U.S. patent application number 15/707401 was filed with the patent office on 2018-06-07 for covalent disulfide-linked diabodies and uses thereof.
The applicant listed for this patent is City of Hope. Invention is credited to Tove Olafsen, Andrew A. Raubitschek, Mark A. Sherman, John E. Shively, Anna M. Wu.
Application Number | 20180155449 15/707401 |
Document ID | / |
Family ID | 47090663 |
Filed Date | 2018-06-07 |
United States Patent
Application |
20180155449 |
Kind Code |
A1 |
Wu; Anna M. ; et
al. |
June 7, 2018 |
COVALENT DISULFIDE-LINKED DIABODIES AND USES THEREOF
Abstract
The present invention provides recombinant antibody fragments
which include a variable domain which has been modified by the
addition of a tail sequence to its C-terminal end. The tail
sequence comprises a terminal cysteine residue and an amino acid
spacer and does not substantially affect the fragment's
target-binding affinity. The present invention also provides
pharmaceutical compositions comprising the described antibody
fragments and a pharmaceutically acceptable carrier and methods of
delivering an agent to cells of interest in a subject using the
fragments as delivery vehicles. The invention further provides
compositions comprising the described antibody fragments for the in
vitro detection and measurement of target molecules which bind to
the fragments and method of determining the presence or amount of
such targets in a biological sample by contacting the sample with
such compositions.
Inventors: |
Wu; Anna M.; (Sherman Oaks,
CA) ; Shively; John E.; (Arcadia, CA) ;
Raubitschek; Andrew A.; (San Marino, CA) ; Sherman;
Mark A.; (Pasadena, CA) ; Olafsen; Tove;
(Sherman Oaks, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
City of Hope |
Duarte |
CA |
US |
|
|
Family ID: |
47090663 |
Appl. No.: |
15/707401 |
Filed: |
September 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13554306 |
Jul 20, 2012 |
9765155 |
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15707401 |
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12788477 |
May 27, 2010 |
9701754 |
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13554306 |
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10690990 |
Oct 23, 2003 |
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12788477 |
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60420271 |
Oct 23, 2002 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/626 20130101;
C07K 16/3007 20130101; C07K 16/46 20130101; A61K 2039/505 20130101;
A61K 39/39558 20130101; C07K 2317/90 20130101; C07K 16/00 20130101;
C07K 2319/00 20130101; C07K 16/30 20130101; C07K 2317/64
20130101 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 16/30 20060101 C07K016/30; C07K 16/00 20060101
C07K016/00 |
Goverment Interests
GOVERNMENT RIGHTS STATEMENT
[0002] The invention described herein was made with Government
support under grant numbers P01 CA 43904 from the National
Institutes of Health and DAMD17-00-1-0150 from the Department of
Defense. Accordingly, the United States Government has certain
rights in this invention.
Claims
1-47. (canceled)
48. A diabody comprising: a first single-chain polypeptide subunit
that comprises: a first heavy chain variable domain polypeptide
connected by a first linker sequence to a first light chain
variable region polypeptide, and a first tail sequence that
comprises a first amino acid spacer a first cysteine residue,
wherein the first amino acid spacer comprises from 1 to about 10
amino acid residues, and wherein the first cysteine residue is at
the C terminus of the first single-chain polypeptide subunit; and a
second single-chain polypeptide subunit that comprises: a second
heavy chain variable domain polypeptide connected by a second
linker sequence to a second light chain variable region
polypeptide, and a second tail sequence that comprises a second
amino acid spacer a second cysteine residue, wherein the second
amino acid spacer comprises from 1 to about 10 amino acid residues,
and wherein the second cysteine residue is at the C terminus of the
second single-chain polypeptide subunit, wherein the first cysteine
residue forms a disulfide bond with the second cysteine
residue.
49. The diabody of claim 48, wherein the first heavy chain variable
domain polypeptide and the second light chain variable region
polypeptide together form a first target binding site, and wherein
the second heavy chain variable domain polypeptide and the first
light chain variable region polypeptide together form a second
target binding site.
50. The diabody of claim 49, wherein the first target binding site
and the second target binding site bind the same target.
51. The diabody of claim 49, wherein the first target binding site
and the second target binding site bind different targets.
52. The diabody of claim 48, wherein the first amino acid spacer
comprises from about 5 to about 10 amino acid residues, and wherein
the second amino acid spacer comprises from about 5 to about 10
amino acid residues.
53. The diabody of claim 48, wherein the first amino acid spacer
comprises from 2 to 5 amino acid residues, and wherein the second
amino acid spacer comprises from 2 to 5 amino acid residues.
54. The diabody of claim 48, wherein the first amino acid spacer
comprises 6, 7, 8, or 9 amino acid residues, and wherein the second
amino acid spacer comprises 6, 7, 8, or 9 amino acid residues.
55. The diabody of claim 48, wherein the first amino acid spacer
comprises 2 amino acid residues, and wherein the second amino acid
spacer comprises 2 amino acid residues.
56. The diabody of claim 48, wherein the amino acid residues of the
first amino acid spacer are glycine residues.
57. The diabody of claim 56, wherein the amino acid residues of the
second amino acid spacer are glycine residues.
58. The diabody of claim 48, wherein the first linker sequence
comprises from about 5 to about 10 amino acid residues, and wherein
the second linker sequence comprises from about 5 to about 10 amino
acid residues.
59. The diabody of claim 58, wherein the first linker sequence
comprises 8 amino acid residues, and wherein the second linker
sequence comprises 8 amino acid residues.
60. The diabody of claim 58, wherein the residues of the first
linker sequence are glycine residues and wherein the residues of
the second linker sequence are glycine residues.
61. The diabody of claim 49, the first target binding site and/or
the second target binding site specifically binds a
carcinoembryonic antigen (CEA).
62. The diabody of claim 61, wherein the first heavy chain variable
domain polypeptide corresponds to a heavy chain variable domain
polypeptide sequence of the murine anti-CEA T84.66 antibody, and
wherein the second light variable domain polypeptide corresponds to
a light chain variable domain polypeptide sequence of the murine
anti-CEA T84.66 antibody.
63. The diabody of claim 62, wherein the second heavy chain
variable domain polypeptide corresponds to a heavy chain variable
domain polypeptide sequence of the murine anti-CEA T84.66 antibody,
and wherein the first light variable domain polypeptide corresponds
to a light chain variable domain polypeptide sequence of the murine
anti-CEA T84.66 antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is related to and claims priority
under 35 U.S.C. .sctn. 119(e) to U.S. provisional patent
application No. 60/420,271 filed on Oct. 23, 2002. This application
is incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to recombinant antibody
fragments, nucleic acids encoding such recombinant antibody
fragments, and methods of using such recombinant antibody
fragments, particularly for in vivo delivery of agents to specific
cells of interest.
BACKGROUND OF THE INVENTION
[0004] Throughout this application, various publications are
referenced. Disclosures of these publications in their entireties
are hereby incorporated by reference into this application to more
fully describe the state of the art to which this invention
pertains. Full bibliographic citations for the references may be
found listed immediately preceding the claims.
