U.S. patent application number 16/684130 was filed with the patent office on 2020-06-04 for novel anti-human muc1 antibody fab fragment.
This patent application is currently assigned to Astellas Pharma Inc.. The applicant listed for this patent is Astellas Pharma Inc.. Invention is credited to Hitoshi DOIHARA, Kazunori HIRAYAMA, Naomi HOSOGAI, Akifumi MORINAKA, Hiroki SHIRAI.
Application Number | 20200171174 16/684130 |
Document ID | / |
Family ID | 62146614 |
Filed Date | 2020-06-04 |
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United States Patent
Application |
20200171174 |
Kind Code |
A1 |
MORINAKA; Akifumi ; et
al. |
June 4, 2020 |
NOVEL ANTI-HUMAN MUC1 ANTIBODY FAB FRAGMENT
Abstract
The problem to be solved is to provide an anti-human MUC1
antibody Fab fragment that is expected to be useful in the
diagnosis and/or treatment of a cancer, particularly, the diagnosis
and/or treatment of breast cancer or bladder cancer, and a
diagnosis approach and/or a treatment approach using a conjugate
comprising the Fab fragment. The solution is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment comprising
a heavy chain variable region consisting of the amino acid sequence
represented by SEQ ID NO: 8 or 10, and a light chain comprising a
light chain variable region consisting of the amino acid sequence
represented by SEQ ID NO: 12, and a conjugate comprising the Fab
fragment.
Inventors: |
MORINAKA; Akifumi; (Tokyo,
JP) ; SHIRAI; Hiroki; (Tokyo, JP) ; HIRAYAMA;
Kazunori; (Tokyo, JP) ; HOSOGAI; Naomi;
(Tokyo, JP) ; DOIHARA; Hitoshi; (Tokyo,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Astellas Pharma Inc. |
Tokyo |
|
JP |
|
|
Assignee: |
Astellas Pharma Inc.
Tokyo
JP
|
Family ID: |
62146614 |
Appl. No.: |
16/684130 |
Filed: |
November 14, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16414895 |
May 17, 2019 |
10507251 |
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16684130 |
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PCT/JP2017/041486 |
Nov 17, 2017 |
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16414895 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/0036 20130101;
A61K 39/395 20130101; A61K 49/0032 20130101; A61K 51/1051 20130101;
A61K 51/106 20130101; C07K 16/18 20130101; C12N 15/09 20130101;
C07K 17/00 20130101; C07K 2317/76 20130101; C07K 16/28 20130101;
C07K 2317/21 20130101; C07K 2317/92 20130101; C07K 16/30 20130101;
G01N 2333/4725 20130101; A61K 47/6855 20170801; A61K 49/16
20130101; C07K 2317/24 20130101; A61K 47/54 20170801; A61K 47/6851
20170801; C07K 2317/55 20130101; C07K 2317/40 20130101; A61K
51/1093 20130101; C12N 15/85 20130101; C12P 21/02 20130101; A61P
35/00 20180101; C07K 16/3092 20130101; G01N 33/57407 20130101; C12N
5/10 20130101; A61K 2039/505 20130101; G01N 33/57415 20130101; A61K
49/0058 20130101; A61K 49/0021 20130101 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C07K 16/30 20060101 C07K016/30; C12N 15/09 20060101
C12N015/09; C12P 21/02 20060101 C12P021/02; C07K 17/00 20060101
C07K017/00; A61K 39/395 20060101 A61K039/395; C12N 5/10 20060101
C12N005/10; A61K 49/16 20060101 A61K049/16; C07K 16/18 20060101
C07K016/18; G01N 33/574 20060101 G01N033/574; C12N 15/85 20060101
C12N015/85; A61K 51/10 20060101 A61K051/10; A61K 47/54 20060101
A61K047/54; A61K 47/68 20060101 A61K047/68; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 18, 2016 |
JP |
2016/224811 |
Claims
1: A conjugate comprising one or more labeling moiety, wherein the
labeling moiety is (i) a fluorescent dye and a linker or (ii) a
fluorescent dye, and an anti-human MUC1 antibody Fab fragment
selected from the group consisting of the following (a) and (b):
(a) an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment comprising a heavy chain variable region consisting
of the amino acid sequence represented by SEQ ID NO: 8 or SEQ ID
NO: 10 and a light chain comprising a light chain variable region
consisting of the amino acid sequence represented by SEQ ID NO: 12;
and (b) an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment comprising a heavy chain variable region derived
from a heavy chain variable region consisting of the amino acid
sequence represented by SEQ ID NO: 8 or SEQ ID NO: 10 by the
modification of glutamine at amino acid position 1 of SEQ ID NO: 8
or SEQ ID NO: 10 into pyroglutamic acid, and a light chain
comprising a light chain variable region consisting of the amino
acid sequence represented by SEQ ID NO: 12.
2: The conjugate according to claim 1 wherein the anti-human MUC1
antibody Fab fragment is selected from the group consisting of the
following (a) and (b): (a) an anti-human MUC1 antibody Fab fragment
comprising a heavy chain fragment consisting of the amino acid
sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and a light
chain consisting of the amino acid sequence represented by SEQ ID
NO: 6; and (b) an anti-human MUC1 antibody Fab fragment comprising
a heavy chain fragment derived from a heavy chain fragment
consisting of the amino acid sequence represented by SEQ ID NO: 2
or SEQ ID NO: 4 by the modification of glutamine at amino acid
position 1 of SEQ ID NO: 2 or SEQ ID NO: 4 into pyroglutamic acid,
and a light chain consisting of the amino acid sequence represented
by SEQ ID NO: 6.
3: The conjugate according to claim 1 wherein the anti-human MUC1
antibody Fab fragment is selected from the group consisting of the
following (a) and (b): (a) an anti-human MUC1 antibody Fab fragment
comprising a heavy chain fragment comprising a heavy chain variable
region consisting of the amino acid sequence represented by SEQ ID
NO: 10 and a light chain comprising a light chain variable region
consisting of the amino acid sequence represented by SEQ ID NO: 12;
and (b) an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment comprising a heavy chain variable region derived
from a heavy chain variable region consisting of the amino acid
sequence represented by SEQ ID NO: 10 by the modification of
glutamine at amino acid position 1 of SEQ ID NO: 10 into
pyroglutamic acid, and a light chain comprising a light chain
variable region consisting of the amino acid sequence represented
by SEQ ID NO: 12.
4: The conjugate according to claim 3 wherein the anti-human MUC1
antibody Fab fragment is selected from the group consisting of the
following (a) and (b): (a) an anti-human MUC1 antibody Fab fragment
comprising a heavy chain fragment consisting of the amino acid
sequence represented by SEQ ID NO: 4 and a light chain consisting
of the amino acid sequence represented by SEQ ID NO: 6; and (b) an
anti-human MUC1 antibody Fab fragment comprising a heavy chain
fragment derived from a heavy chain fragment consisting of the
amino acid sequence represented by SEQ ID NO: 4 by the modification
of glutamine at amino acid position 1 of SEQ ID NO: 4 into
pyroglutamic acid, and a light chain consisting of the amino acid
sequence represented by SEQ ID NO: 6.
5: The conjugate according to claim 4 wherein the anti-human MUC1
antibody Fab fragment is an anti-human MUC1 antibody Fab fragment
comprising a heavy chain fragment consisting of the amino acid
sequence represented by SEQ ID NO: 4 and a light chain consisting
of the amino acid sequence represented by SEQ ID NO: 6.
6: The conjugate according to claim 4 wherein the anti-human MUC1
antibody Fab fragment is an anti-human MUC1 antibody Fab fragment
comprising a heavy chain fragment derived from a heavy chain
fragment consisting of the amino acid sequence represented by SEQ
ID NO: 4 by the modification of glutamine at amino acid position 1
of SEQ ID NO: 4 into pyroglutamic acid, and a light chain
consisting of the amino acid sequence represented by SEQ ID NO:
6.
7: The conjugate according to claim 1, wherein the fluorescent dye
is a fluorescent dye selected from the group consisting of the
following formula (B) and the following formula (C): ##STR00015##
wherein the wavy line represents binding to the anti-human MUC1
antibody Fab fragment or the linker.
8: The conjugate according to claim 7, wherein the wavy line
represents binding to the anti-human MUC1 antibody Fab fragment,
and the anti-human MUC1 antibody Fab fragment is bound via an amino
group thereof to the carbon atom of a labeling moiety terminal
C(.dbd.O) group.
9: The conjugate according to claim 8, wherein the labeling moiety
is a fluorescent dye represented by formula (B).
10: A conjugate selected from the group consisting of the following
(a) to (c): (a) the conjugate according to claim 9 wherein the
anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment consisting
of the amino acid sequence represented by SEQ ID NO: 4 and a light
chain consisting of the amino acid sequence represented by SEQ ID
NO: 6; (b) the conjugate according to claim 9 wherein the
anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment derived
from a heavy chain fragment consisting of the amino acid sequence
represented by SEQ ID NO: 4 by the modification of glutamine at
amino acid position 1 of SEQ ID NO: 4 into pyroglutamic acid, and a
light chain consisting of the amino acid sequence represented by
SEQ ID NO: 6; and (c) a conjugate which is a mixture of (a) and
(b).
11: The conjugate according to claim 8, wherein the labeling moiety
is a fluorescent dye represented by formula (C).
12: A conjugate selected from the group consisting of the following
(a) to (c): (a) the conjugate according to claim 11 wherein the
anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment consisting
of the amino acid sequence represented by SEQ ID NO: 4 and a light
chain consisting of the amino acid sequence represented by SEQ ID
NO: 6; (b) the conjugate according to claim 11 wherein the
anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment derived
from a heavy chain fragment consisting of the amino acid sequence
represented by SEQ ID NO: 4 by the modification of glutamine at
amino acid position 1 of SEQ ID NO: 4 into pyroglutamic acid, and a
light chain consisting of the amino acid sequence represented by
SEQ ID NO: 6; and (c) a conjugate which is a mixture of (a) and
(b).
13: A composition comprising the conjugate according to claim 1,
and a pharmaceutically acceptable carrier.
14. (canceled)
15: A composition comprising the conjugate according to claim 10
and pharmaceutically acceptable carrier.
16: A composition comprising the conjugate according to claim 12
and a pharmaceutically acceptable carrier.
17-25. (canceled)
26: A method for diagnosing breast cancer or bladder cancer,
comprising preoperatively or intraoperatively administering the
conjugate according to claim 1 to a subject in need thereof.
27: A method for treating breast cancer or bladder cancer,
comprising administering a therapeutically effective amount of the
conjugate according to claim 1 to a subject in need thereof.
Description
[0001] This application is a continuation-in-part of international
application No. PCT/JP2017/041486, filed on Nov. 17, 2017.
TECHNICAL FIELD
[0002] The present invention relates to a novel anti-human MUC1
antibody Fab fragment. The present invention also relates to a
composition for diagnosis and/or for treatment comprising the
anti-human MUC1 antibody Fab fragment, and a method for diagnosing
and/or treating a cancer using the Fab fragment.
BACKGROUND ART
[0003] Mucin 1 (MUC1) is a membrane-bound glycoprotcin that is
expressed on the lumen side of epithelial cells constituting the
epithelial tissues of the mammary gland, the trachea and the
gastrointestinal tract, etc. (Nat. Rev. Cancer, 2004 January; 4
(1): 45-60). MUC1 is overexpressed in cancer cells of breast cancer
(Mod. Pathol., 2005 October; 18 (10): 1295-304), lung cancer (Hum.
Pathol., 2008 January; 39 (1): 126-36), colorectal cancer (Int. J.
Oncol., 2000 January; 16 (1): 55-64), bladder cancer (PLoS One,
2014 March; 9 (3): e92742), skin cancer (Histopathology, 2000
September; 37 (3): 218-23), thyroid gland cancer (J. Pathol., 2003
July; 200 (3): 357-69), stomach cancer (J. Pathol., 2000 March; 190
(4): 437-43), pancreatic cancer (Int. J. Oncol., 2004 January; 24
(1): 107-13), kidney cancer (Mod. Pathol., 2004 February; 17 (2):
180-8), ovary cancer (Gynecol. Oncol., 2007 June; 105 (3): 695-702)
and uterine cervical cancer (Am. J. Clin. Pathol., 2004 July; 122
(1): 61-9), etc. MUC1 is useful as a target molecule for detecting
a cancer focus (Nat. Rev. Cancer, 2004 January; 4 (1): 45-60; and
Pathol. Res. Pract., 2010 Aug. 15; 206 (8): 585-9).
[0004] MUC1 undergoes the O-glycosylation of threoninc at position
9 of a 20-amino acid tandem repeat sequence HGVTSAPDTRPAPGSTAPPA
(SEQ ID NO: 15) present in an extracellular domain. In cancer
cells, this O-glycosylation is incomplete, and 0-glycosylation such
as T(Gal.beta.1-3GalNAc.alpha.1-O-Ser/Thr),
Tn(GalNAc.alpha.1-O-Ser/Thr) and
2,3ST(Neu5Ac.alpha.2-3Gal.beta.1-3GalNAc.alpha.-O-Ser/Thr) is known
to occur in a cancer-specific manner (PTL 1 and NPL 1). Since MUC1
in normal tissues does not undergo such cancer-specific
O-glycosylation, human cancer-specific MUC1 is particularly useful
as a target molecule for treating various cancers in humans.
[0005] For example, a 1B2 antibody (PTL 1), a PankoMab antibody
(NPL 2), and a 5E5 antibody (PTL 2) are known as antibodies against
such human cancer-specific MUC1. Among these antibodies, the 1B2
antibody has been reported to have high specificity for human
cancer-specific MUC1 as compared with the PankoMab antibody (PTL
1). It has also been reported that the dissociation constant of the
1B2 antibody is 3.7.times.10.sup.-10 M (PTL 1), and the
dissociation constant of the 5E5 antibody is 1.7.times.10.sup.-9 M
(NPL 1).
[0006] Meanwhile, there are also great needs for the visualization
of cancer lesion. First, there are the needs for the early
detection of cancer lesion. Current diagnostic modalities such as
X-ray photography, echography, computed tomography (CT), magnetic
resonance imaging (MRT), positron emission tomography (PET), and
single photon emission computed tomography (SPECT) cannot
sensitively detect micro cancers. If micro cancers can be detected,
a primary cancer can be cured by operation or radiotherapy or even
a metastatic cancer is curable for a life-sustaining way by early
pharmaceutical intervention. Next, there are the needs for the
differentiation between a cancer lesion and a benign lesion. The
current diagnostic modalities often misdiagnose a benign lesion as
a cancer lesion. If differentiation can be made between a cancer
lesion and a benign lesion, unnecessary biopsy can be decreased.
Furthermore, there are the needs for the intraoperative
visualization of cancer lesion. At present, the position or extent
of a cancer lesion cannot be accurately determined during the
operation of cancers including breast cancer, bladder cancer, and
skin cancer. Therefore, the cancer lesion cannot be completely
resected, and there is a risk of ending the operation while leaving
the cancer cells. Moreover, there are the needs for the correct
determination of the positions of cancer lesion. Even if
postoperative recurrence and metastasis are suspected due to the
elevation of a tumor marker in blood, the current diagnostic
modalities cannot visualize a metastatic micro cancer. Therefore,
the optimum treatment cannot be selected because whether or not the
cancer has actually metastasized or which organ the cancer has
metastasized to cannot be determined. Thus, it is also useful to
visualize cancer lesion by molecular imaging techniques such as
fluorescent imaging and .gamma.-ray imaging (PET and SPECT) using
an antibody specifically binding to human cancer-specific MUC1 as
an in vive diagnostic drug. However, any previous case using an
anti-human cancer-specific MUC1 antibody as an in vivo diagnostic
drug has not been known.
[0007] There is the further needs for a cancer therapeutic drug
such as drug conjugated an antibody. The antibody drug is expected
as a method for treating a cancer with fewer adverse reactions
because of specific delivery to a cancer lesion. Radioimmunotherapy
using an antibody bound to a radioisotope (Takashi Tsuruo,
"Molecular Target Therapy of Cancer", NANZANDO Co., Ltd., published
on Sep. 15, 2008, p. 332-336; J. Nucl. Med., 2016 July; 57 (7):
1105-1111; and Nucl. Mcd. Biol., 2010 November; 37 (8): 949-955),
photoimmunotherapy using an antibody bound to IRDye700DX (Nat.
Med., 2011 December; 17 (12): 1685-91), and the like have been
reported. IRDye700DX is a near-infrared fluorescent dye that can
also be used in diagnosis. It has been reported that cell death can
be induced in a cancer-specific manner through the phototoxic
effect of IRDye700DX by binding this to an antibody against an
antigen expressed on a cancer cell membrane, and allowing the
resultant to specifically accumulate in cancer tissues, followed by
irradiation with near-infrared light (Nat. Med., 2011 December; 17
(12): 1685-91). However, any previous case of clinically applying
an antibody drug of an anti-human cancer-specific MUC1 antibody
bound to a cancer therapeutic drug has not been known.
[0008] In general, antibodies have a long half-life in blood and
require a period as long as 4 days to 5 days for reaching a
tumor-to-blood ratio that confers a signal-to-background ratio
sufficient for visualizing a cancer, after administration into the
body (Clin. Pharmacol. Ther., 2010 May; 87 (5): 586-92). Also, the
Fc regions of antibodies cause a pharmacological effect such as
antibody-dependent cellular cytotoxicity (ADCC) or
complement-dependent cytotoxicity (CDC) (NPL 1; and Curr. Opin.
