NZ616174B2 - Dual variable region antibody-like binding proteins having cross-over binding region orientation - Google Patents
Dual variable region antibody-like binding proteins having cross-over binding region orientation Download PDFInfo
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- NZ616174B2 NZ616174B2 NZ616174A NZ61617412A NZ616174B2 NZ 616174 B2 NZ616174 B2 NZ 616174B2 NZ 616174 A NZ616174 A NZ 616174A NZ 61617412 A NZ61617412 A NZ 61617412A NZ 616174 B2 NZ616174 B2 NZ 616174B2
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Abstract
Discloses antibody-like binding proteins comprising four polypeptide chains that form four antigen binding sites. Each pair of polypeptides forming an antibody-like binding protein possess dual variable domains and have a cross-over orientation. Also disclosed is a method of making said polypeptide. (...)
Description
DUAL VARIABLE REGION ANTIBODY-LIKE BINDING PROTEINS HAVING
CROSS-OVER BINDING REGION ORIENTATION
FIELD OF THE INVENTION
The invention relates to antibody-like binding proteins comprising four
polypeptide chains that form four antigen binding sites, wherein each pair of polypeptides
forming the antibody-like binding protein possesses dual variable domains having a
cross-over orientation. The invention also relates to methods for making such antigen-
like binding proteins.
BACKGROUND OF THE INVENTION
Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific
antibodies having binding specificities for two different antigens can be produced using
recombinant technologies and are projected to have broad clinical applications. It is well
known that complete IgG antibody molecules are Y-shaped molecules comprising four
polypeptide chains: two heavy chains and two light chains. Each light chain consists of
two domains, the N-terminal domain being known as the variable or V domain (or
region) and the C-terminal domain being known as the constant (or C ) domain (constant
kappa (Cκ) or constant lambda (Cλ) domain). Each heavy chain consists of four or five
domains, depending on the class of the antibody. The N-terminal domain is known as the
variable (or V ) domain (or region), which is followed by the first constant (or C )
H H1
domain, the hinge region, and then the second and third constant (or C and C )
H2 H3
domains. In an assembled antibody, the V and V domains associate together to form
an antigen binding site. Also, the C and C domains associate together to keep one
L H1
heavy chain associated with one light chain. The two heavy-light chain heterodimers
associate together by interaction of the C and C domains and interaction between the
H2 H3
hinge regions on the two heavy chains.
It is known that proteolytic digestion of an antibody can lead to the production of
antibody fragments (Fab and Fab2). Such fragments of the whole antibody can exhibit
antigen binding activity. Antibody fragments can also be produced recombinantly. Fv
fragments, consisting only of the variable domains of the heavy and light chains
associated with each other may be obtained. These Fv fragments are monovalent for
antigen binding. Smaller fragments such as individual variable domains (domain
antibodies or dABs; Ward et al., 1989, Nature 341(6242): 544-46), and individual
complementarity determining regions or CDRs (Williams et al., 1989, Proc. Natl. Acad.
Sci. U.S.A. 86(14): 5537-41) have also been shown to retain the binding characteristics of
the parent antibody, although most naturally occurring antibodies generally need both a
V and V to retain full binding potency.
Single chain variable fragment (scFv) constructs comprise a V and a V domain
of an antibody contained in a single polypeptide chain wherein the domains are separated
by a flexible linker of sufficient length (more than 12 amino acid residues), that forces
intramolecular interaction, allowing self-assembly of the two domains into a functional
epitope binding site (Bird et al., 1988, Science 242(4877): 423-26). These small proteins
(MW ~25,000 Da) generally retain specificity and affinity for their antigen in a single
polypeptide and can provide a convenient building block for larger, antigen-specific
molecules.
An advantage of using antibody fragments rather than whole antibodies in
diagnosis and therapy lies in their smaller size. They are likely to be less immunogenic
than whole antibodies and more able to penetrate tissues. A disadvantage associated with
the use of such fragments is that they have only one antigen binding site, leading to
reduced avidity. In addition, due to their small size, they are cleared very fast from the
serum, and hence display a short half-life.
It has been of interest to produce bispecific antibodies (BsAbs) that combine the
antigen binding sites of two antibodies within a single molecule, and therefore, would be
able to bind two different antigens simultaneously. Besides applications for diagnostic
purposes, such molecules pave the way for new therapeutic applications, e.g., by
redirecting potent effector systems to diseased areas (where cancerous cells often develop
mechanisms to suppress normal immune responses triggered by monoclonal antibodies,
like antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent
cytotoxicity (CDC)), or by increasing neutralizing or stimulating activities of antibodies.
This potential was recognized early on, leading to a number of approaches for obtaining
such bispecific antibodies. Initial attempts to couple the binding specificities of two
whole antibodies against different target antigens for therapeutic purposes utilized
chemically fused heteroconjugate molecules (Staerz et al., 1985, Nature 314(6012): 628-
31).
Bispecific antibodies were originally made by fusing two hybridomas, each
capable of producing a different immunoglobulin (Milstein et al., 1983, Nature
305(5934): 537-40), but the complexity of species (up to ten different species) produced
in cell culture made purification difficult and expensive (George et al., 1997, THE
ANTIBODIES 4: 99-141 (Capra et al., ed., Harwood Academic Publishers)). Using this
format, a mouse IgG2a and a rat IgG2b antibody were produced together in the same cell
(e.g., either as a quadroma fusion of two hybridomas, or in engineered CHO cells).
Because the light chains of each antibody associate preferentially with the heavy chains
of their cognate species, three major species of antibody are assembled: the two parental
antibodies, and a heterodimer of the two antibodies comprising one heavy/light chain pair
of each, associating via their Fc portions. The desired heterodimer can be purified from
this mixture because its binding properties to Protein A are different from those of the
parental antibodies: rat IgG2b does not bind to Protein A, whereas the mouse IgG2a
does. Consequently, the mouse-rat heterodimer binds to Protein A but elutes at a higher
pH than the mouse IgG2a homodimer, and this makes selective purification of the
bispecific heterodimer possible (Lindhofer et al., 1995, J. Immunol. 155(1): 219-25). The
resulting bispecific heterodimer is fully non-human, hence highly immunogenic, which
could have deleterious side effects (e.g., "HAMA" or "HARA" reactions), and/or
neutralize the therapeutic. There remained a need for engineered bispecifics with
superior properties that can be readily produced in high yield from mammalian cell
culture.
Despite the promising results obtained using heteroconjugates or bispecific
antibodies produced from cell fusions as cited above, several factors made them
impractical for large scale therapeutic applications. Such factors include: rapid clearance
of heteroconjugates in vivo, the laboratory intensive techniques required for generating
either type of molecule, the need for extensive purification of heteroconjugates away
from homoconjugates or mono-specific antibodies, and the generally low yields obtained.
Genetic engineering has been used with increasing frequency to design, modify,
and produce antibodies or antibody derivatives with a desired set of binding properties
and effector functions. A variety of recombinant methods have been developed for
efficient production of BsAbs, both as antibody fragments (Carter et al., 1995, J.
Hematother. 4(5): 463-70; Pluckthun et al., 1997, Immunotechnology 3(2): 83-105;
Todorovska et al., 2001, J. Immunol. Methods 248(1-2): 47-66) and full length IgG
formats (Carter, 2001, J. Immunol. Methods 248(1-2): 7-15).
Combining two different scFvs results in BsAb formats with minimal molecular
mass, termed sc-BsAbs or Ta-scFvs (Mack et al., 1995, Proc. Natl. Acad. Sci. U.S.A.
92(15): 7021-25; Mallender et al., 1994, J. Biol. Chem. 269(1): 199-206). BsAbs have
been constructed by genetically fusing two scFvs to a dimerization functionality such as a
leucine zipper (Kostelny et al., 1992, J. Immunol. 148(5): 1547-53; de Kruif et al., 1996,
J. Biol. Chem. 271(13): 7630-34).
Diabodies are small bivalent and bispecific antibody fragments. The fragments
comprise a V connected to a V on the same polypeptide chain, by using a linker that is
too short (less than 12 amino acid residues) to allow pairing between the two domains on
the same chain. The domains are forced to pair intermolecularly with the complementary
domains of another chain and create two antigen-binding sites. These dimeric antibody
fragments, or "diabodies," are bivalent and bispecific (Holliger et al., 1993, Proc. Natl.
Acad. Sci. U.S.A. 90(14): 6444-48). Diabodies are similar in size to a Fab fragment.
Polypeptide chains of V and V domains joined with a linker of between 3 and 12 amino
acid residues form predominantly dimers (diabodies), whereas with a linker of between 0
and 2 amino acid residues, trimers (triabodies) and tetramers (tetrabodies) predominate.
In addition to the linker length, the exact pattern of oligomerization seems to depend on
the composition as well as the orientation of the variable domains (Hudson et al., 1999, J.
Immunol. Methods 231(1-2): 177-89). The predictability of the final structure of diabody
molecules is very poor.
Although sc-BsAb and diabody-based constructs display interesting clinical
potential, it was shown that such non-covalently associated molecules are not sufficiently
stable under physiological conditions. The overall stability of a scFv fragment depends
on the intrinsic stability of the V and V domains as well as on the stability of the
domain interface. Insufficient stability of the V -V interface of scFv fragments has
often been suggested as a main cause of irreversible scFv inactivation, since transient
opening of the interface, which would be allowed by the peptide linker, exposes
hydrophobic patches that favor aggregation and therefore instability and poor production
yield (Wörn et al., 2001, J. Mol. Biol. 305(5): 989-1010).
An alternative method of manufacturing bispecific bivalent antigen-binding
proteins from V and V domains is described in U.S. Patent No. 5,989,830. Such
double head and dual Fv configurations are obtained by expressing a bicistronic vector,
which encodes two polypeptide chains. In the Dual-Fv configuration, the variable
domains of two different antibodies are expressed in a tandem orientation on two separate
chains (one heavy chain and one light chain), wherein one polypeptide chain has two
times a V in series separated by a peptide linker (V -linker-V ) and the other
H H1 H2
polypeptide chain consists of complementary V domains connected in series by a
peptide linker (V -linker-V ). In the cross-over double head configuration, the variable
L1 L2
domains of two different antibodies are expressed in a tandem orientation on two separate
polypeptide chains (one heavy chain and one light chain), wherein one polypeptide chain
has two times a V in series separated by a peptide linker (V -linker-V ) and the other
H H1 H2
polypeptide chain consists of complementary V domains connected in series by a
peptide linker in the opposite orientation (V -linker-V ). Molecular modeling of the
L2 L1
constructs suggested the linker size to be long enough to span 30-40 Å (15–20 amino acid
residues).