[0005] Antibodies specific for tumor-associated antigens can
provide effective vehicles for in vivo delivery of agents, such as
radionuclides, for detection or therapy of cancer. The potential
utility of cancer-targeting antibodies can be improved by protein
engineering approaches, which can be used to modify characteristics
such as affinity, immunogenicity, and pharmacokinetic properties.
In particular, recombinant antibody fragments have been produced
with favorable characteristics, including retention of high
affinity for target antigen, rapid, high level accumulation in
xenografts in murine models, and quick clearance from the
circulation, resulting in high tumor:normal activity ratios.
Furthermore, because antibody fragments do not persist in the
circulation, they are less likely to be immunogenic than intact
murine or even chimeric antibodies. With the advent of humanized
and human antibodies, the issue of immunogenicity of recombinant
antibodies is rapidly diminishing.
[0006] Recombinant fragments such as diabodies (e.g., 55 kDa dimers
of single-chain Fv fragments, which self-assemble in a cross-paired
fashion as described by Holliger, et al., 1993) or minibodies
(e.g., 80 kDa scFv-C.sub.H3 fusion proteins as described by Hu et
al., 1996)) have shown promise as in vivo imaging agents in
preclinical studies when radiolabeled with single-photon emitting
radionuclides such as In-111 or 1-123, or positron emitters such as
Cu-64 or 1-124 for positron emission tomography (Sundaresan et al.,
In press; Wu et al. 2000). Targeting and imaging of 1-123
radiolabeled single-chain Fv (scFv, 27 kDa) fragments has been
demonstrated clinically, although the size and monovalency of
scFv's may limit their utility (Begent, et al., 1996). Recent
clinical imaging studies using I-123 radiolabled diabodies appear
promising (Santimaria et al. 2003).
[0007] Most current antibody radiolabeling approaches involve
conjugation to random sites on the surface of the protein. For
example, standard radioiodination methods result in modification of
random surface tyrosine residues. Many antibodies are highly
susceptible to inactivation following iodination, presumably due to
modification of key tyrosines in or near the binding site. (Nikula
et al., 1995; Olafsen et al., 1996). Chemical modification of
lysines located in or near the antigen-binding site could also
potentially interfere with binding through sterical hindrance if a
bulky group is added (Benhar et al., 1994; Olafsen et al., 1995).
Alternative iodination approaches or radiometal labeling through
conjugation of bifunctional chelates direct modifications to -amino
groups of lysine residues, again randomly located on the surface of
antibodies. The issue of inactivation following radiolabeling
becomes more pressing as one moves to smaller and smaller antibody
fragments, if equal reactivity is assumed, because the binding
site(s) represent a larger proportion of the protein surface, and
fewer "safe" sites for conjugation are available.
[0008] Site-specific radiolabeling approaches provide a means for
both directing chemical modification to specific sites on a
protein, located away from the binding site, and for controlling
the stoichiometry of the reaction. Several strategies capitalize on
naturally occurring moieties or structures on antibodies that can
be targeted chemically. For example, the carbohydrate found on
constant domains of immunoglobulins can be oxidized and conjugated
with bifunctional chelates for radiometal labeling. (Rodwell, et
al., 1993). In one instance, an unusual carbohydrate moiety
occurring on a hypervariable loop of a kappa light chain was
modified for site-specific chelation and radiometal labeling
(Leung, et al., 1995). Others have exploited selective reduction of
interchain disulfide bridges to enable modification using
thiol-specific reagents. C-terminal cys residues on antibody Fab or
Fab' fragments have been used for direct labeling using .sup.99mTc
(Behr, et al., 1995; Verhaar, et al., 1996). Novel approaches
include the identification of a purine binding site in antibody Fv
fragments, allowing specific photoaffinity labeling (Rajagopalan,
et al., 1996).
[0009] More recently, genetic engineering approaches have been used
to introduce specific sites for modification or radiolabeling of
proteins and antibodies. Building on the above-mentioned work,
glycosylation sites have been engineered into proteins to provide
novel carbohydrate targets for chemical modification (Leung, et
al., 1995; Qu, et al., 1998). The six-histidine tail commonly
appended to recombinant proteins to provide a purification tag has
been used in a novel 99mTc labeling method (Waibel, et al., 1999).
Alternatively, a popular strategy has been to use site-directed
mutagenesis to place cys residues on the surface of proteins to
provide reactive sulfhydryl groups. This approach has been
implemented by numerous groups to allow site-specific labeling of
antibodies (Lyons, et al. 1990; Stimmel, et al., 2000) and other
proteins (Haran, et al., 1992; Kreitman, et al., 1994).
[0010] Introduction of cys residues into engineered antibody
fragments also has been used for stabilization or multimerization
purposes. For example, introduction of strategically placed cys
residues in the interface between the V.sub.H and V.sub.L domains
of antibody Fv fragments has allowed covalent linkage and
stabilization of these fragments (disulfide-stabilized Fv, or dsFv)
(Glockshuber, et al., 1990; Webber, et al., 1995). Fitzgerald et
al. described a disulfide bonded diabody in which cysteine residues
were introduced into the V.sub.L/V.sub.H interface for stability
and demonstrated its utility for fluorescent imaging of tumors
(Fitzgerald, et al., 1997). Others have appended cys residues to
the C-termini of single-chain Fv fragments (scFv, formed by fusing
V.sub.H and V.sub.L with a synthetic peptide linker) to allow
multimerization into scFv'.sub.2 fragments (Adams, et al., 1993;
Kipriyanov, et al., 1995).
[0011] We have previously produced an anti-carcinoembryonic antigen
(anti-CEA) diabody from the murine anti-CEA T84.66 antibody by
joining V.sub.L--eight amino acid linker--V.sub.H. Tumor targeting,
imaging, and biodistribution studies of a radiolabeled (at random
sites on the protein) anti-CEA diabody demonstrated rapid tumor
uptake, fast clearance from the circulation, and favorable
properties for use as an imaging agent, when evaluated in nude mice
bearing LS14T xenografts (Wu, et al., 1999; Yazaki et al.,
2001b).
[0012] There remains a need in the art, however, for a stable, in
vivo delivery vehicle that can be modified readily in specific
locations without affecting the ability of the vehicle to
specifically target cells of interest. There is also a continuing
need for better in vitro detection methods. The invention provides
a system for adding site-specific functional groups to antibody
fragments that do not interfere with target binding by said
fragment.
