U.S. patent application number 10/957351 was filed with the patent office on 2006-01-12 for c-met kinase binding proteins.
This patent application is currently assigned to Avidia Research Institute. Invention is credited to D. Victor Perlroth, Sanjeev Satyal, Willem P.C. Stemmer.
Application Number | 20060008844 10/957351 |
Document ID | / |
Family ID | 35785714 |
Filed Date | 2006-01-12 |
United States Patent
Application |
20060008844 |
Kind Code |
A1 |
Stemmer; Willem P.C. ; et
al. |
January 12, 2006 |
c-Met kinase binding proteins
Abstract
Polypeptides comprising monomer domains that bind to c-Met, or
portions thereof, are provided.
Inventors: |
Stemmer; Willem P.C.; (Los
Gatos, CA) ; Perlroth; D. Victor; (Palo Alto, CA)
; Satyal; Sanjeev; (San Carlos, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Avidia Research Institute
Mountain View
CA
|
Family ID: |
35785714 |
Appl. No.: |
10/957351 |
Filed: |
September 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10871602 |
Jun 17, 2004 |
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10957351 |
Sep 30, 2004 |
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Current U.S.
Class: |
435/7.1 ; 506/14;
506/18; 506/9; 514/21.3; 514/7.4; 514/9.5; 514/9.6; 530/324 |
Current CPC
Class: |
A61K 38/00 20130101;
G01N 33/566 20130101; A61P 35/02 20180101; C07K 14/705 20130101;
G01N 33/574 20130101; A61P 35/00 20180101; C07K 14/47 20130101;
G01N 2500/00 20130101; G01N 2333/82 20130101; G01N 2333/4753
20130101 |
Class at
Publication: |
435/007.1 ;
530/324; 514/012 |
International
Class: |
G01N 33/53 20060101
G01N033/53; A61K 38/17 20060101 A61K038/17; C07K 14/47 20060101
C07K014/47 |
Claims
1. A polypeptide comprising a monomer domain that binds to c-Met,
wherein the monomer domain: is a non-naturally-occurring monomer
domain consisting of 30 to 50 amino acids; comprises at least one
disulfide bond; and binds to an ion.
2. The polypeptide of claim, wherein binding of at least one
monomer domain to c-Met inhibits dimerization of Met.
3. The polypeptide of claim 1, wherein at least one monomer domain
binds to the Sema domain of c-Met, thereby preventing binding of
Met ligands to c-Met.
4. The polypeptide of claim 1, wherein the polypeptide comprises at
least one and no more than six monomer domains.
5. The polypeptide of claim 1, wherein the polypeptide comprises at
least two monomer domains and the monomer domains are linked by a
linker.
6. The polypeptide of claim 5, wherein the linker is a peptide
linker.
7. The polypeptide of claim 6, wherein the linker is between 4 to
12 amino acids long.
8. The polypeptide of claim 1, wherein the monomer domains are each
between 35 to 45 amino acids.
9. The polypeptide of claim 1, wherein the polypeptide comprises at
least one monomer domain with binding specificity for a blood
factor, thereby increasing the serum half-life of the polypeptide
compared to a polypeptide lacking the blood factor monomer
domain.
10. The polypeptide of claim 9, wherein the blood factor is serum
albumin, an immunoglobulin or an erythrocyte.
11. The polypeptide of claim 1, wherein each monomer domain
comprises two disulfide bonds.
12. The polypeptide of claim 1, wherein each monomer domain
comprises three disulfide bonds.
13. The polypeptide of claim 1, wherein the ion is a metal ion.
14. The polypeptide of claim 1, wherein the ion is a calcium
ion.
15. The polypeptide of claim 1, wherein at least one of the monomer
domains is derived from a LDL-receptor class A domain.
16. The polypeptide of claim 1, wherein at least one of the monomer
domains is derived from an EGF-like domain.
17. The polypeptide of claim 1, wherein the monomer comprises an
amino acid sequence in which: at least 10% of the amino acids in
the sequence are cysteine; and/or at least 25% of the amino acids
are non-naturally-occurring amino acids.
18. A method for identifying a polypeptide that binds to c-Met, the
method comprising, screening a library of polypeptides for affinity
to c-Met; and selecting a polypeptide comprising at least one
monomer domain that binds to c-Met, wherein the monomer domain: is
a non-naturally-occurring monomer domain; comprises at least one
disulfide bond; and binds to an ion.
19. The method of claim 18, wherein the selecting step comprises
selecting a polypeptide that reduces HGF-mediated cell
proliferation and/or migration.
20. The method of claim 18, further comprising selecting a
polypeptide that inhibits tumor growth in an animal.
21. The method of claim 18, wherein the monomer comprises an amino
acid sequence in which: at least 10% of the amino acids in the
sequence are cysteine; and/or at least 25% of the amino acids are
non-naturally-occurring amino acids.
22. The method of claim 18, further comprising linking the monomer
domain in the selected polypeptide 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 c-Met; and selecting a multimer that binds c-Met.
23. The method of claim 18, further comprising linking the monomer
domain in the selected polypeptide 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 a target molecule other than the c-Met; and selecting a
multimer that binds to the target molecule.
24. The method of claim 18, further comprising a step of mutating
at least one monomer domain, thereby providing a library comprising
mutated monomer domains.
25. The method of claim 18, wherein the library of monomer domains
is expressed as a phage display, ribosome display or cell surface
display.
26. The method of claim 18, wherein the polypeptide comprises at
least two monomer domains and the monomer domains are linked by a
linker.
27. The method of claim 26, wherein the linker is a peptide
linker.
28. The method of claim 27, wherein the linker is between 4 to 12
amino acids long.
29. The method of claim 18, wherein the monomer domains are each
between 35 to 45 amino acids.
30. The method of claim 18, wherein each monomer domain comprises
two disulfide bonds.
31. The method of claim 18, wherein each monomer domain comprises
three disulfide bonds.
32. The method of claim 18, wherein the ion is a metal ion.
33. The method of claim 18, wherein the ion is a calcium ion.
34. The method of claim 18, wherein at least one of the monomer
domains is derived from a LDL-receptor class A domain.
35. The method of claim 18, wherein at least one of the monomer
domains is derived from an EGF-like domain.
36. The method of claim 18, wherein the monomer domain comprises an
amino acid sequence in which: at least 10% of the amino acids in
the sequence are cysteine; and/or at least 25% of the amino acids
are non-naturally-occurring amino acids.
37. A polynucleotide encoding the polypeptide of claim 1.
Description
BACKGROUND OF THE INVENTION
[0001] Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is a
mesenchyme-derived pleiotropic factor, which regulates cell growth,
cell motility, and morphogenesis of various types of cells and
mediates epithelial-mesenchymal interactions responsible for
morphogenic tissue interactions during embryonic development and
organogenesis. Although HGF was originally identified as a potent
mitogen for hepatocytes, it has also been identified as an
angiogenic growth factor.
[0002] Met was first identified in the 1980s as an oncogene and is
the receptor for HGF. The proto-oncogene c-Met, was found to encode
a receptor tyrosine kinase. In response to HGF treatment a range of
activities are observed: phosphorylation of receptor, docking of
signaling intermediates Gab-1/Grb2, culminating in activation of
kinases such as P13K, ERK1 and 2, and AKT. These activities aid in
cell growth, survival, migration, and neovascularisation.
[0003] Inappropriate expression or signaling of the receptor
tyrosine kinase Met and its ligand Hepatocyte Growth Factor/Scatter
Factor (HGF/SF) is associated with an aggressive phenotype and poor
clinical prognosis for a wide variety of solid human tumors.
[0004] Four lines of evidence cement the case for a role of c-Met
in cancer:
[0005] First, mouse and human cell lines that ectopically
overexpress HGF and/or Met become tumorigenic and metastatic in
athymic nude mice. Secondly, downregulation of Met or HGF
expression in human tumour cells decreases their tumorigenic
potential. Mouse models that express the receptor or ligand as a
transgene develop various types of tumour and metastatic tumors.
Third, a large number of studies show that HGF and/or Met are
frequently expressed in carcinomas, in other types of human solid
tumours and in their metastases, and that HGF and/or Met over- or
misexpression often correlates with poor prognosis. Fourth,
unequivocal evidence that implicates Met in human cancer is
provided by the activating mutations that have been discovered in
both sporadic and inherited forms of human renal papillary
carcinomas.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention provides a polypeptide comprising a
monomer domain that binds to c-Met. In some embodiments, the
monomer domain: [0007] is a non-naturally-occurring monomer domain
consisting of 30 to 50 amino acids; [0008] comprises at least one
disulfide bond; and [0009] binds to an ion.
[0010] In some embodiments, binding of at least one monomer domain
to c-Met inhibits dimerization of Met. In some embodiments, at
least one monomer domain binds to the Sema domain of c-Met, thereby
preventing binding of Met ligands to c-Met.
[0011] In some embodiments, the polypeptide comprises at least one
and no more than six monomer domains. In some embodiments, the
polypeptide comprises at least two monomer domains and the monomer
domains are linked by a linker. In some embodiments, the linker is
a peptide linker. In some embodiments, the linker is between 4 to
12 amino acids long.
[0012] In some embodiments, the monomer domains are each between 35
to 45 amino acids.
[0013] In some embodiments, the polypeptide comprises at least one
monomer domain with binding specificity for a blood factor, thereby
increasing the serum half-life of the polypeptide compared to a
polypeptide lacking the blood factor monomer domain. In some
embodiments, the blood factor is serum albumin, an immunoglobulin
or an erythrocyte.
[0014] In some embodiments, each monomer domain comprises two
disulfide bonds. In some embodiments, each monomer domain comprises
three disulfide bonds.
[0015] In some embodiments, the ion is a metal ion. In some
embodiments, the ion is a calcium ion.
[0016] In some embodiments, at least one of the monomer domains is
derived from a LDL-receptor class A domain. In some embodiments, at
least one of the monomer domains is derived from an EGF-like
domain.
[0017] In some embodiments, the monomer comprises an amino acid
sequence in which at least 10% of the amino acids in the sequence
are cysteine; and/or at least 25% of the amino acids are
non-naturally-occurring amino acids.
[0018] The present invention also provides methods for identifying
a polypeptide that binds to c-Met. In some embodiments, the method
comprises, [0019] screening a library of polypeptides for affinity
to c-Met; and [0020] selecting a polypeptide comprising at least
one monomer domain that binds to c-Met, wherein the monomer domain:
[0021] is a non-naturally-occurring monomer domain; [0022]
comprises at least one disulfide bond; and [0023] binds to an
ion.
[0024] In some embodiments, the selecting step comprises selecting
a polypeptide that reduces HGF-mediated cell proliferation and/or
migration. In some embodiments, the method further comprises
selecting a polypeptide that inhibits tumor growth in an
animal.
[0025] In some embodiments, the monomer domain comprises an amino
acid sequence in which at least 10% of the amino acids in the
sequence are cysteine; and/or at least 25% of the amino acids are
non-naturally-occurring amino acids.
[0026] In some embodiments, the method further comprises [0027]
linking the monomer domain in the selected polypeptide to a second
monomer domain to form a library of multimers, each multimer
comprising at least two monomer domains; [0028] screening the
library of multimers for the ability to bind to c-Met; and [0029]
selecting a multimer that binds c-Met.
[0030] In some embodiments, the method further comprises [0031]
linking the monomer domain in the selected polypeptide to a second
monomer domain to form a library of multimers, each multimer
comprising at least two monomer domains; [0032] screening the
library of multimers for the ability to bind to a target molecule
other than the c-Met; and [0033] selecting a multimer that binds to
the target molecule.
[0034] In some embodiments, the method further comprises a step of
mutating at least one monomer domain, thereby providing a library
comprising mutated monomer domains.
[0035] In some embodiments, the library of monomer domains is
expressed as a phage display, ribosome display or cell surface
display.
[0036] In some embodiments, the polypeptide comprises at least two
monomer domains and the monomer domains are linked by a linker. In
some embodiments, the linker is a peptide linker. In some
embodiments, the linker is between 4 to 12 amino acids long.
[0037] In some embodiments, the monomer domains are each between 35
to 45 amino acids.
[0038] In some embodiments, each monomer domain comprises two
disulfide bonds. In some embodiments, each monomer domain comprises
three disulfide bonds.
[0039] In some embodiments, the ion is a metal ion. In some
embodiments, the ion is a calcium ion.
[0040] In some embodiments, at least one of the monomer domains is
derived from a LDL-receptor class A domain. In some embodiments, at
least one of the monomer domains is derived from an EGF-like
domain.
