Note: Descriptions are shown in the official language in which they were submitted.
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ENHANCING THE CIRCULATING HALF-LIFE
OF ANTIBODY-BASED FUSION PROTEINS
Cross Reference to Related Application
This incorporates by reference, and claims priority to and the benefit of,
U.S. Provisional
Patent Application Serial Number 60/075,887 which was filed on February 25,
1998.
Field of the Invention
The present invention relates generally to fusion proteins. More specifically,
the present
invention relates to methods of enhancing the circulating half life of
antibody-based fusion
proteins.
Background of the Invention
The use of antibodies for treatment human disease is well established and has
become
more sophisticated with the introduction of genetic engineering. Several
techniques have been
developed to improve the utility of antibodies. These include: ( 1 ) the
generation of monoclonal
antibodies by cell fusion to create "hyridomas", or by molecular cloning of
antibody heavy (H)
and light (L) chains from antibody-producing cells; (2) the conjugation of
other molecules to
antibodies to deliver them to preferred sites in vivo, e.g., radioisotopes,
toxic drugs, protein
toxins, and cytokines; (3) the manipulation of antibody effector functions to
enhance or diminish
biological activity; (4) the joining of other protein such as toxins and
cytokines with antibodies at
the genetic level to produce antibody-based fusion proteins; and (5) the
joining of one or more
sets of antibody combining regions at the genetic level to produce bi-specific
antibodies.
When proteins are joined together through either chemical or genetic
manipulation, it is
often difficult to predict what properties that the end product will retain
from the parent
molecules. With chemical conjugation, the joining process may occur at
different sites on the
molecules, and generally results in molecules with varying degrees of
modification that can
affect the function of one or both proteins. The use of genetic fusions, on
the other hand, makes
the joining process more consistent, and results in the production of
consistent end products that
retain the function of both component proteins. See, for example, Gillies et
al., Pttoc. NATL.
ACRD. Sct. USA 89: 1428-1432 (1992); and U.S. Patent No. 5,650,150.
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However, the utility of recombinantly-produced antibody-based fusion proteins
may be
limited by their rapid in vivo clearance from the circulation. Antibody-
cytokine fusion proteins,
for example, have been shown to have a significantly lower in vtvo circulating
half life than the
free antibody. When testing a variety of antibody-cytokine fusion proteins,
Gillies et al. reported
S that all of the fusion proteins tested had an a phase (distribution phase)
half life of less than
1.5 hour. Indeed, most of the antibody-based fusion protein were cleared to
10% of the serum
concentration of the free antibody by two hours. See, Gillies et al., BIOCONJ.
CHEM. =t: 230-235
(1993). Therefore, there is a need in the art for methods of enhancing the in
vivo circulating half
life of antibody-based fusion proteins.
Summary of the Invention
A novel approach to enhancing the in vivo circulating half life of antibody-
based fusion
proteins has now been discovered. Specifically, the present invention provides
methods for the
production of fusion proteins between an immunoglobulin with a reduced binding
affinity for an
Fc receptor, and a second non-immunoglobulin protein. Antibody-based fusion
proteins with
reduced binding affinity for Fc receptors have a significantly longer in vivo
circulating half life
than the unlinked second non-immunoglobulin protein.
IgG molecules interact with three classes of Fc receptors (FcR) specific for
the IgG class
of antibody, namely FcyRI, FcyRII and FcyRIII. In preferred embodiments, the
immunoglobulin
(Ig) component of the fusion protein has at least a portion of the constant
region of an IgG that
has a reduced binding affinity for at least one of FcyRI, FcyRII or FcyRIII.
In one aspect of the invention, the binding affinity of fusion proteins for Fc
receptors is
reduced by using heavy chain isotypes as fusion partners that have reduced
binding affinity for
Fc receptors on cells. For example, both human IgGI and IgG3 have been
reported to bind to
FcRyI with high affinity, while IgG4 binds 10-fold less well, and IgG2 does
not bind at all. The
important sequences for the binding of IgG to the Fc receptors have been
reported to be located
in the CH2 domain. Thus, in a preferred embodiment, an antibody-based fusion
protein with
enhanced in vivo circulating half life is obtained by linking at least the CH2
domain of IgG2 or
IgG4 to a second non-immunoglobulin protein.
