U.S. patent application number 13/257519 was filed with the patent office on 2012-05-17 for bispecific anti-her antibodies.
Invention is credited to Germaine Fuh, Lauric Haber, Gabriele M. Schaefer, Mark X. Sliwkowski.
Application Number | 20120121596 13/257519 |
Document ID | / |
Family ID | 42111063 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120121596 |
Kind Code |
A1 |
Fuh; Germaine ; et
al. |
May 17, 2012 |
BISPECIFIC ANTI-HER ANTIBODIES
Abstract
The invention provides anti-HER antibodies, including
multispecific anti-HER antibodies, compositions comprising and
methods of using these antibodies. Also provided herein are
EGFR/HER3 multispecific antibodies that are less toxic than
traditional EGFR antagonists.
Inventors: |
Fuh; Germaine; (Pacifica,
CA) ; Haber; Lauric; (San Carlos, CA) ;
Schaefer; Gabriele M.; (San Mateo, CA) ; Sliwkowski;
Mark X.; (San Carlos, CA) |
Family ID: |
42111063 |
Appl. No.: |
13/257519 |
Filed: |
March 19, 2010 |
PCT Filed: |
March 19, 2010 |
PCT NO: |
PCT/US10/28023 |
371 Date: |
February 2, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61210562 |
Mar 20, 2009 |
|
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|
Current U.S.
Class: |
424/136.1 ;
435/252.3; 435/254.11; 435/254.2; 435/328; 435/419; 435/69.6;
530/387.3; 530/391.7; 536/23.53 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 35/00 20180101; C07K 2317/92 20130101; A61P 35/04 20180101;
C07K 16/28 20130101; C07K 2317/73 20130101; A61K 2039/507 20130101;
C07K 2317/732 20130101; C07K 2317/31 20130101; C07K 16/2863
20130101; C07K 2317/56 20130101; C07K 2317/565 20130101; C07K 16/32
20130101 |
Class at
Publication: |
424/136.1 ;
530/387.3; 536/23.53; 435/328; 435/419; 435/252.3; 435/254.11;
435/254.2; 435/69.6; 530/391.7 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 15/13 20060101 C12N015/13; C12N 5/10 20060101
C12N005/10; A61P 35/00 20060101 A61P035/00; C12N 1/15 20060101
C12N001/15; C12N 1/19 20060101 C12N001/19; C12P 21/00 20060101
C12P021/00; C07K 16/40 20060101 C07K016/40; C12N 1/21 20060101
C12N001/21 |
Claims
1. A multispecific antibody comprising an antigen-binding domain
that specifically binds to EGFR and HER3, wherein the toxicity of
the antibody is less than the toxicity of an EGFR antagonist.
2. The antibody of claim 1, wherein the toxicity is dermatological
toxicity.
3. The antibody of claim 1, wherein the EGFR antagonist is
cetuximab.
4. The antibody of claim 1, wherein the antibody inhibits a
biological activity of at least one of EGFR and HER3
5. The antibody of claim 2, wherein the antibody inhibits EGF
binding to EGFR.
6. The antibody of claim 2, wherein the antibody inhibits
TGF-.alpha. induced EGFR phosphorylation.
7. The antibody of claim 1, wherein the antibody inhibits tumor
cell growth.
8. The antibody of claim 1, wherein the antibody binds to domain
III of EGFR.
9. The antibody of claim 1, wherein the antibody binds to EGFR and
HER3 with a Kd of less than 10.sup.-6M.
10. The antibody of claim 9, wherein the antibody binds to EGFR
with a Kd of less than 10.sup.-6 M and binds to HER3 with a Kd of
less than 10.sup.-7 M.
11. The antibody of claim 1, wherein the antibody comprises (a)
HVR-H1 comprising the amino acid sequence of LSGDWIH (SEQ ID NO:
48); (b) HVR-H2 comprising the amino acid sequence of VGEISAAGGYTD
(SEQ ID NO: 51); and (c) HVR-H3 comprising the amino acid sequence
of ARESRVSFEAAMDY (SEQ ID NO: 53); and (d) HVR-L1 comprising the
amino acid sequence of NIATDVA (SEQ ID NO: 55); (e) HVR-L2
comprising the amino acid sequence of SASF (SEQ ID NO: 56); and (f)
HVR-L3 comprising the amino acid sequence of SEPEPYT (SEQ ID NO:
57).
12. The antibody of claim 1, comprising (a) a heavy chain variable
domain having at least 95% sequence identity to the amino acid
sequence of SEQ ID NO: 30; (b) a light chain variable domain having
at least 95% sequence identity to the amino acid sequence of SEQ ID
NO: 29; or (c) a heavy chain variable domain as in (a) and a light
chain variable domain as in (b).
13. The antibody of claim 12, comprising a heavy chain variable
domain of SEQ ID NO: 30.
14. The antibody of claim 12, comprising a light chain variable
domain sequence of SEQ ID NO: 29.
15. A multispecific antibody comprising a heavy chain variable
domain sequence of SEQ ID NO: 30 and a light chain variable domain
sequence of SEQ ID NO: 29.
16. The antibody of claim 1, wherein the antibody is a full length
IgG1 antibody.
17. Isolated nucleic acid encoding the antibody of claim 1.
18. A host cell comprising the nucleic acid of claim 17.
19. A multispecific antibody comprising an antigen-binding domain
that specifically binds to EGFR and HER3, wherein the antibody
comprises (a) HVR-H1 comprising the amino acid sequence of LSGDWIH
(SEQ ID NO: 48); (b) HVR-H2 comprising the amino acid sequence of
VGEISAAGGYTD (SEQ ID NO: 51); and (c) HVR-H3 comprising the amino
acid sequence of ARESRVSFEAAMDY (SEQ ID NO: 53); and (d) HVR-L1
comprising the amino acid sequence of NIATDVA (SEQ ID NO: 55); (e)
HVR-L2 comprising the amino acid sequence of SASF (SEQ ID NO: 56);
and (f) HVR-L3 comprising the amino acid sequence of SEPEPYT (SEQ
ID NO: 57).
20. A multispecific antibody comprising an antigen-binding domain
that specifically binds to EGFR and HER3, wherein the antibody
comprises (a) a heavy chain variable domain having at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 30; (b)
a light chain variable domain having at least 95% sequence identity
to the amino acid sequence of SEQ ID NO: 29; or (c) a heavy chain
variable domain as in (a) and a light chain variable domain as in
(b).
21. The antibody of claim 20 comprising a heavy chain variable
domain of SEQ ID NO: 30.
22. The antibody of claim 20, comprising a light chain variable
domain sequence of SEQ ID NO: 29.
23. A method of producing an antibody comprising culturing the host
cell of claim 18 so that the antibody is produced.
24. An immunoconjugate comprising the antibody of claim 1 and a
cytotoxic agent.
25. A pharmaceutical formulation comprising the antibody of claim 1
and a pharmaceutically acceptable carrier.
26. A method of treating an individual having cancer comprising
administering to the individual an effective amount of the antibody
of claim 1.
27.-28. (canceled)
29. A method of inhibiting a biological activity of a HER receptor
in an individual comprising administering to the individual an
effective amount of the antibody of claim 1 to inhibit a biological
activity of a HER receptor.
30.-37. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35 USC
119(e) of U.S. Provisional Application No. 61/210,562, filed Mar.
20, 2009, the disclosure of which is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention concerns anti-HER antibodies,
including multispecific anti-HER antibodies with binding
specificity for at least two different HER receptors, and use of
the antibodies to treat diseases or disorders.
BACKGROUND OF THE INVENTION
[0003] The HER family of receptor tyrosine kinases are important
mediators of cell growth, differentiation and survival. The
receptor family includes four distinct members including epidermal
growth factor receptor (EGFR, ErbB1, or HER1), HER2 (ErbB2 or
p185.sup.neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).
[0004] EGFR, encoded by the erbB1 gene, has been causally
implicated in human malignancy. In particular, increased expression
of EGFR has been observed in breast, bladder, lung, head, neck and
stomach cancer as well as glioblastomas. Increased EGFR receptor
expression is often associated with increased production of the
EGFR ligand, transforming growth factor alpha (TGF-.alpha.), by the
same tumor cells resulting in receptor activation by an autocrine
stimulatory pathway. Baselga and Mendelsohn Pharmac. Ther.
64:127-154 (1994). Monoclonal antibodies directed against the EGFR
or its ligands, TGF-.alpha. and EGF, have been evaluated as
therapeutic agents in the treatment of such malignancies. See,
e.g., Baselga and Mendelsohn., supra; Masui et al. Cancer Research
44:1002-1007 (1984); and Wu et al. J. Clin. Invest. 95:1897-1905
(1995). The second member of the HER family, HER2 (p185.sup.neu),
was originally identified as the product of the transforming gene
from neuroblastomas of chemically treated rats. The activated form
of the neu proto-oncogene results from a point mutation (valine to
glutamic acid) in the transmembrane region of the encoded protein.
Amplification of the human homolog of neu is observed in breast and
ovarian cancers and correlates with a poor prognosis (Slamon et
al., Science, 235:177-182 (1987); Slamon et al., Science,
244:707-712 (1989); and U.S. Pat. No. 4,968,603). Overexpression of
HER2 (frequently but not uniformly due to gene amplification) has
also been observed in other carcinomas including carcinomas of the
stomach, endometrium, salivary gland, lung, kidney, colon, thyroid,
pancreas and bladder. See, among others, King et al., Science,
229:974 (1985); Yokota et al., Lancet: 1:765-767 (1986); Fukushige
et al., Mol Cell Biol., 6:955-958 (1986); Guerin et al., Oncogene
Res., 3:21-31 (1988); Cohen et al., Oncogene, 4:81-88 (1989);
Yonemura et al., Cancer Res., 51:1034 (1991); Borst et al.,
Gynecol. Oncol., 38:364 (1990); Weiner et al., Cancer Res.,
50:421-425 (1990); Kern et al., Cancer Res., 50:5184 (1990); Park
et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol. Carcinog.,
3:254-257 (1990); Aasland et al. Br. J. Cancer 57:358-363 (1988);
Williams et al. Pathobiology 59:46-52 (1991); and McCann et al.,
Cancer, 65:88-92 (1990). HER2 may be overexpressed in prostate
cancer (Gu et al. Cancer Lett. 99:185-9 (1996); Ross et al. Hum.
Pathol. 28:827-33 (1997); Ross et al. Cancer 79:2162-70 (1997); and
Sadasivan et al. J. Urol. 150:126-31 (1993)).
[0005] Antibodies directed against the rat p185.sup.neu and human
HER2 protein products have been described. Drebin and colleagues
have raised antibodies against the rat neu gene product,
p185.sup.neu See, for example, Drebin et al., Cell 41:695-706
(1985); Myers et al., Meth. Enzym. 198:277-290 (1991); and
WO94/22478. Drebin et al. Oncogene 2:273-277 (1988) report that
mixtures of antibodies reactive with two distinct regions of
p185.sup.neu result in synergistic anti-tumor effects on
neu-transformed NIH-3T3 cells implanted into nude mice. See also
U.S. Pat. No. 5,824,311 issued Oct. 20, 1998.
[0006] Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989)
describe the generation of a panel of HER2 antibodies which were
characterized using the human breast tumor cell line SK-BR-3.
Relative cell proliferation of the SK-BR-3 cells following exposure
to the antibodies was determined by crystal violet staining of the
monolayers after 72 hours. Using this assay, maximum inhibition was
obtained with the antibody called 4D5 which inhibited cellular
proliferation by 56%. Other antibodies in the panel reduced
cellular proliferation to a lesser extent in this assay. The
antibody 4D5 was further found to sensitize HER2-overexpressing
breast tumor cell lines to the cytotoxic effects of TNF-.alpha..
See also U.S. Pat. No. 5,677,171 issued Oct. 14, 1997. The HER2
antibodies discussed in Hudziak et al. are further characterized in
Fendly et al. Cancer Research 50:1550-1558 (1990); Kotts et al. In
Vitro 26(3):59A (1990); Sarup et al. Growth Regulation 1:72-82
(1991); Shepard et al. J. Clin. Immunol. 11(3):117-127 (1991);
Kumar et al. Mol. Cell. Biol. 11(2):979-986 (1991); Lewis et al.
Cancer Immunol. Immunother. 37:255-263 (1993); Pietras et al.
Oncogene 9:1829-1838 (1994); Vitetta et al. Cancer Research
54:5301-5309 (1994); Sliwkowski et al. J. Biol. Chem.
269(20):14661-14665 (1994); Scott et al. J. Biol. Chem. 266:14300-5
(1991); D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994);
Lewis et al. Cancer Research 56:1457-1465 (1996); and Schaefer et
al. Oncogene 15:1385-1394 (1997).
[0007] A recombinant humanized version of the murine HER2 antibody
4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumab or HERCEPTIN.RTM.; U.S.
Pat. No. 5,821,337) is clinically active in patients with
HER2-overexpressing metastatic breast cancers that have received
extensive prior anti-cancer therapy (Baselga et al., J. Clin.
Oncol. 14:737-744 (1996)). Trastuzumab received marketing approval
from the Food and Drug Administration Sep. 25, 1998 for the
treatment of patients with metastatic breast cancer whose tumors
overexpress the HER2 protein.
[0008] Other HER2 antibodies with various properties have been
described in Tagliabue et al. Int. J. Cancer 47:933-937 (1991);
McKenzie et al. Oncogene 4:543-548 (1989); Maier et al. Cancer Res.
51:5361-5369 (1991); Bacus et al. Molecular Carcinogenesis
3:350-362 (1990); Stancovski et al. PNAS (USA) 88:8691-8695 (1991);
Bacus et al. Cancer Research 52:2580-2589 (1992); Xu et al. Int. J.
Cancer 53:401-408 (1993); WO94/00136; Kasprzyk et al. Cancer
Research 52:2771-2776 (1992); Hancock et al., Cancer Res.
51:4575-4580 (1991); Shawver et al. Cancer Res. 54:1367-1373
(1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994); Harwerth et
al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No. 5,783,186;
and Klapper et al. Oncogene 14:2099-2109 (1997).
[0009] Homology screening resulted in the identification of two
other HER receptor family members; HER3 (U.S. Pat. Nos. 5,968,511,
5,183,884 and 5,480,968 as well as Kraus et al. PNAS (USA)
86:9193-9197 (1989)) and HER4 (EP Pat Appln No 599,274; Plowman et
al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman
et al., Nature, 366:473-475 (1993)). Both of these receptors
display increased expression on at least some breast cancer cell
lines.
[0010] The HER receptors are generally found in various
combinations in cells and heterodimerization is thought to increase
the diversity of cellular responses to a variety of HER ligands
(Earp et al. Breast Cancer Research and Treatment 35: 115-132
(1995)). EGFR is bound by six different ligands; epidermal growth
factor (EGF), transforming growth factor alpha (TGF-.alpha.),
amphiregulin, heparin binding epidermal growth factor (HB-EGF),
betacellulin and epiregulin (Groenen et al. Growth Factors
11:235-257 (1994)). A family of heregulin proteins resulting from
alternative splicing of a single gene are ligands for HER3 and
HER4. The heregulin family includes alpha, beta and gamma
heregulins (Holmes et al., Science, 256:1205-1210 (1992); U.S. Pat.
No. 5,641,869; and Schaefer et al. Oncogene 15:1385-1394 (1997));
neu differentiation factors (NDFs), glial growth factors (GGFs);
acetylcholine receptor inducing activity (ARIA); and sensory and
motor neuron derived factor (SMDF). For a review, see Groenen et
al. Growth Factors 11:235-257 (1994); Lemke, G. Molec. & Cell.
Neurosci. 7:247-262 (1996) and Lee et al. Pharm. Rev. 47:51-85
(1995). Three additional HER ligands have been identified;
neuregulin-2 (NRG-2) which is reported to bind either HER3 or HER4
(Chang et al. Nature 387 509-512 (1997); and Carraway et al Nature
387:512-516 (1997)); neuregulin-3 which binds HER4 (Zhang et al.
PNAS (USA) 94(18):9562-7 (1997)); and neuregulin-4 which binds HER4
(Harari et al. Oncogene 18:2681-89 (1999)) HB-EGF, betacellulin and
epiregulin also bind to HER4.
[0011] While EGF and TGF.alpha. do not bind HER2, EGF stimulates
EGFR and HER2 to form a heterodimer, which activates EGFR and
results in transphosphorylation of HER2 in the heterodimer
Dimerization and/or transphosphorylation appears to activate the
HER2 tyrosine kinase. See Earp et al., supra. Likewise, when HER3
is co-expressed with HER2, an active signaling complex is formed
and antibodies directed against HER2 are capable of disrupting this
complex (Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665
(1994)). Additionally, the affinity of HER3 for heregulin (HRG) is
increased to a higher affinity state when co-expressed with HER2.
See also, Levi et al., Journal of Neuroscience 15: 1329-1340
(1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92: 1431-1435
(1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996) with
respect to the HER2-HER3 protein complex. HER4, like HER3, forms an
active signaling complex with HER2 (Carraway and Cantley, Cell
78:5-8 (1994)).
[0012] Therapeutics that target the HER pathway are presently in
use in treating diseases such as breast cancer, non-small cell lung
cancer, colorectal cancer, head and neck cancer and pancreatic
cancer. While these therapeutics have had some success, there
remain issues related to native and induced resistance and
toxicity. Arteaga C L. J Clin Oncol 21:289-91s (2003); Hoshi S, et
al., Gan To Kagaku Ryoho 31:1209-13 (2004); Viloria-Petit A M, and
Kerbel R S. Int J Radiat Oncol Biol Phys 58:914-26 (2004); Bianco
R, et al., Endocr Relat Cancer 12:S159-71 (2005); Engelman J A, and
Janne P A., Clin Cancer Res 14:2895-9 (2008); Davoli A, et al.,
Cancer Chemother Pharmacol. 65(4):611-23 (2010); Pohlmann P R, et
al., Clin Cancer Res. 15(24):7479-7491 (2009). In particular,
therapeutics that target HER1 (EGFR) are often associated with
undesirable side effects, such as significant levels of skin
toxicity. Robert, et al. Lancet Oncology 6:491-500 (2005).
[0013] Accordingly, a need exists to develop improved therapeutics
that target the HER pathway.
SUMMARY OF THE INVENTION
[0014] The invention provides for multispecific antibodies
comprising an antigen-binding domain that specifically binds to at
least two HER receptors selected from the group consisting of (a)
EGFR and HER2, (b) EGFR and HER3, and (c) EGFR and HER4. The
antibody inhibits a biological activity of at least one of the HER
receptors. In particular embodiments, the multispecific antibody
specifically binds to its target HER receptors and does not
specifically bind to the non-target HER receptors. Accordingly, in
one embodiment, the antibody specifically binds to EGFR and HER3
but does not specifically bind to HER2 or HER4. In another
embodiment, the antibody specifically binds to EGFR and HER2 but
does not specifically bind to HER3 or HER4. In another embodiment,
the antibody specifically binds to EGFR and HER4 but does not
specifically bind to HER2 or HER3. The invention also provides for
monospecific antibodies that specifically bind to a target HER
receptor.
[0015] One aspect of the invention provides for multispecific
antibodies that are capable of specifically binding to EGFR and
another HER receptor that are less toxic than traditional EGFR
antagonists, such as cetuximab. In one embodiment, the toxicity is
dermatological toxicity. In one embodiment, the multispecific HER
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER3.
[0016] In one aspect, the invention provides a multispecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR and HER3. In one embodiment, the multispecific
antibody is less toxic than EGFR antagonists. In one embodiment,
the multispecific antibody inhibits a biological activity of at
least one of EGFR and HER3. In one embodiment, the antibody
inhibits EGF binding to EGFR. In another embodiment, the antibody
inhibits TGF-.alpha. induced EGFR phosphorylation. In some
embodiments, the antibody inhibits tumor cell growth. In one
embodiment, the multispecific antibody specifically binds to EGFR
and HER3 but does not specifically bind to HER2 or HER4.
[0017] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
with a Kd of less than 10.sup.-6M. In one embodiment the
multispecific antibody comprises an antigen-binding domain that
specifically binds EGFR with a Kd of less than 10.sup.-6M and
specifically binds HER3 with a Kd of less than 10.sup.-7 M.
[0018] In one embodiment, the multispecific antibody comprising an
antigen-binding domain that specifically binds to EGFR and HER3
comprises (a) HVR-H1 comprising the amino acid sequence of LSGDWIH
(SEQ ID NO: 48); (b) HVR-H2 comprising the amino acid sequence of
VGEISAAGGYTD (SEQ ID NO: 51); and (c) HVR-H3 comprising the amino
acid sequence of ARESRVSFEAAMDY (SEQ ID NO: 53); and (d) HVR-L1
comprising the amino acid sequence of NIATDVA (SEQ ID NO: 55); (e)
HVR-L2 comprising the amino acid sequence of SASF (SEQ ID NO: 56);
and (f) HVR-L3 comprising the amino acid sequence of SEPEPYT (SEQ
ID NO: 57).
[0019] In one embodiment, the multispecific antibody comprising an
antigen-binding domain that specifically binds to EGFR and HER3
comprises (a) a heavy chain variable domain having at least 95%
sequence identity to the amino acid sequence of SEQ ID NO: 30; (b)
a light chain variable domain having at least 95% sequence identity
to the amino acid sequence of SEQ ID NO: 29; or (c) a heavy chain
variable domain sequence as in (a) and a light chain variable
domain sequence as in (b). In one embodiment, the multispecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR and HER3 comprises a heavy chain variable domain
sequence of SEQ ID NO: 30. In one embodiment, the multispecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR and HER3 comprises a light chain variable domain
sequence of SEQ ID NO: 29. In another embodiment, the multispecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR and HER3 comprises a heavy chain variable domain
sequence of SEQ ID NO: 30 and a light chain variable domain
sequence of SEQ ID NO: 29.
[0020] In some embodiments, the multispecific antibody comprising
an antigen-binding domain that specifically binds to EGFR and HER3
is a full length IgG1 antibody.
[0021] One aspect of the invention provides for an isolated nucleic
acid encoding the multispecific HER antibodies. Another aspect
provides for a host cell comprising the nucleic acid encoding the
multispecific HER antibodies. Yet another aspect provides for a
method of producing a multispecific HER antibody comprising
culturing the host cell comprising the nucleic acid encoding the
multispecific HER antibody so that the antibody is produced.
[0022] One aspect of the invention provides for an immunoconjugate
comprising a multispecific HER antibody and a cytotoxic agent.
Another aspect provides a pharmaceutical formulation comprising a
multispecific HER antibody and a pharmaceutically acceptable
carrier.
[0023] One aspect of the invention provides for a method of
treating an individual having cancer comprising administering to
the individual an effective amount of a multispecific HER antibody.
In one embodiment, the multispecific HER antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3. In
one embodiment, the cancer treated by the multispecific HER
antibody comprises cells that express EGFR and HER3. In one
embodiment, the cancer treated by the multispecific HER antibody is
breast cancer, colorectal cancer, pancreatic cancer, head and neck
cancer, melanoma, ovarian cancer, prostate cancer, or non-small
lung cell cancer.
[0024] Another aspect of the invention provides for a method of
inhibiting a biological activity of a HER receptor in an individual
comprising administering to the individual an effective amount of a
multispecific HER antibody. In one embodiment, the multispecific
HER antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER3.
[0025] One aspect of the invention provides for a multispecific HER
antibody for use as a medicament. Another aspect provides for a
multispecific HER antibody for use in treating cancer, such as
breast cancer, colorectal cancer, pancreatic cancer, head and neck
cancer, melanoma, ovarian cancer, prostate cancer, or non-small
lung cell cancer. Another aspect provides for a multispecific HER
antibody for use in inhibiting a biological activity of a HER
receptor. In one embodiment, the multispecific HER antibody
comprises an antigen-binding domain that specifically binds to EGFR
and HER3.
[0026] Another aspect of the invention provides for a multispecific
HER antibody for use in the manufacture of a medicament. The
medicament can be used, in one embodiment, to treat cancer, such as
breast cancer, colorectal cancer, pancreatic cancer, head and neck
cancer, melanoma, ovarian cancer, prostate cancer, or non-small
lung cell cancer. In one embodiment, the medicament is for
inhibiting a biological activity of a HER receptor. In one
embodiment, the multispecific HER antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3. In
one embodiment, the multispecific HER antibody is less toxic than
traditional EGFR antagonists, such as cetuximab.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows inhibition of tumor growth by anti-EGFR
antibody D1.5 in an A431 xenograft model.
[0028] FIG. 2 shows the binding specificity of clones with dual
specificity.
[0029] FIG. 3 shows the inhibition of TGF-.alpha.-induced EGFR
phosphorylation using selected antibodies having dual specificity
(anti-EGFR/HER3 and anti-EGFR/HER2 antibodies).
[0030] FIG. 4 shows that anti-EGFR/HER3 antibodies block heregulin
binding to HER3-ECD-Fc.
[0031] FIG. 5 shows the inhibition of HRG-induced receptor
phosphorylation in MCF7 cells by anti-EGFR/HER3 bispecific
antibodies.
[0032] FIG. 6 shows the inhibition of cellular proliferation of
MDA-175 cells by anti-EGFR/HER3 antibodies.
[0033] FIG. 7 is an image showing the D1.5-100 hot spots mapped on
4D5-Fv structure.
[0034] FIG. 8 outlines a strategy for affinity maturation of
D1.5-100 EGFR-HER3 antibody using shotgun libraries.
[0035] FIG. 9 shows that two selected affinity matured antibodies
(DL7 and DL11) are specific for both EGFR and HER3.
[0036] FIG. 10: A) Shows the comparison of the inhibitory function
of affinity matured antibodies DL7 and DL11 and parental antibody
D1.5 on EGFR. B) Shows the comparison of the inhibitory function of
affinity matured antibodies DL7 and DL11, and monospecific
anti-HER3 antibody DL3.6 on HER3 transactivation.
[0037] FIG. 11A) Provides graphs showing the comparison of growth
inhibitory function of DL11 compared to pertuzumab, an anti-EGFR
antibody, and an anti-HER3 antibody, or DL3.6, D1.5, or the
combination of D1.5 plus DL3.6 on H1666 cells, a NSCLC cell line
that expresses HER2, HER3, EGFR and EGFR ligands, growth stimulated
with HRG. B) Provides graphs showing the comparison of growth
inhibitory function of DL11 compared to pertuzumab, an anti-EGFR
antibody and an anti-HER3 antibody, or DL3.6, D1.5 or the
combination of D1.5 plus DL3.6 on H1666 HSCLC line, growth
stimulated with HRG and TGF.alpha..
[0038] FIG. 12A) Provides graphs showing inhibition of the
proliferation of HCA-7 cells, a colorectal cancer cell line that
expresses HER2, HER3 and EGFR by DL11 as compared to pMab, an
anti-EGFR antibody, and anti-HER3 antibody. Cell growth was
stimulated with HRG. B) Provides graphs showing inhibition of HCA-7
cell growth as in FIG. 12A except that cell growth was stimulated
with HRG plus TGF.alpha..
[0039] FIG. 13 shows inhibition of the proliferation of Calu-3
cells, a NSCLC line that overexpresses HER2 and has normal levels
of HER3 and EGFR. Cell growth was stimulated with HRG and
antibodies were tested in a dose dependent manner.
[0040] FIG. 14 outlines a sorting strategy for the affinity
maturation of D1.5-201.
[0041] FIG. 15 shows inhibition of MDA-175 cell proliferation by
DL11, DL11b and DL11f.
[0042] FIG. 16 shows that DL11f inhibits heregulin-induced HER3
phosphorylation and TGF.alpha.-induced EGFR phosphorylation.
[0043] FIG. 17 is a graph showing inhibition of tumor growth in an
HCA-7 tumor transplant model by DL11 and DL11f.
[0044] FIG. 18 is a graph showing inhibition of tumor growth in an
H358 NSCLC xenograft model by DL11f.
[0045] FIG. 19 shows inhibition of HRG induced HER3 phosphorylation
by DL3-11b, DL3.6b and DL3.6.
[0046] FIG. 20: Inhibition of MDA-175 cell proliferation by
DL3-11b, DL3.6b and DL3.6
[0047] FIG. 21 is a graph showing inhibition of tumor growth in
FaDu cancer model by DL11f.
[0048] FIG. 22 is a graph showing inhibition of tumor growth in
BxPC3 pancreatic cancer model by DL11f.
[0049] FIG. 23 is a graph showing inhibition of tumor growth in
Calu-3 non-small cell lung cancer model by DL11f.
[0050] FIG. 24 is a graph showing inhibition of tumor growth in
A431 epidermal cancer model by DL11f.
[0051] FIG. 25 is a graph showing inhibition of tumor growth in
MAXF44 breast cancer model by DL11f.
[0052] FIG. 26 is a graph showing inhibition of tumor growth in
DU145 prostate cancer model by DL11f.
[0053] FIG. 27 is a graph showing inhibition of tumor growth in
OVXF550 ovarian cancer model by DL11f.
[0054] FIG. 28 shows that DL11f induces ADCC in several cell
lines.
[0055] FIG. 29 shows that DL11f-N297A lacks ADCC activity.
[0056] FIG. 30 shows inhibition of tumor growth in H292 NSCLC
cancer model by DL11f and DL11f-N297A.
[0057] FIG. 31A-C is a table providing information on observed
non-clinical and clinical toxicities for EGFR antagonist
therapies.
[0058] FIG. 32 is a table providing information on grading for
rash/desquamation and acne/acneform rash.
[0059] FIG. 33 provides an amino acid alignment of the heavy chain
variable domain and light chain variable domain of several anti-HER
antibodies with Kabat numbering. FIG. 33A shows the light chain
variable domain of the following antibodies: D1 (SEQ ID NO: 58);
D1.5 (SEQ ID NO: 24); D1.5-100 (SEQ ID NO: 40); DL11 (SEQ ID NO:
27); DL11b (SEQ ID NO: 29); DL11f (SEQ ID NO: 29). FIG. 33B shows
the heavy chain variable domain of the following antibodies: D1
(SEQ ID NO: 25); D1.5 (SEQ ID NO: 25); D1.5-100 (SEQ ID NO: 25);
DL11 (SEQ ID NO: 28); DL11b (SEQ ID NO: 28); DL11f (SEQ ID NO:
30).