[0041] In some embodiments, the monomer domain comprises an amino
acid sequence in which at least 10% of the amino acids in the
sequence are cysteine; and/or at least 25% of the amino acids are
non-naturally-occurring amino acids.
[0042] The present invention also provides polynucleotides encoding
a polypeptide comprising a monomer domain that binds to c-Met,
wherein the monomer domain: [0043] is a non-naturally-occurring
monomer domain consisting of 30 to 50 amino acids; [0044] comprises
at least one disulfide bond; and [0045] binds to an ion.
DEFINITIONS
[0046] Unless otherwise indicated, the following definitions
supplant those in the art.
[0047] "Met" also referred to as "c-Met," refers to the Hepatocyte
Growth Factor/Scatter Factor (HGF/SF)-binding receptor tyrosine
kinase. In response to HGF treatment a range of activities are
observed: phosphorylation of receptor, docking of signaling
intermediates Gab-1/Grb2, culminating in activation of kinases such
as P13K, ERK1 and 2, and AKT. These activities aid in cell growth,
survival, migration, and neovascularisation. See, e.g., Birchmeier
et al., Mol. Cell Biol. 4:915-925 (2003). The amino acid sequence
of Met is known and is displayed in SEQ ID NO:1. See, e.g., Park et
al., Proc. Natl. Acad. Sci. USA 84(18):6379 (1987).
[0048] 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 will
often bind to a target molecule. For example, a polypeptide that
forms a three-dimensional structure that binds to a target molecule
is a monomer domain. As used herein, the term "monomer domain" does
not encompass the complementarity determining region (CDR) of an
antibody.
[0049] 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.
[0050] 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 occurring 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 LDL receptor A-domains, EGF-like domains,
sushi domains, Fibronectin type 1 domains, and the like. Thus, for
example, in the case of LDL receptor A-domains represented by the
consensus sequence, CX.sub.6CX.sub.4CX.sub.6CX.sub.5CX.sub.8C (see
also FIG. 9), wherein X.sub.6, X.sub.4, X.sub.5, and X.sub.8 each
represent a cysteine-defined loop sequence comprising the
designated number of amino acids.
[0051] As used herein, the term "structure-defined loop sequence"
refers to a subsequence of a monomer-domain encoding sequence that
is bound at each end to subsequences that each form a secondary
structure. Secondary structures for proteins with known three
dimensional structures are identified in accordance with the
algorithm STRIDE for assigning protein secondary structure as
described in Frishman, D. and Argos, P. (1995) "Knowledge-based
secondary structure assignment," Proteins, 23(4):566-79 (see also
//hgmp.mrc.ac.uk/Registered/Option/stride.html at the World Wide
Web). Secondary structures for proteins with unknown or
uncharacterized three dimensional structures are identified in
accordance with the algorithm described in Jones, D. T. (1999),
"Protein secondary structure prediction based on position-specific
scoring matrices," J. Mol. Biol., 292:195-202 (see also McGuffin,
L. J., Bryson, K., Jones, D. T. (2000) "The PSIPRED protein
structure prediction server," Bioinformatics, 16:404-405, and
//bioinf.cs.ucl.ac.uk/psipred/ at the World Wide Web). Secondary
structures include, for example, pleated sheets, helices, and the
like. Examples of monomer domains having structure-defined loop
sequences are the C2 domains, Ig domains, Factor 5/8 C domains,
Fibronectin type 3 domains, and the like.
[0052] The term "B-factor-defined loop sequence" refers to a
subsequence of at least three amino acid residues of a
monomer-domain encoding sequence in which the B-factors for the
alpha carbons in the B-factor-defined loop are among the 25%
highest alpha carbon B factors in the entire monomer domain.
Typically the average alpha-carbon B-factor for the subsequence is
at least about 65. As used herein, the term "B-factor" (or
"temperature factor" or "Debye-Waller factor") is derived from
X-ray scattering data. The B-factor is a factor that can be applied
to the X-ray scattering term for each atom, or for groups of atoms,
that describes the degree to which electron density is spread out
B-factors employed in the practice of the present invention may be
either isotropic or anisotropic. The term "average alpha-carbon
B-factor" refers to: ( i = 1 n .times. B .times. - .times. factor C
.times. .times. .alpha. .times. .times. i ) / n ##EQU1## where n
corresponds to the number of residues in the loop, and is at least
3, and B-factor.sub.C.alpha.i is the B-factor for the alpha carbon
of amino acid residue i of the loop.
[0053] The term "multimer" is used herein to indicate a polypeptide
comprising at least two monomer domains. The separate monomer
domains in a multimer can be joined together by a linker. A
multimer is also known as a combinatorial mosaic protein or a
recombinant mosaic protein.
[0054] 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.
[0055] 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. Other 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).
[0056] 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 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.
[0057] 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.
[0058] 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.
[0059] 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 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.
[0060] 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.
[0061] The terms "polypeptide," "peptide," and "protein" are used
herein interchangeably to refer to an amino acid sequence of two or
more amino acids.
[0062] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. "Amino acid mimetics" refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0063] "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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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 (e.g., a cell
or tissue lysate) 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., 2.times., 5.times., 10.times. or more above
background) and does not bind in a significant amount to other
molecules present in the sample.
[0070] 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.
[0071] 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 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
non-naturally occurring fusion protein).
[0072] 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.
[0073] "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.
[0074] 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 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. 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.
[0075] 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 sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0076] 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 from 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, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0077] 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. Software 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 program (for nucleotide
sequences) uses as defaults a wordlength (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.
[0078] 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
[0079] FIG. 1 schematically illustrates the type, number and order
of monomer domains found in members of the LDL-receptor family.
These monomer domains include .beta.-Propeller domains, EGF-like
domains and LDL receptor class A-domains. The members shown include
low-density lipoprotein receptor (LDLR), ApoE Receptor 2 (ApoER2),
very-low-density lipoprotein receptor (VLDLR), LDLR-related protein
2 (LRP2) and LDLR-related protein 1 (LRP1).
[0080] FIG. 2 schematically illustrates the alignment of partial
amino acid sequence from a variety of the LDL-receptor class
A-domains (SEQ ID NOS: 103, 100, 65, 117, 128, 21, 29, 39, 30, 77,
58, 50, and 14, respectively in order of appearance) to demonstrate
the conserved cysteines.
[0081] FIG. 3, panel A schematically illustrates an example of an
A-domain. Panel A schematically illustrates conserved amino acids
in an A-domain of about 40 amino acids long. The conserved cysteine
residues are indicated by C, and the negatively charged amino acids
are indicated by a circle with a minus ("-") sign. Circles with an
"H" indicate hydrophobic residues. Panel B schematically
illustrates two folded A-domains connected via a linker. Panel B
also indicates two calcium binding sites, dark circles with
Ca.sup.+2, and three disulfide bonds within each folded A-domain
for a total of 6 disulfide bonds.
[0082] FIG. 4 indicates some of the ligands recognized by the
LDL-receptor family, which include inhibitors, proteases, protease
complexes, vitamin-carrier complexes, proteins involved in
lipoprotein metabolism, non-human ligands, antibiotics, viruses,
and others.
[0083] FIG. 5 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.
[0084] FIG. 6 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 monomer 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.
[0085] FIG. 7 shows the multimerization process of monomer domains.
The target-binding monomer hits are amplified from a vector. This
mixture of target-binding monomer domains is then cleaved and mixed
with an optimal combination of linker and stopper oligonucleotides.
The multimers that are generated are then cloned into a suitable
vector for the second selection step for identification of
target-binding multimers.
[0086] FIG. 8 depicts common amino acids in each position of the A
domain. The percentages above the amino acid positions refer to the
percentage of naturally-occurring A domains with the inter-cysteine
spacing displayed. Potential amino acid residues in bold depicted
under each amino acid position represent common residues at that
position. The final six amino acids, depicted as lighter-colored
circles, represent linker sequences. The two columns of italicized
amino acid residues at positions 2 and 3 of the linker represent
amino acid residues that do not occur at that position. Any other
amino acid (e.g., A, D, E, G, H, I, K, L, N, P, Q, R, S, T, and V)
may be included at these positions.
[0087] FIG. 9 displays the frequency of occurrence of amino acid
residues in naturally-occurring A domains for A domains with the
following spacing between cysteines:
CX.sub.6CX.sub.4CX.sub.6CX.sub.5CX.sub.8C (SEQ ID NO: 199).
[0088] FIG. 10 depicts an alignment of A domains (SEQ ID NO:
1-197). At the top and the bottom of the figure, small letters
(a-q) indicate conserved residues.
[0089] FIG. 11 depicts linkage of domains via partial linkers.
[0090] FIG. 12 is a graphical representation of the regions of
sequence identity between the sequences of two different selected
clones and known human sequences from a database. The horizontal
bars indicate areas of sequence identity between the sequence of
the selected clone and the human sequence and the numbers indicate
the exact amino acid numbers that define the region of identity.
The vertical arrow depicts an acceptable crossover sequence.
[0091] FIG. 13 illustrates screening a library of monomer domains
against multiple ligands displayed on a cell.
[0092] FIG. 14 illustrates identification of monomers that were
selected to bind to one of a plurality of ligands.
[0093] FIG. 15 illustrates an embodiment for identifying
polynucleotides encoding ligands and monomer domains.
[0094] FIG. 16 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.
[0095] FIG. 17 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.
[0096] FIG. 18 illustrates various possible antibody-monomer or
multimer conformations. In some embodiments, the monomer or
multimer replaces the Fab fragment of the antibody.
[0097] FIG. 19 illustrates a method for intradomain optimization of
monomers.
[0098] FIG. 20 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.
[0099] FIG. 21 illustrates four possible ways to recombine monomer
and/or multimer libraries to introduce new variation.
[0100] FIG. 22 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.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0101] The present invention provides for non-naturally-occurring
proteins that bind to c-Met. Generally, the proteins of the present
invention comprise a domain that binds to c-Met. These domains may
be readily identified using a variety of polypeptide scaffolds to
generate a plurality of polypeptide variants and then selecting a
variant that binds to c-Met. The present invention therefore also
provides for selecting a protein that binds to c-Met. Proteins that
bind c-Met are useful, e.g., for treating individuals with solid
tumors that express c-Met. The polypeptides of the invention are
also useful to detect tissues in which Met is expressed and can be
used to target molecules to those tissues.
[0102] c-Met is inactive in its resting monomer state and dimer
formation results in receptor activation (often even in absence of
ligand binding). The mature form of the receptor consists of a
solely extracellular .alpha. chain and a longer .beta. chain
encompassing the remainder of the extracellular domain, a
transmembrane domain and a cytoplasmic tail. The cytoplasmic tail
contains the juxtamembrane domain, a kinase domain and docking
sites for signaling intermediates. The .alpha. chain and the first
212 amino acids of the .beta. also known as the Sema domain
(Kong-Beltran, et al., Cancer Cell 6:75-84 (2004), are sufficient
for binding to HGF. The rest of the extracellular portion of the
.beta. chain consists of a cysteine-rich C domain and four repeats
of an unusual immunoglobulin domain. Accordingly, in some
embodiments, the polypeptides of the invention comprise at least
one monomer domain that inhibits dimerization of c-Met .alpha. and
.beta. chains and/or functions as an antagonist to prevent ligands
of c-Met from binding and/or activating c-Met.
[0103] While the present invention provides for polypeptides
comprising single domains, multimers of the domains may also be
synthesized and used. In some embodiments, all of the domains of
the multimer bind c-Met. In some of these embodiments, each of the
domains are identical and bind to the same portion (i.e.,
"epitope") of c-Met. FOr example, in some embvodiments, the monomer
domains bind to the Sema domain of c-Met. In other embodiments, at
least some of the domains in the multimer bind to different
portions of c-Met. In yet other embodiments, at least some of the
domains of the polypeptide bind to a molecule or molecules other
than c-Met.
II. Monomers
[0104] 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 can typically maintain a stable
conformation in solution, and are often heat stable, e.g., stable
at 95.degree. C. for at least 10 minutes without losing binding
affinity. Sometimes, monomer domains can fold independently into a
stable conformation. In one embodiment, the stable conformation is
stabilized by ions (e.g., such as metal or calcium 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., LDL A
domains, EGF domains, etc., or otherwise referenced herein.
[0105] The invention provides monomer domains that bind to c-Met or
a portion thereof. A portion of a polypeptide can be, e.g., at
least 5, 10, 15, 20, 30, 50, 100, or more contiguous amino acids of
the polypeptide.
[0106] A publication describing monomer domains and mosaic proteins
include PCT Publication No. WO 2004/044011 and references cited
within.
