U.S. patent application number 13/374202 was filed with the patent office on 2012-05-17 for constructs and libraries comprising antibody surrogate light chain sequences.
Invention is credited to Lawrence Horowitz, Bhatt Ramesh, Li Xu.
Application Number | 20120123098 13/374202 |
Document ID | / |
Family ID | 39765747 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120123098 |
Kind Code |
A1 |
Ramesh; Bhatt ; et
al. |
May 17, 2012 |
Constructs and libraries comprising antibody surrogate light chain
sequences
Abstract
The invention concerns constructs and libraries comprising
antibody surrogate light chain sequences. In particular, the
invention concerns constructs comprising VpreB sequences,
optionally partnered with another polypeptide, such as, for
example, antibody heavy chain variable domain sequences, and
libraries containing the same.
Inventors: |
Ramesh; Bhatt; (Belmont,
CA) ; Horowitz; Lawrence; (Atherton, CA) ; Xu;
Li; (Cupertino, CA) |
Family ID: |
39765747 |
Appl. No.: |
13/374202 |
Filed: |
December 14, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12056151 |
Mar 26, 2008 |
8114967 |
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13374202 |
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60920568 |
Mar 27, 2007 |
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Current U.S.
Class: |
530/387.3 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 2317/34 20130101; C07K 16/005 20130101; C07K 16/22 20130101;
C07K 2319/00 20130101; C07K 2317/52 20130101; C40B 40/10 20130101;
C07K 16/1018 20130101; C07K 16/00 20130101; C07K 2317/56 20130101;
C07K 2317/622 20130101; C07K 16/241 20130101 |
Class at
Publication: |
530/387.3 |
International
Class: |
C07K 19/00 20060101
C07K019/00 |
Claims
1-88. (canceled)
89. A polypeptide comprising a fusion of a VpreB1 sequence of SEQ
ID NO: 1, or a fragment thereof, or a variant thereof having at
least about 70% sequence identity to SEQ ID NO: 1, directly fused
to a .lamda.5 sequence of SEQ ID NO: 6, or a fragment thereof, or a
variant thereof having at least about 85% sequence identity to SEQ
ID NO: 6, conjugated to an antibody heavy chain sequence comprising
a variable region, wherein said conjugate specifically binds to a
target.
90. The polypeptide of claim 89 wherein said VpreB1 sequence is
fused at its C-terminus to the N-terminus of said .lamda.5
sequence.
91. The polypeptide of claim 89 wherein the fusion takes place at
or around the CDR3 analogous regions of said VpreB1 sequence and
.lamda.5 sequence, respectively.
92. The polypeptide of claim 89 wherein the VpreB1 fragment
comprises amino acid residues 20 to 115 of SEQ ID NO: 1.
93. The polypeptide of claim 89 or claim 92 wherein the .lamda.5
fragment comprises amino acid residues 87 to 105 of SEQ ID NO:
6.
94. The polypeptide of claim 89 comprising the fusion of SEQ ID NO:
10 or SEQ ID NO: 12.
95. The polypeptide of claim 89 comprising the VpreB1 sequence of
SEQ ID NO: 1 and the .lamda.5 sequence of SEQ ID NO: 6, wherein the
direct fusion take place between an amino acid residue from
position 116 to position 126 of SEQ ID NO: 1 and an amino acid
residue from position 82 to 93 of SEQ ID NO: 6.
96. The polypeptide of claim 95 wherein the direct fusion take
place at amino acid residue 121 of SEQ ID NO: 1.
97. The polypeptide of claim 89 wherein said fusion is conjugated
to said antibody heavy chain sequence by a peptide linker or by
non-covalent association to dorm a dimeric complex.
98. The polypeptide of claim 89 wherein said target is an
antigen.
99. The polypeptide of claim 98 wherein the antigen is a viral
antigen.
100. The polypeptide of claim 99 wherein the viral antigen is an
influenza virus.
101. The polypeptide of claim 100, wherein said polypeptide
neutralizes said influenza virus.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/920,568, filed Mar. 27, 2007, the entire
disclosure of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention concerns constructs and libraries
comprising antibody surrogate light chain sequences. In particular,
the invention concerns constructs comprising VpreB sequences,
optionally partnered with another polypeptide, such as, for
example, antibody heavy chain variable domain sequences, and
libraries containing the same.
BACKGROUND OF THE INVENTION
[0003] Antibody (Ig) molecules produced by B-lymphocytes are built
of heavy (H) and light (L) chains. The amino acid sequences of the
amino terminal domains of the H and L chains are variable (V.sub.H
and V.sub.L), especially at the three hypervariable regions (CDR1,
CDR2, CDR3) that form the antigen combining site. The assembly of
the H and L chains is stabilized by a disulfide bond between the
constant region of the L chain (C.sub.L) and the first constant
region of the heavy chain (C.sub.HI) and by non-covalent
interactions between the V.sub.H and V.sub.L domains.
[0004] In humans and many animals, such as mice, the genes encoding
the antibody H and L chains are assembled by stepwise somatic
rearrangements of gene fragments encoding parts of the V regions.
Various stages of B lymphocyte development are characterized by the
rearrangement status of the Ig gene loci (see, e.g. Melchers, F.
& Rolink, A., B-Lymphocyte Development and Biology, Paul, W.
E., ed., 1999, Lippincott, Philadelphia).
[0005] Precursors of B cells (pre-B cells) have been identified in
the bone marrow as lymphocytes that produce .mu. heavy chains but
instead of the fully developed light chains express a set of B
lineage-specific genes called VpreB(1-3) and .lamda.5,
respectively.
[0006] The main isoform of human VpreB1 (CAG30495) is a 145 aa-long
polypeptide (SEQ ID NO: 1). It has an Ig V domain-like structure,
but lacks the last .beta.-strand (.beta.7) of a typical V domain,
and has a carboxyl terminal end that shows no sequence homologies
to any other proteins. VpreB2 has several isoforms, including a
142-amino acid mouse VpreB2 polypeptide (P13373; SEQ ID NO: 2), and
a 171-amino acid long splice variant of the mouse VpreB2 sequence
(CAA019641 SEQ ID NO: 3). VpreB1 and VpreB2 sequences have been
disclosed in EP 0 269 127 and U.S. Pat. No. 5,182,205; Collins et
al., Genome Biol, 5(10):R84 (2004); and Hollins et al., Proc. Natl.
Acad. Sci. USA 86(14):5552-5556 (1989). The main isoform of human
VpreB3 (SEQ ID NO: 4) is a 123 amino acid long protein (CAG30496),
disclosed in Collins et al., Genome Biol. 5(10):R84 (2004).
[0007] VpreB(1-3) are non-covalently associated with another
protein, .lamda.5. The human .lamda.5 is a 209-amino acid
polypeptide (CAA01962; SEQ ID NO: 5), that carries an Ig C
domain-like structure with strong homologies to antibody light
chains and, towards its amino terminal end, two functionally
distinct regions, one of which shows strong homology to the .beta.7
strand of the V.lamda. domains. A human .lamda.5-like protein has
213 amino acids (NP.sub.--064455; SEQ ID NO: 6) and shows about 84%
sequence identity to the antibody .lamda. light chain constant
region.
[0008] For further details, see the following review papers:
Karasuyama et al., Adv. Immunol. 63:1-41 (1996); Melchers et al.,
Immunology Today 14:60-68 (1993); and Melchers, Proc. Natl. Acad.
Sci. USA 96:2571-2573 (1999).
[0009] The VpreB and .lamda.5 polypeptides together form a
non-covalently associated, Ig light chain-like structure, which is
called the surrogate light chain or pseudo light chain. On the
surface of early preB cells, the surrogate light chain is
disulfide-linked to membrane-bound Ig .mu. heavy chain in
association with a signal transducer CD79a/CD79b heterodimer to
form a B cell receptor-like structure, the so-called preB cell
receptor (preBCR).
SUMMARY OF THE INVENTION
[0010] In one aspect, the invention concerns polypeptides
comprising a VpreB sequence or a .lamda.5 sequence conjugated to a
heterogeneous amino acid sequence, wherein the polypeptides are
capable of binding to a target.
[0011] In a preferred embodiment; the polypeptide comprises a VpreB
sequence, where VpreB may be any native VpreB, including human
VpreB1 (SEQ ID NO: 1), mouse VpreB2 (SEQ ID NO: 2 and 3) and human
VpreB3 (SEQ ID NO: 4), or a homologue thereof in another mammalian
species, or a fragment or variant thereof, provided that the
polypeptide retains the ability to bind to a target.
[0012] In a preferred embodiment, the heterogeneous amino acid
sequence is a .lamda.5 sequence, which may be any native .lamda.5
sequence, or any fragment or variant thereof, including the native
human .lamda.5 sequence of SEQ ID NO: 5, the human .lamda.5-like
sequence of SEQ ID NO: 6, and fragments and variants thereof.
[0013] The VpreB sequence and the heterogeneous amino acid
sequence, e.g. the .lamda.5 sequence, may be directly fused to each
other, or may be non-covalently associated. In the former case, the
fusion preferably takes place at or around the CDR3 analogous
regions of VpreB and .lamda.5, respectively.
[0014] In another embodiment, the heterogeneous amino acid sequence
is or comprises an antibody light chain variable region sequence.
In a particular embodiment, the antibody light chain variable
region sequence is fused to the VpreB sequence at a site analogous
to an antibody light chain CDR3 region. In another embodiment, the
fusion is between the CDR3 region of an antibody light chain and
the CDR3 analogous region of a VpreB. In all embodiments, the
antibody light chain can be a .lamda. chain or a .kappa. chain.
[0015] In particular embodiments, the polypeptides herein,
including, without limitation, VpreB-.lamda.5 conjugates (including
fusions, other covalent linkage, and non-covalent associations),
and VpreB-antibody light chain conjugates, may be further
associated with a sequence comprising an antibody heavy chain
variable region sequence, such as an antibody heavy chain variable
region, or a complete antibody heavy chain, including a variable
region.
[0016] When the polypeptide comprises a .lamda.5 sequence, .lamda.5
may be any native .lamda.5, including human .lamda.5 of SEQ ID NO:
5 and human .lamda.5-like protein of SEQ ID NO: 6, or a homologue
in another mammalian species, or any fragment or variant thereof,
provided that the polypeptide retains the ability to bind to a
target. In a particular embodiment, the heterogeneous amino acid
sequence conjugated to the .lamda.5 sequence is a VpreB
sequence.
[0017] In the polypeptide constructs of the present invention, the
VpreB and .lamda.5 sequences, if both present, may be conjugates by
any means, including direct fusion, covalent linkage by a linker
sequence (e.g. a peptide linker), and non-covalent association.
[0018] In a particular embodiment, a fusion of a VpreB sequence and
a .lamda.5 sequence is conjugated to an antibody heavy chain
sequence by non-covalent association, to form a dimeric
complex.
[0019] In another embodiment, a trimeric complex is formed by
non-covalent association of a VpreB sequence, a .lamda.5 sequence
and an antibody heavy chain sequence. In certain embodiments, in
these structures, which are also referred to as variant surrogate
light chain structures of"SURROBODY.TM. variants," the
characteristic tails (appendages) of one or both of the VpreB and
.lamda.5 portions may be (but do not have to be) retained. It is
possible to attach additional functionalities to such appendages.
In addition, in various embodiments, beneficial appendage fusions
can be designed and made in order to improve various properties of
the constructs, such as PK and/or potency.
[0020] In all embodiments, when an antibody heavy chain comprising
variable region sequences is present, the polypeptide of the
present invention and the antibody heavy chain variable region
sequences may bind to the same or to different targets.
[0021] In another aspect, the invention concerns a library of such
polypeptides.
[0022] In yet another aspect, the invention concerns a library of
such polypeptides associated with antibody heavy chains or
fragments thereof comprising variable region sequences.
[0023] In a further aspect, the invention concerns a library
comprising a collection of surrogate light chain sequences
optionally associated with antibody heavy chain variable region
sequences.
[0024] In all aspects, the library may be in the form of a display,
such as, for example, a phage display, bacterial display, yeast
display, ribosome display, mRNA display, DNA display, display on
mammalian cells, spore display, viral display, display based on
protein-DNA linkage, or microbead display.
[0025] The invention further concerns various uses of such
polypeptides and libraries containing such polypeptides, for
example, to design or select antibody-like molecules with desired
binding specificities and/or binding affinities, which have
important therapeutic applications.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows the alignment of human VpreB1 (SEQ ID NO: 1)
and human .lamda.5 with antibody .lamda. chain variable and
constant regions. VpreB1 shares some sequence similarity to
antibody .lamda. chain variable regions, while .lamda.5 shares some
similarly to antibody .lamda. chain constant regions and framework
region 4. The boxed regions identify VpreB1 and .lamda.5 sequences
that are similar to antibody .lamda. chain CDR1, CDR2 and CDR3
regions, respectively.
[0027] FIG. 2 is a schematic illustration of a surrogate light
chain formed by VpreB and .lamda.5 sequences, illustrative fusion
polypeptides comprising surrogate light chain sequences, and an
antibody light chain structure derived from V-J joining.
[0028] FIG. 3 is a schematic illustration of various surrogate
light chain deletion and single chain constructs.
[0029] FIG. 4 schematically illustrates the incorporation of
combinatorial functional diversity into surrogate light chain
constructs.
[0030] FIG. 5 shows the gene and protein structures of various
illustrative surrogate light chain constructs.
[0031] FIG. 6 is the alignment of VpreB1 sequences with antibody
.lamda.5 light chain variable region sequences. Regions with the
highest degree of sequence similarity are boxed. As shown in the
figure, VpreB1 shows only 56%-62% (amino acids 2 to 97) sequence
identity to the .lamda.5 light chain variable region germline
sequences.
[0032] FIG. 7 is the alignment of VpreB1 sequences with antibody
.lamda.5 light chain constant region sequences. As shown in the
figure, the aligned VpreB1 sequences show only 62% (amino acids 97
to 209) sequence identity to the corresponding antibody .lamda.5
light chain constant region sequences.
[0033] FIG. 8 is the alignment of VpreB1 sequences with antibody
.kappa. light chain constant region sequences. As shown in the
figure, the aligned VpreB1 sequences show only 35% (amino acids 105
to 209) sequence identity to the corresponding antibody .kappa.
light chain constant region sequences.
[0034] FIG. 9 illustrates various representative ways of adding
functionality to surrogate light chain (SLC) components.
[0035] FIG. 10 shows the human VpreB1 sequence of SEQ ID NO: 1, the
mouse VpreB2 sequences of SEQ ID NOS: 2 and 3; the human VpreB3
sequence of SEQ ID NO: 4, the human .lamda.5 sequence of SEQ ID NO:
5 and the human .lamda.5-like protein sequence of SEQ ID NO: 6, and
sequences of various constructs used in the examples.
[0036] FIG. 11 illustrates various trimeric and dimeric surrogate
light chain constructs of the invention.
[0037] FIG. 12: Detection of surrogate light chains and conjugated
heavy chains. Lane 1: Full Length; Lane 2: Lambda 5 dT; Lane 3:
VpreB dt; Lane 4: Short; Lane 5: SCL fusion 1; Lane 6: SLC fusion
2; Lane 7: Antibody.
[0038] FIG. 13: SLC fusion proteins express and secrete well into
the periplasm of E. coli and are best partnered with heavy chain
CH1 from IgG1 rather than IgM. Panel A: SCL fusion protein
expression in E. coli. Panel B: IgG1 gamma chain partners and
purifies better than IgM .mu. chain with an SLC fusion.
