U.S. patent application number 14/013707 was filed with the patent office on 2013-12-26 for compositions and methods for increasing muscle growth.
This patent application is currently assigned to Novartis AG. The applicant listed for this patent is Novartis AG. Invention is credited to Catrin Berger, Tanja Herrmann, Chris Lu, Kelly-Ann Sheppard, Estelle Trifilieff, Stephanie Urlinger.
Application Number | 20130344091 14/013707 |
Document ID | / |
Family ID | 42306628 |
Filed Date | 2013-12-26 |
United States Patent
Application |
20130344091 |
Kind Code |
A1 |
Berger; Catrin ; et
al. |
December 26, 2013 |
COMPOSITIONS AND METHODS FOR INCREASING MUSCLE GROWTH
Abstract
This disclosure is in the field of anti-Activin receptor IIB
(ActRIIB) antibodies. In particular, it relates to the use of said
antibodies for treating muscle disorders, such as muscle wasting
due to disease or disuse.
Inventors: |
Berger; Catrin; (Germering,
DE) ; Herrmann; Tanja; (Munchen, DE) ; Lu;
Chris; (Shanghai, CN) ; Sheppard; Kelly-Ann;
(Cambridge, MA) ; Trifilieff; Estelle; (Basel,
CH) ; Urlinger; Stephanie; (Munchen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG |
Basel |
|
CH |
|
|
Assignee: |
Novartis AG
Basel
CH
|
Family ID: |
42306628 |
Appl. No.: |
14/013707 |
Filed: |
August 29, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13485399 |
May 31, 2012 |
8551482 |
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14013707 |
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12767509 |
Apr 26, 2010 |
8388968 |
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13485399 |
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61173004 |
Apr 27, 2009 |
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61306137 |
Feb 19, 2010 |
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Current U.S.
Class: |
424/172.1 ;
435/320.1; 435/328; 435/69.6; 530/387.9; 530/389.1; 536/23.53 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 15/00 20180101; A61P 25/16 20180101; A61P 25/14 20180101; A61P
3/02 20180101; C07K 2317/52 20130101; A61P 21/02 20180101; C07K
2317/565 20130101; C07K 16/2863 20130101; C07H 21/04 20130101; C07K
2317/76 20130101; C07K 2317/92 20130101; A61P 19/02 20180101; A61P
43/00 20180101; C07K 16/28 20130101; A61P 13/12 20180101; A61P
19/10 20180101; A61P 21/04 20180101; A61P 35/00 20180101; C07K
2317/34 20130101; A61P 1/16 20180101; A61P 11/00 20180101; C07K
2317/21 20130101; C07K 2317/41 20130101; A61K 45/06 20130101; A61P
13/02 20180101; A61P 21/00 20180101; A61K 39/3955 20130101; A61P
19/00 20180101; A61P 7/06 20180101; C07K 2317/56 20130101; C07K
2317/55 20130101; A61K 2039/505 20130101; A61P 25/28 20180101 |
Class at
Publication: |
424/172.1 ;
530/389.1; 530/387.9; 536/23.53; 435/320.1; 435/328; 435/69.6 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395 |
Claims
1. An anti-ActRIIB therapeutic antibody or functional protein.
2. An anti-ActRIIB antibody or functional protein that binds to the
ligand binding domain of ActRIIB.
3. An anti-ActRIIB antibody or functional protein that binds to
ActRIIB between amino acids 19-134 of SEQ ID NO: 181.
4. An anti-ActRIIB antibody or functional protein according to
claim 1, which binds to ActRIIB with a K.sub.D of 1 nM or less,
preferably 100 pM or less.
5. An anti-ActRIIB antibody according to claim 1, wherein said
antibody inhibits myostatin binding to ActRIIB.
6. An anti-ActRIIB antibody according to claim 1, wherein said
antibody inhibits myostatin induced signalling as measured by a
Smad dependent reporter gene assay.
7. An anti-ActRIIB antibody according to claim 1, wherein said
antibody binds to ActRIIB with a 10-fold or greater affinity than
it binds to ActRIIA.
8. The antibody or functional protein according to claim 1, wherein
said antibody comprises at least one complementarity determining
region having at least 95% identity to at least one of the CDRs
recited in SEQ ID NOs: 1-84.
9. The antibody or functional protein according to claim 1, wherein
said antibody comprises at least one complementarity determining
region having at least 95% identity to at least one CDR3 recited in
SEQ ID NOs: 29-42.
10. The antibody or functional protein according to claim 1,
wherein said antibody comprises a VH polypeptide sequence having at
least 95% sequence identity to at least one of SEQ ID NOs:
99-112.
11. The antibody or functional protein according to claim 1,
wherein said antibody comprises a VL polypeptide sequence having at
least 95% sequence identity to at least one of SEQ ID NOs:
85-98.
12. The antibody or functional protein according to claim 1,
wherein said antibody comprises a VH polypeptide sequence having at
least 95% sequence identity to at least one of SEQ ID NOs: 99-112
and a VL polypeptide sequence having at least 95% sequence identity
to at least one of SEQ ID NOs: 85-98.
13. The antibody or functional protein according to claim 1,
wherein said antibody comprises a heavy chain variable region CDR1
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1-14; a heavy chain variable region CDR2
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 15-28; a heavy chain variable region CDR3
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 29-42; a light chain variable region CDR1
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 43-56; a light chain variable region CDR2
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 57-70; and a light chain variable region
CDR3 comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 71-84.
14. An antibody or functional protein according to claim 1
comprising: (a) a heavy chain variable region CDR1 of SEQ ID NO: 1;
a heavy chain variable region CDR2 of SEQ ID NO: 15; a heavy chain
variable region CDR3 of SEQ ID NO: 29; a light chain variable
region CDR1 of SEQ ID NO: 43; a light chain variable region CDR2 of
SEQ ID NO: 57; and a light chain variable region CDR3 of SEQ ID NO:
71, (b) a heavy chain variable region CDR1 of SEQ ID NO: 2 a heavy
chain variable region CDR2 of SEQ ID NO: 16; a heavy chain variable
region CDR3 of SEQ ID NO: 30; a light chain variable region CDR1 of
SEQ ID NO: 44; a light chain variable region CDR2 of SEQ ID NO: 58;
and a light chain variable region CDR3 of SEQ ID NO: 72, (c) a
heavy chain variable region CDR1 of SEQ ID NO: 3; a heavy chain
variable region CDR2 of SEQ ID NO: 17; a heavy chain variable
region CDR3 of SEQ ID NO: 31; a light chain variable region CDR1 of
SEQ ID NO: 45; a light chain variable region CDR2 of SEQ ID NO: 59;
and a light chain variable region CDR3 of SEQ ID NO: 73, (d) a
heavy chain variable region CDR1 of SEQ ID NO: 4; a heavy chain
variable region CDR2 of SEQ ID NO: 18; a heavy chain variable
region CDR3 of SEQ ID NO: 32; a light chain variable region CDR1 of
SEQ ID NO: 46; a light chain variable region CDR2 of SEQ ID NO: 60;
and a light chain variable region CDR3 of SEQ ID NO: 74, (e) a
heavy chain variable region CDR1 of SEQ ID NO: 5; a heavy chain
variable region CDR2 of SEQ ID NO: 19; a heavy chain variable
region CDR3 of SEQ ID NO: 33; a light chain variable region CDR1 of
SEQ ID NO: 47; a light chain variable region CDR2 of SEQ ID NO: 61;
and a light chain variable region CDR3 of SEQ ID NO: 75, (f) a
heavy chain variable region CDR1 of SEQ ID NO: 6; a heavy chain
variable region CDR2 of SEQ ID NO: 20; a heavy chain variable
region CDR3 of SEQ ID NO: 34; a light chain variable region CDR1 of
SEQ ID NO: 48; a light chain variable region CDR2 of SEQ ID NO: 62;
and a light chain variable region CDR3 of SEQ ID NO: 76, (g) a
heavy chain variable region CDR1 of SEQ ID NO: 7; a heavy chain
variable region CDR2 of SEQ ID NO: 21; a heavy chain variable
region CDR3 of SEQ ID NO: 35; a light chain variable region CDR1 of
SEQ ID NO: 49; a light chain variable region CDR2 of SEQ ID NO: 63;
and a light chain variable region CDR3 of SEQ ID NO: 77, (h) a
heavy chain variable region CDR1 of SEQ ID NO: 8; a heavy chain
variable region CDR2 of SEQ ID NO: 22; a heavy chain variable
region CDR3 of SEQ ID NO: 36; a light chain variable region CDR1 of
SEQ ID NO: 50 a light chain variable region CDR2 of SEQ ID NO: 64;
and a light chain variable region CDR3 of SEQ ID NO: 78, (i) a
heavy chain variable region CDR1 of SEQ ID NO: 9; a heavy chain
variable region CDR2 of SEQ ID NO: 23; a heavy chain variable
region CDR3 of SEQ ID NO: 37; a light chain variable region CDR1 of
SEQ ID NO: 51; a light chain variable region CDR2 of SEQ ID NO: 65;
and a light chain variable region CDR3 of SEQ ID NO: 79, (j) a
heavy chain variable region CDR1 of SEQ ID NO: 10; a heavy chain
variable region CDR2 of SEQ ID NO: 24; a heavy chain variable
region CDR3 of SEQ ID NO: 38; a light chain variable region CDR1 of
SEQ ID NO: 52; a light chain variable region CDR2 of SEQ ID NO: 66;
and a light chain variable region CDR3 of SEQ ID NO: 80, (k) a
heavy chain variable region CDR1 of SEQ ID NO: 11; a heavy chain
variable region CDR2 of SEQ ID NO: 25; a heavy chain variable
region CDR3 of SEQ ID NO: 39; a light chain variable region CDR1 of
SEQ ID NO: 53; a light chain variable region CDR2 of SEQ ID NO: 67;
and a light chain variable region CDR3 of SEQ ID NO: 81, (l) a
heavy chain variable region CDR1 of SEQ ID NO: 12; a heavy chain
variable region CDR2 of SEQ ID NO: 26; a heavy chain variable
region CDR3 of SEQ ID NO: 40; a light chain variable region CDR1 of
SEQ ID NO: 54; a light chain variable region CDR2 of SEQ ID NO: 68;
and a light chain variable region CDR3 of SEQ ID NO: 82, (m) a
heavy chain variable region CDR1 of SEQ ID NO: 13; a heavy chain
variable region CDR2 of SEQ ID NO: 27; a heavy chain variable
region CDR3 of SEQ ID NO: 41; a light chain variable region CDR1 of
SEQ ID NO: 55; a light chain variable region CDR2 of SEQ ID NO: 69;
and a light chain variable region CDR3 of SEQ ID NO: 83, or (n) a
heavy chain variable region CDR1 of SEQ ID NO: 14; a heavy chain
variable region CDR2 of SEQ ID NO: 28; a heavy chain variable
region CDR3 of SEQ ID NO: 42; a light chain variable region CDR1 of
SEQ ID NO: 56; a light chain variable region CDR2 of SEQ ID NO: 70;
and a light chain variable region CDR3 of SEQ ID NO: 84.
15. The antibody or functional protein according to claim 1,
wherein said antibody comprises a full length heavy chain amino
acid sequence having at least 95% sequence identity to at least one
sequence selected from the group consisting of SEQ ID NOs: 146-150
and 156-160.
16. The antibody or functional protein according to claim 1,
wherein said antibody comprises a full length light chain amino
acid sequence having at least 95% sequence identity to at least one
sequence selected from the group consisting of SEQ ID NOs: 141-145
and 151-155.
17. An antibody comprising: (a) the variable heavy chain sequence
of SEQ ID NO: 85 and variable light chain sequence of SEQ ID NO:
99; (b) the variable heavy chain sequence of SEQ ID NO: 86 and
variable light chain sequence of SEQ ID NO: 100; (c) the variable
heavy chain sequence of SEQ ID NO: 87 and variable light chain
sequence of SEQ ID NO: 101; (d) the variable heavy chain sequence
of SEQ ID NO: 88 and variable light chain sequence of SEQ ID NO:
102; (e) the variable heavy chain sequence of SEQ ID NO: 89 and
variable light chain sequence of SEQ ID NO: 103; (f) the variable
heavy chain sequence of SEQ ID NO: 90 and variable light chain
sequence of SEQ ID NO: 104; (g) the variable heavy chain sequence
of SEQ ID NO: 91 and variable light chain sequence of SEQ ID NO:
105; (h) the variable heavy chain sequence of SEQ ID NO: 92 and
variable light chain sequence of SEQ ID NO: 106; (i) the variable
heavy chain sequence of SEQ ID NO: 93 and variable light chain
sequence of SEQ ID NO: 107; (j) the variable heavy chain sequence
of SEQ ID NO: 94 and variable light chain sequence of SEQ ID NO:
108; (k) the variable heavy chain sequence of SEQ ID NO: 95 and
variable light chain sequence of SEQ ID NO: 109; (l) the variable
heavy chain sequence of SEQ ID NO: 96 and variable light chain
sequence of SEQ ID NO: 110; (m) the variable heavy chain sequence
of SEQ ID NO: 97 and variable light chain sequence of SEQ ID NO:
111; or (n) the variable heavy chain sequence of SEQ ID NO: 98 and
variable light chain sequence of SEQ ID NO: 112.
18. An antibody comprising: (a) the heavy chain sequence of SEQ ID
NO: 146 and light chain sequence of SEQ ID NO: 141; (b) the heavy
chain sequence of SEQ ID NO: 147 and light chain sequence of SEQ ID
NO: 142; (c) the heavy chain sequence of SEQ ID NO: 148 and light
chain sequence of SEQ ID NO: 143; (d) the heavy chain sequence of
SEQ ID NO: 149 and light chain sequence of SEQ ID NO: 144; (e) the
heavy chain sequence of SEQ ID NO: 150 and light chain sequence of
SEQ ID NO: 145; (f) the heavy chain sequence of SEQ ID NO: 156 and
light chain sequence of SEQ ID NO: 151; (g) the heavy chain
sequence of SEQ ID NO: 157 and light chain sequence of SEQ ID NO:
152; (h) the heavy chain sequence of SEQ ID NO: 158 and light chain
sequence of SEQ ID NO: 153; (i) the heavy chain sequence of SEQ ID
NO: 159 and light chain sequence of SEQ ID NO: 154; or (j) the
heavy chain sequence of SEQ ID NO: 160 and light chain sequence of
SEQ ID NO: 155.
19. An antibody or functional protein which cross-blocks or is
cross blocked by at least one antibody of claim 18 from binding to
ActRIIB.
20. An antibody or functional protein according to claim 1 which
cross-blocks or is cross blocked by at least one antibody of claim
18 from binding to ActRIIB.
21. An antibody or functional protein that binds to an epitope
recognised by an antibody of claim 17.
22. An antibody or functional protein that binds to an epitope
recognised by an antibody of claim 18.
23. An antibody according to claim 1 which binds to an epitope
comprising or consisting of: (a) amino acids 78-83 of SEQ ID NO:
181 (WLDDFN--SEQ ID NO:188); (b) amino acids 76-84 of SEQ ID NO:
181 (GCWLDDFNC--SEQ ID NO:186); (c) amino acids 75-85 of SEQ ID NO:
181 (KGCWLDDFNCY--SEQ ID NO:190); (d) amino acids 52-56 of SEQ ID
NO: 181 (EQDKR--SEQ ID NO:189); (e) amino acids 49-63 of SEQ ID NO:
181 (CEGEQDKRLHCYASW--SEQ ID NO:187); or (f) amino acids 78-83 of
SEQ ID NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181
(EQDKR).
24. The antibody or functional protein of claim 19 wherein cross
blocking or binding to the same epitope is detected in a BIAcore
assay or an ELISA.
25. An anti-ActRIIB antibody according to claim 1, wherein said
antibody is of the IgG1 isotype.
26. An anti-ActRIIB antibody according to claim 1, which has
altered effector function through mutation of the Fc region.
27. An isolated polynucleotide sequence encoding an antibody or
functional protein according to claim 1.
28. An isolated polynucleotide sequence according to claim 27,
comprising one or more of SEQ ID NOs: 113-140 or 161-180.
29. A cloning or expression vector comprising one or more isolated
polynucleotide sequences according to claim 27.
30. A vector according to claim 29, wherein said vector comprises
one or more of SEQ ID NOs: 113-140 or 161-180, or fragment thereof
encoding at least one CDR region.
31. A host cell comprising one or more vectors according to claim
29.
32. A process for the production of an antibody or functional
protein comprising culturing the host cell of claim 31 and
isolating said antibody or functional protein.
33. A pharmaceutical composition comprising an antibody or
functional protein encoded by a polynucleotide sequence according
to claim 27.
34. A pharmaceutical composition according to claim 33, further
comprising a pharmaceutically acceptable diluent or carrier.
35. A pharmaceutical composition according to claim 33, further
comprising one or more additional active agents.
36. A pharmaceutical composition according to claim 35, wherein
said additional active agent is selected from IGF-1, IGF-2 or
variants of IGF-1 or IGF-2, an anti-myostatin antibody, a myostatin
propeptide, a myostatin decoy protein that binds ActRIIB but does
not activate it, a beta 2 agonist, a Ghrelin agonist, a SARM, GH
agonists/mimetics or follistatin.
37. A method of treating a patient suffering from a musculoskeletal
disease or disorder; acute and/or chronic renal disease or failure;
liver fibrosis or cirrhosis; cancer such as breast cancer;
Parkinson's Disease; conditions associated with neuronal death,
such as ALS, brain atrophy, or dementia and anemia; liver, kidney
and pulmonary fibrosis; age-related conditions; and cancers
examplified by but not restricted to rhabdomyosarcomas, bone-loss
inducing cancers, hepatocellular carcinomas, gastrointestinal
cancers, comprising administering an effective dose of an antibody
or functional protein according to claim 1 to said patient.
38. An antibody or functional protein encoded by the polynucleotide
sequence according to claim 27, or the pharmaceutical composition
comprising the antibody or functional protein for use as a
medicament.
39. Use of an antibody or functional protein encoded by the
polynucleotide sequence according to claim 27, or the
pharmaceutical composition comprising the antibody or functional
protein in the manufacture of a medicament for the treatment of a
musculoskeletal disease or disorder; acute and/or chronic renal
disease or failure; liver fibrosis or cirrhosis; cancer such as
breast cancer; Parkinson's Disease; conditions associated with
neuronal death, such as ALS, brain atrophy, or dementia and anemia;
liver, kidney and pulmonary fibrosis; age-related conditions; and
cancers examplified by but not restricted to rhabdomyosarcomas,
bone-loss inducing cancers, hepatocellular carcinomas,
gastrointestinal cancers.
40. An antibody or functional protein encoded by the polynucleotide
sequence according to claim 27, or the pharmaceutical composition
comprising the antibody or functional protein for use in the
treatment of a musculoskeletal disease or disorder; acute and/or
chronic renal disease or failure; liver fibrosis or cirrhosis;
cancer such as breast cancer; Parkinson's Disease; conditions
associated with neuronal death, such as ALS, brain atrophy, or
dementia and anemia; liver, kidney and pulmonary fibrosis; and
cancers examplified by but not restricted to rhabdomyosarcomas,
bone-loss inducing cancers, hepatocellular carcinomas,
gastrointestinal cancers.
41. The use of claim 39, wherein said muscuoskeletal disease or
disorder is muscle atrophy, for example caused by a myopathy, such
as myotonia, a congential myopathy, including nemalene myopathy,
multi/minicore myopathy and myotubular (centronuclear) myopathy,
mitochondrial myopathy, familial periodic paralysis, inflammatory
myopathy, metabolic myopathy, such as caused by a glycogen or lipid
storage disease, dermatomyositisis, polymyositis, inclusion body
myositis, myositis ossificans, rhabdomyolysis and myoglobinurias; a
dystrophy, such as Duchenne, Becker, myotonic,
fascioscapulohumeral, Emery-Dreifuss, oculopharyngeal,
scapulohumeral, limb girdle, Fukuyama, a congenital muscular
dystrophy, or hereditary distal myopathy; osteoporosis; a bone
fracture; short stature; dwarfism; prolonged bed rest; voluntary
inactivity; or involuntary inactivity.
42. The use according to claim 41, wherein the patient being
treated has been pre-treated with IGF-1, IGF-2 or variants of IGF-1
or IGF-2, an anti-myostatin antibody, a myostatin propeptide, a
myostatin decoy protein that binds ActRIIB but does not activate
it, a beta 2 agonist, a Ghrelin agonist, a SARM, GH
agonists/mimetics or follistatin.
43. The use according to claim 41, wherein the patient being
treated has previously been refractive to treatment with IGF-1,
IGF-2 or variants of IGF-1 or IGF-2, an anti-myostatin antibody, a
myostatin propeptide, a myostatin decoy protein that binds ActRIIB
but does not activate it, a beta 2 agonist, a Ghrelin agonist, a
SARM, GH agonists/mimetics or follistatin.
44. The use according to claim 41, wherein the patient being
treated is elderly, has spent time in a zero gravity environment or
has undergone a period of inactivity.
45. A method of ameliorating the muscle wasting effects of enforced
inactivity or time spent in a zero gravity environment comprising
administering an effective dose of an antibody or functional
protein according to claim 1 prior to said period of enforced
inactivity or time spent in a zero gravity environment.
46. The use according to claim 41, wherein the patient being
treated has a fracture to a limb or joint.
47. The use according to claim 41, wherein the patient has
undergone or is about to undergo, hip or knee replacement
surgery.
48. An antibody encoded by pBW522 (DSM22873) or pBW524 (DSM22874).
Description
TECHNICAL FIELD
[0001] This disclosure is in the field of anti-Activin receptor IIB
(ActRIIB) antibodies. In particular, it relates to the use of said
antibodies for treating muscle disorders, such as muscle wasting
due to disease or disuse.
BACKGROUND ART
[0002] Activins are dimeric growth and differentiation factors
which belong to the transforming growth factor-beta (TGF-beta)
superfamily of structurally related signaling proteins. Activins
signal through a heterodimeric complex of receptor serine kinases
which include at least two type I (I and IB) and two type II (II
and IIB, aka ACVR2A and ACVR2B) receptors. These receptors are all
transmembrane proteins, composed of a ligand-binding extracellular
domain with cysteine-rich region, a transmembrane domain, and a
cytoplasmic domain with predicted serine/threonine specificity.
Type I receptors are essential for signalling while type II
receptors are required for binding ligands and for expression of
type I receptors. Type I and II receptors form a stable complex
after ligand binding resulting in the phosphorylation of type I
receptors by type II receptors.
[0003] The activin receptor II B (ActRIIB) is a receptor for
myostatin. The interaction between myostatin and this receptor
regulates the inhibition of skeletal muscle differentiation via the
Smad-dependent pathway. Thus, by inhibiting or preventing myostatin
from binding to ActRIIB, one can induce the formation of skeletal
muscle.
[0004] Various groups have looked into this. Bogdanovich et al
(Nature, 2002, 420:418-421) describes that anti-myostatin
antibodies were able to block myostatin, resulting in an increase
in muscle mass in a mouse model of Duchenne muscular dystrophy.
Bradley et al (Cell Mol. Life Sci. 2008, 65:2119-2124) have
reviewed the different available approaches for modulating the
myostatin/ActRIIB interaction, including the aforementioned
anti-myostatin antibodies, inhibiting the release of mature
myostatin by administering the myostatin propeptide, administering
follistatin to block the myostatin receptor, administering HDAC
inhibitors to induce follistatin production, administering an
altered myostatin peptide which prevents myostatin from binding the
receptor and administering a soluble decoy receptor for myostatin.
Despite these potential therapies, there is no product available
for the treatment of patients. Indeed, recently one company
cancelled its anti-myostatin antibody project.
[0005] There is therefore a need for a method of increasing muscle
mass and strength in a patient.
DISCLOSURE OF THE INVENTION
[0006] It has been discovered that antibodies directed to the
ActRIIB receptor can prevent myostatin from binding to the
receptor, thus preventing the inhibition of muscle differentiation
by the Smad-dependent pathway. This leads to an increase in muscle
mass and strength in a patient.
[0007] Therefore, in one aspect, the disclosure provides an
anti-ActRIIB antibody, or a functional fragment thereof or
functional protein comprising an antigen-binding portion of an
anti-ActRIIB antibody. In one embodiment, the ActRIIB is human
ActRIIB. The polypeptide sequence of human ActRIIB is recited in
SEQ ID NO: 181 (AAC64515.1, GI:3769443). In one embodiment, the
antibody or functional protein is from a mammal, having an origin
such as human or camelid. Thus the antibody may be a chimeric,
human or a humanized antibody. In a particular embodiment, the
anti-ActRIIB antibody is characterized as having antigen-binding
region that is specific for the target protein ActRIIB and binds to
ActRIIB or a fragment of ActRIIB. In one embodiment, the antibody
is suitable for use in therapy.
[0008] In one embodiment, the antibodies according to the
disclosure are ActRIIB antagonists with no or low agonistic
activity. In another embodiment, the antibody or functional
fragment comprising an antigen-binding portion binds the target
protein ActRIIB and decreases the binding of myostatin to ActRIIB
to a basal level. In one aspect of this embodiment, the antibody or
functional fragment reduces the amount of myostatin that binds to
ActRIIB. In a further aspect of this embodiment, the antibody or
functional fragment completely prevents myostatin from binding to
ActRIIB. In a further embodiment, the antibody or functional
fragment inhibits Smad activation. In a further embodiment, the
antibody or functional fragment inhibits activin receptor type IIB
mediated myostatin-induced inhibition of skeletal differentiation
via the Smad-dependent pathway.
[0009] The binding may be determined by one or more assays that can
be used to measure an activity which is either antagonism or
agonism by the antibody. In one embodiment, the assays measure at
least one of the effects of the antibody on ActRIIB that include:
inhibition of myostatin binding to ActRIIB by ELISA, inhibition of
myostatin induced signalling (for instance by a Smad dependent
reporter gene assay), inhibition of myostatin induced Smad
phosphorylation (P-Smad ELISA) and inhibition of myostatin induced
inhibition of skeletal muscle cell differentiation (for instance by
a creatine kinase assay).
[0010] In one embodiment, the disclosure provides antibodies that
specifically bind to the myostatin binding region (i.e. ligand
binding domain) of ActRIIB. This ligand binding domain consists of
amino acids 19-134 of SEQ ID NO: 181 and has been assigned SEQ ID
NO: 182 herein.
[0011] In one embodiment, the antibodies bind to ActRIIB with a
K.sub.D of 100 nM or less, 10 nM or less, 1 nM or less. In one
embodiment, the antibodies of the disclosure bind to ActRIIB with
an affinity of 100 pM or less (i.e. 100 pM, 50 pM, 10 pM, 1 pM or
less). In one embodiment, the antibodies of the disclosure bind to
ActRIIB with an affinity of between 10 and 20 pM.
[0012] In one embodiment, the antibodies of the disclosure do not
cross-react with an ActRIIB related protein, and more particularly
do not cross-react with human ActRIIA (NP.sub.--001607.1,
GI:4501897).
[0013] In one embodiment, the antibodies of the disclosure in one
embodiment bind to ActRIIB rather than ActRIIA. In one embodiment,
the antibodies of the disclosure bind to ActRIIB with 5-fold
greater affinity than they bind to ActRIIA, more particularly
10-fold, still more particularly 50-fold, still more particularly
100-fold.
[0014] In one embodiment, the antibodies of the disclosure bind to
ActRIIA with an affinity of 100 pM or more (i.e. 250 pM, 500 pM, 1
nM, 5 nM or more).
[0015] In one embodiment the antibodies of the disclosure are of
the IgG2 isotype.
[0016] In another embodiment, the antibodies of the disclosure are
of the IgG1 isotype. In a further embodiment, the antibodies of the
disclosure are of the IgG1 isotype and have an altered effector
function through mutation of the Fc region. In one embodiment, said
altered effector function is reduced ADCC and CDC activity. In one
embodiment, said altered effector function is silenced ADCC and CDC
activity.
[0017] In another related embodiment, the antibodies according to
the disclosure are fully human or humanized IgG1 antibodies with no
antibody dependent cellular cytotoxicity (ADCC) activity or CDC
activity and bind to a region of ActRIIB consisting of amino acids
19-134 of SEQ ID NO:181.
[0018] In another related embodiment, the antibodies according to
the disclosure are fully human or humanized IgG1 antibodies with
reduced antibody dependent cellular cytotoxicity (ADCC) activity or
CDC activity and bind to a region of ActRIIB consisting of amino
acids 19-134 of SEQ ID NO:181.
[0019] The present disclosure relates to isolated antibodies,
particularly human or humanized antibodies that inhibit myostatin
binding to ActRIIB and activate skeletal muscle differentiation in
vitro and in vivo. In certain embodiments, the antibodies of the
disclosure are derived from particular heavy and light chain
sequences and/or comprise particular structural features such as
CDR regions comprising particular amino acid sequences. The
disclosure provides isolated antibodies, methods of making such
antibodies, immunoconjugates and multivalent or multi-specific
molecules comprising such antibodies and pharmaceutical
compositions containing the antibodies, immunoconjugates or
bi-specific molecules of the disclosure. The disclosure also
relates to methods of using the antibodies to inhibit, i.e.
antagonize, function of ActRIIB in order to inhibit Smad activation
and thereby induce skeletal muscle differentiation, for example,
resulting in the treatment of a pathological disorder.
[0020] The pathological disorder may be a musculoskeletal disease
or disorder, such as muscle atrophy. There are many causes of
muscle atrophy, including as a result of treatment with a
glucocorticoid such as cortisol, dexamethasone, betamethasone,
prednisone, methylprednisolone, or prednisolone. The muscle atrophy
can also be a result of denervation due to nerve trauma or a result
of degenerative, metabolic, or inflammatory neuropathy (e.g.,
Guillian-Barre syndrome, peripheral neuropathy, or exposure to
environmental toxins or drugs).
[0021] In addition, the muscle atrophy can be a result of myopathy,
such as myotonia; a congenital myopathy, including nemalene
myopathy, multi/minicore myopathy and myotubular (centronuclear)
myopathy; mitochondrial myopathy; familial periodic paralysis;
inflammatory myopathy; metabolic myopathy, such as caused by a
glycogen or lipid storage disease; dermatomyositisis; polymyositis;
inclusion body myositis; myositis ossificans; rhabdomyolysis and
myoglobinurias.
[0022] The myopathy may be caused by a muscular dystrophy syndrome,
such as Duchenne, Becker, myotonic, fascioscapulohumeral,
Emery-Dreifuss, oculopharyngeal, scapulohumeral, limb girdle,
Fukuyama, a congenital muscular dystrophy, or hereditary distal
myopathy. The musculoskeletal disease can also be osteoporosis, a
bone fracture, short stature, or dwarfism.
[0023] In addition, the muscle atrophy can be a result of an adult
motor neuron disease, infantile spinal muscular atrophy,
amyotrophic lateral sclerosis, juvenile spinal muscular atrophy,
autoimmune motor neuropathy with multifocal conductor block,
paralysis due to stroke or spinal cord injury, skeletal
immobilization due to trauma, prolonged bed rest, voluntary
inactivity, involuntary inactivity, metabolic stress or nutritional
insufficiency, cancer, AIDS, fasting, a thyroid gland disorder,
diabetes, benign congenital hypotonia, central core disease, burn
injury, chronic obstructive pulmonary disease, liver diseases
(examples such as fibrosis, cirrhosis), sepsis, renal failure,
congestive heart failure, ageing, space travel or time spent in a
zero gravity environment.
[0024] Examples of age-related conditions that may be treated
include, sarcopenia, skin atrophy, muscle wasting, brain atrophy,
atherosclerosis, arteriosclerosis, pulmonary emphysema,
osteoporosis, osteoarthritis, immunologic incompetence, high blood
pressure, dementia, Huntington's disease, Alzheimer's disease,
cataracts, age-related macular degeneration, prostate cancer,
stroke, diminished life expectancy, frailty, memory loss, wrinkles,
impaired kidney function, and age-related hearing loss; metabolic
disorders, including Type II Diabetes, Metabolic Syndrome,
hyperglycemia, and obesity.
[0025] Other conditions that may be treated with the antibodies of
the disclosure include acute and/or chronic renal disease or
failure, liver fibrosis or cirrhosis, cancer such as breast cancer,
Parkinson's Disease; conditions associated with neuronal death,
such as ALS, brain atrophy, or dementia and anemia.
[0026] Further conditions include cachexia, cachexia associated
with a rheumatoid arthritis and cachexia associated with
cancer.
[0027] To date, very few reliable or effective therapies have been
developed to treat these disorders.
[0028] Based on reported evidence of a role of activins binding to
ActRIIB amongst other receptors (Werner and Alzheimer, Cytokine
Growth Factors Rev 2006, 17(3):157-171), in contributing to liver,
kidney and pulmonary fibrosis and of a role for myostatin,
activins, or ActRIIB in cancers (Tsuchida et al, Endo J, 2008,
55(1):11-21) the antibodies of the disclosure may be used to treat
liver, kidney, pulmonary fibrosis and cancers exemplified by but
not restricted to rhabdomyosarcomas, bone-loss inducing cancers,
hepatocellular carcinomas, gastrointestinal cancers.
[0029] The prevention may be complete, e.g., the total absence of
an age-related condition or metabolic disorder. The prevention may
also be partial, such that the likelihood of the occurrence of the
age-related condition or metabolic disorder in a subject is less
likely to occur than had the subject not received an antibody of
the present disclosure.
[0030] In order that the present disclosure may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0031] The term "immune response" refers to the action of, for
example, lymphocytes, antigen presenting cells, phagocytic cells,
granulocytes, and soluble macromolecules produced by the above
cells or the liver (including antibodies, cytokines, and
complement) that results in selective damage to, destruction of, or
elimination from the human body of invading pathogens, cells or
tissues infected with pathogens, cancerous cells, or, in cases of
autoimmunity or pathological inflammation, normal human cells or
tissues.
