Note: Descriptions are shown in the official language in which they were submitted.
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HIGH AFFINITY HUMANIZED ANTI-CEA MONOCLONAL ANTIBODIES
~~LD OF THE INVENTION
The present invention relates to humanized monoclonal antibodies and fragments
or
derivatives thereof which specifically bind carcinoembryonic antigen (CEA),
which is an
antigen expressed by various human carcinomas including breast, lung, and
gastrointestinal
carcinomas such as stomach and colon cancers. More specifically, the present
invention
relates to humanized monoclonal antibodies and humanized antibody fragments
and
derivatives thereof which are derived from murine monoclonal antibody COL-1, a
high affinity
anti-CEA antibody. The present invention further relates to methods for
producing such
humanized monoclonal antibodies specific to CEA, pharmaceutical and diagnostic
compositions containing such humanized monoclonal antibodies, and methods of
use
thereof for the treatment or diagnosis of cancer.
BACKGROUND OF THE INVENTION
The identification of antigens expressed by tumor cells and the preparation of
monoclonal antibodies which specifically bind such antigens is well known in
the art. Anti-
tumor monoclonal antibodies exhibit potential application as both therapeutic
and diagnostic
agents. Such monoclonal antibodies have potential application as diagnostic
agents
2 0 because they specifically bind tumor antigens and thereby can detect the
presence of tumor
cells or tumor antigen in an analyte. For example, use of monoclonal
antibodies which bind
tumor antigens for in vitro and in vivo imaging of tumor cells or tumors using
a labeled form
of such a monoclonal antibody is conventional in the art.
Moreover, monoclonal antibodies which bind tumor antigens have well known
2 5 application as therapeutic agents. The usage of monoclonal antibodies
themselves as
therapeutic agents, or as conjugates wherein the monoclonal antibody is
directly or indirectly
attached to an effector moiety, e.g., a drug, cytokine, cytotoxin, etc., is
well known.
Essentially, if the monoclonal antibody is attached to an effector moiety,
then the
monoclonal antibody functions as a targeting moiety, i.e. it directs the
effector moiety (which
3 0 typically possesses therapeutic activity) to the antibody's target, e.g.,
a tumor which
expresses the antigen bound by the monoclonal antibody. In contrast, when the
monoclonal
antibody itself operates as a therapeutic agent, the antibody functions both
as a targeting
moiety -- i.e. it will specifically bind a cell which expresses the antigen --
and as an effector
which mediates therapeutic activity, typically tumor cell lysis. A monoclonal
antibody may
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possess one or more of such effector functions, which include, e.g., antibody-
dependent
cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC),
among others;
these functions are effected by the portion of the antibody molecule generally
referred to in
the literature as the Fc portion.
One specific tumor antigen to which various monoclonal antibodies have been
produced is the carcinoembryonic antigen (CEA). CEA is an antigen complex
having a
molecular weight of about 180,000 D, which is expressed by numerous carcinomas
including
gastrointestinal carcinomas, colorectal carcinomas, breast carcinomas, ovarian
carcinomas,
and lung carcinomas. See, e.g., Robbins et al., !nt'I J. Cancer, x(6):892-897
(1993); Greiner
et aJ., J. Clin. Oncol., x(5):735-746 (1992); Obuchi et al., Cancer Res.,
47(13):3565-3571
{1987); Muraro et al., Cancer Res., _4(11 Pt. 2):5769-5780 (1985).
The use of monoclonal antibodies to detect various, specific CEA epitopes
differentially expressed on human carcinomas has been reported in the
literature. See, e.g.,
Obuchi et al., Cancer Res., x(13):3565-3571 (1987); Muraro et al., Cancer
Res., 45(11 Pt.
2):5769-5780 (1985).
In particular, Muraro et al. (id.) report generation of monoclonal antibodies
designated
COL-1 through COL-15, which exhibit a strong, selective reactivity for human
colon
carcinomas versus normal adult tissues. These antibodies react with distinct,
restricted
epitopes on CEA. Of these antibodies, the COL-1 antibody has been the focus of
considerable attention because of its high affinity for CEA (1.4x109 M'') and
also because it
comprises no detectable reactivity for CEA-related antigens such as the
nonspecific cross-
treating antigen (NCA) and the normal fecal antigen (NFA). Robbins et al.,
Int'I J. Canc.,
x(6):892-897 (1993).
Because of its binding properties, COL-1 is currently being evaluated for use
as a
2 5 therapeutic agent. For example, Siler et al. (Biotech. Ther., 4_(3-4):163-
181 (1993)) report
the administration of'3'I-labeled COL-1 to LS-M4T human colon carcinoma
xenograft-
containing athymic mice. They report that this treatment resulted in reduction
of the rate of
tumor growth, within little or no toxicity, and that their results demonstrate
the potential
therapeutic efficacy of radiolabeled COL-1 in clinical trials. Also, Yu et al.
(J. Clin. Oncol.,
14(6):1798-1809 (1996)) report that'3'I-labeled COL-1 is now in phase 1
clinical trials in
patients having gastrointestinal malignancies. They further indicate that the
antibody
conjugate is well tolerated, except for some hematologic toxicity. In
addition, the use of
conjugates of COL-1 and ~i-galactosidase has been shown to specifically kill
in vitro tumor
cells from a variety of tumor cell lines. Abraham et al., Cell Biophys., 24-
2525:127-133 (1994).
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However, while marine antibodies, such as COL-1 and other anti-CEA marine
antibodies, have applicability as therapeutic agents in humans, they are
disadvantageous in
some respects. Specifically, because marine antibodies are of foreign species
origin, they
may be immunogenic in humans. This may result in a neutralizing antibody
response -- a
human anti-marine antibody (HAMA) response -- which is particularly
problematic if the
antibodies are desired to be administered repeatedly, e.g., for treatment of a
chronic or
recurrent disease condition. This is a significant drawback, as some cancer
treatments are
effected over a prolonged time period, e.g., over several years or longer.
Also, because
these antibody molecules contain marine constant domains they may not exhibit
human
effector functions.
In an effort to eliminate or reduce such problems, chimeric antibodies have
been
disclosed. Chimeric antibodies contain portions of two different antibodies,
typically of two
different species. Generally, such antibodies contain human constant regions
attached to
variable regions from another species, typically marine variable regions. For
example, some
mouse/human chimeric antibodies have been reported which exhibit binding
characteristics w
of the parental mouse antibody and effector functions associated with the
human constant
region. See, e.g., U.S. Patent 4,816,567 to Cabilly et al.; U.S. Patent
4,978,745 to
Shoemaker et al.; U.S. Patent 4,975,369 to Beavers et al.; and U.S. Patent
4,816,397 to
Boss et al. Generally, these chimeric antibodies are constructed by preparing
a genomic
2 0 gene library from DNA extracted from pre-existing marine hybridomas.
Nishimura et al.,
Cancer Res., 47:999 (1987). The library is then screened for variable region
genes from
both heavy and light chains exhibiting the correct antibody fragment
rearrangement patterns.
Alternatively, cDNA libraries are prepared from RNA extracted from the
hybridomas and
then screened, or the variable regions are obtained by polymerase chain
reaction. The
2 5 cloned variable region genes are then ligated into an expression vector
containing cloned
cassettes of the appropriate heavy or light chain human constant region gene.
The chimeric
genes are then expressed in a cell line of choice, usually a marine myeloma
line. Such
chimeric antibodies have been used in human therapy.
Moreover, the production of a chimeric mouse anti-human antibody derived from
3 0 COL-1, which specifically binds CEA, has been reported. See e.g., U.S.
Patent 5,472,693 to
Gourlie et al. {owned by The Dow Chemical Company).
Also, Morrison ef al. report the preparation of several anti-tumor chimeric
monoclonal
antibodies, in Important Advances in Oncology Recombinant Chimeric Monoclonal
Anti-
b ies, pp. 3-18 (S.A. Rosenberg, ed., 1990) (J.B. Lippincott, Philadelphia,
PA). Results of
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clinical trials with chimeric cMAb-17-1 A in patients with metastatic
colorectal carcinoma now
show that this antibody has a 6-fold longer circulation time and significantly
reduced
immunogenicity as compared to the murine monoclonal antibody from which it was
derived.
LoBuglio et al., Proc. Nafl. Acad. Sci. USA, $f:4220-4224 (1989); Meredith ei'
al., J. Nucl.
Med., ?:1162-1168 (1991 ).
However, while such chimerized monoclonal antibodies typically exhibit lesser
immunogenicity, they are still potentially immunogenic in humans because they
contain
murine variable sequences which may elicit antibody responses. Thus, there is
the
possibility that these chimeric antibodies may elicit an anti-idiotypic
response if administered
to patients. Saleh et al., Cancer Immunol. Immunother., X2_:185-190 (1990).
Because of the immunogenicity of chimeric antibodies, methods have been
developed recently for the production of "humanized" antibodies. Ideally,
"humanization°
results in an antibody that is less immunogenic, with complete retention of
the antigen-
binding properties of the original molecule. In order to retain all the
antigen-binding
properties of the original antibody, the structure of its combining-site has
to be faithfully
reproduced in the "humanized" version. This can potentially be achieved by
transplanting
the combining site of the non-human antibody onto a human framework, either:
(a) by
grafting only the non-human complementarity determining regions (CDRsj onto
human
framework regions (FRs) and constant regions, with or without retention of
critical framework
2 0 residues (see, Jones et al., Nature, 3~i,:522 (1986) and Verhoeyen et al.,
Science, ?,~,,~9:1539
(1988); or (b) by transplanting the entire non-human variable domains (to
preserve figand-
binding properties) and also "cloaking" them with a human-like surface through
judicious
replacement of exposed residues (in order to reduce antigenicity) (see,
Padlan, Molec.
ImmunoL, X8_:489 (1991 )).
2 5 Essentially, humanization by CDR-grafting involves transplanting only the
CDRs onto
human framework and constant regions. Theoretically, this should substantially
eliminate
immunogenicity (except if allotypic or idiotypic differences exist). Jones et
al., Nature,
x,:522-525 (1986); Verhoeyen et al., Science, X9:1534-1536 (1988), Riechmann
et al.,
Nature, ?:323-327 {1988). However, CDR-grafting by itself may not yield the
desired
3 0 result. Rather, it has been reported that some framework residues of the
original antibody
may also need to be preserved in order to preserve antigen binding activity.
Riechmann et
al., Nature, ' 2:323-327 (1988); Queen et al., Proc. Natl. Acad. Sci. USA,
$x:10023-10029;
Tempest et aL, Biol. Technology, 9_:266-271 (1991 ); Co et al., Nature, ~5
:501-502 (1991 ).
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As discussed above, in order to presence the antigen-binding properties of the
original antibody, the structure of its combining site must be faithfully
reproduced in the
humanized molecule. X-ray crystallographic studies have shown that the
antibody
combining site is built primarily from CDR residues, although some neighboring
framework
residues have been found to be involved in antigen binding. Amit et al.,
Science, ?~:747-
753 (1986); Colman et al., Nature, x:358-363 (1987); Sheriff et al., Proc.
Natl. Acad. Sci.
USA, X4:8075-8079 (1987); Padlan et al., Proc. Natl. Acad. Sci. USA, 8:5938-
5942 (1989);
Fischmann et al., J. Biol. Chem., x:12915-12920 (1991 ); Tulip et al., J.
Molec. Biol.,
x:122-148 (1992). It has also been found that the structures of the CDR loops
are
significantly influenced by surrounding framework structures. Chothia et al.,
J. Molec. Biota,
1ø:901-917 (1987); Chothia et al., Nature, ~4?:877-883 (1989); Tramomonteno et
al., J.
Molec. BioL, ?5:175-182 (1990).
In addition to the effect of the framework residues on the CDRs, small but
significant
differences from the relative disposition of the variable light chain {V~) and
variable heavy
(VH) domains have been noted (Colman et al., Nature, x:358-363 (1987)) and
those
differences are ostensibly due to variations in the residues involved in the
inter-domain
contact (Padlan et aL, Molec. Immunol., x:169-217 (1994)).
Furthermore, structural studies on the effect of the mutation of interior
residues, in
which changes in side chain volume are involved, have shown that the resulting
local
2 0 deformations are accommodated by shifts in side chain positions that are
propagated to
distant parts of the molecular interior. This suggests that during
humanization the interior
residues in the variable domains and in the interface between these domains,
or at least the
interior volumes, should also be maintained; a humanization protocol in which
an interior
residue is replaced by one of different properties, such as size, charge, or
hydrophobicity,
_ could result in a significant modification of the antigen combining-site
structure. One method
of potentially identifying the framework residues which need to be preserved
is by computer
modeling. Alternatively, critical framework residues may potentially be
identified by
comparing known antibody combining site structures. Padlan, Molec. lmmun.,
~1_(3):169-
217 (1994).