[0107] Monomer domains that are particularly suitable for use in
the practice of the present invention are (1) .beta. sandwich
domains; (2) .beta.-barrel domains; or (3) cysteine-rich domains
comprising disulfide bonds. Cysteine-rich domains employed in the
practice of the present invention typically do not form an .alpha.
helix, a .beta. sheet, or a .beta.-barrel structure. 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. In some embodiments, at least 5, 10, 15 or 20% of the amino
acids in a monomer domain are cysteines.
[0108] Domains can have any number of characteristics. For example,
in some embodiments, the domains have low or no immunogenicity in
an animal (e.g., a human). Domains can have a small size. In some
embodiments, the domains are small enough to penetrate skin or
other tissues. Domains can have a range of in vivo half-lives or
stabilities.
[0109] Illustrative monomer domains suitable for use in the
practice of the present invention include, e.g., an 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 Trefoil (P-type) domain, a von Willebrand factor type C
domain, an Anaphylatoxin-like domain, a CUB domain, a thyroglobulin
type I repeat, LDL-receptor class A domain, a Sushi domain, a Link
domain, a Thrombospondin type I 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, a Laminin-type EGF-like domain, a C2
domain, and other such domains known to those of ordinary skill in
the art, as well as derivatives and/or variants thereof. In some
embodiments, the monomer domain is not the C2 domain. FIG. 1
schematically diagrams various kinds of monomer domains found in
molecules in the LDL-receptor family.
[0110] 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 families of protein domains
that contain .beta.-sandwich or .beta.-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 CATH, (2000) Nucleic
Acids Research 28(1):277-282).
[0111] In another embodiment, monomer domains of the present
invention include domains other than a fibronectin type III domain,
an anticalin domain and a Ig-like domain from CTLA-4. Some aspects
of these domains are described in WO 01/64942 entitled "Protein
scaffolds for antibody mimics and other binding proteins" by
Lipovsek et al., published on Sep. 7, 2001, WO99/16873 entitled
"Anticalins" by Beste et al., published Apr. 8, 1999 and WO
00/60070 entitled "A polypeptide structure for use as a scaffold"
by Desmet, et al., published on Oct. 12, 2000.
[0112] As described supra, monomer domains are optionally cysteine
rich. Suitable cysteine rich monomer domains include, e.g., the LDL
receptor class A domain ("A-domain") or the EGF-like domain. The
monomer domains can also have a cluster of negatively charged
residues. Optionally, the monomer domains contain a repeated
sequence, such as YWTD (SEQ ID NO: 198) as found in the
.beta.-Propeller domain.
[0113] Other features of monomer domains can include the ability to
bind ligands (e.g., as in the LDL receptor class A domain, or the
CUB domain (complement C1r/C1s, Uegf, and bone morphogenic
protein-1 domain)), the ability to participate in endocytosis or
internalization (e.g., as in the cytoplasmic tail of the LDL
receptor or the cytoplasmic tail of Megalin), the ability to bind
an ion (e.g., Ca.sup.2+ binding by the LDL receptor A-domain),
and/or the ability to be involved in cell adhesion (e.g., as in the
EGF-like domain). Monomer domains that bind ions to maintain their
secondary structure include, e.g., A domain, EGF domain, EF Hand
(e.g., such as those found in present in calmodulin and troponin
C), Cadherin domain, C-type lectin, C2 domain, Annexin, Gla-domain,
Trombospondin type 3 domain, 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 provides stability of
secondary structure while providing sufficient flexibility to allow
for numerous binding conformations depending on primary
sequence.
[0114] Characteristics of a monomer domain can include the ability
to fold independently and the ability to form a stable structure.
Thus, the structure of the monomer domain is often conserved,
although the polynucleotide sequence encoding the monomer need not
be conserved. For example, the A-domain structure is conserved
among the members of the A-domain family, while the A-domain
nucleic acid sequence is not. Thus, for example, a monomer domain
is classified as an A-domain by its cysteine residues and its
affinity for calcium, not necessarily by its nucleic acid sequence.
See, FIG. 2.
[0115] As described herein, monomer domains may be 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.
[0116] Specifically, the A-domains (sometimes called
"complement-type repeats") contain about 30-50 or 30-65 amino
acids. In some embodiments, the domains comprise about 35-45 amino
acids and in some cases about 40 amino acids. Within the 30-50
amino acids, there are about 6 cysteine residues. Of the six
cysteines, disulfide bonds typically are found between the
following cysteines: C1 and C3, C2 and C5, C4 and C6. The cysteine
residues of the domain are disulfide linked to form a compact,
stable, functionally independent moiety. See, FIG. 3. Clusters of
these repeats make up a ligand binding domain, and differential
clustering can impart specificity with respect to the ligand
binding.
[0117] Exemplary A domain sequences and consensus sequences are
depicted in FIGS. 2, 3 and 8. FIG. 9 displays location and
occurrence of residues in A domains with the following spacing
between cysteines. In addition, FIG. 10 depicts a number of A
domains and provides a listing of conserved amino acids. One
typical consensus sequence useful to identify A domains is the
following:
C-[VILMA]-X.sub.(5)--C-[DNH]-X.sub.(3)-[DENQHT]--C--X.sub.(3,4)-[STADE]-[-
DEH]-[DE]-X.sub.(1,5)--C (SEQ ID NO: 200), where the residues in
brackets indicate possible residues at one position. "X.sub.(#)"
indicates number of residues. These residues can be any amino acid
residue. Parentheticals containing two numbers refers to the range
of amino acids that can occupy that position (e.g.,
"[DE]-X.sub.(1,5)--C" means that the amino acids DE are followed by
1, 2, 3, 4, or 5 residues, followed by C). This consensus sequence
only represents the portion of the A domain beginning at the third
cysteine. A second consensus is as follows:
C--X.sub.(3-15)--C--X.sub.(4-15)--C--X.sub.(6-7)--C-[N,D]-X.sub.(3)-[D,E,-
N,Q,H,S,T]-C--X.sub.(4-6)-D-E-X.sub.(2-8)--C (SEQ ID NO: 201). The
second consensus predicts amino acid residues spanning all six
cysteine residues. In some embodiments, A domain variants comprise
sequences substantially identical to any of the above-described
sequences.
[0118] Additional exemplary A domains include the following
sequence: [0119]
C.sub.aX.sub.3-15C.sub.bX.sub.3-15C.sub.cX.sub.6-7C.sub.d(D,N)X.s-
ub.4C.sub.eX.sub.4-6DEX.sub.2-8C.sub.f [0120] wherein C is
cysteine, X.sub.n-m represents between n and m number of
independently selected amino acids, and (D,N) indicates that the
position can be either D or N; and wherein C.sub.a-C.sub.c,
C.sub.b-C.sub.e and C.sub.d-C.sub.f form disulfide bonds.
[0121] In some embodiments, the monomer domain is an LDL receptor
class A domain monomer comprising the following sequence: [0122]
CaX.sub.6-7C.sub.bX.sub.4-5C.sub.cX.sub.6C.sub.dX.sub.5C.sub.eX.sub.8-10C-
.sub.f [0123] wherein X is defined as follows: The table above
indicates alternative amino acid residues at each position of the
LDL receptor class A monomer domain. For example, there can be
either 6 or 7 amino acids between cystein C1 and cystein C2. The
upper left box of the table indicates alternative amino acid
residues at each position if there are 6 amino acids between C1 and
C2. The bottom left box in the table indicates alternative amino
acid residues of there are seven amino acids between C1 and C2. In
all cases, the amino acid for one position (e.g., X1) is selected
independently of the amino acids selected for remaining positions
(e.g., X2, X3, etc.)
[0124] To date, at least 190 human A-domains are identified based
on cDNA 10 sequences. See, e.g., FIG. 10. Exemplary proteins
containing A-domains include, e.g., complement components (e.g.,
C6, C7, C8, C9, and Factor I), serine proteases (e.g.,
enteropeptidase, matriptase, and corin), transmembrane proteins
(e.g., ST7, LRP3, LRP5 and LRP6) and endocytic receptors (e.g.,
Sortilin-related receptor, LDL-receptor, VLDLR, LRP1, LRP2, and
ApoER2). A domains and A domain variants can be readily employed in
the practice of the present invention as monomer domains and
variants thereof. Further description of A domains can be found in
the following publications and references cited therein: Howell and
Hertz, The LDL receptor gene family: signaling functions during
development, (2001) Current Opinion in Neurobiology 11:74-81; Herz
(2001), supra; Krieger, The "best" of cholesterols, the "worst" of
cholesterols: A tale of two receptors, (1998) PNAS 95: 4077-4080;
Goldstein and Brown, The Cholesterol Quartet, (2001) Science, 292:
1310-1312; and, Moestrup and Verroust, Megalin-and Cubilin-Mediated
Endocytosis of Protein-Bound Vitamins, Lipids, and Hormones in
Polarized Epithelia, (2001) Ann. Rev. Nutr. 21:407-28.
[0125] Other examples of monomer domains can be found in the
protein Cubilin, which contains EGF-type repeats and CUB domains.
The CUB domains are involved in ligand binding, e.g., some ligands
include intrinsic factor (IF)-vitamin B12, receptor associated
protein (RAP), Apo A-I, Transferrin, Albumin, Ig light chains and
calcium. See, Moestrup and Verroust, supra.
[0126] Megalin also contains multiple monomer domains.
Specifically, megalin possesses LDL-receptor type A-domain,
EGF-type repeat, a transmembrane segment and a cytoplasmic tail.
Megalin binds a diverse set of ligands, e.g., ApoB, ApoE, ApoJ,
clusterin, ApopH/Beta2-glycoprotein-I, PTH, Transthyretin,
Thyroglobulin, Insulin, Aminoglycosides, Polymyxin B, Aprotinin,
Trichosanthin, PAI-1, PAI-1-urokinase, PAI-1-tPA, Pro-urokinase,
Lipoprotein lipase, alpha-Amylase, Albumin, RAP, Ig light chains,
calcium, C1q, Lactoferrin, beta2-microglobulin, EGF, Prolactin,
Lysozyme, Cytochrome c, PAP-1, Odorant-binding protein, seminal
vesicle secretory protein II. See, Moestrup & Verroust,
supra.
[0127] Exemplary EGF monomer domains include the sequence: [0128]
C.sub.aX.sub.3-14C.sub.bX.sub.3-7C.sub.cX.sub.4-16C.sub.dX.sub.1-2C.sub.e-
X.sub.8-23C.sub.f [0129] wherein C is cysteine, X.sub.n-m
represents between n and m number of independently selected amino
acids; and [0130] wherein C.sub.a-C.sub.c, C.sub.b-C.sub.e and
C.sub.d-C.sub.f form disulfide bonds.
[0131] In some embodiments, the monomer domain is an EGF domain
monomer comprising the following sequence: [0132]
C.sub.aX.sub.4-6C.sub.bX.sub.3-5C.sub.cX.sub.8-9C.sub.dX.sub.1C.sub.eX.su-
b.8-12C.sub.f [0133] wherein X is defined as follows: EGF monomer
domains are sometimes between 25-45 amino acids and typically 30-39
amino acids.
[0134] 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. For example, libraries may be designed in which a scaffold
of amino acids remain constant (e.g., an LDL A receptor domain, EGF
domain) while the intervening amino acids in the scaffold comprise
randomly generated amino acids.
[0135] 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 are identified or
selected for a desired property (e.g., binding affinity) and then
the monomer domains are formed into multimers. See, e.g., FIG. 5.
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.
[0136] As mentioned above, monomer domains can be
naturally-occurring or altered (non-natural 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.
[0137] Monomer domains of the present invention can be
naturally-occurring domains or non-naturally occurring variants.
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.
[0138] Monomer domain variants can include ancestral domains,
chimeric domains, randomized domains, mutated domains, and the
like. For example, ancestral domains can be based on phylogenetic
analysis. 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 LDL receptor class A-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. Thus the present invention 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.
[0139] 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.
[0140] 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.
[0141] The present invention also provides recombinant nucleic
acids encoding one or more polypeptides comprising a plurality of
monomer domains and/or immuno-domains, which monomer domains are
altered in order or sequence as compared to a naturally occuring
polypeptide. For example, the naturally occuring polypeptide can be
selected from the group consisting of: an 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 Trefoil (P-type)
domain, a von Willebrand factor type C domain, an
Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I
repeat, LDL-receptor class A domain, a Sushi domain, a Link domain,
a Thrombospondin type I 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, a Laminin-type EGF-like domain, a C2 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.
[0142] All the compositions of the present invention, including the
compositions produced by the methods of the present invention,
e.g., monomer domains and/or immuno-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.
