U.S. patent application number 13/798205 was filed with the patent office on 2013-09-19 for multispecific antigen-binding molecules and uses thereof.
This patent application is currently assigned to REGENERON PHARMACEUTICALS, INC.. The applicant listed for this patent is REGENERON PHARMACEUTICALS, INC.. Invention is credited to Katherine Diana Cygnar, Aris N. Economides, Andrew J. Murphy, Nicholas J. PAPADOPOULOS.
Application Number | 20130243775 13/798205 |
Document ID | / |
Family ID | 48014317 |
Filed Date | 2013-09-19 |
United States Patent
Application |
20130243775 |
Kind Code |
A1 |
PAPADOPOULOS; Nicholas J. ;
et al. |
September 19, 2013 |
MULTISPECIFIC ANTIGEN-BINDING MOLECULES AND USES THEREOF
Abstract
The present invention provides multispecific antigen-binding
molecules and uses thereof. The multispecific antigen-binding
molecules comprise a first antigen-binding domain that specifically
binds a target molecule, and a second antigen-binding domain that
specifically binds an internalizing effector protein. The
multispecific antigen-binding molecules of the present invention
can, in some embodiments, be bispecific antibodies that are capable
of binding both a target molecule and an internalizing effector
protein. In certain embodiments of the invention, the simultaneous
binding of the target molecule and the internalizing effector
protein by the multispecific antigen-binding molecule of the
present invention results in the attenuation of the activity of the
target molecule to a greater extent than the binding of the target
molecule alone. In other embodiments of the invention, the target
molecule is a tumor associated antigen, and the simultaneous
binding of the tumor associated antigen and the internalizing
effector protein by the multispecific antigen-binding molecule of
the present invention causes or facilitates the targeted killing of
tumor cells.
Inventors: |
PAPADOPOULOS; Nicholas J.;
(Lagrangeville, NY) ; Murphy; Andrew J.;
(Croton-on-Hudson, NY) ; Economides; Aris N.;
(Tarrytown, NY) ; Cygnar; Katherine Diana; (New
York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REGENERON PHARMACEUTICALS, INC. |
Tarrytown |
NY |
US |
|
|
Assignee: |
REGENERON PHARMACEUTICALS,
INC.
Tarrytown
NY
|
Family ID: |
48014317 |
Appl. No.: |
13/798205 |
Filed: |
March 13, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61610494 |
Mar 14, 2012 |
|
|
|
61721831 |
Nov 2, 2012 |
|
|
|
61751286 |
Jan 11, 2013 |
|
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Current U.S.
Class: |
424/136.1 ;
424/178.1; 530/387.3 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 14/705 20130101; C07K 16/283 20130101; C07K 16/1203 20130101;
C07K 16/22 20130101; C07K 16/28 20130101; C07K 16/2896 20130101;
A61P 19/08 20180101; C07K 2317/31 20130101; C07K 2317/77 20130101;
C07K 16/2866 20130101 |
Class at
Publication: |
424/136.1 ;
530/387.3; 424/178.1 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. A multispecific antigen-binding molecule comprising: a first
antigen-binding domain (D1); and a second antigen-binding domain
(D2); wherein D1 specifically binds a target molecule (T); and
wherein D2 specifically binds an internalizing effector protein
(E); wherein the simultaneous binding of T and E by the
multispecific antigen-binding molecule attenuates the activity of T
to a greater extent than the binding of T by D1 alone.
2. The multispecific antigen-binding molecule of claim 1, wherein E
is a cell surface-expressed molecule that is directly internalized
into the cell.
3. The multispecific antigen-binding molecule of claim 2, wherein E
is selected from the group consisting of CD63, MHC-I, Kremen-1,
Kremen-2, LRP5, LRP6, transferrin receptor, LDLr, MAL, V-ATPase,
and ASGR.
4. The multispecific antigen-binding molecule of claim 2, wherein
D2 comprises a ligand, or portion of a ligand, that specifically
binds E.
5. The multispecific antigen-binding molecule of claim 1, wherein E
is a soluble ligand that is internalized into a cell via the
interaction between E and an internalizing cell surface-expressed
receptor molecule.
6. The multispecific antigen-binding molecule of claim 5, wherein E
is transferrin or a portion thereof that is capable of binding to
membrane-expressed transferrin receptor.
7. The multispecific antigen-binding molecule of claim 5, wherein
D2 comprises a receptor, or ligand-binding portion of a receptor,
that specifically binds E.
8. The multispecific antigen-binding molecule of claim 1, wherein T
is a cell surface-expressed target molecule.
9. The multispecific antigen-binding molecule of claim 8, wherein T
is selected from the group consisting of IL-4R, IL-6R, PRLR,
Nav1.7, GCGR, and HLA-B27.
10. The multispecific antigen-binding molecule of claim 8, wherein
D1 comprises a ligand, or portion of a ligand, that specifically
binds T.
11. The multispecific antigen-binding molecule of claim 1, wherein
T is an intracellular precursor of a secreted or transmembrane
protein.
12. The multispecific antigen-binding molecule of claim 1, wherein
T is a soluble target molecule.
13. The multispecific antigen-binding molecule of claim 12, wherein
T is selected from the group consisting of IL-4, IL-6, IL-13, SOST,
and DKK1.
14. The multispecific antigen-binding molecule of claim 12, wherein
D1 comprises a receptor, or ligand-binding portion of a receptor,
that specifically binds T.
15. The multispecific antigen-binding molecule of claim 1, wherein
D1 and/or D2 exhibits pH-dependent binding to its antigen.
16. The multispecific antigen-binding molecule of claim 15, wherein
D1 binds T with lower affinity at acidic pH as compared to neutral
pH; and/or wherein D2 binds E with lower affinity at acidic pH as
compared to neutral pH.
17. The multispecific antigen-binding molecule of claim 1, wherein
D1 and/or D2 comprise(s) at least one antibody variable region.
18. The multispecific antigen-binding molecule of claim 17, wherein
D1 and/or D2 comprise(s) a heavy chain variable region (HCVR) and a
light chain variable region (LCVR).
19. The multispecific antigen-binding molecule of claim 18, wherein
the multispecific antigen-binding molecule is a bispecific
antibody.
20. The multispecific antigen-binding molecule of claim 1, wherein
D1 is derived from an antigen-binding molecule that binds but does
not substantially inactivate T on its own.
21. A method for inactivating or attenuating the activity of a
target molecule (T), the method comprising contacting T and an
internalizing effector protein (E) with a multispecific
antigen-binding molecule, wherein the multispecific antigen-binding
molecule comprises a first antigen-binding domain (D1) and a second
antigen-binding domain (D2), wherein D1 specifically binds T, and
wherein D2 specifically binds E; and wherein the simultaneous
binding of T and E by the multispecific antigen-binding molecule
causes inactivation of T to a greater extent than the binding of T
by D1 alone.
22. The method of claim 21, wherein E is a cell surface-expressed
molecule that is directly internalized into the cell.
23. The method of claim 21, wherein E is a soluble ligand that is
internalized into a cell via the interaction between E and an
internalizing cell surface-expressed receptor molecule.
24. The method of claim 21, wherein T is a cell surface-expressed
target molecule.
25. The method of claim 21, wherein T is a soluble target
molecule.
26. The multispecific antigen-binding molecule of claim 1, wherein
D2 comprises an antigen-binding portion of an anti-CD63
antibody.
27. The multispecific antigen-binding molecule of claim 26, wherein
D1 comprises an antigen-binding portion of an anti-IL-4R
antibody.
28. The multispecific antigen-binding molecule of claim 26, wherein
D1 comprises an antigen-binding portion of an anti-SOST
antibody.
29. A multispecific antigen-binding molecule comprising: a first
antigen-binding domain (D1); and a second antigen-binding domain
(D2); wherein D1 specifically binds a target molecule (T); and
wherein D2 binds an internalizing effector protein (E); wherein T
is a tumor-associated antigen; and wherein the simultaneous binding
of T and E by the multispecific antigen-binding molecule causes
internalization of the multispecific antigen-binding molecule into
a tumor cell.
30. The multispecific antigen-binding molecule of claim 29, wherein
the multispecific antigen-binding molecule is conjugated to a drug,
toxin or radioisotope.
31. The multispecific antigen-binding molecule of claim 29, wherein
D2 binds E with low affinity.
32. A method of targeting a tumor in a subject, the method
comprising administering to the subject the multispecific
antigen-binding molecule of claim 29, and a second antigen-binding
protein that specifically binds T at an epitope that is
non-overlapping with the epitope to which D1 binds.
33. The method of claim 32, wherein the second antigen-binding
protein is conjugated to a drug, toxin or radioisotope.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. provisional application Nos. 61/610,494, filed
on Mar. 14, 2012; 61/721,831, filed on Nov. 2, 2012; and
61/751,286, filed on Jan. 11, 2013, the disclosures of which are
herein incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of therapeutic
proteins, and in particular, to the field of therapeutic proteins
that are capable of inactivating, blocking, attenuating,
eliminating and/or reducing the concentration of one or more target
molecules in vitro or in vivo.
BACKGROUND
[0003] Therapeutic treatments often require the inactivation or
blocking of one or more target molecules that act on or in the
vicinity of a cell. For example, antibody-based therapeutics often
function by binding to a particular antigen expressed on the
surface of a cell, or to a soluble ligand, thereby interfering with
the antigen's normal biological activity. Antibodies and other
binding constructs directed against various cytokines (e.g., IL-1,
IL-4, IL-6, IL-13, IL-22, IL-25, IL-33, etc.), or their respective
receptors, for instance, have been shown to be useful in treating a
wide array of human ailments and diseases. Therapeutic agents of
this type typically function by blocking the interaction between
the cytokine and its receptor in order to attenuate or inhibit
cellular signaling. In certain contexts, however, it would be
therapeutically beneficial to inactivate or inhibit the activity of
a target molecule in a manner that does not necessarily involve
blocking its physical interaction with another component. One way
in which such non-blocking attenuation of a target molecule could
be achieved would be to reduce the extracellular or cell surface
concentration of the target molecule. Although genetic and nucleic
acid-based strategies for reducing the amount or concentration of a
given target molecule are known in the art, such strategies are
often fraught with substantial technical complications and
unintended side effects in therapeutic settings. Accordingly,
alternative non-blocking strategies are needed to facilitate the
inactivation or attenuation of various target molecules for
therapeutic purposes.
BRIEF SUMMARY OF THE INVENTION
[0004] The present invention is based, at least in part, on the
concept of attenuating or inactivating a target molecule by
facilitating or bringing about a physical linkage between the
target molecule and an internalizing effector protein. Through this
type of physical intermolecular linkage, the target molecule can be
forced to be internalized into the cell along with the
internalizing effector protein, and processed by the intracellular
degradative machinery, or otherwise attenuated, sequestered, or
inactivated. This mechanism represents a novel and inventive
strategy for inactivating or attenuating the activity of a target
molecule without necessarily blocking the interaction between the
target molecule and its binding partners.
[0005] Accordingly, the present invention provides a multispecific
antigen-binding molecule that is capable of simultaneously binding
a target molecule (T) and an internalizing effector protein (E).