Biotechnol., 2002 December; 13 (6): 609-14). Furthermore,
antibodies highly accumulate in the liver regardless of a target,
and cancer cells such as breast cancer are highly to metastasize to
the liver. The accumulation in the liver interfere with the
detection of hepatic metastasis at the time of diagnosis of
systemic cancer lesion (Clin. Pharmacol. Ther., 2010 May, 87 (5):
586-92).
[0009] For example, low-molecular recombinant antibody fragments
such as Fab, scFv, diabody, and minibody are expected to be
utilized as therapeutic antibodies because of easy reaching to foci
with their high tissue penetration and low cost production by using
an expression system in E. coli or yeast. And also, they are
reported to be utilized as diagnostic drug because of their short
half-lives in blood and the feature of renal excretion (Nat.
Biotechnol., 2005 September; 23 (9): 1126-36).
CITATION LIST
Patent Literature
[0010] PTL 1: WO2010/050528 [0011] PTL 2: WO2008/040362
Non Patent Literature
[0011] [0012] NPL 1: Glycoconj. J., 2013 April; 30 (3): 227-36
[0013] NPL 2: Cancer Immunol Immunother, 2006 November; 55 (11):
1337-47
SUMMARY OF INVENTION
Technical Problem
[0014] Monovalent Fab fragments have a molecular weight of
approximately 50 kDa, which is smaller than antibodies which have a
molecular weight of approximately 150 kDa, are eliminated by renal
excretion, and also have a short half-life in blood. Hence, they
reach a tumor-to-blood ratio that confers a signal-to-background
ratio sufficient for visualizing a cancer, within 2 to 32 hours
after administration. They lack an Fc region and therefore cause
neither ADCC nor CDC. The Fab fragments are typically eliminated by
renal excretion and therefore, do not interfere with the detection
of hepatic metastasis. From these features, the Fab fragments can
be expected to be more effective as in vivo diagnostic drugs as
compared with antibodies.
[0015] However, the binding activity of the Fab fragments is often
attenuated because of being monovalent, not divalent. Antibodies
must be labeled with a detectable substance such as a fluorescent
dye or a contrast medium for their utilization as in vivo
diagnostic drugs or drugs for use in photoimmunotherapy methods. A
further problem is the attenuation of their binding activity due to
labeling with such a substance.
[0016] An object of the present invention is to provide an
anti-human MUC1 antibody Fab fragment that has excellent binding
activity and is expected to accumulate in a cancer focus within a
given time (e.g., 24 hours) after administration. Another object of
the present invention is to provide a composition for diagnosis
comprising the Fab fragment and a diagnosis method using the same,
and to provide a composition for treatment comprising the Fab
fragment and a treatment method using the same.
Solution to Problem
[0017] The present inventors have conducted considerable diligent
studies on the preparation of an anti-human MUC1 antibody Fab
fragment having excellent binding activity against human
cancer-specific MUC1, and consequently prepared an anti-human MUC1
antibody Fab fragment comprising a heavy chain variable region
consisting of the amino acid sequence represented by SEQ ID NO: 8
or SEQ ID NO: 10, and a light chain variable region consisting of
the amino acid sequence represented by SEQ ID NO: 12 (Example 1)
and found that: the anti-human MUC1 antibody Fab fragment has
excellent binding activity against human cancer-specific MUC1
(Example 3) and is free from the attenuation of the binding
activity against human cancer-specific MUC1 by fluorescent labeling
and labeling with a chelating agent (Example 5 and Example 7); and
a conjugate comprising the anti-human MUC1 antibody Fab fragment is
useful in the diagnosis of cancers (Example 8, Example 12 and
Example 13) and exhibits an antitumor effect in subcutaneously
cancer-bearing models (Example 15).
[0018] As a result, a diagnosis approach and a treatment approach
using the anti-human MUC1 antibody Fab fragment and the conjugate
comprising the anti-human MUC1 antibody Fab fragment are
provided.
[0019] The present invention includes aspects given below as
medically or industrially useful substances and methods.
[0020] Specifically, in one aspect, the present invention can be as
follows:
[1] An anti-human MUC1 antibody Fab fragment selected from the
group consisting of the following (a) and (b): (a) an anti-human
MUC1 antibody Fab fragment comprising a heavy chain fragment
comprising a heavy chain variable region consisting of the amino
acid sequence represented by SEQ ID NO: 8 or SEQ ID NO: 10 and a
light chain comprising a light chain variable region consisting of
the amino acid sequence represented by SEQ ID NO: 12; and (b) an
anti-human MUC1 antibody Fab fragment comprising a heavy chain
fragment comprising a heavy chain variable region derived from a
heavy chain variable region consisting of the amino acid sequence
represented by SEQ ID NO: 8 or SEQ ID NO: 10 by the modification of
glutamine at amino acid position 1 of SEQ ID NO: 8 or SEQ ID NO: 10
into pyroglutamic acid, and a light chain comprising a light chain
variable region consisting of the amino acid sequence represented
by SEQ ID NO: 12. [2] The anti-human MUC1 antibody Fab fragment
according to [I] which is selected from the group consisting of the
following (a) and (b): (a) an anti-human MUC1 antibody Fab fragment
comprising a heavy chain fragment consisting of the amino acid
sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 and a light
chain consisting of the amino acid sequence represented by SEQ ID
NO: 6; and (b) an anti-human MUC1 antibody Fab fragment comprising
a heavy chain fragment derived from a heavy chain fragment
consisting of the amino acid sequence represented by SEQ ID NO: 2
or SEQ ID NO: 4 by the modification of glutamine at amino acid
position 1 of SEQ ID NO: 2 or SEQ ID NO: 4 into pyroglutamic acid,
and a light chain consisting of the amino acid sequence represented
by SEQ ID NO: 6. [3] The anti-human MUC1 antibody Fab fragment
according to [1] which is selected from the group consisting of the
following (a) and (b): (a) an anti-human MUC1 antibody Fab fragment
comprising a heavy chain fragment comprising a heavy chain variable
region consisting of the amino acid sequence represented by SEQ ID
NO: 10 and a light chain comprising a light chain variable region
consisting of the amino acid sequence represented by SEQ ID NO: 12;
and (b) an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment comprising a heavy chain variable region derived
from a heavy chain variable region consisting of the amino acid
sequence represented by SEQ ID NO: 10 by the modification of
glutamine at amino acid position 1 of SEQ ID NO: 10 into
pyroglutamic acid, and a light chain comprising a light chain
variable region consisting of the amino acid sequence represented
by SEQ ID NO: 12. [4] The anti-human MUC1 antibody Fab fragment
according to [3] which is selected from the group consisting of the
following (a) and (b): (a) an anti-human MUC1 antibody Fab fragment
comprising a heavy chain fragment consisting of the amino acid
sequence represented by SEQ ID NO: 4 and a light chain consisting
of the amino acid sequence represented by SEQ ID NO: 6; and (b) an
anti-human MUC1 antibody Fab fragment comprising a heavy chain
fragment derived from a heavy chain fragment consisting of the
amino acid sequence represented by SEQ ID NO: 4 by the modification
of glutamine at amino acid position 1 of SEQ ID NO: 4 into
pyroglutamic acid, and a light chain consisting of the amino acid
sequence represented by SEQ ID NO: 6. [5] The anti-human MUC1
antibody Fab fragment according to [4] which is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment consisting
of the amino acid sequence represented by SEQ ID NO: 4 and a light
chain consisting of the amino acid sequence represented by SEQ ID
NO: 6. [6] The anti-human MUC1 antibody Fab fragment according to
[4] which is an anti-human MUC1 antibody Fab fragment comprising a
heavy chain fragment derived from a heavy chain fragment consisting
of the amino acid sequence represented by SEQ ID NO: 4 by the
modification of glutamine at amino acid position 1 of SEQ ID NO: 4
into pyroglutamic acid, and a light chain consisting of the amino
acid sequence represented by SEQ ID NO: 6. [7] A conjugate
comprising one or more labeling moiety and the anti-human MUC1
antibody Fab fragment according to any of [1] to [6]. [8] The
conjugate according to [7], wherein the labeling moiety is (i) a
ligand and a linker, (ii) a ligand, (iii) a fluorescent dye and a
linker, or (iv) a fluorescent dye. [9] The conjugate according to
[8], wherein the labeling moiety is (i) a ligand and a linker or
(ii) a ligand. [10] The conjugate according to [9], wherein the
ligand is a ligand represented by the following formula (A):
##STR00001##
wherein the wavy line represents binding to the anti-human MUC1
antibody Fab fragment or the linker. [11] The conjugate according
to [10], wherein the labeling moiety is a ligand and a linker
represented by the following formula (A'):
##STR00002##
wherein the wavy line represents binding to the anti-human MUC1
antibody Fab fragment. [12] The conjugate according to [11],
wherein the anti-human MUC1 antibody Fab fragment is bound via an
amino group thereof to the carbon atom of a labeling moiety
terminal C(.dbd.S) group. [13] A conjugate selected from the group
consisting of the following (a) to (c): (a) the conjugate according
to [12] wherein the anti-human MUC1 antibody Fab fragment is the
anti-human MUC1 antibody Fab fragment according to [5]; (b) the
conjugate according to [12] wherein the anti-human MUC1 antibody
Fab fragment is the anti-human MUC1 antibody Fab fragment according
to [6]; and (c) a conjugate which is a mixture of (a) and (b). [14]
The conjugate according to any of [9] to [13], further comprising a
metal. [15] The conjugate according to [14], wherein the metal is a
metal radioisotope. [16] The conjugate according to [15], wherein
the metal is .sup.89Zr. [17] The conjugate according to [13],
further comprising .sup.89Zr. [18] The conjugate according to [8],
wherein the labeling moiety is (i) a fluorescent dye and a linker
or (ii) a fluorescent dye. [19] The conjugate according to [18],
wherein the fluorescent dye is a fluorescent dye selected from the
group consisting of the following formula (B) and the following
formula (C):
##STR00003##
wherein the wavy line represents binding to the anti-human MUC1
antibody Fab fragment or the linker. [20] The conjugate according
to [19], wherein the wavy line represents binding to the anti-human
MUC1 antibody Fab fragment, and the anti-human MUC1 antibody Fab
fragment is bound via an amino group thereof to the carbon atom of
a labeling moiety terminal C(.dbd.O) group. [21] The conjugate
according to [20], wherein the labeling moiety is a fluorescent dye
represented by formula (B). [22] A conjugate selected from the
group consisting of the following (a) to (c): (a) the conjugate
according to [21] wherein the anti-human MUC1 antibody Fab fragment
is the anti-human MUC1 antibody Fab fragment according to [5]; (b)
the conjugate according to [21] wherein the anti-human MUC1
antibody Fab fragment is the anti-human MUC1 antibody Fab fragment
according to [6]; and (c) a conjugate which is a mixture of (a) and
(b). [23] The conjugate according to [20], wherein the labeling
moiety is a fluorescent dye represented by formula (C). [24] A
conjugate selected from the group consisting of the following (a)
to (c): (a) the conjugate according to [23] wherein the anti-human
MUC1 antibody Fab fragment is the anti-human MUC1 antibody Fab
fragment according to [5]; (b) the conjugate according to [23]
wherein the anti-human MUC1 antibody Fab fragment is the anti-human
MUC1 antibody Fab fragment according to [6]; and (c) a conjugate
which is a mixture of (a) and (b). [25] A polynucleotide selected
from the group consisting of the following (a) and (b): (a) a
polynucleotide comprising a nucleotide sequence encoding the heavy
chain fragment of the anti-human MUC1 antibody Fab fragment
according to [1]; and (b) a polynucleotide comprising a nucleotide
sequence encoding the light chain of the anti-human MUC1 antibody
Fab fragment according to [1]. [26] A polynucleotide selected from
the group consisting of the following (a) and (b): (a) a
polynucleotide comprising a nucleotide sequence encoding the heavy
chain fragment of the anti-human MUC1 antibody Fab fragment
according to [5]; and (b) a polynucleotide comprising a nucleotide
sequence encoding the light chain of the anti-human MUC1 antibody
Fab fragment according to [5]. [27] An expression vector comprising
the following (a) and/or (b): (a) a polynucleotide comprising a
nucleotide sequence encoding the heavy chain fragment of the
anti-human MUC1 antibody Fab fragment according to [1]; and (b) a
polynucleotide comprising a nucleotide sequence encoding the light
chain of the anti-human MUC1 antibody Fab fragment according to
[1]. [28] An expression vector comprising the following (a) and/or
(b): (a) a polynucleotide comprising a nucleotide sequence encoding
the heavy chain fragment of the anti-human MUC1 antibody Fab
fragment according to [5]; and (b) a polynucleotide comprising a
nucleotide sequence encoding the light chain of the anti-human MUC1
antibody Fab fragment according to [5]. [29] A host cell selected
from the group consisting of the following (a) to (d): (a) a host
cell transformed with an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding the heavy
chain fragment of the anti-human MUC1 antibody Fab fragment
according to [1]; (b) a host cell transformed with an expression
vector comprising a polynucleotide comprising a nucleotide sequence
encoding the light chain of the anti-human MUC1 antibody Fab
fragment according to [1]; (c) a host cell transformed with an
expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the heavy chain fragment of the
anti-human MUC1 antibody Fab fragment according to [1] and a
polynucleotide comprising a nucleotide sequence encoding the light
chain of the anti-human MUC1 antibody Fab fragment according to
[I]; and (d) a host cell transformed with an expression vector
comprising a polynucleotide comprising a nucleotide sequence
encoding the heavy chain fragment of the anti-human MUC1 antibody
Fab fragment according to [1] and an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding the light
chain of the anti-human MUC1 antibody Fab fragment according to
[I]. [30] A host cell selected from the group consisting of the
following (a) to (d): (a) a host cell transformed with an
expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the heavy chain fragment of the
anti-human MUC1 antibody Fab fragment according to [5]; (b) a host
cell transformed with an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding the light
chain of the anti-human MUC1 antibody Fab fragment according to
[5]; (c) a host cell transformed with an expression vector
comprising a polynucleotide comprising a nucleotide sequence
encoding the heavy chain fragment of the anti-human MUC1 antibody
Fab fragment according to [5] and a polynucleotide comprising a
nucleotide sequence encoding the light chain of the anti-human MUC1
antibody Fab fragment according to [5]; and (d) a host cell
transformed with an expression vector comprising a polynucleotide
comprising a nucleotide sequence encoding the heavy chain fragment
of the anti-human MUC1 antibody Fab fragment according to [5] and
an expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the light chain of the anti-human MUC1
antibody Fab fragment according to [5]. [31] A method for producing
an anti-human MUC1 antibody Fab fragment comprising the step of
culturing a host cell selected from the group consisting of the
following (a) to (c) to express the anti-human MUC1 antibody Fab
fragment: (a) a host cell transformed with an expression vector
comprising a polynucleotide comprising a nucleotide sequence
encoding the heavy chain fragment of the anti-human MUC1 antibody
Fab fragment according to [1] and a polynucleotide comprising a
nucleotide sequence encoding the light chain of the anti-human MUC1
antibody Fab fragment according to [1]; (b) a host cell transformed
with an expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the heavy chain fragment of the
anti-human MUC1 antibody Fab fragment according to [1] and an
expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the light chain of the anti-human MUC1
antibody Fab fragment according to [I]; and (c) a host cell
transformed with an expression vector comprising a polynucleotide
comprising a nucleotide sequence encoding the heavy chain fragment
of the anti-human MUC1 antibody Fab fragment according to [1], and
a host cell transformed with an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding the light
chain of the anti-human MUC1 antibody Fab fragment according to
[1]. [32] A method for producing an anti-human MUC1 antibody Fab
fragment comprising the step of culturing a host cell selected from
the group consisting of the following (a) to (c) to express the
anti-human MUC1 antibody Fab fragment: (a) a host cell transformed
with an expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the heavy chain fragment of the
anti-human MUC1 antibody Fab fragment according to [5] and a
polynucleotide comprising a nucleotide sequence encoding the light
chain of the anti-human MUC1 antibody Fab fragment according to
[5]; (b) a host cell transformed with an expression vector
comprising a polynucleotide comprising a nucleotide sequence
encoding the heavy chain fragment of the anti-human MUC1 antibody
Fab fragment according to [5] and an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding the light
chain of the anti-human MUC1 antibody Fab fragment according to
[5]; and (c) a host cell transformed with an expression vector
comprising a polynucleotide comprising a nucleotide sequence
encoding the heavy chain fragment of the anti-human MUC1 antibody
Fab fragment according to [5], and a host cell transformed with an
expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the light chain of the anti-human MUC1
antibody Fab fragment according to [5]. [33] A method for producing
a conjugate comprising a labeling moiety and an anti-human MUC1
antibody Fab fragment, comprising the steps of: producing the
anti-human MUC1 antibody Fab fragment by the method according to
[31] or [32]; and covalently binding the Fab fragment to the
labeling moiety. [34] The method for producing a conjugate
according to [33], wherein the step of covalently binding the Fab
fragment to the labeling moiety is the step of i) binding the Fab
fragment via a linker to a ligand or ii) covalently binding the Fab
fragment directly to a ligand. [35] The method for producing a
conjugate according to [34], further comprising the step of
labeling the ligand of the conjugate with a metal radioisotope.
[36] The method for producing a conjugate according to [33],
wherein the step of covalently binding the Fab fragment to the
labeling moiety is the step of i) binding the Fab fragment via a
linker to a fluorescent dye or ii) covalently binding the Fab
fragment directly to a fluorescent dye. [37] A composition for
diagnosis comprising one or more conjugate according to any of [7]
to [24], and a pharmaceutically acceptable carrier. [38] The
composition for diagnosis according to [37], wherein the conjugate
is the conjugate according to any of [17], [22] and [24]. [39] The
composition for diagnosis according to [38], wherein the conjugate
is the conjugate according to [17]. [40] The composition for
diagnosis according to [38], wherein the conjugate is the conjugate
according to [22]. [41] The composition for diagnosis according to
[38], wherein the conjugate is the conjugate according to [24].
