Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-PirB ANTIBODIES
FIELD OF THE INVENTION
The present invention relates generally to neural development and neurological
disorders. The invention specifically concerns identification of novel
modulators of the
myelin-associated inhibitory system and various uses of the modulators so
identified.
BACKGROUND OF THE INVENTION
Myelin and Myelin-associated proteins
It is known that axons of the adult mammalian CNS neurons have very limited
capacity to regenerate following injury, whereas axons in the peripheral
nervous system
(PNS) regenerate rapidly. It has been known that CNS neuron's limited capacity
to
regenerate is in part to an intrinsic property of CNS axons, but also due to
an impermissible
environment. The CNS myelin, while it is not the only source of inhibitory
cues for neurite
growth, contains numerous inhibitory molecules that actively block axonal
growth and
therefore constitutes a significant barrier to regeneration. Three of such
myelin-associated
proteins (MAPs) have been identified: Nogo (also known as NogoA) is a member
of the
Reticulon family of proteins having two transmembrane domains; myelin-
associated
glycoprotein (MAG) is a transmembrane protein of the Ig superfamily; and OMgp
is a
leucine rich repeat (LRR) protein with a glycosylphosphatidylinositol (GPI)
anchor. Chen et
al., Nature 403:434-39 (2000); GrandPre et al., Nature 417:439-444 (2000);
Prinjha et al.,
Nature 403:383-384 (2000); McKerracher et al, Neuron 13:805-11 (1994); Wang et
al,
Nature 417:941-4 (20020: Kottis et al J. Neurochem 82:1566-9 (2002). A portion
of NogoA,
Nogo66, has been described as a 66-amino acid extracellular polypeptide that
is found in all
three isoforms of Nogo.
Despite their structural differences, all three inhibitory proteins (including
Nogo66)
have been shown to bind the same GPI-anchored receptor, called Nogo receptor
(NgR; also
known as Nogo Receptor-1 or NgR1) , and it has been proposed that NgR might be
required
for mediating the inhibitory actions of Nogo, MAG and OMgp. Fournier et al.,
Nature 409:
341-346 (2001). Two NgR1 homologs (NgR2 and NgR3) have also been identified.
US
2005/0048520 Al (Strittmatter et al.), published March 3, 2005. Given that NgR
is a GPI-
anchored cell surface protein, it is unlikely to be a direct signal
transductor (Zheng et al.,
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Proc. Natl. Acad. Sci. USA 102:1205-1210 (2005)). Others have suggested that
the
neurotrophin receptor p75NTR acts as a co-receptor for NgR and provides the
signal-
transducing moiety in a receptor complex (Wang et al., Nature 420:74-78
(2002); Wong et
al., Nat. Neurosci. 5:1302-1308 (2002)).
PirB and human orthologs
The major histocompatibility complex (MHC) class I was originally identified
as a
region encoding a family of molecules that are important for the immune
system. Recent
evidences have indicated that MHC class I molecules have additional functions
in the
development and adult CNS. Boulanger and Shatz, Nature Rev Neurosci. 5:521-531
(2004);
1 o US 2003/0170690 (Shatz and Syken), published September 11, 2003. Many of
the MHC
class I members and their binding partners are found to be expressed in CNS
neurons.
Recent genetic and molecular studies have focused on the physiological
functions of CNS
MHC class I, and the initial results suggested that MHC class I molecules
might be involved
in activity-dependent synaptic plasticity, a process during which the strength
of existing
synaptic connections increases or decreases in response to neuronal activity,
followed by
long term structural alterations to circuits. Moreover, the MHC class I
encoding region has
also been genetically linked to a wide variety of disorders with neurological
symptoms, and
abnormal functions of MHC class I molecules are thought to contribute to the
disruption of
normal brain development and plasticity.
One of the known MHC class I receptors in the immune setting is PirB, a murine
polypeptide that was first described by Kubagawa et al., Proc. Nat. Acad. Sci.
USA 94:5261-
6 (1997). Mouse PirB has several human orthologs, which are members of the
leukocyte
immunoglobulin-like receptor, subfamily B (LILRB), and are also referred to as
"immunoglobulin-like transcripts" (ILTs) The human orthologs show significant
homology
to the murine sequence, from highest to lowest in the following order:
LILRB3/ILT5,
LILRBI/ILT2, LILRB5/ILT3, LILRB2/ILT4, and, just as PirB, are all inhibitory
receptors.
LILRB3/ILT5 (NP_006855) and LILRBI/ILT2 (NP_006660) were first described by
Samaridis and Colonna, Eur. J. Immunol. 27(3):660-665 (1997) LILRB5/ILT3
(NP_006831) has been identified by Borges et al., J. Immunol. 159(11):5192-
5196 (1997).
LILRB2/ILT4 (also known as MIR10), was identified by Colonna et al., J. Exp.
Med.
186:1809-18 (1997). PirB and its human orthologs show a great degree of
structural
variability. The sequences of various alternatively spliced forms are
available from
EMBL/GenBank, including, for example, the following accession numbers for
human ILT4
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cDNA: ILT4-c 11 AF009634; ILT4-c 117 AF 11566; ILT4-c 126 AF 11565. As noted
above,
the PirB/LILRB polypeptides are MHC Class I (MHCI) inhibitory receptors, and
are known
for their role in regulating immune cell activation (Kubagawa et al., supra;
Hayami et al., J.
Biol. Chem. 272:7320 (1997); Takai et al., Immunology 115:433 (2005); Takai et
al.,
Immunol. Rev. 181:215 (2001); Nakamura et al. Nat. Immunol. 5:623 (2004);
Liang et al.,
Eur. J. Immunol. 32:2418 (2002)).
A recent study by Syken et al. (Science 313:1795-800 (2006)) reported that
PirB is
expressed in subsets of neurons throughout the brain. In mutant mice lacking
functional
PirB, cortical ocular dominance (OD) plasticity is significantly enhanced at
all ages,
suggesting PirB's function in restricting activity-dependent plasticity in
visual cortex.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the finding that
interfering with
PirB activity using function-blocking anti-PirB antibodies helps rescuing
neurite outgrowth
inhibition by Nogo66 and myelin, and that blocking PirB and NgR activities
concurrently
leads to a near-complete release from myelin inhibition.
In one aspect, the invention concerns an isolated anti-PirB/LILRB antibody
that binds
essentially to the same epitope on human PirB (LILRB) as an antibody selected
from the
group consisting of YW259.2, YW259.9 and YW259.12.
In another aspect, the invention concerns an isolated anti-PirB/LILRB antibody
that
competes for binding to human PirB (LILRB) with an antibody selected from the
group
consisting of YW259.2, YW259.9 and YW259.12.
In yet another aspect, the invention concerns an isolated anti-PirB/LILRB
antibody
that comprises one, two, or three, hypervariable region sequences from a heavy
chain
selected from the group consisting of. YW259.2 heavy chain (SEQ ID NO: 4 or
11),
YW259.9 heavy chain (SEQ ID NO: 5 or 12), and YW259.12 heavy chain (SEQ ID NO:
6
or 13).
In an embodiment, the antibody comprises all hypervariable region sequences of
the
YW259.2 antibody heavy chain (SEQ ID NO: 4 or 11).
In another embodiment, the antibody comprises all hypervariable region
sequences of
the YW259.9 antibody heavy chain (SEQ ID NO: 5 or 12).
In yet another embodiment, the antibody comprises all hypervariable region
sequences of the YW259.12 antibody heavy chain (SEQ ID NO: 6 or 13).
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In a further embodiment, the antibody comprises a light chain.
In a still further embodiment, the antibody comprises one, two or three
hypervariable
region sequences of a light chain from the polypeptide sequence of SEQ ID NO:
7.
In yet another embodiment, the antibody comprises all hypervariable region
sequences of a light chain comprising the polypeptide sequence of SEQ ID NO: 7
or 15.
In a specific embodiment, the antibody comprises both a heavy and a light
chain,
where the heavy chain comprises one, two, or three, hypervariable region
sequences from a
heavy chain selected from the group consisting of. YW259.2 heavy chain (SEQ ID
NO: 4 or
11), YW259.9 heavy chain (SEQ ID NO: 5 or 12), and YW259.12 heavy chain (SEQ
ID
1o NO: 6 or 13), and/or the light chain comprises one, two or three
hypervariable region
sequences of a light chain from the polypeptide sequence of SEQ ID NO: 7 or
15.
In a further embodiment, the antibody is selected from the group consisting of
antibodies YW259.2, YW259.9, and YW259.12.
In a further aspect, the invention concerns an isolated anti-PirB antibody
wherein the
full-length IgG form of the antibody specifically binds human PirB with a
binding affinity of
5nM or better, or 1 nM or better.
In an embodiment, the antibody promotes axonal regeneration, such as
regeneration
of CNS neurons.
In another embodiment, the antibody, at least partially, rescues neurite
outgrowth
inhibition by Nogo66 and myelin.
In all aspects, the antibody preferably is a monoclonal antibody, which may,
for
example, be a chimeric antibody, a humanized antibody, an affinity matured
antibody, a
human antibody, or a bispecific antibody, an antibody fragment or an
immunoconjugate.
In a further aspect, the invention concerns a polynucleotide encoding an anti-
PirB/LILRB antibody herein.
In other aspects, the invention concerns vectors and host cells comprising a
polynucleotide encoding an antibody (including coding sequences of one or more
antibody
chains) herein. The host cells include prokaryotic, eukaryotic and mammalian
hosts.
In a further aspect, the invention concerns a method for making an anti-
PirB/LILRB
antibody, comprising (a) expressing a vector comprising nucleic acid encoding
the antibody
in a suitable host cell, and (b) recovering the antibody.
In a still further aspect, the invention concerns a composition comprising an
anti-
PirB/LILRB antibody herein, and a pharmaceutically acceptable excipient.
Optionally, the
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composition comprises a second medicament, wherein the anti-PirB/LILRB
antibody is a
first medicament. The second medicament may, for example, be a NgR inhibitor,
such as an
anti-NgR antibody.
In a different aspect, the invention concerns a kit comprising an anti-
PirB/LILRB
antibody herein.
In another aspect, the invention concerns a method for promoting axon
regeneration
comprising administering to a subject in need an effective amount of an anti-
PirB/LILRB
antibody herein. Preferably, the subject is a human patient.
In embodiments, the treatment method herein enhances survival or neurons
and/or
induces the outgrowth of neurons
In yet another aspect, the invention concerns a method of treating a
neurodegenerative disease, comprising administering to a subject in need an
effective
amount of an anti-PirB/LILRB antibody herein. The neurodegenerative disease
may, for
example, be characterized by physical damage to the central nervous system,
and includes,
without limitation, brain damage associated with stroke.
In a particular embodiment, the neurodegenerative disease is selected from the
group
consisting of trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy,
myasthenia
gravis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), multiple
sclerosis (MS),
progressive muscular atrophy, progressive bulbar inherited muscular atrophy,
peripheral
nerve damage caused by physical injury (e.g., burns, wounds) or disease states
such as
diabetes, kidney dysfunction or by the toxic effects of chemotherapeutics used
to treat cancer
and AIDS, herniated, ruptured or prolapsed invertebrate disk syndromes,
cervical
spondylosis, plexus disorders, thoracic outlet destruction syndromes,
peripheral neuropathies
such as those caused by lead, dapsone, ticks, prophyria, Gullain-Barre
syndrome,
Alzheimer's disease, Huntington's Disease, and Parkinson's disease.
The invention further concerns an anti-idiotype antibody that specifically
binds an
anti-PirB antibody herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures IA and lB show the mouse PirB sequence (SEQ ID NO: 1) and the human
LILRB2 sequence (SEQ ID NO: 2).
Figures 2A and 2B. Blocking PirB reverses inhibition of CGN outgrowth on AP-
Nogo66 or myelin. Dissociated mouse P7 CGN were plated on PDL/laminin
(control), AP-
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Nogo66, or myelin to test inhibition by these substrates. (A) Representative
photomicrographs, (B) a graph measuring average neurite length (+SE) from one
representative experiment. Neurons grown on PDL/laminin, AP-Nogo66, or myelin
were
cultured in the presence or absence of function-blocking antibodies to PirB
(aPB 1; 50
g/ml). aPB l significantly reduced inhibition by either substrate. (*p<0.01;
Scale bars, 50
m)
Figures 3A-3D. Blocking PirB reverses inhibition of CGN outgrowth on AP-Nogo66
or myelin. Dissociated mouse P7 CGN were plated on PDL/laminin (control), AP-
Nogo66,
or myelin to test inhibition by these substrates. Representative
photomicrographs are shown
1o in Figures 3A and 3C and a graph measuring average neurite length ( SE)
from one
representative experiment is shown in Figures 3B and 3D. Neurons grown on
PDL/laminin,
AP-Nogo66, or myelin were cultured in the presence or absence of function-
blocking
antibodies to PirB (aPB 1; 50 g/ml). aPB 1 significantly reduced inhibition
by either
substrate. (*p<0.01; Scale bars, 50 m)
Figures 4A and 4B. Both PirB and NgR are required to mediate growth cone
collapse by myelin inhibitors. Growth cones of postnatal DRG axons were
treated with
medium alone (control), myelin (3 g/ml), or AP-Nogo66 (100 nM) for 30 minutes
to
stimulate collapse, and stained with Rhodamine-phalloidin to visualize growth
cones. (A)
Representative photomicrographs, (B) a graph measuring percent growth cone
collapse
( SEM) from cumulative experiments. Either genetic loss of NgR or inhibition
of PirB by
anti-PirB treatment was sufficient alone to prevent the growth cone collapsing
activity of
myelin or AP-Nogo66. Inhibition of both pathways also fully blocked collapse.
