U.S. patent application number 17/530587 was filed with the patent office on 2022-03-03 for anti-human transferrin receptor antibody permeating blood-brain barrier.
This patent application is currently assigned to JCR Pharmaceuticals Co., Ltd.. The applicant listed for this patent is JCR Pharmaceuticals Co., Ltd.. Invention is credited to Hiroyuki Sonoda, Kenichi Takahashi.
Application Number | 20220064288 17/530587 |
Document ID | / |
Family ID | 1000005962768 |
Filed Date | 2022-03-03 |
United States Patent
Application |
20220064288 |
Kind Code |
A1 |
Sonoda; Hiroyuki ; et
al. |
March 3, 2022 |
Anti-Human Transferrin Receptor Antibody Permeating Blood-Brain
Barrier
Abstract
Disclosed are a means to convert compounds having physiological
or pharmacological activity and unable to pass through the
blood-brain barrier into a form that allows them to pass through
the blood-brain barrier, and compounds converted thereby. The means
is an anti-human transferrin receptor antibody and the converted
compounds are molecular conjugates between physiologically active
protein or pharmacologically active low-molecular-weight compounds
and an anti-human transferrin receptor antibody.
Inventors: |
Sonoda; Hiroyuki; (Hyogo,
JP) ; Takahashi; Kenichi; (Hyogo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JCR Pharmaceuticals Co., Ltd. |
Hyogo |
|
JP |
|
|
Assignee: |
JCR Pharmaceuticals Co.,
Ltd.
Hyogo
JP
|
Family ID: |
1000005962768 |
Appl. No.: |
17/530587 |
Filed: |
November 19, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15739200 |
Dec 22, 2017 |
|
|
|
PCT/JP2016/068738 |
Jun 24, 2016 |
|
|
|
17530587 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/113 20130101;
A61K 2039/505 20130101; A61K 39/395 20130101; C07K 2317/92
20130101; C12N 9/16 20130101; A61K 45/00 20130101; C07K 16/18
20130101; C12Y 302/01076 20130101; C12N 15/09 20130101; C12Y 301/02
20130101; C12Y 304/14009 20130101; C07K 14/7151 20130101; C12N 9/24
20130101; C12N 9/2402 20130101; C07K 16/28 20130101; C07K 14/505
20130101; C07K 16/46 20130101; C07K 2317/24 20130101; C12N 5/10
20130101; C07K 2319/30 20130101; C07K 14/475 20130101; C07K 19/00
20130101; C12Y 310/01001 20130101; A61P 25/00 20180101; C12N 9/10
20130101; C12N 9/48 20130101; C12N 9/64 20130101; C12N 9/14
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C12N 9/16 20060101 C12N009/16; A61K 39/395 20060101
A61K039/395; C12N 9/10 20060101 C12N009/10; C07K 19/00 20060101
C07K019/00; C07K 14/475 20060101 C07K014/475; C12N 5/10 20060101
C12N005/10; A61K 45/00 20060101 A61K045/00; C07K 16/46 20060101
C07K016/46; C07K 16/18 20060101 C07K016/18; C12N 15/09 20060101
C12N015/09; C12N 9/64 20060101 C12N009/64; C12N 9/24 20060101
C12N009/24; C12N 15/113 20060101 C12N015/113; A61P 25/00 20060101
A61P025/00; C07K 14/505 20060101 C07K014/505; C07K 14/715 20060101
C07K014/715; C12N 9/14 20060101 C12N009/14; C12N 9/48 20060101
C12N009/48 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 24, 2015 |
JP |
2015-144379 |
Claims
1-69. (canceled)
70. A fusion protein comprising the amino acid sequences of a
humanized anti-human transferrin receptor antibody and iduronate
2-sulfatase, wherein the amino acid sequence of iduronate
2-sulfatase is linked to the light chain of the antibody on the
C-terminal side or the N-terminal side of the light chain, or
linked to the heavy chain of the antibody on the C-terminal side or
the N-terminal side of the heavy chain, and wherein the light chain
variable region and the heavy chain variable region of the antibody
are selected from (1) or (2) below: (1) the light chain variable
region comprising the amino acid sequence having an identity not
lower than 90% to the amino acid sequence set forth as SEQ ID NO:
191, and comprising the amino acid sequence set forth as SEQ ID
NO:16 as CDR1, the amino acid sequence set forth as SEQ ID NO:18 as
CDR2, and the amino acid sequence set forth as SEQ ID NO:20 as
CDR3, and the heavy chain variable region comprising the amino acid
sequence having an identity not lower than 90% to the amino acid
sequence set forth as SEQ ID NO: 205, and comprising the amino acid
sequence set forth as SEQ ID NO:88 as CDR1, the amino acid sequence
set forth as SEQ ID NO:90 as CDR2, and the amino acid sequence set
forth as SEQ ID NO:92 as CDR3; (2) the light chain variable region
comprising the amino acid sequence having an identity not lower
than 90% to the amino acid sequence set forth as SEQ ID NO: 191,
and comprising the amino acid sequence set forth as SEQ ID NO:16 as
CDR1, the amino acid sequence set forth as SEQ ID NO:18 as CDR2,
and the amino acid sequence set forth as SEQ ID NO:20 as CDR3, and
the heavy chain variable region comprising the amino acid sequence
having an identity not lower than 90% to the amino acid sequence
set forth as SEQ ID NO: 205, and comprising the amino acid sequence
set forth as SEQ ID NO:89 as CDR1, the amino acid sequence set
forth as SEQ ID NO:90 as CDR2, and the amino acid sequence set
forth as SEQ ID NO:92 as CDR3.
71. A fusion protein comprising the amino acid sequences of a
humanized anti-human transferrin receptor antibody and iduronate
2-sulfatase, wherein the amino acid sequence of the antibody is
selected from the group consisting of (1) to (4) below: (1) the
light chain variable region of the antibody comprises the amino
acid sequence set forth as SEQ ID NO:191, and the heavy chain
variable region of the antibody comprises the amino acid sequence
set forth as SEQ ID NO:205; (2) the light chain variable region of
the antibody comprises the amino acid sequence set forth as SEQ ID
NO:193, and the heavy chain variable region of the antibody
comprises the amino acid sequence set forth as SEQ ID NO:205; (3)
the light chain variable region of the antibody comprises the amino
acid sequence set forth as SEQ ID NO:194, and the heavy chain
variable region of the antibody comprises the amino acid sequence
set forth as SEQ ID NO:205; and (4) the light chain variable region
of the antibody comprises the amino acid sequence set forth as SEQ
ID NO:195, and the heavy chain variable region of the antibody
comprises the amino acid sequence set forth as SEQ ID NO:205.
72. A fusion protein comprising the amino acid sequences of a
humanized anti-human transferrin receptor antibody and iduronate
2-sulfatase, wherein the amino acid sequence of the antibody is
selected from the group consisting of (1) to (8) below: (1) the
light chain of the antibody comprises the amino acid sequence set
forth as SEQ ID NO:196, and the heavy chain of the antibody
comprises the amino acid sequence set forth as SEQ ID NO:210; (2)
the light chain of the antibody comprises the amino acid sequence
set forth as SEQ ID NO:198, and the heavy chain of the antibody
comprises the amino acid sequence set forth as SEQ ID NO:210; (3)
the light chain of the antibody comprises the amino acid sequence
set forth as SEQ ID NO:200, and the heavy chain of the antibody
comprises the amino acid sequence set forth as SEQ ID NO:210; (4)
the light chain of the antibody comprises the amino acid sequence
set forth as SEQ ID NO:202, and the heavy chain of the antibody
comprises the amino acid sequence set forth as SEQ ID NO:210; (5)
the light chain of the antibody comprises the amino acid sequence
set forth as SEQ ID NO:196, and the heavy chain of the antibody
comprises the amino acid sequence set forth as SEQ ID NO:212; (6)
the light chain of the antibody comprises the amino acid sequence
set forth as SEQ ID NO:198, and the heavy chain of the antibody
comprises the amino acid sequence set forth as SEQ ID NO:212; (7)
the light chain of the antibody comprises the amino acid sequence
set forth as SEQ ID NO:200, and the heavy chain of the antibody
comprises the amino acid sequence set forth as SEQ ID NO:212; and
(8) the light chain of the antibody comprises the amino acid
sequence set forth as SEQ ID NO:202, and the heavy chain of the
antibody comprises the amino acid sequence set forth as SEQ ID
NO:212.
73. The fusion protein according to claim 70, wherein the antibody
has an affinity to both the extracellular region of human
transferrin receptor and the extracellular region of monkey
transferrin receptor.
74. The fusion protein according to claim 73, wherein the
dissociation constant of the antibody's complex with the
extracellular region of human transferrin receptor is not greater
than 1.times.10.sup.-8 M, and the dissociation constant of the
antibody's complex with the extracellular region of monkey
transferrin receptor is not greater than 5.times.10.sup.-8 M.
75. The fusion protein according to claim 70, wherein the antibody
is Fab antibody, F(ab')2 antibody, or F(ab') antibody.
76. The fusion protein according to claim 70, wherein the antibody
is a single-chain antibody selected from the group consisting of
scFab, scF(ab'), scF(ab')2 and scFv.
77. The fusion protein according to claim 76, wherein the light
chain and the heavy chain thereof are linked via a linker
sequence.
78. The fusion protein according to claim 76, wherein the heavy
chain is linked, via a linker sequence, to the light chain on the
C-terminal side of the light chain.
79. The fusion protein according to claim 76, wherein the light
chain is linked, via a linker sequence, to the heavy chain on the
C-terminal side of the heavy chain.
80. The fusion protein according to claim 77, wherein the linker
sequence consists of 8 to 50 amino acid residues.
81. The fusion protein according to claim 80, wherein the linker
sequence is selected from the group consisting of the amino acid
sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino
acid sequence Gly-Gly-Gly, the amino acid sequences set forth as
SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, and the amino acid
sequence consisting of three consecutively linked amino acid
sequences each set forth as SEQ ID NO:3.
82. The fusion protein according to claim 70, wherein the iduronate
2-sulfatase is linked, via a linker sequence, to the light chain on
the C-terminal side or the N-terminal side of the light chain, or
to the heavy chain on the C-terminal side or the N-terminal side of
the heavy chain.
83: The fusion protein according to claim 81, wherein the linker
sequence consists of 1 to 50 amino acid residues.
84. The fusion protein according to claim 81, wherein the linker
sequence comprises an amino acid sequence selected from the group
consisting of a single glycine, a single serine, the amino acid
sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino
acid sequence set forth as SEQ ID NO:3, the amino acid sequence set
forth as SEQ ID NO:4, the amino acid sequence set forth as SEQ ID
NO:5, and amino acid sequence consisting of 1 to 10 of any of said
amino acid sequences that are consecutively linked.
85. The fusion protein according to claim 70, wherein the iduronate
2-sulfatase is a human iduronate 2-sulfatase.
86. The fusion protein according to claim 85, wherein the light
chain of the antibody has the amino acid sequence set forth as SEQ
ID NO:196, and wherein the heavy chain of the antibody is linked,
on the C-terminal side of the heavy chain and via a linker sequence
Gly-Ser, to the human iduronate 2-sulfatase, and the whole linked
heavy chain has the amino acid sequence set forth as SEQ ID
NO:251.
87. The fusion protein according to claim 85, wherein the fusion
protein comprises the light chain having the amino acid sequence
set forth as SEQ ID NO:196, and the heavy chain having the amino
acid sequence set forth as SEQ ID NO:210 which is linked, on the
C-terminal side of the heavy chain and via a linker sequence
Gly-Ser, to the human iduronate 2-sulfatase set forth as SEQ ID
NO:246.
88. The fusion protein according to claim 85, wherein the fusion
protein comprises a conjugate composed of the human iduronate
2-sulfatase linked via a linker sequence Gly-Ser to the heavy chain
on the C-terminal side of the heavy chain, and the light chain,
wherein the amino acid sequence of the conjugate is set forth as
SEQ ID NO:251, and the amino acid sequence of the light chain is
set forth as SEQ ID NO:196.
89. A DNA fragment encoding the amino acid sequence of the fusion
protein according to claim 70.
90. An expression vector comprising the DNA fragment according to
claim 89 that is incorporated therein.
91. A mammalian cell transformed with the expression vector
according to claim 90.
92. A method of treating a disease condition of the central nervous
system accompanying Hunter syndrome in a subject in need thereof
comprising parenterally administering the fusion protein of claim
70 to the subject.
Description
TECHNICAL FIELD
[0001] The present invention relates to an anti-human transferrin
receptor antibody to be utilized for conjugation with a compound
that needs to exhibit its function in the central nervous system
when administered parenterally (a protein or a low-molecular-weight
compound and the like) in order to make that compound able to pass
through the blood-brain barrier after parenterally administered,
and also a method for production thereof, as well as a method of
use thereof
Background Art
[0002] Unlike the capillaries in other tissues such as muscles, the
capillaries that supply the blood to most of the brain tissues
except some areas including the circumventricular organs (pineal
gland, pituitary body, area postrema, etc.) differ in that the
endothelial cells forming their endothelium are mutually connected
by tight intercellular junctions. Passive transfer of substances
from the capillaries to the brain is thereby restricted, and
although there are some exceptions, substances are unlikely to move
into the brain from the blood except such compounds as are
lipid-soluble or of low-molecular-weight (not greater than 200 to
500 Dalton) and electrically neutral around the physiological pH.
This system, which restricts exchange of substances between the
blood and the tissue fluid of the brain through the endothelium of
capillaries in the brain, is called the blood-brain barrier or BBB.
The blood-brain barrier not only restricts exchange of substances
between the blood and the brain but also between the tissue fluid
of the central nervous system, including the brain and the spinal
chord, and the blood.
[0003] Owing to the blood-brain barrier, most of the cells of the
central nervous system escape the effects of fluctuating
concentrations of substances like hormones and lymphokines in the
blood, and their biochemical homeostasis is thus maintained.
[0004] The blood-brain barrier, however, imposes a problem when it
comes to develop pharmaceutical agents. For mucopolysaccharidosis
type I (Hurler syndrome), an inherited metabolic disease caused by
.alpha.-L-iduronidase deficiency, for example, although an enzyme
replacement therapy is carried out by intravenous supplementation
with a recombinant .alpha.-L-iduronidase as a therapy, the therapy
is not effective for the notable abnormality observed in the
central nervous system (CNS) in Hurler syndrome because the enzyme
cannot pass through the blood-brain barrier.
[0005] Development of various methods has been attempted to make
those macromolecular substances as proteins or the like, which need
to be brought into function in the central nervous system, pass
through the blood-brain barrier. In the case of nerve growth
factor, for example, while attempts have been made for a method to
cause the factor to pass through the blood-brain barrier by
allowing liposomes encapsulating the factor to fuse with the cell
membrane of endothelial cells in brain capillaries, they have not
been reached practical application (Non Patent Literature 1). In
the case of .alpha.-L-iduronidase, an attempt has been made to
enhance the passive transfer of the enzyme through the blood-brain
barrier by raising its blood concentration through an increased
single dose of the enzyme, and it thus has been demonstrated, using
a Hurler syndrome animal model, that the abnormality in the central
nervous system (CNS) is ameliorated by that method (Non Patent
Literature 2).
[0006] Furthermore, circumventing the blood-brain barrier, an
attempt has also been made to administer a macromolecular substance
directly into the medullary cavity or into the brain. For example,
reports have been made about a method in which human
.alpha.-L-iduronidase was intrathecally administered to a patient
with a Hurler syndrome (mucopolysaccharidosis type I) (Patent
Literature 1), a method in which human acid sphingomyelinase was
administered into the brain ventricles of a patient with
Niemann-Pick disease (Patent Literature 2), and a method in which
iduronate 2-sulfatase (I2S) was administered into the brain
ventricles of Hunter syndrome model animals (Patent Literature 3).
While it seems possible by one of such methods to definitely let a
pharmaceutical agent act in the central nervous system, they have a
problem as being highly invasive.
[0007] There have been reported various methods to let a
macromolecular substance get into the brain through the blood-brain
barrier, in which the macromolecular substance is modified to give
it an affinity to membrane proteins existing on the endothelial
cells of the brain capillaries. Examples of those membrane proteins
which exist on the endothelial cells of the brain capillaries
include receptors for compounds such as insulin, transferrin,
insulin-like growth factor (IGF-I, IGF-II), LDL, and leptin.
[0008] For example, a technique has been reported in which nerve
growth factor (NGF) was synthesized in the form of a fusion protein
with insulin, and this fusion protein was allowed to pass through
the blood-brain barrier via its binding to the insulin receptor
(Patent Literatures 4-6). Further, a technique has been reported in
which nerve growth factor (NGF) was synthesized in the form of a
fusion protein with anti-insulin receptor antibody, and this fusion
protein was allowed to pass through the blood-brain barrier via its
binding to the insulin receptor (Patent Literatures 4 and 7).
Further, a technique has been reported in which nerve growth factor
(NGF) was synthesized in the form of a fusion protein with
transferrin, and this fusion protein was allowed to pass through
the blood-brain barrier via its binding to the transferrin receptor
(TfR) (Patent Literature 8). Further, a technique has been reported
in which nerve growth factor (NGF) was synthesized in the form of a
fusion protein with anti-transferrin receptor antibody (anti-TfR
antibody), and this fusion protein is allowed to pass through the
blood-brain barrier via its binding to TfR (Patent Literatures 4
and 9).
[0009] Looking further into the techniques that utilize an
anti-transferrin receptor antibody, there has been reported that in
the field of the technique to make a pharmaceutical agent pass
through the blood-brain barrier by binding it to an anti-TfR
antibody, a single-chain antibody could be used (Non Patent
Literature 3). Further, it has been reported that anti-hTfR
antibodies exhibiting relatively high dissociation constants with
hTfR (low-affinity anti-hTfR antibody) could be favorably used in
the technique to make pharmaceutical agents pass through the
blood-brain barrier (Patent Literatures 10 and 11, and Non Patent
Literature 4). Still further, it has also been reported that an
anti-TfR antibodies whose affinity to hTfR varies depending on pH
could be employed as a carrier for making pharmaceutical agents
pass through the blood-brain barrier (Patent Literature 12, and Non
Patent Literature 5).
CITATION LIST
Patent Literature
[0010] Patent Literature 1: JP2007-504166 A1 [0011] Patent
Literature 2: TP2009-525963 A1 [0012] Patent Literature 3:
JP2012-62312 A1 [0013] Patent Literature 4: U.S. Pat. No. 5,154,924
B1 [0014] Patent Literature 5: JP2011-144178 A1 [0015] Patent
Literature 6: US2004/0101904 A1 [0016] Patent Literature 7:
W2006-511516 A1 [0017] Patent Literature 8: PH06-228199 A1 [0018]
Patent Literature 9: U.S. Pat. No. 5,977,307 B1 [0019] Patent
Literature 10: WO 2012/075037 [0020] Patent Literature 11: WO
2013/177062 [0021] Patent Literature 12: WO 2012/143379
Non Patent Literature
[0021] [0022] Non Patent Literature 1: Xie Y. et al., J Control
Release. 105. 106-19 (2005) [0023] Non Patent Literature 2: Ou L.
et al., Mol Genet Metab. 111. 116-22 (2014) [0024] Non Patent
Literature 3: Li J Y. Protein Engineering. 12. 787-96 (1999) [0025]
Non Patent Literature 4: Bien-Ly N. et al., J Exp Med. 211. 233-44
(2014) [0026] Non Patent Literature 5: Sada H. PLoS ONE. 9. E96340
(2014)
SUMMARY OF INVENTION
Problems to be Solved by the Invention
[0027] Against the above background, it is an objective of the
present invention to provide an anti-TfR antibody that can be
utilized for conjugation with a compound that needs to exhibit its
function in the central nervous system when administered
parenterally (a protein or a low-molecular-weight compound and the
like) in order to make that compound able to pass through the
blood-brain barrier, and also a method for production thereof, as
well as a method of use thereof.
Means for Solving the Problems
[0028] As a result of intense studies aimed at the above objective,
the present inventors have found that anti-human transferrin
receptor antibodies (anti-hTfR antibodies) that recognize the
extracellular region of hTfR which are obtained by the method for
antibody production described in detail in the specification,
efficiently passes through the blood-brain barrier when
administered to the body, and have completed the present invention
thereupon. Thus the present invention provides what follows:
[0029] 1. An anti-human transferrin receptor antibody, wherein the
amino acid sequence of the light chain variable region of the
antibody is selected from the group consisting of (1) to (14)
below:
[0030] (1) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:6 or SEQ ID NO:7 in CDR1, the amino
acid sequence set forth as SEQ ID NO:8 or SEQ ID NO:9 or the amino
acid sequence Trp-Thr-Ser in CDR2, and the amino acid sequence set
forth as SEQ ID NO:10 in CDR3;
[0031] (2) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:11 or SEQ ID NO:12 in CDR1, the
amino acid sequence set forth as SEQ ID NO:13 or SEQ ID NO:14 or
the amino acid sequence Tyr-Ala-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:15 in CDR3;
[0032] (3) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:16 or SEQ ID NO:17 in CDR1, the
amino acid sequence set forth as SEQ ID NO:18 or SEQ ID NO:19 or
the amino acid sequence Lys-Val-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:20 in CDR3;
[0033] (4) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:21 or SEQ ID NO:22 in CDR1, the
amino acid sequence set forth as SEQ ID NO:23 or SEQ ID NO:24 or
the amino acid sequence Asp-Thr-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:25 in CDR3;
[0034] (5) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:26 or SEQ ID NO:27 in CDR1, the
amino acid sequence set forth as SEQ ID NO:28 or SEQ ID NO:29 or
the amino acid sequence Asp-Thr-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:30 in CDR3;
[0035] (6) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:31 or SEQ ID NO:32 in CDR1, the
amino acid sequence set forth as SEQ ID NO:33 or SEQ ID NO:34 or
the amino acid sequence Ala-Ala-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:35 in CDR3;
[0036] (7) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:36 or SEQ ID NO:37 in CDR1, the
amino acid sequence set forth as SEQ ID NO:38 or SEQ ID NO:39 or
the amino acid sequence Gln-Thr-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:40 in CDR3;
[0037] (8) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:41 or SEQ ID NO:42 in CDR1, the
amino acid sequence set forth as SEQ ID NO:43 or SEQ ID NO:44 or
the amino acid sequence Gly-Thr-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:45 in CDR3;
[0038] (9) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:46 or SEQ ID NO:47 in CDR1, the
amino acid sequence set forth as SEQ ID NO:48 or SEQ ID NO:49 or
the amino acid sequence Phe-Thr-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:50 in CDR3;
[0039] (10) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:51 or SEQ ID NO:52 in CDR1, the
amino acid sequence set forth as SEQ ID NO:53 or SEQ ID NO:54 or
the amino acid sequence Ala-Ala-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:55 in CDR3;
[0040] (11) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:56 or SEQ ID NO:57 in CDR1, the
amino acid sequence set forth as SEQ ID NO:58 or SEQ ID NO:59 or
the amino acid sequence Tyr-Ala-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:60 in CDR3;
[0041] (12) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:61 or SEQ ID NO:62 in CDR1, the
amino acid sequence set forth as SEQ ID NO:63 or SEQ ID NO:64 or
the amino acid sequence Trp-Ser-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:65 in CDR3;
[0042] (13) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:66 or SEQ ID NO:67 in CDR1, the
amino acid sequence set forth as SEQ ID NO:68 or SEQ ID NO:69 or
the amino acid sequence Tyr-Ala-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:70 in CDR3; and
[0043] (14) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:71 or SEQ ID NO:72 in CDR1, the
amino acid sequence set forth as SEQ ID NO:73 or SEQ ID NO:74 or
the amino acid sequence Asp-Thr-Ser in CDR2, and the amino acid
sequence set forth as SEQ ID NO:75 in CDR3.
[0044] 2. The anti-human transferrin receptor antibody according to
1 above, wherein the amino acid sequence of the light chain
variable region of the antibody is selected from the group
consisting of (1) to (14) below:
[0045] (1) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:6 in CDR1, the amino acid sequence
set forth as SEQ ID NO:8 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:10 in CDR3;
[0046] (2) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:11 in CDR1, the amino acid sequence
set forth as SEQ ID NO:13 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:15 in CDR3;
[0047] (3) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:16 in CDR1, the amino acid sequence
set forth as SEQ ID NO:18 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:20 in CDR3;
[0048] (4) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:21 in CDR1, the amino acid sequence
set forth as SEQ ID NO:23 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:25 in CDR3;
[0049] (5) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:26 in CDR1, the amino acid sequence
set forth as SEQ ID NO:28 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:30 in CDR3;
[0050] (6) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:31 in CDR1, the amino acid sequence
set forth as SEQ ID NO:33 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:35 in CDR3;
[0051] (7) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:36 in CDR1, the amino acid sequence
set forth as SEQ ID NO:38 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:40 in CDR3;
[0052] (8) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:41 in CDR1, the amino acid sequence
set forth as SEQ ID NO:43 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:45 in CDR3;
[0053] (9) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:46 in CDR1, the amino acid sequence
set forth as SEQ ID NO:48 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:50 in CDR3;
[0054] (10) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:51 in CDR1, the amino acid sequence
set forth as SEQ ID NO:53 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:55 in CDR3;
[0055] (11) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:56 in CDR1, the amino acid sequence
set forth as SEQ ID NO:58 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:60 in CDR3;
[0056] (12) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:61 in CDR1, the amino acid sequence
set forth as SEQ ID NO:63 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:65 in CDR3;
[0057] (13) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:66 in CDR1, the amino acid sequence
set forth as SEQ ID NO:68 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:70 in CDR3; and
[0058] (14) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:71 in CDR1, the amino acid sequence
set forth as SEQ ID NO:73 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:75 in CDR3.
[0059] 3. An anti-human transferrin receptor antibody, wherein the
amino acid sequences of CDR1, CDR2 and CDR3 in the light chain
variable region thereof have a homology not lower than 80% to the
amino acid sequences of CDR1, CDR2 and CDR3, respectively, in the
light chain according to 1 or 2 above.
[0060] 4. An anti-human transferrin receptor antibody, wherein the
amino acid sequences of CDR1, CDR2 and CDR3 in the light chain
variable region thereof have a homology not lower than 90% to the
amino acid sequences of CDR1, CDR2 and CDR3, respectively, in the
light chain according to 1 or 2 above.
[0061] 5. An anti-human transferrin receptor antibody, wherein 1 to
5 amino acids are substituted, deleted or added relative to the
amino acid sequence that forms at least one of the CDRs in the
light chain according to 1 or 2 above.
[0062] 6. An anti-human transferrin receptor antibody, wherein 1 to
3 amino acids are substituted, deleted or added relative to the
amino acid sequence that forms at least one of the CDRs in the
light chain according to 1 or 2 above.
[0063] 7. An anti-human transferrin receptor antibody, wherein the
amino acid sequence of the heavy chain variable region of the
antibody is selected from the group consisting of (1) to (14)
below:
[0064] (1) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:76 or SEQ ID NO:77 in CDR1, the
amino acid sequence set forth as SEQ ID NO:78 or SEQ ID NO:79 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:80 or SEQ
ID NO:81 in CDR3;
[0065] (2) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:82 or SEQ ID NO:83 in CDR1, the
amino acid sequence set forth as SEQ ID NO:84 or SEQ ID NO:85 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:86 or SEQ
ID NO:87 in CDR3;
[0066] (3) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:88 or SEQ ID NO:89 in CDR1, the
amino acid sequence set forth as SEQ ID NO:90 or SEQ ID NO:91 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:92 or SEQ
ID NO:93 in CDR3;
[0067] (4) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:94 or SEQ ID NO:95 in CDR1, the
amino acid sequence set forth as SEQ ID NO:96 or SEQ ID NO:97 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:98 or SEQ
ID NO:99 in CDR3;
[0068] (5) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:100 or SEQ ID NO:101 in CDR1, the
amino acid sequence set forth as SEQ ID NO:102 or SEQ ID NO:103 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:104 or SEQ
ID NO:105 in CDR3;
[0069] (6) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:106 or SEQ ID NO:107 in CDR1, the
amino acid sequence set forth as SEQ ID NO:108 or SEQ ID NO:278 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:109 or SEQ
ID NO:110 in CDR3;
[0070] (7) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:111 or SEQ ID NO:112 in CDR1, the
amino acid sequence set forth as SEQ ID NO:113 or SEQ ID NO:114 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:115 or SEQ
ID NO:116 in CDR3;
[0071] (8) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:117 or SEQ ID NO:118 in CDR1, the
amino acid sequence set forth as SEQ ID NO:119 or SEQ ID NO:279 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:120 or SEQ
ID NO:121 in CDR3;
[0072] (9) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:122 or SEQ ID NO:123 in CDR1, the
amino acid sequence set forth as SEQ ID NO:124 or SEQ ID NO:125 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:126 or SEQ
ID NO:127 in CDR3;
[0073] (10) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:128 or SEQ ID NO:129 or CDR1, the
amino acid sequence set forth as SEQ ID NO:130 or SEQ ID NO:131 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:132 or SEQ
ID NO:133 in CDR3;
[0074] (11) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:134 or SEQ ID NO:135 in CDR1, the
amino acid sequence set forth as SEQ ID NO:136 or SEQ ID NO:137 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:138 or SEQ
ID NO:139 in CDR3;
[0075] (12) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:140 or SEQ ID NO:141 in CDR1, the
amino acid sequence set forth as SEQ ID NO:142 or SEQ ID NO:143 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:144 or SEQ
ID NO:145 in CDR3;
[0076] (13) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:146 or SEQ ID NO:147 in CDR1, the
amino acid sequence set forth as SEQ ID NO:148 or SEQ ID NO:149 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:150 or SEQ
ID NO:151 in CDR3; and
[0077] (14) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:152 or SEQ ID NO:153 in CDR1, the
amino acid sequence set forth as SEQ ID NO:154 or SEQ ID NO:155 in
CDR2, and the amino acid sequence set forth as SEQ ID NO:156 or SEQ
ID NO:157 in CDR3.
[0078] 8. The anti-human transferrin receptor antibody according to
7 above, wherein the amino acid sequence of the heavy chain
variable region of the antibody is selected from the group
consisting of (1) to (14) below:
[0079] (1) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:76 in CDR1, the amino acid sequence
set forth as SEQ ID NO:78 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:80 in CDR3;
[0080] (2) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:82 in CDR1, the amino acid sequence
set forth as SEQ ID NO:84 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:86 in CDR3;
[0081] (3) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:88 in CDR1, the amino acid sequence
set forth as SEQ ID NO:90 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:92 in CDR3;
[0082] (4) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:94 in CDR1, the amino acid sequence
set forth as SEQ ID NO:96 in CDR2, and the amino acid sequence set
forth as SEQ ID NO:98 in CDR3;
[0083] (5) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:100 in CDR1, the amino acid
sequence set forth as SEQ ID NO:102 in CDR2, and the amino acid
sequence set forth as SEQ ID NO:104 in CDR3;
[0084] (6) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:106 in CDR1, the amino acid
sequence set forth as SEQ ID NO:108 in CDR2, and the amino acid
sequence set forth as SEQ ID NO:109 in CDR3;
[0085] (7) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:111 in CDR1, the amino acid
sequence set forth as SEQ ID NO:113 in CDR2, and the amino acid
sequence set forth as SEQ ID NO:115 as CDR3;
[0086] (8) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:117 in CDR1, the amino acid
sequence set forth as SEQ ID NO:119 in CDR2, and the amino acid
sequence set forth as SEQ ID NO:120 in CDR3;
[0087] (9) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:122 in CDR1, the amino acid
sequence set forth as SEQ ID NO:124 in CDR2, and the amino acid
sequence set forth as SEQ ID NO:126 in CDR3;
[0088] (10) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:128 in CDR1, the amino acid
sequence set forth as SEQ ID NO:130 in CDR2, and the amino acid
sequence set forth as SEQ ID NO:132 in CDR3;
[0089] (11) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:134 in CDR1, the amino acid
sequence set forth as SEQ ID NO:136 in CDR2, and the amino acid
sequence set forth as SEQ ID NO:138 in CDR3;
[0090] (12) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:140 in CDR1, the amino acid
sequence set forth as SEQ ID NO:142 in CDR2, and the amino acid
sequence set forth as SEQ ID NO:144 in CDR3;
[0091] (13) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:146 in CDR1, the amino acid
sequence set forth as SEQ ID NO:148 in CDR2, and the amino acid
sequence set forth as SEQ ID NO:150 in CDR3; and
[0092] (14) an amino acid sequence comprising the amino acid
sequence set forth as SEQ ID NO:152 in CDR1, the amino acid
sequence set forth as SEQ ID NO:154 in CDR2, and the amino acid
sequence set forth as SEQ ID NO:156 in CDR3.
[0093] 9. An anti-human transferrin receptor antibody, wherein the
amino acid sequence of CDR1, CDR2 and CDR3 in the heavy chain
variable region thereof have a homology not lower than 80% to the
amino acid sequences of CDR1, CDR2 and CDR3, respectively, in the
heavy chain according to 7 or 8 above.
[0094] 10. An anti-human transferrin receptor antibody, wherein the
amino acid sequence of CDR1, CDR2 and CDR3 in the heavy chain
variable region thereof have a homology not lower than 90% to the
amino acid sequences of CDR1, CDR2 and CDR3, respectively, in the
heavy chain according to 7 or 8 above.
[0095] 11. An anti-human transferrin receptor antibody, wherein 1
to 5 amino acids are substituted, deleted or added relative to the
amino acid sequence that forms at least one of the CDRs in the
heavy chain according to 7 or 8 above.
[0096] 12. An anti-human transferrin receptor antibody, wherein 1
to 3 amino acids are substituted, deleted or added relative to the
amino acid sequence that forms at least one of the CDRs in the
heavy chain according to 7 or 8 above.
[0097] 13. An anti-human transferrin receptor antibody, wherein the
light chain variable region and the heavy chain variable region
thereof are selected from the group consisting of (1) to (14)
below:
[0098] (1) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:6 or SEQ ID NO:7 as CDR1, the
amino acid sequence set forth as SEQ ID NO:8 or SEQ ID NO:9 or the
amino acid sequence Trp-Thr-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:10 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:76 or SEQ ID NO:77 as CDR1, the amino acid sequence set forth
as SEQ ID NO:78 or SEQ ID NO:79 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:80 or SEQ ID NO:81 as CDR3;
[0099] (2) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:11 or SEQ ID NO:12 as CDR1,
the amino acid sequence set forth as SEQ ID NO:13 or SEQ ID NO:14
or the amino acid sequence Tyr-Ala-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:15 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:82 or SEQ ID NO:83 as CDR1, the amino acid sequence set forth
as SEQ ID NO:84 or SEQ ID NO:85 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:86 or SEQ ID NO:87 as CDR3;
[0100] (3) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:16 or SEQ ID NO:17 as CDR1,
the amino acid sequence set forth as SEQ ID NO:18 or SEQ ID NO:19
or the amino acid sequence Lys-Val-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:20 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:88 or SEQ ID NO:89 as CDR1, the amino acid sequence set forth
as SEQ ID NO:90 or SEQ ID NO:91 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:92 or SEQ ID NO:93 as CDR3;
[0101] (4) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:21 or SEQ ID NO:22 as CDR1,
the amino acid sequence set forth as SEQ ID NO:23 or SEQ ID NO:24
or the amino acid sequence Asp-Thr-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:25 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:94 or SEQ ID NO:95 as CDR1, the amino acid sequence set forth
as SEQ ID NO:96 or SEQ ID NO:97 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:98 or SEQ ID NO:99 as CDR3;
[0102] (5) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:26 or SEQ ID NO:27 as CDR1,
the amino acid sequence set forth as SEQ ID NO:28 or SEQ ID NO:29
or the amino acid sequence Asp-Thr-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:30 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:100 or SEQ ID NO:101 as CDR1, the amino acid sequence set
forth as SEQ ID NO:102 or SEQ ID NO:103 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:104 or SEQ ID NO:105 as CDR3;
[0103] (6) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:31 or SEQ ID NO:32 as CDR1,
the amino acid sequence set forth as SEQ ID NO:33 or SEQ ID NO:34
or the amino acid sequence Ala-Ala-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:35 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:106 or SEQ ID NO:107 as CDR1, the amino acid sequence set
forth as SEQ ID NO:108 or SEQ ID NO:278, as CDR2, and the amino
acid sequence set forth as SEQ ID NO:109 or SEQ ID NO:110 as
CDR3;
[0104] (7) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:36 or SEQ ID NO:37 as CDR1,
the amino acid sequence set forth as SEQ ID NO:38 or SEQ ID NO:39
or the amino acid sequence Gln-Thr-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:40 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:111 or SEQ ID NO:112 as CDR1, the amino acid sequence set
forth as SEQ ID NO:113 or SEQ ID NO:114 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:115 or SEQ ID NO:116 as CDR3;
[0105] (8) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:41 or SEQ ID NO:42 as CDR1,
the amino acid sequence set forth as SEQ ID NO:43 or SEQ ID NO:44
or the amino acid sequence Gly-Thr-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:45 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:117 or SEQ ID NO:118 as CDR1, the amino acid sequence set
forth as SEQ ID NO:119 or SEQ ID NO:279 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:120 or SEQ ID NO:121 as CDR3;
[0106] (9) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:46 or SEQ ID NO:47 as CDR1,
the amino acid sequence set forth as SEQ ID NO:48 or SEQ ID NO:49
or the amino acid sequence Phe-Thr-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:50 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:122 or SEQ ID NO:123 as CDR1, the amino acid sequence set
forth as SEQ ID NO:124 or SEQ ID NO:125 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:126 or SEQ ID NO:127 as CDR3;
[0107] (10) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:51 or SEQ ID NO:52 as CDR1,
the amino acid sequence set forth as SEQ ID NO:53 or SEQ ID NO:54
or the amino acid sequence Ala-Ala-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:55 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:128 or SEQ ID NO:129 as CDR1, the amino acid sequence set
forth as SEQ ID NO:130 or SEQ ID NO:131 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:132 or SEQ ID NO:133 as CDR 3;
[0108] (11) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:56 or SEQ ID NO:57 as CDR1,
the amino acid sequence set forth as SEQ ID NO:58 or SEQ ID NO:59
or the amino acid sequence Tyr-Ala-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:60 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:134 or SEQ ID NO:135 as CDR1, the amino acid sequence set
forth as SEQ ID NO:136 or SEQ ID NO:137 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:138 or SEQ ID NO:139 as CDR3;
[0109] (12) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:61 or SEQ ID NO:62 as CDR1,
the amino acid sequence set forth as SEQ ID NO:63 or SEQ ID NO:64
or the amino acid sequence Trp-Ser-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:65 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:140 or SEQ ID NO:141 as CDR1, the amino acid sequence set
forth as SEQ ID NO:142 or SEQ ID NO:143 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:144 or SEQ ID NO:145 as CDR3;
[0110] (13) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:66 or SEQ ID NO:67 as CDR1,
the amino acid sequence set forth as SEQ ID NO:68 or SEQ ID NO:69
or the amino acid sequence Tyr-Ala-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:70 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:146 or SEQ ID NO:147 as CDR1, the amino acid sequence set
forth as SEQ ID NO:148 or SEQ ID NO:149 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:150 or SEQ ID NO:151 as CDR3;
and
[0111] (14) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:71 or SEQ ID NO:72 as CDR1,
the amino acid sequence set forth as SEQ ID NO:73 or SEQ ID NO:74
or the amino acid sequence Asp-Thr-Ser as CDR2, and the amino acid
sequence set forth as SEQ ID NO:75 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:152 or SEQ ID NO:153 as CDR1, the amino acid sequence set
forth as SEQ ID NO:154 or SEQ ID NO:155 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:156 or SEQ ID NO:157 as CDR3.
[0112] 14. The anti-human transferrin receptor antibody according
to 13 above, wherein the light chain variable region and the heavy
chain variable region thereof are selected from the group
consisting of (1) to (14) below:
[0113] (1) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:6 as CDR1, the amino acid
sequence set forth as SEQ ID NO:8 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:10 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:76 as CDR1, the amino acid sequence set forth as SEQ ID NO:78
as CDR2, and the amino acid sequence set forth as SEQ ID NO:80 as
CDR3;
[0114] (2) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:11 as CDR1, the amino acid
sequence set forth as SEQ ID NO:13 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:15 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:82 as CDR1, the amino acid sequence set forth as SEQ ID NO:84
as CDR2, and the amino acid sequence set forth as SEQ ID NO:86 as
CDR3;
[0115] (3) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:16 as CDR1, the amino acid
sequence set forth SEQ ID NO:18 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:20 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:88 as CDR1, the amino acid sequence set forth as SEQ ID NO:90
as CDR2, and the amino acid sequence set forth as SEQ ID NO:92 as
CDR3;
[0116] (4) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:21 as CDR1, the amino acid
sequence set forth as SEQ ID NO:23 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:25 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:94 as CDR1, the amino acid sequence set forth as SEQ ID NO:96
as CDR2, and the amino acid sequence set forth as SEQ ID NO:98 as
CDR3;
[0117] (5) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:26 as CDR1, the amino acid
sequence set forth as SEQ ID NO:28 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:30 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:100 as CDR1, the amino acid sequence set forth as SEQ ID
NO:102 as CDR2, and the amino acid sequence set forth as SEQ ID
NO:104 as CDR3;
[0118] (6) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:31 as CDR1, the amino acid
sequence set forth as SEQ ID NO:33 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:35 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:106 as CDR1, the amino acid sequence set forth as SEQ ID
NO:108 as CDR2, and the amino acid sequence set forth as SEQ ID
NO:109 as CDR3;
[0119] (7) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:36 as CDR1, the amino acid
sequence set forth as SEQ ID NO:38 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:40 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:111 as CDR1, the amino acid sequence set forth as SEQ ID
NO:113 as CDR2, and the amino acid sequence set forth as SEQ ID
NO:115 as CDR3;
[0120] (8) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:41 as CDR1, the amino acid
sequence set forth as SEQ ID NO:43 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:45 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:117 as CDR1, the amino acid sequence set forth as SEQ ID
NO:119 as CDR2, and the amino acid sequence set forth as SEQ ID
NO:120 as CDR 3;
[0121] (9) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:46 as CDR1, the amino acid
sequence set forth as SEQ ID NO:48 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:50 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:122 as CDR1, the amino acid sequence set forth as SEQ ID
NO:124 as CDR2, and the amino acid sequence set forth as SEQ ID
NO:126 as CDR3;
[0122] (10) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:51 as CDR1, the amino acid
sequence set forth as SEQ ID NO:53 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:55 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:128 as CDR1, the amino acid sequence set forth as SEQ ID
NO:130 as CDR2, and the amino acid sequence set forth as SEQ ID
NO:132 as CDR3;
[0123] (11) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:56 as CDR1, the amino acid
sequence set forth as SEQ ID NO:58 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:60 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:134 as CDR1, the amino acid sequence set forth as SEQ ID
NO:136 as CDR2, and the amino acid sequence set forth as SEQ ID
NO:138 as CDR3;
[0124] (12) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:61 as CDR1, the amino acid
sequence set forth as SEQ ID NO:63 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:65 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:140 as CDR1, the amino acid sequence set forth as SEQ ID
NO:142 as CDR2, and the amino acid sequence set forth as SEQ ID
NO:144 as CDR3;
[0125] (13) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:66 as CDR1, the amino acid
sequence set forth as SEQ ID NO:68 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:70 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:146 as CDR1, the amino acid sequence set forth as SEQ ID
NO:148 as CDR2, and the amino acid sequence set forth as SEQ ID
NO:150 as CDR3; and
[0126] (14) the light chain variable region comprising the amino
acid sequence set forth as SEQ ID NO:71 as CDR1, the amino acid
sequence set forth as SEQ ID NO:73 as CDR2, and the amino acid
sequence set forth as SEQ ID NO:75 as CDR3; and the heavy chain
variable region comprising the amino acid sequence set forth as SEQ
ID NO:152 as CDR1, the amino acid sequence set forth as SEQ ID
NO:154 as CDR2, and the amino acid sequence set forth as SEQ ID
NO:156 as CDR3.
[0127] 15. An anti-human transferrin receptor antibody, wherein the
amino acid sequence of each of CDR1, CDR2 and CDR3 in the light
chain and the heavy chain thereof has a homology not lower than 80%
to the amino acid sequence of the CDR1, CDR2 and CDR3, of one of
the combinations of the light chain and the heavy chain according
to 13 or 14 above.
[0128] 16. An anti-human transferrin receptor antibody, wherein the
amino acid sequence of each of CDR1, CDR2 and CDR3 in the light
chain and the heavy chain thereof has a homology not lower than 90%
to the amino acid sequence of the CDR1, CDR2 and CDR3, of one of
the combinations of the light chain and the heavy chain according
to 13 or 14 above.
[0129] 17. An anti-human transferrin receptor antibody, wherein 1
to 5 amino acids are substituted, deleted or added relative to the
amino acid sequence that forms at least one CDR in each of the
light chain and the heavy chain of one of the combinations of the
light chain and the heavy chain according to 13 or 14 above.
[0130] 18. An anti-human transferrin receptor antibody, wherein 1
to 3 amino acids are substituted, deleted or added relative to the
amino acid sequence that forms at least one CDR in each of the
light chain and the heavy chain of one of the combinations of the
light chain and the heavy chain according to 13 or 14 above.
[0131] 19. An anti-human transferrin receptor antibody, wherein the
light chain variable region of the antibody comprises an amino acid
sequence selected from the group consisting of the amino acid
sequences set forth as SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160,
SEQ ID NO:161, SEQ ID NO:162, and SEQ ID NO:163; and wherein the
heavy chain variable region of the antibody comprises an amino acid
sequence selected from the group consisting of the amino acid
sequences set forth as SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168,
SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:171.
[0132] 20. An anti-human transferrin receptor antibody, wherein the
light chain variable region of the antibody comprises an amino acid
sequence selected from the group consisting of the amino acid
sequences set forth as SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176,
SEQ ID NO:177, SEQ ID NO:178, and SEQ ID NO:179; and wherein the
heavy chain variable region of the antibody comprises an amino acid
sequence selected from the group consisting of the amino acid
sequences set forth as SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184,
SEQ ID NO:185, SEQ ID NO:186, and SEQ ID NO:187.
[0133] 21. An anti-human transferrin receptor antibody, wherein the
light chain variable region of the antibody comprises an amino acid
sequence selected from the group consisting of the amino acid
sequences set forth as SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192,
SEQ ID NO:193, SEQ ID NO:194, and SEQ ID NO:195, and wherein the
heavy chain variable region of the antibody comprises an amino acid
sequence selected from the group consisting of the amino acid
sequences set forth as SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206,
SEQ ID NO:207, SEQ ID NO:208, and SEQ ID NO:209.
[0134] 22. An anti-human transferrin receptor antibody selected
from the group consisting of (1) to (6) below:
[0135] (1) an antibody, wherein the light chain variable region
thereof comprises the amino acid sequence set forth as SEQ ID
NO:163; and wherein the heavy chain variable region thereof
comprises the amino acid sequence set forth as SEQ ID NO:171,
[0136] (2) an antibody, wherein the light chain variable region
thereof comprises the amino acid sequence set forth as SEQ ID
NO:179; and wherein the heavy chain variable region thereof
comprises the amino acid sequence set forth as SEQ ID NO:187,
[0137] (3) an antibody, wherein the light chain variable region
thereof comprises the amino acid sequence set forth as SEQ ID
NO:191; and wherein the heavy chain variable region thereof
comprises the amino acid sequence set forth as SEQ ID NO:205,
[0138] (4) an antibody, wherein the light chain variable region
thereof comprises the amino acid sequence set forth as SEQ ID
NO:193; and wherein the heavy chain variable region thereof
comprises the amino acid sequence set forth as SEQ ID NO:205,
[0139] (5) an antibody, wherein the light chain variable region
thereof comprises the amino acid sequence set forth as SEQ ID
NO:194; and wherein the heavy chain variable region thereof
comprises the amino acid sequence set forth as SEQ ID NO:205,
and
[0140] (6) an antibody, wherein the light chain variable region
thereof comprises the amino acid sequence set forth as SEQ ID
NO:195; and wherein the heavy chain variable region thereof
comprises the amino acid sequence set forth as SEQ ID NO:205.
[0141] 23. An anti-human transferrin receptor antibody selected
from the group consisting of (1) to (10) below:
[0142] (1) an antibody, wherein the light chain thereof comprises
the amino acid sequence set forth as SEQ ID NO:164; and wherein the
heavy chain thereof comprises the amino acid sequence set forth as
SEQ ID NO:172,
[0143] (2) an antibody, wherein the light chain thereof comprises
the amino acid sequence set forth as SEQ ID NO:180; and wherein the
heavy chain thereof comprises the amino acid sequence set forth as
SEQ ID NO:188,
[0144] (3) an antibody, wherein the light chain thereof comprises
the amino acid sequence set forth as SEQ ID NO:196; and wherein the
heavy chain thereof comprises the amino acid sequence set forth as
SEQ ID NO:210,
[0145] (4) an antibody, wherein the light chain thereof comprises
the amino acid sequence set forth as SEQ ID NO:198; and wherein the
heavy chain thereof comprises the amino acid sequence set forth as
SEQ ID NO:210,
[0146] (5) an antibody, wherein the light chain thereof comprises
the amino acid sequence set forth as SEQ ID NO:200; and wherein the
heavy chain thereof comprises the amino acid sequence set forth as
SEQ ID NO:210,
[0147] (6) an antibody, wherein the light chain thereof comprises
the amino acid sequence set forth as SEQ ID NO:202; and wherein the
heavy chain thereof comprises the amino acid sequence set forth as
SEQ ID NO:210,
[0148] (7) an antibody, wherein the light chain thereof comprises
the amino acid sequence set forth as SEQ ID NO:196; and wherein the
heavy chain thereof comprises the amino acid sequence set forth as
SEQ ID NO:212,
[0149] (8) an antibody, wherein the light chain thereof comprises
the amino acid sequence set forth as SEQ ID NO:198; and wherein the
heavy chain thereof comprises the amino acid sequence set forth as
SEQ ID NO:212,
[0150] (9) an antibody, wherein the light chain thereof comprises
the amino acid sequence set forth as SEQ ID NO:200; and wherein the
heavy chain thereof comprises the amino acid sequence set forth as
SEQ ID NO:212, and
[0151] (10) an antibody, wherein the light chain thereof comprises
the amino acid sequence set forth as SEQ ID NO:202; and wherein the
heavy chain thereof comprises the amino acid sequence set forth as
SEQ ID NO:212.
[0152] 24. An anti-human transferrin receptor antibody having a
homology not lower than 80% to the anti-human transferrin receptor
antibody according to any one of 19 to 23 above for the amino acid
sequence of the light chain variable region and the amino acid
sequence of the heavy chain variable region.
[0153] 25. An anti-human transferrin receptor antibody having a
homology not lower than 90% to the anti-human transferrin receptor
antibody according to any one of 19 to 23 above for the amino acid
sequence of the light chain variable region and the amino acid
sequence of the heavy chain variable region.
[0154] 26. An anti-human transferrin receptor antibody, wherein 1
to 10 amino acids are substituted, deleted or added relative to the
amino acid sequence that forms the light chain variable region of
the anti-human transferrin receptor antibody according to any one
of 19 to 23 above.
[0155] 27. An anti-human transferrin receptor antibody, wherein 1
to 3 amino acids are substituted, deleted or added relative to the
amino acid sequence that forms the light chain variable region of
the anti-human transferrin receptor antibody according to any one
of 19 to 23 above.
[0156] 28. An anti-human transferrin receptor antibody, wherein 1
to 10 amino acids are substituted, deleted or added relative to the
amino acid sequence that forms the heavy chain variable region of
the anti-human transferrin receptor antibody according to any one
of 19 to 23 above.
[0157] 29. An anti-human transferrin receptor antibody, wherein 1
to 3 amino acids are substituted, deleted or added relative to the
amino acid sequence that forms the heavy chain variable region of
the anti-human transferrin receptor antibody according to any one
of 19 to 23 above.
[0158] 30. An anti-human transferrin receptor antibody, wherein 1
to 10 amino acids are substituted, deleted or added relative to the
respective amino acid sequences that form the light chain variable
region and the heavy chain variable region of the anti-human
transferrin receptor antibody according to any one of 19 to 23
above.
[0159] 31. An anti-human transferrin receptor antibody, wherein 1
to 3 amino acids are substituted, deleted or added relative to the
respective amino acid sequences that form the light chain variable
region and the heavy chain variable region of the anti-human
transferrin receptor antibody according to any one of 19 to 23
above.
[0160] 32. The anti-human transferrin receptor antibody according
to any one of 1 to 31 above, wherein the antibody has an affinity
to both the extracellular region of human transferrin receptor and
the extracellular region of monkey transferrin receptor.
[0161] 33. The anti-human transferrin receptor antibody according
to 32 above, wherein the dissociation constant of its complex with
the extracellular region of human transferrin receptor is not
greater than 1.times.10.sup.-8 M, and the dissociation constant of
its complex with the extracellular region of monkey transferrin
receptor is not greater than 5.times.10.sup.-8M.
[0162] 34. The anti-human transferrin receptor antibody according
to any one of 1 to 33 above, wherein the antibody is Fab antibody,
F(ab').sub.2 antibody, or F(ab') antibody.
[0163] 35. The anti-human transferrin receptor antibody according
to any one of 1 to 33 above, wherein the antibody is a single-chain
antibody selected from the group consisting of scFab, scF(ab'),
scF(ab').sub.2 and scFv.
[0164] 36. The anti-human transferrin receptor antibody according
to 35 above, wherein the light chain and the heavy chain thereof
are linked via a linker sequence.
[0165] 37. The anti-human transferrin receptor antibody according
to 35 above, wherein the heavy chain is linked, via a linker
sequence, to the light chain on the C-terminal side thereof.
[0166] 38. The anti-human transferrin receptor antibody according
to 35 above, wherein the light chain is linked, via a linker
sequence, to the heavy chain on the C-terminal side thereof.
[0167] 39. The anti-human transferrin receptor antibody according
to any one of 36 to 38 above, wherein the linker sequence consists
of 8 to 50 amino acid residues.
[0168] 40. The anti-human transferrin receptor antibody according
to 39 above, wherein the linker sequence is selected from the group
consisting of the amino acid sequence Gly-Ser, the amino acid
sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the
amino acid sequences set forth as SEQ ID NO:3, SEQ ID NO:4, and SEQ
ID NO:5, and the amino acid sequence consisting of three
consecutively linked amino acid sequences each set forth as SEQ ID
NO:3.
[0169] 41. A fusion protein comprising the anti-human transferrin
receptor antibody according to any one of 1 to 40 above and the
amino acid sequence of a different protein (A) linked to the light
chain of the antibody on the C-terminal side or the N-terminal side
thereof.
[0170] 42. A fusion protein comprising an anti-human transferrin
receptor antibody and a different protein (A),
[0171] wherein the anti-human transferrin receptor antibody is the
anti-human transferrin receptor antibody according to any one of 1
to 40 above, and
[0172] wherein the different protein (A) is linked to the light
chain of the anti-human transferrin receptor antibody on the
C-terminal side or the N-terminal side thereof.
[0173] 43. The fusion protein according to 41 or 42 above, wherein
the different protein (A) is linked, via a linker sequence, to the
light chain on the C-terminal side or the N-terminal side
thereof.
[0174] 44. The fusion protein according to 43 above, wherein the
linker sequence consists of 1 to 50 amino acid residues.
[0175] 45. The fusion protein according to 44 above, wherein the
linker sequence comprises an amino acid sequence selected from the
group consisting of a single glycine, a single serine, the amino
acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the
amino acid sequence set forth as SEQ ID NO:3, the amino acid
sequence set forth as SEQ ID NO:4, the amino acid sequence set
forth as SEQ ID NO:5, and the amino acid sequences consisting of 1
to 10 thereof that are consecutively linked.
[0176] 46. A fusion protein comprising the anti-human transferrin
receptor antibody according to any one of 1 to 40 above and the
amino acid sequence of a different protein (A) linked to the heavy
chain of the antibody on the C-terminal side or the N-terminal side
thereof.
[0177] 47. A fusion protein of an anti-human transferrin receptor
antibody and a different protein (A),
[0178] wherein the anti-human transferrin receptor antibody is the
anti-human transferrin receptor antibody according to any one of 1
to 40 above, and
[0179] wherein the different protein (A) is linked to the heavy
chain of the anti-human transferrin receptor antibody on the
C-terminal side or the N-terminal side thereof.
[0180] 48. The fusion protein according to 46 or 47 above, wherein
the different protein (A) is linked, via a linker sequence, to the
heavy chain on the C-terminal side or the N-terminal side
thereof.
[0181] 49. The fusion protein according to 48 above, wherein the
linker sequence consists of 1 to 50 amino acid residues.
[0182] 50. The fusion protein according to 48 above, wherein the
linker sequence comprises an amino acid sequence selected from the
group consisting of a single glycine, a single serine, the amino
acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the
amino acid sequence set forth as SEQ ID NO:3, the amino acid
sequence set forth as SEQ ID NO:4, the amino acid sequence set
forth as SEQ ID NO:5, and the amino acid sequences consisting of 1
to 10 thereof that are consecutively linked.
[0183] 51. The fusion protein according to any one of 41 to 50
above, wherein the different protein (A) is a protein originating
from human.
[0184] 52. The fusion protein according to any one of 41 to 51
above, wherein the different protein (A) is selected from the group
consisting of nerve growth factor (NGF), lysosomal enzymes, ciliary
neurotrophic factor (CNTF), glial cell line-derived neurotrophic
factor (GDNF), neurotrophin-3, neurotrophin-4/5, neurotrophin-6,
neuregulin-1, erythropoietin, darbepoetin, activin, basic
fibroblast growth factor (bFGF), fibroblast growth factor 2 (FGF2),
epidermal growth factor (EGF), vascular endothelial growth factor
(VEGF), interferon .alpha., interferon .beta., interferon .gamma.,
interleukin 6, granulocyte-macrophage colony stimulating factor
(GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage
colony stimulating factor (M-CSF), cytokines, tumor necrosis factor
.alpha. receptor (TNF-.alpha. receptor), PD-1 ligands, enzymes
having .beta.-amyloid-degrading activity, anti-.beta.-amyloid
antibody, anti-BACE antibody, anti-EGFR antibody, anti-PD-1
antibody, anti-PD-L1 antibody, anti-HER2 antibody, anti-TNF-.alpha.
antibody, and other antibody medicines.
[0185] 53. The fusion protein according to any one of 41 to 51
above, wherein the different protein (A) is a lysosomal enzyme, and
wherein the lysosomal enzyme is selected from the group consisting
of .alpha.-L-iduronidase, iduronate 2-sulfatase,
glucocerebrosidase, .beta.-galactosidase, GM2 activator protein,
.beta.-hexosaminidase A, .beta.-hexosaminidase B,
N-acetylglucosamine-1-phosphotransferase, .alpha.-mannosidase,
.beta.-mannosidase, galactosylceramidase, saposin C, arylsulfatase
A, .alpha.-L-fucosidase, aspartylglucosaminidase,
.alpha.-N-acetylgalactosaminidase, acidic sphingomyelinase,
.alpha.-galactosidase A, .beta.-glucuronidase, heparan N-sulfatase,
.alpha.-N-acetylglucosaminidase, acetyl CoA:.alpha.-glucosaminide
N-acetyltransferase, N-Acetylglucosamine-6-sulfate sulfatase, acid
ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein
thioesterase 1, tripeptidyl-peptidase 1, hyaluronidase 1, CLN1 and
CLN2.
[0186] 54. The fusion protein according to any one of 41 to 51
above, wherein the different protein (A) is iduronate
2-sulfatase.
[0187] 55. The fusion protein according to 50 above, wherein the
different protein (A) is human iduronate 2-sulfatase, and wherein
the fusion protein is selected from (1) to (3) below:
[0188] (1) the fusion protein, wherein the light chain of the
humanized anti-hTfR antibody has the amino acid sequence set forth
as SEQ ID NO:164, and wherein the heavy chain of the humanized
anti-hTfR antibody is linked, on the C-terminal side thereof and
via a linker sequence Gly-Ser, to the human iduronate 2-sulfatase,
and the whole linked heavy chain has the amino acid sequence set
forth as SEQ ID NO:247.
[0189] (2) the fusion protein, wherein the light chain of the
humanized anti-hTfR antibody has the amino acid sequence set forth
as SEQ ID NO:180, and wherein the heavy chain of the humanized
anti-hTfR antibody is linked, on the C-terminal side thereof and
via a linker sequence Gly-Ser, to the human iduronate 2-sulfatase,
and the whole linked heavy chain has the amino acid sequence set
forth as SEQ ID NO:249, and
[0190] (3) the fusion protein, wherein the light chain of the
humanized anti-hTfR antibody has the amino acid sequence set forth
as SEQ ID NO:196. and wherein the heavy chain of the humanized
anti-hTfR antibody is linked, on the C-terminal side thereof and
via a linker sequence Gly-Ser, to the human iduronate 2-sulfatase,
and the whole linked heavy chain has the amino acid sequence set
forth as SEQ ID NO:251.
[0191] 56. The fusion protein according to 50 above, wherein the
different protein (A) is human iduronate 2-sulfatase, and wherein
the fusion protein is selected from (1) to (3) below:
[0192] (1) the fusion protein comprising: the humanized anti-hTfR
antibody light chain having the amino acid sequence set forth as
SEQ ID NO:164; and the humanized anti-hTfR antibody heavy chain
having the amino acid sequence set forth as SEQ ID NO:172 which is
linked, on the C-terminal side thereof and via a linker sequence
Gly-Ser, to the human iduronate 2-sulfatase set forth as SEQ ID
NO:246;
[0193] (2) the fusion protein comprising: the humanized anti-hTfR
antibody light chain having the amino acid sequence set forth as
SEQ ID NO:180; and the humanized anti-hTfR antibody heavy chain
having the amino acid sequence set forth as SEQ ID NO:188 which is
linked, on the C-terminal side thereof and via a linker sequence
Gly-Ser, to the human iduronate 2-sulfatase set forth as SEQ ID
NO:246;
[0194] (3) the fusion protein comprising: the humanized anti-hTfR
antibody light chain having the amino acid sequence set forth as
SEQ ID NO:196; and the humanized anti-hTfR antibody heavy chain
having the amino acid sequence set forth as SEQ ID NO:210 which is
linked, on the C-terminal side thereof and via a linker sequence
Gly-Ser, to the human iduronate 2-sulfatase set forth as SEQ ID
NO:246.
[0195] 57. The fusion protein according to 50 above, wherein the
different protein (A) is human iduronate 2-sulfatase, and wherein
the fusion protein is selected from (1) to (3) below:
[0196] (1) the fusion protein comprising: the human iduronate
2-sulfatase linked via a linker sequence Gly-Ser to the heavy chain
on the C-terminal side thereof; and the light chain, wherein the
amino acid sequence of the former is set forth as SEQ ID NO:247,
and the amino acid sequence of the latter is set forth as SEQ ID
NO:164,
[0197] (2) the fusion protein comprising: the human iduronate
2-sulfatase linked via a linker sequence Gly-Ser to the heavy chain
on the C-terminal side thereof; and the light chain, wherein the
amino acid sequence of the former is set forth as SEQ ID NO:249,
and the amino acid sequence of the latter is set forth as SEQ ID
NO:180, and
[0198] (3) the fusion protein comprising: the human iduronate
2-sulfatase linked via a linker sequence Gly-Ser to the heavy chain
on the C-terminal side thereof; and the light chain, wherein the
amino acid sequence of the former is set forth as SEQ ID NO:251,
and the amino acid sequence of the latter is set forth as SEQ ID
NO:196.
[0199] 58. A DNA fragment encoding the amino acid sequence of the
anti-human transferrin receptor antibody according to any one of 1
to 40 above.
[0200] 59. A DNA fragment encoding the amino acid sequence of the
fusion protein according to any one of 41 to 57 above.
[0201] 60. An expression vector comprising the DNA fragment
according to 58 or 59 above that is incorporated therein.
[0202] 61. A mammalian cell transformed with the expression vector
according to 60 above.
[0203] 62. An anti-human transferrin receptor
antibody-pharmacologically active compound complex, wherein the
light chain and/or the heavy chain of the anti-human transferrin
receptor antibody according to any one of 1 to 40 above is linked
to a low-molecular-weight pharmacologically active compound that
needs to be allowed to pass through the blood-brain barrier and
exhibit the function thereof in the brain.
[0204] 63. The anti-human transferrin receptor antibody according
to 62 above, wherein the pharmacologically active compound is
selected from the group consisting of anticancer drug, therapeutic
agent for Alzheimer's disease, therapeutic agent for Parkinson's
disease, therapeutic agent for Huntington's disease, therapeutic
agent for schizophrenia, antidepressant, therapeutic agent for
multiple sclerosis, therapeutic agent for amyotrophic lateral
sclerosis, therapeutic agent for tumors of central nervous system
including brain tumor, therapeutic agent for lysosomal storage
disease accompanied by encephalopathy, therapeutic agent for
glycogenosis, therapeutic agent for muscular dystrophy, therapeutic
agent for cerebral ischemia, therapeutic agent for prion diseases,
therapeutic agent for traumatic central nervous system disorders,
therapeutic agent for viral and bacterial central nervous system
diseases, pharmaceutical agent used for recovery after brain
surgery, pharmaceutical agent used for recovery after spinal
surgery, siRNA, antisense DNA, and peptide.
[0205] 64. Use of the anti-human transferrin receptor antibody
according to any one of 1 to 40 above for allowing the different
protein (A) or a low-molecular-weight pharmacologically active
compound to pass through the blood-brain barrier and exhibit the
function thereof in the brain.
[0206] 65. Use of the anti-human transferrin receptor antibody
according to any one of 1 to 40 above for the manufacture of a
medicament for parenteral administration for the treatment of a
disease condition of the central nervous system, by linking thereto
the molecule of a physiologically active protein or a
pharmacologically active low-molecular-weight compound for the
disease condition.
[0207] 66. A method for treatment of a disorder of the central
nervous system comprising parenterally administering to a patient
with the disorder a therapeutically effective amount of the
physiologically active protein, or pharmacologically active
low-molecular-weight compound, for the disorder, in the form of a
conjugate with the molecule of the anti-human transferrin receptor
antibody according to any one of 1 to 40 above.
[0208] 67. Use of the anti-human transferrin receptor antibody
according to any one of 54 to 57 above for making human iduronate
2-sulfatase pass through the blood-brain barrier and exhibit the
function thereof in the brain.
[0209] 68. Use of the fusion protein according to any one of 54 to
57 for the production of a pharmaceutical agent for parenteral
administration for the treatment of a disease condition of the
central nervous system accompanying Hunter syndrome.
[0210] 69. A method for the treatment of a disease of the central
nervous system accompanying Hunter syndrome comprising parenterally
administering a therapeutically effective amount of the fusion
protein according to any one of 54 to 57 above to a patient with
the disease.
Effects of the Invention
[0211] By the present invention, various compounds, such as
proteins and low-molecular-weight compounds that, although
physiologically or pharmacologically active, have been unusable by
parenteral administration because of their no or little ability to
pass through the blood-brain barrier, can be provided in the form
that allow them to pass through the blood-brain barrier, thus
making them new pharmaceutical agents for parenteral administration
for the treatment of a disease condition of the central nervous
system.
BRIEF DESCRIPTION OF DRAWINGS
[0212] FIG. 1 Substitute photographs for drawings showing the
result of the immunohistochemical staining of the anti-hTfR
antibody in the cerebral cortex of a cynomolgus monkey after a
single intravenous administration of the anti-hTfR antibody. (a)
anti-hTfR antibody not administered, (b) anti-hTfR antibody No. 1
administered, (c) anti-hTfR antibody No. 2 administered, (d)
anti-hTfR antibody No. 3 administered. The bar at the bottom right
in each photograph is a 50-.mu.m gauge.
[0213] FIG. 2 A figure showing the result of the
immunohistochemical staining of the anti-hTfR antibody in the
hippocampus of a cynomolgus monkey after a single intravenous
administration of the anti-hTfR antibody. (a) anti-hTfR antibody
non-administered, (b) anti-hTfR antibody No. 1 administered, (c)
anti-hTfR antibody No. 2 administered, (d) anti-hTfR antibody No. 3
administered. The bar at the bottom right in each photograph is a
50-.mu.m gauge.
[0214] FIG. 3 Substitute photographs for drawings showing the
result of the immunohistochemical staining of the anti-hTfR
antibody in the cerebellum of a cynomolgus monkey after a single
intravenous administration of the anti-hTfR antibody. (a) anti-hTfR
antibody not administered, (b) anti-hTfR antibody No. 1
administered, (c) anti-hTfR antibody No. 2 administered, (d)
anti-hTfR antibody No. 3 administered. The bar at the bottom right
in each photograph is a 50-.mu.m gauge.
[0215] FIG. 4 A figure showing the amount of a humanized anti-hTfR
antibody accumulated in various organs other than the brain of a
cynomolgus monkey after a single intravenous administration. The
vertical axis indicates the amount of the humanized anti-hTfR
antibody (.mu.g/g wet weight) per wet weight of each organ. The
white bars represent, from the left, the amount accumulated in each
organ of the monkey after administration of humanized anti-hTfR
antibody No. 3, humanized anti-hTfR antibody No. 3-2, humanized
anti-hTfR antibody No. 3 (IgG4), and humanized anti-hTfR antibody
No. 3-2 (IgG4), respectively, and the black bars represent the
amount accumulated in respective organs of the monkey after
administration of trastuzumab (Herceptin.TM.). "ND" denotes "not
detected".
[0216] FIG. 5 Substitute photographs for drawings showing the
result of immunohistochemical staining of a humanized anti-hTfR
antibody in the cerebral cortex of a cynomolgus monkey after a
single intravenous administration. (a) Herceptin administered, (b)
humanized anti-hTfR antibody No. 3 administered, (c) humanized
anti-hTfR antibody No. 3-2 administered, (d) humanized anti-hTfR
antibody No. 3 (IgG4) administered, (e) humanized anti-hTfR
antibody No. 3-2 (IgG4) administered. The bar at the bottom right
in each photograph is a 20-.mu.m gauge.
[0217] FIG. 6 Substitute photographs for drawing showing the result
of immunohistochemical staining of a humanized anti-hTfR antibody
in the hippocampus of a cynomolgus monkey after a single
intravenous administration. (a) Herceptin administered, (b)
humanized anti-hTfR antibody No. 3 administered, (c) humanized
anti-hTfR antibody No. 3-2 administered, (d) humanized anti-hTfR
antibody No. 3 (IgG4) administered, (e) humanized anti-hTfR
antibody No. 3-2 (IgG4) administered. The bar at the bottom right
at each photograph is a 20-.mu.m gauge.
[0218] FIG. 7 A figure showing the result of immunohistochemical
staining of humanized anti-hTfR antibody in the cerebellum of a
cynomolgus monkey after a single intravenous administration. (a)
Herceptin administered, (b) humanized anti-hTfR antibody No. 3
administered, (c) humanized anti-hTfR antibody No. 3-2
administered, (d) humanized anti-hTfR antibody No. 3 (IgG4)
administered, (e) humanized anti-hTfR antibody No. 3-2 (IgG4)
administered. The bar at the bottom right at each photograph is a
20-.mu.m gauge.
[0219] FIG. 8 A figure showing the result of immunohistochemical
staining of humanized anti-hTfR antibody in the medulla oblongata
of a cynomolgus monkey after a single intravenous administration.
(a) Herceptin administered, (b) humanized anti-hTfR antibody No. 3
administered, (c) humanized anti-hTfR antibody No. 3-2
administered, (d) humanized anti-hTfR antibody No. 3 (IgG4)
administered, (e) humanized anti-hTfR antibody No. 3-2 (IgG4)
administered. The bar at the bottom right at each photograph is a
20-.mu.m gauge.
[0220] FIG. 9 A figure showing the amount of I2S-anti-hTfR antibody
and rhI2S accumulated in brain tissues of a cynomolgus monkey after
a single intravenous administration. The vertical axis indicates
the amount of I2S-anti-hTfR antibody and rhI2S per wet weight of
the brain tissues (.mu.g/g wet weight). The dotted bars represent
the amount of rhI2S accumulated in the brain tissues of the
cynomolgus monkey. The hatched bars represent the amount of the
I2S-anti-hTfR antibody accumulated in the brain tissues of the
cynomolgus monkey. Vertical line segment indicates the standard
deviation.
[0221] FIG. 10 Graphs showing the amount of the glycosaminoglycan
(GAG) in various organs of a I2S gene-knockout mouse (I2S-KO mouse)
intravenously injected with a humanized anti-hTfR antibody or
rhI2S. (a) brain, (b) liver, (c) lung, (d) heart. For each graph,
the bars represent, from the left, wild-type mouse (WT), control
group (non-administered group), 0.5 mg/kg administered group, 1.0
mg/kg administered group, and 2.0 mg/kg administered group. The
vertical axis indicates the amount of GAG of the dry weight of each
organ (.mu.g/g dry weight). Vertical line segments indicates the
standard deviation, and "**" indicates p<0.01 compared with the
control group according to Dunnett's test.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0222] In the present invention, the term "antibody" refers mainly
to a human antibody, mouse antibody, humanized antibody, as well as
a chimeric antibody between human antibody and non-human mammalian
antibody, and a chimeric antibody between mouse antibody and
non-mouse mammalian antibody, but the meaning of the term is not
limited to them insofar as a substance of interest has a property
to specifically bind to a certain antigen, and there is no specific
limitation as to the animal species of the antibody, either.
[0223] In the present invention, the term "human antibody" refers
to an antibody whose entire protein is encoded by a gene
originating from human. The term "human antibody", however, also
includes an antibody encoded by a gene obtained by introducing a
mutation into an original human gene for a purpose of enhancing
expression efficiency of the gene, for example, without modifying
the original amino acid sequence. The term "human antibody" also
includes an antibody which is obtainable through combining two or
more genes encoding human antibodies by replacing a certain part of
a human antibody with a part of another human antibody. A human
antibody includes three complementarity determining regions (abbr.
CDRs) in the light chain of the immunoglobulin and three
complementarity determining regions (CDRs) in the heavy chain of
the immunoglobulin. The three CDRs in the light chain of the
immunoglobulin are called, from the N-terminal side, CDR1, CDR2 and
CDR3, respectively. The three CDRs in the heavy chain of the
immunoglobulin are also called, from the N-terminal side, CDR1,
CDR2 and CDR3, respectively. The team "human antibody" also
includes a human antibody produced by replacing a CDR of a human
antibody with a CDR of another human antibody to modify such
properties as the antigen specificity and the affinity of the
original human antibodies, etc.
[0224] In the present invention, the tem "human antibody" also
includes an antibody which is produced through modification of the
gene of the original human antibody by introducing a mutation, such
as substitution, deletion, addition, to the amino acid sequence of
the original antibody. When replacing one or more amino acids of
the amino acid sequence of the original antibody with other amino
acids, the number of amino acid replaced may preferably be 1 to 20,
more preferably 1 to 5, and still more preferably 1 to 3. When
deleting one or more amino acids of the amino acid sequence of the
original antibody, the number of amino acids deleted may preferably
be 1 to 20, more preferably 1 to 5, and still more preferably 1 to
3. An antibody produced by a combined mutation of these
substitution and deletion of amino acids is also a "human
antibody". In some cases, one or more amino acids, preferably 1 to
20, more preferably 1 to 5, and still more preferably 1 to 3 amino
acids may be added inside the amino acid sequence of the original
antibody or on its N- or C-terminal side. An antibody produced by a
combined mutation of addition, substitution, and deletion of amino
acids is also a "human antibody". The amino acid sequence of such a
mutated antibody has a homology of preferably not lower than 80%,
more preferably not lower than 90%, still more preferably not lower
than 95%, and even more preferably not lower than 98%, to the amino
acid sequence of the original antibody. Thus, in the present
invention, the term "gene originating from human" includes not only
the unmutated gene originating from human but also a gene produced
by modifying it.
[0225] The homology between the amino acid sequence of an unmutated
antibody and the amino acid sequence of an antibody produced by
introducing a mutation into it may be readily calculated using
well-known homology calculator algorithms. As such algorithms,
there are, for example, BLAST (Altschul S F. J Mol. Biol. 215.
403-10 (1990)), a similarity search by Pearson and Lipman (Proc.
Natl. Acad. Sci. USA. 85. 2444 (1988)), and the local homology
algorithm of Smith and Waterman (Adv. Appl. Math. 2. 482-9 (1981)),
and the like.
[0226] The term "mouse antibody" refers to an antibody whose entire
protein consists of an amino acid sequence which is the same as an
antibody encoded by a gene originating from a mouse. Therefore, the
term "mouse antibody" also includes an antibody that is encoded by
a gene produced by introducing a mutation into the original mouse
gene without causing a change in its amino acid sequence but in
order, for example, to improve the expression efficiency of the
gene. Further, the term "mouse antibody" also includes an antibody
produced through combining two or more genes encoding mouse
antibodies by replacing a part of a mouse antibody with a part of
another mouse antibody. A mouse antibody has three complementarity
determining regions (CDRs) in the light chain of the immunoglobulin
and three complementarity determining regions (CDRs) in the heavy
chain of the immunoglobulin. The three CDRs in the light chain of
the immunoglobulin are called, from the N-terminal side, CDR1, CDR2
and CDR3, respectively. The three CDRs in the heavy chain of the
immunoglobulin are also called, from the N-terminal side, CDR1,
CDR2 and CDR3, respectively. The term "mouse antibody" also
includes an antibody produced by replacing a CDR of a mouse
antibody with a CDR of another mouse antibody to modify the
specificity and affinity of the original mouse antibodies.
[0227] In the present invention, the term "mouse antibody" also
includes an antibody which is produced through modification of the
gene of the original mouse antibody by introducing a mutation, such
as substitution, deletion, addition, to the amino acid sequence of
the original antibody. When replacing one or more amino acids of
the amino acid sequence of the original antibody with other amino
acids, the number of amino acid replaced may preferably be 1 to 20,
more preferably 1 to 5, and still more preferably 1 to 3. When
deleting one or more amino acids of the amino acid sequence of the
original antibody, the number of amino acids deleted may preferably
be 1 to 20, more preferably 1 to 5, and still more preferably 1 to
3. An antibody produced by a combined mutation of these
substitution and deletion of amino acids is also a "mouse
antibody". When adding one or more amino acids, they may be added
inside the amino acid sequence of the original antibody or on its
N- or C-terminal side, preferably 1 to 20, more preferably 1 to 5,
and still more preferably 1 to 3, in number. An antibody produced
by a combined mutation of addition, substitution, and deletion of
amino acids is also a "mouse antibody". The amino acid sequence of
such a mutated antibody has a homology of preferably not lower than
80%, more preferably not lower than 90%, still more preferably not
lower than 95%, and even more preferably not lower than 98%, to the
amino acid sequence of the original antibody. Thus, in the present
invention, the term "gene originating from mouse" includes not only
the unmutated gene originating from mouse but also a gene produced
by modifying it.
[0228] In the present invention, the term "humanized antibody"
refers to an antibody in which part of the amino acid sequence of
its variable region (e.g., especially the whole or part of its
CDRs) originates from a non-human mammal while the rest originates
from human. An example of humanized antibody is an antibody
produced by replacing the three complementarily determining regions
(CDRs) of the light chain of the immunoglobulin and the three
complementarity determining regions (CDRs) of the heavy chain of
the immunoglobulin constituting a human antibody, with CDRs from a
non-human mammal. As far as it originates from a non-human mammal,
there is no particular limitation as to the biological species from
which those CDRs originate that are grafted into a proper position
of the human antibody, though preferred are mouse, rat, rabbit,
horse or non-human primate, more preferred are mouse and rat, and
mouse, for example.
[0229] In the present invention, the term "chimeric antibody"
refers to an antibody produced by connecting fragments of two or
more different antibodies originating from two or more different
species.
