U.S. patent application number 16/787936 was filed with the patent office on 2021-01-07 for antigen binding molecules with increased fc receptor binding affinity and effector function.
This patent application is currently assigned to Roche Glycart AG. The applicant listed for this patent is Roche Glycart AG. Invention is credited to Peter BRUNKER, Claudia FERRARA KOLLER, Ekkehard MOSSNER, Ursula PUNTENER, Tobias SUTER, Pablo UMANA.
Application Number | 20210002382 16/787936 |
Document ID | / |
Family ID | |
Filed Date | 2021-01-07 |
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United States Patent
Application |
20210002382 |
Kind Code |
A1 |
UMANA; Pablo ; et
al. |
January 7, 2021 |
ANTIGEN BINDING MOLECULES WITH INCREASED FC RECEPTOR BINDING
AFFINITY AND EFFECTOR FUNCTION
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 CD20. 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; (Zurich,
CH) ; BRUNKER; Peter; (Hittnau, CH) ; FERRARA
KOLLER; Claudia; (Zug, CH) ; SUTER; Tobias;
(Windisch, CH) ; PUNTENER; Ursula; (Windisch,
CH) ; MOSSNER; Ekkehard; (Kreuzlingen, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Glycart AG |
Schlieren |
|
CH |
|
|
Assignee: |
Roche Glycart AG
Schlieren
CH
|
Appl. No.: |
16/787936 |
Filed: |
February 11, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16455529 |
Jun 27, 2019 |
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16787936 |
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14815562 |
Jul 31, 2015 |
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16455529 |
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10981738 |
Nov 5, 2004 |
9296820 |
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14815562 |
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60517096 |
Nov 5, 2003 |
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Current U.S.
Class: |
1/1 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C12N 9/10 20060101 C12N009/10 |
Claims
1: An isolated polynucleotide comprising a. a sequence selected
from the group consisting of: SEQ ID NO.:5, SEQ ID NO.: 6 and SEQ
ID NO:7; and b. a sequence selected from the group consisting of:
SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO:23; and c. sequence ID
NO:24.
2-259. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/981,738, filed Nov. 5, 2004, which claims the benefit
of U.S. Provisional Application No. 60/517,096, filed Nov. 5, 2003,
the disclosures of which are herein incorporated by reference in
their entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
146392023111SeqList.txt, date recorded: Oct. 1, 2015, size: 58
KB).
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 CD20. 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] The Immune System and Anti-CD20 Antibodies
[0005] The immune system of vertebrates, including humans, consists
of a number of organs and cell types, which have evolved to
accurately and specifically recognize, bind and destroy invading
foreign microorganisms ("antigens"). Lymphocytes are critical for
the proper function of the immune system. These cells are produced
in the thymus, spleen and bone marrow (adult) and represent about
30% of the total white blood cells present in the circulatory
system of adult humans. There are two major sub-populations of
lymphocytes: T cells and B cells. T cells are responsible for cell
mediated immunity, while B cells are responsible for antibody
production (humoral immunity). However, in a typical immune
response, T cells and B cells function interdependently: T cells
are activated when the T cell receptor binds to fragments of an
antigen that are bound to major histocompatability complex ("MHC")
glycoproteins on the surface of an antigen presenting cell; such
activation causes release of biological mediators ("interleukins"),
which stimulate B cells to differentiate and produce antibodies
("immunoglobulins") against the antigen.
[0006] Each B cell within the host expresses an antibody of one
particular type and specificity, and different B cells express
antibodies specific for different antigens. B cell proliferation
and antibody production spike as a reaction to a foreign antigen,
and both typically cease (or substantially decrease) once the
foreign antigen has been neutralized. Occasionally, however,
proliferation of a particular B cell will continue unabated; such
proliferation can result in a cancer referred to as "B cell
lymphoma."
[0007] T cells and B cells both comprise cell surface proteins
which can be utilized as "markers" for differentiation and
identification. One such human B cell marker is the human B
lymphocyte-restricted differentiation antigen Bp35, referred to as
"CD20." CD20 is expressed during early pre-B cell development and
remains until plasma cell differentiation. Specifically, the CD20
molecule may regulate a step in the activation process that is
required for cell cycle initiation and differentiation and is
usually expressed at very high levels on neoplastic ("tumor") B
cells. Because CD20 is present at high levels on "malignant" B
cells, i.e., those B cells whose unabated proliferation can lead to
B cell lymphoma, the CD20 surface antigen has the potential of
serving as a candidate for "targeting" of B cell lymphomas.
[0008] In essence, such targeting can be generalized as follows:
antibodies specific to the CD20 surface antigen of B cells are
introduced into a patient, by injection, for example. These
anti-CD20 antibodies specifically bind to the CD20 cell surface
antigen of (ostensibly) both normal and malignant B cells; the
anti-CD20 antibody bound to the CD20 surface antigen may lead to
the destruction and depletion of neoplastic B cells. Additionally,
chemical agents or radioactive labels having the potential to
destroy the tumor can be conjugated to the anti-CD20 antibody such
that the agent is specifically "delivered" to e.g., the neoplastic
B cells. Irrespective of the approach, a primary goal is to destroy
the tumor: the specific approach can be determined by the
particular anti-CD20 antibody which is utilized and, thus, the
available approaches to targeting the CD20 antigen can vary
considerably.
[0009] 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 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, 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)), 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. The murine monoclonal
antibody, B-Ly1, is another antibody known to be specific to human
CD20. (Poppema, S. and Visser, L., Biotest Bulletin 3: 131-139
(1987)).
[0010] 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 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)).
[0011] Various attempts to target the CD20 surface antigen have
been reported. Murine (mouse) monoclonal antibody 1F5 (an anti-CD20
antibody) was reportedly administered by continuous intravenous
infusion to B cell lymphoma patients. Extremely high levels (>2
grams) of 1F5 were reportedly required to deplete circulating tumor
cells, and the results were described as being "transient." Press
et al., "Monoclonal Antibody 1F5 (Anti-CD2G) Serotherapy of Human
B-Cell Lymphomas." Blood 69/2:584-591 (1987). A potential problem
with this approach is that 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. Furthermore, 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. 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.
[0012] Another reported approach at improving the ability of murine
monoclonal antibodies to be effective in the treatment of B-cell
disorders has been to conjugate a radioactive label or toxin to the
antibody such that the label or toxin is localized at the tumor
site. For example, the above-referenced 1F5 antibody has been
"labeled" with iodine-131 (".sup.131I") and was reportedly
evaluated for biodistribution in two patients. See Eary, J. F, et
al., "Imaging and Treatment of B-Cell Lymphoma" J. Nuc. Med.
31/8:1257-1268 (1990); see also. Press, O. W. et al., "Treatment of
Refractory Non-Hodgkin's Lymphoma with Radiolabeled MB-1
(Anti-CD37) Antibody" J. Clin. One. 7/8:1027-1038 (1989)
(indication that one patient treated with .sup.131I-labeled IF-5
achieved a "partial response"); Goldenberg, D. M. et at,
"Targeting, Dosimetry and Radioinununotherapy of B-Cell Lymphomas
with Iodine-131-Labeled LL2 Monoclonal Antibody" J. Clin. One.
9/4:548-564 (1991) (three of eight patients receiving multiple
injections reported to have developed a HAMA response); Appelbaum,
F. R. "Radiolabeled Monoclonal Antibodies in the Treatment of
Non-Hodgkin's Lymphoma" Hem./Onc. Clinics of N. A. 5/5:1013-1025
(1991) (review article); Press, O. W. et al "Radiolabeled-Antibody
Therapy of B-Cell Lymphoma with Autologous Bone Marrow Support."
New England J. Med. 329/17: 1219-12223 (1993) (iodine-131 labeled
anti-CD20 antibody 1F5 and B1); and Kaminski, M. G. et al
"Radioinimunotherapy of B-Cell Lymphoma with .sup.131I Anti-B1
(Anti-CD2G) Antibody". New England J. Med. 329/7 (1993) (iodine-131
labeled anti-CD20 antibody B1; hereinafter "Kaminski"). Toxins
(i.e., chemotherapeutic agents such as doxorubicin or mitomycin C)
have also been conjugated to antibodies. See, for example, PCT
published application WO 92/07466 (published May 14, 1992).
[0013] 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, Liu, A. Y. et al, "Production of a Mouse-Human Chimeric
Monoclonal Antibody to CD20 with Potent Fc-Dependent Biologic
Activity" J. Immun. 139/10:3521-3526 (1987), describes a
mouse/human chimeric antibody directed against the CD20 antigen.
See also, PCT Publication No. WO 88/04936. For example, rituximab
(RITUXAN.RTM.), a chimeric anti-CD20, antibody has been approved
for the treatment of non-Hodgkins lymphoma.
[0014] Given the expression of CD20 by B cell lymphomas, this
antigen can serve as a candidate for "targeting" of such lymphomas.
In essence, such targeting can be generalized as follows:
antibodies specific for CD20 surface antigen on B cells are
administered to a patient. These anti-CD20 antibodies specifically
bind to the CD20 antigen of (ostensibly) both normal and malignant
B cells, and the antibody bound to the CD20 on the cell surface
results in the destruction and depletion of tumorigenic B cells.
Additionally, chemical agents, cytotoxins or radioactive agents may
be directly or indirectly attached to the anti-CD20 antibody such
that the agent is selectively "delivered" to the CD20 antigen
expressing B cells. With both of these approaches, the primary goal
is to destroy the tumor. The specific approach will depend upon the
particular anti-CD20 antibody that is utilized. Thus, it is
apparent that the various approaches for targeting the CD20 antigen
can vary considerably.
[0015] The rituximab (RITUXAN.RTM.) antibody is a genetically
engineered chimeric human gamma 1 murine constant domain containing
monoclonal antibody directed against tire human CD20 antigen. This
chimeric antibody contains human gamma 1 constant domains and is
identified by the name "C2B8" in U.S. Pat. No. 5,736,137 (Andersen
et. al.) issued on Apr. 17, 1998, assigned to IDEC Pharmaceuticals
Corporation. RITUXAN.RTM. is approved for the treatment of patients
with relapsed or refracting low-grade or follicular, CD20 positive,
B cell non-Hodgkin's lymphoma. In vitro mechanism of action studies
have shown that RITUXAN.RTM. exhibits human complement-dependent
cytotoxicity (CDC) (Reff et. al, Blood 83(2): 435-445 (1994)).
Additionally, it exhibits significant activity in assays that
measure antibody-dependent cellular cytotoxicity (ADCC).
RITUXAN.RTM. has been shown to possess anti-proliferative activity
in thymidine incorporation assays and a limited ability to induce
apoptosis directly, whereas CD20 antibodies do not (Maloney et. al,
Blood 88 (10): 637a (1996)).
[0016] Antibody Glycosylation
[0017] 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)).
[0018] 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.
(Camming 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)).
[0019] 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)).
[0020] 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. 77:176-180 (1999) and U.S. Pat. No.
6,602,684, the entire 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. 765:59-76 (1998); Wright, A. and Morrison, S. L., Trends
Biotechnol. 75:26-32 (1997)).
[0021] The present inventors 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. 77:176-180 (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.
[0022] There remains a need for enhanced therapeutic approaches
targeting the CD20 antigen for the treatment of B cell lymphomas in
primates, including, but not limited to, humans.
BRIEF SUMMARY OF THE INVENTION
[0023] Recognizing the tremendous therapeutic potential of antigen
binding molecules (ABMs) that have the binding specificity of the
murine B-Ly1 antibody 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.
[0024] 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.:5, SEQ ID NO.: 6 and SEQ ID NO.:7.
(CDRs V.sub.H-1); (b) a sequence selected from a group consisting
of: SEQ ID NO.:21, SEQ ID NO.:22 and SEQ ID NO.:23. (CDRs
V.sub.H-2); and SEQ ID NO:24. In another aspect, the invention is
directed to an isolated polynucleotide comprising SEQ ID NO.:8, SEQ
ID NO.: 9 and SEQ ID NO.: 10. (CDRs V.sub.L). In one embodiment,
any of these polynucleotides encodes a fusion polypeptide.
[0025] In a further aspect, the invention is directed to an
isolated polynucleotide comprising SEQ ID No.:3 In another aspect,
the invention is directed to an isolated polynucleotide comprising
SEQ ID No.:4. In a further aspect, the invention is directed to an
Isolated polynucleotide comprising a sequence selected from the
group consisting of SEQ ID No:29; SEQ ID No:31; SEQ ID No:33; SEQ
ID No:35; SEQ ID No:37; SEQ ID No:39; SEQ ID No:41; SEQ ID No:43;
SEQ ID No:45; SEQ ID No:47; SEQ ID No:49; SEQ ID No:51; 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; and SEQ ID
No:71. In another aspect, the invention is directed to an isolated
polynucleotide comprising SEQ ID No.:75. In one embodiment, such
polynucleotides encode fusion polypeptides.
[0026] The invention is further directed to an isolated
polynucleotide comprising a sequence having at least 80% identity
to SEQ ID NO:3, 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 SEQ ID NO:4, wherein said isolated
polynucleotide encodes a fusion polypeptide. The invention is
further 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:29; SEQ ID No:31; SEQ CD No:33;
SEQ ID No:35; SEQ ID No:37; SEQ ID No:39; SEQ ID No:41; SEQ ID
No:43; SEQ ID No:45; SEQ CD No:47; SEQ ID No:49; SEQ ID No:51; SEQ
ID No:53; SEQ ID No:55; SEQ CD 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; and SEQ ID
No:71, 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 SEQ ID NO:75, wherein said isolated polynucleotide
encodes a fusion polypeptide.
[0027] The invention is further directed to a polynucleotide
comprising SEQ ID NO: 11 (whole heavy chain), or to polynucleotides
having 80%, 85%, 90%, 95% or 99% identity to SEQ CD NO:11. The
invention is also directed to a polynucleotide comprising SEQ ID
NO: 12 (whole light chain), or to polynucleotides having 80%, 85%,
90%, 95% or 99% identity to SEQ ID NO:12.
[0028] 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 mouse.
The invention is also directed to an isolated polynucleotide
encoding a chimeric polypeptide having a sequence selected from the
group consisting of SEQ CD No:30; SEQ ID No:32; SEQ ID No:34, SEQ
ID No:36; SEQ ID No:38; SEQ CD No:40; SEQ ID No:42; SEQ ID No:44;
SEQ ID No:46; SEQ ID No:48; SEQ ID No:50; SEQ ID No:52; SEQ ID
No:54; SEQ ID No:56; SEQ ID No:58; SEQ ID No:60; SEQ ID No:62; SEQ
ED No:64; SEQ ID No:66; SEQ ID No:68; SEQ ID No:70; and SEQ ID
No:72. In one embodiment, the polynucleotide comprises a sequence
encoding a polypeptide having a sequence selected from the group
consisting of SEQ ID No:30; SEQ ID No:32; SEQ ID No:34; SEQ ID
No:36; SEQ ID No:38; SEQ ID No:40; SEQ ID No:42; SEQ ID No:44; SEQ
ID No:46; SEQ ID No:48; SEQ ID No:50; SEQ ID No:52; SEQ ED 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; and SEQ ID No:72;
and a sequence encoding a polypeptide having the sequence of an
antibody Fc region, or a fragment thereof, from a species other
than mouse.