III. Multimers
[0143] Methods for generating multimers 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 3 or
at least 3 monomer domains. In some embodiments, the multimers have
no more than 2, 3, 4, 5, 6, 7, or 8 monomer domains. In view of the
possible range of monomer domain sizes, the multimers of the
invention may be, e.g., less than 100 kD, less than 90 kD, less
than 80 kD, less than 70 kD, less than 60 kD, less than 50 kD, less
than 40 kD, less than 30 kD, less than 25 kD, less than 20 kD, less
than 15 kD, less than 10 kD or may be smaller or larger. In some
cases, the monomer domains have been pre-selected for binding to
the target molecule of interest (e.g., Met).
[0144] In some embodiments, each monomer domain specifically binds
to one target molecule (e.g., Met). In some of these embodiments,
each monomer binds to a different position (analogous to an
epitope) on a target molecule. Multiple monomer domains that bind
to the same target molecule results in an avidity effect resulting
in 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 times the avidity of a monomer domain alone. In some
embodiments, at least one, two, three, four or more (e.g., all)
monomers of a multimer bind an ion such as calcium or another ion.
Multimers can comprise a variety of combinations of monomer
domains. For example, in a single multimer, the selected monomer
domains can be identical or different. 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.
[0145] Multimers that are generated in the practice of the present
invention may be any of the following: [0146] (1) A homo-multimer
(a multimer of the same domain, i.e., A1-A1-A1-A1); [0147] (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 A1, A2, A3 and A4 are different non-naturally occurring
variants of a particular LDL-receptor class A domains, or where
some of A1,A2, A3, and A4 are naturally-occurring variants of a
LDL-receptor class A domain (see, e.g., FIG. 10). [0148] (3) A
hetero-multimer of domains from different monomer domain classes,
e.g., A1-B2-A2-B1. For example, where A1 and A2 are two different
monomer domains (either naturally occurring or
non-naturally-occurring) from LDL-receptor class A, and B1 and B2
are two different monomer domains (either naturally occurring or
non-naturally occurring) from class EGF-like domain).
[0149] In another embodiment, the multimer comprises monomer
domains with specificities for different target molecules. For
example, in some embodiments, the multimers of the invention
comprises 1, 2, 3, or more monomer domains that bind to Met and at
least one monomer domain that binds to a second target molecule.
Exemplary target molecules include, e.g., a serum molecule that
extends the serum half-life of the multimer, EGFR gene family
members, VEGF receptors, PDGF receptor, other receptor tyrosine
kinases, integrins, other molecules implicated in tumorigenesis, or
markers of tumor tissue. Exemplary molecule that extends the serum
half-life of a multimer include, e.g., red blood cells, IgG, and
serum albumin such as HSA.
[0150] 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.
[0151] 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, an 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 Trefoil (P-type) domain, a von Willebrand
factor type C domain, an Anaphylatoxin-like domain, a CUB domain, a
thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi
domain, a Link domain, a Thrombospondin type I 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, a Laminin-type
EGF-like domain, a C2 domain, or variants thereof.
[0152] 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 two
domains that bind to one target molecule and a third domain that
binds to a second target molecule.
[0153] 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), at least one, more than one
or all monomer domains may be derived from LDL receptor A domains
and/or EGF-like 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: [0154] a. each monomer domain is between
30-100 amino acids and each of the monomer domains comprise at
least one disulfide linkage; or [0155] b. each monomer domain is
between 30-100 amino acids and is derived from an extracellular
protein; or [0156] c. each monomer domain is between 30-100 amino
acids and binds to a protein target.
[0157] In some embodiments, the multimers comprise at least 4
monomer domains, wherein at least one monomer domain is
non-naturally occurring, and wherein: [0158] a. each monomer domain
is between 35-100 amino acids; or [0159] b. each domain comprises
at least one disulfide bond and is derived from a human protein
and/or an extracellular protein.
[0160] 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: [0161] a.
25-50 amino acids long and comprises at least one disulfide bond;
or [0162] b. 25-50 amino acids long and is derived from an
extracellular protein; or [0163] c. 25-50 amino acids and binds to
a protein target; or [0164] d. 35-50 amino acids long.
[0165] In some embodiments, the multimers comprise at least two
monomer domains, wherein at least one monomer domain is
non-naturally-occurring and: [0166] a. each monomer domain
comprises at least one disulfide bond; or [0167] b. at least one
monomer domain is derived from an extracellular protein; or [0168]
c. at least one monomer domain binds to a target protein.
[0169] 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.
[0170] 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 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.
[0171] 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 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 as a single detection agent capable of
detecting for the possibility of any of a number of pathogens or
contaminants.
[0172] 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, resulting in the formation of non-covalent
interactions between two monomer 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.
[0173] 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. See. e.g., FIGS. 16 and
17.
[0174] 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: [0175] 1. At the N-terminus of the VH1
and/or VL1 domains, optionally just after the leader peptide and
before the domain starts (framework region 1); [0176] 2. At the
N-terminus of the CH1 or CL1 domain, replacing the VH1 or VL1
domain; [0177] 3. At the N-terminus of the heavy chain, optionally
after the CH1 domain and before the cysteine residues in the hinge
(Fc-fusion); [0178] 4. At the N-terminus of the CH3 domain; [0179]
5. At the C-terminus of the CH3 domain, optionally attached to the
last amino acid residue via a short linker; [0180] 6. At the
C-terminus of the CH2 domain, replacing the CH3 domain; [0181] 7.
At the C-terminus of the CL1 or CH1 domain, optionally after the
cysteine that forms the interchain disulfide; or [0182] 8. At the
C-terminus of the VH1 or VL1 domain. See, e.g., FIG. 18.
[0183] 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.
[0184] 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. As
c-Met is expressed at significant levels in the liver, monomer
domains that bind to Met may be used to target other molecules,
including other monomer domains, to the liver. This may be used to
target liver-specific diseases, for example, by targeting
therapeutic or toxic molecules to the liver. An example of a liver
disease that can be treated is hepatocellular carcinoma. 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.
[0185] 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 (e.g., Met) 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.
[0186] 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.
[0187] 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 pET-derived plasmid encoding the
polypeptides are induced to express the protein. After harvesting
the bacteria, they may be lysed and clarified by centrifugation.
The polypeptides may be purified using Ni--NTA agarose elution and
refolded by dialysis. Misfolded proteins may be neutralized by
capping free sulfhydrils with iodoacetic acid. Q sepharose elution,
butyl sepharose FT, SP sepharose elution, Q sepharose elution,
and/or SP sepharose elution may be used to purify the
polypeptides.
IV. Linkers
[0188] Monomer domains can be joined by a linker to form a
multimer. For example, a linker may be positioned between each
separate discrete monomer domain in a multimer.
[0189] 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 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.
[0190] 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.
[0191] A linker can be rigid, or 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.
[0192] 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.
[0193] 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.
[0194] Typically, the linker polypeptide may predominantly include
amino acid residues selected from the group consisting of 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 the group
consisting of 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 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.
[0195] 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.
[0196] 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 minimal 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.
[0197] 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; U.S. Pat. No.
5,856,456).
[0198] 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 (V.sub.L) and a heavy chain (V.sub.H) 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 (SEQ ID NO: 240) amino acid sequence
((Gly.sub.4Ser).sub.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, 1L-5, and
interferon-.gamma.. 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).
[0199] 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 splicing. The use of inteins is
reviewed by F. S. Gimble in Chemistry and Biology, 1998, Vol 5, No.
10 pp. 251-256.
[0200] Still another way of obtaining a suitable linker is by
optimizing a simple linker, e.g. (Gly.sub.4Ser).sub.n, (SEQ ID NO:
240), through random mutagenesis.
[0201] 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.
[0202] The peptide linker may, in addition to the glycine residues,
comprise other residues, in particular residues selected from the
group consisting of 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.sub.x-Xaa-Gly.sub.y-Xaa-Gly.sub.z (SEQ ID NO: 203), 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 the group consisting of Ser, Ala and
Thr, in particular Ser. More particularly, the peptide linker has
the amino acid sequence Gly-Gly-Gly-Xaa-Gly-Gly-Gly-Xaa-Gly-Gly-Gly
(SEQ ID NO: 204), 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 the group
consisting of Ser, Ala and Thr, in particular Ser.
[0203] 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.
[0204] 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).
[0205] 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 the group
consisting of Gly, Ser, Ala, Thr and Cys. In some embodiments, such
a peptide linker comprises one cysteine residue only.
[0206] 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 Gly.sub.n-Cys-Gly.sub.m (SEQ
ID NO: 205), 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 (SEQ ID NO:
206).
[0207] 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
Pro.sub.n-Cys-Pro.sub.m (SEQ ID NO: 207), 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 (SEQ ID NO: 208).
[0208] 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.
[0209] 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.
[0210] 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 the group consisting
of 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.
[0211] A specific example of a peptide linker comprising an in vivo
N-glycosylation site is a peptide linker having the amino acid
sequence Gly.sub.n-Asn-Xaa-Ser/Thr-Gly.sub.m (SEQ ID NO: 209),
preferably Gly.sub.n-Asn-Xaa-Thr-Gly.sub.m (SEQ ID NO: 210),
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.
[0212] Often, the amino acid sequences of all peptide linkers
present in the polypeptide multimer will be identical.
Nevertheless, in certain embodiments the amino acid 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.
[0213] 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.
[0214] 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.
[0215] 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 LDL receptor A domain and the first
cysteine of a second LDL receptor A domain can be used as a linker
sequence. Analysis of various A 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. An
exemplary A domain linker sequence is depicted in FIG. 8. In some
embodiments, the linker is a 6-mer of the following sequence
A.sub.1A.sub.2A.sub.3A.sub.4A.sub.5A.sub.6 (SEQ ID NO: 244),
wherein A1 is selected from the amino acids A, P, T, Q, E and K;
A.sub.2 and A.sub.3 are any amino acid except C, F, Y, W, or M;
A.sub.4 is selected from the amino acids S, G and R; A.sub.5 is
selected from the amino acids H, P, and R; and A.sub.6 is the amino
acid, T.
[0216] Methods for generating multimers from monomer 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 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.
[0217] 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.
[0218] 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.
[0219] 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. A multimer containing such obligate
heterodimers is depicted in FIG. 11. 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 peptide
(i.e., a partial linker) and later combined to form multimers. 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. Pat.
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.
[0220] 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 fragments 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.
[0221] 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. A multimer containing such obligate
heterodimers is depicted in FIG. 11. 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 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. Pat. 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.
[0222] 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 fragments 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.
V. Identifying Monomers or Multimers With Affinity For a Target
Molecule
[0223] Those of skill in the art can readily identify monomer
domains with a desired property (e.g., binding affinity). 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.
[0224] In addition, any method of mutagenesis, such as
site-directed mutagenesis and random mutagenesis (e.g., chemical
mutagenesis) can be used to produce monomer domains, e.g., for a
monomer domain library. 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. Human
chimeric monomer domain libraries can be constructed by combining
loop sequences from different variants of a human monomer domain,
as described above. The loop sequences that are combined may be
sequence-defined loops, structure-defined loops, B-factor-defined
loops, or a combination of any two or more thereof.
[0225] 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. In some
embodiments, 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 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.
[0226] Domains of human monomer variant 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.
[0227] In some embodiments, monomer domains of the invention are
screened for potential immunogenicity by: [0228] providing a
candidate protein sequence; [0229] comparing the candidate protein
sequence to a database of human protein sequences; [0230]
identifying portions of the candidate protein sequence that
correspond to portions of human protein sequences from the
database; and [0231] determining the extent of correspondence
between the candidate protein sequence and the human protein
sequences from the database.
[0232] 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. 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/blast/Blast.cgi?CMD=Web&LAYOUT=TwoWindows&AUTO_FORMAT=Sem-
iauto&ALIGNMENTS=50&ALIGNMENT_VIEW=Pairwise&CLIENT=web&DATABASE=nr&DESCRIP-
TIONS=100&ENTREZ_QUERY=(none)&EXPECT=1000&FORMAT_OBJECT=Alignment&FORMAT_T-
YPE=HTML&NCBI_GI=on&PAGE=Nucleotides&PROGRAM=blastn&SERVICE=plain&SET_DEFA-
ULTS.x=29&SET_DEFAULTS.y=6&SHOW_OVERVIEW=on&WORD_SIZE=7&END_OF_HTTPGET=Yes-
&SHOW_LINKOUT=yes 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. An
example of the comparison step is depicted in FIG. 12, which shows
a comparison of two candidate protein sequences to human sequences
from a database. The horizontal lines indicate where the human
protein sequences from the database are identical to the candidate
protein sequence.
[0233] Information pertaining to portions of human protein
sequences from the database can be used to design a protein library
of human-like chimeric proteins. 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.
[0234] In some embodiments, 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.
[0235] 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.