[0230] A chimeric antibody between a human antibody and a non-human
mammalian antibody is an antibody provided by replacing part of a
human antibody with part of a non-human mammalian antibody. As
explained below, an antibody is made of an Fc region, a Fab region
and a hinge region. A specific example of such chimeric antibodies
is a chimeric antibody whose Fc region originates from a human
antibody while its Fab region originates from a non-human mammalian
antibody. The hinge region either originates from a human antibody
or from a non-human mammalian antibody. On the contrary, the term
chimeric antibody also includes one whose Fc region originates from
a non-human mammalian antibody while its Fab region originates from
a human antibody. In such a case also, the hinge region may either
originate from a human antibody or from a non-human mammalian
antibody. On the contrary, a chimeric antibody is also included as
an example whose Fc region originates from a non-human mammalian
antibody while its Fab region originates from a human antibody. In
this case also, the hinge region may either originate from a human
antibody or from a non-human mammalian antibody.
[0231] An antibody can be viewed as composed of a variable region
and a constant region. Additional examples of chimeric antibodies
include an antibody in which the heavy chain constant region
(C.sub.H) and the light chain constant region (C.sub.1) both
originate from a human antibody while the heavy chain variable
region (V.sub.H) and the light chain variable region (V.sub.L) both
originate from an antibody of a non-human mammal, and conversely,
an antibody in which the heavy chain constant region (C.sub.H) and
the light chain constant region (C.sub.1) both originate from an
antibody of a non-human mammal, while the heavy chain variable
region (V.sub.H) and the light chain variable region (V.sub.L) both
originate from a human antibody. In these, there is no particular
limitation as to the biological species of the non-human mammal, as
far as it is a non-human mammal, though preferred are mouse, rat,
rabbit, horse or non-human primate, and more preferred is
mouse.
[0232] A chimeric antibody between a mouse antibody and a non-mouse
mammalian antibody is an antibody provided by replacing part of a
mouse antibody with part of a non-mouse mammalian antibody.
Specific examples of such chimeric antibodies include a chimeric
antibody whose Fc region originates from a mouse antibody while its
Fab region originates from a non-mouse mammalian antibody, and
conversely, a chimeric antibody whose Fc region originates from a
non-mouse mammal while its Fab region originates from a mouse
antibody. In these, there is no particular limitation as to the
biological species of the non-mouse mammal, as far as it is a
mammal other than mouse, though preferred are rat, rabbit, horse or
non-human primate, and more preferred is human.
[0233] A chimeric antibody between a human antibody and a mouse
antibody is designated in particular "human/mouse chimeric
antibody". Examples of human/mouse chimeric antibodies include a
chimeric antibody in which the Fc region originates from a human
antibody while the Fab region originates from a mouse antibody, and
conversely, a chimeric antibody whose Fc region originates from
mouse antibody, while its Fab region originates from a human
antibody. A hinge region either originate from a human antibody or
a mouse antibody.
[0234] Additional specific examples of human/mouse chimeric
antibodies include those whose heavy chain constant region
(C.sub.H) and light chain constant region (C.sub.1) originate from
a human antibody while its heavy chain variable region (V.sub.H)
and light chain variable region (V.sub.L) originate from a mouse
antibody, and conversely, those whose heavy chain constant region
(C.sub.H) and light chain constant region (C.sub.1) originate from
a mouse antibody while its heavy chain variable region (V.sub.H)
and light chain variable region (V.sub.L) originate from a human
antibody.
[0235] Originally, an antibody is of the basic structure having
four polypeptide chains in total consisting of two immunoglobulin
light chains and two immunoglobulin heavy chains. However, in the
present invention the term "antibody" refers, besides an antibody
having this basic structure, also to:
[0236] (1) one consisting of two polypeptide chains: a single
immunoglobulin light chain and a single immunoglobulin heavy chain,
and also, as explained later,
[0237] (2) a single-chain antibody consisting of an immunoglobulin
light chain which is linked, on the C-terminal side thereof, to a
linker sequence which in turn is linked, on the C-terminal side
thereof, to an immunoglobulin heavy chain,
[0238] (3) single-chain antibodies consisting of an immunoglobulin
heavy chain which is linked, on the C-terminal side thereof, to a
linker sequence which in turn is linked, on the C-terminal side
thereof, to an immunoglobulin light chain, and
[0239] (4) one consisting of a Fab region, i.e., a structure left
behind by removal of the Fc region from an antibody having the
basic structure, as the original meaning, and one consisting of the
Fab region and the whole or part of the hinge region (including
Fab, F(ab'), and F(ab').sub.2) also are included in the term
"antibody" in the present invention.
[0240] The term "Fab" refers to a molecule consisting of a single
light chain comprising the variable region and the C.sub.L region
(light chain constant region) and a single heavy chain comprising
the variable region and the C.sub.H1 region (portion 1 of heavy
chain constant region) which are combined by a disulfide bond
between their respective cysteine residues. While the heavy chain
in a Fab can include part of the hinge region in addition to the
variable region and the C.sub.H1 region (portion 1 of heavy chain
constant region), the hinge region in such a case lacks the
cysteine residue that otherwise is present in the hinge region and
would serve to link two heavy chains of an antibody together. In
Fab, the light chain and the heavy chain are connected by a
disulfide bond formed between the cysteine residue present in the
light chain constant region (C.sub.L region) and the cysteine
residue located in the heavy chain constant region (C.sub.H1
region) or the hinge region. As it lacks the cysteine residue in
the hinge region which serves to bind two heavy chains of an
antibody, Fab consists of a single light chain and a single heavy
chain. The light chain constituting Fab includes a variable region
and a C.sub.L region. The heavy chain as a component of Fab may
either consist of a variable region and a C.sub.H1 region or also
of part of the hinge region in addition to the variable region and
the C.sub.H1 region. However, in the letter case, the hinge region
is so selected as not to include the cysteine residue that could
bind two heavy chains, in order to avoid the formation of a
disulfide bond between two heavy chains at their hinge regions. In
F(ab'), the heavy chain includes, in addition to a variable region
and a C.sub.H1 region, the whole or part of a hinge region
containing a cysteine residue that could bind two heavy chains.
F(ab').sub.2 is a molecule consisting of two F(ab')s bound together
through a disulfide bond Ruined between the cysteine residues
present in their respective hinge regions. Further, a polymer such
as a dimer and a trimer, which consists of two or more antibodies
connected with each other, directly or via a linker, is also
included in the term "antibody". Moreover, in addition to the
aforementioned, any molecule that includes part of an
immunoglobulin molecule and has a property to specifically bind to
the antigen is also included in the term "antibody" in the present
invention. Thus, in the present invention, the term "immunoglobulin
light chain" includes a molecule that is derived from an original
immunoglobulin light chain and having the amino acid sequence of
the whole or part of its variable region. Likewise, the term
"immunoglobulin heavy chain" includes a molecule that is derived
from an original immunoglobulin heavy chain and having the amino
acid sequence of the whole or part of its variable region.
Therefore, insofar as having the whole or part of the amino acid
sequence of the variable region, a molecule is included in the term
"immunoglobulin heavy chain", even if it lacks its Fc region, for
example.
[0241] In the above, the term "Fc" or "Fc region" refers to a
region comprising a fragment consisting of C.sub.H2 region (portion
2 of the heavy chain constant region), and C.sub.H3 region (portion
3 of the heavy chain constant region) in the antibody molecule.
[0242] Furthermore, in the present invention, the term "antibody"
also includes:
[0243] (5) scFab, scF(ab'), and scF(ab').sub.2, which are
single-chain antibodies produced by binding the light chain to the
heavy chain that form, respectively, the Fab, F(ab') and
F(ab').sub.2 mentioned in (4) above, via a linker sequence. Such
scFab, scF(ab') and scF(ab').sub.2 may be a molecule in which
either the light chain is linked, on the C-terminal side thereof,
to a linker sequence, which in turn is linked, on the C-terminal
side thereof, to the heavy chain, or the heavy chain is linked, on
the C-terminal side thereof, to a linker sequence, which in turn is
linked, on the C-terminal side thereof, to the light chain.
Furthermore, a scFv, which is a single-chain antibody provided by
binding the light chain variable region to the heavy chain variable
region, via a linker sequence between them, is also included in the
term "antibody" in the present invention. Such scFv may be a
molecule in which either the light chain variable region is linked,
on the C-terminal side thereof; to a linker sequence, which in turn
is linked, on the C-terminal side thereof, to the heavy chain
variable region, or the heavy chain variable region is linked, on
the C-terminal side thereof, to a linker sequence, which in turn is
linked, on the C-terminal side thereof, to the light chain variable
region.
[0244] Furthermore, in addition to a full-length antibody and those
described in (1) to (5) above, the term "antibody" in the present
specification includes, any form of antigen-binding fragment which
lacks part of the full-length antibody (antibody fragment), a
broader concept which includes (4) and (5) above.
[0245] The term "antigen-binding fragment" refers to an antibody
fragment that retains at least part of the specific binding
activity to its antigen. In addition to those described above in
(4) and (5), examples of binding fragments include Fab, Fab',
F(ab').sub.2, variable region (Fv); a single-chain antibody (scFv)
produced by linking the heavy chain variable region (V.sub.H) and
the light chain variable region (V.sub.L), via a proper linker
between them; a diabody, which is a dimer of a polypeptide that
comprises a heavy chain variable region (V.sub.H) and a light chain
variable region (V.sub.L); a minibody, which is a dimer of a
molecule in which the heavy chain (H chain) of a scFv is linked to
part of the constant region (C.sub.H3), and other
low-molecular-antibodies. However, as far as it has an
antigen-binding ability, the term is not limited to these
molecules. Such binding fragments include not only those produced
by treating a full-length molecule of an antibody protein with a
proper enzyme but also those produced by proper host cells using a
genetically engineered antibody gene.
[0246] In the present invention, the term "single-chain antibody"
refers to a protein in which an amino acid sequence comprising the
whole or part of an immunoglobulin light chain variable region
linked, on the C-terminal side thereof, to a linker sequence, which
in turn is linked, on the C-terminal side thereof, to the amino
acid sequence of the whole or part of an immunoglobulin heavy chain
variable region, and having an ability to specifically bind a
certain antigen. For example, those described in (2), (3) and (5)
are included in "single-chain antibody". Further, a protein in
which an amino acid sequence comprising the whole or part of an
immunoglobulin heavy chain variable region is linked, on the
C-terminal side thereof, to a linker sequence, which in turn is
further linked, on the C-terminal side thereof, to the amino acid
sequence of the whole or part of an immunoglobulin light chain
variable region, and which has an ability to specifically bind to a
certain antigen, is also included in the term "single-chain
antibody" in the present invention. In a single-chain antibody in
which an immunoglobulin heavy chain is linked, on the C-terminal
side thereof and via a linker sequence, to an immunoglobulin light
chain, the immunoglobulin heavy chain generally lacks the Fc
region. An immunoglobulin light chain variable region has three
complementarity determining regions (CDRs) which participate in
determining the antigen specificity of an antibody. Likewise, an
immunoglobulin heavy chain variable region also has three CDRs.
Those CDRs are the primary regions that determine the antigen
specificity of an antibody. Therefore, a single-chain antibody
preferably contains all the three CDRs of the immunoglobulin heavy
chain and all the three CDRs of the immunoglobulin light chain.
However, it is also possible to provide a single-chain antibody in
which one or more of those CDRs are deleted, insofar as the
antigen-specific affinity of the antibody is retained.
[0247] In a single-chain antibody, the linker sequence placed
between the light chain and the heavy chain of the immunoglobulin
is preferably a peptide chain consisting of preferably 2 to 50,
more preferably 8 to 50, still more preferably 10 to 30, even more
preferably 12 to 18, or 15 to 25, for example 15 or 25 amino acid
residues. While there is no particular limitation as to the
specific amino acid sequence of such a linker sequence insofar as
the anti-hTfR antibody comprising the both chains linked thereby
retains the affinity to hTfR, it is preferably made of glycine
only, or of glycine and serine: for example the amino acid sequence
Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid
sequence Gly-Gly-Gly, the amino acid sequence Gly-Gly-Gly-Gly-Ser
(SEQ ID NO:3), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ
ID NO:4), the amino acid sequence Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID
NO:5), or a sequence which includes 2 to 10 or 2 to 5 repeats of
any of those amino acid sequences. For example, in linking the
amino acid sequence of the entire immunoglobulin heavy chain
variable region, on the C-terminal side thereof and via a linker
sequence, to immunoglobulin light chain variable region, the linker
sequence is preferably a linker sequence comprising 15 amino acids
corresponding to three of the amino acid sequence
Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3) consecutively linked.
[0248] In the present invention, the term "human transferrin
receptor" or "hTfR" refers to a membrane protein having the amino
acid sequence set forth as SEQ ID NO:1. The anti-hTfR antibody of
the present invention is, in one of its embodiments, that which
specifically binds to the region from the cysteine residue at the
position 89th from the N-terminal side to the phenylalanine at the
C-terminus in the amino acid sequence set forth as SEQ ID NO:1
(i.e., the extracellular region of the hTfR), though it is not
limited to this embodiment. Further, in the present invention, the
term "monkey transferrin receptor" or "monkey TfR" refers in
particular to the membrane protein having the amino acid sequence
set forth as SEQ ID NO:2, originating from cynomolgus monkey
(Macaca fascicularis). The anti-hTfR antibody of the present
invention is, in one of its embodiments, that which binds also to
region from the cysteine residue at the position 89th from the
N-terminal side to the phenylalanine at the C-terminus in the amino
acid sequence set forth as SEQ ID NO:2 (i.e., the extracellular
region of the monkey TfR), though it is not limited to this
embodiment.
[0249] For preparation of an antibody to hTfR, there is known a
general method according to which a recombinant human transferrin
receptor (rhTfR) is produced using cells which have an introduced
expression vector having an incorporated hTfR gene, and then
animals such as mice are immunized with this rhTfR. By collecting
those cells which produce antibodies to hTfR from the immunized
animals and fusing them with myeloma cells, hybridoma cells can be
obtained having an ability to produce the antibody.
[0250] Further, cells producing an antibody to hTfR can also be
obtained by collecting immunocompetent cells from an animal such as
mouse, and immunizing them with rhTfR by in vitro immunization. In
conducting in vitro immunization, there is no particular limitation
as to the animal species from which the immunocompetent cells are
derived, though preferred are mouse, rat, rabbit, guinea pig, dog,
cat, horse, and primates including human, and more preferred are
mouse, rat and human, and still more preferably mouse and human. As
mouse immunocompetent cells, spleen cells prepared from mouse
spleen may be used, for example. As human immunocompetent cells,
such cells can be used as prepared from human peripheral blood,
bone marrow, spleen, and the like. By immunizing human
immunocompetent cells according to in vitro immunization, a human
antibody to hTfR can be obtained.
[0251] After immunizing the immunocompetent cells according to in
vitro immunization, the cells can be fused with myeloma cells to
prepare hybridoma cells having an ability to produce the antibody.
Further, it is also possible to extract mRNAs from the immunized
cells, synthesize cDNA, perform PCR reaction using the cDNA as a
template to amplify a DNA fragment containing the gene encoding the
light chain and the heavy chain of the immunoglobulin, and
artificially reconstruct the antibody gene using them.
[0252] The hybridoma cells freshly obtained above also include such
cells that produce antibodies that recognize other proteins than
hTfR. Furthermore, not all the hybridoma cells producing an
anti-hTfR antibody necessarily produce an anti-hTfR antibody that
exhibits high affinities to hTfR.
[0253] Likewise, artificially reconstructed antibody genes include
such genes as encode antibodies recognizing other proteins than
hTfR as antigens. Moreover, not all the genes encoding anti-hTfR
antibodies necessarily have desired properties such as encoding an
anti-hTfR antibody exhibiting high affinity to hTfR.
[0254] Therefore, a selection step is necessary to select hybridoma
cells producing an antibody having desired properties (such as high
affinity to hTfR) from the hybridoma cells freshly obtained above.
Further, in the case where antibody genes are artificially
reconstructed, a selection step is necessary to select from the
antibody genes a gene encoding an antibody having desired
properties (such as high affinities to hTfR). For selecting
hybridoma cells that produce antibodies exhibiting high affinities
to hTfR (high affinity antibodies), or for selecting genes encoding
high affinity antibodies, following methods explained in detail
below are effective. Besides, antibodies exhibiting high affinity
to hTfR are those whose dissociation constant (K.sub.D) with hTfR
as measured by the method described in Example 7 is preferably not
greater than 1.times.10.sup.-8 M, more preferably not greater than
1.times.10.sup.-9 M, still more preferably not greater than
1.times.10.sup.-10 M, and even more preferably not greater than
1.times.10.sup.-11 M. For example, those having a dissociation
constant of 1.times.10.sup.-13 M to 1.times.10.sup.-9 M, or
1.times.10.sup.-13 M to 1.times.10.sup.-10 M are preferable.
[0255] For example, for selecting hybridoma cells which produce
high affinity antibodies to anti-hTfR antibody, a method is
employed in which recombinant hTfR is added to a plate and held by
it, then the culture supernatant of the hybridoma cells is added,
and after removing antibody unbound to the recombinant hTfR from
the plate, the amount of the antibody held by the plate is
measured. According to this method, the higher the affinity to hTfR
of the antibody contained in the culture supernatant of the
hybridoma cells added to the plate is, the greater the amount of
antibody held by the plate becomes. Therefore, by measuring the
amount of the antibody held by the plate, it is possible to select
those hybridoma cells corresponding to the plates where the
antibody is held in the greater amount as cell lines producing an
anti-hTfR antibody having the relatively higher affinity to hTfR.
It is also possible to isolate the gene encoding the high-affinity
antibody by extracting mRNAs from each cell line selected in this
manner, synthesizing cDNAs, and amplifying a DNA fragment
containing the gene encoding the anti-hTfR antibody by PCR using
the cDNA as a template.
[0256] In order to select the gene encoding the high-affinity
anti-hTfR antibody from the above artificially reconstructed
antibody genes, the artificially reconstructed antibody genes are
once incorporated into an expression vector, and the expression
vector then is introduced into host cells. Although there is no
particular limitation as to the cells to be employed as host cells,
even whether they are prokaryotic or eukaryotic, insofar as they
can express the antibody gene after introduction of an expression
vector having the incorporated artificially reconstructed antibody
gene, preferred are cells originating mammals such as human, mouse,
Chinese hamster, and the like, and particularly preferred are CHO
cells originating from Chinese hamster ovary cells, or NS/0 cells
originating from mouse myeloma. Further, there is no particular
limitation as to an expression vector to be employed for
incorporation of the antibody encoding gene and expression of it,
and any expression vector may be used as far as it can express the
gene when introduced into mammalian cells. The gene incorporated
into an expression vector is located downstream of a DNA sequence
that can regulate the frequency of transcription of a gene in
mammalian cells (gene expression regulatory site). Examples of gene
expression regulatory sites that may be employed in the present
invention include cytomegalovirus-derived promoter, SV40 early
promoter, human elongation factor-1.alpha. (EF-1.alpha.) promoter,
human ubiquitin C promoter.
[0257] Mammalian cells having such an introduced expression vector
come to express the artificially reconstructed antibody
incorporated in the expression vector. In order to select those
cells that produce a high-affinity antibody to anti-hTfR antibody
from the above obtained cells expressing the artificially
reconstructed antibody, a method is employed in which the
recombinant hTfR is added to a plate and held by it, then the
recombinant hTfR is contacted by the culture supernatant of the
cells, and after the removal of antibody unbound to the recombinant
hTfR from the plate, the amount of the antibody held by the plate
is measured. According to this method, the higher the affinity to
hTfR of the antibody contained in the cells culture supernatant is,
the greater the amount of antibody held by the plate becomes.
Therefore, by measuring the amount of the antibody held by the
plate, one can select those cells corresponding to the plate where
the antibody is held in the greater amount, as a cell line
producing an anti-hTfR antibody having relatively the high-affinity
anti-hTfR antibodyR, and eventually can select a gene encoding an
anti-hTfR antibody having a high-affinity anti-hTfR antibody to
hTfR. Using cell line selected in this manner, one can perform PCR
to amplify a DNA fragment containing the gene encoding the
anti-hTfR antibody to isolate the gene encoding the high-affinity
antibody.
[0258] Selection of the gene encoding a high affinity anti-hTfR
antibody from the above artificially reconstructed antibody genes
can also be carried out by incorporating the artificially
reconstructed antibody genes into an expression vector, introducing
the expression vector into E. coli cells, culturing the E. coli
cells, and selecting the E. coli cells having the desired gene, in
the same manner as in the above selection of hybridoma cells, using
the culture supernatant of the E. coli cells or an
antibody-containing solution prepared by lysing the E. coli cells.
E. coli cells thus selected express the gene encoding an anti-hTfR
antibody having a relatively high affinity to hTfR. From this cell
line, the gene encoding the anti-hTfR antibody having a relatively
the high-affinity anti-hTfR antibody to hTfR can be selected. In
order to allow the antibody to be secreted into the E. coli culture
supernatant, the antibody gene may be incorporated into the
expression vector so that a secretion signal sequence is attached
on the N-terminal side of the gene.
[0259] Another method for selection of the gene encoding a
high-affinity anti-hTfR antibody is a method in which the antibody
encoded by the above artificially reconstructed antibody gene is
expressed and retained on phage particles. For this, the antibody
gene is reconstructed as a gene encoding a single-chain antibody. A
method for retaining the antibody on the surface of phage particles
is disclosed in international publications WO1997/09436 and
WO1995/11317, and the like, and thus well known. In order to select
phages retaining the high-affinity antibody to anti-hTfR antibody
from the phages retaining the antibodies encoded by the
artificially reconstructed antibody genes, a method is employed in
which a recombinant hTfR is added to a plate and held by it,
contacted by the phages, and after removal of the phages unbound to
the recombinant hTfR from the plate, the amount of the phages held
by the plate is measured. According to this method, the higher the
affinity to hTfR of the antibody retained on the phage particles
is, the greater the amount of the phage held by the plate becomes.
Therefore, by measuring the amount of the phage held by the plate,
one can select the phage particles corresponding to the plate where
the phages' were held in the greater amount, as the phage particles
producing anti-hTfR antibody having a relatively high-affinity
anti-hTfR antibody to hTfR, and eventually can select the gene
encoding the high-affinity anti-hTfR antibody to hTfR. Using the
phage particles thus selected, PCR can be performed to amplify a
DNA fragment containing the gene encoding the anti-hTfR antibody
and isolate the gene encoding the high-affinity antibody.
[0260] It is possible to prepare cDNA or phage DNA from the above
cells such as the hybridoma cells producing the high-affinity
antibody to anti-hTfR, or from the above phage particles retaining
high-affinity antibody to anti-hTfR, and perform PCR or the like
using it as a template to amplify and isolate a DNA fragment
containing the gene encoding the whole or part of the anti-hTfR
antibody light chain, the anti-hTfR antibody heavy chain, or a
single-chain antibody. In the same manner, it is also possible to
perform PCR or the like to amplify and isolate a DNA fragment
containing the gene encoding the whole or part of the light chain
variable region of the anti-hTfR antibody, or a DNA fragment
containing the gene encoding the whole or part of the heavy chain
variable region of the anti-hTfR antibody.
[0261] A high-affinity anti-hTfR antibody can be obtained by
incorporating the whole or part of the gene encoding the light
chain and the heavy chain of this high-affinity anti-hTfR antibody
into an expression vector, transforming host cells such as
mammalian cells with this expression vector, and culturing the
obtained transformant cells. Using the nucleotide sequence of the
isolated gene encoding the anti-hTfR antibody, it is also possible
to translate the amino acid sequence of the anti-hTfR antibody, and
artificially synthesize a DNA fragment encoding the same amino acid
sequence. In artificially synthesizing a DNA fragment, the
expression level of the anti-hTfR antibody in the host cells can be
enhanced by proper selection of the codons.
[0262] In order to introduce a mutation such as substitution,
deletion, addition and the like into the amino acid sequence of the
original anti-hTfR antibody, a mutation may be introduced as
desired into the gene encoding the anti-hTfR antibody contained in
the isolated DNA fragment. Though the gene encoding the mutated
anti-hTfR antibody has a homology preferably not lower than 80%,
more preferably not lower than 90%, to the original gene, there is
no particular limitation as to the level of homology. By
introducing a mutation into the amino acid sequence so as to modify
the number or the type of sugar chains bound to the anti-hTfR
antibody, it is also possible to enhance the stability of the
anti-hTfR antibody in the body.
[0263] When introducing a mutation into the gene encoding the whole
or part of the light chain variable region of the anti-hTfR
antibody, the gene thus mutated has a homology that is preferably
not lower than 80%, more preferably not lower than 90%, to the
original gene, though there is no particular limitation as to the
level of homology. When replacing one or more amino acids of the
amino acid sequence of the light chain variable region with other
amino acids, the number of amino acids to be replaced is preferably
1 to 10, more preferably 1 to 5, still more preferably 1 to 3, and
even more preferably 1 or 2. When deleting one or more amino acids
of the amino acid sequence of the light chain variable region, the
number of amino acid to be deleted is preferably 1 to 10, more
preferably 1 to 5, still more preferably 1 to 3, even more
preferably 1 or 2. A combined mutation of these substitution and
deletion of amino acids can also be carried out. When adding one or
more amino acids to the light chain variable region, they may be
added inside, or on the N-terminal side or C-terminal side of, the
amino acid sequence of the light chain variable region, and the
number of amino acids added is preferably 1 to 10, more preferably
1 to 5, still more preferably 1 to 3, and even more preferably 1 or
2. A combined mutation of these addition, substitution, and
deletion of amino acids can also be carried out. The amino acid
sequence of the light chain variable region thus mutated has a
homology that is preferably not lower than 80%, more preferably not
lower than 90%, still more preferably not lower than 95% to the
amino acid sequence of the original light chain variable region. In
particular, when replacing one or more amino acids of the amino
acid sequence of CDR with other amino acids, the number of amino
acid replaced is preferably 1 to 5, more preferable 1 to 3, still
more preferably 1 or 2. When deleting one or more amino acid of the
amino acid sequence of CDR, the number of amino acids to be deleted
is preferably 1 to 5, more preferably 1 to 3, still more preferably
1 or 2. A combined mutation of these substitution and deletion of
the amino acid can also be carried out. When adding one or more
amino acids, they may be added inside, or on the N-terminal side or
C-terminal side of, the amino acid sequence, and the number of
amino acids added is preferably 1 to 5, more preferably 1 to 3,
still more preferably 1 or 2. A combined mutation of these
addition, substitution, and deletion of amino acids can also be
carried out. The amino acid sequence of respective mutated CDR has
a homology that is preferably not lower than 80%, more preferably
not lower than 90%, and still more preferably not lower than 95% to
the amino acid sequence of the original CDR.
[0264] When introducing mutation into the gene encoding the whole
or part of the heavy chain variable region of the anti-hTfR
antibody, the gene thus mutated has a homology that is preferably
not lower than 80%, more preferably not lower than 90%, to the
original gene, though there is no particular limitation as to the
level of homology. When replacing one or more amino acids of the
amino acid sequence of the heavy chain variable region with other
amino acids, the number of amino acids to be replaced is preferably
1 to 10, more preferably 1 to 5, still more preferably 1 to 3, and
even more preferably 1 or 2. When deleting one or more amino acids
of the amino acid sequence of the heavy chain variable region, the
number of amino acids to be deleted is preferably 1 to 10, more
preferably 1 to 5, still more preferably 1 to 3, even more
preferably 1 or 2. A combined mutation of these substitution and
deletion of amino acids can also be carried out. When adding one or
more amino acid to the heavy chain variable region, they may be
added inside, or on the N-terminal side or C-terminal side of, the
amino acid sequence of the heavy chain variable region, and the
number of amino acids added is preferably 1 to 10, more preferably
1 to 5, still more preferably 1 to 3, and even more preferably 1 or
2. A combined mutation of these addition, substitution, and
deletion of amino acids can also be carried out. The amino acid
sequence of the heavy chain variable region thus mutated has a
homology that is preferably not lower than 80%, more preferably not
lower than 90%, still more preferably not lower than 95% to the
amino acid sequence of the original heavy chain variable region. In
particular, when replacing one or more amino acids of the amino
acid sequence of CDR with other amino acids, the number of amino
acid replaced is preferably 1 to 5, more preferable 1 to 3, still
more preferably 1 or 2. When deleting one or more amino acid of the
amino acid sequence of CDR, the number of amino acids to be deleted
is preferably 1 to 5, more preferably 1 to 3, still more preferably
1 or 2. A combined mutation of these substitution and deletion of
the amino acid can also be carried out. When adding one or more
amino acids, they may be added inside, or on the N-terminal side or
C-terminal side of, the amino acid sequence, and the number of
amino acids added is preferably 1 to 5, more preferably 1 to 3,
still more preferably 1 or 2. A combined mutation of these
addition, substitution, and deletion of amino acids can also be
carried out. The amino acid sequence of respective mutated CDR has
a homology that is preferably not lower than 80%, more preferably
not lower than 90%, and still more preferably not lower than 95% to
the amino acid sequence of the original CDR.
[0265] A mutation may be introduced into both the variable regions
of the light chain and the heavy chain of the anti-hTfR antibody,
by combining the above mutation into the light chain variable
region of the anti-hTfR antibody and the above mutation into the
heavy chain variable region of the anti-hTfR antibody.
[0266] Examples of the above mentioned substitution of one or more
amino acids in the amino acid sequence of the light chain and the
heavy chain of the anti-hTfR antibody include substitution between
acidic amino acids, i.e., aspartic acid and glutamic acid,
substitution between amide-type amino acids, i.e., asparagine and
glutamine, substitution between basic amino acids, i.e., lysine and
arginine, substitution between branched amino acids, i.e., valine,
leucine and isoleucine, substitution between aliphatic amino acids,
i.e., glycine and alanine, substitution between hydroxyamino acids,
i.e., serine and threonine, and substitution between aromatic amino
acids, i.e., phenylalanine and tyrosine.
[0267] Besides, in the case where a mutation is introduced into the
anti-hTfR antibody by adding one or more amino acids to the
C-terminus or the N-terminus, if the anti-hTfR antibody and a
different protein (A) are fused via the added amino acids, the
added amino acids constitutes part of a linker. A detailed
explanation will be given later on a linker that is placed between
the anti-hTfR antibody and a different protein (A) in the case
where the anti-hTfR antibody is fused with the different protein
(A).
[0268] The anti-hTfR antibody obtained by culturing the cells
selected by the above methods and the like to produce an anti-hTfR
antibody that has a relatively high-affinity to hTfR, and the
anti-hTfR antibody obtained by expression of the gene encoding a
high-affinity anti-hTfR antibody, may be modified by introducing a
mutation into their amino acid sequences, such as substitution,
deletion, addition to give them desired properties. Introduction of
a mutation into the amino acid sequence of the anti-hTfR antibody
may be performed by introducing a mutation into the gene
corresponding to the amino acid sequence.
[0269] The affinity of an anti-hTfR antibody to hTfR can be
adjusted as desired by introduction of a mutation, such as
substitution, deletion, and addition, into the amino acid sequence
of a variable region of the antibody. For example, if an antibody
has such a high affinity to its antigen that leads to too low a
dissociation constant in an aqueous solution, there is a
possibility that the antibody could, after administered to the
body, fail to dissociate from the antigen, thereby leading to a
functional disadvantage. In such a case, a most preferable antibody
suitable to a given purpose can be obtained by introducing a
mutation into the variable region of the antibody so as to adjust
its dissociation constant stepwise to 2 to 5 times, 5 to 10 times,
10 to 100 times, and so on, that of the original antibody.
Conversely, the dissociation constant can be adjusted stepwise to
1/2 to 1/5 times, 1/5 to 1/10 times, 1/10 to 1/100 times, and so
on, that of the original antibody, by introducing a mutation.
[0270] Introduction of a mutation such as substitution, deletion
and addition to the amino acid sequence of the anti-hTfR antibody
can be performed, for example, either by introducing a mutation
into certain positions of the nucleotide sequence of the gene or by
random introduction of a mutation, by PCR or the like using the
gene encoding the anti-hTfR antibody as a template.
[0271] Introduction of a mutation into the amino acid sequence of
the anti-hTfR antibody for adjusting the affinity of the antibody
to hTfR can be carried out by, for example, incorporating a gene
encoding the anti-hTfR antibody as a single-chain antibody into a
phagemid, preparing with this phagemid a phage with expressed
single-chain antibody on the surface of its capsid, letting the
phage multiply while introducing a mutation into the gene encoding
the single-chain antibody by application of a mutagen or the like,
and selecting, from the multiplied phage, a phage expressing a
single-chain antibody having a desired dissociation constant either
by the method described above or by purification using an antigen
column under a certain condition.
[0272] The antibodies having a relatively high-affinity to hTfR
obtained by the above-mentioned method of selecting the cells
producing a high affinity antibody, are those whose dissociation
constant (K.sub.D) with hTfR as measured by the method described in
Example 7 is preferably not greater than 1.times.10.sup.-8M, more
preferably not greater than 1.times.10.sup.-9 M, still more
preferably not greater than 1.times.10.sup.-10 M, and even more
preferably not greater than 1.times.10.sup.-11 M. For example,
those having a dissociation constant of 1.times.10.sup.-13 M to
1.times.10.sup.-9 M, or 1.times.10.sup.-13 M to 1.times.10.sup.-10
M are preferable. The same also applies if the antibodies are
single-chain antibodies. Once an antibody is obtained, it can be
modified as desired by, e.g., introducing a mutation to give it a
desired property.
[0273] Antibody having affinity both to human and monkey TfRs can
be obtained by selection of antibodies having affinity to monkey
TfR from the antibodies having a relatively high-affinity that have
been obtained by the above described method involving selection of
the cells producing high affinity antibody. Selection of antibodies
having affinity to monkey TfR can be carried out by, for example,
ELISA using a recombinant monkey TfR which is prepared utilizing
recombinant DNA technologies. In such an ELISA, a recombinant
monkey TfR is added to a plate and held by it, and contacted by the
anti-hTfR antibody, and, after removal of antibody unbound to the
recombinant monkey TfR from the plate, the amount of the antibody
held by the plate is measured. The higher the affinity of it to the
recombinant monkey hTfR is, the greater the amount of the antibody
held by the plate becomes. Consequently, the antibody corresponding
to the plate which held the greater amount of antibody can be
selected as the antibody having affinity to monkey TfR. Here, the
term "monkey" is preferably classified as simians except human,
more preferably as Cercopithecidae, still more preferably as
macaques, and for example cynomolgus monkey or Rhesus monkey, among
which cynomolgus monkey is convenient for use in examination.
[0274] An antibody having affinity both to human and monkey hTfRs
offers an advantage that it allows pharmacokinetic observation of
the antibody administered to the body using a monkey. For example,
if a medical drug is being developed utilizing such an anti-hTfR
antibody of the present invention, the progress of its development
can be remarkably accelerated, for its pharmacokinetic study can be
performed using a monkey.
[0275] An antibody having a relatively high-affinity to hTfR and
having affinity both to human and monkey TfRs, simultaneously,
exhibits a dissociation constant with monkey TfR, as measured by
the method described in Example 7, that is preferably not greater
than 5.times.10.sup.-8 M, more preferably not greater than
2.times.10.sup.-8M, and still more preferably not greater than
1.times.10.sup.-8M. For example, one which exhibits a dissociation
constant of 1.times.10.sup.-13 M to 2.times.10.sup.-8M, or
1.times.10.sup.-13 M to 2.times.10.sup.-8 M is preferred. The same
also applies if the antibody is a single-chain antibody.
[0276] If an antibody having a relatively high-affinity to hTfR and
obtained by the above method in which those cells producing a high
affinity antibody were selected, is an antibody of a non-human
animal, it may be converted to a humanized antibody. A humanized
antibody is an antibody produced by replacing a proper region of a
human antibody with an amino acid sequence of part of the variable
region (e.g., the whole or part of the CDRs) of a non-human animal
antibody (implant of the sequence into the proper region of a human
antibody), while maintaining the specificity to the antigen.
Examples of humanized antibodies include an antibody produced by
replacing the three complementarity determining regions (CDRs) in
the immunoglobulin light chain and the three complementarity
determining regions (CDRs) in the immunoglobulin heavy chain, both
constituting a human antibody, with CDRs of a non-human mammal.
Though there is no particular limitation as to the biological
species from which the CDRs to be incorporated into the human
antibody are derived so long as it is a non-human mammal, it
preferably is a mouse, rat, rabbit, horse, and non-human primate,
more preferably a mouse and rat, and still more preferably a
mouse.
[0277] Methods for preparation of humanized antibody are well known
in the art and the most common is a method in which the amino acid
sequence of the complementarity determining regions (CDRs) in the
variable region of a human antibody is replaced with the CDRs of an
antibody of non-human mammal, as devised by Winter et al.
(Verhoeyen M. Science. 239, 1534-1536 (1988)). It is also well
known that in some cases, corresponding part of an acceptor human
antibody needs to be replaced not only with the CDRs of the
non-human mammalian antibody but also amino acid sequences
occurring in regions outside the CDRs that play a role either in
maintaining the structure of the CDRs or in binding to the antigen,
in order to reproduce the activity that the donor antibody
originally possesses (Queen C. Proc. Natl. Acad. Sci. USA. 86.
10029-10033 (1989)). Here, the regions outside the CDRs are called
framework (FR) regions.
[0278] Thus, preparation of humanized antibody involves processes
of implanting the CDRs (and their neighboring FRs, as the case may
be) of non-human mammalian antibody in place of the CDRs (and their
neighboring FRs, as the case may be) in the variable region of a
human antibody. In such processes, the starting framework region of
the variable region of a human antibody can be obtained from a
public DNA database and the like which includes germ line antibody
genes. For example, germ line DNA sequences, as well as amino acid
sequences, of human heavy chain and light chain variable regions
can be selected from "VBase" human germline database (available in
the Internet, at www.mrc-cpe.cam.ac.uk/vbase). Besides, they can be
selected from DNA sequences and amino acid sequences described in
published literatures, such as "Kabat E A. Sequences of Proteins of
Immunological Interest, 5th Ed., U.S. Department of Health and
Human Services, NIH Publication No. 91-3242 (1991)"; "Tomlinson I
M. J. fol. Biol. 227. 776-98 (1992)"; and "Cox J P L. Eur. J
Immunol. 24:827-836 (1994)".
[0279] As aforementioned, in a humanized antibody, the regions of a
non-human mammal antibody to be implanted into the variable regions
of the original human antibody generally include CDRs themselves,
or CDRs and their neighboring part of FRs. However, such FRs
implanted together with CDRs also play a role either in maintaining
the structure of the CDRs or in binding to the antigen, thus having
a substantial function in determining the complementarity of an
antibody, and the term "CDR" in the present invention, therefore,
refers to such regions that are, or could be, taken from a
non-human mammal antibody and grafted into a humanized antibody, in
preparing a humanized antibody. Thus, a region generally considered
to be in a FR region is included in a CDR in the present invention
as far as it takes part either in maintaining the structure of the
CDR or in binding to the antigen, and is thus considered to have a
substantial function in determining the complementarity of the
antigen.
[0280] The anti-hTfR antibody of the present invention, when
administered to the body, e.g., by intravenous injection,
efficiently binds to hTfR existing on the endothelial cells of the
capillaries in the brain. The antibody bound to the hTfR is taken
into the brain across the blood-brain barrier by such mechanisms as
endocytosis, and transcytosis. Therefore, by binding to the
anti-hTfR antibody of the present invention, proteins,
low-molecular-weight compounds and the like that need to be brought
into function in the brain, can be efficiently delivered into the
brain across the blood-brain barrier. Further, the anti-hTfR
antibody of the present invention can, after passing through the
blood-brain barrier, can reach the cerebral parenchyma, and
neuron-like cells in the hippocampus; Purkinje cells and the like
of the cerebellum or at least one of them. And it is also expected
that it reaches to the neuron-like cells in the striatum of the
cerebrum; and the neuron-like cells in the substantia nigra of the
mesencephalon. Therefore, it is possible to make one of those
proteins, low-molecular-weight compounds and the like, which could
act on such tissues or cells, reach the tissues or cells, by
binding it to the anti-hTfR antibody of the present invention.
[0281] The anti-hTfR antibody of the present invention can be an
effective means to make those compounds (proteins,
low-molecular-weight compounds and the like) transfer from the
blood into the brain and function there, which compounds otherwise
cannot pass through the blood-brain barrier when intravenously
administered and therefore cannot or can hardly exhibit their
physiological or pharmacological functions in the brain. In
particular, the anti-hTfR antibody of the present invention can,
after passing through the blood-brain barrier, reach the cerebral
parenchyma, and neuron-like cells in the hippocampus; Purkinje
cells and the like of the cerebellum or at least one of them. And
it is also expected that it reaches to the neuron-like cells in the
striatum of the cerebrum; as well as to the neuron-like cells in
the substantia nigra of the mesencephalon. Therefore, it is
possible to make those compounds function or augment their
function, in those tissues or cells in the brain by administering
those compounds in a combined form with the anti-hTfR antibody
molecule, parenterally, e.g., intravenously.
[0282] For binding an anti-hTfR antibody to such compounds
(proteins, low-molecular-weight compounds and the like), a method
is available to bind them together via a non-peptide linker or a
peptide linker. As non-peptide linkers, there can be used
polyethylene glycol, polypropylene glycol, copolymer of ethylene
glycol and propylene glycol, polyoxyethylated polyol, polyvinyl
alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable
polymer, polymerized lipid, chitins, and hyaluronic acid, or
derivatives thereof, or combinations thereof. A peptide linker is a
peptide chain consisting of 1 to 50 amino acids linked by peptide
bonds or a derivative thereof, whose N-terminus and C-terminus are
to be covalently bonded either to an anti-hTfR antibody or a
compound such as a protein, a low-molecular-weight compound and the
like, respectively, to bind the anti-hTfR antibody to such a
compound like a protein or a low-molecular-weight compound.
[0283] In particular, a conjugate which is formed by binding the
anti-hTfR antibody of the present invention to a desired different
protein (A) via PEG as a non-peptide linker, is designated
"anti-hTfR antibody-PEG-protein". An anti-hTfR antibody-PEG-protein
can be prepared by first binding the anti-hTfR antibody to PEG to
form anti-hTfR antibody-PEG, and then binding the anti-hTfR
antibody-PEG to the different protein (A). Alternatively, an
anti-hTfR antibody-PEG-protein can be prepared by first binding the
different protein (A) to PEG to form "protein-PEG", and then
binding the "protein-PEG" to the anti-hTfR antibody. In order to
bind PEG to the anti-hTfR antibody and the different protein (A), a
PEG is employed which is modified with such functional groups as
carbonate, carbonylimidazole, active ester of carboxylic acid,
azlactone, cyclic imide thione, isocyanate, isothiocyanate,
imidate, aldehyde or the like. Such functional groups introduced to
PEG react mainly with amino groups in the anti-hTfR antibody and a
different protein (A) to covalently bind PEG to the hTfR antibody
and a different protein (A). Though there is no particular
limitation as to the molecular weight and the configuration of PEG
employed here, its mean molecular weight (MW) is as follows:
preferably MW=500 to 60000, more preferably MW=500 to 20000. For
example, such PEG whose mean molecular weight is about 300, about
500, about 1000, about 2000, about 4000, about 10000, about 20000,
and the like. PEG is preferably used as a non-peptide linker. The
anti-hTfR antibody can be bound to a desired low-molecular-weight
compound in the same manner as above.
[0284] For example, "anti-hTfR antibody-PEG" can be prepared by
mixing the anti-hTfR antibody with a polyethylene glycol having
aldehyde groups as functional groups (ALD-PEG-ALD) so that the
molar ratio of ALD-PEG-ALD to the antibody is 11, 12.5, 15, 110,
120 and the like, and then adding to the mixture a reducing agent
such as NaCNBH.sub.3 to let a reaction take place. Then, by
reacting "anti-hTfR antibody-PEG" with a different protein (A) in
the presence of a reducing agent such as NaCNBH.sub.3, "anti-hTfR
antibody-PEG-protein" is obtained. On the contrary, it is also
possible to obtain "anti-hTfR antibody-PEG-protein" by first
binding a different protein (A) to ALD-PEG-ALD to prepare
"protein-PEG", and then binding the "protein-PEG" to the anti-hTfR
antibody.
[0285] The anti-hTfR antibody and a different protein (A) can also
be bound together through peptide bonds by linking the anti-hTfR
antibody heavy chain or light chain, on the C-terminal side or the
N-terminal side thereof, either via a linker sequence or directly,
to the N-terminus or the C-terminus of the different protein (A),
respectively. Thus the fusion protein between the anti-hTfR
antibody and a different protein (A) can be obtained by
incorporating into a mammalian expression vector a DNA fragment in
which a cDNA encoding the different protein (A) is placed in-frame
directly, or via a DNA fragment encoding a linker sequence, on the
3'-end or 5'-end side of a cDNA encoding the heavy chain or light
chain of the anti-hTfR antibody, and culturing mammalian cells into
which the above expression vector has been introduced. Where the
DNA fragment encoding a different protein (A) is linked to the
heavy chain, a mammalian expression vector in which a cDNA fragment
encoding the anti-hTfR antibody light chain is also introduced into
the same host cells, whereas if DNA fragment encoding a different
protein (A) is linked to the light chain, a mammalian expression
vector in which a cDNA fragment encoding the anti-hTfR antibody
heavy chain is also incorporated into the same host cells. In the
case where the anti-hTfR antibody is a single-chain antibody, the
fusion protein comprising the anti-hTfR antibody and a different
protein (A) combined can be obtained by incorporating, into an
expression vector (for eukaryotic cells such as mammalian and
yeast, or for prokaryotic cells such as E. coli.), a DNA fragment
which is formed by linking the cDNA encoding a different protein
(A), on the 5'-end side or on the 3'-end side thereof, directly or
via a DNA fragment encoding a linker sequence, to the cDNA encoding
the single-chain anti-hTfR antibody, and allowing the fusion
protein be expressed in those cells into which the expression
vector has been introduced.
[0286] In a fusion protein of the type in which a different protein
(A) is linked to the anti-hTfR antibody light chain on the
C-terminal side thereof, the anti-human transferrin receptor
antibody comprises an amino acid sequence including the whole or
part of the light chain variable region and an amino acid sequence
including the whole or part of the heavy chain variable region, and
the different protein (A) is linked to the light chain of this
anti-human transferrin receptor antibody on the C-terminal side
thereof. Here, the anti-hTfR antibody light chain and a different
protein (A) may be linked together, directly or via a linker.
[0287] In a fusion protein of the type in which a different protein
(A) is linked to the anti-hTfR antibody heavy chain on the
C-terminal side thereof, the anti-human transferrin receptor
antibody comprises an amino acid sequence including the whole or
part of the light chain variable region and an amino acid sequence
including the whole or part of the heavy chain variable region, and
the different protein (A) is linked to the heavy chain of this
anti-human transferrin receptor antibody on the C-terminal side
thereof. Here, the anti-hTfR antibody heavy chain and a different
protein (A) may be linked together, directly or via a linker.
[0288] In a fusion protein of the type in which a different protein
(A) is linked to the anti-hTfR antibody light chain on the
N-terminal side thereof, the anti-human transferrin receptor
antibody comprises an amino acid sequence including the whole or
part of the light chain variable region and an amino acid sequence
including the whole or part of the heavy chain variable region, and
the different protein (A) is linked to the light chain of this
anti-human transferrin receptor antibody on the N-terminal side
thereof. Here, the anti-hTfR antibody light chain and a different
protein (A) may be linked together, directly or via a linker.
[0289] In a fusion protein of the type in which a different protein
(A) is linked to the anti-hTfR antibody heavy chain on the
N-terminal side thereof, the anti-human transferrin receptor
antibody comprises an amino acid sequence including the whole or
part of the light chain variable region and an amino acid sequence
including the whole or part of the heavy chain variable region, and
the different protein (A) is linked to the heavy chain of this
anti-human transferrin receptor antibody on the N-terminal side
thereof. Here, the anti-hTfR antibody heavy chain and a different
protein (A) may be linked together, directly or via a linker.
[0290] In the above, the linker sequence placed between the
anti-hTfR antibody and a different protein (A) may be a peptide
chain consisting preferably of 1 to 50, more preferably of 1 to 17,
still more preferably of 1 to 10, even more preferably of 1 to 5
amino acids, and in accordance with the different protein (A) to be
linked to the anti-hTfR antibody, the number of amino acids of the
linker sequence may be adjusted to 1, 2, 3, 1 to 17, 1 to 10, 10 to
40, 20 to 34, 23 to 31, 25 to 29, 27, etc., as desired. Though
there is no particular limitation as to amino acid sequence of the
linker sequence insofar as the anti-hTfR antibody linked by it
retains the affinity to hTfR and a different protein (A) linked by
the linker sequence also exhibits the protein's own physiological
activity under a physiological condition, the linker may preferably
be composed of glycine and serine. Examples of such linkers include
one consisting of a single amino acid either glycine or serine, the
amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser,
the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), the
amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4), the
amino acid sequence Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a
sequence which includes 1 to 10 or 2 to 5 of any of those amino
acid sequences consecutively linked. They have sequences consisting
of 1 to 50, 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to
29, or 27 amino acids. For example, those comprising the amino acid
sequence Gly-Ser may preferably be used as linker sequences.
Further, a linker sequence comprising 27 amino acids is preferably
used that is composed of the amino acid sequence Gly-Ser followed
by consecutively linked five copies of the amino acid sequence
Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3).
[0291] In a fusion protein of the anti-hTfR antibody and a
different protein (A), where the anti-hTfR antibody is a
single-chain antibody, the amino acid sequence including the whole
or part of the immunoglobulin light chain variable region and the
amino acid sequence including the whole or part of the
immunoglobulin heavy chain variable region are linked, generally
via a linker sequence. Insofar as the affinity of the anti-hTfR
antibody to hTfR is retained, the amino acid sequence derived from
the light chain may be linked, on the C-terminal side thereof, to a
linker sequence which in turn being linked, on the C-terminal side
thereof, to the amino acid sequence derived from the heavy chain
or, conversely, the amino acid sequence derived from the heavy
chain may be linked, on the C-terminal side thereof, to a linker
sequence which in turn being linked, on the C-terminal side
thereof, to the amino acid sequence derived from the light
chain.
[0292] The linker sequence placed between the light chain and the
heavy chain of the immunoglobulin is a peptide chain consisting
preferably of 2 to 50, more preferably 8 to 50, still more
preferably 10 to 30, even more preferably 12 to 18 or 15 to 25, and
for example 15 or 25 amino acids. Though there is no specific
limitation as to the linker sequence insofar as the anti-hTfR
antibody made of the both chains which are linked via the linker
retains the affinity to hTfR and a different protein (A) linked to
the antibody also exhibits the protein's own physiological activity
under a physiological condition, the linker is preferably composed
of glycine, or glycine and serine. Examples of such linkers include
the amino acid sequence Gly-Ser, the amino acid sequence
Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), the amino acid sequence
Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4), the amino acid sequence
Ser-Gly-Gly-Gly-Gly-Gly) (SEQ ID NO:5), or a sequence which
includes 2 to 10 or 2 to 5 of any of these amino acid sequences
consecutively linked. A preferred embodiment of such a linker
sequence comprises 15 amino acids consisting of consecutively
linked three copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser
(SEQ ID NO:3).
[0293] In the case where the anti-hTfR antibody is a single-chain
antibody, an example of specific embodiments of the fusion protein
between the humanized anti-hTfR antibody of the present invention
and a different protein (A) is a fusion protein consisting of the
different protein (A) which is linked, on the C-terminal side
thereof and via a first linker sequence consisting of 27 amino
acids composed of the amino acid sequence Gly-Ser followed by
consecutively linked five copies of the amino acid sequence
Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), to the single-chain antibody. An
example of a preferred embodiment of single-chain antibodies
employed here is an antibody having the amino acid sequence set
forth as SEQ ID NO:277, which is composed of the amino acid
sequence of the anti-hTfR antibody heavy chain variable region set
forth as SEQ ID NO:205 that is linked, at the C-terminus thereof
and via a first linker sequence consisting of 15 amino acids
consisting of consecutively linked three copies of the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), to the anti-hTfR
antibody light chain variable region having the amino acid sequence
set forth as SEQ ID NO:191.
[0294] Where the anti-hTfR antibody is a single-chain antibody,
such a fusion protein can be produced by, for example, transforming
host cells such as mammalian cells with an expression vector having
an incorporated DNA fragment containing a nucleotide sequence
encoding the fusion protein, and then culturing the host cells.
[0295] Besides, in the present invention, when a peptide chain
includes a plurality of linker sequences, each of those linker
sequences is designated, from the N-terminal side, the first linker
sequence, the second linker sequence, and so on, for
convenience.
[0296] In the case where the anti-hTfR antibody is Fab, an example
of specific embodiments of the fusion protein between a humanized
anti-hTfR antibody and a different protein (A) of the present
invention is a fusion protein which is composed of the different
protein (A) that is fused, on the C-terminal side thereof and via a
linker sequence consisting of 27 amino acids composed of Gly-Ser
followed by consecutively linked five copies of the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), to a region having the
anti-hTfR antibody heavy chain variable region and the C.sub.H1
region. Though part of the hinge region may be included in addition
to the C.sub.H1 region here, the hinge region includes no cysteine
residue which would form a disulfide bond between heavy chains.
[0297] Though there is no particular limitation as to the different
protein (A) to be linked to the anti-hTfR antibody, it is a protein
that can exhibit its physiological activity in the body, and in
particular, such a protein that needs to get inside the brain and
exhibit its function there but, due to its inability to pass
through the blood-brain barrier as it is, cannot be expected to
function in the brain if simply administered intravenously.
Examples of such proteins include lysosomal enzymes such as nerve
growth factor (NGF), .alpha.-L-iduronidase, iduronate 2-sulfatase,
glucocerebrosidase, .beta.-galactosidase, GM2 activator protein,
.beta.-hexosaminidase A, .beta.-hexosaminidase B,
N-acetylglucosamine-1-phosphotransferase, .alpha.-mannosidase,
.beta.-mannosidase, galactosylceramidase, saposin C, arylsulfatase
A, .alpha.-L-fucosidase, aspartylglucosaminidase,
.alpha.-N-acetylgalactosaminidase, acidic sphingomyelinase,
.alpha.-galactosidase A, .beta.-glucuronidase, heparan N-sulfatase,
.alpha.-N-acetylglucosaminidase, acetyl CoA:.alpha.-glucosaminide
N-acetyltransferase, N-Acetylglucosamine-6-sulfate sulfatase, acid
ceramidase, amylo-1,6-glucosidase, sialidase,
aspartylglucosaminidase (PPT1), tripeptidyl-peptidase 1,
hyaluronidase 1, CLN1, and CLN2, and the like.
[0298] The nerve growth factor (NGF) linked to the anti-hTfR
antibody can be used as a therapeutic agent for dementia in
Alzheimer's disease; .alpha.-L-iduronidase linked to the anti-hTfR
antibody as a therapeutic agent for central nervous system
disorders in Hurler syndrome or Hurler-Scheie syndrome; iduronate
2-sulfatase linked to the anti-hTfR antibody as a therapeutic agent
for central nervous system disorders in Hunter syndrome;
glucocerebrosidase as a therapeutic agent for central nervous
system disorders in Gaucher's disease; .beta.-galactosidase as a
therapeutic agent for central nervous system disorders in GM1
gangliosidosis Types 1 to 3; GM2 activator protein as a therapeutic
agent for central nervous system disorders in GM2-gangliosidosis,
AB variant; .beta.-hexosaminidase A as a therapeutic agent for
central nervous system disorders in Sandhoffs disease and Tay-Sachs
disease; .beta.-hexosaminidase B as a therapeutic agent for central
nervous system disorders in Sandhoff s disease;
N-acetylglucosamine-1-phosphotransferase as a therapeutic agent for
central nervous system disorders in I-cell disease;
.alpha.-mannosidase as a therapeutic agent for central nervous
system disorders in .alpha.-mannosidosis; .beta.-mannosidase as a
therapeutic agent for central nervous system disorders in
.beta.-mannosidosis; galactosylceramidase as a therapeutic agent
for central nervous system disorders in Krabbe disease; saposin C
as a therapeutic agent for central nervous system disorders in
Gaucher's disease-like storage disease; arylsulfatase A as a
therapeutic agent for central nervous system disorders in
metachromatic white matter degeneration (metachromatic
leukodystrophy); .alpha.-L-fucosidase as a therapeutic agent for
central nervous system disorders in fucosidosis;
aspartylglucosaminidase as a therapeutic agent for central nervous
system disorders in aspartylglucosaminuria;
.alpha.-N-acetylgalactosaminidase as a therapeutic agent for
central nervous system disorders in Schindler disease and Kawasaki
disease; acidic sphingomyelinase as a therapeutic agent for central
nervous system disorders in Niemann-Pick disease;
.alpha.-galactosidase A as a therapeutic agent for central nervous
system disorders in Fabry disease; .beta.-glucuronidase as a
therapeutic agent for central nervous system disorders in Sly
syndrome; heparan N-sulfatase, .alpha.-N-acetylglucosaminidase,
acetyl CoA: .alpha.-glucosaminide N-acetyltransferase and
N-Acetylglucosamine-6-sulfate sulfatase as therapeutic agents for
central nervous system disorders in Sanfilippo syndrome; acid
ceramidase as a therapeutic agent for central nervous system
disorders in Farber disease; amylo-1,6-glucosidase as a therapeutic
agent for central nervous system disorders in Cori's disease
(Forbes-Cori's disease); sialidase as a therapeutic agent for
central nervous system disorders in sialidase deficiency;
aspartylglucosaminidase as a therapeutic agent for central nervous
system disorders in aspartylglucosaminuria; palmitoyl protein
thioesterase 1 (PPT-1) as a therapeutic agent for central nervous
system disorders in neuronal ceroid lipofuscinosis or
Santavuori-Haltia disease; tripeptidyl-peptidase 1 (TPP-1) as a
therapeutic agent for central nervous system disorders in neuronal
ceroid lipofuscinosis or Jansky-Bielschowsky disease; hyaluronidase
1 as a therapeutic agent for central nervous system disorders in
hyaluronidase deficiency; CLN1 and CLN2 as therapeutic agents for
central nervous system disorders in Batten disease. In particular,
the anti-hTfR antibody of the present invention, after passing
through the blood-brain barrier, reaches the brain parenchyma and
the hippocampus neuron-like cells of the cerebrum, and Purkinje
cells of the cerebellum, and is expected further to reach
neuron-like cells of the striatum of the cerebrum and the
neuron-like cells of the substantia nigra of the mesencephalon.
Therefore, the anti-hTfR antibody can be fused with proteins which
need to exhibit their functions in those tissues or cells to
strength the pharmacological effects of the proteins. Medical
applications of it, however, are not limited thereto.
[0299] Further, examples of proteins that can exhibit their
pharmacological effects when linked to the anti-hTfR antibody
include: lysosomal enzymes, ciliary neurotrophic factor (CNTF),
glial cell line derived neurotrophic factor(GDNF), neurotrophin-3,
neurotrophin-4/5, neurotrophin-6, neuregulin-1, erythropoietin,
darbepoetin, activin, basic fibroblast growth factor (bFGF),
fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF),
vascular endothelial growth factor (VEGF), interferon .alpha.,
interferon .beta., interferon .gamma., interleukin 6,
granulocyte-macrophage colony stimulating factor (GM-CSF),
granulocyte colony stimulating factor (G-CSF), macrophage colony
stimulating factor (M-CSF), cytokines, tumor necrosis factor
.alpha. receptor (TNF-.alpha. receptor), PD-1 ligands, enzymes
having .beta.-amyloid-degrading activity, anti-.beta.-amyloid
antibody, anti-BACE antibody, anti-EGFR antibody, anti-PD-1
antibody, anti-PD-L1 antibody, anti-HER2 antibody, anti-TNF-.alpha.
antibody, and other antibody medicines.
[0300] Lysosomal enzymes linked to the anti-hTfR antibody can be
used as a therapeutic agent for central nervous system disorders in
lysosomal storage diseases; CNTF as a therapeutic agent for
amyotrophic lateral sclerosis; GDNF, neurotrophin-3 and
neurotrophin-4/5 as therapeutic agents for cerebral ischemia; GDNF
as a therapeutic agent for Parkinson's disease; neuregulin-1 as a
therapeutic agent for schizophrenia; erythropoietin and darbepoetin
as therapeutic agents for cerebral ischemia; bFGF and FGF2 as
therapeutic agents for traumatic central nervous system disorders;
for recovery after brain surgery and spinal surgery; enzymes having
f3-amyloid-degrading activity, anti-.beta.-amyloid antibody and
anti-BACE antibody as therapeutic agents for Alzheimer's disease;
anti-EGFR antibody, anti-PD-1 antibody, anti-PD-L1 antibody, and
anti-HER2 antibody as therapeutic agents for tumors of central
nervous system including brain tumor; and TNF.alpha.R-anti-hTfR
antibody as therapeutic agents for a cerebral ischemia and
encephalitis.
[0301] Possible candidates for a "different protein (A)" to be
fused to the anti-hTfR antibody generally include those therapeutic
agents for diseases such as neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, and Huntington's disease;
mental disorders such as schizophrenia and depression; multiple
sclerosis; amyotrophic lateral sclerosis; tumors of the central
nervous system including brain tumor; lysosomal storage diseases
accompanied by encephalopathy; glycogenosis; muscular dystrophy;
cerebral ischemia; encephalitis; prion diseases; traumatic central
nervous system disorders. In addition, therapeutic agent for viral
and bacterial central nervous system diseases can also be
candidates for a different protein (A) to be fused to the anti-hTfR
antibody, in general. Further, pharmaceutical agents that can be
used for recovery after brain surgery or spinal surgery can also be
candidates for a "different protein (A)" to be fused to the
anti-hTfR antibody, in general.
[0302] In addition to the above mentioned natural-type (wild-type)
proteins, a different protein (A) to be linked to the anti-hTfR
antibody may also be one of their analogues in which one or more
amino acids of those natural-type (wild-type) proteins are
modified, e.g., replaced with other amino acids or deleted, insofar
as they fully or partly have the functions of their respective
original proteins. When replacing one or more amino acids with
other amino acids, the number of amino acids to be replaced is
preferably 1 to 10, more preferably 1 to 5, still more preferably 1
to 3. When deleting one or more amino acids, the number of amino
acids to be deleted is preferably 1 to 10, more preferably 1 to 5,
still more preferably 1 to 3. A combination of such substitution
and deletion of amino acids can also be carried out to prepare
desired analogues. Further, amino acid sequences produced by adding
one or more amino acids inside, or on the N-terminal side or on the
C-terminal side of, the amino acid sequence of natural-type
(wild-type) proteins or their analogues, are also included in the
proteins mentioned above insofar as they fully or partly have the
functions of their respective original proteins. The number of
amino acids to be added here is preferably 1 to 10, more preferably
1 to 5, still more preferably 1 to 3. It is also possible to
prepare desired analogues to the original proteins by combining
addition, substitution, and deletion of amino acids.
[0303] Besides, in the case where a mutation is introduced into a
different protein (A) by adding one or more amino acids on its
C-terminus or the N-terminus, if the added amino acids are
positioned between the protein and the anti-hTfR antibody when they
are fused, the added amino acids constitute part of a linker.
[0304] The natural-type human I2S (hI2S) is a lysosomal enzyme
composed of 525 amino acids set forth as SEQ ID NO:246. A specific
example of fusion proteins of the present invention between the
anti-hTfR antibody and a different protein (A) is a type in which
the anti-hTfR antibody heavy chain is fused, on the C-terminus
thereof and via the amino acid sequence Gly-Ser as a linker
sequence, to the natural-type human I2S. Examples of such a type of
fusion proteins include:
[0305] (1) one whose light chain consists of the amino acid
sequence set forth as SEQ ID NO:164, and whose heavy chain linked
by peptide pond, on the C-terminal side thereof and via the linker
sequence Gly-Ser, to human I2S forms the amino acid sequence set
forth as SEQ ID NO:247,
[0306] (2) one whose light chain consists of the amino acid
sequence set forth as SEQ ID NO:180, and whose heavy chain linked
by peptide pond, on the C-terminal side thereof and via the linker
sequence Gly-Ser, to human 12S forms the amino acid sequence set
forth as SEQ ID NO:249, and
[0307] (3) one whose light chain consists of the amino acid
sequence set forth as SEQ ID NO:196, and whose heavy chain linked
by peptide pond, on the C-terminal side thereof and via the linker
sequence Gly-Ser, to human I2S forms the amino acid sequence set
forth as SEQ ID NO:251.
[0308] In the present invention, though the term "human I2S" or
"hI2S" refers, in particular, to the hI2S having the same amino
acid sequence as the natural-type hI2S, it also includes those
amino acid sequences produced by introducing a mutation, such as
substitution, deletion, addition and the like, into the amino acid
sequence of the natural-type hI2S, insofar as they have the I2S
activity. When replacing one or more of the amino acids of the
amino acid sequence of hI2S with other amino acids, the number of
amino acids to be replaced is preferably 1 to 10, more preferably 1
to 5, still more preferably 1 to 3, even more preferably 1 to 2.
When deleting one or more amino acids of the amino acid sequence of
hI2S, the number of amino acids to be deleted is 1 to 10, more
preferably 1 to 5, still more preferably 1 to 3, and even more
preferably 1 or 2. It is also possible to introduce a combined
mutation of such substitution and deletion of amino acids. When
adding one or more amino acids to hI2S, they may be added, inside,
or on the N-terminal side or the C-terminal side of, the amino acid
sequence of hI2S, and the number of amino acids to be added is
preferably 1 to 10, more preferably 1 to 5, still more preferably 1
to 3, even more preferably 1 or 2. It is also possible to introduce
a combined mutation of such addition, substitution, and deletion of
the amino acid. The amino acid sequence of the mutated hI2S has a
homology of preferably not lower than 80%, more preferably not
lower than 90%, still more preferably not lower than 95% to the
amino acid sequence of the original hI2S.
[0309] The statement that hI2S has the I2S activity herein means
that the hI2S fused to anti-hTfR antibody has an activity not lower
than 3% of the activity that the natural-type hI2S intrinsically
has. However, the activity is preferably not lower than 10%, more
preferably not lower than 20%, still more preferably not lower than
50%, even more preferably not lower than 80% of the activity that
the natural-type hI2S intrinsically has. The same also applies if
the hI2S fused to the anti-hTfR antibody is mutated.
[0310] The fusion protein between the anti-hTfR antibody and human
I2S can be produced by, for example, transforming host cells, such
as mammalian cells, with an expression vector having an
incorporated DNA fragment comprising the nucleotide sequence set
forth as SEQ ID NO:250 that encodes the amino acid sequence set
forth as SEQ ID NO:249 and an expression vector having an
incorporated DNA fragment comprising the nucleotide sequence set
forth as SEQ ID NO:181 that encodes the anti-hTfR antibody light
chain having the amino acid sequence set forth as SEQ ID NO:180,
and then culturing the host cells. The fusion protein thus produced
can be used as a therapeutic agent for Hunter disease, in
particular, a therapeutic agent for central nervous system
disorders accompanying Hunter disease.
[0311] Besides, in the case where the anti-hTfR antibody or human
I2S is mutated by adding one or more amino acids to them on the
C-terminus or the N-terminus thereof, if the added amino acids are
positioned between the anti-hTfR antibody and human I2S, the added
amino acids constitute part of a linker.
[0312] Though examples of fusion proteins between the anti-hTfR
antibody and human I2S are described above, there is no particular
limitation as to the amino acid sequence of the CDRs of the
anti-hTfR antibody heavy chain and light chain in preferred
embodiments of fusion protein of the anti-hTfR antibody and human
I2S insofar as the antibody has a specific affinity to hTfR.
However, the anti-hTfR antibody of the present invention exhibits a
dissociation constant (K.sub.D) with hTfR, as measured by the
method described in Example 7, that is preferably not greater than
1.times.10.sup.-8M, more preferably not greater than
1.times.10.sup.-9 M, still more preferably not greater than
1.times.10.sup.-10. For example, one which exhibits a dissociation
constant of 1.times.10.sup.-13M to 1.times.10.sup.-9M or
1.times.10.sup.-13M to 1.times.10.sup.-10 M is preferred. The same
also applies when the antibody is a single-chain antibody. Further,
in the case where the anti-hTfR antibody of the present invention
has also an affinity to monkey TfR, the dissociation constant of
the anti-hTfR antibody with monkey TfR, as measured by the method
described in Example 7, is preferably not greater than
5.times.10.sup.-8M, more preferably not greater than
2.times.10.sup.-8 M, still more preferably not greater than
1.times.10.sup.-8 M. For example, one which exhibits a dissociation
constant of 1.times.10.sup.-13 M to 2.times.10.sup.-8M is
preferred. The same also applies when the antibody is a
single-chain antibody.
[0313] It is also possible to link a relatively short peptide chain
to the anti-hTfR antibody, in the same manner as in linking a
different protein (A) to the anti-hTfR antibody. There is no
particular limitation as to a peptide chain to be linked to the
anti-hTfR antibody, insofar as the peptide chain has a desired
physiological activity. For example, there are peptide chains
comprising the amino acid sequence of such a region of various
proteins that exhibits a physiological activity. Though there is no
particular limitation as to the length of the peptide chain, they
are composed of preferably 2 to 200 amino acids, for example of 5
to 50 amino acids.
[0314] In linking a low-molecular-weight compound to the anti-hTfR
antibody, there is no particular limitation as to candidate
low-molecular-weight compounds, but they are such
low-molecular-weight compound that though needed to get inside the
brain and function there, due to their inability to pass through
the blood-brain barrier as it is, cannot be expected to function in
the brain if simply administered intravenously. Examples of such
low-molecular-weight compounds include anticancer drug such as
cyclophosphamide, Ifosfamide, melphalan, busulfan, thioTEPA,
nimustine, ranimustine, dacarbazine, procarbazine, temozolomide,
carmustine, streptozocin, bendamustine, cisplatin, carboplatin,
oxaliplatin, nedaplatin, 5-fluorouracil, sulfadiazine, sulfamethoxa
vole, methotrexate, trimethoprim, pyrimethamine, fluorouracil,
flucytosine, azathioprine, pentostatin, hydroxyurea, fludarabine,
cytarabine, gemcitabine, irinotecan, doxorubicin, etoposide,
levofloxacin, ciprofloxacin, vinblastine, vincristine, paclitaxel,
docetaxel, Mitomycin C, doxorubicin, epirubicin. Further examples
of low-molecular-weight compound to be linked to the anti-hTfR
antibody include siRNAs, antisense DNAs, and short peptides.
[0315] In linking between the anti-hTfR antibody and a
low-molecular-weight compound, either a low-molecular-weight
compound may be linked only to either the light chain or the heavy
chain, or it may be linked to both the light chain and the heavy
chain, respectively. Further, insofar as it has an affinity to
hTfR, the anti-hTfR antibody may comprise an amino acid sequence
comprising the whole of part of the light chain variable region
and/or an amino acid sequence comprising the whole of part of the
heavy chain variable region.
[0316] Candidates for low-molecular-weight compounds to be fused
with the anti-hTfR antibody can generally be those therapeutic
agents for diseases such as neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, Huntington's disease;
mental disorders such as schizophrenia, depression; multiple
sclerosis; amyotrophic lateral sclerosis; tumor of the central
nervous system including brain tumor; lysosomal storage diseases
with accompanying encephalopathy; glycogenosis; muscular dystrophy;
cerebral ischemia; encephalitis; prion diseases; traumatic
disorders of the central nervous system. Further, therapeutic
agents for viral and bacterial central nervous system diseases can
also be candidates, in general, for low-molecular-weight compounds
to be fused with the anti-hTfR antibody. Still further, those
pharmaceutical agents which can be used for recovery after brain
surgery or spinal surgery can also be candidates, in general, for
low-molecular-weight compounds to be fused.
[0317] If an anti-hTfR antibody originates from a non-human animal,
its administration to human could entail a substantial risk of
causing an antigen-antibody interaction, thereby provoking adverse
side-effects. By converting them to humanized antibodies, the
antigenicity of non-human animal antibodies can be reduced and
therefore the provocation of side-effects due to antigen-antibody
interaction can be suppressed when administered to a human.
Further, it has been reported that according to experiments using
monkeys, humanized antibodies are more stable than mouse antibodies
in the blood, and it is expected that their therapeutic effect can
therefore become longer-lasting accordingly. Provocation of
side-effects due to an antigen-antibody interaction can be
suppressed also by employing a human antibody as the anti-hTfR
antibody.
[0318] A detailed explanation will be given below regarding the
case where the anti-hTfR antibody is a humanized antibody or human
antibody. In human antibody light chain, there are .lamda., and
.kappa. chains. The light chain constituting the human antibody may
either be .lamda. and .kappa. chain. And in human heavy chain,
there are .gamma., .mu., .alpha., .sigma., and .epsilon. chains,
which correspond to IgG, IgM, IgA, IgD and IgE, respectively.
Though the heavy chain constituting the anti-hTfR antibody may be
any one of .gamma., .mu., .alpha., .sigma., and .epsilon. chains,
preferred is a .gamma. chain. Further, in .gamma. chain of human
heavy chain, there are .gamma.1, .gamma.2, .gamma.3 and .gamma.4
chains, which correspond to IgG1, IgG2, IgG3 and IgG4,
respectively. Where the heavy chain constituting the anti-hTfR
antibody is a .gamma. chain, though the .gamma. chain may be any of
.gamma.1, .gamma.2, .gamma.3 and .gamma.4 chains, preferred is a
.gamma.1 or .gamma.4 chain. In the case where the anti-hTfR
antibody is a humanized antibody or human antibody and IgG, the
human antibody light chain may either be .lamda. chain or .kappa.
chain, and though the human antibody heavy chain may either be
.gamma.1, .gamma.2, .gamma.3 and .gamma.4 chains, preferred is a
.gamma.1 or .gamma.4 chain. For example, a preferable embodiment of
anti-hTfR antibody includes one whose light chain is a .lamda.
chain and heavy chain is a .gamma.1 chain.
[0319] In the case where the anti-hTfR antibody is a humanized
antibody or a human antibody, the anti-hTfR antibody and a
different protein (A) may be bound together by linking the
anti-hTfR antibody, at the N-terminus (or the C-terminus) of the
heavy chain or light chain, via a linker sequence or directly, to
the C-terminus (or the N-terminus), respectively, of the different
protein (A), by peptide bonds. When linking the different protein
(A) to the anti-hTfR antibody heavy chain on the N-terminal side
(or to the C-terminal side) thereof, the C-terminus (or the
N-terminus), respectively, of the different protein (A) is linked
to the N-terminus (or the C-terminus) of the .gamma., .mu.,
.alpha., .sigma. or .epsilon. chain of anti-hTfR antibody, via a
linker sequence or directly, by peptide bonds. When linking the
different protein (A) to the anti-hTfR antibody light chain on the
N-terminal side (or the C-terminal side) thereof, the C-terminus
(or the N-terminus), respectively, of the different protein (A) in
linked to the N-terminus (or the C-terminus) of the .lamda. chain
and .kappa. chain of anti-hTfR antibody, via a linker sequence or
directly, by peptide bonds. However, in the case where the
anti-hTfR antibody consists of the Fab region, or of the Fab region
and the whole or part of the hinge region (Fab, F(ab').sub.2, and
F(ab')), the different protein (A) may be linked at the C-terminus
(or the N-terminus) thereof and via a linker sequence or directly,
to the N-terminus (or the C-terminus), respectively, of the heavy
chain or light chain that constitutes the Fab, F(ab').sub.2 and
F(ab'), by peptide bonds.
[0320] In a fusion protein produced by linking the different
protein (A) to the light chain of the anti-hTfR antibody which is a
humanized antibody, or a human antibody, on the C-terminal side
thereof, the anti-human transferrin receptor antibody comprises an
amino acid sequence comprising the whole or part of the light chain
variable region and the amino acid sequence comprising the whole or
part of the heavy chain variable region. The anti-hTfR antibody
light chain and the different protein (A) here may be linked
directly or via a linker.
[0321] In a fusion protein produced by linking the different
protein (A) to the heavy chain of the anti-hTfR antibody which is a
humanized antibody, or human antibody, on the C-terminal side
thereof, the anti-human transferrin receptor antibody comprises an
amino acid sequence comprising the whole or part of the light chain
variable region and the amino acid sequence comprising the whole or
part of the heavy chain variable region. The anti-hTfR antibody
heavy chain and the different protein (A) here may be linked
directly or via a linker.
[0322] In a fusion protein produced by linking the different
protein (A) to the light chain of the anti-hTfR antibody which is a
humanized antibody, or a human antibody, on the N-terminal side
thereof, the anti-human transferrin receptor antibody comprises an
amino acid sequence comprising the whole or part of the light chain
variable region and the amino acid sequence comprising the whole or
part of the heavy chain variable region. The anti-hTfR antibody
light chain and the different protein (A) here may be linked
directly or via a linker.
[0323] In a fusion protein produced by linking the different
protein (A) to the heavy chain of the anti-hTfR antibody which is a
humanized antibody, or human antibody, on the N-terminal side
thereof, the anti-human transferrin receptor antibody comprises an
amino acid sequence comprising the whole or part of the light chain
variable region and the amino acid sequence comprising the whole or
part of the heavy chain variable region. The anti-hTfR antibody
heavy chain and the different protein (A) here may be linked
directly or via a linker.
[0324] When placing a linker sequence between the anti-hTfR
antibody and a different protein (A), the linker sequence is
preferably a peptide chain consisting of 1 to 50 amino acids,
though the number of the amino acids constituting such a linker
sequence may be adjusted as desired in accordance with the
different protein (A) to be linked to the anti-hTfR antibody, like
1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, and so
on. While there is no particular limitation as to the specific
amino acid sequence of such a linker sequence insofar as the
anti-hTfR antibody and the different protein (A) linked by the
linker sequence retain their respective functions (affinity to
hTfR, and activity or function under a physiological condition), it
is preferably composed of glycine or serine, for example, one
consisting of a single amino acid either glycine or serine, the
amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser,
the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), the
amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4), the
amino acid sequence Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a
sequence which includes 1 to 10 or 2 to 5 of any of those amino
acid sequences consecutively linked. For example, a linker sequence
comprising 27 amino acids is preferably used that is composed of
the amino acid sequence Gly-Ser followed by consecutively linked
five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:3).
[0325] Besides, when stated here that a different protein (A) fused
with the anti-hTfR antibody retains its activity or function under
a physiological condition, or simply, it "retains the activity", it
means that in comparison with the intrinsic activity of the
natural-type of the different protein (A), not lower than 3% of the
activity or function is retained. However, such an activity or
function is preferably not lower than 10%, more preferably not
lower than 20%, still more preferably not lower than 50%, and even
more preferably not lower than 80%, in comparison with the
intrinsic activity of the natural-type of the different protein
(A). The same also applies where the different protein (A) fused
with the anti-hTfR antibody is a mutated one.
[0326] A further example of specific embodiments of the fusion
protein between a humanized anti-hTfR antibody and a different
protein (A) of the present invention is one produced by fusing the
anti-hTfR antibody heavy chain, on the C-terminal side thereof,
with a different protein (A), via a linker sequence consisting of
27 amino acids that is composed of the amino acid sequence Gly-Ser
followed by consecutively linked five copies of the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3).
[0327] In the case where the anti-hTfR antibody is Fab, an example
of specific embodiments of fusion proteins between a humanized
anti-hTfR antibody of the present invention and a different protein
(A) is one produced by fusing a different protein (A), on the
C-terminal side thereof via a linker sequences, with the region
consisting of anti-hTfR antibody heavy chain variable region and
its accompanying C.sub.H1 region, wherein the linker sequence
consists of 27 amino acids that is composed of the amino acid
sequence Gly-Ser followed by consecutively linked five copies of
the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3). Though
it is also allowed here that part of the hinge region is also
besides the C.sub.H1 region, the hinge region does not contain a
cysteine residue that would form a disulfide bond between heavy
chains.
[0328] The specific affinity of the anti-hTfR antibody to hTfR
resides mainly in the amino acid sequences of CDRs of the heavy
chain and light chain of the anti-hTfR antibody. There is no
particular limitation as to the amino acid sequences of those CDRs
insofar as the anti-hTfR antibody has a specific affinity to hTfR.
