U.S. patent application number 12/478501 was filed with the patent office on 2010-01-07 for agonist anti-trk-c monoclonal antibodies.
Invention is credited to Brigitte Devaux, Jo-Anne Hongo, Leonard G. Presta, David L. Shelton.
Application Number | 20100003261 12/478501 |
Document ID | / |
Family ID | 26907807 |
Filed Date | 2010-01-07 |
United States Patent
Application |
20100003261 |
Kind Code |
A1 |
Devaux; Brigitte ; et
al. |
January 7, 2010 |
AGONIST ANTI-TRK-C MONOCLONAL ANTIBODIES
Abstract
The invention concerns agonist anti-trkC monoclonal antibodies
which mimic certain biological activities of NT-3, the native
ligand of trkC. The invention further concerns the use of such
antibodies in the prevention andior treatment of cellular
degeneration, including nerve cell damage associated with acute
nervous cell system injury and chronic neurodegenerative diseases,
including peripheral neuropathy.
Inventors: |
Devaux; Brigitte; (Palo
Alto, CA) ; Hongo; Jo-Anne; (Redwood City, CA)
; Presta; Leonard G.; (San Francisco, CA) ;
Shelton; David L.; (Oakland, CA) |
Correspondence
Address: |
GOODWIN PROCTER LLP
135 COMMONWEALTH DRIVE
MENLO PARK
CA
94025
US
|
Family ID: |
26907807 |
Appl. No.: |
12/478501 |
Filed: |
June 4, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11581865 |
Oct 16, 2006 |
7615383 |
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12478501 |
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10312316 |
Oct 30, 2003 |
7384632 |
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PCT/US01/20153 |
Jun 22, 2001 |
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11581865 |
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60213141 |
Jun 22, 2000 |
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60238319 |
Oct 5, 2000 |
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Current U.S.
Class: |
424/141.1 ;
424/130.1; 530/387.1; 530/387.3; 530/388.1 |
Current CPC
Class: |
A61P 21/00 20180101;
A61P 9/00 20180101; C07K 16/32 20130101; C07K 2317/21 20130101;
A61P 25/02 20180101; C07K 2317/34 20130101; A61P 25/00 20180101;
A61K 2039/505 20130101; C07K 16/2863 20130101; A61P 25/28
20180101 |
Class at
Publication: |
424/141.1 ;
530/387.1; 530/387.3; 530/388.1; 424/130.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/00 20060101 C07K016/00 |
Claims
1. An agonist anti-trkC monoclonal antibody which (a) shows no
significant cross-reactivity with trkA or trkB; and (b) recognizes
an epitope in domain 5 of trkC, wherein said agonist anti-trkC
antibody binds trkC effective to activate a trkC receptor tyrosine
kinase.
2. The antibody of claim 1 which further recognizes an epitope in
domain 4 of trkC.
3. The antibody of claim 1 which binds both human and rat trkC.
4. The antibody of claim 1 which is a human antibody.
5. The antibody of claim 1 which is a murine antibody.
6. The antibody of claim 5 which is humanized.
7. The antibody of claim 1 effective in the treatment of cisplatin-
or pyridoxine-induced neuropathy.
8. The antibody of claim 1 effective in the treatment of diabetic
neuropathy.
9. The antibody of claim 1 which does not cause hyperalgesia when
administered to a patient.
10. The antibody of claim 1 which has increased bioavailability as
compared to NT-3.
11. The antibody of claim 1 which has a higher specific activity
than NT-3.
12. An anti-trkC antibody having a heavy chain comprising the
following CDRs: a CDR1 defined by SEQ ID NO: 1; a CDR2 defined by
SEQ ID NO: 6; and a CDR3 defined by SEQ ID NO: 12, and having a
light chain comprising the following CDRs: a CDR1 defined by SEQ ID
NO: 18; a CDR2 defined by SEQ ID NO: 25; and a CDR3 defined by SEQ
ID NO: 31.
13. An anti-trkC antibody having a heavy chain comprising the
following CDRs: a CDR1 defined by SEQ ID NO: 1; a CDR2 defined by
SEQ ID NO: 6; and a CDR3 defined by SEQ ID NO: 12; and having a
light chain comprising the following CDRs: a CDR1 defined by SEQ ID
NO: 19; a CDR2 defined by SEQ ID NO: 26; and a CDR3 defined by SEQ
ID NO: 32.
14-15. (canceled)
16. The antibody of any of claims 12, 13, 70, 71, 72, 73, or 74
which is an agonist anti-trkC antibody.
17. The antibody of claim 16 comprising human framework
residues.
18. The antibody of claim 17 which shows no significant
cross-reactivity with trkA or trkB.
19. The antibody of claim 17 having a homo-tetrameric structure
composed of two disulfide-bonded antibody heavy chain-light chain
pairs.
20. The antibody of claim 17 which is an antibody fragment selected
from the group consisting of Fv, Fab, Fab' and F(ab') 2
fragments.
21. An anti-trkC agonist antibody of claim 16 which is a monoclonal
antibody.
22. The antibody of claim 21 which shows no significant
cross-reactivity with trkA or trkB.
23. The antibody of claim 22 having a homo-tetrameric structure
composed of two disulfide-bonded antibody heavy chain-light chain
pairs.
24. The antibody of claim 22 which is an antibody fragment selected
from the group consisting of Fv, Fab, Fab' and F(ab') 2
fragments.
25. The antibody of claim 22 which is an IgG.
26. The antibody of claim 25 which is an IgG-2 or IgG-4.
27. A murine anti-trkC agonist antibody selected from the group
consisting of antibodies 2248, 2250, 2253 and 2256.
28. (canceled)
29. The antibody of claim 16 that is a human anti-trkC agonist
antibody selected from the group consisting of antibodies of any of
claims 70 to 74.
30-48. (canceled)
49. A pharmaceutical composition comprising an effective amount of
an agonist anti-trkC monoclonal antibody of any one of claims 1,
16, and 21, in admixture with a pharmaceutically acceptable
carrier.
50-69. (canceled)
70. An anti-trkC antibody having a heavy chain comprising the
following CDRs: (a) a CDR1 defined by SEQ ID NO:2; (b) a CDR2
defined by SEQ ID NO:7; and (c) a CDR3 defined by SEQ ID NO: 13,
and having a light chain comprising the following CDRs: (a) a CDR1
defined by SEQ ID NO:20; (b) a CDR2 defined by SEQ ID NO:27; and
(c) a CDR3 defined by SEQ ID NO:33.
71. An anti-trkC antibody having a heavy chain comprising the
following CDRs: (a) a CDR1 defined by SEQ ID NO:3; (b) a CDR2
defined by SEQ ID NO: 10; and (c) a CDR3 defined by SEQ ID NO: 16,
and having a light chain comprising the following CDRs: (a) a CDR1
defined by SEQ ID NO:23; (b) a CDR2 defined by SEQ ID NO:30; and
(c) a CDR3 defined by SEQ ID NO:36.
72. An anti-trkC antibody having a heavy chain comprising the
following CDRs: (a) a CDR1 defined by SEQ ID NO:3; (b) a CDR2
defined by SEQ ID NO:8; and (c) a CDR3 defined by SEQ ID NO:14, and
having a light chain comprising the following CDRs: (a) a CDR1
defined by SEQ ID NO:21; (b) a CDR2 defined by SEQ ID NO:28; and
(c) a CDR3 defined by SEQ ID NO:34.
73. An anti-trkC antibody having a heavy chain comprising the
following CDRs: (a) a CDR1 defined by SEQ ID NO:4; (b) a CDR2
defined by SEQ ID NO:9; and (c) a CDR3 defined by SEQ ID NO: 15,
and having a light chain comprising the following CDRs: (a) a CDR1
defined by SEQ ID NO:22; (b) a CDR2 defined by SEQ ID NO:29; and
(c) a CDR3 defined by SEQ ID NO:35.
74. An anti-trkC antibody having a heavy chain comprising the
following CDRs: (a) a CDR1 defined by SEQ ID NO:5; (b) a CDR2
defined by SEQ ID NO: 11; and (c) a CDR3 defined by SEQ ID NO: 17,
and having a light chain comprising the following CDRs: (a) a CDR1
defined by SEQ ID NO:24; (b) a CDR2 defined by SEQ ID NO:30; and
(c) a CDR3 defined by SEQ ID NO:36.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of co-pending U.S.
application Ser. No. 11/581,865, filed Oct. 16, 2006, which
application is a continuation of co-pending U.S. application Ser.
No. 10/312,316, which application has a 35 U.S.C. .sctn. 371(c)
completion date of Oct. 30, 2003, now U.S. Pat. No. 7,384,632,
which is a national stage application under 35 U.S.C. .sctn.371 of
PCT Application Serial No. PCT/US01/20153 filed Jun. 22, 2001,
which claims priority from U.S. Provisional Application Ser. No.
60/213,141, filed Jun. 22, 2000, and U.S. Provisional Application
Ser. No. 60/238,319, filed Oct. 5, 2000, from which applications
priority is claimed under 35 U.S.C. .sctn. 120 and 35 U.S.C. .sctn.
119(e), the entire disclosures of which are hereby expressly
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention concerns agonist anti-trkC monoclonal
antibodies. It further concerns the use of the agonist antibodies
in the prevention and/or treatment of cellular degeneration,
including nerve cell damage associated with acute nervous cell
system injury and chronic neurodegenerative diseases, including
peripheral neuropathy.
[0004] 2. Description of the Related Art
[0005] Neurotrophins are a family of small, basic proteins, which
play a crucial role in the development and maintenance of the
nervous system. The first identified and probably best understood
member of this family is nerve growth factor (NGF), which has
prominent effects on developing sensory and sympathetic neurons of
the peripheral nervous system (Levi-Montalcini, R. and Angeletti,
P. U., Physiol. Rev. 48, 534-569 [1968]; Thoenen, H. et al., Rev.
Physiol. Biochem. Pharmacol. 109, 145-178 [1987]). Although NGF had
been known for a long time, including a homolog from the mouse
submandibular gland, the mature, active form of which is often
referred to as - or 2.5S NGF, it was only many years later that
sequentially related but distinct polypeptides with similar
functions were identified.
[0006] The first in line was a factor called brain-derived
neurotrophic factor (BDNF), which was cloned and sequenced by
Leibrock, J. et al. (Nature 341, 149.152 [1989]). This factor was
originally purified from pig brain (Barde, Y. A. et al., EMBO J. 1,
549-553 [1982]), but it was not until its cDNA was cloned and
sequenced that its homology with NGF became apparent. The overall
amino acid sequence identity between NGF and BNDF is about 50%. In
view of this finding, Leibrock et al. speculated that there was no
reason to think that BDNF and NGF should be the only members of a
family of neurotrophins having in common structural and functional
characteristics.
[0007] Indeed, further neurotrophins closely related to -NGF and
BDNF have since been discovered. Several groups identified a
neurotrophin originally called neuronal factor (NF), and now
referred to as neurotrophin-3 (NT-3) (Ernfors et al., Proc. Natl.
Acad. Sci. USA 87, 5454-5458 (1990); Hohn et al., Nature 344, 339
1990]; Maisonpierre et al., Science 247, 1446 [1990]; Rosenthal et
al., Neuron 4, 767 [1990]; Jones and Reichardt, Proc. Natl. Acad.
Sci. USA 87, 8060.8064 (1990); Kaisho et al., FEBS Lett. 266, 187
[1990]. NT-3 shares about 50% of its amino acids with both -NGF and
BDNF (NT-2). Neurotrophins-4 and -5 (NT-4 and NT-5), have been
added to the family (U.S. Pat. No. 5,364,769 issued Nov. 15, 1994;
Hallbook, F. et al., Neuron 6, 845.858 [1991]; Berkmeier, L. R. et
al., Neuron 7, 857-866 [1991]; Ip et al., Proc. Natl. Acad. Sci.
USA 89, 3060-3064 [1992]). The mammalian molecule initially
described by Berkmeier et al. supra, which was subsequently seen to
be the homolog of Xenopus NT-4, is usually referred to as NT-4/5.
In addition, there is an acidic homologous protein described in
mammals which is referred to as NT-6 (Berkemeir, et al., Somat.
Cell Mol. Genet. 18(3):233-245 [1992]). More recently, another
homologue protein from the fish, Xiphosphorus has also been labeled
NT-6 (Gotz et al., Nature 372:266.269 [1994]). There are two
proteins described in the literature as NT-7, one cloned from the
carp, Cyprinus, (Lai, et al., Mol. Cell. Neurosci. 11(1-2):64-76
[1998]) and one from the zebrafish, Danio (Nilsson et al., FEBS
Letters 424(3):285-90 [1998]). None of these last three described
fish neurotrophins has been described outside fish, and their
relationship to any mammalian neurotrophins is unclear. The amino
acid sequence of zebrafish neurotrophin-7 (zNT-7) is more closely
related to that of fish nerve growth factor (NGF) and
neurotrophin-6 (NT-6) than to that of any other neurotrophin. zNT-7
is, however, equally related to fish NGF and NT-6 (65% and 63%
amino acid sequence identity, respectively) indicating that it
represents a distinct neurotrophin sequence. zNT-7 contains a 15
amino acid residue in a beta-turn region in the middle of the
mature protein. Recombinant zNT-7 was able to bind to the human p75
neurotrophin receptor and to induce tyrosine phosphorylation of the
rat trkA receptor tyrosine kinase, albeit less efficiently than rat
NGF. zNT-7 did not interact with rat trkB or trkC, indicating a
similar receptor specificity as NGF. We propose that a
diversification of the NGF subfamily in the neurotrophin
evolutionary tree occurred during the evolution of teleost fishes
which in the appearance of several additional members, such as
zNT-7 and NT-6, is structurally and functionally related to
NGF.
[0008] Neurotrophins, similarly to other polypeptide growth
factors, affect their target cells through interactions with cell
surface receptors. According to our current knowledge, two kinds of
transmembrane glycoproteins serve as receptors for neurotrophins.
Equilibrium binding studies have shown that neurotrophin-responsive
neurons possess a common low molecular weight (65-80 kDa), low
affinity receptor (LNGFR), also termed as 7p5.sup.NTR or p75, which
binds NGF, BDNF, and NT-3 with a K.sub.D of 2.times.10.sup.9 M, and
large molecular weight (130-150 kDa), high affinity (K.sub.D in the
10.sup.-11 M) receptors, which are members of the trk family of the
receptor tyrosine kinases.
[0009] The first member of the trk receptor family, trkA, was
initially identified as the result of an oncogenic transformation
caused by the translocation of tropomyosin sequences onto its
catalytic domain (Martin-Zanca et al., Mol. Cell. Biol. 9(1):24-33
[1989]). Later work identified trkA as a signal transducing
receptor for NGF. Subsequently, two other related receptors, mouse
and rat trkB (Klein et al., EMBO J. 8, 3701.3709 [1989]; Middlemas
et al., Mol. Cell. Biol. 11, 143-153 [1991]; EP 455,460 published 6
Nov. 1991) and porcine, mouse and rat trkC (Lamballe et al., Cell
66, 967-979 [1991]; EP 522,530 published 13 Jan. 1993), were
identified as members of the trk receptor family. The structures of
the trk receptors are quite similar, but alternate splicing
increases the complexity of the family by giving rise to two known
forms of trkA, three known forms of trkB (two without functional
tyrosine kinase domains) and at least four forms of trkC (several
without functional tyrosine kinase domain, and two with small
inserts in the tyrosine kinase domain).
[0010] The role of the p75 and trk receptors is controversial. It
is generally accepted that trk receptor tyrosine kinases play an
important role in conferring binding specificity to a particular
neurotrophin, however, cell lines expressing trkA bind not only NGF
but also NT-3 and NT-4/5 (but not BDNF), trkB expressing cells bind
BDNF, NT-3, NT-4, and NT-4/5 (but not NGF), in contrast to
trkC-expressing cells which have been reported to bind NT-3 alone
(but not the other neurotrophins). Furthermore, it has been shown
in model systems that the various forms of trk receptors, arising
from alternate splicing events, can activate different
intracellular signalling pathways, and therefore presumably mediate
different physiological functions in vivo. It is unclear whether
cells expressing a given trk receptor in the absence of p75 bind
neurotrophins with low or high affinity (Meakin and Shooter, Trends
Neurosci. 15, 323-331 [1992]).
[0011] Published results of studies using various cell lines are
confusing and suggest that p75 is either essential or dispensable
for neurotrophin responsiveness. Cell lines that express p75 alone
bind NGF, BDNF, NT-3, and NT-4 with similar low affinity at
equilibrium, but the binding rate constants are remarkably
different. As a result, although p75-binding is a common property
of all neurotrophins, it has been suggested the p75 receptor may
also play a role in ligand discrimination (Rodriguez-Tebar et al.,
EMBO J. 11, 917-922 [19921). While the trk receptors have been
traditionally thought of as the biologically significant
neurotrophin receptors, it has recently been demonstrated that in
melanoma cells devoid of trkA expression, NGF can still elicit
profound changes in biological behavior presumably through p75
(Herrmann et al., Mol. Biol. Cell 4, 1205-1216 (1993). Davies et
al. (Neuron 11, 565-574 (1993) reported the results of studies
investigating the role of p75 in mediating the survival response of
embryonic neurons to neurotrophins in a model of transgenic mice
carrying a null mutation in the p75 gene. They found that p75
enhances the sensitivity of NGF-dependent cutaneous sensory neurons
to NGF. There have now been many studies showing that p75 is
capable of mediating at least some of the biological effects of the
neurotrophins. The field is still somewhat controversial, but p75
signaling has been implicated in controlling cell death, and
neurite outgrowth. (Barker, Pa., Cell Death Diff. 5:346-356 [1998];
Bredesen et al., Cell Death Diff. 5:357-364 (1998);
Casaccia-Bonnefil, et al., Cell Death Diff. 5:357-364 [1998]; Raoul
et al., Curr. Op. Neurobiol. 10:111-117 [2000]; Davies, A M, Curr.
Biol. 10:R198-R200 [2000]). Importantly, stimulation of p75 has
been shown to modify the effects of stimulating trkC (Hapner, et
al., Developm. Biol. 201:90-100 [1998]).
[0012] The extracellular domains of full-length native trkA, trkB
and trkC receptors have five functional domains, that have been
defined with reference to homologous or otherwise similar
structures identified in various other proteins. The domains have
been designated starting at the N-terminus of the amino acid
sequence of the mature trk receptors as 1) a first cysteine-rich
domain extending from amino acid position 1 to about amino acid
position 32 of human trkA, from amino acid position 1 to about
amino acid position 36 of human trkB, and from amino acid position
1 to about amino acid position 48 of human trkC; 2) a leucine-rich
domain stretching from about amino acid 33 to about amino acid to
about amino acid 104 in trkA; from about amino acid 37 to about
amino acid 108 in trkB, and from about amino acid 49 to about amino
acid 120 in trkC; 3) a second cysteine-rich domain from about amino
acid 105 to about amino acid 157 in trkA; from about amino acid 109
to about amino acid 164 in trkB; and from about amino acid 121 to
about amino acid 177 in trkC; 4) a first immunoglobulin-like domain
stretching from about amino acid 176 to about amino acid 234 in
trkA; from about amino acid 183 to about amino acid 239 in trkB;
and from about amino acid 196 to about amino acid 257 in trkC; and
5) a second immunoglobulin-like domain extending from about amino
acid 264 to about amino acid 330 in trkA; from about amino acid 270
to about amino acid 334 in trkB; and from about amino acid 288 to
about amino acid 351 in trkC.
[0013] Neurotrophins exhibit actions on distinct, but overlapping,
sets of peripheral and central neurons. These effects range from
playing a crucial role in ensuring the survival of developing
neurons (NGF in sensory and sympathetic neurons) to relatively
subtle effects on the morphology of neurons (NT-3 on purkinje
cells). These activities have led to interest in using
neurotrophins as treatments of certain neurodegenerative diseases.
NT-3 has also been found to promote proliferation of peripheral
blood leukocytes and, as a result, it has been suggested that NT-3
can be used in the treatment of neutropenia, infectious disease and
tumors (U.S. Pat. No. 6,015,552 issued on Jun. 18, 2000).
[0014] The roles of neurotrophins in regulating cardiovascular
development and modulating the vascular response to injury have
also been investigated (Donovan et al., Nature Genetics 14:210-213
[1996]; Donovan et al., A.J. Path. 147:309-324 [1995]; Kraemer et
al., Arteriol. Thromb. and Vasc. Biol. 19:1041-1050 [1999]).
Neurotrophins have been described as potential therapeutics for
regulating angiogenesis and vascular integrity (PCT Publication WO
00/124415, published May 4, 2000).
[0015] Despite their promise in the treatment of cellular
degeneration, such as occurs due to neurodegenerative disease and
acute neuronal injuries, and potentially angiogenesis,
neurotrophins have several shortcomings. One significant
shortcoming is the lack of specificity. Most neurotrophins
cross-react with more than one receptor. For example NT-3, the
preferred ligand of the trkC receptor tyrosine kinase, also binds
to and activates trkA and trkB (Barbacid, J. Neurobiol.
25:1386-1403 [1994]; Barbarcid, Ann. New York Acad. Sci.
766:442-458 [1995]; Ryden and Ibanez, J. Biol. Chem. 271:5623-5627
[1996]; Belliveau et al., J. Cell. Biol. 136:375-388 [1997];
Farinas et al., Neuron 21:325-334 [1998]). As a result, it is
difficult to devise therapies that target a specific population of
neurons. Another limitation of neurotrophin therapy is that
neurotrophins, including NT-3 are known to elicit hyperalgesia
(Chaudhry, et al., Muscle and Nerve 23:189-192 [2000]). In
addition, some neurotrophins such as NT-3 have poor pharmacokinetic
and bioavailability properties in rodents, which raise serious
questions about their human clinical applications (Haase et al., J.
Neurol. Sci. 160:S97-S105 [1998], dosages used in Helgren et al.,
J. Neurosci. 17(1):372-82 [1997], and data below).
[0016] Accordingly, there is a great need for the development of
new therapeutic agents for the treatment of neurodegenerative
disorders and acute nerve cell injuries that are devoid of the
known shortcomings of neurotrophins.
SUMMARY OF THE INVENTION
[0017] The current invention is based on the development and
characterization of agonist anti-trkC monoclonal antibodies,
directed against epitopes in the extracellular domain of trkC
receptor, which mimic the biological activities of NT-3, the
natural ligand of trkC receptor but are free of some of the known
detriments of NT-3. The invention also demonstrates the usefulness
of these agonist antibodies in the treatment of neuropathy in an
experimental animal model. Anti-trkC agonist antibodies offer
numerous advantages over NT-3 in prophylactic or therapeutic
treatment of cellular degeneration, such as nerve cell damage, in
particular nerve cell injury associated with neurodegenerative
diseases, such as peripheral neuropathies or due to external
factors, such as trauma, toxic agents, surgery, just to mention a
few.
