Note: Descriptions are shown in the official language in which they were submitted.
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PROTEIN SCAFFOLDS AND USES THEREOF
CROSS-REFERENCES TO RELATED APPLICATIONS
{01] The present application claims the benefit of U.S. Provisional Patent
Application No. 60/628,596, filed November 16, 2004 and is a continuation in
part of U.S.S.
N. 10/871602, filed June 17, 2004, which is a continuation-in-part application
of U.S.S.N.
10/840,723, filed May 5, 2004, which is a continuation-in-part application of
U.S.S.N.
10/693,056, filed October 24, 2003 and a continuation-in-part of U.S.S.N.
10/693,057, filed
October 24, 2003, both of which are continuations-in-part of
U.S.S.N.10/289,660, filed
November 6, 2002, which is a continuation-in-part application of U.S.S.N.
10/133,128, filed
April 26, 2002, which claims benefit of priority to U.S.S.N. 60/374,107, filed
April 18, 2002,
U.S.S.N. 60/333,359, filed November 26, 2001, U.S.S.N. 60/337,209, filed
November 19,
2001, and U.S.S.N. 60/286,823, filed April 26, 2001, all of which are
incorporated herein by
reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION
[02] Analysis of protein sequences and three-dimensional structures have
revealed that many proteins are composed of a number of discrete monomer
domains. Such
proteins are often called 'mosaic proteins' because they are a linear mosaic
of recurring
building blocks. The majority of discrete monomer domain proteins is
extracellular or
constitutes the extracellular parts of membrane-bound proteins.
[03] An important characteristic of a discrete monomer domain is its ability
to fold independently of the other domains in the same protein. Folding of
these domains
may require limited assistance from, e.g., a chaperonin(s) (e.g., a receptor-
associated protein
(RAP)), a metal ion(s), or a co-factor. The ability to fold independently
prevents misfolding
of the domain when it is inserted into a new protein or a new environment.
This
characteristic has allowed discrete monomer domains to be evolutionarily
mobile. As a
result, discrete domains have spread during evolution and now occur in
otherwise unrelated
proteins. Some domains, including the fibronectin type III domains and the
immunoglobin-
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like domain, occur in numerous proteins, while other domains are only found in
a limited
number of proteins.
[04] Proteins that contain these domains are involved in a variety of
processes, such as cellular transporters, cholesterol movement, signal
transduction and
signaling functions which are involved in development and neurotransmission.
See Herz,
(2001) Trends in Neurosciences 24(4):193-195; Goldstein and Brown, (2001)
Science 292:
1310-1312. The function of a discrete monomer domain is often specific but it
also
contributes to the overall activity of the protein or polypeptide. For
example, the LDL-
receptor class A domain (also referred to as a class A module, a complement
type repeat or an
A-domain) is involved in ligand binding while the gamma-carboxyglumatic acid
(Gla)
domain which is found in the vitamin-K-dependent blood coagulation proteins is
involved in
high-affinity binding to phospholipid membranes. Other discrete monomer
domains include,
e.g., the epidermal growth factor (EGF)-like domain in tissue-type plasminogen
activator
which mediates binding to liver cells and thereby regulates the clearance of
this fibrinolytic
enzyme from the circulation and the cytoplasmic tail of the LDL-receptor which
is involved
in receptor-mediated endocytosis.
[05] Individual proteins can possess one or more discrete monomer
domains. Proteins containing a large number of recurring domains are often
called mosaic
proteins. For example, members of the LDL-receptor family contain a large
number of
domains belonging to four major families: the cysteine rich A-domain repeats,
epidermal
growth factor precursor-like repeats, a transmembrane domain and a cytoplasmic
domain.
The LDL-receptor family includes members that: 1) are cell-surface receptors;
2) recognize
extracellular ligands; and 3) internalize them for degradation by lysosomes.
See Hussain et
al., (1999) Annu. Rev. Nutr. 19:141-72. For example, some members include very-
low-
density lipoprotein receptors (VLDL-R), apolipoprotein E receptor 2, LDLR-
related protein
(LRP) and megalin. Family members have the following characteristics: 1) cell-
surface
expression; 2) extracellular ligand binding mediated by A-domains; 3)
requirement of
calcium for folding and ligand binding; 4) recognition of receptor-associated
protein and
apolipoprotein (apo) E; 5) epidermal growth factor (EGF) precursor homology
domain
containing YWTD repeats; 6) single membrane-spanning region; and 7) receptor-
mediated
endocytosis of various ligands. See Hussain, supra. These family members bind
several
structurally dissimilar ligands.
[06] It is advantageous to develop methods for generating and optimizing
the desired properties of these discrete monomer domains. However, the
discrete monomer
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domains, while often being structurally conserved, are not conserved at the
nucteotide or
amino acid level, except for certain amino acids, e.g., the cysteine residues
in the A-domain.
Thus, existing nucleotide recombination methods fall short in generating and
optimizing the
desired properties of these discrete monomer domains.
[07] The present invention addresses these and other problems.
BRIEF SUMMARY OF THE INVENTION
[08] The present invention provide proteins comprising monomer domains
that specifically bind to target molecules, polynucleotides encoding the
proteins, methods of
using such proteins, methods of identifying monomer domains for use in such
proteins, and
libraries comprising monomer domains.
[09] One embodiment of the invention provides proteins comprising a non-
naturally occurring monomer domain that specifically binds to a target
molecule. The
monomer domain is 3 0-100 amino acids in length and is selected from a
thrombospondin
monomer domain and a thyroglobulin monomer domain. In some embodiments, the
the
monomer domain comprises at least one, two, three, or more disulfide bonds In
some
embodiments, Cl-C5, C2-C6 and C3-C4 of the thrombospondin monomer domain form
disulfide bonds and Ci-C2a C3-C4 and CS-C6 of the thyroglobulin monomer domain
form
disulfide bonds. In some embodiments, the thrombospondin monomer domain
sequence
comprises no more than three point insertions, mutations, or deletions from
the following
sequence:
(wxxWxx)C1sxtC2xxGxx(x)xRxixC3xxxx(Pxx)xxxxxC4xxxxxx(x)xxxC$(x)xxxxC6, and the
thyroglobulin monomer domain comprises no more than three point insertions,
mutations, or
deletions from the following sequence:
Cixxxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxxyxPxC2xxxGxyxxxQC3x(x)s(xxx)xxgxC4WC5Vd
xx(x)GxxxxGxxxxxgxx(xx)xC6; wherein "x" is any amino acid. In some
embodiments, the
thrombospondin monomer domain comprises the following sequence:
(wxxWxx)C1sxtC2xxGxx(x)xRxrxC3xxxx(Pxx)xxxxxC4xxxxxx(x)xxxCs(x)xxxxC6a and the
thyroglobulin monomer domain comprises n the following sequence:
Clxxxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxxyxPxC2xxxGxyxxxQC3x(x)s(xxx)xxgxC4WC5Vd
xx(x)GxxxxGxxxxxgxx(xx)xC6; wherein "x" is any amino acid. In some
embodiments, the
thrombospondin monomer domain sequence comprises no more than three point
insertions,
mutations, or deletions from the following sequence:
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(WxxWxx)C i [Stnd] [Vkaq]
[Tspl]C2xx[Gq]xx(x)x[Re]x[Rktvm]x[C3vldr]xxxx([Pq]xx)xxxxx[
C41dae]xXxxxx(x)xxxCg(x)xxxxC6, wherein Ci-C5, C2-C6 and C3-C4 form disulfide
bonds; the
thyroglobulin monomer domain sequence comprises no more than three point
insertions,
mutations, or deletions from the following sequence:
Ci[qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxx[ahp]xPxC2xxxGx[a]xx[vkrl]QC3x(x[sa]xx
x
)xx[gas]xC4[a]CSV[Dna]xx(x)Gxxxx[~g]xxxxxgxx(xx)xC6, wherein Cl-C2, C3-C4 and
C5-C6
form disulfide bonds; a is selected from: w, y, f, and 1; ~ is selected from:
d, e, and n; and
"x" is selected from any amino acid. In some embodiments, the thrombospondin
monomer
domain comprises the following sequence:
(WxxWxx)Ci [Stnd] [Vkaq]
[Tspl]C2xx[Gq]xx(x)x[Re]x[Rktvm]x[C3vldr]xxxx([Pq]xx)xxxxx[
C41dae]xxxXxx(x)XxXC5(x)xxxxC6, wherein Ci-C5, C2-C6 and C3-C4 form disulfide
bonds; the
thyroglobulin monomer domain comprises the following sequence:
Cl
[qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxx[ahp]xPxC2xxxGx[a]xx[vkrl]QC3x(x[sa]xxx
)xx[gas]xC4[a]CSV[Dna]xx(x)Gxxxx[~g]xxxxxgxx(xx)xC6, wherein Cl-Ca, C3-C4 and
CS-C6
form disulfide bonds; and a is selected from: w, y, f, and 1; ~ is selected
from: d, e, and n;
and "x" is selected from any amino acid. In some embodiments, the
thrombospondin
monomer domain sequence comprises no more than three point insertions,
mutations, or
deletions from the following sequence:
Cl[nst] [aegiklqrstv]
[adenpqrst]Ca[adetgs]xgx[ikqrstv]x[aqrst]x[ahnrtv]xC3xxxxxxxxx(xxxxx
Xx)C4XXXXxxxxX(xx)CSXxXXC6; the thyroglobulin monomer domain sequence
comprises no
more than three point insertions, mutations, or deletions from the following
sequence:
C1 [qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxx[Yfhp]xPxC2xxxGx[Yf]xx[vkrl]
QC3x(x[sa]x
xx)xx[Gsa]xC4[WyfJCSV[Dnyfl]xx(x)Gxxxx[Gdne]xxxxxgxx(xx)xC6. In some
embodiments, the thrombospondin monomer comprises the following sequence:
Cl[nst][aegiklqrstv][adenpqrst]C2[adetgs]xgx[ikqrstv]x[aqrst]x[almrtv]xC3xxxxxx
xxx(xxxxx
XX)C4XXXXXXXXX(XX)C5XXXXC6; and the thyroglobulin monomer domain sequence
comprises
the following sequence:
C l [qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxx[Yf hp]xPxC2xxxGx[Yf]xx[vkrl]
QC3x(x[sa]x
xx)xx[Gsa]xC4[Wyf] C5V [Dnyfl]xx(x)Gxx.xx[Gdne]xxxxxgxx(xx)xC6.
110] The invention also provides a protein, comprising a non-naturally
occurring monomer domain that specifically binds to a target molecule. The
target molecule
is not bound by a naturally-occurring monomer domain that is at least 75%,
80%, 85%, 90%,
85%, 98%, or 99% identical to the non-naturally occurring monomer domain and
the non-
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naturally occurring monomer domain is selected from a thrombospondin monomer
domain, a
trefoil monomer domain, and a thyroglobulin monomer domain. In some
embodiments, the
monomer domain comprises at least one, two, three, or more disulfide bonds. In
some
embodiments, the monomer domain is 30-100 amino acids in length. In some
embodiments,
the thrombospondin monomer domain comprises the following sequence:
(wxxWxx)C1sxtC2xxGxx(x)xRxrxC3xxxx(Pxx)xxxxxC4xxxxxx(x)xxxCs(x)xxxxC6; the
trefoil monomer domain comprises the following sequence:
Ci(xx)xxxpxxRxnCagx(x)pxibcxxC3xxxgC4C5fdxxx(x)xxxpwC6f; and the thyroglobulin
monomer domain comprises the following sequence:
Cixxxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxxyxPxC2xxxGxyxxxQC3x(x)s(xxx)xxgxC4WCsVd
xx(x)GxxxxGxxxxxgxx(xx)xC and "x" is any amino acid. In some embodiments, Cl-
Cs,
C2-C6 and C3-C4 of the thrombospondin monomer domain form disulfide bonds; and
Cl-C2a
C3-C4 and C5-C6 of the thyroglobulin monomer domain form disulfide bonds. In
some
embodiments, the thrombospondin monomer domain comprises the following
sequence:
(WxxWxx)Cl [Stnd] [Vkaq]
[Tspl]C2xx[Gq]xx(x)x[Re]x[Rktvrn]x[C3vldr]xxxx([Pq]xx)xxxxx[
C41dae]xXxxxx(x)xxxCg(x)xxxxC6, wherein C1-C5, C2-C6 and C3-C4 form disulfide
bonds; the
trefoil monomer domain comprises the following sequence:
Cl(xx)xxx[Pvae]xxRx[ndpm]Ca[Gaiy]
[ypfst]([de]x)[pskq]x[Ivap][Tsa]xx[keqd]C3xx[krln] [G
nk]C4Cs[a][Dnrs][sdpnte]xx(x)xxx[pki][Weash]C[Fy]; the thyroglobulin monomer
domain
comprises the following sequence:
C 1 [qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxXxxxx[ahp]xPxCaxxxGx[a]xx[vkrl]
QC3x(x[sa]xxx
)xx[gas]xC4[a]C5V[Dna]xx(x)Gxxxx[~g]xxxxxgXX(xx)xC6, wherein CI-Ca, C3-C4 and
C5-C6
form disulfide bonds; and a is selected from: w, y, f, and 1; ~ is selected
from: d, e, and n;
and "x" is selected from any amino acid. In some embodiments, the
thrombospondin
monomer comprises the following sequence:
C1 [nst] [aegiklqrstv] [adenpqrst]
C2[adetgs]xgx[ikqrstv]x[aqrst]x[almrtv]xC3xxxxxxxxx(xxxxx
xx)C4xxxxxxxxx(xx)C5xxxxC6; the trefoil monomer domain comprises the following
sequence:
Cl([dnps])[adiklnprstv] [dfilmv] [adenprst] [adelprv] [ehklnqrs] [adegknsv]
[kqr] [fiiklqrtv] [dnpqs
]C2[agiy][flpsvy][dknpqs][adfghlp][aipv][st][aegkpqrs][adegkpqs][deiknqt]C3[ade
flcnqrt][ade
gknqs] [gn]C4C5[wyfh] [deinrs] [adgnpst] [aefgqlrstw] [giknsvmq]([afinprstv]
[degklns] [afiqstv] [
iknpv]w)C6; and the thyroglobulin monomer comprises the following sequence:
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Cl[qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxx[Yflip]xPxC2xxxGx[Yfjxx[vkrl]QC3x(x[sa
]x
xx)xx[Gsa]xC4[Wyf] C5V[Dnyfl]xx(x)Gxxxx[Gdne]xXxxxgxx(xx)xC6.
j11] The invention further provides a composition comprising at least two
monomer domains, wherein at least one monomer domain is a non-naturally
occurring
monomer domain and the monomer domains bind an ion and at least one monomer
domain is
selected from: a thrombospondin monomer domain, a trefoil monomer domain, and
a
thyroglobulin monomer domain. In some embodiments, at least one of the two
monomer
domains is less than about 50 kD. In some embodiments, the two domains are
linked by a
peptide linker. In some embodiments, wherein the linker is heterologous to at
least one of the
monomer domains. In some embodiments, the thrombospondin monomer domain
comprises
the following sequence:
(wxxWxx)CisxtC2xxGxx(x)xRxrxC3xxxx(Pxx)xxxxxC4xxxxxx(x)xxxC5(x)xxxxC6i he
trefoil
monomer domain comprises the following sequence:
Cl(xx)xxxpxxRxnCagx(x)pxitxxxC3xxxgC4C5fdxXx(x)xxxpwC6f; and the thyroglobulin
monomer domain comprises the following sequence:
Clxxxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxxyxPxC2xxxGxyxxxQC3x(x)s(xxx)xxgxC4WC5Vd
xx(x)GxxxxGxxxxxgxx(xx)xC6; and "x" is any amino acid. In some embodiments, Cl-
C5,
C2-C6 and C3-C4 of the thrombospondin monomer domain form disulfide bonds; and
Cl-C2,
C3-C4 and C5-C6 of the thyroglobulin monomer domain form disulfide bonds. In
some
embodiments, the thrombospondin monomer domain comprises the following
sequence:
(WxxWxx)C1 [Stnd] [Vkaq]
[Tspl]C2xx[Gq]xx(x)x[Re]x[Rktvrn]x[C3vldr]xxxx([Pq]xx)xxxxx[
C4ldae]xxxXxx(x)xXxC5(X)xxxxC6a wherein C1-C5, CrC6 and C3-C4 form disulfide
bonds; the
trefoil monomer domain comprises the following sequence:
C1(xx)xxx[Pvae]xxRx[ndpm]Ca[Gaiy] [ypfst]([de]x)[pskq]x[Ivap]
[Tsa]xx[keqd]C3xx[krln][G
nk]C4C5[a][Dnrs][sdpnte]xx(x)xxx[pki][Weash]C6[Fy]; the thyroglobulin monomer
domain
comprises the following sequence:
Cl [qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxxjahp]xPxC2xxxGx[a]xx[vkrl]
QC3x(x[sa]xxx
)xx[gas]xC4[a]C5V[Dna]xx(x)Gxxxx[og]xxxxxgxx(xx)xC6, wherein Ci-C2, C3-C4 and
C5-C6
form disulfide bonds; and and a is selected from: w, y, f, and 1; 0 is
selected from: d, e, and
n; and "x" is selected from any amino acid. In some embodiments, the
thrombospondin
monomer comprises the following sequence:
Cl [nst] [aegiklqrstv]
[adenpqrst]C2[adetgs]xgx[ikqrstv]x[aqrst]x[almrtv]xC3xxxxxxxxx(xxxxx
XX)C4XXXXXXXXX(XX)C5XXXXC6i the trefoil monomer domain comprises the following
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sequence:
C l ([dnps])[ adiklnprstv] [dfilmv] [adenprst] [ adelprv] [ehklnqrs]
[adegknsv] [kqr] [fiklqrtv] [dnpqs
] C2[agiy] [flpsvy] [dknpqs] [adfghlp] [aipv] [st] [aegkpqrs] [adegkpqs]
[deiknqt] C3 [adeflcnqrt] [ade
gknqs] [gn]Ca.C5[wyfh] [deinrs] [adgnpst] [aefgqlrstw]
[giknsvmq]([afinprstv][degklns] [afiqstv] [
iknpv]w)C6i and the thyroglobulin monomer comprises the following sequence:
C i [qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxx[Yfhp]xPxC2xxxGx[Yf]xx[vkrl]
QC3x(x[sa]x
xx)xx[Gsa]xC4[Wyf]C5 V[Dnyfl]xx(x)Gxxxx[Gdne]xxxxxgxx(xx)xC6.
[12] The invention further provides isolated polynucleotides encoding the
proteins described herein and cells comprising the polynucleotides.
[13] The invention also provides methods for identifying a monomer
domain that binds to a target molecule by: (1) providing a library of non-
naturally-occurring
monomer domains, wherein the monomer domain is selected from: a thrombospondin
monomer domain, a trefoil monomer domain, and a thyroglobulin monomer domain,
wherein
the thrombospondin monomer domain comprises the following sequence:
(wxxWxx)CzsxtC2xxGxx(x)xRxrxC3xxxx(Pxx)xxxxxC4xxxxxx(x)xxxC5(x)xxxxC6i the
trefoil monomer domain comprises the following sequence:
Cl(xx)xxxpxxRxnC2gx(x)pxitxxxC3xxxgC4C5fdxxx(x)xxxpwC6f; and the thyroglobulin
monomer domain comprises the following sequence:
C lxxxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxxyxPxCaxxxGxyxxxQC3x(x)s(xxx)xxgxC4WC5Vd
xx(x)GxxxxGxxxxxgxx(xx)xC6; and "x" is any amino acid; (2) screening the
library of
monomer domains for affinity to a first target molecule; and (3)identifying at
least one
monomer domain that binds to at least one target molecule. In some
embodiments, the at
least one monomer domain specifically binds to a target molecule that is not
bound by a
naturally-occurring monomer domain that is at least 90% identical to the non-
naturally
occurring monomer domain. In some embodiments, Cl-C5a C2-C6 and C3-C4 of the
thrombospondin monomer domain form disulfide bonds; and Cl-C2, C3-C4 and C5-C6
of the
thyroglobulin monomer domain form disulfide bonds. In some embodiments, the
thrombospondin monomer domain comprises the following sequence:
(WxxWxx)Ci [Stnd] [Vkaq] [Tspl]
C2xx[Gq]xx(x)x[Re]x[Rktvm]x[C3vldr]xxxx([Pq]xx)xxxxx[
C41dae]xxxxxx(x)xxxCs(x)xxxxC6, wherein Ci-C5, C2-C6 and C3-C4 form disulfide
bonds; the
trefoil monomer domain comprises the following sequence:
C1(xx)xxx[Pvae]xxRx[ndpm] C2[Gaiy] [ypfst]([de]x)[pskq]x[Ivap] [Tsa]xx[keqd]
C3xx[krln] [G
nk]C.aC5[a][Dnrs][sdpnte]xx(x)xxx[pki][Weash]Cg[Fy]; the thyroglobulin monomer
domain
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comprises the following sequence:
Cl
[qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxx[ahp]xPxCaxxxGx[a]xx[vkrl]QC3x(x[sa]xxx
)xx[gas]xC4[a]CsV[Dna]xx(x)Gxxxx[~g]xxxxxgxx(xx)xC6, wherein Cl-Ca, C3-C4 and
C5-C6
form disulfide bonds; and a is selected from: w, y, f, and 1; ~ is selected
from: d, e, and n;
and "x" is selected from any amino acid. In some embodiments, the
thrombospondin
monomer comprises the following sequence:
Ci [nst] [aegiklqrstv]
[adenpqrst]Ca[adetgs]xgx[ikqrstv]x[aqrst]x[almrtv]xC3xxxxxxxxx(xxxxx
XX)C4XXXXXXXXX(XX)C5XXXXC6; the trefoil monomer domain comprises the following
sequence:
Cl ([dnps])[adiklnprstv] [dfilmv] [adenprst] [adelprv] [ehklnqrs] [adegknsv]
[kqr] [fiklqrtv] [dnpqs
]CZ[agiy] [flpsvy] [dknpqs] [adfghlp] [aipv] [st][aegkpqrs] [adegkpqs]
[deiknqt]C3[adeflcnqrt] [ade
gknqs] [gn]C4C5[wyfh] [deinrs] [adgnpst] [aefgqlrstw] [giknsvmq]([afinprstv]
[degklns] [afiqstv] [
iknpv]w)C6; and the thyroglobulin monomer comprises the following sequence:
Ci
[qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxx[Yfhp]xPxCZxxxGx[Yflxx[vkrl]QC3x(x[sa]x
xx)xx[Gsa]xC4[WyfJC5V[Dnyfl]xx(x)Gxxxx[Gdne]xxxxxgxx(xx)xC6. In some
embodiments, the method further comprises linking the identified monomer
domains to a
second monomer domain to form a library of multimers, each multimer comprising
at least
two monomer domains; screening the library of multimers for the ability to
bind to the first
target molecule; and identifying a multimer that binds to the first target
molecule. Each
monomer domain of the selected multimer binds to the same target molecule or
to different
target molecules. In some embodiments, the selected multimer comprises two,
three, four, or
more monomer domains. In some embodiments, the methods further comprises a
step of
mutating at least one monomer domain, thereby providing a library comprising
mutated
monomer domains. In some embodiments, the mutating step comprises recombining
a
plurality of polynucleotide fragments of at least one polynucleotide encoding
a polypeptide
domain. In some embodiments, the methods further comprises screening the
library of
monomer domains for affinity to a second target molecule; identifying a
monomer domain
that binds to a second target molecule; linking at least one monomer domain
with affinity for
the first target molecule with at least one monomer domain with affinity for
the second target
molecule, thereby forming a multimer with affinity for the first and the
second target
molecule. In some embodiments, the library of monomer domains is expressed as
a phage
display, ribosome display or cell surface display. In some embodiments, the
library of
monomer domains is presented on a microarray.
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[14] The invention further comprises a library of proteins comprising non-
naturally-occurring monomer domains, wherein the monomer domain is selected
from: a
thrombospondin monomer domain, a trefoil monomer domain, and a thyroglobulin
monomer
domain. In some embodiments, the thrombospondin monomer domain comprises the
following sequence:
(wxxWxx)C1sxtC2xxGxx(x)xRxrxC3xxxx(Pxx)xxxxxC4xxxxxx(x)xxxC5(x)xxxxC6; the
trefoil monomer domain comprises the following sequence:
Cl(xx)xxxpxxRxnCagx(x)pxitxxxC3xxxgC4Csfdxxx(x)xxxpwC6f; and the thyroglobulin
monomer domain comprises the following sequence:
Clxxxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxxyxPxCaxxxGxyxxxQC3x(x)s(xxx)xxgxC4WC5Vd
xx(x)GxxxxGxxxxxgxx(xx)xC6i and "x" is any amino acid. In some
embodiments,each
monomer domain of the multimers is a non-naturally occurring monomer domain.
In some
embodiments, the library comprises a plurality of multimers, wherein the
multimers comprise
at least two monomer domains linked by a linker. In some embodiments, the
library
comprises at least 100 different proteins comprising different monomer
domains.
[15] The present invention also provides methods for identifying domain
monomers and multimers that bind to a target molecule. In some embodiments,
the method
comprises: providing a library of monomer domains; screening the library of
monomer
domains for affinity to a first target molecule; and identifying at least one
monomer domain
that binds to at least one target molecule. In some embodiments, the monomer
domains each
bind an ion (e.g., calcium).
[16] In some embodiments, the methods further comprise linking the
identified monomer domains to a second, monomer domain to form a library of
multimers,
each multimer comprising at least two monomer domains; screening the library
of multimers
for the ability to bind to the first target molecule; and identifying a
multimer that binds to the
first target molecule.
[17] In some embodiments, each monomer domain of the selected multimer
binds to the same target molecule. In some embodiments, the selected multimer
comprises
three monomer domains. In some embodiments, the selected multimer comprises
four
monomer domains.
[18] In some embodiments, the monomer domains are selected from a
Thrombospondin type I domain, a thyroglobulin type I repeat domain, a Trefoil
(P-type)
domain, and an EGF-like domain (e.g., a Laminin-type EGF-like domain).
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[19] In some embodiments, the methods comprise a further step of mutating
at least one monomer domain, thereby providing a library comprising mutated
monomer
domains. hi some embodiments, the mutating step comprises recombining a
plurality of
polynucleotide fragments of at least one polynucleotide encoding a monomer
domain. In
some embodiments, the mutating step comprises directed evolution; combining
different loop
sequences; site-directed mutagenesis; or site-directed recombination to create
crossovers that
result in the generation of sequences that are identical to human sequences.
[20] In some embodiments, the methods further comprise: screening the
library of monomer domains for affinity to a second target molecule;
identifying a monomer
domain that binds to a second target molecule; linking at least one monomer
domain with
affinity for the first target molecule with at least one monomer domain with
affinity for the
second target molecule, thereby forming a multimer with affinity for the first
and second
target molecule.
[21] In some embodiments, the target molecule is selected from a viral
antigen, a bacterial antigen, a fungal antigen, an enzyme, a cell surface
protein, an
intracellular protein, an enzyme inhibitor, a reporter molecule, a serum
protein, and a
receptor. In some embodiments, the viral antigen is a polypeptide required for
viral
replication.
[22] In some embodiments, the library of monomer domains is expressed as
by phage display, phagemid display, ribosome display, polysome display, or
cell surface
display (e.g., E. coli cell surface display), yeast cell surface display or
display via fusion to a
protein that binds to the polynucleotide encoding the protein. In some
embodiments, the
library of monomer domains is presented on a microarray, including 96-well,
384 well or
higher density microtiter plates.
[23] In some embodiments, the monomer domains are linked by a
polypeptide linker. In some embodiments, the polypeptide linker is a linker
naturally-
associated with the monomer domain. In some embodiments, the polypeptide
linker is a
linker naturally-associated with the family of monomer domains. In some
embodiments, the
polypeptide linker is a variant of a linker naturally-associated with the
monomer domain. In
some embodiments the linker is a gly-ser linker. In some embodiments, the
linking step
comprises linking the monomer domains with a variety of linkers of different
lengths and
composition.
[24] In some embodiments, the domains form a secondary and tertiary
structure by the formation of disulfide bonds. In some embodiments, the
multimers comprise
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an A domain connected to a monomer domain by a polypeptide linker. In some
embodiments, the linker is from 1-20 amino acids inclusive. In some
embodiments, the
linker is made up of 5-7 amino acids. In some embodiments, the linker is 6
amino acids in
length. In some embodiments, the linker comprises the following sequence,
AlA2A3A4A5A6,
wherein Al is selected from the amino acids A, P, T, Q, E and K; A2 and A3 are
any amino
acid except C, F, Y, W, or M; A4 is selected from the amino acids S, G and R;
A5 is selected
from the amino acids H, P, and R; A6 is the amino acid, T. In some
embodiments, the linker
comprises a naturally-occurring sequence between the C-terminal cysteine of a
first A
domain and the N-terminal cysteine of a second A domain. In some embodiments
the linker
comprises glycine and serine.
[25] The present invention also provides methods for identifying a multimer
that binds to at least one target molecule, comprising the steps of= providing
a library of
multimers, wherein each multimer comprises at least two monomer domains and
wherein
each monomer domain exhibits a binding specificity for a target molecule; and
screening the
library of multimers for target molecule-binding multimers. In some
embodiments, the
methods further comprise identifying target molecule-binding multimers having
an avidity
for the target molecule that is greater than the avidity of a single monomer
domain for the
target molecule. In some embodiments, one or more of the multimers comprises a
monomer
domain that specifically binds to a second target molecule.
[26] Alternative methods for identifying a multimer that binds to a target
molecule include methods comprising providing a library of monomer domains
and/or
immuno domains; screening the library of monomer domains and/or immuno domain
for
affinity to a first target molecule; identifying at least one monomer domain
and/or immuno
domain that binds to at least one target molecule; linking the identified
monomer domain
and/or immuno domain to a library of monomer domains and/or immuno domains to
form a
library of multimers, each multimer comprising at least two monomer domains,
immuno
domains or combinations thereof; screening the library of multimers for the
ability to bind to
the first target molecule; and identifying a multimer that binds to the first
target molecule.
[27] In some embodiments, the monomer domains each bind an ion. In
some embodiments, the ion is selected from calcium and zinc.
[28] In some embodiments, the linker comprises at least 3 amino acid
residues. In some embodiments, the linker comprises at least 6 amino acid
residues. In some
embodiments, the linker comprises at least 10 amino acid residues.
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[29] The present invention also provides polypeptides comprising at least
two monomer domains separated by a heterologous linker sequence. In some
embodiments,
each monomer domain specifically binds to a target molecule; and each monomer
domain is a
non-naturally occurring protein monomer domain. In some embodiments, each
monomer
domain binds an ion.
[30] In some embodiments, polypeptides comprise a first monomer domain
that binds a first target molecule and a second monomer domain that binds a
second target
molecule. In some embodiments, the polypeptides comprise two monomer domains,
each
monomer domain having a binding specificity that is specific for a different
site on the same
target molecule. In some embodiments, the polypeptides further comprise a
monomer
domain having a binding specificity for a second target molecule.
[31] In some embodiments, the monomer domains of a library, multimer or
polypeptide are typically about 40% identical to each other, usually about 50%
identical,
sometimes about 60% identical, and frequently at least 70% identical.
[32] The invention also provides polynucleotides encoding the above-
described polypeptides.
[33) The present invention also provides multimers of immuno-domains
having binding specificity for a target molecule, as well as methods for
generating and
screening libraries of such multimers for binding to a desired target
molecule. More
specifically, the present invention provides a method for identifying a
multimer that binds to
a target molecule, the method comprising, providing a library of immuno-
domains; screening
the library of immuno-domains for affinity to a first target molecule;
identifying one or more
(e.g., two or more) immuno-domains that bind to at least one target molecule;
linking the
identified monomer domain to form a library of multimers, each multimer
comprising at least
three immuno-domains (e.g., four or more, five or more, six or more, etc.);
screening the
library of multimers for the ability to bind to the first target molecule; and
identifying a
multimer that binds to the first target molecule. Libraries of multimers of at
least two
immuno-domains that are minibodies, single domain antibodies, Fabs, or
combinations
thereof are also employed in the practice of the present invention. Such
libraries can be
readily screened for multimers that bind to desired target molecules in
accordance with the
invention methods described herein.
[34] The present invention further provides methods of identifying hetero-
immuno multimers that binds to a target molecule. In some embodiments, the
methods
comprise, providing a library of immuno-domains; screening the library of
immuno-domains
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for affinity to a first target molecule; providing a library of monomer
domains; screening the
library of monomer domains for affinity to a first target molecule;
identifying at least one
immuno-domain that binds to at least one target molecule; identifying at least
one monomer
domain that binds to at least one target molecule; linking the identified
immuno-domain with
the identified monomer domains to form a library of multimers, each multimer
comprising at
least two domains; screening the library of multimers for the ability to bind
to the first target
molecule; and identifying a multimer that binds to the first target molecule.
[35] The present invention also provides methods for identifying a laminin-
EGF monomer domain, a thrombospondin type I monomer domain, a thyroglobulin
monomer
domain, or a trefoil monomer domain that binds to a target molecule. In some
embodiments,
the method comprises providing a library of laminin-EGF monomer domains,
thrombospondin type I monomer domains, thyroglobulin monomer domains, or
trefoil
monomer domains; screening the library of laminin-EGF monomer domains,
thrombospondin
type I monomer domains, thyroglobulin monomer domains, or trefoil monomer
domains for
affinity to a target molecule; and identifying a laminin-EGF monomer domain,
thrombospondin type I monomer domain, thyroglobulin monomer domain, or trefoil
monomer domain that binds to the target molecule.
[36] In some embodiments, the method comprises linking each member of a
library of laminin-EGF monomer domains, thrombospondin type I monomer domains,
thyroglobulin monomer domains, or trefoil monomer domains to the identified
monomer
domain to form a library of multimers; screening the library of multimers for
affinity to the
target molecule; and identifying a multimer that binds to the target. In some
embodiments,
the multimer binds to the target with greater affinity than the monomer. In
some
embodiments, the method furrther comprises expressing the library using a
display format
selected from a phage display, a ribosome display, a polysome display, or a
cell surface
display.
[37] In some embodiments, the method further comprises a step of mutating
at least one monomer domain, thereby providing a library comprising mutated
laminin-EGF
monomer domains, thrombospondin type I monomer domains, thyroglobulin monomer
domains, or trefoil monomer domains. In some embodiments, the mutating step
comprises
directed evolution; site-directed mutagenesis; by combining different loop
sequences, or by
site-directed recombination to create crossovers that result in generation of
sequences that are
identical to human sequences.
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[38] The present invention also provides method of producing a polypeptide
comprising the multimer identified in a method comprising providing a library
of laminin-
EGF monomer domains, thrombospondin type I monomer domains, thyroglobulin
monomer
domains, or trefoil monomer domains; screening the library of laminin-EGF
monomer
domains, thrombospondin type I monomer domains, thyroglobulin monomer domains,
or
trefoil monomer domains for affinity to a target molecule; and identifying a
laminin-EGF
monomer domain, thrombospondin type I monomer domain, thyroglobulin monomer
domain,
or trefoil monomer domain that binds to the target molecule. In some
embodiments, the
multimer is produced by recombinant gene expression.