[42] The composition for diagnosis according to any of [37] to [41]
which is used in the diagnosis of a cancer expressing human MUC1.
[43] The composition for diagnosis according to [42], wherein the
cancer is breast cancer or bladder cancer. [44] A pharmaceutical
composition comprising one or more conjugate according to any of
[7] to [24], and a pharmaceutically acceptable carrier. [45] The
pharmaceutical composition according to [44], wherein the conjugate
is the conjugate according to any of [17], [22] and [24]. [46] The
pharmaceutical composition according to [45], wherein the conjugate
is the conjugate according to [24]. [47] The pharmaceutical
composition according to any of [44] to [46] which is a
pharmaceutical composition for treating a cancer expressing human
MUC1. [48] The pharmaceutical composition according to [47],
wherein the cancer is breast cancer or bladder cancer. [49] Use of
the conjugate according to any of [7] to [24] for the production of
a composition for the diagnosis of breast cancer or bladder cancer
and/or a pharmaceutical composition for treating breast cancer or
bladder cancer. [50] The conjugate according to any of [7] to [24]
for use in the diagnosis and/or treatment of breast cancer or
bladder cancer. [51] A method for diagnosing breast cancer or
bladder cancer, comprising preoperatively or intraoperatively
administering the conjugate according to any of [7] to [24] to a
subject. [52] A method for treating breast cancer or bladder
cancer, comprising the step of administering a therapeutically
effective amount of the conjugate according to any of [7] to
[24].
Advantageous Effects of Invention
[0021] The anti-human MUC1 antibody Fab fragment of the present
invention has excellent binding activity against human
cancer-specific MUC1 and is expected to be useful in the diagnosis
and/or treatment of cancers such as breast cancer.
BRIEF DESCRIPTION OF DRAWINGS
[0022] FIG. 1 is a graph and a table showing the binding activity
of P10-1 Fab, P10-2 Fab and 1B2 Fab of Comparative Example against
human cancer-specific MUC1.
[0023] FIG. 2 is a graph and a table showing the binding activity
of P10-1 Fab Dye, P10-2 Fab Dye and 1B2 Fab Dye of Comparative
Example against human cancer-specific MUC1.
[0024] FIG. 3 is a graph and a table showing the binding activity
of P10-2 Fab DFO and P10-2 Fab against human cancer-specific
MUC1.
[0025] FIG. 4 is a graph showing the binding activity of P10-2 Fab
against breast cancer cell line MDA-MB-468 cells (also referred to
as MM-468 cells) and bladder cancer cell line 647-V cells
expressing human cancer-specific MUC1. The horizontal axis depicts
the concentration (Log(mg/mL)) of P10-2 Fab, and the vertical axis
depicts luminescence.
[0026] FIG. 5A is representative photographs taken with a usual
camera (left) and a near-infrared fluorescence camera (center and
right) 6 hours after administration of P10-2 Fab Dye which was
intravenously administered at 3 mg/kg to subcutaneously
cancer-bearing models.
[0027] FIG. 5B is a graph quantifying the tumor/background ratio of
a tumor site and a peritumoral background site and indicates
mean+standard error. The horizontal axis depicts the dose and time
after administration of P10-2 Fab Dye.
[0028] FIG. 6 is a graph quantifying luminescence of the tumor
sites of administering P10-2 Fab IR700 to MM-468 cell-transplanted
mice, euthanizing the animals 2 hours after the administration,
excising tumor, taking photographs with IVIS SPECTRUM. The vertical
axis depicts luminescence.
[0029] FIG. 7 is a graph showing results of measuring cytotoxicity
of reacting P10-2 Fab IR700 to MM-468 cells, 647-V cells or CHO-K1
cells with irradiating light of Comparative Example. The upper
column of the horizontal axis of each graph depicts the
concentration of P10-2 Fab IR700, and the lower column depicts the
exposure of light. The vertical axis depicts luminescence and
indicates mean+standard error.
[0030] FIG. 8 is a graph showing results of measuring an antitumor
effect depending on the presence or absence of light irradiation (0
J or 200 J) at the time of P10-2 Fab IR700 administration using
MM-468 cell-transplanted nude mice. The horizontal axis depicts the
number of days when the day on which tumor volume became 300
mm.sup.3 was defined as day 1. The vertical axis depicts tumor
volume (mm.sup.3) and indicates mean+standard error.
DESCRIPTION OF EMBODIMENTS
[0031] Hereinafter, the present invention will be described in
detail. However, the present invention is not limited thereby.
Scientific terms and technical terms used in relation to the
present invention have meanings generally understood by those
skilled in the art, unless otherwise specified herein.
[0032] The present inventors have conducted considerable diligent
studies on the preparation of an anti-human cancer-specific MUC1
antibody or an antigen binding fragment thereof and consequently
successfully prepared an anti-human MUC1 antibody Fab fragment
having the ability to strongly bind to cancer-specific MUC1.
[0033] The basic structure of an antibody molecule is common among
classes and is constituted by heavy chains having a molecular
weight of 50000 to 70000 and light chains having a molecular weight
of 20000 to 30000. The heavy chain usually consists of a
polypeptide chain comprising approximately 440 amino acids, has a
structure characteristic of each class, and is called .gamma.,
.mu., .alpha., .delta., and .epsilon. chains corresponding to IgG,
IgM, IgA, IgD, and IgE. IgG further has IgG1, IgG2, IgG3, and IgG4
which are called .gamma.1, .gamma.2, .gamma.3, and .gamma.4,
respectively. The light chain usually consists of a polypeptide
chain comprising approximately 220 amino acids and known as two
types, L and K types, which are called .lamda. and .kappa. chains,
respectively. As for the peptide configuration of the basic
structure of the antibody molecule, two homologous heavy chains and
two homologous light chains are linked through disulfide bonds
(S--S bonds) and non-covalent bonds to form a molecular weight of
150000 to 190000. The two light chains can pair with any of the
heavy chains. An individual antibody molecule is constantly made up
of two identical light chains and two identical heavy chains.
[0034] Four (or five for .mu. and .epsilon. chains) and two
intrachain S--S bonds are present in the heavy chain and the light
chain, respectively, and each constitute one loop per 100 to 110
amino acid residues. This conformation is similar among the loops
and is called structural unit or domain. For both the heavy chain
and the light chain, a domain positioned at the N terminus does not
have a constant amino acid sequence even among preparations from
the same classes (subclasses) of animals of the same species, and
is thus called variable region. The respective domains are called
heavy chain variable region (VH domain) and light chain variable
region (VL domain). An amino acid sequence on the C-terminal side
therefrom is almost constant on a class or subclass basis and
called constant region. The respective domains are represented by
CH1, CH2, CH3 and CL.
[0035] The binding specificity of the antibody for an antigen
depends on the amino acid sequence of a moiety constituted by the
heavy chain variable region and the light chain variable region. On
the other hand, biological activity such as binding to complements
or various cells reflects the difference in structure among the
constant regions of Igs of respective classes. It is known that the
variability of the heavy chain and light chain variable regions is
limited substantially by three small hypervariable regions present
in both the chains. These regions are called complementarity
determining regions (CDRs; CDR1, CDR2, and CDR3 in order from the
N-terminal side). The remaining moieties of the variable region are
called framework regions (FRs) and are relatively constant.
[0036] A region between the CH1 domain and the CH2 domain of the
heavy chain constant region of an antibody is called hinge region.
This region is rich in proline residues and contains a plurality of
interchain S--S bonds that connect two heavy chains. For example,
the hinge regions of human IgG1, IgG2, IgG3, and IgG4 contain 2, 4,
11, and 2 cysteine residues, respectively, which constitute S--S
bonds between the heavy chains. The hinge region is a region highly
sensitive to a proteolytic enzyme such as papain or pepsin. In the
case of digesting an antibody with papain, the heavy chains are
cleaved at a position on the N-terminal side from the inter-heavy
chain S--S bonds of the hinge region and thus decomposed into two
Fab fragments and one Fc fragment. The Fab fragment is constituted
by a light chain and a heavy chain fragment comprising a heavy
chain variable region (VH), a CH1 domain and a portion of the hinge
region. The Fab fragment comprises variable regions and has antigen
binding activity.
[0037] <Anti-Human MUC1 Antibody Fab Fragment of Present
Invention>
[0038] The anti-human MUC1 antibody Fab fragment of the present
invention is a Fab fragment having the following feature:
[0039] an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment comprising a heavy chain variable region consisting
of the amino acid sequence represented by SEQ ID NO: 8 or SEQ ID
NO: 10 and a light chain comprising a light chain variable region
consisting of the amino acid sequence represented by SEQ ID NO:
12.
[0040] In one embodiment, the anti-human MUC1 antibody Fab fragment
of the present invention is an anti-human MUC1 antibody Fab
fragment comprising a heavy chain fragment comprising a heavy chain
variable region consisting of the amino acid sequence represented
by SEQ ID NO: 10 and a light chain comprising a light chain
variable region consisting of the amino acid sequence represented
by SEQ TD NO: 12.
[0041] Any constant region of Ig.gamma.1, Ig.gamma.2, Ig.gamma.3 or
Ig.gamma.4, etc. can be selectable as the heavy chain constant
region of the anti-human MUC1 antibody Fab fragment of the present
invention. In one embodiment, the heavy chain constant region of
the anti-human MUC1 antibody Fab fragment of the present invention
is a human Ig.gamma.1 constant region.
[0042] Any constant region of Ig.lamda. or Ig.kappa. can be
selectable as the light chain constant region of the anti-human
MUC1 antibody Fab fragment of the present invention. In one
embodiment, the light chain constant region of the anti-human MUC1
antibody Fab fragment of the present invention is a human Ig.kappa.
constant region.
[0043] In one embodiment, the anti-human MUC1 antibody Fab fragment
of the present invention is the following Fab fragment:
[0044] an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment consisting of the amino acid sequence represented by
SEQ ID NO: 2 or SEQ ID NO: 4 and a light chain consisting of the
amino acid sequence represented by SEQ ID NO: 6.
[0045] In one embodiment, the anti-human MUC1 antibody Fab fragment
of the present invention is an anti-human MUC1 antibody Fab
fragment comprising a heavy chain fragment consisting of the amino
acid sequence represented by SEQ ID NO: 4 and a light chain
consisting of the amino acid sequence represented by SEQ ID NO:
6.
[0046] In the case of expressing an antibody including a Fab
fragment in cells, the antibody is known to undergo a
posttranslational modification. Examples of the posttranslational
modification include the cleavage of heavy chain C-terminal lysine
by carboxypeptidase, the modification of heavy chain and light
chain N-terminal glutamine or glutamic acid into pyroglutamic acid
by pyroglutamylation, glycosylation, oxidation, deamidation, and
glycation. Such a posttranslational modification is known to occur
in various antibodies (J. Pharm. Sci., 2008; 97: 2426-2447).
[0047] The anti-human MUC antibody Fab fragment of the present
invention can also include a Fab fragment resulting from the
posttranslational modification. Examples of the anti-human MUC1
antibody Fab fragment of the present invention resulting from the
posttranslational modification include an anti-human MUC1 antibody
Fab fragment having an N-terminally pyroglutamylated heavy chain.
It is known in the art that such a posttranslational modification
by N-terminal pyroglutamylation has no influence on the activity of
the antibody (Anal. Biochem., 2006; 348: 24-39).
[0048] In one embodiment, the anti-human MUC1 antibody Fab fragment
of the present invention is an anti-human MUC1 antibody Fab
fragment having the following feature:
[0049] an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment comprising a heavy chain variable region derived
from a heavy chain variable region consisting of the amino acid
sequence represented by SEQ ID NO: 8 or SEQ ID NO: 10 by the
modification of glutamine at amino acid position 1 of SEQ ID NO: 8
or SEQ ID NO: 10 into pyroglutamic acid, and a light chain
comprising a light chain variable region consisting of the amino
acid sequence represented by SEQ ID NO: 12.
[0050] In a certain embodiment, the anti-human MUC antibody Fab
fragment of the present invention is an anti-human MUC1 antibody
Fab fragment having the following feature:
[0051] an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment comprising a heavy chain variable region derived
from a heavy chain variable region consisting of the amino acid
sequence represented by SEQ ID NO: 10 by the modification of
glutamine at amino acid position 1 of SEQ ID NO: 10 into
pyroglutamic acid, and a light chain comprising a light chain
variable region consisting of the amino acid sequence represented
by SEQ ID NO: 12.
[0052] In an alternative embodiment, the anti-MUC1 antibody Fab
fragment of the present invention is an anti-human MUC1 antibody
Fab fragment having the following feature:
[0053] an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment derived from a heavy chain fragment consisting of
the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4
by the modification of glutamine at amino acid position 1 of SEQ ID
NO: 2 or SEQ ID NO: 4 into pyroglutamic acid, and a light chain
consisting of the amino acid sequence represented by SEQ ID NO:
6.
[0054] In a certain embodiment, the anti-MUC1 antibody Fab fragment
of the present invention is an anti-human MUC1 antibody Fab
fragment having the following feature:
[0055] an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment derived from a heavy chain fragment consisting of
the amino acid sequence represented by SEQ ID NO: 4 by the
modification of glutamine at amino acid position 1 of SEQ ID NO: 4
into pyroglutamic acid, and a light chain consisting of the amino
acid sequence represented by SEQ ID NO: 6.
[0056] The anti-human MUC1 antibody Fab fragment of the present
invention binds to human cancer-specific MUC1. The cancer-specific
MUC1 is expressed in cancers such as breast cancer, lung cancer,
colorectal cancer, bladder cancer, skin cancer, thyroid gland
cancer, stomach cancer, pancreatic cancer, kidney cancer, ovary
cancer or uterine cervical cancer. A method for measuring the
binding activity of the obtained anti-human MUC1 antibody Fab
fragment against human cancer-specific MUC1 includes methods such
as ELISA and FACS. In the case of using, for example, ELISA, human
cancer-specific MUC1-positive cells (e.g., T-47D cells) are
immobilized onto an ELISA plate, to which the Fab fragment is then
added and reacted, and then, an anti-Ig.kappa. antibody or the like
labeled with horseradish peroxidase or the like is reacted. Then,
the binding of the secondary antibody is identified by activity
measurement using a reagent for detecting the activity thereof
(e.g., a chemiluminescent horseradish peroxidase substrate for the
horseradish peroxidase label) or the like.
[0057] The anti-human MUC1 antibody Fab fragment of the present
invention can be readily prepared by those skilled in the art using
a method known in the art on the basis of sequence information on
the heavy chain fragment and the light chain of the anti-human MUC1
antibody Fab fragment of the present invention disclosed herein.
The anti-human MUC1 antibody Fab fragment of the present invention
can be produced according to, but not particularly limited to, a
method described in, for example, <Method for producing
anti-human MUC1 antibody Fab fragment according to present
invention> mentioned later.
[0058] <Conjugate of Present Invention>
[0059] The conjugate of the present invention is a conjugate
comprising a labeling moiety and the anti-human MUC1 antibody Fab
fragment of the present invention.
[0060] The "labeling moiety" is (i) a ligand and a linker, (ii) a
ligand, (iii) a fluorescent dye and a linker, or (iv) a fluorescent
dye. A certain embodiment is (i) a ligand and a linker, or (ii) a
ligand. A certain embodiment is (i) a fluorescent dye and a linker,
or (ii) a fluorescent dye. The ligand of the "labeling moiety" may
further comprise a metal. A certain embodiment is (i) a ligand and
a linker or (ii) a ligand comprising a metal, and in other words,
is (i) a ligand that has formed a chelate complex with a metal, and
a linker, or (ii) a ligand that has formed a chelate complex with a
metal.
[0061] The conjugate of the present invention comprising a metal or
a fluorescent dye can be used in various contrast media and/or
cancer therapeutic agents and is used in, for example, an MRI
contrast medium, a PET tracer, a fluorescently labeled molecular
imaging agent, and a drug for use in photoimmunotherapy
methods.
[0062] In the present specification, the "metal" means a
paramagnetic metal ion or a metal radioisotope.
[0063] The paramagnetic metal ion is suitably used in an MRI
contrast medium. Examples of the embodiment of the paramagnetic
metal ion include, but are not limited to, Fe.sup.2+, Fe.sup.3+,
Cu.sup.2+, Ni.sup.2+, Rh.sup.2+, Co.sup.2+, Gd.sup.3+, Eu.sup.3+,
Dy.sup.3+, Tb.sup.31, Pm.sup.3+, Nd.sup.3+, Tm.sup.3+, Cc.sup.3+,
Y.sup.3+, Ho.sup.3+, Er.sup.3+, La.sup.3+, Yb.sup.3+, Mn.sup.3+,
and Mn.sup.2+. A certain embodiment is Gd.sup.3+, Mn.sup.3+,
Mn.sup.2+, Fc.sup.2+, or Fe.sup.3+. A certain embodiment is
Mn.sup.3+ or Mn.sup.2+. In this case, halogen or the like can be
used as a counter anion in the conjugate. Alternatively, the
counter anion may be C(.dbd.O)O.sup.- of the ligand. The conjugate
may further have a counter cation such as Na.sup.+.
[0064] The metal radioisotope is used in, for example, a PET
tracer. Examples of a certain embodiment include, but are not
limited to, .sup.89Zr, .sup.51Mn, .sup.52Fe, .sup.60Cu, .sup.67Ga,
.sup.68Ga, .sup.72As, .sup.99mTc, and .sup.111In. A certain
embodiment is .sup.89Zr, .sup.67Cu, .sup.67Ga, .sup.68Ga,
.sup.99mTc, or .sup.111In. A certain embodiment is a radioisotope
of zirconium. A certain embodiment is .sup.89Zr.
