U.S. patent application number 15/965143 was filed with the patent office on 2019-04-11 for antigen binding molecules that bind egfr, vectors encoding same, and uses thereof.
The applicant listed for this patent is Roche GlycArt AG. Invention is credited to Ekkehard MOSSNER, Pablo Umana.
Application Number | 20190106497 15/965143 |
Document ID | / |
Family ID | 36777599 |
Filed Date | 2019-04-11 |
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United States Patent
Application |
20190106497 |
Kind Code |
A1 |
Umana; Pablo ; et
al. |
April 11, 2019 |
Antigen Binding Molecules That Bind EGFR, Vectors Encoding Same,
and Uses Thereof
Abstract
The present invention relates to antigen binding molecules
(ABMs). In particular embodiments, the present invention relates to
recombinant monoclonal antibodies, including chimeric, primatized
or humanized antibodies specific for human EGFR. In addition, the
present invention relates to nucleic acid molecules encoding such
ABMs, and vectors and host cells comprising such nucleic acid
molecules. The invention further relates to methods for producing
the ABMs of the invention, and to methods of using these ABMs in
treatment of disease. In addition, the present invention relates to
ABMs with modified glycosylation having improved therapeutic
properties, including antibodies with increased Fc receptor binding
and increased effector function.
Inventors: |
Umana; Pablo; (Wollerau,
CH) ; MOSSNER; Ekkehard; (Kreuzlingen, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche GlycArt AG |
Schlieren-Zurich |
|
CH |
|
|
Family ID: |
36777599 |
Appl. No.: |
15/965143 |
Filed: |
April 27, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15050821 |
Feb 23, 2016 |
9957326 |
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15965143 |
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14084303 |
Nov 19, 2013 |
9309317 |
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15050821 |
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13315989 |
Dec 9, 2011 |
8614065 |
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14084303 |
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12938180 |
Nov 2, 2010 |
8097436 |
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13315989 |
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11889981 |
Aug 17, 2007 |
7846432 |
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12938180 |
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11348526 |
Feb 7, 2006 |
7722867 |
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11889981 |
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60650115 |
Feb 7, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/179 20130101;
C07K 2317/52 20130101; C07K 2317/565 20130101; A61K 2039/505
20130101; C07K 2317/732 20130101; C07K 2317/41 20130101; A61K
47/6803 20170801; C07K 16/2863 20130101; C07K 2319/00 20130101;
A61K 45/06 20130101; A61P 35/00 20180101; C07K 2317/21 20130101;
A61K 47/6817 20170801 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 47/68 20060101 A61K047/68; A61K 38/17 20060101
A61K038/17; A61K 45/06 20060101 A61K045/06 |
Claims
1. An isolated polynucleotide comprising: (a)(i) a sequence
selected from the group consisting of: SEQ ID NO:56, SEQ ID NO:58,
SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:72, SEQ ID NO:74, SEQ ID NO:122, and SEQ ID NO:124; and (b) ii a
sequence selected from the group consisting of: SEQ ID NO:78, SEQ
ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88,
SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:100, SEQ ID
NO:102, SEQ ID NO:104, SEQ ID NO:106, and SEQ ID NO:126; and (iii)
SEQ ID NO:108; (b)(i) SEQ ID NO:114; and (ii) a sequence selected
from the group consisting of SEQ ID NO:116 and SEQ ID NO:118; and
(iii) SEQ ID NO:119; (c) a sequence having at least 80% identity to
a sequence selected from the group consisting of SEQ ID NO:4; SEQ
ID NO:6; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:12; SEQ ID NO:14; SEQ
ID NO:16; SEQ ID NO:18; SEQ ID NO:20; SEQ ID NO:22; SEQ ID NO:24;
SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID
NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40, and SEQ ID NO:120;
(d) a sequence having at least 80% identity to a sequence selected
from the group consisting of SEQ ID NO:46; SEQ ID NO:50; SEQ ID
NO:52; (e) a sequence that encodes a polypeptide having a sequence
selected from the group consisting of SEQ ID NO:3; SEQ ID NO:5; SEQ
ID NO:7; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ
ID NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25;
SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID
NO:35; SEQ ID NO:37; SEQ ID NO:39; and SEQ ID NO:121; or (f) a
sequence that encodes a polypeptide having a sequence selected from
the group consisting of SEQ ID NO:45, SEQ ID NO:49, and SEQ ID
NO:51.
2-40. (canceled)
41. An expression vector comprising an isolated polynucleotide
according to claim 1.
42. (canceled)
43. A host cell comprising the expression vector of claim 41.
44-46. (canceled)
47. A polypeptide comprising a sequence derived from the rat ICR62
antibody and a sequence derived from a heterologous peptide.
48. An antigen binding molecule comprising the polypeptide of claim
47.
49-54. (canceled)
55. The polypeptide according to claim 47, wherein said polypeptide
comprises: (a)(i) a polypeptide having a sequence selected from the
group consisting of SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ
ID NO:123, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65,
SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, and SEQ ID
NO:125; and (ii) a polypeptide having a sequence selected from the
group consisting of SEQ ID NO:75 SEQ ID NO:77, SEQ ID NO:79, SEQ ID
NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ
ID NO:127, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97,
SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:101, SEQ ID NO:103, SEQ ID
NO:105, and SEQ ID NO:105; and (iii) a polypeptide having the
sequence of SEQ ID NO:107; (b)(i) a polypeptide having a sequence
of SEQ ID NO:111 or SEQ ID NO:113; and (ii) a polypeptide having a
sequence of SEQ ID NO:115; and (iii) a polypeptide having a
sequence of SEQ ID NO:117; (c) a sequence selected from the group
consisting of SEQ ID NO: 1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7;
SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:15; SEQ ID
NO:17; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ
ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35;
SEQ ID NO:37; SEQ ID NO:39; and SEQ ID NO:121, or a variant
thereof; or (d) a sequence selected from the group consisting of
SEQ ID NO: 43, SEQ ID NO:45, SEQ ID NO:49, and SEQ ID NO:51, or a
variant thereof.
56-95. (canceled)
96. A method of producing an antigen binding molecule, which is
capable of competing with the rat ICR62 antibody for binding to
human EGFR, and wherein said antigen binding molecule is chimeric;
said method comprising (a) culturing the host cell of claim 43 in a
medium under conditions allowing the expression of said
polynucleotide encoding said antigen binding molecule; and (b)
recovering said antigen binding molecule.
97-98. (canceled)
99. A pharmaceutical composition comprising the antigen binding
molecule according to claim 48 and a pharmaceutically acceptable
carrier.
100. A method for treating an EGFR-related disorder comprising: (a)
predicting a response to anti-EGFR therapy in a human subject by
assaying a sample from the human subject before therapy with one or
a plurality of reagents that detect expression and/or activation of
predictive biomarkers for cancer; (b) determining a pattern of
expression and/or activation of one or more of said predictive
biomarkers, wherein the pattern predicts the human subject's
response to the anti-EGFR therapy; and (c) administering to a human
subject who is predicted to respond the anti-EGFR directed therapy
a therapeutically effective amount of the composition of claim
99.
101. The method of claim 100, wherein the predictive biomarker is a
growth factor receptor, or a growth factor receptor-related
downstream signaling molecule.
102. The method of claim 101, wherein the growth factor receptor is
EGFR, phosphorylated EGFR, HER2/neu, HER3, or any combination
thereof, and the growth factor receptor-related downstream
signaling molecules is selected from the group consisting of Akt,
RAS, RAF, MAPK, ERK1, ERK2, PKC, STAT3, and STAT5.
103-105. (canceled)
106. A method for targeting cells expressing EGFR in a subject
comprising administering to said subject the pharmaceutical
composition of claim 99.
107-109. (canceled)
110. A method of treating a cell proliferation disorder treatable
by blocking EGFR-mediated signaling comprising administering a
therapeutically effective amount of the pharmaceutical composition
of claim 99 to a human subject in need thereof.
111. A method for detecting in vivo or in vitro the presence of
EGFR in a sample comprising: (a) contacting a sample to be tested,
optionally with a control sample, with the antigen binding molecule
of claim 48 under conditions that allow for formation of a complex
between the antigen binding molecule and EGFR; and (b) detecting
said antigen binding molecule-EGFR complexes.
112. A host cell engineered to express at least one nucleic acid
encoding a polypeptide having
.beta.(1,4)-N-acetylglucosaminyltransferase III activity in an
amount sufficient to modify the oligosaccharides in the Fc region
of a polypeptide produced by said host cell, wherein said
polypeptide is the antigen binding molecule according to claim
48.
113-169. (canceled)
170. A method for producing an antigen binding molecule having
modified oligosaccharides in a host cell, said method comprising:
(a) culturing a host cell engineered to express at least one
nucleic acid encoding a polypeptide having
.beta.(1,4)-N-acetylglucosaminyltransferase III activity under
conditions which permit the production of said antigen binding
molecule, and which permit the modification of the oligosaccharides
present on the Fc region of said antigen binding molecule; and (b)
isolating said antigen binding molecule wherein said antigen
binding molecule is capable of competing with the ICR62 antibody
for binding to EGFR and wherein said antigen binding molecule or
fragment thereof is chimeric.
171-207. (canceled)
208. A method of treating an EGFR-related disorder in a subject in
need of such treatment comprising administering to said subject the
antigen binding molecule of claim 48 in an amount of about 1.0
mg/kg to about 15 mg/kg.
209-238. (canceled)
239. The antigen binding molecule according to claim 48, wherein
said antigen binding molecule, when administered to a mammalian
subject at concentrations above one microgram per milliliter of
serum does not cause a clinically significant level of toxicity in
said mammalian subject.
240-241. (canceled)
242. A method of treating an EGFR-related disorder in a mammal in
need of treatment thereof, said method comprising administering to
said mammal the antigen binding molecule according to claim 48,
wherein said treatment results in serum concentrations of said
antigen binding molecule between about 1 and about 100 .mu.g/ml for
a period of at least 4 weeks, and wherein said treatment does not
cause a clinically significant level of toxicity in said
mammal.
243-244. (canceled)
245. A method of inhibiting ligand binding to EGFR, comprising
contacting EGFR with the antigen binding molecule according to
claim 48.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/050,821, filed Feb. 23, 2016, which is a division of U.S.
application Ser. No. 14/084,303, filed Nov. 19, 2013, now U.S. Pat.
No. 9,309,317, granted on Apr. 12, 2016, which is a division of
U.S. application Ser. No. 13/315,989, filed Dec. 9, 2011, now U.S.
Pat. No. 8,614,065, granted on Dec. 24, 2013, which is a division
of U.S. application Ser. No. 12/938,180, filed Nov. 2, 2010, now
U.S. Pat. No. 8,097,436, granted on Jan. 17, 2012, which is a
division of U.S. application Ser. No. 11/889,981, filed Aug. 17,
2007, now U.S. Pat. No. 7,846,432, granted on Dec. 7, 2010, which
is a division of U.S. application Ser. No. 11/348,526, filed Feb.
7, 2006, now U.S. Pat. No. 7,722,867, granted on May 25, 2010,
which claims the benefit of U.S. Application No. 60/650,115, filed
Feb. 7, 2005, the entire contents of each of which are hereby
incorporated by reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The content of the electronically submitted sequence
listing, file name: substitute_sequencelisting_ascii.txt; Size:
63,840 bytes; and Date of Creation: Jan. 18, 2011, filed herewith,
is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention relates to antigen binding molecules
(ABMs). In particular embodiments, the present invention relates to
recombinant monoclonal antibodies, including chimeric, primatized
or humanized antibodies specific for human epidermal growth factor
receptor (EGFR). In addition, the present invention relates to
nucleic acid molecules encoding such ABMs, and vectors and host
cells comprising such nucleic acid molecules. The invention further
relates to methods for producing the ABMs of the invention, and to
methods of using these ABMs in treatment of disease. In addition,
the present invention relates to ABMs with modified glycosylation
having improved therapeutic properties, including antibodies with
increased Fc receptor binding and increased effector function.
Background Art
[0004] EGFR and Anti-EGFR Antibodies
[0005] Human epidermal growth factor receptor (also known as HER-1
or Erb-B1, and referred to herein as "EGFR") is a 170 kDa
transmembrane receptor encoded by the c-erbB protooncogene, and
exhibits intrinsic tyrosine kinase activity (Modjtahedi et al., Br.
J. Cancer 73:228-235 (1996); Herbst and Shin, Cancer 94:1593-1611
(2002)). SwissProt database entry P00533 provides the sequence of
EGFR. There are also isoforms and variants of EGFR (e.g.,
alternative RNA transcripts, truncated versions, polymorphisms,
etc.) including but not limited to those identified by Swissprot
database entry numbers P00533-1, P00533-2, P00533-3, and P00533-4.
EGFR is known to bind ligands including epidermal growth factor
(EGF), transforming growth factor-.alpha.
(TGf-.quadrature..alpha.), amphiregulin, heparin-binding EGF
(hb-EGF), betacellulin, and epiregulin (Herbst and Shin, Cancer
94:1593-1611 (2002); Mendelsohn and Baselga, Oncogene 19:6550-6565
(2000)). EGFR regulates numerous cellular processes via
tyrosine-kinase mediated signal transduction pathways, including,
but not limited to, activation of signal transduction pathways that
control cell proliferation, differentiation, cell survival,
apoptosis, angiogenesis, mitogenesis, and metastasis (Atalay et
al., Ann. Oncology 14:1346-1363 (2003); Tsao and Herbst, Signal
4:4-9 (2003); Herbst and Shin, Cancer 94:1593-1611 (2002);
Modjtahedi et al., Br. J. Cancer 73:228-235 (1996)).
[0006] Overexpression of EGFR has been reported in numerous human
malignant conditions, including cancers of the bladder, brain, head
and neck, pancreas, lung, breast, ovary, colon, prostate, and
kidney. (Atalay et al., Ann. Oncology 14:1346-1363 (2003); Herbst
and Shin, Cancer 94:1593-1611 (2002) Modjtahedi et al., Br. J.
Cancer 73:228-235 (1996)). In many of these conditions, the
overexpression of EGFR correlates or is associated with poor
prognosis of the patients. (Herbst and Shin, Cancer 94:1593-1611
(2002) Modjtahedi et al., Br. J. Cancer 73:228-235 (1996)). EGFR is
also expressed in the cells of normal tissues, particularly the
epithelial tissues of the skin, liver, and gastrointestinal tract,
although at generally lower levels than in malignant cells (Herbst
and Shin, Cancer 94:1593-1611 (2002)).
[0007] Unconjugated monoclonal antibodies (mAbs) can be useful
medicines for the treatment of cancer, as demonstrated by the U.S.
Food and Drug Administration's approval of Trastuzumab
(Herceptin.TM.; Genentech Inc) for the treatment of advanced breast
cancer (Grillo-Lopez, A.-J., et al., Semin. Oncol. 26:66-73 (1999);
Goldenberg, M. M., Clin. Ther. 21:309-18 (1999)), Rituximab
(Rituxan.TM.; IDEC Pharmaceuticals, San Diego, Calif., and
Genentech Inc., San Francisco, Calif.), for the treatment of CD20
positive B-cell, low-grade or follicular Non-Hodgkin's lymphoma,
Gemtuzumab (Mylotarg.TM., Celltech/Wyeth-Ayerst) for the treatment
of relapsed acute myeloid leukemia, and Alemtuzumab (CAMPATH.TM.,
Millenium Pharmaceuticals/Schering AG) for the treatment of B cell
chronic lymphocytic leukemia. The success of these products relies
not only on their efficacy but also on their outstanding safety
profiles (Grillo-Lopez, A. J., et al., Semin. Oncol. 26:66-73
(1999); Goldenberg, M. M., Clin. Ther. 21:309-18 (1999)). In spite
of the achievements of these drugs, there is currently a large
interest in obtaining higher specific antibody activity than what
is typically afforded by unconjugated mAb therapy.
[0008] The results of a number of studies suggest that
Fc-receptor-dependent mechanisms contribute substantially to the
action of cytotoxic antibodies against tumors and indicate that an
optimal antibody against tumors would bind preferentially to
activation Fc receptors and minimally to the inhibitory partner
Fc.gamma.RIIB. (Clynes, R. A., et al., Nature Medicine 6(4):443-446
(2000); Kalergis, A. M., and Ravetch, J. V., J. Exp. Med.
195(12):1653-1659 (June 2002). For example, the results of at least
one study suggest that polymorphism in the Fc.gamma.RIIIa receptor,
in particular, is strongly associated with the efficacy of antibody
therapy. (Cartron, G., et al., Blood 99(3):754-757 (February
2002)). That study showed that patients homozygous for
Fc.gamma.RIIIa have a better response to Rituximab than
heterozygous patients. The authors concluded that the superior
response was due to better in vivo binding of the antibody to
Fc.gamma.RIIIa, which resulted in better ADCC activity against
lymphoma cells. (Cartron, G., et al., Blood 99(3):754-757 (February
2002)).
[0009] Various strategies to target EGFR and block EGFR signaling
pathways have been reported. Small-molecule tyrosine kinase
inhibitors like gefitinib, erlotinib, and CI-1033 block
autophosphorylation of EGFR in the intracellular tyrosine kinase
region, thereby inhibiting downstream signaling events (Tsao and
Herbst, Signal 4: 4-9 (2003)). Monoclonal antibodies, on the other
hand, target the extracellular portion of EGFR, which results in
blocking ligand binding and thereby inhibits downstream events such
as cell proliferation (Tsao and Herbst, Signal 4: 4-9 (2003)).
[0010] Several murine monoclonal antibodies have been generated
which achieve such a block in vitro and which have been evaluated
for their ability to affect tumor growth in mouse xenograft models
(Masui, et al., Cancer Res. 46:5592-5598 (1986); Masui, et al.,
Cancer Res. 44:1002-1007 (1984); Goldstein, et al., Clin. Cancer
Res. 1: 1311-1318 (1995)). For example, EMD 55900 (EMD
Pharmaceuticals) is a murine anti-EGFR monoclonal antibody that was
raised against human epidermoid carcinoma cell line A431 and was
tested in clinical studies of patients with advanced squamous cell
carcinoma of the larynx or hypopharynx (Bier et al., Eur. Arch.
Otohinolaryngol. 252:433-9 (1995)). In addition, the rat monoclonal
antibodies ICR16, ICR62, and ICR80, which bind the extracellular
domain of EGFR, have been shown to be effective at inhibiting the
binding of EGF and TGF-.alpha. the receptor. (Modjtahedi et al.,
Int. J. Cancer 75:310-316 (1998)). The murine monoclonal antibody
425 is another MAb that was raised against the human A431 carcinoma
cell line and was found to bind to a polypeptide epitope on the
external domain of the human epidermal growth factor receptor.
(Murthy et al., Arch. Biochem. Biophys. 252(2):549-560 (1987). A
potential problem with the use of murine antibodies in therapeutic
treatments is that non-human monoclonal antibodies can be
recognized by the human host as a foreign protein; therefore,
repeated injections of such foreign antibodies can lead to the
induction of immune responses leading to harmful hypersensitivity
reactions. For murine-based monoclonal antibodies, this is often
referred to as a Human Anti-Mouse Antibody response, or "HAMA"
response, or a Human Anti-Rat Antibody, or "HARE" response.
Additionally, these "foreign" antibodies can_be attacked by the
immune system of the host such that they are, in effect,
neutralized before they reach their target site. Furthermore,
non-human monoclonal antibodies (e.g., murine monoclonal
antibodies) typically lack human effector functionality, i.e., they
are unable to, inter alia, mediate complement dependent lysis or
lyse human target cells through antibody dependent cellular
toxicity or Fc-receptor mediated phagocytosis.
[0011] Chimeric antibodies comprising portions of antibodies from
two or more different species (e.g., mouse and human) have been
developed as an alternative to "conjugated" antibodies. For
example, U.S. Pat. No. 5,891,996 (Mateo de Acosta del Rio et al.)
discusses a mouse/human chimeric antibody, R3, directed against
EGFR, and U.S. Pat. No. 5,558,864 discusses generation of chimeric
and humanized forms of the murine anti-EGFR MAb 425. Also, IMC-C225
(Erbitux.RTM.; ImClone) is a chimeric mouse/human anti-EGFR
monoclonal antibody (based on mouse M225 monoclonal antibody, which
resulted in HAMA responses in human clinical trials) that has been
reported to demonstrate antitumor efficacy in various human
xenograft models. (Herbst and Shin, Cancer 94:1593-1611 (2002)).
The efficacy of IMC-C225 has been attributed to several mechanisms,
including inhibition of cell events regulated by EGFR signaling
pathways, and possibly by increased antibody-dependent cellular
toxicity (ADCC) activity (Herbst and Shin, Cancer 94:1593-1611
(2002)). IMC-C225 was also used in clinical trials, including in
combination with radiotherapy and chemotherapy (Herbst and Shin,
Cancer 94:1593-1611 (2002)). Recently, Abgenix, Inc. (Fremont,
Calif.) developed ABX-EGF for cancer therapy. ABX-EGF is a fully
human anti-EGFR monoclonal antibody. (Yang et al., Crit. Rev.
Oncol./Hematol. 38: 17-23 (2001)).
[0012] Antibody Glycosylation
[0013] The oligosaccharide component can significantly affect
properties relevant to the efficacy of a therapeutic glycoprotein,
including physical stability, resistance to protease attack,
interactions with the immune system, pharmacokinetics, and specific
biological activity. Such properties may depend not only on the
presence or absence, but also on the specific structures, of
oligosaccharides. Some generalizations between oligosaccharide
structure and glycoprotein function can be made. For example,
certain oligosaccharide structures mediate rapid clearance of the
glycoprotein from the bloodstream through interactions with
specific carbohydrate binding proteins, while others can be bound
by antibodies and trigger undesired immune reactions. (Jenkins et
al., Nature Biotechnol. 14:975-81 (1996)).
[0014] Mammalian cells are the preferred hosts for production of
therapeutic glycoproteins, due to their capability to glycosylate
proteins in the most compatible form for human application.
(Cumming et al., Glycobiology 1:115-30 (1991); Jenkins et al.,
Nature Biotechnol. 14:975-81 (1996)). Bacteria very rarely
glycosylate proteins, and like other types of common hosts, such as
yeasts, filamentous fungi, insect and plant cells, yield
glycosylation patterns associated with rapid clearance from the
blood stream, undesirable immune interactions, and in some specific
cases, reduced biological activity. Among mammalian cells, Chinese
hamster ovary (CHO) cells have been most commonly used during the
last two decades. In addition to giving suitable glycosylation
patterns, these cells allow consistent generation of genetically
stable, highly productive clonal cell lines. They can be cultured
to high densities in simple bioreactors using serum-free media, and
permit the development of safe and reproducible bioprocesses. Other
commonly used animal cells include baby hamster kidney (BHK) cells,
NS0- and SP2/0-mouse myeloma cells. More recently, production from
transgenic animals has also been tested. (Jenkins et al., Nature
Biotechnol. 14:975-81 (1996)).
[0015] All antibodies contain carbohydrate structures at conserved
positions in the heavy chain constant regions, with each isotype
possessing a distinct array of N-linked carbohydrate structures,
which variably affect protein assembly, secretion or functional
activity. (Wright, A., and Morrison, S. L., Trends Biotech.
15:26-32 (1997)). The structure of the attached N-linked
carbohydrate varies considerably, depending on the degree of
processing, and can include high-mannose, multiply-branched as well
as biantennary complex oligosaccharides. (Wright, A., and Morrison,
S. L., Trends Biotech. 15:26-32 (1997)). Typically, there is
heterogeneous processing of the core oligosaccharide structures
attached at a particular glycosylation site such that even
monoclonal antibodies exist as multiple glycoforms. Likewise, it
has been shown that major differences in antibody glycosylation
occur between cell lines, and even minor differences are seen for a
given cell line grown under different culture conditions. (Lifely,
M. R. et al., Glycobiology 5(8):813-22 (1995)).
[0016] One way to obtain large increases in potency, while
maintaining a simple production process and potentially avoiding
significant, undesirable side effects, is to enhance the natural,
cell-mediated effector functions of monoclonal antibodies by
engineering their oligosaccharide component as described in Umana,
P. et al., Nature Biotechnol. 17:176-180 (1999) and U.S. Pat. No.
6,602,684, the contents of which are hereby incorporated by
reference in their entirety. IgG1 type antibodies, the most
commonly used antibodies in cancer immunotherapy, are glycoproteins
that have a conserved N-linked glycosylation site at Asn297 in each
CH2 domain. The two complex biantennary oligosaccharides attached
to Asn297 are buried between the CH2 domains, forming extensive
contacts with the polypeptide backbone, and their presence is
essential for the antibody to mediate effector functions such as
antibody dependent cellular cytotoxicity (ADCC) (Lifely, M. R., et
al., Glycobiology 5:813-822 (1995); Jefferis, R., et al., Immunol
Rev. 163:59-76 (1998); Wright, A. and Morrison, S. L., Trends
Biotechnol. 15:26-32 (1997)).
[0017] Umana et al. showed previously that overexpression in
Chinese hamster ovary (CHO) cells of
.beta.(1,4)-N-acetylglucosaminyltransferase III ("GnTIII"), a
glycosyltransferase catalyzing the formation of bisected
oligosaccharides, significantly increases the in vitro ADCC
activity of an anti-neuroblastoma chimeric monoclonal antibody
(chCE7) produced by the engineered CHO cells. (See Umana, P. et
al., Nature Biotechnol. 17:176-180 (1999); and International
Publication No. WO 99/54342, the entire contents of which are
hereby incorporated by reference). The antibody chCE7 belongs to a
large class of unconjugated mAbs which have high tumor affinity and
specificity, but have too little potency to be clinically useful
when produced in standard industrial cell lines lacking the GnTIII
enzyme (Umana, P., et al., Nature Biotechnol. 17:176-180 (1999)).
That study was the first to show that large increases of ADCC
activity could be obtained by engineering the antibody-producing
cells to express GnTIII, which also led to an increase in the
proportion of constant region (Fc)-associated, bisected
oligosaccharides, including bisected, nonfucosylated
oligosaccharides, above the levels found in naturally-occurring
antibodies.
[0018] There remains a need for enhanced therapeutic approaches
targeting EGFR for the treatment of cell proliferation disorders in
primates, including, but not limited to, humans, wherein such
disorders are characterized by EGFR expression, particularly
abnormal expression (e.g., overxpression) including, but not
limited to, cancers of the bladder, brain, head and neck, pancreas,
lung, breast, ovary, colon, prostate, and kidney.
BRIEF SUMMARY OF THE INVENTION
[0019] Recognizing the tremendous therapeutic potential of antigen
binding molecules (ABMs) that have the binding specificity of the
rat ICR62 antibody (e.g., bind the same epitope) and that have been
glycoengineered to enhance Fc receptor binding affinity and
effector function, the present inventors developed a method for
producing such ABMs. Inter alia, this method involves producing
recombinant, chimeric antibodies or chimeric fragments thereof. The
efficacy of these ABMs is further enhanced by engineering the
glycosylation profile of the antibody Fc region.
[0020] Accordingly, in one aspect, the invention is directed to an
isolated polynucleotide comprising: (a) a sequence selected from a
group consisting of: SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ
ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68,
SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:122, and SEQ ID
NO:124; (b) a sequence selected from a group consisting of: SEQ ID
NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ
ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94,
SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID
NO:104, SEQ ID NO:106, and SEQ ID NO:126; and (c) SEQ ID NO:108. In
another aspect, the invention is directed to an isolated
polynucleotide comprising (a) a sequence selectd from the group
consisting of SEQ ID NO:112 and SEQ ID NO:114; (b) a sequence
selectd from the group consisting of SEQ ID NO:116 and SEQ ID
NO:118; and (c) SEQ ID NO:119. In one embodiment, any of these
polynucleotides encodes a fusion polypeptide.
[0021] In a further aspect, the invention is directed to an
isolated polynucleotide comprising a sequence selected from the
group consisting of SEQ ID No:2; SEQ ID No:4; SEQ ID No:6; SEQ ID
No:8; SEQ ID No:10; SEQ ID No:12; SEQ ID No:14; SEQ ID No:16; SEQ
ID No:18; SEQ ID No:20; SEQ ID No:22; SEQ ID No:24; SEQ ID No:26;
SEQ ID No:28; SEQ ID No:30; SEQ ID No32; SEQ ID No:34; SEQ ID
No:36; SEQ ID No:38; SEQ ID No:40 and SEQ ID No:120. In another
aspect, the invention is directed to an isolated polynucleotide
comprising a sequence selected from the group consisting of SEQ ID
No:44; SEQ ID No:46; SEQ ID No:50; and SEQ ID No.:52. In one
embodiment, such polynucleotides encode fusion polypeptides.
[0022] The invention is further directed to an isolated
polynucleotide comprising a sequence having at least 80%, 85%, 90%,
95%, or 99% identity to a sequence selected from the group
consisting of SEQ ID No:2; SEQ ID No:4; SEQ ID No:6; SEQ ID No:8;
SEQ ID No:10; SEQ ID No:12; SEQ ID No:14; SEQ ID No:16; SEQ ID
No:18; SEQ ID No:20; SEQ ID No:22; SEQ ID No:24; SEQ ID No:26; SEQ
ID No:28; SEQ ID No:30; SEQ ID No32; SEQ ID No:34; SEQ ID No:36;
SEQ ID No:38; SEQ ID No:40 and SEQ ID No:120, wherein said isolated
polynucleotide encodes a fusion polypeptide. In an additional
aspect, the invention is directed to an isolated polynucleotide
comprising a sequence having at least 80% identity to a sequence
selected from the group consisting of SEQ ID No:44; SEQ ID No:46;
SEQ ID No:50; and SEQ ID No.:52, wherein said isolated
polynucleotide encodes a fusion polypeptide.
[0023] The invention is also directed to an isolated polynucleotide
encoding a chimeric polypeptide having the sequence of SEQ ID
No.:1. In one embodiment, the polynucleotide comprises a sequence
encoding a polypeptide having the sequence of SEQ ID No.:1; and a
sequence encoding a polypeptide having the sequence of an antibody
Fc region, or a fragment thereof, from a species other than rat.
The invention is also directed to an isolated polynucleotide
encoding a chimeric polypeptide having a sequence selected from the
group consisting of SEQ ID No:3; SEQ ID No:5; SEQ ID No:7; SEQ ID
No:9; SEQ ID No:11; SEQ ID No:13; SEQ ID No:15; SEQ ID No:17; SEQ
ID No:19; SEQ ID No:21; SEQ ID No:23; SEQ ID No:25; SEQ ID No:27;
SEQ ID No:29; SEQ ID No:31; SEQ ID No33; SEQ ID No:35; SEQ ID
No:37; SEQ ID No:39; and SEQ ID No:121. In one embodiment, the
polynucleotide comprises a sequence encoding a polypeptide having a
sequence selected from the group consisting of SEQ ID No:3; SEQ ID
No:5; SEQ ID No:7; SEQ ID No:9; SEQ ID No:11; SEQ ID No:13; SEQ ID
No:15; SEQ ID No:17; SEQ ID No:19; SEQ ID No:21; SEQ ID No:23; SEQ
ID No:25; SEQ ID No:27; SEQ ID No:29; SEQ ID No:31; SEQ ID No33;
SEQ ID No:35; SEQ ID No:37; SEQ ID No:39; and SEQ ID No:121; and a
sequence encoding a polypeptide having the sequence of an antibody
Fc region, or a fragment thereof, from a species other than
rat.
[0024] In yet another aspect, the invention is directed to an
isolated polynucleotide encoding a chimeric polypeptide having the
sequence of SEQ ID No.:43. In one embodiment, the polynucleotide
comprises a sequence encoding a polypeptide having the sequence of
SEQ ID No.:43; and a sequence encoding a polypeptide having the
sequence of an antibody Fc region, or a fragment thereof, from a
species other than rat. In yet another aspect, the invention is
directed to an isolated polynucleotide encoding a chimeric
polypeptide having a sequence selected from the group consisting of
SEQ ID No:45; SEQ ID No:49; and SEQ ID No.:51. In one embodiment,
the polynucleotide comprises a sequence encoding a polypeptide
having a sequence selected from the group consisting of SEQ ID
No:45; SEQ ID No:49; and SEQ ID No.:51, and a sequence encoding a
polypeptide having the sequence of an antibody light chain constant
region (CL), or a fragment thereof, from a species other than
rat.
[0025] The invention is also directed to an isolated polynucleotide
comprising a sequence encoding a polypeptide having the VH region
of the ICR62 antibody, or functional variants thereof, and a
sequence encoding a polypeptide having the sequence of an antibody
Fc region, or a fragment thereof, from a species other than rat. In
another aspect, the invention is directed to an isolated
polynucleotide comprising a sequence encoding a polypeptide having
the VL region of the ICR62 antibody, or functional variants
thereof, and a sequence encoding a polypeptide having the sequence
of an antibody CL region, or a fragment thereof, from a species
other than rat.
[0026] The invention is further directed to an expression vector
comprising any of the isolated polynucleotides described above, and
to a host cell that comprises such an expression vector. In a
further aspect, the invention is directed to a host cell comprising
any of the isolated polynucleotides described above.
[0027] In one aspect, the invention is directed to an isolated
polypeptide comprising: (a) a sequence selected from a group
consisting of: SEQ ID NO:53 SEQ ID NO:55, SEQ ID NO:57, SEQ ID
NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ
ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:123, and SEQ ID
NO:125; (b) a sequence selected from a group consisting of: SEQ ID
NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ
ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93,
SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID
NO:103, SEQ ID NO:105, and SEQ ID NO:127; and (c) SEQ ID NO:107.
wherein said polypeptide is a fusion polypeptide. In another
aspect, the invention is directed to an isolated polypeptide
comprising (a) a sequence selected from the group consisting of SEQ
ID NO:111 and SEQ ID NO:113; (b) SEQ ID NO:115; and (c) SEQ ID
NO:117, wherein said polypeptide is a fusion polypeptide.
[0028] The invention is also directed to a chimeric polypeptide
comprising the sequence of SEQ ID NO.:1 or a variant thereof. The
invention is further directed to a chimeric polypeptide comprising
the sequence of SEQ ID NO.:43 or a variant thereof. In one
embodiment, any one of these polypeptides further comprises a human
Fc region and/or a human CL region. The invention is also directed
to a chimeric polypeptide comprising a sequence selected from the
group consisting of SEQ ID No:3; SEQ ID No:5; SEQ ID No:7; SEQ ID
No:9; SEQ ID No:11; SEQ ID No:13; SEQ ID No:15; SEQ ID No:17; SEQ
ID No:19; SEQ ID No:21; SEQ ID No:23; SEQ ID No:25; SEQ ID No:27;
SEQ ID No:29; SEQ ID No:31; SEQ ID No33; SEQ ID No:35; SEQ ID
No:37; SEQ ID No:39; and SEQ ID No:121, or a variant thereof. The
invention is further directed to a chimeric polypeptide comprising
s sequence selected from the group consisting of SEQ ID No:45; SEQ
ID No:49; and SEQ ID No.:51, or a variant thereof. In one
embodiment, any one of these polypeptides further comprises a human
Fc region and/or a human CL region. In one embodiment, the human Fc
region comprises IgG1.
[0029] In another aspect the invention is directed to a polypeptide
comprising a sequence derived from the ICR62 antibody and a
sequence derived from a heterologous polypeptide and to an
antigen-binding molecule comprising such a polypeptide. In one
embodiment the antigen-binding molecule is an antibody. In a
preferred embodiment, the antibody is chimeric. In another
preferred embodiment, the antibody is humanized or primatized.
[0030] In another aspect, the invention is directed to an ABM,
which is capable of competing with the rat ICR62 antibody for
binding to EGFR and which is chimeric. In one embodiment, the ABM
is an antibody or a fragment thereof. In a further embodiment, the
ABM is a recombinant antibody comprising a VH region having an
amino acid sequence selected from the group consisting of SEQ ID
NO.: 1; SEQ ID No:3; SEQ ID No:5; SEQ ID No:7; SEQ ID No:9; SEQ ID
No:11; SEQ ID No:13; SEQ ID No:15; SEQ ID No:17; SEQ ID No:19; SEQ
ID No:21; SEQ ID No:23; SEQ ID No:25; SEQ ID No:27; SEQ ID No:29;
SEQ ID No:31; SEQ ID No33; SEQ ID No:35; SEQ ID No:37; SEQ ID
No:39; and SEQ ID No:121. In another embodiment, the ABM is a
recombinant antibody comprising a VL region having an amino acid
sequence selected from the group consisting of SEQ ID NO:43, SEQ ID
No:45; SEQ ID No:49; and SEQ ID No.:51. In a further embodiment the
ABM is a recombinant antibody that is primatized. In yet a further
embodiment the ABM is a recombinant antibody that is humanized. In
another embodiment, the ABM is a recombinant antibody comprising a
human Fc region. In a further embodiment, any of the ABMs discussed
above may be conjugated to a moiety such as a toxin or a
radiolabel.
[0031] The invention is further related to an ABM of the present
invention, said ABM having modified oligosaccharides. In one
embodiment the modified oligosaccharides have reduced fucosylation
as compared to non-modified oligosaccharides. In other embodiments,
the modified oligosaccharides are hybrid or complex. In a further
embodiment, the ABM has an increased proportion of nonfucosylated
oligosaccharides or bisected, nonfucosylated oligosaccharides in
the Fc region of said molecule. In one embodiment, the bisected,
nonfucosylated oligosaccharides are hybrid. In a further
embodiment, the bisected, nonfucosylated oligosaccharides are
complex. In a one embodiment, at least 20% of the oligosaccharides
in the Fc region of said polypeptide are nonfucosylated or
bisected, nonfucosylated. In more preferred embodiments, at least
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% or more of the
oligosaccharides are nonfucosylated or bisected,
nonfucosylated.
[0032] The invention is further related to a polynucleotide
encoding any of the ABMs discussed above, and to expression vectors
and cells comprising such a polynucleotide.
[0033] The invention is further related to a method of producing an
ABM, which is capable of competing with the rat ICR62 antibody for
binding to EGFR and wherein said ABM is chimeric; said method
comprising: (a) culturing a host cell comprising a polynucleotide
that encodes an ABM of the present invention in a medium under
conditions allowing the expression of said polynucleotide encoding
said ABM; and (b) recovering said ABM from the resultant
culture.
[0034] In another aspect, the invention is related to a
pharmaceutical composition comprising the ABM of the invention. It
is contemplated that the pharmaceutical composition may further
comprise a pharmaceutically acceptable carrier, an adjuvant or a
combination thereof.
[0035] In a further aspect, the invention is related to a method of
treating a disease characterized by expression of EGFR (e.g.,
abnormal or overexpression of EGFR). The method comprises
administering a therapeutically effective amount of the ABM of the
present invention to a subject, preferably a mammalian subject, and
more preferably a human in need thereof. In a preferred embodiment,
the disease is treated by administering an ABM that is a chimeric
(e.g. humanized) antibody, or a chimeric fragment of an antibody.
In one embodiment, the ABM is administered in an amount of about
1.0 mg/kg to about 15.0 mg/kg. In another embodiment, the ABM is
administered in an amount of about 1.5 mg/kg to about 12.0 mg/kg.
In a further embodiment, the ABM is administered in an amount of
about 1.5 mg/kg to about 4.5 mg/kg. In a further embodiment, the
ABM is adminstered in an amount of about 4.5 mg/kg to about 12.0
mg/kg. In a further embodiment, the ABM is administered in an
amount selected from the group consisting of about 1.5, about 4.5,
and about 12.0 mg/kg.
[0036] In yet another aspect, the invention is related to a host
cell engineered to express at least one nucleic acid encoding a
polypeptide having GnTIII activity in an amount sufficient to
modify the oligosaccharides in the Fc region of the ABM produced by
the host cell, wherein the ABM is capable of competing with the rat
ICR62 antibody for binding to EGFR and wherein the ABM is chimeric.
In one embodiment, the polypeptide having GnTIII activity is a
fusion polypeptide. In another embodiment, the ABM produced by the
host cell is an antibody or an antibody fragment. In one
embodiment, the antibody or antibody fragment is humanized. In a
further embodiment, the ABM comprises a region equivalent to the Fc
region of a human IgG.
[0037] The invention is also directed to an isolated polynucleotide
comprising at least one (e.g., one, two, three, four, five, or six)
complementarity determining region of the rat ICR62 antibody, or a
variant or truncated form thereof containing at least the
specificity-determining residues for said complementarity
determining region, wherein said isolated polynucleotide encodes a
fusion polypeptide. Preferably, such isolated polynucleotides
encode a fusion polypeptide that is an antigen binding molecule. In
one embodiment, the polynucleotide comprises three complementarity
determining regions of the rat ICR62 antibody, or variants or
truncated forms thereof containing at least the
specificity-determining residues for each of said three
complementarity determining regions. In another embodiment, the
polynucleotide encodes the entire variable region of the light or
heavy chain of a chimeric (e.g., humanized) antibody. The invention
is further directed to the polypeptides encoded by such
polynucleotides.
[0038] In another embodiment, the invention is directed to an
antigen binding molecule comprising at least one (e.g., one, two,
three, four, five, or six) complementarity determining region of
the rat ICR62 antibody, or a variant or truncated form thereof
containing at least the specificity-determining residues for said
complementarity determining region, and comprising a sequence
derived from a heterologous polypeptide. In one embodiment, the
antigen binding molecule comprises three complementarity
determining regions of the rat ICR62 antibody, or variants or
truncated forms thereof containing at least the
specificity-determining residues for each of said three
complementarity determining regions. In another aspect, the antigen
binding molecule comprises the variable region of an antibody light
or heavy chain. In one particularly useful embodiment, the antigen
binding molecule is a chimeric, e.g., humanized, antibody. The
invention is also directed to methods of making such antigen
binding molecules, and the use of same in the treatment of disease,
including malignancies such as cancers of the bladder, brain, head
and neck, pancreas, lung, breast, ovary, colon, prostate, skin, and
kidney.