In another aspect of the invention, the binding affinity of fusion proteins
for Fc receptors
is reduced by introducing a genetic modification of one or more amino acid in
the constant
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region of the IgG I or IgG3 heavy chains that reduces the binding affinity of
these isotypes for
Fc receptors. Such modifications include alterations of residues necessary for
contacting
Fc receptors or altering others that affect the contacts between other heavy
chain residues and
Fc receptors through induced conformational changes. Thus, in a preferred
embodiment, an
antibody-based fusion protein with enhanced in vivo circulating half life is
obtained by first
introducing a mutation, deletion, or insertion in the IgGI constant region at
one or more amino
acid selected from Leuz,4, Leuz35, Glyz,b, GlYz3>> Asnz9,, and Pro33,, and
then linking the resulting
immunoglobulin, or portion thereof, to a second non-immunoglobulin protein. In
an alternative
preferred embodiment, the mutation, deletion, or insertion is introduced in
the IgG3 constant
I O region at one or more amino acid selected from LeuzB,, Leuz$z, Glyz83,
Glyz84, Asn344, and Pro3,8,
and the resulting immunoglobulin, or portion thereof, is linked to a second
non-immunoglobulin
protein. The resulting antibody-based fusion proteins have a longer in vivo
circulating half life
than the unlinked second non-immunoglobulin protein.
In a preferred embodiment, the second non-immunoglobulin component of the
fusion
protein is a cytokine. The term "cytokine" is used herein to describe
proteins, analogs thereof,
and fragments thereof which are produced and excreted by a cell, and which
elicit a specific
response in a cell which has a receptor for that cytokine. Preferably,
cytokines include
interleukins such as interleukin-2 (IL-2), hematopoietic factors such as
granulocyte-macrophage
colony stimulating factor (GM-CSF), tumor necrosis factor (TNF) such as TNFa,
and
lymphokines such as lymphotoxin. Preferably, the antibody-cytokine fusion
protein of the
present invention displays cytokine biological activity.
In an alternative preferred embodiment, the second non-immunoglobulin
component of
the fusion protein is a ligand-binding protein with biological activity. Such
ligand-binding
proteins may, for example, ( I ) block receptor-ligand interactions at the
cell surface; or
(2) neutralize the biological activity of a molecule (e.g., a cytokine) in the
fluid phase of the
blood, thereby preventing it from reaching its cellular target. Preferably,
ligand-binding proteins
include CD4, CTLA-4, TNF receptors, or interleukin receptors such as the IL-1
and IL-4
receptors. Preferably, the antibody-receptor fusion protein of the present
invention displays the
biological activity of the ligand-binding protein.
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In yet another alternative preferred embodiment, the second non-immunoglobulin
component of the fusion protein is a protein toxin. Preferably, the antibody-
toxin fusion protein
of the present invention displays the toxicity activity of the protein toxin.
In a preferred embodiment, the antibody-based fusion protein comprises a
variable region
specific for a target antigen and a constant region linked through a peptide
bond to a second non-
immunoglobulin protein. The constant region may be the constant region
normally associated
with the variable region, or a different one, e.g., variable and constant
regions from different
species. The heavy chain can include a CH1, CH2, and/or CH3 domains. Also
embraced within
the term "fusion protein" are constructs having a binding domain comprising
framework regions
and variable regions (i. e., complementarity determining regions) from
different species, such as
are disclosed by Winter, et al., GB 2,188, 638. Antibody-based fusion proteins
comprising a
variable region preferably display antigen-binding specificity. In yet another
preferred
embodiment, the antibody-based fusion protein further comprises a light chain.
The invention
thus provides fusion proteins in which the antigen-binding specificity and
activity of an antibody
are combined with the potent biological activity of a second non-
immunoglobulin protein, such
as a cytokine. A fusion protein of the present invention can be used to
deliver selectively the
second non-immunoglobulin protein to a target cell in vivo so that the second
non-
immunoglobulin protein can exert a localized biological effect.
In an alternative preferred embodiment, the antibody-based fusion protein
comprises a
heavy chain constant region linked through a peptide bond to a second non-
immunoglobulin
protein, but does not comprise a heavy chain variable region. The invention
thus further
provides fusion proteins which retain the potent biological activity of a
second non-
immunoglobuiin protein, but which lack the antigen-binding specificity and
activity of an
antibody.
In preferred embodiments, the antibody-based fusion proteins of the present
invention
further comprise sequences necessary for binding to Fc protection receptors
(FcRp), such as
beta-2 microglobulin-containing neonatal intestinal transport receptor (FcRn).
In preferred embodiments, the fusion protein comprises two chimeric chains
comprising
at least a portion of a heavy chain and a second, non-Ig protein are linked by
a disulfide bond.
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The invention also features DNA constructs encoding the above-described fusion
proteins, and cell lines, e.g., myelomas, transfected with these constructs.
These and other objects, along with advantages and features of the invention
disclosed
herein, will be made more apparent from the description, drawings, and claims
that follow.