[0018] In one aspect, the invention concerns an agonist anti-trkC
monoclonal antibody which
[0019] (a) shows no significant cross-reactivity with trkA or trkB;
and
[0020] (b) recognizes an epitope in domain 5 of trkC.
[0021] Certain agonist antibodies of the present invention may
additionally recognize an epitope in domain 4 of trkC. In a
preferred embodiment, the antibodies bind both human and rodent
(e.g. rat or mouse) trkC, and may be murine, chimeric (including
humanized) or human antibodies. The antibodies mimic at least one
activity of the native trkC ligand, NT-3, and may thus be effective
in the prevention and/or treatment of various diseases involving
cellular degeneration, including, for example, neuropathies, such
as cisplatin- or pyridoxine-induced neuropathy, or diabetic
neuropathy, and (where cellular degeneration involves bone marrow
cell degeneration) disorders of insufficient blood cells, such as
leukopenias including eosinopenia and/or basopenia, lymphopenia,
monocytopenia, and neutropenia. In a particularly preferred
embodiment, the agonist antibodies of the present invention show
superior properties over NT-3, for example, do not cause
hyperalgesia when administered to a patient, have increased
bioavailability and/or higher specific activity as compared to
NT-3.
[0022] In another aspect, the invention concerns an anti-trkC
antibody heavy chain comprising the following CDRs: a CDR1 selected
from the group consisting of SEQ ID NOs: 1, 2, 3, 4 and 5; a CDR2
selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9, 10
and 11; and a CDR3 selected from the group consisting of SEQ ID
NOs: 12, 13, 14, 15, 16 and 17.
[0023] In yet another aspect, the invention concerns an anti-trkC
antibody light chain comprising the following CDRs: a CDR1 selected
from the group consisting of SEQ ID NOs: 18, 19, 20, 21, 22, 23 and
24; a CDR2 selected from the group consisting of SEQ ID NOs: 25,
26, 27, 28, 29 and 30; and a CDR3 selected from the group
consisting of SEQ ID NOs: 31, 32, 33, 34, 35 and 36.
[0024] In a further aspect, the invention concerns a murine
anti-trkC antibody heavy chain comprising the following CDRs:
[0025] (a) a CORI of the formula XaaWXaaXaaWVK (SEQ ID NO: 37),
wherein Xaa at position 1 is F or Y; Xaa at position 3 is I or M;
and Xaa at position 4 is E or H;
[0026] (b) a CDR2 of the formula EIXaaPXaaXaaXaaXaaTNYNEKFKXaa (SEQ
ID NO: 38), wherein Xaa at position 3 is L or Y; Xaa at position 5
is G or S; Xaa at position 6 is S or N; Xaa at position 7 is D or
G; Xaa at position 8 is N or R and Xaa at position 16 is G or S;
and
[0027] (c) a CDR3 of the formula KNRNYYGNYVV (SEQ ID NO: 12) or
KYYYGNSYRSWYFDV (SEQ ID NO: 13). In a still further aspect, the
invention relates to a human anti-trkC antibody heavy chain
comprising the following CDRs:
[0028] (a) a CDR1 of the formula XaaXaaXaaYYWXaa (SEQ ID NO: 39),
wherein Xaa at position 1 is S or I; Xaa at position 2 is G or S;
Xaa at position 3 is G, T or Y, and Xaa at position 7 is S or
N;
[0029] (b) a CDR2 of the formula XaalXaaXaaSGSXaaTXaaNPSLKS (SEQ ID
NO: 40), wherein Xaa at position 1 is Y or R; Xaa at position 3 is
Y or F; Xaa at position 4 is Y or T; Xaa at position 8 is S or R;
and Xaa at position 10 is N or Y; and
[0030] (c) a CDR3 of the formula selected from the group consisting
of DRDYDSTGDYYSYYGMDV (SEQ ID NO: 14); DGGYSNPFD (SEQ ID NO: 15);
ERIAAAGXaaDYYYNGLXaaV (SEQ ID NO: 41), wherein Xaa at position 8 is
A or T and Xaa at position 16 is D or A.
[0031] In another aspect, the invention concerns an anti-trkC
agonist monoclonal antibody comprising a heavy chain comprising the
CDRs of the murine anti-trkC antibody heavy chain of claim 14
associated with a light chain. The antibody preferably is human or
comprises human framework residues, and preferably shows no
significant cross-reactivity with trkA or trkB. Throughout the
application, antibodies are defined in the broadest sense, and
specifically include antibody fragment, such as an Fv fragment, Fab
fragment, Fab' or F(ab').sub.2 fragment. Antibodies of all classes
and isotypes are included, but IgG, in particular IgG-2 and IgG-4
are preferred.
[0032] In yet another aspect, the invention concerns isolated
nucleic acid encoding a murine or human anti-trkC agonist antibody
heavy or light chain, or a fragment thereof. In a specific
embodiment, the nucleic acid is a nucleic acid molecule deposited
with ATCC on Jun. 21, 2000 under an accession number selected from
the group consisting of PTA-2133, PTA-2134, PTA-2135, PTA-2136,
PTA-2137, PTA-2138, PTA-2139, PTA-2140, PTA-2141, PTA-2142 and
PTA-2143.
[0033] In a further aspect, the invention concerns a vector
comprising a nucleic acid molecule encoding an antibody heavy
and/or light chain as hereinabove defined. The invention also
concerns cells transformed with such nucleic acid. The invention
further concerns hybridoma cell lines transformed with such nucleic
acid and antibodies produced by such hybridoma cells.
[0034] In a still further aspect, the invention concerns a
pharmaceutical composition comprising an effective amount of an
agonist anti-trkC monoclonal antibody as hereinabove defined in
admixture with a pharmaceutically acceptable carrier.
[0035] In another aspect, the invention concerns a method for
treating a disease or condition involving cell degeneration,
comprising administering to a mammal an effective amount of an
agonist anti-trkC antibody disclosed herein.
[0036] In yet another aspect, the invention concerns a method for
treating a neuropathy or neurodegenerative disease, or repairing a
damaged nerve cell comprising administering to a mammal an
effective amount of an agonist anti-trkC antibody disclosed herein.
The neuropathy may, for example, be a peripheral neuropathy,
including, without limitation, diabetic neuropathy and large-fiber
sensory neuropathies. The neurodegenerative disease may, for
example, be amyotrophic lateral sclerosis (ALS), Alzheimer's
disease, Parkinson's disease, Huntington's disease. The damaged
neurons may be peripheral, such as sensory, e.g. dorsal root
ganglia neurons, motor neurons, e.g. neurons from the spinal cord,
or central neurons, and the injury may be due to a variety of
external and internal factors, including trauma, exposure to
neurotoxins, metabolic diseases, infectious agents, etc.
[0037] In a further aspect, the invention concerns a method for
promoting the development, proliferation, maintenance or
regeneration of peripheral neurons, comprising contacting such
neurons with an effective amount of an antibody of the present
invention.
[0038] In a still further aspect, the invention concerns a method
for the treatment (including prevention) of a disease or condition
involving cell degeneration in a mammalian subject by introducing
nucleic acid encoding an anti-trkC antibody herein into a cell of
such subject. The method (gene therapy) preferably concerns the
treatment of a neuropathy or neurodegenerative disease, or
reparation of a damaged nerve cell. Accordingly, the recipient
cells preferably are nerve cells.
[0039] In yet another aspect, the invention concerns delivery
vehicles containing genetic material (nucleic acid) encoding an
anti-trkC, antibody suitable for gene therapy use.
[0040] In an additional aspect, the invention concerns a method of
inducing angiogenesis by delivering an anti-trkC antibody of the
present invention in an amount effective to induce angiogenesis.
The delivery specifically includes the administration of the
antibodies and the delivery of nucleic acid encoding the antibodies
(e.g. in gene therapy).
[0041] In yet another aspect, the invention concerns an isolated
nucleic acid molecule encoding a murine or human anti-trkC agonist
antibody heavy or light chain selected from the group consisting of
SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID
NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65; SEQ ID NO: 66;
SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 and SEQ
ID NO: 71. The present invention also concerns a polypeptide
encoded by one or more of the isolated nucleic acid molecules.
[0042] In another aspect, the invention concerns a whole cell
transformed with nucleic acid encoding murine or human anti-trkC
agonist antibody heavy chain, light chain or both heavy and light
chain.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIGS. 1A-D show agonist activity of various human (A and C)
and murine (B and D) monoclonal antibodies against trkC receptor
demonstrated using KIRA (A and B) and PC12 neurite outgrowth assay
(C and D). Protein A purified monoclonal antibodies were diluted to
27 .mu.g/ml in KIRA stimulation buffer (F12/DMEM 50:50 containing
2% bovine serum albumin [BSA, Intergen Co., Purchase, N.Y.) and 25
mM Hepes, 0.2 .mu.m filtered). The monoclonal antibodies were then
diluted 1:3 (8 dilutions total; concentrations ranged from 0.01-180
nM Nab) in stimulation media. GD-transfected CHO cells
(5.times.10.sup.4 cells/well) were then stimulated with either NT-3
or Mab (dilutions assayed in duplicate) for 6 hours and the assay
was complete as described in the examples (FIG. 1A, human Mabs;
FIG. 1B, murine Mabs). The purified Mabs were assayed for agonist
activity in the PC12 neurite outgrowth assay as described in the
examples. Rat PC12 cells were transfected with full-length human
trkC and the cells plated at a density of 1000 cells/well. Three
days following transfection, the Mabs were added in triplicate
(concentrations ranging from 0.0002 to 23 nM) to the wells
containing the trkC transfectants and incubated for an additional 3
days at 37.degree. C. The cells were then analyzed by phase
contrast microscopy and cells with neurites exceeding two-times the
diameter of the cell were counted.
[0044] FIG. 2 shows that agonist anti-trkC monoclonal antibodies
bind specifically to trkC using 6.1.2 antibody as a representative
example.
[0045] FIG. 3 demonstrates that agonist anti-trkC monoclonal
antibodies recognize human trkC more efficiently than rat trkC. The
ability of the monoclonal antibodies to bind rat trkC was
determined using an immunoadhesin construct of the receptor. TrkC
(human trkC-gD or rat trkC-IgG) was immobilized on microtiter
plates (100 .mu.l of a 1 .mu.g/ml solution diluted in 50 mM
carbonate buffer, pH 9.5) overnight. The plates were washed and
blocked. The Mabs were then diluted to 1 .mu.g/ml in PBS containing
0.5% BSA and 0.05% Tween 20, added to the appropriate wells (100
.mu.l/well), and incubated for one hour at room temperature. The
plates were washed and the appropriate HRP conjugate was added
(human Mabs: goat anti-human k-HRP, 1:5 K; murine Mabs: goat
anti-molgG (Fc)-HRP, 1:5 K) and incubated for one hour at room
temperature. The plates were then washed, developed and read.
[0046] FIG. 4 shows a representative example of epitope mapping
using competition ELISA. A biotinylated human anti-trkC 6.1.2
monoclonal antibody was incubated with immobilized trkC in the
absence or presence of excess of various unlabeled anti-trkC
monoclonal antibodies.
[0047] FIG. 5 summarizes the results of epitope mapping using
competition ELISA.
[0048] FIGS. 6A-C show a schematic diagram of various trkC chimera
(A) and their use in mapping of epitopes on trkC recognized by
various agonist human (B) and murine (C) anti-trkC monoclonal
antibodies.
[0049] FIG. 7 shows amino acid sequence of human trkC domain 4 and
5 showing residues that were targeted for mutagenesis to decipher
their roles in recognition by agonist anti-trkC monoclonal
antibodies.
[0050] FIG. 8 shows 3-dimensional ribbon diagram of trkC in complex
with anti-trkC monoclonal antibodies. Specifically shown are the
amino acid residues of trkC that are likely to play an important
role in recognition by CDRs of anti-trkC antibodies.
[0051] FIG. 9 shows the amino acid sequence of the heavy chain
variable (V.sub.H) region from murine and human anti trkC agonist
monoclonal antibodies. In addition, the three CDR regions (CDR1,
CDR2 and CDR3) are highlighted in bold. The amino acid sequence of
CDR1 of the 2250 and 2253 heavy chain is SEQ ID NO: 1. The amino
acid sequence of CDR1 of the 2256 heavy chain is SEQ ID NO: 2. The
amino acid sequence of CDR1 of the 6.1.2 and 2345 heavy chain is
SEQ ID NO: 3. The amino acid sequence of CDR1 of the 6.4.1 heavy
chain is SEQ ID NO: 4. The amino acid sequence of CDR1 of the 2349
heavy chain is SEQ ID NO: 5. The amino acid sequence of CDR2 of the
2250 and 2253 heavy chain is SEQ ID NO: 6. The amino acid sequence
of CDR2 of the 2256 heavy chain is SEQ ID NO: 7. The amino acid
sequence of CDR2 of the 6.1.2 heavy chain is SEQ ID NO: 8. The
amino acid sequence of CDR2 of the 6.4.1 heavy chain is SEQ ID NO:
9. The amino acid sequence of CDR2 of the 2345 heavy chain is SEQ
ID NO: 10. The amino acid sequence of CDR2 of the 2349 heavy chain
is SEQ ID NO: 11. The amino acid sequence of CDR3 of the 2250 and
2253 heavy chain is SEQ ID NO: 12. The amino acid sequence of CDR3
of the 2256 heavy chain is SEQ ID NO: 13. The amino acid sequence
of CDR3 of the 6.1.2 heavy chain is SEQ ID NO: 14. The amino acid
sequence of CDR3 of the 6.4.1 heavy chain is SEQ ID NO: 15. The
amino acid sequence of CDR3 of the 2345 heavy chain is SEQ ID NO:
16. The amino acid sequence of CDR3 of the 2349 heavy chain is SEQ
ID NO: 17.
[0052] FIG. 10 shows the amino acid sequence of the light chain
variable (V.sub.L) region from murine and human anti-trkC agonist
monoclonal antibodies. In addition, the three CDR regions (CDR1,
CDR2 and CDR3) are highlighted in bold. The amino acid sequence of
CDR1 of the 2250 light chain is SEQ ID NO: 18. The amino acid
sequence of CDR1 of the 2253 light chain is SEQ ID NO: 19. The
amino acid sequence of CDR1 of the 2256 light chain is SEQ ID NO:
20. The amino acid sequence of CDR1 of the 6.1.2 light chain is SEQ
ID NO: 21. The amino acid sequence of CDR1 of the 6.4.1 light chain
is SEQ ID NO: 22. The amino acid sequence of CDR1 of the 2345 light
chain is SEQ ID NO: 23. The amino acid sequence of CDR1 of the 2349
light chain is SEQ ID NO: 24. The amino acid sequence of CDR2 of
the 2250 light chain is SEQ ID NO: 25. The amino acid sequence of
CDR2 of the 2253 light chain is SEQ ID NO: 26. The amino acid
sequence of CDR2 of the 2256 light chain is SEQ ID NO: 27. The
amino acid sequence of CDR2 of the 6.1.2 light chain is SEQ ID NO:
28. The amino acid sequence of CDR2 of the 6.4.1 light chain is SEQ
ID NO: 29. The amino acid sequence of CDR2 of the 2345 and 2349
light chain is SEQ ID NO: 30. The amino acid sequence of CDR3 of
the 2250 light chain is SEQ ID NO: 31. The amino acid sequence of
CDR3 of the 2253 light chain is SEQ ID NO: 32. The amino acid
sequence of CDR3 of the 2256 light chain is SEQ ID NO: 33. The
amino acid sequence of CDR3 of the 6.1.2 light chain is SEQ ID NO:
34. The amino acid sequence of CDR3 of the 6.4.1 light chain is SEQ
ID NO: 35. The amino acid sequence of CDR3 of the 2345 and 2349
light chain is SEQ ID NO: 36.
[0053] FIG. 11 shows amino acid sequence of CDRs of heavy and light
variable chains of murine and human anti-trkC agonist monoclonal
antibodies. Also shown are the families to which these sequences
belong based on homology with CDR sequences available in
databases.
[0054] FIG. 12 shows that anti-trkC agonist monoclonal antibodies
have improved half-life and bioavailability in vivo.
[0055] FIG. 13 shows effect of anti-trkC agonist monoclonal
antibodies on cisplatin-induced neuropathy.
[0056] FIG. 14 shows decrease in marker expression caused by
pyridoxine neuropathy.
[0057] FIG. 15 shows amelioration of the effects of low doses of
pyridoxine by agonist anti-trkC monoclonal antibodies.
[0058] FIG. 16 shows amelioration of the effects of high doses of
pyridoxine by agonist anti-trkC monoclonal antibodies.
[0059] FIG. 17 shows amelioration of pyridoxine neuropathy by an
anti-trkC agonist monoclonal antibody.
[0060] FIG. 18 shows attenuation of pyridoxine-induced deficit of
ladder by agonist anti-trkC monoclonal antibodies.
[0061] FIG. 19 shows that NT3, but not anti-trkC agonist monoclonal
antibodies, causes hyperalgesi at therapeutic doses.
[0062] FIG. 20 shows the amino acid sequence of human trkC receptor
(SEQ ID NO: 56) where the boundaries of domains 4 and 5 are
indicated.
[0063] FIG. 21 (in 2 pages) shows the nucleotide sequence of human
trkC receptor (SEQ ID NO: 57).
[0064] FIG. 22 shows the nucleotide sequence of the heavy chain (A;
SEQ ID NO: 58) and light chain (B; SEQ ID NO: 59) of the anti-trkC
agonist monoclonal antibody 2250.
[0065] FIG. 23 shows the nucleotide sequence of the heavy chain (A;
SEQ ID NO: 60) and light chain (B; SEQ ID NO: 61) of the anti-trkC
agonist monoclonal antibody 2253.
[0066] FIG. 24 shows the nucleotide sequence of the heavy chain (A;
SEQ ID NO: 62) and light chain (B; SEQ ID NO: 63) of the anti-trkC
agonist monoclonal antibody 2256.
[0067] FIG. 25 shows the nucleotide sequence of the heavy chain (A;
SEQ ID NO: 64) and light chain (B; SEQ ID NO: 65) of the anti-trkC
agonist monoclonal antibody 2345.
[0068] FIG. 26 shows the nucleotide sequence of the heavy chain (A;
SEQ ID NO: 66) and light chain (B; SEQ ID NO: 67) of the anti-trkC
agonist monoclonal antibody 2349.
[0069] FIG. 27 shows the nucleotide sequence of the heavy chain (A;
SEQ ID NO: 68) and light chain (B; SEQ ID NO: 69) of the anti-trkC
agonist monoclonal antibody 6.1.2.
[0070] FIG. 28 shows the nucleotide sequence of the heavy chain (A;
SEQ ID NO: 70) and light chain (B; SEQ ID NO: 71) of the anti-trkC
agonist monoclonal antibody 6.4.1.
7 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
A. Definitions
[0071] The term "neurotrophin" and its grammatical variants are
used interchangeably, and refer to a family of polypeptides
comprising nerve growth factor (NGF) and sequentially related
homologs. NGF, brain-derived growth factor (BDNF, a.k.a. NT-2),
neurotrophin-3 (NT-3), neurotrophins-4 and -5 (NT-4/5),
neurotrophin-6 (NT-6), and neurotrophin-7 (NT-7) have so far been
identified as members of this family.
[0072] The term "neurotrophin" includes native neurotrophins of any
(human or non-human) animal species, and their functional
derivatives, whether purified from a native source, prepared by
methods of recombinant DNA technology, or chemical synthesis, or
any combination of these or other methods. "Native" or "native
sequence" neurotrophins have the amino acid sequence of a
neurotrophin occurring in nature in any human or non-human animal
species, including naturally-occurring truncated and variant forms,
and naturally-occurring allelic variants.
[0073] The terms "trk", "trk polypeptide", "trk receptor" and their
grammatical variants are used interchangeably and refer to
polypeptides of the receptor tyrosine kinase superfamily, which are
capable of binding at least one native neurotrophin. Currently
identified members of this family are trkA (p140.sup.trkA), trkB,
and trkC.
[0074] The expression "extracellular domain" or "ECD" when used
herein refers to any polypeptide sequence that shares a ligand
binding function of the extracellular domain of a naturally
occurring receptor. Ligand binding function of the extracellular
domain refers to the ability of the polypeptide to bind to a
ligand. Accordingly, it is not necessary to include the entire
extracellular domain since smaller segments have been found to be
adequate for ligand binding. The truncated extracellular domain is
generally soluble. The term ECD encompasses polypeptide sequences
in which the hydrophobic transmembrane sequence (and, optionally,
1-20 amino acids C-terminal and/or N-terminal to the transmembrane
domain) of the mature receptor has been deleted.
[0075] The term "agonist anti-trkC antibody" refers to an antibody,
which is able to bind to and activate a native sequence trkC
receptor and/or downstream pathways mediated by the trkC signaling
function thereby mimicking a biological activity of a native ligand
of the receptor, in particular NT-3. For example, the agonist
antibody may bind to the ECD domain of a trkC receptor and thereby
cause dimerization of the receptor, resulting in activation of the
intracellular catalytic kinase domain. Consequently, this may
result in stimulation of growth and/or differentiation of cells
expressing the receptor in vitro and/or in vivo. The agonist
antibodies of the present invention preferably recognize an epitope
that includes at least part of domain 5 (amino acid positions from
about 266 to about 381) and/or domain 4 (amino acid position from
about 178 to about 265) of the human trkC receptor or a
corresponding epitope on a non-human, e.g. murine trkC
receptor.
[0076] "Biological activity", when used in conjunction with the
agonist anti-trkC antibodies of the present invention, generally
refers to having an effector function in common with NT-3, the
native ligand of trkC. The effector function preferably is the
ability to bind and activate the trkC receptor tyrosine kinase
and/or downstream pathways mediated by the trkC signaling function.
Without limitation, preferred biological activities include the
ability to promote the development, proliferation, maintenance
and/or regeneration of damaged cells, in particular neurons in
vitro or in vivo, including peripheral (sympathetic,
parasympathetic, sensory, and enteric) neurons, motorneurons, and
central (brain and spinal cord) neurons, and non-neuronal cells,
e.g. peripheral blood leukocytes. A particularly preferred
biological activity is the ability to treat (including prevention)
a neuropathy, e.g. peripheral neuropathy or other neurodegenerative
disease, or repair a damaged nerve cell. The damaged neurons may be
sensory, sympathetic, parasympathetic, or enteric, e.g. dorsal root
ganglia neurons, motorneurons, and central neurons, e.g. neurons
from the spinal cord, and the damage may be of any cause, including
trauma, toxic agents, surgery, stroke, ischemia, infection,
metabolic disease, nutritional deficiency, and various
malignancies. Another specific biological activity is the ability
to induce angiogenesis.
[0077] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease, delay
or slowing of disease progression, amelioration or palliation of
the disease state, and remission (whether partial or total),
whether detectable or undetectable. "Treatment" can also mean
prolonging survival as compared to expected survival if not
receiving treatment. "Treatment" is an intervention performed with
the intention of preventing the development or altering the
pathology of a disorder. Accordingly, "treatment" refers to both
therapeutic treatment and prophylactic or preventative measures.