[39] The present invention also provides methods for generating a library of
thrombospondin type I monomer domains, thyroglobulin monomer domains, or
trefoil
monomer domains derived from thrombospondin type I monomer domains,
thyroglobulin
monomer domains, or trefoil monomer domains. In some embodiments, the methods
comprise providing loop sequences corresponding to at least one loop from each
of two
different naturally occurring variants of a human laminin-EGF monomer domains,
thrombospondin type I monomer domains, thyroglobulin monomer domains, or
trefoil
monomer domains, wherein the loop sequences are polynucleotide or polypeptide
sequences;
covalently combining loop sequences to generate a library of chimeric monomer
domain
sequences, each chimeric sequence encoding a chimeric thrombospondin type I
monomer
domain, thyroglobulin monomer domain, or trefoil monomer domain having at
least two
loops; expressing the library of chimeric thrombospondin type I monomer
domains,
thyroglobulin monomer domains, or trefoil monomer domains using a display
format selected
from phage display, ribosome display, polysome display, and cell surface
display; screening
the expressed library of chimeric thrombospondin type I monomer domains,
thyroglobulin
monomer domains, or trefoil monomer domains for binding to a target molecule;
and
identifying a chimeric thrombospondin type I monomer domain, thyroglobulin
monomer
domain, or trefoil monomer domain that binds to the target molecule.
[40] In some embodiments, the methods further comprise linking the
identified chimeric thrombospondin type I monomer domain, thyroglobulin
monomer
domain, or trefoil monomer domain to each member of the library of chimeric
thrombospondin type I monomer domains, thyroglobulin monomer domains, or
trefoil
monomer domains to form a library of multimers; screening the library of
multimers for the
ability to bind to the first target molecule with an increased affinity; and
identifying a
multimer of chimeric thrombospondin type I monomer domains, thyroglobulin
monomer
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domains, or trefoil monomer domains that binds to the first target molecule
with an increased
affinity.
[41] The present invention also provides methods of making cbimeric
thrombospondin type I monomer domain, thyroglobulin monomer domain, or trefoil
monomer domain identified in a method comprising providing loop sequences
corresponding
to at least one loop from each of two different naturally occurring variants
of a human
thrombospondin type I monomer domains, thyroglobulin monomer domains, or
trefoil
monomer domains, wherein the loop sequences are polynucleotide or polypeptide
sequences;
covalently combining loop sequences to generate a library of chimeric monomer
domain
sequences, each chimeric sequence encoding a chimeric thrombospondin type I
monomer
domain, thyroglobulin monomer domain, or trefoil monomer domain having at
least two
loops; expressing the library of chimeric thrombospondin type I monomer
domains,
thyroglobulin monomer domains, or trefoil monomer domains using a display
format selected
from phage display, ribosome display, polysome display, and cell surface
display; screening
the expressed library of chimeric thrombospondin type I monomer domains,
thyroglobulin
monomer domains, or trefoil monomer domains for binding to a target molecule;
and
identifying a chimeric thrombospondin type I monomer domain, thyroglobulin
monomer
domain, or trefoil monomer domain that binds to the target molecule. In some
embodiments,
the chimeric thrombospondin type I monomer domain, thyroglobulin monomer
domain, or
trefoil monomer domain is produced by recombinant gene expression.
[42] In some embodiments, the monomer domain binds to a target
molecule. In some embodiments, the polypeptide is 45 or fewer amino acids
long. In some
embodiments, the heterologous amino acid sequence is selected from an affinity
peptide, a
heterologous thrombospondin type I monomer domain, a heterologous
thyroglobulin
monomer domain, or a heterologous trefoil monomer domain, a purification tag,
an enzyme
(e.g., horseradish peroxidase or alkaline phosphatase), and a reporter protein
(e.g., green
fluorescent protein or luciferase). In some embodiments, the target is not a
variable region or
hypervariable region of an antibody.
[43] The present invention provides methods for screening a library of
monomer domains or multimers comprising monomer domains for binding affinity
to
multiple ligands. In some embodiments, the method comprises contacting a
library of
monomer domains or multimers of monomer domains to multiple ligands; and
selecting
monomer domains or multimers that bind to at least one of the ligands.
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[44] In some embodiments, the methods comprise (i.) contacting a library
of monomer domains to multiple ligands; (ii.) selecting monomer domains that
bind to at
least one of the ligands; (iii.) linking the selected monomer domains to a
library of monomer
domains to form a library of multimers, each comprising a selected monomer
domain and a
second monomer domain; (iv.) contacting the library of multimers to the
multiple ligands to
form a plurality of complexes, each complex comprising a multimer and a
ligand; and (v.)
selecting at least one complex. -
[45] In some embodiments, the method further comprises linking the
multimers of the selected complexes to a library of monomer domains or
multimers to form a
second library of multimers, each comprising a selected multimer and at least
a third
monomer domain; contacting the second library of multimers to the multiple
ligands to form
a plurality of second complexes; and selecting at least one second complex.
[46] In some embodiments, the identity of the ligand and the multimer is
determined. In some embodiments, a library of monomer domains is contacted to
multiple
ligands. In some embodiments, a library of multimers is contacted to multiple
ligands.
[47] In some embodiments, the multiple ligands are in a mixture. In some
embodiments, the multiple ligands are in an array. In some embodiments, the
multiple
ligands are in or on a cell or tissue. In some embodiments, the multiple
ligands are
immobilized on a solid support.
[48] In some embodiments, the ligands are polypeptides. In some
embodiments, the polypeptides are expressed on the surface of phage. In some
embodiments,
the monomer domain or multixner library is expressed on the surface of phage.
[49] In some embodiments, the library of multimers is expressed on the
surface of phage to fonn library-expressing phage and the ligands are
expressed on the
surface of phage to form ligand-expressing phage, and the method comprises
contacting
library-expressing phage to the ligand-expressing phage to form ligand-
expressing
phage/library-expressing phage pairs; removing ligand-expressing phage that do
not bind to
library-expressing or removing library-expressing phage that do not bind to
ligand-expressing
phage; and selecting the ligand-expressing phage/library-expressing phage
pairs. In some
embodiments, the methods fiuther comprise isolating polynucleotides from the
phage pairs
and amplifyi.ng the polynucleotides to produce a polynucleotide hybrid
comprising
polynucleotides from the ligand-expressing phage and the library-expressing
phage.
[501 In some embodiments, the methods comprise isolating polynucleotide
hybrids from a plurality of phage pairs, thereby forming a mixture of
polynucleotide hybrids.
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In some embodiments, the methods comprise contacting the mixture of hybrid
polynucleotides to a cDNA library under conditions to allow for polynucleotide
hybridization, thereby hybridizing a hybrid polynucleotide to a cDNA in the
cDNA library;
and determining the nucleotide sequence of the hybridized hybrid
polynucleotide, thereby
identifying a monomer domain that specifically binds to the polypeptide
encoded by the
cDNA. In some embodiments, the monomer domain library is expressed on the
surface of
phage to form library-expressing phage and the ligands are expressed on the
surface of phage
to form ligand-expressing phage, and the selected complexes comprise a library-
expressing
phage bound to a ligand-expressing phage and the method comprises: dividing
the selected
monomer domains or multimers into a first and a second portion, linking the
monomer
domains or multimers of the first portion to a solid surface and contacting a
phage-displayed
ligand library to the monomer domains or multimers of the first portion to
identify target
ligand phage that binds to a monomer domain or multimer of the first portion;
infecting
phage displaying the monomer domains or multimers of the second portion into
bacteria to
express the phage; and contacting the target ligand phage to the expressed
phage to form
phage pairs comprised of a target ligand phage and a phage displaying a
monomer domain or
multimer.
[51] In some embodiments, the methods farther comprise isolating a
polynucleotide from each phage of the phage pair, thereby identifying a
multimer or
monomer domain that binds to the ligand in the phage pair. In some
embodiments, the
methods further comprise amplifyin.g the polynucleotides to produce a
polynucleotide hybrid
comprising polynucleotides from the target ligand phage and the library phage.
[52] In some embodiments, the methods comprise isolating and amplifying
polynucleotide hybrids from a plurality of phage pairs, thereby forming a
mixture of
polynucleotide hybrids. In some embodiments, the methods comprise contacting
the mixture
of hybrid polynucleotides to a cDNA library under conditions to allow for
hybridization,
thereby hybridizing a hybrid polynucleotide to a cDNA in the cDNA library; and
determining
the nucleotide sequence of the associated hybrid polynucleotide, thereby
identifying a
monomer domain that specifically binds to the ligand encoded by the cDNA
associated
cDNA.
[53] The present invention also provides non-naturally-occurring
polypeptides comprising an amino acid sequence in which:
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atleast1%,2%,3%,4%,5%,6%,7%,8%,9%,10%,11 10,12%,13%,14 l0,
15%, 16%, 17%, 18%, 19%, 20% or more of the amino acids in the sequence are
cysteine;
and
the amino acid sequence is at least 10, 20, 30, 45, 50, 55, 60, 70, 80, 90,
100 or
more amino acids long; and/or
the amino acid sequence is less than 150, 140, 130, 120, 110, 100, 90, 80, 70,
60, 50, or 40 amino acids long;and/or
at least 5%, 10%, 15 l0, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of the
amino acids are non-naturally-occurring amino acids. For example, in some
embodiments,
the amino acid sequence comprises at least 10% cysteines and the amino acid
sequence is at
least 50 amino acids long or at least 25% of the amino acids are non-naturally
occurring. In
some embodiments, the amino acid sequence is a non-naturally occurring A
domain.
[54) In some embodiments, the polypeptides of the invention comprise one,
two, three, four, or more monomers with at least 10%,15%, 20%, 25%, 30%, 35
l0, 40 /a,
45%, 50% or more non-naturally-occurring amino acids. In some embodiments, the
one or
more monomer domains comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%,
50% or more amino acids that do not occur at that position in natural human
proteins. In
some embodiments, the monomer domains are derived from a naturally-occurring
human
protein sequence. In some embodiments, the polypeptides of the invention also
have a serum
half-life of at least, e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70 80, 90,
100, 150, 200, 250,
400, 500 or more hours.
DEFINITIONS
[55) Unless otherwise indicated, the following definitions supplant those in
the art.
1561 The term "monomer domain" or "monomer" is used interchangeably
herein refer to a discrete region found in a protein or polypeptide. A monomer
domain forms
a native three-dimensional structure in solution in the absence of flanking
native amino acid
sequences. Monomer domains of the invention can be selected to specifically
bind to a target
molecule. As used herein, the term "monomer domain" does not encompass the
complementarity determining region (CDR) of an antibody.
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[57] The term "monomer domain variant" refers to a domain resulting from
human-manipulation of a monomer domain sequence. Examples of man-manipulated
changes include, e.g., random mutagenesis, site-specific mutagenesis,
recombining, directed
evolution, oligo-directed forced crossover events, direct gene synthesis
incorporation of
mutation, etc. The term "monomer domain variant" does not embrace a
mutagenized
complementarity determining region (CDR) of an antibody.
[58] The term "loop" refers to that portion of a monomer domain that is
typically exposed to the environment by the assembly of the scaffold structure
of the
monomer domain protein, and which is involved in target binding. The present
invention
provides three types of loops that are identified by specific features, such
as, potential for
disulfide bonding, bridging between secondary protein structures, and
molecular dynamics
(i.e., flexibility). The three types of loop sequences are a cysteine-defined
loop sequence, a
structure-defined loop sequence, and a B-factor-defined loop sequence.
[59] As used herein, the term "cysteine-defined loop sequence" refers to a
subsequence of a naturally occurring monomer domain-encoding sequence that is
bound at
each end by a cysteine residue that is conserved with respect to at least one
other naturally
occurring monomer domain of the same family. Cysteine-defined loop sequences
are
identified by multiple sequence alignment of the naturally occuming monomer
domains,
followed by sequence analysis to identify conserved cysteine residues. The
sequence
between each consecutive pair of conserved cysteine residues is a cysteine-
defined loop
sequence. The cysteine-defined loop sequence does not include the cysteine
residues
adjacent to each terminus. Monomer domains having cysteine-defined loop
sequences
include the thrombospondin domains, thyroglobulin domains, trefoil/PD domains,
and the
like. Thus, for example, thrombospondin domains are represented by the
consensus
sequence, CX3CX10CX16CX11CX4C, wherein X3, Xio, X16, Xll, and X4, each
represent a
cysteine-defined loop sequence; trefoil/PD domains are represented by the
consensus
sequence, CXIoCX9CX4CCX1oC, wherein Xio, X9, X4, and Xio, each represent a
cysteine-
defined loop sequence; and thyroglobulin domains are represented by the
consensus
sequence, CX26CX10CX6CXiCX18C, wherein X26, Xlo, X6, Xi, and X18, each
represent a
cysteine-defined loop sequence.
[60] The term "multimer" is used herein to indicate a polypeptide
comprising at least two monomer domains and/or immuno-domains (e.g., at least
two
monomer domains, at least two immuno-domains, or at least one monomer domain
and at
least one imniuno-domain). The separate monomer domains and/or immuno-domains
in a
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multimer can be joined together by a linker. A multimer is also known as a
combinatorial
mosaic protein or a recombinant mosaic protein.
[61] The term "family" and "family class" are used interchangeably to
indicate proteins that are grouped together based on similarities in their
amino acid
sequences. These similar sequences are generally conserved because they are
important for
the function of the protein and/or the maintenance of the three dimensional
structure of the
protein. Examples of such families include the LDL Receptor A-domain family,
the EGF-like
family, and the like.
[62] The term "ligand," also referred to herein as a "target molecule,"
encompasses a wide variety of substances and molecules, which range from
simple molecules
to complex targets. Target molecules can be proteins, nucleic acids, lipids,
carbohydrates or
any other molecule capable of recognition by a polypeptide domain. For
example, a target
molecule can include a chemical compound (i.e., non-biological compound such
as, e.g., an
organic molecule, an inorganic molecule, or a molecule having both organic and
inorganic
atoms, but excluding polynucleotides and proteins), a mixture of chemical
compounds, an
array of spatially localized compounds, a biological macromolecule, a
bacteriophage peptide
display library, a polysome peptide display library, an extract made from a
biological
materials such as bacteria, plants, fungi, or animal (e.g., mammalian) cells
or tissue, a protein,
a toxin, a peptide hormone, a cell, a virus, or the like. Othet target
molecules include, e.g., a
' whole cell, a whole tissue, a mixture of related or unrelated proteins, a
mixture of viruses or
bacterial strains or the like. Target molecules can also be defined by
inclusion in screening
assays described herein or by enhancing or inhibiting a specific protein
interaction (i.e., an
agent that selectively inhibits a binding interaction between two
predetermined polypeptides).
[63] As used herein, the term "immuno-domains" refers to protein binding
domains that contain at least one complementarity determining region (CDR) of
an antibody.
Immuno-domains can be naturally occurring immunological domains (i.e. isolated
from
nature) or can be non-naturally occurring immunological domains that have been
altered by
human-manipulation (e.g., via mutagenesis methods, such as, for example,
random
mutagenesis, site-specific mutagenesis, recombination, and the like, as well
as by directed
evolution methods, such as, for example, recursive error-prone PCR, recursive
recombination, and the like.). Different types of immuno-domains that are
suitable for use in
the practice of the present invention include a minibody, a single-domain
antibody, a single
chain variable fragment (ScFv), and a Fab fragment.
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[64] The term "minibody" refers herein to a polypeptide that encodes only 2
complementarity determining regions (CDRs) of a naturally or non-naturally
(e.g.,
mutagenized) occurring heavy chain.variable domain or light chain variable
domain, or
combination thereof. An example of a minibody is described by Pessi et al,, A
designed
metal-bindingprotein with a novel fold, (1993) Nature 362:367-369.
[65] As used herein, the term "single-domain antibody" refers to the heavy
chain variable domain ("VH") of an antibody, i.e., a heavy chain variable
domain without a
light chain variable domain. Exemplary single-domain antibodies employed in
the practice
of the present invention include, for example, the Camelid heavy chain
variable domain
(about 118 to 136 amino acid residues) as described in Hamers-Casterman, C. et
al.,
Naturally occurring antibodies devoid of light chains (1993) Nature 363:446-
448, and
Dumoulin, et al., Single-domain antibody fi agments with high conformational
stability
(2002) Protein Science 11:500-515.
[66] The terms "single chain variable fragment" or "ScFv" are used
interchangeably herein to refer to antibody heavy and light chain variable
domains that are
joined by a peptide linker having at least 12 amino acid residues. Single
chain variable
fragments contemplated for use in the practice of the present invention
include those
described in Bird, et al., (1988) Science 242(4877):423-426 and Huston et al.,
(1988) PNAS
USA 85(16):5879-83.
[67] As used herein, the term "Fab fragment" refers to an immuno-domain
that has two protein chains, one of which is a light chain consisting of two
light chain
domains (VL variable domain and CL constant domain) and a heavy chain
consisting of two
heavy domains (i.e., a VH variable and a CH constant domain). Fab fragments
employed in
the practice of the present invention include those that have an interchain
disulfide bond at
the C-terminus of each heavy and light component, as well as those that do not
have such a
C-terminal disulfide bond. Each fragment is about 47 kD. Fab fragments are
described by
Pluckthun and Skerra, (1989) Methods Enzymo1178:497-515.
[68] The term "linker" is used herein to indicate a moiety or group of
moieties that joins or connects two or more discrete separate monomer domains.
The linker
allows the discrete separate monomer domains to remain separate when joined
together in a
multimer. The linker moiety is typically a substantially linear moiety.
Suitable linkers
include polypeptides, polynucleic acids, peptide nucleic acids and the like.
Suitable linkers
also include optionally substituted alkylene moieties that have one or more
oxygen atoms
incorporated in the carbon backbone. Typically, the molecular weight of the
linker is less
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than about 2000 daltons. More typically, the molecular weight of the linker is
less than about
1500 daltons and usually is less than about 1000 daltons. The linker can be
small enough to
allow the discrete separate monomer domains to cooperate, e.g., where each of
the discrete
separate monomer domains in a multimer binds to the same target molecule via
separate
binding sites. Exemplary linkers include a polynucleotide encoding a
polypeptide, or a
polypeptide of amino acids or other non-naturally occurring moieties. The
linker can be a
portion of a native sequence, a variant thereof, or a synthetic sequence.
Linkers can
comprise, e.g., naturally occurring, non-naturally occurring amino acids, or a
combination of
both.
[69) The term "separate" is used herein to indicate a property of a moiety
that is independent and remains independent even when complexed with other
moieties,
including for example, other monomer domains. A monomer domain is a separate
domain in
a protein because it has an independent property that can be recognized and
separated from
the protein. For instance, the ligand binding ability of the A-domain in the
LDLR is an
independent property. Other examples of separate include the separate monomer
domains in
a multimer that remain separate independent domains even when complexed or
joined
together in the multimer by a linker. Another example of a separate property
is the separate
binding sites in a multimer for a ligand.
[701 As used herein, "directed evolution" refers to a process by which
polynucleotide variants are generated, expressed, and screened for an activity
(e.g., a
polypeptide with binding activity) in a recursive process. One or more
candidates in the
screen are selected and the process is then repeated using polynucleotides
that encode the
selected candidates to generate new variants. Directed evolution involves at
least two rounds
of variation generation and can include 3, 4, 5, 10, 20 or more rounds of
variation generation
and selection. Variation can be generated by any method known to those of
skill in the art,
including, e.g., by error-prone PCR, gene recombination, chemical mutagenesis
and the like.
[711 The term "shuffling" is used herein to indicate recombination between
non-identical sequences. In some embodiments, shuffling can include crossover
via
homologous recombination or via non-homologous recombination, such as via
cre/lox and/or
flp/frt systems. Shuffling can be carried out by employing a variety of
different formats,
including for example, in vitro and in vivo shuffling formats, in silico
shuffling formats,
shuffling formats that utilize either double-stranded or single-stranded
templates, primer
based shuffling formats, nucleic acid fragmentation-based shuffling formats,
and
oligonucleotide-mediated shuffling formats, all of which are based on
recombination events
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between non-identical sequences and are described in more detail or referenced
herein below,
as well as other similar recombination-based formats. The term "random" as
used herein
refers to a polynucleotide sequence or an amino acid sequence composed of two
or more
amino acids and constructed by a stochastic or random process. The random
polynucleotide
sequence or amino acid sequence can include framework or scaffolding motifs,
which can
comprise invariant sequences.
[72] The term "pseudorandom" as used herein refers to a set of sequences,
polynucleotide or polypeptide, that have limited variability, so that the
degree of residue
variability at some positions is limited, but any pseudorandom position is
allowed at least
some degree of residue variation.
[73] The terms "polypeptide," "peptide," and "protein" are used herein
interchangeably to refer to an amino acid sequence of two or more amino acids.
[74] "Conservative amino acid substitution" refers to the interchangeability
of residues having similar side chains. For example, a group of amino acids
having aliphatic
side chains is glycine, alanine, valine, leucine, and isoleucine; a group of
amino acids having
aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids
having amide-
containing side chains is asparagine and glutamine; a group of amino acids
having aromatic
side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids
having basic
side chains is lysine, arginine, and histidine; and a group of amino acids
having sulfur-
containing side chains is cysteine and methionine. Preferred conservative
amino acids
substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine,
alanine-valine, and asparagine-glutamine.
[75] The phrase "nucleic acid sequence" refers to a single or double-
stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the
5' to the 3'
end. It includes chromosomal DNA, self-replicating plasmids and DNA or RNA
that
performs a primarily structural role.
1761 The term "encoding" refers to a polynucleotide sequence encoding one
or more amino acids. The term does not require a start or stop codon. An amino
acid
sequence can be encoded in any one of six different reading frames provided by
a
polynucleotide sequence.
[77] The term "promoter" refers to regions or sequence located upstream
and/or downstream from the start of transcription that are involved in
recognition and binding
of RNA polymerase and other proteins to initiate transcription.
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[78] A "vector" refers to a polynucleotide, which when independent of the
host chromosome, is capable of replication in a host organism. Examples of
vectors include
plasmids. Vectors typically have an origin of replication. Vectors can
comprise, e.g.,
transcription and translation terminators, transcription and translation
initiation sequences,
and promoters useful for regulation of the expression of the particular
nucleic acid.
[79] The term "recombinant" when used with reference, e.g., to a cell, or
nucleic acid, protein, or vector, indicates that the cell, nucleic acid,
protein or vector, has
been modified by the introduction of a heterologous nucleic acid or protein or
the alteration
of a native nucleic acid or protein, or that the cell is derived from a cell
so modified. Thus,
for example, recombinant cells express genes that are not found within the
native
(nonrecombinant) form of the cell or express native genes that are otherwise
abnormally
expressed, under-expressed or not expressed at all.
[80] The phrase "specifically (or selectively) binds" to a polypeptide, when
referring to a monomer or multimer, refers to a binding reaction that can be
determinative of
the presence of the polypeptide in a heterogeneous population of proteins and
other biologics.
Thus, under standard conditions or assays used in antibody binding assays, the
specified
monomer or multimer binds to a particular target molecule above background
(e.g., 2X, 5X,
10X or more above background) and does not bind in a significant amount to
other molecules
present in the sample.
[81] The terms "identical" or percent "identity," in the context of two or
more nucleic acids or polypeptide sequences, refer to two or more sequences or
subsequences
that are the same. "Substantially identical" refers to two or more nucleic
acids or polypeptide
sequences having a specified percentage of amino acid residues or nucleotides
that are the
same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95%
identity over a
specified region, or, when not specified, over the entire sequence), when
compared and
aligned for maximum correspondence over a comparison window, or designated
region as
measured using one of the following sequence comparison algorithms or by
manual
alignment and visual inspection. Optionally, the identity or substantial
identity exists over a
region that is at least about 50 nucleotides in length, or more preferably
over a region that is
100 to 500 or 1000 or more nucleotides or amino acids in length.
[82] A polynucleotide or amino acid sequence is "heterologous to" a second
sequence if the two sequences are not linked in the same manner as found in
naturally-
occurring sequences. For example, a promoter operably linked to a heterologous
coding
sequence refers to a coding sequence which is different from any naturally-
occurring allelic
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variants. The term "heterologous linker," when used in reference to a
multimer, indicates
that the multimer comprises a linker and a monomer that are not found in the
same
relationship to each other in nature (e.g., they form a fusion protein).
[83] A "non-naturally-occurring amino acid" in a protein sequence refers to
any amino acid other than the amino acid that occurs in the corresponding
position in an
alignment with a naturally-occurring polypeptide with the lowest smallest sum
probability
where the comparison window is the length of the monomer domain queried and
when
compared to the non-redundant ("nr") database of Genbank using BLAST 2.0 as
described
herein.
[84] "Percentage of sequence identity" is determined by comparing two
optimally aligned sequences over a comparison window, wherein the portion of
the
polynucleotide sequence in the comparison window may comprise additions or
deletions (i.e.,
gaps) as compared to the reference sequence (which does not comprise additions
or deletions)
for optimal alignment of the two sequences. The percentage is calculated by
determining the
number of positions at which the identical nucleic acid base or amino acid
residue occurs in
both sequences to yield the number of matched positions, dividing the number
of matched
positions by the total number of positions in the window of comparison and
multiplying the
result by 100 to yield the percentage of sequence identity.
[85] The terms "identical" or percent "identity," in the context of two or
more nucleic acids or polypeptide sequences, refer to two or more sequences or
subsequences
that are the same or have a specified percentage of amino acid residues or
nucleotides that are
the same, when compared and align.ed for maximum correspondence over a
comparison
window, or designated region as measured using one of the following sequence
comparison
algorithms or by manual alignment and visual inspection. Such sequences are
then said to be
"substantially identical." This definition also refers to the complement of a
test sequence.
Optionally, the identity exists over a region that is at least about 50 amino
acids or
nucleotides in length, or more preferably over a region that is 75-100 amino
acids or
nucleotides in length.
[86] For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a sequence
comparison
algorithm, test and reference sequences are entered into a computer,
subsequence coordinates
are designated, if necessary, and sequence algorithm program parameters are
designated.
Default program parameters can be used, or alternative parameters can be
designated. The
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sequence comparison algorithm then calculates the percent sequence identities
for the test
sequences relative to the reference sequence, based on the program parameters.
[87) A"comparison window", as used herein, includes reference to a
segment of any one of the number of contiguous positions selected from the
group consisting
of 20 to 600, usually about 50 to about 200, more usually about 100 to about
150 in which a
sequence may be compared to a reference sequence of the same number of
contiguous
positions after the two sequences are optimally aligned. Methods of alignment
of sequences
for comparison are well-known in the art. Optimal alignment of sequences for
comparison
can be conducted, e.g., by the local homology algorithm of Smith and Waterman
(1970) Adv.
Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and
Wunsch (1970)
J. Mol. Biol. 48:443, by the search for similarity method of Pearson and
Lipman (1988) Proc.
Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these
algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics
Computer Group, 575 Science Dr., Madison, WI), or by manual alignxnent and
visual
inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995
supplement)).
[881 One example of a useful algorithm is the BLAST 2.0 algorithm, which
is described in Altschul et al. (1990) J. Mol. Biol. 215:403-410,
respectively. Sofl.ware for
performing BLAST analyses is publicly available through the National Center
for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm
involves first
identifying high scoring sequence pairs (HSPs) by identifying short words of
length W in the
query sequence, which either match or satisfy some positive-valued threshold
score T when
aligned with a word of the same length in a database sequence. T is referred
to as the
neighborhood word score threshold (Altschul et al., supra). These initial
neighborhood word
hits act as seeds for initiating searches to find longer HSPs containing them.
The word hits
are extended in both directions along each sequence for as far as the
cumulative alignment
score can be increased. Cumulative scores are calculated using, for nucleotide
sequences, the
parameters M (reward score for a pair of matching residues; always > 0) and
N(penalty score
for mismatching,residues; always < 0). For amino acid sequences, a scoring
matrix is used to
calculate the cumulative score. Extension of the word hits in each direction
are halted when:
the cumulative alignment score falls off by the quantity X from its maximum
achieved value;
the cumulative score goes to zero or below, due to the accumulation of one or
more negative-
scoring residue alignments; or the end of either sequence is reached. The
BLAST algorithm
parameters W, T, and X determine the sensitivity and speed of the alignment.
The BLASTN
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program (for nucleotide sequences) uses as defaults a wordlengkh (W) of 11, an
expectation
(E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid
sequences, the
BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10,
and the
BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad.
Sci. USA
89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a
comparison of both
strands.
[89] The BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc.
Natl. Acad. Sci.
USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm
is the
smallest sum probability (P(N)), which provides an indication of the
probability by which a
match between two nucleotide or amino acid sequences would occur by chance.
For
example, a nucleic acid is considered similar to a reference sequence if the
smallest sum
probability in a comparison of the test nucleic acid to the reference nucleic
acid is less than
about 0.2, more preferably less than about 0.01, and most preferably less than
about 0.001.
BRIEF DESCRIPTION OF THE DRAWINGS
[90] Figure 1 schematically illustrates a general scheme for identifying
monomer domains that bind to a ligand, isolating the selected monomer domains,
creating
multimers of the selected monomer domains by joining the selected monomer
domains in
various combinations and screening the multimers to identify multimers
comprising more
than one monomer that binds to a ligand.
1911 Figure 2 is a schematic representation of another selection strategy
(guided selection). A monomer domain with appropriate binding properties is
identified from
a library of monomer domains. The identified monomer domain is then linked to
monomer
domains from another library of monoiner domains to form a library of
multimers. The
multimer library is screened to identify a pair of monomer domains that bind
simultaneously
to the target. This process can then be repeated until the optimal binding
properties are
obtained in the multimer.
[92] Figure 3 illustrates walking selection to generate multimers that bind a
target or targets with increased affinity.
[93) Figure 4 illustrates screening a library of monomer domains against
multiple ligands displayed on a cell.
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1941 Figure 5 illustrates monomer domain and multimer embodiments for
increased avidity. VWhile the figure illustrates specific gene products and
binding affinities, it
is appreciated that these are merely examples and that other binding targets
can be used with
the same or similar conformations.
[95] Figure 6 illustrates monomer domain and multimer embodiments for
increased avidity. While the figure illustrates specific gene products and
binding affinities, it
is appreciated that these are merely examples and that other binding targets
can be used with
the same or similar conformations.
[96] Figure 7 illustrates various possible antibody- monomer or multimer of
the invention) conformations. In some embodiments, the monomer or multimer
replaces the
Fab fragment of the antibody.
[97] Figure 8 illustrates a method for intradomain optimization of
monomers.
[98] Figure 9 illustrates a possible sequence of multimer optimization steps
in which optimal monomers and then multimers are selected followed by
optimization of
monomers, optimization of linkers and then optimization of multimers.
[99] Figure 10 illustrates four exemplary methods to recombine monomer
and/or multimer libraries to introduce new variation. Figure l0A illustrates
one exemplary
embodiment of intra-domain recombination of monomers whereby portions of
different
monomers are recombined to form new monomers. Figure l OB illustrates a second
embodiment of intra-domain recombination whereby portions of monomers
recombined as
set forth in Figure 10A are further recombined to form additional new
monomers. Figure
1 OC illustrates one embodiment of inter-domain recombination, whereby
different
recombined monomers are linked to each other, i.e., to form multimers. Figure
10D
illustrates one embodiment of inter-module recombination whereby linked
recombined
monomers, i.e., multimers that bind to the same target molecule are linked to
other
recombined monomers that recognize a different target molecule to form new
multimers that
simultaneously bind to different target molecules.
[100] Figure 11 depicts a possible conformation of a multimer of the
invention comprising at least one monomer domain that binds to a half-life
extending
molecule and other monomer domains binding to two other different molecules.
In the
Figure, two monomer domains bind to a first target molecule and a separate
monomer
domain binds to a second target molecule.
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DETAILED DESCRIPTION OF THE INVENTION
[101] The invention provides affinity agents comprising monomer domains,
as well as multimers of the monomer domains. The affinity agents can be
selected for the
ability to bind to a desired ligand or mixture of ligands. The monomer domains
and
multimers can be screened to identify those that have an improved
characteristic such as
improved avidity or affinity or altered specificity for the ligand or the
mixture of ligands,
compared to the discrete monomer domain. The monomer domains of the present
invention
include specific variants of the laminin EGF-like domains, the thrombospondin
Type 1
domains, the trefoil domains, and the thyroglobulin domains.
I. Monomer Domains
[102] Many suitable monomer domains can be used in the polypeptides of
the invention. Typically suitable monomer domains comprise three disulfide
bonds, 30 to
100 amino acids and have a binding site for a divalent metal ion, such as,
e.g., calcium. In
some embodiments, thrombospondin type 1 monomer domains, trefoil monomer
domains, or
thyroglobulin monomer domains are used in the scaffolds of the invention. In
other
embodiments, laminin-EGF monomer domains are used.
[103] Monomer domains can have any number of characteristics. For
example, in some embodiments, the monomer domains have low or no
immunogenicity in an
animal (e.g., a human). Monomer domains can have a small size. In some
embodiments, the
monomer domains are small enough to penetrate skin or other tissues. Monomer
domains
can have a range of in vivo half-lives or stabilities. Characteristics of a
monomer domain
include the ability to fold independently and the ability to form a stable
structure.
[104] Monomer domains can be polypeptide chains of any size. In some
embodiments, monomer domains have about 25 to about 500, about 30 to about
200, about
30 to about 100, about 35 to about 50, about 35 to about 100, about 90 to
about 200, about 30
to about 250, about 30 to about 60, about 9 to about 150, about 100 to about
150, about 25 to
about 50, or about 30 to about 150 amino acids. Similarly, a monomer domain of
the present
invention can comprise, e.g., from about 30 to about 200 amino acids; from
about 25 to about
180 amino acids; from about 40 to about 150 amino acids; from about 50 to
about 130 amino
acids; or from about'75 to about 125 amino acids. Monomer domains and immuno-
domains
can typically maintain a stable conformation in solution, and are often heat
stable, e.g., stable
at 95 C for at least 10 minutes without losing binding affinity. Monomer
domains typically
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bind with a Kd of less than about 10"15, 10"14,10-13,10"12, 10"11, 10-10,
10"9, 10"8,10"7, 10'6, 10"
5, 10-4 , 10"3, 10"a, 0.01 gM, about 0.1 gM, or about 1 M. Sometimes, monomer
domains
and immuno-domains can fold independently into a stable conformation. In one
embodiment, the stable conformation is stabilized by metal ions. The stable
conformation
can optionally contain disulfide bonds (e.g., at least one, two, or three or
more disulfide
bonds). The disulfide bonds can optionally be formed between two cysteine
residues. In
some embodiments, monomer domains, or monomer domain variants, are
substantially
identical to the sequences exemplified (e.g., thrombospondin, trefoil, or
thyroglobulin) or
otherwise referenced herein.
[105] Exemplary monomer domains that are particularly suitable for use in
the practice of the present invention are cysteine-rich domains comprising
disulfide bonds.
Typically, the disulfide bonds promote folding of the domain into a three-
dimensional
structure. Usually, cysteine-rich domains have at least two disulfide bonds,
more typically at
least three disulfide bonds. Suitable cysteine rich monomer domains include,
e.g., the
thrombospondin type 1 domain, the trefoil domain, or the thyroglobulin domain.
[106] The monomer domains can also have a cluster of negatively charged
residues. Monomer domains may bind ion to maintain their secondary structure.
Such
monomer domains include, e.g., A domains, EGF domains, EF Hand (e.g., those
present in
calmodulin and troponin C), Cadherin domains, C-type lectins, C2 domains,
Annexin, Gla-
domains, Thrombospondin type 3 domains, all of which bind calcium, and zinc
fingers (e.g.,
C2H2 type C3HC4 type (RING finger), Integrase Zinc binding domain, PHD finger,
GATA
zinc finger, FYVE zinc finger, B-box zinc finger), which bind zinc. Without
intending to
limit the invention, it is believed that ion-binding stabilizes secondary
structure while
providing sufficient flexibility to allow for numerous binding conformations
depending on
primary sequence.