More specifically, the present invention provides a multispecific
antigen-binding molecule comprising a first antigen-binding domain
(D1), and a second antigen-binding domain (D2), wherein D1
specifically binds T, and D2 specifically binds E, and wherein the
simultaneous binding of T and E by the multispecific
antigen-binding molecule attenuates the activity of T to a greater
extent than the binding of T by D1 alone. The enhanced attenuation
of the activity of T may be due to the forced
internalization/degradation of T through its physical linkage to E;
however, other mechanisms of action are possible and are not
excluded from the scope of the present invention.
[0006] In addition, the present invention provides methods of using
the multispecific antigen-binding molecule to inactivate or
attenuate the activity of a target molecule (T). In particular, the
present invention provides a method for inactivating or attenuating
the activity of T by contacting T and an internalizing effector
protein (E) with a multispecific antigen-binding molecule, wherein
the multispecific antigen-binding molecule comprises a first
antigen-binding domain (D1) and a second antigen-binding domain
(D2), wherein D1 specifically binds T, and wherein D2 specifically
binds E; and wherein the simultaneous binding of T and E by the
multispecific antigen-binding molecule attenuates the activity of T
to a greater extent than the binding of T by D1 alone.
[0007] In certain embodiments of the present invention, D1 and/or
D2 comprise(s) at least one antibody variable region. For example,
the multispecific antigen-binding molecule can, in some
embodiments, be a bispecific antibody, wherein D1 comprises an
antibody heavy and light chain variable region (HCVR/LCVR) pair
that specifically binds T, and wherein D2 comprises an HCVR/LCVR
pair that specifically binds E. Alternatively, D1 and/or D2 may
comprise a peptide or polypeptide that specifically interacts with
the target molecule (T) and/or the internalizing effector protein
(E). For example, if the target molecule is a cell surface
receptor, then D1 may comprise a portion of a ligand that
specifically binds the cell surface receptor target molecule.
Similarly, if the internalizing effector protein is a cell surface
internalizing receptor, then D2 may comprise a portion of a ligand
that specifically binds the cell surface internalizing receptor. In
certain embodiments, D1 comprises an antibody variable region that
specifically binds T, and D2 comprises a peptide or polypeptide
that specifically binds E. In yet other embodiments, D1 comprises a
peptide or polypeptide that specifically binds T, and D2 comprises
an antibody variable region that specifically binds E. In any
configuration, however, the end result is that T and E are capable
of being physically linked, directly or indirectly, via the
simultaneous binding of T and E by a multispecific antigen-binding
molecule.
[0008] Other embodiments will become apparent from a review of the
ensuing detailed description.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 (panels A-D) provides schematic representations of
four general exemplary mechanisms of action for the multispecific
antigen binding molecules of the present invention. In each
illustrated configuration D1 is a first antigen-binding domain; D2
is a second antigen binding domain; T is a target molecule; E is an
internalizing effector protein; and R is a receptor which
internalizes upon binding E. Panel A depicts the situation in which
both T and E are membrane-associated. Panel B depicts the situation
in which T is soluble and E is membrane-associated. Panel C depicts
the situation in which T is membrane-associated and E is a soluble
protein that interacts with, and is internalized into the cell via
the interaction of E and R. Panel D depicts the situation in which
T is soluble and E is a soluble protein that interacts with, and is
internalized into the cell via the interaction of E and R.
[0010] FIG. 2 shows the results of an immunoprecipitation
experiment performed on two different cells (Cell-1 expressing
Fc.gamma.R1 alone, and Cell-2 expressing Krm2 and Fc.gamma.R1)
following incubation for different amounts of time (0, 15, 30 and
60 minutes) with a DKK1-mFc multispecific antigen-binding
molecule.
[0011] FIG. 3 shows the relative IL-4-induced luminescence produced
by Stat6-luc reporter HEK293 cells in the presence and absence of
an anti-IL-4R/anti-CD63 multispecific antigen binding protein ("ab
conjugate") or control constructs ("control 1" and "control 2") at
various concentrations of IL-4.
[0012] FIG. 4 shows the results of an experiment carried out in the
same manner as the experiment shown in FIG. 3, except that CD63
expression was significantly reduced in the reporter cell line by
an siRNA directed against CD63.
[0013] FIG. 5 shows the results of an experiment carried out in a
similar manner as the experiments shown in FIGS. 3 and 4, except
that the reporter cells were incubated with the multispecific
antigen binding protein ("Ab conjugate") or control constructs
("control 1" and "control 2") for 2 hours or overnight prior to the
addition of IL-4 ligand. The top row of bar graphs represent the
results of experiments conducted in cells expressing normal levels
of CD63 ("untransfected"), while the bottom row of bar graphs
represents the results of experiments conducted in cells in which
CD63 expression was significantly reduced in the reporter cell line
by an siRNA directed against CD63.
[0014] FIG. 6 shows the results of an experiment carried out in a
similar manner as the experiments shown in FIGS. 3 and 4, except
that the reporter cells were incubated with the
anti-IL-4R/anti-CD63 multispecific antigen binding protein ("Ab
conjugate") or control constructs ("control 1" and "control 2") for
15 minutes, 30 minutes, 1 hour or 2 hours prior to the addition of
IL-4 ligand.
[0015] FIG. 7 shows the results of an experiment in which Stat6-luc
reporter cells were treated with 10 pM IL-4 in the presence of
various dilutions of an anti-IL-4R.times.anti-CD63 bispecific
antibody ("bispecific"), or control constructs (anti-IL-4R
monospecific, or mock bispecific that only binds IL-4R).
[0016] FIG. 8 shows the results of experiments in which HEK293
cells were treated with a SOST construct labeled with a myc tag and
a pH-sensitive label (that produces a fluorescent signal at low
pH), along with the various mono-specific and bispecific antibodies
as shown. Results are expressed in terms of number of fluorescent
spots (i.e., labeled vesicles) per cell. Panel A shows the results
following incubation on ice for 3 hours, panel B shows the results
following 1 hour incubation at 37.degree. C., and panel C shows the
results following 3 hours incubation at 37.degree. C.
[0017] FIG. 9 shows the results of experiments in which HEK293
cells were treated with fluorescently-labeled lipopolysaccharide
(LPS) from E. coli (Panel A) or S. minnesota (Panel B), along with
an anti-CD63.times.anti-LPS bispecific antibody, control
antibodies, or LPS only, for various times, followed by quenching
of non-internalized (i.e., surface bound) fluorophore. Fluorescent
signal therefore reflects internalized LPS under the various
conditions shown. Results are expressed in terms of number of
fluorescent spots (i.e., labeled vesicles) per cell.
DETAILED DESCRIPTION
[0018] Before the present invention is described, it is to be
understood that this invention is not limited to particular methods
and experimental conditions described, as such methods and
conditions may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only by the appended
claims.
[0019] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. As used
herein, the term "about," when used in reference to a particular
recited numerical value, means that the value may vary from the
recited value by no more than 1%. For example, as used herein, the
expression "about 100" includes 99 and 101 and all values in
between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
[0020] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, the preferred methods and materials are now
described. All patents, applications and non-patent publications
mentioned in this specification are incorporated herein by
reference in their entireties.
Multispecific Antigen-Binding Molecules
[0021] The present inventors have surprisingly discovered that a
target molecule's activity can be attenuated by linking the target
molecule to an internalizing effector protein via a multispecific
antigen-binding molecule.
[0022] Accordingly, the present invention provides multispecific
antigen binding molecules comprising a first antigen-binding domain
(also referred to herein as "D1"), and a second antigen-binding
domain (also referred to herein as "D2"). D1 and D2 each bind
different molecules. D1 specifically binds a "target molecule". The
target molecule is also referred to herein as "T". D2 specifically
binds an "internalizing effector protein". The internalizing
effector protein is also referred to herein as "E". According to
the present invention, the simultaneous binding of T and E by the
multispecific antigen-binding molecule attenuates the activity of T
to a greater extent than the binding of T by D1 alone. As used
herein, the expression "simultaneous binding," in the context of a
multispecific antigen-binding molecule, means that the
multispecific antigen-binding molecule is capable of contacting
both a target molecule (T) and an internalizing effector protein
(E) for at least some period of time under physiologically relevant
conditions to facilitate the physical linkage between T and E.
Binding of the multispecific antigen-binding molecule to the T and
E components may be sequential; e.g., the multispecific
antigen-binding molecule may first bind T and then bind E, or it
may first bind E first and then bind T. In any event, so long as T
and E are both bound by the multispecific antigen-binding molecule
for some period of time (regardless of the sequential order of
binding), the multispecific antigen-binding molecule will be deemed
to "simultaneously bind" T and E for purposes of the present
disclosure. Without being bound by theory, the enhanced
inactivation of T is believed to be caused by the internalization
and degradative rerouting of T within a cell due to its physical
linkage to E. The multispecific antigen-binding molecules of the
present invention are thus useful for inactivating and/or reducing
the activity and/or extracellular concentration of a target
molecule without directly blocking or antagonizing the function of
the target molecule.
[0023] According to the present invention, a multispecific
antigen-binding molecule can be a single multifunctional
polypeptide, or it can be a multimeric complex of two or more
polypeptides that are covalently or non-covalently associated with
one another. As will be made evident by the present disclosure, any
antigen binding construct which has the ability to simultaneously
bind a T and an E molecule is regarded as a multispecific
antigen-binding molecule. Any of the multispecific antigen-binding
molecules of the invention, or variants thereof, may be constructed
using standard molecular biological techniques (e.g., recombinant
DNA and protein expression technology), as will be known to a
person of ordinary skill in the art.
Antigen-Binding Domains
[0024] The multispecific antigen-binding molecules of the present
invention comprise at least two separate antigen-binding domains
(D1 and D2). As used herein, the expression "antigen-binding
domain" means any peptide, polypeptide, nucleic acid molecule,
scaffold-type molecule, peptide display molecule, or
polypeptide-containing construct that is capable of specifically
binding a particular antigen of interest. The term "specifically
binds" or the like, as used herein, means that the antigen-binding
domain forms a complex with a particular antigen characterized by a
dissociation constant (K.sub.D) of 500 .mu.M or less, and does not
bind other unrelated antigens under ordinary test conditions.
"Unrelated antigens" are proteins, peptides or polypeptides that
have less than 95% amino acid identity to one another.
[0025] Exemplary categories of antigen-binding domains that can be
used in the context of the present invention include antibodies,
antigen-binding portions of antibodies, peptides that specifically
interact with a particular antigen (e.g., peptibodies), receptor
molecules that specifically interact with a particular antigen,
proteins comprising a ligand-binding portion of a receptor that
specifically binds a particular antigen, antigen-binding scaffolds
(e.g., DARPins, HEAT repeat proteins, ARM repeat proteins,
tetratricopeptide repeat proteins, and other scaffolds based on
naturally occurring repeat proteins, etc., [see, e.g., Boersma and
Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references
cited therein]), and aptamers or portions thereof.