[0065] The "ligand" is a moiety capable of forming a chelate
complex with a metal in the conjugate and means a group constituted
by a chelating agent. The constituted group is a group having a
bond by the removal of a proton from the chelating agent.
[0066] The "chelating agent" is a compound that can form a
coordinate bond with a metal.
[0067] Examples of the "chelating agent" include siderophore and
non-siderophore. Examples of the siderophore include hydroxamic
acid type, catechol type, and mixed ligand type. Examples of the
hydroxamic acid-type siderophore include ferrichrome, deferoxamine
(DFO) represented by the following formula:
##STR00004##
fusarinine C, ornibactin, and rhodotorulic acid. Examples of the
catechol-type siderophore include enterobactin, bacillibactin, and
vibriobactin. Examples of the mixed ligand-type siderophore include
azotobactin, pyoverdine, and yersiniabactin. In the case of the
siderophore, DFO can be reacted via its reactive functional group
--NH.sub.2 with the linker or the Fab fragment, and the siderophore
other than DFO can also be reacted via its reactive functional
group such as a carboxy group, a hydroxy group, or an amino group
with the linker or the Fab fragment by a method usually used by
those skilled in the art.
[0068] Examples of the non-siderophore include DTPA
(diethylenetriaminepentaacetic acid, CAS No: 67-43-6), DTPA-BMA
(1,7-bis(methylcarbamoylmethyl)-1,4,7-triazaheptane-1,4,7-triacetic
acid, CAS No: 119895-95-3), EOB-DTPA (DTPA bound to an ethoxybenzyl
group, CAS No: 158599-72-5), TTHA (triethylenetetraminehexaacetic
acid, CAS No: 869-52-3), DO3A
(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, CAS No:
217973-03-0), HP-DO3A
(10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic
acid, CAS No: 120041-08-9), DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, CAS No:
60239-18-1), and known reactive derivatives thereof.
[0069] Compounds and conjugates described herein also encompass
free forms and salts thereof unless otherwise specified. In this
context, the "salt thereof" is a salt that can be formed by the
compound or the conjugate that may form an acid-addition salt or a
salt with a base depending on the type of a substituent in the
compound or the conjugate. Specific examples thereof include:
acid-addition salts with inorganic acids such as hydrochloric acid,
hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and
phosphoric acid, or organic acids such as formic acid, acetic acid,
propionic acid, oxalic acid, malonic acid, succinic acid, fumaric
acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric
acid, dibenzoyltartaric acid, ditoluoyltartaric acid, citric acid,
methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,
p-toluenesulfonic acid, aspartic acid, and glutamic acid; salts
with inorganic bases such as sodium, potassium, magnesium, calcium,
and aluminum, or organic bases such as methylamine, ethylamine,
ethanolamine, lysine, and ornithine; salts with various amino acids
and amino acid derivatives, such as acetylleucine; and ammonium
salts. For example, DFO exists as deferoxamine methanesulfonate or
exists as other salts. DTPA exits both as a free form and as sodium
salt.
[0070] A certain embodiment of the "chelating agent" for use in an
MRI contrast medium is the siderophore or non-siderophore chelating
agent described above.
[0071] A certain embodiment of the "chelating agent" for use in a
PET tracer is the siderophore or non-siderophore chelating agent
described above. A certain embodiment is MAG3
(mercapto-acetyl-glycine-glycine-glycine, CAS No: 66516-09-4). A
certain embodiment is DFO.
[0072] Examples of a certain embodiment of the "chelating agent"
constituting the ligand contained in the conjugate of the present
invention include DFO, DTPA, DTPA-BMA, EOB-DTPA, DO3A, HP-DO3A, and
DOTA. A certain embodiment is DFO, DTPA, or DOTA. A certain
embodiment is DFO.
[0073] The "linker is a group that creates a distance between the
anti-human MUC1 antibody Fab fragment and the ligand. Examples of a
certain embodiment of the "linker" in the conjugate include the
following formula:
##STR00005##
(hereinafter, referred to as
--C(.dbd.S)--NH-(1,4-phenylene)-NH--C(.dbd.S)--),
--CH.sub.2-(1,4-phenylene)-NH--C(.dbd.S)--, and
--C(.dbd.O)--(C.sub.1-20 alkylene)-C(.dbd.O)--. In this context,
the "C.sub.1-20 alkylene" is linear or branched alkylene having 1
to 20 carbon atoms. A certain embodiment of the C.sub.1-20 alkylene
is C.sub.1-10 alkylene or C.sub.1-2 alkylene. A certain embodiment
of the C.sub.1-20 alkylene is ethylene. A certain embodiment is
--C(.dbd.S)--NH-(1,4-phenylene)-NH--C(.dbd.S)--. A certain
embodiment is --C(.dbd.O)--C.sub.2H.sub.4--C(.dbd.O)--. Examples of
a reagent that can be used as the linker include
HO--C(.dbd.O)--(C.sub.1-20 alkylene)-C(.dbd.O)--OH, succinic acid,
and p-di-NCS-benzene(p-diisocyanobenzene).
[0074] The conjugate of the present invention comprising a
fluorescent dye can be used as a fluorescently labeled molecular
imaging agent, a drug for use in photoimmunotherapy methods, or a
fluorescently labeled molecular imaging agent and a drug for use in
photoimmunotherapy methods.
[0075] A dye having absorption maximum and emission maximum at a
near-infrared wavelength (650 to 1000 nm) usually used in
photoimaging can be used as the fluorescent dye for use in the
conjugate of the present invention. Examples of a certain
embodiment of the fluorescent dye include cyanine and indocyanine
compounds. Examples of a certain embodiment include IRDye800CW and
IRDye700DX (LI-COR Bioscience, Inc.), Cy (Molecular Probes, Inc.),
Alexa Fluor, BODIPY, and DyLight (Thermo Fisher Scientific Inc.),
CF790 (Biotium, Inc.), DY (Dyomics GmbH), HiLyte Fluor 680 and
HiLyte Fluor 750 (AnaSpec Inc.), and PULSAR650 and QUASAR670 (LGC
Biosearch Technologies). A certain embodiment is IRDye800CW
represented by the following formula:
##STR00006##
or IRDye700DX represented by the following formula:
##STR00007##
The fluorescent dye can be reacted via its carboxy group, hydroxy
group, amino group, or the like or via an active group introduced
by a method usually used by those skilled in the art with the Fab
fragment or the linker. A certain embodiment of the fluorescent dye
having an introduced active group is a fluorescent dye esterified
with a N-hydroxysuccinimide (NHS) group. For example, NHS esters of
IRDye800CW and IRDye700DX mentioned above are commercially
available, and they can be utilized.
[0076] The binding of the anti-human MUC1 antibody Fab fragment of
the present invention to the labeling moiety can be appropriately
performed by those skilled in the art using a known approach. For
example, the labeling moiety can be bound to one or more amino
groups (e.g., a N-terminal amino group and an amino group of an
amino acid side chain), one or more thiol groups (e.g., a thiol
group of an amino acid side chain), or one or more carboxyl groups
(e.g., carboxyl groups of the C terminus and an amino acid side
chain) of the anti-human MUC1 antibody Fab fragment of the present
invention. A certain embodiment of the conjugate of the present
invention is a conjugate wherein the labeling moiety is bound to
one or more amino groups of the anti-human MUC1 antibody Fab
fragment of the present invention.
[0077] When the labeling moiety is a ligand and a linker, the
conjugate of the present invention may be produced by reacting the
chelating agent with a substance obtained through the reaction of
the anti-human MUC1 antibody Fab fragment of the present invention
with the linker. It may be produced by reacting the anti-human MUC1
antibody Fab fragment of the present invention with a substance
obtained through the reaction of the chelating agent with the
linker. As a reaction example, a substance obtained through the
reaction of the amino group of the chelating agent with the linker
may be reacted with one or more amino groups (e.g., an N-terminal
amino group and an amino group of a lysine side chain) of the
anti-human MUC1 antibody Fab fragment of the present invention.
Reaction of synthesizing thiourea by adding isothiocyanate to
amine, reaction of synthesizing amide by adding amine and
carboxylic acid, or the like can be used in the production of the
conjugate. The reaction can be performed by the application of a
method known to those skilled in the art. A compound of the
chelating agent bound to the linker in advance may be used as a
starting material. Examples of the compound of the chelating agent
bound to the linker include p-SCN-Bn-DFO (DFO bound to a
p-isothiocyanophenylaminothiocarbonyl group, CAS No: 1222468-90-7)
represented by the following formula:
##STR00008##
DTPA bound to a p-isothiocyanobenzyl group (p-NCS-Bn-DTPA, CAS No:
102650-30-6), DOTA bound to a p-isothiocyanobenzyl group
(p-NCS-Bn-DOTA, CAS No: 127985-74-4), and p-SCN-Bn-CHX-A''-DTPA
([(R)-2-amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1-
,2-diamine-pentaacetic acid, CAS No: 157380-45-5).
[0078] The metal (paramagnetic metal ion or metal radioisotope) can
be added to the anti-human MUC1 antibody Fab fragment of the
present invention bound to one or more labeling moiety thus
produced by the production method to obtain the conjugate of the
present invention comprising the metal.
[0079] Also, the conjugate of the present invention may be produced
as a conjugate which is a Fab fragment bound via an amino group
thereof to one or more labeling moiety by reacting one or more
amino groups (e.g., a N-terminal amino group and an amino group of
an amino acid side chain) of the Fab fragment with the labeling
moiety having a carboxyl group or an isothiocyanic acid group
activated with N-hydroxysuccinimide (NHS).
[0080] The conjugate of the present invention is a conjugate
comprising one or more labeling moiety and the anti-human MUC1
antibody Fab fragment of the present invention. A certain
embodiment is the anti-human MUC1 antibody Fab fragment bound to 1
to 27 labeling moieties. A certain embodiment is the anti-human
MUC1 antibody Fab fragment bound to 1 to 23 labeling moieties. A
certain embodiment is the anti-human MUC1 antibody Fab fragment
bound to 1 to 15 labeling moieties. A certain embodiment is the
anti-human MUC1 antibody Fab fragment bound to 1 to 11 labeling
moieties. A certain embodiment is the anti-human MUC1 antibody Fab
fragment bound to 1 to 9 labeling moieties. A certain embodiment is
the anti-human MUC1 antibody Fab fragment bound to 1 to 7 labeling
moieties. A certain embodiment is the anti-human MUC1 antibody Fab
fragment bound to 1 to 5 labeling moieties. A certain embodiment is
the anti-human MUC1 antibody Fab fragment bound to 1 to 4 labeling
moieties. A certain embodiment is the anti-human MUC1 antibody Fab
fragment bound to one or more labeling moiety further comprising a
metal.
[0081] In one embodiment, the conjugate of the present invention is
a conjugate wherein the labeling moiety is (i) a ligand and a
linker, (ii) a ligand, (iii) a fluorescent dye and a linker, or
(iv) a fluorescent dye.
[0082] In a certain embodiment, examples of the conjugate of the
present invention include the followings:
(1) a conjugate wherein the anti-human MUC1 antibody Fab fragment
is an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment comprising a heavy chain variable region consisting
of the amino acid sequence represented by SEQ ID NO: 10 and a light
chain comprising a light chain variable region consisting of the
amino acid sequence represented by SEQ ID NO: 12; (2) the conjugate
of (1), wherein the anti-human MUC1 antibody Fab fragment is an
anti-human MUC1 antibody Fab fragment comprising a heavy chain
fragment consisting of the amino acid sequence represented by SEQ
ID NO: 4 and a light chain consisting of the amino acid sequence
represented by SEQ ID NO: 6; (3) a conjugate wherein the anti-human
MUC1 antibody Fab fragment is an anti-human MUC1 antibody Fab
fragment comprising a heavy chain fragment comprising a heavy chain
variable region derived from a heavy chain variable region
consisting of the amino acid sequence represented by SEQ ID NO: 10
by the modification of glutamine at amino acid position 1 of SEQ ID
NO: 10 into pyroglutamic acid, and a light chain comprising a light
chain variable region consisting of the amino acid sequence
represented by SEQ ID NO: 12; (4) the conjugate of (2), wherein the
anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment derived
from a heavy chain fragment consisting of the amino acid sequence
represented by SEQ ID NO: 4 by the modification of glutamine at
amino acid position 1 of SEQ ID NO: 4 into pyroglutamic acid, and a
light chain consisting of the amino acid sequence represented by
SEQ ID NO: 6; (5) the conjugate of any of (1) to (4), wherein the
labeling moiety is (i) a ligand and a linker, or (ii) a ligand; (6)
the conjugate of any of (1) to (4), wherein the ligand is a group
constituted by a chelating agent selected from the group consisting
of DFO, DTPA, DTPA-BMA, EOB-DTPA, DO3A, HP-DO3A and DOTA, and the
linker is a linker selected from the group consisting of
--C(.dbd.S)--NH-(1,4-phenylene)-NH--C(.dbd.S)--,
--CH.sub.2-(1,4-phenylene)-NH--C(.dbd.S)-- and
--C(.dbd.O)--(C.sub.1-20 alkylene)-C(.dbd.O)--; (7) the conjugate
of (6), wherein the ligand is a group constituted by a chelating
agent selected from the group consisting of DFO, DTPA, and DOTA;
(8) the conjugate of (6), wherein the ligand is a group constituted
by DFO, and the linker is
--C(.dbd.S)--NH-(1,4-phenylene)-NH--C(.dbd.S)--; (9) the conjugate
according to any of (5) to (8), further comprising a metal; (10)
the conjugate of (9), wherein the metal is a metal radioisotope;
and (11) the conjugate of (10), wherein the metal is .sup.89Zr.
[0083] In a certain embodiment, the conjugate of the present
invention is a conjugate wherein the labeling moiety is (i) a
ligand and a linker, or (ii) a ligand.
[0084] In a certain embodiment, the conjugate of the present
invention is a conjugate wherein the ligand is a ligand represented
by the following formula (A):
##STR00009##
wherein the wavy line represents binding to the anti-human MUC1
antibody Fab fragment or the linker.
[0085] In a certain embodiment, the conjugate of the present
invention wherein the ligand is a ligand represented by formula (A)
is a conjugate wherein the labeling moiety is a ligand and a linker
represented by the following formula (A'):
##STR00010##
wherein the wavy line represents binding to the anti-human MUC1
antibody Fab fragment, and the anti-human MUC1 antibody Fab
fragment is bound via an amino group thereof to the carbon atom of
a labeling moiety terminal C(.dbd.S) group.
[0086] In a certain embodiment, the conjugate of the present
invention is a conjugate wherein the labeling moiety is (i) a
ligand and a linker, or (ii) a ligand, the conjugate further
comprising a metal. A certain embodiment of the metal is a metal
radioisotope. A certain embodiment of the metal radioisotope is
.sup.89Zr.
[0087] In an alternative embodiment, the conjugate of the present
invention is a conjugate wherein the labeling moiety is (i) a
fluorescent dye and a linker, or (ii) a fluorescent dye.
[0088] In a certain embodiment, the conjugate of the present
invention wherein the labeling moiety comprises a fluorescent dye
is a conjugate wherein the fluorescent dye is a fluorescent dye
selected from the group consisting of the following formula (B) and
the following formula (C):
##STR00011##
wherein the wavy line represents binding to the anti-human MUC1
antibody Fab fragment or the linker.
[0089] In a certain embodiment, the conjugate of the present
invention wherein the labeling moiety is a fluorescent dye
represented by formula (B) is a conjugate wherein the wavy line
represents binding to the anti-human MUC1 antibody Fab fragment,
and the anti-human MUC1 antibody Fab fragment is bound via an amino
group thereof to the carbon atom of a labeling moiety terminal
C(.dbd.O) group.
[0090] In a certain embodiment, the conjugate of the present
invention wherein the labeling moiety is a fluorescent dye
represented by formula (C) is a conjugate wherein the wavy line
represents binding to the anti-human MUC1 antibody Fab fragment,
and the anti-human MUC1 antibody Fab fragment is bound via an amino
group thereof to the carbon atom of a labeling moiety terminal
C(.dbd.O) group.