[0029] In yet another aspect, the invention is directed to an
isolated polynucleotide encoding a chimeric polypeptide having the
sequence of SEQ ID No.:2. In one embodiment, the polynucleotide
comprises a sequence encoding a polypeptide having the sequence of
SEQ ID No.:2; and a sequence encoding a polypeptide having the
sequence of an antibody Fc region, or a fragment thereof, from a
species other than mouse. In yet another aspect, the invention is
directed to an isolated polynucleotide encoding a chimeric
polypeptide having the sequence of SEQ ID No.: 76. In one
embodiment, the polynucleotide comprises a sequence encoding a
polypeptide having the sequence of SEQ ID No.:76; and a sequence
encoding a polypeptide having the sequence of an antibody Fc
region, or a fragment thereof, from a species other than mouse.
[0030] The invention is also directed to an isolated polynucleotide
comprising a sequence encoding a polypeptide having the V.sub.H
region of the murine B-Ly1 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 mouse. In another aspect, the invention is directed to an
isolated polynucleotide comprising a sequence encoding a
polypeptide having the V.sub.L region of the murine B-Ly1 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 mouse.
[0031] 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.
[0032] In one aspect, the invention is directed to an isolated
polypeptide comprising: (a) a sequence selected from a group
consisting of: SEQ ID NO.: 15, SEQ ID NO.: 16 and SEQ ID NO.: 17.
(CDRs V.sub.H-1); (b) a sequence selected from a group consisting
of: SEQ ID NO.:25, SEQ ID NO.:26 and SEQ ID NO.:27 (CDRs
V.sub.H-2); and SEQ ID NO:28, wherein said polypeptide is a fusion
polypeptide. In another aspect, the invention is directed to an
isolated polypeptide comprising SEQ ID NO.:18, SEQ ID NO.: 19 and
SEQ ID NO.:20. (CDRs V.sub.L), wherein said polypeptide is a fusion
polypeptide.
[0033] 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.:2 or a variant thereof, in one
embodiment, any one of these polypeptides further comprises a human
Fc region. The invention Is also directed to a chimeric polypeptide
comprising a sequence selected from the group consisting of SEQ ID
No:30; SEQ ID No:32; SEQ ID No:34; SEQ ID No:36; SEQ ID No:38; SEQ
ID No:40; SEQ ID No:42; SEQ ID No:44; SEQ ID No:46; SEQ ID No:48;
SEQ ID No:50; SEQ ID No:52; 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 ED No:70; and SEQ ID No:72, or a variant thereof. The
invention is further directed to a chimeric polypeptide comprising
the sequence of SEQ ID NO.:76 or a variant thereof. In one
embodiment, any one of these polypeptides further comprises a human
Fc region.
[0034] In another aspect the invention is directed to a polypeptide
comprising a sequence derived from the murine B-Ly1 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.
[0035] In an additional aspect, the invention is directed to an
isolated polypeptide comprising SEQ ID NO: 13 or a variant thereof.
In another aspect, the invention is directed to an isolated
polypeptide comprising SEQ ID NO: 14.
[0036] In another aspect, the invention is directed to an ABM,
which is capable of competing with the murine B-Ly1 antibody for
binding to CD20 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 V.sub.H region having an
amino acid sequence selected from the group consisting of SEQ ID
NO.: 1; SEQ ID No:30; SEQ ID No:32; SEQ ID No:34; SEQ ID No:36; SEQ
ID No:38; SEQ ID No:40; SEQ ID No:42; SEQ ID No:44; SEQ ID No:46;
SEQ ID No:48; SEQ ID No:50; SEQ ID No:52; 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; and SEQ ID No:72. In another
embodiment, the ABM is a recombinant antibody comprising a V.sub.L
region having an amino acid sequence selected from the group
consisting of SEQ ID NO.: 2 and SEQ ID NO:76. 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.
[0037] 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 oligosacchardies
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.
[0038] 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.
[0039] The invention is further related to a method of producing an
ABM, which is capable of competing with the murine B-Ly1 antibody
for binding to CD20 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.
[0040] 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.
[0041] In a further aspect, the invention is related to a method of
treating a disease treatable by B-cell depletion. The method
comprises administering a therapeutically effective amount of the
ABM of the present invention to a human subject in need thereof. In
a preferred embodiment, the disease is treated by administering an
ABM that is a chimeric antibody, or a chimeric fragment of an
antibody.
[0042] 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 produced by the
host cell, wherein the ABM is capable of competing with the murine
B-Ly1 antibody for binding to CD20 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 a further
embodiment, the ABM comprises a region equivalent to the Fc region
of a human IgG.
[0043] The invention is also directed to an isolated polynucleotide
comprising at least one complementarity determining region of the
murine B-Ly1 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 murine
B-Ly1 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.
[0044] In another embodiment, the invention is directed to an
antigen combining molecule comprising at least one complementarity
determining region of the murine B-Ly1 antibody, or a variant or
truncated form thereof containing at lest 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
murine B-Ly1 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 B cell lymphomas.
[0045] The present invention is the first known instance in which a
Type II anti-CD20 antibody has been engineered to have increases
effector functions such as ADCC, while still retaining potent
apoptosis ability. Accordingly, the present invention is directed
to an engineered Type 11 anti-CD20 antibody having increased ADCC
as a result of said engineering and without loss of substantial
ability to induces apoptosis. In one embodiment, the Type II
anti-CD20 antibodies have been engineered to have an altered
pattern of glycosylation in the Fc region. In a particular
embodiment, the altered glycosylation comprises an increased level
of bisected complex residues in the Fc region. In another
particular embodiment, the altered glycosylation comprises and
reduced level of fucose residues in the Fc region. In another
embodiment, the Type II anti-CD20 antibodies have undergone
polypeptide engineering, he invention is further directed to
methods of making such engineered Type IT antibodies and to methods
of using such antibodies in the treatment of various B cell
disorders, including B cell lymphomas.
[0046] The host cell of the present invention may be selected from
the group that includes, but is not limited to, 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
V.sub.L region of the murine B-Ly1 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 V.sub.H region of the murine B-Ly1
antibody or variants thereof and a sequence encoding a region
equivalent to the Fc region of a human immunoglobulin.
[0047] 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.
[0048] 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.
[0049] 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 murine B-Ly1 antibody for binding to CD20 and wherein said
ABM is chimeric. 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 ("GnTIII"), and the localization domain of .alpha.1-6 core
fucosyltransferase. Preferably, the Golgi localization domain is
from mannosidase II or GnTI.
[0050] In a further aspect, the invention is directed to a method
for modifying the glycosylation profile of an anti-CD20 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.
[0051] In one aspect, the invention is related to a recombinant,
chimeric antibody, or a fragment thereof, capable of competing with
the murine B-Ly1 antibody for binding to CD20 and having reduced
fucosylation.
[0052] 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.
[0053] 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 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, non fucosylated. In another preferred embodiment, wherein
at least 35% of the oligosaccharides in the Fc region of said
polypeptide are bisected, nonfucosylated.
[0054] 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.
[0055] In another aspect, the invention is directed to a
recombinant, chimeric antibody fragment, having the binding
specificity of the murine B-Ly1 antibody and containing the Fc
region, that is engineered to have increased effector function
produced by any of the methods of the present invention.
[0056] In another aspect, the present invention is directed to a
fusion protein that includes a polypeptide having the sequence of
SEQ ID NO:1 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.
[0057] In another aspect, the present invention is directed to a
fusion protein that includes a polypeptide having the sequence of
SEQ ID NO:2 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.
[0058] In one aspect, the present invention is directed to a
pharmaceutical composition comprising a recombinant, chimeric
antibody, produced by any of the methods of the present invention,
and a pharmaceutically acceptable carrier.
[0059] In another aspect, the present invention is directed to a
pharmaceutical composition comprising a recombinant, chimeric
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.
[0060] The invention is further directed to a method of treating a
disease treatable by B-cell depletion comprising administering a
therapeutically effective amount of the recombinant, chimeric,
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
[0061] FIG. 1. Nucleotide (SEQ ID NO:2), complimentary nucleotide
(SEQ ID NO:85) and amino acid sequence (SEQ ID NO: 1) of the
V.sub.H region of the murine B-Ly1. Also shown are the nucleotide
sequence of the murine B-Ly1 V.sub.H region linker (SEQ ID NO:79),
the complementary nucleotide sequence (SEQ ID NO:80), and the amino
acid sequence (SEQ ID NO:81).
[0062] FIG. 2. Nucleotide (SEQ ID NO:4), complimentary nucleotide
(SEQ ID NO:86) and amino acid sequence (SEQ ID NO:3) of the V.sub.L
region of the murine B-Ly1. Also shown are the nucleotide sequence
of the murine B-Ly1 VL region linker (SEQ ID NO:82), the
complementary nucleotide sequence (SEQ ID NO:83), and the amino
acid sequence (SEQ ID NO:84).
[0063] FIG. 3. Binding of Rituximab.RTM. (O) and ch-B_Ly1 (.DELTA.)
to CD20 on Raji B-lymphoma cells.
[0064] FIG. 4A, 4B, 4C. B-Cell depletion by Rituximab.RTM. (O) and
ch-B_Ly1 (.DELTA.) in whole blood of the three different classes of
Fc.gamma.RIIIa-158 V/F genotype: (FIG. 4A) whole blood from a F/F
donor, homozygous for the lower affinity receptor; (FIG. 4B) whole
blood from a F/V donor, heterozygous for the affinity receptor; and
(FIG. 4C) whole blood from a V/V donor, homozygous for the higher
affinity receptor.
[0065] FIG. 5A, 5B, 5C. Nucleotide (SEQ ID NO: 11) and amino acid
sequence (SEQ ID NO: 13) of the heavy chain of a chimeric,
anti-CD20 antibody.
[0066] FIGS. 6A and 6B. Nucleotide (SEQ ID NO: 12) and amino acid
sequence (SEQ ID NO: 14) of the light chain of a chimeric,
anti-CD20 antibody.
[0067] FIGS. 7A and 7B. Nucleotide and amino acid sequences of the
murine B-Ly1 antibody CDRs. (FIG. 7A) Predicted CDRs for the
V.sub.H region. (FIG. 7B) Predicted CDRs for the V.sub.L
region.
[0068] FIG. 8A, 8B, 8C. MALDI-TOF profile of a glycoengineered,
chimeric B-Ly1 antibody. (FIG. 8A) Table detailing the percentages
of specific peaks; (FIG. 8B) Spectrum for glycoengineered chimeric
B-Ly1; (FIG. 8C) Spectrum for glycoengineered chimeric B-Ly1
treated with Endo-H.
[0069] FIG. 9. Binding of different humanized anti-CD20 antibodies
to Raji B-cells. The differences between the B-HH2 construct and
the B-HL8 and B-HL11 constructs are located in the framework 1 and
2 regions with all three CDRs being identical. B-HL8 and B-HL11
have their FR1 and FR2 sequences derived from the human VH3 class,
whereas the complete B-HH2 framework is human VH1 derived. B-HL11
is a derivative of B-HL8 with the single mutation Glut Gin, with
Gin being the amino acid residue in the B-HH2 construct. This means
that the Glu1Gln exchange does not alter binding affinity or
intensity. The other differences between B-HH2 and B-HL8 are 14 FR
residues, from which one or more will influence the antigen binding
behavior of this antibody.
[0070] FIG. 10. Binding of the humanized anti-CD20 antibody
BHL4-KV1 on Raji target cells. The B-HL4 construct is derived from
the B-HH2 antibody by replacing the FR1 of the B-HH2 with that of
the human germ line sequence VH1_45. This construct shows greatly
diminished antigen binding capacity, despite of having different
amino acids at only three positions within FR1. These residues are
located at positions 2, 14, and 30 according to Rabat numbering. Of
these, position 30 seems to be the most influential position, since
it is part of the Chothia definition of CDR1.
[0071] FIG. 11. Comparison of the binding behavior between B-HH1,
B-HH2, B-HH3, and the parental antibody B-ly1. The data show that
all Abs show a similar EC50 value, but the B-HH1 construct binds
with a lower intensity/stoichiometry than the variants B-HH2 and
B-HH3. B-HH1 can be distinguished from B-HH2 and B-HH3 by its
partially human CDR1 and CDR2 regions (Rabat definition), as well
as the Ala/Thr polymorphism at position 28 (Rabat numbering). This
indicates that either position 28, the complete CDR1, and/or the
complete CDR2 is important for antibody/antigen interaction.
[0072] FIG. 12. The comparison of B-HL1, B-HH1, and the B-ly1
parental antibody. The data showed absence of any binding activity
in the B-HL1 construct, and about half of the binding
intensity/stoichiometry of B-HH1 compared to B-ly1. Both B-HL1, as
well as B-HH1, are designed based on acceptor frameworks derived
from the human VH1 class. Among other differences, position 71
(Rabat numbering) of the B-HL1 construct is a striking difference,
indicating its putative importance for antigen binding.
[0073] FIG. 13. Fluorocytometric analysis of the capacity of the
anti-CD20 antibody to its antigen. The data showed that the B-HL2
and B-HL3 constructs do not display CD-20 binding activity.
[0074] FIG. 14. Apoptosis of anti-CD20 antibodies on Z-138 MCL
cells.
[0075] FIGS. 15A and 15B. Apoptosis of DLBCL cell line by anti-CD20
antibodies (FIG. 15A) and apoptosis of MCL cell line by anti-CD20
antibodies (FIG. 15B). Assay details: 5.times.10.sup.5 cells/well
were seeded in 24-well plates (5.times.10.sup.5 cells/ml) in
culture medium. 10 mg of the respective Ab, PBS for the negative
control or 5 mM Camptothecin (CPT) positive control were added to
the wells. Samples were incubated o/n (16 h), stained with
AnnV-FITC and analysed by FACS. Assay was done in triplicates. (*):
Signal for PBS alone subtracted (PBS alone gave 8% and 2% AnnV+ for
PR-1 and Z-138 cells respectively). Antibodies used were: C2B8
(chimeric, non-glycoengineered); BHH2-KV1 (humanized,
non-glycoengineered). Note: this assay does not involve any
additional effector cells, just targets plus antibody or
controls.
[0076] FIGS. 16A and 16B. Target-cell killing by anti-CD20
antibodies with immune effector cells. Assay details: B-cell
depletion in normal whole blood overnight incubation and analysis
for CD19+/CD3+ by FACS (FIG. 16A). ADCC using PBMCs as effectors, 4
h incubation, 25:1 effector:target ratio, target-killing measured
by Calcein-retention relative to detergent-lysis (100%) and to
lysis without Ab (0%) (FIG. 16B). Antibodies used: C2B8 (chimeric,
non-glycoengineered form); BHH2-KV1-wt (humanized,
non-glycoengineered form of BHH2-KV1); BHH2-KV1-GE (humanized,
glycoengineered form of BHH2-KV1).
[0077] FIG. 17. MALDI/TOF-MS profile of PNGaseF-released
Fc-oligosaccharides of unmodified, nonglycoengineered BHH2-KV1
humanized IgG1 B-ly1 anti-human CD20 antibody.
[0078] FIG. 18. MALDI/TOF-MS profile of PNGaseF-released
Fc-oligosaccharides of glycoengineered BHH2-KV1g1 humanized IgG1
B-ly1 anti-human CD20 antibody. Glycoengineering done by
co-expression in host cells of antibody genes and gene encoding
enzyme with .beta.-1,4-N-acetylglucosaminyltransferase III
(GnT-III) catalytic activity.