[0039] The host cell of the present invention may be selected from
the group that includes, but is not limited to, an HEK293-EBNA
cell, a CHO cell, a BHK cell, a NSO cell, a SP2/0 cell, a YO
myeloma cell, a P3X63 mouse myeloma cell, a PER cell, a PER.C6 cell
or a hybridoma cell. In one embodiment, the host cell of the
invention further comprises a transfected polynucleotide comprising
a polynucleotide encoding the VL region of the rat ICR62 antibody
or variants thereof and a sequence encoding a region equivalent to
the Fc region of a human immunoglobulin. In another embodiment, the
host cell of the invention further comprises a transfected
polynucleotide comprising a polynucleotide encoding the VH region
of the rat ICR62 antibody or variants thereof and a sequence
encoding a region equivalent to the Fc region of a human
immunoglobulin.
[0040] In a further aspect, the invention is directed to a host
cell that produces an ABM that exhibits increased Fc receptor
binding affinity and/or increased effector function as a result of
the modification of its oligosaccharides. In one embodiment, the
increased binding affinity is to an Fc receptor, particularly, the
Fc.gamma.RIIIA receptor. The effector function contemplated herein
may be selected from the group that includes, but is not limited
to, increased Fc-mediated cellular cytotoxicity; increased binding
to NK cells; increased binding to macrophages; increased binding to
polymorphonuclear cells; increased binding to monocytes; increased
direct signaling inducing apoptosis; increased dendritic cell
maturation; and increased T cell priming.
[0041] In a further embodiment, the host cell of the present
invention comprises at least one nucleic acid encoding a
polypeptide having GnTIII activity that is operably linked to a
constitutive promoter element.
[0042] In another aspect, the invention is directed to a method for
producing an ABM in a host cell, comprising: (a) culturing a host
cell engineered to express at least one polynucleotide encoding a
fusion polypeptide having GnTIII activity under conditions which
permit the production of said ABM and which permit the modification
of the oligosaccharides present on the Fc region of said ABM; and
(b) isolating said ABM; wherein said ABM is capable of competing
with the rat ICR62 antibody for binding to EGFR and wherein said
ABM is chimeric (e.g., humanized). In one embodiment, the
polypeptide having GnTIII activity is a fusion polypeptide,
preferably comprising the catalytic domain of GnTIII and the Golgi
localization domain of a heterologous Golgi resident polypeptide
selected from the group consisting of the localization domain of
mannosidase II, the localization domain of
.beta.(1,2)-N-acetylglucosaminyltransferase I ("GnTI"), the
localization domain of mannosidase I, the localization domain of
.beta.(1,2)-N-acetylglucosaminyltransferase II ("GnTII"), and the
localization domain of .alpha.1-6 core fucosyltransferase.
Preferably, the Golgi localization domain is from mannosidase II or
GnTI.
[0043] In a further aspect, the invention is directed to a method
for modifying the glycosylation profile of an anti-EGFR ABM
produced by a host cell comprising introducing into the host cell
at least one nucleic acid or expression vector of the invention. In
one embodiment, the ABM is an antibody or a fragment thereof;
preferably comprising the Fc region of an IgG. Alternatively, the
polypeptide is a fusion protein that includes a region equivalent
to the Fc region of a human IgG.
[0044] In one aspect, the invention is related to a recombinant,
chimeric antibody, or a fragment thereof, capable of competing with
the rat ICR62 antibody for binding to EGFR and having reduced
fucosylation.
[0045] In another aspect, the present invention is directed to a
method of modifying the glycosylation of the recombinant antibody
or a fragment thereof of the invention by using a fusion
polypeptide having GnTIII activity and comprising the Golgi
localization domain of a heterologous Golgi resident polypeptide.
In one embodiment, the fusion polypeptides of the invention
comprise the catalytic domain of GnTIII. In another embodiment, the
Golgi localization domain is selected from the group consisting of:
the localization domain of mannosidase II, the localization domain
of GnTI, the localization domain of mannosidase I, the localization
domain of GnTII and the localization domain of .alpha.1-6 core
fucosyltransferase. Preferably, the Golgi localization domain is
from mannosidase II or GnTI.
[0046] In one embodiment, the method of the invention is directed
towards producing a recombinant, chimeric antibody or a fragment
thereof, with modified oligosaccharides wherein said modified
oligosaccharides have reduced fucosylation as compared to
non-modified oligosaccharides. According to the present invention,
these modified oligosaccharides may be hybrid or complex. In
another embodiment, the method of the invention is directed towards
producing a recombinant, chimeric (e.g., humanized) antibody or a
fragment thereof having an increased proportion of bisected,
nonfucosylated oligosaccharides in the Fc region of said
polypeptide. In one embodiment, the bisected, nonfucosylated
oligosaccharides are hybrid. In another embodiment, the bisected,
nonfucosylated oligosaccharides are complex. In a further
embodiment, the method of the invention is directed towards
producing a recombinant, chimeric antibody or a fragment thereof
having at least 20% of the oligosaccharides in the Fc region of
said polypeptide that are bisected, nonfucosylated. In a preferred
embodiment, at least 30% of the oligosaccharides in the Fc region
of said polypeptide are bisected, nonfucosylated. In another
preferred embodiment, wherein at least 35% of the oligosaccharides
in the Fc region of said polypeptide are bisected,
nonfucosylated.
[0047] In a further aspect, the invention is directed to a
recombinant, chimeric antibody or a fragment thereof, that exhibits
increased Fc receptor binding affinity and/or increased effector
function as a result of the modification of its oligosaccharides.
In one embodiment, the increased binding affinity is to an Fc
activating receptor. In a further embodiment, the Fc receptor is
Fc.gamma. activating receptor, particularly, the Fc.gamma.RIIIA
receptor. The effector function contemplated herein may be selected
from the group that includes, but is not limited to, increased
Fc-mediated cellular cytotoxicity; increased binding to NK cells;
increased binding to macrophages; increased binding to
polymorphonuclear cells; increased binding to monocytes; increased
direct signaling inducing apoptosis; increased dendritic cell
maturation; and increased T cell priming.
[0048] In another aspect, the invention is directed to a
recombinant, chimeric (e.g., humanized) antibody fragment, having
the binding specificity of the rat ICR62 antibody and containing
the Fc region, that is engineered to have increased effector
function produced by any of the methods of the present
invention.
[0049] In another aspect, the present invention is directed to a
fusion protein that includes a polypeptide having a sequence
selected from the group consisting of of SEQ ID NO.: 1; SEQ ID
No:3; SEQ ID No:5; SEQ ID No:7; SEQ ID No:9; SEQ ID No:11; SEQ ID
No:13; SEQ ID No:15; SEQ ID No:17; SEQ ID No:19; SEQ ID No:21; SEQ
ID No:23; SEQ ID No:25; SEQ ID No:27; SEQ ID No:29; SEQ ID No:31;
SEQ ID No33; SEQ ID No:35; SEQ ID No:37; SEQ ID No:39; and SEQ ID
No:121, and a region equivalent to the Fc region of an
immunoglobulin and engineered to have increased effector function
produced by any of the methods of the present invention.
[0050] In another aspect, the present invention is directed to a
fusion protein that includes a polypeptide having a sequence
selected from the group consisting of SEQ ID NO:43, SEQ ID No:45;
SEQ ID No:49; and SEQ ID No.:51 and a region equivalent to the Fc
region of an immunoglobulin and engineered to have increased
effector function produced by any of the methods of the present
invention.
[0051] In one aspect, the present invention is directed to a
pharmaceutical composition comprising a recombinant, chimeric
(e.g., humanized) antibody, produced by any of the methods of the
present invention, and a pharmaceutically acceptable carrier. In
another aspect, the present invention is directed to a
pharmaceutical composition comprising a recombinant, chimeric
(e.g., humanized) antibody fragment produced by any of the methods
of the present invention, and a pharmaceutically acceptable
carrier. In another aspect, the present invention is directed to a
pharmaceutical composition comprising a fusion protein produced by
any of the methods of the present invention, and a pharmaceutically
acceptable carrier.
[0052] In a further aspect, the invention is directed to a method
for targetting in vivo or in vitro cells expressing EGFR. In one
embodiment, the present invention is directed to a method for
targetting cells expressing EGFR in a subject comprising
administering to the subject a composition comprising an ABM of the
invention.
[0053] In yet another aspect, the present invention is directed to
a method for detecting in vivo or in vitro the presence of EGFR in
a sample, e.g., for diagnosing a disorder related to EGFR
expression. In one embodiment, the detection is performed by
contacting a sample to be tested, optionally with a control sample,
with an ABM of the present invention, under conditions that allow
for formation of a complex between the ABM and EGFR. The complex
formation is then detected (e.g., by ELISA or other methods known
in the art). When using a control sample with the test sample,any
statistically significant difference in the formation of ABM-EGFR
complexes when comparing the test and control samples is indicative
of the presence of EGFR in the test sample.
[0054] The invention is further directed to a method of treating a
disorder related to EGFR expression, in particular, a cell
proliferation disorder wherein EGFR is expressed, and more
particularly, wherein EGFR is abnormally expressed (e.g.
overexpressed), including cancers of the bladder, brain, head and
neck, pancreas, lung, breast, ovary, colon, prostate, skin, and
kidney comprising administering a therapeutically effective amount
of the recombinant, chimeric (e.g., humanized) antibody or fragment
thereof, produced by any of the methods of the present invention,
to a human subject in need thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0055] FIG. 1 shows the functional activity of individual heavy and
light chimeric rat-human ICR62 polypeptide chains when combined
with the humanized ICR62 constructs I-HHC (heavy chain) and I-KB
(light chain). rVL represents the chimeric light chain, and rVH
represents the chimeric heavy chain. The "r" designation indicates
that the variable domains are from the original rat antibody.
[0056] FIG. 2 shows binding activity of humanized ICR62 antibodies
comprising heavy chain variable region constructs I-HHC, I-HHA and
I-HLA and humanized light chain variable region constructs I-KA and
I-KB paired in various configurations.
[0057] FIG. 3 shows binding activity of humanized ICR62 antibodies
comprising heavy chain variable region constructs I-HLB, I-HLC and
I-HLA and humanized light chain variable region constructs I-KA and
I-KC paired in various configurations.
[0058] FIG. 4 shows binding activity of humanized ICR62 antibodies
comprising heavy chain variable region constructs I-HLA2, I-HLA3
and I-HLA4 and humanized light chain variable region construct I-KC
as compared to chimeric rat-human ICR62 antibody.
[0059] FIG. 5 shows binding activity of humanized ICR62 antibodies
comprising heavy chain variable region constructs I-HLA1, I-HLA3,
I-HLA5 and I-HLA6 and humanized light chain variable region
construct I-KC as compared to chimeric rat-human ICR62
antibody.
[0060] FIG. 6 shows binding activity of humanized ICR62 antibodies
comprising heavy chain variable region constructs I-HLA7, I-HLA6,
and I-HHB and humanized light chain variable region construct I-KC
as compared to chimeric rat-human ICR62 antibody.
[0061] FIG. 7 shows binding activity of humanized ICR62 antibodies
comprising heavy chain variable region constructs I-HHF, I-HLA9,
and I-HLA8 and humanized light chain variable region construct I-KC
as compared to chimeric rat-human ICR62 antibody.
[0062] FIG. 8 shows binding activity of humanized antibodies
comprising heavy chain varible region constructs I-HHB, I-HHD,
I-HHG, I-HHF, I-HLA7, and I-HLA9 and humanized light chain variable
region construct I-KC.
[0063] FIG. 9 shows a comparison of antibody mediated cellular
cytotoxicity (ADCC) for various glycoforms of the chimeric ICR62
antibody, as well as for the humanized variant I-HLA4. "G1" refers
to glcyoengineering of the antibody by co-expression with GnTIII.
"G2" refers to glycoengineering of the antibody by co-expression
with GnTIII and ManII. "WT" refers to antibodies that were not
glycoengineered. The humanzied heavy chain constructs were paired
with the I-KC light chain construct.
[0064] FIGS. 10A and 10B show a comparison of ADCC for the
non-glycoengineered form (WT) and the G2 glycoform (i.e.,
glycoengineered by co-expression with GnTIII and ManII) of the
humanized ICR62 antibody constructs I-HHB and I-HLA7. The same
antibodies were applied to two different target cell lines: in
Panel A, the target cell line LN229 was used; in Panel B, the cell
line A431 was used. The humanzied heavy chain constructs were
paired with the I-KC light chain construct.
[0065] FIGS. 11A and 11B show a comparison of ADCC for
non-glycoengineered forms (WT) and G2 glcyoforms of chimeric ICR62
and the humanized ICR62 antibody constructs I-HHB and I-HLA7. The
target cell line A431 was used. The humanzied heavy chain
constructs were paired with the I-KC light chain construct.
[0066] FIG. 12 shows a comparison of 72 h ADCC for G2 glcyoforms of
chimeric ICR62 and the humanized ICR62 antibody constructs I-HHB
and I-HLA7. The humanzied heavy chain constructs were paired with
the I-KC light chain construct.
[0067] FIGS. 13A and 13B show an amino acid sequence alignment of
humanized ICR62 heavy chain variable region constructs compared to
the rat ICR62 sequences. Dots represent identity of amino acid
residues at a given position within a given construct.
[0068] FIG. 14 shows an FcgammaRIIIa-Fc binding assay using CHO
cells displaying recombinant human FcgammaRIIIa. A glycoengineered
I-HHB/KC humanized anti-EGFR IgG1 antibody was compared to a
non-glycoengineered (Wt) antibody.
[0069] FIG. 15 shows a MALD/TOF-MS oligosaccharide profile for
glycoengineered humanized anti-EGFR IgG1 antibody, I-HHB/KC.
Glycoengineering achieved by overexpression in the
antibody-producing cells of genes encoding enzymes with GnTIII and
Golgi Mannosidase II activities, yielding over 70% of
non-fucosylated Fc-Asn297-linked oligosaccharides.
[0070] FIG. 16 shows an anti-EGFR precision profile (n=6 replicates
across the calibration range) for the determination of anti-EGFR in
1% monkey serum matrix (monkey serum pool CMS25/31/33, supplied by
HLS).
[0071] FIG. 17 shows a representative anti-EGFR calibration curve
for the determination of anti-EGFR in 1% monkey serum matrix.
[0072] FIG. 18 shows serum concentrations of anti-EGFR on Day 1 of
weekly intravenous administration of anti-EGFR to male cynomolgous
monkeys.
[0073] FIG. 19 shows serum concentrations of anti-EGFR on Day 1 of
weekly intravenous administration of anti-EGFR to female
cynomolgous monkeys.
[0074] FIG. 20 shows the relationship between areas under the serum
anti-EGFR concentration-time curves (AUC.sub.168) and dose level on
Day 1 of weekly intravenous administration of anti-EGFR to
cynomolgous monkeys.
[0075] FIG. 21 shows serum concentrations of anti-EGFR during
weekly intravenous administration of anti-EGFR to male cynomolgous
monkeys.
[0076] FIG. 22 shows serum concentrations of anti-EGFR during
weekly intravenous administration of anti-EGFR to female
cynomolgous monkeys.
[0077] FIG. 23 shows the MALDI/TOF-MS profile of oligosaccharides
from Fc-engineered (glycoengineered) anti-EGFR antibody used for
the in vivo monkey studies described in the Examples herein
below.
[0078] FIG. 24 shows binding to EGFR expressed on the surface of
human A431 epidermoid carcinoma cells. The antibody used for the
binding study was the Fc-engineered anti-EGFR antibody (I-HHB
construct) used for the in vivo monkey studies described in the
Examples herein below.
[0079] FIG. 25 shows binding to EGFR expressed on surface of monkey
COS-7 kidney cells. The antibody used was anti-EGFR antibody (I-HHB
heavy chain; I-KC light chain). For reference, binding to low human
EGFR-expressing cells, MCF-7 breast cancer cells, is shown.
[0080] FIG. 26 shows Fc-FcgammaRIIIa binding using a whole cell
(CHO cells engineered to express human FcgRIIIa on their surface).
The antibody used was the Fc-engineered (glycoengineered) anti-EGFR
antibody used for the in vivo monkey studies described in the
Examples herein below. Binding for a non-Fc-engineered (unmodified)
control IgG1 antibody is shown for comparison.
[0081] FIG. 27 shows ADCC mediated by Fc-engineered
(glycoengineered) anti-EGFR antibody. Target cells are A549 human
lung carcinoma cells. ADCC activity for the non-Fc engineered
(unmodified) form of the antibody is shown for comparison.
[0082] FIG. 28 shows ADCC mediated by Fc-engineered
(glycoengineered) anti-EGFR antibody. Target cells are CYNOM-K1
cynomolgus monkey keratinocyte cell line. ADCC activity for the
non-Fc engineered (unmodified) form of the antibody is shown for
comparison.
[0083] FIG. 29 shows EGFR target binding of various light chain
construct variants based on the I-KC construct paired with the
heavy chain I-HHD construct.
DETAILED DESCRIPTION OF THE INVENTION
[0084] Terms are used herein as generally used in the art, unless
otherwise defined as follows.
[0085] As used herein, the term antibody is intended to include
whole antibody molecules, including monoclonal, polyclonal and
multispecific (e.g., bispecific) antibodies, as well as antibody
fragments having the Fc region and retaining binding specificity,
and fusion proteins that include a region equivalent to the Fc
region of an immunoglobulin and that retain binding specificity.
Also encompassed are antibody fragments that retain binding
specificity including, but not limited to, VH fragments, VL
fragments, Fab fragments, F(ab').sub.2 fragments, scFv fragments,
Fv fragments, minibodies, diabodies, triabodies, and tetrabodies
(see, e.g., Hudson and Souriau, Nature Med. 9: 129-134 (2003)).
Also encompassed are humanized, primatized and chimeric
antibodies.
[0086] As used herein, the term Fc region is intended to refer to a
C-terminal region of an IgG heavy chain. Although the boundaries of
the Fc region of an IgG heavy chain might vary slightly, the human
IgG heavy chain Fc region is usually defined to stretch from the
amino acid residue at position Cys226 to the carboxyl-terminus.
[0087] As used herein, the term region equivalent to the Fc region
of an immunoglobulin is intended to include naturally occurring
allelic variants of the Fc region of an immunoglobulin as well as
variants having alterations which produce substitutions, additions,
or deletions but which do not decrease substantially the ability of
the immunoglobulin to mediate effector functions (such as antibody
dependent cellular cytotoxicity). For example, one or more amino
acids can be deleted from the N-terminus or C-terminus of the Fc
region of an immunoglobulin without substantial loss of biological
function. Such variants can be selected according to general rules
known in the art so as to have minimal effect on activity. (See,
e.g., Bowie, J. U. et al., Science 247:1306-1310 (1990).
[0088] As used herein, the term EGFR refers to the human epidermal
growth factor receptor (also known as HER-1 or Erb-B1) (Ulrich, A.
et al., Nature 309:418-425 (1984); SwissProt Accession #P00533;
secondary accession numbers: 000688, 000732, P06268, Q14225,
Q92795, Q9BZS2, Q9GZX1, Q9H2C9, Q9H3C9, Q9UMD7, Q9UMD8, Q9UMG5), as
well as naturally-occurring isoforms and variants thereof. Such
isoforms and variants include but are not limited to the EGFRvIII
variant, alternative splicing products (e.g., as identified by
SwissProt Accession numbers P00533-1, P00533-2, P00533-3,
P00533-4), variants GLN-98, ARG-266, Lys-521, ILE-674, GLY-962, and
PRO-988 (Livingston, R. J. et al., NIEHS-SNPs, environmental genome
project, NIEHS ES15478, Department of Genome Sciences, Seattle,
Wash. (2004)), and others identified by the following accession
numbers: NM_005228.3, NM_201282.1, NM_201283.1, NM_201284.1 (REFSEQ
mRNAs); AF125253.1, AF277897.1, AF288738.1, AI217671.1, AK127817.1,
AL598260.1, AU137334.1, AW163038.1, AW295229.1, BC057802.1,
CB160831.1, K03193.1, U48722.1, U95089.1, X00588.1, X00663.1;
H5448451, H5448453, H5448452 (MIPS assembly); DT.453606,
DT.86855651, DT.95165593, DT.97822681, DT.95165600, DT.100752430,
DT.91654361, DT.92034460, DT.92446349, DT.97784849, DT.101978019,
DT.418647, DT.86842167, DT.91803457, DT.92446350, DT.95153003,
DT.95254161, DT.97816654, DT.87014330, DT.87079224 (DOTS
Assembly).
[0089] As used herein, the term EGFR ligand refers to a polypeptide
which binds to and/or activates EGFR. The term includes
membrane-bound precursor forms of the EGFR ligand, as well as
proteolytically processed soluble forms of the EGFR ligand.
[0090] As used herein, the term ligand activation of EGFR refers to
signal transduction (e.g., that caused by an intracellular kinase
domain of EGFR receptor phosphorylating tyrosine residues in the
EGFR or a substrate polypeptide) mediated by EGFR ligand
binding.
[0091] As used herein, the term disease or disorder characterized
by abnormal activation or production of EGFR or an EGFR ligand or
disorder related to EGFR expression, refers to a condition, which
may or may not involve malignancy or cancer, where abnormal
activation and/or production of EGFR and/or an EGFR ligand is
occurring in cells or tissues of a subject having, or predisposed
to, the disease or disorder.
[0092] As used herein, the terms overexpress, overexpressed, and
overexpressing, as used in connection with cells expressing EGFR,
refer to cells which have measurably higher levels of EGFR on the
surface thereof compared to a normal cell of the same tissue type.
Such overexpression may be caused by gene amplification or by
increased transcription or translation. EGFR expression (and,
hence, overexpression) may be determined in a diagnostic or
prognostic assay by evaluating levels of EGFR present on the suface
of a cell or in a cell lysate by techniques that are known in the
art: e.g., via an immunohistochemistry assay, immunofluorescence
assay, immunoenzyme assay, ELISA, flow cytometry, radioimmunoassay,
Western blot, ligand binding, kinase activity, etc. (See generally,
CELL BIOLOGY: A LABORATORY HANDBOOK, Celis, J., ed., Academic Press
(2d ed., 1998); CURRENT PROTOCOLS IN PROTEIN SCIENCE, Coligan, J.
E. et al., eds., John Wiley & Sons (1995-2003); see also,
Sumitomo et al., Clin. Cancer Res. 10: 794-801 (2004) (describing
Western blot, flow cytometry, and immunohistochemstry) the entire
contents of which are herein incorporated by reference)).
Alternatively, or additionally, one may measure levels of
EGFR-encoding nucleic acid molecules in the cell, e.g., via
fluorescent in situ hybridization, Southern blotting, or PCR
techniques. The levels of EGFR in normal cells are compared to the
levels of cells affected by a cell proliferation disorder (e.g.,
cancer) to determine if EGFR is overexpressed.
[0093] As used herein, the term antigen binding molecule refers in
its broadest sense to a molecule that specifically binds an
antigenic determinant. More specifically, an antigen binding
molecule that binds EGFR is a molecule which specifically binds to
a transmembrane receptor of 170 kDa, typically designated as the
epidermal growth factor receptor (EGFR), but also known as HER-1 or
ErbB1. By "specifically binds" is meant that the binding is
selective for the antigen and can be discriminated from unwanted or
nonspecific interactions.
[0094] As used herein, the terms fusion and chimeric, when used in
reference to polypeptides such as ABMs refer to polypeptides
comprising amino acid sequences derived from two or more
heterologous polypeptides, such as portions of antibodies from
different species. For chimeric ABMs, for example, the non-antigen
binding components may be derived from a wide variety of species,
including primates such as chimpanzees and humans. The constant
region of the chimeric ABM is most preferably substantially
identical to the constant region of a natural human antibody; the
variable region of the chimeric antibody is most preferably
substantially identical to that of a recombinant anti-EGFR antibody
having the amino acid sequence of the murine variable region.
Humanized antibodies are a particularly preferred form of fusion or
chimeric antibody.
[0095] As used herein, a polypeptide having GnTIII activity refers
to polypeptides that are able to catalyze the addition of a
N-acetylglucosamine (GlcNAc) residue in .beta.-1-4 linkage to the
.beta.-linked mannoside of the trimannosyl core of N-linked
oligosaccharides. This includes fusion polypeptides exhibiting
enzymatic activity similar to, but not necessarily identical to, an
activity of .beta.(1,4)-N-acetylglucosaminyltransferase III, also
known as .beta.-1,4-mannosyl-glycoprotein
4-beta-N-acetylglucosaminyl-transferase (EC 2.4.1.144), according
to the Nomenclature Committee of the International Union of
Biochemistry and Molecular Biology (NC-IUBMB), as measured in a
particular biological assay, with or without dose dependency. In
the case where dose dependency does exist, it need not be identical
to that of GnTIII, but rather substantially similar to the
dose-dependence in a given activity as compared to the GnTIII
(i.e., the candidate polypeptide will exhibit greater activity or
not more than about 25-fold less and, preferably, not more than
about tenfold less activity, and most preferably, not more than
about three-fold less activity relative to the GnTIII.)
[0096] As used herein, the term variant (or analog) refers to a
polypeptide differing from a specifically recited polypeptide of
the invention by amino acid insertions, deletions, and
substitutions, created using, e g., recombinant DNA techniques.
Variants of the ABMs of the present invention include chimeric,
primatized or humanized antigen binding molecules wherein one or
several of the amino acid residues are modified by substitution,
addition and/or deletion in such manner that does not substantially
affect antigen (e.g., EGFR) binding affinity. Guidance in
determining which amino acid residues may be replaced, added or
deleted without abolishing activities of interest, may be found by
comparing the sequence of the particular polypeptide with that of
homologous peptides and minimizing the number of amino acid
sequence changes made in regions of high homology (conserved
regions) or by replacing amino acids with consensus sequence.
[0097] Alternatively, recombinant variants encoding these same or
similar polypeptides may be synthesized or selected by making use
of the "redundancy" in the genetic code. Various codon
substitutions, such as the silent changes which produce various
restriction sites, may be introduced to optimize cloning into a
plasmid or viral vector or expression in a particular prokaryotic
or eukaryotic system. Mutations in the polynucleotide sequence may
be reflected in the polypeptide or domains of other peptides added
to the polypeptide to modify the properties of any part of the
polypeptide, to change characteristics such as ligand-binding
affinities, interchain affinities, or degradation/turnover
rate.
[0098] Preferably, amino acid "substitutions" are the result of
replacing one amino acid with another amino acid having similar
structural and/or chemical properties, i.e., conservative amino
acid replacements. "Conservative" amino acid substitutions may be
made on the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues involved. For example, nonpolar (hydrophobic) amino
acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and methionine; polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine; positively charged (basic) amino acids
include arginine, lysine, and histidine; and negatively charged
(acidic) amino acids include aspartic acid and glutamic acid.
"Insertions" or "deletions" are preferably in the range of about 1
to 20 amino acids, more preferably 1 to 10 amino acids. The
variation allowed may be experimentally determined by
systematically making insertions, deletions, or substitutions of
amino acids in a polypeptide molecule using recombinant DNA
techniques and assaying the resulting recombinant variants for
activity.
[0099] As used herein, the term humanized is used to refer to an
antigen-binding molecule derived from a non-human antigen-binding
molecule, for example, a murine antibody, that retains or
substantially retains the antigen-binding properties of the parent
molecule but which is less immunogenic in humans. This may be
achieved by various methods including (a) grafting the entire
non-human variable domains onto human constant regions to generate
chimeric antibodies, (b) grafting only the non-human CDRs onto
human framework and constant regions with or without retention of
critical framework residues (e.g., those that are important for
retaining good antigen binding affinity or antibody functions), or
(c) transplanting the entire non-human variable domains, but
"cloaking" them with a human-like section by replacement of surface
residues. Such methods are disclosed in Jones et al., Morrison et
al., Proc. Natl. Acad. Sci., 81:6851-6855 (1984); Morrison and Oi,
Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science,
239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991);
Padlan, Molec. Immun., 31(3):169-217 (1994), all of which are
incorporated by reference in their entirety herein. There are
generally 3 complementarity determining regions, or CDRs, (CDR1,
CDR2 and CDR3) in each of the heavy and light chain variable
domains of an antibody, which are flanked by four framework
subregions (i.e., FR1, FR2, FR3, and FR4) in each of the heavy and
light chain variable domains of an antibody:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A discussion of humanized
antibodies can be found, inter alia, in U.S. Pat. No. 6,632,927,
and in published U.S. Application No. 2003/0175269, both of which
are incorporated herein by reference in their entirety.
[0100] Similarly, as used herein, the term primatized is used to
refer to an antigen-binding molecule derived from a non-primate
antigen-binding molecule, for example, a murine antibody, that
retains or substantially retains the antigen-binding properties of
the parent molecule but which is less immunogenic in primates.
[0101] In the case where there are two or more definitions of a
term which is used and/or accepted within the art, the definition
of the term as used herein is intended to include all such meanings
unless explicitly stated to the contrary. A specific example is the
use of the term "complementarity determining region" ("CDR") to
describe the non-contiguous antigen combining sites found within
the variable region of both heavy and light chain polypeptides.
This particular region has been described by Kabat et al., U.S.
Dept. of Health and Human Services, "Sequences of Proteins of
Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol.
196:901-917 (1987), which are incorporated herein by reference,
where the definitions include overlapping or subsets of amino acid
residues when compared against each other. Nevertheless,
application of either definition to refer to a CDR of an antibody
or variants thereof is intended to be within the scope of the term
as defined and used herein. The appropriate amino acid residues
which encompass the CDRs as defined by each of the above cited
references are set forth below in Table I as a comparison. The
exact residue numbers which encompass a particular CDR will vary
depending on the sequence and size of the CDR. Those skilled in the
art can routinely determine which residues comprise a particular
CDR given the variable region amino acid sequence of the
antibody.
TABLE-US-00001 TABLE 1 CDR Definitions.sup.1 Kabat Chothia
AbM.sup.2 V.sub.H CDR1 31-35 26-32 26-35 V.sub.H CDR2 50-65 52-58
50-58 V.sub.H CDR3 95-102 95-102 95-102 V.sub.L CDR1 24-34 26-32
24-34 V.sub.L CDR2 50-56 50-52 50-56 V.sub.L CDR3 89-97 91-96 89-97
.sup.1Numbering of all CDR definitions in Table 1 is according to
the numbering conventions set forth by Kabat et al. (see below).
.sup.2"AbM" refers to the CDRs as defined by Oxford Molecular's
"AbM" antibody modeling software.
[0102] Kabat et al. also defined a numbering system for variable
domain sequences that is applicable to any antibody. One of
ordinary skill in the art can unambigously assign this system of
"Kabat numbering" to any variable domain sequence, without reliance
on any experimental data beyond the sequence itself. As used
herein, "Kabat numbering" refers to the numbering system set forth
by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence
of Proteins of Immunological Interest" (1983). Unless otherwise
specified, references to the numbering of specific amino acid
residue positions in an ABM are according to the Kabat numbering
system. The sequences of the sequence listing (i.e., SEQ ID NO:1 to
SEQ ID NO:127) are not numbered according to the Kabat numbering
system. However, as stated above, it is well within the ordinary
skill of one in the art to determine the Kabat numbering scheme of
any variable region sequence in the Sequence Listing based on the
numbering of the sequences as presented therein.
[0103] By a nucleic acid or polynucleotide having a nucleotide
sequence at least, for example, 95% "identical" to a reference
nucleotide sequence of the present invention, it is intended that
the nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five point mutations per each 100 nucleotides of the
reference nucleotide sequence. In other words, to obtain a
polynucleotide having a nucleotide sequence at least 95% identical
to a reference nucleotide sequence, up to 5% of the nucleotides in
the reference sequence may be deleted or substituted with another
nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the reference sequence may be inserted into the
reference sequence.
[0104] As a practical matter, whether any particular nucleic acid
molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%,
98% or 99% identical to a nucleotide sequence or polypeptide
sequence of the present invention can be determined conventionally
using known computer programs. A preferred method for determining
the best overall match between a query sequence (a sequence of the
present invention) and a subject sequence, also referred to as a
global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag et al., Comp.
App. Biosci. 6:237-245 (1990). In a sequence alignment the query
and subject sequences are both DNA sequences. An RNA sequence can
be compared by converting U's to T's. The result of said global
sequence alignment is in percent identity. Preferred parameters
used in a FASTDB alignment of DNA sequences to calculate percent
identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1,
Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1,
Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length
of the subject nucleotide sequence, whichever is shorter.
[0105] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only bases outside the 5' and 3' bases of the
subject sequence, as displayed by the FASTDB alignment, which are
not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
[0106] For example, a 90 base subject sequence is aligned to a 100
base query sequence to determine percent identity. The deletions
occur at the 5' end of the subject sequence and therefore, the
FASTDB alignment does not show a matched/alignment of the first 10
bases at 5' end. The 10 unpaired bases represent 10% of the
sequence (number of bases at the 5' and 3' ends not matched/total
number of bases in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject
sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the
5' or 3' of the subject sequence which are not matched/aligned with
the query. In this case the percent identity calculated by FASTDB
is not manually corrected. Once again, only bases 5' and 3' of the
subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are to made for the purposes of the present invention.
[0107] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence of the
present invention, it is intended that the amino acid sequence of
the subject polypeptide is identical to the query sequence except
that the subject polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the query amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a query amino acid
sequence, up to 5% of the amino acid residues in the subject
sequence may be inserted, deleted, or substituted with another
amino acid. These alterations of the reference sequence may occur
at the amino or carboxy terminal positions of the reference amino
acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0108] As a practical matter, whether any particular polypeptide is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a
reference polypeptide can be determined conventionally using known
computer programs. A preferred method for determining the best
overall match between a query sequence (a sequence of the present
invention) and a subject sequence, also referred to as a global
sequence alignment, can be determined using the FASTDB computer
program based on the algorithm of Brutlag et al., Comp. App.
Biosci. 6:237-245 (1990). In a sequence alignment the query and
subject sequences are either both nucleotide sequences or both
amino acid sequences. The result of said global sequence alignment
is in percent identity. Preferred parameters used in a FASTDB amino
acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,
Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05,
Window Size=500 or the length of the subject amino acid sequence,
whichever is shorter.
[0109] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N- and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the query sequence, the percent identity is corrected
by calculating the number of residues of the query sequence that
are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence.
[0110] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C-termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are to be made for the purposes of the
present invention.
[0111] As used herein, a nucleic acid that "hybridizes under
stringent conditions" to a nucleic acid sequence of the invention,
refers to a polynucleotide that hybridizes in an overnight
incubation at 42.degree. C. in a solution comprising 50% formamide,
5.times.SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium
phosphate (pH 7.6), 5.times.Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA,
followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0112] As used herein, the term Golgi localization domain refers to
the amino acid sequence of a Golgi resident polypeptide which is
responsible for anchoring the polypeptide in location within the
Golgi complex. Generally, localization domains comprise amino
terminal "tails" of an enzyme.
[0113] As used herein, the term effector function refers to those
biological activities attributable to the Fc region (a native
sequence Fc region or amino acid sequence variant Fc region) of an
antibody. Examples of antibody effector functions include, but are
not limited to, Fc receptor binding affinity, antibody-dependent
cellular cytotoxicity (ADCC), antibody-dependent cellular
phagocytosis (ADCP), cytokine secretion, immune-complex-mediated
antigen uptake by antigen-presenting cells, down-regulation of cell
surface receptors, etc.
[0114] As used herein, the terms engineer, engineered, engineering
and glycosylation engineering are considered to include any
manipulation of the glycosylation pattern of a naturally occurring
or recombinant polypeptide or fragment thereof. Glycosylation
engineering includes metabolic engineering of the glycosylation
machinery of a cell, including genetic manipulations of the
oligosaccharide synthesis pathways to achieve altered glycosylation
of glycoproteins expressed in cells. Furthermore, glycosylation
engineering includes the effects of mutations and cell environment
on glycosylation. In one embodiment, the glycosylation engineering
is an alteration in glycosyltransferase activity. In a particular
embodiment, the engineering results in altered
glucosaminyltransferase activity and/or fucosyltransferase
activity.
[0115] As used herein, the term host cell covers any kind of
cellular system which can be engineered to generate the
polypeptides and antigen-binding molecules of the present
invention. In one embodiment, the host cell is engineered to allow
the production of an antigen binding molecule with modified
glycoforms. In a preferred embodiment, the antigen binding molecule
is an antibody, antibody fragment, or fusion protein. In certain
embodiments, the host cells have been further manipulated to
express increased levels of one or more polypeptides having GnTIII
activity. Host cells include cultured cells, e.g., mammalian
cultured cells, such as CHO cells, HEK293-EBNA cells, BHK cells,
NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma
cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,
insect cells, and plant cells, to name only a few, but also cells
comprised within a transgenic animal, transgenic plant or cultured
plant or animal tissue.
[0116] As used herein, the term Fc-mediated cellular cytotoxicity
includes antibody-dependent cellular cytotoxicity and cellular
cytotoxicity mediated by a soluble Fc-fusion protein containing a
human Fc-region. It is an immune mechanism leading to the lysis of
"antibody-targeted cells" by "human immune effector cells",
wherein:
[0117] The human immune effector cells are a population of
leukocytes that display Fc receptors on their surface through which
they bind to the Fc-region of antibodies or of Fc-fusion proteins
and perform effector functions. Such a population may include, but
is not limited to, peripheral blood mononuclear cells (PBMC) and/or
natural killer (NK) cells.
[0118] The antibody-targeted cells are cells bound by the
antibodies or Fc-fusion proteins. The antibodies or Fc
fusion-proteins bind to target cells via the protein part
N-terminal to the Fc region.
[0119] As used herein, the term increased Fc-mediated cellular
cytotoxicity is defined as either an increase in the number of
"antibody-targeted cells" that are lysed in a given time, at a
given concentration of antibody, or of Fc-fusion protein, in the
medium surrounding the target cells, by the mechanism of
Fc-mediated cellular cytotoxicity defined above, and/or a reduction
in the concentration of antibody, or of Fc-fusion protein, in the
medium surrounding the target cells, required to achieve the lysis
of a given number of "antibody-targeted cells", in a given time, by
the mechanism of Fc-mediated cellular cytotoxicity. The increase in
Fc-mediated cellular cytotoxicity is relative to the cellular
cytotoxicity mediated by the same antibody, or Fc-fusion protein,
produced by the same type of host cells, using the same standard
production, purification, formulation and storage methods, which
are known to those skilled in the art, but that has not been
produced by host cells engineered to express the
glycosyltransferase GnTIII by the methods described herein.
[0120] By antibody having increased antibody dependent cellular
cytotoxicity (ADCC) is meant an antibody, as that term is defined
herein, having increased ADCC as determined by any suitable method
known to those of ordinary skill in the art. One accepted in vitro
ADCC assay is as follows:
[0121] 1) the assay uses target cells that are known to express the
target antigen recognized by the antigen-binding region of the
antibody;
[0122] 2) the assay uses human peripheral blood mononuclear cells
(PBMCs), isolated from blood of a randomly chosen healthy donor, as
effector cells;
[0123] 3) the assay is carried out according to following protocol:
[0124] i) the PBMCs are isolated using standard density
centrifugation procedures and are suspended at 5.times.10.sup.6
cells/ml in RPMI cell culture medium; [0125] ii) the target cells
are grown by standard tissue culture methods, harvested from the
exponential growth phase with a viability higher than 90%, washed
in RPMI cell culture medium, labeled with 100 micro-Curies of 51Cr,
washed twice with cell culture medium, and resuspended in cell
culture medium at a density of 105 cells/ml; [0126] iii) 100
microliters of the final target cell suspension above are
transferred to each well of a 96-well microtiter plate; [0127] iv)
the antibody is serially-diluted from 4000 ng/ml to 0.04 ng/ml in
cell culture medium and 50 microliters of the resulting antibody
solutions are added to the target cells in the 96-well microtiter
plate, testing in triplicate various antibody concentrations
covering the whole concentration range above; [0128] v) for the
maximum release (MR) controls, 3 additional wells in the plate
containing the labeled target cells, receive 50 microliters of a 2%
(V/V) aqueous solution of non-ionic detergent (Nonidet, Sigma, St.
Louis), instead of the antibody solution (point iv above); [0129]
vi) for the spontaneous release (SR) controls, 3 additional wells
in the plate containing the labeled target cells, receive 50
microliters of RPMI cell culture medium instead of the antibody
solution (point iv above); [0130] vii) the 96-well microtiter plate
is then centrifuged at 50.times.g for 1 minute and incubated for 1
hour at 4.degree. C.; [0131] viii) 50 microliters of the PBMC
suspension (point i above) are added to each well to yield an
effector:target cell ratio of 25:1 and the plates are placed in an
incubator under 5% CO.sub.2 atmosphere at 37.degree. C. for 4
hours; [0132] ix) the cell-free supernatant from each well is
harvested and the experimentally released radioactivity (ER) is
quantified using a gamma counter; [0133] x) the percentage of
specific lysis is calculated for each antibody concentration
according to the formula (ER-MR)/(MR-SR).times.100, where ER is the
average radioactivity quantified (see point ix above) for that
antibody concentration, MR is the average radioactivity quantified
(see point ix above) for the MR controls (see point v above), and
SR is the average radioactivity quantified (see point ix above) for
the SR controls (see point vi above);
[0134] 4) "increased ADCC" is defined as either an increase in the
maximum percentage of specific lysis observed within the antibody
concentration range tested above, and/or a reduction in the
concentration of antibody required to achieve one half of the
maximum percentage of specific lysis observed within the antibody
concentration range tested above. The increase in ADCC is relative
to the ADCC, measured with the above assay, mediated by the same
antibody, produced by the same type of host cells, using the same
standard production, purification, formulation and storage methods,
which are known to those skilled in the art, but that has not been
produced by host cells engineered to overexpress GnTIII.
