed under reduced
pressure, then toluene was added, and the solvent was
removed under reduced pressure. The residue obtained
was purified by silica gel column chromatography
[chloroform - partitioned organic layer of chloroform :
methanol water = 7 3 1 (v/v/v)] to yield the
titled compound as a colorless solid (1.66 g, 45%).
1H-NMR (CDCl3) 8: 2.92-3.01 (lH, m), 3.10-3.18 (lH, m),
3.65-3.81 (3H, m), 3.88-3.98 (lH, m), 4.64-4.73 (lH, m),
5.06 (2H, s), 5.87 (lH, brs), 7.10-7.37 (13H, m)
MS (APCI) m/z: 412 (M+H) -
[0310]
Process 3 : N- [(Benzyloxy)carbonyl]glycylglycyl-Dphenylalanyl-
N-{ [2-(benzyloxy)-2-
oxoethoxy]methyl}glycinamide
To a dioxane (25. 0 mL) solution of the compound
(1.25 g, 2.63 mmol) obtained in Process 1 of Example 32,
piperidine (5.00 mL) and N,N-dimethylformamide (5.00
mL) were added and stirred at room temperature for 30
minutes. The solvent was removed under reduced
pressure, and the residue
N,N-dimethylformamide (20.0
compound (1.20 g, 2.90 mmol)
obtained was dissolved in
mL) . After adding the
of Process 2 above and 4-
(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
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chloride (1.03 g, 3.16 mmol) containing 16.4% water, it
was stirred at room temperature for 2 hours. The
reaction solution was charged with chloroform and
washed with water, and then the organic layer was dried
over anhydrous sodium sulfate. The solvent was removed
under reduced pressure, and the residue obtained was
purified by silica gel column
[chloroform - chloroform : methanol = 9
chromatography
1 (v/v)] to
yield the titled compound as a colorless solid (270 mg,
16%) .
1H-NMR (DMSO-dd 8: 2.78 (lH, dd, J = 13.6, 10.0 Hz),
3.05 (lH, dd, J = 13.9, 4.2 Hz), 3.56-3.79 (6H, m),
4.15 (2H, s), 4.47-4.54 (lH, m), 4.63 (2H, d, J = 6.7
Hz), 5.03 (2H, s), 5.15 (2H, s), 7.14-7.39 (15H, m),
7.50 ( lH, t, J 5.7 Hz), 8.02 (lH, t, J 5.4 Hz),
8.16 ( lH, d, J 7.9 Hz), 8.34 (lH, t, J 6.0 Hz),
8.60 (lH, t, J = 7.0 Hz).
MS (APCI) m/z: 648 (M+H) +
[0311]
Process 4: Glycylglycyl-D-phenylalanyl-N-
[(carboxymethoxy)methyl]glycinamide
The compound (200 mg, 0.31 mmol) obtained in
Process 3 above was dissolved in N,N-dimethylformamide
(5. 0 mL). After adding 5% palladium carbon catalyst
(0.12 g), it was stirred under a hydrogen atmosphere at
room temperature for 9 hours. The reaction solution
was filtered through Celite, and the residue was washed
with a mixed solvent of water and N,N-dimethylformamide.
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The filtrate and the wash were combined, and the
solvent was removed under reduced pressure to yield the
tit led compound as a colorless solid ( 0. 15 g,
quantitative).
1H-NMR (DMSO-dd 8: 2.85 (lH, dd, J = 13.3, 9.7 Hz),
3.08 (lH, dd, J 13.9, 5.4 Hz), 3.43-3.52 (4H, m),
3.62-3.89 (7H, m), 4.36-4.44 (lH, m), 4.58-4.67 (2H, m),
7.12-7.29 (SH, m), 8.44 (lH, t, J = 5.7 Hz), 8.67 (lH,
d, J = 7.3 Hz), 8.78 (lH, t, J = 5.4 Hz), 8.91 (lH,
brs).
MS (APCI) m/z: 424 (M+H)+
[0312]
Process 5: N- [6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-D-phenylalanyl-N-
[(carboxymethoxy)methyl]glycinamide
The compound (0.15 g, 0.35 mmol) obtained in
Process 4 above was dissolved in N,N-dimethylformamide
(10 mL). After adding N-succinimidyl 6-maleimide
hexanoate (0.11 g, 0.35 mmol), it was stirred at room
temperature for 1 hour. The reaction solution was
charged with chloroform and washed with water, and then
the organic layer was dried over anhydrous sodium
sulfate. The solvent was removed under reduced
pressure, and
silica gel
the residue obtained
column chromatography
was purified
[chloroform
partitioned organic layer of chloroform methanol
by
water= 7 : 3 : 1 (v/v/v)] to yield the titled compound
as a colorless solid (41 mg, 26%).
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1H-NMR (DMSO-dd 8: 1.13-1.24 (2H, m), 1.42-1.53 (4H,
m), 2.12 (2H, t, J = 7.3 Hz), 2.82 (lH, dd, J 13.9,
10.0 Hz), 3.09 (lH, dd, J = 13.9, 4.8 Hz), 3.17 (2H, d,
J 4.2 Hz), 3.47-3.89 (8H, m), 4.08-4.14 (lH, m),
4.41-4.49 (lH, m), 4.58-4.69 (2H, m), 7.00 (2H, s),
7.14-7.27 (SH, m), 8.31 (lH, t, J = 6.0 Hz), 8.39 (lH,
brs), 8.55 (2H, brs), 8.93 (lH, brs).
MS (APCI) m/z: 615 (M-H) -
[0313]
Process 6: N-[6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-1-
yl)hexanoyl]glycylglycyl-D-phenylalanyl-N- [(2-
{ [(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-
dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]amino}-2-oxoethoxy)methyl]glycinamide
To a N,N-dimethylformamide (10 mL) solution of
methanesulfonic acid salt of exatecan (22 mg, 0. 3 88
mmol) , triethylamine ( 5. 42
compound (29 mg, 0.466 mmol)
μL, 0. 3 88
obtained
mmol), the
in Process 5
above, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride (19 mg, 0.686 mmol)
containing 16.4% water were added at 0°C and stirred at
room temperature for 1 hour. The solvent in the
reaction solution was removed under reduced pressure,
and then the residue obtained was purified by silica
gel column chromatography [chloroform partitioned
organic layer of chloroform : methanol water = 7
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3 : 1 (v/v/v)] to yield the titled compound as a pale
yellow solid (26 mg, 65%).