However, the anti-hTfR antibody of the present invention is one
whose dissociation constant (K.sub.D) with hTfR as measured by the
method described in Example 7 is preferably not greater than
1.times.10.sup.-8M, more preferably not greater than
1.times.10.sup.-9 M, still more preferably not greater than
1.times.10.sup.-10 M, and even more preferably not greater than
1.times.10.sup.-11 M. For example, one having a dissociation
constant of 1.times.10.sup.-13M to 1.times.10.sup.-9M, or
1.times.10.sup.-13M to 1.times.10.sup.-10 M is preferable. The same
also applies when the antibody is a single-chain antibody. Further,
where the anti-hTfR antibody of the present invention has affinity
also to monkey TfR, the dissociation constant of the anti-hTfR
antibody with monkey TfR, as measured by the method described in
Example 7, is preferably not greater than 5.times.10.sup.-8 M, more
preferably not greater than 2.times.10.sup.-8M, and still more
preferably not greater than 1.times.10.sup.-8 M. For example, one
which exhibits a dissociation constant of 1.times.10.sup.-13M to
2.times.10.sup.-8M is preferred. The same also applies if the
antibody is a single-chain antibody.
[0329] Examples of preferable embodiments of the antibody having
affinity to hTfR include those whose light chain CDRs have amino
acid sequences according to one of (1) to (14) below:
[0330] (1) the amino acid sequence set forth as SEQ ID NO:6 or SEQ
ID NO:7 as CDR1; the amino acid sequence set forth as SEQ ID NO:8
or SEQ ID NO:9 or the amino acid sequence Trp-Thr-Ser as CDR2; and
the amino acid sequence set forth as SEQ ID NO:10 as CDR3;
[0331] (2) the amino acid sequence set forth as SEQ ID NO:11 or SEQ
ID NO:12 as CDR1; the amino acid sequence set forth as SEQ ID NO:13
or SEQ ID NO:14 or the amino acid sequence Tyr-Ala-Ser as CDR2; and
the amino acid sequence set forth as SEQ ID NO:15 as CDR3;
[0332] (3) the amino acid sequence set forth as SEQ ID NO:16 or SEQ
ID NO:17 as CDR1; the amino acid sequence set forth as SEQ ID NO:18
or SEQ ID NO:19 or the amino acid sequence Lys-Val-Ser as CDR2; and
the amino acid sequence set forth as SEQ ID NO:20 as CDR3;
[0333] (4) the amino acid sequence set forth as SEQ ID NO:21 or SEQ
ID NO:22 as CDR1; the amino acid sequence set forth as SEQ ID NO:23
or SEQ ID NO:24 or the amino acid sequence Asp-Thr-Ser as CDR2; and
the amino acid sequence set forth as SEQ ID NO:25 as CDR3;
[0334] (5) the amino acid sequence set forth as SEQ ID NO:26 or SEQ
ID NO:27 as CDR1; the amino acid sequence set forth as SEQ ID NO:28
or SEQ ID NO:29 or the amino acid sequence Asp-Thr-Ser as CDR2; and
the amino acid sequence set forth as SEQ ID NO:30 as CDR3;
[0335] (6) the amino acid sequence set forth as SEQ ID NO:31 or SEQ
ID NO:32 as CDR1; the amino acid sequence set forth as SEQ ID NO:33
or SEQ ID NO:34 or the amino acid sequence Ala-Ala-Ser as CDR2; and
the amino acid sequence set forth as SEQ ID NO:35 as CDR3;
[0336] (7) the amino acid sequence set forth as SEQ ID NO:36 or SEQ
ID NO:37 as CDR1; the amino acid sequence set forth as SEQ ID NO:38
or SEQ ID NO:39 or the amino acid sequence Gln-Th-Ser as CDR2; and
the amino acid sequence set forth as SEQ ID NO:40 as CDR3;
[0337] (8) the amino acid sequence set forth as SEQ ID NO:41 or SEQ
ID NO:42 as CDR1; the amino acid sequence set forth as SEQ ID NO:43
or SEQ ID NO:44 or the amino acid sequence Gly-Thr-Ser as CDR2; and
the amino acid sequence set forth as SEQ ID NO:45 as CDR3;
[0338] (9) the amino acid sequence set forth as SEQ ID NO:46 or SEQ
ID NO:47 as CDR1; the amino acid sequence set forth as SEQ ID NO:48
or SEQ ID NO:49 or the amino acid sequence Phe-Thr-Ser as CDR2; and
the amino acid sequence set forth as SEQ ID NO:50 as CDR3;
[0339] (10) the amino acid sequence set forth as SEQ ID NO:51 or
SEQ ID NO:52 as CDR1; the amino acid sequence set forth as SEQ ID
NO:53 or SEQ ID NO:54 or the amino acid sequence Ala-Ala-Ser as
CDR2; and the amino acid sequence set forth as SEQ ID NO:55 as
CDR3;
[0340] (11) the amino acid sequence set forth as SEQ ID NO:56 or
SEQ ID NO:57 as CDR1; the amino acid sequence set forth as SEQ ID
NO:58 or SEQ ID NO:59 or the amino acid sequence Tyr-Ala-Ser as
CDR2; and the amino acid sequence set forth as SEQ ID NO:60 as
CDR3;
[0341] (12) the amino acid sequence set forth as SEQ ID NO:61 or
SEQ ID NO:62 as CDR1; the amino acid sequence set forth as SEQ ID
NO:63 or SEQ ID NO:64 or the amino acid sequence Trp-Ser-Ser as
CDR2; and the amino acid sequence set forth as SEQ ID NO:65 as
CDR3;
[0342] (13) the amino acid sequence set forth as SEQ ID NO:66 or
SEQ ID NO:67 as CDR1; the amino acid sequence set forth as SEQ ID
NO:68 or SEQ ID NO:69 or the amino acid sequence Tyr-Ala-Ser as
CDR2; and the amino acid sequence set forth as SEQ ID NO:70 as
CDR3; and
[0343] (14) the amino acid sequence set forth as SEQ ID NO:71 or
SEQ ID NO:72 as CDR1; the amino acid sequence set forth as SEQ ID
NO:73 or SEQ ID NO:74 or the amino acid sequence Asp-Thr-Ser as
CDR2; and the amino acid sequence set forth as SEQ ID NO:75 as
CDR3.
[0344] Examples of more specific embodiments of the antibody having
affinity to hTfR include those whose light chain CDRs have amino
acid sequences according to one of (1) to (14) below:
[0345] (1) the amino acid sequence set forth as SEQ ID NO:6 as
CDR1; SEQ ID NO:8 as CDR2; and SEQ ID NO:10 as CDR3;
[0346] (2) the amino acid sequence set forth as SEQ ID NO:11 as
CDR1; SEQ ID NO:13 as CDR2; and SEQ ID NO:15 as CDR3;
[0347] (3) the amino acid sequence set forth as SEQ ID NO:16 as
CDR1; SEQ ID NO:18 as CDR2; and SEQ ID NO:20 as CDR3;
[0348] (4) the amino acid sequence set forth as SEQ ID NO:21 as
CDR1; SEQ ID NO:23 as CDR2; and SEQ ID NO:25 as CDR3;
[0349] (5) the amino acid sequence set forth as SEQ ID NO:26 as
CDR1; SEQ ID NO:28 as CDR2; and SEQ ID NO:30 as CDR3;
[0350] (6) the amino acid sequence set forth as SEQ ID NO:31 as
CDR1; SEQ ID NO:33 as CDR2; and SEQ ID NO:35 as CDR3;
[0351] (7) the amino acid sequence set forth as SEQ ID NO:36 as
CDR1; SEQ ID NO:38 as CDR2; and SEQ ID NO:40 as CDR3;
[0352] (8) the amino acid sequence set forth as SEQ ID NO:41 as
CDR1; SEQ ID NO:43 as CDR2; and SEQ ID NO:45 as CDR3;
[0353] (9) the amino acid sequence set forth as SEQ ID NO:46 as
CDR1; SEQ ID NO:48 as CDR2; and SEQ ID NO:50 as CDR3;
[0354] (10) the amino acid sequence set forth as SEQ ID NO:51 as
CDR1; SEQ ID NO:53 as CDR2; and SEQ ID NO:55 as CDR3;
[0355] (11) the amino acid sequence set forth as SEQ ID NO:56 as
CDR1; SEQ ID NO:58 as CDR2; and SEQ ID NO:60 as CDR3;
[0356] (12) the amino acid sequence set forth as SEQ ID NO:61 as
CDR1; SEQ ID NO:63 as CDR2; and SEQ ID NO:65 as CDR3;
[0357] (13) the amino acid sequence set forth as SEQ ID NO:66 as
CDR1; SEQ ID NO:68 as CDR2; and SEQ ID NO:70 as CDR3; and
[0358] (14) the amino acid sequence set forth as SEQ ID NO:71 as
CDR1; SEQ ID NO:73 as CDR2; and SEQ ID NO:75 as CDR3;
[0359] Examples of preferable embodiments of the antibody having
affinity to hTfR include those whose heavy chain CDRs have amino
acid sequences according to one of (1) to (14) below:
[0360] (1) the amino acid sequence set forth as SEQ ID NO:76 or SEQ
ID NO:77 as CDR1; the amino acid sequence set forth as SEQ ID NO:78
or SEQ ID NO:79 as CDR2; and the amino acid sequence set forth as
SEQ ID NO:80 or SEQ ID NO:81 as CDR3;
[0361] (2) the amino acid sequence set forth as SEQ ID NO:82 or SEQ
ID NO:83 as CDR1; the amino acid sequence set forth as SEQ ID NO:84
or SEQ ID NO:85 as CDR2; and the amino acid sequence set forth as
SEQ ID NO:86 or SEQ ID NO:87 as CDR3;
[0362] (3) the amino acid sequence set forth as SEQ ID NO:88 or SEQ
ID NO:89 as CDR1; the amino acid sequence set forth as SEQ ID NO:90
or SEQ ID NO:91 as CDR2; and the amino acid sequence set forth as
SEQ ID NO:92 or SEQ ID NO:93 as CDR3;
[0363] (4) the amino acid sequence set forth as SEQ ID NO:94 or SEQ
ID NO:95 as CDR1; the amino acid sequence set forth as SEQ ID NO:96
or SEQ ID NO:97 as CDR2; and the amino acid sequence set forth as
SEQ ID NO:98 or SEQ ID NO:99 as CDR3;
[0364] (5) the amino acid sequence set forth as SEQ ID NO:100 or
SEQ ID NO:101 as CDR1; the amino acid sequence set forth as SEQ ID
NO:102 or SEQ ID NO:103 as CDR2; and the amino acid sequence set
forth as SEQ ID NO:104 or SEQ ID NO:105 as CDR3;
[0365] (6) the amino acid sequence set forth as SEQ ID NO:106 or
SEQ ID NO:107 as CDR1; the amino acid sequence set forth as SEQ ID
NO:108 or SEQ ID NO:278 as CDR2; and the amino acid sequence set
forth as SEQ ID NO:109 or SEQ ID NO:110 as CDR3;
[0366] (7) the amino acid sequence set forth as SEQ ID NO:111 or
SEQ ID NO:112 as CDR1; the amino acid sequence set forth as SEQ ID
NO:113 or SEQ ID NO:114 as CDR2; and the amino acid sequence set
forth as SEQ ID NO:115 or SEQ ID NO:116 as CDR3;
[0367] (8) the amino acid sequence set forth as SEQ ID NO:117 or
SEQ ID NO:118 as CDR1; the amino acid sequence set forth as SEQ ID
NO:119 or SEQ ID NO:279 as CDR2; and the amino acid sequence set
forth as SEQ ID NO:120 or SEQ ID NO:121 as CDR3;
[0368] (9) the amino acid sequence set forth as SEQ ID NO:122 or
SEQ ID NO:123 as CDR1; the amino acid sequence set forth as SEQ ID
NO:124 or SEQ ID NO:125 as CDR2; and the amino acid sequence set
forth as SEQ ID NO:126 or SEQ ID NO:127 as CDR3;
[0369] (10) the amino acid sequence set forth as SEQ ID NO:128 or
SEQ ID NO:129 as CDR1; the amino acid sequence set forth as SEQ ID
NO:130 or SEQ ID NO:131 as CDR2; and SEQ ID NO:132 or SEQ ID NO:133
as CDR3;
[0370] (11) the amino acid sequence set forth as SEQ ID NO:134 or
SEQ ID NO:135 as CDR1; the amino acid sequence set forth as SEQ ID
NO:136 or SEQ ID NO:137 as CDR2, and the amino acid sequence set
forth as SEQ ID NO:138 or SEQ ID NO:139 as CDR3;
[0371] (12) the amino acid sequence set forth as SEQ ID NO:140 or
SEQ ID NO:141 as CDR1; the amino acid sequence set forth as SEQ ID
NO:142 or SEQ ID NO:143 as CDR2; and the amino acid sequence set
forth as SEQ ID NO:144 or SEQ ID NO:145 as CDR3;
[0372] (13) the amino acid sequence set forth as SEQ ID NO:146 or
SEQ ID NO:147 as CDR1; the amino acid sequence set forth as SEQ ID
NO:148 or SEQ ID NO:149 as CDR2; and the amino acid sequence set
forth as SEQ ID NO:150 or SEQ ID NO:151 as CDR3; and
[0373] (14) the amino acid sequence set forth as SEQ ID NO:152 or
SEQ ID NO:153 as CDR1; the amino acid sequence set forth as SEQ ID
NO:154 or SEQ ID NO:155 as CDR2; and the amino acid sequence set
forth as SEQ ID NO:156 or SEQ ID NO:157 as CDR3.
[0374] Examples of more specific embodiments of the antibody having
affinity to hTfR include those whose heavy chain CDRs have amino
acid sequences according to one of (1) to (14) below:
[0375] (1) the amino acid sequence set forth as SEQ ID NO:76 as
CDR1; SEQ ID NO:78 as CDR2; and SEQ ID NO:80 as CDR3;
[0376] (2) the amino acid sequence set forth as SEQ ID NO:82 as
CDR1; SEQ ID NO:84 as CDR2; and SEQ ID NO:86 as CDR3;
[0377] (3) the amino acid sequence set forth as SEQ ID NO:88 as
CDR1; SEQ ID NO:90 as CDR2; and SEQ ID NO:92 as CDR3;
[0378] (4) the amino acid sequence set forth as SEQ ID NO:94 as
CDR1; SEQ ID NO:96 as CDR2; and SEQ ID NO:98 as CDR3;
[0379] (5) the amino acid sequence set forth as SEQ ID NO:100 as
CDR1; SEQ ID NO:102 as CDR2; and SEQ ID NO:104 as CDR3;
[0380] (6) the amino acid sequence set forth as SEQ ID NO:106 as
CDR1; SEQ ID NO:108 as CDR2; and SEQ ID NO:109 as CDR3;
[0381] (7) the amino acid sequence set forth as SEQ ID NO:111 as
CDR1; SEQ ID NO:113 as CDR2; and SEQ ID NO:115 as CDR3;
[0382] (8) the amino acid sequence set forth as SEQ ID NO:117 as
CDR1; SEQ ID NO:119 as CDR2; and SEQ ID NO:120 as CDR3;
[0383] (9) the amino acid sequence set forth as SEQ ID NO:122 as
CDR1; SEQ ID NO:124 as CDR2; and SEQ ID NO:126 as CDR3;
[0384] (10) the amino acid sequence set forth as SEQ ID NO:128 as
CDR1; SEQ ID NO:130 as CDR2; and SEQ ID NO:132 as CDR3;
[0385] (11) the amino acid sequence set forth as SEQ ID NO:134 as
CDR1; SEQ ID NO:136 as CDR2; and SEQ ID NO:138 as CDR3;
[0386] (12) the amino acid sequence set forth as SEQ ID NO:140 as
CDR1; SEQ ID NO:142 as CDR2; and SEQ ID NO:144 as CDR3;
[0387] (13) the amino acid sequence set forth as SEQ ID NO:146 as
CDR1; SEQ ID NO:148 as CDR2; and SEQ ID NO:150 as CDR3; and
[0388] (14) the amino acid sequence set forth as SEQ ID NO:152 as
CDR1; SEQ ID NO:154 as CDR2; and SEQ ID NO:156 as CDR3.
[0389] Examples of preferable combinations of the light chain and
heavy chain of the antibody having affinity to hTfR include those
having the amino acid sequences as CDRs according to (1) to (14)
below:
[0390] (1) a combination of
[0391] the light chain having the amino acid sequence set forth as
SEQ ID NO:6 or SEQ ID NO:7 as CDR1; the amino acid sequence set
forth as SEQ ID NO:8 or SEQ ID NO:9 or the amino acid sequence
Trp-Thr-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:10 as CDR3; and
[0392] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:76 or SEQ ID NO:77 as CDR1; the amino acid sequence set
forth as SEQ ID NO:78 or SEQ ID NO:79 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:80 or SEQ ID NO:81 as CDR3;
[0393] (2) a combination of
[0394] the light chain having the amino acid sequence set forth as
SEQ ID NO:11 or SEQ ID NO:12 as CDR1; the amino acid sequence set
forth as SEQ ID NO:13 or SEQ ID NO:14 or the amino acid sequence
Tyr Ala Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:15 as CDR3; and
[0395] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:82 or SEQ ID NO:83 as CDR1; the amino acid sequence set
forth as SEQ ID NO:84 or SEQ ID NO:85 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:86 or SEQ ID NO:87 as CDR3;
[0396] (3) a combination of
[0397] the light chain having the amino acid sequence set forth as
SEQ ID NO:16 or SEQ ID NO:17 as CDR1; the amino acid sequence set
forth as SEQ ID NO:18 or SEQ ID NO:19 or the amino acid sequence
Lys-Val-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:20 as CDR3; and
[0398] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:88 or SEQ ID NO:89 as CDR1; the amino acid sequence set
forth as SEQ ID NO:90 or SEQ ID NO:91 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:92 or SEQ ID NO:93 as CDR3;
[0399] (4) a combination of
[0400] the light chain having the amino acid sequence set forth as
SEQ ID NO:21 or SEQ ID NO:22 as CDR1; the amino acid sequence set
forth as SEQ ID NO:23 or SEQ ID NO:24 or the amino acid sequence
Asp-Thr-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:25 as CDR3; and
[0401] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:94 or SEQ ID NO:95 as CDR1; the amino acid sequence set
forth as SEQ ID NO:96 or SEQ ID NO:97 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:98 or SEQ ID NO:99 as CDR3;
[0402] (5) a combination of
[0403] the light chain having the amino acid sequence set forth as
SEQ ID NO:26 or SEQ ID NO:27 as CDR1; the amino acid sequence set
forth as SEQ ID NO:28 or SEQ ID NO:29 or the amino acid sequence
Asp-Thr-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:30 as CDR3;
[0404] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:100 or SEQ ID NO:101 as CDR1; the amino acid sequence set
forth as SEQ ID NO:102 or SEQ ID NO:103 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:104 or SEQ ID NO:105 as CDR3;
[0405] (6) a combination of
[0406] the light chain having the amino acid sequence set forth as
SEQ ID NO:31 or SEQ ID NO:32 as CDR1; the amino acid sequence set
forth as SEQ ID NO:33 or SEQ ID NO:34 or the amino acid sequence
Ala-Ala-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:35 as CDR3; and
[0407] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:106 or SEQ ID NO:107 as CDR1; the amino acid sequence set
forth as SEQ ID NO:108 or SEQ ID NO:278 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:109 or SEQ ID NO:110 as CDR3;
[0408] (7) a combination of
[0409] the light chain having the amino acid sequence set forth as
SEQ ID NO:36 or SEQ ID NO:37 as CDR1; the amino acid sequence set
forth as SEQ ID NO:38 or SEQ ID NO:39 or the amino acid sequence
Gln-Thr-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:40 as CDR3; and
[0410] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:111 or SEQ ID NO:112 as CDR1; the amino acid sequence set
forth as SEQ ID NO:113 or SEQ ID NO:114 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:115 or SEQ ID NO:116 as CDR3;
[0411] (8) a combination of
[0412] the light chain having the amino acid sequence set forth as
SEQ ID NO:41 or SEQ ID NO:42 as CDR1; the amino acid sequence set
forth as SEQ ID NO:43 or SEQ ID NO:44 or the amino acid sequence
Gly-Thr-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:45 as CDR3; and
[0413] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:117 or SEQ ID NO:118 as CDR1; the amino acid sequence set
forth as SEQ ID NO:119 or SEQ ID NO:279 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:120 or SEQ ID NO:121 as CDR3;
[0414] (9) a combination of
[0415] the light chain having the amino acid sequence set forth as
SEQ ID NO:46 or SEQ ID NO:47 as CDR1; the amino acid sequence set
forth as SEQ ID NO:48 or SEQ ID NO:49 or the amino acid sequence
Phe-Thr-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:50 as CDR3; and
[0416] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:122 or SEQ ID NO:123 as CDR1; the amino acid sequence set
forth as SEQ ID NO:124 or SEQ ID NO:125 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:126 or SEQ ID NO:127 as CDR3;
[0417] (10) a combination of
[0418] the light chain having the amino acid sequence set forth as
SEQ ID NO:51 or SEQ ID NO:52 as CDR1; the amino acid sequence set
forth as SEQ ID NO:53 or SEQ ID NO:54 or the amino acid sequence
Ala-Ala-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:55 as CDR3; and
[0419] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:128 or SEQ ID NO:129 as CDR1; the amino acid sequence set
forth as SEQ ID NO:130 or SEQ ID NO:131 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:132 or SEQ ID NO:133 as CDR3;
[0420] (11) a combination of
[0421] the light chain having the amino acid sequence set forth as
SEQ ID NO:56 or SEQ ID NO:57 as CDR1; the amino acid sequence set
forth as SEQ ID NO:58 or SEQ ID NO:59 or the amino acid sequence
Tyr-Ala-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:60 as CDR3; and
[0422] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:134 or SEQ ID NO:135 as CDR1; the amino acid sequence set
forth as SEQ ID NO:136 or SEQ ID NO:137 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:138 or SEQ ID NO:139 as CDR3;
[0423] (12) a combination of
[0424] the light chain having the amino acid sequence set forth as
SEQ ID NO:61 or SEQ ID NO:62 as CDR1; the amino acid sequence set
forth as SEQ ID NO:63 or SEQ ID NO:64 or the amino acid sequence
Trp-Ser-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:65 as CDR3; and
[0425] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:140 or SEQ ID NO:141 as CDR1; the amino acid sequence set
forth as SEQ ID NO:142 or SEQ ID NO:143 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:144 or SEQ ID NO:145 as CDR3;
[0426] (13) a combination of
[0427] the light chain having the amino acid sequence set forth as
SEQ ID NO:66 or SEQ ID NO:67 as CDR1; the amino acid sequence set
forth as SEQ ID NO:68 or SEQ ID NO:69 or the amino acid sequence
Tyr-Ala-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:70 as CDR3; and
[0428] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:146 or SEQ ID NO:147 as CDR1; the amino acid sequence set
forth as SEQ ID NO:148 or SEQ ID NO:149 as CDR2; and the amino acid
sequence set forth as SEQ ID NO:150 or SEQ ID NO:151 as CDR3;
[0429] (14) a combination of
[0430] the light chain having the amino acid sequence set forth as
SEQ ID NO:71 or SEQ ID NO:72 as CDR1; the amino acid sequence set
forth as SEQ ID NO:73 or SEQ ID NO:74 or the amino acid sequence
Asp-Thr-Ser as CDR2; and the amino acid sequence set forth as SEQ
ID NO:75 as CDR3; and
[0431] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:152 or SEQ ID NO:153 as CDR1; the amino acid sequence set
forth as SEQ ID NO:154 or SEQ ID NO:155 as CDR2; and he amino acid
sequence set forth as SEQ ID NO:156 or SEQ ID NO:157 as CDR3.
[0432] Examples of specific embodiments of combinations of the
light chain and heavy chain of the antibody having affinity to hTfR
include those having the amino acid sequences as CDRs according to
one of (1) to (14) below:
[0433] (1) a combination of
[0434] the light chain having the amino acid sequence set forth as
SEQ ID NO:6 as CDR1; SEQ ID NO:8 as CDR2; and SEQ ID NO:10 as CDR3;
and
[0435] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:76 as CDR1; SEQ ID NO:78 as CDR2; and SEQ ID NO:80 as
CDR3;
[0436] (2) a combination of
[0437] the light chain having the amino acid sequence set forth as
SEQ ID NO:11 as CDR1; SEQ ID NO:13 as CDR2; and SEQ ID NO:15 as
CDR3; and
[0438] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:82 as CDR1; SEQ ID NO:84 as CDR2; and SEQ ID NO:86 as
CDR3;
[0439] (3) a combination of
[0440] the light chain having the amino acid sequence set forth as
SEQ ID NO:16 as CDR1; SEQ ID NO:18 as CDR2; and SEQ ID NO:20 as
CDR3; and
[0441] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:88 as CDR1; SEQ ID NO:90 as CDR2; and SEQ ID NO:92 as
CDR3;
[0442] (4) a combination of
[0443] the light chain having the amino acid sequence set forth as
SEQ ID NO:21 as CDR1; SEQ ID NO:23 as CDR2; and SEQ ID NO:25 as
CDR3; and
[0444] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:94 as CDR1; SEQ ID NO:96 as CDR2; and SEQ ID NO:98 as
CDR3;
[0445] (5) a combination of
[0446] the light chain having the amino acid sequence set forth as
SEQ ID NO:26 as CDR1; SEQ ID NO:28 as CDR2; and SEQ ID NO:30 as
CDR3; and
[0447] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:100 as CDR1; SEQ ID NO:102 as CDR2; and SEQ ID NO:104 as
CDR3;
[0448] (6) a combination of
[0449] the light chain having the amino acid sequence set forth as
SEQ ID NO:31 as CDR1; SEQ ID NO:33 as CDR2; and SEQ ID NO:35 as
CDR3; and
[0450] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:106 as CDR1; SEQ ID NO:108 as CDR2; and SEQ ID NO:109 as
CDR3;
[0451] (7) a combination of
[0452] the light chain having the amino acid sequence set forth as
SEQ ID NO:36 as CDR1; SEQ ID NO:38 as CDR2; and SEQ ID NO:40 as
CDR3; and
[0453] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:111 as CDR1; SEQ ID NO:113 as CDR2; and SEQ ID NO:115 as
CDR3;
[0454] (8) a combination of
[0455] the light chain having the amino acid sequence set forth as
SEQ ID NO:41 as CDR1; SEQ ID NO:43 as CDR2; and SEQ ID NO:45 as
CDR3; and
[0456] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:117 as CDR1; SEQ ID NO:119 as CDR2; and SEQ ID NO:120 as
CDR3;
[0457] (9) a combination of
[0458] the light chain having the amino acid sequence set forth as
SEQ ID NO:46 as CDR1; SEQ ID NO:48 as CDR2; and SEQ ID NO:50 as
CDR3; and
[0459] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:122 as CDR1; SEQ ID NO:124 as CDR2; and SEQ ID NO:126 as
CDR3;
[0460] (10) a combination of
[0461] the light chain having the amino acid sequence set forth as
SEQ ID NO:51 as CDR1; SEQ ID NO:53 as CDR2; and SEQ ID NO:55 as
CDR3; and
[0462] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:128 as CDR1; SEQ ID NO:130 as CDR2; and SEQ ID NO:132 as
CDR3;
[0463] (11) a combination of
[0464] the light chain having the amino acid sequence set forth as
SEQ ID NO:56 as CDR1; SEQ ID NO:58 as CDR2; and SEQ ID NO:60 as
CDR3; and
[0465] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:134 as CDR1; SEQ ID NO:136 as CDR2; and SEQ ID NO:138 as
CDR3;
[0466] (12) a combination of
[0467] the light chain having the amino acid sequence set forth as
SEQ ID NO:61 as CDR1; SEQ ID NO:63 as CDR2; and SEQ ID NO:65 as
CDR3; and
[0468] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:140 as CDR1; SEQ ID NO:142 as CDR2; and SEQ ID NO:144 as
CDR3;
[0469] (13) a combination of
[0470] the light chain having the amino acid sequence set forth as
SEQ ID NO:66 as CDR1; SEQ ID NO:68 as CDR2; and SEQ ID NO:70 as
CDR3; and
[0471] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:146 as CDR1; SEQ ID NO:148 as CDR2; and SEQ ID NO:150 as
CDR3; and
[0472] (14) a combination of
[0473] the light chain having the amino acid sequence set forth as
SEQ ID NO:71 as CDR1; SEQ ID NO:73 as CDR2; and SEQ ID NO:75 as
CDR3; and
[0474] the heavy chain having the amino acid sequence set forth as
SEQ ID NO:152 as CDR1; SEQ ID NO:154 as CDR2; and SEQ ID NO:156 as
CDR3.
[0475] As preferred embodiments of humanized antibodies having
affinity to hTfR, there are humanized antibodies produced using the
amino acid sequences of the light chain variable region and the
heavy chain variable region of the mouse anti-human TfR antibody
set forth as SEQ ID NO:218 to SEQ ID NO:245 as CDRs. The humanized
antibodies are produced by replacing proper positions of a human
antibody with the amino acid sequences of CDRs of the light chain
variable region and the heavy chain variable region of mouse
anti-human TfR antibody.
[0476] For example, the light chain of a humanized antibody can be
made by replacing the amino acid sequences of corresponding to CDRs
of the light chain of a human antibody with an amino acid sequence
consisting of not less than 3, or not less than 6, consecutive
amino acids at the positions 24th to 34th of the amino acid
sequence set forth as SEQ ID NO:218 as CDR1; with an amino acid
sequence consisting of not less than 3, or not less than 6,
consecutive amino acids at the positions 50th to 56th of the amino
acid sequence set forth as SEQ ID NO:218 as CDR2; and with an amino
acid sequence consisting of not less than 3, or not less than 8,
consecutive amino acids at the positions 89th to 97th of the amino
acid sequence set forth as SEQ ID NO:218 as CDR3, and
[0477] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding to CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:219 as CDR1; with an amino acid sequence consisting of not
less than 3, or not less than 8, consecutive amino acids at the
positions 50th to 66th of the amino acid sequence set forth as SEQ
ID NO:219 as CDR2; with an amino acid sequence consisting of not
less than 3, or not less than 7, consecutive amino acids at the
positions 97th to 105th of the amino acid sequence set forth as SEQ
ID NO:219 as CDR3.
[0478] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0479] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 24th to 34th of the
amino acid sequence set forth as SEQ ID NO:220 as CDR1; with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 50th to 56th of the
amino acid sequence set forth as SEQ ID NO:220 as CDR2; and with an
amino acid sequence consisting of not less than 3 or not less than
8, consecutive amino acids at the positions 89th to 97th of the
amino acid sequence set forth as SEQ ID NO:220 as CDR3; and
[0480] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding to CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:221 as CDR1; with an amino acid sequence consisting of not
less than 3, or not less than 8, consecutive amino acids at the
positions 50th to 66th of the amino acid sequence set forth as SEQ
ID NO:221 as CDR2; and with an amino acid sequence consisting of
not less than 3, or not less than 14, consecutive amino acids at
the positions 97th to 112nd of the amino acid sequence set forth as
SEQ ID NO:221 as CDR3.
[0481] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0482] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3, or not less than
11, consecutive amino acids at the positions 24th to 39th of the
amino acid sequence set forth as SEQ ID NO:222 as CDR1; with an
amino acid sequence consisting of not less than 3, not less than 6,
consecutive amino acids at the positions 55th to 61st of the amino
acid sequence set forth as SEQ ID NO:222 as CDR2; and with an amino
acid sequence consisting of not less than 3, or not less than 8,
consecutive amino acids at the positions 94th to 102nd of the amino
acid sequence set forth as SEQ ID NO:222 as CDR3; and
[0483] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:223 as CDR1; with an amino acid sequence consisting of not
less than 3, or not less than 8, consecutive amino acids at the
positions 50th to 66th of the amino acid sequence set forth as SEQ
ID NO:223 as CDR2; and with an amino acid sequence consisting of
not less than 3, or not less than 9 consecutive amino acids at the
positions 97th to 107th of the amino acid sequence set forth as SEQ
ID NO:223 as CDR3.
[0484] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0485] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3, or not less than
5, consecutive amino acids at the positions 24th to 33rd of the
amino acid sequence set forth as SEQ ID NO:224 as CDR1; with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 49th to 55th of the
amino acid sequence set forth as SEQ ID NO:224 as CDR2; and with an
amino acid sequence consisting of not less than 3, or not less than
8, consecutive amino acids at the positions 88th to 96th of the
amino acid sequence set forth as SEQ ID NO:224 as CDR3; and
[0486] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:225 as CDR1; with an amino acid sequence consisting of not
less than 3, or not less than 8, consecutive amino acids at the
positions 51st to 66th of the amino acid sequence set forth as SEQ
ID NO:225 as CDR2; and with an amino acid sequence consisting of
not less than 3, or not less than 13, consecutive amino acids at
the positions 97th to 111st of the amino acid sequence set forth as
SEQ ID NO:225 as CDR3.
[0487] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0488] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3, or not less than
5, consecutive amino acids at the positions 24th to 33rd of the
amino acid sequence set forth as SEQ ID NO:226 as CDR1; with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 49th to 55th of the
amino acid sequence set forth as SEQ ID NO:226 as CDR2; and with an
amino acid sequence consisting of not less than 3, or not less than
7, consecutive amino acids at the positions 88th to 95th of the
amino acid sequence set forth as SEQ ID NO:226 as CDR3; and
[0489] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:227 as CDR1; with an amino acid sequence consisting of not
less than 3, or not less than 8, consecutive amino acids at the
positions 51st to 66th of the amino acid sequence set forth as SEQ
ID NO:227 as CDR2; and with an amino acid sequence consisting of
not less than 3, or not less than 9, consecutive amino acids at the
positions 97th to 107th of the amino acid sequence set forth as SEQ
ID NO:227 as CDR3.
[0490] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0491] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 24th to 34th of the
amino acid sequence set forth as SEQ ID NO:228 as CDR1; with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 50th to 56th of the
amino acid sequence set forth as SEQ ID NO:228 as CDR2; and with an
amino acid sequence consisting of not less than 3, or not less than
8, consecutive amino acids at the positions 89th to 97th of the
amino acid sequence set forth as SEQ ID NO:228 as CDR3; and
[0492] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:229 as CDR1; with an amino acid sequence consisting of not
less than 3, or not less than 7, consecutive amino acids at the
positions 50th to 65th of the amino acid sequence set forth as SEQ
ID NO:229 as CDR2; and with an amino acid sequence consisting of
not less than 3, or not less than 4, consecutive amino acids at the
positions 96th to 101st of the amino acid sequence set forth as SEQ
ID NO:229 as CDR3.
[0493] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0494] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3, or not less than
5, consecutive amino acids at the positions 24th to 33rd of the
amino acid sequence set forth as SEQ ID NO:230 as CDR1; with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 49th to 55th of the
amino acid sequence set forth as SEQ ID NO:230 as CDR2; and with an
amino acid sequence consisting of not less than 3, or not less than
8, consecutive amino acids at the positions 88th to 96th of the
amino acid sequence set forth as SEQ ID NO:230 as CDR3; and
[0495] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:231 as CDR1; with an amino acid sequence consisting of not
less than 3, or not less than 8, consecutive amino acids at the
positions 51st to 66th of the amino acid sequence set forth as SEQ
ID NO:231 as CDR2; and with an amino acid sequence consisting of
not less than 3, or not less than 11, consecutive amino acids at
the positions 97th to 109th of the amino acid sequence set forth as
SEQ ID NO:231 as CDR3.
[0496] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0497] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3, or not less than
5, consecutive amino acids at the positions 24th to 33rd of the
amino acid sequence set forth as SEQ ID NO:232 as CDR1; with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 49th to 55th of the
amino acid sequence set forth as SEQ ID NO:232 as CDR2; and with an
amino acid sequence consisting of not less than 3, or not less than
8, consecutive amino acids at the positions 88th to 96th of the
amino acid sequence set forth as SEQ ID NO:232 as CDR3; and
[0498] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:233 as CDR1; with an amino acid sequence consisting of not
less than 3, or not less than 7, consecutive amino acids at the
positions 50th to 65th of the amino acid sequence set forth as SEQ
ID NO:233 as CDR2; and with an amino acid sequence consisting of
not less than 3, or not less than 4, consecutive amino acids at the
positions 96th to 101st of the amino acid sequence set forth as SEQ
ID NO:233 as CDR3.
[0499] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0500] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3, or not less than
5, consecutive amino acids at the positions 24th to 33rd of the
amino acid sequence set forth as SEQ ID NO:234 as CDR1; with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 49th to 55th of the
amino acid sequence set forth as SEQ ID NO:234 as CDR2; and with an
amino acid sequence consisting of not less than 3, or not less than
8, consecutive amino acids at the positions 88th to 96th of the
amino acid sequence set forth as SEQ ID NO:234 as CDR3, and
[0501] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:235 as CDR1; with an amino acid sequence consisting of not
less than 3, or not less than 8, consecutive amino acids at the
positions 51st to 66th of the amino acid sequence set forth as SEQ
ID NO:235 as CDR2, and with an amino acid sequence consisting of
not less than 3, or not less than 8, consecutive amino acids at the
positions 97th to 106th of the amino acid sequence set forth as SEQ
ID NO:235 as CDR3.
[0502] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0503] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 24th to 34th of the
amino acid sequence set forth as SEQ ID NO:236 as CDR1; with an
amino acid sequence consisting of not less than 3, or not less than
6 consecutive amino acids at the positions 50th to 56th of the
amino acid sequence set forth as SEQ ID NO:236 as CDR2; and with an
amino acid sequence consisting of not less than 3, or not less than
8, consecutive amino acids at the positions 89th to 97th of the
amino acid sequence set forth as SEQ ID NO:236 as CDR3; and
[0504] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:237 as CDR1; with an amino acid sequence consisting of not
less than 3, or not less than 8, consecutive amino acids at the
positions 51st to 66th of the amino acid sequence set forth as SEQ
ID NO:237 as CDR2; and with an amino acid sequence consisting of
not less than 3, or not less than 11, consecutive amino acids at
the positions 97th to 109th of the amino acid sequence set forth as
SEQ ID NO:237 as CDR3.
[0505] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0506] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 24th to 34th of the
amino acid sequence set forth as SEQ ID NO:238 as CDR1; with an
amino acid sequence consisting of not less than 3, or not less than
6, consecutive amino acids at the positions 50th to 56th of the
amino acid sequence set forth as SEQ ID NO:238 as CDR2; and with an
amino acid sequence consisting of not less than 3, or not less than
8, consecutive amino acids at the positions 89th to 97th of the
amino acid sequence set forth as SEQ ID NO:238 as CDR3; and
[0507] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:239 as CDR1; with an amino acid sequence consisting of not
less than 3 or not less than 8, consecutive amino acids at the
positions 51st to 66th of the amino acid sequence set forth as SEQ
ID NO:239 as CDR2; and with an amino acid sequence consisting of
not less than 3 or not less than 9, consecutive amino acids at the
positions 97th to 107th of the amino acid sequence set forth as SEQ
ID NO:239 as CDR3.
[0508] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0509] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3 or not less than
6, consecutive amino acids at the positions 24th to 34th of the
amino acid sequence set forth as SEQ ID NO:240 as CDR1; with an
amino acid sequence consisting of not less than 3 or not less than
6, consecutive amino acids at the positions 50th to 56th of the
amino acid sequence set forth as SEQ ID NO:240 as CDR2; and with an
amino acid sequence consisting of not less than 3 or not less than
8, consecutive amino acids at the positions 89th to 97th of the
amino acid sequence set forth as SEQ ID NO:240 as CDR3, and
[0510] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:241 as CDR1; with an amino acid sequence consisting of not
less than 3 or not less than 8, consecutive amino acids at the
positions 51st to 66th of the amino acid sequence set forth as SEQ
ID NO:241 as CDR2; and with an amino acid sequence consisting of
not less than 3 or not less than 10, consecutive amino acids at the
positions 97th to 108th of the amino acid sequence set forth as SEQ
ID NO:241 as CDR3.
[0511] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0512] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3 or not less than
6, consecutive amino acids at the positions 24th to 34th of the
amino acid sequence set forth as SEQ ID NO:242 as CDR1; with an
amino acid sequence consisting of not less than 3 or not less than
6, consecutive amino acids at the positions 50th to 56th of the
amino acid sequence set forth as SEQ ID NO:242 as CDR2; and with an
amino acid sequence consisting of not less than 3 or not less than
8, consecutive amino acids at the positions 89th to 97th of the
amino acid sequence set forth as SEQ ID NO:242 as CDR3; and
[0513] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:243 as CDR1; with an amino acid sequence consisting of not
less than 3 or not less than 8, consecutive amino acids at the
positions 51st to 66th of the amino acid sequence set forth as SEQ
ID NO:243 as CDR2; and with an amino acid sequence consisting of
not less than 3 or not less than 9, consecutive amino acids at the
positions 97th to 107th of the amino acid sequence set forth as SEQ
ID NO:243 as CDR3.
[0514] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0515] Further, for example, the light chain of a humanized
antibody can be made by replacing the amino acid sequences of
corresponding CDRs of the light chain of a human antibody with an
amino acid sequence consisting of not less than 3 or not less than
5, consecutive amino acids at the positions 24th to 33rd of the
amino acid sequence set forth as SEQ ID NO:244 as CDR1; with an
amino acid sequence consisting of not less than 3 or not less than
6, consecutive amino acids at the positions 49th to 55th of the
amino acid sequence set forth as SEQ ID NO:244 as CDR2; and with an
amino acid sequence consisting of not less than 3 or not less than
9, consecutive amino acids at the positions 88th to 96th of the
amino acid sequence set forth as SEQ ID NO:244 as CDR3; and
[0516] the heavy chain of a humanized antibody can be made by
replacing the amino acid sequences of corresponding CDRs of the
heavy chain of a human antibody with an amino acid sequence
consisting of not less than 3 consecutive amino acids at the
positions 26th to 35th of the amino acid sequence set forth as SEQ
ID NO:245 as CDR1; with an amino acid sequence consisting of not
less than 3 or not less than 8, consecutive amino acids at the
positions 51st to 66th of the amino acid sequence set forth as SEQ
ID NO:245 as CDR2; and with an amino acid sequence consisting of
not less than 3, or not less than 9, consecutive amino acids at the
positions 97th to 107th of the amino acid sequence set forth as SEQ
ID NO:245 as CDR3.
[0517] By combining the light chain and the heavy chain of the
humanized antibody thus obtained, the humanized antibody can be
prepared.
[0518] Examples of preferred embodiments of the humanized antibody
having affinity to hTfR include those having an amino acid sequence
according to one of (1) to (3) below:
[0519] (1) An anti-hTfR antibody,
[0520] wherein the light chain variable region thereof comprises an
amino acid sequence selected from the group consisting of the amino
acid sequences set forth as SEQ ID NO:158, SEQ ID NO:159, SEQ ID
NO:160, SEQ ID NO:161, SEQ ID NO:162, and SEQ ID NO:163, and
[0521] wherein the heavy chain variable region thereof comprises an
amino acid sequence selected from the group consisting of the amino
acid sequences set forth as SEQ ID NO:166, SEQ ID NO:167, SEQ ID
NO:168, SEQ ID NO:169, SEQ ID NO:170, and SEQ ID NO:171.
[0522] (2) an anti-hTfR antibody,
[0523] wherein the light chain variable region thereof comprises
any amino acid sequence of SEQ ID NO:174, SEQ ID NO:175, SEQ ID
NO:176, SEQ ID NO:177, SEQ ID NO:178, and SEQ ID NO:179, and
[0524] wherein the heavy chain variable region thereof comprises
any amino acid sequence of SEQ ID NO:182, SEQ ID NO:183, SEQ ID
NO:184, SEQ ID NO:185, SEQ ID NO:186, and SEQ ID NO:187.
[0525] (3) anti-hTfR antibody,
[0526] wherein the light chain variable region thereof comprises an
amino acid sequence selected from the group consisting of the amino
acid sequences set forth as SEQ ID NO:190, SEQ ID NO:191, SEQ ID
NO:192, SEQ ID NO:193, SEQ ID NO:194, and SEQ ID NO:195, and
[0527] wherein the heavy chain variable region thereof comprises an
amino acid sequence selected from the group consisting of the amino
acid sequences set forth as SEQ ID NO:204, SEQ ID NO:205, SEQ ID
NO:206, SEQ ID NO:207, SEQ ID NO:208 and SEQ ID NO:209.
[0528] The amino acid sequences of the light chain variable region
set forth as SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID
NO:161, SEQ ID NO:162 and SEQ ID NO:163 comprise the amino acid
sequence set forth as SEQ ID NO:6 or 7 in CDR1; SEQ ID NO:8 or 9 in
CDR2; and SEQ ID NO:10 in CDR3. However, the term CDRs as used
above in regard to the amino acid sequences of the light chain
variable region set forth as SEQ ID NOs:158 to 162 is not limited
to those specific sequences but may also include a region
containing the amino acid sequences of one of the CDRs or include
an amino acid sequence comprising not less than 3 consecutive amino
acids of one of the above CDRs.
[0529] The amino acid sequences of the heavy chain variable region
set forth as SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID
NO:169, SEQ ID NO:170, and SEQ ID NO:171 comprise the amino acid
sequence set forth as SEQ ID NO:76 or 77 in CDR1; SEQ ID NO:78 or
79 in CDR2; and SEQ ID NO:80 or 81 in CDR3. However, the term CDRs
used above in regard to the amino acid sequences of the heavy chain
variable region set forth as SEQ ID NOs:166 to 171 is not limited
to those specific sequences but may also include a region
containing the amino acid sequences of one of the CDRs or include
an amino acid sequence comprising not less than 3 consecutive amino
acids of one of the above CDRs.
[0530] The amino acid sequences of the light chain variable region
set forth as SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID
NO:177, SEQ ID NO:178, and SEQ ID NO:179 comprise the amino acid
sequence set forth as SEQ ID NO:11 or 12 in CDR1; SEQ ID NO:13 or
14 in CDR2; and SEQ ID NO:15 in CDR3. However, the term CDRs as
used above in regard to the amino acid sequences of the light chain
variable region set forth as SEQ ID NOs:174 to 179 is not limited
to those specific sequences but may also include a region
containing the amino acid sequences of one of the CDRs or include
an amino acid sequence comprising not less than 3 consecutive amino
acids of one of the above CDRs.
[0531] The amino acid sequences of the heavy chain variable region
set forth as SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID
NO:185, SEQ ID NO:186, and SEQ ID NO:187 comprise the amino acid
sequence set forth as SEQ ID NO:82 or 83 in CDR1; SEQ ID NO:84 or
85 in CDR2; and SEQ ID NO:86 or 87 in CDR3. However, the term CDRs
used above in regard to the amino acid sequences of the heavy chain
variable region set forth as SEQ ID NOs:182 to 187 is not limited
to those specific sequences but may also include a region
containing the amino acid sequences of one of the CDRs or include
an amino acid sequence comprising not less than 3 consecutive amino
acids of one of the above CDRs.
[0532] The amino acid sequences of the light chain variable region
set forth as SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID
NO:193, SEQ ID NO:194, and SEQ ID NO:195 comprise the amino acid
sequence set forth as SEQ ID NO:16 or 17 in CDR1, SEQ ID NO:18 or
19 in CDR2, and SEQ ID NO:20 in CDR3. However, the term CDRs as
used above in regard to the amino acid sequences of the light chain
variable region set forth as SEQ ID NOs:190 to 195 is not limited
to those specific sequences but may also include a region
containing the amino acid sequences of one of the CDRs or include
an amino acid sequence comprising not less than 3 consecutive amino
acids of one of the above CDRs.
[0533] The amino acid sequences of the heavy chain variable region
set forth as SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID
NO:207, SEQ ID NO:208 and SEQ ID NO:209 comprise the amino acid
sequence set forth as SEQ ID NO:88 or 89 in CDR1; SEQ ID NO:90 or
91 in CDR2, and SEQ ID NO:92 or 93 in CDR3. However, the term CDRs
used above in regard to the amino acid sequences of the heavy chain
variable region set forth as SEQ ID NOs:204 to 209 is not limited
to those specific sequences but may also include a region
containing the amino acid sequences of one of the CDRs or include
an amino acid sequence comprising not less than 3 consecutive amino
acids of one of the above CDRs.
[0534] Examples of more specific embodiments of the humanized
antibody having affinity to hTfR include:
[0535] the one that comprises the amino acid sequence set forth as
SEQ ID NO:163 in the light chain variable region and comprises the
amino acid sequence set forth as SEQ ID NO:171 in the heavy chain
variable region,
[0536] the one that comprises the amino acid sequence set forth as
SEQ ID NO:179 in the light chain variable region and comprises the
amino acid sequence set forth as SEQ ID NO:187 in the heavy chain
variable region,
[0537] the one that comprises the amino acid sequence set forth as
SEQ ID NO:191 in the light chain variable region and comprises the
amino acid sequence set forth as SEQ ID NO:205 in the heavy chain
variable region,
[0538] the one that comprises the amino acid sequence set forth as
SEQ ID NO:193 in the light chain variable region and comprises the
amino acid sequence set forth as SEQ ID NO:205 in the heavy chain
variable region,
[0539] the one that comprises the amino acid sequence set forth as
SEQ ID NO:194 in the light chain variable region and comprises the
amino acid sequence set forth as SEQ ID NO:205 in the heavy chain
variable region, and
[0540] the one that comprises the amino acid sequence set forth as
SEQ ID NO:195 in the light chain variable region and comprises the
amino acid sequence set forth as SEQ ID NO:205 in the heavy chain
variable region.
[0541] Examples of more specific embodiments of the humanized
antibody having affinity to hTfR include:
[0542] the one that comprises the amino acid sequence set forth as
SEQ ID NO:164 in the light chain and the amino acid sequence set
forth as SEQ ID NO:172 in the heavy chain,
[0543] the one that comprises the amino acid sequence set forth as
SEQ ID NO:180 in the light chain and the amino acid sequence set
forth as SEQ ID NO:188, in the heavy chain,
[0544] the one that comprises the amino acid sequence set forth as
SEQ ID NO:196 in the light chain and the amino acid sequence set
forth as SEQ ID NO:210 in the heavy chain,
[0545] the one that comprises the amino acid sequence set forth as
SEQ ID NO:198 in the light chain and the amino acid sequence set
forth as SEQ ID NO:210 in the heavy chain,
[0546] the one that comprises the amino acid sequence set forth as
SEQ ID NO:200 in the light chain and the amino acid sequence set
forth as SEQ ID NO:210 in the heavy chain,
[0547] the one that comprises the amino acid sequence set forth as
SEQ ID NO:202 in the light chain and the amino acid sequence set
forth as SEQ ID NO:210 in the heavy chain,
[0548] the one that comprises the amino acid sequence set forth as
SEQ ID NO:196 in the light chain and the amino acid sequence set
forth as SEQ ID NO:212 in the heavy chain,
[0549] the one that comprises the amino acid sequence set forth as
SEQ ID NO:198 in the light chain and the amino acid sequence set
forth as SEQ ID NO:212 in the heavy chain,
[0550] the one that comprises the amino acid sequence set forth as
SEQ ID NO:200 in the light chain and the amino acid sequence set
forth as SEQ ID NO:212 in the heavy chain, and
[0551] the one that comprises the amino acid sequence set forth as
SEQ ID NO:202 in the light chain and the amino acid sequence set
forth as SEQ ID NO:212 in the heavy chain.
[0552] Preferred embodiments of the antibody having affinity to
hTfR have been exemplified above. The light chain and heavy chain
of those anti-hTfR antibodies may be mutated as desired, by
substitution, deletion, addition and the like, in their
variable-region amino acid sequences in order to adjust the
affinity of the anti-hTfR antibody to hTfR to a suitable level.
[0553] When replacing on or more amino acids of the light chain
variable-region amino acid sequence with other amino acids, the
number of amino acids to be replaced is preferably 1 to 10, more
preferably 1 to 5, still more preferably 1 to 3, and even more
preferably 1 or 2. When deleting one or more amino acids of the
light chain variable-region amino acid sequence, the number of
amino acids to be deleted is preferably 1 to 10, more preferably 1
to 5, still more preferably 1 to 3, and even more preferably 1 or
2. Introduction of a combined mutation of such substitution and
deletion of amino acids is also allowed.
[0554] When adding one or more amino acids to the light chain
variable region, they may be added inside, or on the N-terminal
side or the C-terminal side of, the light chain variable-region
amino acid sequence, and preferably 1 to 10, more preferably 1 to
5, still more preferably 1 to 3, and even more preferably 1 or 2,
in number. Introduction of a combined mutation of such addition,
substitution, and deletion of amino acids is also allowed. Such a
mutated light chain variable-region amino acid sequence has a
homology preferably not lower than 80%, more preferably not lower
than 90%, still more preferably not lower than 95%, to the amino
acid sequence of the original light chain variable-region.
[0555] In particular, when replacing one or more amino acids of the
amino acid sequence of respective CDRs in the light chain with
other amino acids, the number of amino acids to be replaced is
preferably 1 to 5, more preferably 1 to 3, still more preferably 1
or 2, and even more preferably 1. When deleting one or more amino
acid of the amino acid sequence of the respective CDRs, the number
of amino acids to be deleted is preferably 1 to 5, more preferably
1 to 3, still more preferably 1 or 2, and even more preferably 1.
Introduction of a combined mutation of such substitution and
deletion of amino acids is also allowed.
[0556] When adding one or more amino acids to the amino acid
sequence of respective CDRs in the light chain, they are added
inside, or on the N-terminal side or the C-terminal side of, the
amino acid sequence, and preferably 1 to 5, more preferably 1 to 3,
still more preferably 1 or 2, in number. Introduction of a combined
mutation of such addition, substitution, and deletion of amino
acids is also allowed. The amino acid sequence of each of such
mutated CDRs has a homology preferably not lower than 80%, more
preferably not lower than 90%, and still more preferably not lower
than 95%, to the amino acid sequence of the respective original
CDRs.
[0557] When replacing one or more amino acids of the heavy chain
variable-region amino acid sequence with other amino acids, the
number of amino acids to be replaced is preferably 1 to 10, more
preferably 1 to 5, still more preferably 1 to 3, and even more
preferably 1 or 2. When deleting one or more amino acids of the
heavy chain variable-region amino acid sequence, the number of
amino acids to be deleted is preferably 1 to 10, more preferably 1
to 5, still more preferably 1 to 3, and even more preferably 1 or
2. Introduction of a combined mutation of such substitution and
deletion of amino acids is also allowed.
[0558] When adding one or more amino acids to the heavy chain
variable region, they may be added inside, or on the N-terminal
side or the C-terminal side of, the heavy chain variable-region
amino acid sequence, and preferably 1 to 10, more preferably 1 to
5, still more preferably 1 to 3, and even more preferably 1 or 2,
in number. Introduction of a combined mutation of such addition,
substitution, and deletion of amino acids is also allowed. Such a
mutated heavy chain variable-region amino acid sequence has a
homology preferably not lower than 80%, more preferably not lower
than 90%, still more preferably not lower than 95%, to the amino
acid sequence of the original heavy chain variable-region.
[0559] In particular, when replacing one or more amino acids of the
amino acid sequence of respective CDRs in the heavy chain with
other amino acids, the number of amino acids to be replaced is
preferably 1 to 5, more preferably 1 to 3, still more preferably 1
or 2, and even more preferably 1. When deleting one or more amino
acid of the amino acid sequence of the respective CDRs, the number
of amino acids to be deleted is preferably 1 to 5, more preferably
1 to 3, still more preferably 1 or 2, and even more preferably 1.
Introduction of a combined mutation of such substitution and
deletion of amino acids is also allowed.
[0560] When adding one or more amino acids to the amino acid
sequence of respective CDRs in the heavy chain, they are added
inside, or on the N-terminal side or the C-terminal side of, the
amino acid sequence, and preferably 1 to 5, more preferably 1 to 3,
still more preferably 1 or 2, and even more preferably 1 in number.
Introduction of a combined mutation of such addition, substitution,
and deletion of amino acids is also allowed. The amino acid
sequence of each of such mutated CDRs has a homology preferably not
lower than 80%, more preferably not lower than 90%, and still more
preferably not lower than 95%, to the amino acid sequence of the
respective original CDRs.
[0561] In the above, replacement of one or more amino acids of the
above anti-hTfR antibody variable-region amino acid sequence with
other amino acids is exemplified by interchange between acidic
amino acids, i.e., aspartic acid and glutamic acid; interchange
between amide-type amino acids, i.e., asparagine and glutamine;
interchange between basic amino acids, i.e., lysine and arginine,
interchange between branched amino acids, i.e., valine, leucine and
isoleucine, interchange between aliphatic amino acids, i.e.,
glycine and alanine, interchange between hydroxyamino acids, i.e.,
serine and threonine, and interchange between aromatic amino acids,
i.e., phenylalanine and tyrosine.
[0562] Besides, in the case where introducing a mutation into the
anti-hTfR antibody by adding one or more amino acids on its
C-terminus or the N-terminus, if the added amino acids are
positioned between the anti-hTfR antibody and a different protein
(A) when they are fused, the added amino acids constitute part of a
linker.
[0563] In the above preferred embodiments of the antibody,
including humanized antibody, having affinity to hTfR, there is no
particular limitation as to the amino acid sequence of the
anti-hTfR antibody heavy chain and light chain CDRs, insofar as the
antibody has a specific affinity to hTfR. However, the anti-hTfR
antibody of the present invention exhibits a dissociation constant
(K.sub.D) with hTfR, as measured by the method described in Example
7, which is preferably not greater than 1.times.10.sup.-8M, more
preferably not greater than 1.times.10.sup.-9 M, still more
preferably not greater than 1.times.10.sup.-9 M, and even more
preferably not greater than 1.times.10.sup.-9 M. For example, one
which exhibits a dissociation constant of 1.times.10.sup.-13 M to
1.times.10.sup.-9 M or 1.times.10.sup.-13 M to 1.times.10.sup.-10 M
is preferred. The same applies when the antibody is a single-chain
antibody. Further, in the case where the anti-hTfR antibody of the
present invention has an affinity also to a monkey TfR, the
dissociation constant of the anti-hTfR antibody to the monkey TfR,
as measured by the method described in Example 7, is preferably not
greater than 5.times.10.sup.-8 M, more preferably not greater than
2.times.10.sup.-8M, still more preferably not greater than
1.times.10.sup.-8M. For example, one which exhibits a dissociation
constant of 1.times.10.sup.-13 M to 2.times.10.sup.-8M is
preferred. The same applies when the antibody is a single-chain
antibody.
[0564] Specific embodiments of the above fusion protein between the
humanized antibody, which has affinity to hTfR, and a different
protein (A) described above include: those in which the different
protein (A) is human iduronate 2-sulfatase (hI2S), human
erythropoietin (hEPO), human arylsulfatase A (hARSA), human PPT-1
(hPPT-1), human TPP-1 (hTPP-1), human .alpha.-L-iduronidase
(hIDUA), human TNF.alpha. receptor (hTNF.alpha.R), and human
heparan N-sulfatase (hSGSH).
[0565] Specific examples of the fusion protein where the different
protein (A) is hI2S include;
[0566] (1) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
linker sequence Gly-Ser, to hI2S, and the other portion consisting
of the hTfR light chain, wherein the amino acid sequence of the
former is set forth as SEQ ID NO:247, and the amino acid sequence
of the latter is set forth as SEQ ID NO:164,
[0567] (2) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
linker sequence Gly-Ser, to hI2S, and the other portion consisting
of the hTfR light chain wherein the amino acid sequence of the
former is set forth as SEQ ID NO:249, and the amino acid sequence
of the latter is set forth as SEQ ID NO:180,
[0568] (3) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
linker sequence Gly-Ser, to hI2S, and the other portion consisting
of the hTfR light chain wherein the amino acid sequence of the
former is set forth as SEQ ID NO:251, and the amino acid sequence
of the latter is set forth as SEQ. ID NO:196.
[0569] In (1) above, the amino acid sequence of the hTfR heavy
chain, which is included in SEQ ID NO:247, is the one set forth as
SEQ ID NO:172. Namely, the fusion protein according to (1) above,
includes, as a humanized antibody, the amino acid sequence of the
light chain set forth as SEQ ID NO:164 and the amino acid sequence
of the heavy chain set forth as SEQ ID NO:172.
[0570] In (2) above, the amino acid sequence of the hTfR heavy
chain, which is included in SEQ ID NO:249, is the one set forth as
SEQ ID NO:188. Namely, the fusion protein according to (2) above,
includes, as a humanized antibody, the amino acid sequence of the
light chain set forth as SEQ ID NO:180 and the amino acid sequence
of the heavy chain set forth as SEQ ID NO:188.
[0571] In (3) above, the amino acid sequence of the hTfR heavy
chain, which is included in SEQ ID NO:251, is the one set forth as
SEQ ID NO:210. Namely, the fusion protein according to (3) above,
includes, as a humanized antibody, the amino acid sequence of the
light chain set forth as SEQ ID NO:196 and the amino acid sequence
of the heavy chain set forth as SEQ ID NO:210.
[0572] Specific examples of the fusion protein where the different
protein (A) is human erythropoietin (hEPO) include;
[0573] (1) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hEPO, and the other portion consisting of the hTfR
light chain, wherein the amino acid sequence of hTfR heavy chain of
the former is set forth as SEQ ID NO:172, and the amino acid
sequence of the latter is set forth as SEQ ID NO:164,
[0574] (2) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hEPO, and the other portion consisting of the hTfR
light chain, wherein the amino acid sequence of hTfR heavy chain of
the former is set forth as SEQ ID NO:188, and the amino acid
sequence of the latter is set forth as SEQ ID NO:180, and
[0575] (3) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hEPO, and the other portion consisting of the hTfR
light chain, wherein the amino acid sequence of hTfR heavy chain of
the former is set forth as SEQ ID NO:210, and the amino acid
sequence of the latter is set forth as SEQ ID NO:196.
[0576] In any of (1) to (3) above, hEPO and the hTfR heavy chain
may be linked by peptide bond, either directly or via a linker
sequence. Namely, in any of (1) to (3) above, the meaning of the
phrase "via a peptide bond" and that of the phrase "directly or via
a linker sequence" are the same. The linker sequence employed here
consists of 1 to 50 amino acid residues. Though there is no
particular limitation as to the amino acid sequence of the linker,
it is preferably made of glycine and serine: for example, a single
amino acid either glycine or serine, the amino acid sequence
Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), the amino acid sequence
Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4), the amino acid sequence
Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a sequence consisting 1
to 50, or 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29,
or 27 amino acids which are composed of 1 to 10 or 2 to 5 of any of
those amino acid sequences consecutively linked. For example, an
amino acid sequence comprising the amino acid sequence Gly-Ser may
be preferably used as a linker sequence. Further, a linker sequence
comprising 27 amino acids is preferably used that is composed of
the amino acid sequence Gly-Ser followed by consecutively linked
five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:3).
[0577] More specific embodiments of humanized antibody plus hEPO
include the one that is composed of the part consisting of hEPO
linked, via the linker sequence Gly-Ser, to the C-terminal side of
the hTfR heavy chain, and the other part consisting of the hTfR
light chain, wherein the amino acid sequence of the fowler is set
forth as SEQ ID NO:257, and the amino acid sequence of the latter
is set forth as SEQ ID NO:196.
[0578] Besides, in the present invention, though the term "human
EPO" or "hEPO" refers, in particular to the hEPO having the same
amino acid sequence as the natural-type hEPO set forth as SEQ ID
NO:256, it also includes those amino acid sequences produced by
introducing a mutation such as substitution, deletion, addition and
the like into the amino acid sequence of the natural-type hEPO,
insofar as they have the EPO activity. When replacing one or more
of the amino acids of the amino acid sequence of hEPO with other
amino acids, the number of amino acids to be replaced is preferably
1 to 10, more preferably 1 to 5, still more preferably 1 to 3, even
more preferably 1 or 2. When deleting one or more amino acids of
the amino acid sequence of hEPO, the number of amino acids to be
deleted is preferably 1 to 10, more preferably 1 to 5, still more
preferably 1 to 3, and even more preferably 1 or 2. It is also
possible to introduce a combined mutation of such substitution and
deletion of amino acids. When adding one or more amino acids to
hEPO, they may be added, inside, or on the N-terminal side or the
C-terminal side of, the amino acid sequence of hEPO, and the number
of amino acids to be added is preferably 1 to 10, more preferably 1
to 5, still more preferably 1 to 3, even more preferably 1 or 2. It
is also possible to introduce a combined mutation of such addition,
substitution, and deletion of the amino acid. The amino acid
sequence of each of such mutated hEPO has a homology preferably not
lower than 80%, more preferably not lower than 90%, still more
preferably not lower than 95%, to the amino acid sequence of the
original hEPO. Darbepoetin is an example obtained by mutating
natural-type hEPO.
[0579] Specific examples of the fusion protein where the different
protein (A) is human arylsulfatase A (hARSA) include;
[0580] (1) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hARSA, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:172, and the amino
acid sequence of the latter is set forth as SEQ ID NO:164,
[0581] (2) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hARSA, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:188, and the amino
acid sequence of the latter is set forth as SEQ ID NO:180, and
[0582] (3) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof via a
peptide bond, to hARSA, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:210, and the amino
acid sequence of the latter is set forth as SEQ ID NO:196.
[0583] In any of (1) to (3) above, hARSA and the hTfR heavy chain
may be linked by peptide bond, either directly or via a linker
sequence. Namely, in any of (1) to (3) above, the meaning of the
phrase "via a peptide bond" and that of the phrase "directly or via
a linker sequence" are the same. The linker sequence employed here
consists of 1 to 50 amino acid residues. Though there is no
particular limitation as to the amino acid sequence of the linker,
it is preferably made of glycine and serine: for example, a single
amino acid either glycine or serine, the amino acid sequence
Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), the amino acid sequence
Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4), the amino acid sequence
Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a sequence consisting 1
to 50, or 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29,
or 27 amino acids which are composed of 1 to 10 or 2 to 5 of any of
those amino acid sequences consecutively linked. For example, an
amino acid sequence comprising the amino acid sequence Gly-Ser may
be preferably used as a linker sequence. Further, a linker sequence
comprising 27 amino acids is preferably used that is composed of
the amino acid sequence Gly-Ser followed by consecutively linked
five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:3).
[0584] More specific embodiments of humanized antibody plus hARSA
include the one that is composed of the part consisting of hARSA
linked, via the linker sequence Gly-Ser, to the C-terminal side of
the hTfR heavy chain, and the other part consisting of the hTfR
light chain, wherein the amino acid sequence of the former is set
forth as SEQ ID NO:260, and the amino acid sequence of the latter
is set forth as SEQ ID NO:196.
[0585] Besides, in the present invention, though the term "human
ARSA" or "hARSA" refers, in particular to the hARSA having the same
amino acid sequence as the natural-type hARSA set forth as SEQ ID
NO:259, it also includes those amino acid sequences produced by
introducing a mutation such as substitution, deletion, addition and
the like into the amino acid sequence of the natural-type hARSA,
insofar as they have the ARSA activity. When replacing one or more
of the amino acids of the amino acid sequence of hARSA with other
amino acids, the number of amino acids to be replaced is preferably
1 to 10, more preferably 1 to 5, still more preferably 1 to 3, even
more preferably 1 or 2. When deleting one or more amino acids of
the amino acid sequence of hARSA, the number of amino acids to be
deleted is preferably 1 to 10, more preferably 1 to 5, still more
preferably 1 to 3, and even more preferably 1 or 2. It is also
possible to introduce a combined mutation of such substitution and
deletion of amino acids. When adding one or more amino acids to
hARSA, they may be added, inside, or on the N-terminal side or the
C-terminal side of, the amino acid sequence of hARSA, and the
number of amino acids to be added is preferably 1 to 10, more
preferably 1 to 5, still more preferably 1 to 3, even more
preferably 1 or 2. It is also possible to introduce a combined
mutation of such addition, substitution, and deletion of the amino
acid. The amino acid sequence of each of such mutated hARSA has a
homology preferably not lower than 80%, more preferably not lower
than 90%, and still more preferably not lower than 95%, to the
amino acid sequence of the original hARSA.
[0586] Specific examples of the fusion protein where the different
protein (A) is human PPT-1 (hPPT-1) include;
[0587] (1) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hPPT-1, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:172, and the amino
acid sequence of the latter is set forth as SEQ ID NO:164,
[0588] (2) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hPPT-1, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:188, and the amino
acid sequence of the latter is set forth as SEQ ID NO:180, and
[0589] (3) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hPPT-1, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:210, and the amino
acid sequence of the latter is set forth as SEQ ID NO:196.
[0590] In any of (1) to (3) above, hPPT-1 and the hTfR heavy chain
may be linked by peptide bond, either directly or via a linker
sequence. Namely, in any of (1) to (3) above, the meaning of the
phrase "via a peptide bond" and that of the phrase "directly or via
a linker sequence" are the same. The linker sequence employed here
consists of 1 to 50 amino acid residues. Though there is no
particular limitation as to the amino acid sequence of the linker,
it is preferably made of glycine and serine: for example, a single
amino acid either glycine or serine, the amino acid sequence
Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), the amino acid sequence
Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4), the amino acid sequence
Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a sequence consisting 1
to 50, or 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29,
or 27 amino acids which are composed of 1 to 10 or 2 to 5 of any of
those amino acid sequences consecutively linked. For example, an
amino acid sequence comprising the amino acid sequence Gly-Ser may
be preferably used as a linker sequence. Further, a linker sequence
comprising 27 amino acids is preferably used that is composed of
the amino acid sequence Gly-Ser followed by consecutively linked
five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:3).
[0591] More specific embodiments of humanized antibody plus hPPT-1
include the one that is composed of the part consisting of hPPT-1
linked, via the linker sequence Gly-Ser, to the C-terminal side of
the hTfR heavy chain, and the other part consisting of the hTfR
light chain, wherein the amino acid sequence of the former is set
forth as SEQ ID NO:263, and the amino acid sequence of the latter
is set forth as SEQ ID NO:196.
[0592] Besides, in the present invention, though the term "human
PPT-1" or "hPPT-1" refers, in particular to the hPPT-1 having the
same amino acid sequence as the natural-type hPPT-1 set forth as
SEQ ID NO:262, it also includes those amino acid sequences produced
by introducing a mutation such as substitution, deletion, addition
and the like into the amino acid sequence of the natural-type
hPPT-1, insofar as they have the PPT-1 activity. When replacing one
or more of the amino acids of the amino acid sequence of hPPT-1
with other amino acids, the number of amino acids to be replaced is
preferably 1 to 10, more preferably 1 to 5, still more preferably 1
to 3, even more preferably 1 or 2. When deleting one or more amino
acids of the amino acid sequence of hPPT-1, the number of amino
acids to be deleted is preferably 1 to 10, more preferably 1 to 5,
still more preferably 1 to 3, and even more preferably 1 or 2. It
is also possible to introduce a combined mutation of such
substitution and deletion of amino acids. When adding one or more
amino acids to hPPT-1, they may be added, inside, or on the
N-terminal side or the C-terminal side of, the amino acid sequence
of hPPT-1, and the number of amino acids to be added is preferably
1 to 10, more preferably 1 to 5, still more preferably 1 to 3, even
more preferably 1 or 2. It is also possible to introduce a combined
mutation of such addition, substitution, and deletion of the amino
acid. The amino acid sequence of each of such mutated hPPT-1 has a
homology preferably not lower than 80%, more preferably not lower
than 90%, and still more preferably not lower than 95%, to the
amino acid sequence of the original hPPT-1.
[0593] Specific examples of the fusion protein where the different
protein (A) is human PPT-1 (hPPT-1) include;
[0594] (1) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof via a
peptide bond, to hTPP-1, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:172, and the amino
acid sequence of the latter is set forth as SEQ ID NO:164,
[0595] (2) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof via a
peptide bond, to hTPP-1, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:188, and the amino
acid sequence of the latter is set forth as SEQ ID NO:180, and
[0596] (3) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof via a
peptide bond, to hTPP-1, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:210, and the amino
acid sequence of the latter is set forth as SEQ ID NO:196.
[0597] In any of (1) to (3) above, hTPP-1 and the hTfR heavy chain
may be linked by peptide bond, either directly or via a linker
sequence. Namely, in any of (1) to (3) above, the meaning of the
phrase "via a peptide bond" and that of the phrase "directly or via
a linker sequence" are the same. The linker sequence employed here
consists of 1 to 50 amino acid residues. Though there is no
particular limitation as to the amino acid sequence of the linker,
it is preferably made of glycine and serine: for example, a single
amino acid either glycine or serine, the amino acid sequence
Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), the amino acid sequence
Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4), the amino acid sequence
Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a sequence consisting 1
to 50, or 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29,
or 27 amino acids which are composed of 1 to 10 or 2 to 5 of any of
those amino acid sequences consecutively linked. For example, an
amino acid sequence comprising the amino acid sequence Gly-Ser may
be preferably used as a linker sequence. Further, a linker sequence
comprising 27 amino acids is preferably used that is composed of
the amino acid sequence Gly-Ser followed by consecutively linked
five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:3).
[0598] More specific embodiments of humanized antibody plus hTPP-1
include the one that is composed of the part consisting of hTPP-1
linked, via the linker sequence Gly-Ser, to the C-terminal side of
the hTfR heavy chain, and the other part consisting of the hTfR
light chain, wherein the amino acid sequence of the former is set
forth as SEQ ID NO:266, and the amino acid sequence of the latter
is set forth as SEQ ID NO:196.
[0599] Besides, in the present invention, though the term "human
TPP-1" or "hTPP-1" refers, in particular to the hTPP-1 having the
same amino acid sequence as the natural-type hTPP-1 set forth as
SEQ ID NO:265, it also includes those amino acid sequences produced
by introducing a mutation such as substitution, deletion, addition
and the like into the amino acid sequence of the natural-type
hTPP-1, insofar as they have the TPP-1 activity. When replacing one
or more of the amino acids of the amino acid sequence of hTPP-1
with other amino acids, the number of amino acids to be replaced is
preferably 1 to 10, more preferably 1 to 5, still more preferably 1
to 3, even more preferably 1 or 2. When deleting one or more amino
acids of the amino acid sequence of hTPP-1, the number of amino
acids to be deleted is preferably 1 to 10, more preferably 1 to 5,
still more preferably 1 to 3, and even more preferably 1 or 2. It
is also possible to introduce a combined mutation of such
substitution and deletion of amino acids. When adding one or more
amino acids to hTPP-1, they may be added, inside, or on the
N-terminal side or the C-terminal side of, the amino acid sequence
of hTPP-1, and the number of amino acids to be added is preferably
1 to 10, more preferably 1 to 5, still more preferably 1 to 3, even
more preferably 1 or 2. It is also possible to introduce a combined
mutation of such addition, substitution, and deletion of the amino
acid. The amino acid sequence of each of such mutated hTPP-1 has a
homology preferably not lower than 80%, more preferably not lower
than 90%, and still more preferably not lower than 95%, to the
amino acid sequence of the original hTPP-1.
[0600] Specific examples of the fusion protein where the different
protein (A) is human.alpha.-L-iduronidase (hIDUA) include;
[0601] (1) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hIDUA, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:172, and the amino
acid sequence of the latter is set forth as SEQ ID NO:164,
[0602] (2) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hIDUA, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR. heavy
chain of the former is set forth as SEQ ID NO:188, and the amino
acid sequence of the latter is set forth as SEQ ID NO:180, and
[0603] (3) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hIDUA, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:210, and the amino
acid sequence of the latter is set forth as SEQ ID NO:196.
[0604] In any of (1) to (3) above, hIDUA and the hTfR heavy chain
may be linked by peptide bond, either directly or via a linker
sequence. Namely, in any of (1) to (3) above, the meaning of the
phrase "via a peptide bond" and that of the phrase "directly or via
a linker sequence" are the same. The linker sequence employed here
consists of 1 to 50 amino acid residues. Though there is no
particular limitation as to the amino acid sequence of the linker,
it is preferably made of glycine and serine: for example, a single
amino acid either glycine or serine, the amino acid sequence
Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), the amino acid sequence
Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4), the amino acid sequence
Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a sequence consisting 1
to 50, or 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29,
or 27 amino acids which are composed of 1 to 10 or 2 to 5 of any of
those amino acid sequences consecutively linked. For example, an
amino acid sequence comprising the amino acid sequence Gly-Ser may
be preferably used as a linker sequence. Further, a linker sequence
comprising 27 amino acids is preferably used that is composed of
the amino acid sequence Gly-Ser followed by consecutively linked
five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:3).
[0605] More specific embodiments of humanized antibody plus hIDUA
include the one that is composed of the part consisting of hIDUA
linked, via the linker sequence Gly-Ser, to the C-terminal side of
the hTfR heavy chain, and the other part consisting of the hTfR
light chain, wherein the amino acid sequence of the former is set
forth as SEQ ID NO:269, and the amino acid sequence of the latter
is set forth as SEQ ID NO:196.
[0606] In the present invention, though the term "human IDUA" or
"hIDUA" refers, in particular, to the hIDUA having the same amino
acid sequence as the natural-type hIDUA set forth as SEQ ID NO:268,
it also includes those amino acid sequences produced by introducing
a mutation such as substitution, deletion, addition and the like
into the amino acid sequence of the natural-type hIDUA, insofar as
they have the hIDUA activity. When replacing one or more of the
amino acids of the amino acid sequence of hIDUA with other amino
acids, the number of amino acids to be replaced is preferably 1 to
10, more preferably 1 to 5, still more preferably 1 to 3, even more
preferably 1 or 2. When deleting one or more amino acids of the
amino acid sequence of hIDUA, the number of amino acids to be
deleted is preferably 1 to 10, more preferably 1 to 5, still more
preferably 1 to 3, and even more preferably 1 or 2. It is also
possible to introduce a combined mutation of such substitution and
deletion of amino acids. When adding one or more amino acids to
hIDUA, they may be added, inside, or on the N-terminal side or the
C-terminal side of, the amino acid sequence of hIDUA, and the
number of amino acids to be added is preferably 1 to 10, more
preferably 1 to 5, still more preferably 1 to 3, even more
preferably 1 or 2. It is also possible to introduce a combined
mutation of such addition, substitution, and deletion of the amino
acid. The amino acid sequence of the mutated hIDUA has a homology
of preferably not lower than 80%, more preferably not lower than
90%, still more preferably not lower than 95% to the amino acid
sequence of the original hIDUA.
[0607] Specific examples of the fusion protein where the different
protein (A) is human TNF-.alpha. receptor (hTNF.alpha.R)
include;
[0608] (1) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hTNF.alpha.R, and the other portion consisting of
the hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:172, and the amino
acid sequence of the latter is set forth as SEQ ID NO:164,
[0609] (2) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hTNF.alpha.R, and the other portion consisting of
the hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:188, and the amino
acid sequence of the latter is set forth as SEQ ID NO:180, and
[0610] (3) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hTNF.alpha.R, and the other portion consisting of
the hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:210, and the amino
acid sequence of the latter is set forth as SEQ ID NO:196.
[0611] In any of (1) to (3) above, hTNF.alpha.R and the hTfR heavy
chain may be linked by peptide bond, either directly or via a
linker sequence. Namely, in any of (1) to (3) above, the meaning of
the phrase "via a peptide bond" and that of the phrase "directly or
via a linker sequence" are the same. The linker sequence employed
here consists of 1 to 50 amino acid residues. Though there is no
particular limitation as to the amino acid sequence of the linker,
it is preferably made of glycine and serine: for example, a single
amino acid either glycine or serine, the amino acid sequence
Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), the amino acid sequence
Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4), the amino acid sequence
Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a sequence consisting 1
to 50, or 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29,
or 27 amino acids which are composed of 1 to 10 or 2 to 5 of any of
those amino acid sequences consecutively linked. For example, an
amino acid sequence comprising the amino acid sequence Gly-Ser may
be preferably used as a linker sequence. Further, a linker sequence
comprising 27 amino acids is preferably used that is composed of
the amino acid sequence Gly-Ser followed by consecutively linked
five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:3).
[0612] More specific embodiments of humanized antibody plus
hTNF.alpha.R include the one that is composed of the part
consisting of hTNF.alpha.R linked, via the linker sequence Gly-Ser,
to the C-terminal side of the hTfR heavy chain, and the other part
consisting of the hTfR light chain, wherein the amino acid sequence
of the former is set forth as SEQ ID NO:272, and the amino acid
sequence of the latter is set forth as SEQ ID NO:196.
