U.S. patent application number 14/596874 was filed with the patent office on 2015-08-06 for novel anti-human ngf antibody.
This patent application is currently assigned to Astellas Pharma Inc.. The applicant listed for this patent is Astellas Pharma Inc.. Invention is credited to Masazumi Kamohara, Yukari Koya, Jun Takasaki, Hirotsugu Tanaka, Atsuo Yonezawa, Eiji Yoshimi.
Application Number | 20150218265 14/596874 |
Document ID | / |
Family ID | 47668585 |
Filed Date | 2015-08-06 |
United States Patent
Application |
20150218265 |
Kind Code |
A1 |
Kamohara; Masazumi ; et
al. |
August 6, 2015 |
NOVEL ANTI-HUMAN NGF ANTIBODY
Abstract
To provide an anti-human NGF antibody or an antigen-binding
fragment thereof that is excellent in safety by reducing the risk
of side effects such as effects on a fetus and thrombus formation
while maintaining high neutralizing activity, and to provide means
for preventing or treating various diseases in which human NGF is
involved in the formation of pathological conditions, by using the
antibody or the antibody-binding fragment thereof. An anti-human
NGF antibody Fab' fragment comprising a heavy-chain variable region
consisting of an amino acid sequence shown by SEQ ID NO:6 and a
light-chain variable region consisting of an amino acid sequence
shown by SEQ ID NO:4.
Inventors: |
Kamohara; Masazumi; (Tokyo,
JP) ; Tanaka; Hirotsugu; (Tokyo, JP) ; Koya;
Yukari; (Tokyo, JP) ; Takasaki; Jun; (Tokyo,
JP) ; Yonezawa; Atsuo; (Tokyo, JP) ; Yoshimi;
Eiji; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Astellas Pharma Inc. |
Tokyo |
|
JP |
|
|
Assignee: |
Astellas Pharma Inc.
Tokyo
JP
|
Family ID: |
47668585 |
Appl. No.: |
14/596874 |
Filed: |
January 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13879267 |
Jul 15, 2013 |
8986952 |
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PCT/JP2012/070433 |
Aug 10, 2012 |
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14596874 |
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Current U.S.
Class: |
530/387.3 ;
530/391.1 |
Current CPC
Class: |
C07K 2317/33 20130101;
C07K 2317/565 20130101; A61K 2039/505 20130101; C07K 2317/24
20130101; C07K 2317/76 20130101; C07K 2317/51 20130101; C07K 16/22
20130101; C07K 2317/55 20130101; C07K 2317/21 20130101; C07K
2317/94 20130101; A61K 47/60 20170801; C07K 2317/515 20130101 |
International
Class: |
C07K 16/22 20060101
C07K016/22; A61K 47/48 20060101 A61K047/48 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 11, 2011 |
JP |
2011-176209 |
Dec 8, 2011 |
JP |
2011-269215 |
Claims
1-12. (canceled)
13. An anti-human NGF antibody Fab' fragment comprising: a
heavy-chain fragment comprises a heavy-chain variable region
comprising CDR1 consisting of amino acid sequence at position from
31 to 35 of SEQ ID NO: 6, CDR2 consisting of amino acid sequence at
position from 50 to 65 of SEQ ID NO: 6, and CDR3 consisting of
amino acid sequence at position from 98 to 110 of SEQ ID NO: 6; and
a light chain comprises a light-chain variable region comprising
CDR1 consisting of amino acid sequence at position from 24 to 39 of
SEQ ID NO: 4, CDR2 consisting of amino acid sequence at position
from 55 to 61 of SEQ ID NO: 4, and CDR3 consisting of amino acid
sequence at position from 94 to 102 of SEQ ID NO: 4.
14. The Fab' fragment according to claim 13, wherein the
heavy-chain fragment comprises a heavy-chain constant region which
is a human Igyl constant region.
15. The Fab' fragment according to claim 13, wherein the light
chain comprises a light-chain constant region which is a human
Ig.kappa. constant region.
16. The Fab' fragment according to claim 13, wherein the
heavy-chain fragment comprises a heavy-chain constant region which
is a human Igyl constant region, and the light chain comprises a
light-chain constant region which is a human Ig.kappa. constant
region.
17. The Fab' fragment according to claim 13, wherein the Fab'
fragment is conjugated to polyethylene glycol.
18. The Fab' fragment according to claim 14, wherein the Fab'
fragment is conjugated to polyethylene glycol.
19. The Fab' fragment according to claim 15, wherein the Fab'
fragment is conjugated to polyethylene glycol.
20. The Fab' fragment according to claim 16, wherein the Fab'
fragment is conjugated to polyethylene glycol.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel anti-human NGF
antibody. More specifically, the present invention relates to a
Fab' fragment of an anti-human NGF antibody.
BACKGROUND ART
[0002] A nerve growth factor (NGF) is one of humoral factors called
generally "neurotrophic factors", and plays an important role in
generation and differentiation of neurons and in maintaining
functions of neurons in the body. As NGF receptors, a high affinity
trkA receptor (receptor-type tyrosine kinase) and a low affinity
p75NTR receptor are known. There is a report reporting that among
these, the p75NTR binds to all of the neurotrophic factors and is
involved in apoptosis in the process of neuronal generation.
However, the role of the p75NTR has not yet been sufficiently
explained. Meanwhile, it is known that knockout mice of the NGF and
the trkA receptor express the same phenotype (Non-Patent Document
1), and it is considered that the physiological action of NGF is
expressed mainly via the trkA receptor.
[0003] In 1993, there was a report reporting that the
administration of NGF to rats induced pain (Non-Patent Document 2),
and since then, there has been a report reporting that intravenous
administration of NGF to human beings induces systemic myalgia and
that topical administration of NGF exerts a systemic effect and
induces hyperpathia and allodynia in an injection site (Non-Patent
Document 3). In addition, there is a report reporting that a
knockout mouse of the trkA receptor shows analgesia (Non-Patent
Document 4), so it is considered that NGF is a molecule deeply
involved in the expression of pain. Regarding the correlation
between NGF and the pathological condition of human pain, it has
been demonstrated that expression of NGF/trkA is accelerated in
articular cartilages with osteoarthritis (OA) (Non-Patent Document
6) and that the level of NGF is increased in patients with
rheumatoid arthritis (Non-Patent Document 7) or interstitial
cystitis (Non-Patent Document 8).
[0004] From the above facts, it is expected that if a monoclonal
antibody which specifically binds to NGF and has an inhibitory
activity against the action of NGF can be developed, this will be
useful for treating, preventing, and diagnosing various diseases
including pain relating to NGF.
[0005] As anti-human NGF antibodies which have been clinically
developed so far, tanezumab (Patent Document 1) and PG110 (Patent
Document 2) as humanized anti-human NGF antibodies, and REGN475
(Patent Document 3), fulranumab (Patent Document 4), and MEDI-578
(Patent Document 5) as fully human anti-human NGF antibodies have
been reported. Among these, tanezumab is being most briskly
developed by priority, and there is a report reporting that
according to clinical test results, this antibody exerts a potent
and extensive analgesic effect on pain such as arthralgia
accompanied by osteoarthritis, chronic back pain, and cystalgia
accompanied by interstitial cystitis (Non-Patent Documents 9 to
11).
[0006] Generally, as main factors determining an effective dose of
an antibody drug, the neutralizing activity of an antibody against
an antigen and the amount of antigens present in the body are
exemplified. Improving the neutralizing activity leads to the
decrease of dose, and consequently, this can be mentioned as very
useful amelioration leading to decrease in the financial burden of
patients and medical costs. If the decrease in dose can be
realized, subcutaneous administration can also be carried out.
Subcutaneous administration has a major advantage that a patient
can perform self-injection at home if certain conditions are
satisfied. In addition, while the antibody drug is generally
administered via drips for a certain time in many cases in the
intravenous administration, the drug can be administered as a bolus
in the subcutaneous administration, which is another advantage.
Both the physician and the patient can select a preparation for
intravenous administration and a preparation for subcutaneous
administration, and this is a desirable factor. However, in the
subcutaneous administration, a dose that can be given per
administration is as small as about 1 mL in general, so a
sufficient amount of antibodies need to be included in the dose so
as to express the drug efficacy. Moreover, unlike the intravenous
administration, bioavailability needs to be considered for the
subcutaneous administration. That is, in order to realize a
preparation for subcutaneous administration, it is required to
prepare an antibody which exhibits excellent solubility and
expresses a sufficient drug efficacy even at a small dose.
Accordingly, if an antibody which has a higher neutralizing
activity against NGF compared to the antibodies in the related art
is obtained, this will be useful for treating diseases relating to
NGF and for improving convenience of the treatment.
[0007] As described above, though NGF is an important factor for
growth of neurons, performing sufficient examination in terms of
safety is necessary in developing medical drugs that inhibit the
function of NGF. Particularly, as one of the respects which should
be examined in terms of safety, the effects on a fetus are
exemplified. So far, regarding the functional inhibition of NGF,
there have been reports reporting that NGF mutation is the cause of
congenital analgesia (Non-Patent Document 5), and that in an animal
experiment, when a pregnant guinea pig is caused to produce an
autoantibody to NGF so as to inhibit NGF in the body, the newborn
guinea pig shows symptoms of analgesia (Non-Patent Document 12).
Moreover, in a test using NGF- or trkA-deficient mice, it has been
demonstrated that deficiency of NGF action inhibits the growth of
neurons of sensory nerves and sympathetic nerves in an embryo
(Non-Patent Documents 4 and 13). From these results, it is
understood that NGF is an essential factor of neurodevelopment in
the early stage of development. The NGF-related diseases also
include diseases that women at a child-bearing age suffer from at a
high rate, such as interstitial cystitis (half or more of the
patients are 44 years old or younger, and 90% of patients are
females (Non-Patent Document 14)), chronic back pain (an average
age of 40 to 50, and over 50% of patients are females (Non-Patent
Documents 15 to 17)), and migraine (a peak age of onset ranges from
15 to 40 years, and 80% of patients are female (Non-Patent Document
18)). In this situation, in developing the anti-NGF antibody as a
medical drug, it is very important to avoid the risk of side
effects on a fetus in pregnant women.
[0008] As another risk factor in a case of developing the anti-NGF
antibody as a medical drug, immunocomplex (IC) formation is
exemplified. The immunocomplex which is a combination of an antigen
and an antibody is generally treated in a reticuloendothelial
system such as the spleen or the liver. However, it has been
reported that when a pathological condition such as immune
abnormality is caused or when the size of the formed IC is large,
the IC loses solubility, which relates to the increase of the risk
of thrombus formation and to the onset of nephritis caused by the
glomerular accumulation of the IC. Though IgG is a bivalent
antibody, when an antigen is polyvalent, the IC may have various
sizes due to lattice formation. The size of the IC depends on the
amount of an antibody and an antigen and the ratio therebetween,
affinity of an antibody, and the like. For example, an anti-VEGF
antibody bevacizumab (product name: Avastin) is an IgG1 antibody,
and there is a report reporting that this antibody forms an IC by
binding to a dimer VEGF and induces thrombus formation.
Specifically, when Avastin and VEGF are administered to a human
Fc.gamma.RIIa receptor Tg mouse, formation of a pulmonary artery
thrombus is observed (Non-Patent Document 19). In addition, there
is a report reporting that an arterial thrombus is formed at a
higher rate in patients with metastatic cancer who receive
chemotherapy with Avastin treatment, compared to a placebo group
receiving only chemotherapy (Non-Patent Document 20). Since NGF
also forms a dimer in the body to exert physiological activity, it
is desirable to further improve safety by avoiding the risk of IC
formation in developing a medical drug of the anti-NGF
antibody.
[0009] For the above reasons, for treating or preventing various
NGF-related diseases, it is very important to obtain an anti-NGF
antibody which is excellent in safety by reducing the risk of side
effects such as the effects on a fetus and thrombus formation while
maintaining a high neutralizing activity.
RELATED ART
Patent Document
[0010] [Patent Document 1] WO2004/058184
[0011] [Patent Document 2] WO2005/061540
[0012] [Patent Document 3] WO2009/023540
[0013] [Patent Document 4] WO2005/019266
[0014] [Patent Document 5] WO2006/077441
Non-Patent Document
[0015] [Non-Patent Document 1] Conover J C, et al, Rev Neurosci.
1997, 8:13-27.
[0016] [Non-Patent Document 2] Lewin G R, et al, J Neurosci. 1993,
13:2136-48.
[0017] [Non-Patent Document 3] Petty B G, et al, Ann Neurol. 1994,
36:244-6.
