Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR PRODUCING AND IMPROVING THERAPEUTIC POTENCY
OF BINDING POhYPEPTIDES
BACKGROUND OF THE INVENTION
The present invention relates generally to the
treatment of disease and more specifically to binding
molecules useful as therapeutics.
Modern medicine benefits from increased
manipulation of molecular level interactions that
mediate individual diseases. This is especially the
case for treatment of disease and disease symptoms with
drug therapies. The drug development industry uses
strategies based on molecular level analysis in
attempting to develop therapeutically effective drugs.
One such strategy, used in the drug development
industry, is to identify a target molecule associated
with a disease and to produce a drug that~binds to the
target molecule to either block the target molecule's
activity or to deliver a toxic payload to the site
where the target molecule resides in the diseased
individual. Under such a strategy, the discovery phase
of research utilizes in vitro methods to identify a
lead drug candidate that binds to a target molecule.
The lead drug candidate can then be entered into the
validation phase of research where in vivo tests are
performed to determine if the lead drug candidate
demonstrates therapeutic effectiveness.
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Two commonly used discovery phase approaches
are structure based drug design and screening a pool of
candidate molecules. Structure based drug design uses
the target molecule's three dimensional structure, or
other structure-related property, as a template to
which drug candidates are fit to identify a structural
model for a lead drug candidate. The lead drug
candidate is then synthesized and tested in vitro.
Alternatively, screening uses an isolated target
molecule to select a lead drug candidate from a large
population of drug candidates in vitro. One factor in
both approaches is exploitation of the stability of the
binding interaction between the target molecule and
lead drug candidate. In this regard a large number, of
structure based design algorithms are aimed at
identifying a lead drug candidate that docks with the
target molecule to form a stable complex and a large
number of screens are designed to select lead drug
candidates that form a stable binding complex with the
target molecule.
Genomics, protein engineering and combinatorial
chemistry have been used to identify targets and
potential drug candidates that are input into the in
vitr~ methods of discovery phase research. These and
other methods may allow high throughput identification
and production of therapeutic drugs leading to
increases in both the number of disease targets and the
number of lead drug candidates.
Unfortunately, the production of therapeutic
drugs has not improved in a correlative fashion with
improvements in methods of discovery phase approaches
or the greater number and variety of discovery phase
inputs. In particular, the identified lead drug
candidates too often fail to demonstrate therapeutic
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effectiveness. Diversion of resources to an
unsuccessful drug candidate in the validation phase can
be costly because millions of dollars and numerous
years can be wasted on a failed lead drug candidate.
More importantly, those suffering from devastating
diseases are deprived of a treatment or cure.
Thus, there exists a need for a rapid and
efficient method which accurately predicts successful
lead drug candidates exhibiting therapeutic
effectiveness against a disease. The present invention
satisfies this need and provides related advantages as
well.
SUD~1ARY OF THE INVENTION
The invention provides a binding polypeptide,
or functional fragment thereof, comprising a ko" of at
least about 9 x 10' M-ls-1 for associating with a ligand
and having therapeutic potency. The invention also
provides a method of determining the therapeutic
potency of a binding polypeptide. The methods consist
of (a) contacting a binding polypeptide with a ligand;
(b) measuring association rate for binding between the
binding polypeptide and the ligand, and (c) comparing
the association rate for the binding polypeptide to an
association rate for a therapeutic control, the
relative association rate for the binding polypeptide
compared to the association rate for the therapeutic
control indicating that the binding polypeptide will
exhibit a difference in therapeutic potency correlative
with the difference between the association rates.
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DETAINED DESCRIPTTON OF THE INVENTION
The invention is directed to the discovery that
the therapeutic potency of a molecule correlates with
the rate at which the therapeutic molecule associates
with a ligand that mediates or correlates with a
pathological condition. The invention provides a
binding polypeptide, or functional fragment thereof,
having a kon of at least about 9 x 10' M'ls-1 for
associating with a ligand and having therapeutic
potency. The invention further provides a grafted
antibody, or functional fragment thereof, having a kon
of at least about 1.3 x 106 M'ls'1 to a ligand and having
therapeutic potency.
In one embodiment the methods of the invention
allow for accurate in vitro prediction and
identification of molecules having therapeutic potency.
In one embodiment the methods involve determining the
therapeutic potency of a binding polypeptide by
comparing the association rate for the binding
polypeptide to an association rate for a therapeutic
control. The binding polypeptide will exhibit a
difference in therapeutic potency correlative with the
difference between said association rates. In another
embodiment, the methods of the invention involve
identifying therapeutic potency of a binding
polypeptide by identifying a binding polypeptide
exhibiting a high association rate or ko", correlating
with its therapeutic potency. The methods of the
invention can also be used to change the structure and
ligand binding activity of a parent polypeptide to
create one or more progeny polypeptides, and to
identify progeny polypeptides that are binding
polypeptides having improved therapeutic potency
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resulting from increased association rate with a
ligand.
An advantage of the invention is that a binding
polypeptide having improved therapeutic potency can be
5 distinguished from a binding polypeptide that has an
increased Ka for a ligand but not improved therapeutic
potency. A further advantage of the methods of the
invention is that a means to screen large numbers of
potential therapeutic molecules in vitro is provided,
thereby increasing the rate and efficiency of
identifying effective therapeutics while reducing the
costs associated with in vivo testing of failed
therapeutics.
As used herein, the term "binding polypeptide"
refers to a polymer of amino acids that selectively
associates with a ligand. A binding polypeptide can
have, for example, at least 2, 5, 8, 10, 12, 15, 20,
25, 50, 100, 200 or 400 or more amino acids so long as
the polypeptide retains ability to associate with a
ligand. Therefore, the term binding polypeptide, as
used herein, includes all sizes of amino acid polymers
ranging from a couple to hundreds or even thousands of
amino acids.
A binding polypeptide can be a naturally
occurring polypeptide, for example, a receptor, enzyme
or hormone. A receptor can include, for example, an
immunoglobulin, such as an antibody or T cell receptor;
integrin; hormone receptor; lectin; membrane receptor
or transmitter receptor. An enzyme can include, for
example, a protease, oxidoreductase, kinase, lipase,
phosphatase, DNA modifying enzyme, polymerase, caspase,
transcription factor, GTPase, ATPase, or a membrane
channel. A hormone can include for example, a growth
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factor, insulin, cytokine, neural peptide,
extracellular matrix protein or clotting factor. A
binding polypeptide can be a modified form of a
naturally occurring polypeptide, for example, a
fragment, chimera containing amino acids from a donor
polypeptide, or fusion of fragments from one or more
donor polypeptides so long as such polypeptide retains
ability to associate with a ligand.
A binding polypeptide can be a polypeptide that
contains a non-naturally occurring moiety including,
for example, an amino acid derivative, sterioisomer of
an amino acid, amino acid analogue or functional
mimetic of an amino acid. For example, a derivativea
can include a chemical modification of the polypeptide
such as alkylation, acylation, carbamylation,
iodination, or any modification which derivatizes the
polypeptide. An analogue can include a modified amino
acid, for example, hydroxyproline or carboxyglutamate,
and can include an amino acid that is not linked by a
peptide bond. Mimetics encompass a molecule containing
a chemical moiety that mimics the function of the
polypeptide regardless of a difference in three-
dimensional structure between the binding polypeptide
and mimetic. For example, if a polypeptide contains
two charged chemical moieties in a functional domain, a
mimetic can place two charged chemical moieties in a
spatial orientation and constrained structure so that
the relative location of the charged chemical moieties
is maintained in three-dimensional space independent of
any other differences between the polypeptide and
mimetic.
As used herein, the term "ligand" refers to a
small molecule, compound or macromolecule that can
selectively associate with a binding polypeptide. A
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ligand can be a naturally occurring molecule, compound
or macromolecule including, for example, DNA, RNA,
polypeptide, lipid, carbohydrate, amino acid,
nucleotide or hormone. A ligand can be a derivative of
a naturally occurring molecule, compound or
macromolecule resulting in, for example, an added
moiety, a removed moiety or a rearrangement in the
relative location of moieties. Examples of added
moieties include, for example, a biotin, peptide such
as polyhistidine, radioisotope or chemically reactive
group capable of forming a covalent bond to a second
molecule. A ligand can be a mimetic of naturally
occurring molecule, compound or macromolecule.
Mimetics encompass molecules containing chemical
moieties that mimic the function of the ligand
regardless of differences between three-dimensional
structure of the mimetic and the ligand. A mimetic can
be, for example, a synthetically prepared molecule or a
polypeptide containing a modified form of a naturally
occurring amino acid. A ligand can be an antigen found
on a cell such as a cancer cell, microbe, bacteria,
fungus or virus. A ligand can also be a molecule.that
is a toxic substance.
As used herein, the term "parent polypeptide"
refers to a polymer of amino acids that can be changed
to produce a binding polypeptide. Therefore, a parent
polypeptide is the molecule to be improved using the
methods of the invention. As used herein a parent
polypeptide can have, for example, at least 2, 5, 8,
10, 12, 15, 20, 25, 50, 100, 200 or 400 or more amino
acids. Therefore, the term parent polypeptide, as used
herein, includes all sizes of amino acid polymers
ranging from a couple to hundreds or even thousands of
amino acids.
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A parent polypeptide can be a naturally
occurring polypeptide, for example, a receptor, enzyme
or hormone such as those described above in reference
to a binding polypeptide. A parent polypeptide can be
a polypeptide that contains a non-naturally occurring
moiety including, for example, an amino acid
derivative, a sterioisomers of an amino acid, an amino
acid analogue or a functional mimetic of an amino acid
such as those described above in reference to a binding
polypeptide.
As used herein the term "progeny polypeptide"
refers to a polymer of amino acids that has different
structure compared to the parent polypeptide from which
it was produced. A different structure can include,
for example, addition, deletion, substitution or
chemical modification of one or more amino acids. A
progeny polypeptide can be a different species from the
parent polypeptide. A progeny polypeptide can
associate with a ligand at the same or different
association rate compared to the association rate at
which its parent polypeptide associates with the same
ligand. As used herein a progeny polypeptide can have,
for example, at least 2, 5, 8, 10, 12, 15, 20, 25, 50,
100, 200 or 400 or more amino acids. Therefore, the
term progeny polypeptide, as used herein, includes all
sizes of amino acid polymers ranging from a couple to
hundreds or even thousands of amino acids.
A progeny polypeptide can be a modified form of
a parent polypeptide, for example, a fragment, chimera
containing amino acids from a donor polypeptide, or
fusion of fragments from one or more donor polypeptide.
A progeny polypeptide can be a polypeptide that
contains a non-naturally occurring moiety including,
for example, an amino acid derivative, sterioisomer of
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an amino acid, amino acid analogue or functional
mimetic of an amino acid such as those described above.
As used herein, the term "grafted" when used in
reference to an antibody, or functional fragment
thereof, refers to an antibody, or functional fragment
thereof, having a variable region acceptor framework
from one species containing one or more CDR from a
donor or second species. One skilled in the art will
know that the function of an antibody, or functional
fragment thereof, can be influenced by a change in a
single CDR or more preferably in multiple CDRs. Amino
acids can be added, deleted or substituted at any
position in the acceptor framework or donor CDRs and
can include, for example, changes that modify structure
or function of the grafted antibody, or functional
fragment thereof, whether minor or significant so long
as the antibody, or functional fragment thereof,
contains a variable region acceptor framework from one
species and at least one CDR from another species.
Description of grafted antibodies and methods for their
production are well known in the art and are described,
for example, in U.S. Patent No. 5,225,539; "Protein
Engineering of Antibody Molecules for Prophylactic and
Therapeutic Applications in Man," Clark, M. (ed.),
Nottingham, England: Academic Titles (1993); Winter
and Harris, Immunol. Today, 14:243-246 (1993) Winter
and Harris, Tips, 14:139-143 (1993) and Couto et al.
Cancer Res., 55:1717-1722 (1995) which are incorporated
herein by reference.
As used herein, the term "functional fragment,"
when used in reference to a binding polypeptide, is
intended to refer to a portion of a binding polypeptide
which retains the ability to selectively associate with
a ligand. Functional fragments can include dissociated
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subunits of a binding polypeptide, for example,
individual heavy or light chains of an antibody.
Functional fragments can include portions of a binding
polypeptide having a reduced number of amino acids, for
5 example, Fd, Fab or F(ab)~ portions of an antibody.
Functional fragments can include portions of a
dissociated subunits of a binding polypeptide having a
-reduced number of amino acids including, for example,
Fv, VH, a CDR, or scFv portions of an antibody. Such
10 terms are described in, for example, Harlow and Zane,
Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York (1989); Molec. Biology and
Biotechnology: A Comprehensive Desk Reference (Myers,
R.A. (ed.), New York: VCH Publisher, Inc.); Huston et
al., Cell Biophysics, 22:189-224 (1993); Pliickthun and
Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day,
E.D., Advanced Immunochemistry, Second Ed., Wiley-hiss,
Inc., New York, NY (1990), which are incorporated
herein by reference. Thus, a functional fragment can
include an immunologically active portion, fragment or
segment of an antibody.