[0613] Besides, in the present invention, though the term "human
TNF.alpha.R" or "hTNF.alpha.R" refers, in particular to the
hTNF.alpha.R having the same amino acid sequence as the
natural-type hTNF.alpha.R set forth as SEQ ID NO:271, it also
includes those amino acid sequences produced by introducing a
mutation such as substitution, deletion, addition and the like into
the amino acid sequence of the natural-type hTNF.alpha.R, insofar
as they have the activity or function as TNF.alpha.R. When
replacing one or more of the amino acids of the amino acid sequence
of hTNF.alpha.R with other amino acids, the number of amino acids
to be replaced is preferably 1 to 10, more preferably 1 to 5, still
more preferably 1 to 3, even more preferably 1 or 2. When deleting
one or more amino acids of the amino acid sequence of hTNF.alpha.R,
the number of amino acids to be deleted is preferably 1 to 10, more
preferably 1 to 5, still more preferably 1 to 3, and even more
preferably 1 or 2. It is also possible to introduce a combined
mutation of such substitution and deletion of amino acids. When
adding one or more amino acids to hTNF.alpha.R, they may be added,
inside, or on the N-terminal side or the C-terminal side of, the
amino acid sequence of hTNF.alpha.R, and the number of amino acids
to be added is preferably 1 to 10, more preferably 1 to 5, still
more preferably 1 to 3, even more preferably 1 or 2. It is also
possible to introduce a combined mutation of such addition,
substitution, and deletion of the amino acid. The amino acid
sequence of each of such mutated hTNF.alpha.R has a homology
preferably not lower than 80%, more preferably not lower than 90%,
and still more preferably not lower than 95%, to the amino acid
sequence of the original hTNF.alpha.R.
[0614] Specific examples of the fusion protein where the different
protein (A) is human heparan N-sulfatase (hSGSH) include:
[0615] (1) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hSGSH, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:172, and the amino
acid sequence of the latter is set forth as SEQ ID NO:164,
[0616] (2) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hSGSH, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:188, and the amino
acid sequence of the latter is set forth as SEQ ID NO:180, and
[0617] (3) the one that is composed of: the portion consisting of
hTfR heavy chain linked, on the C-terminal side thereof and via a
peptide bond, to hSGSH, and the other portion consisting of the
hTfR light chain, wherein the amino acid sequence of hTfR heavy
chain of the former is set forth as SEQ ID NO:210, and the amino
acid sequence of the latter is set forth as SEQ ID NO:196.
[0618] In any of (1) to (3) above, hSGSH and the hTfR heavy chain
may be linked by peptide bond, either directly or via a linker
sequence. Namely, in any of (1) to (3) above, the meaning of the
phrase "via a peptide bond" and that of the phrase "directly or via
a linker sequence" are the same. The linker sequence employed here
consists of 1 to 50 amino acid residues. Though there is no
particular limitation as to the amino acid sequence of the linker,
it is preferably made of glycine and serine: for example, a single
amino acid either glycine or serine, the amino acid sequence
Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:3), the amino acid sequence
Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:4), the amino acid sequence
Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:5), or a sequence consisting 1
to 50, or 2 to 17, 2 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29,
or 27 amino acids which are composed of 1 to 10 or 2 to 5 of any of
those amino acid sequences consecutively linked. For example, an
amino acid sequence comprising the amino acid sequence Gly-Ser may
be preferably used as a linker sequence. Further, a linker sequence
comprising 27 amino acids is preferably used that is composed of
the amino acid sequence Gly-Ser followed by consecutively linked
five copies of the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID
NO:3).
[0619] More specific embodiments of humanized antibody plus hSGSH
include the one that is composed of the part consisting of hSGSH
linked, via the linker sequence Gly-Ser, to the C-terminal side of
the hTfR heavy chain, and the other part consisting of the hTfR
light chain, wherein the amino acid sequence of the former is set
forth as SEQ ID NO:275, and the amino acid sequence of the latter
is set forth as SEQ ID NO:196.
[0620] Besides, in the present invention, though the term "human
SGSH" or "hSGSH" refers, in particular to the hSGSH having the same
amino acid sequence as the natural-type hSGSH set forth as SEQ ID
NO:274, it also includes those amino acid sequences produced by
introducing a mutation such as substitution, deletion, addition and
the like into the amino acid sequence of the natural-type hSGSH,
insofar as they have the SGSH activity. When replacing one or more
of the amino acids of the amino acid sequence of hSGSH with other
amino acids, the number of amino acids to be replaced is preferably
1 to 10, more preferably 1 to 5, still more preferably 1 to 3, even
more preferably 1 or 2. When deleting one or more amino acids of
the amino acid sequence of hSGSH, the number of amino acids to be
deleted is preferably 1 to 10, more preferably 1 to 5, still more
preferably 1 to 3, and even more preferably 1 or 2. It is also
possible to introduce a combined mutation of such substitution and
deletion of amino acids. When adding one or more amino acids to
hSGSH, they may be added, inside, or on the N-terminal side or the
C-terminal side of, the amino acid sequence of hSGSH, and the
number of amino acids to be added is preferably 1 to 10, more
preferably 1 to 5, still more preferably 1 to 3, even more
preferably 1 or 2. It is also possible to introduce a combined
mutation of such addition, substitution, and deletion of the amino
acid. The amino acid sequence of each of such mutated hSGSH has a
homology preferably not lower than 80%, more preferably not lower
than 90%, and still more preferably not lower than 95%, to the
amino acid sequence of the original hSGSH.
[0621] In the case where the different protein (A) fused with the
anti-hTfR antibody is human iduronate 2-sulfatase (hI2S), human
erythropoietin (hEPO), human arylsulfatase A (hARSA), human PPT-1
(hPPT-1), human TPP-1 (hTPP-1), human .alpha.-L-iduronidase
(hIDUA), human TNF-.alpha. receptor (hTNF.alpha.R), or human
heparan N-sulfatase (hSGSH), the expression the different protein
(A) fused with the anti-hTfR antibody retains the activity or
function that the different protein (A) exhibits under a
physiological condition, (or simply, it retains the activity or
function), means that the not less than 3% of the activity or
function is retained as compared with the activity or function that
the corresponding natural-type proteins intrinsically have.
However, their activity or function is preferably not less than
10%, more preferably not less than 20%, still more preferably not
less than 50%, and even more preferably not less than 80%, as
compared with the activity or function that the corresponding
natural-type different protein (A) intrinsically have. The same
applies where the different protein (A) fused with the anti-hTfR
antibody in mutated.
[0622] The anti-hTfR antibody according to the present invention
can be used for the production of a pharmaceutical agent for
parenteral administration for the treatment of a disease condition
of the central nervous system by binding it to the molecule of a
physiologically active protein or a pharmacologically active
low-molecular-weight compound. And the anti-hTfR antibody
conjugated with the molecule of a physiologically active protein or
a pharmacologically active low-molecular-weight compound, can be
used in the method of treatment of a patient with a disease
condition of the central nervous system, in which a therapeutically
effective amount of a physiologically active protein or
pharmacologically active low-molecular-weight compound is
administered to the patient with a disease of the central nervous
system parenterally (including intravenous injection such as
intravenous infusion). The anti-hTfR antibody conjugated with the
molecule of a physiologically active protein or a pharmacologically
active low-molecular-weight compound, after parenterally
administered, can not only get inside the brain but also reach
other organs where hTfR is expressed.
[0623] In particular, as the anti-hTfR antibody of the present
invention can, as a conjugate with human iduronate 2-sulfatase
(hI2S), enable hI2S to pass through the blood-brain barrier and
function in the brain, the antibody can be used for the production
of a pharmaceutical agent for parenteral administration for the
treatment of a disease condition of the central nervous system
accompanying Hunter syndrome. Further, the anti-hTfR antibody
conjugated with hI2S can be used in the method of treatment of a
patient with a disease condition of the central nervous system
disorder accompanying Hunter syndrome, in which a therapeutically
effective amount of the antibody is administered to the patient
with Hunter syndrome parenterally (including intravenous injection
such as intravenous infusion). The anti-hTfR antibody conjugated
with hI2S, after parenterally administered, can not only get inside
the brain but also reach other organs where hTfR is expressed.
[0624] Further, as the anti-hTfR antibody of the present invention
can, as a conjugate with human erythropoietin (hEPO), enable hEPO
to pass through the blood-brain barrier and function in the brain,
the antibody can be used for the production of a pharmaceutical
agent for parenteral administration for the treatment of a disease
condition of the central nervous system accompanying cerebral
ischemia. Further, the anti-hTfR antibody conjugated with hEPO can
be used in the method of treatment of a patient with a disease
condition of the central nervous system accompanying cerebral
ischemia, in which a therapeutically effective amount of the
conjugated antibody is administered to the patient with cerebral
ischemia parenterally (including intravenous injection such as
intravenous infusion). The anti-hTfR antibody conjugated with hEPO,
after parenterally administered, can not only get inside the brain
but also reach other organs where hTfR is expressed. The same
applies if human erythropoietin is replaced with human
darbepoetin.
[0625] Further, as the anti-hTfR antibody of the present invention
can, as a conjugate with human arylsulfatase A (hARSA), enable
hARSA to pass through the blood-brain barrier and function in the
brain, the antibody can be used for the production of a
pharmaceutical agent for parenteral administration for the
treatment of a disease condition of the central nervous system
accompanying metachromatic white matter degeneration (metachromatic
leukodystrophy). Further, the anti-hTfR antibody conjugated with
hARSA can be used in the method of treatment of a patient with a
disease condition of the central nervous system accompanying
metachromatic white matter degeneration (metachromatic
leukodystrophy), in which a therapeutically effective amount of the
conjugated antibody is administered to the patient with the disease
parenterally (including intravenous injection such as intravenous
infusion). The anti-hTfR antibody conjugated with hARSA, after
parenterally administered, can not only get inside the brain but
also reach other organs where hTfR is expressed.
[0626] Further, as the anti-hTfR antibody of the present invention
can, as a conjugate with human PPT-1 (hPPT-1), enable hPPT-1 to
pass through the blood-brain barrier and function in the brain, the
antibody can be used for the production of a pharmaceutical agent
for parenteral administration for the treatment of a disease
condition of the central nervous system accompanying neuronal
ceroid lipofuscinosis or Santavuori-Haltia disease. Further, the
anti-hTfR antibody conjugated with hPPT-1 can be used in the method
of treatment of a patient with a disease condition of the central
nervous system accompanying neuronal ceroid lipofuscinosis or
Santavuori-Haltia disease, in which a therapeutically effective
amount of the conjugated antibody is administered to the patient
with any of these diseases parenterally (including intravenous
injection such as intravenous infusion). The anti-hTfR antibody
conjugated with hPPT-1, after parenterally administered, can not
only get inside the brain but also reach other organs where hTfR is
expressed.
[0627] Further, as the anti-hTfR antibody of the present invention
can, as a conjugate with human TPP-1 (hTPP-1), enable hTPP-1 to
pass through the blood-brain barrier and function in the brain, the
antibody can be used for the production of a pharmaceutical agent
for parenteral administration for the treatment of a disease
condition of the central nervous system accompanying neuronal
ceroid lipofuscinosis or Jansky-Bielschowsky disease. Further, the
anti-hTfR antibody conjugated with hPPT-1 can be used in the method
of treatment of a patient with a disease condition of the central
nervous system accompanying neuronal ceroid lipofuscinosis or
Jansky-Bielschowsky disease, in which a therapeutically effective
amount of the conjugated antibody is administered to the patient
with any of these diseases parenterally (including intravenous
injection such as intravenous infusion). The anti-hTfR antibody
conjugated with hTPP-1, after parenterally administered, can not
only get inside the brain but also reach other organs where hTfR is
expressed.
[0628] Further, as the anti-hTfR antibody of the present invention
can, as a conjugate with human .alpha.-L-iduronidase (hIDUA),
enable hIDUA to pass through the blood-brain barrier and function
in the brain, the antibody can be used for the production of a
pharmaceutical agent for parenteral administration for the
treatment of a disease condition of the central nervous system
accompanying Hurler syndrome or Hurler-Scheie syndrome. Further,
the anti-hTfR antibody conjugated with hIDUA can be used in the
method of treatment of a patient with a disease condition of the
central nervous system accompanying Hurler syndrome or
Hurler-Scheie syndrome, in which a therapeutically effective amount
of the conjugated antibody is administered to the patient with any
of these diseases parenterally (including intravenous injection
such as intravenous infusion). The anti-hTfR antibody conjugated
with hIDUA, after parenterally administered, can not only get
inside the brain but also reach other organs where hTfR is
expressed.
[0629] Further, as the anti-hTfR antibody of the present invention
can, as a conjugate with human TNF-.alpha. receptor (hTNF.alpha.R),
enable hTNF.alpha.R to pass through the blood-brain barrier and
function in the brain, the antibody can be used for the production
of a pharmaceutical agent for parenteral administration for the
treatment of a disease condition of the central nervous system
accompanying cerebral ischemia or encephalitis. Further, the
anti-hTfR antibody conjugated with hTNF.alpha.R can be used in the
method of treatment of a patient with a disease condition of the
central nervous system accompanying cerebral ischemia or
encephalitis, in which a therapeutically effective amount of the
conjugated antibody is administered to the patient with any of
these diseases parenterally (including intravenous injection such
as intravenous infusion). The anti-hTfR antibody conjugated with
hTNF.alpha.R, after parenterally administered, can not only get
inside the brain but also reach other organs where hTfR is
expressed.
[0630] Further, as the anti-hTfR antibody of the present invention
can, as a conjugate with human heparan N-sulfatase (hSGSH), enable
hSGSH to pass through the blood-brain barrier and function in the
brain, the antibody can be used for the production of a
pharmaceutical agent for parenteral administration for the
treatment of a disease condition of the central nervous system
accompanying Sanfilippo syndrome. Further, the anti-hTfR antibody
conjugated with hSGSH can be used in the method of treatment of a
patient with a disease condition of the central nervous system
accompanying Sanfilippo syndrome, in which a therapeutically
effective amount of the conjugated antibody is administered to the
patient with any of these diseases parenterally (including
intravenous injection such as intravenous infusion). The anti-hTfR
antibody conjugated with hSGSH, after parenterally administered,
can not only get inside the brain but also reach other organs where
hTfR is expressed.
[0631] The proteins, low-molecular-weight compound and the like
that are conjugated with the anti-hTfR antibody of the present
invention can be used as pharmaceutical agents which are to exhibit
their functions in the central nervous system (CNS) after
parenterally administered. Such pharmaceutical agents may be
administered to patients generally by intravenous injection such as
intravenous injection, subcutaneous injection, intramuscular
injection and the like, though there is no particular limitation as
to the route of their administration.
[0632] The proteins, the low-molecular-weight compounds and the
like that are conjugated with the anti-hTfR antibody of the present
invention can be provided to medical facilities as pharmaceutical
agents in such a form as a lyophilized product or an aqueous
preparation. In the case of an aqueous preparation, it can be
provided in the form of preparations in which one of the
pharmaceutical agents is dissolved in a solution containing a
stabilizer, buffer, and an isotonizer in advance, and sealed in
vials or syringes. A type of preparations sealed in a syringe is
generally called a prefilled syringe-type preparation. Taking the
form of a prefilled syringe-type preparation facilitates patients'
self-administration of the pharmaceutical agent.
[0633] Where an aqueous preparation is provided, the concentration
of the protein, the low-molecular-weight compound or the like
conjugated with the anti-hTfR antibody in the aqueous preparation
is, e.g., 1 to 4 mg/mL, though it is to be adjusted as desired in
accordance with the dosage. Where there is no particular limitation
as to stabilizers to be contained in the aqueous preparation
insofar as they are pharmaceutically available, nonionic
surfactants may preferably be used. Examples of such nonionic
surfactants include polysorbate and poloxamer, either of which may
be used alone or in combination. Among polysorbates, polysorbate 20
and polysorbate 80 are preferably used. As poloxamer, poloxamer 188
(polyoxyethylene (160) polyoxypropylene (30) glycol) is
particularly preferred. Further, the concentration of nonionic
surfactant contained in the aqueous preparation is preferably 0.01
to 1 mg/mL, more preferably, 0.01 to 0.5 mg/mL, and still more
preferably 0.1 to 0.5 mg/mL. As stabilizers, amino acids such as
histidine, arginine, methionine, and glycine may also be used.
Where employed as a stabilizer, the concentration of an amino acid
in the aqueous preparation is preferably 0.1 to 40 mg/mL, more
preferably 0.2 to 5 mg/mL, and still more preferably 0.5 to 4
mg/mL. While there is no particular limitation as to a buffer to be
contained in the aqueous preparation insofar as it is
pharmaceutically available, phosphate buffer is preferred, and more
preferred is sodium phosphate buffer. Where used as a buffer, the
concentration of sodium phosphate is preferably 0.01 to 0.04 M. The
pH of the aqueous preparation adjusted with a buffer is preferably
5.5 to 7.2. While there is no particular limitation as to an
isotonizer to be contained in the aqueous preparation insofar as it
is pharmaceutically available, sodium chloride or mannitol may be
preferably used alone or in combination as an isotonizer.
EXAMPLES
[0634] Though the present invention is described in further detail
below with reference to examples, it is not intended that the
present invention be limited to those examples.
[Example 1] Construction of hTfR Expression Vector
[0635] Employing human spleen Quick Clone cDNA (Clontech Inc.) as a
template and using primer hTfR5' (SEQ ID NO:214) and primer hTfR3'
(SEQ ID NO:215), PCR was performed to amplify the gene fragment
encoding human transferrin receptor (hTfR). The amplified fragment
encoding hTfR was digested with MluI and NotI, and then inserted
between MluI and NotI sites of vector pCI-neo (Promega Corp.). The
vector thus prepared was designated pCI-neo(hTfR). This vector then
was digested with MluI and Nod to cut out the gene fragment
encoding hTfR, and this fragment was inserted between MluI and NotI
sites of pE-mIRES-GS-puro, an expression vector disclosed in an
international publication WO 2012/063799 to construct an hTfR
expression vector, pE-mIRES-GS-puro(hTfR).
[Example 2] Preparation of Recombinant hTfR
[0636] Into CHO-K1 cells was introduced pE-mIRES-GS-puro(hTfR) by
electroporation, and the cells then were subjected to selection
culture in a CD OptiCHO.TM. medium (Invitrogen Inc.) containing
methionine sulfoximine (MSX) and puromycin to prepare recombinant
hTfR expressing cells. The recombinant hTfR expressing cells were
cultured, and recombinant hTfR was prepared.
[Example 3] Immunization of Mouse with Recombinant hTfR
[0637] Mice were Immunized with Recombinant hTfR Prepared in
Example 2 as antigen. Immunization was carried out by intravenously
or intraperitoneally injecting the mice with the antigen.
[Example 4] Preparation of Hybridoma Cells
[0638] About one week after the last injection, the spleens of the
mice were excised and homogenized to isolate spleen cells. The
spleen cells thus obtained were fused with cells of mouse myeloma
cell line (P3.X63.Ag8.653) by the polyethylene glycol method. After
cell fusion, the cells were suspended in a RPMI 1640 medium
containing (1X) HAT supplement (Life Technologies Inc.) and 10%
Ultra low IgG fetal bovine serum (Life Technologies Inc.), and the
cell suspension was dispensed to twenty 96-well plates, each at 200
.mu.L/well. After the cells were cultured for 10 days in a carbon
dioxide gas incubator (37.degree. C., 5% CO.sub.2), each well was
examined under a microscope, and the wells that contain a single
colony were selected.
[0639] When the cells in each well reached near confluence, the
culture supernatant was collected as a culture supernatant of
hybridoma, and subjected to the following screening process.
[Example 5] Screening of High Affinity Antibody Producing Cell
Line
[0640] The recombinant hTfR solution (Sino Biologics Inc.) was
diluted with 50 mM sodium phosphate buffer (pH 9.5 to 9.6) to 5
.mu.g/mL to prepare a solid phase solution. After 50 .mu.L of the
solid phase solution was added to each well of a Nunc MaxiSorp.TM.
flat-bottom 96-well plate (substrate: polystyrene, mfd. by Nunc
Inc.), the plate was left to stand for one hour at room temperature
to let the recombinant hTfR adhere to the plate and become
immobilized. The solid phase solution was discarded, each well was
washed three times with 250 .mu.L of washing solution (PBS
containing 0.05% Tween20), 200 .mu.L of a blocking solution (PBS
containing 1% BSA) then was added to each well, and the plate was
left to stand for one hour at room temperature.
[0641] The blocking solution was discarded, and each well was
washed three times with 250 .mu.L washing solution (PBS containing
0.05% Tween20). To each well was added 50 .mu.L of the hybridoma
culture supernatant, and the plate was left to stand for one hour
at room temperature to let the mouse anti-hTfR antibody contained
in the culture supernatant bind to the recombinant hTfR. At the
same time, to some wells was added 50 .mu.L of culture supernatant
of a hybridoma that did not produce mouse anti-hTfR antibody, as a
control. In addition, 50 .mu.L of the medium for hybridoma culture
was added to the wells, as mock wells, beside the wells to which
the culture supernatant was added. Measurement was conducted in an
n=2 fashion. Then, the solution was discarded, and each well was
washed three times with 250 .mu.L of washing solution (PBS
containing 0.05% Tween20).
[0642] To each of the above wells was added 100 .mu.L of
HRP-labelled goat anti-mouse immunoglobulin antibody solution
(Promega Inc.), and the plate was left to stand for one minute at
room temperature. The solution then was discarded, and each well
was washed three times with 250 .mu.L of washing solution (PBS
containing 0.05% Tween20). To each well as added 50 .mu.L of a
chromogenic substrate solution, TMB Stabilized Substrate for
Horseradish Peroxidase (Promega Inc.), and the wells were left to
stand for 10 to 20 minutes at room temperature. Then, following
addition of 100 .mu.L of a stop solution (2N sulfuric acid), the
absorbance of each well was measured on a plate reader at 450 nm.
Of the two wells for each of the culture supernatant and control,
the mean values were taken, respectively, and from each of the mean
values, the respective mean value for the two mock wells placed
corresponding to each of the culture supernatant and the control,
was subtracted, giving the measurement.
[0643] Fourteen types of hybridoma cells corresponding to culture
supernatants added to the wells which exhibited the higher
measurements were selected as the cell lines (high affinity
antibody producing cell line) that produce antibodies exhibiting
high affinities to hTfR (high affinity anti-hTfR antibody). These
fourteen types of cell lines were designated as Clone 1 line to
Clone 14 line. Further, the anti-hTfR antibodies produced by Clone
1 line to Clone 14 line were designated as anti-hTfR antibodies
Nos. 1 to 14, respectively.
[Example 6] Analysis of the Variable-Region Amino Acid Sequence of
the High Affinity Anti-hTfR Antibodies
[0644] From each of the Clone 1 line to Clone 14 line selected in
Example 5, cDNA were prepared, using which as a template the genes
encoding the light chain and the heavy chain of the antibody were
amplified. By translating the nucleotide sequence of the amplified
genes, the respective amino acid sequences of the light chain and
heavy chain variable regions were determined for the anti-hTfR
antibodies Nos. 1 to 14 produced by the cell lines.
[0645] The anti-hTfR antibody No. 1 was found to include the amino
acid sequence set forth as SEQ ID NO:218 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:219 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:6 or 7 as CDR1; SEQ ID NO:8 or 9 as CDR2, and SEQ ID
NO:10 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:76 or 77 as CDR1, SEQ ID
NO:78 or 79 as CDR2, and SEQ ID NO:80 or 81 as CDR3. However, it
was also considered that CDRs are not limited to those which
consist of these amino acid sequences, but they can also either be
regions of amino acid sequences that include any of the above
sequences, or amino acid sequences consisting of not less than
three consecutive amino acids containing part of the above
sequences.
[0646] The anti-hTfR antibody No. 2 was found to include the amino
acid sequence set forth as SEQ ID NO:220 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:221 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:11 or 12 as CDR1; SEQ ID NO:13 or 14 as CDR2, and SEQ ID
NO:15 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:82 or 83 as CDR1, SEQ ID
NO:84 or 85 as CDR2, and SEQ ID NO:86 or 87 as CDR3. However, it
was also considered that CDRs are not limited to those which
consist of these amino acid sequences, but they can also either be
regions of amino acid sequences that include any of the above
sequences, or amino acid sequences consisting of not less than
three consecutive amino acids containing part of the above
sequences.
[0647] The anti-hTfR antibody No. 3 was found to include the amino
acid sequence set forth as SEQ ID NO:222 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:223 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:16 or 17 as CDR1; SEQ ID NO:18 or 19 as CDR2, and SEQ ID
NO:20 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:88 or 89 as CDR1, SEQ ID
NO:90 or 91 as CDR2, and SEQ ID NO:92 or 93 as CDR3. However, it
was also considered that CDRs are not limited to those which
consist of these amino acid sequences, but they can also either be
regions of amino acid sequences that include any of the above
sequences, or amino acid sequences consisting of not less than
three consecutive amino acids containing part of the above
sequences.
[0648] The anti-hTfR antibody No. 4 was found to include the amino
acid sequence set forth as SEQ ID NO:224 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:225 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:21 or 22 as CDR1; SEQ ID NO:23 or 24 as CDR2, and SEQ ID
NO:25 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:94 or 95 as CDR1, SEQ ID
NO:96 or 97 as CDR2, and SEQ ID NO:98 or 99 as CDR3. However, it
was also considered that CDRs are not limited to those which
consist of these amino acid sequences, but they can also either be
regions of amino acid sequences that include any of the above
sequences, or amino acid sequences consisting of not less than
three consecutive amino acids containing part of the above
sequences.
[0649] The anti-hTfR antibody No. 5 was found to include the amino
acid sequence set forth as SEQ ID NO:226 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:227 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:26 or 27 as CDR1; SEQ ID NO:28 or 29 as CDR2, and SEQ ID
NO:30 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:100 or 101 as CDR1, SEQ
ID NO:102 or 103 as CDR2, and SEQ ID NO:104 or 105 as CDR3.
However, it was also considered that CDRs are not limited to those
which consist of these amino acid sequences, but they can also
either be regions of amino acid sequences that include any of the
above sequences, or amino acid sequences consisting of not less
than three consecutive amino acids containing part of the above
sequences.
[0650] The anti-hTfR antibody No. 6 was found to include the amino
acid sequence set forth as SEQ ID NO:228 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:229 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:31 or 32 as CDR1; SEQ ID NO:33 or 34 as CDR2, and SEQ ID
NO:35 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:106 or 107 as CDR1, SEQ
ID NO:108 or 278 as CDR2, and SEQ ID NO:109 or 110 as CDR3.
However, it was also considered that CDRs are not limited to those
which consist of these amino acid sequences, but they can also
either be regions of amino acid sequences that include any of the
above sequences, or amino acid sequences consisting of not less
than three consecutive amino acids containing part of the above
sequences.
[0651] The anti-hTfR antibody No. 7 was found to include the amino
acid sequence set forth as SEQ ID NO:230 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:231 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:36 or 37 as CDR1; SEQ ID NO:38 or 39 as CDR2, and SEQ ID
NO:40 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:111 or 112 as CDR1, SEQ
ID NO:113 or 114 as CDR2, and SEQ ID NO:115 or 116 as CDR3.
However, it was also considered that CDRs are not limited to those
which consist of these amino acid sequences, but they can also
either be regions of amino acid sequences that include any of the
above sequences, or amino acid sequences consisting of not less
than three consecutive amino acids containing part of the above
sequences.
[0652] The anti-hTfR antibody No. 8 was found to include the amino
acid sequence set forth as SEQ ID NO:232 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:233 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:41 or 42 as CDR1; SEQ ID NO:43 or 44 as CDR2, and SEQ ID
NO:45 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:117 or 118 as CDR1, SEQ
ID NO:119 or 279 as CDR2, and SEQ ID NO:120 or 121 as CDR3.
However, it was also considered that CDRs are not limited to those
which consist of these amino acid sequences, but they can also
either be regions of amino acid sequences that include any of the
above sequences, or amino acid sequences consisting of not less
than three consecutive amino acids containing part of the above
sequences.
[0653] The anti-hTfR antibody No. 9 was found to include the amino
acid sequence set forth as SEQ ID NO:234 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:235 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:46 or 47 as CDR1; SEQ ID NO:48 or 49 as CDR2, and SEQ ID
NO:50 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:122 or 123 as CDR1, SEQ
ID NO:124 or 125 as CDR2, and SEQ ID NO:126 or 127 as CDR3.
However, it was also considered that CDRs are not limited to those
which consist of these amino acid sequences, but they can also
either be regions of amino acid sequences that include any of the
above sequences, or amino acid sequences consisting of not less
than three consecutive amino acids containing part of the above
sequences.
[0654] The anti-hTfR antibody No. 10 was found to include the amino
acid sequence set forth as SEQ ID NO:236 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:237 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:51 or 52 as CDR1; SEQ ID NO:53 or 54 as CDR2, and SEQ ID
NO:55 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:128 or 129 as CDR1, SEQ
ID NO:130 or 131 as CDR2, and SEQ ID NO:132 or 133 as CDR3.
However, it was also considered that CDRs are not limited to those
which consist of these amino acid sequences, but they can also
either be regions of amino acid sequences that include any of the
above sequences, or amino acid sequences consisting of not less
than three consecutive amino acids containing part of the above
sequences.
[0655] The anti-hTfR antibody No. 11 was found to include the amino
acid sequence set forth as SEQ ID NO:238 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:239 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:56 or 57 as CDR1; SEQ ID NO:58 or 59 as CDR2, and SEQ ID
NO:60 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:134 or 135 as CDR1, SEQ
ID NO:136 or 137, as CDR2, and SEQ ID NO:138 or 139 as CDR3.
However, it was also considered that CDRs are not limited to those
which consist of these amino acid sequences, but they can also
either be regions of amino acid sequences that include any of the
above sequences, or amino acid sequences consisting of not less
than three consecutive amino acids containing part of the above
sequences.
[0656] The anti-hTfR antibody No. 12 was found to include the amino
acid sequence set forth as SEQ ID NO:240 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:241 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:61 or 62 as CDR1; SEQ ID NO:63 or 64 as CDR2, and SEQ ID
NO:65 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:140 or 141 as CDR1, SEQ
ID NO:142 or 143 as CDR2, and SEQ ID NO:144 or 145 as CDR3.
However, it was also considered that CDRs are not limited to those
which consist of these amino acid sequences, but they can also
either be regions of amino acid sequences that include any of the
above sequences, or amino acid sequences consisting of not less
than three consecutive amino acids containing part of the above
sequences.
[0657] The anti-hTfR antibody No. 13 was found to include the amino
acid sequence set forth as SEQ ID NO:242 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:243 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:66 or 67 as CDR1; SEQ ID NO:68 or 69 as CDR2, and SEQ ID
NO:70 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:146 or 147 as CDR1, SEQ
ID NO:148 or 149 as CDR2, and SEQ ID NO:150 or 151 as CDR3.
However, it was also considered that CDRs are not limited to those
which consist of these amino acid sequences, but they can also
either be regions of amino acid sequences that include any of the
above sequences, or amino acid sequences consisting of not less
than three consecutive amino acids containing part of the above
sequences.
[0658] The anti-hTfR antibody No. 14 was found to include the amino
acid sequence set forth as SEQ ID NO:244 as the light chain
variable region, and the amino acid sequence set forth as SEQ ID
NO:245 as the heavy chain variable region. The light chain variable
region was found to include the amino acid sequence set forth as
SEQ ID NO:71 or 72 as CDR1; SEQ ID NO:73 or 74 as CDR2, and SEQ ID
NO:75 as CDR3; and the heavy chain variable region to include the
amino acid sequence set forth as SEQ ID NO:152 or 153 as CDR1, SEQ
ID NO:154 or 155 as CDR2, and SEQ ID NO:156 or 157 as CDR3.
However, it was also considered that CDRs are not limited to those
which consist of these amino acid sequences, but they can also
either be regions of amino acid sequences that include any of the
above sequences, or amino acid sequences consisting of not less
than three consecutive amino acids containing part of the above
sequences.
[0659] Table 1 shows collectively the SEQ ID NOs of the respective
amino acid sequences included in the light chain and the heavy
chain variable regions of the anti-hTfR antibody Nos. 1 to 14.
TABLE-US-00001 TABLE 1 Sequence numbers of respective amino acid
sequences included in the light and the heavy chain variable
regions of the anti-hTfR antibodies Nos. 1 to 14 Antibody light
chain heavy chain No. variable region variable region 1 218 219 2
220 221 3 222 223 4 224 225 5 226 227 6 228 229 7 230 231 8 232 233
9 234 235 10 236 237 11 238 239 12 240 241 13 242 243 14 244
245
[0660] Table 2 shows collectively the SEQ ID NOs of the respective
amino acid sequences contained in CDR1 to CDR3 of the light chain
variable region and CDR1 to CDR3 of the heavy chain variable region
of anti-hTfR antibodies Nos. 1 to 14. However, Table 2 shows those
amino acid sequence only as examples and does not limit the amino
acid sequence of each CDR to those in Table 2, but it was
considered that CDRs are not limited to those which consist of
these amino acid sequences, but they can also either be regions of
amino acid sequences that include any of the above sequences, or
amino acid sequences consisting of not less than three consecutive
amino acids containing part of the above sequences.
TABLE-US-00002 TABLE 2 Sequence numbers of respective amino acid
sequences contained in CDR1 to CDR3 of the light chain and the
heavy chain variable regions of anti-hTfR antibodies Nos. 1 to 14
Anti- light chain heavy chain body variable region variable region
No. CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 1 6, 7 8, 9 10 76, 77 78, 79 80,
81 2 11, 12 13, 14 15 82, 83 84, 85 86, 87 3 16, 17 18, 19 20 88,
89 90, 91 92, 93 4 21, 22 23, 24 25 94, 95 96, 97 98, 99 5 26, 27
28, 29 30 100, 101 102, 103 104, 105 6 31, 32 33, 34 35 106, 107
108, 278 109, 110 7 36, 37 38, 29 40 111, 112 113, 114 115, 116 8
41, 42 43, 44 45 117, 118 119, 279 120, 121 9 46, 47 48, 49 50 122,
123 124, 125 126, 127 10 51, 52 53, 54 55 128, 129 130, 131 132,
133 11 56, 57 58, 59 60 134, 135 136, 137 138, 139 12 61, 62 63, 64
65 140, 141 142, 143 144, 145 13 66, 67 68, 69 70 146, 147 148, 149
150, 151 14 71, 72 73, 74 75 152, 153 154, 155 156, 157
[Example 7] Measurement of the Affinity of Anti-hTfR. Antibody to
Human and Monkey TfRs
[0661] The affinity of the anti-hTfR antibody to human and monkey
TfRs were measured on Octet RED96 (ForteBio Inc., a division of
Pall Corporation), a system for analysis of interactions between
biomolecules utilizing bio-layer interferometry (BLI). The basic
principles of bio-layer interferometry are briefly explained below.
When a layer of a biomolecule immobilized on the surface of a
sensor tip is irradiated with light of a certain wavelength, the
light is reflected from two of the surfaces, the one of the
biomolecule and the other of inner, reference layer, producing
interfering light waves. A molecule in the sample being measured
binds to the biomolecule on the surface of the sensor tip and thus
increases the thickness of the layers on the sensor tip, which
results in a shift between the interfering waves. By measuring the
variations of this shift between the interfering waves,
determination of the number of the molecules bound to the layer of
the biomolecules immobilized to the sensor tip surface and kinetic
analysis of it can be performed in real time. The measurement was
performed according generally to the operating manual attached to
Octet RED96. As a human TfR, a recombinant human UR (r human TfR:
Sino Biological Inc.) was used, which had the amino acid sequence
of the hTfR extracellular region, i.e., the cysteine residue at the
position 89th from the N-terminal side to the phenylalanine at the
C-terminus, of the amino acid sequence set forth as SEQ ID NO:1,
with a histidine tag attached to the N-terminus. As a monkey TfR, a
recombinant monkey TfR (r monkey TfR: Sino Biological Inc.) was
used, which had the amino acid sequence of the cynomolgus monkey
TfR extracellular region, i.e., the cysteine residue at the
position 89th from the N-terminal side to the phenylalanine at the
C-terminus, of the amino acid sequence set forth as SEQ ID NO:2,
with a histidine tag attached to the N-terminus.
[0662] Clone 1 line to Clone 14 line selected in Example 5 were
respectively diluted with a RPMI 1640 medium containing (1X) HAT
Supplement (Life Technologies Inc.) and 10% Ultra low IgG fetal
bovine serum (Life Technologies Inc.) so as to adjust the cell
density to approximately 2.times.10.sup.5 cells/mL. To a 1-L
conical flask were added 200 mL of each cell suspension, and the
culture was performed for 6 to 7 days in a humid environment at
37.degree. C., 5% CO.sub.2 and 95% air, with stirring at a rate of
about 70 rpm. The culture supernatant was collected by
centrifugation, and filtered through a 0.22 .mu.m filter (Millipore
Inc.) to prepare the culture supernatant. The culture supernatant
thus collected was loaded onto a Protein G column (column volume: 1
mL, GE Healthcare Inc.) that had been equilibrated in advance with
three column volumes of 20 mM Tris buffer (pH 8.0) containing 150
mM NaCl. After the column was washed with 5 column volumes of the
same buffer, adsorbed antibody was eluted with 4 column volumes of
50 mM glycine buffer (pH 2.8) containing 150 mM NaCl, and eluted
fractions were collected. The eluted fractions were adjusted to pH
7.0 by addition of 1 M Tris buffer (pH 8.0). These were used as
purified products of anti-hTfR antibodies Nos. 1 to 14 in the
experiments described below.
[0663] Each of the antibodies (anti-hTfR antibody Nos. 1 to 14)
purified above was subjected to 2-fold dilution steps with HBS-P+
(10 mM HEPES containing 150 mM NaCl, 50 .mu.M EDTA and 0.05%
Surfactant P20) to prepare antibody solutions of 7 different
concentrations, 0.78125 to 50 nM (0.117 to 7.5 .mu.g/mL). These
antibody solutions were used as the sample solutions. The r human
and r monkey TfRs were respectively diluted with HBS-P+ to prepare
25 .mu.g/mL solutions, which were used as r human TfR-ECD (Histag)
solution and r monkey TfR-ECD (Histag) solution, respectively.
[0664] Each of the sample solutions prepared above by 2-fold
dilution steps was added, 200 .mu.L/well, to a 96-well plate, black
(Greiner Bio-One Inc.). Each of the r human TfR-ECD (Histag)
solution and the r monkey TfR-ECD (Histag) solutions prepared above
was added, 200 .mu.L/well, to predetermined wells. To respective
wells for baseline, dissociation and washing were added HBS-P+, 200
.mu.L/well. To wells for regeneration were added 10 mM Glycine-HCl,
pH 1.7, 200 .mu.L/well. To wells for activation was added 0.5 mM
NiCl.sub.2 solution, 200 .mu.L/well. The plate and biosensor
(Biosensor/Ni-NTA: ForteBio Inc., a division of Pall Corporation)
were set in the prescribed positions of Octet RED96.
[0665] Octet RED96 was run under the conditions shown in Table 3
below to collect data, on which then, using the analyzing software
attached to Octet RED96, and fitting the binding reaction curve to
1:1 binding model or 2:1 binding model, the association rate
constant (k.sub.on) and dissociation rate constant (k.sub.off) of
anti-hTfR antibody to r human TfR and r monkey TfR were measured
and the dissociation constant (Kd) was calculated. The measurement
was performed at 25 to 30.degree. C.
TABLE-US-00003 TABLE 3 Operating conditions of Octet RED96 Contact
time Step (sec) Rate (rpm) Threshold 1 Baseline 1 60 1000 -- 2 Load
600 1000 1.5-2.0 3 Baseline 2 60 1000 4 Association 180 1000 5
Dissociation 540 1000 6 Regeneration 5 1000 7 Washing 5 1000 Steps
6-7 repeated 6 to 7 times 8 Activation 60 1000 -- Steps 1-8
repeated until all the samples measured
[0666] Table 4 shows the results of measurement of association rate
constant (k.sub.on), dissociation rate constant (k.sub.off) of
anti-hTfR antibody Nos. 1 to 14 (corresponding to antibody Nos. 1
to 14, respectively, in the table), and dissociation constant
(k.sub.D) to human TfR.
TABLE-US-00004 TABLE 4 Affinity of anti-hTfR antibodies to human
TfR Antibody No. kon (M.sup.-1s.sup.-1) koff (s.sup.-1) K.sub.D (M)
1 5.00 .times. 10.sup.5 2.55 .times. 10.sup.-6 5.09 .times.
10.sup.-12 2 1.11 .times. 10.sup.6 1.23 .times. 10.sup.-5 1.12
.times. 10.sup.-11 3 6.53 .times. 10.sup.5 <1.0 .times.
10.sup.-7 <1.0 .times. 10.sup.-12 4 1.91 .times. 10.sup.6 2.29
.times. 10.sup.-4 1.20 .times. 10.sup.-10 5 6.71 .times. 10.sup.5
2.44 .times. 10.sup.-5 3.64 .times. 10.sup.-11 6 7.54 .times.
10.sup.5 7.23 .times. 10.sup.-4 9.58 .times. 10.sup.-10 7 3.69
.times. 10.sup.5 3.03 .times. 10.sup.-5 8.22 .times. 10.sup.-11 8
6.96 .times. 10.sup.5 <1.0 .times. 10.sup.-7 <1.0 .times.
10.sup.-12 9 7.82 .times. 10.sup.5 9.46 .times. 10.sup.-5 1.21
.times. 10.sup.-10 10 6.79 .times. 10.sup.5 7.66 .times. 10.sup.-4
1.13 .times. 10.sup.-9 11 2.72 .times. 10.sup.5 2.28 .times.
10.sup.-5 8.37 .times. 10.sup.-11 12 7.54 .times. 10.sup.5 7.23
.times. 10.sup.-4 4.32 .times. 10.sup.-10 13 8.35 .times. 10.sup.5
<1.0 .times. 10.sup.-7 <1.0 .times. 10.sup.-12 14 9.61
.times. 10.sup.5 <1.0 .times. 10.sup.-7 <1.0 .times.
10.sup.-12
[0667] Table 5 shows the results of measurement of association rate
constant (k.sub.on), dissociation rate constant (k.sub.off) of
anti-hTfR antibody Nos. 1 to 14 (corresponding to antibody Nos. 1
to 14, respectively, in the table), and dissociation constant
(k.sub.D) to monkey UR.
TABLE-US-00005 TABLE 5 Affinity of anti-hTfR antibodies to monkey
TfR Antibody No. kon (M.sup.-1s.sup.-1) koff (s.sup.-1) K.sub.D (M)
1 2.80 .times. 10.sup.5 <1.0 .times. 10.sup.-7 <1.0 .times.
10.sup.-12 2 4.18 .times. 10.sup.5 1.75 .times. 10.sup.-6 4.18
.times. 10.sup.-11 3 3.89 .times. 10.sup.5 <1.0 .times.
10.sup.-7 <1.0 .times. 10.sup.-12 4 7.54 .times. 10.sup.5 1.21
.times. 10.sup.-4 1.61 .times. 10.sup.-10 5 5.19 .times. 10.sup.5
7.58 .times. 10.sup.-4 1.46 .times. 10.sup.-9 6 4.95 .times.
10.sup.5 2.36 .times. 10.sup.-4 1.23 .times. 10.sup.-10 7 2.66
.times. 10.sup.5 4.54 .times. 10.sup.-6 1.71 .times. 10.sup.-11 8
5.52 .times. 10.sup.5 5.07 .times. 10.sup.-3 9.18 .times. 10.sup.-9
9 6.99 .times. 10.sup.5 1.47 .times. 10.sup.-4 2.10 .times.
10.sup.-9 10 3.87 .times. 10.sup.5 1.22 .times. 10.sup.-2 3.16
.times. 10.sup.-8 11 1.24 .times. 10.sup.5 4.21 .times. 10.sup.-4
3.38 .times. 10.sup.-9 12 5.05 .times. 10.sup.5 1.26 .times.
10.sup.-4 2.49 .times. 10.sup.-10 13 5.91 .times. 10.sup.5 7.29
.times. 10.sup.-5 1.23 .times. 10.sup.-10 14 7.00 .times. 10.sup.5
3.61 .times. 10.sup.-5 5.16 .times. 10.sup.-11
[0668] As a result of the affinity measurement of those anti-hTfR
antibodies to human TfR, the dissociation constant with human TfR
was not more than 1.times.10.sup.-8 M for all the antibodies; and
for 13 antibodies except antibody No. 10, the dissociation constant
with human TfR was not more than 1.times.10.sup.-9M; and for
antibodies Nos. 3, 8, 13 and 14, in particular, the dissociation
constant was not more than 1.times.10.sup.-12M (Table 4). The
result demonstrates that all of the 14 antibodies are antibodies
having a high-affinity antibody to human TfR. Then looking to the
result of the measurement of the affinity of the anti-hTfR
antibodies to monkey TfR, the dissociation constant with monkey TfR
was not more than 5.times.10.sup.-8M for all the antibodies, and
for antibodies Nos. 1 and 3, in particular, the dissociation
constant with monkey TfR was not more than 1.times.10.sup.-12 M
(Table 5). The result shows that all the 14 antibodies are
antibodies having a high-affinity antibody not only to human TfR
but also to monkey TfR.
[Example 7-2] Evaluation of Brain Uptake of the Anti-hTfR
Antibodies Using Mice
[0669] Then, for 13 antibodies, anti-hTfR antibodies Nos. 1 to 9
and 11 to 14, evaluation was performed about their transfer into
the brain through the BBB, by using hTfR knock-in mice (hTfR-KI
mice) in which the gene encoding the extracellular region of mouse
transferrin receptor has been replaced with a gene encoding the
extracellular region of human transferrin receptor. The hTfR-KI
mice were produced by the method described below as a whole.
Besides, the purified antibodies of Example 7 were used as the
anti-hTfR antibodies.
[0670] A DNA fragment having a nucleotide sequence set forth as SEQ
ID NO:253 was chemically synthesized, in which a neomycin
resistance gene flanked by loxP sequences was placed on the 3'-side
of a cDNA encoding a chimeric hTfR whose intracellular region
consisted of the amino acid sequence of mouse hTfR and the
extracellular region consisted of the amino acid sequence of human
hTfR sequence. This DNA fragment was inserted by a conventional
method into a targeting vector having as the 5'-arm sequence a
nucleotide sequence set forth as SEQ ID NO:254 and as the 3'-arm
sequence a nucleotide sequence set forth as SEQ ID NO:255, and the
construct was introduced into mouse ES cells by electroporation.
The mouse ES cells to which the gene had been introduced were
subjected to selection culture in a medium in the presence of
neomycin to select those mouse ES cells in which the targeting
vector had been incorporated into the chromosome through homologous
recombination. The recombinant mouse ES cells thus obtained were
injected into 8-cell stage embryos (host embryos) of ICR mice, and
the embryos thus prepared were implanted into pseudo pregnant mice
(recipient mice) which had been obtained through mating with mice
having undergone vasoligation. The offspring (chimeric mice)
obtained were examined by their hair color, and those mice which
had the higher proportion of white hairs in their total body hairs
were selected, i.e., those mice in which the ES cells had
contributed at the higher rates in the development of the
individual organisms. Each of these chimeric mice was mated with
ICR mice to generate F1 mice. F1 mice with white hair were
selected, the DNAs extracted from their tail tissue were analyzed,
and those mice whose mouse transferrin receptor gene on their
chromosomes had been replaced with chimeric hTfR, were regarded as
hTfR-KI mice.
[0671] The above 13 anti-hTfR antibodies were fluorescently labeled
with fluorescein isothiocyanate (FITC) using Fluorescein Labeling
Kit-NH.sub.2 (Dojindo Laboratories) according to the attached
manual. PBS solutions were prepared each containing one of the FITC
fluorescent labeled 13 anti-hTfR antibodies. Each of these PBS
antibody solutions was intravenously injected to an hTfR-KI mouse
(male, 10 to 12-week old), at the anti-hTfR antibody dosage of 3
mg/kg. As a control, a PBS solution containing mouse IgG1 (Sigma
Inc.), fluorescently labeled with FITC in the same manner as above,
was intravenously injected to an hTfR-KI mouse (male, 10 to 12-week
old), at the dose of 3 mg/kg. About eight hours after the
intravenous injection, the whole body was perfused with saline, and
the brain (part including the cerebrum and the cerebellum) was
obtained. The brain thus excised was weighed (wet weight), and then
the brain tissues were homogenized with T-PER (Thermo Fisher
Scientific Inc.) containing Protease Inhibitor Cocktail (Sigma
Inc.). The homogenate was centrifuged, the supernatant was
collected, and the amount of the FITC fluorescent labeled antibody
contained in the supernatant was measured in the following manner.
First, 10 .mu.L of anti-FITC Antibody (Bethyl Inc.) was added to
each well of a High Bind Plate (Meso Scale Diagnostics Inc.) and
left to stand for one hour so as to immobilize it to the plate.
Then, the plate was blocked by addition of 150 .mu.L of SuperBlock
Blocking buffer in PBS (Thermo Fisher Scientific Inc.) to each well
and shaking of the plate for one hour. Then, 25 .mu.L of the
supernatant of a brain tissue homogenate was added to each well,
and the plate was shaken for one hour. Then, 25 .mu.L of SULFO-TAG
Anti-Mouse Antibody(Goat)(Meso Scale Diagnostics Inc.) were added
to each well, and shaking was continued for one hour. Then, to each
well was added 150 .mu.L of Read buffer T (Meso Scale Diagnostics
Inc.), and the amount of luminescence from each well was read on a
Sector.TM. Imager 6000 reader. The amount of the antibody contained
per one gram brain (wet weight) (the concentration of the anti-hTfR
antibody in the brain tissues) was calculated, by producing a
standard curve based on measurements of standard samples containing
known concentrations of fluorescently FITC-labeled anti-hTfR
antibody, and then interpolating the measurement of each of the
samples with reference to the standard. The results are shown in
Table 5-2.
[0672] The concentration of any of the antibodies designated
anti-hTfR antibodies Nos. 1 to 9 and 11 to 14 in brain tissues was
over 25 times greater than that of the control. The concentration
of anti-hTfR antibodies Nos. 5 and 6 was both over 100 times
greater than that of the control, with anti-hTfR antibody No. 6, in
particular, the value reached approximately 160 times as high as
that of the control. The results indicate that the antibodies
designated anti-hTfR antibody Nos. 1 to 9 and 11 to 14 transfers
into the brain, actively passing through the BBB.
TABLE-US-00006 TABLE 5-2 Concentration of anti-hTfR antibodies in
brain tissues Antibody Brain tissues Relative value No. (.mu.g/g
wet weight) to the control Control 0.003 1 1 0.141 47.0 2 0.126
42.0 3 0.0833 27.8 4 0.221 73.7 5 0.335 112 6 0.492 164 7 0.0855
28.5 8 0.133 44.3 9 0.112 37.3 11 0.103 34.3 12 0.215 71.7 13 0.127
42.3 14 0.213 71.0
[Example 8] Pharmacokinetic Analysis of Anti-hTfR Antibodies in
Monkey
[0673] Each of anti-hTfR antibodies Nos. 1 to 3 was intravenously
administered once to a male cynomolgus monkey at a dosage of 5.0
mg/kg, and 8 hours after the administration, whole body irrigation
was carried out with physiological saline. As a negative control, a
monkey which had not received anti-hTfR antibody was subjected to
whole body irrigation in the same manner. After the irrigation,
brain tissues including the medulla oblongata were excised. Using
the brain tissues, the concentration of the anti-hTfR antibody was
measured, and immunohistochemical staining was performed. Besides,
the anti-hTfR antibodies employed were purification products of
those described in Example 7.
[0674] Measurement of the concentration of anti-hTfR antibodies in
brain tissues were carried out largely following the procedure
described below. Collected brain tissues were divided into the
cerebrum, the cerebellum, the hippocampus, and the medulla
oblongata, and they were respectively homogenized with RIPA Buffer
(Wako Pure Chemical Industries Inc.) containing Protease Inhibitor
Cocktail (Sigma-Aldrich Inc.), and centrifuged to collect the
supernatant. Affinipure Goat Anti mouse IgG Fc.gamma. pAb (Jackson
ImmunoResearch Inc.) was added, 10 .mu.L each, to the wells of a
High Bind Plate (Meso Scale Diagnostics Inc.), and the plate was
left to stand for one hour to immobilize the antibody. Then, the
plate was blocked by addition of 150 .mu.L of SuperBlock Blocking
buffer in PBS (Thermo Fisher Scientific Inc.) to each well and
shaken for one hour. Then, 25 .mu.L of the supernatant of a brain
tissue homogenate was added to each well, and the plate was shaken
for one hour. Then, 25 .mu.L of Affinipure Goat Anti mouse IgG
Fab-Biotin (Jackson ImmunoResearch Inc.) was added to each well,
and shaking was continued for one hour. Then, 25 .mu.L or
SULFO-Tag-Streptavidin (Meso Scale Diagnostics Inc.) was added to
each well, and shaking was continued for half an hour. To each well
was added 150 .mu.L of Read buffer T (Meso Scale Diagnostics Inc.),
and the amount of luminescence from each well was read on a
Sector.TM. Imager 6000 reader (Meso Scale Diagnostics). The amount
of the antibody contained per one gram of brain (wet weight) (the
concentration of the anti-hTfR antibody in brain tissues) was
calculated, by producing a standard curve based on measurements of
standard samples containing known concentrations of the anti-hTfR
antibody, and then interpolating the measurement of each of the
samples with reference to the standard.
[0675] The result of the measurement of the concentration of the
anti-hTfR antibodies in brain tissues is shown in Table 6. Though
any of anti-hTfR antibodies Nos. 1 to 3 was observed to accumulate
in the cerebrum, the cerebellum, the hippocampus and the medulla
oblongata, in their amount there was a relation of anti-hTfR
antibody No. 1<anti-hTfR antibody No. 3<anti-hTfR antibody
No. 2, showing the lowest with anti-hTfR antibody No. 1 and highest
in anti-hTfR antibody No. 2. In comparison with anti-hTfR antibody
No. 1, the accumulation of anti-hTfR antibody No. 2 was
approximately 4.3 times in the cerebrum, approximately 6.6 times in
the cerebellum, approximately 4.6 times in the hippocampus, and
approximately 2 times in the medulla oblongata. These results
demonstrate that these 3 antibodies had a property to pass through
the blood-brain barrier and accumulate in the brain tissues, and
show that by binding these antibodies to a pharmaceutical agent
which needs to be brought into function in the brain tissues, it is
possible to let those pharmaceutical agents efficiently accumulate
in the brain tissues.
TABLE-US-00007 TABLE 6 Concentration of anti-hTfR antibodies in
brain tissues (.mu.g/g wet weight) Antibody Medulla No. Cerebrum
Cerebellum Hippocampus oblongata 1 0.18 0.15 0.12 0.22 2 0.78 0.99
0.56 0.43 3 0.82 0.6 0.33 0.31
[0676] Immunohistochemical staining of the anti-hTfR antibodies in
these brain tissues was carried out using the following procedures
described below basically. The collected tissues were rapidly
frozen to -80.degree. C. in a Tissue-Tek Cryo 3DM (Sakura Finetek
Inc.) to prepare frozen blocks of tissues. The frozen blocks were
sliced into 4-.mu.m sections, and which were affixed to MAS coated
glass slides (Matsunami Glass Inc.). The tissue sections were
reacted with 4% paraformaldehyde (Wako Pure Chemical Industries
Inc.) for 5 minutes at 4.degree. C. and fixed to glass slides.
Then, the tissue sections were reacted with methanol solution
containing 0.3% hydrogen peroxide (Wako Pure Chemical Industries
Inc.) for 30 min to inactivate endogenous peroxidases. Then, the
glass slides were blocked by reacting SuperBlock blocking buffer in
PBS for 30 min at room temperature. Then, the tissue sections were
reacted with Mouse IgG-heavy and light chain Antibody (Bethyl
Laboratories Inc.) for one hour at room temperature. The tissue
sections were allowed to visualize with DAB substrate
(3,3'-diaminobenzidine, Vector Laboratories Inc.), counterstained
with Mayer's hematoxylin solution (Merck Inc.), embedded after
dehydration and clearing, and observed under an optical
microscope.
[0677] FIG. 1 shows the result of the immunohistochemical staining
of the anti-hTfR antibodies in the cerebral cortex. In the cerebral
cortex of monkeys administered anti-hTfR antibodies Nos. 1 to 3,
specific staining in the blood vessels were observed (FIG. 1b to d,
respectively). In particular, in the cerebral cortex of the monkeys
administered anti-hTfR antibodies No. 2 or 3, specific staining was
also observed extensively in the brain parenchyma region, outside
the blood vessels (FIGS. 1c and 1d, respectively). Besides, no
staining was observed in the cerebral cortex of the control monkey
non-administered anti-hTfR antibody, indicating that there was
almost no background staining (FIG. 1a).
[0678] FIG. 2 shows the result of immunohistochemical staining of
anti-hTfR antibodies in the hippocampus. In the cerebrum of monkeys
administered anti-hTfR antibodies Nos. 1 to 3, specific staining of
blood vessels were observed (FIG. 2b to d, respectively). In
particular, in the hippocampus of the monkeys administered
anti-hTfR antibodies No. 2 or 3, specific staining of neuron-like
cells was also observed (FIGS. 2c and 2d, respectively), and
specific and extensive staining of the brain parenchyma region,
outside the blood vessels, was also observed. Besides, no staining
was observed in the hippocampus of the control monkey
non-administered anti-hTfR antibody, indicating that there was
almost no background staining (FIG. 2a).
[0679] FIG. 3 shows the result of immunohistochemical staining of
the anti-hTfR antibodies in the cerebellum. In the cerebellum of
monkeys administered anti-hTfR antibodies Nos. 1 to 3, specific
staining of blood vessels were observed (FIG. 3b to d,
respectively). In particular, in the cerebellum of the monkeys
administered anti-hTfR antibodies No. 2 or 3, specific staining of
Purkinje cells was also observed (FIGS. 3c and 3d, respectively).
Besides, no staining was observed in the cerebellum of the control
with no anti-hTfR antibody administered, indicating that there was
almost no background staining (FIG. 3a).
[0680] From the above results of immunohistochemical staining in
the cerebrum, the hippocampus, and the cerebellum, it was
considered that while anti-hTfR antibody No. 1 can bind to hTfR
existing on the endothelium of blood vessels in the brain,
relatively small amount of it transfers to the brain parenchyma
compared with anti-hTfR antibodies Nos. 2 and 3. On the other hand,
it was found that anti-hTfR antibodies Nos. 2 and 3 can bind to
hTfR existing on the endothelium of blood vessels of the brain, and
after binding to hTfR, they pass through the blood-brain barrier
and transfer into the brain parenchyma, and further, are taken up
into the brain parenchyma and neuron-like cells in the hippocampus,
and are taken up by Purkinje cells in the cerebellum.
[Example 9] Preparation of Humanized Anti-hTfR Antibodies
[0681] Humanization was tried of the amino acid sequence included
in the light chain and the heavy chain variable regions of
anti-hTfR antibodies Nos. 1 to 3 shown in Table 1. From anti-hTfR
antibody No. 1 were obtained a humanized light chain variable
region having one of the amino acid sequences set forth as SEQ ID
NO:158 to SEQ ID NO:163, and a humanized heavy chain variable
region having one of the amino acid sequences set forth as SEQ ID
NO:166 to SEQ ID NO:171.
[0682] From anti-hTfR antibody No. 2 were obtained a humanized
light chain variable region having one of the amino acid sequences
set forth as SEQ ID NO:174 to SEQ ID NO:179, and a humanized heavy
chain variable region having one of the amino acid sequences set
forth as SEQ ID NO:182 to SEQ ID NO:187.
[0683] From anti-hTfR antibody No. 3 were obtained a humanized
light chain variable region having one of the amino acid sequences
set forth as SEQ ID NO:190 to SEQ ID NO:195, and a humanized heavy
chain variable region having one of the amino acid sequences set
forth as SEQ ID NO:204 to SEQ ID NO:209.
[Example 10] Construction of Genes Encoding Humanized Anti-hTfR
Antibodies
[0684] For each of anti-hTfR antibodies Nos. 1 to 3 above, DNA
fragments were artificially synthesized which contained a gene
encoding the full length of the light chain, and of the heavy
chain, having humanized anti-hTfR antibody light chain and heavy
chain variable regions, respectively. In doing this, a MluI
sequences and a sequence encoding a leader peptide was added, in
this order from the 5' end, on the 5' side of the gene encoding the
full length of the light chain, and on the 3' side was added a NotI
sequence. And, a MluI sequences and a sequence encoding a leader
peptide was added, in this order from the 5' end, on the 5' side of
the gene encoding the full length of the heavy chain, and on the 3'
side was added a Nod sequence. The leader peptide introduced above
is to function as secretion signal when the light chain and heavy
chain of the humanized antibody is expressed in mammalian cells as
host cells so that the light chain and the heavy chain are secreted
out of the cells.
[0685] For the light chain of anti-hTfR antibody No. 1, a DNA
fragment (SEQ ID NO:165) was synthesized, which included a gene
encoding the full length of the light chain (the light chain of
humanized anti-hTfR antibody No. 1) consisting of the amino acid
sequence set forth as SEQ ID NO:164, which had in the variable
region the amino acid sequence set forth as SEQ ID NO:163.
[0686] For the heavy chain of anti-hTfR antibody No. 1, a DNA
fragment (SEQ ID NO:173) was synthesized, which included a gene
encoding the full length of the heavy chain (the heavy chain of
humanized anti-hTfR antibody No. 1) consisting of the amino acid
sequence set forth as SEQ ID NO:172, which had in the variable
region the amino acid sequence set forth as SEQ ID NO:171.
[0687] The heavy chain of the humanized anti-hTfR antibody encoded
by the DNA fragment set forth as SEQ ID NO:173 is IgG1.
[0688] For the light chain of anti-hTfR antibody No. 2, a DNA
fragment (SEQ ID NO:181) was synthesized, which included a gene
encoding the full length of the light chain (the light chain of
humanized anti-hTfR antibody No. 2) consisting of the amino acid
sequence set forth as SEQ ID NO:180, which had in the variable
region the amino acid sequence set forth as SEQ ID NO:179.
[0689] For the heavy chain of anti-hTfR antibody No. 2, a DNA
fragment (SEQ ID NO:189) was synthesized, which included a gene
encoding the full length of the heavy chain (the heavy chain of
humanized anti-hTfR antibody No. 2) consisting of the amino acid
sequence set forth as SEQ ID NO:188, which had in the variable
region the amino acid sequence set forth as SEQ ID NO:187.
[0690] The heavy chain of the humanized anti-hTfR antibody encoded
by the DNA fragment set forth as SEQ ID NO:189 is IgG1.
[0691] For the light chain of anti-hTfR antibody No. 3, a DNA
fragment (SEQ ID NO:197) was synthesized, which included a gene
encoding the full length of the light chain (the light chain of
humanized anti-hTfR antibody No. 3) consisting of the amino acid
sequence set forth as SEQ ID NO:196, which had in the variable
region the amino acid sequence set forth as SEQ ID NO:191.
[0692] For the heavy chain of anti-hTfR antibody No. 3, a DNA
fragment (SEQ ID NO:211) was synthesized, which included a gene
encoding the full length of the heavy chain (the heavy chain of
humanized anti-hTfR antibody No. 3) consisting of the amino acid
sequence set forth as SEQ ID NO:210, which had in the variable
region the amino acid sequence set forth as SEQ ID NO:205.
[0693] The heavy chain of the humanized anti-hTfR antibody encoded
by the DNA fragment set forth as SEQ ID NO:211 is IgG1.
[0694] As to the light chain of anti-hTfR antibody No. 3, also
synthesized were,
[0695] a DNA fragment (SEQ ID NO:199) encoding the full length
amino acid sequence of the light chain (the light chain of
humanized anti-hTfR antibody No. 3-2) consisting of the amino acid
sequence set forth as SEQ ID NO:198, which had in the variable
region the amino acid sequence set forth as SEQ ID NO:193;
[0696] a DNA fragment (SEQ ID NO:201) encoding the full length
amino acid sequence of the light chain (the light chain of
humanized anti-hTfR antibody No. 3-3) consisting of the amino acid
sequence set forth as SEQ ID NO:200, which had in the variable
region the amino acid sequence set forth as SEQ ID NO:194;
[0697] a DNA fragment (SEQ ID NO:203) encoding the full length
amino acid sequence of the light chain (the light chain of
humanized anti-hTfR antibody No. 3-4) consisting of the amino acid
sequence set forth as SEQ ID NO:202, which had in the variable
region the amino acid sequence set forth as SEQ ID NO:195;
[0698] Further, for the heavy chain of anti-hTfR antibody No. 3,
also synthesized was a DNA fragment (SEQ ID NO:213) encoding the
full length amino acid sequence of the heavy chain (the heavy chain
IgG4 of humanized anti-hTfR antibody No. 3) consisting of the amino
acid sequence set forth as SEQ NO:212, which had in the variable
region the amino acid sequence set forth as SEQ ID NO:205;
[0699] The heavy chain of the humanized anti-hTfR antibody encoded
by the DNA fragment set forth as SEQ ID NO:213 is IgG4.
[Example 11] Construction of Humanized Anti-hTiR Antibody
Expression Vector
[0700] Vector pEF/myc/nuc (Invitrogen Inc.) was digested with KpnI
and NcoI to cut out a region including EF-1.alpha., promoter and
its first intron, and this was blunt-ended with T4 DNA polymerase.
A region including the CMV enhancer/promoter and intron was removed
from pCI-neo (Invitrogen Inc.) by digesting it with BgIII and
EcoRI, and the remaining fragment thus left was blunt-ended with T4
DNA polymerase. To this was inserted the above-mentioned region
including EF-1.alpha. promoter and its first intron to construct
pE-neo vector. This vector, pE-neo, was digested with SfiI and
BstXI to remove a region of approximately 1 kb including a neomycin
resistance gene. PCR was performed employing
pcDNA3.1/Hygro(+)(Invitrogen) as a template and using primer
Hyg-Sfi5' (SEQ ID NO:216) and primer Hyg-BstX3' (SEQ ID NO:217) to
amplify hygromycin gene. The hygromycin gene thus amplified was
digested with SfiI and BstXI and inserted into the above pE-neo
vector from which neomycin resistance gene had been removed to
construct a vector pE-hygr.
[0701] Vectors pE-hygr and pE-neo were both digested with MluI and
Nod. The DNA fragment (SEQ ID NO:165) encoding the light chain of
humanized anti-hTfR antibody No. 1 and the DNA fragment (SEQ ID
NO:173) encoding the heavy chain of the antibody, both synthesized
in Example 10, were digested with MluI and NotI, and the fragments
thus obtained were inserted into vector pE-hygr and vector pE-neo,
respectively, between their MluI and NotI sites. The vectors thus
obtained were used as an expression vector for the light chain of
humanized anti-hTfR antibody No. 1, pE-hygr(LC1), and as an
expression vector for the heavy chain of humanized anti-hTfR
antibody No. 1, pE-neo(HC1), in the experiments described
below.
[0702] In an analogous manner, the DNA fragment (SEQ ID NO:181)
encoding the light chain of humanized anti-hTfR antibody No. 2 and
the DNA fragment (SEQ ID NO:189) encoding the heavy chain of the
antibody, both synthesized in Example 10, were both digested with
MluI and NotI, and the fragments thus obtained were inserted into
vector pE-hygr and vector pE-neo, respectively, between their MluI
and NotI sites. The vectors thus obtained were used as an
expression vector for the light chain of humanized anti-hTfR
antibody No. 2, pE-hygr(LC2), and as an expression vector for the
heavy chain of the antibody of humanized anti-hTfR antibody No. 2,
pE-neo(HC2), in the experiments described below.
[0703] Further, in the same manner as above, the DNA fragment (SEQ
ID NO:197) encoding the light chain of humanized anti-hTfR antibody
No. 3 and the DNA fragment (SEQ ID NO:211) encoding the heavy chain
of the antibody, both synthesized in Example 10, were both digested
with MluI and Noll, and the fragments thus obtained were inserted
into vector pE-hygr and vector pE-neo, respectively, between their
MluI and NotI sites. The vectors thus obtained were used as an
expression vector for the light chain of humanized anti-hTfR
antibody No. 3, pE-hygr(LC3), and as an expression vector for the
heavy chain of the antibody, pE-neo(HC3), in the experiments
described below.
[0704] Further, as to the light chain of anti-hTfR antibody No. 3,
the following fragments synthesized in Example 10, namely:
[0705] the DNA fragment (SEQ ID NO:199) encoding the light chain of
humanized anti-hTfR antibody No. 3-2,
[0706] the DNA fragment (SEQ ID NO:201) encoding the light chain of
humanized anti-hTfR antibody No. 3-3, and
[0707] the DNA fragment (SEQ ID NO:203) encoding the light chain of
humanized anti-hTfR antibody No. 3-4,
were digested with MluI and NotI, and inserted into the vector
pE-hygr between the MluI and NotI sites thereof to construct
[0708] pE-hygr(LC3-2), an expression vector for the light chain of
humanized anti-hTfR antibody No. 3-2,
[0709] pE-hygr(LC3-3), an expression vector for the light chain of
humanized anti-hTfR antibody No. 3-3, and
[0710] pE-hygr(LC3-4), an expression vector for the light chain of
humanized anti-hTfR antibody No. 3-4, respectively.
[0711] Further, in the same manner as above, as to the heavy chain
of anti-hTfR antibody No. 3, the DNA fragment (SEQ ID NO:213)
encoding the heavy chain IgG4 of humanized anti-hTfR antibody No. 3
synthesized in Example 10 was digested with MluI and NotI, and
inserted into the vector pE-neo between the MluI and NotI sites
thereof to construct pE-neo(HC3-IgG4), an expression vector for the
heavy chain IgG4 of humanized anti-hTfR antibody No. 3.
[Example 12] Construction of Cells for Expression of Humanized
Anti-hTfR Antibody
[0712] CHO cells (CHO-K1: obtained from American Type Culture
Collection) were transformed with pE-hygr(LC1), the vector for
light chain expression, and pE-neo(HC1), the vector for heavy chain
expression, both constructed in Example 11, as follows, using
GenePulser (Bio-Rad Inc.). Transformation of the cells was
performed in the following manner as a whole. 5.times.10.sup.5 of
CHO-K1 cells were seeded in a 3.5-cm culture dish containing CD
OptiCHO.TM. medium (Life Technologies Inc.) and cultured overnight
at 37.degree. C., 5% CO.sub.2. The medium was replaced with
Opti-MEM.TM. I medium (Life Technologies Inc.), and the cells were
suspended at the density of 5.times.10.sup.6 cells/mL. 100 .mu.l of
the cell suspension were taken, to which was added 5 .mu.L each of
a pE-hygr(LC1) and a pE-neo(HC1) plasmid DNA solution both having
been diluted with Opti-MEM.TM. I medium to 100 .mu.g/mL. These
plasmids were introduced into the cells by electroporation using
GenePulser (Bio-Rad Inc.). The cells then were cultured overnight
under the condition of 37.degree. C., 5% CO.sub.2, and subjected to
selection culture in CD OptiCHO.TM. medium supplemented with 0.5
mg/ml of hygromycin and 0.8 mg/mL of G418.
[0713] Then, the cells selected above through the selection culture
were seeded on 96-well plates so that not more than one cell might
be seeded per well by limiting dilution. The cells then were
cultured for about 10 days so that monoclonal colonies were formed.
Respective culture supernatants of the wells in which monoclonal
colony was formed were collected, the amount of the humanized
antibody contained in culture supernatants was determined by ELISA,
and humanized antibody high-expressing cell lines were
selected.
[0714] The ELISA above was conducted as follows in general. To each
well of 96-well microtiter plates (Nunc Inc.) were added 100 .mu.L
of a goat anti-human IgG polyclonal antibody solution diluted with
0.05 M sodium bicarbonate buffer (pH 9.6) to 4 .mu.g/mL, and the
plate was left to stand for at least one hour at room temperature
so as to allow the antibody to be adsorbed by the plates. Then,
after each well was washed three times with a phosphate-buffered
saline (pH 7.4) supplemented with 0.05% Tween20 (PBS-T), 200 .mu.L
of Starting Block (PBS) Blocking Buffer (Thermo Fisher Scientific
Inc.) was added to each well, and the plates were left to stand for
30 minutes at room temperature. After each well was washed with
PBS-T three times, the culture supernatant or the human IgG
reference standard product which had been diluted with a PBS
supplemented with 0.5% BSA and 0.05% Tween20 (PBS-BT) to
appropriate concentrations, was added to each well, in the amount
of 100 .mu.L, and the plates were left to stand for at least one
hour at room temperature. After the plates were washed three times
with PBS-T, 100 .mu.L of HRP-labeled anti-human IgG polyclonal
antibody solution which had been diluted with PBS-BT, was added to
each well, and the plates were left to stand for at least one hour
at room temperature. After the wells were washed three times with
PBS-T, 0.4 mg/mL o-phenylenediamine in citrate-phosphate buffer (pH
5.0) was added to each well, in the amount of 100 .mu.L, and the
wells were left to stand for 8 to 20 minutes at room temperature.
Then, 1 mol/L sulfuric acid was added to each well, in the amount
of 100 .mu.L to terminate the reaction, and the absorbance for each
well was measured at 490 run using a 96-well plate reader. The
cells corresponding to the wells which exhibited the higher
measurements were regarded as a high-expressing cell line for
humanized anti-hTfR antibody No. 1. This was designated antibody
No. 1 expressing cell line.
[0715] In the same manner, CHO cells were transformed with the
light chain expression vector pE-hygr(LC2) and the heavy chain
expression vector pE-neo(HC2), both constructed in Example 11, and
a high-expressing cell line for humanized anti-hTfR antibody No. 2
was obtained. This was designated antibody No. 2 expressing cell
line.
[0716] Further, in the same manner, CHO cells were transformed with
the light chain expression vector pE-hygr(LC3) and the heavy chain
expression vector pE-neo(HC3), both constructed in Example 11, and
a high-expressing cell line for humanized anti-hTfR antibody No. 3
was obtained. This was designated antibody No. 3 expressing cell
line.
[0717] Further, in the same manner, CHO cells were transformed with
the light chain expression vector pE-hygr(LC3-2) and the heavy
chain expression vector pE-neo(HC3), both constructed in Example
11, and a high-expressing cell line for humanized anti-hTfR
antibody No. 3-2 was obtained. This was designated antibody No. 3-2
expressing cell line.
[0718] Further, in the same manner, CHO cells were transformed with
the light chain expression vector pE-hygr(LC3-3) and the heavy
chain expression vector pE-neo(HC3), both constructed in Example
11, and a high-expressing cell line for humanized anti-hTfR
antibody No. 3-3 was obtained. This was designated antibody No. 3-3
expressing cell line.
[0719] Further, in the same manner, CHO cells were transformed with
the light chain expression vector pE-hygr(LC3-4) and the heavy
chain expression vector pE-neo(HC3) both constructed in Example 11,
and a high-expressing cell line for humanized anti-hTfR antibody
No. 3-4 was obtained. This was designated antibody No. 3-4
expressing cell line.
[0720] Further, in the same manner, CHO cells were transformed with
the light chain expression vector pE-hygr(LC3) and the heavy chain
expression vector pE-neo(HC3-IgG4) both constructed in Example 11,
and a high-expressing cell line for humanized anti-hTfR antibody
No. 3(IgG4) was obtained. This was designated antibody No. 3(IgG4)
expressing cell line.
[0721] Further, in the same manner, CHO cells were transformed with
the light chain expression vector pE-hygr(LC3-2) and the heavy
chain expression vector pE-neo(HC3-IgG4) both constructed in
Example 11, and a high-expressing cell line for humanized anti-hTfR
antibody No. 3-2 (IgG4) was obtained. This was designated antibody
No. 3-2 (IgG4) expressing cell line.
[Example 13] Purification of Humanized Anti-hTfR Antibodies
[0722] Antibody No. 1 expressing cell line, antibody No. 2
expressing cell line, antibody No. 3 expressing cell line, antibody
No. 3-2 expressing cell line, antibody No. 3-3 expressing cell line
and antibody No. 3-4 expressing cell line obtained in Example 12
were respectively diluted with CD OptiCHO.TM. medium to the density
of approximately 2.times.10.sup.5 cells/mL. The cell suspensions,
200 mL, was added to a 1 L-conical flask, and cultured for 6 to 7
days in a humid environment at 37.degree. C., 5% CO.sub.2, 95% air,
with stirring at a rate of about 70 rpm. Each culture supernatant
was collected by centrifugation, and filtered through a 0.22 .mu.m
filter (Millipore Inc.) to prepare the culture supernatant. To each
culture supernatant thus obtained was added five volumes of 20 mM
Tris buffer (pH 8.0) containing 150 mM NaCl, and loaded onto a
Protein A column (column volume: 1 mL, Bio-Rad Inc.) which had been
equilibrated in advance with three column volumes of 20 mM Tris
buffer (pH 8.0) containing 150 mM NaCl. Then, the column was washed
with five column volumes of the same buffer, and the adsorbed
humanized antibody was eluted with four column volumes of 50 mM
glycine buffer (pH 2.8) containing 150 mM NaCl, and the eluted
fraction was collected. The eluted fractions was added and
neutralized with 1 M Tris buffer (pH 8.0) and used as the purified
antibody preparation.
[0723] In the above, the antibody purified from the culture
supernatant of antibody No. 1 expressing cell line was designated
humanized anti-hTfR antibody No. 1. The antibody purified from the
culture supernatant of antibody No. 2 expressing cell line was
designated humanized anti-hTfR antibody No. 2. The antibody
purified from the culture supernatant of antibody No. 3 expressing
cell line was designated humanized anti-hTfR antibody No. 3. The
antibody purified from the culture supernatant of antibody No. 3-2
expressing cell line was designated humanized anti-hTfR antibody
No. 3-2. The antibody purified from the culture supernatant of
antibody No. 3-3 expressing cell line was designated humanized
anti-hTfR antibody No. 3-3. The antibody purified from the culture
supernatant of antibody No. 3-4 expressing cell line was designated
humanized anti-hTfR antibody No. 3-4.
[0724] Further, antibody No. 3(IgG4) expressing cell line and
antibody No. 3-2 (IgG4) expressing cell line obtained in Example 12
also were cultured in the same manner as above, and from their
culture supernatants were obtained purified humanized anti-hTfR
antibody No. 3(IgG4) and humanized anti-hTfR antibody No. 3-2
(IgG4), respectively. These two antibodies were employed in the
pharmacokinetic analysis using monkeys described in Example 15.
[Example 14] Measurement of Affinity of Humanized Anti-hTfR
Antibodies to Human TfR and Monkey TfR
[0725] The affinity of the humanized anti-hTfR antibodies obtained
in Example 13 to human and monkey TfRs was measured by the method
described in Example 7. Table 7 shows the result of the measurement
of the association rate constant (k.sub.on), dissociation rate
constant (k.sub.off), and dissociation constant (k.sub.D) of
humanized anti-hTfR antibodies Nos. 1 to 3-4 (corresponding to Nos.
1 to 3-4, respectively, in the table) to human TfR.
TABLE-US-00008 TABLE 7 Affinity of humanized anti-hTfR antibodies
to human TfR Antibody No. kon (M.sup.-1s.sup.-1) koff (s.sup.-1)
K.sub.D (M) 1 3.93 .times. 10.sup.5 <1.0 .times. 10.sup.-7
<1.0 .times. 10.sup.-12 2 1.97 .times. 10.sup.5 <1.0 .times.
10.sup.-7 <1.0 .times. 10.sup.-12 3 1.19 .times. 10.sup.6
<1.0 .times. 10.sup.-7 <1.0 .times. 10.sup.-12 3-2 6.06
.times. 10.sup.5 1.45 .times. 10.sup.-5 2.39 .times. 10.sup.-11 3-3
6.00 .times. 10.sup.5 1.25 .times. 10.sup.-5 2.09 .times.
10.sup.-11 3-4 1.01 .times. 10.sup.6 <1.0 .times. 10.sup.-7
<1.0 .times. 10.sup.-12
[0726] Table 8 shows the result of the measurement of the
association rate constant (k.sub.on), dissociation rate constant
(k.sub.off), and dissociation constant (k.sub.D) of humanized
anti-hTfR antibodies Nos. 1 to 3-4 (corresponding to Nos. 1 to 3-4,
respectively, in the table) to monkey TfR.