3 0 The residues which potentially affect antigen binding fall into several
groups. The
first group comprises residues that are contiguous with the combining site
surface and which
could therefore make direct contact with antigens. These residues include the
amino-
terminal residues and those adjacent to the CDRs. The second group includes
residues that
could alter the structure or relative alignment of the CDRs by contacting
either the CDRs or
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the opposite chains. The third group comprises amino acids with buried side
chains that
could influence the structural integrity of the variable domains. The residues
in these groups
are usually found in the same positions (io'.) according to the adopted
numbering system.
See Kabat et al., Sequences of Proteins of Immunological Interest, NIH Pub.
No. 91-3242
(5th ed., 1991 ) (U.S. Dept. Health & Human Services, Bethesda, MD) and
Genbank.
Given these effects of changes in amino acid residues, although humanized
antibodies are desirable because of their potential low immunogenicity in
humans, their
production is unpredictable. For example, sequence modification of antibodies
may result in
substantial or even total loss of antigen binding affinity, or loss of binding
specificity.
Alternatively, "humanized antibodies" may still exhibit immunogenicity in
humans,
irrespective of sequence modification.
Thus, there still exists a significant need in the art for novel humanized
antibodies to
desired antigens. More specifically, there exists a need in the art for
humanized antibodies
specific to CEA, because of their potential as improved immunotherapeutic and
immunodiagnostic agents for treatment and diagnosis of cancers expressing CEA,
e.g.,
gastrointestinal and colorectal cancers, breast cancers, lung cancers, and
ovarian cancers,
among others.
OBJECTS OF THE INVENTION
2 0 Toward this end, it is an object of the invention to provide humanized
antibodies
which are specific to human carcinoembryonic antigen (CEA}, i.e. anti-CEA
("aCEA")
antibodies. More specifically, it is an object of the invention to provide
humanized antibodies
derived from murine aCEA antibodies and in particular from COL-1, a specific
murine
antibody of the IgG2a isotype having high affinity for CEA.
It is also an object of the invention to provide pharmaceutical compositions
containing
humanized aCEA antibodies. It is a more specific object of the invention to
provide
pharmaceutical compositions containing humanized antibodies derived from the
high affinity
murine aCEA antibody, COL-1.
It is another specific object of the invention to provide methods of using
humanized
3 0 aCEA antibodies for treatment of cancers which express CEA, in particular
breast, lung,
ovarian, gastrointestinal, and colorectal cancers, among others.
It is another object of the invention to provide immunodiagnostic compositions
for
detecting cancer cells, the compositions containing a humanized aCEA antibody,
preferably
derived from COL-1, which antibody is in labeled or unlabeled form. It is
another object of
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the invention to provide a method of immunodiagnosis of cancer using
compositions which
contain a humanized aCEA antibody, preferably derived from COL-1, which is in
labeled or
unlabeled form.
It is still another object of the invention to provide nucleic acid sequences
which
encode humanized aCEA antibodies or fragments thereof. It is a more specific
object of the
invention to provide nucleic acid sequences which encode humanized antibodies
derived
from the high affinity murine aCEA antibody, COL-1. It is another object of
the invention to
provide vectors which provide for the expression of humanized aCEA antibodies,
in
particular humanized antibodies derived from the high affinity murine aCEA
antibody, COL-1.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 contains an alignment of the amino acid sequences of murine COL-1 V"
(COL1 MuVH), a NEWM FR template, and a humanized NEWM-based VH (COL1 NMVH or
"HuVH"). The CDRs are boxed. Murine FR residues retained in the various
humanized VHs
exemplified herein are indicated by the symbols ( T l , ( A ) , ( T ) , ( s )
, ( ~r ) , ( A. ) ,
and ( ~r ) , according to the table below .
Murine Residues
COL1NMVH Retained at
Version Position
* ?~~~ ?~ $2
?$
HuVH T .
HuVHA T A
HuVHAT T A
HuVHAA T A A
HuVHAY T A ~C
HuVHATAY T A T A 'X
HuVHASTAY T A S T A X
HuVHT T T
HuVHS T s
HuVHSTAY T S 'e' A 'Sr
(* -- Retained murine residues indicated by the symbol (, T ) are F-27, N-28,
I-29, K-30, N
97, and T-98.) HuVHSTAY is the version of COL1 NMVH expressed from the
deposited cell
3 5 line, ATCC CRL-12208.
Figure 2 contains an alignment of the amino acid sequences of murine COL-1 VK
(COL1 MuVK or "HuVK"), an REI FR template, and a humanized REI-based VK
(COL1 REVK). The CDRs are boxed. The murine FR residues retained in the
humanized
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sequence are indicated by the symbols: ( F ) for HuVKF, and ( v ) and ( L )
for HuVKVL.
HuVKVL is the version of COL1 REVK expressed from the deposited cell line,
ATCC CRL-
12208.
Figure 3 shows the IgG1 expression vectors used to express the subject
humanized
antibodies in NSO myeioma cells.
Figure 4 shows binding of different COL-1 antibodies to CEA, as measured by an
ELISA assay.
Figure 5 contains results of ELISA assays with COL-1 antibodies including
those
produced according to the invention.
Figure 6 contains results of ELISA assays with COL-1 antibodies including
those
produced according to the invention.
Figure 7 contains results of ELISA assays with COL-1 antibodies including
those
produced according to the invention.
2 0 Figure 8 contains results of ELISA assays with COL-1 antibodies including
those
produced according to the invention.
Figure 9 contains results of ELISA assays with COL-1 antibodies including
those
produced according to the invention.
Figure 10 contains results of ELISA assays with COL-1 antibodies including
those
produced according to the invention.
Figure 11 contains results of ELISA assays with COL-1 antibodies including
those
3 0 produced according to the invention.
Figure 12 contains results of EL1SA assays with COL-1 antibodies including
those
produced according to the invention.
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Figure 13 contains the amino acid sequences of the humanized V" expressed~by
the
deposited cell line ATCC CRL-12208.
Figure 14 contains the amino acid sequences of the humanized VK expressed by
the
deposited cell line ATCC CRL-12208.
Figure 15 presents the nucleotide sequence of the DNA template use to produce
the
initial humanized COL-1 heavy chain variable region, HuVH.
Figure 16 presents the nucleotide sequence of the DNA template used to produce
a
variety of HuVH derivatives.
Figure 17 presents the nucleotide sequence of the DNA template used to produce
the initial humanized COL-1 light chain variable region, HuVK.
Figure 18 presents the nucleotide sequence of the DNA template used to produce
a
the HuVKVL derivative of HuVK.
DETAILED DESCRIPTION OF THE INVENTION
2 0 Prior to setting forth the invention, definitions of certain terms which
are used in this
disclosure are set forth below.
Anti - This refers to single chain, two-chain, and multi-chain proteins and
giycoproteins belonging to the classes of polyclonal, monoclonal, chimeric,
and hetero
immunoglobulins (monoclonal antibodies being preferred); it also includes
synthetic and
genetically engineered variants of these immunoglobulins. "Antibody fragment"
includes
Fab, Fab', F(ab')2 , and Fv fragments, as well as any portion of an antibody
having specificity
toward a desired target epitope or epitopes.
Humanized antibodx. - This will refer to an antibody derived from a non-human
3 0 antibody, typically murine, that retains or substantially retains the
antigen-binding properties
of the parent antibody but which is less immunogenic in humans. This may be
achieved by
various methods including (a) grafting only the non-human CDRs onto human
framework
and constant regions with or without retention of critical framework residues,
or (b)
transplanting the entire non-human variable domains, but "cloaking" them with
a human-like
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section by replacement of surface residues. Such methods as are useful in
practicing the
present invention include those disclosed in Jones et al., Morrison et al.,
Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984); Morrison and Oi, Adv. lmmunol., 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).
Complementaritv Determinipg Region or CDR - The term CDR, as used herein,
refers to amino acid sequences which together define the binding affinity and
specificity of
the natural Fv region of a native immunoglobulin binding site as delineated by
Kabat et of
(1991 ).
Framework Region - The term FR, as used herein, refers to amino acid sequences
interposed between CDRs. These portions of the antibody serve to hold the CDRs
in an
appropriate orientation for antigen binding.
Constant Region - The portion of the antibody molecule which confers effector
functions. In the present invention, marine constant regions are substituted
with human
constant regions. The constant regions of the subject chimeric or humanized
antibodies are
derived from human immunoglobulins. The heavy chain constant region can be
selected
from any of the five isotypes: alpha, delta, epsilon, gamma or mu. Further,
heavy chains of
various subclasses (such as the IgG subclasses of heavy chains) are
responsible for
different effector functions and thus, by choosing the desired heavy chain
constant region
2 0 chimeric antibodies with desired effector function can be produced.
Preferred constant
regions are gamma 1 (IgG1), gamma 3 (IgG3) and gamma 4 (IgG4). More preferred
is a
constant region of the gamma 1 (IgG1) isotype. The light chain constant region
can be of
the kappa or lambda type, preferably of the kappa type.
Chimeric antibody - This is an antibody containing sequences derived from two
2 5 different antibodies, which typically are of different species. Most
typically chimeric
antibodies comprise human and marine antibody fragments, generally human
constant and
marine variable regions.
Mammals - Animals that nourish their young with milk secreted by mammary
glands,
preferably warm blooded mammals, more preferably humans.
3 0 ImmunQ,genicity - A measure of the ability of a targeting protein or
therapeutic moiety
to elicit an immune response (humoral or cellular) when administered to a
recipient. The
present invention is concerned with the immunogenicity of the subject
humanized antibodies
or fragments thereof.
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Humanized reduced immunogenicitv - This refers to a humanized antibody
exhibiting
reduced immunogenicity relative to the parent antibody, typically a murine
antibody such as
COL-1.
Humanized antibody substantially retainina the binding_~roperties of the ap
rent
an 'b - This refers to a humanized antibody which retains the ability to
specifically bind
the antigen recognized by the parent antibody used to produce such humanized
antibody.
Preferably the humanized antibody will exhibit the same or substantially the
same antigen-
binding affinity and avidity as the parent antibody, e.g., COL-1. Ideally, the
affinity of the
antibody will not be less than 5% of the parent antibody affinity, more
preferably not less
than about 30%, and most preferably the affinity will not be less than 50% of
the parent
antibody. Methods for assaying antigen-binding affinity are well known in the
art and include
half-maximal binding assays, competition assays, and Scatchard analysis.
Suitable antigen
binding assays are described in this application.
In its broadest embodiment, the present invention is directed to humanized
antibodies which specifically bind CEA, an antigen expressed by various human
cancers, in
particular gastrointestinal, colorectal, breast, lung, and ovarian cancers.
Preferably, such
humanized antibodies will be derived from antibodies having good binding
affinity to CEA,
such as COL-1 through COL-15 disclosed by Muraro et al., Cancer Res., 45(11
Pt. 2):5769-
5780 (1985).
Most preferably, such humanized antibodies will be derived from COL-1, a
murine
antibody of the IgG2a isotype, which has been reported to bind to CEA with
high affinity
(1.4x109 M'') with no detectable cross-reactivity for CEA-related antigens,
such as the non-
specific cross-reacting antigen (NCA) and the normal fecal antigen (NFA).
As discussed above, humanized antibodies afford potential advantages over
murine
2 5 and also chimeric antibodies, e.g., reduced immunogenicity in humans. This
is advanta-
geous because it should reduce and potentially eliminate the eliciting of a
HAMA response
when such humanized antibodies are administered in vivo, e.g., either for
treatment of
cancer or for diagnosis of cancer as by tumor imaging. Also, such antibodies
may exhibit
improved plasma clearance, pharmacokinetic, and tumor targeting properties.
3 0 However, as noted above, humanization may in some instances adversely
affect
antigen binding. Preferably, the humanized aCEA antibodies of the present
invention will
possess a binding affinity for CEA of not less than about 5% and more
preferably not less
than about 30%, and most preferably not less than 50% of the CEA binding
antigen affinity
of the parent murine antibody, preferably COL-1. Most preferably, the
humanized antibodies
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of the present invention will possess a binding affinity for CEA of not less
than about 5% and
more preferably not less than about 30% and most preferably not less than
about 50% of the
CEA binding affinity of COL-1, or a chimeric antibody derived therefrom.
Preferably, the humanized antibodies of the present invention will bind the
same
epitope as COL-1. Such antibodies can be identified based on their ability to
compete with
COL-1 for binding to CEA or to CEA-expressing cells.