[0026] In certain embodiments in which the target molecule or the
internalizing effector protein is a receptor molecule, an
"antigen-binding domain," for purposes of the present invention,
may comprise or consist of a ligand or portion of a ligand that is
specific for the receptor. For example, if the target molecule (T)
is IL-4R, the D1 component of the multispecific antigen-binding
molecule may comprise the IL-4 ligand or a portion of the IL-4
ligand that is capable of specifically interacting with IL-4R; or
if the internalizing effector protein (E) is transferrin receptor,
the D2 component of the multispecific antigen-binding molecule may
comprise transferrin or a portion of transferrin that is capable of
specifically interacting with the transferrin receptor.
[0027] In certain embodiments in which the target molecule or the
internalizing effector protein is a ligand that is specifically
recognized by a particular receptor (e.g., a soluble target
molecule), an "antigen-binding domain," for purposes of the present
invention, may comprise or consist of the receptor or a
ligand-binding portion of the receptor. For example, if the target
molecule (T) is IL-6, the D1 component of the multispecific
antigen-binding molecule may comprise the ligand-binding domain of
the IL-6 receptor; or if the internalizing effector protein (E) is
an indirectly internalized protein (as that term is defined
elsewhere herein), the D2 component of the multispecific
antigen-binding molecule may comprise a ligand-binding domain of a
receptor specific for E.
[0028] Methods for determining whether two molecules specifically
bind one another are well known in the art and include, for
example, equilibrium dialysis, surface plasmon resonance, and the
like. For example, an antigen-binding domain, as used in the
context of the present invention, includes polypeptides that bind a
particular antigen (e.g., a target molecule [T] or an internalizing
effector protein [E]) or a portion thereof with a K.sub.D of less
than about 500 pM, less than about 400 pM, less than about 300 pM,
less than about 200 pM, less than about 100 pM, less than about 90
pM, less than about 80 pM, less than about 70 pM, less than about
60 pM, less than about 50 pM, less than about 40 pM, less than
about 30 pM, less than about 20 pM, less than about 10 pM, less
than about 5 pM, less than about 4 pM, less than about 2 pM, less
than about 1 pM, less than about 0.5 pM, less than about 0.2 pM,
less than about 0.1 pM, or less than about 0.05 pM, as measured in
a surface plasmon resonance assay.
[0029] The term "surface plasmon resonance", as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
interactions by detection of alterations in protein concentrations
within a biosensor matrix, for example using the BIAcore.TM. system
(Biacore Life Sciences division of GE Healthcare, Piscataway,
N.J.).
[0030] The term "K.sub.D", as used herein, means the equilibrium
dissociation constant of a particular protein-protein interaction
(e.g., antibody-antigen interaction). Unless indicated otherwise,
the K.sub.D values disclosed herein refer to K.sub.D values
determined by surface plasmon resonance assay at 25.degree. C.
Antibodies and Antigen-Binding Fragments of Antibodies
[0031] As indicated above, an "antigen-binding domain" (D1 and/or
D2) can comprise or consist of an antibody or antigen-binding
fragment of an antibody. The term "antibody," as used herein, means
any antigen-binding molecule or molecular complex comprising at
least one complementarity determining region (CDR) that
specifically binds to or interacts with a particular antigen (e.g.,
T or E). The term "antibody" includes immunoglobulin molecules
comprising four polypeptide chains, two heavy (H) chains and two
light (L) chains inter-connected by disulfide bonds, as well as
multimers thereof (e.g., IgM). Each heavy chain comprises a heavy
chain variable region (abbreviated herein as HCVR or V.sub.H) and a
heavy chain constant region. The heavy chain constant region
comprises three domains, C.sub.H1, C.sub.H2 and C.sub.H3. Each
light chain comprises a light chain variable region (abbreviated
herein as LCVR or V.sub.L) and a light chain constant region. The
light chain constant region comprises one domain (C.sub.L1). The
V.sub.H and V.sub.L regions can be further subdivided into regions
of hypervariability, termed complementarity determining regions
(CDRs), interspersed with regions that are more conserved, termed
framework regions (FR). Each V.sub.H and V.sub.L is composed of
three CDRs and four FRs, arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4. In different embodiments of the invention, the FRs of
the antibodies of the invention (or antigen-binding portion
thereof) may be identical to the human germline sequences, or may
be naturally or artificially modified. An amino acid consensus
sequence may be defined based on a side-by-side analysis of two or
more CDRs.
[0032] The D1 and/or D2 components of the multispecific
antigen-binding molecules of the present invention may comprise or
consist of antigen-binding fragments of full antibody molecules.
The terms "antigen-binding portion" of an antibody,
"antigen-binding fragment" of an antibody, and the like, as used
herein, include any naturally occurring, enzymatically obtainable,
synthetic, or genetically engineered polypeptide or glycoprotein
that specifically binds an antigen to form a complex.
Antigen-binding fragments of an antibody may be derived, e.g., from
full antibody molecules using any suitable standard techniques such
as proteolytic digestion or recombinant genetic engineering
techniques involving the manipulation and expression of DNA
encoding antibody variable and optionally constant domains. Such
DNA is known and/or is readily available from, e.g., commercial
sources, DNA libraries (including, e.g., phage-antibody libraries),
or can be synthesized. The DNA may be sequenced and manipulated
chemically or by using molecular biology techniques, for example,
to arrange one or more variable and/or constant domains into a
suitable configuration, or to introduce codons, create cysteine
residues, modify, add or delete amino acids, etc.
[0033] Non-limiting examples of antigen-binding fragments include:
(i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv)
Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb
fragments; and (vii) minimal recognition units consisting of the
amino acid residues that mimic the hypervariable region of an
antibody (e.g., an isolated complementarity determining region
(CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4
peptide. Other engineered molecules, such as domain-specific
antibodies, single domain antibodies, domain-deleted antibodies,
chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies,
tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies,
bivalent nanobodies, etc.), small modular immunopharmaceuticals
(SMIPs), and shark variable IgNAR domains, are also encompassed
within the expression "antigen-binding fragment," as used
herein.
[0034] An antigen-binding fragment of an antibody will typically
comprise at least one variable domain. The variable domain may be
of any size or amino acid composition and will generally comprise
at least one CDR which is adjacent to or in frame with one or more
framework sequences. In antigen-binding fragments having a V.sub.H
domain associated with a V.sub.L domain, the V.sub.H and V.sub.L
domains may be situated relative to one another in any suitable
arrangement. For example, the variable region may be dimeric and
contain V.sub.H-V.sub.H, V.sub.H-V.sub.L or V.sub.L-V.sub.L dimers.
Alternatively, the antigen-binding fragment of an antibody may
contain a monomeric V.sub.H or V.sub.L domain.
[0035] In certain embodiments, an antigen-binding fragment of an
antibody may contain at least one variable domain covalently linked
to at least one constant domain. Non-limiting, exemplary
configurations of variable and constant domains that may be found
within an antigen-binding fragment of an antibody of the present
invention include: (i) V.sub.H-C.sub.H1; (ii) V.sub.H-C.sub.H2;
(iii) V.sub.H-C.sub.H3; (iv) V.sub.H-C.sub.H1-C.sub.H2; (V)
V.sub.H-C.sub.H1-C.sub.H2-C.sub.H3; (vi) V.sub.H-C.sub.H2-C.sub.H3;
(vii) V.sub.H-C.sub.L; (viii) V.sub.L-C.sub.H1; (ix)
V.sub.L-C.sub.H2; (x) V.sub.L-C.sub.H3; (xi)
V.sub.L-C.sub.H1-C.sub.H2; (xii)
V.sub.L-C.sub.H1-C.sub.H2-C.sub.H3; (xiii)
V.sub.L-C.sub.H2-C.sub.H3; and (xiv) V.sub.L-C.sub.L. In any
configuration of variable and constant domains, including any of
the exemplary configurations listed above, the variable and
constant domains may be either directly linked to one another or
may be linked by a full or partial hinge or linker region. A hinge
region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or
more) amino acids which result in a flexible or semi-flexible
linkage between adjacent variable and/or constant domains in a
single polypeptide molecule. Moreover, an antigen-binding fragment
may comprise a homo-dimer or hetero-dimer (or other multimer) of
any of the variable and constant domain configurations listed above
in non-covalent association with one another and/or with one or
more monomeric V.sub.H or V.sub.L domain (e.g., by disulfide
bond(s)).
[0036] The multispecific antigen-binding molecules of the present
invention may comprise or consist of human antibodies and/or
recombinant human antibodies, or fragments thereof. The term "human
antibody", as used herein, includes antibodies having variable and
constant regions derived from human germline immunoglobulin
sequences. Human antibodies may nonetheless include amino acid
residues not encoded by human germline immunoglobulin sequences
(e.g., mutations introduced by random or site-specific mutagenesis
in vitro or by somatic mutation in vivo), for example in the CDRs
and in particular CDR3. However, the term "human antibody", as used
herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework
sequences.
[0037] The multispecific antigen-binding molecules of the present
invention may comprise or consist of recombinant human antibodies
or antigen-binding fragments thereof. The term "recombinant human
antibody", as used herein, is intended to include all human
antibodies that are prepared, expressed, created or isolated by
recombinant means, such as antibodies expressed using a recombinant
expression vector transfected into a host cell (described further
below), antibodies isolated from a recombinant, combinatorial human
antibody library (described further below), antibodies isolated
from an animal (e.g., a mouse) that is transgenic for human
immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids
Res. 20:6287-6295) or antibodies prepared, expressed, created or
isolated by any other means that involves splicing of human
immunoglobulin gene sequences to other DNA sequences. Such
recombinant human antibodies have variable and constant regions
derived from human germline immunoglobulin sequences. In certain
embodiments, however, such recombinant human antibodies are
subjected to in vitro mutagenesis (or, when an animal transgenic
for human Ig sequences is used, in vivo somatic mutagenesis) and
thus the amino acid sequences of the V.sub.H and V.sub.L regions of
the recombinant antibodies are sequences that, while derived from
and related to human germline V.sub.H and V.sub.L sequences, may
not naturally exist within the human antibody germline repertoire
in vivo.
Bispecific Antibodies
[0038] According to certain embodiments, the multispecific
antigen-binding molecules of the invention are bispecific
antibodies; e.g., bispecific antibodies comprising an
antigen-binding arm that specifically binds a target molecule (T)
and an antigen-binding arm that specifically binds an internalizing
effector protein (E). Methods for making bispecific antibodies are
known in the art and may be used to construct multispecific
antigen-binding molecules of the present invention. Exemplary
bispecific formats that can be used in the context of the present
invention include, without limitation, e.g., scFv-based or diabody
bispecific formats, IgG-scFv fusions, dual variable domain
(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g.,
common light chain with knobs-into-holes, etc.), CrossMab,
CrossFab, (SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual
acting Fab (DAF)-IgG, and Mab.sup.2 bispecific formats (see, e.g.,
Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein,
for a review of the foregoing formats).
Multimerizing Components
[0039] The multispecific antigen-binding molecules of the present
invention, in certain embodiments, may also comprise one or more
multimerizing component(s). The multimerizing components can
function to maintain the association between the antigen-binding
domains (D1 and D2). As used herein, a "multimerizing component" is
any macromolecule, protein, polypeptide, peptide, or amino acid
that has the ability to associate with a second multimerizing
component of the same or similar structure or constitution. For
example, a multimerizing component may be a polypeptide comprising
an immunoglobulin C.sub.H3 domain. A non-limiting example of a
multimerizing component is an Fc portion of an immunoglobulin,
e.g., an Fc domain of an IgG selected from the isotypes IgG1, IgG2,
IgG3, and IgG4, as well as any allotype within each isotype group.