A functional fragment of a binding polypeptide
can have minor structural differences in comparison to
a full length binding polypeptide so long as the
fragment has about the same structure as the
corresponding region of the full length binding
polypeptide and retains the ability to selectively
associate with a ligand.. Minor structural differences
can be at the primary, secondary, tertiary, or
quaternary sequence level. Structural differences at
the primary sequence level include changes in the amino
acid sequence and can be, for example, additions,
deletions or substitutions of amino acids or chemical
modifications of amino acids, such as addition of a
chemical moiety, so long as such a polypeptide retains
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the ability to associate with a ligand. An added
moiety can include, for example, a chemically
derivatized amino acid, D-stereoisomer of an amino
acid, non-naturally occurring amino acid, amino acid
analogue or a mimetic of an amino acid. Structural
differences between a binding polypeptide and
functional fragment thereof at the secondary level
including, for example, a change in alpha helix, loop
or beta sheet structure can occur so long as the
resulting functional fragment retains the ability to
associate with a ligand. A functional fragment of a
binding polypeptide can also have a structural
difference at the tertiary level including, for
example, a change in the relative location of a
secondary structure element or change in overall fold
of the binding polypeptide. Structural differences at
the quaternary level can include, for example, a change
in the number of subunits in a binding polypeptide or a
change in the interfaces at which subunits in a binding
polypeptide interact so long as the functional fragment
retains the ability to associate with a ligand.
As used herein, the term "complimentarity
determining region" or "CDR" is intended to mean a
non-contiguous antigen combining site found within the
variable region of either a heavy or light chain
polypeptides of an immunoglobulin. The term CDR region
is well known in the art and has been defined by Kabat
et al., U.S. Dept. of Health and Human Services,
"Sequences of Proteins of Immunological Interest"
(1983) and by Chothia et al., J. Mol. Biol. 196:901-917
(1987) and additionally by MacCallum et al., J. Mol.
Biol. 262:732-745 (1996), which are incorporated herein
by reference, and include overlapping or subsets of
amino acid residues when compared against each other.
Application of any of the above three definitions to
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refer to a CDR of an antibody, or functional fragment
thereof, is intended to be within the scope of the term
as defined and used herein. The appropriate amino acid
residues which encompass the CDRs, as defined by each
of the above cited references, are set forth below in
Table 1 as a comparison. The exact residue numbers
which encompass a particular CDR will vary depending on
the sequence and size of the CDR. Those skilled in the
art can routinely determine which residues comprise a
particular CDR given the variable region amino acid
sequence of the antibody.
TABLE 1: CDR Definitions
Kabatl Chothia2 MacCallum3
VH CDR1 31-35 26-32 30-35
CDR2 50-65 52-56 47-58
VH
VH CDR3 95-102 95-102 93-101
VL CDR1 24-34 24-34 30-36
Vz CDR2 50-56 50=56 46-55
VL CDR3 89-97 89-97 89-96
~ Residue numbering follows the nomenclature of Kabat
et al . , supra
2 Residue numbering follows the nomenclature of Chothia
et al . , supra
Residue numbering follows the nomenclature of
MacCallum et al., supra
As used herein, the term "association rate"
refers to the time in which binding polypeptide and
ligand become bound to form a complex. Use of the term
herein is intended to be consistent with the meaning of
the term as it is known in the art. The association
rate can be correlated with the time dependent
appearance of a species composed of binding polypeptide
bound to ligand, the time dependent disappearance of
free binding polypeptide or the time dependent
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disappearance of free ligand in a mixture of species
including binding polypeptide and ligand. Free binding
polypeptide species refers to a binding polypeptide
that is competent to bind at least one ligand and free
ligand refers to a ligand that is competent to bind at
least one binding polypeptide. The scope of the term
association rate is intended to include kon. The
association rate is known in the art to be proportional
to kon and proportional to the product of Ka and koff.
As used herein, the term "associating," when
used in reference to a binding polypeptide and ligand,
is intended to refer to the process by which a binding
polypeptide and ligand contact each other in a manner
that results in the species of binding polypeptide
bound to ligand. Use of the term associating is
intended to be consistent. with the meaning of the term
as it is known in the art. The process is different
from and can be distinguished by those skilled in the
art from the reverse process by which the complex of
binding polypeptide bound to ligand dissociates to
yield free binding polypeptide and free ligand.
As used herein, the term "kon" refers to the
association rate constant equating the association rate
with the concentration of the free binding polypeptide
and free ligand. The term ko" is intended to be
consistent with the meaning of the term as it is known
in the art. Therefore, kon is a quantitative measure
of association rate. For example, when binding
polypeptide A and ligand B associate to form the bound
species AB, the association rate will equal the ko"
multiplied by the product of the concentration of free
binding polypeptide A multiplied by the concentration
of free ligand B. A mathematical equation describing
this relationship is: association rate = kon*[A]*[B]
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where [A] is the concentration of polypeptide A and [B]
is the concentration of ligand B.
As used herein, the term "Ka" refers to the
association constant and is intended to be consistent
with the meaning of the term as it is understood in the
art. The Ka is a measure of the strength, affinity and
tightness of binding. Specifically Ka is an
equilibrium constant equating the concentrations of
free binding polypeptide, free ligand and binding
polypeptide bound to ligand occurring at equilibrium.
The Ka can be used to compare the affinity of different
binding polypeptides for various ligands at
equilibrium. For example, a binding polypeptide with a
higher numerical value of Ka for binding a ligand
compared to the Ka for a second binding polypeptide
binding the same ligand is understood in the art to
have higher affinity for that ligand. The Ka relates
the association rate constant (ko") and the
dissociation rate constant (koff) according to the
relationship Ka = kon/koff. The koff 1S the mathematical
constant used in the art to quantitate the time for an
associated binding polypeptide and ligand to separate.
Accordingly, kon is the product of Ka and koff. The
mathematical inverse of Ka is known in the art as the
Kd or dissociation constant. Therefore, Kd = 1/Ka=
koff/kon ~ Thus, a binding polypeptide with a lower
numerical value of Kd for binding a ligand compared to
the Kd for a second binding polypeptide binding the
same ligand is understood in the art to have higher
affinity for that ligand.
As used herein, the term "therapeutic potency"
is intended to refer to a predictive measure of
efficacy or relative efficacy. If a binding
polypeptide has therapeutic potency it produces a
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desired therapeutic effect. Therapeutic potency
includes a kinetic property and is proportional to the
association rate for a binding polypeptide associating
with a ligand. As such, the term reflects the effect
5 of expeditious association between a binding
polypeptide and ligand that cures, alleviates, removes
or lessens the symptoms of, or prevents or reduces the
possibility of contracting a pathological condition. A
binding polypeptide having an increased kon when
10 associating with a ligand will display more expeditious
association with a ligand thereby having improved
therapeutic potency compared to a parent polypeptide or
other polypeptide having a lower ko" when associating
with the same ligand.
15 As used herein, the term "therapeutic control"
refers to a molecule to which a binding polypeptide can
be compared, when determining or identifying therapeutic
potency and which is related to a pathological
condition to which the binding polypeptide is targeted.
A molecule can be related to a pathological condition,
for example, by having demonstrated efficacy in
treating the pathology, having demonstrated interaction
with a ligand associated with a pathological condition,
or having properties identified in the art as holding
promise for treating a pathology. The scope of the
term is intended to include all molecules independent
of structural similarity or difference compared to the
binding polypeptide so long as both can bind the same
ligand. The molecule can be, for example, a naturally
occurring molecule, a synthetic molecule, compound or
macromolecule.
As used herein the term "changing" when used in
reference to a parent polypeptide refers to modifying
the structure of the parent polypeptide. Modification
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of the structure of a parent polypeptide can include,
for example, adding a moiety, deleting an amino acid,
substituting an amino acid or chemically modifying an
amino acid. A moiety that can be substituted includes,
for example, a chemically derivatized amino acid,
D-stereoisomer of an amino acid, non-naturally
occurring amino acid, amino acid analogue or mimetic of
an amino acid. A chemical modification of an amino
acid includes, for example, a covalent change in the
bonding structure of an amino group at the alpha
position, lysine, histidine, arginine, or tryptophan;
covalent change in the bonding structure of a carbonyl
at the alpha position, aspartate or glutamate or
covalent change in the bonding structure of a sulfur at
cysteine, cystine or methionine.
As used herein, the term "measuring," when used
in reference to an association rate, refers to a
determination correlating the appearance of a species
composed of a binding polypeptide bound to ligand with
at least one defined time interval. Therefore, the
term encompasses determination of an amount of time or
rate at which a binding polypeptide binds to a ligand.
Determination of association rate is meaningful when
performed in a non-equilibrium state. Non-equilibrium
states include, for example, pre-equilibrium, which can
occur following mixture of free ligand with free
binding polypeptide and post-equilibrium, which can
occur following altering the concentration of species
in an equilibrated mixture. Post-equilibrium
determination of association rate includes, for example
determination of koff and using the value to calculate
kon from Ka or Kd measured for the binding polypeptide
and ligand.
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Pre-equilibrium determination of association
rate includes a relative determination, quantitative
determination or time based selection. A relative
determination includes a method involving comparing
rates of association for two binding molecules under
similar conditions such that quantitation of individual
rates is not necessary. A quantitative determination
includes a method for determining numerical value for
an association rate or a rate constant such as ko". A
time based selection includes, for example, exploiting
a change in a property of a ligand or binding
polypeptide that occurs when a binding polypeptide
associates with ligand so as to select the bound
species at a specified time interval.
A determination correlating the appearance of a
species composed of a binding polypeptide bound to
ligand involves a time dependent change, from a first
state to a second state, for any property that changes
when the binding polypeptide associates with ligand
including, for example, absorption and emission of
heat, absorption and emission of electromagnetic
radiation, refractive index of surrounding solvent,
affinity for a receptor, molecular weight, density,
electric charge, polarity, molecular shape, or
molecular size. A property that changes when a binding
polypeptide associates with ligand can be transient,
returning to the first state while the binding
polypeptide is bound to ligand, or can remain in the
second state the entire time that the binding
polypeptide and ligand are bound.
As used herein, the team "identifying," when
used in reference to a binding polypeptide with an
increased association rate, refers to recognizing a
binding polypeptide as having an increased association
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rate. A binding polypeptide having increased
association rate can be recognized prior to being
isolated from a population, after being isolated from a
population or the process of isolating the binding
polypeptide from a population can be a form of
recognizing a binding polypeptide with an increased
association rate. A binding polypeptide having
increased association rate can be recognized by
comparing the association rate or kon value with an
association rate or kon value for another binding
molecule or by selecting a binding polypeptide based on
a more rapid association rate. As such, recognizing a
binding polypeptide with an improved association rate
or kon can involve manual methods or automated methods.
As used herein the term "pathological
condition" refers to a disease or abnormal condition
including, for example, an injury of a mammalian cell
or tissue. A pathological condition can be a disease
or abnormal condition that results in unwanted or
abnormal cell growth, viability or proliferation. A
pathological condition characterized by unwanted or
abnormal cell growth includes, for example, cancer or
other neoplastic condition, infectious disease or
autoimmune disease. For example, cancer cells
proliferate in an unregulated manner and consequently
result in tissue destruction. Similarly, the
proliferation of cells mediating autoimmune diseases
are aberrantly regulated which results in, for example,
the continued, proliferation and activation of immune
mechanisms with destruction of the host's cells and
tissue. Specific examples of cancer include prostate,
breast, lung, ovary, uterus, brain and skin cancer.
Specific examples of infectious diseases include DNA or
RNA viral diseases, bacterial diseases, parasitic
diseases whereas autoimmune diseases include, for
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example, diabetes, rheumatoid arthritis and multiple
sclerosis.
The invention provides a binding polypeptide,
or functional fragment thereof, having a kon of at
least about 9 x 10' M-1s-1 for associating with a ligand
and having therapeutic potency.
A binding polypeptide having therapeutic
potency will demonstrate a therapeutic effect and
exhibit expeditious association with a ligand to cure,
alleviate, remove or lessen the symptoms of, or prevent
or reduce the possibility of contracting a pathological
condition. A binding polypeptide of the invention
having therapeutic potency is understood to be a high
potency binding polypeptide. Therapeutic potency can
be identified in vitro according. to a kinetic property,
specifically, the association rate for binding
polypeptide associating with a ligand. A binding
polypeptide having therapeutic potency can be, for
example, a binding polypeptide that prevents or reduces
a pathological condition by associating with a ligand
and preventing its binding to a receptor that is
localized on a cell surface. A binding polypeptide
having an increased association rate when associating
with a ligand will have improved therapeutic potency
compared to a polypeptide, including a binding
polypeptide, that has a lower association rate when
associating with the same ligand. Therefore,
association rate indicates, and correlates with,
therapeutic potency and, as such, provides a predictive
measure of efficacy or relative efficacy.