(Scale bars,
50 m)
Figures 5A-5D. Blocking PirB partially disinhibits neurite outgrowth in DRG
neurons and on the substrate MAG. Representative photomicrographs are shown in
Figures
5A and 5C; graphs showing the average neurite length ( SE) from one
representative
experiment are shown in Figures 5B and 5D. (A) and (B) Dissociated P10 DRG
neurons
were plated on PDL/laminin, AP-Nogo66, or myelin in the presence or absence of
anti-PirB.
There was a significant reduction in inhibition by AP-Nogo66 and myelin by aPB
1. (C) and
(D) Dissociated P7 CGN cultures were plated on PDL/laminin or MAG-Fc, with or
without
aPB 1. Antibodies to PirB reduced the inhibition of neurite outgrowth by MAG-
Fc. (*
p<0.01; Scale bars, 200 pm A, B; 50 m Q.
Figure 6. DNA sequence of anti-PirB antibody YW259.2 heavy chain (SEQ ID NO:
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8).
Figure 7. DNA sequence of anti-PirB antibody YW259.9 heavy chain (SEQ ID NO:
9).
Figure 8 DNA sequence of anti-PirB antibody YW259.12 heavy chain (SEQ ID NO:
3).
Figure 9 Protein sequence of anti-PirB antibody YW259.2 heavy chain (SEQ ID
NO:
4).
Figure 10. Protein sequence of anti-PirB antibody YW259.9 heavy chain (SEQ ID
1o NO: 5).
Figure 11. Protein sequence of anti-PirB antibody YW259.12 heavy chain (SEQ ID
NO: 6).
Figure 12. Protein sequence of the light chain of all YW259 antibodies (SEQ ID
NO:
7).
Figure 13. Ability of anti-PirB antibody YW259.2(IgG) to inhibit the activity
of His-
tagged mouse PirB.
Figure 14. Ability of anti-PirB antibody YW259.9 (IgG) to inhibit the activity
of
His-tagged mouse PirB.
Figure 15. Ability of anti-PirB antibody YW259.12 (IgG) to inhibit the
activity of
His-tagged mouse PirB.
Figure 16. Relative AP-Nogo66 binding of a panel of anti-PirB antibodies,
including
YW259.2, YW259.9, and YW259.12.
Figures 17A-17C. Alignment of heavy chain sequences of anti-PirB antibodies
YW259.2 (SEQ ID NO: 11); YW259.9 (SEQ ID NO: 12) and YW259.12 (SEQ ID NO 13).
The CDR H1, CDR H2 and CDR H3 sequences are boxed, along with the CDR H
domains
according to Kabat, Chothia and the contact CDR H domains. Hum III is
disclosed as SEQ
ID NO: 10.
Figures 18A-18C. Alignment of light chain sequences of anti-PirB antibodies
YW259.2 (SEQ ID NO: 15) ; YW259.9 (SEQ ID NO: 15) and YW259.12 (SEQ ID NO:
15),
and HuKI (SEQ ID NO: 14). The CDR L1, CDR L2 and CDR L3 sequences are boxed,
along with the CDR L domains according to Kabat, Chothia and the contact CDR L
domains. Hum III is disclosed as SEQ ID NO: 10.
Figure 19. C I QTNF5 (CTRP5; NP_05646) inhibits neurite outgrowth of dorsal
root
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ganglion neurons, and this inhibition is reduced when PirB is blocked by PirB
function-
blocking antibody YW259.2.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The terms "paired-immunoglobulin-like receptor B" and "PirB" are used herein
interchangeably, and refer to a native-sequence, 841-amino acid mouse
inhibitory protein of
SEQ ID NO: 1 (Figure 1) (NP_035225), and its native-sequence homologues in rat
and other
non-human mammals, including all naturally occurring variants, such as
alternatively spliced
and allelic variants and isoforms, as well as soluble forms thereof. For
further details see,
Kubagawa et al., Proc Natl Acad Sci USA 94, 5261 (1997).
The terms "LILRB," "ILT" and "MIR," are used herein interchangeably, and refer
to
all members of the human "leukocyte immunoglobulin-like receptor, subfamily
B",
including all naturally occurring variants, such as alternatively spliced and
allelic variants
and isoforms, as well as soluble forms thereof. Individual members within this
B-type sub-
family of LILR receptors are designated by numbers following the acronym, such
as, for
example, LILRB3/ILT5, LILRB1/ILT2, LILRB5/ILT3, and ILIRB2/ILT4, where a
reference
to any individual member, unless otherwise noted, also includes reference to
all naturally
occurring variants, such as alternatively spliced and allelic variants and
isoforms, as well as
soluble forms thereof. Thus, for example, "LILRB2," "LIR2," and "MIRIO" are
used
herein interchangeably and refer to the 598-amino acid polypeptide of SEQ ID
NO:2 (Figure
1) (NP_005865), and its naturally occurring variants, such as alternatively
spliced and allelic
variants and isoforms, as well as soluble forms thereof. For further details,
see Martin et al.,
Trends Immunol. 23, 81 (2002).
The term "PirB/LILRB" is used herein to jointly refer to the corresponding
mouse
and human proteins and native sequence homologues in other non-human mammals,
including all naturally occurring variants, such as alternatively spliced and
allelic variants
and isoforms, as well as soluble forms thereof.
The term "myelin-associated protein" is used in the broadest sense and
includes all
proteins present in CNS myelin that inhibit neuronal regeneration, including
Nogo, MAG
and OMgp.
"Isolated," when used to describe the various proteins disclosed herein, means
protein
that has been identified and separated and/or recovered from a component of
its natural
environment. Contaminant components of its natural environment are materials
that would
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typically interfere with diagnostic or therapeutic uses for the protein, and
may include
enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In
preferred
embodiments, the protein will be purified (1) to a degree sufficient to obtain
at least 15
residues of N-terminal or internal amino acid sequence by use of a spinning
cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions
using
Coomassie blue or, preferably, silver stain, or (3) to homogeneity by mass
spectroscopic or
peptide mapping techniques. Isolated protein includes protein in situ within
recombinant
cells, since at least one component of the natural environment of the protein
in question will
not be present. Ordinarily, however, isolated protein will be prepared by at
least one
purification step.
An "isolated" nucleic acid molecule is a nucleic acid molecule that is
identified and
separated from at least one contaminant nucleic acid molecule with which it is
ordinarily
associated in the natural source of the nucleic acid in question. An isolated
nucleic acid
molecule is other than in the form or setting in which it is found in nature.
Isolated nucleic
acid molecules therefore are distinguished from the nucleic acid molecules as
they exist in
natural cells. However, an isolated nucleic acid molecule includes nucleic
acid molecules
contained in cells that ordinarily express such nucleic acid where, for
example, the nucleic
acid molecule is in a chromosomal location different from that of natural
cells.
As used herein, the term "PirB/LILRB antagonist" is used to refer to an agent
capable
of blocking, neutralizing, inhibiting, abrogating, reducing or interfering
with PirB/LILRB
activities. Particularly, the PirB/LILRB antagonist interferes with myelin
associated
inhibitory activities, thereby enhancing neurite outgrowth, and/or promoting
neuronal
growth, repair and/or regeneration. In a preferred embodiment, the PirB/LILRB
antagonist
inhibits the binding of PirB/LILRB to Nogo66 and/or MAG and/or OMgp by binding
to
PirB/LILRB. PirB/LILRB antagonists include, for example, antibodies to
PirB/LILRB and
antigen binding fragments thereof, truncated or soluble fragments of
PirB/LILRB, Nogo 66,
MAG or OMgp that are capable of sequestering the binding between PirB/LILRB
and Nogo
66, or between PirB/LILRB and MAG, or between PirB/LILRB and OMgp and small
molecule inhibitors of the PirB/LILRB related inhibitory pathway. PirB/LILRB
antagonists
also include short-interfering RNA (siRNA) molecules capable of inhibiting or
reducing the
expression of PirB/LILRB mRNA. A preferred PirB/LILRB antagonist is an anti-
PirB/LILRB antibody.
The term "antibody" herein is used in the broadest sense and specifically
covers intact
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antibodies, monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g.
bispecific antibodies) formed from at least two intact antibodies, and
antibody fragments, so
long as they exhibit the desired biological activity.
The term "monoclonal antibody" as used herein refers to an antibody obtained
from a
population of substantially homogeneous antibodies, i.e., the individual
antibodies
comprising the population are identical except for possible naturally
occurring mutations that
may be present in minor amounts. Monoclonal antibodies are highly specific,
being directed
against a single antigenic site. Furthermore, in contrast to polyclonal
antibody preparations
which include different antibodies directed against different determinants
(epitopes), each
monoclonal antibody is directed against a single determinant on the antigen.
In addition to
their specificity, the monoclonal antibodies are advantageous in that they may
be synthesized
uncontaminated by other antibodies. The modifier "monoclonal" indicates the
character of
the antibody as being obtained from a substantially homogeneous population of
antibodies,
and is not to be construed as requiring production of the antibody by any
particular method.
For example, the monoclonal antibodies to be used in accordance with the
present invention
may be made by the hybridoma method first described by Kohler et al., Nature,
256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No.
4,816,567). The "monoclonal antibodies" may also be isolated from phage
antibody libraries
using the techniques described in Clackson et al., Nature, 352:624-628 (1991)
and Marks et
al., J Mol. Biol., 222:581-597 (1991), for example.
Antibodies specifically include "chimeric" antibodies in which a portion of
the heavy
and/or light chain is identical with or homologous to corresponding sequences
in antibodies
derived from a particular species or belonging to a particular antibody class
or subclass,
while the remainder of the chain(s) is identical with or homologous to
corresponding
sequences in antibodies derived from another species or belonging to another
antibody class
or subclass, as well as fragments of such antibodies, so long as they exhibit
the desired
biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc.
Natl. Acad. Sci.
USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include
primatized
antibodies comprising variable domain antigen-binding sequences derived from a
non-
human primate (e.g. Old World Monkey, Ape etc) and human constant region
sequences.
"Antibody fragments" comprise a portion of an intact antibody, preferably
comprising the antigen-binding or variable region thereof. Examples of
antibody fragments
include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies;
single-chain
CA 02723430 2010-11-03
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antibody molecules; and multispecific antibodies formed from antibody
fragment(s).
An "intact" antibody is one which comprises an antigen-binding variable region
as
well as a light chain constant domain (CL) and heavy chain constant domains,
CH1, CH2 and
CH3. The constant domains may be native sequence constant domains (e.g. human
native
sequence constant domains) or amino acid sequence variant thereof. Preferably,
the intact
antibody has one or more effector functions.
"Humanized" forms of non-human (e.g., rodent) antibodies are chimeric
antibodies
that contain minimal sequence derived from non-human immunoglobulin. For the
most part,
humanized antibodies are human immunoglobulins (recipient antibody) in which
residues
from a hypervariable region of the recipient are replaced by residues from a
hypervariable
region of a non-human species (donor antibody) such as mouse, rat, rabbit or
nonhuman
primate having the desired specificity, affinity, and capacity. In some
instances, framework
region (FR) residues of the human immunoglobulin are replaced by corresponding
non-
human residues. Furthermore, humanized antibodies may comprise residues that
are not
found in the recipient antibody or in the donor antibody. These modifications
are made to
further refine antibody performance. In general, the humanized antibody will
comprise
substantially all of at least one, and typically two, variable domains (Fab,
Fab', F(ab')2, Fabc,
Fv), in which all or substantially all of the hypervariable loops correspond
to those of a non-
human immunoglobulin and all or substantially all of the FRs are those of a
human
immunoglobulin sequence. The humanized antibody optionally also will comprise
at least a
portion of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature 321:522-525
(1986);
Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol. 2:593-596
(1992).
The term "hypervariable region" when used herein refers to the regions of an
antibody variable domain which are hypervariable in sequence and/or form
structurally
defined loops. The hypervariable region comprises amino acid residues from a
"complementarity determining region" or "CDR" (i.e. residues 24-34, 50-56, and
89-97 in
the light chain variable domain and 31-35, 50-65, and 95-102 in the heavy
chain variable
domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health
Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those
residues from a
"hypervariable loop" (i.e. residues 26-32, 50-52, and 91-96 in the light chain
variable
domain and 26-32, 53-55, and 96-101 in the heavy chain variable domain;
Chothia and Lesk
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J. Mol. Biol. 196:901-917 (1987)). In both cases, the variable domain residues
are numbered
according to Kabat et al., supra, as discussed in more detail below.
"Framework" or "FR"
residues are those variable domain residues other than the residues in the
hypervariable
regions as herein defined.