[0079] FIG. 19. MALDI/TOF-MS profile of PNGaseF-released
Fc-oligosaccharides of glycoengineered BHH2-KV1g2 humanized IgG1
B-ly1 anti-human CD20 antibody. Glycoengineering done by
co-expression in host cells of antibody genes and genes encoding
enzyme with .beta.-1,4-N-acetylglucosaminyltransferase III
(GnT-III) catalytic activity and encoding enzyme with Golgi
.alpha.-mannosidase II catalytic activity.
[0080] FIG. 20. Binding of non-glycoengineered and glycoengineered
antibodies to human FcgammaRIIIa receptor displayed on the surface
of recombinant CHO-CD16 cells.
[0081] FIG. 21. Apoptosis of non-Fc engineered and Fc-engineered
anti-CD20 antibodies on Z-138 MCL cells. Assay details: 5.times.105
cells/well were seeded, in 24-well plates (5.times.105 cells/ml) in
culture medium. 10 mg of the respective Ab, PBS for the negative
control were added to the wells. Samples were incubated o/n (16 h),
stained with AnnV-FITC and analysed by FACS. Assay was done in
triplicates. Abs used: C2B8=rituximab (chimeric,
non-glycoengineered form, same as commercial form); BHH2-KV1
(humanized, non-glycoengineered-see FIG. 6 for glycosylation
profile); BHH2-KV1g1 (humanized, glycoengineered--see FIG. 7 for
glycosylation profile); BHH2-KV1g2 (humanized, glycoengineered--see
FIG. 8 for glycosylation profile). Note: this assay does not
involve any additional effector cells, just targets plus antibody
or controls. (*); Signal for PBS alone subtracted.
DETAILED DESCRIPTION OF THE INVENTION
[0082] Terms are used herein as generally used in the art, unless
otherwise defined as follows.
[0083] 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 humanized, primatized and chimeric
antibodies.
[0084] 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.
[0085] 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 247:1306-10 (1990).
[0086] 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 CD20 is a molecule which specifically binds to
a cell surface non-glycosylated phosphoprotein of 35,000 Daltons,
typically designated as the human B lymphocyte restricted
differentiation antigen Bp35, commonly referred to as CD20. By
"specifically binds" is meant that the binding is selective for the
antigen and can be discriminated from unwanted or nonspecific
interactions.
[0087] 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 antiCD-20 antibody
having the amino acid sequence of the murine B-Ly1 variable region.
Humanized antibodies are a particularly preferred form of fusion or
chimeric antibody.
[0088] 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.)
[0089] 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., CD20) 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.
[0090] 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.
[0091] 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.
[0092] 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 arid
light chain variable domains of an antibody:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A discussion of humanized
antibodies can be found, infer 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.
[0093] 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.
[0094] 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
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 V.sub.L
CDR2 50-56 50-52 V.sub.L CDR3 89-97 91-96 .sup.1Numbering of all
CDR definitions in Table 1 is according to the numbering
conventions set forth by Kabat et al. (see below).
[0095] 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 unambiguously 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:78) are not numbered according to the Kabat numbering
system.
[0096] 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. The query sequence may be the entire sequence
shown in either FIG. 24 or FIG. 25.
[0097] 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 ah, 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.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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, 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.
[0109] 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:
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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:
[0114] 1) the assay uses target cells that are known to express the
target antigen recognized by the antigen-binding region of the
antibody;
[0115] 2) the assay uses human peripheral blood mononuclear cells
(PBMCs), isolated from blood of a randomly chosen healthy donor, as
effector cells;
[0116] 3) the assay is carried out according to following protocol:
[0117] 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; [0118] 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
.sup.51Cr, washed twice with cell culture medium, and resuspended
in cell culture medium at a density of 10.sup.5 cells/ml; [0119]
iii) 100 microliters of the final target cell suspension above are
transferred to each well of a 96-well microtiter plate; [0120] 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; [0121] 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); [0122]
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); [0123] 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.; [0124] viii) 50 microliters of the PBMC
suspension (point i above) are added to each well to yield an
effector:target cell ratio of 25: $ and the plates are placed in an
incubator under 5% CO.sub.2 atmosphere at 37.degree. C. for 4
hours; [0125] ix) the cell-free supernatant from each well is
harvested and the experimentally released radioactivity (ER) is
quantified using a gamma counter; [0126] 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);
[0127] 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.
[0128] In one aspect, the present invention is related to antigen
binding molecules having the binding specificity of the murine
B-Ly1 antibody, 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 the CDRs shown in FIG. 7. 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.:5, SEQ ID NO.: 6 and SEQ ID NO.:7. (CDRs
V.sub.H-1); and (b) a sequence selected from a group consisting of:
SEQ ID NO.:21, SEQ ID NO.:22 and SEQ ID NO.:23. (CDRs and SEQ ID
NO:24. In another preferred embodiment, the invention is directed
to an isolated polynucleotide comprising SEQ ID NO.:8, SEQ ID NO.:
9 and SEQ ID NO.:10, (CDRs V.sub.L). In one embodiment, any of
these polynucleotides encodes a fusion polypeptide.
[0129] In another embodiment, the antigen binding molecule
comprises the V.sub.H domain of the murine B-Ly1 antibody shown in
FIG. 1, 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
murine B-Ly1 antibody shown in FIG. 2, or a variant thereof; and a
non-murine polypeptide.
[0130] In another aspect, the invention is directed to antigen
binding molecules comprising one or more truncated CDRs of BLy-1.
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 are hereby
incorporated by reference in their entirety.
[0131] Accordingly, the invention is also directed to an isolated
polynucleotide comprising at least one complementarity determining
region of the murine B-Ly1 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 murine
B-Ly1 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.
[0132] In another embodiment, the invention is directed to an
antigen combining molecule comprising at least one complementarity
determining region of the murine B-Ly1 antibody, or a variant or
truncated form thereof containing at lest 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
murine B-Ly1 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 B cell lymphomas.
[0133] It is known that several mechanism are involved in the
therapeutic efficacy of anti-CD20 antibodies, including antibody
dependent cellular cytotoxicity (ADCC), complement-dependent
cytotoxicity (CDC), and the induction of growth arrest or
apoptosis. For example, the majority of experimental evidence
indicates that rituximab operates through conventional effector
mechanisms measured by CDC and ADCC assays. Similarly, it has been
shown that the resistance of different lymphoma cells to rituximab
in vivo is a function of their sensitivity to CDC in vitro. In
contrast, the mode of action in vivo of another antibody that has
been approved for therapeutic use, B1, requires neither complement
nor natural killer (NK) cell activity. Rather, the efficacy of B1
in vivo is due to its ability to induce potent apoptosis.
[0134] In general, anti-CD20 monoclonal antibodies fall into two
distinct categories based on their mechanism of action in
eradicating lymphoma cells. Type I anti-CD20 antibodies primarily
utilize complement to kill target cells, while Type II antibodies
operate by different mechanisms, primarily apoptosis. Rituximab and
1F5 are examples of Type I anti-CD20 antibodies, whereas B1 is an
example of a Type II antibody. See, e.g., Cragg, M. S. and Glennie,
M. J., Blood 103(7):2738-2743 (April 2004); Teeling, J. L. et al.,
Blood 104(6); 1793-1800 (September 2004), the entire contents of
which are hereby incorporated by reference.
[0135] The present invention is the first known instance in which a
Type II anti-CD20 antibody has been engineered to have increases
effector functions such as ADCC, while still retaining potent
apoptosis ability. Accordingly, the present invention is directed
to an engineered Type II anti-CD20 antibody having increased ADCC
as a result of said engineering and without loss of substantial
ability to induces apoptosis. In one embodiment, the Type II
anti-CD20 antibodies have been engineered to have an altered
pattern of glycosylation in the Fc region. In a particular
embodiment, tire altered glycosylation comprises an increased level
of bisected complex residues in the Fc region. In another
particular embodiment, the altered glycosylation comprises and
reduced level of fucose residues in the Fc region. See U.S. Pat.
Appl. Pub. No. 20040093621 to Shitara et al., the entire contents
of which is incorporated by reference. In another embodiment, the
Type II anti-CD20 antibodies have undergone polypeptide engineering
as taught in U.S. Pat. No. 6,737,056 to Presta or U.S. Pat. Appl.
Pub. No. 2004 0185045 (Macrogenics) or U.S. Pat. Appl. Pub. No.
2004 0132101 (Xencor), the entire contents of each of which are
incorporated by reference. The invention is further directed to
methods of making such engineered Type II antibodies and to methods
of using such antibodies in the treatment of various B cell
disorders, including B cell lymphomas.
[0136] 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, at
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). Robinson et al., in PCT Publication
Number WO/88104936 describe a chimeric antibody with human constant
region and murine variable region, having specificity to an epitope
of CD20; the murine portion of the chimeric antibody of the
Robinson references is derived from the 2H7 mouse monoclonal
antibody (gamma 2b, kappa). While the reference notes that the
described chimeric antibody is a "prime candidate" for the
treatment of B cell disorders, this statement can be viewed as no
more than a suggestion to those in the art to determine whether or
not this suggestion is accurate for this particular antibody,
particularly because the reference lacks any data to support an
assertion of therapeutic effectiveness, and importantly, data using
higher order mammals such as primates or humans.
[0137] Methodologies for generating chimeric antibodies are
available to those in the art. For example, the light and heavy
chains can be expressed separately, using, for example,
immunoglobulin light chain and immunoglobulin heavy chains in
separate plasmids, or on a single (e.g., polycistronic) vector.
These can then be purified and assembled in vitro into complete
antibodies; methodologies for accomplishing such assembly have been
described. See, for example, Scharff, M., Harvey Lectures 69:125
(1974), In vitro reaction parameters for the formation of IgG
antibodies from reduced isolated light and heavy chains have also
been described. See, for example, Sears et al., Biochem.
16(9):2016-25 (1977).
[0138] 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., or U.S. Pat. No. 6,632,927 to Adair et
al., the entire contents of each of which is hereby 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 tire 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-CD20 antibodies
will comprise constant regions of human immunoglobulin.
[0139] 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). 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)).
[0140] 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 are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the 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.
[0141] 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.
[0142] 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.
[0143] 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.131 iodine 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 (eg, 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 eg, .sup.131 iodine,
.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.
[0144] Effective single treatment dosages (i.e., therapeutically
effective amounts) of .sup.90yttrium labeled anti-CD20 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-CD20
antibodies range from between about 5 and about 70 mCi, more
preferably between about 5 and about 40 mCi. Effective single
treatment ablative dosages (ie, may require autologous bone marrow
transplantation) of .sup.131iodine labeled anti-CD20 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-CD20 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-CD20 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.
[0145] With respect to radiolabeled anti-CD20 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 (eg, using .sup.111indium) be
conducted on the patient, a purpose of which is to ensure that the
therapeutically labeled antibody (eg, using .sup.90yttrium) will
not become unnecessarily "concentrated" in any normal organ or
tissue.
[0146] In a preferred embodiment, tire present invention is
directed to an isolated polynucleotide comprising a sequence that
encodes a polypeptide having an amino acid sequence as shown in
Table 3 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 2 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 3. 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 3 with conservative
amino acid substitutions.