A binding polypeptide can also be, for example,
attached to a cytotoxic or cytostatic agent so as to
deliver the agent to a cell experiencing a pathological
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condition by associating with a ligand localized on the
surface of the cell. A binding polypeptide attached to
a cytotoxic or cytostatic agent having an increased
association rate when associating with the ligand will
5 have improved therapeutic potency compared to a
polypeptide that has a lower association rate when
associating with the same ligand.
A binding polypeptide of the invention will be
identified according to its ability to selectively
10 associate with a ligand. Selective binding between a
binding polypeptide and a ligand can be identified by
methods known in the art. Methods of determining
selective binding include, for example, equilibrium
binding analysis, competition assays, and kinetic
15 assays as described in Segel, Enzyme Kinetics John
Wiley and Sons, New York (1975), which is incorporated
herein by reference. Thermodynamic constants can be
used to identify and compare binding polypeptides and
ligands that selectively bind each other and include,
20 for example, dissociation constant or Kd, association
constant or Ka and Michaelis co-nstant or Km.
A binding polypeptide that can be used in the
methods of the invention includes any polypeptide known
to bind a ligand, made to bind a ligand, or known to be
capable of binding a ligand. Therefore, a binding
polypeptide of the invention can be selected from the
group consisting of a receptor, enzyme, hormone,
immunoglobulin, antibody, humanized antibody, human
antibody, T-cell receptor, integrin, hormone receptor,
lectin, membrane receptor, transmitter receptor,
protease, oxidoreductase, kinase, phosphatase, DNA
modifying enzyme, transcription factor, GTPase, ATPase,
membrane channel, growth factor, insulin, cytokine,
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21
neural peptide, extracellular matrix protein and
clotting factor, or functional fragments thereof.
A binding polypeptide can be a naturally or
non-naturally occurring polypeptide. A naturally
occurring binding polypeptide can be obtained, for
example, from a native tissue by directly isolating the
polypeptide or by isolating the nucleotide encoding the
polypeptide and expressing the polypeptide in a
recombinant system. One skilled in the art can isolate
the nucleotide encoding the polypeptide and express the
polypeptide in a recombinant expression system
according to methods known in the art as described, for
example, in Goeddel, Methods in Enzymoloqy, Vol 185,
Academic Press, San Diego (1990); Wu, Methods in
Enzvmoloay, Vol 217, Academic Press, San Diego (1993);
Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York (1992),
and in Ausebel et al., Current protocols in Molecular
Biology, John Wiley and Sons, Baltimore, MD (2000),
which~are incorporated herein by reference. Methods of
isolation of a parent polypeptide from recombinant and
native tissues are well known in the art and are
described, for example, in Scopes, Protein
Purification: Principles and Practice, 3rd Ed.,
Springer-Verlag, New York (1994); Duetscher, Methods in
Enzymology, Vol 182, Academic Press, San Diego (1990),
and Coligan et al., Current,protocols in Protein
Science, John Wiley and Sons, Baltimore, MD (2000),
which are incorporated herein by reference.
A naturally occurring binding polypeptide can
be, for example, synthesized or produced in a
recombinant expression system. For example, a binding
polypeptide can be identified from a polypeptide
sequence or a sequence of a nucleotide encoding a
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22
polypeptide isolated from a natural source or the
nucleotide or polypeptide sequence can be obtained from
a sequence data base including, for example, GenBank or
other databases known in the art. Methods for
isolating and sequencing nucleotides and polypeptides
are well known in the art and are described, for
example, in Sambrook et al., supra and in Ausubel et
al., supra. A binding polypeptide can be expressed in
a recombinant system using methods well known in the
art including, for example, those described herein
below. A binding polypeptide can also be produced by
synthetic methods well known in the art, for example,
Merrifield solid phase synthesis, t-Boc based
synthesis, Fmoc synthesis and variations thereof.
A binding polypeptide of the invention can be
non-naturally occurring. A non-naturally occurring
polypeptide can be selected, for example, from a
randomized population of polypeptides. A randomized
population of non-naturally occurring polypeptides can
be produced by peptide synthesis methods that are well
known in the art including, for example, those
described above. Methods of selecting a parent
polypeptide from a population of polypeptides will be
specific to the parent polypeptide to be selected, and
can be achieved using methods well known by one skilled
in the art based on the physical and chemical
properties of the polypeptide.
A binding polypeptide of the invention can be a
naturally occurring or non-naturally occurring
polypeptide that is modified for use in the methods of
the invention. A modification to facilitate use of a
binding polypeptide in the methods of the invention can
include, for example, incorporation of a label for
detection of the polypeptide, incorporation of a
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23
binding group for capture of a binding polypeptide or
modification to increase stability of the polypeptide.
A label that can be incorporated includes, for example,
a fluorophore, chromophore, paramagnetic spin label, or
radionucleotide. A binding group that can be used to
capture a polypeptide includes, for example, a biotin,
polyhistidine tag (Qiagen; Chatsworth, CA), antibody
epitope such as the flag peptide (Sigma; St Louis, MO),
glutathione-S-transferase (Amersham Pharmacia;
Piscataway, NJ), cellulose binding domain (Novagen;
Madison, WI), calmodulin (Stratagene; San Diego, CA),
staphylococcus protein A (Pharmacia; Uppsala, Sweden),
maltose binding protein (New England BioLabs; Beverley,
MA) or strep-tag (Genosys; Woodlands, TX) or minor
modifications thereof. A modifications to increase
stability can include, for example, incorporation of a
cysteine to form a thioether crosslink, removal of a
protease recognition sequence, addition of a charged
amino acid to promote ionic interactions, or addition
of a hydrophobic amino acid to promote hydrophobic
interactions. The methods of the invention can
accommodate other modifications that can confer
additional properties onto the binding polypeptide of
the invention so long as such modifications do not
inhibit binding activity of the binding polypeptide.
Examples include, addition of amino acids, deletion of
amino acids, substitution of amino acids, chemical
modification of amino acids and incorporation of non-
natural amino acids.
A binding polypeptide of the invention is
intended to include minor structural modifications that
do not significantly change binding activity. For
example, homologs or isotypes of a binding polypeptide
can be isolated or synthesized that have minor
structural modifications and similar binding activity
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24
when compared to the binding polypeptide and are
included in the scope of a binding polypeptide of the
invention. One skilled in the art can identify
homologs or isotypes, for example, by aligning the
sequences with an algorithm such as BLAST (Altschul et
al., J. Mol. Biol. 215:403-410 (1990)), WU-BLAST2
(Altschull and Gish, Meth. Enzymol. 266:460-480
(1996)), FASTA (Pearson, Meth. Enzymol. 266:227-258
(1996)), or SSEARCH (Pearson, supra) to identify
regions of structural homology. One skilled in the art
can also identify homologues or isotypes using an
algorithm that compares polypeptide structure
including, for example, SLOP, CATH, or FSSP which are
reviewed in Hadley and Jones Structure 7:1099-1112
(1999). The publications cited to reference sequence
and structural alignment algorithms are incorporated
herein. Site directed mutagenesis methods including,
for example, those described herein, can be used to
make the appropriate changes to modify homologous
polypeptides to have similar association rate and
therapeutic potency as a binding polypeptide of the
invention. Differences between the homologous binding
polypeptides having an insignificant effect on
association rate and, therefore, therapeutic potency
are considered to be minor modifications. For example,
a second antibody from a second species can be modified
to have similar association rate when associating with
a ligand when compared to a first antibody from a first
species that was produced or used in the methods of the
invention.
Minor modifications that do not significantly
change binding activity include, for example, a change
made in a region of a binding polypeptide that does not
affect the function of a region of the binding
polypeptide that contacts ligand, conservative
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substitution of one or more amino acids that does not
affect interactions between a binding polypeptide and
ligand, and substitution of a functionally equivalent
amino acid. A change made in the region that does not
5 affect the function of a region of the binding
polypeptide that contacts a ligand can include, for
example, addition of one or more amino acid, addition
of one or more moiety, deletion of one or more amino
acid, substitution of one or more amino acid or
10 chemical modification of one or more amino acid. A
minor modification can be conservative substitution of
an amino acid. Conservative substitutions of encoded
amino acids can include, for example, amino acids which
belong within the following groups: (1) non-polar amino
15 acids (Gly, Ala, Val, Leu, and Ile); (2) polar neutral
amino acids (Cys, Met, Ser, Thr, Asn, and Gln); (3)
polar acidic amino acids (Asp and Glu); (4) polar basic
amino acids (Lys, Arg and His); and (5) aromatic amino
acids (Phe, Trp, Tyr, and His). Other minor
20 modifications are included so long as the binding
polypeptide retains binding activity. The substitution
of functionally equivalent amino acids is routine and
can be accomplished by methods known to those skilled
in the art. Briefly, the substitution of functionally
25 equivalent amino acids can be made by identifying the
amino acids which are desired to be changed,
incorporating the changes into the encoding nucleic
acid and then determining the function of the
recombinantly expressed and modified binding
polypeptide.
The invention also provides a grafted antibody,
or functional fragment thereof, having a kon of at
least about 1.3 x 106 M-1 s-1 to a ligand and having
therapeutic potency.
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26
In one embodiment of the invention the binding
polypeptide having therapeutic potency or high potency
can be a grafted antibody. Grafted antibodies and
methods for making grafted antibodies have been
described herein previously. Accordingly, an antibody
or functional fragment thereof can have human constant
regions, or a heavy or light chain framework region at
least a part of which is derived from one or more human
antibody. A heavy or light chain framework regions
used in an antibody or fragment can be derived from a
particular antibody or from a consensus sequence of
human antibodies. A grafted antibody having
therapeutic potency can be produced or identified by
the methods of the invention. An antibody or
functional fragment thereof of the invention can be an
antibody other than vitaxin.
The invention also provides a human antibody,
or functional fragment thereof, comprising a kon of at
least about 9 x 10' M-ls-1 to a ligand and having
therapeutic potency. Methods for identifying and
producing human antibodies are well known in the art
including, for example, those described in Harlow and
Lane, supra.
An antibody or immunoglobulin of the invention
can be a neutralizing antibody or neutralizing
immunoglobulin. The term "neutralizing" refers to the
ability to reduce the replication of microorganisms or
viruses in an organism or in a cell that is cultured.
Thus, an antibody or functional fragment thereof having
therapeutic potency can have specificity for an
antigenic determinant found on a microbe such as a
virus, bacteria or fungus. Examples of viruses to
which an antibody or fragment thereof can have
specificity are respiratory syncytial virus or
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parainfluenza virus. A neutralizing antibody or
neutralizing immunoglobulin the invention including
active fragments thereof can be specific for at least
one protein expressed by a virus such as RSV or PIV. A
protein expressed by the RSV can be the F protein.
The invention provides a method of determining
the therapeutic potency of a binding polypeptide. The
methods consist of (a) contacting a binding polypeptide
with a ligand; (b) measuring association rate for
binding between the binding polypeptide and the ligand,
and (c) comparing the association rate for the binding
polypeptide to an association rate for a therapeutic
control, the relative association rate for the binding
polypeptide compared to the association rate for the
therapeutic control indicating that the binding
polypeptide will exhibit a difference in therapeutic
potency correlative with the difference between the
association rates. The invention further provides a
method where the association rate is indicated by kon.
For example, the ko" for a binding polypeptide of the
invention can be at least about 8 x 106 M-ls-1. A
binding polypeptide of the invention can also have a
kon of at least about 9 x 106 M-ls-1, 1 x 10' M-'s-1, 2 x
10' M-ls-1, 3 x 10' M-ls-', 4 x 10' M-ls-1, 5 x 10' M-ls-'-, 6 x
10' M-ls-1, 7 x 10' M"ls-1, 8 x 10' M-1s"1, 9 x 10' M-1s'1, or
1 x 108 M-ls-'- or higher. Binding polypeptides having
lower kon can also have therapeutic potency. For
example, a therapeutically potent polypeptide can have
kon less than 8 x 106 M-ls-1. Thus, binding polypeptides
having kon of 1 x 106 M-~s-1 can have therapeutic potency
as can polypeptides having ko" of
1 x 10~ M-ls-1. Accordingly, therapeutically potent
polypeptides can have ko" values of about 5 x 104 M-ls-1,
1 x 105 M-ls-1, 2. 5 x 105 M-1 sec-1, 5 x 105 M-1s-1 or 7 . 5 x
105 M-1 sec-1.
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An association rate can be determined in any
non-equilibrium mixture including, for example, one
formed by rapidly contacting a binding polypeptide and
ligand or by rapidly changing temperature. A non-
equilibrium mixture can be a pre-equilibrium mixture.