[0039] FIG. 14: Phage surrogate light chain construct capture ELISA
via anti-phage detection.
[0040] FIG. 15: Purified surrogate light chain constructs expressed
in mammalian cells bind viral target.
[0041] FIG. 16: Purified surrogate light chain constructs expressed
in mammalian cells contain stable complexes that bind viral
antigen.
[0042] FIG. 17: Antigen binding with E. coli periplasmic
lysates.
[0043] FIG. 18: Surrogate light chain fusion construct phage paired
with neutralizing heavy chain readily binds H5 HA antigen.
[0044] FIG. 19: Surrogate light chain construct phage paired with
neutralizing heavy chain binds antigen.
[0045] FIG. 20: Table summarizing the results of phage display
experiments.
[0046] FIGS. 21 and 22: Results of clonal analysis of rounds 1 and
2 of surrogate light chain fusions 1 and 2.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
[0047] Unless defined otherwise, 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
2nd ed., J. Wiley & Sons (New York, N.Y. 1994), provides one
skilled in the art with a general guide to many of the terms used
in the present application.
[0048] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
[0049] The term "surrogate light chain," as used herein, refers to
a dimer formed by the non-covalent association of a VpreB and a
.lamda.5 protein.
[0050] The term "VpreB" is used herein in the broadest sense and
refers to any native sequence or variant VpreB polypeptide,
specifically including, without limitation, human VpreB1 of SEQ ID
NO: 1, mouse VpreB2 of SEQ ID NOS: 2 and 3, human VpreB3 of SEQ ID
NO: 4 and isoforms, including splice variants and variants formed
by posttranslational modifications, other mammalian homologues
thereof, as well as variants of such native sequence
polypeptides.
[0051] The term ".lamda.5" is used herein in the broadest sense and
refers to any native sequence or variant .lamda.5 polypeptide,
specifically including, without limitation, human .lamda.5 of SEQ
ID NO: 5, human .lamda.5-like protein of SEQ ID NO: 6, and their
isoforms, including splice variants and variants formed by
posttranslational modifications, other mammalian homologous
thereof, as well a variants of such native sequence
polypeptides.
[0052] The terms "variant VpreB polypeptide" and "a variant of a
VpreB polypeptide" are used interchangeably, and are defined herein
as a polypeptide differing from a native sequence VpreB polypeptide
at one or more amino acid positions as a result of an amino acid
modification. The "variant VpreB polypeptide," as defined herein,
will be different from a native antibody .lamda. or .kappa. light
chain sequence, or a fragment thereof. The "variant VpreB
polypeptide" will preferably retain at least about 65%, or at least
about 70%, or at least about 75%, or at least about 80%, or at
least about 85%, or at least about 90%, or at least about 95%, or
at least about 98% sequence identity with a native sequence VpreB
polypeptide. In another preferred embodiment, the "variant VpreB
polypeptide" will be less then 95%, or less than 90%, or less then
85%, ore less than 80%, or less than 75%, or less then 70%, or less
than 65%, or less than 60% identical in its amino acid sequence to
a native antibody .lamda. or .kappa. light chain sequence. Variant
VpreB polypeptides specifically include, without limitation, VpreB
polypeptides in which the non-Ig-like unique tail at the C-terminus
of the VpreB sequence is partially or completely removed.
[0053] The terms "variant .lamda.5 polypeptide" and "a variant of a
.lamda.5 polypeptide" are used interchangeably, and are defined
herein as a polypeptide differing from a native sequence .lamda.5
polypeptide at one or more amino acid positions as a result of an
amino acid modification. The "variant .lamda.5 polypeptide," as
defined herein, will be different from a native antibody .lamda. or
.kappa. light chain sequence, or a fragment thereof. The "variant
.lamda.5 polypeptide" will preferably retain at least about 65%, or
at least about 70%, or at least about 75%, or at least about 80%,
or at least about 85%, or at least about 90%, or at least about
95%, or at least about 98% sequence identity with a native sequence
.lamda.5 polypeptide. In another preferred embodiment, the "variant
.lamda.5 polypeptide" will be less then 95%, or less than 90%, or
less then 85%, ore less than 80%, or less than 75%, or less then
70%, or less than 65%, or less than 60% identical in its amino acid
sequence to a native antibody .lamda. or .kappa. light chain
sequence. Variant .lamda.5 polypeptides specifically include,
without limitation, .lamda.5 polypeptides in which the unique tail
at the N-terminus of the .lamda.5 sequence is partially or
completely removed.
[0054] Percent amino acid sequence identity may be determined using
the sequence comparison program NCBI-BLAST2 (Altschul et al.,
Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence
comparison program may be downloaded from
http://www.ncbi.nlm.nih.gov or otherwise obtained from the National
Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0055] The term "VpreB sequence" is used herein to refer to the
sequence of "VpreB," as hereinabove defined, or a fragment
thereof.
[0056] The term ".lamda.5 sequence" is used herein to refers to the
sequence of ".lamda.5," as hereinabove defined, or a fragment
thereof.
[0057] The term "surrogate light chain sequence," as defined
herein, means any polypeptide sequence that comprises a "VpreB
sequence" and/or a ".lamda.5 sequence," as hereinabove defined. The
"surrogate light chain sequence," as defined herein, specifically
includes, without limitation, the human VpreB1 sequence of SEQ ID
NO 1, the mouse VpreB2 sequences of SEQ ID NOS: 2 and 3, and the
human VpreB3 sequence of SEQ ID NO: 4, and their various isoforms,
including splice variants and variants formed by posttranslational
modifications, homologues thereof in other mammalian species, as
well as fragments and variants thereof. The term "surrogate light
chain sequence" additionally includes, without limitation, the
human .lamda.5 sequence of SEQ ID NO: 5, the human .lamda.5-like
sequence of SEQ ID NO: 6, and their isoforms, including splice
variants and variants formed by posttranslational modifications,
homologues thereof in other mammalian species, as well as fragments
and variants thereof. The term "surrogate light chain sequence"
additionally includes a sequence comprising both VpreB and .lamda.5
sequences as hereinabove defined.
[0058] For the three-dimensional structure of the pre-B-cell
receptor (pre-BCR), including the structure of the surrogate light
chain (SCL) and its components see, e.g. Lanig et al., Mol.
Immunol. 40(17):1263-72 (2004).
[0059] The "surrogate light chain sequence" may be optionally
conjugated to a heterogeneous amino acid sequence, or any other
heterogeneous component, to form a "surrogate light chain
construct" herein. Thus, the term, "surrogate light chain
construct" is used in the broadest sense and includes any and all
additional heterogeneous components, including a heterogeneous
amino acid sequence, nucleic acid, and other molecules conjugated
to a surrogate light chain sequence, wherein "conjugation" is
defined below. A "surrogate light chain construct" is also referred
herein as a `SURROBODY.TM.," and the two terms are used
interchangeably.
[0060] In the context of the polypeptides of the present invention,
the term "heterogeneous amino acid sequence," relative to a first
amino acid sequence, is used to refer to an amino acid sequence not
naturally associated with the first amino acid sequence, at least
not in the form it is present in the surrogate light chain
constructs herein. Thus, a "heterogenous amino acid sequence"
relative to a VpreB is any amino acid sequence not associated with
native VpreB in its native environment, including, without
limitation, .lamda.5 sequences that are different from those
.lamda.5 sequences that, together with VpreB, form the surrogate
light chain on developing B cells, such as amino acid sequence
variants, e.g. truncated and/or derivatized .lamda.5 sequences. A
"heterogeneous amino acid sequence" relative to a VpreB also
includes .lamda.5 sequences covalently associated with, e.g. fused
to, VpreB, including native sequence .lamda.5, since in their
native environment, the VpreB and .lamda.5 sequences are not
covalently associated, e.g. fused, to each other. Heterogeneous
amino acid sequences also include, without limitation, antibody
sequences, including antibody and heavy chain sequences and
fragments thereof; such as, for example, antibody light and heavy
chain variable region sequences, and antibody light and heavy chain
constant region sequences.
[0061] The terms "conjugate," "conjugated," and "conjugation" refer
to any and all forms of covalent or non-covalent linkage, and
include, without limitation, direct genetic or chemical fusion,
coupling through a linker or a cross-linking agent, and
non-covalent association, for example through Van der Waals forces,
or by using a leucine zipper.
[0062] The term "fusion" is used herein to refer to the combination
of amino acid sequences of different origin in one polypeptide
chain by in-frame combination of their coding nucleotide sequences.
The term "fusion" explicitly encompasses internal fusions, i.e.,
insertion of sequences of different origin within a polypeptide
chain, in addition to fusion to one of its termini.
[0063] As used herein, the term "target" is a substance that
interacts with a polypeptide herein. Targets, as defined herein,
specifically include antigens with which the VpreB-containing
constructs of the present invention interact. Preferably,
interaction takes-place by direct binding.
[0064] As used herein, the terms "peptide," "polypeptide" and
"protein" all refer to a primary sequence of amino acids that are
joined by covalent "peptide linkages." In general, a peptide
consists of a few amino acids, typically from about 2 to about 50
amino acids, and is shorter than a protein. The term "polypeptide,"
as defined herein, encompasses peptides and proteins.
[0065] The term "amino acid" or "amino acid residue" typically
refers to an amino acid having its art recognized definition such
as an amino acid selected from the group consisting of: alanine
(Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp);
cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine
(Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine
(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine
(Ser); threonine (Thr); tryptophan (Tip); tyrosine (Tyr); and
valine (Val) although modified, synthetic, or rare amino acids may
be used as desired. Thus, modified and unusual amino acids listed
in 37 CFR 1.822(b)(4) are specifically included within this
definition and expressly incorporated herein by reference. Amino
acids can be subdivided into various sub-groups. Thus, amino acids
can be grouped as having a nonpolar side chain (e.g., Ala, Cys,
Ile, Leu, Met, Phe, Pro, Val); a negatively charged side chain
(e.g., Asp, Glu); a positively charged side chain (e.g., Arg, His,
Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gln, Gly,
His, Met, Phe, Ser, Thr, Trp, and Tyr). Amino acids can also be
grouped as small amino acids (Gly, Ala), nucleophilic amino acids
(Ser, His, Thr, Cys), hydrophobic amino acids (Val, Leu, Ile, Met,
Pro), aromatic amino acids (Phe, Tyr, Trp, Asp, Glu), amides (Asp,
Glu), and basic amino acids (Lys, Arg).
[0066] The term "polynucleotide(s)" refers to nucleic acids such as
DNA molecules and RNA molecules and analogs thereof (e.g., DNA or
RNA generated using nucleotide analogs or using nucleic acid
chemistry). As desired, the polynucleotides may be made
synthetically, e.g., using art-recognized nucleic acid chemistry or
enzymatically using, e.g., a polymerase, and, if desired, be
modified. Typical modifications include methylation, biotinylation,
and other art-known modifications. In addition, the nucleic acid
molecule can be single-stranded or double-stranded and, where
desired, linked to a detectable moiety.
[0067] The term "variant" with respect to a reference polypeptide
refers to a polypeptide that possesses at least one amino acid
mutation or modification (i.e., alteration) as compared to a native
polypeptide. Variants generated by "amino acid modifications" can
be produced, for example, by substituting, deleting, inserting
and/or chemically modifying at least one amino acid in the native
amino acid sequence.
[0068] An "amino acid modification" refers to a change in the amino
acid sequence of a predetermined amino acid sequence. Exemplary
modifications include an amino acid substitution, insertion and/or
deletion.
[0069] An "amino acid modification at" a specified position, refers
to the substitution or deletion of the specified residue, or the
insertion of at least one amino acid residue adjacent the specified
residue. By insertion "adjacent" a specified residue is meant
insertion within one to two residues thereof. The insertion may be
N-terminal or C-terminal to the specified residue.
[0070] An "amino acid substitution" refers to the replacement of at
least one existing amino acid residue in a predetermined amino acid
sequence with another different "replacement" amino acid residue.
The replacement residue or residues may be "naturally occurring
amino acid residues" (i.e. encoded by the genetic code) and
selected from the group consisting of: alanine (Ala); arginine
(Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys);
glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine
(His); isoleucine (Ile): leucine (Leu); lysine (Lys); methionine
(Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine
(Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val).
Substitution with one or more non-naturally occurring amino acid
residues is also encompassed by the definition of an amino acid
substitution herein.
[0071] A "non-naturally occurring amino acid residue" refers to a
residue, other than those naturally occurring amino acid residues
listed above, which is able to covalently bind adjacent amino acid
residues(s) in a polypeptide chain. Examples of non-naturally
occurring amino acid residues include norleucine, ornithine,
norvaline, homoserine and other amino acid residue analogues such
as those described in Ellman et al. Meth. Enzym. 202:301 336
(1991). To generate such non-naturally occurring amino acid
residues, the procedures of Noren et al. Science 244:182 (1989) and
Ellman et al., supra, can be used. Briefly, these procedures
involve chemically activating a suppressor tRNA with a
non-naturally occurring amino acid residue followed by in vitro
transcription and translation of the RNA.
[0072] An "amino acid insertion" refers to the incorporation of at
least one amino acid into a predetermined amino acid sequence.
While the insertion will usually consist of the insertion of one or
two amino acid residues, the present application contemplates
larger "peptide insertions", e.g. insertion of about three to about
five or even up to about ten amino acid residues. The inserted
residue(s) may be naturally occurring or non-naturally occurring as
disclosed above.
[0073] An "amino acid deletion" refers to the removal of at least
one amino acid residue from a predetermined amino acid
sequence.
[0074] The term "mutagenesis" refers to, unless otherwise
specified, any art recognized technique for altering a
polynucleotide or polypeptide sequence. Preferred types of
mutagenesis include error prone PCR mutagenesis, saturation
mutagenesis, or other site directed mutagenesis.
[0075] "Site-directed mutagenesis" is a technique standard in the
art, and is conducted using a synthetic oligonucleotide primer
complementary to a single-stranded phage DNA to be mutagenized
except for limited mismatching, representing the desired mutation.
Briefly, the synthetic oligonucleotide is used as a primer to
direct synthesis of a strand complementary to the single-stranded
phage DNA, and the resulting double-stranded DNA is transformed
into a phage-supporting host bacterium. Cultures of the transformed
bacteria are plated in top agar, permitting plaque formation from
single cells that harbor the phage. Theoretically, 50% of the new
plaques will contain the phage having, as a single strand, the
mutated form; 50% will have the original sequence. Plaques of
interest are selected by hybridizing with kinased synthetic primer
at a temperature that permits hybridization of an exact match, but
at which the mismatches with the original strand are sufficient to
prevent hybridization. Plaques that hybridize with the probe are
then selected, sequenced and cultured, and the DNA is
recovered.
[0076] In the context of the present invention, the term "antibody"
(Ab) is used to refer to a native antibody from a classically
recombined heavy chain derived from V(D)J gene recombination and a
classically recombined light chain also derived from VJ gene
recombination, or a fragment thereof.