In general, the subject humanized antibodies are produced by obtaining nucleic
acid
sequences encoding the variable heavy (V") and variable light chains (V~,
e.g., VK) of an
antibody which binds CEA (preferably COL-1 }, identifying the CDRs in said V"
and V
sequences, and grafting such CDR-encoding nucleic acid sequences onto selected
human
framework-encoding nucleic acid sequences.
Preferably, the human framework amino acid sequences are selected such that
the
resulting antibody is likely to be suitable for in vivo administration in
humans. This can be
determined, e.g., based on previous usage of antibodies containing such human
FRs.
Preferably, the human FRs will not themselves be significantly immunogenic.
Examples of
such human frameworks include NEWM and REI.
Alternatively, the amino acid sequences of the FRs of the antibody to be
humanized
(e.g., COL-1) will be compared to those of known human FRs, and the human FRs
to be
used for CDR-grafting will be selected based on their comprising sequences
highly similar to
2 0 those of the parent antibody, e.g., a murine antibody which binds CEA.
Numerous human
FRs have been isolated and their sequences reported in the literature. This
enhances the
likelihood that the resultant CDR-grafted "humanized" antibody, which contains
the CDRs of
the parent (e.g., murine) antibody grafted onto the selected human FRs, will
substantially
retain the antigen binding structure and thus retain the binding affinity of
the parent antibody.
As a result of such studies, the FRs of REI and NEWM antibodies have been
identified as
having amino acid sequences which are likely to allow the CDRs of COL-1 To
retain a
significant degree of antigen binding affinity. As noted, the selected human
framework
regions will preferably be those that are expected to be suitable for in vivo
administration,
i.e., not immunogenic. Based on their amino acid sequences, REI and NEWM human
3 0 framework regions are expected to be substantially non-immunogenic.
In either method, the DNA sequences encoding the VH and V~ regions of the
preferably murine aCEA antibody must be obtained. Methods for cloning nucleic
acid
sequences encoding immunoglobulins are well known in the art. Such methods
will
generally involve the amplification of the immunoglobulin-encoding sequences
to be cloned
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using appropriate primers by polymerase chain reaction (PCR). Primers suitable
for
amplifying immunoglobulin nucleic acid sequences, and specifically murine
variable heavy
and variable light sequences, have been reported in the literature. After such
immunoglobulin-encoding sequences have been cloned, they will be sequenced by
methods
well known in the art. This will be effected in order to identify the V,; and
V~-encoding
sequences, and more specifically the portions thereof which encode the CDRs
and FRs.
This can be effected by well known methods which include, e.g., those
disclosed in U.S.
Patent No. 4,816,397 to Boss et aL and U.S. Patent No. 5, 225,539 to Winter et
al..
Once the DNA sequences encoding the CDRs and FRs of the antibody which is to
be
humanized have been identified, the amino acid sequences encoding the CDRs are
then
identified (deduced based on the nucleic acid sequences and the genetic code
and by
comparison to previous antibody sequences) and the CDR-encoding nucleic acid
sequences
are grafted onto selected human FR-encoding sequences. This may be
accomplished by
use of appropriate primers and linkers. Methods for selecting suitable primers
and linkers to
provide for ligation of desired nucleic acid sequences is well within the
purview of the
ordinary artisan.
As discussed above, the selected human FRs used for humanization will
preferably
be those that are likely to be suitable for in vivo administration, i.e. they
are not in
themselves immunogenic in humans (e.g., because of allotypic differences);
examples
thereof are REI and NEWM human FRs. Alternatively, the human FRs will be
selected such
that they comprise amino acid sequences which are highly similar to those of
the parent
antibody's FR sequences. This may be effected by comparing the amino acid
sequences of
the murine FRs to those of previously reported human FRs (see, e.g., Kabat et
al., id.).
After the CDR-encoding sequences are grafted onto the selected human FR-
2 5 encoding sequences, the resultant DNA sequences encoding the "humanized"
variable
heavy and variable light sequences will then be expressed to produce a
humanized Fv or
humanized antibody which binds CEA. Typically, the humanized V" and V~
sequences will
be expressed as part of a whole aCEA antibody molecule, i.e. as a fusion
protein with
human constant domain sequences whose encoding DNA sequences have been
obtained
3 0 from a commercially available library or which have been obtained using,
e.g., one of the
above-described methods for obtaining DNA sequences. However, the VH and V
sequences can also be expressed in the absence of constant sequences to
produce a
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humanized aCEA Fv. Nevertheless, fusion of human constant sequences is
potentially
desirable because the resultant humanized aCEA antibody may possess human
effector
functions such as CDC and ADCC activity.
Methods for synthesizing DNA encoding a protein of known sequence are well
known
in the art. Using such methods, DNA sequences which encode the subject
humanized V
and V" sequences (with or without constant regions) are synthesized, and then
expressed in
a vector system suitable for expression of recombinant antibodies. This may be
effected in
any vector system which provides for the subject humanized V~ and V" sequences
to be
expressed as a fusion protein with human constant domain sequences and to
associate to
produce functional (antigen binding) antibodies or antibody fragments. Useful
methods are
set forth, e.g., in U.S. Patent 4,816,397 to Boss et al. and U.S. Patent
5,225,539 to Winter et
al.
Expression vectors and host cells suitable for expression of recombinant
antibodies
and humanized antibodies in particular, are well known in the art. The
following references
are representative of methods and vectors suitable for expression of
recombinant immuno-
globulins which may be utilized in carrying out the present invention: Weidle
et al., Gene,
~: 21-29 (1987}; Dorai et aL, J. lmmunoL, x(12}:4232-4241 (1987); De Waele et
al., Eur.
J. Biochem., x:287-295 (1988); Colcher et al., Cancer Res., g~:1738-1745
(1989}; Wood
et al., J. Immunol., 145(a):3011-3016 (1990); Bulens et al., Eur. J. Biochem.,
~9 :235-242
2 0 (1991 ); Beggington et al., Biol. Technology, 10:169 (1992); King et al.,
Biochem. J., ~:317-
323 (1992); Page et al., BioL Technology, 9:64 (1991 ); King et al., Biochem.
J., 290:723-729
(1993); Chaudary et al., Nature, ~Q:394-397 (1989); Jones et al., Nature,
X1:522-525
(1986); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Benhar et al., Proc.
Natl. Acad.
Sci. USA, X1_:12051-12055 (1994); Singer etal., J. ImmunoL, 1_~Q:2844-2857
(1993); Cooto
2 5 et al., Hybridoma, ,~,~(3):215-219 (1994); Queen et aL, Proc. Natl. Acad
Sci. USA, $~:10029-
10033 (1989); Caron et al., Cancer Res., ~:676i-6767 (1992); Cotoma et aL, J.
Immunol.
Meth., 1~?:89-109 (1992). Moreover, vectors suitable for expression of
recombinant
antibodies are commercially available. The vector may, e.g., be a bare nucleic
acid
segment, a carrier-associated nucleic acid segment, a nucleoprotein, a
plasmid, a virus, a
3 0 viroid, or a transposable element.
Host cells known to be capable of expressing functional immunoglobulins
include,
e.g.: mammalian cells such as Chinese Hamster Ovary (CHO) cells; COS cells;
myeloma
cells, such as NSO and SP2/O cells; bacteria such as Escherichia coli; yeast
cells such as
Saccharomyces cerevisiae; and other host cells. Of these, CHO cells are used
by many
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researchers given their ability to effectively express and secrete
immunoglobulins. NSO
cells are one of the preferred types of host cells useful in the present
invention.
Essentially, recombinant expression of humanized antibodies is obtained by one
of
two general methods. In the first method, the host cells are transfected with
a single vector
which provides for the expression of both VH and V~ variable sequences
optionally fused to
selected constant regions. In the second method, host cells are transfected
with two
vectors, each of which provides for expression of either the VH or V~
sequence, each
optionally fused to a selected constant region.
Human constant domain sequences are well known in the art, and have been
reported in the literature. Preferred human constant light chain sequences
(C~) include the
kappa and lambda constant light sequences. Preferred human constant heavy
chain
sequences include human gamma 1, human gamma 2, human gamma 3, human gamma 4,
and mutated versions thereof which provide for altered effect or function,
e.g., enhanced in
vivo half-life, reduced Fc receptor binding, and the like.
After expression, the antigen binding affinity of the resultant humanized
antibody will
be assayed by known methods, e.g., Scatchard analysis. Ideally, the antigen-
binding affinity
of the humanized antibody will approximate that of the parent antibody, e.g.,
COL-1. As
discussed above, ideally the affinity of the humanized antibody will not be
less than 5% of
the parent antibody, more preferably not less than 30%, and most preferably
not less than
2 0 50% of that of the parent antibody, e.g., COL-1.
In some instances, humanized antibodies produced by grafting CDRs (from an
antibody which binds CEA) onto selected human FRs may provide humanized
antibodies
having the desired affinity to CEA. However, it may be necessary or desirable
to further
modify specific residues of the selected human FR in order to enhance antigen
binding. This
2 5 may occur because it is believed that some framework residues are
essential to or at least
affect antigen binding. Preferably, those framework residues of the parent
(e.g., murine)
antibody which maintain or affect combining-site structures will be retained.
These residues
may be identified by X-ray crystallography of the parent antibody or Fab
fragment, thereby
identifying the three-dimensional structure of the antigen-binding site.
3 0 These residues may potentially be identified by X-ray crystallography of
the parent
Fab, thereby identifying the three-dimensional structure of the antigen-
binding site. Also,
framework residues which may be involved in antigen binding may be putatively
selected
based on previously reported humanized murine antibody sequences. Thus, it may
be
beneficial to retain these and other murine framework residues from the parent
murine
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antibody to optimize CEA binding. However, because of inherent
unpredictability associated
with amino acid modification of proteins, and antibodies in particular, the
effects of such
changes, if any, on antigen binding are unpredictable. Nevertheless, such
methodology will
ideally confer a "human-like" character to the resultant humanized antibody
thus rendering it
less immunogenic while retaining the interior and contacting residues which
affect antigen-
binding.
The present invention further embraces variants and equivalents which are
substantially homologous to the humanized antibodies and antibody fragments
set forth
herein. These may contain, e.g., conservative substitution mutations, i.e. the
substitution of
one or more amino acids by similar amino acids. For example, conservative
substitution
refers to the substitution of an amino acid with another within the same
general class, e.g.,
one acidic amino acid with another acidic amino acid, one basic amino acid
with another
basic amino acid, or one neutral amino acid by another neutral amino acid.
What is intended
by a conservative amino acid substitution is well known in the art.
The phrase "substantially homologous" is used in regard to the similarity of a
subject
amino acid sequence (of an oligo- or poly-peptide or protein) to a related,
reference amino
acid sequence. This phrase is defined as at least about 75% "correspondence" --
i.e. the
state of identical amino acid residues being situated in parallel -- between
the subject and
reference sequences when those sequences are in "alignment," i.e. when a
minimal number
2 0 of "null" bases have been inserted in the subject and/or reference
sequences so as to
maximize the number of existing bases in correspondence between the sequences.
"Null"
bases are not part of the subject and reference sequences; also, the minimal
number of
"null" bases inserted in the subject sequence may differ from the minimal
number inserted in
the reference sequence. In this definition, a reference sequence is considered
"related" to a
2 5 subject sequence where both amino acid sequences make up proteins or
portions of
proteins which are either aCEA antibodies or antibody fragments with aCEA
binding affinity.
Each of the proteins comprising these aCEA antibodies or antibody fragments
may
independently be antibodies or antibody fragments or bi- or multi-functional
proteins, e.g.,
such as fusion proteins, bi- and multi-specific antibodies, single chain
antibodies, and the
3 0 like.
The present invention is further directed to nucleic acid sequences from which
such
humanized antibodies and antibody fragments may be expressed, as well as
expression
vectors from which these humanized antibodies and antibody fragments may be
expressed
in transfected host cells.
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In a preferred embodiment, such humanized antibodies and corresponding nucleic
acid sequences will be derived from COL-1. Most preferably, the humanized V"
sequence
and the humanized V~ sequence will have the sequences substantially as
depicted in Figure
1 or 13 or in Figure 2 or 14, respectively, and as discussed in the Examples
set forth below.
However, as discussed, the invention further contemplates other modifications
of these
humanized VH and V~ sequences, e.g., sequences which further comprise one or
more con-
servative amino acid substitutions or which retain one or more additional
murine framework
residues which affect or do not significantly reduce antigen binding.