Those in need of treatment include those already with the disorder
as well as those in which the disorder is to be prevented.
Specifically, the treatment may directly prevent, slow down or
otherwise decrease the pathology of cellular degeneration of
damage, such as the pathology of nerve cells, or may render the
cells, e.g. neurons more susceptible to treatment by other
therapeutic agents. In a preferred embodiment, the treatment
reduces or slows down the decline and/or stimulates the restoration
of the function of target neurons.
[0078] The "pathology" of a (chronic) neurodegenerative disease or
acute nervous system injury includes all phenomena that affect the
well being of the patient including, without limitation, neuronal
disfunction, degeneration, injury and/or death.
[0079] The terms "neurodegenerative disease" and "neurodegenerative
disorder" are used in the broadest sense to include all disorders
the pathology of which involves neuronal degeneration and/or
disfunction, including, without limitation, peripheral
neuropathies; motorneuron disorders, such as amylotrophic lateral
schlerosis (ALS, Lou Gehrig's disease), Bell's palsy, and various
conditions involving spinal muscular atrophy or paralysis; and
other human neurodegenerative diseases, such as Alzheimer's
disease, Parkinson's disease, epilepsy, multiple schlerosis,
Huntington's chorea, Down's Syndrome, nerve deafness, and Meniere's
disease.
[0080] "Peripheral neuropathy" is a neurodegenerative disorder that
affects the peripheral nerves, most often manifested as one or a
combination of motor, sensory, sensorimotor, or autonomic
dysfunction. Peripheral neuropathies may, for example, be
genetically acquired, can result from a systemic disease, or can be
induced by a toxic agent, such as a neurotoxic drug, e.g.
antineoplastic agent, or industrial or environmental pollutant.
"Peripheral sensory neuropathy" is characterized by the
degeneration of peripheral sensory neurons, which may be
idiopathic, may occur, for example, as a consequence of diabetes
(diabetic neuropathy), cytostatic drug therapy in cancer (e.g.
treatment with chemotherapeutic agents such as vincristine,
cisplatin, methotrexate, 3'-azido-3'-deoxythymidine, or taxanes,
e.g. paclitaxel [TAXOL.RTM., Bristol-Myers Squibb Oncology,
Princeton, N.J.] and doxetaxel [TAXOTERE.RTM., Rhone-Poulenc Rorer,
Antony, France]), alcoholism, acquired immunodeficiency syndrom
(AIDS), or genetic predisposition. Genetically acquired peripheral
neuropathies include, for example, Refsum's disease, Krabbe's
disease, Metachromatic leukodystrophy, Fabry's disease,
Dejerine-Sottas syndrome, Abetalipoproteinemia, and
Charcot-Marie-Tooth (CMT) Disease (also known as Proneal Muscular
Atrophy or Hereditary Motor Sensory Neuropathy (HMSN)). Most types
of peripheral neuropathy develop slowly, over the course of several
months or years. In clinical practice such neuropathies are called
chronic. Sometimes a peripheral neuropathy develops rapidly, over
the course of a few days, and is referred to as acute. Peripheral
neuropathy usually affects sensory and motor nerves together so as
to cause a mixed sensory and motor neuropathy, but pure sensory and
pure motor neuropathy are also known.
[0081] The term "toxic agent", as used in the context of the
present invention, is meant to refer to a substance that, through
its chemical action, injures, impairs, or inhibits the activity of
a component of the nervous system. The long list of toxic agents
(also referred to as "neurotoxic agents") includes, without
limitation, chemotherapeutic agents, such as those listed above,
alcohol, metals, industrial toxins, contaminants of food and
medicines, etc.
[0082] "Mammal" for purpose of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sport or pet animals, such as dogs, horses,
sheep, cats, cows, etc. Preferably, the mammal is human.
[0083] The term "trkC immunoadhesin" is used interchangeably with
the expression "trkC-immunoglobulin chimera" and refers to a
chimeric molecule that combines a portion of trkC (generally the
extracellular domain thereof) with an immunoglobulin sequence. The
immunoglobulin sequence preferably, but not necessarily, is an
immunoglobulin constant domain. Chimeras constructed from a
receptor sequence linked to an appropriate immunoglobulin constant
domain sequence (immunoadhesins) are known in the art.
Immunoadhesins reported in the literature include fusions of the T
cell receptor* (Gascoigne et al., Proc. Natl. Acad. Sci. USA, 84:
2936-2940 [1987]); CD4* (Capon et al., Nature 337: 525-531 [1989];
Traunecker et al., Nature, 339: 68-70 [1989]; Zettmeissl et al.,
DNA Cell Biol. 9: 347-353 [1990]; Byrn et al., Nature, 344: 667-670
[1990]); L-selectin (homing receptor) (Watson et al., J. Cell.
Biol., 110:2221-2229 [1990]; Watson et al., Nature, 349: 164-167
[1991]); CD44* (Aruffo et al., Cell, 61: 1303-1313 [1990]); CD28*
and B7* (Linsley et al., J. Exp. Med., 173: 721-730 [1991]);
CTLA-4* (Lisley et al., J. Exp. Med. 174: 561-569 [1991]); CD22*
(Stamenkovic et al., Cell, 66:1133-11144 [1991]); TNF receptor
(Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539
[1991]; Lesslauer et al., Eur. J. Immunol., 27:2883-2886 [1991];
Peppel et al., J. Exp. Med., 174:1483-1489 [1991]); NP receptors
(Bennett et al., J. Biol. Chem. 266:23060-23067 [1991]); and IgE
receptor .alpha.* (Ridgway et al, J. Cell Biol., 115:abstr. 1448
[1991]), where the asterisk (*) indicates that the receptor is
member of the immunoglobulin superfamily.
[0084] "Isolated" nucleic acid or polypeptide in the context of the
present invention is a nucleic acid or polypeptide that is
identified and separated from contaminant nucleic acids or
polypeptides present in the animal or human source of the nucleic
acid or polypeptide. The nucleic acid or polypeptide may be labeled
for diagnostic or probe purposes, using a label as described and
defined further below in discussion of diagnostic assays.
[0085] In general, the term "amino acid sequence variant" refers to
molecules with some differences in their amino acid sequences as
compared to a reference (e.g. native sequence) polypeptide. The
amino acid alterations may be substitutions, insertions, deletions
or any desired combinations of such changes in a native amino acid
sequence.
[0086] The terms "DNA sequence encoding", "DNA encoding" and
"nucleic acid encoding" refer to the order or sequence of
deoxyribonucleotides along a strand of deoxyribonucleic acid. The
order of these deoxyribonucleotides determines the order of amino
acids along the polypeptide chain. The DNA sequence thus codes for
the amino acid sequence.
[0087] The terms "replicable expression vector" and "expression
vector" refer to a piece of DNA, usually double-stranded, which may
have inserted into it a piece of foreign DNA. Foreign DNA is
defined as heterologous DNA, which is DNA not naturally found in
the host cell. The vector is used to transport the foreign or
heterologous DNA into a suitable host cell. Once in the host cell,
the vector can replicate independently of the host chromosomal DNA,
and several copies of the vector and its inserted (foreign) DNA may
be generated. In addition, the vector contains the necessary
elements that permit translating the foreign DNA into a
polypeptide. Many molecules of the polypeptide encoded by the
foreign DNA can thus be rapidly synthesized.
[0088] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, a ribosome binding site, and
possibly, other as yet poorly understood sequences. Eukaryotic
cells are known to utilize promoters, polyadenylation signals, and
enhancer.
[0089] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or a secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, then synthetic oligonucleotide adaptors or linkers are used
in accord with conventional practice.
[0090] In the context of the present invention the expressions
"cell", "cell line", and "cell culture" are used interchangeably,
and all such designations include progeny. Thus, the words
"transformants" and "transformed (host) cells" include the primary
subject cell and cultures derived therefrom without regard for the
number of transfers. It is also understood that all progeny may not
be precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same function
or biological activity as screened for in the originally
transformed cell are included. Where distinct designations are
intended, it will be clear from the context.
[0091] An "exogenous" element is defined herein to mean nucleic
acid sequence that is foreign to the cell, or homologous to the
cell but in a position within the host cell nucleic acid in which
the element is ordinarily not found.
[0092] "Antibodies" (Abs) and "immunoglobulins" (Igs) are
glycoproteins having the same structural characteristics. While
antibodies exhibit binding specificity to a specific antigen,
immunoglobulins include both antibodies and other antibody-like
molecules that lack antigen specificity. Polypeptides of the latter
kind are, for example, produced at low levels by the lymph system
and at increased levels by myelomas.
[0093] "Native antibodies" and "native immunoglobulins" are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed
of two identical light (L) chains and two identical heavy (H)
chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies among
the heavy chains of different immunoglobulin isotypes. Each heavy
and light chain also has regularly spaced intrachain disulfide
bridges. Each heavy chain has at one end a variable domain
(V.sub.H) followed by a number of constant domains. Each light
chain has a variable domain at one end (V.sub.L) and a constant
domain at its other end; the constant domain of the light chain is
aligned with the first constant domain of the heavy chain, and the
light-chain variable domain is aligned with the variable domain of
the heavy chain. Particular amino acid residues are believed to
form an interface between the light- and heavy-chain variable
domains.
[0094] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework region (FR). The variable domains
of native heavy and light chains each comprise four FRs (FR1, FR2,
FR3 and FR4, respectively), largely adopting a-sheet configuration,
connected by three hypervariable regions, which form loops
connecting, and in some cases forming part of, the -sheet
structure. The hypervariable regions in each chain are held
together in close proximity by the FRs and, with the hypervariable
regions from the other chain, contribute to the formation of the
antigen-binding site of antibodies (see Kabat et al, Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991), pages
647-669). The constant domains are not involved directly in binding
an antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody-dependent
cellular toxicity.
[0095] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (i.e.
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (i.e. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the light chain variable domain and 26-32 (H1),
53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;
Chothia and Lesk J. Md. Biol. 196:901-917 (1987)). "Framework" or
"FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined.
[0096] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0097] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This region
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the V.sub.HV.sub.L. dimer. Collectively, the six hypervariable
regions confer antigen-binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three hypervariable regions specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0098] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxyl terminus of the heavy chain CH1 domain
including one or more cysteine(s) from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0099] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa ( ) and lambda ( ), based on the amino acid
sequences of their constant domains.
[0100] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG 3, IgG4, IgA1, and
IgA2. The heavy-chain constant domains that correspond to the
different classes of immunoglobulins are called , , , , and
respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0101] The term "antibody" herein is used in the broadest sense and
specifically covers human, non-human (e.g. murine) and humanized
monoclonal antibodies (including full length monoclonal
antibodies), polyclonal antibodies, multispecific antibodies (e.g.,
bispecific antibodies), and antibody fragments so long as they
exhibit the desired biological activity.
[0102] "Antibody fragments" comprise a portion of a full length
antibody, generally the antigen binding or variable domain thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0103] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. The modifier "monoclonal" indicates the
character of the antibody as being obtained from a substantially
homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, the monoclonal antibodies to be used in accordance with
the present invention may be made by the hybridoma method first
described by Kohler et al., Nature 256:495 (1975), or may be made
by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
The "monoclonal antibodies" may also be isolated from phage
antibody libraries using the techniques described in Clackson et
al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol.
222:581-597 (1991), for example.
[0104] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; and Morrison
et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
[0105] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies which contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which
hypervariable region residues of the recipient are replaced by
hypervariable region residues from a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable regions correspond to those
of a non-human immunoglobulin and all or substantially all of the
His are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0106] "Single-chain Fv" or "sFv" antibody fragments comprise the
V, and V, domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the Fv polypeptide further
comprises a polypeptide linker between the V.sub.H and V.sub.L
domains which enables the sFv to form the desired structure for
antigen binding. For a review of sFv see Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
[0107] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444-6448 (1993).
[0108] The expression "linear antibodies" when used throughout this
application refers to the antibodies described in Zapata et al.
Protein Eng. 8(10):1057-1062 (1995). Briefly, these antibodies
comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0109] The term "epitope" is used to refer to binding sites for
(monoclonal or polyclonal) antibodies on protein antigens.
[0110] Antibodies which bind to domain 5 and/or 4 within the amino
acid sequence of native sequence human trkC, or to an equivalent
epitope in a native sequence non-human trkC receptor, are
identified by "epitope mapping." There are many methods known in
the art for mapping and characterizing the location of epitopes on
proteins, including solving the crystal structure of an
antibody-antigen complex, competition assays, gene fragment
expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999. A competition ELISA assay is
specifically described in Example 1. According to the gene fragment
expression assays, the open reading frame encoding the protein is
fragmented either randomly or by specific genetic constructions and
the reactivity of the expressed fragments of the protein with the
antibody to be tested is determined. The gene fragments may, for
example, be produced by PCR and then transcribed and translated
into protein in vitro, in the presence of radioactive amino acids.
The binding of the antibody to the radioactively labeled protein
fragments is then determined by immunoprecipitation and gel
electrophoresis. Certain epitopes can also be identified by using
large libraries of random peptide sequences displayed on the
surface of phage particles (phage libraries). Alternatively, a
defined library of overlapping peptide fragments can be tested for
binding to the test antibody in simple binding assays. The latter
approach is suitable to define linear epitopes of about 5 to 15
amino acids.
[0111] An antibody binds "essentially the same epitope" as a
reference antibody, when the two antibodies recognize identical or
sterically overlapping epitopes. The most widely used and rapid
methods for determining whether two epitopes bind to identical or
sterically overlapping epitopes are competition assays, which can
be configured in all number of different formats, using either
labeled antigen or labeled antibody. Usually, the antigen is
immobilized on a 96-well plate, and the ability of unlabeled
antibodies to block the binding of labeled antibodies is measured
using radioactive or enzyme labels. A competition ELISA assay is
disclosed in Example 1.
[0112] The term amino acid or amino acid residue, as used herein,
refers to naturally occurring L amino acids or to D amino acids as
described further below with respect to variants. The commonly used
one- and three-letter abbreviations for amino acids are used herein
(Bruce Alberts et al., Molecular Biology of the Cell, Garland
Publishing, Inc., New York (3d ed. 1994)).
[0113] Hybridization is preferably performed under "stringent
conditions" which means (1) employing low ionic strength and high
temperature for washing, for example, 0.015 sodium chloride/0.0015
M sodium citrate/0.1% sodium dodecyl sulfate at 50 C, or (2)
employing during hybridization a denaturing agent, such as
formamide, for example, 50% (vol/vol) formamide with 0.1% bovine
serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM
sodium citrate at 42 C. Another example is use of 50% formamide,
5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6/8), 0.1% sodium pyrophosphate, 5.times.Denhardt's
solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10%
dextran sulfate at 42 C, with washes at 42 C in 0.2.times.SSC and
0.1% SDS.
B. Methods for Carrying Out the Invention
[0114] The present invention concerns agonist human and non-human
monoclonal antibodies (including humanized forms of the latter),
which mimic certain biological properties of NT-3, the native
ligand of the trkC receptor. General techniques for the production
of murine and human anti-trkC antibodies are well known in the art
and are described hereinbelow. Further details, including the
selection of agonist antibodies, are provided in Example 1.
[0115] 1. Antibody Preparation
[0116] (i) Polyclonal Antibodies
[0117] Methods of preparing polyclonal antibodies are known in the
art. Polyclonal antibodies can be raised in a mammal, for example,
by one or more injections of an immunizing agent and, if desired,
an adjuvant. Typically, the immunizing agent and/or adjuvant will
be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. It may be useful to conjugate the
immunizing agent to a protein known to be immunogenic in the mammal
being immunized, such as serum albumin, or soybean trypsin
inhibitor. Examples of adjuvants which may be employed include
Freund's complete adjuvant and MPL-TDM.
[0118] (ii) Monoclonal Antibodies
[0119] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
[0120] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster or macaque monkey, is immunized as
hereinabove described to elicit lymphocytes that produce or are
capable of producing antibodies that will specifically bind to the
protein used for immunization. Alternatively, lymphocytes may be
immunized in vitro. Lymphocytes then are fused with myeloma cells
using a suitable fusing agent, such as polyethylene glycol, to form
a hybridoma cell (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103, [Academic Press, 1986]).
[0121] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
conditions under which the growth of HGPRT-deficient cells is
prevented.
[0122] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOP-21 and M.C.-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63, Marcel
Dekker, Inc., New York, [1987]).
[0123] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0124] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107:220 (1980).
[0125] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the cells
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, DMEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0126] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0127] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences,
Morrison, et al., Proc. Nat. Acad. Sci. 81, 6851 (1984), or by
covalently joining to the immunoglobulin coding sequence all or
part of the coding sequence for a non-immunoglobulin polypeptide.
In that manner, "chimeric" or "hybrid" antibodies are prepared that
have the binding specificity of an anti-trk monoclonal antibody
herein.
[0128] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody of the
invention, or they are substituted for the variable domains of one
antigen-combining site of an antibody of the invention to create a
chimeric bivalent antibody comprising one antigen-combining site
having specificity for an trk receptor and another
antigen-combining site having specificity for a different
antigen.
[0129] Chimeric or hybrid antibodies also may be prepared in vitro
using known methods in synthetic protein chemistry, including those
involving crosslinking agents. For example, immunotoxins may be
constructed using a disulfide exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate.
[0130] Recombinant production of antibodies will be described in
more detail below.
[0131] (iii) Humanized Antibodies
[0132] Generally, a humanized antibody has one or more amino acid
residues introduced into it from a non-human source. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers [Jones et al., Nature, 321:522-525
(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et
al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or
CDR sequences for the corresponding sequences of a human
antibody.
[0133] Accordingly, such "humanized" antibodies are chimeric
antibodies (Cabilly, supra), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0134] It is important that antibodies be humanized with retention
of high affinity for the antigen and other favorable biological
properties. To achieve this goal, according to a preferred method,
humanized antibodies are prepared by a process of analysis of the
parental sequences and various conceptual humanized products using
three dimensional models of the parental and humanized sequences.
Three dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e. the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. In this way, FR residues can be selected and combined from
the consensus and import sequence so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is achieved. In general, the CDR residues are directly
and most substantially involved in influencing antigen binding. For
further details see U.S. application Ser. No. 07/934,373 filed 21
Aug. 1992, which is a continuation-in-part of application Ser. No.
07/715,272 filed 14 Jun. 1991.
[0135] (iv) Human Antibodies
[0136] Human monoclonal antibodies can be made by the hybridoma
method. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described,
for example, by Kozbor, J. Immunol. 133, 3001 (1984), and Brodeur,
et al., Monoclonal Antibody Production Techniques and Applications,
pp. 51-63 (Marcel Dekker, Inc., New York, 1987).
[0137] It is now possible to produce transgenic animals (e.g. mice)
that are capable, upon immunization, of producing a repertoire of
human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy chain joining region (J.sub.H) gene
in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array in such germ-line mutant mice
will result in the production of human antibodies upon antigen
challenge. See, e.g. Jakobovits et al., Proc. Natl. Acad. Sci. USA
90, 2551-255 (1993); Jakobovits et al., Nature 362, 255-258
(1993).
[0138] Mendez et al. (Nature Genetics 15: 146-156 [1997]) have
further improved the technology and have generated a line of
transgenic mice designated as "Xenomouse II" that, when challenged
with an antigen, generates high affinity fully human antibodies.
This was achieved by germ-line integration of megabase human heavy
chain and light chain loci into mice with deletion into endogenous
J.sub.H segment as described above. The Xenomouse II harbors 1,020
kb of human heavy chain locus containing approximately 66 V.sub.H
genes, complete D.sub.H and J.sub.H regions and three different
constant regions (.mu., .delta. and .chi.), and also harbors 800 kb
of human .kappa. locus containing 32 V.kappa. genes, J.kappa.
segments and C.kappa. genes. The antibodies produced in these mice
closely resemble that seen in humans in all respects, including
gene rearrangement, assembly, and repertoire. The human antibodies
are preferentially expressed over endogenous antibodies due to
deletion in endogenous J.sub.H segment that prevents gene
rearrangement in the murine locus.
[0139] Alternatively, the phage display technology (McCafferty et
al., Nature 348, 552-553 [1990]) can be used to produce human
antibodies and antibody fragments in vitro, from immunoglobulin
variable (V) domain gene repertoires from unimmunized donors.
According to this technique, antibody V domain genes are cloned
in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, such as M13 or fd, and displayed as
functional antibody fragments on the surface of the phage particle.
Because the filamentous particle contains a single-stranded DNA
copy of the phage genome, selections based on the functional
properties of the antibody also result in selection of the gene
encoding the antibody exhibiting those properties. Thus, the phage
mimics some of the properties of the B-cell. Phage display can be
performed in a variety of formats; for their review see, e.g.
Johnson, Kevin S, and Chiswell, David J., Current Opinion in
Structural Biology 3, 564-571 (1993). Several sources of V-gene
segments can be used for phage display. Clackson et al., Nature
352, 624-628 (1991) isolated a diverse array of anti-oxazolone
antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes
from unimmunized human donors can be constructed and antibodies to
a diverse array of antigens (including self-antigens) can be
isolated essentially following the techniques described by Marks et
al., J. Mol. Biol. 222, 581-597 (1991), or Griffith et al., EMBO J.
12, 725-734 (1993). In a natural immune response, antibody genes
accumulate mutations at a high rate (somatic hypermutation). Some
of the changes introduced will confer higher affinity, and B cells
displaying high-affinity surface immunoglobulin are preferentially
replicated and differentiated during subsequent antigen challenge.
This natural process can be mimicked by employing the technique
known as "chain shuffling" (Marks et al., Bio/Technol. 10, 779-783
[1992]). In this method, the affinity of "primary" human antibodies
obtained by phage display can be improved by sequentially replacing
the heavy and light chain V region genes with repertoires of
naturally occurring variants (repertoires) of V domain genes
obtained from unimmunized donors. This techniques allows the
production of antibodies and antibody fragments with affinities in
the nM range. A strategy for making very large phage antibody
repertoires (also known as "the mother-of-all libraries") has been
described by Waterhouse et al., Nucl. Acids Res. 21, 2265-2266
(1993), and the isolation of a high affinity human antibody
directly from such large phage library is reported by Griffith et
al., EMBO J. (1994), in press. Gene shuffling can also be used to
derive human antibodies from rodent antibodies, where the human
antibody has similar affinities and specificities to the starting
rodent antibody. According to this method, which is also referred
to as "epitope imprinting", the heavy or light chain V domain gene
of rodent antibodies obtained by phage display technique is
replaced with a repertoire of human V domain genes, creating
rodent-human chimeras. Selection on antigen results in isolation of
human variable capable of restoring a functional antigen-binding
site, i.e. the epitope governs (imprints) the choice of partner.
When the process is repeated in order to replace the remaining
rodent V domain, a human antibody is obtained (see PCT patent
application WO 93/106213, published 1 Apr. 1993). Unlike
traditional humanization of rodent antibodies by CDR grafting, this
technique provides completely human antibodies, which have no
framework or CDR residues of rodent origin.