A "parent antibody" or "wild-type" antibody is an antibody comprising an amino
acid
sequence which lacks one or more amino acid sequence alterations compared to
an antibody
variant as herein disclosed. Thus, the parent antibody generally has at least
one
hypervariable region which differs in amino acid sequence from the amino acid
sequence of
the corresponding hypervariable region of an antibody variant as herein
disclosed. The
parent polypeptide may comprise a native sequence (i.e. a naturally occurring)
antibody
(including a naturally occurring allelic variant), or an antibody with pre-
existing amino acid
sequence modifications (such as insertions, deletions and/or other
alterations) of a naturally
occurring sequence. Throughout the disclosure, "wild type," "WT," "wt," and
"parent" or
"parental" antibody are used interchangeably.
As used herein, "antibody variant" or "variant antibody" refers to an antibody
which
has an amino acid sequence which differs from the amino acid sequence of a
parent
antibody. Preferably, the antibody variant comprises a heavy chain variable
domain or a
light chain variable domain having an amino acid sequence which is not found
in nature.
Such variants necessarily have less than 100% sequence identity or similarity
with the parent
antibody. In a preferred embodiment, the antibody variant will have an amino
acid sequence
from about 75% to less than 100% amino acid sequence identity or similarity
with the amino
acid sequence of either the heavy or light chain variable domain of the parent
antibody, more
preferably from about 80% to less than 100%, more preferably from about 85% to
less than
100%, more preferably from about 90% to less than 100%, and most preferably
from about
95% to less than 100%. The antibody variant is generally one which comprises
one or more
amino acid alterations in or adjacent to one or more hypervariable regions
thereof.
An "amino acid alteration" refers to a change in the amino acid sequence of a
predetermined amino acid sequence. Exemplary alterations include insertions,
substitutions
and deletions. An "amino acid substitution" refers to the replacement of an
existing amino
acid residue in a predetermined amino acid sequence; with another different
amino acid
residue.
A "replacement" amino acid residue refers to an amino acid residue that
replaces or
substitutes another amino acid residue in an amino acid sequence. The
replacement residue
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may be a naturally occurring or non-naturally occurring amino acid residue.
An "amino acid insertion" refers to the introduction of one or more amino acid
residues into a predetermined amino acid sequence. The amino acid insertion
may comprise
a "peptide insertion" in which case a peptide comprising two or more amino
acid residues
joined by peptide bond(s) is introduced into the predetermined amino acid
sequence. Where
the amino acid insertion involves insertion of a peptide, the inserted peptide
may be
generated by random mutagenesis such that it has an amino acid sequence which
does not
exist in nature. An amino acid alteration "adjacent a hypervariable region"
refers to the
introduction or substitution of one or more amino acid residues at the N-
terminal and/or C-
terminal end of a hypervariable region, such that at least one of the inserted
or replacement
amino acid residue(s) form a peptide bond with the N-terminal or C-terminal
amino acid
residue of the hypervariable region in question.
A "naturally occurring amino acid residue" is one encoded by the genetic code,
generally selected from the group consisting of: alanine (Ala); arginine
(Arg); asparagine
(Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid
(Glu); glycine
(Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine (Lys);
methionine (Met);
phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan
(Trp); tyrosine
(Tyr); and valine (Val).
A "non-naturally occurring amino acid residue" herein is an amino acid residue
other
than those naturally occurring amino acid residues listed above, which is able
to covalently
bind adjacent amino acid residues(s) in a polypeptide chain. Examples of non-
naturally
occurring amino acid residues include norleucine, ornithine, norvaline,
homoserine and other
amino acid residue analogues such as those described in Ellman et al. Meth.
Enzym.
202:301-336 (1991). To generate such non-naturally occurring amino acid
residues, the
procedures of Noren et al. Science 244:182 (1989) and Ellman et al., supra,
can be used.
Briefly, these procedures involve chemically activating a suppressor tRNA with
a non-
naturally occurring amino acid residue followed by in vitro transcription and
translation of
the RNA.
Throughout this disclosure, reference is made to the numbering system from
Kabat,
E. A., et al., Sequences of Proteins of Immunological Interest (National
Institutes of Health,
Bethesda, Md. (1987) and (1991). In these compendiums, Kabat lists many amino
acid
sequences for antibodies for each subclass, and lists the most commonly
occurring amino
acid for each residue position in that subclass. Kabat uses a method for
assigning a residue
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WO 2009/140361 PCT/US2009/043757
number to each amino acid in a listed sequence, and this method for assigning
residue
numbers has become standard in the field. The Kabat numbering scheme is
followed in this
description. For purposes of this invention, to assign residue numbers to a
candidate
antibody amino acid sequence which is not included in the Kabat compendium,
one follows
the following steps. Generally, the candidate sequence is aligned with any
immunoglobulin
sequence or any consensus sequence in Kabat. Alignment may be done by hand, or
by
computer using commonly accepted computer programs; an example of such a
program is
the Align 2 program. Alignment may be facilitated by using some amino acid
residues
which are common to most Fab sequences. For example, the light and heavy
chains each
typically have two cysteines which have the same residue numbers; in VL domain
the two
cysteines are typically at residue numbers 23 and 88, and in the VH domain the
two cysteine
residues are typically numbered 22 and 92. Framework residues generally, but
not always,
have approximately the same number of residues, however the CDRs will vary in
size. For
example, in the case of a CDR from a candidate sequence which is longer than
the CDR in
the sequence in Kabat to which it is aligned, typically suffixes are added to
the residue
number to indicate the insertion of additional residues (see, e.g. residues
100abc in Figure
1B). For candidate sequences which, for example, align with a Kabat sequence
for residues
34 and 36 but have no residue between them to align with residue 35, the
number 35 is
simply not assigned to a residue.
As used herein, an antibody with a "high-affinity" is an antibody having a KD,
or
dissociation constant, in the nanomolar (nM) range or better. A KD in the
"nanomolar range
or better" may be denoted by X nM, where Xis a number less than about 10.
An "affinity matured" antibody is one with one or more alterations in one or
more
CDRs thereof which result an improvement in the affinity of the antibody for
antigen,
compared to a parent antibody which does not possess those alteration(s).
Preferred affinity
matured antibodies will have nanomolar or even picomolar affinities for the
target antigen.
Affinity matured antibodies are produced by procedures known in the art. Marks
et al.
Bio/Technology 10:779-783 (1992) describes affinity maturation by VH and VL
domain
shuffling. Random mutagenesis of CDR and/or framework residues is described
by: Barbas
et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene
169:147-155
(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.
Immunol.
154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).
A "functional antigen binding site" of an antibody is one which is capable of
binding
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a target antigen. The antigen binding affinity of the antigen binding site is
not necessarily as
strong as the parent antibody from which the antigen binding site is derived,
but the ability to
bind antigen must be measurable using any one of a variety of methods known
for evaluating
antibody binding to an antigen.
An antibody having a "biological characteristic" of a designated antibody is
one
which possesses one or more of the biological characteristics of that antibody
which
distinguish it from other antibodies that bind to the same antigen.
In order to screen for antibodies which bind to an epitope on an antigen bound
by an
antibody of interest, a routine cross-blocking assay such as that described in
Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane
(1988), can
be performed.
The term "filamentous phage" refers to a viral particle capable of displaying
a
heterogenous polypeptide on its surface, and includes, without limitation, fl,
fd, Pfl, and
M13. The filamentous phage may contain a selectable marker such as
tetracycline (e.g., "fd-
tet"). Various filamentous phage display systems are well known to those of
skill in the art
(see, e.g., Zacher et al. Gene 9: 127-140 (1980), Smith et al. Science 228:
1315-1317 (1985);
and Parmley and Smith Gene 73: 305-318 (1988)).
The term "panning" is used to refer to the multiple rounds of screening
process in
identification and isolation of phages carrying compounds, such as antibodies,
with high
affinity and specificity to a target.
The term "short-interfering RNA (siRNA)" refers to small double-stranded RNAs
that interfere with gene expression. siRNAs are an intermediate of RNA
interference, the
process double-stranded RNA silences homologous genes. siRNAs typically are
comprised
of two single-stranded RNAs of about 15-25 nucleotides in length that form a
duplex, which
may include single-stranded overhang(s). Processing of the double-stranded RNA
by an
enzymatic complex, for example by polymerases, results in the cleavage of the
double-
stranded RNA to produce siRNAs. The antisense strand of the siRNA is used by
an RNA
interference (RNAi) silencing complex to guide mRNA cleavage, thereby
promoting mRNA
degradation. To silence a specific gene using siRNAs, for example in a
mammalian cell, the
base pairing region is selected to avoid chance complementarity to an
unrelated mRNA.
RNAi silencing complexes have been identified in the art, such as, for
example, by Fire et
al., Nature 391:806-811 (1998) and McManus et al., Nat. Rev. Genet. 3(10):737-
47 (2002).
The term "interfering RNA (RNAi)" is used herein to refer to a double-stranded
RNA
CA 02723430 2010-11-03
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that results in catalytic degradation of specific mRNAs, and thus can be used
to inhibit/lower
expression of a particular gene.
The term "polymorphism" is used herein to refer to more than one forms of a
gene or
a portion (e.g., allelic variant) thereof. A portion of a gene of which there
are at least two
different forms is referred to as a "polymorphic region" of the gene. A
specific genetic
sequence at a polymorphic region of a gene is an "allele." A polymorphic
region
can be a single nucleotide, which differs in different alleles, or can be
several
nucleotides long.
As used herein, the term "disorder" in general refers to any condition that
would
benefit from treatment with an antagonists of PirB/LILRB2, such as an anti-
PirB antibody,
including any condition that is expected to benefit from axon regeneration
therapy, and/or an
improvement of synaptic plasticity in the nervous system. Non-limiting
examples of
disorders to be treated herein include, without limitation, diseases and
conditions benefiting
from the enhancement of neurite outgrowth, promotion of neuronal growth,
repair or
regeneration, including neurological disorders, such as physically damaged
nerves and
neurodegenerative diseases. Such disorders specifically include physical
damage to the
central nervous system (e.g. spinal cord injury and head trauma); brain damage
associated
with stroke; and neurological disorders relating to neurodegeneration, such
as, for example,
trigeminal neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia
gravis, muscular
dystrophy, amyotrophic lateral sclerosis (ALS), progressive muscular atrophy,
progressive
bulbar inherited muscular atrophy, multiple sclerosis (MS), herniated,
ruptured or prolapsed
invertebrate disk syndromes, cervical spondylosis, plexus disorders, thoracic
outlet
destruction syndromes, peripheral nerve damage caused by physical injury or
disease states
such as diabetes, peripheral neuropathies such as those caused by lead,
dapsone, ticks,
prophyria, Gullain-Barre syndrome, Alzheimer's disease, Huntington's Disease,
or
Parkinson's disease.
The terms "treating", "treatment" and "therapy" as used herein refer to
curative
therapy, prophylactic therapy, and preventative therapy. Consecutive treatment
or
administration refers to treatment on at least a daily basis without
interruption in treatment
by one or more days. Intermittent treatment or administration, or treatment or
administration
in an intermittent fashion, refers to treatment that is not consecutive, but
rather cyclic in
nature.
The term "preventing neurodegeneration," as used herein includes (1) the
ability to
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inhibit or prevent neurodegeneration in patients newly diagnosed as having a
neurodegenerative disease or at risk of developing a new neurodegenerative
disease and (2)
the ability to inhibit or prevent further neurodegeneration in patients who
are already
suffering from, or have symptoms of, a neurodegenerative disease.
The term "mammal" as used herein refers to any mammal classified as a mammal,
including humans, higher non-human primates, rodents, domestic and farm
animals, such as
cows, horses, dogs and cats. In a preferred embodiment of the invention, the
mammal is a
human.
Administration "in combination with" one or more further therapeutic agents
includes
simultaneous (concurrent) and consecutive administration in any order.
An "effective amount" is an amount sufficient to effect beneficial or desired
therapeutic (including preventative) results. An effective amount can be
administered in one
or more administrations.
As used herein, the expressions "cell," "cell line," and "cell culture" are
used
interchangeably and all such designations include progeny. Thus, the words
"transformants"
and "transformed cells" include the primary subject cell and cultures derived
therefrom
without regard for the number of transfers. It is also understood that all
progeny may not be
precisely identical in DNA content, due to deliberate or inadvertent
mutations. The term
"progeny" refers to any and all offspring of every generation subsequent to an
originally
transformed cell or cell line. Mutant progeny that have the same function or
biological
activity as screened for in the originally transformed cell are included.
Where distinct
designations are intended, it will be clear from the context.
"Percent (%) amino acid sequence identity" with respect to the sequences
identified
herein is defined as the percentage of amino acid residues in a candidate
sequence that are
identical with the amino acid residues in a reference sequence, after aligning
the sequences
and introducing gaps, if necessary, to achieve the maximum percent sequence
identity, and
not considering any conservative substitutions as part of the sequence
identity. Alignment
for purposes of determining percent amino acid sequence identity can be
achieved in various
ways that are within the skill in the art can determine appropriate parameters
for measuring
alignment, including assigning algorithms needed to achieve maximal alignment
over the
full-length sequences being compared. For purposes herein, percent amino acid
identity
values can be obtained using the sequence comparison computer program, ALIGN-
2, which
was authored by Genentech, Inc. and the source code of which has been filed
with user
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documentation in the US Copyright Office, Washington, DC, 20559, registered
under the US
Copyright Registration No. TXU510087. The ALIGN-2 program is publicly
available
through Genentech, Inc., South San Francisco, CA. All sequence comparison
parameters are
set by the ALIGN-2 program and do not vary.