1H-NMR (DMSO-dd 8: 0.87 (3H, t, J = 7.3 Hz), 1.12-1.22
(2H, m), 1.40-1.51 (4H, m), 1.79-1.92 (2H, m), 2.09 (2H,
t, J = 7.6 Hz), 2.13-2.23 (2H, m), 2.39 (3H, s), 2.78
(lH, dd, J 13.6, 9.4 Hz), 2.98-3.05 (lH, m), 3.13-
3.23 (2H, m), 3.54-3.78 (8H, m), 4.02 (2H, s), 4.41-
4.50 (lH, m), 4.61-4.66 (2H, m), 5.21 (2H, s), 5.42 (2H,
s), 5.56-5.64 (lH, m), 6.53 (lH, s), 6.99 (2H, s),
7 . 14 - 7 . 2 7 (SH, m) , 7 . 31 ( lH, s) , 7 . 7 9 ( lH, d, J = 10 . 9
Hz), 8.01 (lH, t, J = 5.4 Hz), 8.07 (lH, t, J = 5.7 Hz),
8.14 (lH, d, J = 7.9 Hz), 8.31 (lH, t, J 5.7 Hz),
8.53 (lH, d, J = 9.1 Hz), 8.63 (lH, t, J = 6.3 Hz).
MS (APCI) m/z: 1034 (M+H)+
[0314]
Process 7: Antibody-drug conjugate (44)
By using the trastuzumab produced in Reference
Example 1 and the compound obtained in Process 6 above,
the titled antibody-drug conjugate was obtained in the
same manner as Process 1 of Example 7.
Antibody concentration: 1.87 mg/mL, antibody yield:
16. 8 mg (84%), and average number of conjugated drug
molecules (n) per antibody molecule: 6.1.
[0315]
Example 45 Intermediate (45)
[Formula 71]
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[0316]
H2N~yo
NH
'-. 0
N-{
F -~N -<. ·_
\\- -( ' 0
H01--< > 0
Process 1: tert-Butyl (2-{ [(1S,9S)-9-ethyl-5-fluoro-9-
hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydrolH,
12H-benzo[de]pyrano[3' ,4' :6,7]indolizino[l,2-
b]quinolin-1-yl]amino}-2-oxoethyl)carbamate
To a dichloromethane (3.00 mL) solution of N(
tert-butoxycarbonyl)-glycine (0.395 g, 2.26 mmol), Nhydroxysuccinimide
(0.260 g, 2.26 mmol) and 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride
( 0. 433 mg, 2. 2 6 mmol) were added and stirred at room
temperature for 1 hour. This solution was added to a
solution consisting of methanesulfonic acid salt of
exatecan (1.00 g, 1.88 mmol), triethylamine (0.315 mL,
2.26 mmol), and N,N-dimethylformamide (3.00 mL) and
stirred at room temperature for 16.5 hours. The
reaction solution was diluted with chloroform and
washed with 10% citric acid solution, and then the
organic layer was dried over anhydrous sodium sulfate.
The solvent was removed under reduced pressure, and the
residue obtained was purified by silica gel column
chromatography [chloroform - chloroform methanol
9 1 (v/v)] to yield the titled compound as a yellow
solid (1.16 g, 99%).
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1H-NMR (400 MHz, DMSO-dd 8: 0.86 (3H, t, J = 7.2 Hz),
1.30 (9H, s), 1.81-1.89 (2H, m), 2.09-2.21 (2H, m),
2.38 (3H, s), 3.15-3.17 (2H, m), 3.55-3.56 (2H, m),
5.15 (lH, d, J = 18.8 Hz), 5.23 (lH, d, J = 19.2 Hz),
5.41 (2H, s), 5.55-5.56 (lH, m), 6.53 (lH, s), 6.95 (lH,
t, J = 5.5 Hz), 7.28 (lH, s), 7.77 (lH, d, J = 11.0 Hz),
8. 3 9 ( lH, d, J = 8. 6 Hz) .
MS (APCI) m/z: 593 (M+H)+
[0317]
Process 2 : N- [(1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-
methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-lH,12Hbenzo[
de]pyrano[3' ,4' :6,7]indolizino[l,2-b]quinolin-1-
yl]glycinamide
The compound (0.513 g, 1.01 mmol) obtained in
Process 1 above was reacted in the same manner as
Process 2 of Example 1 to yield the titled compound as
a yellow solid (0.463 g, 93%).
1H-NMR (400 MHz, CD3OD) 8: 0.96 (3H, t, J = 7.0 Hz),
1.89-1.91 (2H, m), 2.14-2.16 (lH, m), 2.30 (3H, s),
2.40-2.42 (lH, m), 3.15-3.21 (2H, m), 3.79-3.86
4.63-4.67 ( lH, m), 5.00-5.05 ( lH, m), 5.23 ( lH,
16.0 Hz), 5.48 ( lH, d, J = 16.0 Hz), 5.62-5.64
7.40-7.45 (2H, m).
MS (APCI) m/z: 493 (M+H) +
[0318]
Example 46 Antibody-drug conjugate (46)
[Formula 72]
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(2H, m),
d, J =
(lH, m),
Atty. Dkt. No. 111119-0105
Process 1
Trastu:z.umab
--------· OH 0
6.0
[0319]
Process 1: Antibody-drug conjugate (46)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg- 1 cm- 1 was used) . The solution (50 mL) was
placed in a 125 mL polycarbonate Erlenmeyer flask,
charged with an aqueous solution of 1 M dipotassium
hydrogen phosphate at room temperature (0.750 mL) with
stirring using a magnetic stirrer, and then charged
with an aqueous solution of 10 mM TCEP (1.857 mL; 5.4
equivalents per antibody molecule) After confirming
that the solution had a pH of 7.0 ± 0.1, stirring was
terminated, and the disulfide bond at the hinge part in
the antibody was reduced by incubating at 37°C for 1
hour.