In certain embodiments, the multimerizing component is an Fc
fragment or an amino acid sequence of 1 to about 200 amino acids in
length containing at least one cysteine residues. In other
embodiments, the multimerizing component is a cysteine residue, or
a short cysteine-containing peptide. Other multimerizing domains
include peptides or polypeptides comprising or consisting of a
leucine zipper, a helix-loop motif, or a coiled-coil motif.
[0040] In certain embodiments, the multispecific antigen-binding
molecules of the present invention comprise two multimerizing
domains, M1 and M2, wherein D1 is attached to M1 and D2 is attached
to M2, and wherein the association of M1 with M2 facilitates the
physical linkage of D1 and D2 to one another in a single
multispecific antigen-binding molecule. In certain embodiments, M1
and M2 are identical to one another. For example, M1 can be an Fc
domain having a particular amino acid sequence, and M2 is an Fc
domain with the same amino acid sequence as M1. Alternatively, M1
and M2 may differ from one another at one or more amino acid
position. For example, M1 may comprise a first immunoglobulin (Ig)
C.sub.H3 domain and M2 may comprise a second Ig C.sub.H3 domain,
wherein the first and second Ig C.sub.H3 domains differ from one
another by at least one amino acid, and wherein at least one amino
acid difference reduces binding of the targeting construct to
Protein A as compared to a reference construct having identical M1
and M2 sequences. In one embodiment, the Ig C.sub.H3 domain of M1
binds Protein A and the Ig C.sub.H3 domain of M2 contains a
mutation that reduces or abolishes Protein A binding such as an
H95R modification (by IMGT exon numbering; H435R by EU numbering).
The C.sub.H3 of M2 may further comprise a Y96F modification (by
IMGT; Y436F by EU). Further modifications that may be found within
the C.sub.H3 of M2 include: D16E, L18M, N44S, K52N, V57M, and V821
(by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by EU) in
the case of an IgG1 Fc domain; N44S, K52N, and V821 (IMGT; N384S,
K392N, and V422I by EU) in the case of an IgG2 Fc domain; and Q15R,
N44S, K52N, V57M, R69K, E79Q, and V821 (by IMGT; Q355R, N384S,
K392N, V397M, R409K, E419Q, and V422I by EU) in the case of an IgG4
Fc domain.
Internalizing Effector Proteins (E)
[0041] In the context of the present invention, the D2 component of
the multispecific antigen-binding molecule specifically binds an
internalizing effector protein ("E"). An internalizing effector
protein is a protein that is capable of being internalized into a
cell or that otherwise participates in or contributes to retrograde
membrane trafficking. In some instances, the internalizing effector
protein is a protein that undergoes transcytosis; that is, the
protein is internalized on one side of a cell and transported to
the other side of the cell (e.g., apical-to-basal). In many
embodiments, the internalizing effector protein is a cell
surface-expressed protein or a soluble extracellular protein.
However, the present invention also contemplates embodiments in
which the internalizing effector protein is expressed within an
intracellular compartment such as the endosome, endoplasmic
reticulum, Golgi, lysosome, etc. For example, proteins involved in
retrograde membrane trafficking (e.g., pathways from
early/recycling endosomes to the trans-Golgi network) may serve as
internalizing effector proteins in various embodiments of the
present invention. In any event, the binding of D2 to an
internalizing effector protein causes the entire multispecific
antigen-binding molecule, and any molecules associated therewith
(e.g., a target molecule bound by D1), to also become internalized
into the cell. As explained below, internalizing effector proteins
include proteins that are directly internalized into a cell, as
well as proteins that are indirectly internalized into a cell.
[0042] Internalizing effector proteins that are directly
internalized into a cell include membrane-associated molecules with
at least one extracellular domain (e.g., transmembrane proteins,
GPI-anchored proteins, etc.), which undergo cellular
internalization, and are preferably processed via an intracellular
degradative and/or recycling pathway. Specific non-limiting
examples of internalizing effector proteins that are directly
internalized into a cell include, e.g., CD63, MHC-I (e.g.,
HLA-B27), Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin
receptor, LDL-receptor, LDL-related protein 1 receptor, ASGR1,
ASGR2, amyloid precursor protein-like protein-2 (APLP2), apelin
receptor (APLNR), MAL (Myelin And Lymphocyte protein, a.k.a.
VIP17), IGF2R, vacuolar-type H.sup.+ ATPase, diphtheria toxin
receptor, folate receptor, glutamate receptors, glutathione
receptor, leptin receptors, scavenger receptors (e.g., SCARA1-5,
SCARB1-3, CD36), etc.
[0043] In embodiments in which E is a directly internalized
effector protein, the D2 component of the multispecific
antigen-binding molecule can be, e.g., an antibody or
antigen-binding fragment of an antibody that specifically binds E,
or a ligand or portion of a ligand that specifically interacts with
the effector protein. For example, if E is Kremen-1 or Kremen-2,
the D2 component can comprise or consist of a Kremen ligand (e.g.,
DKK1) or Kremen-binding portion thereof. As another example, if E
is a receptor molecule such as ASGR1, the D2 component can comprise
or consist of a ligand specific for the receptor (e.g.,
asialoorosomucoid [ASOR] or Beta-GalNAc) or a receptor-binding
portion thereof.
[0044] Internalizing effector proteins that are indirectly
internalized into a cell include proteins and polypeptides that do
not internalize on their own, but become internalized into a cell
after binding to or otherwise associating with a second protein or
polypeptide that is directly internalized into the cell. Proteins
that are indirectly internalized into a cell include, e.g., soluble
ligands that are capable of binding to an internalizing cell
surface-expressed receptor molecule. A non-limiting example of a
soluble ligand that is (indirectly) internalized into a cell via
its interaction with an internalizing cell surface-expressed
receptor molecule is transferrin. In embodiments wherein E is
transferrin (or another indirectly internalized protein), the
binding of D2 to E, and the interaction of E with transferrin
receptor (or another internalizing cell-surface expressed receptor
molecule), causes the entire multispecific antigen-binding
molecule, and any molecules associated therewith (e.g., a target
molecule bound by D1), to become internalized into the cell
concurrent with the internalization of E and its binding
partner.
[0045] In embodiments in which E is an indirectly internalized
effector protein such as a soluble ligand, the D2 component of the
multispecific antigen-binding molecule can be, e.g., an antibody or
antigen-binding fragment of an antibody that specifically binds E,
or a receptor or portion of a receptor that specifically interacts
with the soluble effector protein. For example, if E is a cytokine,
the D2 component can comprise or consist of the corresponding
cytokine receptor or ligand-binding portion thereof.
Target Molecules (T)
[0046] In the context of the present invention, the D1 component of
the multispecific antigen-binding molecule specifically binds a
target molecule ("T"). A target molecule is any protein,
polypeptide, or other macromolecule whose activity or extracellular
concentration is desired to be attenuated, reduced or eliminated.
In many instances, the target molecule to which D1 binds is a
protein or polypeptide [i.e., a "target protein"]; however, the
present invention also includes embodiments wherein the target
molecule ("T") is a carbohydrate, glycoprotein, lipid, lipoprotein,
lipopolysaccharide, or other non-protein polymer or molecule to
which D1 binds. According to the present invention, T can be a cell
surface-expressed target protein or a soluble target protein.
Target binding by the multispecific antigen-binding molecule may
take place in an extracellular or cell surface context. In certain
embodiments, however, the multispecific antigen-binding molecule
binds a target molecule inside the cell, for example within an
intracellular component such as the endoplasmic reticulum, Golgi,
endosome, lysosome, etc.
[0047] Examples of cell surface-expressed target molecules include
cell surface-expressed receptors, membrane-bound ligands, ion
channels, and any other monomeric or multimeric polypeptide
component with an extracellular portion that is attached to or
associated with a cell membrane. Non-limiting, exemplary cell
surface-expressed target molecules that may be targeted by the
multispecific antigen-binding molecule of the present invention
include, e.g., cytokine receptors (e.g., receptors for IL-1, IL-4,
IL-6, IL-13, IL-22, IL-25, IL-33, etc.), as well as cell surface
targets including other type 1 transmembrane receptors such as
PRLR, G-protein coupled receptors such as GCGR, ion channels such
as Nav1.7, ASIC1 or ASIC2, non-receptor surface proteins such as
MHC-I (e.g., HLA-B*27), etc.
[0048] In embodiments in which T is a cell surface-expressed target
protein, the D1 component of the multispecific antigen-binding
molecule can be, e.g., an antibody or antigen-binding fragment of
an antibody that specifically binds T, or a ligand or portion of a
ligand that specifically interacts with the cell surface-expressed
target protein. For example, if T is IL-4R, the D1 component can
comprise or consist of IL-4 or a receptor-binding portion
thereof.
[0049] Examples of soluble target molecules include cytokines,
growth factors, and other ligands and signaling proteins.
Non-limiting exemplary soluble target protein that may be targeted
by the multispecific antigen-binding molecule of the present
invention include, e.g., IL-1, IL-4, IL-6, IL-13, IL-22, IL-25,
IL-33, SOST, DKK1, etc. Soluble targets molecules also include,
e.g., non-human target molecules such as allergens (e.g., Fel D1,
Betv1, CryJ1), pathogens (e.g., Candida albicans, S. aureus, etc.),
and pathogenic molecules (e.g., lipopolysaccharide [LPS],
lipotechoic acid [LTA], Protein A., toxins, etc.). In embodiments
in which T is a soluble target molecule, the D1 component of the
multispecific antigen-binding molecule can be, e.g., an antibody or
antigen-binding fragment of an antibody that specifically binds T,
or a receptor or portion of a receptor that specifically interacts
with the soluble target molecule. For example, if T is IL-4, the D1
component can comprise or consist of IL-4R or a ligand-binding
portion thereof.
[0050] Target molecules also include tumor-associated antigens, as
described elsewhere herein.
pH-Dependent Binding
[0051] The present invention provides multispecific antigen-binding
molecules comprising a first antigen-binding domain (D1) and a
second antigen-binding domain (D2), wherein one or both of the
antigen-binding domains (D1 and/or D2) binds its antigen (T or E)
in a pH-dependent manner. For example, an antigen-binding domain
(D1 and/or D2) may exhibit reduced binding to its antigen at acidic
pH as compared to neutral pH. Alternatively, an antigen-binding
domain (D1 and/or D2) may exhibit enhanced binding to its antigen
at acidic pH as compared to neutral pH. Antigen-binding domains
with pH-dependent binding characteristics may be obtained, e.g., by
screening a population of antibodies for reduced (or enhanced)
binding to a particular antigen at acidic pH as compared to neutral
pH. Additionally, modifications of the antigen-binding domain at
the amino acid level may yield antigen-binding domains with
pH-dependent characteristics. For example, by substituting one or
more amino acid of an antigen-binding domain (e.g., within a CDR)
with a histidine residue, an antigen-binding domain with reduced
antigen-binding at acidic pH relative to neutral pH may be
obtained.