"Stringency" of hybridization reactions is readily determinable by one of
ordinary
skill in the art, and generally is an empirical calculation dependent upon
probe length,
washing temperature, and salt concentration. In general, longer probes require
higher
temperatures for proper annealing, while shorter probes need lower
temperatures.
Hybridization generally depends on the ability of denatured DNA to re-anneal
when
complementary strands are present in an environment below their melting
temperature. The
higher the degree of desired identity between the probe and hybridizable
sequence, the
higher the relative temperature which can be used. As a result, it follows
that higher relative
temperatures would tend to make the reaction conditions more stringent, while
lower
temperatures less so. For additional details and explanation of stringency of
hybridization
reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience
Publishers, (1995).
"High stringency conditions", as defined herein, are identified by those that:
(1)
employ low ionic strength and high temperature for washing; 0.015 M sodium
chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50 C; (2)
employ during
hybridization a denaturing agent; 50% (v/v) formamide with 0.1 % bovine serum
albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/5OmM sodium phosphate buffer at
pH 6.5
with 750 mM sodium chloride, 75 mM sodium citrate at 42 C; or (3) employ 50%
formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH
6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon
sperm DNA
(50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42 C, with washes at 42 C in
0.2 x SSC
(sodium chloride/sodium citrate) and 50% formamide at 55 C, followed by a high-
stringency
wash consisting of 0.1 x SSC containing EDTA at 55 C.
"Moderately stringent conditions" may be identified as described by Sambrook
et al.,
Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press,
1989, and
include overnight incubation at 37 C in a solution comprising: 20% formamide,
5 x SSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x
Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared
salmon sperm
DNA, followed by washing the filters in 1 x SSC at about 37-50 C. The skilled
artisan will
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recognize how to adjust the temperature, ionic strength, etc. as necessary to
accommodate
factors such as probe length and the like.
The term "control sequences" refers to DNA sequences necessary for the
expression
of an operably linked coding sequence in a particular host organism. The
control sequences
that are suitable for prokaryotes, for example, include a promoter, optionally
an operator
sequence, and a ribosome binding site. Eukaryotic cells are known to utilize
promoters,
polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional
relationship with
another nucleic acid sequence. For example, DNA for a presequence or secretory
leader is
operably linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in
the secretion of the polypeptide; a promoter or enhancer is operably linked to
a coding
sequence if it affects the transcription of the sequence; or a ribosome
binding site is operably
linked to a coding sequence if it is positioned so as to facilitate
translation. Generally,
"operably linked" means that the DNA sequences being linked are contiguous,
and, in the
case of a secretory leader, contiguous and in reading phase. However,
enhancers do not have
to be contiguous. Linking is accomplished by ligation at convenient
restriction sites. If such
sites do not exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance
with conventional practice.
A "small molecule" is defined herein to have a molecular weight below about
1000
Daltons, preferably below about 500 Daltons.
B. Production of Anti-PirB/LILRB Antibodies
The anti-PirB antibodies of the present invention can be produced by methods
known
in the art, including techniques of recombinant DNA technology.
i) Antigen Preparation
Soluble antigens or fragments thereof, optionally conjugated to other
molecules, can
be used as immunogens for generating antibodies. For transmembrane molecules,
such as
receptors, fragments of these (e.g. the extracellular domain of a receptor)
can be used as the
immunogen. Alternatively, cells expressing the transmembrane molecule can be
used as the
immunogen. Such cells can be derived from a natural source (e.g. cancer cell
lines) or may
be cells which have been transformed by recombinant techniques to express the
transmembrane molecule. Other antigens and forms thereof useful for preparing
antibodies
will be apparent to those in the art.
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(ii) Polyclonal Antibodies
Polyclonal antibodies are preferably raised in animals by multiple
subcutaneous (sc)
or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It
may be useful to
conjugate the relevant antigen to a protein that is immunogenic in the species
to be
immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine
thyroglobulin, or
soybean trypsin inhibitor using a bifunctional or derivatizing agent, for
example,
maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine
residues), N-
hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic
anhydride, SOCI2, or
R1N=C=NR, where R and R1 are different alkyl groups.
Animals are immunized against the antigen, immunogenic conjugates, or
derivatives
by combining, e.g., 100 g or 5 g of the protein or conjugate (for rabbits or
mice,
respectively) with 3 volumes of Freund's complete adjuvant and injecting the
solution
intradermally at multiple sites. One month later the animals are boosted with
1/5 to 1/10 the
original amount of peptide or conjugate in Freund's complete adjuvant by
subcutaneous
injection at multiple sites. Seven to 14 days later the animals are bled and
the serum is
assayed for antibody titer. Animals are boosted until the titer plateaus.
Preferably, the animal
is boosted with the conjugate of the same antigen, but conjugated to a
different protein
and/or through a different cross-linking reagent. Conjugates also can be made
in recombinant
cell culture as protein fusions. Also, aggregating agents such as alum are
suitably used to
enhance the immune response.
(iii) Monoclonal Antibodies
Monoclonal antibodies may be made using the hybridoma method first described
by
Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA
methods (U.S.
Pat. No. 4,816,567). In the hybridoma method, a mouse or other appropriate
host animal,
such as a hamster or macaque monkey, is immunized as hereinabove described to
elicit
lymphocytes that produce or are capable of producing antibodies that will
specifically bind
to the protein used for immunization. Alternatively, lymphocytes may be
immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable fusing agent,
such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:
Principles
and Practice, pp.59-103 (Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a suitable culture
medium
that preferably contains one or more substances that inhibit the growth or
survival of the
unfused, parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme
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hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium
for the hybridomas typically will include hypoxanthine, aminopterin, and
thymidine (HAT
medium), which substances prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable high-
level
production of antibody by the selected antibody-producing cells, and are
sensitive to a
medium such as HAT medium. Among these, preferred myeloma cell lines are
murine
myeloma lines, such as those derived from MOPC-21 and MPC-1I mouse tumors
available
from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and
SP-2 or X63-
Ag8-653 cells available from the American Type Culture Collection, Rockville,
Md. USA.
Human myeloma and mouse-human heteromyeloma cell lines also have been
described for
the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001
(1984);
Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.
51-63
(Marcel Dekker, Inc., New York, 1987)).
Culture medium in which hybridoma cells are growing is assayed for production
of
monoclonal antibodies directed against the antigen. Preferably, the binding
specificity of
monoclonal antibodies produced by hybridoma cells is determined by
immunoprecipitation
or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-
linked
immunoabsorbent assay (ELISA).
After hybridoma cells are identified that produce antibodies of the desired
specificity,
affinity, and/or activity, the clones may be subloned by limiting dilution
procedures and
grown by standard methods (Goding, MonoclonalAntibodies: Principles and
Practice, pp.59-
103 (Academic Press, 1986)). Suitable culture media for this purpose include,
for example,
D-MEM or RPMI- 1640 medium. In addition, the hybridoma cells may be grown in
vivo as
ascites tumors in an animal.
The monoclonal antibodies secreted by the subclones are suitably separated
from the
culture medium, ascites fluid, or serum by conventional immunoglobulin
purification
procedures such as, for example, protein A-Sepharose, hydroxylapatite
chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
DNA encoding the monoclonal antibodies is readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that are
capable of binding
specifically to genes encoding the heavy and light chains of the monoclonal
antibodies). The
hybridoma cells serve as a preferred source of such DNA. Once isolated, the
DNA may be
placed into expression vectors, which are then transfected into host cells
such as E. coli cells,
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simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do
not
otherwise produce immunoglobulin protein, to obtain the synthesis of
monoclonal antibodies
in the recombinant host cells. Recombinant production of antibodies will be
described in
more detail below.
In a further embodiment, antibodies or antibody fragments can be isolated from
antibody phage libraries generated using the techniques described in
McCafferty et al.,
Nature, 348:552-554 (1990).
Clackson et al., Nature, 352:624-628 (1991) and Marks etal., J. Mol. Biol.,
222:581-
597 (1991) describe the isolation of murine and human antibodies,
respectively, using phage
libraries. Subsequent publications describe the production of high affinity
(nM range) human
antibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783
(1992)), as well as
combinatorial infection and in vivo recombination as a strategy for
constructing very large
phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)).
Thus, these
techniques are viable alternatives to traditional monoclonal antibody
hybridoma techniques
for isolation of monoclonal antibodies.
The DNA also may be modified, for example, by substituting the coding sequence
for
human heavy- and light-chain constant domains in place of the homologous
murine
sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci.
USA, 81:6851
(1984)), or by covalently joining to the immunoglobulin coding sequence all or
part of the
coding sequence for a non-immunoglobulin polypeptide.
Typically such non-immunoglobulin polypeptides are substituted for the
constant
domains of an antibody, or they are substituted for the variable domains of
one antigen-
combining site of an antibody to create a chimeric bivalent antibody
comprising one antigen-
combining site having specificity for an antigen and another antigen-combining
site having
specificity for a different antigen.
(iv) Humanized and Human Antibodies
A humanized antibody has one or more amino acid residues introduced into it
from a
source which is non-human. These non-human amino acid residues are often
referred to as
"import" residues, which are typically taken from an "import" variable domain.
Humanization can be essentially performed following the method of Winter and
co-workers
(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-
327 (1988);
Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs
or CDR
sequences for the corresponding sequences of a human antibody. Accordingly,
such
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"humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)
wherein
substantially less than an intact human variable domain has been substituted
by the
corresponding sequence from a non-human species. In practice, humanized
antibodies are
typically human antibodies in which some CDR residues and possibly some FR
residues are
substituted by residues from analogous sites in rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in
making
the humanized antibodies is very important to reduce antigenicity. According
to the so-called
"best-fit" method, the sequence of the variable domain of a rodent antibody is
screened
against the entire library of known human variable-domain sequences. The human
sequence
which is closest to that of the rodent is then accepted as the human framework
(FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al.,
J. Mol. Biol.,
196:901 (1987)). Another method uses a particular framework derived from the
consensus
sequence of all human antibodies of a particular subgroup of light or heavy
chains. The same
framework may be used for several different humanized antibodies (Carter et
al., Proc. Natl.
Acad Sci. USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 (1993)).
It is further important that antibodies be humanized with retention of high
affinity for
the antigen and other favorable biological properties. To achieve this goal,
according to a
preferred method, humanized antibodies are prepared by a process of analysis
of the parental
sequences and various conceptual humanized products using three-dimensional
models of
the parental and humanized sequences. Three-dimensional immunoglobulin models
are
commonly available and are familiar to those skilled in the art. Computer
programs are
available which illustrate and display probable three-dimensional
conformational structures
of selected candidate immunoglobulin sequences. Inspection of these displays
permits
analysis of the likely role of the residues in the functioning of the
candidate immunoglobulin
sequence, i.e., the analysis of residues that influence the ability of the
candidate
immunoglobulin to bind its antigen. In this way, FR residues can be selected
and combined
from the recipient and import sequences so that the desired antibody
characteristic, such as
increased affinity for the target antigen(s), is achieved. In general, the CDR
residues are
directly and most substantially involved in influencing antigen binding.
Alternatively, it is now possible to produce transgenic animals (e.g., mice)
that are
capable, upon immunization, of producing a full repertoire of human antibodies
in the
absence of endogenous immunoglobulin production. For example, it has been
described that
the homozygous deletion of the antibody heavy-chain joining region (J<sub>H</sub>)
gene in
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chimeric and germ-line mutant mice results in complete inhibition of
endogenous antibody
production. Transfer of the human germ-line immunoglobulin gene array in such
germ-line
mutant mice will result in the production of human antibodies upon antigen
challenge. See,
e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits
et al., Nature,
362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and
Duchosal et al.
Nature 355:258 (1992). Human antibodies can also be derived from phage-display
libraries
(Hoogenboom et al, J. Mot. Biol., 227:381 (1991); Marks et al, J. MoL Biol.,
222:581-597
(1991); Vaughan et al. Nature Biotech 14:309 (1996)). Generation of human
antibodies
from antibody phage display libraries is further described below.
(v) Antibody Fragments
Various techniques have been developed for the production of antibody
fragments.
Traditionally, these fragments were derived via proteolytic digestion of
intact antibodies
(see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods
24:107-117
(1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments
can now be
produced directly by recombinant host cells. For example, the antibody
fragments can be
isolated from the antibody phage libraries discussed above. Alternatively,
Fab'-SH
fragments can be directly recovered from E. coli and chemically coupled to
form F(ab')2
fragments (Carter et al., Bio/Technology 10:163-167 (1992)). In another
embodiment as
described in the example below, the F(ab')2 is formed using the leucine zipper
GCN4 to
promote assembly of the F(ab')2 molecule. According to another approach,
F(ab')2 fragments
can be isolated directly from recombinant host cell culture. Other techniques
for the
production of antibody fragments will be apparent to the skilled practitioner.