Conjugation between antibody and drug linker: After
cooling the above solution to 15°C, a DMSO solution
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containing
Example 26
molecule)
10 mM of the compound of
(2.958 mL; 8.6 equivalents
was gradually added dropwise
Process 8 of
per antibody
thereto with
stirring. At 15°C, the reaction solution was stirred
for the first 30 minutes and incubated without stirring
for conjugating the drug linker to the antibody for the
next 1 hour. Next, an aqueous solution (0.444 mL; 12.9
equivalents per
added thereto
temperature to
antibody molecule) of 100 mM NAC was
with stirring and stirred at room
terminate the reactivity of unreacted
drug linkers for another 20 minutes.
Purification: By gradually adding 20% aqueous acetic
acid solution (about 0. 25 mL) and ABS (50 mL) to the
above solution with stirring, the pH of the solution
was adjusted to 5.5 ± 0.1. This solution was subjected
to microfiltration (Millipore Corp., Millex-HV filter,
0.45 μm, PVDF membrane) to remove whitish matter. This
solution was subjected to ultrafiltration purification
using an ultrafiltration apparatus composed of an
ultrafiltration membrane (Merck Japan, Pellicon XL
Cassette, Biomax 50 KDa), a tube pump (Cole-Parmer
International, MasterFlex Pump model 77521-40, Pump
Head model 7518-00), and a tube (Cole-Parmer
International, MasterFlex Tube L/S16) Specifically,
while ABS was added dropwise (a total of 800 mL) as a
buffer solution for purification to the reaction
solution, ultrafiltration purification was performed
for removing unconjugated drug linkers and other low-
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molecular-weight reagents, also replacing the buffer
solution with ABS, and further concentrating the
solution. The purified solution obtained was subjected
to microfiltration (0.22 μm (Millipore Corp., Millex-GV
filter, PVDF membrane) and 0.10 μm (Millipore Corp.,
Millex-VV filter, PVDF membrane)) to yield 42.5 mL of a
solution containing the titled antibody-drug conjugate.
Physicochemical characterization: By using the Common
procedures E and F (ED,2so = 5178 (measured value), and
ED,37o = 20217 (measured value) were used), the following
characteristic values were obtained.
Antibody concentration: 10.4 mg/mL, antibody yield: 442
mg (88.5%), average number of conjugated drug molecules
(n) per antibody molecule measured by the Common
procedure E: 6.0, and average number of conjugated drug
molecules (n) per antibody molecule measured by the
Common procedure F: 7.5.
[0320]
Example 47 Antibody-drug conjugate (47)
[ Formula 7 3]
Process 1
Tras1uzumab
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6.2
Atty. Dkt. No. 111119-0105
[0321]
Process 1: Antibody-drug conjugate (47)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg- 1 cm- 1 was used) . The solution ( 15 mL) was
placed in a polypropylene tube and charged with an
aqueous solution of 10 mM TCEP (0.567 mL; 5.5
equivalents per antibody molecule) and an aqueous
solution of 1 M dipotassium hydrogen phosphate ( 0. 225
mL) . After confirming that the solution had a pH of
7.0 ± 0.1, the disulfide bond at the hinge part in the
antibody was reduced by incubating at 37°C for 2 hour.
Conjugation between antibody and drug linker: After
adding DMSO ( 0 .146 mL) and a DMSO solution containing
10 mM of the compound of Process 8 of Example 26 (0.928
mL; 9.0 equivalents per antibody molecule) to the above
solution at room temperature, it was incubated for
conjugating the drug linker to the antibody at 15°C for
30 minutes. Next, an aqueous solution (0.133 mL; 12.9
equivalents per antibody molecule) of 100 mM NAC was
added thereto and stirred at room temperature to
terminate the reactivity of unreacted drug linkers for
another 20 minutes.
Purification: The above solution was subjected to
purification using the Common procedure D (ABS was used
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as buffer solution) to yield 49 mL of a solution
containing the compound of interest.
Physicochemical characterization: By using the Common
procedure E (ED,2so = 5178 and ED,37o 20217 were used),
the following characteristic values were obtained.
Antibody concentration: 2.91 mg/mL, antibody yield: 143
mg ( 95%) , and average number of conjugated drug
molecules (n) per antibody molecule: 6.2.
[0322]
Example 48 Antibody-drug conjugate (48)
[Formula 74]
Process 1
[0323]
Process 1: Antibody-drug conjugate (48)
7
!
0
OHO~ 62
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg- 1 cm- 1 was used) . The solution (280 mL) was
placed in a 1000 mL polycarbonate Erlenmeyer flask,
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charged with an aqueous solution of 1 M dipotassium
hydrogen phosphate at room temperature (4.200 mL) with
stirring using a magnetic stirrer, and then charged
with an aqueous solution of 10 mM TCEP (10.594 mL; 5.5
equivalents per antibody molecule) After confirming
that the solution had a pH of 7.4 ± 0.1, stirring was
terminated, and the disulfide bond at the hinge part in
the antibody was reduced by incubating at 37°C for 2
hour.
Conjugation between antibody and drug linker: After
cooling the above solution to 15°C, a DMSO solution
containing 10 mM of the compound of Process 8 of
Example 26 (17.335 mL; 9.0 equivalents per antibody
molecule) was gradually added dropwise thereto with
stirring. The reaction solution was stirred at 15°C
for conjugating the drug linker to the antibody for 30
minutes. Next, an aqueous solution (2.485 mL; 12.9
equivalents per
added thereto
temperature to
antibody molecule) of 100 mM NAC was
with stirring and stirred at room
terminate the reactivity of unreacted
drug linkers for another 20 minutes.