[0140] (v) Bispecific Antibodies
[0141] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for the trkC receptor to provide an agonist
antibody, the other one is for any other antigen, and preferably
for another receptor or receptor subunit. For example, bispecific
antibodies specifically binding a trkC receptor and a neurotrophin,
or a trkC receptor and another trk receptor are within the scope of
the present invention.
[0142] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the coexpression of two immunoglobulin heavy
chain-light chain pairs, where the two heavy chains have different
specificities (Millstein and Cuello, Nature 305, 537-539 (1983)).
Because of the random assortment of immunoglobulin heavy and light
chains, these hybridomas (quadromas) produce a potential mixture of
different antibody molecules, of which only one has the correct
bispecific structure. The purification of the correct molecule,
which is usually done by affinity chromatography steps, is rather
cumbersome, and the product yields are low. Similar procedures are
disclosed in PCT application publication No. WO 93/08829 (published
13 May 1993), and in Traunecker et al., EMBO 10, 3655-3659
(1991).
[0143] According to a different and more preferred approach,
antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) are fused to immunoglobulin
constant domain sequences. The fusion preferably is with an
immunoglobulin heavy chain constant domain, comprising at least
part of the hinge, CH2 and CH3 regions. It is preferred to have the
first heavy chain constant region (CH1) containing the site
necessary for light chain binding, present in at least one of the
fusions. DNAs encoding the immunoglobulin heavy chain fusions and,
if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are cotransfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains in one expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance. In a preferred embodiment of this approach, the
bispecific antibodies are composed of a hybrid immunoglobulin heavy
chain with a first binding specificity in one arm, and a hybrid
immunoglobulin heavy chain-light chain pair (providing a second
binding specificity) in the other arm. It was found that this
asymmetric structure facilitates the separation of the desired
bispecific compound from unwanted immunoglobulin chain
combinations, as the presence of an immunoglobulin light chain in
only one half of the bispecific molecule provides for a facile way
of separation. This approach is disclosed in PCT Publication No. WO
94/04690, published on Mar. 3, 1994.
[0144] For further details of generating bispecific antibodies see,
for example, Suresh et al., Methods in Enzymology 121, 210
(1986).
[0145] (vi) Heteroconjugate Antibodies
[0146] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980), and for treatment of HIV infection (PCT
application publication Nos. WO 91/00360 and WO 92/200373; EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0147] (vii) Antibody Fragments
[0148] In certain embodiments, the anti-trkC antibody (including
murine, human and humanized antibodies, and antibody variants) is
an antibody fragment. Various techniques have been developed for
the production of antibody fragments. Traditionally, these
fragments were derived via proteolytic digestion of intact
antibodies (see, e.g., Morimoto et al., J. Biochem. Biophys.
Methods 24:107-117 (1992) and Brennan et al., Science 229:81
(1985)). However, these fragments can now be produced directly by
recombinant host cells. For example, Fab'-SH fragments can be
directly recovered from E. coli and chemically coupled to form
F(ab').sub.2 fragments (Carter et al., Bio/Technology 10:163-167
(1992)). In another embodiment, the F(ab').sub.2 is formed using
the leucine zipper GCN4 to promote assembly of the F(ab').sub.2
molecule. According to another approach, Fv, Fab or F(ab').sub.2
fragments can be isolated directly from recombinant host cell
culture. Other techniques for the production of antibody fragments
will be apparent to the skilled practitioner.
[0149] (viii) Amino Acid Sequence Variants of Antibodies
[0150] Amino acid sequence variants of the anti-trkC antibodies are
prepared by introducing appropriate nucleotide changes into the
anti-trkC antibody DNA, or by peptide synthesis. Such variants
include, for example, deletions from, and/or insertions into and/or
substitutions of, residues within the amino acid sequences of the
anti-trkC antibodies of the examples herein. Any combination of
deletion, insertion, and substitution is made to arrive at the
final construct, provided that the final construct possesses the
desired characteristics. The amino acid changes also may alter
post-translational processes of the humanized or variant anti-trkC
antibody, such as changing the number or position of glycosylation
sites.
[0151] A useful method for identification of certain residues or
regions of the anti-trkC antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis," as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
trkC antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed anti-trkC
antibody variants are screened for the desired activity.
[0152] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an anti-trkC antibody with
an N-terminal methionyl residue or the antibody fused to an epitope
tag. Other insertional variants of the anti-trkC antibody molecule
include the fusion to the N- or C-terminus of the anti-trkC
antibody of an enzyme or a polypeptide which increases the serum
half-life of the antibody (see below).
[0153] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
anti-trkC antibody molecule removed and a different residue
inserted in its place. The sites of greatest interest for
substitutional mutagenesis include the hypervariable regions, but
FR alterations are also contemplated. Conservative substitutions
are shown in Table 1 under the heading of "preferred
substitutions". If such substitutions result in a change in
biological activity, then more substantial changes, denominated
"exemplary substitutions" in Table 1, or as further described below
in reference to amino acid classes, may be introduced and the
products screened.
TABLE-US-00001 TABLE 1 Exemplary Preferred Original Residue
Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys;
gln; asn lys Asn (N) gln; his; asp, lys; arg gin Asp (D) glu; asn
glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp
Gly (G) ala ala His (H) asn; gln; lys; arg arg lle (I) leu; val;
met; ala; leu phe; norleucine Leu (L) norleucine; lie; val; lie
met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu
Phe (F) leu; val; lie; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr
Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe; ala; leu norleucine
[0154] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
[0155] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0156] (2) neutral hydrophilic: cys, ser, thr;
[0157] (3) acidic: asp, glu;
[0158] (4) basic: asn, gln, his, lys, arg;
[0159] (5) residues that influence chain orientation: gly, pro;
and
[0160] (6) aromatic: trp, tyr, phe.
[0161] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0162] Any cysteine residue not involved in maintaining the proper
conformation of the humanized or variant anti-trkC antibody also
may be substituted, generally with serine, to improve the oxidative
stability of the molecule and prevent aberrant crosslinking.
Conversely, cysteine bond(s) may be added to the antibody to
improve its stability (particularly where the antibody is an
antibody fragment such as an Fv fragment).
[0163] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g. a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants is affinity maturation using phage display.
Briefly, several hypervariable region sites (e.g. 6-7 sites) are
mutated to generate all possible amino substitutions at each site.
The antibody variants thus generated are displayed in a monovalent
fashion from filamentous phage particles as fusions to the gene III
product of M13 packaged within each particle. The phage-displayed
variants are then screened for their biological activity (e.g.
binding affinity) as herein disclosed. In order to identify
candidate hypervariable region sites for modification, alanine
scanning mutagenesis can be performed to identify hypervariable
region residues contributing significantly to antigen binding.
Alternatively, or in addition, it may be beneficial to analyze a
crystal structure of the antigen antibody complex to identify
contact points between the antibody and human trkC. Such contact
residues and neighboring residues are candidates for substitution
according to the techniques elaborated herein. Once such variants
are generated, the panel of variants is subjected to screening as
described herein and antibodies with superior properties in one or
more relevant assays may be selected for further development.
[0164] (ix) Glycosylation Variants of Antibodies
[0165] Antibodies are glycosylated at conserved positions in their
constant regions (Jefferis and Lund, Chem. Immunol. 65:111-128
[1997]; Wright and Morrison, TibTECH 15:26-32 [1997]). The
oligosaccharide side chains of the immunoglobulins affect the
protein's function (Boyd et al., Mol. Immunol. 32:1311-1318 [1996];
Wittwe and Howard, Biochem. 29:4175-4180 [1990]), and the
intramolecular interaction between portions of the glycoprotein
which can affect the conformation and presented three-dimensional
surface of the glycoprotein (Hefferis and Lund, supra; Wyss and
Wagner, Current Opin. Biotech. 7:409-416 [1996]). Oligosaccharides
may also serve to target a given glycoprotein to certain molecules
based upon specific recognition structures. For example, it has
been reported that in agalactosylated IgG, the oligosaccharide
moiety `flips` out of the inter-CH2 space and terminal
N-acetylglucosamine residues become available to bind mannose
binding protein (Malhotra et al., Nature Med. 1:237-243 [1995]).
Removal by glycopeptidase of the oligosaccharides from CAMPATH-1H
(a recombinant humanized murine monoclonal 1gG1 antibody which
recognizes the CDw52 antigen of human lymphocytes) produced in
Chinese Hamster Ovary (CHO) cells resulted in a complete reduction
in complement mediated lysis (CMCL) (Boyd et al, Mol. Immunol.
32:1311-1318 [1996]), while selective removal of salicylic acid
residues using neuraminidase resulted in no loss of DMCL.
Glycosylation of antibodies has also been reported to affect
antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO
cells with tetracycline-regulated expression of
.beta.(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a
glycosyltransferase catalyzing formation of bisecting GlcNAc, was
reported to have improved ADCC activity (Umana et al, Mature
Biotech. 17:176-180 [1999]).
[0166] Glycosylation variants of antibodies are variants in which
the glycosylation pattern of an antibody is altered. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, adding one or more carbohydrate moieties to the antibody,
changing the composition of glycosylation (glycosylation pattern),
the extent of glycosylation, etc. Glycosylation variants may, for
example, be prepared by removing, changing and/or adding one or
more glycosylation sites in the nucleic acid sequence encoding the
antibody.
[0167] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0168] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0169] Nucleic acid molecules encoding amino acid sequence variants
of the anti-trkC antibody are prepared by a variety of methods
known in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring
amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the anti-trkC antibody.
[0170] The glycosylation (including glycosylation pattern) of
antibodies may also be altered without altering the underlying
nucleotide sequence. Glycosylation largely depends on the host cell
used to express the antibody. Since the cell type used for
expression of recombinant glycoproteins, e.g. antibodies, as
potential therapeutics is rarely the native cell, significant
variations in the glycosylation pattern of the antibodies can be
expected (see, e.g. Hse et al., J. Biol. Chem. 272:9062-9070
[1997]). In addition to the choice of host cells, factors which
affect glycosylation during recombinant production of antibodies
include growth mode, media formulation, culture density,
oxygenation, pH, purification schemes and the like. Various methods
have been proposed to alter the glycosylation pattern achieved in a
particular host organism including introducing or overexpressing
certain enzymes involved in oligosaccharide production (U.S. Pat.
Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain
types of glycosylation, can be enzymatically removed from the
glycoprotein, for example using endoglycosidase H (Endo H). In
addition, the recombinant host cell can be genetically engineered,
e.g. make defective in processing certain types of polysaccharides.
These and similar techniques are well known in the art.
[0171] The glycosylation structure of antibodies can be readily
analyzed by conventional techniques of carbohydrate analysis,
including lectin chromatography, NMR, Mass spectrometry, HPLC, GPC,
monosaccharide compositional analysis, sequential enzymatic
digestion, and HPAEC-PAD, which uses high pH anion exchange
chromatography to separate oligosaccharides based on charge.
Methods for releasing oligosaccharides for analytical purposes are
also known, and include, without limitation, enzymatic treatment
(commonly performed using peptide-N-glycosidase
F/endo-.beta.-galactosidase), elimination using harsh alkaline
environment to release mainly O-linked structures, and chemical
methods using anhydrous hydrazine to release both N- and O-linked
oligosaccharides.
[0172] (x) Other Modifications of Antibodies
[0173] The anti-trkC antibodies disclosed herein may also be
formulated as immunoliposomes. Liposomes containing the antibody
are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et
al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos.
4,485,045 and 4,544,545. Liposomes with enhanced circulation time
are disclosed in U.S. Pat. No. 5,013,556.
[0174] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst.81(19):1484 (1989).
[0175] The antibody of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g., a peptidyl chemotherapeutic agent,
see WO81/01145) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,975,278.
[0176] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active, cytotoxic form.
[0177] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as -galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; -lactamase useful
for converting drugs derivatized with -lactams into free drugs; and
penicillin amidases, such as penicillin V amidase or penicillin G
amidase, useful for converting drugs derivatized at their amine
nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,
into free drugs. Alternatively, antibodies with enzymatic activity,
also known in the art as "abzymes", can be used to convert the
prodrugs of the invention into free active drugs (see, e.g.,
Massey, Nature 328:457-458 (1987)). Antibody-abzyme conjugates can
be prepared as described herein for delivery of the abzyme to a
tumor cell population.
[0178] The enzymes of this invention can be covalently bound to the
anti-trkC antibodies by techniques well known in the art such as
the use of the heterobifunctional crosslinking reagents discussed
above. Alternatively, fusion proteins comprising at least the
antigen binding region of an antibody of the invention linked to at
least a functionally active portion of an enzyme of the invention
can be constructed using recombinant DNA techniques well known in
the art (see, e.g., Neuberger et al., Nature 312:604-608
[1984]).
[0179] In certain embodiments of the invention, it may be desirable
to use an antibody fragment, rather than an intact antibody. In
this case, it may be desirable to modify the antibody fragment in
order to increase its serum half-life. This may be achieved, for
example, by incorporation of a salvage receptor binding epitope
into the antibody fragment (e.g., by mutation of the appropriate
region in the antibody fragment or by incorporating the epitope
into a peptide tag that is then fused to the antibody fragment at
either end or in the middle, e.g., by DNA or peptide synthesis).
See WO96/32478 published Oct. 17, 1996.
[0180] The salvage receptor binding epitope generally constitutes a
region wherein any one or more amino acid residues from one or two
loops of a Fc domain are transferred to an analogous position of
the antibody fragment. Even more preferably, three or more residues
from one or two loops of the Fc domain are transferred. Still more
preferred, the epitope is taken from the CH2 domain of the Fc
region (e.g., of an IgG) and transferred to the CH1, CH3, or
V.sub.H region, or more than one such region, of the antibody.
Alternatively, the epitope is taken from the CH2 domain of the Fc
region and transferred to the C.sub.L region or V.sub.L region, or
both, of the antibody fragment.
[0181] Covalent modifications of the humanized or variant anti-trkC
antibody (including glycosylation variants) are also included
within the scope of this invention. They may be made by chemical
synthesis or by enzymatic or chemical cleavage of the antibody, if
applicable. Other types of covalent modifications of the antibody
are introduced into the molecule by reacting targeted amino acid
residues of the antibody with an organic derivatizing agent that is
capable of reacting with selected side chains or the N- or
C-terminal residues. Exemplary covalent modifications of
polypeptides are described in U.S. Pat. No. 5,534,615, specifically
incorporated herein by reference. A preferred type of covalent
modification of the antibody comprises linking the antibody to one
of a variety of nonproteinaceous polymers, e.g., polyethylene
glycol, polypropylene glycol, or polyoxyalkylenes, in the manner
set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
[0182] 2. Vectors, Host Cells and Recombinant Methods
[0183] The invention also provides isolated nucleic acid encoding
the non-human, e.g. murine and human anti-trkC antibodies of the
present invention (including the humanized versions of the
non-human antibodies), vectors and host cells comprising the
nucleic acid, and recombinant techniques for the production of the
antibodies.
[0184] For recombinant production of an antibody, the nucleic acid
encoding it may be isolated and inserted into a replicable vector
for further cloning (amplification of the DNA) or for expression.
In another embodiment, the antibody may be produced by homologous
recombination, e.g. as described in U.S. Pat. No. 5,204,244,
specifically incorporated herein by reference. DNA encoding the
monoclonal antibody is readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of the antibody). Many vectors are available. The
vector components generally include, but are not limited to, one or
more of the following: a signal sequence, an origin of replication,
one or more marker genes, an enhancer element, a promoter, and a
transcription termination sequence, e.g., as described in U.S. Pat.
No. 5,534,615 issued Jul. 9, 1996 and specifically incorporated
herein by reference.
[0185] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0186] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-trkC antibody-encoding vectors. Saccharomyces cerevisiae,
or common baker's yeast, is the most commonly used among lower
eukaryotic host microorganisms. However, a number of other genera,
species, and strains are commonly available and useful herein, such
as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger.
[0187] Suitable host cells for the expression of glycosylated
anti-trkC antibody are derived from multicellular organisms.
Examples of invertebrate cells include plant and insect cells.
Numerous baculoviral strains and variants and corresponding
permissive insect host cells from hosts such as Spodoptera
frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruitfly), and
Bombyx mori have been identified. A variety of viral strains for
transfection are publicly available, e.g., the L-1 variant of
Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such viruses may be used as the virus herein according to the
present invention, particularly for transfection of Spodoptera
frugiperda cells. Plant cell cultures of cotton, corn, potato,
soybean, petunia, tomato, and tobacco can also be utilized as
hosts.
[0188] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.
36:59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 [1980]); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 [1980]); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);
human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);
TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 [1982]);
MRC 5 cells; and FS4 cells.
[0189] Host cells are transformed with the above-described
expression or cloning vectors for anti-trkC antibody production and
cultured in conventional nutrient media modified as appropriate for
inducing promoters, selecting transformants, or amplifying the
genes encoding the desired sequences.
[0190] The host cells used to produce the anti-trkC antibody of
this invention may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
((MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium (DMEM) (Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used
as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0191] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10:163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an Amicon or Millipore
Pellicon ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0192] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human 1, 2, or 4 heavy chains (Lindmark et al, J.
Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all
mouse isotypes and for human 3 (Guss et al., EMBO J. 5:15671575
(1986)). The matrix to which the affinity ligand is attached is
most often agarose, but other matrices are available. Mechanically
stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the
antibody comprises a CH3 domain, the Bakerbond ABX.TM. resin (J. T.
Baker, Phillipsburg, N.J.) is useful for purification. Other
techniques for protein purification such as fractionation on an
ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE.TM.
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0193] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
[0194] 3. Identification of Aqonist Anti-trkC Antibodies
[0195] Agonist antibodies may be identified, for example, using the
kinase receptor activation (KIRA) assay described in U.S. Pat. Nos.
5,766,863 and 5,891,650. This ELISA-type assay is suitable for
qualitative or quantitative measurement of kinase activation by
measuring the autophosphorylation of the kinase domain of a
receptor protein tyrosine kinase (rPTK, e.g. trk receptor), as well
as for identification and characterization of potential agonist or
antagonists of a selected rPTK. The first stage of the assay
involves phosphorylation of the kinase domain of a kinase receptor,
in the present case a trkC receptor, wherein the receptor is
present in the cell membrane of a eukaryotic cell. The receptor may
be an endogenous receptor or nucleic acid encoding the receptor, or
a receptor construct, may be transformed into the cell. Typically,
a first solid phase (e.g., a well of a first assay plate) is coated
with a substantially homogeneous population of such cells (usually
a mammalian cell line) so that the cells adhere to the solid phase.
Often, the cells are adherent and thereby adhere naturally to the
first solid phase. If a "receptor construct" is used, it usually
comprises a fusion of a kinase receptor and a flag polypeptide. The
flag polypeptide is recognized by the capture agent, often a
capture antibody, in the ELISA part of the assay. An analyte, such
as a candidate agonist, is then added to the wells having the
adherent cells, such that the tyrosine kinase receptor (e.g. trkC
receptor) is exposed to (or contacted with) the analyte. This assay
enables identification of agonist ligands for the tyrosine kinase
receptor of interest (e.g. trkC). It is also possible to use this
assay to detect antagonists of a tyrosine kinase receptor. In order
to detect the presence of an antagonist ligand which blocks binding
of an agonist to the receptor, the adhering cells are exposed to
the suspected antagonist ligand first, and then to the agonist
ligand, so that competitive inhibition of receptor binding and
activation can be measured. Also, the assay can identify an
antagonist which binds to the agonist ligand and thereby reduces or
eliminates its ability to bind to, and activate, the rPTK. To
detect such an antagonist, the suspected antagonist and the agonist
for the rPTK are incubated together and the adhering cells are then
exposed to this mixture of ligands. Following exposure to the
analyte, the adhering cells are solubilized using a lysis buffer
(which has a solubilizing detergent therein) and gentle agitation,
thereby releasing cell lysate which can be subjected to the ELISA
part of the assay directly, without the need for concentration or
clarification of the cell lysate.
[0196] The cell lysate thus prepared is then ready to be subjected
to the ELISA stage of the assay. As a first step in the ELISA
stage, a second solid phase (usually a well of an ELISA microtiter
plate) is coated with a capture agent (often a capture antibody)
which binds specifically to the tyrosine kinase receptor, or, in
the case of a receptor construct, to the flag polypeptide. Coating
of the second solid phase is carried out so that the capture agent
adheres to the second solid phase. The capture agent is generally a
monoclonal antibody, but, as is described in the examples herein,
polyclonal antibodies may also be used. The cell lysate obtained is
then exposed to, or contacted with, the adhering capture agent so
that the receptor or receptor construct adheres to (or is captured
in) the second solid phase. A washing step is then carried out, so
as to remove unbound cell lysate, leaving the captured receptor or
receptor construct. The adhering or captured receptor or receptor
construct is then exposed to, or contacted with, an
anti-phosphotyrosine antibody which identifies phosphorylated
tyrosine residues in the tyrosine kinase receptor. In the preferred
embodiment, the anti-phosphotyrosine antibody is conjugated
(directly or indirectly) to an enzyme which catalyses a color
change of a non-radioactive color reagent. Accordingly,
phosphorylation of the receptor can be measured by a subsequent
color change of the reagent. The enzyme can be bound to the
anti-phosphotyrosine antibody directly, or a conjugating molecule
(e.g., biotin) can be conjugated to the anti-phosphotyrosine
antibody and the enzyme can be subsequently bound to the
anti-phosphotyrosine antibody via the conjugating molecule.
Finally, binding of the anti-phosphotyrosine antibody to the
captured receptor or receptor construct is measured, e.g., by a
color change in the color reagent.
[0197] Following initial identification, the agonist activity can
be further confirmed and refined by bioassays, known to test the
targeted biological activities. For example, the ability of
anti-trkC monoclonal antibodies to mimic the activity of NT-3 can
be tested in the PC12 neurite outgrowth assay as described in
Example 1, and confirmed in known animal models of
neurodegenerative diseases, such as the experimental animal models
of cisplatin- and pyridoxine-induced neuropathies described in
Example 2.
[0198] 3. Therapeutic and Diagnostic Uses of Agonist Anti-TrkC
Antibodies
[0199] The anti-trkC agonist antibodies of the present invention
are believed to be useful in the treatment (including prevention)
of disorders the pathology of which involves cellular degeneration
or disfunction. In particular, the anti-trkC agonist antibodies are
promising candidates for the treatment of various (chronic)
neurodegenerative disorders and acute nerve cell injuries. Such
neurodegenerative disorders include, without limitation, peripheral
neuropathies; motorneuron disorders, such as amylotrophic lateral
schlerosis (ALS, Lou Gehrig's disease), Bell's palsy, and various
conditions involving spinal muscular atrophy or paralysis; and
other human neurodegenerative diseases, such as Alzheimer's
disease, Parkinson's disease, epilepsy, multiple schlerosis,
Huntington's chorea, Down's Syndrome, nerve deafness, and Meniere's
disease, and acute nerve cell injuries, for example due to trauma
or spinal cord injury.