[0032] A "signal transduction pathway" or "signaling activity"
refers to a biochemical causal relationship generally initiated by
a protein-protein interaction such as binding of a growth factor to
a receptor, resulting in transmission of a signal from one portion
of a cell to another portion of a cell. In general, the
transmission involves specific phosphorylation of one or more
tyrosine, serine, or threonine residues on one or more proteins in
the series of reactions causing signal transduction. Penultimate
processes typically include nuclear events, resulting in a change
in gene expression.
[0033] The term ActRIIB or Act IIB receptor refers to human ActRIIB
as defined in SEQ ID NO: 181 (AAC64515.1, GI:3769443). Research
grade polyclonal and monoclonal anti-ActRIIB antibodies are known
in the art, such as those made by R&D Systems.RTM., MN, USA.
Therapeutic anti-ActRIIB antibodies have not previously been
described. Of course, antibodies could be raised against ActRIIB
from other species and used to treat pathological conditions in
those species.
[0034] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e. "antigen-binding
portion") or single chains thereof A naturally occurring "antibody"
is a glycoprotein comprising at least two heavy (H) chains and two
light (L) chains inter-connected by disulfide bonds. Each heavy
chain is comprised of a heavy chain variable region (abbreviated
herein as V.sub.H) and a heavy chain constant region. The heavy
chain constant region is comprised of three domains, CH1, CH2 and
CH3. Each light chain is comprised of a light chain variable region
(abbreviated herein as V.sub.L) and a light chain constant region.
The light chain constant region is comprised of one domain,
C.sub.L. The V.sub.H and V.sub.L regions can be further subdivided
into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR). Each V.sub.H and V.sub.L
is composed of three CDRs and four FRs arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. The
constant regions of the antibodies may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (e.g. effector cells) and the first component
(Clq) of the classical complement system.
[0035] The term "antigen-binding portion" of an antibody (or simply
"antigen portion"), as used herein, refers to full length or one or
more fragments of an antibody that retain the ability to
specifically bind to an antigen (e.g. a portion of ActRIIB). It has
been shown that the antigen-binding function of an antibody can be
performed by fragments of a full-length antibody. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and CH1
domains; a F(ab).sub.2 fragment, a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge region; a
Fd fragment consisting of the V.sub.H and CH1 domains; a Fv
fragment consisting of the V.sub.L and V.sub.H domains of a single
arm of an antibody; a dAb fragment (Ward et al., 1989 Nature
341:544-546), which consists of a V.sub.H domain; and an isolated
complementarity determining region (CDR).
[0036] Furthermore, although the two domains of the Fv fragment,
V.sub.L and V.sub.H, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the
V.sub.L and V.sub.H regions pair to form monovalent molecules
(known as single chain Fv (scFv); see e.g. Bird et al., 1988
Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci.
85:5879-5883). Such single chain antibodies are also intended to be
encompassed within the term "antigen-binding region" of an
antibody. These antibody fragments are obtained using conventional
techniques known to those of skill in the art, and the fragments
are screened for utility in the same manner as are intact
antibodies.
[0037] An "isolated antibody", as used herein, refers to an
antibody that is substantially free of other antibodies having
different antigenic specificities (e.g. an isolated antibody that
specifically binds ActRIIB is substantially free of antibodies that
specifically bind antigens other than ActRIIB). An isolated
antibody that specifically binds ActRIIB may, however, have
cross-reactivity to other antigens, such as ActRIIB molecules from
other species. Moreover, an isolated antibody may be substantially
free of other cellular material and/or chemicals.
[0038] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope.
[0039] The term "human antibody", as used herein, is intended to
include antibodies having variable regions in which both the
framework and CDR regions are derived from sequences of human
origin. Furthermore, if the antibody contains a constant region,
the constant region also is derived from such human sequences, e.g.
human germline sequences, or mutated versions of human germline
sequences or antibody containing consensus framework sequences
derived from human framework sequences analysis, for example, as
described in Knappik, et al. (2000. J Mol Biol 296, 57-86).
[0040] The human antibodies of the disclosure may include amino
acid residues not encoded by human sequences (e.g. mutations
introduced by random or site-specific mutagenesis in vitro or by
somatic mutation in vivo). However, the term "human antibody", as
used herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework
sequences.
[0041] The term "human monoclonal antibody" refers to antibodies
displaying a single binding specificity which have variable regions
in which both the framework and CDR regions are derived from human
sequences. In one embodiment, the human monoclonal antibodies are
produced by a hybridoma which includes a B cell obtained from a
transgenic nonhuman animal, e.g. a transgenic mouse, having a
genome comprising a human heavy chain transgene and a light chain
transgene fused to an immortalized cell.
[0042] The term "recombinant human antibody", as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as antibodies isolated from
an animal (e.g. a mouse) that is transgenic or transchromosomal for
human immunoglobulin genes or a hybridoma prepared therefrom,
antibodies isolated from a host cell transformed to express the
human antibody, e.g. from a transfectoma, antibodies isolated from
a recombinant, combinatorial human antibody library, and antibodies
prepared, expressed, created or isolated by any other means that
involve splicing of all or a portion of a human immunoglobulin
gene, sequences to other DNA sequences. Such recombinant human
antibodies have variable regions in which the framework and CDR
regions are derived from human germline immunoglobulin sequences.
In certain embodiments, however, such recombinant human antibodies
can be subjected to in vitro mutagenesis (or, when an animal
transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the V.sub.H and
V.sub.L regions of the recombinant antibodies are sequences that,
while derived from and related to human germline V.sub.H and
V.sub.L sequences, may not naturally exist within the human
antibody germline repertoire in vivo.
[0043] As used herein, "isotype" refers to the antibody class (e.g.
IgM, IgE, IgG such as IgG1 or IgG2) that is provided by the heavy
chain constant region genes.
[0044] The phrases "an antibody recognizing an antigen" and "an
antibody specific for an antigen" are used interchangeably herein
with the term "an antibody which binds specifically to an
antigen".
[0045] As used herein, an antibody that "specifically binds to
ActRIIB polypeptide" is intended to refer to an antibody that binds
to human ActRIIB polypeptide with a K.sub.D of a 100 nM or less, 10
nM or less, 1 nM or less. An antibody that "cross-reacts with an
antigen other than ActRIIB" is intended to refer to an antibody
that binds that antigen with a K.sub.D of 10.times.10.sup.-9 M or
less, 5.times.10.sup.-9 M or less, or 2.times.10.sup.-9 M or less.
An antibody that "does not cross-react with a particular antigen"
is intended to refer to an antibody that binds to that antigen,
with a K.sub.D of 1.5.times.10.sup.-8 M or greater, or a K.sub.D of
5-10.times.10.sup.-8 M, or 1.times.10.sup.-7 M or greater. In
certain embodiments, such antibodies that do not cross-react with
the antigen exhibit essentially undetectable binding against these
proteins in standard binding assays. K.sub.D may be determined
using a biosensor system, such as a Biacore.RTM. system, or
Solution Equilibrium Titration.
[0046] As used herein, the term "antagonist antibody" is intended
to refer to an antibody that inhibits ActRIIB induced signaling
activity in the presence of myostatin. Examples of an assay to
detect this include inhibition of myostatin induced signalling (for
instance by a Smad dependent reporter gene assay), inhibition of
myostatin induced Smad phosphorylation (P-Smad ELISA) and
inhibition of myostatin induced inhibition of skeletal muscle cell
differentiation (for instance by a creatine kinase assay).
[0047] In some embodiments, the antibodies inhibit myostatin
induced signalling as measured in a Smad dependent reporter gene
assay at an IC50 of 10 nM or less, 1 nM or less, or 100 pM or
less.
[0048] As used herein, an antibody with "no agonistic activity" is
intended to refer to an antibody that does not significantly
increase ActRIIB mediated signaling activity in the absence of
myostatin in a cell-based assay, such as inhibition of myostatin
induced signalling (for instance by a Smad dependent reporter gene
assay), inhibition of myostatin induced Smad phosphorylation
(P-Smad ELISA) and inhibition of myostatin induced inhibition of
skeletal muscle cell differentiation (for instance by a creatine
kinase assay). Such assays are described in more details in the
examples below.
[0049] The term "K.sub.assoc" or "K.sub.a", as used herein, is
intended to refer to the association rate of a particular
antibody-antigen interaction, whereas the term "K.sub.dis" or
"K.sub.d", as used herein, is intended to refer to the dissociation
rate of a particular antibody-antigen interaction. The term
"K.sub.D", as used herein, is intended to refer to the dissociation
constant, which is obtained from the ratio of K.sub.d to K.sub.a
(i.e. K.sub.d/K.sub.a) and is expressed as a molar concentration
(M). K.sub.D values for antibodies can be determined using methods
well established in the art. A method for determining the K.sub.D
of an antibody is by using surface plasmon resonance, such as the
biosensor system of Biacore.RTM., or Solution Equilibrium Titration
(SET) (see Friguet B et al. (1985) J. Immunol Methods; 77(2):
305-319, and Hanel C et al. (2005) Anal Biochem; 339(1):
182-184).
[0050] As used herein, the term "Affinity" refers to the strength
of interaction between antibody and antigen at single antigenic
sites. Within each antigenic site, the variable region of the
antibody "arm" interacts through weak non-covalent forces with
antigen at numerous sites; the more interactions, the stronger the
affinity.
[0051] As used herein, the term "Avidity" refers to an informative
measure of the overall stability or strength of the
antibody-antigen complex. It is controlled by three major factors:
antibody epitope affinity; the valency of both the antigen and
antibody; and the structural arrangement of the interacting parts.
Ultimately these factors define the specificity of the antibody,
that is, the likelihood that the particular antibody is binding to
a precise antigen epitope.
[0052] As used herein, the term "ADCC" or "antibody dependent
cellular cytotoxicity" activity refers to human B cell depleting
activity. ADCC activity can be measured by the human B cell
depleting assays known in the art.
[0053] In order to get a higher avidity probe, a dimeric conjugate
(two molecules of an antibody protein coupled to a FACS marker) can
be constructed, thus making low affinity interactions (such as with
the germline antibody) more readily detected by FACS. In addition,
another means to increase the avidity of antigen binding involves
generating dimers, trimers or multimers of any of the constructs
described herein of the anti-ActRIIB antibodies. Such multimers may
be generated through covalent binding between individual modules,
for example, by imitating the natural C-to-N-terminus binding or by
imitating antibody dimers that are held together through their
constant regions. The bonds engineered into the Fc/Fc interface may
be covalent or non-covalent. In addition, dimerizing or
multimerizing partners other than Fc can be used in ActRIIB hybrids
to create such higher order structures. For example, it is possible
to use multimerizing domains such as the trimerizing domain
described in WO2004/039841 or pentamerizing domain described in
WO98/18943.
[0054] As used herein, the term "selectivity" for an antibody
refers to an antibody that binds to a certain target polypeptide
but not to closely related polypeptides.
[0055] As used herein, the term "high affinity" for an antibody
refers to an antibody having a K.sub.D of 1 nM or less for a target
antigen. As used herein, the term "subject" includes any human or
nonhuman animal.
[0056] The term "nonhuman animal" includes all vertebrates, e.g.
mammals and non-mammals, such as nonhuman primates, sheep, dogs,
cats, horses, cows, chickens, amphibians, reptiles, etc.
[0057] As used herein, the term, "optimized" means that a
nucleotide sequence has been altered to encode an amino acid
sequence using codons that are preferred in the production cell or
organism, generally a eukaryotic cell, for example, a cell of
Pichia, a cell of Trichoderma, a Chinese Hamster Ovary cell (CHO)
or a human cell. The optimized nucleotide sequence is engineered to
retain completely or as much as possible the amino acid sequence
originally encoded by the starting nucleotide sequence, which is
also known as the "parental" sequence. The optimized sequences
herein have been engineered to have codons that are preferred in
CHO mammalian cells, however optimized expression of these
sequences in other eukaryotic cells is also envisioned herein. The
amino acid sequences encoded by optimized nucleotide sequences are
also referred to as optimized.
[0058] Various aspects of the disclosure are described in further
detail in the following subsections.
[0059] Standard assays to evaluate the binding ability of the
antibodies toward ActRIIB of various species are known in the art,
including for example, ELISAs, western blots and RIAs. Suitable
assays are described in detail in the Examples. The binding
affinity of the antibodies also can be assessed by standard assays
known in the art, such as by Biacore analysis or Solution
Equilibrium Titration. Surface plasmon resonance based techniques
such as Biacore can determine the binding kinetics which allows the
calculation of the binding affinity. Assays to evaluate the effects
of the antibodies on functional properties of ActRIIB (e.g.
receptor binding, preventing or inducing human B cell proliferation
or IgG production) are described in further detail in the
Examples.
[0060] Accordingly, an antibody that "inhibits" one or more of
these ActRIIB functional properties (e.g. biochemical,
immunochemical, cellular, physiological or other biological
activities, or the like) as determined according to methodologies
known to the art and described herein, will be understood to relate
to a statistically significant decrease in the particular activity
relative to that seen in the absence of the antibody (e.g. or when
a control antibody of irrelevant specificity is present). An
antibody that inhibits ActRIIB activity effects such a
statistically significant decrease by at least 10% of the measured
parameter, by at least 50%, 80% or 90%, and in certain embodiments
an antibody of the disclosure may inhibit greater than 95%, 98% or
99% of ActRIIB functional activity.
[0061] The terms "cross-block", "cross-blocked" and
"cross-blocking" are used interchangeably herein to mean the
ability of an antibody or other binding agent to interfere with the
binding of other antibodies or binding agents to ActRIIB,
particularly the ligand binding domain, in a standard competitive
binding assay.
[0062] The ability or extent to which an antibody or other binding
agent is able to interfere with the binding of another antibody or
binding molecule to ActRIIB, and therefore whether it can be said
to cross-block according to the disclosure, can be determined using
standard competition binding assays. One suitable assay involves
the use of the Biacore technology (e.g. by using a BIAcore
instrument (Biacore, Uppsala, Sweden)), which can measure the
extent of interactions using surface plasmon resonance technology.
Another assay for measuring cross-blocking uses an ELISA-based
approach. A further assay uses FACS analysis, wherein competition
of various antibodies for binding to ActRIIB expressing cells is
tested (such as described in the Examples).
[0063] According to the disclosure, a cross-blocking antibody or
other binding agent according to the disclosure binds to ActRIIB in
the described BIAcore cross-blocking assay such that the recorded
binding of the combination (mixture) of the antibodies or binding
agents is between 80% and 0.1% (e.g. 80% to 4%) of the maximum
theoretical binding, specifically between 75% and 0.1% (e.g. 75% to
4%) of the maximum theoretical binding, and more specifically
between 70% and 0.1% (e.g. 70% to 4%), and more specifically
between 65% and 0.1% (e.g. 65% to 4%) of maximum theoretical
binding (as defined above) of the two antibodies or binding agents
in combination.
[0064] An antibody is defined as cross-blocking an anti-ActRIIB
antibody of the disclosure in an ELISA assay, if the test antibody
is able to cause a reduction of anti-ActRIIB antibody binding to
ActRIIB of between 60% and 100%, specifically between 70% and 100%,
and more specifically between 80% and 100%, when compared to the
positive control wells (i.e. the same anti-ActRIIB antibody and
ActRIIB, but no "test" cross-blocking antibody). Examples of cross
blocking antibodies as cited herein are MOR08159 and MOR08213.
Thus, the disclosure provides antibodies that cross block MOR08159
or MOR08213 for binding to ActRIIB.
Recombinant Antibodies
[0065] Antibodies of the disclosure include the human recombinant
antibodies, isolated and structurally characterized, as described
in the Examples. The V.sub.H amino acid sequences of isolated
antibodies of the disclosure are shown in SEQ ID NOs: 99-112. The
V.sub.L amino acid sequences of isolated antibodies of the
disclosure are shown in SEQ ID NOs: 85-98 respectively. Examples of
particular full length heavy chain amino acid sequences of
antibodies of the disclosure are shown in SEQ ID NOs: 146-150 and
156-160. Examples of particular full length light chain amino acid
sequences of antibodies of the disclosure are shown in SEQ ID NOs:
141-145 and 151-155 respectively. Other antibodies of the
disclosure include amino acids that have been mutated by amino acid
deletion, insertion or substitution, yet have at least 60, 70, 80,
90, 95, 97 or 99 percent sequence identity in the CDR regions with
the CDR regions depicted in the sequences described above. In some
embodiments, it includes mutant amino acid sequences wherein no
more than 1, 2, 3, 4 or 5 amino acids have been mutated by amino
acid deletion, insertion or substitution in the CDR regions when
compared with the CDR regions depicted in the sequence described
above.
[0066] Further, variable heavy chain parental nucleotide sequences
are shown in SEQ ID NOs: 127-140. Variable light chain parental
nucleotide sequences are shown in SEQ ID NOs: 113-126. Full length
light chain nucleotide sequences optimized for expression in a
mammalian cell are shown in SEQ ID NOs: 161-165 and 171-175. Full
length heavy chain nucleotide sequences optimized for expression in
a mammalian cell are shown in SEQ ID NOs: 166-170 and 176-180.
Other antibodies of the disclosure include amino acids or nucleic
acids that have been mutated, yet have at least 60 or more (i.e.
80, 90, 95, 97, 99 or more) percent sequence identity to the
sequences described above. In some embodiments, it includes mutant
amino acid sequences wherein no more than 1, 2, 3, 4 or 5 amino
acids have been mutated by amino acid deletion, insertion or
substitution in the variable regions when compared with the
variable regions depicted in the sequence described above.
[0067] Since each of these antibodies binds the same epitope and
are progenies from the same parental antibody, the V.sub.H,
V.sub.L, full length light chain, and full length heavy chain
sequences (nucleotide sequences and amino acid sequences) can be
"mixed and matched" to create other anti-ActRIIB binding molecules
of the disclosure. ActRIIB binding of such "mixed and matched"
antibodies can be tested using the binding assays described above
and in the Examples (e.g. ELISAs). When these chains are mixed and
matched, a V.sub.H sequence from a particular V.sub.H/V.sub.L
pairing should be replaced with a structurally similar V.sub.H
sequence. Likewise a full length heavy chain sequence from a
particular full length heavy chain/full length light chain pairing
should be replaced with a structurally similar full length heavy
chain sequence. Likewise, a V.sub.L sequence from a particular
V.sub.H/V.sub.L pairing should be replaced with a structurally
similar V.sub.L sequence. Likewise a full length light chain
sequence from a particular full length heavy chain/full length
light chain pairing should be replaced with a structurally similar
full length light chain sequence. Accordingly, in one aspect, the
disclosure provides an isolated recombinant anti-ActRIIB antibody
or antigen binding region thereof having: a heavy chain variable
region comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 99-112; and a light chain variable region
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 85-98.
In another aspect, the disclosure provides: [0068] (i) an isolated
recombinant anti-ActRIIB antibody having: a full length heavy chain
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 99-112; and a full length light chain
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 85-98, or [0069] (ii) a functional
fragment or functional protein comprising an antigen binding
portion thereof. In another aspect, the disclosure provides: [0070]
(i) an isolated recombinant anti-ActRIIB antibody having a full
length heavy chain encoded by a nucleotide sequence that has been
optimized for expression in the cell of a mammalian selected from
the group consisting of SEQ ID NOs: 127-140, and a full length
light chain encoded by a nucleotide sequence that has been
optimized for expression in the cell of a mammalian selected from
the group consisting of SEQ ID NOs: 113-126, or [0071] (ii) a
functional fragment or functional protein comprising an antigen
binding portion thereof.
[0072] The amino acid sequences of the V.sub.H CDR1s of the
antibodies are shown in SEQ ID NOs: 1-14. The amino acid sequences
of the V.sub.H CDR2s of the antibodies are shown in SEQ ID NOs:
15-28. The amino acid sequences of the V.sub.H CDR3s of the
antibodies are shown in SEQ ID NOs: 29-42. The amino acid sequences
of the V.sub.L CDR1s of the antibodies are shown in SEQ ID NOs:
43-56. The amino acid sequences of the V.sub.L CDR2s of the
antibodies are shown in SEQ ID NOs: 57-70. The amino acid sequences
of the V.sub.L CDR3s of the antibodies are shown in SEQ ID NOs:
71-84. The CDR regions are delineated using the Kabat system
(Kabat, E. A., et al., 1991 Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242). An alternative method of
determining CDR regions uses the method devised by Chothia (Chothia
et al. 1989, Nature, 342:877-883). The Chothia definition is based
on the location of the structural loop regions. However, due to
changes in the numbering system used by Chothia (see e.g.
http://www.biochem.ucl.ac.uk/.about.martin/abs/GeneralInfo.html and
http://www.bioinf.org.uk/abs/), this system is now less commonly
used. Other systems for defining CDRs exist and are also mentioned
in these two websites.
[0073] Given that each of these antibodies can bind to ActRIIB and
that antigen-binding specificity is provided primarily by the CDR1,
2 and 3 regions, the V.sub.H CDR1, 2 and 3 sequences and V.sub.L
CDR1, 2 and 3 sequences can be "mixed and matched" (i.e. CDRs from
different antibodies can be mixed and matched, each antibody
containing a V.sub.H CDR1, 2 and 3 and a V.sub.L CDR1, 2 and 3
create other anti-ActRIIB binding molecules of the disclosure.
ActRIIB binding of such "mixed and matched" antibodies can be
tested using the binding assays described above and in the Examples
(e.g. ELISAs). When V.sub.H CDR sequences are mixed and matched,
the CDR1, CDR2 and/or CDR3 sequence from a particular V.sub.H
sequence should be replaced with a structurally similar CDR
sequence(s). Likewise, when V.sub.L CDR sequences are mixed and
matched, the CDR1, CDR2 and/or CDR3 sequence from a particular
V.sub.L sequence should be replaced with a structurally similar CDR
sequence(s). It will be readily apparent to the ordinarily skilled
artisan that novel V.sub.H and V.sub.L sequences can be created by
substituting one or more V.sub.H and/or V.sub.L CDR region
sequences with structurally similar sequences from the CDR
sequences shown herein for monoclonal antibodies of the present
disclosure.
[0074] An isolated recombinant anti-ActRIIB antibody, or antigen
binding region thereof has: a heavy chain variable region CDR1
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 1-14; a heavy chain variable region CDR2
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 15-28; a heavy chain variable region CDR3
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 29-42; a light chain variable region CDR1
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 43-56; a light chain variable region CDR2
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 57-70; and a light chain variable region
CDR3 comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 71-84.
[0075] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 1; a heavy chain variable region
CDR2 of SEQ ID NO: 15; a heavy chain variable region CDR3 of SEQ ID
NO: 29; a light chain variable region CDR1 of SEQ ID NO: 43; a
light chain variable region CDR2 of SEQ ID NO: 57; and a light
chain variable region CDR3 of SEQ ID NO: 71.
[0076] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 2 a heavy chain variable region
CDR2 of SEQ ID NO: 16; a heavy chain variable region CDR3 of SEQ ID
NO: 30; a light chain variable region CDR1 of SEQ ID NO: 44; a
light chain variable region CDR2 of SEQ ID NO: 58; and a light
chain variable region CDR3 of SEQ ID NO: 72.
[0077] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 3; a heavy chain variable region
CDR2 of SEQ ID NO: 17; a heavy chain variable region CDR3 of SEQ ID
NO: 31; a light chain variable region CDR1 of SEQ ID NO: 45; a
light chain variable region CDR2 of SEQ ID NO: 59; and a light
chain variable region CDR3 of SEQ ID NO: 73.
[0078] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 4; a heavy chain variable region
CDR2 of SEQ ID NO: 18; a heavy chain variable region CDR3 of SEQ ID
NO: 32; a light chain variable region CDR1 of SEQ ID NO: 46; a
light chain variable region CDR2 of SEQ ID NO: 60; and a light
chain variable region CDR3 of SEQ ID NO: 74.
[0079] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 5; a heavy chain variable region
CDR2 of SEQ ID NO: 19; a heavy chain variable region CDR3 of SEQ ID
NO: 33; a light chain variable region CDR1 of SEQ ID NO: 47; a
light chain variable region CDR2 of SEQ ID NO: 61; and a light
chain variable region CDR3 of SEQ ID NO: 75.
[0080] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 6; a heavy chain variable region
CDR2 of SEQ ID NO: 20; a heavy chain variable region CDR3 of SEQ ID
NO: 34; a light chain variable region CDR1 of SEQ ID NO: 48; a
light chain variable region CDR2 of SEQ ID NO: 62; and a light
chain variable region CDR3 of SEQ ID NO: 76.
[0081] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 7; a heavy chain variable region
CDR2 of SEQ ID NO: 21; a heavy chain variable region CDR3 of SEQ ID
NO: 35; a light chain variable region CDR1 of SEQ ID NO: 49; a
light chain variable region CDR2 of SEQ ID NO: 63; and a light
chain variable region CDR3 of SEQ ID NO: 77.
[0082] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 8; a heavy chain variable region
CDR2 of SEQ ID NO: 22; a heavy chain variable region CDR3 of SEQ ID
NO: 36; a light chain variable region CDR1 of SEQ ID NO: 50 a light
chain variable region CDR2 of SEQ ID NO: 64; and a light chain
variable region CDR3 of SEQ ID NO: 78.
[0083] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 9; a heavy chain variable region
CDR2 of SEQ ID NO: 23; a heavy chain variable region CDR3 of SEQ ID
NO: 37; a light chain variable region CDR1 of SEQ ID NO: 51; a
light chain variable region CDR2 of SEQ ID NO: 65; and a light
chain variable region CDR3 of SEQ ID NO: 79.
[0084] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 10; a heavy chain variable
region CDR2 of SEQ ID NO: 24; a heavy chain variable region CDR3 of
SEQ ID NO: 38; a light chain variable region CDR1 of SEQ ID NO: 52;
a light chain variable region CDR2 of SEQ ID NO: 66; and a light
chain variable region CDR3 of SEQ ID NO: 80.
[0085] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 11; a heavy chain variable
region CDR2 of SEQ ID NO: 25; a heavy chain variable region CDR3 of
SEQ ID NO: 39; a light chain variable region CDR1 of SEQ ID NO: 53;
a light chain variable region CDR2 of SEQ ID NO: 67; and a light
chain variable region CDR3 of SEQ ID NO: 81.
[0086] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 12; a heavy chain variable
region CDR2 of SEQ ID NO: 26; a heavy chain variable region CDR3 of
SEQ ID NO: 40; a light chain variable region CDR1 of SEQ ID NO: 54;
a light chain variable region CDR2 of SEQ ID NO: 68; and a light
chain variable region CDR3 of SEQ ID NO: 82.
[0087] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 13; a heavy chain variable
region CDR2 of SEQ ID NO: 27; a heavy chain variable region CDR3 of
SEQ ID NO: 41; a light chain variable region CDR1 of SEQ ID NO: 55;
a light chain variable region CDR2 of SEQ ID NO: 69; and a light
chain variable region CDR3 of SEQ ID NO: 83.
[0088] In one embodiment, the antibody comprises: a heavy chain
variable region CDR1 of SEQ ID NO: 14; a heavy chain variable
region CDR2 of SEQ ID NO: 28; a heavy chain variable region CDR3 of
SEQ ID NO: 42; a light chain variable region CDR1 of SEQ ID NO: 56;
a light chain variable region CDR2 of SEQ ID NO: 70; and a light
chain variable region CDR3 of SEQ ID NO: 84.
[0089] In one embodiment, the disclosure provides an antibody
comprising: (a) the variable heavy chain sequence of SEQ ID NO: 85
and variable light chain sequence of SEQ ID NO: 99; (b) the
variable heavy chain sequence of SEQ ID NO: 86 and variable light
chain sequence of SEQ ID NO: 100; (c) the variable heavy chain
sequence of SEQ ID NO: 87 and variable light chain sequence of SEQ
ID NO: 101; (d) the variable heavy chain sequence of SEQ ID NO: 88
and variable light chain sequence of SEQ ID NO: 102; (e) the
variable heavy chain sequence of SEQ ID NO: 89 and variable light
chain sequence of SEQ ID NO: 103; (f) the variable heavy chain
sequence of SEQ ID NO: 90 and variable light chain sequence of SEQ
ID NO: 104; (g) the variable heavy chain sequence of SEQ ID NO: 91
and variable light chain sequence of SEQ ID NO: 105; (h) the
variable heavy chain sequence of SEQ ID NO: 92 and variable light
chain sequence of SEQ ID NO: 106; (i) the variable heavy chain
sequence of SEQ ID NO: 93 and variable light chain sequence of SEQ
ID NO: 107; (j) the variable heavy chain sequence of SEQ ID NO: 94
and variable light chain sequence of SEQ ID NO: 108; (k) the
variable heavy chain sequence of SEQ ID NO: 95 and variable light
chain sequence of SEQ ID NO: 109; (1) the variable heavy chain
sequence of SEQ ID NO: 96 and variable light chain sequence of SEQ
ID NO: 110; (m) the variable heavy chain sequence of SEQ ID NO: 97
and variable light chain sequence of SEQ ID NO: 111; or (n) the
variable heavy chain sequence of SEQ ID NO: 98 and variable light
chain sequence of SEQ ID NO: 112.
[0090] In one embodiment, the disclosure provides an antibody
comprising: (a) the heavy chain sequence of SEQ ID NO: 146 and
light chain sequence of SEQ ID NO: 141; (b) the heavy chain
sequence of SEQ ID NO: 147 and light chain sequence of SEQ ID NO:
142; (c) the heavy chain sequence of SEQ ID NO: 148 and light chain
sequence of SEQ ID NO: 143; (d) the heavy chain sequence of SEQ ID
NO: 149 and light chain sequence of SEQ ID NO: 144; (e) the heavy
chain sequence of SEQ ID NO: 150 and light chain sequence of SEQ ID
NO: 145; (f) the heavy chain sequence of SEQ ID NO: 156 and light
chain sequence of SEQ ID NO: 151; (g) the heavy chain sequence of
SEQ ID NO: 157 and light chain sequence of SEQ ID NO: 152; (h) the
heavy chain sequence of SEQ ID NO: 158 and light chain sequence of
SEQ ID NO: 153; (i) the heavy chain sequence of SEQ ID NO: 159 and
light chain sequence of SEQ ID NO: 154; or (j) the heavy chain
sequence of SEQ ID NO: 160 and light chain sequence of SEQ ID NO:
155.
[0091] As used herein, a human antibody comprises heavy or light
chain variable regions or full length heavy or light chains that
are "the product of" or "derived from" a particular germline
sequence if the variable regions or full length chains of the
antibody are obtained from a system that uses human germline
immunoglobulin genes. Such systems include immunizing a transgenic
mouse carrying human immunoglobulin genes with the antigen of
interest or screening a human immunoglobulin gene library displayed
on phage with the antigen of interest. A human antibody that is
"the product of" or "derived from" a human germline immunoglobulin
sequence can be identified as such by comparing the amino acid
sequence of the human antibody to the amino acid sequences of human
germline immunoglobulins and selecting the human germline
immunoglobulin sequence that is closest in sequence (i.e. greatest
% identity) to the sequence of the human antibody. A human antibody
that is "the product of" or "derived from" a particular human
germline immunoglobulin sequence may contain amino acid differences
as compared to the germline sequence, due to, for example,
naturally occurring somatic mutations or intentional introduction
of site-directed mutation. However, a selected human antibody
typically is at least 90% identical in amino acids sequence to an
amino acid sequence encoded by a human germline immunoglobulin gene
and contains amino acid residues that identify the human antibody
as being human when compared to the germline immunoglobulin amino
acid sequences of other species (e.g. murine germline sequences).
In certain cases, a human antibody may be at least 80%, 90%, or at
least 95%, or even at least 96%, 97%, 98%, or 99% identical in
amino acid sequence to the amino acid sequence encoded by the
germline immunoglobulin gene. Typically, a human antibody derived
from a particular human germline sequence will display no more than
10 amino acid differences from the amino acid sequence encoded by
the human germline immunoglobulin gene. In certain cases, the human
antibody may display no more than 5, or even no more than 4, 3, 2,
or 1 amino acid difference from the amino acid sequence encoded by
the germline immunoglobulin gene.
[0092] In one embodiment the antibody of the disclosure is that
encoded by pBW522 or pBW524 (deposited at DSMZ, Inhoffenstr. 7B,
D-38124 Braunschweig, Germany on 18 Aug. 2009 under deposit numbers
DSM22873 and DSM22874, respectively).
Homologous Antibodies
[0093] In yet another embodiment, an antibody of the disclosure has
full length heavy and light chain amino acid sequences; full length
heavy and light chain nucleotide sequences, variable region heavy
and light chain nucleotide sequences, or variable region heavy and
light chain amino acid sequences that are homologous to the amino
acid and nucleotide sequences of the antibodies described herein,
and wherein the antibodies retain the desired functional properties
of the anti-ActRIIB antibodies of the disclosure.
[0094] For example, the disclosure provides an isolated recombinant
anti-ActRIIB antibody (or a functional fragment or functional
protein comprising an antigen binding portion thereof) comprising a
heavy chain variable region and a light chain variable region,
wherein: the heavy chain variable region comprises an amino acid
sequence that is at least 80%, or at least 90% (in various
embodiments, at least 95, 97 or 99%) identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 99-112;
the light chain variable region comprises an amino acid sequence
that is at least 80%, or at least 90% (in various embodiments, at
least 95, 97 or 99%) identical to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 85-98; and the antibody
exhibits at least one of the following functional properties: (i)
it inhibits myostatin binding in vitro or in vivo and/or (ii)
decreases inhibition of muscle differentiation through the
Smad-dependent pathway.