[0091] Certain embodiments of the conjugate of the present
invention will be further shown below:
(1) a conjugate wherein the labeling moiety is a ligand and a
linker represented by formula (A'), and the anti-human MUC1
antibody Fab fragment is an anti-human MUC1 antibody Fab fragment
comprising a heavy chain fragment comprising a heavy chain variable
region consisting of the amino acid sequence represented by SEQ ID
NO: 10 and a light chain comprising a light chain variable region
consisting of the amino acid sequence represented by SEQ ID NO: 12;
(2) the conjugate of (1), wherein the anti-human MUC1 antibody Fab
fragment is an anti-human MUC1 antibody Fab fragment comprising a
heavy chain fragment consisting of the amino acid sequence
represented by SEQ ID NO: 4 and a light chain consisting of the
amino acid sequence represented by SEQ ID NO: 6; (3) a conjugate
wherein the labeling moiety is a ligand and a linker represented by
formula (A'), and the anti-human MUC1 antibody Fab fragment is an
anti-human MUC1 antibody Fab fragment comprising a heavy chain
fragment comprising a heavy chain variable region derived from a
heavy chain variable region consisting of the amino acid sequence
represented by SEQ ID NO: 10 by the modification of glutamine at
amino acid position 1 of SEQ ID NO: 10 into pyroglutamic acid, and
a light chain comprising a light chain variable region consisting
of the amino acid sequence represented by SEQ ID NO: 12; (4) the
conjugate of (3), wherein the anti-human MUC1 antibody Fab fragment
is an anti-human MUC1 antibody Fab fragment comprising a heavy
chain fragment derived from a heavy chain fragment consisting of
the amino acid sequence represented by SEQ ID NO: 4 by the
modification of glutamine at amino acid position 1 of SEQ ID NO: 4
into pyroglutamic acid, and a light chain consisting of the amino
acid sequence represented by SEQ ID NO: 6; (5) the conjugate of (2)
or (4) which is the anti-human MUC1 antibody Fab fragment bound to
1 to 11 labeling moieties; (6) the conjugate of (2) or (4) which is
the anti-human MUC1 antibody Fab fragment bound to 1 to 4 labeling
moieties; (7) the conjugate of any of (1) to (6), further
comprising a metal; (8) the conjugate of (7), wherein the metal is
a metal radioisotope; (9) the conjugate of (8), wherein the metal
is .sup.89Zr; (10) a conjugate wherein the labeling moiety is a
fluorescent dye represented by formula (B) or formula (C), and the
anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment comprising
a heavy chain variable region consisting of the amino acid sequence
represented by SEQ ID NO: 10 and a light chain comprising a light
chain variable region consisting of the amino acid sequence
represented by SEQ ID NO: 12; (11) the conjugate of (10), wherein
the anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment consisting
of the amino acid sequence represented by SEQ ID NO: 4 and a light
chain consisting of the amino acid sequence represented by SEQ ID
NO: 6; (12) a conjugate wherein the labeling moiety is a
fluorescent dye represented by formula (B) or formula (C), and the
anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment comprising
a heavy chain variable region derived from a heavy chain variable
region consisting of the amino acid sequence represented by SEQ ID
NO: 10 by the modification of glutamine at amino acid position 1 of
SEQ ID NO: 10 into pyroglutamic acid, and a light chain comprising
a light chain variable region consisting of the amino acid sequence
represented by SEQ ID NO: 12; (13) the conjugate of (12), wherein
the anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment derived
from a heavy chain fragment consisting of the amino acid sequence
represented by SEQ ID NO: 4 by the modification of glutamine at
amino acid position 1 of SEQ ID NO: 4 into pyroglutamic acid, and a
light chain consisting of the amino acid sequence represented by
SEQ ID NO: 6; (14) the conjugate of any of (10) to (13), wherein
the labeling moiety is a fluorescent dye represented by formula
(C); (15) the conjugate of (14) which is the anti-human MUC1
antibody Fab fragment bound to 1 to 11 labeling moieties; (16) the
conjugate of (15) which is the anti-human MUC1 antibody Fab
fragment bound to 1 to 5 labeling moieties; (17) the conjugate of
any of (10) to (13), wherein the labeling moiety is a fluorescent
dye represented by formula (B); (18) the conjugate of (17) which is
the anti-human MUC1 antibody Fab fragment bound to 1 to 11 labeling
moieties; and (19) the conjugate of (18) which is the anti-human
MUC1 antibody Fab fragment bound to 1 to 5 labeling moieties.
[0092] In an alternative embodiment, the conjugate of the present
invention is a conjugate represented by the following formula
(I):
[Formula 14]
(L-X).sub.p-Ab (I)
wherein Ab represents the anti-human MUC1 antibody Fab fragment, L
represents (i) a ligand or (ii) a fluorescent dye, X represents a
linker or a bond, and p is a natural number of 1 to 27. A certain
embodiment of p is a natural number of 1 to 23. A certain
embodiment is a natural number of 1 to 15. A certain embodiment is
a natural number of 1 to 11. A certain embodiment is a natural
number of 1 to 9. A certain embodiment is a natural number of 1 to
7. A certain embodiment is a natural number of 1 to 5. A certain
embodiment is a natural number of 1 to 4.
[0093] In a certain embodiment, the conjugate of the present
invention is a conjugate of formula (I), the conjugate further
comprising a metal. A certain embodiment of the metal is a metal
radioisotope. A certain embodiment of the metal radioisotope is
.sup.89Zr.
[0094] In a certain embodiment, the conjugate of formula (I) is the
conjugate of the present invention wherein the labeling moiety is a
ligand and a linker represented by formula (A'). In a certain
embodiment, this conjugate is a conjugate represented by the
following formula (II):
##STR00012##
wherein Ab represents the anti-human MUC1 antibody Fab fragment,
and p is a natural number of 1 to 27. A certain embodiment of p is
a natural number of 1 to 23. A certain embodiment is a natural
number of 1 to 15. A certain embodiment is a natural number of 1 to
11. A certain embodiment is a natural number of 1 to 9. A certain
embodiment is a natural number of 1 to 7. A certain embodiment is a
natural number of 1 to 5. A certain embodiment is a natural number
of 1 to 4. Ab is bound via an amino group thereof to the carbon
atom of a labeling moiety terminal C(.dbd.S) group.
[0095] In a certain embodiment, the conjugate of the present
invention is a conjugate of formula (II), the conjugate further
comprising a metal. A certain embodiment of the metal is a metal
radioisotope. A certain embodiment of the metal radioisotope is
.sup.89Zr.
[0096] In a certain embodiment, the conjugate of formula (I) is the
conjugate of the present invention wherein the labeling moiety is a
fluorescent dye represented by formula (B). In a certain
embodiment, the conjugate is a conjugate represented by the
following formula (III):
##STR00013##
wherein Ab represents the anti-human MUC1 antibody Fab fragment,
and p is a natural number of 1 to 27. A certain embodiment of p is
a natural number of 1 to 23. A certain embodiment is a natural
number of 1 to 15. A certain embodiment is a natural number of 1 to
11. A certain embodiment is a natural number of 1 to 9. A certain
embodiment is a natural number of 1 to 7. A certain embodiment is a
natural number of 1 to 5. A certain embodiment is a natural number
of 1 to 4. Ab is bound via an amino group thereof to the carbon
atom of a labeling moiety terminal C(.dbd.O) group.
[0097] In a certain embodiment, the conjugate of formula (I) is the
conjugate of the present invention wherein the labeling moiety is a
fluorescent dye represented by formula (C). In a certain
embodiment, this conjugate is a conjugate represented by the
following formula (IV):
##STR00014##
wherein Ab represents the anti-human MUC1 antibody Fab fragment,
and p is a natural number of 1 to 27. A certain embodiment of p is
a natural number of 1 to 23. A certain embodiment is a natural
number of 1 to 15. A certain embodiment is a natural number of 1 to
11. A certain embodiment is a natural number of 1 to 9. A certain
embodiment is a natural number of 1 to 7. A certain embodiment is a
natural number of 1 to 5. A certain embodiment is a natural number
of 1 to 4. Ab is bound via an amino group thereof to the carbon
atom of a labeling moiety terminal C(.dbd.O) group.
[0098] In a certain embodiment, the conjugate of the present
invention is the anti-human MUC1 antibody Fab fragment of the
present invention labeled with a detectable molecule. The
anti-human MUC1 antibody Fab fragment of the present invention
labeled with a detectable molecule is the anti-human MUC1 antibody
Fab fragment of the present invention and the detectable molecule
linked through a covalent bond directly or via an appropriate
linker. In the present specification, the detectable molecule means
every moiety detectable in an imaging diagnosis technique known in
the art. When the imaging diagnosis technique is, for example,
fluorescence imaging, the detectable molecule is a fluorescent dye.
When the imaging diagnosis technique is PET, the detectable
molecule is a compound imagable by PET. A certain embodiment is a
compound comprising a ligand labeled with a radionuclide, or a
sugar residue labeled with a nonmetal radionuclide. A certain
embodiment of the compound comprising a ligand labeled with a
radionuclide is a ligand that has formed a chelate complex with a
metal radioisotope. When the imaging diagnosis technique is MRI,
the detectable molecule is a compound detectable by the MRI
technique. A certain embodiment is a compound comprising a labeled
ligand having a paramagnetic metal ion. A certain embodiment of the
compound comprising a labeled ligand having a paramagnetic metal
ion is a ligand that has formed a chelate complex with a
paramagnetic metal ion.
[0099] When the anti-human MUC1 antibody Fab fragment of the
present invention labeled with a detectable molecule is used in the
context of a detectable molecule, the compound imagable by PET is
referred to as a PET tracer, and the compound detectable by the MRI
technique is referred to as a MRI contrast medium.
[0100] Examples of certain embodiments of the anti-human MUC1
antibody Fab fragment of the present invention labeled with a
detectable molecule include the followings:
(1) an anti-human MUC1 antibody Fab fragment wherein the detectable
molecule is a fluorescent dye, a PET tracer, or a MRI contrast
medium; (2) the anti-human MUC1 antibody Fab fragment of (1),
wherein the detectable molecule is a fluorescent dye; (3) the
anti-human MUC1 antibody Fab fragment of (2), wherein the
detectable molecule is a fluorescent dye represented by formula
(B); (4) the anti-human MUC1 antibody Fab fragment of (2), wherein
the detectable molecule is a fluorescent dye represented by formula
(C); (5) the anti-human MUC1 antibody Fab fragment of (1), wherein
the detectable molecule is a PET tracer or a MRI contrast medium;
(6) an anti-human MUC1 antibody Fab fragment wherein the detectable
molecule is a MRI contrast medium comprising a ligand represented
by formula (A) and a paramagnetic metal ion, or a PET tracer
comprising the ligand and a metal radioisotope; and (7) the
anti-human MUC1 antibody Fab fragment of (6), wherein the
detectable molecule and the linker are a MRI contrast medium
comprising a ligand and a linker represented by formula (A') and a
paramagnetic metal ion, or a PET tracer comprising the ligand and
the linker and a metal radioisotope.
[0101] The method mentioned above can be employed for the linking
of the anti-human MUC1 antibody Fab fragment of the present
invention to the detectable molecule.
[0102] The conjugate of the present invention also includes a
conjugate which is a mixture of a plurality of conjugates of the
present invention. For example, a conjugate which is mixture of a
conjugate comprising a labeling moiety and a
non-posttranslationally-modified anti-human MUC1 antibody Fab
fragment of the present invention, and a conjugate comprising a
labeling moiety and the anti-human MUC1 antibody Fab fragment of
the present invention resulting from the posttranslational
modification of the anti-human MUC1 antibody Fab fragment is also
included in the conjugate of the present invention.
[0103] Certain embodiments of the conjugate of the present
invention which is a mixture of a plurality of conjugates of the
present invention will be shown below:
(1) a conjugate which is a mixture of a conjugate wherein the
labeling moiety is a ligand and a linker represented by formula
(A'), and the anti-human MUC1 antibody Fab fragment is an
anti-human MUC1 antibody Fab fragment comprising a heavy chain
fragment consisting of the amino acid sequence represented by SEQ
ID NO: 4 and a light chain consisting of the amino acid sequence
represented by SEQ ID NO: 6, and a conjugate wherein the labeling
moiety is a ligand and a linker represented by formula (A'), and
the anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment derived
from a heavy chain fragment consisting of the amino acid sequence
represented by SEQ ID NO: 4 by the modification of glutamine at
amino acid position 1 of SEQ ID NO: 4 into pyroglutamic acid, and a
light chain consisting of the amino acid sequence represented by
SEQ ID NO: 6; (2) the conjugate of (1), further comprising a metal;
(3) the conjugate of (2), wherein the metal is a metal
radioisotope; (4) the conjugate of (3), wherein the metal is
.sup.89Zr; (5) a conjugate which is a mixture of a conjugate
wherein the labeling moiety is a fluorescent dye represented by
formula (B), and the anti-human MUC1 antibody Fab fragment is an
anti-human MUC1 antibody Fab fragment comprising a heavy chain
fragment consisting of the amino acid sequence represented by SEQ
ID NO: 4 and a light chain consisting of the amino acid sequence
represented by SEQ ID NO: 6, and a conjugate wherein the labeling
moiety is a fluorescent dye represented by formula (B), and the
anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment derived
from a heavy chain fragment consisting of the amino acid sequence
represented by SEQ ID NO: 4 by the modification of glutamine at
amino acid position 1 of SEQ ID NO: 4 into pyroglutamic acid, and a
light chain consisting of the amino acid sequence represented by
SEQ ID NO: 6; and (6) a conjugate which is a mixture of a conjugate
wherein the labeling moiety is a fluorescent dye represented by
formula (C), and the anti-human MUC1 antibody Fab fragment is an
anti-human MUC1 antibody Fab fragment comprising a heavy chain
fragment consisting of the amino acid sequence represented by SEQ
ID NO: 4 and a light chain consisting of the amino acid sequence
represented by SEQ ID NO: 6, and a conjugate wherein the labeling
moiety is a fluorescent dye represented by formula (C), and the
anti-human MUC1 antibody Fab fragment is an anti-human MUC1
antibody Fab fragment comprising a heavy chain fragment derived
from a heavy chain fragment consisting of the amino acid sequence
represented by SEQ ID NO: 4 by the modification of glutamine at
amino acid position 1 of SEQ ID NO: 4 into pyroglutamic acid, and a
light chain consisting of the amino acid sequence represented by
SEQ ID NO: 6.
[0104] <Polynucleotide of Present Invention>
[0105] The polynucleotide of the present invention includes a
polynucleotide comprising a nucleotide sequence encoding the heavy
chain fragment of the anti-human MUC1 antibody Fab fragment of the
present invention, and a polynucleotide comprising a nucleotide
sequence encoding the light chain of the anti-human MUC1 antibody
Fab fragment of the present invention.
[0106] In one embodiment, the polynucleotide of the present
invention is a polynucleotide comprising a nucleotide sequence
encoding a heavy chain fragment comprising a heavy chain variable
region consisting of the amino acid sequence represented by SEQ ID
NO: 8, or a polynucleotide comprising a nucleotide sequence
encoding a heavy chain fragment comprising a heavy chain variable
region consisting of the amino acid sequence represented by SEQ ID
NO: 10.
[0107] Examples of the polynucleotide comprising a nucleotide
sequence encoding a heavy chain fragment comprising a heavy chain
variable region consisting of the amino acid sequence represented
by SEQ ID NO: 8 include a polynucleotide comprising the nucleotide
sequence represented by SEQ DU) NO: 7. Examples of the
polynucleotide comprising a nucleotide sequence encoding a heavy
chain fragment comprising a heavy chain variable region consisting
of the amino acid sequence represented by SEQ ID NO: 10 include a
polynucleotide comprising the nucleotide sequence represented by
SEQ ID NO: 9.
[0108] In one embodiment, the polynucleotide of the present
invention is a polynucleotide comprising a nucleotide sequence
encoding a heavy chain fragment consisting of the amino acid
sequence represented by SEQ ID NO: 2 or a polynucleotide comprising
a nucleotide sequence encoding a heavy chain fragment consisting of
the amino acid sequence represented by SEQ ID NO: 4.
[0109] Examples of the polynucleotide comprising a nucleotide
sequence encoding a heavy chain fragment consisting of the amino
acid sequence represented by SEQ ID NO: 2 include a polynucleotide
comprising the nucleotide sequence represented by SEQ ID NO: 1.
Examples of the polynucleotide comprising a nucleotide sequence
encoding a heavy chain fragment consisting of the amino acid
sequence represented by SEQ ID NO: 4 include a polynucleotide
comprising the nucleotide sequence represented by SEQ ID NO: 3.
[0110] In one embodiment, the polynucleotide of the present
invention is a polynucleotide comprising a nucleotide sequence
encoding a light chain comprising a light chain variable region
consisting of the amino acid sequence represented by SEQ ID NO:
12.
[0111] Examples of the polynucleotide comprising a nucleotide
sequence encoding a light chain comprising a light chain variable
region consisting of the amino acid sequence represented by SEQ ID
NO: 12 include a polynucleotide comprising the nucleotide sequence
represented by SEQ ID NO: 11.
[0112] 0 In one embodiment, the polynucleotide of the present
invention is a polynucleotide comprising a nucleotide sequence
encoding a light chain consisting of the amino acid sequence
represented by SEQ ID NO: 6.
[0113] Examples of the polynucleotide comprising a nucleotide
sequence encoding a light chain consisting of the amino acid
sequence represented by SEQ ID NO: 6 include a polynucleotide
comprising the nucleotide sequence represented by SEQ ID NO: 5.
[0114] The polynucleotide of the present invention is synthesizable
through the use of a gene synthesis method known in the art on the
basis of nucleotide sequences designed from the amino acid
sequences of the heavy chain fragment and the light chain of the
anti-human MUC1 antibody Fab fragment of the present invention.
Various methods known to those skilled in the art, such as methods
for synthesizing an antibody gene described in International
Publication No. WO 90/07861 can be used as such gene synthesis
methods.
[0115] <Expression Vector of Present Invention>
[0116] The expression vector of the present invention includes an
expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the heavy chain fragment of the
anti-human MUC1 antibody Fab fragment of the present invention, an
expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the light chain of the anti-human MUC
antibody Fab fragment of the present invention, and an expression
vector comprising a polynucleotide comprising a nucleotide sequence
encoding the heavy chain fragment of the anti-human MUC1 antibody
Fab fragment of the present invention and a polynucleotide
comprising a nucleotide sequence encoding the light chain of the
anti-human MUC1 antibody Fab fragment of the present invention.
[0117] The expression vector of the present invention preferably
includes an expression vector comprising a polynucleotide
comprising a nucleotide sequence encoding a heavy chain fragment
consisting of the amino acid sequence represented by SEQ ID NO: 4,
an expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding a light chain consisting of the amino
acid sequence represented by SEQ ID NO: 6, and an expression vector
comprising a polynucleotide comprising a nucleotide sequence
encoding a heavy chain fragment consisting of the amino acid
sequence represented by SEQ ID NO: 4 and a polynucleotide
comprising a nucleotide sequence encoding a light chain consisting
of the amino acid sequence represented by SEQ ID NO: 6.