TABLE-US-00002 TABLE 2 SEQ Con- ID struct NUCLEOTIDE SEQUENCE NO
B-HH1 CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 29
GAAGCCTGGGAGTTCAGTQAAGGTCTCCTGCAAGG
CTTCCGGATACACCTTCAGCTATTCTRTGATGAGC
TGGGTGCGGCAGGCCCCTGGACAAGCFGCTCGAGT
GGATGGGACGGATCTTTCCCGGCGATGGGGATACT
GACTACGCACAGAAATTCCAAGGAAGAGTCACAAT
TACCGCCGACAAATCCACTAGCACAGCUATATGGA
GCTGAGCAGCCTGAGATCTGAGGACACCTGCCGTG
TATTACTGTGCAAGAAATGTCTTTGATGGTTACTG
GCTTGTTTACTQCTGGCCAGGGAACCCTQGTCACC GTCTCCTCA B-HH2
CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 31
GAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG
CTTCCGGATACGCCTTCAGCTATTCTTGGATGAAC
TGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTG
GATGGGACGGATCTTTCCCGGCGATGGGGATACTG
ACTACAATGGGAAATFCAAGGGCAGAGTCACAATT
ACCGCCGACAAATCCACTAGCACAGCCTATATGGA
GCTGAGCAGCCTGAGATCTGAGCTACACGGCCGTG
TATTACTGTGCAAGAAATGTCTTTGATGGTTACTG
GCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA B-HH3
CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 33
GAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG
CTTCCGGATACGCCTTCAGCTATTCTTGGATGAAC
TGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTG
GATGGGACGGATCTTTCCCGGCGATGGGGATACTG
ACTACAATGGGAAATTCAAGGGCAGAGTCACAATT
ACCGCGGACAAATCCACTAGCACAGCCTATATGGA
GCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT
ATCTGTGTGCAAGAAATGTCTTMATGGTTACTGGC
TTGTTTACTGGGGCCAQGGAACCCTGGTCACCGTC TCCTCAGCTAGCACC B-HH4
CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 35
GAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG
TCTCCGGATACGCGTTCAGCTATTCTTGGATGAAC
TGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTG
GATGGGACGGATCTTTCCCGGCGATGGGGATACTG
ACTACAATGGGAAATTCAAGGGCAGAGTCACAATT
ACCGCCGACAAATCCACTAGCACAGCCTATATCGA
GCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT
ATTACTGTGCAAGAAATGTCTTTGATGGTTACTGG
CTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA B-HH5
CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 37
GAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG
CTTCCGGATACCYCGTTCACTCTATCTTGGATGAG
CTGGGTGCGGCAGGCGCCTGGACAAGGGCTCGAGT
GGATGUGACGGATCTTTCCCGGCGATCyGGGATAC
TGACTACAATGGGAAATTCAAGGGCAGAGTCACAA
TFACCGCCGACAAATCCACTAGCACAGCCTATATG
GAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGT
GTATTACTGTGCAAGAAATGTCTTTGATGGTTACT
GGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA B-HH6
CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 39
GAAGCCTGGGACTTTCAGTGAAGGTCTCCTGCAAG
GCTTCCGGATACGCCTTCAGCTATTCTTGGATCAA
TTGGGTGCGGCAGGCGCCTGGACAAGGGCTCGAGT
GGATGGGACGGATCTTTCCCCTGCGATGGGGATAC
TGACTACAATGGGAAATTCAAGGGCAGAGTCACAA
TTACCGCCGACAAATCCACTAGCACAGCCTATATP
GAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGT
GTATTACTGTGCAAGAAATGTCTTTGATGGTTACT
GGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA B-HH7
CAGCTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 41
GAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG
CTTCCGGATACGCCTTCAGCTATTCTTGGATCTCG
TGGGTGCGGCAGGCGCCTGGACAAGGGCTCGAGTG
GATGGGACGCTATCTTTCCCGGCGATGGGGATACT
GACTACAATGGGAAATTCAAGGGCAGAGTCACAAT
TACCGCCGACAAATCCACTAGCACAGCCTATATGG
AGCTGAGCAGCCTGAGATCTGACTGACACGGCCGT
GTATTACTGTGCAAGAAATGTCTTTGATGGTTACT
GGCTTGTTTACTGGGGCCAGGCTAACCCTGGTCAC CGTCTCCTCA B-HH8
CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 43
GAAGCCTGGCGCCTCAGTGAAGGTCTCCZGCAAGG
CTTCCGGATACACCTTCACATACAGCTGGATGAAC
TGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTG
GATGGGACGGATCTTTCCCGGCGATGGGGATACTG
ACTACAATGGGAAATTCAAGGGCAGAGTCACAATT
ACCGCCGACAAATCCACTAGCACAGCCTATATGGA
GCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT
ATTACTGTGCAAGAAATGTCTTTGATGGTTACTGG
CTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA B-HH9
CAGGTGCAATTGPTGCAGTCTGGCGCTGAAGTTAA 45
GAAGCCTGCTCGCCTCAGTGAAGGTCTCCTGCAAG
GCTTCCGGATACACCTTCACCTATTCTTGGATGAA
CTGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGT
GGATGGGACGGATCTTTCCCGGCGATGGGGATACT
GACTACAATGGGAAATTCAAGGGCAGAGTCACAAT
TACCGCCGACAAATCCACTAGCACAGCCTATATGG
AGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTG
TATTACTGTGCAAGAAATGTCTTTGATGGTTACTG
GCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA B-HL1
CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 47
GAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGG
CTTCCGGATACACCTTCACCTATTCTTGGATGCAC
TGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTG
GATGGGACGGATCTTTCCCGGCGATGGGGATACTG
ACTACGCACAGAAATTCCAAGGAAGAGTCACAATG
ACACGGGACACGTCCACTTCCACCGTCTATATGGA
GCTGAGCAGCCTGAGATCTGACTGACACGGCCGTG
TATTACTGTGCAAGAAATGTCTTTGATGGTTACTG
GCTTGTTTACTGGGCTCCAGGGAACCCTGGTCACC GTCTCCTCA B-HL2
GAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 49
GAAGCCTQGGGCCACCGTGAAGATCTCCTGCAAGG
TGTCCGGATACACCTTCACCTATTCTTGGATGCAC
TGGGTGCAGCAGGCCCCTGGAAAGGGGCTCGAGTG
GATGGGACGGATCTTTCCCGGCGATGGGGATACTG
ACTACGCAGAGAAATTCCAAGGAAGAGTCACAATC
ACAGCCGACACGTCCACTGACACCGCCTATATGGA
GCTGACTCAGCCTGAGATCTGAGGACACGGCCGTG
TATTACTGTGCAACCAATGTCTTTGATGGTTACTG
GCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA B-HL3
GAGGTGCAKTTGGTGCAGTCTGGCGCTGAAGTTAA 51
GAAGCCTGGGGCCACCGTGAAQATCTCCTGCAAGG
TGTCCGGATACACCTTCACCTATTCTTGGATGAAC
TGGGTGCAGCAGQCCCCTGGAAAGGGGCTCGAGTG
GATGGGACGGATCTTTCCCGGATGGGGATACTGAC
TACAATGGGAAATTCAAGGGAAGAGTCACAATCAC
AGCCGACACGTCCACTGACACCGCCTATATGGAGC
TGAGCAGCCTGAGATCTGAGGACACGGCCGTGTAT
TACTGTGCAACCAATGTCTTTGATGGTTACTGGCT
TGTTTACTGGGGCCAGGGAACCCTGGTCAcCGTCT CCTCA B-HL4
CAGATGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 53
GAAGACCGGGAGTTCAGTGAAGGTCTCCTGCAAGG
CTTCCGGATACACCTTCACCTATTCTTGGATGAGC
TGGGTGCGGCACTGCCCCTGGACAAGGGCTCGAGT
GGATGGGACGGATCTTTCCCGGCGATGGGGATACT
GACTACGCACAGAAATTCCAAGGAAGAGTCACAAT
TACCGCCGACAAATCCACTAGCACAGCCTATATGG
AGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTG
TATTACTGTGCAAGAAATGTCTTTGATGGTTACTG
GCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCAGCTAGCACC B-HL8
GAAGTGCAGCTGGTGGAGTCTGGAGGAGGCTTCTG 55
TCAAGCCTGGCGGGTCCCTGCGGCTCTCCTGTGCA
GCCTCTGGATTCACATTTAGCTATTCTTGGATGAA
CTGGGTGCGGCAGGCTCCTGGAAAGGGCCTCGAGT
GGGTGGGACGGATCTTTCCCGGCGATCTGGGATAC
TGACTACAATGGGAAATTCAAGGGCAGAGTCACAA
TTACCGCCGACAAATCCACTAGCACAGCCTATATG
GAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGT
GTATTACTGTGCAAGAANTGTCTTTGATGGTTACT
GGCTTGTTTACTGGGGCCAGCKTAACCCTGGTCAC CGTCTCCTCA B-HL10
CGGAATTCGGCCCACCGGTGCTCCACCATGGACTG 57
GACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCA
CAGGAGCCCACTCCGAAGTGCAGCTGGTGGAGTCT
GGAGGAGGCTTGGTCAAGCCTGGCGGGTCCCTGCG
GCTCTCCTGTGCAGCCTCTGGATTCGCATTCAGCT
ATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGA
AAGGGCCTCGAGTGGGTGGACGGATCTTTCCCGGC
GATGGGGATACTGACTACAATGGGAAATTCAAGGG
CAGAGTCACAATTACCGCCGACAAATCCACTAGCA
CAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAG
GACACGGCCGTGTATTACTGTGCAAGAAATGTCTT
TGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAA CCCTGGTCACCGTCTCCTCAGCTAGCGAATT
B-HL11 CAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTFGCT 59
TCAAGCCTGGCGGGTCCCTGCGGCTCTCCTGTGCA
GCCTCTGGATTCACATTTAGCTATTCTTGGATGAA
CTGGGTGCGGCAGGCTCCTGGAAAGGGCCTCGAGT
GGGTGGGACGGATCTTTCCCGGCGATGGGGATACT
GACTACAATGGGAAATTCAAGGGCAGAGTCACAAT
TACCGCCGACAAATCCACTAGCACAGCCTATATGG
AGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTG
TATTACTGTGCAAGAAATGTCTTTGATGGTTACTG
GCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCG TCTCCTCA B-HL12
CGGAATTCGGCCCACCGGTGGCCACCATGCTACTG 61
GACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCA
CAGGAGCTCACTCCGAAGTGCAGCTCGTGGAGTCT
GGAGCAGGCTTGGTCAAGCCTGGCGGGTCCCTGCG
GCTCTCCTGCGCAGCCTCTGGATTCACATTTAGCT
ATTCTTGGATGAACTGGGTGTGCGGCAGGCTCCTG
GAAAGGGCCTCGAGTGGGTGGGACGGATCTTTCCC
GGCGATGGGGATACTGACTACAATGGGAAATTCAA
GGGCAGAGTCACAATTACCGCCGACAAATCCACTA
GCACAGCCTATATGGAGCTGAGCAGCCTGAGATCT
GAGGACACGGCCGTGTATTACTGTGCAAGAAATGT
CTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGG
GAACCCTGGTACCGTCTCCTCAGCTAGCGAATTCT CGA B-HL13
CGGAATTCGGCCCACCGGTGGCCACCATGGACTGG 63
ACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCAC
AGGAGCTCACTCCGAAGTGCAGCTCGTCGAGTCTG
GAGGAGGCGTGGTCAAGCCTGGCGGGTCCCTGCGG
CTCTCCTGCGCAGCCTCTGGATTCACATTTAGCTA
TTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGAA
AGGGCCTCGAGTGGGTGGGACGGATCTTTCCCGGC
GATGGGGATACTGACTACAATQGGAAKTTCAAGGG
CAGAGTCACAATTACCGCCGACAAATCCACTAGCA
CAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAG
GACACGGCCGTGTATTACTGTGCAAGAAATGTCTT
TGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAA CCCTGGTCACTCTCCTCA B-HL14
CGGAATTCGGCCCACCGGTGGCCACCATGGACTGG 65
ACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCAC
AGGAGCTCACTCCGAAGTGCAGCTGGTCGAGTCCG
GAGGAGGCTTGAAGAAGCCTGGCGGGTCCCTGCGG
CTCTCCTGCGCAGCCTCTGGATTCACATTTAGCTA
TTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGAA
AGGGCCTCGAGTGGGTGGGACGGATCTTTCCCGGC
GATGGGGATACTGACTACAATGGGAAATTCAAGGG
CAGAGTCACAATTACCGCCGACAAATCCACTAGCA
CAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAG
GACACGGCCGTGTATTACTCTTGCAAGAAATGTCT
TTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGA
ACCCTGGTCACCGTCTCCTCAGCTAGCGAATTCTC GA B-HL15
CGGAATTCGCTCCCACCGGTGGCCACCATGGACTG 67
GACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCA
CAGGAGCCCACTCCGAAGTGCAGCTGGTGGAGTCT
GGAGGAGCTTGGTCAAGCCTGGCTCTTCCCCTGCG
GCTCTCCTGCGCAGCCTCTGGATTCACATTTAGCT
ATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGA
AAGGGCCTCGAGTGGGTGGGACGGATCTTTCCCGG
CGATGGGGATACTGACTACAATGGGAAATTCAAGG
GCAGAGTCACAATTACCGCCGACAAATCCACTAGC
ACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGA
GGACACGGCCGTGTATTACTGTGCAAGAAATGTCT
TTGATGGTTACTGGCTTQTTTACTGGGGCCAGGGA
ACCCTGGTCACCGTCTCCTCAGCTAGCGAATTCTC GA B-HL16
CGGAATTCGGCCCACCGGTGGCCACCAGGACTCTG 69
GACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCA
CAGGAGCCCACTCCGAAGTGCAGCTGGTGGAGTCT
GGAGGAGGCTTGGTCAAGCCTGGCGGGTCCCTGCG
GGTCAGCTGCGCAGCCTCTGGATTCACATTTAGCT
ATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGA
AAGGGCCTCGAGTGGGTGGGACGGATCTTTCCCGG
CGATGGGGATACTGACTACAATGGGAAATTCAAGG
GCAGAGTCACAATTACCGCCGACAAATCCACTAGC
ACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGA
GGACACGGCCGTGTATTACTGTGCAAGAAATGTCT
TTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGA
ACCCTGGTCACCGTCTCCTCAGCTAGCGAATTCTC GA B-HL17
CGGAATTCGGCCCACCGGTGGCCACCATGGACTGG 71
ACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCAC
AGGAGCCCACTCCGAAGTGCAGCTGGTGGAGTCFG
GAGGAGGCTTGGTCAAGCCTGGCGGGTCCCTGCGG
CTCTCCTGCGCAGCCTCTGGATTCACATTTAGCTA
TTCTTTGGATGAACTGGGTGCGGCAGGCFCCTGGA
AAGGGCCTCCTAGTGGGTGGGACCTGATCTTTCCC
GGCGATGGGGATACTGACTACAATGGGAAATTCAA
GGGCAGAGTCACAATTACCGCCGACAAATCCACTA
GCACAGCCTATATGGAGCTGAGCACTCCTGAGATC
TGAGGACACGGCCGTGTATTACTGTGCAAGAAATG
TCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCCTAGCTAGCGAATTC TCGA VH
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGC 73 Signal
AGCAGCCACAGGAGCCCACTCC Se- quence B-KV1
GATATCGTGATGACCCACTACTCCACTCTCCCTGC 75
CCGTCACCCCTGGAGAGCCCGCCAGCATTAGCTGC
AGGTCTAGCAAGAGCCTCTFGCACAGCAATGGCAT
CACTTATTTGTATTGGTACCTGCAAAAGCCAGGGC
AGTCTCCACAGCTCCTGATTFATCAAATGTCCAAC
CTTGTCTCTGGCGTCCCTGACCGGTTCTCCGGATC
CGGGTCAGGCACTGATTTCACACTGAAAATCAGCA
GGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGC
GCTCAGAATCTAGAACTTCCTTACACCTTCGGCGG AGGGACCAAGGTGGAGATCAAACGTACGGTG
VL ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGCCT 77 Signal
CCTGCTGCTCTGGTTCCCAGTGCCAGGTGT Se- quence
TABLE-US-00003 TABLE 3 SEQ CON- ID STRUCT AMINO ACID SEQUENCE NO
B-HH 1 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSYSWMSWV 30
RQAPGQGLEWMGRIFPGDGDTDYAQKFQGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLVTVSS B-HH2
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWMNWV 32
RQAPGQGLEWMGRIFPGDGTYEDYNGKFKGRVTITAD
KSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWG QGTLVTVSS B-HH3
QVQLVQSGAEVKKPQSSVKVSCKASGYAFSYSWMNWV 34
RQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADK
STSTAYMELSSLRSEDTAVYLCARNVFDGYWLVYWGQ GTLVTVSS B-HH4
QVQLVQSGAEVKKPGASVKVSCKVSGYAFSYSWMNWV 36
RQAPGQGLEWMGRIFPGDGDTDYNGKEKGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLVTVSS B-HH5
QVQLVQSGAEVKKPGASVKVSCKASGYAFSYSWMSWV 38
RQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLVTVSS B-HH6
QVQLVQSGAEVKKPGSSVKVSCKASQYAFSYSWINWV 40
RQAPGQGLEWMGRIFPQDGDTDYNQIUKGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWQQ GTLVTVSS B-HH7
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWISWV 42
RQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVTDGYWLVYWGQ GTLVTVSS B-HH8
QVQLVQSGAEVKKPGASVKVSCKASGYTFSYSWMNWV 44
RQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLVTVSS B-HH9
QVQLVQSGAEVKKPGASVKVSCKASGYTFSYSWMNWV 46
RQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVEDGYWLVYWGQ GTLVTVSS B-HL1
QVQLVQSGAEVKKPGASVKVSCKASGYTFTYSWMHWV 48
RQAPGQGLEWMGRIPPGDGDTDYAQKFQGRVTMTRDT
STSTVYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLVTVSS B-HL2
EVQLVQSGAEVKKPQATVKISCKVSGYTFTYSWMHWV 50
QQAPGKGLEWMQRIFPGDGMDYAEKFQGRVTITADTS
TDTAYMELSSLRSEDTAVYYCATNVFDGYWLVYWGQG TLVTVSS B-HL3
EVQLVQSGAEVKKPGATVKISCKVSGYTFTYSWMNWV 52
QQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADT
STDTAYMELSSLRSEDTAVYYCATNVFDGYWLVYWGQ GTLVTVSS B-HL4
QMQLVQSGAEVKKTGSSVKVSCKASGYTFTYSWMSWV 54
RQAPGQGLEWMGRIFPGDGDTDYAQKFQGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLVTVSS B-HL8
EVQLVESGGGLVKPQGGSLRLSCAASGFTFSYSWMNW 56
VRQAPGKGLEWVGRIFPGDGDTDYNGKFKGRVTITAD
KSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWG QGTLVTVSS B-HL10
EVQLVESGGGLVKPGGSLRLSCAASGFAFSYSWMNWV 58
RQAPGKGLEWVGRIFPGDGDTDYNGKFKGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLVTVSS B-HL11
QVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMNWV 60
RQAPGKQLEWVGRIFPGDGDTDYNGKFKGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLYTVSS B-HL12
EVQLVESGAGLVKPGGSLRLSCAASGFTFSYSWMNWV 62
RQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGWLVYWGQG TLVTVSS B-HL13
EVQLVESGGGVVKPGGSLRLSCAASGFTSYSWMNWVR 64
QAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKS
TSTAYMELSSLRSETAVYYCARNVFDGYWLVYWGQGT LVTVSS B-HL14
EVQLVESGGGLKKPGGSLRLSCAASGFTFSYSWMNWV 66
RQAPGKGLEWMGRIFPGDGDTDYNGKFKQRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLVTVSS B-HL15
EVQLVESGGGLVKPGSSLRLSCAASGFTFSYSWMNWV 68
RQAPQKGLEWMQRIFPGDQDTDYNGICFKGRVTITAD
KSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWG QGTLVTVSS B-HL16
EVQLVESQGGLVKPGGSLRVSCAASGETFSYSWMNWV 70
RQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLVTVSS B-HL17
EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMNWV 72
RQAPGKQLEWMGRIFPGDGDTDYNGKFKGRVTITADK
STSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLVTVSS VH
MDWTWRILFLVAAATGAHS 74 Signal Se- quence B-KV1
DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITY 76
LYWYLQKPGQSPQLLIY.circle-solid.MSNLVSGVPDRFSGSGSGT
DFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEI KRTV VL
MDMRVPAQLLGLLLLWFPGARC 78 Signal Se- quence
[0147] In another preferred embodiment, the present invention is
directed to an isolated polynucleotide comprising a sequence that
encodes a polypeptide having the amino acid sequence shown in FIG.