Brief Description of the Drawings
The foregoing and other objects, features, and advantages of the present
invention, as
well as the invention itself, may be more fully understood from the following
description of
preferred embodiments, when read together with the accompanying drawings, in
which:
FIG. 1 is a homology alignment of the amino acid sequences of the constant
region of
Cyl and Cy3, aligned to maximize amino acid identity, and wherein non-
conserved amino acids
are identified by boxes;
FIG. 2 is a homology alignment of the amino acid sequences of constant region
of Cyl,
Cy2, and Cy 4, aligned to maximize amino acid identity, and wherein non-
conserved amino acids
are identified by boxes;
FIG. 3 is a diagrammatic representation of a map of the genetic construct
encoding an
antibody-based fusion protein showing the relevant restriction sites;
FIG. 4 is a bar graph depicting the binding of antibody hu-KS-1/4 and antibody-
based
fusion proteins, hu-KSyI-IL2 and hu-KSy4-IL2, to Fc receptors on mouse J774
cells in the
presence (solid bars) or absence (stippled bars) of an excess of mouse IgG;
FIG. 5 is a line graph depicting the in vivo plasma concentration of total
antibody (free
antibody and fusion protein) of hu-KSyl-IL2 (closed diamond) and hu-KSy4-IL2
(closed
triangle) and of intact fusion protein of hu-KSyl-IL2 (open diamond) and hu-
KSy4-IL2 (open
triangle) as a function of time;
FIG. 6 is a diagrammatic representation of protocol for constructing an
antibody-based
fusion protein with a mutation that reduces the binding affinity to Fc
receptors;
FIG. 7 is a line graph depicting the in vivo plasma concentration of intact
fusion protein
of hu-KSy 1-IL2 (0); mutated hu-KSy 1-IL2 ('~) and hu-KSy4-IL2 (D) as a
function of time.
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Detailed Description of the Invention
It has now been discovered that fusing a second protein, such as a cytokine,
to an
immunoglobulin may alter the antibody structure, resulting in an increase in
binding affinity for
one or more of the cell-bound Fc receptors and leading to a rapid clearance of
the antibody-based
fusion protein from the circulation. The present invention describes antibody-
based fusion
proteins with enhanced in vivo circulating half lives and involves producing,
through
recombinant DNA technology, antibody-based fusion proteins with reduced
binding affinity for
one or more Fc receptor.
First, an antibody-based fusion protein with an enhanced in vivo circulating
half life can
be obtained by constructing a fusion protein with isotypes having reduced
binding affinity for a
Fc receptor, and avoiding the use of sequences from antibody isotypes that
bind to Fc receptors.
For example, of the four known IgG isotypes, IgGI (Cyl) and IgG3 (Cy3) are
known to bind
FcRyI with high affinity, whereas IgG4 (CY4) has a 10-fold lower binding
affinity, and IgG2
(Cy2) does not bind to FcRyI. Thus, an antibody-based fusion protein with
reduced binding
affinity for a Fc receptor could be obtained by constructing a fusion protein
with a Cy2 constant
region (Fc region) or a Cy4 Fc region, and avoiding constructs with a Cyl Fc
region or a Cy3 Fc
region.
Second, an antibody-based fusion protein with an enhanced in vivo circulating
half life
can be obtained by modifying sequences necessary for binding to Fc receptors
in isotypes that
have binding affinity for an Fc receptor, in order to reduce or eliminate
binding. As mentioned
above, IgG molecules interact with three classes of Fc receptors (FcR), namely
FcYRI, FcyRII,
and FcyRIII. Cyl and C~y3 bind FcR7I with high affinity, whereas Cy4 and Cy2
have reduced or
no binding affinity for FcRyI. A comparison of the Cy l and Cy3 indicates
that, with the
exception of an extended hinge segment in Cy3, the amino acid sequence
homology between
these two isotypes is very high. This is true even in those regions that have
been shown to
interact with the C 1 q fragment of complement and the various FcyR classes.
FIG. 1 provides a
alignment of the amino acid sequences of Cyl and Cy3. The other two isotypes
of human IgG
(CY2 and CY4) have sequence differences which have been associated with FcR
binding. FIG. 2
provides a alignment of the amino acid sequences of Cy 1, Cy2, and Cy4. The
important
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sequences for FcyR binding are Leu-Leu-Gly-Gly (residues 234 through 237 in
Cyl), located in
the CH2 domain adjacent to the hinge. Canfield and Morrison, J. ExP. MED. 173:
1483-1491
( I 991 ). These sequence motifs are conserved in Cy 1 and Cy3, in agreement
with their similar
biological properties, and possibly related to the similarity of
pharmacokinetic behavior when
used to construct IL-2 fusion proteins. Many mutational analyses have been
done to demonstrate
the effect of specific mutations on FcR binding, including those in residues
234-237 as well as
the hinge-proximal bend residue Pro33~ that is substituted by Ser in IgG4.
Another important
structural component necessary for effective FcR binding is the presence of an
N-linked
carbohydrate chain covalently bound to Asnz9,. Enzymatic removal of this
structure or mutation
of the Asn residue effectively abolish. or at least dramatically reduce,
binding to all classes of
FcyR.