[0135] In one aspect, the present invention is related to antigen
binding molecules having the binding specificity of the rat ICR62
(i.e., binds to substantially the same epitope), and to the
discovery that their effector functions can be enhanced by altered
glycosylation. In one embodiment, the antigen binding molecule is a
chimeric antibody. In a preferred embodiment, the invention is
directed to a chimeric antibody, or a fragment thereof, comprising
one or more (e.g., one, two, three, four, five, or six) of the the
CDRs of any of SEQ ID NOs:53-108 and/or SEQ ID NO:s 122-127.
Specifically, in a preferred embodiment, the invention is directed
to an isolated polynucleotide comprising: (a) a sequence selected
from a group consisting of: SEQ ID NO:54 SEQ ID NO:56, SEQ ID
NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ
ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:122,
and SEQ ID NO:124; (b) a sequence selected from a group consisting
of: SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID
NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ
ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,
SEQ ID NO:104, SEQ ID NO:106, and SEQ ID NO:126; and (c) SEQ ID
NO:108. In another preferred embodiment, the invention is directed
to an isolated polynucleotide comprising (a) a sequence selectd
from the group consisting of SEQ ID NO:112 and SEQ ID NO:114; (b) a
sequence selectd from the group consisting of SEQ ID NO:116 and SEQ
ID NO:118; and (c) SEQ ID NO:119. In one embodiment, any of these
polynucleotides encodes a fusion polypeptide.
[0136] In another embodiment, the antigen binding molecule
comprises the V.sub.H domain of the rat ICR62 antibody encoded by
SEQ ID NO:1 or SEQ ID NO:2, or a variant thereof; and a non-murine
polypeptide. In another preferred embodiment, the invention is
directed to an antigen binding molecule comprising the V.sub.L
domain of the rat antibody encoded by SEQ ID NO:43 or SEQ ID NO:44,
or a variant thereof; and a non-murine polypeptide.
[0137] In another aspect, the invention is directed to antigen
binding molecules comprising one or more (e.g., one, two, three,
four, five, or six) truncated CDRs of ICR62. Such truncated CDRs
will contain, at a minimum, the specificity-determining amino acid
residues for the given CDR. By "specificity-determining residue" is
meant those residues that are directly involved in the interaction
with the antigen. In general, only about one-fifth to one-third of
the residues in a given CDR participate in binding to antigen. The
specificity-determining residues in a particular CDR can be
identified by, for example, computation of interatomic contacts
from three-dimensional modeling and determination of the sequence
variability at a given residue position in accordance with the
methods described in Padlan et al., FASEB J. 9(1):133-139 (1995),
the contents of which is hereby incorporated by reference in their
entirety.
[0138] Accordingly, the invention is also directed to an isolated
polynucleotide comprising at least one (e.g., one, two, three,
four, five, or six) complementarity determining region of the rat
ICR62 antibody, or a variant or truncated form thereof containing
at least the specificity-determining residues for said
complementarity determining region, wherein said isolated
polynucleotide encodes a fusion polypeptide. Preferably, such
isolated polynucleotides encode a fusion polypeptide that is an
antigen binding molecule. In one embodiment, the polynucleotide
comprises three complementarity determining regions of the rat
ICR62 antibody, or variants or truncated forms thereof containing
at least the specificity-determining residues for each of said
three complementarity determining regions. In one embodiment, the
polynucleotide comprises at least one of the CDRs set forth in
Tables 2-5, below. In another embodiment, the polynucleotide
encodes the entire variable region of the light or heavy chain of a
chimeric (e.g., humanized) antibody. The invention is further
directed to the polypeptides encoded by such polynucleotides.
[0139] In another embodiment, the invention is directed to an
antigen binding molecule comprising at least one (e.g., one, two,
three, four, five, or six) complementarity determining region of
the rat ICR62 antibody, or a variant or truncated form thereof
containing at least the specificity-determining residues for said
complementarity determining region, and comprising a sequence
derived from a heterologous polypeptide. In one embodiment, the
antigen binding molecule comprises three complementarity
determining regions of the rat ICR62 antibody, or variants or
truncated forms thereof containing at least the
specificity-determining residues for each of said three
complementarity determining regions. In one embodiment, the antigen
binding molecule comprises at least one of the CDRs set forth in
Tables 2-5, below. In another aspect, the antigen binding molecule
comprises the variable region of an antibody light or heavy chain.
In one particularly useful embodiment, the antigen binding molecule
is a chimeric, e.g., humanized, antibody. The invention is also
directed to methods of making such antigen binding molecules, and
the use of same in the treatment of disease, particularly cell
proliferation disorders wherein EGFR is expressed, particularly
wherein EGFR is abnormally expressed (e.g., overexpressed) compared
to normal tissue of the same cell type. Such disorders include, but
are not limited to cancers of the bladder, brain, head and neck,
pancreas, lung, breast, ovary, colon, prostate, skin, and kidney.
EGFR expression levels may be determined by methods known in the
art and those described herein (e.g., via immunohistochemistry
assay, immunofluorescence assay, immunoenzyme assay, ELISA, flow
cytometry, radioimmunoassay, Western blot, ligand binding, kinase
activity, etc.).
[0140] The invention is also directed to a method for targeting in
vivo or in vitro cells expressing EGFR. Cells that express EGFR may
be targeted for therapuetic purposes (e.g., to treat a disorder
that is treatable by disruption of EGFR-mediated signaling, for
example by blocking ligand binding, or by targeting EGFR-expressing
cells for destruction by the immune system). In one embodiment, the
present invention is directed to a method for targeting cells
expressing EGFR in a subject comprising administering to the
subject a composition comprising an ABM of the invention. Cells
that express EGFR may also be targeted for diagnositic purposes
(e.g., to determine if they are expressing EGFR, either normally or
abnormally). Thus, the invention is also directed to methods for
detecting the presence of EGFR or a cell expressing EGFR, either in
vivo or in vitro. One method of detecting EGFR expression according
to the present invention comprises contacting a sample to be
tested, optionally with a control sample, with an ABM of the
present invention, under conditions that allow for formation of a
complex between the ABM and EGFR. The complex formation is then
detected (e.g., by ELISA or other methods known in the art). When
using a control sample with the test sample,any statistically
significant difference in the formation of ABM-EGFR complexes when
comparing the test and control samples is indicative of the
presence of EGFR in the test sample.
TABLE-US-00002 TABLE 2 SEQ CDR Nucleotide Sequence ID NO Heavy
Kabat GACTACAAGATACAC 54 Chain GACTACGCCATCAGC 56 CDR1
GACTACTATATGCAC 58 GACTACAAGATATCC 122 Chothia
GGTTTTACATTCACTGACTAC 60 GGTTACACATTCACTGACTAC 62
GGTTATTCATTCACTGACTAC 64 AbM GGTTTTACATTCACTGACTACAAGATACAC 66
GGTTTTACATTCACTGACTACGCCATCAGC 68 GGTTTTACATTCACTGACTACTATATGCAC 70
GGTTACACATTCACTGACTACTATATGCAC 72 GGTTATTCATTCACTGACTACAAGATACAC 74
GGTTTCACATTCACTGACTACAAGATATCC 124 Heavy Kabat
TATTTTAATCCTAACAGTGGTTATAGTACCTA 76 Chain CAATGAAAAGTTCAAGAGC CDR2
GGGATCAATCCTAACAGTGGTTATAGTACCTA 78 CGCACAGAAGTTCCAGGGC
TATTTCAACCCTAACAGCGGTTATAGTACCTA 80 CGCACAGAAGTTCCAGGGC
TGGATCAATCCTAACAGTGGTTATAGTACCTA 82 CGCACAGAAGTTTCAGGGC
TGGATCAATCCTAACAGTGGTTATAGTACCTA 84 CAGCCCAAGCTTCCAAGGC
TGGATCAATCCTAACAGTGGTTATAGTACCTA 86 CAACGAGAAGTTCCAAGGC
TATTTCAACCCTAACAGCGGTTATTCGAACTA 88 CGCACAGAAGTTCCAGGGC
TATTTCAACCCTAACAGCGGTTATGCCACGTA 90 CGCACAGAAGTTCCAGGGC
TACTTCAATCCTAACAGTGGTTATAGTACCTA 126 CAGCCCAAGCTTCCAAGGC Chothia
AATCCTAACAGTGGTTATAGTACC 92 AACCCTAACAGCGGTTATTCGAAC 94
AACCCTAACAGCGGTTATGCCACG 96 AbM TATTTTAATCCTAACAGTGGTTATAGTACC 98
GGGATCAATCCTAACAGTGGTTATAGTACC 100 TGGATCAATCCTAACAGTGGTTATAGTACC
102 TATTTCAACCCTAACAGCGGTTATTCGAAC 104
TATTTCAACCCTAACAGCGGTTATGCCACG 106 Heavy Kabat
CTATCCCCAGGCGGTTACTATGTTATGGATGC 108 Chain Chothia C CDR3 AbM
TABLE-US-00003 TABLE 3 CDR Amino Acid Sequence SEQ ID NO Heavy
Kabat DYKIH 53 Chain DYAIS 55 CDR1 DYYMH 57 DYKIS 123 Chothia
GFTFTDY 59 GYTFTDY 61 GYSFTDY 63 AbM GFTFTDYKIH 65 GFTFTDYAIS 67
GFTFTDYYMH 69 GYTFTDYYMH 71 GYSFTDYKIH 73 GFTFTDYKIS 125 Heavy
Kabat YFNPNSGYSTYNEKFKS 75 Chain GINPNSGYSTYAQKFQG 77 CDR2
YFNPNSGYSTYAQKFQG 79 WINPNSGYSTYAQKFQG 81 WINPNSGYSTYSPSFQG 83
WINPNSGYSTYNEKFQG 85 YFNPNSGYSNYAQKFQG 87 YFNPNSGYATYAQKFQG 89
YFNPNSGYSTYSPSFQG 127 Chothia NPNSGYST 91 NPNSGYSN 93 NPNSGYAT 95
AbM YFNPNSGYST 97 GINPNSGYST 99 WINPNSGYST 101 YFNPNSGYSN 103
YFNPNSGYAT 105 Heavy Kabat LSPGGYYVMDA 107 Chain Chothia CDR3
AbM
TABLE-US-00004 TABLE 4 SEQ ID CDR Amino Acid Sequence NO Kabat
Light Chain KASQNINNYLN 111 CDR1 RASQGINNYLN 113 Kabat Light Chain
NTNNLQT 115 CDR2 Kabat Light Chain LQHNSFPT 117 CDR3
TABLE-US-00005 TABLE 5 SEQ ID CDR Nucleotide Sequence NO Kabat
Light Chain AAAGCAAGTCAGAATATTAACAATTACTTAAAC 112 CDR1
CGGGCAAGTCAGGGCATTAACAATTACTTAAAT 114 Kabat Light Chain
AATACAAACAATTTGCAAACA 116 CDR2 AATACCAACAACTTGCAGACA 118 Kabat
Light Chain TTGCAGCATAATAGTTTTCCCACG 119 CDR3
[0141] It is known that several mechanism are involved in the
therapeutic efficacy of anti-EGFR antibodies, including blocking of
ligand (e.g., EGF, TGF-.alpha., etc.) binding to EGFR and
subsequent activation of signaling pathways, antibody dependent
cellular cytotoxicity (ADCC), and the induction of growth arrest or
terminal differentiation.
[0142] The rat monoclonal antibody ICR62 (IgG2b) was discussed in
PCT Publication No. WO 95/20045, which is incorporated herein by
reference in its entirety. It was directed to the C epitope of
EGFR, and was shown to inhibit ligand binding, inhibit growth in
vitro of squamous cell carcinomas expressing EGFR, and induce
regression of xenografts of tumors in athymic mice (WO 95/20045;
Modjtahedi et al., Br. J. Cancer 73:228-235 (1996)). As a fully
rodent antibody, administration of ICR62 rat monoclonal antibody to
humans resulted a HARA response in some patients following even a
single dose. (WO 95/20045; Modjtahedi et al., Br. J. Cancer
73:228-235 (1996)).
[0143] Chimeric mouse/human antibodies have been described. See,
for example, Morrison, S. L. et al., PNAS 11:6851-6854 (November
1984); European Patent Publication No. 173494; Boulianna, G. L, et
al., Nature 312:642 (December 1984); Neubeiger, M. S. et al.,
Nature 314:268 (March 1985); European Patent Publication No.
125023; Tan et al., J. Immunol. 135:8564 (November 1985); Sun, L. K
et al., Hybridoma 5(1):517 (1986); Sahagan et al., J. Immunol.
137:1066-1074 (1986). See generally, Muron, Nature 312:597
(December 1984); Dickson, Genetic Engineering News 5(3) (March
1985); Marx, Science 229:455 (August 1985); and Morrison, Science
229:1202-1207 (September 1985). IMC-C225 (Erbitux.RTM., Imclone) is
a chimeric monoclonal antibody directed against EGFR and having a
mouse variable region and a human constant region (See Herbst and
Shin, Cancer 94: 1593-1611 (2002)). The murine portion of IMC-225
is derived from M225, which was found to bind EGFR and inhibit
EGF-induced tyrosine kinase-dependent phosphorylation, as well as
inducing apoptosis in tumor cell lines over-expressing EGFR (Herbst
and Shin, Cancer 94: 1593-1611 (2002)). However, M225 elicited a
HAMA reaction in patients treated with the antibody in Phase I
clinical trials (Herbst and Shin, Cancer 94: 1593-1611 (2002)).
IMC-225 has been tested in vivo and in vitro, and has been used in
combination with radiation therapy and chemotherapy in a number of
tumor types, including those associated with poor prognosis (Herbst
and Shin, Cancer 94: 1593-1611 (2002)). However, IMC-225 has been
associated with toxicities such as allergic and skin reactions in
patients administered the IMC-225 antibody in clincial trials
(Herbst and Shin, Cancer 94: 1593-1611 (2002)).
[0144] In a particularly preferred embodiment, the chimeric ABM of
the present invention is a humanized antibody. Methods for
humanizing non-human antibodies are known in the art. For example,
humanized ABMs of the present invention can be prepared according
to the methods of U.S. Pat. No. 5,225,539 to Winter, U.S. Pat. No.
6,180,370 to Queen et al., U.S. Pat. No. 6,632,927 to Adair et al.,
or U.S. Pat. Appl. Pub. No. 2003/0039649 to Foote, the entire
contents of each of which is herein incorporated by reference.
Preferably, a humanized antibody has one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al., Nature, 321:522-525
(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et
al., Science, 239:1534-1536 (1988)), by substituting hypervariable
region sequences for the corresponding sequences of a human
antibody. Accordingly, such "humanized" antibodies are chimeric
antibodies (U.S. Pat. No. 4,816,567) wherein substantially less
than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent antibodies.
The subject humanized anti-EGFR antibodies will comprise constant
regions of human immunoglobulin.
[0145] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework region (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method of
selecting the human framework sequence is to compare the sequence
of each individual subregion of the full rodent framework (i.e.,
FR1, FR2, FR3, and FR4) or some combination of the individual
subregions (e.g., FR1 and FR2) against a library of known human
variable region sequences that correspond to that framework
subregion (e.g., as determined by Kabat numbering), and choose the
human sequence for each subregion or combination that is the
closest to that of the rodent (Leung, U.S. Patent Application
Publication No. 2003/0040606A1, published Feb. 27, 2003) (the
entire contents of which are hereby incorporated by reference).
Another method uses a particular framework region derived from the
consensus sequence of all human antibodies of a particular subgroup
of light or heavy chains. The same framework may be used for
several different humanized antibodies (Carter et al., Proc. Natl.
Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol.,
151:2623 (1993)) (the entire contents of each of which are herein
incorporated by reference).
[0146] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models can be
generated using computer programs familiar to those skilled in the
art (e.g., InsightII, Accelrys, Inc. (formerly MSI), or at
http://swissmodel.expasy.org). These computer programs can
illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of
the residues in the functioning of the candidate immunoglobulin
sequence, i.e., the analysis of residues that influence the ability
of the candidate immunoglobulin to bind its antigen. In this way,
FR residues can be selected and combined from the recipient and
import sequences so that the desired antibody characteristic, such
as increased affinity for the target antigen(s), is achieved. In
general, the hypervariable region residues are directly and most
substantially involved in influencing antigen binding.
[0147] In one embodiment, the antibodies of the present invention
comprise a human Fc region. In a specific embodiment, the human
constant region is IgG1, as set forth in SEQ ID NOs 109 and 110,
and set forth below:
TABLE-US-00006 IgG1 Nucleotide Sequence (SEQ ID NO: 110)
ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTC
TGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAAC
CGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC
TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA
ATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGG
GGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGA
TCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA
GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAA
TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGG
TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC
AAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAT
CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC
CATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC
AAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA
GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT
CCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA IgG1 Amino Acid Sequence (SEQ
ID NO: 109) TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT
FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK
[0148] However, variants and isoforms of the human Fc region are
also encompassed by the present invention. For example, variant Fc
regions suitable for use in the present invention can be produced
according to the methods taught in U.S. Pat. No. 6,737,056 to
Presta (Fc region variants with altered effector function due to
one or more amino acid modifications); or in U.S. Pat. Appl. Nos.
60/439,498; 60/456,041; 60/514,549; or WO 2004/063351 (variant Fc
regions with increased binding affinity due to amino acid
modification); or in U.S. patent application Ser. No. 10/672,280 or
WO 2004/099249 (Fc variants with altered binding to Fc.gamma.R due
to amino acid modification), the contents of each of which is
herein incorporated by reference in its entirety.
[0149] In another embodiment, the antigen binding molecules of the
present invention are engineered to have enhanced binding affinity
according to, for example, the methods disclosed in U.S. Pat. Appl.
Pub. No. 2004/0132066 to Balint et al., the entire contents of
which are hereby incorporated by reference.
[0150] In one embodiment, the antigen binding molecule of the
present invention is conjugated to an additional moiety, such as a
radiolabel or a toxin. Such conjugated ABMs can be produced by
numerous methods that are well known in the art.
[0151] A variety of radionuclides are applicable to the present
invention and those skilled in the art are credited with the
ability to readily determine which radionuclide is most appropriate
under a variety of circumstances. For example, .sup.131iodine is a
well known radionuclide used for targeted immunotherapy. However,
the clinical usefulness of .sup.131iodine can be limited by several
factors including: eight-day physical half-life; dehalogenation of
iodinated antibody both in the blood and at tumor sites; and
emission characteristics (e.g., large gamma component) which can be
suboptimal for localized dose deposition in tumor. With the advent
of superior chelating agents, the opportunity for attaching metal
chelating groups to proteins has increased the opportunities to
utilize other radionuclides such as .sup.111indium and
.sup.90yttrium. .sup.90Yttrium provides several benefits for
utilization in radioimmunotherapeutic applications: the 64 hour
half-life of .sup.90yttrium is long enough to allow antibody
accumulation by tumor and, unlike e.g., .sup.131iodine,
.sup.90yttrium is a pure beta emitter of high energy with no
accompanying gamma irradiation in its decay, with a range in tissue
of 100 to 1000 cell diameters. Furthermore, the minimal amount of
penetrating radiation allows for outpatient administration of
.sup.90yttrium-labeled antibodies. Additionally, internalization of
labeled antibody is not required for cell killing, and the local
emission of ionizing radiation should be lethal for adjacent tumor
cells lacking the target antigen.
[0152] Effective single treatment dosages (i.e., therapeutically
effective amounts) of .sup.90yttrium labeled anti-EGFR antibodies
range from between about 5 and about 75 mCi, more preferably
between about 10 and about 40 mCi. Effective single treatment
non-marrow ablative dosages of .sup.131iodine labeled anti-EGFR
antibodies range from between about 5 and about 70 mCi, more
preferably between about 5 and about 40 mCi. Effective single
treatment ablative dosages (i.e., may require autologous bone
marrow transplantation) of .sup.131iodine labeled anti-EGFR
antibodies range from between about 30 and about 600 mCi, more
preferably between about 50 and less than about 500 mCi. In
conjunction with a chimeric anti-EGFR antibody, owing to the longer
circulating half life vis-a-vis murine antibodies, an effective
single treatment non-marrow ablative dosages of .sup.131iodine
labeled chimeric anti-EGFR antibodies range from between about 5
and about 40 mCi, more preferably less than about 30 mCi. Imaging
criteria for, e.g., the .sup.111indium label, are typically less
than about 5 mCi.
[0153] With respect to radiolabeled anti-EGFR antibodies, therapy
therewith can also occur using a single therapy treatment or using
multiple treatments. Because of the radionuclide component, it is
preferred that prior to treatment, peripheral stem cells ("PSC") or
bone marrow ("BM") be "harvested" for patients experiencing
potentially fatal bone marrow toxicity resulting from radiation. BM
and/or PSC are harvested using standard techniques, and then purged
and frozen for possible reinfusion. Additionally, it is most
preferred that prior to treatment a diagnostic dosimetry study
using a diagnostic labeled antibody (e.g., using .sup.111indium) be
conducted on the patient, a purpose of which is to ensure that the
therapeutically labeled antibody (e.g., using .sup.90yttrium) will
not become unnecessarily "concentrated" in any normal organ or
tissue.
[0154] In a preferred embodiment, the present invention is directed
to an isolated polynucleotide comprising a sequence that encodes a
polypeptide having an amino acid sequence in Table 7 below. In a
preferred embodiment, the invention is directed to an isolated
polynucleotide comprising a sequence shown in Table 6 below. The
invention is further directed to an isolated nucleic acid
comprising a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99% identical to a nucleotide sequence shown in Table 6 below. In
another embodiment, the invention is directed to an isolated
nucleic acid comprising a sequence that encodes a polypeptide
having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%,
97%, 98% or 99% identical to an amino acid sequence in Table 7. The
invention also encompasses an isolated nucleic acid comprising a
sequence that encodes a polypeptide having the amino acid sequence
of any of the constructs in Table 7 with conservative amino acid
substitutions.
TABLE-US-00007 TABLE 6 CONSTRUCT NUCLEOTIDE SEQUENCE SEQ ID NO
ICR62 VH CAGGTCAACCTACTGCAGTCTGGGGCTGCACTGGT 2
GAAGCCTGGGGCCTCTGTGAAGTTGTCTTGCAAAG
GTTCTGGTTTTACATTCACTGACTACAAGATACAC
TGGGTGAAGCAGAGTCATGGAAAGAGCCTTGAGT
GGATTGGGTATTTTAATCCTAACAGTGGTTATAGT
ACCTACAATGAAAAGTTCAAGAGCAAGGCCACAT
TGACTGCAGACAAATCCACCGATACAGCCTATATG
GAGCTTACCAGTCTGACATCTGAGGACTCTGCAAC
CTATTACTGTACAAGACTATCCCCAGGGGGTTACT
ATGTTATGGATGCCTGGGGTCAAGGAGCTTCAGTC ACTGTCTCCTC I-HHA
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 4
AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG
GCTTCTGGATTTACATTCACTGACTACGCCATCAG
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGAGGGATCAATCCTAACAGTGGTTATAG
TACCTACGCACAGAAGTTCCAGGGCAGGGTCACC
ATTACCGCGGACAAATCCACGAGCACAGCCTACAT
GGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC
GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTA
CTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HHB
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 6
AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG
GGTTCTGGTTTTACATTCACTGACTACAAGATACA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGATATTTCAACCCTAACAGCGGTTATAG
TACCTACGCACAGAAGTTCCAGGGCAGGGTCACC
ATTACCGCGGACAAATCCACGAGCACAGCCTACAT
GGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC
GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTA
CTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HHC
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 8
AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG
GGTTCTGGTTTTACATTCACTGACTACAAGATACA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGATATTTCAACCCTAACAGCGGTTATAG
TACCTACAATGAAAAGTTCAAGAGCAGGGTCACC
ATTACCGCGGACAAATCCACGAGCACAGCCTACAT
GGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC
GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTA
CTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HLA
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 10
AGAAGCCTGGGGCCTCGGTGAAGGTCTCCTGCAA
GGCCTCTGGTTTTACATTCACTGACTACTATATGCA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGCTGGATCAATCCTAACAGTGGTTATAG
TACCTACGCACAGAAGTTTCAGGGCAGGGTCACCA
TGACCGCCGACACGTCCATCAGCACAGCCTACATG
GAGCTGAGCAGGCTGAGATCTGACGACACGGCCG
TGTATTACTGTGCGAGACTATCCCCAGGCGGTTAC
TATGTTATGGATGCCTGGGGCCAAGGGACCACCGT GACCGTCTCCTCA I-HLB
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 12
AGAAGCCTGGAGCCTCGGTGAAGGTCTCCTGCAA
GGGTTCTGGTTTTACATTCACTGACTACAAGATCC
ACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGA
GTGGATGGGATACTTCAACCCTAACAGCGGTTATA
GTACCTACGCACAGAAGTTCCAGGGCAGGGTCAC
CATGACCGCCGACACGTCCATCAGCACAGCCTACA
TGGAGCTGAGCAGGCTGAGATCTGACGACACGGC
CGTGTATTACTGTGCGAGACTATCCCCAGGCGGTT
ACTATGTTATGGATGCCTGGGGCCAAGGGACCACC GTGACCGTCTCCTCA I-HLC
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 14
AGAAGCCTGGAGCCTCAGTGAAGGTCTCCTGCAA
GGGTTCTGGTTTTACATTCACTGACTACAAGATCC
ACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGA
GTGGATGGGATACTTCAACCCTAACAGCGGTTACA
GTACTTACAACGAGAAGTTCAAGAGCCGGGTCAC
CATGACCGCCGACACGTCCATCAGCACAGCCTACA
TGGAGCTGAGCAGGCTGAGATCTGACGACACGGC
CGTGTATTACTGTGCGAGACTATCCCCAGGGGGTT
ACTATGTTATGGATGCCTGGGGCCAAGGGACCACC GTGACCGTCTCCTCA I-HHD
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 16
AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG
GCCTCTGGTTTCACATTCACTGACTACAAGATACA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGATATTTCAACCCTAACAGCGGTTATAG
TACCTACGCACAGAAGTTCCAGGGCAGGGTCACC
ATTACCGCGGACAAATCCACGAGCACAGCCTACAT
GGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC
GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTA
CTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HHE
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 18
AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG
GGTTCTGGTTTCACATTCACTGACTACAAGATATC
CTGGGTGCGACAGGCTCCTGGACAAGGGCTCGAG
TGGATGGGATATTTCAACCCTAACAGCGGTTATAG
TACCTACGCACAGAAGTTCCAGGGCAGGGTCACC
ATTACCGCGGACAAATCCACGAGCACAGCCTACAT
GGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC
GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTA
CTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HHF
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 20
AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG
GGTTCTGGTTTTACATTCACTGACTACAAGATACA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGATATTTCAACCCTAACAGCGGTTATTC
GAACTACGCACAGAAGTTCCAGGGCAGGGTCACC
ATTACCGCGGACAAATCCACGAGCACAGCCTACAT
GGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC
GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTA
CTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HHG
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 22
AGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG
GGTTCTGGTTTTACATTCACTGACTACAAGATACA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGATATTTCAACCCTAACAGCGGTTATGC
CACGTACGCACAGAAGTTCCAGGGCAGGGTCACC
ATTACCGCGGACAAATCCACGAGCACAGCCTACAT
GGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC
GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTA
CTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HLA1
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 24
AGAAGCCTGGAGCCTCGGTGAAGGTCTCCTGCAA
GGCCTCTGGTTTTACATTCACTGACTACTATATGCA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGCTGGATCAATCCTAACAGTGGTTATAG
TACCTACAGCCCAAGCTTCCAAGGCCAGGTCACCA
TCTCAGCCGACAAGTCCATCAGCACCGCCTACCTG
CAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCA
TGTATTACTGTGCGAGACTATCCCCAGGCGGTTAC
TATGTTATGGATGCCTGGGGCCAAGGGACCACCGT GACCGTCTCCTCA I-HLA2
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 26
AGAAGCCTGGAGCCTCGGTGAAGGTCTCCTGCAA
GGCCTCTGGTTTTACATTCACTGACTACTATATGCA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGCTGGATCAATCCTAACAGTGGTTATAG
TACCTACAACGAGAAGTTCCAAGGCCAGGTCACC
ATCTCAGCCGACAAGTCCATCAGCACCGCCTACCT
GCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCC
ATGTATTACTGTGCGAGACTATCCCCAGGCGGTTA
CTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HLA3
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 28
AGAAGCCTGGAGCCTCGGTGAAGGTCTCCTGCAA
GGCCTCTGGTTACACATTCACTGACTACTATATGC
ACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGA
GTGGATGGGCTGGATCAATCCTAACAGTGGTTATA
GTACCTACAGCCCAAGCTTCCAAGGCCAGGTCACC
ATCTCAGCCGACAAGTCCATCAGCACCGCCTACCT
GCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCC
ATGTATTACTGTGCGAGACTATCCCCAGGCGGTTA
CTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HLA4
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGA 30
AGAAGCCTGGAGCCTCGGTGAAGGTCTCCTGCAA
GGCCTCTGGTTACACATTCACTGACTACTATATGC
ACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGA
GTGGATGGGCTGGATCAATCCTAACAGTGGTTATA
GTACCTACAACGAGAAGTTCCAAGGCCAGGTCAC
CATCTCAGCCGACAAGTCCATCAGCACCGCCTACC
TGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGC
CATGTATTACTGTGCGAGACTATCCCCAGGCGGTT
ACTATGTTATGGATGCCTGGGGCCAAGGGACCACC GTGACCGTCTCCTCA I-HLA5
CAGATGCAGCTGGTGCAGTCTGGGCCAGAGGTGA 32
AGAAGCCTGGAACCTCGGTGAAGGTCTCCTGCAA
GGCCTCTGGTTTTACATTCACTGACTACTATATGCA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGCTGGATCAATCCTAACAGTGGTTATAG
TACCTACAGCCCAAGCTTCCAAGGCCAGGTCACCA
TCTCAGCCGACAAGTCCATCAGCACCGCCTACCTG
CAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCA
TGTATTACTGTGCGAGACTATCCCCAGGCGGTTAC
TATGTTATGGATGCCTGGGGCCAAGGGACCACCGT GACCGTCTCCTCA I-HLA6
CAGATGCAGCTGGTGCAGTCTGGGCCAGAGGTGA 34
AGAAGCCTGGAACCTCGGTGAAGGTCTCCTGCAA
GGCCTCTGGTTTTACATTCACTGACTACTATATGCA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGCTGGATCAATCCTAACAGTGGTTATAG
TACCTACAACGAGAAGTTCCAAGGCCAGGTCACC
ATCTCAGCCGACAAGTCCATCAGCACCGCCTACCT
GCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCC
ATGTATTACTGTGCGAGACTATCCCCAGGCGGTTA
CTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HLA7
CAGATGCAGCTGGTGCAGTCTGGGCCAGAGGTGA 36
AGAAGCCTGGAACCTCGGTGAAGGTCTCCTGCAA
GGCCTCTGGTTTTACATTCACTGACTACAAGATCC
ACTGGGTGCGACAGGCCCGCGGACAACGGCTCGA
GTGGATCGGCTGGATCAATCCTAACAGTGGTTATA
GTACCTACAACGAGAAGTTCCAAGGCCAGGTCAC
CATCTCAGCCGACAAGTCCATCAGCACCGCCTACC
TGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGC
CATGTATTACTGTGCGAGACTATCCCCAGGCGGTT
ACTATGTTATGGATGCCTGGGGCCAAGGGACCACC GTGACCGTCTCCTCA I-HLA8
CAGATGCAGCTGGTGCAGTCTGGGCCAGAGGTGA 38
AGAAGCCTGGAACCTCGGTGAAGGTCTCCTGCAA
GGCCTCTGGTTTTACATTCACTGACTACAAGATCC
ACTGGGTGCGACAGGCCCCTGGACAAGGGCTCGA
GTGGATGGGATATTTCAACCCTAACAGCGGTTATA
GTACCTACGCACAGAAGTTCCAGGGCAGGGTCAC
CATTACCGCGGACAAATCCACGAGCACAGCCTAC
ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGG
CCGTGTATTACTGTGCGAGACTATCCCCAGGCGGT
TACTATGTTATGGATGCCTGGGGCCAAGGGACCAC CGTGACCGTCTCCTCA I-HLA9
GAGGTGCAGCTCGTGCAGTCTGGCGCTGAGGTGA 40
AGAAGCCTGGCGAGTCGTTGAAGATCTCCTGCAAG
GGTTCTGGTTATTCATTCACTGACTACAAGATCCA
CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG
TGGATGGGATATTTCAACCCTAACAGCGGTTATAG
TACCTACGCACAGAAGTTCCAGGGCAGGGTCACC
ATTACCGCGGACAAATCCACGAGCACAGCCTACAT
GGAGCTGAGCAGCCTGAGATCTGAGGACACGGCC
GTGTATTACTGTGCGAGACTATCCCCAGGCGGTTA
CTATGTTATGGATGCCTGGGGCCAAGGGACCACCG TGACCGTCTCCTCA I-HLA10
GAGGTGCAGCTCGTGCAGTCTGGCGCTGAGGTGA 120
AGAAGCCTGGCGAGTCGTTGAAGATCTCCTGCAAG
GGTTCTGGTTATTCATTCACTGACTACAAGATCCA
CTGGGTGCGACAGATGCCTGGAAAGGGCCTCGAG
TGGATGGGCTACTTCAATCCTAACAGTGGTTATAG
TACCTACAGCCCAAGCTTCCAAGGCCAGGTCACCA
TCTCAGCCGACAAGTCCATCAGCACCGCCTACCTG
CAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCA
TGTATTACTGTGCGAGACTATCCCCAGGCGGTTAC
TATGTTATGGATGCCTGGGGCCAAGGGACCACCGT GACCGTCTCCTCAG VH Signal
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGC 42 Sequence
AGCAGCCACAGGAGCCCACTCC ICR62 VL
GACATCCAGATGACCCAGTCTCCTTCATTCCTGTCT 44
GCATCTGTGGGAGACAGAGTCACTATCAACTGCAA
AGCAAGTCAGAATATTAACAATTACTTAAACTGGT
ATCAGCAAAAGCTTGGAGAAGCTCCCAAACGCCT
GATATATAATACAAACAATTTGCAAACAGGCATCC
CATCAAGGTTCAGTGGCAGTGGATCTGGTACAGAT
TACACACTCACCATCAGCAGCCTGCAGCCTGAAGA
TTTTGCCACATATTTCTGCTTGCAGCATAATAGTTT
TCCCACGTTTGGAGCTGGGACCAAGCTGGAACTGA AACGTACG I-KC
GATATCCAGATGACCCAGTCTCCATCCTCCCTGTC 46
TGCATCTGTCGGAGACCGGGTCACCATCACCTGCC
GGGCAAGTCAGGGCATTAACAATTACTTAAATTGG
TACCAGCAGAAGCCAGGGAAAGCCCCTAAGCGCC
TGATCTATAATACCAACAACTTGCAGACAGGCGTC
CCATCAAGGTTCAGCGGCAGTGGATCCGGGACAG
AATTCACTCTCACCATCAGCAGCCTGCAGCCTGAA
GATTTTGCCACCTATTACTGCTTGCAGCATAATAG
TTTTCCCACGTTTGGCCAGGGCACCAAGCTCGAGA TCAAGCGTACG VL Signal
ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGCCT 48 Sequence
CCTGCTGCTCTGGTTCCCAGGTGCCAGGTGT 1-KA
GATATCCAGATGACCCAGTCTCCATCCTCCCTGTC 50
TGCATCTGTCGGAGACCGGGTCACCATCACCTGCC
GGGCAAGTCAGGGCATTAACAATTACTTAAATTGG
TACCAGCAGAAGCCAGGGAAAGCCCCTAAGCGCC
TGATCTATAATACCAACAACTTGCAGACAGGCGTC
CCATCAAGGTTCAGCGGCAGTGGATCCGGGACAG
AATACACTCTCACCATCAGCAGCCTGCAGCCTGAA
GATTTTGCCACCTATTACTGCTTGCAGCATAATAG
TTTTCCCACGTTTGGCCAGGGCACCAAGCTCGAGA TCAAGCGTACGGTG I-KB
GATATCCAGATGACCCAGTCTCCATCCTCCCTGTC 52
TGCATCTGTCGGAGACCGGGTCACCATCACCTGCA
AAGCAAGTCAGAATATTAACAATTACTTAAACTGG
TACCAGCAGAAGCCAGGGAAAGCCCCTAAGCGCC
TGATCTATAATACCAACAACTTGCAGACAGGCGTC
CCATCAAGGTTCAGCGGCAGTGGATCCGGGACAG
AATACACTCTCACCATCAGCAGCCTGCAGCCTGAA
GATTTTGCCACCTATTACTGCTTGCAGCATAATAG
TTTTCCCACGTTTGGCCAGGGCACCAAGCTCGAGA TCAAGCGTACGGTG
TABLE-US-00008 TABLE 7 CONSTRUCT AMINO ACID SEQUENCE SEQ ID NO
ICR62 VH QVNLLQSGAALVKPGASVKLSCKGSGFTFTDYKIHWVK 1
QSHGKSLEWIGYFNPNSGYSTYNEKFKSKATLTADKSTD
TAYMELTLTSEDSATYYCTRLSPGGYYVMDAWGQGA SVTVSS I-HHA
QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYAISWVR 3
QAPGQGLEWMGGINPNSGYSTYAQKFQGRVTITADKST
STAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQG TTVTVSS I-HHB
QVQLVQSGAEVKKPGSSVKVSCKGSGFTFTDYKIHWVR 5
QAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKST
STAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQG TTVTVSS I-HHC
QVQLVQSGAEVKKPGSSVKVSCKGSGFTFTDYKIHWVR 7
QAPGQGLEWMGYFNPNSGYSTYNEKFKSRVTITADKST
STAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQG TTVTVSS I-HLA
QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWV 9
RQAPGQGLEWMGWINPNSGYSTYAQKFQGRVTMTADT
SISTAYMELSRLRSDDTAVYYCARLSPGGYYVMDAWGQ GTTVTVSS I-HLB
QVQLVQSGAEVKKPGASVKVSCKGSGFTFTDYKIHWVR 11
QAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTMTADTSI
STAYMELSRLRSDDTAVYYCARLSPGGYYVMDAWGQG TTVTVSS I-HLC
QVQLVQSGAEVKKPGASVKVSCKGSGFTFTDYKIHWVR 13
QAPGQGLEWMGYFNPNSGYSTYNEKFKSRVTMTADTSI
STAYMELSRLRSDDTAVYYCARLSPGGYYVMDAWGQG TTVTVSS I-HHD
QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYKIHWVR 15
QAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKST
STAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQG TTVTVSS I-HHE
QVQLVQSGAEVKKPGSSVKVSCKGSGFTFTDYKISWVR 17
QAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKST
STAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQG TTVTVSS I-HHF
QVQLVQSGAEVKKPGSSVKVSCKGSGFTFTDYKIHWVR 19
QAPGQGLEWMGYFNPNSGYSNYAQKFQGRVTITADKST
STAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQG TTVTVSS I-HHG
QVQLVQSGAEVKKPGSSVKVSCKGSGFTFTDYKIHWVR 21
QAPGQGLEWMGYFNPNSGYATYAQKFQGRVTITADKST
STAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQG TTVTVSS I-HLA1
QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWV 23
RQAPGQGLEWMGWINPNSGYSTYSPSFQGQVTISADKSI
STAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWGQ GTTVTVSS I-HLA2
QVQLVQSGAEVKKPGASVKVSCKASGFTFTDYYMHWV 25
RQAPGQGLEWMGWINPNSGYSTYNEKFQGQVTISADKS
ISTAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWGQ GTTVTVSS I-HLA3
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWV 27
RQAPGQGLEWMGWINPNSGYSTYSPSFQGQVTISADKSI
STAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWGQ GTTVTVSS I-HLA4
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWV 29
RQAPGQGLEWMGWINPNSGYSTYNEKFQGQVTISADKS
ISTAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWGQ GTTVTVSS I-HLA5
QMQLVQSGPEVKKPGTSVKVSCKASGFTFTDYYMHWV 31
RQAPGQGLEWMGWINPNSGYSTYSPSFQGQVTISADKSI
STAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWGQ GTTVTVSS I-HLA6
QMQLVQSGPEVKKPGTSVKVSCKASGFTFTDYYMHWV 33
RQAPGQGLEWMGWINPNSGYSTYNEKFQGQVTISADKS
ISTAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWGQ GTTVTVSS I-HLA7
QMQLVQSGPEVKKPGTSVKVSCKASGFTFTDYKIHWVR 35
QARGQRLEWIGWINPNSGYSTYNEKFQGQVTISADKSIS
TAYLQWSSLKASDTAMYYCARLSPGGYYVMDAWGQG TTVTVSS I-HLA8
QMQLVQSGPEVKKPGTSVKVSCKASGFTFTDYKIHWVR 37
QAPGQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKST
STAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQG TTVTVSS I-HLA9
EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYKIHWVRQ 39
APGQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKSTS
TAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQGT TVTVSS I-HLA10
EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYKIHWVRQ 121
MPGKGLEWMGYFNPNSGYSTYSPSFQGQVTISADKSIST
AYLQWSSLKASDTAMYYCARLSPGGYYVMDAWGQGT TVTVSS VH Signal
MDWTWRILFLVAAATGAHS 41 Sequence ICR62 VL
DIQMTQSPSFLSASVGDRVTINCKASQNINNYLNWYQQK 43
LGEAPKRLIYNTNNLQTGIPSRFSGSGSGTDYTLTISSLQP
EDFATYFCLQHNSFPTFGAGTKLELKRT I-KC
DIQMTQSPSSLSASVGDRVTITCRASQGINNYLNWYQQK 45
PGKAPKRLIYNTNNLQTGVPSRFSGSGSGTEFTLTISSLQP
EDFATYYCLQHNSFPTFGQGTKLEIKRT VL Signal MDMRVPAQLLGLLLLWFPGARC 47
Sequence 1-KA DIQMTQSPSSLSASVGDRVTITCRASQGINNYLNWYQQK 49
PGKAPKRLIYNTNNLQTGVPSRFSGSGSGTEYTLTISSLQ
PEDFATYYCLQHNSFPTFGQGTKLEIKRTV I-KB
DIQMTQSPSSLSASVGDRVTITCKASQNINNYLNWYQQK 51
PGKAPKRLIYNTNNLQTGVPSRFSGSGSGTEYTLTISSLQ
PEDFATYYCLQHNSFPTFGQGTKLEIKRTV
[0155] In another embodiment, the present invention is directed to
an expression vector and/or a host cell which comprise one or more
isolated polynucleotides of the present invention.