Increasing the valency of an antibody is of interest as it enhances the functional
affinity of that antibody due to the avidity effect. Polyvalent protein complexes (PPC)
with an increased valency are described in U.S. Patent Application Publication No. US
2005/0003403 A1. PPCs comprise two polypeptide chains generally arranged laterally to
one another. Each polypeptide chain typically comprises three or four "v-regions," which
comprise amino acid sequences capable of forming an antigen binding site when matched
with a corresponding v-region on the opposite polypeptide chain. Up to about six "v-
regions" can be used on each polypeptide chain. The v-regions of each polypeptide chain
are connected linearly to one another and may be connected by interspersed linking
regions. When arranged in the form of the PPC, the v-regions on each polypeptide chain
form individual antigen binding sites. The complex may contain one or several binding
specificities.
A strategy was proposed by Carter et al. (Ridgway et al., 1996, Protein Eng. 9(7):
617-21; Carter, 2011, J. Immunol. Methods 248(1-2): 7-15) to produce a Fc heterodimer
using a set of "knob-into-hole" mutations in the C domain of Fc. These mutations lead
to the alteration of residue packing complementarity between the C domain interface
within the structurally conserved hydrophobic core so that formation of the heterodimer
is favored as compared with homodimers, which achieves good heterodimer expression
from mammalian cell culture. Although the strategy led to higher heterodimer yield, the
homodimers were not completely suppressed (Merchant et al., 1998, Nat. Biotechnol.
16(7): 677-81.
Gunasekaran et al. explored the feasibility of retaining the hydrophobic core
integrity while driving the formation of Fc heterodimer by changing the charge
complementarity at the C domain interface (Gunasekaran et al., 2010, J. Biol. Chem.
285(25): 19637-46). Taking advantage of the electrostatic steering mechanism, these
constructs showed efficient promotion of Fc heterodimer formation with minimum
contamination of homodimers through mutation of two pairs of peripherally located
charged residues. In contrast to the knob-into-hole design, the homodimers were evenly
suppressed due to the nature of the electrostatic repulsive mechanism, but not totally
avoided.
Davis et al. describe an antibody engineering approach to convert Fc homodimers
into heterodimers by interdigitatingβ-strand segments of human IgG and IgA C
domains, without the introduction of extra interchain disulfide bonds (Davis et al., 2010,
Protein Eng. Des. Sel. 23(4): 195-202). Expression of SEEDbody (Sb) fusion proteins by
mammalian cells yields Sb heterodimers in high yield that are readily purified to
eliminate minor by-products.
U.S. Patent Application Publication No. US 2010/331527 A1 describes a
bispecific antibody based on heterodimerization of the C domain, introducing in one
heavy chain the mutations H95R and Y96F within the C domain. These amino acid
substitutions originate from the C domain of the IgG3 subtype and will heterodimerize
with an IgG1 backbone. A common light chain prone to pair with every heavy chain is a
prerequisite for all formats based on heterodimerization though the C domain. A total
of three types of antibodies are therefore produced: 50% having a pure IgG1 backbone,
one-third having a pure H95R and Y96F mutated backbone, and one-third having two
different heavy chains (bispecific). The desired heterodimer can be purified from this
mixture because its binding properties to Protein A are different from those of the
parental antibodies: IgG3-derived C domains do not bind to Protein A, whereas the
IgG1 does. Consequently, the heterodimer binds to Protein A, but elutes at a higher pH
than the pure IgG1 homodimer, and this makes selective purification of the bispecific
heterodimer possible.
U.S. Patent No. 7,612,181 describes a Dual-Variable-Domain IgG (DVD-IgG)
bispecific antibody that is based on the Dual-Fv format described in U.S. Patent No.
5,989,830. A similar bispecific format was also described in U.S. Patent Application
Publication No. US 2010/0226923 A1. The addition of constant domains to respective
chains of the Dual-Fv (C -Fc to the heavy chain and kappa or lambda constant domain
to the light chain) led to functional bispecific antibodies without any need for additional
modifications (i.e., obvious addition of constant domains to enhance stability). Some of
the antibodies expressed in the DVD-Ig/TBTI format show a position effect on the
second (or innermost) antigen binding position (Fv2). Depending on the sequence and
the nature of the antigen recognized by the Fv2 position, this antibody domain displays a
reduced affinity to its antigen (i.e., loss of on-rate in comparison to the parental
antibody). One possible explanation for this observation is that the linker between V
and V protrudes into the CDR region of Fv2, making the Fv2 somewhat inaccessible
for larger antigens.
The second configuration of a bispecific antibody fragment described in U.S.
Patent No. 5,989,830 is the cross-over double head (CODH), having the following
orientation of variable domains expressed on two chains:
V -linker-V , for the light chain, and
L1 L2
V -linker-V , for the heavy chain
H2 H1
The '830 patent discloses that a bispecific cross-over double-head antibody fragment
(construct GOSA.E) retains higher binding activity than a Dual-Fv (see page 20, lines 20-
50 of the '830 patent), and further discloses that this format is less impacted by the linkers
that are used between the variable domains (see page 20-21 of the '830 patent).
SUMMARY OF THE INVENTION
The invention provides an antibody-like binding protein comprising four
polypeptide chains that form four antigen binding sites, wherein two polypeptide chains
have a structure represented by the formula:
V -L -V -L -C [I]
L1 1 L2 2 L
and two polypeptide chains have a structure represented by the formula:
V -L -V -L -C -Fc [II]
H2 3 H1 4 H1
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain;
H1 H1
Fc is the immunoglobulin hinge region and C , C immunoglobulin heavy chain
H2 H3
constant domains;
L , L , L , and L are amino acid linkers;
1 2 3 4
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
The invention also provides an antibody-like binding protein comprising two
polypeptide chains that form two antigen binding sites, wherein a first polypeptide chain
has a structure represented by the formula:
V -L -V -L -C [I]
L1 1 L2 2 L
and a second polypeptide chain has a structure represented by the formula:
V -L -V -L -C [II]
H2 3 H1 4 H1
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain;
H1 H1
L , L , L , and L are amino acid linkers;
1 2 3 4
and wherein the first and second polypeptides form a cross-over light chain-heavy chain
pair.
The invention further provides a method of making an antibody-like binding
protein comprising four polypeptide chains that form four antigen binding sites,
comprising identifying a first antibody variable domain that binds a first target antigen
and a second antibody variable domain that binds a second target antigen, each
containing a V , and a V ; assigning either the light chain or the heavy chain as template
chain; assigning the V of the first antibody variable domain or the second antibody
variable domain as V ; assigning a V , a V , and a V according to formulas [I] and
L1 L2 H1 H2
[II] below:
V -L -V -L -C [I]
L1 1 L2 2 L
V -L -V -L -C -Fc [II]
H2 3 H1 4 H1
determining maximum and minimum lengths for L , L , L , and L ; generating the
1 2 3 4
polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I
and II that bind the first target antigen and the second target antigen when combined to
form the antibody-like binding protein;
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain;
H1 H1
Fc is the immunoglobulin hinge region and C , C immunoglobulin heavy chain
H2 H3
constant domains; and
L , L , L , and L are amino acid linkers;
1 2 3 4
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
The invention further provides a method of making an antibody-like binding
protein comprising four polypeptide chains that form four antigen binding sites,
comprising identifying a first antibody variable domain that binds a first target antigen
and a second antibody variable domain that binds a second target antigen, each
containing a V , and a V ; assigning either the light chain or the heavy chain as template
chain; assigning the V of the first antibody variable domain or the second antibody
variable domain as V ; assigning a V , a V , and a V according to formulas [I] and
L1 L2 H1 H2
[II] below:
V -L -V -L -C [I]
L1 1 L2 2 L
V -L -V -L -C [II]
H2 3 H1 4 H1
determining maximum and minimum lengths for L , L , L , and L ; generating
1 2 3 4
polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I
and II that bind the first target antigen and the second target antigen when combined to
form the antibody-like binding protein;
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain; and
H1 H1
L , L , L , and L are amino acid linkers;
1 2 3 4
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
Specific embodiments of the invention will become evident from the following
more detailed description of certain embodiments and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Schematic representation of the antigen binding domains Fv1 and Fv2 within
the dual V region configuration and arrangement of their respective peptide linkers L
and L in the TBTI format.
Figure 2. Schematic diagram (2D) of the antigen binding domains Fv1 (anti-IL4) and
Fv2 (anti-IL13) within the cross-over dual variable (CODV) configuration and the
arrangement of their respective peptide linkers.
Figure 3. Schematic representation of the Fv anti-IL4 and Fab anti-IL13 showing one
possible spatial arrangement obtained by protein-protein docking of Fv of anti-IL4 and
the Fv of anti-IL13.
Figure 4. Assessment of tetravalent and bispecific binding ability of the CODV protein
in a BIACORE assay by injecting the two antigens sequentially or simultaneously over a
DVD-Ig protein–coated chip. The maximal signal observed by sequential injection can
be obtained by co-injection of both antigens, demonstrating saturation of all binding sites.
Figure 5. Schematic diagram (2D) of the antigen binding domains within the CODV
configuration and arrangement of their respective peptide linker L (L and L ) and L
L 1 2 H
(L and L ). In panel A, the light chain is kept in a "linear or template" alignment,
whereas the heavy chain is in the "cross-over" configuration. In panel B, the heavy chain
is kept in a "linear or template" alignment and the light chain is in the "cross-over"
configuration.
Figure 6. Schematic representation of CODV-Ig design based on whether the light chain
or heavy chain is used as "template."
Figure 7. Comparison of TBTI/DVD-Ig or CODV-Ig molecules incorporating anti-IL4
and anti-IL13 sequences.
Figure 8. Comparison of CODV-Fab and B-Fab formats in a cytotoxic assay using
NALM-6 cells.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides antibody-like binding proteins comprising four
polypeptide chains that form four antigen binding sites, wherein each pair of polypeptides
forming an antibody-like binding protein possesses dual variable domains having a cross-
over orientation. The invention also provides methods for making such antigen-like
binding proteins.
Computer modeling predicted that the cross-over double-head (CODH) design of
U.S. Patent No. 5,989,830 would yield a complex in which both binding sites face in the
opposite direction, without the restraints suggested for the Dual-Fv configuration of U.S.
Patent No. 7,612,181. In particular, computer modeling indicated that the length of the
amino acid linkers between the variable domains was not critical for the CODH design,
but was important for permitting full access to both antigen binding sites in the Dual-Fv
design. As with the DVD-Ig/TBTI format, antibody-like binding protein constructs were
prepared in which constant domains were attached to the CODH configuration to form
antibody-like binding proteins comprising four polypeptide chains that form four antigen
binding sites, wherein each pair of polypeptides forming an antibody-like binding protein
possesses dual variable domains having a cross-over orientation (i.e., CODH-Ig).
CODH-Ig molecules are expected to possess significantly improved stability as compared
with CODH molecules (as DVD-Ig/TBTI possessed improved stability over Dual-Fv
molecules).