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0060] Unless otherwise defined, all terms of art, notations and
other scientific terminology used herein are intended to have the
meanings commonly understood by those of skill in the art to which
this invention pertains. In some cases, terms with commonly
understood meanings are defined herein for clarity and/or for ready
reference, and the inclusion of such definitions herein should not
necessarily be construed to represent a substantial difference over
what is generally understood in the art. The techniques and
procedures described or referenced herein are generally well
understood and commonly employed using conventional methodology by
those skilled in the art, such as, for example, the widely utilized
molecular cloning methodologies described in Sambrook et al.,
Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As
appropriate, procedures involving the use of commercially available
kits and reagents are generally carried out in accordance with
manufacturer defined protocols and/or parameters unless otherwise
noted.
[0061] Before the present methods, kits and uses therefore are
described, it is to be understood that this invention is not
limited to the particular methodology, protocols, cell lines,
animal species or genera, constructs, and reagents described as
such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention which will be limited only by the appended
claims.
[0062] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise.
[0063] Throughout this specification and claims, the word
"comprise," or variations such as "comprises" or "comprising," will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers.
[0064] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
multispecific antibodies, and antibody fragments so long as they
exhibit the desired biological activity. The term "multispecific
antibody" is used in the broadest sense and specifically covers an
antibody comprising an antigen-binding domain that has polyepitopic
specificity (i.e., is capable of specifically binding to two, or
more, different epitopes on one biological molecule or is capable
of specifically binding to epitopes on two, or more, different
biological molecules). One specific example of an antigen-binding
domain is a V.sub.HV.sub.L unit comprised of a heavy chain variable
domain (V.sub.H) and a light chain variable domain (V.sub.L). Such
multispecific antibodies include, but are not limited to, full
length antibodies, antibodies having two or more V.sub.L and
V.sub.H domains, antibody fragments such as Fab, Fv, dsFv, scFv,
diabodies, bispecific diabodies and triabodies, antibody fragments
that have been linked covalently or non-covalently. A "bispecific
antibody" is a multispecific antibody comprising an antigen-binding
domain that is capable of specifically binding to two different
epitopes on one biological molecule or is capable of specifically
binding to epitopes on two different biological molecules. The
bispecific antibody is also referred to herein as having "dual
specificity" or as being "dual specific".
[0065] In certain embodiments, an antibody of the invention has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g. from 10.sup.-8M to
10.sup.-13M, e.g., from 10.sup.-9M to 10.sup.-13 M) for its target
HER or HERs.
[0066] The basic 4-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains (an IgM antibody consists of 5 of the
basic heterotetramer units along with an additional polypeptide
called J chain, and therefore contains 10 antigen-binding sites,
while secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain). In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to an H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has, at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has, at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain (C.sub.L)
at its other end. The V.sub.L is aligned with the V.sub.H and the
C.sub.L is aligned with the first constant domain of the heavy
chain (C.sub.H1). Particular amino acid residues are believed to
form an interface between the light chain and heavy chain variable
domains. The pairing of a V.sub.H and V.sub.L together forms a
single antigen-binding site. For the structure and properties of
the different classes of antibodies, see, e.g., Basic and Clinical
Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and
Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn.,
1994, page 71 and Chapter 6.
[0067] The L chain from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa and lambda, based
on the amino acid sequences of their constant domains. Depending on
the amino acid sequence of the constant domain of their heavy
chains (C.sub.H), immunoglobulins can be assigned to different
classes or isotypes. There are five classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, having heavy chains designated
.alpha., .delta., .gamma., .epsilon. and .mu., respectively. The
.gamma. and .alpha. classes are further divided into subclasses on
the basis of relatively minor differences in C.sub.H sequence and
function, e.g., humans express the following subclasses: IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2.
[0068] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen-binding and defines
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
110-amino acid span of the variable domains. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called hypervariable regions" or HVR. The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a beta-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the beta-sheet structure. The hypervariable
regions in each chain are held together in close proximity by the
FRs and, with the hypervariable regions from the other chain,
contribute to the formation of the antigen-binding site of
antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in antibody dependent cellular cytotoxicity (ADCC).
[0069] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six HVRs; three in the VH
(HVR-H1, HVR-H2, HVR-H3), and three in the VL (HVR-L1, HVR-L2,
HVR-L3). In native antibodies, H3 and L3 display the most diversity
of the six HVRs, and H3 in particular is believed to play a unique
role in conferring fine specificity to antibodies. See, e.g., Xu et
al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in
Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J.,
2003). Indeed, naturally occurring camelid antibodies consisting of
a heavy chain only are functional and stable in the absence of
light chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448
(1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0070] HVRs generally comprise amino acid residues from the
hypervariable loops and/or from the "complementarity determining
regions" (CDRs), the latter being of highest sequence variability
and/or involved in antigen recognition. A number of HVR
delineations are in use and are encompassed herein. The Kabat
Complementarity Determining Regions (CDRs) are based on sequence
variability and are the most commonly used (Kabat et al., Sequences
of Proteins of Immunological Interest, 5th Ed. Public Health
Service, National Institutes of Health, Bethesda, Md. (1991)).
Chothia refers instead to the location of the structural loops
(Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs
represent a compromise between the Kabat HVRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact" HVRs are based on an analysis of
the available complex crystal structures. The residues from each of
these HVRs are noted below.
TABLE-US-00001 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0071] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and
26-35 (H1), 50-65 or 47-65 (H2) and 93-102, 94-102, or 95-102 (H3)
in the VH. The variable domain residues are numbered according to
Kabat et al., supra, for each of these definitions.
[0072] "Framework" or "FR" residues are those variable domain
residues other than the HVR residues as herein defined.
[0073] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat," and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., supra. Using this numbering system,
the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or HVR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0074] The Kabat numbering system is generally used when referring
to a residue in the variable domain (approximately residues 1-107
of the light chain and residues 1-113 of the heavy chain) (e.g.,
Kabat et al., Sequences of Immunological Interest. 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The "EU numbering system" or "EU index" is generally used
when referring to a residue in an immunoglobulin heavy chain
constant region (e.g., the EU index reported in Kabat et al.,
supra). The "EU index as in Kabat" refers to the residue numbering
of the human IgG1 EU antibody. Unless stated otherwise herein,
references to residue numbers in the variable domain of antibodies
means residue numbering by the Kabat numbering system. Unless
stated otherwise herein, references to residue numbers in the
constant domain of antibodies means residue numbering by the EU
numbering system (e.g., see WO 2006/073941).
[0075] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein.
[0076] An "affinity matured" antibody is one with one or more
alterations in one or more HVRs or framework region thereof which
result in an improvement in the affinity of the antibody for
antigen, compared to a parent antibody which does not possess those
alteration(s). In one embodiment, an affinity matured antibody has
nanomolar or even picomolar affinities for the target antigen.
Affinity matured antibodies may be produced using certain
procedures known in the art. For example, Marks et al.
Bio/Technology 10:779-783 (1992) describes affinity maturation by
VH and VL domain shuffling. Random mutagenesis of HVR and/or
framework residues is described by, for example, Barbas et al. Proc
Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene
169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004
(1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and
Hawkins et al, J. Mol. Biol. 226:889-896 (1992).
[0077] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0078] The term "monoclonal antibody" as used herein refers to an
antibody from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
substantially similar and bind the same epitope(s), except for
possible variants that may arise during production of the
monoclonal antibody, such variants generally being present in minor
amounts. Such monoclonal antibody typically includes an antibody
comprising a variable region that binds a target, wherein the
antibody was obtained by a process that includes the selection of
the antibody from a plurality of antibodies. For example, the
selection process can be the selection of a unique clone from a
plurality of clones, such as a pool of hybridoma clones, phage
clones or recombinant DNA clones. It should be understood that the
selected antibody can be further altered, for example, to improve
affinity for the target, to humanize the antibody, to improve its
production in cell culture, to reduce its immunogenicity in vivo,
to create a multispecific antibody, etc., and that an antibody
comprising the altered variable region sequence is also a
monoclonal antibody of this invention. In addition to their
specificity, the monoclonal antibody preparations are advantageous
in that they are typically uncontaminated by other immunoglobulins.
The modifier "monoclonal" indicates the character of the antibody
as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of
the antibody by any particular method. For example, the monoclonal
antibodies to be used in accordance with the present invention may
be made by a variety of techniques, including the hybridoma method
(e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies
and T-Cell Hybridomas 563-681, (Elsevier, N.Y., 1981), recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display
technologies (see, e.g., Clackson et al., Nature, 352:624-628
(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Sidhu et
al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol.
340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA
101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods
284(1-2):119-132 (2004) and technologies for producing human or
human-like antibodies from animals that have parts or all of the
human immunoglobulin loci or genes encoding human immunoglobulin
sequences (see, e.g., WO98/24893, WO/9634096, WO/9633735, and WO/91
10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551
(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann
et al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806,
5,569,825, 5,591,669 (all of GenPharm); 5,545,807; WO 97/17852,
U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; and 5,661,016, and Marks et al., Bio/Technology, 10:
779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994);
Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature
Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology,
14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13:
65-93 (1995).
[0079] An "intact" antibody is one which comprises an
antigen-binding site as well as a C.sub.L and at least heavy chain
constant domains, C.sub.H1, C.sub.H2, and C.sub.H3. The constant
domains can be native sequence constant domains (e.g., human native
sequence constant domains) or amino acid sequence variant thereof.
Preferably, the intact antibody has one or more effector
functions.
[0080] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen-binding or variable region of the
intact antibody. Examples of antibody fragments include Fv, Fab,
Fab', F(ab').sub.2, Fab'-SH; diabodies; linear antibodies (see U.S.
Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):
1057-1062 (1995)); single-chain antibody molecules (e.g. scFv).
While in the present description, and throughout the specification,
reference is made to antibodies and various properties of
antibodies, the same disclosure also applies to functional antibody
fragments, e.g. dual action Fab fragments.
[0081] The expression "linear antibodies" generally refers to the
antibodies described in Zapata et al., Protein Eng.,
8(10):1057-1062 (1995). These antibodies comprise a pair of tandem
Fd segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which, together
with complementary light chain polypeptides, form a pair of
antigen-binding regions. In a preferred embodiment, the fragment is
"functional," i.e. qualitatively retains the ability of the
corresponding intact antibody to bind to the target HER receptor
and, if the intact antibody also inhibits HER activation or
function, qualitatively retains such inhibitory property as well.
Qualitative retention means that the activity in kind is retained,
but the degree of binding affinity and/or activity might
differ.
[0082] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (V.sub.H), and the first
constant domain of one heavy chain (C.sub.H1). Pepsin treatment of
an antibody yields a single large F(ab').sub.2 fragment which
roughly corresponds to two disulfide linked Fab fragments having
divalent antigen-binding activity and is still capable of
cross-linking antigen. Fab' fragments differ from Fab fragments by
having additional few residues at the carboxy terminus of the
C.sub.H1 domain including one or more cysteines from the antibody
hinge region. Fab'-SH is the designation herein for Fab' in which
the cysteine residue(s) of the constant domains bear a free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments which have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
[0083] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region; this
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[0084] "Fv" consists of a dimer of one heavy- and one light-chain
variable region domain in tight, non-covalent association. From the
folding of these two domains emanate six hypervariable loops (3
loops each from the H and L chain) that contribute the amino acid
residues for antigen-binding and confer antigen-binding specificity
to the antibody. However, even a single variable domain (or half of
an Fv comprising only three HVRs specific for an antigen) has the
ability to recognize and bind antigen, although often at a lower
affinity than the entire binding site.
[0085] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen-binding. For a review of sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994); Borrebaeck 1995.
[0086] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10 residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, resulting in a bivalent fragment,
i.e., fragment having two antigen-binding sites. Diabodies are
described more fully in, for example, EP 404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448
(1993).
[0087] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more.
[0088] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0089] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0090] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0091] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from the
non-human antibody. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
antibody specificity, affinity, and capability. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies can comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0092] An antibody of this invention "which binds" an antigen of
interest is one that binds the antigen with sufficient affinity
such that the antibody is useful as a diagnostic and/or therapeutic
agent in targeting a protein or a cell or tissue expressing the
antigen. With regard to the binding of a antibody to a target
molecule, the term "specific binding" or "specifically binds to" or
is "specific for" a particular polypeptide or an epitope on a
particular polypeptide target means binding that is measurably
different from a non-specific interaction. Specific binding can be
measured, for example, by determining binding of a molecule
compared to binding of a control molecule. For example, specific
binding can be determined by competition with a control molecule
that is similar to the target, for example, an excess of
non-labeled target. In this case, specific binding is indicated if
the binding of the labeled target to a probe is competitively
inhibited by excess non-labeled target. In one particular
embodiment, "specifically binds" refers to binding of an antibody
to its specified target HER receptors and not other specified
non-target HER receptors. For example, the antibody specifically
binds to EGFR and HER3 but does not specifically bind to HER2 or
HER4, or the antibody specifically binds to EGFR and HER2 but does
not specifically bind to HER3 or HER4, or the antibody specifically
binds to EGFR and HER4 but does not specifically bind to HER2 or
HER3.
[0093] A "HER receptor" is a receptor protein tyrosine kinase which
belongs to the HER receptor family and includes EGFR (ErbB1, HER1),
HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) receptors. The HER
receptor will generally comprise an extracellular domain, which may
bind an HER ligand and/or dimerize with another HER receptor
molecule; a lipophilic transmembrane domain; a conserved
intracellular tyrosine kinase domain; and a carboxyl-terminal
signaling domain harboring several tyrosine residues which can be
phosphorylated. The HER receptor may be a "native sequence" HER
receptor or an "amino acid sequence variant" thereof. Preferably
the HER receptor is a native sequence human HER receptor.
[0094] The "HER pathway" refers to the signaling network mediated
by the HER receptor family.
[0095] The terms "ErbB1", "HER1", "epidermal growth factor
receptor" and "EGFR" are used interchangeably herein and refer to
EGFR as disclosed, for example, in Carpenter et al. Ann. Rev.
Biochem. 56:881-914 (1987), including naturally occurring mutant
forms thereof (e.g. a deletion mutant EGFR as in Ullrich et al,
Nature (1984) 309:418425 and Humphrey et al. PNAS (USA)
87:4207-4211 (1990)), as well we variants thereof, such as
EGFRvIII. Variants of EGFR also include deletional, substitutional
and insertional variants, for example those described in Lynch et
al (New England Journal of Medicine 2004, 350:2129), Paez et al
(Science 2004, 304:1497), and Pao et al (PNAS 2004, 101:13306).
[0096] Herein, "EGFR extracellular domain" or "EGFR ECD" refers to
a domain of EGFR that is outside of a cell, either anchored to a
cell membrane, or in circulation, including fragments thereof. In
one embodiment, the extracellular domain of EGFR may comprise four
domains: "Domain I" (amino acid residues from about 1-158, "Domain
II" (amino acid residues 159-336), "Domain III" (amino acid
residues 337-470), and "Domain IV" (amino acid residues 471-645),
where the boundaries are approximate, and may vary by about 1-3
amino acids.
[0097] The expressions "ErbB2" and "HER2" are used interchangeably
herein and refer to human HER2 protein described, for example, in
Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al.
Nature 319:230-234 (1986) (GenBank accession number X03363). The
term "erbB2" refers to the gene encoding human HER2 and "neu"
refers to the gene encoding rat p185''. Preferred HER2 is native
sequence human HER2.
[0098] Herein, "HER2 extracellular domain" or "HER2ECD" refers to a
domain of HER2 that is outside of a cell, either anchored to a cell
membrane, or in circulation, including fragments thereof. In one
embodiment, the extracellular domain of HER2 may comprise four
domains: "Domain I" (amino acid residues from about 1-195, "Domain
II" (amino acid residues from about 196-319), "Domain III" (amino
acid residues from about 320-488), and "Domain IV" (amino acid
residues from about 489-630) (residue numbering without signal
peptide). See Garrett et al. Mol. Cell. 11: 495-505 (2003), Cho et
al. Nature 421: 756-760 (2003), Franklin et al. Cancer Cell
5:317-328 (2004), and Plowman et al. Proc. Natl. Acad. Sci.
90:1746-1750 (1993).
[0099] "ErbB3" and "HER3" refer to the receptor polypeptide as
disclosed, for example, in U.S. Pat. Nos. 5,183,884 and 5,480,968
as well as Kraus et al. PNAS (USA) 86:9193-9197 (1989).
[0100] Herein, "HER3 extracellular domain" or "HER3ECD" refers to a
domain of HER3 that is outside of a cell, either anchored to a cell
membrane, or in circulation, including fragments thereof. In one
embodiment, the extracellular domain of HER3 may comprise four
domains: Domain I, Domain II, Domain III, and Domain IV. In one
embodiment, the HER3 ECD comprises amino acids 1-636 (numbering
including signal peptide). In one embodiment, HER3 domain III
comprises amino acids 328-532 (numbering including signal
peptide.
[0101] The terms "ErbB4" and "HER4" herein refer to the receptor
polypeptide as disclosed, for example, in EP Pat Appin No 599,274;
Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993);
and Plowman et al., Nature, 366:473-475 (1993), including isoforms
thereof, e.g., as disclosed in WO99/19488, published Apr. 22,
1999.
[0102] By "HER ligand" is meant a polypeptide which binds to and/or
activates a HER receptor. The HER ligand of particular interest
herein is a native sequence human HER ligand such as epidermal
growth factor (EGF) (Savage et al., J. Biol. Chem. 247:7612-7621
(1972)); transforming growth factor alpha (TGF-.alpha.) (Marquardt
et al., Science 223:1079-1082 (1984)); amphiregulin also known as
schwanoma or keratinocyte autocrine growth factor (Shoyab et al.
Science 243:1074-1076 (1989); Kimura et al. Nature 348:257-260
(1990); and Cook et al. Mol. Cell. Biol. 11:2547-2557 (1991));
betacellulin (Shing et al., Science 259:1604-1607 (1993); and
Sasada et al. Biochem. Biophys. Res. Commun. 190:1173 (1993));
heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et
al., Science 251:936-939 (1991)); epiregulin (Toyoda et al., J.
Biol. Chem. 270:7495-7500 (1995); and Komurasaki et al. Oncogene
15:2841-2848 (1997)); a heregulin (see below); neuregulin-2 (NRG-2)
(Carraway et al., Nature 387:512-516 (1997)); neuregulin-3 (NRG-3)
(Zhang et al., Proc. Natl. Acad. Sci. 94:9562-9567 (1997));
neuregulin-4 (NRG-4) (Harari et al. Oncogene 18:2681-89 (1999));
and cripto (CR-1) (Kannan et al. J. Biol. Chem. 272(6):3330-3335
(1997)). HER ligands which bind EGFR include EGF, TGF-.alpha.,
amphiregulin, betacellulin, HB-EGF and epiregulin. HER ligands
which bind HER3 include heregulins and NRG-2.
[0103] HER ligands capable of binding HER4 include betacellulin,
epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4, and heregulins.
[0104] "Heregulin" (HRG) when used herein refers to a polypeptide
encoded by the heregulin gene product as disclosed in U.S. Pat. No.
5,641,869, or Marchionni et al., Nature, 362:312-318 (1993).
Examples of heregulins include heregulin-.alpha.,
heregulin-.beta.1, heregulin-.beta.2 and heregulin-.beta.3 (Holmes
et al., Science, 256:1205-1210 (1992); and U.S. Pat. No.
5,641,869); neu differentiation factor (NDF) (Peles et al. Cell 69:
205-216 (1992)); acetylcholine receptor-inducing activity (ARIA)
(Falls et al. Cell 72:801-815 (1993)); glial growth factors (GGFs)
(Marchionni et al., Nature, 362:312-318 (1993)); sensory and motor
neuron derived factor (SMDF) (Ho et al. J. Biol. Chem.
270:14523-14532 (1995)); .gamma.-heregulin (Schaefer et al.
Oncogene 15:1385-1394 (1997)).
[0105] A "HER dimer" herein is a noncovalently associated dimer
comprising at least two HER receptors. Such complexes may form when
a cell expressing two or more HER receptors is exposed to an HER
ligand and can be isolated by immunoprecipitation and analyzed by
SDS-PAGE as described in Sliwkowski et al., J. Biol. Chem.,
269(20):14661-14665 (1994), for example. Other proteins, such as a
cytokine receptor subunit (e.g. gp130) may be associated with the
dimer.
[0106] A "HER heterodimer" herein is a noncovalently associated
heterodimer comprising at least two different HER receptors, such
as EGFR-HER2, EGFR-HER3, EGFR-HER4, HER2-HER3 or HER2-HER4
heterodimers.
[0107] A "HER inhibitor" is an agent which interferes with HER
activation or function. Examples of HER inhibitors include HER
antibodies (e.g. EGFR, HER2, HER3, or HER4 antibodies);
EGFR-targeted drugs; small molecule HER antagonists; HER tyrosine
kinase inhibitors; HER2 and EGFR dual tyrosine kinase inhibitors
such as lapatinib/GW572016; antisense molecules (see, for example,
WO2004/87207); and/or agents that bind to, or interfere with
function of, downstream signaling molecules, such as MAPK or Akt.
Preferably, the HER inhibitor is an antibody which binds to a HER
receptor.
[0108] A "HER dimerization inhibitor" or "HDI" is an agent which
inhibits formation of a HER homodimer or HER heterodimer.
Preferably, the HER dimerization inhibitor is an antibody. However,
HER dimerization inhibitors also include peptide and non-peptide
small molecules, and other chemical entities which inhibit the
formation of HER homo- or heterodimers.
[0109] An antibody which "inhibits HER dimerization" is an antibody
which inhibits, or interferes with, formation of a HER dimer,
regardless of the underlying mechanism. In one embodiment, such an
antibody binds to HER2 at the heterodimeric binding site thereof.
One particular example of a dimerization inhibiting antibody is
pertuzumab (Pmab), or MAb 2C4. Other examples of HER dimerization
inhibitors include antibodies which bind to EGFR and inhibit
dimerization thereof with one or more other HER receptors (for
example EGFR monoclonal antibody 806, MAb 806, which binds to
activated or "untethered" EGFR; see Johns et al., J. Biol. Chem.
279(29):30375-30384 (2004)); antibodies which bind to HER3 and
inhibit dimerization thereof with one or more other HER receptors;
antibodies which bind to HER4 and inhibit dimerization thereof with
one or more other HER receptors; peptide dimerization inhibitors
(U.S. Pat. No. 6,417,168); antisense dimerization inhibitors;
etc.
[0110] As used herein, "EGFR antagonist" or "EGFR inhibitor" refer
to those compounds that specifically bind to EGFR and prevent or
reduce its signaling activity, and do not specifically bind to
HER2, HER3, or HER4. Examples of such agents include antibodies and
small molecules that bind to EGFR. Examples of antibodies which
bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL
HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see,
U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof,
such as chimerized 225 (C225 or Cetuximab; ERBITUX.RTM.) and
reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.);
IMC-11F8, a fully human, EGFR-targeted antibody (Imclone);
antibodies that bind type II mutant EGFR (U.S. Pat. No. 5,212,290);
humanized and chimeric antibodies that bind EGFR as described in
U.S. Pat. No. 5,891,996; and human antibodies that bind EGFR, such
as ABX-EGF or Panitumumab (see WO98/50433, Abgenix/Amgen); EMD
55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996));
EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR
that competes with both EGF and TGF-alpha for EGFR binding
(EMD/Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human
antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6. 3 and
E7.6. 3 and described in U.S. Pat. No. 6,235,883; MDX-447 (Medarex
Inc); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol.
Chem. 279(29):30375-30384 (2004)). The anti-EGFR antibody may be
conjugated with a cytotoxic agent, thus generating an
immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH). EGFR
antagonists include small molecules such as compounds described in
U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307,
5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726,
6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459,
6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and
5,747,498, as well as the following PCT publications: WO98/14451,
WO98/50038, WO99/09016, and WO99/24037. Particular small molecule
EGFR antagonists include OSI-774 (CP-358774, erlotinib,
TARCEVA.RTM. Genentech/OSI Pharmaceuticals); PD 183805 (CI 1033,
2-propenamide,
N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quin-
azolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib
(IRESSA.RTM.)
4-(3'-Chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoli-
ne, AstraZeneca); ZM 105180
((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382
(N-8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-
-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166
((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol)-
;
(R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimi-
dine); CL-387785
(N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569
(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(-
dimethylamino)-2-butenamide) (Wyeth); AG1478 (Sugen); and AG1571
(SU 5271; Sugen).
[0111] A "HER antibody" is an antibody that binds to a HER
receptor. Optionally, the HER antibody further interferes with HER
activation or function. Particular HER2 antibodies include
pertuzumab and trastuzumab. Examples of particular EGFR antibodies
include cetuximab and panitumumab.
[0112] Patent publications related to HER antibodies include: U.S.
Pat. No. 5,677,171, U.S. Pat. No. 5,720,937, U.S. Pat. No.
5,720,954, U.S. Pat. No. 5,725,856, U.S. Pat. No. 5,770,195, U.S.
Pat. No. 5,772,997, U.S. Pat. No. 6,165,464, U.S. Pat. No.
6,387,371, U.S. Pat. No. 6,399,063, US2002/0192211A1, U.S. Pat. No.
6,015,567, U.S. Pat. No. 6,333,169, U.S. Pat. No. 4,968,603, U.S.
Pat. No. 5,821,337, U.S. Pat. No. 6,054,297, U.S. Pat. No.
6,407,213, U.S. Pat. No. 6,719,971, U.S. Pat. No. 6,800,738,
US2004/0236078A1, U.S. Pat. No. 5,648,237, U.S. Pat. No. 6,267,958,
U.S. Pat. No. 6,685,940, U.S. Pat. No. 6,821,515, WO98/17797, U.S.
Pat. No. 6,333,398, U.S. Pat. No. 6,797,814, U.S. Pat. No.
6,339,142, U.S. Pat. No. 6,417,335, U.S. Pat. No. 6,489,447,
WO99/31140, US2003/0147884A1, US2003/0170234A1, US2005/0002928A1,
U.S. Pat. No. 6,573,043, US2003/0152987A1, WO99/48527,
US2002/0141993A1, WO01/00245, US2003/0086924, US2004/0013667A1,
WO00/69460, WO01/00238, WO01/15730, U.S. Pat. No. 6,627,196B1, U.S.
Pat. No. 6,632,979B1, WO01/00244, US2002/0090662A1, WO01/89566,
US2002/0064785, US2003/0134344, WO 04/24866, US2004/0082047,
US2003/0175845A1, WO03/087131, US2003/0228663, WO2004/008099A2,
US2004/0106161, WO2004/048525, US2004/0258685A1, U.S. Pat. No.
5,985,553, U.S. Pat. No. 5,747,261, U.S. Pat. No. 4,935,341, U.S.
Pat. No. 5,401,638, U.S. Pat. No. 5,604,107, WO 87/07646, WO
89/10412, WO 91/05264, EP 412,116 B1, EP 494,135 B1, U.S. Pat. No.
5,824,311, EP 444,181 B1, EP 1,006,194 A2, US 2002/0155527A1, WO
91/02062, U.S. Pat. No. 5,571,894, U.S. Pat. No. 5,939,531, EP
502,812 B1, WO 93/03741, EP 554,441 B1, EP 656,367 A1, U.S. Pat.
No. 5,288,477, U.S. Pat. No. 5,514,554, U.S. Pat. No. 5,587,458, WO
93/12220, WO 93/16185, U.S. Pat. No. 5,877,305, WO 93/21319, WO
93/21232, U.S. Pat. No. 5,856,089, WO 94/22478, U.S. Pat. No.
5,910,486, U.S. Pat. No. 6,028,059, WO 96/07321, U.S. Pat. No.
5,804,396, U.S. Pat. No. 5,846,749, EP 711,565, WO 96/16673, U.S.
Pat. No. 5,783,404, U.S. Pat. No. 5,977,322, U.S. Pat. No.
6,512,097, WO 97/00271, U.S. Pat. No. 6,270,765, U.S. Pat. No.
6,395,272, U.S. Pat. No. 5,837,243, WO 96/40789, U.S. Pat. No.
5,783,186, U.S. Pat. No. 6,458,356, WO 97/20858, WO 97/38731, U.S.
Pat. No. 6,214,388, U.S. Pat. No. 5,925,519, WO 98/02463, U.S. Pat.
No. 5,922,845, WO 98/18489, WO 98/33914, U.S. Pat. No. 5,994,071,
WO 98/45479, U.S. Pat. No. 6,358,682 B1, US 2003/0059790, WO
99/55367, WO 01/20033, US 2002/0076695 A1, WO 00/78347, WO
01/09187, WO 01/21192, WO 01/32155, WO 01/53354, WO 01/56604, WO
01/76630, WO02/05791, WO 02/11677, U.S. Pat. No. 6,582,919,
US2002/0192652A1, US 2003/0211530A1, WO 02/44413, US 2002/0142328,
U.S. Pat. No. 6,602,670 B2, WO 02/45653, WO 02/055106, US
2003/0152572, US 2003/0165840, WO 02/087619, WO 03/006509,
WO03/012072, WO 03/028638, US 2003/0068318, WO 03/041736, EP
1,357,132, US 2003/0202973, US 2004/0138160, U.S. Pat. No.
5,705,157, U.S. Pat. No. 6,123,939, EP 616,812 B1, US 2003/0103973,
US 2003/0108545, U.S. Pat. No. 6,403,630 B1, WO 00/61145, WO
00/61185, U.S. Pat. No. 6,333,348 B1, WO 01/05425, WO 01/64246, US
2003/0022918, US 2002/0051785 A1, U.S. Pat. No. 6,767,541, WO
01/76586, US 2003/0144252, WO 01/87336, US 2002/0031515 A1, WO
01/87334, WO 02/05791, WO 02/09754, US 2003/0157097, US
2002/0076408, WO 02/055106, WO 02/070008, WO 02/089842 and WO
03/86467.
[0113] "HER activation" refers to activation, or phosphorylation,
of any one or more HER receptors. Generally, HER activation results
in signal transduction (e.g. that caused by an intracellular kinase
domain of a HER receptor phosphorylating tyrosine residues in the
HER receptor or a substrate polypeptide). HER activation may be
mediated by HER ligand binding to a HER dimer comprising the HER
receptor of interest. HER ligand binding to a HER dimer may
activate a kinase domain of one or more of the HER receptors in the
dimer and thereby results in phosphorylation of tyrosine residues
in one or more of the HER receptors and/or phosphorylation of
tyrosine residues in additional substrate polypeptides(s), such as
Akt or MAPK intracellular kinases.
