U.S. patent application number 12/931528 was filed with the patent office on 2011-07-07 for gpcr crystalization method using an antibody.
Invention is credited to Peter Brams, Brian Kobilka, Asna Masood, Daniel Rohrer.
Application Number | 20110166330 12/931528 |
Document ID | / |
Family ID | 40567675 |
Filed Date | 2011-07-07 |
United States Patent
Application |
20110166330 |
Kind Code |
A1 |
Kobilka; Brian ; et
al. |
July 7, 2011 |
GPCR crystalization method using an antibody
Abstract
An antibody that specifically binds a three dimensional epitope
on the IC3 loop of a GPCR is provided. The antibody may be employed
in a method that comprises: contacting a GPCR with a monovalent
version of the antibody binding conditions to form a complex; and
crystallizing the complex.
Inventors: |
Kobilka; Brian; (Palo Alto,
CA) ; Rohrer; Daniel; (Los Gatos, CA) ; Brams;
Peter; (Sacramento, CA) ; Masood; Asna;
(Saratoga, CA) |
Family ID: |
40567675 |
Appl. No.: |
12/931528 |
Filed: |
February 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12284245 |
Sep 19, 2008 |
7947807 |
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12931528 |
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60980122 |
Oct 15, 2007 |
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Current U.S.
Class: |
530/387.3 ;
530/388.1 |
Current CPC
Class: |
C07K 16/2869 20130101;
C07K 2317/55 20130101; C07K 2317/34 20130101; A61P 37/04 20180101;
C07K 14/723 20130101; C07K 2299/00 20130101; C07K 2317/92
20130101 |
Class at
Publication: |
530/387.3 ;
530/388.1 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Goverment Interests
GOVERNMENT RIGHTS
[0001] This work was supported in part by federal grant number
NS28471 from the National Institutes of Health. The federal
government has certain rights in this invention.
Claims
1. A monoclonal antibody that specifically binds a three
dimensional epitope on the intracellular region (IC3) loop of a
GPCR, wherein said monoclonal antibody is characterized in that it:
a) stabilizes the relative conformations of the transmembrane
region 5 (TM5) and transmembrane region 6 (TM6) of said GPCR, b)
facilitates crystallization of said GPCR.
2. The monoclonal antibody of claim 1, wherein said antibody
specifically binds the IC3 loop of the .beta.2 adrenoreceptor.
3. The monoclonal antibody of claim 1, wherein said antibody is a
monovalent antibody.
4-17. (canceled)
18. The monoclonal antibody of claim 1, wherein said monoclonal
antibody is a single chain antibody.
19. The monoclonal antibody of claim 1, wherein said monoclonal
antibody is a Fab fragment.
20. The monoclonal antibody of claim 1, wherein said monoclonal
antibody comprises a light chain variable domain of SEQ ID NO. 1
and a heavy chain variable domain of SEQ ID NO:2.
21. The monoclonal antibody of claim 1, wherein said monoclonal
antibody is made by a) reconstituting a GPCR in artificial
phospholipid vesicles to make an antigen; and b) immunizing an
animal with said antigen.
22. The monoclonal antibody of claim 1, wherein said monoclonal
antibody is rabbit, mouse or chicken.
23. The monoclonal antibody of claim 1, wherein said GPCR is a
receptor for a biogenic amine.
24. The monoclonal antibody of claim 1, wherein said GPCR is a
dopamine receptor.
25. The monoclonal antibody of claim 1, wherein said GPCR is not a
.beta.2-adrenergic receptor.
26. The monoclonal antibody of claim 1, wherein said GPCR is a
seratonin receptor.
27. The monoclonal antibody of claim 1, wherein said GPCR is an
adrenergic receptor.
28. The monoclonal antibody of claim 1, wherein said GPCR is a
melanocortin receptor subtype 4.
29. The monoclonal antibody of claim 1, wherein said GPCR is a
ghrelin receptor.
30. The monoclonal antibody of claim 1, wherein said GPCR is a
metabotropic glutamate receptor.
31. The monoclonal antibody of claim 1, wherein said GPCR is a
chemokine receptor.
Description
BACKGROUND
[0002] G protein-coupled receptor (GPCR) signaling plays a vital
role in a number of physiological contexts including, but not
limited to, metabolism, inflammation, neuronal function, and
cardiovascular function. For instance, GPCRs include receptors for
biogenic amines, e.g., dopamine, epinephrine, histamine, glutamate,
acetylcholine, and serotonin; for purines such as ADP and ATP; for
the vitamin niacin; for lipid mediators of inflammation is such as
prostaglandins, lipoxins, platelet activating factor, and
leukotrienes; for peptide hormones such as calcitonin, follicle
stimulating hormone, gonadotropin releasing hormone, ghrelin,
motilin, neurokinin, and oxytocin; for non-hormone peptides such as
beta-endorphin, dynorphin A, Leu-enkephalin, and Met-enkephalin;
for the non-peptide hormone melatonin; for polypeptides such as C5a
anaphylatoxin and chemokines; for proteases such as thrombin,
trypsin, and factor Xa; and for sensory signal mediators, e.g.,
retinal photopigments and olfactory stimulatory molecules. GPCRs
are of immense interest for drug development.
[0003] Efforts to crystallize GPCRs have been frustrated by
intrinsic characteristics of integral membrane proteins. Bovine
rhodopsin is the only GPCR for which a high-resolution structure
has been determined by X-ray crystallography; and this is in part
due to its natural abundance and atypical stability. The seven
hydrophobic transmembrane helices of GPCRs make poor surfaces for
crystal contacts, and the extracellular and intracellular domains
are often relatively short and/or poorly structured.
SUMMARY OF THE INVENTION
[0004] An antibody that specifically binds a three dimensional
epitope of the IC3 loop of a GPCR is provided. In certain
embodiments, the antibody may specifically bind to the IC3 loop of
the .beta.2 adrenoreceptor. Also provided is a complex comprising a
GPCR and a monovalent antibody that binds to a three dimensional
epitope of the IC3 loop of that GPCR. The complex may be in a
crystalline form.
[0005] A method is also provided. In general terms, the method
comprises: a) contacting a GPCR with a monovalent antibody that
specifically binds to a three dimensional epitope of the IC3 loop
of a GPCR under binding conditions to form a complex; and b)
crystallizing the complex. In one embodiment, the GPCR comprises
the .beta.2AR IC3 loop, and the Fab fragment may bind to the
.beta.2AR IC3 loop. The GPCR may also be a hybrid GPCR that
contains the IC3 loop of .beta.2AR.
[0006] Also provided is a method comprising reconstituting a GPCR
in artificial phospholipid vesicles to make an antigen; and
immunizing an animal with the antigen. The animal may be a rabbit,
mouse or chicken, for example.
[0007] A method comprising screening a plurality of hybridoma lines
obtained from an animal immunized with phospholipid vesicles
comprising a GPCR for a hybridoma that produces an antibody that
bind to a three dimensional epitope of the IC3 loop of said GPCR is
also provided. This method may further comprise isolating a
hybridoma line that produces the antibody.
[0008] A method of making GPCR-containing vesicular antigens is
also provided, as is a method of screening hybridomas for the
production of antibodies that bind to GPCR-containing vesicles.
[0009] A method of using the antibody as a treatment for a
GPCR-mediated disorder is also provided.
BRIEF DESCRIPTION OF THE FIGURES
[0010] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0011] FIG. 1 is a schematic illustration of a GPCR, showing the
canonical transmembrane regions (TM1, TM2, TM3, TM4, TM5, TM6, and
TM7), intracellular regions (IC1, IC2, and IC3), and extracellular
regions (EC1, EC2, and EC3).
[0012] FIGS. 2A and 2B. Binding characteristics of
.beta..sub.2AR-specific antibodies. 2A, Dot-blots showing binding
of nine .beta..sub.2AR specific antibodies to denatured receptor.
Equal amounts of .beta..sub.2AR denatured with SDS and
.beta.-mercaptoethanol were spotted in triplicate on nitrocellulose
strips. The strips were blocked with 5% non-fat dry milk in
PBS-Tween (0.05% Tween-20) and then probed with 1 mg/ml of the
indicated antibodies diluted in blocking buffer. Binding of the
primary antibody to the denatured .beta..sub.2AR was detected with
an Alexa-688 labeled anti-mouse secondary antibody. The top panel
is a graphical representation of the average dot intensity from
three independent experiments. The lower panel is a representative
experiment. The binding of all 9 antibodies to denatured
.beta..sub.2AR is reduced compared to M1 binding to the linear Flag
epitope. 2B, Dose-response curves showing the effect on increasing
amounts of is antibodies 4-9 on the fluorescence of .beta..sub.2AR
labeled at C265 with tetramethylrhodamine (.beta..sub.2AR-TMR).
.beta..sub.2AR-TMR was diluted to 4 nM in 500 .mu.L of buffer
consisting of 0.1% dodecylmaltoside, 100 mM sodium chloride, and 20
mM HEPES buffer (pH 7.5). Data represent an n of 3.
[0013] FIGS. 3A and 3B. Fab 5 binds IC3 (labeled as IL3) of the
.beta..sub.2AR but does not effect structural changes associated
with G protein activation. 3A, Western blot analysis of the
.beta..sub.2AR digested with trypsin in the absence and presence of
Fab 5. The fragmented .beta..sub.2AR was visualized using an
Alexa-680 labeled M1 antibody against the N-terminal Flag epitope.