A pre-equilibrium mixture can be formed, for example,
by contacting a soluble binding polypeptide and soluble
ligand in a condition where the amount of total ligand
and total binding polypeptide in the detection chamber
are constant. Measurements of association rates in
pre-equilibrium mixtures can be made in formats
providing rapid mixing of binding polypeptide with
ligand and rapid detection of changing properties of
the binding polypeptide or ligand on a timescale of
milliseconds or faster. Stopped flow and rapid quench
flow instruments such as those described below provide
a convenient means to measure non-equilibrium kinetics.
The association rate can also be measured in non-
equilibrium mixtures including, for example, solutions
containing insoluble species of binding polypeptide,
ligand or binding polypeptide bound to ligand, or
solutions containing variable concentrations of total
ligand or total binding polypeptide. Measurement of an
association rate in a non-equilibrium mixture can be
made in formats providing attachment of a ligand to a
surface and continuous flow of a solution containing
the binding polypeptide over the surface, or vice-
versa, combined with rapid detection of changing
properties of the binding polypeptide, ligand or
surface such that measurements are made on a timescale
of milliseconds or faster. Examples of formats
providing non-equilibrium measurement of association
rates include surface plasmon resonance instruments and
evanescent wave instruments as described below.
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Binding polypeptides and ligands to be
contacted in mixtures for determination of association
rate can be attached to another molecule, ligand or
surface so long as they are capable of binding with
their ligand or binding polypeptide partner
respectively. Molecules that can be attached to a
binding polypeptide or ligand include, for example,
labels and binding groups such as those described
herein previously for incorporation into binding
polypeptide. Attached ligands can include, for
example, an inhibitor that is competitively displaced
when binding occurs between binding polypeptide and
ligand, a second ligand that binds to the binding
polypeptide such that binding can occur between the
binding polypeptide and ligand of interest, or a second
binding polypeptide that binds to ligand such that
binding can occur between the binding polypeptide of
interest and the ligand. Attached surfaces can
include, for example, a dextran surface, polymer bead,
biological membrane, or any biosensor surface.
Association rate measurements can be made by
detecting the change in a property of the binding
polypeptide or ligand that exists between the bound and
unbound state or by detecting a change in the
surrounding environment when binding polypeptide and
ligand associate. Properties of the binding
polypeptide or ligand that can change upon association
and that can be used to measure association rates
include, for example, absorption and emission of heat,
absorption and emission of electromagnetic radiation,
affinity for a receptor, molecular weight, density,
mass, electric charge, conductivity, magnetic moment of
nuclei, spin state of electrons, polarity, molecular
shape, or molecular size. Properties of the surrounding
environment that can change when binding polypeptide
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associates with ligand include, for example,
temperature and refractive index of surrounding
solvent.
Formats for measuring association rates in pre-
y equilibrium mixtures include, for example, stopped flow
kinetic instruments and rapid quench flow instruments.
A stopped flow instrument can be used to push solutions
containing a binding polypeptide and ligand from
separate reservoirs into a mixing chamber just prior to
10 passage into a detection cell. The instrument can then
detect a change in one or more of the above described
properties to monitor progress of the binding event. A
rapid quench flow instrument can be used to rapidly mix
a solution containing a binding polypeptide with a
15 solution containing a ligand followed by quenching the
binding reaction after a finite amount of time. A
change in one or more of the above described properties
can then be detected for quenched mixtures produced by
quenching at different times following mixing.
20 Quenching can be performed for example by freezing or
addition of a chemical quenching agent so long as the
quenching step does not inhibit detection of the
property relied upon for measurement of binding rate.
Thus, a rapid quench instrument can be useful, for
25 example, in situations where spectroscopic detection is
not convenient. A variety of instruments are
commercially available from vendors such as KinTek
Corp. (State College, PA) and Hi-Tech Scientific
(Salisbury, UK).
30 Formats for measuring association rates in non-
equilibrium mixtures include, for example, surface
plasmon resonance and evanescent wave instruments.
Surface plasmon resonance and evanescent wave
technology utilize a ligand or binding polypeptide
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attached to a biosensor surface and a solution
containing either the binding polypeptide or ligand
respectively that is passed over the biosensor surface.
The change in refractive index of the solution that
occurs at the surface of a chip when binding
polypeptide associates with ligand can be measured in a
time dependent fashion. For example, surface plasmon
resonance is based on the phenomenon which occurs when
surface plasmon waves are excited at a metal/liquid
interface. Light is directed at, and reflected from,
the side of the surface not in contact with sample, and
SPR causes a reduction in the reflected light intensity
at a specific combination of angle and wavelength.
Biomolecular binding events cause changes in the
refractive index at the surface layer, which are
detected as changes in the SPR signal. The binding
event can be either binding association or
disassociation between a receptor-ligand pair. The
changes in refractive index can be measured essentially
instantaneously and therefore allows for determination
of the individual components of an affinity constant.
More specifically, the method enables accurate
measurements of association rates (kon) and
disassociation rates (koff). Surface plasmon resonance
instruments are available in the art including, for
example, the BIAcore instrument, IBIS system, SPR-
CELLIA system, Spreeta, and Plasmon SPR and evanescent
wave technology is available in the Iasys system as
described, for example, in Rich and Myszka, Curr. Opin.
Biotech. 11:54-61 (2000).
The association rate can be determined by
measuring a change in a property of a ligand or binding
polypeptide at one or more discreet time intervals
during the binding event using, for example, the
methods described above. Measurements determined at
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discreet time intervals during the binding event can be
used to determine a quantitative measure of association
rate or a relative measure of association rate.
Quantitative measures of association rate can include,
for example, an association rate value or ko" value.
Quanti ative values of association rate or ko" can be
determined from a mathematical or graphical analysis of
a time dependent measurement. Such analyses are well
known in the art and include algorithms for fitting
data to a sum of exponential or linear terms or
algorithms for computer simulation to fit data to a
binding model as described for example in Johnson, Cur.
Opin. Biotech. 9:87-89 (1998), which is incorporated
herein by reference.
Association rates can be determined from
mixtures containing insoluble species or variable
concentrations of total ligand or total binding
polypeptide using mathematical and graphical analyses
such as those described above if effects of mass
transport are accounted for in the reaction. One
skilled in the art can account for mass transport by
comparing association rates under conditions having
similar limitations with respect to mass transport or
by adjusting the calculated association rate according
to models available in the art including, for example
those described in Myszka et al., Biophys. J. 75:583-
594 (1998), which is incorporated herein by reference.
A higher value of either the association rate
or kon is indicative of improved therapeutic potency.
Thus, quantitative determinations provide an advantage
by allowing comparison between an association rate of a
binding polypeptide and a therapeutic control
determined by different methods so long as the methods
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used are understood by one skilled in the art to yield
consistent results.
A relative measure of association rate can
include, for example, comparison of association rate
for two or more binding polypeptides binding to ligand
under similar conditions or comparison of association
rate for a binding polypeptide binding to ligand with a
predefined rate. Comparison of association rate for
two or more binding polypeptides can include a standard
of known association rate or a molecule of known
therapeutic effect. A predefined rate used for
comparison can be determined by calibrating the
measurement to be relative to a previously measured
rate including, for example, one available in the
scientific literature or in a database. An example of
a comparison with a predefined rate is selection of the
species of binding polypeptide bound to ligand at a
discreet time interval defined by the predefined rate
by using a time actuated selection device.
An advantage of the invention is that the
methods can be used with any ligand that mediates or
specifically correlates with a pathological condition.
The methods can also be used with a structurally
modified adduct of a ligand that mediates or
specifically correlates with a pathological condition,
or a ligand that mimics binding function of a ligand
that mediates or specifically correlates with a
pathological condition. Structural modifications can
facilitate use of a ligand in the methods of the
invention and can include, for example, incorporation
of labels for detection of the ligand, incorporation of
binding groups for capture of the ligand or
modifications to increase stability of the ligand.
Labels, binding groups and modifications to increase
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34
stability include, for example, those described herein
previously for incorporation into polypeptides. It can
also be advantageous to use a mimic of the ligand to
bias the binding interaction with respect to a subset
of physical interactions that influence its functional
association with a binding polypeptide. Physical
interactions that allow a ligand and binding
polypeptide to associate include, for example, hydrogen
bonds, ionic forces, van der Waals interactions or
hydrophobic interactions or a combination thereof.
A ligand used with the methods of the invention
can be synthesized or isolated from a natural source by
a variety of methods known in the art. Synthetic
methods for synthesizing a ligand include, for example,
organic synthesis, cell free synthesis using extracted
cellular components, and chemical synthesis. A ligand
that is a polypeptide or nucleic acid can be
synthesized, for example, in a recombinant expression
system using methods similar to those described below.
Additionally, a ligand can be produced in.a recombinant
organism modified to express one or more enzymes that
convert a host intermediate or exogenously supplied
intermediate into the ligand. Isolation of a ligand
from a natural source can be performed by methods known
in the art. For example, a polypeptide.or nucleic acid
based ligand can be isolated as described herein for a
parent polypeptide or binding polypeptide and their
encoding nucleic acids. Small molecule ligands can be
isolated according to methods known in the art
including, for example, extraction, chromatography,
crystallization or distillation. Methods of isolating
small molecules can be found, for example, in Gordon
and Ford, The Chemist's Companion, John Wiley and Sons
(1973) and Vogel, Voc~el's Textbook of Practical Organic.
Chemistry, 5t'' Ed., Addison-Wesley Pub. Co. (1989).
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Binding polypeptides having improved
therapeutic potency can be determined or identified by
comparing an association rate for binding between a
binding polypeptide and ligand with an association rate
5 for a therapeutic control binding to the ligand. Since
the therapeutic potency of the therapeutic control is
correlated with its association rate for associating
with a ligand, the therapeutic control provides a means
of determining therapeutic potency according to
10 association rates measured in vitro.
A therapeutic control can be any molecule so
long as the molecule associates with the same ligand as
the binding polypeptide to be compared. The
therapeutic control of the invention can include, for
15 example, a receptor, enzyme, hormone, immunoglobulin,
antibody, humanized antibody, human antibody, T-cell
receptor, integrin, hormone receptor, lectin, membrane
receptor, transmitter receptor, protease,
oxidoreductase, kinase, phosphatase, DNA modifying
20 enzyme, transcription factor, GTPase, ATPase, membrane
channel, growth factor, insulin, cytokine, neural
peptide, extracellular matrix protein, clotting factor,
or functional fragments thereof.
For purposes of comparison, the association
25 rate of a binding polypeptide and ligand can be
determined relative to association rate for a
therapeutic control and the same ligand. A comparison
can also be made according to a quantitative
association rate for binding polypeptide and ligand
30 compared to a quantitative association rate for a
therapeutic control and ligand. Relative or
quantitative association rates can be determined by the
methods described above. Determination of association
rates for a binding polypeptide associating with a
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36
ligand can be performed simultaneously with a binding
polypeptide and therapeutic control or at separate
times provided conditions are sufficiently similar in
each assay to allow valid comparison. Thus,
association rate determined for a binding polypeptide
by the methods of the invention can be compared to a
previously measured association rate for a therapeutic
control.
The invention provides a method of determining
the therapeutic potency of a binding polypeptide. The
method consists of (a) contacting a binding polypeptide
with a ligand; (b) measuring association rate for
binding between the binding polypeptide and the ligand;
(c) comparing the association rate for the binding
polypeptide to an association rate for a therapeutic
control, the relative association rate for the binding
polypeptide compared to the association rate for the
therapeutic control indicating that the binding
polypeptide will exhibit a difference in therapeutic
potency correlative with the difference between the
association rates, and (d) changing one or more amino
acids in the binding polypeptide and repeating steps
(a) through (c) one or more times. In addition, steps
(a) through (d) can be repeated one or more times and
stopped at step (c). Increased association rate
correlates with improved therapeutic potency where
increases in association rate can be at least 4-fold,
5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold or
more.
Steps (a) through (c), as recited above, can be
performed according to methods described herein
previously for determining therapeutic potency of a
binding polypeptide by measuring association rate.
Step (d), recited above, provides an advantage in
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allowing one skilled in the art to use the methods of
the invention to change the therapeutic potency of a
binding polypeptide by changing the binding polypeptide
and identifying a difference in therapeutic potency of
the changed binding polypeptide from an association
rate. Therapeutic potency of a binding polypeptide can
be altered by changing the binding polypeptide to have
an increased or decreased association rate when binding
a ligand or to have an increased association rate when
binding a new ligand. A binding polypeptide changed by
the methods of the invention to have improved
therapeutic potency by binding to a new ligand can have
substantially unaltered association rate for the
original ligand or can have an increase or decrease in
association rate for the original ligand. Binding of a
new ligand to a changed binding polypeptide can be
competitive with binding of the original ligand, non-
competitive with binding of the original ligand, or
allosteric with binding of the original ligand.