[0077] A "native antibody" is heterotetrameric glycoprotein of
about 150,000 daltons, composed of two identical light (L) chains
and two identical heavy (H) chains. Each light chain is linked to a
heavy chain by covalent disulfide bond(s), while the number of
disulfide linkages varies between the heavy chains of different
immunoglobulin isotypes. Each heavy and light chain also has
regularly spaced intrachain disulfide bridges. Each heavy chain
has, at one end, a variable domain (V.sub.H) followed by a number
of constant domains. Each light chain has a variable domain at one
end (V.sub.L) and a constant domain at its other end; the constant
domain of the light chain is aligned with the first constant domain
of the heavy chain, and the light chain variable domain is aligned
with the variable domain of the heavy chain. Particular amino acid
residues are believed to form an interface between the light- and
heavy-chain variable domains, Chothia et al., J. Mol. Biol. 186:651
(1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592
(1985).
[0078] The term "variable" with reference to antibody chains is
used to refer to Onions of the antibody chains which differ
extensively in sequence among antibodies and participate in the
binding and specificity of each particular antibody for its
particular antigen. Such variability is concentrated in three
segments called hypervariable regions both in the light chain and
the heavy chain variable domains. The more highly conserved
portions of variable domains are called the framework region (FR).
The variable domains of native heavy and light chains each comprise
four FRs (FR1, FR2, FR3 and FR4, respectively), largely adopting a
.beta.-sheet configuration, connected by three hypervariable
regions, which form loops connecting, and in some cases forming
part of, the .beta.-sheet structure. The hypervariable regions in
each chain are held together in close proximity by the FRs and,
with the hypervariable regions from the other chain, contribute to
the formation of the antigen-binding site of antibodies (see Kabat
et al., Sequences of Proteins of immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991), pages 647-669). The constant domains are not involved
directly in binding an antibody to an antigen, but exhibit various
effector functions, such as participation of the antibody in
antibody-dependent cellular toxicity.
[0079] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR"
(i.e., residues 30-36 (L1), 46-55 (L2) and 86-96 (L3) in the light
chain variable domain and 30-35 (H1), 47-58 (H2) and 93-101 (H3) in
the heavy chain variable domain; MacCallum et al., J Mol Biol.
262(5):732-45 (1996).
[0080] The term "framework region" refers to the art recognized
portions of an antibody variable region that exist between the more
divergent CDR regions. Such framework regions are typically
referred to as frameworks 1 through 4 (FR1, FR2, FR3, and FR4) and
provide a scaffold for holding, in three-dimensional space, the
three CDRs found in a heavy or light chain antibody variable
region, such that the CDRs can form an antigen-binding surface.
[0081] Depending on the amino acid sequence of the constant domain
of their heavy chains, antibodies can be assigned to different
classes. There are five major classes of antibodies IgA, IgD, IgE,
IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2.
[0082] The heavy-chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0083] The "light chains" of antibodies from any vertebrate species
can be assigned to one of two clearly distinct types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequences
of their constant domains. Any reference to an antibody light chain
herein includes both .kappa. and .lamda. light chains.
[0084] "Antibody fragments" comprise a portion of a full length
antibody, generally the antigen binding or a variable domain
thereof. Examples of antibody fragments include, but are not
limited to, Fab, Fab', F(ab').sub.2, scFv, and (scFv).sub.2
fragments.
[0085] As used herein the term "antibody binding region" refers to
one or more portions of an immunoglobulin or antibody variable
region capable of binding an antigen(s). Typically, the antibody
binding region is, for example, an antibody light chain (VL) (or
variable region thereof), an antibody heavy chain (VH) (or variable
region thereof), a heavy chain Fd region, a combined antibody light
and heavy chain (or variable region thereof) such as a Fab,
F(ab').sub.2, single domain, or single chain antibody (scFv), or a
full length antibody, for example, an IgG (e.g., an IgG1, IgG2,
IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.
[0086] The term "epitope" as used herein, refers to a sequence of
at least about 3 to 5, preferably at least about 5 to 10, or at
least about 5 to 15 amino acids, and typically not more than about
500, or about 1,000 amino acids, which define a sequence that by
itself, or as part of a larger sequence, binds to an antibody
generated in response to such sequence. An epitope is not limited
to a polypeptide having a sequence identical to the portion of the
parent protein from which it is derived. Indeed, viral genomes are
in a state of constant change and exhibit relatively high degrees
of variability between isolates. Thus the term "epitope"
encompasses sequences identical to the native sequence, as well as
modifications, such as deletions, substitutions and/or insertions
to the native sequence. Generally, such modifications are
conservative in nature but non-conservative modifications are also
contemplated. The term specifically includes "mimotopes," i.e.
sequences that do not identify a continuous linear native sequence
or do not necessarily occur in a native protein, but functionally
mimic an epitope on a native protein. The term "epitope"
specifically includes linear and conformational epitopes.
[0087] The term "vector" is used to refer to a rDNA molecule
capable of autonomous replication in a cell and to which a DNA
segment, e.g., gene or polynucleotide, can be operatively linked so
as to bring about replication of the attached segment. Vectors
capable of directing the expression of genes encoding for one or
more polypeptides are referred to herein as "expression vectors.
"The term "control sequences" refers to DNA sequences necessary for
the expression of an operably linked coding sequence in a
particular host organism. The control sequences that are suitable
for prokaryotes, for example, include a promoter, optionally an
operator sequence, and a ribosome binding site. Eukaryotic cells
are known to utilize promoters, polyadenylation signals, and
enhancers.
[0088] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0089] A "phage display library" is a protein expression library
that expresses a collection of cloned protein sequences as fusions
with a phage coat protein. Thus, the phrase "phage display library"
refers herein to a collection of phage (e.g., filamentous phage)
wherein the phage express an external (typically heterologous)
protein. The external protein is free to interact with (bind to)
other moieties with which the phage are contacted. Each phage
displaying an external protein is a "member" of the phage display
library.
[0090] The term "filamentous phage" refers to a viral particle
capable of displaying a heterogenous polypeptide on its surface,
and includes, without limitation, fl, fd, Pf1, and M13. The
filamentous phage may contain a selectable marker such as
tetracycline (e.g., "fd-tet"). Various filamentous phage display
systems are well known to those of skill in the art (see, e.g.,
Zacher et al. Gene 9: 127-140 (1980), Smith et al. Science 228:
13.15-1317 (1985); and Parmley and Smith Gene 73: 305-318
(1988)).
[0091] The term "panning" is used to refer to the multiple rounds
of screening process in identification and isolation of phages
carrying compounds, such as antibodies, with high affinity and
specificity to a target.
B. Detailed Description
[0092] Techniques for performing the methods of the present
invention are well known in the art and described in standard
laboratory textbooks, including, for example, Ausubel et al.,
Current Protocols of Molecular Biology, John Wiley and Sons (1997);
Molecular Cloning: A Laboratory Manual, Third Edition, J. Sambrook
and D. W. Russell, eds., Cold Spring Harbor, N.Y., USA, Cold Spring
Harbor Laboratory Press, 2001; O'Brian et al., Analytical Chemistry
of Bacillus Thuringiensis, Hickle and Fitch, eds., Am. Chem. Soc.,
1990; Bacillus thuringiensis: biology, ecology and safety, T. R.
Glare and M. O'Callaghan, eds., John Wiley, 2000; Antibody Phage
Display, Methods and Protocols, Humana Press, 2001; and Antibodies,
G. Subramanian, ed., Kluwer Academic, 2004. Mutagenesis can, for
example, be performed using site-directed mutagenesis (Kunkel et
al., Proc. Natl. Acad. Sci. USA 82:488-492 (1985)). PCR
amplification methods are described in U.S. Pat. Nos. 4,683,192,
4,683,202, 4,800,159, and 4,965,188, and in several textbooks
including "PCR Technology: Principles and Applications for DNA
Amplification", H. Erlich, ed., Stockton Press, New York (1989);
and PCR Protocols: A Guide to Methods and Applications, Innis et
al., eds., Academic Press, San Diego, Calif. (1990).
[0093] The present invention concerns constructs and libraries
comprising antibody surrogate light chain sequences.
[0094] Surrogate Light Chain Constructs
[0095] As discussed above, pre-B cells have been identified in the
bone marrow as lymphocytes that produce .mu. heavy chains but
instead of the fully developed light chains express a set of B
lineage-specific genes called VpreB(1-3) and .lamda.5,
respectively. The VpreB and .lamda.5 polypeptides together form a
non-covalently associated, Ig light chain-like structure, which is
called the surrogate light chain. The surrogate light chain,
although not an antibody chain, naturally associates with all
antibody heavy chains, and surrogate light chain-antibody heavy
chain complexes have been shown to bind self-antigens.
[0096] In one aspect, the present invention provides polypeptides
comprising VpreB and/or .lamda.5 sequences and having the ability
to bind a target. The target can be any peptide or polypeptide that
is a binding partner for the VpreB and/or .lamda.5
sequence-containing polypeptides of the present invention. Targets
specifically include all types of targets generally referred to as
"antigens" in the context of antibody binding.
[0097] Thus, the polypeptides of the present invention include,
without limitation, conjugates of VpreB sequences to heterogeneous
amino acid sequences, provided that they retain the ability to bind
a desired target. The binding of the VpreB sequence to the
heterogeneous amino acid sequence can be either covalent or
non-covalent, and may occur directly, or through a linker,
including peptide linkers.
[0098] Specific examples of the polypeptide constructs herein
include polypeptides in which a VpreB sequence, such as a VpreB1,
VpreB2, or VpreB3 sequence, including fragments and variants of the
native sequences, is conjugated to a .lamda.5 sequence, including
fragments and variants of the native sequence. Representative
fusions of this type are illustrated in FIGS. 2 and 11 and
described in the Examples.
[0099] In a direct fusion, typically the C-terminus of a VpreB
sequence (e.g. a VpreB1, VpreB2 or VpreB3 sequence) is fused to the
N-terminus of a .lamda.5 sequence. While it is possible to fuse the
entire length of a native VpreB sequence to a MI-length .lamda.5
sequence (see, e.g. the first diagram in FIG. 3), typically the
fusion takes place at or around a CDR3 analogous site in each of
the two polypeptides. Such CDR3 analogous sites for VpreB1 and
.lamda.5 are illustrated in FIG. 1, and a representative fusion
construct is illustrated in FIG. 2. In this embodiment, the fusion
may take place within, or at a location within about 10 amino acid
residues at either side of the CDR3 analogous region. In a
preferred embodiment, the fusion takes place between about amino
acid residues 116-126 of the native human VpreB1 sequence (SEQ ID
NO: 1) and between about amino acid residues 82 and 93 of the
native human .lamda.5 sequence (SEQ ID NO: 5).
[0100] It is also possible to fuse the VpreB sequence to the CDR3
region of an antibody .lamda. light chain, as shown in FIG. 2.
Further constructs, in which only one of VpreB and .lamda.5 is
truncated are shown in FIG. 3. Similar constructs can be prepared
using antibody .kappa. light chain sequences.
[0101] Further direct fusion structures are illustrated on the
right side of FIG. 11. The structure designated "SLC fusion 1" is a
tetramer, composed of two (timers, in which the fusion of a
truncated V-preB1 sequence (lacking the characteristic "tail" at
the C-terminus of native VpreB1) to a similarly truncated .lamda.5
sequence is non-covalently associated with an antibody heavy chain.
The structure designated "SLC fusion 2" is a tetramer, composed of
two dimers, in which the fusion of a truncated VpreB1 sequence
(lacking the characteristic "tail" at the C-terminus of native
VpreB1) to an antibody light chain constant region is
non-covalently associated with an antibody heavy chain. The
structure designated "SLC fusion 3" is a tetramer, composed of two
dimers, in which the fusion of an antibody light chain variable
region to a truncated .lamda.5 sequence (lacking the characteristic
"tail" at the N-terminus of native .lamda.5) is non-covalently
associated with an antibody heavy chain.
[0102] As noted above, in addition to direct fusions, the
polypeptide constructs of the present invention include
non-covalent associations of a VpreB sequence (including fragments
and variants of a native sequence) with a heterogeneous sequence,
such as a .lamda.5 sequence (including fragments and variants of
the native sequence), and/or an antibody sequence. Thus, for
example, a full-length VpreB sequence may be non-covalently
associated with a truncated .lamda.5 sequence. Alternatively, a
truncated VpreB sequence may be non-covalently associated with a
full-length .lamda.5 sequence.
[0103] Surrogate light chain constructs comprising non-covalently
associated VpreB1 and .lamda.5 sequences, in non-covalent
association with an antibody heavy chain, are shown on the left
side of FIG. 11. As the various illustrations show, the structures
may include, for example, full-length VpreB1 and .lamda.5
sequences, a full-length VpreB1 sequence associated with a
truncated .lamda.5 sequence ("Lambda 5 dT"), a truncated V-reB1
sequence associated with a full-length .lamda.5 sequence (VpreB
dT") and a truncated VpreB1 sequence associated with a truncated
.lamda.5 sequence ("Short").
[0104] Although FIG. 11 illustrates certain specific constructs,
one of ordinary skill will appreciate that a variety of other
constructs can be made and used in a similar fashion. For example,
the structures can be asymmetrical, comprising different surrogate
light chain sequences in each arm, and/or having trimeric or
pentameric structures, as opposed to the structures illustrated in
FIG. 11. It is also possible to include different functionalities
in various portions of the surrogate light chain constructs of the
present invention, thereby producing multi-specific and/or
multivalent constructs.
[0105] If desired, the constructs of the present invention can be
engineered, for example, by incorporating or appending known
sequences or sequence motifs from the CDR1, CDR2 and/or CDR3
regions of antibodies, including known therapeutic antibodies into
the CDR1, CDR2 and/or CDR3 analogous regions of the surrogate light
chain sequences. This allows the creation of molecules that are not
antibodies, but will exhibit binding specificities and affinities
very similar to those of a known therapeutic antibody.
[0106] All surrogate light chain constructs herein may be
associated with antibody sequences. For example, as shown in FIG.
5, a VpreB-.lamda.5 fusion can be linked to an antibody heavy chain
variable region sequence by a peptide linker. In another
embodiment, a VpreB-.lamda.5 fusion is non-covalently associated
with an antibody heavy chain, or a fragment thereof including a
variable region sequence to form a dimeric complex. In yet another
embodiment, the VpreB and .lamda.5 sequences are non-covalently
associated with each other and an antibody heavy chain, or a
fragment thereof including a variable region sequence, thereby
forming a trimeric complex. Exemplary constructs comprising an
antibody heavy chain are illustrated in FIG. 11.
[0107] While the constructs of the present invention are
illustrated by reference to certain embodiments, one of ordinary
skill will understand that numerous further embodiments obtained by
various permutations of surrogate light chain and antibody
sequences are possible, and are within the scope of the present
invention. The present invention includes all constructs that
comprise surrogate light chain sequences and have the ability to
bind a desired target. In certain embodiment, the constructs also
have the ability to associate with antibody heavy chain variable
region sequences.
[0108] The constructs of the present invention may be used to build
libraries of surrogate light chain sequences, which can be used for
various purposes, similarly to antibody libraries, including
selection of constructs with the desired binding specificities and
affinities.