In order to test the above hypothesis, a CODH-Ig molecule was prepared using
the anti-IL4 and anti-IL13 antibody sequences described in U.S. Patent Application
Publication No. US 2010/0226923 A1. The CODH-Ig molecule differed from the CODH
molecule of US 2010/0226923 with respect to the lengths of amino acid linkers
separating the variable domains on the respective polypeptide chains. The CODH-Ig
molecules were expressed in cells following transient transfection and were then purified
by Protein A chromatography. Although their size-exclusion chromatography (SEC)
profiles showed aggregation levels of 5-10%, none of the CODH-Ig molecules were
functional, and thus none of the CODH-Ig molecules was able to bind all of its target
antigens. The lack of antigen binding activity may have been due to a perturbed
dimerization of the Fv-regions of the heavy and light chains due to unsuitable linker
lengths compromising correct paratope formation. As a result, a protocol was developed
to identify suitable amino acid linkers for insertion between the two variable domains and
the second variable domain and constant domain on both the heavy and light polypeptide
chains of an antibody-like binding protein. This protocol was based on protein-protein
docking of homology and experimental models of the FvIL4 and FvIL13 regions,
respectively, inclusion of the Fc1 domain the model, and construction of appropriate
linkers between the FvIL4 and FvIL13 regions and between the Fv and constant Fc1
regions.
Standard recombinant DNA methodologies are used to construct the
polynucleotides that encode the polypeptides which form the antibody-like binding
proteins of the invention, incorporate these polynucleotides into recombinant expression
vectors, and introduce such vectors into host cells. See e.g., Sambrook et al., 2001,
MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press,
3rd ed.). Enzymatic reactions and purification techniques may be performed according to
manufacturer's specifications, as commonly accomplished in the art, or as described
herein. Unless specific definitions are provided, the nomenclature utilized in connection
with, and the laboratory procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry described herein are
those well known and commonly used in the art. Similarly, conventional techniques may
be used for chemical syntheses, chemical analyses, pharmaceutical preparation,
formulation, delivery, and treatment of patients.
1. General Definitions
As utilized in accordance with the present disclosure, the following terms, unless
otherwise indicated, shall be understood to have the following meanings. Unless
otherwise required by context, singular terms shall include pluralities and plural terms
shall include the singular.
The term "polynucleotide" as used herein refers to single-stranded or double-
stranded nucleic acid polymers of at least 10 nucleotides in length. In certain
embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or
deoxyribonucleotides or a modified form of either type of nucleotide. Such
modifications include base modifications such as bromuridine, ribose modifications such
as arabinoside and 2',3'-dideoxyribose, and internucleotide linkage modifications such as
phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term
"polynucleotide" specifically includes single-stranded and double-stranded forms of
DNA.
An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic
origin or some combination thereof, which by virtue of its origin the isolated
polynucleotide: (1) is not associated with all or a portion of a polynucleotide in which
the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it
is not linked in nature, or (3) does not occur in nature as part of a larger sequence.
An "isolated polypeptide" is one that: (1) is free of at least some other
polypeptides with which it would normally be found, (2) is essentially free of other
polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell
from a different species, (4) has been separated from at least about 50 percent of
polynucleotides, lipids, carbohydrates, or other materials with which it is associated in
nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a
polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operably
associated (by covalent or noncovalent interaction) with a polypeptide with which it is
not associated in nature, or (7) does not occur in nature. Such an isolated polypeptide can
be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any
combination thereof. Preferably, the isolated polypeptide is substantially free from
polypeptides or other contaminants that are found in its natural environment that would
interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
The term "human antibody" as used herein includes antibodies having variable
and constant regions substantially corresponding to human germline immunoglobulin
sequences. In some embodiments, human antibodies are produced in non-human
mammals, including, but not limited to, rodents, such as mice and rats, and lagomorphs,
such as rabbits. In other embodiments, human antibodies are produced in hybridoma
cells. In still other embodiments, human antibodies are produced recombinantly.
Naturally occurring antibodies typically comprise a tetramer. Each such tetramer
is typically composed of two identical pairs of polypeptide chains, each pair having one
full-length "light" chain (typically having a molecular weight of about 25 kDa) and one
full-length "heavy" chain (typically having a molecular weight of about 50-70 kDa). The
terms "heavy chain" and "light chain" as used herein refer to any immunoglobulin
polypeptide having sufficient variable domain sequence to confer specificity for a target
antigen. The amino-terminal portion of each light and heavy chain typically includes a
variable domain of about 100 to 110 or more amino acids that typically is responsible for
antigen recognition. The carboxy-terminal portion of each chain typically defines a
constant domain responsible for effector function. Thus, in a naturally occurring
antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable
domain (V ) and three constant domains (C , C , and C ), wherein the V domain is
H H1 H2 H3 H
at the amino-terminus of the polypeptide and the C domain is at the carboxyl-terminus,
and a full-length light chain immunoglobulin polypeptide includes a variable domain
(V ) and a constant domain (C ), wherein the V domain is at the amino-terminus of the
L L L
polypeptide and the C domain is at the carboxyl-terminus.
Human light chains are typically classified as kappa and lambda light chains, and
human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and
define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has
several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has
subclasses including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into
subclasses including, but not limited to, IgA1 and IgA2. Within full-length light and
heavy chains, the variable and constant domains typically are joined by a "J" region of
about 12 or more amino acids, with the heavy chain also including a "D" region of about
10 more amino acids. See, e.g., FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven
Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes.
The variable regions of each light/heavy chain pair typically form an antigen binding site.
The variable domains of naturally occurring antibodies typically exhibit the same general
structure of relatively conserved framework regions (FR) joined by three hypervariable
regions, also called complementarity determining regions or CDRs. The CDRs from the
two chains of each pair typically are aligned by the framework regions, which may
enable binding to a specific epitope. From the amino-terminus to the carboxyl-terminus,
both light and heavy chain variable domains typically comprise the domains FR1, CDR1,
FR2, CDR2, FR3, CDR3, and FR4.
The term "native Fc" as used herein refers to a molecule comprising the sequence
of a non-antigen-binding fragment resulting from digestion of an antibody or produced by
other means, whether in monomeric or multimeric form, and can contain the hinge
region. The original immunoglobulin source of the native Fc is preferably of human
origin and can be any of the immunoglobulins, although IgG1 and IgG2 are preferred.
Native Fc molecules are made up of monomeric polypeptides that can be linked into
dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent
association. The number of intermolecular disulfide bonds between monomeric subunits
of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or
subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of a native Fc is a
disulfide-bonded dimer resulting from papain digestion of an IgG. The term "native Fc"
as used herein is generic to the monomeric, dimeric, and multimeric forms.
The term "Fc variant" as used herein refers to a molecule or sequence that is
modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn
(neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage
receptor, are known in the art. Thus, the term "Fc variant" can comprise a molecule or
sequence that is humanized from a non-human native Fc. Furthermore, a native Fc
comprises regions that can be removed because they provide structural features or
biological activity that are not required for the antibody-like binding proteins of the
invention. Thus, the term "Fc variant" comprises a molecule or sequence that lacks one
or more native Fc sites or residues, or in which one or more Fc sites or residues has be
modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility
with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host
cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor
other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).
The term "Fc domain" as used herein encompasses native Fc and Fc variants and
sequences as defined above. As with Fc variants and native Fc molecules, the term "Fc
domain" includes molecules in monomeric or multimeric form, whether digested from
whole antibody or produced by other means.
The term "antibody-like binding protein" as used herein refers to a non-naturally
occurring (or recombinant) molecule that specifically binds to at least one target antigen,
and which comprises four polypeptide chains that form four antigen binding sites,
wherein two polypeptide chains have a structure represented by the formula:
V -L -V -L -C [I]
L1 1 L2 2 L
and two polypeptide chains have a structure represented by the formula:
V -L -V -L -C -Fc [II]
H2 3 H1 4 H1
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain;
H1 H1
Fc is the immunoglobulin hinge region and C , C immunoglobulin heavy chain
H2 H3
constant domains;
L , L , L , and L are amino acid linkers;
1 2 3 4
and wherein the polypeptides of formula I and the polypeptides of formula II form
a cross-over light chain-heavy chain pair. The term "antibody-like binding protein" as
used herein also refers to a non-naturally occurring (or recombinant) molecule that
specifically binds to at least one target antigen, and which comprises two polypeptide
chains that form two antigen binding sites, wherein a first polypeptide chain has a
structure represented by the formula:
V -L -V -L -C [I]
L1 1 L2 2 L
and a second polypeptide chain has a structure represented by the formula:
V -L -V -L -C [II]
H2 3 H1 4 H1
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain;
H1 H1
L , L , L , and L are amino acid linkers;
1 2 3 4
and wherein the first and second polypeptides form a cross-over light chain-heavy chain
pair. A "recombinant" molecule is one that has been prepared, expressed, created, or
isolated by recombinant means.
One embodiment of the invention provides antibody-like binding proteins having
biological and immunological specificity to between one and four target antigens.
Another embodiment of the invention provides nucleic acid molecules comprising
nucleotide sequences encoding polypeptide chains that form such antibody-like binding
proteins. Another embodiment of the invention provides expression vectors comprising
nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains
that form such antibody-like binding proteins. Yet another embodiment of the invention
provides host cells that express such antibody-like binding proteins (i.e., comprising
nucleic acid molecules or vectors encoding polypeptide chains that form such antibody-
like binding proteins).
The term "swapability" as used herein refers to the interchangeability of variable
domains within the CODV format and with retention of folding and ultimate binding
affinity. "Full swapability" refers to the ability to swap the order of both V and V
H1 H2
domains, and therefore the order of V and V domains, in a CODV-Ig (i.e., to reverse
L1 L2
the order) or CODV-Fab while maintaining full functionality of the antibody-like binding
protein as evidenced by the retention of binding affinity. Furthermore, it should be noted
that the designations V and V within a particular CODV-Ig or CODV-Fab refer only to
the domain's location on a particular protein chain in the final format. For example, V
and V could be derived from V and V domains in parent antibodies and placed into
H2 L1 L2
the V and V positions in the antibody-like binding protein. Likewise, V and V
H1 H2 L1 L2
could be derived from V and V domains in parent antibodies and placed in the V
H1 H2 H1
and V positions in the antibody-like binding protein. Thus, the V and V designations
H2 H L
refer to the present location and not the original location in a parent antibody. V and V
domains are therefore "swappable."
An "isolated" antibody-like binding protein is one that has been identified and
separated and/or recovered from a component of its natural environment. Contaminant
components of its natural environment are materials that would interfere with diagnostic
or therapeutic uses for the antibody-like binding protein, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In preferred
embodiments, the antibody-like binding protein will be purified: (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most preferably more
than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal
or internal amino acid sequence by use of a spinning cup sequenator, or (3) to
homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie
blue or, preferably, silver stain. Isolated antibody-like binding proteins include the
antibody-like binding protein in situ within recombinant cells since at least one
component of the antibody-like binding protein's natural environment will not be present.
The terms "substantially pure" or "substantially purified" as used herein refer to a
compound or species that is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual species in the composition). In some
embodiments, a substantially purified fraction is a composition wherein the species
comprises at least about 50% (on a molar basis) of all macromolecular species present.