[0018] [Non-Patent Document 4] Smeyne R J, et al, Nature. 1994,
368:246-9.
[0019] [Non-Patent Document 5] Indo Y, et al, Nat Genet. 1996,
13:485-8.
[0020] [Non-Patent Document 6] Iannone F, et al, Rheumatology 2002,
41:1413-8.
[0021] [Non-Patent Document 7] Aloe L, et al, Clin Exp Rheumatol.
1997, 15:433-8.
[0022] [Non-Patent Document 8] Lowe E M, et al, Br J Urol. 1997,
79:572-7.
[0023] [Non-Patent Document 9] Lane N E, et al, N Engl J Med. 2010,
363:1521-31.
[0024] [Non-Patent Document 10] Evans R J, et al, J Urol. 2011,
185:1716-21.
[0025] [Non-Patent Document 11] Katz N, et al, Pain. 2011, in
press
[0026] [Non-Patent Document 12] Johnson E M Jr, et al, Science.
1980, 210:916-8.
[0027] [Non-Patent Document 13] Crowley C, et al, Cell. 1994,
76:1001-11.
[0028] [Non-Patent Document 14] Payne C K, et al, J Urol. 2007,
177:2042-9.
[0029] [Non-Patent Document 15] Manchikanti L, et al, Pain
Physician. 2010, 13:E279-92.
[0030] [Non-Patent Document 16] Wilkens P, et al, JAMA. 2010,
304:45-52.
[0031] [Non-Patent Document 17] Buynak R, et al, Expert Opin
Pharmacother. 2010, 11:1787-804.
[0032] [Non-Patent Document 18] Sakai F, et al, Cephalalgia. 1997,
17:15-22.
[0033] [Non-Patent Document 19] Meyer T, et al, J Thromb Haemost.
2009, 7:171-81.
[0034] [Non-Patent Document 20] Scappaticci F A, et al, J Natl
Cancer Inst. 2007, 99:1232-9.
SUMMARY OF INVENTION
Technical Problem
[0035] An object of the present invention is to provide an
anti-human NGF antibody or an antigen-binding fragment thereof that
is excellent in safety by reducing the risk of side effects such as
effects on a fetus and thrombus formation while maintaining high
neutralizing activity.
Solution to Problem
[0036] The present invention includes the following invention as
medically or industrially useful substances and methods.
[0037] [1] An anti-human NGF antibody Fab' fragment comprising: a
heavy-chain variable region consisting of an amino acid sequence
shown by SEQ ID NO:6; and
[0038] a light-chain variable region consisting of an amino acid
sequence shown by SEQ ID NO:4
[0039] [2] The Fab' fragment according to [1], wherein a
heavy-chain constant region of the Fab' fragment is a human
Ig.gamma.1 constant region.
[0040] [3] The Fab' fragment according to [1], wherein a
light-chain constant region of the Fab' fragment is a human
Ig.epsilon. constant region.
[0041] [4] The Fab' fragment according to [1], wherein the
heavy-chain constant region of the Fab' fragment is the human
Ig.gamma.1 constant region, and the light-chain constant region of
the Fab' fragment is the human Ig.kappa. constant region.
[0042] [5] The Fab' fragment according to [1], comprising:
[0043] a heavy-chain fragment consisting of an amino acid sequence
shown by SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:16; and
[0044] a light chain consisting of an amino acid sequence shown by
SEQ ID NO:12.
[0045] [6] The Fab' fragment according to any one of [1] to [5],
wherein the Fab' fragment is conjugated to polyethylene glycol.
[0046] [7] A polynucleotide comprising a sequence that encodes the
heavy-chain fragment of the Fab' fragment according to any one of
[1] to [6].
[0047] [8] A polynucleotide comprising a sequence that encodes the
light chain of the Fab' fragment according to any one of [1] to
[6].
[0048] [9] An expression vector comprising the polynucleotide
according to [7] and/or [8].
[0049] [10] A host cell transformed with the expression vector
according to [9].
[0050] [11] The host cell according to [10], which is selected from
a group consisting of the following (a) and (b),
[0051] (a) a host cell transformed with an expression vector
comprising a polynucleotide comprising a sequence that encodes the
heavy-chain fragment of the Fab' fragment according to any one of
[1] to [6] and a polynucleotide comprising a sequence that encodes
the light chain of the Fab' fragment; and
[0052] (b) a host cell transformed with an expression vector
comprising a polynucleotide comprising a sequence that encodes the
heavy-chain fragment of the Fab' fragment according to any one of
[1] to [6] and with an expression vector comprising a
polynucleotide comprising a sequence that encodes the light chain
of the Fab' fragment.
[0053] [12] A method of producing the anti-human NGF antibody Fab'
fragment according to any one of [1] to [6], comprising expressing
an anti-human NGF antibody Fab' fragment by culturing the host cell
according to [10] or [11].
[0054] [13] An agent for treating pain, which comprises the Fab'
fragment according to any one of [1] to [6].
[0055] [14] The agent for treating pain according to [13], wherein
the pain is osteoarthritis pain.
[0056] [15] A method for preventing or treating pain, comprising
administering the Fab' fragment according to any one of [1] to
[6].
[0057] [16] The method according to [15], wherein the pain is
osteoarthritis pain.
[0058] [17] The Fab' fragment according to any one of [1] to [6]
for use in preventing or treating pain.
[0059] [18] The Fab' fragment according to [17], wherein the pain
is osteoarthritis pain.
Advantage Effects of the Invention
[0060] The anti-human NGF antibody Fab' fragment of the present
invention is useful for preventing or treating various diseases in
which human NGF is involved in the formation of pathological
conditions. Due to its high neutralizing activity, the anti-human
NGF antibody Fab' fragment of the present invention brings about
excellent improvement in clinical applications, such as decreases
in dose, widening of administration intervals, and improvement of
the method of administration (for example, a subcutaneous
injection). Moreover, in reducing the risk of side effects such as
the effects on a fetus and thrombus formation, the anti-human NGF
antibody Fab' fragment of the present invention is significantly
excellent in terms of safety and greatly contributes to the
prevention or treatment of various diseases in which human NGF is
involved in the formation of pathological conditions.
[BRIEF DESCRIPTION OF THE DRAWINGS]
[0061] FIG. 1 shows temporal change in the amount of an antibody
retained in the sole of a collagen-induced arthritis mouse
model.
DESCRIPTION OF EMBODIMENTS
[0062] Hereinbelow, the present invention will be described in
detail.
[0063] The present inventors repeated creative examination to
prepare an anti-human NGF antibody or an antigen-binding fragment
thereof As a result, they succeeded in preparing an anti-human NGF
antibody Fab' fragment which is excellent in safety by reducing the
risk of side effects such as the effects on a fetus and thrombus
formation while maintaining high neutralizing activity.
[0064] The basic structure of an antibody molecule is common among
the respective antibody classes and is constituted with a heavy
chain having a molecular weight of 50000 to 70000 and a light chain
having a molecular weight of 20000 to 30000. The heavy chain
generally consists of a polypeptide chain including about 440 amino
acids, and each class has its characteristic structure. The heavy
chains are called .gamma., .mu., .alpha., .delta., and .epsilon.
chains corresponding to IgG, IgM, IgA, IgD, and IgE. Furthermore,
IgG has subclasses such as IgG1, IgG2, IgG3, and IgG4, and these
chains are called .gamma.1, .gamma.2, .gamma.3, and .gamma.4
respectively. A light chain generally consists of a polypeptide
chain including about 220 amino acids, and two types of the light
chain including an L-type and a K-type light chains are known,
which are called .lamda. and .kappa. chains respectively. Regarding
the peptide constitution of the basic structure of an antibody
molecule, two homologous heavy chains and two homologous light
chains are bound via disulfide bonds (S--S bonds) and non-covalent
bonds, and the molecular weight thereof is 150000 to 190000. The
two kinds of light chains can be paired with any heavy chain. Each
antibody molecule always consists of two identical light chains and
two identical heavy chains.
[0065] There are four intrachain S--S bonds in a heavy chain (five
bonds for .mu. and .epsilon. chains) and two in a light chain. One
loop is formed per 100 to 110 amino acid residues, and this steric
structure is similar among the respective loops and is called a
structural unit or a domain. For both heavy chains and light
chains, the amino acid sequence of the domain positioned at the
N-terminal thereof is not constant, even in a reference standard
from the same class (subclass) of the same animal species, and this
domain is called the variable region. Each of the domains is called
a heavy-chain variable region (V.sub.H) and a light-chain variable
region (V.sub.L) respectively. Since the amino acid sequence of the
C-terminal side from the domain is almost constant in each class or
subclass, this region is called a constant region, and each of the
domains is described as C.sub.H1, C.sub.H2, C.sub.H3 and C.sub.L,
respectively.
[0066] The antigenic determinant site of an antibody is constituted
with V.sub.H and V.sub.L, and the binding specificity depends on
the amino acid sequence of this site. On the other hand, biological
activities such as binding to complements or various cells reflect
the differences in the constant region structure among the various
classes of Ig. It is known that the variability in the variable
regions of the heavy chain and light chain is mostly limited to
three small hypervariable regions present in both chains, and these
regions are called complementarity determining regions (CDRs; CDR1,
CDR2 and CDR3 starting from the N-terminal side). The remaining
portion of the variable region is called a framework region (FR)
and is relatively constant.
[0067] A region between the C.sub.H1 domain and the C.sub.H2 domain
of the heavy-chain constant region of an antibody is called a hinge
region. This region includes lots of proline residues and has a
plurality of inter-chain S--S bonds connecting two heavy-chains.
For example, each hinge region of human IgG1, IgG2, IgG3, and IgG4
includes 2, 4, 11, and 2 cysteine residues respectively which
constitute the inter-heavy-chain S--S bonds. The hinge region is a
region highly sensitive to a proteolytic enzyme such as papain or
pepsin. When an antibody is digested with papain, its heavy chain
is cleaved at a position closer to the N-terminal side than to the
inter-heavy-chain S--S bond of the hinge region, whereby the
antibody is broken down into two Fab fragments and one Fc fragment.
The Fab fragment is constituted with a light-chain and a
heavy-chain fragment including a heavy-chain variable region
(V.sub.H), a C.sub.H1 domain, and a portion of the hinge region.
When an antibody is digested with pepsin, its heavy-chain is
cleaved at a position closer to the C-terminal side than to the
inter-heavy-chain S--S bond of the hinge region, whereby
F(ab').sub.2 fragments is generated. The F(ab').sub.2 fragment is a
fragment having a dimeric structure in which two Fab' fragments
bind to each other via the inter-heavy-chain S--S bond in the hinge
region. The Fab' fragment is constituted with a light-chain and a
heavy-chain fragment including a heavy-chain variable region
(V.sub.H), a C.sub.H1 domain, and a portion of the hinge region.
Cysteine residues constituting the inter-heavy-chain S--S bond are
included in the portion of the hinge region. All of the Fab
fragment, F(ab').sub.2 fragment, and Fab' fragment include the
variable region and have antigen-binding activity.
[0068] The anti-human NGF antibody Fab' fragment of the present
invention that the present inventors successfully prepared is a
Fab' fragment having the following characteristics.
[0069] The anti-human NGF antibody Fab' fragment comprises a
heavy-chain variable region consisting of an amino acid sequence
shown by SEQ ID NO:6 and a light-chain variable region consisting
of an amino acid sequence shown by SEQ ID NO:4.
[0070] Specifically, the present inventors constructed antibodies
using a human monoclonal antibody development technology,
"Veloclmmune " mouse [Veloclmmune antibody technology; Regeneron
Inc. (U.S. Pat. No. 6,596,541)], and screened the antibodies using
tests for various biological activities and physical properties,
thereby succeeding in identifying the anti-human NGF antibody Fab'
fragment of the present invention. In the Veloclmmune technology,
transgenic mice in which the endogenous immunoglobulin heavy and
light chain variable regions are replaced with the corresponding
human variable regions are challenged with the antigen of interest
(for example, human .beta.NGF), and lymphatic cells are recovered
from the mice that express antibodies. The lymphatic cells are
fused with mouse myeloma cells to prepare hybridomas. The hybridoma
cells are screened to identify hybridoma cells that produce those
antibodies that specifically bind to the antigen of interest. The
antibodies that are produced herein are antibodies having the
variable regions of human antibodies and the constant regions of
mouse antibodies (also referred to as chimeric antibodies). Then,
if the antibody that binds specifically to the antigen of interest
and has a desired neutralizing activity is identified, DNAs that
encode the variable regions of the heavy chain and light chain of
the antibody are isolated from the hybridoma cells and linked to
DNAs encoding the constant regions of the heavy chain and light
chain of a desired class of human antibody. The resulting gene
encoding the heavy chain and light chain of the antibody is
expressed in cells (e.g., CHO cells) to produce an antibody
molecule. The heavy chain and light chain of the antibody produced
by the above method are the heavy chain and light chain of a "fully
human" antibody derived from a human immunoglobulin gene.