In other
embodiments, the antibody of choice is a single chain Fv fragment (scFv). See
WO
93/16185.
(vi) Multispecific Antibodies
Multispecific antibodies have binding specificities for at least two different
epitopes,
where the epitopes are usually from different antigens. While such molecules
normally will
only bind two different epitopes (i.e. bispecific antibodies, BsAbs),
antibodies with
additional specificities such as trispecific antibodies are encompassed by
this expression
when used herein. Examples of BsAbs include those with one arm directed
against
PirB/LILRB2 and another arm directed against Nogo or MAG or OMgp. A further
example
of BsABs include those with one arm directed against PirB/LILRB2 and another
arm
directed against NgR.
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Methods for making bispecific antibodies are known in the art. Traditional
production of full length bispecific antibodies is based on the coexpression
of two
immunoglobulin heavy chain-light chain pairs, where the two chains have
different
specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the
random
assortment of immunoglobulin heavy and light chains, these hybridomas
(quadromas)
produce a potential mixture of 10 different antibody molecules, of which only
one has the
correct bispecific structure. Purification of the correct molecule, which is
usually done by
affinity chromatography steps, is rather cumbersome, and the product yields
are low. Similar
procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J.,
10:3655-3659
(1991). According to a different approach, antibody variable domains with the
desired
binding specificities (antibody-antigen combining sites) are fused to
immunoglobulin
constant domain sequences. The fusion preferably is with an immunoglobulin
heavy chain
constant domain, comprising at least part of the hinge, CH2, and CH3 regions.
It is preferred
to have the first heavy-chain constant region (CHI) containing the site
necessary for light
chain binding, present in at least one of the fusions. DNAs encoding the
immunoglobulin
heavy chain fusions and, if desired, the immunoglobulin light chain, are
inserted into
separate expression vectors, and are co-transfected into a suitable host
organism. This
provides for great flexibility in adjusting the mutual proportions of the
three polypeptide
fragments in embodiments when unequal ratios of the three polypeptide chains
used in the
construction provide the optimum yields. It is, however, possible to insert
the coding
sequences for two or all three polypeptide chains in one expression vector
when the
expression of at least two polypeptide chains in equal ratios results in high
yields or when
the ratios are of no particular significance.
In a preferred embodiment of this approach, the bispecific antibodies are
composed of
a hybrid immunoglobulin heavy chain with a first binding specificity in one
arm, and a
hybrid immunoglobulin heavy chain-light chain pair (providing a second binding
specificity)
in the other arm. It was found that this asymmetric structure facilitates the
separation of the
desired bispecific compound from unwanted immunoglobulin chain combinations,
as the
presence of an immunoglobulin light chain in only one half of the bispecific
molecule
provides for a facile way of separation. This approach is disclosed in WO
94/04690. For
further details of generating bispecific antibodies see, for example, Suresh
et al., Methods in
Enzymology, 121:210 (1986).
According to another approach described in W096/27011, the interface between a
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pair of antibody molecules can be engineered to maximize the percentage of
heterodimers
which are recovered from recombinant cell culture. The preferred interface
comprises at
least a part of the CH3 domain of an antibody constant domain. In this method,
one or more
small amino acid side chains from the interface of the first antibody molecule
are replaced
with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or
similar size to the large side chain(s) are created on the interface of the
second antibody
molecule by replacing large amino acid side chains with smaller ones (e.g.
alanine or
threonine). This provides a mechanism for increasing the yield of the
heterodimer over other
unwanted end-products such as homodimers.
Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
For
example, one of the antibodies in the heteroconjugate can be coupled to
avidin, the other to
biotin. Such antibodies have, for example, been proposed to target immune
system cells to
unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection
(WO 91/00360,
WO 92/200373). Heteroconjugate antibodies may be made using any convenient
cross-
linking methods. Suitable cross-linking agents are well known in the art, and
are disclosed
in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
Techniques for generating bispecific antibodies from antibody fragments have
also
been described in the literature. For example, bispecific antibodies can be
prepared using
chemical linkage. Brennan et al., Science 229: 81 (1985) describe a procedure
wherein
intact antibodies are proteolytically cleaved to generate F(ab')2 fragments.
These fragments
are reduced in the presence of the dithiol complexing agent sodium arsenite to
stabilize
vicinal dithiols and prevent intermolecular disulfide formation. The Fab'
fragments
generated are then converted to thionitrobenzoate (TNB) derivatives. One of
the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is
mixed with an equimolar amount of the other Fab'-TNB derivative to form the
bispecific
antibody. The bispecific antibodies produced can be used as agents for the
selective
immobilization of enzymes.
Fab'-SH fragments can also be directly recovered from E. coli, and can be
chemically
coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-
225 (1992)
describe the production of a fully humanized bispecific antibody F(ab')2
molecule. Each Fab'
fragment was separately secreted from E. coli and subjected to directed
chemical coupling in
vitro to form the bispecific antibody.
Various techniques for making and isolating bispecific antibody fragments
directly
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from recombinant cell culture have also been described. For example,
bispecific antibodies
have been produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553
(1992). The leucine zipper peptides from the Fos and Jun proteins were linked
to the Fab'
portions of two different antibodies by gene fusion. The antibody homodimers
were reduced
at the hinge region to form monomers and then re-oxidized to form the antibody
heterodimers. This method can also be utilized for the production of antibody
homodimers.
The "diabody" technology described by Hollinger et al., Proc. Nati. Acad. Sci.
USA,
90:6444-6448 (1993) has provided an alternative mechanism for making
bispecific antibody
fragments. The fragments comprise a heavy-chain variable domain (VH) connected
to a
light-chain variable domain (VL) by a linker which is too short to allow
pairing between the
two domains on the same chain. Accordingly, the VH and VL domains of one
fragment are
forced to pair with the complementary VL and VH domains of another fragment,
thereby
forming two antigen-binding sites. Another strategy for making bispecific
antibody
fragments by the use of single-chain Fv (sFv) dimers has also been reported.
See Gruber et
al, J. Immunol, 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific
antibodies can be prepared. Tuft et al. J. Immunol. 147: 60 (1991).
(vii) Effector Function Engineering
It may be desirable to modify the antibody of the invention with respect to
effector
function, so as to enhance the effectiveness of the antibody. For example
cysteine residue(s)
may be introduced in the Fc region, thereby allowing interchain disulfide bond
formation in
this region. The homodimeric antibody thus generated may have improved
internalization
capability and/or increased complement-mediated cell killing and antibody-
dependent
cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195
(1992) and
Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-
tumor activity may also be prepared using heterobifunctonal cross-linkers as
described in
Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody
can be
engineered which has dual Fc regions and may thereby have enhanced complement
lysis and
ADCC capabilities. See Stevenson et al Anti-Cancer Drug Design 3:219-230
(1989).
(viii) Antibody Salvage Receptor Binding Epitope Fusions.
In certain embodiments of the invention, it may be desirable to use an
antibody
fragment, rather than an intact antibody, to increase tumor penetration, for
example. In this
case, it may be desirable to modify the antibody fragment in order to increase
its serum half
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life. This may be achieved, for example, by incorporation of a salvage
receptor binding
epitope into the antibody fragment (e.g. by mutation of the appropriate region
in the antibody
fragment or by incorporating the epitope into a peptide tag that is then fused
to the antibody
fragment at either end or in the middle, e.g., by DNA or peptide synthesis).
The salvage receptor binding epitope preferably constitutes a region wherein
any one
or more amino acid residues from one or two loops of a Fc domain are
transferred to an
analogous position of the antibody fragment. Even more preferably, three or
more residues
from one or two loops of the Fc domain are transferred. Still more preferred,
the epitope is
taken from the CH2 domain of the Fc region (e.g., of an IgG) and transferred
to the CHI,
CH3, or V<sub>H</sub> region, or more than one such region, of the antibody.
Alternatively, the
epitope is taken from the CH2 domain of the Fc region and transferred to the
CL region or
VL region, or both, of the antibody fragment.
(ix) Other Covalent Modifications of Antibodies
Covalent modifications of antibodies are included within the scope of this
invention.
They may be made by chemical synthesis or by enzymatic or chemical cleavage of
the
antibody, if applicable. Other types of covalent modifications of the antibody
are introduced
into the molecule by reacting targeted amino acid residues of the antibody
with an organic
derivatizing agent that is capable of reacting with selected side chains or
the N- or C-
terminal residues. Examples of covalent modifications are described in U.S.
Pat. No.
5,534,615, specifically incorporated herein by reference. A preferred type of
covalent
modification of the antibody comprises linking the antibody to one of a
variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;
4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
(x) Generation of Antibodies From Synthetic Antibody Phage Libraries
In a preferred embodiment, the invention provides a method for generating and
selecting novel antibodies using a unique phage display approach. The approach
involves
generation of synthetic antibody phage libraries based on single framework
template, design
of sufficient diversities within variable domains, display of polypeptides
having the
diversified variable domains, selection of candidate antibodies with high
affinity to target the
antigen, and isolation of the selected antibodies.
Details of the phage display methods can be found, for example, W003/102157
published December 11, 2003, the entire disclosure of which is expressly
incorporated herein
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by reference.
In one aspect, the antibody libraries used in the invention can be generated
by
mutating the solvent accessible and/or highly diverse positions in at least
one CDR of an
antibody variable domain. Some or all of the CDRs can be mutated using the
methods
provided herein. In some embodiments, it may be preferable to generate diverse
antibody
libraries by mutating positions in CDRH1, CDRH2 and CDRH3 to form a single
library or
by mutating positions in CDRL3 and CDRH3 to form a single library or by
mutating
positions in CDRL3 and CDRHI, CDRH2 and CDRH3 to form a single library.
A library of antibody variable domains can be generated, for example, having
mutations in the solvent accessible and/or highly diverse positions of CDRHl,
CDRH2 and
CDRH3. Another library can be generated having mutations in CDRL 1, CDRL2 and
CDRL3. These libraries can also be used in conjunction with each other to
generate binders
of desired affinities. For example, after one or more rounds of selection of
heavy chain
libraries for binding to a target antigen, a light chain library can be
replaced into the
population of heavy chain binders for further rounds of selection to increase
the affinity of
the binders.
Preferably, a library is created by substitution of original amino acids with
variant
amino acids in the CDRH3 region of the variable region of the heavy chain
sequence. The
resulting library can contain a plurality of antibody sequences, wherein the
sequence
diversity is primarily in the CDRH3 region of the heavy chain sequence.
In one aspect, the library is created in the context of the humanized antibody
4D5
sequence, or the sequence of the framework amino acids of the humanized
antibody 4D5
sequence. Preferably, the library is created by substitution of at least
residues 95-100a of the
heavy chain with amino acids encoded by the DVK codon set, wherein the DVK
codon set is
used to encode a set of variant amino acids for every one of these positions.
An example of
an oligonucleotide set that is useful for creating these substitutions
comprises the sequence
(DVK)7. In some embodiments, a library is created by substitution of residues
95-100a with
amino acids encoded by both DVK and NNK codon sets. An example of an
oligonucleotide
set that is useful for creating these substitutions comprises the sequence
(DVK)6 (NNK). In
another embodiment, a library is created by substitution of at least residues
95-100a with
amino acids encoded by both DVK and NNK codon sets. An example of an
oligonucleotide
set that is useful for creating these substitutions comprises the sequence
(DVK)5 (NNK).
Another example of an oligonucleotide set that is useful for creating these
substitutions
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comprises the sequence (NNK)6. Other examples of suitable oligonucleotide
sequences can
be determined by one skilled in the art according to the criteria described
herein.
In another embodiment, different CDRH3 designs are utilized to isolate high
affinity
binders and to isolate binders for a variety of epitopes. The range of lengths
of CDRH3
generated in this library is 11 to 13 amino acids, although lengths different
from this can also
be generated. H3 diversity can be expanded by using NNK, DVK and NVK codon
sets, as
well as more limited diversity at N and/or C-terminal.
Diversity can also be generated in CDRH1 and CDRH2. The designs of CDR-H1
and H2 diversities follow the strategy of targeting to mimic natural
antibodies repertoire as
1 o described with modification that focus the diversity more closely matched
to the natural
diversity than previous design.
For diversity in CDRH3, multiple libraries can be constructed separately with
different lengths of H3 and then combined to select for binders to target
antigens. The
multiple libraries can be pooled and sorted using solid support selection and
solution sorting
methods as described previously and herein below. Multiple sorting satrategies
may be
employed. For example, one variation involves sorting on target bound to a
solid, followed
by sorting for a tag that may be present on the fusion polypeptide (eg. anti-
gD tag) and
followed by another sort on target bound to solid. Alternatively, the
libraries can be sorted
first on target bound to a solid surface, the eluted binders are then sorted
using solution phase
binding with decreasing concentrations of target antigen. Utilizing
combinations of different
sorting methods provides for minimization of selection of only highly
expressed sequences
and provides for selection of a number of different high affinity clones.
High affinity binders for the target antigen can be isolated from the
libraries.