In other embodiments, a substantially pure composition will comprise more than about
80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition. In
still other embodiments, the species is purified to essential homogeneity (contaminant
species cannot be detected in the composition by conventional detection methods)
wherein the composition consists essentially of a single macromolecular species.
The term "antigen" or "target antigen" as used herein refers to a molecule or a
portion of a molecule that is capable of being bound by an antibody-like binding protein,
and additionally is capable of being used in an animal to produce antibodies capable of
binding to an epitope of that antigen. A target antigen may have one or more epitopes.
With respect to each target antigen recognized by an antibody-like binding protein, the
antibody-like binding protein is capable of competing with an intact antibody that
recognizes the target antigen. A "bivalent" antibody-like binding protein, other than a
"multispecific" or "multifunctional" antibody-like binding protein, is understood to
comprise antigen binding sites having identical antigenic specificity.
A bispecific or bifunctional antibody typically is an artificial hybrid antibody
having two different heavy chain/light chain pairs and two different binding sites or
epitopes. Bispecific antibodies may be produced by a variety of methods including, but
not limited to, fusion of hybridomas or linking of F(ab') fragments.
A F(ab) fragment typically includes one light chain and the V and C domains
H H1
of one heavy chain, wherein the V -C heavy chain portion of the F(ab) fragment cannot
H H1
form a disulfide bond with another heavy chain polypeptide. As used herein, a F(ab)
fragment can also include one light chain containing two variable domains separated by
an amino acid linker and one heavy chain containing two variable domains separated by
an amino acid linker and a C domain.
A F(ab') fragment typically includes one light chain and a portion of one heavy
chain that contains more of the constant region (between the C and C domains), such
H1 H2
that an interchain disulfide bond can be formed between two heavy chains to form a
F(ab') molecule.
The phrases "biological property," "biological characteristic," and the term
"activity" in reference to an antibody-like binding protein of the invention are used
interchangeably herein and include, but are not limited to, epitope affinity and specificity,
ability to antagonize the activity of the antigen target (or targeted polypeptide), the in
vivo stability of the antibody-like binding protein, and the immunogenic properties of the
antibody-like binding protein. Other identifiable biological properties or characteristics
of an antibody-like binding protein include, for example, cross-reactivity, (i.e., with non-
human homologs of the antigen target, or with other antigen targets or tissues, generally),
and ability to preserve high expression levels of protein in mammalian cells. The
aforementioned properties or characteristics can be observed or measured using art-
recognized techniques including, but not limited to ELISA, competitive ELISA, surface
plasmon resonance analysis, in vitro and in vivo neutralization assays, and
immunohistochemistry with tissue sections from different sources including human,
primate, or any other source as the need may be.
The term "immunologically functional immunoglobulin fragment" as used herein
refers to a polypeptide fragment that contains at least the CDRs of the immunoglobulin
heavy or light chains from which the polypeptide fragment was derived. An
immunologically functional immunoglobulin fragment is capable of binding to a target
antigen.
A "neutralizing" antibody-like binding protein as used herein refers to a molecule
that is able to block or substantially reduce an effector function of a target antigen to
which it binds. As used herein, "substantially reduce" means at least about 60%,
preferably at least about 70%, more preferably at least about 75%, even more preferably
at least about 80%, still more preferably at least about 85%, most preferably at least about
90% reduction of an effector function of the target antigen.
The term "epitope" includes any determinant, preferably a polypeptide
determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In
certain embodiments, epitope determinants include chemically active surface groupings
of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl
groups, and, in certain embodiments, may have specific three-dimensional structural
characteristics and/or specific charge characteristics. An epitope is a region of an antigen
that is bound by an antibody or antibody-like binding protein. In certain embodiments,
an antibody-like binding protein is said to specifically bind an antigen when it
preferentially recognizes its target antigen in a complex mixture of proteins and/or
macromolecules. In preferred embodiments, an antibody-like binding protein is said to
specifically bind an antigen when the equilibrium dissociation constant is≤ 10 M, more
preferably when the equilibrium dissociation constant is≤ 10 M, and most preferably
when the dissociation constant is≤ 10 M.
The dissociation constant (K ) of an antibody-like binding protein can be
determined, for example, by surface plasmon resonance. Generally, surface plasmon
resonance analysis measures real-time binding interactions between ligand (a target
antigen on a biosensor matrix) and analyte (an antibody-like binding protein in solution)
by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor;
Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the
analyte (antibody-like binding protein on a biosensor matrix) and presenting the ligand
(target antigen). The term "K ," as used herein refers to the dissociation constant of the
interaction between a particular antibody-like binding protein and a target antigen.
The term "specifically binds" as used herein refers to the ability of an antibody-
like protein or an antigen-binding fragment thereof to bind to an antigen containing an
-6 -7 -8 -9 -
epitope with an Kd of at least about 1 x 10 M, 1 x 10 M, 1 x 10 M, 1 x 10 M, 1 x 10
-11 -12
M, 1 x 10 M, 1 x 10 M, or more, and/or to bind to an epitope with an affinity that
is at least two-fold greater than its affinity for a nonspecific antigen.
The term "linker" as used herein refers to one or more amino acid residues
inserted between immunoglobulin domains to provide sufficient mobility for the domains
of the light and heavy chains to fold into cross over dual variable region
immunoglobulins. A linker is inserted at the transition between variable domains or
between variable and constant domains, respectively, at the sequence level. The
transition between domains can be identified because the approximate size of the
immunoglobulin domains are well understood. The precise location of a domain
transition can be determined by locating peptide stretches that do not form secondary
structural elements such as beta-sheets or alpha-helices as demonstrated by experimental
data or as can be assumed by techniques of modeling or secondary structure prediction.
The linkers described herein are referred to as L , which is located on the light chain
between the N-terminal V and V domains; L , which is also on the light chain is
L1 L2 2
located between the V and C-terminal C domains. The heavy chain linkers are known
L2 L
as L , which is located between the N-terminal V and V domains; and L , which is
3 H2 H1 4
located between the V and C -Fc domains. The linkers L , L , L , and L are
H1 H1 1 2 3 4
independent, but they may in some cases have the same sequence and/or length.
The term "vector" as used herein refers to any molecule (e.g., nucleic acid,
plasmid, or virus) that is used to transfer coding information to a host cell. The term
"vector" includes a nucleic acid molecule that is capable of transporting another nucleic
acid to which it has been linked. One type of vector is a "plasmid," which refers to a
circular double-stranded DNA molecule into which additional DNA segments may be
inserted. Another type of vector is a viral vector, wherein additional DNA segments may
be inserted into the viral genome. Certain vectors are capable of autonomous replication
in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial
origin of replication and episomal mammalian vectors). Other vectors (e.g., non-
episomal mammalian vectors) can be integrated into the genome of a host cell upon
introduction into the host cell and thereby are replicated along with the host genome. In
addition, certain vectors are capable of directing the expression of genes to which they
are operatively linked. Such vectors are referred to herein as "recombinant expression
vectors" (or simply, "expression vectors"). In general, expression vectors of utility in
recombinant DNA techniques are often in the form of plasmids. The terms "plasmid"
and "vector" may be used interchangeably herein, as a plasmid is the most commonly
used form of vector. However, the invention is intended to include other forms of
expression vectors, such as viral vectors (e.g., replication defective retroviruses,
adenoviruses, and adeno-associated viruses), which serve equivalent functions.
The term "operably linked" is used herein to refer to an arrangement of flanking
sequences wherein the flanking sequences so described are configured or assembled so as
to perform their usual function. Thus, a flanking sequence operably linked to a coding
sequence may be capable of effecting the replication, transcription, and/or translation of
the coding sequence. For example, a coding sequence is operably linked to a promoter
when the promoter is capable of directing transcription of that coding sequence. A
flanking sequence need not be contiguous with the coding sequence, so long as it
functions correctly. Thus, for example, intervening untranslated yet transcribed
sequences can be present between a promoter sequence and the coding sequence and the
promoter sequence can still be considered "operably linked" to the coding sequence.
The phrase "recombinant host cell" (or "host cell") as used herein refers to a cell
into which a recombinant expression vector has been introduced. A recombinant host
cell or host cell is intended to refer not only to the particular subject cell, but also to the
progeny of such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but such cells are still included within the scope of the
term "host cell" as used herein. A wide variety of host cell expression systems can be
used to express the antibody-like binding proteins of the invention, including bacterial,
yeast, baculoviral, and mammalian expression systems (as well as phage display
expression systems). An example of a suitable bacterial expression vector is pUC19. To
express an antibody-like binding protein recombinantly, a host cell is transformed or
transfected with one or more recombinant expression vectors carrying DNA fragments
encoding the polypeptide chains of the antibody-like binding protein such that the
polypeptide chains are expressed in the host cell and, preferably, secreted into the
medium in which the host cells are cultured, from which medium the antibody-like
binding protein can be recovered.
The term "transformation" as used herein refers to a change in a cell's genetic
characteristics, and a cell has been transformed when it has been modified to contain a
new DNA. For example, a cell is transformed where it is genetically modified from its
native state. Following transformation, the transforming DNA may recombine with that
of the cell by physically integrating into a chromosome of the cell, or may be maintained
transiently as an episomal element without being replicated, or may replicate
independently as a plasmid. A cell is considered to have been stably transformed when
the DNA is replicated with the division of the cell. The term "transfection" as used
herein refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been
"transfected" when the exogenous DNA has been introduced inside the cell membrane.
A number of transfection techniques are well known in the art. Such techniques can be
used to introduce one or more exogenous DNA molecules into suitable host cells.
The term "naturally occurring" as used herein and applied to an object refers to
the fact that the object can be found in nature and has not been manipulated by man. For
example, a polynucleotide or polypeptide that is present in an organism (including
viruses) that can be isolated from a source in nature and that has not been intentionally
modified by man is naturally-occurring. Similarly, "non-naturally occurring" as used
herein refers to an object that is not found in nature or that has been structurally modified
or synthesized by man.
As used herein, the twenty conventional amino acids and their abbreviations
follow conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty
conventional amino acids; unnatural amino acids such as a-, a-disubstituted amino acids,
N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be
suitable components for the polypeptide chains of the antibody-like binding proteins of
the invention. Examples of unconventional amino acids include: 4-hydroxyproline, g-
carboxyglutamate, e-N,N,N-trimethyllysine, e-N-acetyllysine, O-phosphoserine, N-
acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, s-N-
methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
In the polypeptide notation used herein, the left-hand direction is the amino terminal
direction and the right-hand direction is the carboxyl-terminal direction, in accordance
with standard usage and convention.
Naturally occurring residues may be divided into classes based on common side
chain properties:
(1) hydrophobic: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro;
(2) polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr ;
(3) aliphatic: Ala, Gly, Ile, Leu, Val, Pro;
(4) aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro;
(5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
(6) acidic: Asp, Glu;
(7) basic: His, Lys, Arg;
(8) residues that influence chain orientation: Gly, Pro;
(9) aromatic: His, Trp, Tyr, Phe; and
(10) aromatic hydrophobic: Phe, Trp, Tyr.