[0109] When the VpreB and .lamda.5 surrogate light chain sequences
are non-covalently associated with each other, the free ends of one
or both components (i.e. the C-terminal end of the VpreB sequence
and/or the N-terminal end of the .lamda.5 sequence) are available
for incorporating an additional diversity into the library of such
sequences. For instance, a random peptide library can be appended
or substituted to one of these free ends and panned for specific
binding to a particular target. By combining the surrogate light
chain identified to have the desired binding specificity with a
heavy chain or heavy chain fragment from an antibody to the same
target, a molecule can be created that has the ability to bind to
the cognate target on two distinct places. This tandem binding, or
"chelating" effect, strongly reinforces the binding to a single
target, similarly to the avidity effects seen in dimeric
immunoglobulins. It is also possible to use components binding to
different targets. Thus, for example, the surrogate light chain
component with the desired binding specificity can be combined with
an antibody heavy chain or heavy fragment binding to a different
target. For instance, the surrogate light chain component may bind
a tumor antigen while the antibody heavy chain or heavy chain
fragment may bind to effector cells. This way, a single entity with
targeting and anti-tumor activity can be created. In a particular
embodiment, the appendage or the polypeptide that connects the
VpreB and .lamda.5 sequences can be an antibody or antibody
fragments, such as a Fab or a scFv fragment. The incorporation of
an antibody sequence will not only create a "chelating" effect but
can also generate bispecificity in a single molecule, without the
need of a second independent arm, such as that found in bispecific
antibodies. The two specificities may be to different parts of the
same target, to disparate targets, or to a target antibody complex.
Similarly, multi-specific constructs can be made with any type of
molecule, other than antibodies or antibody fragments, including
peptides, proteins, enzymes, and the like. For example, the
surrogate light chain component with the desired specificity can be
combined with any therapeutic peptide or protein.
[0110] Preparation of Surrogate Light Chain Constructs
[0111] The surrogate light chain constructs of the present
invention can be prepared by methods known in the art, including
well known techniques of recombinant DNA technology.
[0112] Nucleic acid encoding surrogate light chain, e.g. VpreB and
.lamda.5 polypeptides, can be isolated from natural sources, e.g.
developing B cells and/or obtained by synthetic or semi-synthetic
methods. Once this DNA has been identified and isolated or
otherwise produced, it can be ligated into a replicable vector for
further cloning or for expression.
[0113] Cloning and expression vectors that can be used for
expressing the coding sequences of the polypeptides herein are well
known in the art and are commercially available. The vector
components generally include, but are not limited to, one or more
of the following: a signal sequence, an origin of replication, one
or more marker genes, an enhancer element, a promoter, and a
transcription termination sequence. Suitable host cells for cloning
or expressing the DNA encoding the surrogate light chain constructs
in the vectors herein are prokaryote, yeast, or higher eukaryote
(mammalian) cells, mammalian cells are being preferred.
[0114] Examples of suitable mammalian host cell lines include,
without limitation, monkey kidney CV1 line transformed bySV40
(COS-7, ATCC CRL 1651); human embryonic kidney line 293 (293 cells)
subcloned for growth in suspension culture, Graham et al, J. Gen
Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W 138, ATCC CCL 75); human liver cells
(Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);
TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0115] For use in mammalian cells, the control functions on the
expression vectors are often provided by viral material. Thus,
commonly used promoters can be derived from the genomes of polyoma,
Adenovirus2, retroviruses, cytomegalovirus, and Simian Virus 40
(SV40). Other promoters, such as the .beta.-actin protomer,
originate from heterologous sources. Examples of suitable promoters
include, without limitation, the early and late promoters of SV40
virus (Fiers et al., Nature, 273: 113 (1978)), the immediate early
promoter of the human cytomegalovirus (Greenaway et al., Gene, 18:
355-360 (1982)), and promoter and/or control sequences normally
associated with the desired gene sequence, provided such control
sequences are compatible with the host cell system.
[0116] Transcription of a DNA encoding a desired heterologous
polypeptide by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. The enhancer is a cis-acting
element of DNA, usually about from 10 to 300 bp, that acts on a
promoter to enhance its transcription-initiation activity.
Enhancers are relatively orientation and position independent, but
preferably are located upstream of the promoter sequence present in
the expression vector. The enhancer might originate from the same
source as the promoter, such as, for example, from a eukaryotic
cell virus, e.g. the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.
[0117] Expression vectors used in mammalian host cells also contain
polyadenylation sites, such as those derived from viruses such as,
e.g., the SV40 (early and late) or HBV.
[0118] An origin of replication may be provided either by
construction of the vector to include an exogenous origin, such as
may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV,
BPV) source, or may be provided by the host cell.
[0119] The expression vectors usually contain a selectable marker
that encodes a protein necessary for the survival or growth or a
host cell transformed with the vector. Examples of suitable
selectable markers for mammalian cells include dihydrofolate
reductase (DHFR), thymidine kinase (TK), and neomycin.
[0120] Suitable mammalian expression vectors are well known in the
art and commercially available. Thus, for example, the surrogate
light chain constructs of the present invention can be produced in
mammalian host cells using a pa expression vector (Promega),
carrying the human cytomegalovirus (CMV) immediate-early
enhancer/promoter region to promote constitutive expression of a
DNA insert. The vector can contain a neomycin phosphotransferase
gene as a selectable marker.
[0121] The surrogate light chain constructs of the present
invention can also be produced in bacterial host cells. Control
elements for use in bacterial systems include promoters, optionally
containing operator sequences, and ribosome binding sites. Suitable
promoters include, without limitation, galactose (gal), lactose
(lac), maltose, tryptophan (trp), .beta.-lactamase promoters,
bacteriophage .lamda. and T7 promoters. In addition, synthetic
promoters can be used, such as the tac promoter. Promoters for use
in bacterial systems also generally contain a Shine-Dalgarno (SD)
sequence operably linked to the DNA encoding the Fab molecule. The
origin of replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria.
[0122] The coding sequences of the individual chains within a
multi-chain construct comprising antibody surrogate light chain
sequences can be present in the same expression vector, under
control of separate regulatory sequences, or in separate expression
vectors, used to cotransfect a desired host cells, including
eukaryotic and prokaryotic hosts. Thus, multiple genes can be
coexpressed using the Duet.TM. vectors commercially available from
Novagen.
[0123] The transformed host cells may be cultured in a variety of
media. Commercially available media for culturing mammalian host
cells include Ham's F10 (Sigma), Minimal Essential Medium ((MEM),
(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
((DMEM), Sigma). In addition, any of the media described in Ham et
al., Meth. Enz. 58:44 (1979) and Barnes et al., Anal. Biochem.
102:255 (1980) may be used as culture media for the host cells. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and are included in the manufacturer's instructions or will
otherwise be apparent to the ordinarily skilled artisan.
[0124] Further suitable media for culturing mammalian, bacterial
(e.g. E. coli) or other host cells are also described in standard
textbooks, such as, for example, Sambrook et al., supra, or Ausubel
et al., supra.
[0125] Purification can be performed by methods known in the art.
In a preferred embodiment, the surrogate antibody molecules are
purified in a 6.times.His-tagged form, using the Ni-NTA
purification system (Invitrogen).
[0126] Libraries Comprise Surrogate Light Chain Sequences
[0127] The present invention further concerns various libraries of
surrogate light chain sequences and constructs comprising such
sequences. Thus, such libraries may comprise, consist essentially
of, or consist of, displays of surrogate light chain sequences,
such as the VpreB- and/or .lamda.5-containing constructs of the
present invention, including, without limitation, those
specifically described above, illustrated in the figures and/or
described in the Examples.
[0128] The libraries of the present invention are preferably in the
form of a display. Systems for displaying heterologous proteins,
including antibodies and other polypeptides, are well known in the
art. Antibody fragments have been displayed on the surface of
filamentous phage that encode the antibody genes (Hoogenboom and
Winter J. Mol. Blot, 222:381 388 (1992); McCafferty et al., Nature
348(6301):552 554 (1990); Griffiths et al. EMBO J.,
13(14):3245-3260 (1994)). For a review of techniques for selecting
and screening antibody libraries see, e.g., Hoogenboom, Nature
Biotechnol. 23(9):1105-1116 (2005). In addition, there are systems
known in the art for display of heterologous proteins and fragments
thereof on the surface of Escherichia coli (Agterberg et al., Gene
88:37-45 (1990); Charbit et al., Gene 70:181-189 (1988); Francisco
et al., Proc. Natl. Acad. Sci USA 89:2713-2717 (1992)), and yeast,
such as Saccharomyces cerevisiae (Boder and Wittrup, Nat.
Biotechnol. 15:553-557 (1997); Kieke et al., Protein Eng.
10:1303-1310 (1997)). Other known display techniques include
ribosome or mRNA display (Mattheakis et al., Proc. Natl. Acad. Sci.
USA 91:9022-9026 (1994); Hanes and Pluckthun, Proc. Natl. Acad.
Sci. USA 94:4937-4942 (1997)), DNA display (Yonezawa et al., Nucl.
Acid Res. 31(19):e118 (2003)); microbial cell display, such as
bacterial display (Georgiou et al., Nature Biotech. 15:29-34
(1997)), display on mammalian cells, spore display (Isticato et
al., J. Bacteriol. 183:6294-6301 (2001); Cheng et al., Appl.
Environ. Microbiol. 71:3337-3341 (2005) and co-pending provisional
application Ser. No. 60/865,574, filed Nov. 13, 2006), viral
display, such as retroviral display (Urban et al., Nucleic Acids
Res. 33:e35 (2005), display based on protein-DNA linkage (Odegrip
et al., Proc. Acad. Natl. Sci. USA 101:2806-2810 (2004); Rciersen
et al., Nucleic Acids Res. 33:e10 (2005)), and microbead display
(Sepp et al., FEBS Lett. 532:455-458 (2002)).
[0129] For the purpose of the present invention, the surrogate
light chain-containing libraries may be advantageously displayed
using any display technique, including phage display and spore
display.
[0130] In phage display, the heterologous protein, such as a
surrogate light chain polypeptide, is linked to a coat protein of a
phage particle, while the DNA sequence from which it was expressed
is packaged within the phage coat. Details of the phage display
methods can be found, for example, McCafferty et al., Nature 348
552-553 (1990)), describing the production of human antibodies and
antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell.
[0131] Phage display can be performed in a variety of formats; for
their review see, e.g. Johnson, Kevin S. and Chiswell, David J.,
Current Opinion in Structural Biology 3 564-571 (1993). Several
sources of heavy chain V-gene segments can be discovered through
phage display. Clarkson et al., Nature 352, 624-628 (1991) isolated
a diverse array of anti-oxazolone heavy chains and light chains
from a small random combinatorial library of V genes derived from
the spleens of immunized mice. A repertoire of heavy and light
chain V genes from unimmunized human donors can be constructed and
recovered specific to a diverse array of antigens (including
self-antigens) essentially following the techniques described by
Marks et al., J. Mol. Biol. M, 581-597 (1991), or Griffith et al.,
EMBO J. 12, 725-734 (1993). These, and other techniques known in
the art, can be adapted to the display of any polypeptide,
including polypeptides and other constructs comprising surrogate
light chain sequences. Thus, for example, the surrogate light chain
can be supplemented with a collection of heavy chains from either a
naturally diverse source, such as lymphocytes, or a synthetically
generated collection created entirely through techniques of
molecular biology. These collections can be cloned, expressed and
selected, by methods known in the art. The selected resulting
SURROBODY.TM. can be used directly, expressed as multimeric a
molecule, or further optimized through heavy chain optimization, or
surrogate light chain optimization, for example, using random or
nonrandom site specific or regional mutagenesis.
[0132] Spore display systems are based on attaching the sequences
to be displayed to a coat protein, such as a Bacillus subtilis
spore coat protein. The spore protoplast (core) is surrounded by
the cell wall, the cortex, and the spore coat. Depending on the
species, an exosporium may also be present. The core wall is
composed of the same type of peptidoglycan as the vegetative cell
wall. Spore display, including surface display system using a
component of the Bacillus subtilis spore coat (CorB) and Bacillus
thuringiensis (Bt) spore display, is described in Isticato et al.,
J. Bacteria 183:6294-6301 (2001); Cheng et al., Appl. Environ.
Microbial. 71:3337-3341 (2005), the entire disclosures of which is
hereby expressly incorporated by reference. Various spore display
techniques are also disclosed in U.S. Patent Application
Publication Nos. 20020150594; 20030165538; 20040180348;
20040171065; and 20040254364, the entire disclosures are hereby
expressly incorporated by reference herein.
[0133] An advantage of spore display systems is the homogenous
particle surface and particle size of non-eukaryotic nature, which
is expected to provide an ideal non-reactive background. In
addition; the particle size of spores is sufficient to enable
selection by flow cytometry that permits selectable clonal
isolation, based upon interactions.
[0134] Leveraging on the stability of spores, it is possible to
perform various post-sporulation chemical, enzymatic and/or
environmental treatments and modification. Thus, it is possible to
stabilize structural helical structures with chemical treatment
using trifluoroethanol (TFE), when such structures are displayed.
In addition, oxidative stress treatments, such as treatments with
Reactive Oxygen Species (e.g. peroxide) or reactive Nitrogen
Species (e.g. nitrous acid) are possible. It is also possible to
expose defined or crude populations of spore-displayed polypeptides
to enzymatic treatments, such as proteolytic exposure, other
enzymatic processes, phosphorylation, etc. Other possible
treatments include, without limitation, nitrosylation by
peroxynitrite treatment, proteolysis by recombinant, purified, or
serum protease treatment, irradiation, coincubation with known
chaperones, such as heat shock proteins (both bacterial and
mammalian), treatment with folding proteins, such as protein
disulfide isomerase, prolyl isomerase, etc., lyophilization, and
preservative-like treatments, such as treatment with thimerosol.
These treatments can be performed by methods well known in the
art.
[0135] Similar techniques can be used in all spore display systems,
including displays where the attachment is to a spore coat protein,
including, for example, the spore display systems disclosed in
[0136] Uses of Surrogate Light Chain Sequences, Constructs and
Libraries Containing Same
[0137] The libraries of the present invention can be used to
identify surrogate light chain sequences and surrogate light chain
constructs, such as fusions comprising surrogate light chain
sequences, with desired properties. For example, in vitro or in
vivo screening of the libraries herein can yield polypeptides
comprising surrogate light chain sequences binding to desired
targets with high binding specificity and affinity. Thus, the
libraries herein can be used to identify molecules for therapeutic
and diagnostic purposes, such as polypeptides comprising surrogate
light chain sequences that bind to tumor markers or other molecular
targets of therapeutic intervention. In addition, by the techniques
described above, highly diverse libraries of surrogate light chain
polypeptides can be engineered, including libraries comprising a
collection of polypeptides binding to the same target, libraries of
polypeptides binding to different targets, libraries of
polypeptides with multiple specificities, and the like.
[0138] As a result of their ability to bind to any desired target,
the antibody surrogate light chain constructs of the present
invention can be used in analytical and diagnostic assays, to
detect the presence of a desired target molecule, such as a tumor
antigen or any polypeptide associated with a disease state or
condition. In addition, the surrogate light chain constructs of the
present invention can be used as therapeutic agents, such as, for
example, in cancer therapy, to target tumor antigens that have been
determined to associate with the development and/or spread of
cancer.
[0139] Further details of the invention are provided in the
following non-limiting Examples.
Example 1
VpreB as a Binding Domain Protein and Fusions Containing it
[0140] To make a VpreB binding domain a single protein shown in
FIG. 5 is created recombinantly. The SLC binding domain protein
construct is comprised of the amino acids 20 to 121 from VpreB1 and
the amino acids 87 to 105 from .lamda.5. If desired, to create
novel and specific binding capabilities, the molecule is
reengineered according to structural or sequence evidence.