Competitive, non competitive and allosteric binding of
two ligands to a binding polypeptide can be recognized
by methods available in the art as described in Segel,
supra .
Amino acids to be changed in a polypeptide in
order to change therapeutic potency can be incorporated
randomly or incorporated based on knowledge of the
interactions between the binding polypeptide and
ligand. Random incorporation includes, for example,
incorporating each of the twenty naturally occurring
amino acid residues, or a subset thereof, at one or
more defined position or incorporating each of the
twenty naturally occurring amino acid residues, or a
subset thereof, at random positions in the polypeptide
or portion thereof. For example, a portion of a
polypeptide can be randomly changed to incorporate all
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20 natural amino acids or a subset thereof. As an
example of changing random sites in a polypeptide, a
polypeptide can be randomly mutated along its entire
sequence by incorporating all 20 natural amino acids or
a subset thereof.
Knowledge of interactions between a binding
polypeptide and ligand can be used to guide site
directed changes, to bias random changes, or to produce
biased changes. For example, if residues of the
original binding polypeptide are known to interact with
a ligand these residues can be altered to accommodate
or invoke interactions with a second ligand at the same
site. Knowledge of the interactions between binding
polypeptide and ligand can include, for example,
identification of residues in the binding polypeptide
that interact with the ligand, identification of
residues that affect the structure or function of the
binding polypeptide binding site or identification of
residues that are proximal to the binding polypeptide
ligand binding site. Such interactions can be derived
from information on the structure and function of the
binding polypeptide, ligand or binding polypeptide
bound to ligand.
Structure and function information can be used
to identify interactions between a binding polypeptide
and ligand. For example, interactions can be
identified from a structural model, amino acid
sequence, functional binding data, or identification of
sites or regions labeled with reagents that selectively
modify amino acids of a binding polypeptide.
Structural models of a binding polypeptide can be
derived from, for example, X-ray crystallography,
nuclear magnetic resonance spectroscopy, electron
microscopy, atomic force microscopy, X-ray scattering
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or neutron scattering. A structural model can include
structure of a binding polypeptide, structure of a
ligand or structures of both a binding polypeptide and
bound ligand. Molecular modeling can be used in
conjunction with a structural model to identify
potential interactions between a binding polypeptide
and ligand.
The amino acid sequence of a binding
polypeptide or ligand can be used, for example, to
determine binding residues according to homology with
other binding polypeptides and ligands. For example,
amino acids to be changed in a first binding
polypeptide can be chosen based on homology to amino
acids known to interact with a ligand in a second
polypeptide. Again molecular modeling can be used in
conjunction with a homology search to model a putative
structure for the binding polypeptide or ligand thereby
allowing identification of potential interacting amino
acids.
Functional binding studies with modified
binding polypeptides can be useful in identifying
regions to change in the methods of the invention. For
example, a change in binding activity that correlates
with a change in an amino acid of a binding polypeptide
can indicate that the changed amino acid position
potentially interacts with a ligand.
The size of a population of polypeptides
produced from a randomly changed polypeptide can be
minimized by introducing a bias into random mutagenesis
methods. A bias can be introduced with respect to the
particular amino acids to be incorporated, with respect
to the amino acid sites at which a polypeptide is
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changed, or with respect to both the particular amino
acid to be incorporated and the site of incorporation.
A bias can also be introduced into the
randomization at a specified position based on
5 conservative substitutions. Conservative substitutions
of amino acids include, for example, (1) non-polar
amino acids (Gly, Ala, Val, Leu and Ile); (2) polar
neutral amino acids (Cys, Met, Ser, Thr, Asn and Gln);
(3) polar acidic amino acids (Asp and Glu); (4) polar
10 basic amino acids (Lys, Arg and His); and (5) aromatic
amino acids (Phe, Tyr, Trp and His). Additionally,
conservative substitutions of amino acids include, for
example, substitutions based on the frequencies of
amino acid changes between corresponding proteins of
15 homologous organisms as described, for example, in
Principles of Protein Structure, Schulz and Schirmer,
eds., Springer Verlag, New York (1979) which is
incorporated herein by reference.
A subset of residues for randomization within a
20 polypeptide can be chosen based on properties of the
polypeptide. For example, biased mutagenesis of
proteases, protease inhibitors, immunoglobulins, DNA
binding polypeptides and RNA binding polypeptides is
described in Methods in Enzymology 267:52-68 (1996),
25 biased mutagenesis of streptavidin is described in Voss
and Skerra, Prot. EncL 10:975-982 (1997), biased
mutagenesis of binding polypeptides having a lipocalin
fold is described in Beste et al. Proc Natl. Acad. Sci.
USA 96:1898-1903 (1999), biased mutagenesis of growth
30 hormones is described in Ballinger et al., J. Biol.
Chem. 273:11675-11684 (1998) and biased mutagenesis of
an antibody is described in Wu et al., Proc. Natl.
Acad. Sci. USA 95:6037-6042 (1998).
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Random mutagenesis and biased mutagenesis
methods can produce changes at one or more selected
positions without altering the remaining amino acid
positions within a region. For example, a population
of single position changes can contain varied amino
acid residues at each position, incorporated either
randomly or with a biased frequency, while leaving the
remaining positions unchanged. For the specific
example of a ten residue region, a population can
contain species having the first, second and third,
continued through the tenth residue, independently
randomized or represented by a biased frequency of
incorporated amino acid residues while keeping the
remaining positions unchanged. For the specific
example described above, these non-varied positions
would correspond to positions 2-10; 1,3-10; 1,2,4-10,
continued through positions 1-9, respectively.
Therefore, the resultant population will contain
species that represent all single position changes.
Similarly, double, triple quadruple or more
amino acid position changes can be generated within a
region of a polypeptide without altering the remaining
amino acid positions. For example, a population of
double position changes will contain at each set of two
positions the varied amino acid residues while leaving
the remaining positions as unchanged residues. The
sets will correspond to, for example, positions 1 and
2, 1 and 3, 1 and 4, and continued pairwise through the
region until the last set corresponds to the first and
last positions of the region. The population will also
contain sets corresponding to positions 2 and 3, 2 and
4, 2 and 5, through the set corresponding to the second
and last position of the region. Similarly, the
population will contain sets of double position changes
corresponding to all pairs of position changes
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beginning with position three of the region. Similar
pairs of position changes are made with the remaining
sets of amino acid positions. Therefore, the
population will contain species that represent all
pairwise combinations of amino acid position changes.
In a similar fashion, populations corresponding to sets
of changes representing all triple and quadruplet
changes along a region can similarly be targeted using
the methods of the invention.
Because the methods of the invention can employ
the production and screening of diverse populations of
polypeptides, effects on association rate, such as the
neutralization or augmentation of inherently
detrimental changes and the neutralization or
augmentation of beneficial amino acid changes, can
occur due to the combined interactions of two or more
amino acid changes within a single polypeptide. No
prior information is required to assess which amino
acid positions or changes can be inherently beneficial
or detrimental, or which positions or changes can be
further augmented by second site changes. Instead, by
selecting amino acid positions or subsets thereof and
generating a diverse population containing amino acid
variants at these positions, combinations of beneficial
changes occurring at the selected positions will be
identified by screening for increased or optimized
association rate. Such beneficial combinations will
include the unveiling of inherently beneficial residues
and neutralization of inherently detrimental residues.
Methods for efficient synthesis and expression
of populations of changed polypeptides synthesized
using oligonucleotide-directed mutagenesis can be
performed, for example, as previously described in Wu
et al. supra: Wu et al., J. Mol. Biol., 294:151-162
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43
(1999) and Kunkel, Proc. Natl. Acad. Sci. USA,
82:488-492 (1985) which are incorporated herein by
reference. Oligonucleotide-directed mutagenesis is a
well known and efficient procedure for systematically
introducing mutations, independent of their phenotype
and is, therefore, suited for directed evolution
approaches to protein engineering. The methodology is
flexible, permitting precise mutations to be introduced
without the use of restriction enzymes, and is
relatively inexpensive. Briefly, to perform
oligonucleotide directed mutagenesis, a population of
oligonucleotides encoding the desired mutations) is
hybridized to single-stranded uracil-containing
template of the wild type sequence, double-stranded
circular DNA is generated by a polymerase and a ligase,
and the mutant DNA is efficiently recovered following
transformation of a duct ungf bacterial strain which
can not replicate the uracil containing wild-type
template.
Populations of changed polypeptides can also be
generated using gene shuffling. Gene shuffling or DNA
shuffling is a method for directed evolution that
generates diversity by recombination as described, for
example, in Stemmer, Proc. Natl. Acad. Sci. USA
91:10747-10751 (1994); Stemmer, Nature 370:389-391
(1994); Crameri et al., Nature 391:288-291 (1998)
Stemmer et al., U.S. Patent No. 5,830,721, which are
incorporated herein by reference. Gene shuffling or
DNA shuffling is a method using in vitro homologous
recombination of pools of selected mutant genes. For
example, a pool of point mutants of a particular gene
can be used. The genes are randomly fragmented, for
example, using DNase, and reassembled by PCR. If
desired, DNA shuffling can be carried out using
homologous genes from different organisms to generate
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44
diversity (Crameri et al., supra, 1998). The
fragmentation and reassembly can be carried out, for
example, in multiple rounds, if desired. The resulting
reassembled genes are a population of variants that can
be used in the invention.
Simultaneous incorporation of all of the
encoding nucleic acids and all of the selected amino
acid position changes can be accomplished by a variety
of methods known to those skilled in the art, including
for example, recombinant and chemical synthesis.
Simultaneous incorporation can be accomplished by, for
example, chemically synthesizing the nucleotide
sequence for the region and incorporating at the
positions selected for harboring variable amino acid
residues a plurality of corresponding amino acid
codons.
One method well known in the art for rapidly
and efficiently producing a large number of alterations
in a known amino acid sequence or for generating a
diverse population of variable or random sequences is
known as codon-based synthesis or mutagenesis. This
method is the subject matter of U,.S. Patent Nos.
5,264,563 and 5,523,388 and is also described in Glaser
et al. J. Immunoloay 149:3903 (1992), all of which are
incorporated herein by reference. Briefly, coupling
reactions for the randomization of, for example, all
twenty codons which specify the amino acids of the
genetic code are performed in separate reaction vessels
and randomization for a particular codon position
occurs by mixing the products of each of the reaction
vessels. Following mixing, the randomized reaction
products corresponding to codons encoding an equal
mixture of all twenty amino acids are then divided into
separate reaction vessels for the synthesis of each
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randomized codon at the next position. For the
synthesis of equal frequencies of all twenty amino
acids, up to two codons can be synthesized in each
reaction vessel.
5 Variations to this synthesis method also exist
and include, for example, the synthesis of
predetermined codons at desired positions and the
biased synthesis of a predetermined sequence at one or
more codon positions. Biased synthesis involves the
10 use of two reaction vessels where the predetermined or
parent codon is synthesized in one vessel and the
random codon sequence is synthesized in the second
vessel. The second vessel can be divided into multiple
reaction vessels such as that described above for the
15 synthesis of codons specifying totally random amino
acids at a particular position. Alternatively, a
population of degenerate codons can be synthesized in
the second reaction vessel such as through the coupling
of NNG/T nucleotides where N is a mixture of all four
20 nucleotides. Following synthesis of the predetermined
and random codons, the reaction products in each of the
two reaction vessels are mixed and then redivided into
an additional two vessels for synthesis at the next
codon position.
25 A modification to the above-described
codon-based synthesis for producing a diverse number of
changed sequences can similarly be employed for the
production of changed polypeptide populations described
herein. This modification is based on the two vessel
30 method described above which biases synthesis toward
the parent sequence and allows the user to separate the
variants into populations containing a specified number
of codon positions that have random codon changes.
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46
Briefly, this synthesis is performed by
continuing to divide the reaction vessels after the
synthesis of each codon position into two new vessels.
After the division, the reaction products from each
consecutive pair of reaction vessels, starting with the
second vessel, is mixed. This mixing brings together
the reaction products having the same number of codon
positions with random changes. Synthesis proceeds by
then dividing the products of the first and last vessel
and the newly mixed products from each consecutive pair
of reaction vessels and redividing into two new
vessels. In one of the new vessels, the parent codon
is synthesized and in the second vessel, the random
codon is synthesized. For example, synthesis at the
first codon position entails synthesis of the parent
codon in one reaction vessel and synthesis of a random
codon in the second reaction vessel. For synthesis at
the second codon position, each of the first two
reaction vessels is divided into two vessels yielding
two pairs of vessels. For each pair, a parent codon is
synthesized in one of the vessels and a random codon is
synthesized in the second vessel. When arranged
,linearly, the reaction products in the second and third
vessels are mixed to bring together those products
having random codon sequences at single codon
positions. This mixing also reduces the product
populations to three, which are the starting
populations for the next round of synthesis.