[107] The structure of the monomer domain is often conserved, although the
polynucleotide sequence encoding the monomer need not be conserved. For
example,
domain structure may be conserved among the members of the domain family,
while the
domain nucleic acid sequence is not. Thus, for example, a monomer domain is
classified as
an Thrombospondin type 1 domain, a trefoil domain, or a thyroglobulin domain
by its
cysteine residues and its affinity for a metal ion (e.g., calcium,) not
necessarily by its nucleic
acid sequence.
[108] In some embodiments, suitable monomer domains (e.g: domains with
the ability to fold independently or with some limited assistance) can be
selected from the
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families of protein domains that contain (3-sandwich or (3-barrel three
dimensional structures
as defined by such computational sequence analysis tools as Simple Modular
Architecture
Research Tool (SMART), see Shultz et al., SMART. a web-based tool for the
study of
genetically mobile domains, (2000) Nucleic Acids Research 28(1):231-234) or
CATH (see
Pearl et.al., Assigning genomic sequences to CA.TH, (2000) Nucleic Acids
Research
28(1):277-282).
[109] In some embodiments, the monomer domains are modified to bind to
substrates to enhance protein function, including, for example, enzymatic
activity and/or
substrate conversion.
[1101 As described herein, monomer domains maybe selected for the ability
to bind to targets other than the target that a homologous naturally occurring
domain may
bind. Thus, in some embodiments, the invention provides monomer domains (and
multimers
comprising such monomers) that do not bind to the target or the class or
family of target
proteins that a homologous naturally occurring domain may bind.
[111] Each of the domains described herein employ exemplary motifs (i.e.,
scaffolds). Certain positions are marked x, indicating that any amino acid can
occupy the
position. These positions can include a number of different amino acid
possibilities, thereby
allowing for sequence diversity and thus affinity for different target
molecules. Use of
brackets in motifs indicates alternate possible amino acids within a position
(e.g., "[ekq]"
indicates that either E, K or Q may be at that position). Use of parentheses
in a motif
indicates that that the positions within the parentheses may be present or
absent (e.g.,
"([ekq])" indicates that the position is absent or either E, K, or Q may be at
that position).
When more than one "x" is used in parentheses (e.g., "(xx)"), each x
represents a possible
position. Thus "(xx)" indicates that zero, one or two amino acids may be at
that position(s),
where each amino acid is independently selected from any amino acid. a
represents an
aromatic/hydrophobic amino acid such as, e.g., W, Y, F, or L; [3 represents a
hydrophobic
amino acid such as, e.g., V, I, L, A, M, or F; x represents a smallor polar
amino acid such as,
e.g., G, A, S, or T; S represents a charged amino acid such as, e.g., K, R, E,
Q, or D; s
represents a small amino acid such as, e.g.,; V, A, S, or T; and ~ represents
a negatively
charged amino acid such as, e.g., D, E, or N.
[112] Suitable domains include, e.g. thrombospondin type 1 domains, trefoil
domains, and thyroglobulin domains.
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[1131 Thrombospondin type 1("TSPl") domains contain about 30-50 or 30-
65 amino acids. In some embodiments, the domains comprise about 35-55 amino
acids and
in some cases about 50 amino acids. Within the 35-55 amino acids, there are
typically about
4 to about 6 cysteine residues. Of the six cysteines, disulfide bonds
typically are found
between the following cysteines: Cl and C5, C2 and C6, C3 and C4. The cysteine
residues
of the domain are disulfide linked to form a compact, stable, functionally
independent moiety
comprising distorted beta strands. Clusters of these repeats make up a ligand
binding
domain, and differential clustering can impart specificity with respect to the
ligand binding.
[114] Exemplary TSPl domain sequences and consensus sequences are as
follows:
(1) (xxxxxX)C1xxxC2xxxxx(x)xxxxxC3xxxx(xxx)xxxxxC4xxxxxx(x)xxxC5(x)xxxxC6;
(2) (wxxWxx)C1xxxC2xxGxx(x)xRxxxC3xxxX(Pxx)xxxxxC4xxxxxx(x)xxxCs(x)xxxxC6
(3) (wxxWxx)CisxtC2xxGxx(x)xRxrx.C3xxxx(Pxx)xxxxxC4xxxxxx(x)xxxCs(x)xxxxC6
(4)
(WxxWxx)C1[Stnd][Vkaq][Tspl]Caxx[Gq]xx(x)x[Re]x[Rktvm]xC3[vldr]xxXx([Pq]xx)xxxx
x
C4[ldae]xxxxxx(x)xxxCS(x)xxxxC6;
(5)
(WxxWxx)Ci[Stnd] [Vkaq] [Tspl]C2xx[Gq]xx(x)x[Re]X[Rktvm]xC3
[vldr]xxxx([Pq]xx)xxxxx
C4[ldae]xxxxXx(x)xxxC5(x)xxxxC6; and
(6)
CI [nst] [aegiklqrstv]
[adenpqrst]C2[adetgs]xgx[ikqrstv]x[aqrst]x[almrtv]xC3xxxxxXxxx(xxxxx
XX)C4XXXXXXXXX(XX)C5XXXXC6
[115] In some embodiments, thrombospondin type 1 domain variants
comprise sequences substantially identical to any of the above-described
sequences.
1116] To date, at least 1677 naturally occurring thrombospondin domains
have been identified based on cDNA sequences. Exemplary proteins containing
the naturally
occurring thrombospondin domains include, e.g., proteins in the complement
pathway (e.g.,
properdin, C6, C7, C8A, C8B, and C9), extracellular matrix proteins (e.g.,
mindin, F-
spondin, SCO-spondin), circumsporozoite surface protein 2, and TRAP proteins
of
Plasmodium. Thrombospondin type 1 domains are further described in, e.g.,
Roszmusz et al.,
BBRC 296:156 (2002); Higgins et al., Jlmmunol. 155:5777-85 (1995); Schultz-
Cherry et al.,
J. Biol. Chem. 270:7304-7310 (1995); Schultz-Cherry et al., J. Biol. Chem.
269:26783-8
(1994); Bork, FEBSLett 327:125-30 (1993); and Leung-Hagesteijn et al., Cell
71:289-99
(1992).
[117] Another exemplary monomer domain suitable for use in the practice of
the present invention is the trefoil domain. Trefoil monomer domains are
typically about
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about 30-50 or 30-65 amino acids. In some embodiments, the domains comprise
about 35-55
amino acids and in some cases about 45 amino acids. Within the 35-55 amino
acids, there
are typically about 6 cysteine residues. Of the six cysteines, disulfide bonds
typically are
found between the following cysteines: Cl and C5, C2 and C4, C3 and C6.
[118] To date, at least 149 naturally occurring trefoil domains have
identified based on cDNA sequences. Exemplary proteins containing naturally
occurring
trefoil domains include, e.g., protein pS2 (TFFl), spasmolytic peptide SP
(TFF2), intestinal
trefoil factor (TFF3), intestinal surcease-isomaltase, and proteins which may
be involved in
defense against microbial infections by protecting the epithelia (e.g.,
Xenopus xPl, xP4,
integumentary mucins A. 1 and C. 1. Trefoil domains are further described in,
e.g., Sands and
Podoisky, Annu. Rev. Physiol. 58:253-273 (1996); Carr et al., PNAS USA 91:2206-
2210
(1994); DeA et al., PNAS USA 91:1084-1088 (1994); Hoffinan et al., Trends
.Riochem Sci
18:239-243 (1993).
[119] Exemplary trefoil domain sequences and consensus sequences are as
follows:
(1) C1(Xx)XxxXxxxxxC2XX(x)XXXXXXXC3xXXxC4C5xxXXX(x)XxXxxC6
(2) C1(xX)xXXXxxRxxC2xX(X)xxxxxxXC3XXXXC4C5XXXxX(x)XXXXxC6
(3) Cl(xx)xxxpxxRxnC2gx(x)pxitxxxC3xxxgC4C$fdxxx(x)xxxpwC6f
(4)
C1(xX)xxx[Pvae]xxRx[ndpm]Ca[Gaiy] [ypfst]([de]x)[pskq]x[Ivap]
[Tsa]xx[qedk]C3xx[
krln] [Gnk] C4C5[Fwy] [Dnrs] [sdpnte]xx(x)xxx[pki] [Weash] C6[Fy]
(5)
C 1(xx)xxx[Pvae]xxRx[ndpm] C2[Gaiy] [ypfst]([de]x)[pskq]x[Ivap]
[Tsa]xx[keqd]C3x
x[krln][Gnk]C4C5[a][Dnrs] [sdpnte]xx(x)xxx[pki] [Weash] C6[Fy]
(6)
Cl ([dnps])[adiklnprstv] [dfilmv] [adenprst] [adelprv] [ehklnqrs] [adegknsv]
[kqr] [fiklqrtv] [dnpqs
] C2[agiy] [flpsvy] [dknpqs] [adfghlp] [aipv] [st] [aegkpqrs] [adegkpqs]
[deiknqt]C3 [adeflcnqrt] [ade
gknqs] [gn] C4C5 [wyfh] [deinrs] [adgnpst] [aefgqlrstw] jgiknsvmq]
([afinprstv] [degklns] [afiqstv] [
iknpv]w)C6
[120] Another exemplary monomer domain suitable for use in the present
invention is the thyroglobulin domain. Thyroglobulin monomer domains are
typically about
30-85 or 30-80 amino acids. In some embodiments, the domains comprise about 35-
75
amino acids and in some cases about 65 amino acids. Within the 35-75 amino
acids, there
are typically about 6 cysteine residues. Of the six cysteines, disulfide bonds
typically are
found between the following cysteines: C1 and C2, C3 and C4, C5 and C6.
[121] To date at least 251 naturally occurring thyroglobulin domains have
been identified based on cDNA sequences. The N-terminal section of Tg contains
10 repeats
of a domain of about 65 amino acids which is known as the Tg type-1 repeat
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PUBMED:3595599, PUBMED:8797845. Exemplary proteins containing naturally
occurring
thyroglobulin domains include e.g., the HLA class II associated invariant
chain, human
pancreatic carcinoma marker proteins, nidogen (entactin), insulin-like growth
factor binding
proteins (IGFBP), saxiphilin, chum salmon egg cysteine proteinase inhibitor,
and equistatin.
The Thyr-1 and related domains belong to MEROPS proteinase inhibitor family 13
1, clan IX.
Thyroglobulin domains are further described in, e.g., Molina et al., Eur. J.
Biochem. 240:125-
133 (1996); Guncar et aL, EMBO J 18:793-803 (1999); Chong and Speicher,
DW276:5804-
5813 (2001).
[122] Exemplary thyroglobulin domain sequences and consensus sequences
are as follows:
(1)
Clxxxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxxxxxxCaxxxxxxxxxxC3x(x)x(xxx)xxxxC4x
Cgxxxx(x)XXXXxXXXXxXXxX(xx)xC6
(2)
Clxx x(xxxxxxxxxx)xxxxxxxyxPxCaxxxGxxxxxQC3x(x)x(xxx)xxxxC4
WC5 Vxxx(x) GxxxxGxxxxxxxx(xx)xC6
(3)C1 (xxxxxxxxxx)xxxxxxxyxPxCaxxxGxyxxxQC3x(x)s(xxx)xxgxC4WC5
Vdxx(x)GxxxxGxxxxxgxx(xx)xC6
(4)Cl
[qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxx[Yflip]xPxC2xxxGx[Yf]xx[vkrl]QC3x(x[s
a]xxx)xx[Gsa]xC4[Wyf]C5V[Dnyfl]xx(x)Gxxxx[Gdne]xxxxxgxx(xx)xC6
(5)Cl
[qerl]xxxxxxxxxxxxxx(xxxxxxxxxx)xxxxxxx[ahp]xPxC2xxxGx[a]xx[vkrl]QC3x(x[sa]
xxx)xx[gas]xC4[a] C$V [Dna]xx(x)Gxxxx[~g]xxxxxgxx(xx)xC6
[123] Another exemplary monomer domain that can be used in the present
invention is a laminin-EGF domain. Laminin-EGF domains are typically about 30-
85 or 30-
80 amino acids. In some embodiments, the domains comprise about 45-65 amino
acids and
in some cases about 50 amino acids. Within the 45-65 amino acids, there are
typically about
8 cysteine residues which interact to form 4 disulflde bonds. Laminins are a
major
noncollagenous component of basement membranesthat mediate cell adhesion,
growth
migration, and differentiation. They are composed of distinct but related
alpha, beta, and
gamma chains. The three chains form a cross-shaped molecule that consist of a
long arm and
three short globular arms. The long arm consist of a coiled coil structure
contributed by all
three chains and cross-linked by interchain disulphide bonds.
[124] Exemplary laminin EGF domain sequences and consensus sequences
are as follows:
(1)
C 1xC2xxxxxx(xxx)xxC3xxx(xxxxxx)xxxxC4xC5xxxxxxxxC6xxC7xxxxxxx(xxxxx)xxx
xxC8
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(2)
C 1xCZxxxxxx(xxx)xxC3xxx(xxxxxx)xxgxC4xC5xxxxxGxxC6xxC7xxxxxxx(xxxxx)xx
xxxCs
(3)
C1xC2[ndh]xxxxx(xxx)xxC3xxx(xxxxxx)xxgxC4xC5xxxxxGxxC6[denq]xC7xx[gn][yf
ht]xxX(xxxXX)xxxxXCs
[125] As mentioned above, monomer domains can be naturally-occurring or
non-naturally occurring variants. The term "naturally occurring" is used
herein to indicate
that an object can be found in nature. For example, natural monomer domains
can include
human monomer domains or optionally, domains derived from different species or
sources,
e.g., mammals, primates, rodents, fish, birds, reptiles, plants, etc. The
natural occurring
monomer domains can be obtained by a number of methods, e.g., by PCR
amplification of
genomic DNA or cDNA. Libraries of monomer domains employed in the practice of
the
present invention may contain naturally-occurring monomer domain, non-
naturally occurring
monomer domain variants, or a combination thereof.
[126] Monomer domain variants can include ancestral domains, randomized
domains, chimeric domains, mutated domains, and the like. For example,
ancestral domains
can be based on phylogenetic analysis. Randomized domains are domains in which
one or
more regions are randomized. The randomization can be based on full
randomization, or
optionally, partial randomization based on natural distribution of sequence
diversity.
Chimeric domains are domains in which one or more regions are replaced by
corresponding
regions from other domains of the same family. For example, chimeric domains
can be
constructed by combining loop sequences from multiple related domains of the
same family
to form novel domains with potentially lowered immunogenicity. Those of skill
in the art
will recognized the immunologic benefit of constructing modified binding
domain monomers
by combining loop regions from various related domains of the same family
rather than
creating random amino acid sequences. For example, by constructing variant
domains by
combining loop sequences or even multiple loop sequences that occur naturally
in human
thrombospondin type I monomer domains, thyroglobulin monomer domains, or
trefoil
monomer domains, the resulting domains may contain novel binding properties
but may not
contain any immunogenic protein sequences because all of the exposed loops are
of human
origin. The combining of loop amino acid sequences in endogenous context can
be applied to
all of the monomer constructs of the invention.
[127] The non-natural monomer domains or altered monomer domains can
be produced by a number of methods. Any method of mutagenesis, such as site-
directed
mutagenesis and random mutagenesis (e.g., chemical mutagenesis) can be used to
produce
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variants. In some embodiments, error-prone PCR is employed to create variants.
Additional
methods include aligning a plurality of naturally occurring monomer domains by
aligning
conserved amino acids in the plurality of naturally occurring monomer domains;
and,
designing the non-naturally occurring monomer domain by maintaining the
conserved amino
acids and inserting, deleting or altering amino acids around the conserved
amino acids to
generate the non-naturally occurring monomer domain. In one embodiment, the
conserved
amino acids comprise cysteines. In another embodiment, the inserting step uses
random
amino acids, or optionally, the inserting step uses portions of the naturally
occurring
monomer domains. The portions could ideally encode loops from domains from the
same
family. Amino acids are inserted or exchanged using synthetic
oligonucleotides, or by
shuffling, or by restriction enzyme based recombination. Human chimeric
domains of the
present invention are useful for therapeutic applications where minimal
immunogenicity is
desired. The present invention provides methods for generating libraries of
human chimeric
domains.
[128] Multimers or monomer domains of the invention can be produced
according to any methods known in the art. In some embodiments, E. coli
comprising a
plasmid encoding the polypeptides under transcriptional control of a bacterial
promoter are
used to express the protein. After harvesting the bacteria, they may be lysed
by sonication,
heat, or homogenization and clarified by centrifugation. The polypeptides may
be purified
using Ni-NTA agarose elution (if 6xHis tagged) or DEAE sepharose elution (if
untagged) and
refolded by dialysis. Misfolded proteins may be neutralized by capping free
sulthydrils with
iodoacetic acid. Q sepharose elution, butyl sepharose flow-through, SP
sepharose elution,
DEAE sepharose elution, and/or CM sepharose elution may be used to purify the
polypeptides. Equivalent anion and/or cation exchange or hydrophobic
interaction
purification steps may also be employed.
[129] In some embodiments, monomers or multimers are purified using heat
lysis, typically followed by a fast cooling to prevent most proteins from
renaturing. Due to
the heat stability of the proteins of the invention, the desired proteins will
not be denatured by
the heat and therefore will allow for a purification step (i.e., purification
that eliminates
contaminant proteins) resulting in high purity. In some embodiments, a
continuous flow
heating process to purify the monomers or multimers from bacterial cell
cultures is used. For
example, a cell suspension can passed through a stainless steel coil submerged
in a water bath
set to a temperature resulting in lysis of the bacteria (e.g., about 55 C, 60
C, 65 C, 70 C,
75 C, 80 C, 85 C, 90 C, 95 C, or 100 C for about 5, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, or
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60 minutes). The lysed effluent is routed to a cooling bath to obtain rapid
cooling and
prevent renaturation of denatured E. coli proteins. E. coli proteins denature
and are prevented
from renaturing, but the monomer or multimers do not denature under these
conditions due to
the exceptional stability of their scaffold. The heating time is controlled by
adjusting the
flow rate and length of the coil. This approach yields active proteins with
high yield and
exceptionally high purity (e.g., >60%, >65%, >70%, >75%, or >80%) compared to
alternative approaches and is amenable to high throughput (e.g., 96-well or
384-well)
production and large scale (e.g., about 100 gl to about 1, 2, 5, 10, 15, 20,
50, 75, 100, 500, or
1000 liters) production of material including clinical material and material
for screening
assays (e.g., in vitro binding and inhibition assays and cell-based activity
assays) .
[130] In some embodiments, following manufacture of the monomers or
multimers of the invention, the polypeptides are treated in a solution
comprising iodoacetic
acid to cap free -SH moieties of cysteines that have not formed disulfide
bonds. In some
embodiments, 0.1-100 mM (e.g., 1-10 mM) iodoacetic acid is included in the
solutions.
Typically, the iodoacetic acid can be removed before administered to an
individual.
[131] Polynucleotides (also referred to as nucleic acids) encoding the
monomer domains are typically employed to make monomer domains via expression.
Nucleic acids that encode monomer domains can be derived from a variety of
different
sources. Libraries of monomer domains can be prepared by expressing a
plurality of
different nucleic acids encoding naturally occurring monomer domains, altered
monomer
domains (i.e., monomer domain variants), or a combinations thereof.
[132] Nucleic acids encoding fragments of naturally-occurring monomer
domains and/or immuno-domains can also be mixed and/or recombined (e.g., by
using
chemically or enzymatically-produced fragments) to generate full-length,
modified monomer
domains and/or immuno-domains. The fragments and the monomer domain can also
be
recombined by manipulating nucleic acids encoding domains or fragments
thereof. For
example, ligating a nucleic acid constntct encoding fragments of the monomer
domain can be
used to generate an altered monomer domain.
[133] Altered monomer domains can also be generated by providing a
collection of synthetic oligonucleotides (e.g., overlapping oligonucleotides)
encoding
conserved, random, pseudorandom, or a defined sequence of peptide sequences
that are then
inserted by ligation into a predetermined site in a polynucleotide encoding a
monomer
domain. Similarly, the sequence diversity of one or more monomer domains can
be
expanded by mutating the monomer domain(s) with site-directed mutagenesis,
random
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mutation, pseudorandom mutation, defined kemal mutation, codon-based mutation,
and the
like. The resultant nucleic acid molecules can be propagated in a host for
cloning and
amplification. In some embodiments, the nucleic acids are recombined.
[134] The present invention also provides a method for recombining a
plurality of nucleic acids encoding monomer domains and screening the
resulting library for
monomer domains that bind to the desired ligand or mixture of ligands or the
like. Selected
monomer domain nucleic acids can also be back-crossed by recombining with
polynucleotide
sequences encoding neutral sequences (i.e., having insubstantial functional
effect on binding),
such as for example, by back-crossing with a wild-type or naturally-occurring
sequence
substantially identical to a selected sequence to produce native-like
functional monomer
domains. Generally, during back-crossing, subsequent selection is applied to
retain the
property, e.g., binding to the ligand.
[135] In some embodiments, the monomer library is prepared by
recombination. In such a case, monomer domains are isolated and recombined to
combinatorially recombine the nucleic acid sequences that encode the monomer
domains
(recombination can occur between or within monomer domains, or both). The
first step
involves identifying a monomer domain having the desired property, e.g.,
affinity for a
certain ligand. While maintaining the conserved amino acids during the
recombination, the
nucleic acid sequences encoding the monomer domains can be recombined, or
recombined
and joined into multimers.
II. Multimers
[136] Methods for generating multimers (i.e., recombinant mosaic proteins
or combinatorial mosaic proteins) are a feature of the present invention.
Multimers comprise
at least two monomer domains. For example, multimers of the invention can
comprise from
2 to about 10 monomer domains, from 2 and about 8 monomer domains, from about
3 and
about 10 monomer domains, about 7 monomer domains, about 6 monomer domains,
about 5
monomer domains, or about 4 monomer domains. In some embodiments, the multimer
comprises at least 3 monomer domains. In view of the possible range of monomer
domain
sizes, the multimers of the invention may be, e.g., 100 kD, 90kD, 80kD, 70kD,
60kD, 50kd,
40kD, 30kD, 25kD, 20kD, l5kD, lOkD, 5kD or smaller or larger. Typically, the
monomer
domains have been pre-selected for binding to the target molecule of interest.
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[137] In some embodiments, each monomer domain specifically binds to one
target molecule. In some of these embodiments, each monomer binds to a
different position
(analogous to an epitope) on a target molecule. Multiple monomer domains
and/or immuno-
domains that bind to the same target molecule result in an avidity effect
yielding improved
avidity of the multimer for the target molecule compared to each individual
monomer. In
some embodiments, the multimer has an avidity of at least about 1.5, 2, 3, 4,
5, 10, 20, 50 or
100 or 1000 times the avidity of a monomer domain alone. Typically, the
multimer has a Kd
of less than about 10-15, 10-14, 10"13, 10"12, 10-11, 10"10, 10-9, or 10"8. In
some embodiments, at
least one, two, three, four or more (including all) monomers of a multimer
bind an ion such as
calcium or another ion.
[138] In another embodiment, the multimer comprises monomer domains
with specificities for different target molecules. For example, multimers of
such diverse
monomer domains can specifically bind different components of a viral
replication system or
different serotypes of a virus. In some embodiments, at least one monomer
domain binds to a
toxin and at least one monomer domain binds to a cell surface molecule,
thereby acting as a
mechanism to target the toxin. In some embodiments, at least two monomer
domains and/or
immuno-domains of the multimer bind to different target molecules in a target
cell or tissue.
Similarly, therapeutic molecules can be targeted to the cell or tissue by
binding a therapeutic
agent to a monomer of the multimer that also contains other monomer domains
and/or
immuno-domains having cell or tissue binding specificity. In some embodiments,
the
different monomers bind to different components of a signal transduction
pathway, a
metabolic pathway, or components of different metabolic pathways that exert
the same
additive or synergistic physiological or biological effect or effects.
[139] Multimers can comprise a variety of combinations of monomer
domains. For example, in a single multimer, the selected monomer domains can
be the same
or identical, optionally, different or non-identical. In addition, the
selected monomer
domains can comprise various different monomer domains from the same monomer
domain
family, or various monomer domains from different domain families, or
optionally, a
combination of both.
[140] Multimers that are generated in the practice of the present invention
maybe any of the following:
(1) A homo-multimer (a multimer of the same domain, i. e., Al -Al -A1-A1);
(2) A hetero-multimer of different domains of the same domain class, e.g., A1-
A2-A3-
A4. For example, hetero-multimer include multimers where Al, A2, A3 and A4 are
different
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non-naturally occurring variants of a particular thrombospondin type I monomer
domains,
thyroglobulin monomer domains, or trefoil monomer domains, or where some of
Al, A2, A3,
and A4 are naturally-occurring variants of a thrombospondin type I monomer
domain,
thyroglobulin monomer domain, or trefoil monomer domain.
(3) A hetero-multimer of domains from different monomer domain classes, e.g.,
Al-
B2-A2-Bl. For example, where Al and A2 are two different monomer domains
(either
naturally occurring or non-naturally-occurring) from thrombospondin type I,
and B 1 and B2
are two different monomer domains (either naturally occurring or non-naturally
occurring)
from a thyroglobulin.
[141] Multimer libraries employed in the practice of the present invention
may contain homo-multimers, hetero-multimers of different monomer domains
(natural or
non-natural) of the same monomer class, or hetero-multimers of monomer domains
(natural
or non-natural) from different monomer classes, or combinations thereof. Other
exemplary
multimers include, e.g., trimers and higher level (e.g., tetramers).
[142] Monomer domains, as described herein, are also readily employed in a
immuno-domain-containing heteromultimer (i.e., a multimer that has at least
one immuno-
domain variant and one monomer domain variant). Thus, multimers of the present
invention
may have at least one immuno-domain such as a minibody, a single-domain
antibody, a
single chain variable fragment (ScFv), or a Fab fragment; and at least one
monomer domain,
such as, for example, a Thrombospondin type I domain, a thyroglobulin type I
repeat domain,
a Trefoil (P-type) domain, an EGF-like domain (e.g., a Laminin-type EGF-like
domain), a
Kringle-domain, a fibronectin type I domain, a fibronectin type II domain, a
fibronectin type
III domain, a PAN domain, a Gla domain, a SRCR domain, a Kunitz/Bovine
pancreatic
trypsin Inhibitor domain, a Kazal-type serine protease inhibitor domain, a von
Willebrand
factor type C domain, an Anaphylatoxin-like domain, a CUB domain LDL-receptor
class A
domain, a Sushi domain, a Link domain, a Thrombospondin type 3 domain, an
Immunoglobulin-like domain, a C-type lectin domain, a MAM domain, a von
Willebrand
factor type A domain, a Somatomedin B domain, a WAP-type four disulfide core
domain, a
F5/8 type C domain, a Hemopexin domain, an SH2 domain, an SH3 domain, an EF
Hand
domain, a Cadherin domain, an Annexin domain, a zinc finger domain, and a C2
domain, or
variants thereof.
[143] Domains need not be selected before the domains are linked to form
multimers. On the other hand, the domains can be selected for the ability to
bind to a target
molecule before being linked into multimers. Thus, for example, a multimer can
comprise
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two domains that bind to one target molecule and a third domain that binds to
a second target
molecule.
[144] Typically, multimers of the present invention are a single discrete
polypeptide. Multimers of partial linker-domain-partial linker moieties are an
association of
multiple polypeptides, each corresponding to a partial linker-domain-partial
linker moiety.
11451 Accordingly, the multimers of the present invention may have the
following qualities: multivalent, multispecific, single chain, heat stable,
extended serum
and/or shelf half-life. Moreover, at least one, more than one or all of the
monomer domains
may bind an ion (e.g., a metal ion or a calcium ion), atleast one, more than
one or all
monomer domains may be derived from thrombospondin type I monomer domains,
thyroglobulin monomer domains, or trefoil monomer domains, at least one, more
than one or
all of the monomer domains may be non-naturally occurring, and/or at least
one, more than
one or all of the monomer domains may comprise 1, 2, 3, or 4 disulfide bonds
per monomer
domain. In some embodiments, the multimers comprise at least two (or at least
three)
monomer domains, wherein at least one monomer domain is a non-naturally
occurring
monomer domain and the monomer domains bind calcium. In some embodiments, the
multimers comprise at least 4 monomer domains, wherein at least one monomer
domain is
non-naturally occurring, and wherein:
a. each monomer domain is between 30-100 amino acids and each of the monomer
domains comprise at least one disulfide linkage; or
b. each monomer domain is between 30-100 amino acids and is derived from an
extracellular protein; or
c. each monomer domain is between 30-100 amino acids and binds to a protein
target.
[146] In some embodiments, the multimers comprise at least 4 monomer
domains, wherein at least one monomer domain is non-naturally occurring, and
wherein:
a. each monomer domain is between 35-100 amino acids; or
b. each domain comprises at least one disulfide bond and is derived from a
human
protein and/or an extracellular protein.
[147] In some embodiments, the multimers comprise at least two monomer
domains, wherein at least one monomer domain is non-naturally occurring, and
wherein each
domain is:
a. 25-50 amino acids long and comprises at least one disulfide bond; or
b. 25-50 amino acids long and is derived from an extracellular protein; or
c. 25-50 amino acids and binds to a protein target; or
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d. 35-50 amino acids long.
[148] In some embodiments, the multimers comprise at least two monomer
domains, wherein at least one monomer domain is non-naturally-occurring and:
a. each monomer domain comprises at least one disulfide bond; or
b. at least one monomer domain is derived from an extracellular protein; or
c. at least one monomer domain binds to a target protein.
[149] In some embodiments, the multimers of the invention bind to the same
or other multimers to form aggregates. Aggregation can be mediated, for
example, by the
presence of hydrophobic domains on two monomer domains and/or immuno-domains,
resulting in the formation of non-covalent interactions between two monomer
domains and/or
immuno-domains. Alternatively, aggregation may be facilitated by one or more
monomer
domains in a multimer having binding specificity for a monomer domain in
another multimer.
Aggregates can also form due to the presence of affinity peptides on the
monomer domains or
multimers. Aggregates can contain more target molecule binding domains than a
single
multimer.
[150] Multimers with affinity for both a cell surface target and a second
target may provide for increased avidity effects. In some cases, membrane
fluidity can be
more flexible than protein linkers in optimizing (by self-assembly) the
spacing and valency of
the interactions. In some cases, multimers will bind to two different targets,
each on a
different cell or one on a cell and another on a molecule with multiple
binding sites.
III. Linkers
[151] The selected monomer domains may be joined by a linker to form a
single chain multimer. For example, a linker is positioned between each
separate discrete
monomer domain in a multimer. Typically, immuno-domains are also linked to
each other or
to monomer domains via a linker moiety. Linker moieties that can be readily
employed to
link immuno-domain variants together are the same as those described for
multimers of
monomer domain variants. Exemplary linker moieties suitable for joining immuno-
domain
variants to other domains into multimers are described herein.
[152] Joining the selected monomer domains via a linker can be
accomplished using a variety of techniques known in the art. For example,
combinatorial
assembly of polynucleotides encoding selected monomer domains can be achieved
by
restriction digestion and re-ligation, by PCR-based, self-priming overlap
reactions, or other
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recombinant methods. The linker can be attached to a monomer before the
monomer is
identified for its ability to bind to a target multimer or after the monomer
has been selected
for the ability to bind to a target multimer.
[153] The linker can be naturally-occurring, synthetic or a combination of
both. For example, the synthetic linker can be a randomized linker, e.g., both
in sequence
and size. In one aspect, the randomized linker can comprise a fully randomized
sequence, or
optionally, the randomized linker can be based on natural linker sequences.
The linker can
comprise, e.g,. a non-polypeptide moiety, a polynucleotide, a polypeptide or
the like.
[154] A linker can be rigid, or alternatively, flexible, or a combination.of
both. Linker flexibility can be a function of the composition of both the
linker and the
monomer domains that the linker interacts with. The linker joins two selected
monomer
domain, and maintains the monomer domains as separate discrete monomer
domains. The
linker can. allow the separate discrete monomer domains to cooperate yet
maintain separate
properties such as multiple separate binding sites for the same ligand in a
multimer, or e.g.,
multiple separate binding sites for different ligands in a multimer. In some
cases, a disulfide
bridge exists between two linked monomer domains or between a linker and a
monomer
domain. In some embodiments, the monmer domains and/or linkers comprise metal-
binding
centers.
[155] Choosing a suitable linker for a specific case where two or more
monomer domains (i.e. polypeptide chains) are to be connected may depend on a
variety of
parameters including, e.g. the nature of the monomer domains, the structure
and nature of the
target to which the polypeptide multimer should bind and/or the stability of
the peptide linker
towards proteolysis and oxidation.
[156] The present invention provides methods for optimizing the choice of
linker once the desired monomer domains/variants have been identified.
Generally, libraries
of multimers having a composition that is fixed with regard to monomer domain
composition,
but variable in linker composition and length, can be readily prepared and
screened as
described above.
[157] Typically, the linker polypeptide may predominantly include amino
acid residues selected from Gly, Ser, Ala and Thr. For example, the peptide
linker may
contain at least 75% (calculated on the basis of the total number of residues
present in the
peptide linker), such as at least 80%, e.g. at least 85% or at least 90% of
amino acid residues
selected from Gly, Ser, Ala and Thr. The peptide linker may also consist of
Gly, Ser, Ala
and/or Thr residues only. The linker polypeptide should have a length, which
is adequate to
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link two monomer domains in such a way that they assume the correct
conformation relative
to one another so that they retain the desired activity, for example as
antagonists of a given
receptor.
[158] A suitable length for this purpose is a length of at least one and
typically fewer than about 50 amino acid residues, such as 2-25 amino acid
residues, 5-20
amino acid residues, 5-15 amino acid residues, 8-12 amino acid residues or 11
residues.
Similarly, the polypeptide encoding a linker can range in size, e.g., from
about 2 to about 15
amino acids, from about 3 to about 15, from about 4 to about 12, about 10,
about 8, or about
6 amino acids. In methods and compositions involving nucleic acids, such as
DNA, RNA, or
combinations of both, the polynucleotide containing the linker sequence can
be, e.g., between
about 6 nucleotides and about 45 nucleotides, between about 9 nucleotides and
about 45
nucleotides, between about 12 nucleotides and about 36 nucleotides, about 30
nucleotides,
about 24 nucleotides, or about 18 nucleotides. Likewise, the amino acid
residues selected for
inclusion in the linker polypeptide should exhibit properties that do not
interfere significantly
with the activity or function of the polypeptide multimer. Thus, the peptide
linker should on
the whole not exhibit a charge which would be inconsistent with the activity
or function of
the polypeptide multimer, or interfere with internal folding, or form bonds or
other
interactions with amino acid residues in one or more of the monomer domains
which would
seriously impede the binding of the polypeptide multimer to the target in
question.
[159] In another embodiment of the invention, the peptide linker is selected
from a library where the amino acid residues in the peptide linker are
randomized for a
specific set of monomer domains in a particular polypeptide multimer. A
flexible linker
could be used to find suitable combinations of monomer domains, which is then
optimized
using this random library of variable linkers to obtain linkers with optimal
length and
geometry. The optimal linkers may contain the minunal number of amino acid
residues of
the right type that participate in the binding to the target and restrict the
movement of the
monomer domains relative to each other in the polypeptide multimer when not
bound to the
target.