[0114] "Phosphorylation" refers to the addition of one or more
phosphate group(s) to a protein, such as a HER receptor, or
substrate thereof.
[0115] A "heterodimeric binding site" on HER2, refers to a region
in the extracellular domain of HER2 that contacts, or interfaces
with, a region in the extracellular domain of EGFR, HER3 or HER4
upon formation of a dimer therewith. The region is found in Domain
II of HER2. Franklin et al. Cancer Cell 5:317-328 (2004).
[0116] A HER2 antibody that "binds to a heterodimeric binding site"
of HER2, binds to residues in domain II (and optionally also binds
to residues in other of the domains of the HER2 extracellular
domain, such as domains I and III), and can sterically hinder, at
least to some extent, formation of a HER2-EGFR, HER2-HER3, or
HER2-HER4 heterodimer Franklin et al. Cancer Cell 5:317-328 (2004)
characterize the HER2-pertuzumab crystal structure, deposited with
the RCSB Protein Data Bank (ID Code IS78), illustrating an
exemplary antibody that binds to the heterodimeric binding site of
HER2.
[0117] An antibody that "binds to domain II" of HER2 binds to
residues in domain II and optionally residues in other domain(s) of
HER2, such as domains I and III.
[0118] "Isolated," when used to describe the various antibodies
disclosed herein, means an antibody that has been identified and
separated and/or recovered from a cell or cell culture from which
it was expressed. Contaminant components of its natural environment
are materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and can include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In
preferred embodiments, the antibody will be purified (1) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing
conditions using Coomassie blue or, preferably, silver stain.
Isolated antibody includes antibodies in situ within recombinant
cells, because at least one component of the polypeptide natural
environment will not be present. Ordinarily, however, isolated
polypeptide will be prepared by at least one purification step. In
some embodiments, the multispecific anti-HER antibody is an
isolated antibody.
[0119] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0120] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0121] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0122] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
[0123] where X is the number of amino acid residues scored as
identical matches by the sequence alignment program ALIGN-2 in that
program's alignment of A and B, and where Y is the total number of
amino acid residues in B. It will be appreciated that where the
length of amino acid sequence A is not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will
not equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0124] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. For additional details
and explanation of stringency of hybridization reactions, see
Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0125] "Stringent conditions" or "high stringency conditions", as
defined herein, can be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl
sulfate at 50 C; (2) employ during hybridization a denaturing
agent, such as formamide, for example, 50% (v/v) formamide with
0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mM sodium citrate at 42.degree. C.; or (3) overnight
hybridization in a solution that employs 50% formamide, 5.times.SSC
(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH
6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's solution,
sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran
sulfate at 42.degree. C., with a 10 minute wash at 42.degree. C. in
0.2.times.SSC (sodium chloride/sodium citrate) followed by a 10
minute high-stringency wash consisting of 0.1.times.SSC containing
EDTA at 55.degree. C.
[0126] "Moderately stringent conditions" can be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength, and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0127] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: Clq binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor); and B cell activation.
[0128] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or 5,821,337 can be
performed. Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest can be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. (Proc. Natl. Acad. Sci. USA)
95:652-656 (1998).
[0129] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain (see review M. in Daeron,
Annu. Rev. Immunol 15:203-234 (1997)). FcRs are reviewed in Ravetch
and Kinet, Annu. Rev. Immunol 9457-492 (1991); Capel et al.,
Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.
Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)).
[0130] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells, and neutrophils; with PBMCs and NK
cells being preferred. The effector cells can be isolated from a
native source, e.g., from blood.
[0131] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (Clq) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be
performed.
[0132] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of
the invention are used to delay development of a disease or to slow
the progression of a disease.
[0133] The term "therapeutically effective amount" refers to an
amount of an antibody or antibody fragment to treat a disease or
disorder in a subject. In the case of tumor (e.g., a cancerous
tumor), the therapeutically effective amount of the antibody or
antibody fragment may reduce the number of cancer cells; reduce the
primary tumor size; inhibit (i.e., slow to some extent and
preferably stop) cancer cell infiltration into peripheral organs;
inhibit (i.e., slow to some extent and preferably stop) tumor
metastasis; inhibit, to some extent, tumor growth; and/or relieve
to some extent one or more of the symptoms associated with the
disorder. To the extent the antibody or antibody fragment may
prevent growth and/or kill existing cancer cells, it may be
cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo
can, for example, be measured by assessing the duration of
survival, time to disease progression (TTP), the response rates
(RR), duration of response, and/or quality of life.
[0134] By "reduce or inhibit" is meant the ability to cause an
overall decrease preferably of 20% or greater, more preferably of
50% or greater, and most preferably of 75%, 85%, 90%, 95%, or
greater. Reduce or inhibit can refer to the symptoms of the
disorder being treated, the presence or size of metastases, the
size of the primary tumor, or the size or number of the blood
vessels in angiogenic disorders.
[0135] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Included in this definition are benign
and malignant cancers. By "early stage cancer" is meant a cancer
that is not invasive or metastatic or is classified as a Stage 0,
I, or II cancer.
[0136] The term "precancerous" refers to a condition or a growth
that typically precedes or develops into a cancer.
[0137] By "non-metastatic" is meant a cancer that is benign or that
remains at the primary site and has not penetrated into the
lymphatic or blood vessel system or to tissues other than the
primary site. Generally, a non-metastatic cancer is any cancer that
is a Stage 0, I, or II cancer, and occasionally a Stage III
cancer.
[0138] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject., A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0139] The term "anti-cancer therapy" refers to a therapy useful in
treating cancer. Examples of anti-cancer therapeutic agents
include, but are limited to, e.g., chemotherapeutic agents, growth
inhibitory agents, cytotoxic agents, agents used in radiation
therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin
agents, and other agents to treat cancer, anti-CD20 antibodies,
platelet derived growth factor inhibitors (e.g., Gleevec.TM.
(Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib),
interferons, cytokines, antagonists (e.g., neutralizing antibodies)
that bind to one or more of the following targets EGFR, ErbB2,
ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF receptor(s),
TRAIL/Apo2, and other bioactive and organic chemical agents, etc.
Combinations thereof are also included in the invention.
[0140] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.RTM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphoramide and trimethylomelamine; acetogenins
(especially bullatacin and bullatacinone);
delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.);
beta-lapachone; lapachol; colchicines; betulinic acid; a
camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
chlorophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosoureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gammall and calicheamicin omegall (see, e.g., Nicolaou et al.,
Angew. Chem. Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral
alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antibiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including ADRIAMYCIN.RTM., morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin
HCl liposome injection (DOXIL.RTM.), liposomal doxorubicin TLC D-99
(MYOCET.RTM.), peglylated liposomal doxorubicin (CAELYX.RTM.), and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate, gemcitabine (GEMZAR.RTM.), tegafur (UFTORAL.RTM.),
capecitabine (XELODA.RTM.), an epothilone, and 5-fluorouracil
(5-FU); folic acid analogues such as denopterin, methotrexate,
pteropterin, trimetrexate; purine analogs such as fludarabine,
6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such
as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,
dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens
such as calusterone, dromostanolone propionate, epitiostanol,
mepitiostane, testolactone; anti-adrenals such as
aminoglutethimide, mitotane, trilostane; folic acid replenisher
such as frolinic acid; aceglatone; aldophosphamide glycoside;
aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elformithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM.
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine (ELDISINE.RTM., FILDESIN.RTM.); dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); thiotepa; taxoid, e.g., paclitaxel
(TAXOL.RTM.), albumin-engineered nanoparticle formulation of
paclitaxel (ABRAXANE.TM.), and docetaxel (TAXOTERE.RTM.);
chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum
agents such as cisplatin, oxaliplatin (e.g., ELOXATIN.RTM.), and
carboplatin; vincas, which prevent tubulin polymerization from
forming microtubules, including vinblastine (VELBAN.RTM.),
vincristine (ONCOVIN.RTM.), vindesine (ELDISINE.RTM.,
FILDESIN.RTM.), and vinorelbine (NAVELBINE.RTM.); etoposide
(VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid, including bexarotene (TARGRETIN.RTM.);
bisphosphonates such as clodronate (for example, BONEFOS.RTM. or
OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095, zoledronic
acid/zoledronate (ZOMETA.RTM.), alendronate (FOSAMAX.RTM.),
pamidronate (AREDIA.RTM.), tiludronate (SKELID.RTM.), or
risedronate (ACTONEL.RTM.); troxacitabine (a 1,3-dioxolane
nucleoside cytosine analog); antisense oligonucleotides,
particularly those that inhibit expression of genes in signaling
pathways implicated in aberrant cell proliferation, such as, for
example, PKC-alpha, Raf, H-Ras, and epidermal growth factor
receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine and gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; topoisomerase 1
inhibitor (e.g., LURTOTECAN.RTM.); rmRH (e.g., ABARELIX.RTM.);
BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT.RTM.,
Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or
etoricoxib), proteosome inhibitor (e.g. PS341); bortezomib
(VELCADE.RTM.); CCl-779; tipifarnib (R11577); orafenib, ABT510;
Bc1-2 inhibitor such as oblimersen sodium (GENASENSE.RTM.);
pixantrone; EGFR inhibitors (see definition below); tyrosine kinase
inhibitors (see definition below); serine-threonine kinase
inhibitors such as rapamycin (sirolimus, RAPAMUNE.RTM.);
farnesyltransferase inhibitors such as lonafarnib (SCH 6636,
SARASAR.TM.); and pharmaceutically acceptable salts, acids or
derivatives of any of the above; as well as combinations of two or
more of the above such as CHOP, an abbreviation for a combined
therapy of cyclophosphamide, doxorubicin, vincristine, and
prednisolone; and FOLFOX, an abbreviation for a treatment regimen
with oxaliplatin (ELOXATIN.TM.) combined with 5-FU and
leucovorin.
[0141] Chemotherapeutic agents as defined herein include
"anti-hormonal agents" or "endocrine therapeutics" which act to
regulate, reduce, block, or inhibit the effects of hormones that
can promote the growth of cancer. They may be hormones themselves,
including, but not limited to: anti-estrogens with mixed
agonist/antagonist profile, including, tamoxifen (NOLVADEX.RTM.),
4-hydroxytamoxifen, toremifene (FARESTON.RTM.), idoxifene,
droloxifene, raloxifene (EVISTA.RTM.), trioxifene, keoxifene, and
selective estrogen receptor modulators (SERMs) such as SERM3; pure
anti-estrogens without agonist properties, such as fulvestrant
(FASLODEX.RTM.), and EM800 (such agents may block estrogen receptor
(ER) dimerization, inhibit DNA binding, increase ER turnover,
and/or suppress ER levels); aromatase inhibitors, including
steroidal aromatase inhibitors such as formestane and exemestane
(AROMASIN.RTM.), and nonsteroidal aromatase inhibitors such as
anastrazole (ARIMIDEX.RTM.), letrozole (FEMARA.RTM.) and
aminoglutethimide, and other aromatase inhibitors include vorozole
(RIVISOR.RTM.), megestrol acetate (MEGASE.RTM.), fadrozole, and
4(5)-imidazoles; lutenizing hormone-releaseing hormone agonists,
including leuprolide (LUPRON.RTM. and ELIGARD.RTM.), goserelin,
buserelin, and tripterelin; sex steroids, including progestines
such as megestrol acetate and medroxyprogesterone acetate,
estrogens such as diethylstilbestrol and premarin, and
androgens/retinoids such as fluoxymesterone, all transretionic acid
and fenretinide; onapristone; anti-progesterones; estrogen receptor
down-regulators (ERDs); anti-androgens such as flutamide,
nilutamide and bicalutamide; and pharmaceutically acceptable salts,
acids or derivatives of any of the above; as well as combinations
of two or more of the above.
[0142] A "subject" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to,
humans, non-human higher primates, primates, farm animals (such as
cows), sport animals, pets (such as cats, dogs and horses), and
laboratory animals (such as mice and rats).
II. DETAILED DESCRIPTION
[0143] The present invention provides antibodies, and functional
antibody fragments, comprising at least one antigen-binding domain
that has binding specificity for at least two different HER
receptors, in particular EGFR and HER2, EGFR and HER3, or EGFR and
HER4. These multispecific antibodies are distinct from traditional
multispecific antibodies which have antigen-binding domains
(usually two) with different binding specificities. In certain
embodiments, the multispecific antibodies described herein have the
molecular structure of an IgG (or Fab) and thus retain favorable
attributes of an IgG for therapeutic development, such as
predictable pharmacokinetic properties, well established
manufacturing protocols, choice of Fc-mediated effector functions,
and bi- or mono-valencies. These favorable attributes are often
lacking in the traditional multispecific antibodies that are
derived by assembling two distinct antibody fragments into one
molecule.
[0144] The multispecific antibodies described herein, and
functional antibody fragments thereof, are useful for the treatment
of diseases or conditions, such as cancers, that are associated
with HER receptor pathways. In one particular embodiment, the
antigen-binding domain of the multispecific antibody specifically
binds to both EGFR and HER3. In another embodiment, the
antigen-binding domain specifically binds to both EGFR and HER2. In
another embodiment, the antigen-binding domain specifically binds
to both EGFR and HER4.
[0145] One particular aspect of the invention provides for
antibodies comprising two (or more) antigen-binding domains, each
of which has the same binding specificity.
[0146] One embodiment provides for a multispecific antibody
comprised of two antigen-binding domains, where each
antigen-binding domain has the same specificity and specifically
binds to two different HER receptors. In one particular embodiment,
each antigen-binding domain specifically binds to both EGFR and
HER3. In another embodiment, each antigen-binding domain
specifically binds to both EGFR and HER2. In another embodiment,
each antigen-binding domain specifically binds to both EGFR and
HER4. In yet another embodiment, each antigen-binding domain
specifically binds to Domain III of EGFR. In another embodiment,
each antigen-binding domain specifically binds to Domain III of
HER3. In yet another embodiment, each antigen-binding domain is
capable of binding to Domain III of EGFR and Domain III of
HER3.
[0147] In particular embodiments, the multispecific antibody
specifically binds to its target HER receptor or HER receptors and
does not specifically bind to the non-target HER receptors.
Accordingly, in one embodiment, the multispecific antibody
specifically binds to EGFR and HER3 but does not specifically bind
to HER2 or HER4. In another embodiment, the multispecific antibody
specifically binds to EGFR and HER2 but does not specifically bind
to HER3 or HER4. In another embodiment, the multispecific antibody
specifically binds to EGFR and HER4 but does not specifically bind
to HER2 or HER3.
[0148] In certain embodiments, each antigen-binding domain
comprises a heavy chain variable domain (V.sub.H) and a light chain
variable domain (V.sub.L). In one embodiment, the V.sub.HV.sub.L
unit specifically binds to two different HER receptors. In one
particular embodiment, the V.sub.HV.sub.L unit specifically binds
to EGFR and HER3. In another embodiment, the V.sub.HV.sub.L unit
specifically binds to EGFR and HER2. In another embodiment, the
V.sub.HV.sub.L unit specifically binds to EGFR and HER4.
[0149] In particular embodiments, the affinity of the multispecific
antibody for its target HER receptor or receptors is indicated by a
Kd of less than 10.sup.-5 M, less than 10.sup.-6 M, less than
10.sup.-7 M, less than 10.sup.-8 M, less than 10.sup.-9 M, less
than 10.sup.-10 M, less than 10.sup.-11 M, or less than 10.sup.-12
M. In one embodiment, the Kd of the antibody for one of its target
receptors is less than 10.sup.-7 M, less than 10.sup.-8 M, less
than 10.sup.-9 M, less than 10.sup.-10 M, less than 10.sup.-11 M,
or less than 10.sup.-12 M. In another embodiment, the Kd of the
multispecific antibody for all of its target receptors is less than
10.sup.-7 M, less than 10.sup.-8 M, less than 10.sup.-9 M, less
than 10.sup.-10 M, a less than 10.sup.-11 M, or less than
10.sup.-12 M.
[0150] In some embodiments, the affinity of the multispecific
antibody for one of its target HER receptors is greater than for
its other target HER receptor or receptors. In one embodiment, the
affinity of the multispecific antibody for one target HER receptor
is at least 2, 3, 4, 5, 8, 10, 12, 15, 18, 20, 22, 25, 30, 35, 40,
50, 100-fold greater than its affinity for another target HER
receptor. In one embodiment, the multispecific antibody
specifically binds to EGFR and another HER receptor and its binding
affinity for the other HER receptor is at least 2, 3, 4, 5, 8, 10,
12, 15, 18, 20, 22, 25, 30, 35, 40, 50, or 100-fold greater than
its affinity for EGFR. In one embodiment, the multispecific
antibody specifically binds to EGFR and HER3 and its binding
affinity for HER3 is at least 2, 3, 4, 5, 8, 10, 12, 15, 18, 20,
22, 25, 30, 35, 40, 50, or 100-fold greater than its binding
affinity for EGFR. In another embodiment, the multispecific
antibody specifically binds to EGFR and HER2 and its binding
affinity for HER2 is at least 2, 3, 4, 5, 8, 10, 12, 15, 18, 20,
22, 25, 30, 35, 40, 50, or 100-fold greater than its binding
affinity for EGFR. In another embodiment, the multispecific
antibody specifically binds to EGFR and HER4 and its binding
affinity for HER4 is at least 2, 3, 4, 5, 8, 10, 12, 15, 18, 20,
22, 25, 30, 35, 40, 50, or 100-fold greater than its affinity for
EGFR.
[0151] In some embodiments, the multispecific antibodies of the
present invention inhibit a biological activity of at least one of
the HER receptors to which they specifically bind. In some
embodiments, the multispecific antibodies of the present invention
inhibit a biological activity of both of the HER receptors to which
specifically they bind. Thus, for example, a multispecific antibody
of the invention inhibits a biological activity of an EGFR and/or
HER2 and/or HER3 and/or HER4 receptor.
[0152] In one embodiment, the multispecific antibody specifically
binds human EGFR and human HER3, and inhibits a biological activity
of at least the EGFR. In another embodiment, the multispecific
antibody specifically binds human EGFR and human HER3 and inhibits
at least a biological activity of HER3. In another embodiment, the
multispecific antibody specifically binds human EGFR and human HER3
and inhibits a biological activity of both EGFR and HER3.
[0153] In another embodiment, the multispecific antibody
specifically binds human EGFR and human HER2 and inhibits at least
a biological activity of EGFR. In yet another embodiment, the
antibody specifically binds human EGFR and human HER2 and inhibits
at least a biological activity of HER2. In yet another embodiment,
the antibody specifically binds human EGFR and human HER2 and
inhibits a biological activity of both EGFR and HER2.
[0154] In another embodiment, the antibody specifically binds human
EGFR and human HER4, and inhibits a biological activity of at least
the EGFR. In another embodiment, the antibody specifically binds
human EGFR and human HER4 and inhibits at least a biological
activity of HER4. In another embodiment, the antibody specifically
binds human EGFR and human HER4 and inhibits a biological activity
of both EGFR and HER4.
[0155] In certain embodiments, the antibodies herein inhibit a
biological activity driven, at least partially, by a HER receptor
to which they do not bind. For example, antibodies that bind EGFR
and HER3 might still be able to inhibit a HER2-driven biological
activity.
[0156] Inhibition of a biological activity can be measured in
assays well known in the art. Thus, for example, the antibodies
herein may inhibit phosphorylation of one or more of the HER
receptors, and/or may inhibit the binding of a HER ligand to its
receptor, and/or may inhibit ligand induced proliferation of HER
receptor expressing cells and/or may inhibit downstream signaling
pathways that are activated via a HER receptor.
[0157] Two major downstream signaling pathways that are activated
in response to EGFR phosphorylation are the Ras/MAPK and the
phosphatidylinositol 3-kinase (PI3K)/Akt pathways. Therefore, the
ability of an antibody herein to inhibit the biological activity of
a HER receptor can be measured by assessing whether it can block
the activation of these pathways, for example in NR6 cells. Thus,
the ability of the antibodies to block ligand induced
phosphorylation of p44/42MAPK, pAKT or other downstream signaling
molecules can be measured. HER3 signaling has also been implicated
in several other pathways, including c-met and FGFR. The ability of
an antibody herein to inhibit the biological activity of a HER
receptor can be measured by assessing whether it can block the
activation of these pathways.
[0158] One aspect of invention provides for multispecific
antibodies that are generated by diversifying an antibody with
specificity for one HER receptor such that it develops specificity
for a second HER receptor while retaining specificity for the first
HER receptor. In generic terms, this method comprises the steps of
(1) diversifying the amino acid sequence of a light chain variable
domain (V.sub.L) of an antibody, wherein prior to the
diversification, the antibody comprised a V.sub.L and a heavy chain
variable domain (V.sub.H) capable of binding to an epitope on a
first HER receptor and (2) selecting a diversified antibody capable
of binding to the epitope on the first HER receptor and an epitope
on a second HER receptor. These steps can be repeated in order to
generate multi-specific antibodies. A detailed description of this
method is provided in United States Patent Publication No.
20080069820, the entire disclosure of which is expressly
incorporated by reference herein. This method is further
illustrated in the Examples. In the method described in the
Examples, an anti-EGFR antibody is used as a template for
diversification and thus for the preparation of multispecific
anti-HER antibodies, however, other anti-HER antibodies, such as
anti-HER2, anti-HER3, or anti-HER4 antibodies could also serve as a
template.
[0159] The invention further provides for monospecific antibodies
that are capable of specifically binding to one HER receptor and do
not specifically bind to the other HER receptors. In one
embodiment, the antibody specifically binds to EGFR. In one
embodiment, the antibody specifically binds to Domain III of EGFR.
In some embodiments, the antibody specifically binds to EGFR and
inhibits a biologicial activity of EGFR. In another embodiment, the
antibody specifically binds to HER3. In one embodiment, the
antibody specifically binds to Domain III of HER3. In some
embodiments, the antibody specifically binds to HER3 and inhibits a
biologicial activity of HER3.
[0160] The monospecific antibodies can be used as the template
antibody for further diversification to add binding specificity to
other HER receptors or to other target antigens.
Toxicity
[0161] Toxicity of EGFR antagonists is well documented in both
pre-clinical and clinical studies. For example, the anti-EGFR
antibody cetuximab exhibits various forms of toxicity at
therapeutically effective levels. The most common adverse reactions
with cetuximab (ERBITUX.RTM., Imclone) (incidence .gtoreq.25%) are
cutaneous adverse reactions (including rash, pruritus, and nail
changes), headache, diarrhea, and infection. The most serious
adverse reactions associated with cetuximab treatment are infusion
reactions, cardiopulmonary arrest, dermatologic toxicity and
radiation dermatitis, sepsis, renal failure, interstitial lung
disease, and pulmonary embolus. See, Biologics License Agreement
(BLA) for cetuximab (Application No.: 125084) (incorporated by
reference herein). Similar toxicity issues are observed for
panitumumab (VECTIBIX.RTM. Amgen) where dermatologic toxicity
occurred in 89% of patients administered this antibody. These
toxicities were severe, CTC grade 3 and higher. (VECTIBIX.RTM. FDA
label.)
[0162] The anti-EGFR chemotherapeutic agent erlotinib has been
reported to cause, in some instances, acute renal failure or renal
insufficiency, hepatic failure and/or hepatorenal syndrome,
gastrointestinal perforations, bullous and exfoliative skin
disorders, and corneal ulceration and perforation. See, FDA
Warnings and Precautions safety labeling for erlotinib
(TARCEVA.RTM., Genentech, OSI Pharmaceuticals) (2009).
[0163] "Toxic", or "toxicity", refers to any adverse effect caused
by an agent when administered to a subject. Measures of toxicity
include, but are not limited to, mortality, loss of body weight,
organ failure, altered organ function, central nervous system
toxicity, gastrointestinal toxicity (as indicated, for example, by
diarrhea), dermatologic toxicity (as indicated, for example, by
appearance of rash, skin lesion, desquamation, or pruritus),
cardiac toxicity, infection, sepsis, and cytotoxicity.
[0164] Toxicity can be determined by methods known in the art such
as monitoring clinical cage side observations, body weight, food
consumption, respiration rate, pulse oximetry measurements,
physical examination, ophthalmic evaluations, neurological
evaluations, metabolic parameters, cardiovascular parameters,
clinical pathology (including clinical chemistry, hematology,
urinalysis and coagulation parameters), and macroscopic and
microscopic pathology.
[0165] The Common Terminology Criteria for Adverse Events v3.0
(CTCAE) prepared by the National Cancer Institute (incorporated by
reference in its entirety herein) provides information regarding
particular accepted indicators of toxicity in human subjects. FIG.
31 provides information regarding observed non-clinical and
clinical toxicities for EGFR antagonist therapies.
[0166] Toxicity can be measured in terms of total toxic events or
severity of the toxic event/events. Severity of the events can be
described using the grading system set up in the CTCAE. Grades are
assigned each adverse event using unique clinical descriptions of
severity based on the general guideline that Grade 1 refers to a
mild event, Grade 2 refers to a moderate event, Grade 3 refers to a
severe event, Grade 4 refers to a life-threatening or disabling
event, and Grade 5 refers to a death related to the event. The
CTCAE provides the specific clinical descriptions for the toxic
events. The descriptors for dermatologic toxicity are provided
beginning at page 14 of the CTCAE, v.3. As an example, FIG. 32
shows grading for rash/desquamation and acne/acneform rash. (CTCAE,
v.3) There are a number of models known in the art that are used to
monitor for potential indicators of toxicity, including, but not
limited to, in vitro cell-based models and in vivo non-human animal
models. Toxicity is also monitored in human subjects in clinical
trial studies.
[0167] In one particular embodiment, toxicity is measured in
cynomolgus monkeys. The toxic effect of EGFR antagonists in
cynomolgus monkeys is well documented. As set forth in the
Biologics License Agreement (BLA) for cetuximab (Application No.:
125084) (incorporated by reference herein), all monkeys receiving
cetuximab exhibited mild to severe lesions on the skin, consisting
of scale formation, reddening, erythema, dermatitis, fissures,
wounds, and exanthema, and/or hair thinning or loss. The
dermatologic toxicity was dose-dependent in both severity and time
of onset, where severity for high, mid and low doses were severe,
moderate and mild and time of onset occurred on Study Days 15, 22
and 64, respectively. Secondary complications of severe skin
lesions were bacterial infection or sepsis with subsequent
mortality or euthanasia of 50% of the animals in the high dose
group. Other dose-related toxicities included changes in certain
clinical pathology parameters associated with macroscopic and
microscopic evidence of cellular and tissue damage in the liver,
bone marrow, spleen, and lymphoid organs.
[0168] As set forth in the Examples, cynomolgus monkeys dosed with
a bispecific antibody that specifically binds to EGFR and HER3
exhibited fewer incidences of toxicity, as indicated by dermal
lesions, as compared to cynomolgus monkeys dosed with an equal
amount of the EGFR antagonist cetuximab. One of three cynomolgus
monkeys dosed with 25 mg/kg of the bispecific antibody developed
dermal lesions whereas 3 of 3 cynomolgus monkeys dosed with 25
mg/kg of cetuximab developed dermal lesions. The lesion that
occurred in a bispecific antibody dosed monkey was less severe than
the lesions of the cetuximab dosed monkeys and the onset of the
lesion was delayed. The animal dosed with the bispecific antibody
developed the skin lesion one week after the last (sixth) dose
compared to the monkeys dosed with cetuximab where the onset of
dermal lesions occurred after the third dose in all animals.
[0169] In clinical studies of cetuximab, dermatologic toxicities,
including acneiform rash, skin drying and fissuring, and
inflammatory and infectious sequelae were observed. The reported
incidence of dermatologic toxicity was as high as 89% (for those
patients with advanced colorectal cancer).
[0170] Models of skin toxicity are known and can be used to
determine dermatologic toxicity of the antibodies. Examples of such
models include human epidermal keratinocytes (NHEK (Clonetics, San
Diego, Calif.; Lonza Bioscience, Walkersville, Md.); HEKa (Cascade
Biologics, Portland, Oreg.; Invitrogen, Carlsbad, Calif.) and
reconstituted human epidermis (EpiDerm.TM. cultures (MatTek,
Ashland, Mass.). These models can be used to examine the effect of
the antibodies on cellular proliferation, gene expression, protein
expression, receptor phosphorylation, cell viability, and changes
in histopathology. Lacouture, M. E., Nature Rev. Cancer, 6:803-812
(2006).
[0171] It is desirable to provide a less toxic antibody that
targets the EGFR pathway. Dosing of EGFR antagonists such as
cetuximab is limited by toxicity (primarily dermatologic toxicity
and infusion reactions). A less toxic antibody could be
administered at a higher dose than a more toxic EGFR antagonist
which may result in increased antitumor effects. Accordingly, one
aspect of the invention provides a multispecific antibody that
specifically binds to EGFR and at least one other HER receptor,
(HER2, HER3, and/or HER4), where the antibody is less toxic than an
EGFR antagonist when the antibody and EGFR antagonist are
administered at equivalent doses. In one embodiment, the antibody
specifically binds to EGFR and HER3. In another embodiment, the
antibody specifically binds to EGFR and HER2. In yet another
embodiment, the antibody specifically binds to EGFR and HER4.
[0172] In some embodiments the multispecific antibody induces a
lower incidence of toxicities, less severe toxicities, or delayed
onset of toxicities in an in vivo model compared to an EGFR
antagonist. One aspect of the invention provides for a
multispecific antibody that specifically binds to EGFR and at least
one other HER receptor (HER2, HER3, and/or HER4) where the antibody
induces fewer toxicity incidents in subjects administered the
antibody as compared to toxicity incidents in subjects administered
an EGFR antagonist. In particular embodiments, the number of
toxicity incidents in subjects administered the antibody is at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the
number of toxicity incidents in subjects administered an EGFR
antagonist.
[0173] In other embodiments, the rate of toxicity incidents in
subjects administered the antibody is less than 80%, 70%, 60%, 50%,
40%, 30%, 20%, 10%, 5%, 2%, or 1%.