The subject humanized antibodies -- because they specifically bind CEA {an
antigen
expressed on many different cancer cell types, e.g., lung carcinomas, breast
carcinomas,
gastrointestinal carcinomas such as stomach cancers, colorectal carcinomas
such as colon
cancers, ovarian carcinomas, etc.) and further because they will not be
significantly
immunogenic in humans -- should be suitable for use as: therapeutics for the
treatment or
prevention of cancers characterized by CEA expression; diagnostic agents,
e.g., for
diagnosis and evaluating the prognosis of cancers characterized by CEA
expression (based
on levels of CEA expression); tumor imaging agents; or radiolabeled antibodies
in the
Radioimmunoguided Surgery~ System (RIGS~). See Hinkle et al., Antibody,
Immunoconjugates and Radiopharmaceuticals, 4_(3):339-358 {1991 ).
One skilled in the art would be able (by routine experimentation) to determine
what
2 0 amount of antibody would be effective and non-toxic for the purpose of
treating cancer.
Generally, however, an effective dosage will be in the range of about 0.05 to
100 milligrams
per kilogram body weight per day.
The humanized antibodies or humanized antibody fragments of the invention may
be
administered to a human or other animal in accordance with the aforementioned
methods of
treatment in an amount sufficient to produce a therapeutic or prophylactic
effect. The
antibodies of the subject invention can be administered to such human or other
animal in a
conventional dosage form prepared by combining the antibody of the invention
with a
conventional, pharmaceutically acceptable carrier, diluent, and/or excipient
according to
known techniques. It will be recognized by one of ordinary skill in the art
that the form and
3 0 character of the pharmaceutically acceptable carrier, diluent, and/or
excipient is dictated by
the amount of active ingredient with which it is to be combined, the route of
administration,
and other well-known variables.
Pharmaceutically acceptable formulations may include, e.g., a suitable
solvent,
preservatives such as benzyl alcohol if desired, and a buffer. Useful solvent
may include,
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e.g., water, aqueous alcohols, glycols, and phosphonate and carbonate esters.
Such
aqueous solutions contain no more than 50% by volume of organic solvent.
Suspension-
type formulations may include a liquid suspending medium as a carrier, e.g.,
aqueous
polyvinylpyrrolidone, inert oils such as vegetable oils or highly refined
mineral oils, or
aqueous cellulose ethers such as aqueous carboxymethylcellulose. A thickener
such as
gelatin or an alginate may also be present, one or more natural or synthetic
surfactants or
antifoam agents may be used, and one or more suspending agents such as
sorbitoi or
another sugar may be employed therein. Such formations may contain one or more
adjuvants.
The route of administration of the antibodies (or fragment thereof) of the
present
invention may be oral, parenteral, by inhalation, or topical. The term
parenteral as used
herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or
intraperitoneal
administration. The subcutaneous, intravenous, and intramuscular forms of
parenteral
administration are generally preferred.
The daily parenteral and oral dosage regimens for prophyiactically or
therapeutically
employing humanized antibodies of the present invention will generally be in
the range of
about 0.005 to 100, but preferably about 0.5 to 10, milligrams per kilogram
body weight per
day.
The antibodies of the present invention may also be administered by
inhalation. By
2 0 "inhalation" is meant intranasal and oral inhalation administration.
Appropriate dosage forms
for such administration, such as an aerosol formulation or a metered dose
inhaler, may be
prepared by conventional techniques. The preferred dosage amount of a compound
of the
invention to be employed is generally within the range of about 0.1 to about
100, more
preferably about 10 to 100, milligrams per kg body weight.
2 5 The antibody of the invention may also be administered topically. By
topical
administration is meant non-systemic administration. This includes the
administration of a
humanized antibody (or humanized antibody fragment) formulation of the
invention externally
to the epidermis or to the buccal cavity, and instillation of such an antibody
into the ear, eye,
or nose, and wherever it does not significantly enter the bloodstream. By
systemic
3 0 administration is meant oral, intravenous, intraperitoneal, subcutaneous,
and intramuscular
administration. The amount of an antibody required for therapeutic,
prophylactic, or
diagnostic effect will, of course, vary with the antibody chosen, the nature
and severity of the
condition being treated and the animal undergoing treatment, and is ultimately
at the
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discretion of the physician. A suitable topical dose of an antibody of the
invention will
generally be within the range of about 1 to 100 milligrams per kilogram body
weight daily.
Formulations
While it is possible for an antibody or fragment thereof to be administered
alone, it is
preferable to present it as a pharmaceutical formulation. The active
ingredient may
comprise, for topical administration, from 0.001 % to 10% w/w, e.g., from 1 %
to 2% by weight
of the formulation, although it may comprise as much as 10% w/w but preferably
not in
excess of 5% w/w and more preferably from 0.1 % to 1 % w/w of the formulation.
The topical formulations of the present invention, comprise an active
ingredient
together with one or more acceptable carriers) therefor and optionally any
other therapeutic
ingredients(s). The carriers) must be "acceptable" in the sense of being
compatible with the
other ingredients of the formulation and not deleterious to the recipient
thereof.
Formulations suitable for topical administration include liquid or semi-liquid
preparations suitable for penetration through the skin to the site of where
treatment is
required, such as liniments, lotions, creams, ointments or pastes, and drops
suitable for
administration to the eye, ear, or nose.
Drops according to the present invention may comprise sterile aqueous or oily
solutions or suspensions and may be prepared by dissolving the active
ingredient in a
2 0 suitable aqueous solution of a bactericidal and/or fungicidal agent and/or
any other suitable
preservative, and preferably including a surface active agent. The resulting
solution may
then be clarified and sterilized by filtration and transferred to the
container by an aseptic
technique. Examples of bactericidal and fungicidai agents suitable for
inclusion in the drops
are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%)
and
2 5 chlorhexidine acetate (0.01 %). Suitable solvents for the preparation of
an oily solution
include glycerol, diluted alcohol and propylene glycol.
Lotions according to the present invention include those suitable for
application to the
skin or eye. An eye lotion may comprise a sterile aqueous solution optionally
containing a
bactericide and may be prepared by methods similar to those for the
preparation of drops.
3 0 Lotions or liniments for application to the skin may also include an agent
to hasten drying
and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such
as glycerol or
an oil such as castor oil or arachis oil.
Creams, ointments or pastes according to the present invention are semi-solid
formulations of the active ingredient for external application. They may be
made by mixing
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the active ingredient in finely-divided or powdered form, alone or in solution
or suspension in
an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a
greasy or non
greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid
paraffin,
glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such
as almond, corn,
arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid
such as stearic or oleic
acid together with an alcohol such as propylene glycol or macrogels. The
formulation may
incorporate any suitable surface active agent such as an anionic, cationic or
non-ionic
surface active such as sorbitan esters or polyoxyethylene derivatives thereof.
Suspending
agents such as natural gums, cellulose derivatives or inorganic materials such
as silicaceous
silicas, and other ingredients such as lanolin, may also be included.
Kits according to the present invention include frozen or lyophilized
humanized
antibodies or humanized antibody fragments to be reconstituted, respectively,
by thawing
(optionally followed by further dilution) or by suspension in a (preferably
buffered) liquid
vehicle. The kits may also include buffer and/or excipient solutions (in
liquid or frozen form) -
- or buffer and/or excipient powder preparations to be reconstituted with
water - for the
purpose of mixing with the humanized antibodies or humanized antibody
fragments to
produce a formulation suitable for administration. Thus, preferably the kits
containing the
humanized antibodies or humanized antibody fragments are frozen, lyophilized,
pre-diluted,
or pre-mixed at such a concentration that the addition of a predetermined
amount of heat, of
2 0 water, or of a solution provided in the kit will result in a formulation
of sufficient concentration
and pH as to be effective for in vivo or in vitro use in the treatment or
diagnosis of cancer.
Preferably, such a kit will also comprise instructions for reconstituting and
using the
humanized antibody or humanized antibody fragment composition to treat or
detect cancer.
The kit may also comprise two or more component parts for the reconstituted
active
2 5 composition. For example, a second component part - in addition to the
humanized
antibodies or humanized antibody fragments - may be bifunctional chelant,
bifunctional
chelate, or a therapeutic agent such as a radionuclide, which when mixed with
the
humanized antibodies or humanized antibody fragments forms a conjugated system
therewith. The above-noted buffers, excipients, and other component parts can
be sold
3 0 separately or together with the kit.
It will be recognized by one of skill in the art that the optimal quantity and
spacing of
individual dosages of a humanized antibody or humanized antibody fragment of
the invention
will be determined by the nature and extent of the condition being treated,
the form, route
and site of administration, and the particular animal being treated, and that
such optima can
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be determined by conventional techniques. It will also be appreciated by one
of skill in the
art that the optimal course of treatment, i.e., the number of doses of an
antibody or fragment
thereof of the invention given per day for a defined number of days, can be
ascertained by
those skilled in the art using conventional course of treatment determination
tests.
The subject humanized antibodies may also be administered in combination with
other anti-cancer agents, e.g., other antibodies or drugs. Also, the subject
humanized
antibodies or fragments may be directly or indirectly attached to effector
moieties having
therapeutic activity. Suitable effector moieties include by way of example
cytokines (IL-2,
TNF, interferons, colony stimulating factors, IL-1, etc.), cytotoxins
(Pseudomonas exotoxin,
ricin, abrin, etc.), radionuclides, such as ~°Y, '3'I, ~"'Tc, "'In,
'251, among others, drugs
(methotrexate, daunorubicin, doxorubicin, etc.), immunomodulators, therapeutic
enzymes
(e.g., beta-galactosidase), anti-proliferative agents, etc. The attachment of
antibodies to
desired effectors is well known. See, e.g., U.S. Patent No. 5,435,990 to Cheng
et al.
Moreover, bifunctional linkers for facilitating such attachment are well known
and widely
available. Also, chelators (chelants and chelates) providing for attachment of
radionuclides
are well known and available.
Alternatively, the subject humanized aCEA antibodies or fragments may be used
as
immunodiagnostic agents both in vivo and in vitro. A particularly preferred
usage is for in
vivo imaging of cancer cell lesions which express CEA. The subject antibodies
are preferred
2 0 because they should elicit no significant HAMA or allergic response. Thus,
they may be
used repeatedly to monitor the disease status of a patient.
As noted above, another preferred application of the subject humanized
antibodies or
fragments thereof is in the Radioimmunoguided System~ (RIGS~). This technique
involves the intravenous administration of a radiolabeled monoclonal antibody
or its fragment
2 5 prior to surgery. After allowing for tumor uptake and blood clearance of
radioactivity, the
patient is taken to the operating room where surgical exploration is effected
with the aid of a
hand-held gamma activity probe, e.g., the Neoprobe~ 1000. This helps the
surgeon
identify the tumor metastases and lessen the complications of excision.
The RIGS~ system is advantageous because it allows for the detection of tumors
not
3 0 otherwise detectable by visual inspection and/or palpation. See, O'Dwyer
et al., Arch. Surg.,
x:1391-1394 (1986). This technique is described in detail in Hinkle et al.,
Antibody,
Immunoconjugates and Radiopharmaceuticais, 4:(3)339-358 (1991 ). This
reference
discloses the use of this technique with aCEA antibodies including the COL-1
monoclonal
antibody. This technique is particularly useful for cancers of the colon,
breast, pancreas,
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and ovaries. Thus, this technique should be applicable to the subject
humanized antibodies
which react with CEA expressed by colon, breast, and ovarian cancers. Also,
Hinkle et al.
(id.) cite numerous references describing this technique. The subject
humanized antibodies
or fragments thereof may be radiolabeled with radionuclides which are suitable
for in vivo
administration, e.g., iodine radionuclides such as '3' I and 'zsl. Moreover,
"' In and ~"'Tc are
also suitable.
The subject humanized antibodies may be used alone or in combination with
other
antibodies. Also, the subject humanized antibodies may be prepared in the form
of a
diagnostically effective composition. Generally, this will entail the
incorporation of
diagnostically acceptable carriers and excipients, and labels which provide
for detection.
Suitable labels include diagnostic radionuclides, enzymes, etc. Methods for
using antibodies
for tumor imaging are well known in the art.
Without further elaboration, it is believed that one skilled in the art can,
using the
preceding description, utilize the present invention to its fullest extent.
The invention will be
further clarified by a consideration of the following examples, which are
intended to be purely
exemplary of the present invention and thus to be construed as merely
illustrative examples
and not limitations of the scope of the present invention an any way.
EXAMPLES
2 0 Materi ~,s and Methods
DNA Tem lad to Pre aration
All recombination work was performed upon DNA sequences in plasmid M13
vectors.
The source of the NEWM framework regions for producing the initial humanized
COL-1 VH
2 5 was an M13 construct bearing -- between the M13 BamH I and Hind III sites -
- a DNA
segment having the nucleotide sequence shown in figure 13. The source of REI
framework
regions for producing the initial humanized COL-1 VL was an M13 construct
bearing --
between the M13 BamH I and Hind III sites -- a DNA segment encoding the REf
amino acid
sequence of Figure 2.