[0071] The anti-human NGF antibody Fab' fragment of the present
invention can be easily prepared by those skilled in the art on the
basis of the sequence information on the heavy-chain variable
region and light-chain variable region thereof disclosed herein,
using a method commonly known in the art. Preferably, the
anti-human NGF antibody Fab' fragment of the present invention can
be prepared as a fully human antibody Fab' fragment by linking the
heavy-chain variable region and light-chain variable regions
thereof to a part of the heavy-chain constant region (which
includes C.sub.H1 domain and a part of hinge region including hinge
region cysteine) and light-chain constant region of a human
antibody, respectively. Specifically, a heavy-chain variable region
gene fragment having a base sequence that encodes the heavy-chain
variable region amino acid of the Fab' fragment of the present
invention (SEQ ID NO:6), and a light-chain variable region gene
fragment having a base sequence that encodes the light-chain
variable region amino acid of the Fab' fragment of the present
invention (SEQ ID NO:4) are prepared. Then, the variable region
genes of the heavy chain and light chain are linked to each gene of
a part of heavy-chain constant region and a light-chain constant
region in an appropriate class of human antibody to prepare a fully
human antibody Fab' fragment gene. Next, this gene is linked to an
appropriate expression vector and introduced into a cultured cell.
Finally, this cultured cell is cultured, whereby a monoclonal Fab'
fragment can be obtained from the culture supernatant.
[0072] The gene fragments that encode the heavy-chain and
light-chain variable region amino acids of the Fab' fragment of the
present invention can be synthesized using a gene synthesis method
known in the art, on the basis of, for example, base sequences
designed based on the amino acid sequences of the heavy-chain
variable region and the light-chain variable region. Examples of
this gene synthesis method include various methods known to those
skilled in the art, such as the antibody gene synthesis method
described in WO90/07861.
[0073] Then, the above-described variable region gene fragments are
linked to the constant region gene of a human antibody to prepare a
fully human Fab' fragment gene. Although any subclass of the
constant region (for example, the constant region of a heavy chain
such as the .gamma.1, .gamma.2, .gamma.3 or .gamma.4 chain, or the
constant region of a light chain such as the .lamda. or .kappa.
chain) can be chosen as the constant region of the human antibody,
human Ig.gamma.1 as the heavy-chain constant region, and human
Ig.kappa. as the light-chain constant region, can preferably be
used.
[0074] Subsequent to the preparation of this fully human antibody
Fab' fragment gene, introduction of the gene into an expression
vector, introduction of the expression vector into cultured cells,
cultivation of the cultured cells, purification of the Fab'
fragment and the like can be performed using various methods known
in the art.
[0075] Examples of the expression vector that is linked to thus
obtained gene include GS vector pEE6.4 or pEE12.4 (Lonza
Biologics), but are not specifically limited, so long as they can
express such antibody gene. Also, an expression vector already
having a human Ig constant region gene such as AG-.gamma.1 or
AG-.kappa. (for example, see WO94/20632) may be used.
[0076] The above-described expression vector is introduced into
cultured cells by, for example, a calcium phosphate method or an
electroporation method and the like.
[0077] Examples of the cultured cells into which the expression
vector is introduced include cultured cells such as CHO-K1SV cells,
CHO-DG44 cells and 293 cells, and these cells may be cultured by a
conventional method.
[0078] The Fab' fragment accumulated in a culture supernatant after
culturing described above can be purified by various types of
column chromatography. For example, it is possible to use column
chromatography using KappaSelect or the like.
[0079] The Fab' fragment of the present invention can be prepared
using a recombinant expression method as described above. However,
the Fab' fragment may be prepared by performing pepsin digestion
after preparing a full-length antibody first, and treating the
obtained F(ab').sub.2 fragment with a reductant such as
2-mercaptoethanol.
[0080] Preferably, the anti-human NGF antibody Fab' fragment of the
present invention can be easily obtained by synthesizing DNA
comprising a base sequence encoding the heavy-chain variable region
amino acid sequence shown by SEQ ID NO:6 and DNA comprising a base
sequence encoding the light-chain variable region amino acid
sequence shown by SEQ ID NO:4, and linking the DNAs to a suitable
class of human antibody constant region genes, preferably a human
Ig.gamma.1 constant region gene for the heavy chain and a human
Ig.kappa. constant region gene for the light chain, to construct a
fully human antibody Fab' fragment gene by using a method known in
the art, and introducing the gene into an expression vector,
introducing the expression vector into a cultured cell, culturing
the cultured cell, and purifying an Fab' fragment harvested from
the cultured cell by using various methods known in the art.
Preferably, DNA comprising a base sequence encoding the heavy-chain
variable region amino acid sequences shown by SEQ ID NO:6 comprises
the base sequences shown by SEQ ID NO:5. Preferably, DNA comprising
a base sequence encoding the light-chain variable region amino acid
sequences shown by SEQ ID NO:4 comprises the base sequences shown
by SEQ ID NO:3.
[0081] In the present specification, the "Fab' fragment" refers to
a monovalent antibody fragment constituted with a light-chain and a
heavy-chain fragment including a heavy-chain variable region
(V.sub.H), a C.sub.H1 domain, and a portion of a hinge region. In
the portion of the hinge region, at least one cysteine residue
(also called a "hinge region cysteine" in the present
specification) other than cysteine residues constituting the S--S
bond between the heavy chain and the light chain is included. The
hinge region cysteine can be used as a modification site of
polyethylene glycol described later. The number of the hinge region
cysteines in the Fab' fragment is variable within a range of from 1
to several cysteine residues depending on the class of an antibody
used, and is easily adjustable by a person skilled in the art. For
example, when a Fab' fragment of a human IgG1 class (generally
having two hinge region cysteines in a hinge region) is prepared, a
stop codon is inserted between a coding site of the first hinge
region cysteine and a coding site of the second hinge region
cysteine in the hinge region of the heavy chain, whereby a Fab'
fragment having one hinge region cysteine in the hinge region can
be prepared. In addition, if a stop codon is inserted after the
coding site of the second hinge region cysteine, a Fab' fragment
having two hinge region cysteines in the hinge region can be
prepared.
[0082] The preferable heavy-chain fragment of the anti-human NGF
antibody Fab' fragment of the present invention, comprising the
heavy-chain variable region consisting of the amino acid sequence
shown by SEQ ID NO:6 and a portion of a human Ig.gamma.1 constant
region, is a heavy-chain fragment consisting of the amino acid
sequence shown by SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:16.
Preferably, DNA comprising a base sequence that encodes the
heavy-chain fragment of the anti-human NGF antibody Fab' fragment
consisting of the amino acid sequence shown by SEQ ID NO:10, SEQ ID
NO:14, or SEQ ID NO:16 comprises the base sequence shown by SEQ ID
NO:9, SEQ ID NO:13, or SEQ ID NO:15. The preferable light chain of
the anti-human NGF antibody Fab' fragment of the present invention,
comprising the light-chain variable region consisting of the amino
acid sequence shown by SEQ ID NO:4 and a human Ig.kappa. constant
region, is a light chain consisting of the amino acid sequence
shown by SEQ ID NO:12. Preferably, DNA comprising a base sequence
that encodes the light chain of the anti-human NGF antibody Fab'
fragment consisting of the amino acid sequence shown by SEQ ID
NO:12 comprises the base sequence shown by SEQ ID NO:11.
[0083] As a preferable anti-human NGF antibody Fab' fragment of the
present invention that comprises the heavy-chain fragment
consisting of the amino acid sequence shown by SEQ ID NO:10 and the
light-chain consisting of the amino acid sequence shown by SEQ ID
NO:12, a fully human 1-15(N52D) antibody Fab' fragment described
later in examples is exemplified. As a preferable anti-human NGF
antibody Fab' fragment of the present invention that comprises the
heavy-chain fragment consisting of the amino acid sequence shown by
SEQ ID NO:14 and the light chain consisting of the amino acid
sequence shown by SEQ ID NO:12, a fully human 1-15(N52D-A) antibody
Fab' fragment described later in examples is exemplified. As a
preferable anti-human NGF antibody Fab' fragment of the present
invention that comprises the heavy-chain fragment consisting of the
amino acid sequence shown by SEQ ID NO:16 and the light chain
consisting of the amino acid sequence shown by SEQ ID NO:12, a
fully human 1-15(N52D-P) antibody Fab' fragment described later in
examples is exemplified.
[0084] The present invention also comprises an anti-human NGF
antibody Fab' fragment that comprises the heavy-chain variable
region comprising CDR1 consisting of amino acid sequence at
position from 31 to 35 of SEQ ID NO: 6, CDR2 consisting of amino
acid sequence at position from 50 to 65 of SEQ ID NO: 6, and CDR3
consisting of amino acid sequence at position from 98 to 110 of SEQ
ID NO: 6, and the light-chain variable region comprising CDR1
consisting of amino acid sequence at position from 24 to 39 of SEQ
ID NO: 4, CDR2 consisting of amino acid sequence at position from
55 to 61 of SEQ ID NO: 4, and CDR3 consisting of amino acid
sequence at position from 94 to 102 of SEQ ID NO: 4. The Fab'
fragment can be also prepared by those skilled in the art according
to procedures such as ones described above.
[0085] The anti-human NGF antibody Fab' fragment of the present
invention may be modified by being conjugated to polyethylene
glycol (PEG) via the hinge region cysteine thereof. PEG can be
conjugated to the Fab' fragment by using methods known in the art
(for example, EP0948544). In the present invention, linear or
branched PEG having an arbitrary average molecular weight or a
derivative thereof is usable, which can be easily selected by a
person skilled in the art according to the intended use. For
example, in a tumor tissue or at the time of inflammatory response,
vascular permeability is markedly enhanced compared to a normal
tissue, so substances reaching the tissue tend to leak out of the
blood vessel and accumulate in the tumor or the inflammatory tissue
(EPR effect). It is also known that a low molecular weight
substance is easily reabsorbed into blood vessels and that a high
molecular weight substance is not easily reabsorbed. Therefore, in
order to improve retentivity of the Fab' fragment in a lesional
tissue, PEG having a high average molecular weight (for example,
about 40000 Da) may be conjugated to this fragment. When the Fab'
fragment is desired to be rapidly excreted outside the body, PEG
having a low average molecular weight (for example, about 10000 Da)
may be conjugated to this fragment. Moreover, in order to
facilitate the binding of PEG to the hinge region cysteine, a PEG
derivative may be used. For example, as described later in
examples, it is possible to use a PEG derivative to which a
thiol-reactive group such as maleimide has been bound and bind a
thiol group of the hinge region cysteine to the maleimide group via
a covalent bond. Generally, the average molecular weight of PEG
ranges from about 500 Da to about 50000 Da, preferably ranges from
about 5000 Da to about 40000 Da and more preferably ranges from
about 10000 Da to about 40000 Da.
[0086] The anti-human NGF antibody Fab' fragment of the present
invention binds to human NGF. As the method of measuring binding
activity of the obtained anti-human NGF antibody Fab' fragment to
the human NGF, there is a method such as ELISA or FACS. For
example, when ELISA is used, human .beta.NGF is immobilized in an
ELISA plate, the Fab' fragment is added thereto to cause a
reaction, and then a secondary antibody such as an anti-kappa
antibody labeled with an enzyme such as horseradish peroxidase
(HRP) is allowed to react with the reaction mixture. After the
plate is washed, the activity is measured by using a reagent (for
example, a TMB chromogenic reagent in a case of HRP labeling)
detecting the activity, thereby identifying binding of the
secondary antibody. In addition, the anti-human NGF antibody Fab'
fragment of the present invention also includes a Fab' fragment
that binds to NGF derived from another animal (for example, mouse
NGF) as well as human NGF, so binding activity with respect to such
a protein may be measured.
[0087] The anti-human NGF antibody Fab' fragment of the present
invention has neutralizing activity with respect to human NGF. When
being used in the present specification, the term "neutralizing
activity" of the anti-human NGF antibody Fab' fragment refers to an
activity that inhibits any biological activity resulting from NGF
by binding to NGF, and the neutralizing activity can be evaluated
using one or a plurality of biological activities of NGF as an
index. Examples of such neutralizing activity include the
inhibitory activity against binding of NGF to trkA which is the NGF
receptor, the inhibitory activity against intracellular calcium
influx mediated by an NGF-trkA signal, and the inhibitory activity
against NGF-dependent cell survival signaling. The neutralizing
activity can be evaluated using methods described later in
examples.