TABLE-US-00009 TABLE 8 Affinity of humanized anti-hTfR antibodies
to monkey TfR Antibody No. kon (M.sup.-1s.sup.-1) koff (s.sup.-1)
K.sub.D (M) 1 2.53 .times. 10.sup.5 <1.0 .times. 10.sup.-7
<1.0 .times. 10.sup.-12 2 4.87 .times. 10.sup.5 3.67 .times.
10.sup.-5 7.55 .times. 10.sup.-11 3 6.03 .times. 10.sup.5 6.76
.times. 10.sup.-4 1.12 .times. 10.sup.-9 3-2 4.95 .times. 10.sup.5
8.76 .times. 10.sup.-4 1.77 .times. 10.sup.-9 3-3 4.88 .times.
10.sup.5 9.32 .times. 10.sup.-4 1.91 .times. 10.sup.-9 3-4 5.19
.times. 10.sup.5 1.35 .times. 10.sup.-4 2.60 .times. 10.sup.-10
[0727] The result of the measurement of the affinity of humanized
anti-hTfR antibody Nos. 1 to 3-4 to human TfR showed that the
dissociation constant between humanized anti-hTfR antibodies Nos.
1, 2, 3, and 3-4 and human TfR was less than 1.times.10.sup.-12M
(Table 7). And the dissociation constant between humanized
anti-hTfR antibodies Nos. 3-2 and 3-3 and human TfR was
2.39.times.10.sup.-11 M and 2.09.times.10.sup.-11 M, respectively.
At the same time, the dissociation constant between the
pre-humanized anti-hTfR antibodies corresponding to those
antibodies and human TfR was: 5.09.times.10.sup.-12M for antibody
No. 1, 1.12.times.10.sup.-11M for antibody No. 2, and less than
1.times.10.sup.-12 M for antibody No. 3 (Table 4). These results
demonstrate that the high affinity of those pre-humanized anti-hTfR
antibodies to human TfR was maintained after humanization of the
antibodies, and indicate that anti-hTfR antibodies Nos. 4 to 14
would also maintain their affinity to human TfR after their
humanization.
[0728] Then, looking to the result of measurement of the affinity
of humanized anti-hTfR antibodies to monkey DR, it is seen that the
dissociation constant of humanized anti-hTfR antibody No. 1 was
less than 1.times.10.sup.-12 M, indicating that the pre-humanized
affinity was maintained after humanization, and also with regard to
humanized anti-hTfR antibody No. 2, the dissociation constant was
4.18.times.10.sup.-11M before humanization and
7.55.times.10.sup.-11 M after humanization, indicating the affinity
was maintained (Table 5, Table 8). On the other hand, regarding to
humanized anti-hTfR antibodies Nos. 3 to 3-4, while the
dissociation constant of anti-hTfR antibody No. 3, the
pre-humanized antibody corresponding to them, to monkey TfR was
less than 1.times.10.sup.-12 M, their dissociation constant after
humanization was 2.60.times.10.sup.-10M to 1.91.times.10.sup.-9M,
showing a lowering of the affinity to monkey TfR. As to the
humanized anti-hTfR antibody No. 3, although a lowering of affinity
to monkey TfR was observed, the result indicates that the
pre-humanized high affinity of anti-hTfR antibody to monkey TfR was
not lost after its humanization but was maintained as a whole. It
indicates that as to humanized anti-hTfR antibodies Nos. 4 to 14,
too, the pre-humanized affinity to monkey TfR could be maintained
after their humanization.
[Example 15] Pharmacokinetic Analysis of Humanized Anti-hTfR
Antibody in Monkey
[0729] Using monkeys, pharmacokinetic analysis was performed with
four antibodies: humanized anti-hTfR antibody No. 3, humanized
anti-hTfR antibody No. 3-2, humanized anti-hTfR antibody No. 3
(IgG4), and humanized anti-hTfR antibody No. 3-2 (IgG4). Besides,
the heavy chain of humanized anti-hTfR antibody No. 3 was IgG1,
while in humanized anti-hTfR antibody No. 3 (IgG4), the heavy chain
of humanized anti-hTfR antibody No. 3 had been converted into IgG4,
with its variable region kept intact. Further, the heavy chain of
humanized anti-hTfR antibody No. 3-2 was IgG1, while in humanized
anti-hTfR antibody No. 3-2 (IgG4), the heavy chain of humanized
anti-hTfR antibody No. 3-2 had been converted into IgG4 with its
variable region kept intact. These four antibodies were
respectively intravenously administered once to male cynomolgus
monkeys, at a dosage of 5.0 mg/kg, and their peripheral blood was
sampled before the administration, 2 minutes, 30 minutes, 2 hours,
4 hours and 8 hours after the administration, and then they were
subjected to whole body irrigation. As a negative control,
trastuzumab (Herceptin.TM., Chugai Pharmaceutical Co., Ltd.), a
humanized antibody to HER2 protein, was intravenously administered
once to a single monkey in the same manner, and its peripheral
blood was sampled before the administration, 2 minutes, 30 minutes,
2 hours, 4 hours and 8 hours after the administration, and then it
was subjected to the whole body irrigation. After the irrigation,
the brain and spine tissues including the medulla oblongata and
other tissues (liver, heart, spleen and bone marrow) were excised.
Using these brain and spinal tissues and other tissues, the
concentration of the humanized anti-hTfR antibodies was measured
and immunohistochemical staining was carried out.
[0730] Measurement of the concentration of humanized anti-hTfR
antibodies in tissues and peripheral blood was carried out largely
following the procedure described below. Besides, as to the brain,
the collected tissues were separated into the cerebral cortex, the
cerebellum, the hippocampus and the medulla oblongata, and then the
concentration of the humanized anti-hTfR antibodies were measured.
The respective tissues thus obtained were homogenized with RIPA
Buffer (Wako Pure Chemical Industries Inc.) containing Protease
Inhibitor Cocktail (Sigma-Aldrich Inc.), centrifuged, and the
supernatant collected. From the above peripheral blood, serum was
separated. To each well of High Bind Plate (Meso Scale Diagnostics)
was added 10 .mu.L of Affinipure Goat Anti mouse IgG Fc.gamma. pAb
(Jackson ImmunoResearch Inc.), and the plate was left to stand for
one hour to provide a solid phase. Then, 150 .mu.L of SuperBlock
blocking buffer in PBS (Thermo Fisher Scientific Inc.) was added to
each well, and the plate was blocked by one-hour shaking. Then, 25
.mu.L of the supernatant of the homogenate or the serum was added
to each well, and the wells were shaken for one hour. Then, 25
.mu.L of Affinipure Goat Anti mouse IgG Fab-Biotin (Jackson
ImmunoResearch Inc.) was added to each well, and shaking was
continued for one hour. Then, 25 .mu.L of SULFO-Tag-Streptavidin
(Meso Scale Diagnostics Inc.) was added to each well, followed by
shaking for half an hour. To each well was added 150 .mu.L of Read
buffer T (Meso Scale Diagnostics Inc.), and the amount of
luminescence from each well was read on a Sector.TM. Imager 6000
reader. The amount of the antibody contained in each tissue and the
peripheral blood was calculated by producing a standard curve based
on measurements of standard samples containing known concentrations
of the anti-hTfR antibody, and then interpolating the measurement
of each of the samples with reference to the standard. Measurement
of concentration was repeated three times for each sample.
[0731] The result of measurement of the concentration of humanized
anti-hTfR antibodies in the brain and spinal tissues is shown in
Table 9.
TABLE-US-00010 TABLE 9 Concentration of humanized anti-hTfR
antibodies in brain tissues (.mu.g/g wet weight) Antibody Cerebral
Medulla No. cortex Cerebellum Hippocampus oblongata Spinal cord 3
0.67 .+-. 0.12 0.61 .+-. 0.02 0.49 .+-. 0.02 0.59 .+-. 0.10 0.46
.+-. 0.17 3-2 1.05 .+-. 0.07 0.72 .+-. 0.04 0.72 .+-. 0.07 0.69
.+-. 0.03 0.46 .+-. 0.02 3(IgG4) 0.65 .+-. 0.05 0.59 .+-. 0.03 0.56
.+-. 0.02 0.59 .+-. 0.02 0.46 .+-. 0.07 3-2(IgG4) 0.76 .+-. 0.02
0.57 .+-. 0.07 0.62 .+-. 0.05 0.73 .+-. 0.16 0.48 .+-. 0.03
Negative 0.0082 .+-. 0.0032 0.0090 .+-. 0.0067 0.0053 .+-. 0.0009
0.011 .+-. 0.003 0.154 .+-. 0.04 control
[0732] All the antibodies, i.e., humanized anti-hTfR antibody No.
3, humanized anti-hTfR antibody No. 3-2, humanized anti-hTfR
antibody No. 3 (IgG4) and humanized anti-hTfR antibody No. 3-2
(IgG4), were observed to accumulate in the cerebral cortex,
cerebellum, hippocampus, medulla oblongata and spinal cord (Table
9). The respective amount accumulated was as follow:
[0733] with humanized anti-hTfR antibody No. 3, approximately 82
times in the cerebral cortex, approximately 68 times in the
cerebellum, approximately 92 times in the hippocampus,
approximately 54 times in the medulla oblongata, and approximately
3.1 times in the spinal cord, in comparison with the negative
control, trastuzumab (Herceptin.TM.),
[0734] with humanized anti-hTfR antibody No. 3-2, approximately 128
times in the cerebral cortex, approximately 80 times in the
cerebellum, approximately 136 times in the hippocampus,
approximately 63 times in the medulla oblongata, approximately 3.1
times in the spinal cord, in comparison with the negative control,
trastuzumab,
[0735] with humanized anti-hTfR antibody No. 3 (IgG4),
approximately 79 times in the cerebral cortex, approximately 66
times in the cerebellum, approximately 106 times in the
hippocampus, approximately 54 times in the medulla oblongata,
approximately 3.1 times in the spinal cord, in comparison with the
negative control, trastuzumab, and
[0736] with humanized anti-hTfR antibody No. 3-2 (IgG4),
approximately 93 times in the cerebral cortex, approximately 63
times in the cerebellum, approximately 117 times in the
hippocampus, approximately 66 times in the medulla oblongata,
approximately 3.2 times in the spinal cord, in comparison with the
negative control, trastuzumab (Table 10).
[0737] These results indicate that these four humanized anti-hTfR
antibodies have a property that allows them to pass through the
blood-brain barrier and accumulate in the brain tissues, and that
it is now possible to let pharmaceutical agents which need to be
brought into function in the brain tissues efficiently accumulate
there, by binding such pharmaceutical agents to one of these
antibodies.
TABLE-US-00011 TABLE 10 Amount of humanized anti-hTfR antibodies
accumulated in brain tissues (factors in comparison with negative
control) Antibody Cerebral Hippo- Medulla Spinal No. cortex
Cerebellum campus oblongata cord 3 82 68 92 54 3.1 3-2 128 80 136
63 3.1 3(IgG4) 79 66 106 54 3.1 3-2(IgG4) 93 63 117 66 3.2 Negative
1 1 1 1 1 control
[0738] Then, FIG. 4 shows the result of measurement of the
concentration of the humanized anti-hTfR antibodies in the tissues
of the liver, heart, spleen and bone marrow. The four humanized
anti-hTfR antibodies, as well as the negative control, trastuzumab,
were observed to accumulate in the liver and spleen, and their
amount accumulated was equal between the four humanized anti-hTfR
antibodies and trastuzumab. In the heart, the humanized anti-hTfR
antibodies tended to accumulate more than trastuzumab, the negative
control, but the amount was only about 1.5 to 2.8 times that of the
negative control. In bone marrow, the humanized anti-hTfR
antibodies tended to accumulate markedly more than trastuzumab, the
negative control, and the amount was 3.5 to 16 times that of the
negative control. The cause of this accumulation of the humanized
anti-hTfR antibodies in bone marrow is thought to be that TfR is
expressed at high levels in bone marrow, hematopoietic organ, and
more humanized anti-hTfR antibodies, therefore, accumulate through
binding to TfR, than the negative control. These data indicate that
the four humanized anti-hTfR antibodies has a property that allows
them to specifically accumulate the cerebrum, cerebellum,
hippocampus and medulla oblongata, which constitute the central
nervous system, and that it is now possible to let pharmaceutical
agents which need to be brought into function in the brain tissues
efficiently accumulate there, by binding such pharmaceutical agents
to one of these antibodies.
[0739] Then, Table 11 shows the result of pharmacokinetic
measurement of the humanized anti-hTfR antibodies in the blood. As
that of the negative control, trastuzumab, the blood concentration
of the four humanized anti-hTfR antibodies was maintained at high
levels, higher than 60 .mu.g/mL, even eight hours after
administration, indicating that they are stable in the blood.
TABLE-US-00012 TABLE 11 Pharmacokinetics of humanized anti-hTfR
antibodies in blood (.mu.g/mL blood( ) Antibody Time after
administration No. 2 min 30 min 2 hr 4 hr 8 hr 3 173 147 128 117
97.5 3-2 124 99.5 78.5 76.5 61 3(IgG4) 141 113 99 95 83 3-2(IgG4)
132 111 98.5 99 95.5 Negative 124 92.5 96 75.5 60.5 control
[0740] Immunohistochemical staining of the humanized anti-hTfR
antibodies in brain tissues was performed in the following manner.
The collected tissues were rapidly frozen to -80.degree. C. in a
Tissue-Tek Cryo 3DM (Sakura Finetek Inc.) to prepare frozen blocks
of tissues. The frozen blocks were sliced into 4 .mu.m sections,
which were affixed to MAS coated glass slides (Matsunami Glass
Inc.). The tissue sections were reacted with 4% paraformaldehyde
(Wako Pure Chemical Industries Inc.) for 5 minutes at 4.degree. C.
and fixed to glass slides. Then, the tissue sections were reacted
with methanol solution containing 0.3% hydrogen peroxide (Wako Pure
Chemical Industries Inc.) for 30 min to inactivate endogenous
peroxidases. Then, the glass slides were blocked by reacting
SuperBlock blocking buffer in PBS for 30 min at room temperature.
Then, the tissue sections were reacted with Mouse IgG-heavy and
light chain Antibody (Bethyl Laboratories) for one hour at room
temperature. The tissue sections were allowed to visualize with DAB
substrate (3,3'-diaminobenzidine, Vector Laboratories Inc.),
counterstained with Mayer's hematoxylin solution (Merck Inc.),
embedded after dehydration and cleaning, and observed under a
optical microscope.
[0741] FIG. 5 shows the result of immunohistochemical staining of
the humanized anti-hTfR antibodies in the cerebral cortex. Specific
staining of blood vessels and neuron-like cells were observed in
the cerebral cortex of the monkeys administered humanized anti-hTfR
antibody No. 3, humanized anti-hTfR antibody No. 3-2, humanized
anti-hTfR antibody No. 3 (IgG4), and humanized anti-hTfR antibody
No. 3-2 (IgG4) (FIGS. 5b to 5e, respectively). In the cerebral
cortex of the monkey administered humanized anti-hTfR antibody No.
3-2, in particular, (FIG. 5c), the brain parenchyma region, outside
the blood vessels, was also observed specifically stained
extensively. Besides, no staining was observed in the cerebral
cortex of the monkey administered Herceptin as a control,
indicating that the tissue staining observed in FIGS. 5b to 5e was
specific for the humanized anti-hTfR antibodies (FIG. 5a).
[0742] FIG. 6 shows the result of immunohistochemical staining of
the humanized anti-hTfR antibodies in the hippocampus. Specific
staining of blood vessels and neuron-like cells were observed in
the hippocampus of the monkeys administered humanized anti-hTfR
antibody No. 3, humanized anti-hTfR antibody No. 3-2, humanized
anti-hTfR antibody No. 3(IgG4), and humanized anti-hTfR antibody
No. 3-2 (IgG4) (FIGS. 6b to 6e, respectively). Besides, no staining
was observed in the hippocampus of the monkey administered
Herceptin as a control, indicating that the tissue staining
observed in FIGS. 6b to 6e was specific for the humanized anti-hTfR
antibodies (FIG. 6a).
[0743] FIG. 7 shows the result of immunohistochemical staining of
the humanized anti-hTfR antibodies in the cerebellum. Specific
staining of blood vessels and Purkinje cells were observed in the
cerebellum of the monkeys administered humanized anti-hTfR antibody
No. 3, humanized anti-hTfR antibody No. 3-2, humanized anti-hTfR
antibody No. 3(IgG4), and humanized anti-hTfR antibody No. 3-2
(IgG4) (FIGS. 7b to 7e, respectively). Besides, no staining was
observed in the cerebellum of the monkey administered Herceptin as
a control, indicating that the tissue staining observed in FIGS. 7b
to 7e was specific for the humanized anti-hTfR antibodies (FIG.
7a).
[0744] FIG. 8 shows the result of immunohistochemical staining of
the humanized anti-hTfR antibodies in the medulla oblongata.
Specific staining of blood vessels and neuron-like cells were
observed in the medulla oblongata of the monkeys administered
humanized anti-hTfR antibody No. 3, humanized anti-hTfR antibody
No. 3-2, humanized anti-hTfR antibody No. 3(IgG4), and humanized
anti-hTfR antibody No. 3-2 (IgG4) (FIGS. 8b to 8e, respectively).
Besides, no staining was observed in the medulla oblongata of the
monkey administered Herceptin as a control, indicating that the
tissue staining observed in FIGS. 8b to 8e was specific for the
humanized anti-hTfR antibodies (FIG. 8a).
[0745] From the result of immunohistochemical staining of the
cerebrum and cerebellum in Example 8, it had been anticipated that
though the anti-hTfR antibody No. 1, a pre-humanized mouse
antibody, can bind to hTfR existing on the endothelium of blood
vessel in the brain, the amount transferring to the brain
parenchyma would be small. On the other hand, it was shown that
anti-hTfR antibodies Nos. 2 and 3, pre-humanized mouse antibodies,
can bind to hTfR existing on the endothelium of blood vessel in the
brain, and after binding to hTfR, pass through the blood-brain
barrier into the brain parenchyma, and further be taken up into the
brain parenchyma and neuron-like cells in the hippocampus, and into
Purkinje cells in the cerebellum.
[0746] From the result of immunohistochemical staining in the
cerebrum, hippocampus, cerebellum, and medulla oblongata in Example
15, it was revealed that the tested four humanized anti-hTfR
antibodies obtained by humanizing anti-hTfR antibody No. 3
subjected to the experiment can bind to hTfR existing on the
endothelium of blood vessels of the brain, and after binding to
hTfR, pass through the blood-brain barrier and transfer into the
brain parenchyma, and further, be taken up into neuron-like cells
in the cerebral cortex; into the brain parenchyma and the
neuron-like cells in the hippocampus; into Purkinje cells in the
cerebellum; and into neuron-like cells in the medulla
oblongata.
[Example 16] Preparation of hI2S-Humanized Anti-hTfR Antibody
Fusion Protein Expression Cells
[0747] By digesting pEF/myc/nucvector (Invitrogen Inc.) with KpnI
and NcoI, a region including EF-1.alpha. promoter and its first
intron was cut out, which then was blunt-ended with T4 DNA
polymerase. After digesting pCI-neo (Invitrogen Inc.) with BgIII
and EcoRI to remove a region including the enhancer/promoter and
intron of CMV, the vector was blunt-ended with T4 DNA polymerase,
and into which the above mentioned region including EF-1.alpha.
promoter and its first intron was inserted to construct a vector
pE-neo. The vector pE-neo was digested with SfiI and BstXI to cut
out a region of approximately 1 kbp including a neomycin resistance
gene. Employing pcDNA3.1/Hygro(+) (Invitrogen Inc.) as a template
and using primer Hyg-Sfi5' (SEQ ID NO:216) and primer Hyg-BstX3'
(SEQ ID NO:217), PCR was performed to amplify the hygromycin gene.
The hygromycin gene thus amplified was digested with SfiI and BstXI
and inserted into the above vector pE-neo, of which the neomycin
resistance gene had been removed, to construct vector pE-hygr.
[0748] A DNA fragment was artificially synthesized having the
nucleotide sequence set forth as SEQ ID NO:248, which included a
gene encoding a protein in which the humanized anti-hTfR antibody
heavy chain having the amino acid sequence set forth as SEQ ID
NO:172 was linked, on the C-terminal side thereof and via a linker
sequence (Gly Ser), to hI2S having the amino acid sequence set
forth as SEQ ID NO:246. This DNA fragment encoded a protein in
which humanized anti-hTfR antibody heavy chain having the amino
acid sequence set forth as SEQ ID NO:247 was linked, via a linker
sequence (Gly Ser), to hI2S. This DNA fragment had, on its 5' side,
a MluI sequence, and a sequence encoding a leader peptide acting as
a secretion signal in this order from the 5' end, and a NotI
sequence on its 3' side. The DNA fragment was digested with MluI
and NotI, and inserted into the vector pE-neo, between the MluI and
Nod thereof, to construct pE-neo(HC-I2S-1).
[0749] A DNA fragment was artificially synthesized having the
nucleotide sequence set forth as SEQ ID NO:250, which included a
gene encoding a protein in which the humanized anti-hTfR antibody
heavy chain having the amino acid sequence set forth as SEQ ID
NO:188 was linked, on the C-terminal side thereof and via a linker
sequence (Gly Ser), to hI2S having the amino acid sequence set
forth as SEQ ID NO:246. This DNA fragment encoded a protein in
which humanized anti-hTfR antibody heavy chain having the amino
acid sequence set forth as SEQ ID NO:249 was linked, via a linker
sequence (Gly Ser), to hI2S. This DNA fragment had, on its 5' side,
a MluI sequence and a sequence encoding a leader peptide acting as
a secretion signal in this order from the 5' end, and a NotI
sequence on its 3' side. The DNA fragment was digested with MluI
and NotI, and inserted into the vector pE-neo, between the MluI and
Nod thereof, to construct pE-neo(HC-I2S-2).
[0750] A DNA fragment was artificially synthesized having the
nucleotide sequence set forth as SEQ ID NO:252, which included a
gene encoding a protein in which the humanized anti-hTfR antibody
heavy chain having the amino acid sequence set forth as SEQ ID
NO:210 was linked, on the C-terminal side thereof and via a linker
sequence (Gly Ser), to hI2S having the amino acid sequence set
forth as SEQ ID NO:246. This DNA fragment encoded a protein having
the amino acid sequence set forth as SEQ ID NO:251 in which
humanized anti-hTfR antibody heavy chain was linked, via a linker
sequence (Gly Ser), to hI2S. This DNA fragment had, on its 5' side,
a MluI sequence and a sequence encoding a leader peptide acting as
a secretion signal in this order from the 5' end, and a Nod
sequence on its 3' side. The DNA fragment was digested with MluI
and Nod, and inserted into the vector pE-neo, between the MluI and
NotI thereof, to construct pE-neo(HC-I2S-3).
[0751] CHO cells (CHO-K1: obtained from American Type Culture
Collection) were transformed according to the method described in
Example 12 with pE-neo(HC-I2S-1) and pE-hygr(LC1) which had been
prepared in Example 11, to obtain a cell line expressing a fusion
protein between hI2S and a humanized anti-hTfR antibody. This cell
line was designated hI2S-anti-hTfR antibody expressing cell line 1.
The fusion protein between hI2S and a humanized anti-hTfR antibody
expressed by the cell lines was designated I2S-anti-hTfR antibody
1.
[0752] In the same manner, CHO cells were transformed with
pE-neo(HC-I2S-2) and pE-hygr(LC2) which had been prepared in
Example 11, to obtain a cell line expressing a fusion protein
between hI2S and a humanized anti-hTfR antibody. This cell line was
designated hI2S-anti-hTfR antibody expressing cell line 2. The
fusion protein between hI2S and a humanized anti-hTfR antibody
expressed by the cell lines was designated I2S-anti-hTfR antibody
2.
[0753] Further, in the same manner, CHO cells were transformed with
pE-neo(HC-I2S-3) and pE-hygr(LC3) which had been prepared in
Example 11, to obtain a cell line expressing a fusion protein
between hI2S and a humanized anti-hTfR antibody. This cell line was
designated hI2S-anti-hTfR antibody expressing cell line 3. The
fusion protein between hI2S and a humanized anti-hTfR antibody
expressed by the cell lines was designated I2S-anti-hTfR antibody
3.
[Example 17] Production of I2S-Anti-hTfR Antibodies
[0754] I2S-anti-hTfR antibodies were produced by the following
method. With CD OptiCHO.TM. medium, hI2S-anti-hTfR antibody
expressing cell lines 1, 2 and 3 obtained in Example 16 were
diluted to the density of approximately 2.times.10.sup.5 cells/mL,
respectively. The cell suspensions, 200 mL, were added to
corresponding 1 L-conical flasks, and cultured for 6 to 7 days in a
humid environment at 37.degree. C., 5% CO.sub.2, 95% air, with
stirring at a rate of about 70 rpm. Each culture supernatant was
collected by centrifugation, and filtered through a 0.22 .mu.m
filter (Millipore Inc.) to prepare the culture supernatant. To each
culture supernatant thus obtained was added five column volumes of
20 mM Tris buffer (pH 8.0) containing 150 mM NaCl, and loaded onto
a Protein A column (column volume: 1 mL, Bio-Rad Inc.) which had
been equilibrated in advance with three column volumes of 20 mM
Tris buffer (pH 8.0) containing 150 mM NaCl. Then, the column was
washed with five column volumes of the same buffer, and the
adsorbed I2S-anti-hTfR antibody was eluted with four column volumes
of 50 mM glycine buffer (pH 2.8) containing 150 mM NaCl. The pH of
the eluate containing I2S-anti-hTfR antibody was adjusted to pH 7.0
with 1 M Tris buffer (pH 8.0), and then the buffer was replaced
with PBS buffer using Amicon Ultra 30 kDa membrane (Millipore Inc.)
to obtain a I2S-anti-hTfR antibody purified product.
[0755] The affinity of the I2S-anti-hTfR antibodies thus produced
to human and monkey TfRs can be measured by, e.g., the method
described in Example 7. Pharmacokinetic analysis of the
I2S-anti-hTfR antibodies after their intravenous administration can
be conducted by, e.g., the method described in Example 8. The
pharmacological effects of the I2S-anti-hTfR antibodies can be
evaluated by, e.g., intravenously injecting the I2S-anti-hTfR
antibodies to I2S-KO/hTfR KI mice, which can be obtained through
mating of iduronate 2-sulfatasegene knockout mice (I2S-KO mice),
model mice for Hunter syndrome, with hTfR-KI mice described in
Example 7-2, and measuring the reduction of glycosaminoglycans
accumulated in the brain.
[Example 18] Evaluation of Transfer 12S-Anti-hTfR Antibodies into
the Brain-1
[0756] Each of the purification products of the I2S-anti-hTfR
antibody 3 prepared in Example 17 was intravenously injected, at
the dose of 1 mg/kg, to hTfR-KI mice generated by the method
described in Example 7-2 (I2S-anti-hTfR antibody administered
group). As a control, recombinant hI2S (rhI2S) was intravenously
injected, at the dose of 1 mg/kg, to hTfR-KI mice (control group).
Fifteen hTfR-KI mice were administered in I2S-anti-hTfR antibody
administered group, and three in the control group (male, 15 to
18-week old). Besides, the rhI2S used in the above had been
prepared in accordance with a conventional method, which was
described in an international patent publication (WO 2012/102998).
It is also possible to use Elaprase.RTM., a medicinal product
available on the market as the rhI2S.
[0757] In an I2S-anti-hTfR antibody administered group, three mice
were subjected whole body irrigation with physiological saline
after 15 min, 1 hr, 4 hr, 8 hr, and 24 hr, respectively, of the
administration of I2S-anti-hTfR antibody 3, and the brains
(cerebrum and cerebellum) were taken. As for the control group,
whole body irrigation with physiological saline was carried out one
hour after the rhI2S administration, and the brains (cerebrum and
cerebellum) were taken. The weight (wet weight) of the cerebrum and
cerebellum excised was weighed, and then the cerebrum and
cerebellum were homogenized with T-PER (Thermo Fisher Scientific
Inc.) containing Protease Inhibitor Cocktail (Sigma Inc.), and
their supernatants were collected after centrifugation. The amount
of I2S-anti-hTfR antibody contained in the supernatant of
homogenate was measured for I2S-anti-hTfR antibody administered
group, and the amount of rhI2S contained in the supernatant of
homogenate for the control group by ECL method described in
Examples 20 and 21, respectively, and the amount of I2S-anti-hTfR
antibody (concentration of I2S-anti-hTfR antibody in brain tissues)
as well as the amount of rhI2S (concentration of rhI2S in brain
tissues) contained in 1 g of the brain (wet weight) were
calculated. The result is shown in Table 12.
[0758] The concentration of I2S-anti-hTfR antibody and rhI2S in the
brain tissues one hour after administration was 0.368.+-.0.019
.mu.g/g wet weight and 0.00134.+-.0.00232 .mu.g/g wet weight,
respectively, showing that the concentration of I2S-anti-hTfR
antibody reached approximately 270 times that of rhI2S. The result
indicates that rhI2S, which scarcely passes through the blood-brain
barrier, in general, and does not transfer to the brain, can be
made to pass through the blood-brain barrier and transfer into the
brain tissues, by combining it with the anti-hTfR antibody.
Further, the concentration of the I2S-anti-hTfR antibody in the
brain tissues reached 0.263.+-.0.038 .mu.g/g wet weight in only 15
min after administration, which was approximately 200 times that of
rhI2S in the brain tissues one hour after administration. The
result also indicates that by combining with the anti-hTfR
antibody, it becomes possible to let hI2S rapidly transfer into the
brain tissues.
TABLE-US-00013 TABLE 12 Concentration of I2S-anti-hTfR antibody and
rhI2S in brain tissues (.mu.g/g wet weight) Time after
I2S-anti-hTfR administration (hr) antibody rhI2S 0.25 0.263 .+-.
0.038 -- 1 0.368 .+-. 0.019 0.00134 .+-. 0.00232 4 0.440 .+-. 0.033
-- 8 0.382 .+-. 0.011 -- 24 0.245 .+-. 0.012 --
[Example 19] Evaluation of Transfer of I2S-Anti-hTfR Antibodies
into the Brain-2
[0759] The purification product of I2S-anti-hTfR antibody 3
prepared in Example 17 was intravenously administered once to male
cynomolgus monkeys, at a dosage of 5 mg/kg (I2S-anti-hTfR antibody
administered group). In the same manner, rhI2S was administered
once to male cynomolgus monkeys, at a dosage of 5 mg/kg (rhI2S
group). The rhI2S employed here had been prepared in accordance
with a conventional method, which was described in an international
patent publication (WO 2012/102998). It is also possible to use
Elaprase.RTM., a medicinal product available on the market as the
rhI2S. Two monkeys were administered in each group. Eight hours
after administration, whole body irrigation was carried out. After
the irrigation, the brain tissues including the cervical cord were
excised. The brain tissues thus excised were separated into the
cerebral cortex, the cerebellum, the hippocampus and the cervical
cord, and each of them was homogenized with T-PER (Thermo Fisher
Scientific Inc.) containing Protease Inhibitor Cocktail (Sigma
Inc.), centrifuged, and the supernatant was collected. The amount
of the I2S-anti-hTfR antibody contained in the supernatant of
homogenate was measured in I2S-anti-hTfR antibody administered
group, and the amount of rhI2S contained in the supernatant of
homogenate in the control group by ECL method described in Examples
20 and 21, respectively. From the measurement thus obtained, the
amount of I2S-anti-hTfR antibody contained in 1 g (g wet weight) of
the cerebral cortex, cerebellum, hippocampus, and cervical cord
(concentration of I2S-anti-hTfR antibody in those brain tissues),
as well as the amount of rhI2S (concentration of rhI2S in those
brain tissues), were calculated. The result is shown in FIG. 9.
[0760] The concentration of I2S-anti-hTfR antibody in the cerebral
cortex was approximately 0.22 .mu.g/g, whereas that of rhI2S was
0.035 .mu.g/g. The concentration of I2S-anti-hTfR antibody in the
cerebellum was approximately 0.18 .mu.g/g, whereas that of rhI2S
was 0.02 .mu.g. The concentration of I2S-anti-hTfR antibody in the
hippocampus was approximately 0.25 .mu.g/g, whereas that of rhI2S
was 0.017 .mu.g/g. And the concentration of I2S-anti-hTfR antibody
in the cervical cord was approximately 0.15 .mu.g/g, whereas that
of rhI2S was 0.039 .mu.g/g. Thus, the concentration of
I2S-anti-hTfR antibody in the cerebral cortex, cerebellum,
hippocampus, and cervical cord was shown to be approximately 6.3
times, approximately 9.0 times, approximately 14.7 times, and
approximately 3.8 times that of rhI2S, respectively. The result
indicates that by binding hI2S to the anti-hTfR antibody, it
becomes possible to let hI2S actively pass through the blood-brain
barrier and get into the brain efficiently. In particular, that the
concentration of the I2S-anti-hTfR antibody in the hippocampus was
approximately as high as approximately 12.5 times that of rhI2S
indicates that administration of I2S-anti-hTfR antibody could let
I2S exhibit its activity in the hippocampus among others.
Encephalopathy in Hunter syndrome patients cannot be ameliorated by
enzyme replacement therapy with conventional rhI2S because little
rhI2S can pass through the blood-brain barrier. In contrast, as
I2S-anti-hTfR antibody can pass through the blood-brain barrier,
administration of I2S-anti-hTfR antibody could supplement the
activity of I2S in the brain tissues such as the cerebral cortex,
hippocampus, and cerebellum. Therefore, the I2S-anti-hTfR antibody
(especially I2S-anti-hTfR antibody 3) can be used as therapeutic
agents to supplement the I2S activity in the brain of Hunter
syndrome patients. Thus, by administering I2S-anti-hTfR antibody
(especially I2S-anti-hTfR antibody 3), prophylaxis and treatment of
encephalopathy in Hunter syndrome patients are possible, which is
difficult by enzyme replacement therapy with conventional rhI2S. It
is especially promising as a therapeutic agent for patients with
Hunter syndrome accompanied disorders of the hippocampus.
[Example 20] Quantitative Determination of I2S-Anti-hTfR Antibody
by ECL Method
[0761] To each well of a 96-well Streptavidin Gold Plate (Meso
Scale Diagnostics Inc.), a streptavidin-coated plate, was added 150
.mu.L of SuperBlock blocking buffer in PBS (Thermo Fisher
Scientific Inc.), and the plate was left to stand for one hour for
blocking. Anti-Human Kappa Light chain Goat IgG Biotin (Monkey
Absorbed) (IBL Inc.), a biotin-labeled antibody, was diluted with
SuperBlock blocking buffer in PBS to 0.5 .mu.g/mL. SULFO-labeled
anti-human I2S antibody was diluted with SuperBlock blocking buffer
in PBS to 1.0 .mu.g/mL. The diluted solutions of the biotin-labeled
antibody and the SULFO-labeled antibody, 25 .mu.L each, were mixed
with 25 .mu.L of each sample, incubated for one hour to prepare an
antibody reaction sample.
[0762] Each well of the plate after blocking was washed with 200
.mu.L of PBS-T (Sigma Inc.), and 25 .mu.L of the antibody reaction
sample was added, and incubated for one hour. After incubation,
each well of the plate was washed with 200 .mu.L of PBS-T, Read
buffer T (Meso scale Diagnostics Inc.) was added to each well, and
the amount of luminescence from each well was measured on
Sector.TM. Imager 6000 (Meso scale Diagnostics Inc.). A standard
curve was produced on measurements of standard samples containing
known concentrations of I2S-anti-hTfR antibody, and the amount of
I2S-anti-hTfR antibody was determined by interpolating the
measurement of each of the samples with reference to the
standard.
[0763] Besides, the anti-human I2S antibody used in the above was a
monoclonal antibody obtained by immunizing mice with rhI2S, as
antigen, which had been prepared accordance with a conventional
method described in an international patent publication (WO
2012/102998). Multiple monoclonal antibodies had been obtained. As
SULFO-labeled anti-human I2S antibody, an anti-human I2S antibody
was labelled with SULFO using MSD SULFO-TAG NHS-Ester (Meso scale
Diagnostics Inc.) in accordance with the attached manual.
[Example 21] Quantitative Determination of hI2S by ECL Method
[0764] To each well of a 96-well Streptavidin Gold Plate (Meso
Scale Diagnostics Inc.), a streptavidin-coated plate, was added 150
.mu.L of SuperBlock blocking buffer in PBS (Thermo Fisher
Scientific Inc.), and the plate was left to stand for one hour for
blocking. Biotin-labeled anti-human I2S antibody was diluted with
SuperBlock blocking buffer in PBS to 0.5 .mu.g/mL. SULFO-labeled
anti-human I2S antibody was diluted with SuperBlock blocking buffer
in PBS to 1.0 .mu.g/mL. The diluted solutions of the biotin-labeled
antibody and the SULFO-labeled antibody, 25 .mu.L each, were mixed
with 25 .mu.L of each sample, incubated for one hour to prepare an
antibody reaction sample.
[0765] Each well of the plate after blocking was washed with 200
.mu.L of PBS-T (Sigma Inc.), and 25 .mu.L of the antibody reaction
sample was added, and incubated for one hour. After incubation,
each well of the plate was washed with 200 .mu.L of PBS-T, Read
buffer T (Meso scale Diagnostics Inc.) was added to each well, and
the amount of luminescence from each well was measured on
Sector.TM. Imager 6000 (Meso scale Diagnostics Inc.). A standard
curve was produced on measurements of standard samples containing
known concentrations of hI2S, and the amount of rhI2S was
determined by interpolating the measurement of each of the samples
with reference to the standard.
[0766] Besides, the anti-human I2S antibody used in the above was a
monoclonal antibody obtained by immunizing mice with rhI2S, as
antigen, which had been prepared accordance with a conventional
method described in an international patent publication (WO
2012/102998). As SULFO-labeled anti-human I2S antibody, an
anti-human I2S antibody was labelled with SULFO using MSD SULFO-TAG
WS-Ester (Meso scale Diagnostics Inc.) in accordance with the
attached manual. Further, as biotin-labeled anti-human I2S
antibody, another anti-human I2S antibody, except the one that was
employed for labeling with SULFO, was labelled with biotin using
Biotin Labelling Kit-NH.sub.2 (Dojindo Laboratories Inc.) in
accordance with the attached manual.
[Example 22] Assessment of Pharmacological Effect of I2S-Anti-hTfR
Antibody
[0767] The pharmacological effect of I2S-anti-hTfR antibody was
evaluated by measuring the concentration of the glycosaminoglycans
(GAG) that was known to accumulate in the organs of Hunter syndrome
patients, who genetically lacked hI2S activity. The purification
product of I2S-anti-hTfR antibody 3 prepared in Example 17 was
intravenously injected to I2S-KO/hTfR-KI mice at a dosage of 0.5
mg/kg, 1.0 mg/kg, and 2.0 mg/kg (0.5 mg/kg-administered group, 1.0
mg/kg-administered group, and 2.0 mg/kg-administered group).
Administration was made at a frequency of once a week for four
weeks, the mice were euthanized by exsanguination under anesthesia
four weeks after the first administration, and the brain, liver,
lung, and heart were excised. The excised organs then were
lyophilized in a lyophilizer (EYELA Inc.), pulverized, and their
dry weight was measured. To 100 mg (dry weight) of each of the
dried organ preparations was added 1 mL of 0.5 M Tris buffer (pH
7.5), and heated for 10 min at about 100.degree. C. Then, 50 mg/mL
Actinase E solution (Kaken Pharmaceutical Inc.) was added to the
dry preparation so that 1 mg of actinase E was added to 50 mg (dry
weight) the mixture was incubated for 16 hr at about 60.degree. C.
to decompose the protein, and heating was continued for 10 min at
about 100.degree. C. After centrifugation for 10 min at 15,000 rpm,
the supernatant was collected. The amount of GAG contained in the
supernatant was measured using Wieslab.TM. sGAG quantitative kit
(Euro-Diagnostica Inc.), and the amount of GAG contained in 1 g (g
dry weight) of each organ was calculated. Besides, as the control
group, 12S-KO/hTfR-KI mice non-administered I2S-anti-hTfR antibody
was employed. At the same time, the amount of GAG in the organs of
wild-type mice was measured. This experiment was conducted with
three I2S-KO/hTfR-KI mice (male and female, 19 to 25-week old) in
each measurement group. Besides, three wild-type mice, male and
female, 19 to 25-week old, were employed.
[0768] The result is shown in FIG. 10. It was observed that in all
the brain, liver, lung and heart, the concentration of GAG
significantly decreased dose dependently by I2S-anti-hTfR antibody
administration (FIG. 10a to 10d).
[0769] In the brain, the concentration of GAG was approximately
2.66 .mu.g/g in the brain tissues of the control group, whereas the
concentration of GAG in the brain tissues of 0.5 mg/kg, 1.0 mg/kg,
and 2.0 mg/kg administered groups was approximately 2.23 .mu.g/g,
approximately 2.15 .mu.g/g, approximately 2.10 .mu.g/g,
respectively, showing a dose dependent decrease (FIG. 10a).
Abnormal amount of GAG in the brain tissues of I2S-KO/hTfR-KI mice
can be determined as being approximately 0.90 .mu.g/g, i.e., the
amount that remains after the concentration of GAG in the brain
tissues of the wile-type mice (approximately 1.76 .mu.g/g) is
subtracted from the concentration of GAG in the brain tissues of
the control group (approximately 2.66 .mu.g/g). Thus, it can be
concluded that approximately 48%, approximately 57%, and
approximately 62% of GAG abnormally accumulated in the brain
tissues of I2S-KO/hTfR-KI mice were decomposed by the I2S-anti-hTfR
antibody administered at the doses of 0.5 mg/kg, 1.0 mg/kg, and 2.0
mg/kg, respectively. The result indicates that it is possible to
decompose and remove GAG abnormally accumulated in the brain
tissues of a Hunter syndrome patient by administering the
I2S-anti-hTfR antibody to the patient, and that the I2S-anti-hTfR
antibody (I2S-anti-hTfR antibody 3, in particular) could prevent
and treat brain lesions caused by accumulation and the like of GAG
or its fragments observed in Hunter syndrome patients.
[0770] In the liver, the concentration of GAG was approximately
10.3 kg/g in the liver tissue of the control group, whereas the
concentration of GAG in the liver tissue of 0.5 mg/kg, 1.0 mg/kg,
and 2.0 mg/kg administered groups was approximately 2.2 .mu.g/g,
approximately 2.0 .mu.g/g, approximately 1.9 .mu.g/g, respectively,
showing a dose dependent decrease (FIG. 10b). Abnormal portion of
the amount of GAG in the liver tissue of I2S-KO/hTfR-KI mice can be
determined as being approximately 10 .mu.g/g, i.e., the amount that
remains after the concentration of GAG in the liver tissue of the
wild-type mice (approximately 0.3 .mu.g/g) is subtracted from the
concentration of GAG in the liver tissue of the control group
(approximately 10.3 .mu.g/g). Thus, it can be concluded that not
less than 80% of GAG abnormally accumulated in the liver tissue of
I2S-KO/hTfR-KI mice was decomposed by the I2S-anti-hTfR antibody
administered at the doses of 0.5 to 2.0 mg/kg.
[0771] In the lung, the concentration of GAG was approximately 10.5
.mu.g/g in the lung tissue of the control group, whereas the
concentration of GAG in the lung tissue of 0.5 mg/kg, 1.0 mg/kg,
and 2.0 mg/kg administered groups was approximately 7.8 .mu.g/g,
approximately 6.7 .mu.g/g, approximately 5.7 .mu.g/g, respectively,
showing a dose dependent decrease (FIG. 10c). Abnormal portion of
the amount of GAG in the lung tissue of I2S-KO/hTfR-KI mice can be
determined as being approximately 9.0 .mu.g/g, i.e., the amount
that remains after the concentration of GAG in the lung tissue of
the wild-type mice (approximately 1.5 .mu.g/g) is subtracted from
the concentration of GAG in the lung tissue of the control group
(approximately 10.5 .mu.g/g). Thus, it can be concluded that
approximately 30%, approximately 42%, and approximately 53% of GAG
abnormally accumulated in the lung tissue of I2S-KO/hTfR-KI mice
was decomposed by the I2S-anti-hTfR antibody administered at the
doses of 0.5 mg/kg, 1.0 mg/kg, and 2.0 mg/kg, respectively.
[0772] In the heart, the concentration of GAG was approximately 4.6
.mu.g/g in the heart tissue of the control group, whereas the
concentration of GAG in the heart tissue of 0.5 mg/kg, 1.0 mg/kg,
and 2.0 mg/kg administered group was approximately 2.2 .mu.g/g,
approximately 2.0 .mu.g/g, and approximately 1.5 .mu.g/g,
respectively, showing a dose dependent decrease (FIG. 10d).
Abnormal portion of the amount of GAG in the heat tissue of
I2S-KO/hTfR-KI mice can be determined as being approximately 3.8
.mu.g/g, i.e., the amount that remains after the concentration of
GAG in the heart tissue of the wild-type mice (approximately 0.8
.mu.g/g) is subtracted from the concentration of GAG in the heart
tissue of the control group (approximately 4.6 .mu.g/g). Thus, it
can be concluded that approximately 63%, approximately 70%, and
approximately 81% of GAG abnormally accumulated in the heart tissue
of I2S-KO/hTfR-KI mice was decomposed by the I2S-anti-hTfR antibody
administered at the doses of 0.5 mg/kg, 1.0 mg/kg, and 2.0 mg/kg,
respectively.
[0773] The above results in the liver, lung and heart demonstrate
that the I2S-anti-hTfR antibody decomposes accumulated GAG not only
in the brain but also in other organs. This indicates that the
I2S-anti-hTfR antibody (especially I2S-anti-hTfR antibody 3), when
administered to patients with Hunter syndrome as a pharmaceutical
agent in enzyme replacement therapy, could supplement all the
patients' organs including the brain with the enzyme. Also
indicated is that the I2S-anti-hTfR antibody can supplement the all
the patients' organs including the brain with the enzyme through
its intravenous administration.
[Example 23] Method for Measurement of GAG
[0774] Measurement of GAG was conducted as follows in general,
using Wieslab.TM. sGAG quantitative kit (EURO-DIAGNOSTICA Inc.) in
accordance with the attached operating manual. A sample or a
standard solution (blank or water), 50 .mu.L each, was added to a
1.5-mL tube. To each tube was added 50 .mu.L of a GuHCl solution,
and a reaction was allowed to take place for 15 min at room
temperature. A SAT solution, 50 .mu.L, was added to each tube, and
a reaction was allowed to proceed for 15 min at room temperature. A
mixture solution of water/SAT solution/Alcian Blue stock solution
(9/5/1) was prepared and added to the tubes, 750 .mu.L each, and a
reaction was allowed to proceed for 15 min at room temperature. The
reaction mixture liquid thus obtained was centrifuged at
12500.times.g for 15 min to remove the supernatant. To each tube
was added 500 .mu.L of DMSO, and following 15-min shaking for
mixing at room temperature, the mixture was centrifuged to remove
the supernatant. Gu-Prop solution was added to the tubes, 500 .mu.L
each, and mixed by shaking for 15 min at room temperature. The
mixture solution thus prepared was dispensed, 200 .mu.L each, to
the wells of a 96-well plate, and the absorbance of each well was
measured at 600 nm using a plate reader. A standard curve was
produced on measurements of solutions containing known
concentrations of GAG, and the GAG concentration was determined by
interpolating the measurement of each sample with reference to the
standard.
[Example 24] Preparation of Fusion Proteins Between Humanized
Anti-hTfR Antibody and Various Physiologically Active Peptides
[0775] The experiments carried out in Examples 1 to 22 above have
shown that human 12S linked to the humanized anti-hTfR antibody
passes through the blood-brain barrier, gets into the brain
tissues, and exhibits 12S activity in the brain. Then, fusion
proteins were prepared between the humanized anti-hTfR antibody and
various physiologically active peptides to examine whether those
fusion proteins pass through the BBB and get into the brain
tissues. Here, human erythropoietin, human arylsulfatase A, human
PPT-1, human TPP-1, human .alpha.-L-iduronidase, human TNF.alpha.
receptor, and human N-sulphoglucosamine sulphohydrolase (heparan
N-sulfatase) were selected as physiologically active peptides to be
fused with the humanized anti-hTfR antibody. The expression vectors
used to express fusion proteins between the humanized anti-hTfR
antibody and physiologically active peptides, as well as the
resulting fusion proteins, were prepared according to Examples 25
to 31 below.
[Example 25] Method for Preparation of Fusion Protein Between
Humanized Anti-hTfR Antibody and Human Erythropoietin
[0776] A DNA fragment was artificially synthesized comprising the
nucleotide sequence set forth as SEQ ID NO:258, which included the
cDNA encoding a protein consisting of the humanized anti-hTfR
antibody having the amino acid sequence set forth as SEQ ID NO:210
and human erythropoietin (hEPO) having the amino acid sequence set
forth as SEQ ID NO:256 and linked, via a linker sequence Gly-Ser,
on the C-terminal side of the antibody's heavy chain. This DNA
fragment encodes a protein having the amino acid sequence set forth
as SEQ ID NO:257, which consists of the humanized anti-hTfR
antibody and hEPO, which is linked, via a linker sequence Gly-Ser,
to the antibody's heavy chain on the C-terminal side thereof. The
DNA fragment has in the 5' side a MluI sequence and a sequence
encoding a leader peptide acting as a secretion signal, in this
order from the 5' end, and a NotI sequence in the 3' side. The DNA
fragment was digested with MluI and NotI, and inserted into the
vector pE-neo between MluI and NotI sites to construct
pE-neo(HC-hEPO).
[0777] By the method described in Example 12, CHO cells were
transformed with pE-neo(HC-hEPO) and pE-hygr(LC3), which was
constructed in Example 11, a cell line was obtained which expresses
a fusion protein between hEPO and the humanized anti-hTfR antibody.
The cell line was designated hEPO-anti-hTfR antibody expressing
cell line. The fusion protein between hEPO and the humanized
anti-hTfR antibody, expressed by this cell line, were designated
EPO-anti-hTfR antibody.
[0778] Cells of hEPO-anti-hTfR antibody expressing cell line were
diluted to the density of approximately 2.times.10.sup.5 cells/mL
with CD OptiCHO.TM. medium, and 200 mL of the cell suspension was
added to a 1-L conical flask and cultured for 6 to 7 days in a
humid environment at 37.degree. C., 5% CO.sub.2, 95% air, with
stirring at a rate of about 70 rpm. The culture medium was
collected by centrifugation and filtered through a 0.22 .mu.m
filter (Millipore Inc.) to prepare a culture supernatant. To the
culture supernatant thus collected was added five column volumes of
20 mM Tris buffer (pH 8.0) containing 150 mM NaCl, and loaded onto
a Protein A column (column volume 1: mL, Bio-Rad Inc.) that had
been equilibrated in advance with three column volumes of 20 mM
Tris buffer (pH 8.0) containing 150 mM NaCl. Then, five column
volumes of the same buffer was supplied to wash the column, the
adsorbed EPO-anti-hTfR was eluted with four column volumes of 50 mM
glycine buffer (pH 2.8) containing 150 mM NaCl. The eluate
containing EPO-anti-hTfR antibody was adjusted to pH 7.0 with 1 M
Tris buffer (pH 8.0), and then the buffer exchanged for PBS using
Amicon Ultra 30 kDa membrane (Millipore Inc.). The resulting
solution was used as the purification product of EPO-anti-hTfR
antibody.
[Example 26] Method for Preparation of Fusion Protein Between
Humanized Anti-hTfR Antibody and Human Arylsulfatase A
[0779] A DNA fragment was artificially synthesized comprising the
nucleotide sequence set forth as SEQ ID NO:261, which included the
cDNA encoding a protein consisting of the humanized anti-hTfR
antibody having the amino acid sequence set forth as SEQ ID NO:210
and human arylsulfatase A (hARSA) having the amino acid sequence
set forth as SEQ ID NO:259 and linked, via a linker sequence
Gly-Ser, to the antibody's heavy chain on the C-terminal side
thereof. This DNA fragment encodes a protein having the amino acid
sequence set forth as SEQ ID NO:260, which consists of the
humanized anti-hTfR antibody and hARSA, which is linked, via a
linker sequence Gly-Ser, to the antibody's heavy chain on the
C-terminal side thereof. The DNA fragment has in the 5' side a MluI
sequence and a sequence encoding a leader peptide acting as a
secretion signal, in this order from the 5' end, and a Noll
sequence in the 3' side. The DNA fragment was digested with MluI
and NotI, and inserted into the vector pE-neo between MluI and Noll
sites to construct pE-neo(HC-hARSA).
[0780] By the method described in Example 12, CHO cells were
transformed with pE-neo(HC-hARSA) and pE-hygr(LC3), which was
constructed in Example 11, a cell line was obtained which expresses
a fusion protein between hARSA and the humanized anti-hTfR
antibody. The cell line was designated hARSA-anti-hTfR antibody
expressing cell line. The fusion protein between hARSA and the
humanized anti-hTfR antibody, expressed by this cell line, were
designated ARSA-anti-hTfR antibody.
[0781] Cells of hARSA-anti-hTfR antibody expressing cell line were
diluted to the density of approximately 2.times.10.sup.5 cells/mL
with CD OptiCHO.TM. medium, and 200 mL of the cell suspension was
added to a 1-L conical flask and cultured for 6 to 7 days in a
humid environment at 37.degree. C., 5% CO.sub.2, 95% air, with
stirring at a rate of about 70 rpm. The culture medium was
collected by centrifugation and filtered through a 0.22 .mu.m
filter (Millipore Inc.) to prepare a culture supernatant. To the
culture supernatant thus collected was added five column volumes of
20 mM Tris buffer (pH 8.0) containing 150 mM NaCl, and loaded onto
a Protein A column (column volume: 1 mL, Bio-Rad Inc.) that had
been equilibrated in advance with three column volumes of 20 mM
Tris buffer (pH 8.0) containing 150 mM NaCl. Then, five column
volumes of the same buffer was supplied to wash the column, the
adsorbed ARSA-anti-hTfR was eluted with four column volumes of 50
mM glycine buffer (pH 2.8) containing 150 mM NaCl. The eluate
containing ARSA-anti-hTfR antibody was adjusted to pH 7.0 with 1 M
Tris buffer (pH 8.0), and then the buffer exchanged for PBS using
Amicon Ultra 30 kDa membrane (Millipore Inc.). The resulting
solution was used as the purification product of ARSA-anti-hTfR
antibody.
[Example 27] Method for Preparation of Fusion Protein Between
Humanized Anti-hTfR Antibody and Human PPT-1
[0782] A DNA fragment was artificially synthesized comprising the
nucleotide sequence set forth as SEQ ID NO:264, which included the
cDNA encoding a protein consisting of the humanized anti-hTfR
antibody having the amino acid sequence set forth as SEQ ID NO:210
and human PPT-1 (hPPT-1) having the amino acid sequence set forth
as SEQ ID NO:262 and linked, via a linker sequence Gly-Ser, to the
antibody's heavy chain on the C-terminal side thereof. This DNA
fragment encodes a protein having the amino acid sequence set forth
as SEQ ID NO:263, which consists of the humanized anti-hTfR
antibody and hPPT-1, which is linked, via a linker sequence
Gly-Ser, to the antibody's heavy chain on the C-terminal side
thereof. The DNA fragment has in the 5' side a MluI sequence and a
sequence encoding a leader peptide acting as a secretion signal, in
this order from the 5' end, and a NotI sequence in the 3' side. The
DNA fragment was digested with MluI and NotI, and inserted into the
vector pE-neo between MluI and NotI sites to construct
pE-neo(HC-hPPT-1).
[0783] By the method described in Example 12, CHO cells were
transformed with pE-neo(HC-hPPT-1) and pE-hygr(LC3), which was
constructed in Example 11, a cell line was obtained which expresses
a fusion protein between hPPT-1 and the humanized anti-hTfR
antibody. The cell line was designated hPPT-1-anti-hTfR antibody
expressing cell line. The fusion protein between hPPT-1 and the
humanized anti-hTfR antibody, expressed by this cell line, were
designated PPT-1-anti-hTfR antibody.
[0784] Cells of hPPT-1-anti-hTfR antibody expressing cell line were
diluted to the density of approximately 2.times.10.sup.5 cells/mL
with CD OptiCHO.TM. medium, and 200 mL of the cell suspension was
added to a 1-L conical flask and cultured for 6 to 7 days in a
humid environment at 37.degree. C., 5% CO.sub.2, 95% air, with
stirring at a rate of about 70 rpm. The culture medium was
collected by centrifugation and filtered through a 0.22 .mu.m
filter (Millipore Inc.) to prepare a culture supernatant. To the
culture supernatant thus collected was added five column volumes of
20 mM Tris buffer (pH 8.0) containing 150 mM NaCl, and loaded onto
a Protein A column (column volume: 1 mL, Bio-Rad Inc.) that had
been equilibrated in advance with three column volumes of 20 mM
Tris buffer (pH 8.0) containing 150 mM NaCl. Then, five column
volumes of the same buffer was supplied to wash the column, the
adsorbed PPT-1-anti-hTfR was eluted with four column volumes of 50
mM glycine buffer (pH 2.8) containing 150 mM NaCl. The eluate
containing PPT-1-anti-hTfR antibody was adjusted to pH 7.0 with 1 M
Tris buffer (pH 8.0), and then the buffer exchanged for PBS using
Amicon Ultra 30 kDa membrane (Millipore Inc.). The resulting
solution was used as the purification product of PPT-1-anti-hTfR
antibody.
[Example 28] Method for Preparation of Fusion Protein Between
Humanized Anti-hTfR Antibody and Human TPP-1
[0785] A DNA fragment was artificially synthesized comprising the
nucleotide sequence set forth as SEQ ID NO:267, which included the
cDNA encoding a protein consisting of the humanized anti-hTfR
antibody having the amino acid sequence set forth as SEQ ID NO:210
and human TPP-1 (hTPP-1) having the amino acid sequence set forth
as SEQ ID NO:265 and linked, via a linker sequence Gly-Ser, to the
antibody's heavy chain on the C-terminal side thereof. This DNA
fragment encodes a fusion protein having the amino acid sequence
set forth as SEQ ID NO:266, which consists of the humanized
anti-hTfR antibody and hTPP-1 which is linked, via a linker
sequence Gly-Ser, to the antibody's heavy chain on the C-terminal
side thereof. The DNA fragment has in the 5' side a MluI sequence
and a sequence encoding a leader peptide acting as a secretion
signal, in this order from the 5' end, and a NotI sequence in the
3' side. The DNA fragment was digested with MluI and NotI, and
inserted into the vector pE-neo between MluI and NotI sites to
construct pE-neo(HC-hTPP-1).
[0786] By the method described in Example 12, CHO cells were
transformed with pE-neo(HC-hTPP-1) and pE-hygr(LC3), which was
constructed in Example 11, a cell line was obtained which expresses
a fusion protein between hTPP-1 and the humanized anti-hTfR
antibody. The cell line was designated hTPP-1-anti-hTfR antibody
expressing cell line. The fusion protein between hTPP-1 and the
humanized anti-hTfR antibody, expressed by this cell line, were
designated TPP-1-anti-hTfR antibody.
[0787] Cells of hTPP-1-anti-hTfR antibody expressing cell line were
diluted to the density of approximately 2.times.10.sup.5 cells/mL
with CD OptiCHO.TM. medium, and 200 mL of the cell suspension was
added to a 1-L conical flask and cultured for 6 to 7 days in a
humid environment at 37.degree. C., 5% CO.sub.2, 95% air, with
stirring at a rate of about 70 rpm. The culture medium was
collected by centrifugation and filtered through a 0.22 .mu.m
filter (Millipore Inc.) to prepare a culture supernatant. To the
culture supernatant thus collected was added five column volumes of
20 mM Tris buffer (pH 8.0) containing 150 mM NaCl, and loaded onto
a Protein A column (column volume: 1 mL, Bio-Rad Inc.) that had
been equilibrated in advance with three column volumes of 20 mM
Tris buffer (pH 8.0) containing 150 mM NaCl. Then, five column
volumes of the same buffer was supplied to wash the column, the
adsorbed TPP-1-anti-hTfR was eluted with four column volumes of 50
mM glycine buffer (pH 2.8) containing 150 mM NaCl. The eluate
containing TPP-1-anti-hTfR antibody was adjusted to pH 7.0 with 1 M
Tris buffer (pH 8.0), and then the buffer exchanged for PBS using
Amicon Ultra 30 kDa membrane (Millipore Inc.). The resulting
solution was used as the purification product of TPP-1-anti-hTfR
antibody.
[Example 29] Method for Preparation of Fusion Protein Between
Humanized Anti-hTfR Antibody and Human .alpha.-L-Iduronidase
[0788] A DNA fragment was artificially synthesized comprising the
nucleotide sequence set forth as SEQ ID NO:270, which included the
cDNA encoding a protein consisting of the humanized anti-hTfR
antibody having the amino acid sequence set forth as SEQ ID NO:210
and human .alpha.-L-iduronidase (hIDUA) having the amino acid
sequence set forth as SEQ ID NO:268 and linked, via a linker
sequence Gly-Ser, to the antibody's heavy chain on the C-terminal
side thereof. This DNA fragment encodes a fusion protein having the
amino acid sequence set forth as SEQ ID NO:269, which consists of
the humanized anti-hTfR antibody and hIDUA which is linked, via a
linker sequence Gly-Ser, to the antibody's heavy chain on the
C-terminal side thereof. The DNA fragment has in the 5' side a MluI
sequence and a sequence encoding a leader peptide acting as a
secretion signal, in this order from the 5' end, and a Noll
sequence in the 3' side. The DNA fragment was digested with MluI
and NotI, and inserted into the vector pE-neo between MluI and NotI
sites to construct pE-neo(HC-hIDUA).
[0789] By the method described in Example 12, CHO cells were
transformed with pE-neo(HC-hIDUA) and pE-hygr(LC3), which was
constructed in Example 11, a cell line was obtained which expresses
a fusion protein between hIDUA and the humanized anti-hTfR
antibody. The cell line was designated hIDUA-anti-hTfR antibody
expressing cell line. The fusion protein between hIDUA and the
humanized anti-hTfR antibody, expressed by this cell line, were
designated IDUA-anti-hTfR antibody.
[0790] Cells of hIDUA-anti-hTfR antibody expressing cell line were
diluted to the density of approximately 2.times.10.sup.5 cells/mL
with CD OptiCHO.TM. medium, and 200 mL of the cell suspension was
added to a 1-L conical flask and cultured for 6 to 7 days in a
humid environment at 37.degree. C., 5% CO.sub.2, 95% air, with
stirring at a rate of about 70 rpm. The culture medium was
collected by centrifugation and filtered through a 0.22 .mu.m
filter (Millipore Inc.) to prepare a culture supernatant. To the
culture supernatant thus collected was added five column volumes of
20 mM Tris buffer (pH 8.0) containing 150 mM NaCl, and loaded onto
a Protein A column (column volume: 1 mL, Bio-Rad Inc.) that had
been equilibrated in advance with three column volumes of 20 mM
Tris buffer (pH 8.0) containing 150 mM NaCl. Then, five column
volumes of the same buffer was supplied to wash the column, the
adsorbed IDUA-anti-hTfR was eluted with four column volumes of 50
mM glycine buffer (pH 2.8) containing 150 mM NaCl. The eluate
containing IDUA-anti-hTfR antibody was adjusted to pH 7.0 with 1 M
Tris buffer (pH 8.0), and then the buffer exchanged for PBS using
Amicon Ultra 30 kDa membrane (Millipore Inc.). The resulting
solution was used as the purification product of IDUA-anti-hTfR
antibody.
[Example 30] Method for Preparation of Fusion Protein Between
Humanized Anti-hTfR Antibody and Human TNF.alpha. Receptor
[0791] A DNA fragment was artificially synthesized comprising the
nucleotide sequence set forth as SEQ ID NO:273, which included the
cDNA encoding a protein consisting of the humanized anti-hTfR
antibody having the amino acid sequence set forth as SEQ ID NO:210
and human TNF.alpha. receptor (hTNF.alpha.R) having the amino acid
sequence set forth as SEQ ID NO:271 and linked, via a linker
sequence Gly-Ser, to the antibody's heavy chain on the C-terminal
side thereof. This DNA fragment encodes a fusion protein having the
amino acid sequence set forth as SEQ ID NO:272, which consists of
the humanized anti-hTfR antibody and hTNF.alpha.R which is linked,
via a linker sequence Gly-Ser, to the antibody's heavy chain on the
C-terminal side thereof. The DNA fragment has in the 5' side a MluI
sequence and a sequence encoding a leader peptide acting as a
secretion signal, in this order from the 5' end, and a NotI
sequence in the 3' side. The DNA fragment was digested with MluI
and NotI, and inserted into the vector pE-neo between MluI and NotI
sites to construct pE-neo(HC-hTNF.alpha.R).
[0792] By the method described in Example 12, CHO cells were
transformed with pE-neo(HC-hTNF.alpha.R) and pE-hygr(LC3), which
was constructed in Example 11, a cell line was obtained which
expresses a fusion protein between hTNF.alpha.R and the humanized
anti-hTfR antibody. The cell line was designated
hTNF.alpha.R-anti-hTfR antibody expressing cell line. The fusion
protein between hTNF.alpha.R and the humanized anti-hTfR antibody,
expressed by this cell line, were designated TNF.alpha.R-anti-hTfR
antibody.
[0793] Cells of hTNF.alpha.R-anti-hTfR antibody expressing cell
line were diluted to the density of approximately 2.times.10.sup.5
cells/mL with CD OptiCHO.TM. medium, and 200 mL of the cell
suspension was added to a 1-L conical flask and cultured for 6 to 7
days in a humid environment at 37.degree. C., 5% CO.sub.2, 95% air,
with stirring at a rate of about 70 rpm. The culture medium was
collected by centrifugation and filtered through a 0.22 .mu.M
filter (Millipore Inc.) to prepare a culture supernatant. To the
culture supernatant thus collected was added five column volumes of
20 mM Tris buffer (pH 8.0) containing 150 mM NaCl, and loaded onto
a Protein A column (column volume: 1 mL, Bio-Rad Inc.) that had
been equilibrated in advance with three column volumes of 20 mM
Tris buffer (pH 8.0) containing 150 mM NaCl. Then, five column
volumes of the same buffer was supplied to wash the column, the
adsorbed hTNF.alpha.R-anti-hTfR was eluted with four column volumes
of 50 mM glycine buffer (pH 2.8) containing 150 mM NaCl. The eluate
containing TNF.alpha.R-anti-hTfR antibody was adjusted to pH 7.0
with 1 M Tris buffer (pH 8.0), and then the buffer exchanged for
PBS using Amicon Ultra 30 kDa membrane (Millipore Inc.). The
resulting solution was used as the purification product of
TNF.alpha.R-anti-hTfR antibody.
[Example 31] Method for Preparation of Fusion Protein Between
Humanized Anti-hTfR Antibody and Human Heparan N-Sulfatase
[0794] A DNA fragment was artificially synthesized comprising the
nucleotide sequence set forth as SEQ ID NO:276, which included the
cDNA encoding a protein consisting of the humanized anti-hTfR
antibody having the amino acid sequence set forth as SEQ ID NO:210
and human heparan N-sulfatase (hSGSH) having the amino acid
sequence set forth as SEQ ID NO:274 and linked, via a linker
sequence Gly-Ser, to the antibody's heavy chain on the C-terminal
side thereof. This DNA fragment encodes a fusion protein having the
amino acid sequence set forth as SEQ ID NO:275, which consists of
the humanized anti-hTfR antibody and hSGSH which is linked, via a
linker sequence Gly-Ser, to the antibody's heavy chain on the
C-terminal side thereof. The DNA fragment has in the 5' side a MluI
sequence and a sequence encoding a leader peptide acting as a
secretion signal, in this order from the 5' end, and a Nod sequence
in the 3' side. The DNA fragment was digested with MluI and NotI,
and inserted into the vector pE-neo between Mild and NotI sites to
construct pE-neo(HC-hSGSH).
[0795] By the method described in Example 12, CHO cells were
transformed with pE-neo(HC-hSGSH) and pE-hygr(LC3), which was
constructed in Example 11, a cell line was obtained which expresses
a fusion protein between hSGSH and the humanized anti-hTfR
antibody. The cell line was designated hSGSH-anti-hTfR antibody
expressing cell line. The fusion protein between hSGSH and the
humanized anti-hTfR antibody, expressed by this cell line, were
designated SGSH-anti-hTfR antibody.
[0796] Cells of hSGSH-anti-hTfR antibody expressing cell line were
diluted to the density of approximately 2.times.10.sup.5 cells/mL
with CD OptiCHO.TM. medium, and 200 mL of the cell suspension was
added to a 1-L conical flask and cultured for 6 to 7 days in a
humid environment at 37.degree. C., 5% CO.sub.2, 95% air, with
stirring at a rate of about 70 rpm. The culture medium was
collected by centrifugation and filtered through a 0.22 .mu.m
filter (Millipore Inc.) to prepare a culture supernatant. To the
culture supernatant thus collected was added five column volumes of
20 mM Tris buffer (pH 8.0) containing 150 mM NaCl, and loaded onto
a Protein A column (column volume: 1 mL, Bio-Rad Inc.) that had
been equilibrated in advance with three column volumes of 20 mM
Tris buffer (pH 8.0) containing 150 mM NaCl. Then, five column
volumes of the same buffer was supplied to wash the column, the
adsorbed SGSH-anti-hTfR was eluted with four column volumes of 50
mM glycine buffer (pH 2.8) containing 150 mM NaCl. The eluate
containing SGSH-anti-hTfR antibody was adjusted to pH 7.0 with 1 M
Tris buffer (pH 8.0), and then the buffer exchanged for PBS using
Amicon Ultra 30 kDa membrane (Millipore Inc.). The resulting
solution was used as the purification product of SGSH-anti-hTfR
antibody.
[Example 32] Evaluation of Transfer of Fusion Proteins Between
Humanized Anti-hTfR Antibody and Various Physiologically Active
Peptides into the Brain
[0797] Each purification product of EPO-anti-hTfR antibody,
ARSA-anti-hTfR antibody, PPT-1-anti-hTfR antibody, TPP-1-anti-hTfR
antibody, IDUA-anti-hTfR antibody, TNF.alpha.R-anti-hTfR antibody
and SGSH-anti-hTfR antibody prepared in Examples 25 to 31,
respectively, was intravenously administered once to an hTfR-KI
mouse at a dose of 3 mg/kg. As a control, a human immunoglobulin
preparation (Benesis: human immunoglobulin for intramuscular
injection, Mitsubishi Tanabe Pharma Inc.) was intravenously
injected once to an hTfR-KI mouse (17 to 28 week-old) at a dose of
3 mg/kg. A single hTfR-KI mouse (male, 17 to 28 week-old) was used
for the administration of each of the fusion proteins and the
control.
[0798] Eight hours after the intravenous injection, each mouse was
subjected to whole body irrigation with physiological saline, and
its brain tissues then were excised after the irrigation. The
excised brain tissues then were homogenized with T-PER (Thermo
Fisher Scientific Inc.) containing Protease Inhibitor Cocktail
(Sigma Inc.), and the supernatant was collected after
centrifugation. The concentration of the fusion protein contained
in the collected supernatant of the homogenate was measured by the
following method. Besides the biotin-labeled goat anti-human IgG Fe
polyclonal antibody used was prepared by biotin-labeling goat
anti-human IgG Fc polyclonal antibody (Bethyl Inc.) with Biotin
Labelling Kit-NH.sub.2 (Dojindo Laboratories Inc.) in accordance
with the attached manual. Further, the SULFO-labeled goat
anti-human IgG Fc polyclonal antibody used was prepared by
SULFO-labeling goat anti-human IgG Fc polyclonal antibody (Bethyl
Inc.) with MSD SULFO-TAG NHS-Ester (Meso scale Diagnostics Inc.)
according to the attached manual.
[0799] To each well of a Streptavidin Gold Plate 96-well (Meso
scale Diagnostics Inc.) was added 150 .mu.L of SuperBlock blocking
buffer in PBS (Thermo Fisher Scientific Inc.) and left to stand for
one hour at room temperature to block the plate. The biotin-labeled
goat anti-human IgG Fe polyclonal antibody was diluted to 0.5
.mu.g/mL with SuperBlock blocking buffer in PBS. The SULFO-labeled
goat anti-human IgG Fe polyclonal antibody was diluted to 1.0
.mu.g/mL with SuperBlock blocking buffer in PBS. The diluted
solutions of biotin-labeled antibody and SULFO-labeled antibody, 25
.mu.L each, were mixed with 25 .mu.L of each sample, and incubated
for one hour to prepare samples for antibody reaction.
[0800] After each well of the blocked plate was washed with 200 pt
of PBS-T (Sigma Inc.), 25 .mu.L of a sample for antibody reaction
was added to the well, and incubated for one hour. Following the
incubation, each well of the plate was washed with 200 .mu.L of
PBS-T, and Read buffer T (Meso scale Diagnostics Inc.) was added
and the amount of luminescence from each well was measured on
Sector.TM. Imager 6000 (Meso scale Diagnostics Inc.). The amount of
the fusion protein contained per one gram of brain tissues (g wet
weight) (the concentration of the fusion protein in the brain
tissues) was calculated by producing a standard curve on
measurements of standard samples containing known concentrations of
the sample, and then interpolating the measurement of each of the
samples with reference to the standard. The result is shown in
Table 13.
[0801] When the concentration of the human immunoglobulin, the
control, in the brain tissues is taken as a unit value, the
relative values of concentration of EPO-anti-hTfR antibody,
ARSA-anti-hTfR antibody, PPT-1-anti-hTfR antibody, TPP-1-anti-hTfR
antibody, IDUA-anti-hTfR antibody, TNF.alpha.R-anti-hTfR antibody
and SGSH-anti-hTfR antibody in the brain tissues are 4.33, 3.39,
4.87, 6.48, 5.62, 7.44, and 2.24, respectively, demonstrating that
these physiologically active peptides linked to the anti-hTfR
antibody actively transfer into the brain tissues. The result thus
indicates that these physiologically active proteins, which usually
do not pass through the blood-brain barrier, can be made to pass
through the blood-brain barrier and get into the brain tissues by
fusing them to the anti-hTfR antibody.
[0802] Thus, the above results indicate that EPO-anti-hTfR antibody
can be used as a therapeutic agent for cerebral ischemia,
ARSA-anti-hTfR antibody, arylsulfatase A as a therapeutic agent for
central nervous system disorders in metachromatic white matter
degeneration (metachromatic leukodystrophy), PPT-1-anti-hTfR
antibody as a therapeutic agent for central nervous system
disorders in neuronal ceroid lipofuscinosis or Santavuori-Haltia
disease, TPP-1-anti-hTfR antibody as a therapeutic agent for
central nervous system disorders in neuronal ceroid lipofuscinosis
or Jansky-Bielschowsky disease, IDUA-anti-hTfR antibody as a
therapeutic agent for central nervous system disorders in Hurler
syndrome or Hurler-Scheie syndrome, SGSH-anti-hTfR antibody as a
therapeutic agent for central nervous system disorders in
Sanfilippo syndrome, IDUA-anti-hTfR antibody as a therapeutic agent
for central nervous system disorders in Hurler syndrome or
Hurler-Scheie syndrome, and TNF.alpha.R-anti-hTfR antibody as a
therapeutic agent for cerebral ischemia and encephalitis. Besides,
the above results also indicate physiologically active proteins of
interest which do not usually pass through the blood-brain barrier
can be made to pass through the blood-brain barrier and get into
the brain tissues, by fusing them with the anti-hTfR antibody.
TABLE-US-00014 TABLE 13 Concentration of fusion proteins in brain
tissues (.mu.g/g wet weight) Relative value Fusion protein
Concentration to control Control 0.0199 1 EPO-anti-hTfR antibody
0.0862 4.33 ARSA-anti-hTfR antibody 0.0675 3.39 PPT-1-anti-hTfR
antibody 0.0970 4.87 TPP-1-anti-hTfR antibody 0.129 6.48
IDUA-1-anti-hTfR antibody 0.112 5.62 TNF.alpha.R-anti-hTfR antibody
0.148 7.44 SGSH-anti-hTfR antibody 0.0445 2.24
INDUSTRIAL APPLICABILITY
[0803] The anti-hTfR antibody of the present invention, when fused
with physiologically active proteins, low-molecular-weight
compounds and the like of interest, can make them able to pass
through the blood-brain barrier, and is, therefore, highly useful
in providing a means to deliver physiologically active proteins to
the brain, low-molecular-weight compounds and the like which are
needed to act in the central nervous system.