In the absence of Fab 5, two fragments at approximately 27 and 29
kDa appear corresponding to cleavage in the third intracellular
loop. In contrast, the presence of Fab 5 protects the N-terminal
end of the loop thus leaving the larger of the two fragments. The
diagram shows the third intracellular loop connecting TM5 and TM6.
The loop is marked with MW indicators corresponding to N-terminal
fragments containing the Flag epitope. Residues sensitive to
trypsin digest are shown in red. 3B, The change in the bimane
fluorescence of .beta..sub.2AR labeled with monobromobimane at
H271C at the cytoplasmic end of TM6. Bimane fluorescence is
quenched by W135 at the cytoplasmic end of TM3 upon agonist
binding.sup.9. The response to the full agonist isoproterenol,
isoproterenol plus Fab 5, and Fab 5 alone are shown. Fluorescence
intensity was corrected for background fluorescence from buffer and
ligands in all experiments. The data are the mean.+-.S.E. of two
independent experiments performed in triplicate.
[0014] FIG. 4 Fluorescence images showing monoclonal antibody
detection of flag tagged .beta..sub.2AR stably expressed in HEK-293
cells. HEK-293 cells stably expressing a flag tagged version of the
.beta..sub.2AR were cultured on coverslips and processed for
immunocytochemistry. After fixation, with 4% paraformaldehyde, the
cells were washed and blocked with 2.5% goat serum in PBS alone
(nonpermeabilized) or with 0.5% NP-40 in PBS (permeabilized). The
cells were incubated with the .beta..sub.2AR specific antibody
indicated on the left side of the figure. After several washes, the
.beta..sub.2AR monoclonal antibodies were detected with a Texas Red
conjugated anti-mouse secondary antibody. To demonstrate that all
of the cells are expressing the receptor they were subsequently
stained with an Alexa-488 conjugated M1 antibody (M1 column), which
recognizes the amino terminal flag tag on the .beta..sub.2AR.
Antibodies 1, 3, 4 and 8 stain cells in the absence of
permeabilization and therefore bind an epitope on the extracellular
face of the receptor. In contrast, antibodies 2, 5, 6, 7 and 9 only
stain cells that have been permeabilized indicative of an
intracellular epitope. The images shown are representative of at
least 3 experiments. All images were acquired using an Axioplan 2
microscope (Carl Zeiss MicroImaging), fitted with a camera
(RTE/CCD-1300-Y/HS; Roper Scientific) controlled using IPLab
software (BD Bioscience).
[0015] FIG. 5 Affinity of Fab 5 for purified .beta..sub.2AR
determined by isothermal titration calorimetry (ITC). ITC
measurements were performed at 25.degree. C. using a VP-ITC
calorimeter (Microcal, Inc.). The ITC cell contained 11 .mu.M
detergent-solubilized .beta..sub.2AR or .beta..sub.2AR buffer
(blank titration), and the injection syringe contained 70 .mu.M Fab
5. Titrations were initiated with a 3 .mu.l injection, followed by
a series of 8 .mu.l injections (37 .beta..sub.2AR and 31 for
.beta..sub.2AR buffer blank), with 240 s between injections. After
subtracting the blank titration, the measured heat released upon
binding was plotted against the Fab: .beta..sub.2AR molar ratio,
and the data were fitted to a single-site binding model to obtain
the association constant Ka, enthalpy change .DELTA.H, and
stoichiometry n using the Origin software package.sup.1 (Microcal,
Inc.)
[0016] FIG. 6 Image of Fab 5-.beta..sub.2AR-TMR crystals.
.beta..sub.2AR was labeled with tetramethylrhodamine
(.beta..sub.2AR-TMR) and mixed with an excess of Fab 5. The complex
was purified by size exclusion chromatography. Crystals were grown
in bicelles and ammonium sulfate (Rasmussen et al manuscript).
Images were obtained by light (panel A) and fluorescence (panel B)
microscopy. The fluorescence confirms the presence of
.beta..sub.2AR-TMR in the crystals. Bar represents 50 .mu.m. All
images were acquired using an Axioplan 2 microscope (Carl Zeiss
MicroImaging), fitted with a camera (RTE/CCD-1300-Y/HS; Roper
Scientific) controlled using IPLab software (BD Bioscience).
[0017] is FIG. 7 Figure illustrates packing of .beta.2AR-Fab
complex in a crystal.
[0018] FIG. 8 Figure illustrates the .beta.2AR-Fab complex. As
shown, the Fab5 antibody binds a three dimensional epitope that
contains amino acids from both ends of the IC3 loop.
DEFINITIONS
[0019] Unless defined otherwise herein, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY
AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York
(1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF
BIOLOGY, Harper Perennial, NY (1991) provide one of skill with
general dictionaries of many of the terms used in this disclosure.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, the preferred methods and materials are
described.
[0020] All patents and publications, including all sequences
disclosed within such patents and publications, referred to herein
are expressly incorporated by reference.
[0021] Numeric ranges are inclusive of the numbers defining the
range. Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation; amino acid sequences are written
left to right in amino to carboxy orientation, respectively.
[0022] The headings provided herein are not limitations of the
various aspects or embodiments of the invention which can be had by
reference to the specification as a whole. Accordingly, the terms
defined immediately below are more fully defined by reference to
the specification as a whole.
[0023] "G-protein coupled receptors", or "GPCRs" are polypeptides
that share a common structural motif, having seven regions of
between 22 to 24 hydrophobic amino acids that form to seven alpha
helices, each of which spans a membrane. As illustrated in FIG. 1,
each span is identified by number, i.e., transmembrane-1 (TM1),
transmembrane-2 (TM2), etc. The transmembrane helices are joined by
regions of amino acids between transmembrane-2 and transmembrane-3,
transmembrane-4 and transmembrane-5, and transmembrane-6 and
transmembrane-7 on the exterior, or "extracellular" side, of the
cell membrane, referred to as "extracellular" regions 1, 2 and 3
(EC1, EC2 and EC3), respectively. The transmembrane helices are
also joined by regions of amino acids between transmembrane-1 and
transmembrane-2, transmembrane-3 and transmembrane-4, and
transmembrane-5 and transmembrane-6 on the interior, or
"intracellular" side, of the cell membrane, referred to as
"intracellular" regions 1, 2 and 3 (IC1, IC2 and IC3),
respectively. The "carboxy" ("C") terminus of the receptor lies in
the intracellular space within the cell, and the "amino" ("N")
terminus of the receptor lies in the extracellular space outside of
the cell. GPCR structure and classification is generally well known
in the art, and further discussion of GPCRs may be found in Probst,
DNA Cell Biol. 1992 11:1-20; Marchese et al Genomics 23: 609-618,
1994; and the following books: Jurgen Wess (Ed) Structure-Function
Analysis of G Protein-Coupled Receptors published by Wiley-Liss
(1st edition; Oct. 15, 1999); Kevin R. Lynch (Ed) Identification
and Expression of G Protein-Coupled Receptors published by John
Wiley & Sons (March 1998) and Tatsuya Haga (Ed), G
Protein-Coupled Receptors, published by CRC Press (Sep. 24, 1999);
and Steve Watson (Ed) G-Protein Linked Receptor Factsbook,
published by Academic Press (1st edition; 1994). A schematic
representation of an exemplary GPCR is shown in FIG. 1. A GPCR may
be naturally occurring or non-naturally occurring (i.e., altered my
man).
[0024] The term "naturally-occurring" in reference to a GPCR means
a GPCR that is naturally produced (for example and not limitation,
by a mammal or by a human). Such GPCRs are found in nature. The
term "non-naturally occurring" in reference to a GPCR means a GPCR
that is not naturally-occurring. Wild-type GPCRs that have been
made constitutively active through mutation, and variants of
naturally-occurring GPCRs are examples of non-naturally occurring
GPCRs. Non-naturally occurring GPCR may have an amino acid sequence
that is at least 80% identical to, e.g., at least 90% identical to,
at least 95% identical to or at lest 99% identical to, a
naturally-occurring GPCR.
[0025] The term "ligand" means a molecule that specifically binds
to a GPCR. A ligand may be, for example a polypeptide, a lipid, a
small molecule, an antibody. A "native ligand" is a ligand that is
an endogenous, natural ligand for a native GPCR. A ligand may be a
GPCR "antagonist", "agonist", "partial agonist" or "inverse
agonist", or the like.
[0026] A "modulator" is a ligand that increases or decreases a GPCR
intracellular response when it is in contact with, e.g., binds, to
a GPCR that is expressed in a cell. This term includes agonists,
including partial agonists and inverse agonists, and
antagonists.
[0027] A "deletion" is defined as a change in either amino acid or
nucleotide sequence in which one or more amino acid or nucleotide
residues, respectively, are absent as compared to an amino acid
sequence or nucleotide sequence of a parental GPCR polypeptide or
nucleic acid. In the context of a GPCR or a fragment thereof, a
deletion can involve deletion of about 2, about 5, about 10, up to
about 20, up to about 30 or up to about 50 or more amino acids. A
GPCR or a fragment thereof may contain more than one deletion.