[0052] In certain embodiments, the present invention includes
multispecific antigen-binding molecules comprising a D1 and/or D2
component that binds its respective antigen (T or E) at acidic pH
with a K.sub.D that is at least about 3, 5, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, or more times greater than the K.sub.D of the D1
and/or D2 component for binding to its respective antigen at
neutral pH. pH dependent binding may also be expressed in terms of
the t1/2 of the antigen-binding domain for its antigen at acidic pH
compared to neutral pH. For example, the present invention includes
multispecific antigen-binding molecules comprising a D1 and/or D2
component that binds its respective antigen (T or E) at acidic pH
with a t1/2 that is at least about 5, 6, 7, 8, 9, 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, or more times shorter than the t1/2 of
the D1 and/or D2 component for binding to its respective antigen at
neutral pH.
[0053] Multispecific antigen-binding molecules of the present
invention that comprise a D1 and/or D2 component with reduced
antigen binding at acidic pH as compared to neutral pH, when
administered to animal subjects, may in certain embodiments exhibit
slower clearance from circulation as compared to comparable
molecules that do not exhibit pH-dependent binding characteristics.
According to this aspect of the invention, multispecific
antigen-binding molecules with reduced antigen binding to either T
and/or E at acidic pH as compared to neutral pH are provided which
exhibit at least 2 times slower clearance from circulation relative
to comparable antigen-binding molecules that do not possess reduced
antigen binding at acidic pH as compared to neutral pH. Clearance
rate can be expressed in terms of the half-life of the antibody,
wherein a slower clearance correlates with a longer half-life.
[0054] As used herein, the expression "acidic pH" means a pH of 6.0
or less. The expression "acidic pH" includes pH values of about
6.0, 5.95, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35,
5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, the
expression "neutral pH" means a pH of about 7.0 to about 7.4. The
expression "neutral pH" includes pH values of about 7.0, 7.05, 7.1,
7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.
Attenuation of Target Molecule Activity
[0055] As noted elsewhere herein, and as demonstrated by the
working Examples herein below, the present inventors have
discovered that the simultaneous binding of a target molecule (T)
and an internalizing effector protein (E) by a multispecific
antigen-binding molecule attenuates the activity of T to a greater
extent than the binding of T by the first antigen-binding domain
(D1) component of the multispecific antigen-binding molecule alone.
As used herein, the expression "attenuates the activity of T to a
greater extent than the binding of T by D1 alone" means that, in an
assay in which the activity of T can be measured using cells that
express E, the level of T activity measured in the presence of a
multispecific antigen-binding molecule is at least 10% lower than
the level of T activity measured in the presence of a control
construct containing D1 by itself (i.e., not physically linked to
the second antigen-binding domain (D2)). For instance, the level of
T activity measured in the presence of the multispecific
antigen-binding molecule may be about 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%
lower than the level of T activity measured in the presence of a
control construct containing D1 by itself.
[0056] A non-limiting, illustrative assay format for determining
whether a multispecific antigen-binding molecule attenuates the
activity of a target molecule to a greater extent than the binding
of the target molecule by the D1 domain alone is shown in working
Examples 1 and 2, herein below. In Example 1, for instance, "T" is
the interleukin-4 receptor (IL-4R), and "E" is CD63. The
multispecific antigen-binding molecule of Example 1 is a 2-antibody
conjugate comprising an anti-IL-4R mAb linked to an anti-CD63 mAb
via a streptavidin/biotin linker. Thus, "D1" in this exemplary
construct is the antigen-binding domain (HCVR/LCVR pair) of the
anti-IL-4R antibody, and "D2" is the antigen-binding domain
(HCVR/LCVR pair) of the anti-CD63 antibody. For the experiments of
Examples 1 and 2, a cell-based assay format was used that produces
a reporter signal when IL-4R activity is stimulated by the addition
of exogenous IL-4 ligand. The amount of IL-4-induced reporter
activity detected in the presence of the multispecific
antigen-binding molecule was compared to the amount of IL-4-induced
reporter activity detected in the presence of control constructs
containing the anti-IL-4R antibody either connected to an
irrelevant control immunoglobulin (control 1), or combined with,
but not physically connected to, the anti-CD63 antibody (control
2). The control constructs thus produce the condition in which T is
bound by D1 alone (i.e., wherein D1 is not a part of the
multispecific antigen-binding molecule per se). If the extent of
target molecule activity (represented by the reporter signal)
observed in the presence of the multispecific antigen-binding
molecule is at least 10% less than the amount of target molecule
activity observed in the presence of a control construct comprising
the D1 component not physically linked to the D2 component (e.g.,
control 1 or control 2), then for purposes of the present
disclosure, it is concluded that "the simultaneous binding of T and
E by the multispecific antigen-binding molecule attenuates the
activity of T to a greater extent than the binding of T by D1
alone."
[0057] The binding of T by D1 alone may, in some embodiments,
result in partial attenuation of the activity of T (as in the case
of Example 1 where the treatment of reporter cells with an
anti-IL-4R antibody alone [i.e., controls 1 and 2] caused a small
level of attenuation of IL-4 signaling relative to untreated
cells). In other embodiments, the binding of T by D1 alone will
result in no detectable attenuation of the activity of T; that is,
the biological activity of T may be unaffected by the binding of T
by D1 alone. In any event, however, the simultaneous binding of T
and E by a multispecific antigen-binding molecule of the invention
will attenuate the activity of T to a greater extent than the
binding of T by D1 alone.
[0058] Alternative assay formats and variations on the assay
format(s) exemplified herein will be apparent to persons of
ordinary skill in the art, taking into account the nature of the
specific target molecule and effector proteins to which any given
multispecific antigen-binding molecule may be directed. Any such
format can be used in the context of the present invention to
determine whether the simultaneous binding of T and E by a
multispecific antigen-binding molecule attenuates the activity of T
to a greater extent than the binding of T by D1 alone.
Tumor Targeting
[0059] In another aspect of the invention, the multispecific
antigen-binding molecules are useful for targeting tumor cells.
According to this aspect of the invention, the target molecule "T"
to which D1 binds is a tumor-associated antigen. In certain
instances, the tumor-associated antigen is an antigen that is not
ordinarily internalized. The internalizing effector protein "E" to
which D2 binds may be tumor specific, or it may be expressed on
both tumor and non-tumor cells of an individual. Any of the
internalizing effector proteins mentioned elsewhere herein may be
targeted for anti-tumor applications of the invention.
[0060] As used herein, the term "tumor-associated antigen" includes
proteins or polypeptides that are preferentially expressed on the
surface of a tumor cell. The expression "preferentially expressed,"
as used in this context, means that the antigen is expressed on a
tumor cell at a level that is at least 10% greater (e.g., 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 150%, 200%, 400%, or
more) than the expression level of the antigen on non-tumor cells.
In certain embodiments, the target molecule is an antigen that is
preferentially expressed on the surface of a tumor cell selected
from the group consisting of a renal tumor cell, a colon tumor
cell, a breast tumor cell, an ovarian tumor cell, a skin tumor
cell, a lung tumor cell, a prostate tumor cell, a pancreatic tumor
cell, a glioblastoma cell, a head and neck tumor cell and a
melanoma cell. Non-limiting examples of specific tumor-associated
antigens include, e.g., AFP, ALK, BAGE proteins, .beta.-catenin,
brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CD40,
CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2/Her2,
ErbB3, ErbB4, ETV6-AML, EphA2, Fra-1, FOLR1, GAGE proteins (e.g.,
GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2,
HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, LMP2, MAGE proteins
(e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin,
ML-IAP, Muc1, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85,
NY-ES01, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR,
PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3,
Steap-1, Steap-2, survivin, TAG-72, TGF-.beta., TMPRSS2, Tn, TRP-1,
TRP-2, tyrosinase, and uroplakin-3.
[0061] The multispecific antigen-binding molecule, according to
this aspect of the invention, may be conjugated to a drug, toxin,
radioisotope, or other substance which is detrimental to the
viability of a cell. Alternatively, the drug or toxin may be a
substance which does not directly kill a cell, but renders a cell
more susceptible to killing by other external agents. In yet other
embodiments involving tumor targeting, the multispecific
antigen-binding molecule of the invention is not itself conjugated
to a drug, toxin or radioisotope, but instead is administered in
combination with a second antigen-binding molecule specific for the
target (T) (herein referred to as an "accomplice molecule"),
wherein the accomplice molecule is conjugated to a drug, toxin or
radioisotope. In such embodiments, the multispecific antigen
binding molecule will preferably bind to an epitope on the target
molecule (T) that is distinct from and/or non-overlapping with the
epitope recognized by the accomplice molecule (i.e., to allow for
simultaneous binding of the multispecific antigen-binding molecule
and the accomplice molecule to the target).
[0062] In a related embodiment, the present invention also includes
anti-tumor combinations, and therapeutic methods, comprising: (a) a
toxin- or drug-conjugated antigen-binding molecule that
specifically binds a tumor-associated antigen; and (b) a
multispecific antigen-binding molecule comprising (i) a first
binding domain that specifically binds an internalizing effector
protein (e.g., with low affinity) and (ii) a second binding domain
that specifically binds the toxin- or drug-conjugated
antigen-binding molecule. In this embodiment, the multispecific
antigen-binding molecule functions to link the toxin- or
drug-conjugated antigen-binding molecule to the internalizing
effector protein, which thereby functions to physically link the
tumor associated antigen to the internalizing effector protein.
Internalization of the toxin-labeled anti-tumor-associated antigen
antibody via its connection to the internalizing effector protein
would consequently result in targeted tumor cell killing.
[0063] According to certain embodiments of the tumor-targeting
aspects of the invention, the multispecific antigen-binding
molecule (or accomplice antibody) may be conjugated to one or more
cytotoxic drugs selected from the group consisting of:
calicheamicin, esperamicin, methotrexate, doxorubicin, melphalan,
chlorambucil, ARA-C, vindesine, mitomycin C, cis-platinum,
etoposide, bleomycin, 5-fluorouracil, estramustine, vincristine,
etoposide, doxorubicin, paclitaxel, larotaxel, tesetaxel, orataxel,
docetaxel, dolastatin 10, auristatin E, auristatin PHE and
maytansine-based compounds (e.g., DM1, DM4, etc.). The
multispecific antigen-binding molecule (or accomplice antibody) may
also, or alternatively, be conjugated to a toxin such as diphtheria
toxin, Pseudomonas aeruginosa exotoxin A, ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins, etc. The
multispecific antigen-binding molecule (or accomplice antibody) may
also, or alternatively, be conjugated to one or more radioisotope
selected from the group consisting of .sup.225Ac, .sup.211At,
.sup.212Bi, .sup.213Bi, .sup.186Rh, .sup.188Rh, .sup.177Lu,
.sup.80Y, .sup.131I, .sup.67Cu, .sup.125I, .sup.123I, .sup.77Br,
.sup.153Sm, .sup.166Ho, .sup.64Cu, .sup.121Pb, .sup.224Ra and
.sup.223Ra. Thus, this aspect of the invention includes
multispecific antigen-binding molecules that are antibody-drug
conjugates (ADCs) or antibody-radioisotope conjugates (ARCs).