Purification: By gradually adding 20% aqueous acetic
acid solution (about 1. 4 mL) and ABS (280 mL) to the
above solution with stirring, the pH of the solution
was adjusted to 5.5 ± 0.1. This solution was subjected
to microfiltration (0.45 μm, PVDF membrane) to remove
whitish matter while yielding about 600 mL of a
filtrate. This solution was subjected to
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ultrafiltration purification using an ultrafiltration
apparatus composed of an ultrafiltration membrane
(Merck Japan, Pell icon XL Cassette, Biomax 50 KDa) , a
tube pump ( Cole-Parmer International, MasterFlex Pump
model 77521-40, Pump Head model 7518-00), and a tube
(Cole-Parmer International, MasterFlex Tube L/S16).
Specifically, while ABS was added dropwise (a total of
4800 mL) as a buffer solution for purification to the
reaction solution, ultrafiltration purification was
performed for removing unconjugated drug linkers and
other low-molecular-weight reagents, also replacing the
buffer solution with ABS, and further concentrating the
solution. The purified solution obtained was subjected
to microfiltration (twice with 0. 22 μm and 0 .10 μm,
PVDF membrane) to yield 70 mL of a solution containing
the titled antibody-drug conjugate.
Physicochemical characterization: By using the Common
procedure E (ED,2so = 5178 and ED,37o 20217 were used),
the following characteristic values were obtained.
Antibody concentration: 35.96 mg/mL, antibody yield:
2517 mg (90%), and average number of conjugated drug
molecules (n) per antibody molecule: 6.2.
[0324]
Example 49 Antibody-drug conjugate (49)
[Formula 75]
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Process 1 Trastuzumab
[0325]
Process 1: Antibody-drug conjugate (49)
Reduction of the antibody: The trastuzumab produced in
Reference Example 1 was prepared to have an antibody
concentration of 10 mg/mL by replacing the medium with
PBS6.0/EDTA by using the Common procedure C-1 and
Common procedure B (as absorption coefficient at 280 nm,
1. 4 8 mLmg- 1 cm- 1 was used) . The solution (280 mL) was
placed in a 1000 mL polycarbonate Erlenmeyer flask,
charged with an aqueous solution of 1 M dipotassium
hydrogen phosphate at room temperature (4.200 mL)with
stirring using a magnetic stirrer, and then charged
with an aqueous solution of 10 mM TCEP (10.594 mL; 5.5
equivalents per antibody molecule) After confirming
that the solution had a pH of 7.0 ± 0.1, stirring was
terminated, and the disulfide bond at the hinge part in
the antibody was reduced by incubating at 37°C for 2
hour.
Conjugation between antibody and drug linker: After
cooling the above solution to 15°C, a DMSO solution
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containing
Example 26
molecule)
10 mM of the compound of Process 8 of
per antibody
thereto with
( 1 7 . 3 3 5 mL ; 9 . 0 equiv a 1 en t s
was gradually added dropwise
stirring. The reaction solution was stirred at 15°C
for conjugating the drug linker to the antibody for 30
minutes. Next, an aqueous solution (2.485 mL; 12.9
equivalents per
added thereto
temperature to
antibody molecule) of 100 mM NAC was
with stirring and stirred at room
terminate the reactivity of unreacted
drug linkers for another 20 minutes.
Purification: By gradually adding 20% aqueous acetic
acid solution (about 1. 4 mL) and ABS (280 mL) to the
above solution with stirring, the pH of the solution
was adjusted to 5.5 ± 0.1. This solution was subjected
to microfiltration (0.45 μm, PVDF membrane) to remove
whitish matter while yielding about 600 mL of a
filtrate. This solution was subjected to
ultrafiltration purification using an ultrafiltration
apparatus composed of an ultrafiltration membrane
(Merck Japan, Pellicon XL Cassette, Ultracell 30 KDa),
a tube pump (Cole-Parmer International, MasterFlex Pump
model 77521-40, Pump Head model 7518-00), and a tube
(Cole-Parmer International, MasterFlex Tube L/S16).
Specifically, while ABS was added dropwise (a total of
4800 mL) as a buffer solution for purification to the
reaction solution, ultrafiltration purification was
performed for removing unconjugated drug linkers and
other low-molecular-weight reagents, also replacing the
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buffer solution with ABS, and further concentrating the
solution. The purified solution obtained was subjected
to microfiltration (twice with 0. 22 μm and 0 .10 μm,
PVDF membrane) to yield 130 mL of a solution containing
the titled antibody-drug conjugate.
Physicochemical characterization: By using the Common
procedure E (ED,2so = 5178 and ED,37o 20217 were used),
the following characteristic values were obtained.
Antibody concentration: 21.00 mg/mL, antibody yield:
2730 mg (97.5%), and average number of conjugated drug
molecules (n) per antibody molecule: 6.3.
[0326]
Example 50 Antibody-drug conjugate (50)
[0327]
Process 1: Antibody-drug conjugate (50)
The antibody-drug conjugates (47), (48), and (49)
produced in Examples 47, 48, and 49 were mixed (243 mL)
and further charged with ABS (39.75 mL) to yield 283 mL
of a solution containing the titled antibody-drug
conjugate.
Physicochemical characterization: By using the Common
procedures E and F (ED,2so = 5178, and ED,37o = 20217 were
used), the following characteristic values were
obtained.
Antibody
5655 mg,
(n) per
concentration: 20.0 mg/mL, antibody yield:
average number of conjugated drug molecules
antibody molecule measured by the Common
procedure E: 6.3, and average number of conjugated drug
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molecules (n) per antibody molecule measured by the
Common procedure F: 7.8.