[0028] An "insertion" or "addition" is that change in an amino acid
or nucleotide sequence which has resulted in the addition of one or
more amino acid or nucleotide residues, respectively, as compared
to an amino acid sequence or nucleotide sequence of a parental
GPCR. "Insertion" generally refers to addition to one or more amino
acid residues within an amino acid sequence of a polypeptide, while
"addition" can be an insertion or refer to amino acid residues
added at an N- or C-terminus, or both termini. In the context of a
GPCR or fragment thereof, an insertion or addition is usually of
about 1, about 3, about 5, about 10, up to about 20, up to about 30
or up to about 50 or more amino acids. A GPCR or fragment thereof
may contain more than one insertion.
[0029] A "substitution" results from the replacement of one or more
amino acids or nucleotides by different amino acids or nucleotides,
respectively as compared to an amino acid sequence or nucleotide
sequence of a parental GPCR or a fragment thereof. It is understood
that a GPCR or a fragment thereof may have conservative amino acid
substitutions which have substantially no effect on GPCR activity.
By conservative substitutions is intended combinations such as gly,
ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and
phe, tyr.
[0030] The term "biologically active", with respect to a GPCR,
refers to a GPCR having a biochemical function (e.g., a binding
function, a signal transduction function, or an ability to change
conformation as a result of ligand binding) of a naturally
occurring GPCR.
[0031] As used herein, the terms "determining," "measuring,"
"assessing," and "assaying" are used interchangeably and include
both quantitative and qualitative determinations. Reference to an
"amount" of a GPCR in these contexts is not intended to require
quantitative assessment, and may be either qualitative or
quantitative, unless specifically indicated otherwise.
[0032] The terms "polypeptide" and "protein", used interchangeably
herein, refer to a polymeric form of amino acids of any length,
which can include coded and non-coded amino acids, chemically or
biochemically modified or derivatized amino acids, and polypeptides
having modified peptide backbones.
[0033] The term "fusion protein" or grammatical equivalents thereof
is meant a protein composed of a plurality of polypeptide
components, that while typically unjoined in their native state,
are joined by their respective amino and carboxyl termini through a
peptide linkage to form a single continuous polypeptide. Fusion
proteins may be a combination of two, three or even four or more
different proteins. The term polypeptide includes fusion proteins,
including, but not limited to, fusion proteins with a heterologous
amino acid sequence, fusions with heterologous and homologous
leader sequences, with or without N-terminal methionine residues;
immunologically tagged proteins; fusion proteins with detectable
fusion partners, e.g., fusion proteins including as a fusion
partner a fluorescent protein, .beta.-galactosidase, luciferase,
etc.; and the like.
[0034] The terms "nucleic acid molecule" and "polynucleotide" are
used interchangeably and refer to a polymeric form of nucleotides
of any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. Polynucleotides may have any three-dimensional
structure, and may perform any function, known or unknown.
Non-limiting examples of polynucleotides include a gene, a gene
fragment, exons, introns, messenger RNA (mRNA), transfer RNA,
ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides,
branched polynucleotides, plasmids, vectors, isolated DNA of any
sequence, control regions, isolated RNA of any sequence, nucleic
acid probes, and primers. The nucleic acid molecule may be linear
or circular.
[0035] As used herein the term "isolated," when used in the context
of an isolated compound, refers to a compound of interest that is
in an environment different from that in which the compound
naturally occurs. "Isolated" is meant to include compounds that are
within samples that are substantially enriched for the compound of
interest and/or in which the compound of interest is partially or
substantially purified.
[0036] As used herein, the term "substantially pure" refers to a
compound that is removed from its natural environment and is at
least 60% free, at least 75% free, or at least 90% free from other
components with which it is naturally associated.
[0037] A "coding sequence" or a sequence that "encodes" a selected
polypeptide, is a nucleic acid molecule which can be transcribed
(in the case of DNA) and translated (in the case of mRNA) into a
polypeptide, for example, in a host cell when placed under the
control of appropriate regulatory sequences (or "control
elements"). The boundaries of the coding sequence are typically
determined by a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxy) terminus. A coding
sequence can include, but is not limited to, cDNA from viral,
procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or
prokaryotic DNA, and synthetic DNA sequences. A transcription
termination sequence may be located 3' to the coding sequence.
Other "control elements" may also be associated with a coding
sequence. A DNA sequence encoding a polypeptide can be optimized
for expression in a selected cell by using the codons preferred by
the selected cell to represent the DNA copy of the desired
polypeptide coding sequence.
[0038] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. In the case of a promoter, a promoter that is
operably linked to a coding sequence will effect the expression of
a coding sequence. The promoter or other control elements need not
be contiguous with the coding sequence, so long as they function to
direct the expression thereof. For example, intervening
untranslated yet transcribed sequences can be present between the
promoter sequence and the coding sequence and the promoter sequence
can still be considered "operably linked" to the coding
sequence.
[0039] By "nucleic acid construct" it is meant a nucleic acid
sequence that has been constructed to comprise one or more
functional units not found together in nature. Examples include
circular, linear, double-stranded, extrachromosomal DNA molecules
(plasmids), cosmids (plasmids containing COS sequences from lambda
phage), viral genomes comprising non-native nucleic acid sequences,
and the like.
[0040] A "vector" is capable of transferring gene sequences to a
host cell. Typically, "vector construct," "expression vector," and
"gene transfer vector," mean any nucleic acid construct capable of
directing the expression of a gene of interest and which can
transfer gene sequences to host cells, which can be accomplished by
genomic integration of all or a portion of the vector, or transient
or inheritable maintenance of the vector as an extrachromosomal
element. Thus, the term includes cloning, and expression vehicles,
as well as integrating vectors.
[0041] An "expression cassette" comprises any nucleic acid
construct capable of directing the expression of a gene/coding
sequence of interest, which is operably linked to a promoter of the
expression cassette. Such cassettes can be constructed into a
"vector," "vector construct," "expression vector," or "gene
transfer vector," in order to transfer the expression cassette into
a host cell. Thus, the term includes cloning and expression
vehicles, as well as viral vectors.
[0042] A first polynucleotide is "derived from" or "corresponds to"
a second polynucleotide if it has the same or substantially the
same nucleotide sequence as a region of the second polynucleotide,
its cDNA, complements thereof, or if it displays sequence identity
as described above.
[0043] A first polypeptide is "derived from" or "corresponds to" a
second polypeptide if it is (i) encoded by a first polynucleotide
derived from a second polynucleotide, or (ii) displays sequence
identity to the second polypeptides as described above.
[0044] The terms "antibodies" and "immunoglobulin" include
antibodies or immunoglobulins of any isotype, fragments of
antibodies which retain specific binding to antigen, including, but
not limited to, Fab, Fv, scFv, and Fd fragments, chimeric
antibodies, humanized antibodies, single-chain antibodies, and
fusion proteins comprising an antigen-binding portion of an
antibody and a non-antibody protein. The antibodies may be
detectably labeled, e.g., with a radioisotope, an enzyme which
generates a detectable product, a fluorescent protein, and the
like. The antibodies may be further conjugated to other moieties,
such as members of specific binding pairs, e.g., biotin (member of
biotin-avidin specific binding pair), and the like. The antibodies
may also be bound to a solid support, including, but not limited
to, polystyrene plates or beads, and the like. Also encompassed by
the terms are Fab', Fv, F(ab').sub.2, and or other antibody
fragments that retain specific binding to antigen.
[0045] Antibodies may exist in a variety of other forms including,
for example, Fv, Fab, and (Fab').sub.2, as well as bi-functional
(i.e. bi-specific) hybrid antibodies (e.g., Lanzavecchia et al.,
Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston
et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and
Bird et al., Science, 242, 423-426 (1988), which are incorporated
herein by reference). (See, generally, Hood et al., "Immunology",
Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature,
323, 15-16 (1986)). This term also encompasses so-called "phage
display" antibodies.
[0046] A "monovalent" antibody is an antibody that has a single
antigen binding region. Fab fragments, scFv antibodies, and phage
display antibodies are types of monovalent antibodies, although
others are known. A "Fab" fragment of an antibody has a single
binding region, and may be made by papain digestion of a full
length monoclonal antibody. A single chain variable (or "scFv")
fragment of an antibody is an antibody fragment containing the
variable regions of the heavy and light chains of immunoglobulins,
linked together with a short flexible linker.
[0047] A "hybridoma" is a cell that is a hybrid of a spleen cell or
other antibody producing cell and an immortal cell (e.g., a myeloma
cell). Hybridomas are both immortal and capable of producing the
genetically coded antibody. Hybridomas and methodologies for making
hybridomas may be found in U.S. Pat. No. 6,420,140 and Harlow,
(Antibodies: A Laboratory Manual, First Edition (1988) Cold Spring
Harbor, N.Y.) for example.
[0048] A "complex" between an antibody an antigen is characterized
by a K.sub.D (dissociation constant) of less than 10.sup.-6 M, less
than 10.sup.-7 M, less than 10.sup.-8 M, less than 10.sup.-9 M, or
less than 10.sup.-9 M.
[0049] The "IC3 loop" of a GPCR is the intracellular loop that is
between transmembrane region 5 (TM5) and transmembrane region 6
(TM6) of the GPCR. Such regions are readily identifiable by
analysis of the primary amino acid sequence of a GPCR.
[0050] The terms "specifically binds" and "specific binding" refer
to the ability of an antibody to preferentially bind to a
particular antigen that is present in a homogeneous mixture of
different antigens. In certain embodiments, a specific binding
interaction will discriminate between desirable and undesirable
antigens in a sample, in some embodiments more than about 10 to
100-fold or more (e.g., more than about 1000- or 10,000-fold).