[0064] In the context of tumor killing applications, the D2
component may, in certain circumstances, bind with low affinity to
the internalizing effector protein "E". Thus, the multispecific
antigen-binding molecule will preferentially target tumor cells
that express the tumor-associated antigen. As used herein, "low
affinity" binding means that the binding affinity of the D2
component for the internalizing effector protein (E) is at least
10% weaker (e.g., 15% weaker, 25% weaker, 50% weaker, 75% weaker,
90% weaker, etc.) than the binding affinity of the D1 component for
the target molecule (T). In certain embodiments, "low affinity"
binding means that the D2 component interacts with the
internalizing effector protein (E) with a K.sub.D of greater than
about 10 nM to about 1 .mu.M, as measured in a surface plasmon
resonance assay at about 25.degree. C.
[0065] The simultaneous binding of a multispecific antigen-binding
molecule to an internalizing effector protein and a
tumor-associated antigen will result in preferential
internalization of the multispecific antigen-binding molecule into
tumor cells. If, for example, the multispecific antigen-binding
molecule is conjugated to a drug, toxin or radioisotope (or if the
multispecific antigen-binding molecule is administered in
combination with an accomplice antibody that is conjugated to a
drug, toxin or radioisotope), the targeted internalization of the
tumor-associated antigen into the tumor cell via its linkage to the
multispecific antigen-binding molecule, will result in extremely
specific tumor cell killing.
Pharmaceutical Compositions and Administration Methods
[0066] The present invention includes pharmaceutical compositions
comprising a multispecific antigen-binding molecule. The
pharmaceutical compositions of the invention can be formulated with
suitable carriers, excipients, and other agents that provide
improved transfer, delivery, tolerance, and the like.
[0067] The present invention also includes methods for inactivating
or attenuating the activity of a target molecule (T). The methods
of the present invention comprise contacting a target molecule with
a multispecific antigen-binding molecule as described herein. In
certain embodiments, the methods according to this aspect of the
invention comprise administering a pharmaceutical composition
comprising a multispecific antigen-binding molecule to a patient
for whom it is desirable and/or beneficial to inactivate,
attenuate, or otherwise decrease the extracellular concentration of
a target molecule.
[0068] Various delivery systems are known in the art and can be
used to administer the pharmaceutical compositions of the present
invention to a patient. Methods of administration that can be used
in the context of the present invention include, but are not
limited to, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes.
The pharmaceutical compositions of the invention may be
administered by any convenient route, for example by infusion or
bolus injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.),
and may be administered together with other biologically active
agents. Administration can be systemic or local. For example, a
pharmaceutical composition of the present invention can be
delivered subcutaneously or intravenously with a standard needle
and syringe. In addition, with respect to subcutaneous delivery, a
pen delivery device can be used to administer a pharmaceutical
composition of the present invention to a patient.
EXAMPLES
[0069] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the methods and compositions of
the invention, and are not intended to limit the scope of what the
inventors regard as their invention. Efforts have been made to
ensure accuracy with respect to numbers used (e.g., amounts,
temperature, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, molecular weight is average molecular weight,
temperature is in degrees Centigrade, and pressure is at or near
atmospheric.
Example 1
Use of a Multispecific Antigen-Binding Molecule to Induce
Degradation of a Cell Surface Receptor Via Linkage with an
Internalizing Effector Protein
[0070] As an initial proof-of-concept experiment, a multispecific
antigen-binding molecule was created which is capable of binding
(a) an internalizing effector molecule and (b) a cell surface
receptor target molecule. In this Example, the internalizing
effector protein is Kremen-2 (Krm2), and the cell surface receptor
target molecule is an Fc receptor (Fc.gamma.R1 [Fc-gamma-R1]).
[0071] Kremen molecules (Krm1 and Krm2) are cell-surface proteins
known to mediate WNT signaling by directing the internalization and
degradation of the WNT pathway signaling molecules LRP5 and LRP6.
Internalization of LRP5/6 is accomplished via the soluble
interacting protein DKK1. In particular, DKK1 links Kremen to
LRP5/6 on the cell surface, and because of this linkage, the
internalization of Kremen drives the internalization and
degradation of LRP5 and LRP6. (See Li et al., PLoS One
5(6):e11014).
[0072] The present inventors sought to exploit the Kremen-binding
properties of DKK1 and the internalization properties of Kremen to
induce the internalization of Fc.gamma.R1. To facilitate
Kremen-mediated internalization/degradation of Fc.gamma.R1, a
multispecific antigen-binding molecule was constructed consisting
of DKK1 fused to a mouse Fc (DKK1-mFc, having the amino acid
sequence of SEQ ID NO:1). As explained elsewhere herein, a
multispecific antigen-binding molecule is defined as a molecule
comprising a first antigen-binding domain (D1) which specifically
binds a target molecule, and a second antigen-binding domain (D2)
which specifically binds an internalizing effector protein. In this
proof-of-concept Example, the "first antigen-binding domain" is the
mFc component which specifically binds the target molecule
Fc.gamma.R1, and the "second antigen-binding domain" is the DKK1
component which specifically binds the internalizing effector
protein Kremen.
[0073] An experiment was first conducted to determine whether
DKK1-mFc can be endocytosed into cells in a Kremen-dependent
manner. For this experiment, two cell lines were used: Cell-1, an
HEK293 cell line engineered to express Fc.gamma.R1 but not
Kremen-2, and Cell-2, an HEK293 cell line engineered to express
both Fc.gamma.R1 and Kremen-2. A 1:10 dilution of DKK1-mFc
conditioned medium was added to the respective cell lines and
allowed to incubate at 37.degree. C. for 90 minutes. After the 90
minute incubation, cells were stained with Alexa-488-labeled
anti-mouse IgG antibody to detect the DKK1-mFc molecule. Using
fluorescence microscopy, it was observed that virtually no DKK1-mFc
was localized inside Cell-1 (lacking Kremen); however, substantial
amounts of DKK1-mFc were detected within Cell-2 which expresses
Kremen-2. Thus, these results show that the multispecific
antigen-binding molecule DKK1-mFc can be internalized into cells in
a Kremen-dependent manner.
[0074] Next, a time-course experiment was conducted to determine
whether DKK1-mFc can induce Fc.gamma.R1 degradation in a
Kremen-dependent manner. A brief description of the experimental
protocol is as follows: Cell-1 (expressing only Fc.gamma.R1) and
Cell-2 (expressing Kremen-2 and Fc.gamma.R1) were treated with 2
mg/ml NHS-Sulfo-Biotin for 15 minutes on ice to label all cell
surface expressed proteins. Cells were then washed and resuspended
in 400 .mu.l of medium and divided into four-100 .mu.l aliquots
which were treated with DKK1-mFc for varying amounts of time (0
min, 15 min, 30 min and 60 min) at 37.degree. C. Following DKK1-mFc
incubation, cells were pelleted and treated with protease
inhibitors. Lysates of the cells from the different incubation time
points were subjected to Fc.gamma.R1 immunoprecipitation. For the
Fc.gamma.R1 immunoprecipitation, mouse anti-Fc.gamma.R1 antibody
was added to cell lysates and incubated for 1 hour at 4.degree. C.
Then Protein-G beads were added and the mixture was incubated for 1
hour at 4.degree. C. The beads were then washed and the proteins
eluted and subjected to SDS-PAGE. Proteins were transferred to
membrane and probed with HRP-labeled streptavidin to reveal
relative amounts of remaining surface-exposed Fc.gamma.R1 protein
in each sample. Results are shown in FIG. 2.
[0075] As illustrated in FIG. 2, the amount of surface-exposed
Fc.gamma.R1 protein in Cell-1 samples (expressing Fc.gamma.R1 but
not Kremen-2) remained relatively constant regardless of the amount
of time the cells were exposed to DKK1-mFc. By contrast, the amount
of surface-exposed Fc.gamma.R1 protein in Cell-2 samples
(expressing both Kremen-2 and Fc.gamma.R1) decreased substantially
with increasing incubation times with DKK1-mFc. Thus, this
experiment demonstrates that DKK1-mFc induces degradation of cell
surface expressed Fc.gamma.R1 in a Kremen-2-dependent manner.
[0076] Taken together, the foregoing results show that a
multispecific antigen-binding molecule that simultaneously binds a
cell surface target molecule (Fc.gamma.R1) and an internalizing
effector protein (Kremen-2), can induce degradation of the target
molecule in an effector protein-dependent manner.
Example 2
IL-4R Activity is Attenuated Using a Multispecific Antigen-Binding
Molecule with Specificity for IL-4R and CD63
[0077] In a further set of proof-of-concept experiments, a
multispecific antigen-binding molecule was constructed which is
capable of simultaneously binding a cell surface-expressed target
molecule (i.e., IL-4R) and a cell surface-expressed internalizing
effector protein (i.e., CD63). The purpose of these experiments was
to determine whether IL-4R activity on a cell can be attenuated to
a greater extent by physically linking IL-4R to an effector
molecule that is internalized and targeted for degradation within
the lysosome (in this case, CD63). In other words, this Example was
designed to test whether the normal internalization and degradation
of CD63 could be used to force the internalization and degradative
rerouting of IL-4R within a cell.
[0078] First, a multispecific antigen-binding molecule was
constructed that is able to bind both IL-4R and CD63. Specifically,
a streptavidin-conjugated anti-IL-4R antibody and a biotinylated
anti-CD63 antibody were combined in a 1:1 ratio to produce an
anti-IL-4R:anti-CD63 conjugate (i.e., a multispecific
antigen-binding molecule that specifically binds both IL-4R and
CD63). The anti-IL-4R antibody used in this Example is a fully
human mAb raised against the IL-4R extracellular domain. (The
anti-IL-4R antibody comprised a heavy chain variable region having
SEQ ID NO:3 and a light chain variable region having SEQ ID NO:4).
The anti-CD63 antibody used in this Example is the mouse anti-human
CD63 mAb clone MEM-259, obtained from Biolegend (San Diego,
Calif.), catalog. No. 312002.
[0079] Two control constructs were also created:
Control-1=streptavidin-conjugated anti-IL-4R antibody combined in a
1:1 ratio with biotinylated control mouse IgG1kappa antibody; and
Control-2=streptavidin-conjugated anti-IL-4R antibody combined in a
1:1 ratio with non-biotinylated anti-CD63 antibody. The anti-IL-4R
antibody used in the experimental and control constructs for this
Example is an antibody that is known to specifically bind IL-4R and
only partially block IL-4-mediated signaling.
[0080] The experimental cell line used in this Example is an HEK293
cell line containing a STAT6-luciferase reporter construct and
additional STAT6 ("HEK293/STAT6-luc cells"). The cells used in this
experiment express both IL-4R and CD63 on their surface. When
treated with IL-4 in the absence of any inhibitors, this cell line
produces a dose-dependent detectable chemiluminescence signal which
reflects the extent of IL-4-mediated signaling.