[0088] In order to more specifically evaluate the effect of the
anti-human NGF antibody Fab' fragment of the present invention, an
in vivo test may be performed. For example, as described later in
examples, the in vivo drug efficacy of the Fab' fragment can be
evaluated by an analgesic effect test or the like that uses a mouse
arthritis model. It is also possible to evaluate the retention
effect in a lesional tissue by using a test for distribution
property into a lesion.
[0089] Further, the anti-human NGF antibody Fab' fragment of the
present invention may also be evaluated in terms of the risk of
side effects. For example, as described later in examples, by using
a placental transfer test performed after administration of the
Fab' fragment to pregnant animals, it is possible to evaluate the
possibility that the anti-human NGF antibody Fab' fragment of the
present invention may exert effects on a fetus. In addition, as
described later in examples, the size of an immunocomplex (IC)
formed between the anti-human NGF antibody Fab' fragment of the
present invention and NGF is measured by using a test for IC
formation with NGF, whereby the possibility of the induction of
thrombus formation can be evaluated.
[0090] In addition, as the method of evaluating various types of
stability (for example, thermal stability, long-term storage
stability, and high-concentration stability) of the anti-human NGF
antibody Fab' fragment of the present invention, a method of using
differential scanning calorimetry and a method of measuring the
formation of aggregates during storage are exemplified.
[0091] The anti-human NGF antibody Fab' fragment of the present
invention is optionally purified and then formulated according to
common methods, and can be used for treating pain such as
osteoarthritis pain (OA pain), rheumatic pain, cancer pain,
neuropathic pain, chronic low back pain, postoperative pain,
postherpetic neuralgia, painful diabetic neuropathy, fracture pain,
and painful bladder syndrome and diseases in which NGF is involved
in the formation of pathological conditions, such as interstitial
cystitis, acute pancreatitis, chronic pancreatitis, and
endometriosis.
[0092] The anti-human NGF antibody Fab' fragment of the present
invention can be used preferably as an agent for treating pain and
more preferably as an agent for treating osteoarthritis pain.
Examples of the formulation of this treating agent and the like
include parenteral formulations such as injectable agents and
infusion agents, which are preferably administered by intravenous
administration, subcutaneous administration and the like. In the
formulation process, carriers or additives that match these
formulations can be used within a pharmaceutically acceptable
range.
[0093] The amount of inventive anti-human NGF antibody Fab'
fragment added in the above-described formulation varies depending
on the patient's symptom severity or age, the dosage form of the
formulation used or the binding titer of the antibody and the like;
for example, about 0.001 mg/kg to 100 mg/kg of the antibody may be
used.
[0094] The present invention also provides a polynucleotide
comprising a sequence encoding the anti-human NGF antibody Fab'
fragment of the present invention, and an expression vector
comprising the same. The present invention also provides a
polynucleotide comprising a sequence encoding the heavy-chain
variable region of the anti-human NGF antibody Fab' fragment of the
present invention, and a polynucleotide comprising a sequence
encoding the light-chain variable region of the anti-human NGF
antibody Fab' fragment of the present invention, and expression
vector comprising either or both of them. The expression vector of
the present invention is not specifically limited, so long as it
can express a gene that encodes the Fab' fragment of the present
invention or its heavy-chain variable region and/or light-chain
variable region in various host cells of prokaryotic cells and/or
eukaryotic cells, and produce these polypeptides. Examples thereof
include plasmid vectors, viral vectors (for example, adenovirus,
retrovirus) and the like. Preferably, the expression vector of the
present invention comprises a polynucleotide comprising either a
sequence encoding the heavy chain fragment or light chain fragment
of the above-described Fab' fragment of the present invention, or
both a polynucleotide comprising a sequence encoding the heavy
chain fragment of the Fab' fragment of the present invention and a
polynucleotide comprising a sequence encoding the light chain of
the Fab' fragment of the present invention.
[0095] The expression vector of the present invention can comprise
a gene that encodes the anti-human NGF antibody Fab' fragment of
the present invention, or a gene that encodes the heavy-chain
variable region and/or light-chain variable region of the
anti-human NGF antibody Fab' fragment of the present invention, and
a promoter operably linked to the gene. Examples of a promoter for
expressing a gene encoding the Fab' fragment of the present
invention or its heavy-chain variable region and/or light-chain
variable region in a bacterium include Trp promoter, lac promoter,
recA promoter, .lamda.PL promoter, 1pp promoter, tac promoter and
the like, when the host is a bacterium of the genus Escherichia.
Examples of a promoter for expression in yeast include PHO5
promoter, PGK promoter, GAP promoter and ADH promoter, and some
examples of a promoter for expression in the genus Bacillus include
SL01 promoter, SP02 promoter, penP promoter and the like. When the
host is a eukaryotic cell such as a mammalian cell, examples of the
promoter include SV40-derived promoter, retrovirus promoter, heat
shock promoter and the like.
[0096] When a bacterium, particularly Escherichia coli, is used as
the host cell, the expression vector of the present invention can
further comprise an initiation codon, a stop codon, a terminator
region and a replicable unit. When yeast, an animal cell or insect
cell is used as the host, the expression vector of the present
invention can comprise an initiation codon and a stop codon. In
this case, it may comprise an enhancer sequence, noncoding regions
on the 5' side and 3' side of a gene that encodes the Fab' fragment
of the present invention or the heavy-chain variable region or
light-chain variable region thereof, a secretion signal sequence, a
splicing junction, a polyadenylation region, a replicable unit or
the like. Also, it may comprise a selection marker that is in
common use (for example, tetracycline-resistant gene,
ampicillin-resistant gene, kanamycin-resistant gene,
neomycin-resistant gene, dihydrofolic acid reductase gene)
according to the intended use.
[0097] The present invention also provides a transformant
introduced with a gene encoding the anti-human NGF antibody Fab'
fragment of the present invention or a gene encoding the
heavy-chain variable region and/or light-chain variable region of
the anti-human NGF antibody Fab' fragment of the present invention.
Such a transformant can be prepared by, for example, transforming a
host cell with the expression vector of the present invention. A
host cell that is used to prepare the transformant is not
specifically limited, so long as it is suitable for the
aforementioned expression vector and is transformable; examples
thereof include various cells such as natural cells or artificially
established lines of cells commonly being used in the technical
field of the present invention (for example, bacteria (bacteria of
the genus Escherichia, bacteria of the genus Bacillus), yeasts (the
genus Saccharomyces, the genus Pichia and the like), animal cells
or insect cells (for example, Sf9) and the like. The transformation
can be performed by any known method per se.
[0098] Preferably, the transformant of the present invention is
either a host cell transformed with an expression vector comprising
a polynucleotide comprising a sequence encoding the heavy-chain
variable region of the Fab' fragment of the present invention and a
polynucleotide comprising a sequence encoding the light-chain
variable region of the Fab' fragment, or a host cell transformed
with an expression vector comprising a polynucleotide comprising a
sequence encoding the heavy-chain variable region of the Fab'
fragment of the present invention and an expression vector
comprising a polynucleotide comprising a sequence encoding the
light-chain variable region of the Fab' fragment. More preferably,
the transformant of the present invention is either a host cell
transformed with an expression vector comprising a polynucleotide
comprising a sequence encoding the heavy chain fragment of the
above-described Fab' fragment of the present invention and a
polynucleotide comprising a sequence encoding the light chain of
the Fab' fragment, or a host cell transformed with an expression
vector comprising a polynucleotide comprising a sequence encoding
the heavy chain fragment of the above-mentioned Fab' of the present
invention and an expression vector comprising a polynucleotide
comprising a sequence encoding the light chain of the Fab'
fragment.
[0099] The present invention also provides a method for producing
the anti-human NGF antibody Fab' fragment of the present invention,
comprising expressing in a host cell a gene encoding the anti-human
NGF antibody Fab' fragment of the present invention or a gene
encoding the heavy-chain variable region and/or light-chain
variable region of the anti-human NGF antibody Fab' fragment of the
present invention, that is, using such a transformant. Preferably,
the host cell that is used in the above method is a host cell
transformed with the above-described expression vector of the
present invention, and it may separately or simultaneously comprise
a polynucleotide comprising a sequence encoding the heavy-chain
variable region of the Fab' fragment of the present invention and a
polynucleotide comprising a sequence encoding the light-chain
variable region of the Fab' fragment.
[0100] When producing the anti-human NGF antibody Fab' fragment of
the present invention, the transformant may be cultured in a
nutrient medium. The nutrient medium preferably contains a carbon
source and an inorganic nitrogen source or organic nitrogen source,
which are required for the growth of the transformant. Examples of
the carbon source include glucose, dextran, soluble starch, sucrose
and the like; examples of the inorganic nitrogen source or organic
nitrogen source include ammonium salts, nitrates, amino acids, corn
steep liquor, peptone, casein, meat extract, soybean cake, potato
extract and the like. If desired, other nutrients (for example,
inorganic salts (for example, calcium chloride, sodium dihydrogen
phosphate, magnesium chloride), vitamins, antibiotics (for example,
tetracycline, neomycin, ampicillin, kanamycin and the like) and the
like) may be contained.
[0101] Culture of the transformant is performed by a method known
per se. Culture conditions, for example, temperature, pH of the
medium, and culture time are suitably selected. For example, when
the host is an animal cell, an MEM medium containing about 5% to
20% fetal bovine serum (Science, Vol. 122, p. 501, 1952), DMEM
medium (Virology, Vol. 8, p. 396, 1959), RPMI1640 medium (J. Am.
Med. Assoc., Vol. 199, p. 519, 1967), 199 medium (Proc. Soc. Exp.
Biol. Med., Vol. 73, p. 1, 1950) and the like can be used as the
medium. The pH of the medium is preferably about 6 to 8, culture is
normally performed at about 30.degree. C. to 40.degree. C. for
about 15 to 72 hours, and aeration or agitation may be performed as
necessary. When the host is an insect cell, for example, Grace's
medium comprising fetal bovine serum (Proc. Natl. Acad. Sci. USA,
Vol. 82, p. 8404, 1985) and the like can be mentioned, and the pH
thereof is preferably about 5 to 8. Culturing is normally performed
at about 20.degree. C. to 40.degree. C. for 15 to 100 hours, and
aeration or agitation may be performed as necessary. When the host
is a bacterium, an actinomyces, yeast, or a filamentous fungus, for
example, a liquid medium comprising the above-described nutrient
sources is appropriate. A medium having a pH of 5 to 8 is
preferable. When the host is E. coli, preferred examples of the
medium include LB medium, M9 medium (Miller et al., Exp. Mol.
Genet, Cold Spring Harbor Laboratory, p. 431, 1972) and the like.
In this case, culture can be normally performed at 14.degree. C. to
43.degree. C. for about 3 to 24 hours, while aeration or agitation
is performed as necessary. When the host is a bacterium of the
genus Bacillus, cultivation can be normally performed at 30.degree.
C. to 40.degree. C. for about 16 to 96 hours, while aeration or
agitation is performed as necessary. When the host is yeast,
examples of the medium include Burkholder's minimal medium
(Bostian, Proc. Natl. Acad. Sci. USA, Vol. 77, p. 4505, 1980), and
the pH of the medium is desirably 5 to 8. Culturing is normally
performed at about 20.degree. C. to 35.degree. C. for about 14 to
144 hours, and aeration or agitation may be performed as
necessary.
[0102] The anti-human NGF antibody Fab' fragment of the present
invention can be recovered, preferably isolated and purified, from
a cultured transformant as described above. Examples of the method
of isolation and purification include methods based on differences
in solubility, such as salting-out and solvent precipitation;
methods based on differences in molecular weight, such as dialysis,
ultrafiltration, gel filtration, and sodium dodecyl
sulfate-polyacrylamide gel electrophoresis; methods based on
differences in electric charge, such as ion exchange chromatography
and hydroxyl apatite chromatography; methods based on specific
affinity, such as affinity chromatography; methods based on
differences in hydrophobicity, such as reverse phase high
performance liquid chromatography; methods based on differences in
isoelectric point, such as isoelectric focusing; and the like.
[0103] Although the present invention has been generally described
above, specific examples are provided herein only for a better
understanding of the present invention. These examples are for
illustrative purposes only and do not limit the scope of the
present invention.
EXAMPLS
[0104] In steps using a commercially available kit or reagent,
experiments were performed according to the attached protocols
unless otherwise specified.