Similarly, for the third, fourth and each remaining
position, each reaction product population for the
preceding position are divided and a parent and random
codon synthesized.
Following the above modification of codon-based
synthesis, populations containing random codon changes
at one, two, three and four positions as well as others
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47
can be conveniently separated out and used based on the
need of the individual. Moreover, this synthesis
scheme also allows enrichment of the populations for
the randomized sequences over the parent sequence since
the vessel containing only the parent sequence
synthesis is similarly separated out from the random
codon synthesis.
Other methods well known in the art for
producing a large number of alterations in a known
amino acid sequence or for generating a diverse
population of variable or random sequences include, for
example, degenerate or partially degenerate
oligonucleotide synthesis. Codons specifying equal
mixtures of all four nucleotide monomers, represented
as NNN, results in degenerate synthesis. Whereas
partially degenerate synthesis can be accomplished
using, for example, the NNG/T codon described
previously. Other methods well known in the art can
alternatively be used such as the use of statistically
predetermined, or variegated, codon synthesis which is
the subject matter of U.S. Patent Nos. 5,223,409 and
5,403,484, which are incorporated herein by reference.
Once the populations of changed polypeptides
encoding nucleic acids have been constructed as
described above, they can be expressed to generate a
population of changed polypeptides that can be screened
for association rate. For example, the nucleic acids
encoding the changed polypeptides can be cloned into an
appropriate vector for propagation, manipulation and
expression. Such vectors are known or can be
constructed by those skilled in the art and should
contain all expression elements sufficient for the
transcription, translation, regulation, and if desired,
sorting and secretion of the altered polypeptide or
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48
polypeptides. The vectors also can be for use in
either procaryotic or eukaryotic host systems so long
as the expression and regulatory elements are of
compatible origin. The expression vectors can
additionally included regulatory elements for inducible
or cell type-specific expression. One skilled in the
art will know which host systems are compatible with a
particular vector and which regulatory or functional
elements are sufficient to achieve expression of a
polypeptide in soluble, secreted or cell surface forms.
Suitable expression vectors are well-known in
the art and include vectors capable of expressing
nucleic acid operatively linked to a regulatory
sequence or element such as a promoter region or
enhancer region that is capable of regulating
expression of such nucleic acid. Promoters or
enhancers, depending upon the nature of the regulation,
can be constitutive or inducible. The reaulatorv
sequences or regulatory elements are operatively linked
to a nucleic acid of the invention or population of
changed nucleic acids as described above in an
appropriate orientation to allow transcription of the
nucleic acid.
Appropriate expression vectors include those
that are replicable in eukaryotic cells and/or
prokaryotic cells and those that remain episomal or
those which integrate into the host cell genome.
Suitable vectors for expression in prokaryotic or
eukaryotic cells are well known to those skilled in the
art as described, for example, in Ausubel et al.,
supra. Vectors useful for expression in eukaryotic
cells can include, for example, regulatory elements
including the SV40 early promoter, the cytomegalovirus
(CMV) promoter, the mouse mammary tumor virus (MMTV)
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49
steroid-inducible promoter, Moloney murine leukemia
virus (MMLV) promoter, and the like. A vector useful
in the methods of the invention can include, for
example, viral vectors such as a bacteriophage, a
baculovirus or a retrovirus; cosmids or plasmids; and,
particularly for cloning large nucleic acid molecules,
bacterial artificial chromosome vectors (BACs) and
yeast artificial chromosome vectors (YACs). Such
vectors are commercially available, and their uses are
well known in the art. One skilled in the art will
know or can readily determine an appropriate promoter
_ for expression in a particular host cell.
Appropriate host cells, include for example,
bacteria and corresponding bacteriophage expression
systems, yeast, avian, insect and mammalian cells and
compatible expression systems known in the art
corresponding to each host species. Methods for
recombinant expression of populations of progeny
polypeptides or progeny polypeptides within such
populations in various host systems are well known in
the art and are described, for example, in Sambrook et
al., supra and in Ansubel et al., supra. The choice of
a particular vector and host system for expression and
screening of progeny polypeptides will be known by
those skilled in the art and will depend on the
preference of the user. Expression of diverse
populations of hetereomeric receptors in either soluble
or cell surface form using filamentous bacteriophage
vector/host systems is well known in the art and is the
subject matter of U.S. Patent No. 5,871,974 which are
incorporated herein by reference.
The recombinant cells are generated by
introducing into a host cell a vector or population of
vectors containing a nucleic acid molecule encoding a
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binding polypeptide. The recombinant cells are
transducted, transfected or otherwise genetically
modified by any of a variety of methods known in the
art to incorporate exogenous nucleic acids into a cell
5 or its genome. Exemplary host cells that can be used
to express a binding polypeptide include mammalian
primary cells; established mammalian cell lines, such
as COS, CHO, HeLa, NIH3T3, HEK 293 and PC12 cells;
amphibian cells, such as Xenopus embryos and oocytes;
10 and other vertebrate cells. Exemplary host cells also
include insect cells such as Drosophila, yeast cells
such as Saccharomyces cerevisiae, Saccharomyces pombe,
or Pichia pastor.is, and prokaryotic cells such as
Escherichia coli.
15 In one embodiment, a nucleic acids encoding a
polypeptide can be delivered into mammalian cells,
either in v.ivo or in vitro using suitable vectors well-
known in the art. Suitable vectors for delivering a
nucleic acid encoding a polypeptide to a mammalian
20 cell, include viral vectors such as retroviral vectors,
adenovirus, adeno-associated virus, lentivirus,
herpesvirus, as well as non-viral vectors such as
plasmid vectors.
Viral based systems provide the advantage of
25 being able to introduce relatively high levels of the
heterologous nucleic acid into a variety of cells.
Suitable viral vectors for introducing a nucleic acid
encoding a polypeptide into mammalian cells are well
known in the art. These viral vectors include, for
30 example, Herpes simplex virus vectors (teller et al.,
Science, 241:1667-1669 (1988)); vaccinia virus vectors
(Piccini et al., Meth. EnzymolocLy, 153:545-563 (1987));
cytomegalovirus vectors (Mocarski et al., in Viral
Vectors, Y. Gluzman and S.H. Hughes, Eds., Cold Spring
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51
Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp.
78-84)); Moloney murine leukemia virus vectors (Danos
et al., Proc. Natl. Acad. Sci. USA, 85:6460-6464
(1988); Blaese et al., Science, 270:475-479 (1995);
Onodera et al., J. Virol., 72:1769-1774 (1998));
adenovirus vectors (Berkner, Biotechniaues, 6:616-626
(1988); Cotten et al., Proc. Natl. Acad. Sci. USA,
89:6094-6098 (1992); Graham et al., Meth. Mol. Biol.,
7:109-127 (1991); Li et al., Human Gene Therapy, 4:403-
409 (1993); Zabner et al., Nature Genetics, 6:75-83
(1994)); adeno-associated virus vectors (Goldman et
al., Human Gene Therapy. 10:2261-2268 (1997); Greelish
et al., Nature Med., 5:439-443 (1999); Wang et al.,
Proc. Natl. Acad. Sci. USA, 96:3906-3910 (1999); Snyder
et al., Nature Med., 5:64-70 (1999); Herzog et al.,
Nature Med., 5:56-63 (1999)); retrovirus vectors
(Donahue et al., Nature Med., 4:181-186 (1998);
Shackleford et al., Proc. Natl. Acad. Sci. USA,
85:9655-9659 (1988); U.S. Patent Nos. 4,405,712,
4,650,764 and 5,252,479, and WIPO publications WO
92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO
92/14829; and lentivirus vectors (Kafri et al., Nature
Genetics, 17:314-317 (1997)). The above publications
describing vectors or their use are incorporated herein
by reference.
In addition to mutagenesis methods described
above, a polypeptide can be changed by chemical
modifications. Chemical modifications can be made to
change the binding properties of a polypeptide, to
benefit measurement of association rates or to benefit
identification of a binding polypeptide. A chemical
modification of an amino acid includes, for example,
modification of amino groups by amidination,
guanidination, reductive methylation, carbamylation,
acetylation, trinitrobenzoylation, succinylation or
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52
formylation; modification of arginine by butanedione
reaction, phenylglyoxal reaction, or
nitromalondialdehyde reaction; modification of
carbonyls by esterification or carbodiimide coupling;
sulfenylation of tryptophan; modification of tyrosine
by nitration or iodination; modification of sulfhydrils
by reduction, oxidation, carboxymethylation,
carboxyerthylation, aminoethylation, methylation,
sulphonation, addition of thiols, or cyanylation. One
skilled in the art can chemically modify a parent
polypeptide by methods described in Means and Feeney,
Chemical Modification of Proteins Holden-Day Inc., San
Francisco (1971) and Glazer et al., Chemical
Modification of Proteins: Selected methods and
analytical procedures Elsevier Biomedical Press, New
York (1975) which are incorporated herein by reference.
Changing the structure of a binding polypeptide
by the methods described herein provides a means to
alter therapeutic potency by increasing or decreasing
the association rate or by increasing the association
rate for a new ligand. Therefore, binding polypeptides
having increased or decreased therapeutic potency can
be identified according to the needs of the,
practitioner. The methods of the invention provide for
production of a population of progeny polypeptides that
has sufficient size and diversity to yield a likely
probability of obtaining a binding polypeptide having
desired changes in therapeutic potency, whether it be
an increases or decrease. As described previously, the
size and diversity of the population can be adjusted
according to the chosen method of mutagenesis. For
example, if random mutagenesis methods are to be
employed then a large population of high diversity can
be produced. The size or diversity of the population
can be reduced by using biased mutagenesis, focused
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53
mutagenesis or site directed mutagenesis.° One skilled
in the art will be able to determine the size and
diversity of the population of progeny pnolypeptides
based on the properties of the particL~rlar polypeptide
to be changed and which method is to be used for
changing the polypeptide.
The methods of the invention provide for
repetition of steps to further optimize the therapeutic
potency of a binding polypeptide. The therapeutic
potency of a binding polypeptide can be optimized by
isolating a binding polypeptide having altered
therapeutic potency and repeating the steps of the
method described herein. Specifically, the therapeutic
potency of the isolated binding polypeptide having
altered therapeutic potency can be determined by
changing one or more amino acid, contacting the
isolated binding polypeptide having altered therapeutic
potency with a ligand, measuring association rate for
binding between the isolated binding polypeptide having
altered therapeutic potency and the ligand, and
comparing the association rate for the binding
polypeptide to an association rate for a therapeutic
control. The steps can be repeated once, twice, or
many times until a desired therapeutic potency is
obtained.
An example of a binding polypeptide that can be
made and used with the methods of the invention is an
antibody, or functional fragment thereof. For example,
often grafted antibodies are observed to have reduced
affinity when compared to the donor antibody from which
the CDRs were derived. The methods of the invention
can be used to improve the association rate for a
grafted antibody~binding to a ligand and, therefore,
therapeutic potency of the grafted antibody. The
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54
grafted antibody binding site can be identified by any
or all of the criteria specified previously and in the
examples and the methods of the invention described
previously with respect to binding polypeptides can be
utilized. A grafted antibody can have at least 1, 2,
3, 4, 5 or 6 CDRs from a therapeutically potent
antibody or other functional antibody. A CDR of an
antibody or fragment thereof can be selected from a
light chain CDR such as L1, L2 or L3 or heavy chain CDR
such as H1, H2 and H3.
The invention further provides a method to
determine therapeutic potency of a binding polypeptide
where the difference between the kon for a binding
polypeptide and the kon for a therapeutic control is
independent of an effect of a difference between Ka for
the binding polypeptide and Ka for the therapeutic
control. Also provided is a method where the
difference between the kon for the binding polypeptide
and the kon for the therapeutic control can be an
increase and Ka for the binding polypeptide can be a
similar value to Ka for the therapeutic control.
Similarly, a method is provided where the difference
between the kon for the binding.polypeptide and the kon
for the therapeutic control can be an increase and Ka
for the binding polypeptide can be a lower value than
Ka for the therapeutic control.
An advantage of the invention is that a binding
polypeptide having improved therapeutic potency can be
distinguished from a binding polypeptide that has an
increased Ka for a ligand but not improved therapeutic
potency. Methods for identifying a therapeutic binding
polypeptide based on Ka rely on an equilibrium
measurement which, absent time dependent measurements
made in a non-equilibrium condition, are inaccurate for
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identifying a binding polypeptide having increased
association rate and therefore improved therapeutic
potency. According to the relationship Ka = kon/koff. an
increased Ka for association of a binding polypeptide
5 and ligand can be due to changes in ko" or koff. For
example, a binding polypeptide having improved
therapeutic potency can have a reduced Ka if a
reduction in koff occurs that over compensates for an
increase in kon. Thus, changes in Ka, being influenced
10 by changes in koff, do not unambiguously correlate with
changes in therapeutic potency since binding
polypeptides having improved therapeutic potency can
display either reduced or increased Ka.