SUMMARY OF THE INVENTION
[0013] The present invention provides recombinant antibody
fragments for use in in vivo delivery of agents for detection and
treatment of diseases, primarily cancers. The present invention
also provides recombinant antibody fragments for the in vitro
detection of certain targets of interest. Preferred antibody
fragments comprise at least two single chain polypeptide subunits,
each subunit having a heavy-chain variable domain polypeptide
sequence connected by a linker sequence to a light-chain variable
domain polypeptide sequence. One of the variable domain polypeptide
sequences in each subunit is modified at its C-terminal end by
addition of a tail sequence. The tail sequence comprises a terminal
cysteine residue and an amino acid spacer. The selection of the
C-terminal end as the modification site for introduction of the
tail sequence provides a location at the end of the variable region
opposite the target combining site. This configuration avoids
interference with antigen binding and thus, the addition of the
tail sequence does not substantially affect the antibody fragment's
target-binding affinity. A "target" in the context of the present
invention is a molecule of interest that can bind to or complex
with the antibody fragments of the present invention and includes
any molecule against which an antibody can be isolated. Examples of
a target include an antigen, an anti-antibody, a self antigen or a
hapten. Accordingly, "target binding sites" in the context of the
present invention are sites of antibody fragments that bind
"targets" and include, but are not limited to, antigen binding
sites.
[0014] The subunits assemble such that each heavy chain domain is
bound to a light chain domain, thereby providing a specific
target-binding site with each such light chain/heavy chain pairing.
Moreover, in preferred embodiments of the invention, the addition
of the tail sequence provides a disulfide covalent bond, or bridge,
between the heavy chain variable regions or between the light chain
variable regions, depending on to which variable region the tail
sequence was added. Advantages of the bond include, but are not
limited to, added stability and the presence of thiol groups in an
internal, protected location, which then can be released when
desired for site-specific chemical modification.
[0015] The present invention also provides pharmaceutical
compositions comprising the described antibody fragments and a
pharmaceutically acceptable carrier. The invention further provides
methods of delivering an agent to cells of interest in a subject.
Preferred delivery methods involve conjugating the agent to the
recombinant antibody fragment and administering the conjugate to
the subject under conditions permitting specific binding between
the fragment and the cell of interest in the subject.
[0016] The present invention also provides in vitro diagnostic
methods for detecting in a biological sample at least one target of
interest. In such an diagnostic method complexes between at least
one recombinant antibody fragment described herein and at least one
target are detected.
[0017] Finally, it surprisingly has been found that the addition of
a tail sequence having a terminal cysteine residue and an amino
acid spacer provides advantages over known antibody fragment
structures, including the formation of a stable disulfide bond and
ease of site-specific chemical modification.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1a shows the design of anti-CEA cys-diabodies with a
parental diabody with an 8 amino acid linker sequence (SEQ ID NO:
9) between the V domains and a C-terminus 5 amino acid sequence
present in the V.sub.H domain (SEQ ID NO: 10). (1) and (2) show
cys-diabody variants made with the added amino acids highlighted
and the cysteine residue underlined in each construct (SEQ ID NO: 2
and SEQ ID NO: 4). FIG. 1b is a schematic drawing of a non-covalent
bound diabody.
[0019] FIG. 2 shows SDS-PAGE analysis of purified antibody fragment
proteins in accordance with the invention.
[0020] FIG. 3 shows results of a competition ELISA between an
embodiment of the present invention, a known antibody, and a known
antibody fragment.
[0021] FIGS. 4a and 4b show traverse slices of serial microPET
scans of a mouse bearing bilateral C6 (arrow) and LS174T
(arrowhead) xenografts, injected with .sup.64Cu cys-diabody (57
.mu.Ci) and imaged at 4 hrs (4a) and 18 hrs (4b).
[0022] FIGS. 5a and 5b show the published crystal structure of the
parental anti-CEA diabody (5a), and a model of the structure of
-SGGC cys-diabody (5b) where the Fvs have been rotated to bring the
C-termini close enough for disulfide bridge formation.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention provides a recombinant antibody
fragment comprising at least two single chain polypeptide subunits.
Each subunit comprises a heavy-chain variable domain polypeptide
sequence connected by a linker sequence to a light-chain variable
domain polypeptide sequence. The linker sequence is preferably a
glycine sequence typically having from about 5 to about 10 glycine
residues, preferably 6, 7, 8 or 9 residues and most preferably 8
residues. One of the variable domain polypeptide sequences in each
subunit is modified at its C-terminal end by addition of a tail
sequence. See FIG. 1.a. The tail sequence comprises an amino acid
spacer and a terminal cysteine residue. The spacer, positioned
between the terminal cysteine residue and the end of the sequence
to which the tail is added, is preferably between 1 and about 10
residues in length, more preferably 2 to 5 residues in length, most
preferably 2 residues in length. However, depending on the distance
of the C-termini, a longer tail sequence might be advantageous,
e.g. a tail sequence having 11, 12 or more amino acid residues. In
a preferred embodiment, the residues are glycine residues. The
subunits assemble such that each heavy chain domain is bound to a
light chain domain, wherein each such light chain/heavy chain
pairing provides a target-binding site. Following addition of the
tail sequence, each target-binding site retains binding affinity
substantially similar or equivalent to that of a recombinant
fragment without addition of the tail sequence, as described above
and illustrated by FIG. 3. "Substantially similar binding affinity"
in the context of the antibody fragments according to the present
invention describes an in vitro binding affinity that a person
skilled in the art would consider not inferior when compared to the
in vitro binding affinity of the same antibody fragment without a
tail sequence.
[0024] In a preferred embodiment, the tail sequence is added to the
heavy chain variable domain of each subunit and provides a
disulfide covalent bond between the heavy chain variable domains.
Alternatively, the tail sequence is added to the light chain
variable domain of each subunit and provides a disulfide covalent
bond between the light chain variable domains.
[0025] In a preferred embodiment, at least one of the target
binding sites of the recombinant antibody fragment specifically
binds a carcinoembryonic antigen (CEA). A preferred diabody is an
anti-CEA diabody. In a preferred embodiment, at least one light
chain and at least one heavy chain of the fragment correspond to a
light chain variable domain and a heavy chain variable domain of
the murine anti-CEA T84.66 antibody. The tail sequence preferably
is added to the heavy chain variable domain of each subunit and
provides a disulfide covalent bond between the heavy chain variable
domains. Alternatively, the tail sequence is added to the light
chain variable domain of each subunit and provides a disulfide
covalent bond between the light chain variable domains.
[0026] In another preferred embodiment, the recombinant antibody
fragment further comprises an agent, such as a diagnostic or
therapeutic agent, conjugated to the cysteine residue of the tail
sequence. This conjugation can be achieved readily by methods known
in the art. The agent can be conjugated to the fragment via a
thiol-specific bifunctional chelating agent or other suitable
chelating agent. The agent can be, without limitation, a
radionuclide label such as In-111 or 1-123, a positron emitter,
such as Cu-64 or 1-124 or, in particular for the in vitro
applications, a dye, such as a fluorescent dye for direct detection
by colorimetric assays, for fluorescent detection including
Fluorescence Activated Cell Sorting (FACS) or for Fluorescence
Resonance Energy Transfer (FRET); a protein such as horseradish
peroxidase or alkaline phosphatase, that generates a colored
product with an appropriate substrate for ELISA type assays, or a
luciferase that generates light upon addition of an appropriate
substrate. Alternatively, the agent can be a cytotoxic agent such
as a chemotherapeutic drug.
[0027] Another embodiment comprises an in vitro detection method in
which an antibody fragment that has formed a complex with a target
is detected via a labeled antibody or antibody fragment that is
specific for a site of the fragment, attachment to which does not
interfere with the fragment's ability to bind a target. Such a site
is preferably the terminal LGGC or SGGC sequence.