[0174] In particular embodiments the multispecific antibody induces
a lower incidence of dermatologic toxicities, less severe
dermatologic toxicities, or delayed onset of dermatologic
toxicities in an in vivo model compared to an EGFR antagonist. One
aspect of the invention provides for a multispecific antibody that
specifically binds to EGFR and at least one other HER receptor
(HER2, HER3, and/or HER4) where the antibody induces fewer total
dermatologic toxicity incidents in subjects administered the
antibody as compared to total dermatologic toxicity incidents in
subjects administered an EGFR antagonist. In particular
embodiments, the number of total dermatologic toxicity incidents in
subjects administered the antibody is at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, or 90% less than the number of total
dermatologic toxicity incidents in subjects administered an EGFR
antagonist.
[0175] In other embodiments, the rate of total dermatologic
toxicity incidents in subjects administered the antibody is less
than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, or 1%.
[0176] Another aspect of the invention provides for a multispecific
antibody that specifically binds to EGFR and at least one other HER
receptor (HER2, HER3, and/or HER4) where the antibody induces fewer
grade 3 or higher toxicity incidents in subjects administered the
antibody as compared to grade 3 or higher toxicity incidents in
subjects administered the EGFR antagonist. In particular
embodiments, the number of grade 3 or higher toxicity incidents in
subjects administered the antibody is at least 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, or 90% less than the number of grade 3 or
higher toxicity incidents in subjects administered an EGFR
antagonist.
[0177] In other embodiments, the rate of grade 3 or higher toxicity
incidents in subjects administered the multispecific antibody is
less than 70%, 60%, 50%, 40%, 30%, 20%, 15%, 12%, 11%, 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2%, or 1%.
[0178] In particular embodiments, the multispecific antibody
induces fewer grade 3 or higher dermatologic toxicity incidents in
subjects administered the bispecific antibody as compared to grade
3 or higher dermatologic toxicity incidents in subjects
administered an EGFR antagonist. In particular embodiments, the
number of grade 3 or higher dermatologic toxicity incidents in
subjects administered the multispecific antibody is at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than the number of
grade 3 or higher dermatologic toxicity incidents in subjects
administered an EGFR antagonist.
[0179] In other embodiments, the rate of grade 3 or higher
dermatologic toxicity incidents in subjects administered the
multispecific antibody is less than 70%, 60%, 50%, 40%, 30%, 20%,
15%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.
[0180] In other embodiments, the antibody induces fewer incidences
of altered organ function in an in vivo model compared to an EGFR
antagonist. In other embodiments, the antibody induces fewer, or
less severe, gastrointestinal toxicities in an in vivo model
compared to an EGFR antagonist.
[0181] In some embodiment, the EGFR antagonist is an anti-EGFR
antibody. In one embodiment, the EGFR antagonist is cetuximab. In
another embodiment, the EGFR antagonist is panitumumab. In another
embodiments, the EGFR antagonist is a small molecule. In one
embodiment, the EGFR antagonist is erlotinib. In one embodiment,
the in vivo model is a monkey, such as a cynomolgus monkey. In
another embodiment, the in vivo model is a human.
Antibody and Antibody Variants
[0182] In some embodiments, the invention provides a multispecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR and HER3. In some embodiments, the antigen-binding
domain does not specifically bind to other targets, including other
HER receptors.
[0183] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H (heavy chain variable
domain) comprising the amino acid sequence of SEQ ID NO: 25. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER3 where the antibody
comprises a V.sub.L (light chain variable domain) comprising the
amino acid sequence of SEQ ID NO: 40. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER3 where the antibody comprises a
V.sub.H comprising the amino acid sequence of SEQ ID NO: 25 and a
V.sub.L comprising the amino acid sequence of SEQ ID NO: 40.
[0184] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 25. In
one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.L comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 40. In
one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 25 and a
V.sub.L comprising one, two, and/or three of the HVRs of the amino
acid sequence of SEQ ID NO: 40. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER3 where the antibody comprises a
V.sub.H comprising all three HVRs of the amino acid sequence of SEQ
ID NO: 25 and a V.sub.L comprising all three of the HVRs of the
amino acid sequence of SEQ ID NO: 40. In some embodiments, the HVRs
are extended HVRs.
[0185] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising the amino acid
sequence of SEQ ID NO: 64. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER3 where the antibody comprises a V.sub.L
comprising the amino acid sequence of SEQ ID NO: 26. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER3 where the antibody
comprises a V.sub.H comprising the amino acid sequence of SEQ ID
NO: 64 and a V.sub.L comprising the amino acid sequence of SEQ ID
NO: 26. In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 64. In
one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.L comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 26. In
one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 64 and a
V.sub.L comprising one, two, and/or three of the HVRs of the amino
acid sequence of SEQ ID NO: 26. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER3 where the antibody comprises a
V.sub.H comprising all three HVRs of the amino acid sequence of SEQ
ID NO: 64 and a V.sub.L comprising all three of the HVRs of the
amino acid sequence of SEQ ID NO: 26. In some embodiments, the HVRs
are extended HVRs.
[0186] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising the amino acid
sequence of SEQ ID NO: 28. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER3 where the antibody comprises a V.sub.L
comprising the amino acid sequence of SEQ ID NO: 27. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER3 where the antibody
comprises a V.sub.H comprising the amino acid sequence of SEQ ID
NO: 28 and a V.sub.L comprising the amino acid sequence of SEQ ID
NO: 27.
[0187] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 28. In
one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.L comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 27. In
one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 28 and a
V.sub.L comprising one, two, and/or three of the HVRs of the amino
acid sequence of SEQ ID NO: 27. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER3 where the antibody comprises a
V.sub.H comprising all three HVRs of the amino acid sequence of SEQ
ID NO: 28 and a V.sub.L comprising all three of the HVRs of the
amino acid sequence of SEQ ID NO: 27. In some embodiments, the HVRs
are extended HVRs. In one specific embodiment, HVR-H1 comprises the
amino acid sequence LSGDWIH (SEQ ID NO: 48), HVR-H2 comprises the
amino acid sequence LGEISAAGGYTD (SEQ ID NO: 50), HVR-H3 comprises
the amino acid sequence ARESRVSFEAAMDY (SEQ ID NO: 53), HVR-L1
comprises the amino acid sequence DLATDVA (SEQ ID NO: 54), HVR-L2
comprises the amino acid sequence SASF (SEQ ID NO: 56), and HVR-L3
comprises the amino acid sequence SEPEPYT (SEQ ID NO: 57).
[0188] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.L comprising the amino acid
sequence of SEQ ID NO: 29. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER3 where the antibody comprises a V.sub.H
comprising the amino acid sequence of SEQ ID NO: 28 and a V.sub.L
comprising the amino acid sequence of SEQ ID NO: 29.
[0189] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.L comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 29. In
one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 28 and a
V.sub.L comprising one, two, and/or three of the HVRs of the amino
acid sequence of SEQ ID NO: 29. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER3 where the antibody comprises a
V.sub.H comprising all three HVRs of the amino acid sequence of SEQ
ID NO: 28 and a V.sub.L comprising all three of the HVRs of the
amino acid sequence of SEQ ID NO: 29. In some embodiments, the HVRs
are extended HVRs. In one specific embodiment, HVR-H1 comprises the
amino acid sequence LSGDWIH (SEQ ID NO: 48), HVR-H2 comprises the
amino acid sequence LGEISAAGGYTD (SEQ ID NO: 50), HVR-H3 comprises
the amino acid sequence ARESRVSFEAAMDY (SEQ ID NO: 53), HVR-L1
comprises the amino acid sequence NIATDVA (SEQ ID NO: 55), HVR-L2
comprises the amino acid sequence SASF (SEQ ID NO: 56), and HVR-L3
comprises the amino acid sequence SEPEPYT (SEQ ID NO: 57).
[0190] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising the amino acid
sequence of SEQ ID NO: 30. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER3 where the antibody comprises a V.sub.H
comprising the amino acid sequence of SEQ ID NO: 30 and a V.sub.L
comprising the amino acid sequence of SEQ ID NO: 29.
[0191] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 30. In
one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 30 and a
V.sub.L comprising one, two, and/or three of the HVRs of the amino
acid sequence of SEQ ID NO: 29. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER3 where the antibody comprises a
V.sub.H comprising all three HVRs of the amino acid sequence of SEQ
ID NO: 30 and a V.sub.L comprising all three of the HVRs of the
amino acid sequence of SEQ ID NO: 29. In some embodiments, the HVRs
are extended HVRs. In one specific embodiment, HVR-H1 comprises the
amino acid sequence LSGDWIH (SEQ ID NO: 48), HVR-H2 comprises the
amino acid sequence VGEISAAGGYTD (SEQ ID NO: 51), HVR-H3 comprises
the amino acid sequence ARESRVSFEAAMDY (SEQ ID NO: 53), HVR-L1
comprises the amino acid sequence NIATDVA (SEQ ID NO: 55), HVR-L2
comprises the amino acid sequence SASF (SEQ ID NO: 56), and HVR-L3
comprises the amino acid sequence SEPEPYT (SEQ ID NO: 57).
[0192] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.L comprising the amino acid
sequence of SEQ ID NOs: 40, 41, 42, 43, 44, 45, or 46. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER3 where the antibody
comprises a V.sub.H comprising the amino acid sequence of SEQ ID
NO: 25 and a V.sub.L comprising the amino acid sequence of SEQ ID
NOs: 40, 41, 42, 43, 44, 45, or 46.
[0193] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.L comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NOs: 40, 41,
42, 43, 44, 45, or 46. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER3 where the antibody comprises a V.sub.H
comprising one, two, and/or three of the HVRs of the amino acid
sequence of SEQ ID NO: 25 and a V.sub.L comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NOs: 40, 41,
42, 43, 44, 45, or 46. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER3 where the antibody comprises a V.sub.H
comprising all three HVRs of the amino acid sequence of SEQ ID NO:
25 and a V.sub.L comprising all three of the HVRs of the amino acid
sequence of SEQ ID NOs: 40, 41, 42, 43, 44, 45, or 46. In some
embodiments, the HVRs are extended HVRs.
[0194] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER2
where the antibody comprises a light chain variable domain
comprising the amino acid sequence of SEQ ID NOs: 36, 37, or 38. In
one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER2
where the antibody comprises a heavy chain variable domain
comprising the amino acid sequence of SEQ ID NO: 25 and a light
chain variable domain comprising the amino acid sequence of SEQ ID
NOs: 36, 37, or 38.
[0195] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER2
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 25. In
one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER2
where the antibody comprises a V.sub.L comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NOs: 36, 37,
or 38. In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER2
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 25 and a
V.sub.L comprising one, two, and/or three of the HVRs of the amino
acid sequence of SEQ ID NOs: 36, 37, or 38. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER2 where the antibody comprises a
V.sub.H comprising all three HVRs of the amino acid sequence of SEQ
ID NO: 25 and a V.sub.L comprising all three of the HVRs of the
amino acid sequence of SEQ ID NOs: 36, 37, or 38. In some
embodiments, the HVRs are extended HVRs.
[0196] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER4
where the antibody comprises a V.sub.H comprising the amino acid
sequence of SEQ ID NO: 25. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER4 where the antibody comprises a V.sub.L
comprising the amino acid sequence of SEQ ID NO: 39. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER4 where the antibody
comprises a heavy chain variable domain comprising the amino acid
sequence of SEQ ID NO: 25 and a light chain variable domain
comprising the amino acid sequence of SEQ ID NO: 39.
[0197] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER4
where the antibody comprises a V.sub.L comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 39. In
one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER4
where the antibody comprises a V.sub.H comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 25 and a
V.sub.L comprising one, two, and/or three of the HVRs of the amino
acid sequence of SEQ ID NO: 39. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER4 where the antibody comprises a
V.sub.H comprising all three HVRs of the amino acid sequence of SEQ
ID NO: 25 and a V.sub.L comprising all three of the HVRs of the
amino acid sequence of SEQ ID NO: 39. In some embodiments, the HVRs
are extended HVRs.
[0198] In one embodiment, the invention provides for a monospecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR where the antibody comprises a V.sub.H comprising the
amino acid sequence of SEQ ID NO: 25. In one embodiment, the
monospecific antibody comprises an antigen-binding domain that
specifically binds to EGFR where the antibody comprises a V.sub.L
comprising the amino acid sequence of SEQ ID NO: 58 or SEQ ID NO:
24. In one embodiment, monospecific antibody comprises an
antigen-binding domain that specifically binds to EGFR where the
antibody comprises a V.sub.H comprising the amino acid sequence of
SEQ ID NO: 25 and a V.sub.L comprising the amino acid sequence of
SEQ ID NO: 58 or SEQ ID NO: 24.
[0199] In one embodiment, the invention provides for a monospecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR where the antibody comprises a V.sub.H comprising
one, two, and/or three of the HVRs of the amino acid sequence of
SEQ ID NO: 25. In one embodiment, the monospecific antibody
comprises an antigen-binding domain that specifically binds to EGFR
where the antibody comprises a V.sub.L comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 24. In
one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to EGFR where the
antibody comprises a V.sub.H comprising one, two, and/or three of
the HVRs of the amino acid sequence of SEQ ID NO: 25 and a V.sub.L
comprising one, two, and/or three of the HVRs of the amino acid
sequence of SEQ ID NO: 24. In one embodiment, the monospecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR where the antibody comprises a V.sub.H comprising all
three HVRs of the amino acid sequence of SEQ ID NO: 25 and a
V.sub.L comprising all three of the HVRs of the amino acid sequence
of SEQ ID NO: 24. In some embodiments, the HVRs are extended HVRs.
In one specific embodiment, HVR-H1 comprises the amino acid
sequence FTGNWIH (SEQ ID NO: 47), HVR-H2 comprises the amino acid
sequence VGEISPSGGYTD (SEQ ID NO: 49), HVR-H3 comprises the amino
acid sequence ARESRVSYEAAMDY (SEQ ID NO: 52), HVR-L1 comprises the
amino acid sequence DVSTAVA (SEQ ID NO: 78), HVR-L2 comprises the
amino acid sequence SASF (SEQ ID NO: 56), and HVR-L3 comprises the
amino acid sequence SYPTPYT (SEQ ID NO: 79).
[0200] In one embodiment, the invention provides for a monospecific
antibody comprising an antigen-binding domain that specifically
binds to HER3 where the antibody comprises a V.sub.H comprising the
amino acid sequence of SEQ ID NO: 29. In one embodiment, the
monospecific antibody comprises an antigen-binding domain that
specifically binds to HER3 where the antibody comprises a V.sub.L
comprising the amino acid sequence of SEQ ID NOs: 33, 34, or 35. In
one embodiment, monospecific antibody comprises an antigen-binding
domain that specifically binds to HER3 where the antibody comprises
a V.sub.H comprising the amino acid sequence of SEQ ID NO: 29 and a
V.sub.L comprising the amino acid sequence of SEQ ID NOs: 33, 34,
or 35.
[0201] In one embodiment, the invention provides for a monospecific
antibody comprising an antigen-binding domain that specifically
binds to HER3 where the antibody comprises a V.sub.H comprising
one, two, and/or three of the HVRs of the amino acid sequence of
SEQ ID NO: 29. In one embodiment, the monospecific antibody
comprises an antigen-binding domain that specifically binds to HER3
where the antibody comprises a V.sub.L comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NOs: 33, 34,
or 35. In one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to HER3 where the
antibody comprises a V.sub.H comprising one, two, and/or three of
the HVRs of the amino acid sequence of SEQ ID NO: 29 and a V.sub.L
comprising one, two, and/or three of the HVRs of the amino acid
sequence of SEQ ID NOs: 33, 34, or 35. In one embodiment, the
monospecific antibody comprises an antigen-binding domain that
specifically binds to HER3 where the antibody comprises a V.sub.H
comprising all three HVRs of the amino acid sequence of SEQ ID NO:
29 and a V.sub.L comprising all three of the HVRs of the amino acid
sequence of SEQ ID NOs: 33, 34, or 35. In some embodiments, the
HVRs are extended HVRs. In one specific embodiment, HVR-H1
comprises the amino acid sequence FTGDWIH (SEQ ID NO: 62), HVR-H2
comprises the amino acid sequence VGEISPAGAYTD (SEQ ID NO: 60),
HVR-H3 comprises the amino acid sequence AREAKVSFEAAMDY (SEQ ID NO:
61), HVR-L1 comprises the amino acid sequence NIATDVA (SEQ ID NO:
55), HVR-L2 comprises the amino acid sequence SASF (SEQ ID NO: 56),
and HVR-L3 comprises the amino acid sequence SEPEPYT (SEQ ID NO:
57).
[0202] In one embodiment, the invention provides for a monospecific
antibody comprising an antigen-binding domain that specifically
binds to HER3 where the antibody comprises a V.sub.H comprising the
amino acid sequence of SEQ ID NO: 32. In one embodiment, the
monospecific antibody comprises an antigen-binding domain that
specifically binds to HER3 where the antibody comprises a V.sub.L
comprising the amino acid sequence of SEQ ID NO: 31. In one
embodiment, monospecific antibody comprises an antigen-binding
domain that specifically binds to HER3 where the antibody comprises
a V.sub.H comprising the amino acid sequence of SEQ ID NO: 32 and a
V.sub.L comprising the amino acid sequence of SEQ ID NO: 31.
[0203] In one embodiment, the invention provides for a monospecific
antibody comprising an antigen-binding domain that specifically
binds to HER3 where the antibody comprises a V.sub.H comprising
one, two, and/or three of the HVRs of the amino acid sequence of
SEQ ID NO: 32. In one embodiment, the monospecific antibody
comprises an antigen-binding domain that specifically binds to HER3
where the antibody comprises a V.sub.L comprising one, two, and/or
three of the HVRs of the amino acid sequence of SEQ ID NO: 31. In
one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to HER3 where the
antibody comprises a V.sub.H comprising one, two, and/or three of
the HVRs of the amino acid sequence of SEQ ID NO: 32 and a V.sub.L
comprising one, two, and/or three of the HVRs of the amino acid
sequence of SEQ ID NO: 31. In one embodiment, the monospecific
antibody comprises an antigen-binding domain that specifically
binds to HER3 where the antibody comprises a V.sub.H comprising all
three HVRs of the amino acid sequence of SEQ ID NO: 32 and a
V.sub.L comprising all three of the HVRs of the amino acid sequence
of SEQ ID NO: 31. In some embodiments, the HVRs are extended HVRs.
In one specific embodiment, HVR-H1 comprises the amino acid
sequence FSGDWIH (SEQ ID NO: 59), HVR-H2 comprises the amino acid
sequence VGEISPAGAYTD (SEQ ID NO: 60), HVR-H3 comprises the amino
acid sequence AREAKVSFEAAMDY (SEQ ID NO: 61), HVR-L1 comprises the
amino acid sequence DLATDVA (SEQ ID NO: 54), HVR-L2 comprises the
amino acid sequence SASF (SEQ ID NO: 56), and HVR-L3 comprises the
amino acid sequence SEPEPYT (SEQ ID NO: 57).
[0204] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a heavy chain comprising the amino
acid sequences of SEQ ID NOs: 2, 12, or 14. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER3 where the antibody comprises a
light chain comprising the amino acid sequence of SEQ ID NOs: 4, 5,
6, 7, 8, 9, 10, 11, or 13. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER3 where the antibody comprises a heavy chain
comprising the amino acid sequence of SEQ ID NO: 2 and a light
chain comprising the amino acid sequence of SEQ ID NO: 4. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER3 where the antibody
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO: 12 and a light chain comprising the amino acid sequence of
SEQ ID NO: 11. In one embodiment, the multispecific antibody
comprises an antigen-binding domain that specifically binds to EGFR
and HER3 where the antibody comprises a heavy chain comprising the
amino acid sequence of SEQ ID NO: 12 and a light chain comprising
the amino acid sequence of SEQ ID NO: 13. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER3 where the antibody comprises a
heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and
a light chain comprising the amino acid sequence of SEQ ID NO:
13.
[0205] In some embodiments, the invention provides a multispecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR and HER2. In some embodiments, the antigen-binding
domain does not specifically bind to other targets, including other
HER receptors. In one embodiment, the multispecific antibody
comprises an antigen-binding domain that specifically binds to EGFR
and HER2 where the antibody comprises a heavy chain comprising the
amino acid sequence of SEQ ID NO: 2. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER2 where the antibody comprises a
light chain comprising the amino acid sequence of SEQ ID NOs: 21,
22, or 23.
[0206] In some embodiments, the invention provides a multispecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR and HER4. In some embodiments, the antigen-binding
domain does not specifically bind to other targets, including other
HER receptors. In one embodiment, the multispecific antibody
comprises an antigen-binding domain that specifically binds to EGFR
and HER4 where the antibody comprises a heavy chain comprising the
amino acid sequence of SEQ ID NO: 2. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER4 where the antibody comprises a
light chain comprising the amino acid sequence of SEQ ID NO:
18.
[0207] In one embodiment, the invention provides a monospecific
antibody that specifically binds to EGFR. In some embodiments, the
antigen-binding domain does not specifically bind to other targets,
including other HER receptors. In one embodiment, the monospecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR where the antibody comprises a heavy chain comprising
the amino acid sequence of SEQ ID NO: 2. In one embodiment, the
monospecific antibody comprises an antigen-binding domain that
specifically binds to EGFR where the antibody comprises a light
chain comprising the amino acid sequence of SEQ ID NOs: 1 or 3. In
one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to EGFR where the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO: 2 and a light chain comprising the amino acid
sequence of SEQ ID NO: 1. In one embodiment, the monospecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR where the antibody comprises a heavy chain comprising
the amino acid sequence of SEQ ID NO: 2 and a light chain
comprising the amino acid sequence of SEQ ID NO: 3.
[0208] In one embodiment, the invention provides a monospecific
antibody that specifically binds to HER3. In some embodiments, the
antigen-binding domain does not specifically bind to other targets,
including other HER receptors. In one embodiment, the monospecific
antibody comprises an antigen-binding domain that specifically
binds to HER3 where the antibody comprises a heavy chain comprising
the amino acid sequence of SEQ ID NOs: 16, 17, 19, or 20. In one
embodiment, the monospecific antibody comprises an antigen-binding
domain that specifically binds to HER3 where the antibody comprises
a light chain comprising the amino acid sequence of SEQ ID NOs: 13
or 15. In one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to HER3 where the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NOs: 16, 17, 19, or 20 and a light chain comprising the
amino acid sequence of SEQ ID NOs: 13 or 15.
[0209] In one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to HER3 where the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO: 16 and a light chain comprising the amino acid
sequence of SEQ ID NO: 15. In one embodiment, the monospecific
antibody comprises an antigen-binding domain that specifically
binds to HER3 where the antibody comprises a heavy chain comprising
the amino acid sequence of SEQ ID NO: 17 and a light chain
comprising the amino acid sequence of SEQ ID NO: 13. In one
embodiment, the monospecific antibody comprises an antigen-binding
domain that specifically binds to HER3 where the antibody comprises
a heavy chain comprising the amino acid sequence of SEQ ID NO: 19
and a light chain comprising the amino acid sequence of SEQ ID NO:
13. In one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to HER3 where the
antibody comprises a heavy chain comprising the amino acid sequence
of SEQ ID NO: 20 and a light chain comprising the amino acid
sequence of SEQ ID NO: 13.
[0210] In some embodiments, amino acid sequence modification(s) of
the antibodies described herein are contemplated. For example, it
may be desirable to improve the binding affinity and/or other
biological properties of the antibody Amino acid sequence variants
of the antibody may be prepared by introducing appropriate changes
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of, residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. The amino acid alterations may be introduced in
the subject antibody amino acid sequence at the time that sequence
is made.
[0211] In some embodiments, an amino acid sequence having at least
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity contains substitutions, insertions, or deletions
relative to the reference sequence, but an antibody comprising that
amino acid sequence retains the ability to bind to the original
target or targets of the reference sequence. In some embodiments,
an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity contains
substitutions, insertions, or deletions relative to the reference
sequence, but an antibody comprising that amino acid sequence
retains the ability to bind to the original target or target of the
reference sequence and does not specifically bind to any other
target, including other HER receptors. In some embodiments, a total
of 1 to 10 amino acids have been substituted, inserted, or deleted
in the amino acid sequence of a reference sequence. In some
embodiments, the substitutions, insertions, or deletions occur in
regions outside the HVRs (i.e., in the FRs).
[0212] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 25.
[0213] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.L having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 40 In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER3 where the antibody
comprises a V.sub.H having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 25 and a V.sub.L having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 40.
[0214] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 64. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER3 where the antibody
comprises a V.sub.L having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 26. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER3 where the antibody comprises a V.sub.H
having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the amino acid sequence of SEQ ID
NO: 64 and a V.sub.L having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 26.
[0215] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.L having the amino acid
sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% sequence identity to SEQ ID NO: 29. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER3 where the antibody
comprises a V.sub.H having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 28 and a V.sub.L having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 29.
[0216] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 30. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER3 where the antibody
comprises a V.sub.H having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 30 and a V.sub.L having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 29.
[0217] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.L having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to any one of the amino acid sequences of SEQ ID NOs: 40,
41, 42, 43, 44, 45, or 46. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER3 where the antibody comprises a V.sub.H
having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the amino acid sequence of SEQ ID
NO: 25 and a V.sub.L having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NOs: 40, 41, 42, 43, 44, 45, or 46.
[0218] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER2
where the antibody comprises a V.sub.H having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 25. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER2 where the antibody
comprises a V.sub.L having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 36. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER2 where the antibody comprises a V.sub.H
having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the amino acid sequence of SEQ ID
NO: 25 and a V.sub.L having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 36. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER2 where the antibody comprises a V.sub.L
having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the amino acid sequence of SEQ ID
NO: 37. In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER2
where the antibody comprises a V.sub.H domain having at least 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 25 and a V.sub.L
having the amino acid sequence of SEQ ID NO: 37. In one embodiment,
the multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER2 where the antibody comprises a
V.sub.L having the amino acid sequence of SEQ ID NO: 38. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER2 where the antibody
comprises a V.sub.H having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 25 and a V.sub.L having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 38.
[0219] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER4
where the antibody comprises a V.sub.H having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 25. In one
embodiment, the multispecific antibody comprises an antigen-binding
domain that specifically binds to EGFR and HER4 where the antibody
comprises a V.sub.L having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 39. In one embodiment, the multispecific
antibody comprises an antigen-binding domain that specifically
binds to EGFR and HER4 where the antibody comprises a V.sub.H
having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the amino acid sequence of SEQ ID
NO: 25 and a V.sub.L having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 39.
[0220] In one embodiment, the invention provides for a monospecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR where the antibody comprises a V.sub.H having at
least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity the amino acid sequence of SEQ ID NO: 25. In one
embodiment, the monospecific antibody comprises an antigen-binding
domain that specifically binds to EGFR where the antibody comprises
a V.sub.L having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% sequence identity to the amino acid sequence
of SEQ ID NO: 24. In one embodiment, the monospecific antibody
comprises an antigen-binding domain that specifically binds to EGFR
where the antibody comprises a V.sub.H having at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the amino acid sequence of SEQ ID NO: 25 and a V.sub.L
having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the amino acid sequence of SEQ ID
NO: 24.
[0221] In one embodiment, the invention provides for a monospecific
antibody comprising an antigen-binding domain that specifically
binds to HER3 where the antibody comprises a V.sub.H having at
least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the amino acid sequence of SEQ ID NO: 32. In
one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to HER3 where the
antibody comprises a V.sub.L having at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
amino acid sequence of SEQ ID NO: 31. In one embodiment, the
monospecific antibody comprises an antigen-binding domain that
specifically binds to HER3 where the antibody comprises a V.sub.H
having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the amino acid sequence of SEQ ID
NO: 32 and a V.sub.L having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 31.
[0222] In one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to HER3 where the
antibody comprises a V.sub.H having at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
amino acid sequence of SEQ ID NO: 33. In one embodiment, the
monospecific antibody comprises an antigen-binding domain that
specifically binds to HER3 where the antibody comprises a V.sub.L
having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the amino acid sequence of SEQ ID
NO: 29. In one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to HER3 where the
antibody comprises a V.sub.H having at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
amino acid sequence of SEQ ID NO: 33 and a V.sub.L having at least
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the amino acid sequence of SEQ ID NO: 29. In
one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to HER3 where the
antibody comprises a V.sub.H having at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
amino acid sequence of SEQ ID NO: 34. In one embodiment, the
monospecific antibody comprises an antigen-binding domain that
specifically binds to HER3 where the antibody comprises a V.sub.H
having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the amino acid sequence of SEQ ID
NO: 34 and a V.sub.L having at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid
sequence of SEQ ID NO: 29. In one embodiment, the monospecific
antibody comprises an antigen-binding domain that specifically
binds to HER3 where the antibody comprises a V.sub.H having at
least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the amino acid sequence of SEQ ID NO: 35. In
one embodiment, the monospecific antibody comprises an
antigen-binding domain that specifically binds to HER3 where the
antibody comprises a V.sub.H having at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
amino acid sequence of SEQ ID NO: 35 and a V.sub.L having at least
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the amino acid sequence of SEQ ID NO: 29.
[0223] An exemplary alignment showing the Kabat numbering for the
heavy chain variable domain and light chain variable domain of
several anti-HER antibodies is shown in FIG. 33.
[0224] Another aspect of the invention provides for a multispecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR and HER3 where the antibody comprises a V.sub.H
comprising the amino acid sequence of SEQ ID NO: 25 where the
V.sub.H of SEQ ID NO: 25 comprises an amino acid substitution at
F29(V.sub.H), T30(V.sub.H), N32(V.sub.H), V48(V.sub.H),
P52a(V.sub.H), S53(V.sub.H), T57(V.sub.H), S96(V.sub.H), or
Y100(V.sub.H), numbered according to the Kabat numbering system. In
one embodiment, the antibody comprises more than one of these
substitutions. In one embodiment, the antibody comprises all of
these substitutions. In one embodiment, the multispecific antibody
comprises an antigen-binding domain that specifically binds to EGFR
and HER3 where the antibody comprises a V.sub.H comprising the
amino acid sequence of SEQ ID NO: 25 where the V.sub.H of SEQ ID
NO: 25 comprises one or more amino acid substitutions selected from
the group consisting of F29(V.sub.H)L; T30(V.sub.H)S;
N32(V.sub.H)D, V48(V.sub.H)L, P52a(V.sub.H)A, S53(V.sub.H)A,
T57(V.sub.H)S, S96(V.sub.H)A, and Y100(V.sub.H)F, numbered
according to the Kabat numbering system. In one embodiment, the
multispecific antibody comprises an antigen-binding domain that
specifically binds to EGFR and HER3 where the antibody comprises a
V.sub.H comprising the amino acid sequence of SEQ ID NO: 25 where
the V.sub.H of SEQ ID NO: 25 comprises the amino acid substitutions
F29(V.sub.H)L, T30(V.sub.H)S, N32(V.sub.H)D, P52a(V.sub.H)A, and
S53(V.sub.H)A, and Y100(V.sub.H)F, numbered according to the Kabat
numbering system.