REFERENCE SIGNS LIST
[0804] 1 Blood vessel [0805] 2 Brain parenchyma [0806] 3
Neuron-like cells [0807] 4 Purkinje cells
SEQUENCE LISTING FREE TEXT
[0808] SEQ ID NO:3: Amino acid sequence of exemplified linker 1
[0809] SEQ ID NO.4: Amino acid sequence of exemplified linker 2
[0810] SEQ ID NO:5: Amino acid sequence of exemplified linker 3
[0811] SEQ ID NO:6: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 1
[0812] SEQ ID NO:7: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 1
[0813] SEQ ID NO:8: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 1
[0814] SEQ ID NO:9: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 1
[0815] SEQ ID NO:10: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 1
[0816] SEQ ID NO:11: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 2
[0817] SEQ ID NO:12: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 2
[0818] SEQ ID NO:13: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 2
[0819] SEQ ID NO:14: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 2
[0820] SEQ ID NO:15: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 2
[0821] SEQ ID NO:16: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 3
[0822] SEQ ID NO:17: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 3
[0823] SEQ ID NO:18: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 3
[0824] SEQ ID NO:19: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 3
[0825] SEQ ID NO:20: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 3
[0826] SEQ ID NO:21: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 4
[0827] SEQ ID NO:22: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 4
[0828] SEQ ID NO:23: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 4
[0829] SEQ ID NO:24: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 4
[0830] SEQ ID NO:25: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 4
[0831] SEQ ID NO:26: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 5
[0832] SEQ ID NO:27: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 5
[0833] SEQ ID NO:28: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 5
[0834] SEQ ID NO:29: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 5
[0835] SEQ ID NO:30: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 5
[0836] SEQ ID NO:31: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 6
[0837] SEQ ID NO:32: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 6
[0838] SEQ ID NO:33: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 6
[0839] SEQ ID NO:34: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 6
[0840] SEQ ID NO:35: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 6
[0841] SEQ ID NO:36: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 7
[0842] SEQ ID NO:37: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 7
[0843] SEQ ID NO:38: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 7
[0844] SEQ ID NO:39: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 7
[0845] SEQ ID NO:40: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 7
[0846] SEQ ID NO:41: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 8
[0847] SEQ ID NO:42: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 8
[0848] SEQ ID NO:43: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 8
[0849] SEQ ID NO:44: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 8
[0850] SEQ ID NO:45: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 8
[0851] SEQ ID NO:46: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 9
[0852] SEQ ID NO:47: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 9
[0853] SEQ ID NO:48: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 9
[0854] SEQ ID NO:49: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 9
[0855] SEQ ID NO:50: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 9
[0856] SEQ ID NO:51: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 10
[0857] SEQ ID NO:52: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 10
[0858] SEQ ID NO:53: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 10
[0859] SEQ ID NO:54: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 10
[0860] SEQ ID NO:55: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 10
[0861] SEQ ID NO:56: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 11
[0862] SEQ ID NO:57: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 11
[0863] SEQ ID NO:58: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 11
[0864] SEQ ID NO:59: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 11
[0865] SEQ ID NO:60: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 11
[0866] SEQ ID NO:61: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 12
[0867] SEQ ID NO:62: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 12
[0868] SEQ ID NO:63: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 12
[0869] SEQ ID NO:64: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 12
[0870] SEQ ID NO:65: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 12
[0871] SEQ ID NO:66: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 13
[0872] SEQ ID NO:67: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 13
[0873] SEQ ID NO:68: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 13
[0874] SEQ ID NO:69: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 13
[0875] SEQ ID NO:70: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 13
[0876] SEQ ID NO:71: Amino acid sequence 1 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 14
[0877] SEQ ID NO:72: Amino acid sequence 2 of CDR1 in the light
chain of mouse anti-hTfR antibody No. 14
[0878] SEQ ID NO:73: Amino acid sequence 1 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 14
[0879] SEQ ID NO:74: Amino acid sequence 2 of CDR2 in the light
chain of mouse anti-hTfR antibody No. 14
[0880] SEQ ID NO:75: Amino acid sequence of CDR3 in the light chain
of mouse anti-hTfR antibody No. 14
[0881] SEQ ID NO:76: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 1
[0882] SEQ ID NO:77: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 1
[0883] SEQ ID NO:78: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 1
[0884] SEQ ID NO:79: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 1
[0885] SEQ ID NO:80: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 1
[0886] SEQ ID NO:81: Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 1
[0887] SEQ ID NO:82: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 2
[0888] SEQ ID NO:83: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 2
[0889] SEQ ID NO:84: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 2
[0890] SEQ ID NO:85: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 2
[0891] SEQ ID NO:86: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 2 SEQ ID NO:87: Amino acid
sequence 2 of CDR3 in the heavy chain of mouse anti-hTfR antibody
No. 2
[0892] SEQ ID NO:88: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 3
[0893] SEQ ID NO:89: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 3
[0894] SEQ ID NO:90: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 3
[0895] SEQ ID NO:91: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 3
[0896] SEQ ID NO:92: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 3
[0897] SEQ ID NO:93: Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 3
[0898] SEQ ID NO:94: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 4
[0899] SEQ ID NO:95: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 4
[0900] SEQ ID NO:96: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 4
[0901] SEQ ID NO:97: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 4
[0902] SEQ ID NO:98: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 4
[0903] SEQ ID NO:99: Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 4
[0904] SEQ ID NO:100: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 5
[0905] SEQ ID NO:101: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 5
[0906] SEQ ID NO:102: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 5
[0907] SEQ ID NO:103: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 5
[0908] SEQ ID NO:104: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 5
[0909] SEQ ID NO:105: Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 5
[0910] SEQ ID NO:106: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 6
[0911] SEQ ID NO:107: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 6
[0912] SEQ ID NO:108: Amino acid sequence of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 6
[0913] SEQ ID NO:109: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 6
[0914] SEQ ID NO:110: Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 6
[0915] SEQ ID NO:111: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 7
[0916] SEQ ID NO:112: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 7
[0917] SEQ ID NO:113: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 7
[0918] SEQ ID NO:114: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 7
[0919] SEQ ID NO:115: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 7
[0920] SEQ ID NO:116: Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 7
[0921] SEQ ID NO:117. Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 8
[0922] SEQ ID NO:118: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 8
[0923] SEQ ID NO:119: Amino acid sequence of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 8
[0924] SEQ ID NO:120: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 8
[0925] SEQ ID NO:121. Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 8
[0926] SEQ ID NO:122: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 9
[0927] SEQ ID NO:123: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 9
[0928] SEQ ID NO:124: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 9
[0929] SEQ ID NO:125: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 9
[0930] SEQ ID NO:126: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 9
[0931] SEQ ID NO:127: Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 9
[0932] SEQ ID NO:128: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 10
[0933] SEQ ID NO:129: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 10
[0934] SEQ ID NO:130: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 10
[0935] SEQ ID NO:131: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 10
[0936] SEQ ID NO:132: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 10
[0937] SEQ ID NO:133. Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 10
[0938] SEQ ID NO:134: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 11
[0939] SEQ ID NO:135: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 11
[0940] SEQ ID NO:136: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 11
[0941] SEQ ID NO:137: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 11
[0942] SEQ ID NO:138: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 11
[0943] SEQ ID NO:139: Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 11
[0944] SEQ ID NO:140: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 12
[0945] SEQ ID NO:141: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 12
[0946] SEQ ID NO:142: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 12
[0947] SEQ ID NO:143: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 12
[0948] SEQ ID NO:144: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 12
[0949] SEQ ID NO:145: Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 12
[0950] SEQ ID NO:146: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 13
[0951] SEQ ID NO:147: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 13
[0952] SEQ ID NO:148: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 13
[0953] SEQ ID NO:149: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 13
[0954] SEQ ID NO:150: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 13
[0955] SEQ ID NO:151: Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 13
[0956] SEQ ID NO:152: Amino acid sequence 1 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 14
[0957] SEQ ID NO:153: Amino acid sequence 2 of CDR1 in the heavy
chain of mouse anti-hTfR antibody No. 14
[0958] SEQ ID NO:154: Amino acid sequence 1 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 14
[0959] SEQ ID NO:155: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 14
[0960] SEQ ID NO:156: Amino acid sequence 1 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 14
[0961] SEQ ID NO:157: Amino acid sequence 2 of CDR3 in the heavy
chain of mouse anti-hTfR antibody No. 14
[0962] SEQ ID NO:158: Amino acid sequence 1 of the light chain
variable region of humanized anti-hTfR antibody No. 1
[0963] SEQ ID NO:159: Amino acid sequence 2 of the light chain
variable region of humanized anti-hTfR antibody No. 1
[0964] SEQ ID NO:160: Amino acid sequence 3 of the light chain
variable region of humanized anti-hTfR antibody No. 1
[0965] SEQ ID NO:161: Amino acid sequence 4 of the light chain
variable region of humanized anti-hTfR antibody No. 1
[0966] SEQ ID NO:162: Amino acid sequence 5 of the light chain
variable region of humanized anti-hTfR antibody No. 1
[0967] SEQ ID NO:163: Amino acid sequence 6 of the light chain
variable region of humanized anti-hTfR antibody No. 1
[0968] SEQ ID NO:164: Amino acid sequence of the light chain of
humanized anti-hTfR antibody No. 1 containing amino acid sequence 6
as the variable region, synthetic sequence
[0969] SEQ ID NO:165: Nucleotide sequence encoding the amino acid
sequence of the light chain of humanized anti-hTfR antibody No. 1
containing amino acid sequence 6 as the variable region, synthetic
sequence
[0970] SEQ ID NO:166: Amino acid sequence 1 of the heavy chain
variable region of humanized anti-hTfR antibody No. 1
[0971] SEQ ID NO:167: Amino acid sequence 2 of the heavy chain
variable region of humanized anti-hTfR antibody No. 1
[0972] SEQ ID NO:168: Amino acid sequence 3 of the heavy chain
variable region of humanized anti-hTfR antibody No. 1
[0973] SEQ ID NO:169: Amino acid sequence 4 of the heavy chain
variable region of humanized anti-hTfR antibody No. 1
[0974] SEQ ID NO:170: Amino acid sequence 5 of the heavy chain
variable region of humanized anti-hTfR antibody No. 1
[0975] SEQ ID NO:171: Amino acid sequence 6 of the heavy chain
variable region of humanized anti-hTfR antibody No. 1
[0976] SEQ ID NO:172: Amino acid sequence of the heavy chain of
humanized anti-hTfR antibody No. 1 containing amino acid sequence 6
as the variable region
[0977] SEQ ID NO:173: Nucleotide sequence encoding the amino acid
sequence of the heavy chain of humanized anti-hTfR antibody No. 1
containing amino acid sequence 6 as the variable region, synthetic
sequence
[0978] SEQ ID NO:174: Amino acid sequence 1 of the light chain
variable region of humanized anti-hTfR antibody No. 2 SEQ ID
NO:175: Amino acid sequence 2 of the light chain variable region of
humanized anti-hTfR antibody No. 2
[0979] SEQ ID NO:176: Amino acid sequence 3 of the light chain
variable region of humanized anti-hTfR antibody No. 2
[0980] SEQ ID NO:177: Amino acid sequence 4 of the light chain
variable region of humanized anti-hTfR antibody No. 2
[0981] SEQ ID NO:178: Amino acid sequence 5 of the light chain
variable region of humanized anti-hTfR antibody No. 2
[0982] SEQ ID NO:179: Amino acid sequence 6 of the light chain
variable region of humanized anti-hTfR antibody No. 2
[0983] SEQ ID NO:180: Amino acid sequence of the light chain of
humanized anti-hTfR antibody No. 2 containing amino acid sequence 6
as the variable region
[0984] SEQ ID NO:181: Nucleotide sequence comprising a nucleotide
sequence encoding the amino acid sequence of the light chain of
humanized anti-hTfR antibody No. 2 containing amino acid sequence 6
as the variable region, synthetic sequence
[0985] SEQ ID NO:182: Amino acid sequence 1 of the heavy chain
variable region of humanized anti-hTfR antibody No. 2
[0986] SEQ ID NO:183: Amino acid sequence 2 of the heavy chain
variable region of humanized anti-hTfR antibody No. 2
[0987] SEQ ID NO:184: Amino acid sequence 3 of the heavy chain
variable region of humanized anti-hTfR antibody No. 2
[0988] SEQ ID NO:185: Amino acid sequence 4 of the heavy chain
variable region of humanized anti-hTfR antibody No. 2
[0989] SEQ ID NO:186: Amino acid sequence 5 of the heavy chain
variable region of humanized anti-hTfR antibody No. 2
[0990] SEQ ID NO:187: Amino acid sequence 6 of the heavy chain
variable region of humanized anti-hTfR antibody No. 2
[0991] SEQ ID NO:188: Amino acid sequence of the heavy chain of
humanized anti-hTfR antibody No. 2 containing amino acid sequence 6
as the variable region
[0992] SEQ ID NO:189: Nucleotide sequence comprising a nucleotide
sequence encoding the amino acid sequence of the heavy chain of
humanized anti-hTfR antibody No. 2 containing amino acid sequence 6
as the variable region, synthetic sequence
[0993] SEQ ID NO:190: Amino acid sequence 1 of the light chain
variable region of humanized anti-hTfR antibody No. 3
[0994] SEQ ID NO:191: Amino acid sequence 2 of the light chain
variable region of humanized anti-hTfR antibody No. 3
[0995] SEQ ID NO:192: Amino acid sequence 3 of the light chain
variable region of humanized anti-hTfR antibody No. 3
[0996] SEQ ID NO:193: Amino acid sequence 4 of the light chain
variable region of humanized anti-hTfR antibody No. 3
[0997] SEQ ID NO:194: Amino acid sequence 5 of the light chain
variable region of humanized anti-hTfR antibody No. 3
[0998] SEQ ID NO:195: Amino acid sequence 6 of the light chain
variable region of humanized anti-hTfR antibody No. 3
[0999] SEQ ID NO:196: Amino acid sequence of the light chain of
humanized anti-hTfR antibody No. 3 containing amino acid sequence 2
as the variable region
[1000] SEQ ID NO:197: Nucleotide sequence encoding the amino acid
sequence of the light chain of humanized anti-hTfR antibody No. 3
containing amino acid sequence 2 as the variable region, synthetic
sequence
[1001] SEQ ID NO:198: Amino acid sequence of the light chain of
humanized anti-hTfR antibody No. 3 containing amino acid sequence 4
as the variable region
[1002] SEQ ID NO:199: Nucleotide sequence encoding the amino acid
sequence of the light chain of humanized anti-hTfR antibody No. 3
containing amino acid sequence 4 as the variable region, synthetic
sequence
[1003] SEQ ID NO:200: Amino acid sequence of the light chain of
humanized anti-hTfR antibody No. 3 containing amino acid sequence 5
as the variable region
[1004] SEQ ID NO:201: Nucleotide sequence encoding the amino acid
sequence of the light chain of humanized anti-hTfR antibody No. 3
containing amino acid sequence 5 as the variable region, synthetic
sequence
[1005] SEQ ID NO:202: Humanized anti-hTfR antibody No. 3 containing
amino acid sequence 6 as the variable region
[1006] SEQ ID NO:203: Nucleotide sequence encoding the amino acid
sequence of the light chain of humanized anti-hTfR antibody No. 3
containing amino acid sequence 6 as the variable region, synthetic
sequence
[1007] SEQ ID NO:204: Amino acid sequence 1 of the heavy chain
variable region of humanized anti-hTfR antibody No. 3
[1008] SEQ ID NO:205: Amino acid sequence 2 of the heavy chain
variable region of humanized anti-hTfR antibody No. 3
[1009] SEQ ID NO:206: Amino acid sequence 3 of the heavy chain
variable region of humanized anti-hTfR antibody No. 3
[1010] SEQ ID NO:207: Amino acid sequence 4 of the heavy chain
variable region of humanized anti-hTfR antibody No. 3
[1011] SEQ ID NO:208: Amino acid sequence 5 of the heavy chain
variable region of humanized anti-hTfR antibody No. 3
[1012] SEQ ID NO:209: Amino acid sequence 6 of the heavy chain
variable region of humanized anti-hTfR antibody No. 3
[1013] SEQ ID NO:210: Amino acid sequence of the heavy chain of
humanized anti-hTfR antibody No. 3 containing amino acid sequence 2
as the variable region
[1014] SEQ ID NO:211: Nucleotide sequence encoding the amino acid
sequence of the heavy chain of humanized anti-hTfR antibody No. 3
containing amino acid sequence 2 as the variable region, synthetic
sequence
[1015] SEQ ID NO:212: Amino acid sequence of the heavy chain (IgG4)
of humanized anti-hTfR antibody No. 3 containing amino acid
sequence 2 as the variable region
[1016] SEQ ID NO:213: Nucleotide sequence encoding the amino acid
sequence of the heavy chain (IgG4) of humanized anti-hTfR antibody
No. 3 containing amino acid sequence 2 as the variable region,
synthetic sequence
[1017] SEQ ID NO:214: Primer hTfR5', synthetic sequence
[1018] SEQ ID NO:215: Primer hTfR3', synthetic sequence
[1019] SEQ ID NO:216: Primer Hyg-Sfi5', synthetic sequence
[1020] SEQ ID NO:217: Primer Hyg-BstX3', synthetic sequence
[1021] SEQ ID NO:218: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 1
[1022] SEQ ID NO:219: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 1
[1023] SEQ ID NO:220: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 2
[1024] SEQ ID NO:221: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 2
[1025] SEQ ID NO:222: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 3
[1026] SEQ ID NO:223: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 3
[1027] SEQ ID NO:224: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 4
[1028] SEQ ID NO:225: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 4
[1029] SEQ ID NO:226: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 5
[1030] SEQ ID NO:227: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 5
[1031] SEQ ID NO:228: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 6
[1032] SEQ ID NO:229: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 6
[1033] SEQ ID NO:230: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 7
[1034] SEQ ID NO:231: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 7
[1035] SEQ ID NO:232: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 8
[1036] SEQ ID NO:233: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 8
[1037] SEQ ID NO:234: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 9
[1038] SEQ ID NO:235: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 9
[1039] SEQ ID NO:236: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 10
[1040] SEQ ID NO:237: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 10
[1041] SEQ ID NO:238: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 11
[1042] SEQ ID NO:239: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 11
[1043] SEQ ID NO:240: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 12
[1044] SEQ ID NO:241: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 12
[1045] SEQ ID NO:242: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 13
[1046] SEQ ID NO:243: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 13
[1047] SEQ ID NO:244: Amino acid sequence of the light chain
variable region of mouse anti-hTfR antibody No. 14
[1048] SEQ ID NO:245: Amino acid sequence of the heavy chain
variable region of mouse anti-hTfR antibody No. 14
[1049] SEQ ID NO:247: Amino acid sequence of the fusion protein of
heavy chain of anti-hTfR antibody No. 1 (humanized 6) and hI2S
[1050] SEQ ID NO:248: Nucleotide sequence encoding the amino acid
sequence of fusion protein of heavy chain of anti-hTfR antibody No.
1 (humanized 6) and hI2S, synthetic sequence
[1051] SEQ ID NO:249: Amino acid sequence of the fusion protein of
heavy chain of anti-hTfR antibody No. 2 (humanized 6) and hI2S
[1052] SEQ ID NO:250: Nucleotide sequence encoding the amino acid
sequence of fusion protein of heavy chain of anti-hTfR antibody No.
2 (humanized 6) and hI2S, synthetic sequence
[1053] SEQ ID NO:251: Amino acid sequence of fusion protein of
heavy chain of anti-hTfR antibody No. 3 (humanized 2) and hI2S,
synthetic sequence
[1054] SEQ ID NO:252: Nucleotide sequence encoding the amino acid
sequence of fusion protein of heavy chain of anti-hTfR antibody No.
3 (humanized 2) and hI2S, synthetic sequence
[1055] SEQ ID NO:253: Nucleotide sequence of the DNA in which a
neomycin resistance gene flanked by loxP sequences was placed on
the cDNA's 3' side of a cDNA encoding chimeric hTfR, synthetic
sequence
[1056] SEQ ID NO:254: Nucleotide sequence of the 5'-arm of
targeting vector, synthetic sequence
[1057] SEQ ID NO:255: Nucleotide sequence of the 3'-arm of
targeting vector, synthetic sequence
[1058] SEQ ID NO:257: Amino acid sequence of the fusion protein of
heavy chain of anti-hTfR antibody No. 3 (humanized 2) and hEPO
[1059] SEQ ID NO:258: Nucleotide sequence encoding the amino acid
sequence of fusion protein of heavy chain of anti-hTfR antibody No.
3 (humanized 2) and hEPO, synthetic sequence
[1060] SEQ ID NO:260: Amino acid sequence of the fusion protein of
heavy chain of anti-hTfR antibody No. 3 (humanized 2) and hARSA
[1061] SEQ ID NO:261: Nucleotide sequence encoding the amino acid
sequence of fusion protein of heavy chain of anti-hTfR antibody No.
3 (humanized 2) and hARSA, synthetic sequence
[1062] SEQ ID NO:263: Amino acid sequence of the fusion protein of
heavy chain of anti-hTfR antibody No. 3 (humanized 2) and
hPPT-1
[1063] SEQ ID NO:264: Nucleotide sequence encoding the amino acid
sequence of fusion protein of heavy chain of anti-hTfR antibody No.
3 (humanized 2) and hPPT-1, synthetic sequence
[1064] SEQ ID NO:266: Amino acid sequence of the fusion protein of
heavy chain of anti-hTfR antibody No. 3 (humanized 2) and
hTPP-1
[1065] SEQ ID NO:267: Nucleotide sequence encoding the amino acid
sequence of fusion protein of heavy chain of anti-hTfR antibody No.
3 (humanized 2) and hTPP-1, synthetic sequence
[1066] SEQ ID NO:269: Amino acid sequence of the fusion protein of
heavy chain of anti-hTfR antibody No. 3 (humanized 2) and hIDUA
[1067] SEQ ID NO:270: Nucleotide sequence encoding the amino acid
sequence of fusion protein of heavy chain of anti-hTfR antibody No.
3 (humanized 2) and hIDUA, synthetic sequence
[1068] SEQ ID NO:272: Amino acid sequence of the fusion protein of
heavy chain of anti-hTfR antibody No. 3 (humanized 2) and
hTNF.alpha.R
[1069] SEQ ID NO:273: Nucleotide sequence encoding the amino acid
sequence of fusion protein of heavy chain of anti-hTfR.alpha.
antibody No. 3 (humanized 2) and hTNF.alpha.R, synthetic
sequence
[1070] SEQ ID NO:275: Amino acid sequence of the fusion protein of
heavy chain of anti-hTfR antibody No. 3 (humanized 2) and hSGSH
[1071] SEQ ID NO:276: Nucleotide sequence encoding the amino acid
sequence of fusion protein of heavy chain of anti-hTfR antibody No.
3 (humanized 2) and hSGSH, synthetic sequence
[1072] SEQ ID NO:277: Amino acid sequence of anti-hTfR single-chain
antibody
[1073] SEQ ID NO:278: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 6
[1074] SEQ ID NO:279: Amino acid sequence 2 of CDR2 in the heavy
chain of mouse anti-hTfR antibody No. 8
Sequence CWU 1
1
2791760PRTHomo sapiens 1Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn
Leu Phe Gly Gly Glu1 5 10 15Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala
Arg Gln Val Asp Gly Asp 20 25 30Asn Ser His Val Glu Met Lys Leu Ala
Val Asp Glu Glu Glu Asn Ala 35 40 45Asp Asn Asn Thr Lys Ala Asn Val
Thr Lys Pro Lys Arg Cys Ser Gly 50 55 60Ser Ile Cys Tyr Gly Thr Ile
Ala Val Ile Val Phe Phe Leu Ile Gly65 70 75 80Phe Met Ile Gly Tyr
Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr 85 90 95Glu Cys Glu Arg
Leu Ala Gly Thr Glu Ser Pro Val Arg Glu Glu Pro 100 105 110Gly Glu
Asp Phe Pro Ala Ala Arg Arg Leu Tyr Trp Asp Asp Leu Lys 115 120
125Arg Lys Leu Ser Glu Lys Leu Asp Ser Thr Asp Phe Thr Gly Thr Ile
130 135 140Lys Leu Leu Asn Glu Asn Ser Tyr Val Pro Arg Glu Ala Gly
Ser Gln145 150 155 160Lys Asp Glu Asn Leu Ala Leu Tyr Val Glu Asn
Gln Phe Arg Glu Phe 165 170 175Lys Leu Ser Lys Val Trp Arg Asp Gln
His Phe Val Lys Ile Gln Val 180 185 190Lys Asp Ser Ala Gln Asn Ser
Val Ile Ile Val Asp Lys Asn Gly Arg 195 200 205Leu Val Tyr Leu Val
Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys 210 215 220Ala Ala Thr
Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys225 230 235
240Lys Asp Phe Glu Asp Leu Tyr Thr Pro Val Asn Gly Ser Ile Val Ile
245 250 255Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala Asn
Ala Glu 260 265 270Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met Asp
Gln Thr Lys Phe 275 280 285Pro Ile Val Asn Ala Glu Leu Ser Phe Phe
Gly His Ala His Leu Gly 290 295 300Thr Gly Asp Pro Tyr Thr Pro Gly
Phe Pro Ser Phe Asn His Thr Gln305 310 315 320Phe Pro Pro Ser Arg
Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr 325 330 335Ile Ser Arg
Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp 340 345 350Cys
Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Arg Met Val Thr Ser 355 360
365Glu Ser Lys Asn Val Lys Leu Thr Val Ser Asn Val Leu Lys Glu Ile
370 375 380Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe Val Glu
Pro Asp385 390 395 400His Tyr Val Val Val Gly Ala Gln Arg Asp Ala
Trp Gly Pro Gly Ala 405 410 415Ala Lys Ser Gly Val Gly Thr Ala Leu
Leu Leu Lys Leu Ala Gln Met 420 425 430Phe Ser Asp Met Val Leu Lys
Asp Gly Phe Gln Pro Ser Arg Ser Ile 435 440 445Ile Phe Ala Ser Trp
Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr 450 455 460Glu Trp Leu
Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr465 470 475
480Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys Val
485 490 495Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr Met
Gln Asn 500 505 510Val Lys His Pro Val Thr Gly Gln Phe Leu Tyr Gln
Asp Ser Asn Trp 515 520 525Ala Ser Lys Val Glu Lys Leu Thr Leu Asp
Asn Ala Ala Phe Pro Phe 530 535 540Leu Ala Tyr Ser Gly Ile Pro Ala
Val Ser Phe Cys Phe Cys Glu Asp545 550 555 560Thr Asp Tyr Pro Tyr
Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu 565 570 575Ile Glu Arg
Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu 580 585 590Val
Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Val Glu Leu Asn 595 600
605Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Ser Phe Val Arg Asp
610 615 620Leu Asn Gln Tyr Arg Ala Asp Ile Lys Glu Met Gly Leu Ser
Leu Gln625 630 635 640Trp Leu Tyr Ser Ala Arg Gly Asp Phe Phe Arg
Ala Thr Ser Arg Leu 645 650 655Thr Thr Asp Phe Gly Asn Ala Glu Lys
Thr Asp Arg Phe Val Met Lys 660 665 670Lys Leu Asn Asp Arg Val Met
Arg Val Glu Tyr His Phe Leu Ser Pro 675 680 685Tyr Val Ser Pro Lys
Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser 690 695 700Gly Ser His
Thr Leu Pro Ala Leu Leu Glu Asn Leu Lys Leu Arg Lys705 710 715
720Gln Asn Asn Gly Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu Ala
725 730 735Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu Ser
Gly Asp 740 745 750Val Trp Asp Ile Asp Asn Glu Phe 755
7602760PRTMacaca fascicularis 2Met Met Asp Gln Ala Arg Ser Ala Phe
Ser Asn Leu Phe Gly Gly Glu1 5 10 15Pro Leu Ser Tyr Thr Arg Phe Ser
Leu Ala Arg Gln Val Asp Gly Asp 20 25 30Asn Ser His Val Glu Met Lys
Leu Ala Val Asp Asp Glu Glu Asn Ala 35 40 45Asp Asn Asn Thr Lys Ala
Asn Gly Thr Lys Pro Lys Arg Cys Gly Gly 50 55 60Asn Ile Cys Tyr Gly
Thr Ile Ala Val Ile Ile Phe Phe Leu Ile Gly65 70 75 80Phe Met Ile
Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr 85 90 95Glu Cys
Glu Arg Leu Ala Gly Thr Glu Ser Pro Ala Arg Glu Glu Pro 100 105
110Glu Glu Asp Phe Pro Ala Ala Pro Arg Leu Tyr Trp Asp Asp Leu Lys
115 120 125Arg Lys Leu Ser Glu Lys Leu Asp Thr Thr Asp Phe Thr Ser
Thr Ile 130 135 140Lys Leu Leu Asn Glu Asn Leu Tyr Val Pro Arg Glu
Ala Gly Ser Gln145 150 155 160Lys Asp Glu Asn Leu Ala Leu Tyr Ile
Glu Asn Gln Phe Arg Glu Phe 165 170 175Lys Leu Ser Lys Val Trp Arg
Asp Gln His Phe Val Lys Ile Gln Val 180 185 190Lys Asp Ser Ala Gln
Asn Ser Val Ile Ile Val Asp Lys Asn Gly Gly 195 200 205Leu Val Tyr
Leu Val Glu Asn Pro Gly Gly Tyr Val Ala Tyr Ser Lys 210 215 220Ala
Ala Thr Val Thr Gly Lys Leu Val His Ala Asn Phe Gly Thr Lys225 230
235 240Lys Asp Phe Glu Asp Leu Asp Ser Pro Val Asn Gly Ser Ile Val
Ile 245 250 255Val Arg Ala Gly Lys Ile Thr Phe Ala Glu Lys Val Ala
Asn Ala Glu 260 265 270Ser Leu Asn Ala Ile Gly Val Leu Ile Tyr Met
Asp Gln Thr Lys Phe 275 280 285Pro Ile Val Lys Ala Asp Leu Ser Phe
Phe Gly His Ala His Leu Gly 290 295 300Thr Gly Asp Pro Tyr Thr Pro
Gly Phe Pro Ser Phe Asn His Thr Gln305 310 315 320Phe Pro Pro Ser
Gln Ser Ser Gly Leu Pro Asn Ile Pro Val Gln Thr 325 330 335Ile Ser
Arg Ala Ala Ala Glu Lys Leu Phe Gly Asn Met Glu Gly Asp 340 345
350Cys Pro Ser Asp Trp Lys Thr Asp Ser Thr Cys Lys Met Val Thr Ser
355 360 365Glu Asn Lys Ser Val Lys Leu Thr Val Ser Asn Val Leu Lys
Glu Thr 370 375 380Lys Ile Leu Asn Ile Phe Gly Val Ile Lys Gly Phe
Val Glu Pro Asp385 390 395 400His Tyr Val Val Val Gly Ala Gln Arg
Asp Ala Trp Gly Pro Gly Ala 405 410 415Ala Lys Ser Ser Val Gly Thr
Ala Leu Leu Leu Lys Leu Ala Gln Met 420 425 430Phe Ser Asp Met Val
Leu Lys Asp Gly Phe Gln Pro Ser Arg Ser Ile 435 440 445Ile Phe Ala
Ser Trp Ser Ala Gly Asp Phe Gly Ser Val Gly Ala Thr 450 455 460Glu
Trp Leu Glu Gly Tyr Leu Ser Ser Leu His Leu Lys Ala Phe Thr465 470
475 480Tyr Ile Asn Leu Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Lys
Val 485 490 495Ser Ala Ser Pro Leu Leu Tyr Thr Leu Ile Glu Lys Thr
Met Gln Asp 500 505 510Val Lys His Pro Val Thr Gly Arg Ser Leu Tyr
Gln Asp Ser Asn Trp 515 520 525Ala Ser Lys Val Glu Lys Leu Thr Leu
Asp Asn Ala Ala Phe Pro Phe 530 535 540Leu Ala Tyr Ser Gly Ile Pro
Ala Val Ser Phe Cys Phe Cys Glu Asp545 550 555 560Thr Asp Tyr Pro
Tyr Leu Gly Thr Thr Met Asp Thr Tyr Lys Glu Leu 565 570 575Val Glu
Arg Ile Pro Glu Leu Asn Lys Val Ala Arg Ala Ala Ala Glu 580 585
590Val Ala Gly Gln Phe Val Ile Lys Leu Thr His Asp Thr Glu Leu Asn
595 600 605Leu Asp Tyr Glu Arg Tyr Asn Ser Gln Leu Leu Leu Phe Leu
Arg Asp 610 615 620Leu Asn Gln Tyr Arg Ala Asp Val Lys Glu Met Gly
Leu Ser Leu Gln625 630 635 640Trp Leu Tyr Ser Ala Arg Gly Asp Phe
Phe Arg Ala Thr Ser Arg Leu 645 650 655Thr Thr Asp Phe Arg Asn Ala
Glu Lys Arg Asp Lys Phe Val Met Lys 660 665 670Lys Leu Asn Asp Arg
Val Met Arg Val Glu Tyr Tyr Phe Leu Ser Pro 675 680 685Tyr Val Ser
Pro Lys Glu Ser Pro Phe Arg His Val Phe Trp Gly Ser 690 695 700Gly
Ser His Thr Leu Ser Ala Leu Leu Glu Ser Leu Lys Leu Arg Arg705 710
715 720Gln Asn Asn Ser Ala Phe Asn Glu Thr Leu Phe Arg Asn Gln Leu
Ala 725 730 735Leu Ala Thr Trp Thr Ile Gln Gly Ala Ala Asn Ala Leu
Ser Gly Asp 740 745 750Val Trp Asp Ile Asp Asn Glu Phe 755
76035PRTArtificial SequenceAmino acid sequence of exemplified
linker 1 3Gly Gly Gly Gly Ser1 546PRTArtificial SequenceAmino acid
sequence of exemplified linker 2 4Gly Gly Gly Gly Gly Ser1
555PRTArtificial SequenceAmino acid sequence of exemplified linker
3 5Ser Gly Gly Gly Gly1 566PRTArtificial SequenceAmino acid
sequence 1 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.1 6Gln Asp Val Asn Ser Ala1 5711PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.1 7Lys Ala Ser Gln Asp Val Asn Ser Ala Val Ala1 5
1086PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.1 8Trp Thr Ser Thr Arg
His1 597PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.1 9Trp Thr Ser Thr Arg
His Thr1 5109PRTArtificial SequenceAmino acid sequence of CDR 3 in
the light chain of mouse anti-hTfR antibody No.1 10Gln Gln His Tyr
Ser Thr Pro Arg Thr1 5116PRTArtificial SequenceAmino acid sequence
1 of CDR 1 in the light chain of mouse anti-hTfR antibody No.2
11Gln Ser Ile Ser Asn Asn1 51211PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.2 12Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu Gln1 5
10136PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.2 13Tyr Ala Ser Gln Ser
Ile1 5147PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in
the light chain of mouse anti-hTfR antibody No.2 14Tyr Ala Ser Gln
Ser Ile Ser1 5159PRTArtificial SequenceAmino acid sequence of CDR 3
in the light chain of mouse anti-hTfR antibody No.2 15Gln Gln Ser
Asn Ser Trp Pro Arg Thr1 51611PRTArtificial SequenceAmino acid
sequence 1 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.3 16Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr1 5
101716PRTArtificial SequenceAmino acid sequence 2 of CDR 1 in the
light chain of mouse anti-hTfR antibody No.3 17Arg Ser Ser Gln Ser
Leu Val His Ser Asn Gly Asn Thr Tyr Leu His1 5 10
15186PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.3 18Lys Val Ser Asn Arg
Phe1 5197PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in
the light chain of mouse anti-hTfR antibody No.3 19Lys Val Ser Asn
Arg Phe Ser1 5209PRTArtificial SequenceAmino acid sequence of CDR 3
in the light chain of mouse anti-hTfR antibody No.3 20Ser Gln Ser
Thr His Val Pro Trp Thr1 5215PRTArtificial SequenceAmino acid
sequence 1 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.4 21Ser Asn Val Asn Tyr1 52210PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.4 22Ser Ala Ser Ser Asn Val Asn Tyr Met His1 5
10236PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.4 23Asp Thr Ser Lys Leu
Ala1 5247PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in
the light chain of mouse anti-hTfR antibody No.4 24Asp Thr Ser Lys
Leu Ala Ser1 5259PRTArtificial SequenceAmino acid sequence of CDR 3
in the light chain of mouse anti-hTfR antibody No.4 25Phe Gln Gly
Asn Gly Asn Pro Tyr Thr1 5265PRTArtificial SequenceAmino acid
sequence 1 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.5 26Ser Ser Ile Ser Ser1 52710PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.5 27Ser Ala Ser Ser Ser Ile Ser Ser Ile His1 5
10286PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.5 28Asp Thr Ser Thr Leu
Ala1 5297PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in
the light chain of mouse anti-hTfR antibody No.5 29Asp Thr Ser Thr
Leu Ala Ser1 5308PRTArtificial SequenceAmino acid sequence of CDR 3
in the light chain of mouse anti-hTfR antibody No.5 30His Gln Arg
Ser Ser Tyr Pro Thr1 5316PRTArtificial SequenceAmino acid sequence
1 of CDR 1 in the light chain of mouse anti-hTfR antibody No.6
31Gln Glu Ile Ser Gly Tyr1 53211PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.6 32Arg Ala Ser Gln Glu Ile Ser Gly Tyr Leu Ser1 5
10336PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.6 33Ala Ala Ser Thr Leu
Asp1 5347PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in
the light chain of mouse anti-hTfR antibody No.6 34Ala Ala Ser Thr
Leu Asp Ser1 5359PRTArtificial SequenceAmino acid sequence of CDR 3
in the light chain of mouse anti-hTfR antibody No.6 35Leu Gln Tyr
Ser Ser Tyr Pro Arg Thr1 5365PRTArtificial SequenceAmino acid
sequence 1 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.7 36Ser Ser Val Asn Tyr1 53710PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.7 37Ser Ala Ser Ser Ser Val Asn Tyr Ile His1 5
10386PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.7 38Gln Thr Ser Asn Leu
Ala1 5397PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in
the light chain of mouse anti-hTfR antibody No.7 39Gln Thr Ser Asn
Leu Ala Ser1 5409PRTArtificial SequenceAmino acid sequence of CDR 3
in the light chain of mouse anti-hTfR antibody No.7 40His Gln Trp
Ser Ser Tyr Pro Trp Thr1 5415PRTArtificial SequenceAmino acid
sequence 1 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.8 41Ser Ser Val Ser Tyr1 54210PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.8 42Ser Ala Ser Ser Ser Val Ser Tyr Met His1 5
10436PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.8 43Gly Thr Ser Asn Leu
Ala1 5447PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in
the light chain of mouse anti-hTfR antibody No.8 44Gly Thr Ser Asn
Leu Ala Ser1 5459PRTArtificial
SequenceAmino acid sequence of CDR 3 in the light chain of mouse
anti-hTfR antibody No.8 45His Gln Trp Ser Ser Tyr Pro Trp Thr1
5465PRTArtificial SequenceAmino acid sequence 1 of CDR 1 in the
light chain of mouse anti-hTfR antibody No.9 46Ser Ser Val Ser Leu1
54710PRTArtificial SequenceAmino acid sequence 2 of CDR 1 in the
light chain of mouse anti-hTfR antibody No.9 47Ser Ala Ser Ser Ser
Val Ser Leu Met Phe1 5 10486PRTArtificial SequenceAmino acid
sequence 1 of CDR 2 in the light chain of mouse anti-hTfR antibody
No.9 48Phe Thr Ser Tyr Arg Ala1 5497PRTArtificial SequenceAmino
acid sequence 2 of CDR 2 in the light chain of mouse anti-hTfR
antibody No.9 49Phe Thr Ser Tyr Arg Ala Ser1 5509PRTArtificial
SequenceAmino acid sequence of CDR 3 in the light chain of mouse
anti-hTfR antibody No.9 50Gln Gln Trp Thr Ser Asp Pro Pro Thr1
5516PRTArtificial SequenceAmino acid sequence 1 of CDR 1 in the
light chain of mouse anti-hTfR antibody No.10 51Gln Glu Ile Ser Gly
Tyr1 55211PRTArtificial SequenceAmino acid sequence 2 of CDR 1 in
the light chain of mouse anti-hTfR antibody No.10 52Arg Pro Ser Gln
Glu Ile Ser Gly Tyr Leu Ser1 5 10536PRTArtificial SequenceAmino
acid sequence 1 of CDR 2 in the light chain of mouse anti-hTfR
antibody No.10 53Ala Ala Ser Thr Leu Asp1 5547PRTArtificial
SequenceAmino acid sequence 2 of CDR 2 in the light chain of mouse
anti-hTfR antibody No.10 54Ala Ala Ser Thr Leu Asp Ser1
5559PRTArtificial SequenceAmino acid sequence of CDR 3 in the light
chain of mouse anti-hTfR antibody No.10 55Leu Gln Tyr Ala Ser Tyr
Pro Arg Thr1 5566PRTArtificial SequenceAmino acid sequence 1 of CDR
1 in the light chain of mouse anti-hTfR antibody No.11 56Gln Ser
Ile Arg Asn Tyr1 55711PRTArtificial SequenceAmino acid sequence 2
of CDR 1 in the light chain of mouse anti-hTfR antibody No.11 57Arg
Ala Ser Gln Ser Ile Arg Asn Tyr Leu His1 5 10586PRTArtificial
SequenceAmino acid sequence 1 of CDR 2 in the light chain of mouse
anti-hTfR antibody No.11 58Tyr Ala Ser Gln Ser Ile1
5597PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.11 59Tyr Ala Ser Gln Ser
Ile Ser1 5609PRTArtificial SequenceAmino acid sequence of CDR 3 in
the light chain of mouse anti-hTfR antibody No.11 60Gln Gln Thr Asn
Ser Trp Pro Tyr Thr1 5616PRTArtificial SequenceAmino acid sequence
1 of CDR 1 in the light chain of mouse anti-hTfR antibody No.12
61His Asp Val Lys Thr Ala1 56211PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.12 62Lys Ala Ser His Asp Val Lys Thr Ala Val Ala1 5
10636PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.12 63Trp Ser Ser Thr Arg
His1 5647PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in
the light chain of mouse anti-hTfR antibody No.12 64Trp Ser Ser Thr
Arg His Thr1 5659PRTArtificial SequenceAmino acid sequence of CDR 3
in the light chain of mouse anti-hTfR antibody No.12 65Gln Gln His
Phe Ser Thr Pro Leu Thr1 5666PRTArtificial SequenceAmino acid
sequence 1 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.13 66Gln Ser Ile Arg Asn Tyr1 56711PRTArtificial SequenceAmino
acid sequence 2 of CDR 1 in the light chain of mouse anti-hTfR
antibody No.13 67Arg Ala Ser Gln Ser Ile Arg Asn Tyr Leu His1 5
10686PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.13 68Tyr Ala Ser Gln Ser
Ile1 5697PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in
the light chain of mouse anti-hTfR antibody No.13 69Tyr Ala Ser Gln
Ser Ile Ser1 5709PRTArtificial SequenceAmino acid sequence of CDR 3
in the light chain of mouse anti-hTfR antibody No.13 70Gln Gln Thr
Asn Ser Trp Pro Tyr Thr1 5715PRTArtificial SequenceAmino acid
sequence 1 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.14 71Ser Asn Ile Asn Ser1 57210PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the light chain of mouse anti-hTfR antibody
No.14 72Ser Ala Ser Ser Asn Ile Asn Ser Ile His1 5
10736PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
light chain of mouse anti-hTfR antibody No.14 73Asp Thr Ser Asn Leu
Ala1 5747PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in
the light chain of mouse anti-hTfR antibody No.14 74Asp Thr Ser Asn
Leu Ala Ser1 5759PRTArtificial SequenceAmino acid sequence of CDR 3
in the light chain of mouse anti-hTfR antibody No.14 75His Gln Arg
Ser Gly Tyr Pro Phe Thr1 5765PRTArtificial SequenceAmino acid
sequence 1 of CDR 1 in the heavy chain of mouse anti-hTfR antibody
No.1 76Asn Tyr Gly Met Ser1 5778PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the heavy chain of mouse anti-hTfR antibody
No.1 77Gly Leu Thr Phe Ser Asn Tyr Gly1 5788PRTArtificial
SequenceAmino acid sequence 1 of CDR 2 in the heavy chain of mouse
anti-hTfR antibody No.1 78Ile Asn Thr Asn Gly Gly Ser Thr1
57917PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in the
heavy chain of mouse anti-hTfR antibody No.1 79Asn Ile Asn Thr Asn
Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys1 5 10
15Gly807PRTArtificial SequenceAmino acid sequence 1 of CDR 3 in the
heavy chain of mouse anti-hTfR antibody No.1 80Asn Arg Tyr Asp Glu
Asp Tyr1 5819PRTArtificial SequenceAmino acid sequence 2 of CDR 3
in the heavy chain of mouse anti-hTfR antibody No.1 81Thr Asn Asn
Arg Tyr Asp Glu Asp Tyr1 5825PRTArtificial SequenceAmino acid
sequence 1 of CDR 1in the heavy chain of mouse anti-hTfR antibody
No.2 82Asp Tyr Val Met His1 5838PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the heavy chain of mouse anti-hTfR antibody
No.2 83Gly Tyr Thr Phe Thr Asp Tyr Val1 5848PRTArtificial
SequenceAmino acid sequence 1 of CDR 2 in the heavy chain of mouse
anti-hTfR antibody No.2 84Ile Ser Thr Tyr Tyr Gly His Gly1
58517PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in the
heavy chain of mouse anti-hTfR antibody No.2 85Val Ile Ser Thr Tyr
Tyr Gly His Gly Ser Tyr Asn Gln Arg Phe Lys1 5 10
15Gly8614PRTArtificial SequenceAmino acid sequence 1 of CDR 3 in
the heavy chain of mouse anti-hTfR antibody No.2 86Gly Gly Tyr Gly
Ser Ser Ser Leu Ala Gly Asn Phe Asp Val1 5 108716PRTArtificial
SequenceAmino acid sequence 2 of CDR 3 in the heavy chain of mouse
anti-hTfR antibody No.2 87Val Arg Gly Gly Tyr Gly Ser Ser Ser Leu
Ala Gly Asn Phe Asp Val1 5 10 15885PRTArtificial SequenceAmino acid
sequence 1 of CDR 1 in the heavy chain of mouse anti-hTfR antibody
No.3 88Asn Tyr Trp Leu Gly1 5898PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the heavy chain of mouse anti-hTfR antibody
No.3 89Gly Tyr Ser Phe Thr Asn Tyr Trp1 5908PRTArtificial
SequenceAmino acid sequence 1 of CDR 2 in the heavy chain of mouse
anti-hTfR antibody No.3 90Ile Tyr Pro Gly Gly Asp Tyr Pro1
59117PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in the
heavy chain of mouse anti-hTfR antibody No.3 91Asp Ile Tyr Pro Gly
Gly Asp Tyr Pro Thr Tyr Ser Glu Lys Phe Lys1 5 10
15Val929PRTArtificial SequenceAmino acid sequence 1 of CDR 3 in the
heavy chain of mouse anti-hTfR antibody No.3 92Ser Gly Asn Tyr Asp
Glu Val Ala Tyr1 59311PRTArtificial SequenceAmino acid sequence 2
of CDR 3 in the heavy chain of mouse anti-hTfR antibody No.3 93Ala
Arg Ser Gly Asn Tyr Asp Glu Val Ala Tyr1 5 10945PRTArtificial
SequenceAmino acid sequence 1 of CDR 1 in the heavy chain of mouse
anti-hTfR antibody No.4 94Asn Tyr Leu Ile Glu1 5958PRTArtificial
SequenceAmino acid sequence 2 of CDR 1 in the heavy chain of mouse
anti-hTfR antibody No.4 95Gly Tyr Ala Phe Thr Asn Tyr Leu1
5968PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
heavy chain of mouse anti-hTfR antibody No.4 96Ile Asn Pro Gly Ser
Gly Gly Ile1 59716PRTArtificial SequenceAmino acid sequence 2 of
CDR 2 in the heavy chain of mouse anti-hTfR antibody No.4 97Ile Asn
Pro Gly Ser Gly Gly Ile Ile Tyr Asn Glu Lys Phe Thr Asp1 5 10
159813PRTArtificial SequenceAmino acid sequence 1 of CDR 3 in the
heavy chain of mouse anti-hTfR antibody No.4 98Ser Asn Tyr Tyr Gly
Thr Thr Tyr Trp His Phe Asp Val1 5 109915PRTArtificial
SequenceAmino acid sequence 2 of CDR 3 in the heavy chain of mouse
anti-hTfR antibody No.4 99Ala Arg Ser Asn Tyr Tyr Gly Thr Thr Tyr
Trp His Phe Asp Val1 5 10 151005PRTArtificial SequenceAmino acid
sequence 1 of CDR 1 in the heavy chain of mouse anti-hTfR antibody
No.5 100Asn Phe Val Ile His1 51018PRTArtificial SequenceAmino acid
sequence 2 of CDR 1 in the heavy chain of mouse anti-hTfR antibody
No.5 101Gly Tyr Thr Phe Thr Asn Phe Val1 51028PRTArtificial
SequenceAmino acid sequence 1 of CDR 2 in the heavy chain of mouse
anti-hTfR antibody No.5 102Phe Asn Pro His Lys Asn Gly Ala1
510316PRTArtificial SequenceAmino acid sequence 2 of CDR 2 in the
heavy chain of mouse anti-hTfR antibody No.5 103Phe Asn Pro His Lys
Asn Gly Ala Glu Tyr Asn Glu Lys Phe Gln Val1 5 10
151049PRTArtificial SequenceAmino acid sequence 1 of CDR 3 in the
heavy chain of mouse anti-hTfR antibody No.5 104Ser Phe Tyr Tyr Tyr
Ser Met Asp Tyr1 510511PRTArtificial SequenceAmino acid sequence 2
of CDR 3 in the heavy chain of mouse anti-hTfR antibody No.5 105Ala
Arg Ser Phe Tyr Tyr Tyr Ser Met Asp Tyr1 5 101065PRTArtificial
SequenceAmino acid sequence 1 of CDR 1 in the heavy chain of mouse
anti-hTfR antibody No.6 106Thr Tyr Gly Val Tyr1 51078PRTArtificial
SequenceAmino acid sequence 2 of CDR 1 in the heavy chain of mouse
anti-hTfR antibody No.6 107Gly Phe Ser Leu Ser Thr Tyr Gly1
51087PRTArtificial SequenceAmino acid sequence of CDR 2 in the
heavy chain of mouse anti-hTfR antibody No.6 108Ile Trp Gly Asp Gly
Ser Thr1 51094PRTArtificial SequenceAmino acid sequence 1 of CDR 3
in the heavy chain of mouse anti-hTfR antibody No.6 109Pro Asp Asp
Val11106PRTArtificial SequenceAmino acid sequence 2 of CDR 3 in the
heavy chain of mouse anti-hTfR antibody No.6 110Ala Lys Pro Asp Asp
Val1 51115PRTArtificial SequenceAmino acid sequence 1 of CDR 1 in
the heavy chain of mouse anti-hTfR antibody No.7 111Asn Tyr Phe Met
Ser1 51128PRTArtificial SequenceAmino acid sequence 2 of CDR 1 in
the heavy chain of mouse anti-hTfR antibody No.7 112Gly Ile Thr Phe
Arg Asn Tyr Phe1 51138PRTArtificial SequenceAmino acid sequence 1
of CDR 2 in the heavy chain of mouse anti-hTfR antibody No.7 113Ile
Ser Ser Ala Gly Gly Tyr Thr1 511416PRTArtificial SequenceAmino acid
sequence 2 of CDR 2 in the heavy chain of mouse anti-hTfR antibody
No.7 114Ile Ser Ser Ala Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys
Gly1 5 10 1511511PRTArtificial SequenceAmino acid sequence 1 of CDR
3 in the heavy chain of mouse anti-hTfR antibody No.7 115Gln Glu
Val Pro Tyr Pro Tyr Ala Met Asp Tyr1 5 1011613PRTArtificial
SequenceAmino acid sequence 2 of CDR 3 in the heavy chain of mouse
anti-hTfR antibody No.7 116Ala Arg Gln Glu Val Pro Tyr Pro Tyr Ala
Met Asp Tyr1 5 101175PRTArtificial SequenceAmino acid sequence 1 of
CDR 1 in the heavy chain of mouse anti-hTfR antibody No.8 117Thr
Tyr Gly Val Ser1 51188PRTArtificial SequenceAmino acid sequence 2
of CDR 1 in the heavy chain of mouse anti-hTfR antibody No.8 118Gly
Phe Ser Leu Thr Thr Tyr Gly1 51197PRTArtificial SequenceAmino acid
sequence of CDR 2 in the heavy chain of mouse anti-hTfR antibody
No.8 119Ile Trp Gly Asp Gly Ser Thr1 51204PRTArtificial
SequenceAmino acid sequence 1 of CDR 3 in the heavy chain of mouse
anti-hTfR antibody No.8 120Pro Asp Asp Tyr11216PRTArtificial
SequenceAmino acid sequence 2 of CDR 3 in the heavy chain of mouse
anti-hTfR antibody No.8 121Ala Lys Pro Asp Asp Tyr1
51225PRTArtificial SequenceAmino acid sequence 1 of CDR 1 in the
heavy chain of mouse anti-hTfR antibody No.9 122Asn Tyr Gly Val
Ser1 51238PRTArtificial SequenceAmino acid sequence 2 of CDR 1 in
the heavy chain of mouse anti-hTfR antibody No.9 123Gly Tyr Thr Phe
Thr Asn Tyr Gly1 51248PRTArtificial SequenceAmino acid sequence 1
of CDR 2 in the heavy chain of mouse anti-hTfR antibody No.9 124Ile
Tyr Thr Phe Thr Gly Glu Ala1 512516PRTArtificial SequenceAmino acid
sequence 2 of CDR 2 in the heavy chain of mouse anti-hTfR antibody
No.9 125Ile Tyr Thr Phe Thr Gly Glu Ala Thr Tyr Ile Asp Asp Phe Arg
Gly1 5 10 151268PRTArtificial SequenceAmino acid sequence 1 of CDR
3 in the heavy chain of mouse anti-hTfR antibody No.9 126Arg Asn
Gly Ala Trp Phe Glu Asp1 512710PRTArtificial SequenceAmino acid
sequence 2 of CDR 3 in the heavy chain of mouse anti-hTfR antibody
No.9 127Ser Arg Arg Asn Gly Ala Trp Phe Glu Asp1 5
101285PRTArtificial SequenceAmino acid sequence 1 of CDR 1 in the
heavy chain of mouse anti-hTfR antibody No.10 128Asn Tyr Ala Met
Ser1 51298PRTArtificial SequenceAmino acid sequence 2 of CDR 1 in
the heavy chain of mouse anti-hTfR antibody No.10 129Gly Ile Thr
Phe Arg Asn Tyr Ala1 51308PRTArtificial SequenceAmino acid sequence
1 of CDR 2 in the heavy chain of mouse anti-hTfR antibody No.10
130Ile Ser Ser Tyr Gly Gly Tyr Thr1 513116PRTArtificial
SequenceAmino acid sequence 2 of CDR 2 in the heavy chain of mouse
anti-hTfR antibody No.10 131Ile Ser Ser Tyr Gly Gly Tyr Thr Tyr Tyr
Ala Asp Ser Val Lys Gly1 5 10 1513211PRTArtificial SequenceAmino
acid sequence 1 of CDR 3 in the heavy chain of mouse anti-hTfR
antibody No.10 132Gln Glu Val Pro Tyr Pro Tyr Pro Met Asp Asn1 5
1013313PRTArtificial SequenceAmino acid sequence 2 of CDR 3 in the
heavy chain of mouse anti-hTfR antibody No.10 133Ala Arg Gln Glu
Val Pro Tyr Pro Tyr Pro Met Asp Asn1 5 101345PRTArtificial
SequenceAmino acid sequence 1 of CDR 1 in the heavy chain of mouse
anti-hTfR antibody No.11 134Ile Tyr Trp Ile Asn1 51358PRTArtificial
SequenceAmino acid sequence 2 of CDR 1 in the heavy chain of mouse
anti-hTfR antibody No.11 135Gly Tyr Ala Phe Ser Ile Tyr Trp1
51368PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
heavy chain of mouse anti-hTfR antibody No.11 136Ile Tyr Pro Gly
Asn Gly Asp Thr1 513716PRTArtificial SequenceAmino acid sequence 2
of CDR 2 in the heavy chain of mouse anti-hTfR antibody No.11
137Ile Tyr Pro Gly Asn Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly1
5 10 151389PRTArtificial SequenceAmino acid sequence 1 of CDR 3 in
the heavy chain of mouse anti-hTfR antibody No.11 138Trp Gly Asp
Asp Tyr Ala Met Asp Phe1 513911PRTArtificial SequenceAmino acid
sequence 2 of CDR 3 in the heavy chain of mouse anti-hTfR antibody
No.11 139Gly Arg Trp Gly Asp Asp Tyr Ala Met Asp Phe1 5
101405PRTArtificial SequenceAmino acid sequence 1 of CDR 1 in the
heavy chain of mouse anti-hTfR antibody No.12 140Asn Tyr Leu Ile
Glu1 51418PRTArtificial SequenceAmino acid sequence 2 of CDR 1 in
the heavy chain of mouse anti-hTfR antibody No.12 141Gly Tyr Val
Phe Ile Asn Tyr Leu1 51428PRTArtificial SequenceAmino acid sequence
1 of CDR 2 in the heavy chain of mouse anti-hTfR antibody No.12
142Ile His Ser Gly Ser Gly Gly Thr1 514316PRTArtificial
SequenceAmino acid sequence 2 of CDR 2 in the heavy chain of mouse
anti-hTfR antibody No.12 143Ile His Ser Gly Ser Gly Gly Thr Asn Tyr
Asn Asp Asn Phe Lys Asp1 5 10 1514410PRTArtificial SequenceAmino
acid sequence 1 of CDR 3 in the heavy chain of mouse anti-hTfR
antibody No.12 144Arg Asn Phe Gly Asn Tyr Tyr Phe Asp Tyr1 5
1014512PRTArtificial SequenceAmino acid sequence 2 of CDR 3 in the
heavy chain of mouse anti-hTfR antibody No.12 145Ala Arg Arg Asn
Phe Gly Asn Tyr Tyr Phe Asp Tyr1 5 101465PRTArtificial
SequenceAmino acid sequence 1 of CDR 1 in the heavy
chain of mouse anti-hTfR antibody No.13 146Ile Tyr Trp Ile Asn1
51478PRTArtificial SequenceAmino acid sequence 2 of CDR 1 in the
heavy chain of mouse anti-hTfR antibody No.13 147Gly Tyr Ala Phe
Ser Ile Tyr Trp1 51488PRTArtificial SequenceAmino acid sequence 1
of CDR 2 in the heavy chain of mouse anti-hTfR antibody No.13
148Ile Tyr Pro Gly Asn Gly Asp Thr1 514916PRTArtificial
SequenceAmino acid sequence 2 of CDR 2 in the heavy chain of mouse
anti-hTfR antibody No.13 149Ile Tyr Pro Gly Asn Gly Asp Thr Asn Tyr
Asn Gly Lys Phe Lys Gly1 5 10 151509PRTArtificial SequenceAmino
acid sequence 1 of CDR 3 in the heavy chain of mouse anti-hTfR
antibody No.13 150Trp Gly Asp Asp Tyr Ala Met Asp Phe1
515111PRTArtificial SequenceAmino acid sequence 2 of CDR 3 in the
heavy chain of mouse anti-hTfR antibody No.13 151Gly Arg Trp Gly
Asp Asp Tyr Ala Met Asp Phe1 5 101525PRTArtificial SequenceAmino
acid sequence 1 of CDR 1 in the heavy chain of mouse anti-hTfR
antibody No.14 152Gly Tyr Val Ile His1 51538PRTArtificial
SequenceAmino acid sequence 2 of CDR 1 in the heavy chain of mouse
anti-hTfR antibody No.14 153Gly Tyr Ala Phe Thr Gly Tyr Val1
51548PRTArtificial SequenceAmino acid sequence 1 of CDR 2 in the
heavy chain of mouse anti-hTfR antibody No.14 154Leu Asn Pro His
Lys Asp Asp Ser1 515516PRTArtificial SequenceAmino acid sequence 2
of CDR 2 in the heavy chain of mouse anti-hTfR antibody No.14
155Leu Asn Pro His Lys Asp Asp Ser Glu Tyr Asn Glu Lys Phe Arg Gly1
5 10 151569PRTArtificial SequenceAmino acid sequence 1 of CDR 3 in
the heavy chain of mouse anti-hTfR antibody No.14 156Gly Tyr Tyr
Tyr Tyr Ser Met Asp Tyr1 515711PRTArtificial SequenceAmino acid
sequence 2 of CDR 3 in the heavy chain of mouse anti-hTfR antibody
No.14 157Ala Arg Gly Tyr Tyr Tyr Tyr Ser Met Asp Tyr1 5
10158107PRTArtificial SequenceAmino acid sequence 1 of the variable
region of light chain of humanized anti-hTfR antibody No.1 158Asp
Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10
15Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Gln Asp Val Asn Ser Ala
20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
Ile 35 40 45Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Ala65 70 75 80Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His
Tyr Ser Thr Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105159107PRTArtificial SequenceAmino acid sequence 2 of the
variable region of light chain of humanized anti-hTfR antibody No.1
159Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Ser
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Leu 35 40 45Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
His Tyr Ser Thr Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105160107PRTArtificial SequenceAmino acid sequence 3 of
the variable region of light chain of humanized anti-hTfR antibody
No.1 160Asp Ile Gln Val Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Asn
Ser Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln His Tyr Ser Thr Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105161107PRTArtificial SequenceAmino acid sequence
4 of the variable region of light chain of humanized anti-hTfR
antibody No.1 161Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
Asp Val Asn Ser Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Thr Ser Thr Arg His Thr Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg 85 90 95Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105162107PRTArtificial SequenceAmino
acid sequence 5 of the variable region of light chain of humanized
anti-hTfR antibody No.1 162Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala
Ser Gln Asp Val Asn Ser Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Thr Ser Thr Arg His
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105163107PRTArtificial
SequenceAmino acid sequence 6 of the variable region of light chain
of humanized anti-hTfR antibody No.1 163Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Gln Asp Val Asn Ser Ala 20 25 30Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Thr Ser
Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105164214PRTArtificial SequenceAmino acid sequence of the light
chain of humanized anti-hTfR antibody No.1 containing amino acid
sequence 6 as the variable region 164Asp Ile Gln Val Thr Gln Ser
Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Lys Ala Ser Gln Asp Val Asn Ser Ala 20 25 30Val Ala Trp Phe Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Trp Thr Ser
Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Val Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Arg 85 90
95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 210165725DNAArtificial SequenceNucleotide
sequence encoding the amino acid sequence of the light chain of
humanized anti-hTfR antibody No.1 containing amino acid sequence 6
as the variable region, synthetic sequence 165acgcgtgccg ccaccatggg
ctggagctgg attctgctgt tcctcctgag cgtgacagca 60ggagtgcaca gcgacatcca
ggtcacacag tcaccaagtt ttctgagcgc aagcgtgggc 120gacagggtca
ctatcacatg caaggcaagc caggacgtga actccgcagt ggcctggttc
180cagcagaagc cagggaaagc acccaagctg ctgatctatt ggacctctac
aaggcacacc 240ggtgtcccag atcggttctc aggttccggc agcggaacag
tgtatactct gaccatttcc 300agcctgcagc ctgaagactt cgctacttac
tattgccagc agcattactc caccccaaga 360acatttggcg gagggactaa
agtggagatc aagaggaccg tggccgctcc ctccgtcttc 420atttttcccc
ctagcgacga acagctgaag agtggcacag cctcagtggt ctgtctgctg
480aacaatttct accctaggga ggctaaagtg cagtggaagg tcgataacgc
actgcagtct 540ggaaatagtc aggagtcagt gacagaacag gactccaaag
atagcactta ttctctgtct 600agtacactga ctctgagcaa ggccgattac
gaaaagcaca aagtgtatgc ttgcgaagtc 660acccatcagg ggctgtcatc
accagtcacc aagtcattca atagaggcga gtgctaagcg 720gccgc
725166116PRTArtificial SequenceAmino acid sequence 1 of the
variable region of the heavy chain of humanized anti-hTfR antibody
No.1 166Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Asn Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr
Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asn Arg Tyr Asp Glu Asp
Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115167116PRTArtificial SequenceAmino acid sequence 2 of the
variable region of the heavy chain of humanized anti-hTfR antibody
No.1 167Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Asn Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr
Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asn Arg Tyr Asp Glu Asp
Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115168116PRTArtificial SequenceAmino acid sequence 3 of the
variable region of the heavy chain of humanized anti-hTfR antibody
No.1 168Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser
Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Tyr Val 35 40 45Ser Asn Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr
Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Lys Asn Arg Tyr Asp Glu Asp
Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115169116PRTArtificial SequenceAmino acid sequence 4 of the
variable region of the heavy chain of humanized anti-hTfR antibody
No.1 169Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ala Asn Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr
Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Arg Tyr Asp Glu Asp
Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115170116PRTArtificial SequenceAmino acid sequence 5 of the
variable region of the heavy chain of humanized anti-hTfR antibody
No.1 170Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Val Trp Val 35 40 45Ser Asn Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr
Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Arg Tyr Asp Glu Asp
Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115171116PRTArtificial SequenceAmino acid sequence 6 of the
variable region of the heavy chain of humanized anti-hTfR antibody
No.1 171Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser
Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Leu Val 35 40 45Ala Asn Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr
Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Asn Asn Arg Tyr Asp Glu Asp
Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser
115172446PRTArtificial SequenceAmino acid sequence of the heavy
chain of humanized anti-hTfR antibody No.1 containing amino acid
sequence 6 as the variable region 172Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Leu Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45Ala Asn Ile Asn
Thr Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Thr Asn Asn Arg Tyr Asp Glu Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala 115 120 125Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu 130 135 140Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 180 185 190Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 195 200
205Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val 260 265 270Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315
320Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro 340 345 350Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
4451731421DNAArtificial SequenceNucleotide sequence encoding the
amino acid sequence of the heavy chain of humanized anti-hTfR
antibody No.1 containing amino acid sequence 6 as the variable
region, synthetic sequence 173acgcgtgccg ccaccatggg ctggagctgg
attctgctgt tcctcctgag cgtgacagca 60ggagtgcaca gcgaagtgca gctggtcgaa
tcaggggggg ggctggtgca gcctggaggc 120agcctgagac tgtcctgcgc
cgcttctggc ttgaccttta gcaactacgg gatgtcctgg 180gtgcggcagg
ctcctggcaa gggactggag ttggtggcca acatcaatac caacggcgga
240agtacatact atcccgattc agtgaagggc cggttcacca tcagcaggga
caacgccaag 300aacagcctgt atctgcagat gaactctctg agggccgagg
atacagccgt gtactattgc 360actaacaacc ggtacgacga ggactattgg
ggccagggca ccctggtgac agtgtctagc 420gcctctacca agggcccaag
cgtgtttcct ctggctccat cctctaaatc cacctctggc 480ggcacagccg
ctctgggctg tctggtgaag gattacttcc cagagcccgt gacagtgtct
540tggaacagcg gcgccctgac ctccggcgtg cacacatttc ctgctgtgct
gcagagctcc 600ggcctgtaca gcctgtctag cgtggtgacc gtgccatcct
ctagcctggg cacccagaca 660tatatctgca acgtgaatca caagcccagc
aatacaaagg tggataagaa ggtggagcca 720aagtcctgtg acaagaccca
cacatgcccc ccttgtcctg ctccagagct gctgggagga 780ccaagcgtgt
tcctgtttcc acccaagccc aaggataccc tgatgatctc tcggacccca
840gaggtgacat gcgtggtggt ggatgtgagc cacgaggacc ccgaggtgaa
gttcaactgg 900tatgtggacg gcgtggaggt gcacaatgct aagaccaagc
ccagggagga gcagtacaac 960tccacctata gagtggtgtc tgtgctgaca
gtgctgcacc aggattggct gaacggcaag 1020gagtataagt gcaaggtgtc
caataaggcc ctgcccgctc ctatcgagaa gaccatctct 1080aaggccaagg
gccagcccag agagcctcag gtgtacacac tgcctccatc ccgggatgag
1140ctgaccaaga accaggtgtc tctgacatgt ctggtcaagg gcttctatcc
ctctgacatc 1200gccgtggagt gggagagcaa tggccagcct gagaacaatt
acaagaccac accccctgtg 1260ctggattccg acggctcttt ctttctgtat
agcaagctga ccgtggacaa gtcccggtgg 1320cagcagggca acgtgttcag
ctgttccgtg atgcacgaag ctctgcataa tcactatact 1380cagaaatccc
tgtcactgtc acctggtaaa taagcggccg c 1421174107PRTArtificial
SequenceAmino acid sequence 1 of the variable region of the light
chain of humanized anti-hTfR antibody No.2 174Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30Leu Gln Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Tyr
Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Arg
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys 100
105175107PRTArtificial SequenceAmino acid sequence 2 of the
variable region of the light chain of humanized anti-hTfR antibody
No.2 175Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro
Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser
Asn Asn 20 25 30Leu Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu Ile 35 40 45Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Ser Asn Ser Trp Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105176107PRTArtificial SequenceAmino acid sequence
3 of the variable region of the light chain of humanized anti-hTfR
antibody No.2 176Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser
Val Thr Pro Lys1 5 10 15Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Ser Asn Asn 20 25 30Leu Gln Trp Tyr Gln Gln Lys Pro Asp Gln
Ser Pro Lys Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Asn Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln Ser Asn Ser Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105177107PRTArtificial SequenceAmino
acid sequence 4 of the variable region of the light chain of
humanized anti-hTfR antibody No.2 177Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30Leu Gln Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Tyr Ala Ser
Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Arg 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105178107PRTArtificial SequenceAmino acid sequence 5 of the
variable region of the light chain of humanized anti-hTfR antibody
No.2 178Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Asn Asn 20 25 30Leu Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45Tyr Tyr Ala Ser Gln Ser Ile Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Ser Asn Ser Trp Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105179107PRTArtificial SequenceAmino acid sequence
6 of the variable region of the light chain of humanized anti-hTfR
antibody No.2 179Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser
Val Thr Pro Lys1 5 10 15Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Ser Asn Asn 20 25 30Leu Gln Trp Tyr Gln Gln Lys Pro Asp Gln
Ser Pro Lys Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly
Ile Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Asn Ser Leu Glu Ala65 70 75 80Glu Asp Ala Ala Thr Tyr
Leu Cys Gln Gln Ser Asn Ser Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105180214PRTArtificial SequenceAmino
acid sequence of the light chain of humanized anti-hTfR antibody
No.2 containing amino acid sequence 6 as the variable region 180Glu
Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys1 5 10
15Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn
20 25 30Leu Gln Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu
Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser
Leu Glu Ala65 70 75 80Glu Asp Ala Ala Thr Tyr Leu Cys Gln Gln Ser
Asn Ser Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210181725DNAArtificial
SequenceNucleotide sequence encoding the amino acid sequence of the
light chain of humanized anti-hTfR antibody No.2 containing amino
acid sequence 6 as the variable region, synthetic sequence
181acgcgtgccg ccaccatggg ctggagctgg attctgctgt tcctcctgag
cgtgacagca 60ggagtgcaca gcgaaattgt gctgacccag tctcccgatt tccagtccgt
gacccccaag 120gagaaagtca ccatcacatg cagagcatca cagtccatta
gcaacaatct gcagtggtac 180cagcagaagc cagaccagag ccccaagctg