[0328]
Evaluation Example 1 Cell growth inhibitory effect (1)
of antibody-drug conjugate
breast
cells
Human
positive
Medical School,
cancer line KPL-4 of HER2
(Dr. Junichi
British Journal
Kurebayashi,
of Cancer,
antigenKawasaki
(1999) 79
(5/6). 707-717) or human breast cancer line MCF7 of
antigen-negative cells (European Collection of Cell
Cultures; ECACC) was cultured in RPMI1640 (GIBCO)
containing 10% fetal bovine serum (MOREGATE)
(hereinafter, referred to as medium) . The KPL-4 or
MCF7 was prepared to have a concentration of 2.5 X 10 4
cells/mL by using medium, added at a concentration of
100 μL/well to a 96-well microplate for cell culture,
and cultured overnight.
On the next day, trastuzumab or the antibody-drug
conjugate diluted into 1000 nM, 200 nM, 40 nM, 8 nM,
1.6 nM, 0.32 nM, and 0.064 nM by using medium was added
at a concentration of 10 μL/well to the microplate.
Medium was added at a concentration of 10 μL/well to
antibody non-supplemented wells. The cells were
cultured under 5 % CO2 at 3 7°C for 5 to 7 days. After
the culture, the microplate was taken out from the
incubator and left standing at room temperature for 30
minutes. The culture solution was charged with an
equal amount of CellTiter-Glo Luminescent Cell
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Viability Assay (Promega) and stirred. After the
microplate was left standing at room temperature for 10
minutes, luminescence intensity of each well was
measured by using a plate reader (PerkinElmer). The
IC50 value was calculated according to the following
equation:
IC50 (nM) = antilog((5O - d) X (LOG10 (b) - LOG10 (a)) / (d
- c) + LOG1o(b))
a: Concentration of sample a
b: Concentration of sample b
c: Cell viability of sample a
d: Cell viability of sample b
The cell viability at each concentration was
calculated according to the following equation:
Cell viability (%) =a/bx 100
a: Average luminescence intensity of the sample wells
(n = 2)
b: Average luminescence intensity of the antibody nonsupplemented
wells (n = 10)
[0329]
The antibody-drug conjugates (2), (3), (5), (7),
(10), (12), (13), (16), (18), (40), and (42) exhibited
a cell growth inhibitory effect of IC50 < 0 .1 (nM)
against the KPL-4 cells.
The antibody-drug conjugates (4), (6), (9), (15),
( 1 7 ) , ( 21 ) , ( 2 2 ) , ( 2 5 ) , ( 3 6 ) , ( 3 7 ) , ( 3 9 ) , ( 4 1 ) , and
(43) exhibited a cell growth inhibitory effect of 0.1 <
IC50 < 1 (nM) against the cells.
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The antibody-drug conjugates (20), (24), and (27)
exhibited a cell growth inhibitory effect of 1 < IC50 <
100 (nM) against the cells. Neither of the antibodydrug
conjugates (19) or (26) exhibited a cell growth
inhibitory effect against the cells (> 100 (nM)).
On the other hand, the antibody-drug conjugates
(5), (13), and (43) exhibited a cell growth inhibitory
effect of 1 < IC50 < 100 (nM) against the MCF7 cells,
whereas none of the antibody-drug conjugates (2), (3),
(4), (6), (7), (9), (10), (12), (15), (16), (17), (18),
( 2 5) , ( 2 6) , ( 2 7) , ( 3 9) , ( 4 0) , ( 41) , ( 4 2) , and ( 4 4)
exhibited a cell growth inhibitory effect against the
c e 11 s ( > 1 0 0 ( nM) ) .
Trastuzumab
effect against
exhibited
neither the
c e 11 s ( > 1 0 0 ( nM) ) .
[0330]
a cell
KPL-4
growth inhibitory
cells nor the MCF7
Evaluation Example 2 Antitumor test (1)
Mouse: 5- to 6-week-old female nude mice (Charles
River Laboratories Japan, Inc.) were acclimatized for 4
to 7 days under SPF conditions before use in the
experiment. The mice were fed with sterilized solid
feed (FR-2, Funabashi Farms Co., Ltd) and given
sterilized tap water (prepared by the addition of 5 to
15 ppm sodium hypochlorite solution).
Assay and calculation expression: In all studies,
the major axis and minor axis of a tumor were measured
twice a week by using an electronic digital caliper
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(CD-15CX, Mitutoyo Corp.), and the tumor volume (mm3
)
was calculated. The calculation expression is as shown
below.
Tumor volume (mm3
)
axis (mm)] 2
[0331]
1/2 X Major axis (mm) X [Minor
All of the antibody-drug conjugates and the
antibody were diluted with physiological saline (Otsuka
Pharmaceutical Factory, Inc.) and used at a volume of
10 mL/kg for intravenous administration to the tail
vein of each mouse.
KPL-4 cells were suspended in physiological saline,
and 1.5 X 10 7 cells were subcutaneously transplanted to
the right side of the body of each female nude mouse
(Day 0), and the mice were randomly grouped on Day 15.
The antibody-drug conjugate (27) or the anti-HER2
antibody trastuzumab (Reference Example 1) for a
control group was intravenously administered at a dose
of 10 mg/kg to the tail vein of each mouse at Days 15
and 22. An untreated group was established as a
control group.
[0332]
The results are shown
administration
whereas the
of trastuzumab
administration
in Fig. 3 . The
inhibited
of the
tumor growth,
antibody-drug
conjugate (27) produced a more significant tumor growth
inhibitory effect. In the drawing, the abscissa
depicts days after tumor inoculation, and the ordinate
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depicts tumor volume. In addition, the mice that
received trastuzumab or the antibody-drug conjugate
(27) were free from notable signs such as weight loss,
suggesting that the antibody-drug conjugate (27) is
highly safe. In the Evaluation Examples below
regarding the antitumor test, the test was conducted by
the procedure used in this Evaluation Example, unless
otherwise specified.
[0333]
Evaluation Example 3 Antitumor test (2)
Human gastric cancer line NCI-N87 cells purchased
from ATCC (American Type Culture Collection) were
suspended in physiological saline, and 1 X 10 7 cells
were subcutaneously transplanted to the right side of
the body of each female nude mouse (Day O) , and the
mice were randomly grouped on Day 7. The antibody-drug
conjugate ( 8) or ( 2 8) , or trastuzumab emtansine
(Reference Example 2) was intravenously administered at
a dose of 10 mg/kg to the tail vein of each mouse on
Day 7. An untreated group was established as a control
group.