Example 1
Immunization of Veloclmmune Mouse
[0105] An anti-human NGF antibody was obtained by immunizing
VelocImmune mice. In order to increase diversity of the obtained
antibody, the present inventors examined a plurality of
immunization methods, routes of administration, adjuvants,
immunization periods, and the like. By using a human .beta.NGF
(R&D Systems, Inc.) as an immunogen, the present inventors
examined a method of immunization in which the human .beta.NGF is
used for immunization by mixing it with an adjuvant after
dissolution, and a method of immunization in which the human
.beta.NGF is used by mixing it with an adjuvant after thermally
denaturing (treating at 80.degree. C. for 10 minutes in a 0.5% SDS
solution). As the route of administration, footpad administration
and intraperitoneal administration were examined. As the adjuvant,
TiterMax Gold (CytRx Corporation), complete Freund's Adjuvant
(Sigma), incomplete Freund's Adjuvant (Sigma), and RIBI Adjuvant
(Corixa Corporation) were examined. As an immunoactivator to be
added, CpG oligonucleotide and Aluminum Phosphate Gel (BRENNTAG)
were examined. As the immunization period, 3 to 14 weeks were
examined. After the animals was immunized several times, blood was
collected from the caudal vein of the mice, and the titer was
monitored. In this manner, Veloclmmune mice producing an antibody
binding to the human NGF were selected.
[0106] The titer was measured using the following standard ELISA
method. The human I3NGF was added to a Maxisorp 384 plate (Nunc) at
10 ng/well and immobilized by being incubated overnight at
4.degree. C. The next day, the plate was washed once with a wash
solution (TBST: a tris buffer (TBS) containing 0.05% Tween-20), a
blocking agent (TBST containing 20% Blocking One (Nacalai Tesque,
Inc.)) was then added thereto, and the plate was left to stand at
room temperature for an hour. After the plate was washed once with
the TBST wash solution, the collected blood was serially diluted
and added to the plate. After an hour of incubation at room
temperature, the plate was washed three times with the TBST wash
solution, and an HRP-goat anti-mouse Ig antibody (Zymed) which was
2000-fold diluted with the TBST wash solution containing 5%
Blocking One was added thereto. After an hour of incubation at room
temperature, the plate was washed three times with the TBST wash
solution. The plate was supplemented with a TMB chromogenic reagent
(SUMITOMO BAKELITE CO., LTD) and left to stand at room temperature
for 10 minutes, a stop solution (2 mol/L sulfuric acid) was then
added thereto to stop the reaction, and an absorbance at 450 nm was
measured.
Example 2
Preparation of Anti-Human NGF Antibody-Producing Hybridoma
[0107] The mice selected by confirming the increase in antibody
titer were finally immunized (intravenous or intraperitoneal
administration of an antigen). The spleen, lymph node, or the like
of the immunized mice was extracted according to a normal method so
as to collect lymphocytes, and the lymphocytes were fused with
mouse myeloma cells SP2/0, thereby preparing a hybridoma. The
hybridoma was subjected to limiting dilution and monocloning, and
the antibody was purified from the supernatant by using a protein A
or protein G column (GE Healthcare Japan).
Example 3
Evaluation of NGF-trkA Binding Inhibition
[0108] The human .beta.NGF (R&D Systems, Inc.) was allowed to
react with EZ-LINK 5-(biotinamido)pentylamine (Pierce) at room
temperature for 30 minutes in a dark place to perform biotin
labeling, and the excess biotin was removed using a desalting
column, thereby obtaining biotin-labeled human .beta.NGF. In the
following Examples 6 and 7, the prepared biotin-labeled human
.beta.NGF was confirmed to have the same biological activity as
that of the original human .beta.NGF.
[0109] Inhibitory activity was measured by the following method.
The human trkA (R&D Systems, Inc.) was added to a white
Maxisorp 384 plate (Nunc) at 60 ng/well and immobilized by being
incubated overnight at 4.degree. C. The next day, the plate was
washed once with the TBST wash solution, a blocking agent (TBST
containing 20% Blocking One (Nacalai Tesque, Inc.)) was then added
thereto, and the plate was left to stand at room temperature for an
hour. Subsequently, a mixture obtained by mixing the biotin-labeled
human .beta.NGF (0.2 .mu.g/ml) prepared as above with the antibody
prepared in Example 2 was added to the trkA-immobilized plate
having undergone blocking. After an hour of incubation at room
temperature, the plate was washed three times with the TBST wash
solution, and alkaline phosphatase-labeled streptavidin (Pierce)
was added thereto. After an hour of incubation at room temperature,
the plate was washed three times with the TBST wash solution and
then supplemented with APU4 (BioFx) which is a reagent detecting
chemiluminescence, and the amount of chemiluminescence was measured
by an EnVision counter (PerkinElmer Co., Ltd.).
Example 4
Evaluation of Species Cross-Reactivity
[0110] When an antibody has cross-reactivity with respect to a
mouse .beta.NGF, it is possible to perform drug efficacy evaluation
in a mouse pathological model by using the antibody. Consequently,
a biotin-labeled mouse .beta.NGF was prepared in the method of
Example 3 by using a mouse .beta.NGF (R&D Systems, Inc.),
whereby the cross-reactivity of the antibody to the mouse .beta.NGF
was evaluated.
Example 5
Evaluation of Binding Specificity
[0111] The binding specificity of the antibody to NOF was evaluated
by using the ELISA method described in Example 1. Specifically,
NT-3 as a family molecule showing the highest homology to NGF was
used. Human NT-3 (PeproTech) was added to the plate in the method
of Example 1 at 20 ng/well and immobilized in the plate, thereby
allowing performance of evaluation.
Example 6
Evaluation of NGF-trkA Signaling Inhibition
[0112] The inhibitory activity of the antibody against NGF-trkA
signaling was evaluated. NGF increases intracellular calcium
(Ca.sup.2+) concentration via trkA as the NGF receptor. Generally,
the change in Ca.sup.2+ concentration can be evaluated in the
presence of a calcium indicator by using an intracellular calcium
(Ca.sup.2+) concentration measurement system (FLIPR; Molecular
Devices, LLC.).
[0113] The inhibitory activity was measured by the following
method. HEK293 cells (WO2009/054468) caused to stably express the
human trkA were dispensed in a 96-well poly-D-lysine-coated plate
(Becton, Dickinson and Company, Japan) at 2.times.10.sup.4
cells/well the day before experiment and cultured overnight. The
next day, the culture medium was replaced with a DMEM culture
medium (containing 3.6 mM sodium hydroxide (NaOH) and 2.5 mM
probenecid (Sigma)) containing a calcium indicator (Fluo4-AM;
Dojindo) and left to stand at 37.degree. C. for 30 minutes.
Thereafter, the cells were washed twice with a wash solution
(Hank's balanced salt solution) (HBSS) (20 mM
2-[4-(2-hydroxyethyl)-1-piperazinyl]ethane sulfonic acid (HEPES),
3.6 mM sodium hydroxide, 2.5 mM probenecid (Sigma), and 0.1% bovine
serum albumin), and the culture medium was replaced with this wash
solution at 150 .mu.l/well. The cell plate was set in the FLIPR. By
operating the FLIPR, a mixed solution of the antibody obtained in
Example 2 and NGF was added to the plate at 50 .mu.l/well (final
NGF concentration of 100 ng/ml), and the change in intracellular
Ca.sup.2+ concentration was measured. The difference between
maximum and minimum values of the change in intracellular Ca.sup.2+
concentration was calculated and stored as measurement data.
Example 7
Evaluation of NGF-Dependent Cell Survival Signaling Inhibition
[0114] When PC12 cells naturally expressing the trkA and p75
receptors are cultured in a serum-free condition, NGF enables the
cells to survive for several days. By the following method, the
inhibitory activity of the antibody against the NGF-dependent cell
survival signaling was evaluated.
[0115] PC12 cells were seeded in a 96-well collagen-coated plate
(ASAHI TECHNO CO., LTD.) at lx10.sup.4 cells/well and incubated
overnight in an F12K culture medium (Invitrogen) containing 2.5%
fetal bovine serum and 15% inactivated horse serum (Invitrogen) at
37.degree. C. under 5% CO.sub.2. The next day, the culture medium
was replaced with only F12K in a serum-free condition. After an
hour, the antibody and the human .beta.NGF (final concentration of
50 ng/ml) were added thereto, followed by culturing for 72 hours.
Subsequently, the culture solution was removed by an aspirator, and
cell viability was measured using a reagent (CellTiter Glo; Promega
Corporation) quantitating endogenous ATP of cells.
Example 8
Preparation of Fab Fragment
[0116] A digestive enzyme papain-bound gel was added to 1 mg/ml of
the antibody by using a Fab preparation kit (Pierce), followed by
treatment at 37.degree. C. for 3 hours. The treated reaction
solution was added to a protein G column (GE Healthcare Japan),
cleaved Fc and unreacted IgG were removed by being adsorbed onto
the column, and the eluted fraction was collected, thereby
obtaining Fab fragments. The obtained Fab fragments were evaluated
by the tests described in Examples 3, 6, and 7.
[0117] As a result of the evaluation of Examples 3 to 8, it was
confirmed that the antibody named 1-15 (chimeric antibody) had high
neutralizing activity, species cross-reactivity, and binding
specificity and maintained high neutralizing activity even though
this antibody is in the form of a monovalent antibody fragment.
Example 9
Determining Antibody Gene Sequence)
[0118] For the identified antibody 1-15, the present inventors
cloned genes encoding the heavy chains and light chains of the
antibody from hybridomas. Specifically, a hybridoma clone was
prepared in an amount of 1.times.10.sup.5 or more and suspended in
RLT buffer which is included in RNeasy Mini Kit (QIAGEN), and then
the cells were shredded with QlAshredder (QIAGEN). Subsequently,
RNA was extracted according to the protocol, and by using the
extracted RNA as a template, cDNA was synthesized using a DNA
amplification kit (SMARTer RACE cDNA Amplification Kit; Clontech).
PCR was carried out using the obtained cDNA, thereby elongating and
amplifying the variable region of the heavy chains and light
chains. Sequence analysis was performed directly on the PCR
products by using a sequencer (ABI PRISM 3100; Applied Biosystems).
In addition, the PCR products were recombined with a PCR product
subcloning vector such as pCR3.1-TOPO (Invitrogen), followed by
gene sequence analysis, thereby determining the sequence.
[0119] The determined base sequence of the heavy-chain variable
region of the antibody 1-15 is shown by SEQ ID NO:1, and the amino
acid sequence thereof is shown by SEQ ID NO:2. Moreover, the base
sequence of the light-chain variable region of the antibody is
shown by SEQ ID NO:3, and the amino acid sequence thereof is shown
by SEQ ID NO:4. The CDR1, CDR2 and CDR3 of the heavy-chain variable
region of the antibody 1-15 is a region of position from 31 to 35,
50 to 65, and 95 to 102 of the heavy-chain variable region based on
Kabat numbering, respectively, which consists of the amino acid
sequence at position from 31 to 35, 50 to 65, and 98 to 110 of SEQ
ID NO:2, respectively. The CDR1, CDR2 and CDR3 of the light-chain
variable region of the antibody 1-15 is a region of position from
24 to 34, 50 to 56, and 89 to 97 of the light-chain variable region
based on Kabat numbering, respectively, which consists of the amino
acid sequence at position from 24 to 39, 55 to 61, and 94 to 102 of
SEQ ID NO:4, respectively.
Example 10
Preparation of Mutant of Glycosylation Site of Variable Region
[0120] The amino acid (SEQ ID NO:2) of the heavy-chain variable
region of the antibody 1-15 described above includes an N-type
glycosylation motif sequence as N-X-(T/S). Specifically, in the
heavy-chain variable region shown by SEQ ID NO:2, Asn (N52) at
position 52 based on Kabat numbering corresponds to the
glycosylation site. It is known that though glycosylation of an
antibody occurs during cell culturing if the antibody has
glycosylation site, the glycosylation depends on the culturing
conditions or the host expressing the antibody. In other words,
even among the same antibody-producing cells established, the
degree of glycosylation is likely to vary with the culturing
conditions (such as the culture medium and cell density), which
leads to a possibility that it may be difficult to obtain antibody
drugs having uniform quality. Therefore, the present inventors
prepared 1-15(N52D) which was obtained by introducing a mutation to
N52 in the heavy-chain variable region of the antibody 1-15.