3 0 When overlap-extension procedures were used to introduce mutations into a
given
DNA sequence, double stranded M13 DNA was utilized. In contrast, when
extension-iigation
procedures were used instead, the oiigonucleotides were designed to anneal to
only one of
the two DNA strands. In this latter procedure, the M13 DNA was first treated
to substitute
uridine for every thymidine base in the DNA, to produce uridinylated DNA. This
was
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accomplished by transfecting the M13 plasmid DNA into competent cells lacking
dUTPase
and uracil glycosylase, normally RZ1032 cells (though CJ236 cells available
from Bio-Rad of
Hercules, CA, are also suitable) by combining the following ingredients.
1 p.L of M13 plasmid DNA
4 mL of LB broth
40 wL of competent RZ1032 cells.
The culture was shaken for 5 hours at 37°C and the resulting single-
stranded plasmid
DNA (ssDNA) was isolated and dissolved in 50 ~.L Tris-EDTA buffer. The DNA was
then
treated with uracil glycosylase by mixing together:
1 p,L uridinylated ssDNA
1 pL 10x glycosylase buffer
1 U uracil glycosylase (Gibco BRL, Gathersburg, MD)
40 p.L 25mM MgCIZ.
20
This mixture was then incubated at 37°C.for one hour and then 6.6 p.L
25mM MgClz and
9.9p,L 1 M NaOH. The mixture was then further incubated for 5 minutes at
37°C and 16.5 ~L
of 0.6M HCI was then added to neutralize the mixture. The DNA was then ethanol
precipitated and dissolved in water.
13 Oligonucleotide Primers
The following oligonucleotide primers were used throughout the process of
preparing
the humanized COL-1 VHs and VLs exemplified below.
10. 5'-CTAAAACGACGGCCAGT-3' and
2 5 11. 5'-AACAGCTATGACCATG-3' (both for using in producing VHs); and
385. 5'-GCGGGCCTCTTCGCTATTACGC-3' and
391. 5'-CTCTCTCAGGGCCAGGCGGTGA-3' (both for use in producing VKs).
These primers are complementary to regions of the plasmid M13 which are
external both to
the (NEWM or REI) target framework sequences and to the BamH I site -to- Hind
111 site
3 0 section of M13.
Murine Variable Regions
1n order to compare the antibody binding characteristics of the antibodies
produced
according to the examples set forth below, antibodies having a chimeric heavy
35 chain (i.e. a heavy chain having a murine COL-1 VH region and a human IgG1
constant
region) and/or a chimeric light chain (i.e. a light chain having a murine COL-
1 VL and a
human x constant region) were expressed. The source of these chimeric chains
was the
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ATCC-deposited cell line CRL 11217 (Budapest) which expresses a chimeric COL-1
antibody having both chains. The heavy chain of this antibody was termed
"MuVH" and the
light chain thereof was termed "MuVL."
Oligonucleotide Phospho~yrlation Protocol
Mutating oligonucleotides used in non-overlap extensions were phosphorylated
according to the procedure below. In a final volume of 25p.L, the following
ingredients were
combined:
pmol of each oligonucleotide,
10 5p.L of a 5x polynucleotide kinase buffer, and
5U of T4 polynucleotide kinase (Gibco BRL).
The phosphorylation reaction was started with the addition of the enzyme and
allowed to
proceed for one hour at 37°C.
Annealing Protocol for Non-Overlap Extension-Legations
The annealing step for non-overlap extension-legations involved performing one
annealing in which all mutation-carrying oligonucleotides and one primer
oligonucleotide
were annealed to a single stranded DNA template in which all thymidine bases
had been
2 0 replaced with uridine bases. The mutating oligonucleotides were first
phosphorylated
according to the above oligonucleotide phosphorylation protocol. In a final
volume of 20 pL,
the following ingredients were combined:
1 pmol of each mutation-carrying phosphorylated oligonucleotide
1 pmol of a primer oligonucleotide
4 p.L 5x annealing buffer
0.2 pmol ssU-DNA template
The mixture was then heated to 90°C for 30 sec., then quickly cooled to
70°C, and finally
allowed to slowly cool to 37°C.
3 0 Extension-Legation Protocol for Non-Overlap Extension-Legations
After completion of the annealing step in which the primer and phosphorylated
mutating oligonucleotides were annealed to the ssU-DNA template, extension-
legation was
performed as follows. In a final volume of 30 p.L, the following ingredients
were combined:
20 p.L annealed ssU-DNA (i.e. the contents of the above annealing procedure)
3 5 2 p.L 5x annealing buffer
2 p.L 0.1 M dithiothreitol
0.3 p.L 0.1 M ATP
1 pL 6.25mM dNTP mixture of equimolar amounts of dATP, dTTP, dGTP, dCTP
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2.5U T7 DNA polymerase (USB, now Amersham Life Sciences, Cleveland, OH)
0.5U T4 DNA ligase (Gibco BRL)
Water to 30~CL
This mixture was then incubated at room temperature for 1 hour.
Standard PCR Protocols
The following procedure was used, alternately, both to amplify the non-overlap
extension-ligation DNA sequences and to perform extension of each overlap DNA
sequence.
In a final volume of 50p.L, the following ingredients were combined:
2 p.L template DNA (either annealed ssU-DNA or non-annealed ssDNA)
5 pL 10x Vent buffer (NEB, 1. e. New England Biolabs, Beverly, MA) or
10x Thermalase buffer (IBI of New Haven, CT)
2 p,L 6.25mM dNTP mixture of equimolar amounts of dATP, dTTP, dGTP, dCTP
25 pmol of one oligonucleotide primer
pmol of either a mutation-carrying oligonucleotide (for overlap-extension) or
a second oiigonucleotide primer
1 U Vent DNA polymerase (NEB) or Thermalase DNA polymerase (IBI)
2 0 Reactions were initiated with the addition of the DNA polymerase and then
treated with
about 15 cycles of: (1 ) 94°C for 30 sec., (2) 50°C for 30 sec.,
and (3) 30-60 seconds at
either 75°C (for Vent DNA polymerase) or 72°C (for Thermalase).
Reactions were brought
to completion with 5 minutes at a constant temperature of either 75°C
(for Vent DNA
polymerase) or 72°C (for Thermalase).
PCR Overla -p Extension Amplification Protocol
After a pair of PCR reactions were performed - one for each of the two
(partially
complementary) overlapping DNA segments, the two resulting segments were
joined
according to the following PCR procedure. In a final volume of 50 wL, the
following
3 0 ingredients were mixed:
1 p.L of each overlap DNA (from the above overlap PCR extension reactions)
5 p.L 10x Vent buffer (NEB) or Thermalase buffer (IBI)
2 p.L 6.25mM dNTP mixture of equimolar amounts of dATP, dTTP, dGTP, dCTP
25 pmof of each oligonucleotide primer used in the overlap PCR extensions
1U Vent DNA polymerase (NEB) or Thermaiase DNA poiymerase (IBI)
Reactions were initiated with the addition of the DNA polymerase and then
treated with
about 15 cycles of: (1 ) 94°C for 30 sec., (2) 50°C for 30 sec.,
and (3) 30-60 seconds at
either 75°C (for Vent DNA polymerase) or 72°C (for Thermalase).
Reactions were brought
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to completion with 5 minutes at a constant temperature of either 75°C
(for Vent DNA
polymerase) or 72°C (for Thermalase).
i V i A 1 V
and Subseo~nt Antibod~,~x~~ r~ssion
Humanized antibodies were expressed in pSV vectors grown in NSO cells as
follows.
The humanized variable region constructs which were produced in the plasmid,
M13, were
obtained by ethanol precipitating the M13 plasmids (as cytosolic DNA),
redissolving them in
aqueous solution, and digesting them with 10U each of Hind III and BamH I
(both from BRL,
i.e. Gibco BRL) for 1 hour at 37°C in a final volume of 100 wL with
Tris-EDTA buffer. The
resulting DNA fragments were then run on a low melting point agarose get, the
band
containing the humanized construct DNA was cut out, and the DNA was purified
using an
ELUTIP 'd' column with 20 p.L Tris-EDTA buffer. 10 ~.L of the purified DNA
preparation was
then combined with 1 p,L of a Hind III and BamH I-digested pSV preparation, 3
p.L of 5x
ligase buffer, and 1 U of T4 DNA ligase (BRL), in order to insert the
construct into a pSV
plasmid. Humanized COL-1 VH constructs were inserted into pSVgpt vectors
bearing a
human IgG1 heavy chain constant region; the pSVgpt vector used is the "aLYS-
30" shown in
Figure 5. Humanized COL-1 VL constructs were inserted into pSVhyg vectors
bearing a
2 0 human K light chain constant domain; the pSVhyg vector used in the "aLys-
17" shown in
Figure 5. Each humanized variable region construct was inserted adjacent to
the respective
constant region, i.e. so as to replace either the HuVHLYS or the HuVLLys
segment
illustrated in Figure 5.
The resulting vectors were transfected into NSO cells as follows. About 3 pg
of the
VH vector, or about 6p.g of the VL vector, produced by the pSV-insertion
procedures, was
then linearized by digestion with 10U Pvul (Gibco BRL). The digested DNA was
then
precipitated with ethanol and redissofved in 50 p.L of water. NSO cells were
collected by
centrifugation and resuspended in 0.5mL Dulbecco's Modified Eagle's Medium
(DMEM) and
then transferred to a Gene Pulser cuvette (Bio-Rad). The DNA from both one VH
and one
3 0 VL construct was gently mixed with the cells by pipetting and the cuvette
was left on ice for 5
minutes. Next, the cuvette was inserted between the electrodes of the Bio-Rad
Gene Pulser
and a single pulse of 170V at 960 wF was applied. The contents of the cuvette
were then
transferred to a flask containing 20mL DMEM and the cells were allowed to rest
for 1-2 days
at 37°C. Cells were again harvested by centrifugation and resuspended
in 36mL selective
3 5 DMEM. 1.SmL aliquots of this resuspension were placed in each well of a 24-
well plate and
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incubated at 37°C for 4 days, at which time the medium in each well was
replaced with
l.SmL of fresh selective DMEM. After 6 more days of incubation at 37°C,
surviving cell
colonies were visible to the naked eye and the supernatants of each well were
assayed for
antibody production. Both whole antibody production (i.e. without
purification) and purified
antibody production were assayed. To obtain purified antibodies, the
supernatants were
passed through a protein A column.
ELISA Assay Protocols
Antibody concentrations and antibody binding characteristics were tested using
enzyme-linked immunosorbent assay (EL1SA) procedures which are set forth as
follows
Measurement of IgG concentration
The concenBration of IgG secreted from transfected cells was measured using an
enzyme-linked immunosorbent assay (ELISA) procedure which is set forth as
follows.
Polyvinyl chloride (PVC) microtiter plates (Dynatech Laboratories, Chantilly,
VA,
catalog # 001-010-2101 ) were coated with goat anti-human IgG (lOmg/mL, GAHIG,
Southern Biotechnology Associates, Inc., Birmingham, AL, catalog # 2010-01 )
diluted with
Milli-C~~ water and placed on the plates using 50mUwell. Plates were air-dried
overnight at
ambient temperature or at 37°C for 3 hours. Prior to use, non-specific
binding was blocked
2 0 the addition of 0.2 mUwell of 1 % (w/v) bovine serum albumin (Sigma, St.
Louis, MO catalog
# A7888) in phosphate buffered saline (Sigma, catalog # 1000-3) (PBS/BSA). All
incubations were carried out in a humidified container. Pfates were incubated
for 1-2 hours
at 37°C and the blocking solution removed prior to sample addition. Two-
fold serial dilutions
of samples or a standard IgG solution set at 500 ng/mL (50 p.l/well) were made
in triplicate in
2 5 the PBS/BSA solution. The plate was incubated at 37°C for 3 hours
or overnight at 4°C.
The plate was washed 3-times with 0.025% Tween-20 (v/v, Sigma) using an
automatic plate
washer. 50 pl/well of 1:1000 dilution of a goat anti-human IgG conjugated to
Horseradish
Peroxidase (Southern Biotechnology Associates Inc.) was added and incubated at
37°C for
1.5 hours. The wells were washed 3 times with 0.025% Tween-20 (v/v, Sigma)
using an
3 0 automatic plate washer and 50 p.l/well OPD substrate buffer added. The
color was
developed for 4 minutes, stopped with 12.5 p,l 12.5% HZSO, and the absorbance
at 492 nm
read. The concentration of IgG in the test sample was estimated by comparison
of the mean
of the optical densities to a standard curve constructed from the standard
IgG.