[0156] Generally, any type of cultured cell line can be used to
express the ABM of the present invention. In a preferred
embodiment, HEK293-EBNA cells, CHO cells, BHK cells, NSO cells,
SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER
cells, PER.C6 cells or hybridoma cells, other mammalian cells,
yeast cells, insect cells, or plant cells are used as the
background cell line to generate the engineered host cells of the
invention.
[0157] The therapeutic efficacy of the ABMs of the present
invention can be enhanced by producing them in a host cell that
further expresses a polynucleotide encoding a polypeptide having
GnTIII activity. In a preferred embodiment, the polypeptide having
GnTIII activity is a fusion polypeptide comprising the Golgi
localization domain of a Golgi resident polypeptide. In another
preferred embodiment, the expression of the ABMs of the present
invention in a host cell that expresses a polynucleotide encoding a
polypeptide having GnTIII activity results in ABMs with increased
Fc receptor binding affinity and increased effector function.
Accordingly, in one embodiment, the present invention is directed
to a host cell comprising (a) an isolated nucleic acid comprising a
sequence encoding a polypeptide having GnTIII activity; and (b) an
isolated polynucleotide encoding an ABM of the present invention,
such as a chimeric, primatized or humanized antibody that binds
human EGFR. In a preferred embodiment, the polypeptide having
GnTIII activity is a fusion polypeptide comprising the catalytic
domain of GnTIII and the Golgi localization domain is the
localization domain of mannosidase II. Methods for generating such
fusion polypeptides and using them to produce antibodies with
increased effector functions are disclosed in U.S. Provisional Pat.
Appl. No. 60/495,142 and U.S. Pat. Appl. Publ. No. 2004/0241817 A1,
the entire contents of each of which are expressly incorporated
herein by reference. In another preferred embodiment, the chimeric
ABM is a chimeric antibody or a fragment thereof, having the
binding specificity of the rat ICR62 antibody. In a particularly
preferred embodiment, the chimeric antibody comprises a human Fc.
In another preferred embodiment, the antibody is primatized or
humanized.
[0158] In one embodiment, one or several polynucleotides encoding
an ABM of the present invention may be expressed under the control
of a constitutive promoter or, alternately, a regulated expression
system. Suitable regulated expression systems include, but are not
limited to, a tetracycline-regulated expression system, an
ecdysone-inducible expression system, a lac-switch expression
system, a glucocorticoid-inducible expression system, a
temperature-inducible promoter system, and a metallothionein
metal-inducible expression system. If several different nucleic
acids encoding an ABM of the present invention are comprised within
the host cell system, some of them may be expressed under the
control of a constitutive promoter, while others are expressed
under the control of a regulated promoter. The maximal expression
level is considered to be the highest possible level of stable
polypeptide expression that does not have a significant adverse
effect on cell growth rate, and will be determined using routine
experimentation. Expression levels are determined by methods
generally known in the art, including Western blot analysis using
an antibody specific for the ABM or an antibody specific for a
peptide tag fused to the ABM; and Northern blot analysis. In a
further alternative, the polynucleotide may be operatively linked
to a reporter gene; the expression levels of a chimeric (e.g.,
humanized) ABM having substantially the same binding specificity of
the rat ICR62 antibody are determined by measuring a signal
correlated with the expression level of the reporter gene. The
reporter gene may be transcribed together with the nucleic acid(s)
encoding said fusion polypeptide as a single mRNA molecule; their
respective coding sequences may be linked either by an internal
ribosome entry site (IRES) or by a cap-independent translation
enhancer (CITE). The reporter gene may be translated together with
at least one nucleic acid encoding a chimeric (e.g., humanized) ABM
having substantially the same binding specificity of the rat ICR62
antibody such that a single polypeptide chain is formed. The
nucleic acids encoding the AMBs of the present invention may be
operatively linked to the reporter gene under the control of a
single promoter, such that the nucleic acid encoding the fusion
polypeptide and the reporter gene are transcribed into an RNA
molecule which is alternatively spliced into two separate messenger
RNA (mRNA) molecules; one of the resulting mRNAs is translated into
said reporter protein, and the other is translated into said fusion
polypeptide.
[0159] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing the coding
sequence of an ABM having substantially the same binding
specificity of the rat ICR62 antibody along with appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques
and in vivo recombination/genetic recombination. See, for example,
the techniques described in Maniatis et al., Molecular Cloning A
Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989) and
Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates and Wiley Interscience, N.Y (1989).
[0160] A variety of host-expression vector systems may be utilized
to express the coding sequence of the ABMs of the present
invention. Preferably, mammalian cells are used as host cell
systems transfected with recombinant plasmid DNA or cosmid DNA
expression vectors containing the coding sequence of the protein of
interest and the coding sequence of the fusion polypeptide. Most
preferably, HEK293-EBNA cells, CHO cells, BHK cells, NSO cells,
SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER
cells, PER.C6 cells or hybridoma cells, other mammalian cells,
yeast cells, insect cells, or plant cells are used as host cell
system. Some examples of expression systems and selection methods
are described in the following references, and references therein:
Borth et al., Biotechnol. Bioen. 71(4):266-73 (2000-2001), in
Werner et al., Arzneimittelforschung/Drug Res. 48(8):870-80 (1998),
in Andersen and Krummen, Curr. Op. Biotechnol. 13:117-123 (2002),
in Chadd and Chamow, Curr. Op. Biotechnol. 12:188-194 (2001), and
in Giddings, Curr. Op. Biotechnol. 12: 450-454 (2001). In alternate
embodiments, other eukaryotic host cell systems may be used,
including yeast cells transformed with recombinant yeast expression
vectors containing the coding sequence of an ABM of the present
invention, such as the expression systems taught in U.S. Pat. Appl.
No. 60/344,169 and WO 03/056914 (methods for producing human-like
glycoprotein in a non-human eukaryotic host cell) (the contents of
each of which are incorporated by reference in their entirety);
insect cell systems infected with recombinant virus expression
vectors (e.g., baculovirus) containing the coding sequence of a
chimeric ABM having substantially the same binding specificity of
the rat ICR62 antibody; plant cell systems infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing the coding sequence of the ABM of the invention,
including, but not limited to, the expression systems taught in
U.S. Pat. No. 6,815,184 (methods for expression and secretion of
biologically active polypeptides from genetically engineered
duckweed); WO 2004/057002 (production of glycosylated proteins in
bryophyte plant cells by introduction of a glycosyl transferase
gene) and WO 2004/024927 (methods of generating extracellular
heterologous non-plant protein in moss protoplast); and U.S. Pat.
Appl. Nos. 60/365,769, 60/368,047, and WO 2003/078614 (glycoprotein
processing in transgenic plants comprising a functional mammalian
GnTIII enzyme) (the contents of each of which are herein
incorporated by reference in its entirety); or animal cell systems
infected with recombinant virus expression vectors (e.g.,
adenovirus, vaccinia virus) including cell lines engineered to
contain multiple copies of the DNA encoding a chimeric ABM having
substantially the same binding specificity of the rat ICR62
antibody either stably amplified (CHO/dhfr) or unstably amplified
in double-minute chromosomes (e.g., murine cell lines). In one
embodiment, the vector comprising the polynucleotide(s) encoding
the ABM of the invention is polycistronic. Also, in one embodiment
the ABM discussed above is an antibody or a fragment thereof. In a
preferred embodiment, the ABM is a humanized antibody.
[0161] For the methods of this invention, stable expression is
generally preferred to transient expression because it typically
achieves more reproducible results and also is more amenable to
large-scale production. Rather than using expression vectors which
contain viral origins of replication, host cells can be transformed
with the respective coding nucleic acids controlled by appropriate
expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker. Following the introduction of foreign DNA,
engineered cells may be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The selectable
marker in the recombinant plasmid confers resistance to the
selection and allows selection of cells which have stably
integrated the plasmid into their chromosomes and grow to form foci
which in turn can be cloned and expanded into cell lines.
[0162] A number of selection systems may be used, including, but
not limited to, the herpes simplex virus thymidine kinase (Wigler
et al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:2026 (1962)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes,
which can be employed in tk-, hgprt- or aprt-cells, respectively.
Also, antimetabolite resistance can be used as the basis of
selection for dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:3567 (1989); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981)); and hygro, which confers resistance to hygromycin
(Santerre et al., Gene 30:147 (1984) genes. Recently, additional
selectable genes have been described, namely trpB, which allows
cells to utilize indole in place of tryptophan; hisD, which allows
cells to utilize histinol in place of histidine (Hartman &
Mulligan, Proc. Natl. Acad. Sci. USA 85:8047 (1988)); the glutamine
synthase system; and ODC (ornithine decarboxylase) which confers
resistance to the ornithine decarboxylase inhibitor,
2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, in: Current
Communications in Molecular Biology, Cold Spring Harbor Laboratory
ed. (1987)).
[0163] The present invention is further directed to a method for
modifying the glycosylation profile of the ABMs of the present
invention that are produced by a host cell, comprising expressing
in said host cell a nucleic acid encoding an ABM of the invention
and a nucleic acid encoding a polypeptide with GnTIII activity, or
a vector comprising such nucleic acids. Preferably, the modified
polypeptide is IgG or a fragment thereof comprising the Fc region.
In a particularly preferred embodiment the ABM is a humanized
antibody or a fragment thereof.
[0164] The modified ABMs produced by the host cells of the
invention exhibit increased Fc receptor binding affinity and/or
increased effector function as a result of the modification. In a
particularly preferred embodiment the ABM is a humanized antibody
or a fragment thereof containing the Fc region. Preferably, the
increased Fc receptor binding affinity is increased binding to a
Fc.gamma. activating receptor, such as the Fc.gamma.RIIIa receptor.
The increased effector function is preferably an increase in one or
more of the following: increased antibody-dependent cellular
cytotoxicity, increased antibody-dependent cellular phagocytosis
(ADCP), increased cytokine secretion, increased
immune-complex-mediated antigen uptake by antigen-presenting cells,
increased Fc-mediated cellular cytotoxicity, increased binding to
NK cells, increased binding to macrophages, increased binding to
polymorphonuclear cells (PMNs), increased binding to monocytes,
increased crosslinking of target-bound antibodies, increased direct
signaling inducing apoptosis, increased dendritic cell maturation,
and increased T cell priming.
[0165] The present invention is also directed to a method for
producing an ABM of the present invention, having modified
oligosaccharides in a host cell comprising (a) culturing a host
cell engineered to express at least one nucleic acid encoding a
polypeptide having GnTIII activity under conditions which permit
the production of an ABM according to the present invention,
wherein said polypeptide having GnTIII activity is expressed in an
amount sufficient to modify the oligosaccharides in the Fc region
of said ABM produced by said host cell; and (b) isolating said ABM.
In a preferred embodiment, the polypeptide having GnTIII activity
is a fusion polypeptide comprising the catalytic domain of GnTIII.
In a particularly preferred embodiment, the fusion polypeptide
further comprises the Golgi localization domain of a Golgi resident
polypeptide.
[0166] Preferably, the Golgi localization domain is the
localization domain of mannosidase II or GnTI. Alternatively, the
Golgi localization domain is selected from the group consisting of:
the localization domain of mannosidase I, the localization domain
of GnTII, and the localization domain of a 1-6 core
fucosyltransferase. The ABMs produced by the methods of the present
invention have increased Fc receptor binding affinity and/or
increased effector function. Preferably, the increased effector
function is one or more of the following: increased Fc-mediated
cellular cytotoxicity (including increased antibody-dependent
cellular cytotoxicity), increased antibody-dependent cellular
phagocytosis (ADCP), increased cytokine secretion, increased
immune-complex-mediated antigen uptake by antigen-presenting cells,
increased binding to NK cells, increased binding to macrophages,
increased binding to monocytes, increased binding to
polymorphonuclear cells, increased direct signaling inducing
apoptosis, increased crosslinking of target-bound antibodies,
increased dendritic cell maturation, or increased T cell priming.
The increased Fc receptor binding affinity is preferably increased
binding to Fc activating receptors such as Fc.gamma.RIIIa. In a
particularly preferred embodiment the ABM is a humanized antibody
or a fragment thereof.
[0167] In another embodiment, the present invention is directed to
a chimeric ABM having substantially the same binding specificity of
the rat ICR62 antibody produced by the methods of the invention
which has an increased proportion of bisected oligosaccharides in
the Fc region of said polypeptide. It is contemplated that such an
ABM encompasses antibodies and fragments thereof comprising the Fc
region. In a preferred embodiment, the ABM is a humanized antibody.
In one embodiment, the percentage of bisected oligosaccharides in
the Fc region of the ABM is at least 50%, more preferably, at least
60%, at least 70%, at least 80%, or at least 90%, and most
preferably at least 90-95% of the total oligosaccharides. In yet
another embodiment, the ABM produced by the methods of the
invention has an increased proportion of nonfucosylated
oligosaccharides in the Fc region as a result of the modification
of its oligosaccharides by the methods of the present invention. In
one embodiment, the percentage of nonfucosylated oligosaccharides
is at least 50%, preferably, at least 60% to 70%, most preferably
at least 75%. The nonfucosylated oligosaccharides may be of the
hybrid or complex type. In a particularly preferred embodiment, the
ABM produced by the host cells and methods of the invention has an
increased proportion of bisected, nonfucosylated oligosaccharides
in the Fc region. The bisected, nonfucosylated oligosaccharides may
be either hybrid or complex. Specifically, the methods of the
present invention may be used to produce ABMs in which at least
15%, more preferably at least 20%, more preferably at least 25%,
more preferably at least 30%, more preferably at least 35% of the
oligosaccharides in the Fc region of the ABM are bisected,
nonfucosylated. The methods of the present invention may also be
used to produce polypeptides in which at least 15%, more preferably
at least 20%, more preferably at least 25%, more preferably at
least 30%, more preferably at least 35% of the oligosaccharides in
the Fc region of the polypeptide are bisected hybrid
nonfucosylated.
[0168] In another embodiment, the present invention is directed to
a chimeric ABM having substantially the same binding specificity of
the rat ICR62 antibody engineered to have increased effector
function and/or increased Fc receptor binding affinity, produced by
the methods of the invention. Preferably, the increased effector
function is one or more of the following: increased Fc-mediated
cellular cytotoxicity (including increased antibody-dependent
cellular cytotoxicity), increased antibody-dependent cellular
phagocytosis (ADCP), increased cytokine secretion, increased
immune-complex-mediated antigen uptake by antigen-presenting cells,
increased binding to NK cells, increased binding to macrophages,
increased binding to monocytes, increased binding to
polymorphonuclear cells, increased direct signaling inducing
apoptosis, increased crosslinking of target-bound antibodies,
increased dendritic cell maturation, or increased T cell priming.
In a preferred embodiment, the increased Fc receptor binding
affinity is increased binding to a Fc activating receptor, most
preferably Fc.gamma.RIIIa. In one embodiment, the ABM is an
antibody, an antibody fragment containing the Fc region, or a
fusion protein that includes a region equivalent to the Fc region
of an immunoglobulin. In a particularly preferred embodiment, the
ABM is a humanized antibody.
[0169] The present invention is further directed to pharmaceutical
compositions comprising the ABMs of the present invention and a
pharmaceutically acceptable carrier.
[0170] The present invention is further directed to the use of such
pharmaceutical compositions in the method of treatment of cancer.
Specifically, the present invention is directed to a method for the
treatment of cancer comprising administering a therapeutically
effective amount of the pharmaceutical composition of the
invention.
[0171] The present invention further provides methods for the
generation and use of host cell systems for the production of
glycoforms of the ABMs of the present invention, having increased
Fc receptor binding affinity, preferably increased binding to Fc
activating receptors, and/or having increased effector functions,
including antibody-dependent cellular cytotoxicity. The
glycoengineering methodology that can be used with the ABMs of the
present invention has been described in greater detail in U.S. Pat.
No. 6,602,684, U.S. Pat. Appl. Publ. No. 2004/0241817 A1, U.S. Pat.
Appl. Publ. No. 2003/0175884 A1, Provisional U.S. Patent
Application No. 60/441,307 and WO 2004/065540, the entire contents
of each of which is incorporated herein by reference in its
entirety. The ABMs of the present invention can alternatively be
glycoengineered to have reduced fucose residues in the Fc region
according to the techniques disclosed in U.S. Pat. Appl. Pub. No.
2003/0157108 (Genentech), or in EP 1 176 195 A1, WO 03/084570, WO
03/085119 and U.S. Pat. Appl. Pub. Nos. 2003/0115614, 2004/093621,
2004/110282, 2004/110704, 2004/132140 (Kyowa). The contents of each
of these documents are herein incorporated by reference in their
entireties. Glycoengineered ABMs of the invention may also be
produced in expression systems that produce modified glycoproteins,
such as those taught in U.S. Pat. Appl. Pub. No. 60/344,169 and WO
03/056914 (GlycoFi, Inc.) or in WO 2004/057002 and WO 2004/024927
(Greenovation), the contents of each of which are hereby
incorporated by reference in their entirety.
Generation of Cell Lines for the Production of Proteins with
Altered Glycosylation Pattern
[0172] The present invention provides host cell expression systems
for the generation of the ABMs of the present invention having
modified glycosylation patterns. In particular, the present
invention provides host cell systems for the generation of
glycoforms of the ABMs of the present invention having an improved
therapeutic value. Therefore, the invention provides host cell
expression systems selected or engineered to express a polypeptide
having GnTIII activity. In one embodiment, the polypeptide having
GnTIII activity is a fusion polypeptide comprising the Golgi
localization domain of a heterologous Golgi resident polypeptide.
Specifically, such host cell expression systems may be engineered
to comprise a recombinant nucleic acid molecule encoding a
polypeptide having GnTIII, operatively linked to a constitutive or
regulated promoter system.
[0173] In one specific embodiment, the present invention provides a
host cell that has been engineered to express at least one nucleic
acid encoding a fusion polypeptide having GnTIII activity and
comprising the Golgi localization domain of a heterologous Golgi
resident polypeptide. In one aspect, the host cell is engineered
with a nucleic acid molecule comprising at least one gene encoding
a fusion polypeptide having GnTIII activity and comprising the
Golgi localization domain of a heterologous Golgi resident
polypeptide.
[0174] Generally, any type of cultured cell line, including the
cell lines discussed above, can be used as a background to engineer
the host cell lines of the present invention. In a preferred
embodiment, HEK293-EBNA cells, CHO cells, BHK cells, NSO cells,
SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER
cells, PER.C6 cells or hybridoma cells, other mammalian cells,
yeast cells, insect cells, or plant cells are used as the
background cell line to generate the engineered host cells of the
invention.
[0175] The invention is contemplated to encompass any engineered
host cells expressing a polypeptide having GnTIII activity,
including a fusion polypeptide that comprises the Golgi
localization domain of a heterologous Golgi resident polypeptide as
defined herein.
[0176] One or several nucleic acids encoding a polypeptide having
GnTIII activity may be expressed under the control of a
constitutive promoter or, alternately, a regulated expression
system. Such systems are well known in the art, and include the
systems discussed above. If several different nucleic acids
encoding fusion polypeptides having GnTIII activity and comprising
the Golgi localization domain of a heterologous Golgi resident
polypeptide are comprised within the host cell system, some of them
may be expressed under the control of a constitutive promoter,
while others are expressed under the control of a regulated
promoter. Expression levels of the fusion polypeptides having
GnTIII activity are determined by methods generally known in the
art, including Western blot analysis, Northern blot analysis,
reporter gene expression analysis or measurement of GnTIII
activity. Alternatively, a lectin may be employed which binds to
biosynthetic products of the GnTIII, for example, E.sub.4-PHA
lectin. Alternatively, a functional assay which measures the
increased Fc receptor binding or increased effector function
mediated by antibodies produced by the cells engineered with the
nucleic acid encoding a polypeptide with GnTIII activity may be
used.
Identification of Transfectants or Transformants that Express the
Protein Having a Modified Glycosylation Pattern
[0177] The host cells which contain the coding sequence of a
chimeric (e.g., humanized) ABM having substantially the same
binding specificity of the rat ICR62 antibody and which express the
biologically active gene products may be identified by at least
four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b)
the presence or absence of "marker" gene functions; (c) assessing
the level of transcription as measured by the expression of the
respective mRNA transcripts in the host cell; and (d) detection of
the gene product as measured by immunoassay or by its biological
activity.
[0178] In the first approach, the presence of the coding sequence
of a chimeric (e.g., humanized) ABM having substantially the same
binding specificity of the rat ICR62 antibody and the coding
sequence of the polypeptide having GnTIII activity can be detected
by DNA-DNA or DNA-RNA hybridization using probes comprising
nucleotide sequences that are homologous to the respective coding
sequences, respectively, or portions or derivatives thereof.
[0179] In the second approach, the recombinant expression
vector/host system can be identified and selected based upon the
presence or absence of certain "marker" gene functions (e.g.,
thymidine kinase activity, resistance to antibiotics, resistance to
methotrexate, transformation phenotype, occlusion body formation in
baculovirus, etc.). For example, if the coding sequence of the ABM
of the invention, or a fragment thereof, and the coding sequence of
the polypeptide having GnTIII activity are inserted within a marker
gene sequence of the vector, recombinants containing the respective
coding sequences can be identified by the absence of the marker
gene function. Alternatively, a marker gene can be placed in tandem
with the coding sequences under the control of the same or
different promoter used to control the expression of the coding
sequences. Expression of the marker in response to induction or
selection indicates expression of the coding sequence of the ABM of
the invention and the coding sequence of the polypeptide having
GnTIII activity.
[0180] In the third approach, transcriptional activity for the
coding region of the ABM of the invention, or a fragment thereof,
and the coding sequence of the polypeptide having GnTIII activity
can be assessed by hybridization assays. For example, RNA can be
isolated and analyzed by Northern blot using a probe homologous to
the coding sequences of the ABM of the invention, or a fragment
thereof, and the coding sequence of the polypeptide having GnTIII
activity or particular portions thereof. Alternatively, total
nucleic acids of the host cell may be extracted and assayed for
hybridization to such probes.
[0181] In the fourth approach, the expression of the protein
products can be assessed immunologically, for example by Western
blots, immunoassays such as radioimmuno-precipitation,
enzyme-linked immunoassays and the like. The ultimate test of the
success of the expression system, however, involves the detection
of the biologically active gene products.
Generation and Use of ABMs Having Increased Effector Function
Including Antibody-Dependent Cellular Cytotoxicity
[0182] In preferred embodiments, the present invention provides
glycoforms of chimeric (e.g., humanized) ABMs having substantially
the same binding specificity of the rat ICR62 antibody and having
increased effector function including antibody-dependent cellular
cytotoxicity. Glycosylation engineering of antibodies has been
previously described. See e.g., U.S. Pat. No. 6,602,684,
incorporated herein by reference in its entirety.
[0183] Clinical trials of unconjugated monoclonal antibodies (mAbs)
for the treatment of some types of cancer have recently yielded
encouraging results. Dillman, Cancer Biother. & Radiopharm.
12:223-25 (1997); Deo et al., Immunology Today 18:127 (1997). A
chimeric, unconjugated IgG1 has been approved for low-grade or
follicular B-cell non-Hodgkin's lymphoma. Dillman, Cancer Biother.
& Radiopharm. 12:223-25 (1997), while another unconjugated mAb,
a humanized IgG1 targeting solid breast tumors, has also been
showing promising results in phase III clinical trials. Deo et al.,
Immunology Today 18:127 (1997). The antigens of these two mAbs are
highly expressed in their respective tumor cells and the antibodies
mediate potent tumor destruction by effector cells in vitro and in
vivo. In contrast, many other unconjugated mAbs with fine tumor
specificities cannot trigger effector functions of sufficient
potency to be clinically useful. Frost et al., Cancer 80:317-33
(1997); Surfus et al., J. Immunother. 19:184-91 (1996). For some of
these weaker mAbs, adjunct cytokine therapy is currently being
tested. Addition of cytokines can stimulate antibody-dependent
cellular cytotoxicity (ADCC) by increasing the activity and number
of circulating lymphocytes. Frost et al., Cancer 80:317-33 (1997);
Surfus et al., J. Immunother. 19:184-91 (1996). ADCC, a lytic
attack on antibody-targeted cells, is triggered upon binding of
leukocyte receptors to the constant region (Fc) of antibodies. Deo
et al., Immunology Today 18:127 (1997).
[0184] A different, but complementary, approach to increase ADCC
activity of unconjugated IgGls is to engineer the Fc region of the
antibody. Protein engineering studies have shown that Fc.gamma.Rs
interact with the lower hinge region of the IgG CH2 domain. Lund et
al., J. Immunol. 157:4963-69 (1996). However, Fc.gamma.R binding
also requires the presence of oligosaccharides covalently attached
at the conserved Asn 297 in the CH2 region. Lund et al., J.
Immunol. 157:4963-69 (1996); Wright and Morrison, Trends Biotech.
15:26-31 (1997), suggesting that either oligosaccharide and
polypeptide both directly contribute to the interaction site or
that the oligosaccharide is required to maintain an active CH2
polypeptide conformation. Modification of the oligosaccharide
structure can therefore be explored as a means to increase the
affinity of the interaction.
[0185] An IgG molecule carries two N-linked oligosaccharides in its
Fc region, one on each heavy chain. As any glycoprotein, an
antibody is produced as a population of glycoforms which share the
same polypeptide backbone but have different oligosaccharides
attached to the glycosylation sites. The oligosaccharides normally
found in the Fc region of serum IgG are of complex bi-antennary
type (Wormald et al., Biochemistry 36:130-38 (1997), with a low
level of terminal sialic acid and bisecting N-acetylglucosamine
(GlcNAc), and a variable degree of terminal galactosylation and
core fucosylation. Some studies suggest that the minimal
carbohydrate structure required for Fc.gamma.R binding lies within
the oligosaccharide core. Lund et al., J. Immunol. 157:4963-69
(1996).
[0186] The mouse- or hamster-derived cell lines used in industry
and academia for production of unconjugated therapeutic mAbs
normally attach the required oligosaccharide determinants to Fc
sites. IgGs expressed in these cell lines lack, however, the
bisecting GlcNAc found in low amounts in serum IgGs. Lifely et al.,
Glycobiology 318:813-22 (1995). In contrast, it was recently
observed that a rat myeloma-produced, humanized IgG1 (CAMPATH-1H)
carried a bisecting GlcNAc in some of its glycoforms. Lifely et
al., Glycobiology 318:813-22 (1995). The rat cell-derived antibody
reached a similar maximal in vitro ADCC activity as CAMPATH-1H
antibodies produced in standard cell lines, but at significantly
lower antibody concentrations.
[0187] The CAMPATH antigen is normally present at high levels on
lymphoma cells, and this chimeric mAb has high ADCC activity in the
absence of a bisecting GlcNAc. Lifely et al., Glycobiology
318:813-22 (1995). In the N-linked glycosylation pathway, a
bisecting GlcNAc is added by GnTIII. Schachter, Biochem. Cell Biol.
64:163-81 (1986).
[0188] Previous studies used a single antibody-producing CHO cell
line, that was previously engineered to express, in an
externally-regulated fashion, different levels of a cloned GnT III
gene enzyme (Umana, P., et al., Nature Biotechnol. 17:176-180
(1999)). This approach established for the first time a rigorous
correlation between expression of GnTIII and the ADCC activity of
the modified antibody. Thus, the invention contemplates a
recombinant, chimeric antibody or a fragment thereof with the
binding specificity of the rat ICR62 antibody, having altered
glycosylation resulting from increased GnTIII activity. The
increased GnTIII activity results in an increase in the percentage
of bisected oligosaccharides, as well as a decrease in the
percentage of fucose residues, in the Fc region of the ABM. This
antibody, or fragment thereof, has increased Fc receptor binding
affinity and increased effector function. In addition, the
invention is directed to antibody fragment and fusion proteins
comprising a region that is equivalent to the Fc region of
immunoglobulins. In a preferred embodiment, the antibody is
humanized.
Therapeutic Applications of ABMs Produced According to the Methods
of the Invention.
[0189] In the broadest sense, the ABMs of the present invention can
be used target cells in vivo or in vitro that express EGFR. The
cells expressing EGFR can be targetted for diagnostic or
therapeutic purposes. In one aspect, the ABMs of the present
invention can be used to detect the presence of EGFR in a sample.
In another aspect, the ABMs of the present invention can be used to
inhibit or reduce EGFR-mediated signal transduction in cells
expressing EGFR on the surface. EGFR is abnormally expressed (e.g.,
overexpressed) in many human tumors compared to non-tumor tissue of
the same cell type. Thus, the ABMs of the invention are
particularly useful in the prevention of tumor formation,
eradication of tumors and inhibition of tumor growth. By blocking
the binding of EGFR ligands to EGFR, the ABMs of the invention
inhibit EGF-dependent tumor cell activation, including EGFR
tyrosine phosphorylation, increased extracellular acidification
rate, and cell proliferation. The ABMs of the invention also act to
arrest the cell cycle, cause apoptosis of the target cells (e.g.,
tumor cells), and inhibit angiogenesis and/or differentiation of
target cells. The ABMs of the invention can be used to treat any
tumor expressing EGFR. Particular malignancies that can be treated
with the ABMs of the invention include, but are not limited to,
epidermal and squamous cell carcinomas, non-small cell lung
carcinomas, gliomas, pancreatic cancer, ovarian cancer, prostate
cancer, breast cancer, bladder cancer, head and neck cancer, and
renal cell carcinomas.
[0190] The ABMs of the present can be used alone to target and kill
tumor cells in vivo. The ABMs can also be used in conjunction with
an appropriate therapeutic agent to treat human carcinoma. For
example, the ABMs can be used in combination with standard or
conventional treatment methods such as chemotherapy, radiation
therapy or can be conjugated or linked to a therapeutic drug, or
toxin, as well as to a lymphokine or a tumor-inhibitory growth
factor, for delivery of the therapeutic agent to the site of the
carcinoma. The conjugates of the ABMs of this invention that are of
prime importance are (1) immunotoxins (conjugates of the ABM and a
cytotoxic moiety) and (2) labeled (e.g. radiolabeled,
enzyme-labeled, or fluorochrome-labeled) ABMs in which the label
provides a means for identifying immune complexes that include the
labeled ABM. The ABMs can also be used to induce lysis through the
natural complement process, and to interact with antibody dependent
cytotoxic cells normally present.
[0191] The cytotoxic moiety of the immunotoxin may be a cytotoxic
drug or an enzymatically active toxin of bacterial or plant origin,
or an enzymatically active fragment ("A chain") of such a toxin.
Enzymatically active toxins and fragments thereof used are
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, Phytolacca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, and enomycin. In another embodiment, the
ABMs are conjugated to small molecule anticancer drugs. Conjugates
of the ABM and such cytotoxic moieties are made using a variety of
bifunctional protein coupling agents. Examples of such reagents are
SPDP, IT, bifunctional derivatives of imidoesters such a 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
tolylene 2,6-diisocyanate, and bis-active fluorine compounds such
as 1,5-difluoro-2,4-dinitrobenzene. The lysing portion of a toxin
may be joined to the Fab fragment of the ABMs. Additional
appropriate toxins are known in the art, as evidenced in e.g.,
published U.S. Patent Application No. 2002/0128448, incorporated
herein by reference in its entirety.
[0192] In one embodiment, a chimeric (e.g., humanized),
glycoengineered ABM having substantially the same binding
specificity of the rat ICR62 antibody, is conjugated to ricin A
chain. Most advantageously, the ricin A chain is deglycosylated and
produced through recombinant means. An advantageous method of
making the ricin immunotoxin is described in Vitetta et al.,
Science 238, 1098 (1987), hereby incorporated by reference.
[0193] When used to kill human cancer cells in vitro for diagnostic
purposes, the conjugates will typically be added to the cell
culture medium at a concentration of at least about 10 nM. The
formulation and mode of administration for in vitro use are not
critical. Aqueous formulations that are compatible with the culture
or perfusion medium will normally be used. Cytotoxicity may be read
by conventional techniques to determine the presence or degree of
cancer.
[0194] As discussed above, a cytotoxic radiopharmaceutical for
treating cancer may be made by conjugating a radioactive isotope
(e.g., I, Y, Pr) to a chimeric, glycoengineered ABM having
substantially the same binding specificity of the rat ICR62
antibody. The term "cytotoxic moiety" as used herein is intended to
include such isotopes.
[0195] In another embodiment, liposomes are filled with a cytotoxic
drug and the liposomes are coated with the ABMs of the present
invention. Because there are many EGFR molecules on the surface of
the EGFR-expressing malignant cell, this method permits delivery of
large amounts of drug to the correct cell type.
[0196] Techniques for conjugating such therapeutic agents to
antibodies are well known (see, e.g., Amon et al., "Monoclonal
Antibodies for Immunotargeting of Drugs in Cancer Therapy", in
Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and
Thorpe et al., "The Preparation And Cytotoxic Properties Of
Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982)).
[0197] Still other therapeutic applications for the ABMs of the
invention include conjugation or linkage, e.g., by recombinant DNA
techniques, to an enzyme capable of converting a prodrug into a
cytotoxic drug and the use of that antibody-enzyme conjugate in
combination with the prodrug to convert the prodrug to a cytotoxic
agent at the tumor site (see, e.g., Senter et al., "Anti-Tumor
Effects of Antibody-alkaline Phosphatase", Proc. Natl. Acad. Sci.
USA 85:4842-46 (1988); "Enhancement of the in vitro and in vivo
Antitumor Activites of Phosphorylated Mitocycin C and Etoposide
Derivatives by Monoclonal Antibody-Alkaline Phosphatase
Conjugates", Cancer Research 49:5789-5792 (1989); and Senter,
"Activation of Prodrugs by Antibody-Enzyme Conjugates: A New
Approach to Cancer Therapy," FASEB J. 4:188-193 (1990)).
[0198] Still another therapeutic use for the ABMs of the invention
involves use, either unconjugated, in the presence of complement,
or as part of an antibody-drug or antibody-toxin conjugate, to
remove tumor cells from the bone marrow of cancer patients.
According to this approach, autologous bone marrow may be purged ex
vivo by treatment with the antibody and the marrow infused back
into the patient [see, e.g., Ramsay et al., "Bone Marrow Purging
Using Monoclonal Antibodies", J. Clin. Immunol., 8(2):81-88
(1988)].
[0199] Furthermore, it is contemplated that the invention comprises
a single-chain immunotoxin comprising antigen binding domains that
allow substantially the same specificity of binding as the rat
ICR62 antibody (e.g., polypeptides comprising the CDRs of the rat
ICR62 antibody) and further comprising a toxin polypeptide. The
single-chain immunotoxins of the invention may be used to treat
human carcinoma in vivo.
[0200] Similarly, a fusion protein comprising at least the
antigen-binding region of an ABM of the invention joined to at
least a functionally active portion of a second protein having
anti-tumor activity, e.g., a lymphokine or oncostatin, can be used
to treat human carcinoma in vivo.
[0201] The present invention provides a method for selectively
killing tumor cells expressing EGFR. This method comprises reacting
the immunoconjugate (e.g., the immunotoxin) of the invention with
said tumor cells. These tumor cells may be from a human
carcinoma.
[0202] Additionally, this invention provides a method of treating
carcinomas (for example, human carcinomas) in vivo. This method
comprises administering to a subject a pharmaceutically effective
amount of a composition containing at least one of the
immunoconjugates (e.g., the immunotoxin) of the invention.
[0203] In a further aspect, the invention is directed to an
improved method for treating cell proliferation disorders wherein
EGFR is expressed, particularly wherein EGFR is abnormally
expressed (e.g. overexpressed), including cancers of the bladder,
brain, head and neck, pancreas, lung, breast, ovary, colon,
prostate, skin, and kidney, comprising administering a
therapeutically effective amount of an ABM of the present invention
to a human subject in need thereof. In a preferred embodiment, the
ABM is a glycoengineered anti-EGFR antibody with a binding
specificity substantially the same as that of the rat ICR62
antibody. In another preferred embodiment the antibody is
humanized. Examples of cell proliferation disorders that can be
treated by an ABM of the present invention include, but are not
limited to neoplasms located in the: abdomen, bone, breast,
digestive system, liver, pancreas, peritoneum, endocrine glands
(adrenal, parathyroid, pituitary, testicles, ovary, thymus,
thyroid), eye, head and neck, nervous system (central and
peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,
thoracic region, and urogenital system.
[0204] Similarly, other cell proliferation disorders can also be
treated by the ABMs of the present invention. Examples of such cell
proliferation disorders include, but are not limited to:
hypergammaglobulinemia, lymphoproliferative disorders,
paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,
Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis,
and any other cell proliferation disease, besides neoplasia,
located in an organ system listed above.
[0205] In accordance with the practice of this invention, the
subject may be a human, equine, porcine, bovine, murine, canine,
feline, and avian subjects. Other warm blooded animals are also
included in this invention.
[0206] The subject invention further provides methods for
inhibiting the growth of human tumor cells, treating a tumor in a
subject, and treating a proliferative type disease in a subject.
These methods comprise administering to the subject an effective
amount of the composition of the invention.
[0207] The invention is further directed to methods for treating
non-malignant diseases or disorders in a mammal characterized by
abnormal activation or production of EGFR or one or more EGFR
ligands, comprising administering to the mammal a therapeutically
effective amount of the ABMs of the invention. The subject will
generally have EGFR-expressing cells, for instance in diseased
tissue thereof, such that the ABMs of the invention are able to
bind to cells within the subject.
[0208] Abnormal activation or expression of EGFR or an EGFR ligand
may be occurring in cells of the subject, e.g. in diseased tissue
of the subject. Abnormal activation of EGFR may be attributable to
amplification, overexpression or aberrant production of the EGFR
and/or EGFR ligand. In one embodiment of the invention, a
diagnostic or prognostic assay will be performed to determine
whether abnormal production or activation of EGFR (or EGFR ligand)
is occurring the subject. For example, gene amplification and/or
overexpression of EGFR and/or ligand may be determined. Various
assays for determining such amplification/overexpression are
available in the art and include the IHC, FISH and shed antigen
assays described above. Alternatively, or additionally, levels of
an EGFR ligand, such as TGF-.alpha. in or associated with the
sample may be determined according to known procedures. Such assays
may detect protein and/or nucleic acid encoding it in the sample to
be tested. In one embodiment, EGFR ligand levels in a sample may be
determined using immunohistochemistry (IHC); see, for example,
Scher et al. Clin. Cancer Research 1:545-550 (1995). Alternatively,
or additionally, one may evaluate levels of EGFR-encoding nucleic
acid in the sample to be tested; e.g. via FISH, southern blotting,
or PCR techniques.
[0209] Moreover, EGFR or EGFR ligand overexpression or
amplification may be evaluated using an in vivo diagnostic assay,
e.g. by administering a molecule (such as an antibody) which binds
the molecule to be detected and is tagged with a detectable label
(e.g. a radioactive isotope) and externally scanning the patient
for localization of the label.
[0210] Alternatively, one may detect EGFR heterodimers, especially
EGFR-ErbB2, EGFR-ErbB3 or EGFR-ErbB4 heterodimers, in the patient,
e.g. in diseased tissue thereof, prior to therapy. Various methods
to detect noncovalent protein-protein interactions or otherwise
indicate proximity between proteins of interest are available.
Exemplary methods for detecting EGFR heterodimers include, without
limitation, immunoaffinity-based methods, such as
immunoprecipitation; fluorescence resonance energy transfer (FRET)
(Selvin, Nat. Struct. Biol. 7:730-34 (2000); Gadella & Jovin,
J. Cell Biol. 129:1543-58 (1995); and Nagy et al., Cytometry
32:120-131 (1998)); co-localization of EGFR with either ErbB2 or
ErbB3 using standard direct or indirect immunofluorescence
techniques and confocal laser scanning microscopy; laser scanning
imaging (LSI) to detect antibody binding and co-localization of
EGFR with either ErbB2 or ErbB3 in a high-throughput format, such
as a microwell plate (Zuck et al, Proc. Natl. Acad. Sci. USA
96:11122-11127 (1999)); or eTag/m assay system (Aclara Bio
Sciences, Mountain View, Calif.; and U.S. Patent Application
2001/0049105, published Dec. 6, 2001).
[0211] It is apparent, therefore, that the present invention
encompasses pharmaceutical compositions, combinations and methods
for treating human malignancies such as cancers of the bladder,
brain, head and neck, pancreas, lung, breast, ovary, colon,
prostate, skin, and kidney. For example, the invention includes
pharmaceutical compositions for use in the treatment of human
malignancies comprising a pharmaceutically effective amount of an
antibody of the present invention and a pharmaceutically acceptable
carrier.
[0212] The ABM compositions of the invention can be administered
using conventional modes of administration including, but not
limited to, intravenous, intraperitoneal, oral, intralymphatic or
administration directly into the tumor. Intravenous administration
is preferred.