Additionally, or alternatively, a collection of variants is created
either randomly, for example by error-prone PCR, or directly by
single or multi-site specific mutagenesis with a collection of
amino acids. The resulting clones or collections are then cloned in
frame with pIII for use in phage or phagemid display. This phagemid
construct is transformed into TG1 cells. Next a single colony is
propagated in Luria Broth (L13) supplemented with 50 .mu.g/ml
Ampicillin and 2% glucose until it reached 00600.about.0.3, and
infected with MK307 helper phage at 37.degree. C. for 30 minutes
without shaking. The cells are then pelleted and then resuspended
in LB containing 50 .mu.g/ml ampicillin and 75 .mu.g/ml kanamycin
and allowed to grow overnight with vigorous aeration at 30.degree.
C. The next day the supernatant containing phagemid expressed
SLC-HC fusion protein is used in Phage ELISA to determine targeted
binding. Briefly the ELISA entails coating and blocking of an ELISA
plate with human TNF-.alpha., followed by incubation of the SLC-HC
phage for 2 hours at 4.degree. C., washing with PBS-Tween-20
(0.05%) and direct detection with anti-m13-HRP antibody.
Alternatively binding is assessed by directly amplifying or eluting
the bound phage and determining phage titers using XL-1Blue cells.
This example describes a SLC binding domain fusion as a single
clone, but this SLC can be recombinantly recombined with other
heterologous sequences that recognize a common target and screened
as a library. Furthermore, this SLC binding protein can be combined
with a previously selected collection of heavy chains and screened
directly on the same target of interest or a second target of
interest to create a bispecific molecule. Alternatively this
reinforced binding or bispecific binding can be discovered by
screening in conjunction with unselected collections of heavy
chains. In addition, while this example refers to antibody heavy
chains, it should be understood that a complete heavy chain is not
needed. Single-chain fusions comprising heavy chain variable region
sequences, in the absence of a heavy chain constant region, or a
complete heavy chain constant region, can be made in an analogous
manner and are within the scope of this example.
Example 2
VpreB Fusions as a Variable Heavy Chain (VH) Partner
[0141] A functional VpreB-.lamda.5 fusion protein shown in the
second diagram of FIG. 5 (designated "VpreB protein fusion-dimeric
complex") is recombinantly created. The VpreB-.lamda.5 fusion
protein is comprised of an m13 gene III signal sequence, the amino
acids 20 to 115 from VpreB1, and the amino acids 83 to 209 from
.lamda.5. This construct is coexpressed with a variable heavy
chain-CH1 fusion in frame with pIII for use in phage or phagemid
display. As a VH coding sequence the VH coding sequence from the
anti-TNF-.alpha. antibody, D2E7, is used, and CH1 is the CH1 region
of human IgG1. This phagemid construct is transformed into TG1
cells. Next, a single colony is propagated in Luria Broth (LB)
supplemented with 50 .mu.g/ml Ampicillin and 2% glucose until it
reached OD600.about.0.3, and infected with MK307 helper phage at 37
degrees for 30 minutes, without shaking. The cells are then
pelleted and then resuspended in LB containing 50 .mu.g/ml
ampicillin and 75 .mu.g/ml kanamycin and allowed to grow overnight
with vigorous aeration at 30 degrees. The next day the supernatant
containing phagemid expressed SLC-HC fusion protein is used in
Phage ELISA to determine targeted binding. Briefly the ELISA
entails coating and blocking of an ELISA plate with human
TNF-.alpha., followed by incubation of the SLC-HC phage for 2 hours
at 4 degrees, washing with PBS-Tween-20 (0.05%) and direct
detection with anti-m13-HRP antibody. Alternatively binding can be
assessed by directly amplifying or eluting the bound phage and
determining phage titers using XL-1Blue cells. This example
describes a SLC fusion partnered with a heavy chain variable-CH1
fusion as a single clone, but this SLC can also be combined with a
focused collection of heavy chain variable regions that recognize a
common target and screened as a library. Furthermore, this SLC
fusion can be combined with an unselected collection of heavy
chains and screened directly on a target of interest. As a
reasonable SLC fusion alternative, VH association can be reinforced
by fusing the constant lambda region from a traditional antibody
light chain instead of the .lamda.5 protein fragment.
Example 3
VpreB and Lambda5 as an Associated Variable Heavy Chain (VH)
Partner
[0142] The VpreB-.lamda.5 coexpressed protein shown in the third
diagram of FIG. 5 (designated "VpreB and lambda 5-trimeric
complex") is made of an m13 gene III signal sequence and the
corresponding amino acids of the predicted mature, processed VpreB1
(amino acids 20 to 146) and lambda 5 (amino acids 31 to 209). These
are coexpressed with a variable heavy chain-CH1 fusion in frame
with pIII for use in phage or phagemid display. As a VH coding
sequence the VH coding sequence from the anti-TNF-.alpha. antibody,
D2E7, is used, and CH1 is the CH1 region from human IgG1. This
phagemid construct is transformed into TG1 cells. Next a single
colony is propagated, in Luria Broth (LB) supplemented with 50
.mu.g/ml Ampicillin and 2% glucose until it reached
OD600.about.0.3, and is then infected with MK307 helper phage at 37
degrees for 30 minutes, without shaking. The cells are then
pelleted and then resuspended in LB containing 50 .mu.g/ml
ampicillin and 75 .mu./ml kanamycin and allowed to grow overnight
with vigorous aeration at 30 degrees. The next day the supernatant
containing phagemid expressed SLC HC trimeric protein complexes is
used in Phage ELISA to determine targeted binding. Briefly the
ELISA entails coating and blocking of an ELISA plate with human
TNF-.alpha., followed by incubation of the SLC-HC phage for 2 hours
at 4 degrees, washing with PBS-Tween-20 (0.05%) and direct
detection with anti-m13-HRP antibody. Alternatively binding can be
assessed by directly amplifying or eluting the bound phage and
determining phage titers using XL-1Blue cells. This example
describes a SLC partnered with a heavy chain variable-CH1 fusion as
a single clone, but this SLC can be combined with a focused
collection of heavy chain variable regions that recognize a common
target and screened as a library. Furthermore, this SLC fusion can
be combined with an unselected collection of heavy chains and
screened directly on a target of interest.
Example 4
[0143] Engineering Diversity into VpreB1 CDR3 Analogous Regions
[0144] As the CDR analogous regions of the surrogate light chain
(SLC) will have similar functions to the CDR's of an antibody light
chain, it is important to determine the fusion points between the
VpreB and .lamda.5. According to one approach the most suitable
fusion point for a particular purpose is determined starting with
the CDR3 analogous site containing all VpreB amino acids and
incrementally substituting amino acids position by position from
.lamda.5 encoded in clonable oligonucleotides. This incremental
substitution continues until the CDR analogous site is entirely
composed of a .lamda.5 source sequence. At some point during this
process, it might be desirable to add a complementary heavy chain
and allow/facilitate its antigen binding and recognition. To
further enhance or enable this complementation random diversity can
be used in any of the CDR analogous sites, as well as diversity
based upon matched CDR length analysis. Alternatively, or in
addition, antibody V.lamda.5 sequences can be used to add
diversity, as their CDR lengths match well with VpreB CDR analogous
site lengths.
Example 5
Adding Functionalities to SLC Components
[0145] As the SLC is comprised of two independent polypeptides this
creates natural opportunities to append or embed secondary
functionalities. In the present Example, in the first instance an
anti-VEGF scFv is inserted to create a fusion protein linking VpreB
and .lamda.5 (FIG. 9A). This resulting engineered SLC-constrained
scFv is paired with the heavy chain of an anti-TNF-.alpha.
antibody. The resulting construct is co-expressed with the heavy
chain cloned in frame with pIII for use in phage or phagemid
display. This phagemid construct is transformed into TG1 cells and
a single colony is propagated in Luria Broth (LB) supplemented with
50 .mu.g/ml Ampicillin and 2% glucose until it reached
OD600.about.0.3, and infected with MK307 helper phage at 37.degree.
C. for 30 minutes without shaking. The cells are then be pelleted
and then resuspended in LB containing 50 .mu.g/ml ampicillin and 75
.mu.g/ml kanamycin and allowed to grow overnight with vigorous
aeration at 30.degree. C. The next day the supernatant containing
phagemid expressed SLC-HC fusion protein is used in Phage ELISA to
determine targeted binding. Briefly the ELISA entails coating and
blocking of an ELISA plate with human TNF-.alpha. or human VEGF,
followed by incubation of the SLC-HC phage for 2 hours at 4.degree.
C., washing with PBS-Tween-20 (0.05%) and direct detection with
anti-m13-HRP antibody.
[0146] Next a fusion of the anti-VEGF scFv to the C-terminus of
VpreB is created, and the resulting tripartite protein complex
construct assessed similarly to the phagemid ELISA described
above.
[0147] Alternatively the an anti-ovalbumin scFv is fused to the
amino terminus of .lamda.5 and the tripartite protein complex
tested for binding to both TNF-.alpha. and ovalbumin.
[0148] Finally, these two fusion constructs (VpreB-antiVEGF scFv
and the .lamda.5-anti-ovalbumin) are combined with the heavy chain
of the anti-TNF-.alpha. antibody to create a trispecific molecule,
which is then confirmed in phagemid ELISA as described above.
[0149] In the description scFv against disparate targets are
incorporated, however one can combine functional binders to the
same target to create tandem "super-binders." These tandem binders
can either provide reinforced binding, or even in some instances
cross-linking function. Fab cross-linking will be beneficial in
instances where whole antibodies provide undesirable and prolonged
cross-linking. For instance, it may be undesirable for whole
immunoglobulin insulin receptor antibodies that act as insulin
substitutes to require 3-4 weeks for scrum clearance. As insulin
usually has a half-life of minutes, a Fab would be more in tune
with this scale of half-life and the tandem functionality could
appropriately address this application.
[0150] The above descriptions describe only antibodies as secondary
functional groups, but one can also similarly incorporate relevant
peptides (e.g., erythropoietin (EPO) mimetics), receptors (e.g.,
TNF-R1), binding proteins (e.g., IL-1ra), and any therapeutic
protein, such as interferons, to the appended and constrained
constructs to create molecules of similar functions.
[0151] Also one might utilize the two sites to incorporate
heterodimeric proteins, such as heavy and light chains to create a
secondary Fab-like molecule.
[0152] Finally, we have described only singular instances, but the
incorporation of combinatorially diverse phage antibody libraries
and peptide diversity libraries is also included herein, to screen
with SLC candidate antibodies against their directed and desired
targets.
Example 6
Expression of Surrogate Light Chain Constructs (SURROBODY.TM.) in
Mammalian Cells
[0153] Coding sequences of the surrogate light chain components of
the structures designated in FIG. 11 as "trimers" (also referred to
as "SURROBODY.TM. variants") were cotransfected with a full-length
IgG1 antibody heavy chain into CHO-K1 cells (ATCC CCL-61) to
transiently produce surrogate light chain constructs for
biochemical analysis. Specifically, full length human VpreB1 and
.lamda.5 were cloned into the mammalian expression vector pCI
(Promega, Madison Wis.). These constructs contained their native
predicted secretion signals. In the case of VpreB1 the predicted
signal peptide is amino acids 1-20 of SEQ ID NO: 1, for .lamda.5
the predicted signal sequence is amino acids 1-30 of SEQ ID NO: 5.
For both of these proteins portions of their predicted
nonstructural tails were deleted. For VpreB1 this included the
C-terminal amino acids 122-146 of SEQ ID NO: 1 and for lambda 5
this included the N-terminal amino acids 30-86 of SEQ ID NO: 5.
[0154] The sequence of the truncated .lamda.5 sequence in the
"trimer" designated in FIG. 11 as "Lambda 5 dT" is shown as SEQ ID
NO: 7. The sequence of the truncated VpreB1 sequence in the
"trimer" designated in FIG. 11 as "VpreB dT" is shown as SEQ ID NO:
8.
[0155] Each of the four combinatorial surrogate light chain
possibilities were cotransfected with a known human anti-influenza
heavy chain, containing a C-terminal hexahistidine (His6) tag (SEQ
ID NO: 9), and expressed according to manufacturer's suggestions
(Invitrogen, Carlsbad Calif.) in low serum media. After 3 days the
supernatants were collected, filtered, and purified by nickel
chelate chromatography (Qiagen, Germany). The purified proteins
were then examined by western blot analysis with either
anti-peptide rabbit serum (VpreB and .lamda.5) or anti-histidine
antibodies (Serotec, Raleigh N.C.). Detection of proteins was
visualized following anti-rabbit HRP (VpreB and .lamda.5) or
anti-mouse HRP (heavy chain) and colorimetric development with TMB
substrate. (FIG. 12, lanes 1-4)
[0156] Additionally, surrogate light chain fusions (see FIG. 11)
were created by engineering a chimeric protein composed of the
VpreB1 gene and either the .lamda.5 gene or the light chain
constant lambda domain. Specifically a recombinantly fused protein
was produced that contained amino acids 1-87 from VpreB (SEQ ID NO:
1) to .lamda.5 protein amino acids 121-209 (SEQ ID NO: 5) (SEQ ID
NO: 10). Additionally, a second fusion was made that contained
amino acids 1-87 from VpreB (SEQ ID NO: 1) to the C-terminal 121
amino acid of the antibody .lamda. light chain constant (SEQ ID NO:
11). Each surrogate light chain fusion was transiently expressed,
harvested, purified, and examined by western blot analysis,
essentially as described above. Notably, as both fusions contained
the epitope to the anti-VpreB1 anti-peptide serum, it was used for
western blot analysis. (FIG. 12, lanes 5-6)
Example 7
[0157] Expression of Surrogate Light Chain Constructs
(SURROBODY.TM.) in E. coli
[0158] As recombinant proteins are often beneficially expressed in
bacteria the ability of producing soluble surrogate light chain
constructs in prokaryotic systems was tested. To address this, the
surrogate light chain fusions designated as "dimers" in FIG. 11
were clones into E. coli expression/secretion systems. A plac
repressible expression system was used, where the mature mammalian
proteins were expressed and secreted into the periplasm by
recombinant fusion to prokaryotic leader sequences. Specifically,
the surrogate light chain fusions were directed to the periplasm by
fusing the coding sequence of the mature protein to the C-terminus
of the m13 gill leader coding sequence (SEQ ID NOs: 12 and 13). The
heavy chain was expressed by fusing the IgG1 heavy chain variable
region and heavy chain constant region domain of an anti-influenza
antibody to the C-terminus of the pelB leader sequence (SEQ ID NO:
14). The plasmids expressing both proteins were transformed into
HB2151 E. coli cells (Stratagene) and expressed overnight in LB
media containing 100 mcg/ml ampicillin, and 200 micromolar IPTG at
30 degrees. The cells were harvested and periplasmic lysates were
prepared, following methods known in the art. The periplasmic
lysates were tested directly by western blot analysis or purified
as described above (FIG. 13, panel A).
[0159] As the surrogate light chain is traditionally a component of
the membrane bound preB cell receptor, it is normally found paired
with an IgM class heavy chain. For our utilitarian purposes we
wished to compare the ability to pair a surrogate light chain
fusion with an IgM versus a IgG based constant heavy domain 1
region. To examine this we substituted a .mu. constant heavy domain
1 (SEQ ID NO: 15) for the gamma constant heavy domain region of the
anti-influenza antibody described above. We found, from western
blot analysis of the periplasmic lysates that the IgG
(.gamma.)-based constant heavy domain expressed better and purified
to greater levels than a .mu.-based constant heavy domain based
system (FIG. 13, panel B).