[0051] An antibody that specifically binds to a "three dimensional"
epitope or "conformational" epitope is an antibody that
specifically binds to a tertiary (i.e., three dimensional)
structure a folded protein. Such an antibody binds at much reduced
(i.e., by a factor of at least 2, 5, 10, 50 or 100) affinity to the
linear (i.e., unfolded, denatured) form of the protein. The
structure to which such an antibody binds contains amino acids that
are discontiguous in the protein. In other words, binding of such
an antibody to a polypeptide is dependent upon the polypeptide
being folded into a particular three dimensional conformation.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0052] As noted above, various compositions are provided, including
an antibody that specifically binds to a three dimensional epitope
of the IC3 loop of a GPCR, a complex containing a monovalent
fragment of the antibody and a GPCR, and a crystal containing the
complex. Various crystallization methods are also provided. In
particular embodiments, the GPCR to be complexed with the antibody
may contain the .beta.2AR IC3 loop, and, in certain embodiments,
may be a hybrid GPCR that contains the IC3 loop of .beta.2AR.
Methods of preparing an antigen, and screening for an antibody are
also provided. These embodiments are described in greater detail
below.
[0053] Antibodies
[0054] A monoclonal antibody that that specifically binds to a
three dimensional epitope of the IC3 loop of a GPCR is provided.
Such antibodies may be made, in general terms, by reconstituting an
active GPCR in phospholipid vesicles, immunizing a suitable animal
with the phospholipid vesicles, and screening antibody-producing
cells of the animal (or hybridomas thereof) for the antibody.
[0055] A GPCR may be produced and purified using conventional
methods that may employ expressing a recombinant form of the GPCR
in a host cell, and purifying the GPCR using affinity
chromatography and/or antibody-based methods. In particular
embodiments, the bactulovirus/Sf-9 system may be employed for
expression, although other expression systems (e.g., bacterial,
yeast or mammalian cell systems) may also be used. Exemplary
methods for expressing and purifying GCPRs are described in, for
example, Kobilka (Anal Biochem 1995 231, 269-71), Eroglu et al
(EMBO 2002 3: 491-496), Chelikani et al (Protein Sci. 2006
15:1433-40) and the book "Identification and Expression of G
Protein-Coupled Receptors" (Kevin R. Lynch (Editor), Wiley-Liss
(March 1998)), among many others.
[0056] Likewise, methods for reconstituting an active GPCR in
phospholipid vesicles are known, and are described in: Luca et al
(Proc. Natl. Acad. Sci. 2003 100:10706-11); Mansoor et al (Proc.
Natl. Acad. Sci. 2006 103: 3060-3065); Niu et al, (Biophys J. 2005
89: 1833-1840); Shimada et al (J. Biol. Chem. 2002 277:31774-80);
and Eroglu et al, (Proc. Natl. Acad. Sci. 2003 100: 10219-10224),
among others. In certain cases, the GPCR and phospholipids may be
reconstituted at high density (e.g., 1 mg receptor per mg of
phospholipid). In particular embodiments, the phospholipids
vesicles may be tested to confirm that the GPCR is active. In many
cases, a GPCR may be present in the phospholipid vesicle in both
orientations (in the normal orientation, and in the "upside down"
orientation in which the intracellular loops are on the outside of
the vesicle).
[0057] Any suitable animal, e.g., a warm-blooded animal, in
particular a mammal such as a rabbit, mouse, rat, camel, sheep, cow
or pig or a bird such as a chicken or turkey, may be immunized with
the reconstituted GPCR using any of the techniques well known in
the art suitable for generating an immune response. Procedures for
immunizing animals are well known in the art, and are described in
Harlow (Antibodies: A Laboratory Manual, First Edition (1988) Cold
Spring Harbor, N.Y.) and Weir (Handbook of Experimental Immunology
Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). As
will be appreciated by one of ordinary skill in the art, the
immunogen may be admixed with an adjuvant or hapten in order to
increase the immune response (for example, complete or incomplete
Freund's or lipid A adjuvant), or with a carrier such as keyhole
limpet hemocyanin (KLH).
[0058] Once a suitable animal has been immunized and an immune
response against the antigen has been established by the animal,
antibody producing cells from the animal are screened to identify
cells that produce antibodies having a desired activity. In many
embodiments, these methods may employ hybridoma technology in which
cells from the spleen of the immunized animal are fused with a
suitable immortal cell to produce hybridoma cells. Supernatants
from these hybridoma cells may be screened, and positive clones are
expanded according to standard procedures (Harlow et al.
Antibodies: A Laboratory Manual, First Edition (1988) Cold spring
Harbor, N.Y.; and Spieker-Polet et al., supra).
[0059] The antibodies may be screened for binding to the GPCR
folded into a native conformation by, e.g., cell staining to
identify those antibodies that bind a three dimensional epitope in
the GPCR. In certain cases, the antibodies may also be screened for
binding to denatured GPCR. Antibodies that bind to the IC3 loop of
the polypeptide may be identified using any of a variety of
different means, depending on the GPCR. For example, the antibody
can be tested to determine if it can block interactions with a
G-protein, or it can be tested on a GPCR containing amino acid
substitutions in the IC3 loop. Other methods are described in the
examples section of this disclosure.
[0060] In alternative embodiments, a phage display antibody may be
employed, methods for the production of which are well known (see,
e.g., Scott et al. Science 1990 249: 386; Devlin et al., Science
1990 249: 404; U.S. Pat. Nos. 5,223,409, 5,733,731, 5,498,530,
5,432,018, 5,338,665, and 5,922,545, for example).
[0061] Any known GPCR is suitable for use in the subject methods. A
disclosure of the sequences and phylogenetic relationships between
277 GPCRs is provided in Joost et al. (Genome Biol. 2002
3:RESEARCH0063, the entire contents of which is incorporated by
reference) and, as such, at least 277 GPCRs are suitable for the
subject methods. A more recent disclosure of the sequences and
phylogenetic relationships between 367 human and 392 mouse GPCRs is
provided in Vassilatis et al. (Proc Natl Acad Sci 2003 100:4903-8
and www.primalinc.com, each of which is hereby incorporated by
reference in its entirety) and, as such, at least 367 human and at
least 392 mouse GPCRs are suitable for the subject methods. GPCR
families are also described in Fredriksson et al (Mol. Pharmacol.
2003 63, 1256-72). Since the amino acid sequences of many GPCRs are
available and their code sequences are known, the subject antibody
may be made for any GPCR. For example, a human, mouse, rat, insect
or plant GPCR may be used in the subject method.
[0062] The methods may be used, by way of exemplification, for
purinergic receptors, vitamin receptors, lipid receptors, peptide
hormone receptors, non-hormone peptide receptors, non-peptide
hormone receptors, polypeptide receptors, protease receptors,
receptors for sensory signal mediator, and biogenic amine receptors
not including .beta.2-adrenergic receptor. In certain embodiments,
said biogenic amine receptor does not include an adrenoreceptor.
.alpha.-type adrenoreceptors (e.g. .alpha..sub.1A, .alpha..sub.1B
or .alpha..sub.1C adrenoreceptors) and .beta.-type adrenoreceptors
(e.g. .beta..sub.1, .beta..sub.2, or .beta..sub.3 adrenoreceptors)
are discussed in Singh et al., J. Cell Phys. 189:257-265, 2001.
[0063] It is recognized that both naturally occurring and altered
native (non-naturally occurring) GPCRs may be used in the subject
methods. In certain embodiments, therefore, an altered native GPCR
(e.g. a native GPCR that is altered by an amino acid substitution,
deletion and/or insertion) such that it binds the same ligand as a
corresponding native GPCR may be used in the subject methods.
[0064] As such, the following GPCRs (native or altered) find
particular use as parental GPCRs in the subject methods:
cholinergic receptor, muscarinic 3; melanin-concentrating hormone
receptor 2; cholinergic receptor, muscarinic 4; niacin receptor,
histamine 4 receptor; ghrelin receptor; CXCR3 chemokine receptor;
motilin receptor; 5-hydroxytryptamine (serotonin) receptor 2A;
5-hydroxytryptamine (serotonin) receptor 2B; 5-hydroxytryptamine
(serotonin) receptor 2C; dopamine receptor D3; dopamine receptor
D4; dopamine receptor D1; histamine receptor H2; histamine receptor
H3; galanin receptor 1; neuropeptide Y receptor Y1; angiotensin II
receptor 1; neurotensin receptor 1; melanocortin 4 receptor;
glucagon-like peptide 1 receptor; adenosine A1 receptor;
cannabinoid receptor 1; and melanin-concentrating hormone receptor
1.