1 or FIG. 2. 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 the nucleotide sequence shown in
FIG. 5 or FIG. 6. 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 the amino acid sequence FIG.
5 or FIG. 6. The invention also encompasses an isolated nucleic
acid comprising a sequence that encodes a polypeptide having the
amino acid sequence of any of FIG. 1, FIG. 2, FIG. 5 or FIG. 6 with
conservative amino acid substitutions.
[0148] 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.
[0149] Generally, any type of cultured cell line can be used to
express the ABM of the present invention. In a preferred
embodiment, CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO
myeloma cells, P3X63 mouse myeloma cells, PER cells, PRR.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.
[0150] 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 CD20, 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, the entire contents 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 murine B-LY1 antibody. In a
particularly preferred embodiment, the chimeric antibody comprises
a human Fc. In another preferred embodiment, the antibody is
primatized or humanized.
[0151] 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 ABM having
substantially the same binding specificity of the murine B-Ly1
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 ABM having substantially the same binding
specificity of the murine B-Ly1 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.
[0152] 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 murine B-Ly1 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).
[0153] 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, CHO cells, BHK cells, NS0 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
Charaow, 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 contemplated, including
yeast cells transformed with recombinant yeast expression vectors
containing the coding sequence of an ABM of the present invention;
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 murine B-Ly1 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; 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 murine B-Ly1 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.
[0154] 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.
[0155] A number of selection systems may be used, including, but
not limited to, the herpes simplex virus thymidine kinase (Wigter
et al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybaiski, 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.sup.-, hgprt.sup.- or aprt.sup.- 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 75:1527 (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-omithine, DFMO (McConlogue, in: Current
Communications in Molecular Biology, Cold Spring Harbor Laboratory
ed. (1987)).
[0156] 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.
[0157] 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.
[0158] 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.
[0159] 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 .alpha. 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.
[0160] In another embodiment, the present invention is directed to
a chimeric ABM having substantially the same binding specificity of
the murine B-Ly1 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.
[0161] In another embodiment, the present invention is directed to
a chimeric ABM having substantially the same binding specificity of
the murine B-Ly1 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.
[0162] The present invention is further directed to pharmaceutical
compositions comprising the ABMs of the present invention and a
pharmaceutically acceptable carrier.
[0163] 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.
[0164] 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 and 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 EP 1 176 195 A1, the entire contents of
which is incorporated by reference herein.
[0165] Generation of Cell Lines for the Production of Proteins with
Altered Glycosylation Pattern
[0166] 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.
[0167] 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.
[0168] 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, 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.
[0169] 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.
[0170] 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.
[0171] Identification of Transfectants or Transformants that
Express the Protein Having a Modified Glycosylation Pattern
[0172] The host cells which contain the coding sequence of a
chimeric ABM having substantially the same binding specificity of
the murine B-Ly1 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.
[0173] In the first approach, the presence of the coding sequence
of a chimeric ABM having substantially the same binding specificity
of the murine B-Ly1 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.
[0174] 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 cm 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.
[0175] 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.
[0176] 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 tire
success of the expression system, however, involves the detection
of the biologically active gene products.
[0177] Generation and Use of ABMs Having Increased Effector
Function Including Antibody-Dependent Cellular Cytotoxicity
[0178] In preferred embodiments, the present invention provides
glycoforms of chimeric ABMs having substantially the same binding
specificity of the murine B-Ly1 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.
[0179] Clinical trials of unconjugated monoclonal antibodies (mAbs)
for the treatment of some types of cancer have recently yielded
encouraging results. Diltman, 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. Diltman, Cancer Biother.
& Radiopharm. 12:223-25 (1997), while another unconjugated mAh,
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).
[0180] A different, but complementary, approach to increase ADCC
activity of unconjugated IgG1s 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.
75:26-31 (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.
[0181] 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)
[0182] 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 315:813-22 (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.
[0183] 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).
[0184] 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 are
recombinant, chimeric antibody or a fragment thereof with the
binding specificity of the murine B-Ly1 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.
[0185] Therapeutic Applications of ABMs Produced According to the
Methods of the Invention.
[0186] 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.
[0187] 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
bi functional 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, his-azido compounds
such as bis (p-azidobcnzoyl) hexanediamine, bis-diazonium
derivatives such as bis-(p-diazoniumbenzoyl)-etliylenediamine,
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.
[0188] In one embodiment, a chimeric, glycoengineered ABM having
substantially the same binding specificity of the murine B-Ly1
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.
[0189] 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.
[0190] 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 murine B-Ly1
antibody. The term "cytotoxic moiety" as used herein is intended to
include such isotopes.
[0191] 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 CD20 molecules on the surface of
the malignant B-cell, this method permits delivery of large amounts
of drug to the correct cell type.
[0192] Techniques for conjugating such therapeutic agents to
antibodies are well known (see, e.g., Arnon 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 at, "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 at, "The Preparation And Cytotoxic Properties Of
Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982)).
[0193] 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. Set.
USA 85:4842-46 (1988); "Enhancement of the in vitro and in vivo
Antitumor Activities 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)).
[0194] 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)].
[0195] 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 tire murine
B-Ly1 antibody (e.g., polypeptides comprising the CDRs of the
murine B-Ly1 antibody) and further comprising a toxin polypeptide.
The single-chain immunotoxins of the invention may be used to treat
human carcinoma in vivo.
[0196] 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.
[0197] The present invention provides a method for selectively
killing tumor cells expressing CD20. 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.
[0198] 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.
[0199] In a further aspect, the invention is directed to an
improved method for treating B-cell proliferative disorders
including B-cell lymphoma, as well as an autoimmune disease
produced in whole or in part by pathogenic autoantibodies, based on
B-cell depletion 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-CD20 antibody with a binding specificity
substantially the same as that of the murine B-Ly1 antibody. In
another preferred embodiment the antibody is humanized. Examples of
autoimmune diseases or disorders include, but are not limited to,
immune-mediated thrombocytopenias, such as acute idiopathic
thrombocytopenic purpurea and chronic idiopathic thrombocytopenic
purpurea, dermatomyositis, Sydenham's chorea, lupus nephritis,
rheumatic fever, polyglandular syndromes, Henoch-Schoniein purpura,
post-streptococcal nephritis, erythema nodosum, Takayasu's
arteritis, Addison's disease, erythema multiforme, polyarteritis
nodosa, ankylosing spondylitis, Goodpasture's syndrome,
thromboangitis ubiterans, primary biliary cirrhosis, Hashimoto's
thyroiditis, thyrotoxicosis, chronic active hepatitis,
polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,
Wegener's granulomatosis, membranous nephropathy, amyotrophic
lateral sclerosis, tabes dorsalis, potymyaglia, pernicious anemia,
rapidly progressive glomerulonephritis and fibrosing alveolitis,
inflammatory responses such as inflammatory skin diseases including
psoriasis and dermatitis (e.g. atopic dermatitis); systemic
scleroderma and sclerosis; responses associated with inflammatory
bowel disease (such as Crohn's disease and ulcerative colitis);
respiratory distress syndrome (including adult respiratory distress
syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis;
colitis; glomerulonephritis; allergic conditions such as eczema and
asthma and other conditions involving infiltration of T cells and
chronic inflammatory responses; atherosclerosis; leukocyte adhesion
deficiency; rheumatoid arthritis; systemic lupus erythematosus
(SLE); diabetes mellitus (e g. Type 1 diabetes mellitus or insulin
dependent diabetes mellitus); multiple sclerosis; Reynaud's
syndrome; autoimmune thyroiditis; allergic encephalomyelitis;
Sjorgen's syndrome; juvenile onset diabetes; and immune responses
associated with acute and delayed hypersensitivity mediated by
cytokines and T-lymphocytes typically found in tuberculosis,
sarcoidosis, polymyositis, granulomatosis and vasculitis;
pernicious amenia (Addison's disease); diseases involving leukocyte
diapedesis; central nervous system (CNS) inflammatory disorder;
multiple organ injury syndrome; hemolytic anemia (including, but
not limited to cryoglobinemia or Coombs positive anemia);
myasthenia gravis; antigen-antibody complex mediated diseases;
anti-glomerular basement membrane disease; antiphospholipid
syndrome; allergic neuritis; Graves' disease; Lambert-Eaton
myasthenic syndrome; pemphigoid bullous; pemphigus; autoimmune
polyendccrinopathies; Reiter's disease; stiff-man syndrome; Behcet
disease; giant cell arteritis; immune complex nephritis; IgA
nephropathy; IgM polyneuropathies; immune thrombocytopenic purpura
(FTP) or autoimmune thrombocytopenia etc. In this aspect of the
invention, the ABMs of the invention are used to deplete the blood
of normal B-cells for an extended period.
[0200] 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.
[0201] 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.
[0202] It is apparent, therefore, that the present invention
encompasses pharmaceutical compositions, combinations and methods
for treating human carcinomas, such as a B cell lymphoma. For
example, the invention includes pharmaceutical compositions for use
in the treatment of human carcinomas comprising a pharmaceutically
effective amount of an antibody of the present invention and a
pharmaceutically acceptable carrier.
[0203] 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.
[0204] 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 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0205] Exemplary anti-CD20 ABM formulations are described in
WO98/56418, expressly incorporated herein by reference. This
publication describes a liquid multidose formulation comprising 40
mg/mL rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl
alcohol, 0.02% polysorbate 20 at pH 5.0 that has a minimum shelf
life of two years storage at 2-8.degree. C. Another anti-CD20
formulation of interest comprises 10 mg/mL rituximab in 9.0 mg/mL
sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL
polysorbate 80, and Sterile Water for Injection, pH6.5. in the
present invention, RITUXAN.RTM. will be substituted by an ABM of
the present invention.
[0206] 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.
[0207] 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.
[0208] 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).
[0209] 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 tire 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 .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0210] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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.
[0215] 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, tire 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.
[0216] 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.
[0217] 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.3 to about 1000 mg/m.sup.2. In one embodiment, the dosage
of the antibody differs from that presently recommended for
RITUXAN.RTM.. For example, one may administer to tire patient one
or more doses of substantially less titan 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.
[0218] 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 dosc(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.
[0219] 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.
[0220] 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.
[0221] 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.
[0222] It would be clear that the dose of the composition of the
invention required to achieve cures may be further reduced with
schedule optimization.
[0223] 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.
[0224] 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).
[0225] Alternatively, the detergent maybe an ionic detergent. An
example of an ionic detergent includes, but is not limited to,
alkyltrimethylammonium bromide.
[0226] 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.
[0227] 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.
EXAMPLES
[0228] [NOTE: Unless otherwise specified, references to the
numbering of specific amino acid residue positions in the following
Examples are according to the Rabat numbering system.]
Example 1
Materials and Methods
Cloning and Expression of Recombinant Antibody B-Ly1
[0229] B-Ly1 expressing hybridoma cells were grown in RPMI
containing 10% FBS and 4 mM L-glutamine. 6.times.10.sup.6 cells
with a viability >90% were harvested and total RNA was isolated
using a Qiagen RNAeasy midi kit. cDNAs encoding the variable light
and heavy chains of B-Ly1 were amplified by RT-PCR. The RT-PCR
reaction was performed using the following conditions: 30 min
50.degree. C. for the first strand cDNA synthesis; 15 min
95.degree. C. initial denaturation; 30 cycles of 1 min 94.degree.
C.>1 min 45.degree. C., 1.5 min 72.degree. C.; and a final
elongation step for 10 min at 72.degree. C. The expected size of
the PCR products was confirmed by gel electrophoresis. The PCR
products were cloned into suitable E. coli vectors and DNA
sequencing confirmed that the variable light and heavy chain
encoding genes were isolated.
[0230] For construction of chimeric B-Ly1 expression vectors,
synthetic signal sequences and appropriate restriction sites were
fused to the variable chains by additional PCR reactions. After a
final confirmation of the correct DNA sequence of the variable
chains, they were combined with the corresponding human IgG1
constant regions. Once the genes were constructed, they were cloned
under control of the MPSV promoter and upstream of a synthetic
potyA site, using two separate vectors, one for each chain,
resulting in the plasmids pETR1808 (heavy chain expression vector)
and pETR1813 (light chain expression vector). Each vector carried
an EBV OriP sequence.
[0231] Chimeric B-Ly1 was produced by co-transfecting HEK293-EBNA
cells with vectors pETR1808 and pETR1813 using a calcium
phosphate-transfection approach. Exponentially growing HEK293-EBNA
cells were transfected by the calcium phosphate method. 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% CO.sub.2 atmosphere overnight. For each T75
flask to be transfected, a solution of DNA, CaCl.sub.2 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 CaCl.sub.2 solution, and adding water to a final
volume of 469 .mu.l. To this solution, 469 .mu.l of a 50 mM HEPES,
280 mM NaCl, 1.5 mM Na.sub.2HPO.sub.4 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%
CO.sub.2 for about 17 to 20 hours, then medium was replaced with 12
ml DMEM, 10% FCS. For the production of unmodified antibody
"chB-Ly1", the cells were transfected only with antibody expression
vectors pETR1808 and pETR1813 in a 1:1 ratio. For the production of
the glycoengineered antibody "chB-Ly1-ge", the cells were
co-transfected with four plasmids, two for antibody expression
(pETR1808 and pETR1813), one for a fusion GnTIII polypeptide
expression (pETR1519), and one for mannosidase 11 expression
(pCLF9) at a ratio of 4:4:1:1, respectively. At day 5
post-transfection, supernatant was harvested, centrifuged for 5 min
at 1200 rpm, followed by a second centrifugation for 10 min at 4000
rpm and kept at 4.degree. C.
[0232] chB-Ly1 and chB-Ly1-ge were purified from culture
supernatant using three sequential chromatographic steps, Protein A
chromatography, cation exchange chromatography, and a size
exclusion chromatography step on a Superdex 200 column (Amersham
Pharmacia) exchanging the buffer to phosphate buffer saline and
collecting the monomeric antibody peak from this last step.
Antibody concentration was estimated using a spectrophotometer from
the absorbance at 280 ran,
Oligosaccharide Analysis
[0233] Oligosaccharides were enzymatically released from the
antibodies by PNGaseF digestion, with the antibodies being either
immobilized on a PVDF membrane or in solution.
[0234] 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
[0235] 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.
[0236] 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.
[0237] 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.