[0081] In an initial experiment, the experimental
anti-IL-4R/anti-CD63 multispecific molecule, or the control
constructs, were added to the HEK293/STAT6-luc cells so that the
final concentration of anti-IL-4R antibody in the media was 12.5
nM. Reporter signal was measured at increasing concentrations of
IL-4 in the presence and absence of the experimental and control
constructs (FIG. 3). As seen in FIG. 3, The anti-IL-4R/anti-CD63
multispecific molecule ("ab conjugate") inhibited IL-4-mediated
signaling to a significantly greater extent than either control
construct.
[0082] To confirm that the effect observed in FIG. 3 was dependent
on CD63, the same experiment described above was carried out,
except that CD63 expression was significantly reduced in the
reporter cell line using an siRNA directed against CD63. With CD63
expression significantly reduced, the enhanced inhibitory activity
of the anti-IL-4R/anti-CD63 multispecific molecule was no longer
observed (FIG. 4). This result suggests that the ability of the
anti-IL-4R/anti-CD63 multispecific molecule to attenuate
IL-4-mediated signaling is due to the simultaneous binding of the
multispecific molecule to IL-4R and CD63 and the consequent
internalization and degradation of the entire antibody-IL-4R-CD63
complex.
[0083] Similar experiments were next carried out in which the
anti-IL-4R/anti-CD63 multispecific molecule, or the control
constructs, were allowed to incubate with the HEK293/STAT6-luc
reporter cell line for various amounts of time prior to the
addition of IL-4. In a first set of such experiments, the molecules
were allowed to incubate with the reporter cell line for 0 hours
(i.e., added concurrently with IL-4), 2 hours, or overnight prior
to the addition of 50 pM IL-4. Luciferase activity was measured six
hours after the addition of IL-4. Results are shown in FIG. 5, top
panel ("untransfected"). In a further set of experiments, a similar
protocol was carried out, except that the experimental or control
molecules were allowed to incubate with the reporter cell line for
15 minutes, 30 minutes, 1 hour or 2 hours prior to the addition of
50 pM IL-4. Results are shown in FIG. 6.
[0084] The results summarized in FIGS. 5 and 6 show that the
anti-IL-4R/anti-CD63 multispecific molecule is able to inhibit
IL-4-mediated signaling, and that this inhibitory effect is
enhanced with longer incubation times. As with the initial set of
experiments, it was confirmed using CD63 siRNA that the inhibitory
effect of the anti-IL-4R/anti-CD63 multispecific molecule was
dependent on CD63 expression (FIG. 5 bottom panel ["CD63
siRNA"]).
[0085] In summary, this Example provides further proof-of-concept
for the inhibition of a target molecule activity through the use of
a multispecific antigen-binding molecule that is capable of
simultaneously binding both the target molecule (in this case
IL-4R) and an internalizing effector protein (in this case CD63) to
thereby cause the internalization and degradative rerouting of the
target molecule within a cell. Stated differently, the simultaneous
binding of IL-4R and CD63 by the exemplary multispecific
antigen-binding molecule attenuated the activity of IL-4R to a
substantially greater extent (i.e., >10%) than the binding of
IL-4R by the control constructs alone.
Example 3
An Anti-IL-4R.times.Anti-CD63 Bispecific Antibody Attenuates IL-4R
Activity in a CD63-Dependent Manner
[0086] The experiments of Example 2, herein, show that an
anti-IL-4R/anti-CD63 multispecific molecule inhibits IL-4-mediated
signaling in a CD63-dependent manner. In those experiments, the
multispecific antigen-binding molecule consisted of two separate
monoclonal antibodies (anti-IL-4R and anti-CD63) that were
connected via a biotin-streptavidin linkage. To confirm that the
results observed with that proof-of-concept multispecific
antigen-binding molecule are generalizable to other multispecific
antigen-binding molecule formats, a true bispecific antibody was
constructed.
[0087] Standard bispecific antibody technology was used to
construct a bispecific antibody consisting of a first arm specific
for IL-4R and a second arm specific for CD63. The IL-4R-specific
arm contained an anti-IL-4R heavy chain paired with a CD63-specific
light chain. The CD63-specific light chain was paired with the
IL-4R specific heavy chain solely for purposes of convenience of
construction; nevertheless, the pairing of the anti-IL-4R heavy
chain with the anti-CD63 light chain retained full specificity for
IL-4R and did not exhibit binding to CD63. The CD63-specific arm
contained an anti-CD63 heavy chain paired with an anti-CD63 light
chain (the same light chain as used in the IL-4R arm). The
anti-IL-4R heavy chain (comprising SEQ ID NO:3) was derived from
the full anti-IL-4R antibody as used in Example 2; However, the
anti-CD63 heavy and light chains were derived from the anti-CD63
antibody designated H5C6, obtained from the Developmental Studies
Hybridoma Bank (University of Iowa Department of Biology, Iowa
City, Iowa). As with the full anti-IL-4R antibody used in Example
2, the anti-IL-4R component of the bispecific antibody used in this
Example exhibited only moderate IL-4R blocking activity on its
own.
[0088] An IL-4 luciferase assay was carried out to assess the
blocking activity of the anti-IL-4R.times.anti-CD63 bispecific
antibody. Briefly, serial dilutions of anti-IL-4R.times.anti-CD63
bispecific antibody or control molecules were added to
HEK293/STAT6-luc reporter cells (see Example 2). Under normal
conditions, these cells produce a detectable luciferase signal when
treated with IL-4. For this experiment, 10 pM IL-4 was then added
to the cells, and luciferase activity was quantified for each
dilution of antibody used. The controls used in this assay were:
(a) mock bispecific antibody that binds IL-4R with one arm and has
a non-functional anti-CD63 arm (i.e., containing one anti-IL-4R
heavy chain and one anti-CD63 heavy chain, both paired with the
anti-IL-4R light chain); (b) anti-IL-4R monospecific antibody; and
(c) buffer (PBS) only (without antibody). Results are shown in FIG.
7. As shown in FIG. 7, for the control samples used, luciferase
activity remained relatively high even at the highest antibody
concentrations, whereas for the bispecific antibody, luciferase
activity declined significantly as antibody concentration
increased. These results confirm that simultaneous binding of IL-4R
and CD63 by a bispecific antibody causes substantial inhibition of
IL-4R activity.
Example 4
Internalization of SOST Using a Multispecific Antigen-Binding
Molecule that Simultaneously Binds SOST and CD63
[0089] In this Example, the ability of multispecific
antigen-binding molecules to promote the internalization of the
soluble target molecule SOST (sclerostin) was assessed. For these
experiments, the target molecule was a fusion protein consisting of
a human SOST protein tagged with a pHrodo.TM. moiety (Life
Technologies, Carlsbad, Calif.) and a myc tag. The pHrodo.TM.
moiety is a pH-sensitive dye that is virtually non-fluorescent at
neutral pH and brightly fluorescent in an acidic environment such
as the endosome. The fluorescent signal, therefore, can be used as
an indicator of cellular internalization of the SOST fusion
protein. The multispecific antigen-binding molecules for these
experiments were bispecific antibodies with binding specificity for
both CD63 (an internalizing effector protein) and the SOST fusion
protein (a soluble target molecule), as described in more detail
below.
[0090] The experiments were conducted as follows: Briefly, HEK293
cells were plated at 10,000 cells/well in poly-D-lysine coated 96
well plates (Greiner Bio-One, Monroe, N.C.). After allowing the
cells to settle overnight, the media was replaced with media
containing antibody (5 .mu.g/mL, as described below),
pHrodo.TM.-myc-tagged-SOST (5 .mu.g/mL), heparin (10 .mu.g/mL), and
Hoechst 33342. The cells were then incubated for either 3 hours on
ice or 3 hours at 37.degree. C. All cells were washed twice prior
to imaging in PBS, and the number of fluorescent spots per cell, as
well as the corresponding fluorescence intensity, was counted to
establish the extent of pHrodo-myc-tagged-SOST cellular
internalization in the presence of the various antibody
constructs.
[0091] The antibodies used in this Example were as follows: (1)
anti-CD63 monospecific antibody (clone H5C6, Developmental Studies
Hybridoma Bank, University of Iowa Department of Biology, Iowa
City, Iowa); (2) anti-myc antibody (clone 9E10, Schiweck et al.,
1997, FEBS Lett. 414(1):33-38); (3) anti-SOST antibody (an antibody
having the heavy and light chain variable regions of the antibody
designated "Ab-B" in U.S. Pat. No. 7,592,429); (4)
anti-CD63.times.anti-myc bispecific antibody (i.e., a multispecific
antigen-binding molecule comprising an anti-CD63 arm derived from
the antibody H5C6 and an anti-myc arm derived from 9E10); (5)
anti-CD63.times.anti-SOST bispecific antibody #1 (i.e., a
multispecific antigen-binding molecule comprising an anti-CD63 arm
derived from the antibody H5C6 and an anti-SOST arm derived from
"Ab-B"); and (6) anti-CD63.times.anti-SOST bispecific antibody #2
(i.e., a multispecific antigen-binding molecule comprising an
anti-CD63 arm derived from the antibody H5C6 and an anti-SOST arm
derived from the antibody designated "Ab-20" in U.S. Pat. No.
7,592,429). The bispecific antibodies used in these experiments
were assembled using the so-called "knobs-into-holes" methodology
(see, e.g., Ridgway et al., 1996, Protein Eng. 9(7):617-621).
[0092] Results of the internalization experiments are shown in FIG.
8. FIG. 8 shows the number of spots (labeled vesicles) per cell,
under the various treatment conditions tested. Taken together, the
results of these experiments demonstrate that the bispecific
constructs, which simultaneously bind CD63 and SOST (either
directly or via the myc tag), caused the greatest amount of SOST
internalization as reflected by the fluorescence intensity and
number of fluorescent spots per cell over time at 37.degree. C.
Thus, the multispecific antigen-binding molecules used in this
Example are able to effectively direct the internalization of a
soluble target molecule.
Example 5
Changes in Bone Mineral Density in Mice Treated with a
Multispecific Antigen-Binding Molecule that Binds CD63 and SOST
[0093] An anti-CD63.times.anti-SOST multispecific antigen-binding
molecule, as described in Example 4, is next tested for its ability
to increase bone mineral density in mice. Five groups of mice
(about 6 mice per group) are used in these experiments. The
treatment groups are as follows: (I) untreated negative control
mice; (II) mice treated with a blocking anti-SOST monospecific
antibody that is known to increase bone mineral density on its own
(positive control); (Ill) mice treated with a bispecific antibody
that specifically binds CD63 and SOST but does not inhibit SOST
activity on its own or only slightly inhibits SOST activity on its
own; (IV) mice treated with an anti-CD63 parental antibody (i.e., a
monospecific antibody containing the same anti-CD63 antigen-binding
domain as in the bispecific antibody); and (V) mice treated with an
anti-SOST parental antibody (i.e., a monospecific antibody
containing the same anti-SOST antigen-binding domain as in the
bispecific antibody). The amount of antibody administered to the
mice in each group is about 10 to 25 mg/kg.