Conservative amino acid substitutions may involve exchange of a member of one
of these classes with another member of the same class. Conservative amino acid
substitutions may encompass non-naturally occurring amino acid residues, which are
typically incorporated by chemical peptide synthesis rather than by synthesis in
biological systems. These include peptidomimetics and other reversed or inverted forms
of amino acid residues. Non-conservative substitutions may involve the exchange of a
member of one of these classes for a member from another class.
A skilled artisan will be able to determine suitable variants of the polypeptide
chains of the antibody-like binding proteins of the invention using well-known
techniques. For example, one skilled in the art may identify suitable areas of a
polypeptide chain that may be changed without destroying activity by targeting regions
not believed to be important for activity. Alternatively, one skilled in the art can identify
residues and portions of the molecules that are conserved among similar polypeptides. In
addition, even areas that may be important for biological activity or for structure may be
subject to conservative amino acid substitutions without destroying the biological activity
or without adversely affecting the polypeptide structure.
The term "patient" as used herein includes human and animal subjects.
A "disorder" is any condition that would benefit from treatment using the
antibody-like binding proteins of the invention. "Disorder" and "condition" are used
interchangeably herein and include chronic and acute disorders or diseases, including
those pathological conditions that predispose a patient to the disorder in question.
The terms "treatment" or "treat" as used herein refer to both therapeutic treatment
and prophylactic or preventative measures. Those in need of treatment include those
having the disorder as well as those prone to have the disorder or those in which the
disorder is to be prevented.
The terms "pharmaceutical composition" or "therapeutic composition" as used
herein refer to a compound or composition capable of inducing a desired therapeutic
effect when properly administered to a patient.
The term "pharmaceutically acceptable carrier" or "physiologically acceptable
carrier" as used herein refers to one or more formulation materials suitable for
accomplishing or enhancing the delivery of an antibody-like binding protein.
The terms "effective amount" and "therapeutically effective amount" when used
in reference to a pharmaceutical composition comprising one or more antibody-like
binding proteins refer to an amount or dosage sufficient to produce a desired therapeutic
result. More specifically, a therapeutically effective amount is an amount of an antibody-
like binding protein sufficient to inhibit, for some period of time, one or more of the
clinically defined pathological processes associated with the condition being treated. The
effective amount may vary depending on the specific antibody-like binding protein that is
being used, and also depends on a variety of factors and conditions related to the patient
being treated and the severity of the disorder. For example, if the antibody-like binding
protein is to be administered in vivo, factors such as the age, weight, and health of the
patient as well as dose response curves and toxicity data obtained in preclinical animal
work would be among those factors considered. The determination of an effective
amount or therapeutically effective amount of a given pharmaceutical composition is well
within the ability of those skilled in the art.
One embodiment of the invention provides a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and a therapeutically effective amount
of an antibody-like binding protein.
2. Antibody-like Binding Proteins
In one embodiment of the invention, the antibody-like binding proteins comprise
four polypeptide chains that form four antigen binding sites, wherein two polypeptide
chains have a structure represented by the formula:
V -L -V -L -C [I]
L1 1 L2 2 L
and two polypeptide chains have a structure represented by the formula:
V -L -V -L -C -Fc [II]
H2 3 H1 4 H1
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain;
H1 H1
Fc is the immunoglobulin hinge region and C , C immunoglobulin heavy chain
H2 H3
constant domains;
L , L , L , and L are amino acid linkers;
1 2 3 4
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
In another embodiment of the invention, the antibody-like binding proteins
comprise two polypeptide chains that form two antigen binding sites, wherein a first
polypeptide chain has a structure represented by the formula:
V -L -V -L -C [I]
L1 1 L2 2 L
and a second polypeptide chain has a structure represented by the formula:
V -L -V -L -C [II]
H2 3 H1 4 H1
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain;
H1 H1
L , L , L , and L are amino acid linkers;
1 2 3 4
and wherein the first and second polypeptides form a cross-over light chain-heavy chain
pair.
The antibody-like binding proteins of the invention may be prepared using
domains or sequences obtained or derived from any human or non-human antibody,
including, for example, human, murine, or humanized antibodies.
In some antibody-like binding proteins of the invention, the length of L is at least
twice the length of L . In other antibody-like binding proteins of the invention, the length
of L is at least twice the length of L . In some antibody-like binding proteins of the
invention, the length of L is at least twice the length of L . In other antibody-like
binding proteins of the invention, the length of L is at least twice the length of L .
In some antibody-like binding proteins of the invention, L is 3 to 12 amino acid
residues in length, L is 3 to 14 amino acid residues in length, L is 1 to 8 amino acid
residues in length, and L is 1 to 3 amino acid residues in length. In other antibody-like
binding proteins, L is 5 to 10 amino acid residues in length, L is 5 to 8 amino acid
residues in length, L is 1 to 5 amino acid residues in length, and L is 1 to 2 amino acid
residues in length. In a preferred antibody-like binding protein, L is 7 amino acid
residues in length, L is 5 amino acid residues in length, L is 1 amino acid residues in
length, and L is 2 amino acid residues in length.
In some antibody-like binding proteins of the invention, L is 1 to 3 amino acid
residues in length, L is 1 to 4 amino acid residues in length, L is 2 to 15 amino acid
residues in length, and L is 2 to 15 amino acid residues in length. In other antibody-like
binding proteins, L is 1 to 2 amino acid residues in length, L is 1 to 2 amino acid
residues in length, L is 4 to 12 amino acid residues in length, and L is 2 to 12 amino
acid residues in length. In a preferred antibody-like binding protein, L is 1 amino acid
residue in length, L is 2 amino acid residues in length, L is 7 amino acid residues in
length, and L is 5 amino acid residues in length.
In some antibody-like binding proteins of the invention, L , L , or L may be
1 3 4
equal to zero. However, in antibody-like binding proteins wherein L , L , or L is equal
1 3 4
to zero, the corresponding transition linker between the variable region and constant
region or between the dual variable domains on the other chain cannot be zero. In some
embodiments, L is equal to zero and L is 2 or more amino acid residues, L is equal to
1 3 3
zero and L is equal to 1 or more amino acid residues, or L is equal to 0 and L is 3 or
1 4 2
more amino acid residues.
In some antibody-like binding proteins of the invention, at least one of the linkers
selected from the group consisting of L , L , L , and L contains at least one cysteine
1 2 3 4
residue.
Examples of suitable linkers include a single glycine (Gly) residue; a diglycine
peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues (Gly-
Gly-Gly-Gly; SEQ ID NO: 25); a peptide with five glycine residues (Gly-Gly-Gly-Gly-
Gly; SEQ ID NO: 26); a peptide with six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly;
SEQ ID NO: 27); a peptide with seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly;
SEQ ID NO: 28); a peptide with eight glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-
Gly; SEQ ID NO: 29). Other combinations of amino acid residues may be used such as
the peptide Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 30) and the peptide Gly-Gly-Gly-Gly-
Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 31). Other suitable linkers include a single Ser,
and Val residue; the dipeptide Arg-Thr, Gln-Pro, Ser-Ser, Thr-Lys, and Ser-Leu; Thr-
Lys-Gly-Pro-Ser (SEQ ID NO: 52), Thr-Val-Ala-Ala-Pro (SEQ ID NO: 53), Gln-Pro-
Lys-Ala-Ala (SEQ ID NO: 54), Gln-Arg-Ile-Glu-Gly (SEQ ID NO: 55); Ala-Ser-Thr-
Lys-Gly-Pro-Ser (SEQ ID NO: 48), Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO: 49),
Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 50), and His-Ile-Asp-Ser-Pro-Asn-Lys
(SEQ ID NO: 51). The examples listed above are not intended to limit the scope of the
invention in any way, and linkers comprising randomly selected amino acids selected
from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine,
arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine, and proline have
been shown to be suitable in the antibody-like binding proteins of the invention (see
Example 12).
The identity and sequence of amino acid residues in the linker may vary
depending on the type of secondary structural element necessary to achieve in the linker.
For example, glycine, serine, and alanine are best for linkers having maximum flexibility.
Some combination of glycine, proline, threonine, and serine are useful if a more rigid and
extended linker is necessary. Any amino acid residue may be considered as a linker in
combination with other amino acid residues to construct larger peptide linkers as
necessary depending on the desired properties.
In some antibody-like binding proteins of the invention, V comprises the amino
acid sequence of SEQ ID NO: 1; V comprises the amino acid sequence of SEQ ID NO:
3; V comprises the amino acid sequence of SEQ ID NO: 2; and V comprises the
H1 H2
amino acid sequence of SEQ ID NO: 4.
In some embodiments of the invention, the antibody-like binding protein is
capable of specifically binding one or more antigen targets. In preferred embodiments of
the invention, the antibody-like binding protein is capable of specifically binding at least
one antigen target selected from the group consisting of B7.1, B7.2, BAFF, BlyS, C3, C5,
CCL11 (eotaxin), CCL15 (MIP-1d), CCL17 (TARC), CCL19 (MIP-3b), CCL2 (MCP-1),
CCL20 (MIP-3a), CCL21 (MIP-2), SLC, CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK),
CCL26 (eotaxin-3), CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-
3), CCL8 (mcp-2), CD3, CD19, CD20, CD24, CD40, CD40L, CD80, CD86, CDH1 (E-
cadherin), Chitinase, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA4,
CX3CL1 (SCYD1), CXCL12 (SDF1), CXCL13, EGFR, FCER1A, FCER2, HER2,
IGF1R, IL-1, IL-12, IL13, IL15, IL17, IL18, IL1A, IL1B, IL1F10, IL1β, IL2, IL4, IL6,
IL7, IL8, IL9, IL12/23, IL22, IL23, IL25, IL27, IL35, ITGB4 (b 4 integrin), LEP
(leptin), MHC class II, TLR2, TLR4, TLR5, TNF, TNF-a, TNFSF4 (OX40 ligand),
TNFSF5 (CD40 ligand), Toll-like receptors, TREM1, TSLP, TWEAK, XCR1
(GPR5/CCXCR1), DNGR-1(CLEC91), and HMGB1. In other embodiments of the
invention, the antibody-like binding protein is capable of inhibiting the function of one or
more of the antigen targets.
In some embodiments of the invention, the antibody-like binding protein is
bispecific and capable of binding two different antigen targets or epitopes. In a preferred
embodiment of the invention, the antibody-like binding protein is bispecific and each
light chain-heavy chain pair is capable of binding two different antigen targets or
epitopes. In a more preferred embodiment, the antibody-like binding protein is capable
of binding two different antigen targets that are selected from the group consisting of IL4
and IL13, IGF1R and HER2, IGF1R and EGFR, EGFR and HER2, BK and IL13, PDL-1
and CTLA-4, CTLA4 and MHC class II, IL-12 and IL-18, IL-1α and IL-1β, TNFα and
IL12/23, TNFα and IL-12p40, TNFα and IL-1β, TNFα and IL-23, and IL17 and IL23. In
an even more preferred embodiment, the antibody-like binding protein is capable of
binding the antigen targets IL4 and IL13.