[0028] The tail sequence for at least one of the variable domains
preferably can comprise the sequence set forth in SEQ ID NO: 2.
Alternatively, the tail sequence preferably can comprise the
sequence set forth in SEQ ID NO: 4. The invention further provides
nucleotide sequences encoding the antibody fragments described
herein (SEQ ID NO: 5 and SEQ ID NO: 7).
[0029] The invention moreover provides pharmaceutical compositions
which comprise a recombinant antibody fragment as described herein
and a pharmaceutically acceptable carrier. These pharmaceutical
compositions can be administered in accordance with the present
invention as a bolus injection or infusion or by continuous
infusion. Pharmaceutical carriers suitable for facilitating such
means of administration are well known in the art. Examples of
suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. Liquid formulations can
be solutions or suspensions and can include vehicles such as
suspending agents, solubilizers, surfactants, preservatives, and
chelating agents.
[0030] The present invention also provides a method of delivering
an agent to cells of interest in a subject which includes
conjugating the agent to a recombinant antibody fragment and
administering the conjugate, (or "conjugated recombinant fragment")
to the subject. As described above, the recombinant antibody
fragment comprises at least two single chain polypeptide subunits
in which each subunit comprises a heavy-chain variable domain
polypeptide sequence connected by a linker sequence to a
light-chain variable domain polypeptide sequence. One of the
variable domain polypeptide sequences in each subunit is modified
at its C-terminal end by addition of a tail sequence comprising a
terminal cysteine residue and an amino acid spacer. The spacer,
positioned between the terminal cysteine residue and the end of the
sequence to which the tail is added, is preferably between 1 and
about 10 residues in length, most preferably about 2 residues in
length. In a preferred embodiment, the residues are glycine
residues. The single chain subunits assemble such that each heavy
chain domain associates with a light chain domain, each such light
chain/heavy chain pairing thereby providing a target-binding site.
At least one target-binding site specifically binds the cells of
interest.
[0031] The conjugate is then administered to the subject under
conditions permitting the conjugate to specifically bind targets on
the cells of interest, which in turn thereby achieves delivery of
the agent to cells of interest in the subject. Methods of in vivo
targeting which are suitable for delivering conjugated antibody
fragments are known in the art and described in the example
provided herein. The subject is preferably a human patient. The
amount to be administered to a human patient can be determined
readily through methods known in the art, including those based on
animal data. The cells of interest are preferably cancer cells and
can be, without limitation, colon cancer cells, breast cancer
cells, lung cancer cells, lymphoma cells, or cells from other human
malignancies and other human diseases or conditions. The agent is
preferably a detectable label. In a preferred embodiment of the
invention, following administration of the conjugate to the
subject, the detectable label can be detected to determine the
location of the cells of interest in the subject. The detectable
label can be a radioisotope, a thiol-specific label, an optical or
fluorescent probe, or any other suitable label known in the
art.
[0032] Alternatively, the agent can be a cytotoxic agent, such as a
chemotherapeutic drug or radionuclide. Methods in accordance with
the present invention also can be used to diagnose, without
limitation, autoimmune, inflammatory, or angiogenic processes by
selecting an appropriate antibody from which to derive the antibody
fragment.
[0033] The invention also provides methods of detecting and
quantitatively determining the concentration of a target in a
biological fluid sample. In one embodiment the method comprises
contacting a solid support with an excess of a certain type of
antibody fragment which specifically forms a complex with a target,
such as a tumor associated antigen, e.g., CEA, under conditions
permitting the antibody fragments to attach to the surface of the
solid support. The resulting solid support to which the antibody
fragments are attached is then contacted with a biological fluid
sample so that the target in the biological fluid binds to the
antibody fragments and forms a target-antibody complex. The complex
can be labeled with a detectable marker. Alternatively, either the
target or the antibody fragments can be labeled before the
formation the complex. For example, a detectable marker can be
conjugated to the antibody fragments as described elsewhere herein.
The complex then can be detected and quantitatively determined
thereby detecting and quantitatively determining the concentration
of the target in the biological fluid sample.
[0034] A biological fluid according to the present invention
includes, but is not limited to tissue extract, urine, blood,
serum, and phlegm. Further, the detectable marker includes but is
not limited to an enzyme, biotin, a fluorophore, a chromophore, a
heavy metal, a paramagnetic isotope, or a radioisotope.
[0035] Further, the invention provides a diagnostic kit comprising
an antibody fragment that recognizes and binds an antibody or
antibody fragment against a target; and a conjugate of a detectable
label and a specific binding partner of the antibody or antibody
fragment against the target. In accordance with the practice of the
invention the label includes, but is not limited to, enzymes,
radiolabels, chromophores and fluorescers.
[0036] In light of the preceding description, one of ordinary skill
in the art can practice the invention to its fullest extent. The
following example, therefore, is merely illustrative and should not
be construed to limit in any way the invention as set forth in the
claims which follow.
EXAMPLE
[0037] In the present example, cysteine residues were introduced
into an anti-CEA diabody at different locations, in order to
provide specific thiol groups for subsequent chemical modification.
Modified proteins carrying an added C-terminal gly-gly-cys sequence
were shown to exist exclusively as a disulfide-bonded dimer. This
"cys-diabody" retained high binding to CEA and demonstrated tumor
targeting and biodistribution properties identical to the
non-covalent diabody. Furthermore, following reduction of the
disulfide bond, the cys-diabody could be chemically modified using
a thiol-specific bifunctional chelating agent, to allow labeling
with radiometal. Thus, the disulfide-linked cys-diabody provides a
covalently linked alternative to conventional diabodies, and,
following reduction, generates specific thiol groups that are
readily modified chemically. This format provides a useful platform
for targeting a variety of agents to cells, such as for example,
CEA-positive tumors.
[0038] In order to allow site-specific radiolabeling using
thiol-specific reagents, the present example describes mutant
anti-CEA diabodies engineered by substitution or addition of unique
cys residues. Two variants, namely a variant having the C-terminal
sequence-LGGC, and a variant having the C-terminal sequence-SGGC,
were found to exist as a stable disulfide-linked dimer. The former
demonstrated equivalent antigen binding in vitro and tumor
targeting in vivo compared to the parental diabody shown in FIG. 1.
a., and had the added advantage of allowing site-specific chemical
modification following reduction of the interchain disulfide
bridge. SEQ ID NO: 5 represents the nucleotide sequence of the
VTVS-SGGC Cys diabody used in the examples. SEQ ID NO: 6 represents
the respective amino acid sequence. SEQ ID NO: 7 represents the
nucleotide sequence of the VTVS-LGGC Cys diabody used in the
examples. SEQ ID NO: 8 represents the respective amino acid
sequence.