[0200] The anti-trkC antibodies of the present invention are
believed to be particularly suited for the treatment of peripheral
neuropathy, a neurodegenerative disorder that affects the
peripheral nerves, most often manifested as one or a combination of
motor, sensory, sensorimotor, or autonomic dysfunction. Peripheral
neuropathies may, for example, be genetically acquired, can result
from a systemic disease, can be induced by a toxic agent, such as a
neurotoxic drug, e.g. antineoplastic agent, or industrial or
environmental pollutant, or can be idiopathic. Thus, peripheral
sensory neuropathy is characterized by the degeneration, decrease
or failure of function of peripheral sensory neurons, which may
occur, for example, as a consequence of diabetes (diabetic
neuropathy), cytostatic drug therapy in cancer (e.g. treatment with
chemotherapeutic agents such as vincristine, cisplatin,
methotrexate, or 3'-azido-3'-deoxythymidine), alcoholism, acquired
immunodeficiency syndrome (AIDS), or genetic predisposition.
Genetically acquired peripheral neuropathies include, for example,
Refsum's disease, Krabbe's disease, Metachromatic leukodystrophy,
Fabry's disease, Dejerine-Sottas syndrome, Abetalipoproteinemia,
and Charcot-Marie-Tooth (CMT) Disease (also known as Proneal
Muscular Atrophy or Hereditary Motor Sensory Neuropathy
(HMSN)).
[0201] Based on the demonstrated ability of NT-3, the native ligand
of the trkC receptor, to promote proliferation of peripheral blood
leukocytes, the anti-trkC agonist antibodies of the present
invention may be used also as therapeutic agents for the treatment
of neutropenia, various infections, and tumors. Since the
expression of trkC is not limited to neurons, anti-trkC agonist
antibodies are expected to find utility in the prevention or
treatment of disorders characterized by cellular degeneration in
general, without restriction to neural cells.
[0202] The anti-trkC antibodies of the present invention may also
be used to induce angiogenesis, or treat pathological
conditions/diseases in which the induction of angiogenesis is
desirable. Such pathological conditions include, for example,
cardiac ischemia regardless of the underlying pathology, including
cerebrovascular disorders caused by insufficient cerebral
circulation. Angiogenesis may also be desirable in the treatment of
wounds, including ulcers, diabetic complications of sickle cell
disease, and post surgical wounds.
[0203] The anti-trkC antibodies of the present invention may also
be useful in the diagnosis of diseases involving cellular
degeneration, in particular the neurodegenerative diseases listed
above.
[0204] For diagnostic applications, the antibody typically will be
labeled with a detectable moiety. Numerous labels are available
which can be generally grouped into the following categories:
[0205] (a) Radioisotopes, such as .sup.35S, .sup.14C, .sup.125I
.sup.3H, and .sup.131I. The antibody can be labeled with the
radioisotope using the techniques described in Current Protocols in
Immunology, Volumes 1 and 2, Coligen et al., Ed.
Wiley-Interscience, New York, N.Y., Pubs. (1991) for example and
radioactivity can be measured using scintillation counting.
[0206] (b) Fluorescent labels such as rare earth chelates (europium
chelates) or fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are
available. The fluorescent labels can be conjugated to the antibody
using the techniques disclosed in Current Protocols in Immunology,
supra, for example. Fluorescence can be quantified using a
fluorimeter.
[0207] (c) Various enzyme-substrate labels are available and U.S.
Pat. No. 4,275,149 provides a review of some of these. The enzyme
generally catalyzes a chemical alteration of the chromogenic
substrate which can be measured using various techniques. For
example, the enzyme may catalyze a color change in a substrate,
which can be measured spectrophotometrically. Alternatively, the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are
described above. The chemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit
light which can be measured (using a chemiluminometer, for example)
or donates energy to a fluorescent acceptor. Examples of enzymatic
labels include luciferases (e.g., firefly luciferase and bacterial
luciferase; U.S. Pat. No. 4,737,456), luciferin,
2,3-dihydrophthalazinediones, malate dehydrogenase, urease,
peroxidase such as horseradish peroxidase (HRPO), alkaline
phosphatase, -galactosidase, glucoamylase, lysozyme, saccharide
oxidases (e.g., glucose oxidase, galactose oxidase, and
glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as
uricase and xanthine oxidase), lactoperoxidase, microperoxidase,
and the like. Techniques for conjugating enzymes to antibodies are
described in O'Sullivan et al., Methods for the Preparation of
Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in
Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic
press, New York, 73:147-166 (1981).
[0208] Examples of enzyme-substrate combinations include, for
example: [0209] (i) Horseradish peroxidase (HRPO) with hydrogen
peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes
a dye precursor (e.g., orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB)); [0210] (ii)
alkaline phosphatase (AP) with para-Nitrophenyl phosphate as
chromogenic substrate; and [0211] (iii)-D-galactosidase (-D-Gal)
with a chromogenic substrate (e.g., p-nitrophenyl-D-galactosidase)
or fluorogenic substrate 4-methylumbelliferyl-D-galactosidase.
[0212] Numerous other enzyme-substrate combinations are available
to those skilled in the art. For a general review of these, see
U.S. Pat. Nos. 4,275,149 and 4,318,980.
[0213] Sometimes, the label is indirectly conjugated with the
antibody. The skilled artisan will be aware of various techniques
for achieving this. For example, the antibody can be conjugated
with biotin and any of the three broad categories of labels
mentioned above can be conjugated with avidin, or vice versa.
Biotin binds selectively to avidin and thus, the label can be
conjugated with the antibody in this indirect manner.
Alternatively, to achieve indirect conjugation of the label with
the antibody, the antibody is conjugated with a small hapten (e.g.,
digoxin) and one of the different types of labels mentioned above
is conjugated with an anti-hapten antibody (e.g., anti-digoxin
antibody). Thus, indirect conjugation of the label with the
antibody can be achieved.
[0214] In another embodiment of the invention, the anti-trkC
antibody need not be labeled, and the presence thereof can be
detected using a labeled antibody which binds to the anti-trkC
antibody.
[0215] The antibodies of the present invention may be employed in
any known assay method, such as competitive binding assays, direct
and indirect sandwich assays, and immunoprecipitation assays. Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc. 1987).
[0216] Competitive binding assays rely on the ability of a labeled
standard to compete with the test sample analyte for binding with a
limited amount of antibody. The amount of trkC protein in the test
sample is inversely proportional to the amount of standard that
becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies generally are
insolubilized before or after the competition, so that the standard
and analyte that are bound to the antibodies may conveniently be
separated from the standard and analyte which remain unbound.
[0217] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the protein to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody which is immobilized on a
solid support, and thereafter a second antibody binds to the
analyte, thus forming an insoluble three-part complex. See, e.g.,
U.S. Pat. No. 4,376,110. The second antibody may itself be labeled
with a detectable moiety (direct sandwich assays) or may be
measured using an anti-immunoglobulin antibody that is labeled with
a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA assay, in which case the
detectable moiety is an enzyme.
[0218] The antibodies may also be used for in vivo diagnostic
assays. Generally, the antibody is labeled with a radionuclide
(such as .sup.111In, .sup.99Tc, .sup.14C, .sup.131I, .sup.125I
.sup.3H, .sup.32P or .sup.35S) so that the cells or tissue of
interest can be localized using immunoscintiography.
[0219] The antibodies may also be used as staining reagents in
pathology, following techniques well known in the art.
[0220] The anti-trkC agonist antibodies of the present invention
are believed to possess numerous advantages over NT-3 as
therapeutic agents, including improved efficacy, improved
pharmacokinetic properties (pK) and bioavailability, and lack of
hyperalgesia following administration.
[0221] 4. Pharmaceutical Formulations
[0222] Therapeutic formulations of the antibodies of the present
invention are prepared for storage by mixing the antibody having
the desired degree of purity with optional physiologically
acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the
form of lyophilized formulations or aqueous solutions. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at
the dosages and concentrations employed, and include buffers such
as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0223] The formulations herein may also contain more than one
active compound as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0224] The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0225] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0226] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsule. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the Lupron
Depot.TM. (injectable microspheres composed of lactic acid glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0227] An effective amount of an antibody of the present invention
to be employed therapeutically will depend, for example, upon the
therapeutic objectives, the route of administration, and the
condition of the patient. Accordingly, it will be necessary for the
therapist to titer the dosage and modify the route of
administration as required to obtain the optimal therapeutic
effect. A typical daily dosage might range from about 1 g/kg to up
to 100 mg/kg or more, depending on the factors mentioned above.
Typically, the clinician will administer a molecule of the present
invention until a dosage is reached that provides the required
biological effect. The progress of this therapy is easily monitored
by conventional assays.
[0228] Administration may be by any conventional route known in the
art including, without limitation, intravenous, subcutaneous,
topical, intramuscular, intratracheal, intracerebral, intranasal,
intrapulmonary, and intraparyncal administration.
[0229] 5. Gene Therapy
[0230] The nucleic acid encoding the antibodies of the present
invention may also be used in gene therapy of various (chronic)
neurodegenerative disorders and acute nerve cell injuries,
especially genetically acquired peripheral neuropathies. Two basic
approaches to gene therapy have evolved: ex vivo gene therapy and
in vivo gene therapy. In ex vivo gene therapy, cells are removed
from a subject and cultured in vitro. A functional replacement gene
is introduced into the cells in vitro, the modified cells are
expanded in culture, and then reimplanted in the subject. In in
vivo gene therapy, the target cells are not removed from the
subject. Rather, the transferred gene is introduced into cells of
the recipient in situ, that is, within the recipient.
[0231] Several ex vivo gene therapy studies in humans have been
reported and are reviewed, for example, in Anderson, Science
256:808-813 (1992), and Miller, Nature 357:455-460 (1992).
[0232] The viability of in vivo gene therapy has been demonstrated
in several animal models, as reviewed in Feigner et al., Nature
349:351-352 (1991). Direct gene transfer has been reported, for
example, into muscle tissue (Ferry et al., Proc Natl. Acad. Sci.
88:8377-8781 (1991); Guantin et al., Proc. Natl. Acad. Sci. USA
89:2581-2584 [1992]); the arterial wall (Nabel et al., Science
244:1342-1344 [1989]); and the nervous system (Price et al., Proc.
Natl. Acad. Sci. 84:156-160 [1987]).
[0233] Accordingly, the present invention also provides delivery
vehicles suitable for delivery of a polynucleotide encoding an
agonist anti-trkC antibody into cells (whether in vivo or ex vivo).
Generally, a polynucleotide encoding an antibody (e.g. linear
antibody or antibody chains) will be operably linked to a promoter
and a heterologous polynucleotide. Delivery vehicles suitable for
incorporation of a polynucleotide encoding an antibody of the
present invention for introduction into a host cell include
non-viral vehicles and viral vectors. Verma and Somia, Nature
389:239-242 (1997).
[0234] A wide variety of non-viral vehicles for delivery of a
polynucleotide encoding an antibody of the present invention are
known in the art and are encompassed in the present invention. A
polynucleotide encoding an anti-trkC antibody can be delivered to a
cell as naked DNA (U.S. Pat. No. 5,692,622; WO 97/40163).
Alternatively, a polynucleotide encoding an anti-trkC antibody
herein can be delivered to a cell associated in a variety of ways
with a variety of substances (forms of delivery) including, but not
limited to cationic lipids; biocompatible polymers, including
natural polymers and synthetic polymers; lipoproteins;
polypeptides; polysaccharides; lipopolysaccharides; artificial
viral envelopes; metal particles; and bacteria. A delivery vehicle
can be a microparticle. Mixtures or conjugates of these various
substances can also be used as delivery vehicles. A polynucleotide
encoding an antibody herein can be associated non-covalently or
covalently with these various forms of delivery. Liposomes can be
targeted to a particular cell type, e.g., to a glomerular
epithelial cell.
[0235] Viral vectors include, but are not limited to, DNA viral
vectors such as those based on adenoviruses, herpes simplex virus,
poxviruses such as vaccinia virus, and parvoviruses, including
adeno-associated virus; and RNA viral vectors, including, but not
limited to, the retroviral vectors. Retroviral vectors include
murine leukemia virus, and lentiviruses such as human
immunodeficiency virus. Naldini et al., Science 272:263-267
(1996).
[0236] Non-viral delivery vehicles comprising a polynucleotide
encoding an anti-trkC antibody can be introduced into host cells
and/or target cells by any method known in the art, such as
transfection by the calcium phosphate coprecipitation technique;
electroporation; electropermeabilization; liposome-mediated
transfection; ballistic transfection; biolistic processes including
microparticle bombardment, jet injection, and needle and syringe
injection; or by microinjection. Numerous methods of transfection
are known to the skilled worker in the field.
[0237] Viral delivery vehicles can be introduced into cells by
infection. Alternatively, viral vehicles can be incorporated into
any of the non-viral delivery vehicles described above for delivery
into cells. For example, viral vectors can be mixed with cationic
lipids (Hodgson and Solaiman, Nature Biotechnol. 14:339-342
[1996]); or lamellar liposomes (Wilson et al. Proc. Natl. Acad.
Sci. USA 74:3471 [1977]; and Faller et al. J. Virol. 49:269
[1984]).
[0238] In a preferred embodiment, nucleic acid encoding both the
heavy and the light chains (including fragments) of an anti-trkC
antibody of the present invention will be present in the same
polycistronic expression vector, such as those disclosed in U.S.
Pat. Nos. 4,965,196 and 4,713,339. Polycistronic expression vectors
contain sequences coding for a secondary protein and a desired
protein, wherein both the desired and secondary sequences are
governed by the same promoter. The coding sequences are separated
by translational stop and start signal colons. The expression of
the secondary sequence effects control over the expression of the
sequence for the desired protein, and the secondary protein
functions as a marker for selection of transfected cells.
[0239] In in vivo gene therapy, the vector may be administered to
the recipient, for example, by intravenous (i.v.) injection.
Suitable titers will depend on a variety of factors, such as the
particular vector chosen, the host, strength of promoter used, and
the severity of the disease being treated.
[0240] The invention will be further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Production and Characterization of Agonist Anti-trkC Monoclonal
Antibodies
[0241] Production and Isotyping of Antibodies
[0242] Wild type Balb/C mice and transgenic mice producing human
IgG2 or IgG4 (Xenomice, described in Mendez et al., Nature Genetics
15: 146-156 [1997]) were hyperimmunized either intraperitoneally,
via rear footpad, or subcutaneously with 20 .mu.g of human trkC-IgG
(Shelton et al., J. Neurosci. 15: 477-491 [1995]) in either
Frieund's or Ribi adjuvant as described in Mendez et al. (supra).
Spleen cells from the immune mice were fused with myeloma cells
(X63.Ag8.653, ATCC Rockville, Md.). A total of 33 fusions were
performed using 253 Xenomice and 35 wild type Balb/C mice. Plates
(21,734 wells total) were initially screened by direct ELISA using
trkC-IgG. The ELISA screen identified 684 trkC positive hybridomas,
all of which were then evaluated for agonist activity in trkC KIRA
(Kinase activated Receptor Assay). The KIRA identified 14 Xenomouse
derived and 22 wild type Balb/C mice derived hybridomas secreting
anti-trkC agonist antibodies. These hybridomas were subcloned by
limiting dilution, reassayed to confirm agonist activity, and were
used to induce ascites by injecting into Pristane-primed Balb/C or
nude mice (Hongo et al., Hybridoma 14: 253-260 [1995]). The
monoclonal antibodies present in ascites were purified by Protein A
affinity chromatography (Hongo et al., supra). Specific fusion
efficiency (number of positives/number of wells screened) was 3%
for both the Xenomouse and wild type Balb/C mouse fusions. The
incidence of agonist monoclonal antibodies (agonists/number of trkC
ELISA positives) was 3% and 8% for the Xenomouse and wild type
Balb/C mouse fusions, respectively. Isotypes of the murine
monoclonal antibodies were determined using either GIBCO BRL
dipstick or Zymed mouse-typer isotyping kit, following supplier's
instructions. The Xenomice were either IgG.sub.2 or IgG.sub.4
strain, producing corresponding isotypes of antibodies. Table 1
shows isotypes of various human and murine anti-trkC monoclonal
antibodies. A total of 8 human IgG.sub.2, 6 human IgG.sub.4, 7
murine IgG.sub.1, 10 murine IgG.sub.2, and 5 murine IgG.sub.2b
monoclonal antibodies were identified. The monoclonal antibodies
with the most potent agonist activity (depicted by asterisk in
Table 2), as determined by KIRA assay, were selected for in-depth
characterization.
TABLE-US-00002 TABLE 2 Human Mabs (14 Total) IgG.sub.2 Isotype (8
Mabs) IgG.sub.4 Isotype (6 Mabs) 2.5.1* 4.8 6.1.2* 2337 6.4.1* 2338
2342 2339 2343 2348 2344* 2349* 2345* 2346 Murine Mabs (22 Total)
IgG.sub.1 IgG.sub.2a IgG.sub.2b (7) (10) (5) IgG.sub.3 2249 2248*
2252 2250* 2272 2273 2253* 2251 2277 2254 2255 2279 2256* 2274 2280
2257 2275 2260 2276 2278 2281 2282
[0243] Determination of Agonist Activity
[0244] a. KIRA Assay
[0245] Two bioassays were used to determine NT-3 agonist activity
of anti-trkC monoclonal antibodies. The Kinase activated receptor
assay (KIRA), which has been discussed in greater detail
hereinabove, measures tyrosine phosphorylation of trkC in
transfected cells in response to stimulation with a ligand, such as
NT-3, or agonist monoclonal antibodies (Sadick et al., Exp. Cell
Res. 234: 354-361 [1997]). The monoclonal antibodies were diluted
to 27 .mu.g/ml in KIRA stimulation buffer (F12/DMEM 50:50
containing 2% bovine serum albumin (BSA; Intergen Co., Purchase,
N.Y.) and 25 mM Hepes, 0.2 .mu.m filtered). The monoclonal
antibodies were further diluted serially 1:3 (8 dilutions total;
concentrations ranging from 0.01-180 nM) in stimulation medium.
Chinese Hamster Ovary (CHO) cells stably transfected with trkC
fused with a 26 amino acid polypeptide flag epitope derived from
HSV glycoprotein D (gD) were seeded (5.times.10.sup.4 cells/well)
and grown in 96-well cell culture plates. The cells were then
stimulated with either NT-3 (as a positive control) or various
anti-trkC monoclonal antibodies, using serial dilutions of 0.1;
1.56; 3.13; 6.25; 12.5; 25; 50 and 100 ng/ml. All dilutions were
assayed in duplicate for 6 hours. The assay was carried out
essentially as described in Sadick et al. (supra). Briefly, cells
were lysed using Triton X-100 and trkC present in lysate captured
in ELISA using antibodies against the gD epitope and phosphorylated
trkC detected and quantitated using anti-phosphotyrosine antibodies
suitably conjugated with enzyme. A monoclonal antibody not directed
against trkC (anti-IL8 IgG.sub.2 Xenomous-derived human antibody or
anti-gp120 IgG.sub.1 murine monoclonal antibody) was used as a
negative control. As shown in FIG. 1 (A and B), all the selected
anti-trkC monoclonal antibodies could mimic the activity of NT-3
inasmuch as they could stimulate tyrosine phosphorylation of trkC
receptor. The human anti-trkC monoclonal antibodies (FIG. 1A)
showed more potent agonistic activity than the murine anti-trkC
monoclonal antibodies (FIG. 1B). For example, the best human
anti-trkC monoclonal antibody is 10-fold more potent than the best
murine anti-trkC monoclonal antibody. Furthermore, the human
monoclonals were nearly as efficient as NT-3 especially in the
tower range of concentration.
[0246] b. PC12 Neurite Outgrowth Assay
[0247] Another assay used to determine NT-3 mimetic activity of
anti-trkC monoclonal antibodies was PC12 neurite outgrowth assay.
This assay measures the outgrowth of neurite processes by rat
pheocytochroma cells (PC12) in response to stimulation by
appropriate ligands. These cells express endogenous trkA and are
therefore responsive to NGF. However, they do not express
endogenous trkC and are therefore transfected with trkC expression
construct in order to elicit response to NT-3 and its agonists.
PC12 cells were transfected (Urfer et al, Biochem. 36:4775-4781
[1997]; Tsoulfas et al., Neuron 10:975-990 [1993]) with full-length
human trkC and plated in 96-well cell culture plates (1000
cells/well). Three days following transfection, anti-trkC
monoclonal antibodies were added in triplicate (concentration
ranging from 0.0002 to 2.7 nM) and incubated for an additional 3
days at 37.degree. C. The cells were then analyzed by phase
contrast microscopy and cells with neurites exceeding 2 times the
diameter of the cell were counted. The human as well as the murine
anti-trkC monoclonal antibodies could stimulate neurite outgrowth
in PC12 cells as shown in FIGS. 1C and D. The human anti-trkC
monoclonal antibodies (FIG. 1C) exhibited far more potent activity
than the murine anti-trkC monoclonal antibodies (FIG. 1D) thus
corroborating the results obtained in the KIRA assay. Furthermore,
consistent with the KIRA assay results, the human anti-trkC
monoclonal antibodies showed roughly similar stimulation as
obtained with NT-3. The results obtained with the two bioassays
described above demonstrate the ability of anti-trkC monoclonal
antibodies to mimic the activity of NT-3, the natural ligand of
trkC receptor.
[0248] Agonist activity of the monoclonal antibodies was ranked
according to maximum induction of tyrosine phosphorylation and
calculated EC50 of the phosphorylation curves in the KIRA assay and
PC12 neurite outgrowth assay. Table 3 summarizes characteristics of
various anti-trkC agonist monoclonal antibodies.
TABLE-US-00003 TABLE 3 Agonist Immunoblot Activity Binds NR/Red.