[0213] In one aspect of the invention, therapeutic formulations
containing the ABMs of the invention are prepared for storage by
mixing an antibody having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (REMINGTON'S PHARMACEUTICAL SCIENCES, 16.sup.th
edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions. Acceptable carriers, excipients,
or stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid and methionine; preservatives (such as octadecyldimethylbenzyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0214] The ABMs of the present invention may be administered to a
subject to treat a disease or disorder characterized by abnormal
EGFR or EGFR ligand activity, such as a tumor, either alone or in
combination therapy with, for example, a chemotherapeutic agent
and/or radiation therapy. Exemplary anti-EGFR antibody formulations
are described in Herbst and Shen, Cancer 94(5):1593-1611. Suitable
chemotherapeutic agents include cisplatin, doxorubicin, topotecan,
paclitaxel, vinblastine, carboplatin, and etoposide.
[0215] Lyophilized formulations adapted for subcutaneous
administration are described in WO97/04801. Such lyophihized
formulations may be reconstituted with a suitable diluent to a high
protein concentration and the reconstituted formulation may be
administered subcutaneously to the mammal to be treated herein.
[0216] 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. For example, it may be desirable to
further provide a cytotoxic agent, chemotherapeutic agent, cytokine
or immunosuppressive agent (e.g. one which acts on T cells, such as
cyclosporin or an antibody that binds T cells, e.g., one which
binds LFA-1). The effective amount of such other agents depends on
the amount of antagonist present in the formulation, the type of
disease or 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.
[0217] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0218] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antagonist,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. 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 yethyl-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.
[0219] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0220] The compositions of the invention may be in a variety of
dosage forms which include, but are not limited to, liquid
solutions or suspension, tablets, pills, powders, suppositories,
polymeric microcapsules or microvesicles, liposomes, and injectable
or infusible solutions. The preferred form depends upon the mode of
administration and the therapeutic application.
[0221] The compositions of the invention also preferably include
conventional pharmaceutically acceptable carriers and adjuvants
known in the art such as human serum albumin, ion exchangers,
alumina, lecithin, buffer substances such as phosphates, glycine,
sorbic acid, potassium sorbate, and salts or electrolytes such as
protamine sulfate.
[0222] The most effective mode of administration and dosage regimen
for the pharmaceutical compositions of this invention depends upon
the severity and course of the disease, the patient's health and
response to treatment and the judgment of the treating physician.
Accordingly, the dosages of the compositions should be titrated to
the individual patient. Nevertheless, an effective dose of the
compositions of this invention will generally be in the range of
from about 0.01 to about 2000 mg/kg. In one embodiment, the
effective dose is in the range of from about 1.0 mg/kg to about
15.0 mg/kg. In a more specific embodiment, the dose is in the range
of from about 1.5 mg/kg to about 12 mg/kg. In other embodiments,
the dose is in the range of from about 1.5 mg/kg to about 4.5
mg/kg, or from about 4.5 mg/kg to about 12 mg/kg. The dose of the
present invention may also be any dose within these ranges,
including, but not limited to, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5
mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5
mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5
mg/kg, 9.0 mg/kg, 9.5 mg/kg, 10.0 mg/kg, 10.5 mg/kg, 11.0 mg/kg,
11.5 mg/kg, 12.0 mg/kg, 12.5 mg/kg, 13.0 mg/kg, 13.5 mg/kg, 14.0
mg/kg, 14.5 mg/kg, or 15.0 mg/kg.
[0223] The molecules described herein may be in a variety of dosage
forms which include, but are not limited to, liquid solutions or
suspensions, tablets, pills, powders, suppositories, polymeric
microcapsules or microvesicles, liposomes, and injectable or
infusible solutions. The preferred form depends upon the mode of
administration and the therapeutic application.
[0224] The dosages of the present invention may, in some cases, be
determined by the use of predictive biomarkers. Predictive
biomarkers are molecular markers that are used to determine (i.e.,
observe and/or quanitate) a pattern of expression and/or activation
of tumor related genes or proteins, or cellular components of a
tumor related signalling pathway. Elucidating the biological
effects of targeted-therapies in tumor tissue and correlating these
effects with clinical response helps identify the predominant
growth and survival pathways operative in tumors, thereby
establishing a profile of likely responders and conversely
providing a rational for designing strategies to overcoming
resistance. For example, biomarkers for anti-EGFR therapy may
comprise molecules that are in the EGFR downstream signalling
pathway that leads to a cell proliferation disorder including, but
not limited to, Akt, RAS, RAF, MAPK, ERK1, ERK2, PKC, STAT3, STAT5
(Mitchell, Nature Biotech. 22: 363-364 (2004); Becker, Nature
Biotech 22:15-18 (2004); Tsao and Herbst, Signal 4:4-9 (2003)).
Biomarkers for anti-EGFR therapy may also comprise growth factor
receptors such as EGFR, ErbB-2 (HER2/neu), and ErbB-3 (HER3), and
may be positive or negative predictors of patient response to
anti-EGFR therapy. For example, the growth factor receptor ErbB-3
(HER3) was determined to be a negative predictive biomarker for the
anti-EGFR antibody ABX-EGF (U.S. Pat. Appl. Pub. No. 2004/0132097
A1).
[0225] Predictive biomarkers may be measured by cellular assays
that are well known in the art including, but not limited to
immunohistochemistry, flow cytometry, immunofluorescence,
capture-and-detection assays, and reversed phase assays, and/or
assays set forth in U.S. Pat. Appl. Pub. No. 2004/0132097 A1, the
entire contents of which are herein incorporated by reference.
Predictive biomarkers of anti-EGFR therapy, themselves, can be
identified according to the techniques set forth in U.S. Pat. Appl.
Pub. No. 2003/0190689A1, the entire contents of which are hereby
incorporated by reference.
[0226] In one aspect, the present invention provides for a method
for treating an EGFR-related disorder comprising predicting a
response to anti-EGFR therapy in a human subject in need of
treatment by assaying a sample from the human subject prior to
therapy with one or a plurality of reagents that detect expression
and/or activiation of predictive biomarkers for an EGFR-related
disorder such as cancer; determining a pattern of expression
and/opr activation of one or more of the predictive biomarkers,
wherein the pattern predicts the human subject's response to the
anti-EGFR therapy; and administering to a human subject who is
predicted to respond positively to anti-EGFR treatment a
therapeutically effective amount of a composition comprising an ABM
of the present invention. As used herein, a human subject who is
predicted to respond positively to anti-EGFR treatment is one for
whom anti-EGFR will have a measurable effect on the EGFR-related
disorder (e.g., tumor regression/shrinkage) and for whom the
benefits of anti-EGFR therapy are not outweighed by adverse effects
(e.g., toxicity). As used herein, a sample means any biological
sample from an organism, particularly a human, comprising one or
more cells, including single cells of any origin, tissue or biopsy
samples which has been removed from organs such as breast, lung,
gastrointestinal tract, skin, cervix, ovary, prostate, kidney,
brain, head and neck,or any other other organ or tissue of the
body, and other body samples including, but not limited to, smears,
sputum, secretions, cerebrospinal fluid, bile, blood, lymph fluid,
urine and feces.
[0227] The composition comprising an ABM of the present invention
will be formulated, dosed, and administered in a fashion consistent
with good medical practice. Factors for consideration in this
context include the particular disease or disorder being treated,
the particular mammal being treated, the clinic condition of the
individual patient, the cause of the disease or 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
antagonist to be administered will be governed by such
considerations.
[0228] As a general proposition, the therapeutically effective
amount of the antibody administered parenterally per dose will be
in the range of about 0.1 to 20 mg/kg of patient body weight per
day, with the typical initial range of antagonist used being in the
range of about 2 to 10 mg/kg. In one embodiment, the
therapeutically effective amount is in the range of from about 1.0
mg/kg to about 15.0 mg/kg. In a more specific embodiment, the dose
is in the range of from about 1.5 mg/kg to about 12 mg/kg. In other
embodiments, the dose is in the range of from about 1.5 mg/kg to
about 4.5 mg/kg, or from about 4.5 mg/kg to about 12 mg/kg. The
dose of the present invention may also be any dose within these
ranges, including, but not limited to, 1.0 mg/kg, 1.5 mg/kg, 2.0
mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0
mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0
mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, 10.0 mg/kg, 10.5 mg/kg,
11.0 mg/kg, 11.5 mg/kg, 12.0 mg/kg, 12.5 mg/kg, 13.0 mg/kg, 13.5
mg/kg, 14.0 mg/kg, 14.5 mg/kg, or 15.0 mg/kg.
[0229] In a preferred embodiment, the ABM is an antibody,
preferably a humanized antibody. Suitable dosages for such an
unconjugated antibody are, for example, in the range from about 20
mg/m.sup.2 to about 1000 mg/m.sup.2. For example, one may
administer to the patient one or more doses of substantially less
than 375 mg/m.sup.2 of the antibody, e.g., where the dose is in the
range from about 20 mg/m.sup.2 to about 250 mg/m.sup.2, for example
from about 50 mg/m.sup.2 to about 200 mg/m.sup.2.
[0230] Moreover, one may administer one or more initial dose(s) of
the antibody followed by one or more subsequent dose(s), wherein
the mg/m.sup.2 dose of the antibody in the subsequent dose(s)
exceeds the mg/m.sup.2 dose of the antibody in the initial dose(s).
For example, the initial dose may be in the range from about 20
mg/m.sup.2 to about 250 mg/m.sup.2 (e.g., from about 50 mg/m.sup.2
to about 200 mg/m.sup.2) and the subsequent dose may be in the
range from about 250 mg/m.sup.2 to about 1000 mg/m.sup.2.
[0231] As noted above, however, these suggested amounts of ABM are
subject to a great deal of therapeutic discretion. The key factor
in selecting an appropriate dose and scheduling is the result
obtained, as indicated above. For example, relatively higher doses
may be needed initially for the treatment of ongoing and acute
diseases. To obtain the most efficacious results, depending on the
disease or disorder, the antagonist is administered as close to the
first sign, diagnosis, appearance, or occurrence of the disease or
disorder as possible or during remissions of the disease or
disorder.
[0232] In the case of anti-EGFR antibodies used to treat tumors,
optimum therapeutic results have generally been achieved with a
dose that is sufficient to completely saturate the EGF receptors on
the target cells. The dose necessary to achieve saturation will
depend on the number of EGF receptors expressed per tumor cell
(which can vary significantly between different tumor types). Serum
concentrations as low as 30 nM have been effective in treating some
tumors, while concentrations above 100 nM may be necessary to
achieve optimum therapeutic effect with other tumors. The dose
necessary to achieve saturation for a given tumor can be readily
determined in vitro by radioimmunoassay or immunoprecipiation.
[0233] In general, for combination therapy with radiation, one
suitable therapeutic regimen involves eight weekly infusions of an
anti-EGFR ABM of the invention at a loading dose of 100-500
mg/m.sup.2 followed by maintenance doses at 100-250 mg/m.sup.2 and
radiation in the amount of 70.0 Gy at a dose of 2.0 Gy daily. For
combination therapy with chemotherapy, one suitable therapeutic
regimen involves administering an anti-EGFR ABM of the invention as
loading/maintenance doses weekly of 100/100 mg/m.sup.2, 400/250
mg/m.sup.2, or 500/250 mg/m.sup.2 in combination with cisplatin at
a dose of 100 mg/m.sup.2 every three weeks. Alternatively,
gemcitabine or irinotecan can be used in place of cisplatin.
[0234] The ABM of the present invention is administered by any
suitable means, including parenteral, subcutaneous,
intraperitoneal, intrapulinonary, and intranasal, and, if desired
for local immunosuppressive treatment, intralesional
administration. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous
administration. In addition, the antagonist may suitably be
administered by pulse infusion, e.g., with declining doses of the
antagonist. Preferably the dosing is given by injections, most
preferably intravenous or subcutaneous injections, depending in
part on whether the administration is brief or chronic.
[0235] One may administer other compounds, such as cytotoxic
agents, chemotherapeutic agents, immunosuppressive agents and/or
cytokines with the antagonists herein. The combined administration
includes coadministration, using separate formulations or a single
pharmaceutical formulation, and consecutive administration in
either order, wherein preferably there is a time period while both
(or all) active agents simultaneously exert their biological
activities.
[0236] It would be clear that the dose of the composition of the
invention required to achieve cures may be further reduced with
schedule optimization.
[0237] In accordance with the practice of the invention, the
pharmaceutical carrier may be a lipid carrier. The lipid carrier
may be a phospholipid. Further, the lipid carrier may be a fatty
acid. Also, the lipid carrier may be a detergent. As used herein, a
detergent is any substance that alters the surface tension of a
liquid, generally lowering it.
[0238] In one example of the invention, the detergent may be a
nonionic detergent. Examples of nonionic detergents include, but
are not limited to, polysorbate 80 (also known as Tween 80 or
(polyoxyethylenesorbitan monooleate), Brij, and Triton (for example
Triton WR-1339 and Triton A-20).
[0239] Alternatively, the detergent may be an ionic detergent. An
example of an ionic detergent includes, but is not limited to,
alkyltrimethylammonium bromide.
[0240] Additionally, in accordance with the invention, the lipid
carrier may be a liposome. As used in this application, a
"liposome" is any membrane bound vesicle which contains any
molecules of the invention or combinations thereof.
[0241] In yet another embodiment, the invention relates to an ABM
according to the present invention for use as a medicament, in
particular for use in the treatment or prophylaxis of cancer or for
use in a precancerous condition or lesion. The cancer may be, for
example, lung cancer, non small cell lung (NSCL) cancer,
bronchioalviolar cell lung cancer, bone cancer, pancreatic cancer,
skin cancer, cancer of the head or neck, cutaneous or intraocular
melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of
the anal region, stomach cancer, gastric cancer, colon cancer,
breast cancer, uterine cancer, carcinoma of the fallopian tubes,
carcinoma of the endometrium, carcinoma of the cervix, carcinoma of
the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of
the esophagus, cancer of the small intestine, cancer of the
endocrine system, cancer of the thyroid gland, cancer of the
parathyroid gland, cancer of the adrenal gland, sarcoma of soft
tissue, cancer of the urethra, cancer of the penis, prostate
cancer, cancer of the bladder, cancer of the kidney or ureter,
renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma,
hepatocellular cancer, biliary cancer, chronic or acute leukemia,
lymphocytic lymphomas, neoplasms of the central nervous system
(CNS), spinal axis tumors, brain stem glioma, glioblastoma
multiforme, astrocytomas, schwannomas, ependymomas,
medulloblastomas, meningiomas, squamous cell carcinomas, pituitary
adenomas, including refractory versions of any of the above
cancers, or a combination of one or more of the above cancers. The
precancerous condition or lesion includes, for example, the group
consisting of oral leukoplakia, actinic keratosis (solar
keratosis), precancerous polyps of the colon or rectum, gastric
epithelial dysplasia, adenomatous dysplasia, hereditary
nonpolyposis colon cancer syndrome (HNPCC), Barrett's esophagus,
bladder dysplasia, and precancerous cervical conditions.
[0242] Preferably, said cancer is selected from the group
consisting of breast cancer, bladder cancer, head & neck
cancer, skin cancer, pancreatic cancer, lung cancer, ovarian
cancer, colon cancer, prostate cancer, kidney cancer, and brain
cancer.
[0243] Yet another embodiment is the use of the ABM according to
the present invention for the manufacture of a medicament for the
treatment or prophylaxis of cancer. Cancer is as defined above.
[0244] Preferably, said cancer is selected from the group
consisting of breast cancer, bladder cancer, head & neck
cancer, skin cancer, pancreatic cancer, lung cancer, ovarian
cancer, colon cancer, prostate cancer, kidney cancer, and brain
cancer.
[0245] Also preferably, said antigen binding molecule is used in a
therapeutically effective amount from about 1.0 mg/kg to about 15
mg/kg.
[0246] Also more preferably, said antigen binding molecule is used
in a therapeutically effective amount from about 1.5 mg/kg to about
12 mg/kg.
[0247] Also more preferably, said antigen binding molecule is used
in a therapeutically effective amount from about 1.5 mg/kg to about
4.5 mg/kg.
[0248] Also more preferably, said antigen binding molecule is used
in a therapeutically effective amount from about 4.5 mg/kg to about
12 mg/kg.
[0249] Most preferably, said antigen binding molecule is used in a
therapeutically effective amount of about 1.5 mg/kg.
[0250] Also most preferably, said antigen binding molecule is used
in a therapeutically effective amount of about 4.5 mg/kg.
[0251] Also most preferably, said antigen binding molecule is used
in a therapeutically effective amount of about 12 mg/kg.
[0252] In another embodiment, the present invention is directed to
method of treating an EGFR-related disorder in a mammal in need of
treatment thereof comprising administering to the mammal an ABM of
of the present invention, wherein the treatment results in serum
concentrations of said ABM between about 1 and about 500 .mu.g/ml,
for a period of at least 4 weeks, and wherein the treatment does
not cause a clinically significant level of toxicity in said
mammal. In other embodiments, the serum concentration is an amount
selected from the group consisting of above 1 .mu.g/ml, above 25
.mu.g/ml, above 50 .mu.g/ml, above 100 .mu.g/ml, above 200
.mu.g/ml, above 300 .mu.g/ml, above 400 .mu.g/ml, above 500
.mu.g/ml, between about 1 and about 100 .mu.g/ml, between about 1
and about 200 .mu.g/ml, between about 1 and about 300 .mu.g/ml,
between about 1 and about 400 .mu.g/ml, and between about 1 and
about 500 .mu.g/ml. In a preferred embodiment, the mammal is a
human. In one embodiment the ABM is an antibody. In a preferred
embodiment, the antibody is glycoengineered and has increased
FcgammaRIII binding compared to a non-glycoengineered form of the
antibody.
Articles of Manufacture
[0253] In another embodiment of the invention, an article of
manufacture containing materials useful for the treatment of the
disorders described above is provided. 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 an anti-EGFR antibody. The label
or package insert indicates that the composition is used for
treating the condition of choice, such as a non-malignant disease
or disorder, where the disease or disorder involves abnormal
activation or production of an EGFR receptor and/or a EGFR-ligand,
for example a benign hyperproliferative disease or disorder.
Moreover, the article of manufacture may comprise (a) a first
container with a composition contained therein, wherein the
composition comprises a first antibody which binds EGFR and
inhibits growth of cells which overexpress EGFR; and (b) a second
container with a composition contained therein, wherein the
composition comprises a second antibody which binds EGFR and blocks
ligand activation of an EGFR receptor. The article of manufacture
in this embodiment of the invention may further comprises a package
insert indicating that the first and second antibody compositions
can be used to treat a non-malignant disease or disorder from the
list of such diseases or disorders in the definition section above.
Moreover, the package insert may instruct the user of the
composition (comprising an antibody which binds EGFR and blocks
ligand activation of an EGFR receptor) to combine therapy with the
antibody and any of the adjunct therapies described in the
preceding section (e.g. a chemotherapeutic agent, an EGFR-targeted
drug, an anti-angiogenic agent, an immunosuppressive agent,
tyrosine kinase inhibitor, an anti-hormonal compound, a
cardioprotectant and/or a cytokine). Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) 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.
[0254] The examples below explain the invention in more detail. The
following preparations and examples are given to enable those
skilled in the art to more clearly understand and to practice the
present invention. The present invention, however, is not limited
in scope by the exemplified embodiments, which are intended as
illustrations of single aspects of the invention only, and methods
which are functionally equivalent are within the scope of the
invention. Indeed, various modifications of the invention in
addition to those described herein will become apparent to those
skilled in the art from the foregoing description and accompanying
drawings. Such modifications are intended to fall within the scope
of the appended claims.
[0255] All patents, applications, and publications cited in this
application are hereby incorporated by reference in their
entirety.
EXAMPLES
[0256] Unless otherwise specified, references to the numbering of
specific amino acid residue positions in the following Examples are
according to the Kabat numbering system. Unless otherwise noted,
the materials and methods used to make the antibodies described in
these working examples are in accordance with those set forth in
the examples of U.S. patent application Ser. No. 10/981,738, which
is herein incorporated by reference in its entirety.
Example 1
Materials and Methods
[0257] High Homology Acceptor Approach
[0258] The high homology antibody acceptor framework search was
performed by aligning the rat ICR62 protein sequence to a
collection of human germ-line sequences and choosing that human
sequence that showed the highest sequence identity, while
conserving all canonical residues on a functional level. Here, the
sequence 1-e from the VH1 family within the VBase database was
chosen as the heavy chain framework acceptor sequence, and the A30
sequence from the VK1 family of the VBase database was chosen to be
the framework acceptor for the light chain. On these two acceptor
frameworks the three complementarity determining regions (CDRs)
and/or specificity-determining residues of those CDRs of the rat
ICR62 heavy and light variable domains were grafted. Since the
framework 4 region is not part of the variable region of the germ
line gene, the alignment for that position was performed
individually. The JH6 region was chosen for the heavy chain, and
the JK2 region was chosen for the light chain. Molecular modelling
of the designed immunoglobulin domain revealed one position outside
of the Kabat CDR1 potentially requiring the murine amino acid
residues instead of the human ones outside of the CDR.
Reintroduction of murine amino acid residues into the human
framework would generate so-called "back mutations." For example,
the human acceptor amino acid residue at Kabat position 27 (Glycine
in 1-e) was back mutated to a tyrosine residue. To show the
importance of the residues for antigen binding, humanized antibody
variants were designed that either included or omitted the back
mutations. The humanized antibody light chain did not require any
back mutations. After designing the protein sequences, DNA
sequences encoding these proteins were synthesized as detailed
below.
[0259] Mixed Framework Approach
[0260] To avoid the need for introducing back mutations at critical
positions (critical to retain good antigen binding affinity or
antibody functions) of the human acceptor framework, it was
investigated whether framework region 1 (FR1), framework regions 1
(FR1) and 2 (FR2) together, or framework region 3 (FR3) of a
functionally humanized antibody could be replaced by human antibody
sequences already having donor residues, or amino acid residues
that are functionally equivalent to donor residues, at important
residue positions in the natural human germline sequence. For this
purpose, the VH frameworks 1, 2 and 3 of the rat ICR62 VH sequence
were aligned individually to human germ-line sequences. Here,
highest sequence identity was not used for choosing acceptor
frameworks; instead matching of several critical residues was
performed. Those critical residues comprise the so-called canonical
residues, and also those residues at position 27, 28, and 30 (Kabat
numbering), which lie outside of the CDR1 definition by Kabat, but
often are involved in antigen binding. In addition, critical
residues are those that show important interaction with the CDRs,
as can be determined using molecular modelling. The IMGT sequence
IGHV1-58 (Accession No. M29809), IGHV5-51 (Accession No. M99686),
as well as the VBase sequence 1-02 from the VH1 family were chosen
as suitable ones for replacing either FR1, 2, or 3. In brief:
IGHV1-58 showed a promising pattern in the Kabat positions 27 to
30, but does not fulfill the criteria for the canonical position
71. The IGHV5-51 has a matching residue 71, so its FR3 could be
used. Also its FR1 is close to the desired FR1 sequence.
[0261] The 1-e of VH1 fulfilled all criteria apart from position
27. Sequence 1-02 was considered acceptable for the FR1 and FR2
regions, but would require a back mutation in FR3.
[0262] After designing the protein sequences, DNA sequences
encoding these proteins were synthesized as detailed below. Using
this approach back mutations were not necessary in most of the
constructs of the heavy chain, in order to retain good levels of
antigen binding. The chronology and the reasoning of the mixed
framework constructs is explained in the results section.
[0263] Synthesis of the Antibody Genes
[0264] After having designed the amino acid sequence of the
humanized antibody V region, the DNA sequence was generated. The
DNA sequence data of the individual frame work regions was found in
the databases (e.g. IMGT or VBase) for human germ line sequences.
The DNA sequence information of the CDR regions was taken from the
published sequence of the rat ICR62 antibody (see, e.g., PCT
Publication WO 95/20045). With these sequences, the whole DNA
sequence was virtually assembled. Having this DNA sequence data,
diagnostic restriction sites were introduced in the virtual
sequence by introducing silent mutations, creating recognition
sites for restriction endonucleases. To obtain the physical DNA
chain, gene synthesis was performed (see, e.g., Wheeler et al.
1995). In this method, oligonucleotides are designed from the genes
of interest, such, that a series of oligonucleotides is derived
from the coding strand, and one other series is from the non-coding
strand. The 3' and 5' ends of each oligonucleotide (except the very
first and last in the row) always show complementary sequences to
two primers derived from the opposite strand. When putting these
oligonucleotides into a reaction buffer suitable for any heat
stable polymerase, and adding Mg.sup.2+, dNTPs and a DNA
polymerase, each oligonucleotide is extended from its 3' end. The
newly formed 3' end of one primer then anneals with the next primer
of the opposite strand, and extending its sequence further under
conditions suitable for template dependant DNA chain elongation.
The final product was cloned into a conventional vector for
propagation in E. coli.
[0265] Antibody Production
[0266] For construction of the chimeric (i.e., fully rat V region
and human C region) and humanized anti-EGFR light and heavy chain
chain expression vectors, human heavy and light chain leader
sequences (for secretion) were added upstream of the variable
region DNA sequences. Downstream of the variable regions, the
constant regions of IgG1 for the heavy chain were added, and the
kappa constant region for the light chain using standard molecular
biology techniques. The resulting full antibody heavy and light
chain DNA sequences were subcloned into mammalian expression
vectors (one for the light chain and one for the heavy chain) under
the control of the MPSV promoter and upstream of a synthetic polyA
site, each vector carrying an EBV OriP sequence.
[0267] Antibodies were produced by co-transfecting HEK293-EBNA
cells with the mammalian antibody heavy and light chain expression
vectors using a calcium phosphate-transfection approach.
Exponentially growing HEK293-EBNA cells were transfected by the
calcium phosphate method. For the production of unmodified
antibody, the cells were transfected only with antibody heavy and
light chain expression vectors in a 1:1 ratio. For the production
of the glycoengineered antibody, the cells were co-transfected with
four plasmids, two for antibody expression, one for a fusion GnTIII
polypeptide expression, and one for mannosidase II expression at a
ratio of 4:4:1:1, respectively. Cells were grown as adherent
monolayer cultures in T flasks using DMEM culture medium
supplemented with 10% FCS, and were transfected when they were
between 50 and 80% confluent. For the transfection of a T75 flask,
8 million cells were seeded 24 hours before transfection in 14 ml
DMEM culture medium supplemented with FCS (at 10% V/V final), 250
.mu.g/ml neomycin, and cells were placed at 37.degree. C. in an
incubator with a 5% CO2 atmosphere overnight. For each T75 flask to
be transfected, a solution of DNA, CaCl2 and water was prepared by
mixing 47 .mu.g total plasmid vector DNA divided equally between
the light and heavy chain expression vectors, 235 .mu.l of a 1M
CaCl2 solution, and adding water to a final volume of
469.quadrature. .mu.l. To this solution, 469 .mu.l of a 50 mM
HEPES, 280 mM NaCl, 1.5 mM Na2HPO4 solution at pH 7.05 were added,
mixed immediately for 10 sec and left to stand at room temperature
for 20 sec. The suspension was diluted with 12 ml of DMEM
supplemented with 2% FCS, and added to the T75 in place of the
existing medium. The cells were incubated at 37.degree. C., 5% CO2
for about 17 to 20 hours, then medium was replaced with 12 ml DMEM,
10% FCS. The conditioned culture medium was harvested 5 to 7 days
post-transfection centrifuged for 5 min at 1200 rpm, followed by a
second centrifugation for 10 min at 4000 rpm and kept at 4.degree.
C.
[0268] The secreted antibodies were purified by Protein A affinity
chromatography, followed by cation exchange chromatography and a
final size exclusion chromatographic step on a Superdex 200 column
(Amersham Pharmacia) exchanging the buffer to phosphate buffer
saline and collecting the pure monomeric IgG1 antibodies. Antibody
concentration was estimated using a spectrophotometer from the
absorbance at 280 nm. The antibodies were formulated in a 25 mM
potassium phosphate, 125 mM sodium chloride, 100 mM glycine
solution of pH 6.7.
[0269] Glycoengineered variants of the humanized antibody were
produced by co-transfection of the antibody expression vectors
together with a GnT-III glycosyltransferase expression vector, or
together with a GnT-III expression vector plus a Golgi mannosidase
II expression vector. Glycoengineered antibodies were purified and
formulated as described above for the non-glycoengineered
antibodies. The oligosaccharides attached to the Fc region of the
antibodies were analysed by MALDI/TOF-MS as described below.
Oligosaccharide Analysis
[0270] Oligosaccharides were enzymatically released from the
antibodies by PNGaseF digestion, with the antibodies being either
immobilized on a PVDF membrane or in solution.
[0271] The resulting digest solution containing the released
oligosaccharides either prepared directly for MALDI/TOF-MS analysis
or was further digested with EndoH glycosidase prior to sample
preparation for MALDI/TOF-MS analysis.
Oligosaccharide Release Method for PVDF Membrane-Immobilized
Antibodies
[0272] The wells of a 96-well plate made with a PVDF (Immobilon P,
Millipore, Bedford, Mass.) membrane were wetted with 100 .mu.l
methanol and the liquid was drawn through the PVDF membrane using
vacuum applied to the Multiscreen vacuum manifold (Millipore,
Bedford, Mass.). The PVDF membranes were washed three times with
300 .mu.l of water. The wells were then washed with 50 .mu.l RCM
buffer (8M Urea, 360 mM Tris, 3.2 mM EDTA, pH 8.6). Between 30-40
.mu.g antibody was loaded in a well containing 10 .mu.l RCM buffer.
The liquid in the well was drawn through the membrane by applying
vacuum, and the membrane was subsequently washed twice with 50
.mu.l RCM buffer. The reduction of disulfide bridges was performed
by addition of 50 .mu.l of 0.1M dithiothreitol in RCM and
incubation at 37.degree. C. for 1 h.
[0273] Following reduction, a vacuum was applied to remove the
dithiothreitol solution from the well. The wells were washed three
times with 300 .mu.l water before performing the carboxymethylation
of the cysteine residues by addition of 50 .mu.l 0.1M iodoacetic
acid in RCM buffer and incubation at room temperature in the dark
for 30 min.
[0274] After carboxymethylation, the wells were drawn with vacuum
and subsequently washed three times with 300 .mu.l water. The PVDF
membrane was then blocked, to prevent adsorption of the
endoglycosidase, by incubating 100 .mu.l of a 1% aqueous solution
of polyvinylpyrrolidone 360 at room temperature for 1 hour. The
blocking reagent was then removed by gentle vacuum followed by
three washes with 300 .mu.l water.
[0275] N-linked oligosaccharides were released by addition of 2.5
mU peptide-N-glycosydase F (recombinat N-Glycanase, GLYKO, Novato,
Calif.) and 0.1 mU Sialidase (GLYKO, Novato, Calif.), to remove any
potential charged monosaccharide residues, in a final volume of 25
.mu.l in 20 mM NaHCO.sub.3, pH7.0). Digestion was performed for 3
hours at 37.degree. C.
Oligosaccharide Release Method for Antibodies in Solution
[0276] Between 40 and 50 .mu.g of antibody were mixed with 2.5 mU
of PNGaseF (Glyko, U.S.A.) in 2 mM Tris, pH7.0 in a final volume of
25 microliters, and the mix was incubated for 3 hours at 37.degree.
C.
Use of Endoglycosidase H Digestion of PNGaseF-Released
Oligosaccharides for the Assignment of Hybrid Bisected
Oligosaccharide Structures to MALDI/TOF-MS Neutral Oligosaccharide
Peaks
[0277] The PNGaseF released oligosaccharides were subsequently
digested with Endoglycosidase H (EC 3.2.1.96). For the EndoH
digestion, 15 mU of EndoH (Roche, Switzerland) were added to the
PNGaseF digest (antibody in solution method above) to give a final
volume of 30 microliters, and the mix was incubated for 3 hours at
37.degree. C. EndoH cleaves between the N-acetylglucosamine
residues of the chitobiose core of N-linked oligosaccharides. The
enzyme can only digest oligomannose and most hybrid type glycans,
whereas complex type oligosaccharides are not hydrolyzed.
Sample Preparation for MALDI/TOF-MS
[0278] The enzymatic digests containing the released
oligosaccharides were incubated for a further 3 h at room after the
addition of acetic acid to a final concentration of 150 mM, and
were subsequently passed through 0.6 ml of cation exchange resin
(AG50W-X8 resin, hydrogen form, 100-200 mesh, BioRad, Switzerland)
packed into a micro-bio-spin chromatography column (BioRad,
Switzerland) to remove cations and proteins. One microliter of the
resulting sample was applied to a stainless steel target plate, and
mixed on the plate with 1 .mu.l of sDHB matrix. sDHB matrix was
prepared by dissolving 2 mg of 2,5-dihydroxybenzoic acid plus 0.1
mg of 5-methoxysalicylic acid in 1 ml of ethanol/10 mM aqueous
sodium chloride 1:1 (v/v). The samples were air dried, 0.2 .mu.l
ethanol was applied, and the samples were finally allowed to
re-crystallize under air.
MALDI/TOF-MS
[0279] The MALDI-TOF mass spectrometer used to acquire the mass
spectra was a Voyager Elite (Perspective Biosystems). The
instrument was operated in the linear configuration, with an
acceleration of 20 kV and 80 ns delay. External calibration using
oligosaccharide standards was used for mass assignment of the ions.
The spectra from 200 laser shots were summed to obtain the final
spectrum.
[0280] Antigen Binding Assay
[0281] The purified, monomeric humanized antibody variants were
tested for binding to human epidermal growth factor receptor (EGFR,
also referred to in the literature as HER-1 or ErbB1) on the A431
human epidermal cell line, using a flow cytometry-based assay.
200,000 cells (e.g., from human A431 cell line) in 180 .mu.l FACS
buffer (PBS containing 2% FCS and 5 mM EDTA) were transferred to 5
ml polystyrene tubes and 20 .mu.l 10 fold concentrated anti-EGFR
antibody (primary antibody) samples (1-5000 ng/ml final
concentration) or PBS only were added. After gently mixing the
samples, the tubes were incubated at 4.degree. C. for 30 min in the
dark. Subsequently, samples were washed twice with FACS buffer and
pelleted at 300.times.g for 3 min. Supernatant was aspirated off
and cells were taken up in 50 .mu.l FACS buffer and 2 .mu.l
secondary antibody (anti-Fc-specific F(ab')2-FITC fragments
(Jackson Immuno Research Laboratories, USA)) was added and the
tubes were incubated at 4.degree. C. for 30 min. Samples were
washed twice with FACS buffer and taken up in 500 .mu.l of FACS
buffer for analysis by Flow Cytometry. Binding was determined by
plotting the geometric mean fluorescence against the antibody
concentrations.
Binding of Monomeric IgG1 Glycovariants to
Fc.gamma.RIIIA-Expressing CHO Cell Line
[0282] CHO cells were transfected by electroporation (280 V, 950
.mu.f, 0.4 cm) with an expression vector coding for the
FcgammaRIIIA-Val158 .alpha.-chain and the .gamma.-chain.
Transfectants were selected by addition of 6 .mu.g/ml puromycin and
stable clones were analyzed by FACS using 10 .mu.l
FITC-conjugated-anti-FcgammaRIII 3G8 monoclonal antibody (BD
Biosciences, Allschwil/Switzerland) for 106 cells. Binding of IgG1
to FcgammaRIIIA-Val158-expressing CHO cells was performed. Briefly,
the anti-FcgammaRIIIA 3G8 F(ab')2 fragments (Ancell, Bayport,
Minn./USA) were added at a concentration of 10 .mu.g/ml to compete
binding of antibody glycovariants (3 .mu.g/ml). The fluorescence
intensity referring to the bound antibody variants was determined
on a FACSCalibur (BD Biosciences, Allschwil/Switzerland).
[0283] ADCC Assay
[0284] Human peripheral blood mononuclear cells (PBMC) were used as
effector cells and were prepared using Histopaque-1077 (Sigma
Diagnostics Inc., St. Louis, M063178 USA) following essentially the
manufacturer's instructions. In brief, venous blood was taken with
heparinized syringes from healthy volunteers. The blood was diluted
1:0.75-1.3 with PBS (not containing Ca.sup.++ or Mg.sup.+) and
layered on Histopaque-1077. The gradient was centrifuged at
400.times.g for 30 min at room temperature (RT) without breaks. The
interphase containing the PBMC was collected and washed with PBS
(50 ml per cells from two gradients) and harvested by
centrifugation at 300.times.g for 10 minutes at RT. After
resuspension of the pellet with PBS, the PBMC were counted and
washed a second time by centrifugation at 200.times.g for 10
minutes at RT. The cells were then resuspended in the appropriate
medium for the subsequent procedures.
[0285] The effector to target ratio used for the ADCC assays was
25:1 and 10:1 for PBMC and NK cells, respectively. The effector
cells were prepared in AIM-V medium at the appropriate
concentration in order to add 50 .mu.l per well of round bottom 96
well plates. Target cells were human EGFR expressing cells (e.g.,
A431, EBC-1, or LN229) grown in DMEM containing 10% FCS. Target
cells were washed in PBS, counted and resuspended in AIM-V at 0.3
million per ml in order to add 30,000 cells in 100 .mu.l per
microwell. Antibodies were diluted in AIM-V, added in 50 .mu.l to
the pre-plated target cells and allowed to bind to the targets for
10 minutes at RT. Then the effector cells were added and the plate
was incubated for 4 hours at 37.degree. C. in a humidified
atmosphere containing 5% CO2. Killing of target cells was assessed
by measurement of lactate dehydrogenase (LDH) release from damaged
cells using the Cytotoxicity Detection kit (Roche Diagnostics,
Rotkreuz, Switzerland). After the 4-hour incubation the plates were
centrifuged at 800.times.g. 100 .mu.l supernatant from each well
was transferred to a new transparent flat bottom 96 well plate. 100
.mu.l color substrate buffer from the kit were added per well. The
Vmax values of the color reaction were determined in an ELISA
reader at 490 nm for at least 10 min using SOFTmax PRO software
(Molecular Devices, Sunnyvale, Calif.94089, USA). Spontaneous LDH
release was measured from wells containing only target and effector
cells but no antibodies. Maximal release was determined from wells
containing only target cells and 1% Triton X-100. Percentage of
specific antibody-mediated killing was calculated as follows:
((x-SR)/(MR-SR)*100, where x is the mean of Vmax at a specific
antibody concentration, SR is the mean of Vmax of the spontaneous
release and MR is the mean of Vmax of the maximal release.
Example 2
Results and Discussion
[0286] Comparison of the binding to human EGF-receptor of antibody
variants I-HHA, I-HHB, I-HHC, I-HLA, I-HLB, I-HLC, I-HLA1, I-HLA2,
I-HLA3, I-HLA4, I-HLA5, I-HLA6, I-HLA7, I-HLA8, I-HLA-9, I-HHD,
I-HHE, I-HHF, and I-HHG, either complexed with the chimeric ICR62
light chain or with the humanized ICR62 light chains (I-KA, I-KB,
or I-KC) and the parental, chimeric antibody ch-ICR62 shows that
all antibodies have within one log unit similar EC50 values. Only
the I-HHA has strongly diminished binding activity (see FIG. 2).
FIG. 1 shows the functional activity of the individual chimeric
ICR62 (ch-ICR62) polypeptide chains when combined with the
humanized constructs I-HHC and I-KB, respectively. In this
experiment, eiher the light chain, the heavy chain or both chains
simultaneously of the ch-ICR62 were replaced by the above mentioned
humanized constructs. This shows that the VH/VL interface formation
seems to work as well in the rodent antibody as well as in the
humanized constructs.
[0287] As shown in FIG. 2, the humanized heavy chain I-HHA could
not restore binding activity with either the I-KA, or the I-KB
light chain. Since the I-HLA did show binding with both the I-KA,
and the I-KB, the present inventors concluded that the heavy chain
of I-HHA is not functional in antigen binding. FIGS. 1 and 2,
combined with FIG. 3, show that the light chain constructs I-KA,
I-KB, and I-KC show binding behavior indistinguishable from the
rodent counterpart. Variant I-KC does not possess any back
mutations, and additionally has its CDR1 partially humanized, such
that residues 24-29 can be derived from the human acceptor sequence
(A30 of VK1, as mentioned before).
[0288] In the series I-HHA, I-HHB, and I-HHC, only the latter two
variants showed satisfactory binding behavior (FIGS. 2 and 3).
Sequence analysis of the I-HHA revealed three potential amino acid
residues responsible for this behavior: Lys33, His35, and Tyr50.
Constructs that have Lys33 replaced by tyrosine showed good
binding, as well as constructs having the Tyr50 replaced by
tryptophane. Only when these two residues were replaced by alanine
and glycine, respectively, was the binding lost. Since I-HHC did
not show better binding than I-HHB, the present inventors concluded
that residues Asn60, Glu61, Lys64, and Ser65 need not be of rodent
origin; or they can be replaced by Ala, Gln, Gln, and Gly,
respectively. This procedure leads to a construct in which the CDR2
is more humanized, since amino acid positions 60 to 65 are part of
the Kabat CDR definition, but there is no need to graft the rodent
donor residues for this antibody.
[0289] FIGS. 4 and 5 compare the constructs of the series I-HLA1,
I-HLA2, I-HLA3, I-HLA4, I-HLA5, and I-HLA6. The best binding
behavior was observed in the constructs ch-ICR62, I-HLA1, and
IHLA2, with an EC50 value of approx. 300 ng/ml. The other
constructs had this value increased by a factor of two, and
therefore have slightly reduced binding activity. The first
conclusion from this data is that, within the Kabat CDR1, the
Lys33Tyr, and the Ile34Met substitutions were tolerated. These two
positions are located within the Kabat definition of CDR1, but
outside of the Chothia CDR boundaries (that were based on
structural rather than sequence analysis). In the latter part of
CDR1, then, at least some promiscuity is permitted.