Limiting diversity in the H1/H2 region decreases degeneracy about 104 to 105
fold and
allowing more H3 diversity provides for more high affinity binders. Utilizing
libraries with
different types of diversity in CDRH3 (eg. utilizing DVK or NVT) provides for
isolation of
binders that may bind to different epitopes of a target antigen.
Of the binders isolated from the pooled libraries as described above, it has
been
discovered that affinity may be further improved by providing limited
diversity in the light
chain. Light chain diversity is generated in this embodiment as follows in
CDRL1: amino
acid position 28 is encoded by RDT; amino acid position 29 is encoded by RKT;
amino acid
position 30 is encoded by RVW; amino acid position 31 is encoded by ANW; amino
acid
position 32 is encoded by THT; optionally, amino acid position 33 is encoded
by CTG ; in
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CDRL2: amino acid position 50 is encoded by KBG; amino acid position 53 is
encoded by
AVC; and optionally, amino acid position 55 is encoded by GMA ; in CDRL3:
amino acid
position 91 is encoded by TMT or SRT or both; amino acid position 92 is
encoded by DMC;
amino acid position 93 is encoded by RVT; amino acid position 94 is encoded by
NHT; and
amino acid position 96 is encoded by TWT or YKG or both.
In another embodiment, a library or libraries with diversity in CDRHI, CDRH2
and
CDRH3 regions is generated. In this embodiment, diversity in CDRH3 is
generated using a
variety of lengths of H3 regions and using primarily codon sets XYZ and NNK or
NNS.
Libraries can be formed using individual oligonucleotides and pooled or
oligonucleotides
can be pooled to form a subset of libraries. The libraries of this embodiment
can be sorted
against target bound to solid. Clones isolated from multiple sorts can be
screened for
specificity and affinity using ELISA assays. For specificity, the clones can
be screened
against the desired target antigens as well as other nontarget antigens. Those
binders to the
target antigen can then be screened for affinity in solution binding
competition ELISA assay
or spot competition assay. High affinity binders can be isolated from the
library utilizing
XYZ codon sets prepared as described above. These binders can be readily
produced as
antibodies or antigen binding fragments in high yield in cell culture.
In some embodiments, it may be desirable to generate libraries with a greater
diversity in lengths of CDRH3 region. For example, it may be desirable to
generate libraries
with CDRH3 regions ranging from about 7 to 19 amino acids.
High affinity binders isolated from the libraries of these embodiments are
readily
produced in bacterial and eukaryotic cell culture in high yield. The vectors
can be designed
to readily remove sequences such as gD tags, viral coat protein component
sequence, and/or
to add in constant region sequences to provide for production of full length
antibodies or
antigen binding fragments in high yield.
A library with mutations in CDRH3 can be combined with a library containing
variant versions of other CDRs, for example CDRLI, CDRL2, CDRL3, CDRHI and/or
CDRH2. Thus, for example, in one embodiment, a CDRH3 library is combined with
a
CDRL3 library created in the context of the humanized 4D5 antibody sequence
with variant
amino acids at positions 28, 29, 30,31, and/or 32 using predetermined codon
sets. In another
embodiment, a library with mutations to the CDRH3 can be combined with a
library
comprising variant CDRH 1 and/or CDRH2 heavy chain variable domains. In one
embodiment, the CDRHI library is created with the humanized antibody 4D5
sequence with
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variant amino acids at positions 28, 30, 31, 32 and 33. A CDRH2 library may be
created
with the sequence of humanized antibody 4D5 with variant amino acids at
positions 50, 52,
53, 54, 56 and 58 using the predetermined codon sets.
(xi) Antibody Mutants
The novel antibodies generated from phage libraries can be further modified to
generate antibody mutants with improved physical, chemical and or biological
properties
over the parent antibody. Where the assay used is a biological activity assay,
the antibody
mutant preferably has a biological activity in the assay of choice which is at
least about 10
fold better, preferably at least about 20 fold better, more preferably at
least about 50 fold
better, and sometimes at least about 100 fold or 200 fold better, than the
biological activity of
the parent antibody in that assay. For example, an anti-PirB/LILRB antibody
mutant
preferably has a binding affinity for PirB/LILRB which is at least about 10
fold stronger,
preferably at least about 20 fold stronger, more preferably at least about 50
fold stronger, and
sometimes at least about 100 fold or 200 fold stronger, than the binding
affinity of the parent
antibody.
To generate the antibody mutant, one or more amino acid alterations (e.g.
substitutions) are introduced in one or more of the hypervariable regions of
the parent
antibody. Alternatively, or in addition, one or more alterations (e.g.
substitutions) of
framework region residues may be introduced in the parent antibody where these
result in an
improvement in the binding affinity of the antibody mutant for the antigen
from the second
mammalian species. Examples of framework region residues to modify include
those which
non-covalently bind antigen directly (Amit et al. (1986) Science 233:747-753);
interact
with/effect the conformation of a CDR (Chothia et al. (1987) J Mol. Biol.
196:901-917);
and/or participate in the VL - VH interface (EP 239 400B1). In certain
embodiments,
modification of one or more of such framework region residues results in an
enhancement of
the binding affinity of the antibody for the antigen from the second mammalian
species. For
example, from about one to about five framework residues may be altered in
this
embodiment of the invention. Sometimes, this may be sufficient to yield an
antibody mutant
suitable for use in preclinical trials, even where none of the hypervariable
region residues
have been altered. Normally, however, the antibody mutant will comprise
additional
hypervariable region alteration(s).
The hypervariable region residues which are altered may be changed randomly,
especially where the starting binding affinity of the parent antibody is such
that such
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randomly produced antibody mutants can be readily screened.
One useful procedure for generating such antibody mutants is called "alanine
scanning mutagenesis" (Cunningham and Wells (1989) Science 244:1081-1085).
Here, one
or more of the hypervariable region residue(s) are replaced by alanine or
polyalanine
residue(s) to affect the interaction of the amino acids with the antigen from
the second
mammalian species. Those hypervariable region residue(s) demonstrating
functional
sensitivity to the substitutions then are refined by introducing further or
other mutations at or
for the sites of substitution. Thus, while the site for introducing an amino
acid sequence
variation is predetermined, the nature of the mutation per se need not be
predetermined. The
ala-mutants produced this way are screened for their biological activity as
described herein.
Normally one would start with a conservative substitution such as those shown
below
under the heading of "preferred substitutions". If such substitutions result
in a change in
biological activity (e.g. binding affinity), then more substantial changes,
denominated
"exemplary substitutions" in the following table, or as further described
below in reference
to amino acid classes, are introduced and the products screened.
Preferred substitutions:
Original Residue Exemplary Preferred
Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gin; asn lys
Asn (N) gln; his; iys; arg gln
Asp (D) glu giu
Cys (C) ser ser
Gln (Q) asn asn
Glu (E) asp asp
Gly (G) pro; ala ala
His (H) asn; gin; lys; arg arg
lie (I) leu; val; met; aia; phe; leu
norleucine
Leu (L) norleucine; ile; val; met; ala; ile
phe
Lys (K) arg; gin; asn arg
Met (M) leu; phe; iie ieu
Phe (F) ieu; val; iie; ala; tyr leu
Pro (P) aia ala
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Ser(S) thr thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe; ala; leu
norleucine
Even more substantial modifications in the antibodies biological properties
are
accomplished by selecting substitutions that differ significantly in their
effect on maintaining
(a) the structure of the polypeptide backbone in the area of the substitution,
for example, as a
sheet or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target
site, or (c) the bulk of the side chain. Naturally occurring residues are
divided into groups
based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr, asn, g1n;
(3) acidic: asp, glu;
(4) basic: his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these
classes for another class.
In another embodiment, the sites selected for modification are affinity
matured using
phage display (see above).
Nucleic acid molecules encoding amino acid sequence mutants are prepared by a
variety of methods known in the art. These methods include, but are not
limited to,
oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and
cassette
mutagenesis of an earlier prepared mutant or a non-mutant version of the
parent antibody.
The preferred method for making mutants is site directed mutagenesis (see,
e.g., Kunkel
(1985) Proc. Natl. Acad. Sci. USA 82:488).
In certain embodiments, the antibody mutant will only have a single
hypervariable
region residue substituted. In other embodiments, two or more of the
hypervariable region
residues of the parent antibody will have been substituted, e.g. from about
two to about ten
hypervariable region substitutions.
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Ordinarily, the antibody mutant with improved biological properties will have
an
amino acid sequence having at least 75% amino acid sequence identity or
similarity with the
amino acid sequence of either the heavy or light chain variable domain of the
parent
antibody, more preferably at least 80%, more preferably at least 85%, more
preferably at
least 90%, and most preferably at least 95%. Identity or similarity with
respect to this
sequence is defined herein as the percentage of amino acid residues in the
candidate
sequence that are identical (i. e same residue) or similar (i.e. amino acid
residue from the
same group based on common side-chain properties, see above) with the parent
antibody
residues, after aligning the sequences and introducing gaps, if necessary, to
achieve the
maximum percent sequence identity. None of N-terminal, C-terminal, or internal
extensions,
deletions, or insertions into the antibody sequence outside of the variable
domain shall be
construed as affecting sequence identity or similarity.
Following production of the antibody mutant, the biological activity of that
molecule
relative to the parent antibody is determined. As noted above, this may
involve determining
the binding affinity and/or other biological activities of the antibody. In a
preferred
embodiment of the invention, a panel of antibody mutants is prepared and
screened for
binding affinity for the antigen or a fragment thereof. One or more of the
antibody mutants
selected from this initial screen are optionally subjected to one or more
further biological
activity assays to confirm that the antibody mutant(s) with enhanced binding
affinity are
indeed useful, e.g. for preclinical studies.
The antibody mutant(s) so selected may be subjected to further modifications,
oftentimes depending on the intended use of the antibody. Such modifications
may involve
further alteration of the amino acid sequence, fusion to heterologous
polypeptide(s) and/or
covalent modifications such as those elaborated below. With respect to amino
acid sequence
alterations, exemplary modifications are elaborated above. For example, any
cysteine
residue not involved in maintaining the proper conformation of the antibody
mutant also may
be substituted, generally with serine, to improve the oxidative stability of
the molecule and
prevent aberrant cross linking. Conversely, cysteine bond(s) may be added to
the antibody to
improve its stability (particularly where the antibody is an antibody fragment
such as an Fv
fragment). Another type of amino acid mutant has an altered glycosylation
pattern. This may
be achieved by deleting one or more carbohydrate moieties found in the
antibody, and/or
adding one or more glycosylation sites that are not present in the antibody.
Glycosylation of
antibodies is typically either N-linked or O-linked. N-linked refers to the
attachment of the
CA 02723430 2010-11-03
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carbohydrate moiety to the side chain of an asparagine residue. The tripeptide
sequences
asparagine-X-serine and asparagine-X-threonine, where X is any amino acid
except proline,
are the recognition sequences for enzymatic attachment of the carbohydrate
moiety to the
asparagine side chain. Thus, the presence of either of these tripeptide
sequences in a
polypeptide creates a potential glycosylation site. 0-linked glycosylation
refers to the
attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to
a
hydroxyamino acid, most commonly serine or threonine, although 5-
hydroxyproline or 5-
hydroxylysine may also be used. Addition of glycosylation sites to the
antibody is
conveniently accomplished by altering the amino acid sequence such that it
contains one or
more of the above-described tripeptide sequences (for N-linked glycosylation
sites). The
alteration may also be made by the addition of, or substitution by, one or
more serine or
threonine residues to the sequence of the original antibody (for O-linked
glycosylation sites).
(xii) Recombinant Production of Antibodies
For recombinant production of an antibody, the nucleic acid encoding it is
isolated
and inserted into a replicable vector for further cloning (amplification of
the DNA) or for
expression. DNA encoding the monoclonal antibody is readily isolated and
sequenced using
conventional procedures (e.g., by using oligonucleotide probes that are
capable of binding
specifically to genes encoding the heavy and light chains of the antibody).
Many vectors are
available. The vector components generally include, but are not limited to,
one or more of
the following: a signal sequence, an origin of replication, one or more marker
genes, an
enhancer element, a promoter, and a transcription termination sequence (e.g.
as described in
U.S. Pat. No. 5,534,615, specifically incorporated herein by reference).
Suitable host cells for cloning or expressing the DNA in the vectors herein
are the
prokaryote, yeast, or higher eukaryote cells described above. Suitable
prokaryotes for this
purpose include eubacteria, such as Gram-negative or Gram-positive organisms,
for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,
Erwinia,
Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serrafia, e.g,
Serratia
marcescans, and Shigeila, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B.
licheniformis 41P disclosed in DD 266,710 published Apr. 12, 1989),
Pseudomonas such as
P. aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli
294 (ATCC
31,446), although other strains such as E. coli B, E. coli X 1776 (ATCC
31,537), and E coil
W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than
limiting.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are
36
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WO 2009/140361 PCT/US2009/043757
suitable cloning or expression hosts for antibody-encoding vectors.
Saccharomyces
cerevisiae, or common baker's yeast, is the most commonly used among lower
eukaryotic
host microorganisms. However, a number of other genera, species, and strains
are commonly
available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces
hosts
such as, e.g., K. lactic, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC
16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC
36,906), K.
thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP
183,070);
Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces
such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora,
Penicillium,
to Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
Suitable host cells for the expression of glycosylated antibody are derived
from
multicellular organisms. Examples of invertebrate cells include plant and
insect cells.