[0095] In a further example, the disclosure provides an isolated
recombinant anti-ActRIIB antibody, (or a functional fragment or
functional protein comprising an antigen binding portion thereof)
comprising a full length heavy chain and a full length light chain,
wherein: the full length heavy chain comprises an amino acid
sequence that is at least 80%, or at least 90% (in various
embodiments, at least 95, 97 or 99%) identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 146-150
and 156-160; the full length light chain comprises an amino acid
sequence that is at least 80%, or at least 90% (in various
embodiments, at least 95, 97 or 99%) identical to an amino acid
sequence selected from the group consisting of SEQ ID NOs: 141-145
and 151-155; and the antibody exhibits at least one of the
following functional properties: (i) it inhibits myostatin binding
in vitro or in vivo and/or (ii) decreases inhibition of muscle
differentiation through the Smad-dependent pathway. In one
embodiment, such an antibody binds to the ligand binding domain of
ActRIIB.
[0096] In another example, the disclosure provides an isolated
recombinant anti-ActRIIB antibody (or a functional fragment or
functional protein comprising an antigen binding portion thereof),
comprising a full length heavy chain and a full length light chain,
wherein: the full length heavy chain is encoded by a nucleotide
sequence that is at least 80%, or at least 90% (in various
embodiments, at least 95, 97 or 99%) identical to a nucleotide
sequence selected from the group consisting of SEQ ID NOs: 166-170
and 176-180; the full length light chain is encoded by a nucleotide
sequence that is at least 80%, or at least 90% (in various
embodiments, at least 95, 97 or 99%) identical to a nucleotide
sequence selected from the group consisting of SEQ ID NOs: 161-165
and 171-175; and the antibody exhibits at least one of the
following functional properties: (i) it inhibits myostatin binding
in vitro or in vivo and/or (ii) decreases inhibition of muscle
differentiation through the Smad-dependent pathway. In various
embodiments such an antibody binds to the ligand binding domain of
ActRIIB.
[0097] In various embodiments, the antibody may exhibit one or
more, two or more, or three of the functional properties discussed
above. The antibody can be, for example, a human antibody, a
humanized antibody or a chimeric antibody. In various embodiments,
the antibody is a fully human IgG1 antibody.
[0098] In other embodiments, the V.sub.H and/or V.sub.L amino acid
sequences may be 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to
the sequences set forth above. In other embodiments, the V.sub.H
and/or V.sub.L amino acid sequences may be identical except an
amino acid substitution in no more than 1, 2, 3, 4 or 5 amino acid
position. An antibody having V.sub.H and V.sub.L regions having
high (i.e. 80% or greater) sequence identity to the V.sub.H and
V.sub.L regions of SEQ ID NOs 99-112 and SEQ ID NOs: 85-98
respectively, can be obtained by mutagenesis (e.g. site-directed or
PCR-mediated mutagenesis) of nucleic acid molecules SEQ ID NOs:
127-140 and 113-126 respectively, followed by testing of the
encoded altered antibody for retained function (i.e. the functions
set forth above) using the functional assays described herein.
[0099] In other embodiments, the full length heavy chain and/or
full length light chain amino acid sequences may be 80%, 90%, 95%,
96%, 97%, 98% or 99% identical to the sequences set forth above. An
antibody having a full length heavy chain and full length light
chain having high (i.e. 80% or greater) identity to the full length
heavy chains of any of SEQ ID NOs: 146-150 and 156-160 and full
length light chains of any of SEQ ID NOs: 141-145 and 151-155
respectively, can be obtained by mutagenesis (e.g. site-directed or
PCR-mediated mutagenesis) of nucleic acid molecules SEQ ID NOs:
166-170 and 176-180 and SEQ ID NOs: 161-165 and 171-175
respectively, followed by testing of the encoded altered antibody
for retained function (i.e. the functions set forth above) using
the functional assays described herein.
[0100] In other embodiments, the full length heavy chain and/or
full length light chain nucleotide sequences may be 80%, 90%, 95%,
96%, 97%, 98% or 99% identical to the sequences set forth
above.
[0101] In other embodiments, the variable regions of heavy chain
and/or light chain nucleotide sequences may be 80%, 90%, 95%, 96%,
97%, 98% or 99% identical to the sequences set forth above.
[0102] As used herein, the percent identity between the two
sequences is a function of the number of identical positions shared
by the sequences (i.e. % identity=# of identical positions/total #
of positions.times.100), taking into account the number of gaps,
and the length of each gap, which need to be introduced for optimal
alignment of the two sequences. The comparison of sequences and
determination of percent identity between two sequences can be
accomplished using a mathematical algorithm, as described
below.
[0103] The percent identity between two amino acid sequences can be
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4:11-17, 1988) which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4. In addition, the
percent identity between two amino acid sequences can be determined
using the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970)
algorithm which has been incorporated into the GAP program in the
GCG software package (available at http://www.gcg.com), using
either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6.
Antibodies with Conservative Modifications
[0104] In certain embodiments, an antibody of the disclosure has a
heavy chain variable region comprising CDR1, CDR2, and CDR3
sequences and a light chain variable region comprising CDR1, CDR2,
and CDR3 sequences, wherein one or more of these CDR sequences have
specified amino acid sequences based on the antibodies described
herein or conservative modifications thereof, and wherein the
antibodies retain the desired functional properties of the
anti-ActRIIB antibodies of the disclosure. Accordingly, the
disclosure provides an isolated recombinant anti-ActRIIB antibody,
or a functional fragment or functional protein comprising an
antigen binding portion thereof, consisting of a heavy chain
variable region comprising CDR1, CDR2, and CDR3 sequences and a
light chain variable region comprising CDR1, CDR2, and CDR3
sequences, wherein: the heavy chain variable region CDR1 amino acid
sequences are selected from the group consisting of SEQ ID NOs:
1-14, and conservative modifications thereof; the heavy chain
variable region CDR2 amino acid sequences are selected from the
group consisting of SEQ ID NOs: 15-28, and conservative
modifications thereof; the heavy chain variable region CDR3 amino
acid sequences are selected from the group consisting of SEQ ID
NOs: 29-42, and conservative modifications thereof; the light chain
variable regions CDR1 amino acid sequences are selected from the
group consisting of SEQ ID NOs: 43-56, and conservative
modifications thereof; the light chain variable regions CDR2 amino
acid sequences are selected from the group consisting of SEQ ID
NOs: 57-70, and conservative modifications thereof; the light chain
variable regions of CDR3 amino acid sequences are selected from the
group consisting of SEQ ID NOs: 71-84, and conservative
modifications thereof. In various embodiments, the antibody
exhibits at least one of the following functional properties: (i)
it inhibits myostatin binding in vitro or in vivo and/or (ii)
decreases inhibition of muscle differentiation through the
Smad-dependent pathway.
[0105] In various embodiments, the antibody may exhibit one or both
of the functional properties listed above. Such antibodies can be,
for example, human antibodies, humanized antibodies or chimeric
antibodies.
[0106] In other embodiments, an antibody of the disclosure
optimized for expression in a mammalian cell has a full length
heavy chain sequence and a full length light chain sequence,
wherein one or more of these sequences have specified amino acid
sequences based on the antibodies described herein or conservative
modifications thereof, and wherein the antibodies retain the
desired functional properties of the anti-ActRIIB antibodies of the
disclosure. Accordingly, the disclosure provides an isolated
monoclonal anti-ActRIIB antibody optimized for expression in a
mammalian cell consisting of a full length heavy chain and a full
length light chain wherein: the full length heavy chain has amino
acid sequences selected from the group of SEQ ID NOs: 146-150 and
156-160, and conservative modifications thereof; and the full
length light chain has amino acid sequences selected from the group
of SEQ ID NOs: 141-145 and 151-155, and conservative modifications
thereof; and the antibody exhibits at least one of the following
functional properties: (i) it inhibits myostatin binding in vitro
or in vivo and/or (ii) decreases inhibition of muscle
differentiation through the Smad-dependent pathway.
[0107] In various embodiments, the antibody may exhibit one or both
of the functional properties listed above. Such antibodies can be,
for example, human antibodies, humanized antibodies or chimeric
antibodies.
[0108] As used herein, the term "conservative sequence
modifications" is intended to refer to amino acid modifications
that do not significantly affect or alter the binding
characteristics of the antibody containing the amino acid sequence.
Such conservative modifications include amino acid substitutions,
additions and deletions. Modifications can be introduced into an
antibody of the disclosure by standard techniques known in the art,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
[0109] Conservative amino acid substitutions are ones in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g. lysine, arginine,
histidine), acidic side chains (e.g. aspartic acid, glutamic acid),
uncharged polar side chains (e.g. glycine, asparagine, glutamine,
serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side
chains (e.g. alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine), beta-branched side chains (e.g.
threonine, valine, isoleucine) and aromatic side chains (e.g.
tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more
amino acid residues within the CDR regions of an antibody of the
disclosure can be replaced with other amino acid residues from the
same side chain family, and the altered antibody can be tested for
retained function using the functional assays described herein.
Antibodies that Bind to the Same Epitope as Anti-ActRIIB
Antibodies
[0110] In another embodiment, the disclosure provides antibodies
that bind to the same epitope as the various specific anti-ActRIIB
antibodies of the disclosure described herein. All the antibodies
described in the Examples that are capable of blocking myostatin
binding to ActRIIB bind the same epitope in ActRIIB with high
affinity, said epitope being comprised between amino acids 19-134
of SEQ ID NO:181.
[0111] Additional antibodies can therefore be identified based on
their ability to cross-compete (e.g. to competitively inhibit the
binding of, in a statistically significant manner) with other
antibodies of the disclosure in standard ActRIIB binding assays.
The ability of a test antibody to inhibit the binding of antibodies
of the present disclosure to human ActRIIB demonstrates that the
test antibody can compete with that antibody for binding to human
ActRIIB; such an antibody may, according to non-limiting theory,
bind to the same or a related (e.g. a structurally similar or
spatially proximal) epitope on human ActRIIB as the antibody with
which it competes. In a certain embodiment, the antibody that binds
to the same epitope on human ActRIIB as the antibodies of the
present disclosure is a human recombinant antibody. Such human
recombinant antibodies can be prepared and isolated as described in
the Examples.
[0112] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 85, and the variable light chain
sequence recited in SEQ ID NO: 99.
[0113] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 86, and the variable light chain
sequence recited in SEQ ID NO: 100.
[0114] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 87, and the variable light chain
sequence recited in SEQ ID NO: 101.
[0115] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 88, and the variable light chain
sequence recited in SEQ ID NO: 102.
[0116] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 89, and the variable light chain
sequence recited in SEQ ID NO: 103.
[0117] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 90, and the variable light chain
sequence recited in SEQ ID NO: 104.
[0118] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 91, and the variable light chain
sequence recited in SEQ ID NO: 105.
[0119] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 92, and the variable light chain
sequence recited in SEQ ID NO: 106.
[0120] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 93, and the variable light chain
sequence recited in SEQ ID NO: 107.
[0121] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 94, and the variable light chain
sequence recited in SEQ ID NO: 108.
[0122] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 95, and the variable light chain
sequence recited in SEQ ID NO: 109.
[0123] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 96, and the variable light chain
sequence recited in SEQ ID NO: 110.
[0124] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 97, and the variable light chain
sequence recited in SEQ ID NO: 111.
[0125] Thus, the disclosure provides an antibody that binds to an
epitope recognised by an antibody having the variable heavy chain
sequence recited in SEQ ID NO: 98, and the variable light chain
sequence recited in SEQ ID NO: 112.
[0126] Following more detailed epitope mapping experiments, the
binding regions of particular antibodies of the disclosure have
been more clearly defined.
[0127] Thus, the disclosure provides an antibody that binds to an
epitope comprising amino acids 78-83 of SEQ ID NO: 181 (WLDDFN--SEQ
ID NO:188).
[0128] The disclosure also provides an antibody that binds to an
epitope comprising amino acids 76-84 of SEQ ID NO: 181
(GCWLDDFNC--SEQ ID NO:186).
[0129] The disclosure also provides an antibody that binds to an
epitope comprising amino acids 75-85 of SEQ ID NO: 181
(KGCWLDDFNCY--SEQ ID NO:190).
[0130] The disclosure also provides an antibody that binds to an
epitope comprising amino acids 52-56 of SEQ ID NO: 181 (EQDKR--SEQ
ID NO:189).
[0131] The disclosure also provides an antibody that binds to an
epitope comprising amino acids 49-63 of SEQ ID NO: 181
(CEGEQDKRLHCYASW--SEQ ID NO:187).
[0132] The disclosure also provides antibodies that bind to
epitopes consisting of these sequences or epitopes comprising
combinations of these epitope regions.
[0133] Thus, the disclosure also provides an antibody that binds to
an epitope comprising or consisting of amino acids 78-83 of SEQ ID
NO: 181 (WLDDFN) and amino acids 52-56 of SEQ ID NO: 181
(EQDKR).
Engineered and Modified Antibodies
[0134] An antibody of the disclosure further can be prepared using
an antibody having one or more of the V.sub.H and/or V.sub.L
sequences shown herein as starting material to engineer a modified
antibody, which modified antibody may have altered properties from
the starting antibody. An antibody can be engineered by modifying
one or more residues within one or both variable regions (i.e.
V.sub.H and/or V.sub.L), for example within one or more CDR regions
and/or within one or more framework regions. Additionally or
alternatively, an antibody can be engineered by modifying residues
within the constant region(s), for example to alter the effector
function(s) of the antibody.
[0135] One type of variable region engineering that can be
performed is CDR grafting. Antibodies interact with target antigens
predominantly through amino acid residues that are located in the
six heavy and light chain complementarity determining regions
(CDRs). For this reason, the amino acid sequences within CDRs are
more diverse between individual antibodies than sequences outside
of CDRs. Because CDR sequences are responsible for most
antibody-antigen interactions, it is possible to express
recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody
with different properties (see, e.g. Riechmann, L. et al., 1998
Nature 332:323-327; Jones, P. et al., 1986 Nature 321:522-525;
Queen, C. et al., 1989 Proc. Natl. Acad. Sci. U.S.A.
86:10029-10033; U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.).
[0136] Accordingly, another embodiment of the disclosure pertains
to an isolated monoclonal anti-ActRIIB antibody, or a functional
fragment or functional protein comprising an antigen binding
portion thereof, comprising a heavy chain variable region
comprising CDR1 sequences having an amino acid sequence selected
from the group consisting of SEQ ID NOs: 1-14; CDR2 sequences
having an amino acid sequence selected from the group consisting of
SEQ ID NOs: 15-28; CDR3 sequences having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 29-42,
respectively; and a light chain variable region having CDR1
sequences having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 43-56; CDR2 sequences having an amino
acid sequence selected from the group consisting of SEQ ID NOs:
57-70; and CDR3 sequences consisting of an amino acid sequence
selected from the group consisting of SEQ ID NOs: 71-84,
respectively. Thus, such antibodies contain the V.sub.H and V.sub.L
CDR sequences of monoclonal antibodies, yet may contain different
framework sequences from these antibodies.
[0137] Such framework sequences can be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for human heavy
and light chain variable region genes can be found in the "VBase"
human germline sequence database (available on the Internet at
www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al.,
[supra]; Tomlinson, I. M., et al., 1992 J. fol. Biol. 227:776-798;
and Cox, J. P. L. et al., 1994 Eur. J. Immunol 24:827-836.
[0138] An example of framework sequences for use in the antibodies
of the disclosure are those that are structurally similar to the
framework sequences used by selected antibodies of the disclosure,
e.g. consensus sequences and/or framework sequences used by
monoclonal antibodies of the disclosure. The V.sub.H CDR1, 2 and 3
sequences, and the V.sub.L CDR1, 2 and 3 sequences, can be grafted
onto framework regions that have the identical sequence as that
found in the germline immunoglobulin gene from which the framework
sequence derive, or the CDR sequences can be grafted onto framework
regions that contain one or more mutations as compared to the
germline sequences. For example, it has been found that in certain
instances it is beneficial to mutate residues within the framework
regions to maintain or enhance the antigen binding ability of the
antibody (see e.g. U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762
and 6,180,370 to Queen et al). Another type of variable region
modification is to mutate amino acid residues within the V.sub.H
and/or V.sub.L CDR1, CDR2 and/or CDR3 regions to thereby improve
one or more binding properties (e.g. affinity) of the antibody of
interest, known as "affinity maturation." Site-directed mutagenesis
or PCR-mediated mutagenesis can be performed to introduce the
mutation(s) and the effect on antibody binding, or other functional
property of interest, can be evaluated in in vitro or in vivo
assays as described herein and provided in the Examples.
Conservative modifications (as discussed above) can be introduced.
The mutations may be amino acid substitutions, additions or
deletions. Moreover, typically no more than one, two, three, four
or five residues within a CDR region are altered.
[0139] Accordingly, in another embodiment, the disclosure provides
isolated anti-ActRIIB monoclonal antibodies, or a functional
fragment or functional protein comprising an antigen binding
portion thereof, consisting of a heavy chain variable region
having: a V.sub.H CDR1 region consisting of an amino acid sequence
selected from the group having SEQ ID NOs: 1-14 or an amino acid
sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID NOs:
1-14; a V.sub.H CDR2 region having an amino acid sequence selected
from the group consisting of SEQ ID NOs: 15-28, or an amino acid
sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID NOs:
15-28; a V.sub.H CDR3 region having an amino acid sequence selected
from the group consisting of SEQ ID NOs: 29-42, or an amino acid
sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID NOs:
29-42; a V.sub.L CDR1 region having an amino acid sequence selected
from the group consisting of SEQ ID NOs: 43-56, or an amino acid
sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID NOs:
43-56; a V.sub.L CDR2 region having an amino acid sequence selected
from the group consisting of SEQ ID NOs: 52-70, or an amino acid
sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID NOs:
52-70; and a V.sub.L CDR3 region having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 71-84, or an
amino acid sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID NOs:
71-84.
Grafting Antigen-Binding Domains into Alternative Frameworks or
Scaffolds
[0140] A wide variety of antibody/immunoglobulin frameworks or
scaffolds can be employed so long as the resulting polypeptide
includes at least one binding region which specifically binds to
ActRIIB. Such frameworks or scaffolds include the 5 main idiotypes
of human immunoglobulins, or fragments thereof (such as those
disclosed elsewhere herein), and include immunoglobulins of other
animal species, for example, having humanized aspects. Single
heavy-chain antibodies such as those identified in camelids are of
particular interest in this regard. Novel frameworks, scaffolds and
fragments continue to be discovered and developed by those skilled
in the art.
[0141] In one aspect, the disclosure pertains to generating
non-immunoglobulin based antibodies using non-immunoglobulin
scaffolds onto which CDRs of the disclosure can be grafted. Known
or future non-immunoglobulin frameworks and scaffolds may be
employed, as long as they comprise a binding region specific for
the target protein of SEQ ID NO: 181 (in various embodiments, the
ligand binding domain thereof as shown in SEQ ID NO: 182). Such
compounds are known herein as "polypeptides comprising a
target-specific binding region". Examples of non-immunoglobulin
framework are further described in the sections below (camelid
antibodies and non-antibody scaffold).
Camelid Antibodies
[0142] Antibody proteins obtained from members of the camel and
dromedary family (Camelus bactrianus and Camelus dromaderius)
including new world members such as llama species (Lama paccos,
Lama glama and Lama vicugna) have been characterized with respect
to size, structural complexity and antigenicity for human subjects.
Certain IgG antibodies from this family of mammals as found in
nature lack light chains, and are thus structurally distinct from
the typical four chain quaternary structure having two heavy and
two light chains, for antibodies from other animals (see
WO94/04678).
[0143] A region of the camelid antibody which is the small single
variable domain identified as V.sub.HH can be obtained by genetic
engineering to yield a small protein having high affinity for a
target, resulting in a low molecular weight antibody-derived
protein known as a "camelid nanobody" (see U.S. Pat. No. 5,759,808;
Stijlemans, B. et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin,
M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003
Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int
J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J. 17:
3512-3520). Engineered libraries of camelid antibodies and antibody
fragments are commercially available, for example, from Ablynx,
Ghent, Belgium. As with other antibodies of non-human origin, an
amino acid sequence of a camelid antibody can be altered
recombinantly to obtain a sequence that more closely resembles a
human sequence, i.e. the nanobody can be "humanized". Thus the
natural low antigenicity of camelid antibodies to humans can be
further reduced.
[0144] The camelid nanobody has a molecular weight approximately
one-tenth that of a human IgG molecule, and the protein has a
physical diameter of only a few nanometers. One consequence of the
small size is the ability of camelid nanobodies to bind to
antigenic sites that are functionally invisible to larger antibody
proteins, i.e. camelid nanobodies are useful as reagents detect
antigens that are otherwise cryptic using classical immunological
techniques, and as possible therapeutic agents. Thus yet another
consequence of small size is that a camelid nanobody can inhibit as
a result of binding to a specific site in a groove or narrow cleft
of a target protein, and hence can serve in a capacity that more
closely resembles the function of a classical low molecular weight
drug than that of a classical antibody.
[0145] The low molecular weight and compact size further result in
camelid nanobodies being extremely thermostable, stable to extreme
pH and to proteolytic digestion, and poorly antigenic. Another
consequence is that camelid nanobodies readily move from the
circulatory system into tissues, and even cross the blood-brain
barrier and can treat disorders that affect nervous tissue.
Nanobodies can further facilitate drug transport across the blood
brain barrier (see US2004/0161738). These features combined with
the low antigenicity to humans indicate great therapeutic
potential. Further, these molecules can be fully expressed in
prokaryotic cells such as E. coli and are expressed as fusion
proteins with bacteriophage and are functional.
[0146] Accordingly, a feature of the present disclosure is a
camelid antibody or nanobody having high affinity for ActRIIB. In
certain embodiments herein, the camelid antibody or nanobody is
naturally produced in the camelid animal, i.e. is produced by the
camelid following immunization with ActRIIB or a peptide fragment
thereof, using techniques described herein for other antibodies.
Alternatively, the anti-ActRIIB camelid nanobody is engineered,
i.e. produced by selection for example from a library of phage
displaying appropriately mutagenized camelid nanobody proteins
using panning procedures with ActRIIB as a target as described in
the examples herein. Engineered nanobodies can further be
customized by genetic engineering to have a half life in a
recipient subject of from 45 minutes to two weeks. In a specific
embodiment, the camelid antibody or nanobody is obtained by
grafting the CDRs sequences of the heavy or light chain of the
human antibodies of the disclosure into nanobody or single domain
antibody framework sequences, as described for example in
WO94/04678.
Non-Antibody Scaffold
[0147] Known non-immunoglobulin frameworks or scaffolds include,
but are not limited to, Adnectins (fibronectin) (Compound
Therapeutics, Inc., Waltham, Mass.), ankyrin (Molecular Partners
AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd
(Cambridge, Mass.) and Ablynx nv (Zwijnaarde, Belgium)), lipocalin
(Anticalin) (Pieris Proteolab AG, Freising, Germany), small modular
immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle,
Wash.), maxybodies (Avidia, Inc. (Mountain View, Calif.)), Protein
A (Affibody AG, Sweden) and affilin (gamma-crystallin or ubiquitin)
(Scil Proteins GmbH, Halle, Germany), protein epitope mimetics
(Polyphor Ltd, Allschwil, Switzerland).
(i) Fibronectin Scaffold
[0148] The fibronectin scaffolds are based in various embodiments
on fibronectin type III domain (e.g. the tenth module of the
fibronectin type III (10 Fn3 domain)). The fibronectin type III
domain has 7 or 8 beta strands which are distributed between two
beta sheets, which themselves pack against each other to form the
core of the protein, and further containing loops (analogous to
CDRs) which connect the beta strands to each other and are solvent
exposed. There are at least three such loops at each edge of the
beta sheet sandwich, where the edge is the boundary of the protein
perpendicular to the direction of the beta strands (U.S. Pat. No.
6,818,418).
[0149] These fibronectin-based scaffolds are not an immunoglobulin,
although the overall fold is closely related to that of the
smallest functional antibody fragment, the variable region of the
heavy chain, which comprises the entire antigen recognition unit in
camel and llama IgG. Because of this structure, the
non-immunoglobulin antibody mimics antigen binding properties that
are similar in nature and affinity to those of antibodies. These
scaffolds can be used in a loop randomization and shuffling
strategy in vitro that is similar to the process of affinity
maturation of antibodies in vivo. These fibronectin-based molecules
can be used as scaffolds where the loop regions of the molecule can
be replaced with CDRs of the disclosure using standard cloning
techniques.
(ii) Ankyrin--Molecular Partners
[0150] The technology is based on using proteins with ankyrin
derived repeat modules as scaffolds for bearing variable regions
which can be used for binding to different targets. The ankyrin
repeat module is a 33 amino acid polypeptide consisting of two
anti-parallel .alpha.-helices and a .beta.-turn. Binding of the
variable regions is mostly optimized by using ribosome display.
(iii) Maxybodies/Avimers--Avidia
[0151] Avimers are derived from natural A-domain containing protein
such as LRP-1. These domains are used by nature for protein-protein
interactions and in human over 250 proteins are structurally based
on A-domains. Avimers consist of a number of different "A-domain"
monomers (2-10) linked via amino acid linkers. Avimers can be
created that can bind to the target antigen using the methodology
described in, for example, US2004/0175756; US2005/0053973;
US2005/0048512; and US2006/0008844.
(vi) Protein A--Affibody
[0152] Affibody.RTM. affinity ligands are small, simple proteins
composed of a three-helix bundle based on the scaffold of one of
the IgG-binding domains of Protein A. Protein A is a surface
protein from the bacterium Staphylococcus aureus. This scaffold
domain consists of 58 amino acids, 13 of which are randomized to
generate Affibody.RTM. libraries with a large number of ligand
variants (See e.g. U.S. Pat. No. 5,831,012). Affibody.RTM.
molecules mimic antibodies, they have a molecular weight of 6 kDa,
compared to the molecular weight of antibodies, which is 150 kDa.
In spite of its small size, the binding site of Affibody.RTM.
molecules is similar to that of an antibody.
(v) Anticalins--Pieris
[0153] Anticalins.RTM. are products developed by the company Pieris
ProteoLab AG. They are derived from lipocalins, a widespread group
of small and robust proteins that are usually involved in the
physiological transport or storage of chemically sensitive or
insoluble compounds. Several natural lipocalins occur in human
tissues or body liquids.
[0154] The protein architecture is reminiscent of immunoglobulins,
with hypervariable loops on top of a rigid framework. However, in
contrast with antibodies or their recombinant fragments, lipocalins
are composed of a single polypeptide chain with 160 to 180 amino
acid residues, being just marginally bigger than a single
immunoglobulin domain.
[0155] The set of four loops, which makes up the binding pocket,
shows pronounced structural plasticity and tolerates a variety of
side chains. The binding site can thus be reshaped in a proprietary
process in order to recognize prescribed target molecules of
different shape with high affinity and specificity.
[0156] One protein of lipocalin family, the bilin-binding protein
(BBP) of Pieris brassicae has been used to develop anticalins by
mutagenizing the set of four loops. One example of a patent
application describing "anticalins" is WO1999/16873.
(vi) Affilin--Scil Proteins
[0157] Affilin.TM. molecules are small non-immunoglobulin proteins
which are designed for specific affinities towards proteins and
small molecules. New Affilin.TM. molecules can be very quickly
selected from two libraries, each of which is based on a different
human derived scaffold protein.
[0158] Affilin.TM. molecules do not show any structural homology to
immunoglobulin proteins. Scil Proteins employs two Affilin.TM.
scaffolds, one of which is gamma crystalline, a human structural
eye lens protein and the other is "ubiquitin" superfamily proteins.
Both human scaffolds are very small, show high temperature
stability and are almost resistant to pH changes and denaturing
agents. This high stability is mainly due to the expanded beta
sheet structure of the proteins. Examples of gamma crystalline
derived proteins are described in WO2001/004144 and examples of
"ubiquitin-like" proteins are described in WO2004/106368.
(vii) Protein Epitope Mimetics (PEM)
[0159] PEM are medium-sized, cyclic, peptide-like molecules (MW 1-2
kDa) mimicking beta-hairpin secondary structures of proteins, the
major secondary structure involved in protein-protein
interactions.
Framework or Fc Engineering
[0160] Engineered antibodies of the disclosure include those in
which modifications have been made to framework residues within
V.sub.H and/or V.sub.L, e.g. to improve the properties of the
antibody. Typically such framework modifications are made to
decrease the immunogenicity of the antibody. For example, one
approach is to "backmutate" one or more framework residues to the
corresponding germline sequence. More specifically, an antibody
that has undergone somatic mutation may contain framework residues
that differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived. To return the framework region sequences to
their germline configuration, the somatic mutations can be
"backmutated" to the germline sequence by, for example,
site-directed mutagenesis or PCR-mediated mutagenesis. Such
"backmutated" antibodies are also intended to be encompassed by the
disclosure.
[0161] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T-cell epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in US2003/0153043.
[0162] In addition or alternative to modifications made within the
framework or CDR regions, antibodies of the disclosure may be
engineered to include modifications within the Fc region, typically
to alter one or more functional properties of the antibody, such as
serum half-life, complement fixation, Fc receptor binding, and/or
antigen-dependent cellular cytotoxicity. Furthermore, an antibody
of the disclosure may be chemically modified (e.g. one or more
chemical moieties can be attached to the antibody) or be modified
to alter its glycosylation, again to alter one or more functional
properties of the antibody. Each of these embodiments is described
in further detail below. The numbering of residues in the Fc region
is that of the EU index of Kabat.
[0163] In one embodiment, the hinge region of CH1 is modified such
that the number of cysteine residues in the hinge region is
altered, e.g. increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425. The number of cysteine residues
in the hinge region of CH1 is altered to, for example, facilitate
assembly of the light and heavy chains or to increase or decrease
the stability of the antibody.
[0164] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half-life of the antibody. More
specifically, one or more amino acid mutations are introduced into
the CH2-CH3 domain interface region of the Fc-hinge fragment such
that the antibody has impaired Staphylococcyl protein A (SpA)
binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Pat. No.
6,165,745.
[0165] In another embodiment, the antibody is modified to increase
its biological half-life. Various approaches are possible. For
example, one or more of the following mutations can be introduced:
T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375.
Alternatively, to increase the biological half life, the antibody
can be altered within the CH1 or CL region to contain a salvage
receptor binding epitope taken from two loops of a CH2 domain of an
Fc region of an IgG, as described in U.S. Pat. No. 5,869,046 and
U.S. Pat. No. 6,121,022.
[0166] In yet other embodiments, the Fc region is altered by
replacing at least one amino acid residue with a different amino
acid residue to alter the effector functions of the antibody. For
example, one or more amino acids can be replaced with a different
amino acid residue such that the antibody has an altered affinity
for an effector ligand but retains the antigen-binding ability of
the parent antibody. The effector ligand to which affinity is
altered can be, for example, an Fc receptor or the C1 component of
complement. This approach is described in further detail in U.S.
Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, both by Winter et
al. In particular, residues 234 and 235 may be mutated. In
particular, these mutations may be to alanine. Thus in one
embodiment the antibody of the disclosure has a mutation in the Fc
region at one or both of amino acids 234 and 235. In another
embodiment, one or both of amino acids 234 and 235 may be
substituted to alanine. Substitution of both amino acids 234 and
235 to alanine results in a reduced ADCC activity.
[0167] In another embodiment, one or more amino acids selected from
amino acid residues can be replaced with a different amino acid
residue such that the antibody has altered Clq binding and/or
reduced or abolished complement dependent cytotoxicity (CDC). This
approach is described in further detail in U.S. Pat. No.
6,194,551.
[0168] In another embodiment, one or more amino acid residues are
altered to thereby alter the ability of the antibody to fix
complement. This approach is described further in WO94/29351.
[0169] In yet another embodiment, the Fc region is modified to
increase the ability of the antibody to mediate antibody dependent
cellular cytotoxicity (ADCC) and/or to increase the affinity of the
antibody for an Fc.gamma. receptor by modifying one or more amino
acids. This approach is described further in WO00/42072. Moreover,
the binding sites on human IgG1 for Fc.gamma.RI, Fc.gamma.RII,
Fc.gamma.RIII and FcRn have been mapped and variants with improved
binding have been described (see Shields, R. L. et al., 2001 J.
Biol. Chen. 276:6591-6604).
[0170] In still another embodiment, the glycosylation of an
antibody is modified. For example, an aglycoslated antibody can be
made (i.e. the antibody lacks glycosylation). Glycosylation can be
altered to, for example, increase the affinity of the antibody for
the antigen. Such carbohydrate modifications can be accomplished
by; for example, altering one or more sites of glycosylation within
the antibody sequence. For example, one or more amino acid
substitutions can be made that result in elimination of one or more
variable region framework glycosylation sites to thereby eliminate
glycosylation at that site. Such aglycosylation may increase the
affinity of the antibody for antigen. Such an approach is described
in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co
et al.