Affinity MAb ID Isotype KIRA/PC12 Rat trkC NR Red. Kd (nM) Human
MAbs 2.5.1 G2 (+++/+++) NO ++ ++ 12 6.1.2 G2 (++++/++++) NO + +
12.5 6.4.1 G2 (++++/++++) YES + + 12 2344 G2 (+++/+++) NO ++ + 19
2345 G2 (++++/++++) NO ++ ++ 12.1 2349 G4 (++++/++++) NO ++ + 23
Murine MAbs 2248 G2a (+/+) NO +++ 5.9 2250 G1 (++/++) NO ++ ++ 8.7
2253 G1 (++/+) NO ++ ++ 42 2256 G1 (+/+) YES ++ + 62
[0249] Testing Specificity of Anti-trkC Antibodies
[0250] The specificity of anti-trkC monoclonal antibodies was
tested using direct ELISA. The microtiter plates were coated
overnight with immunoadhesin construct of the receptor trkA-IgG,
trkB-IgG or trkC-IgG as capture antigens (described in Shelton et
al., J. Neurosci. 15: 477-491 [1995]) using 100 .mu.l of 1 .mu.g/ml
solution diluted in 50 mM carbonate buffer, pH 9.5. CD4-IgG (Capon
et al., Nature 337: 525-531 [1989]) was used in place of capture
antigen as a negative control. The coated plates were incubated for
1 hr at room temperature with various concentration of anti-trkC
monoclonal antibodies (100 .mu.l of 0.01 to 1 .mu.g/ml) diluted in
PBS containing 0.5% BSA and 0.05% Tween 20. After washing to remove
excess unbound antibodies, appropriate HRP conjugate (human
monoclonal antibodies: goat anti-human .kappa.-HRP, 1:5000 diluted;
murine monoclonal antibodies: goat anti-mouse IgG (Fc)-HRP, 1:5000
diluted) was added and incubated for 1 hr at room temperature. The
plates were then washed, developed and read as previously described
(Hongo et al., Hybridoma 14: 253-260 [1995]). FIG. 2 shows a
representative example using a human anti-trkC monoclonal antibody
6.1.2. The binding was highly specific to trkC, and no significant
cross-reaction was observed with either trkA or trkB. Similarly,
other human and mouse anti-trkC monoclonal antibodies showed
specific recognition of trkC.
[0251] The binding of various anti-trkC agonist monoclonal
antibodies to human trkC and rat trkC was compared using a direct
ELISA essentially as described above except the capture antigen
used for human trkC was trkC-gD instead of trkC-IgG. Results shown
in FIG. 3 indicate that among human anti-trkC monoclonal
antibodies, only 6.4.1 significantly recognized rat trkC, rest were
specific for human trkC. Similarly, among murine monoclonal
antibodies, only 2256 recognized rat trkC to a significant extent
while others showed specific recognition of human trkC only.
[0252] Affinity Studies
[0253] Affinities of anti-trkC agonist monoclonal antibodies were
determined using BIAcore-2000.TM. surface plasmon resonance (SPR)
system (BIAcore, Inc., Piscataway, N.J.). CM5 biosensor chips were
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. In the first series of binding
experiments, the antigen, gD-tagged trkC, was diluted into 10 mM
sodium acetate buffer (pH 4.8), and injected over the activated
chip at a concentration of 0.09 mg/mL. Using variable exposure
times, four ranges of antigen density were achieved: 14,000-17,000
response units (RU), 7000-9000 RU, 2000-3000 RU, and 400-600 RU.
The chip was blocked with ethanolamine.
[0254] In the first series of kinetic measurements, anti-trkC
antibodies (IgG's) were diluted into running buffer (PBS containing
0.05% Tween-20 and 0.01% sodium azide) and 0.03 mL (667 nM) was
injected over the biosensor chip at 25.degree. C. at a flow rate of
0.01 mL/min. Regeneration was achieved with a 30 sec pulse of 10 mM
HCl, followed by a 1 min pulse of 100 mM Tris-HCl pH 8.0 and two
wash steps.
[0255] In a second series of experiments, the IgG's (0.1 mg/mL in
10 mM sodium acetate, pH 4.8) were immobilized as described above,
except that antibody density was limited to 1000-2000 RU. Two-fold
serial dilutions of gD-trkC in the range of 3.7 M to 29 nM were
then injected over the biosensor chip for kinetics measurements as
described above.
[0256] The dissociation phase of each kinetic curve were fit to a
single exponential dissociation rate (k.sub.off), and these rates
were used in the calculation of the association rate (k.sub.on)
from the injection phase, using a simple 1:1 Langmuir binding model
(Lofas & Johnsson, 1990).
[0257] Equilibrium dissociation constants, K.sub.d's, from SPR
measurements were calculated as the ratio k.sub.off/k.sub.on.
[0258] The affinities of anti-trkC antibodies for gD-trkC were
measured in SPR kinetics experiments with either antigen or
antibody immobilized. Apparent affinities determined from
experiments using low densities of immobilized antigen (0.4 to 0.6
ng/mm2), were generally consistent with those determined in
experiments using immobilized IgG (see Table 2). However, at higher
densities of immobilized gD-trkC, the apparent binding affinity of
each antibody became progressively tighter by factors of as much as
10 fold, probably because of an avidity effect of binding by the
bivalent IgG (data not shown). In some cases, no binding could be
detected when trkC was injected over immobilized IgG. This may have
occurred because immobilization of the IgG led to steric blocking
of the antigen-binding site. Under all conditions tested, the
antibody 2248 had the highest apparent affinity (K.sub.d=5.6 to 8.5
nM) of all antibodies tested.
TABLE-US-00004 TABLE 4 Binding affinities determined by SPR.
Results are shown for IgG's binding to immobilized gD-trkC (400-600
RU) and for gD-trkC binding to immobilized IgG's (1000-3000 RU).
K.sub.d(nM) Antibody (IgG) Immobilized gD-trkC Immobilized IgG 2248
5.9 8.5 2250 8.7 28 2253 42 51 2256 62 300 2344 19 NDB 2345 12 NDB
2349 23 NDB 6.4.1 12 28 6.1.2 13 16 2.5.1 12 NDB NDB = no
detectable binding.
[0259] Competition ELISA
[0260] A competition ELISA was used to get preliminary information
about various groups to which these antibodies belong depending on
the epitope(s) on trkC they recognize. In this assay, trkC-gD (1
.mu.g/ml) was used as a capture antigen to coat microtiter plate. A
specific biotinylated anti-trkC monoclonal antibody (1 .mu.g/ml)
was added to the coated plate either alone or in presence of
another anti-trkC monoclonal antibody that was unlabeled and used
in excess (50 .mu.g/ml) as compared to the labeled antibody. If
biotinylated antibody and unlabeled antibody both recognize the
same or overlapping epitope, they will compete for binding to the
immobilized trkC, resulting in decreased binding of the labeled
antibody. If they recognize different and non-overlapping epitopes,
there will be no competition between them, and the binding of the
labeled antibody to the immobilized trkC will not be affected.
Unlabeled human IgG2 and mouse IgG were used as negative control. A
representative data in FIG. 4 shows that all anti-trkC monoclonal
antibodies, except murine anti-trkC 2248 monoclonal antibody,
compete with labeled human anti-trkC 6.1.2 monoclonal antibody for
binding to the immobilized trkC, suggesting that murine 2248
antibody recognizes an epitope on trkC that is different from the
epitope(s) recognized by all other anti-trkC antibodies.
[0261] It is interesting to note that when unlabeled murine
monoclonal antibody 2248 is bound first to immobilized trkC, none
of the other (biotinylated) antibodies can access their binding
site, suggesting that even though the epitopes are distinct, steric
hinderance may play a role. Such pairwise comparison gives valuable
information and helps in classifying antibodies directed against
the same antigen into different groups based on epitope
recognition. A summary of such comparison is shown in FIG. 5. The
results indicate that the antibodies can be divided into two
distinct groups: Group 1 encompasses all monoclonal antibodies
except 2248, whereas Group 2 is composed of 2248.
[0262] Epitope Mapping with Domain Swap Mutants
[0263] Further epitope mapping was performed utilizing chimeric
trkC in which various domains were replaced with corresponding
domains from trkA or trkB. This approach was made possible by the
fact that anti-trkC antibodies do not significantly cross-react
with trkA or trkB. The use of such domain-swap mutants has a
distinct advantage over deletion mutants. The deletion of a domain
might disrupt the secondary structure of protein whereas
substitution of a domain with a corresponding domain, of similar
size and substantially similar amino acid sequence, from a related
protein in domain-swap mutants is likely to retain the secondary
structure. The extracellular domain of trk receptors is composed of
5 domains as shown in FIG. 6A. D1 and D3 are cysteine-rich domains,
D2 is a leucine-rich domain, and D4 and D5 are immunoglobulin-like
domains. Domain-swap mutants of trkC containing replacement of D1,
D4 and D5 with the corresponding domains from trkB or trkA were
made (Urfer et al., EMBO J. 14:2795-2805 [1995]). Wild type trkC
and wild type trkA were used as positive and negative controls
respectively. The domain-swap mutants of trkC are designated
according to the source of the replaced domain. For example, s1B
has D1 domain from trkB, s4B has D4 domain from trkB, s5B has D5
domain from trkB, and s5A has D5 domain from trkA. All of the
mutants were expressed as immunoadhesin, i.e. fused to IgG, and
purified.
[0264] The binding of each of the agonist anti-trkC monoclonal
antibody to various domain-swap mutants was evaluated by ELISA.
F(ab').sub.2 fragment from goat anti-human IgG was used for coating
microtiter plates to capture serial dilutions (100 .mu.g/ml to 2.4
ng/ml, 100 .mu.l/well, one hour at room temperature) of
immunoadhesins (trkC-IgG, trkA-IgG and domain-swap mutants of trkC
as immunoadhesin). Either unlabeled human or biotinylated murine
anti-trkC monoclonal antibodies were added (100 .mu.l per well; 1
.mu.g/ml, one hour at room temperature) to the plates containing
immobilized immunoadhesins, washed to remove unbound excess
reagents, and incubated with goat anti-human .kappa. or
streptavidin conjugated to HRP. As shown in FIG. 6B, all human
anti-trkC monoclonal antibodies were able to recognize trkC
domain-swap mutants with replacement of domain D1 or D4. However,
replacement of domain D5 with the corresponding domain derived from
either trkB or trkA destroyed recognition by anti-trkC antibodies.
The extent of binding was reduced to the same low level as that
observed with a negative control, trkA. The results suggest that
all the human anti-trkC monoclonal antibodies tested recognize an
epitope located somewhere in domain D5.
[0265] Similar analysis was performed with murine anti-trkC
monoclonal antibodies essentially the same way except the secondary
antibody used was goat anti-mouse IgG Fc coupled to HRP. As with
the human anti-trkC antibodies, the replacement of domain D5
abolished binding to all the murine anti-trkC monoclonal antibodies
tested (FIG. 6B). Additionally, the replacement of domain D4 also
destroyed the binding of 2248 murine anti-trkC antibody. The human
as well as murine anti-trkC agonist monoclonal antibodies all seem
to recognize an epitope in domain 5 with the exception of 2248
murine antibody, which seems to additionally recognize a
determinant in domain 4. It appears that 2248 epitope may be a
linear epitope overlapping the boundary of domain 4 and 5.
Alternatively, 2248 antibody might recognize a secondary structure
formed by discontiguous epitope with determinants derived from both
domain 4 and domain 5. Interestingly, Urfer et al. (J. Biol. Chem.
273: 5829-5840 [1998]) have earlier established the prominent role
of domain 5 in trkC receptor for mediating the interaction with
NT-3. Surprisingly, the antibodies described herein also bind to an
epitope of trkC which is largely overlapping with that recognized
by NT-3. This is surprising because of the relative sizes and
shapes of NT-3 and immunoglobulin molecules. The likely mode of
action of these activators is to crosslink the extracellular
domains of two trkC molecules in such a way to bring together their
intracellular tyrosine kinase domains and cross phosphorylate and
activate them.
[0266] In homodimeric NT-3, it has been established that the two
areas of the molecule which interact with trkC are diametrically
opposed on opposite sides of the molecule, 180 degrees apart from
each other. The distance between these areas is on the order of 16
A. On the other hand, the two trkC interacting sites in the
immunoglobulin molecules described here are not diametrically
opposed. In addition to displaying the trkC binding domains at a
different angle than NT-3, immunoglobulins will have the trkC
binding domains separated from each other by a much wider distance
than they are in NT-3. This will vary with the exact angle of the
two Fab domains, but is in the range of 50 A to 150 A. It would
have been difficult to have foreseen that two such very different
crosslinkers as NT-3 and the agonist Mabs act as agonists when
bound to the same site on trkC.
[0267] Site-Directed Mutagenesis
[0268] Site-directed mutagenesis approach was used to determine the
contribution of selected individual amino acid residues of domain 5
in the recognition by anti-trkC antibodies. FIG. 7 shows the amino
acid sequence of human trkC domains 4 and 5. All dotted residues
were mutagenized to alanine except residues L284, L286 and E287
which were changed to E, H, and K respectively (Urfer et al., J.
Biol. Chem. 273: 5829-5840 [1998]). A total of 26 single amino acid
mutations were made and evaluated for their effect on binding to
anti-trkC monoclonal antibodies. The values shown in Table 5
represent the ratio of binding to anti-trkC antibody of mutant vs
wildtype trkC. In order to minimize variation and provide effective
comparison, EC50 values were determined for each mutant for each
antibody and divided by the EC50 value obtained with wildtype
trkC.
TABLE-US-00005 TABLE 5 ##STR00001##
[0269] The gray areas indicate that the designated mutants did not
have an initial effect on monoclonal antibody binding, and were
therefore not re-assayed. Mutations that completely obliterated
monoclonal antibody binding are shown as NB ("no binding
observed"). The analysis indicates the major contribution of amino
acid residues L284, E287 and N335 of trkC in recognition by
anti-trkC agonist monoclonal antibodies tested. A model of the
complex of trkC domain 5 with NT3 shows the position of these
residues in close contact with CDRs of antibody (FIG. 8). This
model is based on the crustal structure of the complex of trkA
domain 5 with NGF. For further details see, e.g. Urfer et al. J.
Biol. Chem. (1998), supra, or Ultsch et al., J. Mol. Biol.
290:149-159 (1999).
[0270] Cloning and Sequencing of Antibody Variable Regions
[0271] In order to better understand the molecular basis of
interaction between trkC and anti-trkC monoclonal antibodies, the
heavy and light chain variable sequences of agonist antibodies were
cloned and DNA sequence determined. Total RNA was isolated from
hybridoma cells producing the human and murine anti-trkC antibodies
using RNA isolation kit from Stratagene (La Jolla, Calif.). RNA was
reverse transcribed into cDNA using SuperScript II system (Life
Technologies, Inc., Gaithersburg, Md.) and specific 3' primers
based on framework 4 sequences derived from the respective heavy or
light chain subgroup (Kabat and Wu, J. Immunol. 147: 1709-1719
[1991]). Subsequent PCR amplification was performed using AmpliTaq
DNA polymerase (Perkin Elmer, Foster City, Calif.) in presence of
2.5 M DMSO with specific forward primers based on the N-terminal
amino acid sequences of heavy and light chains and the same 3'
primers used for cDNA synthesis. PCR products were subcloned into
an F(ab)'.sub.2 vector containing both human heavy and light chain
constant regions (Carter et al., Bio/Technology 10: 163-167
[1992]). Five clones each of the V.sub.H and V.sub.L domains were
sequenced and a consensus sequence was obtained.
[0272] FIG. 9 shows the deduced amino acid sequences of heavy chain
of anti-trkC agonist monoclonal antibodies (2250, SEQ ID NO: 42;
2253, SEQ ID NO: 43; 2256, SEQ ID NO: 44; 6.1.2, SEQ ID NO: 45;
6.4.1, SEQ ID NO: 46; 2345, SEQ ID NO: 47; and 2349, SEQ ID NO:
48). The deduced amino acid sequences of light chain of anti-trkC
agonist monoclonal antibodies are shown in FIG. 10 (2250, SEQ ID
NO: 49; 2253, SEQ ID NO: 50; 2256, SEQ ID NO: 51; 6.1.2, SEQ ID NO:
53; 6.4.1, SEQ ID NO: 53; 2345, SEQ ID NO: 54; and 2349, SEQ ID NO:
55). In both FIG. 9 and FIG. 10 the Complementarity Determining
Regions (CDRs) are labeled as CDR1, CDR2 and CDR3, and the
corresponding amino acid residues are shown in bold face. FIG. 11
summarizes the sequences of CDRs of heavy chain as well as light
chain of various anti-trkC monoclonal antibodies along with
designation of respective heavy and light chain variable family to
which they belong.
[0273] Based on the determined amino acid sequences of the CDRs of
the heavy and light chains of the anti-trkC agonist monoclonal
antibodies, it is possible to provide a general formula for several
of these regions. For the murine antibodies, the heavy chain CDR1
may be represented by the formula XaaWXaaXaaWVK (SEQ ID NO:37),
wherein Xaa at position 1 is F or Y, Xaa at position 3 is I or M
and Xaa at position 4 is E or H. The murine heavy chain CDR2 may be
represented by the formula EIXaaPXaaXaaXaaXaaTNYNEKFKXaa (SEQ ID
NO:38), wherein Xaa at position 3 is L or Y, Xaa at position 5 is G
or S, Xaa at position 6 is S or N, Xaa at position 7 is D or G, Xaa
at position 8 is N or R and Xaa at position 17 is G or S. The
murine heavy chain CDR3 may be represented by the formula
KNRNYYGNYVV (SEQ ID NO: 12) or KYYYGNSYRSWYFDV (SEQ ID NO:13). For
the human antibodies, the heavy chain CDR1 may be represented by
the formula XaaXaaXaaYYWXaa (SEQ ID NO:39), wherein Xaa at position
1 is S or I, Xaa at position 2 is G or S and Xaa at position 3 is
G, T or Y and Xaa at position 7 is S or N. The human heavy chain
CDR2 may be represented by the formula XaalXaaXaaSGSXaaTXaaNPSLKS
(SEQ ID NO:40), wherein Xaa at position 1 is Y or R, Xaa at
position 3 is Y or F, Xaa at position 4 is Y or T, Xaa at position
8 is S or R and Xaa at position 10 is N or Y. The human heavy chain
CDR3 may be represented by DRDYDSTGDYYSYYGMDV (SEQ ID NO:14),
DGGYSNPFD (SEQ ID NO:15) or the formula ERIAAAGXaaDYYYNGLXaaV (SEQ
ID NO:41) wherein Xaa at position 8 is A or T and Xaa at position
16 is D or A.
[0274] The deduced amino acid sequence of heavy and light chain
variable regions was confirmed by determination of N-terminal
peptide sequence of these antibodies. Electroblotting onto
Millipore Immobilon-PSQ membranes was carried out for 1 hr at 250
mA constant current in a BioRad Trans-Blot transfer cell
(Matsudaira, J. Biol. Chem. 262:10035-10038 [1987]). The PVDF
membrane was stained with 0.1% Coomassie Blue R-250 in 50%
methanol, 0.5 min. and destained for 2-3 min. with 10% acetic acid
in 50% methanol. The membrane was thoroughly washed with water and
allowed to dry before storage at 20.degree. C. Automated protein
sequencing was performed on model 494A Perkin-Elmer sequencer
(Perkin-Elmer Corporation, Foster City, Calif.) equipped with
on-line PTH analyzer. Protein electroblotted onto PVDF membrane
were sequenced in 6 mm micro glass cartridge. Peaks were integrated
with Justice Innovation software using Nelson Analytical 760
interfaces. Sequence interpretation was performed on a DEC Alpha
(Henzel et al., J. Chromatography 404: 41-52 [1987]). Table 6
summarizes the classification of human and murine anti-trkC agonist
monoclonal antibodies based on their N-terminal sequences.
TABLE-US-00006 TABLE 6 Heavy chain Light chain Human anti-trkC
agonist mAbs 6.1.2 Subgroup II Kappa I 6.4.1 Subgroup II Kappa I
2345 Subgroup II Kappa III 2349 Subgroup II Kappa III 2.5.1
Subgroup II Kappa I 2344 Subgroup II Kappa I Murine anti-trkC
agonist mAbs 2248 Subgroup IIA Kappa I 2250 Subgroup IIA Kappa I
2253 Subgroup IIA Kappa IV 2256 Subgroup IIA Kappa III
Example 2
Effect of Agonist anti-trkC Monoclonal Antibodies on Neuropathies
in Experimental Animal Model
[0275] The principal use of NT-3 agonists is in the treatment
and/or prevention of peripheral neuropathies. It is known that
large fiber myelinated sensory neurons, which are involved in
mediating proprioception and vibration sense, express trkC that
acts as a high affinity receptor for NT-3. Neuropathies involving
these large fibers are common in diabetes and are also induced in
response to certain chemotherapeutic agents particularly cisplatin
and pyridoxine. NT-3 has shown efficacy in animal models of
experimental diabetic neuropathy and cisplatin induced neuropathy.
However, the use of NT-3 is severely hampered by its poor
bioavailability as shown in a rodent model. The use of anti-trkC
monoclonal antibodies as agonist of NT-3 offers numerous advantages
and obviates a number of potential problems associated with the use
of NT-3.
[0276] The in vivo half-life of agonist anti-trk monoclonal
antibodies was determined by injecting either intravenously or
subcutaneously in experimental animals. Shown on FIG. 12 are serum
levels of monoclonal antibody 2256 at various times after
intravenous (IV) injection of 1 mg/kg or subcutaneous (subQ)
injection of 5 mg/kg in rats. The serum levels were determined by
using the KIRA assay to measure the amount of fully functional
antibody 2256 by its ability to increase tyrosine
autophosphorylation of trkC. These data indicate that monoclonal
antibody 2256 in the rat has a half-life of 9 days and a
bioavailability of 69% after subcutaneous administration. These
values are consistent with those obtained with other antibodies,
and are distinctly different from those obtained with NT3. Also
shown in FIG. 12 is data obtained after injection of NT-3 at the
same doses and routes as shown for Mab 2256 (1 mg/kg, IV; 5 mg/kg
subQ). These data indicate a serum half-life on the order of 4-5
minutes for NT-3, and a subcutaneous bioavailability of 7%. These
data indicate that the antibodies are a significant improvement
over NT-3 in terms of the very important properties of
bioavailability and in vivo serum half-life.
[0277] It has been shown in two animal models of large fiber
sensory neuropathy that NT-3 can protect or reverse the effects of
chemical insult. Very high doses of NT3 have been shown to protect
large fiber sensory neurons from the toxic effects of high doses of
pyridoxine, and more moderate doses of NT3 have been shown to
reverse the effects of cisplatinum administration. Since there
might be many differences in the tissue distribution of NT-3 and
the agonist Mabs described here, it is important to determine
whether the in vitro activity of the Mabs translates into efficacy
in animal models.
[0278] In order to create an animal model of cisplatinum induced
neuropathy, adult rats were dosed with cisplatinum twice a week for
sixteen weeks with 1 mg/kg intraperitoneally (IP). At this point,
rats were split into four groups. All four groups continued
receiving cisplatinum twice weekly. In addition to the continued
cisplatinum, one group received NT-3 at a dose of 1 mg/kg, three
times per week, one group received Mab 2256 at a dose of 1 mg/kg
once a week, one group received Mab 6.4.1 at a dose of 1 mg/kg once
a week, and one group received saline three times a week. The NT-3
doses were given subcutaneously, while the Mabs and saline were
administered IV. This treatment regime was continued for an
additional four weeks, for a total of twenty weeks of cisplatinum
administration.
[0279] The function of large fiber sensory neurons was assessed in
these animals electrophysiologically, by use of H-wave recording
(Gao et al., Ann. Neurol. 38(1):30-7 [1995]). As can be seen from
the data shown in FIG. 13, the sensory conduction velocity was very
low in the animals treated with cisplatinum with saline alone. NT-3
treatment three times a week caused an improvement of this lowered
conduction velocity, as did treatment with either Mab 2256 or Mab
6.4.1 once a week. The magnitude of the improvement seen with the
monoclonal antibodies used once a week was at least as great as
that seen with three times a week treatment with NT-3.