Numerous baculoviral strains and variants and corresponding permissive insect
host cells
from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti
(mosquito), Aedes
albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori
have been
identified. A variety of viral strains for transfection are publicly
available, e.g., the L-1
variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,
and such
viruses may be used as the virus herein according to the present invention,
particularly for
transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton,
corn, potato,
soybean, petunia, tomato, and tobacco can also be utilized as hosts.
However, interest has been greatest in vertebrate cells, and propagation of
vertebrate
cells in culture (tissue culture) has become a routine procedure. Examples of
useful
mammalian host cell lines are monkey kidney CV 1 line transformed bySV40 (COS-
7,
ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subloned for
growth in
suspension culture, Graham et al, J. Gen Virol. 36:59 (1977)); baby hamster
kidney cells
(BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al.,
Proc.
Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.
Reprod.
23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green
monkey
kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA,
ATCC
CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL
3A,
ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep
G2,
HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et
al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human
hepatoma
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line (Hep G2).
Host cells are transformed with the above-described expression or cloning
vectors for
antibody production and cultured in conventional nutrient media modified as
appropriate for
inducing promoters, selecting transformants, or amplifying the genes encoding
the desired
sequences.
The host cells used to produce the antibody of this invention may be cultured
in a
variety of media. Commercially available media such as Ham's F 10 (Sigma),
Minimal
Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified
Eagle's
Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition,
any of the
media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.
Biochem.
102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or
5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used as culture
media for
the host cells. Any of these media may be supplemented as necessary with
hormones and/or
other growth factors (such as insulin, transferrin, or epidermal growth
factor), salts (such as
sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleotides
(such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM), trace
elements
(defined as inorganic compounds usually present at final concentrations in the
micromolar
range), and glucose or an equivalent energy source. Any other necessary
supplements may
also be included at appropriate concentrations that would be known to those
skilled in the art.
The culture conditions, such as temperature, pH, and the like, are those
previously used with
the host cell selected for expression, and will be apparent to the ordinarily
skilled artisan.
When using recombinant techniques, the antibody can be produced
intracellularly, in
the periplasmic space, or directly secreted into the medium. If the antibody
is produced
intracellularly, as a first step, the particulate debris, either host cells or
lysed cells, is
removed, for example, by centrifugation or ultrafiltration. Where the antibody
is secreted
into the medium, supernatants from such expression systems are generally first
concentrated
using a commercially available protein concentration filter, for example, an
Amicon or
Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may
be included in
any of the foregoing steps to inhibit proteolysis and antibiotics may be
included to prevent
the growth of adventitious contaminants.
The antibody composition prepared from the cells can be purified using, for
example,
hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity
chromatography,
with affinity chromatography being the preferred purification technique. The
suitability of
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CA 02723430 2010-11-03
WO 2009/140361 PCT/US2009/043757
protein A as an affinity ligand depends on the species and isotype of any
immunoglobulin Fc
domain that is present in the antibody. Protein A can be used to purify
antibodies that are
based on human.gamma. 1, .gamma.2, or.gamma.4 heavy chains (Lindmark et al.,
J.
Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse
isotypes and for
human.gamma.3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which
the
affinity ligand is attached is most often agarose, but other matrices are
available.
Mechanically stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene
allow for faster flow rates and shorter processing times than can be achieved
with agarose.
Where the antibody comprises a CH 3 domain, the Bakerbond ABXTM resin (J. T.
Baker,
Phillipsburg, N.J.) is useful for purification. Other techniques for protein
purification such as
fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase
HPLC,
chromatography on silica, chromatography on heparin SEPHAROSETM chromatography
on
an anion or cation exchange resin (such as a polyaspartic acid column),
chromatofocusing,
SDS-PAGE, and ammonium sulfate precipiation are also available depending on
the
antibody to be recovered.
C. Uses of anti-PirB/LILRB antibodies
The anti-PirB/LILRB antibodies of the present invention are believed to find
use as
agents for enhancing the survival or inducing the outgrowth of nerve cells.
They are,
therefore, useful in the therapy of degenerative disorders of the nervous
system
("neurodegenerative diseases"), including, for example, physical damage to the
central
nervous system (spinal cord and brain); brain damage associated with stroke;
and
neurological disorders relating to neurodegeneration, such as, for example,
trigeminal
neuralgia, glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis,
muscular dystrophy,
amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), progressive
muscular atrophy,
progressive bulbar inherited muscular atrophy, peripheral nerve damage caused
by physical
injury (e.g., burns, wounds) or disease states such as diabetes, kidney
dysfunction or by the
toxic effects of chemotherapeutics used to treat cancer and AIDS, herniated,
ruptured or
prolapsed invertebrate disk syndromes, cervical spondylosis, plexus disorders,
thoracic outlet
destruction syndromes, peripheral neuropathies such as those caused by lead,
dapsone, ticks,
prophyria, Gullain-Barre syndrome, Alzheimer's disease, Huntington's Disease,
or
Parkinson's disease.
The anti-PirB/LILRB antibodies herein are also useful as components of culture
39
CA 02723430 2010-11-03
WO 2009/140361 PCT/US2009/043757
media for use in culturing nerve cells in vitro.
Finally, preparations comprising the anti-PirB/LILRB antibodies herein are
useful as
standards in competitive binding assays when labeled with radioiodine,
enzymes,
fluorophores, spin labels, and the like.
Therapeutic formulations of the anti-PirB/LILRB antibodies herein are prepared
for
storage by mixing the compound identified (such as an antibody) having the
desired degree
of purity with optional physiologically acceptable carriers, excipients or
stabilizers
(Remington's Pharmaceutical Sciences, supra), in the form of lyophilized cake
or aqueous
solutions. Acceptable carriers, excipients or stabilizers are nontoxic to
recipients at the
dosages and concentrations employed, and include buffers such as phosphate,
citrate and
other organic acids; antioxidants including ascorbic acid; low molecular
weight (less than
about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino
acids such as
glycine, glutamine, asparagine, arginine or lysine; monosaccharides,
disaccharides and other
carbohydrates including glucose, mannose, or dextrins; chelating agents such
as EDTA;
sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as
sodium; and/or
nonionic surfactants such as Tween, Pluronics or PEG.
The anti-PirB/LILRB antibodies to be used for in vivo administration must be
sterile.
This is readily accomplished by filtration through sterile filtration
membranes, prior to or
following lyophilization and reconstitution.
Therapeutic compositions may be placed into a container having a sterile
access port,
for example, an intravenous solution bag or vial having a stopper pierceable
by a hypodermic
injection needle.
The anti-PirB/LILRB antibodies of the present invention may be optionally
combined
with or administered in combination with neurotrophic factors including NGF,
NT-3, and/or
BDNF and used with other conventional therapies for degenerative nervous
disorders. In
addition, the anti-PirB/LILRB antibodies of the present invention can be
advantageously
administered in combination with NgR inhibitors, such as antibodies, small
molecules or
peptides, blocking the binding of Nogo-66, MAG and/or OMgp to NgR.
The route of administration is in accord with known methods, e.g. injection or
infusion by intravenous, intraperitoneal, intracerebral, intramuscular,
intraocular, intraarterial
or intralesional routes, topical administration, or by sustained release
systems as noted
below.
CA 02723430 2010-11-03
WO 2009/140361 PCT/US2009/043757
For intracerebral use, the compounds may be administered continuously by
infusion
into the fluid reservoirs of the CNS, although bolus injection is acceptable.
The compounds
are preferably administered into the ventricles of the brain or otherwise
introduced into the
CNS or spinal fluid. Administration may be performed by an indwelling catheter
using a
continuous administration means such as a pump, or it can be administered by
implantation,
e.g., intracerebral implantation, of a sustained-release vehicle. More
specifically, the
compounds can be injected through chronically implanted cannulas or
chronically infused
with the help of osmotic minipumps. Subcutaneous pumps are available that
deliver proteins
through a small tubing to the cerebral ventricles. Highly sophisticated pumps
can be refilled
through the skin and their delivery rate can be set without surgical
intervention. Examples of
suitable administration protocols and delivery systems involving a
subcutaneous pump
device or continuous intracerebroventricular infusion through a totally
implanted drug
delivery system are those used for the administration of dopamine, dopamine
agonists, and
cholinergic agonists to Alzheimer patients and animal models for Parkinson's
disease
described by Harbaugh, J. Neural Transm. Suppl., 24:271 (1987); and DeYebenes,
et al.,
Mov. Disord. 2:143 (1987).
Suitable examples of sustained release preparations include semipermeable
polymer
matrices in the form of shaped articles, e.g. films, or microcapsules.
Sustained release
matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919,
EP 58,481),
copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman, et al.,
1983,
Biopolymers 22:547), poly (2-hydroxyethyl-methacrylate) (Langer, et al., 1981,
J. Biomed.
Mater. Res. 15:167; Langer, 1982, Chem. Tech. 12:98), ethylene vinyl acetate
(Langer, et al.,
Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988A) Sustained release
compositions also
include liposomally entrapped compounds, which can be prepared by methods
known per se.
(Epstein, et al., Proc. Natl. Acad. Sci. 82:3688 (1985); Hwang, et al., Proc.
Natl. Acad. Sci.
USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324A).
Ordinarily the liposomes are of the small (about 200-800 Angstroms) unilamelar
type in
which the lipid content is greater than about 30 mol. % cholesterol, the
selected proportion
being adjusted for the optimal therapy.
An effective amount of an active compound to be employed therapeutically will
depend, for example, upon the therapeutic objectives, the route of
administration, and the
condition of the patient. Accordingly, it will be necessary for the therapist
to titer the dosage
and modify the route of administration as required to obtain the optimal
therapeutic effect. A
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WO 2009/140361 PCT/US2009/043757
typical daily dosage might range from about 1 g/kg to up to 100 mg/kg or
more, depending
on the factors mentioned above. Typically, the clinician will administer an
active compound
until a dosage is reached that repairs, maintains, and, optimally,
reestablishes neuron
function. The progress of this therapy is easily monitored by conventional
assays.
Further details of the invention are illustrated by the following non-limiting
examples.
Example 1
Expression cloning LILRB2
To identify novel receptors for inhibitory myelin proteins, an expression
cloning
approach was taken. As bait, constructs were generated that fused Alkaline
Phosphatase
(AP) to the N- and/or C-terminus of the following characterized myelin
inhibitors (human
cDNA used): Nogo66, two additional inhibitory domains of NogoA (NiR<delta>D2
and
NiG<delta>20) (Oertle T, J Neurosci. 2003, 23(13): 5393-406), MAG, and OMgp.
These
constructs were transfected into 293 cells to produce conditioned medium (in
DMEM/2%
FBS) containing the bait proteins. The cDNA library used in the screen was
comprised of
full-length human cDNA clones in expression-ready vectors generated by
Origene. These
cDNAs were compiled, arrayed, and pooled. Pools of approximately 100 cDNA's
were
transiently transfected into COS7 cells.
In particular, on Day 1, COST cells were plated at a density of 85, 000 cells
per well
in 12-well plates. On Day 2, 1 mg of pooled cDNA's were transfected per well
using the
lipid-based transfection reagent FuGENE 6 (Roche). On Day 4, screening was
performed.
Briefly, culture medium was removed from cells and replaced with 0.5 ml of 293
cell-
conditioned medium containing AP-fusion bait proteins (20-50 nM). Cells were
incubated at
room temperature for 90 minutes. The cells were then washed 3 times with
phosphate-
buffered saline (PBS), fixed for 7 minutes with 4% paraformaldehyde, washed 3
times in
HEPES-buffered saline (HBS), and heat inactivated at 65 C for 90 minutes to
destroy
endogenous AP activity. The cells were washed once in AP Buffer (100 mM NaCl,
5 mM
MgCl2, 100 mM Tris pH 9.5), and incubated in chromogenic substrate (Western
Blue,
Promega), and analyzed for presence of reaction product one hour after
incubation, and again
after overnight incubation. Positive cells were identified by the presence of
dark blue
precipitate over the surface of the membrane. Positive pools were further
broken down to
identify individual positive clones by subsequent rounds of screening.
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From the screening, the following positive hits were identified:
MAG-AP bait yielded 4 positive hits. One was the previously characterized Nogo
Receptor (Fournier et al., Nature 409, 342-346 (2001). Two of these hits were
glycolytic
processing enzymes, and deemed unlikely to be of relevance. The fourth was
annotated as
"Homo sapiens hypothetical protein from clone 643 (LOC57228), mRNA". Closer
analysis
of the cDNA revealed an alternative ORF that was homologous to the previously
described
protein SMAG.
AP-Nogo66 bait yielded 2 positive hits. One was the previously characterized
Nogo
Receptor. The other was "Homo sapiens leukocyte immunoglobulin-like receptor,
subfamily
B (with TM and ITIM domains), member 2 (LILRB2), mRNA" (SEQ ID NO: 2). This
gene
is also known by multiple alternative nomenclatures, including MIG10, ILT4,
and LIR2
(.Kubagawa et al., Proc. Natl. Acad. Sci. USA 94:5261-6 (1997); Colonna et
al., J. Exp.