[0171] Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, such as a hypofucosylated
antibody having reduced amounts of fucosyl residues or an antibody
having increased bisecting GlcNac structures. Such altered
glycosylation patterns have been demonstrated to increase the ADCC
ability of antibodies. Such carbohydrate modifications can be
accomplished by, for example, expressing the antibody in a host
cell with altered glycosylation machinery. Cells with altered
glycosylation machinery have been described in the art and can be
used as host cells in which to express recombinant antibodies of
the disclosure to thereby produce an antibody with altered
glycosylation. For example, EP 1,176,195 by Hang et al. describes a
cell line with a functionally disrupted FUT8 gene, which encodes a
fucosyl transferase, such that antibodies expressed in such a cell
line exhibit hypofucosylation. Therefore, in one embodiment, the
antibodies of the disclosure are produced by recombinant expression
in a cell line which exhibit hypofucosylation pattern, for example,
a mammalian cell line with deficient expression of the FUT8 gene
encoding fucosyltransferase. WO03/035835 describes a variant CHO
cell line, Lecl3 cells, with reduced ability to attach fucose to
Asn(297)-linked carbohydrates, also resulting in hypofucosylation
of antibodies expressed in that host cell (see also Shields, R. L.
et al., 2002 J. Biol. Chem. 277:26733-26740). WO99/54342 describes
cell lines engineered to express glycoprotein-modifying glycosyl
transferases (e.g. beta(1,4)-N acetylglucosaminyltransferase III
(GnTIII)) such that antibodies expressed in the engineered cell
lines exhibit increased bisecting GlcNac structures which results
in increased ADCC activity of the antibodies (see also Umana et
al., 1999 Nat. Biotech. 17:176-180). Alternatively, the antibodies
of the disclosure can be produced in a yeast or a filamentous fungi
engineered for mammalian-like glycosylation pattern, and capable of
producing antibodies lacking fucose as glycosylation pattern (see
for example EP1297172B1).
[0172] Another modification of the antibodies herein that is
contemplated by the disclosure is pegylation. An antibody can be
pegylated to, for example, increase the biological (e.g. serum)
half-life of the antibody. To pegylate an antibody, the antibody,
or fragment thereof, typically is reacted with polyethylene glycol
(PEG), such as a reactive ester or aldehyde derivative of PEG,
under conditions in which one or more PEG groups become attached to
the antibody or antibody fragment. The pegylation can be carried
out by an acylation reaction or an alkylation reaction with a
reactive PEG molecule (or an analogous reactive water-soluble
polymer). As used herein, the term "polyethylene glycol" is
intended to encompass any of the forms of PEG that have been used
to derivatize other proteins, such as mono (C1-C10) alkoxy- or
aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In
certain embodiments, the antibody to be pegylated is an
aglycosylated antibody. Methods for pegylating proteins are known
in the art and can be applied to the antibodies of the disclosure
(see for example, EP0154316 and EP0401384).
[0173] Another modification of the antibodies that is contemplated
by the disclosure is a conjugate or a protein fusion of at least
the antigen-binding region of the antibody of the disclosure to
serum protein, such as human serum albumin or a fragment thereof to
increase half-life of the resulting molecule (see, for example,
EP0322094).
[0174] Another possibility is a fusion of at least the
antigen-binding region of the antibody of the disclosure to
proteins capable of binding to serum proteins, such human serum
albumin to increase half life of the resulting molecule (see, for
example, EP0486525).
Methods of Engineering Altered Antibodies
[0175] As discussed above, the anti-ActRIIB antibodies having
V.sub.H and V.sub.L sequences or full length heavy and light chain
sequences shown herein can be used to create new anti-ActRIIB
antibodies by modifying full length heavy chain and/or light chain
sequences, V.sub.H and/or V.sub.L sequences, or the constant
region(s) attached thereto. Thus, in another aspect of the
disclosure, the structural features of an anti-ActRIIB antibody of
the disclosure are used to create structurally related anti-ActRIIB
antibodies that retain at least one functional property of the
antibodies of the disclosure, such as binding to human ActRIIB but
also inhibit one or more functional properties of ActRIIB (for
example, the inhibition of Smad activation).
[0176] For example, one or more CDR regions of the antibodies of
the present disclosure, or mutations thereof, can be combined
recombinantly with known framework regions and/or other CDRs to
create additional, recombinantly-engineered, anti-ActRIIB
antibodies of the disclosure, as discussed above. Other types of
modifications include those described in the previous section. The
starting material for the engineering method is one or more of the
V.sub.H and/or V.sub.L sequences provided herein, or one or more
CDR regions thereof. To create the engineered antibody, it is not
necessary to actually prepare (i.e. express as a protein) an
antibody having one or more of the V.sub.H and/or V.sub.L sequences
provided herein, or one or more CDR regions thereof. Rather, the
information contained in the sequence(s) is used as the starting
material to create a "second generation" sequence(s) derived from
the original sequence(s) and then the "second generation"
sequence(s) is prepared and expressed as a protein.
[0177] Accordingly, in another embodiment, the disclosure provides
a method for preparing an anti-ActRIIB antibody consisting of: a
heavy chain variable region antibody sequence having a CDR1
sequence selected from the group consisting of SEQ ID NOs: 1-14, a
CDR2 sequence selected from the group consisting of SEQ ID NOs:
15-28 and/or a CDR3 sequence selected from the group consisting of
SEQ ID NOs: 29-42; and a light chain variable region antibody
sequence having a CDR1 sequence selected from the group consisting
of SEQ ID NOs: 43-56, a CDR2 sequence selected from the group
consisting of SEQ ID NOs: 57-70 and/or a CDR3 sequence selected
from the group consisting of SEQ ID NOs: 71-84; altering at least
one amino acid residue within the heavy chain variable region
antibody sequence and/or the light chain variable region antibody
sequence to create at least one altered antibody sequence; and
expressing the altered antibody sequence as a protein.
[0178] Accordingly, in another embodiment, the disclosure provides
a method for preparing an anti-ActRIIB antibody optimized for
expression in a mammalian cell consisting of: a full length heavy
chain antibody sequence having a sequence selected from the group
of SEQ ID NOs: 146-150 and 156-160; and a full length light chain
antibody sequence having a sequence selected from the group of SEQ
ID NOs: 141-145 and 151-155; altering at least one amino acid
residue within the full length heavy chain antibody sequence and/or
the full length light chain antibody sequence to create at least
one altered antibody sequence; and expressing the altered antibody
sequence as a protein.
[0179] The altered antibody sequence can also be prepared by
screening antibody libraries having fixed CDR3 sequences selected
among the group consisting of SEQ ID NO: 29-42 and SEQ ID NO: 71-84
or minimal essential binding determinants as described in
US2005/0255552 and diversity on CDR1 and CDR2 sequences. The
screening can be performed according to any screening technology
appropriate for screening antibodies from antibody libraries, such
as phage display technology.
[0180] Standard molecular biology techniques can be used to prepare
and express the altered antibody sequence. The antibody encoded by
the altered antibody sequence(s) is one that retains one, some or
all of the functional properties of the anti-ActRIIB antibodies
described herein, which functional properties include, but are not
limited to, specifically binding to human ActRIIB and inhibition of
Smad activation.
[0181] The altered antibody may exhibit one or more, two or more,
or three or more of the functional properties discussed above.
[0182] The functional properties of the altered antibodies can be
assessed using standard assays available in the art and/or
described herein, such as those set forth in the Examples (e.g.
ELISAs).
[0183] In certain embodiments of the methods of engineering
antibodies of the disclosure, mutations can be introduced randomly
or selectively along all or part of an anti-ActRIIB antibody coding
sequence and the resulting modified anti-ActRIIB antibodies can be
screened for binding activity and/or other functional properties as
described herein. Mutational methods have been described in the
art. For example, WO02/092780 describes methods for creating and
screening antibody mutations using saturation mutagenesis,
synthetic ligation assembly, or a combination thereof.
Alternatively, WO03/074679 describes methods of using computational
screening methods to optimize physiochemical properties of
antibodies.
Nucleic Acid Molecules Encoding Particular Antibodies
[0184] Another aspect of the disclosure pertains to nucleic acid
molecules that encode the antibodies of the disclosure. Examples of
full length light chain nucleotide sequences optimized for
expression in a mammalian cell are shown in SEQ ID NOs: 161-165 and
171-175. Examples of full length heavy chain nucleotide sequences
optimized for expression in a mammalian cell are shown in SEQ ID
NOs: 166-170 and 176-180.
[0185] The nucleic acids may be present in whole cells, in a cell
lysate, or may be nucleic acids in a partially purified or
substantially pure form. A nucleic acid is "isolated" or "rendered
substantially pure" when purified away from other cellular
components or other contaminants, e.g. other cellular nucleic acids
or proteins, by standard techniques, including alkaline/SDS
treatment, CsCl banding, column chromatography, agarose gel
electrophoresis and others well known in the art. See, F. Ausubel,
et al., ed. 1987 Current Protocols in Molecular Biology, Greene
Publishing and Wiley Interscience, New York. A nucleic acid of the
disclosure can be, for example, DNA or RNA and may or may not
contain intronic sequences. In an embodiment, the nucleic acid is a
cDNA molecule. The nucleic acid may be present in a vector such as
a phage display vector, or in a recombinant plasmid vector. The
disclosure also provides the vectors referred to as pBW522 and
pBW524 (deposited at DSMZ, Inhoffenstr. 7B, D-38124 Braunschweig,
Germany on 18 Aug. 2009 under deposit numbers DSM22873 and
DSM22874, respectively).
[0186] Nucleic acids of the disclosure can be obtained using
standard molecular biology techniques. For antibodies expressed by
hybridomas (e.g. hybridomas prepared from transgenic mice carrying
human immunoglobulin genes as described further below), cDNAs
encoding the light and heavy chains of the antibody made by the
hybridoma can be obtained by standard PCR amplification or cDNA
cloning techniques. For antibodies obtained from an immunoglobulin
gene library (e.g. using phage display techniques), nucleic acid
encoding the antibody can be recovered from various phage clones
that are members of the library.
[0187] Also included within the scope of the disclosure are variant
nucleic acid sequences that comprise one or more deletions,
additions or substitutions. In one embodiment, the disclosure
comprises one or more of SEQ ID NOs: 113-140 or 161-180, which
comprises a conservative nucleotide substitution. Due to the
degeneracy of the genetic code, an amino acid may be encoded by
more than one codon. Thus, it is possible to amend the nucleotide
sequence, while the translated amino acid sequence remains the
same.
[0188] Once DNA fragments encoding V.sub.H and V.sub.L segments are
obtained, these DNA fragments can be further manipulated by
standard recombinant DNA techniques, for example to convert the
variable region genes to full-length antibody chain genes, to Fab
fragment genes or to an scFv gene. In these manipulations, a
V.sub.L- or V.sub.H-encoding DNA fragment is operatively linked to
another DNA molecule, or to a fragment encoding another protein,
such as an antibody constant region or a flexible linker. The term
"operatively linked", as used in this context, is intended to mean
that the two DNA fragments are joined in a functional manner, for
example, such that the amino acid sequences encoded by the two DNA
fragments remain in-frame, or such that the protein is expressed
under control of a desired promoter.
[0189] The isolated DNA encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the V.sub.H-encoding DNA to another DNA molecule encoding heavy
chain constant regions (CH1, CH2 and CH3). The sequences of human
heavy chain constant region genes are known in the art (see e.g.
Kabat, E. A., et al. [supra]) and DNA fragments encompassing these
regions can be obtained by standard PCR amplification. The heavy
chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE,
IgM or IgD constant region. In some embodiments, the heavy chain
contstant region is selected among IgG1 isotypes. For a Fab
fragment heavy chain gene, the V.sub.H-encoding DNA can be
operatively linked to another DNA molecule encoding only the heavy
chain CH1 constant region.
[0190] The isolated DNA encoding the V.sub.L region can be
converted to a full-length light chain gene (as well as to a Fab
light chain gene) by operatively linking the V.sub.L-encoding DNA
to another DNA molecule encoding the light chain constant region,
CL. The sequences of human light chain constant region genes are
known in the art (see e.g. Kabat, E. A., et al. [supra]) and DNA
fragments encompassing these regions can be obtained by standard
PCR amplification. The light chain constant region can be a kappa
or a lambda constant region.
[0191] To create an scFv gene, the V.sub.H- and V.sub.L-encoding
DNA fragments are operatively linked to another fragment encoding a
flexible linker, e.g. encoding the amino acid sequence
(Gly4-Ser).sub.3, such that the V.sub.H and V.sub.L sequences can
be expressed as a contiguous single-chain protein, with the V.sub.L
and V.sub.H regions joined by the flexible linker (see e.g. Bird et
al., 1988 Science 242:423-426; Huston et al., 1988 Proc. Natl.
Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature
348:552-554).
[0192] The nucleic acids of the disclosure may be used in gene
delivery. That is, nucleic acid encoding the polypeptides
(antibodies or functional proteins) of the disclosure may be
directly delivered to a patient for translation in the patient.
[0193] The nucleic acid is typically "packaged" for administration
to a patient. Gene delivery vehicles may be non-viral, such as
liposomes, or replication-deficient viruses, such as adenovirus as
described by Berkner, K. L., in Curr. Top. Microbiol. Immunol.,
158, 39-66 (1992) or adeno-associated virus (AAV) vectors as
described by Muzyczka, N., in Curr. Top. Microbiol. Immunol., 158,
97-129 (1992) and U.S. Pat. No. 5,252,479. Alternatively a
retrovirus, such as a lentivirus may be used. For example, a
nucleic acid molecule encoding a polypeptide of the disclosure may
be engineered for expression in a replication-defective retroviral
vector. This expression construct may then be isolated and
introduced into a packaging cell transduced with a retroviral
plasmid vector containing RNA encoding the polypeptide, such that
the packaging cell now produces infectious viral particles
containing the gene of interest. These producer cells may be
administered to a subject for engineering cells in vivo and
expression of the polypeptide in vivo (see Chapter 20, Gene Therapy
and other Molecular Genetic-based Therapeutic Approaches, (and
references cited therein) in Human Molecular Genetics (1996), T
Strachan and A P Read, BIOS Scientific Publishers Ltd).
[0194] Another approach is the administration of "naked DNA" in
which the therapeutic gene is directly injected into the
bloodstream or muscle tissue.
Generation of Particular Monoclonal Antibodies
[0195] Monoclonal antibodies (mAbs) can be produced by a variety of
techniques, including conventional monoclonal antibody methodology
e.g. the standard somatic cell hybridization technique of Kohler
and Milstein (1975 Nature 256: 495). Many techniques for producing
monoclonal antibody can be employed e.g. viral or oncogenic
transformation of B lymphocytes.
[0196] An animal system for preparing hybridomas is the murine
system. Hybridoma production in the mouse is a well established
procedure. Immunization protocols and techniques for isolation of
immunized splenocytes for fusion are known in the art. Fusion
partners (e.g. murine myeloma cells) and fusion procedures are also
known.
[0197] Chimeric or humanized antibodies of the present disclosure
can be prepared based on the sequence of a murine monoclonal
antibody prepared as described above. DNA encoding the heavy and
light chain immunoglobulins can be obtained from the murine
hybridoma of interest and engineered to contain non-murine (e.g.
human) immunoglobulin sequences using standard molecular biology
techniques. For example, to create a chimeric antibody, the murine
variable regions can be linked to human constant regions using
methods known in the art (see e.g. U.S. Pat. No. 4,816,567). To
create a humanized antibody, the murine CDR regions can be inserted
into a human framework using methods known in the art (see e.g.
U.S. Pat. Nos. 5,225,539; 5530101; 5585089; 5693762 and
6180370).
[0198] In a certain embodiment, the antibodies of the disclosure
are human monoclonal antibodies. Such human monoclonal antibodies
directed against ActRIIB can be generated using transgenic or
transchromosomic mice carrying parts of the human immune system
rather than the mouse system. These transgenic and transchromosomic
mice include mice referred to herein as HuMAb mice and KM mice,
respectively, and are collectively referred to herein as "human Ig
mice."
[0199] The HuMAb Mouse.RTM. (Medarex, Inc.) contains human
immunoglobulin gene miniloci that encode un-rearranged human heavy
(.mu. and .gamma.) and .kappa. light chain immunoglobulin
sequences, together with targeted mutations that inactivate the
endogenous .mu. and .kappa. chain loci (see e.g. Lonberg, et al.,
1994 Nature 368(6474): 856-859). Accordingly, the mice exhibit
reduced expression of mouse IgM or .kappa., and in response to
immunization, the introduced human heavy and light chain transgenes
undergo class switching and somatic mutation to generate high
affinity human IgG.kappa. monoclonal (Lonberg, N. et al., 1994
[supra]; reviewed in Lonberg, N., 1994 Handbook of Experimental
Pharmacology 113:49-101; Lonberg, N. and Huszar, D., 1995 Intern.
Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N., 1995 Ann.
N.Y. Acad. Sci. 764:536-546). The preparation and use of HuMAb
mice, and the genomic modifications carried by such mice, is
further described in Taylor, L. et al., 1992 Nucleic Acids Research
20:6287-6295; Chen, J. et al., 1993 International Immunology 5:
647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA
94:3720-3724; Choi et al., 1993 Nature Genetics 4:117-123; Chen, J.
et al., 1993 EMBO J. 12: 821-830; Tuaillon et al., 1994 J. Immunol.
152:2912-2920; Taylor, L. et al., 1994 International Immunology
579-591; and Fishwild, D. et al., 1996 Nature Biotechnology 14:
845-851, the contents of all of which are hereby specifically
incorporated by reference in their entirety. See further, U.S. Pat.
Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650;
5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and
5,545,807; as well as WO92/103918, WO93/12227, WO94/25585,
WO97/113852, WO98/24884; WO99/45962; and WO01/14424.
[0200] In another embodiment, human antibodies of the disclosure
can be raised using a mouse that carries human immunoglobulin
sequences on transgenes and transchomosomes such as a mouse that
carries a human heavy chain transgene and a human light chain
transchromosome. Such mice, referred to herein as "KM mice", are
described in detail in WO02/43478.
[0201] Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-ActRIIB antibodies of the disclosure. For
example, an alternative transgenic system referred to as the
Xenomouse (Abgenix, Inc.) can be used. Such mice are described in,
e.g. U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and
6,162,963.
[0202] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-ActRIIB antibodies of the disclosure. For
example, mice carrying both a human heavy chain transchromosome and
a human light chain tranchromosome, referred to as "TC mice" can be
used; such mice are described in Tomizuka et al., 2000 Proc. Natl.
Acad. Sci. USA 97:722-727. Furthermore, cows carrying human heavy
and light chain transchromosomes have been described in the art
(Kuroiwa et al., 2002 Nature Biotechnology 20:889-894) and can be
used to raise anti-ActRIIB antibodies of the disclosure.
[0203] Human recombinant antibodies of the disclosure can also be
prepared using phage display methods for screening libraries of
human immunoglobulin genes. Such phage display methods for
isolating human antibodies are established in the art or described
in the examples below. See for example: U.S. Pat. Nos. 5,223,409;
5,403,484; 5,571,698; 5,427,908; 5,580,717; 5,969,108; 6,172,197;
5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and
6,593,081.
[0204] Human monoclonal antibodies of the disclosure can also be
prepared using SCID mice into which human immune cells have been
reconstituted such that a human antibody response can be generated
upon immunization. Such mice are described in, for example, U.S.
Pat. Nos. 5,476,996 and 5,698,767.
Generation of Hybridomas Producing Human Monoclonal Antibodies
[0205] To generate hybridomas producing human monoclonal antibodies
of the disclosure, splenocytes and/or lymph node cells from
immunized mice can be isolated and fused to an appropriate
immortalized cell line, such as a mouse myeloma cell line. The
resulting hybridomas can be screened for the production of
antigen-specific antibodies. For example, single cell suspensions
of splenic lymphocytes from immunized mice can be fused to
one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma
cells (ATCC, CRL 1580) with 50% PEG. Cells are plated at
approximately 2.times.145 in flat bottom microtiter plates,
followed by a two week incubation in selective medium containing
20% fetal Clone Serum, 18% "653" conditioned media, 5% origen
(IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0:055
mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml
streptomycin, 50 mg/ml gentamycin and 1.times.HAT (Sigma; the HAT
is added 24 hours after the fusion). After approximately two weeks,
cells can be cultured in medium in which the HAT is replaced with
HT. Individual wells can then be screened by ELISA for human
monoclonal IgM and IgG antibodies. Once extensive hybridoma growth
occurs, medium can be observed usually after 10-14 days. The
antibody secreting hybridomas can be replated, screened again, and
if still positive for human IgG, the monoclonal antibodies can be
subcloned at least twice by limiting dilution. The stable subclones
can then be cultured in vitro to generate small amounts of antibody
in tissue culture medium for characterization.
[0206] To purify human monoclonal antibodies, selected hybridomas
can be grown in two-liter spinner-flasks for monoclonal antibody
purification. Supernatants can be filtered and concentrated before
affinity chromatography with protein A-sepharose (Pharmacia).
Eluted IgG can be checked by gel electrophoresis and high
performance liquid chromatography to ensure purity. The buffer
solution can be exchanged into PBS, and the concentration can be
determined by OD.sub.280 using 1.43 extinction coefficient. The
monoclonal antibodies can be aliquoted and stored at -80.degree.
C.
Generation of Transfectomas Producing Monoclonal Antibodies
[0207] Antibodies of the disclosure also can be produced in a host
cell transfectoma using, for example, a combination of recombinant
DNA techniques and gene transfection methods as is well known in
the art (e.g. Morrison, S. (1985) Science 229:1202).
[0208] For example, to express the antibodies, or antibody
fragments thereof, DNAs encoding partial or full-length light and
heavy chains, can be obtained by standard molecular biology
techniques (e.g. PCR amplification or cDNA cloning using a
hybridoma that expresses the antibody of interest) and the DNAs can
be inserted into expression vectors such that the genes are
operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is
intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene
and the antibody heavy chain gene can be inserted into separate
vector or, more typically, both genes are inserted into the same
expression vector. The antibody genes are inserted into the
expression vector by standard methods (e.g. ligation of
complementary restriction sites on the antibody gene fragment and
vector, or blunt end ligation if no restriction sites are present).
The light and heavy chain variable regions of the antibodies
described herein can be used to create full-length antibody genes
of any antibody isotype by inserting them into expression vectors
already encoding heavy chain constant and light chain constant
regions of the desired isotype such that the V.sub.H segment is
operatively linked to the CH segment(s) within the vector and the
V.sub.L segment is operatively linked to the CL segment within the
vector. Additionally or alternatively, the recombinant expression
vector can encode a signal peptide that facilitates secretion of
the antibody chain from a host cell. The antibody chain gene can be
cloned into the vector such that the signal peptide is linked in
frame to the amino terminus of the antibody chain gene. The signal
peptide can be an immunoglobulin signal peptide or a heterologous
signal peptide (i.e. a signal peptide from a non-immunoglobulin
protein).
[0209] In addition to the antibody chain genes, the recombinant
expression vectors of the disclosure carry regulatory sequences
that control the expression of the antibody chain genes in a host
cell. The term "regulatory sequence" is intended to include
promoters, enhancers and other expression control elements (e.g.
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel (Gene Expression Technology.
Methods in Enzymology 185, Academic Press, San Diego, Calif. 1990).
It will be appreciated by those skilled in the art that the design
of the expression vector, including the selection of regulatory
sequences, may depend on such factors as the choice of the host
cell to be transformed, the level of expression of protein desired,
etc. Regulatory sequences for mammalian host cell expression
include viral elements that direct high levels of protein
expression in mammalian cells, such as promoters and/or enhancers
derived from cytomegalovirus (CMV), Simian Virus 40 (SV40),
adenovirus (e.g. the adenovirus major late promoter (AdMLP)), and
polyoma. Alternatively, nonviral regulatory sequences may be used,
such as the ubiquitin promoter or P-globin promoter. Still further,
regulatory elements composed of sequences from different sources,
such as the SRa promoter system, which contains sequences from the
SV40 early promoter and the long terminal repeat of human T cell
leukemia virus type 1 (Takebe, Y. et al., 1988 Mol. Cell. Biol.
8:466-472).
[0210] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the disclosure may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g. origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see, e.g. U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017). For example, typically the selectable marker gene
confers resistance to drugs, such as G418, hygromycin or
methotrexate, on a host cell into which the vector has been
introduced. Selectable marker genes include the dihydrofolate
reductase (DHFR) gene (for use in dhfr-host cells with methotrexate
selection/amplification) and the neo gene (for G418 selection).
[0211] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g. electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. It is theoretically possible to express the antibodies of the
disclosure in either prokaryotic or eukaryotic host cells.
Expression of antibodies in eukaryotic cells, in particular
mammalian host cells, is discussed because such eukaryotic cells,
and in particular mammalian cells, are more likely than prokaryotic
cells to assemble and secrete a properly folded and immunologically
active antibody. Prokaryotic expression of antibody genes has been
reported to be ineffective for production of high yields of active
antibody (Boss, M. A. and Wood, C. R., 1985 Immunology Today
6:12-13).
[0212] Mammalian host cells for expressing the recombinant
antibodies of the disclosure include Chinese Hamster Ovary (CHO
cells) (including dhfr-CHO cells, described Urlaub and Chasin, 1980
Proc. Natl. Acad. Sci. USA 77:4216-4220 used with a DH FR
selectable marker, e.g. as described in R. J. Kaufman and P. A.
Sharp, 1982 Mol. Biol. 159:601-621), NSO myeloma cells, COS cells
and SP2 cells. In one embodiment the host cells are CHO K1PD cells.
In particular, for use with NSO myeloma cells, another expression
system is the GS gene expression system shown in WO87/04462,
WO89/01036 and EP 338,841. In one embodiment, mammalian host cells
for expressing the recombinant antibodies of the disclosure include
mammalian cell lines deficient for FUT8 gene expression, for
example as described in U.S. Pat. No. 6,946,292B2. When recombinant
expression vectors encoding antibody genes are introduced into
mammalian host cells, the antibodies are produced by culturing the
host cells for a period of time sufficient to allow for expression
of the antibody in the host cells or secretion of the antibody into
the culture medium in which the host cells are grown. Antibodies
can be recovered from the culture medium using standard protein
purification methods.
Immunoconjugates
[0213] In another aspect, the present disclosure features an
anti-ActRIIB antibody, or a fragment thereof, conjugated to a
therapeutic moiety, such as a cytotoxin, a drug (e.g. an
immunosuppressant) or a radiotoxin. Such conjugates are referred to
herein as "immunoconjugates" Immunoconjugates that include one or
more cytotoxins are referred to as "immunotoxins." A cytotoxin or
cytotoxic agent includes any agent that is detrimental to (e.g.
kills) cells.
[0214] Cytotoxins can be conjugated to antibodies of the disclosure
using linker technology available in the art. Examples of linker
types that have been used to conjugate a cytotoxin to an antibody
include, but are not limited to, hydrazones, thioethers, esters,
disulfides and peptide-containing linkers. A linker can be chosen
that is, for example, susceptible to cleavage by low pH within the
lysosomal compartment or susceptible to cleavage by proteases, such
as proteases, in various embodiments, expressed in tumor tissue
such as cathepsins (e.g. cathepsins B, C, D).
[0215] For further discussion of types of cytotoxins, linkers and
methods for conjugating therapeutic agents to antibodies, see also
Saito, G. et al., 2003 Adv. Drug Deliv. Rev. 55:199-215; Trail, P.
A. et al., 2003 Cancer Immunol. Immunother. 52:328-337; Payne, G.
2003 Cancer Cell 3:207-212; Allen, T. M., 2002 Nat. Rev. Cancer
2:750-763; Pastan, I. and Kreitman, R. J., 2002 Curr. Opin.
Investig. Drugs 3:1089-1091; Senter, P. D. and Springer, C. J.,
2001 Adv. Drug Deliv. Rev. 53:247-264.
[0216] Antibodies of the present disclosure also can be conjugated
to a radioactive isotope to generate cytotoxic
radiopharmaceuticals, also referred to as radioimmunoconjugates.
Examples of radioactive isotopes that can be conjugated to
antibodies for use diagnostically or therapeutically include, but
are not limited to, iodine.sup.131, indium.sup.111, yttrium.sup.90,
and lutetium.sup.177. Methods for preparing radioimmunconjugates
are established in the art. Examples of radioimmunoconjugates are
commercially available, including Zevalin.TM. (DEC Pharmaceuticals)
and Bexxar.TM. (Corixa Pharmaceuticals), and similar methods can be
used to prepare radioimmunoconjugates using the antibodies of the
disclosure.
[0217] The antibody conjugates of the disclosure can be used to
modify a given biological response, and the drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, an enzymatically active toxin, or active
fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor or
interferon-.gamma.; or, biological response modifiers such as, for
example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating
factor ("G-CSF"), or other growth factors.
[0218] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g. Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
Bispecific Molecules
[0219] In another aspect, the present disclosure features
bispecific or multispecific molecules comprising an anti-ActRIIB
antibody, or a fragment thereof, of the disclosure. An antibody of
the disclosure, or antigen-binding regions thereof, can be
derivatized or linked to another functional molecule, e.g. another
peptide or protein (e.g. another antibody or ligand for a receptor)
to generate a bispecific molecule that binds to at least two
different binding sites or target molecules. The antibody of the
disclosure may in fact be derivatized or linked to more than one
other functional molecule to generate multi-specific molecules that
bind to more than two different binding sites and/or target
molecules; such multi-specific molecules are also intended to be
encompassed by the term "bispecific molecule" as used herein. To
create a bispecific molecule of the disclosure, an antibody of the
disclosure can be functionally linked (e.g. by chemical coupling,
genetic fusion, noncovalent association or otherwise) to one or
more other binding molecules, such as another antibody, antibody
fragment, peptide or binding mimetic, such that a bispecific
molecule results.
[0220] Accordingly, the present disclosure includes bispecific
molecules comprising at least one first binding specificity for
ActRIIB and a second binding specificity for a second target
epitope. For example, the second target epitope may be another
epitope of ActRIIB different from the first target epitope.
[0221] Additionally, for the disclosure in which the bispecific
molecule is multi-specific, the molecule can further include a
third binding specificity, in addition to the first and second
target epitope.
[0222] In one embodiment, the bispecific molecules of the
disclosure comprise as a binding specificity at least one antibody,
or an antibody fragment thereof, including, e.g. an Fab, Fab',
F(ab').sub.2, Fv, or a single chain Fv. The antibody may also be a
light chain or heavy chain dimer, or any minimal fragment thereof
such as a Fv or a single chain construct as described in Ladner et
al. U.S. Pat. No. 4,946,778, the contents of which is expressly
incorporated by reference.
[0223] Other antibodies which can be employed in the bispecific
molecules of the disclosure are murine, chimeric and humanized
monoclonal antibodies.
[0224] The bispecific molecules of the present disclosure can be
prepared by conjugating the constituent binding specificities,
using methods known in the art. For example, each binding
specificity of the bispecific molecule can be generated separately
and then conjugated to one another. When the binding specificities
are proteins or peptides, a variety of coupling or cross-linking
agents can be used for covalent conjugation. Examples of
cross-linking agents include protein A, carbodiimide,
N-succinimidyl-5-acetyl-thioacetate (SATA),
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide
(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate
(sulfo-SMCC) (see e.g. Karpovsky et al., 1984 J. Exp. Med.
160:1686; Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA
82:8648). Other methods include those described in Paulus, 1985
Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science
229:81-83), and Glennie et al., 1987 J. Immunol. 139: 2367-2375).
Conjugating agents are SATA and sulfo-SMCC, both available from
Pierce Chemical Co. (Rockford, Ill.).
[0225] When the binding specificities are antibodies, they can be
conjugated by sulfhydryl bonding of the C-terminus hinge regions of
the two heavy chains. In a particularly embodiment, the hinge
region is modified to contain an odd number of sulfhydryl residues,
for example one, prior to conjugation.
[0226] Alternatively, both binding specificities can be encoded in
the same vector and expressed and assembled in the same host cell.
This method is particularly useful where the bispecific molecule is
a mAb.times.mAb, mAb.times.Fab, Fab.times.F(ab').sub.2 or ligand x
Fab fusion protein. A bispecific molecule of the disclosure can be
a single chain molecule comprising one single chain antibody and a
binding determinant, or a single chain bispecific molecule
comprising two binding determinants. Bispecific molecules may
comprise at least two single chain molecules. Methods for preparing
bispecific molecules are described for example in U.S. Pat. Nos.
5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;
5,013,653; 5,258,498; and 5,482,858.
[0227] Binding of the bispecific molecules to their specific
targets can be confirmed by, for example, enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis,
bioassay (e.g. growth inhibition), or Western Blot assay. Each of
these assays generally detects the presence of protein-antibody
complexes of particular interest by employing a labeled reagent
(e.g. an antibody) specific for the complex of interest.
Multivalent Antibodies
[0228] In another aspect, the present disclosure provides
multivalent antibodies comprising at least two identical or
different antigen-binding portions of the antibodies of the
disclosure binding to ActRIIB. In one embodiment, the multivalent
antibodies provides at least two, three or four antigen-binding
portions of the antibodies. The antigen-binding portions can be
linked together via protein fusion or covalent or non covalent
linkage. Alternatively, methods of linkage have been described for
the bispecific molecules. Tetravalent compounds can be obtained for
example by cross-linking antibodies of the antibodies of the
disclosure with an antibody that binds to the constant regions of
the antibodies of the disclosure, for example the Fc or hinge
region.
Pharmaceutical Compositions
[0229] In another aspect, the present disclosure provides a
composition, e.g. a pharmaceutical composition, containing one or a
combination of monoclonal antibodies, or antigen-binding portion(s)
thereof, of the present disclosure, formulated together with a
pharmaceutically acceptable carrier. Such compositions may include
one or a combination of (e.g. two or more different) antibodies, or
immunoconjugates or bispecific molecules of the disclosure. For
example, a pharmaceutical composition of the disclosure can
comprise a combination of antibodies that bind to different
epitopes on the target antigen or that have complementary
activities.
[0230] Pharmaceutical compositions of the disclosure also can be
administered in combination therapy, i.e. combined with other
agents. For example, the combination therapy can include an
anti-ActRIIB antibody of the present disclosure combined with at
least one other muscle mass/strength increasing agent, for example,
IGF-1, IGF-2 or variants of IGF-1 or IGF-2, an anti-myostatin
antibody, a myostatin propeptide, a myostatin decoy protein that
binds ActRIIB but does not activate it, a beta 2 agonist, a Ghrelin
agonist, a SARM, GH agonists/mimetics or follistatin. Examples of
therapeutic agents that can be used in combination therapy are
described in greater detail below in the section on uses of the
antibodies of the disclosure.