[0118] The expression vector of the present invention is not
particularly limited as long as a polypeptide encoded by the
polynucleotide of the present invention can be produced in various
host cells of prokaryotic cells and/or eukaryotic cells. Examples
of such an expression vector include plasmid vectors and virus
vectors (e.g., adenovirus and retrovirus). Preferably, pEE6.4 or
pEE12.4 (Lonza Ltd.) can be used.
[0119] The expression vector of the present invention can comprise
a promoter operably linked to a gene encoding the heavy chain
fragment and/or the light chain in the polynucleotide of the
present invention. Examples of the promoter for expressing the Fab
fragment of the present invention in a host cell include Trp
promoter, lac promoter, recA promoter, .lamda.PL promoter, lpp
promoter, and tac promoter when the host cell is a bacterium of the
genus Escherichia. Examples of the promoter for expression in
yeasts include PH05 promoter, PGK promoter, GAP promoter, and ADH
promoter. Examples of the promoter for expression in bacteria of
the genus Bacillus include SL01 promoter, SP02 promoter, and penP
promoter. Examples thereof include promoters derived from viruses
such as CMV, RSV, and SV40, retrovirus promoter, actin promoter, EF
(elongation factor) la promoter, and heat shock promoter when the
host is a eukaryotic cell such as a mammalian cell.
[0120] In the case of using a bacterium, particularly, E. coli, as
a host cell, the expression vector of the present invention can
further comprise a start codon, a stop codon, a terminator region
and a replicable unit. On the other hand, in the case of using a
yeast, an animal cell or an insect cell as a host, the expression
vector of the present invention can comprise a start codon and a
stop codon. In this case, an enhancer sequence, 5' and 3'
untranslated regions of a gene encoding the heavy chain fragment
and/or the light chain of the present invention, a secretion signal
sequence, a splicing junction, a polyadenylation site, or a
replicable unit, etc. may be contained therein. Also, a selective
marker usually used (e.g., tetracycline resistance gene, ampicillin
resistance gene, kanamycin resistance gene, neomycin resistance
gene, dihydrofolate reductase gene) may be contained therein
according to a purpose.
[0121] <Transformed Host Cell of Present Invention>
[0122] The transformed host cell of the present invention includes
a host cell transformed with the expression vector of the present
invention, selected from the group consisting of the following (a)
to (d):
(a) a host cell transformed with an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding the heavy
chain fragment of the anti-human MUC1 antibody Fab fragment of the
present invention; (b) a host cell transformed with an expression
vector comprising a polynucleotide comprising a nucleotide sequence
encoding the light chain of the anti-human MUC1 antibody Fab
fragment of the present invention; (c) a host cell transformed with
an expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the heavy chain fragment of the
anti-human MUC1 antibody Fab fragment of the present invention and
a polynucleotide comprising a nucleotide sequence encoding the
light chain of the anti-human MUC1 antibody Fab fragment of the
present invention; and (d) a host cell transformed with an
expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the heavy chain fragment of the
anti-human MUC1 antibody Fab fragment of the present invention and
an expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the light chain of the anti-human MUC1
antibody Fab fragment of the present invention.
[0123] In one embodiment, the transformed host cell of the present
invention is a host cell transformed with the expression vector of
the present invention, selected from the group consisting of the
following (a) to (d):
(a) a host cell transformed with an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding a heavy
chain fragment consisting of the amino acid sequence represented by
SEQ ID NO: 4; (b) a host cell transformed with an expression vector
comprising a polynucleotide comprising a nucleotide sequence
encoding a light chain consisting of the amino acid sequence
represented by SEQ ID NO: 6; (c) a host cell transformed with an
expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding a heavy chain fragment consisting of
the amino acid sequence represented by SEQ ID NO: 4 and a
polynucleotide comprising a nucleotide sequence encoding a light
chain consisting of the amino acid sequence represented by SEQ ID
NO: 6; and (d) a host cell transformed with an expression vector
comprising a polynucleotide comprising a nucleotide sequence
encoding a heavy chain fragment consisting of the amino acid
sequence represented by SEQ ID NO: 4 and an expression vector
comprising a polynucleotide comprising a nucleotide sequence
encoding a light chain consisting of the amino acid sequence
represented by SEQ ID NO: 6.
[0124] The host cell to be transformed is not particularly limited
as long as it is compatible with the expression vector used and can
be transformed with the expression vector to express the Fab
fragment. Examples thereof include various cells such as natural
cells and artificially established cells usually used in the
technical field of the present invention (e.g., bacteria (bacteria
of the genus Escherichia and bacteria of the genus Bacillus),
yeasts (the genus Saccharomyces, the genus Pichia, etc.), animal
cells and insect cells (e.g., Sf9)), and mammalian cell lines
(e.g., cultured cells such as CHO-K1SV cells, CHO-DG44 cells, and
293 cells). The transformation itself can be performed by a known
method, for example, a calcium phosphate method or an
electroporation method.
[0125] <Method for Producing Anti-Human MUC1 Antibody Fab
Fragment According to Present Invention>
[0126] The method for producing an anti-human MUC1 antibody Fab
fragment according to the present invention comprises the step of
culturing the transformed host cell of the present invention to
express the anti-human MUC1 antibody Fab fragment.
[0127] In one embodiment, the transformed host cell of the present
invention to be cultured in the method for producing an anti-human
MUC1 antibody Fab fragment according to the present invention is
selected from the group consisting of the following (a) to (c):
(a) a host cell transformed with an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding the heavy
chain fragment of the anti-human MUC1 antibody Fab fragment of the
present invention and a polynucleotide comprising a nucleotide
sequence encoding the light chain of the anti-human MUC1 antibody
Fab fragment of the present invention; (b) a host cell transformed
with an expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the heavy chain fragment of the
anti-human MUC1 antibody Fab fragment of the present invention and
an expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding the light chain of the anti-human MUC1
antibody Fab fragment of the present invention; and (c) a host cell
transformed with an expression vector comprising a polynucleotide
comprising a nucleotide sequence encoding the heavy chain fragment
of the anti-human MUC1 antibody Fab fragment of the present
invention, and a host cell transformed with an expression vector
comprising a polynucleotide comprising a nucleotide sequence
encoding the light chain of the anti-human MUC1 antibody Fab
fragment of the present invention.
[0128] A certain form of the transformed host cell of the present
invention to be cultured in the method for producing an anti-human
MUC1 antibody Fab fragment according to the present invention is
selected from the group consisting of the following (a) to (c):
(a) a host cell transformed with an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding a heavy
chain fragment consisting of the amino acid sequence represented by
SEQ ID NO: 4 and a polynucleotide comprising a nucleotide sequence
encoding a light chain consisting of the amino acid sequence
represented by SEQ ID NO: 6; (b) a host cell transformed with an
expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding a heavy chain fragment consisting of
the amino acid sequence represented by SEQ ID NO: 4 and an
expression vector comprising a polynucleotide comprising a
nucleotide sequence encoding a light chain consisting of the amino
acid sequence represented by SEQ ID NO: 6; and (c) a host cell
transformed with an expression vector comprising a polynucleotide
comprising a nucleotide sequence encoding a heavy chain fragment
consisting of the amino acid sequence represented by SEQ ID NO: 4,
and a host cell transformed with an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding a light
chain consisting of the amino acid sequence represented by SEQ ID
NO: 6.
[0129] Preferably, the transformed host cell of the present
invention used is a host cell transformed with an expression vector
comprising a polynucleotide comprising a nucleotide sequence
encoding the heavy chain fragment of the anti-human MUC1 antibody
Fab fragment of the present invention and a polynucleotide
comprising a nucleotide sequence encoding the light chain of the
anti-human MUC1 antibody Fab fragment of the present invention, or
a host cell transformed with an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding the heavy
chain fragment of the anti-human MUC1 antibody Fab fragment of the
present invention and an expression vector comprising a
polynucleotide comprising a nucleotide sequence encoding the light
chain of the anti-human MUC1 antibody Fab fragment of the present
invention.
[0130] In the method for producing an anti-human MUC1 antibody Fab
fragment according to the present invention, the transformed host
cell can be cultured in a nutrient medium. The nutrient medium
preferably contains a carbon source, an inorganic nitrogen source
or an organic nitrogen source necessary for the growth of the
transformed host cell. Examples of the carbon source include
glucose, dextran, soluble starch, and sucrose. Examples of the
inorganic nitrogen source or the organic nitrogen source include
ammonium salts, nitrates, amino acids, corn steep liquor, peptone,
casein, meat extracts, soymeal, and potato extracts. Also, other
nutrients (e.g., inorganic salts (e.g., calcium chloride, sodium
dihydrogen phosphate, and magnesium chloride), vitamins, and
antibiotics (e.g., tetracycline, neomycin, ampicillin, and
kanamycin)) may be contained therein, if desired.
[0131] The culture itself of the transformed host cell is performed
by a known method. Culture conditions, for example, temperature,
medium pH and culture time, are appropriately selected. When the
host is, for example, an animal cell, MEM medium (Science; 1952;
122: 501), DMEM medium (Virology; 1959; 8: 396-97), RPMI1640 medium
(J. Am. Med. Assoc.; 1967; 199: 519-24), 199 medium (Proc. Soc.
Exp. Biol. Med.; 1950; 73:1-8), or the like containing
approximately 5 to 20% of fetal bovine serum can be used as a
medium. The medium pH is preferably approximately 6 to 8. The
culture is usually performed at approximately 30 to 40.degree. C.
for approximately 15 to 336 hours, and aeration or stirring can
also be performed, if necessary. When the host is an insect cell,
examples thereof include Grace's medium (PNAS; 1985; 82: 8404-8)
containing fetal bovine serum. Its pH is preferably approximately 5
to 8. The culture is usually performed at approximately 20 to
40.degree. C. for 15 to 100 hours, and aeration or stirring can
also be performed, if necessary. When the host is a bacterium, an
actinomycete, a yeast, or a filamentous fungus, for example, a
liquid medium containing the nutrient source described above is
appropriate. A medium of pH 5 to 8 is preferred. When the host is
E. coli, preferred examples of the medium include LB medium and M9
medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor
Laboratory; 1972: 431). In such a case, the culture can usually be
performed at 14 to 43.degree. C. for approximately 3 to 24 hours
with aeration or stirring, if necessary. When the host is a
bacterium of the genus Bacillus, it can usually be performed at 30
to 40.degree. C. for approximately 16 to 96 hours with aeration or
stirring, if necessary. When the host is a yeast, examples of the
medium include Burkholder minimum medium (PNAS; 1980; 77: 4505-8).
Its pH is desirably 5 to 8. The culture is usually performed at
approximately 20 to 35.degree. C. for approximately 14 to 144
hours, and aeration or stirring can also be performed, if
necessary.
[0132] The method for producing an anti-human MUC1 antibody Fab
fragment according to the present invention can comprise the step
of recovering, preferably isolating or purifying, the expressed
anti-human MUC1 antibody Fab fragment, in addition to the step of
culturing the transformed host cell of the present invention to
express the anti-human MUC1 antibody Fab fragment. Examples of the
isolation or purification method include: methods exploiting
solubility, such as salting out and a solvent precipitation method;
methods exploiting difference in molecular weight, such as
dialysis, ultrafiltration, gel filtration, and sodium dodecyl
sulfate-polyacrylamide gel electrophoresis; methods exploiting
charge, such as ion-exchange chromatography and hydroxylapatite
chromatography; methods exploiting specific affinity, such as
affinity chromatography; methods exploiting difference in
hydrophobicity, such as reverse-phase high-performance liquid
chromatography; and methods exploiting difference in isoelectric
point, such as isoelectric focusing.
[0133] <Method for Producing Conjugate According to Present
Invention>
[0134] The method for producing a conjugate according to the
present invention comprises the step of covalently binding the
anti-human MUC1 antibody Fab fragment of the present invention to a
labeling moiety. The method for producing a conjugate according to
the present invention may also comprise the steps of: culturing the
transformed host cell of the present invention to express the
anti-human MUC1 antibody Fab fragment; and covalently binding the
Fab fragment to a labeling moiety. The method for producing a
conjugate according to the present invention may also comprise the
steps of: culturing the transformed host cell of the present
invention to express the anti-human MUC1 antibody Fab fragment;
recovering the expressed Fab fragment; and covalently binding the
Fab fragment to a labeling moiety. The method for producing a
conjugate according to the present invention may further comprise
the step of adding a metal. The linker, chelating agent, metal, or
fluorescent dye, etc., and linking method used can employ those
described in <Conjugate of present invention>.
[0135] In one embodiment, the method for producing a conjugate
according to the present invention is a method comprising the steps
of: culturing the transformed host cell of the present invention to
express the anti-human MUC1 antibody Fab fragment; and covalently
binding the Fab fragment to a labeling moiety. A certain embodiment
of the method for producing a conjugate according to the present
invention is a method comprising the steps of: culturing the
transformed host cell of the present invention to express the
anti-human MUC1 antibody Fab fragment; recovering the expressed Fab
fragment; and covalently binding the Fab fragment to a labeling
moiety.
[0136] A certain embodiment of the method for producing a conjugate
according to the present invention is a method comprising the steps
of: culturing the transformed host cell of the present invention to
express the anti-human MUC1 antibody Fab fragment; and i) binding
the Fab fragment via a linker to a ligand or ii) covalently binding
the Fab fragment directly to a ligand. A certain embodiment of the
method for producing a conjugate according to the present invention
is a method comprising the steps of: culturing the transformed host
cell of the present invention to express the anti-human MUC1
antibody Fab fragment; recovering the expressed Fab fragment; and
i) binding the Fab fragment via a linker to a ligand or ii)
covalently binding the Fab fragment directly to a ligand.
[0137] A certain embodiment of the method for producing a conjugate
according to the present invention is a method comprising the steps
of: culturing the transformed host cell of the present invention to
express the anti-human MUC1 antibody Fab fragment; i) binding the
Fab fragment via a linker to a ligand or ii) covalently binding the
Fab fragment directly to a ligand; and labeling the ligand of the
conjugate with a metal (i.e., forming a chelate complex). A certain
embodiment of the method for producing a conjugate according to the
present invention is a method comprising the steps of: culturing
the transformed host cell of the present invention to express the
anti-human MUC1 antibody Fab fragment; recovering the expressed Fab
fragment; i) binding the Fab fragment via a linker to a ligand or
ii) covalently binding the Fab fragment directly to a ligand; and
labeling the ligand of the conjugate with a metal.
[0138] A certain embodiment of the method for producing a conjugate
according to the present invention is a method comprising the steps
of: culturing the transformed host cell of the present invention to
express the anti-human MUC1 antibody Fab fragment; i) binding the
Fab fragment via a linker to a ligand or ii) covalently binding the
Fab fragment directly to a ligand; and labeling the ligand of the
conjugate with a metal radioisotope (i.e., forming a chelate
complex). A certain embodiment of the method for producing a
conjugate according to the present invention is a method comprising
the steps of: culturing the transformed host cell of the present
invention to express the anti-human MUC1 antibody Fab fragment;
recovering the expressed Fab fragment; i) binding the Fab fragment
via a linker to a ligand or ii) covalently binding the Fab fragment
directly to a ligand; and labeling the ligand of the conjugate with
a metal radioisotope.
[0139] A certain embodiment of the method for producing a conjugate
according to the present invention is a method comprising the steps
of: culturing the transformed host cell of the present invention to
express the anti-human MUC1 antibody Fab fragment; and i) binding
the Fab fragment via a linker to a fluorescent dye or ii)
covalently binding the Fab fragment directly to a fluorescent dye.
A certain embodiment of the method for producing a conjugate
according to the present invention is a method comprising the steps
of: culturing the transformed host cell of the present invention to
express the anti-human MUC1 antibody Fab fragment; recovering the
expressed Fab fragment; and i) binding the Fab fragment via a
linker to a fluorescent dye or ii) covalently binding the Fab
fragment directly to a fluorescent dye.
[0140] A certain embodiment of the method for producing a conjugate
according to the present invention is a method comprising the steps
of: culturing the transformed host cell of the present invention to
express the anti-human MUC1 antibody Fab fragment; and labeling the
Fab fragment with a detectable molecule. A certain embodiment of
the method for producing a conjugate according to the present
invention is a method comprising the steps of: culturing the
transformed host cell of the present invention to express the
anti-human MUC1 antibody Fab fragment; recovering the expressed Fab
fragment; and labeling the Fab fragment with a detectable
molecule.
[0141] The method for producing a conjugate according to the
present invention may be carried out as a method comprising two or
more of the steps defined above as a series of steps or may be
carried out as a method comprising at least one of the steps
defined above. For example, a method comprising the step of binding
the anti-human MUC1 antibody Fab fragment of the present invention
to a labeling moiety, and a method comprising the step of labeling
the anti-human MUC1 antibody Fab fragment of the present invention
bound to the labeling moiety with a metal are also included in the
method for producing a conjugate according to the present
invention. Also, the method for producing a conjugate according to
the present invention includes a method having a different order of
steps. For example, a method comprising labeling a chelating agent
with a metal, and then covalently binding the chelating agent to
the anti-human MUC1 antibody Fab fragment of the present invention
is also included in the method for producing a conjugate according
to the present invention.