[0225] Another aspect of the invention provides for a multispecific
antibody comprising an antigen-binding domain that specifically
binds to EGFR and HER3 where the antibody comprises a V.sub.L
comprising the amino acid sequence of SEQ ID NO: 58 where the
V.sub.L of SEQ ID NO: 58 comprises an amino acid substitution at
D28(V.sub.L), V29(V.sub.L), S30(V.sub.L), T31(V.sub.L),
A32(V.sub.L), V33(V.sub.L), S50(V.sub.L), A51(V.sub.L),
F53(V.sub.L), S91(V.sub.L), Y92(V.sub.L), T93(V.sub.L),
T94(V.sub.L), or P96(V.sub.L), numbered according to the Kabat
numbering system. In one embodiment, the antibody comprises more
than one of these substitutions. In one embodiment, the antibody
comprises all of these substitutions. In one embodiment, the
antibody comprises amino acid insertions between amino acid 31 and
amino acid 32.
[0226] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.L comprising the amino acid
sequence of SEQ ID NO: 58 where the V.sub.L of SEQ ID NO: 58
comprises one or more amino acid substitutions selected from the
group consisting of D28(V.sub.L)N, V29(V.sub.L)I, V29(V.sub.L)L,
S30(V.sub.L)A, A32(V.sub.L)D, Y92(V.sub.L)E, T93(V.sub.L)P,
T94(V.sub.L)E, and P96(V.sub.L)Y, numbered according to the Kabat
numbering system. In one embodiment, the multispecific antibody
comprises an antigen-binding domain that specifically binds to EGFR
and HER3 where the antibody comprises a V.sub.L comprising the
amino acid sequence of SEQ ID NO: 58 where the V.sub.L of SEQ ID
NO: 58 comprises the amino acid substitutions D28(V.sub.L)N,
V29(V.sub.L)I, S30(V.sub.L)A, A32(V.sub.L)D, Y92(V.sub.L)E,
T93(V.sub.L)P, T94(V.sub.L)E, and P96(V.sub.L)Y, numbered according
to the Kabat numbering system.
[0227] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising the amino acid
sequence of SEQ ID NO: 25 and a V.sub.L comprising the amino acid
sequence of SEQ ID NO: 58, where the V.sub.H of SEQ ID NO: 25
comprises an amino acid substitution at F29(V.sub.H), T30(V.sub.H),
N32(V.sub.H), V48(V.sub.H), P52a(V.sub.H), S53(V.sub.H),
T57(V.sub.H), S96(V.sub.H), or Y100(V.sub.H), and where the V.sub.L
of SEQ ID NO: 58 comprises an amino acid substitution at
D28(V.sub.L), V29(V.sub.L), S30(V.sub.L), T31(V.sub.L),
A32(V.sub.L), V33(V.sub.L), S50(V.sub.L), A51(V.sub.L),
F53(V.sub.L), S91(V.sub.L), Y92(V.sub.L), T93(V.sub.L),
T94(V.sub.L), or P96(V.sub.L), numbered according to the Kabat
numbering system. In one embodiment, the antibody comprises more
than one of these substitutions. In one embodiment, the antibody
comprises all of these substitutions.
[0228] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising the amino acid
sequence of SEQ ID NO: 25 and a V.sub.L comprising the amino acid
sequence of SEQ ID NO: 58, where the V.sub.H of SEQ ID NO: 25
comprises one or more amino acid substitutions selected from the
group consisting of F29(V.sub.H)L; T30(V.sub.H)S; N32(V.sub.H)D,
V48(V.sub.H)L, P52a(V.sub.H)A, S53(V.sub.H)A, T57(V.sub.H)S,
S96(V.sub.H)A, and Y100(V.sub.H)F, the V.sub.L of SEQ ID NO: 58
comprises one or more amino acid substitutions selected from the
group consisting of D28(V.sub.L)N, V29(V.sub.L)I, V29(V.sub.L)L,
S30(V.sub.L)A, A32(V.sub.L)D, Y92(V.sub.L)E, T93(V.sub.L)P,
T94(V.sub.L)E, and P96(V.sub.L)Y, numbered according to the Kabat
numbering system.
[0229] In one embodiment, the multispecific antibody comprises an
antigen-binding domain that specifically binds to EGFR and HER3
where the antibody comprises a V.sub.H comprising the amino acid
sequence of SEQ ID NO: 25 where the V.sub.H of SEQ ID NO: 25
comprises the amino acid substitutions F29(V.sub.H)L,
T30(V.sub.H)S, N32(V.sub.H)D, P52a(V.sub.H)A, and S53(V.sub.H)A,
and Y100(V.sub.H)F, and a V.sub.L comprising the amino acid
sequence of SEQ ID NO: 58 where the V.sub.L of SEQ ID NO: 58
comprises the amino acid substitutions D28(V.sub.L)N,
V29(V.sub.L)I, S30(V.sub.L)A, A32(V.sub.L)D, Y92(V.sub.L)E,
T93(V.sub.L)P, T94(V.sub.L)E, and P96(V.sub.L)Y, numbered according
to the Kabat numbering system.
[0230] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (e.g., alanine or
polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
immunoglobulins are screened for the desired activity.
[0231] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
Glycosylation Variants
[0232] In certain embodiments, an antibody of the invention is
altered to increase or decrease the extent to which the antibody is
glycosylated. Glycosylation of polypeptides is typically either
N-linked or O-linked. N-linked refers to the attachment of a
carbohydrate moiety to the side chain of an asparagine residue. The
tripeptide sequences asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars N-aceylgalactosamine,
galactose, or xylose to a hydroxyamino acid, most commonly serine
or threonine, although 5-hydroxyproline or 5-hydroxylysine may also
be used.
[0233] Addition or deletion of glycosylation sites to the antibody
is conveniently accomplished by altering the amino acid sequence
such that one or more of the above-described tripeptide sequences
(for N-linked glycosylation sites) is created or removed. The
alteration may also be made by the addition, deletion, or
substitution of one or more serine or threonine residues to the
sequence of the original antibody (for O-linked glycosylation
sites).
[0234] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
(1997) TIBTECH 15:26-32. The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody of the invention may be made in order to create antibody
variants with certain improved properties.
[0235] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e.g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (Eu numbering of Fc region residues);
however, Asn297 may also be located about .+-.3 amino acids
upstream or downstream of position 297, i.e., between positions 294
and 300, due to minor sequence variations in antibodies. Such
fucosylation variants may have improved ADCC function. See, e.g.,
US Patent Publication Nos. US 2003/0157108 (Presta, L.); US
2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications
related to "defucosylated" or "fucose-deficient" antibody variants
include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US
2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742;
WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004);
Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of
cell lines capable of producing defucosylated antibodies include
Lec13 CHO cells deficient in protein fucosylation (Ripka et al.
Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US
2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,
especially at Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0236] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by
GlcNAc.
[0237] Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
Fc Region Variants
[0238] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0239] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting
examples of in vitro assays to assess ADCC activity of a molecule
of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.
Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063
(1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA
82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp.
Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays
methods may be employed (see, for example, ACTI.TM. non-radioactive
cytotoxicity assay for flow cytometry (CellTechnology, Inc.
Mountain View, Calif.; and CytoTox 96.RTM. non-radioactive
cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells
for such assays include peripheral blood mononuclear cells (PBMC)
and Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. Proc.
Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also
be carried out to confirm that the antibody is unable to bind C1q
and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA
in WO 2006/029879 and WO 2005/100402. To assess complement
activation, a CDC assay may be performed (see, for example,
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg,
M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S., and M.
J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo
clearance/half life determinations can also be performed using
methods known in the art (see, e.g., Petkova, S. B. et al., Intl.
Immunol. 18(12):1759-1769 (2006)).
[0240] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0241] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001).)
[0242] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0243] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0244] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
[0245] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
Cysteine Engineered Antibody Variants
[0246] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
5400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
Antibody Derivatives
[0247] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0248] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed. See, for example,
Petkova, S. B. et al., Intl. Immunol. 18(12):1759-1769 (2006)).
[0249] Other antibody variants having one or more amino acid
substitutions are provided. Sites of interest for substitutional
mutagenesis include the hypervariable regions, but FR alterations
are also contemplated. Conservative substitutions are shown in
Table 1 under the heading of "preferred substitutions." More
substantial changes, denominated "exemplary substitutions" are
provided in Table 4, or as further described below in reference to
amino acid classes Amino acid substitutions may be introduced into
an antibody of interest and the products screened, e.g., for a
desired activity, such as improved antigen-binding, decreased
immunogenicity, improved ADCC or CDC, etc.
TABLE-US-00002 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; Ile; Val; Ile
Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine
[0250] Modifications in the biological properties of an antibody
may be accomplished by selecting substitutions that affect (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain Amino acids may be grouped according
to similarities in the properties of their side chains (in A. L.
Lehninger, in Biochemistry, second ed., pp. 73-75, Worth
Publishers, New York (1975)):
[0251] (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P),
Phe (F), Trp (W), Met (M)
[0252] (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr
(Y), Asn (N), Gln (.RTM.)
[0253] (3) acidic: Asp (D), Glu (E)
[0254] (4) basic: Lys (K), Arg (R), His(H)
[0255] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties:
[0256] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0257] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0258] (3) acidic: Asp, Glu;
[0259] (4) basic: H is, Lys, Arg;
[0260] (5) residues that influence chain orientation: Gly, Pro;
[0261] (6) aromatic: Trp, Tyr, Phe.
[0262] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, into the remaining (non-conserved) sites.
[0263] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further development will have modified (e.g.,
improved) biological properties relative to the parent antibody
from which they are generated. An exemplary substitutional variant
is an affinity matured antibody, which may be conveniently
generated using phage display-based affinity maturation techniques.
Briefly, several hypervariable region sites (e.g. 6-7 sites) are
mutated to generate all possible amino acid substitutions at each
site. The antibodies thus generated are displayed from filamentous
phage particles as fusions to at least part of a phage coat protein
(e.g., the gene III product of M13) packaged within each particle.
The phage-displayed variants are then screened for their biological
activity (e.g. binding affinity). In order to identify candidate
hypervariable region sites for modification, scanning mutagenesis
(e.g., alanine scanning) can be performed to identify hypervariable
region residues contributing significantly to antigen-binding.
Alternatively, or additionally, it may be beneficial to analyze a
crystal structure of the antigen-antibody complex to identify
contact points between the antibody and antigen. Such contact
residues and neighboring residues are candidates for substitution
according to techniques known in the art, including those
elaborated herein. Once such variants are generated, the panel of
variants is subjected to screening using techniques known in the
art, including those described herein, and variants with superior
properties in one or more relevant assays may be selected for
further development.
[0264] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
Immunoconjugates
[0265] The invention also provides immunoconjugates
(interchangeably referred to as "antibody-drug conjugates," or
"ADCs") comprising an antibody conjugated to one or more cytotoxic
agents, such as a chemotherapeutic agent, a drug, a growth
inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically
active toxin of bacterial, fungal, plant, or animal origin, or
fragments thereof), or a radioactive isotope, such as At.sup.211,
I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu
(i.e., a radioconjugate).
[0266] Immunoconjugates have been used for the local delivery of
cytotoxic agents, i.e., drugs that kill or inhibit the growth or
proliferation of cells, in the treatment of cancer (Lambert, J.
(2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al (2005)
Nature Biotechnology 23(9):1137-1146; Payne, G. (2003) i 3:207-212;
Syrigos and Epenetos (1999) Anticancer Research 19:605-614;
Niculescu-Duvaz and Springer (1997) Adv. Drug Deliv. Rev.
26:151-172; U.S. Pat. No. 4,975,278) Immunoconjugates allow for the
targeted delivery of a drug moiety to a tumor, and intracellular
accumulation therein, where systemic administration of unconjugated
drugs may result in unacceptable levels of toxicity to normal cells
as well as the tumor cells sought to be eliminated (Baldwin et al.,
Lancet (Mar. 15, 1986) pp. 603-05; Thorpe (1985) "Antibody Carriers
Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal
Antibodies '84: Biological And Clinical Applications (A. Pinchera
et al., eds) pp. 475-506. Both polyclonal antibodies and monoclonal
antibodies have been reported as useful in these strategies
(Rowland et al., (1986) Cancer Immunol. Immunother. 21:183-87).
Drugs used in these methods include daunomycin, doxorubicin,
methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins
used in antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst.
92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.
Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.
13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc.
Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al
(1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.
53:3336-3342). The toxins may exert their cytotoxic effects by
mechanisms including tubulin binding, DNA binding, or topoisomerase
inhibition. Some cytotoxic drugs tend to be inactive or less active
when conjugated to large antibodies or protein receptor
ligands.
[0267] Trastuzumab-DM1 (or T-DM1) has been shown to be efficacious
in trastuzumab-sensitive and trastuzumab-insensitive models of
HER2-overexpressing cancer. (U.S. Pat. No. 7,097,840). ZEVALIN.RTM.
(ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotope
conjugate composed of a murine IgG1 kappa monoclonal antibody
directed against the CD.sub.20 antigen found on the surface of
normal and malignant B lymphocytes and 111In or 90Y radioisotope
bound by a thiourea linker-chelator (Wiseman et al (2000) Eur.
Jour. Nucl. Med. 27(7):766-77; Wiseman et al (2002) Blood
99(12):4336-42; Witzig et al (2002) J. Clin. Oncol. 20(10):2453-63;
Witzig et al (2002) J. Clin. Oncol. 20(15):3262-69). Although
ZEVALIN has activity against B-cell non-Hodgkin's Lymphoma (NHL),
administration results in severe and prolonged cytopenias in most
patients. MYLOTARG.TM. (gemtuzumab ozogamicin, Wyeth
Pharmaceuticals), an antibody-drug conjugate composed of a huCD33
antibody linked to calicheamicin, was approved in 2000 for the
treatment of acute myeloid leukemia by injection (Drugs of the
Future (2000) 25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233;
5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285; 5,773,001).
Cantuzumab mertansine (Immunogen, Inc.), an antibody-drug conjugate
composed of the huC242 antibody linked via the disulfide linker SPP
to the maytansinoid drug moiety, DM1, is advancing into Phase II
trials for the treatment of cancers that express CanAg, such as
colon, pancreatic, gastric, and other cancers. MLN-2704 (Millennium
Pharm., BZL Biologics, Immunogen Inc.), an antibody-drug conjugate
composed of the anti-prostate specific membrane antigen (PSMA)
monoclonal antibody linked to the maytansinoid drug moiety, DM1, is
under development for the potential treatment of prostate tumors.
The auristatin peptides, auristatin E (AE) and monomethylauristatin
(MMAE), synthetic analogs of dolastatin, were conjugated to
chimeric monoclonal antibodies cBR96 (specific to Lewis Y on
carcinomas) and cAC10 (specific to CD30 on hematological
malignancies) (Doronina et al (2003) Nature Biotechnol.
21(7):778-784) and are under therapeutic development.
[0268] In certain embodiments, an immunoconjugate comprises an
antibody and a chemotherapeutic agent or other toxin.
Chemotherapeutic agents useful in the generation of
immunoconjugates are described herein (e.g., above). Enzymatically
active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active fragments of diphtheria
toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A
chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin, and the tricothecenes. See,
e.g., WO 93/21232 published Oct. 28, 1993. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic
agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al.,
Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0269] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, dolastatins,
aurostatins, a trichothecene, and CC1065, and the derivatives of
these toxins that have toxin activity, are also contemplated
herein.
Maytansine and Maytansinoids
[0270] In some embodiments, the immunoconjugate comprises an
antibody (full length or fragments) conjugated to one or more
maytansinoid molecules.
[0271] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.
[0272] Maytansinoid drug moieties are attractive drug moieties in
antibody drug conjugates because they are: (i) relatively
accessible to prepare by fermentation or chemical modification,
derivatization of fermentation products, (ii) amenable to
derivatization with functional groups suitable for conjugation
through the non-disulfide linkers to antibodies, (iii) stable in
plasma, and (iv) effective against a variety of tumor cell
lines.
[0273] Immunoconjugates containing maytansinoids, methods of making
same, and their therapeutic use are disclosed, for example, in U.S.
Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1,
the disclosures of which are hereby expressly incorporated by
reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.105 HER-2 surface antigens per
cell. The drug conjugate achieved a degree of cytotoxicity similar
to the free maytansinoid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
[0274] Antibody-maytansinoid conjugates are prepared by chemically
linking an antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. See, e.g., U.S. Pat. No.
5,208,020 (the disclosure of which is hereby expressly incorporated
by reference). An average of 3-4 maytansinoid molecules conjugated
per antibody molecule has shown efficacy in enhancing cytotoxicity
of target cells without negatively affecting the function or
solubility of the antibody, although even one molecule of
toxin/antibody would be expected to enhance cytotoxicity over the
use of naked antibody. Maytansinoids are well known in the art and
can be synthesized by known techniques or isolated from natural
sources. Suitable maytansinoids are disclosed, for example, in U.S.
Pat. No. 5,208,020 and in the other patents and nonpatent
publications referred to hereinabove. Preferred maytansinoids are
maytansinol and maytansinol analogues modified in the aromatic ring
or at other positions of the maytansinol molecule, such as various
maytansinol esters.
[0275] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1,
Chari et al., Cancer Research 52:127-131 (1992), and U.S. patent
application Ser. No. 10/960,602, filed Oct. 8, 2004, the
disclosures of which are hereby expressly incorporated by
reference. Antibody-maytansinoid conjugates comprising the linker
component SMCC may be prepared as disclosed in U.S. patent
application Ser. No. 10/960,602, filed Oct. 8, 2004. The linking
groups include disulfide groups, thioether groups, acid labile
groups, photolabile groups, peptidase labile groups, or esterase
labile groups, as disclosed in the above-identified patents,
disulfide and thioether groups being preferred. Additional linking
groups are described and exemplified herein.
[0276] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 (1978)) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0277] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
Auristatins and Dolastatins
[0278] In some embodiments, the immunoconjugate comprises an
antibody conjugated to dolastatins or dolostatin peptidic analogs
and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483;
5,780,588). Dolastatins and auristatins have been shown to
interfere with microtubule dynamics, GTP hydrolysis, and nuclear
and cellular division (Woyke et al (2001) Antimicrob. Agents and
Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No.
5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob.
Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug
moiety may be attached to the antibody through the N (amino)
terminus or the C (carboxyl) terminus of the peptidic drug moiety
(WO 02/088172).
[0279] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, disclosed in
"Monomethylvaline Compounds Capable of Conjugation to Ligands",
U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of
which is expressly incorporated by reference in its entirety.
[0280] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lake, "The Peptides", volume 1, pp 76-136, 1965, Academic Press)
that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug moieties may be prepared according to
the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;
Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al
(1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al.
Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.
Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol
21(7):778-784; "Monomethylvaline Compounds Capable of Conjugation
to Ligands", U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, hereby
incorporated by reference in its entirety (disclosing, e.g.,
linkers and methods of preparing monomethylvaline compounds such as
MMAE and MMAF conjugated to linkers).
Calicheamicin
[0281] In other embodiments, the immunoconjugate comprises an
antibody conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma.1I, .alpha.2I, .alpha.3I,
N-acetyl-.gamma.1I, PSAG and .theta.11 (Hinman et al., Cancer
Research 53:3336-3342 (1993), Lode et al., Cancer Research
58:2925-2928 (1998) and the aforementioned U.S. patents to American
Cyanamid). Another anti-tumor drug that the antibody can be
conjugated is QFA which is an antifolate. Both calicheamicin and
QFA have intracellular sites of action and do not readily cross the
plasma membrane. Therefore, cellular uptake of these agents through
antibody mediated internalization greatly enhances their cytotoxic
effects.
Other Cytotoxic Agents
[0282] Other antitumor agents that can be conjugated to the
antibodies include BCNU, streptozoicin, vincristine and
5-fluorouracil, the family of agents known collectively LL-E33288
complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well
as esperamicins (U.S. Pat. No. 5,877,296).
[0283] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0284] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0285] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
antibodies. Examples include At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu. When the conjugate is
used for detection, it may comprise a radioactive atom for
scintigraphic studies, for example tc99m or I123, or a spin label
for nuclear magnetic resonance (NMR) imaging (also known as
magnetic resonance imaging, mri), such as iodine-123 again,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
[0286] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Ree.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods in detail.
[0287] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat.
No. 5,208,020) may be used.
[0288] The compounds expressly contemplate, but are not limited to,
ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A). See pages 467-498, 2003-2004 Applications
Handbook and Catalog.
Preparation of Antibody Drug Conjugates
[0289] In the antibody drug conjugates (ADC), an antibody (Ab) is
conjugated to one or more drug moieties (D), e.g. about 1 to about
20 drug moieties per antibody, through a linker (L). The ADC of
Formula I may be prepared by several routes, employing organic
chemistry reactions, conditions, and reagents known to those
skilled in the art, including: (1) reaction of a nucleophilic group
of an antibody with a bivalent linker reagent, to form Ab-L, via a
covalent bond, followed by reaction with a drug moiety D; and (2)
reaction of a nucleophilic group of a drug moiety with a bivalent
linker reagent, to form D-L, via a covalent bond, followed by
reaction with the nucleophilic group of an antibody. Additional
methods for preparing ADC are described herein.
Ab-(L-D).sub.p I
[0290] The linker may be composed of one or more linker components.
Exemplary linker components include 6-maleimidocaproyl ("MC"),
maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"),
alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl
("PAB"), N-Succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"),
N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate
("SMCC`), and N-Succinimidyl (4-iodo-acetyl) aminobenzoate
("STAB"). Additional linker components are known in the art and
some are described herein. See also "Monomethylvaline Compounds
Capable of Conjugation to Ligands", U.S. Ser. No. 10/983,340, filed
Nov. 5, 2004, the contents of which are hereby incorporated by
reference in its entirety.
[0291] In some embodiments, the linker may comprise amino acid
residues. Exemplary amino acid linker components include a
dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
Exemplary dipeptides include: valine-citrulline (vc or val-cit),
alanine-phenylalanine (af or ala-phe). Exemplary tripeptides
include: glycine-valine-citrulline (gly-val-cit) and
glycine-glycine-glycine (gly-gly-gly) Amino acid residues which
comprise an amino acid linker component include those occurring
naturally, as well as minor amino acids and non-naturally occurring
amino acid analogs, such as citrulline Amino acid linker components
can be designed and optimized in their selectivity for enzymatic
cleavage by a particular enzymes, for example, a tumor-associated
protease, cathepsin B, C and D, or a plasmin protease.
[0292] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine,
and (iv) sugar hydroxyl or amino groups where the antibody is
glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic
and capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents including: (i) active
esters such as NHS esters, HOBt esters, haloformates, and acid
halides; (ii) alkyl and benzyl halides such as haloacetamides;
(iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain
antibodies have reducible interchain disulfides, i.e. cysteine
bridges. Antibodies may be made reactive for conjugation with
linker reagents by treatment with a reducing agent such as DTT
(dithiothreitol). Each cysteine bridge will thus form,
theoretically, two reactive thiol nucleophiles. Additional
nucleophilic groups can be introduced into antibodies through the
reaction of lysines with 2-iminothiolane (Traut's reagent)
resulting in conversion of an amine into a thiol. Reactive thiol
groups may be introduced into the antibody (or fragment thereof) by
introducing one, two, three, four, or more cysteine residues (e.g.,
preparing mutant antibodies comprising one or more non-native
cysteine amino acid residues).
[0293] Antibody drug conjugates may also be produced by
modification of the antibody to introduce electrophilic moieties,
which can react with nucleophilic substituents on the linker
reagent or drug. The sugars of glycosylated antibodies may be
oxidized, e.g. with periodate oxidizing reagents, to form aldehyde
or ketone groups which may react with the amine group of linker
reagents or drug moieties. The resulting imine Schiff base groups
may form a stable linkage, or may be reduced, e.g. by borohydride
reagents to form stable amine linkages. In one embodiment, reaction
of the carbohydrate portion of a glycosylated antibody with either
glactose oxidase or sodium meta-periodate may yield carbonyl
(aldehyde and ketone) groups in the protein that can react with
appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment, proteins containing N-terminal
serine or threonine residues can react with sodium meta-periodate,
resulting in production of an aldehyde in place of the first amino
acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146;
U.S. Pat. No. 5,362,852). Such aldehyde can be reacted with a drug
moiety or linker nucleophile.
[0294] Likewise, nucleophilic groups on a drug moiety include, but
are not limited to: amine, thiol, hydroxyl, hydrazide, oxime,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide groups capable of reacting to form covalent bonds
with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups.
[0295] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g., by recombinant techniques or
peptide synthesis. The length of DNA may comprise respective
regions encoding the two portions of the conjugate either adjacent
one another or separated by a region encoding a linker peptide
which does not destroy the desired properties of the conjugate.
[0296] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
Recombinant Methods and Compositions
[0297] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an anti-HER antibody
described herein is provided. Such nucleic acid may encode an amino
acid sequence comprising the VL and/or an amino acid sequence
comprising the VH of the antibody (e.g., the light and/or heavy
chains of the antibody). In a further embodiment, one or more
vectors (e.g., expression vectors) comprising such nucleic acid are
provided. In a further embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and an amino acid sequence
comprising the VH of the antibody, or (2) a first vector comprising
a nucleic acid that encodes an amino acid sequence comprising the
VL of the antibody and a second vector comprising a nucleic acid
that encodes an amino acid sequence comprising the VH of the
antibody. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an anti-HER
antibody is provided, wherein the method comprises culturing a host
cell comprising a nucleic acid encoding the antibody, as provided
above, under conditions suitable for expression of the antibody,
and optionally recovering the antibody from the host cell (or host
cell culture medium).
For recombinant production of an anti-HER antibody, nucleic acid
encoding an antibody, e.g., as described above, is isolated and
inserted into one or more vectors for further cloning and/or
expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0298] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular
Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.,
2003), pp. 245-254, describing expression of antibody fragments in
E. coli.) After expression, the antibody may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified. In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0299] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0300] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0301] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TR1 cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells
(Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and
myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
Therapeutic Uses
[0302] The antibodies and antibody fragments described herein can
be used for the treatment of cancer, including pre-cancerous,
non-metastatic, metastatic, and cancerous tumors (e.g., early stage
cancer), or for the treatment of a subject at risk for developing
cancer, for example, breast cancer. The antibodies and antibody
fragments can also be used to treat or prevent non-maligant
diseases, such as neurodegenerative disorders, psychiatric
disorders, or autoimmune diseases.
[0303] The term cancer embraces a collection of proliferative
disorders, including but not limited to pre-cancerous growths,
benign tumors, and malignant tumors. Benign tumors remain localized
at the site of origin and do not have the capacity to infiltrate,
invade, or metastasize to distant sites. Malignant tumors will
invade and damage other tissues around them. They can also gain the
ability to break off from where they started and spread to other
parts of the body (metastasize), usually through the bloodstream or
through the lymphatic system where the lymph nodes are located.
Primary tumors are classified by the type of tissue from which they
arise; metastatic tumors are classified by the tissue type from
which the cancer cells are derived. Over time, the cells of a
malignant tumor become more abnormal and appear less like normal
cells. This change in the appearance of cancer cells is called the
tumor grade and cancer cells are described as being
well-differentiated, moderately-differentiated,
poorly-differentiated, or undifferentiated. Well-differentiated
cells are quite normal appearing and resemble the normal cells from
which they originated. Undifferentiated cells are cells that have
become so abnormal that it is no longer possible to determine the
origin of the cells.
[0304] The tumor can be a solid tumor or a non-solid or soft tissue
tumor. Examples of soft tissue tumors include leukemia (e.g.,
chronic myelogenous leukemia, acute myelogenous leukemia, adult
acute lymphoblastic leukemia, acute myelogenous leukemia, mature
B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia,
polymphocytic leukemia, or hairy cell leukemia), or lymphoma (e.g.,
non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's
disease). A solid tumor includes any cancer of body tissues other
than blood, bone marrow, or the lymphatic system. Solid tumors can
be further separated into those of epithelial cell origin and those
of non-epithelial cell origin. Examples of epithelial cell solid
tumors include tumors of the gastrointestinal tract, colon, breast,
prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral
cavity, stomach, duodenum, small intestine, large intestine, anus,
gall bladder, labium, nasopharynx, skin, uterus, male genital
organ, urinary organs, bladder, and skin (including melanoma).
Solid tumors of non-epithelial origin include sarcomas, brain
tumors, and bone tumors.
[0305] Epithelial cancers generally evolve from a benign tumor to a
preinvasive stage (e.g., carcinoma in situ), to a malignant cancer,
which has penetrated the basement membrane and invaded the
subepithelial stroma.
[0306] In one embodiment, the multispecific antibodies specifically
bind EGFR and at least one other HER receptor, such as HER2 or HER3
or HER4, and find utility in the prevention and/or treatment of
solid tumors, in particular colorectal, lung (such as non-small
cell lung cancer and squamous cell carcinoma), head and neck,
ovarian, skin, pancreatic, and/or breast tumors.
[0307] The multispecific antibodies also find use in reducing or
preventing resistance to HER pathway targeted treatment. A
significant limitation in using treatments that target the HER
pathway is the resistance many cancer patients exhibit to the
therapeutic effects of the treatment. Some cancer patients show no
response to HER pathway targeted treatment. Other cancer patients
may show an initial response but then become resistant to the
treatment. A cancer is resistant to a treatment if the cancer has
progressed while receiving the treatment (refractory) or if the
cancer has progressed within 12 months after completing a treatment
regimen (relapse).