[0231] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
The carrier should be suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.
by injection or infusion). Depending on the route of
administration, the active compound, i.e. antibody, immunoconjuage,
or bispecific molecule, may be coated in a material to protect the
compound from the action of acids and other natural conditions that
may inactivate the compound.
[0232] The pharmaceutical compounds of the disclosure may include
one or more pharmaceutically acceptable salts. A "pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any
undesired toxicological effects (see e.g. Berge, S. M., et al.,
1977 J. Pharm. Sci. 66:1-19). Examples of such salts include acid
addition salts and base addition salts. Acid addition salts include
those derived from nontoxic inorganic acids, such as hydrochloric,
nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous
and the like, as well as from nontoxic organic acids such as
aliphatic mono- and di-carboxylic acids, phenyl-substituted
alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic
and aromatic sulfonic acids and the like. Base addition salts
include those derived from alkaline earth metals, such as sodium,
potassium, magnesium, calcium and the like, as well as from
nontoxic organic amines, such as N,N'-dibenzylethylenediamine,
N-methylglucamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, procaine and the like.
[0233] A pharmaceutical composition of the disclosure also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like;
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and metal chelating
agents, such as citric acid, ethylenediamine tetraacetic acid
(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[0234] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the
disclosure include water, ethanol, polyols (such as glycerol,
propylene glycol, polyethylene glycol, and the like), and suitable
mixtures thereof, vegetable oils, such as olive oil, and injectable
organic esters, such as ethyl oleate. Proper fluidity can be
maintained, for example, by the use of coating materials, such as
lecithin, by the maintenance of the required particle size in the
case of dispersions, and by the use of surfactants.
[0235] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as, aluminum monostearate and gelatin.
[0236] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the disclosure is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0237] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, one can
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought
about by including in the composition an agent that delays
absorption for example, monostearate salts and gelatin.
[0238] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of agents enumerated
above, as required, followed by sterilization microfiltration.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other agents from those enumerated above.
In the case of sterile powders for the preparation of sterile
injectable solutions, the methods of preparation are vacuum drying
and freeze-drying (lyophilization) that yield a powder of the
active agent plus any additional desired agent from a previously
sterile-filtered solution thereof.
[0239] The amount of active agent which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active agent which can be combined
with a carrier material to produce a single dosage form will
generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 0.01 percent to about ninety-nine
percent of active agent, from about 0.1 percent to about 70
percent, or from about 1 percent to about 30 percent of active
agent in combination with a pharmaceutically acceptable
carrier.
[0240] Dosage regimens are adjusted to provide the optimum desired
response (e.g. a therapeutic response). For example, a single bolus
may be administered, several divided doses may be administered over
time or the dose may be proportionally reduced or increased as
indicated by the exigencies of the therapeutic situation. It is
especially advantageous to formulate parenteral compositions in
dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the subjects to be
treated; each unit contains a predetermined quantity of active
compound calculated to produce the desired therapeutic effect in
association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the disclosure are
dictated by and directly dependent on the unique characteristics of
the active compound and the particular therapeutic effect to be
achieved, and the limitations inherent in the art of compounding
such an active compound for the treatment of sensitivity in
individuals.
[0241] For administration of the antibody, the dosage ranges from
about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the
host body weight. For example dosages can be 0.3 mg/kg body weight,
1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10
mg/kg body weight or within the ranges of 1-10 mg/kg or 3-7 mg/kg.
An example treatment regime entails administration once per week,
once every two weeks, once every three weeks, once every four
weeks, once a month, once every 3 months or once every three to 6
months. Alternatively, the antibody may be administered about once
a year or once only. Such administration may be carried out
intraveneously or subcutaneously. Dosage regimens for an
anti-ActRIIB antibody of the disclosure include 1 mg/kg body weight
or 3 mg/kg body weight by intravenous administration, with the
antibody being given using one of the following dosing schedules:
every four weeks for six dosages, then every three months; every
three weeks; 3 mg/kg body weight once followed by 1 mg/kg body
weight every three weeks.
[0242] The dosage should be one that causes an upregulation of
muscle mass and/or strength. In various embodiments the effect is
on skeletal muscle. In various embodiments, the dosage causes
muscle hypertrophy with no more than a proportional increase in the
size of internal organs (e.g. heart, lungs, liver, kidneys). Such a
proportional increase may be compared by measuring either mass or
volume.
[0243] In some methods, two or more monoclonal antibodies with
different binding specificities are administered simultaneously, in
which case the dosage of each antibody administered falls within
the ranges indicated. Antibody is usually administered on multiple
occasions. Intervals between single dosages can be, for example,
weekly, monthly, every three months, every six months or yearly.
Intervals can also be irregular as indicated by measuring blood
levels of antibody to the target antigen in the patient. In some
methods, dosage is adjusted to achieve a plasma antibody
concentration of about 1-1000 .mu.g/ml and in some methods about
25-300 .mu.g/ml. For example, an ActRIIB antibody of the disclosure
could be co-administered with an anti-myostatin antibody.
[0244] Alternatively, antibody can be administered as a sustained
release formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of
the antibody in the patient. In general, human antibodies show the
longest half-life, followed by humanized antibodies, chimeric
antibodies, and nonhuman antibodies. The dosage and frequency of
administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some patients continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated or until the patient shows partial or
complete amelioration of symptoms of disease. Thereafter, the
patient can be administered a prophylactic regime.
[0245] Actual dosage levels of the active agents in the
pharmaceutical compositions of the present disclosure may be varied
so as to obtain an amount of the active agent which is effective to
achieve the desired therapeutic response for a particular patient,
composition, and mode of administration, without being toxic to the
patient. The selected dosage level will depend upon a variety of
pharmacokinetic factors including the activity of the particular
compositions of the present disclosure employed, or the ester, salt
or amide thereof, the route of administration, the time of
administration, the rate of excretion of the particular compound
being employed, the duration of the treatment, other drugs,
compounds and/or materials used in combination with the particular
compositions employed, the age, sex, weight, condition, general
health and prior medical history of the patient being treated, and
like factors well known in the medical arts.
[0246] A "therapeutically effective dosage" of an anti-ActRIIB
antibody of the disclosure can result in a decrease in severity of
disease symptoms, an increase in frequency and duration of disease
symptom-free periods, or a prevention of impairment or disability
due to the disease affliction i.e. an increase in muscle mass
and/or strength.
[0247] A composition of the present disclosure can be administered
by one or more routes of administration using one or more of a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. Routes of administration for
antibodies of the disclosure include intravenous, intramuscular,
intradermal, intraperitoneal, subcutaneous, spinal or other
parenteral routes of administration, for example by injection or
infusion. The phrase "parenteral administration" as used herein
means modes of administration other than enteral and topical
administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrastemal injection and infusion. In one embodiment the
antibody is administered intravenously. In another embodiment the
antibody is administered subcutaneously.
[0248] Alternatively, an antibody of the disclosure can be
administered by a nonparenteral route, such as a topical, epidermal
or mucosal route of administration, for example, intranasally,
orally, vaginally, rectally, sublingually or topically.
[0249] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g. Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0250] Therapeutic compositions can be administered with medical
devices known in the art. For example, in one embodiment, a
therapeutic composition of the disclosure can be administered with
a needleless hypodermic injection device, such as the devices shown
in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413;
4,941,880; 4,790,824 or 4,596,556. Examples of well known implants
and modules useful in the present disclosure include: U.S. Pat. No.
4,487,603, which shows an implantable micro-infusion pump for
dispensing medication at a controlled rate; U.S. Pat. No.
4,486,194, which shows a therapeutic device for administering
medicants through the skin; U.S. Pat. No. 4,447,233, which shows a
medication infusion pump for delivering medication at a precise
infusion rate; U.S. Pat. No. 4,447,224, which shows a variable flow
implantable infusion apparatus for continuous drug delivery; U.S.
Pat. No. 4,439,196, which shows an osmotic drug delivery system
having multi-chamber compartments; and U.S. Pat. No. 4,475,196,
which shows an osmotic drug delivery system. Many other such
implants, delivery systems, and modules are known to those skilled
in the art and include those made by MicroCHIPS.TM. (Bedford,
Mass.).
[0251] In certain embodiments, the human monoclonal antibodies of
the disclosure can be formulated to ensure proper distribution in
vivo. For example, the blood-brain barrier (BBB) excludes many
highly hydrophilic compounds. To ensure that the therapeutic
compounds of the disclosure cross the BBB (if desired), they can be
formulated, for example, in liposomes. For methods of manufacturing
liposomes, see, e.g. U.S. Pat. Nos. 4,522,811; 5,374,548; and
5,399,331. The liposomes may comprise one or more moieties which
are selectively transported into specific cells or organs, thus
enhance targeted drug delivery (see, e.g. V. V. Ranade, 1989 J.
Clin Pharmacol. 29:685). Example targeting moieties include folate
or biotin (see, e.g. U.S. Pat. No. 5,416,016); mannosides (Umezawa
et al., 1988 Biochem. Biophys. Res. Commun 153:1038); antibodies
(P. G. Bloeman et al., 1995 FEBS Lett. 357:140; M. Owais et al.,
1995 Antimicrob. Agents Chemother. 39:180); surfactant protein A
receptor (Briscoe et al., 1995 Am. J. Physiol. 1233:134); p120
(Schreier et al., 1994 J. Biol. Chem. 269:9090); see also K.
Keinanen; M. L. Laukkanen, 1994 FEBS Lett. 346:123; J. J. Killion;
I. J. Fidler, 1994 Immunomethods 4:273.
Uses and Methods of the Antibodies
[0252] The antibodies of the present disclosure have in vitro and
in vivo diagnostic and therapeutic utilities. For example, these
molecules can be administered to cells in culture, e.g. in vitro or
ex vivo, or in a subject, e.g. in vivo, to treat, prevent or
diagnose a variety of disorders. Thus the antibodies may be used in
both the treatment of disease, prophylaxis and for delaying the
onset of disease symptoms. The term "subject" as used herein is
intended to include human and non-human animals. Non-human animals
include all vertebrates, e.g. mammals and non-mammals, such as
non-human primates, sheep, dogs, cats, cows, horses, chickens,
amphibians, and reptiles.
[0253] The disclosure provides a method of treating a patient
suffering from a pathological disorder (such as a muscle wasting
disease or disorder) comprising administering a therapeutically
effective amount of an anti-ActRIIB antibody.
[0254] The disclosure also provides an anti-ActRIIB antibody for
use in therapy.
[0255] The disclosure also provides use of an anti-ActRIIB antibody
in the manufacture of a medicament for the treatment of a
pathological disorder.
[0256] The methods are particularly suitable for treating,
preventing, ameliorating or diagnosing pathological disorders.
[0257] As used herein, a "pathological disorder" includes, but is
not limited to, musculoskeletal diseases or disorders, such as
muscle atrophy. There are many causes of muscle atrophy, including
as a result of treatment with a glucocorticoid such as cortisol,
dexamethasone, betamethasone, prednisone, methylprednisolone, or
prednisolone. The muscle atrophy can also be a result of
denervation due to nerve trauma or a result of degenerative,
metabolic, or inflammatory neuropathy (e.g., Guillian-Barre
syndrome, peripheral neuropathy, or exposure to environmental
toxins or drugs).
[0258] In addition, the muscle atrophy can be a result of myopathy,
such as myotonia; a congential myopathy, including nemalene
myopathy, multi/minicore myopathy and myotubular (centronuclear)
myopathy; mitochondrial myopathy; familial periodic paralysis;
inflammatory myopathy; metabolic myopathy, such as caused by a
glycogen or lipid storage disease; dermatomyositisis; polymyositis;
inclusion body myositis; myositis ossificans; rhabdomyolysis and
myoglobinurias.
[0259] The myopathy may be caused by a muscular dystrophy syndrome,
such as Duchenne, Becker, myotonic, fascioscapulohumeral,
Emery-Dreifuss, oculopharyngeal, scapulohumeral, limb girdle,
Fukuyama, a congenital muscular dystrophy, or hereditary distal
myopathy. The musculoskeletal disease can also be osteoporosis, a
bone fracture, short stature, or dwarfism.
[0260] In addition, the muscle atrophy can be a result of an adult
motor neuron disease, infantile spinal muscular atrophy,
amyotrophic lateral sclerosis, juvenile spinal muscular atrophy,
autoimmune motor neuropathy with multifocal conductor block,
paralysis due to stroke or spinal cord injury, skeletal
immobilization due to trauma, prolonged bed rest, voluntary
inactivity, involuntary inactivity, metabolic stress or nutritional
insufficiency, cancer, AIDS, fasting, a thyroid gland disorder,
diabetes, benign congenital hypotonia, central core disease, burn
injury, chronic obstructive pulmonary disease, liver diseases
(examples such as fibrosis, cirrhosis), sepsis, renal failure,
congestive heart failure, ageing, space travel or time spent in a
zero gravity environment.
[0261] Examples of age-related conditions that may be treated
include, sarcopenia, skin atrophy, muscle wasting, brain atrophy,
atherosclerosis, arteriosclerosis, pulmonary emphysema,
osteoporosis, osteoarthritis, immunologic incompetence, high blood
pressure, dementia, Huntington's disease, Alzheimer's disease,
cataracts, age-related macular degeneration, prostate cancer,
stroke, diminished life expectancy, frailty, memory loss, wrinkles,
impaired kidney function, and age-related hearing loss; metabolic
disorders, including Type II Diabetes, Metabolic Syndrome,
hyperglycemia, and obesity. Of course, patients may simultaneously
suffer from one or more of these conditions, for example,
sarcopenia and pulmonary emphysema, or sarcopenia and impaired
kidney function.
[0262] Other conditions that are considered to be "pathological
disorders" as recited herein include acute and/or chronic renal
disease or failure, liver fibrosis or cirrhosis, cancer such as
breast cancer, Parkinson's Disease; conditions associated with
neuronal death, such as ALS, brain atrophy, or dementia and
anemia.
[0263] Further conditions include cachexia, cachexia associated
with a rheumatoid arthritis and cachexia associated with
cancer.
[0264] To date, very few reliable or effective therapies have been
developed to treat these disorders.
[0265] Based on reported evidence of a role of activins binding to
ActRIIB amongst other receptors (Werner and Alzheimer, Cytokine
Growth Factors Rev 2006, 17(3):157-171), in contributing to liver,
kidney and pulmonary fibrosis and of a role for myostatin,
activins, or ActRIIB in cancers (Tsuchida et al, Endo J, 2008) the
"pathological disorders" recited herein include liver, kidney and
pulmonary fibrosis and cancers examplified by but not restricted to
rhabdomyosarcomas, bone-loss inducing cancers, hepatocellular
carcinomas, gastrointestinal cancers.
[0266] The prevention may be complete, e.g., the total absence of
an age-related condition or metabolic disorder. The prevention may
also be partial, such that the likelihood of the occurrence of the
age-related condition or metabolic disorder in a subject is less
likely to occur than had the subject not received an antibody of
the present disclosure.
[0267] An age-related condition as referred to herein may begin at
the age of 50 years or older (i.e. 60, 70, 80 or older).
[0268] In one embodiment, a patient may be pre-treated with an
anti-ActRIIB antibody prior to an anticipated period of enforced
rest/inactivity. Such a period may occur when a patient is admitted
to hospital, for example for surgery to the hip or leg. The
inactivity may be localised, such as by casting of a broken limb or
joint, or by administration of a paralytic agent.
[0269] In one embodiment, the patient being treated has a fracture
to a limb (i.e. leg or arm) or joint (i.e. knee or hip). Thus, in
one embodiment, the patient being treated has a fracture to one or
more of the humerus, radius, ulnar, a carpal, a metacarpal, the
clavical, scapular, femur, os coxae, patella, tibia, fibula, talus,
calcaneus, a tarsal, a metatarsal, the ischium or the ileum. In
another embodiment, the patient being treated has undergone, or
will undergo surgery on one or more of the following joints: knee,
hip, ankle, shoulder, elbow. Such surgery includes hip replacement
and knee replacement.
[0270] Atrophy due to immobilisation may occur quickly, but
normally occurs slowly. Therefore, in one embodiment, the patient,
joint or limb has been immobilised, or will be immobilised, for 2
weeks or longer (i.e. 3 weeks, 4 weeks, 6 weeks, 8 weeks or
longer). In one embodiment, the patient, joint or limb has been
immobilised, or will be immobilised, for 1-8 weeks, 2-6 weeks or
3-5 weeks.
[0271] In a further embodiment, the patient may be one who has not
responded to previous bone anabolic treatments. For example, the
patient may not have responded to treatment with IGF-1, IGF-2 or
variants of IGF-1 or IGF-2, an anti-myostatin antibody, a myostatin
propeptide, a myostatin decoy protein that binds ActRIIB but does
not activate it, a beta 2 agonist, a Ghrelin agonist, a SARM, GH
agonists/mimetics or follistatin. A simple way of measuring a
patient's response to treatment may be timing how long it takes for
a patient to climb a known height of stairs and comparing the
results both before and after treatment.
[0272] The antibodies of the disclosure may be administered as the
sole active agent or in conjunction with, e.g. as an adjuvant to or
in combination to, other drugs e.g. IGF-1, IGF-2 or variants of
IGF-1 or IGF-2, an anti-myostatin antibody, a myostatin propeptide,
a myostatin decoy protein that binds ActRIIB but does not activate
it, a beta 2 agonist, a Ghrelin agonist, a SARM, GH
agonists/mimetics or follistatin. For example, the antibodies of
the disclosure may be used in combination with an IGF-1 mimetic as
disclosed in WO2007/146689.
[0273] In accordance with the foregoing the present disclosure
provides in a yet further aspect:
[0274] A method as defined above comprising co-administration, e.g.
concomitantly or in sequence, of a therapeutically effective amount
of an ActRIIB antagonist, e.g. an antibody of the disclosure, and
at least one second drug substance, said second drug substance
being IGF-1, IGF-2 or variants of IGF-1 or IGF-2, an anti-myostatin
antibody, a myostatin propeptide, a myostatin decoy protein that
binds ActRIIB but does not activate it, a beta 2 agonist, a Ghrelin
agonist, a SARM, GH agonists/mimetics or follistatin.
[0275] The disclosure further provides a therapeutic combination,
e.g. a kit, comprising of a therapeutically effective amount of a)
an ActRIIB antagonist, e.g. an antibody of the disclosure, and b)
at least one second substance selected from an IGF-1, IGF-2 or
variants of IGF-1 or IGF-2, an anti-myostatin antibody, a myostatin
propeptide, a myostatin decoy protein that binds ActRIIB but does
not activate it, a beta 2 agonist, a Ghrelin agonist, a SARM, GH
agonists/mimetics or follistatin, e.g. as indicated above. The kit
may further comprise instructions for its administration.
[0276] Where the antibodies of the disclosure are administered in
conjunction with another active agent, dosages of the
co-administered combination compound will of course vary depending
on the type of co-drug employed, on the specific drug employed, on
the condition being treated and so forth.
[0277] In another embodiment, the antibodies of the disclosure are
administered only to a patient population which is selected among
patients suffering from muscle atrophy. In another embodiment, the
antibodies of the disclosure are administered to patient
populations suffering from skeletal muscle atrophy. In another
embodiment, the antibodies of the disclosure are administered only
to a patient population which is selected among a group of patients
which respond to anti-ActRIIB treatment. Biomarkers that identify
patients that have an increased likelihood of responding to
anti-ActRIIB treatment may be any of the following without being
limited to these: high levels of serum myostatin, GDF-11 or
activins compared to a control patient.
[0278] In one embodiment, the antibodies of the disclosure can be
used to detect levels of ActRIIB, or levels of cells that contain
ActRIIB. This can be achieved, for example, by contacting a sample
(such as an in vitro sample) and a control sample with the
anti-ActRIIB antibody under conditions that allow for the formation
of a complex between the antibody and ActRIIB. Any complexes formed
between the antibody and ActRIIB are detected and compared in the
sample and the control. For example, standard detection methods,
well known in the art, such as ELISA and flow cytometic assays, can
be performed using the compositions of the disclosure.
[0279] Accordingly, in one aspect, the disclosure further provides
methods for detecting the presence of ActRIIB (e.g. human ActRIIB)
in a sample, or measuring the amount of ActRIIB, comprising
contacting the sample, and a control sample, with an antibody of
the disclosure, or an antigen binding region thereof, which
specifically binds to ActRIIB, under conditions that allow for
formation of a complex between the antibody or portion thereof and
ActRIIB. The formation of a complex is then detected, wherein a
difference in complex formation between the sample compared to the
control sample is indicative of the presence of ActRIIB in the
sample.
[0280] Also within the scope of the disclosure are kits consisting
of the compositions (e.g. antibodies, human antibodies and
bispecific molecules) of the disclosure and instructions for use.
The kit can further contain at least one additional reagent, or one
or more additional antibodies of the disclosure (e.g. an antibody
having a complementary activity which binds to an epitope on the
target antigen distinct from the first antibody). Kits typically
include a label indicating the intended use of the contents of the
kit. The term label includes any writing, or recorded material
supplied on or with the kit, or which otherwise accompanies the
kit. The kit may further comprise tools for diagnosing whether a
patient belongs to a group that will response to an anti-ActRIIB
antibody treatment, as defined above. Such kits may comprise an
antibody of the disclosure in lyophilised form, a diluent and
instructions for use.
[0281] The disclosure having been fully described, it is further
illustrated by the following examples and claims, which are
illustrative and are not meant to be further limiting.
General
[0282] The term "comprising" means "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0283] The term "about" in relation to a numerical value x means,
for example, x.+-.10%.
DESCRIPTION OF THE FIGURES
[0284] FIG. 1 shows EC50 determination of MOR07079 by FACS
titration on parental and ActRIIB transfected HEK293T/17 cell
lines.
[0285] FIG. 2 shows inhibition of myostatin-induced luciferase
expression in a reporter gene assay by multiples of anti-ActRIIB
Fabs at 2, 10 and 50 .mu.g/ml.
[0286] FIG. 3 shows IC50 determination of Fabs in myostatin-induced
luciferase reporter gene assay.
[0287] FIG. 4 shows antibody binding to primary human skeletal
muscle cells.
[0288] FIG. 5 shows IC50 determination of IgG in myostatin-induced
inhibition of skeletal muscle differentiation assay.
[0289] FIG. 6 shows mouse study: in vivo efficacy study in naive
animals--6 weeks treatment with MOR08159 or MOR08213 at 10 mg/kg
increased body and muscle weight. Changes are shown for (A) body
weight, (B) Tibialis, (C) Gastrocnemius with plantaris, (D)
Quadriceps and (E) Pectoralis.
[0290] FIG. 7 shows mouse study: dose response in vivo efficacy
study in naive animals--6 weeks treatment with MOR08213 at 25, 5, 1
mg/kg, dose-dependently increases body and muscle weight. Changes
are shown for (A) body weight, (B) Tibialis, (C) Gastrocnemius with
plantaris, (D) Quadriceps and (E) Pectoralis.
[0291] FIG. 8 shows a FACS output demonstrating cross-blocking
between MOR08159 in the presence of MOR08213 (bold dashed) and
MOR08159 alone (bold black), compared to isotype control alone
(black) or isotype control in the presence of MOR08213
(dashed).
[0292] FIG. 9 shows an overview of ActRIIB residues (SEQ ID NO:181)
to which MOR08159 binds, using various epitope determination
techniques.
MODES FOR CARRYING OUT PARTICULAR METHODS
Functional Assays
Reporter Gene Assay (RGA)
Cultivation of HEK293T/17 Cell Lines
[0293] Parental HEK293T/17 cells are maintained in DMEM containing
10% FBS, 2 mM L-glutamine, penicillin (50 IE/ml), and streptomycin
(50 .mu.g/ml). Cells are grown in an incubator at 37.degree. C. and
5% CO.sub.2 and subcultured every 3-4 days. Cells are detached
using Accutase.TM. and then transferred into a new flask containing
fresh medium.
[0294] HEK293T/17 cells stably transfected with CAGA-12 luc are
cultured as described above for parental HEK293T/17 cells but cell
growth medium is supplemented with 4 mM L-glutamine and 3 .mu.g/ml
blasticidin in addition to FBS, penicillin and streptomycin.
Myostatin-Induced Luciferase Reporter Gene Assay
[0295] To determine the capacity of anti-ActRIIB antibodies to
inhibit myostatin-induced signaling, a reporter gene assay using
the stable reporter cell line HEK293T/17 CAGA-12 luc is performed.
The CAGA-12 luciferase reporter construct carries the luciferase
gene downstream of a minimal promoter and multiple CAGA boxes which
are specific for phosphorylated Smad-2 and Smad-3. Addition of
purified myostatin (but also of GDF-11, activin or TGF.beta.)
induces Smad phosphorylation and thus binding to the CAGA-12
reporter and leads to luciferase gene expression.
[0296] At 90% confluency of HEK293T/17 CAGA-12 luc cells, cells are
detached as described and diluted in culture medium to a
concentration of 2.5.times.10.sup.5 cells/ml. Subsequently, 100
.mu.l cells per well are seeded into flat-bottomed 96-well plates
and incubated at 37.degree. C. and 5% CO.sub.2 overnight.
[0297] The next day, the antibodies (Fab or IgG) and the
recombinant human ActRIIB/Fc, which served as the positive control,
are diluted in PBS to the desired concentrations. 20 .mu.l of the
antibody solutions are added to the seeded wells of the previous
day and the cells cultivated for 1 hr to allow binding of the
antibodies. Finally, 50 ng/ml myostatin is added to the wells and
the cells further cultivated over night.
[0298] The next morning, 120 .mu.l. 1 Bright-Glo luciferase reagent
(Promega) is added to each well. After 2 min incubation time, the
luminescence is read in a luminometer. The half maximal inhibitory
concentration (IC50 values) is calculated after full titration of
the respective antibodies.
Specificity Elisas
[0299] The specificity of anti-ActRIIB Fab antibodies to human
ActRIIB and crossreactivity to human ActRIIA and mouse ActRIIB is
evaluated in an ELISA setting. Additionally, binding to related
receptors (counter-targets: human TGF-.beta.RII/Fc (R&D
systems), mouse TGF-.beta.RI (ALK-5)/Fc (R&D systems), human
Activin RIB (ALK-4)/Fc (R&D systems)) is determined. For this,
5 .mu.g/ml (if not stated otherwise) of the recombinant proteins
diluted in PBS are added to a black 96-well flat-bottom
MaxiSorp.TM. plate and incubated over night at 4.degree. C. for
coating.
[0300] The next morning, the plates are washed with TBST and
blocked with MTBST. After washing the plates several times, 5
.mu.g/ml anti-ActRIIB Fab are added and incubated for 2.5 hrs.
Subsequently antigen bound Fabs are detected by incubation with
alkaline phosphatase conjugated goat-anti-human IgG Fab-specific,
followed by addition of AttoPhos fluorescence substrate.
Fluorescence emission at 535 nm is recorded with excitation at 430
nm in a TECAN Spectrafluor plate reader.
ActRIIB/Fc-Myostatin Binding Interaction ELISA
[0301] To assess whether the inhibitory Fabs act via blocking the
myostatin binding site of human ActRIIB a hActRIIB/Fc-myostatin
interaction ELISA is performed. For this, recombinant myostatin is
diluted to 5 .mu.g/ml in PBS and coated onto a black 96-well
flat-bottom Maxisorp plate. The next morning the wells are blocked
with MTBST. Meanwhile 50 .mu.g/ml anti-ActRIIB Fabs are
pre-incubated with 10 .mu.g/ml ActRIIB/Fc in TBST for 1.5 hrs at
room temperature and finally added to the coated and blocked wells
(1.5 hr at room temperature). After washing with TBST buffer,
detection of bound ActRIIB/Fc is performed using an unlabelled
mouse anti-human Ig Fc-specific antibody and a POD-labelled sheep
anti-mouse IgG detection antibody. After washing the wells several
times with TBST buffer Quanta Blu.TM. Fluorogenic Peroxidase
substrate is added. The fluorescence is read in a GENiosPro.TM.
reader (excitation 320 nm, emission 430 nm).
Binding to Cells
Cells
[0302] Stable human ActRIIA- and human ActRIIB-transfected
HEK293T/17 cells, generated using HEK293T/17 cells (ATCC)
transfected with linearized pEGFP (Clontech)-ActRIIB(ECD) or
-ActRIIA(ECD) and pPGK-puro (AddGene) using FuGENE6 (Roche), are
maintained in DMEM containing 10% FBS, 2 mM L-glutamine, penicillin
(50 IE/ml), streptomycin (50 .mu.g/ml) and puromycin (2 .mu.g/ml).
Cells are grown in an incubator at 37.degree. C. and 5% CO.sub.2
and subcultured every 3-4 days. Cells are detached using
Accutase.TM. and then transferred into a new flask containing fresh
media.
[0303] Human skeletal muscle cells (huSkMC) (Cambrex) are harvested
at a confluency of about 70-90%. For those cells, culture medium,
growth medium (GM) consisting of skeletal muscle basal medium
(skBM; Lonza) supplemented with 20% FCS (Amimed), is aspirated, and
the cells are washed with HEPES-BSS and incubated with
Trypsin/EDTA. After the cells are detached, trypsin is neutralized
with trypsin neutralizing solution. Cells are centrifuged at
220.times.g for 5 min and the pellet is resuspended in Skeletal
Muscle Growth Media. Cells are then used for experiments or seeded
for subculturing at a cell density of .about.3500 cell/cm.sup.2.
Cells are grown in an incubator at 37.degree. C. and 5% CO.sub.2
and subcultured every 5-6 days.
FACS Titration on hActRIIB- and hActRIIA-Expressing Cells
[0304] The half maximal effective concentration (EC50) of the
anti-ActRIIB antibodies is determined via binding to cellular
hActRIIA and hActRIIB by FACS.
[0305] For this, serial dilutions of anti-ActRIIB Fab or IgG are
incubated with 1.times.10.sup.4 hActRIIA-transfected,
hActRIIB-transfected or parental HEK293T/17 cells per well for 1 h
at 4.degree. C. After several washing steps cell-bound Fabs or IgGs
are detected with phycoerythrin-conjugated goat anti-human IgG
(H+L) secondary antibody. After one hour incubation at 4.degree.
C., the cells are washed again and resuspended in FACS buffer and
fluorescence intensity of the cells is determined in a
FACSArray.TM. instrument.
Binding to Primary Human Skeletal Muscle Cells
[0306] Anti-ActRIIB Fab or IgG as well as isotype control Fab or
IgG (10 .mu.g) are incubated with 10.sup.5 huSkMC in FACS buffer
(PBS, 2% FCS, 1 mM EDTA) per tube for 1 h at 4.degree. C. After
washing steps, cell-bound Fabs or IgGs are detected with
phycoerythrin-conjugated goat anti-human IgG (H+L) secondary
antibody which had been diluted 1:200 in FACS buffer. After one
hour incubation at 4.degree. C. on a shaker, the cells are washed
again and resuspended in FACS buffer and fluorescence intensity of
the cells determined in the FACSCaliber.TM. instrument.
Affinity Determination
Affinity Determination of Selected Anti-Human ActRIIB Fabs Using
Surface Plasmon Resonance (Biacore)
[0307] For direct antigen immobilisation standard EDC-NHS amine
coupling chemistry is used. CMS chips (Biacore, Sweden) are coated
with approximately 6000 RU human- or mouse-ActRIIB/Fc, or
approximately 1500 RU human-ActRIIA/Fc (according to the activity
of the antigens) in 10 mM acetate buffer, pH 4.5. For the reference
flow cell, a respective amount of HSA is used. Regeneration is done
with 5 .mu.l 10 mM Glycine/HCl buffer pH1.5.
[0308] Alternatively, the antigens are not immobilized directly,
but captured on a CMS chip, which is modified with an anti-human-Fc
antibody (Fc capture kit, GE Healthcare/Biacore). On the reference
flow cell, capture antibody is immobilized, but no antigen
captured. Regeneration is achieved using 2 injections of 5 .mu.L 3M
MgCl.sub.2.
[0309] Kinetic measurements are done in Dulbecco's PBS at a flow
rate of 20 .mu.l/min using a serial dilution row of Fab samples.
The Fab concentrations ranged from 15.6 to 500 nM. Injection time
for each concentration is 1 min. The dissociation time is set to at
least 2 min (or more, according to determined affinity). A blank
injection of running buffer is used for double referencing. All
sensorgrams are fitted globally using BIA evaluation software 3.2
(Biacore, Sweden).
CK Assay
[0310] Differentiation is initiated 24 hours after seeding by
changing cells from GM to serum-free differentiation medium
consisting of skeletal muscle basal medium (skBM). Cells are
differentiated for 3 days in the absence and presence of myostatin
(R&D systems) or other TGF-b proteins and tested antibodies at
given concentrations. Cells are washed with PBS and then lysed with
Reporter lysis buffer (Promega) and stored till measurement at
-80.degree. C. CK activity is measured using the CK (IFCC) reagent
(Thermo Electron). The CK reagent is prepared according to the
manufacturers instructions. Cell lysates are adjusted to room
temperature, CK reagent is added and absorbance is immediately read
at 340 nm for 20 min, reading interval 1 min. CK standard curves
are freshly prepared using CK from rabbit muscle (Roche
Diagnostics). Protein content is determined using BCA kit.
Animal Models
[0311] Nine-week-old female CB17/ICR-Prkdc.sup.scid/Crl mice (n=10
per group, Charles River, Germany) are randomized with body weight
and then treated intraperitoneally with anti-human ActRIIB
antibodies (MOR8159, MOR8213) or IgG control antibody at a dose of
10 mg/kg (Study 1; comparison study), or with MOR8213 at doses of
25, 5, or 1 mg/kg (Study 2; dose response study) on day 0, 3, 7,
14, 21, 28 and 35 (once weekly with day 3). Body weights are
determined two times per week. Six weeks (42 days) after
administration, mice are euthanized with CO.sub.2. Tibialis,
gastrocnemius with plantaris, quadriceps and pectoralis are
collected and weighed.