ctgatcaaat atgcctctca gagtatttca 240ggcatacctt ctaggttctc
cggtagcggc tctggaaccg actttactct gaccatcaac 300agtctggagg
ctgaagatgc cgctacatac ttgtgccagc agagtaattc atggcctagg
360acctttggcc aggggacaaa ggtggagatc aaaaggactg tggcagcccc
aagtgtcttc 420atttttcccc cttcagacga acagctgaag agcggcacag
catctgtggt ctgtctgctg 480aacaatttct acccacggga ggctaaggtg
cagtggaaag tcgataacgc actgcagtcc 540ggaaatagcc aggagtctgt
gactgaacag gacagtaagg attcaaccta ttccctgtcc 600agcacactga
ctctgagcaa agccgattac gagaagcaca aagtgtatgc ttgcgaagtc
660acacatcagg ggctgtctag tcccgtgact aagtctttta ataggggtga
atgttaagcg 720gccgc 725182120PRTArtificial SequenceAmino acid
sequence 1 of the variable region of the heavy chain of humanized
anti-hTfR antibody No.2 182Gln 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 Asp Tyr 20 25 30Val Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Val Ile Ser Thr Tyr Tyr
Gly His Gly Ser Tyr Asn Gln Arg Phe 50 55 60Lys Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly
Gly Tyr Gly Ser Ser Ser Leu Ala Gly Asn Phe Asp Val 100 105 110Trp
Gly Gln Gly Thr Leu Val Thr 115 120183120PRTArtificial
SequenceAmino acid sequence 2 of the variable region of the heavy
chain of humanized anti-hTfR antibody No.2 183Gln 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 Asp Tyr 20 25 30Val Met His
Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45Gly Val
Ile Ser Thr Tyr Tyr Gly His Gly Ser Tyr Asn Gln Arg Phe 50 55 60Lys
Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala 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 Gly Gly Tyr Gly Ser Ser Ser Leu Ala Gly Asn Phe Asp
Val 100 105 110Trp Gly Gln Gly Thr Leu Val Thr 115
120184120PRTArtificial SequenceAmino acid sequence 3 of the
variable region of the heavy chain of humanized anti-hTfR antibody
No.2 184Gln 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
Asp Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45Gly Val Ile Ser Thr Tyr Tyr Gly His Gly Ser Tyr
Asn Gln Arg Phe 50 55 60Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser
Thr Ser Ala Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Gly Gly Tyr Gly Ser Ser
Ser Leu Ala Gly Asn Phe Asp Val 100 105 110Trp Gly Gln Gly Thr Leu
Val Thr 115 120185120PRTArtificial SequenceAmino acid sequence 4 of
the variable region of the heavy chain of humanized anti-hTfR
antibody No.2 185Gln 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 Asp Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Val Ile Ser Thr Tyr Tyr Gly His
Gly Ser Tyr Asn Gln Arg Phe 50 55 60Lys Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr
Gly Ser Ser Ser Leu Ala Gly Asn Phe Asp Val 100 105 110Trp Gly Gln
Gly Thr Thr Val Thr 115 120186120PRTArtificial SequenceAmino acid
sequence 5 of the variable region of the heavy chain of humanized
anti-hTfR antibody No.2 186Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Asp Tyr 20 25 30Val Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Val Ile Ser Thr Tyr Tyr
Gly His Gly Ser Tyr Asn Gln Arg Phe 50 55 60Lys Gly Arg Val Thr Ile
Thr Ala Asp Lys 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 Gly
Gly Tyr Gly Ser Ser Ser Leu Ala Gly Asn Phe Asp Val 100 105 110Trp
Gly Gln Gly Thr Val Val Thr 115 120187120PRTArtificial
SequenceAmino acid sequence 6 of the variable region of the heavy
chain of humanized anti-hTfR antibody No.2 187Gln 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 Asp Tyr 20 25 30Val Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Val
Ile Ser Thr Tyr Tyr Gly His Gly Ser Tyr Asn Gln Arg Phe 50 55 60Lys
Gly Arg Ala Thr Met Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75
80Met Glu Leu Val Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95Val Arg Gly Gly Tyr Gly Ser Ser Ser Leu Ala Gly Asn Phe Asp
Val 100
105 110Trp Gly Gln Gly Thr Thr Val Thr 115 120188453PRTArtificial
SequenceAmino acid sequence of the heavy chain of humanized
anti-hTfR antibody No.2 containing amino acid sequence 6 as the
variable region 188Gln 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 Asp Tyr 20 25 30Val Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Ser Thr Tyr Tyr Gly His
Gly Ser Tyr Asn Gln Arg Phe 50 55 60Lys Gly Arg Ala Thr Met Thr Val
Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Val Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg Gly Gly Tyr
Gly Ser Ser Ser Leu Ala Gly Asn Phe Asp Val 100 105 110Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135
140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu225 230 235 240Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250
255Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val 275 280 285Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser 290 295 300Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu305 310 315 320Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375
380Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr385 390 395 400Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu 405 410 415Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser 420 425 430Val Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser 435 440 445Leu Ser Pro Gly Lys
4501891442DNAArtificial SequenceNucleotide sequence encoding the
amino acid sequence of the heavy chain of humanized anti-hTfR
antibody No.2 containing amino acid sequence 6 as the variable
region, synthetic sequence 189acgcgtgccg ccaccatggg ctggagctgg
attctgctgt tcctcctgag cgtgacagca 60ggagtgcaca gccaggtgca gctggtccag
tcaggagccg aagtgaaaaa gcccggagcc 120tcagtcaaag tgtcttgtaa
agcatcaggt tatacattta cagactacgt catgcactgg 180gtgaggcagg
cacctggaca gggtctggaa tggatcggcg tgatctccac ttactatggc
240catggaagct acaaccagag attcaagggc agggcgacaa tgactgtaga
caaatcaatt 300tccactgctt atatggagct ggtaaggctg cggtccgacg
ataccgctgt gtactattgc 360gtacgaggag gatacggctc cagctctctg
gctggtaatt tcgatgtgtg ggggcagggt 420accacagtca ccgtgagttc
agcaagcaca aagggcccat ctgtgtttcc actggccccc 480tccagcaaaa
gcacctctgg gggtacagcc gctctgggat gtctggtgaa ggattatttc
540ccagagccag tcaccgtgtc ctggaacagc ggagccctga catctggagt
ccacactttt 600ccagctgtgc tgcagtctag tgggctgtac tccctgtcat
ccgtggtcac tgtccccagc 660tctagtctgg gtacccagac atatatctgc
aacgtgaatc acaagccatc taataccaaa 720gtcgacaaga aagtggaacc
caagtcctgt gataaaactc atacctgccc cccttgtcct 780gcaccagagc
tgctgggagg accatccgtg ttcctgtttc cacccaagcc taaagacacc
840ctgatgatta gccgaactcc cgaagtcacc tgcgtggtcg tggacgtgtc
tcacgaggac 900cctgaagtca agtttaactg gtacgtggat ggcgtcgagg
tgcataatgc taagacaaaa 960ccccgagagg aacagtacaa cagtacatat
cgtgtcgtgt cagtgctgac cgtcctgcat 1020caggactggc tgaacgggaa
ggaatataag tgcaaagtgt ccaataaggc actgcccgcc 1080cctatcgaga
aaaccattag caaggccaaa ggacagccta gggaaccaca ggtgtacaca
1140ctgcctccat cccgggacga gctgactaag aaccaggtca gcctgacctg
tctggtgaaa 1200ggcttctatc cttcagatat cgctgtggag tgggaaagta
atggacagcc agagaacaat 1260tacaagacta ccccccctgt gctggactct
gatgggagtt tctttctgta ttctaagctg 1320accgtggata aaagtcggtg
gcagcagggt aatgtcttta gttgttcagt gatgcacgaa 1380gcactgcaca
accactacac ccagaaatca ctgtcactgt caccagggaa ataagcggcc 1440gc
1442190112PRTArtificial SequenceAmino acid sequence 1 of the
variable region of the light chain of humanized anti-hTfR antibody
No.3 190Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu
Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro
Gly Gln Ser 35 40 45Pro Arg Arg Leu Ile Tyr Lys 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 Ser Gln Ser 85 90 95Thr His Val Pro Trp Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 105 110191112PRTArtificial
SequenceAmino acid sequence 2 of the variable region of the light
chain of humanized anti-hTfR antibody No.3 191Asp Ile 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 Leu Val His Ser 20 25 30Asn Gly Asn
Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln
Leu Leu Ile Tyr Lys 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 Ser Gln Ser
85 90 95Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 110192112PRTArtificial SequenceAmino acid sequence 3 of
the variable region of the light chain of humanized anti-hTfR
antibody No.3 192Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro
Val Thr Leu Gly1 5 10 15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Leu Gln
Gln Arg Pro Gly Gln Pro 35 40 45Pro Arg Leu Leu Ile Tyr Lys Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ala
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Val Pro Trp
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110193112PRTArtificial SequenceAmino acid sequence 4 of the
variable region of the light chain of humanized anti-hTfR antibody
No.3 193Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro
Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys 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 Ser Gln Ser 85 90 95Thr His Val Pro Trp Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105 110194112PRTArtificial
SequenceAmino acid sequence 5 of the variable region of the light
chain of humanized anti-hTfR antibody No.3 194Asp Ile Val Met Thr
Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn
Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln
Leu Leu Ile Tyr Lys 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 Ser Gln Ser
85 90 95Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile
Lys 100 105 110195112PRTArtificial SequenceAmino acid sequence 6 of
the variable region of the light chain of humanized anti-hTfR
antibody No.3 195Asp Ile 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 Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys 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 Phe Phe Cys Ser Gln Ser 85 90 95Thr His Val Pro Trp
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
110196219PRTArtificial SequenceAmino acid sequence of the light
chain of humanized anti-hTfR antibody No.3 containing amino acid
sequence 2 as the variable region 196Asp Ile 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 Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr
Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu
Ile Tyr Lys 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 Ser Gln Ser 85 90
95Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val 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 215197740DNAArtificial
SequenceNucleotide sequence encoding the amino acid sequence of the
light chain of humanized anti-hTfR antibody No.3 containing amino
acid sequence 2 as the variable region, synthetic sequence
197acgcgtgccg ccaccatggg ctggagctgg attctgctgt tcctcctgag
cgtgacagca 60ggagtgcaca gcgacatcgt gatgacccag actcccctga gcctgagcgt
gacacctggc 120cagcctgcca gcatcagctg cagaagctct cagagcctgg
tgcacagcaa cggcaacacc 180tacctgcact ggtatctgca gaagcccggc
cagagccctc agctgctgat ctacaaggtg 240tccaacagat tcagcggcgt
gcccgacaga ttctccggca gcggctctgg caccgacttc 300accctgaaga
tttccagagt ggaagccgag gacgtgggcg tgtactactg cagccagagc
360acccacgtgc cctggacatt cggccagggc accaaggtgg aaatcaagag
aaccgtggcc 420gctcccagcg tgttcatctt cccacctagc gacgagcagc
tgaagtccgg cacagcctct 480gtcgtgtgcc tgctgaacaa cttctacccc
cgcgaggcca aggtgcagtg gaaggtggac 540aacgccctgc agagcggcaa
cagccaggaa agcgtgaccg agcaggactc caaggacagc 600acctacagcc
tgagcagcac cctgaccctg agcaaggccg actacgagaa gcacaaggtg
660tacgcctgcg aagtgaccca ccagggcctg tctagccccg tgaccaagag
cttcaacaga 720ggcgagtgct aagcggccgc 740198219PRTArtificial
SequenceAmino acid sequence of the light chain of humanized
anti-hTfR antibody No.3 containing amino acid sequence 4 as the
variable region 198Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys 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 Ser Gln Ser 85 90 95Thr His Val Pro Trp
Thr Phe Gly Gln Gly Thr Lys Val 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 215199740DNAArtificial SequenceNucleotide
sequence encoding the amino acid sequence of the light chain of
humanized anti-hTfR antibody No.3 containing amino acid sequence 4
as the variable region, synthetic sequence 199acgcgtgccg ccaccatggg
ctggagctgg attctgctgt tcctcctgag cgtgacagca 60ggagtgcaca gcgacatcgt
gatgacccag agccccctga gcctgcctgt gacacctggc 120gagcctgcca
gcatcagctg cagatctagc cagagcctgg tgcacagcaa cggcaacacc
180tacctgcact ggtatctgca gaagcccggc cagagccctc agctgctgat
ctacaaggtg 240tccaacagat tcagcggcgt gcccgacaga ttctccggca
gcggctctgg caccgacttc 300accctgaaga tctccagagt ggaagccgag
gacgtgggcg tgtactactg cagccagagc 360acccacgtgc cctggacatt
cggccagggc accaaggtgg aaatcaagag aaccgtggcc 420gctcccagcg
tgttcatctt cccacctagc gacgagcagc tgaagtccgg cacagcctct
480gtcgtgtgcc tgctgaacaa cttctacccc cgcgaggcca aggtgcagtg
gaaggtggac 540aacgccctgc agagcggcaa cagccaggaa agcgtgaccg
agcaggactc caaggacagc 600acctacagcc tgagcagcac cctgaccctg
agcaaggccg actacgagaa gcacaaggtg 660tacgcctgcg aagtgaccca
ccagggcctg tctagccccg tgaccaagag cttcaacaga 720ggcgagtgct
aagcggccgc 740200219PRTArtificial SequenceAmino acid sequence of
the light chain of humanized anti-hTfR antibody No.3 containing
amino acid sequence 5 as the variable region 200Asp Ile Val Met Thr
Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn
Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln
Leu Leu Ile Tyr Lys 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 Ser Gln Ser
85 90
95Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Arg 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 215201740DNAArtificial
SequenceNucleotide sequence encoding the amino acid sequence of the
light chain of humanized anti-hTfR antibody No.3 containing amino
acid sequence 5 as the variable region, synthetic sequence
201acgcgtgccg ccaccatggg ctggagctgg attctgctgt tcctcctgag
cgtgacagca 60ggagtgcaca gcgacatcgt gatgacccag acacccctga gcctgcctgt
gacacctggc 120gagcctgcca gcatcagctg cagatctagc cagagcctgg
tgcacagcaa cggcaacacc 180tacctgcact ggtatctgca gaagcccggc
cagagccctc agctgctgat ctacaaggtg 240tccaacagat tcagcggcgt
gcccgacaga ttctccggca gcggctctgg caccgacttc 300accctgaaga
tctccagagt ggaagccgag gacgtgggcg tgtactactg cagccagagc
360acccacgtgc cctggacatt cggccagggc accaggctgg aaatcaagag
aaccgtggcc 420gctcccagcg tgttcatctt cccacctagc gacgagcagc
tgaagtccgg cacagcctct 480gtcgtgtgcc tgctgaacaa cttctacccc
cgcgaggcca aggtgcagtg gaaggtggac 540aacgccctgc agagcggcaa
cagccaggaa agcgtgaccg agcaggactc caaggacagc 600acctacagcc
tgagcagcac cctgaccctg agcaaggccg actacgagaa gcacaaggtg
660tacgcctgcg aagtgaccca ccagggcctg tctagccccg tgaccaagag
cttcaacaga 720ggcgagtgct aagcggccgc 740202219PRTArtificial
SequenceAmino acid sequence of the light chain of humanized
anti-hTfR antibody No.3 containing amino acid sequence 6 as the
variable region 202Asp Ile 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 Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys 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 Phe Phe Cys Ser Gln Ser 85 90 95Thr His Val Pro Trp
Thr Phe Gly Gln Gly Thr Lys Val 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 215203740DNAArtificial SequenceNucleotide
sequence encoding the amino acid sequence of the light chain of
humanized anti-hTfR antibody No.3 containing amino acid sequence 6
as the variable region, synthetic sequence 203acgcgtgccg ccaccatggg
ctggagctgg attctgctgt tcctcctgag cgtgacagca 60ggagtgcaca gcgacatcgt
gatgacccag actcccctga gcctgagcgt gacacctggc 120cagcctgcca
gcatcagctg cagatccagc cagagcctgg tgcacagcaa cggcaacacc
180tacctgcact ggtatctgca gaagcccggc cagagccctc agctgctgat
ctacaaggtg 240tccaacagat tcagcggcgt gcccgacaga ttctccggca
gcggctctgg caccgacttc 300accctgaaga tttccagagt ggaagccgag
gacgtgggcg tgttcttctg cagccagagc 360acccacgtgc cctggacatt
cggccagggc accaaggtgg aaatcaagag aaccgtggcc 420gctcccagcg
tgttcatctt cccacctagc gacgagcagc tgaagtccgg cacagcctct
480gtcgtgtgcc tgctgaacaa cttctacccc cgcgaggcca aggtgcagtg
gaaggtggac 540aacgccctgc agagcggcaa cagccaggaa agcgtgaccg
agcaggactc caaggacagc 600acctacagcc tgagcagcac cctgaccctg
agcaaggccg actacgagaa gcacaaggtg 660tacgcctgcg aagtgaccca
ccagggcctg tctagccccg tgaccaagag cttcaacaga 720ggcgagtgct
aagcggccgc 740204118PRTArtificial SequenceAmino acid sequence 1 of
the variable region of the heavy chain of humanized anti-hTfR
antibody No.3 204Gln 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 30Trp Leu Gly Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr
Pro Thr Tyr Ser Glu Lys Phe 50 55 60Lys Val Arg Val Thr Met Thr Arg
Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Asn
Tyr Asp Glu Val Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr
Val Ser Ser 115205118PRTArtificial SequenceAmino acid sequence 2 of
the variable region of the heavy chain of humanized anti-hTfR
antibody No.3 205Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr
Ser Phe Thr Asn Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln Met Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr
Pro Thr Tyr Ser Glu Lys Phe 50 55 60Lys Val Gln Val Thr Ile Ser Ala
Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Asn
Tyr Asp Glu Val Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr
Val Ser Ser 115206118PRTArtificial SequenceAmino acid sequence 3 of
the variable region of the heavy chain of humanized anti-hTfR
antibody No.3 206Gln 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 30Trp Leu Gly Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr
Pro Thr Tyr Ser Glu Lys Phe 50 55 60Lys Val Arg Val Thr Met Thr Arg
Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Asn
Tyr Asp Glu Val Ala Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr
Val Ser Ser 115207118PRTArtificial SequenceAmino acid sequence 4 of
the variable region of the heavy chain of humanized anti-hTfR
antibody No.3 207Gln 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 30Trp Leu Gly Trp Val Arg Gln Ala Pro Gly
Gln Arg Leu Glu Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr
Pro Thr Tyr Ser Glu Lys Phe 50 55 60Lys Val Arg Val Thr Ile Thr Arg
Asp Thr Ser Ala 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 Gly Asn
Tyr Asp Glu Val Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr
Val Ser Ser 115208118PRTArtificial SequenceAmino acid sequence 5 of
the variable region of the heavy chain of humanized anti-hTfR
antibody No.3 208Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
Thr Phe Ser Asn Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr
Pro Thr Tyr Ser Glu Lys Phe 50 55 60Lys Val Arg Val Thr Ile Thr Ala
Asp Lys 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 Gly Asn
Tyr Asp Glu Val Ala Tyr Trp Gly Gln Gly Thr 100 105 110Val Val Thr
Val Ser Ser 115209118PRTArtificial SequenceAmino acid sequence 6 of
the variable region of the heavy chain of humanized anti-hTfR
antibody No.3 209Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr
Ser Phe Met Asn Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln Met Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr
Pro Thr Tyr Ser Glu Lys Phe 50 55 60Lys Val Lys Ala Ile Ile Ser Ala
Asp Thr Ser Ile Ser Thr Val Tyr65 70 75 80Leu Gln Leu Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met Tyr Phe Cys 85 90 95Ala Arg Ser Gly Asn
Tyr Asp Glu Val Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr
Val Ser Ser 115210448PRTArtificial SequenceAmino acid sequence of
the heavy chain of humanized anti-hTfR antibody No.3 containing
amino acid sequence 2 as the variable region 210Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile
Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30Trp Leu Gly
Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Asp
Ile Tyr Pro Gly Gly Asp Tyr Pro Thr Tyr Ser Glu Lys Phe 50 55 60Lys
Val Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75
80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Ser Gly Asn Tyr Asp Glu Val 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 Lys Thr
210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
4452111427DNAArtificial SequenceNucleotide sequence encoding the
amino acid sequence of the heavy chain of humanized anti-hTfR
antibody No.3 containing amino acid sequence 2 as the variable
region, synthetic sequence 211acgcgtgccg ccaccatggg ctggagctgg
attctgctgt tcctcctgag cgtgacagca 60ggagtgcaca gcgaggtgca actagtgcag
tctggagcag aggtgaaaaa gcccggggag 120tctctgaaga tttcctgtaa
gggttctgga tacagcttta ccaactactg gctgggatgg 180gtgcgccaga
tgcccgggaa aggcctggag tggatggggg acatctaccc cggcggagac
240taccctacat acagcgagaa gttcaaggtc caggtcacca tctcagccga
caagtccatc 300agcaccgcct acctgcagtg gagcagcctg aaggcctcgg
acaccgccat gtattactgt 360gcgagatcag gcaattacga cgaagtggcc
tactggggcc aaggaaccct ggtcaccgtc 420tcctcagcta gcaccaaggg
cccatcggtc ttccccctgg caccctcctc caagagcacc 480tctgggggca
cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg
540gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc
tgtcctacag 600tcctcaggac tctactccct cagcagcgtg gtgaccgtgc
cctccagcag cttgggcacc 660cagacctaca tctgcaacgt gaatcacaag
cccagcaaca ccaaggtgga caagaaagtt 720gagcccaaat cttgtgacaa
aactcacacg tgcccaccgt gcccagcacc tgaactcctg 780ggaggtccgt
cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg
840acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga
ggtcaagttc 900aactggtacg tggacggcgt ggaggtgcat aatgccaaga
caaagccgcg ggaggagcag 960tacaacagca cgtaccgggt ggtcagcgtc
ctcaccgtcc tgcaccagga ctggctgaat 1020ggcaaggagt acaagtgcaa
ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1080atctccaaag
ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg
1140gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt
ctatcccagc 1200gacatcgccg tggagtggga gagcaatggg cagccggaga
acaactacaa gaccacgcct 1260cccgtgctgg actccgacgg ctccttcttc
ctctacagca agctcaccgt ggacaagagc 1320aggtggcagc aggggaacgt
cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1380tacacgcaga
agagcctctc cctgtctccg ggtaaataag cggccgc 1427212445PRTArtificial
SequenceAmino acid sequence of the heavy chain (IgG4) of humanized
anti-hTfR antibody No.3 containing amino acid sequence 2 as the
variable region 212Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr
Ser Phe Thr Asn Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln Met Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr
Pro Thr Tyr Ser Glu Lys Phe 50 55 60Lys Val Gln Val Thr Ile Ser Ala
Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu
Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Asn
Tyr Asp Glu Val 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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr
Cys Asn Val
Asp His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro Cys 210 215 220Pro Pro Cys Pro Ala Pro Glu
Phe Leu Gly Gly Pro Ser Val Phe Leu225 230 235 240Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265
270Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
Val Leu 290 295 300Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys305 310 315 320Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly385 390
395 400Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln 405 410 415Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn 420 425 430His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 435 440 4452131418DNAArtificial SequenceNucleotide sequence
encoding the amino acid sequence of the heavy chain (IgG4) of
humanized anti-hTfR antibody No.3 containing amino acid sequence 2
as the variable region, synthetic sequence 213acgcgtgccg ccaccatggg
ctggagctgg attctgctgt tcctcctgag cgtgacagca 60ggagtgcaca gcgaggtgca
actagtgcag tctggagcag aggtgaaaaa gcccggggag 120tctctgaaga
tttcctgtaa gggttctgga tacagcttta ccaactactg gctgggatgg
180gtgcgccaga tgcccgggaa aggcctggag tggatggggg acatctaccc
cggcggagac 240taccctacat acagcgagaa gttcaaggtc caggtcacca
tctcagccga caagtccatc 300agcaccgcct acctgcagtg gagcagcctg
aaggcctcgg acaccgccat gtattactgt 360gcgagatcag gcaattacga
cgaagtggcc tactggggcc aaggaaccct ggtcaccgtc 420tcctcagcta
gcaccaaggg cccatcggtc ttccccctgg cgccctgctc caggagcacc
480tccgagagca cagccgccct gggctgcctg gtcaaggact acttccccga
accggtgacg 540gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca
ccttcccggc tgtcctacag 600tcctcaggac tctactccct cagcagcgtg
gtgaccgtgc cctccagcag cttgggcacg 660aagacctaca cctgcaacgt
agatcacaag cccagcaaca ccaaggtgga caagagagtt 720gagtccaaat
atggtccccc atgcccacca tgcccagcac ctgagttcct ggggggtcca
780tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg
gacccctgag 840gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg
aggtccagtt caactggtac 900gtggatggcg tggaggtgca taatgccaag
acaaagccgc gggaggagca gttcaacagc 960acgtaccgtg tggtcagcgt
cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 1020tacaagtgca
aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa
1080gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca
ggaggagatg 1140accaagaacc aggtcagcct gacctgcctg gtcaaaggct
tctaccccag cgacatcgcc 1200gtggagtggg agagcaatgg gcagccggag
aacaactaca agaccacgcc tcccgtgctg 1260gactccgacg gctccttctt
cctctacagc aggctcaccg tggacaagag caggtggcag 1320gaggggaatg
tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag
1380aagagcctct ccctgtctcc gggtaaataa gcggccgc
141821443DNAArtificial SequencePrimer hTfR5', synthetic sequence
214ccgacgcgtc gccaccatga tggatcaagc tagatcagca ttc
4321559DNAArtificial SequencePrimer hTfR3', synthetic sequence
215ataatgcggc cgcttaatga tgatgatgat gatgaaactc attgtcaatg tcccaaacg
5921620DNAArtificial SequencePrimer Hyg-Sfi5', synthetic sequence
216gaggccgcct cggcctctga 2021729DNAArtificial SequencePrimer
Hyg-BstX3', synthetic sequence 217aaccatcgtg atgggtgcta ttcctttgc
29218108PRTArtificial SequenceAmino acid sequence of the variable
region of the light chain of anti-hTfR antibody No.1 218Asp Ile Val
Leu Thr Gln Ser Ser Lys Phe Met Ser Thr Ser Val Gly1 5 10 15Asp Arg
Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asn Ser Ala 20 25 30Val
Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40
45Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60Ser Gly Ser Gly Thr Val Tyr Thr Leu Thr Ile Ser Ser Val Gln
Ala65 70 75 80Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser
Thr Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105219116PRTArtificial SequenceAmino acid sequence of the
variable region of the heavy chain of anti-hTfR antibody No.1
219Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Asn
Tyr 20 25 30Gly Met Ser Trp Ile Arg Gln Thr Pro Asp Lys Arg Leu Glu
Leu Val 35 40 45Ala Asn Ile Asn Thr Asn Gly Gly Ser Thr Tyr Tyr Pro
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ser Glu Asp
Thr Ala Ile Tyr Tyr Cys 85 90 95Thr Asn Asn Arg Tyr Asp Glu Asp Tyr
Trp Gly Gln Gly Thr Ser Val 100 105 110Thr Val Ser Ser
115220108PRTArtificial SequenceAmino acid sequence of the variable
region of the light chain of anti-hTfR antibody No.2 220Asp Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10 15Asp Ser
Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30Leu
Gln Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40
45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu
Thr65 70 75 80Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser
Trp Pro Arg 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
100 105221123PRTArtificial SequenceAmino acid sequence of the
variable region of the heavy chain of anti-hTfR antibody No.2
221Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Val1
5 10 15Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30Val Met His Trp Val Arg Gln Ser His Ala Lys Ser Leu Glu
Trp Ile 35 40 45Gly Val Ile Ser Thr Tyr Tyr Gly His Gly Ser Tyr Asn
Gln Arg Phe 50 55 60Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser
Ser Thr Ala Tyr65 70 75 80Leu Glu Leu Val Arg Leu Thr Ser Glu Asp
Ser Ala Ile Tyr Tyr Cys 85 90 95Val Arg Gly Gly Tyr Gly Ser Ser Ser
Leu Ala Gly Asn Phe Asp Val 100 105 110Trp Gly Ala Gly Thr Thr Val
Thr Val Ser Ser 115 120222113PRTArtificial SequenceAmino acid
sequence of the variable region of the light chain of anti-hTfR
antibody No.3 222Asp Ile Val Met Thr Gln Thr Thr Leu Ser Leu Pro
Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Val His Ser 20 25 30Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Leu Leu Ile Tyr Lys 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 Leu Gly Val Phe Phe Cys Ser Gln Ser 85 90 95Thr His Val Pro Trp
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
110Arg223118PRTArtificial SequenceAmino acid sequence of the
variable region of the heavy chain of anti-hTfR antibody No.3
223Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Thr1
5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Met Asn
Tyr 20 25 30Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu
Trp Ile 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr Pro Thr Tyr Ser
Glu Lys Phe 50 55 60Lys Val Lys Ala Ile Leu Thr Ala Asp Thr Ser Ser
Ser Ser Val Tyr65 70 75 80Leu His Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Ser Gly Asn Tyr Asp Glu Val
Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Thr
115224106PRTArtificial SequenceAmino acid sequence of the variable
region of the light chain of anti-hTfR antibody No.4 224Asp Ile Val
Leu Thr Gln Thr Pro Ala Ile Met Ser Ala Ser Pro Gly1 5 10 15Glu Lys
Val Thr Met Thr Cys Ser Ala Ser Ser Asn Val Asn Tyr Met 20 25 30His
Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 35 40
45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser
50 55 60Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala
Glu65 70 75 80Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Asn Gly Asn
Pro Tyr Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105225122PRTArtificial SequenceAmino acid sequence of the variable
region of the heavy chain of anti-hTfR antibody No.4 225Glu Val Gln
Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Thr1 5 10 15Ser Val
Arg Val Phe Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30Leu
Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Leu Ile Asn Pro Gly Ser Gly Gly Ile Ile Tyr Asn Glu Lys Phe
50 55 60Thr Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val
Tyr Phe Cys 85 90 95Ala Arg Ser Asn Tyr Tyr Gly Thr Thr Tyr Trp His
Phe Asp Val Trp 100 105 110Gly Ala Gly Thr Thr Val Thr Val Ser Ser
115 120226105PRTArtificial SequenceAmino acid sequence of the
variable region of the light chain of anti-hTfR antibody No.5
226Asp Ile Val Met Thr Gln Thr Pro Ala Ile Met Ser Ala Ser Pro Gly1
5 10 15Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser
Ile 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp
Ile Tyr 35 40 45Asp Thr Ser Thr Leu Ala Ser Gly Val Pro Val Arg Phe
Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser
Met Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg
Ser Ser Tyr Pro Thr Phe 85 90 95Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105227118PRTArtificial SequenceAmino acid sequence of the
variable region of the heavy chain of anti-hTfR antibody No.5
227Glu Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1
5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn
Phe 20 25 30Val Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Phe Phe Asn Pro His Lys Asn Gly Ala Glu Tyr Asn
Glu Lys Phe 50 55 60Gln Val Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser
Thr Thr Ala Tyr65 70 75 80Met Glu Leu Asn Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Phe Tyr Tyr Tyr Ser Met
Asp Tyr Trp Gly Gln Gly Thr 100 105 110Ser Val Thr Val Ser Ser
115228107PRTArtificial SequenceAmino acid sequence of the variable
region of the light chain of anti-hTfR antibody No.6 228Asp Ile Val
Leu Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Glu Arg
Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr 20 25 30Leu
Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile 35 40
45Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu
Ser65 70 75 80Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ser Ser
Tyr Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105229112PRTArtificial SequenceAmino acid sequence of the variable
region of the heavy chain of anti-hTfR antibody No.6 229Glu Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu
Ser Ile Thr Cys Ser Val Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30Gly
Val Tyr Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45Gly Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile
50 55 60Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe
Leu65 70 75 80Lys Leu Asn Ser Leu Gln Thr Ala Asp Thr Ala Thr Tyr
Tyr Cys Ala 85 90 95Lys Pro Asp Asp Val Trp Gly Ala Gly Thr Thr Val
Thr Val Ser Ser 100 105 110230106PRTArtificial SequenceAmino acid
sequence of the variable region of the light chain of anti-hTfR
antibody No.7 230Asp Ile Val Leu Thr Gln Ser Pro Val Ile Met Ser
Ala Ser Leu Gly1 5 10 15Glu Glu Ile Thr Leu Thr Cys Ser Ala Ser Ser
Ser Val Asn Tyr Ile 20 25 30His Trp Tyr Gln Gln Lys Ser Gly Thr Ser
Pro Lys Val Leu Ile Tyr 35 40 45Gln Thr Ser Asn Leu Ala Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser 50 55 60Arg Ser Gly Thr Phe Tyr Ser Leu
Lys Ile Ser Ser Val Glu Ala Glu65 70 75 80Asp Ala Ala Asp Tyr Tyr
Cys His Gln Trp Ser Ser Tyr Pro Trp Thr 85 90 95Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105231120PRTArtificial SequenceAmino acid
sequence of the variable region of the heavy chain of anti-hTfR
antibody No.7 231Glu Val Gln Leu Gln Glu Ser Gly Gly Ala Leu Val
Lys Pro Gly Gly1 5 10 15Ser Leu Lys Val Ser Cys Glu Ala Ser Gly Ile
Thr Phe Arg Asn Tyr 20 25 30Phe Met Ser Trp Val Arg Gln Thr Pro Glu
Lys Arg Leu Glu Trp Val 35 40 45Ala Thr Ile Ser Ser Ala Gly Gly Tyr
Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Gly Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu
Arg Ser Glu Asp Thr Ala Met Tyr Phe Cys 85 90 95Ala Arg Gln Glu Val
Pro Tyr Pro Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Ser
Val Ile Val Ser Ser 115 120232106PRTArtificial SequenceAmino acid
sequence of the variable region of the light chain of anti-hTfR
antibody No.8 232Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser
Ala Ser Leu Gly1 5 10 15Glu Glu Ile Thr Leu Thr Cys Ser Ala Ser Ser
Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Ser Gly Thr Ser
Pro Lys Val Leu Ile Tyr 35 40 45Gly Thr Ser Asn Leu Ala Ser Glu
Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Phe Tyr Ser
Leu Thr Ile Ser Ser Val Glu Ala Glu65 70 75 80Asp Ala Ala Asp Tyr
Tyr Cys His Gln Trp Ser Ser Tyr Pro Trp Thr 85 90 95Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 105233112PRTArtificial SequenceAmino
acid sequence of the variable region of the heavy chain of
anti-hTfR antibody No.8 233Glu Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser
Gly Phe Ser Leu Thr Thr Tyr 20 25 30Gly Val Ser Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Ile Ile Trp Gly Asp Gly
Ser Thr Asn Tyr Arg Ser Ala Leu Ile 50 55 60Ser Arg Leu Ser Ile Ser
Lys Asp Asn Ser Lys Asn Gln Val Phe Leu65 70 75 80Lys Leu Asn Ser
Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95Lys Pro Asp
Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 100 105
110234106PRTArtificial SequenceAmino acid sequence of the variable
region of the light chain of anti-hTfR antibody No.9 234Asp Ile Val
Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly1 5 10 15Glu Thr
Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Leu Met 20 25 30Phe
Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40
45Phe Thr Ser Tyr Arg Ala Ser Gly Val Pro Ile Arg Phe Ser Gly Ser
50 55 60Gly Ser Gly Thr Ser Phe Ser Leu Thr Ile Ser Asn Leu Glu Ala
Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asp
Pro Pro Thr 85 90 95Phe Gly Ser Gly Thr Lys Leu Glu Ile Glu 100
105235117PRTArtificial SequenceAmino acid sequence of the variable
region of the heavy chain of anti-hTfR antibody No.9 235Glu Val Gln
Leu Gln Glu Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly
Val Ser Trp Val Lys Gln Ala Pro Gly Lys Asp Leu Lys Trp Met 35 40
45Gly Trp Ile Tyr Thr Phe Thr Gly Glu Ala Thr Tyr Ile Asp Asp Phe
50 55 60Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Asn Thr Ala
Tyr65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr
Tyr Phe Cys 85 90 95Ser Arg Arg Asn Gly Ala Trp Phe Glu Asp Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ala
115236107PRTArtificial SequenceAmino acid sequence of the variable
region of the light chain of anti-hTfR antibody No.10 236Asp Ile
Val Met Thr Gln Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Glu
Arg Val Ser Leu Thr Cys Arg Pro Ser Gln Glu Ile Ser Gly Tyr 20 25
30Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser
Gly 50 55 60Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu
Glu Ser65 70 75 80Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala
Ser Tyr Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105237120PRTArtificial SequenceAmino acid sequence of the
variable region of the heavy chain of anti-hTfR antibody No.10
237Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1
5 10 15Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Ile Thr Phe Arg Asn
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu
Trp Val 35 40 45Ala Thr Ile Ser Ser Tyr Gly Gly Tyr Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Gly
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg Ser Glu Asp
Thr Ala Met Tyr His Cys 85 90 95Ala Arg Gln Glu Val Pro Tyr Pro Tyr
Pro Met Asp Asn Trp Gly Gln 100 105 110Gly Thr Ser Val Ile Val Ser
Ser 115 120238107PRTArtificial SequenceAmino acid sequence of the
variable region of the light chain of anti-hTfR antibody No.11
238Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1
5 10 15Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Arg Asn
Tyr 20 25 30Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu
Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn
Ser Val Glu Thr65 70 75 80Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln
Thr Asn Ser Trp Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105239118PRTArtificial SequenceAmino acid sequence of
the variable region of the heavy chain of anti-hTfR antibody No.11
239Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ser1
5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ile
Tyr 20 25 30Trp Ile Asn Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Gln Ile Tyr Pro Gly Asn Gly Asp Thr Asn Tyr Asn
Gly Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
Asn Thr Val Tyr65 70 75 80Met Arg Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Phe Cys 85 90 95Gly Arg Trp Gly Asp Asp Tyr Ala Met
Asp Phe Trp Gly Gln Gly Thr 100 105 110Ser Val Thr Val Ser Ser
115240107PRTArtificial SequenceAmino acid sequence of the variable
region of the light chain of anti-hTfR antibody No.12 240Asp Ile
Val Leu Thr Gln Ser His Lys Phe Met Ser Thr Val Ile Gly1 5 10 15Asp
Arg Val Ser Ile Thr Cys Lys Ala Ser His Asp Val Lys Thr Ala 20 25
30Val Ala Trp Tyr Gln Gln Thr Pro Gly Gln Ser Pro Lys Leu Leu Thr
35 40 45Tyr Trp Ser Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val
Gln Ala65 70 75 80Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Phe
Ser Thr Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105241119PRTArtificial SequenceAmino acid sequence of the
variable region of the heavy chain of anti-hTfR antibody No.12
241Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ala Arg Pro Gly Thr1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ala Gly Tyr Val Phe Ile Asn
Tyr 20 25 30Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Val Ile His Ser Gly Ser Gly Gly Thr Asn Tyr Asn
Asp Asn Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Arg Asp Val Ser Ser
Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Asp Asp
Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Arg Asn Phe Gly Asn Tyr Tyr
Phe Asp Tyr Trp Gly Gln Gly 100 105 110Thr Thr Leu Thr Val Ser Ser
115242107PRTArtificial SequenceAmino acid sequence of the variable
region of the light chain of anti-hTfR antibody No.13 242Asp Ile
Val Ile Thr Gln Thr Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10 15Asp
Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Arg Asn Tyr 20 25
30Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
35 40 45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val
Glu Thr65 70 75 80Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Thr Asn
Ser Trp Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105243118PRTArtificial SequenceAmino acid sequence of the
variable region of the heavy chain of anti-hTfR antibody No.13
243Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ser1
5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ile
Tyr 20 25 30Trp Ile Asn Trp Leu Lys Gln Arg Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Gln Ile Tyr Pro Gly Asn Gly Asp Thr Asn Tyr Asn
Gly Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
Asn Thr Val Tyr65 70 75 80Met Arg Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Phe Cys 85 90 95Gly Arg Trp Gly Asp Asp Tyr Ala Met
Asp Phe Trp Gly Gln Gly Thr 100 105 110Ser Val Thr Val Ser Ser
115244106PRTArtificial SequenceAmino acid sequence of the variable
region of the light chain of anti-hTfR antibody No.14 244Asp Ile
Val Leu Thr Gln Thr Pro Val Ile Met Ser Ala Tyr Pro Gly1 5 10 15Glu
Lys Val Thr Leu Thr Cys Ser Ala Ser Ser Asn Ile Asn Ser Ile 20 25
30His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly
Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
Ala Glu65 70 75 80Asp Ala Ala Ser Tyr Tyr Cys His Gln Arg Ser Gly
Tyr Pro Phe Thr 85 90 95Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100
105245118PRTArtificial SequenceAmino acid sequence of the variable
region of the heavy chain of anti-hTfR antibody No.14 245Glu Val
Gln Leu Gln Glu Ser Gly Pro Glu Leu Leu Lys Pro Gly Ala1 5 10 15Ser
Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Gly Tyr 20 25
30Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45Gly Phe Leu Asn Pro His Lys Asp Asp Ser Glu Tyr Asn Glu Lys
Phe 50 55 60Arg Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr
Ala Tyr65 70 75 80Met Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Gly Tyr Tyr Tyr Tyr Ser Met Asp Tyr
Trp Gly Gln Gly Thr 100 105 110Ser Val Thr Val Ser Ser
115246525PRTHomo sapiens 246Ser Glu Thr Gln Ala Asn Ser Thr Thr Asp
Ala Leu Asn Val Leu Leu1 5 10 15Ile Ile Val Asp Asp Leu Arg Pro Ser
Leu Gly Cys Tyr Gly Asp Lys 20 25 30Leu Val Arg Ser Pro Asn Ile Asp
Gln Leu Ala Ser His Ser Leu Leu 35 40 45Phe Gln Asn Ala Phe Ala Gln
Gln Ala Val Cys Ala Pro Ser Arg Val 50 55 60Ser Phe Leu Thr Gly Arg
Arg Pro Asp Thr Thr Arg Leu Tyr Asp Phe65 70 75 80Asn Ser Tyr Trp
Arg Val His Ala Gly Asn Phe Ser Thr Ile Pro Gln 85 90 95Tyr Phe Lys
Glu Asn Gly Tyr Val Thr Met Ser Val Gly Lys Val Phe 100 105 110His
Pro Gly Ile Ser Ser Asn His Thr Asp Asp Ser Pro Tyr Ser Trp 115 120
125Ser Phe Pro Pro Tyr His Pro Ser Ser Glu Lys Tyr Glu Asn Thr Lys
130 135 140Thr Cys Arg Gly Pro Asp Gly Glu Leu His Ala Asn Leu Leu
Cys Pro145 150 155 160Val Asp Val Leu Asp Val Pro Glu Gly Thr Leu
Pro Asp Lys Gln Ser 165 170 175Thr Glu Gln Ala Ile Gln Leu Leu Glu
Lys Met Lys Thr Ser Ala Ser 180 185 190Pro Phe Phe Leu Ala Val Gly
Tyr His Lys Pro His Ile Pro Phe Arg 195 200 205Tyr Pro Lys Glu Phe
Gln Lys Leu Tyr Pro Leu Glu Asn Ile Thr Leu 210 215 220Ala Pro Asp
Pro Glu Val Pro Asp Gly Leu Pro Pro Val Ala Tyr Asn225 230 235
240Pro Trp Met Asp Ile Arg Gln Arg Glu Asp Val Gln Ala Leu Asn Ile
245 250 255Ser Val Pro Tyr Gly Pro Ile Pro Val Asp Phe Gln Arg Lys
Ile Arg 260 265 270Gln Ser Tyr Phe Ala Ser Val Ser Tyr Leu Asp Thr
Gln Val Gly Arg 275 280 285Leu Leu Ser Ala Leu Asp Asp Leu Gln Leu
Ala Asn Ser Thr Ile Ile 290 295 300Ala Phe Thr Ser Asp His Gly Trp
Ala Leu Gly Glu His Gly Glu Trp305 310 315 320Ala Lys Tyr Ser Asn
Phe Asp Val Ala Thr His Val Pro Leu Ile Phe 325 330 335Tyr Val Pro
Gly Arg Thr Ala Ser Leu Pro Glu Ala Gly Glu Lys Leu 340 345 350Phe
Pro Tyr Leu Asp Pro Phe Asp Ser Ala Ser Gln Leu Met Glu Pro 355 360
365Gly Arg Gln Ser Met Asp Leu Val Glu Leu Val Ser Leu Phe Pro Thr
370 375 380Leu Ala Gly Leu Ala Gly Leu Gln Val Pro Pro Arg Cys Pro
Val Pro385 390 395 400Ser Phe His Val Glu Leu Cys Arg Glu Gly Lys
Asn Leu Leu Lys His 405 410 415Phe Arg Phe Arg Asp Leu Glu Glu Asp
Pro Tyr Leu Pro Gly Asn Pro 420 425 430Arg Glu Leu Ile Ala Tyr Ser
Gln Tyr Pro Arg Pro Ser Asp Ile Pro 435 440 445Gln Trp Asn Ser Asp
Lys Pro Ser Leu Lys Asp Ile Lys Ile Met Gly 450 455 460Tyr Ser Ile
Arg Thr Ile Asp Tyr Arg Tyr Thr Val Trp Val Gly Phe465 470 475
480Asn Pro Asp Glu Phe Leu Ala Asn Phe Ser Asp Ile His Ala Gly Glu
485 490 495Leu Tyr Phe Val Asp Ser Asp Pro Leu Gln Asp His Asn Met
Tyr Asn 500 505 510Asp Ser Gln Gly Gly Asp Leu Phe Gln Leu Leu Met
Pro 515 520 525247973PRTArtificial SequenceAmino acid sequence of
the fusion protein of heavy chain of anti-hTfR antibody No.1
(humanized 6) and hI2S 247Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Leu Thr Phe Ser Asn Tyr 20 25 30Gly Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Leu Val 35 40 45Ala Asn Ile Asn Thr Asn Gly
Gly Ser Thr Tyr Tyr Pro Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Asn Asn
Arg Tyr Asp Glu Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120
125Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
130 135 140Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly145 150 155 160Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu 180 185 190Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser
Asn Thr 195 200 205Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr 210 215 220Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val 260 265 270Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 290 295
300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys305 310 315 320Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser 325 330 335Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro 340 345 350Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp385 390 395 400Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410
415Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
420 425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
Gly Ser 435 440 445Ser Glu Thr Gln Ala Asn Ser Thr Thr Asp Ala Leu
Asn Val Leu Leu 450 455 460Ile Ile Val Asp Asp Leu Arg Pro Ser Leu
Gly Cys Tyr Gly Asp Lys465 470 475 480Leu Val Arg Ser Pro Asn Ile
Asp Gln Leu Ala Ser His Ser Leu Leu 485 490 495Phe Gln Asn Ala Phe
Ala Gln Gln Ala Val Cys Ala Pro Ser Arg Val 500 505 510Ser Phe Leu
Thr Gly Arg Arg Pro Asp Thr Thr Arg Leu Tyr Asp Phe 515 520 525Asn
Ser Tyr Trp Arg Val His Ala Gly Asn Phe Ser Thr Ile Pro Gln 530 535
540Tyr Phe Lys Glu Asn Gly Tyr Val Thr Met Ser Val Gly Lys Val
Phe545 550 555 560His Pro Gly Ile Ser Ser Asn His Thr Asp Asp Ser
Pro Tyr Ser Trp 565 570 575Ser Phe Pro Pro Tyr His Pro Ser Ser Glu
Lys Tyr Glu Asn Thr Lys 580 585 590Thr Cys Arg Gly Pro Asp Gly Glu
Leu His Ala Asn Leu Leu Cys Pro 595 600 605Val Asp Val Leu Asp Val
Pro Glu Gly Thr Leu Pro Asp Lys Gln Ser 610 615 620Thr Glu Gln Ala
Ile Gln Leu Leu Glu Lys Met Lys Thr Ser Ala Ser625 630 635 640Pro
Phe Phe Leu Ala Val Gly Tyr His Lys Pro His Ile Pro Phe Arg 645 650
655Tyr Pro Lys Glu Phe Gln Lys Leu Tyr Pro Leu Glu Asn Ile Thr Leu
660 665 670Ala Pro Asp Pro Glu Val Pro Asp Gly Leu Pro Pro Val Ala
Tyr Asn 675 680 685Pro Trp Met Asp Ile Arg Gln Arg Glu Asp Val Gln
Ala Leu Asn Ile 690 695 700Ser Val Pro Tyr Gly Pro Ile Pro Val Asp
Phe Gln Arg Lys Ile Arg705 710 715 720Gln Ser Tyr Phe Ala Ser Val
Ser Tyr Leu Asp Thr Gln Val Gly Arg 725 730 735Leu Leu Ser Ala Leu
Asp Asp Leu Gln Leu Ala Asn Ser Thr Ile Ile 740 745 750Ala Phe Thr
Ser Asp His Gly Trp Ala Leu Gly Glu His Gly Glu Trp 755 760 765Ala
Lys Tyr Ser Asn Phe Asp Val Ala Thr His Val Pro Leu Ile Phe 770 775
780Tyr Val Pro Gly Arg Thr Ala Ser Leu Pro Glu Ala Gly Glu Lys
Leu785 790 795 800Phe Pro Tyr Leu Asp Pro Phe Asp Ser Ala Ser Gln
Leu Met Glu Pro 805 810 815Gly Arg Gln Ser Met Asp Leu Val Glu Leu
Val Ser Leu Phe Pro Thr 820 825 830Leu Ala Gly Leu Ala Gly Leu Gln
Val Pro Pro Arg Cys Pro Val Pro 835 840 845Ser Phe His Val Glu Leu
Cys Arg Glu Gly Lys Asn Leu Leu Lys His 850 855 860Phe Arg Phe Arg
Asp Leu Glu Glu Asp Pro Tyr Leu Pro Gly Asn Pro865 870 875 880Arg
Glu Leu Ile Ala Tyr Ser Gln Tyr Pro Arg Pro Ser Asp Ile Pro 885 890
895Gln Trp Asn Ser Asp Lys Pro Ser Leu Lys Asp Ile Lys Ile Met Gly
900 905 910Tyr Ser Ile Arg Thr Ile Asp Tyr Arg Tyr Thr Val Trp Val
Gly Phe 915 920 925Asn Pro Asp Glu Phe Leu Ala Asn Phe Ser Asp Ile
His Ala Gly Glu 930 935 940Leu Tyr Phe Val Asp Ser Asp Pro Leu Gln
Asp His Asn Met Tyr Asn945 950 955 960Asp Ser Gln Gly Gly Asp Leu
Phe Gln Leu Leu Met Pro 965 9702483002DNAArtificial
SequenceNucleotide sequence encoding the amino acid sequence of
fusion protein of heavy chain of anti-hTfR antibody No.1 (humanized
6) and hI2S, synthetic sequence 248acgcgtgccg ccaccatggg ctggagctgg
attctgctgt tcctcctgag cgtgacagca 60ggagtgcaca gcgaagtgca gctggtcgaa
tcaggggggg ggctggtgca gcctggaggc 120agcctgagac tgtcctgcgc
cgcttctggc ttgaccttta gcaactacgg gatgtcctgg 180gtgcggcagg
ctcctggcaa gggactggag ttggtggcca acatcaatac caacggcgga
240agtacatact atcccgattc agtgaagggc cggttcacca tcagcaggga
caacgccaag 300aacagcctgt atctgcagat gaactctctg agggccgagg
atacagccgt gtactattgc 360actaacaacc ggtacgacga ggactattgg
ggccagggca ccctggtgac agtgtctagc 420gcctctacca agggcccaag
cgtgtttcct ctggctccat cctctaaatc cacctctggc 480ggcacagccg
ctctgggctg tctggtgaag gattacttcc cagagcccgt gacagtgtct
540tggaacagcg gcgccctgac ctccggcgtg cacacatttc ctgctgtgct
gcagagctcc 600ggcctgtaca gcctgtctag cgtggtgacc gtgccatcct
ctagcctggg cacccagaca 660tatatctgca acgtgaatca caagcccagc
aatacaaagg tggataagaa ggtggagcca 720aagtcctgtg acaagaccca
cacatgcccc ccttgtcctg ctccagagct gctgggagga 780ccaagcgtgt
tcctgtttcc acccaagccc aaggataccc tgatgatctc tcggacccca
840gaggtgacat gcgtggtggt ggatgtgagc cacgaggacc ccgaggtgaa
gttcaactgg 900tatgtggacg gcgtggaggt gcacaatgct aagaccaagc
ccagggagga gcagtacaac 960tccacctata gagtggtgtc tgtgctgaca
gtgctgcacc aggattggct gaacggcaag 1020gagtataagt gcaaggtgtc
caataaggcc ctgcccgctc ctatcgagaa gaccatctct 1080aaggccaagg
gccagcccag agagcctcag gtgtacacac tgcctccatc ccgggatgag
1140ctgaccaaga accaggtgtc tctgacatgt ctggtcaagg gcttctatcc
ctctgacatc 1200gccgtggagt gggagagcaa tggccagcct gagaacaatt
acaagaccac accccctgtg 1260ctggattccg acggctcttt ctttctgtat
agcaagctga ccgtggacaa gtcccggtgg 1320cagcagggca acgtgttcag
ctgttccgtg atgcacgaag ctctgcataa tcactatact 1380cagaaatccc
tgtcactgtc acctggtaaa ggatcttccg aaacgcaggc caactcgacc
1440acagatgctc tgaacgttct tctcatcatc gtggatgacc tgcgcccctc
cctgggctgt 1500tatggggata agctggtgag gtccccaaat attgaccaac
tggcatccca cagcctcctc 1560ttccagaatg cctttgcgca gcaagcagtg
tgcgccccga gccgcgtttc tttcctcact 1620ggcaggagac ctgacaccac
ccgcctgtac gacttcaact cctactggag ggtgcacgct 1680ggaaacttct
ccaccatccc ccagtacttc aaggagaatg gctatgtgac catgtcggtg
1740ggaaaagtct ttcaccctgg gatatcttct aaccataccg atgattctcc
gtatagctgg 1800tcttttccac cttatcatcc ttcctctgag aagtatgaaa
acactaagac atgtcgaggg 1860ccagatggag aactccatgc caacctgctt
tgccctgtgg atgtgctgga tgttcccgag 1920ggcaccttgc ctgacaaaca
gagcactgag caagccatac agttgttgga aaagatgaaa 1980acgtcagcca
gtcctttctt cctggccgtt gggtatcata agccacacat ccccttcaga
2040taccccaagg aatttcagaa gttgtatccc ttggagaaca tcaccctggc
ccccgatccc 2100gaggtccctg atggcctacc ccctgtggcc tacaacccct
ggatggacat caggcaacgg 2160gaagacgtcc aagccttaaa catcagtgtg
ccgtatggtc caattcctgt ggactttcag 2220cggaaaatcc gccagagcta
ctttgcctct gtgtcatatt tggatacaca ggtcggccgc 2280ctcttgagtg
ctttggacga tcttcagctg gccaacagca ccatcattgc atttacctcg
2340gatcatgggt gggctctagg tgaacatgga gaatgggcca aatacagcaa
ttttgatgtt 2400gctacccatg ttcccctgat attctatgtt cctggaagga
cggcttcact tccggaggca 2460ggcgagaagc ttttccctta cctcgaccct
tttgattccg cctcacagtt gatggagcca 2520ggcaggcaat ccatggacct
tgtggaactt gtgtctcttt ttcccacgct ggctggactt 2580gcaggactgc
aggttccacc tcgctgcccc gttccttcat ttcacgttga gctgtgcaga
2640gaaggcaaga accttctgaa gcattttcga ttccgtgact tggaagaaga
tccgtacctc 2700cctggtaatc cccgtgaact gattgcctat agccagtatc
cccggccttc agacatccct 2760cagtggaatt ctgacaagcc gagtttaaaa
gatataaaga tcatgggcta ttccatacgc 2820accatagact ataggtatac
tgtgtgggtt ggcttcaatc ctgatgaatt tctagctaac 2880ttttctgaca
tccatgcagg ggaactgtat tttgtggatt ctgacccatt gcaggatcac
2940aatatgtata atgattccca aggtggagac cttttccagt tgttgatgcc
ttaagcggcc 3000gc 3002249980PRTArtificial SequenceAmino acid
sequence of the fusion protein of heavy chain of anti-hTfR antibody
No.2 (humanized 6) and hI2S 249Gln 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 Asp Tyr 20 25 30Val Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Val Ile Ser Thr Tyr
Tyr Gly His Gly Ser Tyr Asn Gln Arg Phe 50 55 60Lys Gly Arg Ala Thr
Met Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Val Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Arg
Gly Gly Tyr Gly Ser Ser Ser Leu Ala Gly Asn Phe Asp Val 100 105
110Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly 130 135 140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu225 230
235 240Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr 245 250 255Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val 260 265 270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val 275 280 285Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser 290 295 300Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu305 310 315 320Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345
350Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
355 360 365Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala 370 375 380Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr385 390 395 400Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu 405 410 415Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445Leu Ser Pro
Gly Lys Gly Ser Ser Glu Thr Gln Ala Asn Ser Thr Thr 450 455 460Asp
Ala Leu Asn Val Leu Leu Ile Ile Val Asp Asp Leu Arg Pro Ser465 470
475 480Leu Gly Cys Tyr Gly Asp Lys Leu Val Arg Ser Pro Asn Ile Asp
Gln 485 490 495Leu Ala Ser His Ser Leu Leu Phe Gln Asn Ala Phe Ala
Gln Gln Ala 500 505 510Val Cys Ala Pro Ser Arg Val Ser Phe Leu Thr
Gly Arg Arg Pro Asp 515 520 525Thr Thr Arg Leu Tyr Asp Phe Asn Ser
Tyr Trp Arg Val His Ala Gly 530 535 540Asn Phe Ser Thr Ile Pro Gln
Tyr Phe Lys Glu Asn Gly Tyr Val Thr545 550 555 560Met Ser Val Gly
Lys Val Phe His Pro Gly Ile Ser Ser Asn His Thr 565 570 575Asp Asp
Ser Pro Tyr Ser Trp Ser Phe Pro Pro Tyr His Pro Ser Ser 580 585
590Glu Lys Tyr Glu Asn Thr Lys Thr Cys Arg Gly Pro Asp Gly Glu Leu
595 600 605His Ala Asn Leu Leu Cys Pro Val Asp Val Leu Asp Val Pro
Glu Gly 610 615 620Thr Leu Pro Asp Lys Gln Ser Thr Glu Gln Ala Ile
Gln Leu Leu Glu625 630 635 640Lys Met Lys Thr Ser Ala Ser Pro Phe
Phe Leu Ala Val Gly Tyr His 645 650 655Lys Pro His Ile Pro Phe Arg
Tyr Pro Lys Glu Phe Gln Lys Leu Tyr 660 665 670Pro Leu Glu Asn Ile
Thr Leu Ala Pro Asp Pro Glu Val Pro Asp Gly 675 680 685Leu Pro Pro
Val Ala Tyr Asn Pro Trp Met Asp Ile Arg Gln Arg Glu 690 695 700Asp
Val Gln Ala Leu Asn Ile Ser Val Pro Tyr Gly Pro Ile Pro Val705 710
715 720Asp Phe Gln Arg Lys Ile Arg Gln Ser Tyr Phe Ala Ser Val Ser
Tyr 725 730 735Leu Asp Thr Gln Val Gly Arg Leu Leu Ser Ala Leu Asp
Asp Leu Gln 740 745 750Leu Ala Asn Ser Thr Ile Ile Ala Phe Thr Ser
Asp His Gly Trp Ala 755 760 765Leu Gly Glu His Gly Glu Trp Ala Lys
Tyr Ser Asn Phe Asp Val Ala 770 775 780Thr His Val Pro Leu Ile Phe
Tyr Val Pro Gly Arg Thr Ala Ser Leu785 790 795 800Pro Glu Ala Gly
Glu Lys Leu Phe Pro Tyr Leu Asp Pro Phe Asp Ser 805 810 815Ala Ser
Gln Leu Met Glu Pro Gly Arg Gln Ser Met Asp Leu Val Glu 820 825
830Leu Val Ser Leu Phe Pro Thr Leu Ala Gly Leu Ala Gly Leu Gln Val
835 840 845Pro Pro Arg Cys Pro Val Pro Ser Phe His Val Glu Leu Cys
Arg Glu 850 855 860Gly Lys Asn Leu Leu Lys His Phe Arg Phe Arg Asp
Leu Glu Glu Asp865 870 875 880Pro Tyr Leu Pro Gly Asn Pro Arg Glu
Leu Ile Ala Tyr Ser Gln Tyr 885 890 895Pro Arg Pro Ser Asp Ile Pro
Gln Trp Asn Ser Asp Lys Pro Ser Leu 900 905 910Lys Asp Ile Lys Ile
Met Gly Tyr Ser Ile Arg Thr Ile Asp Tyr Arg 915 920 925Tyr Thr Val
Trp Val Gly Phe Asn Pro Asp Glu Phe Leu Ala Asn Phe 930 935 940Ser
Asp Ile His Ala Gly Glu Leu Tyr Phe Val Asp Ser Asp Pro Leu945 950
955 960Gln Asp His Asn Met Tyr Asn Asp Ser Gln Gly Gly Asp Leu Phe
Gln 965 970 975Leu Leu Met Pro 9802503023DNAArtificial
SequenceNucleotide sequence encoding the amino acid sequence of
fusion protein of heavy chain of anti-hTfR antibody No.2 (humanized
6) and hI2S, synthetic sequence 250acgcgtgccg ccaccatggg ctggagctgg
attctgctgt tcctcctgag cgtgacagca 60ggagtgcaca gccaggtgca gctggtccag
tcaggagccg aagtgaaaaa gcccggagcc 120tcagtcaaag tgtcttgtaa
agcatcaggt tatacattta cagactacgt catgcactgg 180gtgaggcagg
cacctggaca gggtctggaa tggatcggcg tgatctccac ttactatggc
240catggaagct acaaccagag attcaagggc agggcgacaa tgactgtaga
caaatcaatt 300tccactgctt atatggagct ggtaaggctg cggtccgacg
ataccgctgt gtactattgc 360gtacgaggag gatacggctc cagctctctg
gctggtaatt tcgatgtgtg ggggcagggt 420accacagtca ccgtgagttc
agcaagcaca aagggcccat ctgtgtttcc actggccccc 480tccagcaaaa
gcacctctgg gggtacagcc gctctgggat gtctggtgaa ggattatttc
540ccagagccag tcaccgtgtc ctggaacagc ggagccctga catctggagt
ccacactttt 600ccagctgtgc tgcagtctag tgggctgtac tccctgtcat
ccgtggtcac tgtccccagc 660tctagtctgg gtacccagac atatatctgc
aacgtgaatc acaagccatc taataccaaa 720gtcgacaaga aagtggaacc
caagtcctgt gataaaactc atacctgccc cccttgtcct 780gcaccagagc
tgctgggagg accatccgtg ttcctgtttc cacccaagcc taaagacacc
840ctgatgatta gccgaactcc cgaagtcacc tgcgtggtcg tggacgtgtc
tcacgaggac 900cctgaagtca agtttaactg gtacgtggat ggcgtcgagg
tgcataatgc taagacaaaa 960ccccgagagg aacagtacaa cagtacatat
cgtgtcgtgt cagtgctgac cgtcctgcat 1020caggactggc tgaacgggaa
ggaatataag tgcaaagtgt ccaataaggc actgcccgcc 1080cctatcgaga
aaaccattag caaggccaaa ggacagccta gggaaccaca ggtgtacaca
1140ctgcctccat cccgggacga gctgactaag aaccaggtca gcctgacctg
tctggtgaaa 1200ggcttctatc cttcagatat cgctgtggag tgggaaagta
atggacagcc agagaacaat 1260tacaagacta ccccccctgt gctggactct
gatgggagtt tctttctgta ttctaagctg 1320accgtggata aaagtcggtg
gcagcagggt aatgtcttta gttgttcagt gatgcacgaa 1380gcactgcaca
accactacac ccagaaatca ctgtcactgt caccagggaa aggatcttcc
1440gaaacgcagg ccaactcgac cacagatgct ctgaacgttc ttctcatcat
cgtggatgac 1500ctgcgcccct ccctgggctg ttatggggat aagctggtga
ggtccccaaa tattgaccaa 1560ctggcatccc acagcctcct cttccagaat
gcctttgcgc agcaagcagt gtgcgccccg 1620agccgcgttt ctttcctcac
tggcaggaga cctgacacca cccgcctgta cgacttcaac 1680tcctactgga
gggtgcacgc tggaaacttc tccaccatcc cccagtactt caaggagaat
1740ggctatgtga ccatgtcggt gggaaaagtc tttcaccctg ggatatcttc
taaccatacc 1800gatgattctc cgtatagctg gtcttttcca ccttatcatc
cttcctctga gaagtatgaa 1860aacactaaga catgtcgagg gccagatgga
gaactccatg ccaacctgct ttgccctgtg 1920gatgtgctgg atgttcccga
gggcaccttg cctgacaaac agagcactga gcaagccata 1980cagttgttgg
aaaagatgaa aacgtcagcc agtcctttct tcctggccgt tgggtatcat
2040aagccacaca tccccttcag ataccccaag gaatttcaga agttgtatcc
cttggagaac 2100atcaccctgg cccccgatcc cgaggtccct gatggcctac
cccctgtggc ctacaacccc 2160tggatggaca tcaggcaacg ggaagacgtc
caagccttaa acatcagtgt gccgtatggt 2220ccaattcctg tggactttca
gcggaaaatc cgccagagct actttgcctc tgtgtcatat 2280ttggatacac
aggtcggccg cctcttgagt gctttggacg atcttcagct ggccaacagc
2340accatcattg catttacctc ggatcatggg tgggctctag gtgaacatgg
agaatgggcc 2400aaatacagca attttgatgt tgctacccat gttcccctga
tattctatgt tcctggaagg 2460acggcttcac ttccggaggc aggcgagaag
cttttccctt acctcgaccc ttttgattcc 2520gcctcacagt tgatggagcc
aggcaggcaa tccatggacc ttgtggaact tgtgtctctt 2580tttcccacgc
tggctggact tgcaggactg caggttccac ctcgctgccc cgttccttca
2640tttcacgttg agctgtgcag agaaggcaag aaccttctga agcattttcg
attccgtgac 2700ttggaagaag atccgtacct ccctggtaat ccccgtgaac
tgattgccta tagccagtat 2760ccccggcctt cagacatccc tcagtggaat
tctgacaagc cgagtttaaa agatataaag 2820atcatgggct attccatacg
caccatagac tataggtata ctgtgtgggt tggcttcaat 2880cctgatgaat
ttctagctaa cttttctgac atccatgcag gggaactgta ttttgtggat
2940tctgacccat tgcaggatca caatatgtat aatgattccc aaggtggaga
ccttttccag 3000ttgttgatgc cttaagcggc cgc 3023251975PRTArtificial
SequenceAmino acid sequence of fusion protein of heavy chain of
anti-hTfR antibody No.3 (humanized 2) and hI2S, synthetic sequence
251Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1
5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn
Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr Pro Thr Tyr Ser
Glu Lys Phe 50 55 60Lys Val Gln Val Thr Ile Ser Ala Asp Lys Ser Ile
Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp
Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Asn Tyr Asp Glu Val
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 Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 445Gly Ser Ser Glu Thr Gln Ala Asn Ser Thr
Thr Asp Ala Leu Asn Val 450 455 460Leu Leu Ile Ile Val Asp Asp Leu
Arg Pro Ser Leu Gly Cys Tyr Gly465 470 475 480Asp Lys Leu Val Arg
Ser Pro Asn Ile Asp Gln Leu Ala Ser His Ser 485 490 495Leu Leu Phe
Gln Asn Ala Phe Ala Gln Gln Ala Val Cys Ala Pro Ser 500 505 510Arg
Val Ser Phe Leu Thr Gly Arg Arg Pro Asp Thr Thr Arg Leu Tyr 515 520
525Asp Phe Asn Ser Tyr Trp Arg Val His Ala Gly Asn Phe Ser Thr Ile
530 535 540Pro Gln Tyr Phe Lys Glu Asn Gly Tyr Val Thr Met Ser Val
Gly Lys545 550 555 560Val Phe His Pro Gly Ile Ser Ser Asn His Thr
Asp Asp Ser Pro Tyr 565 570 575Ser Trp Ser Phe Pro Pro Tyr His Pro
Ser Ser Glu Lys Tyr Glu Asn 580 585 590Thr Lys Thr Cys Arg Gly Pro
Asp Gly Glu Leu His Ala Asn Leu Leu 595 600 605Cys Pro Val Asp Val
Leu Asp Val Pro Glu Gly Thr Leu Pro Asp Lys 610 615 620Gln Ser Thr
Glu Gln Ala Ile Gln Leu Leu Glu Lys Met Lys Thr Ser625 630 635
640Ala Ser Pro Phe Phe Leu Ala Val Gly Tyr His Lys Pro His Ile Pro
645 650 655Phe Arg Tyr Pro Lys Glu Phe Gln Lys Leu Tyr Pro Leu Glu
Asn Ile 660 665 670Thr Leu Ala Pro Asp Pro Glu Val Pro Asp Gly Leu
Pro Pro Val Ala 675 680 685Tyr Asn Pro Trp Met Asp Ile Arg Gln Arg
Glu Asp Val Gln Ala Leu 690 695 700Asn Ile Ser Val Pro Tyr Gly Pro
Ile Pro Val Asp Phe Gln Arg Lys705 710 715 720Ile Arg Gln Ser Tyr
Phe Ala Ser Val Ser Tyr Leu Asp Thr Gln Val 725 730 735Gly Arg Leu
Leu Ser Ala Leu Asp Asp Leu Gln Leu Ala Asn Ser Thr 740 745 750Ile
Ile Ala Phe Thr Ser Asp His Gly Trp Ala Leu Gly Glu His Gly 755 760
765Glu Trp Ala Lys Tyr Ser Asn Phe Asp Val Ala Thr His Val Pro Leu
770 775 780Ile Phe Tyr Val Pro Gly Arg Thr Ala Ser Leu Pro Glu Ala
Gly Glu785 790 795 800Lys Leu Phe Pro Tyr Leu Asp Pro Phe Asp Ser
Ala Ser Gln Leu Met 805 810 815Glu Pro Gly Arg Gln Ser Met Asp Leu
Val Glu Leu Val Ser Leu Phe 820 825 830Pro Thr Leu Ala Gly Leu Ala
Gly Leu Gln Val Pro Pro Arg Cys Pro 835 840 845Val Pro Ser Phe His
Val Glu Leu Cys Arg Glu Gly Lys Asn Leu Leu 850 855 860Lys His Phe
Arg Phe Arg Asp Leu Glu Glu Asp Pro Tyr Leu Pro Gly865 870 875
880Asn Pro Arg Glu Leu Ile Ala Tyr Ser Gln Tyr Pro Arg Pro Ser Asp
885 890 895Ile Pro Gln Trp Asn Ser Asp Lys Pro Ser Leu Lys Asp Ile
Lys Ile 900 905 910Met Gly Tyr Ser Ile Arg Thr Ile Asp Tyr Arg Tyr
Thr Val Trp Val 915 920 925Gly Phe Asn Pro Asp Glu Phe Leu Ala Asn
Phe Ser Asp Ile His Ala 930 935 940Gly Glu Leu Tyr Phe Val Asp Ser
Asp Pro Leu Gln Asp His Asn Met945 950 955 960Tyr Asn Asp Ser Gln
Gly Gly Asp Leu Phe Gln Leu Leu Met Pro 965 970
9752523011DNAArtificial SequenceNucleotide sequence encoding the
amino acid sequence of fusion protein of heavy chain of anti-hTfR
antibody No.3 (humanized 2) and hI2S, synthetic sequence
252acgcgtgccg ccaccatggg ctggagctgg attctgctgt tcctcctgag
cgtgacagca 60ggagtgcaca gcgaggtgca actagtgcag tctggagcag aggtgaaaaa
gcccggggag 120tctctgaaga tttcctgtaa gggttctgga tacagcttta
ccaactactg gctgggatgg 180gtgcgccaga tgcccgggaa aggcctggag
tggatggggg acatctaccc cggcggagac 240taccctacat acagcgagaa
gttcaaggtc caggtcacca tctcagccga caagtccatc 300agcaccgcct
acctgcagtg gagcagcctg aaggcctcgg acaccgccat gtattactgt
360gcgagatcag gcaattacga cgaagtggcc tactggggcc aaggaaccct
ggtcaccgtc 420tcctcagcta gcaccaaggg cccatcggtc ttccccctgg
caccctcctc caagagcacc 480tctgggggca cagcggccct gggctgcctg
gtcaaggact acttccccga accggtgacg 540gtgtcgtgga actcaggcgc
cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 600tcctcaggac
tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc
660cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga
caagaaagtt 720gagcccaaat cttgtgacaa aactcacacg tgcccaccgt
gcccagcacc tgaactcctg 780ggaggtccgt cagtcttcct cttcccccca
aaacccaagg acaccctcat gatctcccgg 840acccctgagg tcacatgcgt
ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900aactggtacg
tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
960tacaacagca cgtaccgggt ggtcagcgtc ctcaccgtcc tgcaccagga
ctggctgaat 1020ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc
cagcccccat cgagaaaacc 1080atctccaaag ccaaagggca gccccgagaa
ccacaggtgt acaccctgcc cccatcccgg 1140gatgagctga ccaagaacca
ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1200gacatcgccg
tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
1260cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt
ggacaagagc 1320aggtggcagc aggggaacgt cttctcatgc tccgtgatgc
atgaggctct gcacaaccac 1380tacacgcaga agagcctctc cctgtctccg
ggtaaaggat cttccgaaac gcaggccaac 1440tcgaccacag atgctctgaa
cgttcttctc atcatcgtgg atgacctgcg cccctccctg 1500ggctgttatg
gggataagct ggtgaggtcc ccaaatattg accaactggc atcccacagc
1560ctcctcttcc agaatgcctt tgcgcagcaa gcagtgtgcg ccccgagccg
cgtttctttc 1620ctcactggca ggagacctga caccacccgc ctgtacgact
tcaactccta ctggagggtg 1680cacgctggaa acttctccac catcccccag
tacttcaagg agaatggcta tgtgaccatg 1740tcggtgggaa aagtctttca
ccctgggata tcttctaacc ataccgatga ttctccgtat 1800agctggtctt
ttccacctta tcatccttcc tctgagaagt atgaaaacac taagacatgt
1860cgagggccag atggagaact ccatgccaac ctgctttgcc ctgtggatgt
gctggatgtt 1920cccgagggca ccttgcctga caaacagagc actgagcaag
ccatacagtt gttggaaaag 1980atgaaaacgt cagccagtcc tttcttcctg
gccgttgggt atcataagcc acacatcccc 2040ttcagatacc ccaaggaatt
tcagaagttg tatcccttgg agaacatcac cctggccccc 2100gatcccgagg
tccctgatgg cctaccccct gtggcctaca acccctggat ggacatcagg
2160caacgggaag acgtccaagc cttaaacatc agtgtgccgt atggtccaat
tcctgtggac 2220tttcagcgga aaatccgcca gagctacttt gcctctgtgt
catatttgga tacacaggtc 2280ggccgcctct tgagtgcttt ggacgatctt
cagctggcca acagcaccat cattgcattt 2340acctcggatc atgggtgggc
tctaggtgaa catggagaat gggccaaata cagcaatttt 2400gatgttgcta
cccatgttcc cctgatattc tatgttcctg gaaggacggc ttcacttccg
2460gaggcaggcg agaagctttt cccttacctc gacccttttg attccgcctc
acagttgatg 2520gagccaggca ggcaatccat ggaccttgtg gaacttgtgt
ctctttttcc cacgctggct 2580ggacttgcag gactgcaggt tccacctcgc
tgccccgttc cttcatttca cgttgagctg 2640tgcagagaag gcaagaacct
tctgaagcat tttcgattcc gtgacttgga agaagatccg 2700tacctccctg
gtaatccccg tgaactgatt gcctatagcc agtatccccg gccttcagac
2760atccctcagt ggaattctga caagccgagt ttaaaagata taaagatcat
gggctattcc 2820atacgcacca tagactatag gtatactgtg tgggttggct
tcaatcctga tgaatttcta 2880gctaactttt ctgacatcca tgcaggggaa
ctgtattttg tggattctga cccattgcag 2940gatcacaata tgtataatga
ttcccaaggt ggagaccttt tccagttgtt gatgccttaa 3000taagcggccg c
30112534460DNAArtificial SequenceNucleotide sequence of the DNA
formed of a cDNA encoding chimeric hTfR and a loxP-flanked neomycin
resistance gene placed on the cDNA's 3' side, synthetic sequence
253gtttatcctc ccttgtagca gctgagaatg atggatcaag ccagatcagc
attctctaac 60ttgtttggtg gggaaccatt gtcatacacc cggtttagcc ttgctcggca
agtagatgga 120gataacagtc atgtggagat gaaactggct gcagatgaag
aagaaaatgc cgacaataac 180atgaaggcta gtgtcagaaa acccaagagg
tttaatggaa gactctgctt tgcagctatt 240gcactagtca ttttcttctt
gattggattc atgagtggct acctgggcta ttgtaaaggg 300gtagaaccaa
aaactgagtg tgagagactg gcaggaaccg agtctccagt gagggaggag
360ccaggagagg acttccctgc agcacgtcgc ttatattggg atgacctgaa
gagaaagttg 420tcggagaaac tggacagcac agacttcacc ggcaccatca
agctgctgaa tgaaaattca 480tatgtccctc gtgaggctgg atctcaaaaa
gatgaaaatc ttgcgttgta tgttgaaaat 540caatttcgtg aatttaaact
cagcaaagtc tggcgtgatc aacattttgt taagattcag 600gtcaaagaca
gcgctcaaaa ctcggtgatc atagttgata agaacggtag acttgtttac
660ctggtggaga atcctggggg ttatgtggcg tatagtaagg ctgcaacagt
tactggtaaa 720ctggtccatg ctaattttgg tactaaaaaa gattttgagg
atttatacac tcctgtgaat 780ggatctatag tgattgtcag agcagggaaa
atcacctttg cagaaaaggt tgcaaatgct 840gaaagcttaa atgcaattgg
tgtgttgata tacatggacc agactaaatt tcccattgtt 900aacgcagaac
tttcattctt tggacatgct catctgggga caggtgaccc ttacacacct
960ggattccctt ccttcaatca cactcagttt ccaccatctc ggtcatcagg
attgcctaat 1020atacctgtcc agacaatctc cagagctgct gcagaaaagc
tgtttgggaa tatggaagga 1080gactgtccct ctgactggaa aacagactct
acatgtagga tggtaacctc agaaagcaag 1140aatgtgaagc tcactgtgag
caatgtgctg aaagagataa aaattcttaa catctttgga 1200gttattaaag
gctttgtaga accagatcac tatgttgtag ttggggccca gagagatgca
1260tggggccctg gagctgcaaa atccggtgta ggcacagctc tcctattgaa
acttgcccag 1320atgttctcag atatggtctt aaaagatggg tttcagccca
gcagaagcat tatctttgcc 1380agttggagtg ctggagactt tggatcggtt
ggtgccactg aatggctaga gggatacctt 1440tcgtccctgc atttaaaggc
tttcacttat attaatctgg ataaagcggt tcttggtacc 1500agcaacttca
aggtttctgc cagcccactg ttgtatacgc ttattgagaa aacaatgcaa
1560aatgtgaagc atccggttac tgggcaattt ctatatcagg acagcaactg
ggccagcaaa 1620gttgagaaac tcactttaga caatgctgct ttccctttcc
ttgcatattc tggaatccca 1680gcagtttctt tctgtttttg cgaggacaca
gattatcctt atttgggtac caccatggac 1740acctataagg aactgattga
gaggattcct gagttgaaca aagtggcacg agcagctgca 1800gaggtcgctg
gtcagttcgt gattaaacta acccatgatg ttgaattgaa cctggactat
1860gagaggtaca acagccaact gctttcattt gtgagggatc tgaaccaata
cagagcagac 1920ataaaggaaa tgggcctgag tttacagtgg ctgtattctg
ctcgtggaga cttcttccgt 1980gctacttcca gactaacaac agatttcggg