Example 8
Expression of Surrogate Light Chain Constructs (SURROBODY.TM.) m13
Phagemid
[0160] As recombinant proteins are not only usefully expressed in
bacteria but also individually and in diverse library collections
on the surface of bacterial virus particles we wished to produce
soluble surrogate light chain constructs on the surface of m13
phagemids. To address this, surrogate light chain fusions ("dimers"
in FIG. 11) were clones into E. coli expression/secretion systems.
For all systems a pLac repressible expression system described
above was used. However, in this case we appended an E-tag epitope
(GAPVPYPDPLEPR) (SEQ ID NO: 16) to the surrogate light chain
fusions, as well as to a light chain control protein. The sequences
of the geneIII VpreB1-lambda5-E tag fusion (Fusion 1) and the
geneIII VpreB1-CI-E tag fusion (Fusion 2) are shown as SEQ ID NOs:
12 and 13, respectively. To anchor the heavy chain constructs to
the m13 phagemid the heavy chain constructs were recombinantly
cloned the variable heavy chains and gamma constant heavy domain 1
regions in frame with the m13 gene III product. Specifically the
recombinant proteins contained an intervening, a hexahistidine
peptide, the peptide epitope for the anti-c-myc antibody
(GEQKLISLEEDL) (SEQ ID NO: 17), and amber stop codon. We examined
the fidelity of protein expression and complex formation
respectively by anti-histidine and anti-E capture ELISA.
[0161] Phagemid expression of antibodies and surrobodies were
accomplished by standard methods well known in the art.
Essentially, TG-1 cells transformed with expression plasmids were
grown to mid log (OD 600.about.0.3) in 2-YT media supplemented with
100 mcg/ml ampicillin and 2% glucose repression and then infected
with m13K07 helper phage and then grown overnight in 2-YT media
supplemented with 100 mcg ampicillin, 70 mcg/ml kanamycin, and 200
micromolar IPTG. Phage containing supernatants or precipitated and
PBS resuspended phage were used for phage capture ELISA. The phage
capture ELISA was accomplished by coating microtiter plates with
either anti-histidine (Serotec) or anti-E antibodies (Abcam) and
then detecting binding with anti-m13 peroxidase antibodies
(Pharmacia), followed by colorimetric visualization with TMB
substrate. In these instances we found specific capture of the
phage by both methods, supporting high fidelity protein expression
fusion to phage by the heavy chains and stable surrogate light
chain association. The results are shown in FIG. 14.
Example 9
Antigen Binding of Surrogate Light Chain Constructs Expressed in
Mammalian Cells
[0162] As it appeared that the surrogate light chain variants
formed readily detectable complexes following nickel chelate
chromatography, their ability to bind the parent antigen of cognate
heavy chain partner was tested. Transient expression and
purification were performed as described above. Antigen binding was
tested by ELISA. Briefly, microtiter wells were coated with
inactivated H5N1 Vietnam 12-3/04 virus preparations (USFDA-CBER,
antigen standard), blocked and then incubated with quantified
serially diluted purified proteins. After washing, the complexes
were detected with anti-human Fc peroxidase conjugated antibodies.
Finally, binding was colorimetrially visualized and quantitated
with TMB substrate development (see FIG. 15).
[0163] Additionally, supernatants from transient transfections were
similarly tested undiluted for antigen binding and shown in FIG.
16. After washing, the surrogate light chain complexes were
detected with either anti-VpreB1 anti-peptide sera and anti-rabbit
peroxidase conjugated secondary or anti-human Fc peroxidase
conjugated antibodies and then colorimetrically visualized and
quantitated with TMB substrate development.
Example 10
[0164] Antigen Binding of Surrogate Light Chain Constructs
Expressed in E. coli
[0165] Because the surrogate light chain fusions appeared to form
stable complexes we wanted to establish whether such fusions paired
with a heavy chain from and anti-influenza antibody would bind the
antibody's cognate virus. To test for binding periplasmic lysates
were prepared as described above. The lysates were then subjected
to ELISA antigen binding, essentially as described above, except
binding was detected with either a monoclonal antibody to an
appended hexahistidine epitope at the C-terminus of the heavy chain
or to an appended E-tag at the C-terminus of the surrogate light
chain fusion via polyclonal affinity purified antibodies. Epitope
detection was accomplished by either anti-mouse or anti-rabbit
peroxidase conjugated antibodies. Finally, binding was
colorimetrically visualized and quantitated with TMB substrate
development. The results are shown in FIG. 17.
Example 11
Antigen Binding of Phage Displayed Surrogate Light Chain
Constructs
[0166] Because it was possible to make surrogate light chain
variants in heterologous systems, and as phage displayed
collections are desirable to future protein discovery and
engineering, we wanted to determine whether the surrogate light
chain variants and/or fusions were readily displayed on the surface
of m13 phage as gene III-associated complexes. The variants (SEQ ID
NOs: 18-22) and previously described fusions (SEQ ID NOs: 12 and
13) were coexpressed with either of two anti-influenza antibody
heavy chains (SEQ ID NOs: 19 and 14) as described above and binding
followed essentially the conditions also described above. Briefly,
microtiter wells were coated with H5N1 Vietnam 1203/04 virus and
phagemid were allowed to bind and then washed and detected directly
through anti-m13 peroxidase conjugated antibodies. Binding was
quantitatively determined following colorimetric substrate product
formation with TMB. The results are shown in FIGS. 18 and 19.
Example 12
Phage Surrogate Light Chain Construct Library Constructions,
Selection, and Clonal ELISA
[0167] An iterative approach using combinatorial antibody libraries
was employed to generate and test surrogate light chain constructs
that bound antigen. Briefly, combinatorial antibody libraries
prepared from the bone marrow of H5N1 avian influenza survivors
were created. These libraries were screened against H5N1 viral
hemagglutinin protein for two rounds of selection. Next the
phagemid plasmid was amplified and purified. Heavy chain variable
regions isolated by restriction digest from this plasmid
preparation and cloned in frame with the constant heavy domain 1 to
form a recombinant fusion to the m13 gene III coat protein for
phagemid display. Importantly, we used two recipient plasmids that
either coexpressed a surrogate light chain fusion comprised of
VpreB1 and lambda 5 (SLC fusion 1) (SEQ ID NO: 10) or VpreB1 and a
constant lambda domain (SEQ ID NO: 11) from the classical lambda
light chain (SLC fusion 2). The fusion 1 library produced
3.84.times.10.sup.7 independent transformants, while the fusion 2
library produced 7.8.times.10.sup.7 transformants. Both libraries
were screened independently through two rounds and both showed
significant enrichment over background (Fusion 1=5.times., Fusion
2=20.times.) that increased in a second round of panning (Fusion
1=97.times., Fusion 2=48.times.).
[0168] To test by ELISA for clonal antigen binding phage from both
rounds and both libraries were transferred into the HB2151 E. coli
strain to produce soluble surrobody fusion proteins. Briefly,
HB2151 clones were grown and induced to produce soluble
surrobodies. Specifically, colonies were cultured overnight in 2-YT
media supplemented with 100 mcg/ml ampicillin and 200 micromolar
IPTG overnight at 30 degrees the periplasmic lysates, as described
above. The resulting periplasmic lysates were tested by ELISA,
essentially as outlined above.
[0169] The number of transformants and percent positive clones for
the two fusions, at rounds 1 and 2 of panning are shown in FIG. 20,
and the clonal analysis data for Rounds 1 and 2 of Fusion 1 and
Fusion 2 library clones are shown in FIGS. 21 and 22.
Example 13
Surrogate Light Chain Fusions to Increase Serum Half-Life
[0170] The half-life of an antibody fragment in vivo is extended
considerably when it is part of a fusion to an intact and complete
heavy chain that includes all heavy chain constant domains, not
just those necessary to form a stable antigen binding fragment. In
the case of IgG this means the inclusion of domains CH1, CH2, and
CH3. In particular it is well established that CH2 and CH3 confer
the majority of this effect in vivo. In fact fusion of these CH2
and CH3 domains to heterologous proteins is typically sufficient to
improve the potencies and PK/PD of these chimeric molecules
compared to the parent molecules. Similarly functional fusions to
the either or both VpreB and .lamda.5 benefit by this association
with the constant domains of the heavy chain.
[0171] For the treatment of type II diabetes administration of
glucagon-like peptide 1 (or GLP-1) benefits individuals by inducing
glucose-dependent insulin secretion in the pancreas, thereby
improving glucose management in those patients. However, a
long-lived GLP-1 peptide is a desirable goal. As the tails of the
surrogate light chains are distinct and accessible, we could
accomplish this goal by either recombinantly fusing the active
GLP-1 moiety to either the C-terminus of the VpreB1 tail (SEQ ID
NOs: 23 and 24) or the N-terminal tail of .lamda.5 (SEQ ID NOs: 25
and 26). In the case of a .lamda.5 fusion we may express this in
the presence or absence of VpreB1 and even in the presence or
absence of the Variable heavy domain, as depicted in FIG. 11.
Fusions to VpreB1 can similarly be made in the presence or absence
of .lamda.5, and possibly with or without the CH1 domain of the
heavy chain. Similarly, other beneficial growth factor, cytokine,
receptor, and enzyme fusions may be created. In all of these cases
binding is not requisite of the surrogate light chain, or
SURROBODY.TM. components, but rather may be conferred either
entirely or in large part by the heterologous surrogate light chain
fused element.
[0172] Although in the foregoing description the invention is
illustrated with reference to certain embodiments, it is not so
limited. Indeed, various modifications of the invention in addition
to those shown and described herein will become apparent to those
skilled in the an from the foregoing description and fall within
the scope of the appended claims.
Example 14
[0173] Affinity determination of Hemagglutinin-Binding Surrogate
Light Chain Constructs
[0174] To determine the affinities of fusion surrogate light chain
constructs (SURROBODIES.TM., see, FIG. 11) we overexpressed and
purified various surrobodies in E coli and compared them to the
parental Fabs from which the heavy chains were first identified.
Affinities were determined by Bio-Layer Interferometry on a
BioForte Octet essentially as follows. First, 100 .mu.g of purified
hemagglutinin protein was biotinlyated at a 20:1 molar excess using
Pierce No-Weigh PEO4 biotin (cat#21329) according to manufacturer's
instructions, incubated at room temperature for 1-3 hours with
intermittent mixing and then incubated overnight at 4C. The excess
biotin was removed by size exclusion spin column and exchanged into
PBS. Next, HA binding surrogate light chain constructs and Fabs
were purified by FPLC using Ni.sup.2+ affinity chromatography,
desalted to remove excess imidazole, concentrated, and quantitated
by quantitative light chain ELISAs (Bethel Labs,
cat#E80-115-.kappa., and E80-116-.lamda.) are performed according
to the manufacturer's instructions. Finally affinities were
determined by analyzing a range of sample concentrations that are
typically 15 nM-500 nM in serial 2 fold dilutions. The samples were
incubated with biosensors coated with HA protein for up to 15
minutes, then incubated in sample diluent for up to 1 hour. All of
these steps were done with sample plate rotation at 1500 RPM.
Association was measured during the Fab incubation with the
HA-coated biosensors and dissociation is measured in the sample
diluent incubation following binding. Affinities are shown in the
following Table
TABLE-US-00001 Fusion 1 Fusion 1 Clone VpreB1-Lambda5
VpreB1-constant lambda Fab F5 250-400 pM 150-270 pM 1 pM B11 31-180
pM Not determined 13 pM
[0175] All references cited throughout the specification, and the
references cited therein, are hereby expressly incorporated by
reference in their entirety.