Treatment Protocol
[0312] Control antibody: anti-chicken lysozyme-hIgG, Concentration:
2 mg/mL (study 1), 5 mg/mL (study 2), application volume: 5 mL/kg
Vehicle: 50 mM Citrate, 140 mM NaCl or PBS anti-human ActRIIB
antibodies: anti-ActRIIB-MOR8159 and MOR8213, hIgG, Concentration:
2 mg/mL (study 1), 5 mg/mL (study 2), 1 mg/mL (study 2), 0.2 mg/mL
(study 2), application volume: 5 mL/kg
Vehicle: 50 mM Citrate, 140 mM NaCl
Treatment Groups:
Study 1; Comparison of MOR08159 and MOR08213
[0313] 1 IgG control, i.p. (anti-chicken lysozyme-IgG), 10 mg/kg 2
anti-ActRIIB-MOR8159, i.p., 10 mg/kg 3 anti-ActRIIB-MOR8213, i.p,
10 mg/kg Study 2; dose response of MOR08213 1 IgG control, i.p.
(anti-chicken lysozyme IgG), 25 mg/kg 2 anti-ActRIIB-MOR8213, i.p.,
25 mg/kg 3 anti-ActRIIB-MOR8213, i.p., 5 mg/kg 4
anti-ActRIIB-MOR8213, i.p., 1 mg/kg
Maintenance Conditions
[0314] Animals are housed in groups of four to five animals at
25.degree. C. with a 12:12 h light-dark cycle. They are fed a
standard laboratory diet containing 18.2% protein and 3.0% fat with
energy of 15.8 MJ/kg (NAFAG 3890, Kliba). Food and water are
provided ad libitum. Animal experimentation is carried out
according to the regulations effective in the Canton of Basel-City,
Switzerland.
Methods
Statistical Analysis
[0315] Results are expressed as mean+/-SEM. Statistical analysis is
carried out using Dunnett's multiple comparison test following
one-way analysis of variance. Treatment (anti-ActRIIB antibodies
MOR8159 and MOR8213 are tested for difference to control (control
antibody) and differences are considered to be significant when the
probability value is <0.05: *: P<0.05, **: P<0.01, NS: no
significance versus IgG control. Statistical analyses are performed
by GraphPad Prism version 5.0 (GraphPad Software, Inc). Body weight
are calculated by subtracting body weight at day 0, and muscle
weight is normalized by body weight at day 0 (initial body
weight).
Pannings, Antibody Identification and Characterization
[0316] Therapeutic antibodies against human ActRIIB protein are
generated by selection of clones having high binding affinities,
using as the source of antibody variant proteins a commercially
available phage display library, the MorphoSys HuCAL GOLD.RTM.
library.
[0317] HuCAL GOLD.RTM. library is a Fab library (Knappik et al.,
2000) in which all six CDRs are diversified by appropriate methods,
and which employs the CysDisplay.TM. technology for linking the Fab
to the phage surface (WO01/05950).
[0318] HuCAL GOLD.RTM. phagemid library (Rothe et al., 2008) is
used to select specific Fab antibody fragments.
Selection by Panning of ActRIIB-Specific Antibodies from the
Library
[0319] For the selection of antibodies recognizing human ActRIIB
several panning strategies are applied.
[0320] In summary, HuCAL GOLD.RTM. antibody-phages are divided into
several pools comprising different VH master genes.
[0321] These pools are individually subjected to differential cell
pannings whereby selection rounds on transiently human ActRIIB
transfected cells alternated with selection rounds on recombinant
human ActRIIB/Fc protein.
[0322] i. Whole Cell Panning
[0323] For the pannings, phage particles diluted in PBS are mixed
with an equal volume of PBS/BSA and blocked. In parallel, also in
pre-blocked tubes, 1.times.10.sup.7 of the respective hActRIIB
expressing cells per phage pool are resuspended in PBS/3% FCS/0.04%
NaN.sub.3 and blocked for one hour at 4.degree. C. on a shaker. The
blocked cells are spun down, resuspended in the pre-blocked phage
particles and incubated for three hours. In the meantime,
1.times.10.sup.7 hActRIIB knock-down cells per phage pool are
prepared.
[0324] The phage-cell complexes are washed in PBS/BSA, followed by
washing in PBS. Elution of phage particles from the hActRIIB
expressing cells is performed by acidic elution with glycine
buffer, pH 2.2. After centrifugation, the eluate is neutralized by
adding unbuffered Tris.
[0325] After infection and subsequent centrifugation, the bacterial
pellets are resuspended in 2xYT medium, plated onto LB/CAM/Glc agar
plates and incubated overnight at 37.degree. C. The next morning,
the colonies are scraped off the plates and the phages are rescued
and amplified.
[0326] ii. Solid phase Panning
[0327] For solid phase panning recombinant human ActRIIB/Fc is
coated onto a MaxiSorp.TM. plate at 4.degree. C. over night. After
washing with PBS the coated wells are blocked with 5% MPBST.
[0328] Prior to the selections, HuCAL GOLD.RTM. phage are
pre-adsorbed in blocking buffer. The blocked phage are added to the
coated antigen and incubated for 2 hrs at room temperature.
Unspecific phage are washed off with PBST and PBS. Bound phage are
eluted by addition of 20 mM DTT. The eluates are used for infection
of an E. coli TG-1 culture. After infection, the bacteria are
plated onto LB/CAM/Glc agar plates and incubated overnight at
37.degree. C. The next morning, the colonies are scraped off the
plates and the phage are rescued and amplified.
[0329] The most successful panning approach proved to be
differential cell/protein pannings with first panning round on
ActRIIB transfected HEK293T/17 cells followed by selection round on
recombinant human ActRIIB/Fc and again on transfected cells.
[0330] Selected Fabs are analyzed for binding to parental or
rhActRIIB transfected HEK293 cells.
[0331] MOR07079 Fab binds in various embodiments to
ActRIIB-transfected cells with an EC50 of 20 nM (FIG. 1). In a
myostatin binding inhibition ELISA, MOR07079 Fab showed inhibitory
activity and blocked rhActRIIB/Fc binding to myostatin. Strong
myostatin binding inhibition in ELISA is reflected by myostatin
inhibition in the reporter gene assay using HEK293-CAGA12 for
MOR07079. Using the specificity ELISA, MOR07079 is shown to bind
specifically to human and murine ActRIIB and not to the unrelated
TGF.beta.RII, ALK4 and ALK5 receptors. MOR7079 is also shown to
bind in various embodiments to ActRIIB compared to ActRIIA.
Production of HuCAL.RTM. Immunoglobulins
[0332] i. Conversion of Fabs into the IgG Format
[0333] In order to express full length immunoglobulin (Ig), the
variable domain fragments of heavy (VH) and light chains (VL) are
subcloned from the pMORPH.RTM.X9_FH Fab expression vectors into the
pMORPH.RTM.2_h_Ig vector series for human IgG2. Selected clones are
also converted into the silent IgG1LALA format in which leucines at
positions 234 and 235 are mutated to alanines to abrogate
FcR.gamma. binding and attenuate effector functions.
[0334] Appropriate restriction enzymes (Knappik et al., 2000) are
used for subcloning of the VH and VL domain fragments into
pMORPH.RTM.2_h_IgG2, pMORPH.RTM.2_h_IgG1LALA,
pMORPH.RTM.2_h_Ig.kappa., and pMORPH.RTM.2_h_Ig.lamda.2.
[0335] All DNA preparations are subjected to sequence analysis
before transfection into HKB11 cells.
[0336] ii. Transient Expression and Purification of Human IgG
[0337] Eukaryotic HKB11 cells are transfected with IgG heavy and
light chain expression vector DNA. Cell culture supernatant is
harvested at 3 or 7 days post transfection and subjected to
standard protein A affinity chromatography. If not otherwise
stated, buffer exchange is performed to 1.times. Dulbecco's PBS (pH
7.2) and samples are sterile filtered (0.2 .mu.m).
CDR-L3 and CDR-H2 Maturation Libraries
[0338] To increase affinity and biological activity of selected
antibody fragments, CDR-L3 and CDR-H2 regions are optimized in
parallel by cassette mutagenesis using trinucleotide directed
mutagenesis (Virnekas et al., 1994, Nucleic Acids Res.
22:5600-5607), whereby the framework regions are kept constant
(Nagy et al., 2002, Nature Medicine, 8:801-807). Prior to cloning
of the maturation libraries, all parental Fab fragments are
transferred from the expression vector pMORPH.RTM.X9 into the
CysDisplay.TM. maturation vector pMORPH.RTM.25 via the XbaI/EcoRI
restriction sites. This vector provides the phage protein pIII
fused N-terminally to a cysteine residue as well as a C-terminal
cysteine fused to the Fd antibody chain and thus allows
disulfide-linked display of the respective Fab fragments on the
phage surface.
[0339] For generation of the CDR-H2 libraries the CDR-H2 region of
each parental Fab is excised and replaced by the highly diversified
CDR-H2 maturation cassette.
[0340] In parallel, the CDR-L3 region of the parental clones is
replaced by a diversified CDR-L3 maturation cassette.
[0341] The sizes of the maturation libraries ranged from
4.times.10.sup.5 to 1.times.10.sup.8 clones. The vector background
is below 1% in all cases. The quality control by sequencing of
single clones revealed a high quality of each library.
[0342] For each CDR-L3 and CDR-H2 maturation library,
antibody-displaying phage are prepared and phage titers determined
by spot titration.
Panning Strategies for Affinity Maturation
[0343] The antibody-displaying phages from the following maturation
libraries are subjected to separate pannings and screenings:
Lead 1: MOR07079 (L-CDR3 maturation) Lead 1: MOR07079 (H-CDR2
maturation)
[0344] Maturation pannings using the respective antibodies are
performed on biotinylated hActRIIB/Fc and on huSkMC.
[0345] Either 2.times.10.sup.10 or 1.times.10.sup.11 phages per
subcode, rescued from the newly generated maturation libraries are
used for the first selection rounds.
[0346] Several differential pannings are performed whereby
selection rounds on recombinant biotinylated hActRIIB/Fc alternated
with a selection round on huSkMC.
[0347] For the first and third round of the solution panning
biotinylated recombinant hActRIIB/Fc is captured onto
Streptavidin-coated Dynabeads. The following protocol is applied:
for each phage pool, Streptavidin beads are washed with PBS and
resuspended in blocking buffer. Phage particles diluted in PBS are
mixed blocking buffer containing 0.1% Tween20 and kept on a
rotating wheel. Preclearing of phage particles for removal of
Streptavidin- or bead-binding phages is performed twice: Per phage
pool blocked Streptavidin beads are added to the blocked phage
particles and incubated on a rotating wheel. After separation of
the beads via a magnetic device the phage supernatant is
transferred to a fresh, pre-blocked reaction tube and
pre-adsorption is repeated.
[0348] After the blocking procedure, the biotinylated hActRIIB/Fc
antigen is added to the precleared and blocked phage particles and
incubated on a rotating wheel. The phage-antigen complexes are
captured using blocked streptavidin beads, added to the phage
panning pools and incubated further. Phage particles bound to the
streptavidin beads are collected. Beads are then washed with PBST
and PBS. Elution of phage particles from the streptavidin beads is
performed by addition of 20 mM DTT. The eluate is collected and
used for infection of an E. coli TG-1 culture grown to an
OD.sub.600nm of 0.6-0.8.
[0349] After infection and subsequent centrifugation, the bacterial
pellets are resuspended in 2xYT medium, plated onto LB/CAM/Glc agar
plates and incubated overnight at 37.degree. C. The next morning,
the colonies are scraped off the plates and the phages are rescued
and amplified mainly as described (Krebs et al., 2001) with the
exception that helper phage infected cells are grown at 22.degree.
C. over night in medium containing 0.25 mM IPTG. The third rounds
of the solution pannings on biotinylated hActRIIB/Fc are performed
according to the protocol of the first round except for decreasing
amounts of antigen used and increased stringency of the washing
conditions.
[0350] For the second round of panning (on huSkMC expressing
endogenous hActRIIB), phage particles diluted in PBS are mixed with
an equal volume of PBS/BSA and blocked. In parallel, for each
subcode 9.times.10.sup.5 huSkMC are blocked with PBS/FCS/0.02%
NaN.sub.3 at 4.degree. C. The blocked cells are spun down,
resuspended together with the pre-blocked phage particles and
incubated further.
[0351] The phage-cell complexes are washed with PBS/BSA, followed
by washing in PBS. Cells are centrifuged at 410.times.g for 2 min
at 4.degree. C. Acidic elution of phage particles from the hActRIIB
expressing huSkMC is performed by a 10 min incubation step with
glycine buffer, pH 2.2. After centrifugation, the eluate is
neutralized by adding unbuffered Tris. The phage containing
supernatant is used for infection of an E. coli TG-1 culture grown
to an OD.sub.600nm of 0.6-0.8.
[0352] After infection and subsequent centrifugation, the bacterial
pellets are resuspended in 2xYT medium, plated onto LB/CAM/Glc agar
plates and incubated overnight at 37.degree. C. The next morning,
the colonies are scraped off the plates and the phages are rescued
and amplified mainly as described (Krebs et al., 2001) with the
exception that helper phage infected cells are grown at 22.degree.
C. over night in medium containing 0.25 mM IPTG.
[0353] The most successful panning approach which resulted in very
potent binders proved to be the differential panning with the first
and third round performed on biotinylated ActRIIB/Fc and the second
round on huSkMC.
[0354] After sequencing, Fabs are selected for expression and
purification, and the most promising further characterized.
[0355] Most anti-ActRIIB antibodies showed binding to
hActRIIB-transfected HEK293T/17 cells with EC50 values in the
single up to low double digit nanomolar range. Several Fabs could
displace myostatin from ActRIIB/Fc in a myostatin binding
inhibition ELISA, but amongst those only MOR08067 displayed full
inhibition of myostatin-induced activity in the reporter gene assay
(FIG. 2).
[0356] Summarized affinities of the most promising Fab to human and
mouse ActRIIB/Fc are listed in the table below (Table 1).
TABLE-US-00001 TABLE 1 Affinity data of anti-ActRIIB Fab-FH to
ActRIIB antigens KD determination (Biacore) human mouse ActRIIB-Fc
ActRIIB-Fc Fab KD [nM] KD [nM] MOR07079 51 62 MOR08047 23 22
MOR08062 15 17 MOR08067 <0.1 <0.1 MOR08077 11 13 MOR08078 9
10
[0357] The Fab clone MOR08067 exhibited good inhibition in the
myostatin induced RGA as well as binding to rhActRIIB transfected
HEK293 cells. Affinity determination by Biacore revealed KD values
to human and mouse ActRIIB/Fc below 100 pM. MOR08067 and other
candidates are selected for further optimization by a cross cloning
approach, while MOR08067, containing a potential N-linked
glycolsylation site is also subjected to a deglycosylation
approach.
Optimization of Antibodies Derived from First Affinity
Maturation
[0358] a) Deglycosylation of MOR08067
[0359] According to sequence analysis this antibody contained a
potential N-linked glycosylation site within the CDR-H2 of the
heavy chain. This site is removed to yield MOR08156 and MOR08159.
The characterization of these MOR08067-derivatives is described
below.
[0360] b) Cross Cloning of Optimized Fabs
[0361] For a further functional improvement and removal of
potential N-linked glycosylation sites in CDR-H2s and/or CDR-L3s,
the independently optimized CDR-H2 and CDR-L3 regions from single
affinity matured Fabs resulting from the first affinity maturation
are combined while keeping each family separate. Descendants of
MOR07079 entered cross cloning. Roughly 200 bacterial lysates are
tested in FACS affinity ranking on HEK293T/17/ActRIIB and the most
promising Fab clones, MOR08144 and MOR08213 are expressed, and
purified.
[0362] c) Characterization of Optimized Antibodies
[0363] In the following sections, the deglycosylated progenies of
MOR08067 (MOR08156, MOR08159) and the two cross clones derived from
MOR08067 (MOR08144 and MOR08213) are described in detail.
[0364] The ability of optimized Fabs to inhibit myostatin signaling
in the reporter gene assay is determined, with all binders being
able to induce >95% inhibition at the highest concentration
(FIG. 3).
[0365] In affinity determination experiments using Biacore,
MOR08159 and MOR08213 are identified as highly potent binders to
both human and mouse ActRIIB (Table 2). It became obvious the
increased affinity of matured and optimized Fabs reflected
increased potency in the myostatin-induced reporter gene assay.
TABLE-US-00002 TABLE 2 Affinity data of anti-ActRIIB Fabs to
ActRIIB antigens KD determination (Biacore) human mouse ActRIIB-Fc
ActRIIB-Fc Fab KD [pM] KD [pM] MOR08159 3.8 3.1 MOR08213 13.2
13.5
IgG2 Conversion of Affinity Matured Fabs (1.sup.st Maturation)
[0366] The most promising Fabs derived from the first affinity
maturation are selected for IgG2 conversion.
[0367] IgG2 expression is performed by transient transfection of
HKB11 cells and the full length immunoglobulins are purified from
the cell culture supernatants.
[0368] Upon conversion to IgG, all candidates retained their
ability to dose-dependently inhibit myostatin-induced activity in
the reporter gene assay (Table 3).
TABLE-US-00003 TABLE 3 IC50 determination of anti-ActRIIB IgGs in
myostatin-induced luciferase reporter gene assay IgG IC50 [nM] %
inhibition MOR08067 2.57 86.5 MOR08144 0.5 94.9 MOR08156 0.19 97.4
MOR08159 0.32 99 MOR08213 0.32 98.6
[0369] MOR08159 and MOR08213 are tested for their ability to bind
to human primary muscle myoblasts by FACS, and specific binding to
those cells is reported, in line with low expression of ActRIIB on
those cells (FIG. 4).
[0370] MOR08159 and MOR08213 displayed ability to fully reverse the
myostatin-induced inhibition of primary skeletal myoblasts
differentiation (FIG. 5). Those antibodies also increased
differentiation above basal level in the absence of exogenous
myostatin, due to their ability to neutralize endogenously produced
ActRIIB ligands.
Second Affinity Maturation
[0371] Selection of Candidates for Second Affinity Maturation to
further improve the efficacy.
[0372] i. Construction of the CDR-L3 and CDR-H2 Maturation
Libraries
[0373] To increase both affinity and biological activity of the
selected antibody fragments (e.g. MOR08067), CDR-L1 and CDR-H2
regions are optimized by cassette mutagenesis using trinucleotide
directed mutagenesis (Virnekas et al. [supra]), whereby the
framework regions are kept constant (Nagy et al. [supra]). Prior to
cloning of the maturation libraries, all parental Fab fragments are
transferred from the expression vector pMORPH.RTM.X9 into the
CysDisplay.TM. maturation vector pMORPH.RTM.25 via the XbaI/EcoRI
restriction sites.
[0374] The sizes of all maturation libraries yielded always a
minimum of 1.times.10.sup.7 independent clones. The vector
background is below 1% in all cases. Quality control by sequencing
of single clones revealed a high quality of each library.
[0375] For each CDR-L1 and CDR-H2 maturation library,
antibody-displaying phages are prepared and phage titers are
determined by spot titration.
[0376] ii. Panning Strategies, Affinity Ranking and Screening for
Improved Antibodies
[0377] Differential pannings for the second round of affinity
maturation included parental HEK293T/17 cells and huSkMC expressing
human ActRIIB endogeneously at low levels. Additionally,
recombinant biotinylated hActRIIB/Fc antigen is included in all
panning strategies.
[0378] For ranking of the anti-ActRIIB Fabs, approximately 2700
bacterial lysates (.about.88 clones for each panning subcode) are
affinity ranked on recombinant biotinylated hActRIIB/Fc antigen and
membrane vesicle preparations of hActRIIB-transfected HEK293T/17
cells in an MSD-based method. Hits with high affinity ranking
factor are sequenced.
[0379] In addition, randomly selected clones which did not show up
as hits are evaluated in FACS affinity ranking. For this, bacterial
lysates are screened using parental HEK293T/17 and/or
hActRIIB-transfected HEK293T/17 cells. Cell-bound Fabs are detected
with phycoerythrin-conjugated goat anti-human IgG (H+L) secondary
antibody. The quantification of Fab expression in the lysates is
performed in parallel.
[0380] All panning strategies yielded anti-ActRIIB specific
antibodies. MOR08067 progenies could be identified after sequence
analysis. All binders are matured in CDR-H2.
IgG2 Conversion and Characterization of IgG2 (2.sup.nd
Maturation)
[0381] Again, the most promising Fabs derived from the second
affinity maturation are selected for IgG2 conversion. IgG2
expression is performed by transient transfection of HKB11 cells
and the full length immunoglobulins are purified from the cell
culture supernatants.
[0382] All IgGs tested are able to fully reverse the
myostatin-induced inhibition of primary skeletal myoblasts
differentiation (table 4).
TABLE-US-00004 TABLE 4 IC50 determination of anti-ActRIIB IgGs in
myostatin-induced inhibition of skeletal muscle differentiation
assay IgG CK assay IC50 [nM] MOR08159 1.89 MOR08213 1.7 MOR08806
0.52 MOR08807 5.02 MOR09032 1.02 MOR09058 2.3
[0383] We evaluated the ability of the anti-ActRIIB Ab to
neutralize binding of myostatin as well as other TGF.beta. family
ligands to ActRIIB on primary human skeletal myoblasts. In the
myoblast differentiation assay, we assessed the various ligands
potential to inhibit differentiation in the absence or presence of
either MOR08159 or MOR08213.
TABLE-US-00005 TABLE 5 IC50 and Emax determination of various
ligand-induced inhibition of skeletal muscle differentiation assay
in the presence or absence of MOR08159/MOR08213 (10 .mu.g/ml) no Ab
MOR08159 MOR08213 TGF.beta. family IC.sub.50 E.sub.max IC.sub.50
E.sub.max IC.sub.50 E.sub.max ligands (ng/ml) (% control) (ng/ml)
(% control) (ng/ml) (% control) Myostatin 8.5 .+-. 0.6 25.7 .+-.
1.5 42.7 .+-. 5.8 28.9 .+-. 4.8 35.1 .+-. 5.1 35.3 .+-. 4.5 GDF-11
7.0 .+-. 1.2 23.2 .+-. 3.8 13.3 .+-. 0.9 22.1 .+-. 2.1 12.0 .+-.
1.0 27.1 .+-. 2.6 Activin A 14.7 .+-. 2.9 37.1 .+-. 3.9 34.7 .+-.
9.0 61.9 .+-. 5.4 41.9 .+-. 3.4 57.2 .+-. 1.9 BMP-2 26.9 .+-. 2.6
2.6 .+-. 4.2 34.0 .+-. 2.6 5.1 .+-. 3.4 32.3 .+-. 1.4 4.8 .+-.
1.9
[0384] Myostatin and GDF-11 are able to inhibit human myoblasts
differentiation with similar efficiencies and to similar extents.
In the presence of a single concentration of MOR08159 or MOR08213,
myostatin and GDF-11 dose responses are shifted in a parallel
manner. Activin A is also able to inhibit differentiation, however
in the presence of MOR08159 or MOR08213, we observed a non parallel
shift accompanied by a change in Emax and potency. BMP-2 response
is unaffected by the presence of MOR08159 or MOR08213, suggesting
that it does not occur via ActRIIB binding.
Characterization of Anti-ActRIIB Antibodies in In Vivo Murine
Studies.
[0385] The ability of anti-ActRIIB antibodies to induce muscle
hypertrophy is evaluated in SCID mice administered MOR08159 or
MOR08213, 10 mg/kg i.p. weekly for 6 weeks (FIG. 6).
[0386] Both antibodies are able to induce a profound hypertrophy of
all examined muscles at study end. Significant increase in overall
bodyweight of anti-ActRIIB antibody treated mice is detected as
early as after 1 week of treatment.
[0387] MOR08213 is able to induce a dose-dependent profound
hypertrophy of all examined muscles at 5 and 25 mg/kg while no
significant changes are noticed at 1 mg/kg dose (FIG. 7).
Cross Blocking Studies
[0388] Stable human ActRIIB-transfected HEK293T/17 cells are
maintained in DMEM containing 10% FBS, 2 mM L-glutamine, penicillin
(50 IE/ml), streptomycin (50 .mu.g/ml) and puromycin (2 .mu.g/ml).
Cells are grown in an incubator at 37.degree. C. and 5% CO.sub.2
and subcultured every 3-4 days. Cells are detached using
Accutase.TM. and then transferred into a new flask containing fresh
media.
[0389] The ability of the anti-ActRIIB antibodies to bind to the
same epitope of human ActRIIB is assessed by FACS using
hActRIIB-expressing cells.
[0390] For this, an anti-ActRIIB IgG is incubated with
1.times.10.sup.5 hActRIIB-transfected cells per well for 1 h at
4.degree. C. After washing, a different biotinylated anti-ActRIIB
IgG or a control biotinylated IgG are incubated at equimolar
concentration to the first anti-ActRIIB IgG for 1 h at 4.degree. C.
After washing, cell-bound biotonylated IgG are detected with
streptavidin-APC (Biolegend). After one hour incubation at
4.degree. C., the cells are washed again and resuspended in FACS
buffer and fluorescence intensity of the cells is determined in a
FACSArray.TM. instrument.
[0391] MOR08159 and MOR08213 are tested for their ability to
jointly bind to human ActRIIB-transfected cells by FACS, and
specific binding of MOR08159 alone (bold black) or in the presence
of MOR08213 (bold dashed) is reported compared to isotype control
(black) or isotype control in the presence of MOR08213 (dashed)
(FIG. 8).
[0392] In the presence of MOR08213, binding of MOR08159 is
significantly reduced suggesting that those two antibodies either
bind to the same sites or to sites that may have some degree of
overlap or that binding of MOR08213 to a distinct, but nearby site,
might sterically hinder binding of MOR08159.
Epitope Mapping
[0393] Several complementary methods are used to determine the
epitope to which the antibody MOR08159 binds. In this example,
residue numbering is with reference to the full length ActRIIB
amino acid sequence (SEQ ID NO: 181).
Dot Blot
[0394] A dot blot analysis of the MOR08159 epitope is carried out.
Native and denatured (reduced and heat-denatured) ActRIIB is
spotted on a nitrocellulose membrane, probed with MOR08159, and
detected with a labeled anti-human antibody. Only native ActRIIB,
but not reduced and heat-denatured ActRIIB is detected. The results
indicated that the epitope is a conformational epitope.
Mutational Studies
[0395] A library of the extracellular domain of ActRIIB (aa 21-120)
is generated by error-prone PCR and the variants are expressed in
the periplasm of E. coli. The binding of about 30'000 (small
fraction of the theoretical library size) of those variants to
MOR08159 is tested by colony filter screening and western staining.
Variants which showed only weak or no binding to MOR08159 are
further confirmed by ELISA. Expression level (detected with an
anti-Flag antibody) and MOR08159 binding of the ActRIIB variants
are compared to wild-type ActRIIB. If expression is at least 75% of
wild type and binding to MOR08159 is less than 25%, the mutation is
rated to be involved in MOR08159 binding. Only variants having a
single point mutation, which is not obviously structure distorting
(as e.g. the mutation of an S--S bridging cysteine would be) are
considered.
[0396] Most mutations which prevented MOR08159 binding are found in
a stretch from position K75 to D81, which indicates that this
region is important for antibody binding. Mutations at positions
W78, D80 and D81 are found to reduce MOR08159 binding
significantly.
Cyclic Peptide Arrays
[0397] A collection of antigen derived cyclic peptides displayed on
peptide microarrays are incubated with antibodies of interest. The
determination of peptide-antibody binding is performed by
RepliTope-analysis where the peptide microarray is incubated with
the primary antibody followed by a fluorescently labelled secondary
antibody directed against the Fc-part of the primary one. After
several washing steps the peptide microarrays where dried using a
microarray centrifuge and scanned in a high resolution microarray
scanning system with appropriate wavelength settings.
[0398] The microarray is composed of three subarrays, each
displaying cyclic peptides derived from ActRIIB (with Cys residue
exchanged to Ser), which are scanned (peptide scan format 15/12).
As control experiment, one incubation with unrelated antibody
(ACE18543, isotope control) followed by fluorescently labelled
secondary antibody (Cy-5 labelled anti-human IgG) is performed to
determine false positive signals. Additionally, incubation with
target antibody, followed by fluorescently labelled secondary
antibody, is performed.
[0399] Antibody MOR08159 (ACE19819) is shown to recognise one
epitope, which is found in three of the tested peptides (nos.
18-20).
TABLE-US-00006 (SEQ ID NO: 183) 18 IELVKKGSWLDDFNS (SEQ ID NO: 184)
19 VKKGSWLDDFNSYDR (SEQ ID NO: 185) 20 GSWLDDFNSYDRQES
[0400] The sequence common to these peptides to which MOR08159 is
considered to bind is 76GCWLDDFNC84 (SEQ ID NO: 186).
[0401] A second region with weaker binding characteristics is also
identified using this method. This second region has the sequence
49CEGEQDKRLHCYASW63 (SEQ ID NO: 187).
X-Ray Crystallography
[0402] Human ActRIIB aa20-120 and aa24-117 are expressed. In
addition, MOR08159 Fab and Fv regions are expressed and purified
(all expression is carried out in E. coli). Using these proteins,
four protein complexes are prepared, purified and crystallised
(MOR08159Fab-ActRIIB 20-120, MOR08159Fab-ActRIIB 24-117,
MOR08159Fv-ActRIIB 20-120, MOR08159Fv-ActRIIB 24-117).
[0403] The x-ray structure of free MOR08159 Fab is resolved to 1.78
.ANG. resolution. The x-ray structure of the Fv complex with
ActRIIB-LBD is resolved to 3.35 .ANG. resolution. Using the
standard 3.9 .ANG. distance cut-off to determine contact residues,
it is confirmed that the sequence 76GCWLDDFNC84 is an important
region with dominant binding contribution from the 78WLDDFN83
sequence (SEQ ID NO: 188). In addition, interaction is also found
with the peptide region 49CEGEQDKRLHCYASW63.
[0404] The results for the various epitope mapping experiments are
summarised in FIG. 9.
Confirmation of Affinity by SET
[0405] Serial dilutions of antigen (extracellular domain of ActRIIB
or ActRIIA) are prepared in PBS0.5% (w/v)BSA/0.02% (w/v)Tween 20
and antibody (MOR08159) is added to each antigen concentration to
reach a constant antibody concentration. 100 .mu.l/well of each
dilution mix is distributed in duplicates to a 96-well
polypropylene MTP (Greiner). Assay buffer served as negative
control and a sample containing no antigen as positive control
(Bmax). The plate is sealed and incubated overnight. A 96-well High
Bind MTP (Meso Scale Discovery) is coated with 25 .mu.l of 0.1
.mu.g/ml mouse ActRIIB-Fc diluted in PBS. Also this plate is sealed
and incubated over night at 4.degree. C. After the incubation the
antigen-coated High Bind MTP is washed with PBS/0.05% (w/v)Tween
20. Subsequently, the plate is blocked with PBS/5% (w/v) BSA. The
washing steps are repeated and 50 .mu.l/well of the
antibody-antigen preparation from the polypropylene MTP is
transferred into the antigen-coated High Bind MTP. The High Bind
MTP is incubated for 25 min at room temperature. After three
additional washing steps, 25 .mu.l of 1 .mu.g/ml Sulfo-Tag-labeled
goat anti-human-detection antibody (Meso Scale Discovery) diluted
in assay buffer is added to each well and incubated one hour at
room temperature. After washing the plate, 50 .mu.l of Read Buffer
(Meso Scale Discovery) is transferred into each well.
Electrochemiluminescence (ECL) signals are generated and detected
by a Sector Imager 6000 reader (Meso Scale Discovery).
[0406] ECL values are plotted against the corresponding antigen
concentrations. K.sub.D is determined by fitting the plot with the
fit model described by Piehler J, et al. (J Immunol Methods; 1997,
201(2): 189-206).
[0407] The reported K.sub.D values and standard deviations are
determined from the individual K.sub.D values obtained from
independent experiments.
[0408] From these experiments a mean value for the dissociation
equilibrium constant K.sub.D of 1.73 (.+-.0.31) pM is determined
for human ActRIIB, while a mean value for the dissociation
equilibrium constant K.sub.D of 434 (.+-.25) pM is determined for
ActRIIA.
[0409] It will be understood that the disclosure has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the disclosure.