aatgctgaga aaacagacag atttgtcatg 2040aagaaactca atgatcgtgt
catgagagtg gagtatcact tcctctctcc ctacgtatct 2100ccaaaagagt
ctcctttccg acatgtcttc tggggctccg gctctcacac gctgccagct
2160ttactggaga acttgaaact gcgtaaacaa aataacggtg cttttaatga
aacgctgttc 2220agaaaccagt tggctctagc tacttggact attcagggag
ctgcaaatgc cctctctggt 2280gacgtttggg acattgacaa tgagttttaa
cgtggctcgc tgatcagcct cgactgtgcc 2340ttctagttgc cagccatctg
ttgtttgccc ctcccccgtg ccttccttga ccctggaagg 2400tgccactccc
actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
2460gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
attgggaaga 2520caatagcagg catgctgggg atgcggtggg ctctatggct
tctgaggcgg aaagaaccag 2580ctggggctcg atcctctagt taagcttccc
agcggccgct atcgaattcc gatcatattc 2640aataaccctt aatataactt
cgtataatgt atgctatacg aagttattag gtctgaagag 2700gagtttacgt
ccagccaagc taattctacc gggtagggga ggcgcttttc ccaaggcagt
2760ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt
ggcctctggc 2820ctcgcacaca ttccacatcc accggtaggc gccaaccggc
tccgttcttt ggtggcccct 2880tcgcgccacc ttctactcct cccctagtca
ggaagttccc ccccgccccg cagctcgcgt 2940cgtgcaggac gtgacaaatg
gaagtagcac gtctcactag tctcgtgcag atggacagca 3000ccgctgagca
atggaagcgg gtaggccttt ggggcagcgg ccaatagcag ctttgctcct
3060tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc
aggggcgggc 3120tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc
ggcattctgc acgcttcaaa 3180agcgcacgtc tgccgcgctg ttctcctctt
cctcatctcc gggcctttcg acctgcagcc 3240aatatgggat cggccattga
acaagatgga ttgcacgcag gttctccggc cgcttgggtg 3300gagaggctat
tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
3360ttccggctgt cagcgcaggg
gcgcccggtt ctttttgtca agaccgacct gtccggtgcc 3420ctgaatgaac
tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
3480tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
attgggcgaa 3540gtgccggggc aggatctcct gtcatctcac cttgctcctg
ccgagaaagt atccatcatg 3600gctgatgcaa tgcggcggct gcatacgctt
gatccggcta cctgcccatt cgaccaccaa 3660gcgaaacatc gcatcgagcg
agcacgtact cggatggaag ccggtcttgt cgatcaggat 3720gatctggacg
aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
3780cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
gccgaatatc 3840atggtggaaa atggccgctt ttctggattc atcgactgtg
gccggctggg tgtggcggac 3900cgctatcagg acatagcgtt ggctacccgt
gatattgctg aagagcttgg cggcgaatgg 3960gctgaccgct tcctcgtgct
ttacggtatc gccgctcccg attcgcagcg catcgccttc 4020tatcgccttc
ttgacgagtt cttctgaggg gatccgctgt aagtctgcag aaattgatga
4080tctattaaac aataaagatg tccactaaaa tggaagtttt tcctgtcata
ctttgttaag 4140aagggtgaga acagagtacc tacattttga atggaaggat
tggagctacg ggggtggggg 4200tggggtggga ttagataaat gcctgctctt
tactgaaggc tctttactat tgctttatga 4260taatgtttca tagttggata
tcataattta aacaagcaaa accaaattaa gggccagctc 4320attcctccca
ctcatgatct atagatccct cgatcgagat ccggaaccct taatataact
4380tcgtataatg tatgctatac gaagttatta ggtccctcga agaggttcac
tagttctaga 4440gcatttaaat acgtgctagc 44602545003DNAArtificial
SequenceNucleotide sequence of the 5'-arm of targeting vector
254aaagacattc ccagggtccc taagggctcc atctgggagc gcgctatctg
tagtcctgtt 60gtggtttaag ataccactct ctttgttatt tgtaaattta ccacaggctg
ggcagtaaag 120tacagatagg aagaaaagag tgtgaacgtt tgcagtttta
acagaactcc ttttatttgc 180tatgcattgc aacaaccctc taaaagcaat
aatggcgata aacatgcaag ttaatctaca 240actttatcac cctagtaaat
cattggttcc tatttccctt ccataggctg aacatagtga 300gcactggtag
ctctttgctt ggcttaagcg ctgcttgtta aaatctaaaa aaaaaaaaaa
360aaaagatttg cagttttgct ttaaagtttt tcctttaaag catctctgag
ctgcaaaaac 420tcgtctttag gatatggttt tggcggagac ctaagcctgc
aagtaggaat attttaaaga 480gtaaaggctg atactttcga acttgcaccg
cagggttggg tgtcggtcaa ggttaggcgg 540agggcgacag gccaactcca
aagacacggc gggggagggg tgcggtggag aaagcgagcc 600gcagccaatc
gcacgcgctc tcccgacacc tgccgccttg gcgccttttc taggctactc
660cgcgcacgca ctggctgcgc gcgcagttcg cctccagcgg tcttggggga
gcacctcggt 720aggtgtacgt gcggaaggaa gtgacgtaga tccagagggc
cggccggggg gtggggccga 780gctataagct ttgggtggga ggcagcgctg
ccttcagaag gcgtgcggag cgcgggctgc 840tgcattgcgg actgtagagg
cgcttcctag tgagtgactc ccttgtcagc ggcacggccc 900atcgtggtcc
tcgcgtggcg ggcggaccag agcgagacgc cagggcctgg gtggtgcggg
960cggggaggcg gaggggtgtc gcggagtccg gggctgagga gcgcgggttg
caggtgcagc 1020gcggtgggtg tggggagccg ctgtaccctg cgcccctcgg
gtcctccggg ccttcgcagg 1080ccagtgctag gccgcgggtt cgagagtcac
cacgctgagg cgcaggcttg ttccgccggg 1140agcacgtggt ggcggctgga
ggaagtcgcc ccagggaacg gctgtcgggg tacgtgggtg 1200accttggggc
ccctcgcagg agggcgtcac agctgaaaag gacaaagctg ttttctattc
1260ggttactagt gtcacggaca tttagagggg cggggggagc ttccaataac
tgcacgttgg 1320aacttcggca ccacctggtc ggtttttttg ccagtctctc
cctcttggcc cagcgtgtgg 1380aatctcattt ttctagggca gaataggtct
gaacgctgca ggtaatacta agaacgtctc 1440tagcatctcc taagatggga
gaacgtagaa atacgacctc tttgtacgag ctcttttaga 1500actagctgta
gagaaccagc gtgcaccctg gtgttggaca gctctctaaa atggtgtttg
1560agggtaagaa aactgcattt gcaaattttt cagttagcac actttgtccc
gagcgtaaaa 1620tgaaatgatc tccttatact taggcagaag actggactgg
attgctgttc agtttctgtg 1680ctattttttt aaaataggat ttaagtggga
aatagttgtg ttacagaaat tctcggctat 1740tctgtgctat ttttttttaa
ataggattta agtgggaaat agttgtgtta cagaaattct 1800cggctacctg
atacttttat tctaagatta gatgagttgg ctctgagctg tgaaatatga
1860cctctttgac aaagacattt aagctgattc aggatgttat ctacaaagaa
aacgggattt 1920agcttgtgtg ggtccacttg catttatttt ctgttaagga
ttataataaa ctgctttata 1980caggaatcca atatcagctg ttttttatat
agagagcata atacttttta ctttgagaga 2040ggatgttgtc aaggaatggt
ggctgtgaac ctggcctgtg ttgactggtt agatctgtct 2100gcctaacccc
accctaaggc taagtagtta tatgcttgtg gcaatgtgct tatttataat
2160agggcaagat tatgggctaa atttgggtta gacaacaatg aaagttaatt
aaacgaccct 2220caggccttgg gtctactatg tgtaagtgat ttccttctct
cccagatgag tgctatacaa 2280aataaacttc agtgacctca agtggttttg
accttttggt tgctattcag aaaactatgg 2340aaatgaaaac ctgctaccta
tttcctattg ccttttcaat ttcccaaaga aggtctcccc 2400tatgaatcca
tgggtagcct tgaactcaga tccacctgct tctgtttgga gagtgataag
2460attaaaggca agtgccacca cacccagcaa agtaggctct taaaactaaa
acctttgcag 2520tcgggcatgg tgccacacac ctttggtccc agcactgggg
ggtaaggcag aggcaggtgg 2580atcactgagt ttgaggtcag ctagtgctga
agagtaagag cctgtcttta aaacatctca 2640acagctgggc agtcgtggtc
cattccttta atcccagcac ttgggaggca gaggcaagtg 2700gatttttgag
ttcgaggcca gcctggtcta cagagtgagt tccaggacag ccagggatac
2760acagagaaac cctgtctcaa aaaaccaaaa tataaataaa taaataaaga
ttgggagaga 2820agtcaaggat ctcataggtg gtcagggagc tacaactgca
gtagtaaaga agtaggactt 2880aaaagaacag ggccggcact aattttgagg
atctagatcg ggccctcaat taaggaactt 2940ccttttgtgt gaactcagaa
tttgaaatga aatgtgcttg tcagaaccat tgcatggctt 3000attttttaat
gaaaagtctg gctagtatct gcttatcttc cagcttccag ctcaaagtta
3060aggtcataga tcaaaagaac tatgtcttta tcttagttgt atcttaattt
ttattagaat 3120tgaatggttt tcctaatgtt tggtaacatc aaaggtgtgt
aagtaaaagt gagaaatcaa 3180gattaacttt ctcttggcaa agattgttga
cattggtgac atcttggacc aaatgagaat 3240tgttttactt ttaaatgtcc
catcaacagc tctcagttag gctgttctat ctggtttgtc 3300ttgccatgct
tgcagagtat agatttgaca atttgaaaat tcaaaaagct atataaatag
3360gtatgttgct atatgtaaga ttttaaatga gtcagttaag acttaaagaa
taactgggtt 3420tattttatct tgtcaggtta tcactgtgta gaccaggttg
accttgaaaa caaattctct 3480gcttcccaag tgctgggatt aaaggcgtgt
gtcactagct ctgacactgg ctactttgga 3540actactatgg tgttcacaaa
tgcagagttg agtgttggga ttaaatggaa atttcatgtc 3600ttttttttta
actttccctt ctacacaggg cttctctgtg tagtcctggc tgtccttgta
3660gctctagact aggctgaatt caaactcaga tccacccacc taagtgctga
gattaaaggc 3720atgtgccacc actgcccagt tctgaattgt tggggttttt
tttttgttgt tattcttaat 3780tttagttcga tgaattaaaa tcgaaataac
ttgtttctta gaaaaataag gtgtaattgg 3840gttataaagc caaatttaga
cattaatacc aacagcctgt ttaggctcaa aattgttcaa 3900tcattttatt
agtattatta ttaatcatat caacttgaga cctgtttggg aaagcagaat
3960attttaggga tagctatttc agacaagcat taatgtgtta gctgtttttt
tccccctaga 4020atatgattaa aattggctca gggtggggcc ttctagttct
ggctctagcg attgggtctg 4080tttctggtga ggtggtagtg ataaactgta
acagaaggga caagagattg ggcttctgag 4140aacatgtatg atctggtatg
tgactttaat cattaaagca tggggattca aaaatactaa 4200tgaataggcc
ttagaactag tcctgagtgt tttgtaaaat aacagtctta attctcctag
4260tttctggatt tttttctttg tctttggata ctaagtttaa gcatttattt
ggacagagtg 4320gtgcccacta gctgttctat tctagtactt gggaggcaag
aggcagaacg aacatggcca 4380gcttggacaa cttaagaaaa ctgaaaggct
tgccatccta gtttttcttt tgattaaaca 4440cacttatatt ctaactagtt
ttctctatcc tttgggtttt gttttgtttt ttatttgttt 4500tgtagctcta
gctggctggc ttcggaattg cctgcctccc aagtgctagg attaaaggca
4560tcacagtcac tacccaattg atatcatgat tcttaattca acttctaaga
acaaattatc 4620acactctgaa tctaacatgg aatagcatta tccatgttca
gatatctttg tctcaaggct 4680caggtttatc ctttgtagct ttctttttgc
tatcccacct ctattcagca tccagtcaag 4740gatcactgag ttggttatca
gtaaattaaa cattttaatt aatgtctagg agacaggtta 4800tggtatagtt
ctcagtgctg ggaagttgac atggtaggat ctcagttcat ggccagccag
4860aactttctgg tgagatcctg gttcaagcac tacagagctt ttctagaaaa
gtaactatat 4920ttaggagtaa gtttgatata atgacaatcc catcgtaagc
cttcagtaac ctgatgcatt 4980ggtctctgtt ttaatatcag gta
50032552604DNAArtificial SequenceNucleotide sequence of the 3'-arm
of targeting vector 255gcattctcta acttggtaag gtactttcat ctatctgaaa
aaatgttcag taaaaaacaa 60aacaaaaaca tgacttcacc cagtgagtac aagatgatac
ccccaatttt tttttttttt 120attatatgta tgtacactgt agctgtcttc
agacacacca gaagagggcg tcagatctca 180tcagatggtt gtgagccacc
atgtggttgc tgggacttga acactggacc tttggaaaag 240cagttgggta
ctcttaccca ctgagctatc acaccagccc caatacccca aattttgacc
300agcctttttt tttttttttt tttttttttt aaatctgaac taatggtatt
aaggtgaaac 360agattgcaca aaagaggtac taggtttttc tttgaggcaa
ggtctttcta tattgcctca 420ggttgacctt aaactccaac ttcctttttt
tccccaagtg ctggagattg tagacagata 480tatacaacac cccaaaacaa
atatgtttag tttcgattaa gattcattat gtggggctag 540agggatgact
tagaggttaa gaacactgcc tgctcttcca gaggtcctga gttcaattcc
600cagcaaccac gtggtggctc acaaccatct gtaatgggat ccgatgccct
ctttctggtg 660tgtttgaaga cagctacagt gtcctcatac ataaaaaaaa
taaacaaaca gatcttttaa 720aaaaaaaggt acagtgtact tatacattat
ataaatgaat gattctttaa aaaattaatt 780atgggaaata cttatgaaga
atagggtagc tttggctgtt ttggaaacgt tatataacaa 840ggtagaacta
aaatgtatgc cagtaatccc agaggaatca ttagccagtc agggctagtc
900tgagcaatgt ggcaagataa acccatctct ttaaaaaaaa aaaaaagtat
tataaataga 960aatgttatag gaaacaggaa atagaaaccc tcgaaaggct
gaatgaaaga gtattagtgg 1020gctggagaga tggttcagcg gttaagagca
ctatctcctg agttcagttc ccagtgacca 1080catggtggct cacagccatc
tgtaatgaga tctgacgccc tcttctgggg tgtctgaaga 1140cagcgacagt
gtactcacat aaaataaata cataaagact gttagttagc cttcatctac
1200catttacaga actgggcaca gaaaggagtt catcagttat aaagggtaac
tttccatatg 1260aatgtttgtc atattattat gcatatagta taatgaccaa
actactgtaa tgtcttaata 1320tttgtatctc ttttctcttt tttaaaaata
tcagtttggt ggggaaccat tgtcatacac 1380ccggtttagc cttgctcggc
aagtagatgg agataacagt catgtggaga tgaaactggc 1440tgcagatgaa
gaagaaaatg ccgacaataa catgaaggct agtgtcagaa aacccaagag
1500gtttaatgga agactctgct ttgcagctat tgcactagtc attttcttct
tgattggtaa 1560gaatgagtgg ccattcagaa ggatttctta tgactaacta
gttcttagac tagctagttc 1620ttagactagc tagttcttgt ttcttttgga
tgaggagatg ctttgtactt taaatggcac 1680tggggctccc tacctgccgg
cagattaggt cctgcaagat gggaaacgtt tacattatgg 1740atgttttatt
agagatatgc aggacatttg gaatagtact aagaaaggct tccagtaaga
1800caaggtgtgc acccatgtct taaacaggtc actgtaaaat atgacttagt
tgtggtaatt 1860taaattccat taaactcagg ttcataattt tctattagat
tctcatagtt taattaaaag 1920ttttcaggga taagttaaaa atgagttctg
tgagtttagc tctaaaactt cctgttttta 1980ggattcatga gtggctacct
gggctattgt aagcgtgtag aacaaaaaga ggagtgtgtg 2040aaactggctg
aaacggagga gacagacaag tcagaaacca tggaaacaga ggatgttcct
2100acatcatctc gcttatattg ggcagacctc aaaacactgt tgtcagagaa
gttgaactcc 2160atagagtttg ctgacaccat caagtaagct caacttccca
agttcagtcc tgatggaaac 2220gtttttgttg ggggggtagg gagacttgaa
aggctttcag agggtcctcc tgacaatgtg 2280gaactatgct gacaggaaat
taggacttac ctggagagcc tcatagcctc ctttttcttc 2340agaatcgttt
tatcagttgt agtttagtgt gggatgtcag attttcttct gttctaatat
2400tccttttaaa aattttttaa aattaaaatt actttttatg tattatgagt
atagcctgca 2460tatatgaatg aatgtgcatt actaattaca cttatctcct
agtgcctaca taagccttat 2520agatggttgt gagccagcat gtgggtgctg
gaatccaaaa tggttcttgc aagaccaaat 2580atgttacatc ccagagccat taca
2604256166PRTHomo sapiens 256Ala Pro Pro Arg Leu Ile Cys Asp Ser
Arg Val Leu Glu Arg Tyr Leu1 5 10 15Leu Glu Ala Lys Glu Ala Glu Asn
Ile Thr Thr Gly Cys Ala Glu His 20 25 30Cys Ser Leu Asn Glu Asn Ile
Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45Tyr Ala Trp Lys Arg Met
Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55 60Gln Gly Leu Ala Leu
Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu65 70 75 80Leu Val Asn
Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95Lys Ala
Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105
110Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala
115 120 125Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe
Arg Val 130 135 140Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr
Thr Gly Glu Ala145 150 155 160Cys Arg Thr Gly Asp Arg
165257616PRTArtificial SequenceAmino acid sequence of the fusion
protein of heavy chain of anti-hTfR antibody No.3 (humanized 2) and
hEPO 257Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr
Asn Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr Pro Thr Tyr
Ser Glu Lys Phe 50 55 60Lys Val Gln Val Thr Ile Ser Ala Asp Lys Ser
Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser
Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Asn Tyr Asp Glu
Val 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 Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 445Gly Ser Ala Pro Pro Arg Leu Ile Cys Asp
Ser Arg Val Leu Glu Arg 450 455 460Tyr Leu Leu Glu Ala Lys Glu Ala
Glu Asn Ile Thr Thr Gly Cys Ala465 470 475 480Glu His Cys Ser Leu
Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val 485 490 495Asn Phe Tyr
Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu 500 505 510Val
Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln 515 520
525Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His
530 535 540Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu
Leu Arg545 550 555 560Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser Pro
Pro Asp Ala Ala Ser 565 570 575Ala Ala Pro Leu Arg Thr Ile Thr Ala
Asp Thr Phe Arg Lys Leu Phe 580 585 590Arg Val Tyr Ser Asn Phe Leu
Arg Gly Lys Leu Lys Leu Tyr Thr Gly 595 600 605Glu Ala Cys Arg Thr
Gly Asp Arg 610 6152581931DNAArtificial SequenceNucleotide sequence
encoding the amino acid sequence of fusion protein of heavy chain
of anti-hTfR antibody No. 3 (humanized 2) and hEPO, synthetic
sequence 258acgcgtgccg ccaccatggg ctggagctgg attctgctgt tcctcctgag
cgtgacagca 60ggagtgcaca gcgaggtgca actagtgcag tctggagcag aggtgaaaaa
gcccggggag 120tctctgaaga tttcctgtaa gggttctgga tacagcttta
ccaactactg gctgggatgg 180gtgcgccaga tgcccgggaa aggcctggag
tggatggggg acatctaccc cggcggagac 240taccctacat acagcgagaa
gttcaaggtc caggtcacca tctcagccga caagtccatc 300agcaccgcct
acctgcagtg gagcagcctg aaggcctcgg acaccgccat gtattactgt
360gcgagatcag gcaattacga cgaagtggcc tactggggcc aaggaaccct
ggtcaccgtc 420tcctcagcta gcaccaaggg cccatcggtc ttccccctgg
caccctcctc caagagcacc 480tctgggggca cagcggccct gggctgcctg
gtcaaggact acttccccga accggtgacg 540gtgtcgtgga actcaggcgc
cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 600tcctcaggac
tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc
660cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga
caagaaagtt 720gagcccaaat cttgtgacaa aactcacacg tgcccaccgt
gcccagcacc tgaactcctg 780ggaggtccgt cagtcttcct cttcccccca
aaacccaagg acaccctcat gatctcccgg 840acccctgagg tcacatgcgt
ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900aactggtacg
tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
960tacaacagca cgtaccgggt ggtcagcgtc ctcaccgtcc tgcaccagga
ctggctgaat 1020ggcaaggagt
acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc
1080atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc
cccatcccgg 1140gatgagctga ccaagaacca ggtcagcctg acctgcctgg
tcaaaggctt ctatcccagc 1200gacatcgccg tggagtggga gagcaatggg
cagccggaga acaactacaa gaccacgcct 1260cccgtgctgg actccgacgg
ctccttcttc ctctacagca agctcaccgt ggacaagagc 1320aggtggcagc
aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac
1380tacacgcaga agagcctctc cctgtctccg ggtaaaggat ctgccccacc
acgcctcatc 1440tgtgacagcc gagtcctgga gaggtacctc ttggaggcca
aggaggccga gaatatcacg 1500acgggctgtg ctgaacactg cagcttgaat
gagaatatca ctgtcccaga caccaaagtt 1560aatttctatg cctggaagag
gatggaggtc gggcagcagg ccgtagaagt ctggcagggc 1620ctggccctgc
tgtcggaagc tgtcctgcgg ggccaggccc tgttggtcaa ctcttcccag
1680ccgtgggagc ccctgcagct gcatgtggat aaagccgtca gtggccttcg
cagcctcacc 1740actctgcttc gggctctggg agcccagaag gaagccatct
cccctccaga tgcggcctca 1800gctgctccac tccgaacaat cactgctgac
actttccgca aactcttccg agtctactcc 1860aatttcctcc ggggaaagct
gaagctgtac acaggggagg cctgcaggac aggggacaga 1920taagcggccg c
1931259489PRTHomo sapiens 259Arg Pro Pro Asn Ile Val Leu Ile Phe
Ala Asp Asp Leu Gly Tyr Gly1 5 10 15Asp Leu Gly Cys Tyr Gly His Pro
Ser Ser Thr Thr Pro Asn Leu Asp 20 25 30Gln Leu Ala Ala Gly Gly Leu
Arg Phe Thr Asp Phe Tyr Val Pro Val 35 40 45Ser Leu Cys Thr Pro Ser
Arg Ala Ala Leu Leu Thr Gly Arg Leu Pro 50 55 60Val Arg Met Gly Met
Tyr Pro Gly Val Leu Val Pro Ser Ser Arg Gly65 70 75 80Gly Leu Pro
Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala Ala Arg 85 90 95Gly Tyr
Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val Gly Pro 100 105
110Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe Leu Gly
115 120 125Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr
Cys Phe 130 135 140Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln
Gly Leu Val Pro145 150 155 160Ile Pro Leu Leu Ala Asn Leu Ser Val
Glu Ala Gln Pro Pro Trp Leu 165 170 175Pro Gly Leu Glu Ala Arg Tyr
Met Ala Phe Ala His Asp Leu Met Ala 180 185 190Asp Ala Gln Arg Gln
Asp Arg Pro Phe Phe Leu Tyr Tyr Ala Ser His 195 200 205His Thr His
Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu Arg Ser 210 215 220Gly
Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala Ala Val225 230
235 240Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu Glu
Thr 245 250 255Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Thr Met
Arg Met Ser 260 265 270Arg Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly
Lys Gly Thr Thr Tyr 275 280 285Glu Gly Gly Val Arg Glu Pro Ala Leu
Ala Phe Trp Pro Gly His Ile 290 295 300Ala Pro Gly Val Thr His Glu
Leu Ala Ser Ser Leu Asp Leu Leu Pro305 310 315 320Thr Leu Ala Ala
Leu Ala Gly Ala Pro Leu Pro Asn Val Thr Leu Asp 325 330 335Gly Phe
Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser Pro Arg 340 345
350Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg Gly Val
355 360 365Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr
Gln Gly 370 375 380Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys
His Ala Ser Ser385 390 395 400Ser Leu Thr Ala His Glu Pro Pro Leu
Leu Tyr Asp Leu Ser Lys Asp 405 410 415Pro Gly Glu Asn Tyr Asn Leu
Leu Gly Gly Val Ala Gly Ala Thr Pro 420 425 430Glu Val Leu Gln Ala
Leu Lys Gln Leu Gln Leu Leu Lys Ala Gln Leu 435 440 445Asp Ala Ala
Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly Glu Asp 450 455 460Pro
Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg Pro Ala465 470
475 480Cys Cys His Cys Pro Asp Pro His Ala 485260939PRTArtificial
SequenceAmino acid sequence of the fusion protein of heavy chain of
anti-hTfR antibody No.3 (humanized 2) and hARSA 260Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys
Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30Trp Leu
Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly
Asp Ile Tyr Pro Gly Gly Asp Tyr Pro Thr Tyr Ser Glu Lys Phe 50 55
60Lys Val Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65
70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr
Cys 85 90 95Ala Arg Ser Gly Asn Tyr Asp Glu Val 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 Lys Thr
210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445Gly Ser Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly
450 455 460Tyr Gly Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr
Pro Asn465 470 475 480Leu Asp Gln Leu Ala Ala Gly Gly Leu Arg Phe
Thr Asp Phe Tyr Val 485 490 495Pro Val Ser Leu Cys Thr Pro Ser Arg
Ala Ala Leu Leu Thr Gly Arg 500 505 510Leu Pro Val Arg Met Gly Met
Tyr Pro Gly Val Leu Val Pro Ser Ser 515 520 525Arg Gly Gly Leu Pro
Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala 530 535 540Ala Arg Gly
Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val545 550 555
560Gly Pro Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe
565 570 575Leu Gly Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn
Leu Thr 580 585 590Cys Phe Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys
Asp Gln Gly Leu 595 600 605Val Pro Ile Pro Leu Leu Ala Asn Leu Ser
Val Glu Ala Gln Pro Pro 610 615 620Trp Leu Pro Gly Leu Glu Ala Arg
Tyr Met Ala Phe Ala His Asp Leu625 630 635 640Met Ala Asp Ala Gln
Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala 645 650 655Ser His His
Thr His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu 660 665 670Arg
Ser Gly Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala 675 680
685Ala Val Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu
690 695 700Glu Thr Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Thr
Met Arg705 710 715 720Met Ser Arg Gly Gly Cys Ser Gly Leu Leu Arg
Cys Gly Lys Gly Thr 725 730 735Thr Tyr Glu Gly Gly Val Arg Glu Pro
Ala Leu Ala Phe Trp Pro Gly 740 745 750His Ile Ala Pro Gly Val Thr
His Glu Leu Ala Ser Ser Leu Asp Leu 755 760 765Leu Pro Thr Leu Ala
Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr 770 775 780Leu Asp Gly
Phe Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser785 790 795
800Pro Arg Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg
805 810 815Gly Val Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe
Phe Thr 820 825 830Gln Gly Ser Ala His Ser Asp Thr Thr Ala Asp Pro
Ala Cys His Ala 835 840 845Ser Ser Ser Leu Thr Ala His Glu Pro Pro
Leu Leu Tyr Asp Leu Ser 850 855 860Lys Asp Pro Gly Glu Asn Tyr Asn
Leu Leu Gly Gly Val Ala Gly Ala865 870 875 880Thr Pro Glu Val Leu
Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala 885 890 895Gln Leu Asp
Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly 900 905 910Glu
Asp Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg 915 920
925Pro Ala Cys Cys His Cys Pro Asp Pro His Ala 930
9352612900DNAArtificial SequenceNucleotide sequence encoding the
amino acid sequence of fusion protein of heavy chain of anti-hTfR
antibody No.3 (humanized 2) and hARSA, synthetic sequence
261acgcgtgccg ccaccatggg ctggagctgg attctgctgt tcctcctgag
cgtgacagca 60ggagtgcaca gcgaggtgca actagtgcag tctggagcag aggtgaaaaa
gcccggggag 120tctctgaaga tttcctgtaa gggttctgga tacagcttta
ccaactactg gctgggatgg 180gtgcgccaga tgcccgggaa aggcctggag
tggatggggg acatctaccc cggcggagac 240taccctacat acagcgagaa
gttcaaggtc caggtcacca tctcagccga caagtccatc 300agcaccgcct
acctgcagtg gagcagcctg aaggcctcgg acaccgccat gtattactgt
360gcgagatcag gcaattacga cgaagtggcc tactggggcc aaggaaccct
ggtcaccgtc 420tcctcagcta gcaccaaggg cccatcggtc ttccccctgg
caccctcctc caagagcacc 480tctgggggca cagcggccct gggctgcctg
gtcaaggact acttccccga accggtgacg 540gtgtcgtgga actcaggcgc
cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 600tcctcaggac
tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc
660cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga
caagaaagtt 720gagcccaaat cttgtgacaa aactcacacg tgcccaccgt
gcccagcacc tgaactcctg 780ggaggtccgt cagtcttcct cttcccccca
aaacccaagg acaccctcat gatctcccgg 840acccctgagg tcacatgcgt
ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900aactggtacg
tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
960tacaacagca cgtaccgggt ggtcagcgtc ctcaccgtcc tgcaccagga
ctggctgaat 1020ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc
cagcccccat cgagaaaacc 1080atctccaaag ccaaagggca gccccgagaa
ccacaggtgt acaccctgcc cccatcccgg 1140gatgagctga ccaagaacca
ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1200gacatcgccg
tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
1260cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt
ggacaagagc 1320aggtggcagc aggggaacgt cttctcatgc tccgtgatgc
atgaggctct gcacaaccac 1380tacacgcaga agagcctctc cctgtctccg
ggtaaaggat ctcgtccgcc caacatcgtg 1440ctgatctttg ccgacgacct
cggctatggg gacctgggct gctatgggca ccccagctct 1500accactccca
acctggacca gctggcggcg ggagggctgc ggttcacaga cttctacgtg
1560cctgtgtctc tgtgcacacc ctctagggcc gccctcctga ccggccggct
cccggttcgg 1620atgggcatgt accctggcgt cctggtgccc agctcccggg
ggggcctgcc cctggaggag 1680gtgaccgtgg ccgaagtcct ggctgcccga
ggctacctca caggaatggc cggcaagtgg 1740caccttgggg tggggcctga
gggggccttc ctgccccccc atcagggctt ccatcgattt 1800ctaggcatcc
cgtactccca cgaccagggc ccctgccaga acctgacctg cttcccgccg
1860gccactcctt gcgacggtgg ctgtgaccag ggcctggtcc ccatcccact
gttggccaac 1920ctgtccgtgg aggcgcagcc cccctggctg cccggactag
aggcccgcta catggctttc 1980gcccatgacc tcatggccga cgcccagcgc
caggatcgcc ccttcttcct gtactatgcc 2040tctcaccaca cccactaccc
tcagttcagt gggcagagct ttgcagagcg ttcaggccgc 2100gggccatttg
gggactccct gatggagctg gatgcagctg tggggaccct gatgacagcc
2160ataggggacc tggggctgct tgaagagacg ctggtcatct tcactgcaga
caatggacct 2220gagaccatgc gtatgtcccg aggcggctgc tccggtctct
tgcggtgtgg aaagggaacg 2280acctacgagg gcggtgtccg agagcctgcc
ttggccttct ggccaggtca tatcgctccc 2340ggcgtgaccc acgagctggc
cagctccctg gacctgctgc ctaccctggc agccctggct 2400ggggccccac
tgcccaatgt caccttggat ggctttgacc tcagccccct gctgctgggc
2460acaggcaaga gccctcggca gtctctcttc ttctacccgt cctacccaga
cgaggtccgt 2520ggggtttttg ctgtgcggac tggaaagtac aaggctcact
tcttcaccca gggctctgcc 2580cacagtgata ccactgcaga ccctgcctgc
cacgcctcca gctctctgac tgctcatgag 2640cccccgctgc tctatgacct
gtccaaggac cctggtgaga actacaacct gctggggggt 2700gtggccgggg
ccaccccaga ggtgctgcaa gccctgaaac agcttcagct gctcaaggcc
2760cagttagacg cagctgtgac cttcggcccc agccaggtgg cccggggcga
ggaccccgcc 2820ctgcaaatct gctgtcatcc tggctgcacc ccccgcccag
cttgctgcca ttgcccagat 2880ccccatgcct aagcggccgc 2900262279PRTHomo
sapiens 262Asp Pro Pro Ala Pro Leu Pro Leu Val Ile Trp His Gly Met
Gly Asp1 5 10 15Ser Cys Cys Asn Pro Leu Ser Met Gly Ala Ile Lys Lys
Met Val Glu 20 25 30Lys Lys Ile Pro Gly Ile Tyr Val Leu Ser Leu Glu
Ile Gly Lys Thr 35 40 45Leu Met Glu Asp Val Glu Asn Ser Phe Phe Leu
Asn Val Asn Ser Gln 50 55 60Val Thr Thr Val Cys Gln Ala Leu Ala Lys
Asp Pro Lys Leu Gln Gln65 70 75 80Gly Tyr Asn Ala Met Gly Phe Ser
Gln Gly Gly Gln Phe Leu Arg Ala 85 90 95Val Ala Gln Arg Cys Pro Ser
Pro Pro Met Ile Asn Leu Ile Ser Val 100 105 110Gly Gly Gln His Gln
Gly Val Phe Gly Leu Pro Arg Cys Pro Gly Glu 115 120 125Ser Ser His
Ile Cys Asp Phe Ile Arg Lys Thr Leu Asn Ala Gly Ala 130 135 140Tyr
Ser Lys Val Val Gln Glu Arg Leu Val Gln Ala Glu Tyr Trp His145 150
155 160Asp Pro Ile Lys Glu Asp Val Tyr Arg Asn His Ser Ile Phe Leu
Ala 165 170 175Asp Ile Asn Gln Glu Arg Gly Ile Asn Glu Ser Tyr Lys
Lys Asn Leu 180 185 190Met Ala Leu Lys Lys Phe Val Met Val Lys Phe
Leu Asn Asp Ser Ile 195 200 205Val Asp Pro Val Asp Ser Glu Trp Phe
Gly Phe Tyr Arg Ser Gly Gln 210 215 220Ala Lys Glu Thr Ile Pro Leu
Gln Glu Thr Ser Leu Tyr Thr Gln Asp225 230 235 240Arg Leu Gly Leu
Lys Glu Met Asp Asn Ala Gly Gln Leu Val Phe Leu 245 250 255Ala Thr
Glu Gly Asp His Leu Gln Leu Ser Glu Glu Trp Phe Tyr Ala 260 265
270His Ile Ile Pro Phe Leu Gly 275263729PRTArtificial SequenceAmino
acid sequence of the fusion protein of heavy chain of anti-hTfR
antibody No.3 (humanized 2) and hPPT-1 263Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser
Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30Trp Leu Gly Trp
Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Asp Ile
Tyr Pro Gly Gly Asp Tyr Pro Thr Tyr Ser Glu
Lys Phe 50 55 60Lys Val Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser
Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Asn Tyr Asp Glu Val 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 Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410
415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 435 440 445Gly Ser Asp Pro Pro Ala Pro Leu Pro Leu Val Ile
Trp His Gly Met 450 455 460Gly Asp Ser Cys Cys Asn Pro Leu Ser Met
Gly Ala Ile Lys Lys Met465 470 475 480Val Glu Lys Lys Ile Pro Gly
Ile Tyr Val Leu Ser Leu Glu Ile Gly 485 490 495Lys Thr Leu Met Glu
Asp Val Glu Asn Ser Phe Phe Leu Asn Val Asn 500 505 510Ser Gln Val
Thr Thr Val Cys Gln Ala Leu Ala Lys Asp Pro Lys Leu 515 520 525Gln
Gln Gly Tyr Asn Ala Met Gly Phe Ser Gln Gly Gly Gln Phe Leu 530 535
540Arg Ala Val Ala Gln Arg Cys Pro Ser Pro Pro Met Ile Asn Leu
Ile545 550 555 560Ser Val Gly Gly Gln His Gln Gly Val Phe Gly Leu
Pro Arg Cys Pro 565 570 575Gly Glu Ser Ser His Ile Cys Asp Phe Ile
Arg Lys Thr Leu Asn Ala 580 585 590Gly Ala Tyr Ser Lys Val Val Gln
Glu Arg Leu Val Gln Ala Glu Tyr 595 600 605Trp His Asp Pro Ile Lys
Glu Asp Val Tyr Arg Asn His Ser Ile Phe 610 615 620Leu Ala Asp Ile
Asn Gln Glu Arg Gly Ile Asn Glu Ser Tyr Lys Lys625 630 635 640Asn
Leu Met Ala Leu Lys Lys Phe Val Met Val Lys Phe Leu Asn Asp 645 650
655Ser Ile Val Asp Pro Val Asp Ser Glu Trp Phe Gly Phe Tyr Arg Ser
660 665 670Gly Gln Ala Lys Glu Thr Ile Pro Leu Gln Glu Thr Ser Leu
Tyr Thr 675 680 685Gln Asp Arg Leu Gly Leu Lys Glu Met Asp Asn Ala
Gly Gln Leu Val 690 695 700Phe Leu Ala Thr Glu Gly Asp His Leu Gln
Leu Ser Glu Glu Trp Phe705 710 715 720Tyr Ala His Ile Ile Pro Phe
Leu Gly 7252642270DNAArtificial SequenceNucleotide sequence
encoding the amino acid sequence of fusion protein of heavy chain
of anti-hTfR antibody No.3 (humanized 2) and hPPT-1, synthetic
sequence 264acgcgtgccg ccaccatggg ctggagctgg attctgctgt tcctcctgag
cgtgacagca 60ggagtgcaca gcgaggtgca actagtgcag tctggagcag aggtgaaaaa
gcccggggag 120tctctgaaga tttcctgtaa gggttctgga tacagcttta
ccaactactg gctgggatgg 180gtgcgccaga tgcccgggaa aggcctggag
tggatggggg acatctaccc cggcggagac 240taccctacat acagcgagaa
gttcaaggtc caggtcacca tctcagccga caagtccatc 300agcaccgcct
acctgcagtg gagcagcctg aaggcctcgg acaccgccat gtattactgt
360gcgagatcag gcaattacga cgaagtggcc tactggggcc aaggaaccct
ggtcaccgtc 420tcctcagcta gcaccaaggg cccatcggtc ttccccctgg
caccctcctc caagagcacc 480tctgggggca cagcggccct gggctgcctg
gtcaaggact acttccccga accggtgacg 540gtgtcgtgga actcaggcgc
cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 600tcctcaggac
tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc
660cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga
caagaaagtt 720gagcccaaat cttgtgacaa aactcacacg tgcccaccgt
gcccagcacc tgaactcctg 780ggaggtccgt cagtcttcct cttcccccca
aaacccaagg acaccctcat gatctcccgg 840acccctgagg tcacatgcgt
ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900aactggtacg
tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
960tacaacagca cgtaccgggt ggtcagcgtc ctcaccgtcc tgcaccagga
ctggctgaat 1020ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc
cagcccccat cgagaaaacc 1080atctccaaag ccaaagggca gccccgagaa
ccacaggtgt acaccctgcc cccatcccgg 1140gatgagctga ccaagaacca
ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1200gacatcgccg
tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
1260cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt
ggacaagagc 1320aggtggcagc aggggaacgt cttctcatgc tccgtgatgc
atgaggctct gcacaaccac 1380tacacgcaga agagcctctc cctgtctccg
ggtaaaggat ctgacccgcc ggcgccgctg 1440ccgttggtga tctggcatgg
gatgggagac agctgttgca atcccttaag catgggtgct 1500attaaaaaaa
tggtggagaa gaaaatacct ggaatttacg tcttatcttt agagattggg
1560aagaccctga tggaggacgt ggagaacagc ttcttcttga atgtcaattc
ccaagtaaca 1620acagtgtgtc aggcacttgc taaagatcct aaattgcagc
aaggctacaa tgctatggga 1680ttctcccagg gaggccaatt tctgagggca
gtggctcaga gatgcccttc acctcccatg 1740atcaatctga tctcggttgg
gggacaacat caaggtgttt ttggactccc tcgatgccca 1800ggagagagct
ctcacatctg tgacttcatc cgaaaaacac tgaatgctgg ggcgtactcc
1860aaagttgttc aggaacgcct cgtgcaagcc gaatactggc atgaccccat
aaaggaggat 1920gtgtatcgca accacagcat cttcttggca gatataaatc
aggagcgggg tatcaatgag 1980tcctacaaga aaaacctgat ggccctgaag
aagtttgtga tggtgaaatt cctcaatgat 2040tccattgtgg accctgtaga
ttcggagtgg tttggatttt acagaagtgg ccaagccaag 2100gaaaccattc
ccttacagga gacctccctg tacacacagg accgcctggg gctaaaggaa
2160atggacaatg caggacagct agtgtttctg gctacagaag gggaccatct
tcagttgtct 2220gaagaatggt tttatgccca catcatacca ttccttggat
aagcggccgc 2270265544PRTHomo sapiens 265Ser Tyr Ser Pro Glu Pro Asp
Gln Arg Arg Thr Leu Pro Pro Gly Trp1 5 10 15Val Ser Leu Gly Arg Ala
Asp Pro Glu Glu Glu Leu Ser Leu Thr Phe 20 25 30Ala Leu Arg Gln Gln
Asn Val Glu Arg Leu Ser Glu Leu Val Gln Ala 35 40 45Val Ser Asp Pro
Ser Ser Pro Gln Tyr Gly Lys Tyr Leu Thr Leu Glu 50 55 60Asn Val Ala
Asp Leu Val Arg Pro Ser Pro Leu Thr Leu His Thr Val65 70 75 80Gln
Lys Trp Leu Leu Ala Ala Gly Ala Gln Lys Cys His Ser Val Ile 85 90
95Thr Gln Asp Phe Leu Thr Cys Trp Leu Ser Ile Arg Gln Ala Glu Leu
100 105 110Leu Leu Pro Gly Ala Glu Phe His His Tyr Val Gly Gly Pro
Thr Glu 115 120 125Thr His Val Val Arg Ser Pro His Pro Tyr Gln Leu
Pro Gln Ala Leu 130 135 140Ala Pro His Val Asp Phe Val Gly Gly Leu
His His Phe Pro Pro Thr145 150 155 160Ser Ser Leu Arg Gln Arg Pro
Glu Pro Gln Val Thr Gly Thr Val Gly 165 170 175Leu His Leu Gly Val
Thr Pro Ser Val Ile Arg Lys Arg Tyr Asn Leu 180 185 190Thr Ser Gln
Asp Val Gly Ser Gly Thr Ser Asn Asn Ser Gln Ala Cys 195 200 205Ala
Gln Phe Leu Glu Gln Tyr Phe His Asp Ser Asp Leu Ala Gln Phe 210 215
220Met Arg Leu Phe Gly Gly Asn Phe Ala His Gln Ala Ser Val Ala
Arg225 230 235 240Val Val Gly Gln Gln Gly Arg Gly Arg Ala Gly Ile
Glu Ala Ser Leu 245 250 255Asp Val Gln Tyr Leu Met Ser Ala Gly Ala
Asn Ile Ser Thr Trp Val 260 265 270Tyr Ser Ser Pro Gly Arg His Glu
Gly Gln Glu Pro Phe Leu Gln Trp 275 280 285Leu Met Leu Leu Ser Asn
Glu Ser Ala Leu Pro His Val His Thr Val 290 295 300Ser Tyr Gly Asp
Asp Glu Asp Ser Leu Ser Ser Ala Tyr Ile Gln Arg305 310 315 320Val
Asn Thr Glu Leu Met Lys Ala Ala Ala Arg Gly Leu Thr Leu Leu 325 330
335Phe Ala Ser Gly Asp Ser Gly Ala Gly Cys Trp Ser Val Ser Gly Arg
340 345 350His Gln Phe Arg Pro Thr Phe Pro Ala Ser Ser Pro Tyr Val
Thr Thr 355 360 365Val Gly Gly Thr Ser Phe Gln Glu Pro Phe Leu Ile
Thr Asn Glu Ile 370 375 380Val Asp Tyr Ile Ser Gly Gly Gly Phe Ser
Asn Val Phe Pro Arg Pro385 390 395 400Ser Tyr Gln Glu Glu Ala Val
Thr Lys Phe Leu Ser Ser Ser Pro His 405 410 415Leu Pro Pro Ser Ser
Tyr Phe Asn Ala Ser Gly Arg Ala Tyr Pro Asp 420 425 430Val Ala Ala
Leu Ser Asp Gly Tyr Trp Val Val Ser Asn Arg Val Pro 435 440 445Ile
Pro Trp Val Ser Gly Thr Ser Ala Ser Thr Pro Val Phe Gly Gly 450 455
460Ile Leu Ser Leu Ile Asn Glu His Arg Ile Leu Ser Gly Arg Pro
Pro465 470 475 480Leu Gly Phe Leu Asn Pro Arg Leu Tyr Gln Gln His
Gly Ala Gly Leu 485 490 495Phe Asp Val Thr Arg Gly Cys His Glu Ser
Cys Leu Asp Glu Glu Val 500 505 510Glu Gly Gln Gly Phe Cys Ser Gly
Pro Gly Trp Asp Pro Val Thr Gly 515 520 525Trp Gly Thr Pro Asn Phe
Pro Ala Leu Leu Lys Thr Leu Leu Asn Pro 530 535
540266994PRTArtificial SequenceAmino acid sequence of the fusion
protein of heavy chain of anti-hTfR antibody No.3 (humanized 2) and
hTPP-1 266Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe
Thr Asn Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly
Leu Glu Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr Pro Thr
Tyr Ser Glu Lys Phe 50 55 60Lys Val Gln Val Thr Ile Ser Ala Asp Lys
Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala
Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Asn Tyr Asp
Glu Val 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 Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265
270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val 290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser 405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 435 440 445Gly Ser Ser Tyr Ser Pro Glu Pro Asp
Gln Arg Arg Thr Leu Pro Pro 450 455 460Gly Trp Val Ser Leu Gly Arg
Ala Asp Pro Glu Glu Glu Leu Ser Leu465 470 475 480Thr Phe Ala Leu
Arg Gln Gln Asn Val Glu Arg Leu Ser Glu Leu Val 485 490 495Gln Ala
Val Ser Asp Pro Ser Ser Pro Gln Tyr Gly Lys Tyr Leu Thr 500 505
510Leu Glu Asn Val Ala Asp Leu Val Arg Pro Ser Pro Leu Thr Leu His
515 520 525Thr Val Gln Lys Trp Leu Leu Ala Ala Gly Ala Gln Lys Cys
His Ser 530 535 540Val Ile Thr Gln Asp Phe Leu Thr Cys Trp Leu Ser
Ile Arg Gln Ala545 550 555 560Glu Leu Leu Leu Pro Gly Ala Glu Phe
His His Tyr Val Gly Gly Pro 565 570 575Thr Glu Thr His Val Val Arg
Ser Pro His Pro Tyr Gln Leu Pro Gln 580 585 590Ala Leu Ala Pro His
Val Asp Phe Val Gly Gly Leu His His Phe Pro 595 600 605Pro Thr Ser
Ser Leu Arg Gln Arg Pro Glu Pro Gln Val Thr Gly Thr 610 615 620Val
Gly Leu His Leu Gly Val Thr Pro Ser Val Ile Arg Lys Arg Tyr625 630
635 640Asn Leu Thr Ser Gln Asp Val Gly Ser Gly Thr Ser Asn Asn Ser
Gln 645 650 655Ala Cys Ala Gln Phe Leu Glu Gln Tyr Phe His Asp Ser
Asp Leu Ala 660 665 670Gln Phe Met Arg Leu Phe Gly Gly Asn Phe Ala
His Gln Ala Ser Val 675 680 685Ala Arg Val Val Gly Gln Gln Gly Arg
Gly Arg Ala Gly Ile Glu Ala 690 695 700Ser Leu Asp Val Gln Tyr Leu
Met Ser Ala Gly Ala Asn Ile Ser Thr705 710 715 720Trp Val Tyr Ser
Ser Pro Gly Arg His Glu Gly Gln Glu Pro Phe Leu 725 730 735Gln Trp
Leu Met Leu Leu Ser Asn Glu Ser Ala Leu Pro His Val His 740 745
750Thr Val Ser Tyr Gly Asp Asp Glu Asp Ser Leu Ser Ser Ala Tyr Ile
755 760 765Gln Arg Val Asn Thr Glu Leu Met Lys Ala Ala Ala Arg Gly
Leu Thr 770 775 780Leu Leu Phe Ala Ser Gly Asp Ser Gly Ala Gly Cys
Trp Ser Val Ser785 790 795 800Gly Arg His Gln Phe Arg Pro Thr Phe
Pro Ala Ser Ser Pro Tyr Val 805 810 815Thr Thr Val Gly Gly Thr Ser
Phe Gln Glu Pro Phe Leu Ile Thr Asn 820 825 830Glu
Ile Val Asp Tyr Ile Ser Gly Gly Gly Phe Ser Asn Val Phe Pro 835 840
845Arg Pro Ser Tyr Gln Glu Glu Ala Val Thr Lys Phe Leu Ser Ser Ser
850 855 860Pro His Leu Pro Pro Ser Ser Tyr Phe Asn Ala Ser Gly Arg
Ala Tyr865 870 875 880Pro Asp Val Ala Ala Leu Ser Asp Gly Tyr Trp
Val Val Ser Asn Arg 885 890 895Val Pro Ile Pro Trp Val Ser Gly Thr
Ser Ala Ser Thr Pro Val Phe 900 905 910Gly Gly Ile Leu Ser Leu Ile
Asn Glu His Arg Ile Leu Ser Gly Arg 915 920 925Pro Pro Leu Gly Phe
Leu Asn Pro Arg Leu Tyr Gln Gln His Gly Ala 930 935 940Gly Leu Phe
Asp Val Thr Arg Gly Cys His Glu Ser Cys Leu Asp Glu945 950 955
960Glu Val Glu Gly Gln Gly Phe Cys Ser Gly Pro Gly Trp Asp Pro Val
965 970 975Thr Gly Trp Gly Thr Pro Asn Phe Pro Ala Leu Leu Lys Thr
Leu Leu 980 985 990Asn Pro2673065DNAArtificial SequenceNucleotide
sequence encoding the amino acid sequence of fusion protein of
heavy chain of anti-hTfR antibody No.3 (humanized 2) and hTPP-1,
synthetic sequence 267acgcgtgccg ccaccatggg ctggagctgg attctgctgt
tcctcctgag cgtgacagca 60ggagtgcaca gcgaggtgca actagtgcag tctggagcag
aggtgaaaaa gcccggggag 120tctctgaaga tttcctgtaa gggttctgga
tacagcttta ccaactactg gctgggatgg 180gtgcgccaga tgcccgggaa
aggcctggag tggatggggg acatctaccc cggcggagac 240taccctacat
acagcgagaa gttcaaggtc caggtcacca tctcagccga caagtccatc
300agcaccgcct acctgcagtg gagcagcctg aaggcctcgg acaccgccat
gtattactgt 360gcgagatcag gcaattacga cgaagtggcc tactggggcc
aaggaaccct ggtcaccgtc 420tcctcagcta gcaccaaggg cccatcggtc
ttccccctgg caccctcctc caagagcacc 480tctgggggca cagcggccct
gggctgcctg gtcaaggact acttccccga accggtgacg 540gtgtcgtgga
actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag
600tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag
cttgggcacc 660cagacctaca tctgcaacgt gaatcacaag cccagcaaca
ccaaggtgga caagaaagtt 720gagcccaaat cttgtgacaa aactcacacg
tgcccaccgt gcccagcacc tgaactcctg 780ggaggtccgt cagtcttcct
cttcccccca aaacccaagg acaccctcat gatctcccgg 840acccctgagg
tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc
900aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg
ggaggagcag 960tacaacagca cgtaccgggt ggtcagcgtc ctcaccgtcc
tgcaccagga ctggctgaat 1020ggcaaggagt acaagtgcaa ggtctccaac
aaagccctcc cagcccccat cgagaaaacc 1080atctccaaag ccaaagggca
gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1140gatgagctga
ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc
1200gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa
gaccacgcct 1260cccgtgctgg actccgacgg ctccttcttc ctctacagca
agctcaccgt ggacaagagc 1320aggtggcagc aggggaacgt cttctcatgc
tccgtgatgc atgaggctct gcacaaccac 1380tacacgcaga agagcctctc
cctgtctccg ggtaaaggat ctagttacag cccggagccc 1440gaccagcgga
ggacgctgcc cccaggctgg gtgtccctgg gccgtgcgga ccctgaggaa
1500gagctgagtc tcacctttgc cctgagacag cagaatgtgg aaagactctc
ggagctggtg 1560caggctgtgt cagatcccag ctctcctcaa tacggaaaat
acctgaccct agagaatgtg 1620gctgatctgg tgaggccatc cccactgacc
ctccacacgg tgcaaaaatg gctcttggca 1680gccggagccc agaagtgcca
ttctgtgatc acacaggact ttctgacttg ctggctgagc 1740atccgacaag
cagagctgct gctccctggg gctgagtttc atcactatgt gggaggacct
1800acggaaaccc atgttgtaag gtccccacat ccctaccagc ttccacaggc
cttggccccc 1860catgtggact ttgtgggggg actgcaccat tttcccccaa
catcatccct gaggcaacgt 1920cctgagccgc aggtgacagg gactgtaggc
ctgcatctgg gggtaacccc ctctgtgatc 1980cgtaagcgat acaacttgac
ctcacaagac gtgggctctg gcaccagcaa taacagccaa 2040gcctgtgccc
agttcctgga gcagtatttc catgactcag acctggctca gttcatgcgc
2100ctcttcggtg gcaactttgc acatcaggca tcagtagccc gtgtggttgg
acaacagggc 2160cggggccggg ccgggattga ggccagtcta gatgtgcagt
acctgatgag tgctggtgcc 2220aacatctcca cctgggtcta cagtagccct
ggccggcatg agggacagga gcccttcctg 2280cagtggctca tgctgctcag
taatgagtca gccctgccac atgtgcatac tgtgagctat 2340ggagatgatg
aggactccct cagcagcgcc tacatccagc gggtcaacac tgagctcatg
2400aaggctgctg ctcggggtct caccctgctc ttcgcctcag gtgacagtgg
ggccgggtgt 2460tggtctgtct ctggaagaca ccagttccgc cctaccttcc
ctgcctccag cccctatgtc 2520accacagtgg gaggcacatc cttccaggaa
cctttcctca tcacaaatga aattgttgac 2580tatatcagtg gtggtggctt
cagcaatgtg ttcccacggc cttcatacca ggaggaagct 2640gtaacgaagt
tcctgagctc tagcccccac ctgccaccat ccagttactt caatgccagt
2700ggccgtgcct acccagatgt ggctgcactt tctgatggct actgggtggt
cagcaacaga 2760gtgcccattc catgggtgtc cggaacctcg gcctctactc
cagtgtttgg gggtatccta 2820tccttgatca atgagcacag aatccttagt
ggccgccccc ctcttggctt tctcaaccca 2880aggctctacc agcagcatgg
ggcaggactc tttgatgtaa cccgtggctg ccatgagtcc 2940tgtctggatg
aagaggtaga gggccagggt ttctgctctg gtcctggctg ggaccctgta
3000acaggctggg gaacacccaa cttcccagct ttgctgaaga ctctactcaa
cccctaagcg 3060gccgc 3065268628PRTHomo sapiens 268Ala Glu Ala Pro
His Leu Val His Val Asp Ala Ala Arg Ala Leu Trp1 5 10 15Pro Leu Arg
Arg Phe Trp Arg Ser Thr Gly Phe Cys Pro Pro Leu Pro 20 25 30His Ser
Gln Ala Asp Gln Tyr Val Leu Ser Trp Asp Gln Gln Leu Asn 35 40 45Leu
Ala Tyr Val Gly Ala Val Pro His Arg Gly Ile Lys Gln Val Arg 50 55
60Thr His Trp Leu Leu Glu Leu Val Thr Thr Arg Gly Ser Thr Gly Arg65
70 75 80Gly Leu Ser Tyr Asn Phe Thr His Leu Asp Gly Tyr Leu Asp Leu
Leu 85 90 95Arg Glu Asn Gln Leu Leu Pro Gly Phe Glu Leu Met Gly Ser
Ala Ser 100 105 110Gly His Phe Thr Asp Phe Glu Asp Lys Gln Gln Val
Phe Glu Trp Lys 115 120 125Asp Leu Val Ser Ser Leu Ala Arg Arg Tyr
Ile Gly Arg Tyr Gly Leu 130 135 140Ala His Val Ser Lys Trp Asn Phe
Glu Thr Trp Asn Glu Pro Asp His145 150 155 160His Asp Phe Asp Asn
Val Ser Met Thr Met Gln Gly Phe Leu Asn Tyr 165 170 175Tyr Asp Ala
Cys Ser Glu Gly Leu Arg Ala Ala Ser Pro Ala Leu Arg 180 185 190Leu
Gly Gly Pro Gly Asp Ser Phe His Thr Pro Pro Arg Ser Pro Leu 195 200
205Ser Trp Gly Leu Leu Arg His Cys His Asp Gly Thr Asn Phe Phe Thr
210 215 220Gly Glu Ala Gly Val Arg Leu Asp Tyr Ile Ser Leu His Arg
Lys Gly225 230 235 240Ala Arg Ser Ser Ile Ser Ile Leu Glu Gln Glu
Lys Val Val Ala Gln 245 250 255Gln Ile Arg Gln Leu Phe Pro Lys Phe
Ala Asp Thr Pro Ile Tyr Asn 260 265 270Asp Glu Ala Asp Pro Leu Val
Gly Trp Ser Leu Pro Gln Pro Trp Arg 275 280 285Ala Asp Val Thr Tyr
Ala Ala Met Val Val Lys Val Ile Ala Gln His 290 295 300Gln Asn Leu
Leu Leu Ala Asn Thr Thr Ser Ala Phe Pro Tyr Ala Leu305 310 315
320Leu Ser Asn Asp Asn Ala Phe Leu Ser Tyr His Pro His Pro Phe Ala
325 330 335Gln Arg Thr Leu Thr Ala Arg Phe Gln Val Asn Asn Thr Arg
Pro Pro 340 345 350His Val Gln Leu Leu Arg Lys Pro Val Leu Thr Ala
Met Gly Leu Leu 355 360 365Ala Leu Leu Asp Glu Glu Gln Leu Trp Ala
Glu Val Ser Gln Ala Gly 370 375 380Thr Val Leu Asp Ser Asn His Thr
Val Gly Val Leu Ala Ser Ala His385 390 395 400Arg Pro Gln Gly Pro
Ala Asp Ala Trp Arg Ala Ala Val Leu Ile Tyr 405 410 415Ala Ser Asp
Asp Thr Arg Ala His Pro Asn Arg Ser Val Ala Val Thr 420 425 430Leu
Arg Leu Arg Gly Val Pro Pro Gly Pro Gly Leu Val Tyr Val Thr 435 440
445Arg Tyr Leu Asp Asn Gly Leu Cys Ser Pro Asp Gly Glu Trp Arg Arg
450 455 460Leu Gly Arg Pro Val Phe Pro Thr Ala Glu Gln Phe Arg Arg
Met Arg465 470 475 480Ala Ala Glu Asp Pro Val Ala Ala Ala Pro Arg
Pro Leu Pro Ala Gly 485 490 495Gly Arg Leu Thr Leu Arg Pro Ala Leu
Arg Leu Pro Ser Leu Leu Leu 500 505 510Val His Val Cys Ala Arg Pro
Glu Lys Pro Pro Gly Gln Val Thr Arg 515 520 525Leu Arg Ala Leu Pro
Leu Thr Gln Gly Gln Leu Val Leu Val Trp Ser 530 535 540Asp Glu His
Val Gly Ser Lys Cys Leu Trp Thr Tyr Glu Ile Gln Phe545 550 555
560Ser Gln Asp Gly Lys Ala Tyr Thr Pro Val Ser Arg Lys Pro Ser Thr
565 570 575Phe Asn Leu Phe Val Phe Ser Pro Asp Thr Gly Ala Val Ser
Gly Ser 580 585 590Tyr Arg Val Arg Ala Leu Asp Tyr Trp Ala Arg Pro
Gly Pro Phe Ser 595 600 605Asp Pro Val Pro Tyr Leu Glu Val Pro Val
Pro Arg Gly Pro Pro Ser 610 615 620Pro Gly Asn
Pro6252691078PRTArtificial SequenceAmino acid sequence of the
fusion protein of heavy chain of anti-hTfR antibody No.3 (humanized
2) and hIDUA 269Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser
Phe Thr Asn Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys
Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp Tyr Pro
Thr Tyr Ser Glu Lys Phe 50 55 60Lys Val Gln Val Thr Ile Ser Ala Asp
Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys
Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Gly Asn Tyr
Asp Glu Val 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 Lys Thr 210 215 220His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230 235 240Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250
255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375
380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445Gly Ser Ala Glu Ala Pro
His Leu Val His Val Asp Ala Ala Arg Ala 450 455 460Leu Trp Pro Leu
Arg Arg Phe Trp Arg Ser Thr Gly Phe Cys Pro Pro465 470 475 480Leu
Pro His Ser Gln Ala Asp Gln Tyr Val Leu Ser Trp Asp Gln Gln 485 490
495Leu Asn Leu Ala Tyr Val Gly Ala Val Pro His Arg Gly Ile Lys Gln
500 505 510Val Arg Thr His Trp Leu Leu Glu Leu Val Thr Thr Arg Gly
Ser Thr 515 520 525Gly Arg Gly Leu Ser Tyr Asn Phe Thr His Leu Asp
Gly Tyr Leu Asp 530 535 540Leu Leu Arg Glu Asn Gln Leu Leu Pro Gly
Phe Glu Leu Met Gly Ser545 550 555 560Ala Ser Gly His Phe Thr Asp
Phe Glu Asp Lys Gln Gln Val Phe Glu 565 570 575Trp Lys Asp Leu Val
Ser Ser Leu Ala Arg Arg Tyr Ile Gly Arg Tyr 580 585 590Gly Leu Ala
His Val Ser Lys Trp Asn Phe Glu Thr Trp Asn Glu Pro 595 600 605Asp
His His Asp Phe Asp Asn Val Ser Met Thr Met Gln Gly Phe Leu 610 615
620Asn Tyr Tyr Asp Ala Cys Ser Glu Gly Leu Arg Ala Ala Ser Pro
Ala625 630 635 640Leu Arg Leu Gly Gly Pro Gly Asp Ser Phe His Thr
Pro Pro Arg Ser 645 650 655Pro Leu Ser Trp Gly Leu Leu Arg His Cys
His Asp Gly Thr Asn Phe 660 665 670Phe Thr Gly Glu Ala Gly Val Arg
Leu Asp Tyr Ile Ser Leu His Arg 675 680 685Lys Gly Ala Arg Ser Ser
Ile Ser Ile Leu Glu Gln Glu Lys Val Val 690 695 700Ala Gln Gln Ile
Arg Gln Leu Phe Pro Lys Phe Ala Asp Thr Pro Ile705 710 715 720Tyr
Asn Asp Glu Ala Asp Pro Leu Val Gly Trp Ser Leu Pro Gln Pro 725 730
735Trp Arg Ala Asp Val Thr Tyr Ala Ala Met Val Val Lys Val Ile Ala
740 745 750Gln His Gln Asn Leu Leu Leu Ala Asn Thr Thr Ser Ala Phe
Pro Tyr 755 760 765Ala Leu Leu Ser Asn Asp Asn Ala Phe Leu Ser Tyr
His Pro His Pro 770 775 780Phe Ala Gln Arg Thr Leu Thr Ala Arg Phe
Gln Val Asn Asn Thr Arg785 790 795 800Pro Pro His Val Gln Leu Leu
Arg Lys Pro Val Leu Thr Ala Met Gly 805 810 815Leu Leu Ala Leu Leu
Asp Glu Glu Gln Leu Trp Ala Glu Val Ser Gln 820 825 830Ala Gly Thr
Val Leu Asp Ser Asn His Thr Val Gly Val Leu Ala Ser 835 840 845Ala
His Arg Pro Gln Gly Pro Ala Asp Ala Trp Arg Ala Ala Val Leu 850 855
860Ile Tyr Ala Ser Asp Asp Thr Arg Ala His Pro Asn Arg Ser Val
Ala865 870 875 880Val Thr Leu Arg Leu Arg Gly Val Pro Pro Gly Pro
Gly Leu Val Tyr 885 890 895Val Thr Arg Tyr Leu Asp Asn Gly Leu Cys
Ser Pro Asp Gly Glu Trp 900 905 910Arg Arg Leu Gly Arg Pro Val Phe
Pro Thr Ala Glu Gln Phe Arg Arg 915 920 925Met Arg Ala Ala Glu Asp
Pro Val Ala Ala Ala Pro Arg Pro Leu Pro 930 935 940Ala Gly Gly Arg
Leu Thr Leu Arg Pro Ala Leu Arg Leu Pro Ser Leu945 950 955 960Leu
Leu Val His Val Cys Ala Arg Pro Glu Lys Pro Pro Gly Gln Val 965 970
975Thr Arg Leu Arg Ala Leu Pro Leu Thr Gln Gly Gln Leu Val Leu Val
980 985 990Trp Ser Asp Glu His Val Gly Ser Lys Cys Leu Trp Thr Tyr
Glu Ile 995 1000 1005Gln Phe Ser Gln Asp Gly Lys Ala Tyr Thr Pro
Val Ser Arg Lys 1010 1015 1020Pro Ser Thr Phe Asn Leu Phe Val Phe
Ser Pro Asp Thr Gly Ala 1025 1030 1035Val Ser Gly Ser Tyr Arg Val
Arg Ala Leu Asp Tyr Trp Ala Arg 1040 1045 1050Pro Gly Pro Phe Ser
Asp Pro Val Pro Tyr Leu Glu Val Pro Val 1055 1060 1065Pro Arg Gly
Pro Pro Ser Pro Gly Asn Pro 1070 10752703317DNAArtificial
SequenceNucleotide sequence encoding the amino acid sequence of
fusion protein of heavy chain of anti-hTfR antibody No.3 (humanized
2) and hIDUA, synthetic sequence 270acgcgtgccg ccaccatggg
ctggagctgg attctgctgt tcctcctgag cgtgacagca 60ggagtgcaca gcgaggtgca
actagtgcag tctggagcag aggtgaaaaa gcccggggag
120tctctgaaga tttcctgtaa gggttctgga tacagcttta ccaactactg
gctgggatgg 180gtgcgccaga tgcccgggaa aggcctggag tggatggggg
acatctaccc cggcggagac 240taccctacat acagcgagaa gttcaaggtc
caggtcacca tctcagccga caagtccatc 300agcaccgcct acctgcagtg
gagcagcctg aaggcctcgg acaccgccat gtattactgt 360gcgagatcag
gcaattacga cgaagtggcc tactggggcc aaggaaccct ggtcaccgtc
420tcctcagcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc
caagagcacc 480tctgggggca cagcggccct gggctgcctg gtcaaggact
acttccccga accggtgacg 540gtgtcgtgga actcaggcgc cctgaccagc
ggcgtgcaca ccttcccggc tgtcctacag 600tcctcaggac tctactccct
cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 660cagacctaca
tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt
720gagcccaaat cttgtgacaa aactcacacg tgcccaccgt gcccagcacc
tgaactcctg 780ggaggtccgt cagtcttcct cttcccccca aaacccaagg
acaccctcat gatctcccgg 840acccctgagg tcacatgcgt ggtggtggac
gtgagccacg aagaccctga ggtcaagttc 900aactggtacg tggacggcgt
ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960tacaacagca
cgtaccgggt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat
1020ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat
cgagaaaacc 1080atctccaaag ccaaagggca gccccgagaa ccacaggtgt
acaccctgcc cccatcccgg 1140gatgagctga ccaagaacca ggtcagcctg
acctgcctgg tcaaaggctt ctatcccagc 1200gacatcgccg tggagtggga
gagcaatggg cagccggaga acaactacaa gaccacgcct 1260cccgtgctgg
actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc
1320aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct
gcacaaccac 1380tacacgcaga agagcctctc cctgtctccg ggtaaaggat
ctgccgaggc cccgcacctg 1440gtgcacgtgg acgcggcccg cgcgctgtgg
cccctgcggc gcttctggag gagcacaggc 1500ttctgccccc cgctgccaca
cagccaggct gaccagtacg tcctcagctg ggaccagcag 1560ctcaacctcg
cctatgtggg cgccgtccct caccgcggca tcaagcaggt ccggacccac
1620tggctgctgg agcttgtcac caccaggggg tccactggac ggggcctgag
ctacaacttc 1680acccacctgg acgggtactt ggaccttctc agggagaacc
agctcctccc agggtttgag 1740ctgatgggca gcgcctcggg ccacttcact
gactttgagg acaagcagca ggtgtttgag 1800tggaaggact tggtctccag
cctggccagg agatacatcg gtaggtacgg actggcgcat 1860gtttccaagt
ggaacttcga gacgtggaat gagccagacc accacgactt tgacaacgtc
1920tccatgacca tgcaaggctt cctgaactac tacgatgcct gctcggaggg
tctgcgcgcc 1980gccagccccg ccctgcggct gggaggcccc ggcgactcct
tccacacccc accgcgatcc 2040ccgctgagct ggggcctcct gcgccactgc
cacgacggta ccaacttctt cactggggag 2100gcgggcgtgc ggctggacta
catctccctc cacaggaagg gtgcgcgcag ctccatctcc 2160atcctggagc
aggagaaggt cgtcgcgcag cagatccggc agctcttccc caagttcgcg
2220gacaccccca tttacaacga cgaggcggac ccgctggtgg gctggtccct
gccacagccg 2280tggagggcgg acgtgaccta cgcggccatg gtggtgaagg
tcatcgcgca gcatcagaac 2340ctgctactgg ccaacaccac ctccgccttc
ccctacgcgc tcctgagcaa cgacaatgcc 2400ttcctgagct accacccgca
ccccttcgcg cagcgcacgc tcaccgcgcg cttccaggtc 2460aacaacaccc
gcccgccgca cgtgcagctg ttgcgcaagc cggtgctcac ggccatgggg
2520ctgctggcgc tgctggatga ggagcagctc tgggccgaag tgtcgcaggc
cgggaccgtc 2580ctggacagca accacacggt gggcgtcctg gccagcgccc
accgccccca gggcccggcc 2640gacgcctggc gcgccgcggt gctgatctac
gcgagcgacg acacccgcgc ccaccccaac 2700cgcagcgtcg cggtgaccct
gcggctgcgc ggggtgcccc ccggcccggg cctggtctac 2760gtcacgcgct
acctggacaa cgggctctgc agccccgacg gcgagtggcg gcgcctgggc
2820cggcccgtct tccccacggc agagcagttc cggcgcatgc gcgcggctga
ggacccggtg 2880gccgcggcgc cccgcccctt acccgccggt ggccgcctga
ccctgcgccc cgcgctgcgg 2940ctgccgtcgc ttttgctggt gcacgtgtgt
gcgcgccccg agaagccgcc cgggcaggtc 3000acgcggctcc gcgccctgcc
cctgacccaa gggcagctgg ttctggtctg gtcggatgaa 3060cacgtgggct
ccaagtgcct gtggacatac gagatccagt tctctcagga cggtaaggcg
3120tacaccccgg tcagcaggaa gccatcgacc ttcaacctct ttgtgttcag
cccagacaca 3180ggtgctgtct ctggctccta ccgagttcga gccctggact
actgggcccg accaggcccc 3240ttctcggacc ctgtgccgta cctggaggtc
cctgtgccaa gagggccccc atccccgggc 3300aatccataag cggccgc
3317271235PRTHomo sapiens 271Leu Pro Ala Gln Val Ala Phe Thr Pro
Tyr Ala Pro Glu Pro Gly Ser1 5 10 15Thr Cys Arg Leu Arg Glu Tyr Tyr
Asp Gln Thr Ala Gln Met Cys Cys 20 25 30Ser Lys Cys Ser Pro Gly Gln
His Ala Lys Val Phe Cys Thr Lys Thr 35 40 45Ser Asp Thr Val Cys Asp
Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55 60Trp Asn Trp Val Pro
Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser65 70 75 80Asp Gln Val
Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 85 90 95Thr Cys
Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys 100 105
110Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala
115 120 125Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys
Ala Pro 130 135 140Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile
Cys Arg Pro His145 150 155 160Gln Ile Cys Asn Val Val Ala Ile Pro
Gly Asn Ala Ser Met Asp Ala 165 170 175Val Cys Thr Ser Thr Ser Pro
Thr Arg Ser Met Ala Pro Gly Ala Val 180 185 190His Leu Pro Gln Pro
Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 195 200 205Pro Glu Pro
Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly 210 215 220Pro
Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp225 230
235272685PRTArtificial SequenceAmino acid sequence of the fusion
protein of heavy chain of anti-hTfR antibody No.3 (humanized 2) and
hTNF-alpha receptor 272Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly
Tyr Ser Phe Thr Asn Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln Met Pro
Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly Gly Asp
Tyr Pro Thr Tyr Ser Glu Lys Phe 50 55 60Lys Val Gln Val Thr Ile Ser
Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser
Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Ser Gly
Asn Tyr Asp Glu Val 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 Lys Thr 210 215 220His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445Gly Ser Leu Pro Ala
Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro 450 455 460Gly Ser Thr
Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met465 470 475
480Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr
485 490 495Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr
Tyr Thr 500 505 510Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys
Gly Ser Arg Cys 515 520 525Ser Ser Asp Gln Val Glu Thr Gln Ala Cys
Thr Arg Glu Gln Asn Arg 530 535 540Ile Cys Thr Cys Arg Pro Gly Trp
Tyr Cys Ala Leu Ser Lys Gln Glu545 550 555 560Gly Cys Arg Leu Cys
Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly 565 570 575Val Ala Arg
Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys 580 585 590Ala
Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg 595 600
605Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met
610 615 620Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala
Pro Gly625 630 635 640Ala Val His Leu Pro Gln Pro Val Ser Thr Arg
Ser Gln His Thr Gln 645 650 655Pro Thr Pro Glu Pro Ser Thr Ala Pro
Ser Thr Ser Phe Leu Leu Pro 660 665 670Met Gly Pro Ser Pro Pro Ala
Glu Gly Ser Thr Gly Asp 675 680 6852732138DNAArtificial
SequenceNucleotide sequence encoding the amino acid sequence of
fusion protein of heavy chain of anti-hTfR antibody No.3 (humanized
2) and hTNF-alpha receptor, synthetic sequence 273acgcgtgccg
ccaccatggg ctggagctgg attctgctgt tcctcctgag cgtgacagca 60ggagtgcaca
gcgaggtgca actagtgcag tctggagcag aggtgaaaaa gcccggggag
120tctctgaaga tttcctgtaa gggttctgga tacagcttta ccaactactg
gctgggatgg 180gtgcgccaga tgcccgggaa aggcctggag tggatggggg
acatctaccc cggcggagac 240taccctacat acagcgagaa gttcaaggtc
caggtcacca tctcagccga caagtccatc 300agcaccgcct acctgcagtg
gagcagcctg aaggcctcgg acaccgccat gtattactgt 360gcgagatcag
gcaattacga cgaagtggcc tactggggcc aaggaaccct ggtcaccgtc
420tcctcagcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc
caagagcacc 480tctgggggca cagcggccct gggctgcctg gtcaaggact
acttccccga accggtgacg 540gtgtcgtgga actcaggcgc cctgaccagc
ggcgtgcaca ccttcccggc tgtcctacag 600tcctcaggac tctactccct
cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 660cagacctaca
tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt
720gagcccaaat cttgtgacaa aactcacacg tgcccaccgt gcccagcacc
tgaactcctg 780ggaggtccgt cagtcttcct cttcccccca aaacccaagg
acaccctcat gatctcccgg 840acccctgagg tcacatgcgt ggtggtggac
gtgagccacg aagaccctga ggtcaagttc 900aactggtacg tggacggcgt
ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960tacaacagca
cgtaccgggt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat
1020ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat
cgagaaaacc 1080atctccaaag ccaaagggca gccccgagaa ccacaggtgt
acaccctgcc cccatcccgg 1140gatgagctga ccaagaacca ggtcagcctg
acctgcctgg tcaaaggctt ctatcccagc 1200gacatcgccg tggagtggga
gagcaatggg cagccggaga acaactacaa gaccacgcct 1260cccgtgctgg
actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc
1320aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct
gcacaaccac 1380tacacgcaga agagcctctc cctgtctccg ggtaaaggat
ctttgcccgc ccaggtggca 1440tttacaccct acgccccgga gcccgggagc
acatgccggc tcagagaata ctatgaccag 1500acagctcaga tgtgctgcag
caagtgctcg ccgggccaac atgcaaaagt cttctgtacc 1560aagacctcgg
acaccgtgtg tgactcctgt gaggacagca catacaccca gctctggaac
1620tgggttcccg agtgcttgag ctgtggctcc cgctgtagct ctgaccaggt
ggaaactcaa 1680gcctgcactc gggaacagaa ccgcatctgc acctgcaggc
ccggctggta ctgcgcgctg 1740agcaagcagg aggggtgccg gctgtgcgcg
ccgctgcgca agtgccgccc gggcttcggc 1800gtggccagac caggaactga
aacatcagac gtggtgtgca agccctgtgc cccggggacg 1860ttctccaaca
cgacttcatc cacggatatt tgcaggcccc accaaatctg taacgtggtg
1920gccatccctg ggaatgcaag catggatgca gtctgcacgt ccacgtcccc
cacccggagt 1980atggccccag gggcagtaca cttaccccag ccagtgtcca
cacgatccca acacacgcag 2040ccaactccag aacccagcac tgctccaagc
acctccttcc tgctcccaat gggccccagc 2100cccccagctg aagggagcac
tggcgactaa gcggccgc 2138274482PRTHomo sapiens 274Arg Pro Arg Asn
Ala Leu Leu Leu Leu Ala Asp Asp Gly Gly Phe Glu1 5 10 15Ser Gly Ala
Tyr Asn Asn Ser Ala Ile Ala Thr Pro His Leu Asp Ala 20 25 30Leu Ala
Arg Arg Ser Leu Leu Phe Arg Asn Ala Phe Thr Ser Val Ser 35 40 45Ser
Cys Ser Pro Ser Arg Ala Ser Leu Leu Thr Gly Leu Pro Gln His 50 55
60Gln Asn Gly Met Tyr Gly Leu His Gln Asp Val His His Phe Asn Ser65
70 75 80Phe Asp Lys Val Arg Ser Leu Pro Leu Leu Leu Ser Gln Ala Gly
Val 85 90 95Arg Thr Gly Ile Ile Gly Lys Lys His Val Gly Pro Glu Thr
Val Tyr 100 105 110Pro Phe Asp Phe Ala Tyr Thr Glu Glu Asn Gly Ser
Val Leu Gln Val 115 120 125Gly Arg Asn Ile Thr Arg Ile Lys Leu Leu
Val Arg Lys Phe Leu Gln 130 135 140Thr Gln Asp Asp Arg Pro Phe Phe
Leu Tyr Val Ala Phe His Asp Pro145 150 155 160His Arg Cys Gly His
Ser Gln Pro Gln Tyr Gly Thr Phe Cys Glu Lys 165 170 175Phe Gly Asn
Gly Glu Ser Gly Met Gly Arg Ile Pro Asp Trp Thr Pro 180 185 190Gln
Ala Tyr Asp Pro Leu Asp Val Leu Val Pro Tyr Phe Val Pro Asn 195 200
205Thr Pro Ala Ala Arg Ala Asp Leu Ala Ala Gln Tyr Thr Thr Val Gly
210 215 220Arg Met Asp Gln Gly Val Gly Leu Val Leu Gln Glu Leu Arg
Asp Ala225 230 235 240Gly Val Leu Asn Asp Thr Leu Val Ile Phe Thr
Ser Asp Asn Gly Ile 245 250 255Pro Phe Pro Ser Gly Arg Thr Asn Leu
Tyr Trp Pro Gly Thr Ala Glu 260 265 270Pro Leu Leu Val Ser Ser Pro
Glu His Pro Lys Arg Trp Gly Gln Val 275 280 285Ser Glu Ala Tyr Val
Ser Leu Leu Asp Leu Thr Pro Thr Ile Leu Asp 290 295 300Trp Phe Ser
Ile Pro Tyr Pro Ser Tyr Ala Ile Phe Gly Ser Lys Thr305 310 315
320Ile His Leu Thr Gly Arg Ser Leu Leu Pro Ala Leu Glu Ala Glu Pro
325 330 335Leu Trp Ala Thr Val Phe Gly Ser Gln Ser His His Glu Val
Thr Met 340 345 350Ser Tyr Pro Met Arg Ser Val Gln His Arg His Phe
Arg Leu Val His 355 360 365Asn Leu Asn Phe Lys Met Pro Phe Pro Ile
Asp Gln Asp Phe Tyr Val 370 375 380Ser Pro Thr Phe Gln Asp Leu Leu
Asn Arg Thr Thr Ala Gly Gln Pro385 390 395 400Thr Gly Trp Tyr Lys
Asp Leu Arg His Tyr Tyr Tyr Arg Ala Arg Trp 405 410 415Glu Leu Tyr
Asp Arg Ser Arg Asp Pro His Glu Thr Gln Asn Leu Ala 420 425 430Thr
Asp Pro Arg Phe Ala Gln Leu Leu Glu Met Leu Arg Asp Gln Leu 435 440
445Ala Lys Trp Gln Trp Glu Thr His Asp Pro Trp Val Cys Ala Pro Asp
450 455 460Gly Val Leu Glu Glu Lys Leu Ser Pro Gln Cys Gln Pro Leu
His Asn465 470 475 480Glu Leu275932PRTArtificial SequenceAmino acid
sequence of the fusion protein of heavy chain of anti-hTfR antibody
No.3 (humanized 2) and hSGSH 275Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys Gly
Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30Trp Leu Gly Trp Val Arg Gln
Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Asp Ile Tyr Pro Gly
Gly Asp Tyr Pro Thr Tyr Ser Glu Lys Phe 50 55 60Lys Val Gln Val Thr
Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp
Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg
Ser Gly Asn Tyr Asp Glu Val 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 Lys Thr 210 215
220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445Gly
Ser Arg Pro Arg Asn Ala Leu Leu Leu Leu Ala Asp Asp Gly Gly 450 455
460Phe Glu Ser Gly Ala Tyr Asn Asn Ser Ala Ile Ala Thr Pro His
Leu465 470 475 480Asp Ala Leu Ala Arg Arg Ser Leu Leu Phe Arg Asn
Ala Phe Thr Ser 485 490 495Val Ser Ser Cys Ser Pro Ser Arg Ala Ser
Leu Leu Thr Gly Leu Pro 500 505 510Gln His Gln Asn Gly Met Tyr Gly
Leu His Gln Asp Val His His Phe 515 520 525Asn Ser Phe Asp Lys Val
Arg Ser Leu Pro Leu Leu Leu Ser Gln Ala 530 535 540Gly Val Arg Thr
Gly Ile Ile Gly Lys Lys His Val Gly Pro Glu Thr545 550 555 560Val
Tyr Pro Phe Asp Phe Ala Tyr Thr Glu Glu Asn Gly Ser Val Leu 565 570
575Gln Val Gly Arg Asn Ile Thr Arg Ile Lys Leu Leu Val Arg Lys Phe
580 585 590Leu Gln Thr Gln Asp Asp Arg Pro Phe Phe Leu Tyr Val Ala
Phe His 595 600 605Asp Pro His Arg Cys Gly His Ser Gln Pro Gln Tyr
Gly Thr Phe Cys 610 615 620Glu Lys Phe Gly Asn Gly Glu Ser Gly Met
Gly Arg Ile Pro Asp Trp625 630 635 640Thr Pro Gln Ala Tyr Asp Pro
Leu Asp Val Leu Val Pro Tyr Phe Val 645 650 655Pro Asn Thr Pro Ala
Ala Arg Ala Asp Leu Ala Ala Gln Tyr Thr Thr 660 665 670Val Gly Arg
Met Asp Gln Gly Val Gly Leu Val Leu Gln Glu Leu Arg 675 680 685Asp
Ala Gly Val Leu Asn Asp Thr Leu Val Ile Phe Thr Ser Asp Asn 690 695
700Gly Ile Pro Phe Pro Ser Gly Arg Thr Asn Leu Tyr Trp Pro Gly
Thr705 710 715 720Ala Glu Pro Leu Leu Val Ser Ser Pro Glu His Pro
Lys Arg Trp Gly 725 730 735Gln Val Ser Glu Ala Tyr Val Ser Leu Leu
Asp Leu Thr Pro Thr Ile 740 745 750Leu Asp Trp Phe Ser Ile Pro Tyr
Pro Ser Tyr Ala Ile Phe Gly Ser 755 760 765Lys Thr Ile His Leu Thr
Gly Arg Ser Leu Leu Pro Ala Leu Glu Ala 770 775 780Glu Pro Leu Trp
Ala Thr Val Phe Gly Ser Gln Ser His His Glu Val785 790 795 800Thr
Met Ser Tyr Pro Met Arg Ser Val Gln His Arg His Phe Arg Leu 805 810
815Val His Asn Leu Asn Phe Lys Met Pro Phe Pro Ile Asp Gln Asp Phe
820 825 830Tyr Val Ser Pro Thr Phe Gln Asp Leu Leu Asn Arg Thr Thr
Ala Gly 835 840 845Gln Pro Thr Gly Trp Tyr Lys Asp Leu Arg His Tyr
Tyr Tyr Arg Ala 850 855 860Arg Trp Glu Leu Tyr Asp Arg Ser Arg Asp
Pro His Glu Thr Gln Asn865 870 875 880Leu Ala Thr Asp Pro Arg Phe
Ala Gln Leu Leu Glu Met Leu Arg Asp 885 890 895Gln Leu Ala Lys Trp
Gln Trp Glu Thr His Asp Pro Trp Val Cys Ala 900 905 910Pro Asp Gly
Val Leu Glu Glu Lys Leu Ser Pro Gln Cys Gln Pro Leu 915 920 925His
Asn Glu Leu 9302762879DNAArtificial SequenceNucleotide sequence
encoding the amino acid sequence of fusion protein of heavy chain
of anti-hTfR antibody No.3 (humanized 2) and hSGSH, synthetic
sequence 276acgcgtgccg ccaccatggg ctggagctgg attctgctgt tcctcctgag
cgtgacagca 60ggagtgcaca gcgaggtgca actagtgcag tctggagcag aggtgaaaaa
gcccggggag 120tctctgaaga tttcctgtaa gggttctgga tacagcttta
ccaactactg gctgggatgg 180gtgcgccaga tgcccgggaa aggcctggag
tggatggggg acatctaccc cggcggagac 240taccctacat acagcgagaa
gttcaaggtc caggtcacca tctcagccga caagtccatc 300agcaccgcct
acctgcagtg gagcagcctg aaggcctcgg acaccgccat gtattactgt
360gcgagatcag gcaattacga cgaagtggcc tactggggcc aaggaaccct
ggtcaccgtc 420tcctcagcta gcaccaaggg cccatcggtc ttccccctgg
caccctcctc caagagcacc 480tctgggggca cagcggccct gggctgcctg
gtcaaggact acttccccga accggtgacg 540gtgtcgtgga actcaggcgc
cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 600tcctcaggac
tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc
660cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga
caagaaagtt 720gagcccaaat cttgtgacaa aactcacacg tgcccaccgt
gcccagcacc tgaactcctg 780ggaggtccgt cagtcttcct cttcccccca
aaacccaagg acaccctcat gatctcccgg 840acccctgagg tcacatgcgt
ggtggtggac gtgagccacg aagaccctga ggtcaagttc 900aactggtacg
tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag
960tacaacagca cgtaccgggt ggtcagcgtc ctcaccgtcc tgcaccagga
ctggctgaat 1020ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc
cagcccccat cgagaaaacc 1080atctccaaag ccaaagggca gccccgagaa
ccacaggtgt acaccctgcc cccatcccgg 1140gatgagctga ccaagaacca
ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1200gacatcgccg
tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
1260cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt
ggacaagagc 1320aggtggcagc aggggaacgt cttctcatgc tccgtgatgc
atgaggctct gcacaaccac 1380tacacgcaga agagcctctc cctgtctccg
ggtaaaggat ctcgtccccg gaacgcactg 1440ctgctcctcg cggatgacgg
aggctttgag agtggcgcgt acaacaacag cgccatcgcc 1500accccgcacc
tggacgcctt ggcccgccgc agcctcctct ttcgcaatgc cttcacctcg
1560gtcagcagct gctctcccag ccgcgccagc ctcctcactg gcctgcccca
gcatcagaat 1620gggatgtacg ggctgcacca ggacgtgcac cacttcaact
ccttcgacaa ggtgcggagc 1680ctgccgctgc tgctcagcca agctggtgtg
cgcacaggca tcatcgggaa gaagcacgtg 1740gggccggaga ccgtgtaccc
gtttgacttt gcgtacacgg aggagaatgg ctccgtcctc 1800caggtggggc
ggaacatcac tagaattaag ctgctcgtcc ggaaattcct gcagactcag
1860gatgaccggc ctttcttcct ctacgtcgcc ttccacgacc cccaccgctg
tgggcactcc 1920cagccccagt acggaacctt ctgtgagaag tttggcaacg
gagagagcgg catgggtcgt 1980atcccagact ggacccccca ggcctacgac
ccactggacg tgctggtgcc ttacttcgtc 2040cccaacaccc cggcagcccg
agccgacctg gccgctcagt acaccaccgt cggccgcatg 2100gaccaaggag
ttggactggt gctccaggag ctgcgtgacg ccggtgtcct gaacgacaca
2160ctggtgatct tcacgtccga caacggtatc cccttcccca gcggcaggac
caacctgtac 2220tggccgggca ctgctgaacc cttactggtg tcatccccgg
agcacccaaa acgctggggc 2280caagtcagcg aggcctacgt gagcctccta
gacctcacgc ccaccatctt ggattggttc 2340tcgatcccgt accccagcta
cgccatcttt ggctcgaaga ccatccacct cactggccgg 2400tccctcctgc
cggcgctgga ggccgagccc ctctgggcca ccgtctttgg cagccagagc
2460caccacgagg tcaccatgtc ctaccccatg cgctccgtgc agcaccggca
cttccgcctc 2520gtgcacaacc tcaacttcaa gatgcccttt cccatcgacc
aggacttcta cgtctcaccc 2580accttccagg acctcctgaa ccgcaccaca
gctggtcagc ccacgggctg gtacaaggac 2640ctccgtcatt actactaccg
ggcgcgctgg gagctctacg accggagccg ggacccccac 2700gagacccaga
acctggccac cgacccgcgc tttgctcagc ttctggagat gcttcgggac
2760cagctggcca agtggcagtg ggagacccac gacccctggg tgtgcgcccc
cgacggcgtc 2820ctggaggaga agctctctcc ccagtgccag cccctccaca
atgagctgta agcggccgc 2879277245PRTArtificial SequenceAmino acid
sequence of anti-hTfR single-chain antibody 277Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile
Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30Trp Leu Gly
Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Asp
Ile Tyr Pro Gly Gly Asp Tyr Pro Thr Tyr Ser Glu Lys Phe 50 55 60Lys
Val Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75
80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Ser Gly Asn Tyr Asp Glu Val Ala Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln
Thr Pro Leu Ser Leu 130 135 140Ser Val Thr Pro Gly Gln Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln145 150 155 160Ser Leu Val His Ser Asn
Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln 165 170 175Lys Pro Gly Gln
Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg 180 185 190Phe Ser
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200
205Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
210 215 220Tyr Cys Ser Gln Ser Thr His Val Pro Trp Thr Phe Gly Gln
Gly Thr225 230 235 240Lys Val Glu Ile Lys 24527816PRTArtificial
SequenceAmino acid sequence 2 of CDR 2 in the heavy chain of mouse
anti-hTfR antibody No. 6 278Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr
His Ser Ala Leu Ile Ser1 5 10 1527916PRTArtificial SequenceAmino
acid sequence 2 of CDR 2 in the heavy chain of mouse anti-hTfR
antibody No. 8 279Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr Arg Ser
Ala Leu Ile Ser1 5 10 15
* * * * *
References