[0334]
The results are shown in Fig. 4. The antibodydrug
conjugates (8) and (28) were confirmed to have a
strong anti tumor effect with tumor regression
equivalent to
addition, the
4833-0752-7733.1
that of trastuzumab emtansine. In
administration of the antibody-drug
- 269 -
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conjugate (8) or (28), or trastuzumab emtansine was
found to be free from weight loss of the mice.
[0335]
Evaluation Example 4 Antitumor test (3)
Human breast cancer line JIMT-1 cells purchased
from DSMZ (Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH) were suspended in physiological
saline, and 3 X 10 6 cells were subcutaneously
transplanted to the right side of the body of each
female nude mouse (Day 0), and the mice were randomly
grouped on Day 12. The antibody-drug conjugate (8),
(29), or (30), trastuzumab, or trastuzumab emtansine
was intravenously administered at a dose of 10 mg/kg to
the tail vein of each mouse on Days 12 and 19. A
physiological saline administration
established as a control group.
[0336]
The results are shown in Fig.
group was
5. The
administration of trastuzumab or trastuzumab emtansine
did not inhibit the growth of the JIMT-1 tumor. On the
other hand, the administration of the antibody-drug
conjugate (8), (29), or (30) inhibited the growth of
the tumor. In addition, the administration of the
antibody-drug conjugate (8), (29), or (30), trastuzumab,
or trastuzumab emtansine was found to be free from
weight loss of the mice.
[0337]
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Evaluation Example 5 Cell growth inhibitory effect (2)
of antibody-drug conjugate
Human non-small cell lung cancer line Calu-3
(ATCC) was cultured in Eagle's Minimum Essential Medium
(GIBCO) containing 10% fetal bovine serum (MOREGATE)
(hereinafter, referred to as MEM medium).
Human gastric cancer line NCI-N87 (ATCC) or human
gastric cancer line MKN-45 (Health Science Research
Resources Bank) was cultured in RPMI1640 Medium (GIBCO)
containing 10% fetal bovine serum (hereinafter,
referred to as RPMI medium)
Human breast cancer line MDA-MB-453 (ATCC) or
human breast cancer line MDA-MB-468 (ATCC) was cultured
in Leibovitz's L-15 Medium (GIBCO) containing 10% fetal
bovine serum (hereinafter, referred to as Leibovi tz 's
medium)
Among these 5 types of cell lines, Calu-3, NCI-N87,
and MDA-MB-453 are HER2-positive cells, and MKN-45 and
MDA-MB-468 are HER2-negative cells.
Calu-3, NCI-N87, or MKN-45 was prepared to have a
concentration of 4 X 104 cells/mL by using MEM medium
or RPMI medium, added at a concentration of 25 μL/well
to a 96-well microplate for cell culture charged with
65 μL/well of a medium, and cultured overnight under 5%
CO2 at 37°C. Also, MDA-MB-453 or MDA-MB-468 was
prepared to have a concentration of 4 X 104 cells/mL by
using Leibovitz's medium, added at a concentration of
25 μL/well to a 96-well microplate for cell culture
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charged with 65 μL/well of medium, and cultured
overnight at 37°C without setting CO2 concentration.
On the next day, a test substance diluted into
100 0 nM, 2 0 0 nM, 4 0 nM , 8 nM, 1 . 6 nM, 0 . 3 2 nM , and
0.064 nM by using RPMI medium or Leibovitz's medium, or
RPMI medium or Leibovitz's medium was added at a
concentration of 10 μL/well to the microplate. The
cells were cultured under 5% CO2 at 37°C or at 37°C
without setting CO2 concentration for 6 days.
For Calu-3, NCI-N87, and MDA-MB-468, the antibodydrug
conjugate (46) was added as the test substance.
For the other cells, the antibody-drug conjugate (50)
was added as the test substance. After the culture,
the microplate was taken out from the incubator and
left standing at room temperature for 30 minutes. The
culture solution was charged with an equal amount of
CellTiter-Glo Luminescent Cell Viability Assay
(Promega) and stirred with a plate mixer to completely
lyse the cells. After the microplate was left standing
at room temperature for 10 minutes, luminescence
intensity was measured by using a plate reader.
Cell viability was calculated according to the
following equation:
Cell viability (%) a/bx 100
a: Average luminescence intensity
substance-supplemented wells
b: Average luminescence
supplemented wells
intensity of
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The IC50 value was calculated according to the
following equation:
IC50 (nM) = anti log ( (50 - d) X (LOG10 (b) - LOG10 (a)) / (d
- c) + LOG1o(b))
a: Concentration a of the test substance
b: Concentration b of the test substance
c: Cell viability supplemented with the test substance
having the concentration a
d: Cell viability supplemented with the test substance
having the concentration b
The concentrations a and b establish the relation
a> b crossing 50% of cell viability.
[0338]
The antibody-drug conjugate (46) exhibited a cell
growth inhibitory effect of ICso < 1 (nM) against the
HER2-positive cells Calu-3 and NCI-N87. On the other
hand, the antibody-drug conjugate (46) exhibited no
cell growth inhibitory effect against the HER2-negative
cells MDA-MB-468 (> 100 (nM)).
[0339]
The antibody-drug conjugate (50) exhibited a cell
growth inhibitory effect of IC50 < 1 (nM) against the
HER2-positive cells MDA-MB-453. On the other hand, the
antibody-drug conjugate (50) exhibited no cell growth
inhibitory effect against the HER2-negative cells MKN-
4 5 ( > 1 0 0 ( nM ) ) .
[0340]
Evaluation Example 6 Antitumor test (4)
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Human pancreatic cancer line Capan-1 cells (ATCC)
weakly expressing HER2 were suspended in physiological
saline, and 4 X 10 7 cells were subcutaneously
transplanted to the right side of the body of each
female nude mouse to generate Capan-1 solid tumor.