Materials and Methods
Design of Cysteine-Modified Diabodies (Cys Diabodies)
[0039] Two variants of anti-CEA diabodies (FIG. 1, I & II;
Table 1) were constructed by PCR-based mutagenesis (QuickChange
Site Directed Mutagenesis Kit, Strategene, LaJolla, Calif.) of the
pEE12 expression vector (Lonza Biologics, Slough, UK) containing
the original anti-CEA diabody constructed with an 8 amino acid
glycine-serine linker (GS8) (Wu, et al., 1999). The two constructs
contained a cysteine at the C-terminus of the V.sub.H and two
glycines inserted in front of the cysteine as a spacer. One
retained the original C-terminal sequence of the V.sub.H with GGC
appended (VTVS-SGGC) and in the other, serine 113 was exchanged to
a leucine (VTVS-LGGC).
Mammalian Expression, Selection and Purification
[0040] 1.times.10.sup.7 NSO cells (provided by Lonza
Biologics)(Galfe and Milstein, 1981) were transfected with 40 .mu.g
of linearized vector DNA by electroporation and selected in
glutamine-deficient media as described (Yazaki, et al., 2001a).
Clones were screened for expression by ELISA, in which the desired
protein was captured either by protein L or by a recombinant CEA
fragment, N-A3 (You, et al., 1998) and detected using alkaline
phosphatase-conjugated goat anti-mouse Fab antibodies (Sigma, St.
Louis, Mo.). Supernatants also were examined by Western blot for
size analysis, using the alkaline phosphatase-conjugated goat
anti-mouse Fab antibodies. The best producing clones were expanded.
Cys-diabodies were purified from cell culture supernatants, using a
BioCad 700E chromatography system (Applied Biosystems, Foster City,
Calif.) as described (Yazaki et al. 2001a). Briefly, the
supernatants were treated with 5% AG1.RTM.-X8 (Bio-Rad Labs,
Hercules, Calif.) overnight to remove phenol red and cell debris
and then dialyzed versus 50 mM Tris-HCl, pH 7.4. Treated
supernatant was loaded onto an anion exchange chromatography column
(Source 15Q, Amersham Pharmacia Biotech AB, Uppsala, Sweden), and
proteins were eluted with a NaCl gradient to 0.2 M in the presence
of 50 mM HEPES, pH 7.4. Eluted fractions, containing the desired
protein, were subsequently loaded onto a Ceramic Hydroxyapatite
(Bio-Rad Laboratories, Hercules, Calif.) column and eluted with a
KPi gradient to 0.15 M in the presence of 50 mM MES, pH 6.5.
Fractions containing pure proteins were pooled and concentrated by
Centriprep 10 (Amicon Inc., Beverly, Mass.). Elution was monitored
by absorption at 280 nm. The concentration of purified protein per
ml was determined by OD.sub.280, but also by applying a small
sample on protein L using known amounts of parental diabody and
later cys-diabody standards quantitated by amino acid composition
analysis (Wu, et al., Immunotechnology, 1996).
Characterization of Purified Cys-Diabodies
[0041] Purified proteins were analyzed by SDS-PAGE pre-cast 4-20%
polyacrylamide Ready gels (Bio-Rad Laboratories under non-reducing
and reducing (1 mM DTT) conditions and stained using
MicrowaveBlue.TM. (Protiga Inc., Frederick, Md.). Samples were also
subjected to size-exclusion HPLC on Superdex 75 (Amersham
Biosciences). Retention time was compared to a standard of parental
diabody. Binding to CEA was initially assessed by ELISA as
described above. Competition/Scatchard was also carried out in
ELISA microtiter plates wells coated with N-A3, using a fixed
concentration (1 nM) of biotinylated chimeric T84.66 antibody, and
increasing concentration of non-biotinylated competitors (0.01-100
nM). Displacement was monitored with alkaline
phosphatase-conjugated streptavidin (1:5000 dilution) (Jackson
ImmunoResearch Labs, West Grove, Pa.) and color was developed with
Phosphatase substrate tablets (Sigma, St. Louis, Mo.) dissolved in
diethanolamine buffer, pH 9.8. All experiments were carried out in
triplicate.
Radioiodination
[0042] 70 .mu.g of purified cys-diabody was radiolabeled with 140
.mu.Ci Na.sup.131I (Perkin Elmer Life Sciences, Inc., Boston,
Mass.) in 0.1 phosphate buffer at pH 7.5, using 1.5 ml
polypropylene tubes coated with 10 .mu.g Iodogen (Pierce, Rockford,
Ill.). Following a 5-7 min. incubation at room temperature, the
sample was purified by HPLC on Superdex 75. Peak fractions were
selected and diluted in normal saline/1% human serum albumin to
prepare doses for injection. The labeling efficiency was 85%.
Immunoreactivity and valency were determined by incubation of
radiolabeled protein with a 20-fold excess of CEA at 37.degree. C.
for 15 min., followed by HPLC size-exclusion chromatography on a
calibrated Superose 6 column (Amersham Biosciences).
Biodistribution in Tumor-Bearing Mice
[0043] 7-8 week-old female athymic mice were injected
subcutaneously in the flank with 10.sup.6 LS174T human colon
carcinoma cells (ATCC #CL-188). At 7 days post inoculation, mice
bearing LS174T xenografts were injected with 1 .mu.g of
.sup.131I-labeled cys-diabody (specific activity, 1.7 .mu.Ci/.mu.g)
via the tail vein. Groups of five mice were sacrificed and
dissected at 0, 2, 4, 6, and 24 h post injection. Major organs were
weighed and counted in a gamma scintillation counter. Radiouptakes
in organs were corrected for decay and expressed as percentage of
injected dose per gram of tissue (% ID/g) and as percentage of
injected dose per organ (% ID/organ). Tumor masses ranged from an
average of 0.580 mg (0 h group) to 1.058 mg (24 h group).
Biodistribution data are summarized as means and corresponding
standard errors (sem). Animal blood curves were calculated using
ADAPT II software (D' Argenio and Schumitzky, 1979) to estimate two
rate constants (k.sub.i) and associated amplitudes (A.sub.i).
Conjugation and Radiometal Labeling of Cys-Diabodies
[0044] The VTVS-LGGC cys-diabody was reduced and conjugated with a
novel bifunctional chelating agent comprised of the macrocyclic
chelate DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid), a tetrapeptide linker, and hexanevinylsulfone group for
chemical attachment to thiol groups. This compound,
DOTA-glycylleucylglycyl-( -amino-bis-1,6 hexanevinylsufone)lysine,
abbreviated DOTA-GLGK-HVS, is described in greater detail elsewhere
(Li, et al., 2002). VTVS-LGGC cys-diabody (2 mg in 0.5 mL PBS) was
reduced by treatment with 20 .mu.L of 20 .mu.M
tris-(carboxyethyl)phosphine (TCEP) (Pierce) in PBS for 2 h at
37.degree. under Ar and centrifuged through a Sephadex G25 spin
column. DOTA-GLGK-HVS, (58 .mu.L of 20 mM in PBS) was added and the
solution rotated at 10 rpm for 4 h at 25.degree. C. The conjugate
was dialyzed against 0.25 M NH4OAc, pH 7.0. Extent of modification
was evaluated by isoelectric focusing gels (Li, et al., 2002).