[0121] The base sequence of the heavy-chain variable region of the
prepared 1-15(N52D) is shown by SEQ ID NO:5, and the amino acid
sequence thereof is shown by SEQ ID NO:6. The CDR1, CDR2 and CDR3
of the heavy-chain variable region of the antibody 1-15(N52D) is a
region of position from 31 to 35, 50 to 65, and 95 to 102 of the
heavy-chain variable region based on Kabat numbering, respectively,
which consists of the amino acid sequence at position from 31 to
35, 50 to 65, and 98 to 110 of SEQ ID NO:6, respectively.
Example 11
Preparation of Fully Human Antibody Fab' Fragment
[0122] By using the heavy-chain variable regions of 1-15 and
1-15(N52D) described above and the light-chain variable region of
1-15, the respective fully human antibody Fab' fragments were
prepared.
[0123] A signal sequence was linked to the 5' side of the
respective heavy-chain variable region genes of 1-15 and
1-15(N52D), and the constant region gene of human Ig.gamma.1 (Man
Sung Co. et al., (1992) J Immunol. Vol. 148(4):1149-1154) was
linked to the 3' side thereof respectively. This heavy-chain
fragment gene was inserted into GS vector pEE6.4 (Lonza Biologics).
At this time, in order to express the genes as a Fab' fragment, a
stop codon was inserted after the codon of Cys at position 226
(corresponding to Cys at position 230 in the amino acid sequence of
SEQ ID NO:8 and SEQ ID NO:10 described later) based on the EU index
in the heavy-chain constant region gene. In addition, a signal
sequence was linked to the 5' side of the light-chain variable
region gene of 1-15, and the constant region gene of human .kappa.
chain (Man Sung Co. et al., described above) was linked to the 3'
side thereof respectively. This light-chain gene was inserted into
GS vector pEE12.4 (Lonza Biologics).
[0124] The Fab' fragment was expressed in two manners including
transient expression and constant expression. In the transient
expression, FreeStyle 293 cells (Invitrogen) cultured in FreeStyle
293 Expression Medium (Invitrogen) at about 1,000,000 cells/mL were
transfected with the above-described GS vectors of the heavy-chain
fragment and the light-chain by using 293fectin (Invitrogen),
followed by culturing for seven days. In the constant expression,
both the GS vectors described above were cleaved with restriction
enzymes NotI and PvuI, followed by ligation by using a DNA ligation
kit (TAKARA BIO INC), thereby constructing a GS vector into which
genes of both the heavy-chain fragment and the light-chain were
inserted. This expression vector encodes the heavy-chain fragment,
the light chain, and glutamine synthetase and was expressed by
being transfected to CHO-K1-SV cells. After the vectors were
expressed in the respective manners, the culture supernatant was
purified by using KappaSelect (GE Healthcare Japan), thereby
obtaining the respective Fab' fragments.
[0125] The base sequence of the heavy-chain fragment of the
prepared fully human 1-15 antibody Fab' fragment (also referred to
as 1-15-Fab') is shown by SEQ ID NO:7, and the amino acid sequence
thereof is shown by SEQ ID NO:8 respectively.
[0126] The base sequence of the heavy-chain fragment of the
prepared fully human 1-15(N52D) antibody Fab' fragment (also
referred to as 1-15(N52D)-Fab') is shown by SEQ ID NO:9, and the
amino acid sequence thereof is shown by SEQ ID NO:10
respectively.
[0127] The light chain of the respective Fab' fragments are the
same and the base sequence thereof is shown by SEQ ID NO:11, and
the amino sequence thereof is shown by SEQ ID NO:12
respectively.
Example 12
Evaluation of Neutralizing Activity and Expression Level of Fully
Human Antibody Fab' Fragment)
[0128] The 1-15-Fab' and the 1-15(N52D)-Fab' obtained in Example 11
were evaluated by the tests described in Examples 3 and 6. In the
test of Example 3, IC50 of the 1-15-Fab' and the 1-15(N52D)-Fab'
was 0.17 .mu.g/ml and 0.18 .mu.g/ml respectively. In the test of
Example 6, IC50 of the 1-15-Fab' and the 1-15(N52D)-Fab' was 0.021
.mu.g/m1 and 0.018 .mu.g/ml respectively. From these results, it
was confirmed that the neutralizing activity of the 1-15(N52D)-Fab'
was maintained to almost the same degree as that of the unmodified
1-15-Fab', and that the neutralizing activity was not influenced
even if a mutation was introduced.
[0129] In addition, the respective Fab' fragments were expressed by
the constant expression, and the amount of antibody produced in the
culture supernatant of a stable expression cell pool was measured.
As a result, the concentrations of the respective culture
supernatants of the 1-15-Fab' and the 1-15(N52D)-Fab' were 86 mg/L
and 106 mg/L respectively, which showed that the 1-15(N52D)-Fab' is
an antibody produced in a higher amount than the unmodified
1-15-Fab'.
Example 13
Preparation of PEGylated Fab' Fragment and Evaluation of
Neutralizing Activity
[0130] Next, the present inventors introduced PEG to the
1-15(N52D)-Fab'. After being purified by KappaSelect, the Fab'
fragment was subjected to a reduction reaction by using TCEP
hydrochloride (Tris(2-carboxyethyl)phosphine HCl), whereby the Fab'
fragment was made into a PEGylatable structure.
[0131] Specifically, TCEP was added to a Fab' fragment solution of
which the concentration was adjusted to 1.2 mg/ml by a 20 mM of
sodium phosphate buffer (pH 6.8), such that the TCEP became 1 mM,
followed by a reaction at 37.degree. C. for 2 hours, and then the
resultant was diluted with a 20 mM sodium acetate buffer (pH 5.0)
to adjust pH. This solution was adsorbed onto a cation exchange
resin (SP-5PW; TOSOH CORPORATION) and subjected to NaC1 gradient
elution, and the main peak was collected. The obtained Fab'
fragment was diluted with a 20 mM sodium phosphate buffer (pH 6.8)
so as to yield 1 mg/ml, the pH was adjusted to 6.8, and then the
solution was left to stand at 4.degree. C. for a night or longer so
as to be naturally oxidized. 40 kDa PEG (SUNBRIGHT GL2-400MA; NOF
CORPORATION) was added to the solution to yield a final
concentration of 0.1 mM, and the solution was left to stand at room
temperature for 2 hours and then at 4.degree. C. overnight. Having
a maleimide group on the terminal thereof, this PEG rapidly reacts
with Cys (C226 based on EU index; Cys at position 230 of SEQ ID
NO:10) of the carboxyl terminal of the heavy-chain fragment. The
solution was diluted with a 20 mM sodium acetate buffer (pH 4.5) to
adjust pH and then adsorbed again onto a cation exchange resin
(SP-5PW; TOSOH CORPORATION), the resultant was subjected to NaC1
gradient elution, and the main peak was collected. The resultant
PEGylated Fab' fragment was purified. This PEGylated
1-15(N52D)-Fab' is also called 1-15(N52D)-Fab'-PEG.
[0132] The neutralizing activity of the non-PEGylated and PEGylated
1-15(N52D)-Fab's was evaluated by the method shown in Example 3. As
a result, while IC50 of the 1-15(N52D)-Fab' was 0.15 .mu.g/ml, IC50
of the 1-15(N52D)-Fab'-PEG was 0.12 .mu.g/ml (in terms of Fab'
fragment concentration), whereby it was confirmed that the
neutralizing activity of the 1-15(N52D)-Fab' was not influenced
even if PEG was added.
[0133] In addition, by using the method of Example 6, the
1-15(N52D)-Fab'-PEG was compared to the anti-human NGF antibody
tanezumab of the prior art, in terms of the neutralizing activity
with respect to human and mouse NGFs. As a result, while IC50 of
the 1-15(N52D)-Fab'-PEG was 0.051 .mu.g/ml for the human NGF and
0.069 .mu.g/ml for the mouse NGF, IC50 of the tanezumab was 0.17
.mu.g/ml for the human NGF and 0.23 .mu.g/ml for the mouse NGF.
Therefore, it was confirmed that the neutralizing activity of the
1-15(N52D)-Fab'-PEG was about 3.3 times stronger than the
tanezumab, with respect to any of the human and mouse NGFs.
Example 14
Analgesic Effect Test using Mouse Model of Adjuvant-Induced
Arthritis
[0134] The present inventors evaluated an analgesic effect of the
above 1-15(N52D)-Fab'-PEG on a mouse model of adjuvant-induced
arthritis.
[0135] The 1-15(N52D)-Fab'-PEG was intravenously administered (0.03
mg/kg, 0.1 mg/kg, and 0.3 mg/kg; the dose was 10 mL/kg) to mice,
and 1 mg/mL Freund's complete adjuvant (Sigma) was administered in
an amount of 25 .mu.L to the hindlimb footpad to induce pain. 24
hours after the pain induction, a rearing behavior for 20 minutes
was measured. Specifically, by using SUPERMEX spontaneous activity
monitoring system (Muromachi Kikai Co., Ltd.), the number of times
of spontaneous rearing behavior of the mice was automatically
measured for 20 minutes by using an infrared beam sensor (Matson et
al., JPET 320:194-201, 2007). As a comparative control, a prior art
antibody tanezumab was used. As a result, while the intravenous
administration of the tanezumab produced an analgesic effect of
ED50=0.27 mg/kg, the 1-15(N52D)-Fab'-PEG produced an analgesic
effect of ED50=0.11 mg/kg which showed an effectiveness greater by
about 3 times.
Example 15
Rat Placental Transfer Test
[0136] The 1-15(N52D)-Fab'-PEG or the tanezumab was intravenously
administered (100 mg/kg, the dose was 10 mL/kg) to female rats in
the 17th day of pregnancy. Three days later, antibody concentration
in the blood of the mother and fetus was measured.
[0137] The antibody concentration was measured in the following
manner. The human .beta.NGF (R&D Systems, Inc.) was added to a
MULTI-ARRAY Plate (Standard) 96 plate (Meso Scale Discovery) at 25
ng/well and immobilized by being left to stand at room temperature
for an hour. The plate was washed three times with the TBST wash
solution, and a blocking agent (1% casein TBS; Thermo Fisher) was
added thereto and left to stand at room temperature for an hour.
Subsequently, a blood sample obtained by diluting blood collected
over time was added to the human .beta.NGF-immobilized plate having
undergone blocking. After the mixture was reacted at room
temperature for 60 minutes under stirring, the plate was washed
three times with the TBST wash solution, and then a biotin-labeled
anti-human Kappa antibody (Immuno-Biological Laboratories Co.,
Ltd.) was added thereto. After the mixture was reacted for 60
minutes at room temperature under stirring, the plate was washed
three times with the TBST wash solution, and SULFO-TAG-labeled
streptavidin (Meso Scale Discovery) was added thereto. After the
mixture was reacted for 60 minutes at room temperature under
stirring, the plate was washed three times with the TBST wash
solution, Read Buffer T (Meso Scale Discovery) was added thereto,
and the amount of electrochemical luminescence was measured with
SECTOR Imager 6000 (Meso Scale Discovery).
[0138] This test was performed on three mother rats. Three days
later, the antibody concentration of the 1-15(N52D)-Fab'-PEG and
the tanezumab in the blood of the mother rats was 12.1 .mu.g/ml and
7.1 .mu.g/m1 on average respectively. Meanwhile, regarding the
antibody concentration in the blood of 3 fetuses extracted from
each mother rat (9 fetuses in total), while the concentration of
1-15(N52D)-Fab'-PEG in the blood was 0.01 .mu.g/ml (quantitation
limit) or less in all fetuses, the concentration of tanezumab in
the blood was 5.39 .mu.g/ml on average. That is, while the
tanezumab was transferred to the fetus at a rate of 75.9%, the
1-15(N52D)-Fab'-PEG was transferred to the fetus at a rate of 0.08%
(detection limit) or less. These results suggested that the
1-15(N52D)-Fab'-PEG is a medical agent which is excellent in safety
by avoiding the risk of side effects caused in a fetus due to NGF
inhibition.
Example 16
Formation of Immunocomplex (IC)
[0139] Whether or not the 1-15(N52D)-Fab'-PEG formed an IC, or how
large the size of the formed IC was evaluated. Specifically, 1
mg/ml of the 1-15(N52D)-Fab'-PEG was mixed with the human .beta.NGF
(R&D Systems, Inc.) at a molar ratio of 1:1, followed by
incubation at room temperature for 3 hours, thereby forming an IC.
The particle size and distribution of the IC in this reaction
solution were measured using Zetasizer Nano (Malvern) as an
instrument measuring dynamic light scattering. For the analysis, a
Zetasizer v6.01 (Malvern) was used, and the particle size was
indicated by a value (d. nm) analyzed in terms of Intensity
(%).