Thereafter, this solid tumor was maintained at several
passages by transplantation to female nude mice and
used in this test. A tumor section of the solid tumor
was subcutaneously transplanted to
the body of each female nude mouse
mice were randomly grouped on Day 20.
the right
(Day O) ,
side of
and the
The antibody-drug conjugate (31), trastuzumab, or
trastuzumab emtansine was intravenously administered at
a dose of 10 mg/kg to the tail vein of each mouse on
Day 20. A physiological saline administration group
was established as a control group.
The results are shown in Fig. 6. The
administration of trastuzumab or trastuzumab emtansine
did not inhibit the growth of
contrast, the administration
conjugate ( 31) inhibited the
the Capan-1 tumor. By
of the antibody-drug
growth of the tumor,
demonstrating that the antibody-drug conjugate (31) is
effective for even tumor with low HER2 expression. The
antibody-drug conjugate (31) did not inhibit the growth
of HER2 non-expressing gastric cancer line GCIY tumor.
As for the expression of HER2 in tumor, on the
basis of measurement results of immunohistochemical
staining described in the 3rd edition of the guidelines
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for HER2 testing (developed by the Japanese Pathology
Board for
Society of
such that
Optimal Use
Pathology) ,
score of 3+:
of Trastuzumab,
classification
high expression,
The
was
2+:
Japanese
performed
moderate
expression, and 1+: low expression. Even if the score
was O in this measurement method, tumor found HER2-
positive by other measurement methods such as a
measurement method using a flow cytometer was
classified as low expressing tumor.
[0341]
Evaluation Example 7 Antitumor test (5)
Human gastric cancer line NCI-N87 cells purchased
from ATCC were suspended in physiological saline, and 1
X 10 7 cells were subcutaneously transplanted to the
right side of the body of each female nude mouse (Day
0), and the mice were randomly grouped on Day 6. The
antibody-drug conjugate (50) was intravenously
administered at each dose of 0.3, 1, 3, or 10 mg/kg to
the tail vein of each mouse on Day 6. An acetate
buffer solution administration group was established as
a control group.
The results are shown in Fig. 7. The antibodydrug
conjugate (50) exhibited a dose-dependent
anti tumor effect. In addition, the administration of
the antibody-drug conjugate (50) was found to be free
from weight loss of the mice.
[0342]
Evaluation Example 8 Antitumor test (6)
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This test was carried out by the following method.
Mouse: 6- to 12-week-old female nude mice (Charles
River Laboratories Japan, Inc.) were subjected to the
experiment.
Assay and calculation expression: The major axis
and minor axis of tumor were measured twice a week by
using an electronic digital caliper, and the tumor
volume (mm3
) was calculated. The calculation
expression is as shown below.
Tumor volume (mm3
) 0.52 x Major axis (mm) x
[Minor axis (mm)] 2
The antibody-drug conjugate, trastuzumab, and
trastuzumab emtansine were diluted with an acetate
buffer solution and used at a volume of 10 mL/kg for
intravenous administration to the tail vein of each
mouse.
[0343]
Tumor (ST225; South Texas
Therapeutics (START)) excised
patient and maintained at
transplantation to female nude
test. This tumor moderately
Accelerated Research
from a breast cancer
several passages by
mice was used in this
expressed HER2 (which
received a score of 2+ in immunohistochemical staining).
A tumor section of the solid tumor was
subcutaneously transplanted to the side of the body of
each female nude mouse, and the mice were randomly
grouped when the tumor volume reached 100 to 300 mm3
•
The date of grouping was defined as Day 0. The
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antibody-drug conjugate ( 5 0) , trastuzumab, or
trastuzumab emtansine was intravenously administered at
a dose of 10 mg/kg to the tail vein of each mouse on
Day 0. An acetate buffer solution administration group
was established as a control group.
The results are shown in Fig. 8. The
administration of trastuzumab did not inhibit the
growth of
expressing
the breast cancer ST225
HER2. By contrast, the
tumor moderately
administration of
trastuzumab emtansine or the antibody-drug conjugate
(50) remarkably inhibited the growth of the tumor.
[0344]
Evaluation Example 9 Antitumor test (7)
Tumor (ST910; START) excised from a breast cancer
patient and maintained at several passages by
transplantation to female nude mice was used in this
test. This tumor low expressed HER2 (which received a
score of 1+ in
A tumor
immunohistochemical staining)
section of the solid tumor was
subcutaneously transplanted to the side of the body of
each female nude mouse, and the mice were randomly
grouped when the tumor volume reached 100 to 300 mm3
•
The date of grouping was defined as Day 0. The
antibody-drug conjugate ( 5 0) , trastuzumab, or
trastuzumab emtansine was intravenously administered at
a dose of 10 mg/kg to the tail vein of each mouse on
Day 0. An acetate buffer solution administration group
was established as a control group.
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The results are shown in Fig. 9. The
administration of trastuzumab or trastuzumab emtansine
did not inhibit the growth of the breast cancer ST910
tumor low expressing HER2. By contrast, the
administration of the antibody-drug conjugate (50)
remarkably inhibited the growth of the tumor,
demonstrating that the antibody-drug conjugate (50) is
effective for breast cancer tumor low expressing HER2.
This Evaluation Example 9 was carried out by the same
procedure as Evaluation Example 8.
[0345]
Evaluation Example 10 Antitumor test
This test was carried out
procedure. Evaluation Examples 11
carried out by this procedure.
Mouse: 5- to 8 -week-old female
( 8)
by the following
to 13 were also
nude mice (Harlan
Laboratories Ltd.) were subjected to the experiment.
Assay and calculation expression: The major axis
and minor axis of tumor were measured twice a week by
using an electronic digital caliper, and the tumor
volume (mm3
) was calculated. The calculation
expression is as shown below.