[0236] Nucleic acids encoding fragments of naturally-occurring
monomer domains can also be mixed and/or recombined (e.g., by using
chemically or enzymatically-produced fragments) to generate
full-length, modified monomer 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 construct encoding fragments of the monomer domain can
be used to generate an altered monomer domain.
[0237] 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 mutation, pseudorandom
mutation, defined kernal 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 shuffled.
[0238] 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 shuffling 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.
[0239] In some embodiments, the monomer library is prepared by
shuffling. In such a case, monomer domains are isolated and
shuffled 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.
[0240] 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 to which they bind.
[0241] 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 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.
[0242] 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.
[0243] The present invention also provides libraries of monomer
domains and libraries of nucleic acids that encode monomer domains.
The libraries can include, e.g., about 100, 250, 500 or more
nucleic acids encoding monomer domains, or the library can include,
e.g., about 100, 250, 500 or more polypeptides that encode monomer
domains. Libraries can include monomer domains containing the same
cysteine frame, e.g., A-domains or EGF-like domains.
[0244] 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: an 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 Trefoil (P-type)
domain, a von Willebrand factor type C domain, an
Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I
repeat, LDL-receptor class A domain, a Sushi domain, a Link domain,
a Thrombospondin type I 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, a Laminin-type EGF-like domain, 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.
[0245] 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.
[0246] The libraries can be screened for a desired property such as
binding of a desired ligand or mixture of ligands. 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. 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).
VI. Selection of Monomer Domains that Bind c-Met
[0247] Preliminary screens can be conducted by screening for agents
capable of binding to c-Met, as at least some of the agents so
identified are likely c-Met modulators (e.g., antagonists or
agonists). The binding assays usually involve contacting a c-Met
protein (or a fragment thereof such as a fragment comprising the
SEMA rdomain or the .alpha. chain) with one or more test agents
(i.e., monomers or multimers of the invention) and allowing
sufficient time for the protein and test agents to form a binding
complex. Any binding complexes formed can be detected using any of
a number of established analytical techniques. Protein binding
assays include, but are not limited to, immunohistochemical binding
assays, flow cytometry or other assays. The c-Met protein utilized
in such assays can be naturally expressed, cloned or
synthesized.
[0248] The screening methods of the invention can be performed as
in vitro or cell-based assays. Cell based assays can be performed
in any cells in which c-Met is expressed. Cell-based assays may
involve whole cells or cell fractions containing a c-Met receptor
to screen for agent binding or modulation of activity of c-Met by
the agent. Exemplary cell types that can be used according to the
methods of the invention include, e.g., any mammalian cells, as
well as fungal cells, including yeast, and bacterial cells. Cells
can be primary cells or tumor cells or other types of immortal cell
lines. Of course, c-Met can be expressed in cells that do not
endogenously contain c-Met.
[0249] c-Met activity assays may also be used to identify a
modulator (antagonist or agonist) of c-Met. In these embodiments,
one or more test agents are contacted to a cell expressing c-Met
and then tested for an activity of c-Met. Exemplary c-Met
activities include HGF-dependent or constitutive kinase activity.
See, e.g., Christensen et al., Cancer Res. 63:7345-7355 (2003). In
other embodiments, down stream molecular events can also be
monitored to determine signaling activity. For example, c-Met
induces cell growth (proliferation and survival), cell motility,
invasion and morphology changes. In addition, c-Met indirectly
mediates phosphorylation of Gab-1, Akt, signal transducer and
activator of transcription 3, phospholipase C .gamma., and focal
adhesions kinase, among others. See, e.g., Christensen et al.,
Cancer Res. 63:7345-7355 (2003).
[0250] In some embodiments, activity assays are also used to
confirm that identified antagonist monomers or multimers (i.e.,
that compete with HGF) lack agonist activity (i.e., that they do
not activate c-Met in the absence of HGF or another agonist).
[0251] Agents that are initially identified by any of the foregoing
screening methods can be further tested to validate the apparent
activity. Such studies may be conducted with suitable animal
models. The basic format of such methods involves administering a
lead compound identified during an initial screen to an animal that
serves as a model for humans and then determining if c-Met is in
fact modulated and/or the disease or condition is ameliorated. The
animal models utilized in validation studies generally are mammals
of any kind. Specific examples of suitable animals include, but are
not limited to, primates, mice and rats.
[0252] Selection of monomer domains that bind c-Met from a library
of domains can be accomplished by a variety of procedures. For
example, one method of identifying monomer domains which have a
desired property (e.g., binding c-Met) involves translating a
plurality of nucleic acids, where each nucleic acid encodes a
monomer domain, screening the polypeptides encoded by the plurality
of nucleic acids, and identifying those monomer domains that, e.g.,
bind to a desired ligand or mixture of ligands, thereby producing a
selected monomer domain. The monomer 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).
[0253] As mentioned above, selection of monomer domains can be
based on binding to a ligand such as c-Met, or a fragment therof or
other target molecule (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.sup.+2.
[0254] When a monomer domain of the invention 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. 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.
[0255] A variety of reporting display vectors or systems can be
used to express nucleic acids encoding the monomer 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 on
the surface of a filamentous bacteriophage, typically as a fusion
with a bacteriophage coat protein.
[0256] 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 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 acids.
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 particle for
detection and selection by the methods described above and below.
For example, the phage library can be incubated with a
predetermined ligand such as c-Met or a fragment thereof, 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.
[0257] Examples of other display systems include ribosome displays,
a nucleotide-linked display (see, e.g., U.S. Pat. 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.
[0258] The monomer and multimer libraries of the invention can be
screened for a desired property such as binding of a desired ligand
(e.g., c-Met) or mixture of ligands. 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. 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 ligands, 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).
[0259] 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. FIG. 7 illustrates a generic cycle of selection
and generation of variation.
[0260] All the compositions 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.
[0261] When multimers capable of binding relatively large targets
are desired, they can be generated by a "walking" selection method.
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 monomer is covalently linked to a
new library or each remaining member of the original library of
monomer domains. This new library of multimers (dimers) 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.
[0262] 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. See, e.g., FIG. 6. 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.
[0263] The methods of the present invention also include methods of
evolving monomers or multimers. As illustrated in FIG. 21,
intra-domain recombination can be introduced into monomers across
the entire monomer or by taking portions of different monomers to
form new recombined units. Interdomain recombination (e.g.,
recombining different monomers into or between multimers) or
recombination of modules (e.g., multiple monomers within a
multimer) may be achieved. Inter-library recombination is also
contemplated.
[0264] 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 (e.g., c-Met) 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 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.
[0265] Variation can be introduced into either monomers or
multimers. 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).
[0266] 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., FIG. 21.
[0267] 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.
[0268] Methods of the present invention also include generating one
or more selected multimers by providing a plurality of monomer
domains. The plurality of monomer 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.
[0269] 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. coli, library size
(10.sup.9-10.sup.10) 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.
[0270] For example, the Cre-loxP system may be used to generate
dimer libraries with 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13, 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. coli cells (carrying the
other library). For example, infecting 10.sub.12 E. coli cells (1 L
at OD600=1) with >10.sup.12 phage could produce as many as
10.sup.12 dimer combinations.
[0271] 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 acid sequence of the
individual monomer domains or the linker domain or the nucleotide
sequence encoding such domains, as mentioned in the present
invention.
[0272] 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. Pat. 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.
[0273] 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 Gal4 protein (the DNA binding domain). A third plasmid
contains a reporter gene downstream of the DNA sequence of the Gal4
binding site. A monomer that can bind to the target protein brings
with it the Gal4 activation domain, thus reconstituting a
functional Gal4 protein. This functional Gal4 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. Pat. 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
(Germino et al. (1993) Proc. Nat. Acad. Sci. (U.S.A.) 90:993;
Guarente L. (1993) Proc. Nat. Acad. Sci. (U.S.A.) 90:1639).
[0274] 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 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.
[0275] 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.
[0276] 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. 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.
[0277] 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. See, e.g., FIG. 13.
[0278] 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. See, e.g., FIG. 14. The first portion is infected into
bacteria, resulting in either plaques or bacterial colonies,
depending on the type of phage used. The expressed phage are
immobilized and then probed with ligands displayed in phage
selected as described below.
[0279] 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.
[0280] 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., FIG. 15.
[0281] 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 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), 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. For
example, the inventors have found that trimers of monomer domains
that bind CD28 have a higher affinity than dimmers, which in turn
have a higher affinity than single CD28-binding monomer domains
alone. 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.
[0282] 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 have an enhanced property compared to the selected
multimer, thereby producing selected altered multimers.
[0283] 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
IC.sub.50 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.
[0284] 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.
[0285] 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.
VII. Therapeutic and Prophylactic Treatment Methods
[0286] 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.
[0287] Antagonists of c-Met are useful treating cancers in which
Met is expressed. Exemplary cancers include bladder, breast,
cervical, colorectal, oesophageal, gastric, head and neck, kidney,
liver, lung, nasopharyngeal, ovarian, pancreatic, gall bladder,
prostate or thyroid cancer, osteosarcoma, synovial sarcoma,
rhabdomosarcoma, MFH/fibrosarcoma, Kaposi's sarcoma, multiple
myeloma, lymphomas, adult T-cell leukemia, glioblastomas,
astrocytomas, melanoma, mesothelioma, and Wilm's tumor.
[0288] 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.
[0289] 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, 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.
[0290] 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.
[0291] 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 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.
[0292] 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.
[0293] 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.
[0294] 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 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.
[0295] 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.
[0296] 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).
[0297] 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 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-On.TM. Gene Expression
System and Tet-Off.TM. 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.).
VIII. Additional Multimer Uses
[0298] The potential applications of multimers of the present
invention are diverse and include any use where an affinity agent
is desired.
[0299] 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. 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 albumin (e.g., human
serum albumin), IgG, red blood cells, etc. The half-life
extender-binding monomer is then covalently linked to another
monomer domain that has a binding affinity for the protein of
interest (e.g., Met). 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. 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.
[0300] The half-life extender-binding multimers are typically
multimers of at least two domains, chimeric domains, or mutagenized
domains (i.e., one that binds to Met and one that binds to the
blood-carried molecule or cell). Suitable domains include all of
those described herein, that are 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. 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.
[0301] 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: [0302] (1) immobilizing the phage or
protein mixture that was previously selected to bind the target
protein [0303] (2) contacting the target protein to the immobilized
phage or protein and washing; [0304] (3) contacting the phage or
protein mixture to the bound target and washing; and [0305] (4)
eluting the bound phage or protein without eluting the immobilized
phage or protein.
[0306] 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 embodiments, 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, monomer domain or multimer) representing the sample 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.
[0307] 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, e.g., the
.alpha. and .beta. chains of Met and/or between Met and HGF.
Optionally, the invention can generate agonists. For example,
multimers binding two different proteins, e.g., enzyme and
substrate, can enhance protein function, including, for example,
enzymatic activity and/or substrate conversion.
[0308] In some embodiments, the monomer domains are used for ligand
inhibition, ligand clearance or ligand stimulation. Possible
ligands in these methods, include, e.g., HGF.
[0309] If inhibition of ligand binding to a receptor is desired, a
monomer domain is selected that binds to the ligand (e.g., HGF) 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.
[0310] 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 by clearing the
ligand through the kidney so long as the complex is no larger than
the maximum size able to pass through the kidney (less than about
50 or 40 kD). The portion of the ligand (e.g., HGF) 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 (e.g., Met), thereby further inhibiting the
ligand's effect. This method is useful for reducing the
concentration of any molecule in the bloodstream.
[0311] 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.
[0312] 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.
[0313] 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.
[0314] Further examples of potential uses of the invention include
monomer domains, and multimers thereof, that are capable of drug
binding (e.g., binding 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 plgR for trans-mucosal
transport, and blood-brain transfer receptors such as
transferring).
[0315] 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.).
[0316] Monomers or multimers of the invention that bind to Met may
also be used in diagnostic and predictive applications in which is
is useful to detect Met. For example, detection of Met can be used
to predict prognosis of breast cancer, wherein higher abundance of
Met than in a normal tissue indicates a poor prognosis. See, e.g.,
U.S. Pat. No. 6,673,559.
IX. Further Manipulating Monomer Domains and/or Multimer Nucleic
Acids And Polypeptides
[0317] 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 herein and the
references cited therein.
[0318] 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. 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
Enzymology volume 152 Academic Press, Inc., San Diego, Calif.
(Berger); Sambrook et al., Molecular Cloning--A Laboratory Manual
(2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 1989 ("Sambrook") and Current Protocols in Molecular
Biology, 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. Pat.
No. 4,683,202; PCR Protocols A Guide to Methods and Applications
(Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990)
(Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The
Journal Of NIH Research (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. Improved methods of amplifying 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
40 kb 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.