Brumbell et al. postulated the existence of a protection receptor (FcRp) that
would slow
the rate of catabolism of circulating antibodies by binding to the Fc portion
of antibodies and,
following their pinocytosis into cells, would redirect them back into the
circulation. Brumbell et
IS al., NATURE 203: 1352-1355 (1964). The beta-2 microglobulin-containing
neonatal intestinal
transport receptor (FcRn) has recently been identified as a FcRp. See,
Junghans et al., PROC.
NATL. ACRD. Sct. USA 93: 5512-5516 (1996). The sequences necessary for binding
to this
receptor are conserved in all four classes of human IgG and are located at the
interface between
the CH2 and CH3 domains. See, Medesan et al., J. IMMUNOL. 158: 2211-2217
(1997). These
sequences have been reported to be important for the in vivo circulating half
life of antibodies.
See, International PCT publication WO 97/34631. Thus, preferred antibody-based
fusion
proteins of the present invention will have the sequences necessary for
binding to FcRp.
Methods for synthesizing useful embodiments of the invention are described, as
well as
assays useful for testing their pharmacokinetic activities, both in vitro and
in pre-clinical in vivo
animal models. The preferred gene construct encoding a chimeric chain
includes, in 5' to 3'
orientation, a DNA segment which encodes at least a portion of an
immunoglobulin and DNA
which encodes a second, non-immunoglobulin protein. An alternative preferred
gene construct
includes, in 5' to 3' orientation, a DNA segment which encodes a second, non-
immunoglobulin
protein and DNA which encodes at least a portion of an immunoglobulin. The
fused gene is
assembled in or inserted into an expression vector for transfection of the
appropriate recipient
cells where it is expressed.
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The invention is illustrated further by the following non-limiting examples:
Example 1 Improving the in vivo circulating half life of an antibody-IL2
fusion protein
by class switching from Cyl to Cy4 IgG constant regions.
According to the present invention, antibody-based fusion proteins with
enhanced in vivo
circulating half lives can be obtained by constructing antibody-based fusion
proteins using
sequences from antibody isotypes that have reduced or no binding affinity for
Fc receptors.
In order to assess whether the in vivo circulating half life of the antibody-
based fusion
protein can be enhanced by using sequences from antibody isotypes with reduced
or no binding
affinity for Fc receptors, an antibody-IL2 fusion protein with a human Cyl
constant region (Fc
region) was compared to an antibody-IL2 fusion protein with a human Cy4 Fc
region.
1.1 Construction of antibody-ILZ fusion proteins with a Cyp4 IgG constant
region.
The construction of antibody-IL2 fusion proteins with a Cyl constant region
has been
described in the prior art. See, for example, Gillies et al., Pttoc. NATL.
ACRD. Sct. USA 89:
1428-1432 (1992); and U.S. Patent No 5,650,150, the disclosure of which is
incorporated herein
by reference.
To construct antibody-IL2 fusion proteins with a Cy4 constant region, a
plasmid vector,
capable of expressing a humanized antibody-IL2 fusion protein with variable
(V) regions specific
for a human pancarcinoma antigen (KSA) and the human Cyl heavy chain fused to
human IL-2,
was modified by removing the Cyl gene fragment and replacing it with the
corresponding
sequence from the human Cy4 gene. A map of some of the relevant restriction
sites and the site
of insertion of the Cy4 gene fragment is provided in FIG. 3. These plasmid
constructs contain
the cytomegalovirus (CMV) early promoter for transcription of the mRNA
encoding the light (L)
and heavy (H) chain variable (V) regions derived from the mouse antibody KS-
1/4. The mouse
V regions were humanized by standard methods and their encoding DNA sequences
were
chemically synthesized. A functional splice donor site was added at the end of
each V region so
that it could be used in vectors containing H and L chain constant region
genes. The human Cx
light chain gene was inserted downstream of the cloning site for the VL gene
and was followed
by its endogenous 3' untranslated region and poly adenylation site. This
transcription unit was
followed by a second independent transcription unit for the heavy chain-IL2
fusion protein. It is
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also driven by a CMV promoter. The VH encoding sequence was inserted upstream
of the DNA
encoding the Cy heavy chain gene of choice, fused to human IL-2 encoding
sequences. Such Cy
genes contain splice acceptor sites for the first heavy chain exon (CHl ),
just downstream from a
unique Hind III common to all human Cy genes. A 3' untranslated and
polyadenylation site from
SV40 virus was inserted at the end of the IL-2 encoding sequence. The
remainder of the vector
contained bacterial plasmid DNA necessary for propagation in E. toll and a
selectable marker
gene (dihydrofolate reductase - dhfr) for selection of transfectants of
mammalian cells.