[0065] In certain embodiments, the antibody is a monovalent
antibody that contains a single antigen binding region comprising
variable heavy and variable light domains. Such antibodies,
including Fab antibodies and scFv antibodies, may be produced from
a monoclonal antibody after it is identified. In one embodiment,
the monovalent antibody is an antibody containing the heavy and
light variable regions from antibody 5, which is described in the
examples section of this disclosure. Such antibodies include Fab
fragment of antibody 5 (i.e., the Fab5 antibody) or a scFv antibody
comprising the same variable domains as the Fab5 antibody, where
the heavy and light variable domains of the Fab5 antibody have the
following amino acid sequence:
TABLE-US-00001 Fab5 Light Chain: (SEQ ID NO: 1)
DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLI
YRANRLVDGVPSRFIGTGSGQDYSLITSSLDYEDMGIYYCLQYDEFPY
TFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDI
NVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSY
TCEATHKTSTSPIVKSFNRNEC Fab5 Heavy chain: (SEQ ID NO: 2)
EVQLQQSGAELARPGASVKLSCKASGYIFTDYYINWVRQRTGQGFEWI
GEIYPGSGNIDYNERFKDKATLTADKSSSTAYMQLSSLTSEDSAVYFC
VRGFGYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKG
YFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSET
VTCNVAHPASSTKVDKKIVPRDCGC
[0066] In certain cases, a subject antibody may contain: a) a heavy
chain containing the CDR1, CDR2 and CDR3 regions of the heavy chain
of an above-described antibody, separated by framework sequence and
b) a light chain containing the CDR1, CDR2 and CDR3 regions of the
light chain of that antibody, separated by framework sequence.
[0067] Also provided is a complex containing the GPCR and a single
antigen binding region of the antibody. Such a complex may be
formed under antibody binding conditions described in Harlow and
Weir, supra. A crystal of the complex is also provided, and methods
of making of which are described in greater detail below.
[0068] Crystallization Methods
[0069] A crystallization method is also provided. The method
includes contacting a GPCR with a monovalent antibody that
specifically binds to a three dimensional epitope of the IC3 loop
of a GPCR under binding conditions to form a complex; and
crystallizing the complex.
[0070] A subject complex may be crystallized using any of a variety
of crystallization methods, many of which are reviewed in Caffrey
Membrane protein crystallization. J. Struct. Biol. 2003 142:108-32.
In general terms, the methods are lipid-based methods that include
adding lipid to the fusion protein prior to crystallization. Such
methods have previously been used to crystallize other membrane
proteins. Many of these methods including the exploit the
spontaneous self-assembling properties of lipids and detergent as
vesicles (vesicle-fusion method), discoidal micelles (bicelle
method), and liquid crystals or mesophases (in meso or cubic-phase
method). Lipidic cubic phases crystallization methods are described
in, for example: Landau et al, Lipidic cubic phases: a novel
concept for the crystallization of membrane proteins. Proc. Natl.
Acad. Sci. 1996 93:14532-5; Gouaux, It's not just a phase:
crystallization and X-ray structure determination of
bacteriorhodopsin in lipidic cubic phases. Structure. 1998 6:5-10;
Rummel et al, Lipidic Cubic Phases: New Matrices for the
Three-Dimensional Crystallization of Membrane Proteins. J. Struct.
Biol. 1998 121:82-91; and Nollert et al Lipidic cubic phases as
matrices for membrane protein crystallization Methods. 2004
34:348-53, which publications are incorporated by reference for
disclosure of those methods. Bicelle crystallization methods are
described in, for example: Faham et al Crystallization of
bacteriorhodopsin from bicelle formulations at room temperature.
Protein Sci. 2005 14:836-40. 2005 and Faham et al, Bicelle
crystallization: a new method for crystallizing membrane proteins
yields a monomeric bacteriorhodopsin structure. J Mol Biol. 2002
Feb. 8; 316(1):1-6, each of which are incorporated by reference for
disclosure of those methods.
[0071] Also provided is a method of determining a crystal
structure. This method may so comprise receiving an above described
complex, crystallizing the complex to produce a crystal, and
obtaining atomic coordinates of the GPCR from the crystal. The
complex may be received from a remote location (e.g., a different
laboratory in the same building or campus, or from a different
campus or city), and, in certain embodiments, the method may also
comprise transmitting the atomic coordinates, e.g., by mail, e-mail
or, using the internet, to the remote location or to a third
party.
[0072] In other embodiments, the method may comprise forwarding a
complex to a remote location, where the complex may be crystallized
and analyzed, and receiving the atomic coordinates of the GPCR.
[0073] In certain cases, the GPCR to be crystallized may be a GPCR
that has been modified to contain a three dimensional epitope from
the IC3 loop from a different GPCR (e.g., the .beta.2 GPCR). In
these embodiments, once a suitable antibody has been identified for
a first GPCR, the antigen binding region for that antibody can be
swapped from the first GPCR into a second GPCR to make a chimeric
GPCR. The chimeric GPCR can then be bound to a monovalent antibody,
and crystallized using a method described herein. In one
embodiment, the entire IC3 loop, including the three dimensional
epitope contained in the loop, may be grafted from one GPCR to
another to produce an active GPCR. Such methods have been
successfully performed by others, e.g., Kim et al (Biochemistry
2005 44:2284-92); Yamashita et al (J. Biol. Chem. 2000
275:34272-9); Geiser et at (Protein Sci. 2006 15:1679-90); Tumova
et al (J. Biol. Chem. 2003 278:8146-53); Wong et al (J. Biol. Chem.
1990 265:6219-24) and Wess et al (FEBS Lett. 1989 258:133-6).
[0074] The IC3 region of a GPCR lies in between transmembrane
regions TM5 and TM6 and, may be about 12 amino acids (CXCR3 and
GPR40) to about 235 amino acids (cholinergic receptor, muscarinic
3) in length, for example. The TM5, IC3, and TM6 regions are
readily discernable by one of skill in the art using, for example,
a program for identifying transmembrane regions; once transmembrane
regions TM5 and TM6 regions are identified, the IC3 region will be
apparent. The TM5, IC3, and TM6 regions may also be identified
using such methods as pairwise or multiple sequence alignment (e.g.
using the GAP or BESTFIT of the University of Wisconsin's GCG
program, or CLUSTAL alignment programs, Higgins et al., Gene. 1988
73:237-44), using a target GPCR and, for example, GPCRs of known
structure.
[0075] Suitable programs for identifying transmembrane regions
include those described by Moller et al., (Bioinformatics,
17:646-653, 2001). A particularly suitable program is called
"TMHMM" Krogh et al., (Journal of Molecular Biology, 305:567-580,
2001). To use these programs via a user interface, a sequence
corresponding to a GPCR or a fragment thereof is entered into the
user interface and the program run. Such programs are currently
available over the world wide web, for example at the website of
the Center for Biological Sequence Analysis at
cbs.dtu.dk/services/. The output of these programs may be variable
in terms its format, however they usually indicate transmembrane
regions of a GPCR using amino acid coordinates of a GPCR.
[0076] When TM regions of a GPCR polypeptide are determined using
TMHMM, the prototypical GPCR profile is usually obtained: an
N-terminus that is extracellular, followed by a segment comprising
seven TM regions, and further followed by a C-terminus that is
intracellular. TM numbering for this prototypical GPCR profile
begins with the most N-terminally disposed TM region (TM1) and
concludes with the most C-terminally disposed TM region (TM7).
[0077] Accordingly, in certain embodiments, the amino acid
coordinates of the TM5, IC-3, and TM6 regions of a GPCR are
identified by a suitable method such as TMHMM.
[0078] In certain cases, once the TM5-IC3-TM6 segment is identified
for a GPCR, a suitable region of amino acids is chosen for
substitution with the amino acid sequence of an IC3 region of
another GPCR, e.g. the .beta.2 GPCR. In certain embodiments, the
substituted region may be identified using conserved or
semi-conserved amino acids in the TM5 and TM6 transmembrane
regions.
[0079] For GPCRs that contain no conserved proline residues in TM5
and TM6, positions for inserting a different IC3 loop may be based
on two considerations: a) alignment of the sequence of the GPCR
with receptor members of the same subfamily (which contained
conserved proline residues in TM5 or TM6; b) by identifying the
juxtaposition to the TM5/TM6 regions by hydrophobicity
analysis.
[0080] In addition to substituting IC3 region of a GPCR with a
stable, folded protein insertion, as described above, in certain
cases, the C-terminal region of the GPCR (which is C-terminal to
the cysteine palmitoylation site that is approximately 10 to 25
amino acid residues downstream of a conserved NPXXY motif), may be
deleted. In certain cases, the 20-30 amino acids immediately
C-terminal to the cysteine palmitoylation site are not deleted.
[0081] In one embodiment, a region containing the binding region of
antibody 5 described below (i.e., a region containing nine amino
acids at the amino-terminal end of the .beta.2AR IC3 loop
(I233-V242), having the amino acid sequence IDKSEGRFHV; SEQ ID NO:3
and also containing L266 and/or K270 from the C-terminal end of the
.beta.2AR IC3 loop) may be substituted into a different
(non-(.beta.2AR) GPCR at the same position, to produce a chimeric
GPCR. That chimeric GPCR may be complexed with a monovalent version
of antibody 5 (e.g., a Fab fragment of antibody 5 or a scFv
antibody) to make a complex that can be crystallized. In certain
embodiments, the antibody 5 binding site sequences may be
transferred along with amino acids that flank the binding site
(e.g., up to 2, 3, 4, or 5 amino acids from each side). In
particular embodiments, the entire IC3 region of the non-.beta.2AR
GPCR may be substituted with the IC3 region of PAR. In certain
cases, the transferred may contain I233 to K270 of .beta.2AR GPCR,
and may contain amino acids that flank that sequence (e.g., up to
2, 3, 4, or 5 amino acids from each side). In other cases, two
regions may be transferred, one containing the amino acid sequence
of I233 to V242, and the other containing the amino acid sequence
of L266 to K270. These sequences can be transferred to the
N-terminus and the C-terminus of the recipient GPCR's IC3,
respectively.