Sequence CWU 1
1
351145PRTHomo sapiens 1Met Ser Trp Ala Pro Val Leu Leu Met Leu Phe
Val Tyr Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro Val Leu His Gln Pro
Pro Ala Met Ser Ser Ala 20 25 30Leu Gly Thr Thr Ile Arg Leu Thr Cys
Thr Leu Arg Asn Asp His Asp 35 40 45Ile Gly Val Tyr Ser Val Tyr Trp
Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu Leu Arg Tyr
Phe Ser Gln Ser Asp Lys Ser Gln Gly65 70 75 80Pro Gln Val Pro Pro
Arg Phe Ser Gly Ser Lys Asp Val Ala Arg Asn 85 90 95Arg Gly Tyr Leu
Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Met 100 105 110Tyr Tyr
Cys Ala Met Gly Ala Arg Ser Ser Glu Lys Glu Glu Arg Glu 115 120
125Arg Glu Trp Glu Glu Glu Met Glu Pro Thr Ala Ala Arg Thr Arg Val
130 135 140Pro1452142PRTMus sp. 2Met Ala Trp Thr Ser Val Leu Leu
Met Leu Leu Ala His Leu Thr Gly1 5 10 15Cys Gly Pro Gln Pro Met Val
His Gln Pro Pro Ser Ala Ser Ser Ser 20 25 30Leu Gly Ala Thr Ile Arg
Leu Ser Cys Thr Leu Ser Asn Asp His Asn 35 40 45Ile Gly Ile Tyr Ser
Ile Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu
Leu Arg Tyr Phe Ser His Ser Asp Lys His Gln Gly65 70 75 80Pro Asp
Ile Pro Pro Arg Phe Ser Gly Ser Lys Asp Thr Ala Arg Asn 85 90 95Leu
Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Val 100 105
110Tyr Tyr Cys Ala Val Gly Leu Arg Ser His Glu Lys Lys Arg Met Glu
115 120 125Arg Glu Trp Glu Gly Glu Lys Ser Tyr Thr Asp Leu Gly Ser
130 135 1403171PRTMus sp. 3Met Ala Trp Thr Ser Val Leu Leu Met Leu
Leu Ala His Leu Thr Gly1 5 10 15Lys Gly Thr Leu Gly Val Gln Gly Phe
Leu Ala Pro Pro Val Ala Leu 20 25 30Leu Cys Pro Ser Asp Gly His Ala
Ser Ile Phe Ser Gly Cys Gly Pro 35 40 45Gln Pro Met Val His Gln Pro
Pro Ser Ala Ser Ser Ser Leu Gly Ala 50 55 60Thr Ile Arg Leu Ser Cys
Thr Leu Ser Asn Asp His Asn Ile Gly Ile65 70 75 80Tyr Ser Ile Tyr
Trp Tyr Gln Gln Arg Pro Gly His Pro Pro Arg Phe 85 90 95Leu Leu Arg
Tyr Phe Ser His Ser Asp Lys His Gln Gly Pro Asp Ile 100 105 110Pro
Pro Arg Phe Ser Gly Ser Lys Asp Thr Ala Arg Asn Leu Gly Tyr 115 120
125Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Val Tyr Tyr Cys
130 135 140Ala Val Gly Leu Arg Ser His Glu Lys Lys Arg Met Glu Arg
Glu Trp145 150 155 160Glu Gly Glu Lys Ser Tyr Thr Asp Leu Gly Ser
165 1704123PRTHomo sapiens 4Met Ala Cys Arg Cys Leu Ser Phe Leu Leu
Met Gly Thr Phe Leu Ser1 5 10 15Val Ser Gln Thr Val Leu Ala Gln Leu
Asp Ala Leu Leu Val Phe Pro 20 25 30Gly Gln Val Ala Gln Leu Ser Cys
Thr Leu Ser Pro Gln His Val Thr 35 40 45Ile Arg Asp Tyr Gly Val Ser
Trp Tyr Gln Gln Arg Ala Gly Ser Ala 50 55 60Pro Arg Tyr Leu Leu Tyr
Tyr Arg Ser Glu Glu Asp His His Arg Pro65 70 75 80Ala Asp Ile Pro
Asp Arg Phe Ser Ala Ala Lys Asp Glu Ala His Asn 85 90 95Ala Cys Val
Leu Thr Ile Ser Pro Val Gln Pro Glu Asp Asp Ala Asp 100 105 110Tyr
Tyr Cys Ser Val Gly Tyr Gly Phe Ser Pro 115 1205209PRTHomo sapiens
5Met Lys Leu Arg Val Gly Gln Thr Leu Gly Thr Ile Pro Arg Gln Cys1 5
10 15Glu Val Leu Leu Leu Leu Leu Leu Leu Gly Leu Val Asp Gly Val
His 20 25 30His Ile Leu Ser Pro Ser Ser Ala Glu Arg Ser Arg Ala Val
Gly Pro 35 40 45Gly Ala Ser Val Gly Ser Asn Arg Pro Ser Leu Trp Ala
Leu Pro Gly 50 55 60Arg Leu Leu Phe Gln Ile Ile Pro Arg Gly Ala Gly
Pro Arg Cys Ser65 70 75 80Pro His Arg Leu Pro Ser Lys Pro Gln Phe
Trp Tyr Val Phe Gly Gly 85 90 95Gly Thr Gln Leu Thr Ile Leu Gly Gln
Pro Lys Ser Asp Pro Leu Val 100 105 110Thr Leu Phe Leu Pro Ser Leu
Lys Asn Leu Gln Pro Thr Arg Pro His 115 120 125Val Val Cys Leu Val
Ser Glu Phe Tyr Pro Gly Thr Leu Val Val Asp 130 135 140Trp Lys Val
Asp Gly Val Pro Val Thr Gln Gly Val Glu Thr Thr Gln145 150 155
160Pro Ser Lys Gln Thr Asn Asn Lys Tyr Met Val Ser Ser Tyr Leu Thr
165 170 175Leu Ile Ser Asp Gln Trp Met Pro His Ser Arg Tyr Ser Cys
Arg Val 180 185 190Thr His Glu Gly Asn Thr Val Glu Lys Ser Val Ser
Pro Ala Glu Cys 195 200 205Ser6213PRTHomo sapiens 6Met Arg Pro Gly
Thr Gly Gln Gly Gly Leu Glu Ala Pro Gly Glu Pro1 5 10 15Gly Pro Asn
Leu Arg Gln Arg Trp Pro Leu Leu Leu Leu Gly Leu Ala 20 25 30Val Val
Thr His Gly Leu Leu Arg Pro Thr Ala Ala Ser Gln Ser Arg 35 40 45Ala
Leu Gly Pro Gly Ala Pro Gly Gly Ser Ser Arg Ser Ser Leu Arg 50 55
60Ser Arg Trp Gly Arg Phe Leu Leu Gln Arg Gly Ser Trp Thr Gly Pro65
70 75 80Arg Cys Trp Pro Arg Gly Phe Gln Ser Lys His Asn Ser Val Thr
His 85 90 95Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu Ser Gln Pro
Lys Ala 100 105 110Thr Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
Glu Leu Gln Ala 115 120 125Asn Lys Ala Thr Leu Val Cys Leu Met Asn
Asp Phe Tyr Pro Gly Ile 130 135 140Leu Thr Val Thr Trp Lys Ala Asp
Gly Thr Pro Ile Thr Gln Gly Val145 150 155 160Glu Met Thr Thr Pro
Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175Ser Tyr Leu
Ser Leu Thr Pro Glu Gln Trp Arg Ser Arg Arg Ser Tyr 180 185 190Ser
Cys Gln Val Met His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 195 200
205Pro Ala Glu Cys Ser 2107158PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
construct" 7Met Arg Pro Gly Thr Gly Gln Gly Gly Leu Glu Ala Pro Gly
Glu Pro1 5 10 15Gly Pro Asn Leu Arg Gln Arg Trp Pro Leu Leu Leu Leu
Gly Leu Ala 20 25 30Val Val Thr His Gly Ser Val Thr His Val Phe Gly
Ser Gly Thr Gln 35 40 45Leu Thr Val Leu Ser Gln Pro Lys Ala Thr Pro
Ser Val Thr Leu Phe 50 55 60Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
Lys Ala Thr Leu Val Cys65 70 75 80Leu Met Asn Asp Phe Tyr Pro Gly
Ile Leu Thr Val Thr Trp Lys Ala 85 90 95Asp Gly Thr Pro Ile Thr Gln
Gly Val Glu Met Thr Thr Pro Ser Lys 100 105 110Gln Ser Asn Asn Lys
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro 115 120 125Glu Gln Trp
Arg Ser Arg Arg Ser Tyr Ser Cys Gln Val Met His Glu 130 135 140Gly
Ser Thr Val Glu Lys Thr Val Ala Pro Ala Glu Cys Ser145 150
1558119PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic construct" 8Met Ser Trp Ala Pro Val Leu Leu Met
Leu Phe Val Tyr Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro Val Leu His
Gln Pro Pro Ala Met Ser Ser Ala 20 25 30Leu Gly Thr Thr Ile Arg Leu
Thr Cys Thr Leu Arg Asn Asp His Asp 35 40 45Ile Gly Val Tyr Ser Val
Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu Leu
Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln Gly65 70 75 80Pro Gln Val
Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg Asn 85 90 95Arg Gly
Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala Met 100 105
110Tyr Tyr Cys Ala Met Gly Ala 1159480PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
construct" 9Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp
Val Pro1 5 10 15Gly Ser Thr Gly Asp Ala Gln Met Gln Leu Gln Glu Ser
Gly Pro Gly 20 25 30Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly 35 40 45Tyr Ser Phe Asp Ser Gly Tyr Tyr Trp Gly Trp
Leu Arg Gln Pro Pro 50 55 60Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile
Tyr His Ser Arg Asn Thr65 70 75 80Tyr Tyr Asn Pro Ser Leu Lys Ser
Arg Val Thr Ile Ser Val Asp Thr 85 90 95Ser Lys Asn Gln Phe Ser Leu
Gln Leu Ser Ser Val Thr Ala Ala Asp 100 105 110Thr Ala Val Tyr Tyr
Cys Ala Arg Gly Thr Trp Tyr Ser Ser Asn Leu 115 120 125Arg Tyr Trp
Phe Asp Pro Trp Gly Lys Gly Thr Leu Val Arg Val Ser 130 135 140Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser145 150
155 160Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys
Asp 165 170 175Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr 180 185 190Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr 195 200 205Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln 210 215 220Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp225 230 235 240Lys Arg Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 245 250 255Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 260 265
270Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
275 280 285Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn 290 295 300Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg305 310 315 320Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val 325 330 335Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser 340 345 350Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 355 360 365Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 370 375 380Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe385 390
395 400Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 405 410 415Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe 420 425 430Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly 435 440 445Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr 450 455 460Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly His His His His His His465 470 475
48010242PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic construct" 10Met Ser Trp Ala Pro Val
Leu Leu Met Leu Phe Val Tyr Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro
Val Leu His Gln Pro Pro Ala Met Ser Ser Ala 20 25 30Leu Gly Thr Thr
Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His Asp 35 40 45Ile Gly Val
Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg
Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln Gly65 70 75
80Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg Asn
85 90 95Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala
Met 100 105 110Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Val Thr His
Val Phe Gly 115 120 125Ser Gly Thr Gln Leu Thr Val Leu Ser Gln Pro
Lys Ala Thr Pro Ser 130 135 140Val Thr Leu Phe Pro Pro Ser Ser Glu
Glu Leu Gln Ala Asn Lys Ala145 150 155 160Thr Leu Val Cys Leu Met
Asn Asp Phe Tyr Pro Gly Ile Leu Thr Val 165 170 175Thr Trp Lys Ala
Asp Gly Thr Pro Ile Thr Gln Gly Val Glu Met Thr 180 185 190Thr Pro
Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu 195 200
205Ser Leu Thr Pro Glu Gln Trp Arg Ser Arg Arg Ser Tyr Ser Cys Gln
210 215 220Val Met His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro
Ala Glu225 230 235 240Cys Ser11242PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
construct" 11Met Ser Trp Ala Pro Val Leu Leu Met Leu Phe Val Tyr
Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro Val Leu His Gln Pro Pro Ala
Met Ser Ser Ala 20 25 30Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu
Arg Asn Asp His Asp 35 40 45Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln
Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu Leu Arg Tyr Phe Ser
Gln Ser Asp Lys Ser Gln Gly65 70 75 80Pro Gln Val Pro Pro Arg Phe
Ser Gly Ser Lys Asp Val Ala Arg Asn 85 90 95Arg Gly Tyr Leu Ser Ile
Ser Glu Leu Gln Pro Glu Asp Glu Ala Met 100 105 110Tyr Tyr Cys Ala
Met Gly Ala Arg Ser Ser Val Thr His Val Phe Gly 115 120 125Ser Gly
Thr Gln Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser 130 135
140Val Thr Leu Phe Pro Pro Ser Ser Xaa Glu Leu Gln Ala Asn Lys
Ala145 150 155 160Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly
Ala Val Thr Val 165 170 175Ala Trp Lys Ala Asp Ser Ser Pro Val Lys
Ala Gly Val Glu Thr Thr 180 185 190Thr Pro Ser Lys Gln Ser Asn Asn
Lys Tyr Ala Ala Ser Ser Tyr Leu 195 200 205Ser Leu Thr Pro Glu Gln
Trp Lys Ser His Arg Ser Tyr Ser Cys Gln 210 215 220Val Thr His Glu
Gly Ser Thr Val Glu Lys Thr Val Ala Pro Ala Glu225 230 235 240Cys
Ser12256PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic construct" 12Val Lys Lys Leu Leu Leu
Phe Ala Ile Pro Leu Val Val Pro Phe Tyr1 5 10 15Ser His Ser Ala Gln
Pro Val Leu His Gln Pro Pro Ala Met Ser Ser 20 25 30Ala Leu Gly Thr
Thr Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His 35 40 45Asp Ile Gly
Val Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His 50 55 60Pro Pro
Arg Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln65 70 75
80Gly Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg
85 90 95Asn Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu
Ala 100 105 110Met Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Val Thr
His Val Phe 115 120 125Gly Ser Gly Thr Gln Leu Thr Val Leu Ser
Gln
Pro Lys Ala Thr Pro 130 135 140Ser Val Thr Leu Phe Pro Pro Ser Ser
Glu Glu Leu Gln Ala Asn Lys145 150 155 160Ala Thr Leu Val Cys Leu
Met Asn Asp Phe Tyr Pro Gly Ile Leu Thr 165 170 175Val Thr Trp Lys
Ala Asp Gly Thr Pro Ile Thr Gln Gly Val Glu Met 180 185 190Thr Thr
Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr 195 200
205Leu Ser Leu Thr Pro Glu Gln Trp Arg Ser Arg Arg Ser Tyr Ser Cys
210 215 220Gln Val Met His Glu Gly Ser Thr Val Glu Lys Thr Val Ala
Pro Ala225 230 235 240Glu Cys Ser Gly Ala Pro Val Pro Tyr Pro Asp
Pro Leu Glu Pro Arg 245 250 25513256PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
construct" 13Val Lys Lys Leu Leu Leu Phe Ala Ile Pro Leu Val Val
Pro Phe Tyr1 5 10 15Ser His Ser Ala Gln Pro Val Leu His Gln Pro Pro
Ala Met Ser Ser 20 25 30Ala Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr
Leu Arg Asn Asp His 35 40 45Asp Ile Gly Val Tyr Ser Val Tyr Trp Tyr
Gln Gln Arg Pro Gly His 50 55 60Pro Pro Arg Phe Leu Leu Arg Tyr Phe
Ser Gln Ser Asp Lys Ser Gln65 70 75 80Gly Pro Gln Val Pro Pro Arg
Phe Ser Gly Ser Lys Asp Val Ala Arg 85 90 95Asn Arg Gly Tyr Leu Ser
Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala 100 105 110Met Tyr Tyr Cys
Ala Met Gly Ala Arg Ser Ser Val Thr His Val Phe 115 120 125Gly Ser
Gly Thr Gln Leu Thr Val Leu Arg Gln Pro Lys Ala Ala Pro 130 135
140Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
Lys145 150 155 160Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro
Gly Ala Val Thr 165 170 175Val Ala Trp Lys Ala Asp Gly Ser Pro Val
Lys Ala Gly Val Glu Thr 180 185 190Thr Thr Pro Ser Lys Gln Ser Asn
Asn Lys Tyr Ala Ala Ser Ser Tyr 195 200 205Leu Ser Leu Thr Pro Glu
Gln Trp Lys Ser His Lys Ser Tyr Ser Cys 210 215 220Gln Val Thr His
Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr225 230 235 240Glu
Cys Ser Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg 245 250
25514269PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic construct" 14Met Lys Tyr Leu Leu Pro
Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met
Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Gly 20 25 30Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45Phe Pro Phe