[0290] The second conclusion is that, within CDR2, in addition to
the above-mentioned replacement of residues Asn60 and Glu61 by
non-donor residues, Asn60Ser, Glu61Pro, and Lys62Ser non-donor
substitutions within the Kabat CDR were also allowed. These
residues were derived from the human germ-line IGHV5-51 acceptor
sequence, which was used as an FR3 acceptor sequence. Constructs
I-HLA3 and I-HLA4 differ from I-HLA1 and I-HLA2 only by the removal
of the Phe27Tyr back mutation, and both the I-HLA3 and I-HLA4
constructs lose affinity compared to their parental construct.
Therefore, the third conclusion of the comparison of I-HLA1,
I-HLA2, I-HLA3, I-HLA4, I-HLA5, and I-HLA6 is the involvement of
Phe27 in antigen binding, either directly or indirectly, via
modifying the loop conformation of CDR1.
[0291] Variants I-HLA5 and I-HLA6 have the FR1 of I-HLA1 and
I-HLA2, respectively, replaced by another germ-line acceptor
sequence with the Phe27 naturally present (i.e., IGHV1-58). This
could only be achieved by simultaneously introducing several other
mutations which are: Val2Met, Ala9Pro, and Ala16Thr. By doing so,
the beneficial effect of (re-)introducing the Phe27 was again
abrogated.
[0292] The I-HLA7 construct was assessed to determine whether the
restoration of additional donor residues in the heavy chain CDR1
and CDR2 of the I-HLA6 construct would restore full binding
activity as compared to ICR62. As shown in FIG. 6, this was not the
case.
[0293] As shown in FIGS. 7 and 8, two additional constructs, I-HLA8
and I-HLA9, were tested to determine if the full binding activity
compared to ch-ICR62 could be achieved. Starting from the I-HHB
construct, the FR1 regions were replaced by FR1 regions having
maximal homology within the Chothia CDR1 region. The I-HLA8
construct has the FR1 of the IGHV1-58 sequence, and the I-HLA9 has
the IGHV5-51 FR1 region. Both constructs bound the antigen at least
as well as the ch-ICR62 antibody. The I-HLA8 construct may, in
fact, be even better, with the EC50 lowered by a factor of 2. The
I-HLA8 construct has the same FR1 sequence as the I-HLA5 and I-HLA6
constructs and therefore has the same non-donor residues (i.e.,
Val2Met, Ala9Pro, and Ala16Thr), suggesting that the presence of
these non-donor residues does not have a negative effect on
binding. Non-beneficial mutations occurring in the I-HLA5 and 6
arise from the combination of a VH1 FR1 paired with a VH5 FR3,
which could potentially be compensated for by having a FR1 and a
FR3 of the same VH family.
[0294] Shown in FIG. 8 are constructs that contain non-donor
residues within the CDRs. Thus, these constructs are even further
humanized within the CDRs because the non-donor residues occur in
the human framework regions that were chosen for these constructs.
The I-HHE (His35Ser), I-HHF (Thr58Asn) and I-HHG (Ser57Ala)
constructs all have one residue within the CDR1 or CDR2 that is
humanized (compared to the I-HHB construct). Construct I-HHD
(Gly24A1a) was also assayed. I-HHF showed reduced binding
indicating the importance of Thr58. In contrast to the Kabat CDR
residue 58, amino acid 57 is more tolerant to substitutions, since
the Ser57Ala mutation apparently has no influence on binding (FIG.
8).
[0295] Since the FR3 region of IGHV5-51 seemed to show promising
properties in the I-HLA1 and 2 constructs, and the FR1 of the same
germ-line sequence proved to be useful in the I-HLA9 construct, the
FR1, FR2, and FR3 of IGHV5-51 was designed to be used together as
an acceptor for loop grafting.
Summary of the analysis of the canonical residues in humanized
ICR62 constructs:
[0296] VL: Kabat position 2: Ile probably required. [0297] Kabat
position 71: Ile or Phe allowed.
[0298] VH: Pos. 24, Gly, Thr, Ala, Val, Ser allowed. [0299] Pos.
26, Gly allowed. [0300] Pos. 29, Phe, Ile, Leu, Val, Ser allowed.
[0301] Pos. 34, Ile, Met, Leu, Val, Trp, Tyr, Thr allowed. [0302]
Pos. 71, Ala, Leu, Val, Thr allowed. [0303] Pos 94, Arg, Gly, Asn,
Lys, Ser, His, Thr, Ala allowed.
Results of the ADCC Experiments
[0304] FIG. 9 shows a comparison of the antibody mediated cellular
cyotoxicity (ADCC) is shown for the various glycoforms of the
chimeric ICR62 antibody, as well as for the humanized variant
I-HLA4. The different glycoforms are marked by a label that either
indicates not-glycoengineered (WT), Glycoform 1 (G1), or Glycoform
2 (G2). "G1" refers to glcyoengineering of the antibody by
co-expression with GnTIII. "G2" refers to glycoengineering of the
antibody by co-expression with GnTIII and ManII. "WT" refers to
antibodies that were not glycoengineered. The light chain for all
the humanized constructs is the I-KC variant, and was not labeled
explicitly.
[0305] The chimeric, as well as the humanized antibody were
improved in their potency and efficacy by the two different
glycoengineering approaches. The ch-ICR62 construct performed
slightly better than I-HLA4 for the wild-type or the glycoforms,
respective. As seen in FIG. 4, when comparing the affinities of the
two antibodies towards their antigen, the ch-ICR62 had a twofold
lower EC50 value. This difference in affinity is here reflected in
differences in efficacy.
[0306] FIG. 10 shows a comparison of the antibody mediated cellular
cyotoxicity (ADCC) for the non-glycoengineered ("wild-type") and
the G2 glycoform of the humanized ICR62 antibody constructs I-HHB
and I-HLA7. The same antibodies were applied to two different
target cell lines. In panel A of FIG. 10, the target cell line
LN229 is used; and in panel B of FIG. 10, the cell line A431 was
used. The A431 cells are apparently more susceptible towards
antibody mediated cell killing than the LN229 cells. More
importantly, the glycoengineering enhanced the potency of both
antibodies. This effect seemed to be more pronounced for the I-HLA7
than for the I-HHB. The percentage of cell killing at maximal
antibody concentration for the I-HHB could be shifted from
.about.30% to -40% by introducing the G2 glycoengineered variant,
when using the LN229 target cell line. When using the A431 cell
line, this value was apparently unchanged. This behavior was
completely different for the I-HLA7 antibody. Target cell killing
at maximal antibody concentration was shifted from about 10% to
about 50%, for the LN229 cells, and from about 40% to about 70% for
the A431 cells by introducing the G2 glycoengineering variants. In
this case, despite having lower activity in the non glycoengineered
antibody for the I-HLA7 relative to I-HHB, the ranking of activity
is reversed for the glycoengineered antibodies. FIGS. 11 and 12
also show comparisons of non-glycoengineered forms (WT) and G2
glcyoforms of chimeric ICR62 and the humanized ICR62 antibody
constructs I-HHB and I-HLA7.
Example 3
Preliminary Toxicity Study by Intravenous (Bolus) Administration to
Cynomolgus Monkeys--Bioanalytical Analysis
Introduction
[0307] Glyco-Engineered Anti-EGFR Assay
[0308] This Bioanalytical Analysis describes the measurement of
anti-EGFR in samples originating from cynomolgus monkeys following
intravenous (bolus) administration of anti-EGFR (recombinant,
glycoengineered anti-EGFR antibody produced from transfected
mammalian cells in culture with antibody expression vectors
harboring the heavy chain I-HHB and the light chain I-KC genes as
described above, and purified as described above) as described in
the protocol set forth herein below. A total of 78 monkey serum
samples were stored frozen at about -20.degree. C. until use.
[0309] The Bioanalytical methods used for the determination of
anti-EGFR used an ELISA method to measure serum concentrations of
anti-EGFR. Acceptance criteria were set at .+-.20% (.+-.25% low QC)
for precision and inaccuracy.
Materials and Methods
[0310] Objective:
[0311] The objective of this study was the assessment of systemic
toxic potential of Glyco-mAb (anti-EGFR) intravenous (bolus)
administration to Cynomolgus Monkeys followed by an 8-week recovery
period.
TABLE-US-00009 TABLE 8 Animal model Cynomolgus Monkeys, accepted by
regulatory agencies, background data available. Justification for
use The primate was the non-rodent species of of the primate choice
because it alone conserves two critical parameters: EGFR antigen
recognition by the test antibody, and test antibody Fc region
recognition by immune system Fc receptors. Route Intravenous
(Bolus), to simulate the conditions of clinical administration.
TABLE-US-00010 TABLE 9 Treatment groups and dosages Group 1 2 3
Compound Glyco-mAb(Anti-EGFR) Dosage (mg/kg/day) 1.5 4.5 12
Rationale for Dosage Level Selection
[0312] 1.5-7.5 mg/kg is the expected range for human studies (7.5
mg/kg being the corresponding dose for a similar compound in
humans).
TABLE-US-00011 TABLE 10 Identity of treatment groups Dosage Number
of Animal ID (mg/kg/day) animals Numbers Group Treatment # Male
Female Male Female 1 Glyco-mAb 1.5 1 1 623 590 (Anti-EGFR) 2
Glyco-mAb 4.5 1 1 461 462 (Anti-EGFR) 3 Glyco-mAb 12 1 1 463 612
(Anti-EGFR) # Expressed in terms of the test substance as
supplied.
TABLE-US-00012 TABLE 11 Animals Species Cynomolgus monkey (purpose
bred). Age received Approximately 15 months. Weight range ordered
1.5 to 2.5 kg.
TABLE-US-00013 TABLE 12 Administration of Anti-EGFR Route
Intravenous injection. Treated at Constant dosages in
mg/kg/occasion. Volume dosage Calculated in advance, based on the
most recently recorded bodyweight. Individual dose 1 ml/kg/day
volume Frequency Days 1, 8, 15 and 22, immediately before feeding.
Sequence By group. Dose sites Using left saphenous veins. Injection
Bolus, new sterile disposable needle per animal. Formulation A
record of the usage of formulation was maintained based on weights.
This balance is compared with the expected usage as a check of
correct administration.
TABLE-US-00014 TABLE 13 Clinical observations Animals and their
cage trays Visually inspected at least twice daily for evidence of
reaction to treatment or ill-health. Deviations from normal Nature
and severity. recorded at the time in Date and time of onset.
respect of Duration and progress of the observed condition.
Physical examination Once each week for all animals. Daily records
of cage trays For vomitus, blood, diarrhoea, etc.
TABLE-US-00015 TABLE 14 Dosing Frequency: Frequency 1. Immediately
pre-dose. 2. 1/2 to 2 hours after completion of dosing. 3. As late
as possible in the working day. Injection sites Daily.
TABLE-US-00016 TABLE 15 Toxicokinetics Day Animals Sample times
hours after dosing. 1 All animals 1, 4, 12, 24, 72, 120. 8 All
animals Predose, 1 hour post-dose (169 hours post Day 1 dose). 15
All animals Pre-dose, 1 hour post-dose (337 hours post Day 1 dose).
22 All animals Pre-dose, 1 hour post-dose (505 hours post Day 1
dose). 29 All animals 672 hours post Day 1 dose.
TABLE-US-00017 TABLE 16 Samples Sample site Suitable vein.
Anticoagulant/Sample No anticoagulant/0.7 ml. volume Total number
of samples 104. taken Separation of serum By centrifugation at
ambient temperature unless otherwise indicated to provide a minimum
of 0.3 ml, where possible. Storage of serum Appropriately labeled
plastic tubes. Deep frozen (approximately -20.degree. C.), while
awaiting bioanalysis.
Histology
TABLE-US-00018 [0313] TABLE 17 Tissue Fixation Standard 10% Neutral
Buffered Formalin. Others Testes and epididymides: Initially in
Bonin's fluid. Eyes: Initially in Davidson's fluid.
TABLE-US-00019 TABLE 18 Histology Processing All animals. Routine
staining 4-5 .mu.m sections stained with haematoxylin and
eosin.
Immunoassay Procedure
[0314] A plate was coated with 100 .mu.l per well of coating
solution (5 .mu.l sheep anti-human IgG (monkey adsorbed IgG, the
Binding Site, UK) added to 11495 .mu.l bicarbonate buffer (0.05M,
pH9.6) and incubated for approximately 2 hours at room temperature.
The plate was washed 3 times with 400 .mu.l per well of wash
solution (PBS (Sigma Chemical Co., UK) 0.01% (v/v) Triton-X100
(Sigma Chemical Co., UK)) and tapped dry.
[0315] Assay buffer (1% w/v BSA, Sigma Chemical Co., UK) was added
at 200 .mu.l per well and incubated for approximately 1 hour at
room temperature. The plate was washed 3 times with 400 .mu.l per
well of wash solution and tapped dry. [0316] The calibration
standards, Quality Controls (QC) and/or samples were added at 100
.mu.l per well and incubated for approximately 2 hours at room
temperature, after which the plate was washed 3 times with 400
.mu.l per well of wash solution and tapped dry.
[0317] The conjugate solution (6 .mu.l goat anti-human IgG
kappa-HRP conjugate (Bethyl Laboratories Inc., USA) added to 12 ml
assay buffer) was added at 100 .mu.l per well and incubated for
approximately 1 hour at room temperature. The plate was washed 3
times with 400 .mu.l per well of wash solution and tapped dry.
[0318] Trimethylbenzidine (TMB; Europa Bioproducts, Ely, UK) was
added at 100 .mu.l per well. The plate was covered and incubated
for approximately 15 minutes at room temperature. 100 .mu.l of stop
solution (0.5M HCl, Fisher, UK) was then added to each well.
Absorbances were read at 450 nm (reference filter 630 nm) on a
DYNATECH MRX microplate reader (Mettler Instruments, UK).
Results and Discussion
Test Sample Analysis
[0319] Concentrations of anti-EGFR were measured by an immunoassay
method (ELISA) in 78 monkey serum samples generated according to
the protocol described herein above. These results are presented in
Tables 19-21, below.
TABLE-US-00020 TABLE 29 Serum concentration of anti-EGFR in monkey
serum (pg/ml) following Intravenous administration of 1.5 mg/kg
anti-EGFR on days 1, 8, 15 and 22 (GROUP 1) Animal number Timepoint
1M 623 1F 590 Mean sd Day 1 1 hour 33.42 30.86 32.14 1.8 Day 1 4
hours 27.33 27.49 27.41 0.1 Day 1 12 hours 13.09 17.01 15.05 2.8
Day 1 24 hours 9.656 9.468 9.562 0.1 Day 1 72 hours 2.528 0.786
1.657 1.2 Day 1 120 hours 0.845 0.431 0.638 0.3 Day 8 predose 0.538
0.287 0.413 0.2 Day 8 1 hour 30.02 19.07 24.55 7.7 Day 15 predose
0.902 0.382 0.642 0.4 Day 15 1 hour 17.91 33.08 25.50 10.7 Day 22
predose 1.065 0.595 0.830 0.3 Day 22 1 hour 19.41 33.00 26.21 9.6
Day 1 672 hours 1.202 0.362 0.782 0.6 sd standard deviation
TABLE-US-00021 TABLE 20 Serum concentration of anti-EGFR in monkey
serum (pg/ml) following Intravenous administration of 5.0 mg/kg
anti-EGFR on days 1, 8, 15 and 22 (GROUP 2) Animal number Timepoint
2M 461 2F 462 Mean sd Day 1 1 hour 32.45 29.51 30.98 2.1 Day 1 4
hours 32.39 29.57 30.98 2.0 Day 1 12 hours 28.05 25.88 26.97 1.5
Day 1 24 hours 23.70 23.78 23.74 0.1 Day 1 72 hours 14.03 14.38
14.21 0.2 Day 1 120 hours 10.42 8.137 9.279 1.6 Day 8 predose 4.672
3.683 4.178 0.7 Day 8 1 hour 25.91 31.06 28.49 3.6 Day 15 predose
5.752 5.450 5.601 0.2 Day 15 1 hour 32.20 35.38 33.79 2.2 Day 22
predose BLQ 6.497 3.249 -- Day 22 1 hour 26.98 30.23 28.61 2.3 Day
1 672 hours BLQ 4.845 2.423 -- BLQ below limit of quantification
(<0.195 .mu.g/ml) sd standard deviation Note: BLQ entered as
zero in calculations
TABLE-US-00022 TABLE 21 Serum concentration of anti-EGFR in monkey
serum (.mu.g/ml) following Intravenous administration of 15 mg/kg
anti-EGFR on days 1, 8, 15 and 22 (GROUP 3) Animal number Timepoint
3 M 463 3 F 612 Mean sd Day 1 1 hour 262.2 168.0 215.1 66.6 Day 1 4
hours 223.3 174.5 198.9 34.5 Day 1 12 hours 164.9 165.7 165.3 0.6
Day 1 24 hours 141.7 146.0 143.9 3.0 Day 1 72 hours 99.54 86.64
93.09 9.1 Day 1 120 hours 86.64 69.08 77.86 12.4 Day 8 predose
65.86 45.21 55.54 14.6 Day 8 1 hour 282.1 209.9 246.0 51.1 Day 15
predose 98.43 71.21 84.82 19.2 Day 15 1 hour 385.9 231.4 308.7
109.2 Day 22 predose 117.3 105.6 111.5 8.3 Day 22 1 hour 234.1
402.5 318.3 119.1 Day 1 672 hours 127.5 122.9 125.2 3.3 sd standard
deviation
Example 4
Preliminary Toxicity Study by Intravenous (Bolus) Administration to
Cynomolgus Monkeys--Toxicokinetics
Summary
[0320] Three groups of cynomolgus monkeys (1 male and 1 female per
group) were administered intravenous bolus doses of anti-EGFR on
Days 1, 8, 15 and 22 of a 28-day toxicity study in order to assess
the systemic exposure of the animals to anti-EGFR. Serum
concentrations of anti-EGFR in samples collected up to 672 hours
after the first dose were determined by means of an immunoassay
method. Pharmacokinetic analysis of serum concentration-time data
resulted in the following pharmacokinetic parameters:
TABLE-US-00023 TABLE 22 Dose C.sub.max T.sub.max AUC.sub.t AUC CL
V.sub.ss k t.sub.1/2 (mg/kg) Animal (.mu.g/mL) (h) (.mu.g h/mL)
(.mu.g h/mL) (mL/h/kg) (mL/kg) (1/h) (h) 1.5 1M623 33.42 1 830.4
849.4 1.778 60.79 0.0214 32.5 1.5 1F590 30.86 1 748.4 .sup.
774.9.sup.a 1.962.sup.a 57.85.sup.a 0.0105.sup.a 66.0.sup.a 4.5
2M461 32.45 1 2537 .sup. 3005 1.488 133.6 0.0110 63.1 4.5 2F462
29.57 4 2378 .sup. 2719 1.663 133.2 0.0121 57.4 12 3M463 262.2 1
18310 29870.sup.a 0.4058.sup.a 71.33.sup.a 0.0056.sup.a 124.3.sup.a
12 3F612 174.5 4 15980 21400.sup.a 0.5552.sup.a 66.94.sup.a
0.0082.sup.a 84.4.sup.a .sup.aValue is an estimate as the data did
not meet all the acceptance criteria defined in Data Processing and
should be treated with caution
[0321] The relationships between areas under the serum anti-EGFR
concentration-time curves (AUC.sub.168) and dose level on Day 1 are
presented below:
TABLE-US-00024 TABLE 23 Dose level Dose level AUC.sub.168 ratio
(mg/kg/occasion) ratio Males Females 1.5 1 1 1 4.5 3.0 3.1 3.2 12
8.0 22.0 21.4
[0322] The rate and extent of systemic exposure of monkeys to
anti-EGFR, characterised by AUC.sub.168, increased approximately
proportionately with increasing dose over the dose range 1.5 to 4.5
mg/kg/occasion but by more than the proportionate dose increase
over the dose range 4.5 to 12 mg/kg/occasion on Day 1. At the
highest dose (12 mg/kg/occasion) the AUC.sub.168 was ca 2.8-fold
higher than that predicted by a linear relationship.
[0323] The extent (AUC.sub.168) of systemic exposure of female
monkeys to anti-EGFR was generally similar to the exposure in male
monkeys.
[0324] After repeated intravenous doses, the pre-dose serum
concentrations of anti-EGFR were generally higher than those values
after a single dose and indicated accumulation of anti-EGFR in
serum throughout the period of the study.
[0325] The terminal half-life could not be estimated adequately for
all animals, but where it could be estimated was in the range 32.5
to 63.1 hours, and appeared to increase with dose in male animals.
Total serum clearance of anti-EGFR appeared to be independent of
dose over the range 1.5-4.5 mg/kg/occasion but was reduced at the
top dose level in male and female monkeys.
[0326] In conclusion, the extent of systemic exposure of cynomolgus
monkeys to anti-EGFR appeared to be characterised by non-linear
(dose-dependent) kinetics over the dose range 1.5 to 12
mg/kg/occasion on Day 1 of the intravenous toxicity study.
Increasing the dose of anti-EGFR above 4.5 mg/kg/occasion is likely
to result in a higher systemic exposure than would be predicted
from a linear relationship, which is consistent with the
possibility of a capacity limited process for the elimination of
anti-EGFR.
[0327] In addition, the study also provided evidence that in
general there were no differences in the systemic exposure of male
and female monkeys to anti-EGFR and that there was accumulation
after repeated intravenous administration.
Introduction
[0328] Three groups of one male and one female cynomolgus monkey
were administered anti-EGFR by intravenous bolus injection, at dose
levels of 1.5, 4.5 and 12 mg/kg/occasion on Days 1, 8, 15 and 22 of
a preliminary toxicity study. Blood samples were taken from each
animal at the following time-points following administration on Day
1: 1, 4, 12, 24, 72 and 120 hours post-dose. In addition, samples
were taken pre-dose and at 1 hour post-dose on Days 8, 15 and 22
and at 672 hours after the first dose on Day 1. The separated serum
was frozen at ca -20.degree. C. prior to analysis of serum
concentrations of anti-EGFR by an immunoassay method.
ABBREVIATIONS
[0329] AUC Area under the serum concentration-time curve to
infinite time [0330] AUC.sub.168 Area under the serum
concentration-time curve during a 168-hour dosing interval [0331]
BLQ Below the limit of quantification [0332] ca Approximately
[0333] CL Total serum clearance [0334] Cmax Maximum serum
concentration [0335] F Female [0336] k Terminal rate constant
[0337] M Male [0338] t.sub.1/2 Terminal half-life [0339] Tmax Time
at which Cmax occurred [0340] Vss Volume of distribution at
steady-state
Antibody Used for Study
[0341] Glyco-mAb (Anti-EGFR), an anti-EGFR antibody Fc-engineered
for increased Fc-FcgammaRIII receptor binding affinity and
increased ADCC, was produced, purified and characterized as
described above. Briefly, antibody was produced by co-transfection
of HEK-293-EBNA cells with plasmid DNA vectors for expression of
I-HHB antibody heavy chain, I-KC antibody light chain, GnT-III and
ManII. A linearly scaled-up version of the transfection method
described above was employed, transfecting cell monolayers cultured
in roller bottles instead of T-flasks. An additional flow-through
anion-exchange chromatographic step using Q-sepharose matrix was
included in the purification process immediately before the size
exclusion chromatographic step described above.
[0342] The glycosylation pattern of the Fc-engineered antibody was
analyzed as described above using MALDI/TOF-MS spectrometry of
enzymatically released Fc-derived oligosaccharides. The
oligosaccharide profile is shown in FIG. 23.
[0343] Binding to human EGFR and monkey EGFR was demonstrated by
whole-cell binding as described above using A431 and COS-7 cells,
respectively, and FACS-based analysis. Binding curves are shown in
FIGS. 24 and 25 respectively.
[0344] Increased FcgammaRIII receptor binding resulting from the
applied Fc engineering was demonstrated as described above using
whole cell binding to CHO cells engineered for surface expression
of human FcgammaRIII and FACS-based analysis. Results are shown in
FIG. 26. Additionally, the engineered antibody has equivalent
degree of Fc-engineering to the "Glyco-2" antibody (75% on
Fc-oligosaccharides being of non-fucosylated type) described
elsewhere (Ferrara, C. et al., J Biol Chem. 2005 Dec. 5;
[E-publication ahead of print]). Such Fc-engineered antibodies have
up to 50-fold increased binding affinity for human FcgammaRIII
relative to a standard non-Fc engineered antibody (Equilibrium
dissociation constanst of 15 and 150 nM for the 158V and 158F
polymorphic variants of the human receptor vs. 750 and 5000 nM for
the same receptor variants, respectively, when binding to non-Fc
engineered human IgG1 antibodies).
[0345] ADCC was measured as described aboved using two target cell
lines: A549 human lung carcinoma cells and CYNOM-K1 cynomolgus
monkey keratinocyte cells. Results are shown in FIGS. 27 and 28,
respectively.
Data Processing
[0346] Pharmacokinetic parameters were calculated using the
computer program WinNonlin Pro version 3.3 (Pharsight Corporation,
USA).
[0347] All serum concentrations supplied as part of this study were
reported to 4 significant figures or 3 decimal places.
Pharmacokinetic parameters were reported as follows: Cmax,
AUC.sub.168, CL and Vss to 4 significant figures; k to 4 decimal
places; t.sub.1/2 to 1 decimal place.
[0348] Values that were BLQ (<0.195 pg/mL) were entered as zero
in the pharmacokinetic processing.
Toxicokinetics
[0349] Maximum serum concentrations of anti-EGFR (Cmax) and their
times of occurrence (Tmax) were the observed values. Areas under
the serum anti-EGFR concentration-time curves within a 168-hour
dosing interval (AUC.sub.168), were estimated by the linear
trapezoidal rule. In the calculation of AUC.sub.168 values the
serum anti-EGFR concentrations at zero hours were estimated by back
extrapolation using log-linear regression analysis, based on the
first two sampling times, however, if the serum concentration did
not decline during this period then the serum concentration at zero
hours was considered to be equivalent to the concentration at the
first sampling time. Areas under the serum anti-EGFR
concentration-time curves to infinite time (AUC), were estimated by
the following expression:
AUC=AUC.sub.168+Clast/k
Where Clast is the predicted serum concentration at the last
quantifiable sample point and k is the terminal rate constant.
[0350] Terminal rate constants (k) were estimated by fitting a
linear regression of log concentration against time. For the
estimate of k to be accepted as reliable, the following criteria
were imposed:
[0351] 1. The terminal data points were apparently randomly
distributed about a single straight line (on visual inspection)
[0352] 2. A minimum of 3 data points was available for the
regression
[0353] 3. The regression coefficient was .gtoreq.0.95, and the
fraction of the variance accounted for was .gtoreq.0.90
[0354] 4. The interval including the data points chosen for the
regression was at least two-fold greater than the half-life
itself
[0355] Terminal half-lives (t.sub.1/2) were calculated as ln 2/k.
Total serum clearance (CL) was calculated as Dose/AUC. Volume of
distribution at steady-state (Vss) was calculated as
Dose.AUMC/AUC2. Accumulation (R) was assessed as the ratio of the
trough concentration following the last dose (Day 22) to the trough
concentration following the first dose (Day 1) i.e. serum
concentration at 672 hours/serum concentration at 168 hours
(pre-dose on Day 8).
Results and Discussion
[0356] Blood samples were taken up to 120 hours after dosing on Day
1; at pre-dose and 1 hour post-dose on Days 8, 15 and 22, and at
672 hours post dosing on Day 1 during a toxicity study to assess
the systemic exposure of male and female monkeys to anti-EGFR
following intravenous bolus administration of anti-EGFR at dose
levels of 1.5, 4.5 and 12 mg/kg/occasion on Days 1, 8, 15 and 22 of
the study. Serum concentrations of anti-EGFR in samples taken up to
168 hours post-dose are presented in Tables 27-29, and the mean
serum concentration-time profiles are illustrated in FIGS. 18 and
19.
[0357] Pharmacokinetic parameters of anti-EGFR are presented in
Table 50, and the AUC.sub.168 values are summarised below:
TABLE-US-00025 TABLE 24 Dose level AUC.sub.168 (.mu.g h/mL)
(mg/kg/occasion) Males Females 1.5 830.4 748.4 4.5 2537 2378 12
18310 15980
[0358] The times at which the maximum serum concentrations occurred
(T.sub.max) were generally 1 hour post-dose (the first sample
point) but occurred at 4 hours post-dose (the second sample point)
in females 2F462 (4.5 mg/kg) and 3F612 (12 mg/kg). However, for
both these females, the concentrations at 4 hours post-dose were
very similar to those concentrations at 1 hour post-dose and were
probably within the variability of the assay. Therefore the
apparent delay in Tmax is unlikely to be of any significance.
[0359] Serum concentrations of anti-EGFR prior to the succeeding
dose were quantifiable in all animals except male 2M461 on Day 22
(4.5 mg/kg/occasion dose level) therefore, in general, animals were
continuously exposed to quantifiable concentrations of anti-EGFR
during a dosing interval.
[0360] The relationships between areas under the serum anti-EGFR
concentration-time curves (AUC.sub.168) and dose level on Day 1 are
presented below:
TABLE-US-00026 TABLE 25 Dose level Dose level AUC.sub.168 ratio
(mg/kg/occasion) ratio Males Females 1.5 1 1 1 4.5 3.0 3.1 3.2 12
8.0 22.0 21.4
[0361] The rate and extent of systemic exposure of monkeys to
anti-EGFR, characterised by AUC.sub.168, increased approximately
proportionately with increasing dose over the dose range 1.5 to 4.5
mg/kg/occasion but by more than the proportionate dose increase
over the dose range 4.5 to 12 mg/kg/occasion on Day 1. At the
highest dose (12 mg/kg/occasion) the AUC.sub.168 was ca 2.8-fold
higher than that predicted by a linear relationship (FIG. 20).
[0362] The extent (AUC.sub.168) of systemic exposure of female
monkeys to anti-EGFR was generally similar to the exposure in male
monkeys.
[0363] After repeated intravenous doses, the pre-dose serum
concentrations of anti-EGFR were generally higher than those values
after a single dose (FIGS. 21-22) and indicated accumulation of
anti-EGFR in serum throughout the period of the study. This
accumulation was generally lower in females than in males, except
at the highest dose level. The ratios of the trough (pre-dose)
concentrations following the last dose on Day 22 (672 hours post
Day 1 dose) to the trough concentration following the first dose on
Day 1 are presented in the Table 26, below:
TABLE-US-00027 TABLE 26 Dose level R (mg/kg/occasion) Males Females
1.5 2.23 1.26 4.5 * 1.32 12 1.94 2.72 *Could not be calculated as
trough concentration was BLQ
[0364] The terminal rate constants (k), and corresponding terminal
half-lives (t.sub.1/2), of anti-EGFR on Day 1 are presented in
Table 30. The terminal half-life could not be estimated adequately
for all animals, but where it could be estimated was in the range
32.5 to 63.1 hours, and appeared to increase with dose in male
animals. Total serum clearance of anti-EGFR appeared to be
independent of dose over the range 1.5-4.5 mg/kg/occasion but was
reduced at the highest dose level in male and female monkeys.
[0365] In conclusion, the extent of systemic exposure of cynomolgus
monkeys to anti-EGFR appeared to be characterised by non-linear
(dose-dependent) kinetics over the dose range 1.5 to 12
mg/kg/occasion on Day 1 of the intravenous toxicity study.
Increasing the dose of anti-EGFR above 4.5 mg/kg/occasion is likely
to result in a higher systemic exposure than would be predicted
from a linear relationship, which is consistent with the
possibility of a capacity limited process for the elimination of
anti-EGFR.
[0366] In addition, the study also provided evidence that in
general there were no differences in the systemic exposure of male
and female monkeys to anti-EGFR and that there was accumulation
after repeated intravenous administration.
TABLE-US-00028 TABLE 27 Serum concentrations (.mu.g/ml) of
anti-EGFR in monkey serum following intravenous administration of
1.5 mg/kg anti-EGFR on Days 1, 8, 15 and 22 Animal number Timepoint
1 M 623 1 F 590 Day 1 1 hour 33.42 30.86 Day 1 4 hours 27.33 27.49
Day 1 12 hours 13.09 17.01 Day 1 24 hours 9.656 9.468 Day 1 72
hours 2.528 0.786 Day 1 120 hours 0.845 0.431 Day 8 pre-dose 0.538
0.287 Day 8 1 hour 30.02 19.07 Day 15 pre-dose 0.902 0.382 Day 15 1
hour 17.91 33.08 Day 22 pre-dose 1.065 0.595 Day 22 1 hour 19.41
33.00 Day 1 672 hours 1.202 0.362
TABLE-US-00029 TABLE 28 Serum concentrations (.mu.g/ml) of
anti-EGFR in cynomolgus monkey serum following intravenous
administration of 4.5 mg/kg anti-EGFR on Days 1, 8, 15 and 22
Animal number Timepoint 2 M 461 2 F 462 Day 1 1 hour 32.45 29.51
Day 1 4 hours 32.39 29.57 Day 1 12 hours 28.05 25.88 Day 1 24 hours
23.70 23.78 Day 1 72 hours 14.03 14.38 Day 1 120 hours 10.42 8.137
Day 8 pre-dose 4.672 3.683 Day 8 1 hour 25.91 31.06 Day 15 pre-dose
5.752 5.450 Day 15 1 hour 32.20 35.38 Day 22 pre-dose BLQ 6.497 Day
22 1 hour 26.98 30.23 Day 1 672 hours BLQ 4.845
TABLE-US-00030 TABLE 29 Serum concentrations (.mu.g/ml) of
anti-EGFR in cynomolgus monkey serum following intravenous
administration of 12 mg/kg anti-EGFR on Days 1, 8, 15 and 22 Animal
number Timepoint 1 M 623 1 F 590 Day 1 1 hour 262.2 168.0 Day 1 4
hours 223.3 174.5 Day 1 12 hours 164.9 165.7 Day 1 24 hours 141.7
146.0 Day 1 72 hours 99.54 86.64 Day 1 120 hours 86.64 69.08 Day 8
pre-dose 65.86 45.21 Day 8 1 hour 282.1 209.9 Day 15 pre-dose 98.43
71.21 Day 15 1 hour 385.9 231.4 Day 22 pre-dose 117.3 105.6 Day 22
1 hour 234.1 402.5 Day 1 672 hours 127.5 122.9
TABLE-US-00031 TABLE 30 Pharmacokinetic parameters of anti-EGFR on
Day 1 of weekly intravenous administration of anti-EGFR to
cynomolgus monkeys Dose C.sub.max T.sub.max AUC.sub.t AUC CL
V.sub.ss k t.sub.1/2 (mg/kg) Animal (.mu.g/mL) (h) (.mu.g h/mL)
(.mu.g h/mL) (mL/h/kg) (mL/kg) (1/h) (h) 1.5 1M623 33.42 1 830.4
849.4 1.778 60.79 0.0214 32.5 1.5 1F590 30.86 1 748.4 .sup.
774.9.sup.a 1.962.sup.a 57.85.sup.a 0.0105.sup.a 66.0.sup.a 4.5
2M461 32.45 1 2537 .sup. 3005 1.488 133.6 0.0110 63.1 4.5 2F462
29.57 4 2378 .sup. 2719 1.663 133.2 0.0121 57.4 12 3M463 262.2 1
18310 29870.sup.a 0.4058.sup.a 71.33.sup.a 0.0056.sup.a 124.3.sup.a
12 3F612 174.5 4 15980 21400.sup.a 0.5552.sup.a 66.94.sup.a
0.0082.sup.a 84.4.sup.a .sup.aValue is an estimate as the data did
not meet all the acceptance criteria defined in Data Processing and
should be treated with caution
Blood Chemistry and Haematology
[0367] Blood samples were taken from the femoral vein of
cynomolgous monkeys that had been administered an intravenous bolus
injection of GlycoMAB anti-EGFR on days 1, 8, 15, and 22. Samples
were taken from the limb not used for dose administration,
following overnight deprivation of food (not decedents). Samples
were examined at pretreatment, three days after the second dose,
and on termination for the following parameters, using lithium
heparin as anticoagulant:
[0368] Alkaline phosphatase
[0369] Alanine amino-transferase
[0370] Aspartate amino-transferase
[0371] Bilirubin--total
[0372] Urea
[0373] Creatinine
[0374] Glucose
[0375] Cholesterol--total
[0376] Triglycerides
[0377] Sodium
[0378] Potassium
[0379] Chloride
[0380] Calcium
[0381] Phosphorus
[0382] Total protein
[0383] Protein electrophoretogram
[0384] Albumin/globulin ratio
[0385] Average normal cynomolgus monkey blood chemistry analysis
data are presented in Table 31.
TABLE-US-00032 TABLE 31 Cynomolgus monkeys (origin
Mauritius)--Blood Chemistry Parameter sex n 1% 5% 50% 95% 99% mean
s.d. ALP M 949 837 1339 2147 3201 3899 2175.5 565.44 F 881 946 1342
2144 3163 3740 2164.1 552.82 ALP-N M 511 481 579 881 1453 1771
928.0 260.88 F 499 427 546 846 1362 1694 879.5 240.24 ALT M 1489 24
30 50 87 127 53.7 19.07 F 1407 23 28 46 84 111 49.3 18.65 AST M
1487 26 30 41 63 101 43.6 17.57 F 1404 25 29 39 61 89 41.7 13.59
gGT M 663 95 118 178 292 342 188.5 52.47 F 641 81 102 153 232 266
158.6 38.38 LAP M 207 18 26 40 79 217 45.1 28.25 F 205 15 20 35 62
89 37.1 12.73 GLDH M 159 8 10 17 35 126 20.1 16.46 F 159 6 8 15 27
35 16.3 5.97 Bilirubin M 1494 1 1 3 8 11 3.8 2.04 F 1413 1 2 4 8 11
4.1 2.07 LDH M 99 160 596 808 1166 2029 838.3 218.75 F 82 477 529
711 945 1021 715.0 117.07 CPK M 331 68 83 179 713 1867 287.4 464.03
F 335 57 77 184 925 2628 309.7 534.02 Indir Bili M 59 1 2 4 10 11
4.2 2.15 F 57 1 2 4 5 7 3.6 1.13 Direct M 59 0 0 0 2 3 0.2 0.65
Bilirubin F 57 0 0 0 1 3 0.2 0.52 Bile Acids M 386 0.9 2.5 6.4 15.3
23.4 7.38 4.574 F 380 1.4 3.0 7.0 12.9 17.8 7.41 3.474 Urea M 1457
3.01 3.66 5.50 8.81 10.53 5.775 1.5710 F 1379 2.77 3.42 5.33 8.56
9.90 5.559 1.5432 Creatinine M 1458 55 59 71 87 94 71.6 8.67 F 1383
56 60 72 87 85 72.7 8.36 Glucose M 1455 2.22 2.64 3.71 5.21 6.37
3.809 0.8135 F 1380 2.23 2.65 3.63 5.18 6.34 3.735 0.7990
Cholesterol M 1455 1.69 1.93 2.68 3.55 3.96 2.706 1.4909 F 1382
1.83 2.15 2.86 3.69 4.05 2.885 0.4813 Chol HDL M 45 1.26 1.34 1.79
2.23 2.59 1.784 0.3128 F 45 1.09 1.31 1.82 2.43 2.55 1.844 0.3179
Chol VLDL M 45 0.00 0.00 0.00 0.03 0.11 0.004 0.0175 F 45 0.00 0.00
0.00 0.12 0.19 0.012 0.0370 NEFA M 132 0.10 0.28 0.98 1.84 2.44
0.994 0.4700 F 132 0.14 0.22 1.06 1.90 2.18 1.070 0.4704
Triglycerides M 1453 0.26 0.32 0.53 0.86 1.30 0.561 0.2051 F 1374
0.26 0.34 0.57 0.90 1.14 0.587 0.1778 Ph Lipid M 64 1.65 1.68 2.18
2.91 3.15 2.254 0.3769 F 49 1.77 1.83 2.44 2.91 2.98 2.405 0.3267
Uric Acid M 17 0 0 0 8 8 1.1 2.34 F 17 0 0 0 1 1 0.4 0.51 Na M 1461
141 142 147 152 155 146.8 3.00 F 1382 140 142 147 153 157 147.3
3.34 K M 1460 3.2 3.4 4.0 5.0 5.6 4.08 0.511 F 1382 3.2 3.3 4.0 4.9
5.4 4.01 0.484 Cl M 1461 102 104 108 112 115 107.7 2.71 F 1382 102
104 108 113 116 108.4 2.83 Ca M 1462 2.31 2.39 2.56 2.76 2.87 2.568
0.1176 F 1382 2.32 2.39 2.57 2.77 2.89 2.572 0.1168 Phos M 1172
1.16 1.40 1.93 2.43 2.69 1.921 0.3126 F 1098 1.17 1.37 1.84 2.35
2.60 1.844 0.2978 Chol LDL M 45 0.54 0.62 1.20 1.87 1.92 1.253
0.3694 F 45 0.69 0.78 1.19 1.83 1.88 1.233 0.2906 Bicarbonate M 288
7 10 17 22 25 16.5 3.51 F 283 6 10 16 22 25 15.9 3.81 Total Protein
M 1455 71 74 80 87 90 80.2 4.06 F 1381 71 74 81 89 92 81.2 4.32
Albumin M 346 34 38 43 46 49 42.6 2.67 (Chemical) F 342 36 38 43 47
49 42.7 2.60 Albumin M 1089 33 36 45 51 54 44.3 4.46 F 1019 34 37
45 52 55 44.7 4.58 Globulin M 289 31 32 36 41 45 36.3 2.83 F 285 30
32 37 43 48 37.5 3.59 A/G Ratio M 340 0.79 0.98 1.17 1.39 1.45
1.172 0.1215 (Chemical) F 336 0.84 0.95 1.14 1.34 1.42 1.143 0.1274
A/G Ratio M 1068 0.68 0.80 1.26 1.65 1.82 1.252 0.2522 F 998 0.67
0.79 1.26 1.66 1.81 1.247 0.2647 A1 Globulin M 1105 2 2 3 4 4 2.8
0.62 F 1035 2 2 3 4 5 2.8 0.64 A2 Globulin M 1105 3 3 4 6 7 4.2
0.92 F 1035 3 3 4 6 7 4.2 1.01 beta M 1105 12 13 16 22 24 16.6 2.63
Globulin F 1035 12 13 16 22 25 16.8 2.85 gamma M 1105 8 9 12 17 18
12.6 2.26 Globulin F 1035 8 9 13 17 20 13.1 2.52 Aldolase M 96 9 14
21 38 57 22.0 7.48 F 97 10 13 19 48 84 21.9 11.91 Plasm CHE M 17
4159 4159 5745 9160 9160 5919.8 1181.68 F 17 3371 3371 5869 8367
8367 5689.2 1512.98 CRP M 57 0.000 0.000 0.002 0.013 0.026 0.0032
0.00414 F 56 0.000 0.000 0.002 0.004 0.007 0.0017 0.00167 T3 M 40
1.90 1.90 2.50 3.38 3.58 2.537 0.4011 F 40 1.71 1.96 2.59 3.06 4.02
2.631 0.3799 T4 M 40 35 42 59 86 92 59.7 11.84 F 40 38 40 56 81 107
58.6 14.37
[0386] Samples for haematological, peripheral blood analysis were
taken from the femoral vein of cynomolgous monkey that had been
administered an intravenous bolus injection of GlycoMAB anti-EGFR
on days 1, 8, 15, and 22. Samples were taken from the limb not used
for dose administration, following overnight deprivation of food
(not decedents). Samples were examined at pretreatment, three days
after the second dose, and on termination for the following
parameters:
[0387] 1) Using EDTA as anticoagulant-- [0388]
HaematocritHaemoglobin concentration [0389] Erythrocyte count
[0390] Reticulocytes [0391] Mean cell haemoglobin [0392] Mean cell
haemoglobin concentration [0393] Mean cell volume [0394] Total
leucocyte count [0395] Differential leucocyte count [0396] Platelet
count [0397] Abnormalities of the blood morphology [0398]
Anisocytosis [0399] Microcytosis [0400] Macrocytosis [0401]
Hypochromasia [0402] Hyperchromasia
[0403] 2) Using citrate as anticoagulant-- [0404] Prothrombin time
[0405] Activated partial thromboplastin time
[0406] Average normal cynomolgus monkey hematology analysis data
are presented in Table 32.