A binding polypeptide having therapeutic
15 potency such as an antibody or functional fragment
thereof can have a Ka of at least about 1 x 109 M-1, 1 x
101° M-1 or 1 x 1011 M-1. A binding polypeptide of the
invention such as an antibody or functional fragment
thereof can be evaluated by other known measures such
20 as EC5°. A binding polypeptide can have an ECS° of less
than about 6.0 nM, 3.0 nM, or 1.0 nM.
The invention provides a method of determining
therapeutic potency of a binding polypeptide. The
25 method consists of (a) contacting two or more binding
polypeptides with a ligand; (b) measuring kon for
binding between the two or more binding polypeptides
and the ligand, and (c) identifying a binding
polypeptide exhibiting a high kon, the kon value
30 correlating with the therapeutic potency of the
identified binding polypeptide.
The invention further provides a method of
determining therapeutic potency of a binding
polypeptide where the method consists of(a) contacting
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56
two or more binding polypeptides of a population with a
ligand; (b) measuring association rates for the two or
more binding polypeptides binding to the ligand; (c)
comparing the association rates for the two or more
binding polypeptides binding to the ligand, and (d)
identifying a binding polypeptide exhibiting a higher
association rate for binding to said ligand than one or
more other binding polypeptides of the population, said
higher association rate correlating with the
therapeutic potency of said identified binding
polypeptide. The association rate identified by the
method can be indicated by kon. The ko" of a binding
polypeptide exhibiting a higher association rate for a
ligand can be at least about 1. 5 x 106 M-ls-~ . A binding
polypeptide exhibiting a higher association rate for a
ligand can also have a kon of at least about 2 x 106 M-
'-s-1, 4 x 106 M-ls-1, 6 x 106 M-ls-1, 8 x 106 M-ls-1, 1 x 10'
M-ls-1, 2 x 10' M-1s-'-, 4 x 10' M-'-s-l, 6 x 10' M-ls-1, 8 x 10'
M-ls-1, or 1 x 10e M-ls-1 or higher. Preferably, a high kon
is larger than kon for a therapeutic control.
The step of contacting two or more binding
polypeptides of a population with a ligand can be
performed with binding polypeptides.isolated from the
population prior to being contacted with the ligand or
a mixture containing two or more binding polypeptides
from the population. The step of measuring association
rates for a binding polypeptide isolated from the
population can be performed according to essentially
any of the methods described herein previously.
Measuring association rates for binding polypeptides in
a mixture containing two or more binding polypeptides
can be performed by relative methods including, for
example, selection of a binding polypeptide bound to
ligand at a discreet time interval by using a time
actuated collection device.
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Comparing the association rates for two or more
binding polypeptides isolated from a population can be
achieved essentially as described previously.
Comparing the association rates for two or more binding
polypeptides in the same mixture can be achieved by
selection methods, for example, using a time actuated
device as described above. Such methods of comparison
can be made with a population of binding polypeptides
containing one or more binding polypeptide that are
standards of known association rate or therapeutic
potency. Additionally a population containing binding
polypeptides of unknown association rate can be
measured such that one or more binding polypeptides is
identified as having increased association rate and
improved therapeutic potency compared to the average
for the population.
A population of polypeptides used in the
methods of the invention can include 2, 10, 100, 1 x
103, 1 x 104, 1 x 105, 1 x 106, 1 x 10', 1 x 108, 1 x
10g, or 1 x 101° or more different binding polypeptides.
As described previously one skilled in the art will be
able to determine the size and diversity of the
population of binding polypeptides based on the
properties of the particular polypeptide to be changed
and which method is used to change the polypeptide.
One skilled in the art can also alter the number of
binding polypeptides to be measured from a population
such that a sub-population can be measured. The number
of polypeptides to be measured can be based on factors
such as the diversity of the population, the magnitude
of change desired in the therapeutic potency, or the
degree of bias incorporated during mutagenesis.
Accordingly, association rates can be measured for 2,
10, 100, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 10', 1 x
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108, 1 x 109, or 1 x 101° or more different binding
polypeptides from a population.
The invention provides a method for producing
one or more binding polypeptides with improved
therapeutic potency. The method consists of (a)
changing one or more amino acids in a parent
polypeptide to produce one or more different progeny
polypeptides; (b) measuring the association rate for
the one or more different progeny polypeptides
associating with a ligand, and (c) identifying a
binding polypeptide from one or more progeny
polypeptides having at least a 4-fold increase in
association rate to a ligand compared to the parent
polypeptide, the increased association rate resulting
in improved therapeutic potency toward a pathological
condition. Further provided is a method where the fold
increase in association rate is indicated by an
increase in ko". Therefore, kon can increase by 4-fold,
5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or
more in the methods of the invention. The increased
kon can be at least about 3 x 105 M-ls-1. The increased
kon can also be at least about 5 x 105 M-~s'1, 7 x 105 M'
1s-1, 9 x 105 M'ls''-, 1 x 106 M'1s'l, 3 x 106 M'ls-'-, 5 x 106
M-ls-1, 7 x 106 M'1s-1, 9 x 106 M'~s-1 or 1 x 10' M'ls-1 or
more. Furthermore, the increase in kon resulting in
improved therapeutic potency can be independent of an
effect of a change in Ka for the binding polypeptide.
The binding polypeptide having an increase in kon can
have a Ka value similar to Ka for its parent
polypeptide or a Ka value lower than Ka for its parent
polypeptide.
A polypeptide changed by the methods of the
invention can be a parent polypeptide. A parent
polypeptide is one example of a peptide described
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herein and therefore can have any of the properties
thereof and be made and used according to the
description provided herein. For example, one or more
amino acids in a parent polypepticie can be changed
according to the previously described methods to
produce one or more different progeny polypeptides. A
progeny polypeptide is one example of the changed
polypeptides described herein and can therefore be made
and used according to the previous descriptions herein.
Accordingly, the step of measuring an association rate
for one or more different progeny polypeptides
associating with a ligand, can be performed as
described herein previously. In addition, the step of
identifying a binding polypeptide from one or more
progeny polypeptides having at least a 4-fold increase
in association rate when binding to a ligand compared
to its parent polypeptide can be performed according to
the methods described previously herein for determining
association rates and therapeutic potency.
The step of identifying a binding polypeptide
from one or more progeny polypeptides having at least a
4-fold increase in association rate to a ligand
compared to the parent polypeptide can be performed to
identify a binding polypeptide from one or more progeny
polypeptides having at least a 5-fold, 6-fold, 7-fold,
8-fold, 9-fold, or 10-fold or greater increase in
association rate resulting in improved therapeutic
potency toward a pathological condition. Binding
polypeptides having a larger fold increase in
association rate will have an increased therapeutic
potency. Additionally the increased kon can be at
least about 1 x 10B M-ls-1, 1.5 x 10B M-1s-1, 2 x 108 M-ls-1,
2.5 x 108 M-ls-1, 3 x 10$ M-ls-1, 4 x 10$ M-ls-l, 5 x 108 M-
ls-~, or 1 x 109 M-ls-1 or more.
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Therefore, the invention further provides a
method for producing a binding polypeptide with
improved therapeutic potency. The method consists of
(a) changing one or more amino acids in a parent
5 polypeptide to produce one or more different progeny
polypeptides; (b) measuring the association rate for
the one or more different progeny polypeptides
associating with a ligand, and (c) identifying a
binding polypeptide from the one or more different
10 progeny polypeptides having a kon of at least about 1.5
x 106 M-ls-'- for binding polypeptide associating with a
ligand, thereby having improved therapeutic potency.
The method can also involve the step of identifying a
binding polypeptide from the one or more different
15 progeny polypeptides having improved therapeutic
potency and a kon of at least about 3 x 106 M-ls-z, 5 x
106 M-ls-~, 7 x 106 M-ls-1, 9 x 106 M-1s-1, 1 x 10' M-ls-1, 3 x
10' M-ls-1, 5 x 10' M-ls-1, 7 x 10' M-1s-1 or 9 x 10' M-ls-1 or
higher for binding polypeptide associating with a
20 ligand.
A binding polypeptide that associates with a
ligand and that is produced from a parent polypeptide
having no measurable association rate with the ligand
has an improved association rate. Specifically, a
25 binding polypeptide improved by the methods of the
invention having a measurable value for an association
rate or kon that is at least 4-fold greater than the
limits of detection available to the art constitutes at
least a 4-fold increase in association rate or konfor
30 the ligand. Thus, a binding polypeptide that
associates with a ligand and that is produced from a
parent polypeptide having no measurable association
rate with the ligand is understood to have improved
therapeutic potency.
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The efficacy of a binding polypeptide having
improved therapeutic potency can be observed in an
individual to be treated or, as an alternative, in an
in vivo model system including, for example, a cell
based assay, a tissue based assay, or a whole organism
assay. One skilled in the art will know how to
determine efficacy in a model according to the
conditions specific to the assay and disease under
study. For example, the chick chorioallantoic membrane
(CAM) assay measures angiogenesis and is a well
recognized model for in vivo angiogenesis. The assay
has -been described in detail and has been used to
measure neovascularization as well as the
neovascularization of tumor tissue (Ausprunk et al.,
Am. J. Pathol., 79:597-618 (1975); Ossonski et al.
Cancer Res., 40:2300-2309 (1980); Brooks et al.
Science, 264:569-571 (1994a) and Brooks et al. Cell,
79:1157-1164 (1994b) which are incorporated herein by
reference.
A binding polypeptide identified, determined or
produced by the methods of the invention and having
improved therapeutic potency can have improved
efficacy. For example, a binding polypeptide having
improved therapeutic potency as identified or
determined relative to a therapeutic control of known
efficacy will show improved efficacy. The methods of
the invention can also be used to identify a binding
polypeptide having improved therapeutic potency
relative to a therapeutic control for which efficacy
has not been determined as described previously.
Efficacy of a binding polypeptide .having improved
therapeutic potency relative to a therapeutic control
of unknown efficacy can be tested in an in vivo model
as described above. In cases where the efficacy is
less than desired the binding polypeptide can be
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further improved by the methods of the invention and re
tested in the in vivo model. Repetition of the methods
of the invention and testing in an in vivo model can be
used to iteratively improve therapeutic potency of a
binding polypeptide until a binding polypeptide
yielding the desired efficacy is produced.
One skilled in the art will recognize that the
methods of the invention, which have been exemplified
herein with respect to a binding polypeptide, can be
performed with any binding molecule. In this regard,
one skilled in the art will know that a ligand is a
binding molecule. Accordingly, a binding molecule can
be identified and produced according to methods
described herein with respect to identifying and
producing a ligand. Thus the invention provides a
method of determining the therapeutic potency of a
binding molecule. The method can consist of (a)
contacting a binding molecule with a ligand; (b)
measuring association rate for binding between the
binding molecule and the ligand; and (c) comparing the
association rate for the binding molecule to an
association rate for a therapeutic control, the
relative association rate for the binding molecule
compared to the association rate for the therapeutic
control indicating that the binding molecule will
exhibit a difference in therapeutic potency correlative
with the difference between the association rates. The
method can further consist of (d) changing one or more
moiety in the binding molecule and repeating steps (a)
through (c) one or more times. One skilled in the art
will know that methods of combinatorial chemistry can
be used in the methods of the invention to produce or
change any binding molecule.
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The invention further provides a method of
preventing or treating a virus related disease. The
method can include administering to a patient at risk
thereof, or afflicted therewith, a therapeutically
effective amount of an antibody or active fragment
thereof of the invention.
The invention further provides a process for
producing a high potency neutralizing antibody. The
process includes the steps of (a) producing a
recombinant antibody, including immunologically active
fragments thereof, having heavy and light chain
variable regions containing one or more framework
and/or CDR having preselected amino acid sequences; (b)
screening the recombinant antibodies for high kon when
the antibody reacts in vitro with a selected antigen;
and (c) selecting antibodies with the high kon. The Ka
or kon of the antibody can be any of the values
described above.
The invention further provides a method of
increasing the potency of an antibody or functional
fragment thereof by selectively changing one or more
amino acids within the variable region framework and/or
CDR regions so as to increase the measured Ka or kon
values. Amino acid changes can be restricted to either
the variable region framework or CDR regions. The Ka
or kon of the antibody prior to or after changing amino
acids can be any of the values described above and can
be increased to at least the above-described values.
EXAMPLE I
Synthesis of focused libraries of
butyrylcholinesterase variants by codon-based
mutagenesis.
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This example describes the design and synthesis
of butyrylcholinesterase variant libraries.
A variety of information can be used to focus
the synthesis of the initial libraries of
butyrylcholinesterase variants to discreet regions.