In some embodiments of the invention, the antibody-like binding protein
specifically binds IL4 with an on-rate of 2.97 E+07 and an off-rate of 3.30 E-04 and
specifically binds IL13 with an on-rate of 1.39 E+06 and an off-rate of 1.63 E-04. In
other embodiments of the invention, the antibody-like binding protein specifically binds
IL4 with an on-rate of 3.16 E+07 and an off-rate of 2.89 E-04 and specifically binds IL13
with an on-rate of 1.20 E+06 and an off-rate of 1.12 E-04.
In one embodiment of the invention, an antibody-like binding protein comprising
four polypeptide chains that form four antigen binding sites is prepared by identifying a
first antibody variable domain that binds a first target antigen and a second antibody
variable domain that binds a second target antigen, each containing a V , and a V ;
assigning either the light chain or the heavy chain as template chain; assigning the V of
the first antibody variable domain or the second antibody variable domain as V ;
assigning a V , a V , and a V according to formulas [I] and [II] below:
L2 H1 H2
V -L -V -L -C [I]
L1 1 L2 2 L
V -L -V -L -C -Fc [II]
H2 3 H1 4 H1
determining maximum and minimum lengths for L , L , L , and L ; generating
1 2 3 4
polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I
and II that bind the first target antigen and the second target antigen when combined to
form the antibody-like binding protein;
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain;
H1 H1
Fc is the immunoglobulin hinge region and C , C immunoglobulin heavy chain
H2 H3
constant domains; and
L , L , L , and L are amino acid linkers;
1 2 3 4
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
In another embodiment of the invention, an antibody-like binding protein
comprising four polypeptide chains that form four antigen binding sites is prepared by
identifying a first antibody variable domain that binds a first target antigen and a second
antibody variable domain that binds a second target antigen, each containing a V , and a
V ; assigning either the light chain or the heavy chain as template chain; assigning the V
of the first antibody variable domain or the second antibody variable domain as V ;
assigning a V , a V , and a V according to formulas [I] and [II] below:
L2 H1 H2
V -L -V -L -C [I]
L1 1 L2 2 L
V -L -V -L -C [II]
H2 3 H1 4 H1
determining maximum and minimum lengths for L , L , L , and L ; generating
1 2 3 4
polypeptide structures of formulas I and II; selecting polypeptide structures of formulas I
and II that bind the first target antigen and the second target antigen when combined to
form the antibody-like binding protein;
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain; and
H1 H1
L , L , L , and L are an amino acid linkers;
1 2 3 4
and wherein the polypeptides of formula I and the polypeptides of formula II form a
cross-over light chain-heavy chain pair.
In other embodiments of the invention, an antibody-like binding protein in which
the first antibody variable domain and the second antibody variable domain are the same
is prepared.
One embodiment of the invention provides a method for making an antibody-like
binding protein, comprising expressing in a cell one or more nucleic acid molecules
encoding polypeptides having structures represented by the formulas [I] and [II] below:
V -L -V -L -C [I]
L1 1 L2 2 L
V -L -V -L -C -Fc [II]
H2 3 H1 4 H1
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain;
H1 H1
Fc is the immunoglobulin hinge region and C , C immunoglobulin heavy chain
H2 H3
constant domains; and
L , L , L , and L are amino acid linkers;
1 2 3 4
and wherein the polypeptides of formula I and the polypeptides of formula II form
a cross-over light chain-heavy chain pair.
Another embodiment of the invention provides a method for making an antibody-
like binding protein, comprising expressing in a cell one or more nucleic acid molecules
encoding polypeptides having structures represented by the formulas [I] and [II] below:
V -L -V -L -C [I]
L1 1 L2 2 L
V -L -V -L -C [II]
H2 3 H1 4 H1
wherein:
V is a first immunoglobulin light chain variable domain;
V is a second immunoglobulin light chain variable domain;
V is a first immunoglobulin heavy chain variable domain;
V is a second immunoglobulin heavy chain variable domain;
C is an immunoglobulin light chain constant domain;
C is the immunoglobulin C heavy chain constant domain; and
H1 H1
L , L , L , and L are amino acid linkers;
1 2 3 4
and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-
over light chain-heavy chain pair.
3. Uses for Antibody-like Binding Proteins
The antibody-like binding proteins of the invention can be employed in any
known assay method, such as competitive binding assays, direct and indirect sandwich
assays, and immunoprecipitation assays for the detection and quantitation of one or more
target antigens. The antibody-like binding proteins will bind the one or more target
antigens with an affinity that is appropriate for the assay method being employed.
For diagnostic applications, in certain embodiments, antibody-like binding
proteins can be labeled with a detectable moiety. The detectable moiety can be any one
that is capable of producing, either directly or indirectly, a detectable signal. For
3 14 32 35 125 99
example, the detectable moiety can be a radioisotope, such as H, C, P, S, I, Tc,
111 67
In, or Ga; a fluorescent or chemiluminescent compound, such as fluorescein
isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-
galactosidase, or horseradish peroxidase.
The antibody-like binding proteins of the invention are also useful for in vivo
imaging. An antibody-like binding protein labeled with a detectable moiety can be
administered to an animal, preferably into the bloodstream, and the presence and location
of the labeled antibody in the host assayed. The antibody-like binding protein can be
labeled with any moiety that is detectable in an animal, whether by nuclear magnetic
resonance, radiology, or other detection means known in the art.
The invention also relates to a kit comprising an antibody-like binding protein and
other reagents useful for detecting target antigen levels in biological samples. Such
reagents can include a detectable label, blocking serum, positive and negative control
samples, and detection reagents.
4. Antibody-Like Binding Protein Therapeutic Compositions and
Administration Thereof
Therapeutic or pharmaceutical compositions comprising antibody-like binding
proteins are within the scope of the invention. Such therapeutic or pharmaceutical
compositions can comprise a therapeutically effective amount of an antibody-like binding
protein, or antibody-like binding protein-drug conjugate, in admixture with a
pharmaceutically or physiologically acceptable formulation agent selected for suitability
with the mode of administration.
Acceptable formulation materials preferably are nontoxic to recipients at the
dosages and concentrations employed.
The pharmaceutical composition can contain formulation materials for modifying,
maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color,
isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or
penetration of the composition. Suitable formulation materials include, but are not
limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine),
antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-
sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other
organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as
ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers,
monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or
dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring,
flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as
polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions
(such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic
acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic
acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene
glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or
wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate
or polysorbate 80; triton; tromethamine; lecithin; cholesterol or tyloxapal), stability
enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali
metal halides – preferably sodium or potassium chloride – or mannitol sorbitol), delivery
vehicles, diluents, excipients and/or pharmaceutical adjuvants (see, e.g., REMINGTON'S
PHARMACEUTICAL SCIENCES (18th Ed., A.R. Gennaro, ed., Mack Publishing Company
1990), and subsequent editions of the same, incorporated herein by reference for any
purpose).
The optimal pharmaceutical composition will be determined by a skilled artisan
depending upon, for example, the intended route of administration, delivery format, and
desired dosage. Such compositions can influence the physical state, stability, rate of in
vivo release, and rate of in vivo clearance of the antibody-like binding protein.
The primary vehicle or carrier in a pharmaceutical composition can be either
aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier for injection
can be water, physiological saline solution, or artificial cerebrospinal fluid, possibly
supplemented with other materials common in compositions for parenteral
administration. Neutral buffered saline or saline mixed with serum albumin are further
exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer
of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include
sorbitol or a suitable substitute. In one embodiment of the invention, antibody-like
binding protein compositions can be prepared for storage by mixing the selected
composition having the desired degree of purity with optional formulation agents in the
form of a lyophilized cake or an aqueous solution. Further, the antibody-like binding
protein can be formulated as a lyophilizate using appropriate excipients such as sucrose.
The pharmaceutical compositions of the invention can be selected for parenteral
delivery. Alternatively, the compositions can be selected for inhalation or for delivery
through the digestive tract, such as orally. The preparation of such pharmaceutically
acceptable compositions is within the skill of the art.
The formulation components are present in concentrations that are acceptable to
the site of administration. For example, buffers are used to maintain the composition at
physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to
about 8.
When parenteral administration is contemplated, the therapeutic compositions for
use in this invention can be in the form of a pyrogen-free, parenterally acceptable,
aqueous solution comprising the desired antibody-like binding protein in a
pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral
injection is sterile distilled water in which an antibody-like binding protein is formulated
as a sterile, isotonic solution, properly preserved. Yet another preparation can involve the
formulation of the desired molecule with an agent, such as injectable microspheres, bio-
erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid),
beads, or liposomes, that provides for the controlled or sustained release of the product
which can then be delivered via a depot injection. Hyaluronic acid can also be used, and
this can have the effect of promoting sustained duration in the circulation. Other suitable
means for the introduction of the desired molecule include implantable drug delivery
devices.
In one embodiment, a pharmaceutical composition can be formulated for
inhalation. For example, an antibody-like binding protein can be formulated as a dry
powder for inhalation. Antibody-like binding protein inhalation solutions can also be
formulated with a propellant for aerosol delivery. In yet another embodiment, solutions
can be nebulized.
It is also contemplated that certain formulations can be administered orally. In
one embodiment of the invention, antibody-like binding proteins that are administered in
this fashion can be formulated with or without those carriers customarily used in the
compounding of solid dosage forms such as tablets and capsules. For example, a capsule
can be designed to release the active portion of the formulation at the point in the
gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is
minimized. Additional agents can be included to facilitate absorption of the antibody-
like binding protein. Diluents, flavorings, low melting point waxes, vegetable oils,
lubricants, suspending agents, tablet disintegrating agents, and binders can also be
employed.
Another pharmaceutical composition can involve an effective quantity of
antibody-like binding proteins in a mixture with non-toxic excipients that are suitable for
the manufacture of tablets. By dissolving the tablets in sterile water, or another
appropriate vehicle, solutions can be prepared in unit-dose form. Suitable excipients
include, but are not limited to, inert diluents, such as calcium carbonate, sodium
carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch,
gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
Additional pharmaceutical compositions of the invention will be evident to those
skilled in the art, including formulations involving antibody-like binding proteins in
sustained- or controlled-delivery formulations. Techniques for formulating a variety of
other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible
microparticles or porous beads and depot injections, are also known to those skilled in the
art. Additional examples of sustained-release preparations include semipermeable
polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained
release matrices can include polyesters, hydrogels, polylactides, copolymers of L-
glutamic acid and gamma ethyl-L-glutamate, poly(2-hydroxyethyl-methacrylate),
ethylene vinyl acetate, or poly-D(-)hydroxybutyric acid. Sustained-release
compositions can also include liposomes, which can be prepared by any of several
methods known in the art.