[0142] <Composition for Diagnosis and Diagnosis Method>
[0143] The present invention relates to a composition for diagnosis
comprising the conjugate of the present invention comprising a
metal or a fluorescent dye (hereinafter, referred to as the
detectable conjugate of the present invention). The composition for
diagnosis of the present invention may comprise one or more
conjugate of the present invention. Specifically, the composition
for diagnosis of the present invention may comprise one conjugate
of the present invention, or may comprise two or more conjugates of
the present invention in combination. The detectable conjugate of
the present invention can be formulated according to a routine
method and utilized as an early diagnostic drug, a staging drug or
a intraoperative diagnostic drug (particularly, a cancer diagnostic
drug). The intraoperative diagnostic drug means a diagnostic drug
capable of identifying a lesion site during operation such as
surgery or endoscopic operation and examining its nature. In the
case of using the composition for diagnosis of the present
invention as an intraoperative diagnostic drug, the composition for
diagnosis is administered to a patient, for example, 2 to 32 hours,
6 to 24 hours in a certain embodiment, or 2 hours in another
embodiment, before operation.
[0144] The early diagnostic drug means a diagnostic drug aimed at
performing diagnosis when no condition is observed or at an early
stage. For example, for cancers, it means a diagnostic drug that is
used when no condition is observed or at stage 0 or stage 1.
[0145] The staging drug means a diagnostic drug capable of
examining the degree of progression of a condition. For example,
for cancers, it means a diagnostic drug capable of examining the
stage thereof.
[0146] The cancer expected to be able to be diagnosed by the
composition for diagnosis of the present invention is a cancer
expressing human MUC1. Examples of a certain embodiment include
breast cancer, lung cancer, colorectal cancer, bladder cancer, skin
cancer, thyroid gland cancer, stomach cancer, pancreatic cancer,
kidney cancer, ovary cancer and uterine cervical cancer.
Preferably, the cancer is breast cancer or bladder cancer.
[0147] The amount of the conjugate of the present invention added
for the formulation of the composition for diagnosis of the present
invention differs depending on the degree of symptoms or age of a
patient, the dosage form of a preparation used, or the binding
titer of the Fab fragment, etc. For example, approximately 0.001
mg/kg to 100 mg/kg based on the mass of the Fab fragment can be
used per unit body weight of a patient.
[0148] Examples of the dosage form of the composition for diagnosis
of the present invention can include parenteral agents such as
injections and agents for drip infusion. Administration can be
performed by intravenous injection, local intramuscular injection
to a target tissue, subcutaneous injection, intravesical
administration, or the like. For the formulation, a carrier or an
additive suitable for these dosage forms can be used in a
pharmaceutically acceptable range. The type of the pharmaceutically
acceptable carrier or additive is not particularly limited, and a
carrier or an additive well known to those skilled in the art can
be used.
[0149] The present invention also relates to use of the detectable
conjugate of the present invention for the production of a
composition for the early diagnosis, a composition for the staging
or a composition for the intraoperative diagnosis of a cancer. The
present invention also relates to the detectable conjugate of the
present invention for use in the early diagnosis, staging or
intraoperative diagnosis of a cancer.
[0150] Further, the present invention also relates to a method for
diagnosing a cancer, comprising preoperatively or intraoperatively
administering the detectable conjugate of the present invention to
a subject. In this context, the "subject" is a human or any of
other mammals in need of receiving the diagnosis. A certain
embodiment is a human in need of receiving the diagnosis. The
effective amount of the detectable conjugate of the present
invention in the diagnosis method of the present invention may be
the same amount as the effective amount of the conjugate of the
present invention for the formulation described above. In the
diagnosis method of the present invention, the detectable conjugate
of the present invention is preferably administered by local
intramuscular injection to a target tissue, subcutaneous injection,
or the like. In the diagnosis method of the present invention, in
the case of preoperatively administering the conjugate of the
present invention, the conjugate is administered to a patient, for
example, 2 to 48 hours, 6 to 24 hours in a certain embodiment, and
2 hours in another embodiment before operation.
[0151] In an alternative embodiment, the present invention also
relates to use of the anti-human MUC1 antibody Fab fragment of the
present invention for the production of the conjugate of the
present invention. In a certain embodiment, the present invention
also relates to use of the anti-human MUC1 antibody Fab fragment of
the present invention for the production of a composition for
diagnosis comprising the conjugate of the present invention.
[0152] As for a embodiment in which the composition for diagnosis
of the present invention comprising a metal radioisotope is
provided, it may be labeled with the metal radioisotope immediately
before use or may be provided as a composition for diagnosis
comprising the metal radioisotope.
[0153] <Composition for Treatment and Treatment Method>
[0154] The present invention includes a pharmaceutical composition
comprising one or more conjugate of the present invention and a
pharmaceutically acceptable excipient. Specifically, the
composition for treatment of the present invention may comprise one
conjugate of the present invention, or may comprise two or more
conjugates of the present invention in combination. The conjugate
of the present invention can be used in the preparation of a
pharmaceutical composition by a method usually used using an
excipient usually used in the art, i.e., a pharmaceutical
excipient, a pharmaceutical carrier, or the like. Examples of the
dosage forms of these pharmaceutical compositions include
parenteral agents such as injections and agents for drip infusion.
Administration can be performed by intravenous injection,
subcutaneous injection, intravesical administration, or the like.
For the formulation, an excipient, a carrier, an additive, or the
like suitable for these dosage forms can be used in a
pharmaceutically acceptable range.
[0155] The amount of the conjugate of the present invention added
for the formulation described above differs depending on the degree
of symptoms or age of a patient, the dosage form of a preparation
used, or the binding titer of the Fab fragment, etc. For example,
approximately 0.001 mg/kg to 100 mg/kg based on the mass of the Fab
fragment can be used per unit body weight of a patient.
[0156] The pharmaceutical composition comprising the conjugate of
the present invention can be used for the treatment of a cancer.
The cancer expected to be able to be treated by the pharmaceutical
composition comprising the conjugate of the present invention is a
cancer expressing human MUC1. Examples thereof include breast
cancer, lung cancer, colorectal cancer, bladder cancer, skin
cancer, thyroid gland cancer, stomach cancer, pancreatic cancer,
kidney cancer, ovary cancer and uterine cervical cancer.
[0157] A certain embodiment of the pharmaceutical composition
comprising the conjugate of the present invention is a
pharmaceutical composition comprising the conjugate comprising a
fluorescent dye and can be used in the treatment of a cancer by
application to a photoimmunotherapy method. The photoimmunotherapy
method is a method of allowing the conjugate comprising a
fluorescent dye to specifically accumulate in cancer tissues,
followed by irradiation with light having a wavelength exciting the
fluorescent dye contained in the conjugate to induce cell death in
a cancer-specific manner through the phototoxic effect of the
fluorescent dye. In the case of using the conjugate of the present
invention comprising a fluorescent dye in the photoimmunotherapy
method, the wavelength of the light for irradiation can be a
near-infrared wavelength (650 to 1000 nm).
[0158] The present invention includes a pharmaceutical composition
for treating breast cancer or bladder cancer, comprising the
conjugate of the present invention. The present invention also
includes a method for treating breast cancer or bladder cancer,
comprising the step of administering a therapeutically effective
amount of the conjugate of the present invention, which can
comprise the step of performing irradiation with light having a
near-infrared wavelength (650 to 1000 nm, for example, 660 to 740
nm, for example, 680 nm), in addition to the step described above.
A certain embodiment of the dose of light irradiation is at least 1
J/cm.sup.2. A certain embodiment is at least 10 J/cm.sup.2. A
certain embodiment is at least 100 J/cm.sup.2. A certain embodiment
is 1 to 500 J/cm.sup.2. A certain embodiment is 50 to 200
J/cm.sup.2. In a certain embodiment, the irradiation may be carried
out a plurality of times after administration of the conjugate of
the present invention. The present invention also includes a method
for inducing the cell death of cancer cells of breast cancer or
bladder cancer, comprising the step of administering a
therapeutically effective amount of the conjugate of the present
invention.
[0159] The pharmaceutical composition for treating a cancer can
also be used in the diagnosis of a cancer. For example, the
pharmaceutical composition for treating breast cancer or bladder
cancer can also be used in the diagnosis of the cancer.
[0160] The present invention also includes the conjugate of the
present invention for use in the treatment of breast cancer or
bladder cancer. The present invention further includes use of the
conjugate of the present invention for the production of a
pharmaceutical composition for treating breast cancer or bladder
cancer.
[0161] In an alternative embodiment, the present invention also
relates to use of the anti-human MUC1 antibody Fab fragment of the
present invention for the production of a pharmaceutical
composition comprising the conjugate of the present invention.
[0162] The present invention is generally described above.
Particular Examples will be provided here for reference in order to
obtain further understanding. However, these are given for
illustrative purposes and do not limit the present invention.
EXAMPLES
Example 1: Preparation of Anti-Human MUC1 Antibody Fab Fragment
[0163] Two anti-human MUC1 antibody Fab fragments designated as
P10-1 Fab and P10-2 Fab were prepared.
[0164] The amino acid sequences of the heavy chain variable regions
and the light chain variable regions of P10-1 Fab and P10-2 Fab
were specifically designed as sequences expected to improve
affinity and not to attenuate affinity even by the binding of a
labeling moiety, by using a molecular model of a humanized antibody
constructed in accordance with the literature (Proteins, 2014
August; 82 (8): 1624-35) after humanization of a 1B2 antibody,
which is a mouse-derived anti-human cancer-specific MUC1 antibody,
with reference to the method described in the literature (Front
Biosci., 2008 Jan. 1; 13: 1619-33).
[0165] GS vector pEE6.4 (Lonza Ltd.) having an insert of a heavy
chain fragment gene formed by connecting a gene encoding a signal
sequence (MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 13)) to the 5' side of
the heavy chain variable region gene of P10-1 Fab or P10-2 Fab and
connecting a human Ig.gamma.1 constant region gene (consisting of a
nucleotide sequence from nucleotide positions 355 to 669 of SEQ ID
NO: 1 or 3) to the 3' side thereof was prepared. Here, in order to
express each Fab fragment, a strop codon was inserted to downstream
of a codon of Asp at position 221 based on the EU index provided by
Kabat et al. (corresponding to Asp at position 222 in the amino
acid sequences of SEQ ID NOs: 2 and 4 mentioned later) in the heavy
chain constant region gene. Also, GS vector pEE12.4 (Lonza Ltd.)
having an insert of a light chain gene formed by connecting a gene
encoding a signal sequence (MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 14))
to the 5' side of the common light chain variable region gene of
P10-1 Fab and P10-2 Fab and connecting a human K chain constant
region gene (consisting of a nucleotide sequence from nucleotide
positions 340 to 660 of SEQ ID NO: 5) to the 3' side thereof was
prepared.
[0166] The expression of each Fab fragment was performed by the
method of transient expression. Expi293F cells (Thermo Fisher
Scientific Inc.) cultured into approximately 2500000 cells/mL in
Expi293 Expression Medium (Thermo Fisher Scientific Inc.) were
transfected with the GS vectors of the heavy chain fragment and the
light chain mentioned above using ExpiFectamine 293 Transfection
Kit (Thermo Fisher Scientific Inc.), and cultured for 8 days. After
expression, the culture supernatant was purified using KappaSelect
(GE Healthcare Japan Corp.) to obtain each Fab fragment.
[0167] The nucleotide sequence of the heavy chain fragment of P10-1
Fab is shown in SEQ ID NO: 1, and the amino acid sequence encoded
thereby is shown in SEQ ID NO: 2. The nucleotide sequence of the
heavy chain variable region of P10-1 Fab is shown in SEQ ID NO: 7.
The amino acid sequence encoded thereby is shown in SEQ ID NO:
8.
[0168] The nucleotide sequence of the heavy chain fragment of P10-2
Fab is shown in SEQ ID NO: 3. The amino acid sequence encoded
thereby is shown in SEQ ID NO: 4. The nucleotide sequence of the
heavy chain variable region of P10-2 Fab is shown in SEQ ID NO: 9.
The amino acid sequence encoded thereby is shown in SEQ ID NO:
10.
[0169] The light chain is common in P10-1 Fab and P10-2 Fab. The
nucleotide sequence thereof is shown in SEQ ID NO: 5. The amino
acid sequence encoded thereby is shown in SEQ ID NO: 6. The
nucleotide sequence of the light chain variable region of P10-1 Fab
and P10-2 Fab is shown in SEQ ID NO: 11. The amino acid sequence
encoded thereby is shown in SEQ ID NO: 12.
Example 2: Amino Acid Modification Analysis of Fab Fragment
[0170] As a result of analyzing the amino acid modification of
purified P10-2 Fab, it was suggested that heavy chain N-terminal
glutamine was modified into pyroglutamic acid in a great majority
of purified antibodies.
Example 3: Binding Activity Evaluation of Fab Fragment
[0171] Binding activity against human cancer-specific MUC1 was
compared as to P10-1 Fab and P10-2 Fab expressed by the method
mentioned above with a chimeric 1B2 antibody Fab fragment
(hereinafter, referred to as 1B2 Fab; prepared by linking a human
IgG1 CH1 domain and a .kappa. chain CL domain to the VH domain and
the VL domain (their sequence information was quoted from Patent
Literature 1), respectively, of the 1B2 antibody (Patent Literature
1); for the convenience of linking of the CH1 domain and the CL
domain, an alanine residue at position 113 based on the EU index
(Kabat et al.) in the VH domain was substituted by a serine
residue, and an alanine residue at position 109 based on the EU
index (Kabat et al.) in the VL domain was substituted by a
threonine residue) by Cell ELISA. Specifically, breast cancer cell
line T-47D cells (purchasable from ATCC; HTB-133) expressing human
cancer-specific MUC1 were inoculated at 0.75.times.10.sup.4 cells
per well to a 96-well ELISA plate coated with collagen I, and
cultured overnight. Then, the cells were fixed in formalin, and
P10-1 Fab, P10-2 Fab or 1B2 Fab described above was reacted
therewith. Then, a horseradish peroxidase (HRP)-labeled goat
anti-human Ig.kappa. antibody (Southern Biotechnology Associates,
Inc.) was reacted as a secondary antibody. ECL Prime Western
Blotting Detection Reagent (GE Healthcare Japan Corp.) was added
thereto for luminescence, and the degree of the luminescence was
examined. As a result, as shown in FIG. 1, P10-1 Fab and P10-2 Fab
were confirmed to have approximately 10 or more times the binding
activity against human cancer-specific MUC1 compared to 1B2
Fab.
Example 4: Fluorescent Labeling of Fab Fragment
[0172] Subsequently, the present inventors labeled P10-1 Fab, P10-2
Fab and 1B2 Fab with a fluorescent dye mentioned above.
[0173] Specifically, each Fab fragment solution adjusted to
approximately 1 mg/mL with phosphate-buffered saline (pH 7.4) was
adjusted to pH 8.5 by the addition of a 1/10 amount of a 1 M
dipotassium hydrogen phosphate solution (pH 9). IRDye800CW NHS
Ester (LI-COR Bioscience, Inc.) was added thereto at a final
concentration of 310.8 .mu.g/mL, and the resultant was stirred at
room temperature under shading for 2 hours. IRDye800CW NHS Ester
has a N-hydroxysuccinimide group and therefore reacts immediately
with Lys of the Fab fragment. This was recovered through Amicon
Ultra 3K-0.5 mL centrifugal filter (Merck Millipore) to purify a
fluorescently labeled Fab fragment. P10-1 Fab, P10-2 Fab and 1B2
Fab harboring this fluorescent dye were designated as P10-1 Fab
Dye, P10-2 Fab Dye and 1B2 Fab Dye.
Example 5: Binding Activity Evaluation of Fluorescently Labeled Fab
Fragment
[0174] Binding activity against human cancer-specific MUC1 was
compared as to P10-1 Fab Dye and P10-2 Fab Dye labeled by the
method mentioned above with 1B2 Fab Dye by Cell ELISA.
Specifically, breast cancer cell line T-47D cells expressing human
cancer-specific MUC1 were inoculated at 0.75.times.10.sup.4 cells
per well to a 96-well ELISA plate coated with collagen I, and
cultured overnight. Then, the cells were fixed in formalin, and
P10-1 Fab Dye, P10-2 Fab Dye or 1B2 Fab Dye described above was
reacted therewith. Then, a HRP-labeled goat anti-human Ig.kappa.
antibody (Southern Biotechnology Associates, Inc.) was reacted as a
secondary antibody. ECL Prime Western Blotting Detection Reagent
(GE Healthcare Japan Corp.) was added thereto for luminescence, and
the degree of the luminescence was examined. As a result, as shown
in FIG. 2, the binding activity of 1B2 Fab Dye was attenuated by
labeling, whereas P10-1 Fab Dye and P10-2 Fab Dye were confirmed to
be free from the attenuation of the binding activity by
labeling.
Example 6: Labeling of Fab Fragment with Chelating Agent
[0175] Subsequently, the present inventors labeled P10-2 Fab with a
chelating agent mentioned above.
[0176] Specifically, a Fab fragment solution adjusted to 12.5 mg/mL
with phosphate-buffered saline (pH 7.4) was adjusted to pH 9.0 by
the addition of a 100 M sodium carbonate solution at 10 mM.
p-SCN-Bn-deferoxamine (Macrocyclics, Inc.) was added thereto at a
final concentration of 1 mM, and the resultant was reacted at
37.degree. C. for 2 hours. p-SCN-Bn-deferoxamine has an
isothiocyanate group and therefore reacts immediately with Lys of
the Fab fragment. This was recovered through Amicon Ultra 10K-0.5
mL centrifugal filter to purify a chelating agent-labeled Fab
fragment. This chelating agent-labeled P10-2 Fab was designated as
P10-2 Fab DFO.