Sequence CWU 1
1
190110PRTArtificialCDR 1Gly Tyr Thr Phe Thr Ser Ser Tyr Ile Asn 1 5
10 210PRTArtificialCDR 2Gly Tyr Thr Phe Thr Ser Ser Tyr Ile Asn 1 5
10 310PRTArtificialCDR 3Gly Tyr Thr Phe Thr Ser Ser Tyr Ile Asn 1 5
10 410PRTArtificialCDR 4Gly Tyr Thr Phe Thr Ser Ser Tyr Ile Asn 1 5
10 510PRTArtificialCDR 5Gly Tyr Thr Phe Thr Ser Ser Tyr Ile Asn 1 5
10 610PRTArtificialCDR 6Gly Tyr Thr Phe Thr Ser Ser Tyr Ile Asn 1 5
10 710PRTArtificialCDR 7Gly Tyr Thr Phe Thr Ser Ser Tyr Ile Asn 1 5
10 810PRTArtificialCDR 8Gly Tyr Thr Phe Thr Ser Ser Tyr Ile Asn 1 5
10 910PRTArtificialCDR 9Gly Tyr Thr Phe Thr Ser Ser Tyr Ile Asn 1 5
10 1010PRTArtificialCDR 10Gly Tyr Thr Phe Thr Ser Ser Tyr Ile Asn 1
5 10 1110PRTArtificialCDR 11Gly Tyr Thr Phe Thr Ser Ser Tyr Ile Asn
1 5 10 1210PRTArtificialCDR 12Gly Tyr Thr Phe Thr Ser Ser Tyr Ile
Asn 1 5 10 1310PRTArtificialCDR 13Gly Tyr Thr Phe Thr Ser Ser Tyr
Ile Asn 1 5 10 1410PRTArtificialCDR 14Gly Tyr Thr Phe Thr Ser Ser
Tyr Ile Asn 1 5 10 1517PRTArtificialCDR 15Thr Ile Asn Pro Val Ser
Gly Asn Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly
1617PRTArtificialCDR 16Thr Ile Asn Pro Val Ser Gly Asn Thr Ser Tyr
Ala Gln Lys Phe Gln 1 5 10 15 Gly 1717PRTArtificialCDR 17Thr Ile
Asn Pro Val Ser Gly Asn Thr Ser Tyr Ala Gln Lys Phe Gln 1 5 10 15
Gly 1817PRTArtificialCDR 18Thr Ile Asn Pro Val Ser Gly Asn Thr Ser
Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 1917PRTArtificialCDR 19Met
Ile Asn Ala Pro Ile Gly Thr Thr Arg Tyr Ala Gln Lys Phe Gln 1 5 10
15 Gly 2017PRTArtificialCDR 20Gln Ile Asn Ala Ala Ser Gly Met Thr
Arg Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 2117PRTArtificialCDR
21Met Ile Asn Ala Pro Ile Gly Thr Thr Arg Tyr Ala Gln Lys Phe Gln 1
5 10 15 Gly 2217PRTArtificialCDR 22Thr Ile Asn Pro Val Ser Gly Asn
Thr Arg Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 2317PRTArtificialCDR
23Thr Ile Asn Pro Val Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln 1
5 10 15 Gly 2417PRTArtificialCDR 24Gln Ile Asn Ala Ala Ser Gly Met
Thr Arg Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 2517PRTArtificialCDR
25Asn Ile Asn Ala Ala Ala Gly Ile Thr Leu Tyr Ala Gln Lys Phe Gln 1
5 10 15 Gly 2617PRTArtificialCDR 26Thr Ile Asn Pro Pro Thr Gly Gly
Thr Tyr Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 2717PRTArtificialCDR
27Gly Ile Asn Pro Pro Ala Gly Thr Thr Ser Tyr Ala Gln Lys Phe Gln 1
5 10 15 Gly 2817PRTArtificialCDR 28Asn Ile Asn Pro Ala Thr Gly His
Ala Asp Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 296PRTArtificialCDR
29Gly Gly Trp Phe Asp Tyr 1 5 306PRTArtificialCDR 30Gly Gly Trp Phe
Asp Tyr 1 5 316PRTArtificialCDR 31Gly Gly Trp Phe Asp Tyr 1 5
326PRTArtificialCDR 32Gly Gly Trp Phe Asp Tyr 1 5
336PRTArtificialCDR 33Gly Gly Trp Phe Asp Tyr 1 5
346PRTArtificialCDR 34Gly Gly Trp Phe Asp Tyr 1 5
356PRTArtificialCDR 35Gly Gly Trp Phe Asp Tyr 1 5
366PRTArtificialCDR 36Gly Gly Trp Phe Asp Tyr 1 5
376PRTArtificialCDR 37Gly Gly Trp Phe Asp Tyr 1 5
386PRTArtificialCDR 38Gly Gly Trp Phe Asp Tyr 1 5
396PRTArtificialCDR 39Gly Gly Trp Phe Asp Tyr 1 5
406PRTArtificialCDR 40Gly Gly Trp Phe Asp Tyr 1 5
416PRTArtificialCDR 41Gly Gly Trp Phe Asp Tyr 1 5
426PRTArtificialCDR 42Gly Gly Trp Phe Asp Tyr 1 5
4314PRTArtificialCDR 43Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn
Tyr Val Asn 1 5 10 4414PRTArtificialCDR 44Thr Gly Thr Ser Ser Asp
Val Gly Ser Tyr Asn Tyr Val Asn 1 5 10 4514PRTArtificialCDR 45Thr
Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn Tyr Val Asn 1 5 10
4614PRTArtificialCDR 46Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn
Tyr Val Asn 1 5 10 4714PRTArtificialCDR 47Thr Gly Thr Ser Ser Asp
Val Gly Ser Tyr Asn Tyr Val Asn 1 5 10 4814PRTArtificialCDR 48Thr
Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn Tyr Val Asn 1 5 10
4914PRTArtificialCDR 49Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn
Tyr Val Asn 1 5 10 5014PRTArtificialCDR 50Thr Gly Thr Ser Ser Asp
Val Gly Ser Tyr Asn Tyr Val Asn 1 5 10 5114PRTArtificialCDR 51Thr
Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn Tyr Val Asn 1 5 10
5214PRTArtificialCDR 52Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn
Tyr Val Asn 1 5 10 5314PRTArtificialCDR 53Thr Gly Thr Ser Ser Asp
Val Gly Ser Tyr Asn Tyr Val Asn 1 5 10 5414PRTArtificialCDR 54Thr
Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn Tyr Val Asn 1 5 10
5514PRTArtificialCDR 55Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr Asn
Tyr Val Asn 1 5 10 5614PRTArtificialCDR 56Thr Gly Thr Ser Ser Asp
Val Gly Ser Tyr Asn Tyr Val Asn 1 5 10 5711PRTArtificialCDR 57Leu
Met Ile Tyr Gly Val Ser Lys Arg Pro Ser 1 5 10 5811PRTArtificialCDR
58Leu Met Ile Tyr Gly Val Ser Lys Arg Pro Ser 1 5 10
5911PRTArtificialCDR 59Leu Met Ile Tyr Gly Val Ser Lys Arg Pro Ser
1 5 10 6011PRTArtificialCDR 60Leu Met Ile Tyr Gly Val Ser Lys Arg
Pro Ser 1 5 10 6111PRTArtificialCDR 61Leu Met Ile Tyr Gly Val Ser
Lys Arg Pro Ser 1 5 10 6211PRTArtificialCDR 62Leu Met Ile Tyr Gly
Val Ser Lys Arg Pro Ser 1 5 10 6311PRTArtificialCDR 63Leu Met Ile
Tyr Gly Val Ser Lys Arg Pro Ser 1 5 10 6411PRTArtificialCDR 64Leu
Met Ile Tyr Gly Val Ser Lys Arg Pro Ser 1 5 10 6511PRTArtificialCDR
65Leu Met Ile Tyr Gly Val Ser Lys Arg Pro Ser 1 5 10
6611PRTArtificialCDR 66Leu Met Ile Tyr Gly Val Ser Lys Arg Pro Ser
1 5 10 6711PRTArtificialCDR 67Leu Met Ile Tyr Gly Val Ser Lys Arg
Pro Ser 1 5 10 6811PRTArtificialCDR 68Leu Met Ile Tyr Gly Val Ser
Lys Arg Pro Ser 1 5 10 6911PRTArtificialCDR 69Leu Met Ile Tyr Gly
Val Ser Lys Arg Pro Ser 1 5 10 7011PRTArtificialCDR 70Leu Met Ile
Tyr Gly Val Ser Lys Arg Pro Ser 1 5 10 719PRTArtificialCDR 71Gln
Ala Trp Thr Ser Lys Met Ala Gly 1 5 729PRTArtificialCDR 72Ser Ser
Tyr Thr Arg Met Gly His Pro 1 5 7310PRTArtificialCDR 73Ala Thr Tyr
Gly Lys Gly Val Thr Pro Pro 1 5 10 7410PRTArtificialCDR 74Gly Thr
Phe Ala Gly Gly Ser Tyr Tyr Gly 1 5 10 759PRTArtificialCDR 75Gln
Ala Trp Thr Ser Lys Met Ala Gly 1 5 769PRTArtificialCDR 76Gln Ala
Trp Thr Ser Lys Met Ala Gly 1 5 7710PRTArtificialCDR 77Gly Thr Phe
Ala Gly Gly Ser Tyr Tyr Gly 1 5 10 7810PRTArtificialCDR 78Gly Thr
Phe Ala Gly Gly Ser Tyr Tyr Gly 1 5 10 7910PRTArtificialCDR 79Gly
Thr Phe Ala Gly Gly Ser Tyr Tyr Gly 1 5 10 8010PRTArtificialCDR
80Gly Thr Phe Ala Gly Gly Ser Tyr Tyr Gly 1 5 10
8110PRTArtificialCDR 81Gly Thr Phe Ala Gly Gly Ser Tyr Tyr Gly 1 5
10 8210PRTArtificialCDR 82Gly Thr Phe Ala Gly Gly Ser Tyr Tyr Gly 1
5 10 8310PRTArtificialCDR 83Gly Thr Phe Ala Gly Gly Ser Tyr Tyr Gly
1 5 10 8410PRTArtificialCDR 84Gly Thr Phe Ala Gly Gly Ser Tyr Tyr
Gly 1 5 10 85112PRTArtificialVL 85Asp Ile Ala Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val Asn
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile
Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Thr
Ser Lys 85 90 95 Met Ala Gly Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln 100 105 110 86112PRTArtificialVL 86Asp Ile Ala Leu
Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile
Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30
Asn Tyr Val Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35
40 45 Met Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg
Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser
Ser Tyr Thr Arg Met 85 90 95 Gly His Pro Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu Gly Gln 100 105 110 87113PRTArtificialVL 87Asp
Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10
15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr
20 25 30 Asn Tyr Val Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Met Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val
Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Thr Tyr Gly Lys Gly 85 90 95 Val Thr Pro Pro Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
88113PRTArtificialVL 88Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val Asn Trp Tyr Gln
Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gly Val
Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala Gly Gly 85 90
95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110 Gln 89112PRTArtificialVL 89Asp Ile Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val
Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Thr
Ser Lys 85 90 95 Met Ala Gly Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln 100 105 110 90112PRTArtificialVL 90Asp Ile Ala Leu
Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile
Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30
Asn Tyr Val Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35
40 45 Met Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg
Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln
Ala Trp Thr Ser Lys 85 90 95 Met Ala Gly Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu Gly Gln 100 105 110 91113PRTArtificialVL 91Asp
Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10
15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr
20 25 30 Asn Tyr Val Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Met Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val
Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Tyr Cys Gly Thr Phe Ala Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
92113PRTArtificialVL 92Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val Asn Trp Tyr Gln
Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gly Val
Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala Gly Gly 85 90
95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110 Gln 93113PRTArtificialVL 93Asp Ile Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val
Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala
Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gln 94113PRTArtificialVL 94Asp Ile Ala
Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser
Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25
30 Asn Tyr Val Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35
40 45 Met Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg
Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly
Thr Phe Ala Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly
Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln 95113PRTArtificialVL
95Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1
5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser
Tyr 20 25 30 Asn Tyr Val Asn Trp Tyr Gln Gln His Pro Gly Lys Ala
Pro Lys Leu 35 40 45 Met Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly
Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala
Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp
Tyr Tyr Cys Gly Thr Phe Ala Gly Gly 85 90 95 Ser Tyr Tyr Gly Val
Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
96113PRTArtificialVL 96Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val Asn Trp Tyr Gln
Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gly Val
Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala Gly Gly 85 90
95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110 Gln 97113PRTArtificialVL 97Asp Ile Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val
Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala
Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gln 98113PRTArtificialVL 98Asp Ile Ala
Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser
Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25
30 Asn Tyr Val Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45 Met Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn
Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Gly Thr Phe Ala Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln
99115PRTArtificialVH 99Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asn Pro
Val Ser Gly Asn Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110 Val Ser Ser 115 100115PRTArtificialVH 100Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25
30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 Gly Thr Ile Asn Pro Val Ser Gly Asn Thr Ser Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile
Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
101115PRTArtificialVH 101Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asn
Pro Val Ser Gly Asn Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110 Val Ser Ser 115 102115PRTArtificialVH 102Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20
25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Thr Ile Asn Pro Val Ser Gly Asn Thr Ser Tyr Ala
Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser
Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
103115PRTArtificialVH 103Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile Asn
Ala Pro Ile Gly Thr Thr Arg Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110 Val Ser Ser 115 104115PRTArtificialVH 104Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20
25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Gln Ile Asn Ala Ala Ser Gly Met Thr Arg Tyr Ala
Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser
Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
105115PRTArtificialVH 105Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Met Ile Asn
Ala Pro Ile Gly Thr Thr Arg Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110 Val Ser Ser 115 106115PRTArtificialVH 106Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20
25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Thr Ile Asn Pro Val Ser Gly Asn Thr Arg Tyr Ala
Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser
Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
107115PRTArtificialVH 107Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asn
Pro Val Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110 Val Ser Ser 115 108115PRTArtificialVH 108Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20
25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Gln Ile Asn Ala Ala Ser Gly Met Thr Arg Tyr Ala
Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser
Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
109115PRTArtificialVH 109Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Asn
Ala Ala Ala Gly Ile Thr Leu Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110 Val Ser Ser 115 110115PRTArtificialVH 110Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20
25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Thr Ile Asn Pro Pro Thr Gly Gly Thr Tyr Tyr Ala
Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser
Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
111115PRTArtificialVH 111Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn
Pro Pro Ala Gly Thr Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110 Val Ser Ser 115 112115PRTArtificialVH 112Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20
25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Asn Ile Asn Pro Ala Thr Gly His Ala Asp Tyr Ala
Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser
Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
113336DNAArtificialVL 113gatatcgcac tgacccagcc agcttcagtg
agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt
tcttataatt atgtgaattg gtaccagcag 120catcccggga aggcgccgaa
acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg 180agcaaccgtt
ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg
240caagcggaag acgaagcgga ttattattgc caggcttgga cttctaagat
ggctggtgtg 300tttggcggcg gcacgaagtt aaccgttctt ggccag
336114336DNAArtificialVL 114gatatcgcac tgacccagcc agcttcagtg
agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt
tcttataatt atgtgaattg gtaccagcag 120catcccggga aggcgccgaa
acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg 180agcaaccgtt
ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg
240caagcggaag acgaagcgga ttattattgc tcttcttata ctcgtatggg
tcatcctgtg 300tttggcggcg gcacgaagtt aaccgttctt ggccag
336115339DNAArtificialVL 115gatatcgcac tgacccagcc agcttcagtg
agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt
tcttataatt atgtgaattg gtaccagcag 120catcccggga aggcgccgaa
acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg 180agcaaccgtt
ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg
240caagcggaag acgaagcgga
ttattattgc gctacttatg gtaagggtgt tactcctcct 300gtgtttggcg
gcggcacgaa gttaaccgtt cttggccag 339116339DNAArtificialVL
116gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag
cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt tcttataatt atgtgaattg
gtaccagcag 120catcccggga aggcgccgaa acttatgatt tatggtgttt
ctaagcgtcc ctcaggcgtg 180agcaaccgtt ttagcggatc caaaagcggc
aacaccgcga gcctgaccat tagcggcctg 240caagcggaag acgaagcgga
ttattattgc ggtacttttg ctggtggttc ttattatggt 300gtgtttggcg
gcggcacgaa gttaaccgtt cttggccag 339117336DNAArtificialVL
117gatatcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag
cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt tcttataatt atgtgaattg
gtaccagcag 120catcccggga aggcgccgaa acttatgatt tatggtgttt
ctaagcgtcc ctcaggcgtg 180agcaaccgtt ttagcggatc caaaagcggc
aacaccgcga gcctgaccat tagcggcctg 240caagcggaag acgaagcgga
ttattattgc caggcttgga cttctaagat ggctggtgtg 300tttggcggcg
gcacgaagtt aaccgttctt ggccag 336118336DNAArtificialVL 118gatatcgcac
tgacccagcc agcttcagtg agcggctcac caggtcagag cattaccatc 60tcgtgtacgg
gtactagcag cgatgttggt tcttataatt atgtgaattg gtaccagcag
120catcccggga aggcgccgaa acttatgatt tatggtgttt ctaagcgtcc
ctcaggcgtg 180agcaaccgtt ttagcggatc caaaagcggc aacaccgcga
gcctgaccat tagcggcctg 240caagcggaag acgaagcgga ttattattgc
caggcttgga cttctaagat ggctggtgtg 300tttggcggcg gcacgaagtt
aaccgttctt ggccag 336119339DNAArtificialVL 119gatatcgcac tgacccagcc
agcttcagtg agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag
cgatgttggt tcttataatt atgtgaattg gtaccagcag 120catcccggga
aggcgccgaa acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg
180agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat
tagcggcctg 240caagcggaag acgaagcgga ttattattgc ggtacttttg
ctggtggttc ttattatggt 300gtgtttggcg gcggcacgaa gttaaccgtt cttggccag
339120339DNAArtificialVL 120gatatcgcac tgacccagcc agcttcagtg
agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt
tcttataatt atgtgaattg gtaccagcag 120catcccggga aggcgccgaa
acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg 180agcaaccgtt
ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg
240caagcggaag acgaagcgga ttattattgc ggtacttttg ctggtggttc
ttattatggt 300gtgtttggcg gcggcacgaa gttaaccgtt cttggccag
339121339DNAArtificialVL 121gatatcgcac tgacccagcc agcttcagtg
agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt
tcttataatt atgtgaattg gtaccagcag 120catcccggga aggcgccgaa
acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg 180agcaaccgtt
ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg
240caagcggaag acgaagcgga ttattattgc ggtacttttg ctggtggttc
ttattatggt 300gtgtttggcg gcggcacgaa gttaaccgtt cttggccag
339122339DNAArtificialVL 122gatatcgcac tgacccagcc agcttcagtg
agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt
tcttataatt atgtgaattg gtaccagcag 120catcccggga aggcgccgaa
acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg 180agcaaccgtt
ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg
240caagcggaag acgaagcgga ttattattgc ggtacttttg ctggtggttc
ttattatggt 300gtgtttggcg gcggcacgaa gttaaccgtt cttggccag
339123339DNAArtificialVL 123gatatcgcac tgacccagcc agcttcagtg
agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt
tcttataatt atgtgaattg gtaccagcag 120catcccggga aggcgccgaa
acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg 180agcaaccgtt
ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg
240caagcggaag acgaagcgga ttattattgc ggtacttttg ctggtggttc
ttattatggt 300gtgtttggcg gcggcacgaa gttaaccgtt cttggccag
339124339DNAArtificialVL 124gatatcgcac tgacccagcc agcttcagtg
agcggctcac caggtcagag cattaccatc 60tcgtgtactg gtactagcag cgatgttggt
tcttataatt atgtgaattg gtaccagcag 120catcccggga aggcgccgaa
acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg 180agcaaccgtt
ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg
240caagcggaag acgaagcgga ttattattgc ggtacttttg ctggtggttc
ttattatggt 300gtgtttggcg gcggcacgaa gttaaccgtt cttggccag
339125339DNAArtificialVL 125gatatcgcac tgacccagcc agcttcagtg
agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt
tcttataatt atgtgaattg gtaccagcag 120catcccggga aggcgccgaa
acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg 180agcaaccgtt
ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg
240caagcggaag acgaagcgga ttattattgc ggtacttttg ctggtggttc
ttattatggt 300gtgtttggcg gcggcacgaa gttaaccgtt cttggccag
339126339DNAArtificialVL 126gatatcgcac tgacccagcc agcttcagtg
agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt
tcttataatt atgtgaattg gtaccagcag 120catcccggga aggcgccgaa
acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg 180agcaaccgtt
ttagcggatc caaaagcggc aacaccgcga gcctgaccat tagcggcctg
240caagcggaag acgaagcgga ttattattgc ggtacttttg ctggtggttc
ttattatggt 300gtgtttggcg gcggcacgaa gttaaccgtt cttggccag
339127345DNAArtificialVH 127caggtgcaat tggttcagag cggcgcggaa
gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata tacctttact
tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg gtctcgagtg
gatgggcact atcaatccgg tttctggcaa tacgtcttac 180gcgcagaagt
ttcagggccg ggtgaccatg acccgtgata ccagcattag caccgcgtat
240atggaactga gcagcctgcg tagcgaagat acggccgtgt attattgcgc
gcgtggtggt 300tggtttgatt attggggcca aggcaccctg gtgacggtta gctca
345128345DNAArtificialVH 128caggtgcaat tggttcagag cggcgcggaa
gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata tacctttact
tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg gtctcgagtg
gatgggcact atcaatccgg tttctggcaa tacgtcttac 180gcgcagaagt
ttcagggccg ggtgaccatg acccgtgata ccagcattag caccgcgtat
240atggaactga gcagcctgcg tagcgaagat acggccgtgt attattgcgc
gcgtggtggt 300tggtttgatt attggggcca aggcaccctg gtgacggtta gctca
345129345DNAArtificialVH 129caggtgcaat tggttcagag cggcgcggaa
gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata tacctttact
tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg gtctcgagtg
gatgggcact atcaatccgg tttctggcaa tacgtcttac 180gcgcagaagt
ttcagggccg ggtgaccatg acccgtgata ccagcattag caccgcgtat
240atggaactga gcagcctgcg tagcgaagat acggccgtgt attattgcgc
gcgtggtggt 300tggtttgatt attggggcca aggcaccctg gtgacggtta gctca
345130345DNAArtificialVH 130caggtgcaat tggttcagag cggcgcggaa
gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata tacctttact
tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg gtctcgagtg
gatgggcact atcaatccgg tttctggcaa tacgtcttac 180gcgcagaagt
ttcagggccg ggtgaccatg acccgtgata ccagcattag caccgcgtat
240atggaactga gcagcctgcg tagcgaagat acggccgtgt attattgcgc
gcgtggtggt 300tggtttgatt attggggcca aggcaccctg gtgacggtta gctca
345131345DNAArtificialVH 131caggtgcaat tggttcagag cggcgcggaa
gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata tacctttact
tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg gtctcgagtg
gatgggcatg attaatgctc ctattggtac tactcgttat 180gctcagaagt
ttcagggtcg ggtgaccatg acccgtgata ccagcattag caccgcgtat
240atggaactga gcagcctgcg tagcgaagat acggccgtgt attattgcgc
gcgtggtggt 300tggtttgatt attggggcca aggcaccctg gtgacggtta gctca
345132345DNAArtificialVH 132caggtgcaat tggttcagag cggcgcggaa
gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata tacctttact
tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg gtctcgagtg
gatgggccag attaatgctg cttctggtat gactcgttat 180gctcagaagt
ttcagggtcg ggtgaccatg acccgtgata ccagcattag caccgcgtat
240atggaactga gcagcctgcg tagcgaagat acggccgtgt attattgcgc
gcgtggtggt 300tggtttgatt attggggcca aggcaccctg gtgacggtta gctca
345133345DNAArtificialVH 133caggtgcaat tggttcagag cggcgcggaa
gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata tacctttact
tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg gtctcgagtg
gatgggcatg attaatgctc ctattggtac tactcgttat 180gctcagaagt
ttcagggtcg ggtgaccatg acccgtgata ccagcattag caccgcgtat
240atggaactga gcagcctgcg tagcgaagat acggccgtgt attattgcgc
gcgtggtggt 300tggtttgatt attggggcca aggcaccctg gtgacggtta gctca
345134345DNAArtificialVH 134caggtgcaat tggttcagag cggcgcggaa
gtgaaaaaac cgggcgcgag cgtgaaagtg 60 agctgcaaag cctccggata
tacctttact tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg
gtctcgagtg gatgggcact atcaatccgg tttctggcaa tacgcgttac
180gcgcagaagt ttcagggccg ggtgaccatg acccgtgata ccagcattag
caccgcgtat 240atggaactga gcagcctgcg tagcgaagat acggccgtgt
attattgcgc gcgtggtggt 300tggtttgatt attggggcca aggcaccctg
gtgacggtta gctca 345135345DNAArtificialVH 135caggtgcaat tggttcagag
cggcgcggaa gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata
tacctttact tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg
gtctcgagtg gatgggcact atcaatccgg tttctggctc tacgtcttac
180gcgcagaagt ttcagggccg ggtgaccatg acccgtgata ccagcattag
caccgcgtat 240atggaactga gcagcctgcg tagcgaagat acggccgtgt
attattgcgc gcgtggtggt 300tggtttgatt attggggcca aggcaccctg
gtgacggtta gctca 345136345DNAArtificialVH 136caggtgcaat tggttcagag
cggcgcggaa gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata
tacctttact tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg
gtctcgagtg gatgggccag attaatgctg cttctggtat gactcgttat
180gctcagaagt ttcagggtcg ggtcaccatg acccgtgata ccagcattag
caccgcgtat 240atggaactga gcagcctgcg tagcgaagat acggccgtgt
attattgcgc gcgtggtggt 300tggtttgatt attggggcca aggcaccctg
gtgacggtta gctca 345137345DNAArtificialVH 137caggtgcaat tggttcagag
cggcgcggaa gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata
tacctttact tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg
gtctcgagtg gatgggcaat attaatgctg ctgctggtat tactctttat
180gctcagaagt ttcagggtcg ggtcaccatg acccgtgata ccagcattag
caccgcgtat 240atggaactga gcagcctgcg tagcgaagat acggccgtgt
attattgcgc gcgtggtggt 300tggtttgatt attggggcca aggcaccctg
gtgacggtta gctca 345138345DNAArtificialVH 138caggtgcaat tggttcagag
cggcgcggaa gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata
tacctttact tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg
gtctcgagtg gatgggcact attaatcctc ctactggagg tacttattat
180gctcagaagt ttcagggtcg ggtgaccatg acccgtgata ccagcattag
caccgcgtat 240atggaactga gcagcctgcg tagcgaagat acggccgtgt
attattgcgc gcgtggtggt 300tggtttgatt attggggcca aggcaccctg
gtgacggtta gctca 345139345DNAArtificialVH 139caggtgcaat tggttcagag
cggcgcggaa gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata
tacctttact tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg
gtctcgagtg gatgggcggt attaatcctc ctgctggtac tacttcttat
180gctcagaagt ttcagggtcg ggtcaccatg acccgtgata ccagcattag
caccgcgtat 240atggaactga gcagcctgcg tagcgaagat acggccgtgt
attattgcgc gcgtggtggt 300tggtttgatt attggggcca aggcaccctg
gtgacggtta gctca 345140345DNAArtificialVH 140caggtgcaat tggttcagag
cggcgcggaa gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata
tacctttact tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg
gtctcgagtg gatgggcaat attaatcctg ctactggtca tgctgattat
180gctcagaagt ttcagggtcg ggtgaccatg acccgtgata ccagcattag
caccgcgtat 240atggaactga gcagcctgcg tagcgaagat acggccgtgt
attattgcgc gcgtggtggt 300tggtttgatt attggggcca aggcaccctg
gtgacggtta gctca 345141217PRTArtificiallight chain 141Gln Ser Ala
Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser
Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25
30 Asn Tyr Val Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu
35 40 45 Met Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn
Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr
Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys
Gly Thr Phe Ala Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro
Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala
Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155
160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp
Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210
215 142217PRTArtificiallight chain 142Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val
Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala
Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185
190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215
143217PRTArtificiallight chain 143Gln Ser Ala Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val Asn
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile
Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala
Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185
190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215
144217PRTArtificiallight chain 144Gln Ser Ala Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val Asn
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile
Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala
Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155
160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp
Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly
Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210
215 145217PRTArtificiallight chain 145Gln Ser Ala Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val
Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala
Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185
190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215
146445PRTArtificialheavy chain 146Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr
Ile Asn Pro Val Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185
190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310
315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
147445PRTArtificialheavy chain 147Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gln
Ile Asn Ala Ala Ser Gly Met Thr Arg Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185
190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310
315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
148445PRTArtificialheavy chain 148Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn
Ile Asn Ala Ala Ala Gly Ile Thr Leu Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185
190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310
315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
149445PRTArtificialheavy chain 149Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly
Ile Asn Pro Pro Ala Gly Thr Thr Ser Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185
190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310
315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
150445PRTArtificialheavy chain 150Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn
Ile Asn Pro Ala Thr Gly His Ala Asp Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185
190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205 Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys 210 215 220 Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250
255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375
380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 435 440 445 151217PRTArtificiallight chain 151Gln
Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10
15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr
20 25 30 Asn Tyr Val Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Met Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val
Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser
Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Tyr Cys Gly Thr Phe Ala Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala
Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu
Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140
Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145
150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn
Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu
Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His
Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys
Ser 210 215 152217PRTArtificiallight chain 152Gln Ser Ala Leu Thr
Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr
Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn
Tyr Val Asn Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40
45 Met Ile Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser
Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr
Phe Ala Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val
Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys
Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala
Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170
175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr
Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215
153217PRTArtificiallight chain 153Gln Ser Ala Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val Asn
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile
Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala
Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185
190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215
154217PRTArtificiallight chain 154Gln Ser Ala Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val Asn
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile
Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala
Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185
190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215
155217PRTArtificiallight chain 155Gln Ser Ala Leu Thr Gln Pro Ala
Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys
Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Tyr Val Asn
Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile
Tyr Gly Val Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Phe Ala
Gly Gly 85 90 95 Ser Tyr Tyr Gly Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr
Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr
Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly
Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr
Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185
190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215
156441PRTArtificialheavy chain 156Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr
Ile Asn Pro Val Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly 180 185
190 Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
195 200 205 Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro
Pro Cys 210 215 220 Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val 245 250 255 Val Val Asp Val Ser His Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270 Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285 Gln Phe Asn
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His 290 295 300 Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305 310
315 320 Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
Gln 325 330 335 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met 340 345 350 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 355 360 365 Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 405 410 415 Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430
Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
157441PRTArtificialheavy chain 157Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gln
Ile Asn Ala Ala Ser Gly Met Thr Arg Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly 180 185
190 Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys
195 200 205 Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro
Pro Cys 210 215 220 Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val 245 250 255 Val Val Asp Val Ser His Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270 Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285 Gln Phe Asn
Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His 290 295 300 Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305 310
315 320 Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
Gln 325 330 335 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met 340 345 350 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro 355 360 365 Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 405 410 415 Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430
Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
158441PRTArtificialheavy chain 158Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn
Ile Asn Ala Ala Ala Gly Ile Thr Leu Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr
Tyr Cys
85 90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly 180 185 190 Thr
Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200
205 Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys
210 215 220 Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys 225 230 235 240 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val 245 250 255 Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr 260 265 270 Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285 Gln Phe Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His 290 295 300 Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305 310 315 320
Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 325
330 335 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met 340 345 350 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro 355 360 365 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 405 410 415 Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430 Lys Ser
Leu Ser Leu Ser Pro Gly Lys 435 440 159441PRTArtificialheavy chain
159Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Ser Ser 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Pro Ala Gly Thr Thr
Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly
Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130
135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Asn Phe Gly 180 185 190 Thr Gln Thr Tyr Thr Cys Asn Val
Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Thr Val Glu
Arg Lys Cys Cys Val Glu Cys Pro Pro Cys 210 215 220 Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 225 230 235 240 Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250
255 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
260 265 270 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 275 280 285 Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
Thr Val Val His 290 295 300 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 305 310 315 320 Gly Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Thr Lys Gly Gln 325 330 335 Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 340 345 350 Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365 Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375
380 Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
385 390 395 400 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 160441PRTArtificialheavy chain 160Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Tyr Ile
Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Asn Ile Asn Pro Ala Thr Gly His Ala Asp Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Trp Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly 180
185 190 Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys 195 200 205 Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys
Pro Pro Cys 210 215 220 Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val 245 250 255 Val Val Asp Val Ser His
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 260 265 270 Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285 Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His 290 295 300
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305
310 315 320 Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
Gly Gln 325 330 335 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met 340 345 350 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro 355 360 365 Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 405 410 415 Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425
430 Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
161651DNAArtificiallight chain 161cagagcgccc tgacccagcc cgccagcgtg
tccggcagcc caggccagtc tatcacaatc 60agctgcaccg gcacctccag cgacgtgggc
agctacaact acgtgaactg gtatcagcag 120caccccggca aggcccccaa
gctgatgatc tacggcgtga gcaagaggcc cagcggcgtg 180tccaacaggt
tcagcggcag caagagcggc aacaccgcca gcctgacaat cagtgggctg
240caggctgagg acgaggccga ctactactgc ggcacctttg ccggcggatc
atactacggc 300gtgttcggcg gagggaccaa gctgaccgtg ctgggccagc
ctaaggctgc ccccagcgtg 360accctgttcc cccccagcag cgaggagctg
caggccaaca aggccaccct ggtgtgcctg 420atcagcgact tctacccagg
cgccgtgacc gtggcctgga aggccgacag cagccccgtg 480aaggccggcg
tggagaccac cacccccagc aagcagagca acaacaagta cgccgccagc
540agctacctga gcctgacccc cgagcagtgg aagagccaca ggtcctacag
ctgccaggtg 600acccacgagg gcagcaccgt ggaaaagacc gtggccccaa
ccgagtgcag c 651162651DNAArtificiallight chain 162cagagcgccc
tgacccagcc cgccagcgtg tccggcagcc caggccagtc tatcacaatc 60agctgcaccg
gcacctccag cgacgtgggc agctacaact acgtgaactg gtatcagcag
120caccccggca aggcccccaa gctgatgatc tacggcgtga gcaagaggcc
cagcggcgtg 180tccaacaggt tcagcggcag caagagcggc aacaccgcca
gcctgacaat cagtgggctg 240caggctgagg acgaggccga ctactactgc
ggcacctttg ccggcggatc atactacggc 300gtgttcggcg gagggaccaa
gctgaccgtg ctgggccagc ctaaggctgc ccccagcgtg 360accctgttcc
cccccagcag cgaggagctg caggccaaca aggccaccct ggtgtgcctg
420atcagcgact tctacccagg cgccgtgacc gtggcctgga aggccgacag
cagccccgtg 480aaggccggcg tggagaccac cacccccagc aagcagagca
acaacaagta cgccgccagc 540agctacctga gcctgacccc cgagcagtgg
aagagccaca ggtcctacag ctgccaggtg 600acccacgagg gcagcaccgt
ggaaaagacc gtggccccaa ccgagtgcag c 651163651DNAArtificiallight
chain 163cagagcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag
cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt tcttataatt atgtgaattg
gtaccagcag 120catcccggga aggcgccgaa acttatgatt tatggtgttt
ctaagcgtcc ctcaggcgtg 180agcaaccgtt ttagcggatc caaaagcggc
aacaccgcga gcctgaccat tagcggcctg 240caagcggaag acgaagcgga
ttattattgc ggtacttttg ctggtggttc ttattatggt 300gtgtttggcg
gcggcacgaa gttaaccgtc ctaggtcagc ccaaggctgc cccctcggtc
360actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact
ggtgtgtctc 420ataagtgact tctacccggg agccgtgaca gtggcctgga
aggcagatag cagccccgtc 480aaggcgggag tggagaccac cacaccctcc
aaacaaagca acaacaagta cgcggccagc 540agctatctga gcctgacgcc
tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600acgcatgaag
ggagcaccgt ggagaagaca gtggccccta cagaatgttc a
651164651DNAArtificiallight chain 164cagagcgcac tgacccagcc
agcttcagtg agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag
cgatgttggt tcttataatt atgtgaattg gtaccagcag 120catcccggga
aggcgccgaa acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg
180agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat
tagcggcctg 240caagcggaag acgaagcgga ttattattgc ggtacttttg
ctggtggttc ttattatggt 300gtgtttggcg gcggcacgaa gttaaccgtc
ctaggtcagc ccaaggctgc cccctcggtc 360actctgttcc cgccctcctc
tgaggagctt caagccaaca aggccacact ggtgtgtctc 420ataagtgact
tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc
480aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta
cgcggccagc 540agctatctga gcctgacgcc tgagcagtgg aagtcccaca
gaagctacag ctgccaggtc 600acgcatgaag ggagcaccgt ggagaagaca
gtggccccta cagaatgttc a 651165651DNAArtificiallight chain
165cagagcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag
cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt tcttataatt atgtgaattg
gtaccagcag 120catcccggga aggcgccgaa acttatgatt tatggtgttt
ctaagcgtcc ctcaggcgtg 180agcaaccgtt ttagcggatc caaaagcggc
aacaccgcga gcctgaccat tagcggcctg 240caagcggaag acgaagcgga
ttattattgc ggtacttttg ctggtggttc ttattatggt 300gtgtttggcg
gcggcacgaa gttaaccgtc ctaggtcagc ccaaggctgc cccctcggtc
360actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact
ggtgtgtctc 420ataagtgact tctacccggg agccgtgaca gtggcctgga
aggcagatag cagccccgtc 480aaggcgggag tggagaccac cacaccctcc
aaacaaagca acaacaagta cgcggccagc 540agctatctga gcctgacgcc
tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600acgcatgaag
ggagcaccgt ggagaagaca gtggccccta cagaatgttc a
6511661335DNAArtificialheavy chain 166caggtgcagc tggtgcagag
cggagctgag gtgaagaagc caggcgccag cgtcaaggtg 60tcctgcaagg ccagcggcta
caccttcacc agcagctaca tcaactgggt ccgccaggct 120cctgggcagg
gactggagtg gatgggcacc atcaaccccg tgtccggcag caccagctac
180gcccagaagt tccagggcag agtcaccatg accagggaca ccagcatcag
caccgcctac 240atggagctgt ccaggctgag aagcgacgac accgccgtgt
actactgcgc caggggcggc 300tggttcgact actggggcca gggcaccctg
gtgaccgtgt cctcagctag caccaagggc 360cccagcgtgt tccccctggc
ccccagcagc aagagcacct ccggcggcac agccgccctg 420ggctgcctgg
tgaaggacta cttccccgag cccgtgaccg tgtcctggaa cagcggagcc
480ctgaccagcg gcgtgcacac cttccccgcc gtgctgcaga gcagcggcct
gtacagcctg 540tccagcgtgg tgacagtgcc cagcagcagc ctgggcaccc
agacctacat ctgcaacgtg 600aaccacaagc ccagcaacac caaggtggac
aagagagtgg agcccaagag ctgcgacaag 660acccacacct gccccccctg
cccagccccc gaagctgcag gcggcccttc cgtgttcctg 720ttccccccca
agcccaagga caccctgatg atcagcagga cccccgaggt gacctgcgtg
780gtggtggacg tgagccacga ggacccagag gtgaagttca actggtacgt
ggacggcgtg 840gaggtgcaca acgccaagac caagcccaga gaggagcagt
acaacagcac ctacagggtg 900gtgtccgtgc tgaccgtgct gcaccaggac
tggctgaacg gcaaagaata caagtgcaag 960gtctccaaca aggccctgcc
tgcccccatc gaaaagacca tcagcaaggc caagggccag 1020ccacgggagc
cccaggtgta caccctgccc ccttctcggg aggagatgac caagaaccag
1080gtgtccctga cctgtctggt gaagggcttc taccccagcg acatcgccgt
ggagtgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc
cagtgctgga cagcgacggc 1200agcttcttcc tgtacagcaa gctgaccgtg
gacaagagca ggtggcagca gggcaacgtg 1260ttcagctgca gcgtgatgca
cgaggccctg cacaaccact acacccagaa gagcctgagc 1320ctgtcacccg gcaag
13351671335DNAArtificialheavy chain 167caggtgcagc tggtgcagag
cggagctgag gtgaagaagc caggcgccag cgtcaaggtg 60tcctgcaagg ccagcggcta
caccttcacc agcagctaca tcaactgggt gcgccaggct 120ccagggcagg
gactggagtg gatgggccag atcaacgccg ccagcggcat gaccagatac
180gcccagaagt tccagggcag agtcacaatg accagggaca cctctatcag
caccgcctac 240atggagctgt ccaggctgag aagcgacgac accgccgtgt
actactgcgc caggggcggc 300tggttcgact actggggcca gggcaccctg
gtgaccgtgt cctcagctag caccaagggc 360cccagcgtgt tccccctggc
ccccagcagc aagagcacct ccggcggcac agccgccctg 420ggctgcctgg
tgaaggacta cttccccgag cccgtgaccg tgtcctggaa cagcggagcc
480ctgaccagcg gcgtgcacac cttccccgcc gtgctgcaga gcagcggcct
gtacagcctg 540tccagcgtgg tgacagtgcc cagcagcagc ctgggcaccc
agacctacat ctgcaacgtg 600aaccacaagc ccagcaacac caaggtggac
aagagagtgg agcccaagag ctgcgacaag 660acccacacct gccccccctg
cccagccccc gaagctgcag gcggcccttc cgtgttcctg 720ttccccccca
agcccaagga caccctgatg atcagcagga cccccgaggt gacctgcgtg
780gtggtggacg tgagccacga ggacccagag gtgaagttca actggtacgt
ggacggcgtg 840gaggtgcaca acgccaagac caagcccaga gaggagcagt
acaacagcac ctacagggtg 900gtgtccgtgc tgaccgtgct gcaccaggac
tggctgaacg gcaaagaata caagtgcaag 960gtctccaaca aggccctgcc
tgcccccatc gaaaagacca tcagcaaggc caagggccag 1020ccacgggagc
cccaggtgta caccctgccc ccttctcggg aggagatgac caagaaccag
1080gtgtccctga cctgtctggt gaagggcttc taccccagcg acatcgccgt
ggagtgggag 1140agcaacggcc agcccgagaa caactacaag accacccccc
cagtgctgga cagcgacggc 1200agcttcttcc tgtacagcaa gctgaccgtg
gacaagagca ggtggcagca gggcaacgtg 1260ttcagctgca gcgtgatgca
cgaggccctg cacaaccact acacccagaa gagcctgagc 1320ctgtcacccg gcaag
13351681335DNAArtificialheavy chain 168caggtgcaat tggttcagag
cggcgcggaa gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata
tacctttact tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg
gtctcgagtg gatgggcaat attaatgctg ctgctggtat tactctttat
180gctcagaagt ttcagggtcg ggtcaccatg acccgtgata ccagcattag
caccgcgtat 240atggaactga gccgcctgcg tagcgatgat acggccgtgt
attattgcgc gcgtggtggt 300tggtttgatt attggggcca aggcaccctg
gtgacggtta gctcagcctc caccaagggt 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaagcagcgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgggtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 1140agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 1200tccttcttcc tctacagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320ctgtctccgg gtaaa
13351691335DNAArtificialheavy chain 169caggtgcaat tggttcagag
cggcgcggaa gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata
tacctttact tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg
gtctcgagtg gatgggcggt attaatcctc ctgctggtac tacttcttat
180gctcagaagt ttcagggtcg ggtcaccatg acccgtgata ccagcattag
caccgcgtat 240atggaactga gccgcctgcg tagcgatgat acggccgtgt
attattgcgc gcgtggtggt 300tggtttgatt attggggcca aggcaccctg
gtgacggtta gctcagcctc caccaagggt 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaagcagcgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgggtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 1140agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 1200tccttcttcc tctacagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320ctgtctccgg gtaaa
13351701335DNAArtificialheavy chain 170caggtgcaat tggttcagag
cggcgcggaa gtgaaaaaac cgggcgcgag cgtgaaagtg 60agctgcaaag cctccggata
tacctttact tcttcttata ttaattgggt ccgccaagcc 120cctgggcagg
gtctcgagtg gatgggcaat attaatcctg ctactggtca tgctgattat
180gctcagaagt ttcagggtcg ggtgaccatg acccgtgata ccagcattag
caccgcgtat 240atggaactga gccgcctgcg tagcgatgat acggccgtgt
attattgcgc gcgtggtggt 300tggtttgatt attggggcca aggcaccctg
gtgacggtta gctcagcctc caccaagggt 360ccatcggtct tccccctggc
accctcctcc aagagcacct ctgggggcac agcggccctg 420ggctgcctgg
tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc
480ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact
ctactccctc 540agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc
agacctacat ctgcaacgtg 600aatcacaagc ccagcaacac caaggtggac
aagagagttg agcccaaatc ttgtgacaaa 660actcacacat gcccaccgtg
cccagcacct gaagcagcgg ggggaccgtc agtcttcctc 720ttccccccaa
aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg
780gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt
ggacggcgtg 840gaggtgcata atgccaagac aaagccgcgg gaggagcagt
acaacagcac gtaccgggtg 900gtcagcgtcc tcaccgtcct gcaccaggac
tggctgaatg gcaaggagta caagtgcaag 960gtctccaaca aagccctccc
agcccccatc gagaaaacca tctccaaagc caaagggcag 1020ccccgagaac
cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag
1080gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt
ggagtgggag 1140agcaatgggc agccggagaa caactacaag accacgcctc
ccgtgctgga ctccgacggc 1200tccttcttcc tctacagcaa gctcaccgtg
gacaagagca ggtggcagca ggggaacgtc 1260ttctcatgct ccgtgatgca
tgaggctctg cacaaccact acacgcagaa gagcctctcc 1320ctgtctccgg gtaaa
1335171651DNAArtificiallight chain 171cagagcgccc tgacccagcc
cgccagcgtg tccggcagcc caggccagtc tatcacaatc 60agctgcaccg gcacctccag
cgacgtgggc agctacaact acgtgaactg gtatcagcag 120caccccggca
aggcccccaa gctgatgatc tacggcgtga gcaagaggcc cagcggcgtg
180tccaacaggt tcagcggcag caagagcggc aacaccgcca gcctgacaat
cagtgggctg 240caggctgagg acgaggccga ctactactgc ggcacctttg
ccggcggatc atactacggc 300gtgttcggcg gagggaccaa gctgaccgtg
ctgggccagc ctaaggctgc ccccagcgtg 360accctgttcc cccccagcag
cgaggagctg caggccaaca aggccaccct ggtgtgcctg 420atcagcgact
tctacccagg cgccgtgacc gtggcctgga aggccgacag cagccccgtg
480aaggccggcg tggagaccac cacccccagc aagcagagca acaacaagta
cgccgccagc 540agctacctga gcctgacccc cgagcagtgg aagagccaca
ggtcctacag ctgccaggtg 600acccacgagg gcagcaccgt ggaaaagacc
gtggccccaa ccgagtgcag c 651172651DNAArtificiallight chain
172cagagcgccc tgacccagcc cgccagcgtg tccggcagcc caggccagtc
tatcacaatc 60agctgcaccg gcacctccag cgacgtgggc agctacaact acgtgaactg
gtatcagcag 120caccccggca aggcccccaa gctgatgatc tacggcgtga
gcaagaggcc cagcggcgtg 180tccaacaggt tcagcggcag caagagcggc
aacaccgcca gcctgacaat cagtgggctg 240caggctgagg acgaggccga
ctactactgc ggcacctttg ccggcggatc atactacggc 300gtgttcggcg
gagggaccaa gctgaccgtg ctgggccagc ctaaggctgc ccccagcgtg
360accctgttcc cccccagcag cgaggagctg caggccaaca aggccaccct
ggtgtgcctg 420atcagcgact tctacccagg cgccgtgacc gtggcctgga
aggccgacag cagccccgtg 480aaggccggcg tggagaccac cacccccagc
aagcagagca acaacaagta cgccgccagc 540agctacctga gcctgacccc
cgagcagtgg aagagccaca ggtcctacag ctgccaggtg 600acccacgagg
gcagcaccgt ggaaaagacc gtggccccaa ccgagtgcag c
651173651DNAArtificiallight chain 173cagagcgcac tgacccagcc
agcttcagtg agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag
cgatgttggt tcttataatt atgtgaattg gtaccagcag 120catcccggga
aggcgccgaa acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg
180agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat
tagcggcctg 240caagcggaag acgaagcgga ttattattgc ggtacttttg
ctggtggttc ttattatggt 300gtgtttggcg gcggcacgaa gttaaccgtc
ctaggtcagc ccaaggctgc cccctcggtc 360actctgttcc cgccctcctc
tgaggagctt caagccaaca aggccacact ggtgtgtctc 420ataagtgact
tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc
480aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta
cgcggccagc 540agctatctga gcctgacgcc tgagcagtgg aagtcccaca
gaagctacag ctgccaggtc 600acgcatgaag ggagcaccgt ggagaagaca
gtggccccta cagaatgttc a 651174651DNAArtificiallight chain
174cagagcgcac tgacccagcc agcttcagtg agcggctcac caggtcagag
cattaccatc 60tcgtgtacgg gtactagcag cgatgttggt tcttataatt atgtgaattg
gtaccagcag 120catcccggga aggcgccgaa acttatgatt tatggtgttt
ctaagcgtcc ctcaggcgtg 180agcaaccgtt ttagcggatc caaaagcggc
aacaccgcga gcctgaccat tagcggcctg 240caagcggaag acgaagcgga
ttattattgc ggtacttttg ctggtggttc ttattatggt 300gtgtttggcg
gcggcacgaa gttaaccgtc ctaggtcagc ccaaggctgc cccctcggtc
360actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact
ggtgtgtctc 420ataagtgact tctacccggg agccgtgaca gtggcctgga
aggcagatag cagccccgtc 480aaggcgggag tggagaccac cacaccctcc
aaacaaagca acaacaagta cgcggccagc 540agctatctga gcctgacgcc
tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600acgcatgaag
ggagcaccgt ggagaagaca gtggccccta cagaatgttc a
651175651DNAArtificiallight chain 175cagagcgcac tgacccagcc
agcttcagtg agcggctcac caggtcagag cattaccatc 60tcgtgtacgg gtactagcag
cgatgttggt tcttataatt atgtgaattg gtaccagcag 120catcccggga
aggcgccgaa acttatgatt tatggtgttt ctaagcgtcc ctcaggcgtg
180agcaaccgtt ttagcggatc caaaagcggc aacaccgcga gcctgaccat
tagcggcctg 240caagcggaag acgaagcgga ttattattgc ggtacttttg
ctggtggttc ttattatggt 300gtgtttggcg gcggcacgaa gttaaccgtc
ctaggtcagc ccaaggctgc cccctcggtc 360actctgttcc cgccctcctc
tgaggagctt caagccaaca aggccacact ggtgtgtctc 420ataagtgact
tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc
480aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta
cgcggccagc 540agctatctga gcctgacgcc tgagcagtgg aagtcccaca
gaagctacag ctgccaggtc 600acgcatgaag ggagcaccgt ggagaagaca
gtggccccta cagaatgttc a 6511761323DNAArtificialheavy chain
176caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggcgccag
cgtcaaggtg 60tcctgcaagg ccagcggcta caccttcacc agcagctaca tcaactgggt
ccgccaggct 120cctgggcagg gactggagtg gatgggcacc atcaaccccg
tgtccggcag caccagctac 180gcccagaagt tccagggcag agtcaccatg
accagggaca ccagcatcag caccgcctac 240atggagctgt ccaggctgag
aagcgacgac accgccgtgt actactgcgc caggggcggc 300tggttcgact
actggggcca gggcaccctg gtgaccgtgt cctcagctag caccaagggc
360cccagcgtgt tccccctggc cccctgcagc agaagcacca gcgagagcac
agccgccctg 420ggctgcctgg tgaaggacta cttccccgag ccagtgaccg
tgtcctggaa cagcggagcc 480ctgaccagcg gcgtgcacac cttccccgcc
gtgctgcaga gcagcggcct gtacagcctg 540tccagcgtgg tgaccgtgcc
cagcagcaac ttcggcaccc agacctacac ctgcaacgtg 600gaccacaagc
ccagcaacac caaggtggac aagaccgtgg agaggaagtg ctgcgtggag
660tgccccccct gcccagcccc cccagtggcc ggaccctccg tgttcctgtt
cccccccaag 720cccaaggaca ccctgatgat cagcaggacc cccgaggtga
cctgcgtggt ggtggacgtg 780agccacgagg acccagaggt gcagttcaac
tggtacgtgg acggcgtgga ggtgcacaac 840gccaagacca agcccagaga
ggaacagttt aacagcacct tcagggtggt gtccgtgctg 900accgtggtgc
accaggactg gctgaacggc aaagagtaca agtgcaaggt ctccaacaag
960ggcctgccag cccccatcga gaaaaccatc agcaagacca agggccagcc
acgggagccc 1020caggtgtaca ccctgccccc cagccgggag gaaatgacca
agaaccaggt gtccctgacc 1080tgtctggtga agggcttcta ccccagcgac
atcgccgtgg agtgggagag caacggccag 1140cccgagaaca actacaagac
cacccccccc atgctggaca gcgacggcag cttcttcctg 1200tacagcaagc
tgacagtgga caagagcagg tggcagcagg gcaacgtgtt cagctgcagc
1260gtgatgcacg aggccctgca caaccactac acccagaaga gcctgagcct
gtcccccggc 1320aag 13231771323DNAArtificialheavy chain
177caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggcgccag
cgtcaaggtg 60tcctgcaagg ccagcggcta caccttcacc agcagctaca tcaactgggt
gcgccaggct 120ccagggcagg gactggagtg gatgggccag atcaacgccg
ccagcggcat gaccagatac 180gcccagaagt tccagggcag agtcacaatg
accagggaca cctctatcag caccgcctac 240atggagctgt ccaggctgag
aagcgacgac accgccgtgt actactgcgc caggggcggc 300tggttcgact
actggggcca gggcaccctg gtgaccgtgt cctcagctag caccaagggc
360cccagcgtgt tccccctggc cccctgcagc agaagcacca gcgagagcac
agccgccctg 420ggctgcctgg tgaaggacta cttccccgag ccagtgaccg
tgtcctggaa cagcggagcc 480ctgaccagcg gcgtgcacac cttccccgcc
gtgctgcaga gcagcggcct gtacagcctg 540tccagcgtgg tgaccgtgcc
cagcagcaac ttcggcaccc agacctacac ctgcaacgtg 600gaccacaagc
ccagcaacac caaggtggac aagaccgtgg agaggaagtg ctgcgtggag
660tgccccccct gcccagcccc cccagtggcc ggaccctccg tgttcctgtt
cccccccaag 720cccaaggaca ccctgatgat cagcaggacc cccgaggtga
cctgcgtggt ggtggacgtg 780agccacgagg acccagaggt gcagttcaac
tggtacgtgg acggcgtgga ggtgcacaac 840gccaagacca agcccagaga
ggaacagttt aacagcacct tcagggtggt gtccgtgctg 900accgtggtgc
accaggactg gctgaacggc aaagagtaca agtgcaaggt ctccaacaag
960ggcctgccag cccccatcga gaaaaccatc agcaagacca agggccagcc
acgggagccc 1020caggtgtaca ccctgccccc cagccgggag gaaatgacca
agaaccaggt gtccctgacc 1080tgtctggtga agggcttcta ccccagcgac
atcgccgtgg agtgggagag caacggccag 1140cccgagaaca actacaagac
cacccccccc atgctggaca gcgacggcag cttcttcctg 1200tacagcaagc
tgacagtgga caagagcagg tggcagcagg gcaacgtgtt cagctgcagc
1260gtgatgcacg aggccctgca caaccactac acccagaaga gcctgagcct
gtcccccggc 1320aag 13231781323DNAArtificialheavy chain
178caggtgcaat tggttcagag cggcgcggaa gtgaaaaaac cgggcgcgag
cgtgaaagtg 60agctgcaaag cctccggata tacctttact tcttcttata ttaattgggt
ccgccaagcc 120cctgggcagg gtctcgagtg gatgggcaat attaatgctg
ctgctggtat tactctttat 180gctcagaagt ttcagggtcg ggtcaccatg
acccgtgata ccagcattag caccgcgtat 240atggaactga gccgcctgcg
tagcgatgat acggccgtgt attattgcgc gcgtggtggt 300tggtttgatt
attggggcca aggcaccctg gtgacggtta gctcagcttc caccaagggc
360cccagcgtgt tccccctggc cccctgcagc agaagcacca gcgagagcac
agccgccctg 420ggctgcctgg tgaaggacta cttccccgag cccgtgaccg
tgagctggaa cagcggagcc 480ctgaccagcg gcgtgcacac cttccccgcc
gtgctgcaga gcagcggcct gtacagcctg 540agcagcgtgg tgaccgtgcc
cagcagcaac ttcggcaccc agacctacac ctgcaacgtg 600gaccacaagc
ccagcaacac caaggtggac aagaccgtgg agcggaagtg ctgcgtggag
660tgccccccct gccctgcccc tcctgtggcc ggaccctccg tgttcctgtt
cccccccaag 720cccaaggaca ccctgatgat cagccggacc cccgaggtga
cctgcgtggt ggtggacgtg 780agccacgagg accccgaggt gcagttcaac
tggtacgtgg acggcgtgga ggtgcacaac 840gccaagacca agccccggga
ggaacagttc aacagcacct tccgggtggt gtccgtgctg 900accgtggtgc
accaggactg gctgaacggc aaagaataca agtgcaaggt gtccaacaag
960ggcctgcctg cccccatcga gaaaaccatc agcaagacaa agggccagcc
cagggaaccc 1020caggtgtaca ccctgccccc cagccgggag gaaatgacca
agaaccaggt gtccctgacc 1080tgtctggtga agggcttcta ccccagcgac
atcgccgtgg agtgggagag caacggccag 1140cccgagaaca actacaagac
cacccccccc atgctggaca gcgacggcag cttcttcctg 1200tacagcaagc
tgacagtgga caagagccgg tggcagcagg gcaacgtgtt cagctgcagc
1260gtgatgcacg aggccctgca caaccactac acccagaaga gcctgagcct
gtcccccggc 1320aaa 13231791323DNAArtificialheavy chain
179caggtgcaat tggttcagag cggcgcggaa gtgaaaaaac cgggcgcgag
cgtgaaagtg 60agctgcaaag cctccggata tacctttact tcttcttata ttaattgggt
ccgccaagcc 120cctgggcagg gtctcgagtg gatgggcggt attaatcctc
ctgctggtac tacttcttat 180gctcagaagt ttcagggtcg ggtcaccatg
acccgtgata ccagcattag caccgcgtat 240atggaactga gccgcctgcg
tagcgatgat acggccgtgt attattgcgc gcgtggtggt 300tggtttgatt
attggggcca aggcaccctg gtgacggtta gctcagcttc caccaagggc
360cccagcgtgt tccccctggc cccctgcagc agaagcacca gcgagagcac
agccgccctg 420ggctgcctgg tgaaggacta cttccccgag cccgtgaccg
tgagctggaa cagcggagcc 480ctgaccagcg gcgtgcacac cttccccgcc
gtgctgcaga gcagcggcct gtacagcctg 540agcagcgtgg tgaccgtgcc
cagcagcaac ttcggcaccc agacctacac ctgcaacgtg 600gaccacaagc
ccagcaacac caaggtggac aagaccgtgg agcggaagtg ctgcgtggag
660tgccccccct gccctgcccc tcctgtggcc ggaccctccg tgttcctgtt
cccccccaag 720cccaaggaca ccctgatgat cagccggacc cccgaggtga
cctgcgtggt ggtggacgtg 780agccacgagg accccgaggt gcagttcaac
tggtacgtgg acggcgtgga ggtgcacaac 840gccaagacca agccccggga
ggaacagttc aacagcacct tccgggtggt gtccgtgctg 900accgtggtgc
accaggactg gctgaacggc aaagaataca agtgcaaggt gtccaacaag
960ggcctgcctg cccccatcga gaaaaccatc agcaagacaa agggccagcc
cagggaaccc 1020caggtgtaca ccctgccccc cagccgggag gaaatgacca
agaaccaggt gtccctgacc 1080tgtctggtga agggcttcta ccccagcgac
atcgccgtgg agtgggagag caacggccag 1140cccgagaaca actacaagac
cacccccccc atgctggaca gcgacggcag cttcttcctg 1200tacagcaagc
tgacagtgga caagagccgg tggcagcagg gcaacgtgtt cagctgcagc
1260gtgatgcacg aggccctgca caaccactac acccagaaga gcctgagcct
gtcccccggc 1320aaa 13231801323DNAArtificialheavy chain
180caggtgcaat tggttcagag cggcgcggaa gtgaaaaaac cgggcgcgag
cgtgaaagtg 60agctgcaaag cctccggata tacctttact tcttcttata ttaattgggt
ccgccaagcc 120cctgggcagg gtctcgagtg gatgggcaat attaatcctg
ctactggtca tgctgattat 180gctcagaagt ttcagggtcg ggtgaccatg
acccgtgata ccagcattag caccgcgtat 240atggaactga gccgcctgcg
tagcgatgat acggccgtgt attattgcgc gcgtggtggt 300tggtttgatt
attggggcca aggcaccctg gtgacggtta gctcagcttc caccaagggc
360cccagcgtgt tccccctggc cccctgcagc agaagcacca gcgagagcac
agccgccctg 420ggctgcctgg tgaaggacta cttccccgag cccgtgaccg
tgagctggaa cagcggagcc 480ctgaccagcg gcgtgcacac cttccccgcc
gtgctgcaga gcagcggcct gtacagcctg 540agcagcgtgg tgaccgtgcc
cagcagcaac ttcggcaccc agacctacac ctgcaacgtg 600gaccacaagc
ccagcaacac caaggtggac aagaccgtgg agcggaagtg ctgcgtggag
660tgccccccct gccctgcccc tcctgtggcc ggaccctccg tgttcctgtt
cccccccaag 720cccaaggaca ccctgatgat cagccggacc cccgaggtga
cctgcgtggt ggtggacgtg 780agccacgagg accccgaggt gcagttcaac
tggtacgtgg acggcgtgga ggtgcacaac 840gccaagacca agccccggga
ggaacagttc aacagcacct tccgggtggt gtccgtgctg 900accgtggtgc
accaggactg gctgaacggc aaagaataca agtgcaaggt gtccaacaag
960ggcctgcctg cccccatcga gaaaaccatc agcaagacaa agggccagcc
cagggaaccc 1020caggtgtaca ccctgccccc cagccgggag gaaatgacca
agaaccaggt gtccctgacc 1080tgtctggtga agggcttcta ccccagcgac
atcgccgtgg agtgggagag caacggccag 1140cccgagaaca actacaagac
cacccccccc atgctggaca gcgacggcag cttcttcctg 1200tacagcaagc
tgacagtgga caagagccgg tggcagcagg gcaacgtgtt cagctgcagc
1260gtgatgcacg aggccctgca caaccactac acccagaaga gcctgagcct
gtcccccggc 1320aaa 1323181512PRTHomo sapiens 181Met Thr Ala Pro Trp
Val Ala Leu Ala Leu Leu Trp Gly Ser Leu Cys 1 5 10 15 Ala Gly Ser
Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr 20 25 30 Asn
Ala Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg 35 40
45 Cys Glu Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg
50 55 60 Asn Ser Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp
Leu Asp 65 70 75 80 Asp Phe Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala
Thr Glu Glu Asn 85 90 95 Pro Gln Val Tyr Phe Cys Cys Cys Glu Gly
Asn Phe Cys Asn Glu Arg 100 105 110 Phe Thr His Leu Pro Glu Ala Gly
Gly Pro Glu Val Thr Tyr Glu Pro 115 120 125 Pro Pro Thr Ala Pro Thr
Leu Leu Thr Val Leu Ala Tyr Ser Leu Leu 130 135 140 Pro Ile Gly Gly
Leu Ser Leu Ile
Val Leu Leu Ala Phe Trp Met Tyr 145 150 155 160 Arg His Arg Lys Pro
Pro Tyr Gly His Val Asp Ile His Glu Asp Pro 165 170 175 Gly Pro Pro
Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu Gln Leu 180 185 190 Leu
Glu Ile Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Gln 195 200
205 Leu Met Asn Asp Phe Val Ala Val Lys Ile Phe Pro Leu Gln Asp Lys
210 215 220 Gln Ser Trp Gln Ser Glu Arg Glu Ile Phe Ser Thr Pro Gly
Met Lys 225 230 235 240 His Glu Asn Leu Leu Gln Phe Ile Ala Ala Glu
Lys Arg Gly Ser Asn 245 250 255 Leu Glu Val Glu Leu Trp Leu Ile Thr
Ala Phe His Asp Lys Gly Ser 260 265 270 Leu Thr Asp Tyr Leu Lys Gly
Asn Ile Ile Thr Trp Asn Glu Leu Cys 275 280 285 His Val Ala Glu Thr
Met Ser Arg Gly Leu Ser Tyr Leu His Glu Asp 290 295 300 Val Pro Trp
Cys Arg Gly Glu Gly His Lys Pro Ser Ile Ala His Arg 305 310 315 320
Asp Phe Lys Ser Lys Asn Val Leu Leu Lys Ser Asp Leu Thr Ala Val 325
330 335 Leu Ala Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro
Pro 340 345 350 Gly Asp Thr His Gly Gln Val Gly Thr Arg Arg Tyr Met
Ala Pro Glu 355 360 365 Val Leu Glu Gly Ala Ile Asn Phe Gln Arg Asp
Ala Phe Leu Arg Ile 370 375 380 Asp Met Tyr Ala Met Gly Leu Val Leu
Trp Glu Leu Val Ser Arg Cys 385 390 395 400 Lys Ala Ala Asp Gly Pro
Val Asp Glu Tyr Met Leu Pro Phe Glu Glu 405 410 415 Glu Ile Gly Gln
His Pro Ser Leu Glu Glu Leu Gln Glu Val Val Val 420 425 430 His Lys
Lys Met Arg Pro Thr Ile Lys Asp His Trp Leu Lys His Pro 435 440 445
Gly Leu Ala Gln Leu Cys Val Thr Ile Glu Ala Cys Trp Asp His Asp 450
455 460 Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Glu Glu Arg Val Ser
Leu 465 470 475 480 Ile Arg Arg Ser Val Asn Gly Thr Thr Ser Asp Cys
Leu Val Ser Leu 485 490 495 Val Thr Ser Val Thr Asn Val Asp Leu Pro
Pro Lys Glu Ser Ser Ile 500 505 510 182116PRTHomo sapiens 182Ser
Gly Arg Gly Glu Ala Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala 1 5 10
15 Asn Trp Glu Leu Glu Arg Thr Asn Gln Ser Gly Leu Glu Arg Cys Glu
20 25 30 Gly Glu Gln Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg
Asn Ser 35 40 45 Ser Gly Thr Ile Glu Leu Val Lys Lys Gly Cys Trp
Leu Asp Asp Phe 50 55 60 Asn Cys Tyr Asp Arg Gln Glu Cys Val Ala
Thr Glu Glu Asn Pro Gln 65 70 75 80 Val Tyr Phe Cys Cys Cys Glu Gly
Asn Phe Cys Asn Glu Arg Phe Thr 85 90 95 His Leu Pro Glu Ala Gly
Gly Pro Glu Val Thr Tyr Glu Pro Pro Pro 100 105 110 Thr Ala Pro Thr
115 18315PRTHomo sapiens 183Ile Glu Leu Val Lys Lys Gly Ser Trp Leu
Asp Asp Phe Asn Ser 1 5 10 15 18415PRTHomo sapiens 184Val Lys Lys
Gly Ser Trp Leu Asp Asp Phe Asn Ser Tyr Asp Arg 1 5 10 15
18515PRTHomo sapiens 185Gly Ser Trp Leu Asp Asp Phe Asn Ser Tyr Asp
Arg Gln Glu Ser 1 5 10 15 1869PRTHomo sapiens 186Gly Cys Trp Leu
Asp Asp Phe Asn Cys 1 5 18715PRTHomo sapiens 187Cys Glu Gly Glu Gln
Asp Lys Arg Leu His Cys Tyr Ala Ser Trp 1 5 10 15 1886PRTHomo
sapiens 188Trp Leu Asp Asp Phe Asn 1 5 1895PRTHomo sapiens 189Glu
Gln Asp Lys Arg 1 5 19011PRTHomo sapiens 190Lys Gly Cys Trp Leu Asp
Asp Phe Asn Cys Tyr 1 5 10
* * * * *
References