The swapping of the Cyl and Cy4 fragments was accomplished by digesting the
original
Cyl-containing plasmid DNA with Hind III and Xho I and purifying the large 7.8
kb fragment by
agarose gel electrophoresis. A second plasmid DNA containing the Cy4 gene was
digested with
Hind III and Nsi I and the 1.75 kb fragment was purified. A third plasmid
containing the human
IL-2 cDNA and SV40 poly A site, fused to the carboxyl terminus of the human
Cyl gene, was
digested with Xho I and Nsi I and the small 470 by fragment was purified. All
three fragments
were ligated together in roughly equal molar amounts and the ligation product
was used to
transform competent E. toll. The ligation product was used to transform
competent E. toll and
colonies were selected by growth on plates containing ampicillin. Correctly
assembled
recombinant plasmids were identified by restriction analyses of plasmid DNA
preparations from
isolated transformants and digestion with Fsp I was used to discriminate
between the Cyl (no
Fsp I) and Cy4 (one site) gene inserts. The final vector, containing the Cy4-
IL2 heavy chain
replacement, was introduced into mouse myeloma cells and transfectants were
selected by
growth in medium containing methotrexate (0.1 ~M). Cell clones expressing high
levels of the
antibody-IL2 fusion protein were expanded and the fusion protein was purified
from culture
supernatants using protein A Sepharose chromatography. The purity and
integrity of the Cy4
fusion protein was determined by SDS-polyacrylamide gel electrophoresis. IL-2
activity was
measured in a T-cell proliferation assay and found to be identical to that of
the Cyl construct.
1.2 Binding to Fc receptors by antibody and antibody-IL2 fusion proteins with
Cyl
and Cy4 IgG constant region.
Various mouse and human cell lines express one or more Fc receptor. For
example, the
mouse J774 macrophage-like cell line expresses FcRyI that is capable of
binding mouse or
human IgG of the appropriate subclasses. Likewise, the human K562
erythroleukemic cell line
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expresses FcRyII but not FcRyI. In order to assess the potential contribution
of Fc receptor
binding to clearance of antibody-based fusion proteins from the circulation,
the binding affinities
of an antibody, a Cyl-IL2 fusion protein, and a Cy4-IL2 fusion protein for
FcRyI were compared
in the mouse J774 cell line.
The two antibody-IL-2 fusion proteins described in Example 1, hu-KSy 1-IL2 and
hu-KSy4-IL2, were diluted to 2 ~.g/ml in PBS containing 0.1% bovine serum
albumin (BSA),
together with 2x105 J774 cells in a final volume of 0.2 ml. After incubation
on ice for 20 min, a
FITC-conjugated anti-human IgG Fc antibody (Fab2) was added and incubation was
continued
for an additional 30 min. Unbound antibodies were removed by two washes with
PBS-BSA, and
the cells were analyzed in a fluorescence-activated cell sorter (FRCS).
Control reactions
contained the same cells mixed with just the FITC-labeled secondary antibody
or with the
humanized KSyI antibody (without IL-2).
As expected, the binding of the Cy4-IL2 fusion protein to J774 cells was
significantly
lower than the binding of the Cyl-IL2 fusion protein. See FIG. 4.
Unexpectedly, however, both
I S the Cyl-IL2 and Cy4-IL2 fusion proteins had significantly higher binding
to J774 cells than the
KSyI antibody (without IL-2). This suggests that fusing a second protein, such
as a cytokine, to
an immunoglobulin may alter the antibody structure, resulting in an increase
in binding affinity
for one or more of the cell-bound Fc receptors, thereby leading to a rapid
clearance from the
circulation.
In order to determine whether the greater binding observed with IL-2 fusion
proteins was
due to the presence of IL-2 receptors or FcRyI receptors on the cells, excess
mouse IgG (mIgG)
was used to compete the binding at the Fc receptors. As illustrated in FIG. 4,
background levels
of binding were observed with the antibody and both antibody-IL2 fusion
proteins in the
presence of a 50-fold molar excess of mIgG. This suggests that the increased
signal binding of
antibody-IL2 fusion proteins was due to increased binding to the Fc receptor.
Cell lines expressing Fc receptors are useful for testing the binding
affinities of candidate
fusion proteins to Fc receptors in order to identify antibody-based fusion
proteins with enhanced
in vivo half lives. Candidate antibody-based fusion proteins can be tested by
the above-described
methods. Candidate antibody-based fusion proteins with substantially reduced
binding affinity
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for an Fc receptor will be identified as antibody-based fusion proteins with
enhanced in vivo half
lives.
1.3 Measuring the circulating half-life of antibody-IL2 fusion proteins with
Cyl
and Cy~4 IgG constant region.
In order to assess whether using the Fc region of an IgG isotype having
reduced affinity
for Fc receptors will enhance the in vivo circulating half life, fusion
proteins containing the Cy 1
isotype heavy chain (i.e., hu-KSyl-IL2) were compared to fusion proteins
containing the Cy4
isotype heavy chain (i. e., hu-KSy4-IL2).