For example, butyrylcholinesterase and Torpedo
acetylcholinesterase (AChE) share a high degree of
homology (53% identity). Furthermore, residues 4 to
534 of Torpedo AChE can be aligned with residues 2 to
532 of butyrylcholinesterase without deletions or
insertions. The catalvtic triad residues
(butyrylcholinesterase residues Ser198, G1u325, and
His438) and the intrachain disulfides are all in the
same positions. Due to the high degree of similarity
I5 between these proteins, a refined 2.8 A x-ray structure
of Torpedo AChE (Sussman et al., Science 253: 872-879
'(1991)) has been used to model butyrylcholinesterase
structure (Harel et al., Proc. Nat. Acad. Sci. USA 89:
10827-10831 (1992)).
Studies with cholinesterases have revealed that
the catalytic triad and other residues involved in
ligand binding are positioned within a deep, narrow,
active-site gorge rich in hydrophobic residues
(reviewed in Soreq et al., Trends Biochem. Sci. 17:353-
358 (1992)). The sites of seven focused libraries of
butyrylcholinesterase variants were selected to include
amino acids determined to be lining the active site
gorge.
In addition to the structural modeling of
butyrylcholinesterase, butyrylcholinesterase
biochemical data was integrated into the library design
process. For example, characterization of naturally
occurring butyrylcholinesterases with altered cocaine
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hydrolysis activity and site-directed mutagenesis
studies provide information regarding amino acid
positions and segments important for cocaine
hydrolysis activity (reviewed in Schwartz et al.,
5 Pharmac. Ther. 67: 283-322(1995)). Moreover,
comparison of sequence and cocaine hydrolysis data of
butyrylcholinesterases from different species can also
provide information regarding regions important for
cocaine hydrolysis activity of the molecule based on
10 comparison of the cocaine hydrolysis activities of
these butyrylcholinesterases. The A328Y mutant is
present in the library and serves as a control to
demonstrate the quality of the library synthesis and
expression in mammalian cells.
15 The seven regions of butyrylcholinesterase
selected for focused library synthesis (summarized in
Table 2) span residues that include the 8 hydrophobic
active site gorge residues as well as two of the
catalytic triad residues. The integrity of intrachain
20 disulfide bonds, located between 65Cys-9zCys,
zs2Cys-zs3Cys, and 4°°Cys-5'-9Cys is maintained to ensure
functional butyrylcholinesterase structure. In
addition, putative glycosylation sites (N-X-S/T)
located at residues 17, 57, 106, 241, 256, 341, 455,
25 481, 485, and 486 also are avoided in the library
syntheses. In total, the seven focused libraries span
79 residues, representing approximately 140 of the
butyrylcholinesterase linear sequence, and result in
the expression of about 1500 distinct
30 butyrylcholinesterase variants.
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TABLE 2. Summary of Butyrylcholinesterase Libraries
Region Locatio Length # Species Diversity
n Variants
1 68-82 15 285 3
2 110-121 12 228 3
3 194-201 8 152 1
4 224-234 11 209 2
5 277-289 13 247 8
6 327-332 6 114 0
7 429-442 14 266 0
Total 79 1,501
13.8s
Libraries of nucleic acids corresponding to the
seven regions of human butyrylcholinesterase to be
mutated are synthesized by codon-based mutagenesis, as
described above. Briefly, multiple DNA synthesis
columns are used for synthesizing the oligonucleotides
by ~3-cyanoethyl phosphoramidite chemistry, as described
previously by Glaser et al., supra, 1992. In the first
step, trinucleotides encoding for the amino acids of
butyrylcholinesterase are synthesized on one column
while a second column is used to synthesize the
trinucleotide NN(G/T), where. N is a mixture of dA, dG,
dC, and dT cyanoethyl phosphoramadites. Using the
trinucleotide NN(G/T) results in thorough mutagenesis
with minimal degeneracy, accomplished through the
systematic expression of all twenty amino acids at
every position.
Following the synthesis of the first codon,
resins from the two columns are mixed together,
divided, and replaced in four columns. By adding
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additional synthesis columns for each codon and mixing
the column resins, pools of degenerate oligonucleotides
will be segregated based on the extent of mutagenesis.
The resin mixing aspect of codon-based mutagenesis
makes the process rapid and cost-effective because it
eliminates the need to synthesize multiple
oligonucleotides. In the present study, the pool of
oligonucleotides encoding single amino acid mutations
are used to synthesize focused butyrylcholinesterase
libraries.
The oligonucleotides encoding the
butyrylcholinesterase variants containing a single
amino acid mutation is cloned into the doublelox
targeting vector using oligonucleotide-directed
mutagenesis (Kunkel, supra, 1985). To improve the
mutagenesis efficiency and diminish the number of
clones expressing wild-type butyrylcholinesterase, the
libraries are synthesized in a two-step process. In
the first step, the butyrylcholinesterase DNA sequenee
corresponding to each library site is deleted by
hybridization mutagenesis. In the second step,
uracil-containing single-stranded DNA for each deletion
mutant, one deletion mutant corresponding to each
library, is isolated and used as template for synthesis
of the libraries by oligonucleotide-directed
mutagenesis. This approach has been used routinely for
the synthesis of antibody libraries and results in more
uniform mutagenesis by removing annealing biases that
potentially arise from the differing DNA sequence of
the mutagenic oligonucleotides. In addition, the
two-step process decreases the frequency of wild-type
sequences relative to the variants in the libraries,
and consequently makes library screening more efficient
by eliminating repetitious screening of clones encoding
wild-type butyrylcholinesterase.
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The quality of the libraries and the efficiency
of mutagenesis is characterized by obtaining DNA
sequence from approximately 20 randomly selected clones
from each library. The DNA sequences demonstrate that
mutagenesis occurs at multiple positions within each
library and that multiple amino acids were expressed at
each position. Furthermore, DNA sequence of randomly
selected clones demonstrates that the libraries contain
diverse clones and are not dominated by a few clones.
Optimization of Transfection Parameters for Site-
Specific Integration
Optimization of transfection parameters for
Cre-mediated site-specific integration was achieved
utilizing Bleomycin Resistance Protein (BRP) DNA as a
model system.
Cre recombinase is a well-characterized 38-kDa
DNA recombinase (Abremski et al., Cell 32:1301-1311
(1983)) that is both necessary and sufficient for
sequence-specific recombination in bacteriophage P1.
Recombination occurs between two 34-base pair loxP
sequences each consisting of two inverted 13-base pair
recombinase recognition sequences that surround a core
region (Sternberg and Hamilton, J. Mol. Biol.
150:467-486 (1981a); Sternberg and Hamilton, J. Mol.
Biol., 150:487-507 (1981b)). DNA cleavage and strand
exchange occurs on the top or bottom strand at the
edges of the core region. Cre recombinase also
catalyzes site-specific recombination in eukaryotes,
including both yeast (Saner, Mol. Cell. Biol.
7:2087-2096 (1987)) and mammalian cells (Saner and
Henderson, Proc. Natl. Acad. Sci. USA, 85:5166-5170
(1988); Fukushige and Saner, Proc. Natl. Acad. Sci.
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U.S.A. 89:7905-7909 (1992); Bethke and Sauer, Nuc.
Acids Res., 25:2828-2834 (1997)).
Calcium phosphate transfection of 13-1 cells
was previously demonstrated to result in targeted
integration in 10 of the viable cells plated (Bethke
and Sauer, Nuc. Acids Res., 25:2828-2834 (1997)).
Therefore, initial studies were conducted using calcium
phosphate to transfect 13-1 cells with 4 ~g pBS185 and
10, 20, 30, or 40 ~g of pBS397-fl(+)/BRP. The total
level of DNA per transfection was held constant using
unrelated pBluescript II KS DNA (Stratagene; La Jolla,
CA), and transformants were selected 48 hours later by
replating in media containing 400 ~.g/ml geneticin.
Colonies were counted 10 days later to determine the
efficiency of targeted integration. Optimal targeted
integration was typically observed using 30 Ng of
targeting vector and 4 ~g of Cre recombinase vector
pBS185, consistent with the 20 ~Zg targeting vector and
5 ug of pBS185 previously reported (Bethke and Sauer,
Nuc. Acids Res., 25:2828-2834 (1997)). The frequency
of targeted integration observed was generally less
than lo. Despite the sensitivity of the oalcium
phosphate methodology to the amount of DNA used and the
buffer pH, targeted integration efficiencies observed
were sufficient to express the protein libraries.
As shown in Table 3, several cell lines as well
as other transfection methods were also characterized.
In general, lipid-mediated transfection methods are
more efficient than methods that alter the chemical
environment, such as calcium phosphate and DEAE-dextran
transfection. In addition, lipid-mediated
transfections are less affected by contaminants in the
DNA preparations, salt concentration, and pH and thus
generally provide more reproducible results (Felgner et
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al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
Consequently, a formulation of the neutral lipid
dioleoyl phosphatidylethanolamine and a cationic lipid,
termed GenePORTER transfection reagent (Gene Therapy
5 Systems; San Diego, CA), was evaluated as an
alternative transfection approach. Briefly,
endotoxin-free DNA was prepared for both the targeting
vector pBS397-fl(+)/BRP and the Cre recombinase vector
pBS185 using the EndoFree Plasmid Maxi kit (QIAGEN;
10 Valencia, CA). Next, 5 ~g pBSl85 and varying amounts
of pBS397-fl(+)/BRP were diluted in serum-free medium
and mixed with the GenePORTER transfection reagent.
The DNA/lipid mixture was then added to a 60-70a
confluent monolayer of 13-1 cells consisting of
15 approximately 5 x 105 cells/100-mm dish and incubated
at 37°C. Five hours later, fetal calf serum was added
to 100, and the next day the transfection media was
removed and replaced with fresh media.
Transfection of the cells with variable
20 quantities of the targeting vector yielded targeted
integration efficiencies ranging from 0.1o to 1.0o,
with the optimal targeted integration efficiency
observed using 5 pg each of the targeting vector and
the Cre recombinase vector. Lipid-based transfection of
25 the 13-1 host cells under the optimized conditions
resulted in 0.5o targeted integration efficiency being
consistently observed. A 0.5o targeted integration is
slightly less than the previously reported 1.0o
efficiency (Bethke and Sauer, Nuc. Acids Res.,
30 25:2828-2834 (1997)), and is sufficient to express
large protein libraries and allows expressing libraries
of protein variants in mammalian cells.
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TABZE 3. Expression of a single butyrylcholinesterase
variant per cell using either stable or transient cell
transfecti0n.
Cell Expression Integration Integration? Integration?
Zine Method (PCR) (Activity)
NIH3T3 Transient N/A N/A Transient,
(13-l) (lipid- very low
based) activity
NIH3T3 Stable Cre Yes No measurable
(13-1) recombinase activity
CHO Transient N/A N/A Transient,
(lipid- measurable
based) activity
(colorimetric
and cocaine
hydrolysis)
293 Transient N/A N/A Transient,
(lipid- measurable
based) activity
(colorimetric
and cocaine
hydrolysis)
293 Stable Flp Yes Measurable
recombinase activity
(colorimetric
and cocaine
hydrolysis)
These results demonstrate optimization of
transfection conditions for targeted insertion in
N1H3T3 13-1 cells. Conditions for a simple,
lipid-based transfection method that required a small
amount of DNA and generated reproducible 0.5o targeting
efficiency were established.
Expression of butyrylcholinesterase variant libraries
in mammalian cells
Each of the seven libraries of
butyrylcholinesterase variants are transformed into a
host mammalian cell line using the doublelox targeting
vector and the optimized transfection conditions
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described above. Following Cre-mediated transformation
the host cells are plated at limiting dilutions to
isolate distinct clones in a 96-well format. Cells
with the butyrylcholinesterase variants integrated in
the Crellox targeting site are selected with geneticin.
Subsequently, the DNA encoding butyrylcholinesterase
variants from 20-30 randomly selected clones from each
library are sequenced and analyzed as described above.
Briefly, total cellular DNA is isolated from about 104
cells of each clone of interest using DNeasy Tissue
Kits (Qiagen, Valencia, CA). Next, the
butyrylcholinesterase gene is amplified using Pfu Turbo
DNA polymerase (Stratagene; Za Jolla, CA) and an
aliquot of the PCR product is then used for sequencing
the DNA encoding butyrylcholinesterase variants from
randomly selected clones by the fluorescent
dideoxynucleotide termination method (Perkin-Elmer,
Norwalk, CT) using a nested oligonucleotide primer.
As described previously, the sequencing
demonstrates uniform introduction of the library and
the diversity of mammalian transformants resembles the
diversity of the library in the doublelox targeting
vector following transformation of bacteria.
Although the invention has been described with
reference to the disclosed embodiments, those skilled
in the art will readily appreciate that the specific
experiments detailed are only illustrative of the
invention. It should be understood that various
modifications can be made without departing from the
spirit of the invention. Accordingly, the invention is
limited only by the following claims.