[0319] 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, 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 transformants, 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, 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 of Animal Cells, a Manual of Basic
Technique, third edition, Wiley-Liss, New York and the references
cited therein.
[0320] 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, N.Y.; 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, Fla.
[0321] 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.
X. Animal Models
[0322] 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.
[0323] 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 LDL
receptor class A-domain family, the EGF-like domain family, as well
as the other domain families described herein.
[0324] 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, 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.
[0325] An example of a mouse model useful for screening LDL
receptor class A-domain derived binding proteins is described as
follows. Gene clusters encoding the wild type human LDL receptor
class A-domain monomers are amplified from human cells using PCR.
Almost all of the 200 different A-domains can be amplified with
only three separate PCR amplification reactions of about 7 kb each.
These fragments are then used to generate transgenic mice according
to the method described above. The transgenic mice will recognize
the human A-domains as "self", thus mimicking the "selfness" of a
human with regard to A-domains. Individual A-domain-derived
monomers or multimers are tested in these mice by injecting the
A-domain-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.
[0326] 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.
XI. Kits
[0327] 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 may contain a multimer library, or a
single type of monomer or 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.
[0328] 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.
[0329] 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.
[0330] 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.
XII. Integrated Systems
[0331] 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.
[0332] 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
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.
[0333] 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).
[0334] 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
alignments. It can also plot a tree showing the clustering
relationships used to create the alignment. PILEUP uses a
simplification of the progressive alignment 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 alignment 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.
[0335] 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 Feb. 5, 1999 (U.S. Ser. No. 60/118,854) and "Methods
For Making Character Strings, Polynucleotides & Polypeptides
Having Desired Characteristics" by Selifonov and Stemmer, filed
Oct. 12, 1999 (U.S. Ser. No. 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.
[0336] 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).
[0337] 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., 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
[0338] The following example is offered to illustrate, but not to
limit the claimed invention.
Example 1
[0339] This example describes selection of monomer domains and the
creation of multimers.
[0340] 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.
[0341] 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 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.
[0342] The selected monomer domain 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 affinity for the desired or target ligand. The
diversification and selection process is optionally repeated until
a desired affinity is obtained.
[0343] 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.
[0344] 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
[0345] This example describes the selection of monomer domains that
are capable of binding to Human Serum Albumin (HSA).
[0346] 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 as a fusions to the pIII
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 .mu.l of
the cell suspension, incubated on ice and transferred into the
cuvette (electrode gap 1 mm). After pulsing, 2 ml of SOC medium (2%
w/v tryptone, 0.5% w/v yeast extract, 10 mM NaCl, 10 mM MgSO.sub.4,
10 mM MgCl.sub.2) were added and the transformation mixture was
incubated at 37 C for 1 h. Multiple transformations were combined
and diluted in 500 ml 2xYT medium containing 201 g/m tetracycline
and 2 mM CaCl.sub.2. With 10 electroporations using a total of 10
.mu.g ligated DNA 1.2.times.10.sup.8 independent clones were
obtained.
[0347] 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.degree. C.).
The supernatant containing the phage particles was mixed with 1/5
volumes 20% w/v PEG 8000, 15% w/v NaCl, and was incubated for
several hours at 4.degree. C. After centrifugation (20 minutes,
10000 g, 4.degree. C.) the precipitated phage particles were
dissolved in 2 ml of cold TBS (50 mM Tris, 100 mM NaCl, pH 8.0)
containing 2 mM CaCl.sub.2. 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.degree. 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.degree. C.) and
removal of the supernatants, the precipitated phage particles were
dissolved in a total of 1 ml TBS containing 2 mM CaCl.sub.2. 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.
[0348] 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 .mu.l of a solution of 100 .mu.g/ml
human serum albumin (HSA, Sigma) in TBS. Binding sites remaining
after HSA incubation were saturated by incubation with 250 .mu.l 2%
w/v bovine serum albumin (BSA) in TBST (TBS with 0.1% v/v Tween 20)
for 2 hours at RT. Afterwards, 40 .mu.l of the phage solution,
containing approximately 5.times.10.sup.11 phage particles, were
mixed with 80 .mu.l TBST containing 3% BSA and 2 mM CaCl.sub.2 for
1 hour at RT. In order to remove non binding phage particles, the
wells were washed 5 times for 1 min using 130 .mu.l TBST containing
2 mM CaCl.sub.2.
[0349] Phage bound to the well surface were eluted either by
incubation for 15 minutes with 130 .mu.l 0.1 M glycine/HCl pH 2.2
or in a competitive manner by adding 130 .mu.l of 500 .mu.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 .mu.l 1 M Tris/HCl pH 8.0.
[0350] For the amplification of phage, the eluate was used to
infect E. coli K91BluKan cells (F.sup.+). 50 .mu.l of the eluted
phage solution were mixed with 50 .mu.l of a preparation of cells
and incubated for 10 minutes at RT. Afterwards, 20 ml LB medium
containing 20 .mu.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.degree. 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.
Example 3
[0351] This example describes the determination of biological
activity of monomer domains that are capable of binding to HSA.
[0352] In order to show the ability of an HSA binding domain to
extend the serum half life of a 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.
[0353] 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 used to produce protein in E. coli. After refolding and
affinity tag mediated purification the proteins were dialysed
several times against 150 mM NaCl, 5 mM Tris pH 8.0, 100 .mu.M
CaCl.sub.2 and sterile filtered (0.45 .mu.M).
[0354] Two sets of animal experiments were performed. In a first
set, 1 ml of each prepared protein solution with a concentration of
2.5 .mu.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.
[0355] 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 .mu.g
anti-His.sub.6-antibody in TBS containing 2 mM CaCl.sub.2 for 1 h
at 4 C. After blocking remaining binding sites with casein (Sigma)
solution for 1 h, wells were washed three times with TBS containing
0.1% Tween and 2 mM CaCl.sub.2. 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 .mu.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.
[0356] 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.
Example 4
[0357] This example describes experiments demonstrating extension
of half-life of proteins in blood.
[0358] 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.
[0359] The following list provides sequences of monomer domains
analyzed in this example.
[0360] Blood aliquots containing monomer protein were then added to
individual dialysis bags (25,000 MWCO), sealed, and stirred in 4 L
of Tris-buffered saline at room temperature overnight.
[0361] Anti-6.times.His 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 .alpha.-HA-HRP to detect monomer.
[0362] 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.
[0363] 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-6.times.His
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
.alpha.-HA-HRP, .alpha.-FLAG-HRP, .alpha.-ETag-HRP, and
.alpha.-myc-HRP to detect the monomer.
[0364] The following illustrates a comparison between commercial
antibodies and an anti-IgG multimer: TABLE-US-00001 Drug Mol. Wt.
Human T1/2 Dosing Rebif rIFN-b 23 kD 69 hrs Weekly 3.times. 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 2.times. Multimer
Anti-IgG 5 kD 120 hrs Weekly 1-2.times. Herceptin Her2 Antibody 150
kD 144 hrs Weekly Remicade TNFa Antibody 150 kD 216 hrs Monthly
.5.times. Humira TNFa Antibody 150 kD 336 hrs Monthly 2.times.
Example 5
[0365] This example describes in vivo intra-protein recombination
to generate libraries of greater diversity.
[0366] 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.
[0367] Reagents and Experimental Procedures
[0368] 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 P.sub.lac.
Cre was PCR-amplified from p705-cre (from GeneBridges) with
cre-specific primers that incorporated XbaI (5') and SfiI (3') at
the ends of the PCR product. This product was digested with XbaI
and SfiI and cloned into the identical sites of pCK, a bla.sup.-,
Cm.sup.R derivative of pCK110919-HC-Bla (pACYC ori), yielding
pCK-cre.
[0369] 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 EcoRI
and HinDIII-digested pCK, were hybridized together and ligated to
pCK-cre in a standard ligation reaction (T4 ligase; overnight at 16
C).
[0370] The resulting plasmid was digested with EcoRI and SphI and
ligated to the hybridized, 5'-phosphorylated oligos loxP(L) and
loxP (L_rc), which carry loxP(FAS) and EcoRI and SphI-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 CsC1 gradient centrifugation for further
purification. This construct was then digested with SphI and BglII
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.
[0371] 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(I)_rc, carrying
loxP(WT), a string of stop codons and XmaI and SfiI-compatible
overhangs, were hybridized together and ligated to XmaI- and
SfiI-digested fUSE5HA in a standard ligation reaction (New England
Biolabs T4 ligase; overnight at 16 C).
[0372] The resulting phage vector was next digested with XmaI and
SphI and ligated to the hybridized oligos loxP(J) and loxP(J)_rc,
which carry loxP(FAS) and overhangs compatible with XmaI and SphI.
This construct was digested with XmaI/SfiI and then ligated to
pre-cut (XmaI/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.
[0373] 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.
[0374] pCK-gIII. This plasmid carries gIII under the control of its
native promoter. It was constructed by PCR-amplifying gIII and its
promoter from VCSM13 helper phage (Stratagene) with primers
gIIIPromoter_EcoRI and gIIIPromoter_HinDIII. This product was
digested with EcoRI and HinDIII and cloned into the same sites of
pCK110919-HC-Bla. As gIII is under the control of its own promoter,
gIII expression is presumably constitutive. pCK-gIII was
transformed into E. coli EC100 (Epicentre).
[0375] In vivo recombination procedure. In summary, the procedure
involves the following key steps: a) Production of infective (i.e.
rescue) of fUSE5HA-Mb-lox-lox library with an E. coli host
expressing gIII from a plasmid; b) Cloning of 2.sup.nd library
(pCK) and transformation into F.sup.+ TG1 E. coli; c) Infection of
the culture carrying the 2.sup.nd library with the rescued
fUSE5HA-Mb-lox-lox phage library.
[0376] a. Rescue of phage vector. Electrocompetent cells carrying
pCK-gIII were prepared by a standard protocol. These cells had a
transformation frequency of 4.times.10.sup.8/.mu.g DNA and were
electroporated with large-scale ligations (.about.5 .mu.g vector
DNA) of fUSE5HA-lox-lox vector and the naive A domain library
insert. After individual electroporations (100 ng
DNA/electroporation) with .about.70 .mu.L cells/cuvette, 930 .mu.L
warm SOC media were added, and the cells were allowed to recover
with shaking at 37 C for 1 hour. Next, tetracycline was added to a
final concentration of 0.2 .mu.g/mL, and the cells were shaken for
.about.45 minutes at 37 C. An aliquot of this culture was removed,
10-fold serially diluted and plated to determine the resulting
library size (1.8.times.10.sup.7). The remaining culture was
diluted into 2.times.500 mL 2xYT (with 20 .mu.g/mL chloramphenicol
and 20 .mu.g/mL tetracycline to select for pCK-gIII and the
fUSE5HA-based vector, respectively) and grown overnight at 30
C.
[0377] Rescued phage were harvested using a standard PEG/NaCl
precipitation protocol. The titer was approximately
1.times.10.sup.12 transducing units/mL.
[0378] b. Cloning of the 2.sup.nd library and transformation into
an E. coli host. The ligated pCK/naive A domain library is
electroporated into a bacterial F.sup.+ host, with an expected
library size of approximately 10.sup.8. After an hour-long recovery
period at 37 C with shaking, the electroporated cells are diluted
to OD.sub.600.about.0.05 in 2.times.YT (plus 20 .mu.g/mL
chloramphenicol) and grown to mid-log phase at 37 C before
infection by fUSEHA-Mb-lox-lox.
[0379] c. Infection of the culture carrying the 2.sup.nd library
with the rescued fUSE5HA-Mb-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 TG1 (carrying an
F') and K91 (an Hfr strain) were hosts for the recombination
system. TABLE-US-00002 Oligonucleotides loxP(K) [P-5'
agcttataacttcgtatagaaaggtatatacgaagttatagatctcgtgctgcatgcggtgcg]
loxP(K_rc) [P-5'
aattcgcaccgcatgcagcacgagatctataacttcgtatatacctttctatacgaagttataagct]
loxP(L) [P-5' ataacttcgtatagcatacattatacgaagttatcgag] loxP(L_rc)
[P-5' ctcgataacttcgtataatgtatgctatacgaagttatg] loxP(I) [P-5'
ccgggagcagggcatgctaagtgagtaataagtgagtaaataacttcgtatatacctttctatacgaa-
gttatcgtctg] loxP(I)_rc [P-5'
acgataacttcgtatagaaaggtatatacgaagttatttactcacttattactcacttagcatgccct-
gctc] loxP(J) [5'
ccgggaccagtggcctctggggccataacttcgtatagcatacattatacgaagttatg]
loxP(J)_rc [5'
cataacttcgtataatgtatgctatacgaagttatggccccagaggccactggtc]
gIIIPromoter_EcoRI [5' atggcgaattctcattgtcggcgcaactat
gIIIPromoter_HinDIII [5' gataagctttcattaagactccttattacgcag]
Example 6
[0380] This example describes construction of an EGF-based monomer
library.