Radiolabeling of Cys-Diabody Conjugates with Copper-64
[0045] Copper-64 (copper chloride in 0.1 N HCL; radionuclide purity
>99%) was produced in a cyclotron from enriched .sup.64Ni
targets, at the Mallinckrodt Institute of Radiology, Washington
University Medical Center (McCarthy, et al., 1997).
DOTA-GLGK-HVS-conjugated cys-diabody (200 .mu.g) was incubated with
7.3 mCi of .sup.64Cu in 0.1 M NH.sub.4 citrate, pH 5.5 for 1 h at
43.degree. C. The reaction was terminated by addition of EDTA to 1
mM. Labeled protein was purified by size exclusion HPLC on a
TSK2000 column (30 cm.times.7.5 mm I.D.; Toso-Haas;
Montgomeryville, Pa.). The radiolabeling efficiency was 56% and the
specific activity was 1.7 .mu.Ci/.mu.g.
MicroPET Imaging
[0046] CEA-positive (LS174T) and CEA-negative (C6 rat glioma)
xenografts were established in nude mice by subcutaneous injection
of 1-2.times.10.sup.6 cells subcutaneously into the shoulder area,
10-14 days prior to imaging. Mice were imaged using the dedicated
small animal microPET scanner developed at the Crump Institute for
Biological Imaging (UCLA) (Chatziioannou, et al., 1999). Mice were
injected in the tail vein with 57 .mu.Ci of .sup.64Cu-diabody.
After the appropriate time had elapsed, mice were anesthetized with
a 4:1 mixture of ketamine (80 mg/kg body weight) and xylazine (10
mg/kg body weight, injected intraperitoneally), placed in a prone
position, and imaged using the microPET scanner with the long axis
of the mouse parallel to the long axis of the scanner. Acquisition
time was 56 min. (8 min. per bed position; 7 bed positions), and
images were reconstructed using MAP reconstruction algorithm (Qi,
et al., 1998).
Results
Expression, Purification, and Characterization of Cys-Diabodies
[0047] The cys-diabodies were expressed in the mouse myeloma cell
line, NSO. The expression levels for the VTVS-SGGC and VTVS-LGGC
constructs were between 5-20 .mu.g/mL as determined by ELISA.
Cultures were expanded in T flasks and supernatants collected. The
VTVS-SGGC and VTVS-LGGC cys-diabodies were purified essentially as
described (Yazaki, et al., 2001).
[0048] Analysis of the purified proteins on SDS-PAGE demonstrated
that the two-step purification scheme yielded VTVS-SGGC and
VTVS-LGGC diabodies that were >95% pure (FIG. 2). Under
non-reducing conditions both proteins migrated as single species in
the range of 55-60 kDa, with the VTVS-LGGC version exhibiting
slightly lower mobility (FIG. 2, lanes 1 and 2). Under reducing
conditions both proteins demonstrated the presence of the expected
25 kDa monomer (FIG. 2, lanes 4 and 5). Thus, when purified under
native conditions, both of these cys-diabodies existed as
essentially pure disulfide-bonded homodimers. Incorporation of a
serine (polar) versus a leucine (nonpolar) residue made no
difference at position 113. One of ordinary skill in the art would
recognize that most other amino acid substitutions at this position
would be suitable with the probable exception of cys, (which would
introduce another thiol) and proline (which would restrict
flexibility).
[0049] Size exclusion chromatography demonstrated that the
covalently linked cys-diabody was slightly smaller than the regular
diabody, as it eluted at 20.42 min. (average of 5 experiments) as
opposed to 20.13 min. (average of 4 experiments) (not shown).
[0050] The binding-activity of the cys-diabody to CEA was initially
demonstrated by ELISA. Affinity was measured by competition ELISA
in the presence of competitors at different concentrations. As
shown in FIG. 3, by competition assay the affinities of the
cys-diabody and parental diabody were essentially the same as that
of the parental intact murine T84.66 monoclonal antibody.
[0051] The immunoreactivity and valency of the cys-diabody were
analyzed following radioiodination by solution-phase incubation in
the presence of excess CEA. Size-exclusion HPLC analysis
demonstrated that 90% of the cys-diabody shifted to high molecular
weight complexes indicated by two peaks, suggesting that the
cys-diabody was bound to one and two CEA molecules (not shown).
In Vivo Biodistribution and Targeting
[0052] The .sup.131I-labeled cys-diabody was assessed for its
ability to target tumor in athymic mice bearing xenografts of
LS174T human colon carcinoma cells. As can be seen in Table 2 the
accumulation of the .sup.131I-labeled cys-diabody reached 9.32%
ID/g at 2 h and this level of localization was maintained at 4 and
6 hours post injection. Blood clearance was rapid and nearly
complete by 18 h (0.55% ID/g), with the half life in the beta phase
being 2.68 hrs., essentially the same as that observed with the
non-covalently bound diabody (2.89 hrs.) (Yazaki et al., 2001 b).
Activities in other normal organs (liver, spleen, lung, kidney)
fell rapidly as well and were below 1% ID/g by 18 h. These
biodistribution results were essentially identical to those
observed for the parental anti-CEA diabody (Wu, et al., 1999).
In Vivo Imaging by microPET
[0053] Cys-diabody was conjugated with the macrocyclic chelate DOTA
using a novel peptide-hexanevinylsufone derivative described in
detail elsewhere (Li, et al., 2002). This allowed efficient
radiolabeling with .sup.64Cu, a positron-emitting radionuclide with
a 12.7 h half-life, well-matched to the targeting and clearance
kinetics observed for diabodies in murine systems in vivo. MicroPET
imaging studies were conducted on athymic mice bearing LS174T
xenografts (CEA-positive human colorectal carcinoma) or C6
xenografts (CEA-negative rat glioma). Specific targeting to the
CEA-positive xenograft was observed at 4 and 18 h post injection,
with little evidence of activity in the CEA-negative tumor. The
results are shown in FIGS. 4a and 4b. However, this particular
combination of protein/chelate/radionuclide resulted in elevated
liver activity (19.4% ID/g at 4 hrs) in addition to kidney activity
(55.1% ID/g at 4 hrs) (Li et al. 2002).
Discussion
[0054] The present example provides the design, production and
evaluation of a novel antibody format, a covalently-linked
(disulfide-bonded) diabody which is referred to as the cys-diabody.
Two constructs were made. The initial intent was to introduce
cysteine residues into the anti-CEA diabody in order to provide
specific sites for chemical modification including conjugation and
radiolabeling. In course of these experiments, it was unexpectedly
discovered that addition of the sequence GGC to the end of the
protein resulted in a diabody in which the C-termini of the
V.sub.L-V.sub.H subunits came together and formed a disulfide bond.
Two slightly different versions of the cys-diabody (with C-terminal
sequences of LGGC or SGGC) resulted in essentially 100% formation
of the disulfide linkage. This protein provides various
improvements over a standard diabody, including, but without
limitation, covalent linkage for greater potential stability, and
the feasibility of site-specific modification following reduction
of the disulfide bond and generation of free reactive thiols.