[0308] In one embodiment, the HER pathway targeted treatment
comprises treatment with antibodies that target the HER pathway
(for example, EGFR antibodies, HER2 antibodies, HER3 antibodies,
and/or HER4 antibodies). In another embodiment, the HER pathway
targeted treatment comprises treatment with a chemotherapeutic
agent.
Dosages and Formulations
[0309] The antibody or antibody fragment compositions herein will
be formulated, dosed, and administered in a fashion consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual
patient, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The "therapeutically effective amount" of the antibody or antibody
fragment to be administered will be governed by such
considerations, and is the minimum amount necessary to prevent,
ameliorate, or treat a cancer. The antibody or antibody fragment
need not be, but is optionally formulated with one or more agents
currently used to prevent or treat cancer or a risk of developing a
cancer. The effective amount of such other agents depends on the
amount of antibody or antibody fragment present in the formulation,
the type of disorder or treatment, and other factors discussed
above. These are generally used in the same dosages and with
administration routes as used hereinbefore or about from 1 to 99%
of the heretofore employed dosages. Generally, alleviation or
treatment of a cancer involves the lessening of one or more
symptoms or medical problems associated with the cancer. The
therapeutically effective amount of the drug can accomplish one or
a combination of the following: reduce (by at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100% or more) the number of cancer
cells; reduce or inhibit the tumor size or tumor burden; inhibit
(i.e., to decrease to some extent and/or stop) cancer cell
infiltration into peripheral organs; reduce hormonal secretion in
the case of adenomas; reduce vessel density; inhibit tumor
metastasis; reduce or inhibit tumor growth; and/or relieve to some
extent one or more of the symptoms associated with the cancer. In
some embodiments, the antibody or antibody fragment is used to
prevent the occurrence or reoccurrence of cancer in the
subject.
[0310] In one embodiment, the present invention can be used for
increasing the duration of survival of a human patient susceptible
to or diagnosed with a cancer. Duration of survival is defined as
the time from first administration of the drug to death. Duration
of survival can also be measured by stratified hazard ratio (HR) of
the treatment group versus control group, which represents the risk
of death for a patient during the treatment.
[0311] In yet another embodiment, the treatment of the present
invention significantly increases response rate in a group of human
patients susceptible to or diagnosed with a cancer who are treated
with various anti-cancer therapies. Response rate is defined as the
percentage of treated patients who responded to the treatment. In
one aspect, the combination treatment of the invention using an
antibody or antibody fragment and surgery, radiation therapy, or
one or more chemotherapeutic agents significantly increases
response rate in the treated patient group compared to the group
treated with surgery, radiation therapy, or chemotherapy alone, the
increase having a Chi-square p-value of less than 0.005.
[0312] Additional measurements of therapeutic efficacy in the
treatment of cancers are described in U.S. Patent Application
Publication No. 20050186208.
[0313] Therapeutic formulations are prepared using standard methods
known in the art by mixing the active ingredient having the desired
degree of purity with optional physiologically acceptable carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences
(20.sup.th edition), ed. A. Gennaro, 2000, Lippincott, Williams
& Wilkins, Philadelphia, Pa.). Acceptable carriers, include
saline, or buffers such as phosphate, citrate and other organic
acids; antioxidants including ascorbic acid; low molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone, amino acids such as glycine, glutamine,
asparagines, arginine or lysine; monosaccharides, disaccharides,
and other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN.TM., PLURONICS.TM., or PEG.
[0314] Optionally, but preferably, the formulation contains a
pharmaceutically acceptable salt, preferably sodium chloride, and
preferably at about physiological concentrations. Optionally, the
formulations of the invention can contain a pharmaceutically
acceptable preservative. In some embodiments the preservative
concentration ranges from 0.1 to 2.0%, typically v/v. Suitable
preservatives include those known in the pharmaceutical arts.
Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben
are preferred preservatives. Optionally, the formulations of the
invention can include a pharmaceutically acceptable surfactant at a
concentration of 0.005 to 0.02%.
[0315] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0316] The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, supra.
[0317] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsule. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0318] The antibodies and antibody fragments described herein are
administered to a human subject, in accord with known methods, such
as intravenous administration as a bolus or by continuous infusion
over a period of time, by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. Local
administration may be particularly desired if extensive side
effects or toxicity is associated with HER (e.g. EGFR, HER2, HER3,
HER4 etc.) antagonism. An ex vivo strategy can also be used for
therapeutic applications. Ex vivo strategies involve transfecting
or transducing cells obtained from the subject with a
polynucleotide encoding an antibody or antibody fragment. The
transfected or transduced cells are then returned to the subject.
The cells can be any of a wide range of types including, without
limitation, hemopoietic cells (e.g., bone marrow cells,
macrophages, monocytes, dendritic cells, T cells, or B cells),
fibroblasts, epithelial cells, endothelial cells, keratinocytes, or
muscle cells.
[0319] In one example, the antibody or antibody fragment is
administered locally, e.g., by direct injections, when the disorder
or location of the tumor permits, and the injections can be
repeated periodically. The antibody or antibody fragment can also
be delivered systemically to the subject or directly to the tumor
cells, e.g., to a tumor or a tumor bed following surgical excision
of the tumor, in order to prevent or reduce local recurrence or
metastasis.
[0320] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with one or more other additional therapeutic agents)
will depend on the type of disease to be treated, the type of
antibody, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 20 mg/kg (e.g. 0.1 mg/kg-15 mg/kg)
of antibody can be an initial candidate dosage for administration
to the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody would be in the range from about 0.05 mg/kg
to about 20 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, or 20 mg/kg (or any
combination thereof) may be administered to the patient. Such doses
may be administered intermittently, e.g. every week, every two
weeks, or every three weeks (e.g. such that the patient receives
from about two to about twenty, or e.g. about six doses of the
antibody). An initial higher loading dose, followed by one or more
lower doses may be administered. An exemplary dosing regimen
comprises administering an initial loading dose of about 4 mg/kg,
followed by a weekly maintenance dose of about 2 mg/kg of the
antibody. However, other dosage regimens may be useful. The
progress of this therapy is easily monitored by conventional
techniques and assays.
Combination Therapy
[0321] An antibody of the invention may be combined in a
pharmaceutical combination formulation, or dosing regimen as
combination therapy, with a second compound having anti-cancer
properties. The second compound of the pharmaceutical combination
formulation or dosing regimen may have complementary activities to
the antibody of the combination such that they do not adversely
affect each other. In one embodiment, the multispecific antibody is
used in combination with another anti-HER antibody, such as
HERCEPTIN.RTM., pertuzumab, and/or cetuximab. Antibodies of the
invention can also be used in combination with radiation
therapy.
[0322] The second compound may be a chemotherapeutic agent,
cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal
agent, and/or cardioprotectant. Such molecules are suitably present
in combination in amounts that are effective for the purpose
intended. A pharmaceutical composition containing an antibody of
the invention may also have a therapeutically effective amount of a
chemotherapeutic agent such as a tubulin-forming inhibitor, a
topoisomerase inhibitor, a DNA intercalator, or a DNA binder.
[0323] Other therapeutic regimens may be combined with the
administration of an anticancer agent identified in accordance with
this invention. The combination therapy may be administered as a
simultaneous or sequential regimen. When administered sequentially,
the combination may be administered in two or more administrations.
The combined administration includes coadministration, using
separate formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein there is a time
period while both (or all) active agents simultaneously exert their
biological activities.
[0324] Examples of such combination therapy include combinations
with chemotherapeutic agents such as erlotinib (TARCEVA.RTM.,
Genentech/OSI Pharm.), bortezomib (VELCADE.RTM., Millenium Pharm.),
fulvestrant (FASLODEX.RTM., AstraZeneca), sutent (SU11248, Pfizer),
letrozole (FEMARA.RTM., Novartis), imatinib mesylate (GLEEVEC.RTM.,
Novartis), PTK787/ZK 222584 (Novartis), oxaliplatin (Eloxatin.RTM.,
Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin (Sirolimus,
RAPAMUNE.RTM., Wyeth), lapatinib (TYKERB.RTM., GSK572016,
GlaxoSmithKline), lonafarnib (SCH 66336), sorafenib (BAY43-9006,
Bayer Labs.), and gefitinib (IRESSA.RTM., AstraZeneca), AG1478,
AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and
CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates such as busulfan,
improsulfan and piposulfan; antifolate antineoplastic such as
pemetrexed (ALIMTA.RTM. Eli Lilly), aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosoureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics, calicheamicin, calicheamicin
gamma1I and calicheamicin omegaI1; dynemicin, including dynemicin
A; bisphosphonates, such as clodronate; an esperamicin; as well as
neocarzinostatin chromophore and related chromoprotein enediyne
antibiotic chromophores, aclacinomysins, actinomycin, anthramycin,
azaserine, bleomycins, cactinomycin, carabicin, caminomycin,
carzinophilin, chromomycinis, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM.
doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., paclitaxel (TAXOL.RTM., Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.TM. Cremophor-free, albumin,
nanoparticle formulation of paclitaxel (American Pharmaceutical
Partners, Schaumberg, Ill.), and TAXOTERE.RTM. doxetaxel
(Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR.RTM.
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; platinum;
etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;
NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate;
daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; capecitabine; and pharmaceutically acceptable
salts, acids or derivatives of any of the above.
[0325] Such combination therapy also includes: (i) anti-hormonal
agents that act to regulate or inhibit hormone action on tumors
such as anti-estrogens and selective estrogen receptor modulators
(SERMs), including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and
FARESTON.cndot.toremifene; (ii) aromatase inhibitors that inhibit
the enzyme aromatase, which regulates estrogen production in the
adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, MEGACE.RTM. megestrol acetate, AROMASIN.RTM.
exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole,
FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole; (iii)
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; as well as troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase
inhibitors; (v) lipid kinase inhibitors; (vi) antisense
oligonucleotides, particularly those which inhibit expression of
genes in signaling pathways implicated in abherant cell
proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;
(vii) ribozymes such as a VEGF expression inhibitor (e.g.,
ANGIOZYME.RTM. ribozyme) and a HER2 expression inhibitor; (viii)
vaccines such as gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; PROLEUKIN.RTM. rIL-2; LURTOTECAN.RTM. topoisomerase 1
inhibitor; ABARELIX.RTM. rmRH; (ix) anti-angiogenic agents such as
bevacizumab (AVASTIN.RTM., Genentech); and (x) pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0326] Preparation and dosing schedules for such chemotherapeutic
agents may be used according to manufacturer's instructions or as
determined empirically by the skilled practitioner. Preparation and
dosing schedules for such chemotherapy are also described in
Chemotherapy Service, (1992) Ed., M. C. Perry, Williams &
Wilkins, Baltimore, Md.
[0327] The combination therapy may provide "synergy" and prove
"synergistic", i.e. the effect achieved when the active ingredients
used together is greater than the sum of the effects that results
from using the compounds separately. A synergistic effect may be
attained when the active ingredients are: (1) co-formulated and
administered or delivered simultaneously in a combined, unit dosage
formulation; (2) delivered by alternation or in parallel as
separate formulations; or (3) by some other regimen. When delivered
in alternation therapy, a synergistic effect may be attained when
the compounds are administered or delivered sequentially, e.g. by
different injections in separate syringes. In general, during
alternation therapy, an effective dosage of each active ingredient
is administered sequentially, i.e. serially, whereas in combination
therapy, effective dosages of two or more active ingredients are
administered together.
Articles of Manufacture and Kits
[0328] Another embodiment of the invention is an article of
manufacture containing materials useful for the treatment of
cancers. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
etc. The containers may be formed from a variety of materials such
as glass or plastic. The container holds a composition which is
effective for treating the condition and may have a sterile access
port (for example the container may be an intravenous solution bag
or a vial having a stopper pierceable by a hypodermic injection
needle). At least one active agent in the composition is a
multispecific antibody or antibody fragment antibody of the
invention. The label or package insert indicates that the
composition is used for treating the particular condition. The
label or package insert will further comprise instructions for
administering the antibody composition to the patient. Articles of
manufacture and kits comprising combinatorial therapies described
herein are also contemplated.
[0329] Package insert refers to instructions customarily included
in commercial packages of therapeutic products that contain
information about the indications, usage, dosage, administration,
contraindications and/or warnings concerning the use of such
therapeutic products. In one embodiment, the package insert
indicates that the composition is used for treating a solid tumor,
such as, for example, colorectal, lung and/or breast cancer.
[0330] Additionally, the article of manufacture may further
comprise a second container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0331] Kits are also provided that are useful for various purposes,
e.g., for purification or immunoprecipitation of HER receptors from
cells. For isolation and purification of EGFR and/or HER2 and/or
HER3 and/or HER4, the kit can contain an EGFR/HER2 and/or EGFR/HER3
and/or EGFR/HER4 antibody coupled to beads (e.g., SEPHAROSE.RTM.
beads). Kits can be provided which contain the antibodies for
detection and quantitation of the desired HER receptor in vitro,
e.g., in an ELISA or a Western blot. As with the article of
manufacture, the kit comprises a container and a label or package
insert on or associated with the container. The container holds a
composition comprising at least one multispecific antibody or
antibody fragment of the invention. Additional containers may be
included that contain, e.g., diluents and buffers or control
antibodies. The label or package insert may provide a description
of the composition as well as instructions for the intended in
vitro or diagnostic use.
[0332] The foregoing written description is considered to be
sufficient to enable one skilled in the art to practice the
invention. The following Examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
[0333] Commercially available reagents referred to in the Examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
Examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Unless otherwise noted, the present invention uses standard
procedures of recombinant DNA technology, such as those described
hereinabove and in the following textbooks: Sambrook et al., supra;
Ausubel et al., Current Protocols in Molecular Biology (Green
Publishing Associates and Wiley Interscience, N.Y., 1989); Innis et
al., PCR Protocols: A Guide to Methods and Applications (Academic
Press, Inc.: N.Y., 1990); Harlow et al., Antibodies: A Laboratory
Manual (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait,
Oligonucleotide Synthesis (IRL Press: Oxford, 1984); Freshney,
Animal Cell Culture, 1987; Coligan et al., Current Protocols in
Immunology, 1991.
EXAMPLES
Example 1
Isolation and Characterization of Antibodies Binding Human EGFR
Materials
[0334] Enzymes and M13-KO7 helper phage were purchased from New
England Biolabs. E. coli XL1-Blue was from Stratagene. Bovine serum
albumin (BSA), ovalbumin, and Tween 20 were purchased from Sigma.
Neutravidin, casein and Superblock were purchased from Pierce.
anti-M13 conjugated horse-radish peroxidase (HRP) was purchased
from Amersham Pharmacia. Maxisorp immunoplates were purchased from
NUNC (Roskilde, Denmark). Tetramethylbenzidine (TMB) substrate was
purchased from Kirkegaard and Perry Laboratories (Gaithersburg,
Md.).
Library Construction
[0335] Phage displayed antibody libraries were generated based on
human antibody framework from humanized 4D5 (h4D5, trastuzumab),
where side chain and length diversity were incorporated into heavy
chain complementarity determining regions (CDR1, CDR2, CDR3) in the
first library (Library 1), and into heavy chain CDRs and light
chain CDR3 in the second library (Library 2) still focusing on the
diverstification in heavy chain as described (Lee et al., J. Mol.
Biol, 340: 1073-1093 (2004)). Libraries were constructed as
described (Lee et al., J. Mol. Biol, 340: 1073-1093 (2004)) except
that the degenerate oligonucleotides used were modified
modestly.
Sorting of the Two Libraries.
[0336] Library 1 and Library 2 were directly subjected to target
(hEGFR-ECD-Fc (human EGFR extracellular domain fused to an Fc
region of human IgG1) and EGFRvIII-Fc) binding selection.
EGFRvIII-Fc protein is a variant of EGFR missing most of domain II
(E1-P353 (not including the signal peptide)) fused to an Fc region
of hIgG1. See Kuan, C-T, et al., Endocrine-Related Canc. 8: 83-96
(2001); Bigner, S H, et al., Cancer Research, 50: 8017-8022 (1990).
96-well Nunc Maxisorp plates were coated with 100W/well of target
antigen (hEGFR-ECD-Fc, EGFRvIII-Fc) (5 m/ml) in PBS (0.05M Sodium
Carbonate buffer, pH9.6) at 4 C overnight or room temperature for 2
hours. The plates were blocked with alternating blocking agents.
Phage solutions of 10.sup.13 phage/ml (3-50D/ml) were incubated
with the coated antigens for 18 h in the first round of selection.
Phage input was decreased in each round of selection as following:
1.sup.st round 3-5 O.D/ml, 2.sup.nd round 3 O.D/int, 3.sup.rd round
0.5.about.1 O.D/int and 4.sup.th round input 0.1.about.0.5 O.D/int.
The diluted phage was incubated for 30 minutes on ice. 1 .mu.M of
an Fc-Fusion protein was added to the blocked phage from 3.sup.rd
round to remove Fc binders. Following incubation of the phage
solutions (100 .mu.l/well to 8 target antigen-coated wells and 2
uncoated wells) on the immunoplates to allow binding to the
immobilized antigen (overnight for 1.sup.st round, 2 hours for
remaining rounds), immunoplates were washed at least ten times
continuously with PBS and 0.05% Tween 20. Bound phage was eluted
with 100 ul/well of 100 mM HCl at room temperature for 20 minutes.
The eluted phage (from coated wells) and background phage (from
uncoated wells) were neutralized by adding 1/10 volume 1M Tris pH
11.0 and BSA to final 0.1%. The recovery of phage per
antigen-coated immunoplate well was calculated and compared to that
of a blocked well without coated antigen to study the enrichment of
phage clones displaying Fabs that specifically bound the target
antigen. Eluted phage were amplified in E. coli and used for
further rounds of selection.
High-Throughput Characterization of hEGFR Binding Clones
[0337] Random clones from round 4 were selected for screening and
assayed using phage ELISA in which binding to target and anti-gD
was compared to binding of non-relevant proteins (BSA, HER2, an
anti-EGFR antibody, trastuzumab).
[0338] 384-well format immunoplates were coated with 1 .mu.g/ml of
target, anti-gD and non-relevant proteins at 4.degree. C. overnight
or room temperature for 2 hours and blocked 1 h with 1% BSA in PBS.
Phagemid clones in E. coli XL1-Blue were grown in 150 ul of 2YT
broth supplemented with carbenicillin and M13-KO7 helper phage; the
cultures were grown with shaking overnight at 37.degree. C. in a
96-well format. Culture supernatants containing phage were diluted
five fold in PBST (PBS with 0.05% Tween 20 and 0.5% (w/v) BSA). 30
.mu.l of mixture was added to each quadrant of 384-well coated
plate and incubated at room temperature for 1 hour. The plate was
washed with PBT (PBS with 0.05% Tween 20) and incubated for 30
minutes with anti-M13 antibody horse-radish peroxidase conjugate
diluted 5000-fold to 1 nM in PBST. The plates were washed,
developed with TMB substrate for approximately five minutes,
quenched with 1.0 M H.sub.3PO.sub.4, and read
spectrophotometrically at 450 nm
[0339] The clones that bound the anti-gD antibody and target but
not the non-specific proteins were considered specific positives.
VH library enabled the isolation of specific binders for both
EGFR-ECD-Fc and EGFRvIII-Fc.
Solution Binding Competition ELISA
[0340] The binding affinity of selected binding clones was
determined by solution binding competition ELISA.
[0341] Selected phagemid clones in E. coli XL1-Blue were grown in
20 ml of 2YT broth supplemented with carbenicillin, Kanamycin and
M13-KO7 helper phage; the cultures were grown with shaking
overnight at 30.degree. C. Supernatant of phage was purified by
double precipitation in 20% PEG/2.5M NaCl, resuspended in PBS, and
read spectrophotometrically at 268 nM for concentration
determination (in OD/ml).
[0342] Purified phage was titered on immunoplates coated with 1
.mu.g/ml of hEGFR-ECD-Fc to determine optimal concentration for
solution competition ELISA.
[0343] The dilution that gave a 10D/ml signal at 450 nM was used in
the solution binding assay in which phage were first incubated with
increasing concentration of antigen (hEGFR-ECD) for one hour and
then transferred to antigen-coated immunoplates for 15 minutes to
capture unbound phage. IC.sub.50 was calculated as the
concentration of antigen in solution-binding stage that inhibited
50% of the phage from binding to immobilized antigen. The solution
binding competition ELISA was performed at room temperature. The
IC.sub.50 values for selected clones ranged from 36.2 nM to
>1000 nM.
Conversion of Phage Displaying F(ab)zip to Human IgG
[0344] To accurately determine affinity, specificity and other
properties, selected clones were expressed as free hIgG.
[0345] The variable domains of light chain and heavy chain were
cloned into vectors previously designed for transient human IgG
expression in mammalian cells. (Leet et al., J. Mol. Biol.
23:340:1073-1093 (2004)).
[0346] The V.sub.L region of phagemid DNA was digested with
restriction enzymes, which cleaved the DNA upstream of the region
encoding for CDR L1 (EcoRV) and downstream of the region encoding
for CDR L3 (KpnI).
[0347] The V.sub.H region of phagemid DNA was digested with
restriction enzymes, which cleaved the DNA upstream of the region
encoding for CDR H1 (ApaI) and downstream of the region encoding
for CDR H3 (Bsiwl).
[0348] Secreted free IgG were purified with protein A affinity
chromatography and tested in direct binding ELISA on hEGFR-coated
immunoplates.
Comparison of anti-hEGFR Epitopes
[0349] The ability of the isolated anti-EGFR antibodies to compete
with another anti-hEGFR antibody known to bind to domain III of
EGFR was studied in order to determine the epitopes recognized by
the anti-hEGFR antibodies.
[0350] The assays were done in a competitive ELISA format. For the
competitive ELISA, EGFR-ECD-Fc was immobilized on Maxisorp
immunoplates at 2 .mu.g/ml. A fixed concentration of the anti-EGFR
antibody (or an unspecific antibody control) was captured by coated
EGFR-ECD-Fc and the purified selected anti-EGFR phage-Fabs were
added, and detected with .alpha.-M13-conjugated HRP. Antibodies
that were blocked from binding EGFR-ECD-Fc coated plates are likely
to share over-lapping epitopes.
[0351] For the TGF-.alpha. competitive ELISA, EGFR-ECD-Fc was
captured first by coated anti-human Fc antibody, then a fixed
concentration of TGF-.alpha. was captured by EGFR-ECD-Fc. The
purified phage-Fabs were added, and detected with
.alpha.-M13-conjugated HRP.
[0352] Finally, the binding of selected clones to constructs with
exposed and deleted domains of EGFR was assessed to confirm
previous competitive ELISA. Clone designated D1 competes with the
anti-EGFR antibody and is likely to bind EGFR at domain III.
Example 2
Characterization of antibodies against human Epidermal Growth
Factor Receptor
Inhibition of Ligand Binding
[0353] order to determine if selected anti-EGFR antibody D1
inhibits .sup.125I-EGF binding to H1666 cells (ATCC CRL-5885,
Manassas, Va.), the purified IgGs version of D1 was tested in a
ligand binding assay as follows. H1666 cells were plated in 12 well
plates. The next day growth medium was removed and cells were
pretreated with 200 nM antibody for 2 hours at room RT. 20
.mu.l/well of radiolabelled EGF was added (use conc. below Kd of
cell line) and cells were treated for an additional 2 hours at room
temperature. Cells were washed with binding buffer and solubilized,
transferred and samples were counted using an iso Data
.gamma.-counter. Unlabelled EGF was used as a cold competitor. The
results demonstrate that D1 inhibits .sup.125I-EGF ligand binding
to H1666 cells.
Inhibition of TGF-.alpha. induced EGFR Phosphorylation in Stably
Transfected EGFR-NR6 Cells
[0354] To determine if anti-EGFR antibody D1 selectively blocks
TGF-.alpha. induced EGFR phosphorylation, stably transfected
EGFR-NR6 cells were serum starved for 2-3 hours and pre-incubated
with various concentrations of D1 for 2 hours. Cells were
stimulated with 1 nM TGF-.alpha.. Whole cell lysates were subjected
to SDS-PAGE analysis, and immunoblots were probed with
anti-phosphotyrosine and anti-EGFR as a loading control. A
commercially available anti-EGFR antibody was used as a positive
control. The data demonstrated that D1 inhibits TGF-.alpha. induced
EGFR phosphorylation in a cell based assay.
Example 3
Affinity Maturation of D1
Library Construction
[0355] Two phage-displayed libraries (L3/H3 and L3/H1H2 libraries)
were created using oligonucleotide-directed mutagenesis as
described (Lee et al., Blood, 108, 3103-3111, 2006). The library
template vectors contained a stop codon (TAA) embedded in CDR-L3,
which was repaired during the mutagenesis reaction using degenerate
oligonucleotides that annealed over the sequences encoding CDR-L3
(all libraries), CDR-H3 (L3/H3 library), CDR-H2 and CDR H1 (L3/H1H2
library). The library mutagenesis reactions were performed
according to the method of Kunkel et al (Methods Enzymol. 1987;
154:367-82). The oligonucleotides were combined in different ratios
to fine-tune the diversity to reflect the amino acid frequency in
natural repertoire at selected positions. The mutagenesis products
were pooled into one reaction per library and electroporated in to
E. coli SS320 cells and grown supplemented with KO7 helper phage as
described (Lee et al., 2004, supra).
Library Sorting and Screening
[0356] For affinity improvement selection, phage libraries were
subjected to plate sorting against hEGFR-ECD-Fc for the first
round, followed by three rounds of solution sorting.
[0357] Three rounds of solution sorting were performed with
increasing stringency of selection Immunoplates were coated with 5
ug/ml Neutravidin overnight at 4.degree. C. and blocked with
Superblock (Pierce) and PBST. 3-50D/ml of propagated phage was
pre-incubated with 100 nM of biotinylated EGFR-ECD in Superblock,
then diluted 10.times. and added to blocked immunoplates for 5-10
minutes. Plates were washed 8 times, the phage were eluted and
propagated for next round of solution sorting, decreasing phage
input (0.5 OD/ml) and concentration of biotinylated EGFR-ECD down
to 1 nM.
High throughput Affinity Screening ELISA (Single Spot
Competition)
[0358] Random clones from last round of solution sorting were
picked for screening and assayed using phage single point
competition ELISA in which binding to target of phage supernatant
pre-incubated (or not) with hEGFR-ECD is compared.
[0359] Phagemid clones in E. coli XL1-Blue were grown in 150 ul of
2YT broth supplemented with carbenicillin and M13-KO7 helper phage;
the cultures were grown with shaking overnight at 37.degree. C. in
a 96-well format. Culture supernatants containing phage were
diluted five fold in PBST (PBS with 0.05% Tween 20 and 0.5% (w/v)
BSA) with or without 5 nM EGFR-EC). The OD reduction (%) was
calculated by the following equation:
OD.sub.450nm reduction (%)=(OD.sub.450nm of wells with
competitor)/(OD.sub.450nm of well with no competitor)*100
[0360] Clones with OD reduction greater that 25% were selected for
solution binding competition ELISA.
Solution Binding Competition ELISA
[0361] The binding affinity of selected binding clones by single
spot ELISA was determined by solution binding competition ELISA as
described above.
[0362] The phage IC50s of one clone selected from D1 parent clone
affinity maturation (D1.5) was determined as described above. The
IC50 for D1.5 was 0.39 nM. D1.5 was reformatted into mIgG2A for
further characterization, using same restriction site described
above and vectors designed for transient murine IgG expression in
mammalian cells.
Example 4
Characterization of Anti-EGFR Improved Antibodies as Free mIgG
BIAcore Measurement
[0363] Surface plasmon resonance assays on a BIAcore.TM.-2000 was
used to determine the affinity of anti-EGFR mIgG2A. Immobilized
mIgG D1.5 on CM5 chips at .about.150 response units (RU) were used
in the BIAcore assays. Increasing concentration from 12.5 nM to 200
nM of EGFR-ECD were injected at 30 .mu.l/min at 25.degree. C.
Binding responses were corrected by subtracting RU from a blank
flow cell. For kinetics analysis, 1:1 Languir model of simultaneous
fitting of k.sub.on and k.sub.off was used. The KD determined by
this method for D1.5 was 1.2 nM.
Epitope Mapping
[0364] The binding of selected clones to constructs with exposed
and deleted domains of EGFR was assessed to confirm inherited
binding epitope from parent clones. D1.5, the affinity improved
clone selected from D1 sorting, binds EGFR at domain III.
Inhibition of TGF-.alpha. Induced EGFR Phosphorylation in EGFR-NR6
Cells
[0365] D1.5 was tested to determine if it inhibited TGF-.alpha.
induced EGFR phosphorylation with higher potency compared to the
parental clone. The antibodies were tested on EGFR-NR6 cells and
the assay was performed as described above in Example 2. A
commercially available anti-EGFR antibody was used as a positive
control.
[0366] The results demonstrate that D1.5 showed a greater
inhibition of ligand induced EGFR phosphorylation when compared
with parent clone D1.
Inhibition of Cell Proliferation in H1666 Cells
[0367] An assay was performed to determine whether D1.5 inhibits
cell proliferation of a NSCLC cancer cell line, H1666, that
expresses EGFR, HER2 and HER3 at moderate levels. H1666 cells (ATCC
CRL-5885, Manassas, Va.) were seeded in 96 well plates at a density
of 5000 cells. The following day cells were simultaneously treated
with various concentrations of antibody (up to 50 ug/ml) in 1%
serum containing medium. After 3 days Alamar Blue was added and
fluorescence was detected using a fluorometer. The results were
expressed in RFUs.
[0368] The results demonstrate that D1.5 showed a greater
inhibition of cell growth than D1.
A431 Xenograft Study
[0369] An A431 xenograft model was used to validate the in-vivo
efficacy of affinity matured anti-EGFR antibody D1.5. A431 cells
are EGFR amplified and respond very well to anti-EGFR agents. The
study was conducted in nu/nu mice and a commercially available
anti-EGFR antibody was used as a reference.