[0140] The measured particle sizes are shown in the following Table
1. In this experiment, the particle size of only the NGF was 6.2 nm
on average. In the case of only the tanezumab, a peak size was
shown at 11.7 nm. When the IC formed by incubating the tanezumab
and the NGF was measured, the peak size shifted to 91.3 nm. On the
other hand, when an antibody not binding to the NGF was used as a
control antibody, the peak size was still 11.7 nm. In consideration
of the shifting width, it was assumed that each of the tanezumab
and the NGF became a macromolecule as a combination of a plurality
of molecules, whereby a large-sized IC was formed. Contrary to
this, when IC formation of the 1-15(N52D)-Fab'-PEG and the NGF was
measured, the peak size shifted from 18.1 nm to 24.4 nm. In
consideration of the shifting width, this result reflected only
one-to-one binding and suggested that lattice formation did not
occur in the 1-15(N52D)-Fab'-PEG
TABLE-US-00001 TABLE 1 Particle size Sample Peak (d. nm) Average
(d. nm) Control IgG IgG 11.7 12.8 IgG + rhNGF 11.7 12.6 Tanezumab
IgG 11.7 13.0 IgG + rhNGF 91.3 99.2 1-15(N52D)-Fab'-PEG IgG 18.1
19.8 IgG + rhNGF 24.4 26.0
Example 17
Distribution Property into Lesional Tissue
[0141] An emulsion including collagen (bovine joint-derived type 2
collagen, 10 mg/mL; Collagen technique workshop) and a complete
Freund's adjuvant (0.5 mg/mL; DIFCO) at a ratio of 1:1 was
subcutaneously administered to the ankle joint of male DBA/1 mice,
thereby preparing collagen-induced arthritis models. Four weeks
after the induction of arthritis, the emulsion was administered
again to cause arthritis. The degree of development (score and the
size of swelling) of the arthritis in hindlimbs was observed to
group the mice. Fluorescent labeling was performed on 1 mg/mL PBS
solutions of the 1-15(N52D)-Fab'-PEG and the tanezumab by using
SAIVI.sup.TM Rapid Antibody Labeling Kit, Alexa Fluor (registered
trademark) 680 (Life Technologies Corporation). Each solution was
administered to the caudal vein at 2 mg/kg (N=4). The fluorescence
accumulated in the swollen footpad was analyzed for 50 hours from
an hour after the administration by using an IVIS Spectrum
(Caliper/Xenogen), and the fluorescence intensity was indicated as
numerical values.
[0142] FIG. 1 shows temporal change of the amount of antibody
retained in the sole. The 1-15(N52D)-Fab'-PEG more clearly showed
the retention effect in a lesional tissue compared to the
tanezumab, and this effect lasted for 48 hours. From this result,
the 1-15(N52D)-Fab'-PEG is considered to efficiently exert an
analgesic effect, and it is expected that this antibody will be
able to exert an analgesic effect equal to or stronger than the
strength of the drug efficacy with a low dose. It is also expected
that the 1-15(N52D)-Fab'-PEG can be a medical agent excellent in
safety since this antibody is selectively accumulated in a lesional
site.
Example 18
Preparation of Amino Acid Adduct of Fab' Fragment
[0143] In order to improve the efficiency of PEG introduction into
the 1-15(N52D)-Fab', the present inventors prepared Fab' fragments
that were obtained by adding two alanines (A) or prolines (P) after
the Cys residue in the carboxyl terminal of the heavy-chain
fragment and performed expression and purification. The same method
as in Example 11 was used to prepare these Fab' fragments. In this
method, the codon of the two alanines or prolines were inserted
after the codon of the Cys residue of the carboxyl terminal of the
heavy-chain fragment of the 1-15(N52D)-Fab', and a stop codon was
inserted after this codon.
[0144] The base sequence of the heavy-chain fragment of
alanine-added 1-15(N52D)-Fab' (a fully human 1-15(N52D-A) antibody
Fab' fragment; also referred to as a 1-15(N52D-A)-Fab') is shown by
SEQ ID NO:13, and the amino acid sequence thereof is shown by SEQ
ID NO:14 respectively. The base sequence of the heavy-chain
fragment of proline-added 1-15(N52D)-Fab' (a fully human
1-15(N52D-P) antibody Fab' fragment; also referred to as a
1-15(N52D-P)-Fab') is shown by SEQ ID NO:15, and the amino acid
sequence thereof is shown by SEQ ID NO:16 respectively. The light
chain of the respective Fab' fragments is the same as the light
chain of the 1-15(N52D)-Fab', and the base sequence and the amino
acid sequence thereof are shown by SEQ ID NO:11 and SEQ ID NO:12
respectively.
Example 19
Preparation of PEGylated 1-15(N52D-A)-Fab' and Evaluation of
Neutralizing Activity and Pharmacological Evaluation
[0145] 40 kDa PEG was conjugated to the 1-15(N52D-A)-Fab' in the
same manner as in Example 13, thereby obtaining PEGylated
1-15(N52D-A)-Fab' (hereinbelow, also referred to as
1-15(N52D-A)-Fab'-PEG).
[0146] The neutralizing activity of the 1-15(N52D-A)-Fab'-PEG was
evaluated in the method described in Example 3. As a result, while
IC50 of the 1-15(N52D)-Fab'-PEG was 0.081.+-.0.034 IC50 of the
1-15(N52D-A)-Fab'-PEG was 0.074.+-.0.021 In addition, IC50 of the
tanezumab at this time was 0.410.+-.0.099 .mu.g/ml.
[0147] Next, the neutralizing activity was compared using the
method described in Example 6. As a result, while IC50 of the
1-15(N52D)-Fab'-PEG was 0.061.+-.0.011 .mu.g/ml for human NGF, IC50
of the 1-15(N52D-A)-Fab'-PEG was 0.064.+-.0.028 .mu.g/ml.
[0148] Moreover, the analgesic effect in the adjuvant-induced
arthritis model was evaluated using the method described in Example
14. As a result, the 1-15(N52D-A)-Fab'-PEG showed the analgesic
effect with respect to the arthritis model.
[0149] From the above results, it was confirmed that even if two
alanines are added after the Cys residue of the carboxyl terminal,
the neutralizing activity and the pharmacological activity are not
influenced.
Example 20
Evaluation of Binding Affinity of 1-15(N52D-A)-Fab'-PEG
[0150] Thermodynamics in binding of 1-15(N52D-A)-Fab'-PEG and
tanezumab to an NGF antigen was examined by Isothermal titration
calorimetry (ITC) (Scappaticci FA, J Natl Cancer Inst. 2007,
99:1232-9. Velazquez-Compoy, A., et al, Curr Protoc Cell Biol.
2004, Chapter 17, Unit 17-18). The entire measurement was performed
using Auto-iTC 200 manufactured by GE healthcare. During the
experiment, a test was performed at the following concentration so
as to evaluate binding of a monovalent Fab' fragment to one
molecule of antigen, and the entire test was performed in a PBS
solution. Specifically, 44 .mu.M of human .beta.NGF (R&D
Systems, Inc.) contained in a titration syringe was titrated to
calorimeter cells filled with an antibody sample (3 .mu.M of
1-15(N52D-A)-Fab'-PEG or 1.5 .mu.M of tanezumab) at 1.4 .mu.L for
30 times, and the amount of heat produced thereby was detected. The
obtained data was analyzed by a Single site binding model by using
software attached to the instrument, whereby binding affinity (Kd),
a binding ratio (n), binding free energy (AG), binding enthalpy
(AH), and binding entropy (-TAS) associated with antigen-antibody
binding were estimated. The results are shown in Table 2.
[0151] As a result, while the value of Kd of tanezumab was 20.41
nM, the value of Kd of 1-15(N52D-A)-Fab'-PEG was 1.49 nM, which
showed that the binding affinity of 1-15(N52D-A)-Fab'-PEG was
stronger by 10 times or more than that of tanezumab (Table 2).
TABLE-US-00002 TABLE 2 .DELTA.G .DELTA.H -T.DELTA.S Kd (nM)
(cal/mol) (cal/mol) (cal/mol) Tanezumab 20.41 -10490 -4759 -5731
1-15(N52D-A)-Fab'-PEG 1.49 -12041 -20806 8765
Example 21
Preparation of PEGylated 1-15(N52D-A)-Fab' having Various PEG Sizes
and Evaluation of Neutralizing Activity
[0152] The 1-15(N52D-A)-Fab' prepared in Example 18 was conjugated
to 5 kDa PEG or 10 kDa PEG by using the similar procedure to that
of Example 13. Specifically, Fab' fragment solution prepared by
using 20 mM Tris-HCl buffer (pH 7.4) was subjected to reduction
treatment by using TCEP. Then, Fab' fragment was collected by using
a desalting column. PEG (SUNBRIGHT GL2-SOMA or SUNBRIGHT GL2-100MA;
NOF CORPORATION) was added to the obtained Fab' fragment, and the
solution was left to stand at 4.degree. C. for a night. The
1-15(N52D-A)-Fab' fragments conjugated to 5 kDa PEG or to 10 kDa
PEG which were obtained in this manner are called
1-15(N52D-A)-Fab'-SkPEG and 1-15(N52D-A)-Fab'-10kPEG,
respectively.
[0153] Thereafter, by using the method shown in Example 6, the
respective PEGylated Fab' fragments were compared with each other
in terms of the neutralizing activity. As a comparative control,
the 1-15(N52D-A)-Fab'-PEG (conjugated to 40 kDa PEG; hereinbelow,
also referred to as 1-15(N52D-A)-Fab'-40kPEG) prepared in Example
19 was used. At this time, the test was performed at a final NGF
concentration of 50 ng/ml.
[0154] As a result, IC50 of the 1-15(N52D-A)-Fab'-5kPEG,
1-15(N52D-A)-Fab'-10kPEG, and 1-15(N52D-A)-Fab'-40kPEG were 0.030
.mu.g/ml, 0.028 .mu.g/ml, and 0.023 .mu.g/ml, respectively. From
these results, it was understood that a PEG size ranging from 5 kDa
to 40 kDa did not influence the neutralizing activity of Fab'
fragments.
Example 22
Mouse PK Evaluation for PEGylated 1-15(N52D-A)-Fab' having Various
PEG Sizes
[0155] Mouse PK evaluation was performed for various types of
PEGylated 1-15(N52D-A)-Fab'. Specifically, 0.3 mg/kg of various
types of PEGylated 1-15(N52D-A)-Fab' were intravenously
administered, and blood was collected 1, 4, 8, 12, 24, 48, 72, 96,
and 168 hours after the administration. The amount of tested
antibody in the obtained blood was measured by using the sandwich
ELISA. Specifically, the tested antibody was added to MSD plate
(Meso Scale Discovery) which NGF was immobilized. The antibody
bound to the plate was recognized by a biotin-labeled anti-human
Kappa antibody, which was then detected by SULFO-TAG-labeled
streptavidin. The concentration of the antibody in the blood was
calculated by creating a calibration curve by using the respective
standards. From the calculated concentration of the antibody in the
blood, the antibody half-life in the blood (T1/2: hour) was
calculated. As a result, T1/2 of the 1-15(N52D-A)-Fab'-5kPEG,
1-15(N52D-A)-Fab'-10kPEG, and 1-15(N52D-A)-Fab'-40kPEG were
13.8.+-.2.2 hours, 17.7.+-.0.4 hours, and 39.2.+-.3.7 hours,
respectively.
Example 23
Analgesic Effect Test using Rat Plantar Incision Model
[0156] By using a rat post-plantar incision pain model (Brennan et
al, Current Protocols in Pharmacology 2004; 5.34.1-5.34.8) which is
considered to reflect postoperative pain in clinical practice, the
analgesic effect of the 1-15(N52D-A)-Fab'-5kPEG and the
1-15(N52D-A)-Fab'-10kPEG on postoperative pain was evaluated.
[0157] Specifically, 8 rats were assigned to each group, and the
1-15(N52D-A)-Fab'-5kPEG or -10kPEG was intravenously administered
to the rats (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg, the dose was 1
mL/kg). Thereafter, in the sole of right hindlimb, a straight
incision was made which extended 10 mm toward the toe from a
starting point at a position distant by 5 mm from the end of the
heel, and then mattress sutures were immediately made with a nylon
thread at two sites, thereby inducing pain. Pain thresholds around
the operation site were measured after 5 hours and the first,
second, third, fourth, and fifth days after the pain induction. For
the measurement, a Dynamic plantar anesthesiometer manufactured by
Ugo Basile was used to measure a pressure at which the rats showed
avoidance behavior when pressure was applied to the sole. As a
comparative control, the antibody tanezumab in the prior art was
used.