Additionally, the introduced cysteine residues are essentially
"protected" through the internal linkage and prevented from forming
random disulfide bonds with small molecules (such as glutathione)
or other sulfhydryl-containing proteins present in the cell. As a
result, the cys-diabody can be obtained in higher amounts and with
greater purity than might be expected for proteins containing
engineered cysteine residues that are unpaired and accessible to
random chemical modification and fortuitous disulfide
formation.
[0055] The crystal structure of the parental T84.66/GS8 diabody has
recently been solved (Carmichael, et al., 2003). In the crystal,
the Fv units of the diabody assumed a very compact, twisted
structure, with the binding sites oriented in a skewed orientation
at a tight 70.degree. angle. The C-termini of the heavy chain
variable regions (where we have appended the cys residues in the
present example) are about 60 .ANG. apart. However, the structure
that was solved is likely to represent one of many conformations
that the parental diabody can adopt. The fact that the cys-diabody
forms with such high efficiency implies that the parental diabody
is in fact quite flexible, and that the Fv domains can swivel such
that the C-termini are juxtaposed. FIG. 5a shows the crystal
structure of the diabody and FIG. 5b shows a model where the Fv
domains have been rotated to bring the C termini close enough for
disulfide bridge formation (the disulfide bridge is the
light-colored loop protruding from the model in the central bottom
part of the Figure). Size exclusion HPLC analysis confirms the
notion that the native, non-covalent diabody is quite flexible; it
elutes at an earlier retention time suggesting a larger Stokes
radius. By contrast, the covalently-linked cys-diabody elutes at a
later retention time, implying a more compact, more highly
constrained molecule. Taken together, these results suggest that
native diabody has a more open and flexible structure.
TABLE-US-00001 TABLE 1 Cys-diabody constructs VTVS-SGGC: 5'-GTC ACC
GTC TCC TCA GGT GGA TGT-3' (SEQ ID NO: 1) Val Thr Val Ser Ser Gly
Gly Cys (SEQ ID NO: 2) VTVS-LGGC: 5'-GTC ACC GTC TCC TTA GGT GGA
TGT-3' (SEQ ID NO: 3) Val Thr Val Ser Leu Gly Gly Cys (SEQ ID NO:
4)
TABLE-US-00002 TABLE 2 Biodistribution of .sup.131I-labeled T84.66
cys-diabody in athymic mice bearing LS174T xenografts.sup.a Organ
(% ID/g) 0 2 4 6 18 24 Tumor 2.49 .+-. 0.33 9.32 .+-. 1.73 10.02
.+-. 0.69 9.15 .+-. 0.77 6.43 .+-. 1.05 4.79 .+-. 1.28 Blood 36.34
.+-. 2.70 5.87 .+-. 0.63 4.04 .+-. 0.53 3.28 .+-. 0.27 0.55 .+-.
0.13 0.36 .+-. 0.09 Liver 8.40 .+-. 0.61 3.16 .+-. 0.81 2.66 .+-.
0.64 1.67 .+-. 0.27 0.64 .+-. 0.24 0.32 .+-. 0.06 Spleen 5.67 .+-.
0.80 2.54 .+-. 0.39 1.57 .+-. 0.20 1.30 .+-. 0.14 0.34 .+-. 0.11
0.19 .+-. 0.04 Kidney 18.58 .+-. 1.22 4.03 .+-. 0.57 2.99 .+-. 0.51
2.09 .+-. 0.25 0.47 .+-. 0.09 0.37 .+-. 0.04 Lung 9.90 .+-. 0.69
3.40 .+-. 0.48 2.50 .+-. 0.39 1.94 .+-. 0.17 0.46 .+-. 0.10 0.27
.+-. 0.07 Ratios.sup.b T:B 0.07 1.59 2.48 2.79 11.69 13.31 T:kidney
0.13 2.31 3.35 4.38 13.68 12.95 T:Liver 0.30 2.95 3.77 5.48 10.05
14.97 Tumor Tumor weight 0.58 .+-. 0.28 0.7 .+-. 0.20 0.69 .+-.
0.09 0.63 .+-. 0.19 1.02 .+-. 0.54 1.06 .+-. 0.53 Groups of five
mice were analyzed at each time point. Tumor and normal organ
uptake are expressed as percent injected dose per gram (% ID/g).
Table values are the means with corresponding standard errors of
the means (s.e.m.). The ratios presented are the averages of the
T:B, Tumor:kidney and Tumor:Liver ratios for the individual mice.
.sup.aTime given in hours .sup.bRatios were determined for each
individual mouse, and then averages were calculated
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Sequence CWU 1
1
10124DNAHomo sapiensCDS(1)..(24)misc_feature(13)..(24) 1gtc acc gtc
tcc tca ggt gga tgt 24Val Thr Val Ser Ser Gly Gly Cys 1 5 28PRTHomo
sapiens 2Val Thr Val Ser Ser Gly Gly Cys 1 5 324DNAHomo
sapiensCDS(1)..(24)misc_feature(13)..(24) 3gtc acc gtc tcc tta ggt
gga tgt 24Val Thr Val Ser Leu Gly Gly Cys 1 5 48PRTHomo sapiens
4Val Thr Val Ser Leu Gly Gly Cys 1 5 5729DNAHomo
sapiensCDS(1)..(729) 5gac att gtg ctg acc caa tct cca gct tct ttg
gct gtg tct ctt ggg 48Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu Gly 1 5 10 15 cag agg gcc acc atg tcc tgc aga gcc
ggt gaa agt gtt gat att ttt 96Gln Arg Ala Thr Met Ser Cys Arg Ala
Gly Glu Ser Val Asp Ile Phe 20 25 30 ggc gtt ggg ttt ttg cac tgg
tac cag cag aaa cca gga cag cca ccc 144Gly Val Gly Phe Leu His Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 aaa ctc ctc atc tat
cgt gca tcc aac cta gaa tct ggg atc cct gtc 192Lys Leu Leu Ile Tyr
Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Val 50 55 60 agg ttc agt
ggc act ggg tct agg aca gac ttc acc ctc atc att gat 240Arg Phe Ser
Gly Thr Gly Ser Arg Thr Asp Phe Thr Leu Ile Ile Asp 65 70 75 80 cct
gtg gag gct gat gat gtt gcc acc tat tac tgt cag caa act aat 288Pro
Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Thr Asn 85 90
95 gag gat ccg tac acg ttc gga ggg ggg acc aag ctg gaa ata aaa ggt
336Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110 gga ggc agt gga ggc ggt gga gag gtt cag ctg cag cag tcc
ggg gca 384Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Gln Gln Ser
Gly Ala 115 120 125 gag ctt gtg gag cca ggg gcc tca gtc aag ttg tcc
tgc aca gct tct 432Glu Leu Val Glu Pro Gly Ala Ser Val Lys Leu Ser
Cys Thr Ala Ser 130 135 140 ggc ttc aac att aaa gac acc tat atg cac