[0082] Thus, a chimeric GPCR used in the method may contain a) an
IC3 loop of one GPCR (e.g., the .beta.2 GPCR), and b) TM1-TM7, IC1
and IC2 regions of another GPCR.
[0083] Methods of Treatment
[0084] Certain of the above-described antibodies may have
therapeutic utility and may be administered to a subject having a
condition in order to treat the subject for the condition. The
therapeutic utility for an antibody may be determined by the GPCR
to which the antibody binds in that signaling via that GPCR is
linked to the condition. In certain cases, the GPCR may be
activated in the condition via by binding by a ligand. In other
embodiments, the GPCR may be mutated to make it constitutively
active, for example. A subject antibody may be employed for the
treatment of a GPCR-mediated condition such as schizophrenia,
migraine headache, reflux, asthma, bronchospasm, prostatic
hypertrophy, ulcers, epilepsy, angina, allergy, rhinitis, cancer
e.g. prostate cancer, glaucoma and stroke. Further exemplary
GPCR-related conditions at the On-line Mendelian Inheritance in Man
database found at the world wide website of the NCBI.
[0085] In certain embodiments as noted above, the antibody may bind
to the .beta.2-adrenoreceptor, in which case it may be employed in
the treatment of a condition requiring relaxation of smooth muscle
of the uterus or vascular system. Such an antibody may be thus used
for the prevention or alleviation of premature labour pains in
pregnancy, or in the treatment of chronic or acute asthma,
urticaria, psoriasis, rhinitis, allergic conjunctivitis, actinitis,
hay fever, or mastocytosis, which conditions have been linked to
the .beta.2-adrenoreceptor. In these embodiments, the antibody may
be employed as co-therapeutic agents for use in combination with
other drug substances such as anti-inflammatory, bronchodilatory or
antihistamine drug substances, particularly in the treatment of
obstructive or inflammatory airways diseases such as those
mentioned hereinbefore, for example as potentiators of therapeutic
activity of such drugs or as a means of reducing required dosaging
or potential side effects of such drugs. A subject antibody may be
mixed with the other drug substance in a fixed pharmaceutical
composition or it may be administered separately, before,
simultaneously with or after the other drug substance. Such
anti-inflammatory drugs include steroids, in particular
glucocorticosteroids such as budesonide, beclamethasone,
fluticasone, ciclesonide or mometasone or steroids described in WO
0200679 especially those of Examples 3, 11, 14, 17, 19, 26, 34, 37,
39, 51, 60, 67, 72, 73, 90, 99 and 101, LTB4 antagonists such as
those described in U.S. Pat. No. 5,451,700, LTD4 antagonists such
as montelukast and zafirlukast, and PDE4 inhibitors such as
Ariflo.RTM. (GlaxoSmith Kline), Roflumilast (Byk Gulden), V-11294A
(Napp), BAY19-8004 (Bayer), SCH-351591 (Schering-Plough),
Arofylline (Almirall Prodesfarma), PD189659 (Parke-Davis),
AWD-12-281 (Asta Medica), CDC-801 (Celgene) and KW-4490 (Kyowa
Hakko Kogyo) and A2a agonists such as those described in EP
1052264, EP 1241176, WO 0023457, WO0077018, WO 0123399, WO 0160835,
WO 0194368, WO 0200676, WO 0222630, WO 0296462, WO 0127130, WO
0127131, WO 9602543, WO 9602553, WO 9828319, WO 9924449, WO
9924450, WO 9924451, WO 9938877, WO 9941267, WO 9967263, WO
9967264, WO 9967265, WO 9967266, WO 9417090, EP 409595A2 and WO
0078774 and A2b antagonists such as those described in WO 02/42298.
Such bronchodilatory drugs include anticholinergic or
antimuscarinic agents, in particular ipratropium bromide,
oxitropium bromide and tiotropium bromide. In general terms, these
protocols involve administering to an individual suffering from a
GPCR-related disorder an effective amount of an antibody that
modulates a GPCR to modulate the GPCR in the host and treat the
individual for the disorder.
[0086] In some embodiments, where a reduction in activity of a
certain GPCR is desired, one or more compounds that decrease the
activity of the GPCR may be administered, whereas when an increase
in activity of a certain GPCR is desired, one or more compounds
that increase the activity of the GPCR activity may be
administered.
[0087] A variety of individuals are treatable according to the
subject methods. Generally such individuals are mammals or
mammalian, where these terms are used broadly to describe organisms
which are within the class mammalia, including the orders carnivore
(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and
rats), and primates (e.g., humans, chimpanzees, and monkeys). In
many embodiments, the individuals will be humans. Subject treatment
methods are typically performed on individuals with such disorders
or on individuals with a desire to avoid contracting such
disorders.
[0088] A variety of publications describe treatment regimens using
the antibodies including, for example, published patent
applications 20080159981, 20080102069, 20070142346, 20060018899,
20050148607 and 20030228663, as well as U.S. Pat. Nos. 7,306,801,
6,399,063, 6,387,371 and 6,165,464, which are each incorporated by
reference in their entirety. In general terms, these documents
described methods including administering a therapeutic monoclonal
antibody to a subject, singly or in combination with another agent.
The subject may then be monitored for a clinically beneficial
response, where a beneficial response to the antibody can be
assessed according to whether an individual patient experiences a
desirable change in disease status.
[0089] In particular embodiments, the antibody may have a
translocation sequence (e.g., an HIV TAT, AntP, or VP22
traslocation sequence, or other short sequence rich in basic amino
acids) which facilitates translocation of the antibody across the
plasma membrane into the cytoplasm of a target cell. Such
translocation sequences and their use are known and reviewed in,
for example, Gupta (Drug Deliv. Rev. 2005 57:637-51), Steffen
(Methods Mol. Biol. 2001 161:141-8) and Hansen (Scientific World
Journal. 2005 5:782-8).
[0090] In order to further illustrate the present invention, the
following specific examples are given with the understanding that
they are being offered to illustrate the present invention and
should not be construed in any way as limiting its scope.
Example 1
Antigen Production
[0091] Antigen was prepared by reconstituting purified, functional
.beta..sub.2AR at high density into phospholipid vesicles (1 mg
receptor per mg of phospholipids) using the following method.
[0092] .beta..sub.2AR having an amino terminal Flag epitope tag and
a carboxyl hexahistidine tag was expressed in Sf9 insects cells and
purified by sequential M1 antibody and alprenolol affinity
chromatography as previously described (Kobilka Anal Biochem 1995
231, 269-71). Purified .beta..sub.2AR was immobilized on a Ni
column and equilibrated with 10 column volumes of a mixture of 5
mg/ml DOPC (Avanti Polar Lipids) and 0.5 mg/ml Lipid A (Sigma) in
1% (w/v) octylglucoside (Anatrace), 100 mM NaCl, 20 mM Hepes pH 7.5
and 1 .mu.M carazolol (a .beta..sub.2AR antagonist). The
.beta..sub.2AR was then eluted in the same buffer containing 200 mM
imidazole. The concentration of the eluted .beta..sub.2AR was
adjusted to 5 mg/ml. This usually involved diluting the protein
with the same buffer, but occasionally required concentrating the
protein up to two-fold with an Amicon ultrafiltration cell (100 kDa
pore). The protein was then dialyzed against phosphate buffered
saline containing 1 .mu.M carazolol at 4.degree. C. to remove
detergent. The reconstituted protein was stored at -80.degree. C.
prior to use for immunization.
[0093] The phospholipid environment ensures the functional
integrity of the protein after injection into mice. To provide
additional stability, the .beta..sub.2AR was liganded with
carazolol, a high affinity inverse agonist. To facilitate the
immune response, phospholipid vesicles consisted of a 10:1 mixture
(by weight) of DOPC and the adjuvant Lipid A. The generated
vesicles contained randomly oriented .beta..sub.2AR, so that both
cytoplasmic and extracellular domains are presented to immune
cells.
Example 2
Antibody Production
[0094] Monoclonal antibodies (MABs) were generated using a
conventional fusion protocol, as follows:
Myeloma Cell Lines
[0095] The P3x63Ag8.653 myeloma cell line (ATCC # CRL-1580; Lot No.
1131010) was used for the fusion experiment performed with
splenocytes from immunized mice.
Cell Culture
[0096] Myeloma cells were propagated in Dulbecco's Modified Eagle's
Medium (DMEM-high glucose) supplemented with 10% Fetal Bovine Serum
(FBS), 0.15 g/L oxaloacetate, 0.05 g/L pyruvate, 0.0082 g/L bovine
insulin (OPI supplement), 10% NCTC 109 medium and 4 mM L-glutamine.
This formulation is referred to the SDMEM formulation. Newly formed
hybridoma were selected in SDMEM supplemented with HAT mixture
(1.0.times.10.sup.-4 M Hypoxanthine, 4.0.times.10.sup.-6 M
Aminopterin, 1.6.times.10.sup.-5M thymidine), 5% hybridoma cloning
factor, gentamicin (50 .mu.g/ml) and .beta.-mercaptoethanol at
0.055 mM. This formulation is referred as SDMEM-HAT. After
selection, the newly formed hybridomas were propagated in SDMEM
supplemented with 2% hybridoma cloning factor HT mixture
(1.0.times.10.sup.-4 M hypoxanthine, 1.6.times.10.sup.-5 M
thymidine). This formulation is referred as DMEM-HT. For in vitro
antibody production, the hybridomas were propagated in SDMEM
supplemented with 1% hybridoma cloning factor and 5-10% low-IgG
FBS.