TABLE-US-00033 TABLE 32 Cynomolgus monkeys (origin
Mauritius)--Hematology Parameter sex n 1% 5% 50% 95% 99% mean s.d.
HCT M 1495 0.385 0.401 0.443 0.488 0.512 0.4435 0.02776 F 1426
0.376 0.399 0.442 0.489 0.508 0.4424 0.02947 Haemoglobin M 1495
11.4 12.1 13.3 14.5 15.1 13.31 0.769 F 1426 11.2 11.9 13.2 14.5
15.1 13.21 0.855 RBC M 1495 5.67 6.04 6.74 7.51 7.92 6.744 0.4792 F
1426 5.58 5.96 6.71 7.45 7.73 6.707 0.4894 Retic (1) % M 20 0.1 0.1
0.4 1.8 1.8 0.48 0.438 F 20 0.1 0.1 0.3 0.9 0.9 0.42 0.268 Retic
(2) % M 1476 0.21 0.27 0.49 0.95 1.58 0.551 0.3804 F 1408 0.22 0.28
0.54 1.06 1.60 0.595 0.2934 MCH M 1495 17.0 17.8 19.8 21.6 22.5
19.80 1.432 F 1425 16.7 17.7 19.7 21.8 22.6 19.74 1.216 MCHC M 1495
27.2 28.2 30.1 31.9 32.7 30.06 1.801 F 1425 27.0 27.9 29.9 31.8
32.6 29.88 1.174 MCV M 1495 57.9 60.5 65.8 71.4 73.5 65.88 3.278 F
1425 58.3 60.4 66.0 71.7 74.5 66.07 3.353 RDW M 280 12.6 12.9 14.4
16.1 16.7 14.40 0.934 F 285 12.4 12.8 14.2 15.7 16.3 14.21 0.879
WBC M 1507 5.61 6.62 10.52 18.59 30.24 11.372 4.7766 F 1432 5.39
6.58 10.62 19.55 28.79 11.637 4.7307 Neutrophils M 1507 0.88 1.28
3.49 10.24 16.27 4.319 3.1741 F 1432 1.06 1.62 4.45 12.27 17.41
5.392 3.4777 Lymphocytes M 1507 2.19 2.96 5.53 9.81 15.91 6.021
3.4066 F 1432 2.16 2.67 4.86 8.55 13.95 5.265 2.9997 Eosinophils M
1507 0.00 0.01 0.17 0.81 1.49 0.254 0.3127 F 1432 0.00 0.01 0.14
0.73 1.55 0.232 0.3188 Basophils M 1507 0.01 0.02 0.04 0.10 0.25
0.053 0.0627 F 1432 0.01 0.02 0.04 0.10 0.21 0.051 0.0540 Monocytes
M 1507 0.17 0.25 0.51 1.03 1.45 0.562 0.2575 F 1432 0.16 0.23 0.49
1.04 1.56 0.547 0.2705 Large Unstained M 1507 0.04 0.06 0.14 0.32
0.60 0.163 0.1330 Cells F 1432 0.04 0.06 0.13 0.29 0.50 0.148
0.1147 Platelets M 1495 158 238 359 497 575 362.1 81.69 F 1426 181
234 359 496 560 360.5 80.04 PT M 1481 9.6 9.9 10.8 12.0 14.8 10.88
0.877 F 1406 9.7 10.0 10.8 12.1 14.1 10.93 0.847 Act PTT M 1483
23.1 24.4 29.1 37.9 50.1 30.06 5.267 F 1408 22.8 24.4 29.4 37.4
47.2 30.19 5.185 Fibrinogen M 265 1.61 1.86 2.61 3.51 4.88 2.664
0.6178 F 252 1.58 1.84 2.43 3.27 3.89 2.487 0.4545
[0407] The Biochemistry Cumulative Individual Values for the
monkeys are presented in Tables 33a-h, below:
TABLE-US-00034 TABLE 33a Animal Group/ Occn. ALP ALT AST Bili Urea
Creat Gluc Chol Number Sex Code U/L U/L U/L .mu.mol/L mmol/L
.mu.mol/L mmol/L mmol/L 615 1M PT 740 36 39 3 5.35 69 3.65 2.34 D11
743 35 33 3 5.43 73 3.66 2.00 TERM 597 29 31 2 5.16 76 3.74 2.30
465 2M PT 647 44 33 3 5.73 70 4.69 2.50 PD 775 47 36 2 3.60 73 4.50
2.72 D11 655 74 46 5 4.34 74 3.35 2.64 TERM 768 48 38 3 4.42 73
3.67 2.54 639 2M PD 741 29 26 2 4.41 87 3.31 3.22 D11 629 29 28 3
3.87 99 2.64 2.99 TERM 599 34 22 2 3.62 83 3.46 2.45 613 3M PT 1003
37 31 5 3.80 90 5.72 2.61 D11 793 36 29 4 4.45 80 3.28 2.70 TERM
931 34 32 5 5.16 83 2.78 2.46 631 4M PD 590 34 37 2 3.43 83 3.92
2.49 D11 508 38 36 3 3.33 82 3.38 2.30 TERM 578 25 25 2 4.00 89
3.70 2.26
TABLE-US-00035 TABLE 33b Total Animal Group/ Occn. Trig Na K Cl Ca
Phos Prot Alb Number Sex Code mmol/L mmol/L mmol/L mmol/L mmol/L
mmol/L g/L g/L 615 1M PT 0.24 146 4.7 108 2.54 1.77 91 35 D11 0.39
145 3.9 106 2.55 1.85 94 41 TERM 0.34 147 4.2 107 2.47 1.63 87 38
465 2M PT 0.74 147 3.7 108 2.71 1.32 81 37 PD 0.93 147 4.1 109 2.70
1.54 84 46 D11 0.66 151 3.9 111 2.70 1.98 83 39 TERM 0.51 146 3.6
105 2.67 1.85 80 40 639 2M PD 0.27 146 4.2 107 2.68 1.85 84 44 D11
0.32 150 4.8 108 2.81 2.07 85 42 TERM 0.38 146 4.5 107 2.70 2.03 74
39 613 3M PT 0.47 151 4.5 106 2.75 1.85 83 41 D11 0.44 147 4.2 106
2.69 1.76 80 45 TERM 0.69 147 4.3 106 2.63 1.62 76 40 631 4M PD
0.45 149 4.2 107 2.67 2.00 87 46 D11 0.77 151 5.0 112 2.68 2.00 84
39 TERM 0.64 150 4.7 107 2.77 1.89 86 46
TABLE-US-00036 TABLE 33c Ani- mal Gam- Num- Group/ Occn. a1 a2 Beta
ma A/G Alb a1 a2 ber Sex Code g/L g/L g/L g/L Ratio % % % 615 1M PT
3 5 25 23 0.63 39.0 3.4 5.1 D11 3 4 22 25 0.77 43.1 3.0 4.5 TERM 3
4 21 21 0.78 43.5 3.3 4.9 465 2M PT 4 5 22 13 0.84 45.7 4.5 5.9 PD
3 4 18 12 1.21 54.9 3.6 5.0 D11 4 5 20 15 0.89 46.7 4.9 6.4 TERM 3
5 20 11 1.00 50.5 4.0 5.8 639 2M PD 3 5 17 14 1.10 52.9 3.5 5.8 D11
3 6 18 16 0.98 49.1 4.1 6.6 TERM 3 4 17 11 1.11 52.2 3.7 6.0 613 3M
PT 4 4 20 14 0.98 49.5 5.0 4.7 D11 3 3 17 13 1.29 55.8 3.7 3.7 TERM
3 3 15 14 1.11 53.0 4.6 3.9 631 4M PD 3 4 18 16 1.12 53.3 3.6 4.3
D11 4 4 19 18 0.87 46.4 4.4 4.4 TERM 3 4 19 15 1.15 53.1 3.4
4.3
TABLE-US-00037 TABLE 33d Animal Group/ Clem. Beta Gamma Number Sex
Code % % 615 1 M PT 27.3 25.3 D11 23.2 26.2 TERM 24.4 23.9 465 2 M
PT 27.4 16.6 PD 21.8 14.7 D11 24.0 17.9 TERM 25.5 14.2 639 2 M PD
20.7 17.0 D11 21.5 18.7 TERM 23.2 14.9 613 3 M PT 24.5 16.3 D11
21.0 15.8 TERM 20.0 18.5 631 4 M PD 20.7 18.0 D11 22.8 22.0 TERM
22.1 17.1
TABLE-US-00038 TABLE 33e Animal Group/ Occn. ALP ALT AST Bili Urea
Creat Glue Chol Number Sex Code U/L U/L U/L .mu.mol/L mmol/L
.mu.mol/L mmol/L mmol/L 614 1F PT 687 71 50 3 4.92 60 4.45 2.77 D11
576 63 44 2 5.22 63 4.32 2.73 TERM 517 59 41 3 4.78 70 3.98 2.83
652 1F PT 598 43 25 3 7.26 69 3.26 2.42 D11 540 40 28 3 6.94 78
3.28 2.46 TERM 511 40 27 4 6.78 80 3.46 2.38 624 2F PD 533 56 35 3
3.85 73 3.91 2.31 D11 410 40 34 13 4.18 78 2.72 2.56 TERM 432 41 25
3 3.70 78 3.30 2.16 632 3F PT 559 35 34 5 5.44 80 3.08 2.47 D11 510
37 31 5 4.36 85 3.89 2.61 TERM 428 37 34 5 5.32 88 3.10 2.63 640 4F
PD 343 23 32 4 4.09 65 3.46 1.24 D11 292 25 28 4 4.12 63 2.69 1.13
TERM 266 22 27 2 4.55 69 3.69 1.00
TABLE-US-00039 TABLE 33f Total Animal Group/ Occn. Trig Na K Cl Ca
Phos Prot Alb Number Sex Code mmol/L mmol/L mmol/L mmol/L mmol/L
mmol/L g/L g/L 614 1F PT 0.37 148 4.6 109 2.69 2.03 80 39 D11 0.33
147 4.1 109 2.72 2.08 82 43 TERM 0.54 148 3.6 108 2.59 1.84 80 41
652 1F PT 0.57 147 4.3 105 2.58 1.52 78 35 D11 0.43 149 4.7 108
2.74 1.80 88 43 TERM 0.59 149 4.4 108 2.59 1.51 80 40 624 2F PD
0.60 145 4.1 108 2.54 1.50 77 40 D11 0.51 148 4.0 111 2.58 1.42 77
39 TERM 0.43 146 3.9 108 2.56 1.46 76 44 632 3F PT 0.31 151 4.5 109
2.48 1.72 76 34 D11 0.34 149 4.6 109 2.58 1.85 80 39 TERM 0.49 150
4.5 111 2.55 1.47 78 38 640 4F PD 0.36 144 4.8 111 2.31 1.45 68 29
D11 0.31 145 4.3 112 2.24 1.53 63 25 TERM 0.27 144 4.7 108 2.20
1.33 60 25
TABLE-US-00040 TABLE 33g Ani- mal Gam- Num- Group/ Occn. a1 a2 Beta
ma A/G Alb a1 a2 ber Sex Code g/L g/L g/L g/L Ratio % % % 614 1F PT
4 5 20 13 0.95 49.2 4.4 5.9 D11 3 4 19 13 1.10 52.7 3.3 4.9 TERM 3
4 18 13 1.05 51.7 3.8 5.2 652 1F PT 4 5 19 15 0.81 45.1 4.6 5.9 D11
3 5 20 17 0.96 49.3 3.4 5.5 TERM 4 5 18 14 1.00 49.4 4.7 6.2 624 2F
PD 3 4 16 14 1.08 52.2 4.1 5.4 D11 4 4 17 14 1.03 50.1 4.9 5.5 TERM
3 4 15 10 1.38 58.5 3.4 5.3 632 3F PT 4 4 19 15 0.81 44.7 5.1 5.0
D11 3 4 17 16 0.95 49.2 4.2 4.7 TERM 4 4 17 15 0.95 49.2 4.6 4.9
640 4F PD 4 4 17 15 0.74 42.2 5.7 5.6 D11 4 3 17 13 0.66 40.1 6.6
5.5 TERM 4 3 16 13 0.71 41.4 6.4 4.6
TABLE-US-00041 TABLE 33h Animal Group/ Clem. Beta Gamma Number Sex
Code % % 614 1 F PT 24.8 15.7 D11 23.1 16.0 TERM 22.8 16.5 652 1 F
PT 24.7 19.7 D11 22.3 19.4 TERM 22.1 17.6 624 2 F PD 20.7 17.6 D11
21.6 18.0 TERM 19.9 12.9 632 3 F PT 24.9 20.2 D11 21.5 20.4 TERM
21.8 19.5 640 4 F PD 24.9 21.6 D11 26.9 20.9 TERM 26.5 21.1
[0408] The Haematology Cumulative Individual Values for the monkeys
are presented in Table 34a-1, below:
TABLE-US-00042 TABLE 34a Animal Group/ Occn. Hct Hb RBC .times.
Retic MCH MCHC MCV Number Sex Code L/L g/dL 10.sup.12/L % pg g/dL
fL 615 1M PT 0.389 12.4 5.94 0.38 20.9 31.9 65.5 D11 0.366 11.5
5.59 0.76 20.6 31.4 65.5 TERM 0.381 11.8 5.91 0.25 20.0 31.0 64.5
465 2M PT 0.439 13.2 6.76 0.56 19.5 30.1 65.0 PTR PD 0.460 13.7
7.22 0.50 19.0 29.9 63.8 D11 0.391 11.7 6.11 1.62 19.1 29.9 64.0
TERM 0.441 13.6 6.93 0.65 19.7 30.9 63.7 639 2M PD 0.419 12.7 6.23
0.51 20.4 30.3 67.2 D11 0.400 11.7 5.99 0.52 19.6 29.3 66.7 TERM
0.388 12.2 5.70 1.14 21.4 31.4 68.1 613 3M PT 0.461 14.1 6.79 0.48
20.7 30.5 67.8 PTR D11 0.396 12.7 6.05 0.92 21.0 32.1 65.4 TERM
0.410 12.9 6.23 0.49 20.8 31.5 65.9
TABLE-US-00043 TABLE 34b Baso- Mono- Animal Group/ Occn. WBC
.times. N .times. L .times. E .times. phil .times. cyte .times. LUC
.times. Plt .times. Number Sex Code 10.sup.9/L 10.sup.9/L
10.sup.9/L 10.sup.9/L 10-9/L 10-9/L 10.sup.9/L 10.sup.9/L 615 1M PT
7.57 3.06 3.68 0.06 0.03 0.53 0.20 236 D11 7.56 2.78 4.35 0.06 0.02
0.21 0.13 284 TERM 7.93 3.77 3.52 0.06 0.02 0.49 0.07 254 465 2M PT
14.19 1.78 10.37 1.24 0.05 0.55 0.20 302 PTR 13.69 3.69 8.25 0.93
0.04 0.47 0.29 PD 13.36 1.55 9.95 1.01 0.04 0.58 0.25 325 D11 12.26
4.70 5.63 1.27 0.04 0.51 0.11 403 TERM 15.45 1.54 11.57 1.65 0.07
0.42 0.20 356 639 2M PD 10.02 5.21 3.42 0.90 0.01 0.39 0.10 306 D11
8.26 4.06 2.47 1.17 0.01 0.45 0.10 371 TERM 8.70 2.55 4.20 1.04
0.02 0.80 0.09 253 613 3M PT 20.21 12.99 6.45 0.02 0.04 0.50 0.21
438 PTR 16.85 8.87 6.90 0.08 0.06 0.67 0.26 D11 10.85 6.11 4.15
0.03 0.02 0.41 0.12 441 TERM 23.26 17.70 4.27 0.05 0.04 1.11 0.08
434
TABLE-US-00044 TABLE 34c Animal Group/ Clem. PT APTT Number Sex
Code sec sec 615 1 M PT 11.3 37.3 D11 10.3 32.7 TERM 11.3 33.3 465
2 M PT 10.0 33.9 PTR PD 9.9 26.1 D11 10.0 31.6 TERM 10.2 29.4 639 2
M PD 10.6 22.6 D11 10.5 26.3 TERM 10.3 28.9 613 3 M PT 10.3 35.5
PTR D11 11.4 26.7 TERM 10.3 30.8
TABLE-US-00045 TABLE 34d Animal Group/ Occn. Aniso- Micro- Macro-
Hypo- Hyper- Number Sex Code cytosis cytosis cytosis chromasia
chromasia 615 1M PT - - - - + D11 - - - - - TERM - - - - + 465 2M
PT - - - - - PTR PD - - - - - D11 - - - - - TERM - - - - - 639 2M
PD - - - - - D11 - - - - - TERM - - - - - 613 3M PT - - - - - PTR
D11 - - - - + TERM - - - - -
TABLE-US-00046 TABLE 34e Animal Group/ Occn. Hct Hb RBC .times.
Retic MCH MCHC MCV Number Sex Code L/L g/dL 10.sup.12/L % pg g/dL
fL 631 4M PD 0.460 13.6 7.11 0.43 19.1 29.5 64.6 D11 0.395 11.9
6.36 0.45 18.7 30.2 62.1 TERM 0.449 13.7 6.97 0.30 19.6 30.5 64.4
614 1F PT CTD CTD CTD CTD CTD CTD CTD PTR D11 0.404 13.1 6.33 0.57
20.6 32.3 63.8 TERM 0.424 13.0 6.59 0.68 19.7 30.7 64.3 652 1F PT
0.390 11.3 5.72 1.15 19.8 29.1 68.2 D11 0.374 11.9 5.55 1.06 21.4
31.7 67.4 TERM 0.384 11.5 5.71 0.58 20.2 30.0 67.2 624 2F PD 0.407
11.8 7.14 0.73 16.6 29.0 57.1 D11 0.377 10.8 6.69 0.48 16.1 28.6
56.3 TERM 0.401 11.7 6.99 1.07 16.7 29.1 57.4
TABLE-US-00047 TABLE 34f Animal Group/ Occn. WBC .times. N .times.
L .times. E .times. Baso- Mono- LUC .times. Plt .times. Number Sex
Code 10.sup.9/L 10.sup.9/L 10.sup.9/L 10.sup.9/L phil cyte
10.sup.9/L 10.sup.9/L 631 4M PD 10.53 2.82 6.36 0.62 0.02 0.58 0.13
378 D11 7.97 2.86 3.81 0.61 0.01 0.53 0.16 424 TERM 8.08 1.73 5.04
0.63 0.02 0.60 0.05 384 614 1F PT CTD CTD CTD CTD CTD CTD CTD CTD
PTR 11.32 3.47 6.36 0.17 0.05 0.96 0.31 D11 9.70 4.74 3.73 0.09
0.02 0.84 0.28 429 TERM 9.64 3.65 5.09 0.13 0.01 0.62 0.13 414 652
1F PT 10.66 3.21 6.05 0.42 0.03 0.80 0.15 373 D11 12.01 5.53 5.20
0.34 0.02 0.78 0.13 380 TERM 11.88 7.59 3.24 0.35 0.02 0.61 0.08
338 624 2F PD 9.06 3.10 5.02 0.41 0.02 0.33 0.18 362 D11 7.82 5.14
2.06 0.14 0.02 0.34 0.13 353 TERM 11.69 4.33 5.46 0.96 0.02 0.76
0.16 426
TABLE-US-00048 TABLE 34g Animal Group/ Clem. PT APTT Number Sex
Code sec sec 631 4 M PD 10.9 29.3 D11 11.3 27.9 TERM 10.8 32.1 614
1 F PT CTD CTD PTR D11 10.2 32.1 TERM 10.5 29.3 652 1 F PT 10.2
33.0 D11 9.6 27.9 TERM 10.3 30.3 624 2 F PD 10.6 27.1 D11 10.6 29.7
TERM 10.8 33.3
TABLE-US-00049 TABLE 34h Animal Group/ Occn. Aniso- Micro- Macro-
Hypo- Hyper- Number Sex Code cytosis cytosis cytosis chromasia
Chromasia 631 4M PD - - - - - D11 - - - - - TERM - - - - - 614 1F
PT CTD CTD CTD CTD CTD PTR D11 - - - - - TERM - - - - - 652 1F PT -
- - - - D11 - - - - - TERM - - - - - 624 2F PD - + - - - D11 - + -
- - TERM + + - - -
TABLE-US-00050 TABLE 34i Animal Group/ Occn. Hct Hb RBC .times.
Retic MCH MCHC MCV Number Sex Code L/L g/dL 10.sup.12/L % pg g/dL
fL 632 3F PT 0.416 12.4 6.36 0.83 19.6 29.9 65.4 PTR 0.410 12.2
6.29 0.64 19.5 29.8 65.3 D11 0.392 12.2 6.19 0.73 19.7 31.0 63.4
TERM 0.412 12.3 6.46 0.55 19.1 29.9 63.8 640 4F PD 0.398 11.8 5.81
0.95 20.3 29.7 68.5 D11 0.369 11.0 5.30 1.17 20.7 29.8 69.6 TERM
0.401 11.9 5.58 0.83 21.3 29.6 71.9
TABLE-US-00051 TABLE 34j Animal Group/ Occn. WBC .times. N .times.
L .times. E .times. Baso- Mono- LUC .times. Plt .times. Number Sex
Code 10.sup.9/L 10.sup.9/L 10.sup.9/L 10.sup.9/L phil cyte
10.sup.9/L 10.sup.9/L 632 3F PT 18.12 10.58 5.61 1.07 0.04 0.61
0.21 335 PTR 15.16 5.99 7.24 0.95 0.03 0.63 0.31 308 D11 10.23 4.69
4.15 0.66 0.01 0.48 0.23 348 TERM 13.04 6.89 4.62 0.44 0.03 0.93
0.13 321 640 4F PD 12.49 8.29 2.91 0.46 0.02 0.64 0.17 567 D11
13.71 10.31 2.18 0.34 0.01 0.74 0.12 578 TERM 11.49 5.41 4.18 0.63
0.03 1.12 0.12 555
TABLE-US-00052 TABLE 34k Animal Group/ Oeen. PT APTT Number Sex
Code sec sec 632 3 F PT 10.6 47.8 PTR 10.6 44.7 D11 10.2 33.1 TERM
10.6 37.2 640 4 F PD 12.0 25.8 D11 12.4 28.0 TERM 12.8 30.1
TABLE-US-00053 TABLE 34l Animal Group/ Occn. Aniso- Micro- Macro-
Hypo- Hyper- Number Sex Code cytosis cytosis cytosis chromasia
chromasia 632 3F PT - - - - - PTR - - - - - D11 - - - - - TERM - -
- - - 640 4F PD - - - - - D11 - - - - - TERM - - - - -
Microscopic Pathology--Treatment-Related Findings
[0409] Pericholangitis (inflammation of connective tissue around
the bile duct) was reported in the female monkey dosed at 12
mg/kg/day, but not in any other female or male monkeys. This
finding may be related to treatment with Glyco-mAb (Anti-EGFR), but
with such small numbers of animals the significance is uncertain.
All other findings were considered to be incidental and of no
toxicological significance.
Macropathology and Histopathology
[0410] The summary of histopatholigical for all animals tested is
set forth in Table 35, below:
TABLE-US-00054 TABLE 35 Histopathology - group distribution and
severity of findings for all animals Group 1 2 3 Compound
-GLYCO-MAB (ANTI-EGFR)- Dosage 1.5 4.5 12 Number of Animals
Affected Sex Male Female Group 1 2 3 1 2 3 Organ/Tissue Examined
Number 1 1 1 1 1 1 Colon No. Examined 1 1 1 1 1 1 Heart No.
Examined 1 1 1 1 1 1 Kidneys No. Examined 1 1 1 1 1 1 Cortical
Lymphocytic Minimal 1 1 0 0 1 1 Infiltration Slight 0 0 0 1 0 [?]
Total 1 1 0 1 1 1 Left Cephalic No. Examined 0 0 0 0 0 0 Left
Saphenous No. Examined 1 1 1 1 1 1 Epidermal Hyperplasia Minimal 0
0 0 1 0 0 Total 0 0 0 1 0 0 Liver No. Examined 1 1 1 1 1 1
Inflammatory Cell Foci Minimal 1 1 1 1 1 1 Total 1 1 1 1 1 1 Bile
Duct Proliferation Minimal 0 0 0 0 0 1 Total 0 0 0 0 0 1 Hepatocyte
Vacuolation - Minimal 0 1 0 0 0 0 Median Cleft Total 0 1 0 0 0 0
Pericholangitis Minimal 0 0 0 0 0 1 Total 0 0 0 0 0 1 Lungs &
Bronchi No. Examined 1 1 1 1 1 1 Bronchi/Bronchioles - Slight 1 0 0
0 0 0 Mucosal/Submucosal Inflammatory Cells Total 1 0 0 0 0 0
Alveolar Macrophages Minimal 0 1 0 0 0 1 Total 0 1 0 0 0 1
Perivascular Minimal 0 0 1 0 0 1 Inflammatory/Lymphoid Cells Total
0 0 1 0 0 1 Lymphoid Aggregates Minimal 0 0 1 0 0 0 Total 0 0 1 0 0
0 Oesophagus No. Examined 1 1 1 1 1 1 Lymphoid Aggregates Minimal 0
0 0 0 0 1 Total 0 0 0 0 0 1 Ovaries No. Examined 0 0 0 1 1 1
Follicular Cyst(S) Present 0 0 0 1 0 0 Total 0 0 0 1 0 0 Prominent
Corpora Present 0 0 0 0 1 0 Lutea Total 0 0 0 0 1 0 Pancreas No.
Examined 1 1 1 1 1 1 Acinar Atrophy Minimal 0 0 1 1 0 1 Total 0 0 1
1 0 1 Lymphoid Aggregates Minimal 0 0 0 1 0 0 Total 0 0 0 1 0 0
Right Cephalic No. Examined 0 0 0 0 0 0 Right Saphenous No.
Examined 0 0 0 0 0 0 Skin (Protocol) No. Examined 1 1 1 1 1 1
Epidermal Hyperplasia Minimal 0 0 0 0 0 1 Moderate 0 0 0 1 0 0
Total 0 0 0 1 0 1 Spinal Cord No. Examined 1 1 1 1 1 1 Haemorrhage
Minimal 0 0 1 1 1 1 Slight 0 1 0 0 0 0 Total 0 1 1 1 1 1 Spleen No.
Examined 1 1 1 1 1 1 Sternum & Marrow No. Examined 0 0 0 0 0 0
Stomach No. Examined 1 1 1 1 1 1 Testes No. Examined 1 1 1 0 0 0
Immaturity Present 1 1 1 0 0 0 Total 1 1 1 0 0 0 Thymus No.
Examined 1 1 1 1 1 1 Cyst(S) Present 0 0 0 0 1 0 Total 0 0 0 0 1 0
Involution/ Atrophy Minimal 0 0 0 0 1 0 Total 0 0 0 0 1 0 Urinary
Bladder No. Examined 1 1 1 1 1 1 Uterine Cervix No. Examined 0 0 0
1 1 1 Epithelial Mucification Present 0 0 0 1 1 1 Total 0 0 0 1 1 1
Uterus No. Examined 0 0 0 1 1 1 Congestion Minimal 0 0 0 0 1 0
Total 0 0 0 0 1 0 Caecum No. Examined 1 0 0 1 0 0 Prominent
Submucosal Minimal 1 0 0 1 0 0 Adipose Tissue Total 1 0 0 1 0 0
Fallopian Tube No. Examined 0 0 0 1 1 1 Ln Mesenteric No. Examined
0 0 0 1 0 0 Increased Pigmented Slight 0 0 0 1 0 0 Macrophages
Total 0 0 0 1 0 0
Individual Findings for All Animals
[0411] The pathology observations for individual animals are set
forth in Table 36, below:
TABLE-US-00055 TABLE 36 Macropathology and
histopathology--individual findings for all animals Group 1 2 3
Compound -GLYCO-MAB (ANTI-EGFR)- Dosage 1.5 4.5 12 Pathology
Observations Sex Male Dose Group 1 Animal No. 0623 Study week of
Sacrifice 11 Terminal body weight 2715.0 grams Study day of
sacrifice 77 NECROPSY HISTOPATHOLOGY Caecum: Caecum: Raised
Area(S); Mucosal Aspect, Prominent Submucosal Adipose Tissue,
Multiple, Up To 3 mm. Minimal, Focal Colon: Colon: Raised Area(S);
Mucosal Aspect, No Significant Lesion Multiple, Up To 2 mm.
Kidneys: Cortical Lymphocytic Infiltration, Minimal Liver: Liver:
Median Cleft Pale Area(S); One, Inflammatory Cell Foci, Minimal
Subcapsular, 3 mm. Lungs & Bronchi: Bronchi/Bronchioles
Mucosal/Submucosal Inflammatory Cells, Slight Stomach: Stomach:
Corpus Raised Area(S); Mucosa, >No Significant Lesion One, Near
To Antrum, 3 mm. Testes: Immaturity, Present Sex Male Dose Group 2
Animal No. 0461 Study week of Sacrifice 18 Terminal body weight
2573.0 grams Study day of sacrifice 125 NECROPSY HISTOPATHOLOGY
Kidneys Cortical Lymphocytic Infiltration, Minimal Liver: Liver
Median Cleft Pale Area(S); One, Inflammatory Cell Foci, Minimal
Subcapsular, 4 mm. Hepatocyte Vacuolation Median Cleft, Minimal
Lungs & Bronchi: Lungs & Bronchi: Incomplete Collapse;
Right Lobes. Alveolar Macrophages, Minimal Spinal Cord:
Haemorrhage, Slight, Multi-Focal Testes: Immaturity, Present Sex
Male Dose Group 3 Animal No. 0463 Study week of Sacrifice 18
Terminal body weight 2919.0 grams Study day of sacrifice 125
NECROPSY HISTOPATHOLOGY Liver: Inflammatory Cell Foci, Minimal
Lungs & Bronchi: Perivascular Inflammatory/Lymphoid Cells,
Minimal Lymphoid Aggregates, Minimal, Focal Pancreas: Acinar
Atrophy, Minimal, Focal Spinal Cord: Haemorrhage, Minimal Testes:
Immaturity, Present ***Animal has no gross observations recorded***
Sex Female Dose Group 1 Animal No. 0590 Study week of Sacrifice 11
Terminal body weight 3176.0 grams Study day of sacrifice 77
NECROPSY HISTOPATHOLOGY Caecum: Caecum: Raised Area(S); Mucosal
Aspect, Prominent Submucosal Adipose Tissue, Multiple, Up To 2mm.
Minimal, Multi-Focal Colon: Colon: -Raised Area(S); Mucosal Aspect,
>No Significant Lesion Multiple, Up To 2 mm. Kidneys: Cortical
Lymphocytic Infiltration, Slight, Focal Left Saphenous: Epidermal
Hyperplasia, Minimal Liver: Liver: Median Cleft Pale Area(S); One,
Inflammatory Cell Foci, Minimal Subcapsular, 3 mm. Ln Mesenteric:
Ln Mesenteric : Congested, Minimal Increased Pigmented Macrophages,
Slight Ovaries: Ovaries : Cyst(S); Left, One, Clear Fluid-Filled,
Follicular Cyst(S), Present 4 mm. Pancreas: Acinar Atrophy, Minimal
Lymphoid Aggregates, Minimal Skin (Protocol) : Epidermal
Hyperplasia, Moderate Spinal Cord: Haemorrhage, Minimal Spleen:
Spleen: Capsule Thickened; Area, Diffuse. >No Significant Lesion
Uterine Cervix: Epithelial Mucification, Present Sex Female Dose
Group 2 Animal No. 0462 Study week of Sacrifice 18 Terminal body
weight 2910.0 grams Study day of sacrifice 125 NECROPSY
HISTOPATHOLOGY Kidneys: Cortical Lymphocytic Infiltration, Minimal,
Focal Liver: Inflammatory Cell Foci, Minimal Lungs & Bronchi:
Lungs & Bronchi: Incomplete Collapse; Left Lobes. No
Significant Lesion Ovaries: Ovaries : Raised Area(S); One On Each,
Left, Prominent Corpora Lutea, Present 3 mm; Right, 2 mm.
(Follicles) Spinal Cord: Haemorrhage, Minimal Thymus: Thymus:
Small; 1.066 g. Cyst(S), Present Involution/Atrophy, Minimal
Uterine Cervix: Epithelial Mucification, Present Uterus: Uterus :
Congested, Minimal Congestion, Minimal Sex Female Dose Group 3
Animal No. 0612 Study week of Sacrifice 18 Terminal body weight
2934.0 grams Study day of sacrifice 125 NECROPSY HISTOPATHOLOGY
Kidneys: Cortical Lymphocytic Infiltration, Minimal, Focal Liver:
Liver: Median Cleft Pale Area(S); One, Inflammatory Cell Foci,
Minimal Subcapsular, 3 mm. Bile Duct Proliferation, Minimal
Cyst(S); Within Cleft, one, Dark Pericholangitis, Minimal
Fluid-Filled, Green, 2 mm. Lungs & Bronchi: Lungs &
Bronchi: Incomplete Collapse; Left Lobes. Alveolar Macrophages,
Minimal Perivascular Inflammatory/Lymphoid Cells, Minimal
Oesophagus: Lymphoid Aggregates, Minimal Pancreas: Acinar Atrophy,
Minimal, Focal Skin (Protocol): Epidermal Hyperplasia, Minimal
Spinal Cord: Haemorrhage, Minimal Uterine Cervix: Epithelial
Mucification, Present
[0412] Individual body weights of the cynomolgus monkeys are
presented in Table 37, below:
TABLE-US-00056 TABLE 37 Bodyweights: Individual Values Animal Body
weight (kg) on Day No. -17 -9 1* 8* 15* 22* 29 36 50 Group 1:
GA201-ge, 1.5 mg/kg/occasion 623m 2.67 2.65 2.69 2.72 2.76 2.62
2.72 2.65 2.71 2.73 (-0.02) (+0.04) (+0.03) (+0.04) (-0.14) (+0.10)
(-0.07) (+0.06) (+0.02) 590f 2.98 2.92 2.97 3.14 3.13 3.01 3.06
3.00 3.19 3.16 (-0.06) (+0.05) (+0.17) (-0.01) (-0.12) (+0.05)
(-0.06) (+0.19) (-0.03) Group 2: GA201-ge, 4.5 mg/kg/occasion 461m
2.50 2.56 2.53 2.47 2.53 2.53 2.53 2.61 2.53 2.45 (+0.06) (-0.03)
(-0.06) (+0.06) NC NC (+0.08) (-0.08) (-0.08) 462f 2.87 2.96 2.91
2.82 2.95 2.89 2.88 2.79 2.91 2.95 (+0.09) (-0.05) (-0.09) (+0.13)
(-0.06) (-0.01) (-0.09) (+0.12) (+0.04) Group 3: GA201-ge, 12
mg/kg/occasion 463m 2.69 2.81 2.86 2.74 2.81 2.81 2.81 2.81 2.75
2.71 (+0.12) (+0.05) (-0.12) (+0.07) NC NC NC (-0.06) (-0.04) 612f
2.84 2.96 2.92 2.98 3.06 3.01 3.01 2.94 2.91 2.81 (+0.12) (-0.04)
(+0.06) (+0.08) (-0.05) NC (-0.07) (-0.03) (-0.10) Body weight (kg)
on Day Animal Weight change (kg) No. 57 64 71 77 D 1 to 71 D 29 to
71 Group 1: GA201-ge, 1.5 mg/kg/occasion 623m 2.61 2.74 2.71 2.75
+0.06 +0.03 (-0.12) (+0.13) (-0.03) (+0.04) 590f 3.11 3.18 3.27
3.23 +0.27 +0.17 (-0.05) (+0.07) (+0.09) (-0.04) Group 2: GA201-ge,
4.5 mg/kg/occasion 461m 2.54 2.60 2.59 +0.03 +0.06 (+0.09) (+0.06)
(-0.01) 462f 3.05 3.02 2.96 +0.05 +0.08 (+0.10) (-0.03) (-0.06)
Group 3: GA201-ge, 12 mg/kg/occasion 463m 2.92 2.98 3.09 +0.23
+0.28 (+0.21) (+0.06) (+0.11) 612f 3.04 3.05 3.10 +0.18 +0.09
(+0.23) (+0.01) (0.05)
CONCLUSIONS
[0413] There was no effect of treatment at the injection sites and
no clinical findings considered to be related to treatment with
Glyco-mAb (anti-EGFR). Bodyweight changes were within normal
expected ranges. There were no findings considered to be related to
treatment at macroscopic examination and organ weights of animals
were within normal expected ranges. In conclusion, treatment at
1.5, 4.5 or 12 mg/kg/occasion was well tolerated with no clear
findings of systemic toxicity.
[0414] EGFR is not a tumor specific target, since it is present on
the surface of various normal tissues including liver, kidney and
skin. Anti-EGFR antibodies with human IgG1 Fc region have
previously been administered to humans and have shown a tolerable
side-effect profile (Vanhoefer, U. et al., Clin. Oncol. 2004 Jan.
1; 22(1):175-84; Needle M N, Semin Oncol. 2002 October; 29 (5 Suppl
14):55-60). Clearly, there would be significant concerns for
administering to a human or other mammal an anti-EGFR antibody with
significantly increased ADCC, due to enhanced killing activity that
could be displayed against critical normal tissues such as liver,
kidney and skin. Surprisingly, the present inventors have found
that administering such an anti-EGFR antibody, Fc engineered as
described above and with up to 1000-fold increased ADCC activity,
in vivo to mammals did not lead to significant toxicities. The
concentrations of antibody were kept above 1 microgram per
milliliter for at least 4 weeks (and above 100 micrograms per
milliliter for some animals). Such exposure levels are typical for
antibody therapy. Maximal ADCC for the antibody of this study is
already achieved at concentrations of 1 microgram per milliliter.
Single dose administrations of doses of 40 and 100 mg of anti-EGFR
antibody (the parental rat ICR62 antibody) to human cancer patients
have shown specific targeting of tumors in vivo (Modjtahedi, H. et
al., Br J Cancer. 1996 January; 73(2):228-35.). Cynomolgus monkey
effector cells have highly-homologous FcgammaRIII receptor and have
been shown to mediate enhanced ADCC with Fc engineered antibodies
(and with antibodies glycoengineered for increased levels of
non-fucosylated oligosaccharides in the Fc region). The level of
ADCC increase is very similar to that observed with human effector
cells (PBMCs).
[0415] In summary, we have found that anti-EGFR antibodies Fc
engineered for increased Fc-FcgammaRIII binding affinity and for
increased ADCC can be administered to mammals to give
concentrations above 1 microgram of antibody per milliliter of
serum for a period of at least 4 weeks in order to give drug
exposures normally associated with significant accumulation of
antibody on target cells in vivo, without leading to significant
toxicity.
[0416] Toxicity of an antigen binding molecule of the present
invention can be measured and/or determined using any of the
methods and/or parameters (e.g. blood chemistry values,
histopathological indicators, etc.) described herein above, or by
any means known to those of skill in the art. A clinically
significant level of toxicity is understood by one of skill in the
art to be a level that exceeds levels generally accepted by the
U.S. Food and Drug Administration for antibodies administered
clinically.