[0158] As a result, while intravenous administration of tanezumab
resulted in an analgesic effect of ED50=0.26 mg/kg on postoperative
day 1, both the 1-15(N52D-A)-Fab'-5kPEG and -10kPEG exerted an
analgesic effect of ED50=0.15 mg/kg which was about twice as
efficacious. In addition, a significant analgesic effect of the
1-15(N52D-A)-Fab'-5kPEG and -10kPEG was still observed on
postoperative day 3 and 4, respectively.
Example 24
Evaluation of Aggregation Stability
[0159] The 1-15(N52D-A)-Fab'-40kPEG was dissolved at 1 mg/ml and 10
mg/ml under conditions of pH 5, pH 6, pH 7.4, and pH 9. Each of
these solutions were placed under a condition of 50.degree. C. so
as to evaluate aggregation stability observed after 2 weeks. To
evaluate the aggregation property, size exclusion chromatography
was performed by using Agilent 1100 manufactured by Agilent. As
measurement conditions, 0.1 M sodium phosphate containing 0.2 M
arginine (pH 6.8) was used as a buffer of mobile phase, and TSK gel
Super Sw3000 (TOSOH, 2.0 mm IDx300 mm) was used as a column. The
detection wavelength was 280 nm. In the test at 1 mg/ml, tanezumab
was used as comparative antibody, and the results are shown in
Table 3. In the test at 10 mg/ml, tanezumab and REGN 475 were used
as comparative antibodies, and the results are shown in Table
4.
[0160] As a result, for tanezumab and REGN 475, marked increase in
the amount of produced aggregates was observed after two weeks.
Contrary to this, for the 1-15(N52D-A)-Fab'-40kPEG, aggregates were
almost not detected. This result suggests that the PEGylated
1-15(N52D-A)-Fab' is highly likely to be a drug having excellent
storage stability.
TABLE-US-00003 TABLE 3 1-15(N52D-A)- Fab'-40kPEG Tanezumab Time
Aggregation (%) Aggregation (%) pH (day) Polymer Dimer Polymer
Dimer pH 5 0 0.0 0.7 0.2 2.5 14 0.0 0.7 11.7 5.6 pH 6 0 0.0 0.7 0.3
2.7 14 0.0 0.6 1.3 3.8 pH 7.4 0 0.0 0.7 0.3 2.8 14 0.0 0.6 3.1 3.8
pH 9 0 0.0 0.6 0.3 2.8 14 0.4 1.1 5.7 4.7
TABLE-US-00004 TABLE 4 1-15(N52D-A)- Fab'-40kPEG Tanezumab REGN 475
Time Aggregation (%) Aggregation (%) Aggregation (%) pH (day)
Polymer Dimer Polymer Dimer Polymer Dimer pH 5 0 0.0 0.4 0.9 4.8
0.3 1.7 14 0.1 0.5 23.5 8.4 8.7 3.9 pH 6 0 0.0 0.4 -- -- 1.2 3.0 14
0.0 0.7 -- -- 2.1 3.6 pH 7.4 0 0.0 0.5 1.7 6.1 0.3 2.0 14 0.0 0.8
5.6 7.8 4.0 2.6 pH 9 0 0.0 0.6 1.8 6.4 0.3 1.8 14 0.0 1.5 7.1 7.5
12.9 2.9 -- not tested
INDUSTRIAL APPLICABILITY
[0161] The anti-human NGF antibody, more specifically, the
anti-human NGF antibody Fab' fragment of the present invention is
useful for preventing or treating various diseases in which human
NGF is involved in the formation of pathological conditions.
Sequence CWU 1
1
161363DNAArtificial SequenceVH gene of anti-human NGF antibody
1caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc
60acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctgggt ccgccagccc
120ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac
caacaacaac 180ccgtccctca agagtcgagt caccatatca gtaggcacgt
ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc cgcggacacg
gctgtgtatt actgttcgag agatgggggc 300cccgaatcgg ggatgggggc
ttttgatatc tggggccaag ggacaatggt caccgtctcc 360tca
3632121PRTArtificial SequenceVH of anti-human NGF antibody 2Gln Val
Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20
25 30 Tyr Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Asn Asn Pro
Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Gly Thr Ser Lys
Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Arg Asp Gly Gly Pro Glu Ser
Gly Met Gly Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 3339DNAArtificial SequenceVL gene of
anti-human NGF antibody 3gatattgtga tgactcagtc tccactctcc
ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg
catagtaatg gattcaacta tttgggttgg 120tacctgcaga agccagggca
gtctccacag ctcctgatct atttgggttc taatcgggcc 180tccggggtcc
ctgacaggtt cagtggcagt ggatcaggca cagattttac tctgaaaatc
240agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct
acaaactccg 300tacacttttg gccaggggac caagctggag atcaaacgg
3394113PRTArtificial SequenceVL of anti-human NGF antibody 4Asp Ile
Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20
25 30 Asn Gly Phe Asn Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser
Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Tyr Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg
5363DNAArtificial SequenceVH gene of anti-human NGF antibody
5caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc
60acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctgggt ccgccagccc
120ccagggaagg ggctggagtg gattggggaa atcgaccata gtggaagcac
caacaacaac 180ccgtccctca agagtcgagt caccatatca gtaggcacgt
ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc cgcggacacg
gctgtgtatt actgttcgag agatgggggc 300cccgaatcgg ggatgggggc
ttttgatatc tggggccaag ggacaatggt caccgtctcc 360tca
3636121PRTArtificial SequenceVH of anti-human NGF antibody 6Gln Val
Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20
25 30 Tyr Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Asn Asn Pro
Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Gly Thr Ser Lys
Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Arg Asp Gly Gly Pro Glu Ser
Gly Met Gly Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val
Thr Val Ser Ser 115 120 7693DNAArtificial SequenceH-chain fragment
gene of anti-human NGF antibody 7caggtgcagc tacagcagtg gggcgcagga
ctgttgaagc cttcggagac cctgtccctc 60acctgcgctg tctatggtgg gtccttcagt
ggttactact ggagctgggt ccgccagccc 120ccagggaagg ggctggagtg
gattggggaa atcaatcata gtggaagcac caacaacaac 180ccgtccctca
agagtcgagt caccatatca gtaggcacgt ccaagaacca gttctccctg
240aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgttcgag
agatgggggc 300cccgaatcgg ggatgggggc ttttgatatc tggggccaag
ggacaatggt caccgtctcc 360tcagcctcca ccaagggccc atcggtcttc
cccctggcac cctcctccaa gagcacctct 420gggggcacag cggccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480tcgtggaact
caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc
540tcaggactct actcccttag tagcgtggtg accgtgccct ccagcagctt
gggcacccag 600acctacatct gcaacgtgaa tcacaagccc agcaacacca
aggtggacaa gaaagttgag 660cccaaatctt gtgacaaaac tcacacatgc tga
6938230PRTArtificial SequenceH-chain fragment of anti-human NGF
antibody 8Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Val Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser
Thr Asn Asn Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser
Val Gly Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Arg Asp
Gly Gly Pro Glu Ser Gly Met Gly Ala Phe Asp Ile Trp Gly 100 105 110
Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115
120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys
Thr His Thr Cys 225 230 9693DNAArtificial SequenceH-chain fragment
gene of anti-human NGF antibody 9caggtgcagc tacagcagtg gggcgcagga
ctgttgaagc cttcggagac cctgtccctc 60acctgcgctg tctatggtgg gtccttcagt
ggttactact ggagctgggt ccgccagccc 120ccagggaagg ggctggagtg
gattggggaa atcgaccata gtggaagcac caacaacaac 180ccgtccctca
agagtcgagt caccatatca gtaggcacgt ccaagaacca gttctccctg
240aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgttcgag
agatgggggc 300cccgaatcgg ggatgggggc ttttgatatc tggggccaag
ggacaatggt caccgtctcc 360tcagcctcca ccaagggccc atcggtcttc
cccctggcac cctcctccaa gagcacctct 420gggggcacag cggccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480tcgtggaact
caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc
540tcaggactct actcccttag tagcgtggtg accgtgccct ccagcagctt
gggcacccag 600acctacatct gcaacgtgaa tcacaagccc agcaacacca
aggtggacaa gaaagttgag 660cccaaatctt gtgacaaaac tcacacatgc tga
69310230PRTArtificial SequenceH-chain fragment of anti-human NGF
antibody 10Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Val Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser
Thr Asn Asn Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser
Val Gly Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Arg Asp
Gly Gly Pro Glu Ser Gly Met Gly Ala Phe Asp Ile Trp Gly 100 105 110
Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115
120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys
Thr His Thr Cys 225 230 11660DNAArtificial SequenceL-chain gene of
anti-human NGF antibody 11gatattgtga tgactcagtc tccactctcc
ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg
catagtaatg gattcaacta tttgggttgg 120tacctgcaga agccagggca
gtctccacag ctcctgatct atttgggttc taatcgggcc 180tccggggtcc
ctgacaggtt cagtggcagt ggatcaggca cagattttac tctgaaaatc
240agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct
acaaactccg 300tacacttttg gccaggggac caagctggag atcaaacgga
ctgtggctgc accatctgtc 360ttcatcttcc cgccatctga tgagcagttg
aaatctggaa ctgcctctgt tgtgtgcctg 420ctgaataact tctatcccag
agaggccaaa gtacagtgga aggtggataa cgccctccaa 480tcgggtaact
cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc
540agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta
cgcctgcgaa 600gtcacccatc agggcctgag ctcgcccgtc acaaagagct
tcaacagggg agagtgttag 66012219PRTArtificial SequenceL-chain of
anti-human NGF antibody 12Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Phe Asn Tyr Leu
Gly Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu
Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85
90 95 Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
13699DNAArtificial SequenceH-chain fragment gene of anti-human NGF
antibody 13caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac
cctgtccctc 60acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctgggt
ccgccagccc 120ccagggaagg ggctggagtg gattggggaa atcgaccata
gtggaagcac caacaacaac 180ccgtccctca agagtcgagt caccatatca
gtaggcacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc
cgcggacacg gctgtgtatt actgttcgag agatgggggc 300cccgaatcgg
ggatgggggc ttttgatatc tggggccaag ggacaatggt caccgtctcc
360tcagcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa
gagcacctct 420gggggcacag cggccctggg ctgcctggtc aaggactact
tccccgaacc ggtgacggtg 480tcgtggaact caggcgccct gaccagcggc
gtgcacacct tcccggctgt cctacagtcc 540tcaggactct actcccttag
tagcgtggtg accgtgccct ccagcagctt gggcacccag 600acctacatct
gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag
660cccaaatctt gtgacaaaac tcacacatgc gcagcctga 69914232PRTArtificial
SequenceH-chain fragment of anti-human NGF antibody 14Gln Val Gln
Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr
Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25
30 Tyr Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45 Gly Glu Ile Asp His Ser Gly Ser Thr Asn Asn Asn Pro Ser
Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Gly Thr Ser Lys Asn
Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr Tyr Cys Ser 85 90 95 Arg Asp Gly Gly Pro Glu Ser Gly
Met Gly Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155
160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Ala Ala
225 230 15699DNAArtificial SequenceH-chain fragment gene of
anti-human NGF antibody 15caggtgcagc tacagcagtg gggcgcagga
ctgttgaagc cttcggagac cctgtccctc 60acctgcgctg tctatggtgg gtccttcagt
ggttactact ggagctgggt ccgccagccc 120ccagggaagg ggctggagtg
gattggggaa atcgaccata gtggaagcac caacaacaac 180ccgtccctca
agagtcgagt caccatatca gtaggcacgt ccaagaacca gttctccctg
240aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgttcgag
agatgggggc 300cccgaatcgg ggatgggggc ttttgatatc tggggccaag
ggacaatggt caccgtctcc 360tcagcctcca ccaagggccc atcggtcttc
cccctggcac cctcctccaa gagcacctct 420gggggcacag cggccctggg
ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480tcgtggaact
caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc
540tcaggactct actcccttag tagcgtggtg accgtgccct ccagcagctt
gggcacccag 600acctacatct gcaacgtgaa tcacaagccc agcaacacca
aggtggacaa gaaagttgag 660cccaaatctt gtgacaaaac tcacacatgc ccaccgtga
69916232PRTArtificial SequenceH-chain fragment of anti-human NGF
antibody 16Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Val Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp His Ser Gly Ser
Thr Asn Asn Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser
Val Gly Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Arg Asp
Gly Gly Pro Glu Ser Gly Met Gly Ala Phe Asp Ile Trp Gly 100 105 110
Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115
120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220 Asp Lys Thr His Thr Cys Pro Pro 225 230
* * * * *