Purified humanized KS-1/4-IL2 fusion proteins containing either the Cyl or Cy4
isotype
heavy chain were buffer-exchanged by diafiltration into phosphate buffered
saline (PBS) and
diluted further to a concentration of 100 pg/ml. Approximately 20 pg of the
antibody-based
fusion protein (0.2 ml) was injected into 6-8 week old Balb/c mice in the tail
vein using a slow
push. Four mice were injected per group. At various time points, small blood
samples were
taken by retro-orbital bleeding from anaesthetized animals and collected in
tubes containing
citrate buffer to prevent clotting. Cells were removed by centrifugation in an
Eppendorf
high-speed tabletop centrifuge for 5 rnin. The plasma was removed with a
micropipettor and
frozen at -70°C. The concentration of human antibody determinants in
the mouse blood was
measured by ELISA. A capture antibody specific for human H and L antibody
chains was used
for capture of the fusion proteins from the diluted plasma samples. After a
two hour incubation
in antibody-coated 96-well plates, the unbound material was removed by three
washes with
ELISA buffer (0.01 % Tween 80 in PBS). A second incubation step used either an
anti-human Fc
antibody (for detection of both antibody and intact fusion protein), or an
anti-human IL-2
antibody (for detection of only the intact fusion protein). Both antibodies
were conjugated to
horse radish peroxidase (HRP). After a one hour incubation, the unbound
detecting antibody
was removed by washing with ELISA buffer and the amount of bound HPR was
determined by
incubation with substrate and measuring in a spectrophotometer.
As depicted in FIG. 5, the a phase half life of the hu-KSy4-IL2 fusion protein
was
significantly longer than the a phase half life of the hu-KSyI-IL2 fusion
protein. The increased
half life is best exemplified by the significantly higher concentrations of
the hu-KSy4-IL2 fusion
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protein (3.3 pg/ml) compared to the hu-KSyI-IL2 fusion protein (60 ng/ml)
found in mice after
24 hours.
The hu-KSyI-IL2 protein had a rapid distribution (a) phase followed by a
slower
catabolic ((3) phase, as reported earlier for the chimeric 14.18-IL2 fusion
protein. See, Gillies et
S al., BIOCONJ. CHEM. 4: 230-235 ( 1993}. In the Gillies et al. study, only
antibody determinants
were measured, so it was not clear if the clearance represented the clearance
of the intact fusion
protein or the clearance of the antibody component of the fusion protein. In
the present Example,
samples were assayed using both ( 1 ) an antibody-specific ELISA, and (2) a
fusion protein-
specific ELISA (i.e., an ELISA that requires that both the antibody and IL-2
components be
physically linked). As illustrated in FIG. 5, in animals injected with the hu-
KSyI-IL2 fusion
protein, the amount of circulating fusion protein was lower than the total
amount of circulating
antibody, especially at the 24 hr time point. This suggests that the fusion
protein is being
proteolytically cleaved in vivo and that the released antibody continues to
circulate. Surprisingly,
in animals injected with the hu-KSy4-IL2 fusion protein, there was no
significant differences
between the amount of circulating fusion protein and the total amount of
circulating antibody.
This suggests the hu-KSy4-IL2 fusion protein was not being proteolytically
cleaved in these
animals during the 24 hour period measured.
As discussed above, Cyl and Cy3 have binding affinity for Fc receptors,
whereas while
Cy4 has reduced binding affinity and Cy2 has no binding affinity for Fc
receptors. The present
Example described methods for producing antibody-based fusion proteins using
the Cy4 Fc
region, an IgG isotype having reduced affinity for Fc receptors, and
established that such
antibody-based fusion proteins have enhanced in vivo circulating half life.
Accordingly, a skilled
artisan can use these methods to produce antibody-based fusion proteins with
the Cy2 Fc region,
instead of the Cy4 Fc region, in order to enhance the circulating half life of
fusion proteins. A
Hu-KS-IL2 fusion protein utilizing the human Cy2 region can be constructed
using the same
restriction fragment replacement and the above-described methods for Cy4-IL2
fusion protein.
and tested using the methods described herein to demonstrate increased
circulating half life.
Antibody-based fusion proteins with the Cy2 Fc region, or any other Fc region
having reduced
binding affinity or lacking binding affinity for a Fc receptor will have
enhanced in vivo
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circulating half life compared to antibody-based fusion proteins having
binding affinity for a Fc
receptor.
Example 2 Mutating the human Cyl or Cy3 gene in antibody-based fusion protein
constructs to improve their in vivo circulating half life.