[160] The use of naturally occurring as well as artificial peptide linkers to
connect polypeptides into novel linked fusion polypeptides is well known in
the literature
(Hallewell et al. (1989), J. Biol. Chem. 264, 5260-5268; Alfthan et al.
(1995), Protein Eng. 8,
725-731; Robinson & Sauer (1996), Biochemistry 35, 109-116; Khandekar et al.
(1997), J.
Biol. Chem. 272, 32190-32197; Fares et al. (1998), Endocrinology 139, 2459-
2464;
Smallshaw et al. (1999), Protein Eng. 12, 623-630; US 5,856,456).
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[161] One example where the use of peptide linkers is widespread is for
production of single-chain antibodies where the variable regions of a light
chain (VL) and a
heavy chain (VH) are joined through an artificial linker, and a large number
of publications
exist within this particular field. A widely used peptide linker is a 15mer
consisting of three
repeats of a Gly-Gly-Gly-Gly-Ser amino acid sequence ((G1y4Ser)3). Other
linkers have been
used, and phage display technology, as well as, selective infective phage
technology has been
used to diversify and select appropriate linker sequences (Tang et al. (1996),
J. Biol. Chem.
271, 15682-15686; Hennecke et al. (1998), Protein Eng. 11, 405-410). Peptide
linkers have
been used to connect individual chains in hetero- and homo-dimeric proteins
such as the T-
cell receptor, the lambda Cro repressor, the P22 phage Arc repressor, IL- 12,
TSH, FSH, IL-5,
and interferon-y. Peptide linkers have also been used to create fusion
polypeptides. Various
linkers have been used and in the case of the Arc repressor phage display has
been used to
optimize the linker length and composition for increased stability of the
single-chain protein
(Robinson and Sauer (1998), Proc. Natl. Acad. Sci. USA 95, 5929-5934).
[162] Another type of linker is an intein, i.e. a peptide stretch which is
expressed with the single-chain polypeptide, but removed post-translationally
by protein
splicirig. The use of inteins is reviewed by F.S. Gimble in Chemistry and
Biology, 1998, Vol
5, No. 10 pp. 251-256.
[163] Still another way of obtaining a suitable linker is by optimizing a
simple linker, e.g. (G1y4Ser),,, through random mutagenesis.
[164] As mentioned above, it is generally preferred that the peptide linker
possess at least some flexibility. Accordingly, in some embodiments, the
peptide linker
contains 1-25 glycine residues, 5-20 glycine residues, 5-15 glycine residues
or 8-12 glycine
residues. The peptide linker will typically contain at least 50% glycine
residues, such as at
least 75% glycine residues. In some embodiments of the invention, the peptide
linker
comprises glycine residues only.
11651 The peptide,linker may, in addition to the glycine residues, comprise
other residues, in particular residues selected from Ser, Ala and Thr, in
particular Ser. Thus,
one example of a specific peptide linker includes a peptide linker having the
amino acid
sequence Gly,-Xaa-Glyy-Xaa-GlyZ, wherein each Xaa is independently selected
from the
group consisting Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys,
Tyr, Asn, Gln,
Lys, Arg, His, Asp and Glu, and wherein x, y and z are each integers in the
range from 1-5.
In some embodiments, each Xaa is independently selected from Ser, Ala and Thr,
in
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particular Ser. More particularly, the peptide linker has the amino acid
sequence Gly-Gly-
Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly, wherein each Xaa is independently
selected from
the group consisting Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Gly, Ser, Thr,
Cys, Tyr, Asn, Gln,
Lys, Arg, His, Asp and Glu. In some embodiments, each Xaa is independently
selected from
Ser, Ala and Thr, in particular Ser.
11661 In some cases it may be desirable or necessary to provide some rigidity
into the peptide linker. This may be accomplished by including proline
residues in the amino
acid sequence of the peptide linker. Thus, in another embodiment of the
invention, the
peptide linker comprises at least one proline residue in the amino acid
sequence of the
peptide linker. For example, the peptide linker has an amino acid sequence,
wherein at least
25%, such as at least 50%, e.g. at least 75%, of the amino acid residues are
proline residues.
In one particular embodiment of the invention, the peptide linker comprises
proline residues
only.
[167] In some embodiments of the invention, the peptide linker is modified
in such a way that an amino acid residue comprising an attachment group for a
non-
polypeptide moiety is introduced. Examples of such amino acid residues may be
a cysteine
residue (to which the non-polypeptide moiety is then subsequently attached) or
the amino
acid sequence may include an in vivo N-glycosylation site (thereby attaching a
sugar moiety
(in vivo) to the peptide linker). An additional option is to genetically
incorporate non-natural
amino acids using evolved tRNAs and tRNA synthetases (see, e.g., U.S. Patent
Application
Publication 2003/0082575) into the monomer domains or linkers. For example,
insertion of
keto-tyrosine allows for site-specific coupling to expressed monomer domains
or multimers.
[168] In some embodiments of the invention, the peptide linker comprises at
least one cysteine residue, such as one cysteine residue. Thus, in some
embodiments of the
invention the peptide linker comprises amino acid residues selected from Gly,
Ser, Ala, Thr
and Cys. In some embodiments, such a peptide linker comprises one cysteine
residue only.
11691 In a further embodiment, the peptide linker comprises glycine residues
and cysteine residue, such as glycine residues and cysteine residues only.
Typically, only one
cysteine residue will be included per peptide linker. Thus, one example of a
specific peptide
linker comprising a cysteine residue, includes a peptide linker having the
amino acid
sequence Glyn-Cys-Glym, wherein n and m are each integers from 1-12, e.g.,
from 3-9, from
4-8, or from 4-7. More particularly, the peptide linker may have the amino
acid sequence
GGGGG-C-GGGGG.
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[170] This approach (i.e. introduction of an amino acid residue comprising
an attachment group for a non-polypeptide moiety) may also be used for the
more rigid
proline-containing linkers. Accordingly, the peptide linker may comprise
proline and
cysteine residues, such as proline and cysteine residues only. An example of a
specific
proline-containing peptide linker comprising a cysteine residue, includes a
peptide linker
having the amino acid sequence Proõ-Cys-Prom, wherein n and m are each
integers from 1-12,
preferably from 3-9, such as from 4-8 or from 4-7. More particularly, the
peptide linker may
have the amino acid sequence PPPPP-C-PPPPP.
[171] In some embodiments, the purpose of introducing an amino acid
residue, such as a cysteine residue, comprising an attachment group for a non-
polypeptide
moiety is to subsequently attach a non-polypeptide moiety to said residue. For
example, non-
polypeptide moieties can improve the serum half-life of the polypeptide
multimer. Thus, the
cysteine residue can be covalently attached to a non-polypeptide moiety.
Preferred examples
of non-polypeptide moieties include polymer molecules, such as PEG or mPEG, in
particular
mPEG as well as non-polypeptide therapeutic agents.
[172] The skilled person will acknowledge that amino acid residues other
than cysteine may be used for attaching a non-polypeptide to the peptide
linker. One
particular example of such other residue includes coupling the non-polypeptide
moiety to a
lysine residue.
. [173] Another possibility of introducing a site-specific attachment group
for
a non-polypeptide moiety in the peptide linker is to introduce an in vivo N-
glycosylation site,
such as one in vivo N-glycosylation site, in the peptide linker. For example,
an in vivo N-
glycosylation site may be introduced in a peptide linker comprising amino acid
residues
selected from Gly, Ser, Ala and Thr. It will be understood that in order to
ensure that a sugar
moiety is in fact attached to said in vivo N-glycosylation site, the
nucleotide sequence
encoding the polypeptide multimer must be inserted in a glycosylating,
eukaryotic expression
host.
[174] A specific exainple of a peptide linker comprising an in vivo N-
glycosylation site is a peptide linker having the amino acid sequence Glyn-Asn-
Xaa-Ser/Thr-
Glym, preferably Gly,-Asn-Xaa-Thr-G1ym, wherein Xaa is any amino acid residue
except
proline, and wherein n and m are each integers in the range from 1-8,
preferably in the range
from 2-5.
[175] Often, the amino acid sequences of all peptide linkers present in the
polypeptide multimer will be identical. Nevertheless, in certain embodiments
the amino acid
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sequences of all peptide linkers present in the polypeptide multimer may be
different. The
latter is believed to be particular relevant in case the polypeptide multimer
is a polypeptide
tri-mer or tetra-mer and particularly in such cases where an amino acid
residue comprising an
attachment group for a non-polypeptide moiety is included in the peptide
linker.
[176] Quite often, it will be desirable or necessary to attach only a few,
typically only one, non-polypeptide moieties/moiety (such as mPEG, a sugar
moiety or a
non-polypeptide therapeutic agent) to the polypeptide multimer in order to
achieve the
desired effect, such as prolonged serum-half life. Evidently, in case of a
polypeptide tri-mer,
which will contain two peptide linkers, only one peptide linker is typically
required to be
modified, e.g: by introduction of a cysteine residue, whereas modification of
the other peptide
linker will typically not be necessary not. In this case all (both) peptide
linkers of the
polypeptide multimer (tri-mer) are different.
[177] Accordingly, in a further embodiment of the invention, the amino acid
sequences of all peptide linkers present in the polypeptide multimer are
identical except for
one, two or three peptide linkers, such as except for one or two peptide
linkers, in particular
except for one peptide linker, which has/have an amino acid sequence
comprising an amino
acid residue comprising an attachment group for a non-polypeptide moiety.
Preferred
examples of such amino acid residues include cysteine residues of in vivo N-
glycosylation
sites.
[178] A linker can be a native or synthetic linker sequence. An exemplary
native linker includes, e.g., the sequence between the last cysteine of a
first thrombospondin
type I monomer domain, thyroglobulin monomer domain, or trefoil monomer domain
and the
first cysteine of a second thrombospondin type I monomer domain, thyroglobulin
monomer
domain, or trefoil monomer domain can be used as a linker sequence. Analysis
of various
domain linkages reveals that native linkers range from at least 3 amino acids
to fewer than 20
amino acids, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18
amino acids long.
However, those of skill in the art will recognize that longer or shorter
linker sequences can be
used. In some embodiments, the linker is a 6-mer of the following sequence
AlA2A3A4A5A6,
wherein Al is selected from the amino acids A, P, T, Q, E and K; A2 and A3 are
any amino
acid except C, F, Y, W, or M; A4 is selected from the amino acids S, G and R;
A5 is selected
from the amino acids H, P, and R; and A6 is the amino acid, T.
[179] Methods for generating multimers from monomer domains and/or
immuno-domains can include joining the selected domains with at least one
linker to generate
at least one multimer, e.g., the multimer can comprise at least two of the
monomer domains
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and/or immuno-domains and the linker. The multimer(s) is then screened for an
improved
avidity or affinity or altered specificity for the desired ligand or mixture
of ligands as
compared to the selected monomer domains. A composition of the multimer
produced by the
method is included in the present invention.
[180] In other methods, the selected multimer domains are joined with at
least one linker to generate at least two multimers, wherein the two multimers
comprise two
or more of the selected monomer domains and the linker. The two or more
multimers are
screened for an improved avidity or affinity or altered specificity for the
desired ligand or
mixture of ligands as compared to the selected monomer domains. Compositions
of two or
more multimers produced by the above method are also features of the
invention.
[181] Linkers, multimers or selected multimers produced by the methods
indicated above and below are features of the present invention. Libraries
comprising
multimers, e.g, a library comprising about 100, 250, 500 or more members
produced by the
methods of the present invention or selected by the methods of the present
invention are
provided. In some embodiments, one or more cell comprising members of the
libraries, are
also included. Libraries of the recombinant polypeptides are also a feature of
the present
invention, e.g., a library comprising about 100, 250, 500 or more different
recombinant
polypetides.
[182] Suitable linkers employed in the practice of the present invention
include an obligate heterodimer of partial linker moieties. The term "obligate
heterodimer"
(also referred to as "affinity peptides") refers herein to a dimer of two
partial linker moieties
that differ from each other in composition, and which associate with each
other in a non-
covalent, specific manner to join two domains together. The specific
association is such that
the two partial linkers associate substantially with each other as compared to
associating with
other partial linkers. Thus, in contrast to multimers of the present invention
that are
expressed as a single polypeptide, multimers of domains that are linked
together via
heterodimers are assembled from discrete partial linker-monomer-partial linker
units.
Assembly of the heterodimers can be achieved by, for example, mixing. Thus, if
the partial
linkers are polypeptide segments, each partial linker-monomer-partial linker
unit may be
expressed as a discrete peptide prior to multimer assembly. A disulfide bond
can be added to
covalently lock the peptides together following the correct non-covalent
pairing. Partial
linker moieties that are appropriate for forming obligate heterodimers
include, for example,
polynucleotides, polypeptides, and the like. For example, when the partial
linker is a
polypeptide, binding domains are produced individually along with their unique
linking
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peptide (i.e., a partial linker) and later combined to form multimers. See,
e.g., Madden, M.,
Aldwin, L., Gallop, M. A., and Stemmer, W. P. C. (1993) Peptide linkers:
Unique self-
associative high-affinity peptide linkers. Thirteenth American Peptide
Symposium,
Edmonton, Canada (abstract). The spatial order of the binding domains in the
multimer is
thus mandated by the heterodimeric binding specificity of each partial linker.
Partial linkers
can contain terminal amino acid sequences that specifically bind to a defined
heterologous
amino acid sequence. An example of such an amino acid sequence is the Hydra
neuropeptide
head activator as described in Bodenmuller et al., The neuropeptide head
activator loses its
biological activity by dimerization, (1986) EMBO J 5(8):1825-1829. See, e.g.,
U.S. Patent
No. 5,491,074 and WO 94/28173. These partial linkers allow the multimer to be
produced
first as monomer-partial linker units or partial linker-monomer-partial linker
units that are
then mixed together and allowed to assemble into the ideal order based on the
binding
specificities of each partial linker. Alternatively, monomers linked to
partial linkers can be
contacted to a surface, such as a cell, in which multiple monomers can
associate to form
higher avidity complexes via partial linkers. In some cases, the association
will form via
random Brownian motion.
[183] When the partial linker comprises a DNA binding motif, each
monomer domain has an upstream and a downstream partial linker (i.e., Lp-
domain-Lp,
where "Lp" is a representation of a partial linker) that contains a DNA
binding protein with
exclusively unique DNA binding specificity. These domains can be produced
individually
and then assembled into a specific multimer by the mixing of the domains with
DNA
fraglnents containing the proper nucleotide sequences (i.e., the specific
recognition sites for
the DNA binding proteins of the partial linkers of the two desired domains) so
as to join the
domains in the desired order. Additionally, the same domains may be assembled
into many
different multimers by the addition of DNA sequences containing various
combinations of
DNA binding protein recognition sites. Further randomization of the
combinations of DNA
binding protein recognition sites in the DNA fragments can allow the assembly
of libraries of
multimers. The DNA can be synthesized with backbone analogs to prevent
degradation in
vivo.
[184] In some embodiments, the multimer comprises monomer domains with
specificities for different proteins. The different proteins can be related or
unrelated.
Examples of related proteins including members of a protein family or
different serotypes of
a virus. Alternatively, the monomer domains of a multimer can target different
molecules in
a physiological pathway (e.g., different blood coagulation proteins). In yet
other
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embodiments, monomer domains bind to proteins in unrelated pathways (e.g., two
domains
bind to blood factors, two other domains bind to inflammation-related proteins
and a fifth
binds to serum albumin). In another embodiment, a multimer is comprised of
monomer
domains that bind to different pathogens or contaminants of interest. Such
multimers are
useful to as a single detection agent capable of detecting for the possibility
of any of a
number of pathogens or contaminants.
IV. Methods of Identifying Monomer Domains and/or Multimers with a Desired
Binding Affmity
[185] The invention provides methods of identifying monomer domains that
bind to a selected or desired ligand or mixture of ligands. In some
embodiments, monomer
domains and/or immuno-domains are identified or selected for a desired
property (e.g.,
binding affinity) and then the monomer domains and/or immuno-domains are
formed into
multimers. For those embodiments, any method resulting in selection of domains
with a
desired property (e.g., a specific binding property) can be used. For example,
the methods
can comprise providing a plurality of different nucleic acids, each nucleic
acid encoding a
monomer domain; translating the plurality of different nucleic acids, thereby
providing a
plurality of different monomer domains; screening the plurality of different
monomer
domains for binding of the desired ligand or a mixture of ligands; and,
identifying members
of the plurality of different monomer domains that bind the desired ligand or
mixture of
ligands.
11861 Selection of monomer domains and/or immuno-domains from a library
of domains can be accomplished by a variety of procedures. For example, one
method of
identifying monomer domains and/or immuno-domains which have a desired
property
involves translating a plurality of nucleic acids, where each nucleic acid
encodes a monomer
domain and/or immuno-domain, screening the polypeptides encoded by the
plurality of
nucleic acids, and identifying those monomer domains and/or immuno-domains
that, e.g.,
bind to a desired ligand or mixture of ligands, thereby producing a selected
monomer domain
and/or immuno-domain. The monomer domains and/or immuno-domains expressed by
each
of the nucleic acids can be tested for their ability to bind to the ligand by
methods known in
the art (i.e. panning, affinity chromatography, FACS analysis).
[187] As mentioned above, selection of monomer domains and/or immuno-
domains can be based on binding to a ligand such as a target protein or other
target molecule
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(e.g., lipid, carbohydrate, nucleic acid and the like). Other molecules can
optionally be
included in the methods along with the target, e.g., ions such as Ca 2. The
ligand can be a
known ligand, e.g., a ligand known to bind one of the plurality of monomer
domains, or e.g.,
the desired,ligand can be an unknown monomer domain ligand. Other selections
of monomer
domains and/or immuno-domains can be based, e.g., on inhibiting or enhancing a
specific
function of a target protein or an activity. Target protein activity can
include, e.g.,
endocytosis or internalization, induction of second messenger system, up-
regulation or down-
regulation of a gene, binding to an extracellular matrix, release of a
molecule(s), or a change
in conformation. In this case, the ligand does not need to be known. The
selection can also
include using high-throughput assays.
[188] When a monomer domain and/or immuno-domain is selected based on
its ability to bind to a ligand, the selection basis can include selection
based on a slow
dissociation rate, which is usually predictive of high affinity. The valency
of the ligand can
also be varied to control the average binding affinity of selected monomer
domains and/or
immuno-domains. The ligand can be bound to a surface or substrate at varying
densities,
such as by including a competitor compound, by dilution, or by other method
known to those
in the art. High density (valency) of predetermined ligand can be used to
enrich for monomer
domains that have relatively low affinity, whereas a low density (valency) can
preferentially
enrich for higher affinity monomer domains.
[189] A variety of reporting display vectors or systems can be used to
express nucleic acids encoding the monomer domains immuno-domains and/or
multimers of
the present invention and to test for a desired activity. For example, a phage
display system
is a system in which monomer domains are expressed as fusion proteins on the
phage surface
(Pharmacia, Milwaukee Wis.). Phage display can involve the presentation of a
polypeptide
sequence encoding monomer domains and/or immuno-domains on the surface of a
filamentous bacteriophage, typically as a fusion with a bacteriophage coat
protein.
[1901 Generally in these methods, each phage particle or cell serves as an
individual library member displaying a single species of displayed polypeptide
in addition to
the natural phage or cell protein sequences. The plurality of nucleic acids
are cloned into the
phage DNA at a site which results in the transcription of a fusion protein, a
portion of which
is encoded by the plurality of the nucleic aoids. The phage containing a
nucleic acid
molecule undergoes replication and transcription in the cell. The leader
sequence of the
fusion protein directs the transport of the fusion protein to the tip of the
phage particle. Thus,
the fusion protein that is partially encoded by the nucleic acid is displayed
on the phage
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particle for detection and selection by the methods described above and below.
For example,
the phage library can be incubated with a predetermined (desired) ligand, so
that phage
particles which present a fusion protein sequence that binds to the ligand can
be differentially
partitioned from those that do not present polypeptide sequences that bind to
the
predetermined ligand. For example, the separation can be provided by
immobilizing the
predetermined ligand. The phage particles (i.e., library members) which are
bound to the
immobilized ligand are then recovered and replicated to amplify the selected
phage
subpopulation for a subsequent round of affinity enrichment and phage
replication. After
several rounds of affinity enrichment and phage replication, the phage library
members that
are thus selected are isolated and the nucleotide sequence encoding the
displayed polypeptide
sequence is determined, thereby identifying the sequence(s) of polypeptides
that bind to the
predetermined ligand. Such methods are further described in PCT patent
publication Nos.
91/17271, 91/18980, and 91/19818 and 93/08278.
[1911 Examples of other display systems include ribosome displays, a
nucleotide-linked display (see, e.g., U.S. Patent Nos. 6,281,344; 6,194,550,
6,207,446,
6,214,553, and 6,258,558), polysome display, cell surface displays and the
like. The cell
surface displays include a variety of cells, e.g., E. coli, yeast and/or
mammalian cells. When
a cell is used as a display, the nucleic acids, e.g., obtained by PCR
amplification followed by
digestion, are introduced into the cell and translated. Optionally,
polypeptides encoding the
monomer domains or the multimers of the present invention can be introduced,
e.g., by
injection, into the cell.
[1921 Those of skill in the art will recognize that the steps of generating
variation and screening for a desired property can be repeated (i.e.,
performed recursively) to
optimize results. For example, in a phage display library or other like
format, a first
screening of a library can be performed at relatively lower stringency,
thereby selected as
many particles associated with a target molecule as possible. The selected
particles can then
be isolated and the polynucleotides encoding the monomer or multimer can be
isolated from
the particles. Additional variations can then be generated from these
sequences and
subsequently screened at higher affinity.
[193] Monomer domains may be selected to bind any type of target
molecule, including protein targets. Exemplary targets include, but are not
limited to, e.g.,
IL-6, Alpha3, cMet, ICOS, IgE, IL-1-R11, BAFF, CD40L, CD28, Her2, TRAIL-R,
VEGF,
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TPO-R, TNFaa, LFA-l, TACI, IL-1b, B7.1, B7.2, or OX40. When the target is a
receptor for
a ligand, the monomer domains may act as antagonists or agonists of the
receptor.
[194] When multimers capable of binding relatively large targets are desired,
they can be generated by a "walking" selection method. As shown in Figure 3,
this method is
carried out by providing a library of monomer domains and screening the
library of monomer
domains for affinity to a first target molecule. Once at least one monomer
that binds to the
target is identified, that particular monomer is covalently linked to a new
library or each
remaining member of the original library of monomer domains. The new library
members
each comprise one common domain and at least one domain that that is
different, i.e.,
randomized. Thus, in some embodiments, the invention provides a library of
multimers
generated using the "walking" selection method. This new library of multimers
(e.g., dimers,
trimers, tetramers, and the like) is then screened for multimers that bind to
the target with an
increased affinity, and a multimer that binds to the target with an increased
affinity can be
identified. The "walking" monomer selection method provides a way to assemble
a multimer
that is composed of monomers that can act additively or even synergistically
with each other
given the restraints of linker length. This walking technique is very useful
when selecting for
and assembling multimers that are able to bind large target proteins with high
affinity. The
walking method can be repeated to add more monomers thereby resulting in a
multimer
comprising 2, 3, 4, 5, 6, 7, 8 or more monomers linked together.
[195] In some embodiments, the selected multimer comprises more than two
domains. Such multimers can be generated in a step fashion, e.g., where the
addition of each
new domain is tested individually and the effect of the domains is tested in a
sequential
fashion. In an alternate embodiment, domains are linked to form multimers
comprising more
than two domains and selected for binding without prior knowledge of how
smaller
multimers, or alternatively, how each domain, bind.
[196] The methods of the present invention also include methods of evolving
monomers or multimers. As illustrated in Figure 10, intra-domain recombination
can be
introduced into monomers across the entire monomer or by taking portions of
different
monomers to form new recombined units. The different monomers may bind the
same target
or different targets. For example, in some embodiments portions of different
thrombospondin
monomers may be recombined. In some embdiments, a portion of a thrombospondin
monomer may be combined with a portion of a thyroglobulin monomer and/or a
portion of a
trefoil/PD monomer. Interdomain recombination (e.g., recombining different
monomers into
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or between multimers) or recombination of modules (e.g., multiple monomers
within a
multimer) may be achieved. Inter-library recombination is also contemplated.
[197] Figure 8 illustrates the process of intradomain optimization by
recombination. Shown is a three-fragment PCR overlap reaction, which
recombines three
segments of a single domain relative to each other. One can use two, three,
four, five or more
fragment overlap'reactions in the same way as illustrated. This recombination
process has
many applications. One application is to recombine a large pool of hundreds of
previously
selected clones without sequence information. All that is needed for each
overlap to work is
one known region of (relatively) constant sequence that exists in the same
location in each of
the clones (fixed site approach). The intra-domain recombination method can
also be
performed on a pool of sequence-related monomer domains by standard DNA
recombination
(e.g., Stemmer, Nature 370:389-391 (1994)) based on random fragmentation and
reassembly
based on DNA sequence homology, which does not require a fixed overlap site in
all of the
clones that are to be recombined.
[198] Another application of this process is to create multiple separate,
naive
(meaning unpanned) libraries in each of which only one of the intercysteine
loops is
randomized, to randomize a different loop in each library. After panning of
these libraries
separately against the target, the selected clones are then recombined. From
each panned
library only the randomized segment is amplified by PCR and multiple
randomized segments
are then combined into a single domain, creating a shuffled library which is
panned and/or
screened for increased potency. This process can also be used to shuffle a
small number of
clones of known sequence.
[199] Any common sequence may be used as cross-over points. For
cysteine-containing monomers, the cysteine residues are logical places for the
crossover.
However, there are other ways to determine optimal crossover sites, such as
computer
modeling. Alternatively, residues with highest entropy, or the least number of
intramolecular
contacts, may also be good sites for crossovers.
[200] Methods for evolving monomers or multimers can comprise, e.g., any
or all of the following steps: providing a plurality of different nucleic
acids, where each
nucleic acid encoding a monomer domain; translating the plurality of different
nucleic acids,
which provides a plurality of different monomer domains; screening the
plurality of different
monomer domains for binding of the desired ligand or mixture of ligands;
identifying
members of the plurality of different monomer domains that bind the desired
ligand or
mixture of ligands, which provides selected monomer domains; joining the
selected monomer
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domains with at least one linker to generate at least one multimer, wherein
the at least one
multimer comprises at least two of the selected monomer domains and the at
least one linker;
and, screening the at least one multimer for an improved affinity or avidity
or altered
specificity for the desired ligand or mixture of ligands as compared to the
selected monomer
domains.
[201) Variation can be introduced into either monomers or multimers. As
discussed above, an example of improving monomers includes intra-domain
recombination in
which two or more (e.g., three, four, five, or more ) portions of the monomer
are amplified
separately under conditions to introduce variation (for example by shuffling
or other
recombination method) in the resulting amplification products, thereby
synthesizing a library
of variants for different portions of the monomer. By locating the 5' ends of
the middle
primers in a "middle" or 'overlap' sequence that both of the PCR fragments
have in common,
the resulting "left" side and "right" side libraries may be combined by
overlap PCR to
generate novel variants of the original pool of monomers. These new variants
may then be
screened for desired properties, e.g., panned against a target or screened for
a functional
effect. The "middle" primer(s) may be selected to correspond to any segment of
the
monomer, and will typically be based on the scaffold or one or more concensus
amino acids
within the monomer (e.g., cysteines such as those found in A domains).
[202] Similarly, multimers may be created by introducing variation at the
monomer level and then recombining monomer variant libraries. On a larger
scale,
multimers (single or pools) with desired properties may be recombined to form
longer
multimers. In some cases variation is introduced (typically synthetically)
into the monomers
or into the linkers to form libraries. This may be achieved, e.g., with two
different multimers
that bind to two different targets, thereby eventually selecting a multimer
with a portion that
binds to one target and a portion that binds a second target. See, e.g.,
Figure 9.
[203] Additional variation can be introduced by inserting linkers of different
length and composition between domains. This allows for the selection of
optimal linkers
between domains. In some embodiments, optimal length and composition of
linkers will
allow for optimal binding of domains. In some embodiments, the domains with a
particular
binding affinity(s) are linked via different linkers and optimal linkers are
selected in a binding
assay. For example, domains are selected for desired binding properties and
then formed into
a library comprising a variety of linkers. The library can then be screened to
identify optimal
linkers. Alternatively, multimer libraries can be formed where the effect of
domain or linker
on target molecule binding is not known.
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[204] Methods of the present invention also include generating one or more
selected multimers by providing a plurality of monomer domains and/or immuno-
domains.
The plurality of monomer domains and/or immuno-domains is screened for binding
of a
desired ligand or mixture of ligands. Members of the plurality of domains that
bind the
desired ligand or mixture of ligands are identified, thereby providing domains
with a desired
affinity. The identified domains are joined with at least one linker to
generate the multimers,
wherein each multimer comprises at least two of the selected domains and the
at least one
linker; and, the multimers are screened for an improved affinity or avidity or
altered
specificity for the desired ligand or mixture of ligands as compared to the
selected domains,
thereby identifying the one or more selected multimers.
[205] Multimer libraries may be generated, in some embodiments, by
combining two or more libraries or monomers or multimers in a recombinase-
based
approach, where each library member comprises as recombination site (e.g., a
lox site). A
larger pool of molecularly diverse library members in principle harbor more
variants with
desired properties, such as higher target-binding affinities and functional
activities. When
libraries are constructed in phage vectors, which may be transformed into E.
eoli, library size
(109 - 1010) is limited by the transformation efficiency of E. coli. A
recombinase/recombination site system (e.g., the Cre-loxP system) and in vivo
recombination
can be exploited to generate libraries that are not limited in size by the
transformation
efficiency of E. coli.
[206] For example, the Cre-loxP system may be used to generate dimer
libraries with 1010, 1011, 1012, 1013, or greater diversity. In some
embodiments, E. coli as a
host for one naive monomer library and a filamentous phage that carries a
second naive
monomer library are used. The library size in this case is limited only by the
number of
infective phage (carrying one library) and the number of infectible E. cali
cells (carrying the
other library). For example, infecting 1012 E. coli cells (1L at OD600=1) with
>1012 phage
could produce as many as 1012 dimer combinations.
[207] Selection of multimers can be accomplished using a variety of
techniques including those mentioned above for identifying monomer domains.
Other
selection methods include, e.g., a selection based on an improved affinity or
avidity or altered
specificity for the ligand compared to selected monomer domains. For example,
a selection
can be based on selective binding to specific cell types, or to a set of
related cells or protein
types (e.g., different virus serotypes). Optimization of the property selected
for, e.g., avidity
of a ligand, can then be achieved by recombining the domains, as well as
manipulating amino
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acid sequence of the individual monomer domains or the linker domain or the
nucleotide
sequence encoding such domains, as mentioned in the present invention.
[208] One method for identifying multimers can be accomplished by
displaying the multimers. As with the monomer domains, the multimers are
optionally
expressed or displayed on a variety of display systems, e.g., phage display,
ribosome display,
polysome display, nucleotide-linked display (see, e.g., U.S. Patent Nos.
6,281,344;
6,194,550, 6,207,446, 6,214,553, and 6,258,558) and/or cell surface display,
as described
above. Cell surface displays can include but are not limited to E. coli, yeast
or mammalian
cells. In addition, display libraries of multimers with multiple binding sites
can be panned for
avidity or affinity or altered specificity for a ligand or for multiple
ligands.
[209] Monomers or multimers can be screened for target binding activity in
yeast cells using a two-hybrid screening assay. In this type of screen the
monomer or
multimer library to be screened is cloned into a vector that directs the
formation of a fusion
protein between each monomer or multimer of the library and a yeast
transcriptional activator
fragment (i.e., Gal4). Sequences encoding the "target" protein are cloned into
a vector that
results in the production of a fusion protein between the target and the
remainder of the Ga14
protein (the DNA binding domain). A third plasmid contains a reporter gene
downstream of
the DNA sequence of the Ga14 binding site. A monomer that can bind to the
target protein
brings with it the Ga14 activation domain, thus reconstituting a functional
Ga14 protein. This
functional Ga14 protein bound to the binding site upstream of the reporter
gene results in the
expression of the reporter gene and selection of the monomer or multimer as a
target binding
protein. (see Chien et.al. (1991) Proc. Natl. Acad. Sci. (USA) 88:9578; Fields
S. and Song O.
(1989) Nature 340: 245) Using a two-hybrid system for library screening is
further
described in U.S. Patent No. 5,811,238 (see also Silver S.C. and Hunt S.W.
(1993) Mol. Biol.
Rep. 17:155; Durfee et al. (1993) Genes Devel. 7:555; Yang et al. (1992)
Science 257:680;
Luban et al. (1993) Cell 73:1067; Hardy et al. (1992) Genes Devel. 6:801;
Bartel et al. (1993)
Biotechniques 14:920; and Vojtek et al. (1993) Cell 74:205). Another useful
screening
system for carrying out the present invention is the E.coli/BCCP interactive
screening system
(Gennino et al. (1993) Proc. Nat. Acad. Sci. (U.S.A.) 90:993; Guarente L.
(1993) Proc. Nat.
Acad. Sci. (U.S.A.) 90:1639).
[210] Other variations include the use of multiple binding compounds, such
that monomer domains, multimers or libraries of these molecules can be
simultaneously
screened for a multiplicity of ligands or compounds that have different
binding specificity.
Multiple predetermined ligands or compounds can be concomitantly screened in a
single
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library, or sequential screening against a number of monomer domains or
multimers. In one
variation, multiple ligands or compounds, each encoded on a separate bead (or
subset of
beads), can be mixed and incubated with monomer domains, multimers or
libraries of these
molecules under suitable binding conditions. The collection of beads,
comprising multiple
ligands or compounds, can then be used to isolate, by affinity selection,
selected monomer
domains, selected multimers or library members. Generally, subsequent affinity
screening
rounds can include the same mixture of beads, subsets thereof, or beads
containing only one
or two individual ligands or compounds. This approach affords efficient
screening, and is
compatible with laboratory automation, batch processing, and high throughput
screening
methods.
[211] In another embodiment, multimers can be simultaneously screened for
the ability to bind multiple ligands, wherein each ligand comprises a
different label. For
example, each ligand can be labeled with a different fluorescent label,
contacted
simultaneously with a multimer or multimer library. Multimers with the desired
affinity are
then identified (e.g., by FACS sorting) based on the presence of the labels
linked to the
desired labels.
[212] Libraries of either monomer domains or multimers (referred in the
following discussion for convenience as "affinity agents") can be screened
(i.e., panned)
simultaneously against multiple ligands in a number of different formats. For
example,
multiple ligands can be screened in a simple mixture, in an array, displayed
on a cell or tissue
(e.g., a cell or tissue provides numerous molecules that can be bound by the
monomer
domains or multimers of the invention), and/or immobilized. See, e.g., Figure
4. The
libraries of affinity agents can optionally be displayed on yeast or phage
display systems.
Similarly, if desired, the ligands (e.g., encoded in a cDNA library) can be
displayed in a yeast
or phage display system.
[213] Initially, the affinity agent library is panned against the multiple
ligands. Optionally, the resulting "hits" are panned against the ligands one
or more times to
enrich the resulting population of affinity agents.
[214] If desired, the identity of the individual affinity agents and/or
ligands
can be determined. In some embodiments, affinity agents are displayed on
phage. Affinity
agents identified as binding in the initial screen are divided into a first
and second portion.
The first portion is infected into bacteria, resulting in either plaques or
bacterial colonies,
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depending on the type of phage used. The expressed phage are immobilized and
then probed
with ligands displayed in phage selected as described below.