[0238] N-linked oligosaccharides were released by addition of 2.5
mU peptide-N-glycosydase F (recombinant 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
[0239] 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
[0240] 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
[0241] 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
[0242] 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.
Whole Blood B Cell Depletion
[0243] 495 ul heparinized blood from a healthy donor was aliquoted
into 5 ml polystyrene tubes, 5 .mu.l 100-fold concentrated antibody
samples (1-1000 ng/ml final concentration) or PBS only were added
and the tubes were incubated at 37.degree.. After 24 h 50 .mu.l
blood was transferred to a fresh tube and stained with
anti-CD3-FITC, anti-CD19-PE and anti-CD45CyChrome
(Becton-Dickinson) for 15 min at room temperature in the dark.
Before analysis, 500 .mu.l FACS buffer (PBS containing 2% FCS and 5
mM EDTA) was added to the tubes. The CD3-FITC and CD19-PE
fluorescence of the blood samples were flowcytometrically analyzed
by setting a threshold on CD45-CyChrome. B cell-depletion was
determined by plotting the ratio of CD19.sup.+ B cells to CD3.sup.+
T cells.
Binding of anti-CD20 Antibodies to Raji Cells
[0244] 500.000 in 180 .mu.l FACS buffer (PBS containing 2% FCS and
5 mM EDTA) were transferred to 5 ml polystyrene tubes and 20 ul 10
fold concentrated anti-CD20 antibody samples (1-5000 ng/ml final
concentration) or PBS only were added and the tubes were incubated
at 4.degree. C. for 30 min. 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 100 .mu.l
FACS buffer and 1 .mu.l 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
containing 0.5 .mu.g/ml PI for analysis by Flow Cytometry. Binding
was determined by plotting the geometric mean fluorescence against
the antibody concentrations.
Example 2
High Homology Acceptor Approach
[0245] The high homology antibody acceptor framework search was
performed by aligning the mouse B-ly1 protein sequence to a
collection of human germ-line sequences and picking that human
sequence that showed the highest sequence identity. Here, the
sequence VH1_10 from the VBase database was chosen as the heavy
chain framework acceptor sequence, and the VK_2_40 sequence was
chosen to be the framework acceptor for the light chain. Onto these
two acceptor frameworks, the three complementary determining
regions (CDRs) of the mouse heavy and light variable domains were
grafted. Since the framework 4 region is not part of the variable
region of the germ line V gene, the alignment for that position was
done individually. The JH4 region was chosen for the heavy chain,
and the JK4 region was chosen for the light chain. Molecular
modelling of the designed immunoglobulin domain revealed one spot
potentially requiring the murine amino acid residues instead of the
human ones outside of the CDR. Re-introducing murine amino acid
residues into the human framework would generate the so-called back
mutations. For example, the human acceptor amino acid residue at
Kabat position 27 was back mutated to a tyrosine residue. 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 having designed the protein sequences,
DNA sequences encoding these proteins were synthesized as detailed
below.
Mixed Framework Approach
[0246] In order to avoid introducing back mutations at critical
amino acid residue positions (critical to retain good antigen
binding affinity or antibody functions) of the human acceptor
framework, it was investigated whether either the whole framework
region 1 (FR1), or framework regions 1 (FR1) and 2 (FR2) together,
could be replaced by human antibody sequences already having donor
residues, or functionally equivalent ones, at those important
positions in the natural human germline sequence. For this purpose,
the VH frameworks 1 and 2 of the mouse Bly1 sequence were aligned
individually to human germ-line sequences. Here, highest sequence
identity was not important, and was not used, for choosing acceptor
frameworks, but instead matching of several critical residues was
assumed to be more important. Those critical residues comprise
residues 24, 71, and 94 (Kabat numbering), 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. The IMGT sequence VH_3_15 was chosen as a suitable one.
After having designed the protein sequences, DNA sequences encoding
these proteins were synthesized as detailed below. Using this
approach no back mutations were required either for the light or
heavy chain, in order to retain good levels of antigen binding.
Synthesis of the Antibody Genes
[0247] After having designed the amino acid sequence of the
humanized antibody V region, the DNA sequence had to be generated.
The DNA sequence data of the individual framework regions was found
in the databases for human germ line sequences. The DNA sequence of
the CDR regions was taken from the corresponding murine cDNA data.
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 (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.
Antibody Production
[0248] Human heavy and light chain leader sequences (for secretion)
were added upstream of the above variable region sequences and
these were then joined upstream of human IgG1 kappa constant heavy
and light chain sequences, respectively, 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, as described in
Example 1 above. Antibodies were produced as described in Example 1
above, namely by co-transfecting HEK293-EBNA with the mammalian
antibody heavy and light chain expression vectors, harvesting the
conditioned culture medium 5 to 7 days post-transfection, and
purifying the secreted antibodies by Protein A affinity
chromatography, followed by cation exchange chromatography and a
final size exclusion chromatographic step to isolate pure monomeric
IgG1 antibodies. The antibodies were formulated in a 25 mM
potassium phosphate, 125 mM sodium chloride, 100 mM glycine
solution of pH 6.7. Glycoengineered variants of the humanized
antibody variants were produced by co-transfection of the antibody
expression vectors together with a GnT-III glycosyltransferase
expression vectors, or together with a GnT-III expression vector
plus a Golgi mannosidase II expression vector, as described for the
chimeric antibody in Example 1 above. Glycoengineered antibodies
were purified and formulated as described above for the
non-glycoengineered antibodies. The oligosaccharides attached to
the Fc region of the antibodies was analysed by MALDI/TOF-MS as
described below.
Oligossacharide Analysis
[0249] Oligosaccharide release method for antibodies in solution
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.
Sample Preparation for MALDI/TOF-MS
[0250] The enzymatic digests containing the released
oligosaccharides were incubated for a further 3 h at room
temperature 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
[0251] 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.
Antigen Binding Assay
[0252] The purified, monomeric humanized antibody variants were
tested for binding to human CD20 on Raji B-cell lymphoma target
cells using a flow cytometry-based assay, as described for the
chimeric B-ly1 antibody in Example 1 above.
Binding of Monomeric IgG1 Glycovariants to NK Cells and
Fc.quadrature.RIIIA-Expressing CHO Cell Line
[0253] Human NK cells were isolated from freshly isolated
peripheral blood mononuclear cells (PBMC) applying a negative
selection enriching for CD16- and CD56-positive cells (MACS system,
Miltenyi Biotec GmbH, Bergisch Gladbach/Germany). The purity
determined by CD56 expression was between 88-95%. Freshly isolated
NK cells were incubated in PBS without calcium and magnesium ions
(3.times.105 cells/ml) for 20 minutes at 37.degree. C. to remove NK
cell-associated IgG. Cells were incubated at 106 cells/ml at
different concentrations of anti-CD20 antibody (0, 0.1, 0.3, 1, 3,
10 .mu.g/ml) in PBS, 0.1% BSA. After several washes antibody
binding was detected by incubating with 1:200 FITC-conjugated
F(ab').sub.2 goat anti-human, F(ab')2 specific IgG (Jackson
ImmunoReasearch, West Grove, Pa./USA) and anti-human CD56-PE (BD
Biosciences, Allschwil/Switzerland). 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 for CD56-positive cells
on a FACSCalibur (BD Biosciences, Allschwil/Switzerland). 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 10.sup.6
cells. Binding of IgG1 to FcgammaRIIIA-Val158-expressing CHO cells
was performed analogously to the NK cell binding described
above.
ADCC Assay
[0254] Human peripheral blood mononuclear cells (PBMC) were used as
effector cells and were prepared using Histopaque-1077 (Sigma
Diagnostics Inc., St. Louis, Mo. 63178 USA) and following
essentially the manufacturer's instructions. In brief, venous blood
was taken with heparinized syringes from volunteers. The blood was
diluted 1:0.75-1.3 with PBS (not containing Ca++ or Mg++) 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.
[0255] 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 B lymphoma cells (e.g., Raji
cells) grown in DMEM containing 10% FCS. Target cells were washed
in PBS, counted and resuspended in A1M-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
horns at 37.degree. C. in a humified atmoshpere containing 5%
CO.sub.2. 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.
Complement Dependent Cytotoxicity Assay
[0256] Target cells were counted, washed with PBS, resuspended in
AIM-V (Invitrogen) at 1 million cells per ml. 50 .mu.l cells were
plated per well in a flat bottom 96 well plate. Antibody dilutions
were prepared in AIM-V and added in 50 .mu.l to the cells.
Antibodies were allowed to bind to the cells for 10 minutes at room
temperature. Human serum complement (Quidel) was freshly thawed,
diluted 3-fold with AIM-V and added in 50 .mu.l to the wells.
Rabbit complement (Cedarlane Laboratories) was prepared as
described by the manufacturer, diluted 3-fold with AIM-V and added
in 50 .mu.l to the wells. As a control, complement sources were
heated for 30 min at 56.degree. C. before addition to the assay.
The assay plates were incubated for 2 h at 37.degree. C. Killing of
cells was determined by measuring LDH release. Briefly, the plates
were centrifuged at 300.times.g for 3 min. 50 .mu.l supernatant per
well were transferred to a new 96 well plate and 50 .mu.l of the
assay reagent from the Cytotoxicity Kit (Roche) were added. A
kinetic measurement with the ELISA reader determined the Vmax
corresponding with LDH concentration in the supernatant. Maximal
release was determined by incubating the cells in presence of 1%
Trition X-1G0.
Whole Blood B-Cell Depletion Assay
[0257] Normal B-cell depletion in whole blood by the anti-CD20
antibodies was carried out as described in Example 1 above.
Apoptosis Assay
[0258] The apoptotic potency of the antibodies was assayed by
incubating the antibody at 10 .mu.g/ml (saturating conditions in
respect to antigen binding) with the target cells (at a target cell
concentration of 5.times.105 cells/ml) overnight (16-24 h). Samples
were stained with Ann V-FITC and analyzed by FAGS. Assay was done
in triplicates.
[0259] Detection is performed by flow cytometry by following the
appearance of apoptotic markers like annexin V and phosphatidy
serine. Negative control (no apoptosis induced) does not contain
any antibody, but only phosphate buffered saline. Positive control
(maximal apoptosis) contains 5 micromolar of the strong apoptosis
inducer Camptothecin (CPT).
Results and Discussion
[0260] Comparison of the binding to human CD20 antigen of antibody
variants B-HH1, B-HH2, B-HH3, either complexed with the chimeric
B-ly1 light chain (mVL, as described in Example 1 above) or with
the humanized B-ly1 light chain (KV1), and the parental, chimeric
antibody chB-ly1 (described in Example 1 above) shows that all
antibodies have a similar EC50 value, but the B-HH1 construct binds
with a tower intensity/stoichiometry than the variants B-HH2 and
B-HH3 (FIG. 11). B-F1H1 can be distinguished from B-HH2 and B-HH3
by its partially human CDR1 and CDR2 regions (Kabat definition), as
well as the Ala/Thr polymorphism at position 28 (Kabat numbering).
This indicates that either position 28, the complete CDR1, and/or
the complete CDR2 are important for antibody/antigen
interaction.
[0261] The comparison of the B-HL1, B-HH1, and the chimeric chB-ly1
parental antibody showed absence of any binding activity in the
B-HL1 construct, and about half of the binding
intensity/stoichiometry of the B-HH1 compared to B-ly1 (FIG. 12).
Both the B-HL1 as well as the B-HH1 are designed based on acceptor
frameworks derived from the human VH1 class. Among other
differences, position 71 (Kabat numbering; Kabat position 71
corresponds to position 72 of SEQ ID NO:48) of the B-HL1 construct
is a striking difference, indicating its putative importance for
antigen binding.
[0262] When comparing the antigen binding data of FIGS. 9 to 13,
the BBH2-KV1, BHL8-KV1, and BHL11-KV1 variants show the best
binding affinity, among the different humanized antibody variants
tested, to human CD20 on the surface of human cells. The
differences between B-HH2, on one hand, and B-HL8 and B-HL11 on the
other hand are located in the FR1 and FR2 regions only, with all
three CDRs being identical (compare, e.g., SEQ ID NOs: 32, 56, and
60, which are not numbered according to Kabat, but whose Kabat
numbering can be readily determined by one of ordinary skill).
B-HL8 and B-HL11 have their FR1 and FR2 sequences derived from the
human VH3 class, whereas the complete B-HH2 framework is human VH1
derived. B-HL11 is a derivative of B-HL8 with the single mutation
Glu1 Gin (position 1 is the same in both Kabat numbering and the
conventional numbering system used in the sequence listing), with
Gin being the amino acid residue in the B-HH2 construct. This means
that Glut Gin exchange does not alter binding affinity nor
intensity. The other differences between B-HH2 and B-HL8 are 14
framework residues, of which one or more will influence the antigen
binding behavior of this antibody.
[0263] The B-HL4 construct is derived from the B-HH2 antibody by
replacing the FR1 of the B-HH2 with that of the human germ line
sequence VH1_45. This construct shows greatly diminished antigen
binding capacity, despite having different amino acids at only
three positions within FR1. These residues are located at positions
2, 14, and 30 (Kabat numbering). Of these, position 30 could be an
influential position, since it is part of the Chothia definition of
CDR1. Overall analysis of all the binding curves from FIGS. 9 to 13
indicates that the following humanized B-ly1 heavy chain residues
(Kabat numbering) are important for binding to CD20: N35 (end of
Rabat CDR1), foil Rabat CDR1, foil Rabat CDR2 and foil Rabat CDR3,
residues A71 and R94 (in this case R94 cannot be replaced by a
threonine) and Y27. A28 and S30 also contribute to a lesser extent.
In addition, Rabat CDR3 and all canonical residues are important
for antigen binding. No back mutations were introduced in the
humanized light chain, which had the full Rabat CDR1, CDR2 and CDR3
grafted, in induction of apoptosis (FIGS. 14,15 and 21), the most
potent variant was humanized B-ly1 variant BHH2-KV1 (even more
potent than the original chB-ly1 and a lot more potent than an
antibody with a sequence identical to rituximab, C2B8). Other
humanized variants (derivatives of BHL8) that can recover the
increased apoptosis are: B-HL12 to B-HL17 (see Table) and BHH8
(mixed frameworks) and BHH9 ("mixed frameworks" with one back
mutation, S30T). Positions 9 and 48 (Rabat numbering) can contact
the antigen. Variants BHH4 to BHH7 are other humanized B-ly1
variants that do not introduce additional non-human sequences.
[0264] Important properties of tire humanized B-ly1 antibody are
that it is a type II anti-CD20 antibody as defined in Cragg, M S.
and Glennie, M. J., Blood 103(7):2738-2743 (April 2004). It
therefore did not induce, upon binding to CD20, any significant
resistance to non-ionic detergent extraction of CD20 from the
surface of CD20+ human cells, using the assay described for this
purposes in Polyak, M. J. and Deans, J. P., Blood 99(9):3256-3262
(2002). It certainly induced significantly less resistance to
non-ionic detergent extraction of CD20 than the C2B8 antibody does
(another anti-CD20 antibody with identical sequence to rituximab,
(See U.S. Pat. Pub, No. 2003 0003097 to Reff). As expected of a
type II anti-CD2Q antibody, the humanized B-ly1 did not have any
significant complement mediated lysis activity and certainly a lot
complement mediated lysis activity than the anti-CD20 antibody C2B8
(chimeric IgG1 with identical sequence to rituximab). Another
important property of the humanized B-ly1 antibody was that it was
very potent in the homotypic aggregation assay. In this assay
CD20-positive human cells, Daudi cells, were incubated in cell
culture medium for up to 24 hours at 37.degree. C. in a 5% CO2
atmosphere in a mammalian cell incubator as described in detail in
(Deans reference), with the antibody at a concentration of 1
microgram per ml and in parallel at a concentration of 5 micrograms
per ml. As a comparison, control, parallel incubation of the cells
were done under identical conditions but using the anti-CD20
antibody C2B8. At different time points, including 8 hours and 24
hours of incubation, the cells were inspected visually using a
microscope. It was found that the humanized B-ly1 antibody led to
strong homotypic aggregation, with aggregates being significantly
larger that those induced by addition of the C2B8 control antibody.