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Determinat~gn of relative affinities of humanized antibodies
Antibody binding characteristics were tested in an ELISA using partially
purified CEA
antigen immobilized on Polyvinyl chloride (PVC) microtiter plates (Dynatech
Laboratories,
Chantilly, VA, catalog # 001-010-2101 )
PVC plates were coated with 50 p,l/well CEA (Dow Chemical, lot #040191 ),
diluted
1:300 in Milli-Q water. Plates were air-dried overnight at ambient temperature
or at 37°C for
3 hours. Prior to use, non-specific binding was blocked by the addition of 0.2
mUwell of 1
(w/v) bovine serum albumin (Sigma, St. Louis, MO catalog # A7888) in phosphate
buffered
saline (Sigma, catalog # 1000-3) (PBS/BSA). Plates were incubated for 1-2
hours at 37°C
and the blocking solution removed prior to sample addition. All incubations
were carried out
in a humidified container. Two-fold serial dilutions (starting concentration
range of l.Op.g/ml -
10~g/ml) of the samples to be tested in the PBS/BSA solution were added to
triplicate wells
of the TAG-coated plate (50 p,llwell). The plate was incubated overnight at
4°C or 1-2 hours
at 37°C. The plate was washed 3-times with 0.025% Tween-20 (v/v, Sigma)
using an
automatic plate washer. 50 pl/well of 1:1000 dilution of a goat anti-human IgG
conjugated to
Horseradish Peroxidase (Southern Biotechnology Associates Inc.) was added and
incubated
at 37°C for 1.5 hours. The wells were washed 3 times with 0.025% Tween-
20 (v/v, Sigma)
using an automatic plate washer and 50 ~I/well OPD substrate buffer added. The
color was
developed for 4 minutes, stopped with 12.5 p.l 12.5% H2S0, and the absorbance
at 492 nm
2 0 read.
Determination of Affinity Constants for binding to CEA
Two-fold dilutions of purified Hu-COL-1 were prepared in PBS/BSA over a range
of
1.Opg/ml - 0.003 ~.g/ml and samples (20 ~.I/well) were applied in triplicate
to TAG coated
PVC prepared and blocked as described supra. Plates were incubated overnight
at 4°C.
Following this incubation, samples were transferred from the plate to the
corresponding wells
on the GAHIG-coated trap plate. The original TAG plate was washed 3-times with
0.025%
Tween-20 (v/v, Sigma, catalog # P1379) using an automatic plate washer. An
'251-labeled
goat anti-human IgG probe (ICN Biomedicals, Inc., catalog # 68088) was diluted
to 75,000
cpm/25~.1 in PBS1BSA and added (25~1/well) to all wells. This TAG plate was
incubated for 1
3 0 hour at 37°C.
After a 1 hour incubation at 37°C, the trap plate was washed as
described above and
'251-labeled GAHIG probe was added. This plate was incubated for 1 hour at
37°C. Both
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plates (TAG and GAHIG-trap) containing probe were then washed with a
microplate washer
to remove the unbound probe. A plate cutter (D. Lee, Sunnyvale, CA, Model HWC-
4) was
used to separate the wells from the plate frame. The radioactivity in each
well was
quantified by a gamma counter. The resulting data was analyzed according to
the method
of Scatchard (Ann. NYAcad, 51:600-672 (1946)).
Synthesis of Initial CDR-Grafted (Humanized)
Antibody from Murine COL-1
We describe in this Example the construction of humanized COL-1 monoclonal
antibodies ("Mabs") -- COL-1 HuVH/HuVK -- using the V~ and V" frameworks of
human Mabs
REI and NEWM, respectively. The CDRs for murine COL-1 were grafted onto human
frame-
works according to known methods as discussed supra. In particular, human
frameworks
were selected from antibodies which, based on previous studies, were predicted
to be
suitable, i.e. which should not adversely affect antigen binding and not
exhibit significant
immunogenicity in humans. The human frameworks selected for the variable heavy
and
variable light chains, respectively, were NEWM and REI.
In the production of the initial version of the humanized VH, certain murine
framework
residues were also retained which, based on previous studies, might allow
retention of
2 0 antigen binding properties. Specifically, residues F27, N28, t29, K30,
N97, and T98 of the
murine heavy chain were initially retained.
The production of this NEWM-grafted humanized COL-1 VH was accomplished using
two rounds of a dual PCR procedure: the standard PCR protocol (for overlap-
extension)
followed by PCR amplification with oligonucleotide primers 10 and 11. This
procedure was
2 5 carried out as described above using a single-stranded M13 DNA template
bearing, between
the Hind III and BamH I sites thereof, a DNA segment having the nucleotide
sequence
shown in Figure 13. The mutating oligonucleotides used in the first round
(with Vent DNA
polymerase)were designed and synthesized with the following sequences:
3 0 836. 5'-TGAGAATGGTGATACTGAATATGCCCCGAAGT; and
837. 5'-TCGGGGCATATTCAGTATCACCATTCTCAGGATC-3'.
Those for the second round (with Thermalase) were:
3 5 838. 5'-ACTATGATTACGACGCGTTGGTTCTTCGATGTCTGGGGCCAAGGGTCCTT
GGTCACCGTC-3'; and
839. 5'-ACGCGTCGTAATCATAGTAGATAGACCCCGTGTATTACAGTAATAGACCGCG
GTG-3'.
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The resulting humanized VH was named COL1 NMVH or "HuVH."
In the production of the initial version of the humanized VL, no uniquely
murine
framework residues were retained. The production of the REI-grafted humanized
COL-1 V
was accomplished by using two rounds of a procedure involving performing the
annealing
and extension-ligation protocols described above - followed by amplification
by the standard
Thermalase PCR protocol -- using a ssMl3 template bearing, between the Hind
III and
BamH I sites thereof, a ssU-DNA segment produced by the above procedure using
the
REIVK sequence shown in Figure 17 (or in the second round, the mutated Mi 3
template
resulting from the first round). The mutating oligonucleotides used in round
number 1 were
designed and synthesized with the following sequences:
842. 5'-TATAGCCAGATGCACTGACACTTTTGCTGGCCCTACAGGTGATG-3';
844. 5'-GCTCTGGGTCATCTGGATGTCGG-3';
849.5'-TTCTACTCACGTGTGATTTGCAGCTTGGTCCCTTGGCCGAACGTAGGAAG
CTCCCTACTGTGCTGGCAGTAG-3';
850. 5'-ATGGTGAAGGTGTAGTCGGTACCGC-3'; and
851. 5'-GCCTTACCTGGCGTCTGCTGGTACC-3'.
2 0 The mutating oligonucleotide used in the second round was:
841. 5'-GCACACCAGATTGTAGGTTGGATGCAAGG-3'.
The resulting humanized VL was called "COL1 REVK" or "HuVK."
Concurrently, a second humanized COL-1 VL was made by the same procedure using
the
following mutating oligonucleotides.
For round 1:
842. 5'-TATAGCCAGATGCACTGACACTTTTGCTGGCCCTACAGGTGATG-3';
844. 5'-GCTCTGGGTCATCTGGATGTCGG-3';
849. 5'-TTCTACTCACGTGTGATTTGCAGCTTGGTCCCTTGGCCGAACGTAGGAAG
CTCCCTACTGTGCTGGCAGTAG-3'; and
3 5 851. 5'-GCCTTACCTGGCGTCTGCTGGTACC-3'.
For round 2:
841. 5'-GCACACCAGATTGTAGGTTGGATGCAAGG-3'.
The resulting VL was termed "HuVKF."
A number of amino acid substituted-versions of HuVH and HuVK were also
constructed,
using the above-described overlap extension technique employing
oligonucleotide primers
10 and 11 or primers 385 and 391, followed by PCR amplification with Vent DNA
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polymerase. The mutating oligonucleotides and DNA templates are described
below under
their resulting humanized variable region name:
HuVHT (using the HuVH DNA shown in Figure 16 as a template)
954. 5'-GACAATGCTGACAGACACCAGCAA-3' and
955. 5'-TGCTGGTGTCTGTCAGCATTGTCA-3';
HuVHS (using the HuVH DNA as a template)
684. 5'-CACCAGCAGCAACCAGTTCAG-3' and
IO 683. 5'-ACTGGTTGCTCGTGGTGTCTA-3',
HuVHSTAY (using the HuVHS DNA as a template)
1026. 5'-ACCAGCAGCAACACAGCCTACCTGAGACTCAGCAG-3' and
1028. 5'-TGCTGAGTCTCAGGTAGGCTGTGTTGCTGCTGGTGT-3';
HuVHA (using the HuVH DNA as a template)
745. 5'-TGACCTGCACCGCGTCTGGCTTCAAC-3' and
746. 5'-TTGAAGCCAGACGCGGTGCAGGTCAG-3';
2 0 HuVHAA (using the HuVHA DNA as a template)
1071. 5'-GAGACTCAGCAGCGTGACAG-3' and
1072. 5'-CGCTGCTGAGTCTCAGGCTGAATGTGTTCTTGCTGGTGTC-3';
HuVHAT (using the HuVHA DNA as a template)
2 5 1071. 5'-CCTGAGACTCAGCAGCGTGACAG-3' and
1074. 5'-CGCTGCTGAGTCTCAGGCTGGCCTGGTTCTTGCTGGTG-3';
HuVHAY (using the HuVHA DNA as a template)
1071. 5'-CCTGAGACTCAGCAGCGTGACAG-3' and
3 0 1073. 5'-CGCTGCTGAGTCTCAGGTAGAACTGGTTCTTGC-3';
HuVHATAY (using the HuVHA DNA as a template)
1071. 5'-CCTGAGACTCAGCAGCGTGACAG-3' and
1075. 5'-CGCTGCTGAGTCTCAGGTAGGCTGTGTTCTTGCTGGTGTC-3';
HuVHASTAY (using the HuVHS DNA as a template)
745. 5'-TGACCTGCACCGCGTCTGGCTTCAAC-3' and
746. 5'-TTGAAGCCAGACGCGGTGCAGGTCAG-3'; and
HuVKVL (using the HuVK DNA shown in Figure 18 as a template)
1010. 5'-ACTCCGACATCGTGCTGACCCAGAG-3' and
1011. 5'-CTCTGGGTCAGCACGATGTCGGAG-3'.
The above-produced M13 DNA constructs were transferred to pSV vectors which
were then
transfected into PJSO host cells, according to the above-described procedures.
The VH
construct- and VL construct-containing pSV vectors were transfected into NSO
cells in the
following combinations (to produce antibodies having the indicated
combinations of VH and
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VL regions):
MuVH/MuVK;
HuVH/HuVK;
HuVH/MuVK;
MuVH/HuVK;
HuVHA/HuVK;
HuVHT/HuVK;
HuVHT/HuVKF;
HuVH/HuVKVL;
HuVHS/HuVKVL;
HuVHSTAY/HuVK;
HuVH/HuVKF;
MuVH/HuVKF; and
HuVHSTAY/HuVKVL.
Combinations of the other VHs with the various VKs are expected to behave in
an analogous
manner, though having an unpredictable variation in degree of binding. Final
selection of the
optimum combination will be a function of obtaining an antibody having the
best, selective
antigen binding properties with the fewest murine amino acid substitutions.
The amino acid sequences of the initial humanized (CDR-grafted) COL-1 variable
2 0 heavy (V") and variable light (VK) regions, HuVH and HuVK, are show in
Figures 1 and 2,
respectively. NSO transfectants were screened for whole antibody expression
and the
antigen binding characteristics of the antibodies produced thereby were
measured by an
ELISA test against CEA. Results are presented in Figures 4-9. This data shows
that - of
the graft-humanized and the mix-and-match clones - MuVH/HuVK outperformed both
HuVH/MuVK and HuVH/HuVK, but did not perform as well as the chimeric MuVH/MuVK
antibodies.
These results indicate that the initial fully humanized antibody (HuVH/HuVK)
exhibits
a 20-fold loss in CEA antigen binding affinity. Thus, it exhibits about 5% the
binding affinity
of murine COL-1. Based on analysis of CEA binding of mix-and-match antibodies
3 0 (HuVH/HuVK) and (MuVH/HuVK), it was determined that the reduction in CEA
antigen
binding was apparently largely attributable to the amino acid sequence of
humanized heavy
chain. However, an approximate 2-fold reduction in antigen binding also
occurred
apparently because of the amino acid sequence of the humanized kappa chain.
This can be
appreciated from the ELISA data in Figure 4.
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As a result of this research, an antibody comprising the VHSTAY- and VKVL-
containing heavy and light chains was deposited on October 16, 1996 with the
American
Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 and
accorded
Accession Number ATCC CRL-12208 (this is a murine plasmacytoma cell line).
This
deposit was made in accordance with the Budapest Treaty. This deposited cell
line wiH be
made irrevocably available, without restriction, upon issuance of a patent to
this application,
or any patent claiming benefit of priority to this application under 35 U.S.C.