[0280] Pyridoxine is also known to induce a sensory neuropathy that
primarily damages the large myelinated subpopulation of sensory
neurons (Helgren et al., J. Neurosci. 17(1):372-82 [1997]). High
doses of NT3 have been shown to block the development of this
neuropathy (Helgren et al., supra). Treatment of animals with two
different doses of pyridoxine (either 400 mg/kg or 600 mg/kg daily,
IP) for two weeks causes damage to the large neurons of the DRG.
This damage can be detected by a decrease in the expression of
several proteins known to be expressed either preferentially or
exclusively by large neurons in the DRG. The expression level of
these markers was assessed by measuring the level of the mRNA
encoding them by use of the TAQMAN RT-PCR technique.
[0281] Taqman RT-PCR for trkC Agonist Effects:
A. Probes and Primers
TABLE-US-00007 [0282] NFL (SEQ ID NO: 72) F-CAGCAGAACAAGGTCCTGGAA
21 MER (SEQ ID NO: 73) R-AGCGGGAAGGCTCTGAGTG 19 MER (SEQ ID NO: 74)
P-AGCTGTTGGTGCTGCGCCAGAA 22 MER NSE (SEQ ID NO: 75)
F-TCCATTGAAGACCCATTCGAC 21 MER (SEQ ID NO: 76)
R-GCCGACATTGGCTGTGAAC 19 MER (SEQ ID NO: 77)
P-AGGATGACTGGGCAGCTTGGTCCA 24 MER TRKC (SEQ ID NO: 78)
F-CAGCCCACTGCACCATATCA 20 MER (SEQ ID NO: 79) R-CTGTATCCGGCCCAGCAT
18 MER (SEQ ID NO: 80) P-CCATGGCATCACTACACCTTCATCGCT 27 MER CALRET
(SEQ ID NO: 81) F-TGGGAAAATTGAGATGGCAGA 21 MER (SEQ ID NO: 82)
R-GCTGCCTGAAGCACAAAAGG 20 MER (SEQ ID NO: 83)
P-CGCAGATCCTGCCMCCGAAGAGA 24 MER PARVALB. (SEQ ID NO: 84)
F-GACACCACTCTTCTGGAAAATGC 23 MER (SEQ ID NO: 85)
R-TTGCCAAACCAACACCTACCA 21 MER (SEQ ID NO: 86)
P-ATCGGACACCACCTGTAGGGAGGACC 26 MER GAPDI-I (SEQ ID NO: 87)
F-CAGTGGCAAAGTGGAGATTGT 21 MER (SEQ ID NO: 88)
R-AATTTGCCGTGAGTGGAGTC 20 MER (SEQ ID NO: 89)
P-CCATCAACGACCCCTTCATTGACCTC 26 MER
[0283] Probes and primers were designed using Primer Express,
(ABI-Perkin-Elmer). Guidelines for primer probe selection are
included in Williams and Tucker (1999) PCR applications, pp. 365-75
(Academic Press).
B. Total RNA Preparation and Quantification
[0284] L4 and L5 were dissected from phosphate buffered saline
perfused rats. Left and right sides were isolated in separate
tubes. For total RNA used in standard curves, all DRG were
dissected from control rats. Total RNA was isolated using the
Qiagen Rneasy mini columns. Tissue was homogenized as per
manufacturers instructions. Total RNA was quantified utilizing the
Ribogreen Quantitation Kit (Molecular Probes) and following the
manufacturers instructions.
C. RT-PCR
[0285] Twenty five nanograms of total RNA was used per 50 ul
reaction, except in standard curve reactions where 500, 250, 25 or
2.5 nanograms per reaction was used. Each reaction contained 25
.mu.mol of each oligonucleotide primer, 0.2 mM of each dNTP, 100 nM
fluorescently labelled oligonucleotide probe, 1.times.RT-PCR buffer
(PE biosystems), 2.0 mM MgCl.sub.2, 20 U RNAse inhibitor, 12.5 MuLV
reverse transcriptase (RT, PE biosystems) and 2.5 U Amplitaq Gold
polymerase (PE biosystems). Reverse transcription was performed for
30 min at 48 degrees C. followed by 95 degrees C. for 10 min for
Amplitaq Gold activation and RT inactivation, then PCR; 40 cycles
of 95 degrees C. for 15 sec and 60 degrees C. for one and a half
minutes.
D. Gene Expression Quantitation
[0286] Control RNA was used to generate standard curves for a
housekeeping gene and the genes of interest with 5 each taqman run.
A standard curve was obtained by plotting the threshold cycle (Ct)
value obtained from the Taqman run versus the log of the quantity
of control total RNA added. The resultant linear equation was
solved for the log RNA value. Plugging in the experimental Ct value
produced the log of the experimental gene expression value. Ten
raised to the power of this value gives the experimental gene
expression in nanograms.
[0287] As can be seen from FIG. 14, pyridoxine treatment for two
weeks resulted in a dose dependent decrease in 10 neurofilament
light chain (NFL), neuron specific enolase (NSE), trkC, and
calretinin expression. Both the dose dependency and magnitude of
these decreases varies from marker to marker, indicating a
differential sensitivity of these proteins as markers of the
neuronal damage.
[0288] In FIG. 15 the results of treating animals with two doses of
Mab 2256 along with the low dose (400 mg/kg daily) of pyridoxine
are shown. NFL and NSE show a significant decrease in expression at
this level of 15 pyridoxine treatment. Cotreatment of animals with
5 mg/kg of Mab 2256 (subQ weekly) completely blocked this decrease
in expression. A Mab 2256 dose of 1 mg/kg had no appreciable effect
on the expression of these proteins. Neither trkC nor calretinin
expression is significantly affected by this low dose pyridoxine
treatment, but treatment with 5 mg/kg Mab 2256 actually increases
trkC expression over control level.
[0289] When animals are treated with the higher pyridoxine dose of
600 mg/kg daily, the expression of NFL, NSE and 20 calretinin falls
to very low levels, while trkC expression falls to about 50% of
control values (FIG. 16). Cotreatment with Mab 2256 at either 1
mg/kg or 5 mg/kg significantly but not completely blocks the
decrease in expression seen in trkC and calretinin. There is a
slight trend towards protection seen with NFL and NSE expression in
animals treated with Mab 2256, but it did not attain statistical
significance. Thus, using multiple biochemical markers of damage to
large sensory neurons, Mab 2256 is seen to be capable of
ameliorating the toxicity of pyridoxine treatment.
[0290] In order to examine the electrophysiolgical and behavioral
effects of pyridoxine neuropathy, rats were treated with twice
daily injections of 400 mg/kg pyridoxine for 8 days. The function
of their large diameter sensory afferents were tested
electrophysiologically by recording the M-wave (direct motor) and
H-wave (reflex sensory) response in the muscles of the foot after
stimulation of the sciatic nerve at the thigh and the calf (Gao et
al., Ann. Neurol. 38(1):30-7 [1995]). Treatment with pyridoxine for
8 days resulted in a large decrease in the amplitude of the sensory
response compared to the motor response as seen in FIG. 18.
Cotreatment with Mab 2256 significantly blocked the
pyridoxine-induced decrease in the sensory amplitude. This is
similar to effects published using very high doses (20 mg/kg daily)
of NT3 (Helgren et al., supra).
[0291] Animals treated with this regime of pyridoxine were also
behaviorally tested for their proprioceptive function.
[0292] They were trained to walk across a horizontal ladder in
order to escape a bright light and white noise stimulus into a dark
box. The animals were videotaped from below, and the quality of the
placement of their hindpaws on the rungs of the ladder was read by
an observer blind to their treatment. Each paw placement was scored
as a good placement (paw lands on forward part of metatarsals,
immediately behind toes, with toes wrapping the rung immediately),
solid landing (paw hits other than immediately behind toes, but
solidly on rung, toes often not wrapping), near footfault (paw
barely hits rung, either on the extreme forward part of toes or
rearward aspect of heel, but does support weight) or footfault (paw
either misses rung entirely or poor enough placement that foot does
not support weight and falls through ladder upon weight bearing).
Normal rats very quickly learn to place their hindpaws correctly,
which requires an excellent proprioceptive sense of where the
hindpaw is in space. After treatment with pyridoxine (400 mg/kg
twice daily for 8 days), the performance on this task had declined,
with an almost thirty percent decline in good placements and an
increase in both footfaults and near footfaults (FIG. 18).
Cotreatment of the animals with Mab 2256 during this time, allowed
the animals to maintain a much higher degree of performance, with a
smaller decline in good placements and smaller increases in
footfaults and near footfaults.
[0293] In summary, cotreatment with Mab 2256 ameliorates the toxic
effects of pyridoxine as measured biochemically,
electrophysiologically, and by performance on a behavioral
task.
[0294] After establishing that the trkC Mabs were therapeutically
at least as effective as NT-3, the observed adverse event of
hyperalgesia was examined. This side effect of NT-3 administration
has been seen in rodents (see FIG. 19) and in humans (Chaudhary et
al., Muscle and Nerve 23:189-192 [2000]). Rats were trained and
tested for thermal sensitivity of the hind paws using a Hargreaves
device and then administered 1 mg/kg of Mab 2256 IV, or 1 mg/kg
NT-3 subcutaneously in the scruff. At two, four, and six hours
after administration, the rats were again tested for their thermal
withdrawal times. As can be seen from FIG. 19, NT-3 administration
caused a significant heat hyperalgesia at four and six hours post
dosing, while the trkC Mab 2256 was without any effect on thermal
pain sensation. So, at doses known to be effective in reversing or
preventing neuropathy, NT-3 does cause an increase in sensitivity
to pain, while the Mab 2256 does not.
[0295] Cisplatin, a widely used chemotherapeutic agent, induces a
sensory neuropathy with selective loss of vibration sense and
proprioception. Here we demonstrate that neurotrophin-3 (NT-3), a
member of the nerve growth factor family of neurotrophic factors,
restored to normal levels the reduced H-reflex-related sensory
nerve conduction velocity induced by cisplatin in rats. NT-3
treatment corrected an abnormal cytoplasmic distribution of
neurofilament protein in large sensory neurons in dorsal root
ganglia and the reduction in the numbers of myelinated fibers in
sural nerves caused by cisplatin. The NT-3-dependent reversal of
cisplatin neurotoxicity thus suggests the possible use of NT-3 in
the treatment of peripheral sensory neuropathy.
[0296] Chronic treatment of adult rats for 2-3 weeks with high
doses of pyridoxine (Vitamin B6) produced a profound proprioceptive
loss, similar to that found in humans overdosed with this vitamin
or treated with the chemotherapeutic agent cisplatin. Pyridoxine
toxicity was manifest as deficits in simple and precise locomotion
and sensory nerve function and as degeneration of
large-diameter/large-fiber spinal sensory neurons. As assessed
quantitatively in a beam-walking task and by EMG recording of H
waves evoked by peripheral nerve stimulation, coadministration of
the neurotrophic factor neurotrophin-3 (NT-3; 5-20 mg/kg/day, s.c.)
during chronic pyridoxine treatment largely attenuated the
behavioral and electrophysiological sequelae associated with
pyridoxine toxicity.
[0297] Furthermore, NT-3 administration prevented degeneration of
sensory fibers in the dorsal column of the spinal cord. These data
are consistent with the evidence that NT-3 is a target-derived
neurotrophic factor for muscle sensory afferents and suggest that
pharmacological doses of NT-3 may be beneficial in the treatment of
large-fiber sensory neuropathies.
Deposit of Biological Material
[0298] The following hybridoma cell lines and plasmids have been
deposited with the American Type Culture Collection; 10801
University Boulevard, Manassas, Va. 20110-2209, USA (ATCC) on Jun.
21, 2000:
TABLE-US-00008 Hybridoma/Plasmid Designation ATCC No. 2.5.1
PTA-2151 61.2 PTA-2148 6.4.1 PTA-2150 2344 PTA-2144 2345 PTA-2146
2349 PTA-2153 2248 PTA-2147 2250 PTA-2149 2253 PTA-2145 2256
PTA-2152 DNA pXCA-2250HL PTA-2136 DNA pXCA-2253HL PTA-2137 DNA
pXCA-2256HL PTA-2138 DNA pXCA-6.L2H PTA-2141 DNA pXCA-6.4.1H
PTA-2143 25 DNA pXCA-2345H PTA-2142 DNA pXCA-2349H PTA-2133 DNA
vegf4chim-6.1.2L PTA-2134 DNA vegf4chim-6.4.1L PTA-2135 DNA
vegf4chim-2345L PTA-2139 30 DNA vegf4chim-2349L PTA-2140
[0299] This deposit was made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty).
[0300] This assures maintenance of viable cultures for 30 years
from the date of the deposit. The organisms will be made available
by ATCC under the terms of the Budapest Treaty, and subject to an
agreement between Genentech, Inc. and ATCC, which assures permanent
and unrestricted availability of the progeny of the cultures to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn. 122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn.1.12
with particular reference to 886 OG 638).
[0301] In respect of those designations in which a European patent
is sought, a sample of the deposited microorganism will be made
available until the publication of the mention of the grant of the
European patent or until the date on which the application has been
refused or withdrawn or is deemed to be withdrawn, only by the
issue of such a sample to an expert nominated by the person
requesting the sample. (Rule 28(4) EPC)
[0302] The assignee of the present application has agreed that if
the cultures on deposit should die or be lost or destroyed when
cultivated under suitable conditions, they will be promptly
replaced on notification with a viable specimen of the same
culture. Availability of the deposited strain is not to be
construed as a license to practice the invention in contravention
of the rights granted under the authority of any government in
accordance with its patent laws.
[0303] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the constructs deposited, since the deposited embodiments are
intended to illustrate only certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that they represent. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
[0304] It is understood that the application of the teachings of
the present invention to a specific problem or situation will be
within the capabilities of one having ordinary skill in the art in
light of the teachings contained herein. Examples of the products
of the present invention and representative processes for their
isolation, use, and manufacture appear below, but should not be
construed to limit the invention.
[0305] All references cited throughout the specification and the
references cited therein are hereby expressly incorporated by
reference.
Sequence CWU 1
1
8917PRTMurine 1Phe Trp Ile Glu Trp Val Lys 1 527PRTMurine 2Tyr Trp
Met His Trp Val Lys 1 537PRTHomo sapiens 3Ser Gly Gly Tyr Tyr Trp
Ser 1 547PRTHomo sapiens 4Ile Ser Thr Tyr Tyr Trp Asn 1 557PRTHomo
sapiens 5Ser Gly Tyr Tyr Tyr Trp Ser 1 5617PRTMurine 6Glu Ile Leu
Pro Gly Ser Asp Asn Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10
15Gly717PRTMurine 7Glu Ile Tyr Pro Ser Asn Gly Arg Thr Asn Tyr Asn
Glu Lys Phe Lys 1 5 10 15Ser816PRTHomo sapiens 8Tyr Ile Tyr Tyr Ser
Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15916PRTHomo
sapiens 9Arg Ile Tyr Thr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu
Lys Ser 1 5 10 151016PRTHomo sapiens 10Tyr Ile Phe Tyr Ser Gly Arg
Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 151116PRTHomo sapiens
11Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1
5 10 151211PRTMurine 12Lys Asn Arg Asn Tyr Tyr Gly Asn Tyr Val Val
1 5 101315PRTMurine 13Lys Tyr Tyr Tyr Gly Asn Ser Tyr Arg Ser Trp
Tyr Phe Asp Val 1 5 10 151418PRTHomo sapiens 14Asp Arg Asp Tyr Asp
Ser Thr Gly Asp Tyr Tyr Ser Tyr Tyr Gly Met 1 5 10 15Asp
Val159PRTHomo sapiens 15Asp Gly Gly Tyr Ser Asn Pro Phe Asp 1
51617PRTHomo sapiens 16Glu Arg Ile Ala Ala Ala Gly Ala Asp Tyr Tyr
Tyr Asn Gly Leu Asp 1 5 10 15Val1717PRTHomo sapiens 17Glu Arg Ile
Ala Ala Ala Gly Thr Asp Tyr Tyr Tyr Asn Gly Leu Ala 1 5 10
15Val1815PRTMurine 18Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Tyr
Ser Tyr Met His 1 5 10 151910PRTMurine 19Ser Ala Ser Ser Ser Val
Ser Tyr Met Tyr 1 5 102015PRTMurine 20Arg Ala Ser Glu Ser Val Asp
Asn Tyr Gly Ile Ser Phe Met Asn 1 5 10 152111PRTHomo sapiens 21Arg
Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly 1 5 102217PRTHomo sapiens
22Lys Ser Ser Gln Ser Val Ser Tyr Ser Ser Asn Asn Lys Asn Tyr Leu 1
5 10 15Ala2312PRTHomo sapiens 23Arg Ala Ser Gln Ser Val Ser Ser Asn
Tyr Leu Thr 1 5 102412PRTHomo sapiens 24Arg Ala Ser Gln Ser Gly Ser
Ser Thr Tyr Leu Ala 1 5 10257PRTMurine 25Leu Val Ser Asn Leu Glu
Ser 1 5267PRTMurine 26Ser Thr Ser Asn Leu Ala Ser 1 5277PRTMurine
27Ala Ala Ser Asn Gln Gly Ser 1 5287PRTHomo sapiens 28Ala Ala Ser
Ser Leu Gln Ser 1 5297PRTHomo sapiens 29Trp Ala Ser Thr Arg Glu Ser
1 5307PRTHomo sapiens 30Gly Ala Ser Ser Arg Ala Thr 1 5319PRTMurine
31Gln His Ile Arg Glu Leu Thr Arg Ser 1 5329PRTMurine 32Gln Gln Arg
Ser Ser Tyr Pro Leu Thr 1 5339PRTMurine 33Gln Gln Ser Lys Glu Val
Pro Arg Thr 1 5349PRTHomo sapiens 34Leu Gln His Asn Ser Leu Pro Leu
Thr 1 5359PRTHomo sapiens 35Gln Gln His Tyr Asn Thr Pro Leu Thr 1
53610PRTHomo sapiens 36Gln Gln Tyr Gly Arg Ser Pro Pro Ile Thr 1 5
10377PRTMurineUNSURE1Xaa = F or Y 37Xaa Trp Xaa Xaa Trp Val Lys 1
53817PRTMurineUNSURE3Xaa = L or Y 38Glu Ile Xaa Pro Xaa Xaa Xaa Xaa
Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15Xaa397PRTHomo
sapiensUNSURE1Xaa = S or I 39Xaa Xaa Xaa Tyr Tyr Trp Xaa 1
54016PRTHomo sapiensUNSURE1Xaa = Y or R 40Xaa Ile Xaa Xaa Ser Gly
Ser Xaa Thr Xaa Asn Pro Ser Leu Lys Ser 1 5 10 154117PRTHomo
sapiensUNSURE8Xaa = A or T 41Glu Arg Ile Ala Ala Ala Gly Xaa Asp
Tyr Tyr Tyr Asn Gly Leu Xaa 1 5 10 15Val42122PRTMurine 42Asn Gln
Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Gln Pro Gly 1 5 10
15Ala Ser Val Lys Ile Ser Cys Lys Ser Thr Gly Tyr Thr Phe Ser Asn
20 25 30Phe Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu
Trp 35 40 45Ile Gly Glu Ile Leu Pro Gly Ser Asp Asn Thr Asn Tyr Asn
Glu Lys 50 55 60Phe Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser
Asn Thr Ala65 70 75 80Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr 85 90 95Cys Ala Arg Lys Asn Arg Asn Tyr Tyr Gly
Asn Tyr Val Val Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser
Ala Cys 115 12043122PRTMurine 43Asn Gln Val Gln Leu Gln Gln Ser Gly
Ala Glu Leu Met Gln Pro Gly 1 5 10 15Ala Ser Val Lys Ile Ser Cys
Lys Ser Thr Gly Tyr Thr Phe Ser Asn 20 25 30Phe Trp Ile Glu Trp Val
Lys Gln Arg Pro Gly His Gly Leu Glu Trp 35 40 45Ile Gly Glu Ile Leu
Pro Gly Ser Asp Asn Thr Asn Tyr Asn Glu Lys 50 55 60Phe Lys Gly Lys
Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala65 70 75 80Tyr Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 85 90 95Cys
Ala Arg Lys Asn Arg Asn Tyr Tyr Gly Asn Tyr Val Val Trp Gly 100 105
110Ala Gly Thr Thr Leu Thr Val Ser Ser Cys 115 12044126PRTMurine
44Asn Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly 1
5 10 15Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Ser 20 25 30Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu Trp 35 40 45Ile Gly Glu Ile Tyr Pro Ser Asn Gly Arg Thr Asn Tyr
Asn Glu Lys 50 55 60Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser
Ser Ser Thr Ala65 70 75 80Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu
Asp Ser Ala Val Tyr Tyr 85 90 95Cys Ala Arg Lys Tyr Tyr Tyr Gly Asn
Ser Tyr Arg Ser Trp Tyr Phe 100 105 110Asp Val Trp Gly Ala Gly Thr
Thr Leu Thr Val Ser Ser Cys 115 120 12545130PRTHomo sapiens 45Asn
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10
15Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser
20 25 30Gly Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly
Leu 35 40 45Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr
Asn Pro 50 55 60Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser
Lys Asn Gln65 70 75 80Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Arg Asp Arg Asp Tyr Asp Ser
Thr Gly Asp Tyr Tyr Ser 100 105 110Tyr Tyr Gly Met Asp Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser 115 120 125Ser Cys 13046119PRTHomo