Cell Fusion Protocol
[0097] Balb/c mice were immunized weekly with 30 .mu.g of
.beta..sub.2AR in liposomes. The mice were injected
intraperitoneally with 15 .mu.g of antigen and subcutaneously at 2
sites with 7.5 mg of antigen. Final boosts of 10 .mu.g of
.beta..sub.2AR were given intraperitoneally and intravenously on
day 3 and 4 before fusion. Spleens were harvested aseptically,
placed in cold DMEM, diced and homogenized. The homogenate was
centrifuged at 1000 rpm for 5 minutes. The cell pellet was
incubated with 5 ml of Red cell lysis buffer (Sigma R7757) for 3-5
mins. DMEM (20 ml) was added and the cells were centrifuged at 1000
rpm for 5 minutes. The pellet was rinsed twice in 20 ml DMEM. The
final pellet was resuspended in 30 mL DMEM. Spleen and myeloma
cells were combined at a 3:1 ratio, centrifuged (1000 rpm) and
resuspend in DMEM. The centrifugation was repeated and 1 mL
PEG-1500 (50% solution) was added to the cell pellet over 3 min
with gentle mixing. 4 mL of SDMEM was slowly added to cells over 2
min. After 1 min incubation, 20 mL of SDMEM was added over 1 min.
The suspension was centrifuge (1000 rpm) and the pellet was
resuspended in a total of 30 mL HAT-SDMEM and incubate at
37.degree. C. for 2 hours. HAT-SDMEM media was added to achieve a
cell density of 5.times.10.sup.4 cells/well and dispensed into
96-well plates at 200 .mu.L per well. Cells were incubated for 11
days before screening media for antibody production.
[0098] Fusions from two mice yielded seventeen hybridoma clones
that produced antibody against .beta..sub.2AR as determined by an
ELISA assay on immobilized phospholipid vesicles containing
purified .beta..sub.2AR. Nine of these hybridomas produced
sufficient quantities of .beta..sub.2AR-specific antibody for
further characterization. The antibodies were characterized as
binding the intracellular or extracellular surface of the receptor
in immunofluorescence experiments with HEK-293 cells stably
expressing an N-terminally FLAG-tagged version of the
.beta..sub.2AR. Antibodies that recognize an intracellular epitope
only stain permeabilized cells. Staining of cells that were either
fixed or fixed and permeabilized indicated that five of the
antibodies bound to the intracellular face and four bound to the
extracellular surface (FIG. 4).
Example 3
Antibody Screening
[0099] Antibodies were screened for binding to a three dimensional
epitope on the three-dimensional surface on the IC3 of the native
.beta..sub.2AR, rather than a flexible linear epitope. Nine MABs
were screened, along with positive (M1 antibody) and negative
(9E10) controls, for binding to .beta..sub.2AR denatured with
sodium dodecyl sulfate (SDS) and spotted on nitrocellulose (FIG.
2A). Antibody 5 and antibody 9 showed the weakest binding to
denatured protein, even though they showed immunostaining
comparable to M1 on binding to native receptor in fixed cells (see
FIG. 4). The reduced binding of antibody 5 and antibody 9 to SDS
denatured .beta..sub.2AR showed that these antibodies bind to a
three-dimensional epitope.
[0100] Antibodies 2, 5, 6, 7 and 9 all reacted with intracellular
epitopes. To select for antibodies that may interact with IC3, we
examined the effect of antibody binding on the fluorescence of
.beta..sub.2AR labeled at C265 (at the cytoplasmic end of TM6) with
tetramethylrhodamine. Tetramethylrhodamine bound to C265 is
predicted to lie at the interface between TM5 and TM6. Previous
studies have shown that tetramethylrhodamine binding to C265 is
sensitive to ligand-induced three dimensional changes in the
.beta..sub.2AR (Swaminath et al, J. Biol. Chem. 2004 279: 686-691).
As such, antibodies binding to IC3 may stabilize a specific
conformation of TM5 relative to TM6 that would be detected by a
change in tetramethylrhodamine fluorescence. It can be seen that
antibody 5 induced the largest fluorescence response and had the
highest affinity for the .beta..sub.2AR (FIG. 2B). The fluorescence
change induced by antibody 5 was significantly greater than the
response to antibody 9, the other intracellular binder that reacted
weakly to SDS denatured receptor. It is notable that there was a
response to antibody 8 and antibody 4, two antibodies that bind to
an extracellular epitope. This suggests that binding of these
antibodies to the extracellular surface of the .beta..sub.2AR
influences the structure around the cytoplasmic end of TM6.
[0101] Based on the results of assays shown in FIGS. 2A and 2B
antibody 5 was chosen for crystallography experiments. Antibody 5
bound to an intracellular epitope, it exhibited relatively high
affinity for native .beta..sub.2AR (FIG. 2B), but bound weakly to
SDS denatured protein (FIG. 2A). Antibody 5 also induced the
largest fluorescence response in .beta..sub.2AR labeled at C265
with tetramethylrhodamine. Based on these experiments, antibody 5
binds to a three dimensional epitope on the native third
intracellular loop.
Example 4
Antibody Characterization
[0102] Fab 5 fragments were generated from purified antibody 5 by
papain cleavage and purified by ion exchange chromatography. The
dissociation constant for Fab 5 binding to purified .beta..sub.2AR
was determined to be 150 nM by isothermal titration calorimetry
(FIG. 5). As expected, this value is higher than the EC50 (23 nM)
observed for the intact antibody in the fluorescence experiments
(FIG. 2B).
[0103] To localize the intracellular epitope of the .beta..sub.2AR
that interacts with Fab 5, limited tryptic digestions of purified
.beta..sub.2AR were performed. Cleavage of purified .beta..sub.2AR
with trypsin yielded two bands with molecular weights of
approximately 27 and 29 kDa on a Western blot probed with the M1
anti-FLAG antibody (FIG. 3A). The sizes of these fragments indicate
trypsin digestion at two of ten potential sites in IC3 (FIG. 3A).
When preincubated with Fab 5, the 27 kDa band disappears suggesting
that the antibody binds to one of the amino-terminal trypsin sites
in IC3 (FIG. 3A).
[0104] The effect of Fab binding on ligand binding properties and
on agonist-induced three dimensional changes was determined. Fab 5
had no significant effect on antagonist or agonist binding affinity
(Table 1 below).
TABLE-US-00002 TABLE 1 Antagonist and agonist binding properties of
the .beta..sub.2AR with and without prebound Fab 5.
[.sup.3H]dihydroalprenolol Isoproterenol K.sub.d .+-. S.E. K.sub.i
[S.E. interval] (nM) (nM) .beta.2AR 1.02 .+-. 0.09 290 [269-311]
.beta.2AR-Fab5 1.08 .+-. 0.05 329 [310-348] Saturation and
competition binding assays were performed on purified
.beta..sub.2AR reconstituted in phospholipid vesicles in the
absence and presence of prebound Fab 5. Values are from three
independent experiments performed in triplicate. The IC.sub.50
values used for calculations of K.sub.i values were obtained from
means of pIC.sub.50 values determined by nonlinear regression
analysis and the S.E. interval from pIC.sub.50 .+-. S.E.
[0105] IC3 is known to be important for G protein activation. Fab 5
prevented coupling of purified .beta..sub.2AR to purified G protein
(data not shown), most likely due to steric competition. However,
it is possible that Fab 5 restricts the conformational changes
associated with receptor activation. The effect of Fab binding on
agonist-induced conformational changes was determined using a
fluorescence-based assay (Yao et al. Nat. Chem. Biol. 2006 2,
417-422).
[0106] Purified receptor was reacted with 1:1 equivalent of
monobromobimane (mBBr, Invitrogen) in buffer A (100 mM NaCl, 20 mM
HEPES, pH 7.5, 0.1% dodecyl maltoside) and incubated overnight on
ice in the dark. The fluorophore-labeled receptor was purified
right before use by gel filtration on a desalting column
equilibrated with buffer B. Fluorescence spectroscopy experiments
were performed on a Spex FluoroMax-3 spectrofluorometer (Jobin Yvon
Inc, NJ) with photon counting mode by using an excitation and
emission bandpass of 4 nm. All experiments were performed at
25.degree. C. For emission scans, excitation was set at 370 nm and
emission was measured from 430-530 nm with an integration time of 1
s/nm. To determine the effect of Fab5 antibody and drug, three
individual labeled protein samples were incubated with antibody (1
.mu.M Fab5) or 100 .mu.M Isoproterenol or both. Emission spectra of
the samples were taken after 15 minutes incubation. Fluorescence
intensity was corrected for background fluorescence from buffer and
ligands in all experiments. The data are the mean.+-.S.E. of two
independent experiments performed in triplicate.
[0107] Agonist binding induces a change that brings the fluorophore
monobromobimane bound to C271 at the cytoplasmic end of TM6 closer
to W135 at the cytoplasmic end of TM3 resulting in a decrease in
bimane fluorescence. This fluorescence change was not affected by
binding of antibody 5 (FIG. 3B). In conclusion, while Fab 5
required a native .beta..sub.2AR to bind, it did not restrict the
movement of transmembrane segments involved in ligand binding and
agonist activation.