[0381] The CaEGF domain library, E3, encodes a protein domain of
36-43 amino acids having the following pattern: [0382]
X(5)C1-X(4/6)--C2-X(4,5)--C3-X(8)--C4-X(1)--C5-X(8/12)--C6
[0383] The table below describes for each position which amino
acids are encoded in the library based upon the natural diversity
of human calcium binding EGF domains:
[0384] The library of DNA sequences, E3, encoding monomeric calcium
binding EGF domains, was created by assembly PCR as described in
Stemmer et al., Gene 164:49-53 (1995). The oligonucleotides used in
this PCR reaction are in two groups, 1 and 2. They are:
TABLE-US-00003 Group 1: 1. 5'-AAAAGGCCTCGAGGGCCTGGGTGGCAATGGT-3' 2.
5'-CCTGAACCACCACAKHKACCGYKSNBGCACGGAYYCGRCRMACATTC
ATYAAYATCTDYACCATTGCCACCC-3' 3.
5'-CCTGAACCACCACAKNTGSCGYYGYKMHSGCACGGAYYCGRCRMACATTC
ATYAAYATCTDYACCATTGCCACCC-3' 4.
5'-CCTGAACCACCACAKHKACCGYKSNBGCAARBAYBCGVAHYCWSKBYAC
ATTCATYAAYATCTDYACCATTGCCACCC-3' 5.
5'-CCTGAACCACCACAKNTGSCGYYGYKMHSGCAARBAYBCGVAHYCWSKBY
ACATTCATYAAYATCTDYACCATTGCCACCC-3' 6.
5'-TGAATTTTCTGTATGAGGTTTTGCTAAACAACTTTCAACAGTTTCGGCCC
CAGAGGCCCTGGAGCCACCTGAACCACCACA-3' Group 2: 1.
5'-ACGGTGCCTACCCGTATGATGTTCCGGATTATGCCCCG GGTGGCAATGGT-3' 2.
5'-CCTGAACCACCACAGHKTDBACCGGHAWAGCCTKSCRSGCASHBACAK
YKAWAGCYACCCDSTRWATYTWBACCATTGCCACCC-3' 3.
5'-CCTGAACCACCACAKBYKBTKCYGKYCBSABYCNGCDBAWAGCCTK
BGBKGCASHBACAKYKAWAGCYACCCDSTRWATYTWBACCATTGCCACCC-3' 4.
5'-AAAAGGCCCCAGAGGCCCCTGAACCACCACA-3'
[0385] 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.
[0386] Following the separate PCRs of the Group 1 and 2
oligonucleotides, the Group 1 PCR fragments were digested with BpmI
and group 2 PCR fragments were digested with BsrDI. Digestion
products were purified using Qiagen Qiaquick columns and then
ligated together. The ligated DNA was then amplified in a PCR using
two primers. These are: TABLE-US-00004
5'-AAAAGGCCTCGAGGGCCTGGGTGGCAATGGT-3'
5'-AAAAGGCCCCAGAGGCCCCTGAACCACCACA-3'
[0387] The PCR products were purified with Qiagen Qiaquick columns
and digested with SfiI. The digested product was purified with
Qiagen Qiaquick columns. The DNA fragments were ligated into the
SfiI restriction sites of phage display vector fuse5-HA(G4S)4, a
derivative of fuse5 carrying an in-frame HA-epitope and a glycine,
serine flexible linker. The amino acids which comprise the flexible
linker are: S-G-G-G-G-S-G-G-G-G-S-G-G-G-G-S-G-G-G-G. The ligation
mixture was electroporated into TransforMax.TM. EC100.TM.
electrocompetent E. coli cells. Transformed E. coli cells were
grown overnight at 37.degree. C. in 2xYT medium containing 20
.mu.g/ml tetracycline. The resulting library contained
2.times.10.sup.9 independent clones. Phage particles were purified
from the culture medium by PEG-precipitation. The titer of the
phage was 1.3.times.10.sup.12/ml. The sequences of 24 individual
clones were determined and these were consistent with the library
design.
Example 7
[0388] This example describes construction of an EGF-based monomer
library.
[0389] FIG. 19 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).
For A domains, typically these clones would have been derived from
a library in which 20-25 amino acids distributed over all five
inter-cysteine segments were randomized. 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.
[0390] 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.
[0391] Any common sequence may be used as cross-over points. For A
domains or other 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.
[0392] An exemplary method of generating libraries comprised of
proteins with randomized inter-cysteine loops is presented below.
In this example, in contrast to the separate loop, separate library
approach described above, multiple intercysteine loops are
randomized simultaneously in the same library.
[0393] An A domain NNK library encoding a protein domain of 39-45
amino acids having the following pattern was constructed: [0394] C1
--X(4,6)-E1-F-R1-C2-A-X(2,4)-G1-R2-C3I-P--S1-S2-W-V-C4-D1-G2-E2-D2-D3-C5--
G3-D4-G4-S3-D5-E3-X(4,6)--C6; where, [0395] C1-C6: cysteines;
[0396] X(n): sequence of n amino acids with any residue at each
position; [0397] E1-E3: glutamine; [0398] F: phenylalanine; [0399]
R1-R2: arginine; [0400] A: alanine; [0401] G1-G4: glycine; [0402]
I: isoleucine; [0403] P: proline; [0404] S1-S3: serine; [0405] W:
tryptophan; [0406] V: valine; [0407] D1-D5: aspartic acid; and
[0408] C1-C3, C2-C5 & C4-C6 form disulfides.
[0409] 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 A1
(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 A1 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: TABLE-US-00005 1.
5'-ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKNNKNNKNNKGAATTCCGA-3' 2.
5'-ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKNNKNNKNNKNNKGAATTCCGA-3'
3.
5'-ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKNNKNNKNNKNNKNNKGAATTCCGA-
3' 4.
5'-ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTTATNNKNNKNNKGAATTCCGA-3' 5.
5'-ATATCCCGGGTCTGGAGGCGTCTGGTGGTTCGTGTNNKTATNNKNNKNNKGAATTCCGA-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'-ATACCCAAGAAGACGGTATACATCGTCCATANNNMNNTGCACATCGGAATTC-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' 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
[0410] 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 400 pmols of DNA. Pool 1
contained oligonucleotides 1-9, pool 2 contained 10-17, pool 3
contained only 18 and pool 4 contained 19-27. The fully assembled
library was obtained through an additional 8 cycles of PCR using
pool 1 and 4. The library fragments were digested with XmaI 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 TransforMax.TM. EC100.TM. electrocompetent E.
coli cells resulting in a library of 2.times.10.sup.9 individual
clones. Transformed E. coli cells were grown overnight at
37.degree. C. in 2.times.YT medium containing 20 .mu.g/ml
tetracycline. Phage particles were purified from the culture medium
by PEG-precipitation and a titer of 1.1.times.10.sup.13/ml was
determined. Sequences of 24 clones were determined and were
consistent with the expectations of the library design.
Example 8
[0411] This example describes optimization of multimers by
optimizing monomers and/or linkers for binding to a target.
[0412] FIG. 20 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., Met). 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.
[0413] 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.
[0414] 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.
[0415] 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.
Example 9
[0416] This example describes a structural analysis of A
domains.
[0417] As with virtually all proteins, only a small fraction of the
total surface of an A-domain participates in binding a single
target. Based on the solution structure of the domain, adjacent
residue positions can be identified which are likely to be able to
cooperate in binding to a given target. Herein, such groups of
adjacent residues are referred to as structural categories. As an
example, four such categories have been identified through
examination of the A-domain structure, designated Top, Bottom, Loop
1, and Loop 2. By designing libraries which only allow diversity
within a given category, the theoretical sequence space allowed by
a library can be significantly reduced, allowing for better
coverage of the theoretical space by the physical library. Further,
in the case of non-overlapping categories such as the Top and
Bottom categories, half-domain sequences selected against different
targets can be combined into a single sequence which would be able
to bind simultaneously or alternatively to the selected targets. In
either case, creating binding sites that occupy only half a domain
allows for the creation of molecules that are half as large and
would have half the number of immunogenic epitopes, reducing the
risk of immunogenicity.
Structural Classification of A-domain Positions
[0418] A canonical A-domain sequence is shown below with
high-diversity positions represented as an X. Positions that belong
to either the Top, Bottom, Loop 1, or Loop 2 categories are
designated with a star. TABLE-US-00006 2 3 4 5 6 7 8 9 10 11 12 13
14 15 16 17 18 19 20 C X X X X F X C X X X X C I X X X W X Top
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. .cndot. Bottom .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. Loop 1 .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
Loop 2 .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. 21
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 C D G X X D C X D X S
D E X X C Top .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. Bottom .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
Loop 1 .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. Loop 2 .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
.cndot. .cndot. .cndot. .cndot. .cndot. .cndot. .cndot.
Example 10
[0419] This example describes screening for monomers or multimers
that bind c-Met.
[0420] Phage libraries are panned through several rounds either on
solid support (i.e. Maxisorp plates) or in solution (i.e. Dynal
Streptavidin beads). The panning is repeated on each enriched
library several times in order to select for a pool with the
enrichment and maximal diversity.
I. Round 1 (Maxisorp Plates or Dynal Beads)
[0421] 1. Coating Target
[0422] A. Coating plates: Six wells/library are coated with c-Met
(approximately 0.5 .mu.g/well) using 100 .mu.L/well of 5 .mu.g/mL
pure c-Met diluted in TBS[pH 7.5]/2 mM CaCl.sub.2. In addition, one
negative control well/library is coated with TBS[pH 7.5]/2 mM
CaCl.sub.2 only. The plates are then incubated for 1.5 hrs. at room
temperature with shaking.
[0423] B. Coating beads: 20 .mu.L Dynal streptavidin beads (M-280)
plus 5 .mu.g biotinylated target are incubated in 500 .mu.L TBS[pH
7.5]/2 mM CaCl.sub.2 and rotated at room temperature for 1 hr. in
Eppendorf tubes. As a negative control, 20 uL Dynal streptavidin
beads without target are incubated in 500 uL of TBS[pH 7.5]/2 mM
CaCl.sub.2 and rotated at room temperature for 1 hr. Dynal beads
are typically washed at least twice with TBS [pH 7.5]/2 mM
CaCl.sub.2 before adding target and beads are coated in bulk.
[0424] 2. Blocking
[0425] A. Blocking Plates: Coating solution is removed and wells
are washed one time with 200 .mu.L/well of TBS[pH 7.5]/2 mM
CaCl.sub.2. 250 .mu.l/well of 1% BSA (protease free) in TBS[pH
7.5]/2 mM CaCl.sub.2 is added and incubated for 1 hr. at room
temperature with shaking. Alternative reagents can be used to block
if BSA contains proteins closely related to the target of interest
(for example reagents such as casein or milk).
[0426] B. Blocking e Beads: Coating solution is removed and beads
are washed twice with TBS [pH 7.5]/2 mM CaCl.sub.2; add 500 .mu.l
1% BSA (protease free) in TBS[pH 7.5]/2 mM CaCl.sub.2 and rotated
for 1 hr. at room temperature.
[0427] 3. Washes
[0428] A. Wash Plates: Wells are washed three times with 200
.mu.L/well of TBS[pH 7.5]/2 mM CaCl.sub.2 to remove excess
target.
[0429] B. Wash Beads: Beads are washed three times with 1000 .mu.L
of TBS[pH 7.5]/2 mM CaCl.sub.2 to remove excess target. Beads are
allowed to collect on a magnet for a few minutes after each wash to
avoid bead loss.
[0430] 4. Phage Addition
[0431] A. Phaze addition to Plates: About 1000 library equivalents
are added in 1% nonfat dry milk/0.2% BSA (protease free) (or
appropriate blocking agent) in TBS [pH 7.5]/2 mM CaCl.sub.2 and
incubated at room temperature for 2 hours with shaking. In rounds
2-3, 100 .mu.L total of harvested phage is added to 7 wells (6
target+1 negative control) diluted in phage addition buffer.
[0432] B. Phage addition to Beads: About 1000 library equivalents
are added in 500 .mu.l 1% non-fat dry milk+100 .mu.l 1% BSA
(protease free) in TBS [pH 7.5]/2 mM CaCl.sub.2 and incubated with
rotation at room temperature for 2 hr. In rounds 2-3, 100 .mu.L
total of harvested phage are added to beads.