[215] The second portion are coupled to beads or otherwise immobilized and
a phage display library containing at least some of the ligands in the
original mixture is
contacted to the immobilized second portion. Those phage that bind to the
second portion are
subsequently eluted and contacted to the immobilized phage described in the
paragraph
above. Phage-phage interactions are detected (e.g., using a monoclonal
antibody specific for
the ligand-expressing phage) and the resulting phage polynucleotides can be
isolated.
[216] In some embodiments, the identity of an affinity agent-ligand pair is
determined. For example, when both the affinity agent and the ligand are
displayed on a
phage or yeast, the DNA from the pair can be isolated and sequenced. In some
embodiments,
polynucleotides specific for the ligand and affinity agent are amplified.
Amplification
primers for each reaction can include 5' sequences that are complementary such
that the
resulting amplification products are fused, thereby forming a hybrid
polynucleotide
comprising a polynucleotide encoding at least a portion of the affinity agent
and at least a
portion of the ligand. The resulting hybrid can be used to probe affinity
agent or ligand (e.g.,
cDNA-encoded) polynucleotide libraries to identify both affinity agent and
ligand. See, e.g.,
Figure 10.
[217] The above-described methods can be readily combined with "walking"
to simultaneous generate and identify multiple multimers, each of which bind
to a ligand in a
mixture of ligands. In these embodiments, a first library of affinity agents
(monomer
domains, imnluno domains or multimers) are panned against multiple ligands and
the eluted
affinity agents are linked to the first or a second library of affinity agents
to form a library of
multimeric affinity agents (e.g., comprising 2, 3, 4, 5, 6, 7, 8, 9, or more
monomer or immuno
domains), which are subsequently panned against the multiple ligands. This
method can be
repeated to continue to generate larger multimeric affinity agents. Increasing
the number of
monomer domains may result in increased affinity and avidity for a particular
target. Of
course, at each stage, the panning is optionally repeated to enrich for
significant binders. In
some cases, walking will be facilitated by inserting recombination sites
(e.g., lox sites) at the
ends of monomers and recombining monomer libraries by a recombinase-mediated
event.
[218] The selected multimers of the above methods can be further
manipulated, e.g., by recombining or shuffling the selected multimers
(recombination can
occur between or within multimers or both), mutating the selected multimers,
and the like.
This results in altered multimers which then can be screened and selected for
members that
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have an enhanced property compared to the selected multimer, thereby producing
selected
altered multimers.
12191 In view of the description herein, it is clear that the following
process
may be followed. Naturally or non-naturally occurring monomer domains may be
recombined or variants may be formed. Optionally the domains initially or
later are selected
for those sequences that are less likely to be immunogenic in the host for
which they are
intended. Optionally, a phage library comprising the recombined domains is
panned for a
desired affinity. Monomer domains or multimers expressed by the phage may be
screened
for IC50 for a target. Hetero- or homo-meric multimers may be selected. The
selected
polypeptides may be selected for their affinity to any target, including,
e.g., hetero- or homo-
multimeric targets.
[220] A significant advantage of the present invention is that known ligands,
or unknown ligands can be used to select the monomer domains and/or multimers.
No prior
information regarding ligand structure is required to isolate the monomer
domains of interest
or the multimers of interest. The monomer domains and/or multimers identified
can have
biological activity, which is meant to include at least specific binding
affinity for a selected or
desired ligand, and, in some instances, will further include the ability to
block the binding of
other compounds, to stimulate or inhibit metabolic pathways, to act as a
signal or messenger,
to stimulate or inhibit cellular activity, and the like. Monomer domains can
be generated to
function as ligands for receptors where the natural ligand for the receptor
has not yet been
identified (orphan receptors). These orphan ligands can be created to either
block or activate
the receptor top which they bind.
[221] A single ligand can be used, or optionally a variety of ligands can be
used to select the monomer domains and/or multimers. A monomer domain and/or
immuno-
domain of the present invention can bind a single ligand or a variety of
ligands. A multimer
of the present invention can have multiple discrete binding sites for a single
ligand, or
optionally, can have multiple binding sites for a variety of ligands.
V. Libraries
[222] The present invention also provides libraries of monomer domains and
libraries of nucleic acids that encode monomer domains and/or immuno-domains.
The
libraries can include, e.g., about 10, 100, 250, 500, 1000, or 10,000 or more
nucleic acids
encoding monomer domains, or the library can include, e.g., about 10, 100,
250, 500, 1000 or
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10,000 or more polypeptides that encode monomer domains. Libraries can include
monomer
domains containing the same cysteine frame, e.g., thrombosponding domains,
thyroglobulin
domains, or trefoil/PD domains.
[2231 In some embodiments, variants are generated by recombining two or
more different sequences from the same family of monomer domains (e.g., the
LDL receptor
class A domain). Alternatively, two or more different monomer domains from
different
families can be combined to form a multimer. In some embodiments, the
multimers are
formed from monomers or monomer variants of at least one of the following
family classes: a
thrombospondin type I domain, a thyroglobulin type I repeat domain, a Trefoil
(P-type)
domain, an EGF-like domain (e.g., a Laminin-type EGF-like domain), a Kringle-
domain, a
fibronectin type I domain, a fibronectin type II domain, a fibronectin type
III domain, a PAN
domain, a Gla domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin
.Inhibitor domain,
a Kazal-type serine protease inhibitor domain, a von Willebrand factor type C
domain, an
Anaphylatoxin-like domain, a CUB domain LDL-receptor class A domain, a Sushi
domain, a
Link domain, a Thrombospondin type 3 domain, an linmunoglobulin-like domain, a
C-type
lectin domain, a MAM domain, a von Willebrand factor type A domain, a
Somatomedin B
domain, a WAP-type four disulfide core domain, a F5/8 type C domain, a
Hemopexin
domain, an SH2 domain, an SH3 domain, an EF Hand domain, a Cadherin domain, an
Annexin domain, a zinc finger domain, and a C2 domain and derivatives thereof.
In another
embodiment, the monomer domain and the different monomer domain can include
one or
more domains found in the Pfam database and/or the SMART database. Libraries
produced
by the methods above, one or more cell(s) comprising one or more members of
the library,
and one or more displays comprising one or more members of the library are
also included in
the present invention.
12241 Optionally, a data set of nucleic acid character strings encoding
monomer domains can be generated e.g., by mixing a first character string
encoding a
monomer domain, with one or more character string encoding a different monomer
domain,
thereby producing a data set of nucleic acids character strings encoding
monomer domains,
including those described herein. In another embodiment, the monomer domain
and the
different monomer domain can include one or more domains found in the Pfam
database
and/or the SMART database. The methods can further comprise inserting the
first character
string encoding the monomer domain and the one or more second character string
encoding
the different monomer domain in a computer and generating a multimer character
string(s) or
library(s), thereof in the computer.
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[225] The libraries can be screened for a desired property such as binding of
a desired ligand or mixture of ligands or otherwise exposed to selective
conditions. For
example, members of the library of monomer domains can be displayed and
prescreened for
binding to a known or unknown ligand or a mixture of ligands or incubated in
serum to
remove those clones that are sensitive to serum proteases. The monomer domain
sequences
can then be mutagenized (e.g., recombined, chemically altered, etc.) or
otherwise altered and
the new monomer domains can be screened again for binding to the ligand or the
mixture of
ligands with an improved affinity. The selected monomer domains can be
combined or
joined to form multimers, which can then be screened for an improved affinity
or avidity or
altered specificity for the ligand or the mixture of ligands. Altered
specificity can mean that
the specificity is broadened, e.g., binding of multiple related viruses, or
optionally, altered
specificity can mean that the specificity is narrowed, e.g., binding within a
specific region of
a ligand. Those of skill in the art will recognize that there are a number of
methods available
to calculate avidity. See, e.g., Mammen et al., Angew Chem Int. Ed. 37:2754-
2794 (1998);
Muller et al., Anal. Biochem. 261:149-158 (1998).
[226] The present invention also provides a method for generating a library
of chimeric monomer domains derived from human proteins, the method
comprising:
providing loop sequences corresponding to at least one loop from each of at
least two
different naturally occurring variants of a human protein, wherein the loop
sequences are
polynucleotide or polypeptide sequences; and covalently combining loop
sequences to
generate a library of at least two different chimeric sequences, wherein each
chimeric
sequence encodes a chimeric monomer domain having at least two loops.
Typically, the
chimeric domain has at least four loops, and usually at least six loops. As
described above,
the present invention provides three types of loops that are identified by
specific features,
such as, potential for disulfide bonding, bridging between secondary protein
structures, and
molecular dynamics (i, e., flexibility). The three types of loop sequences are
a cysteine-
defined loop sequence, a structure-defined loop sequence, and a B-factor-
defined loop
sequence.
[227] Alternatively, a human chimeric domain library can be generated by
modifying naturally occurring human monomer domains at the amino acid level,
as compared
to the loop level. To minimize the potential for immunogenicity, only those
residues that
naturally occur in protein sequences from the same family of human monomer
domains are
utilized to create the chimeric sequences. This can be achieved by providing a
sequence
alignment of at least two human monomer domains from the same family of
monomer
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domains, identifying amino acid residues in corresponding positions in the
human monomer
domain sequences that differ between the human monomer domains, generating two
or more
human chimeric monomer domains, wherein each human chimeric monomer domain
sequence consists of amino acid residues that correspond in type and position
to residues
from two or more human monomer domains from the same family of monomer
domains.
Libraries of human chimeric monomer domains can be employed to identify human
chimeric
monomer domains that bind to a target of interest by: screening the library of
human chimeric
monomer domains for binding to a target molecule, and identifying a human
chimeric
monomer domain that binds to the target molecule. Suitable naturally occurring
human
monomer domain sequences employed in the initial sequence alignment step
include those
corresponding to any of the naturally occurring monomer domains described
herein.
[228] Human chimeric domain libraries of the present invention (whether
generated by varying loops or single amino acid residues) can be prepared by
methods known
to those having ordinary skill in the art. Methods particularly suitable for
generating these
libraries are split-pool format and trinucleotide synthesis format as
described in
WO01/23401.
VI. Fusion Proteins
[229] In some embodiments, the monomers or multimers of the present
invention are linked to another polypeptide to form a fusion protein. Any
polypeptide in the
art may be used as a fusion partner, though it can be useful if the fusion
partner forms
multimers. For example, monomers or multimers of the invention may, for
example, be
fused to the following locations or combinations of locations of an antibody:
1. At the N-terminus of the VHl and/or VLl domains, optionally just after the
leader peptide and before the domain starts (framework region 1);
2. At the N-terminus of the CHl or CLl domain, replacing the VH1 or VLl
domain;
3. At the N-terminus of the heavy chain, optionally after the CH1 domain and
before the cysteine residues in the hinge (Fc-fusion);
4. At the N-terminus of the CH3 domain;
5. At the C-terminu's of the CH3 domain, optionally attached to the last amino
acid residue via a short linker;
6. At the C-terminus of the CH2 domain, replacing the CH3 domain;
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7. At the C-terminus of the CLI or CH1 domain, optionally after the cysteine
that forms the interchain disulfide; or
8. At the C-terminus of the VH1 or VL1 domain. See, e.g., Figure 7.
[230] In some embodiments, the monomer or multimer domain is linked to a
molecule (e.g., a protein, nucleic acid, organic small molecule, etc.) useful
as a
pharmaceutical. Exemplary pharmaceutical proteins include, e.g., cytokines,
antibodies,
chemokines, growth factors, interleukins, cell-surface proteins, extracellular
domains, cell
surface receptors, cytotoxins, etc. Exemplary small molecule pharmaceuticals
include small
molecule toxins or therapeutic agents.
[231] In some embodiments, the monomer or multimers are selected to bind
to a tissue- or disease-specific target protein. Tissue-specific proteins are
proteins that are
expressed exclusively, or at a significantly higher level, in one or several
particular tissue(s)
compared to other tissues in an animal. Similarly, disease-specific proteins
are proteins that
are expressed exclusively, or at a significantly higher level, in one or
several diseased cells or
tissues compared to other non-diseased cells or tissues in an animal. Examples
of such
diseases include, but are not limited to, a cell proliferative disorder such
as actinic keratosis,
arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed
connective tissue disease
(MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,
psoriasis,
primary thrombocythemia, and cancers including adenocarcinoma, leukemia,
lymphoma,
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of
the adrenal
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, autoimmune polyendacrinopathycandidiasis-ectodermal dystrophy
(APECED),
bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis,
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis,
myocardial or pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,
scleroderrna, Sjogren's
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syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic
sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of
cancer, hemodialysis, and extracorporeal circulation, viral, bacterial,
fungal, parasitic,
protozoal, and helminthic infections, and trauma; a cardiovascular disorder
such as
congestive heart failure, ischemic heart disease, angina pectoris, myocardial
infarction,
hypertensive heart disease, degenerative valvular heart disease, calcific
aortic valve stenosis,
congenitally bicuspid aortic valve, mitral annular calcification, mitral valve
prolapse,
rheumatic fever and rheumatic heart disease, infective endocarditis,
nonbacterial thrombotic
endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart
disease,
cardiomyopathy, myocarditis, pericarditis, neoplastic heart disease,
congenital heart disease,
complications of cardiac transplantation, arteriovenous fistula,
atherosclerosis, hypertension,
vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose
veins,
thrombophlebitis and phlebothrombosis, vascular tumors, and complications of
thrombolysis,
balloon angioplasty, vascular replacement, and coronary artery bypass graft
surgery; a
neurological disorder such as epilepsy, ischemic cerebrovascular disease,
stroke, cerebral
neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease,
dementia, Parkinson's
disease and other extrapyramidal disorders, amyotrophic lateral. sclerosis and
other motor
neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa,
hereditary
ataxias, multiple sclerosis and other demyelinating diseases, bacterial and
viral meningitis,
brain abscess, subdural empyema, epidural abscess, suppurative intracranial
thrombophlebitis, myelitis and radiculitis, viral central nervous system
disease, prion diseases
including kuru, Creutzfeldt-Jakob disease, and GerstmannStraussler-Scheinker
syndrome,
fatal familial insomnia, nutritional and metabolic diseases of the nervous
system,
neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the
central nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders,
autonomic nervous system disorders, cranial nerve disorders, spinal cord
diseases, muscular
dystrophy and other neuromuscular disorders, peripheral nervous system
disorders,
dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic
myopathies,
myasthenia gravis, periodic paralysis, mental disorders including mood,
anxiety, and
schizophrenic disorders, seasonal affective disorder (SAD), akathesia,
amnesia, catatonia,
diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses,
postherpetic
neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and
familial frontotemporal dementia; and a developmental disorder such as renal
tubular
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acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenne and
Becker
muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor,
aniridia,
genitourinary abnormalities, and mental retardation), Smith-Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas,
hereditary neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis,
hypothyroidism, hydrocephalus, seizure disorders such as Syndenham's chorea
and cerebral
palsy, spina bifida, anencephaly,. craniorachischisis, congenital glaucoma,
cataract, and
sensorineural hearing loss. Exemplary disease or conditions include, e.g., MS,
SLE, ITP,
IDDM, MG, CLL, CD, RA, Factor VIII Hemophilia, transplantation,
arteriosclerosis,
Sjogren's Syndrome, Kawasaki Disease, anti-phospholipid Ab, AHA, ulcerative
colitis,
multiple myeloma, Glomerulonephritis, seasonal allergies, and IgA Nephropathy.
[232] In some embodiments, the monomers or multimers that bind to the
target protein are linked to the pharmaceutical protein or small molecule such
that the
resulting complex or fusion is targeted to the specific tissue or disease-
related cell(s) where
the target protein is expressed. Monomers or multimers for use in such
complexes or fusions
can be initially selected for binding to the target protein and may be
subsequently selected by
negative selection against other cells or tissue (e.g., to avoid targeting
bone marrow or other
tissues that set the lower limit of drug toxicity) where it is desired that
binding be reduced or
eliminated in other non-target cells or tissues. By keeping the pharmaceutical
away from
sensitive tissues, the therapeutic window is increased so that a higher dose
may be
administered safely. In another alternative, in vivo panning can be performed
in animals by
injecting a library of monomers or multimers into an animal and then isolating
the monomers
or multimers that bind to a particular tissue or cell of interest.
[233] The fusion proteins described above may also include a linker peptide
between the pharmaceutical protein and the monomer or multimers. A peptide
linker
sequence may be employed to separate, for example, the polypeptide components
by a
distance sufficient to ensure that each polypeptide folds into its secondary
and tertiary
structures. Fusion proteins may generally be prepared using standard
techniques, including
chemical conjugation. Fusion proteins can also be expressed as recombinant
proteins in an
expression system by standard techniques.
[234] Exemplary tissue-specific or disease-specific proteins can be found in,
e.g., Tables I and II of U.S. Patent Publication No 2002/0107215. Exemplary
tissues where
target proteins may be specifically expressed include, e.g., liver, pancreas,
adrenal gland,
thyroid, salivary gland, pituitary gland, brain, spirial cord, lung, heart,
breast, skeletal
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muscle, bone marrow, thymus, spleen, lymph node, colorectal, stomach, ovarian,
small
intestine, uterus, placenta, prostate, testis, colon, colon, gastric, bladder,
trachea, kidney, or
adipose tissue.
VII. Compositions
[235] The invention also includes compositions that are produced by
methods of the present invention. For example, the present invention includes
monomer
domains selected or identified from a library and/or libraries comprising
monomer domains
produced by the methods of the present invention.
[236] Compositions of nucleic acids and polypeptides are included in the
present invention. For example, the present invention provides a plurality of
different nucleic
acids wherein each nucleic acid encodes at least one monomer domain or immuno-
domain.
In some embodiments, at least one monomer domain is selected from: an EGF-like
domain
(e.g., a laminin EGF domain), a Trefoil (P-type) domain, a thyroglobulin type
I repeat, a
Thrombospondin type I domain, and variants of one or more thereof. Suitable
monomer
domains also include those listed in the Pfam database and/or the SMART
database.
[237] The present invention also provides recombinant nucleic acids
encoding one or more polypeptides comprising a plurality of monomer domains,
which
monomer domains are altered in order or sequence as compared to a naturally
occuring
polypeptide. For exaxriple, the naturally occuring polypeptide can be selected
from: an EGF-
like domain (e.g., a laminin EGF domain), a Trefoil (P-type) domain, a
thyroglobulin type I
repeat domain, a Thrombospondin type I domain, and variants of one or more
thereof. In
another embodiment, the naturally occuring polypeptide encodes a monomer
domain found in
the Pfam database and/or the SMART database.
[238] All the compositions of the present invention, including the
compositions produced by the methods of the present invention, e.g., monomer
domains as
well as multimers and libraries thereof can be optionally bound to a matrix of
an affinity
material. Examples of affinity material include beads, a column, a solid
support, a
microarray, other pools of reagent-supports, and the like. In some
embodiments, screening in
solution uses a target that has been biotinylated. In these embodiments, the
target is incubated
with the phage library and the targets with the bound phage, are captured
using streptavidin
beads.
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[239] Compositions of the present invention can be bound to a matrix of an
affinity material, e.g., the recombinant polypeptides. Examples of affinity
material include,
e.g., beads, a column, a solid support, and/or the like.
VIII. Therapeutic and Prophylactic Treatment Methods
[240] The present invention also includes methods of therapeutically or
prophylactically treating a disease or disorder by administering in vivo or ex
vivo one or more
nucleic acids or polypeptides of the invention described above (or
compositions comprising a
pharmaceutically acceptable excipient and one or more such nucleic acids or
polypeptides) to
a subject, including, e.g., a mammal, including a human, primate, mouse, pig,
cow, goat,
rabbit, rat, guinea pig, hamster, horse, sheep; or a non-mammalian vertebrate
such as a bird
(e.g., a chicken or duck), fish, or invertebrate.
[241] In one aspect of the invention, in ex vivo methods, one or more cells or
a population of cells of interest of the subject (e.g., tumor cells, tumor
tissue sample, organ
cells, blood cells, cells of the skin, lung, heart, muscle, brain, mucosae,
liver, intestine,
spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth, tongue,
etc.) are obtained
or removed from the subject and contacted with an amount of a selected monomer
domain
and/or multimer of the invention that is effective in prophylactically or
therapeutically
treating the disease, disorder, or other condition. The contacted cells are
then returned or
delivered to the subject to the site from which they were obtained or to
another site (e.g.,
including those defined above) of interest in the subject to be treated. If
desired, the
contacted cells can be grafted onto a tissue, organ, or system site (including
all described
above) of interest in the subject using standard and well-known grafting
techniques or, e.g.,
delivered to the blood or lymph system using standard delivery or transfusion
techniques.
[242] The invention also provides in vivo methods in which one or more cells
or a population of cells of interest of the subject are contacted directly or
indirectly with an
amount of a selected monomer domain and/or multimer of the invention effective
in
prophylactically or therapeutically treating the disease, disorder, or other
condition. In direct
contact/administration formats, the selected monomer domain and/or multimer is
typically
administered or transferred directly to the cells to be treated or to the
tissue site of interest
(e.g., tumor cells, tumor tissue sample, organ cells, blood cells, cells of
the skin, lung, heart,
muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphatic system,
cervix, vagina,
prostate, mouth, tongue, etc.) by any of a variety of formats, including
topical administration,
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injection (e.g., by using a needle or syringe), or vaccine or gene gun
delivery, pushing into a
tissue, organ, or skin site. The selected monomer domain and/or multimer can
be delivered,
for example, intramuscularly, intradermally, subdermally, subcutaneously,
orally,
intraperitoneally, intrathecally, intravenously, or placed within a cavity of
the body
(including, e.g., during surgery), or by inhalation or vaginal or rectal
administration. In some
embodiments, the proteins of the invention are prepared at concentrations of
at least 25
mglml, 50 mg/ml, 75 mg/ml, 100 mg/ml, 150 mg/ml or more. Such concentrations
are
useful, for example, for subcutaneous formulations.
[243] In in vivo indirect contact/administration formats, the selected
monomer domain and/or multimer is typically administered or transferred
indirectly to the
cells to be treated or to the tissue site of interest, including those
described above (such as,
e.g., skin cells, organ systems, lymphatic system, or blood cell system,
etc.), by contacting or
administering the polypeptide of the invention directly to one or more cells
or population of
cells from which treatment can be facilitated. For example, tumor cells within
the body of
the subject can be treated by contacting cells of the blood or lymphatic
system, skin, or an
organ with a sufficient amount of the selected monomer domain and/or multimer
such that
delivery of the selected monomer domain and/or multimer to the site of
interest (e.g., tissue,
organ, or cells of interest or blood or lymphatic system within the body)
occurs and effective
prophylactic or therapeutic treatment results. Such contact, administration,
or transfer is
typically made by using one or more of the routes or modes of administration
described
above.
[244] In another aspect, the invention provides ex vivo methods in which one
or more cells of interest or a population of cells of interest of the subject
(e.g., tumor cells,
tumor tissue sample, organ cells, blood cells, cells of the skin, lung, heart,
muscle, brain,
mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina,
prostate, mouth,
tongue, etc.) are obtained or removed from the subject and transformed by
contacting said
one or more cells or population of cells with a polynucleotide construct
comprising a nucleic
acid sequence of the invention that encodes a biologically active polypeptide
of interest (e.g.,
a selected monomer domain and/or multimer) that is effective in
prophylactically or
therapeutically treating the disease, disorder, or other condition. The one or
more cells or
population of cells is contacted with a sufficient amount of the
polynucleotide construct and a
promoter controlling expression of said nucleic acid sequence such that uptake
of the
polynucleotide construct (and promoter) into the cell(s) occurs and sufficient
expression of
the target nucleic acid sequence of the invention results to produce an amount
of the
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biologically active polypeptide, encoding a selected monomer domain and/or
multimer,
effective to prophylactically or therapeutically treat the disease, disorder,
or condition. The
polynucleotide construct can include a promoter sequence (e.g., CMV promoter
sequence)
that controls expression of the nucleic acid sequence of the invention and/or,
if desired, one
or more additional nucleotide sequences encoding at least one or more of
another polypeptide
of the invention, a cytokine, adjuvant, or co-stimulatory molecule, or other
polypeptide of
interest.
[245] Following transfection, the transformed cells are returned, delivered,
or
transferred to the subject to the tissue site or system from which they were
obtained or to
another site (e.g., tumor cells, tumor tissue sample, organ cells, blood
cells, cells of the skin,
lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach,
lymphatic system,
cervix, vagina, prostate, mouth, tongue, etc.) to be treated in the subject.
If desired, the cells
can be grafted onto a tissue, skin, organ, or body system of interest in the
subject using
standard and well-known grafting techniques or delivered to the blood or
lymphatic system
using standard delivery or transfusion techniques. Such delivery,
administration, or transfer
of transformed cells is typically made by using one or more of the routes or
modes of
administration described above. Expression of the target nucleic acid occurs
naturally or can
be induced (as described in greater detail below) and an amount of the encoded
polypeptide is
expressed sufficient and effective to treat the disease or condition at the
site or tissue system.
[246] In another aspect, the invention provides in vivo methods in which one
or more cells of interest or a population of cells of the subject (e.g.,
including those cells and
cells systems and subjects described above) are transformed in the body of the
subject by
contacting the cell(s) or population of cells with (or administering or
transferring to the cell(s)
or population of cells using one or more of the routes or modes of
administration described
above) a polynucleotide construct comprising a nucleic acid sequence of the
invention that
encodes a biologically active polypeptide of interest (e.g., a selected
monomer domain and/or
multimer) that is effective in prophylactically or therapeutically treating
the disease, disorder,
or other condition.
[247] The polynucleotide construct can be directly administered or
transferred to cell(s) suffering from the disease or disorder (e.g., by direct
contact using one
or more of the routes or modes of administration described above).
Alternatively, the
polynucleotide construct can be indirectly administered or transferred to
cell(s) suffering
from the disease or disorder by first directly contacting non-diseased cell(s)
or other diseased
cells using one or more of the routes or modes of administration described
above with a
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sufficient amount of the polynucleotide construct comprising the nucleic acid
sequence
encoding the biologically active polypeptide, and a promoter controlling
expression of the
nucleic acid sequence, such that uptake of the polynucleotide construct (and
promoter) into
the cell(s) occurs and sufficient expression of the nucleic acid sequence of
the invention
results to produce an amount of the biologically active polypeptide effective
to
prophylactically or therapeutically treat the disease or disorder, and whereby
the
polynucleotide construct or the resulting expressed polypeptide is transferred
naturally or
automatically from the initial delivery site, system, tissue or organ of the
subject's body to
the diseased site, tissue, organ or system of the subject's body (e.g., via
the blood or
lymphatic system). Expression of the target nucleic acid occurs naturally or
can be induced
(as described in greater detail below) such that an amount of expressed
polypeptide is
sufficient and effective to treat the disease or condition at the site or
tissue system. The
polynucleotide construct can include a promoter sequence (e.g., CMV promoter
sequence)
that controls expression of the nucleic acid sequence and/or, if desired, one
or more
additional nucleotide sequences encoding at least one or more of another
polypeptide of the
invention, a cytokine, adjuvant, or co-stimulatory molecule, or other
polypeptide of interest.
[248] In each of the in vivo and ex vivo treatment methods as described
above, a composition comprising an excipient and the polypeptide or nucleic
acid of the
invention can be administered or delivered. In one aspect, a composition
comprising a
pharmaceutically acceptable excipient and a polypeptide or nucleic acid of the
invention is
administered or delivered to the subject as described above in an amount
effective to treat the
disease or disorder.
[249] In another aspect, in each in vivo and ex vivo treatment method
described above, the amount of polynucleotide administered to the cell(s) or
subject can be an
amount such that uptake of said polynucleotide into one or more cells of the
subject occurs
and sufficient expression of said nucleic acid sequence results to produce an
amount of a
biologically active polypeptide effective to enhance an immune response in the
subject,
including an immune response induced by an immunogen (e.g., antigen). In
another aspect,
for each such method, the amount of polypeptide administered to cell(s) or
subject can be an
amount sufficient to enhance an immune response in the subject, including that
induced by an
immunogen (e.g., antigen).
[250] In yet another aspect, in an in vivo or in vivo treatment method in
which a polynucleotide construct (or composition comprising a polynucleotide
construct) is
used to deliver a physiologically active polypeptide to a subject, the
expression of the
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polynucleotide construct can be induced by using an inducible on- and off-gene
expression
system. Examples of such on- and off-gene expression systems include the Tet-
OnTM Gene
Expression System and Tet-OffTM Gene Expression System (see, e.g., Clontech
Catalog
2000, pg. 110-111 for a detailed description of each such system),
respectively. Other
controllable or inducible on- and off-gene expression systems are known to
those of ordinary
skill in the art. With such system, expression of the target nucleic of the
polynucleotide
construct can be regulated in a precise, reversible, and quantitative manner.
Gene expression
of the target nucleic acid can be induced, for example, after the stable
transfected cells
containing the polynucleotide construct comprising the target nucleic acid are
delivered or
transferred to or made to contact the tissue site, organ or system of
interest. Such systems are
of particular benefit in treatment methods and formats in which it is
advantageous to delay or
precisely control expression of the target nucleic acid (e.g., to allow time
for completion of
surgery and/or healing following surgery; to allow time for the polynucleotide
construct
comprising the target nucleic acid to reach the site, cells, system, or tissue
to be treated; to
allow time for the graft containing cells transformed with the construct to
become
incorporated into the tissue or organ onto or into which it has been spliced
or attached, etc.).
IX. Additional Multimer Uses
[2511 The potential applications of multimers of the present invention are
diverse and include any use where an affinity agent is desired. For example,
the invention
can be used in the application for creating antagonists, where the selected
monomer domains
or multimers block the interaction between two proteins. Optionally, the
invention can
generate agonists. For example, multirners binding two different proteins,
e.g., enzyme and
substrate, can enhance protein function, including, for example, enzymatic
activity and/or
substrate conversion.
[252) Other applications include cell targeting. For example, multimers
consisting of monomer domains and/or immuno-domains that recognize specific
cell surface
proteins can bind selectively to certain cell types. Applications involving
monomer domains
and/or immuno-domains as antiviral agents are also included. For example,
multimers
binding to different epitopes on the virus particle can be useful as antiviral
agents because of
the polyvalency. Other applications can include, but are not limited to,
protein purification,
protein detection, biosensors, ligand-affinity capture experiments and the
like. Furthermore,
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domains or multimers can be synthesized in bulk by conventional means for any
suitable use,
e.g., as a therapeutic or diagnostic agent.
[253] In some embodiments, the monomer domains are used for ligand
inhibition, ligand clearance or ligand stimulation. Possible ligands in these
methods, include,
e.g., cytokines, chemokines, or growth factors.
[254] If inhibition of ligand binding to a receptor is desired, a monomer
domain is selected that binds to the ligand at a portion of the ligand that
contacts the ligand's
receptor, or that binds to the receptor at a portion of the receptor that
binds contacts the
ligand, thereby preventing the ligand-receptor interaction. The monomer
domains can
optionally be linked to a half-life extender, if desired.
[255] Ligand clearance refers to modulating the half-life of a soluble ligand
in bodily fluid. For example, most monomer domains, absent a half-life
extender, have a
short half-life. Thus, binding of a monomer domain to the ligand will reduce
the half-life of
the ligand, thereby reducing ligand concentration. The portion of the ligand
bound by the
monomer domain will generally not matter, though it may be beneficial to bind
the ligand at
the portion of the ligand that binds to its receptor, thereby further
inhibiting the ligand's
effect. This method is useful for reducing the concentration of any molecule
in the
bloodstream. In some embodiments, the concentration of a molecule in the
bloodstream is
reduced by enhancing the rate of kidney clearance of the molecule. Typically
the monomer
domain-molecule complex is less than about 40 KDa, less than about 50 KDa, or
less than
about 60 KDa.
[2561 Alternatively, a multimer comprising a first monomer domain that
binds to a half-life extender and a second monomer domain that binds to a
portion of the
ligand that does not bind to the ligand's receptor can be used to increase the
half-life of the
ligand.
[257] The invention further provide monomer domains that bind to a blood
factor (e.g., serum albumin, immunoglobulin, or erythrocytes).
[258] In some embodiments, the the monomer domains bind to an
immunoglobulin polypeptide or a portion thereof.
[259] Four families (i.e., Families 1, 2, 3 and 4) of monomer domains that
bind to immunoglobulin have been identified.
1260] Sequences for Family I are set forth below. Dashes are included only
for spacing.
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Fannl
CASGQFQCRSTSICVPMWWRCDGVPDCPDNSDEK--SCEPP----T-------
CASGQFQCRSTSICVPMWWRCDGVPDCVDNSDET--SCTST----VHT-----
CASGQFQCRSTSICVPMWWRCDGVPDCADGSDEK--DCQQH----T-------
CASGQFQCRSTSICVPMWWRCDGVNDCGDGSDEA--DCGRPGPGATSAPAA--
CASGQFQCRSTSICVPMWWRCDGVPDCLDSSDEK--SCNAP----ASEPPGSL
CASGQFQCRSTSICVPMWWRCDGVPDCRDGSDEAPAHCSAP---- ASEPPGSL
CASGQFQCRSTSICVPQWWVCDGVPDCRDGSDEP-EQCTPP----T-------
CLSSQFRCRDTGICVPQWWVCDGVPDCGDGSDEKG--CGRT----GHT-----
CLSSQFRCRDTGICVPQWWVCDGVPDCRDGSDEAAV-CGRP----GHT-----
CLSSQFRCRDTGICVPQWWVCDGVPDCRDGSDEAPAHCSAP----ASEPPGSL
[261] Family 2 has the following motif
[EQ]FXCRX[ST]XRC[IV]XXXW[ILV]CDGXXDCXD[DN]SDE
[262] Exemplary sequences comprising the IgG Family 2 motif are set forht
below. Dashes are included only for spacing.
Fam2
CGAS-EFTCRSSSRCIPQAWVCDGENDCRDNSDE--ADCSAPASEPPGSL
CRSN-EFTCRSSERCIPLAWVCDGDNDCRDDSDE--ANCSAPASEPPGSL
CVSN-EFQCRGTRRCIPRTWLCDGLPDCGDNSDEAPANCSAPASEPPGSL
CHPTGQFRCRSSGRCVSPTWVCDGDNDCGDNSDE--ENCSAPASEPPGSL
CQAG-EFQC-GNGRCISPAWVCDGENDCRDGSDE--ANCSAPASEPPGSL
[263] Family 3 has either of the two following motifs:
CXSSGRCIPXXWVCDGXXDCRDXSDE; or
CXSSGRCIPXXWLCDGXXDCRDXSDE
[264] Exemplary sequences comprising the IgG Family 3 motif are set forth
below. Dashes are included only for spacing.
Fam3
CPPSQFTCKSNDKCIPVHWLCDGDNDCGDSSDE--ANCGRPGPGATSAPAA
CPSGEFPCRSSGRCIPLAWLCDGDNDCRDNSDEPPALCGRPGPGATSAPAA
CAPSEFQCRSSGRCIPLPWVCDGEDDCRDGSDES-AVCGAPAP--T-----
CQASEFTCKSSGRCIPQEWLCDGEDDCRDSSDE--KNCQQPT---------
CLSSEFQCQSSGRCIPLAWVCDGDNDCRDDSDE--KSCKPRT---------
[265] Based on family 3 alignments, additional non-naturally occurring
monomer domains that bind IgG and that has the sequence SSGR immediately
preceding the
third cysteine in an A domain scaffold. The sequences of these monomer domains
are set
forth below. Dashes are included only for spacing.