[0108] To more easily identify crystals of the Fab 5-.beta..sub.2AR
complex, we labeled purified .beta..sub.2AR at C265 with
tetramethylrhodamine (.beta..sub.2AR-TMR). The Fab
5-.beta..sub.2AR-TMR complex was formed by mixing
.beta..sub.2AR-TMR with a stoichiometric excess of Fab 5 and
isolating the complex by size exclusion chromatography. Small
fluorescent crystals formed by vapor phase diffusion using ammonium
sulfate as the precipitant (FIG. 6). Importantly, no crystals
formed from .beta..sub.2AR alone or from Fab 5 alone under these
conditions, showing that the additional protein interactions and
the stabilizing effects of the antibody were critical for
successful crystallization of the .beta..sub.2AR. Further
refinement of the crystallography conditions has since produced
diffraction quality crystals and a 3.4 .ANG. structure of the Fab
5-.beta..sub.2AR complex.
Example 5
Preparation of Fab Fragments
[0109] Monoclonal mouse immunoglobulins against the .beta.2AR
reconstituted in liposomes were raised, as described above. Mab5
IgGs from mouse hybridoma cell culture supernatant were purified
using a Protein G column (Pierce). Fab5 fragments were generated by
papain proteolysis (Worthington) and purified by Mono Q
chromatography. The fragments were concentrated to .about.100 mg
ml.sup.-1 with a Millipore concentrator (5 kDa molecular weight cut
off) in a solution of 10 mM HEPES pH 7.5 and 100 mM NaCl.
Example 6
Preparation of .beta.2AR365-Fab5 Complexes
[0110] The .beta..sub.2AR365 was expressed in Sf-9 insect cells
infected with .beta..sub.2AR365 baculovirus, and solubilized
according to previously described methods. Functional protein was
obtained by M1 FLAG affinity chromatography (Sigma) prior to and
following alprenolol-Sepharose chromatography. Receptor bound
alprenolol was exchanged for carazolol on the second M1 resin.
N-linked glycolsylations were removed by treatment with PNGaseF
(NEB), and the FLAG epitope was removed by treatment with AcTEV
protease (Invitrogen). Protein was concentrated to .about.50 mg/ml
with a 100 kDa molecular weight cut off Vivaspin concentrator
(Vivascience) and mixed in stoichiometric excess of Fab5. The
complex was purified on a Superdex-200 (10/300GL) column in a
solution of 10 mM HEPES pH 7.5, 100 mM NaCl, 0.1% dodecylmaltoside,
and 10 .mu.M carazolol. The purified .beta..sub.2AR365-Fab5
complexes were concentrated to .about.60 mg ml.sup.-1 using a
Vivaspin concentrator.
Example 7
Crystallization
[0111] The .beta..sub.2AR365-Fab5 complexes were mixed with
bicelles (10% w/v 3:1 DMPC:CHAPSO in 10 mM HEPES pH 7.5, 100 mM
NaCl) at a 1:5 (protein:bicelle) ratio, and crystals were grown in
sitting- and hanging-drop formats at 22.degree. C. using equal
volumes of protein mixture and reservoir solutions. Initial
crystallization leads were identified using multiple 96-well
sitting-drop screens from Nextal (Qiagen). After extensive
optimization, crystals for data collection were grown in
hanging-drop format over a reservoir solution of to 1.85-2.0 M
ammonium sulfate, 180 mM sodium acetate, 5 mM EDTA, 100 mM MES or
HEPES pH 6.5-7.5. Crystals grew to full size within 7 to 10 days.
Crystals were flash frozen and stored in liquid nitrogen, with
reservoir solution plus 20% glycerol as cryoprotectant.
Example 8
Microcrystallography Data Collection and Processing
[0112] Microbeams were employed for data collection. The shape of
the crystals permitted complete data to be measured from a single
crystal. A small wedge of data, typically 5-10.degree., (1.degree.
per frame) could be measured before significant radiation damage
was observed. The crystal was then translated to a new, undamaged
position to collect the next wedge of data. A total of 182.degree.
of data collected in this manner, measured at beamline ID23-2 of
the ESRF, were used for the final .beta..sub.2AR365-Fab5 data set
(Table 2). The .beta..sub.2AR24/365-Fab5 data set was obtained from
225.degree. of data measured on beamline 23ID-B of the APS (Table
2).
TABLE-US-00003 TABLE 2 X-ray data collection and refinement
statistics .beta..sub.2AR365-Fab5 .beta..sub.2AR24/365-Fab5 Data
collection Space group C2 C2 Cell dimensions a, b, c (.ANG.) 338.4,
48.5, 89.4 338.4, 48.5, 89.4 .alpha., .beta., .gamma. (.degree.)
90., 104.6, 90. 90., 104.6, 90. Resolution (.ANG.) 86.4.-3.4
(3.49-3.40) * 50-3.4 (3.52-3.40) * R.sub.merge 0.117 (0.407) 0.120
(0.456) I/.sigma.I 9.9 (2.3) 7.8 (2.6) Completeness (%) 98.9 (94.9)
99.4 (98.4) Multiplicity 3.3 (2.9) 4.1 (3.4) Refinement Resolution
(.ANG.) 20.-3.4 20.-3.4 No. reflections work/free 17658/1902
17458/1886 R.sub.work/R.sub.free 0.216/0.269 0.226/0.279 No. atoms
4905 4887 Average B values (.ANG..sup.2) Receptor 156. 187. Fab5
67. 91. Overall anisotropic B (.ANG..sup.2)
B.sub.11/B.sub.22/B.sub.33/B.sub.13 -27.1/31.3/-4.2/4.4
-16.8/20.4/-3.5/12.4 R.m.s deviations Bond lengths (.ANG.) 0.007
0.008 Bond angles (.degree.) 1.4 1.5 Ramachandran plot **
receptor/Fab5 % most favored 76.3/71.5 75.8/71.8 allowed 22.1/27.2
22.1/26.6 generously allowed 1.6/1.3 2.1/1.6 disallowed 0.0/0.0
0.0/0.0 * Highest resolution shell is shown in parenthesis. ** As
defined in PROCHECK
[0113] ESRF data were processed with MOSFLM and SCALA
(Collaborative Computational Project, N. Acta Cryst 1994 D50,
760-763) and data measured at the APS were processed with HKL2000
(Otwinowski et al, Methods Enzymol. 1997 276, 307-326). In many
cases it was necessary to reindex the crystal after moving to a new
position on the crystal, which may have been due to bending of the
frozen crystals such that the indexing matrix from the previous
volume could not accurately predict the diffraction pattern from a
new volume. This problem precluded global postrefinement of the
unit cell parameters. The unit cell parameters used for subsequent
analysis (Table 1) were obtained from initial indexing and
refinement from one wedge of the ESRF data, and were subsequently
found to be sufficient for processing the remaining data without
unit cell constant refinement. Using a partial specific volume of
1.21 .ANG..sup.3/Da for protein, the unit cell would have 66%
lipid, detergent and aqueous solvent for is one .beta..sub.2AR-Fab5
complex in the asymmetric unit. The structure of the
.beta..sub.2AR365-Fab5 complex was solved by molecular replacement,
by searching with separate constant and variable domain models
against a low resolution (4.1 .ANG.) data set measured at ESRF
beamline ID-13.
[0114] The crystal structure confirms that Fab 5 binds to a three
dimensional epitope consisting of nine amino acids at the
amino-terminal end of IC3 (I233-V242). The antibody also binds two
amino acids at the carboxyl-terminal end (L266 and K270), but at a
different region of the antibody. FIG. 7 illustrating packing of
.beta.2AR-Fab complex in a crystal, and FIG. 8 schematically
illustrates the three-dimensional epitope to which the Fab 5
antibody binds.
Sequence CWU 1
1
31214PRTmus musculisUNSURE(0)...(0)Fab5 light chain 1Asp Ile Lys
Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly1 5 10 15Glu Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30Leu
Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40
45Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ile Gly
50 55 60Thr Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp
Tyr65 70 75 80Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu
Phe Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
Ala Asp Ala Ala 100 105 110Pro Thr Val Ser Ile Phe Pro Pro Ser Ser
Glu Gln Leu Thr Ser Gly 115 120 125Gly Ala Ser Val Val Cys Phe Leu
Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140Asn Val Lys Trp Lys Ile
Asp Gly Ser Glu Arg Gln Asn Gly Val Leu145 150 155 160Asn Ser Trp
Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175Ser
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185
190Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205Phe Asn Arg Asn Glu Cys 2102217PRTmus
musculisUNSURE(0)...(0)Fab 5 Heavy chain 2Glu Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser
Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr 20 25 30Tyr Ile Asn Trp
Val Arg Gln Arg Thr Gly Gln Gly Phe Glu Trp Ile 35 40 45Gly Glu Ile
Tyr Pro Gly Ser Gly Asn Ile Asp Tyr Asn Glu Arg Phe 50 55 60Lys Asp
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95Val Arg Gly Phe Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
Ser 100 105 110Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala
Pro Gly Ser 115 120 125Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly
Cys Leu Val Lys Gly 130 135 140Tyr Phe Pro Glu Pro Val Thr Val Thr
Trp Asn Ser Gly Ser Leu Ser145 150 155 160Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr 165 170 175Leu Ser Ser Ser
Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr 180 185 190Val Thr
Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys 195 200
205Lys Ile Val Pro Arg Asp Cys Gly Cys 210 215310PRThomo sapien
3Ile Asp Lys Ser Glu Gly Arg Phe His Val1 5 10
* * * * *
References