Ser Ser Tyr Val Met Ile Trp Val Arg Gln Val Pro Gly 50 55 60Lys Gly
Leu Glu Trp Val Ser Ala Ile Gly Gly Ser Gly Gly Ser Thr65 70 75
80Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
85 90 95Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp
Asp 100 105 110Thr Ala Val Tyr Tyr Cys Val Leu Ser Pro Lys Ser Tyr
Tyr Asp Asn 115 120 125Ser Gly Ile Tyr Phe Asp Phe Trp Gly Lys Gly
Thr Leu Val Arg Val 130 135 140Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser145 150 155 160Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 165 170 175Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 180 185 190Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 195 200
205Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
210 215 220Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val225 230 235 240Asp Lys Arg Val Glu Pro Lys Ser Cys Ala Ala
Ala His His His His 245 250 255His His Gly Glu Gln Lys Leu Ile Ser
Glu Glu Asp Leu 260 26515271PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
construct" 15Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu
Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Glu
Ser Gly Gly Gly 20 25 30Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly 35 40 45Phe Pro Phe Ser Ser Tyr Val Met Ile Trp
Val Arg Gln Val Pro Gly 50 55 60Lys Gly Leu Glu Trp Val Ser Ala Ile
Gly Gly Ser Gly Gly Ser Thr65 70 75 80Tyr Tyr Ala Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95Ser Lys Asn Thr Leu Tyr
Leu Gln Met Asn Ser Leu Arg Ala Asp Asp 100 105 110Thr Ala Val Tyr
Tyr Cys Val Leu Ser Pro Lys Ser Tyr Tyr Asp Asn 115 120 125Ser Gly
Ile Tyr Phe Asp Phe Trp Gly Lys Gly Thr Leu Val Arg Val 130 135
140Ser Ser Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu Val Ser
Cys145 150 155 160Glu Asn Ser Pro Ser Asp Thr Ser Ser Val Ala Val
Gly Cys Leu Ala 165 170 175Gln Asp Phe Leu Pro Asp Ser Ile Thr Phe
Ser Trp Lys Tyr Lys Asn 180 185 190Asn Ser Asp Ile Ser Ser Thr Arg
Gly Phe Pro Ser Val Leu Arg Gly 195 200 205Gly Lys Tyr Ala Ala Thr
Ser Gln Val Leu Leu Pro Ser Lys Asp Val 210 215 220Met Gln Gly Thr
Asp Glu His Val Val Cys Lys Val Gln His Pro Asn225 230 235 240Gly
Asn Lys Glu Lys Asn Val Pro Leu Pro Val Ala Ala Ala His His 245 250
255His His His His Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 260
265 2701613PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 16Gly Ala Pro Val Pro Tyr
Pro Asp Pro Leu Glu Pro Arg1 5 101712PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 17Gly Glu Gln Lys Leu Ile Ser Leu Glu Glu Asp Leu1 5
1018159PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic construct" 18Val Lys Lys Leu Leu Leu Phe Ala Ile
Pro Leu Val Val Pro Phe Tyr1 5 10 15Ser His Ser Ala Gln Pro Val Leu
His Gln Pro Pro Ala Met Ser Ser 20 25 30Ala Leu Gly Thr Thr Ile Arg
Leu Thr Cys Thr Leu Arg Asn Asp His 35 40 45Asp Ile Gly Val Tyr Ser
Val Tyr Trp Tyr Gln Gln Arg Pro Gly His 50 55 60Pro Pro Arg Phe Leu
Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln65 70 75 80Gly Pro Gln
Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg 85 90 95Asn Arg
Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala 100 105
110Met Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Glu Lys Glu Glu Arg
115 120 125Glu Arg Glu Trp Glu Glu Glu Met Glu Pro Thr Ala Ala Arg
Thr Arg 130 135 140Val Pro Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu
Glu Pro Arg145 150 15519120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
construct" 19Val Lys Lys Leu Leu Leu Phe Ala Ile Pro Leu Val Val
Pro Phe Tyr1 5 10 15Ser His Ser Ala Gln Pro Val Leu His Gln Pro Pro
Ala Met Ser Ser 20 25 30Ala Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr
Leu Arg Asn Asp His 35 40 45Asp Ile Gly Val Tyr Ser Val Tyr Trp Tyr
Gln Gln Arg Pro Gly His 50 55 60Pro Pro Arg Phe Leu Leu Arg Tyr Phe
Ser Gln Ser Asp Lys Ser Gln65 70 75 80Gly Pro Gln Val Pro Pro Arg
Phe Ser Gly Ser Lys Asp Val Ala Arg 85 90 95Asn Arg Gly Tyr Leu Ser
Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala 100 105 110Met Tyr Tyr Cys
Ala Met Gly Ala 115 12020133PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
construct" 20Val Lys Lys Leu Leu Leu Phe Ala Ile Pro Leu Val Val
Pro Phe Tyr1 5 10 15Ser His Ser Ala Gln Pro Val Leu His Gln Pro Pro
Ala Met Ser Ser 20 25 30Ala Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr
Leu Arg Asn Asp His 35 40 45Asp Ile Gly Val Tyr Ser Val Tyr Trp Tyr
Gln Gln Arg Pro Gly His 50 55 60Pro Pro Arg Phe Leu Leu Arg Tyr Phe
Ser Gln Ser Asp Lys Ser Gln65 70 75 80Gly Pro Gln Val Pro Pro Arg
Phe Ser Gly Ser Lys Asp Val Ala Arg 85 90 95Asn Arg Gly Tyr Leu Ser
Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala 100 105 110Met Tyr Tyr Cys
Ala Met Gly Ala Gly Ala Pro Val Pro Tyr Pro Asp 115 120 125Pro Leu
Glu Pro Arg 13021198PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic construct" 21Met Lys Lys Thr Ala
Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala1 5 10 15Thr Val Ala Gln
Ala Ala Leu Leu Arg Pro Thr Ala Ala Ser Gln Ser 20 25 30Arg Ala Leu
Gly Pro Gly Ala Pro Gly Gly Ser Ser Arg Ser Ser Leu 35 40 45Arg Ser
Arg Trp Gly Arg Phe Leu Leu Gln Arg Gly Ser Trp Thr Gly 50 55 60Pro
Arg Cys Trp Pro Arg Gly Phe Gln Ser Lys His Asn Ser Val Thr65 70 75
80His Val Phe Gly Ser Gly Thr Gln Leu Thr Val Leu Ser Gln Pro Lys
85 90 95Ala Thr Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu
Gln 100 105 110Ala Asn Lys Ala Thr Leu Val Cys Leu Met Asn Asp Phe
Tyr Pro Gly 115 120 125Ile Leu Thr Val Thr Trp Lys Ala Asp Gly Thr
Pro Ile Thr Gln Gly 130 135 140Val Glu Met Thr Thr Pro Ser Lys Gln
Ser Asn Asn Lys Tyr Ala Ala145 150 155 160Ser Ser Tyr Leu Ser Leu
Thr Pro Glu Gln Trp Arg Ser Arg Arg Ser 165 170 175Tyr Ser Cys Gln
Val Met His Glu Gly Ser Thr Val Glu Lys Thr Val 180 185 190Ala Pro
Ala Glu Cys Ser 19522143PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
construct" 22Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala
Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Ala Ser Val Thr His Val Phe
Gly Ser Gly Thr 20 25 30Gln Leu Thr Val Leu Ser Gln Pro Lys Ala Thr
Pro Ser Val Thr Leu 35 40 45Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala
Asn Lys Ala Thr Leu Val 50 55 60Cys Leu Met Asn Asp Phe Tyr Pro Gly
Ile Leu Thr Val Thr Trp Lys65 70 75 80Ala Asp Gly Thr Pro Ile Thr
Gln Gly Val Glu Met Thr Thr Pro Ser 85 90 95Lys Gln Ser Asn Asn Lys
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr 100 105 110Pro Glu Gln Trp
Arg Ser Arg Arg Ser Tyr Ser Cys Gln Val Met His 115 120 125Glu Gly
Ser Thr Val Glu Lys Thr Val Ala Pro Ala Glu Cys Ser 130 135
14023175PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic construct" 23Met Ser Trp Ala Pro Val
Leu Leu Met Leu Phe Val Tyr Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro
Val Leu His Gln Pro Pro Ala Met Ser Ser Ala 20 25 30Leu Gly Thr Thr
Ile Arg Leu Thr Cys Thr Leu Arg Asn Asp His Asp 35 40 45Ile Gly Val
Tyr Ser Val Tyr Trp Tyr Gln Gln Arg Pro Gly His Pro 50 55 60Pro Arg
Phe Leu Leu Arg Tyr Phe Ser Gln Ser Asp Lys Ser Gln Gly65 70 75
80Pro Gln Val Pro Pro Arg Phe Ser Gly Ser Lys Asp Val Ala Arg Asn
85 90 95Arg Gly Tyr Leu Ser Ile Ser Glu Leu Gln Pro Glu Asp Glu Ala
Met 100 105 110Tyr Tyr Cys Ala Met Gly Ala Arg Ser Ser Glu Lys Glu
Glu Arg Glu 115 120 125Arg Glu Trp Glu Glu Glu Met Glu Pro Thr Ala
Ala Arg Thr Arg Val 130 135 140Pro His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu145 150 155 160Gly Gln Ala Ala Lys Glu
Phe Ile Ala Trp Leu Val Lys Gly Arg 165 170 17524149PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
construct" 24Met Ser Trp Ala Pro Val Leu Leu Met Leu Phe Val Tyr
Cys Thr Gly1 5 10 15Cys Gly Pro Gln Pro Val Leu His Gln Pro Pro Ala
Met Ser Ser Ala 20 25 30Leu Gly Thr Thr Ile Arg Leu Thr Cys Thr Leu
Arg Asn Asp His Asp 35 40 45Ile Gly Val Tyr Ser Val Tyr Trp Tyr Gln
Gln Arg Pro Gly His Pro 50 55 60Pro Arg Phe Leu Leu Arg Tyr Phe Ser
Gln Ser Asp Lys Ser Gln Gly65 70 75 80Pro Gln Val Pro Pro Arg Phe
Ser Gly Ser Lys Asp Val Ala Arg Asn 85 90 95Arg Gly Tyr Leu Ser Ile
Ser Glu Leu Gln Pro Glu Asp Glu Ala Met 100 105 110Tyr Tyr Cys Ala
Met Gly Ala His Ala Glu Gly Thr Phe Thr Ser Asp 115 120 125Val Ser
Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp 130 135
140Leu Val Lys Gly Arg14525243PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
construct" 25Met Arg Pro Gly Thr Gly Gln Gly Gly Leu Glu Ala Pro
Gly Glu Pro1 5 10 15Gly Pro Asn Leu Arg Gln Arg Trp Pro Leu Leu Leu
Leu His Ala Glu 20 25 30Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly Gln Ala Ala 35 40 45Lys Glu Phe Ile Ala Trp Leu Val Lys Gly
Arg Gly Leu Ala Val Val 50 55 60Thr His Gly Leu Leu Arg Pro Thr Ala
Ala Ser Gln Ser Arg Ala Leu65 70 75 80Gly Pro Gly Ala Pro Gly Gly
Ser Ser Arg Ser Ser Leu Arg Ser Arg 85 90 95Trp Gly Arg Phe Leu Leu
Gln Arg Gly Ser Trp Thr Gly Pro Arg Cys 100 105 110Trp Pro Arg Gly
Phe Gln Ser Lys His Asn Ser Val Thr His Val Phe 115 120 125Gly Ser
Gly Thr Gln Leu Thr Val Leu Ser Gln Pro Lys Ala Thr Pro 130 135
140Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn
Lys145 150 155 160Ala Thr Leu Val Cys Leu Met Asn Asp Phe Tyr Pro
Gly Ile Leu Thr 165 170 175Val Thr Trp Lys Ala Asp Gly Thr Pro Ile
Thr Gln Gly Val Glu Met 180 185 190Thr Thr Pro Ser Lys Gln Ser Asn
Asn Lys Tyr Ala Ala Ser Ser Tyr 195 200 205Leu Ser Leu Thr Pro Glu
Gln Trp Arg Ser Arg Arg Ser Tyr Ser Cys 210 215 220Gln Val Met His
Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Ala225 230 235 240Glu
Cys Ser26188PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic construct" 26Met Arg Pro Gly Thr Gly
Gln Gly Gly Leu Glu Ala Pro Gly Glu
Pro1 5 10 15Gly Pro Asn Leu Arg Gln Arg Trp Pro Leu Leu Leu Leu Gly
His Ala 20 25 30Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
Gly Gln Ala 35 40 45Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
Leu Ala Val Val 50 55 60Thr His Gly Ser Val Thr His Val Phe Gly Ser
Gly Thr Gln Leu Thr65 70 75 80Val Leu Ser Gln Pro Lys Ala Thr Pro
Ser Val Thr Leu Phe Pro Pro 85 90 95Ser Ser Glu Glu Leu Gln Ala Asn
Lys Ala Thr Leu Val Cys Leu Met 100 105 110Asn Asp Phe Tyr Pro Gly
Ile Leu Thr Val Thr Trp Lys Ala Asp Gly 115 120 125Thr Pro Ile Thr
Gln Gly Val Glu Met Thr Thr Pro Ser Lys Gln Ser 130 135 140Asn Asn
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln145 150 155
160Trp Arg Ser Arg Arg Ser Tyr Ser Cys Gln Val Met His Glu Gly Ser
165 170 175Thr Val Glu Lys Thr Val Ala Pro Ala Glu Cys Ser 180
18527105PRTHomo sapiens 27Gly Ser Gln Ser Val Leu Thr Gln Pro Pro
Ser Val Ser Ala Ala Pro1 5 10 15Gly Gln Lys Val Thr Ile Ser Cys Ser
Gly Ser Ser Ser Asn Ile Gly 20 25 30Asn Asn Tyr Val Ser Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys 35 40 45Leu Leu Ile Tyr Asp Asn Asn
Lys Arg Pro Ser Gly Ile Pro Asp Arg 50 55 60Phe Ser Gly Ser Lys Ser
Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly65 70 75 80Leu Gln Thr Gly
Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser 85 90 95Ser Leu Ser
Ala Val Val Phe Gly Gly 100 10528114PRTHomo sapiens 28Gly Gly Thr
Lys Leu Thr Val Leu Arg Gln Pro Lys Ala Ala Pro Ser1 5 10 15Val Thr
Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala 20 25 30Thr
Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val 35 40
45Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly Val Glu Thr Thr
50 55 60Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr
Leu65 70 75 80Ser Leu Thr Pro Glu Gln Trp Lys Ser His Lys Ser Tyr
Ser Cys Gln 85 90 95Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
Ala Pro Thr Glu 100 105 110Cys Ser29105PRTHomo sapiens 29Gln Pro
Val Leu Thr Gln Pro Ser Ser His Ser Ala Ser Ser Gly Ala1 5 10 15Ser
Val Arg Leu Thr Cys Met Leu Ser Ser Gly Phe Ser Val Gly Asp 20 25
30Phe Trp Ile Arg Trp Tyr Gln Gln Lys Pro Gly Asn Pro Pro Arg Tyr
35 40 45Leu Leu Tyr Tyr His Ser Asp Ser Asn Lys Gly Gln Gly Ser Gly
Val 50 55 60Pro Ser Arg Phe Ser Gly Ser Asn Asp Ala Ser Ala Asn Ala
Gly Ile65 70 75 80Leu Arg Ile Ser Gly Leu Gln Pro Glu Asp Glu Ala
Asp Tyr Tyr Cys 85 90 95Gly Thr Trp His Ser Asn Ser Lys Thr 100
10530104PRTHomo sapiens 30Gln Ala Val Leu Thr Gln Pro Ala Ser Leu
Ser Ala Ser Pro Gly Ala1 5 10 15Ser Ala Ser Leu Thr Cys Thr Leu Arg
Ser Gly Ile Asn Val Gly Thr 20 25 30Tyr Arg Ile Tyr Trp Tyr Gln Gln
Lys Pro Gly Ser Pro Pro Gln Tyr 35 40 45Leu Leu Arg Tyr Lys Ser Asp
Ser Asp Lys Gln Gln Gly Ser Gly Val 50 55 60Pro Ser Arg Phe Ser Gly
Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile65 70 75 80Leu Leu Ile Ser
Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90 95Met Ile Trp
His Ser Ser Ala Ser 10031104PRTHomo sapiens 31Gln Pro Val Leu Thr
Gln Pro Pro Ser Ser Ser Ala Ser Pro Gly Glu1 5 10 15Ser Ala Arg Leu
Thr Cys Thr Leu Pro Ser Asp Ile Asn Val Gly Ser 20 25 30Tyr Asn Ile
Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Arg Tyr 35 40 45Leu Leu
Tyr Tyr Tyr Ser Asp Ser Asp Lys Gly Gln Gly Ser Gly Val 50 55 60Pro
Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Thr Gly Ile65 70 75
80Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
85 90 95Met Ile Trp Pro Ser Asn Ala Ser 10032113PRTHomo sapiens
32Gly Thr Lys Leu Thr Val Leu Arg Gln Pro Lys Ala Ala Pro Ser Val1
5 10 15Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala
Thr 20 25 30Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr
Val Ala 35 40 45Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly Val Glu
Thr Thr Thr 50 55 60Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser
Ser Tyr Leu Ser65 70 75 80Leu Thr Pro Glu Gln Trp Lys Ser His Lys
Ser Tyr Ser Cys Gln Val 85 90 95Thr His Glu Gly Ser Thr Val Glu Lys
Thr Val Ala Pro Thr Glu Cys 100 105 110Ser33107PRTHomo sapiens
33Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1
5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 100 105346PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic 6xHis tag" 34His His His His His His1
53515PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 35Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser1 5 10 15
* * * * *
References