In addition, and consistent with the antibody being anti-CD20 type
II, it induced higher levels of apoptosis when CD20-positive human
cells were incubated with the humanized B-ly1 antibody, relative to
a control under identical conditions using the C2B8 chimeric IgG1
antibody with identical sequence to rituximab.
[0265] Glycoengineered variants of the humanized antibodies were
produced by co-expression of GnTIII glycosyltransferase, together
with the antibody genes, in mammalian cells. This led to an
increase in the fraction of non-fucosylated oligosaccharides
attached to the Fc region of the antibodies, including bisected
non-fucosylated oligosaccharides, as has been described in WO
2004/065540 (FIGS. 17-19). The glycoengineered antibodies had
significantly higher levels of binding to human FcgammaRIII
receptors (FIG. 20) and ADCC activity as well (FIG. 16), relative
to the non-glycoengineered antibody and relative to the C2B8
antibody. The humanized B-ly1 antibody was also more potent at
inducing human B-cell depletion in a whole blood assay (FIG. 16)
than the control C2B8 antibody. This was true both for the
non-glycoengineered B-ly1 antibody and for the glycoengineered
version of it. The glycoengineered antibody was approximately
1000-fold more potent than the C2B8 control anti-CD20 antibody in
depleting B-cells in the whole blood assay. This comparison is
important both for the non-glycoengineered and for the
glycoengineered humanized forms of B-ly1 antibody, because it
showed that in assays that combined Fc receptor-dependent
activities, such as ADCC, plus complement mediated lysis, plus
induction of apoptosis, that both forms of B-ly1 were significantly
more potent that C2B8, although both forms of B-ly1 have
dramatically lower complement mediated lysis activity. The ADCC, Fc
receptor-dependent cell killing activities and apoptosis induction
were present in this superior activity of the humanized B-ly1
antibody variants. Furthermore, in the apoptosis assay, both the
glycoengineered and non-glycoengineered forms of this type 11
anti-CD20 antibody were potent, with the Fc-engineered variants
with increased binding affinity to Fcgamma receptors being even
more potent in apoptosis induction than the non-Fc-engineered
variant, and with all variants being significantly more potent than
the control antibody C2B8. The exact mechanism for enhanced
homotypic aggregation and induction of apoptopsis mediated by type
H anti-CD20 antibodies is not known and concomitant binding to
other molecules on the surface of CD20-positive cells, such as Fc
gamma receptors, can influence this important property. It was
therefore important to demonstrate that anti-CD20 antibodies of
type II that have been engineered in their Fc region for increased
binding affinity to Fc gamma receptors, including FcgammaRIII and
with an associated increase in ADCC activity, were still able to
induce strong apoptosis, even higher than the non-Fc-engineered,
and homotypic aggregation. Apoptopsis induction is important as in
vivo, as there are locations in the body where the target
CD20-positive cells can be found, but were access to
FcgammaRIII-positive cells is more difficult than in blood, such
locations are, for example, lymph nodes. In those locations, the
induction of apoptosis by the anti-CD20 antibody itself can be
crucial for good efficacy of the anti-CD20 antibody therapy in
humans, both for the treatment of haematological malignancies such
as non-Hodgkins lymphomas and B-cell chronic lymphocytic leukaemia,
and for the treatment of autoimmune diseases such as rheumatoid
arthritis and lupus via a B-cell depletion approach. The increased
binding affinity to FcgammaRIII and higher ADCC of the humanized,
Fc-engineered type II anti-CD20 antibody can also be a very
important attribute for such therapies. Finally, tire reduced or
negligible complement mediated lysis activity of this type II
anti-CD20 antibodies, including humanized and Fc-engineered
variants, can also be important higher complement activation by
anti-CD20 antibodies has been correlated with increased,
undesirable side-effects
Sequence CWU 1
1
861112PRTMus sp. 1Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys
Ile Ser Cys Lys1 5 10 15Ala Ser Gly Tyr Ala Phe Ser Tyr Ser Trp Met
Asn Trp Val Lys Leu 20 25 30Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly
Arg Ile Phe Pro Gly Asp 35 40 45Gly Asp Thr Asp Tyr Asn Gly Lys Phe
Lys Gly Lys Ala Thr Leu Thr 50 55 60Ala Asp Lys Ser Ser Asn Thr Ala
Tyr Met Gln Leu Thr Ser Leu Thr65 70 75 80Ser Val Asp Ser Ala Val
Tyr Leu Cys Ala Arg Asn Val Phe Asp Gly 85 90 95Tyr Trp Leu Val Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 1102336DNAMus
sp. 2ggacctgaac tggtgaagcc tggggcctca gtgaagattt cctgcaaagc
ttctggctac 60gcattcagtt actcttggat gaactgggtg aaactgaggc ctggacaggg
tcttgagtgg 120attggacgga tttttcctgg agatggggat actgactaca
atgggaaatt caagggcaag 180gccacactga ctgctgacaa atcctccaac
acagcctaca tgcaactcac cagcctgacc 240tctgtggact ctgcggtcta
tttatgtgca agaaatgtct ttgatggtta ctggttagtt 300tactggggcc
aagggactct ggtcactgtc tctgca 3363103PRTMus sp. 3Asn Pro Val Thr Leu
Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser1 5 10 15Lys Ser Leu Leu
His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu 20 25 30Gln Lys Pro
Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn 35 40 45Leu Val
Ser Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr 50 55 60Asp
Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val65 70 75
80Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly
85 90 95Thr Lys Leu Glu Ile Lys Arg 1004309DNAMus sp. 4aatccagtca
ctcttggaac atcagcttcc atctcctgca ggtctagtaa gagtctccta 60catagtaatg
gcatcactta tttgtattgg tatctgcaga agccaggcca gtctcctcag
120ctcctgattt atcagatgtc caaccttgtc tcaggagtcc cagacaggtt
cagtagcagt 180gggtcaggaa ctgatttcac actgagaatc agcagagtgg
aggctgagga tgtgggtgtt 240tattactgtg ctcaaaatct agaacttccg
tacacgttcg gaggggggac caagctggaa 300ataaaacgg 309515DNAMus sp.
5tactcttgga tgaac 15618DNAMus sp. 6ggctacgcat tcagttac 18730DNAMus
sp. 7ggctacgcat tcagttactc ttggatgaac 30848DNAMus sp. 8aggtctagta
agagtctcct acatagtaat ggcatcactt atttgtat 48921DNAMus sp.
9cagatgtcca accttgtctc a 211027DNAMus sp. 10gctcaaaatc tagaacttcc
gtacacg 27111407DNAArtificial SequenceMouse-human chimeric cDNA
11atgggttgga gcctcatctt gctcttcctt gtcgctgttg ctacgcgtgt cctgtccgag
60gtcaagctgc agcagtctgg acctgaactg gtgaagcctg gggcctcagt gaagatttcc
120tgcaaagctt ctggctacgc attcagttac tcttggatga actgggtgaa
actgaggcct 180ggacagggtc ttgagtggat tggacggatt tttcctggag
atggggatac tgactacaat 240gggaaattca agggcaaggc cacactgact
gctgacaaat cctccaacac agcctacatg 300caactcacca gcctgacctc
tgtggactct gcggtctatt tatgtgcaag aaatgtcttt 360gatggttact
ggttagttta ctggggccaa gggactctgg tcactgtctc tgcagctagc
420accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc
tgggggcaca 480gcggccctgg gctgcctggt caaggactac ttccccgaac
cggtgacggt gtcgtggaac 540tcaggcgccc tgaccagcgg cgtgcacacc
ttcccggctg tcctacagtc ctcaggactc 600tactccctca gcagcgtggt
gaccgtgccc tccagcagct tgggcaccca gacctacatc 660tgcaacgtga
atcacaagcc cagcaacacc aaggtggaca agaaagcaga gcccaaatct
720tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg
gggaccgtca 780gtcttcctct tccccccaaa acccaaggac accctcatga
tctcccggac ccctgaggtc 840acatgcgtgg tggtggacgt gagccacgaa
gaccctgagg tcaagttcaa ctggtacgtg 900gacggcgtgg aggtgcataa
tgccaagaca aagccgcggg aggagcagta caacagcacg 960taccgtgtgg
tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac
1020aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat
ctccaaagcc 1080aaagggcagc cccgagaacc acaggtgtac accctgcccc
catcccggga tgagctgacc 1140aagaaccagg tcagcctgac ctgcctggtc
aaaggcttct atcccagcga catcgccgtg 1200gagtgggaga gcaatgggca
gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260tccgacggct
ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag
1320gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
cacgcagaag 1380agcctctccc tgtctccggg taaatga 140712720DNAArtificial
SequenceMouse-human chimeric cDNA 12atggattttc aggtgcagat
tatcagcttc ctgctaatca gtgcttcagt cataatgtcc 60agaggagaca ttgtgctcac
ccaaactaca aatccagtca ctcttggaac atcagcttcc 120atctcctgca
ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg
180tatctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc
caaccttgtc 240tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa
ctgatttcac actgagaatc 300agcagagtgg aggctgagga tgtgggtgtt
tattactgtg ctcaaaatct agaacttccg 360tacacgttcg gaggggggac
caagctggaa ataaaacgta cggtggctgc accatctgtc 420ttcatcttcc
cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg
480ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa
cgccctccaa 540tcgggtaact cccaggagag tgtcacagag caggacagca
aggacagcac ctacagcctc 600agcagcaccc tgacgctgag caaagcagac
tacgagaaac acaaagtcta cgcctgcgaa 660gtcacccatc agggcctgag
ctcgcccgtc acaaagagct tcaacagggg agagtgttag 72013468PRTArtificial
SequenceMouse-human chimeric polypeptide 13Met Gly Trp Ser Leu Ile
Leu Leu Phe Leu Val Ala Val Ala Thr Arg1 5 10 15Val Leu Ser Glu Val
Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30Pro Gly Ala Ser
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe 35 40 45Ser Tyr Ser
Trp Met Asn Trp Val Lys Leu Arg Pro Gly Gln Gly Leu 50 55 60Glu Trp
Ile Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn65 70 75
80Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn
85 90 95Thr Ala Tyr Met Gln Leu Thr Ser Leu Thr Ser Val Asp Ser Ala
Val 100 105 110Tyr Leu Cys Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu
Val Tyr Trp 115 120 125Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala
Ser Thr Lys Gly Pro 130 135 140Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr145 150 155 160Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200
205Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
210 215 220His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro
Lys Ser225 230 235 240Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu 245 250 255Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 260 265 270Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 275 280 285His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr305 310 315
320Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
325 330 335Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro 340 345 350Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 355 360 365Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val 370 375 380Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val385 390 395 400Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440
445Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
450 455 460Ser Pro Gly Lys46514239PRTArtificial SequenceMouse-human
chimeric polypeptide 14Met Asp Phe Gln Val Gln Ile Ile Ser Phe Leu
Leu Ile Ser Ala Ser1 5 10 15Val Ile Met Ser Arg Gly Asp Ile Val Leu
Thr Gln Thr Thr Asn Pro 20 25 30Val Thr Leu Gly Thr Ser Ala Ser Ile
Ser Cys Arg Ser Ser Lys Ser 35 40 45Leu Leu His Ser Asn Gly Ile Thr
Tyr Leu Tyr Trp Tyr Leu Gln Lys 50 55 60Pro Gly Gln Ser Pro Gln Leu
Leu Ile Tyr Gln Met Ser Asn Leu Val65 70 75 80Ser Gly Val Pro Asp
Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe 85 90 95Thr Leu Arg Ile
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110Cys Ala
Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys 115 120
125Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
130 135 140Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val
Cys Leu145 150 155 160Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp 165 170 175Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp 180 185 190Ser Lys Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205Ala Asp Tyr Glu Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220Gly Leu Ser
Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230 235155PRTMus
sp. 15Tyr Ser Trp Met Asn1 5166PRTMus sp. 16Gly Tyr Ala Phe Ser
Tyr1 51710PRTMus sp. 17Gly Tyr Ala Phe Ser Tyr Ser Trp Met Asn1 5
101816PRTMus sp. 18Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile
Thr Tyr Leu Tyr1 5 10 15197PRTMus sp. 19Gln Met Ser Asn Leu Val
Ser1 5209PRTMus sp. 20Ala Gln Asn Leu Glu Leu Pro Tyr Thr1
52151DNAMus sp. 21cggatttttc ctggagatgg ggatactgac tacaatggga
aattcaaggg c 512224DNAMus sp. 22tttcctggag atggggatac tgac
242330DNAMus sp. 23cggatttttc ctggagatgg ggatactgac 302430DNAMus
sp. 24aatgtctttg atggttactg gttagtttac 302517PRTMus sp. 25Arg Ile
Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe Lys1 5 10
15Gly268PRTMus sp. 26Phe Pro Gly Asp Gly Asp Thr Asp1 52710PRTMus
sp. 27Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp1 5 102810PRTMus sp.
28Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr1 5
1029357DNAArtificialMouse-human chimeric DNA 29caggtgcaat
tggtgcagtc tggcgctgaa gttaagaagc ctgggagttc agtgaaggtc 60tcctgcaagg
cttccggata caccttcagc tattcttgga tgagctgggt gcggcaggcc
120cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180gcacagaaat tccaaggaag agtcacaatt accgccgaca
aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35730119PRTArtificialMouse-human chimeric polypeptide 30Gln 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 Tyr Thr Phe Ser Tyr Ser 20 25 30Trp
Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11531357DNAArtificialMouse-human chimeric DNA 31caggtgcaat
tggtgcagtc tggcgctgaa gttaagaagc ctgggagttc agtgaaggtc 60tcctgcaagg
cttccggata cgccttcagc tattcttgga tgaactgggt gcggcaggcc
120cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180aatgggaaat tcaagggcag agtcacaatt accgccgaca
aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35732119PRTArtificialMouse-human chimeric polypeptide 32Gln 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 Tyr Ala Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11533366DNAArtificialMouse-human chimeric DNA 33caggtgcaat
tggtgcagtc tggcgctgaa gttaagaagc ctgggagttc agtgaaggtc 60tcctgcaagg
cttccggata cgccttcagc tattcttgga tgaactgggt gcggcaggcc
120cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180aatgggaaat tcaagggcag agtcacaatt accgccgaca
aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt atctgtgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctcagct 360agcacc
36634119PRTArtificialMouse-human chimeric polypeptide 34Gln 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 Tyr Ala Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Leu Cys 85 90 95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11535357DNAArtificialMouse-human chimeric DNA 35caggtgcaat
tggtgcagtc tggcgctgaa gttaagaagc ctggagcttc agtgaaggtc 60tcctgcaagg
tctccggata cgcgttcagc tattcttgga tgaactgggt gcggcaggcc
120cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180aatgggaaat tcaagggcag agtcacaatt accgccgaca
aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35736119PRTArtificialMouse-human chimeric polypeptide 36Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Val Ser Gly Tyr Ala Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11537357DNAArtificialMouse-human chimeric DNA 37caggtgcaat
tggtgcagtc tggcgctgaa gttaagaagc ctgggagttc agtgaaggtc 60tcctgcaagg
cttccggata
cgcgttcagc tattcttgga tgagctgggt gcggcaggcg 120cctggacaag
ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac
180aatgggaaat tcaagggcag agtcacaatt accgccgaca aatccactag
cacagcctat 240atggagctga gcagcctgag atctgaggac acggccgtgt
attactgtgc aagaaatgtc 300tttgatggtt actggcttgt ttactggggc
cagggaaccc tggtcaccgt ctcctca 35738119PRTArtificialMouse-human
chimeric polypeptide 38Gln 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
Tyr Ala Phe Ser Tyr Ser 20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Phe Pro Gly Asp Gly
Asp Thr Asp Tyr Asn Gly Lys Phe 50 55 60Lys 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 Asn Val
Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser 11539357DNAArtificialMouse-human chimeric DNA
39caggtgcaat tggtgcagtc tggcgctgaa gttaagaagc ctgggagttc agtgaaggtc
60tcctgcaagg cttccggata cgccttcagc tattcttgga tcaattgggt gcggcaggcg
120cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180aatgggaaat tcaagggcag agtcacaatt accgccgaca
aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35740119PRTArtificialMouse-human chimeric polypeptide 40Gln 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 Tyr Ala Phe Ser Tyr Ser 20 25 30Trp
Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11541357DNAArtificialMouse-human chimeric DNA 41caggtgcaat
tggtgcagtc tggcgctgaa gttaagaagc ctgggagttc agtgaaggtc 60tcctgcaagg
cttccggata cgccttcagc tattcttgga tctcgtgggt gcggcaggcg
120cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180aatgggaaat tcaagggcag agtcacaatt accgccgaca
aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35742119PRTArtificialMouse-human chimeric polypeptide 42Gln 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 Tyr Ala Phe Ser Tyr Ser 20 25 30Trp
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11543357DNAArtificialMouse-human chimeric DNA 43caggtgcaat
tggtgcagtc tggcgctgaa gttaagaagc ctggcgcctc agtgaaggtc 60tcctgcaagg
cttccggata caccttcaca tacagctgga tgaactgggt gcggcaggcc
120cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180aatgggaaat tcaagggcag agtcacaatt accgccgaca
aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35744119PRTArtificialMouse-human chimeric polypeptide 44Gln 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 Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11545357DNAArtificialMouse-human chimeric DNA 45caggtgcaat
tggtgcagtc tggcgctgaa gttaagaagc ctggcgcctc agtgaaggtc 60tcctgcaagg
cttccggata caccttcagc tattcttgga tgaactgggt gcggcaggcc
120cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180aatgggaaat tcaagggcag agtcacaatt accgccgaca
aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35746119PRTArtificialMouse-human chimeric polypeptide 46Gln 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 Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11547357DNAArtificialMouse-human chimeric DNA 47caggtgcaat
tggtgcagtc tggcgctgaa gttaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg
cttccggata caccttcacc tattcttgga tgcactgggt gcggcaggcc
120cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180gcacagaaat tccaaggaag agtcacaatg acacgggaca
cgtccacttc caccgtctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35748119PRTArtificialMouse-human chimeric polypeptide 48Gln 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 Tyr Ser 20 25 30Trp
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Ala Gln Lys Phe
50 55 60Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11549357DNAArtificialMouse-human chimeric DNA 49gaggtgcaat
tggtgcagtc tggcgctgaa gttaagaagc ctggggccac cgtgaagatc 60tcctgcaagg
tgtccggata caccttcacc tattcttgga tgcactgggt gcagcaggcc
120cctggaaagg ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180gcagagaaat tccaaggaag agtcacaatc acagccgaca
cgtccactga caccgcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aaccaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35750119PRTArtificialMouse-human chimeric polypeptide 50Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val
Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Tyr Ser 20 25 30Trp
Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Ala Glu Lys Phe
50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Thr Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11551357DNAArtificialMouse-human chimeric DNA 51gaggtgcaat
tggtgcagtc tggcgctgaa gttaagaagc ctggggccac cgtgaagatc 60tcctgcaagg
tgtccggata caccttcacc tattcttgga tgaactgggt gcagcaggcc
120cctggaaagg ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180aatgggaaat tcaagggaag agtcacaatc acagccgaca
cgtccactga caccgcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aaccaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35752119PRTArtificialMouse-human chimeric polypeptide 52Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val
Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Tyr Ser 20 25 30Trp
Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Thr Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11553366DNAArtificialMouse-human chimeric DNA 53cagatgcaat
tggtgcagtc tggcgctgaa gttaagaaga ccgggagttc agtgaaggtc 60tcctgcaagg
cttccggata caccttcacc tattcttgga tgagctgggt gcggcaggcc
120cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga
tactgactac 180gcacagaaat tccaaggaag agtcacaatt accgccgaca
aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctcagct 360agcacc
36654119PRTArtificialMouse-human chimeric polypeptide 54Gln Met Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Thr Gly Ser1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Tyr Ser 20 25 30Trp
Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11555357DNAArtificialMouse-human chimeric DNA 55gaagtgcagc
tggtggagtc tggaggaggc ttggtcaagc ctggcgggtc cctgcggctc 60tcctgtgcag
cctctggatt cacatttagc tattcttgga tgaactgggt gcggcaggct
120cctggaaagg gcctcgagtg ggtgggacgg atctttcccg gcgatgggga
tactgactac 180aatgggaaat tcaagggcag agtcacaatt accgccgaca
aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35756119PRTArtificialMouse-human chimeric polypeptide 56Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11557456DNAArtificialMouse-human chimeric DNA 57cggaattcgg
cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60cagcagccac
aggagcccac tccgaagtgc agctggtgga gtctggagga ggcttggtca
120agcctggcgg gtccctgcgg ctctcctgtg cagcctctgg attcgcattc
agctattctt 180ggatgaactg ggtgcggcag gctcctggaa agggcctcga
gtgggtggga cggatctttc 240ccggcgatgg ggatactgac tacaatggga
aattcaaggg cagagtcaca attaccgccg 300acaaatccac tagcacagcc
tatatggagc tgagcagcct gagatctgag gacacggccg 360tgtattactg
tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa
420ccctggtcac cgtctcctca gctagcgaat tctcga
45658119PRTArtificialMouse-human chimeric polypeptide 58Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11559357DNAArtificialMouse-human chimeric DNA 59caggtgcagc
tggtggagtc tggaggaggc ttggtcaagc ctggcgggtc cctgcggctc 60tcctgtgcag
cctctggatt cacatttagc tattcttgga tgaactgggt gcggcaggct
120cctggaaagg gcctcgagtg ggtgggacgg atctttcccg gcgatgggga
tactgactac 180aatgggaaat tcaagggcag agtcacaatt accgccgaca
aatccactag cacagcctat 240atggagctga gcagcctgag atctgaggac
acggccgtgt attactgtgc aagaaatgtc 300tttgatggtt actggcttgt
ttactggggc cagggaaccc tggtcaccgt ctcctca
35760119PRTArtificialMouse-human chimeric polypeptide 60Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11561456DNAArtificialMouse-human chimeric DNA 61cggaattcgg
cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60cagcagccac
aggagctcac tccgaagtgc agctcgtgga gtctggagca ggcttggtca
120agcctggcgg gtccctgcgg ctctcctgcg cagcctctgg attcacattt
agctattctt 180ggatgaactg ggtgcggcag gctcctggaa agggcctcga
gtgggtggga cggatctttc 240ccggcgatgg ggatactgac tacaatggga
aattcaaggg cagagtcaca attaccgccg 300acaaatccac tagcacagcc
tatatggagc tgagcagcct gagatctgag gacacggccg 360tgtattactg
tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa
420ccctggtcac cgtctcctca gctagcgaat tctcga
45662119PRTArtificialMouse-human chimeric polypeptide 62Glu Val Gln
Leu Val Glu Ser Gly Ala Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55
60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly
Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11563456DNAArtificialMouse-human chimeric DNA 63cggaattcgg
cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60cagcagccac
aggagctcac tccgaagtgc agctcgtcga gtctggagga ggcgtggtca
120agcctggcgg gtccctgcgg ctctcctgcg cagcctctgg attcacattt
agctattctt 180ggatgaactg ggtgcggcag gctcctggaa agggcctcga
gtgggtggga cggatctttc 240ccggcgatgg ggatactgac tacaatggga
aattcaaggg cagagtcaca attaccgccg 300acaaatccac tagcacagcc
tatatggagc tgagcagcct gagatctgag gacacggccg 360tgtattactg
tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa
420ccctggtcac cgtctcctca gctagcgaat tctcga
45664119PRTArtificialMouse-human chimeric polypeptide 64Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11565456DNAArtificialMouse-human chimeric DNA 65cggaattcgg
cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60cagcagccac
aggagctcac tccgaagtgc agctggtcga gtccggagga ggcttgaaga
120agcctggcgg gtccctgcgg ctctcctgcg cagcctctgg attcacattt
agctattctt 180ggatgaactg ggtgcggcag gctcctggaa agggcctcga
gtgggtggga cggatctttc 240ccggcgatgg ggatactgac tacaatggga
aattcaaggg cagagtcaca attaccgccg 300acaaatccac tagcacagcc
tatatggagc tgagcagcct gagatctgag gacacggccg 360tgtattactg
tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa
420ccctggtcac cgtctcctca gctagcgaat tctcga
45666119PRTArtificialMouse-human chimeric polypeptide 66Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Lys Lys Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11567456DNAArtificialMouse-human chimeric DNA 67cggaattcgg
cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60cagcagccac
aggagcccac tccgaagtgc agctggtgga gtctggagga ggcttggtca
120agcctggctc ttccctgcgg ctctcctgcg cagcctctgg attcacattt
agctattctt 180ggatgaactg ggtgcggcag gctcctggaa agggcctcga
gtgggtggga cggatctttc 240ccggcgatgg ggatactgac tacaatggga
aattcaaggg cagagtcaca attaccgccg 300acaaatccac tagcacagcc
tatatggagc tgagcagcct gagatctgag gacacggccg 360tgtattactg
tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa
420ccctggtcac cgtctcctca gctagcgaat tctcga
45668119PRTArtificialMouse-human chimeric polypeptide 68Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Ser1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11569456DNAArtificialMouse-human chimeric DNA 69cggaattcgg
cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60cagcagccac
aggagcccac tccgaagtgc agctggtgga gtctggagga ggcttggtca
120agcctggcgg gtccctgcgg gtcagctgcg cagcctctgg attcacattt
agctattctt 180ggatgaactg ggtgcggcag gctcctggaa agggcctcga
gtgggtggga cggatctttc 240ccggcgatgg ggatactgac tacaatggga
aattcaaggg cagagtcaca attaccgccg 300acaaatccac tagcacagcc
tatatggagc tgagcagcct gagatctgag gacacggccg 360tgtattactg
tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa
420ccctggtcac cgtctcctca gctagcgaat tctcga
45670119PRTArtificialMouse-human chimeric polypeptide 70Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Arg Val Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11571456DNAArtificialMouse-human chimeric DNA 71cggaattcgg
cccaccggtg gccaccatgg actggacctg gaggatcctc ttcttggtgg 60cagcagccac
aggagcccac tccgaagtgc agctggtgga gtctggagga ggcttggtca
120agcctggcgg gtccctgcgg ctctcctgcg cagcctctgg attcacattt
agctattctt 180ggatgaactg ggtgcggcag gctcctggaa agggcctcga
gtgggtggga cggatctttc 240ccggcgatgg ggatactgac tacaatggga
aattcaaggg cagagtcaca attaccgccg 300acaaatccac tagcacagcc
tatatggagc tgagcagcct gagatctgag gacacggccg 360tgtattactg
tgcaagaaat gtctttgatg gttactggct tgtttactgg ggccagggaa
420ccctggtcac cgtctcctca gctagcgaat tctcga
45672119PRTArtificialMouse-human chimeric polypeptide 72Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Ser 20 25 30Trp
Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40
45Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe
50 55 60Lys 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 Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
1157357DNAHomo sapiens 73atggactgga cctggaggat cctcttcttg
gtggcagcag ccacaggagc ccactcc 577419PRTHomo sapiens 74Met Asp Trp
Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly1 5 10 15Ala His
Ser75345DNAArtificialMouse-human chimeric DNA 75gatatcgtga
tgacccagac tccactctcc ctgcccgtca cccctggaga gcccgccagc 60attagctgca
ggtctagcaa gagcctcttg cacagcaatg gcatcactta tttgtattgg
120tacctgcaaa agccagggca gtctccacag ctcctgattt atcaaatgtc
caaccttgtc 180tctggcgtcc ctgaccggtt ctccggatcc gggtcaggca
ctgatttcac actgaaaatc 240agcagggtgg aggctgagga tgttggagtt
tattactgcg ctcagaatct agaacttcct 300tacaccttcg gcggagggac
caaggtggag atcaaacgta cggtg 34576115PRTArtificialMouse-human
chimeric polypeptide 76Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Lys Ser Leu Leu His Ser 20 25 30Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr
Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Gln Met
Ser Asn Leu Val Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala
Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85 90 95Leu Glu Leu Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr
Val 1157766DNAHomo sapiens 77atggacatga gggtccccgc tcagctcctg
ggcctcctgc tgctctggtt cccaggtgcc 60aggtgt 667822PRTHomo sapiens
78Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp1
5 10 15Phe Pro Gly Ala Arg Cys 207921DNAArtificialnucleic acids
encoding linker peptides 79gaggtcaagc tgcagcagtc t
218021DNAArtificialcomplementary nucleic acid sequence of SEQ ID NO
79, which encodes a linker peptide 80agactgctgc agcttgacct c
21817PRTArtificiallinker peptides 81Glu Val Lys Leu Gln Gln Ser1
58224DNAArtificialnucleic acids encoding linker peptides
82gacattgtgc tcacccaaac taca 248324DNAArtificialcomplementary
nucleic acid sequence of SEQ ID NO 82, which encodes a linker
peptide 83tgtagtttgg gtgagcacaa tgtc 24848PRTArtificiallinker
peptides 84Asp Ile Val Leu Thr Gln Thr Thr1 585336DNAMus sp.
85tgcagagaca gtgaccagag tcccttggcc ccagtaaact aaccagtaac catcaaagac
60atttcttgca cataaataga ccgcagagtc cacagaggtc aggctggtga gttgcatgta
120ggctgtgttg gaggatttgt cagcagtcag tgtggccttg cccttgaatt
tcccattgta 180gtcagtatcc ccatctccag gaaaaatccg tccaatccac
tcaagaccct gtccaggcct 240cagtttcacc cagttcatcc aagagtaact
gaatgcgtag ccagaagctt tgcaggaaat 300cttcactgag gccccaggct
tcaccagttc aggtcc 33686309DNAMus sp. 86ccgttttatt tccagcttgg
tcccccctcc gaacgtgtac ggaagttcta gattttgagc 60acagtaataa acacccacat
cctcagcctc cactctgctg attctcagtg tgaaatcagt 120tcctgaccca
ctgctactga acctgtctgg gactcctgag acaaggttgg acatctgata
180aatcaggagc tgaggagact ggcctggctt ctgcagatac caatacaaat
aagtgatgcc 240attactatgt aggagactct tactagacct gcaggagatg
gaagctgatg ttccaagagt 300gactggatt 309
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