Med. 186:1809-18 (1997)).
Example 2
Preparation and testing of PirB function-blocking antibodies
PirB function-blocking antibodies
Antibodies against PirB were generated by panning a synthetic phage antibody
library against the PirB extracellular domain (W. C. Liang et al., J Mol Biol
366, 815
(2007)). Antibody clones (10 g/ml) were then tested in vitro for their
ability to block
binding of AP-Nogo66 (50 nM) to PirB-expressing COST cells. The nucleotide and
amino
acid sequences of the heavy and light chain sequences of various YW259 anti-
mouse PirB
(anti-mPirB) antibodies are shown in Figures 6-16, and 17 and 18. Figures 17
and 18 also
show the hypervariable region sequences within the heavy and light chains of
YW259.2,
YW250.9 and YW259.12, respectively.
Neurite Outgrowth Assay
96-well plates pre-coated with poly-D-lysine (Biocoat, BD) were coated with
myelin
(0.75 g/ml) overnight or with AP-Nogo66 or MAG-Fc (150-300 ng/spot) for two
hours, and
then treated with laminin (10 g/ml in F-12) for 2 hours (CGN cultures) or 4
hours (DRG
cultures). Mouse P7 cerebellar neurons were cultured as previously described
(B. Zheng et
at., Proc Natl Acad Sci U S A 102, 1205 (2005)) and plated at 2X104 cells per
well. Mouse
P 10 DRG neurons were cultured as previously described (Zheng et al.,
2005,supra) and
plated at 5X103 cells per well. Cultures were grown for 22 hours at 37 C with
5% C02,
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and then fixed with 4% paraformaldehyde/10% sucrose and stained with anti -
Plll-tubulin
(TuJI, Covance). For each experiment, all conditions were performed in six
replicate wells,
from which maximum neurite lengths were measured and averages were determined
between
the six wells. Each experiment was performed at least three times with similar
results. p-
values were determined using Student's t test.
Growth Cone Collapse Assay
DRG explants were isolated by dissecting out DRG from 3-week-old mice and
slicing
them into thirds. Each DRG explant was then cultured in an individual PDL (100
g/ml)-
and laminin (10 g/ml)-coated well from an eight-well plate. At 72-hours-post-
plating,
l0 explants were incubated with AP-Nogo66 (100 nM) or myelin (3 g/ml) for 30
minutes to
stimulate collapse. Cultures were fixed with 4%paraformaldehyde/10% sucrose,
and growth
cones were then visualized by rhodamine-phalloidin (Molecular Probes) staining
and scored
for collapse. Average growth cone collapse was determined by averaging at
least 3 replicate
wells.
Results
To address whether PirB is a functional receptor for Nogo66, we focused on
juvenile
(P7) cerebellar granule neurons (CGN), for which neurite outgrowth is
inhibited when grown
on AP-Nogo66 (K.C. Wang et al., Nature 420,74 (2002)). Adult CGN have been
shown to
express PirB (J. Syken et al., Science 313,1795 (2006)), and we found that is
also the case
for juvenile CGN as assessed by RT-PCR, immunohistochemistry and in situ
hybridization
(data not shown).
First, the ability of a soluble ectodomain of PirB (PirB-His) to interfere
with AP-
Nogo66 inhibition was tested in vitro. As shown in Figure 2A, AP-Nogo66
inhibits neurite
outgrowth of P7 CGN to approximately 66% of untreated control levels.
Inclusion of PirB-
His in this assay reversed AP-Nogo66 inhibition, with neurite outgrowth
returning
essentially to control levels. These results are similar to those reported
using the ectodomain
of NgR to block inhibition by Nogo66 (B Zgeng et al., Proc. Natl. Acad. Sci.
USA 102, 1205
(2005); A. E. Fournier, et al., J. Neurosci. 22, 8876 (200); Z. L. He et al.,
Neuron 38, 177
(2003)), and indicate that PirB can bind the functionally inhibitory domain of
Nogo66, but
do not address whether endogenous PirB in CGN mediates inhibition by AP-
Nogo66.
Therefore, antibodies were generated to PirB (anti-PirB) capable of
interfering with
the PirB-Nogo66 interaction. Using a phage display platform (W.C. Liang et
al., J. Mol.
Biol. 366, 815 (2007)) directed against the extracellular domain of PirB,
multiple clones
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WO 2009/140361 PCT/US2009/043757
were screened for their ability to block binding of AP-Nogo66 to PirB. Clone
YW259.2
(hereafter referred to as aPB1), which interfered best with AP-Nogo66-PirB
binding, had a
Kd of 5 nM for PirB (see, Figures 13-16).
aPB 1 had no effect on the baseline axon growth of CGNs. However, aPB 1
significantly reduced inhibition by AP-Nogo66 or myelin in cultured CGN (Fig.
2B),
rescuing neurite outgrowth to 59% from 41% on AP-Nogo66, and 62% from 47% on
myelin.
Similar results were seen using MAG as an inhibitory substrate, or using a
different cell type
(dorsal root ganglion (DRG) neurons) (Fig. 5). These results argue that PirB
is a functional
receptor mediating long-term inhibition of neurite outgrowth.
To confirm this result, it was tested whether genetic removal of cell surface
PirB also
reversed inhibition by AP-Nogo66 or myelin, by culturing neurons from PirBTM
mice, in
which four exons encoding the transmembrane domain and part of the PirB
intracellular
domain have been removed (J. Syken et al., Science 313,1795 (2006))). CGN were
cultured
from PirBTM mice or wild-type (WT) littermates on control substrate, AP-Nogo66
or
15' myelin. On control substrate (PDL/laminin), PirBTM neurons behaved
similarly to WT
neurons (Fig. 2C). However, neurite outgrowth from PirBTM neurons was markedly
less
inhibited than from WT neurons on either AP-Nogo66 or myelin. On AP-Nogo66,
outgrowth
from WT neurons was inhibited to 50% of control levels, whereas PirBTM neurons
were
inhibited to only 66%. Similarly, on myelin, WT neurons were inhibited to 52%
of control
levels, whereas PirBTM neurons were inhibited to only 70%. Again, we saw
similar partial
disinhibition of PirBTM DRG neurons on both myelin and AP-Nogo66 (Fig. 5).
These
findings indicate that PirB is indeed a functional receptor for AP-Nogo66 and
myelin-
mediated inhibition of neurite growth. However, loss of PirB activity does not
fully rescue
outgrowth.
Since NgR has previously been described as a receptor for myelin inhibitors,
it is
possible that PirB and NgR function together to mediate inhibition of neurite
outgrowth. To
address this, both PirB and NgR function were blocked together in CGN's by
culturing
neurons from NgR-null mice in the presence of anti-PirB. As we have reported
previously
(B. Cheng et al., PNAS 2005, supra), NgR-/- CGN neurite outgrowth is inhibited
by AP-
Nogo66 or myelin to the same extent as that in WT neurons (50% and 49%; Figure
3). aPB 1
antibody treatment of NgR+/- neurons partially reversed inhibition by either
AP-Nogo66 or
myelin, as seen above for aPB 1 treatment of WT neurons. Similarly, aPB 1
treatment of
NgR-/- neurons partially reversed inhibition by AP-Nogo66, but did not provide
any further
CA 02723430 2010-11-03
WO 2009/140361 PCT/US2009/043757
rescue than that seen with aPB 1 treatment of NgR+/- neurons or WT neurons. In
contrast,
aPB I treatment of NgR-/- neurons restored neurite outgrowth on myelin to
nearly control
levels. Thus, it appears that PirB, but not NgR, is required for substrate
inhibition by
APNogo66 in CGN, but only in part. Moreover both PirB and NgR together
contribute to the
substrate inhibition imparted by myelin.
Since NgR is thought to be required for growth cone collapse in response to
various
myelin inhibitors (J.E. Kim et al., Neuron 44, 439 (2004), O.Chivatakarn et
al., J. Neurosci.
27, 7117 (2007)), it is possible that PirB is also involved in this more acute
response.
Sensory neurons from the dorsal root ganglia (DRG) of 3-week-old mice,
confirmed to
express PirB, were used for this experiment. It has been found that growth
cones in this
culture system have a high baseline level of collapse (-30%), which is further
increased by
incubation with APNogo66 or myelin (Fig. 4). This collapse was largely
abolished in NgR-/-
neurons. In addition, blocking PirB function with aPB 1 was also sufficient to
reverse growth
cone collapse by these inhibitors. Inhibiting both PirB and NgR pathways
together (using
aPB 1 treatment on neurons from NgR-/- mice) also fully reversed growth cone
collapse, but
this result was not informative since either treatment alone gave full rescue
in this assay.
In another experiment, C I QTNF5 inhibited neurite outgrowth of cereberral
granule
neurone (CGN), and this inhibition was reversed by PirB function-blocking
antibody
YW259.2. The results are shown in Figure 19.
Together, these results support a novel role for PirB as a necessary receptor
for
neurite inhibition by myelin extracts, and more specifically by the myelin-
associated
inhibitors Nogo66 and MAG. Indeed, PirB appears to be a more significant
mediator of
substrate inhibition than NgR, since removal of PirB function alone (either
genetically or
using antibodies) partially disinhibits growth on both myelin extracts- and
myelin inhibitors,
whereas genetic removal of NgR alone does not disinhibit on any of these
substrates.
However, NgR appears to play an adjunct role in mediating inhibition by myelin
extracts
(but not Nogo66), since genetic removal of NgR can augment the disinhibition
caused by
anti-PirB antibodies on myelin (but not on Nogo66). Our findings may help to
explain the
surprising lack of enhanced CST regeneration in NgR knockout mice (J.E. Kim et
al., supra,
B. Zheng et al., Proc. Natl. Acad. Sci. USA 102, 1205 (2005)), despite the
reported
regeneration or sprouting seen in rodents infused with the NgR ectodomain (S.
Li et al., J.
Neurosci. 24, 10511 (2004)). Thus, it might be necessary to remove both PirB
and NgR to
achieve significant regeneration in vivo. In addition, since on Nogo66
substrate the genetic
46
CA 02723430 2010-11-03
WO 2009/140361 PCT/US2009/043757
removal of NgR does not further augment the partial disinhibitory effect of
PirB removal, it
is likely that there are additional binding receptor(s) for Nogo66.
Although PirB appears to be a more significant receptor for substrate
inhibition than
NgR, inactivation of either PirB or NgR alone is sufficient to block the acute
growth cone
collapse caused by addition of myelin inhibitors. This observation suggests
that collapse is a
more demanding process, requiring both PirB and NgR activities, acting either
in parallel or
together. In this context, it is of interest that PirB and NgR receptors have
recently been
shown to play similar roles in limiting plasticity of synaptic connections in
the visual cortex:
in mice lacking either receptor, eye closure during a critical developmental
period results in
excessive strengthening of connections via the open eye (J. Syken et al.,
2006, supra, A.W.
McGee et al., Science 309, 2222 (2005), supra). The mechanisms responsible for
the effect
of both receptors in mediating growth cone collapse could also underlie the
commonality of
their role in ocular dominance plasticity.
The inability of adult axons to regenerate following injury is a major
obstacle to
regaining function after traumatic insults to the CNS. It has been speculated
that regeneration
potential declines as the capacity for synaptic plasticity becomes limited
with age, in an
effort to restrict the development of excess or exuberant synaptic
connections. This
speculation gains support from the finding that PirB, previously implicated in
limiting
synaptic plasticity both during development and in adulthood (J. Syken et al.,
2006, supra),
is also a mediator of axonal inhibition by myelin, providing a parallel with
the finding that
NgR, initially implicated in axonal inhibition, similarly regulates synaptic
plasticity (S. Li et
al., J Neurosci. 24, 10511 (2004)).
Our findings also broaden the repertoire of potential PirB ligands beyond the
scope of
Class I MHC molecules, to include neuronal regrowth inhibitors. Conversely,
since genetic
deletion of the known myelin inhibitor Nogo or MAG results in only a modest
decrease in
inhibition by myelin - implying that other inhibitors are present - our
findings raise the
possibility that MIICI molecules, which are normally expressed at low levels
by
oligodendrocytes, may be upregulated following injury and contribute to
outgrowth
inhibition in concert with Nogo and MAG in central myelin.
The mechanism by which PirB signals to inhibit axon growth in response to
myelin
inhibitors is not clear. However, PirB has been shown to antagonize the
function of integrin
receptors (S. Pereira et al., J Immunol. 173:5757 (2004)), and to recruit both
SKIP-1 and
SHP-2 phosphatases); either or both of these events could attenuate normal
neurite
47
CA 02723430 2010-11-03
WO 2009/140361 PCT/US2009/043757
outgrowth. Blockade of PirB activity, using the anti-PirB antibodies herein or
by other
means, provides an important new target for therapeutic interventions to
stimulate axonal
regeneration.
All references cited throughout the disclosure are hereby expressly
incorporated by
reference in their entirety
While the present invention has been described with reference to what are
considered
to be the specific embodiments, it is to be understood that the invention is
not limited to such
embodiments. To the contrary, the invention is intended to cover various
modifications and
equivalents included within the spirit and scope of the appended claims.
48