Tumor volume (mm3
) 0.52 x Major axis (mm) x
[Minor axis (mm)] 2
The antibody-drug conjugate, trastuzumab, and
trastuzumab emtansine were diluted with an acetate
buffer solution and used at a volume of 10 mL/kg for
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intravenous administration to the tail vein of each
mouse.
[0346]
Tumor (CTG-0401; Champions Oncology Inc.) excised
from a colorectal cancer patient and maintained at
several passages by transplantation to female nude mice
was used in this test. This tumor low or moderately
expressed HER2 (which received a score of 1+ or 2+ in
immunohistochemical staining)
A tumor section of the solid tumor was
subcutaneously transplanted to the left side of the
body of each female nude mouse, and the mice were
randomly grouped when the tumor volume reached 100 to
300 mm3
• The date of grouping was defined as Day O.
The antibody-drug conjugate (50), trastuzumab, or
trastuzumab emtansine was intravenously administered at
a dose of 10 mg/kg to the tail vein of each mouse on
Day 0. An acetate buffer solution administration group
was established as a control group.
The results are shown in Fig. 10. The
administration of trastuzumab or trastuzumab emtansine
did not inhibit the growth of the colorectal cancer
CTG-0401 tumor low or moderately expressing HER2. By
contrast, the administration of the antibody-drug
conjugate (50) remarkably inhibited the growth of the
tumor.
[0347]
Evaluation Example 11 Antitumor test (9)
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Tumor (CTG-0860; Champions Oncology Inc.) excised
from a non-small cell lung cancer patient and
maintained at several passages by
female nude mice was used in this
transplantation to
test. This tumor
moderately expressed HER2 (which received a score of 2+
in immunohistochemical staining).
A tumor section of the solid tumor was
subcutaneously transplanted to the left side of the
body of each female nude mouse, and the mice were
randomly grouped when the tumor volume reached 100 to
300 mm3
• The date of grouping was defined as Day O.
The antibody-drug conjugate (50), trastuzumab, or
trastuzumab emtansine was intravenously administered at
a dose of 10 mg/kg to the tail vein of each mouse on
Day 0. An acetate buffer solution administration group
was established as a control group.
The results are shown in Fig. 11. The
administration of trastuzumab or trastuzumab emtansine
did not inhibit the growth of the non-small cell lung
cancer CTG-0860
contrast, the
tumor moderately expressing HER2. By
administration of the antibody-drug
conjugate (50) remarkably inhibited the growth of the
tumor.
[0348]
Evaluation Example 12 Antitumor test (10)
Tumor (CTG-0927; Champions Oncology Inc.) excised
from a bile duct cancer patient and maintained at
several passages by transplantation to female nude mice
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was used in this test. This tumor highly expressed
HER2 (which received a score of 3+ in
immunohistochemical staining)
A tumor section of the solid tumor was
subcutaneously transplanted to the left side of the
body of each female nude mouse, and the mice were
randomly grouped when the tumor volume reached 100 to
300 mm3
• The date of grouping was defined as Day O.
The antibody-drug conjugate (50), trastuzumab, or
trastuzumab emtansine was intravenously administered at
a dose of 10 mg/kg to the tail vein of each mouse on
Day 0. An acetate buffer solution administration group
was established as a control group.
The results are shown in Fig. 12. The
administration of trastuzumab did not
growth of the bile duct cancer CTG-0927
inhibit the
tumor highly
expressing HER2. By contrast, the administration of
trastuzumab emtansine inhibited the growth of the tumor.
Furthermore, the administration of the antibody-drug
conjugate (50) induced the regression of the tumor.
[0349]
Evaluation Example 13 Antitumor test (11)
Tumor (CTG-0137; Champions Oncology Inc.) excised
from an esophageal cancer patient and maintained at
several passages by transplantation to female nude mice
was used in this test. This tumor highly expressed
HER2 (which received a score of 3+ in
immunohistochemical staining).
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A tumor section of the solid tumor was
subcutaneously transplanted to the left side of the
body of each female nude mouse, and the mice were
randomly grouped when the tumor volume reached 100 to
300 mm3
• The date of grouping was defined as Day O.
The antibody-drug conjugate (50), trastuzumab, or
trastuzumab emtansine was intravenously administered at
a dose of 10 mg/kg to the tail vein of each mouse on
Day 0. An acetate buffer solution administration group
was established as a control group.
The results are shown in Fig. 13. The
administration of trastuzumab did not inhibit the
growth of the esophageal cancer CTG-0137 tumor highly
expressing HER2. By contrast, the administration of
trastuzumab emtansine or the antibody-drug conjugate
(50) remarkably inhibited the growth of the tumor.
[0350]
Evaluation Example 14 Antitumor test (12)
Human ovarian cancer line SK-OV-3 cells highly
expressing HER2 purchased from ATCC were suspended in
physiological saline, and 4 x 10 7 cells were
subcutaneously transplanted to the right side of the
body of each female nude mouse to prepare SK-OV-3 solid
tumor. Thereafter, this solid tumor was maintained at
several passages by transplantation to female nude mice
and used in this test.
A tumor section of the solid tumor was
subcutaneously transplanted to the right side of the
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body of each female nude mouse, and the mice were
randomly grouped when the tumor volume reached 100 to
300 mm3
• The date of grouping was defined as Day O.
The antibody-drug conjugate (50), trastuzumab, or
trastuzumab emtansine was intravenously administered at
a dose of 10 mg/kg to the tail vein of each mouse on
Day 0. A physiological saline administration group was
established as a control group.
The results are shown in Fig. 14. The
administration of trastuzumab did not inhibit the
growth of the SK-OV-3 tumor.
administration of trastuzumab
By contrast,
emtansine or
the
the
antibody-drug conjugate (50) remarkably inhibited the
growth of the tumor.
Free Text of Sequence Listing
[0351]
SEQ ID NO: 1 - Amino acid sequence of a heavy chain of
the humanized anti-HER2 monoclonal antibody
SEQ ID NO: 2 - Amino acid sequence of a light chain of
the humanized anti-HER2 monoclonal antibody
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