Example 7: Binding Activity Evaluation of Chelating Agent-Labeled
Fab Fragment
[0177] Binding activity against human cancer-specific MUC1 was
compared as to P10-2 Fab DFO labeled by the method mentioned above
with P10-2 Fab by Cell ELISA. Specifically, T-47D cells expressing
human cancer-specific MUC1 were inoculated at 0.75.times.10.sup.4
cells to a 96-well ELISA plate coated with collagen I, and cultured
overnight. Then, the cells were fixed in formalin, and P10-2 Fab
DFO or P10-2 Fab described above was reacted therewith. Then, a
HRP-labeled goat anti-human Ig.kappa. antibody (Southern
Biotechnology Associates, Inc.) was reacted as a secondary
antibody. ECL Prime Western Blotting Detection Reagent (GE
Healthcare Japan Corp.) was added thereto for luminescence, and the
degree of the luminescence was examined. As a result, as shown in
FIG. 3, P10-2 Fab DFO and P10-2 Fab had equivalent binding
activity. P10-2 Fab was confirmed to be free from the attenuation
of the binding activity by labeling with a chelating agent.
Example 8: Reactivity of P10-2 in Human Bladder Cancer Tissue
Sample
[0178] In order to study the reactivity of P10-2 Fab in human
bladder cancer, the study was made by immunostaining using a human
bladder cancer tissue array (US Biomax, Inc., BC12011b). When P10-2
Fab is used as a primary antibody, an anti-human antibody used as a
secondary antibody cross-reacts with human tissues. Therefore,
mouse chimeric P10-2 IgG (antibody in which the VH domain and the
VL domain of P10-2 Fab were fused with mouse IgG2a CH1 to CH3
domains and a mouse .kappa. chain CL domain, respectively) was
prepared and used as a primary antibody. The human bladder cancer
tissue array sample was reacted with a 3% hydrogen peroxide
solution for 5 minutes and then washed with phosphate-buffered
saline of pH 7.4. Then, mouse chimeric P10-2 IgG (0.25 .mu.g/mL)
was reacted, and then, One-Step Polymer-HRP antibody (BioGenex) was
reacted as a secondary antibody. Super Sensitive DAB (BioGenex) was
added thereto for color development. The positive reaction of
staining for cancer tissues was determined as .+-.: very mildly
positive, +: positive, ++: moderately positive, and +++: highly
positive, and one case whose cancer tissues were not clear was
excluded. As a result, positive reaction was found in 57 out of 59
studied cases with 11 cases for .+-., 24 cases for +, 17 cases for
++, and 5 cases for +++(positive rate: 97%).
Example 9: Labeling of Chelated Fab Fragment with .sup.89Zr
[0179] .sup.89Zr used was dissolved in a 1 M aqueous oxalic acid
solution and produced as .sup.89Zr-Oxalate (Okayama University). 20
.mu.L of .sup.89Zr-Oxalate (21.4 MBq) was neutralized with 10 .mu.L
of a 2 M aqueous sodium carbonate solution. Then, 70 .mu.L of a 5
mg/mL solution of gentisic acid dissolved in 250 mM aqueous sodium
acetate solution was added thereto. Further, 200 .mu.L of a 500 mM
aqueous HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
solution containing 0.1% polysorbate 80 and 20% glycerol was added
thereto. To this solution containing .sup.89Zr, 100 .mu.L of 9.5
mg/mL P10-2 Fab DFO was added and reacted at room temperature for
30 minutes. The obtained reaction mixture was purified using Amicon
Ultra 10K-0.5 mL centrifugal filter (Merck Millipore) and further
filtered through a membrane filter (Millex-GV 0.22 .mu.m 13 mm;
Merck Millipore) to obtain .sup.89Zr-labeled P10-2 Fab DFO (16.1
MBq) of interest. This .sup.89Zr-labeled P10-2 Fab DFO was
designated as P10-2 Fab DFO .sup.89Zr. The obtained P10-2 Fab DFO
.sup.89Zr solution was analyzed using high-performance liquid
chromatography (20AD series; Shimadzu Corp.). The retention time
(UV: 10.192 min) of P10-2 Fab DFO was compared with the retention
time (UV: 10.178 min, RI: 10.515 min) of P10-2 Fab DFO .sup.89Zr.
The respective retention times were equivalent, confirming that
P10-2 Fab DFO was labeled with .sup.89Zr. The HPLC analysis was
carried out under the following conditions: column: BioSep SEC
s3000 300.times.7.8 mm (Phenomenex Inc.), column temperature: room
temperature, UV detector wavelength: 280 nm, mobile phase:
phosphate buffer solution (Gibco, 10010-023), flow rate: 1
ml/min.
Example 10: Labeling of Fab Fragment with IRDye700DX
[0180] IRDye700DX NHS Ester (LI-COR Bioscience, Inc.) was used to
label P10-2 Fab with IRDye700DX.
[0181] Specifically, a P10-2 Fab solution adjusted to 1 mg/mL with
phosphate-buffered saline (pH 7.4) was supplemented with a 1/10
amount of a 1 M dipotassium hydrogen phosphate solution (pH 9).
IRDye700DX NHS Ester was added thereto at a final concentration of
154 .mu.g/mL, and the resultant was stirred at room temperature for
2 hours. After reaction, IRDye700DX-labeled P10-2 Fab was purified
by recovery through Amicon Ultra 10K-15 mL centrifugal filter
(Merck Millipore). This IRDye700DX-labeled P10-2 Fab was designated
as P10-2 Fab IR700.
Example 11: Binding Activity Evaluation of Fab Fragment Against
Breast Cancer Cell Line and Bladder Cancer Cell Line
[0182] The binding activity of P10-2 Fab in human breast cancer and
bladder cancer was studied.
[0183] Specifically, human breast cancer cell line MDA-MB-468 cells
(purchasable from ATCC; HTB-132; hereinafter, referred to as MM-468
cells) or human bladder cancer cell line 647-V cells (purchasable
from DSMZ; ACC 414) expressing human cancer-specific MUC1 were
inoculated at 1.times.10.sup.4 cells per well to a 96-well ELISA
plate and cultured overnight. Then, P10-2 Fab described above was
reacted at a concentration of 0.0000003 to 0.001 mg/mL. Then, a
horseradish peroxidase (HRP)-labeled goat anti-human IgG antibody
(Medical & Biological Laboratories Co., Ltd.) was reacted as a
secondary antibody. ECL Prime Western Blotting Detection Reagent
(GE Healthcare Japan Corp.) was added thereto for luminescence, and
the degree of the luminescence was examined. As a result, as shown
in FIG. 4, P10-2 Fab was found to have binding activity against the
human breast cancer cell line MM-468 cells and the human bladder
cancer cell line 647-V cells.
Example 12: Contrast Evaluation of Fluorescently Labeled Fab
Fragment in Subcutaneously Cancer-Bearing Model
[0184] 3.times.10.sup.6 human breast cancer cell line MM-468 cells
were subcutaneously transplanted as human cancer-specific
MUC1-positive cells to the right back of each immunodeficient mouse
(SCID mouse; Charles River Laboratories Japan, Inc.). Mice having
tumorigenesis were selected approximately one month after the
transplantation. P10-2 Fab Dye dissolved in phosphate-buffered
saline (pH 7.4) was intravenously administered at a dose of 0.1,
0.3, 1 or 3 mg/kg (N=2 for the 0.1 mg/kg administration group, and
N=3 for the 0.3, 1 and 3 mg/kg administration groups). Photographs
were taken with a usual camera and a near-infrared fluorescence
camera (Fluobeam (800 nm filter); Fluoptics) 6 hours and 24 hours
after the P10-2 Fab Dye administration. The fluorescent brightness
of a tumor site and a peritumoral background site in the image
taken with the infrared fluorescence camera was measured. As a
result, as shown in FIG. 5A, P10-2 Fab Dye was found to accumulate
in the human cancer-specific MUC1-positive MM-468 tumor site and be
clearly visible in the fluorescent image 6 hours after
administration. As shown in FIG. 5B, the tumor/background ratio was
found to elevate in a P10-2 Fab Dye dose-dependent manner in the
range of 0.1 to 3 mg/kg. It was further obvious that the effect was
sustained even 24 hours after administration. These results
demonstrated that P10-2 Fab Dye permits detection of human
MUC1-positive cancer cells from 6 hours to 24 hours after
administration.
Example 13: Detection of Tumor with IRDye700DX Labeled Fab
Fragment
[0185] 5.times.10.sup.6 MM-468 cells were subcutaneously
transplanted to the right back of each immunodeficient mouse (nude
mouse; Charles River Laboratories Japan, Inc.). This test was
conducted at N=2. P10-2 Fab IR700 (3 mg/kg) or a vehicle
(phosphate-buffered saline) was intravenously administered 40 days
after the transplantation. The animals were euthanized 2 hours
after the P10-2 Fab IR700 administration. Tumor was excised, and
the luminescence of the tumor sites was measured by excitation at a
wavelength of 675 nm and detection at a wavelength of 740 nm in
IVIS SPECTRUM (PerkinElmer, Inc.). As a result, as shown in FIG. 6,
P10-2 Fab IR700 was found to accumulate in MM-468 cells 2 hours
after administration.
Example 14: Cytotoxicity Evaluation of IRDye700DX Labeled Fab
Fragment
[0186] In order to study the utilization of P10-2 Fab IR700 in
cancer treatment, cytotoxicity in photoimmunotherapy was
evaluated.
[0187] Specifically, human breast cancer cell line MM-468 cells,
human bladder cancer cell line 647-V cells or CHO-K1 cells of
Comparative Example (purchasable from ATCC; CCL-61) were inoculated
at 5.times.10.sup.3 cells per well to a 384-well white plate and
cultured overnight. Then, P10-2 Fab IR700 was reacted at 0, 0.01,
0.1, or 1 .mu.g/mL and then irradiated with 0 to 30 J/cm.sup.2 of
light having a wavelength of 680 nm. After the light irradiation,
overnight culture was performed. CellTiter-Glo (Promega Corp.) was
added thereto for luminescence, and the luminescence was measured
to measure the number of live cells. This test was conducted using
3 wells under each condition. As a result, as shown in FIG. 7, it
was obvious that P10-2 Fab IR700 exhibits cytotoxicity in a manner
specific for the expression of human cancer-specific MUC1 by light
irradiation.
Example 15: Antitumor Activity Evaluation of IRDye700DX Labeled Fab
Fragment
[0188] 5.times.10.sup.6 MM-468 cells were subcutaneously
transplanted to the right back of each immunodeficient mouse (nude
mouse; Charles River Laboratories Japan, Inc.). This test was
conducted at N=4. After tumor volume became 300 mm.sup.3, P10-2 Fab
IR700 (0.3, 1, or 3 mg/kg) was intravenously administered at days
1, 5, 8, 12 and 15. Phosphate-buffered saline was similarly
administered to a vehicle group. On the day following the drug
administration, irradiation with 0 or 200 J/cm.sup.2 of light
having a wavelength of 680 nm was performed, and the tumor volume
was measured over time. As a result, as shown in FIG. 8, it was
obvious that P10-2 Fab IR700 showed an antitumor effect in a manner
dependent on a dose and light irradiation.
INDUSTRIAL APPLICABILITY
[0189] The anti-human MUC1 antibody Fab fragment of the present
invention is expected to be useful in the diagnosis and/or
treatment of cancers such as breast cancer, lung cancer, colorectal
cancer, bladder cancer, skin cancer, thyroid gland cancer, stomach
cancer, pancreatic cancer, kidney cancer, ovary cancer or uterine
cervical cancer.
SEQUENCE LISTING FREE TEXT
[0190] SEQ ID NO: 1: Nucleotide sequence of DNA encoding a P10-1
Fab heavy chain fragment
[0191] SEQ ID NO: 2: Amino acid sequence of the P10-1 Fab heavy
chain fragment
[0192] SEQ ID NO: 3: Nucleotide sequence of DNA encoding a P10-2
Fab heavy chain fragment
[0193] SEQ ID NO: 4: Amino acid sequence of the P10-2 Fab heavy
chain fragment
[0194] SEQ ID NO: 5: Nucleotide sequence of DNA encoding an
antibody light chain
[0195] SEQ ID NO: 6: Amino acid sequence of the antibody light
chain
[0196] SEQ ID NO: 7: Nucleotide sequence of DNA encoding a P10-1
Fab heavy chain variable region
[0197] SEQ ID NO: 8: Amino acid sequence of the P10-1 Fab heavy
chain variable region
[0198] SEQ ID NO: 9: Nucleotide sequence of DNA encoding a P10-2
Fab heavy chain variable region
[0199] SEQ ID NO: 10: Amino acid sequence of the P10-2 Fab heavy
chain variable region
[0200] SEQ ID NO: 11: Nucleotide sequence of DNA encoding an
antibody light chain variable region
[0201] SEQ ID NO: 12: Amino acid sequence of the antibody light
chain variable region
[0202] SEQ ID NO: 13: Heavy chain signal sequence
[0203] SEQ ID NO: 14: Light chain signal sequence
[0204] SEQ ID NO: 15: Tandem repeat sequence of the extracellular
domain of MUC1
Sequence CWU 1
1
151669DNAArtificial SequenceDNA encoding P10-1 Fab heavy chain
fragment 1caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac caggcgccag
cgtgaaggtg 60tcctgcaagg ccagcggcta caccttcacc aactacggcc tgagctgggt
gcgccaggct 120cctggacagg gactggaatg gatgggcgag aaccaccctg
gcagcggcat catctaccac 180aacgagaagt tccggggcag agtgaccctg
accgccgacc ggagcaccag caccgcctac 240atggaactga gcagcctgcg
gagcgaggac accgccgtgt actactgtgc cagaagcagc 300ggcaccagag
gctttgccta ttggggacag ggcaccctcg tgaccgtgtc ctcagcctcc
360accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc
tgggggcaca 420gcggccctgg gctgcctggt caaggactac ttccccgaac
cggtgacggt gtcgtggaac 480tcaggcgccc tgaccagcgg cgtgcacacc
ttcccggctg tcctacagtc ctcaggactc 540tactccctta gtagcgtggt
gaccgtgccc tccagcagct tgggcaccca gacctacatc 600tgcaacgtga
atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 660tgtgactga
6692222PRTArtificial SequenceP10-1 Fab heavy chain fragment 2Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30Gly Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Glu Asn His Pro Gly Ser Gly Ile Ile Tyr His Asn Glu
Lys Phe 50 55 60Arg Gly Arg Val Thr Leu Thr Ala Asp Arg Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Ser Gly Thr Arg Gly Phe Ala
Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 2203669DNAArtificial SequenceDNA encoding P10-2 Fab
heavy chain fragment 3caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac
caggcgccag cgtgaaggtg 60tcctgcaagg ccagcggcta caccttcacc aactacggcc
tgagctgggt gcgccaggct 120cctggacagg gactggaatg gatgggcgag
aaccaccctg gcagcggcat catctaccac 180aacgagaagt tccggggcag
agtgaccctg accgccgacc ggagcaccag caccgcctac 240atggaactga
gcagcctgcg gagcgaggac accgccgtgt actactgtgc cagaagcagc
300ggcaccagag gctttgacta ttggggacag ggcaccctcg tgaccgtgtc
ctcagcctcc 360accaagggcc catcggtctt ccccctggca ccctcctcca
agagcacctc tgggggcaca 420gcggccctgg gctgcctggt caaggactac
ttccccgaac cggtgacggt gtcgtggaac 480tcaggcgccc tgaccagcgg
cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540tactccctta
gtagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc
600tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga
gcccaaatct 660tgtgactga 6694222PRTArtificial SequenceP10-2 Fab
heavy chain fragment 4Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr 20 25 30Gly Leu Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Glu Asn His Pro Gly Ser Gly
Ile Ile Tyr His Asn Glu Lys Phe 50 55 60Arg Gly Arg Val Thr Leu Thr
Ala Asp Arg Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Ser
Gly Thr Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 2205660DNAArtificial
SequenceDNA encoding antibody light chain 5gacgtcgtga tgacccagac
ccctctgagc ctgagcgtga cacctggaca gcctgccagc 60atcagctgca gatccagcca
gagcatcgtg cacagcaacg gcaacaccta cctggaatgg 120tatctgcaga
agcccggcca gagcccccag ctgctgatct acagggtgtc caaccggttc
180agcggcgtgc ccgacagatt ttctggcagc ggctccggca ccgacttcac
cctgaagatc 240tcccgggtgg aagccgagga cgtgggcgtg tactactgtt
ttcaaggcag ccacggcccc 300tggacctttg gccagggaac aaagctggaa
atcaagcgta cggtggctgc accatctgtc 360ttcatcttcc cgccatctga
tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420ctgaataact
tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa
480tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac
ctacagcctg 540agcagcaccc tgacgctgag caaagcagac tacgagaaac
acaaagtcta cgcctgcgaa 600gtcacccatc agggcctgag ctcgcccgtc
acaaagagct tcaacagggg agagtgttag 6606219PRTArtificial
SequenceAntibody light chain 6Asp Val Val Met Thr Gln Thr Pro Leu
Ser Leu Ser Val Thr Pro Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Ile Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr
Arg Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95Ser His
Gly Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe 130 135 140Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln145 150 155 160Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 210 2157354DNAArtificial
SequenceDNA encoding P10-1 Fab heavy chain variable region
7caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac caggcgccag cgtgaaggtg
60tcctgcaagg ccagcggcta caccttcacc aactacggcc tgagctgggt gcgccaggct
120cctggacagg gactggaatg gatgggcgag aaccaccctg gcagcggcat
catctaccac 180aacgagaagt tccggggcag agtgaccctg accgccgacc
ggagcaccag caccgcctac 240atggaactga gcagcctgcg gagcgaggac
accgccgtgt actactgtgc cagaagcagc 300ggcaccagag gctttgccta
ttggggacag ggcaccctcg tgaccgtgtc ctca 3548118PRTArtificial
SequenceP10-1 Fab heavy chain variable region 8Gln Val Gln Leu Val
Gln Ser Gl