Fam4
CPANEFQCSNGRCISPAWLCDGENDCVDGSDE--KGCTPRT
CPPSEFQCGNGRCISPAWLCDGDNDCVDGSDE--TNCTTSGPT
CPPGEFQCGNGRCISAGWVCDGENDCVDDSDE--KDCPART
CGSGEFQCSNGRCISLGWVCDGEDDCPDGSDE--TNCGDSHILPFSTPGPST
CPADEFTCGNGRCISPAWVCDGEPDCRDGSDE-AA.VCETHT
CPSNEFTCGNGRCISLAWLCDGEPDCRDSSDESLAICSQDPEFHKV
[266] Monomer domains that bind to red blood cells (RBC) or serum
albumin (CSA) are described in U.S. Patent Publication No. 2005/0048512, and
include,
e.g.,:
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RBCA CRSSQFQCNDSRICIPGRWRCDGDNDCQDGSDETGCGDSHILPFSTPGPST
RBCB CPAGEFPCKNGQCLPVTWLCDGVNDCLDGSDEKGCGRPGPGATSAPAA
RBC11 CPPDEFPCKNGQCIPQDWLCDGVNDCLDGSDEKDCGRPGPGATSAPAA
CSA-A8 CGAGQFPCKNGHCLPLNLLCDGVNDCEDNSDEPSELCKALT
[267] The present invention provides a method for extending the serum half-
life of a protein, including, e.g., a multimer of the invention or a protein
of interest in an
animal. The protein of interest can be any protein with therapeutic,
prophylactic, or
otherwise desirable functionality (including another monomer domain or
multimer of the
present invention). This method comprises first providing a monomer domain
that has been
identified as a binding protein that specifically binds to a half-life
extender such as a blood-
carried molecule or cell, such as serum proteins such as albumin (e.g., human
serum albumin)
or transferrin, IgG or a portion thereof, red blood cells, etc. In some
embodiments, the half-
life extender-binding monomer can be covalently linked to another monomer
domain that has
a binding affinity for the protein of interest. This multimer, optionally
binding the protein of
interest, can be administered to a mammal where they will associate with the
half-life
extender(e.g., HSA, transferrin, IgG, red blood cells, etc.) to form a
complex. This complex
formation results in the half-life extension protecting the multimer and/or
bound protein(s)
from proteolytic degradation and/or other removal of the multimer and/or
protein(s) and
thereby extending the half-life of the protein and/or multimer (see, e.g.,
example 3 below).
One variation of this use of the invention includes the half-life extender-
binding monomer
covalently linked to the protein of interest. The protein of interest may
include a monomer
domain, a multimer of monomer domains, or a synthetic drug. Alternatively,
monomers that
bind to either immunoglobulins or erythrocytes could be generated using the
above method
and could be used for half-life extension.
[268] The half-life extender-binding multimers are typically multimers of at
least two domains, chimeric domains, or mutagenized domains two domains,
chimeric
domains, or mutagenized domains (i.e., one that binds to a target of interest
and one that
binds to the blood-carried molecule or cell). Suitable domains, e.g., those
described herein,
can be further screened and selected for binding to a half-life extender. The
half-life
extender-binding multimers are generated in accordance with the methods for
making
multimers described herein, using, for example, monomer domains pre-screened
for half-life
extender -binding activity. For example, some half-life extender-binding LDL
receptor class
A-domain monomers are described in Example 2 below.
[269] In some embodiments, the multimers comprise at least one domain that
binds to HSA, transferrin, IgG, a red blood cell or other half-life extender
wherein the domain
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comprises a trefoil/PD domain motif, a thrombospondin domain motif, or a
thyroglobulin
domain motif as provided herein, and the multimer comprises at least a second
domain that
binds a target molecule, wherein the second domain comprises a trefoil/PD
domain motif, a
thrombospondin domain motif, or a thyroglobulin domain motif as provided
herein. The
serum half-life of a molecule can be extended to be, e.g., at least 1, 2, 3,
4, 5, 10, 20, 30, 40,
50, 60, 70 80, 90, 100, 150, 200, 250, 400, 500 or more hours.
[270] The present invention also provides a method for the suppression of or
lowering of an immune response in a mammal. This method comprises first
selecting a
monomer domain that binds to an immunosuppressive target. Such an
"immunosuppressive
target" is defined as any protein that when bound by another protein produces
an
immunosuppressive result in a mammal. The immunosuppressive monomer domain can
then
be either administered directly or can be covalently linked to another monomer
domain or to
another protein that will provide the desired targeting of the
immunosuppressive monomer.
The immunosuppressive multimers are typically multimers of at least two
domains, chimeric
domains, or mutagenized domains. Suitable domains include all of those
described herein
and are further screened and selected for binding to an immunosuppressive
target.
Immunosuppressive multimers are generated in accordance with the methods for
making
multimers described herein, using, for example, trefoil/PD monomer domains,
thrombospondin monomer domains, or thyroglobulin monomer domains.
[271] In another embodiment, a multimer comprising a first monomer
domain that binds to the ligand and a second monomer domain that binds to the
receptor can
be used to increase the effective affinity of the ligand for the receptor.
[272] In another embodiment, multimers comprising at least two monomers
that bind to receptors are used to bring two receptors into proximity by both
binding the
multimer, thereby activating the receptors.
[273] In some embodiments, multimers with two different monomers can be
used to employ a target-driven avidity increase. For example, a first monomer
can be
targeted to a cell surface molecule on a first cell type and a second monomer
can be targeted
to a surface molecule on a second cell type. By linking the two monomers to
forma a
multimer and then adding the multimer to a mixture of the two cell types,
binding will occur
between the cells once an initial binding event occurs between one multimer
and two cells,
other multimers will also bind both cells.
[274] Further examples of potential uses of the invention include monomer
domains, and multimers thereof, that are capable of drug binding (e.g.,
binding
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radionucleotides for targeting, pharmaceutical binding for half-life extension
of drugs,
controlled substance binding for overdose treatment and addiction therapy),
immune function
modulating (e.g., immunogenicity blocking by binding such receptors as CTLA-4,
immunogenicity enhancing by binding such receptors as CD80,or complement
activation by
Fc type binding), and specialized delivery (e.g., slow release by linker
cleavage,
electrotransport domains, dimerization domains, or specific binding to: cell
entry domains,
clearance receptors such as FcR, oral delivery receptors such as p1gR for
trans-mucosal
transport, and blood-brain transfer receptors such as transferrinR).
[275] In further embodiments, monomers or multimers can be linked to a
detectable label (e.g., Cy3, Cy5, etc.) or linked to a reporter gene product
(e.g., CAT,
luciferase, horseradish peroxidase, alkaline phosphotase, GFP, etc.).
[276] In some embodiments, the monomers of the invention are selected for
the ability to bind antibodies from specific animals, e.g., goat, rabbit,
mouse, etc., for use as a
secondary reagent in detection assays.
[277] In some cases, a pair of monomers or multimers are selected to bind to
the same target (i.e., for use in sandwich-based assays). To select a matched
monomer or
multimer pair, two different monomers or multimers typically are able to bind
the target
protein simultaneously. One approach to identify such pairs involves the
following:
(1) immobilizing the phage or protein mixture that was previously selected to
bind the
target protein
(2) contacting the target protein to the immobilized phage or protein and
washing;
(3) contacting the phage or protein mixture to the bound target and washing;
and
(4) eluting the bound phage or protein without eluting the immobilized phage
or
protein.
In some embodiments, different phage populations with different drug markers
are used.
[278] One use of the multimers or monomer domains of the invention is use
to replace antibodies or other affinity agents in detection or other affinity-
based assays. Thus,
in some embodixnents, monomer domains or multimers are selected against the
ability to bind
components other than a target in a mixture. The general approach can include
performing
the affinity selection under conditions that closely resemble the conditions
of the assay,
including mimicking the composition of a sample during the assay. Thus, a step
of selection
could include contacting a monomer domain or multimer to a mixture not
including the target
ligand and selecting against any monomer domains or multimers that bind to the
mixture.
Thus, the mixtures (absent the target ligand, which could be depleted using an
antibody,
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monomer domain or multimer) representing the sarnple in an assay (serum,
blood, tissue,
cells, urine, semen, etc) can be used as a blocking agent. Such subtraction is
useful, e.g., to
create pharmaceutical proteins that bind to their target but not to other
serum proteins or non-
target tissues.
X. Further Manipulating Monomer Domains and/or Multimer Nucleic Acids and
Polypeptides
[2791 As mentioned above, the polypeptide of the present invention can be
altered. Descriptions of a variety of diversity generating procedures for
generating modified
or altered nucleic acid sequences encoding these polypeptides are described
above and below
in the following publications and the references cited therein: Soong et al.,
(2000) Nat Genet
25(4):436-439; Stemmer, et al., (1999) Tumor Targeting 4:1-4; Ness et al.,
(1999) Nat.
Biotech. 17:893-896; Chang et al., (1999) Nat. Biotech. 17:793-797; Minshull
and Stemmer,
(1999) Curr. Op. Chem. Biol. 3:284-290; Christians et al., (1999) Nat.
Biotech. 17:259-264;
Crameri et al., (1998) Nature 391:288-291; Crarneri et al., (1997) Nat.
Biotech. 15:436-438;
Zhang et al., (1997) PNAS USA 94:4504-4509; Patten et al., (1997) Curr. Op.
Biotech.
8:724-733; Crameri et al., (1996) Nat. Med. 2:100-103; Crameri et al., (1996)
Nat. Biotech.
14:315-319; Gates et al., (1996) J. Mol. Biol. 255:373-386; Stemmer, (1996)
In: The
Encyclopedia of Molecular Biology. VCH Publishers, New York. pp.447-457;
Crameri and
Stemmer, (1995) BioTechniques 18:194-195; Stemmer et al., (1995) Gene, 164:49-
53;
Stemmer, (1995) Science 270: 15110; Stemmer, (1995) Bio/Technolo~y 13:549-553;
Stemmer, (1994) Nature 370:389-391; and Stemmer, (1994) PNAS USA 91:10747-
10751.
[280] Mutational methods of generating diversity include, for example, site-
directed mutagenesis (Ling et al., (1997) Anal Biochem. 254(2): 157-178; Dale
et al., (1996)
Methods Mol. Biol. 57:369-374; Smith, (1985) Ann. Rev. Genet. 19:423-462;
Botstein &
Shortle, (1985) Science 229:1193-1201; Carter, (1986) Biochem. J. 237:1-7; and
Kunkel,
(1987) in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D.M.J.
eds., Springer
Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel,
(1985) PNAS USA
82:488-492; Kunkel et al., (1987) Methods in Enzymol. 154, 367-382; and Bass
et al., (1988)
Science 242:240-245); oligonucleotide-directed mutagenesis ((1983) Methods in
Enzymol.
100: 468-500; (1987) Methods in Enz r~nol. 154: 329-350; Zoller & Smith,
(1982) Nucleic
Acids Res. 10:6487-6500; Zoller & Smith, (1983) Methods in Enzymol. 100:468-
500; and
Zoller & Smith, (1987) Methods in Enzyrnol. 154:329-350); phosphorothioate-
modified
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DNA mutagenesis (Taylor et al., (1985) Nucl. Acids Res. 13: 8749-8764; Taylor
et al.,
(1985) Nucl. Acids Res. 13: 8765-8787; Nakamaye & Eckstein, (1986) Nucl. Acids
Res. 14:
9679-9698; Sayers et al., (1988) Nucl. Acids Res. 16:791-802; and Sayers et
al., (1988) Nucl.
Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA (Kramer et al.,
(1984)
Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987) Methods in Enz3nol.
154:350-367;
Kramer et al., (1988) Nucl. Acids Res. 16: 7207; and Fritz et al., (1988)
Nucl. Acids Res. 16:
6987-6999).
[281] Additional suitable methods include point mismatch repair (Kramer et
al., Point Mismatch Repair, (1984) Ce1138:879-887), mutagenesis using repair-
deficient host
strains (Carter et al., (1985) Nucl. Acids Res. 13: 4431-4443; and Carter,
(1987) Methods in
Enzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh & Henikoff, (1986)
Nucl.
Acids Res. 14: 5115), restriction-selection and restriction-purification
(Wells et al., (1986)
Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis by total gene
synthesis (Nambiar et
al., (1984) Science 223: 1299-1301; Sakamar and Khorana, (1988) Nucl. Acids
Res. 14:
6361-6372; Wells et al., (1985) Gene 34:315-323; and Grundstrom et al., (1985)
Nucl. Acids
Res. 13: 3305-3316), double-strand break repair (Mandecki, (1986) PNAS USA,
83:7177-
7181; and Arnold, (1993) Curr. Op. Biotech. 4:450-455). Additional details on
many of the
above methods can be found in Methods in Enzymoloay Volume 154, which also
describes
useful controls for trouble-shooting problems with various mutagenesis
methods.
[282] Additional details regarding various diversity generating methods can
be found in U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252;
5,837,458; WO
95/22625; WO 96/33207; WO 97/20078; WO 97/35966; WO 99/41402; WO 99/41383; WO
99/41369; WO 99/41368; EP 752008; EP 0932670; WO 99/23107; WO 99/21979; WO
98/31837; WO 98/27230; WO 98/27230; WO 00/00632; WO 00/09679; WO 98/42832; WO
99/29902; WO 98/41653; WO 98/41622; WO 98/42727; WO 00/18906; WO 00/04190; WO
00/42561; WO 00/42559; WO 00/42560; WO 01/23401; PCT/US01/06775.
[283] Another aspect of the present invention includes the cloning and
expression of monomer domains, selected monomer domains, multimers and/or
selected
multimers coding nucleic acids. Thus, multimer domains can be synthesized as a
single
protein using expression systems well known in the art. In addition to the
many texts noted
above, general texts which describe molecular biological techniques useful
herein, including
the use of vectors, promoters and many other topics relevant to expressing
nucleic acids such
as monomer domains, selected monomer domains, multimers and/or selected
multimers,
include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in
CA 02587463 2007-05-11
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Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger); Sambrook
et al.,
Molecular Cloning,= A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring
Harbor
Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook") and Current
Protocols in
Molecular BioloQV, F.M. Ausubel et al., eds., Current Protocols, a joint
venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented
through
1999) ("Ausubel")). Examples of techniques sufficient to direct persons of
skill through in
vitro amplification methods, useful in identifying, isolating and cloning
monomer domains
and multimers coding nucleic acids, including the polymerase chain reaction
(PCR) the ligase
chain reaction (LCR), Q-replicase amplification and other RNA polymerase
mediated
techniques (e.g., NASBA), are found in Berger, Sambrook, and Ausubel, as well
as Mullis et
al., (1987) U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and
Applications
(Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis); Arnheim &
Levinson
(October 1, 1990) C&EN 36-47; The Journal OfNIHResearch (1991) 3, 81-94; (Kwoh
et al.
(1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl.
Acad. Sci. USA
87, 1874; Lomell et al. (1989) J. Clin. Chem 35, 1826; Landegren et al.,
(1988) Science 241,
1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989)
Gene 4,
560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek (1995)
Biotechnology
13: 563-564. Improved methods of cloning in vitro amplified nucleic acids are
described in
Wallace et al., U.S. Pat. No. 5,426,039. Im.proved methods of amplifyin.g
large nucleic acids
by PCR are summarized in Cheng et al. (1994) Nature 369: 684-685 and the
references
therein, in which PCR amplicons of up to 40kb are generated. One of skill will
appreciate
that essentially any RNA can be converted into a double stranded DNA suitable
for
restriction digestion, PCR expansion and sequencing using reverse
transcriptase and a
polymerase. See, Ausubel, Sambrook and Berger, all supra.
[284] The present invention also relates to the introduction of vectors of the
invention into host cells, and the production of monomer domains, selected
monomer
domains immuno-domains, multimers and/or selected multimers of the invention
by
recombinant techniques. Host cells are genetically engineered (i.e.,
transduced, transformed
or transfected) with the vectors of this invention, which can be, for example,
a cloning vector
or an expression vector. The vector can be, for example, in the form of a
plasmid, a viral
particle, a phage, etc. The engineered host cells can be cultured in
conventional nutrient
media modified as appropriate for activating promoters, selecting
transfonnants, or
amplifying the monomer domain, selected monomer domain, multimer and/or
selected
multimer gene(s) of interest. The culture conditions, such as temperature, pH
and the like,
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are those previously used with the host cell selected for expression, and will
be apparent to
those skilled in the art and in the references cited herein, including, e.g.,
Freshney (1994)
Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-
Liss, New York
and the references cited therein.
[285] As mentioned above, the polypeptides of the invention can also be
produced in non-animal cells such as plants, yeast, fungi, bacteria and the
like. Indeed, as
noted throughout, phage display is an especially relevant technique for
producing such
polypeptides. In addition to Sambrook, Berger and Ausubel, details regarding
cell culture
can be found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid
Systems John
Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds) (1995) Plant Cell,
Tissue
and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag
(Berlin
Heidelberg New York) and Atlas and Parks (eds) The Handbook of.Microbiological
Media
(1993) CRC Press, Boca Raton, FL.
[286] The present invention also includes alterations of monomer domains,
immuno-domains and/or multimers to improve pharmacological properties, to
reduce
immunogenicity, or to facilitate the transport of the multimer and/or monomer
domain into a
cell or tissue (e.g., through the blood-brain barrier, or through the skin).
These types of
alterations include a variety of modifications (e.g., the addition of sugar-
groups or
glycosylation), the addition of PEG, the addition of protein domains that bind
a certain
protein (e.g., HSA or other serum protein), the addition of proteins fragments
or sequences
that signal movement or transport into, out of and through a cell. Additional
components can
also be added to a multimer and/or monomer domain to manipulate the properties
of the
multimer and/or monomer domain. A variety of components can also be added
including,
e.g., a domain that binds a known receptor (e.g., a Fc-region protein domain
that binds a Fc
receptor), a toxin(s) or part of a toxin, a prodomain that can be optionally
cleaved off to
activate the multimer or monomer domain, a reporter molecule (e.g., green
fluorescent
protein), a component that bind a reporter molecule (such as a radionuclide
for radiotherapy,
biotin or avidin) or a combination of modifications.
XI. Additional Methods of Screening
[287] The present invention also provides a method for screening a protein
for potential immunogenicity by:
providing a candidate protein sequence;
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comparing the candidate protein sequence to a database of human protein
sequences;
identifying portions of the candidate protein sequence that correspond to
portions of
human protein sequences from the database; and
determining the extent of correspondence between the candidate protein
sequence and
the human protein sequences from the database.
12881 In general, the greater the extent of correspondence between the
candidate protein sequence and one or more of the human protein sequences from
the
database, the lower the potential for immunogenicity is predicted as compared
to a candidate
protein having little correspondence with any of the human protein sequences
from the
database. Removal or limitation of the number of immunogenic amino acids
and/or
sequences may also be used to reduce immunogenicity of the monomer domains,
e.g., either
before or after the libraries are screened. Immunogenic sequences include,
e.g., HLA type I
or type II sequences or proteasome sites. A variety of commercial products and
computer
programs are available to identify these amino acids, e.g., Tepitope (Roche),
the Parker
Matrix, ProPred-I matrix, Biovation, Epivax, Epimatrix.
[289] A database of human protein sequences that is suitable for use in the
practice of the invention method for screening candidate proteins can be found
at
ncbi.nlm.nih.gov/blast/Blast.cgi at the World Wide Web (in addition, the
following web site
can be used to search short, nearly exact matches:
cbi.nlm.nih.gov/blastBlast.cgi?CMD=Web&LAYOUT=TwoWindows&AUTO FORMAT=
Semiauto&ALIGNMENTS=50&ALIGNMENT VIEW=Pairwise&CLIENT=web&DATAB
ASE=nr&DESCRIPTIONS=100&ENTREZ QUERY=(none)&EXPECT=1000&FORMAT_
OBJECT=Alignment&FORMAT TYPE=HTML&NCBI Gl=on&PAGE=Nucleotides&PRO
GRAM=blastn&SERVICE=plain&SET DEFAULTS.x=29&SET DEFAULTS.y=6&SHO
W OVERVIEW=on&WORD SIZE=7&END OF HTTPGET=Yes&SHOW LINKOUT=y
es at the World Wide Web). The method is particularly useful in determining
whether a
crossover sequence in a chimeric protein, such as, for example, a chimeric
monomer domain,
is likely to cause an immunogenic event. If the crossover sequence corresponds
to a portion
of a sequence found in the database of human protein sequences, it is believed
that the
crossover sequence is less likely to cause an immunogenic event.
[290] Human chimeric domain libraries prepared in accordance to the
methods of the present invention can be screened for potential immunogenicity,
in addition to
binding affinity. Furthermore, information pertaining to portions of human
protein sequences
from the database can be used to design a protein library of human-like
chimeric proteins.
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Such library can be generated by using information pertaining to "crossover
sequences" that
exist in naturally occurring human proteins. The term "crossover sequence"
refers herein to a
sequence that is found in its entirety in at least one naturally occurring
human protein, in
which portions of the sequence are found in two or more naturally occurring
proteins. Thus,
recombination of the latter two or more naturally occurring proteins would
generate a
chimeric protein in which the chimeric portion of the sequence actually
corresponds to a
sequence found in another naturally occurring protein. The crossover sequence
contains a
chimeric junction of two consecutive amino acid residue positions in which the
first amino
acid position is occupied by an amino acid residue identical in type and
position found in a
first and second naturally occurring human protein sequence, but not a third
naturally
occurring human protein sequence. The second amino acid position is occupied
by an amino
acid residue identical in type and position found in a second and third
naturally occurring
human protein sequence, but not the first naturally occurring human protein
sequence. In
other words, the "second" naturally occurring human protein sequence
corresponds to the
naturally occurring human protein in which the crossover sequence appears in
its entirety, as
described above.
[291] In accordance with the present invention, a library of human-like
chimeric proteins is generated by: identifying human protein sequences from a
database that
correspond to proteins from the same family of proteins; aligning the human
protein
sequences from the same family of proteins to a reference protein sequence;
identifying a set
of subsequences derived from different human protein sequences of the same
family, wherein
each subsequence shares a region of identity with at least one other
subsequence derived from
a different naturally occurring human protein sequence; identifying a chimeric
junction from
a first, a second, and a third subsequence, wherein each subsequence is
derived from a
different naturally occurring human protein sequence, and wherein the chimeric
junction
comprises two consecutive amino acid residue positions in which the first
amino acid
position is occupied by an amino acid residue common to the first and second
naturally
occurring human protein sequence, but not the third naturally occurring human
protein
sequence, and the second amino acid position is occupied by an amino acid
residue common
to the second and third naturally occurring human protein sequence, and
generating human-
like chimeric protein molecules each corresponding in sequence to two or more
subsequences
from the set of subsequences, and each comprising one of more of the
identified chimeric
junctions.
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[292] Thus, for example, if the first naturally occurring human protein
sequence is, A-B-C, and the second is, B-C-D-E, and the third is, D-E-F, then
the chimeric
junction is C-D. Alternatively, if the first naturally occurring human protein
sequence is D-
E-F-G, and the second is B-C-D-E-F, and the third is A-B-C-D, then the
chimeric junction is
D-E. Human-like chimeric protein molecules can be generated in a variety of
ways. For
example, oligonucleotides comprising sequences encoding the chimeric junctions
can be
recombined with oligonucleotides corresponding in sequence to two or more
subsequences
from the above-described set of subsequences to generate a human-like chimeric
protein, and
libraries thereof. The reference sequence used to align the naturally
occurring human
proteins is a sequence from the same family of naturally occurring human
proteins, or a
chimera or other variant of proteins in the family.
XII. Animal Models
[293] Another aspect of the invention is the development of specific non-
human animal models in which to test the immunogenicity of the monomer or
multimer
domains. The method of producing such non-human animal model comprises:
introducing
into at least some cells of a recipient non-human animal, vectors comprising
genes encoding
a plurality of human proteins from the same family of proteins, wherein the
genes are each
operably linked to a promoter that is functional in at least some of the cells
into which the
vectors are introduced such that a genetically modified non-human animal is
obtained that
can express the plurality of human proteins from the same family of proteins.
[294] Suitable non-human animals employed in the practice of the present
invention include all vertebrate animals, except humans (e.g., mouse, rat,
rabbit, sheep, and
the like). Typically, the plurality of members of a family of proteins
includes at least two
members of that family, and usually at least ten family members. In some
embodiments, the
plurality includes all known members of the family of proteins. Exemplary
genes that can be
used include those encoding monomer domains, such as, for example, members of
the
thrombospondin type I domain family, thyroglobulin domain family, or trefoil
domain
family, as well as the other domain families described herein.
[295] The non-human animal models of the present invention can be used to
screen for immunogenicity of a monomer or multimer domain that is derived from
the same
family of proteins expressed by the non-human animal model. The present
invention
includes the non-human animal model made in accordance with the method
described above,
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as well as transgenic non-human animals whose somatic and germ cells contain
and express
DNA molecules encoding a plurality of human proteins from the same family of
proteins
(such as the monomer domains described herein), wherein the DNA molecules have
been
introduced into the transgenic non-human animal at an embryonic stage, and
wherein the
DNA molecules are each operably linked to a promoter in at least some of the
cells in which
the DNA molecules have been introduced.
[296] An exa.mple of a mouse model useful for screening thrombospondin
type I domain, thyroglobulin domain, or trefoil domain derived binding
proteins is described
as follows. Gene clusters encoding the wild type human thrombospondin type I
monomer
domains, thyroglobulin monomer domains, or trefoil monomer domains are
amplified from
human cells using PCR. These fragments are then used to generate transgenic
mice
according to the method described above. The transgenic mice will recognize
the human
thrombospondin type I domains, thyroglobulin domains, or trefoil domains as
"self', thus
mimicking the "selfness" of a human with regard to thrombospondin type I
domains,
thyroglobulin domains, or trefoil domains. Individual thrombospondin type I
derived
monomers, thyroglobulin derived monomers, or trefoil derived monomers or
multimers are
tested in these mice by injecting the thrombospondin type I derived monomers
or multimers,
thyroglobulin derived monomers or multimers, or trefoil derived monomers or
multimers into
the mice, then analyzing the immune response (or lack of response) generated.
The mice are
tested to determine if they have developed a mouse anti-human response
(.MAHR).
Monomers and multimers that do not result in the generation of a MAHR are
likely to be
non-immunogenic when administered to humans.
[297] Historically, MAHR test in transgenic mice is used to test individual
proteins in mice that are transgenic for that single protein. In contrast, the
above described
method provides a non-human animal model that recognizes an entire family of
human
proteins as "self," and that can be used to evaluate a huge number of variant
proteins that
each are capable of vastly varied binding activities and uses.
XIII. Kits
[298] Kits comprising the components needed in the methods (typically in an
unmixed form) and kit components (packaging materials, instructions for using
the
components and/or the methods, one or more containers (reaction tubes,
columns, etc.)) for
holding the components are a feature of the present invention. Kits of the
present invention
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may contain a multimer library, or a single type of multimer. Kits can also
include reagents
suitable for promoting target molecule binding, such as buffers or reagents
that facilitate
detection, including detectably-labeled molecules. Standards for calibrating a
ligand binding
to a monomer domain or the like, can also be included in the kits of the
invention.
[299] The present invention also provides commercially valuable binding
assays and kits to practice the assays. In some of the assays of the
invention, one or more
ligand is employed to detect binding of a monomer domain, immuno-domains
and/or
multimer. Such assays are based on any known method in the art, e.g., flow
cytometry,
fluorescent microscopy, plasmon resonance, and the like, to detect binding of
a ligand(s) to
the monomer domain and/or multimer.
[300] Kits based on the assay are also provided. The kits typically include a
container, and one or more ligand. The kits optionally comprise directions for
performing the
assays, additional detection reagents, buffers, or instructions for the use of
any of these
components, or the like. Alternatively, kits can include cells, vectors,
(e.g., expression
vectors, secretion vectors comprising a polypeptide of the invention), for the
expression of a
monomer domain and/or a multimer of the invention.
[301] In a further aspect, the present invention provides for the use of any
composition, monomer domain, immuno-domain, multimer, cell, cell culture,
apparatus,
apparatus component or kit herein, for the practice of any method or assay
herein, and/or for
the use of any apparatus or kit to practice any assay or method herein and/or
for the use of
cells, cell cultures, compositions or other features herein as a therapeutic
formulation. The
manufacture of all components herein as therapeutic formulations for the
treatments
described herein is also provided.
XIV. Integrated Systems
[302] The present invention provides computers, computer readable media
and integrated systems comprising character strings corresponding to monomer
domains,
selected monomer domains, multimers and/or selected multimers and nucleic
acids encoding
such polypeptides. These sequences can be manipulated by in silico
recombination methods,
or by standard sequence alignment or word processing software.
[303] For example, different types of similarity and considerations of various
stringency and character string length can be detected and recognized in the
integrated
systems herein. For example, many homology determination methods have been
designed
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for comparative analysis of sequences of biopolymers, for spell checking in
word processing,
and for data retrieval from various databases. With an understanding of double-
helix pair-
wise complement interactions among 4 principal nucleobases in natural
polynucleotides,
models that simulate annealing of complementary homologous polynucleotide
strings can
also be used as a foundation of sequence alignment or other operations
typically performed
on the character strings corresponding to the sequences herein (e.g., word-
processing
manipulations, construction of figures comprising sequence or subsequence
character strings,
output tables, etc.). An example of a software package with GOs for
calculating sequence
similarity is BLAST, which can be adapted to the present invention by
inputting character
strings corresponding to the sequences herein.
[304] BLAST is described in Altschul et al., (1990) J. Mol. Biol. 215:403-
410. Software for performing BLAST analyses is publicly available through the
National
Center for Biotechnology Information (available on the World Wide Web at
ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring
sequence pairs
(HSPs) by identifying short words of length W in the query sequence, which
either match or
satisfy some positive-valued threshold score T when aligned with a word of the
same length
in a database sequence. T is referred to as the neighborhood word score
threshold (Altschul
et al., supra). These initial neighborhood word hits act as seeds for
initiating searches to find
longer HSPs containing them. The word hits are then extended in both
directions along each
sequence for as far as the cumulative alignment score can be increased.
Cumulative scores
are calculated using, for nucleotide sequences, the parameters M (reward score
for a pair of
matching residues; always > 0) and N (penalty score for mismatching residues;
always < 0).
For amino acid sequences, a scoring matrix is used to calculate the cumulative
score.
Extension of the word hits in each direction are halted when: the cumulative
alignment score
falls off by the quantity X from its maximum achieved value; the cumulative
score goes to
zero or below, due to the accumulation of one or more negative-scoring residue
alignments;
or the end of either sequence is reached. The BLAST algorithm parameters W, T,
and X
determine the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10,
a cutoff of 100,
M=5, N=-4, and a comparison of both strands. For amino acid sequences, the
BLASTP
program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and
the
BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci.
USA
89:10915).
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[305] An additional example of a useful sequence alignment algorithm is
PILEUP. PILEUP creates a multiple sequence alignment from a group of related
sequences
using progressive, pairwise aligrrments. It can also plot a tree showing the
clustering
relationships used to create the alignment. PILEUP uses a simplification of
the progressive
aligiiment method of Feng & Doolittle, (1987) J. Mol. Evol. 35:351-360. The
method used is
similar to the method described by Higgins & Sharp, (1989) CABIOS 5:151-153.
The
program can align, e.g., up to 300 sequences of a maximum length of 5,000
letters. The
multiple alignment procedure begins with the pairwise alignment of the two
most similar
sequences, producing a cluster of two aligned sequences. This cluster can then
be aligned to
the next most related sequence or cluster of aligned sequences. Two clusters
of sequences
can be aligned by a simple extension of the pairwise aligrJment of two
individual sequences.
The final alignment is achieved by a series of progressive, pairwise
alignments. The program
can also be used to plot a dendogram or tree representation of clustering
relationships. The
program is run by designating specific sequences and their amino acid or
nucleotide
coordinates for regions of sequence comparison. For example, in order to
determine
conserved amino acids in a monomer domain family or to compare the sequences
of
monomer domains in a family, the sequence of the invention, or coding nucleic
acids, are
aligned to provide structure-function information.
[306] In one aspect, the computer system is used to perform "in silico"
sequence recombination or shuffling of character strings corresponding to the
monomer
domains. A variety of such methods are set forth in "Methods For Making
Character Strings,
Polynucleotides & Polypeptides Having Desired Characteristics" by Selifonov
and Stemmer,
filed February 5, 1999 (USSN 60/118854) and "Methods For Making Character
Strings,
Polynucleotides & Polypeptides Having Desired Characteristics" by Selifonov
and Stemmer,
filed October 12, 1999 (USSN 09/416,375). In brief, genetic operators are used
in genetic
algorithms to change given sequences, e.g., by mimicking genetic events such
as mutation,
recombination, death and the like. Multi-dimensional analysis to optimize
sequences can be
also be performed in the computer system, e.g., as described in the '375
application.
13071 A digital system can also instruct an oligonucleotide synthesizer to
synthesize oligonucleotides, e.g., used for gene reconstruction or
recombination, or to order
oligonucleotides from commercial sources (e.g., by printing appropriate order
forms or by
linking to an order form on the Internet).
[308] The digital system can also include output elements for controlling
nucleic acid synthesis (e.g., based upon a sequence or an alignment of a
recombinant, e.g.,
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recombined, monomer domain as herein), i. e., an integrated system of the
invention
optionally includes an oligonucleotide synthesizer or an oligonucleotide
synthesis controller.
The system can include other operations that occur downstream from an
alignment or other
operation performed using a character string corresponding to a sequence
herein, e.g., as
noted above with reference to assays.
EXAMPLES
[3091 The following examples are offered to illustrate, but not to limit the
claimed invention.
Example 1
[310] This example describes selection of monomer domains and the
creation of multimers.
[311] Starting materials for identifying monomer domains and creating
multimers from the selected monomer domains and procedures can be derived from
any of a
variety of human and/or non-human sequences. For example, to produce a
selected monomer
domain with specific binding for a desired ligand or mixture of ligands, one
or more
monomer domain gene(s) are selected from a family of monomer domains that bind
to a
certain ligand. The nucleic acid sequences encoding the one or more monomer
domain gene
can be obtained by PCR amplification of genomic DNA or cDNA, or optionally,
can be
produced synthetically using overlapping oligonucleotides.
[312] Most commonly, these sequences are then cloned into a cell surface
display format (i.e., bacterial, yeast, or mammalian (COS) cell surface
display; phage
display) for expression and screening. The recombinant sequences are
transfected
(transduced or transformed) into the appropriate host cell where they are
expressed and
displayed on the cell surface. For example, the cells can be stained with a
labeled (e.g.,
fluorescently labeled), desired ligand. The stained cells are sorted by flow
cytometry, and the
selected monomer domains encoding genes are recovered (e.g., by plasmid
isolation, PCR or
expansion and cloning) from the positive cells. The process of staining and
sorting can be
repeated multiple times (e.g., using progressively decreasing concentrations
of the desired
ligand until a desired level of enricbment is obtained). Alternatively, any
screening or
detection method known in the art that can be used to identify cells that bind
the desired
ligand or mixture of ligands can be employed.
[313] The selected monomer domain encoding genes recovered from the
desired ligand or mixture of ligands binding cells can be optionally
recombined according to
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any of the methods described herein or in the cited references. The
recombinant sequences
produced in this round of diversification are then screened by the same or a
different method
to identify recombinant genes with improved affinity for the desired or target
ligand. The
diversification and selection process is optionally repeated until a desired
affinity is obtained.