[0370] In a first step, the cross-reactivity of D1.5 with murine
EGFR was assessed in a competitive ELISA. Immunoplates were coated
with hEGFR-ECD-Fc. Serial dilutions of mEGFR-ECD-Fc were incubated
with fixed concentration of D1.5. The results show that D1.5
cross-reacted with murine EGFR. To assess the dosing needed for the
in vivo efficacy study, D1.5 was first injected at a single dose
(50 mg/kg) and serum IgG concentration were measured by ELISA. D1.5
was cleared more rapidly and diminishing concentrations were seen
after seven days post injection. The efficacy study (A431 xenograft
model) revealed that D1.5 inhibited tumor growth completely. D1.5
was as effective as the anti-EGFR antibody at 50 mg/kg. Its reduced
potency in the lower dose group (25 mg/kg) was due to the faster
clearance of the antibody. (FIG. 1.)
Example 5
Light Chain Library Design and Screening for Bispecific
Antibodies
Libraries Design and Construction
[0371] Libraries based on a D1.5 template were designed for
diversifying the amino acid composition and CDR length of antibody
light chain. A subset of the randomized positions were tailored to
represent amino acids which are part of the natural repertoire at
these sites, whereas the remaining sites were randomized to include
all 20 naturally occurring amino acids. In addition, randomized
positions in CDR L3 were tailored and biased toward the native
sequence of the template because this CDR is considered important
for the binding of D1.5. For CDR L1, each length was a mix of three
oligonucleotides containing codons for positions 28-33. For longer
L1, NNK was inserted in between position 30 and 31.
TABLE-US-00003 Positions 28 29 30 31 32 33 CDR-L1
G.sub.70A.sub.70C.sub.70 RTT NNK NNK TAC STA
G.sub.70A.sub.70C.sub.70 RTT NNK NNK DGG STA
G.sub.70A.sub.70C.sub.70 RTT NNK NNK NMT STA
For CDR L2, four oligonucleotides were mixed 1:1:2:10.
TABLE-US-00004 50 51 52 53 CDR-L2 NKK GST TCC NNK TGG GST TCC NNK
KGG GST TCC TMT NKK GST TCC TMT N = A/C/G/T, D = A/G/T, V = A/C/G,
B = C/G/T, H = A/C/T, K = G/T, M = A/C, R = A/G, S = G/C, W = A/T,
Y = C/T. *G.sub.70A.sub.70C.sub.70 allows 70% of the designated
nucleotide and 10% each of the other three to encode approximately
50% Glu and 50% of the other amino acids. See U.S. Patent
Publication No. 20080069820 and Bostrom, J. et al., Science 323:
1610-1614(2009).
[0372] The diversity for CDR L3 is derived from the mixture of the
following oligonucleotides. D1.5 CDR_L3 oligonucleotides
TABLE-US-00005 (SEQ ID: 70) F693 ACTTATTAC TGT CAG CAA 878 NNK 776
RST CCT TAC ACG TTC GGA (SEQ ID: 71) F694 ACTTATTAC TGT CAG CAA 878
TAC 776 RST CCT TAC ACG TTC GGA (SEQ ID: 72) F695 ACTTATTAC TGT CAG
CAA DGG NNK 776 577 CCT TAC ACG TTC GGA (SEQ ID: 73) F696 ACTTATTAC
TGT CAG CAA KMT NNK 776 577 CCT TAC ACG TTC GGA (SEQ ID: 74) F697
ACTTATTAC TGT CAG CAA 878 NNK 776 NNK CCT TAC ACG TTC GGA (SEQ ID:
75) F698 ACTTATTAC TGT CAG CAA DGG NNK NNK RST CCT TAC ACG TTC GGA
(SEQ ID: 76) F699 ACTTATTAC TGT CAG CAA KMT NNK NNK RST CCT TAC ACG
TTC GGA 5 = 70%A, 6 = 70%G, 7 = 70%C, 8 = 70%T (10% for the rest of
three nucleotides)
[0373] In all of the libraries the heavy chain was held constant
with the parent clone sequence. All library templates contained a
stop codon embedded in CDR L1 preventing the presence of template
light chain among the phage-displayed antibody library members.
[0374] Libraries were constructed by mutagenesis method (Kunkel
mutagenesis) using the single strand DNA template containing stop
codon to anneal with the sets of oligocleotides for three CDR L1,
L2 and L3 simultaneously. Library DNA from mutagenesis were
transformed into bacterial cell strain SS320 by electroporation and
grown up with helper phage KO7. Single colonies from constructed
libraries were evaluated for display levels by the detection of gD
Tag at the c-terminus of light chain and for the retention of
binding for primary antigen (EGFR) in a single spot ELISA format.
An average of 35% of evaluated single colonies from D1.5 libraries,
show a high level of display and retained EGFR binding
property.
Library Sorting and Screening for the Isolation of Dual Specific
Clones
[0375] The libraries were subject to an initial round of binding
selection with anti-gD antibody as the capture target to eliminate
clones in which the Fab gene had been deleted or were not
expressed, and binding selection with hEGFR-ECD-Fc, followed by 4
rounds of plated antigen selection (HER2-ECD, HER2-ECD-Fc,
HER2-I-III-Fc, HER3-ECD-Fc, HER4-ECD-Fc (in each case the Fc in the
fusion constructs is the complement binding fragment from hIgG1).
Alternatively, they were directly subjected to target binding
selection without pre-selection with anti-gD and hEGFR-ECD-Fc.
[0376] Each round of plate selection was performed as previously
described in Example 3. Random clones from round 3 and 4 were
selected for screening and assayed using phage ELISA in which
binding to target and anti-gD was compared to binding of a
non-relevant protein (BSA) for checking non-specific binding.
Clones that bound the anti-gD antibody and target but not the
non-targeted protein controls (such as bovine serum albumin, other
IgGs) were considered specific positives.
[0377] The light chain variable domain regions of the positive
clones were amplified by PCR and sequenced. The DNA sequence
analysis of the positive specific binders revealed 1 unique binder
for both EGFR and HER2 (D1.5-201 (SEQ ID NO: 36)), 7 unique binders
for both EGFR and HER3 (D1.5-100 (SEQ ID NO: 40), D1.5-103 (SEQ ID
NO: 41), D1.5-113 (SEQ ID NO: 42), D1.5-115 (SEQ ID NO: 43),
D1.5-116 (SEQ ID NO: 44), D1.5-121 (SEQ ID NO: 45), D1.5-122 (SEQ
ID NO: 46)), 1 unique binder for both EGFR and HER4 (D1.5-400, (SEQ
ID NO: 39)).
[0378] Eight out of the nine bispecific clones retained the proline
at position 93 in HVR L3, Since the adoption of proline at this
position (with tyrosine at position 96) dramatically increased D1.5
affinity for EGFR compared to its parent clone D1, it is likely
that it is a contributor in retention of binding for EGFR.
Evaluation of Bispecific hEGFR/HER2, hEGFR/HER3, hEGFR/HER4 Phage
Clones Specificity.
[0379] To determine if the nine clones with dual activities were
specific to their cognate targets, their binding to a panel of
antigens in a direct plate ELISA format was evaluated.
[0380] 2 .mu.g/ml of several proteins were coated on immunoplates
overnight at 4.degree. C. The plates were blocked with 1% BSA in
PBST, and a dilution of phage-Fab supernatant grown as described in
Example 1 was applied to the plate for 30 minutes.
[0381] The plates were washed and binding signals recorded and
analyzed as described in Example 1. The results show that all
clones with dual specificities were specific to their cognate
target. Clones D1.5-4 and parent clone D1.5 were used as control.
(FIG. 2.)
Example 6
Characterization of Antibodies with Dual Specificity as Free
mIgG
[0382] All clones with dual specificity were reformatted into
mIgG2A for further characterization, using same restriction site
described above, and vectors previously designed for transient
murine IgG expression in mammalian cells.
Inhibition of TGF-.alpha.1 induced EGFR phosphorylation in EGFR-NR6
Cells
[0383] To determine if the seven selected antibodies with dual
specificity block TGF-.alpha. induced EGFR phosphorylation, stably
transfected EGFR-NR6 cells were treated as described in Example 2.
The data in FIG. 3 demonstrate that EGFR/HER3 and EGFR/HER2
antibodies inhibited TGF-.alpha. induced EGFR phosphorylation at
high concentration. A commercially available anti-EGFR antibody was
used as a positive control.
[0384] A dose response on clones D1.5-100 and D1.5-103 showed that
clone D1.5-100 lost its parent clone's (D1.5) high potency to
inhibit EGFR phosphorylation. D1.5-103, however, had a similar
potency to D1.5 in this assay.
Inhibition of Heregulin Binding to HER3 by anti-EGFR/HER3
Antibodies
[0385] To determine whether the selected anti-EGFR/HER3 antibodies
could inhibit heregulin binding to HER3-ECD-Fc protein, purified
IgGs were tested in a radiolabeled ligand binding assay.
[0386] Binding assays were performed in Nunc break-apart strip
wells. Plates were coated at 4.degree. C. overnight with 100 .mu.l
of 5 mg/mL goat anti-human Ab in 50 mM carbonate buffer (pH 9.6).
Plates were rinsed twice with wash buffer (PBS/0.005% Tween20) and
blocked with 100 .mu.l 1% BSA/PBS for 30 min. Buffer was removed
and each well was incubated with 200 ng of HER3-ECD-Fc in 1%
BSA/PBS for 1.5 h. Plates were rinsed three times with wash buffer
and antibodies in 1% BSA/PBS were pre-bound to HER3-IgG at
4.degree. C. overnight. .sup.125I-HRG was added and plates were
incubated for 2 hours at room temperature. Plates were rinsed three
times and individual wells were counted using a 100 Series Iso Data
.gamma.-counter. (FIG. 4.) The results demonstrate that all seven
antibodies with dual specificity for EGFR and HER3 can inhibit
heregulin binding to HER3-ECD-Fc.
Inhibition of Heregulin Binding to HER4 by anti-EGFR/HER4
Antibody
[0387] To determine whether the selected anti-EGFR/HER4 antibody
D1.5-400 could inhibit heregulin binding to HER4-ECD-Fc a ligand
binding assay as described above was performed. 25 ng HER4-ECD-Fc
was used instead of HER3-ECD-Fc. The results demonstrate that
D1.5-400 inhibited heregulin binding to HER4.
Inhibition of Heregulin Induced HER2/HER3 Phosphorylation in MCF7
Cells.
[0388] To determine whether the seven selected antibodies with dual
specificity for EGFR and HER3 could inhibit heregulin induced
HER2/HER3 phosphorylation in MCF7 cells, they were tested in a
receptor phosphorylation assay: MCF-7 cells (ATCC HTB 22, Manassas,
Va.) were plated in 12 well plates. Following serum starvation,
cells were incubated with indicated antibodies (75 ug/ml or 150
ug/ml) for 2 hours. Cells were stimulated with 0.5 nM HRG for 8
minutes and total cell lysates were run on SDS-PAGE and Western
blots were probed with anti-phospho-HER3, anti-pTyr or anti-tubulin
as loading control. (FIG. 5.) The results demonstrate that all
anti-EGFR/HER3 antibodies inhibited heregulin induced HER2/HER3
phosphorylation at high concentration. Pertuzumab (Pmab) was used
as a positive control.
Inhibition of MDA-175 Cell Proliferation
[0389] To determine the growth inhibitory potential of
anti-EGFR/HER3 antibodies on HRG driven cell growth, MDA-175 cells
(ATCC HTB 25, Manassas, Va.) (20000 cells/well) were plated in 96
well plates. The following day cells were simultaneously treated
with various concentrations of antibody (up to 50 ug/ml) in 1%
serum containing medium. After either 4 days or 5 days, Alamar Blue
was added and fluorescence was detected using a fluorometer. The
results were expressed in RFUs. MDA-175 cells were chosen since
their growth is the result of an autocrine stimulation by HRG. The
anti-EGFR/HER3 antibody D1.5-100, and clone D1.5-122 showed
inhibition of MDA-175 cell growth. Pertuzumab (Pmab) was used as a
positive control. (FIG. 6.)
Example 7
Mutagenesis Mapping Study of D1.5-100 and D1.5-103, anti-hEGFR/HER3
bi-Specific Antibodies
[0390] An alanine and homolog shotgun scanning analysis was
performed using combinatorial phage displayed libraries (Vajdos et
al., J. Biol. Biol. 320:415-28 (2002)) to investigate the
interaction between each antibody with its two antigens, EGFR and
HER3.
[0391] Binding selections on the antigens (hEGFR and HER3) to
isolate functional clones followed by DNA sequencing enabled
calculations of wild-type/mutant ratios at each varied position.
These ratios were then used to determine the contribution of each
scanned side-chain to EGFR and HER3 binding
[0392] The results enabled mapping of the functional paratope for
binding EGFR and HER3.
D1.5-100 and D1.5-103 Shotgun Libraries Design
[0393] Solvent exposed residues in the CDRs were scanned using
phage-displayed libraries in which the wild type residues were
allowed to vary as either alanine or wild type (Alanine Scan) or as
a homolog residue or wild type (Homolog Scan). The nature of the
genetic code required some other substitutions to be included in
the library in addition to Wt/Alanine or Wt/Homlog residues.
Separate heavy chain and light chain alanine and homolog scanning
libraries were constructed. The degeneracy ranged from
1.3.times.10.sup.5 to 7.5.times.10.sup.7.
Construction of Shotgun Scanning Libraries
[0394] Libraries were constructed as previously described except
that a single Fab was expressed on the surface of bacteriophage
fused to C-terminal domain of the M13 gene-3 minor coat protein,
after removal of leucine zipper from original plasmid by kunkel
mutagenesis (Oligo F220: TCT TGT GAC AAA ACT CAC AGT GGC GGT GGC
TCT GGT). (SEQ ID NO: 77)
[0395] The light chain alanine and homolog scanning library had
stop codons in HVR-L1, HVR-L2 and HVR-L3 and the heavy chain
alanine and homolog libraries contained stop codons in each heavy
chain HVRs. The libraries were constructed by previously described
methods (Sidhu et al., J. Mol. Biol. 338:299-310 (2004)), using
Kunkel mutagenesis (Kunkel et al., 1987, supra) on the respective
stop templates. Alanine scanning libraries are phage displayed
libraries that allow selected side chains to vary as wild-type or
alanine. Homolog scan means that the phage displayed libraries
allow selected side chains to vary as wild-type or similar amino
acids.
Library Selection
[0396] NUNC 96-well Maxisorp immunoplates were coated with 5 mg/ml
capture target (EGFR-ECD-Fc, HER3-ECD-Fc or Protein-L) and blocked
with 1% BSA (w/v) in PBS. Phage from the above-described libraries
were propagated with KO7 helper phage (NEB) as described (Lee et
al., 2004, supra). The library phage solutions were added to the
coated plates at a concentration of 10.sup.13 phage particles/ml
and incubated for 1-2 h in RT. The plates were washed 8 times with
PBST and followed by elution of bound phage with 0.1 M HCl for 30
min. Enrichment after each round of selection was determined as
described previously. After 2 rounds of target selection, a number
of random clones from each library were selected for sequencing as
described (Sidhu et al., 2004, supra).
[0397] The DNA sequences of binding clones were used to determine
wild-type/mutation ratios at each varied position. The ratios were
used to assess binding contribution to antigen of each selected
side chain. Dividing wt/mut ratio from antigen selection by wt/mut
ratio from display selection provides quantitative estimate of each
mutation's effect on antigen-binding affinity (Function ratio
F.sub.wt/mut). If the ratio is greater than 1, the mutation is
deleterious. If the ratio is less than 1, the mutation is
beneficial. 2500 clones were sequenced.
[0398] Based on the alanine and homolog scan results, hot spots of
the antibody D1.5-100 for the binding of EGFR and HER3 were mapped
on the structure of known anti-HER2 antibody 4D5-Fv. As shown in
FIG. 7 the mapping suggests that for EGFR binding, the heavy chain
dominates, and heavy and light chains work together for the binding
of HER3. Acidic (E, D) residues play an important role in binding
both EGFR and HER3, and especially HER3.
Affinity Maturation
[0399] FIG. 8 illustrates affinity maturation of D1.5-100, using
the shotgun libraries described above. In this affinity maturation
strategy, D1.5-100 heavy chain homolog library was sorted as
previously described. After two rounds of plate sorting was
performed on HER3-ECD-Fc coated plates, and three rounds of
solution alternating targets were sorted (EGFR-ECD and HER3-ECD-Fc)
under increasing stringency. Single clones were picked and assessed
in a single spot ELISA to isolate clones that retained dual binding
activity. Among the clones assessed, 79 of 94 show binding for both
EGFR/HER3, while 11 lost EGFR binding and show specific binding for
HER3.
Isolation of Clones by Single Spot Competition
[0400] Single colonies from the last round of sort were picked and
phage were grown as described in Example 1. 384 wells were coated
with EGFR-ECD-Fc at 1 .mu.l/ml. For each colony grown, 25 ml of
phage supernatant or ELISA buffer were incubated with 25 .mu.l of
EGFR-ECD (50 nM) and HER3-ECD-Fc (10 nM). 4 .mu.l of incubation was
added to EGFR-ECD-Fc coated plate, and the plate was washed eight
times. 60 .mu.l of 1/5000 anti-M13-HRP conjugates antibody was
added, the plate was washed eight times and developed with
TMB+H.sub.3PO.sub.4.
[0401] Eight unique clones were selected that show greater
inhibition than D1.5-100 for EGFR and HER3, using the following
single spot competition protocol:
[0402] 1. Single colonies from last round of sort were picked and
phage growth as previously described;
[0403] 2. 384 wells plate was coated with EGFR-ECD-Fc at 1
mg/ml;
[0404] 3. For each colony grown, 25 ml of phage supernatant or
ELISA buffer were incubated with 25 .mu.l of EGFR-ECD (50 nM) and
HER3-ECD-Fc (10 nM);
[0405] 4. 4 .mu.l of incubation was added to EGFR-ECD-Fc coated
plate;
[0406] 5. Plate was washed 8 times;
[0407] 6. Added 60 ml of 1/5000 anti-M13-HRP conjugates
antibody;
[0408] 7. Plate was washed 8 times;
[0409] 8. Plate was developed with TMB+H.sub.3PO.sub.4.
[0410] 8 unique clones were selected that show greater inhibition
than D1.5-100 for both EGFR and HER3 (DL6, SEQ ID NO: 63; DL7, SEQ
ID NO: 64; DL8, SEQ ID NO: 65; DL9, SEQ ID NO: 66; DL10, SEQ ID NO:
67; DL11, SEQ ID NO: 28; DL12, SEQ ID NO: 68; DL13, SEQ ID NO: 69).
For anti-HER3 antibodies, 7 unique clones were selected that show
greater inhibition than D1.5-100 for HER3.
Characterization of Affinity-Matured Bispecific and anti-HER3
Antibodies
[0411] Specificity of binding of selected affinity matured
bispecific antibodies was assessed by direct binding to a panel of
various proteins as described in Example 5. As shown in FIG. 9, the
selected antibodies showed specificity of binding for both EGFR and
HER3.
[0412] IC50 values were calculated for two selected bispecific
antibodies (DL7 and DL11) as described in Example 1 and compared to
D1.5-100 parent clone. Both selected bispecific antibodies show
similar affinity for HER3-ECD-Fc and increased affinity for EGFR.
DL1.5-100 had an IC50 of 44 nM for EGFR-ECD, 0.1 nM for HER3-FC;
DL7 had an IC50 of 6.1 nM for EGFR-ECD, 0.25 nM for HER3-FC; DL11
had an IC50 of 5.7 nM for EGFR-ECD, 0.43 nM for HER3-FC.
[0413] The specificity of binding of selected anti-HER3 antibodies
was assessed by direct binding to a panel of various proteins as
previously described. The selected antibodies (DL3.5, DL3.6, DL3.7)
show binding specificity for HER3 only.
[0414] Phage IC50 values were calculated for selected antibodies as
described above and compared to the D1.5-100 parent clone. All
antibodies (DL3.1-3.7) show increased affinity for HER3-ECD-Fc with
IC50s of between 1 and 3.8 nM. The parent DL1.5-100 had an IC50 of
4.6 nM.
[0415] Binding of selected bispecific antibodies and anti-HER3
antibodies was compared to the binding of HER3 domain III protein
(N-term His tag) using the competition ELISA described above.
EGFR/HER3 and monospecific anti-HER3 antibodies have similar
affinities for HER3ECD-Fc and HER3 domain III constructs (Phage
IC50).
[0416] Selected affinity matured bispecific antibodies and
anti-HER3 antibodies were reformatted into mIgG2A and validated in
a competition ELISA, as described above. mIgG2A bispecific
antibodies show increased affinity for both EGFR-ECD and HER3
domain III compared to the D1.5-100 parent clone.
[0417] Using BIAcore 300 for the kinetic analysis of affinity
matured EGFR/HER3 antibodies DL7 and DL11, and anti-HER3 specific
antibodies DL3.6 and DL3.7, purified mIgG2A of each antibody
(DL1.5-100, DL7, DL11, DL3.6, DL3.7) was coupled onto a CM5 chip,
and several dilutions of antigen (EGFR-ECD, HER3 domainIII,
HER3-ECD) were flowed over the coated chip under the conditions
described in Example 4. The CM5 chip was regenerated between each
injection of antigen. Finally, KD was determined using a 1:1
binding analysis with mass transfer. The affinity matured EGFR/HER3
antibodies DL7 and DL11 have improved KD (M) for both targets.
[0418] In order to assess the inhibitory function of affinity
matured antibodies DL7, DL11 and parental antibody D1.5 on EGFR,
EGFR-NR6 cells that only express EGFR were pretreated with various
amounts of antibodies (up to 20 ug/ml) for one hour and,
subsequently, phosphorylation of EGFR was induced by TGF.alpha. (5
nM). Inhibition of receptor phosphorylation by the antibodies was
detected using an anti-Phosphotyrosine antibody. Inhibitions of
MAPK activation was also seen in a dose dependent manner. Antibody
DL11 was more potent than DL7 in inhibiting EGFR and ERK1/2
phosphorylation. (FIG. 10A.)
[0419] The inhibitory function of DL7, DL11 and monospecific
anti-HER3 antibody DL3.6 on HER3 transactivation was compared.
(FIG. 10B.) MCF-7 cells that express HER2, HER3 and EGFR were
pretreated with indicated amounts of antibody (up to 50 ug/ml) for
one hour, and activation of HER3 and transphosphorylation of HER3
was induced by HRG. Inhibition of HER3 phosphorylation was detected
using an anti-phospho HER3 antibody. Inhibition of downstream
signaling molecules, ERK1/2 as well as Akt, was seen in a dose
dependent manner. DL11 again was more potent than DL7.
[0420] The growth inhibitory function of DL11 was compared to that
of a commercially available anti-EGFR antibody, pertuzumab, and an
anti-HER3 antibody, or to that of DL3.6, D1.5, or the combination
of D1.5 plus DL3.6. H1666 cells (ATCC CRL-5885, Manassas, Va.) (an
NSCLC cell line that expresses HER2, HER3, EGFR and EGFR ligands)
(6000 cells/well) were growth stimulated with HRG (3 nM). The
antibodies were tested in a dose dependent manner and growth
inhibitory characteristics compared to all other monospecific
antibodies. As shown in FIG. 11A, DL11 inhibited cell growth to a
greater extent than the monospecific antibodies, or combinations
thereof.
[0421] Similar results were obtained when the assay was repeated
using H1666 cells growth stimulated with HRG (3 nM)+TGF.alpha. (6
nM). (FIG. 11B).
[0422] The growth inhibitory function of DL11 was compared to
pertuzumab, an anti-EGFR antibody, and an anti-HER3 antibody, or
DL3.6, D1.5 or to the combination of D1.5 plus DL3.6 in HCA-7
cells. HCA-7 is a colorectal cell line that expresses HER2, HER3
and EGFR. Cell growth was stimulated with HRG (3 nM) in the
presence of 1% serum. The antibodies were tested in a dose
dependent manner and cell viability was detected after 3 days,
using Alamar Blue reagent. As shown in FIG. 12A, DL11 showed
superior growth inhibitory characteristics compared to all other
treatments.
[0423] Inhibition of HCA-7 cell growth was investigated as
described in connection with FIG. 12A, except growth was stimulated
with HRG (3 nM) plus TGF.alpha. (5 nM). As shown in FIG. 12B, DL11
showed superior growth inhibitory characteristics compared to all
other treatments.
[0424] Inhibition of Calu-3 growth by DL11 as compared to an
anti-EGFR antibody, pertuzumab, and an anti-HER3 antibody was
investigated The NSCLC cell line Calu-3 (ATCC HTB-55, Manassas,
Va.) over-expresses HER2 and has normal levels of HER3 and EGFR.
Cell growth (10,000 cells/well) was stimulated with HRG (3 nM) in
the presence of 1% serum, and antibodies were tested in a dose
dependent manner. As shown in FIG. 13, DL11 showed superior
activity compared to the monospecific antibodies.
Example 8
Mutagenesis Mapping Study of D1.5-201 and D1.5-201-2,
anti-hEGFR/HER2 bi-Specific Antibodies
[0425] For affinity maturation of D1.5-201, a light chain
soft/homolog library was designed on selected amino acids. Some
soft residues were soft randomized, where wild-type residue
frequency was 50%. Some residues were randomized using codons
encoding for wild-type residue or homolog residue. Some homolog
randomization allows 1 or 2 extra residues besides wild-type and
homolog. Finally, some residues were unchanged.
[0426] FIG. 14 illustrates another sorting strategy for the
affinity maturation of D1.5-201. In this strategy, D1.5-201 light
chain soft/homolog library was sorted as previously described.
After three rounds of alternating plate/solution sorting was
performed on HER2/ECD under increasing stringency, single clones
were picked and assessed in a single spot ELISA to isolate clones
that retained dual binding activity for EGFR and HER2. Among the
clones assessed, 77/192 show binding for both EGFR and HER2.
[0427] The sequences of light chain variable region for the
selected affinity matured EGFR/HER2 bispecific antibodies were
determined (D1.5-201 (SEQ ID NO: 36, D1.5-201-2 (SEQ ID NO: 37,
D1.5-201-3 SEQ ID NO: 38). Phage IC50 were calculated for two
selected affinity matured bispecific antibodies (D1.5-201-2,
D1.5-201-3) as previously described and compared to D1.5-201 parent
clone. Both affinity matured bispecific antibodies show increased
affinity for HER2-ECD and EGFR-ECD.
Example 9
DL11 Affinity Maturation
[0428] DL11 was further affinity matured as described above
resulting in two additional bispecific antibodies with specificity
for EGFR and HER3 (DL11b and DL11f) and two antibodies specific for
HER3 (DL3-11fb and DL3-11f). The heavy chain and light chain amino
acid sequence of DL11b are shown in SEQ ID NOs: 12 and 13,
respectively. The heavy chain and light chain amino acid sequence
of DL11f are shown in SEQ ID NOs: 14 and 13, respectively. The
heavy chain and light chain amino acid sequence of DL3-11b are
shown in SEQ ID NOs: 19 and 13, respectively and the heavy chain
and light chain amino acid sequence of DL3-11f are shown in SEQ ID
NOs: 20 and 13, respectively.
Biacore Affinity Assay
[0429] The binding affinities for DL11b and DL11f their EGFR and
HER3 targets were determined in the following Biocore assay. Both
DL11b and DL11f showed improved affinity for their targets as
compared to DL11.
[0430] Measurements were done using surface plasmon resonance on a
BIAcore.TM. 2000 instrument (GE Healthcare, BIAcore Life Sciences,
Piscataway, N.J.). cDNAs encoding the extracellular domains (ECDs)
of human EGFR (amino acids 1-637) and human HER3 (amino acids
1-640) were cloned into a mammalian expression vector containing
sequences encoding the Fc region of human IgG1 to generate human Fc
fusion protein. Recombinant human EGFR-IgG1 (2.65 mg/ml), human
HER3-IgG1 (3.35 mg/ml) were produced by transiently transfecting
Chinese hamster ovary cells and were purified via protein A
affinity chromatography. cDNA encoding the extracellular domain of
human EGFR (amino acids 1-637) was cloned into a mammalian
expression vector containing a N-terminal flag sequence.
[0431] The binding affinities for DL11f as both a Fab and IgG
antibody were determined. For the Fab assay, the DL11f Fab was the
analyte and was flowed over a CM5 chip where the different
ligands--human EGFR-Fc and human HER3-Fc--were first captured using
the BIAcore human Antibody Capture Kit (BR-1008-39, Lot 10020611).
A 2-fold dilution series of DL11f Fab was injected in a range of
0.244-250 nM in PBS, 0.05% Tween20 at a flow rate of 30
.mu.l/minute at 25.degree. C. Between each injection of Fc fusion
ligands and analyte, 3M Magnesium chloride was used to regenerate
the sensor chip (5 .mu.l at a flow rate of 10 .mu.l/mn). To
determine the affinity constants of DL11f Fab to human EGFR and
human HER3Fc fusion proteins, the signal from the reference cell
was subtracted from the observed test sensorgram. Kinetic constants
were calculated by non-linear regression fitting of the data
according to a 1:1 Langmuir binding model using BIAcore evaluation
software (GE Healthcare), version 4.1, supplied by the
manufacturer. Two replicates of a representative concentration of
DL11f Fab (125 nM) gave very similar fitting and kinetics constants
for all Fc fusions proteins. DL11f Fab bound to human EGFR-Fc with
a KD value of 1.92 nM and to human HER3-Fc with a KD value of 0.39
nM.
[0432] A second experimental condition was explored to obtain
binding kinetics from DL11f as IgG. Here, DL11f human IgG1 was
immobilized on the sensor chip CM5, and monomeric human EGFR-ecd
and human HER3-ecd were used as the analyte. A 2-fold dilution
series of human EGFR-ecd and human HER3-ecd was injected in a range
of 0.244-250 nM in PBS, 0.05% Tween20 at a flow rate of 30
.mu.l/minute at 25.degree. C. Binding kinetics were determined as
for the Fab. DL11f bound to human EGFR-ecd, and human HER3-Fc with
K.sub.D values of 19.9 nM and 2.63 nM respectively. In both
experiments, we observed that DL11f antibody has a consistent 5-8
fold higher affinity for HER3 than for EGFR. The weaker affinities
found in experiment 2 for both receptors when compared to
experiment 1 could be due to a difference between having EGFR ecd
or HER3 ecd as analytes in solution and the receptor as Fc fusion
immobilized on the flow cell chip. It is possible that these
multi-domain receptors as free ECD may encounter more entropic
penalty for binding energy when in solution thus resulting in
weaker affinity.