tgg gtg aag cag agg cct 480Gly Phe Asn Ile Lys Asp Thr Tyr Met His
Trp Val Lys Gln Arg Pro 145 150 155 160 gaa cag ggc ctg gaa tgg att
gga agg att gat cct gcg aat ggt aat 528Glu Gln Gly Leu Glu Trp Ile
Gly Arg Ile Asp Pro Ala Asn Gly Asn 165 170 175 agt aaa tat gtc ccg
aag ttc cag ggc aag gcc act ata aca gca gac 576Ser Lys Tyr Val Pro
Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp 180 185 190 aca tcc tcc
aac aca gcc tac ctg cag ctc acc agc ctg aca tct gag 624Thr Ser Ser
Asn Thr Ala Tyr Leu Gln Leu Thr Ser Leu Thr Ser Glu 195 200 205 gac
act gcc gtc tat tat tgt gct ccg ttt ggt tac tac gtg tct gac 672Asp
Thr Ala Val Tyr Tyr Cys Ala Pro Phe Gly Tyr Tyr Val Ser Asp 210 215
220 tat gct atg gcc tac tgg ggt caa gga acc tca gtc acc gtc tcc tca
720Tyr Ala Met Ala Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
225 230 235 240 ggt gga tgt 729Gly Gly Cys 6243PRTHomo sapiens 6Asp
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10
15 Gln Arg Ala Thr Met Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe
20 25 30 Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln
Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser
Gly Ile Pro Val 50 55 60 Arg Phe Ser Gly Thr Gly Ser Arg Thr Asp
Phe Thr Leu Ile Ile Asp 65 70 75 80 Pro Val Glu Ala Asp Asp Val Ala
Thr Tyr Tyr Cys Gln Gln Thr Asn 85 90 95 Glu Asp Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110 Gly Gly Ser Gly
Gly Gly Gly Glu Val Gln Leu Gln Gln Ser Gly Ala 115 120 125 Glu Leu
Val Glu Pro Gly Ala Ser Val Lys Leu Ser Cys Thr Ala Ser 130 135 140
Gly Phe Asn Ile Lys Asp Thr Tyr Met His Trp Val Lys Gln Arg Pro 145
150 155 160 Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn
Gly Asn 165 170 175 Ser Lys Tyr Val Pro Lys Phe Gln Gly Lys Ala Thr
Ile Thr Ala Asp 180 185 190 Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu
Thr Ser Leu Thr Ser Glu 195 200 205 Asp Thr Ala Val Tyr Tyr Cys Ala
Pro Phe Gly Tyr Tyr Val Ser Asp 210 215 220 Tyr Ala Met Ala Tyr Trp
Gly Gln Gly Thr Ser Val Thr Val Ser Ser 225 230 235 240 Gly Gly Cys
7729DNAHomo sapiensCDS(1)..(729) 7gac att gtg ctg acc caa tct cca
gct tct ttg gct gtg tct ctt ggg 48Asp Ile Val Leu Thr Gln Ser Pro
Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 cag agg gcc acc atg tcc
tgc aga gcc ggt gaa agt gtt gat att ttt 96Gln Arg Ala Thr Met Ser
Cys Arg Ala Gly Glu Ser Val Asp Ile Phe 20 25 30 ggc gtt ggg ttt
ttg cac tgg tac cag cag aaa cca gga cag cca ccc 144Gly Val Gly Phe
Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 aaa ctc
ctc atc tat cgt gca tcc aac cta gaa tct ggg atc cct gtc 192Lys Leu
Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Val 50 55 60
agg ttc agt ggc act ggg tct agg aca gac ttc acc ctc atc att gat
240Arg Phe Ser Gly Thr Gly Ser Arg Thr Asp Phe Thr Leu Ile Ile Asp
65 70 75 80 cct gtg gag gct gat gat gtt gcc acc tat tac tgt cag caa
act aat 288Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln
Thr Asn 85 90 95 gag gat ccg tac acg ttc gga ggg ggg acc aag ctg
gaa ata aaa ggt 336Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys Gly 100 105 110 gga ggc agt gga ggc ggt gga gag gtt cag
ctg cag cag tcc ggg gca 384Gly Gly Ser Gly Gly Gly Gly Glu Val Gln
Leu Gln Gln Ser Gly Ala 115 120 125 gag ctt gtg gag cca ggg gcc tca
gtc aag ttg tcc tgc aca gct tct 432Glu Leu Val Glu Pro Gly Ala Ser
Val Lys Leu Ser Cys Thr Ala Ser 130 135 140 ggc ttc aac att aaa gac
acc tat atg cac tgg gtg aag cag agg cct 480Gly Phe Asn Ile Lys Asp
Thr Tyr Met His Trp Val Lys Gln Arg Pro 145 150 155 160 gaa cag ggc
ctg gaa tgg att gga agg att gat cct gcg aat ggt aat 528Glu Gln Gly
Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Asn 165 170 175 agt
aaa tat gtc ccg aag ttc cag ggc aag gcc act ata aca gca gac 576Ser
Lys Tyr Val Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp 180 185
190 aca tcc tcc aac aca gcc tac ctg cag ctc acc agc ctg aca tct gag
624Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Thr Ser Leu Thr Ser Glu
195 200 205 gac act gcc gtc tat tat tgt gct ccg ttt ggt tac tac gtg
tct gac 672Asp Thr Ala Val Tyr Tyr Cys Ala Pro Phe Gly Tyr Tyr Val
Ser Asp 210 215 220 tat gct atg gcc tac tgg ggt caa gga acc tca gtc
acc gtc tcc tta 720Tyr Ala Met Ala Tyr Trp Gly Gln Gly Thr Ser Val
Thr Val Ser Leu 225 230 235 240 ggt gga tgt 729Gly Gly Cys
8243PRTHomo sapiens 8Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Met Ser Cys Arg Ala
Gly Glu Ser Val Asp Ile Phe 20 25 30 Gly Val Gly Phe Leu His Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr
Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Val 50 55 60 Arg Phe Ser
Gly Thr Gly Ser Arg Thr Asp Phe Thr Leu Ile Ile Asp 65 70 75 80 Pro
Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Thr Asn 85 90
95 Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110 Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Gln Gln Ser
Gly Ala 115 120 125 Glu Leu Val Glu Pro Gly Ala Ser Val Lys Leu Ser
Cys Thr Ala Ser 130 135 140 Gly Phe Asn Ile Lys Asp Thr Tyr Met His
Trp Val Lys Gln Arg Pro 145 150 155 160 Glu Gln Gly Leu Glu Trp Ile
Gly Arg Ile Asp Pro Ala Asn Gly Asn 165 170 175 Ser Lys Tyr Val Pro
Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp 180 185 190 Thr Ser Ser
Asn Thr Ala Tyr Leu Gln Leu Thr Ser Leu Thr Ser Glu 195 200 205 Asp
Thr Ala Val Tyr Tyr Cys Ala Pro Phe Gly Tyr Tyr Val Ser Asp 210 215
220 Tyr Ala Met Ala Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Leu
225 230 235 240 Gly Gly Cys 98PRTHomo sapiens 9Gly Gly Gly Ser Gly
Gly Gly Gly 1 5 105PRTHomo sapiens 10Val Thr Val Ser Ser 1 5
* * * * *