[314] The selected monomer domain nucleic acids selected by the methods
can be joined together via a linker sequence to create multimers, e.g., by the
combinatorial
assembly of nucleic acid sequences encoding selected monomer domains by DNA
ligation, or
optionally, PCR-based, self-priming overlap reactions. The nucleic acid
sequences encoding
the multimers are then cloned into a cell surface display format (i.e.,
bacterial, yeast, or
mammalian (COS) cell surface display; phage display) for expression and
screening. The
recombinant sequences are transfected (transduced or transformed) into the
appropriate host
cell where they are expressed and displayed on the cell surface. For example,
the cells can be
stained with a labeled, e.g., fluorescently labeled, desired ligand or mixture
of ligands. The
stained cells are sorted by flow cytometry, and the selected multimers
encoding genes are
recovered (e.g., by PCR or expansion and cloning) from the positive cells.
Positive cells
include multimers with an improved avidity or affinity or altered specificity
to the desired
ligand or mixture of ligands compared to the selected monomer domain(s). The
process of
staining and sorting can be repeated multiple times (e.g., using progressively
decreasing
concentrations of the desired ligand or mixture of ligands until a desired
level of enrichment
is obtained). Alternatively, any screening or detection method known in the
art that can be
used to identify cells that bind the desired ligand or mixture of ligands can
be employed.
[3151 The selected multimer encoding genes recovered from the desired
ligand or mixture of ligands binding cells can be optionally recombined
according to any of
the methods described herein or in the cited references. The recombinant
sequences
produced in this round of diversification are then screened by the same or a
different method
to identify recombinant genes with improved avidity or affinity or altered
specificity for the
desired or target ligand. The diversification and selection process is
optionally repeated until
a desired avidity or affinity or altered specificity is obtained.
Example 2
[316] This example describes the selection of monomer domains that are
capable of binding to Human Serum Albumin (HSA).
[317] For the production of phages, E. coli DH10B cells (Invitrogen) were
transformed with phage vectors encoding a library of LDL receptor class A-
domain variants
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as a fusions to the pITl phage protein. To transform these cells, the
electroporation system
MicroPulser (Bio-Rad) was used together with cuvettes provided by the same
manufacturer.
The DNA solution was mixed with 100 l of the cell suspension, incubated on
ice and
transferred into the cuvette (electrode gap lmm). After pulsing, 2 ml of SOC
medium (2 %
w/v tryptone, 0.5 % w/v yeast extract, 10 mM NaCI, 10 mM MgSO4, 10 mM MgC12)
were
added and the transformation mixture was incubated at 37 C for 1 h. Multiple
transformations
were combined and diluted in 500 m12xYT medium containing 20 g/m tetracycline
and 2
mM CaC12. With 10 electroporations using a total of 10 g ligated DNA 1.2x108
independent
clones were obtained.
[318] 160 ml of the culture, containing the cells which were transformed
with the phage vectors encoding the library of the A-domain variant phages,
were grown for
24 h at 22 C, 250 rpm and afterwards transferred in sterile centrifuge tubes.
The cells were
sedimented by centrifugation (15 minutes, 5000 g, 4 C). The supernatant
containing the
phage particles was mixed with 1/5 volumes 20 % w/v PEG 8000, 15 % w/v NaC1,
and was
incubated for several hours at 4 C. After centrifugation (20 minutes, 10000 g,
4 C) the
precipitated phage particles were dissolved in 2 ml of cold TBS (50 mM Tris,
100 mM NaCI,
pH 8.0) containing 2 mM CaCl2. The solution was incubated on ice for 30
minutes and was
distributed into two 1.5 ml reaction vessels. After centrifugation to remove
undissolved
components (5 minutes, 18500 g, 4 C) the supernatants were transferred to a
new reaction
vessel. Phage were reprecipitated by adding 1/5 volumes 20 % w/v PEG 8000, 15
% w/v
NaCl and incubation for 60 minutes on ice. After centrifugation (30 minutes,
18500 g, 4 C)
and removal of the supernatants, the precipitated phage particles were
dissolved in a total of 1
ml TBS containing 2 mM CaC12. After incubation for 30 minutes on ice the
solution was
centrifuged as described above. The supernatant containing the phage particles
was used
directly for the affinity enrichment.
[319] Affinity enrichment of phage was performed using 96 well plates
(Maxisorp, NUNC, Denmark). Single wells were coated for 12 h at RT by
incubation with
150 l of a solution of 100 g/ml human serum albumin (HSA, Sigma) in TBS.
Binding sites
remaining after HSA incubation were saturated by incubation with 250 12% w/v
bovine
serum albumin (BSA) in TBST (TBS with 0.1 % v/v Tween 20) for 2 hours at RT.
Afterwards, 40 l of the phage solution, containing approximately 5x1011 phage
particles,
were mixed with 80 l TBST containing 3 % BSA and 2 mM CaC12 for 1 hour at RT.
In
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order to remove non binding phage particles, the wells were washed 5 times for
1 min using
130 41 TBST containing 2 mM CaC12.
[320] Phage bound to the well surface were eluted either by incubation for 15
minutes with 130 10.1 M glycine/HCl pH 2.2 or in a competitive manner by
adding 130 l
of 500 g/ml HSA in TBS. In the first case, the pH of the elution fraction was
immediately
neutralized after removal from the well by mixing the eluate with 30 l 1 M
Tris/HCl pH 8Ø
[321] For the amplification of phage, the eluate was used to infect E. coli
K91BluKan cells (F). 50 l of the eluted phage solution were mixed with 50 l
of a
preparation of cells and incubated for 10 minutes at RT. Afterwards, 20 ml LB
medium
containing 20 g/ml tetracycline were added and the infected cells were grown
for 36 h at 22
C, 250 rpm. Afterwards, the cells were sedimented (10 minutes, 5000 g, 4 C).
Phage were
recovered from the supernatant by precipitation as described above. For the
repeated affinity
enrichment of phage particles the same procedure as described in this example
was used.
After two subsequent rounds of panning against HSA, random colonies were
picked and
tested for their binding properties against the used target protein.
[322] While this example demonstrates the use of LDL-receptor A domains,
those of skill in the art will appreciate that the same techniques can be used
to generate
desired binding properties in monomer domains of the present invention.
Example 3
[323] This example describes the determination of biological activity of
monomer domains that are capable of binding to HSA.
[324] In order to show the ability of an HSA binding domain to extend the
serum half life of an protein in vivo, the following experimental setup was
performed. A
multimeric A-domain, consisting of an A-domain which was evolved for binding
HSA (see
Example 2) and a streptavidin binding A-domain was compared to the
streptavidin binding
A-domain itself. The proteins were injected into mice, which were either
loaded or not loaded
(as control) with human serum albumin (HSA). Serum levels of a-domain proteins
were
monitored.
[325] Therefore, an A-domain, which was evolved for binding HSA (see
Example 1) was fused on the genetic level with a streptavidin binding A-domain
multimer
using standard molecular biology methods (see Maniatis et al.). The resulting
genetic
construct, coding for an A-domain multimer as well as a hexahistidine tag and
a HA tag, were
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used to produce protein in E. coli. After refolding and affinity tag mediated
purification the
proteins were dialysed several times against 150 mM NaC1, 5 mM Tris pH 8.0,
100 M
CaC12 and sterile filtered (0.45 M).
[326] Two sets of animal experiments were performed. In a first set, 1 ml of
each prepared protein solution with a concentration of 2.5 M were injected
into the tail vein
of separate mice and serum samples were taken 2, 5 and 10 minutes after
injection. In a
second set, the protein solution described before was supplemented with 50
mg/ml human
serum albumin. As described above, 1 ml of each solution was injected per
animal. In case of
the injected streptavidin binding A-domain dimer, serum samples were taken 2,
5 and 10
minutes after injection, while in case of the trimer, serum samples were taken
after 10, 30 and
120 minutes. All experiments were performed as duplicates and individual
animals were
assayed per time point.
[327] In order to detect serum levels of A-domains in the serum samples, an
enzyme linked immunosorbent assay (ELISA) was performed. Therefore, wells of a
maxisorp 96 well microtiter plate (NUNC, Denmark) were coated with each 1 g
anti-Hisb-
antibody in TBS containing 2 mM CaC12 for 1 h at 4 C. After blocking remaining
binding
sites with casein (Sigma) solution for I h, wells were washed three times with
TBS
containing 0.1 % Tween and 2 mM CaC12. Serial concentration dilutions of the
serum
samples were prepared and incubated in the wells for 2 h in order to capture
the a-domain
proteins. After washing as before, anti-HA-tag antibody coupled to horse
radish peroxidase
(HRP) (Roche Diagnostics, 25 g/ml) was added and incubated for 2 h. After
washing as
described above, HRP substrate (Pierce) was added and the detection reaction
developed
according to the instructions of the manufacturer. Light absorption,
reflecting the amount of
a-domain protein present in the serum samples, was measured at a wavelength of
450 nm.
Obtained values were normalized and plotted against a time scale.
[328] Evaluation of the obtained values showed a serum half life for the
streptavidin binding A-domain of about 4 minutes without presence of HSA
respectively 5.2
minutes when the animal was loaded with HSA. The trimer of A-domains, which
contained
the HSA binding A-domain, exhibited a serum half life of 6.3 minutes without
the presence
of HSA but a significantly increased half life.of 38 minutes when HSA was
present in the
animal. This clearly indicates that the HSA binding A-domain can be used as a
fusion
partner to increase the serum half life of any protein, including protein
therapeuticals.
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Example 4
[329] This example describes experiments demonstrating extension of half-
life of proteins in blood.
[330] To further demonstrate that blood half-life of proteins can be extended
using monomer domains of the invention, individual monomer domain proteins
selected
against monkey serum albumin, human serum albumin, human IgG, and human red
blood
cells were added to aliquots of whole, heparinized human or monkey blood.
[331] The following list provides sequences of monomer domains analyzed
in this example.
IG156 CLSSEFQCQSSGRCIPLAWVCDGDNDCRDDSDEKSCKPRT
RBCA CRSSQFQCNDSRICIPGRWRCDGDNDCQDGSDETG.CGDSHILPFSTPGPST
RBCB CPAGEFPCKNGQ~LPVTWLCDGVNDCLDGSDEKGCGRPGPGATSAPAA
RBC11 CPPDEFPCKNGQCIPQDWLCDGVNDCLDGSDEKDCGRPGPGATSAPAA
CSA-A8 CGAGQFPCKNGHCLPLNLLCDGVND~CEDNSDEPSELCKALT
_ ._.:
[332] Blood aliquots containing monomer protein were then added to
individual dialysis bags (25,000 MWCQ), sealed, and stirred in 4 L of Tris-
buffered saline at
room temperature overnight.
[333] Anti-6xHis antibody was immobilized by hydrophobic interaction to a
96-well plate (Nunc). Serial dilutions of serum from each blood sample were
incubated with
the immobilized antibody for 3 hours. Plates were washed to remove unbound
protein and
probed with a-HA-HRP to detect monomer.
[334] Monomers identified as having long half-lives in dialysis experiments
were constructed to contain either an HA, FLAG, E-Tag, or myc epitope tag.
Four
monomers were pooled, containing one protein for each tag, to make two pools.
[335] One monkey was injected subcutaneously per pool, at a dose of 0.25
mg/kg/monomer in 2.5 mL total volume in saline. Blood samples were drawn at
24, 48, 96,
and 120 hours. Anti-6xHis antibody was immobilized by hydrophobic interaction
to a 96-
well plate (Nunc). Serial dilutions of serum from each blood sample were
incubated with the
immobilized antibody for 3 hours. Plates were washed to remove unbound protein
and
separately probed with a-HA-HRP, a-FLAG-HRP, a-ETag-HRP, anda-myc-HRP to
detect
the monomer.
[336] The following illustrates a comparison between commercial antibodies
and an anti-IgG multimer:
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Drug Mol. Wt. Human T112 Dosing
Rebif rIFN-b 23 kD 69 hrs Weekly 3x
Pegasys rIFN-a-PEG 40 kD 78 hrs Weekly
Rituxan CD20 Antibody 150 kD 78 hrs Weekly
Enbrel sTNF-R-Fc 150 kD 103 hrs Weekly 2x
Multimer Anti-IgG 5 kD 120 hrs Weekly 1-2x
Herceptin Her2 Antibody 150 kD 144 hrs Weekly
Remicade TNFa Antibody 150 kD 216 hrs Monthly.5x
Humira TNFa Antibody 150 kD 336 hrs Monthiy 2x
Example 5
[337] This example describes the development of protein-specific monomer
domains and dimers by "walking."
13381 A library of DNA sequences encoding monomeric domains is created
by assembly PCR as described in Stemmer et al., Gene 164:49-53 (1995).
[339] PCR fragments were digested with appropriate restriction enzymes
(e.g., Xmal and SfiI). Digestion products were separated on 3% agarose gel and
domain
fragments are purified from the gel. The DNA fragments are ligated into the
corresponding
restriction sites of phage display vector fuse5-HA, a derivative of fuse5
carrying an in-frame
HA-epitope. The ligation mixture is electroporated into TransforMaxTM EC100TM
electrocompetent E. coli cells. Transformed E. coli cells are grown overnight
at 37 C in
2xYT medium containing 20 g/ml tetracycline and 2 mM CaC12.
[340] Phage particles are purified from the culture medium by PEG-
precipitation. Individual wells of a 96-well microtiter plate (Maxisorp) are
coated with target
protein (1 g/well) in 0.1 M NaHCO3. After blocking the wells with TBS buffer
containing
10 mg/ml casein, purified phage is added at a typical number of -1-3 x 1011.
The microtiter
plate is incubated at 4 C for 4 hours, washed 5 times with washing buffer
(TBS/Tween) and
bound phages are eluted by adding glycine-HCl buffer pH 2.2. The eluate is
neutralized by
adding 1 M Tris-HCI (pH 9.1). The phage eluate is amplified using E. coli
K9IBlueKan cells
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and after purification used as input to a second and a third round of affinity
selection
(repeating the steps above).
[341] Phage from the final eluate is used directly, without purification, as a
template to PCR amplify domain encoding DNA sequences.
[342] The PCR products are purified and subsequently digested with suitable
restriction enzymes (e.g., 50% with Bpml and 50% with BsrDI).
[343] The digested monomer fragments are 'walked' to dimers by attaching a
library of naive domain fragments using DNA ligation. Naive domain sequences
are
obtained by PCR amplification of the initial domain library (resulting from
the PEG
purification described above) using primers suitable for amplifying the
domains. The PCR
fxagments are purified, split into 2 equal amounts and then digested with
suitable restriction
enzymes (e.g., either Bpml or BsrDI).
[344] Digestion products are separated on a 2% agarose gel and domain
fragments were purified from the gel. The purified fragments are combined into
2 separate
pools (e.g., naive/Bpml + selected/BsrD1 & naive/BsrD1 + selected/Bpml) and
then ligated
overnight at 16 C.
[345] The dimeric domain fragments are PCR amplified (5 cycles), digested
with suitable restriction enzymes (e.g., XmaI and SfiI) and purified from a 2%
agarose gel.
Screening steps are repeated as described above except for the washing, which
is done more
stringently to obtain high-affinity binders. After infection, the K91 BlueKan
cells are plated
on 2xYT agar plates containing 40 g/ml tetracycline and grown overnight.
Single colonies
are picked and grown overnight in 2xYT medium containing 20 g/ml tetracycline
and 2 mM
CaC12. Phage particles are purified from these cultures.
[346) Binding of the individual phage clones to their target proteins was
analyzed by ELISA. Clones yielding the highest ELISA signals were sequenced
and
subsequently recloned into a protein expression vector.
[347] Protein production is induced in the expression vectors with IPTG and
purified by metal chelate affinity chromatography. Protein-specific monomers
are
characterized as follows.
BiacoYe
[348] Two hundred fifty RU protein are immobilized by NHS/EDC coupling
to a CM5 chip (Biacore). 0.5 and 5 M solutions of monomer protein are flowed
over the
derivatized chip, and the data is analyzed using the standard Biacore software
package.
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ELISA
[349] Ten nanograms of protein per well is immobilized by hydrophobic
interaction to 96-well plates (Nunc). Plates were blocked with 5 mg/mL casein.
Serial
dilutions of monomer protein were added to each well and incubated for 3
hours. Plates were
washed to remove unbound protein and probed with a-HA-HRP to detect monomers.
Functional Assays
[350] Functional assays to determine the biological activity of the monomers
can also be conducted and include, e.g., assays to determine the binding
specificity of the
monomers, assays to determine whether the monomers antagonize or stimulate a
metabolic
pathway by binding to their target molecule, and the like.
Example 6
[351] This example describes in vivo intra-protein recombination to generate
libraries of greater diversity.
. [352] A monomer-encoding plasmid vector (pCK-derived vector; see below),
flanked by orthologous loxP sites, was recombined in a Cre-dependent manner
with a phage
vector via its compatible loxP sites. The recombinant phage vectors were
detected by PCR
using primers specific for the recombinant construct. DNA sequencing indicated
that the
correct recombinant product was generated.
Reagents and experin2ental procedures
[353] pCK-cre-lox-Mb-loxP. This vector has two particularly relevant
features. First, it carries the cre gene, encoding the site-specific DNA
recombinase Cre,
under the control of Plac. Cre was PCR-amplified from p705-cre (from
GeneBridges) with
cre-specific primers that incorporatedXbal (5') and Sfil (3') at the ends of
the PCR product.
This product was digested with Xbal and Sfil and cloned into the identical
sites of pCK, a bla
CmR derivative of pCK110919-HC-B1a (pACYC ori), yielding pCK-cre.
13541 The second feature is the naive A domain library flanked by two
orthologous loxP sites, loxP(wild-type) and loxP(FAS), which are required for
the site-
specific DNA recombination catalyzed by Cre. See, e.g., Siegel, R.W., et al.,
FEBS Letters
505:467-473 (2001). These sites rarely recombine with another. loxP sites were
built into
pCK-cre sequentially. 5'-phosphorylated oligonucleotides loxP(K) and loxP(K
rc), carrying
loxP(WT) and EcoRI and HinDIII-compatible overhangs to allow ligation to
digested EcoR1
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and HinDIII-digested pCK, were hybridized together and ligated to pCK-cre in a
standard
ligation reaction (T4 ligase; overnight at 16 C).
[355] The resulting plasmid was digested with EcoRI and Sphl and ligated to
the hybridized, 5'-phosphorylated oligos loxP(L) and loxP (L rc), which carry
loxP(FAS)
and EcoRI and Sphl-compatible overhangs. To prepare for library construction,
a large-scale
purification (Qiagen MAXI prep) of pCK-cre-lox-P(wt)-loxP(FAS) was performed
according
to Qiagen's protocol. The Qiagen-purified plasmid was subjected to CsCI
gradient
centrifugation for further purification. This construct was then digested with
Sphl and BglI1
and ligated to digested naive A domain library insert, which was obtained via
a PCR-
amplification of a preexisting A domain library pool. By design, the loxP
sites and Mb are
in-frame, which generates Mbs with loxP-encoded linkers. This library was
utilized in the in
vivo recombination procedure as detailed below.
[356] fUSE5HA-Mb-lox-lox vector. The vector is a derivative of fUSE5
from George Smith's laboratory (University of Missouri). It was subsequently
modified to
carry an HA tag for immunodetection assays. loxP sites were built into fUSE5HA
sequentially. 5'phosphorylated oligonucleotides loxP(I) and loxP(])__rc,
carrying loxP(WT), a
string of stop codons and Xmal and Sfal-compatible overhangs, were hybridized
together and
ligated to XmaI- and SfaI-digested fUSE5HA in a standard ligation reaction
(New England
Biolabs T4 ligase; overnight at 16C).
[357] The resulting phage vector was next digested with XmaI and Sphl and
ligated to the hybridized oligos loxP(J) and loxP(J)_rc, which carry loxP(FAS)
and overhangs
compatible with XrnaI and SphI. This construct was digested with.Xmal/Sfil and
then ligated
to pre-cut (Xmal/SfiI) naive A domain library insert (PCR product). The stop
codons are
located between the loxP sites, preventing expression of gIII and
consequently, the
production of infectious phage.
[358] The ligated vector/library was subsequently transformed into an E. coli
host bearing a gIII-expressing plasmid that allows the rescue of the fUSE5HA-
Mb-lox-lox
phage, as detailed below.
[359] pCK-gIII. This plasmid carries glll under the control of its native
promoter. It was constructed by PCR-amplifying glll and its promoter from
VCSM13 helper
phage (Stratagene) with primers gIIIPromoter EcoRI and gIIIPromoter HinDIII.
This
product was digested with EcoRI and HinDI1I and cloned into the same sites of
pCK1 10919-
HC-Bla. As glll is under the control of its own promoter, glll expression is
presumably
constitutive. pCK-gIII was transformed into E. coli EC100 (Epicentre).
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13601 In vivo recombination procedure. In summary, the procedure
involves the following key steps: a) Production of infective (i.e. rescue) of
fUSE5HA-Mb-
lox-1ox library with an E. coli host expressing gIll from a plasmid; b)
Cloning of 2nd library
(pCK) and transformation into F+ TGl E. coli; c) Infection of the culture
carrying the 2na
library with the rescued fUSESHA-Mb-lox-lox phage library.
[361] a. Rescue ofphage vector. Electrocompetent cells carrying pCK gIII
were prepared by a standard protocol. These cells had a transformation
frequency of 4 x
108 / g DNA and were electroporated with large-scale ligations (-5 g vector
DNA) of
fUSE5HA-lox-lox vector and the naive A domain library insert. After individual
electroporations (100 ng DNA/electroporation) with - 70 L cells/cuvette, 930
L warm
SOC media were added, and the cells were allowed to recover with shaking at
37C for 1
hour. Next, tetracycline was added to a final concentration of 0.2 g/mL, and
the cells were
shaken for - 45 minutes at 37C. An aliquot of this culture was removed, 10-
fold serially
diluted and plated to determine the resulting library size (1.S x 107). The
remaining culture
was diluted into 2 x 500 mL 2xYT (with 20 g/mL chloramphenicol and 20 g/mL
tetracycline to select for pCK-glll and the fT.TSE5HA-based vector,
respectively) and grown
overnight at 30C.
[362] Rescued phage were harvested using a standard PEG/NaCl
precipitation protocol. The titer was approximately 1 x 1012 transducing
units/mL.
[363] b. Cloning of the 2"d library and transformation into an E. coli host.
The ligated pCK/ naive A domain library is electroporated into a bacterial F+
host, with an
expected library size of approximately 10&. After an hour-long recovery period
at 37C with
shaking, the electroporated cells are diluted to OD600- 0.05 in 2xYT (plus 20
g/mL
chloramphenicol) and grown to mid-log phase at 37C before infection by fUSEHA-
Mb-lox-
lox.
[364] c. Infection of the culture carrying the 2nd librar.y with the rescued
f USE5HA 1llb-lox-lox phage library. To maximize the generation of
recombinants, a high
infection rate (> 50%) of E. coli within a culture is desirable. The
infectivity of E. coli
depends on a number of factors, including the expression of the F pilus and
growth
conditions. E. coli backgrounds TGl (carrying an F) and K91 (an Hfr strain)
were hosts for
the recombination system.
[365] Oligonucleotides:
loxP(K)
[P-5' agcttataacttcgtatagaaaggtatatacgaagttatagatctcgtgctgcatgcggtgcg]
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loxP(K rc)
[P-5' aattcgcaccgcatgcagcacgagatctataacttcgtatatacctttctatacgaagttataagct]
IoxP(L)
[P-5' ataacttcgtatagcatacattatacgaagttatcgag]
loxP (L rc)
[P-5' ctcgataacttcgtataatgtatgctatacgaagttatg]
loxP(I)
[P5'
ccgggagcagggcatgctaagtgagtaataagtgagtaaataacttcgtatatacctttctatacgaagttatcgtctg
]
loxP(I) rc
[P-5'
acgataacttcgtatagaaaggtatatacgaagttatttactcacttattactcacttagcatgccctgctc]
loxP(J)
[5' ccgggaccagtggcctctggggccataacttcgtatagcatacattatacgaagttatg]
loxP(J) rc
[5' cataacttcgtataatgtatgctatacgaagttatggccccagaggccactggtc]
gIIIPromoter_EcoRI
[5' atggcgaattctcattgtcggcgcaactat
gIIIPromoterHinDIII
[5' gataagctttcattaagactccttattacgcag]
Example 7
[366] This example describes optimization of multimers by optimizing
monomers and/or linkers for binding to a target.
[367] Figure 8 illustrates an approach for optimizing multimer binding to
targets, as exemplified with a trimeric multimer. In the figure, first a
library of monomers is
panned for binding to the target (e.g., BAFF). However, some of the monomers
may bind at
locations on the target that are far away from each other, such that the
domains that bind to
these sites cannot be connected by a linker peptide. It is therefore useful to
create and screen
a large library of homo- or heterotrimers from these monomers before
optimization of the
monomers. These trimer libraries can be screened, e.g., on phage (typical for
heterotrimers
created from a large pool of monomers) or made and assayed separately (e.g.,
for
homotrimers). By this method, the best trimer is identified. The assays may
include binding
assays to a target or agonist or antagonist potency determination of the
multimer in functional
protein- or cell-based assays.
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[368] The monomeric domain(s) of the single best trimer are then optimized
as a second step. Homomultimers are easiest to optimize, since only one domain
sequence
exists, though heteromultimers may also be synthesized. For homomultimers, an
increase in
binding by the multimer compared to the monomer is an avidity effect.
[369] After optimization of the domain sequence itself (e.g., by recombining
or NNK randomization) and phage panning, the improved monomers are
used to construct a dimer with a linker library. Linker libraries may be
formed, e.g., from
linkers with an NNK composition and/or variable sequence length.
[370] After panning of this linker library, the best clones (e.g., determined
by
potency in the inhibition or other functional assay) are converted into
multimers composed of
multiple (e.g., two, three, four, five, six, seven, eight, etc.) sequence-
optimized domains and
length- and sequence-optimized linkers.
[371] To demonstrate this method, a multimer is optimized for binding to
BAFF. The BAFF binding clone, anti-BAFF 2, binds to BAFF with nearly equal
affinity as a
trimer or as a monomer. The linker sequences that separate the monomers within
the trimer
are four amino acids in length, which is unusually short. It was proposed that
expansion of
the linker length between monomers will allow multiple binding contacts of
each monomer in
the trimer, greatly enhancing the affinity of the trimer compared to the
monomer molecule.
[372] To test this, libraries of linker sequences are created between two
monomers, creating potentially higher affinity dimer molecules. The identified
optimum
linker motif is then used to create a potentially even higher affinity trimer
BAFF binding
molecule.
[373] These libraries consist of random codons, NNK, varying in length from
4 to 18 amino acids. The linker oligonucleotides for these libraries are:
1. 5'-AAAACTGCAATGAC ACAGCCTGCTTCATCCGA-3'
2. 5'-AAAACTGCAATGAC ACAGCCTGCTTCATCCGA-3'
3. 5'-AAAACTGCAATGAC ACAGCCTGCTTCATCCGA-3'
4. 5'AAAACTGCAATGAC ACAGC
CTGCTTCATCCGA-3'
5. 5'-AAAACTGCAATGAC ACA
GCCTGCTTCATCCGA-3'
6. 5'-AAAACTGCAATGAC
NwNNINNACAGCCTGCTTCATCCGA-3'
7. 5'-AAAACTGCAATGAC
NMNNMNNMNNACAGCCTGCTTCATCCGA-3'
8. 5'-AAAACTGCAATGAC
MNNMNNMNNMNNMNNACAGCCTGCTTCATCCGA-3'
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[374] Libraries of these sequences are created by PCR. A generic primer,
SfiI (5'-TCAACAGTTTCGGCCCCAGA-3'), is used with the linker oligonucleotides in
a
PCR with the clone anti-BAFF2 as template. The PCR products are purified with
Qiagen
Qiaquick columns and then digested with BsrDI. The parent anti-BAFF 2 clone is
digested
with BpmI. These digests are purified with Qiagen Qiaquick columns and ligated
together.
The ligation is amplified by 10 cycles of PCR with the SfiI primer and the
primer Bpml (5'-
ATGCCCCGGGTCTGGAGGCGT-3'). After purification with Qiagen Qiaquick columns,
the DNAs are digested with Xmal and SfiI. Digestion products are separated on
3% agarose
gel and the Dimeric BAFF domain fragments are purified from the gel. The DNA
fragments
are ligated into the corresponding restriction sites of phage display vector
fuse5-HA, a
derivative of fuse5 carrying an in-frame HA-epitope. The ligation mixture is
electroporated
into TransforMaxTM EC100TM electrocompetent E. coli cells. Transformed E. coli
cells are
grown overnight at 37 C in 2xYT medium containing 20 g/ml tetracycline. Phage
particles
are purified from the culture medium by PEG-precipitation and used for
pan.ning.
Example 8
[375] This example describes intra-domain recombination to identify
monomer domains with improved function.
[376] Monomer sequences were generated by several steps of panning and
one step of recombination to identify monomers that bind to either the
CD401igand or human
serum albumin. CD40L and HSA was panned against three different A-domain phage
libraries. After two rounds of panning, the eluted phage pools were PCR
amplified with two
sets of oligonucleotides to produce two overlapping fragments. The two
fragments were then
fused together and cloned into the phagemid vector, pID, to fuse the products
of two-
fragment recombination. The recombined libraries (1010 size each) were then
panned two
rounds against CD40L and HSA targets using solution panning and streptavidin
magnetic
bead capture.
[377] The selected phagemid pools were then recloned into the protein
expression vector, pET, a T7 polyinerase driven vector, for high protein
expression. Almost
1400 clones were screened for anti-CD40L binding monomers by standard ELISA
and about
2000 clones were screened for HSA. All clones were unique sequences.
[378] ELISA plate wells were coated with 0.2 g of CD40L or 0.5 g of
HAS, and 5 1 of the monomer expression clone lysate was applied to each well.
The bound
monomers (which were produced as a hemagglutinin (HA) fusion) were then
detected by
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anti-HA-HRP conjugated antibody, developed by horse-radish peroxidase enzyme
activity,
and read at an OD of 450 nm. The positive clones were selected by comparing
the ELISA
reading to the existing trimer anti-CD40L 2.2 and were selected and sequenced
with the T7
primer.
[379] For the anti-CD40L samples, two anti-CD40L 2.21g clones were
grown in the same plate with selected monomer clones and processed side by
side as the
positive control. Two empty pET vector clones transformed were grown and
processed as
negative controls. The ELISA reading at OD450 and the corresponding clone
sequences are
shown.
[380] The same selection and screen processes apply to HSA. Existing anti-
HSA monomer and trimer were used as positive controls, empty pET vector were
used as
negative controls. Positive binders were selected as those with an ELISA
signal equal or
better than the anti-HSA trimer.
[381] The positive rate of clones with an OD450 greater or equal to the anti-
CD40L2.2Ig binding was about 0.7% for CD40L and 0.4% for HSA.
[382] Identified sequences are listed below:
Anti-CD40L positive clones after 2 fragments recombination and solution
panning
pmA2_84 CRPNQFT CGNGH CLPRTWL CDGVPD CQDSSDETPIP CKSSVPTSLQ
A5C1 CQSSQFR CRDNST CLPLRLR CDGVND CRDGSDESPAL CGRPGPGATSAPAASLQ
pmA2_18 CPADQFQ CKNGS CIPRPLR CDGVED CADGSDEGQD CGRPGPGATSAPAASLQ
pmA5_79 CARDGEFR CAMNGR CIPSSWV CDGEDD CGDGSDESQVY CGGGGSLQ
A2F10 CLPSQFP CQNSSI CVPPALV CDGDAD CGDDSDEAS CAPPGSLSLQ
A1E9 CAPGEFT CGNGH CLSRALR CDGDDG CLDNSDEKN CPQRTSLQ
pmA11_40 CLANECT CDSGR CLPLPLV CDGVPD CEDDSDEKN CTKPTSLQ
Anti-HSA positive clones after 2 fragments recombination and solution
panning
A5B_10 CRPSQFR CGSGK CIPQPWG CDGVPD CEDNSDETD CKTPVRTSLQ
A5.2_68 CPASQFR CENGH CVPPEWL CDGVDD CQDDSDESSAT CQPRTSLQ
A5893 CAPGQFR CRNYGT CISLRWG CDGVND CGDGSDEQN CTPHTSLQ
A14 CLANQFK CESGH CLPPALV CDGVDD CQDSSDEASAN C
A134 CNPTGKFK CRSGR CVPRESCR CDGVDD CEDNSDEKD CQPHTSLQ
A2 10 CESSEFQ CENGH CLPVPWL CDGVND CADGSDEKN CPKPTSLQ
[383] While this example demonstrates the use of LDL-receptor A domains,
those of skill in the art will appreciate that the same techniques can be used
to generate
desired binding properties in monomer domains of the present invention.
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Example 9
[384] This example describes an exemplary method for the design and
analysis of libraries comprising monomers that comprise only residues observed
in natural
domains at any given sequence position. To this end, a sequence alignment of
all natural
domains of a given family is constructed. Since the cysteine residues tend to
be the most
conserved feature of the alignment, these residues are used as a guide for fiu-
ther design.
Each stretch of sequence between two cysteines is considered separately to
account for
structural variability due to length variations. For each inter-cysteine
sequence, a histogram
of lengths is constructed. Lengths observed at roughly 10% or greater
frequency in known
domains are considered for use in the library design. A separate alignment of
sequences is
constructed for each length, and amino acids which occur at greater than
approximately 5% at
a given position in the sub-alignment are allowed in the final library design
for that lengtli.
This process is repeated for each inter-cysteine sequence segment to generate
the final library
design. Oligonucleotides with degenerate codons designed to optimally express
the desired
protein diversity are then synthesized and assembled using standard methods to
create the
final library.
13851 Typically four sets of overlapping oligonucleotides are designed with a
9-base overlap between sets 1 and 2, sets 2 and 3, as well as sets 3 and 4 for
PCR assembly.
In some cases, two sets of overlapping oligonucleotides are designed with a 9-
base overlap
between the two sets. The libraries are constructed with the following
protocol:
[3861 Oligonuleotides: A 10 M working solution of each oligonucleotide is
prepared. Equal molar amounts of oligos for each set are mixed (sets 1, 2, 3
and 4). The
oligonucleotides are assembled in two PCR assembly steps: the first round of
PCR assembles
sets 1 and 2, as well as sets 3 and 4 and the the second round of PCR uses the
first round PCR
products to assemble the full length of each library.
[387] PCR assembly - Round 1: Separate PCR reactions are performed done
using the following pairs of oligos: each oligo from set 1 vs. pooled set 2;
each oligo from set
2 vs. pooled set 1; each oligo from set 3 vs. pooled set 4; each oligo from
set 4 vs. pooled set
3. PCR reaction mixtures are 50 L in volume and comprise 5 L l OX PCR
buffer, 8 L 2.5
mM dNTPs, 5 L each of oligo and its pairing oligo pool, 0.5 L LA Taq
polymerase and
26.5 L water. PCR reaction conditions are as follows: 18 cycles of [94 C/10",
25 C/30",
72 C/30"] and 2 cycles of [94 C/30", 25 C/30", 72 C /1']. 5 L of each PCR
reaction is run
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on 3% low-melting Agrose gel in TBE buffer to verify the presence of expected
PCR
product.
[388] PCR assembly - Round 2: All Round 1 PCR products are pooled with
gL from each PCR reaction. The full length product of each library scaffold is
assembled
5 by PCR using a reaction volume of 50 L comprising 4 L l OX PCR buffer, 8
L 2.5 mM
dNTPs, 10 L pooled Round 1 PCR products, 0.5 L LA Taq and 27.5 L water and
the
following reaction conditions: 8 cycles of [94 C/10", 25 C/30", 72 C/30"] and
2 cycles of
[94 C/30", 25 C/30", 72 C/1 '].