IgG molecules interact with several molecules in the circulation, including
members of
the complement system of proteins (e.g., C 1 q fragment), as well as the three
classes of FcR. The
important residues for C 1 q binding are residues Glu3, 8, Lys32o, and Lys3u
which are located in the
CH2 domains of human heavy chains. Tao et al., J. ExP. MED. 178: 661-667
(1993). In order to
discriminate between FcR and C 1 q binding as mechanisms for rapid clearance,
we substituted
the more drastically altered Cy2 hinge-proximal segment into the Cyl heavy
chain. This
mutation is expected to affect FcR binding but not complement fixation.
The mutation was achieved by cloning and adapting the small region between the
hinge
and the beginning of the CH2 exon of the germ line Cyl gene using overlapping
polymerase
chain reactions (PCR). The PCR primers were designed to substitute the new
sequence at the
1 S junction of two adjacent PCR fragments spanning a Pst I to Drd I fragment
(see FIG. 6). In the
first step, two separate PCR reactions with primers l and 2 (SEQ ID NOS: 5 and
6, respectively),
or primers 3 and 4 (SEQ ID NOS: 7 and 8, respectively), were prepared using
the Cyl gene as
the template. The cycle conditions for the primary PCR were 35 cycles of:
94°C for 45 sec,
annealing at 48°C for 45 seconds, and primer extension at 72°C
for 45 sec. The products of each
PCR reaction were used as template for the second, joining reaction step. One
tenth of each
primary reaction was mixed together and combined with primers 1 and 4 to
amplify only the
combined product of the two initial PCR products. The conditions for the
secondary PCR were:
94°C for 1 min, annealing at S 1 °C for 1 min, and primer
extension at 72°C for 1 min. Joining
occurs as a result of the overlapping between the two individual fragments
which pairs with the
end of the other, following denaturation and annealing. The fragments that
form hybrids get
extended by the Taq polymerase, and the complete, mutated product was
selectively amplified by
the priming of the outer primers, as shown in FIG. 6. The final PCR product
was cloned in a
plasmid vector and its sequence verified by DNA sequence analysis.
The assembly of the mutated gene was done in multiple steps. In the first
step, a cloning
vector containing the human Cyl gene was digested with Pst I and Xho I to
remove the
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non-mutated hinge-CH2-CH3 coding sequences. A Drd I to Xho I fragment encoding
part of
CH2, all of CH3 and the fused human IL-2 coding sequences was prepared from
the Cy 1-IL2
vector, described above. A third fragment was prepared from the subcloned PCR
product by
digestion with Pst I and Drd I. All three fragments were purified by agarose
gel electrophoresis
and ligated together in a single reaction mixture. The ligation product was
used to transform
competent E. coli and colonies were selected by growth on plates containing
ampicillin.
Correctly assembled recombinant plasmids were identified by restriction
analyses of plasmid
DNA preparations from isolated transformants and mutated genes were confirmed
by DNA
sequence analysis. The Hind III to Xho I fragment from the mutated Cyl-IL2
gene was used to
reassemble the complete hu-KS antibody-IL2 fusion protein expression vector.
In order to assess the enhancement of the in viva circulating half life
induced by a
mutation of an important amino acid for FcR binding, and to discriminate
between FcR and C 1 q
binding as mechanisms for rapid clearance, the in viva plasma concentration of
the mutated
hu-KSyl-IL2 was compared to the plasma concentration of hu-KSyl-IL2 at various
specified
times. As illustrated in FIG. 7, the in viva clearance rates of the mutated hu-
KSyI-IL2 and
hu-KSy4-IL2 were significantly lower than the clearance rate of hu-KSyI-IL2.
These results
suggests that an antibody-based fusion protein with enhanced in viva
circulating half life can be
obtained by modifying sequences necessary for binding to Fc receptors in
isotypes that have
binding affinity for an Fc receptor. Further, the results suggests that the
mechanisms for rapid
clearance involve FcR binding rather than Clq binding.
The skilled artisan will understand, from the teachings of the present
invention, that
several other mutations to the C~y 1 or Cy3 genes can be introduced in order
to reduce binding to
FcR and enhance the in viva circulating half life of an antibody-based fusion
protein. Moreover,
mutations can also be introduced into the Cy4 gene in order to further reduce
the binding of Cy4
fusion proteins to FcR. For example, additional possible mutations include
mutations in the
hinge proximal amino acid residues, mutating Pro33,, or by mutating the single
N-linked
glycosylation site in all IgG Fc regions. The latter is located at Asnz9, as
part of the canonical
sequence: Asn-X-Thr/Ser, where the second position can be any amino acid (with
the possible
exception of Pro), and the third position is either Thr or Ser. A conservative
mutation to the
amino acid Gln, for example, would have little effect on the protein but would
prevent the
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attachment of any carbohydrate side chain. A strategy for mutating this
residue might follow the
general procedure, just described, for the hinge proximal region. Methods for
generating point
mutations in cloned DNA sequences are well established in the art and
commercial kits are
available from several vendors for this purpose.