~120.
Based on the foregoing, it will be appreciated that the humanized antibodies
disclosed herein exhibit antigen-binding characteristics, i.e., CEA affinities
comparable to the
parent monoclonal antibody, nCOL-1 (murine antibody), and to chimeric
antibodies derived
from nCOL-1, e.g., ChCOL-1y1 (ATCC No. CRL 11217). Moreover, based on the
foregoing
results, these antibodies possess properties which wilt render them well
suited for usage as
in vivo diagnostics or therapeutics, e.g., improved serum clearance, metabolic
properties,
and little or no immunogenicity in humans.
These properties are highly significant because they will enable the subject
humanized antibodies to be administered repeatedly, in large dosages, and over
a prolonged
period of time without significant adverse effects, e.g., a HAMA response or
non-specific
cytotoxicity. This is important for cancer treatment as well as for cancer
diagnosis as it
enables these antibodies to be used over prolonged time periods. Moreover, the
clearance
2 0 properties of the subject human antibodies will enable these antibodies to
effectively target
desired target sites, e.g., CEA expressing carcinomas (because of the effects
of serum
clearance on targeting efficiency). Therefore, the humanized antibodies of the
present
invention comprise a substantial improvement in relation to previously
disclosed antibodies
specific to CEA.
2 5 Other embodiments of the invention will be apparent to those skilled in
the art from a
consideration of this specification or practice of the invention disclosed
herein. It is intended
that the specification and examples be considered as exemplary only, with the
true scope
and spirit of the invention being indicated by the following claims.
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SEQUENCE LISTING
GENERAL INFORMATION:
APPLICANT:
NAME: The Dow Chemical Company
STREET: 1790 Bldg. Washington Street
CITY: Midland
STATE: MI
COUNTRY: U.S.A.
POSTAL CODE: 48674
TELEPHONE: 517-636-1687
TELEFAX: 517-638-9786
TITLE OF INVENTION: High Affinity Humanized Anti-CEA
Monoclonal Antibodies
NUMBER OF SEQUENCES: 53
COMPUTER READABLE FORM:
MEDIUM TYPE: 3-1/2" diskette
COMPUTER: IBM PC Compatible
OPERATING SYSTEM: MS-DOS
SOFTWARE: PatentIn
INFORMATION FOR SEQ ID N0:1
SEQUENCE CHARACTERISTICS:
LENGTH: 124
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Murine COL-1 VH
LOCATION: 1..124
SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala
10 15
Ser Val Lys Met Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Met Thr Thr Asp Tyr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Thr Arg Gly Leu Ser Thr Met Ile Thr Thr Arg Trp Phe Phe Asp
100 105 110
Val Trp Gly Ala Gly Thr Thr Val Ala Val Ser Ser
115 120
-34
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
INFORMATION FOR SEQ ID N0:2
SEQUENCE CHARACTERISTICS:
LENGTH: 124
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: NEWM VH FR template
LOCATION: 1..124
SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Xaa Xaa Xaa Xaa Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val.Thr Ala Ala Asp Thr Ala Val Tyr Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
100 105 110
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Thr Val Ser Ser
115 120
INFORMATION FOR SEQ ID N0:3:
SEQUENCE CHARACTERISTICS:
LENGTH: 124
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VH, HuVH
LOCATION: 1..124
SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
-35-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Thr Arg Gly Leu Ser Thr Met Ile Thr Thr Arg Trp Phe Phe Asp
100 105 110
Val Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser
115 120
INFORMATION FOR SEQ ID N0:4:
SEQUENCE CHARACTERISTICS:
. LENGTH: 124
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VH, HuVHA
LOCATION: 1..124
SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
10 25
Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Thr Arg Gly Leu Ser Thr Met Ile Thr Thr Arg Trp Phe Phe Asp
100 105 110
Val Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser
115 120
INFORMATION FOR SEQ ID N0:5:
SEQUENCE CHARACTERISTICS:
LENGTH: 124
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
-36-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
FEATURE:
NAME/KEY: Humanized COL-1
VH,
HuVHAT
LOCATION: 1..124
SEQUENCE DESCRIPTION: ID NO:5:
SEQ
GlnVal Gln Leu Glu Ser Pro GlyLeu Val Arg Pro Ser
Gln Gly Gln
5 10 15
ThrLeu Ser Leu Cys Thr Ser GlyPhe Asn Ile Lys Asp
Thr Ala Tyr
20 25 30
TyrMet His Trp Arg Gln Pro GlyArg Gly Leu Glu Trp
Val Pro Ile
35 40 45
GlyTrp Ile Asp Glu Asn Asp ThrGlu Tyr Ala Pro Lys
Pro Gly Phe
50 55 60
GlnGly Arg Val Met Leu Asp ThrSer Lys Asn Thr Phe
Thr Val Ser
65 60 75 80
LeuArg Leu Ser Val Thr Ala AspThr Ala Val Tyr Tyr
Ser Ala Cys
85 90 95
AsnThr Arg Gly Ser Thr Ile ThrThr Arg Trp Phe Phe
Leu Met Asp
100 105 110
ValTrp Gly Gln Ser Leu Thr ValSer Ser
Gly Val
115 120
INFORMATION ID N0:6:
FOR
SEQ
SEQUENCE CHARACTERISTICS:
LENGTH: 124
TYPE: amino
acid
STRANDEDNESS:
single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL -1
VH,
HuVHAA
LOCATION: 1..124
SEQUENCE DESCR IPTION: ID NO:6:
SEQ
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
10 15
Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Ala Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
-37-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
Asn Thr Arg Gly Leu Ser Thr Met Ile Thr Thr Arg Trp Phe Phe Asp
100 105 110
Val Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser
115 120
INFORMATION FOR SEQ ID N0:7:
SEQUENCE CHARACTERISTICS:
LENGTH: 124
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VH, HuVHAY
LOCATION: 1..124
SEQUENCE DESCRIPTION: SEQ ID N0: 7:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
10 15
Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln Phe Tyr
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Thr Arg Gly Leu Ser Thr Met Ile Thr Thr Arg Trp Phe Phe Asp
100 105 110
Val Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser
115 120
INFORMATION FOR SEQ ID N0:8:
SEQUENCE CHARACTERISTICS:
LENGTH: 124
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VH, HuVHATAY
LOCATION: 1..124
SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg P.ro Ser Gln
5 10 15
-38-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Thr Arg Gly Leu Ser Thr Met Ile Thr Thr Arg Trp Phe Phe Asp
100 105 110
Val Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser
115 120
INFORMATION FOR SEQ ID N0:9:
SEQUENCE CHARACTERISTICS:
LENGTH: 124
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VH, HuVHASTAY
LOCATION: 1..124
SEQUENCE DESCRIPTION: SEQ ID N0: 9:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
10 15
Thr Leu Ser Leu Thr Cys Thr A1a Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Thr Arg Gly Leu Ser Thr Met Ile Thr Thr Arg Trp Phe Phe Asp
100 105 110
Val Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser
115 120
-39-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
INFORMATION FOR SEQ ID NO:10
SEQUENCE CHARACTERISTICS:
LENGTH: 124
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VH, HuVHT
LOCATION: 1..124
SEQUENCE DESCRIPTION: SEQ ID NO:10:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Leu Thr Asp Thr Ser Lys Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Thr Arg Gly Leu Ser Thr Met Ile Thr Thr Arg Trp Phe Phe Asp
100 105 110
Val Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser
115 120
INFORMATION FOR SEQ ID N0:11:
SEQUENCE CHARACTERISTICS:
LENGTH: 124
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VH, HuVHS
LOCATION: 1..124
SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
-40-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser Ser Asn Gln Phe Ser
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Thr Arg Gly Leu Ser Thr Met Ile Thr Thr Arg Trp Phe Phe Asp
100 105 110
Val Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser
115 120
INFORMATION FOR SEQ ID N0:12:
SEQUENCE CHARACTERISTICS:
LENGTH: 124
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VH, HuVHSTAY
LOCATION: 1..124
SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Leu Vai Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Thr Arg Gly Leu Ser Thr Met Ile Thr Thr Arg Trp Phe Phe Asp
100 105 110
Val Trp Gly GIn Gly Ser Leu Val Thr Val Ser Ser
115 120
INFORMATION FOR SEQ ID N0:13:
SEQUENCE CHARACTERISTICS:
LENGTH: 110
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
-41-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
FEATURE:
NAME/KEY: Murine COL-1 VK
LOCATION: 1..110
SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Thr Val Ser Leu Gly
10 15
Leu Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Ala Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Arg Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Gln Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly THr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu .Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
INFORMATION FOR SEQ ID N0:14:
SEQUENCE CHARACTERISTICS:
LENGTH: 110
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: REI VK FR template
LOCATION: 1..110
SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Xaa Xaa Xaa
85 90 95
Xaa Xaa Xaa Thr Phe Gly Gln Gly Thr Lys Xaa Xaa Xaa Xaa
100 105 110
-42-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
INFORMATION FOR SEQ ID N0:15:
SEQUENCE CHARACTERISTICS:
LENGTH: 110
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VK, HuVK
LOCATION: 1..110
SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ala Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr
100 105 110
INFORMATION FOR SEQ ID N0:16:
SEQUENCE CHARACTERISTICS:
LENGTH: 110
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VK, HuVKVL
LOCATION: 1..110
SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ala Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
-43-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
Arg Phe Ser Gly Ser Gly Ser ThrAspTyr Thr Phe Thr Ile
Gly Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Ile ThrTyrTyr Cys Gln His Ser
Ala Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gln ThrLysLeu Gln Ile Thr
Gly
100 105 110
INFORMATION
FOR
SEQ
ID
N0:17:
SEQUENCE CHARACTERISTICS:
LENGTH: 110
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1
VK,
HuVKF
LOCATION: 1..110
SEQUENCE DESCRIPTION: SEQ ID NO:17:
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ala Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr
100 105 110
INFORMATION FOR SEQ ID N0:18:
SEQUENCE CHARACTERISTICS:
LENGTH: 125
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VH, ATCC CRL-12208 VH
LOCATION: 1..125
SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln
5 10 15
-44-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Asn Ile Lys Asp Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Gly Phe