[389] Rescue PCR and Sfi digestion: The fully assembled library scaffolds
are amplified via PCR to generate sufficient material for library production.
Four separate 50
L- PCR reactions are performed. Each reaction mixture comprises: 2.5 L l OX
PCR
buffer, 8 L 2.5 mM dNTPs, 25 L Round-2 PCR products, 0.5 L LA Taq, 5 L
each of 10
M 5' and 3' Rescue PCR primers (Table 2), and 4 L water. The reaction
conditions are as
follows: 8 cycles of [94 C/10", 25 C/30", 72 C/30"] and 2 cycles of [94 C/30",
45 Cl30",
72 C/1 ']. 5 L of the reaction mixture is run on a 3% low-melting Agrose gel
in TBE buffer
to confirm that the amplification product is the correct size. The
amplification product is then
purified by QIAGEN QIAquick columns, eluted in EB buffer, and digested with
Sfi
restriction enzyme for cloning to Sfi-digested ARI 2 vector. Twenty g of the
assembled
library scaffold is digested with 200 units of Sfz restriction enzyme in 1,000
L total volume
and 3 hrs at 50 C. The digested DNA is purified with QIAGEN QlAquick columns
and
eluted in water.
[390] Test ligation: To determine the optimal library insert/vector ratio for
ligation, 1 L of each a dilution series of Sfi-digested library insert (1/1,
1/5, 1/25, 1/125 and
1/625) is used for ligation with 1 L Sfi-digested ARI 2 vector, 1 L T4 DNA
ligase, .1 gL
I OX ligase buffer and 7 L water. The ligation reaction mixture is incubated
at room
temperature for 2 hours to generate a ligated product. 1 L ligated product is
mixed with 40
L EC 100 cells in 0.1 cm cuvette, incubated on ice for 5 minutes,
electroporated, and
recovered in 1 mL SOC for 1 hour at 37 C. For each electroporation, 5 L each
of dilution
series (1/1, 1/10, 1/100, 1/1,000) is spotted on Agar plate with Tetracycline
to determine the
optimal inert/vector ratio. In addition, 50 L of each of dilution is plated
to grow single
colonies for library QC.
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[391] Sequence Analysis and Protein Expression: Individual clones are
picked and grown overnight in 0.4 mL 2xYT with 20 g/mL tetracycline in 96-
well plates.
The overnight grown cells are spun down, and 0.5 L 115 dilute supernatant is
used to
amplify the library inserts using 5' and 3' rescue primer for sequencing. DNA
sequence
analyses is used to verify the presence of the expected library inserts. To
examine the protein
expression, the library inserts are transferred to a pEVE expression vector.
The 0.5 L of
pooled supernatants of selected clones from overnight-culture are amplified
using a pair of
PCR primers with Sfi restriction sites that are in-frame with HA epitope at
the N-terminus
and HisB Tag at the C-terminus. The PCR reaction mixture comprises: 0.5 L
phage (pool of
32 supernatants), 5 L l Ox LA Taq buffer, 8 L 2.5 mM dNTPs, 5 L each of 10
M EGF
Eve 5 and 10 M 3 Sfi N primers, and 0.5 L LA Taq polymerase. The PCR
reaction.
conditions are as follows: 23 cycles of [94 C/10", 45 C/30", 72 C/30"] and 2
cycles of
[94 C/", 45 C/30", 72 C/1 ']. The amplification product is purified by
QTAquick columns and
digested with Sfi enzyme, and ligated with Sfi-digested pEVE vector for 2
hours at room
temperature according to manufacture's specifications. 1 L of the ligated
product is
transformed in 40 gL BL21 cells by electroporation, plated on Kanamycin plate,
and grown
in the 37 C incubator overnight. Colonies are picked and cultured overnight in
0.5 mL 2xYT
media. The following day, 50 .L of overnight culture is inoculated to 1 mL
2xYT media and
grown for about 2.5 hours until OD600 reached about 0.8, at which point IPTG
is added to a
final concentration of 1 mM for protein expression. The cells are spun down at
3,600 rpm for
15 minutes, the pellets are suspended in 100 L TBS/2 mM Ca+, heated at 65 C
for 5
minutes to release the protein, and spun down at 3,600 rpm for 15 minutes. The
supernatant
from each clone is run on a 4-12% NuPAGE gel, 10 L each with or without
reducing agent
(Invitrogen). Shift in band position between reduced and unreduced samples
indicates that
the expressed proteins are likely to fold properly.
[392] Library Scale-up: The full library is ligated in a ARI 2 vector,
transformed in EC100 cells, then expanded in K91 cells. The ligation is
performed overnight
at room temperature in a final volume of 2.5 mL with 25 g of Sfi-digested
vector, 2.5 g Sfi-
digested library insert, 5 L T4 DNA ligase, and 250 L lOx DNA ligase buffer.
The ligated
product is precipitated with sodium acetate and ethanol, suspended in 400 L
water,
reprecipitated with NaAc/EtOH and resuspended in 50 L H20. The library is
electroporated
in a vessel comprising 10 L DNA and 200 gL EC100 cells, transferred to 50 mL
SOC
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media, and grown at 37 C for 1 hour at 300 rpm. A 5 L aliquot is removed and
(1) serially
diluted to determine the library size; and (2) plated out for sequence
verification. The
transformed EC100 in 50 mL SOC is divided equally, added to six 500 mL culture
of K91
cells with OD600 of 0.5, and incubated for 30 minutes at 37 C without shaking.
Tetracycline
is added to a concentration 0.2 g/mL, and the cultures are grown for 30
minutes at 37 C at
300 rpm. Finally, tetracycline is added to a final concentration 20 g/mL, and
the cultures
are grown overnight at 37 C at 300 rpm. Cells are centrifuged at 8,000 rpm for
10 minutes.
Phages in the supematant are precipitated by adding 40 g PEG and 30 g NaCl
/1000 mL, and
centrifugation at 8,000 rpm for 10 minutes. Phages are resuspended in 50 mL
TBS/2 mM
Ca and centrifuged at 5,000 rpm for 10 minutes to remove the cell debris. The
supernatant
is added with a final concentration of 20% PEG and 1.5 M NaCI, and placed on
ice for 40
minutes, and phages are spun down at 5,000 rpm for 10 minutes, and resuspended
in 10 mL
TBS/2 mM Ca+. Phage titer is determined by serial dilution.
Example 10
[393] This example describes design and analysis of a library from trefoil/PD
domains using the methods set forth in Example 9 above.
[394] Based on sequence alignments of naturally occurring trefoil/PD
domains, a panel of degenerate oligonucleotides were designed that encode
trefoil/PD
domains that comprise amino acids at each position that are found only in
naturally occurring
trefoil/PD domains. The trefoil/PD library design is set forth below.
L E A S G G S QD A D A A E D K F D QA F D A A S A A D QA A I GMF D A A G A D A
I W QF F G 0 A A D A D T S L Q A
N D F D D H E R I N G L K D I T E D E D D K N W E D E I F E F K Y H P K I E E
D E
P I I E E K N K P I P N F P G E I E E L Y I N F K M G I N K S L N H G E K
5 K L N L L S L S Y S P G V K G K F G N N P G N P K Q P Y V N P I P G L
L M P P N V Q V Q H P K N K K R R S Q S R L S V P Q K Q N Q
N V R R Q R Y S L Q P Q N N S T L V S N T Q S N T Q R
P S V R T p R Q T Q Q R T S V R T V 5 S
R T 5 V S 5 R S S V T Y T T
S T T W
T W
V
- A M A A K A K F D F D G Q M---=-
D V D D L D Q I N H N R Q
E E P N E R K Q S
S P V R G V
V S K
S
V
[395] The degenerate oligonucleotide sequences are set forth in the table
below:
PD1 1 1 CTG GAG GCG TCT GGT GGT TCG TGT YCN SYA WTK RAY GWB MRY GWS ARR AVA
GAC TGC GCG
PD1 1 2 CTG GAG GCG TCT GGT GGT TCG TGT RAY ANM GWY MSY CBN CWR ARY ARR CWA
GAC TGC GCG
PDI 1 3 CTG GAG GCG TCT GGT GGT TCG TGT RAY ANM WTK GMR CBN RAR GWS ARR DTC
GAC TGC GCG
PD1 1 4 CTG GAG GCG TCT GGT GGT TCG TGT RAY SYA GWY GMR GWB RAR ARY ARR DTC
GAC TGC GCG
PD1 2 1 CTG GAG GCG TCT GGT GGT TCG TGT TCN RTG SCN GWY CTN KCN MRR AWA GAC
TGC GCG
PD1 2 2 CTG GAG GCG TCT GGT GGT TCG TGT GVS RTG GAD SCN ARN GDY MRR KTY GAC
TGC GCG
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PD1 2 3 CTG GAG GCG TCT GGT GGT TCG TGT GVS RTG SCN SCN CTN RAR MRR KTY GAC
TGC GCG
PD1 2 4 CTG GAG GCG TCT GGT GGT TCG TGT TCN RTG GAD GWY ARN RAR MRR AWA GAC
TGC GCG
PD2 1 GCA GCA CCC TMK YTB RAA RCA WRT YYB YYB RST DAY AAR NGR DGR CGC GCA GTC
PD2 2 GCA GCA CCC NTT BGC YYG RCA YTB NGR CGV RST NGS RBC RTY RWA CGC GCA GTC
PD2 3 GCA GCA CCC NTT RYY WKY RCA RTY RBC YYB RST NGS RKG YTK YAM CGC GCA GTC
PD2 4 GCA GCA CCC TMK RYY WKY RCA RTY RBC CGV RST DAY RKG YTK YAM CGC GCA GTC
PD3 11 GGG TGC TGC TWY MGY HCN DSG RKY KYY RAR DYY AAH TGG TGC TAC
PD3 1 2 GGG TGC TGC TGG AWY RMY SAR AAH ABG YTR CAR RTH TGG TGC TAC
PD3 1 3 GGG TGC TGC TWY GAS RMY YTT RKY BCN RRY CAR CCN TGG TGC TAC
PD3 1 4 GGG TGC TGC TWY GAS HCN YTT AAH BCN RRY DYY RTH TGG TGC TAC
PD3 2 1 GGG TGC TGC TTY RAY GGA CRR ATG TGG TGC TAC
PD3 2 2 GGG TGC TGC AAY RAY GGA CRR CAR TGG TGC TAC
PD3 2 3 GGG TGC TGC AAY RAY GGA CRR TCN TGG TGC TAC
PD3 2 4 GGG TGC TGC TTY RAY GGA CRR TCN TGG TGC TAC
PD4 1 GGC CTG CAA TGA CGT CSW RBY NGK RTD YKG YMG NGR YTT GTA GCA CCA
PD4 2 GGC CTG CAA TGA CGT YWK YTS YTS YDC RHT RTY NMC RAA GTA GCA CCA
PD4 3 GGC CTG CAA TGA CGT STY YTS RYC TWT NGY YKK NGR RTR GTA GCA CCA
PD4 4 GGC CTG CAA TGA CGT STY RBY RYC TWT NGY YKK NMC RTR GTA GCA CCA
5' Rescue 5' AAAAGGCCTCGAGGGCCTGGAGGCGTCTGGTGGTTCGTGT 3'
3' Rescue 5' AAAAGGCCCCAGAGGCCTGCAATGACGT 3'
[3961 N represesents A, T, G, or C: B represents G, C, or T; D represents G,
A, or T; H represents A, T, or C; K represents G or T; M represents A or C; R
represents A or
G; S represents G or C; V represents G, A, or C; W represents A or T; and Y
represents T or
C.
[3971 Thirty two individual phages from each library were amplified by PCR
and the amplification products were sequenced. The results of sequencing
confirmed that the
phage contained inserts of the expected sizes and sequences for the library.
The library
comprised 2.31 x 109 monomer domains comprising 57, 58, 61, or 62 amino acids.
The
sequencing results are shown in the table below. Clones 5 and 6 were
identified as clones
that do not contain a domain insert, but instead represent empty vector
background from the
transformation.
PD 1 PGLEGLEASGGSCDANEVKNKFDCAYDAATPSQCRAKGCCWINQNTLQIWCYFGNNEEEQTSLQASGA
PD 2 PGLEGLEASGGSCDIDSRLNKQDCAVKPPSEGDCENNGCCFNGQMWCYFGNSEKKKTSLQASGA
PD 3 PGLEGLEASGGSCGVEPNGQVDCAFDGPTSSKCQANGCCNNGRS*CYFVNNAKQKTSLQASGA
PD 4 PGLEGLEASGGSCDMEAKGRVDCAFNGASASECRANGCCNNGQQWCYKSRPYTASTSLQASGA
PD 5 PGLEGH**LCYEASGA
PD 6 PGLEGH**LCYEASGA
PD 7 PGLEGLEASGGSCAVPALKRFDCALKPVSPADCAGRGCCNNGQQWCYKSLQYTGSTSLQASGA
PD 8 PGLEGLEASGGSCNRDRLLNRLDCAYDAASPPKCRANGCCFNGQMWCYYPPTIGEDTSLQASGA
PD 9 PGLEGLEASGGSCDNLAREVKIDCAVKHASETDCDNNGCCWNDENRLQVWCYFGNSEQKKTSLQASGA
PD 10 PGLEGLEASGGSCSMAVLAQKDCAVQHPTKADCENKGCCNNGRSWCYKPLQNTNWTSLQASGA
PD 12 PGLEGLEASGGSCAVAPLERFDCALQHATRADCANKGCCFGQMWCYKSRQNPDTTSLQASGA
PD 13 PGLEGLEASGGSCGVEPKGKVDCAPPLVSEQTCFKRGCCFDGQMWCYYGKTKDNNTSLQASGA
PD 15 PGLEGLEASGGSCDAVEKENKFDCAVQHASRANCENNGCCNNGQSWCYHVTAKDANTSLQASGA
PD 16 PGLEGLEASGGSCSVPDLAKKDCALKPITAANCEDIGCCFDGRQWCYFGDNAEQKTSLQASGA
PD 17 PGLEGLEASGGSCPPINEHERRDCAVKHATKADCDGNGCCFDDLGADQPWCYFVDNAEKKTSLQASGA
PD 19 PGLEGLEASGGSCSVPVLSKIDCAVKHPSRANCENNGCCNNGQSWCYYVQTKGNKTSLQASGA
PD 20 PGLEGLEASGGSCDKDSPLSKLDCAPSLITRRTCFELGCCNNGRQWCYFGNNAEQlTSLQASGA
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PD 21 PGLEGLEASGGSCEVPALEKFDCAYDDPSAPKCQAKGCCFNGQMWCYYGKTKDTDTSLQASGA
PD 22 PGLEGLEASGGSCDMEAKVRFDCAVQHPTRDNCDSKGCCNNGQSWCYFGNNAQQKTSLQASGA
PD 23 PGLEGLEASGGSCGVAALEQFDCALKHPSGDNCDSNGCCFDGRMWCYHSQTKGQETSLQASGA
PD 25 PGLEGLEASGGSCSAINVSVRTDCAVKHVSPGDCNDLGCCNNGQSWCYHVPAIGNETSLQASGA
PD 27 PGLEGLEASGGSCAMPPLEQFDCAVKPITADDCANRGCCFNGQMWCYYPPTINEDTSLQASGA
PD 29 PGLEGLEASGGSCGMEARVKVDCAYDDATPPKCQANGCCNNGQSWCYFGNNAQQQTSLQASGA
PD 30 PGLEGLEASGGSCGVAALERVDCAVKHPTEGNCTSNGCCFDGQMWCYKPRQNTDSTSLQASGA
PD 31 PGLEGLEASGGSCDVEANGQVDCALKHATGNDCASNGCCFDGQSWCYHPKA(NENTSLQASGA
PD 32 PGLEGLEASGGSCDANENESKVDCALQHVTSGDCTDIGCCFNGQSWCYYVQAIGANTSLQASGA
[398] Clones from the trefoil/PD library were tested for their ability to
produce folded protein. SDS-PAGE verified that the clones produced full-length
soluble
protein following heat lysis.
Exam lpel1
[399] This example describes design and analysis of a library from
thrombospondin domains using the methods set forth in Example 9 above.
[400] Based on sequence alignments of naturally occurring thrombospondin
domains, a panel of degenerate oligonucleotides were designed that encode
thrombospondin
domains that comprise amino acids at each position that are found only in
naturally
occurring thrombospondin domains. The thrombospondin library design is set
forth below.
L E A S G G S QN A A QA A G A I E A A A A QL A A F A E G A A ------- QA E A A
A D A A AQD A A A QA A A A A F A A T S L q A
S E D D E E K F G D L E Q G G G D G L G D E G D D E E E E D E D D D E D T E D
W D D
T G E E G F q H R E M G R K L L F P S R G L E E F q F I E G E E E G E V G E E
E
{ N G K H R f S H R H S P P q H S S P N L G K R H K K H G F G K H W N K G K
K P S L I S K T I T I V Q q P S P N I. L I q L L I G K 5 K Y S L S L
L q M K T M K V K T S R T S P N R K R N M L H L T L T N T P
q R N L V N L L T T q R S S T P N M K N V N P q
R S P M Q p M V R S T T R Q N N P W P q R
S T Q 0 R 0 N S T V V S R P P R Q S S
T R R S R P I D A F G A A A G E------ T V Y T S q Q S 5 T T
V S S T S Q L K E N L D D G S G V T R R T V
T T V T R Q S H Q P E L S T L V S V
V V Y V S R M R Q Q P V M Y T Y
w Y T V 0 R T S P A A A A K P A A A E A V
Y V T R D E E L R E D E K P
Y V E H H P N E K Q Q
G L L q P 1 L R R
L P Q S Q P Q 5 S
D A P A A A F G G A A----- N S S T S S R T
I H Q E G G G N K D P V T T
M I V H P K H N E q
N L K L N Q F S
S N L q R R I
T Q P R Y S P
V R R S V
S S
T
V
I D A A A D E A A D D E----
K F D D F E I L D E E F
L G H F G G P S E F q N
M H K G N H q V N G S O
q K N I V K R P H T S
R N P N Q S Q S
V R S q R T S
5 R S V
T T
I E K K A A A E E E A L D D D A
L K P L P D G K L I S V E E K K
q q 5 F Q T K T H H R L
V R N R L P 5 R
p S P S Y
q S
S
[401] The degenerate oligonucleotide sequences are set forth in the table
below:
T1_1 CTG GAG GCG TCT GGT GGT TCG TGT AVY RSH GMN TGT GRN ARY GGT WBB RTH WHY
DCN BMY CKN
GGC TGC GAC
T1_2 CTG GAG GCG TCT GGT GGT TCG TGT AVY VDA AVY TGT KCN VNG GGT VAR WCN RWG
CRR SWA RYG
GGC TGC GAC
T1_3 CTG GAG GCG TCT GGT GGT TCG TGT AVY VDA CVR TGT KCN ARY GGT YWY MRR CRS
CRR ANA RYG
GGC TGC GAC
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T1_4 CTG GAG GCG TCT GGT GGT TCG TGT AVY RSH CVR TGT KCN VNG GGT YWY MRR CRS
CRR ANA CKN
GGC TGC GAC
T2 1 1 CTC CGG GCA NGD BGM NCC NGR RKS NCC HSC NSC GTC GCA GCC
T2 1 2 CTC CGG GCA RBC NCB YRA YYC RAA RAA YDG YKK GTC GCA GCC
T2 1 3 CTC CGG GCA RBC NCB YRA YYC YST YWG YGA YKK GTC GCA GCC
T2 2 1 CTC CGG GCA CAY NYC NSC RKC YTS YCS RTT YKC RTC GTC GCA GCC
T2 2 2 CTC CGG GCA YYC NGW RCT YRR RKC YDG YYG MTG NGA GTC GCA GCC
T2 2 3 CTC CGG GCA CVG NGW RCT YRR NGT YDG RAA CRT YTT GTC GCA GCC
T2 3 1 CTC CGG GCA RWW YYK NCC NCC YCS YRA NCC YKY YKG YTG GTC GCA GCC
T2 3 2 CTC CGG GCA YKS RYT NCC RTT RTK HSC NGS RBK NAC RHK GTC GCA GCC
T2 3 3 CTC CGG GCA NTM NGC NCC RTT RWA YYK NGS YRM NAC NGC GTC GCA GCC
T2 4 1 CTC CGG GCA RAA RTC RRA YKS YRM DAY HYS NSC RBY RTB YKT GTC GCA GCC
T2 4 2 CTC CGG GCA YTS YTS YYC RYT YRM RSK YGW RTT YYG NGV RYB GTC GCA GCC
T2 4 3 CTC CGG GCA RYT NGW RTS RTY YRM YTS YGW RAM RWW YTT RAA GTC GCA GCC
T2 5 1 CTC CGG GCA VWR YYT YTC BTC NAS KGH KMT YTC YGT NSC RRM NGV YTT NGG YCK
GTC GCA GCC
T2 5 2 CTC CGG GCA NGC RTC RTS RDG NAS KGH YTY YAR YTY YTG YKS NGV YAR YTT YTB
GTC GCA GCC
T2 5 3 CTC CGG GCA YYT NGA NGR RCT NAS KGH YTY YAR YTY YTG RKY NGV YAR YTT YTB
GTC GCA GCC
T3 1 1 TGC CCG GAG CNR CKN GHR GAN THY CRR RAK TGT WMY MBG VAN GCC TGC GGC
T3 1 2 TGC CCG GAG GMY GWR AVR CRR RHA ATA KYR TGT SRN SMR SVK GCC TGC GGC
T3 1 3 TGC CCG GAG AVY RVY TYW CRR RHA RMR MSS TGT SRN RNY SVK GCC TGC GGC
T3 2 1 TGC CCG GAG SAR GYN ARR CCG SMR GMN CDR VAR CVR TGT WMY MBG VAN GCC TGC
GGC
T3 2 2 TGC CCG GAG KCN WCN ARR CCG ARY NCN RMR AGB DCN TGT SRN SMR SVK GCC TGC
GGC
T3 2 3 TGC CCG GAG CWY CHR ARR CCG ARY ATY RMR AGB DCN TGT SRN RNY SVK GCC TGC
GGC
T4 1 GGC CTG CAA TGA CGT YKK HTC CCA YDG RBT CCA BWS GCC GCA GGC
T4 2 GGC CTG CAA TGA CGT YRM RSY RAA NKY YBC RTA RKN GCC GCA GGC
T4 3 GGC CTG CAA TGA CGT HTC HTC RAA YDG YBC SRY BWS GCC GCA GGC
T4 4 GGC CTG CAA TGA CGT YKK RSY CCA NKY RBT SRY RKN GCC GCA GGC
5' Rescue 5' AAAAGGCCTCGAGGGCCTGGAGGCGTCTGGTGGTTCGTGT 3'
3' Rescue 5' AAAAGGCCCCAGAGGCCTGCAATGACGT 3'
[402] N represesents A, T, G, or C: B represents G, C, or T; D represents G,
A, or T; H represents A, T, or C; K represents G or T; M represents A or C; R
represents A or
G; S represents G or C; V represents G, A, or C; W represents A or T; and Y
represents T or
C.
[403] Thirty two individual phages from the library were amplified by PCR
and the amplification products were sequenced. The results of sequencing
confinned that the
phage contained inserts of the expected sizes and sequences for the library.
The library
comprised 1.98 x 109 monomer domains comprising 60-70 amino acids. The
sequencing
results are shown in the table below. Clones 1, 4, 8, 11, 12, 22, 26, and 30
were identified as
clones that do not contain a domain insert, but instead represent empty vector
background
from the transformation.
Ts 1 1 PGLEGH**LCYEASGA
Ts 1 2 PGLEGLEASGGSCNDPCSRRYQQQNSGCYHENRQAGDMCPETSFXTKTCRVGACGQWNPWDTTSLQASGA
Ts 1 3
PGLEGLEASGGSCTSECDNGSVYSYLGCDFKIFSQSNDSSCPESDLRKKTCRVRACGHWSLWETTSLQASGA
Ts 1 4 PGLEGH**LCYEASGA
Ts 1 5 PGLEGLEASGGSCNGSCSVGESERVMGCDPSQTESSDCPENNSQETRCGGAACGHTNTWTQTSLQASGA
Ts 1 6 PGLEGLEASGGSCTESCSAGQSVRQMGCDDENRQAADMCPESAFRTTSCGIQACGLWNQWEQTSLQASGA
Ts 1 7 PGLEGLEASGGSCSTQCSRGHQRQRLGCDPSQRESRGCPEQLADSRKCTPEACGNYETFGSTSLQASGA
Ts 1 8 PGLEGH**LCYEASGA
Ts 1 9 PGLEGLEASGGSCNSPCARGYRHQTLGCDKTFQTLSSPCPENSFQETRCDDGACGTMSNWAPTSLQASGA
Ts 1 10 PGLEGLEASGGSCGGAACGQVPPFEETSLQASGA
Ts 1 11 PGLEGH**LCYEASGA
Ts 1 12 PGLEGH**LCYEASGA
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Ts 1 13 PGLEGLEASGGSCSRSCSLGKSERETGCDDANRQDGKMCPERLEEFRKCNRKACGVPEPFEETSLQASGA
Ts 1 14
PGLEGLEASGGSCTTQCAMGYRRRKLGCDLVTAGHNGNECPELLKPNIASACDVRPCGPYATFXLTSLHASGA
Ts1 15 PGLEGLEASGGSCSGPCAMGLQRQTLGCDDENRQAANMCPESNLRVKRCHVAACGTYEKFAATSLQASGA
Ts 1 16
PGLEGLEASGGSCTGPCAMGLKRQILGCDKLFFGSRACPEHLRPSIARTCGGGACGAYGTFTATSLQASGA
Ts 1 17
PGLEGLEASGGSCSXNCSLGKSERLAGCDQKLPEQKLETVHHDACPESGFREKRXDVGACGHYXKFCFDVIAGIWG
Ts 1 18
PGLEGLEASGGSCSIRCSKGYRHQILGCDKTFQTLSTPCPEEARPAAREPCYRKACGPATTWTQTSLQASGA
Ts1 19 PGLEGLEASGGSCSKNCSTGQSMRQVGCDAAGDPGSSCPESGSRVKRCGSPACGLTEQFEKTSLQASGA
Ts 1 20
PGLEGLEASGGSCSKRCAPGHRRRTLGCDDENREDADMCPEEARPPDLQRCSRKACGQVEPFXKTSLQASGA
Ts 1 21 PGLEGLEASGGSCSVSCSLGESVREMGCDKTFLTLSSLCPESGFQTKRCGDRACGATNNWTPTSLQASGA
Ts 1 22 PGLEGH**LCYEASGA
Ts1 23 PGLEGLEASGGSCSGRCAKGYRRQKRGCDPQFFELRACPEEARPAEQEPCSMDACGDVNTWAKTSLQASGA
Ts 1 24
PGLEGLEASGGSCSGTCAVGESERQMGCDSVNAGNKGSECPESNFRVKRCRGAACGPYETFTSTSLQASGA
Ts1 25 PGLEGLEASGGSCTKNCSGGETKRQTGCDEANREDAEMCRENNSRPEMCGIGACGACGGRGPHL{AX
Ts 1 26 PGLEGH**LCYEASGA
Ts 1 27 PGLEGLEASGGSCNPNCAGGKTLQLMSCYPPFFDSRACPESDLQVXPCHGGLXWRXSRXXWGX
Ts 1 28
PGLEGLEASGGSCSGPCAKGLQRRKLGCDNSNREXAEMCPELLRPNIKRTCGNGACYQWXQWEQTSLQASGA
Ts1 29 PGLEGLEASGGSCNVTCATGESKRVMGCDQPTGSGGGKICPESDLQIEPCRVGACGDVNAWTKTSLQASGA
Ts 1 30 PGLEGH**LCYEASGA
Ts1 31 PGLEGLEASGGSCSTQCAMGYRQRKRGCDTSQTESRGCPENALRKTPCRTGAYGNANNWTPTSLQASGA
Ts 1 32
PGLEGLEASGGSCTGPCSMGFKRQILGCDFAYMNNANCPEXXEPADPNRCNARACGHSNACSHTSLQASGA
[404] Clones from the thrombospondin library were tested for their ability to
produce folded protein. SDS-PAGE verified that the clones produced full-length
soluble
protein following heat lysis.
Example 12
[405] This example describes an exemplary method of generating libraries
comprised of proteins with randomized inter-cysteine loops. In this example,
in contrast to the separate loop, separate library approach described above,
multiple intercysteine loops are
randomized simultaneously in the same library.
[406] An A domain NNK library encoding a protein domain of 39-45 amino
acids having the following pattern was constructed:
C1-X(4,6)-E1-F-R1-C2-A-X(2,4)-G1-R2-C3-I-P-S 1-S2-W-V-C4-D1-G2-E2-D2-D3-C5-
G3-D4-G4-S3-D5-E3-X(4,6)-C6;
where,
C 1-C6: cysteines;
X(n): sequence of n amino acids with any residue at each position;
E1-E3: glutamine;
F: phenylalanine;
Rl-R2: arginine;
A: alanine;
Gl-G4: glycine;
1: isoleucine;
P: proline;
S1-S3: serine;
W: tryptophan;
V: valine;
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D 1-D5: aspartic acid; and
C1-C3, C2-C5 & C4-C6 form disulfides.
[407] The library was constructed by creating a library of DNA sequences,
containing tyrosine codons (TAT) or variable non-conserved codons (NNK), by
assembly
PCR as described in Stemmer et aL, Gene 164:49-53 (1995). Compared to the
native A-
domain scaffold and the design that was used to construct library Al
(described previously)
this approach: 1) keeps more of the existing residues in place instead of
randomizing these
potentially critical residues, and 2) inserts a string of amino acids of
variable length of all 20
amino acids (NNK codon), such that the average number of inter-cysteine
residues is
extended beyond that of the natural A domain or the Al library. The rate of
tyrosine residues
was increased by including tyrosine codons in the oligonucleotides, because
tyrosines were
found to be overrepresented in antibody binding sites, presumably because of
the large
number of different contacts that tyrosine can make. The oligonucleotides used
in this PCR
reaction are:
1. 5' -ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKNNKNNKNNKGAATTCCGA- 3'
2. 5' -ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKNNKNNKNNKNNKGAATTCCGA- 3'
3. 5' -ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKNNKNNKNNKNNKNNKGAATTCCGA-
3'
4. 5' -ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTTATNNKNNKNNKGAATTCCGA- 3'
5. 5' -ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKTATI3NKNNKNI3KGAATTCCGA- 3'
6. 5' -ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKTATNNKNNKGAATTCCGA- 3'
7. 5' -ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKNNKTATNNKGAATTCCGA- 3'
8. 5' -ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKNNKNNKTATGAATTCCGA- 3'
9. 5' -ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKNNKNNKTATNNKGAATTCCGA- 3'
10. 5' -ATACCCAAGAAGACGGTATACATCGTCCMNNMNNTGCACATCGGAATTC- 3'
11. 5' -ATACCCAAGAAGACGGTATACATCGTCCMNNMNNMNNTGCACATCGGAATTC- 3'
12. 5' -ATACCCAAGAAGACGGTATACATCGTCCMNNMNNMNNMNNTGCACATCGGAATTC- 3'
13. 5' -ATACCCAAGAAGACGGTATACATCGTCCATAMNNMNNTGCACATCGGAATTC- 3'
14. 5' -ATACCCAAGAAGACGGTATACATCGTCCMNNATAMNNMNNTGCACATCGGAATTC- 3'
15. 5' -ATACCCAAGAAGACGGTATACATCGTCCMNNATAMNNTGCACATCGGAATTC- 3'
16. 5' -ATACCCAAGAAGACGGTATACATCGTCCMNNMNNATATGCACATCGGAATTC- 3'
17. 5' -ATACCCAAGAAGACGGTATACATCGTCCMNNMNNATAMNNTGCACATCGGAATTC- 3'
18. 5' -ACCGTCTTCTTGGGTATGTGACGGGGAGGACGATTGTGGTGACGGATCTGACGAG- 3'
19. 5' -ATATGGCCCCAGAGGCCTGCAATGATCCACCGCCCCCACAMNNMNNMNNMNNCTCGTCAG
ATCCGT- 3'
20. 5' -ATATGGCCCCAGAGGCCTGCAATGATCCACCGCCCCCACAMNNMNNMNNMNNMNNCTCGTCA
GATCCGT- 3'
21. 5' -ATATGGCCCCAGAGGCCTGCAATGATCCACCGCCCCCACAMNNMNNMNNMNNMNNMNNC
TCGTCAGATCCGT- 3'
22. 5' -ATATGGCCCCAGAGGCCTGCAATGATCCACCGCCCCCACAATAMNNMNNMNNCTCGTC
AGATCCGT- 3'
23. 5' -ATATGGCCCCAGAGGCCTGCAATGATCCACCGCCCCCACAMNNATAMNNMNNMNNCT
CGTCAGATCCGT- 3'
24. 5' -ATATGGCCCCAGAGGCCTGCAATGATCCACCGCCCCCACAMNNATAMNNMNNCTCGT
CAGATCCGT- 3'
25. 5' -ATATGGCCCCAGAGGCCTGCAATGATCCACCGCCCCCACAMNNMNNATAMNNCTCG
TCAGATCCGT- 3'
26. 5' -ATATGGCCCCAGAGGCCTGCAATGATCCACCGCCCCCACAMNNMNNMNNATACTCG
TCAGATCCGT- 3'
113
CA 02587463 2007-05-11
WO 2006/127040 PCT/US2005/041639
27. 5' -
ATATGGCCCCAGAGGCCTGCAATGATCCACCGCCCCCACAMNNMNNMNNATAMNNCTCGTCAGATCCGT- 3'
where R=A/G, Y=C/T, M=A/C, K=G/T, S=C/G, W=A/T, B=C/G/T, D=A/G/T, H=A/C/T,
V=A/C/G, and N=A/C/G/T
[408] The library was constructed though an initial round of 10 cycles of
PCR amplification using a mixture of 4 pools of oligonucleotides, each pool
containing
400pmols of DNA. Pool 1 contained oligonucleotides 1-9, pool 2 contained 10-
17, pool 3
contained only 18 and poo14 contained 19-27. The fully assembled library was
obtained
through an additional 8 cycles of PCR using pool I and 4. The library
fragments were
digested with Xmal and SfiI. The DNA fragments were ligated into the
corresponding
restriction sites of phage display vector fuse5-HA, a derivative of fuse5
carrying an in-frame
HA-epitope. The ligation mixture was electroporated into TransforMaxTM EC100TM
electrocompetent E. coli cells resulting in a library of 2X109 individual
clones. Transformed
E. coli cells were grown overnight at 37 C in 2xYT medium containing 20 g/ml
tetracycline. Phage particles were purified from the culture medium by PEG-
precipitation
and a titer of 1.1X1013/ml was determined. Sequences of 24 clones were
determined and
were consistent with the expectations of the library design.
[409] While the foregoing invention has been described in some detail for
purposes of clarity and understanding, it will be clear to one skilled in the
art from a reading
of this disclosure that various changes in form and detail can be made without
departing from
the true scope of the invention. For example, all the techniques, methods,
compositions,
apparatus and systems described above can be used in various combinations. All
publications, patents, patent applications, or other documents cited in this
application are
incorporated by reference in their entirety for all purposes to the same
extent as if each
individual publication, patent, patent application, or other document were
individually
indicated to be incorporated by reference for all purposes.
114