[0433] DL11b showed similar binding affinities for EGFR and HER3 as
DL11f under similar conditions in a separate Biacore analysis.
[0434] DL3-11b and DL3-11f lost the ability to specifically bind to
EGFR while retaining the ability to specifically bind to HER3.
Inhibition of MDA-175 Cell Proliferation
[0435] Inhibition of MDA-175 cell proliferation by DL11b and DL11f
was investigated as described above. Both DL11b and DL11f inhibited
proliferation of MDA-175 cells to a similar degree as the DL11
antibody. FIG. 15. The IC50s of DL11, DL11f, and DL11b were all
around 0.8-1.0 ug/ml.
Inhibition of Heregulin Induced HER3 Phosphorylation in MCF7
Cells.
[0436] To determine whether DL11f could inhibit heregulin induced
HER3 phosphorylation in MCF7 cells, a receptor phosphorylation
assay was performed as described Example 6. The results demonstrate
that DL11f inhibited heregulin induced HER3 phosphorylation in a
dose dependent manner. FIG. 16.
Inhibition of TGF-.alpha. induced EGFR Phosphorylation in Stably
Transfected EGFR-NR6 cells
[0437] To determine if DL11f selectively blocks TGF-.alpha. induced
EGFR phosphorylation, a receptor phosphorylation assay was
performed as described in Example 2. The data in FIG. 16
demonstrate that DL11f inhibits TGF-.alpha. induced EGFR
phosphorylation in a dose dependent manner in this cell based
assay.
[0438] DL11 and DL11f Inhibit Tumor Growth in an In Vivo Model
[0439] A HCA-7 tumor transplant model assay was used to determine
the effect of DL11 and DL11f on in vivo tumor growth. The assay was
performed as follows.
[0440] SCID beige mice (Charles River Laboratories, San Diego,
Calif. were transplanted subcutaneously with HCA-7 tumor pieces.
When tumors reached a mean volume of 100 to 250 mm3, mice with
similarly sized tumors were randomized into treatment cohorts
(n=9/group) as follows: Vehicle (PBS), Pertuzumab (10 mg/kg), D1.5
(25 mg/kg), D1.5+DL3.6 (25 mg/kg each), DL11 (25 mg/kg), or DL11f
(25 mg/kg). Treatments were administered intraperitoneally,
beginning with a 2.times. loading dose (20 or 50 mg/kg) on the day
of randomization and continuing weekly for a total of three
treatments. Tumors were measured with calipers twice a week for the
duration of the study. Mice were housed in standard rodent
microisolator cages. Environmental controls for the animal rooms
were set to maintain a temperature of approximately 70.degree. F.,
a relative humidity of approximately 40%-60%, and an approximate
14-hour light/10-hour dark cycle. Mice were maintained according to
the ILAR Guide for the Care and Use of Laboratory Animals, and the
study was reviewed and approved by the Institutional Animal Care
and Use Committee (IACUC) at Genentech.
[0441] DL11 and DL11f were equally effective in reducing mean tumor
volume in this xenograft model. FIG. 17.
[0442] DL11f is Active in a Non-Small Lung Cell Cancer Model
[0443] The antibodies were tested in mice with established tumors
derived from the human NSCLC line H358 (ATCC CRL-5807, Manassas,
Va.). 5.times.10.sup.6 H358 cells were inoculated subcutaneously
with matrigel in CB17 SCID mice. Animals with similarly sized
tumors were randomized into treatment cohorts (n=9/group) as
follows: Vehicle (DL11f formulation buffer), D1.5 (25 mg/kg),
DL3.11b (25 mg/kg), DL11f (30 mg/kg), or D1.5+.quadrature.L3.11b
(25 mg/kg each). Treatments were administered intraperitoneally,
beginning with a 2.times. loading dose (50 or 60 mg/kg) on the day
of randomization and continuing weekly for a total of four
treatments. Tumors were measured with calipers twice a week for the
duration of the study.
[0444] As shown in FIG. 18, the bispecific antibody DL11f is active
in this NSCLC model and is more effective in inhibiting tumor
growth than a combination of an anti-EGFR specific and an anti-HER3
specific antibody (D1.5+DL3.11b).
Example 10
DL3.6 Affinity Maturation
[0445] DL3.6 was further affinity matured as described above
resulting in additional anti-HER3 antibodies. DL3.6b exhibited an
increase in affinity for its target as compared to parent antibody
DL3.6. The heavy and light chain amino acid sequences of DL3.6b are
shown in SEQ ID NOs: 17 and 13, respectively.
[0446] As shown in FIGS. 19 and 20, DL3-11b, DL3.6, and DL3.6 b
inhibited HRG induced HER3 phosphorylation and MDA-175 cell
proliferation. Assays were performed as described above.
Example 11
In Vivo Activity in Fadu Xenograft Model, a Head and Neck Squamous
Cell Carcinoma Model
[0447] DL11f, a commercially available anti-EGFR antibody, and an
anti-HER3 antibody were tested in mice with established tumors
derived from Fadu cells (ATCC HTB-43, Manassas, Va.)
5.times.10.sup.6 FaDu cells were inoculated subcutaneously in CB17
SCID mice. Animals with similarly sized tumors were randomized into
treatment cohorts (n=9/group) as follows: Vehicle (DL11f
formulation buffer), anti-EGFR antibody (25 mg/kg), anti-HER3
antibody (50 mg/kg), and DL11f (25 mg/kg). Treatments were
administered intraperitoneally, beginning with a 2.times. loading
dose (50 or 100 mg/kg respectively) on the day of randomization and
continuing weekly for a total of four treatments. Tumors were
measured with calipers twice a week for the duration of the
study.
[0448] As shown in FIG. 21, DL11f is active in the FaDu head and
neck cancer model and is more effective in inhibiting tumor growth
than either an anti-EGFR specific or an anti-HER3 specific
antibody.
Example 12
In Vivo Activity in BxPC3 Xenograft Model, a HER3 driven Pancreatic
Model
[0449] DL11f, an anti-EGFR antibody, pertuzumab, and an anti-HER3
antibody were tested in mice with established tumors derived from
the pancreatic cell line BxPC3 (ATCC CRL-1687, Manassas, Va.).
10.times.10.sup.6 BxPC3 cells were inoculated subcutaneously in
CB17 SCID mice. Animals with similarly sized tumors were randomized
into treatment cohorts (n=8/group) as follows: Vehicle (DL11f
formulation buffer), anti-EGFR antibody (25 mg/kg), pertuzumab (25
mg/kg), anti-HER3 antibody (50 mg/kg), and DL11f (25 mg/kg),
Treatments were administered intraperitoneally, beginning with a
2.times. loading dose (50 or 100 mg/kg) on the day of randomization
and continuing weekly for a total of four treatments. Tumors were
measured with calipers twice a week for the duration of the
study.
[0450] DL11f is active in the BxPC3 pancreatic cancer model and is
more effective in delaying tumor growth than either an anti-EGFR
specific or an anti-HER3 specific antibody. (FIG. 22.)
Example 13
In Vivo Activity in NSCLC Calu-3 Xenograft Model
[0451] DL11f, a commercially available anti-EGFR antibody, and an
anti-HER3 antibody were tested in mice with established tumors
derived from the NSCLC line Calu-3 (ATCC HTB-55, Manassas, Va.).
5.times.10.sup.6 Calu-3 cells were inoculated subcutaneously in
SCID Beige mice. Animals with similarly sized tumors were
randomized into treatment cohorts (n=9/group) as follows: Vehicle
(DL11f formulation buffer), anti-EGFR antibody (25 mg/kg),
anti-HER3 antibody (25 mg/kg), and DL11f (25 mg/kg), Treatments
were administered intraperitoneally, beginning with a 2.times.
loading dose (50) on the day of randomization and continuing weekly
(anti-HER3 biweekly) for a total of four (eight) treatments. Tumors
were measured with calipers twice a week for the duration of the
study.
[0452] DL11f is active in the Calu-3 non-small cell lung cancer
model and is more effective in delaying tumor growth than either an
anti-EGFR specific or an anti-HER3 specific antibody. (FIG.
23.)
Example 14
In Vivo Activity in Epidermal A431 Xenograft Model
[0453] DL11f, a commercially available anti-EGFR antibody, and an
anti-HER3 antibody were tested in mice with established tumors
derived from the epidermoid cell line A431 (ATCC CRL-2592,
Manassas, Va.). 5.times.10.sup.6 A431 cells were inoculated
subcutaneously in SCID Beige mice Animals with similarly sized
tumors were randomized into treatment cohorts (n=8/group) as
follows: Vehicle (DLI If formulation buffer), an anti-EGFR antibody
(12.5 mg/kg), anti-HER3 (50 mg/kg), and DLI If (12.5 and 25 mg/kg),
Treatments were administered once intraperitoneally on the day of
randomization. Tumors were measured with calipers twice a week for
the duration of the study.
[0454] Due to the faster clearance of DL11f compared to the
anti-EGFR antibody in mice, DL11f was dosed at 2.times.
concentration compared to the anti-EGFR antibody in order to
achieve comparable exposure levels. Taken together, DL11f inhibits
tumor growth in the A431 epidermal cancer model as well as the
anti-EGFR antibody. (FIG. 24.)
Example 15
In Vivo Activity in Nude Mice Bearing Xenografts of the
Patient-Derived Breast Cancer MAXF449
[0455] DL11f, a commercially available anti-EGFR antibody, and an
anti-HER3 antibody were tested at Oncotest GmbH, Freiburg, Germany.
Oncotest passages patient tumors, like the mammary cancer MAXF 449,
as subcutaneous xenografts in nude mice, following direct
transplantation of tumors from donor patients. According to
Oncotest's protocols, animals with similarly sized tumors were
randomized into treatment cohorts (n=10/group) as follows: DL11f
(30 mg), anti-EGFR antibody (30 mg/kg), anti-HER3 (60 mg/kg) and
Vehicle (DL11f formulation buffer). Treatments were administered
intraperitoneally, beginning with a 2.times. loading dose (60 or
120 mg/kg respectively) on the day of randomization and continuing
weekly for a total of four treatments.
[0456] DL11f and anti-HER3 inhibits tumor growth in the MAXF44
breast cancer model whereas the anti-EGFR antibody has no effect
(FIG. 25).
Example 16
[0457] In vivo activity in prostate DU145 xenograft model
[0458] DL11f, a commercially available anti-EGFR antibody, and an
anti-HER3 antibody were tested at Piedmont Research Center,
Morrisville according to Piedmont's protocols. Animals with
similarly sized tumors were randomized into treatment cohorts
(n=10/group) as follows: DL11f (25 mg), anti-EGFR antibody (25
mg/kg), anti-HER3 (50 mg/kg) and Vehicle (DL11f formulation
buffer). Treatments were administered intraperitoneally, beginning
with a 2.times. loading dose (50 or 100 mg/kg respectively) on the
day of randomization and continuing weekly for a total of four
treatments. DL11f is active in DU145 prostate cancer model and is
more effective in inhibiting tumor growth than either an anti-EGFR
specific or an anti-HER3 specific antibody. (FIG. 26.)
Example 17
In Vivo Activity in Nude Mice Bearing Xenografts of the
Patient-Derived Ovarian Cancer OVXF550
[0459] DL11f, a commercially available anti-EGFR antibody, and an
anti-HER3 antibody were tested at Oncotest GmbH, Freiburg, Germany.
Oncotest passages patient tumors, like the ovarian cancer OVXF550,
as subcutaneous xenografts in nude mice, following direct
transplantation of tumors from donor patients. According to
Oncotest's protocols animals with similarly sized tumors were
randomized into treatment cohorts (n=10/group) as follows: DL11f
(30 mg), anti-EGFR antibody (30 mg/kg), anti-HER3 antibody (60
mg/kg) and Vehicle (DL11f formulation buffer). Treatments were
administered intraperitoneally, beginning with a 2.times. loading
dose (60 or 120 mg/kg respectively) on the day of randomization and
continuing weekly for a total of five treatments.
[0460] DL11f is active in the OVFX550 ovarian cancer model. (FIG.
27.)
Example 18
DL11f Mediates Antibody Dependent Cellular Cytotoxicity (ADCC) In
Vitro and In Vivo
[0461] DL11f mediates ADCC in vitro. A431, H292 (ATCC CRL-1848,
Manassas, Va.), FaDu, BxPC3 and MDA 468 (ATCC HTB-132, Manassas,
Va.) cells (all from ATCC) were plated in 96 well plates in the
presence of indicated concentrations of antibodies. After
pre-incubation for 30 minutes at 37.degree. C. isolated peripheral
blood mononuclear cells (PBMC) were added and the incubation
continued for 4 more hours at 37.degree. C. After 4 hours, the
plates were centrifuged and the supernatants were harvested. The
LDH activity in the supernatants was determined according to the
Promega CytoTox-One homogeneous membrane integrity assay procedure.
To determine the percentage cell mediated cytotoxicity the average
absorbance were calculated and the background was subtracted. As
shown in FIG. 28, DL11f mitigates ADCC in EGFR expressing cell
lines in a dose dependent manner.
[0462] An amino acid substitution of N297A was introduced into
DL11f to delete the effector function. N297 is required for
FcR.gamma. and/or complement binding. DL11f-N297A exhibits a lack
of ADCC in vitro. As expected, the growth inhibitory function of
DL11f in vitro is not affected by the mutation. (FIG. 29.)
[0463] DL11f and DL11f-N297A were tested in mice with established
tumors derived from the NSCLC cell line H292 (ATCC CRL-1848,
Manassas, Va.). 5.times.10.sup.6 A431 cells were inoculated
subcutaneously in C.B17-SCID mice Animals with similarly sized
tumors were randomized into treatment cohorts (n=10/group) as
follows: Vehicle (DL11f formulation buffer), DL11f (6 mg/kg) and
DL11f-N297A (6 mg/kg). Treatments were administered once
intraperitoneally on the day of randomization. Tumors were measured
with calipers twice a week for the duration of the study.
Initially, DL11f and DL11f N297A inhibited tumor growth
equivalently by inhibiting HER pathway signaling. But as doses
diminished DL11f exhibited prolonged anti-tumor activity compared
to DL11f N297A due to its ADCC capability. (FIG. 30.)
Example 19
DL11f is Less Toxic than Cetuximab in Cynomolgus Monkeys
[0464] A study was conducted to determine the relative toxicity of
DL11f and cetuximab. Cynomolgus monkeys were assigned into three
Groups and dosed with either DL11f or cetuximab (Capital Wholesale
Drug, Columbus, Ohio) once weekly for six weeks as follows:
[0465] Group 1: Cetuximab 25 mg/kg;
[0466] Group 2: DL11f 25 mg/kg;
[0467] Group 3: DL11f 12.5 mg/kg.
[0468] All 3 cetuximab dosed animals developed skin lesions between
the 3rd and 4th dose, indicating toxicity. This result was expected
based on prior Cynomolgus studies conducted during FDA approval of
cetuximab. None of the DL11f dosed animals showed signs of toxicity
at this point in the study.
[0469] One of the animals receiving the 25 mg/kg dose of DL11f
developed a skin lesion one week following the 6th dose. This
lesion measured approximately 4 cm.times.7 cm and was very mild and
limited to a smaller area when compared to lesions observed in the
cetuximab treated animals.
[0470] Based on the analysis of toxicokinetic parameters in this
toxicology study, exposure of cetuximab and DL11f were similar in
the animals dosed at 25 mg/kg of each test compound.
[0471] A second, larger scale study was performed under the
following conditions.
[0472] Cynomolgus monkeys were assigned into six Groups and dosed,
by intravenous administration, with either DL11f or a vehicle
control once weekly for twelve weeks as follows:
TABLE-US-00006 Group 1: 10 monkeys (5 male/5 female)- Vehicle
Control Group 2: 10 monkeys (5 male/5 female) DL11f 5 mg/kg Group
3: 10 monkeys (5 male/5 female) DL11f 15 mg/kg Group 4: 10 monkeys
(5 male/5 female) DL11f 30 mg/kg Group 5: 4 monkeys (2 male/2
female) Vehicle Control Group 6: 4 monkeys (2 male/2 female) DL11f
30 mg/kg
[0473] None of the animals exhibited any apparent skin toxicities
during the study or during the recovery period following the final
dosing with the DL11f.
[0474] A single dose, IV administration PK study was also conducted
in cynomolgus monkeys. In this study, 3 monkeys per group were
dosed intravenously with 1, 10, or 30 mg/kg of DL11f. The PK over
the dose range explored were non-linear, consistent with a
saturable clearance as has been seen with other EGFR-targeting
antibodies.
[0475] All patents, patent applications, patent application
publications, and other publications cited or referred to in this
specification are herein incorporated by reference in their
entirety to the same extent as if each independent patent, patent
application, patent application publication or publication was
specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
791214PRTArtificial Sequencesynthetic 1Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser
Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 2102228PRTArtificial Sequencesynthetic 2Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Gly Asn 20 25
30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Gly Glu Ile Ser Pro Ser Gly Gly Tyr Thr Asp Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ser Arg Val Ser Tyr Glu Ala Ala
Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His2253214PRTArtificial
Sequencesynthetic 3Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Tyr Pro Thr Pro Tyr 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
2104214PRTArtificial Sequencesynthetic 4Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Leu Ala Thr Asp 20 25 30Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser
Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Glu Pro Glu Pro Tyr 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 2105220PRTArtificial Sequencesynthetic 5Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Leu Gly Asp Ser 20 25
30Glu Asn Gly Tyr Ala Asp Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45Ala Pro Lys Leu Leu Ile Tyr Glu Gly Ser Ser Leu Tyr Ser Gly
Val 50 55 60Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr65 70 75 80Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln 85 90 95Ala Ala Pro Ser Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile 100 105 110Lys Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp 115 120 125Glu Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140Phe Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu145 150 155 160Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170
175Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser 195 200 205Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 2206220PRTArtificial Sequencesynthetic 6Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Leu Leu Ala Ser 20 25 30His Asp Gly
Thr Pro Trp Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45Ala Pro
Lys Leu Leu Ile Tyr Asp Ala Ser Tyr Leu Tyr Ser Gly Val 50 55 60Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75
80Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95Ser Thr Phe Ala Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile 100 105 110Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp 115 120 125Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn 130 135 140Phe Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu145 150 155 160Gln Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190Glu Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200
205Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
2207214PRTArtificial Sequencesynthetic 7Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Glu Ile Phe Pro Asp 20 25 30Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser
Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Ala Pro Thr Pro Tyr 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 2108214PRTArtificial Sequencesynthetic 8Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Ala Ser Asp 20 25
30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Glu
Pro Glu Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 2109214PRTArtificial
Sequencesynthetic 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Gly Ser Asp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Tyr Leu Tyr Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Asp Pro Glu Pro Tyr 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21010214PRTArtificial Sequencesynthetic 10Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asn Ile Ala Ser Asp 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Pro Glu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21011214PRTArtificial Sequencesynthetic
11Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Leu Ala Thr
Asp 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Glu Pro Glu Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21012228PRTArtificial Sequencesynthetic 12Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Glu Ile
Ser Ala Ala Gly Gly Tyr Thr Asp
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr
Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ser Arg Val Ser
Phe Glu Ala Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150
155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His22513214PRTArtificial Sequencesynthetic 13Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asn Ile Ala Thr Asp 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Glu Pro Glu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21014228PRTArtificial Sequencesynthetic
14Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly
Asp 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Glu Ile Ser Ala Ala Gly Gly Tyr Thr Asp Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ser Arg Val Ser Phe Glu
Ala Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His22515214PRTArtificial Sequencesynthetic 15Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Leu Ala Thr Asp 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Glu Pro Glu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21016228PRTArtificial Sequencesynthetic
16Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly
Asp 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Glu Ile Ser Pro Ala Gly Ala Tyr Thr Asp Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ala Lys Val Ser Phe Glu
Ala Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His22517228PRTArtificial Sequencesynthetic 17Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Gly Asp 20 25 30Trp Ile His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu
Ile Ser Pro Ala Gly Ala Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ala Lys Val Ser Phe Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His22518216PRTArtificial Sequencesynthetic
18Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Leu Phe
Tyr 20 25 30Gly Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu 35 40 45Leu Ile Tyr Asp Gly Ser Tyr Leu Tyr Ser Gly Val Pro
Ser Arg Phe 50 55 60Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu65 70 75 80Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Tyr Pro Ala 85 90 95Pro Tyr Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val 100 105 110Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145 150 155
160Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
165 170 175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys 180 185 190Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr 195 200 205Lys Ser Phe Asn Arg Gly Glu Cys 210
21519228PRTArtificial Sequencesynthetic 19Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Glu Ile
Ser Ala Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ser Lys Val Ser Phe Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His22520228PRTArtificial Sequencesynthetic
20Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly
Asp 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Glu Ile Ser Ala Ala Gly Gly Tyr Thr Asp Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ser Lys Val Ser Phe Glu
Ala Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His22521218PRTArtificial Sequencesynthetic 21Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ala Val Trp Gly Gly 20 25 30Tyr Ile Ala
Pro Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu
Leu Ile Tyr Asp Gly Ser Tyr Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Leu
85 90 95Pro Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21522218PRTArtificial Sequencesynthetic 22Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Asn Gln Ser Ile Ala Gly Ala 20 25 30Tyr Tyr Ala Pro
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu
Ile Tyr Asp Gly Tyr Phe Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Leu
85 90 95Pro Ser Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200
205Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21523218PRTArtificial Sequencesynthetic 23Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Leu Trp Ala Ala 20 25 30Tyr Phe Ala Pro
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu
Ile
Tyr Asp Gly Ser Tyr Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75 80Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Leu 85 90
95Pro Ser Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 110Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln 115 120 125Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr 130 135 140Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser145 150 155 160Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 21524108PRTArtificial
Sequencesynthetic 24Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Tyr Pro Thr Pro Tyr 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 10525121PRTArtificial
Sequencesynthetic 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Thr Gly Asn 20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile Ser Pro Ser Gly Gly Tyr
Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala
Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Ser Arg
Val Ser Tyr Glu Ala Ala Met Asp Tyr Trp Gly 100 105 110Gln Gly Thr
Leu Val Thr Val Ser Ser 115 12026108PRTArtificial Sequencesynthetic
26Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Leu Ala Thr
Asp 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Glu Pro Glu Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 10527108PRTArtificial Sequencesynthetic 27Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Leu Ala Thr Asp 20 25
30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Glu
Pro Glu Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 10528121PRTArtificial Sequencesynthetic 28Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Asp 20 25 30Trp Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly
Glu Ile Ser Ala Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Glu Ser Arg Val Ser Phe Glu Ala Ala Met Asp Tyr
Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12029108PRTArtificial Sequencesynthetic 29Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asn Ile Ala Thr Asp 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Glu Pro Glu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
10530121PRTArtificial Sequencesynthetic 30Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Ala Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ser Arg Val Ser Phe Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12031108PRTArtificial Sequencesynthetic 31Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asp Leu Ala Thr Asp 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Glu Pro Glu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
10532121PRTArtificial Sequencesynthetic 32Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Pro Ala Gly Ala Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ala Lys Val Ser Phe Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12033121PRTArtificial Sequencesynthetic 33Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Thr Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Pro Ala Gly Ala Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ala Lys Val Ser Phe Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12034121PRTArtificial Sequencesynthetic 34Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Glu Ile
Ser Ala Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ser Lys Val Ser Phe Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12035121PRTArtificial Sequencesynthetic 35Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Ala Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ser Lys Val Ser Phe Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12036112PRTArtificial Sequencesynthetic 36Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ala Val Trp Gly Gly 20 25 30Tyr Ile Ala Pro
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu
Ile Tyr Asp Gly Ser Tyr Leu Tyr Ser Gly Val Pro Ser 50 55 60Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser65 70 75
80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Leu
85 90 95Pro Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 11037112PRTArtificial Sequencesynthetic 37Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Asn Gln Ser Ile Ala Gly Ala 20 25 30Tyr
Tyr Ala Pro Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40
45Lys Leu Leu Ile Tyr Asp Gly Tyr Phe Leu Tyr Ser Gly Val Pro Ser
50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Arg Leu 85 90 95Pro Ser Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 105 11038112PRTArtificial Sequencesynthetic
38Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Leu Trp Ala
Ala 20 25 30Tyr Phe Ala Pro Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro 35 40 45Lys Leu Leu Ile Tyr Asp Gly Ser Tyr Leu Tyr Ser Gly
Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser65 70 75 80Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Arg Leu 85 90 95Pro Ser Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 11039110PRTArtificial
Sequencesynthetic 39Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Leu Phe Tyr 20 25 30Gly Asn Val Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu 35 40 45Leu Ile Tyr Asp Gly Ser Tyr Leu Tyr
Ser Gly Val Pro Ser Arg Phe 50 55 60Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu65 70 75 80Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Ser Tyr Pro Ala 85 90 95Pro Tyr Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 11040108PRTArtificial
Sequencesynthetic 40Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Leu Ala Thr Asp 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Glu Pro Glu Pro Tyr 85 90 95Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 10541114PRTArtificial
Sequencesynthetic 41Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ala Leu Gly Asp Ser 20 25 30Glu Asn Gly Tyr Ala Asp Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys 35 40 45Ala Pro Lys Leu Leu Ile Tyr Glu Gly
Ser Ser Leu Tyr Ser Gly Val 50 55 60Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95Ala Ala Pro Ser Pro
Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110Lys
Arg42114PRTArtificial Sequencesynthetic 42Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Leu Leu Ala Ser 20 25 30His Asp Gly Thr
Pro Trp Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45Ala Pro Lys
Leu Leu Ile Tyr Asp Ala Ser Tyr Leu Tyr Ser Gly Val 50 55 60Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75
80Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95Ser Thr Phe Ala Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile 100 105 110Lys Arg43108PRTArtificial Sequencesynthetic 43Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Glu Ile Phe
Pro Asp 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45Tyr Ser Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Ala Ala Pro Thr Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys Arg 100 10544108PRTArtificial Sequencesynthetic 44Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Ala Ser Asp
20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Glu Pro Glu Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 10545108PRTArtificial Sequencesynthetic 45Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Gly Ser Asp 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ser Ala Ser Tyr Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Pro
Glu Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 10546108PRTArtificial Sequencesynthetic 46Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asn Ile Ala Ser Asp 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Ala Ser Ser Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Pro Glu Pro Tyr
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
105477PRTArtificial Sequencesynthetic 47Phe Thr Gly Asn Trp Ile
His1 5487PRTArtificial Sequencesynthetic 48Leu Ser Gly Asp Trp Ile
His1 54912PRTArtificial Sequencesynthetic 49Val Gly Glu Ile Ser Pro
Ser Gly Gly Tyr Thr Asp1 5 105012PRTArtificial Sequencesynthetic
50Leu Gly Glu Ile Ser Ala Ala Gly Gly Tyr Thr Asp1 5
105112PRTArtificial Sequencesynthetic 51Val Gly Glu Ile Ser Ala Ala
Gly Gly Tyr Thr Asp1 5 105214PRTArtificial Sequencesynthetic 52Ala
Arg Glu Ser Arg Val Ser Tyr Glu Ala Ala Met Asp Tyr1 5
105314PRTArtificial Sequencesynthetic 53Ala Arg Glu Ser Arg Val Ser
Phe Glu Ala Ala Met Asp Tyr1 5 10547PRTArtificial Sequencesynthetic
54Asp Leu Ala Thr Asp Val Ala1 5557PRTArtificial Sequencesynthetic
55Asn Ile Ala Thr Asp Val Ala1 5564PRTArtificial Sequencesynthetic
56Ser Ala Ser Phe1577PRTArtificial Sequencesynthetic 57Ser Glu Pro
Glu Pro Tyr Thr1 558108PRTArtificial Sequencesynthetic 58Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25
30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr
Thr Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105597PRTArtificial Sequencesynthetic 59Phe Ser Gly Asp Trp
Ile His1 56012PRTArtificial Sequencesynthetic 60Val Gly Glu Ile Ser
Pro Ala Gly Ala Tyr Thr Asp1 5 106114PRTArtificial
Sequencesynthetic 61Ala Arg Glu Ala Lys Val Ser Phe Glu Ala Ala Met
Asp Tyr1 5 10627PRTArtificial Sequencesynthetic 62Phe Thr Gly Asp
Trp Ile His1 563121PRTArtificial Sequencesynthetic 63Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Gly Asp 20 25 30Trp
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Gly Glu Ile Ser Pro Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Glu Ala Arg Val Ser Phe Glu Ala Ala Met
Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12064121PRTArtificial Sequencesynthetic 64Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Ala Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ala Arg Val Ser Tyr Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12065121PRTArtificial Sequencesynthetic 65Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Pro Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ser Arg Val Ser Phe Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12066121PRTArtificial Sequencesynthetic 66Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Asn 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Pro Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ala Arg Val Ser Tyr Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12067121PRTArtificial Sequencesynthetic 67Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Ala Ala Gly Gly Tyr Ser Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ala Arg Val Ser Tyr Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12068121PRTArtificial Sequencesynthetic 68Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Leu Ser Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Ala Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ala Arg Val Ser Phe Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12069121PRTArtificial Sequencesynthetic 69Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Leu Thr Gly Asp 20 25 30Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Pro Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Glu Ala Arg Val Ser Tyr Glu Ala Ala Met Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
1207045DNAArtificial Sequencesynthetic 70acttattact gtcagcaann
nnnnnnnnst ccttacacgt tcgga 457145DNAArtificial Sequencesynthetic
71acttattact gtcagcaann ntacnnnnnt ccttacacgt tcgga
457245DNAArtificial Sequencesynthetic 72acttattact gtcagcaang
gnnnnnnnnn ccttacacgt tcgga 457345DNAArtificial Sequencesynthetic
73acttattact gtcagcaann tnnnnnnnnn ccttacacgt tcgga
457445DNAArtificial Sequencesynthetic 74acttattact gtcagcaann
nnnnnnnnnn ccttacacgt tcgga 457545DNAArtificial Sequencesynthetic
75acttattact gtcagcaang gnnnnnnnnt ccttacacgt tcgga
457645DNAArtificial Sequencesynthetic 76acttattact gtcagcaann
tnnnnnnnnt ccttacacgt tcgga 457736DNAArtificial Sequencesynthetic
77tcttgtgaca aaactcacag tggcggtggc tctggt 36787PRTArtificial
sequencesynthetic 78Asp Val Ser Thr Ala Val Ala1 5797PRTArtificial
sequencesynthetic 79Ser Tyr Pro Thr Pro Tyr Thr1 5
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