[0433] 5. Washes
[0434] A. Washing Plates: The plates are washed eight times with
200 .mu.l/well of TBS [pH 7.5]/2 mM CaCl.sub.2/0.1% Tween-20 over a
period of 10 minutes.
[0435] B. Washing Beads: The beads are washed with 800 .mu.l of TBS
[pH 7.5]/2 mM CaCl.sub.2/0.1% Tween-20 over a period of 30-45 mins.
Bead resuspension is facilitated by dispensing wash buffer directly
onto collected beads or by pipetting up and down.
[0436] Conditions for Stringent Washes (Options)
[0437] a. 800 .mu.l of TBS [pH 7.5]/2 mM CaCl.sub.2/0.1% Tween-20
at 37 C;
[0438] b. 800 .mu.l of TBS [450 mM NaCl, pH 7.5]/2 mM
CaCl.sub.2/0.1% Tween-20 at room temperature;
[0439] c. Wash beads normally 6-8.times., then add unlabeled 1
.mu.g of c-Met for 1 hour at room temperature or 37 C. Phage that
survive this wash are retained for elution/infection;
[0440] d. 1% milk/0.2% BSA/with or without 1 M urea/37 C (high
stringency).
[0441] 6. Competition (Optional):
[0442] A. Competition on Plates: Phage are incubated with 100
.mu.L/well of 50 .mu.g/mL or 5 .mu.g/well (10.times. over target)
of c-Met competitor in TBS [pH 7.5]/2 mM CaCl.sub.2 for 1 hr. at
room temperature, with shaking.
[0443] B. Competition on Beads: Phage are incubated with 10 .mu.g
c-Met in 500 .mu.L TBS [pH 7.5]/2 mM CaCl.sub.2 for 1 hr. at room
temperature with shaking.
[0444] 7. Phage Elution
[0445] A. Elution off of Plates: 100 .mu.L/well of 10 mg/mL trypsin
in TBS [pH 7.5]/2 mM CaCl.sub.2 is added and incubated at 37 C for
30 min; with shaking.
[0446] B. Elution off of Beads: 100 .mu.L 10 mg/ml trypsin TBS [pH
7.5]/2 mM CaCl.sub.2 is added to beads and incubated at 37.degree.
C. for 30 min; shaking in Eppendorf rack at 37.degree. C.
[0447] C. Alternative elution/infection: 200 uL of log-phase
BlueKan K91 cells at OD600.about.0.5 are added to each well (for
plates) or to aspirated beads. The infection is allowed to proceed
for 30 minutes at 37 C without shaking. Next, the 200 uL volumes
are pooled and added to .about.3 mL of 2.times.YT/0.2 .mu.g/mL
tetracycline and shaken for 15 minutes.
[0448] 8. Infection: (Same for Plate and Bead Protocol)
[0449] An appropriate volume of BlueKan K91 (in 2xYT/40 .mu.g/mL
kanamycin) are grown to OD.sub.600 .about.0.5-0.6. When the culture
reaches this OD.sub.600, culture may be placed on ice prior to use,
although the time on ice is generally minimized.
[0450] A. In 50 mL sterile conical tube, the eluted phage is mixed
with 5 mL of BlueKan K91 culture and incubated at 37.degree. C. for
25 minutes without shaking, covering sterile conical tubes with
AirPore tape in order to allow for aeration.
[0451] B. Tetracycline is added to a final concentration of 0.2
.mu.g/mL and shaken for 15 minutes at 37.degree. C.
[0452] C. A 30 .mu.L aliquot is sampled for titering and diluted
10-fold (10.sup.-1 to 10.sup.-6) in 2.times.YT, plated in 8
.mu.L/dilution in spots on 2.times.YT/20 .mu.g/ml tetracycline
plates and incubated plates overnight at 30.degree. C. or
37.degree. C. The remaining volume of 10.sup.-2-10.sup.-4 dilutions
is plated to obtain single colonies for phage ELISAs.
[0453] D. Infected cultures are diluted .about.10-fold into 50 mL
2.times.YT/20 .mu.g/mL tetracycline and incubated with shaking at
30.degree. C. overnight to saturation.
[0454] 9. Titer sample of input phage used in the current round of
panning (same for Plate and Bead Protocol)
[0455] A. 100-fold serial dilutions of harvested phage (10.sup.-4
to 10.sup.-1) are made in 2.times.YT.
[0456] B. 100 .mu.L/well of OD.sub.600 0.5-0.6 BlueKan K91 is added
to 6 wells of 96-well polypropylene plate.
[0457] C. 10 .mu.L of diluted phage is added to wells containing
100 .mu.L of BlueKan K91 cells.
[0458] D. Phage are incubated at 37 C for 25 minutes without
shaking, with the plates covered with AirPore tape to allow for
aeration.
[0459] E. Tetracycline is added to a final concentration of 0.2
.mu.g/mL and the plate is shaen for 15 minutes at 37.degree. C.
[0460] F. 8 .mu.L of each dilution (10.sup.-4 to 10.sup.-10) is
plated onto a dry 2.times.YT agar/20 .mu.g/mL tetracycline
plate.
[0461] G. Plates are incubated at 30.degree. C. or 37.degree. C.
overnight.
[0462] 10. Harvesting Phage (Same for Plate and Bead Protocols)
[0463] A. Cultures are centrifuged overnight at 7000 rpm in
disposable 50 mL tubes for 25 min to pellet cells.
[0464] B. A standard PEG/NaCl precipitation protocol is performed
by adding 1/5 volume of a 20% PEG/15% NaCl stock to culture
supernatant; mixing and incubating on ice for 45 minutes .about.1
hour.
[0465] C. The culture is then centrifuged at 7000 rpm for 40
minutes to pellet phage and the supernatant is removed.
[0466] D. The phage pellet is resuspended in 1 mL TBS [pH 7.5]/2 mM
CaCl.sub.2 and transfered to an Eppendorf tube and centrifuged at
13 K rpm for at least 2 minutes to pellet insoluble material.
[0467] E. Supernatant is transferred to fresh tube and 1/5 volume
of PEG/NaCl is added, vortexed and incubated on ice for 5 mins.
[0468] F. The supernatant is then centrifuged at 13 K rpm for at
least 2 minutes, removed and purified phage is resuspended in up to
1 mL TBS [pH 7.5]/2 mM CaCl.sub.2 and stored at 4.degree. C.
II. Round 2 and Round 3 Panning
[0469] The 2.sup.nd and 3.sup.rd round panning conditions are
generally the same as round 1 described above, except the coated
target (i.e., c-Met) amount is decreased 2 to 4 fold for each next
round, and the plates (and beads) are washed more times for each
next round of panning.
III. Optional Intra-Domain Recombination
[0470] The final pool of monomer panning is optionally recombined
by dividing the sequences of monomers into at least two fragments
and mixing the two fragments. Two sets of oligos are used to PCR
each fragment of the monomer, and then the entire monomer is fused
by overlapping PCR and amplified. Once the fragments are fused
together and amplified the DNAs are then cut by a restriction
enzyme to allow for cloning and ligated into cut fuse5 phage
vector. Recombined monomer libraries are then panned two more
rounds.
IV. Analysis of Panning Output (Same for Plate and Bead
Protocols)
[0471] Phage ELISAs: For each output to be analyzed (typically
Rounds 2, 3 and 4, if applicable), independent clones ae inoculated
in 1 mL (2xYT/20 .mu.g/mL tetracycline) cultures grown in costar
96-well polypropylene deep-well plates, leaving inoculating tips in
and shaken overnight at 37.degree. C. Cells are pelleted by
centrifugation at 3600 rpm for 15 minutes. Culture supernatants are
retained and ELISAs are performed as discussed below.
[0472] 96-well Maxisorp plate are coated with 50 .mu.L/well of 50
.mu.g/mL (2.5 .mu.g/well). Streptavidin is diluted in TBS [pH
7.5]/2 mM CaCl.sub.2 and the plate is incubated at 37.degree. C.
for 1 hr with shaking. Plates are washed three times with 200
.mu.L/well of TBS [pH 7.5]/2 mM CaCl.sub.2. Wells are blocked with
200 .mu.L/well of 1% BSA (fraction V) and the covered plate is
incubated at RT for 1 hr with shaking. The plate is washed three
times with TBS [pH 7.5]/2 mM CaCl.sub.2. The 96-well Maxisorp plate
is coated with 100 .mu.L/well of 1 .mu.g/mL (0.1 .mu.g/well) c-Met
diluted in TBS [pH 7.5]/2 mM CaCl.sub.2 or 100 .mu.L/well buffer
only (negative control). The plate is incubated at RT for 1 hr.
with shaking. Plates are washed three times with TBS [pH 7.5]/2 mM
CaCl.sub.2. Phage is added to wells. First, 70 .mu.L of 1%
Milk/0.2% BSA/[pH 7.5]/2 mM CaCl.sub.2/0.02% Tween-20 is added to
the wells, then 30 .mu.L each of phage supernatant is added from
spun down overnight 1 mL culture to 1 positive (+SA-bn-CD40 L) well
and to 1 negative (+SA only) well. Covered plates are incubated at
RT for 1.5 hrs with shaking.
[0473] Plates are washed four times with TBS [pH 7.5]/2 mM
CaCl.sub.2/0.02% Tween-20. Note: 100 .mu.L/well of .alpha.-M13-HRP
detection antibody diluted 1:5000 in TBS [pH 7.5]/2 mM
CaCl.sub.2+0.02% Tween-20 is added. Covered plates are incubated at
4.degree. C. for 1 hr with shaking. Plates are washed three times
with cold TBS [pH 7.5]/2 mM CaCl.sub.2/0.02% Tween-20. 100
.mu.L/well of TMB/H.sub.2O.sub.2 mixture diluted 1:1 is added.
[0474] Color is allowed to turn blue until OD.sub.650 reaches
.about.1.0. The reaction is stopped with 100 .mu.L/well 2N
H.sub.2SO.sub.4. Positive wells should change in color from blue to
yellow. Once the reaction is stopped it can be read on ELISA plate
reader OD.sub.450 using SoftMaxPro software.
[0475] Selected phage are amplified overnight by infection of K91
cells. The phage are then purified by PEG/NaCl precipitation of
culture supernatants. To clone the monomer or multimer sequences
into the expression vector, pEve, approximately 10.sup.10 phage are
amplified by 20 cycles of PCR as follows: [0476] 5 ul 10.times.
Buffer [0477] 8 ul 2.5 mM dNTPs [0478] 5 ul 10 uM VS--For primer
(5'-ATCATCTGGCCGGTCCGGCCTACCCGTATGATGTTCCGGA-3') [0479] 5 ul 10 uM
EveNut primer (5'-AAAAGGCCCCAGAGGCCTTCTGCAATGAC-3') [0480] 1 ul
Phage [0481] 26 ul H.sub.2O [0482] 0.5 ul LA Taq (1 unit) TAKARA
[0483] Cycle 20.times.[94.degree. C./10 sec.-45.degree. C./30
sec.-72.degree. C./30 sec.]
[0484] The PCR is then run on a 3% Agarose gel, the approximately
200 bp product is purified with a QIAquick column (Qiagen),
digested with Sfi I (NEB), purified again with a QIAquick column
and then ligated with T4 DNA Ligase (NEB) to the Sfi I digested
vector, pEve. The ligation is used to transform electro-competent
BL21 (DE3) E. coli and plated onto 2.times.YT plates containing
Kanamycin at 40 .mu.g/ml. Following growth, approximately 6000
individual clones are picked into 1.times.YT/Kan and grown
overnight;--included on the palte are wells that will serve as
positive and negative controls.
[0485] Monomers may be purified in parallel in E. coli BL21DE3
(transformed with monomer-variants cloned in pETHC) in a parallel
manner and subsequently purified by boiling.
[0486] Optionally, the selected pEve/monomer clone plasmid DNAs are
digested in separate reactions with BsrDI and BpmI (NEB). The 10.1
kb BsrDI and 2.9 kb BpmI bands are isolated from 1% Agarose gels
and purified with QIAquick columns. The DNAs are ligated together
with T4 DNA Ligase (NEB). The ligation is purified with a QIAquick
column and amplified as described for Phage Sub-cloning. The 400 bp
product is then purified, ligated and transformed as described for
Phage Sub-cloning. Protein can be expressed and purified from these
and screened using the same methods described above.
[0487] Additional libraries referred to as "walking libraries" are
generated by ligating the selected monomers with the full
representation of random domains (generated using the same methods
used to generate the monomer libraries). These "walking libraries"
are then panned and screened as described above.
[0488] 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.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20060008844A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20060008844A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References