50 55 60
Gln Gly Arg Val Thr Met Leu Val Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr A1 Val Tyr Tyr Cys
85 90 95
Asn Thr Arg Gly Leu Ser Thr Met Ile Thr Thr Arg Trp Phe Phe Asp
100 105 110
Val Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser
115 120 125
INFORMATION FOR SEQ ID N0:19:
SEQUENCE CHARACTERISTICS:
LENGTH: 110
TYPE: amino acid
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Humanized COL-1 VK, ATCC CRL-12208 VK
LOCATION: 1..110
SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ala Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr
100 105 110
INFORMATION FOR SEQ ID N0:20:
SEQUENCE CHARACTERISTICS:
LENGTH: 348
TYPE: DNA
STRANDEDNESS: double
-45-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCTNS98/03680
TOPOLOGY: linear
FEATURE:
NAME/KEY: Template used to produce HuVH
LOCATION: 1..348
SEQUENCE DESCRIPTION: SEQ ID NO: 20:
CAG GTC CAA CTG CAG GAG AGC GGT CCA GGT CTT GTG AGA CCT AGC CAG 48
ACC CTG AGC CTG ACC TGC ACC GTG TCT GGC TTC AAC ATT AAA GAC TAC 96
TAT ATG CAC TGG GTG AGA CAG CCA CCT GGA CGA GGT CTT GAG TGG ATT 144
GGA TGG AT'r GAT CCT GAG AAT NNN NNN NNN NNN TAT GCC CCG AAG TTC 192
CAG GGC AGA GTG ACA ATG CTG GTA GAC ACC AGC AAG AAC CAG TTC AGC 240
CTG AGA CTC AGC AGC GTG ACA GCC GCC GAC ACC GCG GTC TAT NNN NNN 288
rINN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN GTC 336
ACC GTC TCC TCA 348
INFORMATION FOR SEQ ID N0:21:
SEQUENCE CHARACTERISTICS:
LENGTH: 372
TYPE: DNA
STRANDEDNESS: double
TOPOLOGY: linear
FEATURE:
NAME/KEY: Template used to produce HuVH variants
LOCATION: 1..372
SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CAG GTC CAA CTG CAG GAG AGC GGT CCA GGT CTT GTG AGA CCT AGC CAG 48
ACC CTG AGC CTG ACC TGC ACC GTG TCT GGC TTC AAC ATT AAA GAC TAC 96
TAT ATG CAC TGG GTG AGA CAG CCA CCT GGA CGA GGT CTT GAG TGG ATT 144
GGA TGG ATT GAT CCT GAG AAT GGT GAT ACT GAA TAT GCC CCG AAG TTC 192
CAG GGC AGA GTG ACA ATG CTG GTA GAC ACC AGC AAG AAC CAG TTC AGC 240
CTG AGA CTC AGC AGC GTG ACA GCC GCC GAC ACC GCG GTC TAT TAC TGT 28$
AAT ACA CGG GGT CTA TCT ACT ATG ATT ACG ACG GCT TGG TTC TTC GAT 336
GTC TGG GGC CAA GGG TCC TTG GTC ACC GTC TCC TCA 372
-46
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
INFORMATION FOR SEQ ID N0:22:
SEQUENCE CHARACTERISTICS:
LENGTH: 318
TYPE: DNA
STRANDEDNESS: double
TOPOLOGY: linear
FEATURE:
NAME/KEY: Template used to produce HuVK
LOCATION: 1..318
SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GAC ATC CAR CTG ACC CAG AGC CCA AGC AGC CTG AGC GCC AGC GTG GGT 48
GAC AGA GTG ACC ATC ACC TGT AGG NNN NNN NNN NNN NNN NNN NNN NNN 96
GGC TAT AGT TAT ATG CAC TTG TAC CAG CAG ACG CCA GGT AAG GCT CCA 144
AAG CTG CTG ATC TAC NNN NNN NNN NNN NNN NNN TCT GGT GTG CCA AGC 192
AGA TTC AGC GGT AGC GGT AGC GGT ACC GAC TTY ACC TTC ACC ATC AGC 240
AGC CTC CAG CCA GAG GAC ATC GCC ACC TAC TAC TGC CAG NNN NNN NNN 288
NNN NNN NNN ACG TTC GGC CAA GGG ACC AAG 318
INFORMATION FOR SEQ ID N0:23:
SEQUENCE CHARACTERISTICS:
LENGTH: 330
TYPE: DNA
STRANDEDNESS: double
TOPOLOGY: linear
FEATURE:
NAME/KEY: Template used to produce HuVK variants
LOCATION: 1..330
SEQUENCE DESCRIPTION: SEQ ID NO: 23:
GAC ATC CAR ATG ACC CAG AGC CCA AGC AGC CTG AGC GCC AGC GTG GGT 48
GAC AGA GTG ACC ATC ACC TGT AGG GCC AGC AAA AGT GTC AGT GCA TGT 96
GGC TAT AGT TAT ATG CAC TGG TAC CAG CAG ACG CCA GGT AAG GCT CCA 144
AAG CTG CTG ATC TAC CTT GCA TCC AAC CTA CAA TCT GGT GTG CCA AGC 192
AGA TTC AGC GGT AGC GGT AGC GGT ACC GAC TAC ACC TTC ACC ATC AGC 240
AGC CTC CAG CCA GAG GAC ATC GCC ACC TAC TAC TGC CAG CAC AGT AGG 288
GAG CTT CCT ACG TTC GGC CAA GGG ACC AAG CTG CAA ATC ACA 330
-47
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 . PCT/US98/03680
INFORMATION FOR SEQ ID N0:24:
SEQUENCE CHARACTERISTICS:
LENGTH: 17
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 10
LOCATION: 1..17
SEQUENCE DESCRIPTION: SEQ ID NO: 24:
CTAAAACGAC GGCCAGT 17
INFORMATION FOR SEQ ID N0:25:
SEQUENCE CHARACTERISTICS:
LENGTH: 16
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 11
LOCATION: 1..16
SEQUENCE DESCRIPTION: SEQ ID NO: 25:
AACAGCTATG ACCATG 16
INFORMATION FOR SEQ ID N0:26:
SEQUENCE CHARACTERISTICS:
LENGTH: 22
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 385
LOCATION: 1..22
SEQUENCE DESCRIPTION: SEQ ID NO: 26:
GCGGGCCTCT TCGCTATTAC GC 22
INFORMATION FOR SEQ ID N0:27:
SEQUENCE CHARACTERISTICS:
LENGTH: 22
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 391
LOCATION: 1..22
-48-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
SEQUENCE DESCRIPTION: SEQ ID NO: 27:
CTCTCTCAGG GCCAGGCGGT GA 22
INFORMATION FOR SEQ ID N0:28:
SEQUENCE CHARACTERISTICS:
LENGTH : 3 2
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 836
LOCATION: 1..32
SEQUENCE DESCRIPTION: SEQ ID NO: 28:
TGAGAATGGT GATACTGAAT ATGCCCCGAA GT 32
INFORMATION FOR SEQ ID N0:29:
SEQUENCE CHARACTERISTICS:
LENGTH: 34
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 837
LOCATION: 1..34
SEQUENCE DESCRIPTION: SEQ ID NO: 29:
TCGGGGCATA TTCAGTATCA CCATTCTCAG GATC 34
INFORMATION FOR SEQ ID N0:30:
SEQUENCE CHARACTERISTICS:
LENGTH: 59
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 838
LOCATION: 1..59
SEQUENCE DESCRIPTION: SEQ ID NO: 30:
ACTATGATTA CGACGCGTTG GTTCTTCGAT GTCTGGGGCC AAGGGTCCTT GGTCACGTC 59
-49-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
INFORMATION FOR SEQ ID N0:31:
SEQUENCE CHARACTERISTICS:
LENGTH: 55
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 839
LOCATION: 1..55
SEQUENCE DESCRIPTION: SEQ ID NO: 31:
ACGCGTCGTA ATCATAGTAG ATAGACCCCG TGTATTACAG TAATAGACCG CGGTG 55
INFORMATION FOR SEQ ID N0:32:
SEQUENCE CHARACTERISTICS:
LENGTH: 44
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 842
LOCATION: 1..44
SEQUENCE DESCRIPTION: SEQ ID NO: 32:
TATAGCCAGA TGCACTGACA CTTTTGCTGG CCCTACAGGT GATG 44
INFORMATION FOR SEQ ID N0:33:
SEQUENCE CHARACTERISTICS:
LENGTH: 23
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 844
LOCATION: 1..23
SEQUENCE DESCRIPTION: SEQ ID NO: 33:
GCTCTGGGTC ATCTGGATGT CGG 23
INFORMATION FOR SEQ ID N0:34:
SEQUENCE CHARACTERISTICS:
LENGTH: 72
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 849
LOCATION: 1..72
-50-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
SEQUENCE DESCRIPTION: SEQ ID N0: 34:
TTCTACTCAC GTGTGATTTG CAGCTTGGTC CCTTGGCCGA ACGTAGGAAG CTCCCTACTG 60
TGCTGGCAGT AG 72
INFORMATION FOR SEQ ID N0:35:
SEQUENCE CHARACTERISTICS:
LENGTH: 25
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 850
LOCATION: 1..25
SEQUENCE DESCRIPTION: SEQ ID NO: 35:
ATGGTGAAGG TGTAGTCGGT ACCGC 25
INFORMATION FOR SEQ ID N0:36:
SEQUENCE CHARACTERISTICS:
LENGTH: 25
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 851
LOCATION: 1..25
SEQUENCE DESCRIPTION: SEQ ID NO: 36:
GCCTTACCTG GCGTCTGCTG GTACC 25
INFORMATION FOR SEQ ID N0:37:
SEQUENCE CHARACTERISTICS:
LENGTH: 29
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 841
LOCATION: 1..29
SEQUENCE DESCRIPTION: SEQ ID NO: 37:
GCACACCAGA TTGTAGGTTG GATGCAAGG 29
-51-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
INFORMATION FOR SEQ ID N0:38:
SEQUENCE CHARACTERISTICS:
LENGTH: 44
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 842
LOCATION: 1..44
SEQUENCE DESCRIPTION: SEQ ID NO: 38:
TATAGCCAGA TGCACTGACA CTTTTGCTGG CCCTACAGGT GATG 44
INFORMATION FOR SEQ ID N0:39:
SEQUENCE CHARACTERISTICS:
LENGTH: 24
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 954
LOCATION: 1..24
SEQUENCE DESCRIPTION: SEQ ID NO: 39:
GACAATGCTG ACAGACACCA GCAA 24
INFORMATION FOR SEQ ID N0:40:
SEQUENCE CHARACTERISTICS:
LENGTH: 24
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 955
LOCATION: 1..24
SEQUENCE DESCRIPTION: SEQ ID NO: 40:
TGCTGGTGTC TGTCAGCATT GTCA 24
INFORMATION FOR SEQ ID N0:41:
SEQUENCE CHARACTERISTICS:
LENGTH: 21
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 684
LOCATION: 1..21
-52-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
SEQUENCE DESCRIPTION: SEQ ID N0: 41:
CACCAGCAGC AACCAGTTCA G 21
INFORMATION FOR SEQ ID N0:42:
SEQUENCE CHARACTERISTICS:
LENGTH: 21
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 683
LOCATION: 1..21
SEQUENCE DESCRIPTION: SEQ ID NO: 42:
ACTGGTTGCT CGTGGTCTCT A 21
INFORMATION FOR SEQ ID N0:43:
SEQUENCE CHARACTERISTICS:
LENGTH: 35
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 1026
LOCATION: 1..35
SEQUENCE DESCRIPTION: SEQ ID NO: 43:
ACCAGCAGCA ACACAGCCTA CCTGAGACTC AGCAG 35
INFORMATION FOR SEQ ID N0:44:
SEQUENCE CHARACTERISTICS:
LENGTH: 36
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 1028
LOCATION: 1..36
SEQUENCE DESCRIPTION: SEQ ID NO: 44:
TGCTGAGTCT CAGGTAGGCT GTGTTGCTGC TGGTGT 36
-53-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
INFORMATION FOR SEQ ID N0:45:
SEQUENCE CHARACTERISTICS:
LENGTH: 26
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 745
LOCATION: 1..26
SEQUENCE DESCRIPTION: SEQ ID NO: 45:
TGACCTGCAC CGCGTCTGGC TTCAAC 26
INFORMATION FOR SEQ ID N0:46:
SEQUENCE CHARACTERISTICS:
LENGTH: 26
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 746
LOCATION: 1..26
SEQUENCE DESCRIPTION: SEQ ID N0: 46:
TTGAAGCCAG ACGCGGTGCA GGTCAG 26
INFORMATION FOR SEQ ID N0:47:
SEQUENCE CHARACTERISTICS:
LENGTH: 20
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 1071
LOCATION: 1..20
SEQUENCE DESCRIPTION: SEQ ID NO: 47:
GAGACTCAGC AGCGTGACAG 20
INFORMATION FOR SEQ ID N0:48:
SEQUENCE CHARACTERISTICS:
LENGTH: 40
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
-54-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCT/US98/03680
FEATURE:
NAME/KEY: Oligonucleotide 1072
LOCATION: 1..40
SEQUENCE DESCRIPTION: SEQ ID NO: 48:
CGCTGCTGAG TCTCAGGCTG AATGTGTTCT TGCTGGTGTC 40
INFORMATION FOR SEQ ID N0:49:
SEQUENCE CHARACTERISTICS:
LENGTH: 38
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 1074
LOCATION: 1..38
SEQUENCE DESCRIPTION: SEQ ID NO: 49:
CGCTGCTGAG TCTCAGGCTG GCCTGGTTCT TGCTGGTG 38
INFORMATION FOR SEQ ID N0:50:
SEQUENCE CHARACTERISTICS:
LENGTH: 33
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 1073
LOCATION: 1..33
SEQUENCE DESCRIPTION: SEQ ID NO: 50:
CGCTGCTGAG TCTCAGGTAG AACTGGTTCT TGC 33
INFORMATION FOR SEQ ID N0:51:
SEQUENCE CHARACTERISTICS:
LENGTH: 40
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 1075
LOCATION: 1..40
SEQUENCE DESCRIPTION: SEQ ID NO: 51:
CGCTGCTGAG TCTCAGGTAG GCTGTGTTCT TGCTGGTGTC 40
-55-
SUBSTITUTE SHEET (RULE 26)
CA 02321947 2000-08-24
WO 99/43817 PCTNS98/03680
INFORMATION FOR SEQ ID N0:52:
SEQUENCE CHARACTERISTICS:
LENGTH: 25
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 1010
LOCATION: 1..25
SEQUENCE DESCRIPTION: SEQ ID NO: 52:
ACTCCGACAT CGTGCTGACC CAGAG 25
INFORMATION FOR SEQ ID N0:53:
SEQUENCE CHARACTERISTICS:
LENGTH: 24
TYPE: DNA
STRANDEDNESS: single
TOPOLOGY: linear
FEATURE:
NAME/KEY: Oligonucleotide 1011
LOCATION: 1..24
SEQUENCE DESCRIPTION: SEQ ID NO: 53:
CTCTGGGTCA GCACGATGTC GGAG 24
-56-
SUBSTITUTE SHEET (RULE 26)