Pharmaceutical compositions of the invention to be used for in vivo
administration typically must be sterile. This can be accomplished by filtration through
sterile filtration membranes. Where the composition is lyophilized, sterilization using
this method can be conducted either prior to, or following, lyophilization and
reconstitution. The composition for parenteral administration can be stored in lyophilized
form or in a solution. In addition, parenteral compositions generally are placed into a
container having a sterile access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
Once the pharmaceutical composition has been formulated, it can be stored in
sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or
lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a
form (e.g., lyophilized) requiring reconstitution prior to administration.
The invention also encompasses kits for producing a single-dose administration
unit. The kits can each contain both a first container having a dried protein and a second
container having an aqueous formulation. Also included within the scope of this
invention are kits containing single and multi-chambered pre-filled syringes (e.g., liquid
syringes and lyosyringes).
The effective amount of an antibody-like binding protein pharmaceutical
composition to be employed therapeutically will depend, for example, upon the
therapeutic context and objectives. One skilled in the art will appreciate that the
appropriate dosage levels for treatment will thus vary depending, in part, upon the
molecule delivered, the indication for which the antibody-like binding protein is being
used, the route of administration, and the size (body weight, body surface, or organ size)
and condition (the age and general health) of the patient. Accordingly, the clinician can
titer the dosage and modify the route of administration to obtain the optimal therapeutic
effect. A typical dosage can range from about 0.1 mg/kg to up to about 100 mg/kg or
more, depending on the factors mentioned above. In other embodiments, the dosage can
range from 0.1 mg/kg up to about 100 mg/kg; or 1 mg/kg up to about 100 mg/kg; or 5
mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg,
50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, up to about 100 mg/kg.
Dosing frequency will depend upon the pharmacokinetic parameters of the
antibody-like binding protein in the formulation being used. Typically, a clinician will
administer the composition until a dosage is reached that achieves the desired effect. The
composition can therefore be administered as a single dose, as two or more doses (which
may or may not contain the same amount of the desired molecule) over time, or as a
continuous infusion via an implantation device or catheter. Further refinement of the
appropriate dosage is routinely made by those of ordinary skill in the art and is within the
ambit of tasks routinely performed by them. Appropriate dosages can be ascertained
through use of appropriate dose-response data.
The route of administration of the pharmaceutical composition is in accord with
known methods, e.g., orally; through injection by intravenous, intraperitoneal,
intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular,
intraarterial, intraportal, or intralesional routes; by sustained release systems; or by
implantation devices. Where desired, the compositions can be administered by bolus
injection or continuously by infusion, or by implantation device.
The composition can also be administered locally via implantation of a
membrane, sponge, or other appropriate material onto which the desired molecule has
been absorbed or encapsulated. Where an implantation device is used, the device can be
implanted into any suitable tissue or organ, and delivery of the desired molecule can be
via diffusion, timed-release bolus, or continuous administration.
. Examples
The Examples that follow are illustrative of specific embodiments of the
invention, and various uses thereof. They are set forth for explanatory purposes only, and
should not be construed as limiting the scope of the invention in any way.
Example 1. Design and Engineering of Bispecific Cross-over Dual Variable Region
Antibody-like Binding Proteins
The cross-over dual variable region in an Fv format was described in U.S. Patent
No. 5,989,830 and was referred to as a cross-over double head (CODH) configuration.
Molecular modeling predicted that the cross-over double-head (CODH) design results in
a complex with both binding sites facing in opposite directions, without the restraints
suggested for the Dual-Fv configuration. The CODH Fv format was examined to
determine whether it could be converted into complete antibody-like molecules by adding
a C domain to the light chain and an Fc region to the heavy chain. A similar conversion
was successful for the corresponding dual variable domains (DVD-Ig) and TBTI as
described in U.S. Patent No. 7,612,181 and International Publication No. WO
2009/052081. The arrangement of the variable regions in the CODH format is shown in
the structures below, which indicate the amino to carboxyl orientation of the peptide
chains:
(a) light chain: NH -V -Linker-V -COOH
2 L1 L2
(b) heavy chain: NH -V -Linker-V -COOH
2 H2 H1
The amino to carboxyl terminal arrangement of the variable regions in (a) and (b)
above can be distinguished from the arrangement in the Dual-Fv configuration shown in
(c) and (d) below:
(c) light chain: NH -V -Linker-V -COOH
2 L1 L2
(d) heavy chain: NH -V -Linker-V -COOH
2 H1 H2
The main difference to note is the distinct placement of the corresponding light
chain and heavy chain variable regions (V /V and V /V ) with respect to each other
H1 L1 H2 L2
in the two dual variable region configurations. The corresponding V and V domains
L1 H1
were both at the N-terminus of the light and heavy chains in dual variable region
configuration. In contrast, in the cross-over configuration, one half of one pair of an
antibody variable region was separated spatially within the protein chain in the cross-over
configuration. In the cross-over configuration, the V domain would be at the N-
terminus of the protein light chain but the pairing V domain is at the C-terminus of the
cross-over configuration heavy chain. The spatial relationship between V and V
L1 H1
found in the dual variable region configuration is the arrangement found in natural
antibodies.
One potential disadvantage of the dual Fv configuration is that the linker L
separating the two variable regions protrudes into the antigen binding site of the Fv2
domain (see Figure 1). This protrusion may interfere with antigen binding and result in a
perturbed accessibility of Antigen 2 to Fv2. This perturbed accessibility or interference
may prevent antigen binding. In addition, this interference might be more pronounced
where the size of Antigen 2 is larger. Indeed, it has been documented in U.S. Patent No.
7,612,181 that the binding affinity and neutralization ability of a DVD-Ig molecule
depends on which antigen specificity is presented at the N-terminus or C-terminus. See
U.S. Patent No. 7,612,181, Example 2.
Therefore, to create more stable antibody-like binding proteins that are not subject
to loss of antigen affinity as compared to the parental antibody, cross-over dual variable
region molecules having a C domain on the light chain and an Fc region on the heavy
chain were designed and constructed. The polypeptides that form these antibody-like
proteins have the structures shown below, in which the amino to carboxyl terminal
orientation of the polypeptide chains is indicated:
(e) light chain: NH -V -Linker-V -C -COOH
2 L1 L2 L
(f) heavy chain: NH -V -Linker-V -C -Fc-COOH
2 H2 H1 H1
To evaluate whether this bispecific antibody-like protein design would bind to
two different antigens, two previously generated and humanized variable regions from
antibodies specific for IL4 (parental humanized anti-IL4) and IL13 (parental humanized
anti-IL13) were used to construct the bispecific antibody-like molecules shown in Table
1. Sequencing of the mouse antibodies and the humanization process were described in
International Publication No. (TBTI). Briefly, amino acid sequences
of the variable heavy and light chains of the murine anti-IL13 clone B-B13 and the
murine anti-IL4 clone 8D4-8 were determined by amino acid sequencing. The murine
sequences were humanized and then back-translated into nucleotide sequences as
described in Example 5 of International Publication No. , which is
incorporated herein by reference in its entirety. The parental humanized anti-IL4 V and
V , and parental humanized anti-IL13 V and V sequences were combined and arranged
L H L
as shown in Table 1. The shorthand codes in column one of Table 1 were created to
simplify discussion of these antibody-like binding proteins. The antibody-like binding
proteins differ in the size of the linker inserted between the two variable regions as shown
in Table 1. DNA molecules encoding the polypeptides shown in Table 1 were generated
from the back-translated parental anti-IL4 and anti-IL13 antibodies. C , C , and Fc
H1 L
domains were obtained from IGHG1 (GenBank Accession No. 569F4) and IGKC
(GenBank Accession No. Q502W4).
Table 1. Cross-over Double Head Immunoglobulins
Shorthand Code for Protein Protein Description ID NO:
Parental anti-IL4 Light Chain anti-IL4 V 1
Parental anti-IL4 Heavy Chain anti-IL4 V 2
Parental anti-IL13 Light Chain anti-IL13 V 3
Parental anti-IL13 Heavy Chain anti-IL13 V 4
Heavy Chain Codes
IL13(G4S)IL4CH1-Fc anti-IL13 V -(G S)-anti-IL4 V -C -Fc 5
H 4 H H1
IL13(G4S2)IL4CH1-Fc anti-IL13 V -(G S) -anti-IL4 V -C -Fc 6
H 4 2 H H1
IL4(G4S)IL13CH1-Fc anti-IL4 V -(G S)-anti-IL13 V -C -Fc 7
H 4 H H1
IL4(G4S2)IL13CH1-Fc anti-IL4 V -(G S) -anti-IL13 V -C -Fc 8
H 4 2 H H1
Light Chain Codes
IL13(G4S)IL4CL anti-IL13 V -(G S)-anti-IL4 V -CL 9
L 4 L
IL13(G4S2)IL4CL anti-IL13 V -(G S) -anti-IL4 V -CL 10
L 4 2 L
IL4(G4S)IL13CL anti-IL4 V -(G S)-anti-IL13 V -CL 11
L 4 L
IL4(G4S2)IL13CL anti-IL4 V -(G S) -anti-IL13 V -CL 12
L 4 2 L
The protein combinations shown in Table 2 were expressed by transient
transfection and purified by Protein A chromatography. In each case, size exclusion
chromatography revealed less than 12% aggregation, with most having less than 7%
aggregation; but none of the cross-over double head immunoglobulins were found to
display any ability to bind either IL4 or IL13. However, no functional antibody-like
binding could be detected and the reasons for this lack of activity could not be
ascertained. It was previously predicted that this arrangement would show superior
stability over the dual variable region domain antibodies described in U.S. Patent No.
7,612,181 and International Publication No. .