[0094] It is expected that mice in group III (treated with an
anti-SOST.times.anti-CD63 bispecific antibody) will exhibit an
increase in bone mineral density that is at least comparable to
that which is observed in the mice of group II (treated with a
known blocking anti-SOST antibody), even though the anti-SOST
component of the bispecific antibody does not inhibit SOST activity
on its own (as confirmed by the mice in Group V which are expected
to not exhibit an increase in bone mineral density). The increase
in bone mineral density that is expected in the mice of group III
is believed to be driven by CD63-mediated internalization of SOST,
as observed in the cellular experiments of Example 4, above.
Example 6
Cellular Internalization of Lipopolysaccharide (LPS) Mediated by a
Multispecific Antigen-Binding Molecule that Simultaneously Binds
LPS and CD63
[0095] This Example illustrates the use of a multispecific
antigen-binding molecule of the invention to direct the
internalization of a non-protein target molecule, namely
lipopolysaccharide (LPS). LPS is a component of the outer membrane
of Gram-negative bacteria and is known to contribute to septic
shock. Anti-LPS antibodies have been investigated as possible
treatment agents for sepsis. The experiments of the present Example
were designed to assess the ability of a multispecific
antigen-binding molecule to promote the internalization of LPS.
[0096] The multispecific antigen-binding molecule used in this
Example was a bispecific antibody with one arm directed to LPS
(target) and the other arm directed to CD63 (internalizing effector
protein). The anti-LPS arm was derived from the antibody known as
WN1 222-5. (DiPadova et al., 1993, Infection and Immunity
61(9):3863-3872; Muller-Loennies et al., 2003, J. Biol. Chem.
278(28):25618-25627; Gomery et al., 2012, Proc. Natl. Acad. Sci.
USA 109(51):20877-20882; U.S. Pat. No. 5,858,728). The anti-CD63
arm was derived from the H5C6 antibody (see Example 4). The
anti-LPS.times.anti-CD63 bispecific antibody (i.e., multispecific
antigen-binding molecule) was assembled using the so-called
"knobs-into-holes" methodology (see, e.g., Ridgway et al., 1996,
Protein Eng. 9(7):617-621).
[0097] Two LPS species were used in these experiments: E. coli LPS
and Salmonella minnesota LPS. Both versions were obtained as
fluorescent-labeled molecules (ALEXA-FLUOR.RTM.-488-labeled LPS,
Life Technologies, Carlsbad, Calif.).
[0098] Experiments were conducted as follows: HEK293 cells were
plated in 96-well PDL-coated imaging plates. After overnight rest,
media was replaced with fresh medium. Fluorescently labeled LPS
(either E. coli- or S. minnesota-derived) was added in regular
medium. Next, the anti-LPS.times.anti-CD63 bispecific antibody, or
control half-antibodies paired with dummy Fc, were added to the
samples. Following various incubation times at 37.degree. C. (1
hour and 3 hours) or on ice (3 hours), cells from the LPS-treated
samples were processed as follows: washed--quenched with
anti-ALEXA-FLUOR.RTM.-488 antibody--washed & fixed. The
anti-ALEXA-FLUOR.RTM.-488 antibody quenches fluorescence from
non-internalized (i.e., surface bound) fluorophore. Thus, any
fluorescence observed in the quenching antibody-treated samples is
due to internalized LPS. The level of fluorescence from each sample
at the various time points was measured.
[0099] FIG. 9 expresses the results of these experiments in terms
of the number of labeled vesicles per cell. As shown in FIG. 9,
only cells treated with the anti-CD63.times.anti-LPS bispecific
antibody demonstrated significant numbers of labeled vesicles that
increased over time. Cells treated with labeled LPS and the control
antibodies did not exhibit appreciable numbers of fluorescent
vesicles, indicating that LPS was not internalized under those
treatment conditions.
[0100] This Example therefore demonstrates that an
anti-LPS.times.anti-CD63 bispecific antibody causes internalization
of LPS into cells in a manner that requires simultaneous binding of
LPS and CD63. Accordingly, these results support the use of
multispecific antigen-binding molecules of the invention to promote
cellular internalization of target molecules such as LPS for the
treatment of diseases and disorders such as sepsis.
[0101] The present invention is not to be limited in scope by the
specific embodiments describe herein. Indeed, various modifications
of the invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and the accompanying figures. Such modifications are intended to
fall within the scope of the appended claims.
Sequence CWU 1
1
41502PRTArtificial SequenceSynthetic 1Met Met Ala Leu Gly Ala Ala
Gly Ala Thr Arg Val Phe Val Ala Met1 5 10 15 Val Ala Ala Ala Leu
Gly Gly His Pro Leu Leu Gly Val Ser Ala Thr 20 25 30 Leu Asn Ser
Val Leu Asn Ser Asn Ala Ile Lys Asn Leu Pro Pro Pro 35 40 45 Leu
Gly Gly Ala Ala Gly His Pro Gly Ser Ala Val Ser Ala Ala Pro 50 55
60 Gly Ile Leu Tyr Pro Gly Gly Asn Lys Tyr Gln Thr Ile Asp Asn
Tyr65 70 75 80 Gln Pro Tyr Pro Cys Ala Glu Asp Glu Glu Cys Gly Thr
Asp Glu Tyr 85 90 95 Cys Ala Ser Pro Thr Arg Gly Gly Asp Ala Gly
Val Gln Ile Cys Leu 100 105 110 Ala Cys Arg Lys Arg Arg Lys Arg Cys
Met Arg His Ala Met Cys Cys 115 120 125 Pro Gly Asn Tyr Cys Lys Asn
Gly Ile Cys Val Ser Ser Asp Gln Asn 130 135 140 His Phe Arg Gly Glu
Ile Glu Glu Thr Ile Thr Glu Ser Phe Gly Asn145 150 155 160 Asp His
Ser Thr Leu Asp Gly Tyr Ser Arg Arg Thr Thr Leu Ser Ser 165 170 175
Lys Met Tyr His Thr Lys Gly Gln Glu Gly Ser Val Cys Leu Arg Ser 180
185 190 Ser Asp Cys Ala Ser Gly Leu Cys Cys Ala Arg His Phe Trp Ser
Lys 195 200 205 Ile Cys Lys Pro Val Leu Lys Glu Gly Gln Val Cys Thr
Lys His Arg 210 215 220 Arg Lys Gly Ser His Gly Leu Glu Ile Phe Gln
Arg Cys Tyr Cys Gly225 230 235 240 Glu Gly Leu Ser Cys Arg Ile Gln
Lys Asp His His Gln Ala Ser Asn 245 250 255 Ser Ser Arg Leu His Thr
Cys Gln Arg His Gly Pro Gly Glu Pro Arg 260 265 270 Gly Pro Thr Ile
Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn 275 280 285 Leu Leu
Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp 290 295 300
Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp305
310 315 320 Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val
Asn Asn 325 330 335 Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
Glu Asp Tyr Asn 340 345 350 Ser Thr Leu Arg Val Val Ser Ala Leu Pro
Ile Gln His Gln Asp Trp 355 360 365 Met Ser Gly Lys Glu Phe Lys Cys
Lys Val Asn Asn Lys Asp Leu Pro 370 375 380 Ala Pro Ile Glu Arg Thr
Ile Ser Lys Pro Lys Gly Ser Val Arg Ala385 390 395 400 Pro Gln Val
Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys 405 410 415 Gln
Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile 420 425
430 Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn
435 440 445 Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
Ser Lys 450 455 460 Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
Ser Tyr Ser Cys465 470 475 480 Ser Val Val His Glu Gly Leu His Asn
His His Thr Thr Lys Ser Phe 485 490 495 Ser Arg Thr Pro Gly Lys 500
2496PRTArtificial SequenceSynthetic 2Met Met Ala Leu Gly Ala Ala
Gly Ala Thr Arg Val Phe Val Ala Met1 5 10 15 Val Ala Ala Ala Leu
Gly Gly His Pro Leu Leu Gly Val Ser Ala Thr 20 25 30 Leu Asn Ser
Val Leu Asn Ser Asn Ala Ile Lys Asn Leu Pro Pro Pro 35 40 45 Leu
Gly Gly Ala Ala Gly His Pro Gly Ser Ala Val Ser Ala Ala Pro 50 55
60 Gly Ile Leu Tyr Pro Gly Gly Asn Lys Tyr Gln Thr Ile Asp Asn
Tyr65 70 75 80 Gln Pro Tyr Pro Cys Ala Glu Asp Glu Glu Cys Gly Thr
Asp Glu Tyr 85 90 95 Cys Ala Ser Pro Thr Arg Gly Gly Asp Ala Gly
Val Gln Ile Cys Leu 100 105 110 Ala Cys Arg Lys Arg Arg Lys Arg Cys
Met Arg His Ala Met Cys Cys 115 120 125 Pro Gly Asn Tyr Cys Lys Asn
Gly Ile Cys Val Ser Ser Asp Gln Asn 130 135 140 His Phe Arg Gly Glu
Ile Glu Glu Thr Ile Thr Glu Ser Phe Gly Asn145 150 155 160 Asp His
Ser Thr Leu Asp Gly Tyr Ser Arg Arg Thr Thr Leu Ser Ser 165 170 175
Lys Met Tyr His Thr Lys Gly Gln Glu Gly Ser Val Cys Leu Arg Ser 180
185 190 Ser Asp Cys Ala Ser Gly Leu Cys Cys Ala Arg His Phe Trp Ser
Lys 195 200 205 Ile Cys Lys Pro Val Leu Lys Glu Gly Gln Val Cys Thr
Lys His Arg 210 215 220 Arg Lys Gly Ser His Gly Leu Glu Ile Phe Gln
Arg Cys Tyr Cys Gly225 230 235 240 Glu Gly Leu Ser Cys Arg Ile Gln
Lys Asp His His Gln Ala Ser Asn 245 250 255 Ser Ser Arg Leu His Thr
Cys Gln Arg His Gly Pro Gly Asp Lys Thr 260 265 270 His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 275 280 285 Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 290 295 300
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro305
310 315 320 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala 325 330 335 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 340 345 350 Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr 355 360 365 Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr 370 375 380 Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu385 390 395 400 Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 405 410 415 Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 420 425
430 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
435 440 445 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser 450 455 460 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala465 470 475 480 Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 485 490 495 3122PRTArtificial
SequenceSynthetic 3Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val
Gln Pro Gly Arg1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly
Phe Thr Phe Ser Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp
Gly Ser Ile Lys Tyr Phe Arg Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Val Glu Asp Thr Ala His Tyr Tyr Cys 85 90 95
Ala Lys Glu Ser Ser Ser Trp Tyr Phe Tyr His Gly Leu Asp Val Trp 100
105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
4114PRTArtificial SequenceSynthetic 4Glu Ile Val Met Thr Gln Ser
Pro Leu Ser Leu Pro Val Ser Pro Gly1 5 10 15 Glu Pro Ala Ser Ile
Ser Cys Arg Ser Asn Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Asn
Asn Tyr Leu Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro
His Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55
60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile65 70 75 80 Ser Arg Val Glu Ala Gly Asp Val Gly Thr Tyr Phe Cys
Met Gln Ser 85 90 95 Leu Gln Ala Pro Pro Phe Thr Phe Gly Pro Gly
Thr Lys Val Glu Ile 100 105 110 Lys Arg
* * * * *