Example 3 Increasing the circulating half life of receptor-antibody-based
fusion
proteins.
Several references have reported that the Fc portion of human IgG can serve as
a useful
carrier for many ligand-binding proteins, or receptors, with biological
activity. Some of these
ligand-binding proteins have been fused to the N-terminal of the Fc portion of
an Ig, such as
CD4, CTLA-4, and TNF receptors. See, for example, Capon et al., NATURE 337:
525-531
(1989); Linsley et al., J. ExP. MED. 174: 561-569 (1991); Wooley et al., J.
IMMLJNOL. 151: 6602-
6607 (1993). Increasing the circulating half life of receptor-antibody-based
fusion proteins may
permit the ligand-binding protein partner (i. e., the second non-Ig protein)
to more effectively
( 1 ) block receptor-ligand interactions at the cell surface; or (2)
neutralize the biological activity
of a molecule (e.g., a cytokine) in the fluid phase of the blood, thereby
preventing it from
reaching its cellular target. In order to assess whether reducing the ability
of receptor-antibody-
based fusion proteins to bind to IgG receptors will enhance their in vivo
circulating half life,
receptor-antibody-based fusion proteins with human Cyl Fc regions are compared
to antibody-
based fusion proteins with human Cy4 Fc regions.
To construct CD4-antibody-based fusion proteins, the ectodomain of the human
CD4 cell
surface receptor is cloned using PCR from human peripheral blood monocytic
cells (PBMC).
The cloned CD4 receptor includes compatible restriction sites and splice donor
sites described in
Example 1. The expression vector contains a unique Xba I cloning site
downstream of the CMV
early promoter, and the human Cyl or Cy4 gene downstream of their endogenous
Hind III site.
The remainder of the plasmid contains bacterial genetic information for
propagation in E. coli, as
well as a dhfr selectable marker gene. Ligated DNAs are used to transform
competent bacteria
and recombinant plasmids are identified from restriction analyses from
individual bacterial
colonies. Two plasmid DNA constructs are obtained: CD4-Cyl and CD4-Cy4.
The expression plasmids are used to transfect mouse myeloma cells by
electroporation
and transfectants are selected by growth in culture medium containing
methotrexate (0.1 ~M).
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Transfectants expressing the fusion proteins are identified by ELISA analyses
and are expanded
in culture in order to generate fusion protein for purification by binding to
and elution from
protein A Sepharose. Purified proteins in chromatography elution buffer are
diafiltered into PBS
and diluted to a final concentration of 100 pg/ml. Balb/c mice are injected
with 0.2 ml (20 pg)
of either the CD4-Cyl or CD4-Cy4 fusion protein and the pharmacokinetics are
tested as
described in Example 1.3. The CD4-CY4 fusion protein has a significantly
greater half life than
the CD4-Cyl fusion protein.
Equivalents
The invention may be embodied in other specific forms without departing from
the spirit
or essential characteristics thereof. The foregoing embodiments are therefore
to be considered in
all respects illustrative rather than limiting on the invention described
herein. Scope of the
invention is thus indicated by the appended claims rather than by the
foregoing description, and
all changes which come within the meaning and range of equivalency of the
claims are intended
to be embraced therein.
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<110> GILLIES, Stephen D
L0, Kin-Ming
LAN, Yan
WESOLOWSKI, John
<120> Enhancing the Circulating Half-life of
Antibody-based Fusion Proteins
<130> LEX-003PC
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Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
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Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
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Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
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Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
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Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
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Xaa Xaa Xaa Xaa Xaa Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
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Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
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Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
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Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
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Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
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Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
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Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
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Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
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<223> IGG-2 CHAIN C REGION
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Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
100 105 110
Xaa Xaa Xaa Xaa Xaa Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
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295 250 255
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn
260 265 270
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275 280 285
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
290 295 300
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
305 310 315 320
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<213> Homo sapiens
<220>
<223> IGG-3 CHAIN C REGION
<400> 3
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 25
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
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Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
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<210> 4
<211> 444
<212> PRT
<213> Homo Sapiens
<220>
<223> IGG-4 CHAIN C REGION
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1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
100 105 110
Xaa Xaa Xaa Xaa Xaa Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
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180 185 190
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210 215 220
Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
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340 345 350
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935 940
<210> 5
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer 1
<900> 5
catcggtctt ccccctg 17
<210> 6
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer 2
<400> 6
cggtcctgcg acgggaggtg ctgaggaaga gatgg 35
<210> 7
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer 3
<400> 7
tcttcctcag cacctcccgt cgcaggaccg tcagtcttcc tcttc 95
<210> 8
<211> 17
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer 9
<400> 8
gaggcgtggt cttgtag
17
SUBSTITUTE SHEET (RULE 26)