Table 2. Binding of CODH-Ig to IL4 and IL13
Protein Combination Aggregation IL4 binding IL13 binding
anti-IL13 V -(G S)-anti-IL4 V -C -Fc 5.4% ND* ND
H 4 H H1
anti-IL4 V -(G S)-anti-IL13 V -C
L 4 L L
anti-IL13 V -(G S)-anti-IL4 V - C -Fc 6.3% ND ND
H 4 H H1
anti-IL4 V -(G S) -anti-IL13 V -CL
L 4 2 L
anti-IL13 V -(G S) -anti-IL4 V - C -Fc 11.5% ND ND
H 4 2 H H1
anti-IL4 V -(G S)-anti-IL13 V -CL
L 4 L
anti-IL13 V -(G S) -anti-IL4 V - C -Fc 10.1% ND ND
H 4 2 H H1
anti-IL4 V -(G S) -anti-IL13 V -CL
L 4 2 L
anti-IL4 V -(G S)-anti-IL13 V - C -Fc 2.7% ND ND
H 4 H H1
anti-IL13 V -(G S)-anti-IL4 V -CL
L 4 L
anti-IL4 V -(G S)-anti-IL13 V - C -Fc 3.6% ND ND
H 4 H H1
anti-IL13 V -(G S) -anti-IL4 V -CL
L 4 2 L
anti-IL4 V -(G S) -anti-IL13 V - C -Fc 2.9% ND ND
H 4 2 H H1
anti-IL13 V -(G S)-anti-IL4 V -CL
L 4 L
anti-IL4 V -(G S) -anti-IL13 V - C -Fc 10.8% ND ND
H 4 2 H H1
anti-IL13 V -(G S) -anti-IL4 V -CL
L 4 2 L
* ND means none detected
Example 2. Design of CODV-Ig Proteins by Molecular Modeling
To obtain fully functional antibody-like proteins utilizing the cross-over double
head configuration that are amendable to incorporation of the Fc and C domains, a
molecular modeling protocol was developed for the inclusion and evaluation of different
linkers between the constant and variable domains and between the dual variable domains
on both the heavy and light chains. The question was whether the addition of unique
linkers between each constant/variable domain interface and between the two
variable/variable domain interfaces on both the heavy and light chains would allow
proper protein folding to occur and produce functional antibody-like molecules in the
cross-over dual variable region configuration (see Figure 2). In other words, a total of
four independent and unique linkers were evaluated (see Figure 2). This molecular
modeling protocol was based on protein-protein docking of homology models and
experimental models of the Fv and Fv regions, respectively, in combination with
IL4 IL13
appropriate linkers between the Fv and Fv regions and between the Fv and constant
IL4 IL13
or Fc regions.
The independent linkers were assigned unique names as follows: L refers to the
linker between N-terminal V and the C-terminal V on the light chain; L refers to the
L L 2
linker between the C-terminal V and C on the light chain; L refers to the linker
L L 3
between N-terminal V and the C-terminal V on the heavy chain; L refers to the linker
H H 4
between the C-terminal V and C (and Fc) on the heavy chain. It should be noted that
H H1
the designations V and V refer only to the domain's location on a particular protein
chain in the final format. For example, V and V could be derived from V and V
H1 H2 L1 L2
domains in parent antibodies and placed into the V and V positions in a CODV-Ig.
H1 H2
Likewise, V and V could be derived from V and V domains in parent antibodies
L1 L2 H1 H2
and placed into the V and V positions in a CODV-Ig. Thus, V and V designations
H1 H2 H L
refer to present location and not the original location in a parent antibody.
In more detail, a homology model of Fv was constructed on PDB entries 1YLD
(light chain) and 1IQW (heavy chain). The Fv dimer was recomposed on an in-house
crystal structure of the IL13/anti-IL13 Fab complex and optimized. In order to obtain
IL13
an estimate of the volume required by IL4 when bound to Fv , the crystal structure of
IL4 (1RCB.pdb) was docked to the homology model of Fv . Next, twenty-two putative
models of the complex were generated that merited further consideration.
In parallel, the homology model of Fv was docked to Fv extracted from an
IL4 IL13
in-house crystal structure of the IL13/Fab complex. One superior solution was found
IL13
that permitted construction of relatively short linkers while showing no steric interference
for antigen binding and placement of the constant domains as was the case for dual
variable region immunoglobulins (see Figure 3). In this arrangement Fv (V ) was
IL4 L1
placed at the N-terminus of the light chain, followed by Fv (V ) and Fc (C ) on the
IL13 L2 L1
light chain C-terminus. On the heavy chain, Fv (V ) was placed N-terminally,
IL13 H2
followed by Fv (V ) and the constant regions (C - C - C ).
IL4 H1 H1 H2 H3
As shown in Table 3, the models of the light chain suggested that the linker L
between the V and V domains and the linker L between the V and C domains
L1 L2 2 L2 L1
should be between one to three and zero to two glycine residues long, respectively.
Models of the heavy chain suggested that the linker L between the V and V domains
3 H2 H1
and the linker L between the V and C domains should be between two to six and
4 H1 H1
four to seven glycine residues long, respectively (see Table 3 and Figure 2). In this
example, glycine was used as a prototypical amino acid for the linkers but other amino
acid residues may also serve as linkers. The structural stability of the proposed models
was verified by optimization of the linker conformations, minimization, and molecular
dynamics calculations. Systematic combination between four light chain and six heavy
chain constructs resulted in 24 possible cross-over dual variable region bispecific anti-
IL4 and anti-IL13 antibody-like binding proteins (see Table 4).
Table 3. Proposed Linker Lengths
Linker Between Maximal Linker Minimal Linker Linker
Insertion Insertion Name
V -V Gly Gly L
L1 L2 3 1
V -C Gly None L
L2 L 2 2
V -V Gly Gly L
H2 H1 6 2 3
V -C Gly Gly L
H1 H1 7 4 4
Table 4. CODV-Ig for Expression
Code* Heavy Chains (N to C terminal) ID NO:
HC1 IL13 V -(Gly6)-IL4 V -(Gly7)-C -Fc 13
H H H1
HC2 IL13 V -(Gly6)-IL4 V -(Gly4)- C -Fc 14
H H H1
HC3 IL13 V -(Gly2)-IL4 V -(Gly7)- C -Fc 15
H H H1
HC4 IL13 V -(Gly2)-IL4 V -(Gly4)- C -Fc 16
H H H1
HC5 IL13 V -(Gly4)-IL4 V -(Gly7)- C -Fc 17
H H H1
HC6 IL13 V -(Gly4)-IL4 V -(Gly4)- C -Fc 18
H H H1
Code* Light Chains (N to C terminal)
LC1 IL4 V -(Gly3)-IL13 V -C 19
L L L1
LC2 IL4 V -(Gly)-IL13 V - C 20
L L L1
LC3 IL4 V -(Gly3)-IL13 V -(Gly2)- C 21
L L L1
LC4 IL4 V -(Gly)-IL13 V -(Gly2)- C 22
L L L1
C C human light chain constant domain 23
L1 L1
C -Fc C human heavy chain constant domain and Fc region 24
H1 H1
Gly4 peptide linker with 4 glycines (GGGG) 25
Gly5 peptide linker with 5 glycines (GGGGG) 26
Gly6 peptide linker with 6 glycines (GGGGGG) 27
Gly7 peptide linker with 7 glycines (GGGGGGG) 28
Gly8 peptide linker with 8 glycines (GGGG GGGG) 29
*A short hand code was devised to represent the associated structures. Codes beginning
with HC represent the adjacent heavy chain and codes beginning with LC represent the
adjacent light chains.
In Table 4, the prefix "anti" is not included but it is intended to mean that IL13 refers to
anti-IL13 and IL4 refers to anti-IL4.
Example 3. Generation of CODV-Ig Expression Plasmids
Nucleic acid molecules encoding the variable heavy and light chains of the six
heavy chains and four light chains described in Table 4 were generated by gene synthesis
at Geneart (Regensburg, Germany). The variable light chain domains were fused to the
constant light chain (IGKC, GenBank Accession No. Q502W4) by digestion with the
restriction endonucleases ApaLI and BsiWI and subsequently ligated into the
ApaLI/BsiWI sites of the episomal expression vector pFF, an analogon of the pTT vector
described by Durocher et al., (2002, Nucl. Acids Res. 30(2): E9), creating the mammalian
expression plasmid for expression of the light chains.
The variable heavy chain domains were fused to the "Ted" variant of the human
constant heavy chain (IGHG1, GenBank Accession No. 569F4), or alternatively, to a 6x
His tagged C domain from the human constant IGHG1 in order to create a bispecific
Fab. Next, the V domain was digested with the restriction endonucleases ApaLI and
ApaI and then fused to the IGHG1 or His tagged C domain respectively, by ligation
into the ApaLI/ApaI sites of the episomal expression vector pFF, creating the mammalian
expression plasmids for expression of the heavy chains (IgG1 or Fab respectively).
Example 4. Expression of CODV-Ig
The expression plasmids encoding the heavy and light chains of the
corresponding constructs were propagated in E. coli DH5a cells. Plasmids used for
transfection were prepared from E. coli using the Qiagen EndoFree Plasmid Mega Kit.
HEK 293-FS cells growing in Freestyle Medium (Invitrogen) were transfected
with indicated LC and HC plasmids encoding the heavy chains and light chains shown in
Table 4 using 293fectin (Invitrogen) transfection reagent as described by the
manufacturer. After 7 days, cells were removed by centrifugation and the supernatant
was passed over a 0.22 µm filter to remove particles.
CODV-IgG1 constructs were purified by affinity chromatography on Protein A
columns (HiTrap Protein A HP Columns, GE Life Sciences). After elution from the
column with 100 mM acetate buffer and 100 mM NaCl, pH 3.5, the CODV-IgG1
constructs were desalted using HiPrep 26/10 Desalting Columns, formulated in PBS at a
concentration of 1 mg/mL and filtered using a 0.22 µm membrane.
Bispecific CODV Fab constructs were purified by IMAC on HiTrap IMAC HP
Columns (GE Life Sciences). After elution from the column with a linear gradient
(Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 50 - 500 mM imidazole, pH 7.4),
the protein containing fractions were pooled and desalted using HiPrep 26/10 Desalting
Columns, formulated in PBS at a concentration of 1 mg/mL and filtered using a 0.22 µm
membrane.
Protein concentration was determined by measurement of absorbance at 280 nm.
Each batch was analyzed by SDS-PAGE under reducing and non-reducing conditions to
determine the purity and molecular weight of each subunit and of the monomer.
A Nunc F96-MaxiSorp-Immuno plate was coated with goat anti-Human IgG (Fc
specific) [NatuTec A80-104A]. The antibody was diluted to 10 µg/ml in carbonate
coating buffer (50 mM sodium carbonate, pH 9.6) and dispensed at 50 µL per well. The
plate was sealed with adhesive tape, and stored overnight at 4 C. The plate was washed
three times with Wash buffer (PBS, pH 7.4 and 0.1% Tween20). 150 µL of blocking
solution (1% BSA / PBS) was dispensed into each well to cover the plate. After 1 hour at
room temperature, the plate was washed three times with Wash buffer. 100 µL of sample
or standards (in a range from 1500 ng/ml to 120 ng/ml) were added and allowed to sit for
1 hour at room temperature. The plate was washed three times with Wash buffer. 100
µL of goat anti-Human IgG-FC – HRP conjugate [NatuTec A80-104P-60] diluted
1:10.000 were added using incubation solution (0.1% BSA, PBS, pH 7.4, and 0.05%
Tween20). After 1 hour incubation at room temperature, the plate was washed three
times with Wash buffer. 100 µL of ABTS substrate (10 mg ABTS tablet (Pierce 34026)
in 0.1 M Na HPO , 0.05 M citric acid solution, pH 5.0). Addition of 10 µL of 30% H O
2 4 2
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FR1160311 | 2011-11-14 | ||
PCT/US2012/030948 WO2012135345A1 (en) | 2011-03-28 | 2012-03-28 | Dual variable region antibody-like binding proteins having cross-over binding region orientation |
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