Example 5
Modifications to the Light Chain CDRs
[0417] Using methods described above, anti-EGFR light chain
variable region variants were generated from the I-KC light chain
variable region construct (SEQ ID NO:43 and SEQ ID NO:45), wherein
the sequence encoding the amino acid residue at varous positions in
the rat ICR62 CDRs were replaced with the corresponding amino acid
residue from a human germline variable gene sequence. Table 38
shows the substitutions that were made within the CDRs of the I-KC
light chain variable region construct (SEQ ID NO:45):
TABLE-US-00057 TABLE 38 Minimized Light Chain CDRs AMINO ACID LIGHT
CHAIN CDR SUBSTITUTION IN WHICH NAME OF MADE IN SUBSTITUTION WAS
CONSTRUCT SEQ ID NO: 45 MADE I-KC1 N30R* CDR1 I-KC2 Y32W CDR1 I-KC3
N34G CDR1 I-KC4 N50T CDR2 I-KC5 T51A CDR2 I-KC6 N52S CDR2 I-KC7
N53S CDR2 I-KC8 T56S CDR2 I-KC9 F94Y CDR3 * Identified according to
standard nomenclature (e.g., "N30R" means the Asparagine (N)
residue at position 30 of SEQ ID NO: 45 is replaced with an
argininen (R) residue).
[0418] All substitution residues identified above were derived from
the human VK1_6 acceptor sequence except for the Y32W exchange,
wherein the W of a related human germline sequence was substituted
for the Y at position 32 in SEQ ID NO:45.
[0419] Each of the I-KC variant constructs (I-KC1 to I-KC9) was
paired with a heavy chain variable region comprising construct
I-HHD (SEQ ID NO:16 and SEQ ID NO:15) and a binding assay performed
according to the methods described in the previous examples.
Constructs I-KC1 to I-KC9 were compared to the I-KC construct (SEQ
ID NO:46 and SEQ ID NO:45) for binding affinity to EGFR in A431
target cells (FIG. 29). As seen in FIG. 29, only the modfication of
residue 34 to its corresponding human sequence (N34G) resulted in a
slight decrease in binding affinity (EC50 value increased by a
factor of 10). All other constructs retained binding activity
comparable to the I-KC construct (SEQ ID NO:45). Therefore, when
paired with a chimeric (e.g., humanized) heavy chain construct
specific for EGFR, the light chain can be entirely human (e.g.,
from a human light chain V gene sequence) and still retain specific
binding for EGFR. In particular, CDR2 and CDR3 can be entirely in
human germline form.
[0420] Antigen Binding Molecules Comprising EGFR-Specific CDRs
[0421] The present invention therefore contemplates an antigen
binding molecule comprising a chimeric (e.g., humanized) heavy
chain variable region comprising EGFR-specific CDRs paired with a
light chain variable region, wherein the light chain variable
region has fewer than ten non-human amino acid residues. In other
embodiments, the light chain variable region has fewer than nine,
eight seven, six, five, four, three, two, or one non-human amino
acid residue(s). In preferred embodiments, the light chain variable
region has fewer than two or fewer than one (i.e., no) non-human
amino acid residues. In one embodiment, the light chain variable
region comprises one or more human germline variable region gene
sequences. Human germline variable region gene sequences encoding
light chain variable regions are known in the art, and can be
found, for example, in the IMGT database, available at
http://imgt.cines.fr/home.html. In a preferred embodiment, the
human germline sequence is derived from the VK1_6 germline
sequence. In other embodiments, amino acid residues within the
human germline light chain variable region amino acid sequence can
be substituted with one or more residues from another human
germline light chain variable region sequence.
[0422] In one embodiment, the present invention is directed to an
antigen binding molecule comprising a sequence selected from the
group consisting of SEQ ID NO.:1; SEQ ID No:3; SEQ ID No:5; SEQ ID
No:7; SEQ ID No:9; SEQ ID No:11; SEQ ID No:13; SEQ ID No:15; SEQ ID
No:17; SEQ ID No:19; SEQ ID No:21; SEQ ID No:23; SEQ ID No:25; SEQ
ID No:27; SEQ ID No:29; SEQ ID No:31; SEQ ID No33; SEQ ID No:35;
SEQ ID No:37; SEQ ID No:39; and SEQ ID No:121, and a light chain
comprising a polypeptide encoded by one or more human germline
variable gene sequence. In a preferred embodiment, the human
germline sequence is derived from the VK1_6 germline sequence.
[0423] In another embodiment, the present invention is directed to
an antigen binding molecule comprising a sequence selected from the
group consisting of: SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ
ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68,
SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:122, and SEQ ID
NO:124; (b) a sequence selected from a group consisting of: SEQ ID
NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ
ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94,
SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID
NO:104, SEQ ID NO:106, and SEQ ID NO:126; (c) SEQ ID NO:108; and
(d) a polypeptide comprising a human light chain variable region
encoded by one or more human germline gene sequences. In a
particular embodiment, the human germline sequence is derived from
the VK1_6 germline sequence. In another embodiment, the human
germline variable region gene sequence comprises the VK1_6 germline
gene sequence with a substitution of one or more amino acid codons
with a sequence from a different human germline light chain
variable region gene sequence.
[0424] In other embodiments, the antigen binding molecule of the
present invention comprises an EGFR-specific heavy chain variable
region of the present invention, and a variant of SEQ ID NO:45. In
one embodiment, the variant of SEQ ID NO:45 comprises an amino acid
substitution at one or more positions in the complementarity
determining regions (CDRs). In specific embodiments, the
substitution is of an amino acid residue at a position selected
from the group consisting of: amino acid position 30 of SEQ ID
NO:45; amino acid position 32 of SEQ ID NO:45; amino acid position
34 of SEQ ID NO:45; amino acid position 50 of SEQ ID NO:45; amino
acid position 51 of SEQ ID NO:45; amino acid position 52 of SEQ ID
NO:45; amino acid position 53 of SEQ ID NO:45; amino acid position
56 of SEQ ID NO:45; amino acid position 94 of SEQ ID NO:45; and any
combination of substitutions thereof. In more specific embodiments,
the substitution in SEQ ID NO:45 is selected from the group
consisting of: substitution of an arginine (R) for the asparagine
(N) at position 30 of SEQ ID NO:45; substitution of a tryptophan
(W) for the tyrosine (Y) at position 32 of SEQ ID NO:45;
substitution of a glycine (G) for the asparagine (N) at position 34
of SEQ ID NO:45; substitution of a threonine (T) for the asparagine
(N) at position 50 of SEQ ID NO:45; substitution of an alanine (A)
for the threonine (T) at position 51 of SEQ ID NO:45; substitution
of a serine (S) for the asparagine (N) at position 52 of SEQ ID
NO:45; substitution of a serine (S) for the asparagine (N) at
position 53 of SEQ ID NO:45; substitution of a serine (S) for the
threonine (T) at position 56 of SEQ ID NO:45; substitution of a
tyrosine (Y) for the phenylalanine (F) at position 94 of SEQ ID
NO:45; and any combination thereof. In a particular embodiment, all
of these substitutions of amino acid residues in SEQ ID NO:45 are
incorporated in a single light chain variant. In preferred
embodiments, antigen binding molecules comprising the light chain
variants with amino acid substitutions for the ICR62 CDRs retain
specific binding to EGFR (as compared to an antigen binding
molecule comprising a light chain variable region comprising the
sequence of SEQ ID NO:45) when the light chain variant is paired
with a polypeptide comprising a heavy chain variable region of the
present invention.
[0425] The present invention is also directed to polynucleotides
that encode any of the above polypeptides and/or antigen binding
molecules. The present invention is further directed to the antigen
binding molecules described above, with a pharmaceutically
acceptable carrier.
[0426] All publications such as textbooks, journal articles,
GenBank or other sequence database entries, published applications,
and patent applications mentioned in this specification are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference.
Sequence CWU 1
1
1271120PRTRattus sp.ICR62 VH 1Gln Val Asn Leu Leu Gln Ser Gly Ala
Ala Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Gly
Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Lys Ile His Trp Val Lys Gln
Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45Gly Tyr Phe Asn Pro Asn
Ser Gly Tyr Ser Thr Tyr Asn Glu Lys Phe 50 55 60Lys Ser Lys Ala Thr
Leu Thr Ala Asp Lys Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu
Thr Ser Leu Thr Ser Glu Asp Ser Ala Thr Tyr Tyr Cys 85 90 95Thr Arg
Leu Ser Pro Gly Gly Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105
110Gly Ala Ser Val Thr Val Ser Ser 115 1202359DNARattus sp.ICR62 VH
2caggtcaacc tactgcagtc tggggctgca ctggtgaagc ctggggcctc tgtgaagttg
60tcttgcaaag gttctggttt tacattcact gactacaaga tacactgggt gaagcagagt
120catggaaaga gccttgagtg gattgggtat tttaatccta acagtggtta
tagtacctac 180aatgaaaagt tcaagagcaa ggccacattg actgcagaca
aatccaccga tacagcctat 240atggagctta ccagtctgac atctgaggac
tctgcaacct attactgtac aagactatcc 300ccagggggtt actatgttat
ggatgcctgg ggtcaaggag cttcagtcac tgtctcctc 3593120PRTArtificial
SequenceSynthetic heavy chain variable region construct 3Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Gly Ile Asn Pro Asn Ser Gly Tyr Ser Thr Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met
Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
1204360DNAArtificial SequenceSynthetic heavy chain variable region
construct 4caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc
ggtgaaggtc 60tcctgcaagg cttctggatt tacattcact gactacgcca tcagctgggt
gcgacaggcc 120cctggacaag ggctcgagtg gatgggaggg atcaatccta
acagtggtta tagtacctac 180gcacagaagt tccagggcag ggtcaccatt
accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagactatcc 300ccaggcggtt
actatgttat ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca
3605120PRTArtificial SequenceSynthetic heavy chain variable region
construct 5Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Gly Ser Gly Phe Thr Phe
Thr Asp Tyr 20 25 30Lys Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Tyr Phe Asn Pro Asn Ser Gly Tyr Ser Thr
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly
Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr
Val Ser Ser 115 1206360DNAArtificial SequenceSynthetic heavy chain
variable region construct 6caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg gttctggttt tacattcact
gactacaaga tacactgggt gcgacaggcc 120cctggacaag ggctcgagtg
gatgggatat ttcaacccta acagcggtta tagtacctac 180gcacagaagt
tccagggcag ggtcaccatt accgcggaca aatccacgag cacagcctac
240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc
gagactatcc 300ccaggcggtt actatgttat ggatgcctgg ggccaaggga
ccaccgtgac cgtctcctca 3607120PRTArtificial SequenceSynthetic heavy
chain variable region construct 7Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Gly Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Lys Ile His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Phe Asn Pro
Asn Ser Gly Tyr Ser Thr Tyr Asn Glu Lys Phe 50 55 60Lys Ser Arg Val
Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 1208360DNAArtificial
SequenceSynthetic heavy chain variable region construct 8caggtgcagc
tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg
gttctggttt tacattcact gactacaaga tacactgggt gcgacaggcc
120cctggacaag ggctcgagtg gatgggatat ttcaacccta acagcggtta
tagtacctac 180aatgaaaagt tcaagagcag ggtcaccatt accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagactatcc 300ccaggcggtt actatgttat
ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca 3609120PRTArtificial
SequenceSynthetic heavy chain variable region construct 9Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Tyr Ser Thr Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Ile Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met
Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
12010360DNAArtificial SequenceSynthetic heavy chain variable region
construct 10caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc
ggtgaaggtc 60tcctgcaagg cctctggttt tacattcact gactactata tgcactgggt
gcgacaggcc 120cctggacaag ggctcgagtg gatgggctgg atcaatccta
acagtggtta tagtacctac 180gcacagaagt ttcagggcag ggtcaccatg
accgccgaca cgtccatcag cacagcctac 240atggagctga gcaggctgag
atctgacgac acggccgtgt attactgtgc gagactatcc 300ccaggcggtt
actatgttat ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca
36011120PRTArtificial SequenceSynthetic heavy chain variable region
construct 11Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Gly Ser Gly Phe Thr Phe
Thr Asp Tyr 20 25 30Lys Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Tyr Phe Asn Pro Asn Ser Gly Tyr Ser Thr
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Ala Asp Thr
Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly
Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr
Val Ser Ser 115 12012360DNAArtificial SequenceSynthetic heavy chain
variable region construct 12caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctggagcctc ggtgaaggtc 60tcctgcaagg gttctggttt tacattcact
gactacaaga tccactgggt gcgacaggcc 120cctggacaag ggctcgagtg
gatgggatac ttcaacccta acagcggtta tagtacctac 180gcacagaagt
tccagggcag ggtcaccatg accgccgaca cgtccatcag cacagcctac
240atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc
gagactatcc 300ccaggcggtt actatgttat ggatgcctgg ggccaaggga
ccaccgtgac cgtctcctca 36013120PRTArtificial SequenceSynthetic heavy
chain variable region construct 13Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Gly Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Lys Ile His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Phe Asn Pro
Asn Ser Gly Tyr Ser Thr Tyr Asn Glu Lys Phe 50 55 60Lys Ser Arg Val
Thr Met Thr Ala Asp Thr Ser Ile Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 12014360DNAArtificial
SequenceSynthetic heavy chain variable region construct
14caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggagcctc agtgaaggtc
60tcctgcaagg gttctggttt tacattcact gactacaaga tccactgggt gcgacaggcc
120cctggacaag ggctcgagtg gatgggatac ttcaacccta acagcggtta
cagtacttac 180aacgagaagt tcaagagccg ggtcaccatg accgccgaca
cgtccatcag cacagcctac 240atggagctga gcaggctgag atctgacgac
acggccgtgt attactgtgc gagactatcc 300ccagggggtt actatgttat
ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca 36015120PRTArtificial
SequenceSynthetic heavy chain variable region construct 15Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30Lys Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Tyr Phe Asn Pro Asn Ser Gly Tyr Ser Thr Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met
Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
12016360DNAArtificial SequenceSynthetic heavy chain variable region
construct 16caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc
ggtgaaggtc 60tcctgcaagg cctctggttt cacattcact gactacaaga tacactgggt
gcgacaggcc 120cctggacaag ggctcgagtg gatgggatat ttcaacccta
acagcggtta tagtacctac 180gcacagaagt tccagggcag ggtcaccatt
accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagactatcc 300ccaggcggtt
actatgttat ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca
36017120PRTArtificial SequenceSynthetic heavy chain variable region
construct 17Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Gly Ser Gly Phe Thr Phe
Thr Asp Tyr 20 25 30Lys Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Tyr Phe Asn Pro Asn Ser Gly Tyr Ser Thr
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly
Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr
Val Ser Ser 115 12018360DNAArtificial SequenceSynthetic heavy chain
variable region construct 18caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg gttctggttt cacattcact
gactacaaga tatcctgggt gcgacaggct 120cctggacaag ggctcgagtg
gatgggatat ttcaacccta acagcggtta tagtacctac 180gcacagaagt
tccagggcag ggtcaccatt accgcggaca aatccacgag cacagcctac
240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc
gagactatcc 300ccaggcggtt actatgttat ggatgcctgg ggccaaggga
ccaccgtgac cgtctcctca 36019120PRTArtificial SequenceSynthetic heavy
chain variable region construct 19Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Gly Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Lys Ile His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Phe Asn Pro
Asn Ser Gly Tyr Ser Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 12020360DNAArtificial
SequenceSynthetic heavy chain variable region construct
20caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60tcctgcaagg gttctggttt tacattcact gactacaaga tacactgggt gcgacaggcc
120cctggacaag ggctcgagtg gatgggatat ttcaacccta acagcggtta
ttcgaactac 180gcacagaagt tccagggcag ggtcaccatt accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagactatcc 300ccaggcggtt actatgttat
ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca 36021120PRTArtificial
SequenceSynthetic heavy chain variable region construct 21Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser
Val Lys Val Ser Cys Lys Gly Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30Lys Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Tyr Phe Asn Pro Asn Ser Gly Tyr Ala Thr Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met
Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
12022360DNAArtificial SequenceSynthetic heavy chain variable region
construct 22caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc
ggtgaaggtc 60tcctgcaagg gttctggttt tacattcact gactacaaga tacactgggt
gcgacaggcc 120cctggacaag ggctcgagtg gatgggatat ttcaacccta
acagcggtta tgccacgtac 180gcacagaagt tccagggcag ggtcaccatt
accgcggaca aatccacgag cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgtgt attactgtgc gagactatcc 300ccaggcggtt
actatgttat ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca
36023120PRTArtificial SequenceSynthetic heavy chain variable region
construct 23Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
Thr Asp Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Tyr Ser Thr
Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys
Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala
Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly
Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr
Val Ser Ser 115 12024360DNAArtificial SequenceSynthetic heavy chain
variable region construct 24caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctggagcctc ggtgaaggtc 60tcctgcaagg cctctggttt tacattcact
gactactata tgcactgggt gcgacaggcc 120cctggacaag ggctcgagtg
gatgggctgg atcaatccta acagtggtta tagtacctac 180agcccaagct
tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac
240ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc
gagactatcc 300ccaggcggtt actatgttat ggatgcctgg ggccaaggga
ccaccgtgac cgtctcctca 36025120PRTArtificial SequenceSynthetic heavy
chain variable region construct 25Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro
Asn Ser Gly Tyr Ser Thr Tyr Asn Glu
Lys Phe 50 55 60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser
Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr
Ala Met Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly Tyr Tyr Val
Met Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser
115 12026360DNAArtificial SequenceSynthetic heavy chain variable
region construct 26caggtgcagc tggtgcagtc tggggctgag gtgaagaagc
ctggagcctc ggtgaaggtc 60tcctgcaagg cctctggttt tacattcact gactactata
tgcactgggt gcgacaggcc 120cctggacaag ggctcgagtg gatgggctgg
atcaatccta acagtggtta tagtacctac 180aacgagaagt tccaaggcca
ggtcaccatc tcagccgaca agtccatcag caccgcctac 240ctgcagtgga
gcagcctgaa ggcctcggac accgccatgt attactgtgc gagactatcc
300ccaggcggtt actatgttat ggatgcctgg ggccaaggga ccaccgtgac
cgtctcctca 36027120PRTArtificial SequenceSynthetic heavy chain
variable region construct 27Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser
Gly Tyr Ser Thr Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile
Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser
Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Leu
Ser Pro Gly Gly Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105 110Gly
Thr Thr Val Thr Val Ser Ser 115 12028360DNAArtificial
SequenceSynthetic heavy chain variable region construct
28caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggagcctc ggtgaaggtc
60tcctgcaagg cctctggtta cacattcact gactactata tgcactgggt gcgacaggcc
120cctggacaag ggctcgagtg gatgggctgg atcaatccta acagtggtta
tagtacctac 180agcccaagct tccaaggcca ggtcaccatc tcagccgaca
agtccatcag caccgcctac 240ctgcagtgga gcagcctgaa ggcctcggac
accgccatgt attactgtgc gagactatcc 300ccaggcggtt actatgttat
ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca 36029120PRTArtificial
SequenceSynthetic heavy chain variable region construct 29Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Tyr Ser Thr Tyr Asn Glu Lys
Phe 50 55 60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr
Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala
Met Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met
Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
12030360DNAArtificial SequenceSynthetic heavy chain variable region
construct 30caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggagcctc
ggtgaaggtc 60tcctgcaagg cctctggtta cacattcact gactactata tgcactgggt
gcgacaggcc 120cctggacaag ggctcgagtg gatgggctgg atcaatccta
acagtggtta tagtacctac 180aacgagaagt tccaaggcca ggtcaccatc
tcagccgaca agtccatcag caccgcctac 240ctgcagtgga gcagcctgaa
ggcctcggac accgccatgt attactgtgc gagactatcc 300ccaggcggtt
actatgttat ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca
36031120PRTArtificial SequenceSynthetic heavy chain variable region
construct 31Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro
Gly Thr1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
Thr Asp Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Tyr Ser Thr
Tyr Ser Pro Ser Phe 50 55 60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys
Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala
Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly
Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr
Val Ser Ser 115 12032360DNAArtificial SequenceSynthetic heavy chain
variable region construct 32cagatgcagc tggtgcagtc tgggccagag
gtgaagaagc ctggaacctc ggtgaaggtc 60tcctgcaagg cctctggttt tacattcact
gactactata tgcactgggt gcgacaggcc 120cctggacaag ggctcgagtg
gatgggctgg atcaatccta acagtggtta tagtacctac 180agcccaagct
tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac
240ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc
gagactatcc 300ccaggcggtt actatgttat ggatgcctgg ggccaaggga
ccaccgtgac cgtctcctca 36033120PRTArtificial SequenceSynthetic heavy
chain variable region construct 33Gln Met Gln Leu Val Gln Ser Gly
Pro Glu Val Lys Lys Pro Gly Thr1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asn Pro
Asn Ser Gly Tyr Ser Thr Tyr Asn Glu Lys Phe 50 55 60Gln Gly Gln Val
Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75 80Leu Gln
Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala
Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 12034360DNAArtificial
SequenceSynthetic heavy chain variable region construct
34cagatgcagc tggtgcagtc tgggccagag gtgaagaagc ctggaacctc ggtgaaggtc
60tcctgcaagg cctctggttt tacattcact gactactata tgcactgggt gcgacaggcc
120cctggacaag ggctcgagtg gatgggctgg atcaatccta acagtggtta
tagtacctac 180aacgagaagt tccaaggcca ggtcaccatc tcagccgaca
agtccatcag caccgcctac 240ctgcagtgga gcagcctgaa ggcctcggac
accgccatgt attactgtgc gagactatcc 300ccaggcggtt actatgttat
ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca 36035120PRTArtificial
SequenceSynthetic heavy chain variable region construct 35Gln Met
Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25
30Lys Ile His Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile
35 40 45Gly Trp Ile Asn Pro Asn Ser Gly Tyr Ser Thr Tyr Asn Glu Lys
Phe 50 55 60Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr
Ala Tyr65 70 75 80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala
Met Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met
Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
12036360DNAArtificial SequenceSynthetic heavy chain variable region
construct 36cagatgcagc tggtgcagtc tgggccagag gtgaagaagc ctggaacctc
ggtgaaggtc 60tcctgcaagg cctctggttt tacattcact gactacaaga tccactgggt
gcgacaggcc 120cgcggacaac ggctcgagtg gatcggctgg atcaatccta
acagtggtta tagtacctac 180aacgagaagt tccaaggcca ggtcaccatc
tcagccgaca agtccatcag caccgcctac 240ctgcagtgga gcagcctgaa
ggcctcggac accgccatgt attactgtgc gagactatcc 300ccaggcggtt
actatgttat ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca
36037120PRTArtificial SequenceSynthetic heavy chain variable region
construct 37Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro
Gly Thr1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe
Thr Asp Tyr 20 25 30Lys Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Tyr Phe Asn Pro Asn Ser Gly Tyr Ser Thr
Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly Gly
Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val Thr
Val Ser Ser 115 12038360DNAArtificial SequenceSynthetic heavy chain
variable region construct 38cagatgcagc tggtgcagtc tgggccagag
gtgaagaagc ctggaacctc ggtgaaggtc 60tcctgcaagg cctctggttt tacattcact
gactacaaga tccactgggt gcgacaggcc 120cctggacaag ggctcgagtg
gatgggatat ttcaacccta acagcggtta tagtacctac 180gcacagaagt
tccagggcag ggtcaccatt accgcggaca aatccacgag cacagcctac
240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc
gagactatcc 300ccaggcggtt actatgttat ggatgcctgg ggccaaggga
ccaccgtgac cgtctcctca 36039120PRTArtificial SequenceSynthetic heavy
chain variable region construct 39Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser Cys Lys
Gly Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Lys Ile His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Phe Asn Pro
Asn Ser Gly Tyr Ser Thr Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 12040360DNAArtificial
SequenceSynthetic heavy chain variable region construct
40gaggtgcagc tcgtgcagtc tggcgctgag gtgaagaagc ctggcgagtc gttgaagatc
60tcctgcaagg gttctggtta ttcattcact gactacaaga tccactgggt gcgacaggcc
120cctggacaag ggctcgagtg gatgggatat ttcaacccta acagcggtta
tagtacctac 180gcacagaagt tccagggcag ggtcaccatt accgcggaca
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc gagactatcc 300ccaggcggtt actatgttat
ggatgcctgg ggccaaggga ccaccgtgac cgtctcctca 3604119PRTArtificial
SequenceSignal Sequence 41Met Asp Trp Thr Trp Arg Ile Leu Phe Leu
Val Ala Ala Ala Thr Gly1 5 10 15Ala His Ser4257DNAArtificial
SequenceSignal Sequence 42atggactgga cctggaggat cctcttcttg
gtggcagcag ccacaggagc ccactcc 5743108PRTRattus sp.ICR62 VL 43Asp
Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Asn Cys Lys Ala Ser Gln Asn Ile Asn Asn Tyr
20 25 30Leu Asn Trp Tyr Gln Gln Lys Leu Gly Glu Ala Pro Lys Arg Leu
Ile 35 40 45Tyr Asn Thr Asn Asn Leu Gln Thr Gly Ile Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Phe Cys Leu Gln His
Asn Ser Phe Pro Thr 85 90 95Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
Arg Thr 100 10544324DNARattus sp.ICR62 VL 44gacatccaga tgacccagtc
tccttcattc ctgtctgcat ctgtgggaga cagagtcact 60atcaactgca aagcaagtca
gaatattaac aattacttaa actggtatca gcaaaagctt 120ggagaagctc
ccaaacgcct gatatataat acaaacaatt tgcaaacagg catcccatca
180aggttcagtg gcagtggatc tggtacagat tacacactca ccatcagcag
cctgcagcct 240gaagattttg ccacatattt ctgcttgcag cataatagtt
ttcccacgtt tggagctggg 300accaagctgg aactgaaacg tacg
32445108PRTArtificial SequenceSynthetic light chain variable region
construct 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 Gly Ile
Asn Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Arg Leu Ile 35 40 45Tyr Asn Thr Asn Asn Leu Gln Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Leu Gln His Asn Ser Phe Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg Thr 100 10546324DNAArtificial SequenceSynthetic
light chain variable region construct 46gatatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtcggaga ccgggtcacc 60atcacctgcc gggcaagtca
gggcattaac aattacttaa attggtacca gcagaagcca 120gggaaagccc
ctaagcgcct gatctataat accaacaact tgcagacagg cgtcccatca
180aggttcagcg gcagtggatc cgggacagaa ttcactctca ccatcagcag
cctgcagcct 240gaagattttg ccacctatta ctgcttgcag cataatagtt
ttcccacgtt tggccagggc 300accaagctcg agatcaagcg tacg
3244722PRTArtificial SequenceSignal Sequence 47Met Asp Met Arg Val
Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro Gly Ala
Arg Cys 204866DNAArtificial SequenceSignal Sequence 48atggacatga
gggtccccgc tcagctcctg ggcctcctgc tgctctggtt cccaggtgcc 60aggtgt
6649109PRTArtificial SequenceSynthetic light chain variable region
construct 49Asp 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 Gly Ile
Asn Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Arg Leu Ile 35 40 45Tyr Asn Thr Asn Asn Leu Gln Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Leu Gln His Asn Ser Phe Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg Thr Val 100 10550327DNAArtificial SequenceSynthetic
light chain variable region construct 50gatatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtcggaga ccgggtcacc 60atcacctgcc gggcaagtca
gggcattaac aattacttaa attggtacca gcagaagcca 120gggaaagccc
ctaagcgcct gatctataat accaacaact tgcagacagg cgtcccatca
180aggttcagcg gcagtggatc cgggacagaa tacactctca ccatcagcag
cctgcagcct 240gaagattttg ccacctatta ctgcttgcag cataatagtt
ttcccacgtt tggccagggc 300accaagctcg agatcaagcg tacggtg
32751109PRTArtificial SequenceSynthetic light chain variable region
construct 51Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Ile
Asn Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Arg Leu Ile 35 40 45Tyr Asn Thr Asn Asn Leu Gln Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Leu Gln His Asn Ser Phe Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg Thr Val 100 10552327DNAArtificial SequenceSynthetic
light chain variable region construct 52gatatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtcggaga ccgggtcacc 60atcacctgca aagcaagtca
gaatattaac aattacttaa actggtacca gcagaagcca 120gggaaagccc
ctaagcgcct gatctataat accaacaact tgcagacagg cgtcccatca
180aggttcagcg gcagtggatc cgggacagaa tacactctca ccatcagcag
cctgcagcct 240gaagattttg ccacctatta ctgcttgcag cataatagtt
ttcccacgtt tggccagggc 300accaagctcg agatcaagcg tacggtg
327535PRTArtificial SequenceHeavy Chain CDR1 Kabat 53Asp Tyr Lys
Ile His1 55415DNAArtificial SequenceHeavy Chain CDR1 Kabat
54gactacaaga tacac 15555PRTArtificial SequenceHeavy Chain CDR1
Kabat 55Asp Tyr Ala Ile Ser1 55615DNAArtificial SequenceHeavy Chain
CDR1 Kabat 56gactacgcca tcagc 15575PRTArtificial SequenceHeavy
Chain CDR1 Kabat 57Asp Tyr Tyr Met His1 55815DNAArtificial
SequenceHeavy Chain CDR1 Kabat 58gactactata tgcac
15597PRTArtificial SequenceHeavy Chain CDR1 Chothia 59Gly Phe Thr
Phe Thr Asp Tyr1 56021DNAArtificial SequenceHeavy Chain CDR1
Chothia 60ggttttacat tcactgacta c 21617PRTArtificial SequenceHeavy
Chain CDR1 Chothia 61Gly Tyr Thr Phe Thr Asp Tyr1
56221DNAArtificial SequenceHeavy Chain CDR1 Chothia 62ggttacacat
tcactgacta c 21637PRTArtificial SequenceHeavy Chain CDR1 Chothia
63Gly Tyr Ser Phe Thr Asp Tyr1 56421DNAArtificial SequenceHeavy
Chain CDR1 Chothia 64ggttattcat tcactgacta c 216510PRTArtificial
SequenceHeavy Chain CDR1 AbM 65Gly Phe Thr Phe Thr Asp Tyr Lys Ile
His1 5 106630DNAArtificial SequenceHeavy Chain CDR1 AbM
66ggttttacat tcactgacta caagatacac 306710PRTArtificial
SequenceHeavy Chain CDR1 AbM 67Gly Phe Thr Phe Thr Asp Tyr Ala Ile
Ser1 5 106830DNAArtificial SequenceHeavy Chain CDR1 AbM
68ggttttacat tcactgacta cgccatcagc 306910PRTArtificial
SequenceHeavy Chain CDR1 AbM 69Gly Phe Thr Phe Thr Asp Tyr Tyr Met
His1 5 107030DNAArtificial SequenceHeavy Chain CDR1 AbM
70ggttttacat tcactgacta ctatatgcac 307110PRTArtificial
SequenceHeavy Chain CDR1 AbM 71Gly Tyr Thr Phe Thr Asp Tyr Tyr Met
His1 5 107230DNAArtificial SequenceHeavy Chain CDR1 AbM
72ggttacacat tcactgacta ctatatgcac 307310PRTArtificial
SequenceHeavy Chain CDR1 AbM 73Gly Tyr Ser Phe Thr Asp Tyr Lys Ile
His1 5 107430DNAArtificial SequenceHeavy Chain CDR1 AbM
74ggttattcat tcactgacta caagatacac 307517PRTArtificial
SequenceHeavy Chain CDR2 Kabat 75Tyr Phe Asn Pro Asn Ser Gly Tyr
Ser Thr Tyr Asn Glu Lys Phe Lys1 5 10 15Ser7651DNAArtificial
SequenceHeavy Chain CDR2 Kabat 76tattttaatc ctaacagtgg ttatagtacc
tacaatgaaa agttcaagag c 517717PRTArtificial SequenceHeavy Chain
CDR2 Kabat 77Gly Ile Asn Pro Asn Ser Gly Tyr Ser Thr Tyr Ala Gln
Lys Phe Gln1 5 10 15Gly7851DNAArtificial SequenceHeavy Chain CDR2
Kabat 78gggatcaatc ctaacagtgg ttatagtacc tacgcacaga agttccaggg c
517917PRTArtificial SequenceHeavy Chain CDR2 Kabat 79Tyr Phe Asn
Pro Asn Ser Gly Tyr Ser Thr Tyr Ala Gln Lys Phe Gln1 5 10
15Gly8051DNAArtificial SequenceHeavy Chain CDR2 Kabat 80tatttcaacc
ctaacagcgg ttatagtacc tacgcacaga agttccaggg c 518117PRTArtificial
SequenceHeavy Chain CDR2 Kabat 81Trp Ile Asn Pro Asn Ser Gly Tyr
Ser Thr Tyr Ala Gln Lys Phe Gln1 5 10 15Gly8251DNAArtificial
SequenceHeavy Chain CDR2 Kabat 82tggatcaatc ctaacagtgg ttatagtacc
tacgcacaga agtttcaggg c 518317PRTArtificial SequenceHeavy Chain
CDR2 Kabat 83Trp Ile Asn Pro Asn Ser Gly Tyr Ser Thr Tyr Ser Pro
Ser Phe Gln1 5 10 15Gly8451DNAArtificial SequenceHeavy Chain CDR2
Kabat 84tggatcaatc ctaacagtgg ttatagtacc tacagcccaa gcttccaagg c
518517PRTArtificial SequenceHeavy Chain CDR2 Kabat 85Trp Ile Asn
Pro Asn Ser Gly Tyr Ser Thr Tyr Asn Glu Lys Phe Gln1 5 10
15Gly8651DNAArtificial SequenceHeavy Chain CDR2 Kabat 86tggatcaatc
ctaacagtgg ttatagtacc tacaacgaga agttccaagg c 518717PRTArtificial
SequenceHeavy Chain CDR2 Kabat 87Tyr Phe Asn Pro Asn Ser Gly Tyr
Ser Asn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly8851DNAArtificial
SequenceHeavy Chain CDR2 Kabat 88tatttcaacc ctaacagcgg ttattcgaac
tacgcacaga agttccaggg c 518917PRTArtificial SequenceHeavy Chain
CDR2 Kabat 89Tyr Phe Asn Pro Asn Ser Gly Tyr Ala Thr Tyr Ala Gln
Lys Phe Gln1 5 10 15Gly9051DNAArtificial SequenceHeavy Chain CDR2
Kabat 90tatttcaacc ctaacagcgg ttatgccacg tacgcacaga agttccaggg c
51918PRTArtificial SequenceHeavy Chain CDR2 Chothia 91Asn Pro Asn
Ser Gly Tyr Ser Thr1 59224DNAArtificial SequenceHeavy Chain CDR2
Chothia 92aatcctaaca gtggttatag tacc 24938PRTArtificial
SequenceHeavy Chain CDR2 Chothia 93Asn Pro Asn Ser Gly Tyr Ser Asn1
59424DNAArtificial SequenceHeavy Chain CDR2 Chothia 94aaccctaaca
gcggttattc gaac 24958PRTArtificial SequenceHeavy Chain CDR2 Chothia
95Asn Pro Asn Ser Gly Tyr Ala Thr1 59624DNAArtificial SequenceHeavy
Chain CDR2 Chothia 96aaccctaaca gcggttatgc cacg 249710PRTArtificial
SequenceHeavy Chain CDR2 AbM 97Tyr Phe Asn Pro Asn Ser Gly Tyr Ser
Thr1 5 109830DNAArtificial SequenceHeavy Chain CDR2 AbM
98tattttaatc ctaacagtgg ttatagtacc 309910PRTArtificial
SequenceHeavy Chain CDR2 AbM 99Gly Ile Asn Pro Asn Ser Gly Tyr Ser
Thr1 5 1010030DNAArtificial SequenceHeavy Chain CDR2 AbM
100gggatcaatc ctaacagtgg ttatagtacc 3010110PRTArtificial
SequenceHeavy Chain CDR2 AbM 101Trp Ile Asn Pro Asn Ser Gly Tyr Ser
Thr1 5 1010230DNAArtificial SequenceHeavy Chain CDR2 AbM
102tggatcaatc ctaacagtgg ttatagtacc 3010310PRTArtificial
SequenceHeavy Chain CDR2 AbM 103Tyr Phe Asn Pro Asn Ser Gly Tyr Ser
Asn1 5 1010430DNAArtificial SequenceHeavy Chain CDR2 AbM
104tatttcaacc ctaacagcgg ttattcgaac 3010510PRTArtificial
SequenceHeavy Chain CDR2 AbM 105Tyr Phe Asn Pro Asn Ser Gly Tyr Ala
Thr1 5 1010630DNAArtificial SequenceHeavy Chain CDR2 AbM
106tatttcaacc ctaacagcgg ttatgccacg 3010711PRTArtificial
SequenceHeavy Chain CDR3 Kabat Chothia AbM 107Leu Ser Pro Gly Gly
Tyr Tyr Val Met Asp Ala1 5 1010833DNAArtificial SequenceHeavy Chain
CDR3 Kabat Chothia AbM 108ctatccccag gcggttacta tgttatggat gcc
33109328PRTHomo sapiensIgG1 constant region 109Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr1 5 10 15Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 20 25 30Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 35 40 45His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 50 55 60Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile65 70 75
80Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala
85 90 95Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala 100 105 110Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro 115 120 125Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val 130 135 140Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val145 150 155 160Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 165 170 175Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 180 185 190Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 195 200
205Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
210 215 220Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr225 230 235 240Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser 245 250 255Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr 260 265 270Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr 275 280 285Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 290 295 300Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys305 310 315
320Ser Leu Ser Leu Ser Pro Gly Lys 325110987DNAHomo sapiensIgG1
constant region 110accaagggcc catcggtctt ccccctggca ccctcctcca
agagcacctc tgggggcaca 60gcggccctgg gctgcctggt caaggactac ttccccgaac
cggtgacggt gtcgtggaac 120tcaggcgccc tgaccagcgg cgtgcacacc
ttcccggctg tcctacagtc ctcaggactc 180tactccctca gcagcgtggt
gaccgtgccc tccagcagct tgggcaccca gacctacatc 240tgcaacgtga
atcacaagcc cagcaacacc aaggtggaca agaaagcaga gcccaaatct
300tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg
gggaccgtca 360gtcttcctct tccccccaaa acccaaggac accctcatga
tctcccggac ccctgaggtc 420acatgcgtgg tggtggacgt gagccacgaa
gaccctgagg tcaagttcaa ctggtacgtg 480gacggcgtgg aggtgcataa
tgccaagaca aagccgcggg aggagcagta caacagcacg 540taccgtgtgg
tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac
600aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat
ctccaaagcc 660aaagggcagc cccgagaacc acaggtgtac accctgcccc
catcccggga tgagctgacc 720aagaaccagg tcagcctgac ctgcctggtc
aaaggcttct atcccagcga catcgccgtg 780gagtgggaga gcaatgggca
gccggagaac aactacaaga ccacgcctcc cgtgctggac 840tccgacggct
ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag
900gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
cacgcagaag 960agcctctccc tgtctccggg taaatga 98711111PRTArtificial
SequenceKabat Light Chain CDR1 111Lys Ala Ser Gln Asn Ile Asn Asn
Tyr Leu Asn1 5 1011233DNAArtificial SequenceKabat Light Chain CDR1
112aaagcaagtc agaatattaa caattactta aac 3311311PRTArtificial
SequenceKabat Light Chain CDR1 113Arg Ala Ser Gln Gly Ile Asn Asn
Tyr Leu Asn1 5 1011433DNAArtificial SequenceKabat Light Chain CDR1
114cgggcaagtc agggcattaa caattactta aat 331157PRTArtificial
SequenceKabat Light Chain CDR2 115Asn Thr Asn Asn Leu Gln Thr1
511621DNAArtificial SequenceKabat Light Chain CDR2 116aatacaaaca
atttgcaaac a 211178PRTArtificial SequenceKabat Light Chain CDR3
117Leu Gln His Asn Ser Phe Pro Thr1 511821DNAArtificial
SequenceKabat Light Chain CDR2 118aataccaaca acttgcagac a
2111924DNAArtificial SequenceKabat Light Chain CDR3 119ttgcagcata
atagttttcc cacg 24120361DNAArtificial SequenceSynthetic heavy chain
variable region construct 120gaggtgcagc tcgtgcagtc tggcgctgag
gtgaagaagc ctggcgagtc gttgaagatc 60tcctgcaagg gttctggtta ttcattcact
gactacaaga tccactgggt gcgacagatg 120cctggaaagg gcctcgagtg
gatgggctac ttcaatccta acagtggtta tagtacctac 180agcccaagct
tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac
240ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc
gagactatcc 300ccaggcggtt actatgttat ggatgcctgg ggccaaggga
ccaccgtgac cgtctcctca 360g 361121120PRTArtificial SequenceSynthetic
heavy chain variable region construct 121Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Lys Ile Ser
Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30Lys Ile His Trp
Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Tyr Phe
Asn Pro Asn Ser Gly Tyr Ser Thr Tyr Ser Pro Ser Phe 50 55 60Gln Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75
80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Leu Ser Pro Gly Gly Tyr Tyr Val Met Asp Ala Trp Gly
Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
12012215DNAArtificial SequenceKabat Heavy Chain CDR1 122gactacaaga
tatcc 151235PRTArtificial SequenceKabat Heavy Chain CDR1 123Asp Tyr
Lys Ile Ser1 512430DNAArtificial SequenceAbM Heavy Chain CDR1
124ggtttcacat tcactgacta caagatatcc 3012510PRTArtificial
SequenceAbM Heavy Chain CDR1 125Gly Phe Thr Phe Thr Asp Tyr Lys Ile
Ser1 5 1012651DNAArtificial SequenceKabat Heavy Chain CDR2
126tacttcaatc ctaacagtgg ttatagtacc tacagcccaa gcttccaagg c
5112717PRTArtificial SequenceKabat Heavy Chain CDR2 127Tyr Phe Asn
Pro Asn Ser Gly Tyr Ser Thr Tyr Ser Pro Ser Phe Gln1 5 10 15Gly
* * * * *
References