sapiens 46Asn Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg
Pro Ser 1 5 10 15Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly
Ser Ile Ser Thr 20 25 30Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Ala Gly
Lys Gly Leu Glu Trp 35 40 45Ile Gly Arg Ile Tyr Thr Ser Gly Ser Thr
Asn Tyr Asn Pro Ser Leu 50 55 60Lys Ser Arg Val Thr Met Ser Val Asp
Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Gly Tyr
Ser Asn Pro Phe Asp Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val
Ser Ser Cys 11547129PRTHomo sapiens 47Asn Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15Gln Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser 20 25 30Gly Gly Tyr Tyr
Trp Ser Trp Ile Arg Gln His Pro Glu Lys Gly Leu 35 40 45Glu Trp Ile
Gly Tyr Ile Phe Tyr Ser Gly Arg Thr Tyr Tyr Asn Pro 50 55 60Ser Leu
Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln65 70 75
80Phe Ser Leu Lys Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
85 90 95Tyr Cys Ala Arg Glu Arg Ile Ala Ala Ala Gly Ala Asp Tyr Tyr
Tyr 100 105 110Asn Gly Leu Asp Val Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser 115 120 125Cys48129PRTHomo sapiens 48Asn Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15Gln Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser 20 25 30Gly
Tyr Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu 35 40
45Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro
50 55 60Ser Leu Lys Ser Arg Leu Thr Ile Ser Val Asp Thr Ser Lys Asn
Gln65 70 75 80Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
Ala Val Tyr 85 90 95Tyr Cys Ala Arg Glu Arg Ile Ala Ala Ala Gly Thr
Asp Tyr Tyr Tyr 100 105 110Asn Gly Leu Ala Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120 125Cys49112PRTMurine 49Asn Asp Ile
Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu 1 5 10 15Gly
Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr 20 25
30Ser Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro
35 40 45Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val
Pro 50 55 60Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Asn Ile65 70 75 80His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr
Cys Gln His Ile 85 90 95Arg Glu Leu Thr Arg Ser Ala Arg Gly Gln Ser
Trp Lys Lys Arg Cys 100 105 11050109PRTMurine 50Asn Gln Ile Val Leu
Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro 1 5 10 15Gly Glu Lys
Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr 20 25 30Met Tyr
Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile 35 40 45Tyr
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala65
70 75 80Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro
Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Cys 100
10551114PRTMurine 51Asn Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu 1 5 10 15Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala
Ser Glu Ser Val Asp Asn 20 25 30Tyr Gly Ile Ser Phe Met Asn Trp Phe
Gln Gln Lys Pro Gly Gln Pro 35 40 45Pro Lys Leu Leu Ile Tyr Ala Ala
Ser Asn Gln Gly Ser Gly Val Pro 50 55 60Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Ser Leu Asn Ile65 70 75 80His Pro Met Glu Glu
Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser 85 90 95Lys Glu Val Pro
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys 100 105 110Arg
Cys52110PRTHomo sapiens 52Asn Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val 1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Gly Ile Arg Asn 20 25 30Asp Leu Gly Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Arg Leu 35 40 45Ile Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp
Phe Ala Thr Phe Tyr Cys Leu Gln His Asn Ser Leu Pro 85 90 95Leu Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Cys 100 105
11053116PRTHomo sapiens 53Asn Asp Ile Gln Met Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu 1 5 10 15Gly Glu Arg Ala Thr Ile Asn Cys
Lys Ser Ser Gln Ser Val Ser Tyr 20 25 30Ser Ser Asn Asn Lys Asn Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40 45Gln Pro Pro Lys Leu Leu
Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly 50 55 60Val Pro Asp Arg Ile
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu65 70 75 80Thr Ile Ser
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln 85 90 95Gln His
Tyr Asn Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu 100 105
110Ile Lys Arg Cys 11554112PRTHomo sapiens 54Asn Glu Ile Val Leu
Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro 1 5 10 15Gly Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser 20 25 30Asn Tyr
Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45Leu
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe 50 55
60Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu65
70 75 80Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Arg
Ser 85 90 95Pro Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Arg Cys 100 105 11055112PRTHomo sapiens 55Asn Gly Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro 1 5 10 15Gly Glu Arg Ala
Thr Phe Ser Cys Arg Ala Ser Gln Ser Gly Ser Ser 20 25 30Thr Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45Leu Ile
Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe 50 55 60Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu65 70 75
80Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Arg Ser
85 90 95Pro Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg
Cys 100 105 11056808PRTHomo sapiens 56Cys Pro Ala Asn Cys Val Cys
Ser Lys Thr Glu Ile Asn Cys Arg Arg 1 5 10 15Pro Asp Asp Gly Asn
Leu Phe Pro Leu Leu Glu Gly Gln Asp Ser Gly 20 25 30Asn Ser Asn Gly
Asn Ala Asn Ile Asn Ile Thr Asp Ile Ser Arg Asn 35 40 45Ile Thr Ser
Ile His Ile Glu Asn Trp Arg Ser Leu His Thr Leu Asn 50 55 60Ala Val
Asp Met Glu Leu Tyr Thr Gly Leu Gln Lys Leu Thr Ile Lys65 70 75
80Asn Ser Gly Leu Arg Ser Ile Gln Pro Arg Ala Phe Ala Lys Asn Pro
85 90 95His Leu Arg Tyr Ile Asn Leu Ser Ser Asn Arg Leu Thr Thr Leu
Ser 100 105 110Trp Gln Leu Phe Gln Thr Leu Ser Leu Arg Glu Leu Gln
Leu Glu Gln 115 120 125Asn Phe Phe Asn Cys Ser Cys Asp Ile Arg Trp
Met Gln Leu Trp Gln 130 135 140Glu Gln Gly Glu Ala Lys Leu Asn Ser
Gln Asn Leu Tyr Cys Ile Asn145 150 155 160Ala Asp Gly Ser Gln Leu
Pro Leu Phe Arg Met Asn Ile Ser Gln Cys 165 170 175Asp Leu Pro Glu
Ile Ser Val Ser His Val Asn Leu Thr Val Arg Glu 180 185 190Gly Asp
Asn Ala Val Ile Thr Cys Asn Gly Ser Gly Ser Pro Leu Pro 195 200
205Asp
Val Asp Trp Ile Val Thr Gly Leu Gln Ser Ile Asn Thr His Gln 210 215
220Thr Asn Leu Asn Trp Thr Asn Val His Ala Ile Asn Leu Thr Leu
Val225 230 235 240Asn Val Thr Ser Glu Asp Asn Gly Phe Thr Leu Thr
Cys Ile Ala Glu 245 250 255Asn Val Val Gly Met Ser Asn Ala Ser Val
Ala Leu Thr Val Tyr Tyr 260 265 270Pro Pro Arg Val Val Ser Leu Glu
Glu Pro Glu Leu Arg Leu Glu His 275 280 285Cys Ile Glu Phe Val Val
Arg Gly Asn Pro Pro Pro Thr Leu His Trp 290 295 300Leu His Asn Gly
Gln Pro Leu Arg Glu Ser Lys Ile Ile His Val Glu305 310 315 320Tyr
Tyr Gln Glu Gly Glu Ile Ser Glu Gly Cys Leu Leu Phe Asn Lys 325 330
335Pro Thr His Tyr Asn Asn Gly Asn Tyr Thr Leu Ile Ala Lys Asn Pro
340 345 350Leu Gly Thr Ala Asn Gln Thr Ile Asn Gly His Phe Leu Lys
Glu Pro 355 360 365Phe Pro Glu Ser Thr Asp Asn Phe Ile Leu Phe Asp
Glu Val Ser Pro 370 375 380Thr Pro Pro Ile Thr Val Thr His Lys Pro
Glu Glu Asp Thr Phe Gly385 390 395 400Val Ser Ile Ala Val Gly Leu
Ala Ala Phe Ala Cys Val Leu Leu Val 405 410 415Val Leu Phe Val Met
Ile Asn Lys Tyr Gly Arg Arg Ser Lys Phe Gly 420 425 430Met Lys Gly
Pro Val Ala Val Ile Ser Gly Glu Glu Asp Ser Ala Ser 435 440 445Pro
Leu His His Ile Asn His Gly Ile Thr Thr Pro Ser Ser Leu Asp 450 455
460Ala Gly Pro Asp Thr Val Val Ile Gly Met Thr Arg Ile Pro Val
Ile465 470 475 480Glu Asn Pro Gln Tyr Phe Arg Gln Gly His Asn Cys
His Lys Pro Asp 485 490 495Thr Tyr Val Gln His Ile Lys Arg Arg Asp
Ile Val Leu Lys Arg Glu 500 505 510Leu Gly Glu Gly Ala Phe Gly Lys
Val Phe Leu Ala Glu Cys Tyr Asn 515 520 525Leu Ser Pro Thr Lys Asp
Lys Met Leu Val Ala Val Lys Ala Leu Lys 530 535 540Asp Pro Thr Leu
Ala Ala Arg Lys Asp Phe Gln Arg Glu Ala Glu Leu545 550 555 560Leu
Thr Asn Leu Gln His Glu His Ile Val Lys Phe Tyr Gly Val Cys 565 570
575Gly Asp Gly Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly
580 585 590Asp Leu Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Met
Ile Leu 595 600 605Val Asp Gly Gln Pro Arg Gln Ala Lys Gly Glu Leu
Gly Leu Ser Gln 610 615 620Met Leu His Ile Ala Ser Gln Ile Ala Ser
Gly Met Val Tyr Leu Ala625 630 635 640Ser Gln His Phe Val His Arg
Asp Leu Ala Thr Arg Asn Cys Leu Val 645 650 655Gly Ala Asn Leu Leu
Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp 660 665 670Val Tyr Ser
Thr Asp Tyr Tyr Arg Leu Phe Asn Pro Ser Gly Asn Asp 675 680 685Phe
Cys Ile Trp Cys Glu Val Gly Gly His Thr Met Leu Pro Ile Arg 690 695
700Trp Met Pro Pro Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu
Ser705 710 715 720Asp Val Trp Ser Phe Gly Val Ile Leu Trp Glu Ile
Phe Thr Tyr Gly 725 730 735Lys Gln Pro Trp Phe Gln Leu Ser Asn Thr
Glu Val Ile Glu Cys Ile 740 745 750Thr Gln Gly Arg Val Leu Glu Arg
Pro Arg Val Cys Pro Lys Glu Val 755 760 765Tyr Asp Val Met Leu Gly
Cys Trp Gln Arg Glu Pro Gln Gln Arg Leu 770 775 780Asn Ile Lys Glu
Ile Tyr Lys Ile Leu His Ala Leu Gly Lys Ala Thr785 790 795 800Pro
Ile Tyr Leu Asp Ile Leu Gly 805572607DNAHomo sapiens 57tgccctgcaa
attgtgtctg cagcaagact gagatcaatt gccggcggcc ggacgatggg 60aacctcttcc
ccctcctgga agggcaggat tcagggaaca gcaatgggaa cgccaatatc
120aacatcacgg acatctcaag gaatatcact tccatacaca tagagaactg
gcgcagtctt 180cacacgctca acgccgtgga catggagctc tacaccggac
ttcaaaagct gaccatcaag 240aactcaggac ttcggagcat tcagcccaga
gcctttgcca agaaccccca tttgcgttat 300ataaacctgt caagtaaccg
gctcaccaca ctctcgtggc agctcttcca gacgctgagt 360cttcgggaat
tgcagttgga gcagaacttt ttcaactgca gctgtgacat ccgctggatg
420cagctctggc aggagcaggg ggaggccaag ctcaacagcc agaacctcta
ctgcatcaat 480gctgatggct cccagcttcc tctcttccgc atgaacatca
gtcagtgtga ccttcctgag 540atcagcgtga gccacgtcaa cctgaccgta
cgagagggtg acaatgctgt tatcacttgc 600aatggctctg gatcacccct
tcctgatgtg gactggatag tcactgggct gcagtccatc 660aacactcacc
agaccaatct gaactggacc aatgttcatg ccatcaactt gacgctggtg
720aatgtgacga gtgaggacaa tggcttcacc ctgacgtgca ttgcagagaa
cgtggtgggc 780atgagcaatg ccagtgttgc cctcactgtc tactatcccc
cacgtgtggt gagcctggag 840gagcctgagc tgcgcctgga gcactgcatc
gagtttgtgg tgcgtggcaa ccccccacca 900acgctgcact ggctgcacaa
tgggcagcct ctgcgggagt ccaagatcat ccatgtggaa 960tactaccaag
agggagagat ttccgagggc tgcctgctct tcaacaagcc cacccactac
1020aacaatggca actataccct cattgccaaa aacccactgg gcacagccaa
ccagaccatc 1080aatggccact tcctcaagga gccctttcca gagagcacgg
ataactttat cttgtttgac 1140gaagtgagtc ccacacctcc tatcactgtg
acccacaaac cagaagaaga cacttttggg 1200gtatccatag cagttggact
tgctgctttt gcctgtgtcc tgttggtggt tctcttcgtc 1260atgatcaaca
aatatggtcg acggtccaaa tttggaatga agggtcccgt ggctgtcatc
1320agtggtgagg aggactcagc cagcccactg caccacatca accacggcat
caccacgccc 1380tcgtcactgg atgccgggcc cgacactgtg gtcattggca
tgactcgcat ccctgtcatt 1440gagaaccccc agtacttccg tcagggacac
aactgccaca agccggacac gtatgtgcag 1500cacattaaga ggagagacat
cgtgctgaag cgagaactgg gtgagggagc ctttggaaag 1560gtcttcctgg
ccgagtgcta caacctcagc ccgaccaagg acaagatgct tgtggctgtg
1620aaggccctga aggatcccac cctggctgcc cggaaggatt tccagaggga
ggccgagctg 1680ctcaccaacc tgcagcatga gcacattgtc aagttctatg
gagtgtgcgg cgatggggac 1740cccctcatca tggtctttga atacatgaag
catggagacc tgaataagtt cctcagggcc 1800catgggccag atgcaatgat
ccttgtggat ggacagccac gccaggccaa gggtgagctg 1860gggctctccc
aaatgctcca cattgccagt cagatcgcct cgggtatggt gtacctggcc
1920tcccagcact ttgtgcaccg agacctggcc accaggaact gcctggttgg
agcgaatctg 1980ctagtgaaga ttggggactt cggcatgtcc agagatgtct
acagcacgga ttattacagg 2040ctctttaatc catctggaaa tgatttttgt
atatggtgtg aggtgggagg acacaccatg 2100ctccccattc gctggatgcc
tcctgaaagc atcatgtacc ggaagttcac tacagagagt 2160gatgtatgga
gcttcggggt gatcctctgg gagatcttca cctatggaaa gcagccatgg
2220ttccaactct caaacacgga ggtcattgag tgcattaccc aaggtcgtgt
tttggagcgg 2280ccccgagtct gccccaaaga ggtgtacgat gtcatgctgg
ggtgctggca gagggaacca 2340cagcagcggt tgaacatcaa ggagatctac
aaaatcctcc atgctttggg gaaggccacc 2400ccaatctacc tggacattct
tggctagtgg tggctggtgg tcatgaattc atactctgtt 2460gcctcctctc
tccctgcctc acatctccct tccacctcac aactccttcc atccttgact
2520gaagcgaaca tcttcatata aactcaagtg cctgctacac atacaacact
gaaaaaagga 2580aaaaaaaaga aaaaaaaaaa aaaccgc 260758360DNAMurine
58caggtccaac tgcagcagtc tggggctgag ctgatgcagc ctggggcctc agtgaagata
60tcctgcaagt ctactggcta cacattcagt aacttctgga tagagtgggt aaagcagagg
120cctggacatg gccttgagtg gattggagag attttacctg gcagtgataa
tactaactac 180aatgagaagt tcaagggcaa ggccacattc actgcagata
catcctccaa cacagcctac 240atgcaactca gcagcctgac atctgaggac
tctgccgtct attactgtgc aagaaagaat 300cgtaactact atggtaacta
cgttgtatgg ggccaaggga ctctggtcac tgtctctgca 36059330DNAMurine
59gacattgtga tgacccagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc
60atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac
120caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa
cctagaatct 180ggggtccctg ccaggttcag tggcagtggg tctgggacag
acttcaccct caacatccat 240cctgtggagg aggaggatgc tgcaacctat
tactgtcagc acattaggga gcttacacgt 300tcggctcggg gacaaagttg
gaaaaaacgg 33060360DNAMurine 60caggtccagc tgcagcagtc tggagctgag
ctgatgcagc ctggggcctc agtgaagata 60tcctgcaagt ctactggcta cacattcagt
aacttctgga tagagtgggt aaagcagagg 120cctggacatg gccttgagtg
gattggagag attttacctg gcagtgataa tactaactac 180aatgagaagt
tcaagggcaa ggccacattc actgcagata catcctccaa cacagcctac
240atgcaactca gcagcctgac atctgaggac tctgccgtct attactgtgc
aagaaagaat 300cgtaactact atggtaacta cgttgtctgg ggcgcaggca
ccactctcac agtctcctca 36061321DNAMurine 61caaattgtgc tgacccagtc
tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60ataacctgca gtgccagctc
aagtgtaagt tacatgtact ggttccagca gaagccaggc 120acttctccca
aactctggat ttatagtaca tccaacctgg cttctggagt ccctgctcgc
180ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagccgaat
ggaggctgaa 240gatgctgcca cttattactg ccagcaaagg agtagttacc
cgctcacgtt cggtgctggg 300accaagctgg aactaaaacg g 32162372DNAMurine
62caggtccagc tgcagcagcc tggggctgaa ctggtgaagc ctggggcttc agtgaagctg
60tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg
120cctggacaag gccttgagtg gattggagag atttatccta gcaacggtcg
tactaactac 180aatgagaagt tcaagagcaa ggccacactg actgtagaca
aatcctccag cacagcctac 240atgcaactca gcagcctgac atctgaggac
tctgcggtct attactgtgc aagaaaatat 300tactacggta atagctatcg
ttcctggtac ttcgatgtct ggggcgcagg caccactctc 360acagtctcct ca
37263336DNAMurine 63gacattgtgc tgacccagtc tccagcttct ttggctgtgt
ctctagggca gagggccacc 60atctcctgca gagccagcga aagtgttgat aattatggca
ttagttttat gaactggttc 120caacagaaac caggacagcc acccaaactc
ctcatctatg ctgcatccaa ccaaggatcc 180ggggtccctg ccaggtttag
tggcagtggg tctgggacag acttcagcct caacatccat 240cctatggagg
aggatgatac tgcaatgtat ttctgtcagc aaagtaagga ggttcctcgg
300acgttcggtg gaggcaccaa gctggagatg aaacgg 33664381DNAHomo sapiens
64caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc
60acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc
120cagcacccag aaaagggcct ggagtggatt gggtacatct tttacagtgg
gaggacctac 180tacaacccgt ccctcaagag tcgagttacc atatcagtag
acacgtctaa gaaccagttc 240tccctgaagc tgaactctgt gactgccgcg
gacacggccg tgtattactg tgcgagagag 300cggatagcag cagctggtgc
ggactactac tacaacggtt tggacgtctg gggccaaggg 360accacggtca
ccgtctcctc a 38165330DNAHomo sapiens 65gaaattgtgt tgacgcagtc
tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca gggccagtca
gagtgttagc agcaactact taacctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca
180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag
cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagtatggtc
gctcacctcc gatcaccttc 300ggccaaggga cacgactgga gattaaacga
33066381DNAHomo sapiens 66caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcacagac cctgtccctc 60acctgcactg tctctggtgg ctccatcagc
agtggttatt attattggag ctggatccgc 120cagcacccag ggaagggcct
ggagtggatt gggtacatct attacagtgg gagcacctac 180tacaacccgt
ccctcaagag tcgacttacc atatcagtag acacgtctaa gaaccagttc
240tccctgaagc tgagctctgt gactgccgcg gacacggccg tgtattactg
tgcgagagag 300cggatagcag cagctggaac ggactactac tacaacggtt
tggccgtctg gggccaaggg 360accacggtca ccgtctcctc a 38167330DNAHomo
sapiens 67ggaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aagagccact 60ttctcctgca gggccagtca gagtggtagc agcacctact tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta ggtcacctcc gatcaccttc 300ggccaaggga
cacgactgga gattaaacga 33068384DNAHomo sapiens 68caggtgcagc
tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60acctgcactg
tctctggtgg ctccatcagc agtggtggtt actactggag ctggatccgc
120cagcacccag ggaagggcct ggagtggatt gggtacatct attacagtgg
gagcaccaac 180tacaacccgt ccctcaagag tcgagttacc atatcagtgg
acacgtctaa gaaccagttc 240tccctgaagc tgagctctgt gactgccgcg
gacacggccg tgtattactg tacgagagat 300cgggactatg atagtaccgg
ggattactac tcctactacg gtatggacgt ctggggccaa 360gggaccacgg
tcaccgtctc ctca 38469324DNAHomo sapiens 69gatatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca
gggcattaga aatgatttag gctggtatca gcagaaacca 120gggaaagccc
ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag
cctgcagcct 240gaagattttg caacttttta ctgtctacag cataatagtc
ttccgctcac tttcggcgga 300gggaccaagg tggagatcaa acga 32470354DNAHomo
sapiens 70caggtgcagc tgcaggagtc gggcccagga ctggtgaggc cttcggagac
cctgtccctc 60acctgcactg tctctggtgg ctccatcagt acttactact ggaactggat
ccggcagccc 120gccgggaagg gactggagtg gattgggcgt atctatacca
gtgggagcac caactacaac 180ccctccctca agagtcgagt caccatgtca
gtagacacgt ccaagaacca gttctccctg 240aagctgagct ctgtgaccgc
cgcggacacg gccgtgtatt actgtgcgag agatgggggc 300tacagtaacc
cttttgacta ctggggccag ggaaccctgg tcaccgtctc ctca 35471342DNAHomo
sapiens 71gatatccaga tgacccagtc tccagactcc ctggctgtgt ctctgggcga
gagggccacc 60atcaactgca agtccagcca gagtgtttca tacagctcca acaataagaa
ctacttagct 120tggtaccagc agaaacctgg acagcctcct aagctgctca
tttactgggc atctacccgg 180gaatccgggg tccctgaccg aatcagtggc
agcgggtctg ggacagattt cactctcacc 240atcagcagcc tgcaggctga
agatgtggca gtttattact gtcaacaaca ttataatact 300ccactcactt
tcggcggagg gaccaaggtg gagatcaaac ga 3427221DNAArtificial
SequenceSynthetic oligonucleotide 72cagcagaaca aggtcctgga a
217319DNAArtificial SequenceSynthetic oligonucleotide 73agcgggaagg
ctctgagtg 197422DNAArtificial SequenceSynthetic oligonucleotide
74agctgttggt gctgcgccag aa 227521DNAArtificial SequenceSynthetic
oligonucleotide 75tccattgaag acccattcga c 217619DNAArtificial
SequenceSynthetic oligonucleotide 76gccgacattg gctgtgaac
197724DNAArtificial SequenceSynthetic oligonucleotide 77aggatgactg
ggcagcttgg tcca 247820DNAArtificial SequenceSynthetic
oligonucleotide 78cagcccactg caccatatca 207918DNAArtificial
SequenceSynthetic oligonucleotide 79ctgtatccgg cccagcat
188027DNAArtificial SequenceSynthetic oligonucleotide 80ccatggcatc
actacacctt catcgct 278121DNAArtificial SequenceSynthetic
oligonucleotide 81tgggaaaatt gagatggcag a 218220DNAArtificial
SequenceSynthetic oligonucleotide 82gctgcctgaa gcacaaaagg
208324DNAArtificial SequenceSynthetic oligonucleotide 83cgcagatcct
gccaaccgaa gaga 248423DNAArtificial SequenceSynthetic
oligonucleotide 84gacaccactc ttctggaaaa tgc 238521DNAArtificial
SequenceSynthetic oligonucleotide 85ttgccaaacc aacacctacc a
218626DNAArtificial SequenceSynthetic oligonucleotide 86atcggacacc
acctgtaggg aggacc 268721DNAArtificial SequenceSynthetic
oligonucleotide 87cagtggcaaa gtggagattg t 218820DNAArtificial
SequenceSynthetic oligonucleotide 88aatttgccgt gagtggagtc
208926DNAArtificial SequenceSynthetic oligonucleotide 89ccatcaacga
ccccttcatt gacctc 26
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